Role Of Truncated ApoE4 In Alzheimer's Disease And The Possible Mechanism

It is well-known that apolipoproteinE4 (apoE4) is a major risk factor of Alzheimer's disease (AD). However, the mechanism of increasing the risk of AD and its role in the pathogenesis of AD are not fully understood. It is widely recognized that the major neuropathological hallmarks in AD brains are the intracellular neurofibrillary tangles (NFTs) and extracellular senile plaques (SP). Studies both in vitro and in vivo suggest that apoE4 can be cleaved by a chymotrypsin-like serine protease to produce a kind of carboxyl-terminal amino acids 272-299-truncated fragments (A272-299) in cultured neurons and in AD brains. And this kind of truncated apoE4 fragments (A 272-299) also exists in NFTs and SP. Therefore, studing the role of full length apoE4 in the two neuropathology changes of AD becomes the focus in AD research recently. We speculate that this kind of truncated-apoE4 fragments maybe involved in the pathological process of AD. In present study, we have systemically explored the effect of truncated-apoE4 fragments on the tau and NF phosphorylation in vitro and in vivo and its possible underlying mechanisms.Part 1 The effects of overexpression of truncated-apoE4 fragments on tau and neurofilaments in N2a cellsHuman apolipoprotein (apo) E4 is one of proverbial major risk factors for Alzheimer's disease (AD). However, the mechanism of increasing the risk of AD and its role in the pathogenesis of AD are not fully understood. It is widely recognized that the intracellular neurofibrillary tangles (NFTs) is one of the major neuropathological hallmarks in AD brains. The NFTs are largely composed of the hyperphosphorylated microtubule-associated protein tau (p-tau) and hyperphosphorylated neurofilaments (p-NF). Studies both in vitro and in vivo suggested that apoE4 can be cleaved by a chymotrypsin-like serine protease to produce a kind of carboxyl-terminal amino acids 272-299-truncated fragments (A272-299) in cultured neurons and in AD brains. And this kind of truncated apoE4 fragments (A272-299) also exists in NFTs. So we speculate that this kind of truncated-apoE4 fragments maybe involved in the phosphorylation of tau and neurofilaments, resulting in the pathological process of AD.To investigate the role of truncated apoE4 fragments on tau and neurofilament phosphorylation, we constructed the recombinant plasmid of truncated apoE4 fragment (pEGFP-T-apoE4) and overexpressed the full length-apoE4 and truncated-apoE4 fragments in N2a cells respectively by transient transfection. It was observed that overexpression both of the full length-apoE4 and truncated apoE4 fragments in N2a cells induced a dramatic increase in phosphorylation of tau and NFs at several sites compared to untransfected cells. The most efficiently increasing phosphorylation of tau and NFs is in transfected pEGFP-T-apoE4 cells. These results indicated that overexpression truncated-apoE4 has the higher ability to phosphorylate tau and NFs than overexpression full length-apoE4. GSK-3 and CDK5 are putative key kinases for phosphorylating tau and NFs. Our results show that the activity of GSK-3 is significantly increased in N2a transfected pEGFP-apoE4 or pEGFP-T-apoE4 compared with control (p<0.05), and the most efficiently increasing the activity of GSK-3 is also in transfected pEGFP-T-apoE4 cells. But the activity of CDK5 has no change after transfected pEGFP-apoE4 or pEGFP-T-apoE4. These data indicated that overexpression full length-apoE4 and truncated-apoE4 can phosphorylate tau and NF and these effects maybe involved in activating GSK-3.Besides, we prepared the condition medium respectively containing full length-apoE4 or truncated-apoE4. We detected the correspondial changes of concentration of the phophorylated tau, NFs and dab1. At the same time we also detected the activities of GSK-3 and CDK5. These data showed that neither extracellular full length-apoE4 nor extracellular truncated-apoE4 fragments can increase the correspondial changes of concentration of phosphorylated tau and NFs and can activate GSK-3 and CDK5 activities. It was observed that the correspondial change of concentration of phophorylated dab1 was increased in either transfected pEGFP-apoE4 or pEGFP-T-apoE4 cells. We suggest that intracellular but not extracellular apoE4 can activate GSK-3 resulting in hyperphosphorylation of tau and NFs.In conclusion, our data demonstrate that overexpression of truncated-apoE4 fragments can induce tau and NF hyperphosphorylation and it maybe involved in activating GSK-3. These results imply that truncated-apoE4 fragments take part in the pathology process of AD. We propose that the production of truncated-apoE4 fragments could contribute to the increased susceptibility of human apoE4 carriers to AD.Part 2 Truncated-apoE4 gene transfection in vivoIt is well-known that AD is a kind of neurodegenerative disease and the major neuropathological hallmarks in AD brains are the NFTs and senile plaques (SP). Phosphorylation of tau is correlated with learning and memory. So studing the relationship between neuropathology of AD and memory impairment is still high light. Our previous research showed that overexpression of full length apoE4 and truncated-apoE4 induced tau and NF hyperphosphorylation. We