Mitigation of TDP-43 toxic phenotype by an RGNEF fragment in amyotrophic lateral sclerosis models.

Aggregation of the RNA-binding protein TAR DNA binding protein (TDP-43) is a hallmark of TDP-proteinopathies including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As TDP-43 aggregation and dysregulation are causative of neuronal death, there is a special interest in targeting this protein as a therapeutic approach. Previously, we found that TDP-43 extensively co-aggregated with the dual function protein GEF (guanine exchange factor) and RNA-binding protein rho guanine nucleotide exchange factor (RGNEF) in ALS patients. Here, we show that an N-terminal fragment of RGNEF (NF242) interacts directly with the RNA recognition motifs of TDP-43 competing with RNA and that the IPT/TIG domain of NF242 is essential for this interaction. Genetic expression of NF242 in a fruit fly ALS model overexpressing TDP-43 suppressed the neuropathological phenotype increasing lifespan, abolishing motor defects and preventing neurodegeneration. Intracerebroventricular injections of AAV9/NF242 in a severe TDP-43 murine model (rNLS8) improved lifespan and motor phenotype, and decreased neuroinflammation markers. Our results demonstrate an innovative way to target TDP-43 proteinopathies using a protein fragment with a strong affinity for TDP-43 aggregates and a mechanism that includes competition with RNA sequestration, suggesting a promising therapeutic strategy for TDP-43 proteinopathies such as ALS and FTD.

Assessing the Impact of Novel BRCA1 Exon 11 Variants on Pre-mRNA Splicing.

Our study focused on assessing the effects of three newly identified BRCA1 exon 11 variants (c.1019T>C, c.2363T>G, and c.3192T>C) on breast cancer susceptibility. Using computational predictions and experimental splicing assays, we evaluated their potential as pathogenic mutations. Our in silico analyses suggested that the c.2363T>G and c.3192T>C variants could impact both splicing and protein function, resulting in the V340A and V788G mutations, respectively. We further examined their splicing effects using minigene assays in MCF7 and SKBR3 breast cancer cell lines. Interestingly, we found that the c.2363T>G variant significantly altered splicing patterns in MCF7 cells but not in SKBR3 cells. This finding suggests a potential influence of cellular context on the variant's effects. While attempts to correlate in silico predictions with RNA binding factors were inconclusive, this observation underscores the complexity of splicing regulation. Splicing is governed by various factors, including cellular contexts and protein interactions, making it challenging to predict outcomes accurately. Further research is needed to fully understand the functional consequences of the c.2363T>G variant in breast cancer pathogenesis. Integrating computational predictions with experimental data will provide valuable insights into the role of alternative splicing regulation in different breast cancer types and stages.

Comprehensive splicing analysis of the alternatively spliced CHEK2 exons 8 and 10 reveals three enhancer/silencer-rich regions and 38 spliceogenic variants.

Splicing is controlled by a large set of regulatory elements (SREs) including splicing enhancers and silencers, which are involved in exon recognition. Variants at these motifs may dysregulate splicing and trigger loss-of-function transcripts associated with disease. Our goal here was to study the alternatively spliced exons 8 and 10 of the breast cancer susceptibility gene CHEK2. For this purpose, we used a previously published minigene with exons 6-10 that produced the expected minigene full-length transcript and replicated the naturally occurring events of exon 8 [Δ(E8)] and exon 10 [Δ(E10)] skipping. We then introduced 12 internal microdeletions of exons 8 and 10 by mutagenesis in order to map SRE-rich intervals by splicing assays in MCF-7 cells. We identified three minimal (10-, 11-, 15-nt) regions essential for exon recognition: c.863_877del [ex8, Δ(E8): 75%] and c.1073_1083del and c.1083_1092del [ex10, Δ(E10): 97% and 62%, respectively]. Then 87 variants found within these intervals were introduced into the wild-type minigene and tested functionally. Thirty-eight of them (44%) impaired splicing, four of which (c.883G>A, c.883G>T, c.884A>T, and c.1080G>T) induced negligible amounts (<5%) of the minigene full-length transcript. Another six variants (c.886G>A, c.886G>T, c.1075G>A, c.1075G>T, c.1076A>T, and c.1078G>T) showed significantly strong impacts (20-50% of the minigene full-length transcript). Thirty-three of the 38 spliceogenic variants were annotated as missense, three as nonsense, and two as synonymous, underlying the fact that any exonic change is capable of disrupting splicing. Moreover, c.883G>A, c.883G>T, and c.884A>T were classified as pathogenic/likely pathogenic variants according to ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based criteria. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Changes in the expression of COI1, TIR1, and ERF1 genes and respective MiRNAs in Fusarium basal Rot-Stressed onion.

