Regulation of Endoplasmic Reticulum–Mitochondria Tethering and Ca2+ Fluxes by TDP-43 via GSK3β

Mitochondria–ER contacts (MERCs), tightly regulated by numerous tethering proteins that act as molecular and functional connections between the two organelles, are essential to maintain a variety of cellular functions. Such contacts are often compromised in the early stages of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). TDP-43, a nuclear protein mainly involved in RNA metabolism, has been repeatedly associated with ALS pathogenesis and other neurodegenerative diseases. Although TDP-43 neuropathological mechanisms are still unclear, the accumulation of the protein in cytoplasmic inclusions may underlie a protein loss-of-function effect. Accordingly, we investigated the impact of siRNA-mediated TDP-43 silencing on MERCs and the related cellular parameters in HeLa cells using GFP-based probes for MERCs quantification and aequorin-based probes for local Ca2+ measurements, combined with targeted protein and mRNA profiling. Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca2+ uptake after ER Ca2+ release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca2+-mediated ER–mitochondria cross-talk, nor alterations of mitochondrial density and morphology were observed by confocal microscopy. Further mechanistic inspections, however, suggested that the observed cellular alterations are correlated to increased expression/activity of GSK3β, previously associated with MERCs disruption.

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The Impact of ALS-Associated Genes hnRNPA1, MATR3, VCP and UBQLN2 on the Severity of TDP-43 Aggregation

Cells ◽

10.3390/cells9081791 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1791

Author(s):

Ana Bajc Česnik ◽

Helena Motaln ◽

Boris Rogelj

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Rna Binding ◽

Neurodegenerative Disorder ◽

Low Complexity ◽

Wild Type ◽

Disease Heterogeneity ◽

Cytoplasmic Inclusions ◽

The Impact ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder, characterized by cytoplasmic inclusions of RNA-binding protein TDP-43. Despite decades of research and identification of more than 50 genes associated with amyotrophic lateral sclerosis (ALS), the cause of TDP-43 translocation from the nucleus and its aggregation in the cytoplasm still remains unknown. Our study addressed the impact of selected ALS-associated genes on TDP-43 aggregation behavior in wild-type and aggregation prone TDP-43 in vitro cell models. These were developed by deleting TDP-43 nuclear localization signal and stepwise shortening its low-complexity region. The SH-SY5Y cells were co-transfected with the constructs of aggregation-prone TDP-43 and wild-type or mutant ALS-associated genes hnRNPA1, MATR3, VCP or UBQLN2. The investigated genes displayed a unique impact on TDP-43 aggregation, generating distinct types of cytoplasmic inclusions, similar to those already described as resembling prion strains, which could represent the basis for neurodegenerative disease heterogeneity.

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TDP-43 stabilizes transcripts encoding stress granule protein G3BP1: potential relevance to ALS/FTD

10.1101/2020.09.15.298455 ◽

2020 ◽

Author(s):

Hadjara Sidibé ◽

Yousra Khalfallah ◽

Shangxi Xiao ◽

Nicolás B. Gómez ◽

Elizabeth M.H. Tank ◽

...

Keyword(s):

Regulatory Element ◽

Stress Granule ◽

Loss Of Function ◽

Cytoplasmic Inclusions ◽

Protein Levels ◽

Assembly Factor ◽

Direct Binding ◽

Lateral Sclerosis

ABSTRACTTDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. G3BP1 (Ras-GAP SH3-domain-binding protein 1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3’UTR. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data suggest that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease.

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FUS ALS-causative mutations impair FUS autoregulation and splicing factor networks through intron retention

Nucleic Acids Research ◽

10.1093/nar/gkaa410 ◽

2020 ◽

Vol 48 (12) ◽

pp. 6889-6905 ◽

Cited By ~ 7

Author(s):

Jack Humphrey ◽

Nicol Birsa ◽

Carmelo Milioto ◽

Martha McLaughlin ◽

Agnieszka M Ule ◽

...

