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1.
Mol Neurobiol ; 58(11): 5682-5702, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34390468

ABSTRACT

The nuclear RNA-binding protein TDP-43 forms abnormal cytoplasmic aggregates in the brains of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients and several molecular mechanisms promoting TDP-43 cytoplasmic mislocalization and aggregation have been proposed, including defects in nucleocytoplasmic transport, stress granules (SG) disassembly and post-translational modifications (PTM). SUMOylation is a PTM which regulates a variety of cellular processes and, similarly to ubiquitination, targets lysine residues. To investigate the possible regulatory effects of SUMOylation on TDP-43 activity and trafficking, we first assessed that TDP-43 is SUMO-conjugated in the nuclear compartment both covalently and non-covalently in the RRM1 domain at the predicted lysine 136 and SUMO-interacting motif (SIM, 106-110 residues), respectively. By using the SUMO-mutant TDP-43 K136R protein, we demonstrated that SUMOylation modifies TDP-43 splicing activity, specifically exon skipping, and influences its sub-cellular localization and recruitment to SG after oxidative stress. When promoting deSUMOylation by SENP1 enzyme over-expression or by treatment with the cell-permeable SENP1 peptide TS-1, the cytoplasmic localization of TDP-43 increased, depending on its SUMOylation. Moreover, deSUMOylation by TS-1 peptide favoured the formation of small cytoplasmic aggregates of the C-terminal TDP-43 fragment p35, still containing the SUMO lysine target 136, but had no effect on the already formed p25 aggregates. Our data suggest that TDP-43 can be post-translationally modified by SUMOylation which may regulate its splicing function and trafficking, indicating a novel and druggable mechanism to explore as its dysregulation may lead to TDP-43 pathological aggregation in ALS and FTD.


Subject(s)
Cell Nucleus/chemistry , Cytoplasm/chemistry , DNA-Binding Proteins/metabolism , Nerve Tissue Proteins/metabolism , Protein Processing, Post-Translational , Cell Line, Tumor , DNA-Binding Proteins/analysis , HEK293 Cells , Humans , Models, Molecular , Molecular Dynamics Simulation , Nerve Tissue Proteins/analysis , Neuroblastoma , Peptide Fragments/pharmacology , Potassium Chloride/pharmacology , Protein Conformation , Protein Transport , RNA Interference , RNA Splicing , RNA, Small Interfering/pharmacology , Sequence Alignment , Sequence Homology, Amino Acid , Stress Granules , Sumoylation
3.
Neurobiol Dis ; 145: 105051, 2020 11.
Article in English | MEDLINE | ID: mdl-32827688

ABSTRACT

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases characterized by the presence of neuropathological aggregates of phosphorylated TDP-43 (P-TDP-43) protein. The RNA-binding protein TDP-43 participates also to cell stress response by forming stress granules (SG) in the cytoplasm to temporarily arrest translation. The hypothesis that TDP-43 pathology directly arises from SG has been proposed but is still under debate because only sub-lethal stress conditions have been tested experimentally so far. In this study we reproduced a mild and chronic oxidative stress by sodium arsenite to better mimic the persistent and subtle alterations occurring during the neurodegenerative process in primary fibroblasts and induced pluripotent stem cell-derived motoneurons (iPSC-MN) from ALS patients carrying mutations in TARDBP and C9ORF72 genes. We found that not only the acute sub-lethal stress usually used in literature, but also the chronic oxidative insult was able to induce SG formation in both primary fibroblasts and iPSC-MN. We also observed the recruitment of TDP-43 into SG only upon chronic stress in association to the formation of distinct cytoplasmic P-TDP-43 aggregates and a significant increase of the autophagy marker p62. A quantitative analysis revealed differences in both the number of cells forming SG in mutant ALS and healthy control fibroblasts, suggesting a specific genetic contribution to cell stress response, and in SG size, suggesting a different composition of these cytoplasmic foci in the two stress conditions. Upon removal of arsenite, the recovery from chronic stress was complete for SG and P-TDP-43 aggregates at 72 h with the exception of p62, which was reduced but still persistent, supporting the hypothesis that autophagy impairment may drive pathological TDP-43 aggregates formation. The gene-specific differences observed in fibroblasts in response to oxidative stress were not present in iPSC-MN, which showed a similar formation of SG and P-TDP-43 aggregates regardless their genotype. Our results show that SG and P-TDP-43 aggregates may be recapitulated in patient-derived neuronal and non-neuronal cells exposed to prolonged oxidative stress, which may be therefore exploited to study TDP-43 pathology and to develop individualized therapeutic strategies for ALS/FTD.