Fusarium oxysporum f.sp. cepae (FOC), as basal rot fungus, is the most detrimental pathogen causing a serious threat to onion productivity in the world. In this study, we first determined FOC tolerance in seven Iranian onion cultivars, two known international onions (Texas Early Grano and Sweet Yellow Spanish), and an Allium species related to the onion (Allium asarence) based on the infection severity. Then, a transcriptional screen was performed by comparing the transcript levels of some pathogen-responsive genes (ERF1, COI1, and TIR1) and their predicted miRNAs in the sensitive (Ghermeze Azarshahr Cv.) and the resistant (A. asarence) onions to determine key genes and their miRNAs involved in the defense responses of onions to FOC. From our results, a difference was found in the COI1 and ERF1 expression 48 h after inoculation with FOC as compared to the respective 24 and 72 h. It can be explained by either special mechanisms involved in raising energy consumption efficiency or the interactive effects of other genes in the jasmonic acid (JA) and ethylene (ET) signaling pathways. Moreover, expression analysis of the pathogen-responsive genes and their targeting miRNAs identified the miR-5629, which targets the COI1 gene as a likely key factor in conferring resistance in the FOC-resistant onion, i.e., A. asarence. However, exploring the function of the miRNA/target pair is highly recommended to deeply understand the effect of the miRNA/target pair-associated pathway in the control of A. asarense-FOC interaction.

Cross-seeding by prion protein inactivates TDP-43.

A common pathological denominator of various neurodegenerative diseases is the accumulation of protein aggregates. Neurotoxic effects are caused by a loss of the physiological activity of the aggregating protein and/or a gain of toxic function of the misfolded protein conformers. In transmissible spongiform encephalopathies or prion diseases, neurodegeneration is caused by aberrantly folded isoforms of the prion protein (PrP). However, it is poorly understood how pathogenic PrP conformers interfere with neuronal viability. Employing in vitro approaches, cell culture, animal models and patients' brain samples, we show that misfolded PrP can induce aggregation and inactivation of TAR DNA-binding protein-43 (TDP-43). Purified PrP aggregates interact with TDP-43 in vitro and in cells and induce the conversion of soluble TDP-43 into non-dynamic protein assemblies. Similarly, mislocalized PrP conformers in the cytosol bind to and sequester TDP-43 in cytosolic aggregates. As a consequence, TDP-43-dependent splicing activity in the nucleus is significantly decreased, leading to altered protein expression in cells with cytosolic PrP aggregates. Finally, we present evidence for cytosolic TDP-43 aggregates in neurons of transgenic flies expressing mammalian PrP and Creutzfeldt-Jakob disease patients. Our study identified a novel mechanism of how aberrant PrP conformers impair physiological pathways by cross-seeding.

Detection of TDP-43 seeding activity in the olfactory mucosa from patients with frontotemporal dementia.

INTRODUCTION: We assessed TAR DNA-binding protein 43 (TDP-43) seeding activity and aggregates detection in olfactory mucosa of patients with frontotemporal lobar degeneration with TDP-43-immunoreactive pathology (FTLD-TDP) by TDP-43 seeding amplification assay (TDP43-SAA) and immunocytochemical analysis. METHODS: The TDP43-SAA was optimized using frontal cortex samples from 16 post mortem cases with FTLD-TDP, FTLD with tau inclusions, and controls. Subsequently, olfactory mucosa samples were collected from 17 patients with FTLD-TDP, 15 healthy controls, and three patients carrying MAPT variants. RESULTS: TDP43-SAA discriminated with 100% accuracy post mortem cases presenting or lacking TDP-43 neuropathology. TDP-43 seeding activity was detectable in the olfactory mucosa, and 82.4% of patients with FTLD-TDP tested positive, whereas 86.7% of controls tested negative (P < 0.001). Two out of three patients with MAPT mutations tested negative. In TDP43-SAA positive samples, cytoplasmatic deposits of phosphorylated TDP-43 in the olfactory neural cells were detected. DISCUSSION: TDP-43 aggregates can be detectable in olfactory mucosa, suggesting that TDP43-SAA might be useful for identifying and monitoring FTLD-TDP in living patients.

