Neuron Specific ApoE4(1-272) Fragment Triggers ER Stress And Contributes To The Pathogenesis Of Alzheimer’s Diseases

Background:Alzheimer’s disease (AD) is the most common form of dementia in the elderly, characterized clinically by memory loss, cognitive decline, pathologically by the occurrence of brain senile plaques and neurofibrillary tangles (NFTs). At present, the pathogenesis of AD is unknown.The human apolipoprotein E4(ApoE4), has been identified as the major risk or susceptibility factor for Alzheimer’s disease. The ApoE4allele, which is found in40-65%of cases of sporadic and familial AD, increases the occurrence and lowers the age of onset of the disease in a gene dose-dependent manner. Lots of stdies have suggested that ApoE4facilitated amyloid-β aggregation and the formation of plaques, phosphorylation of tau and the formation of NFTs, disrupted cytoskeletal structure and function, impaired learning and memory, and so on.Under physiological conditions, ApoE is synthesized and secreted by astrocytes and microglia in the central nervous system, located in high-density-like lipoprotein particles(HDL). A major function of ApoE is to transport lipids among various cells and tissues of the body, by binding to the ApoE receptors of cell surface and mediating the interaction of ApoE-containing lipoproteins and lipid complexes. However, it has been reported that neuron could synthesize and secret ApoE, when they were suffered with stress or injury. Especially, the neuron specific ApoE4can be cleaved by a protease to generate bio-active C-terminal-truncated forms of ApoE4(residues1-272), refereed as ApoE4(l-272) fragment. Meanwhile, the fragment is found in the brains of AD patients and transgenic mice expressing human ApoE. When expressed in cultured neuronal cells or added exogenously to the cultures, the ApoE4(1-272) fragment, cause cell death, and when expressed in transgenic mice, it cause AD-like neurodegeneration and behavioral deficits. These lines of evidence suggest it is neurotoxic and can enhance the neuropathology and promote neurodegeneration in AD brains, but its roles in the pathogenesis of these lesions is unclear.Meanwhile, the premise results of our laboratory indicated that Neuro-2a cells overexpressing human ApoE4or ApoE4(1-272) fragment caused the hyperphosphorylation of tau and NFs, especially the ApoE4(1-272) fragment. Moreover, we also found that compared to ApoE4, the affinity of the ApoE4(1-272) fragment reduced the binding and uptake to A β in the Neuro-2a cells.The other lines of evidence has been suggested that endoplasmic reticulum (ER) stress was involved in familial and sporadic AD. Meanwhile, ApoE4are also closely associated with AD. However, the exact mechanisms are still not completely understood and some of the conclusions even remain controversial.Objective:In vitro study, determine the expression of ER Stress related marker and the ER Stress specific apopotosis marker after overexpessing the ApoE4(1-272) Fragment in Neuro-2a cells. Next, using neuron specific expression ApoE4(1-272) Fragment construct and its transgenic Mice, to verify the results from in vitro study, and then further examine the affects of neuron-specific-expression ApoE4(1-272) fragment to the learning and memory of the transgenic mice.Methods:Firstly, after overexpressing the ApoE4(1-272) fragment in Neuro-2a cells, we used the real-time PCR and Western blotting to determine the expression of the ER Stress related marker, GRP78/Bip and CHOP, and by RT-PCR to detect the splicing of xbp-1mRNA. Next, we further examined the expression of the ER Stress specific apopotosis marker Caspase-12and its downstream effectors Caspase-3by real-time PCR and Western blotting. Then, using the human neuron specific promoter SYN1, we established the neuron specific expression human ApoE4(1-272) fragment construct and generated ApoE4(1-272) fragment transgenic mice(referred to the transgenic mice), to verify the results from in vitro study. Meanwhile, by the Morris water maze to further evaluate the learning and memory of the transgenic mice, and by the nissl staining to observe the morphology of hippocampus and cortex.Results:Firstly, in vitro study, by overexpressing the human apoE4, ApoE4(1-272) fragment constructs or vector in Neuro-2a cells, we found the ApoE4(1-272) fragment triggered obviously ER Stress, as the expression of the ER Stress related marker, GRP78/Bip and CHOP, were upreglulated, and the splicing of xbp-1mRNA happened. However, the full-length ApoE4did not observed these changes. Then, we found after overexpression of the ApoE4(1-272) fragment in Neuro-2a cells, the expression of the ER Stress specific apopotosis marker Caspase-12and its downstream effectors Cleaced-Caspase-3were also increased, indicated the