Divergent single cell transcriptome and epigenome alterations in ALS and FTD patients with C9orf72 mutation

Li, Junhao; Jaiswal, Manoj K.; Chien, Jo-Fan; Kozlenkov, Alexey; Jung, Jinyoung; Zhou, Ping; Gardashli, Mahammad; Pregent, Luc J.; Engelberg-Cook, Erica; Dickson, Dennis W.; Belzil, Veronique V.; Mukamel, Eran A.; Dracheva, Stella

doi:10.1038/s41467-023-41033-y

Cited by 14 publications

(13 citation statements)

References 124 publications

(118 reference statements)

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“…Differential expression analysis for sporadic ALS data was carried out following the procedures outlined in the previous RNA sequencing section. Meanwhile, the differential expression analysis for snRNA-seq data was downloaded from Supplementary Dataset 3 of the corresponding study [ 50 ].…”

Section: Methodsmentioning

confidence: 99%

Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs

Zhu,

Burg,

Neyrinck

et al. 2024

Acta Neuropathol

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Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder, characterized by selective loss of motor neurons (MNs). A number of causative genetic mutations underlie the disease, including mutations in the fused in sarcoma (FUS) gene, which can lead to both juvenile and late-onset ALS. Although ALS results from MN death, there is evidence that dysfunctional glial cells, including oligodendroglia, contribute to neurodegeneration. Here, we used human induced pluripotent stem cells (hiPSCs) with a R521H or a P525L mutation in FUS and their isogenic controls to generate oligodendrocyte progenitor cells (OPCs) by inducing SOX10 expression from a TET-On SOX10 cassette. Mutant and control iPSCs differentiated efficiently into OPCs. RNA sequencing identified a myelin sheath-related phenotype in mutant OPCs. Lipidomic studies demonstrated defects in myelin-related lipids, with a reduction of glycerophospholipids in mutant OPCs. Interestingly, FUSR521H OPCs displayed a decrease in the phosphatidylcholine/phosphatidylethanolamine ratio, known to be associated with maintaining membrane integrity. A proximity ligation assay further indicated that mitochondria-associated endoplasmic reticulum membranes (MAM) were diminished in both mutant FUS OPCs. Moreover, both mutant FUS OPCs displayed increased susceptibility to ER stress when exposed to thapsigargin, and exhibited impaired mitochondrial respiration and reduced Ca2+ signaling from ER Ca2+ stores. Taken together, these results demonstrate a pathological role of mutant FUS in OPCs, causing defects in lipid metabolism associated with MAM disruption manifested by impaired mitochondrial metabolism with increased susceptibility to ER stress and with suppressed physiological Ca2+ signaling. As such, further exploration of the role of oligodendrocyte dysfunction in the demise of MNs is crucial and will provide new insights into the complex cellular mechanisms underlying ALS.

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Section: Methodsmentioning

confidence: 99%

Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs

Zhu,

Burg,

Neyrinck

et al. 2024

Acta Neuropathol

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“…As others have shown, unique cell types respond differently to pathology [ 76 ]. Future single-cell/nuclei studies in the cerebellum will be essential for revealing cell-type-specific disease mechanisms and vulnerable cell populations, similar to a recent study of the frontal cortex [ 47 ]. This would help to determine whether a given event is mainly present in a specific type of cell and to elucidate whether differences in cellular proportions might, in part, contribute to some of the observed findings (e.g., splicing events).…”

Section: Discussionmentioning

confidence: 99%

Abundant transcriptomic alterations in the human cerebellum of patients with a C9orf72 repeat expansion

