Similar Datasets
Project description:Understanding the pathogenic mechanisms of disease mutations is critical to advancing treatments. ALS-associated mutations in the gene encoding the microtubule motor KIF5A result in skipping of exon 27 (KIF5AΔExon27) and the encoding of a protein with a novel 39 amino acid residue C-terminal sequence. Here, we report that expression of ALS-linked mutant KIF5A results in dysregulated motor activity, cellular mislocalization, altered axonal transport, and decreased neuronal survival. Single-molecule analysis revealed that the altered C terminus of mutant KIF5A results in a constitutively active state. Furthermore, mutant KIF5A possesses altered protein and RNA interactions and its expression results in altered gene expression/splicing. Taken together, our data support the hypothesis that causative ALS mutations result in a toxic gain of function in the intracellular motor KIF5A that disrupts intracellular trafficking and neuronal homeostasis.
| S-EPMC9134378 | biostudies-literature
Project description:Understanding the pathogenic mechanisms of disease mutations is critical to advancing treatments. ALS-associated mutations in the gene encoding the microtubule motor KIF5A result in skipping of exon 27 (KIF5AΔExon27) and the encoding of a protein with a novel 39-amino acid residue C-terminal sequence. Here, we report that expression of ALS-linked mutant KIF5A results in dysregulated motor activity, cellular mislocalization, altered axonal transport, and decreased neuronal survival. Single-molecule analysis revealed that the altered C-terminus of mutant KIF5A results in a constitutively active state. Further, mutant KIF5A possesses novel protein and RNA interactions and its expression results in altered gene expression/splicing. Taken together, our data support the hypothesis that causative ALS mutations result in a toxic gain of function in the intracellular motor KIF5A that disrupts intracellular trafficking and neuronal homeostasis.
2022-06-09 | PXD031012 | Pride
Project description:Purpose: The goal of this study is to identify and compare the mRNA interactomes of WT and ALS-related mutant (d27) KIF5A Methods: RNA samples were extracted and sequenced on an Illumina NovaSeq6000 instrument at the Yale Center for Genome Analysis. The sample sequence quality control check was performed using FastQc. The STAR aligner program was used for read mapping with reference file and HTSeq was used to count the reads for each transcript in strand specific mode. The Differential expression analysis was performed using the DESeq2 package to compare the gene expression profiles between wild type and mutant was performed using the Wald test to generate p-values and Log2 fold changes. The STAR aligned RNA Sequence sample bam files were used to detect differential alternative splicing events with the Multivariate Analysis of Transcript Splicing (MATS) method. Pathway analysis was performed with Metascape Results: With the RNA RIP Sequence pipeline, we analyzed the RNA Sequence from SKNAS cells expressing WT KIF5A and cells epxressing the KIF5a ALS delta27 mutant. We identified 1,184 transcripts that displayed enriched binding to KIF5AΔExon27 and 303 that showed decreased binding to KIF5A ΔExon27. Pathway analysis revealed several categories related to ALS pathogenesis. KIF5AΔExon27 enriched pathways included NGF-stimulated transcription, which is related to neurite outgrowth. Pathways that were underrepresented in KIF5A ΔExon27 samples compared to KIF5AWT included interactions between L1 and Ankyrins, synapse assembly, and potassium channels. The results demonstrate that mutations in KIF5A can result in altered interaction with proteins and RNA associated with ALS-related pathways. Conclusions: Our analysis demonstates that ALS-related mutant KIF5A (d27) has altered RNA interactions compared to the WT protein.
2022-02-17 | GSE196538 | GEO
Project description:Purpose: The goal of this study was to identify gene expression changes in iMNs containing a patient KIF5a mutation compared to an isogenic control. Further, splicing changes were evaluated in the mutant vs WT sample. Methods: RNA samples were extracted and sequenced on an Illumina NovaSeq6000 instrument at the Yale Center for Genome Analysis. The sample sequence quality control check was performed using FastQc. The STAR aligner program was used for read mapping with reference file and HTSeq was used to count the reads for each transcript in strand specific mode. The Differential expression analysis was performed using the DESeq2 package to compare the gene expression profiles between wild type and mutant was performed using the Wald test to generate p-values and Log2 fold changes. The STAR aligned RNA Sequence sample bam files were used to detect differential alternative splicing events with the Multivariate Analysis of Transcript Splicing (MATS) method. Pathway analysis was performed with Metascape Results: With the RNA Sequence pipeline, we analyzed the RNA Sequence from wild-type parental line (Iso Control) and the heterozygous mutant line (KIF5AR1007K). The RNA Sequence analysis of the iMNs revealed 57 genes displaying altered expression. Using the method, Multivariate Analysis of Transcript Splicing (MATS), we identified 1,919 transcripts with altered splicing compared to the isogenic control iMNs. Of these, 1,000 exons showed decreased skipping, while 919 showed increased skipping in KIF5AR1007K lines. Pathway analysis of each of these groups suggests that this group of altered genes, as a whole, represents cytoskeletal and transport defects in the cell, both of which are hallmarks of neurodegenerative disease.