Overlapping Neuroimmune Mechanisms and Therapeutic Targets in Neurodegenerative Disorders.

Many potential immune therapeutic targets are similarly affected in adult-onset neurodegenerative diseases, such as Alzheimer's (AD) disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD), as well as in a seemingly distinct Niemann-Pick type C disease with primarily juvenile onset. This strongly argues for an overlap in pathogenic mechanisms. The commonly researched immune targets include various immune cell subsets, such as microglia, peripheral macrophages, and regulatory T cells (Tregs); the complement system; and other soluble factors. In this review, we compare these neurodegenerative diseases from a clinical point of view and highlight common pathways and mechanisms of protein aggregation, neurodegeneration, and/or neuroinflammation that could potentially lead to shared treatment strategies for overlapping immune dysfunctions in these diseases. These approaches include but are not limited to immunisation, complement cascade blockade, microbiome regulation, inhibition of signal transduction, Treg boosting, and stem cell transplantation.

TDP-43 Epigenetic Facets and Their Neurodegenerative Implications.

Since its initial involvement in numerous neurodegenerative pathologies in 2006, either as a principal actor or as a cofactor, new pathologies implicating transactive response (TAR) DNA-binding protein 43 (TDP-43) are regularly emerging also beyond the neuronal system. This reflects the fact that TDP-43 functions are particularly complex and broad in a great variety of human cells. In neurodegenerative diseases, this protein is often pathologically delocalized to the cytoplasm, where it irreversibly aggregates and is subjected to various post-translational modifications such as phosphorylation, polyubiquitination, and cleavage. Until a few years ago, the research emphasis has been focused particularly on the impacts of this aggregation and/or on its widely described role in complex RNA splicing, whether related to loss- or gain-of-function mechanisms. Interestingly, recent studies have strengthened the knowledge of TDP-43 activity at the chromatin level and its implication in the regulation of DNA transcription and stability. These discoveries have highlighted new features regarding its own transcriptional regulation and suggested additional mechanistic and disease models for the effects of TPD-43. In this review, we aim to give a comprehensive view of the potential epigenetic (de)regulations driven by (and driving) this multitask DNA/RNA-binding protein.

Understanding the phenotypic variability in Niemann-Pick disease type C (NPC): a need for precision medicine.

Niemann-Pick type C (NPC) disease is a lysosomal storage disease (LSD) characterized by the buildup of endo-lysosomal cholesterol and glycosphingolipids due to loss of function mutations in the NPC1 and NPC2 genes. NPC patients can present with a broad phenotypic spectrum, with differences at the age of onset, rate of progression, severity, organs involved, effects on the central nervous system, and even response to pharmacological treatments. This article reviews the phenotypic variation of NPC and discusses its possible causes, such as the remaining function of the defective protein, modifier genes, sex, environmental cues, and splicing factors, among others. We propose that these factors should be considered when designing or repurposing treatments for this disease. Despite its seeming complexity, this proposition is not far-fetched, considering the expanding interest in precision medicine and easier access to multi-omics technologies.

Emerging Trends in the Field of Inflammation and Proteinopathy in ALS/FTD Spectrum Disorder.

Proteinopathy and neuroinflammation are two main hallmarks of neurodegenerative diseases. They also represent rare common events in an exceptionally broad landscape of genetic, environmental, neuropathologic, and clinical heterogeneity present in patients. Here, we aim to recount the emerging trends in amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) spectrum disorder. Our review will predominantly focus on neuroinflammation and systemic immune imbalance in ALS and FTD, which have recently been highlighted as novel therapeutic targets. A common mechanism of most ALS and ~50% of FTD patients is dysregulation of TAR DNA-binding protein 43 (TDP-43), an RNA/DNA-binding protein, which becomes depleted from the nucleus and forms cytoplasmic aggregates in neurons and glia. This, in turn, via both gain and loss of function events, alters a variety of TDP-43-mediated cellular events. Experimental attempts to target TDP-43 aggregates or manipulate crosstalk in the context of inflammation will be discussed. Targeting inflammation, and the immune system in general, is of particular interest because of the high plasticity of immune cells compared to neurons.

