Prophylactic Effects Of P75ECD On Alzheimer’s Disease And The Related Mechanisms

Background and objectiveAlzheimer’s disease(AD) is the most common form of neurodegenerative disease that affects the cognitive functions of the elderly. Currently no therapeutics is available to stop halt or reverse the disease progression. Amyloid-beta(Aβ) plays a pivotal or causative role in the pathogenesis of AD and has become the major therapeutic target. However, the regulation of its production and clearance is not fully understood. The dysregulation of neurotrophins and their receptors in AD is a key pathological process in the development of sporadic AD, where neurotrophic signals consisting of mature neurotrophins and Trk receptors are down-regulated. Conversely, neurodegenerative signals such as Aβ, the precursor of nerve growth factor(proNGF) and their receptors, the p75 neurotrophin receptor(p75NTR) and sortilin, are increased. p75 NTR is the neurotrophin receptor responsible for mediating mediates the survival or apoptosis of neurons depending on the activation of its co-receptors. The ectodomain(p75ECD) shedding of p75 NTR is physiologically processed shed regulated by an alpha-secretase tumor necrosis factor-alpha-converting enzyme(TACE) to release its ectodomain(p75ECD) from cell surface, and its C-terminal fragment is further under thefollowed by regulated- intramembranous proteolysis by gamma-secretase to generate the intracellular domain(ICD). Previous studies showed p75 NTR plays two facets of roles in the pathogenesis of Alzheimer’s disease. On one hand, p75 NTR positively regulates amyloidogensis by neurons; on the other hand p75 ECD is an physiological neuroprotective factor against Aβ generation, neurotoxicity and deposition in the brain. In a more recent study, we found that the expression of p75 NTR in the AD postmortem brain and in the brain of AD transgenic mice was upregulated but the shedding of its and levels of p75 ECD is dramatically down-regulated, leading to the remarkable reduction of p75 ECD in the CSF of AD subjects are reduced in the brains of AD cases. This abnormal regulation in p75 NTR proteolytic processing in AD leads to the imbalance between the level of p75 ECD and full length p75NTR(p75FL), resulting in several fold increase in the ratio of p75FL/p75 ECD in AD. Because p75 FL and p75 ECD plays the opposing roles in AD pathogenesis and amyloid metabolism, this imbalance between the two p75 species is likely plays a key role in propagating AD pathogenic progression. However, the physiological and pathological significance of p75 NTR shedding is not known. The regulation of p75 NTR shedding and the function of diffusible p75 ECD after shedding in vivo are yet to be determined. One of the mechanisms underpinning the therapeutic role of p75 ECD may be related to the clearance of Aβ. In our recent studies we have found that about 50% of brain amyloid peptide can efflux into the circulation and peripheral organs can clear Aβ from the brain. Peripheral clearance of Aβ is an important mechanism that maintains homoestasis of Aβ in the brain. Therefore, we investigated the efficacy of peripheral injection of p75 ECD on brain amyloid burden and associated pathogenesis.Matierals and methodsP75ECD in both human CSF and mouse brain was quantified by ELISA and/or Western blots. Vectors of AAV8-CBA-p75NTR-ECD-Fc(AAV-ECD-Fc) and AAV8-CBA-GFP(AAV-GFP) were generated, produced and purified by Virovek(Hayward, CA), and injected into the left thigh muscle of APPswe/PS1dE9(AD) mice. ECD-Fc in brain tissues and peripheral organs was detected by Western blot, immunohistochemistry and immunofluorescence, or quantified by ELISA. Female AD mice and their wild type littermates were used in the present study, as there are gender-specific differences in AD mouse models. AAV-ECD-Fc or AAV-GFP was injected to 3 months old AD mice. By 12 months of age, all mice were subjected to behavioural tests using Morris water-maze, Y-maze, and open-field protocols. Aβ burdens were analysed by Congo red staining, Aβ immunohistochemistry(6E10 antibody) and ELISA assays. Microglia(CD45), astrocyte(GFAP), and tau phosphorylation(anti-pS396), MAP2 in the brain were stained with immunohistochemistry. β-secretase and TACE activities were detected by commercial kits. The protein levels of TACE, full length APP, carboxyl terminal fragments of APP, BACE1, IDE, LRP, NEP, RAGE, pS396, pT231, pS262, pS199 were analysed with Western blots. Concentrations of IL-6, IL-1β, INF-γ and TNF-α were quantified by ELISA. Cultured primary cortical neurons from neonatal mice of different genotypes and Wt and human SH-SY5 Y cell line in vitro were applied for detection of neurite outgrowth, cell death, p75 ECD shedding and the mechanistic studies.Statistical comparisons between groups were assayed using Tukey’s test, Students t-test, one-way analysis of variance, or two-way repeated-measures analysis of variance for testing the significance of values. P-values < 0.05 were considered significant. All these analyses were performed using SPSS for Windows version 13.0(SPSS Inc., Chicago, IL, USA).Resultsp75ECD inhibits Aβ aggregation and promotes Aβ disaggregation in vitro. To study the effection of p75 ECD with Aβ aggregation, we did the Thioflavin T(ThT) fluorescence assay. we found that p75 ECD, when incubated with Aβ monomers or preformed Aβ fibrils, reduced Aβ fibrillation-induced fluorescence intensity; when pre-incubated with Aβ, increased the soluble Aβ oligomer species. Moreover, TEM assays visually confirmed that p75 ECD suppressed the fibrillation of Aβ and disaggregated the preformed Aβ fibrils, as reflected by the twisted long fibrils, short fibrils, and some small and relatively