Subject(s)
Amyotrophic Lateral Sclerosis/pathology , DNA-Binding Proteins/metabolism , Fibroblasts/pathology , Motor Neurons/pathology , Oxidative Stress/physiology , Cells, Cultured , Humans , Induced Pluripotent Stem Cells
4.
J Cell Mol Med ; 24(7): 4051-4060, 2020 04.
Article in English | MEDLINE | ID: mdl-32125773

ABSTRACT

As for the majority of neurodegenerative diseases, pathological mechanisms of amyotrophic lateral sclerosis (ALS) have been challenging to study due to the difficult access to alive patients' cells. Induced pluripotent stem cells (iPSCs) offer a useful in vitro system for modelling human diseases. iPSCs can be theoretically obtained by reprogramming any somatic tissue although fibroblasts (FB) remain the most used cells. However, reprogramming peripheral blood cells (PB) may offer significant advantages. In order to investigate whether the choice of starting cells may affect reprogramming and motor neuron (MNs) differentiation potential, we used both FB and PB from a same C9ORF72-mutated ALS patient to obtain iPSCs and compared several hallmarks of the pathology. We found that both iPSCs and MNs derived from the two tissues showed identical properties and features and can therefore be used interchangeably, giving the opportunity to easily obtain iPSCs from a more manageable source of cells, such as PB.


Subject(s)
Amyotrophic Lateral Sclerosis/blood , C9orf72 Protein/genetics , Cellular Reprogramming/genetics , Neurodegenerative Diseases/blood , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Blood Cells/cytology , Blood Cells/metabolism , C9orf72 Protein/blood , Cell Differentiation/genetics , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Induced Pluripotent Stem Cells/metabolism , Motor Neurons/metabolism , Motor Neurons/pathology , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/pathology
5.
Int J Mol Sci ; 20(23)2019 Nov 22.
Article in English | MEDLINE | ID: mdl-31766750

ABSTRACT

Cytoplasmic aggregates and nuclear depletion of the ubiquitous RNA-binding protein TDP-43 have been described in the autoptic brain tissues of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD) patients and both TDP-43 loss-of-function and gain-of-function mechanisms seem to contribute to the neurodegenerative process. Among the wide array of RNA targets, TDP-43 regulates progranulin (GRN) mRNA stability and sortilin (SORT1) splicing. Progranulin is a secreted neurotrophic and neuro-immunomodulatory factor whose endocytosis and delivery to the lysosomes are regulated by the neuronal receptor sortilin. Moreover, GRN loss-of-function mutations are causative of a subset of FTLD cases showing TDP-43 pathological aggregates. Here we show that TDP-43 loss-of-function differently affects the progranulin-sortilin axis in murine and human neuronal cell models. We demonstrated that although TDP-43 binding to GRN mRNA occurs similarly in human and murine cells, upon TDP-43 depletion, a different control of sortilin splicing and protein content may determine changes in extracellular progranulin uptake that account for increased or unchanged secreted protein in murine and human cells, respectively. As targeting the progranulin-sortilin axis has been proposed as a therapeutic approach for GRN-FTLD patients, the inter-species differences in TDP-43-mediated regulation of this pathway must be considered when translating studies from animal models to patients.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , DNA-Binding Proteins/metabolism , Models, Biological , Neurodegenerative Diseases/metabolism , Progranulins/metabolism , Signal Transduction , Adaptor Proteins, Vesicular Transport/genetics , Animals , Cell Line, Tumor , DNA-Binding Proteins/genetics , Humans , Mice , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/pathology , Neurodegenerative Diseases/therapy , Progranulins/genetics , Species Specificity
6.
Nat Commun ; 10(1): 3827, 2019 08 23.
Article in English | MEDLINE | ID: mdl-31444357

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown etiology. Although defects in nucleocytoplasmic transport (NCT) may be central to the pathogenesis of ALS and other neurodegenerative diseases, the molecular mechanisms modulating the nuclear pore function are still largely unknown. Here we show that genetic and pharmacological modulation of actin polymerization disrupts nuclear pore integrity, nuclear import, and downstream pathways such as mRNA post-transcriptional regulation. Importantly, we demonstrate that modulation of actin homeostasis can rescue nuclear pore instability and dysfunction caused by mutant PFN1 as well as by C9ORF72 repeat expansion, the most common mutation in ALS patients. Collectively, our data link NCT defects to ALS-associated cellular pathology and propose the regulation of actin homeostasis as a novel therapeutic strategy for ALS and other neurodegenerative diseases.


