Mechanism Underlying ABCA7 P.A696S Variant Disturbed Microglial Response In Alzheimers Disease

Alzheimer’s disease（AD）is the most common age-related neurodegenerative disease,which contributes to 60-80%of dementia patients.AD is pathologically characterized by the deposition of amyloid-β（Aβ）peptides in plaques and hyperphosphorylated tau aggregates as neurofibrillary tangles.In addition,amyloid plaques are often surrounded by microglia and dystrophic neurites accompanied with synaptic loss in AD brains.Indeed,multiple studies have confirmed that variants in the ATP-binding cassette（ABC）transporter A7（ABCA7）gene are significantly associated with AD risk.Scientists have confirmed that ABCA7 is expressed to varying degrees in different types of cells in the brain,and the main cell type that expresses ABCA7 is neurons or microglia.ABCA7 is a homologues of ABCA1,sharing 54%sequence identity and the structural similarity,which mediates the transport of lipids across the cellular membrane.However,further studies are needed to understand its biology and clinical applicability.In recent years,the role of microglia in AD has attracted more and more attention.It has two roles in the occurrence and development of AD,which is closely related to the nature of its main activities:clearing Aβor releasing pro-inflammatory factors.On the one hand,microglia scavenge Aβand damaged/dead cells by phagocytosis,and on the other hand,microglia surrounding plaques may form a physical barrier preventing their further spread.Therefore,microglial dysfunction may be a triggering or aggravating factor for Aβaccumulation in the brain.At present,scientists have confirmed in different ethnic groups that many common or rare mutations of ABCA7 are associated with the risk of AD,which may be achieved by affecting the function or expression of ABCA7.Our team uncovered that ABCA7 p.A696S variant is associated with the increased AD risk.However,our understanding of this ABCA7 mutant on AD disease progression and its underlying molecular mechanisms is limited.Therefore,we generated a knock-in mouse model with ABCA7-A696S substitution(Abca7^KI/KI),which were crossed with the 5x FAD amyloid model mice to gain insights into the fundamental pathogenic mechanisms in AD and gain insights to new targets for AD treatment.Part 1 Effects of ABCA7 p.A696S on Aβdeposition-related pathology in AD miceObjective:To investigate the pathological changes of ABCA7 p.A696S on Aβdeposition in AD mice.Methods:We purchased the novel ABCA7-A696S knockin mice（ABCA7 p.A696S）,which were generated through CRISPR/Cas9-mediated gene editing.The ABCA7 p.A696S knockin mice were crossed with the 5x FAD amyloid model mice,and male littermates of control(5x FAD^+/-;Abca7^WT/WT),heterozygous knockin(5x FAD^+/-;Abca7^WT/KI),and homozygous knockin(5x FAD^+/-;Abca7^KI/KI)mice with 5x FAD background were used at 3 and 10 months of ages.Using immunohistochemical staining,we first detected the effect of ABCA7 p.A696S on the expression of pan-Aβ,amyloid plaques,and dystrophic neurites in AD mouse brain.Besides,soluble and insoluble Aβ40,Aβ42 levels,APP metabolite C terminal fragment-β（CTF-β）levels,andβ-site amyloid precursor protein cleaving enzyme 1（BACE1）gene expression levels were detected by q PCR or enzyme-linked immunosorbent assay（ELISA）technology to evaluate the impact of ABCA7 p.A696S on key AD phenotypes and APP processing.Results:We first confirmed that ABCA7 p.A696S had no significant effect on the expression of ABCA7 protein in mouse brain.We did not find a statistically significant difference in pan-Aβdeposition between the two groups at 3 or 10 months of age;however,by co-staining of X-34 and dystrophic neurites in brain tissue of 10-month-old mice,we observed that ABCA7 p.A696S promoted amyloid plaque deposition and the accumulation of dystrophic neurites around plaques in cortex and hippocampus of5x FAD mice.In addition,the soluble Aβ42 level of TBSX in 5x FAD;Abca7^KI/KI group was significantly decreased compared with the other two groups in 3-month-old mice,while the level of insoluble Aβ42 in 5x FAD;Abca7^KI/KIgroup was significantly higher than that of 5x FAD;Abca7^WT/WT group.At 10 months of age,Aβ42 level in TBSX was significantly increased in 5x FAD;Abca7^KI/KI group compared with 5x FAD;Abca7^WT/WTgroup,while insoluble Aβlevel was not significantly changed.However,CTF-βand BACE1 levels were not significantly different between groups of mice with different genotypes.Conclusion:ABCA7 p.A696S significantly promotes amyloid plaque deposition and dystrophic neurites aggregation around plaques.Part 2 ABCA7 p.A696S alters cerebral cortex genomics in AD miceObjective:To clarify the changes of ABCA7 p.A696S on the brain transcriptional profile of AD mice.Methods:We purchased the novel ABCA7-A696S knockin mice（ABCA7 p.A696S）,which were generated through CRISPR/Cas9-mediated