Early neurotransmitters changes in prodromal frontotemporal dementia: A GENFI study.

BACKGROUND: Neurotransmitters deficits in Frontotemporal Dementia (FTD) are still poorly understood. Better knowledge of neurotransmitters impairment, especially in prodromal disease stages, might tailor symptomatic treatment approaches. METHODS: In the present study, we applied JuSpace toolbox, which allowed for cross-modal correlation of Magnetic Resonance Imaging (MRI)-based measures with nuclear imaging derived estimates covering various neurotransmitter systems including dopaminergic, serotonergic, noradrenergic, GABAergic and glutamatergic neurotransmission. We included 392 mutation carriers (157 GRN, 164 C9orf72, 71 MAPT), together with 276 non-carrier cognitively healthy controls (HC). We tested if the spatial patterns of grey matter volume (GMV) alterations in mutation carriers (relative to HC) are correlated with specific neurotransmitter systems in prodromal (CDR® plus NACC FTLD = 0.5) and in symptomatic (CDR® plus NACC FTLD≥1) FTD. RESULTS: In prodromal stages of C9orf72 disease, voxel-based brain changes were significantly associated with spatial distribution of dopamine and acetylcholine pathways; in prodromal MAPT disease with dopamine and serotonin pathways, while in prodromal GRN disease no significant findings were reported (p < 0.05, Family Wise Error corrected). In symptomatic FTD, a widespread involvement of dopamine, serotonin, glutamate and acetylcholine pathways across all genetic subtypes was found. Social cognition scores, loss of empathy and poor response to emotional cues were found to correlate with the strength of GMV colocalization of dopamine and serotonin pathways (all p < 0.01). CONCLUSIONS: This study, indirectly assessing neurotransmitter deficits in monogenic FTD, provides novel insight into disease mechanisms and might suggest potential therapeutic targets to counteract disease-related symptoms.

TDP-43 Proteinopathy Specific Biomarker Development.

TDP-43 is the primary or secondary pathological hallmark of neurodegenerative diseases, such as amyotrophic lateral sclerosis, half of frontotemporal dementia cases, and limbic age-related TDP-43 encephalopathy, which clinically resembles Alzheimer's dementia. In such diseases, a biomarker that can detect TDP-43 proteinopathy in life would help to stratify patients according to their definite diagnosis of pathology, rather than in clinical subgroups of uncertain pathology. For therapies developed to target pathological proteins that cause the disease a biomarker to detect and track the underlying pathology would greatly enhance such undertakings. This article reviews the latest developments and outlooks of deriving TDP-43-specific biomarkers from the pathophysiological processes involved in the development of TDP-43 proteinopathy and studies using biosamples from clinical entities associated with TDP-43 pathology to investigate biomarker candidates.

Reviewing the Potential Links between Viral Infections and TDP-43 Proteinopathies.

Transactive response DNA binding protein 43 kDa (TDP-43) was discovered in 2001 as a cellular factor capable to inhibit HIV-1 gene expression. Successively, it was brought to new life as the most prevalent RNA-binding protein involved in several neurological disorders, such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite the fact that these two research areas could be considered very distant from each other, in recent years an increasing number of publications pointed out the existence of a potentially important connection. Indeed, the ability of TDP-43 to act as an important regulator of all aspects of RNA metabolism makes this protein also a critical factor during expression of viral RNAs. Here, we summarize all recent observations regarding the involvement of TDP-43 in viral entry, replication and latency in several viruses that include enteroviruses (EVs), Theiler's murine encephalomyelitis virus (TMEV), human immunodeficiency virus (HIV), human endogenous retroviruses (HERVs), hepatitis B virus (HBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), West Nile virus (WNV), and herpes simplex virus-2 (HSV). In particular, in this work, we aimed to highlight the presence of similarities with the most commonly studied TDP-43 related neuronal dysfunctions.

NOS1AP is a novel molecular target and critical factor in TDP-43 pathology.