Subject(s)
Actins/metabolism , Amyotrophic Lateral Sclerosis/pathology , Motor Neurons/pathology , Nuclear Pore/pathology , Profilins/metabolism , Acrylamides/pharmacology , Actins/ultrastructure , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/genetics , Amyotrophic Lateral Sclerosis/genetics , Biopsy , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Cell Line , Cerebral Cortex/cytology , Cerebral Cortex/pathology , Embryo, Mammalian , Fibroblasts , Humans , Microscopy, Electron, Transmission , Motor Neurons/cytology , Mutation , Nuclear Pore/drug effects , Nuclear Pore/ultrastructure , Primary Cell Culture , Profilins/genetics , Protein Multimerization/drug effects , Protein Multimerization/genetics , Skin/cytology , Skin/pathology , Thiazoles/pharmacology , Thiazolidines/pharmacology
7.
Biochim Biophys Acta Gene Regul Mech ; 1862(9): 194413, 2019 09.
Article in English | MEDLINE | ID: mdl-31382054

ABSTRACT

The RNA-binding protein TDP-43, associated to amyotrophic lateral sclerosis and frontotemporal dementia, regulates the alternative splicing of several genes, including the skipping of TNIK exon 15. TNIK, a genetic risk factor for schizophrenia and causative for intellectual disability, encodes for a Ser/Thr kinase regulating negatively F-actin dynamics. Here we show that in the human adult nervous system TNIK exon 15 is mostly included compared to the other tissues and that, during neuronal differentiation of human induced pluripotent stem cells and of human neuroblastoma cells, TNIK exon 15 inclusion increases independently of TDP-43 protein content. By studying the possible molecular interplay of TDP-43 with brain-specific splicing factors, we found that the neuronal NOVA-1 protein competitively inhibits both TDP-43 and hnRNPA2/B1 skipping activity on TNIK by means of a RNA-dependent interaction and that this competitive mechanism is common to other TDP-43 RNA targets. We also show that the TNIK protein isoforms including/excluding exon 15 differently regulate cell spreading in non-neuronal cells and neuritogenesis in primary cortical neurons. Our data suggest a complex regulation between the ubiquitous TDP-43 and the neuron-specific NOVA-1 splicing factors in the brain that may help better understand the pathobiology of both neurodegenerative diseases and schizophrenia.


Subject(s)
DNA-Binding Proteins/genetics , Protein Serine-Threonine Kinases/genetics , RNA-Binding Proteins/genetics , Schizophrenia/genetics , Alternative Splicing/genetics , Cell Line , DNA-Binding Proteins/chemistry , Exons/genetics , Humans , Neuro-Oncological Ventral Antigen , Neurons/metabolism , Neurons/pathology , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Serine-Threonine Kinases/chemistry , RNA, Messenger/genetics , RNA-Binding Proteins/chemistry , Schizophrenia/pathology
8.
Stem Cell Res ; 30: 61-68, 2018 07.
Article in English | MEDLINE | ID: mdl-29800782

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease, mainly affecting the motor neurons (MNs) and without effective therapy. Drug screening is hampered by the lack of satisfactory experimental and pre-clinical models. Induced pluripotent stem cells (iPSCs) could help to define disease mechanisms and therapeutic strategies as they could be differentiated into MNs, otherwise inaccessible from living humans. In this study, given the seminal role of TDP-43 in ALS pathophysiology, MNs were obtained from peripheral blood mononuclear cells-derived iPSCs of an ALS patient carrying a p.A382T TARDBP mutation and a healthy donor. Venous samples were preferred to fibroblasts for their ease of collection and no requirement for time consuming extended cultures before experimentation. iPSCs were characterized for expression of specific markers, spontaneously differentiated into primary germ layers and, finally, into MNs. No differences were observed between the mutated ALS patient and the control MNs with most of the cells displaying a nuclear localization of the TDP-43 protein. In conclusion, we here demonstrated for the first time that human TARDBP mutated MNs can be successfully obtained exploiting the reprogramming and differentiation ability of peripheral blood cells, an easily accessible source from any patient.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Induced Pluripotent Stem Cells/metabolism , Motor Neurons/metabolism , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Cell Differentiation , Female , Humans , Induced Pluripotent Stem Cells/cytology , Middle Aged , Motor Neurons/cytology , Mutation
9.
Acta Neuropathol Commun ; 4(1): 47, 2016 05 05.
Article in English | MEDLINE | ID: mdl-27151080

