DISC1 Attenuates Cognitive Deficits In An APP/PS1 Transgenic Mouse Model By Reduction Of BACE1 Levels

Alzheimer’s disease (AD) is a progressive, degenerative brain disease, which is also the most common cause of dementia. AD leads to death within 3 to 9 years after diag-nosis. There are more than 9 million patients with AD, and the diagnosis of new cases are more the 3000 thousands per year. Thus, AD is becoming a serious social burden which must be paid more attention.The amyloid hypothesis of AD maintains that the accumulation of amyloid-β (Aβ), especially its oligomeric forms, is a key event in disease pathogenesis. The generation of Aβ is initiated by the proteolytic cleavage of amyloid precursor protein (APP) by β-secretase, which generates P-CTF, a C-terminal membrane-retained fragment of APP. The latter is further cleaved by y-secretase and thus produces Aβ. APP is also under sequential cleavage by a-secretase and y-secretase, which precludes generation of Aβ. Thus, β-secretase mediates the initial and rate-limiting processing step during Aβ generation, β-site APP-cleaving enzyme 1 (BACE1), a type I transmembrane aspartyl protease, is the sole β-secretase in the brain. BACE1-deficient neurons do not produce detectable levels of Aβ40, Aβ42 or β-CTF, and AD mouse models with BACE1 deficiency do not generate amyloid deposition. Moreover, protein levels and enzymatic activity of BACE1 are both increased in the brains of AD patients. Therefore, inhibition of BACE1 is a potential therapeutic target for AD. Identification of molecules which reduce the increased levels/activity of BACE1 in the brains of AD patients may provide a worthwhile strategy in AD therapy.Disrupted-in-Schizophrenia-1 (DISCI) is a genetic risk factor for a wide range of major mental disorders. Familial mutations in the DISCI gene, including the balanced chromosomal (1; 11) (q42.1; q14.3) translocation, cosegregate with schizophrenia, major depression and bipolar disorders. This chromosomal translocation generates a C-terminal truncated, or a fusion protein with DISCI or a haploinsufficient DISCI protein, which all cause dysfunctions of DISCI. DISCI plays essential roles in nervous system development, such as proliferation and differentiation of neural progenitor cells, neuronal migration, neurite outgrowth, synapse formation and mitochondrial trafficking. Interestingly, substantial evidence suggests that DISCI is linked to AD. DISCI interacts with APP, from which Aβ is derived by proteolytic cleavage through a BACE1-dependent mechanism. A genome-wide association study indicates an association of a single nucleotide polymorphism in a DISCI intron and late onset of AD. However, the function of DISCI in the pathogenesis of AD remains unknown. In our study, we observe decreased levels of DISCI in the cortex and hippocampus of 8-month-old APP/PS1 transgenic mice, which express a chimeric mouse/human Swedish mutant APP (APPswe) and a mutant human presenilin 1 (PS1). We further provide evidence that DISCI interacts with BACE1 and promotes lysosomal degradation of BACE1, thus reducing the generation of Aβ. Overexpression of DISCI via an adeno-associated virus in the hippocampus reduces the levels of soluble Aβ40 and Aβ42, the density of Aβ plaques and, importantly rescues cognitive deficits in APP/PS1 transgenic mice. Therefore, we propose that DISCI, a genetic risk factor for mental disorders, plays essential roles in the pathology of AD.Chapter I. Exploration of DISCI expression levels in the APPswe/PSldE9 transgenic miceObjective:To exam the expression levels of DISCI protein in APP/PS1 transgenic mice and wild type mice.Methods:4 and 8 months-old APP/PS1 transgenic mice and wild type mice were used to collect the cortex and hippocampus to extract the total protein. The samples were subjected to Western Blotting for examining the DISCI protein level.Results:The levels of DISCI in the hippocampus and cortex of APP/PS1 transgenic mice were investigated. Decreased levels of DISCI were detected in the hippocampus and cortex of APP/PS1 transgenic mice at 8 months of age; In contrast, DISCI exhibited identical levels in the hippocampus and cortex of APP/PS1 transgenic mice, in comparison to wild type littermates at 4 months of age.Conclusion:The level of DISCI protein is decreased in APP/PS1 transgenic mice; And there maybe is a relationship between AD and schizophrenia.Chapter II. Overexpression of DISCI decreases BACE1 at the protein levelObjective:To explore the relationship between DISCI and BACE1.Methods:DISC1-Flag and BACE1-HA plasmids were cotransfected into HEK293 cells. The cells cotransfected with a plasmid expressing GFP and BACE1-HA plasmid were taken as control cells. Cell lysates were collected 48 h after transfection and subjected to Western blot analysis. HEK293 cells were transfected with the DISC1-Flag plasmid, and then probed by Western blot analysis with an antibody against BACE1. Small interference RNA (siRNA) targeting DISCI was transfected into HEK293 cells to examine the level of DISCI and BACE1 by western blot analysis. RT-PCR were used to examine the RNA level of BACE1 and DISC1 of all the transfected cells above.Results:BACE1-HA protein levels were decreased in DISC1-cotransfected cells compared to GFP-cotransfected control cells. The levels of endogenous BACE1 protein were decreased in DISC1-transfected cells compared to those in GFP-transfected cells, small interference RNA (siRNA) targeting DISC1 transfection led to downregulation of DISCI levels in HEK293 cells, but enhanced the levels of BACE1. However, BACE1 mRNA levels were similar in both DISC1-transfected-and DISCI siRNA-transfected-HEK293 cells, compared to control cells. These results indicate that DISCI reduces BACE1 protein levels in a