gene editing.The ABCA7 p.A696S knockin mice were crossed with the 5x FAD amyloid model mice,and male littermates of control(5x FAD^+/-;Abca7^WT/WT),heterozygous knockin(5x FAD^+/-;Abca7^WT/KI),and homozygous knockin(5x FAD^+/-;Abca7^KI/KI)mice with 5x FAD background were used at 3 and 10 months of ages.First,we used next-generation sequencing technology to analyze the differentially expressed genes（DEGs）in cortex of 3-month-old ABCA7^KI/KI mice,ABCA7 knockout（ABCA7-KO）mice and wildtype mice,and screened and identified ABCA7 p.A696S-related weighted gene co-expression network analysis（WGCNA）modules.Meanwhile,we performed enrichment analysis of DEGs in the cortex tissue of 3-month-old and 10-month-old5x FAD;ABCA7^KI/KI mice and 5x FAD;ABCA7^WT/WT mice,and then further carried out WGCNA to analyze the ABCA7 p.A696S-related gene modules in AD.Results:Principal component analysis（PCA）demonstrated clear separation between Abca7^WT/WT and Abca7^KI/KI mice in the cortical transcriptome profiles at 3months of age through bulk RNA-sequencing（RNA-seq）.We identified one up-regulated module（MEturquoise）and two down-regulated module（MEbrown and MEskyblue）in Abca7^KI/KI mice compared with Abca7^WT/WT mice by WGCNA.While MEturquoise was enriched for genes related to regulation of cell size,MEbrown and MEskyblue genes were correlated with the pathways for“Glutamine family amino acid metabolic process”,“Interleukin-1 beta secretion and production”,and“Acetyl-Co A biosynthetic process”.Enrichment analysis of bulk-RNASeq from 5x FAD;Abca7^WT/WTand 5x FAD;Abca7^KI/KI cortex samples at different ages showed that5x FAD;Abca7^WT/WT mouse specific gene ontologies were“Interferon signaling”and“CTLA4 signaling in cytotoxic T lymphocytes”while 5x FAD;Abca7^KI/KI mouse specific gene ontologies was“Inflammasome pathway”with increasing months of age.We identified one up-regulated module（MEsalmon）and one down-regulated module（MEgreenyellow）in 5x FAD;Abca7^KI/KI mice compared with 5x FAD;Abca7^WT/WT mice by WGCNA.While MEsalmon was enriched for genes related to extracellular region or extracellular matrix,MEgreenyellow genes were correlated with the pathways for innate immunity including“beta2-macroglobulin binding”and“MHC class I protein complex”.The WGCNA also found two up-regulated modules（MEbrown and MEgrey60）and two down-regulated modules（MEmidnightblue and MEblack）in mice at 10 months old compared with those at 3 months old,in which the top-ranked pathways include“Immune system process”,“Neutrophil aggregation”,“Nervous system development”,and“Negative regulation of muscle contraction”.Conclusion:With the increase of months,neuroimmune-related changes were manifested to different degrees in the 5x FAD;Abca7^KI/KI mice and the 5x FAD;Abca7^WT/WT mice.Part3 ABCA7 p.A696S regulates microglial function and inflammatory response in AD miceObjective:To clarify the critical role of ABCA7 p.A696S in regulating microglial function in AD mice.Methods:We purchased the novel ABCA7-A696S knockin mice（ABCA7 p.A696S）,which were generated through CRISPR/Cas9-mediated gene editing.The ABCA7 p.A696S knockin mice were crossed with the 5x FAD amyloid model mice,and male littermates of control(5x FAD^+/-;Abca7^WT/WT),heterozygous knockin(5x FAD^+/-;Abca7^WT/KI),and homozygous knockin(5x FAD^+/-;Abca7^KI/KI)mice with 5x FAD background were used at 3 and 10 months of ages.Using immunohistochemical staining,we determined the immunoreactivity of astrocytes and microglia in the cortex and hippocampus to determine their activation status.Using immunofluorescence staining,we analyzed the aggregation of microglia around plaques,and the morphological changes of microglia.Using genomic analysis and q PCR technology,we explored the expression of homeostasis-related microglial genes and disease-associated microglial genes.By meso scale discovery multi-cytokine detection technique,we explored the effects of ABCA7 p.A696S on inflammatory factor levels in AD mouse brains.Results:ABCA7 p.A696S resulted in decreased microglial activation,but no significant changes in astrocyte activation in the brains of 10-month-old 5x FAD mice.Meanwhile,ABCA7 p.A696S may inhibit the accumulation of microglia around plaques.Besides,microglia in 5x FAD;Abca7^KI/KI mice displayed a dystrophic morphology,such as a marked reduction in the total branch length and endpoint numbers,especially in 10-month-old mice.In addition,we observed that ABCA7-A696S substitution disturbs the induction of proinflammatory cytokines in the brains of 5x FAD mice,although some of disease-associated microglia（DAM）gene expressions are rather upregulated to some extent.Conclusion:ABCA7 p.A696S may impair the innate immune response to amyloid pathology in AD,especially the microglial barrier dysfunction,which may make amyloid plaques more diffuse,leading to aggravation of neuroinflammatory dystrophy.