Noncoding RNAs induce TDP43 pathology in the ALS-iPSC models
Ontology highlight
ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron (MN) degenerative disease with a major pathological feature of cytoplasmic TDP-43 aggregation. However, the mechanisms underlying TDP-43 proteinopathy are still largely unknown. We performed in vitro differentiation of ALS-induced pluripotent stem cells (ALS-iPSCs; carrying the TDP-43M337V mutation) and isogenic controls and found upregulation of paraspeckle-associated lncRNA NEAT1 isoforms in the ALS-iPSC-derived MNs (ALS-iPSC-MNs). Intriguingly, the upregulated NEAT1 isoforms were mislocalized to the cytoplasm of ALS-iPSC-MNs, and the cytoplasmic NEAT1 provoked TDP-43 and TDP-43M337V liquid-liquid phase separation, generating long-lived protein condensates. These condensates had reduced mobility and were converted into aggregates, finally co-aggregating with phospho-TDP-43. Disruption of NEAT1 expression reduced its cytoplasmic levels and also reduced the levels of TDP-43/TDP-43M337V condensates. In 3D neuromuscular organoids with the TDP-43M337V mutation, treatment with NEAT1-antisense oligonucleotides (NEAT1-ASO) promoted neuromuscular junction formation and function, as well as muscle contractility. Furthermore, treatment of TDP-43Q331K mice with Neat1-ASO attenuated TDP-43 pathology in spinal cord and preserved motor function. These findings suggest that NEAT1 plays an important role in TDP-43-associated pathology, and NEAT1-ASO may attenuate pathological TDP-43 aggregation to prevent motor neuron degeneration and muscle weakness in ALS.
Project description:Amyotrophic lateral sclerosis (ALS) is an incurable neurological disease featuring progressive loss of motor neuron (MN) function in the brain and spinal cord. Mutations in TARDBP, encoding the RNA-binding protein TDP-43, are one cause of ALS and TDP-43 mislocalization in MNs is a key pathological feature of >95% of ALS cases. While numerous studies support altered RNA regulation by TDP-43 as a major cause of disease, specific changes within MNs that trigger disease onset remain unclear. Here, we combined Translating Ribosome Affinity Purification (TRAP) with RNA sequencing to identify molecular changes in spinal MNs of TDP-43–driven ALS at motor symptom onset. By comparing the MN translatome of hTDP-43A315T mice to littermate controls and to mice expressing wildtype hTDP-43, we identify hundreds of mRNAs that were selectively up- or downregulated in MNs. We validated effects on Tex26, Syngr4, and Plekhb1 mRNAs in an independent TRAP experiment. Moreover, by quantitative immunostaining of spinal cord MNs we found corresponding protein level changes for SYNGR4 and PLEKHB1. We also observed these changes in spinal MNs of an independent ALS mouse model caused by a different patient mutant allele of TDP-43, suggesting that they may be a general feature of TDP-43-driven ALS. Thus, we have identified two new proteins deregulated in MNs at motor symptom onset in TDP-43-driven ALS models. This spatial and temporal pattern suggests that deregulation of these proteins could be functionally important for driving the transition to the symptomatic phase of disease.
Project description:Through splicing analysis of a publicly available RNA-Seq dataset, we discovered TDP-43 represses a cryptic exon splicing event in UNC13A, a gene that had been associated with FTD/ALS through GWA studies. To confirm the sequences of the cryptic exons, we used shRNA to reduce TDP-43 levels in iPSC-derived motor neurons (iPSC-MNs) and by amplicon sequencing the RT-PCR product, we observed the insertion in cells with TDP-43 depletion but not in control shRNA-treated cells. Through sequence alignment, we verified the sequences of the cryptic exons.
Project description:Sporadic amyotrophic lateral sclerosis (sALS) is the most common (~90%) form of ALS. There are no animal models of sALS and exact molecular mechanisms remain elusive. Here, we elucidate gene-expression profiles in laser capture microdissected enriched surviving motor neurons (MNs) from sALS lumbar spinal cords in patients who had rostral onset and caudal progression. A strong signature was detected and immunological signals were computationally filtered. The filtered dataset showed clustering groups that were significantly explained by levels of phosphorylated TDP-43 (pTDP-43). Transcriptome-pathology correlations and enhanced crosslinking and immunoprecipitation combined with sequencing (eCLIP-seq) identified that Casein kinase 1ε (CSNK1E) had the highest correlation with pTDP-43 status and TDP-43 binding in its 3’UTR. Furthermore, CSNK1E interacted with TDP-43 on protein level and its overexpression lead to increased cytoplasmic pTDP-43 accumulations in iPSC-MNs, suggesting CSNK1E directly mediates TDP-43 phosphorylation. Therefore, we report an essential framework for molecular disease classification and transcriptome – pathology correlation in sALS to identify candidate genes for elucidating disease mechanisms and potential therapeutic interventions.