ABSTRACT

Dysregulation of RNA metabolism represents an important pathogenetic mechanism in both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) due to the involvement of the DNA/RNA-binding proteins TDP-43 and FUS and, more recently, of C9ORF72. A potential link between dysregulation of RNA metabolism and mitochondrial dysfunction is recently emerged in TDP-43 disease models. To further investigate the possible relationship between these two pathogenetic mechanisms in ALS/FTD, we studied mitochondria functionality in human mutant TARDBP(p.A382T) and C9ORF72 fibroblasts grown in galactose medium to induce a switch from a glycolytic to an oxidative metabolism. In this condition we observed significant changes in mitochondria morphology and ultrastructure in both mutant cells with a fragmented mitochondria network particularly evident in TARDBP(p.A382T) fibroblasts. From analysis of the mitochondrial functionality, a decrease of mitochondria membrane potential with no alterations in oxygen consumption rate emerged in TARDBP fibroblasts. Conversely, an increased oxygen consumption and mitochondria hyperpolarization were observed in C9ORF72 fibroblasts in association to increased ROS and ATP content. We found evidence of autophagy/mitophagy in dynamic equilibrium with the biogenesis of novel mitochondria, particularly in mutant C9ORF72 fibroblasts where an increase of mitochondrial DNA content and mass, and of PGC1-α protein was observed. Our imaging and biochemical data show that wild-type and mutant TDP-43 proteins do not localize at mitochondria so that the molecular mechanisms responsible for such mitochondria impairment remain to be further elucidated. For the first time our findings assess a link between C9ORF72 and mitochondria dysfunction and indicate that mitochondria functionality is affected in TARDBP and C9ORF72 fibroblasts with gene-specific features in oxidative conditions. As in neuronal metabolism mitochondria are actively used for ATP production, we speculate that TARDBP and C9ORF72 mutations might trigger cell death by impairing not only RNA metabolism, but also mitochondria activity in ALS/FTD neurons.


Subject(s)
DNA-Binding Proteins/metabolism , Mitochondria/metabolism , Mitochondria/ultrastructure , Mutation , Proteins/metabolism , Adenosine Triphosphate/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Apoptosis/physiology , C9orf72 Protein , Cell Line, Tumor , Cell Survival/physiology , DNA-Binding Proteins/genetics , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Frontotemporal Lobar Degeneration/genetics , Frontotemporal Lobar Degeneration/metabolism , Frontotemporal Lobar Degeneration/pathology , Humans , Membrane Potential, Mitochondrial/physiology , Mitochondrial Diseases/genetics , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/pathology , Oxygen/metabolism , Proteins/genetics , Reactive Oxygen Species/metabolism
10.
Biochim Biophys Acta ; 1849(12): 1398-410, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26514432

ABSTRACT

The full definition of the physiological RNA targets regulated by TDP-43 and FUS RNA-binding proteins (RBPs) represents an important issue in understanding the pathogenic mechanisms associated to these two proteins in amyotrophic lateral sclerosis and frontotemporal dementia. In the last few years several high-throughput screenings have generated a plethora of data, which are difficult to compare due to the different experimental designs and models explored. In this study by using the Affymetrix Exon Arrays, we were able to assess and compare the effects of both TDP-43 and FUS loss-of-function on the whole transcriptome using the same human neuronal SK-N-BE cell model. We showed that TDP-43 and FUS depletion induces splicing and gene expression changes mainly distinct for the two RBPs, although they may regulate common pathways, including neuron differentiation and cytoskeleton organization as evidenced by functional annotation analysis. In particular, TDP-43 and FUS were found to regulate splicing and expression of genes related to neuronal (SEPT6, SULT4A1, TNIK) and RNA metabolism (DICER, ELAVL3/HuC, POLDIP3). Our extended analysis at protein level revealed that these changes have also impact on the protein isoform ratio and content, not always in a direct correlation with transcriptomic data. Contrarily to a loss-of-function mechanism, we showed that mutant TDP-43 proteins maintained their splicing activity in human ALS fibroblasts and experimental cell lines. Our findings further contribute to define the biological functions of these two RBPs in physiological and disease state, strongly encouraging the evaluation of the identified transcriptomic changes at protein level in neuronal experimental models.


Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation, Neoplastic/genetics , Neoplasm Proteins/physiology , Neurons/metabolism , Proteome , RNA Precursors/genetics , RNA, Messenger/genetics , RNA, Neoplasm/genetics , RNA-Binding Protein FUS/physiology , Transcriptome , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Cell Line, Tumor , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Exons/genetics , Fibroblasts/metabolism , Humans , Mice , Mice, Inbred BALB C , Models, Genetic , Molecular Sequence Data , Neoplasm Proteins/deficiency , Neoplasm Proteins/genetics , Neuroblastoma/pathology , Protein Isoforms/metabolism , RNA Interference , RNA Precursors/metabolism , RNA, Messenger/metabolism , RNA, Neoplasm/metabolism , RNA, Small Interfering/genetics , RNA-Binding Protein FUS/deficiency , RNA-Binding Protein FUS/genetics , Sequence Alignment , Sequence Homology
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