|
Size: 76889
Comment:
|
Size: 90850
Comment:
|
| Deletions are marked like this. | Additions are marked like this. |
| Line 15: | Line 15: |
| . The ɛ4 allele of apolipoprotein E (APOE) is the major genetic risk factor for Alzheimer's disease (AD). Although there have been numerous studies attempting to elucidate theunderlying mechanism for this increased risk, how apoE4 influences AD onset and | . The ɛ4 allele of apolipoprotein E (APOE) is the major genetic risk factor for Alzheimer's disease (AD). Although there have been numerous studies attempting to elucidate theunderlying mechanism for this increased risk, how apoE4 influences AD onset and progression has yet to be proven. However, prevailing evidence suggests that the differential effects of apoE isoforms on Aβ aggregation and clearance play the major role in AD pathogenesis. Other potential mechanisms, such as the differential modulation of neurotoxicity and tau phosphorylation by apoE isoforms as well as its role in synaptic plasticity and neuroinflammation, have not been ruled out. Inconsistent results among studies have made it difficult to define whether the APOE ε4 allele represents a gain of toxic function, a loss of neuroprotective function, or both. Therapeutic strategies based on apoE propose to reduce the toxic effects of apoE4 or to restore the physiological, protective functions of apoE. |
| Line 17: | Line 17: |
| .Along with amyloid plaque formation, hyperphosphorylation of the microtubule-binding protein tau and subsequent formation of neurofibrillary tangles are hallmarks of AD pathology. The relationship between apoE and tau has not been thoroughly investigated and the results are much less clear than the association between APOE ε4 allele dose and amyloid plaque burden. Although some studies reported a positive relationship between the neurofibrillary tangle density and APOE 34 allele dosage (Ghebremedhin et al., 1998; Nagy et al., 1995; Ohm et al., 1995; Polvikoski et al., 1995), others found no clear correlation (Itoh and Yamada, 1996; Landen et al., 1996b; Morris et al., 1995; Olichney et al., 1996; Oyama et al., 1995). Unlike studies with human subjects, data from in vitro and animal models appear to be more consistent among studies. Under in vitro conditions, apoE3 binds tightly to tau and forms an SDS-stable complex through the interaction of the N-terminal domain of apoE3 and the microtubule-binding repeat regions of tau, whereas apoE4 does not interact significantly with tau (Strittmatter et al., 1994). The interaction between apoE3 and tau was prevented by the phosphorylation of tau, suggesting that apoE3 binds preferentially to nonphosphorylated tau. However, there is currently no conclusive evidence demonstrating localization of apoE to the neuronal cytosol, where the majority of tau exists under normal conditions ('''!DeMattos et al., 1999'''). Therefore, the physiological relevance of the direct interaction between apoE and tau remains to be determined. Of note, more recent data suggests the possibility that a fragment of apoE4 (1–272 amino acids), but not full-length apoE4, the secretory pathway, translocates to the cytosolic compartment, and interacts with cytoskeletal components, including tau and neurofilament (Chang et al., 2005). Alternatively, the effects of apoE on tau phosphorylation could be explained by an intracellular signaling pathway induced by apoE (Harris et al., 2004; Hoe et al., 2005a), rather than the direct interaction between apoE and tau. Although in vitro studies have provided some insights, in vivo studies proving whether an apoE-isoform dependent effect on tau even exists will be critical. | . '''p294 /8:''' Along with amyloid plaque formation, hyperphosphorylation of the microtubule-binding protein tau and subsequent formation of neurofibrillary tangles are hallmarks of AD pathology. The relationship between apoE and tau has not been thoroughly investigated and the results are much less clear than the association between APOE ε4 allele dose and amyloid plaque burden. Although some studies reported a positive relationship between the neurofibrillary tangle density and APOE ε4 allele dosage (Ghebremedhin et al., 1998; Nagy et al., 1995; Ohm et al., 1995; Polvikoski et al., 1995), others found no clear correlation (Itoh and Yamada, 1996; Landen et al., 1996b; Morris et al., 1995; Olichney et al., 1996; Oyama et al., 1995). Unlike studies with human subjects, data from in vitro and animal models appear to be more consistent among studies. Under in vitro conditions, apoE3 binds tightly to tau and forms an SDS-stable complex through the interaction of the N-terminal domain of apoE3 and the microtubule-binding repeat regions of tau, whereas apoE4 does not interact significantly with tau (Strittmatter et al., 1994). The interaction between apoE3 and tau was prevented by the phosphorylation of tau, suggesting that apoE3 binds preferentially to nonphosphorylated tau. However, there is currently no conclusive evidence demonstrating localization of apoE to the neuronal cytosol, where the majority of tau exists under normal conditions ('''!DeMattos et al., 1999'''). Therefore, the physiological relevance of the direct interaction between apoE and tau remains to be determined. Of note, more recent data suggests the possibility that a fragment of apoE4 (1–272 amino acids), but not full-length apoE4, the secretory pathway, translocates to the cytosolic compartment, and interacts with cytoskeletal components, including tau and neurofilament (Chang et al., 2005). Alternatively, the effects of apoE on tau phosphorylation could be explained by an intracellular signaling pathway induced by apoE (Harris et al., 2004; Hoe et al., 2005a), rather than the direct interaction between apoE and tau. Although in vitro studies have provided some insights, in vivo studies proving whether an apoE-isoform dependent effect on tau even exists will be critical. |
| Line 25: | Line 25: |
| . PNAS-2001-Huang-8838-43-1.pdf ref 23 MoreLater '''needs evaluation''' | . PNAS-2001-Huang-8838-43-1.pdf ref 23 '''needs evaluation''' |
| Line 30: | Line 30: |
| . I Tesseur, J Van Dorpe, K Spittaels… - The American journal of …, 2000 . Epidemiological studies have established that the epsilon 4 allele of the ApoE gene (!ApoE4 ) constitutes an important risk factor for Alzheimer's disease and might influence the outcome of central nervous system injury. The mechanism by which !ApoE4 contributes to the |
. I Tesseur, J Van Dorpe, K Spittaels, C van den Haute, D Moechars, and F van Leuven, The American journal of Pathology, 2000 . Epidemiological studies have established that the epsilon 4 allele of the ApoE gene (ApoE4) constitutes an important risk factor for Alzheimer's disease and might influence the outcome of central nervous system injury. The mechanism by which !ApoE4 contributes to thedevelopment of neurodegeneration remains unknown. To test one hypothesis or mode of action of ApoE, we generated transgenic mice that overexpressed human !ApoE4 in different cell types in the brain, using four distinct gene promoter constructs. Many transgenic mice expressing !ApoE4 in neurons developed motor problems accompanied by muscle wasting, loss of body weight, and premature death. Overexpression of human !ApoE4 in neurons resulted in hyperphosphorylation of the microtubule-associated protein tau. In three independent transgenic lines from two different promoter constructs, increased phosphorylation of protein tau was correlated with !ApoE4 expression levels. Hyperphosphorylation of protein tau increased with age. In the hippocampus, astrogliosis and ubiquitin-positive inclusions were demonstrated. These findings demonstrate that expression of ApoE in neurons results in hyperphosphorylation of protein tau and suggests a role for ApoE in neuronal cytoskeletal stability and metabolism. |
| Line 33: | Line 33: |
| . Most recently, it was shown that direct expression of ApoE in the cytosol of Neuro-2a cells is toxic. '''74''' The !ApoE4 transgenic mice are, however, not expected to express ApoE directly in the cytosol, and we also did not observe obvious signs of neurotoxicity. In addition, the hyperphosphorylation of protein tau was not restricted to brain regions expressing human ApoE as shown by immunohistochemistry. Therefore, we favor a mechanism involving an indirect interaction between ApoE and protein tau. In addition, this implies that hyper-phosphorylation of protein tau is not simply a nonspecific downstream event marking degeneration. | . '''p 961 /11''' To explain the genetic association of !ApoE4 to AD, two types of mechanisms have been proposed. First, ApoE could function as a “pathological chaperone,” affecting clearance of β-amyloid and causing amyloid deposition. (70–72) We obtained no evidence for the presence of amyloid plaques in any of our !ApoE4 transgenic mice, which of course might be because only endogenous mouse APP is present, which is less amyloidogenic. (39) The second hypothesis states that ApoE interacts with the microtubule-associated protein tau, thereby altering its phosphorylation state, and hence is involved in stabilizing the neuronal cytoskeleton. (2,73) The results presented here support this hypothesis. However, the route by which ApoE gains access to the neuronal cytoplasm has been the major criticism against this hypothesis and subject of much speculation. Most recently, it was shown that direct expression of ApoE in the cytosol of Neuro-2a cells is toxic. '''(74)''' The !ApoE4 transgenic mice are, however, not expected to express ApoE directly in the cytosol, and we also did not observe obvious signs of neurotoxicity. In addition, the hyperphosphorylation of protein tau was not restricted to brain regions expressing human ApoE as shown by immunohistochemistry. Therefore, we favor a mechanism involving an indirect interaction between ApoE and protein tau. In addition, this implies that hyper-phosphorylation of protein tau is not simply a nonspecific downstream event marking degeneration. |
| Line 37: | Line 37: |
| . K Horsburgh, MO !McCarron, F White, JAR Nicoll - Neurobiology of aging, 2000 - Elsevier . The ϵ4 allele of apolipoprotein E (APOE denotes gene; apoE denotes protein) is a major risk factor for Alzheimer's disease (AD). More recent evidence indicates an association with a poor outcome after acute brain injury including that due to head trauma and intracerebral |
. K Horsburgh, MO !McCarron, F White, JAR Nicoll - Neurobiology of aging, 2000 . The ϵ4 allele of apolipoprotein E (APOE denotes gene; apoE denotes protein) is a major risk factor for Alzheimer's disease (AD). More recent evidence indicates an association with a poor outcome after acute brain injury including that due to head trauma and intracerebral hemorrhage. APOE gene polymorphism also influences the risk of hemorrhage in cerebral amyloid angiopathy. These diverse brain disorders seem to have some mechanisms in common. The multiplicity of the roles of apoE within the central nervous system is currently being unraveled. For example, apoE can interact with amyloid β-protein and tau, proteins central to the pathogenesis of AD. In addition to these effects, it is proposed that one of the major functions of apoE is to mediate neuronal protection, repair and remodeling. In all of the different roles proposed, there are marked apoE-isoform specific differences. Although it remains to be clarified which is the most important mechanism(s) in each disorder in which apoE is involved, these isoform specific differences seem to underly a genetically determined susceptibility to outcome from acute brain injury and to AD with APOE ε4 conferring relative vulnerability. This review focuses on apoE research, from clinical studies to animal models, in AD, acute brain injury and cerebrovascular disease and explores the common mechanisms that may explain some of the complex underlying neurobiology. |
| Line 40: | Line 40: |
| . In vitro evidence is consistent with an isoform-dependent neurotrophic role of apoE. Rabbit dorsal root ganglion neurons and neuroblastoma cells incubated with lipoproteins alone have enhanced neurite outgrowth that is further enhanced in the presence of apoE E3 lipoproteins and inhibited in the presence of apoE E4 lipoproteins. [33,86]. Similar results are obtained when cells are stably transfected to secrete apoE E3 or apoE E4; in the presence of lipoproteins, cells expressing APOE ε3 have extensive neurite outgrowth whereas neurite extension is suppressed in cells expressing APOE ε4 [7]. One of the cellular events associated with neurite outgrowth is an apoE-isoform specific effect on the cytoskeleton. ApoE E3 stabilizes the formation of microtubules in vitro in contrast to apoE E4 that is associated with a destabilization of the microtubule assembly [23]. ApoE E3 can interact with tau, a microtubule associated protein, whereas apoE E4 is unable to bind tau. One hypothesis is that apoE E3 by binding to tau protects it from hyperphosphorylation and the generation of intracellular neurofibrillary tangles in AD brain. ApoE is present at high concentrations in neurons after injury in rodent and human brain [30,41– 44,56] and may be expressed at low levels normally in human brain [134] providing functional relevance between the interactions of apoE with intracellular cytoskeletal proteins. In vitro studies have also indicated that there may be a preferential accumulation of apoE E3 and E2 in neurons as compared to apoE E4 [51]. '''Some groups have proposed that apoE E3 escapes lysosomal degradation and enters the cytosol''' where it is able to interact with cytosolic proteins such as tau and microtubule associated proteins. This mechanism may account for the apoE isoform specific effect of apoE E3 in promoting neurite outgrowth and microtubule stability and the lack of such an effect with apoE E4. '''However, it has been shown that apoE remains within the endocytic pathway''' and does not escape into the cytosol and argues that the effects of apoE are mediated by actions on the cell surface and not via cytosolic interactions ['''20''']. The intracellular fate of the apoE isoforms remains a critical question. | . p251/7 In vitro evidence is consistent with an isoform-dependent neurotrophic role of apoE. Rabbit dorsal root ganglion neurons and neuroblastoma cells incubated with lipoproteins alone have enhanced neurite outgrowth that is further enhanced in the presence of apoE E3 lipoproteins and inhibited in the presence of apoE E4 lipoproteins. [33,86]. Similar results are obtained when cells are stably transfected to secrete apoE E3 or apoE E4; in the presence of lipoproteins, cells expressing APOE ε3 have extensive neurite outgrowth whereas neurite extension is suppressed in cells expressing APOE ε4 [7]. One of the cellular events associated with neurite outgrowth is an apoE-isoform specific effect on the cytoskeleton. ApoE E3 stabilizes the formation of microtubules in vitro in contrast to apoE E4 that is associated with a destabilization of the microtubule assembly [23]. ApoE E3 can interact with tau, a microtubule associated protein, whereas apoE E4 is unable to bind tau. One hypothesis is that apoE E3 by binding to tau protects it from hyperphosphorylation and the generation of intracellular neurofibrillary tangles in AD brain. ApoE is present at high concentrations in neurons after injury in rodent and human brain [30,41– 44,56] and may be expressed at low levels normally in human brain [134] providing functional relevance between the interactions of apoE with intracellular cytoskeletal proteins. In vitro studies have also indicated that there may be a preferential accumulation of apoE E3 and E2 in neurons as compared to apoE E4 [51]. '''Some groups have proposed that apoE E3 escapes lysosomal degradation and enters the cytosol''' where it is able to interact with cytosolic proteins such as tau and microtubule associated proteins. This mechanism may account for the apoE isoform specific effect of apoE E3 in promoting neurite outgrowth and microtubule stability and the lack of such an effect with apoE E4. '''However, it has been shown that apoE remains within the endocytic pathway''' and does not escape into the cytosol and argues that the effects of apoE are mediated by actions on the cell surface and not via cytosolic interactions ['''20''']. The intracellular fate of the apoE isoforms remains a critical question. |
| Line 52: | Line 52: |
| . I Tesseur, J Van Dorpe, K Bruynseels… - The American journal of …, 2000 - Elsevier . The epsilon 4 allele of the human apolipoprotein E gene (!ApoE4. constitutes an important genetic risk factor for Alzheimer's disease. Recent experimental evidence suggests that human ApoE is expressed in neurons, in addition to being synthesized in glial cells. |
. I Tesseur, J Van Dorpe, K Bruynseels, F Bronfman, R Sciot, A van Lommel, and F van Leuven, The American journal of Pathology, 2000 . The epsilon 4 allele of the human apolipoprotein E gene (!ApoE4. constitutes an important genetic risk factor for Alzheimer's disease. Recent experimental evidence suggests that human ApoE is expressed in neurons, in addition to being synthesized in glial cells. Moreover, brain regions in which neurons express ApoE seem to be most vulnerable to neurofibrillary pathology. The hypothesis that the expression pattern of human ApoE might be important for the pathogenesis of Alzheimer’s disease was tested by generating transgenic mice that express human !ApoE4 in neurons or in astrocytes of the central nervous system. Transgenic mice expressing human !ApoE4 in neurons developed axonal degeneration and gliosis in brain and in spinal cord, resulting in reduced sensorimotor capacities. In these mice, axonal dilatations with accumulation of synaptophysin, neurofilaments, mitochondria, and vesicles were documented, suggesting impairment of axonal transport. In contrast, transgenic mice expressing human !ApoE4 in astrocytes remained normal throughout life. These results suggest that expression of human ApoE in neurons of the central nervous system could contribute to impaired axonal transport and axonal degeneration. The possible contribution of hyperphosphorylation of protein Tau to the resulting phenotype is discussed. |
| Line 55: | Line 55: |
| . Epidemiological data do not provide evidence for a direct role of ApoE in central nervous system disorders, and its mechanism of action within the central nervous system is not clear. Originally, it was thought that ApoE present in neurons originated only from endocytosis. [20–22] Numerous cell culture experiments demonstrated receptor-mediated uptake of ApoE as well as effects on neurite outgrowth and cell-morphology. ['''23'''–29] However, more and more evidence indicates that intraneuronal ApoE might also originate from synthesis by neurons. ''In situ'' hybridization of brain sections of transgenic mice expressing genomic fragments containing the entire human ApoE locus, including its promoter sequences, revealed expression in neurons, besides astrocytes. [30–32] Human neuroblastoma cells were shown to synthesize both ApoE mRNA and protein. [33,34] Moreover, in situ hybridization of human brain sections conclusively demonstrated ApoE mRNA in neurons. [35] Taken together these results suggest that besides its well-known synthesis by glia cells and its role in lipid transport and metabolism, not only uptake but also synthesis of ApoE by neurons might be important. Interestingly, the brain regions expressing ApoE in neurons seemed most vulnerable to the development of neurofibrillary pathology, [36] raising the possibility that the neuronal expression pattern of human ApoE might be important in the pathogenesis of AD. | . '''p1495 /1''' Epidemiological data do not provide evidence for a direct role of ApoE in central nervous system disorders, and its mechanism of action within the central nervous system is not clear. Originally, it was thought that ApoE present in neurons originated only from endocytosis. [20–22] Numerous cell culture experiments demonstrated receptor-mediated uptake of ApoE as well as effects on neurite outgrowth and cell-morphology. ['''23'''–29] However, more and more evidence indicates that intraneuronal ApoE might also originate from synthesis by neurons. ''In situ'' hybridization of brain sections of transgenic mice expressing genomic fragments containing the entire human ApoE locus, including its promoter sequences, revealed expression in neurons, besides astrocytes. [30–32] Human neuroblastoma cells were shown to synthesize both ApoE mRNA and protein. [33,34] Moreover, in situ hybridization of human brain sections conclusively demonstrated ApoE mRNA in neurons. [35] Taken together these results suggest that besides its well-known synthesis by glia cells and its role in lipid transport and metabolism, not only uptake but also synthesis of ApoE by neurons might be important. Interestingly, the brain regions expressing ApoE in neurons seemed most vulnerable to the development of neurofibrillary pathology, [36] raising the possibility that the neuronal expression pattern of human ApoE might be important in the pathogenesis of AD. |