Project description:Recent studies exploring the underlying pathomechanisms of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder, have focused on biomolecular condensates, such as stress granules (SGs) and TDP-43 condensates. Our comprehension of the physicochemical processes that control the behavior of these condensates is still evolving. In this work, we reveal an unexpected function for YAP, a central component of the Hippo pathway, in regulating the dynamic behavior of SGs and TDP-43 condensates, a role that is independent of its transcriptional activity in Hippo pathway. We found that YAP can directly bind to the TB domain of TDP-43 through its N-terminal domain. This interaction promotes the homotypic multimerization and phase separation of TDP-43, while inhibiting its hyper- phosphorylation and solidification under stress conditions. Remarkably, YAP, whose mRNA level is reduced in ALS patients, is found to co-localize with pathological pTDP-43 aggregates in the cytoplasmic foci of ALS patient brains. Additionally, elevating YAP/Yorkie expression substantially reduces neurotoxicity in a TDP-43 transgenic fly model of ALS. Our findings highlight an unexpected chaperone-like role of YAP in managing ALS-associated biomolecular condensates, presenting significant implications for potential ALS treatments.
Project description:Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are associated with loss of nuclear TDP-43. Here we identify that TDP-43 regulates expression of the neuronal growth-associated factor stathmin-2. Lowered TDP-43 levels, which reduce its binding to sites within the first intron of stathmin-2 pre-mRNA, uncover a cryptic polyadenylation site whose utilization produces a truncated, non-functional mRNA. Reduced stathmin-2 expression is found in neurons trans-differentiated from patient fibroblasts expressing an ALS-causing TDP-43 mutation, in motor cortex and spinal motor neurons from sporadic ALS patients and familial ALS patients with expansion in C9orf72, and in induced pluripotent stem cell (iPSC)-derived motor neurons depleted of TDP-43. Remarkably, while reduction in TDP-43 is shown to inhibit axonal regeneration of iPSC-derived motor neurons, rescue of stathmin-2 expression restores axonal regenerative capacity. Thus, premature polyadenylation-mediated reduction in stathmin-2 is a hallmark of ALS/FTD that functionally links reduced nuclear TDP-43 function to enhanced neuronal vulnerability.
Project description:TDP-43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP, the gene encoding TDP-43, are one of the common causes of familial ALS. In this study, we investigate TDP-43 protein behavior in induced pluripotent stem cell (iPSC)-derived motor neurons from three ALS patients with different TARDBP mutations and three healthy controls. TARDPB mutations induce several TDP-43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP-43, C-terminal fragments and phospho-TDP-43. By generating iPSC lines with allele-specific tagging of TDP-43, we find that mutant TDP-43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry-based proteomics. Our findings also indicate that TDP-43 proteinopathy results in a defect in mitochondrial transport. Lastly, proteomics analyses also show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP-43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP-43 pathology.
Project description:Single cell dataset of human motor neurons laser-captured from postmortem ALS and control tissues
(1) ALS Pilot dataset
(2) TDP43 stratified dataset
Summary: Unbiased proteomics has been employed to interrogate central nervous system (CNS) tissues (brain, spinal cord) and fluid matrices (CSF, plasma) from amyotrophic lateral sclerosis (ALS) patients; yet, a limitation of conventional bulk tissue studies is that motor neuron (MN) proteome signals may be confounded by admixed non-MN proteins. Recent advances in trace sample proteomics have enabled quantitative protein abundance datasets from single human MNs (Cong et al., 2020b). In this study, we leveraged laser capture microdissection (LCM) and nanoPOTS (Zhu et al., 2018c) single-cell mass spectrometry (MS)-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control donor spinal cord tissues, leading to the identification of 2515 proteins across MNs samples (>900 per single MN) and quantitative comparison of 1870 proteins between disease groups. Furthermore, we studied the impact of enriching/stratifying MN proteome samples based on the presence and extent of immunoreactive, cytoplasmic TDP-43 inclusions, allowing identification of 3368 proteins across MNs samples and profiling of 2238 proteins across TDP-43 strata. We found extensive overlap in differential protein abundance profiles between MNs with or without obvious TDP-43 cytoplasmic inclusions that together point to early and sustained dysregulation of oxidative phosphorylation, mRNA splicing and translation, and retromer-mediated vesicular transport in ALS. Our data are the first unbiased quantification of single MN protein abundance changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein abundance changes in human neurologic diseases.
