Project description:ATP6V1A plays a unique role in synapse function in neurons and we found decreased neuronal activity in ATP6V1A-deficient neurons. To characterize the molecular pathways regulated by ATP6V1A under both normal and stressed conditions, we generated hiPSC-derived NGN2-neurons with reduced ATP6V1A expression by CRISPRi knock-down (KD) and performed RNA-seq analysis on the wild-type and KD neurons which were subject to amyloid-beta or vehicle treatment. A number of gene ontology (GO)/pathways were identified in KD neurons without amyloid-beta treatment (proton transporting V-ATPase complex, phagosome acidification and trivalent inorganic cation transport were down-regulated, and mitochondrial protein complex up-regulated), while KD in amyloid-beta treated cells specifically resulted in down-regulation of cell adhesion, synapse assembly and structure/activity and up-regulation of UPR and ER stress response.
Project description:Complement protein C1q is induced after injury in the brain and during Alzheimer's disease and has been shown to protect against amyloid-beta induced neuronal death. In this study, we used microarray approach to identify the pathways modulated by C1q that are associated with neuroprotection. Immature rat cortical primary neurons are treated with fibrillar amyloid-beta peptides and/or C1q for 3h before RNA extraction and hybridization on rat Affymetrix microarrays. Supplementary file: Processed/normalized, probe-level signal intensities from neurons treated with amyloid-beta or C1q. Median signal intensity used as global normalization method, done with JMP genomics (v5.0) software.
Project description:To determine targets of PTBP2-dependent alternative splicing, we depleted PTBP2 in human neurons derived from induced-pluripotent stem cells (iPSC-neurons) using an LNA gapmer and performed RNA-seq on untreated, negative control-treated, and knock-down samples.
Project description:A novel model of amyloid-beta pathology in C. elegans has been generated which allows for substoichiometric fluorescent labeling of amyloid-beta species in living nematodes. By microscopy, FLIM, physiological and biochemical studies, the progress of neurodegeneration and general pathogenicity of amyloidogenic peptides is confirmed. Notably, a single set of neurons was shown to be most vulnerable to Amyloid-beta overexpression and putatively act as a seed for systemic pathogeny. A neuronal version of the amyloid-beta reporter strain was used to perform proteomics analysis from worm lysates after enrichment by immunoprecipitation, using anti-amyloid-beta antibodies.
Project description:A novel model of amyloid-beta pathology in C. elegans has been generated which allows for substoichiometric fluorescent labeling of amyloid-beta species in living nematodes. By microscopy, FLIM, physiological and biochemical studies, the progress of neurodegeneration and general pathogenicity of amyloidogenic peptides is confirmed. Notably, a single set of neurons was shown to be most vulnerable to Amyloid-beta overexpression and putatively act as a seed for systemic pathogeny. A muscular version of the amyloid-beta reporter strain was used to perform proteomics analysis from worm lysates after enrichment by immunoprecipitation, using anti-amyloid-beta antibodies.
Project description:Clusterin (CLU) is one of the most significant genetic risk factors for late onset Alzheimer’s disease. Numerous studies have now demonstrated that CLU-AD mutations and amyloid-β (Aβ) treatment alter the trafficking and localisation of glycosylated CLU. iPSCs with altered CLU trafficking were generated following the removal of CLU exon 2 by CRISPR/Cas9 gene editing. Neurons were generated from control, unedited and exon 2 -/- iPSCs and were incubated with aggregated Aβ peptides. Changes in cell death and neurite length were quantified to determine if altered CLU protein trafficking influenced neuronal sensitivity to Aβ.
Project description:Early-onset Alzheimer’s disease-like pathology in Down syndrome (DS, trisomy 21) is commonly attributed to an increased dosage of the amyloid precursor protein (APP) gene. To test this central tenet of the amyloid-cascade hypothesis we deleted the supernumerary copy of the APP gene in trisomic DS iPSC, or upregulated APP expression in euploid human pluripotent stem cell lines with dCas9-VP64, and subjected these lines to prolonged cortical neural differentiation. Our data reveal that increased APP gene dosage and expression is necessary and sufficient for increased beta-amyloid production and pyroglutamate(E3)-containing plaque deposition, but is neither sufficient nor required for tau hyperphosphorylation, neurofibrillary tangle formation, or increased oxidative stress-induced apoptosis in neurons. Transcriptome comparisons of the isogenic neurons demonstrates that the supernumerary APP gene copy has profound temporally-modulated genome-wide effects on gene expression during differentiation and maturation of DS neuronal cultures that link APP function to regulation of genes involved in neuronal synaptic function and outgrowth of neuronal processes. Collectively, our data reveal that APP plays an important role in the amyloidogenic aspects of Alzheimer’s disease, but challenge the hypothesis that increased APP levels are solely responsible for hyperphosphorylation of tau or enhanced oxidative stress-induced neuronal cell death in Down syndrome associated AD-pathogenesis.