| Line 66: | Line 66: |
| . FE Thorngate, LL Rudel, RL Walzem… - … , and vascular biology, 2000 - Am Heart Assoc . The prevention of atherosclerosis by apolipoprotein E (apoE) is generally attributed to the removal of plasma lipoprotein remnant particles. We developed transgenic apoE-knockout mice expressing apoE specifically in the adrenal gland and found that only |
. FE Thorngate, LL Rudel, RL Walzem, DL Williams, Journal of Arteriosclosis, Thromobosis, and Vascular Biology, 2000 . The prevention of atherosclerosis by apolipoprotein E (apoE) is generally attributed to the removal of plasma lipoprotein remnant particles. We developed transgenic apoE-knockout mice expressing apoE specifically in the adrenal gland and found that only 3% of the wild-type plasma level of apoE was sufficient to normalize plasma cholesterol levels in the apoE-deficient mouse. As expected, mice expressing apoE at levels that correct hypercholesterolemia had almost no cholesteryl ester deposition in their aortas. In contrast, their nontransgenic siblings had significant atherosclerosis. Unexpectedly, we found that atherosclerosis was also reduced in 2 transgenic lines expressing too little apoE (< 1% to 2% of wild type) to correct their hypercholesterolemia. Gel exclusion chromatographic profiles of plasma lipoproteins and the size distributions of lipoproteins with density < 1.063 (low density and very low density lipoproteins), as determined by dynamic laser light scattering, were the same in mice expressing < 2 μg/mL plasma apoE and their nontransgenic littermates. We conclude that the antiatherogenic action of low levels of plasma apoE is not due to the clearance of remnant lipoproteins. Thus, low levels of apoE provided systemically, but not made in the liver or in macrophages, can block atherogenesis in the vascular wall independently of normalizing the plasma concentration of atherogenic remnant lipoprotein particles. |
| Line 69: | Line 69: |
| . Samples were electrophoretically transferred to nitrocellulose and immunostained as described 27 by use of an affinity-purified goat anti-rat apoE. | . '''p1940 /2:''' Samples were electrophoretically transferred to nitrocellulose and immunostained as described 27 by use of an affinity-purified goat anti-rat apoE. |
| Line 74: | Line 74: |
| . Neurons are unique polarized cells in which the growing axon is often located up to a meter or more from the cell body. Consequently, the intracellular movement of membrane lipids and proteins between cell bodies and axons poses a special challenge. The mechanisms of | . Neurons are unique polarized cells in which the growing axon is often located up to a meter or more from the cell body. Consequently, the intracellular movement of membrane lipids and proteins between cell bodies and axons poses a special challenge. The mechanisms of lipid transport within neurons are, for the most part, unknown although lipid transport via vesicles and via cholesterol- and sphingolipid-rich `rafts' are considered likely mechanisms. Very active anterograde and retrograde transport of lipid-containing vesicles occurs between the cell body and distal axons. However, it is becoming clear that the axon need not obtain all of its membrane constituents from the cell body. For example, the synthesis of phosphatidylcholine, the major membrane phospholipid, occurs in axons, and its synthesis at this location is required for axonal elongation. In contrast, cholesterol synthesis appears to occur only in cell bodies, and cholesterol is efficiently delivered from cell bodies to axons by anterograde transport. Cholesterol that is required for axonal growth can also be exogenously supplied from lipoproteins to axons of cultured neurons. Several studies have suggested a role for apolipoprotein E in lipid delivery for growth and regeneration of axons after a nerve injury. Alternatively, or in addition, apolipoprotein E has been proposed to be a ligand for receptors that mediate signal transduction cascades. Lipids are also transported from axons to myelin, although the importance of this process for myelination is not clear. |
| Line 76: | Line 76: |
| .''pg 91'' Another complexity regarding the role of apo E in neuronal extension is that in humans the most common isoform of apo E is apo E3 [77] and a less common isoform is apo E4. Inheritance of the allele encoding apo E4, rather than apo E3, has been implicated as a strong risk factor for the development of Alzheimer's disease [84-86]. A clue to why apo E3 and apo E4 play di¡erent roles in the development of this disease might be the observation that in several different model systems these two isoforms modulate axonal growth differently, with apo E3 stimulating growth and apo E4 either inhibiting, or not affecting, growth [81,87-90]. The mechanism by which apo E3 and apo E4 differentially mediate neuron growth is being actively investigated, but is probably not due to a different affnity of lipoprotein receptors for apo E3- versus apo E4-containing lipoproteins, since the LDL receptor and the LDL receptor-like protein bind these two apo E isoforms equally [77]. Recent studies suggest that apo E need not be present intracellularly in neurons for it to elicit its effects on growth ''' [91] ''' . Nor are the differential effects of apo E3 and E4 on neuron growth likely to be associated solely with lipid delivery, since lipid-poor apo E3 and apo E4 have the same e¡ects on growth as do their lipid-rich counterparts [90,92,93]. An exciting recent proposal is that the effect of apo E on neuron growth is mediated by signal transduction events since members of the LDL receptor superfamily - for example, the LDL receptor [94], the LDL receptor-like protein [94], the very low density lipoprotein receptor [95] and the brain-specific receptor apo ER2 [96] [also called LR7/8B [95,97,98]] - all of which bind apo E, contain a NPxY motif which associates with known signaling proteins. This motif binds to the neuronal adaptor proteins FE65 and Disabled and most likely activates an intracellular signal transduction cascade in the brain [94,95]. Therefore, a strong possibility exists that apo E in£uences axonal growth through receptor-mediated signaling events in addition to, or perhaps instead of, acting as a ligand for receptor-mediated endocytosis of lipoproteins. The mechanism by which apo E3 alike protein [94], the very low density lipoprotein receptor [95] and the brain-specific receptor apo ER2 [96] [also called LR7/8B [95,97,98]] . . . all of which bind apo E, contain a NPxY motif which associates with known signaling proteins. This motif binds to the neuronal adaptor proteins FE65 and Disabled and most likely activates an intracellular signal transduction cascade in the brain [94,95]. Therefore, a strong possibility exists that apo E in£uences axonal growth through receptor-mediated signaling events in addition to, or perhaps instead of, acting as a ligand for receptor-mediated endocytosis of lipoproteins. The mechanism by which apo E3 and E4 a¡ect these processes differently remains to be established. | .'''pg 91/8:''' Another complexity regarding the role of apo E in neuronal extension is that in humans the most common isoform of apo E is apo E3 [77] and a less common isoform is apo E4. Inheritance of the allele encoding apo E4, rather than apo E3, has been implicated as a strong risk factor for the development of Alzheimer's disease [84-86]. A clue to why apo E3 and apo E4 play di¡erent roles in the development of this disease might be the observation that in several different model systems these two isoforms modulate axonal growth differently, with apo E3 stimulating growth and apo E4 either inhibiting, or not affecting, growth [81,87-90]. The mechanism by which apo E3 and apo E4 differentially mediate neuron growth is being actively investigated, but is probably not due to a different affnity of lipoprotein receptors for apo E3- versus apo E4-containing lipoproteins, since the LDL receptor and the LDL receptor-like protein bind these two apo E isoforms equally [77]. Recent studies suggest that apo E need not be present intracellularly in neurons for it to elicit its effects on growth ''' [91] ''' . Nor are the differential effects of apo E3 and E4 on neuron growth likely to be associated solely with lipid delivery, since lipid-poor apo E3 and apo E4 have the same e¡ects on growth as do their lipid-rich counterparts [90,92,93]. An exciting recent proposal is that the effect of apo E on neuron growth is mediated by signal transduction events since members of the LDL receptor superfamily - for example, the LDL receptor [94], the LDL receptor-like protein [94], the very low density lipoprotein receptor [95] and the brain-specific receptor apo ER2 [96] [also called LR7/8B [95,97,98]] - all of which bind apo E, contain a NPxY motif which associates with known signaling proteins. This motif binds to the neuronal adaptor proteins FE65 and Disabled and most likely activates an intracellular signal transduction cascade in the brain [94,95]. Therefore, a strong possibility exists that apo E in£uences axonal growth through receptor-mediated signaling events in addition to, or perhaps instead of, acting as a ligand for receptor-mediated endocytosis of lipoproteins. The mechanism by which apo E3 alike protein [94], the very low density lipoprotein receptor [95] and the brain-specific receptor apo ER2 [96] [also called LR7/8B [95,97,98]] . . . all of which bind apo E, contain a NPxY motif which associates with known signaling proteins. This motif binds to the neuronal adaptor proteins FE65 and Disabled and most likely activates an intracellular signal transduction cascade in the brain [94,95]. Therefore, a strong possibility exists that apo E in£uences axonal growth through receptor-mediated signaling events in addition to, or perhaps instead of, acting as a ligand for receptor-mediated endocytosis of lipoproteins. The mechanism by which apo E3 and E4 a¡ect these processes differently remains to be established. |
| Line 84: | Line 84: |
| .Uncertainty remains, however, regarding the mechanism(s) underlying the differential effects of apo E isoforms on axonal extension. One possible mechanism is that the apo E isoforms bind with different affinities to one of the LDL receptor family members on neurons, thereby differentially initiating a signaling pathway that mediates axonal growth. Another possible mechanism is that apo E3 accumulates within neurons to a greater extent than does apo E4 [223,224]. In this case, the apo E isoform-specific effects on axonal growth might be attributable to differences in the amounts of intracellular apo E which have been proposed to affect the structure of the cytoskeleton and microtubule assembly. Indeed, microtubule formation was enhanced by apo E3 in cultured neuronal cells whereas apo E4 destabilized microtubule assembly [223]. This proposal is supported by the finding that apo E3 interacts more readily than apo E4 with tau and prevents tau hyperphosphorylation which, as a result, might reduce the formation of intracellular neurofibrillary tangles (deposits of tau), a hallmark of AD disease [225]. Nevertheless, this mechanism is controversial since it would imply that apo E is present in the cytosol of neurons and that endocytosed apo E escapes from degradation in the endosomal–lysosomal system [226]. Another study found no evidence that apo E escapes from the endocytic pathway into the cytosol, and also reported that the expression of apo E in the cytosol is toxic ['''227''']. | . '''p811 /6:''' Uncertainty remains, however, regarding the mechanism(s) underlying the differential effects of apo E isoforms on axonal extension. One possible mechanism is that the apo E isoforms bind with different affinities to one of the LDL receptor family members on neurons, thereby differentially initiating a signaling pathway that mediates axonal growth. Another possible mechanism is that apo E3 accumulates within neurons to a greater extent than does apo E4 [223,224]. In this case, the apo E isoform-specific effects on axonal growth might be attributable to differences in the amounts of intracellular apo E which have been proposed to affect the structure of the cytoskeleton and microtubule assembly. Indeed, microtubule formation was enhanced by apo E3 in cultured neuronal cells whereas apo E4 destabilized microtubule assembly [223]. This proposal is supported by the finding that apo E3 interacts more readily than apo E4 with tau and prevents tau hyperphosphorylation which, as a result, might reduce the formation of intracellular neurofibrillary tangles (deposits of tau), a hallmark of AD disease [225]. Nevertheless, this mechanism is controversial since it would imply that apo E is present in the cytosol of neurons and that endocytosed apo E escapes from degradation in the endosomal–lysosomal system [226]. Another study found no evidence that apo E escapes from the endocytic pathway into the cytosol, and also reported that the expression of apo E in the cytosol is toxic ['''227''']. |
| Line 88: | Line 88: |
| . Y Hashimoto, H Jiang, T Niikura, Y Ito… - The Journal of …, 2000 - Soc Neuroscience . The ε4 genotype of apolipoprotein E (!apoE4) is the most established predisposing factor in Alzheimer's disease (AD); however, it remains unclear how apoE4 contributes to the pathophysiology. Here, we report that the !apoE4 protein (!ApoE4) evokes apoptosis inneuronal cells through the low-density lipoprotein receptor-related protein (LRP) and heterotrimeric GTPases. We examined neuron/neuroblastoma hybrid F11 cells and found that these cells were killed by 30 μg/ml !ApoE4, but not by 30 μg/ml !ApoE3. !ApoE4-induced death occurred with typical features for apoptosis in time- and dose-dependent manners, and was observed in SH-SY5Y neuroblastomas, but not in glioblastomas or non-neuronal Chinese hamster ovary cells. Activated, but not native, α2-macroglobulin suppressed this ApoE4 toxicity. Suppression by the antisense oligonucleotide to LRP and inhibition by low nanomolar concentrations of LRP-associated protein RAP provided evidence for the involvement of LRP. The involvement of heterotrimeric GTPases was demonstrated by the findings that (1) ApoE4-induced death was suppressed by pertussis toxin (PTX), but not by heat-inactivated PTX; and (2) transfection with PTX-resistant mutant cDNAs of Gα₁ restored the toxicity of !ApoE4 restricted by PTX. We thus conclude that one of the neurotoxic mechanisms triggered by ApoE4 is to activate a cell type-specific apoptogenic program involving LRP and the G₁ class of GTPases and that the apoE4 gene may play a direct role in the pathogenesis of AD and other forms of dementia. . 8401.full.pdf ref as Demattos et al. (1999) '''Read Tmis One Carefully''' . !ApoE4 binds Aβ and facilitates its aggregation (Strittmatter et al., 1993; !LaDu et al., 1994, 1995). However, it is unlikely that this action is implicated in the !ApoE4 neurotoxicity, because (1) !ApoE3 binds Aβ at 20-fold higher levels than does !ApoE4 (!LaDu et al., 1994); (2) binding of Aβ to rabbit ApoE decreases Aβ toxicity in rat hippocampal neurons (Whitson et al., 1994); and (3) the N-terminal 22 kDa fragment of !ApoE4, which lacks the Aβ binding domain (Pillot et al., 1999), exhibits isoform-specific neurotoxicity (Marques et al., 1996, 1997; Tolar et al., 1997, 1999). Also, '''Demattos et al. (1999)''' demonstrated that !ApoE4 exerts neurotoxicity not through interaction with intracellular Aβ or tau. The present study was conducted to examine whether !ApoE4 has a direct action on neuronal death, and if so, with what molecular mechanism. We find that !ApoE4 exerts isoform-specific neurotoxicity through LRP and the G₁ class of GTPases. |
. Y Hashimoto, H Jiang, T Niikura, Y Ito… - The Journal of Neuroscience, 2000 . The ε4 genotype of apolipoprotein E (!apoE4) is the most established predisposing factor in Alzheimer's disease (AD); however, it remains unclear how apoE4 contributes to the pathophysiology. Here, we report that the !apoE4 protein (!ApoE4) evokes apoptosis inneuronal cells through the low-density lipoprotein receptor-related protein (LRP) and heterotrimeric GTPases. We examined neuron/neuroblastoma hybrid F11 cells and found that these cells were killed by 30 μg/ml !ApoE4, but not by 30 μg/ml !ApoE3. !ApoE4-induced death occurred with typical features for apoptosis in time- and dose-dependent manners, and was observed in SH-SY5Y neuroblastomas, but not in glioblastomas or non-neuronal Chinese hamster ovary cells. Activated, but not native, α2-macroglobulin suppressed this !ApoE4 toxicity. Suppression by the antisense oligonucleotide to LRP and inhibition by low nanomolar concentrations of LRP-associated protein RAP provided evidence for the involvement of LRP. The involvement of heterotrimeric GTPases was demonstrated by the findings that (1) !ApoE4-induced death was suppressed by pertussis toxin (PTX), but not by heat-inactivated PTX; and (2) transfection with PTX-resistant mutant cDNAs of Gα₁ restored the toxicity of !ApoE4 restricted by PTX. We thus conclude that one of the neurotoxic mechanisms triggered by !ApoE4 is to activate a cell type-specific apoptogenic program involving LRP and the G₁ class of GTPases and that the apoE4 gene may play a direct role in the pathogenesis of AD and other forms of dementia. . 8401.full.pdf ref as Demattos et al. (1999) '''Read This One Carefully''' . '''pg 8401/1''' !ApoE4 binds Aβ and facilitates its aggregation (Strittmatter et al., 1993; !LaDu et al., 1994, 1995). However, it is unlikely that this action is implicated in the !ApoE4 neurotoxicity, because (1) !ApoE3 binds Aβ at 20-fold higher levels than does !ApoE4 (!LaDu et al., 1994); (2) binding of Aβ to rabbit ApoE decreases Aβ toxicity in rat hippocampal neurons (Whitson et al., 1994); and (3) the N-terminal 22 kDa fragment of !ApoE4, which lacks the Aβ binding domain (Pillot et al., 1999), exhibits isoform-specific neurotoxicity (Marques et al., 1996, 1997; Tolar et al., 1997, 1999). Also, '''!DeMattos et al. (1999)''' demonstrated that !ApoE4 exerts neurotoxicity not through interaction with intracellular Aβ or tau. The present study was conducted to examine whether !ApoE4 has a direct action on neuronal death, and if so, with what molecular mechanism. We find that !ApoE4 exerts isoform-specific neurotoxicity through LRP and the G₁ class of GTPases. |
| Line 93: | Line 93: |