Project description:Transactive Response DNA-binding protein 43 (TDP-43) is a multifunctional nuclear protein ubiquitously expressed in all tissues. Although it is primarily nuclear, Amyotrophic Lateral Sclerosis (ALS) patients frequently present with cytoplasmic aggregates of TDP-43 in motor neurons on post-mortem examination. Although only about 5% of all ALS patients have a TARDBP mutation (Ghasemi and Brown 2018; Ingre et al. 2015), which is known to cause these insoluble cytoplasmic aggregates, 97% of ALS patients will develop cytoplasmic, insoluble TDP-43 aggregates regardless of whether they have a mutation in TARDBP or not (Ling et al. 2013). Overexpression of wild-type TDP-43 has previously been shown to be a valid model of inducing neurotoxicity as well as a limited amount of cytoplasmic re-localization and aggregation characteristic of ALS (Elden et al. 2010; Wils et al. 2010). In the search for modifiers of toxicity as conferred by TDP-43 overexpression in these models, knockout of Ataxin-2 (ATXN2) was proposed as a protective intervention in the context of TDP-43 perturbation in non-human model systems (Elden et al. 2010, Becker et al. 2017). This promising finding had not previously been validated in a human motor neuron system, nor had a thorough investigation been conducted to determine the mechanism of neuroprotection that ATXN2 knockout employs to drive the therapeutic effect. This sequencing study examines both ATXN2 intact and ATXN2 knockout motor neurons in the context of TDP-43 perturbation to explore global transcriptomic changes that arise from these changes in cellular state that are associated with progression of ALS (ATXN2 intact) and what pathways could be implicated in protection from ALS-associated neurodegeneration (ATXN2 Knockout).
Project description:TDP-43, a DNA/RNA binding protein involved in RNA transcription and splicing has been associated with the pathophysiology of neurodegenerative diseases, including ALS. However, the function of TDP-43 in motor neurons remains undefined. Here, we employ both gain- and loss-of-function approaches to determine roles of TDP-43 in motor neurons. Mice expressing human TDP-43 in neurons exhibited growth retardation and premature death that are characterized by abnormal intranuclear inclusions comprised of TDP-43 and Fused in Sarcoma (FUS), and massive accumulation of mitochondria in TDP-43-negative cytoplasmic inclusions in motor neurons, lack of mitochondria in motor axon terminals and immature neuromuscular junctions. Whereas elevated level of TDP-43 disrupts the normal nuclear distribution of Survival Motor Neuron (SMN)-associated Gemini of coiled bodies (GEMs) in motor neurons, its absence prevents the formation of GEMs in the nuclei of these cells. Moreover, transcriptome-wide deep sequencing analysis revealed that decrease in abundance of neurofilament transcripts contributed to the reduction of caliber of motor axons in TDP-43 mice. In concert, our findings indicate that TDP-43 participates in pathways critical for motor neuron physiology, including those that regulate the normal distributions of SMN-associated GEMs in the nucleus and mitochondria in the cytoplasm. Human TDP-43 coding region were inserted into pThy1.2 expression cassette and subsequently injected into C57BL/6;SJL hybrid mouse embryos to make human TDP-43 transgenic mice
Project description:Aims: Loss of nuclear TDP-43 characterises sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether 1) RNA splicing dysregulation is present in lower motor neurons in ALS and in a motor neuron-like cell model, and 2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Methods: Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurons obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by qRT-PCR and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. Results: We found altered expression of spliceosome components in motor neurons and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43 depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, that were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Conclusion: Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurons, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism. RNA was extracted from lower motor neurons obtained by laser capture microdissection from autopsy material from neurologically healthy controls (n=6) and cases of sporadic ALS (n=3) and ALS due to C9ORF72 mutations (n=3).