Many lines of evidence have highlighted the role played by heterogeneous nuclear ribonucleoproteins in amyotrophic lateral sclerosis. In this study, we have aimed to identify transcripts co-regulated by TAR DNA-binding protein 43 kDa and highly conserved heterogeneous nuclear ribonucleoproteins which have been previously shown to regulate TAR DNA-binding protein 43 kDa toxicity (deleted in azoospermia-associated protein 1, heterogeneous nuclear ribonucleoprotein -Q, -D, -K and -U). Using the transcriptome analyses, we have uncovered that Nitric Oxide Synthase 1 Adaptor Protein mRNA is a direct TAR DNA-binding protein 43 kDa target, and in flies, its modulation alone can rescue TAR DNA-binding protein 43 kDa pathology. In primary mouse cortical neurons, we show that TAR DNA-binding protein 43 kDa mediated downregulation of Nitric Oxide Synthase 1 Adaptor Protein expression strongly affects the NMDA-receptor signalling pathway. In human patients, the downregulation of Nitric Oxide Synthase 1 Adaptor Protein mRNA strongly correlates with TAR DNA-binding protein 43 kDa proteinopathy as measured by cryptic Stathmin-2 and Unc-13 homolog A cryptic exon inclusion. Overall, our results demonstrate that Nitric Oxide Synthase 1 Adaptor Protein may represent a novel disease-relevant gene, potentially suitable for the development of new therapeutic strategies.

Distinguishing post-translational modifications in dominantly inherited frontotemporal dementias: FTLD-TDP Type A (GRN) vs Type B (C9orf72).

AIMS: Frontotemporal dementias are neuropathologically characterised by frontotemporal lobar degeneration (FTLD). Intraneuronal inclusions of transactive response DNA-binding protein 43 kDa (TDP-43) are the defining pathological hallmark of approximately half of the FTLD cases, being referred to as FTLD-TDP. The classification of FTLD-TDP into five subtypes (Type A to Type E) is based on pathologic phenotypes; however, the molecular determinants underpinning the phenotypic heterogeneity of FTLD-TDP are not well known. It is currently undetermined whether TDP-43 post-translational modifications (PTMs) may be related to the phenotypic diversity of the FTLDs. Thus, the investigation of FTLD-TDP Type A and Type B, associated with GRN and C9orf72 mutations, becomes essential. METHODS: Immunohistochemistry was used to identify and map the intraneuronal inclusions. Sarkosyl-insoluble TDP-43 was extracted from brains of GRN and C9orf72 mutation carriers post-mortem and studied by Western blot analysis, immuno-electron microscopy and mass spectrometry. RESULTS: Filaments of TDP-43 were present in all FTLD-TDP preparations. PTM profiling identified multiple phosphorylated, N-terminal acetylated or otherwise modified residues, several of which have been identified for the first time as related to sarkosyl-insoluble TDP-43. Several PTMs were specific for either Type A or Type B, while others were identified in both types. CONCLUSIONS: The current results provide evidence that the intraneuronal inclusions in the two genetic diseases contain TDP-43 filaments. The discovery of novel, potentially type-specific TDP-43 PTMs emphasises the need to determine the mechanisms leading to filament formation and PTMs, and the necessity of exploring the validity and occupancy of PTMs in a prognostic/diagnostic setting.

Unraveling the toxic effects mediated by the neurodegenerative disease-associated S375G mutation of TDP-43 and its S375E phosphomimetic variant.

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein found in the nucleus that accumulates in the cytoplasm under pathological conditions, leading to proteinopathies, such as frontotemporal dementia and ALS. An emerging area of TDP-43 research is represented by the study of its post-translational modifications, the way they are connected to disease-associated mutations, and what this means for pathological processes. Recently, we described a novel mutation in TDP-43 in an early onset ALS case that was affecting a potential phosphorylation site in position 375 (S375G). A preliminary characterization showed that both the S375G mutation and its phosphomimetic variant, S375E, displayed altered nuclear-cytoplasmic distribution and cellular toxicity. To better investigate these effects, here we established cell lines expressing inducible WT, S375G, and S375E TDP-43 variants. Interestingly, we found that these mutants do not seem to affect well-studied aspects of TDP-43, such as RNA splicing or autoregulation, or protein conformation, dynamics, or aggregation, although they do display dysmorphic nuclear shape and cell cycle alterations. In addition, RNA-Seq analysis of these cell lines showed that although the disease-associated S375G mutation and its phosphomimetic S375E variant regulate distinct sets of genes, they have a common target in mitochondrial apoptotic genes. Taken together, our data strongly support the growing evidence that alterations in TDP-43 post-translational modifications can play a potentially important role in disease pathogenesis and provide a further link between TDP-43 pathology and mitochondrial health.