| . We have herein shown that ApoE causes death in neuronal cells in an isoform-specific manner and that at least one mechanism for neurotoxic actions of !ApoE4 is apoptosis mediated by LRP. The toxicity of !ApoE4 was observed in cells of neuroblast origin, but not in glial cells or non-neuronal CHO cells. The observed resistance of glial cells is consistent not only with the study of Crutcher et al. (1994), indicating that glial cells are resistant to neurotoxic ApoE peptides, but also with the well established finding that LRP is found abundantly in neurons but not in glial cells (Wolf et al., 1992; Lopes et al., 1994; Tooyama et al., 1995; Fabrizi et al., 1997). Whereas this study provides additional evidence that !ApoE4 is toxic in neuronal cells, discrepancies have existed in the literature. In some studies, !ApoE4 causes neurotoxic effects (Marques et al., 1997; Tolar et al., 1997, 1999; Jordan et al., 1998; Michikawa and Yanagisawa, 1998; DeMattos et al., 1999), whereas in others, no toxicity has been found (Bellosta et al., 1995; Nathan et al., 1995; DeMattos et al., 1998). This variability could be attributed to several possibilities. One is that !ApoE4 sensitivity of the neuronal cells used may be different not only in cell preparations but in cell conditions. Michikawa and Yanagisawa (1998) found that same neurons exhibit different responses to !ApoE4 toxicity, in the presence or absence of compactin. Also, the tissue distribution (Bu et al., 1994; Zheng et al., 1994) suggests that RAP expression is differentially regulated from LRP expression, whereas their expression is mutually related (Willnow et al., 1995). It is thus conceivable that the ratio of cellular expression of RAP versus LRP, which could vary among neuronal cells and by cellular conditions, influences the toxic effects of !ApoE4. Another possibility is that the culture conditions may affect !ApoE4 neurotoxicity. The aforementioned studies reporting negative effects of !ApoE4 were performed under conditions with serum or serum supplements including 5 μg/ml of insulin, which could suppress apoptosis. In contrast, our study was performed in the complete absence of serum or other supplements. | . We have herein shown that ApoE causes death in neuronal cells in an isoform-specific manner and that at least one mechanism for neurotoxic actions of !ApoE4 is apoptosis mediated by LRP. The toxicity of !ApoE4 was observed in cells of neuroblast origin, but not in glial cells or non-neuronal CHO cells. The observed resistance of glial cells is consistent not only with the study of Crutcher et al. (1994), indicating that glial cells are resistant to neurotoxic ApoE peptides, but also with the well established finding that LRP is found abundantly in neurons but not in glial cells (Wolf et al., 1992; Lopes et al., 1994; Tooyama et al., 1995; Fabrizi et al., 1997). Whereas this study provides additional evidence that !ApoE4 is toxic in neuronal cells, discrepancies have existed in the literature. In some studies, !ApoE4 causes neurotoxic effects (Marques et al., 1997; Tolar et al., 1997, 1999; Jordan et al., 1998; Michikawa and Yanagisawa, 1998; !DeMattos et al., 1999), whereas in others, no toxicity has been found (Bellosta et al., 1995; Nathan et al., 1995; !DeMattos et al., 1998). This variability could be attributed to several possibilities. One is that !ApoE4 sensitivity of the neuronal cells used may be different not only in cell preparations but in cell conditions. Michikawa and Yanagisawa (1998) found that same neurons exhibit different responses to !ApoE4 toxicity, in the presence or absence of compactin. Also, the tissue distribution (Bu et al., 1994; Zheng et al., 1994) suggests that RAP expression is differentially regulated from LRP expression, whereas their expression is mutually related (Willnow et al., 1995). It is thus conceivable that the ratio of cellular expression of RAP versus LRP, which could vary among neuronal cells and by cellular conditions, influences the toxic effects of !ApoE4. Another possibility is that the culture conditions may affect !ApoE4 neurotoxicity. The aforementioned studies reporting negative effects of !ApoE4 were performed under conditions with serum or serum supplements including 5 μg/ml of insulin, which could suppress apoptosis. In contrast, our study was performed in the complete absence of serum or other supplements. |
| Line 99: | Line 99: |
| .'''Introduction:''' We have known for nearly a decade that the inheritance of the APOE-ε4 allele is a risk factor for Alzheimer’s disease (AD) [131]. Despite this relatively long period of time, '''we still do not know whether expression of apoE4 per se is detrimental to the AD process'''. This debate focuses on whether it is the absence of apoE2 and apoE3 or the presence of apoE4 that puts ε4 individuals at risk for AD. The basis of our argument is that apoE performs neuroprotective and neurotrophic functions in the normal, aging brain. ApoE3 (and apoE2) perform these functions more efficiently than apoE4. Thus, individuals without apoE2 or apoE3 are at risk for CNS damage and AD. | .'''Introduction:''' We have known for nearly a decade that the inheritance of the APOE-ε4 allele is a risk factor for Alzheimer’s disease (AD) [131]. Despite this relatively long period of time, '''we still do not know whether expression of apoE4 per se is detrimental to the AD process'''. This debate focuses on whether it is the absence of apoE2 and apoE3 or the presence of apoE4 that puts ε4 individuals at risk for AD. The basis of our argument is that apoE performs neuroprotective and neurotrophic functions in the normal, aging brain. !ApoE3 (and apoE2) perform these functions more efficiently than apoE4. Thus, individuals without apoE2 or apoE3 are at risk for CNS damage and AD. |
| Line 105: | Line 105: |
| . T Nakamura, A Watanabe… - Molecular …, 2009 - molecularneurodegeneration. … . ''' ''Abstract'' Background:''' Apolipoprotein E allele ε4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1–272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. '''Results:''' To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2) and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1), which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1–272) more strongly than intact apoE4(1–299). Further analysis showed that in Neuro-2a cells expressing apoE4(1–272), the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1–299). '''Conclusion:''' ApoE4(1–272) fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1–272) fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration. |
. T Nakamura, A Watanabe, TFujino, T Hosono and M Michikawa - Molecular Neurodegeneration, 2009 . ''' ''Abstract'' Background:''' Apolipoprotein E allele ε4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1–272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. '''Results:''' To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2) and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1), which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1–272) more strongly than intact apoE4(1–299). Further analysis showed that in Neuro-2a cells expressing apoE4(1–272), the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1–299). '''Conclusion:''' !ApoE4(1–272) fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1–272) fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration. |
| Line 108: | Line 109: |
| . One may consider that apoE is synthesized as a secretory protein; however, '''how apoE enters the cytosol remains unclear and controversial'''. Previous studies have shown that apoE escapes the secretory or endosomal internalization pathway, and enters the cytosol of neuronal cells [25,29,40] and non-neuronal cells [41], whereas another study has failed to show this ['''42''']. Therefore, the physiological relevance of the three mitochondrial proteins that we have identified in this study, which are associated with apoE, remains to be confirmed under physiological conditions. | . '''p4:35 /8''' One may consider that apoE is synthesized as a secretory protein; however, '''how apoE enters the cytosol remains unclear and controversial'''. Previous studies have shown that apoE escapes the secretory or endosomal internalization pathway, and enters the cytosol of neuronal cells [25,29,40] and non-neuronal cells [41], whereas another study has failed to show this ['''42''']. Therefore, the physiological relevance of the three mitochondrial proteins that we have identified in this study, which are associated with apoE, remains to be confirmed under physiological conditions. |
| Line 115: | Line 116: |
| . RB !DeMattos - Journal of Molecular Neuroscience, 2004 - Springer | . RB !DeMattos - Journal of Molecular Neuroscience, 2004 |
| Line 121: | Line 122: |
| . MMM Wilhelmus, I Otte-Höller, J Davis… - The Journal of …, 2005 - Soc Neuroscience . The ϵ4 allele of apolipoprotein E (ApoE) is a risk factor for Alzheimer's disease (AD), whereas the ϵ2 allele may be relatively protective. Both alleles are risk factors for cerebral amyloid angiopathy (CAA)-related hemorrhages. CAA is associated with |
. MMM Wilhelmus, I Otte-Höller, J Davis, WE Van Nostrand, RMW de Waal, and MM Verbeek - The Journal of Neuroscience, 2005 . The ϵ4 allele of apolipoprotein E (ApoE) is a risk factor for Alzheimer's disease (AD), whereas the ϵ2 allele may be relatively protective. Both alleles are risk factors for cerebral amyloid angiopathy (CAA)-related hemorrhages. CAA is associated with degeneration of smooth muscle cells and pericytes. Previously, we described that synthetic amyloid-β,,1–40,, peptide (Aβ,,1–40,,) with the ^22^Glu → Gln “Dutch” mutation caused pericyte death in vitro by a mechanism that involves Aβ fibril-like assembly at the cell surface. It is known that ApoE binds to Aβ and may modify its biological activities. In the present study, we evaluated the effect of ApoE on Aβ-mediated toxicity of cerebrovascular cells. We observed that cultured cells with an ε4/ε4 genotype were more vulnerable to Aβ than cultures with an ε3/ε3 or ε3/ε4 genotype. The one cell culture with the ε2/ε3 genotype was relatively resistant to Aβ compared with other cultures. Furthermore, we observed a dose-dependent protective effect of native ApoE against Aβ-mediated toxicity of cerebrovascular cells and, in addition, ApoE ε2/ε3 cells secreted more ApoE protein compared with cells with other ApoE genotypes, in particular, compared with ε4/ε4 cells. Thus, the disparity between ApoE genotype and Aβ-mediated toxicity might be related to differences in the cellular capacity to secrete ApoE. The present data suggest that one mechanism by which ApoE may alter the risk for AD is a genotype-dependent regulation of Aβ cytotoxicity, possibly via variations in its secretion levels, whereby extracellular ApoE may bind to A  and thereby modify Aβ-mediated cell death. |
| Line 124: | Line 125: |
| . Partial purification of ApoE from culture supernatant. Purification of extracellular ApoE protein from medium of cultured HBP was performed as described by !DeMattos et al. (1999) with minor modifications. | . '''p3623 /3:''' ''Partial purification of ApoE from culture supernatant.'' Purification of extracellular ApoE protein from medium of cultured HBP was performed as described by '''!DeMattos et al. (1999)''' with minor modifications. ... |
| Line 138: | Line 139: |
| . '''''Paywall'''' | . '''''Paywall''''' |
| Line 144: | Line 145: |
| . PNAS-2001-Huang-883843-1.pdf |
. '''''Paywall''''' |
| Line 152: | Line 151: |
| . '''''Paywall''''' | |
| Line 157: | Line 157: |
| . '''''Paywall''''' | |
| Line 160: | Line 160: |
| . J Grootendorst, M Mulder, E Haasdijk, ER de Kloet… - Brain research, 2000 - Elsevier . Apolipoprotein E (apoE) is a major apolipoprotein in the central nervous system (CNS) that may play a role in various CNS disorders. ApoE is primarily localised in astrocytes, but neuronal apoE mRNA expression has been demonstrated in normal and diseased human . 1-s2.0-S0006899300022502-main.pdf ref 10 vague reference to "several lines of evidence' .It has been proposed that apoE has a function in the removal of degeneration products e.g. membrane fragments of neurones and possibly astrocytes. Previously, it has been found that after lesioning of peripheral nerves, macrophages infiltrate the site of injury and produce huge amounts of apoE, which associates with membrane fragments of degenerating cells. These fragments are thought to be internalised by the macrophages and redistributed amongst regenerating cells [6,31,44]. The disappearance of apoE-ir neurones as well as astrocytes, observed in severely injured areas following increases in apoE levels, therefore suggests that apoE has a role in the removal of cell debris. Recently, evidence for such a role of apoE was obtained by Fagan et al. [12] who found following an entorhinal cortex lesion, a persistence of degeneration products in the deafferented hippocampus of apoE-deficient mice compared with wild type mice. Another observation supporting this role for apoE is the accumulation of cholesterol in the ageing brain of apoE-deficient mice [67,68]. In the CNS, microglia may be involved in the removal of membrane fragments associated with apoE. Alternatively, a protective role for apoE against injury is suggested by the observation that apoE-deficient mice are more susceptible to injury than wild type mice. They display an abnormal compensatory response after ECL, show age-related neurodegeneration and worsened outcome from global ischaemia [7,33,54]. However, recently it has been shown that cytosolic apoE is incompatible with neuronal survival ['''10''']. The precise role of apoE in the CNS is not yet fully understood. However, several lines of evidence indicate that in conditions of neuronal injury it acts as a chaperone involved in the redistribution of lipids. |
. J Grootendorst, M Mulder, E Haasdijk, ER de Kloet, D Jaarsma, Brain research, 2000 . Apolipoprotein E (apoE) is a major apolipoprotein in the central nervous system (CNS) that may play a role in various CNS disorders. ApoE is primarily localised in astrocytes, but neuronal apoE mRNA expression has been demonstrated in normal and diseased human brain, as well as in ischaemic rat brain. To obtain further insight into the role of apoE in neuronal degeneration in the CNS and conditions of neuronal apoE localisation, we have investigated in mice the distribution of apoE following neuronal injury induced by kainic acid (n535, 25 or 35 mg kainic acid / kg BW). Consecutive series of brain sections were immunostained for apoE and markers for astroglia (GFAP) and microglia / macrophage cells (CR3). Degenerating neurones were identified with a silver-degeneration staining technique. The intensity and cellular distribution of apoE-immunoreactivity (apoE-ir) was dependent on the severity of neuronal injury. Mice that developed mild neuronal degeneration, restricted to a subset of neurones in the hippocampus, showed increased apoE-ir in astrocytes concomitant with increased GFAP-ir and mild microgliosis. In these mice, no neuronal apoE-ir was detected. In contrast, mice developing severe neuronal injury in the hippocampus — frequently also showing degeneration in other brain regions including cortex, thalamus, striatum and amygdala — showed intense apoE-ir in degenerating neurones. Surrounding the lesion, apoE-ir was increased in neuropil recurrently whereas GFAP-ir astrocytes disappeared. Thus, in mice apoE accumulates in degenerating neurones in conditions of severe neuronal injury putatively in association with disruption of the glial network. . 1-s2.0-S0006899300022502-main.pdf ref 10 vague reference to "several lines of evidence" . '''p172 /8:''' It has been proposed that apoE has a function in the removal of degeneration products e.g. membrane fragments of neurones and possibly astrocytes. Previously, it has been found that after lesioning of peripheral nerves, macrophages infiltrate the site of injury and produce huge amounts of apoE, which associates with membrane fragments of degenerating cells. These fragments are thought to be internalised by the macrophages and redistributed amongst regenerating cells [6,31,44]. The disappearance of apoE-ir neurones as well as astrocytes, observed in severely injured areas following increases in apoE levels, therefore suggests that apoE has a role in the removal of cell debris. Recently, evidence for such a role of apoE was obtained by Fagan et al. [12] who found following an entorhinal cortex lesion, a persistence of degeneration products in the deafferented hippocampus of apoE-deficient mice compared with wild type mice. Another observation supporting this role for apoE is the accumulation of cholesterol in the ageing brain of apoE-deficient mice [67,68]. In the CNS, microglia may be involved in the removal of membrane fragments associated with apoE. Alternatively, a protective role for apoE against injury is suggested by the observation that apoE-deficient mice are more susceptible to injury than wild type mice. They display an abnormal compensatory response after ECL, show age-related neurodegeneration and worsened outcome from global ischaemia [7,33,54]. However, recently it has been shown that cytosolic apoE is incompatible with neuronal survival ['''10''']. The precise role of apoE in the CNS is not yet fully understood. However, several lines of evidence indicate that in conditions of neuronal injury it acts as a chaperone involved in the redistribution of lipids. |
| Line 180: | Line 179: |
| . Since an apolipoprotein E4 (!ApoE4) peptide composed of the low-density lipoprotein (LDL) receptor-related protein (LRP)-binding domain [!ApoE4 (141–149) 2 or ApoE (141–155) 2] exerts neurotoxicity in primary neurons and neuronal cell lines, it has been controversial | . Since an apolipoprotein E4 (!ApoE4) peptide composed of the low-density lipoprotein (LDL) receptor-related protein (LRP)-binding domain [!ApoE4 (141–149) 2 or ApoE (141–155) 2] exerts neurotoxicity in primary neurons and neuronal cell lines, it has been controversial whether these effects are mediated by LRP. Here, we examined whether !ApoE4(141–149)^2^-induced toxicity is mediated by LRP in a neuronal cell system where ApoE4 toxicity is mediated by LRP: serum-deprived F11 neuronal cells. In these cells, where !ApoE4 exerted toxicity by apoptosis in a manner sensitive to both caspase inhibitors and pertussis toxin (PTX), !ApoE4(141–149) 2 also caused cell death by apoptosis but in a caspase-inhibitor-resistant, PTX-resistant manner. !ApoE4(141–149) 2 -induced death was not inhibited by antisense oligonucleotides to LRP. Therefore, we conclude that !ApoE4(141–149) 2 is able to exert neurotoxicity without involving LRP |
| Line 182: | Line 181: |
| . An important clue is the finding that !ApoE4 exerts in vitro neurotoxicity in primary neurons and immortalized neuronal cells (15–'''19'''). In clarifying the molecular mechanism, the first and most important question is whether LRP mediates this action. LRP was identified as a molecule structurally related to LDLR and has been established as the primary ApoE receptor, particularly in neurons. Despite the fact that multiple groups have made efforts to address this question, no consistent answer has been obtained. Jordan et al. (20) argued against the mediation by LRP of !ApoE4-induced neurotoxicity in rat hippocampal neurons. In contrast, Tolar et al. (17, 18) provided evidence that the toxicity by 22-kDa N-terminal fragments of !ApoE4, as well as full-length !!ApoE4, is mediated by LRP in chick lumbar sympathetic ganglion neurons and rat hippocampal neurons. | . '''p633/ 1''' An important clue is the finding that !ApoE4 exerts in vitro neurotoxicity in primary neurons and immortalized neuronal cells (15–'''19'''). In clarifying the molecular mechanism, the first and most important question is whether LRP mediates this action. LRP was identified as a molecule structurally related to LDLR and has been established as the primary ApoE receptor, particularly in neurons. Despite the fact that multiple groups have made efforts to address this question, no consistent answer has been obtained. Jordan et al. (20) argued against the mediation by LRP of !ApoE4-induced neurotoxicity in rat hippocampal neurons. In contrast, Tolar et al. (17, 18) provided evidence that the toxicity by 22-kDa N-terminal fragments of !ApoE4, as well as full-length !ApoE4, is mediated by LRP in chick lumbar sympathetic ganglion neurons and rat hippocampal neurons. |
| Line 188: | Line 187: |
| . '''''Paywall''''' | |
| Line 190: | Line 190: |
| . S Lovestone, B Anderton, J Betts… - Biochemical …, 2001 - symposia.biochemistry.org . The finding that APOE (the gene encoding apolipoprotein E) polymorphic variation was associated with an altered risk of developing Alzheimer's disease (AD) was a significant advance and immediately prompted a search for the mechanisms responsible for this |
. S Lovestone, B Anderton, J Betts, R Dayanandan, G Gibb, C Ljungberg and J Pearce - Biochemical Society Symposia, 2001 . The finding that APOE (the gene encoding apolipoprotein E) polymorphic variation was associated with an altered risk of developing Alzheimer's disease (AD) was a significant advance and immediately prompted a search for the mechanisms responsible for this alteration. Some 6 years later, a number of different hypotheses remain that might account for this influence on pathogenesis with no single mechanism being unequivocally accepted. The different approaches to understanding these mechanisms can be broadly categorized as: those suggesting a remote effect, such as different rates of vascular risk factors in those with the different APOE alleles; those proposing altered neuronal vulnerability, perhaps due to apolipoprotein E (ApoE)-isoform-specific differences in local cholesterol transport; and those hypotheses postulating an ApoE interaction with the two key lesions of AD, plaques and tangles. In this chapter we will review the evidence for and against an interaction between ApoE and the neuronal cytoskeleton, in particular with the microtubule-associated protein tau. |