begin insight into the role of the full length apoE4 and truncated-apoE4 fragment in AD in vivo.The full length apoE4 gene and truncated-apoE4 gene were transfected into the rat hippocampus respectively. The results of Morris Water Maze showed that the ability of spatial memory decreased in both of full length apoE4 gene and truncated-apoE4 gene transfection groups. The most efficiently decreasing was in transfected pEGFP-T-apoE4 rats. It was observed that overexpression both of the full length-apoE4 and truncated apoE4 fragments induced a dramatic increase in phosphorylation of tau at Ser202 site compared to untransfected rats. The most efficiently increasing phosphorylation of tau is in transfected pEGFP-T-apoE4 rats.Enzyme assay showed that the activity of GSK-3 was significantly increased in rat hippocampi homogenates transfected pEGFP-apoE4 or pEGFP-T-apoE4 compared with control (p<0.05), and the most efficiently increasing the activity of GSK-3 is also in transfected pEGFP-T-apoE4 rats. But the activity of CDK5 has no change after transfected pEGFP-apoE4 or pEGFP-T-apoE4. These data indicated that overexpression both of full length-apoE4 or truncated-apoE4 fragments can phosphorylate tau and NF and it maybe involved in activating GSK-3 in vivo.We also detected the accumulation of Aβin rat hippocampi homogenates. It was observed that overexpression both of the full length-apoE4 and truncated apoE4 fragments induced a dramatic increase in Aβaggregation compared to untransfected rats. The most efficiently increasing Aβaggregation is in transfected pEGFP-T-apoE4 rats.In conclusion, our data showed that truncated-apo4 gene transfection could induce neurodegenerative changes and memory impairment of rats. Inhibition of truncated-apoE4 production may be a new target against AD.Part 3 The effects of truncated-apoE4 fragments on the metabolism of AβThe aggragation of extrocellular amyloid β-peptide (Aβ) to form senile plaques is one of the main pathology changes in AD brains. We proposed that a water-soluble form of Aβ (sAβ) is an early marker of amyloid formation because it is detectable only in brains of subjects with AD or at risk for AD, whereas it is undetectable in normal brain tissue. Aβ is the major component of AD amyloid. The presence of apoE in amyloid plaques, the positive correlation between amyloid burden and the frequency of allele ε4, and the in vitro binding of apoE to Aβ, strongly suggest that apoE influences the rate of cerebral amyloidogenesis. So apoE is the best candidate to play a role in sequestering and clearing sAβ in brain. However, it is still debatable whether apoE 4 promotes or inhibits the aggregation and polymerization of Aβ.Our previouse research shows that both full-length apoE4 overexpression and truncated-apoE4 overexpression can induce the aggregation of Aβ in rat hippocampus tissue,whereas the latter has stronger ability in inducing Aβ aggregation .The 4kD Aβ is generated from its precursor, the Aβ peptide precursor (APP). Most Aβ is comprised of a peptide designated Aβ40 (95%), while Aβ42(5%) is much more aggregatable than Aβ40. So we investigate the different abilities of the two apoE4 in binding with Aβ42 in vitro. We also detect the effects on binding and uptaking of Aβ42 in N2a cells.We prepared full-length apoE4-Aβ42 complex and truncated-apoE4-Aβ42 complex by using Aβ42 incubation with full-length apoE4 condition medium and truncated-apoE4 condition medium respectively. Immunodetection with the monoclonal anti-Aβ antibody 6E10 shows that there is only one band (approximate 40kDa) in SDS-free-PAGE, while there are two bands (approximate 40kDa and 4kDa) in SDS/PAGE. these results reveal that Aβ 42 is released from the complex after SDS/PAGE. Analysis shows that the ratio of the 4 kDa band versus the 40-kDa band is 50% in full-length apoE4 group and 80% in truncated-apoE4 group . These results demonstrate that the release of A β 42 from the truncated-apoE4-Aβ42 complex is more than the full-length apoE4-Aβ42 complex (P < 0.05).This unstable complex can increase the concentration of free Aβ. The unbound Aβ, although soluble, is in an aggregated state. So our results imply that truncated-apoE4 has a stronger ability in promoting Aβ aggregation than full-length apoE4.Some research indicate that the Aβ bound to apoE3 is more sensitive to protease digestion than the Aβ unbound to apoE3. The higher sensitivity to proteinase K of the Aβ pool complexed with apoE3 suggests that apoE3 maintains the Aβ peptides in a soluble aggregate that is sensitive to proteases or might be clearanced by some way. So apoE3 efficiently binds and sequesters Aβ, preventing Aβ aggregation. We investigated the different effects on N2a cells binding and uptaking Aβ42 of full-length apoE4 and truncated-apoE4 in vitro. Our results indicate that the ability of N2a binding and uptaking Aβ42 is sharply impaired after co-incubation with truncated-apoE4-Aβ42 complex compared with full-length apoE4-Aβ42 complex.In conclusion, our results imply that truncated-apoE4 fragment has higher ability to promote Aβ aggregation than full-length apoE4. So we speculate that production of truncated-apoE4 may be one of the pathogenesis of AD induced by allele ε4. Inhibition of truncated-apoE4 production may be a new target against AD.