Optineurin Deficiency and Insufficiency Lead to Higher Microglial TDP-43 Protein Levels.

Mutations in optineurin, a ubiquitin-binding adaptor protein, cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease of motor neurons linked to chronic inflammation and protein aggregation. The majority of ALS patients, including those carrying the optineurin mutations, exhibit cytoplasmic mislocalization, ubiquitination, and aggregation of nuclear TAR DNA-binding protein 43 kDa (TDP-43). To address the crosstalk between optineurin and TDP-43, we generated optineurin knockout (KO) neuronal and microglial cell lines using the CRISPR/Cas9 approach. Interestingly, we observed that loss of optineurin resulted in elevated TDP-43 protein expression in microglial BV2 but not neuronal Neuro 2a and NSC-34 cell lines. No changes were observed at the mRNA level, suggesting that this increase was post-translationally regulated. To confirm this observation in primary cells, we then used microglia and macrophages from an optineurin loss-of-function mouse model that lacks the C-terminal ubiquitin-binding region (Optn470T), mimicking optineurin truncations in ALS patients. As observed in the BV2 cells, we also found elevated basal levels of TDP-43 protein in Optn470T microglia and bone marrow-derived macrophages. To test if inflammation could further enhance TDP-43 accumulation in cells lacking functional optineurin, we stimulated them with lipopolysaccharide (LPS), and we observed a significant increase in TDP-43 expression following LPS treatment of WT cells. However, this was absent in both BV2 Optn KO and primary Optn470T microglia, which exhibited the same elevated TDP-43 levels as in basal conditions. Furthermore, we did not observe nuclear TDP-43 depletion or cytoplasmic aggregate formation in either Optn470T microglia or LPS-treated WT or Optn470T microglia. Taken together, our results show that optineurin deficiency and insufficiency post-translationally upregulate microglial TDP-43 protein levels and that elevated TDP-43 levels in cells lacking functional optineurin could not be further increased by an inflammatory stimulus, suggesting the presence of a plateau.

Cell environment shapes TDP-43 function with implications in neuronal and muscle disease.

TDP-43 (TAR DNA-binding protein 43) aggregation and redistribution are recognised as a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. As TDP-43 inclusions have recently been described in the muscle of inclusion body myositis patients, this highlights the need to understand the role of TDP-43 beyond the central nervous system. Using RNA-seq, we directly compare TDP-43-mediated RNA processing in muscle (C2C12) and neuronal (NSC34) mouse cells. TDP-43 displays a cell-type-characteristic behaviour targeting unique transcripts in each cell-type, which is due to characteristic expression of RNA-binding proteins, that influence TDP-43's performance and define cell-type specific splicing. Among splicing events commonly dysregulated in both cell lines, we identify some that are TDP-43-dependent also in human cells. Inclusion levels of these alternative exons are altered in tissues of patients suffering from FTLD and IBM. We therefore propose that TDP-43 dysfunction contributes to disease development either in a common or a tissue-specific manner.

Sirtuin-1 sensitive lysine-136 acetylation drives phase separation and pathological aggregation of TDP-43.

Trans-activation response DNA-binding protein of 43 kDa (TDP-43) regulates RNA processing and forms neuropathological aggregates in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Investigating TDP-43 post-translational modifications, we discovered that K84 acetylation reduced nuclear import whereas K136 acetylation impaired RNA binding and splicing capabilities of TDP-43. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. Introduction of acetyl-lysine at the identified sites via amber suppression confirmed the results from site-directed mutagenesis. K84-acetylated TDP-43 showed cytoplasmic mislocalization, and the aggregation propensity of K136-acetylated TDP-43 was confirmed. We generated antibodies selective for TDP-43 acetylated at these lysines, and found that sirtuin-1 can potently deacetylate K136-acetylated TDP-43 and reduce its aggregation propensity. Thus, distinct lysine acetylations modulate nuclear import, RNA binding and phase separation of TDP-43, suggesting regulatory mechanisms for TDP-43 pathogenesis.