| Line 193: | Line 193: |
| . In an important recent challenge to these findings !DeMattos and colleagues [65] generated a construct in which a nuclear localization signal (NLS) was tagged to ApoE. Neuro-2a (neuroblastoma) cells were incubated with lipidated ApoE-NLS derived from transiently transfected cells. This ApoE was taken up into a vesicular compartment as previously observed in other cell models. If ApoE were to escape from this compartment and enter the cytosol then it would be transferred to the nucleus. As nuclear fractions contained no ApoE it was concluded that ApoE remains within the vesicles and does not reach the cytoplasm. The authors also emphasize that previous studies reporting intracellular ApoE have not fully clarified whether the ApoE is truly cytoplasmic. Furthermore, in initial studies, transfection of cells with !ApoE3 lacking the normal signal peptide, and therefore cytosolic on expression, resulted in low expression and few clones in attempts to generate cell lines. The authors interpret this as excitotoxicity. However, despite the findings of this important study the cytosolic hypothesis of the mechanism of action of ApoE cannot be entirely rejected. Firstly, the intracellular fate of ApoE is clearly both cell and ApoE-type dependent. We reported that ApoE from CSF remains, even visually, restricted to a vesicular compartment in the absence of tau. It was only when tau was massively overexpressed in COS cells (when the tau becomes one of the major proteins produced by the cell) that a redistribution of ApoE, occurs. Neuro-2a cells, a neuroblastoma cell line, express tau, at relatively low levels. As we found no redistribution of ApoE, even in COS cells expressing low amounts of tau, it is not surprising that !DeMattos and colleagues failed to find ApoE entering a cytosolic compartment. Secondly, in ongoing studies we find that the intracellular fate of ApoE is highly dependent upon ApoE type. We have compared different recombinant !ApoEs combined with different lipid preparations to CSF and to ApoE derived from different cell lines stably expressing ApoE. It is apparent from these experiments that the fate of recombinant, lipidated ApoE, on incubation with cells expressing the LDL receptor, is entirely different from that of CSF ApoE. ApoE from cell lines is taken up by cells but less effectively than ApoE from CSF. It may be that there are important differences between the entirely physiological ApoE in CSF and the ApoE used in the experiments of !DeMattos and colleagues. Finally, the finding that there was a reduced transfection efficiency for !ApoE3 directed to the cytosol cannot be accepted as evidence for cell toxicity. Indeed the generation of cell lines stably expressing tau is difficult and neuroblastoma cell lines tend to express low levels of tau (and even less before differentiation). One plausible explanation of these observations is that the expression of tau stabilizes the microtubule network and that, therefore, cell division is inhibited, rendering the generation of stable cell lines expressing high levels of tau difficult. If the cytosolic tau-interaction hypothesis of the ApoE mechanism is correct then this is exactly what would be expected if !ApoE3 were expressed in the cytosol of cells expressing some tau – that the tau would be relatively less phosphorylated, that it would be more effective in stabilizing microtubules, that cell division would be inhibited and that, therefore, the generation of high-expressing cell lines would be prevented. | . '''p116/6:''' In an important recent challenge to these findings !DeMattos and colleagues [65] generated a construct in which a nuclear localization signal (NLS) was tagged to ApoE. Neuro-2a (neuroblastoma) cells were incubated with lipidated ApoE-NLS derived from transiently transfected cells. This ApoE was taken up into a vesicular compartment as previously observed in other cell models. If ApoE were to escape from this compartment and enter the cytosol then it would be transferred to the nucleus. As nuclear fractions contained no ApoE it was concluded that ApoE remains within the vesicles and does not reach the cytoplasm. The authors also emphasize that previous studies reporting intracellular ApoE have not fully clarified whether the ApoE is truly cytoplasmic. Furthermore, in initial studies, transfection of cells with !ApoE3 lacking the normal signal peptide, and therefore cytosolic on expression, resulted in low expression and few clones in attempts to generate cell lines. The authors interpret this as excitotoxicity. However, despite the findings of this important study the cytosolic hypothesis of the mechanism of action of ApoE cannot be entirely rejected. Firstly, the intracellular fate of ApoE is clearly both cell and ApoE-type dependent. We reported that ApoE from CSF remains, even visually, restricted to a vesicular compartment in the absence of tau. It was only when tau was massively overexpressed in COS cells (when the tau becomes one of the major proteins produced by the cell) that a redistribution of ApoE, occurs. Neuro-2a cells, a neuroblastoma cell line, express tau, at relatively low levels. As we found no redistribution of ApoE, even in COS cells expressing low amounts of tau, it is not surprising that !DeMattos and colleagues failed to find ApoE entering a cytosolic compartment. Secondly, in ongoing studies we find that the intracellular fate of ApoE is highly dependent upon ApoE type. We have compared different recombinant !ApoEs combined with different lipid preparations to CSF and to ApoE derived from different cell lines stably expressing ApoE. It is apparent from these experiments that the fate of recombinant, lipidated ApoE, on incubation with cells expressing the LDL receptor, is entirely different from that of CSF ApoE. ApoE from cell lines is taken up by cells but less effectively than ApoE from CSF. It may be that there are important differences between the entirely physiological ApoE in CSF and the ApoE used in the experiments of !DeMattos and colleagues. Finally, the finding that there was a reduced transfection efficiency for !ApoE3 directed to the cytosol cannot be accepted as evidence for cell toxicity. Indeed the generation of cell lines stably expressing tau is difficult and neuroblastoma cell lines tend to express low levels of tau (and even less before differentiation). One plausible explanation of these observations is that the expression of tau stabilizes the microtubule network and that, therefore, cell division is inhibited, rendering the generation of stable cell lines expressing high levels of tau difficult. If the cytosolic tau-interaction hypothesis of the ApoE mechanism is correct then this is exactly what would be expected if !ApoE3 were expressed in the cytosol of cells expressing some tau – that the tau would be relatively less phosphorylated, that it would be more effective in stabilizing microtubules, that cell division would be inhibited and that, therefore, the generation of high-expressing cell lines would be prevented. |
| Line 206: | Line 206: |
| . MC Ljungberg, A Asuni, J Pearce, R Dayanandan… - Neuroscience …, 2003 - Elsevier . As part of investigations of the cellular uptake of apolipoprotein E (apoE) relevant to Alzheimer's disease we have found that different preparations of apoE are handled differently by cells expressing the LDL-receptor. Comparing recombinant, cellular and native |
. MC Ljungberg, A Asuni, J Pearce, R Dayanandan, W Marz, MM Hoffmann, P Bertrand, G Siest, HTR Rupniak, BH Anderton, M Huettinger, S Lovestone, Neuroscience Letters, 2003 . As part of investigations of the cellular uptake of apolipoprotein E (apoE) relevant to Alzheimer's disease we have found that different preparations of apoE are handled differently by cells expressing the LDL-receptor. Comparing recombinant, cellular and native apoE, complexed with different preparations of lipid we find that only cellular and native apoE enter a vesicular compartment. Some, but not all of these apoE containing vesicles are lysosomes. In order to further examine the intracellular fate of apoE we demonstrate that apoE-Enhanced green fluorescent protein chimeric protein can be taken up from medium by recipient cells and tracked within these cells for extended periods. |
| Line 209: | Line 209: |
| . However, in order for this to occur apoE would have to be taken up from the extracellular space into the cytoplasm as even neuronally expressed apoE would be secreted and not be present in the same intra-cellular compartment as tau. Two independent studies suggested such uptake could occur – Nathan et al. [15] reported that exogenously applied apoE3 preferentially accumulated in the cytoplasm of neuroblastoma cells whilst we observed a similar phenomenon with apoE derived from CSF when applied to COS-7 cells expressing LDL receptors [13]. However, in direct contrast to these studies, '''De Mattos et al. [3]''' failed to observe escape of apoE from the endosomal-lysosomal pathway. These apparent contradictions might result from the different assays or from different preparations of apoE utilised. We therefore examined apoE uptake and intra-cellular localisation and trafficking using a number of different sources of apoE in both fixed and in living nonneuronal cell models. | . '''p69 /1:''' However, in order for this to occur apoE would have to be taken up from the extracellular space into the cytoplasm as even neuronally expressed apoE would be secreted and not be present in the same intra-cellular compartment as tau. Two independent studies suggested such uptake could occur – Nathan et al. [15] reported that exogenously applied apoE3 preferentially accumulated in the cytoplasm of neuroblastoma cells whilst we observed a similar phenomenon with apoE derived from CSF when applied to COS-7 cells expressing LDL receptors [13]. However, in direct contrast to these studies, '''De Mattos et al. [3]''' failed to observe escape of apoE from the endosomal-lysosomal pathway. These apparent contradictions might result from the different assays or from different preparations of apoE utilised. We therefore examined apoE uptake and intra-cellular localisation and trafficking using a number of different sources of apoE in both fixed and in living nonneuronal cell models. |
| Line 228: | Line 228: |
| . Cholesterol 24-hydroxylase (CYP46A1) belongs to the cytochrome P450 super family and is responsible for conversion of cholesterol to the oxysterol 24S-hydroxycholesterol (24S-OHC). This structural modification allows 24S-OHC to traverse the blood brain barrier and | . Cholesterol 24-hydroxylase (CYP46A1) belongs to the cytochrome P450 super family and is responsible for conversion of cholesterol to the oxysterol 24S-hydroxycholesterol (24S-OHC). This structural modification allows 24S-OHC to traverse the blood brain barrier and this pathway is the major one for elimination of cholesterol from the mammalian brain. CYP46A1 is almost exclusively located to neurons in the brain and retina. The promoter region of CYP46A1 shows classical hallmarks of a gene with putative housekeeping function. Oxidative stress was the only factor initially shown to cause signicant increase in CYP46A1 reporter activity. The aim of this thesis was to obtain a more profound knowledge about regulation of CYP46A1 and the regulatory importance of its product. In addition the possibility was investigated that the levels of 24S-OHC in cerebrospinal fluid may be used diagnostically. |
| Line 230: | Line 230: |
| . There is evidence suggesting that APOE4 is somehow involved in the Aȕ formation. As mentioned above, APOE is present in the neuritic plaques and it has been reported that Aβ levels are elevated in brains of AD patients carrying APOE4 allele. It is not yet known if APOE4 has an active role in aggregation and/or deposition of Aβ. APOE4, in a lipid- free form has a greater avidity to Aβ than APOE3 (34, 39, 40). Post mortem studies have shown increased Aβ deposition in APOE4 carriers both in sporadic and genetic AD cases (39, 41). Hyper-phosphorylation of microtubule-associated protein known as tau protein leads to insoluble aggregation and form neurofibrillary tangles in the neuronal cells. This is common in connection with neurodegenerative diseases, in particular AD. Different APOE isoforms may influence formation of tau. ''In vitro'' studies have shown that APOE3 forms a stable complex with tau. The interaction between APOE3 and tau was prevented by phosphorylation of tau, suggesting that APOE3 preferentially binds to nonphosphorylated tau (42). However there is no evidence demonstrating localization of APOE to the neuronal cytosol, where the majority of tau exists under normal conditions (29, '''43'''). In the peripheral nervous system and CNS the levels of APOE increases following neuronal injuries. It has been suggested that this increase may be required for repair of the nervous system by redistribution of lipids and cholesterol for membrane repair and synaptic plasticity. Most studies have shown that APOE3 augment neurite outgrowth to a greater extent than APOE4 (29, 44). The effect of APOE2 has not yet been fully examined in connection to neurite sprouting. | . '''pg 7''' The APOE isoforms differ only by a single amino acid substitution of Cys to Arg at position 112 and 158 (32) leading to different biological properties (33). APOE 3 is the most common isoform (77-78%) in the general population while APOE2 is found in 7-8% and APOE4 in 14-16% of individuals (34, 35). After findings of immunoreactivity of APOE in the amyloid plaques (36) the APOE4 allele was discovered to be the most important genetic risk factor for sporadic Alzheimer’s disease (AD) (32, 37). In contrast, the APOE2 allele has been associated with a lower risk for AD (38). Amyloid beta (Aȕ) is formed by proteolytic cleavage of the amyloid precursor protein (APP) and plays an important role in the AD pathology. There is evidence suggesting that APOE4 is somehow involved in the Aȕ formation. As mentioned above, APOE is present in the neuritic plaques and it has been reported that Aβ levels are elevated in brains of AD patients carrying APOE4 allele. It is not yet known if APOE4 has an active role in aggregation and/or deposition of Aβ. APOE4, in a lipid- free form has a greater avidity to Aβ than APOE3 (34, 39, 40). Post mortem studies have shown increased Aβ deposition in APOE4 carriers both in sporadic and genetic AD cases (39, 41). Hyper-phosphorylation of microtubule-associated protein known as tau protein leads to insoluble aggregation and form neurofibrillary tangles in the neuronal cells. This is common in connection with neurodegenerative diseases, in particular AD. Different APOE isoforms may influence formation of tau. ''In vitro'' studies have shown that APOE3 forms a stable complex with tau. The interaction between APOE3 and tau was prevented by phosphorylation of tau, suggesting that APOE3 preferentially binds to nonphosphorylated tau (42). However there is no evidence demonstrating localization of APOE to the neuronal cytosol, where the majority of tau exists under normal conditions (29, '''43'''). In the peripheral nervous system and CNS the levels of APOE increases following neuronal injuries. It has been suggested that this increase may be required for repair of the nervous system by redistribution of lipids and cholesterol for membrane repair and synaptic plasticity. Most studies have shown that APOE3 augment neurite outgrowth to a greater extent than APOE4 (29, 44). The effect of APOE2 has not yet been fully examined in connection to neurite sprouting. |
Cytotoxic ApoE Cannot Reach Nucleus from Cytosol
Hence No Effect on Gene Expression, or Effect on Microtubules
a page of research shared by an associate of Dr. Glenn
A test of the cytosolic apolipoprotein E hypothesis fails to detect the escape of apolipoprotein E from the endocytic pathway into the cytosol and shows that direct expression of apolipoprotein E in the cytosol is cytotoxic. J Neurosci 1999, 19:2464–2473
DeMattos RB, Thorngate FE, Williams DL
What's happened since?
- Journal research in progress
36 Citations on Google Scholar, 25 available
The role of apolipoprotein E in Alzheimer's disease
- J Kim, JM Basak, DM Holtzman - Neuron, 2009 - Elsevier
- The ɛ4 allele of apolipoprotein E (APOE) is the major genetic risk factor for Alzheimer's disease (AD). Although there have been numerous studies attempting to elucidate theunderlying mechanism for this increased risk, how apoE4 influences AD onset and progression has yet to be proven. However, prevailing evidence suggests that the differential effects of apoE isoforms on Aβ aggregation and clearance play the major role in AD pathogenesis. Other potential mechanisms, such as the differential modulation of neurotoxicity and tau phosphorylation by apoE isoforms as well as its role in synaptic plasticity and neuroinflammation, have not been ruled out. Inconsistent results among studies have made it difficult to define whether the APOE ε4 allele represents a gain of toxic function, a loss of neuroprotective function, or both. Therapeutic strategies based on apoE propose to reduce the toxic effects of apoE4 or to restore the physiological, protective functions of apoE.
1-s2.0-S0896627309005492-main.pdf "DeMattos et al., 1999"
p294 /8: Along with amyloid plaque formation, hyperphosphorylation of the microtubule-binding protein tau and subsequent formation of neurofibrillary tangles are hallmarks of AD pathology. The relationship between apoE and tau has not been thoroughly investigated and the results are much less clear than the association between APOE ε4 allele dose and amyloid plaque burden. Although some studies reported a positive relationship between the neurofibrillary tangle density and APOE ε4 allele dosage (Ghebremedhin et al., 1998; Nagy et al., 1995; Ohm et al., 1995; Polvikoski et al., 1995), others found no clear correlation (Itoh and Yamada, 1996; Landen et al., 1996b; Morris et al., 1995; Olichney et al., 1996; Oyama et al., 1995). Unlike studies with human subjects, data from in vitro and animal models appear to be more consistent among studies. Under in vitro conditions, apoE3 binds tightly to tau and forms an SDS-stable complex through the interaction of the N-terminal domain of apoE3 and the microtubule-binding repeat regions of tau, whereas apoE4 does not interact significantly with tau (Strittmatter et al., 1994). The interaction between apoE3 and tau was prevented by the phosphorylation of tau, suggesting that apoE3 binds preferentially to nonphosphorylated tau. However, there is currently no conclusive evidence demonstrating localization of apoE to the neuronal cytosol, where the majority of tau exists under normal conditions (DeMattos et al., 1999). Therefore, the physiological relevance of the direct interaction between apoE and tau remains to be determined. Of note, more recent data suggests the possibility that a fragment of apoE4 (1–272 amino acids), but not full-length apoE4, the secretory pathway, translocates to the cytosolic compartment, and interacts with cytoskeletal components, including tau and neurofilament (Chang et al., 2005). Alternatively, the effects of apoE on tau phosphorylation could be explained by an intracellular signaling pathway induced by apoE (Harris et al., 2004; Hoe et al., 2005a), rather than the direct interaction between apoE and tau. Although in vitro studies have provided some insights, in vivo studies proving whether an apoE-isoform dependent effect on tau even exists will be critical.
- Chang, S., ran Ma, T., Miranda, R.D., Balestra, M.E., Mahley, R.W., and Huang, Y. (2005). Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity. Proc. Natl. Acad. Sci. USA 102, 18694–18699.
- Harris, F.M., Brecht, W.J., Xu, Q., Mahley, R.W., and Huang, Y. (2004). Increased tau phosphorylation in apolipoprotein E4 transgenic mice is associated with activation of extracellular signal-regulated kinase: modulation by zinc. J. Biol. Chem. 279, 44795–44801.
- Hoe, H.S., Harris, D.C., and Rebeck, G.W. (2005a). Multiple pathways of apolipoprotein E signaling in primary neurons. J. Neurochem. 93, 145–155.
Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons
- Y Huang, XQ Liu, T Wyss-Coray, WJ Brecht, DA Sanan, and RW Mahley- Proceedings of the National Academy of Sciences, 2001
Human apolipoprotein (apo) E4, a major risk factor for Alzheimer's disease (AD), occurs in amyloid plaques and neurofibrillary tangles (NFTs) in AD brains; however, its role in the pathogenesis of these lesions is unclear. Here we demonstrate that carboxyl-terminal-truncated forms of apoE, which occur in AD brains and cultured neurons, induce intracellular NFT-like inclusions in neurons. These cytosolic inclusions were composed of phosphorylated tau, phosphorylated neurofilaments of high molecular weight, and truncated apoE. Truncated apoE4, especially apoE4(⌬272–299), induced inclusions in up to 75% of transfected neuronal cells, but not in transfected nonneuronal cells. ApoE4 was more susceptible to truncation than apoE3 and resulted in much greater intracellular inclusion formation. These results suggest that apoE4 preferentially undergoes intracellular processing, creating a bioactive fragment that interacts with cytoskeletal components and induces NFT-like inclusions containing phosphorylated tau and phosphorylated neurofilaments of high molecular weight in neurons.
PNAS-2001-Huang-8838-43-1.pdf ref 23 needs evaluation
p8840/3 Carboxyl-Terminal-Truncated ApoE Induces Intracellular NFT-Like Inclusions. To determine whether carboxyl-terminal-truncated apoE induces intracellular NFT-like inclusions, we expressed apoE3 or apoE4 constructs possessing carboxyl-terminal truncations in Neuro-2a cells. Expression of apoE4 lacking the first 28 amino acids of the carboxyl terminus [apoE4(Δ272–299)] resulted in intracellular NFT-like inclusions in 78 ± 8% of transfected Neuro-2a cells (Fig. 2e). The inclusions were recognized by anti-p-tau (Fig. 2f ), which colocalized with apoE (Fig. 2g), and anti-p-NF-H (data not shown). Expression of apoE3(Δ272–299) also induced intracellular inclusions, but they were smaller (Fig. 2h) and occurred in significantly fewer cells (32 ± 5% versus 78 ± 8%, P < 0.001). Importantly, exogenous apoE4 (Δ272–299), which had been complexed with β-very low density lipoproteins as a lipid transport vehicle and incubated with Neuro-2a cells, also induced intracellular NFT-like inclusions (Fig. 2d). Thus, both endogenously expressed and exogenously added apoE with the carboxyl-terminal truncation induced NFT-like inclusions in Neuro-2a cells. The truncated apoE probably escapes the secretory or the endosomal-lysosomal internalization pathway, enters the cytosol, and interacts with p-tau and p-NF-H. There is evidence that apoE can appear in the cytosol of various cells (20–22), although another study failed to show this (23).
Expression of human apolipoprotein E4 in neurons causes hyperphosphorylation of protein tau in the brains of transgenic mice
- I Tesseur, J Van Dorpe, K Spittaels, C van den Haute, D Moechars, and F van Leuven, The American journal of Pathology, 2000
Epidemiological studies have established that the epsilon 4 allele of the ApoE gene (ApoE4) constitutes an important risk factor for Alzheimer's disease and might influence the outcome of central nervous system injury. The mechanism by which ApoE4 contributes to thedevelopment of neurodegeneration remains unknown. To test one hypothesis or mode of action of ApoE, we generated transgenic mice that overexpressed human ApoE4 in different cell types in the brain, using four distinct gene promoter constructs. Many transgenic mice expressing ApoE4 in neurons developed motor problems accompanied by muscle wasting, loss of body weight, and premature death. Overexpression of human ApoE4 in neurons resulted in hyperphosphorylation of the microtubule-associated protein tau. In three independent transgenic lines from two different promoter constructs, increased phosphorylation of protein tau was correlated with ApoE4 expression levels. Hyperphosphorylation of protein tau increased with age. In the hippocampus, astrogliosis and ubiquitin-positive inclusions were demonstrated. These findings demonstrate that expression of ApoE in neurons results in hyperphosphorylation of protein tau and suggests a role for ApoE in neuronal cytoskeletal stability and metabolism.
- 1-s2.0-S0002944010649632-main.pdf Ref 74
p 961 /11 To explain the genetic association of ApoE4 to AD, two types of mechanisms have been proposed. First, ApoE could function as a “pathological chaperone,” affecting clearance of β-amyloid and causing amyloid deposition. (70–72) We obtained no evidence for the presence of amyloid plaques in any of our ApoE4 transgenic mice, which of course might be because only endogenous mouse APP is present, which is less amyloidogenic. (39) The second hypothesis states that ApoE interacts with the microtubule-associated protein tau, thereby altering its phosphorylation state, and hence is involved in stabilizing the neuronal cytoskeleton. (2,73) The results presented here support this hypothesis. However, the route by which ApoE gains access to the neuronal cytoplasm has been the major criticism against this hypothesis and subject of much speculation. Most recently, it was shown that direct expression of ApoE in the cytosol of Neuro-2a cells is toxic. (74) The ApoE4 transgenic mice are, however, not expected to express ApoE directly in the cytosol, and we also did not observe obvious signs of neurotoxicity. In addition, the hyperphosphorylation of protein tau was not restricted to brain regions expressing human ApoE as shown by immunohistochemistry. Therefore, we favor a mechanism involving an indirect interaction between ApoE and protein tau. In addition, this implies that hyper-phosphorylation of protein tau is not simply a nonspecific downstream event marking degeneration.
The role of apolipoprotein E in Alzheimer's disease, acute brain injury and cerebrovascular disease: evidence of common mechanisms and utility of animal models
K Horsburgh, MO McCarron, F White, JAR Nicoll - Neurobiology of aging, 2000
- The ϵ4 allele of apolipoprotein E (APOE denotes gene; apoE denotes protein) is a major risk factor for Alzheimer's disease (AD). More recent evidence indicates an association with a poor outcome after acute brain injury including that due to head trauma and intracerebral hemorrhage. APOE gene polymorphism also influences the risk of hemorrhage in cerebral amyloid angiopathy. These diverse brain disorders seem to have some mechanisms in common. The multiplicity of the roles of apoE within the central nervous system is currently being unraveled. For example, apoE can interact with amyloid β-protein and tau, proteins central to the pathogenesis of AD. In addition to these effects, it is proposed that one of the major functions of apoE is to mediate neuronal protection, repair and remodeling. In all of the different roles proposed, there are marked apoE-isoform specific differences. Although it remains to be clarified which is the most important mechanism(s) in each disorder in which apoE is involved, these isoform specific differences seem to underly a genetically determined susceptibility to outcome from acute brain injury and to AD with APOE ε4 conferring relative vulnerability. This review focuses on apoE research, from clinical studies to animal models, in AD, acute brain injury and cerebrovascular disease and explores the common mechanisms that may explain some of the complex underlying neurobiology.
- 1-s2.0-S019745800000097X-main.pdf ref 20
p251/7 In vitro evidence is consistent with an isoform-dependent neurotrophic role of apoE. Rabbit dorsal root ganglion neurons and neuroblastoma cells incubated with lipoproteins alone have enhanced neurite outgrowth that is further enhanced in the presence of apoE E3 lipoproteins and inhibited in the presence of apoE E4 lipoproteins. [33,86]. Similar results are obtained when cells are stably transfected to secrete apoE E3 or apoE E4; in the presence of lipoproteins, cells expressing APOE ε3 have extensive neurite outgrowth whereas neurite extension is suppressed in cells expressing APOE ε4 [7]. One of the cellular events associated with neurite outgrowth is an apoE-isoform specific effect on the cytoskeleton. ApoE E3 stabilizes the formation of microtubules in vitro in contrast to apoE E4 that is associated with a destabilization of the microtubule assembly [23]. ApoE E3 can interact with tau, a microtubule associated protein, whereas apoE E4 is unable to bind tau. One hypothesis is that apoE E3 by binding to tau protects it from hyperphosphorylation and the generation of intracellular neurofibrillary tangles in AD brain. ApoE is present at high concentrations in neurons after injury in rodent and human brain [30,41– 44,56] and may be expressed at low levels normally in human brain [134] providing functional relevance between the interactions of apoE with intracellular cytoskeletal proteins. In vitro studies have also indicated that there may be a preferential accumulation of apoE E3 and E2 in neurons as compared to apoE E4 [51]. Some groups have proposed that apoE E3 escapes lysosomal degradation and enters the cytosol where it is able to interact with cytosolic proteins such as tau and microtubule associated proteins. This mechanism may account for the apoE isoform specific effect of apoE E3 in promoting neurite outgrowth and microtubule stability and the lack of such an effect with apoE E4. However, it has been shown that apoE remains within the endocytic pathway and does not escape into the cytosol and argues that the effects of apoE are mediated by actions on the cell surface and not via cytosolic interactions [20]. The intracellular fate of the apoE isoforms remains a critical question.
Truncated apolipoprotein E (ApoE) causes increased intracellular calcium and may mediate ApoE neurotoxicity
- M Tolar, JN Keller, S Chan, MP Mattson, MA Marques, andKA Crutcher- The Journal of Neurosicience, 1999
- Apolipoprotein E (apoE)-related synthetic peptides, the 22 kDa N-terminal thrombin-cleavage fragment of a!poE (truncated !apoE), and full-length !apoE have all been shown to exhibit neurotoxic activity under certain culture conditions. In the present study, protease inhibitors reduced the neurotoxicity and proteolysis of full-length apoE but did not block the toxicity of truncated apoE or a synthetic apoE peptide, suggesting that fragments of apoE may account for its toxicity. Additional experiments demonstrated that both truncated apoE and the apoE peptide elicit an increase in intracellular calcium levels and subsequent death of embryonic rat hippocampal neurons in culture. Similar effects on calcium were found when the apoE peptide was applied to chick sympathetic neurons. The rise in intracellular calcium and the hippocampal cell death caused by the apoE peptide were significantly reduced by receptor-associated protein, removal of extracellular calcium, or administration of the specific NMDA glutamate receptor antagonist MK-801. These results suggest that apoE may be a source of both neurotoxicity and calcium influx that involves cell surface receptors. Such findings strengthen the hypothesis that apoE plays a direct role in the pathology of Alzheimer’s disease.
7100.full.pdf Ref as DeMattos et al., 1999 may disagree
p7105 / 6: As found previously (Crutcher et al., 1994; Marques et al., 1996, 1997), a synthetic apoE-related peptide, the N-terminal portion of apoE4 (truncated apoE4), and f ull-length apoE4 exhibited neurotoxic activity. Protease inhibitors reduced both the toxicity of full-length apoE4 and the appearance of truncated apoE fragments, suggesting that proteolysis of apoE may mediate its toxic effects. If so, this might help explain the variability that exists in the literature. In some studies, apoE causes neurotoxic effects (Marques et al., 1997; Jordan et al., 1998; DeMattos et al., 1999), but in other studies, no toxicity has been found (Bellosta et al., 1995; Nathan et al., 1995; DeMattos et al., 1998). In one study, cytosolic expression of apoE in neuroblastoma cells was found to be toxic (DeMattos et al., 1999), but no information was provided on whether apoE was degraded under these conditions. The present results suggest that conditions may be required in which truncated apoE is generated to observe toxic effects of apoE. We have also observed that serum blocks apoE peptide toxicity (M. Marques and K. Crutcher, unpublished observations), indicating that composition of the medium may be critical. The contribution of apoE proteolysis to neurotoxicity may also have some bearing on understanding the role of apoE in vivo. Fragments of apoE are present in both the brain and CSF, and the most abundant fragment, with an apparent molecular weight of 22 kDa, likely represents the N-terminal thrombin cleavage fragment of apoE (Marques et al., 1996).
Prominent axonopathy and disruption of axonal transport in transgenic mice expressing human apolipoprotein E4 in neurons of brain and spinal cord
- I Tesseur, J Van Dorpe, K Bruynseels, F Bronfman, R Sciot, A van Lommel, and F van Leuven, The American journal of Pathology, 2000
The epsilon 4 allele of the human apolipoprotein E gene (ApoE4. constitutes an important genetic risk factor for Alzheimer's disease. Recent experimental evidence suggests that human ApoE is expressed in neurons, in addition to being synthesized in glial cells. Moreover, brain regions in which neurons express ApoE seem to be most vulnerable to neurofibrillary pathology. The hypothesis that the expression pattern of human ApoE might be important for the pathogenesis of Alzheimer’s disease was tested by generating transgenic mice that express human ApoE4 in neurons or in astrocytes of the central nervous system. Transgenic mice expressing human ApoE4 in neurons developed axonal degeneration and gliosis in brain and in spinal cord, resulting in reduced sensorimotor capacities. In these mice, axonal dilatations with accumulation of synaptophysin, neurofilaments, mitochondria, and vesicles were documented, suggesting impairment of axonal transport. In contrast, transgenic mice expressing human ApoE4 in astrocytes remained normal throughout life. These results suggest that expression of human ApoE in neurons of the central nervous system could contribute to impaired axonal transport and axonal degeneration. The possible contribution of hyperphosphorylation of protein Tau to the resulting phenotype is discussed.
1-s2.0-S0002944010647888-main.pdf ref 23 Read more ... synthesis by neurons?
p1495 /1 Epidemiological data do not provide evidence for a direct role of ApoE in central nervous system disorders, and its mechanism of action within the central nervous system is not clear. Originally, it was thought that ApoE present in neurons originated only from endocytosis. [20–22] Numerous cell culture experiments demonstrated receptor-mediated uptake of ApoE as well as effects on neurite outgrowth and cell-morphology. [23–29] However, more and more evidence indicates that intraneuronal ApoE might also originate from synthesis by neurons. In situ hybridization of brain sections of transgenic mice expressing genomic fragments containing the entire human ApoE locus, including its promoter sequences, revealed expression in neurons, besides astrocytes. [30–32] Human neuroblastoma cells were shown to synthesize both ApoE mRNA and protein. [33,34] Moreover, in situ hybridization of human brain sections conclusively demonstrated ApoE mRNA in neurons. [35] Taken together these results suggest that besides its well-known synthesis by glia cells and its role in lipid transport and metabolism, not only uptake but also synthesis of ApoE by neurons might be important. Interestingly, the brain regions expressing ApoE in neurons seemed most vulnerable to the development of neurofibrillary pathology, [36] raising the possibility that the neuronal expression pattern of human ApoE might be important in the pathogenesis of AD.
Apolipoprotein E: a major piece in the Alzheimer's disease puzzle
- A Cedazo‐Mínguez, RF Cowburn - Journal of cellular and …, 2001 - Wiley Online Library
Alzheimer's disease (AD) is a complex neurodegenerative disorder with multiple etiologies. The presence of the E4 isoform of apolipoprotein E (apoE) has been shown to increase the risk and to decrease the age of onset for AD and is the major susceptibility factor known for the disease. ApoE4 has been shown to intensify all the biochemical disturbances characteristic of AD, including beta amyloid (Aβ) deposition, tangle formation, neuronal cell death, oxidative stress, synaptic plasticity and dysfunctions of lipid homeostasis and cholinergic signalling. In contrast, other apoE isoforms are protective. Here we review and discuss these major hypotheses of the apoE4-AD association.
- j.1582-4934.2001.tb00159.x.pdf ref 30
p259/6The hypothesis that apoE isoforms may influence differently tau pathology derives from in vitro studies demonstrating that the apoE2 and E3 isoforms bind more readily to tau, as compared to apoE4, and thereby prevent abnormal tau hyperphosphorylation and destabilisation of the neuronal cytoskeleton [25]. Several neuropathology studies have found either a correlation or lack of correlation between apoE4 and NFT. Differences in the subjects studied (such as diagnosis criteria, severity of the disease, etc) could account for these inconsistent observations. In support of the hypothesis that the apoE genotype may influence NFT formation, it has been shown that apoE4 decreases neuronal extension and branching and induces microtubule collapse in vitro [26]. In addition, transgenic mice studies have shown that both apoE deficiency [27] and human apoE4 expression in neurons [28] results in tau hyperphosphorylation. However, such direct molecular interactions between the apoE and tau molecules would require that both meet in the same cytosolic compartment. The question of how apoE accesses the neuronal cytoskeleton has been a major criticism against this hypothesis. Although low levels of neuronal expression of apoE have been shown, it remains puzzling as to how the apoE protein should reach the cytosol, since the normal signal peptide within the apoE gene would direct newly synthesised protein to secretory pathways. In cells expressing the LDL-receptor, it was found that apoE from CSF was taken up and then remained in a vesicular compartment unless tau was also expressed. In this case, apoE appeared to redistribute while apoE4 did not [29]. In addition, a recent study in neuro-2a cells has shown that apoE does not escape from the secretory pathway to the cytosol and also that direct expression of apoE in the cytosol is neurotoxic [30]. An alternative mechanism for apoE effects on tau phosphorylation is that the molecule modulates cellular signalling pathways involving tau kinases and phosphatases. ApoE effects on signal transduction pathways and their importance in AD neuropathology modulation are reviewed below.
Low levels of extrahepatic nonmacrophage ApoE inhibit atherosclerosis without correcting hypercholesterolemia in !ApoE-deficient mice
- FE Thorngate, LL Rudel, RL Walzem, DL Williams, Journal of Arteriosclosis, Thromobosis, and Vascular Biology, 2000
The prevention of atherosclerosis by apolipoprotein E (apoE) is generally attributed to the removal of plasma lipoprotein remnant particles. We developed transgenic apoE-knockout mice expressing apoE specifically in the adrenal gland and found that only 3% of the wild-type plasma level of apoE was sufficient to normalize plasma cholesterol levels in the apoE-deficient mouse. As expected, mice expressing apoE at levels that correct hypercholesterolemia had almost no cholesteryl ester deposition in their aortas. In contrast, their nontransgenic siblings had significant atherosclerosis. Unexpectedly, we found that atherosclerosis was also reduced in 2 transgenic lines expressing too little apoE (< 1% to 2% of wild type) to correct their hypercholesterolemia. Gel exclusion chromatographic profiles of plasma lipoproteins and the size distributions of lipoproteins with density < 1.063 (low density and very low density lipoproteins), as determined by dynamic laser light scattering, were the same in mice expressing < 2 μg/mL plasma apoE and their nontransgenic littermates. We conclude that the antiatherogenic action of low levels of plasma apoE is not due to the clearance of remnant lipoproteins. Thus, low levels of apoE provided systemically, but not made in the liver or in macrophages, can block atherogenesis in the vascular wall independently of normalizing the plasma concentration of atherogenic remnant lipoprotein particles.
1939.full.pdf ref 27 NOT RELEVANT
p1940 /2: Samples were electrophoretically transferred to nitrocellulose and immunostained as described 27 by use of an affinity-purified goat anti-rat apoE.