induction of ER Stress-induced apopotosis. Meanwhile, when we used lmM the chemical chaperone4-PBA treatment, both the ER Stress related marker(Bip and CHOP) and ER Stress induced apopotosis molecules Caspase-12and Cleaced-Caspase-3were decreased, compared to the only overexpressing the human ApoE4(1-272) fragment. Next, we used the ApoE4(1-272) fragment transgenic mice to verify these results from in vitro study. We found that compared to the non transgenic mice, the expression of the ER Stress related marker(Bip and CHOP) of the hippocampus and cortex were also upregulated both at mRNA and protein levels in the transgenic mice. Simultaneously, we observed the splicing of xbp-1mRNA. Further, we found the ER Stress specific apopotosis marker Caspase-12and its downstream effectors Cleaved-Caspase-3were also increased in the hippocampus and cortex of the transgenic mice, compared to the non transgenic mice. Moreover, the results of the Morris water maze showed that, compared to the non transgenic mice, the transgenic mice required longer times to reach the hidden target platform and spent shorter time in the targeted quadrant, indicated its Learning and Memory impairment. Then, using the nissl staining, we found the transgenic mice existed obviously neuron loss and structure loose, especially in CA3and CA1of the hippocampus.Conclusion:We found neuron specific human ApoE4(1-272) fragment induced ER Stress, and further caused the ER Stress-induced apopotosis both in vitro and in vivo. Moreover, we also observed the spatial learning and memory impairment in the transgenic mice, which was involved with the expression down-regulation of the synapse-associated protein and neuron loss induced by the ER Stress-induced apopotosis in hippocampus and cortex. Backgroud:Axonal transport (AT) is an active, directional transport of organelles and molecules along the axon driven by specific motor proteins running along microtubules. Anterograde and retrograde transport move cargoes away from and towards the cell body, respectively. In this context, axonal transport is critical to maintain neuronal function and synaptic activity. Because of the axonal transport distance is extremely long, any malfunction of this process may result in axonal transport impairment, which causes neuronal dysfunction and pathological changes.Recently, it has been reported the axonal transport (AT) defects play an important role in the pathogenesis of Alzheimer’disease (AD). Prior to the deposition of Ap and synapse dysfunction, AT deficits which consisted of axonal swellings that accumulated abnormal amounts of molecular motor proteins, organelles, and vesicles occurred in transgenic mice overexpressing wild-type APR Meanwhile, in the AD brain, the axonopathy happened in the early stage of Braak. It has suggested that such deficits might be early events in AD. Moreover, the axonal transport deficits could cause synapse reduction, dysfunction and neuronal degeneration. However, the mechanism of axonal transport (AT) defects in AD is not clerar.Tau protein, a typical microtubules associated protein(MAP), contributes to the stabilization and spacing of microtubules, and the hyperphosphorylation of tau is closely associated with AD. Menwhile, emerging evidence indicates that endoplasmic reticulum (ER) stress was involved in AD. ER stress can promote the activity of GSK-3β, a key kinase in the phosphorylation of tau. At the same time, some researchers find out the hyperphosphorylation of tau in AD, could lower the affinity of tau for the microtubules, which most likely leads to destabilization. Certainly, the destabilization of microtubules can cause structural disruption and the impairment of AT, even resulting in the synapse dysfunction and neuronal degeneration.The human apolipoprotein E4(ApoE4), has been identified as the major risk or susceptibility factor for Alzheimer’s disease. Especially, it has been reported that neuron specific ApoE4can be cleaved by a protease to generate bio-active C-terminal-truncated forms of ApoE4(residues1-272), refereed as ApoE4(1-272) fragment, which could be a key events in the pathogenesis of ApoE4for AD. However, its roles in the pathogenesis of AD is unclear.Combined with the results of the part1, in which we found that neuron-specific human ApoE4(1-272) fragment triggered ER Stress, and further caused the ER Stress-induced apopotosis, contributed to the pathogenesis of AD, we speculate that whether the ER Stress-triggered by human ApoE4(1-272) fragment may also affects the protein kinase or phosphatase, and then promotes the hyperphosphorylation of tau? Meanwhile, the hyperphosphorylation of tau further caused cytoskeletal structural disruption and the impairment of axonal transport, even synapse dysfunction and impairment of learning