Keyword(s):

Rna Processing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Intron Retention ◽

Mrna Splicing ◽

Loss Of Function ◽

Induced Changes ◽

The Impact ◽

High Depth ◽

Lateral Sclerosis

Abstract Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.

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Pathway from TDP-43-Related Pathology to Neuronal Dysfunction in Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration

International Journal of Molecular Sciences ◽

10.3390/ijms22083843 ◽

2021 ◽

Vol 22 (8) ◽

pp. 3843

Author(s):

Yuichi Riku ◽

Danielle Seilhean ◽

Charles Duyckaerts ◽

Susana Boluda ◽

Yohei Iguchi ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Frontotemporal Lobar Degeneration ◽

Nuclear Protein ◽

Dna Binding Protein ◽

Neuronal Dysfunction ◽

Loss Of Function ◽

Cytoplasmic Inclusions ◽

Disease Modifying Therapy ◽

Basic Studies ◽

Lateral Sclerosis

Transactivation response DNA binding protein 43 kDa (TDP-43) is known to be a pathologic protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TDP-43 is normally a nuclear protein, but affected neurons of ALS or FTLD patients exhibit mislocalization of nuclear TDP-43 and cytoplasmic inclusions. Basic studies have suggested gain-of-neurotoxicity of aggregated TDP-43 or loss-of-function of intrinsic, nuclear TDP-43. It has also been hypothesized that the aggregated TDP-43 functions as a propagation seed of TDP-43 pathology. However, a mechanistic discrepancy between the TDP-43 pathology and neuronal dysfunctions remains. This article aims to review the observations of TDP-43 pathology in autopsied ALS and FTLD patients and address pathways of neuronal dysfunction related to the neuropathological findings, focusing on impaired clearance of TDP-43 and synaptic alterations in TDP-43-related ALS and FTLD. The former may be relevant to intraneuronal aggregation of TDP-43 and exocytosis of propagation seeds, whereas the latter may be related to neuronal dysfunction induced by TDP-43 pathology. Successful strategies of disease-modifying therapy might arise from further investigation of these subcellular alterations.

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FUS inclusions disrupt RNA localization by sequestering kinesin-1 and inhibiting microtubule detyrosination

The Journal of Cell Biology ◽

10.1083/jcb.201608022 ◽

2017 ◽

Vol 216 (4) ◽

pp. 1015-1034 ◽

Cited By ~ 44

Author(s):

Kyota Yasuda ◽

Sarah F. Clatterbuck-Soper ◽

Meredith E. Jackrel ◽

James Shorter ◽

Stavroula Mili

Keyword(s):

Rna Binding ◽

Fibroblast Cell ◽

Rna Localization ◽

Cellular Functions ◽

Cytoplasmic Inclusions ◽

Inclusion Formation ◽

Fused In Sarcoma ◽

Lateral Sclerosis ◽

Tubulin Carboxypeptidase ◽

Dependent Mechanism

Cytoplasmic inclusions of the RNA-binding protein fused in sarcoma (FUS) represent one type of membraneless ribonucleoprotein compartment. Formation of FUS inclusions is promoted by amyotrophic lateral sclerosis (ALS)–linked mutations, but the cellular functions affected upon inclusion formation are poorly defined. In this study, we find that FUS inclusions lead to the mislocalization of specific RNAs from fibroblast cell protrusions and neuronal axons. This is mediated by recruitment of kinesin-1 mRNA and protein within FUS inclusions, leading to a loss of detyrosinated glutamate (Glu)–microtubules (MTs; Glu-MTs) and an inability to support the localization of RNAs at protrusions. Importantly, dissolution of FUS inclusions using engineered Hsp104 disaggregases, or overexpression of kinesin-1, reverses these effects. We further provide evidence that kinesin-1 affects MT detyrosination not through changes in MT stability, but rather through targeting the tubulin carboxypeptidase enzyme onto specific MTs. Interestingly, other pathological inclusions lead to similar outcomes, but through apparently distinct mechanisms. These results reveal a novel kinesin-dependent mechanism controlling the MT cytoskeleton and identify loss of Glu-MTs and RNA mislocalization as common outcomes of ALS pathogenic mutations.

