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Functional characterization of a single nucleotide polymorphism associated with Alzheimer’s Disease in a hiPSC-based neuron model.

ABSTRACT: Neurodegenerative diseases encompass a group of debilitating conditions resulting from progressive nerve cell death. Of these, Alzheimer’s disease (AD) occurs most frequently, but is currently incurable and has limited treatment success. Late onset AD, the most common form, is highly heritable but is caused by a combination of non-genetic risk factors and many low-effect genetic variants whose disease-causing mechanisms remain unclear. By mining the FinnGen study database of phenome-wide association studies, we identified a rare variant, rs148726219, enriched in the Finnish population that is associated with AD risk and dementia, and appears to have arisen on a common haplotype with older AD-associated variants such as rs429358. The rs148726219 variant lies in an overlapping intron of the (FosB proto-oncogene) FOSB and (ERCC excision repair 1) ERCC1 genes. To understand the impact of this SNP on disease phenotypes, we performed CRISPR/Cas9 editing in a human induced pluripotent stem cell (hiPSC) line to generate isogenic clones harboring heterozygous and homozygous alleles of rs148726219. hiPSC clones differentiated into induced excitatory neurons (iNs) did not exhibit detectable molecular or morphological variation in differentiation potential compared to isogenic controls. However, global transcriptome analysis showed differential regulation of nearby genes and upregulation of several biological pathways related to neuronal function, particularly synaptogenesis and calcium signaling, specifically in mature iNs harboring rs148726219 homozygous and heterozygous alleles. Functional differences in iN circuit maturation as measured by calcium imaging were observed across genotypes. Edited mature iNs also displayed downregulation of unfolded protein response and cell death pathways. This study implicates a phenotypic impact of rs148726219 in the context of mature neurons, consistent with its identification in late onset AD, and underscores a hiPSC-based experimental model to functionalize GWAS-identified variants.

ORGANISM(S): Homo sapiens

PROVIDER: GSE239367 | GEO | 2023/08/28

REPOSITORIES: GEO

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Project description:Among fos family genes modulating the cell fates such as cell proliferation, differentiation and cell death, only the fosB gene produces two forms of mature mRNA for FosB and deltaFosB proteins by alternative splicing of an exonic intron in the exon 4. FosB dramatically enhances the transcription regulation of AP-1 dependent promoters by Jun, while deltaFosB, a truncated form of FosB lacking its C-terminal transactivation domain, suppresses the function of Jun. We and other have shown that FosB and deltaFosB have a distinct function to control the cell fate as well as neuronal functions based on their exogenous expression. To elucidate the authentic function of each protein, it is essential to control each expression separately. We established a mutant mouse embryonic stem (ES) cell line carrying homozygous fosBd/d alleles which encode exclusively deltaFosB, and compared its gene expression profile and phenotypes with fosB-null and wild-type ES cell lines. Both mutant ES cells are devoid of FosB, therefore the common phenotypes between the two mutant ES cells in comparison to wild type depict the effects of FosB deficiency. Opposite phenotypes between the two are considered to be determined by deltaFosB itself. We analyzed the gene expression and cellular function among these two mutant and wild-type ES cells. Experiment Overall Design: Total RNA was purified from ES cells grown in the absence of feeder cells using the Isogen kit (Nippon Gene), according to the manufacturerâs instructions. For DNA microarray experiments, Total RNA (10 microg) were labeled using the Agilent Linear Amplification/Labeling Kit (Agilent Technologies) according to the manufacturer's instructions. One microgram of each Cy3-labeled wild-type and Cy5-labeled mutant cDNA or each Cy3-labeled mutant and Cy5-labeled wild-type cDNA, respectively, were mixed, then hybridized to Agilent Mouse cDNA Microarrays (G4104A, design file number: 000522R000679, Agilent Technologies) with 8500 unique clones from Incyteâs mouse UniGene 1 clone set, according to the manufacturer's hybridization protocol. After the washing step, the microarray slides were analyzed with an Agilent G2565AA microarray scanner system. These experiments were carried out in duplicate using exchanged dye-labeled cDNA probes (i.e., Cy3 and Cy5 dye-swapping experiments).

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Functional characterization of a single nucleotide polymorphism associated with Alzheimer’s Disease in a hiPSC-based neuron model.

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