The synthesis and transport of lipids for axonal growth and nerve regeneration
- JE Vance, RB Campenot, DE Vance - … (BBA)-Molecular and Cell Biology of …, 2000 - Elsevier
- Neurons are unique polarized cells in which the growing axon is often located up to a meter or more from the cell body. Consequently, the intracellular movement of membrane lipids and proteins between cell bodies and axons poses a special challenge. The mechanisms of lipid transport within neurons are, for the most part, unknown although lipid transport via vesicles and via cholesterol- and sphingolipid-rich `rafts' are considered likely mechanisms. Very active anterograde and retrograde transport of lipid-containing vesicles occurs between the cell body and distal axons. However, it is becoming clear that the axon need not obtain all of its membrane constituents from the cell body. For example, the synthesis of phosphatidylcholine, the major membrane phospholipid, occurs in axons, and its synthesis at this location is required for axonal elongation. In contrast, cholesterol synthesis appears to occur only in cell bodies, and cholesterol is efficiently delivered from cell bodies to axons by anterograde transport. Cholesterol that is required for axonal growth can also be exogenously supplied from lipoproteins to axons of cultured neurons. Several studies have suggested a role for apolipoprotein E in lipid delivery for growth and regeneration of axons after a nerve injury. Alternatively, or in addition, apolipoprotein E has been proposed to be a ligand for receptors that mediate signal transduction cascades. Lipids are also transported from axons to myelin, although the importance of this process for myelination is not clear.
- 1-s2.0-S1388198100000500-main.pdf ref 91
pg 91/8: Another complexity regarding the role of apo E in neuronal extension is that in humans the most common isoform of apo E is apo E3 [77] and a less common isoform is apo E4. Inheritance of the allele encoding apo E4, rather than apo E3, has been implicated as a strong risk factor for the development of Alzheimer's disease [84-86]. A clue to why apo E3 and apo E4 play di¡erent roles in the development of this disease might be the observation that in several different model systems these two isoforms modulate axonal growth differently, with apo E3 stimulating growth and apo E4 either inhibiting, or not affecting, growth [81,87-90]. The mechanism by which apo E3 and apo E4 differentially mediate neuron growth is being actively investigated, but is probably not due to a different affnity of lipoprotein receptors for apo E3- versus apo E4-containing lipoproteins, since the LDL receptor and the LDL receptor-like protein bind these two apo E isoforms equally [77]. Recent studies suggest that apo E need not be present intracellularly in neurons for it to elicit its effects on growth [91] . Nor are the differential effects of apo E3 and E4 on neuron growth likely to be associated solely with lipid delivery, since lipid-poor apo E3 and apo E4 have the same e¡ects on growth as do their lipid-rich counterparts [90,92,93]. An exciting recent proposal is that the effect of apo E on neuron growth is mediated by signal transduction events since members of the LDL receptor superfamily - for example, the LDL receptor [94], the LDL receptor-like protein [94], the very low density lipoprotein receptor [95] and the brain-specific receptor apo ER2 [96] [also called LR7/8B [95,97,98]] - all of which bind apo E, contain a NPxY motif which associates with known signaling proteins. This motif binds to the neuronal adaptor proteins FE65 and Disabled and most likely activates an intracellular signal transduction cascade in the brain [94,95]. Therefore, a strong possibility exists that apo E in£uences axonal growth through receptor-mediated signaling events in addition to, or perhaps instead of, acting as a ligand for receptor-mediated endocytosis of lipoproteins. The mechanism by which apo E3 alike protein [94], the very low density lipoprotein receptor [95] and the brain-specific receptor apo ER2 [96] [also called LR7/8B [95,97,98]] . . . all of which bind apo E, contain a NPxY motif which associates with known signaling proteins. This motif binds to the neuronal adaptor proteins FE65 and Disabled and most likely activates an intracellular signal transduction cascade in the brain [94,95]. Therefore, a strong possibility exists that apo E in£uences axonal growth through receptor-mediated signaling events in addition to, or perhaps instead of, acting as a ligand for receptor-mediated endocytosis of lipoproteins. The mechanism by which apo E3 and E4 a¡ect these processes differently remains to be established.
- Apart from any role in signaling events, the ability of lipoproteins to supply cholesterol for axonal growth has been demonstrated. When cholesterol synthesis was inhibited in the cell bodies of compartmented cultures of sympathetic neurons by the addition of pravastatin, axonal growth essentially ceased, presumably because of the reduced supply of cholesterol. Normal axonal growth in the presence of pravastatin was restored by the addition of cholesterol to either cell bodies or distal axons [58]. Presentation of LDL or high density lipoproteins (both of which lacked apo E) to the distal axons also restored normal growth to pravastatin-treated neurons, as did the addition of LDL to the cell bodies. In contrast, when high density lipoproteins were provided to the cell bodies axonal growth was not restored [58].
Formation and function of apolipoprotein E-containing lipoproteins in the nervous system
- JE Vance, H Hayashi - Biochimica et Biophysica Acta (BBA)-Molecular and …, 2010 - Elsevier
- The strongest known genetic risk factor for the development of late-onset Alzheimer disease is inheritance of the apolipoprotein (apo) E4 (ε4 allele) although the mechanisms underlying this connection are still not entirely clear. In this review, we shall discuss the role of apo E in the brain, particularly in relation to Alzheimer disease. Cholesterol transport and homeostasis in the central nervous system (CNS) are separated from that in the peripheral circulation by the blood–brain barrier. However, the brain operates its own lipoprotein transport system that is mediated by high density lipoprotein-sized, apo E-containing lipoproteins that are synthesized and secreted by glial cells (primarily astrocytes). Several ATP-binding cassette (ABC) transporters are expressed in the brain, including ABCA1 and ABCG1 which play important roles in the transfer of phos- pholipids and cholesterol to apo E. The astrocyte-derived apo E-containing lipoproteins can bind to, and be internalized by, receptors of the low density lipoprotein receptor superfamily that are located on the surface of neurons. In addition to these receptors serving as endocytosis receptors for lipoproteins, several of these receptors also act as signaling receptors in neurons and activate pathways involved in axonal growth, as well as neuronal survival. These beneficial pathways appear to be enhanced to a greater extent by apo E3 than by apo E4. Apo E has also been implicated in the deposition of amyloid plaques since apo E3, more readily than apo E4, forms a complex with Aß peptides, and mediates the degradation of amyloid deposits.
- 1-s2.0-S1388198110000429-main.pdf ref 227
p811 /6: Uncertainty remains, however, regarding the mechanism(s) underlying the differential effects of apo E isoforms on axonal extension. One possible mechanism is that the apo E isoforms bind with different affinities to one of the LDL receptor family members on neurons, thereby differentially initiating a signaling pathway that mediates axonal growth. Another possible mechanism is that apo E3 accumulates within neurons to a greater extent than does apo E4 [223,224]. In this case, the apo E isoform-specific effects on axonal growth might be attributable to differences in the amounts of intracellular apo E which have been proposed to affect the structure of the cytoskeleton and microtubule assembly. Indeed, microtubule formation was enhanced by apo E3 in cultured neuronal cells whereas apo E4 destabilized microtubule assembly [223]. This proposal is supported by the finding that apo E3 interacts more readily than apo E4 with tau and prevents tau hyperphosphorylation which, as a result, might reduce the formation of intracellular neurofibrillary tangles (deposits of tau), a hallmark of AD disease [225]. Nevertheless, this mechanism is controversial since it would imply that apo E is present in the cytosol of neurons and that endocytosed apo E escapes from degradation in the endosomal–lysosomal system [226]. Another study found no evidence that apo E escapes from the endocytic pathway into the cytosol, and also reported that the expression of apo E in the cytosol is toxic [227].
Neuronal apoptosis by apolipoprotein E4 through low-density lipoprotein receptor-related protein and heterotrimeric GTPases
- Y Hashimoto, H Jiang, T Niikura, Y Ito… - The Journal of Neuroscience, 2000
The ε4 genotype of apolipoprotein E (!apoE4) is the most established predisposing factor in Alzheimer's disease (AD); however, it remains unclear how apoE4 contributes to the pathophysiology. Here, we report that the !apoE4 protein (ApoE4) evokes apoptosis inneuronal cells through the low-density lipoprotein receptor-related protein (LRP) and heterotrimeric GTPases. We examined neuron/neuroblastoma hybrid F11 cells and found that these cells were killed by 30 μg/ml ApoE4, but not by 30 μg/ml ApoE3. ApoE4-induced death occurred with typical features for apoptosis in time- and dose-dependent manners, and was observed in SH-SY5Y neuroblastomas, but not in glioblastomas or non-neuronal Chinese hamster ovary cells. Activated, but not native, α2-macroglobulin suppressed this ApoE4 toxicity. Suppression by the antisense oligonucleotide to LRP and inhibition by low nanomolar concentrations of LRP-associated protein RAP provided evidence for the involvement of LRP. The involvement of heterotrimeric GTPases was demonstrated by the findings that (1) ApoE4-induced death was suppressed by pertussis toxin (PTX), but not by heat-inactivated PTX; and (2) transfection with PTX-resistant mutant cDNAs of Gα₁ restored the toxicity of ApoE4 restricted by PTX. We thus conclude that one of the neurotoxic mechanisms triggered by ApoE4 is to activate a cell type-specific apoptogenic program involving LRP and the G₁ class of GTPases and that the apoE4 gene may play a direct role in the pathogenesis of AD and other forms of dementia.
8401.full.pdf ref as Demattos et al. (1999) Read This One Carefully
pg 8401/1 ApoE4 binds Aβ and facilitates its aggregation (Strittmatter et al., 1993; LaDu et al., 1994, 1995). However, it is unlikely that this action is implicated in the ApoE4 neurotoxicity, because (1) ApoE3 binds Aβ at 20-fold higher levels than does ApoE4 (LaDu et al., 1994); (2) binding of Aβ to rabbit ApoE decreases Aβ toxicity in rat hippocampal neurons (Whitson et al., 1994); and (3) the N-terminal 22 kDa fragment of ApoE4, which lacks the Aβ binding domain (Pillot et al., 1999), exhibits isoform-specific neurotoxicity (Marques et al., 1996, 1997; Tolar et al., 1997, 1999). Also, DeMattos et al. (1999) demonstrated that ApoE4 exerts neurotoxicity not through interaction with intracellular Aβ or tau. The present study was conducted to examine whether ApoE4 has a direct action on neuronal death, and if so, with what molecular mechanism. We find that ApoE4 exerts isoform-specific neurotoxicity through LRP and the G₁ class of GTPases.
LRP = lipoprotein receptor-related protein . . . α2M = α2-macroglobulin
We have herein shown that ApoE causes death in neuronal cells in an isoform-specific manner and that at least one mechanism for neurotoxic actions of ApoE4 is apoptosis mediated by LRP. The toxicity of ApoE4 was observed in cells of neuroblast origin, but not in glial cells or non-neuronal CHO cells. The observed resistance of glial cells is consistent not only with the study of Crutcher et al. (1994), indicating that glial cells are resistant to neurotoxic ApoE peptides, but also with the well established finding that LRP is found abundantly in neurons but not in glial cells (Wolf et al., 1992; Lopes et al., 1994; Tooyama et al., 1995; Fabrizi et al., 1997). Whereas this study provides additional evidence that ApoE4 is toxic in neuronal cells, discrepancies have existed in the literature. In some studies, ApoE4 causes neurotoxic effects (Marques et al., 1997; Tolar et al., 1997, 1999; Jordan et al., 1998; Michikawa and Yanagisawa, 1998; DeMattos et al., 1999), whereas in others, no toxicity has been found (Bellosta et al., 1995; Nathan et al., 1995; DeMattos et al., 1998). This variability could be attributed to several possibilities. One is that ApoE4 sensitivity of the neuronal cells used may be different not only in cell preparations but in cell conditions. Michikawa and Yanagisawa (1998) found that same neurons exhibit different responses to ApoE4 toxicity, in the presence or absence of compactin. Also, the tissue distribution (Bu et al., 1994; Zheng et al., 1994) suggests that RAP expression is differentially regulated from LRP expression, whereas their expression is mutually related (Willnow et al., 1995). It is thus conceivable that the ratio of cellular expression of RAP versus LRP, which could vary among neuronal cells and by cellular conditions, influences the toxic effects of ApoE4. Another possibility is that the culture conditions may affect ApoE4 neurotoxicity. The aforementioned studies reporting negative effects of ApoE4 were performed under conditions with serum or serum supplements including 5 μg/ml of insulin, which could suppress apoptosis. In contrast, our study was performed in the complete absence of serum or other supplements.
We also found that α2M* suppresses ApoE4-induced neuronal death. As this suppression was observed for α2M*, but not native α2M, this effect is highly likely mediated by LRP. However, it is unlikely that this suppression occurs only through inhibition by α2M* of ApoE4 binding to LRP, because Hussain et al. (1991) demonstrated only partial cross-competition between α2M* and ApoE-activated β-migrating very low-density lipoproteins for binding to LRP. Another possibility is that α2M* binding evokes internalization of LRP (Gliemann, 1998) and decreases the amount of cell surface LRP, resulting in impaired toxicity of ApoE4. The third possibility is that α2M* binding to LRP may suppress the function of LRP stimulated by another ligand ApoE4, without inhibiting the binding of ApoE4 to LRP. Such a phenomenon has been observed for another multiligand receptor, the mannose 6-phosphate/insulin-like growth factor-II receptor (Murayama et al., 1990; Takahashi et al., 1993; Ikezu et al., 1995). These possibilities are not mutually exclusive and could help to explain the nearly compete suppression of the ApoE4 effect, if they occur in combination. Whereas this is the first report that α2M negatively interferes with neuronal cell death caused by AD gene products, this α2M antagonism against ApoE4 concurs with recent reports (Blacker et al., 1998; Liao et al., 1998; Alvarez et al., 1999; Dodel et al., 2000; Romas et al., 2000) that polymorphisms of α2M are genetically associated with AD, although this association controversial (Kovacs et al., 1999; Gibson et al., 2000; Higuchi et al., 2000; Sodeyama et al., 2000).
Apolipoprotein E and Alzheimer's disease: the protective effects of ApoE2 and E3
GW Rebeck, M Kindy, MJ LaDu - Journal of Alzheimer's Disease, 2002 - Amsterdam: IOS Press
Introduction: We have known for nearly a decade that the inheritance of the APOE-ε4 allele is a risk factor for Alzheimer’s disease (AD) [131]. Despite this relatively long period of time, we still do not know whether expression of apoE4 per se is detrimental to the AD process. This debate focuses on whether it is the absence of apoE2 and apoE3 or the presence of apoE4 that puts ε4 individuals at risk for AD. The basis of our argument is that apoE performs neuroprotective and neurotrophic functions in the normal, aging brain. ApoE3 (and apoE2) perform these functions more efficiently than apoE4. Thus, individuals without apoE2 or apoE3 are at risk for CNS damage and AD.
rebeck_kindy_ladu.PDF.pdf ref 24 ... noncommital about cytosol
pg 3 / 147: Changes in neurite outgrowth have been associated with changes in the neuronal cytoskeleton, suggesting that apoE may affect the cytoskeletal pathology in the AD brain [95]. NFT composed of aggregates of hyperphosphorylated tau, are the intracellular pathological hallmark of AD. Some NFT-containing neurons are apoE immuno-positive [93,113,122,131], although the temporal appearance and density of NFT are not correlated with APOE-ε4 [35,99,122,153]. ApoE is a secretory protein and undergoes receptor-mediated endocytosis. Therefore, in normal cellular processing, apoE and tau are not in the same intracellular compartment [24], suggesting that a physical interaction between apoE and tau may occur only after cellular damage has led to the breakdown of normal protein trafficking [9]. In apparently contradictory findings, both the presence and absence of apoE appears linked to NFT-like inclusions in transgenic mouse models. ApoE deficient mice exhibit an increase in AT8 (phosphorylated tau)-positive deposits in response to lysosomal dysfunction [10], while over-expression of C-terminal fragments of apoE in neurons induces the formation of NFT-like inclusions [52]. In vitro, Strittmatter and co-workers have demonstrated that purified apoE3 but not apoE4 binds several species of tau [50,51,129,130]. The authors hypothesize that apoE3:tau complex formation protects tau from phosphorylation and prevents self-assembly into paired helical filaments [132]. However, as apoE binds tau at its microtubule-binding domain, tau bound to apoE cannot perform its normal function of binding microtubules [29]. Together, these arguments suggest that it is unlikely that an association between apoE and NFT underlies the link between ε4 and AD.
[HTML] Apolipoprotein E4 (1–272) fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells
- T Nakamura, A Watanabe, TFujino,
T Hosono and M Michikawa - Molecular Neurodegeneration, 2009
Abstract Background: Apolipoprotein E allele ε4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1–272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. Results: To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2) and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1), which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1–272) more strongly than intact apoE4(1–299). Further analysis showed that in Neuro-2a cells expressing apoE4(1–272), the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1–299). Conclusion: ApoE4(1–272) fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1–272) fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration.
art%3A10.1186%2F1750-1326-4-35.pdf ref 42 no contradiction
p4:35 /8 One may consider that apoE is synthesized as a secretory protein; however, how apoE enters the cytosol remains unclear and controversial. Previous studies have shown that apoE escapes the secretory or endosomal internalization pathway, and enters the cytosol of neuronal cells [25,29,40] and non-neuronal cells [41], whereas another study has failed to show this [42]. Therefore, the physiological relevance of the three mitochondrial proteins that we have identified in this study, which are associated with apoE, remains to be confirmed under physiological conditions.
- It is well known that apoE4 is a strong risk factor for AD development, and the regulation of apoE4 function may be a therapeutic target for AD. Our findings indicate that if we could modulate the generation of apoE4(1–272) and/or modulate its translocation to mitochondria, the apoE4-associated induction of neurodegeneration could be prevented or attenuated. Because it has been shown that the C-terminus of the apoE-cleaving enzyme is a neuron-specific, chymotrypsin-like serine protease [25-27], the characterization and modulation of this enzyme activity would be a therapeutic target for AD.
- [25] Huang 2001 [29] Chang 2005
- [40] Lovestone S, Anderton BH, Hartley C, Jensen TG, Jorgensen AL: The intracellular fate of apolipoprotein E is tau dependent and apoe allele-specific. Neuroreport 1996, 7:1005-8.