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Granulins modulate liquid-liquid phase separation and aggregation of TDP-43 C-terminal domain

10.1101/812115 ◽

2019 ◽

Author(s):

Anukool A. Bhopatkar ◽

Vladimir N. Uversky ◽

Vijayaraghavan Rangachari

Keyword(s):

Direct Interaction ◽

Loss Of Function ◽

Granule Formation ◽

Cytoplasmic Inclusions ◽

Factors Affecting ◽

Amyotropic Lateral Sclerosis ◽

Significant Attention ◽

Lateral Sclerosis ◽

Liquid Liquid Phase Separation

ABSTRACTTar DNA binding protein (TDP-43) has emerged as a key player in many neurodegenerative pathologies including frontotemporal lobar degeneration (FTLD) and amyotropic lateral sclerosis (ALS). Important hallmarks of FTLD and ALS are the toxic cytoplasmic inclusions of C-terminal fragments of TDP-43 (TDP-43CTD), which are formed upon proteolytic cleavage of full-length TDP-43 in the nucleus and subsequent transport to the cytoplasm. TDP-43CTD is also known to form stress granules (SGs) by coacervating with RNA in cytoplasm under stress conditions and are believed to be involved in modulating the pathologies. Among other factors affecting these pathologies, the pleiotropic protein called progranulin (PGRN) has gained significant attention lately. The haploinsufficiency of PGRN, caused by autosomal dominant mutations in GRN gene, results in its loss-of-function linked to FTLD and ALS. But precisely how the protein contributes to the pathology remains unknown. Recently, cleavage to GRNs were observed to be a significant part of FTLD and ALS progression with specific GRNs exacerbating TDP-43-induced toxicity in C.elegans. In this report, we show that GRNs −3 and −5 directly interact with TDP-43CTD to modulate latter’s aggregation or stress granule formation in disparate ways in vitro. These results constitute the first observation of direct interaction between GRNs and TDP-43 and suggest a mechanism by which the loss of PGRN function could lead to FTLD and ALS.

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GuidePro: A multi-source ensemble predictor for prioritizing sgRNAs in CRISPR/Cas9 protein knockouts

10.1101/2020.07.10.197996 ◽

2020 ◽

Author(s):

Wei He ◽

Helen Wang ◽

Yanjun Wei ◽

Zhiyun Jiang ◽

Yitao Tang ◽

...

Keyword(s):

Amino Acids ◽

Dna Sequences ◽

Web Application ◽

Protein Structures ◽

Superior Performance ◽

Loss Of Function ◽

Protein Loss ◽

Protein Coding ◽

Cas9 Protein ◽

The Impact

AbstractThe efficiency of CRISPR/Cas9-mediated protein knockout is determined by three factors: sequence-specific sgRNA activity, frameshift probability, and the characteristics of targeted amino acids. A number of computational methods have been developed for predicting sgRNA efficiency from different perspectives. We propose GuidePro, a two-layer ensemble predictor that enables the integration of multiple predictive methods and feature sets. GuidePro leverages information from DNA sequences, amino acids, and protein structures, and reduces the impact of dataset-specific biases. Tested on independent datasets, GuidePro demonstrated consistent superior performance in predicting phenotypes caused by protein loss-of-function. GuidePro is implemented as a web application for prioritizing sgRNAs that target protein-coding genes in human, monkey and mouse genomes, available at https://bioinformatics.mdanderson.org/apps/GuidePro.

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ATP13A2 Regulates Cellular α-Synuclein Multimerization, Membrane Association, and Externalization

International Journal of Molecular Sciences ◽

10.3390/ijms22052689 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2689

Author(s):

Jianmin Si ◽

Chris Van den Haute ◽

Evy Lobbestael ◽

Shaun Martin ◽

Sarah van Veen ◽

...