Udine,

DeJesus-Hernandez,

Tian

et al. 2024

Acta Neuropathol

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The most prominent genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a repeat expansion in the gene C9orf72. Importantly, the transcriptomic consequences of the C9orf72 repeat expansion remain largely unclear. Here, we used short-read RNA sequencing (RNAseq) to profile the cerebellar transcriptome, detecting alterations in patients with a C9orf72 repeat expansion. We focused on the cerebellum, since key C9orf72-related pathologies are abundant in this neuroanatomical region, yet TDP-43 pathology and neuronal loss are minimal. Consistent with previous work, we showed a reduction in the expression of the C9orf72 gene and an elevation in homeobox genes, when comparing patients with the expansion to both patients without the C9orf72 repeat expansion and control subjects. Interestingly, we identified more than 1000 alternative splicing events, including 4 in genes previously associated with ALS and/or FTLD. We also found an increase of cryptic splicing in C9orf72 patients compared to patients without the expansion and controls. Furthermore, we demonstrated that the expression level of select RNA-binding proteins is associated with cryptic splice junction inclusion. Overall, this study explores the presence of widespread transcriptomic changes in the cerebellum, a region not confounded by severe neurodegeneration, in post-mortem tissue from C9orf72 patients.

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“…snRNAseq localizes cryptic splicing in cortex to L2/3, L5, and L6 IT excitatory neurons Finally, we sought to determine the cell-type enrichment of our cryptic splices, utilizing snRNAseq datasets from two recent studies profiling (1) motor cortex from sporadic and C9orf72 ALS and FTLD patients (Pineda et al) [27] and (2) motor and frontal cortex (analyzed separately) from C9orf72 ALS and FTLD patients (Li et al) [15]. After QC and mutual-nearest-neighbor batch correction [12], 415,741 nuclei were retained from Pineda yielding 29 clusters (Figure 5a).…”

Section: Validation Of Cryptic Splices By Rt-qpcr and Rnaseqmentioning

confidence: 99%

“…Alignment and splice variant quantification were performed as described above. were analyzed separately, using the scuttle, scran, and scater packages [15,19,21,27]. snRNA-seq quality control metrics (total number of unique molecular identifiers (UMIs), number of detected genes, and mitochondrial read percentage) were computed for each nucleus, and sample-wise outlier-based filtering was performed to remove nuclei with any metric >3 median absolute deviations from the sample median.…”

Section: Bulk-rnaseq Of Lumbar Spinal Cord Samples From Als and Controlsmentioning

confidence: 99%

TDP43 proteinopathy exhibits disease, tissue, and context-specific cryptic splicing signatures

Newton,

Yang,

Gutierrez

et al. 2024

Preprint

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Mislocalization of the nuclear protein TAR DNA-binding protein 43 (TDP43) is a hallmark of ALS and FTD which leads to de-repression and inclusion of cryptic exons. These cryptic exons represent promising biomarkers of TDP43 pathology in a spectrum of neurodegenerative diseases. However, most cryptic exons to date have been identified from in vitro models or a single cortical FTD dataset, limiting our understanding of tissue and cell-specific splices as well as differences between in vitro and in vivo processes. We meta-analyzed published bulk RNAseq datasets representing 1,778 RNAseq profiles of ALS and FTD post-mortem tissue, and in vitro models with experimentally depleted TDP43. We identified 142 cryptic splices, including 76 novel events, and identified cryptic splicing signatures with distinct cortical and spinal cord enrichment, among other context-specific profiles. Using RNAseq and RT-qPCR, we validated a subset of these splices in an independent spinal cord cohort, and demonstrated a correlation of TDP43 pathology severity with degree of cryptic splicing. Leveraging multiple public single-nucleus RNAseq datasets of ALS and FTD motor and frontal cortex, we confirmed the elevation of cortical-enriched splices in disease and localized them to layer-specific neuronal populations. This catalog of cryptic splices could inform efforts to develop biomarkers for tissue-specific and cell type-specific TDP43 pathology.

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Divergent single cell transcriptome and epigenome alterations in ALS and FTD patients with C9orf72 mutation

Cited by 14 publications

References 124 publications

Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs

Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs

Abundant transcriptomic alterations in the human cerebellum of patients with a C9orf72 repeat expansion

TDP43 proteinopathy exhibits disease, tissue, and context-specific cryptic splicing signatures

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