post-transcriptional manner. Conclusion:DISCI reduces BACE1 at the protein level.Chapter Ⅲ. The Mechanism of Overexpression of DISCI decreasing BACE1 protein levelObjective:To explore the mechanism of DISCI reducing BACE1 protein.Methods:DISC1-Flag and BACE1-HA plasmids were cotransfected into HEK293 cells and subjected to immunofluorescence and co-immunoprecipitation to examine the interaction of DISCI and BACE1 in vitro. Mouse Hippocampus was use to examine the interaction of DISCI and BACE1 by co-immunoprecipitation in vivo. HEK293 cells transfected with the DISC1-Flag plasmid or GFP plasmid were treated with CHX and subjected to western blot analysis to examine the degradation rate of BACE1. HEK293 cells transfected with the DISC1-Flag plasmid or GFP plasmid were also treated with CQ, MG132 and vehicle to examine the manner of degradation of BACE1.Results:BACE1 immunoreactivity is detectable in perinuclear regions. DISCI exhibited a staining pattern similar to BACE1 and colocalized with BACE1 in the perinuclear regions. Co-immunoprecipitation was performed to investigate a potential interaction between these two molecules. Antibodies against HA and Flag immunoprecipitated DISC1-Flag and BACE1-HA respectively, from HEK293 cells cotransfected with BACE1-HA and DISC1-Flag plasmids. Moreover, antibodies against BACE1 and DISCI immunoprecipitated DISCI and BACE1 respectively, from the lysates of adult mouse hippocampus. These results indicate that DISC1 associates with BACE1 in cells and brains. The transfected HEK293 cells were treated withCHX and the cell lysates were collected at 0,8 and 16 h. Levels of BACE1 in DISC1-Flag-transfected HEK293 cells were decreased after being treated by CHX for 8 or 16 h, compared to those in GFP-transfected cells. BACE1 levels in DISC1-transfected HEK293 cells declined at a faster rate compared to those in GFP-transfected cells. These results suggest that DISCI promotes the degradation of BACE1 protein. HEK293 cells were transfected with either DISCI-Flag or GFP and treated with CQ or MG-132. The downregulation of BACE1 levels by transfection with DISC-Flag was blocked by treatment with chloroquine, but not with MG-132. These results indicate that DISCI promotes degradation of BACE1 through lysosomes.Conclusion:DISCI promotes the degradation of BACE1 protein through lysosome.Chapter IV. Overexpression of DISCI ameliorates neuropatholoty and cognitive deficits of APP/PS1 transgenic miceObjective:To explore the effects of DISCI in the brain of APP/PS1 transgenic mice.Methods:APP/PS1 transgenic mice and the wild-type littermates were randomly divided into three groups:TG+DISC1(TG mice injected with DISC1-Flag-AAV8), TG+GFP(TG mice injected with GFP-AAV8), and WT-GFP(WT mice injected with GFP-AAV8). All the mice were injected with AAV8 into the hippocampus when they were 4 month old and were subjected to Morris Water Maze 3.5 month after virus injection. Then mice were killed and brains were decapitated for coronal section and other biochemical analysises.Brain sections were subjected to Immunofluorescence with 6E10 antibody for detecting amyloid plaques. The level of AP 40 and AB 42 were evaluated by ELISA. BACE1, APP, andα/β-CTF were evaluated by western blot analysis.Results:Consistent with the results from cultured HEK293 cells, levels of BACE1 decreased in DISC1-AAV8 injected, compared with those in GFP-AAV8 injected hippocampus. In contrast, BACE1 mRNA levels were similar in DISC1-Flag-AAV8 injected and GFP-AAV8 injected hippocampus.β-CTF exhibited decreased levels in the hippocampus of DISCI-AAV8 injected APP/PS1 transgenic mice at 8 months of age, i.e.4 months after injection of virus, in comparison to transgenic mice injected with GFP-AAV8. In contrast, a-CTF and full length APP were found at similar levels in the hippocampus of DISC-injected and GFP-injected APP/PS1 mice. Consistent with the results that DISCI decreases BACE1-cleavage of APP, a key step for Aβ generation, decreased levels of soluble Aβ40 and Aβ42 were detected by ELISA in the hippocampus, but not in the cortex of DISC1-injected APP/PS1 mice. The ratio of Aβ40/Aβ42 levels were similar in the hippocampus of DISCI-injected and GFP-injected APP/PS1 mice, suggesting that DISCI may have minimal effect on y-secretase cleavage of CTFs. Moreover, amyloid plaques exhibited reduced numbers and smaller size in the hippocampus, but not in the cortex, of DISCI-injected, compared to those in GFP-injected APP/PS1 transgenic mice. Wild type littermates injected into the hippocampus with AAV8 encoding GFP were used as controls. GFP injected transgenic mice were impaired in learning to use the available visuospatial cues to locate the submerged platform, as indicated by their longer escape latency compared to AAV8-GFP injected wild type mice. As expected, the escape latency of DISCI injected transgenic mice was reduced compared to that of GFP-injected transgenic mice, and even approached a level comparable to GFP injected wild type mice. The changes in escape latency were not due to the differences in swimming speed, which were identical in the groups. Consistently, DISCI overexpressing transgenic mice exhibited shorter swimming paths to locate the target, when compared to GFP injected transgenic mice. In the reversal trials, where the platform was moved to the opposite quadrant, DISCI injected transgenic mice also exhibited shorter escape latencies, being comparable to those of GFP injected wild type mice.Conclusion:Overexpression of DISCI rescues cognitive deficits of APP/PS1 transgenic mice by reducing the levels of BACE1 protein and thus reducing the generation of Aβ and the density of amyloid plaques in APP/PS1 transgenic mice; DISCI decreases the cleavage of APP by BACE1 rather than by a-secretase.