Keyword(s):

Membrane Integrity ◽

Ubiquitin Proteasome System ◽

Regulatory Function ◽

Membrane Association ◽

Loss Of Function ◽

Atypical Form ◽

Polyamine Transport ◽

Independent Functions ◽

Ubiquitin Proteasome ◽

The Impact

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson’s disease and Kufor–Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.

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Neuroligin-2 as a central organizer of inhibitory synapses in health and disease

Science Signaling ◽

10.1126/scisignal.abd8379 ◽

2020 ◽

Vol 13 (663) ◽

pp. eabd8379

Author(s):

Heba Ali ◽

Lena Marth ◽

Dilja Krueger-Burg

Keyword(s):

Synaptic Transmission ◽

Synapse Formation ◽

Current Knowledge ◽

Protein Complexes ◽

Inhibitory Synapses ◽

Molecular Architecture ◽

Cellular Functions ◽

Altered Expression ◽

Health And Disease ◽

Flow Of Information

Postsynaptic organizational protein complexes play central roles both in orchestrating synapse formation and in defining the functional properties of synaptic transmission that together shape the flow of information through neuronal networks. A key component of these organizational protein complexes is the family of synaptic adhesion proteins called neuroligins. Neuroligins form transsynaptic bridges with presynaptic neurexins to regulate various aspects of excitatory and inhibitory synaptic transmission. Neuroligin-2 (NLGN2) is the only member that acts exclusively at GABAergic inhibitory synapses. Altered expression and mutations in NLGN2 and several of its interacting partners are linked to cognitive and psychiatric disorders, including schizophrenia, autism, and anxiety. Research on NLGN2 has fundamentally shaped our understanding of the molecular architecture of inhibitory synapses. Here, we discuss the current knowledge on the molecular and cellular functions of mammalian NLGN2 and its role in the neuronal circuitry that regulates behavior in rodents and humans.

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A Melanocortin-4 Receptor Agonist Induces Skin and Hair Pigmentation in Patients with Monogenic Mutations in the Leptin-Melanocortin Pathway

Skin Pharmacology and Physiology ◽

10.1159/000516282 ◽

2021 ◽

pp. 1-10

Author(s):

Varvara Kanti ◽

Lia Puder ◽

Irina Jahnke ◽

Philipp Maximilian Krabusch ◽

Jan Kottner ◽

...

Keyword(s):

Leptin Receptor ◽

Skin Pigmentation ◽

Gene Mutations ◽

Continuous Treatment ◽

Whole Body ◽

Hair Color ◽

Phase 2 ◽

Loss Of Function ◽

The Impact ◽

Over Time

Background and Objectives: Gene mutations within the leptin-melanocortin signaling pathway lead to severe early-onset obesity. Recently, a phase 2 trial evaluated new pharmacological treatment options with the MC4R agonist setmelanotide in patients with mutations in the genes encoding proopiomelanocortin (POMC) and leptin receptor (LEPR). During treatment with setmelanotide, changes in skin pigmentation were observed, probably due to off-target effects on the closely related melanocortin 1 receptor (MC1R). Here, we describe in detail the findings of dermatological examinations and measurements of skin pigmentation during this treatment over time and discuss the impact of these changes on patient safety. Methods: In an investigator-initiated, phase 2, open-label pilot study, 2 patients with loss-of-function POMC gene mutations and 3 patients with loss-of-function variants in LEPR were treated with the MC4R agonist setmelanotide. Dermatological examination, dermoscopy, whole body photographic documentation, and spectrophotometric measurements were performed at screening visit and approximately every 3 months during the course of the study. Results: We report the results of a maximum treatment duration of 46 months. Skin pigmentation increased in all treated patients, as confirmed by spectrophotometry. During continuous treatment, the current results indicate that elevated tanning intensity levels may stabilize over time. Lips and nevi also darkened. In red-haired study participants, hair color changed to brown after initiation of setmelanotide treatment. Discussion: Setmelanotide treatment leads to skin tanning and occasionally hair color darkening in both POMC- and LEPR-deficient patients. No malignant skin changes were observed in the patients of this study. However, the results highlight the importance of regular skin examinations before and during MC4R agonist treatment.

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