Apolipoprotein E dose-dependent modulation of β-amyloid deposition in a transgenic mouse model of Alzheimer's disease
RB DeMattos - Journal of Molecular Neuroscience, 2004
- Susceptibility to the development of Alzheimer's disease (AD) is increased for individuals harboring one or more apolipoprotein E4 (!apoE4) alleles. Even though several isoform-specific effects of !apoE have been identified, the relationship between biochemical
PAYWALL
Apolipoprotein E genotype regulates amyloid-β cytotoxicity
- MMM Wilhelmus, I Otte-Höller, J Davis, WE Van Nostrand, RMW de Waal, and MM Verbeek - The Journal of Neuroscience, 2005
The ϵ4 allele of apolipoprotein E (ApoE) is a risk factor for Alzheimer's disease (AD), whereas the ϵ2 allele may be relatively protective. Both alleles are risk factors for cerebral amyloid angiopathy (CAA)-related hemorrhages. CAA is associated with degeneration of smooth muscle cells and pericytes. Previously, we described that synthetic amyloid-β1–40 peptide (Aβ1–40) with the 22Glu → Gln “Dutch” mutation caused pericyte death in vitro by a mechanism that involves Aβ fibril-like assembly at the cell surface. It is known that ApoE binds to Aβ and may modify its biological activities. In the present study, we evaluated the effect of ApoE on Aβ-mediated toxicity of cerebrovascular cells. We observed that cultured cells with an ε4/ε4 genotype were more vulnerable to Aβ than cultures with an ε3/ε3 or ε3/ε4 genotype. The one cell culture with the ε2/ε3 genotype was relatively resistant to Aβ compared with other cultures. Furthermore, we observed a dose-dependent protective effect of native ApoE against Aβ-mediated toxicity of cerebrovascular cells and, in addition, ApoE ε2/ε3 cells secreted more ApoE protein compared with cells with other ApoE genotypes, in particular, compared with ε4/ε4 cells. Thus, the disparity between ApoE genotype and Aβ-mediated toxicity might be related to differences in the cellular capacity to secrete ApoE. The present data suggest that one mechanism by which ApoE may alter the risk for AD is a genotype-dependent regulation of Aβ cytotoxicity, possibly via variations in its secretion levels, whereby extracellular ApoE may bind to A  and thereby modify Aβ-mediated cell death.
3621.full.pdf ref DeMattos et al. (1999) NOT RELEVANT?
p3623 /3: Partial purification of ApoE from culture supernatant. Purification of extracellular ApoE protein from medium of cultured HBP was performed as described by DeMattos et al. (1999) with minor modifications. ...
Biochemical analysis of cell-derived !apoE3 particles active in stimulating neurite outgrowth
RB DeMattos, LL Rudel, DL Williams - Journal of lipid research, 2001 - ASBMB
Susceptibility to the development of late-onset Alzheimer's disease is increased for individuals harboring one or more apolipoprotein E4 (apoE4) alleles. Although several isoform-specific effects of apoE have been identified, the relationship between biochemical mechanism responsible for the stimulatory effects of apoE3 on neurite outgrowth, we performed a detailed physical characterization of cell-derived apoE3 and apoE4 particles. Immunoaffinity chromatography followed by SDS-PAGE illustrated homogeneity in protein content (apoE >95%). The affinity-purified particles contained phospholipid and 1 mol of cholesterol per mole of apoE but no core lipids. Nondenaturing gradient gel electrophoresis identified two major particle populations with hydrated diameters of 8.0 and 9.2 nm. Neurite outgrowth assays performed with the affinity-purified particles resulted in similar isoform-specific differences as seen previously, apoE3 stimulatory and apoE4 neutral. Interestingly, we did not observe a reduction in apoE medium concentrations over the duration of the neurite outgrowth assays, suggesting little or no endocytic uptake. Ligand blot analysis demonstrated that the affinity-purified apoE particles bind to several Neuro-2a membrane proteins. Western blots of the Neuro-2a membrane proteins indicated that the LDL receptor, gp330, and LR8B might be involved in the apoE-binding event. These results discriminate against the lipid delivery hypothesis and suggest that the biological activity of the phospholipid apoE3 particles may be due to cell surface signaling.
J. Lipid Res.-2001-DeMattos-976-87.pdf ref 33 READ ME
p980/5: Biological activity The biological activity of the affinity-purified apoE3 and apoE4 phospholipid particles was evaluated in neurite outgrowth assays. Parental Neuro-2a cells were incubated with a 30 μg/ml concentration of either apoE3 or apoE4 particles for 48 h and subsequently analyzed for neurite outgrowth. Figure 3A shows that Neuro-2a cells incubated in the presence of apoE3 maintain longer neurites over the entire population, whereas cells incubated with apoE4 were similar to the control. When analyzed for average neurite lengths (Fig. 3B), the apoE3-treated cells had significantly longer neurites (164%, P = 0.0001 vs. control). No significant change in neurite outgrowth was detected for cells treated with the affinity-purified apoE4, confirming our previous observation in stably transfected Neuro-2a cells that apoE4 is neutral for neurite outgrowth (37). ELISA measurement of apoE mass in the medium at the conclusion of the 48-h assay showed no difference from initial concentration (data not shown). We also analyzed the apoE particle populations present at the conclusion of a 48- and 96-h assay by NDGGE apoE Western blot ( Fig. 4 ). Neither particle concentration nor the distribution of the particle populations for apoE3 and apoE4 was altered after incubation with cells for either 48 or 96 h. Note that we have shown previously that Neuro-2a-derived apoE3 is effective in mediating rapid endocytosis when the apoE is purified and reconstituted into large dimyristoylphosphatidylcholine disks or added to β-VLDL particles from apoE-deficient mice (33). The lack of endocytosis observed here, therefore, likely reflects the presence of apoE in small particles that are not efficiently taken up by receptor-mediated endocytosis.
Apolipoprotein E is a prime suspect, not just an accomplice, in Alzheimer's disease
- KA Crutcher - Journal of Molecular Neuroscience, 2004 - Springer
- There is now a large body of evidence suggesting that apolipoprotein E (apoE) genotype is the single most important genetic risk factor for the most common (sporadic) form of Alzheimer's disease. Yet in proportion to the total number of investigations in this
Paywall
Truncated !apoE forms tangle-like structures in a neuronal cell line
- MC Ljungberg, R Dayanandan, A Asuni… - …, 2002 - journals.lww.com
- Apolipoprotein E is the predominant brain lipoprotein and polymorphic variation in the APOE gene the major genetic susceptibly factor for late onset Alzheimer's disease (AD). Recently it was reported that carboxyl-truncated ApoE fragments induce tangle-like
Paywall
Progress toward identification of protease activity involved in proteolysis of apolipoprotein e in human brain
- MA Marques, PA Owens, KA Crutcher - Journal of Molecular Neuroscience, 2004 - Springer
- Apolipoprotein E (apoE) genotype is the single most important genetic risk factor for the most common (sporadic) form of Alzheimer's disease (AD). Increasing evidence supports the hypothesis that the presence of the E4 isoform of this cholesterol-binding protein
Paywall
Endogenously expressed apolipoprotein E has different effects on cell lipid metabolism as compared to exogenous apolipoprotein E carried on triglyceride-rich …
- YY Ho, M Al-Haideri, T Mazzone, T Vogel… - Biochemistry, 2000 - ACS Publications
- Apolipoprotein E (apoE) on model triglyceride-rich particles (TGRP) increases triglyceride (TG) utilization and cholesteryl ester (CE) hydrolysis, independent of its effect on enhancing particle uptake. We questioned whether, under physiological concentrations, endogenously
Paywall
Presence of apolipoprotein E immunoreactivity in degenerating neurones of mice is dependent on the severity of kainic acid-induced lesion
- J Grootendorst, M Mulder, E Haasdijk, ER de Kloet, D Jaarsma, Brain research, 2000
- Apolipoprotein E (apoE) is a major apolipoprotein in the central nervous system (CNS) that may play a role in various CNS disorders. ApoE is primarily localised in astrocytes, but neuronal apoE mRNA expression has been demonstrated in normal and diseased human brain, as well as in ischaemic rat brain. To obtain further insight into the role of apoE in neuronal degeneration in the CNS and conditions of neuronal apoE localisation, we have investigated in mice the distribution of apoE following neuronal injury induced by kainic acid (n535, 25 or 35 mg kainic acid / kg BW). Consecutive series of brain sections were immunostained for apoE and markers for astroglia (GFAP) and microglia / macrophage cells (CR3). Degenerating neurones were identified with a silver-degeneration staining technique. The intensity and cellular distribution of apoE-immunoreactivity (apoE-ir) was dependent on the severity of neuronal injury. Mice that developed mild neuronal degeneration, restricted to a subset of neurones in the hippocampus, showed increased apoE-ir in astrocytes concomitant with increased GFAP-ir and mild microgliosis. In these mice, no neuronal apoE-ir was detected. In contrast, mice developing severe neuronal injury in the hippocampus — frequently also showing degeneration in other brain regions including cortex, thalamus, striatum and amygdala — showed intense apoE-ir in degenerating neurones. Surrounding the lesion, apoE-ir was increased in neuropil recurrently whereas GFAP-ir astrocytes disappeared. Thus, in mice apoE accumulates in degenerating neurones in conditions of severe neuronal injury putatively in association with disruption of the glial network.
- 1-s2.0-S0006899300022502-main.pdf ref 10 vague reference to "several lines of evidence"
p172 /8: It has been proposed that apoE has a function in the removal of degeneration products e.g. membrane fragments of neurones and possibly astrocytes. Previously, it has been found that after lesioning of peripheral nerves, macrophages infiltrate the site of injury and produce huge amounts of apoE, which associates with membrane fragments of degenerating cells. These fragments are thought to be internalised by the macrophages and redistributed amongst regenerating cells [6,31,44]. The disappearance of apoE-ir neurones as well as astrocytes, observed in severely injured areas following increases in apoE levels, therefore suggests that apoE has a role in the removal of cell debris. Recently, evidence for such a role of apoE was obtained by Fagan et al. [12] who found following an entorhinal cortex lesion, a persistence of degeneration products in the deafferented hippocampus of apoE-deficient mice compared with wild type mice. Another observation supporting this role for apoE is the accumulation of cholesterol in the ageing brain of apoE-deficient mice [67,68]. In the CNS, microglia may be involved in the removal of membrane fragments associated with apoE. Alternatively, a protective role for apoE against injury is suggested by the observation that apoE-deficient mice are more susceptible to injury than wild type mice. They display an abnormal compensatory response after ECL, show age-related neurodegeneration and worsened outcome from global ischaemia [7,33,54]. However, recently it has been shown that cytosolic apoE is incompatible with neuronal survival [10]. The precise role of apoE in the CNS is not yet fully understood. However, several lines of evidence indicate that in conditions of neuronal injury it acts as a chaperone involved in the redistribution of lipids.
Apolipoprotein E-related neurotoxicity as a therapeutic target for Alzheimer's disease
- MA Marques, KA Crutcher - Journal of Molecular Neuroscience, 2003 - Springer
- Apolipoprotein E (apoE) remains the most important genetic risk factor for the development of Alzheimer's disease (AD). Still elusive, the role of apoE is under intense investigation. We propose that proteolysis of apoE in the brain leads to two major fragments,
Following activation of the amyloid cascade, apolipoprotein E4 drives the in vivo oligomerization of amyloid-β resulting in neurodegeneration
- H Belinson, Z Kariv-Inbal, R Kayed… - Journal of …, 2010 - content.iospress.com
- According to the amyloid hypothesis, the accumulation of oligomerized amyloid-β (Aβ) is a primary event in the pathogenesis of Alzheimer's disease (AD). The trigger of the amyloid cascade and of Aβ oligomerization in sporadic AD, the most prevalent form of the
Neuronal cell apoptosis by a receptor-binding domain peptide of ApoE4, not through low-density lipoprotein receptor-related protein
- A Hagiwara, Y Hashimoto, T Niikura, Y Ito… - Biochemical and …, 2000 - Elsevier
Since an apolipoprotein E4 (ApoE4) peptide composed of the low-density lipoprotein (LDL) receptor-related protein (LRP)-binding domain [ApoE4 (141–149) 2 or ApoE (141–155) 2] exerts neurotoxicity in primary neurons and neuronal cell lines, it has been controversial whether these effects are mediated by LRP. Here, we examined whether ApoE4(141–149)2-induced toxicity is mediated by LRP in a neuronal cell system where ApoE4 toxicity is mediated by LRP: serum-deprived F11 neuronal cells. In these cells, where ApoE4 exerted toxicity by apoptosis in a manner sensitive to both caspase inhibitors and pertussis toxin (PTX), ApoE4(141–149) 2 also caused cell death by apoptosis but in a caspase-inhibitor-resistant, PTX-resistant manner. ApoE4(141–149) 2 -induced death was not inhibited by antisense oligonucleotides to LRP. Therefore, we conclude that ApoE4(141–149) 2 is able to exert neurotoxicity without involving LRP
1-s2.0-S0006291X00938415-main.pdf ref 19 IRRELEVANT?
p633/ 1 An important clue is the finding that ApoE4 exerts in vitro neurotoxicity in primary neurons and immortalized neuronal cells (15–19). In clarifying the molecular mechanism, the first and most important question is whether LRP mediates this action. LRP was identified as a molecule structurally related to LDLR and has been established as the primary ApoE receptor, particularly in neurons. Despite the fact that multiple groups have made efforts to address this question, no consistent answer has been obtained. Jordan et al. (20) argued against the mediation by LRP of ApoE4-induced neurotoxicity in rat hippocampal neurons. In contrast, Tolar et al. (17, 18) provided evidence that the toxicity by 22-kDa N-terminal fragments of ApoE4, as well as full-length ApoE4, is mediated by LRP in chick lumbar sympathetic ganglion neurons and rat hippocampal neurons.
[CITATION] 'Lipoproteins in the brain: a new frontier'
- S Koch, U Beisiegel - Lipids and vascular disease, 2000 - Martin Dunitz London
Paywall
Apolipoprotein E gene and Alzheimer's disease: is tau the link?
- S Lovestone, B Anderton, J Betts, R Dayanandan, G Gibb, C Ljungberg and J Pearce - Biochemical Society Symposia, 2001
- The finding that APOE (the gene encoding apolipoprotein E) polymorphic variation was associated with an altered risk of developing Alzheimer's disease (AD) was a significant advance and immediately prompted a search for the mechanisms responsible for this alteration. Some 6 years later, a number of different hypotheses remain that might account for this influence on pathogenesis with no single mechanism being unequivocally accepted. The different approaches to understanding these mechanisms can be broadly categorized as: those suggesting a remote effect, such as different rates of vascular risk factors in those with the different APOE alleles; those proposing altered neuronal vulnerability, perhaps due to apolipoprotein E (ApoE)-isoform-specific differences in local cholesterol transport; and those hypotheses postulating an ApoE interaction with the two key lesions of AD, plaques and tangles. In this chapter we will review the evidence for and against an interaction between ApoE and the neuronal cytoskeleton, in particular with the microtubule-associated protein tau.
- 111.full.pdf ref 65
p116/6: In an important recent challenge to these findings DeMattos and colleagues [65] generated a construct in which a nuclear localization signal (NLS) was tagged to ApoE. Neuro-2a (neuroblastoma) cells were incubated with lipidated ApoE-NLS derived from transiently transfected cells. This ApoE was taken up into a vesicular compartment as previously observed in other cell models. If ApoE were to escape from this compartment and enter the cytosol then it would be transferred to the nucleus. As nuclear fractions contained no ApoE it was concluded that ApoE remains within the vesicles and does not reach the cytoplasm. The authors also emphasize that previous studies reporting intracellular ApoE have not fully clarified whether the ApoE is truly cytoplasmic. Furthermore, in initial studies, transfection of cells with ApoE3 lacking the normal signal peptide, and therefore cytosolic on expression, resulted in low expression and few clones in attempts to generate cell lines. The authors interpret this as excitotoxicity. However, despite the findings of this important study the cytosolic hypothesis of the mechanism of action of ApoE cannot be entirely rejected. Firstly, the intracellular fate of ApoE is clearly both cell and ApoE-type dependent. We reported that ApoE from CSF remains, even visually, restricted to a vesicular compartment in the absence of tau. It was only when tau was massively overexpressed in COS cells (when the tau becomes one of the major proteins produced by the cell) that a redistribution of ApoE, occurs. Neuro-2a cells, a neuroblastoma cell line, express tau, at relatively low levels. As we found no redistribution of ApoE, even in COS cells expressing low amounts of tau, it is not surprising that DeMattos and colleagues failed to find ApoE entering a cytosolic compartment. Secondly, in ongoing studies we find that the intracellular fate of ApoE is highly dependent upon ApoE type. We have compared different recombinant ApoEs combined with different lipid preparations to CSF and to ApoE derived from different cell lines stably expressing ApoE. It is apparent from these experiments that the fate of recombinant, lipidated ApoE, on incubation with cells expressing the LDL receptor, is entirely different from that of CSF ApoE. ApoE from cell lines is taken up by cells but less effectively than ApoE from CSF. It may be that there are important differences between the entirely physiological ApoE in CSF and the ApoE used in the experiments of DeMattos and colleagues. Finally, the finding that there was a reduced transfection efficiency for ApoE3 directed to the cytosol cannot be accepted as evidence for cell toxicity. Indeed the generation of cell lines stably expressing tau is difficult and neuroblastoma cell lines tend to express low levels of tau (and even less before differentiation). One plausible explanation of these observations is that the expression of tau stabilizes the microtubule network and that, therefore, cell division is inhibited, rendering the generation of stable cell lines expressing high levels of tau difficult. If the cytosolic tau-interaction hypothesis of the ApoE mechanism is correct then this is exactly what would be expected if ApoE3 were expressed in the cytosol of cells expressing some tau – that the tau would be relatively less phosphorylated, that it would be more effective in stabilizing microtubules, that cell division would be inhibited and that, therefore, the generation of high-expressing cell lines would be prevented.
- ...
Further work is needed to clarify whether ApoE does indeed pass from endosomes into Golgi, endoplasmic reticulum and thence to the cytosol. However, even if exogenous ApoE does not enter the cytosol, the finding that human ApoE message is present in neurons [64], suggests that a tau–ApoE interaction within the cytosol is plausible.
- Ref 64: Roses, A.D., Gilbert, J., Xu, P.T., Sullivan, P., Popko, B., Burkhart, D.S., Christian-Rothrock, T., Saunders, A.M., Maeda, N. and Schmechel, D.E. (1998) Neurobiol. Aging 19 (Suppl.), S53–S58
[HTML] Fleshing out the amyloid cascade hypothesis: the molecular biology of Alzheimer's disease
- S Lovestone - Dialogues Clin Neurosci, 2000 - dialogues-cns.com
- Alzheimer's disease (AD) is a disorder of two pathologies-plaques and tangles. The former have as a key constituent amyloid protein and the latter the microtubule-associaied protein tau. Genetics has demonstrated that changes in either protein are sufficient to cause dementia. The amyloid cascade hypothesis proposes that plaque-related changes precede tangle-related changes and positions amyloid as central to the degeneration of AD. All the evidence suggests this is correct, including evidence that presenilins alter the processing of the amyloid precursor protein and evidence that disrupting the normal properties of tau underlies the related frontotemporal dementias. The amyloid cascade hypothesis has provided the basis for nearly a decade of intensive basic science—the skeleton of that hypothesis can now be fleshed out, and confidence is growing that this will result in useful disease-modifying therapies in the future.