amorphous aggregates mixed with fibrils after treatment with p75 ECD.Expression of p75ECD-Fc distribution after intramuscular delivery of AAV8-p75 ECD. Adeno-associated virus 8(AAV8) vector was used to deliver the fusion gene of human p75 ECD and human IgG Fc fragment(AAV-ECD-Fc) by injection at vastus medialis muscle of AD mice at 3 months. Expression of the ECD-Fc transgenes was detected at one week after delivery, and remained stable in muscle, but not in brain regions, up to 12 months of age. The levels of p75 ECD in serum were significantly higher,so we can say p75 ECD distributed in periphery.Intramuscular delivery of p75 ECD improves cognitive decline in AD mice. Compared with GFP mice, the mice treated with ECD-Fc performed better in the Morris Water Maze test, Y-maze and open-field tests. This was reflected by a significant reduction in the escape latency time and the distance travelled to escape onto the platform, greater number of platform area crossings in Morris water maze test, more entries into the novel arm and higher spontaneous alternation in the Y-maze test, a longer travelling distance in open-field test. These data indicates that the ECD-Fc treatment significantly improves spatial learning, working memory memory and general locomotor activity in AD mice.Intramuscular delieverydelivery of p75 ECD reduces Aβ burdernburden in the brain and blood of AD mice. The total amyloid plaque load in the HC and NC, identified by immunohistochemistry, was also reduced in ECD-Fc treated AD mice groups relative to the controls. Consistent with the histological results, ELISA tests also showed a significant reduction in total Aβ and Aβ40 or Aβ42 levels in Tris-buffered saline(TBS), sodium dodecyl sulfate(SDS) and formic acid(FA) fractions of brain homogenates and serum in ECD-Fc treated AD mice. The ECD-Fc treatment significantly reduced BACE1 expression and activity, and β-cleavage product(CTFβ) of APP and Aβ production in treatment group. The level of production of the Aβ degrading enzymes, neprilysin(NEP) and insulin degrading enzyme(IDE), and the Aβ blood-brain barrier(BBB)-transporting molecules, LRP and receptor for RAGE, did not change. BACE1 expression and the sAPP level in the cultured primary neurons dose-dependently increased in response to Aβ42. This increase was abolished by the presence of recombinant ECD-Fc or in p75 NTR knockout neurons. This suggests that p75 NTR is required for amyloidogenic APP processing, promoted by Aβ. These results indicate that p75 NTR is a key receptor in mediating Aβ-induced BACE1 upregulation and plays a critical role in the Aβ-BACE1 vicious cycle, driving the pathogenesis of AD, and ECD-Fc treatment could break this vicious cycle.Intramuscular delivery of p75 ECD attenuates Tau hyperphosphorylation. The fraction area of Tau-phospho-Ser396 positive neurons in the hippocampus was lower in ECD-Fc group compared with the control. The levels of Tau-phosphorylation at multiple sites including serine 396, 262, 199 and threonine 231 were consistently and significantly diminished in the brain of ECD-Fc group. We reason that the effect of p75 ECD on Tau phosphorylation occurs via blocking Aβ which activates upstream GSK3β. Indeed, we found that both the recombinant ECD-Fc and p75 NTR neutralising antibodies could block the increased phosphorylation of Ser262 of Tau in response to Aβ in the human SH-SY5 Y cell line. The recombinant ECD-Fc dose-dependently suppressed the phosphorylation of Ser262 and increased p-Ser9-GSK3β phosphorylation, triggered by Aβ42. This data suggests that the elevation of p75 ECD levels can protect against Aβ-induced Tau hyperphosphorylation.Intramuscular delivery of p75 ECD levels attenuates microgliosis, astrocytosis and inflammation in the brain of AD mice. The fraction area of GFAP(astrocytosis) and CD45(microgliosis) staining in the neocortex and hippocampus were significantly reduced in treatment groups. The levels of pro-inflammatory cytokines including interleukin(IL-1β), IL-6, interferon-gamma(IFN-γ), and tumour necrosis factor-alpha(TNF-α) in the serum and TBS fraction were also lower in p75 ECD treatment group compared with the control group. These data indicate that ECD-Fc attenuates microgliosis, astrocytosis and inflammation in the brain of AD mice.Intramuscular delivery of the p5ECD-Fc gene using AAV8 is well tolerated. Throughout the animal studies, all animals were healthy without premature death and obvious abnormal behavior. In the present study, we specifically assessed the possible side effects of p75 ECD on peripheral organs and the brain. Moreover, we also assessed the autoimmune effects of p75 ECD, the signals of ELISA to detect p75ECD-Ab were very low, and there was no significant difference among the groups. All organs looked normal by gross anatomical examination. No discernible pathological morphology was observed in major organs including brain, muscle(non-injected), heart, liver, lung, kidney and intestine. The gene delivery did not significantly influence animal body weight or liver enzyme activities, including aspartate aminotransferase(AST) and Alanine aminotransferase(ALT). The data suggest that long-term expression of the p75ECD-Fc fusion gene in the muscle and liver is well tolerated by AD mice, demonstrating the safety of AAV-ECD-Fc treatment.Conclusions1. p75 ECD inhibits Aβ aggregation and promotes Aβ fibrils disaggregation in vitro. 2. Peripheral injection of p75 ECD gene alleviated AD pathology and improves learning and memory in early phases of AD in a mouse model. 3. Peripheral injection of p75 ECD gene is safe for the prevention of AD. So, we think that peripheral delivery of p75 ECD gene represents a safe and effective prophylactic strategy for AD.