and memory?Objective:In order to confirm the above hypothesis, in vitro study, we determined the phosphorylation levels of tau protein and the activity of GSK-3, Cdk5and PP2A, after overexpressing the ApoE4(1-272) fragment in Neuro-2a cells. Next, using neuron specific ApoE4(1-272) fragment transgenic mice, to verify the results from in vitro study, and then using neuronal tracing by biotinylated dextran amine (BDA), to further examine the axonal structure of the neuron specific expression ApoE4(1-272) fragment transgenic mice. Methods:Firstly, after overexpressing the ApoE4(1-272) fragment in Neuro-2a cells, we used Western blotting to determine the phosphorylation levels of tau at Ser404, Ser262and Ser202. Next, we examined the activity of the major kinase or phosphatase of tau phosphorylation, GSK-3, Cdk5and PP2A, by y-32P-labeling assay using specific substrates. Then, using the neuron specific expression human ApoE4(l-272) fragment transgenic mice(referred as the transgenic mice), to verify the results from in vitro study. Meanwhile, using neuronal tracing by biotinylated dextran amine (BDA) to further observe the axonal structure of the neuron specific ApoE4(1-272) fragment transgenic mice.Results:In vitro study, we found the phosphorylation levels of tau at Ser404, Ser262and Ser202was increased, after overexpressing the human ApoE4(1-272) fragment in Neuro-2a cells. By y-32P-labeling assay using specific substrates, we found the activity of the GSK-3, a major kinase of tau phosphorylation, was obviously increased, but the activity of Cdk5and PP2A were not changed. Meanwhile, after1mM the chemical chaperone4-PBA treatment, the phosphorylation levels of tau and the activity of the GSK-3were all deceased compared to the only overexpressing the human ApoE4(1-272) fragment, which indicated that ER stress are involved in the hyperphosphorylation of tau. Next, we used the ApoE4(1-272) fragment transgenic mice, to verify these results from in vitro study. Compared with the non transgenic mice, tau became hyperphosphorylated at all of the three sites in the hippocampus and cortex of the transgenic mice. Simultaneously, by γ-2P-labeling assay using specific substrates, we also found the activity of the GSK-3was obviously increased, but the activity of Cdk5and PP2A were not changed, in the hippocampus and cortex of the transgenic mice. Using the neuronal tracing by BDA, we found the obviously swollen axons, even the swollen varicosities in hippocampus and cortex of the transgenic mice, and the axonal structure of the non transgenic mice is almost normal.Conclusion:We found ER Stress triggered by neuron specific human ApoE4(1-272) fragment caused the hyperphosphorylation of tau by increased the activity of the GSK-3. Moreover, we observed the obviously axonophathy, including swollen axons and varicosities, in the hippocampus and cortex of the transgenic mice, which may be involved with impairment of axonal transport caused by the hyperphosphorylation of tau. Background:Berberine is a primary component of the most functional extracts of Coptidis rhizome used in traditional Chinese medicine for centuries. Recent reports indicate that Berberine has the potential to prevent and treat Alzheimer’s disease (AD). The previous studies reported that Calyculin A (CA) impaired the axonal transport in neuroblastoma-2a (Neuro-2a) cells. Berberine attenuated tau hyperphosphorylation and cytotoxicity induced by CA.Objective:Our study aimed at investigating the effects of Berberine on the axonal transport impairment induced by CA in Neuro-2a cellsMethods:The cultured Neuro-2a cells were transiently transfected with pEGFP-NFM, which enabled the direct observation of NF axonal transport by live fluorescence imaging. When Neuro-2a cells were transfected with pEGFP-NFM for24h, and added2.5nM CA or2.5nM CA+25ug/mL Berberine, then we used TIRFM to see the numerous fluorescence particles moving along axons.Results:The results showed that Berberine could protect the cell from CA-induced toxicity in metabolism and viability, as well as hyperphosphorylation of tau and neurofilaments (NFs). Furthermore, Berberine could reverse CA-induced axonal transport impairment significantly. Berberine also partially reversed the phosphorylation of the catalytic subunit of PP-2A at Tyrosine307, a crucial site negatively regulating the activity of PP-2A, and reduced the levels of malondialdehyde and the activity of superoxide dismutase, markers of oxidative stress, induced by CA.Conclusion:The present work for the first time demonstrates that Berberine may play a role in protecting against CA-induced axonal transport impairment by modulating the activity of PP-2A and oxidative stress. Our findings also suggest that Berberine may be a potential therapeutic drug for AD.