DialoguesClinNeurosci-2-101.pdf ref 121 MoreLater
p106/6: What else is known about AD that impacts upon the cascade? Most obviously omitted from this scheme is apolipoprotein E (apo E), the only confirmed genetic association with late-onset AD. [111,112] Studies of the biology of apo E have proved very difficult to conduct, with disparate results partly accounted for by technical differences in the preparation of apo E protein. Apo E has been shown to interact with amyloid peptide, but some studies show greater interaction with apo E2 and others with apo E4. [113-115] Depending upon the true result in vivo, apo E binding might enhance amyloid fibrillization and hence plaque formation, or enhance amyloid clearance and hence plaque destruction. Alternatively, apo E might affect tau phosphorylation. Tau binds apo E in an isoorm-dependent manner, and it was hypothesized that such binding would alter the phosphorylation state of tau. [116-118] We have confirmed this is in fact the case (unpublished observations), although whether this occurs in vivo is uncertain. Indeed it is not even known if tau and apo E would meet in vivo. Some studies suggest extracellular apo E is internalized into the cytoplasm compartment. [119,120] At least one study suggests it is not. [121] In neurons, apo E appears to be in the cytoplasm, but this might result from expression of apo E in a form that is not immediately secreted. [122-124] Other cellular approaches do suggest tau alters microtubules and affects neuronal growth, both compatible with, but not proving, an effect of apo E on tau. [120,125-127] It might be that apo E has no effect on either tau or amyloid, affecting instead local cholesterol transport, neuronal viability, and resilience to damage. At present, apo E can be slotted into the cascade in too many places to be sure which is the most likely.
Apolipoprotein E (!apoE) uptake and distribution in mammalian cell lines is dependent upon source of !apoE and can be monitored in living cells
- MC Ljungberg, A Asuni, J Pearce, R Dayanandan, W Marz, MM Hoffmann, P Bertrand, G Siest, HTR Rupniak, BH Anderton, M Huettinger, S Lovestone, Neuroscience Letters, 2003
- As part of investigations of the cellular uptake of apolipoprotein E (apoE) relevant to Alzheimer's disease we have found that different preparations of apoE are handled differently by cells expressing the LDL-receptor. Comparing recombinant, cellular and native apoE, complexed with different preparations of lipid we find that only cellular and native apoE enter a vesicular compartment. Some, but not all of these apoE containing vesicles are lysosomes. In order to further examine the intracellular fate of apoE we demonstrate that apoE-Enhanced green fluorescent protein chimeric protein can be taken up from medium by recipient cells and tracked within these cells for extended periods.
1-s2.0-S0304394003000648-main.pdf ref 3 Look More At This One!!!
p69 /1: However, in order for this to occur apoE would have to be taken up from the extracellular space into the cytoplasm as even neuronally expressed apoE would be secreted and not be present in the same intra-cellular compartment as tau. Two independent studies suggested such uptake could occur – Nathan et al. [15] reported that exogenously applied apoE3 preferentially accumulated in the cytoplasm of neuroblastoma cells whilst we observed a similar phenomenon with apoE derived from CSF when applied to COS-7 cells expressing LDL receptors [13]. However, in direct contrast to these studies, De Mattos et al. [3] failed to observe escape of apoE from the endosomal-lysosomal pathway. These apparent contradictions might result from the different assays or from different preparations of apoE utilised. We therefore examined apoE uptake and intra-cellular localisation and trafficking using a number of different sources of apoE in both fixed and in living nonneuronal cell models.
Purification of Mg2+-dependent phosphatidate phosphohydrolase from rat liver: new steps and aspects
- EA Siess, MM Hofstetter - Biological chemistry, 2005 - degruyter.com
A new procedure for the partial purification of Mg 2+-dependent, N-ethylmaleimide- sensitive phosphatidate phosphohydrolase (Mg 2+-PAP; EC 3.1. 3.4) from rat liver cytosol is described, using protein precipitation with MgCl 2, gel filtration on Sephacryl S-400,chromatography on DEAE-cellulose and affinity chromatography on cal-modulin-agarose. From the parallel change in staining intensity and in the level of the specific activity of enzyme fractions, a relationship between a 90-kDa SDS gel band, identified as the b-isoform of the 90-kDa heat shock protein, and Mg 2 q -PAP could be detected.
bc.2005.137.pdf , ref as DeMattos et al., 1999 noteworthy
p 1198: Regarding enzyme stability, the activity of the KSCN extract decreased only slowly within 3–4 weeks at 4 deg C; however, further purification markedly destabilised the enzyme activity. One possible reason might be the separation of a stabilising component(s) from Mg2+ -PAP duing the purification procedure. In this context, it seems noteworthy that a 34-kDa band identified by amino acid sequencing as apolipoprotein E lacking the signal peptide (DeMattos et al., 1999) was markedly diminished on further purification of the KSCN extract, as the involvement of apolipoprotein E in hepatic triglyceride synthesis has been documented (Huang et al., 1998; Mensenkamp et al., 1999). However, our recombination experiments failed to improve the enzyme stability. Thus, for this marked increase in instability concomitant with the increase in specific activity, the activity of the overall enzyme purification should be estimated from the factors yielded from step to step, rather than from the final absolute value of the specific activity to avoid gross under-estimation. Compared on this basis, our purification procedure seems to be at least as effective as that of others who reported a 416-fold purification (Butterwith et al., 1984).
Alterations in the cerebrospinal fluid relating to apolipoprotein E after traumatic brain injury and subarachnoid haemorrhage.
- AD Kay - 2004 - discovery.ucl.ac.uk
Background: The human gene coding for apolipoprotein E is polymorphic, and the APOE4 allele has been associated with less favourable outcome after acute brain injury including traumatic brain injury (TBI) and subarachnoid haemorrhage (SAH). Experimental studies identify key roles for apoE in the central nervous system such as the scavenging and recycling of lipids for cellular maintenance and repair and formation of cerebral amyloid aggregate. Human in-vivo evidence supporting the concept that apoE is involved in the response of the brain to acute injury is sparse. Objectives: This study tests the hypothesis that apoE is involved in the response of the human brain to injury, and this role is reflected by changes in cerebrospinal fluid (CSF) apoE concentration after brain injury which correlate with injury severity and outcome. In addition it was hypothesised that changes in apoE concentration would be paralleled by changes in the composition of CSF lipoprotein particles (Lps) of which apoE is a major component. Lastly, apoE is reported to chaperone amyloid-beta peptide (AP), therefore we hypothesised that alteration in CSF apoE after brain injury would parallel alterations in Ap. Methods: Enzyme linked immunosorbant assay (ELISA) was used to determine the concentration of apoE, Ap, S100B and Tau (as surrogate markers of brain injury) in CSF from TBI and SAH patients and a non-brain injured control group. Lipoprotein particles were isolated from CSF using size exclusion chromatography and characterised in relation to cholesterol, phospholipid, apolipoprotein E, and apolipoprotein AI composition. Injury severity was determined using the Glasgow Coma Score, and clinical outcome using the Glasgow Outcome Score. Results: Compared to controls there was a sustained decrease in the concentration of apoE in the CSF after TBI and SAH which was paralleled by a depletion of apoE containing lipoprotein particles. Furthermore, CSF Ap also decreased, and the decrease correlated with injury severity and clinical outcome. In contrast the levels of S100B and Tau in brain injury CSF was substantially elevated. Conclusion: Despite the likely leakage of plasma apolipoprotein E into the subarachnoid space at the time of brain injury, apoE in the form of LpE is cleared from the CSF within days of injury. In addition, indirect evidence suggesting apoE-Ap interactions in-vivo support the concept that apoE may form insoluble aggregates with Ap soon after brain injury. The finding that these alterations in the CSF correlate with injury severity and outcome provides novel indirect in-vivo evidence that apoE is important to the response of the human brain to injury.
- U602809.pdf
1.3.2 Apolipoprotein E and neuronal plasticity In contrast to the CNS, peripheral nerves have the capacity to regenerate. Following rat sciatic nerve crush injury, macrophage apoE secretion increases one hundred fold, reaching a peak one week after injury and normalises within a few months when regeneration is largely complete. The secreted apoE facilitates the recycling of cholesterol from the degenerating neuron, which may then be used by the growth cones of neurites via upregulated LDL receptors at their tips. (Goodrum, 1990; Goodrum, 1991; Goodrum et al. 2000a; Goodrum et al. 2000b; Ignatius et al. 1987a; Ignatius et al. 1987b). However, when APOE -/- (knockout) mice undergo peripheral nerve injury, there is no apparent reduction in regeneration suggesting that in the mouse peripheral nervous system, lipoproteins other than apoE (e.g. apoJ) may be utilised for the same purpose. (Goodrum et al. 1995; Popko et al. 1993) The CNS of APOE knockout mice are reported to have synapto-dendritic structural abnormalities, measured using Microtubule-Associated Protein (MAP-2) and synaptophysin immunoreactivity, in hippocampal and cortical areas, which increase with age. Amelioration of these changes by intraventricular infusion of exogenous apoE or endogenous expression of human apoE3 (but not human apoE4) suggests there is a degree of synaptic plasticity that is apoE dependent. (Masliah et al. 1996; Masliah et al. 1997) If these apoE knockout mice experience hippocampal injury, then clearance of lipid-laden products of neurodegeneration is impaired supporting the concept that apoE plays a role in lipid and cholesterol recycling. (Fagan et al. 1998; White et al. 2001a) A number of in-vitro studies utilising peripheral and central nervous system cell cultures, and more recently organotypic hippocampal cultures from human APOE transgenic mice, have shown that apoE3 promotes neurite extension to a greater extent than apoE4. The effect requires the apoE to be associated with a lipoprotein transport vehicle directing it to the HSPG-LRP pathway. The mechanism by which apoE promotes neurite extensionand the reason for greater effect with apoE3 has not been conclusively elucidated. (Bellosta et al. 1995; DeMattos et al. 1998; Fagan et al. 1996; Holtzman et al. 1995; Nathan et al. 1994; Nathan et al. 1995; Sun et al. 1998) In vitro studies support a role for apoE in the stabilisation and remodelling of the neuronal cytoskeleton. Binding of apoE3 to the microtubule associated protein Tau stabilises the microtubules and prohibits hyperphosphorylation. It has been postulated that deficient apoE4- Tau interactions result in Tau self association and hyperphosphorylation, laying down the foundations for Paired Helical Filament (PHF) formation and thereafter neurofibrillary tangles (NFT). If cultured Neuro-2a cells are treated with neurite outgrowth inhibiting lipidated apoE4 there are fewer microtubules, and a lower ratio of polymerised to monomeric tubulin, compared to cultures treated with neurite outgrowth promoting lipidated apoE3 treated cells. (Nathan et al. 1995) There is additional evidence to support a role for apoE in cytoskeletal remodelling from the APOE transgenic and knockout mice. In addition to cytoskeletal abnormalities, APOE knockout mice have increased phosphorylated Tau immunoreactivity compared to wild-type mice. (Genis et al. 1995; Masliah et al. 1995) The transgenic mice expressing human APOE&4 have more cytoskeletal abnormalities than those expressing APOEz3. (Buttini et al. 1999) However, the relevance of these cytoskeletal-remodelling observations to human pathology such as Alzheimer's Disease (AD) is uncertain given the failure to demonstrate apoE in the neuronal cytosol, and the lack of consistent evidence supporting an association between APOE genotype and NFT burden in AD brain. (Beffert et al. 1998; DeMattos et al. 1999)
[Thesis] 24s-Hydroxycholesterol. Studies on regulatory mechanisms behind its formation in the brain and its potential use as a marker for neurodegeneration
- M Shafaati - 2010 - openarchive.ki.se
- Cholesterol 24-hydroxylase (CYP46A1) belongs to the cytochrome P450 super family and is responsible for conversion of cholesterol to the oxysterol 24S-hydroxycholesterol (24S-OHC). This structural modification allows 24S-OHC to traverse the blood brain barrier and this pathway is the major one for elimination of cholesterol from the mammalian brain. CYP46A1 is almost exclusively located to neurons in the brain and retina. The promoter region of CYP46A1 shows classical hallmarks of a gene with putative housekeeping function. Oxidative stress was the only factor initially shown to cause signicant increase in CYP46A1 reporter activity. The aim of this thesis was to obtain a more profound knowledge about regulation of CYP46A1 and the regulatory importance of its product. In addition the possibility was investigated that the levels of 24S-OHC in cerebrospinal fluid may be used diagnostically.
ShafaaiThesis.pdf ref 43
pg 7 The APOE isoforms differ only by a single amino acid substitution of Cys to Arg at position 112 and 158 (32) leading to different biological properties (33). APOE 3 is the most common isoform (77-78%) in the general population while APOE2 is found in 7-8% and APOE4 in 14-16% of individuals (34, 35). After findings of immunoreactivity of APOE in the amyloid plaques (36) the APOE4 allele was discovered to be the most important genetic risk factor for sporadic Alzheimer’s disease (AD) (32, 37). In contrast, the APOE2 allele has been associated with a lower risk for AD (38). Amyloid beta (Aȕ) is formed by proteolytic cleavage of the amyloid precursor protein (APP) and plays an important role in the AD pathology. There is evidence suggesting that APOE4 is somehow involved in the Aȕ formation. As mentioned above, APOE is present in the neuritic plaques and it has been reported that Aβ levels are elevated in brains of AD patients carrying APOE4 allele. It is not yet known if APOE4 has an active role in aggregation and/or deposition of Aβ. APOE4, in a lipid- free form has a greater avidity to Aβ than APOE3 (34, 39, 40). Post mortem studies have shown increased Aβ deposition in APOE4 carriers both in sporadic and genetic AD cases (39, 41). Hyper-phosphorylation of microtubule-associated protein known as tau protein leads to insoluble aggregation and form neurofibrillary tangles in the neuronal cells. This is common in connection with neurodegenerative diseases, in particular AD. Different APOE isoforms may influence formation of tau. In vitro studies have shown that APOE3 forms a stable complex with tau. The interaction between APOE3 and tau was prevented by phosphorylation of tau, suggesting that APOE3 preferentially binds to nonphosphorylated tau (42). However there is no evidence demonstrating localization of APOE to the neuronal cytosol, where the majority of tau exists under normal conditions (29, 43). In the peripheral nervous system and CNS the levels of APOE increases following neuronal injuries. It has been suggested that this increase may be required for repair of the nervous system by redistribution of lipids and cholesterol for membrane repair and synaptic plasticity. Most studies have shown that APOE3 augment neurite outgrowth to a greater extent than APOE4 (29, 44). The effect of APOE2 has not yet been fully examined in connection to neurite sprouting.
Role of Genetic Background: Influence of Apolipoprotein E Genotype in Alzheimer's Disease and After Head Injury
ME Kerr, ST DeKosky, A Kay, DW Marion - Brain Injury, 2001 - Springer
- Severe traumatic brain injury (TBI) represents an emergent situation whereby health team members are challenged to optimize cerebral perfusion and prevent secondary brain injury. Despite intense interest in furthering our understanding of the pathogenesis of
PAYWALL
[CITATION] Targeting !ApoE in Alzheimer's Disease: Liver X Receptor Agonists as Potential
- DR RIDDELL, DJ O'NEILL - Emerging Drugs and Targets for Alzheimer's Disease
can't find
[BOOK] small heat shock proteins and apolipoprotein E in Alzheimer's disease
- MM Wilhelmus - 2006 - repository.ubn.ru.nl
- Small heat shock proteins (sHsps) are professional chaperones that have a specific function in helping the normal folding process of proteins and in the intracellular handling of misfolded proteins. The pathological lesions of Alzheimer's disease (AD), such as senile
50411.pdf ref 396 NOT RELEVANT, procedure only
p102 Partial purification of ApoE from culture supernatant
Purification of extracellular ApoE protein from medium of cultured HBP was performed as described by DeMattos et al (396) with minor modifications. Cells were incubated with serum-free EMEM medium for 6 days. Cells remained viable during this period. Supernatant was collected and passed through a D100 weakly basic anion exchange filter (Sartorius, Goettingen, Germany), and eluted with 1 M ammonium bicarbonate. The eluted ApoE was recirculated over a HiTrap heparin column (Amersham Pharmacia). Fractions containing ApoE were pooled and dialyzed against PBS. Using the above-described ApoE sandwich ELISA and comparing the ApoE concentration with the total protein amount, it was demonstrated that in the purified fractions 25% of the total protein amount consisted of ApoE.
Regulierung und Verfügbarkeit von Apolipoprotein E in Astrozyten
- U Hamker - 2005 - edoc.hu-berlin.de
- Page 1. i Regulierung und Verfügbarkeit von Apolipoprotein E in Astrozyten Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der
Hamker.pdf ref 81 supportive
- Verglich man den ApoE-Gehalt der Zytosol-Fraktionen, so konnte ApoE hier weder bei der PBS-Kontrolle noch bei den β/A4(1-40) inkubierte Astrozyten nachgewiesen werden. Letzteres steht in Übereinstimmung mit der Literatur [81]. In den Debris-Fraktionen fand sich ApoE sowohl bei der PBS-Kontrolle als auch bei den β/A4(1-40) inkubierten Astrozyten, wobei die Menge an ApoE in der β/A4(1-40)-Debris-Fraktion um ein Vielfaches höher war. Dies war zu erwarten, da in dieser Fraktion auch Amyloid enthalten ist, welches an den Zellmembranen anhaftet und in großer Menge ApoE bindet (siehe 4.5.5).
If the apoE content of the cytosol fractions was compared, ApoE could not be detected here either in the PBS control nor in the β / A4 (1-40) incubated astrocytes. The latter is consistent with the literature [81]. In the debris fractions ApoE was found both in the PBS control and in the β / A4 (1-40) incubated astrocytes, the amount of ApoE in the β / A4 (1-40) debris fraction being reduced by one Many times higher. This was to be expected, since this fraction also contains amyloid, which adheres to the cell membranes and binds ApoE in large amounts (see 4.5.5).