2022-02-17 | GSE196537 | GEO
Project description:ALS-Associated KIF5A Mutations Abolish Autoinhibition Resulting in a Toxic Gain of Function
| PRJNA805280 | ENA
Project description:ALS-Associated KIF5A Mutations Abolish Autoinhibition Resulting in a Toxic Gain of Function [RNA-seq]
| PRJNA805277 | ENA
Project description:ALS-Associated KIF5A Mutations Abolish Autoinhibition Resulting in a Toxic Gain of Function [RIP-seq]
| PRJNA805281 | ENA
Project description:Mutations in FUS cause amyotrophic lateral sclerosis (ALS), including some of the most aggressive, juvenile-onset forms of the disease. FUS loss-of-function and toxic gain-of-function mechanisms have been proposed to explain how mutant FUS leads to motor neuron degeneration, but neither has been firmly established in the pathogenesis of ALS. Here we characterize a series of transgenic FUS mouse lines that manifest progressive, mutant-dependent motor neuron degeneration preceded by early, structural and functional abnormalities at the neuromuscular junction. A novel, conditional FUS knockout mutant reveals that postnatal elimination of FUS has no effect on motor neuron survival or function. Moreover, endogenous FUS does not contribute to the onset of the ALS phenotype induced by mutant FUS. These findings demonstrate that FUS-dependent motor degeneration is not due to loss of FUS function, but to the gain of toxic properties conferred by ALS mutations.
| S-EPMC4742863 | biostudies-other
Project description:Mutations in Fused in Sarcoma/Translocated in Liposarcoma (FUS) cause familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by progressive axonal degeneration mainly affecting motor neurons. Evidence from transgenic mouse models suggests mutant forms of FUS exert an unknown gain-of-toxic function in motor neurons, but mechanisms underlying this effect remain unknown. Towards this end, we studied the effect of wild type FUS (FUS WT) and three ALS-linked variants (G230C, R521G and R495X) on fast axonal transport (FAT), a cellular process critical for appropriate maintenance of axonal connectivity. All ALS-FUS variants impaired anterograde and retrograde FAT in squid axoplasm, whereas FUS WT had no effect. Misfolding of mutant FUS is implicated in this process, as the molecular chaperone Hsp110 mitigated these toxic effects. Interestingly, mutant FUS-induced impairment of FAT in squid axoplasm and of axonal outgrowth in mammalian primary motor neurons involved aberrant activation of the p38 MAPK pathway, as also reported for ALS-linked forms of Cu, Zn superoxide dismutase (SOD1). Accordingly, increased levels of active p38 MAPK were detected in post-mortem human ALS-FUS brain tissues. These data provide evidence for a novel gain-of-toxic function for ALS-linked FUS involving p38 MAPK activation.
| S-EPMC5428330 | biostudies-literature
Project description:BackgroundMotor neurons (MNs), which are primarily affected in amyotrophic lateral sclerosis (ALS), are a specialized type of neurons that are long and non-dividing. Given their unique structure, these cells heavily rely on transport of organelles along their axons and the process of autophagy to maintain their cellular homeostasis. It has been shown that disruption of the autophagy pathway is sufficient to cause progressive neurodegeneration and defects in autophagy have been associated with various subtypes of ALS, including those caused by hexanucleotide repeat expansions in the C9orf72 gene. A more comprehensive understanding of the dysfunctional cellular mechanisms will help rationalize the design of potent and selective therapies for C9orf72-ALS.MethodsIn this study, we used induced pluripotent stem cell (iPSC)-derived MNs from C9orf72-ALS patients and isogenic control lines to identify the underlying mechanisms causing dysregulations of the autophagy-lysosome pathway. Additionally, to ascertain the potential impact of C9orf72 loss-of-function on autophagic defects, we characterized the observed phenotypes in a C9orf72 knockout iPSC line (C9-KO).ResultsDespite the evident presence of dysfunctions in several aspects of the autophagy-lysosome pathway, such as disrupted lysosomal homeostasis, abnormal lysosome morphology, inhibition of autophagic flux, and accumulation of p62 in C9orf72-ALS MNs, we were surprised to find that C9orf72 loss-of-function had minimal influence on these phenotypes. Instead, we primarily observed impairment in endosome maturation as a result of C9orf72 loss-of-function. Additionally, our study shed light on the pathological mechanisms underlying C9orf72-ALS, as we detected an increased TBK1 phosphorylation at S172 in MNs derived from C9orf72 ALS patients.ConclusionsOur data provides further insight into the involvement of defects in the autophagy-lysosome pathway in C9orf72-ALS and strongly indicate that those defects are mainly due to the toxic gain-of-function mechanisms underlying C9orf72-ALS.
| S-EPMC10506245 | biostudies-literature