Deciphering Distinct Genetic Risk Factors for FTLD-TDP Pathological Subtypes via Whole-Genome Sequencing.

Frontotemporal lobar degeneration with neuronal inclusions of the TAR DNA-binding protein 43 (FTLD-TDP) is a fatal neurodegenerative disorder with only a limited number of risk loci identified. We report our comprehensive genome-wide association study as part of the International FTLD-TDP Whole-Genome Sequencing Consortium, including 985 cases and 3,153 controls, and meta-analysis with the Dementia-seq cohort, compiled from 26 institutions/brain banks in the United States, Europe and Australia. We confirm UNC13A as the strongest overall FTLD-TDP risk factor and identify TNIP1 as a novel FTLD-TDP risk factor. In subgroup analyses, we further identify for the first time genome-wide significant loci specific to each of the three main FTLD-TDP pathological subtypes (A, B and C), as well as enrichment of risk loci in distinct tissues, brain regions, and neuronal subtypes, suggesting distinct disease aetiologies in each of the subtypes. Rare variant analysis confirmed TBK1 and identified VIPR1 , RBPJL , and L3MBTL1 as novel subtype specific FTLD-TDP risk genes, further highlighting the role of innate and adaptive immunity and notch signalling pathway in FTLD-TDP, with potential diagnostic and novel therapeutic implications.

MAPT H2 haplotype and risk of Pick's disease in the Pick's disease International Consortium: a genetic association study.

BACKGROUND: Pick's disease is a rare and predominantly sporadic form of frontotemporal dementia that is classified as a primary tauopathy. Pick's disease is pathologically defined by the presence in the frontal and temporal lobes of Pick bodies, composed of hyperphosphorylated, three-repeat tau protein, encoded by the MAPT gene. MAPT has two distinct haplotypes, H1 and H2; the MAPT H1 haplotype is the major genetic risk factor for four-repeat tauopathies (eg, progressive supranuclear palsy and corticobasal degeneration), and the MAPT H2 haplotype is protective for these disorders. The primary aim of this study was to evaluate the association of MAPT H2 with Pick's disease risk, age at onset, and disease duration. METHODS: In this genetic association study, we used data from the Pick's disease International Consortium, which we established to enable collection of data from individuals with pathologically confirmed Pick's disease worldwide. For this analysis, we collected brain samples from individuals with pathologically confirmed Pick's disease from 35 sites (brainbanks and hospitals) in North America, Europe, and Australia between Jan 1, 2020, and Jan 31, 2023. Neurologically healthy controls were recruited from the Mayo Clinic (FL, USA, or MN, USA between March 1, 1998, and Sept 1, 2019). For the primary analysis, individuals were directly genotyped for the MAPT H1-H2 haplotype-defining variant rs8070723. In a secondary analysis, we genotyped and constructed the six-variant-defined (rs1467967-rs242557-rs3785883-rs2471738-rs8070723-rs7521) MAPT H1 subhaplotypes. Associations of MAPT variants and MAPT haplotypes with Pick's disease risk, age at onset, and disease duration were examined using logistic and linear regression models; odds ratios (ORs) and ß coefficients were estimated and correspond to each additional minor allele or each additional copy of the given haplotype. FINDINGS: We obtained brain samples from 338 people with pathologically confirmed Pick's disease (205 [61%] male and 133 [39%] female; 338 [100%] White) and 1312 neurologically healthy controls (611 [47%] male and 701 [53%] female; 1312 [100%] White). The MAPT H2 haplotype was associated with increased risk of Pick's disease compared with the H1 haplotype (OR 1·35 [95% CI 1·12 to 1·64], p=0·0021). MAPT H2 was not associated with age at onset (ß -0·54 [95% CI -1·94 to 0·87], p=0·45) or disease duration (ß 0·05 [-0·06 to 0·16], p=0·35). Although not significant after correcting for multiple testing, associations were observed at p less than 0·05: with risk of Pick's disease for the H1f subhaplotype (OR 0·11 [0·01 to 0·99], p=0·049); with age at onset for H1b (ß 2·66 [0·63 to 4·70], p=0·011), H1i (ß -3·66 [-6·83 to -0·48], p=0·025), and H1u (ß -5·25 [-10·42 to -0·07], p=0·048); and with disease duration for H1x (ß -0·57 [-1·07 to -0·07], p=0·026). INTERPRETATION: The Pick's disease International Consortium provides an opportunity to do large studies to enhance our understanding of the pathobiology of Pick's disease. This study shows that, in contrast to the decreased risk of four-repeat tauopathies, the MAPT H2 haplotype is associated with an increased risk of Pick's disease in people of European ancestry. This finding could inform development of isoform-related therapeutics for tauopathies. FUNDING: Wellcome Trust, Rotha Abraham Trust, Brain Research UK, the Dolby Fund, Dementia Research Institute (Medical Research Council), US National Institutes of Health, and the Mayo Clinic Foundation.

A multi-omics dataset for the analysis of frontotemporal dementia genetic subtypes.

Understanding the molecular mechanisms underlying frontotemporal dementia (FTD) is essential for the development of successful therapies. Systematic studies on human post-mortem brain tissue of patients with genetic subtypes of FTD are currently lacking. The Risk and Modyfing Factors of Frontotemporal Dementia (RiMod-FTD) consortium therefore has generated a multi-omics dataset for genetic subtypes of FTD to identify common and distinct molecular mechanisms disturbed in disease. Here, we present multi-omics datasets generated from the frontal lobe of post-mortem human brain tissue from patients with mutations in MAPT, GRN and C9orf72 and healthy controls. This data resource consists of four datasets generated with different technologies to capture the transcriptome by RNA-seq, small RNA-seq, CAGE-seq, and methylation profiling. We show concrete examples on how to use the resulting data and confirm current knowledge about FTD and identify new processes for further investigation. This extensive multi-omics dataset holds great value to reveal new research avenues for this devastating disease.

Phenylalanine-tRNA aminoacylation is compromised by ALS/FTD-associated C9orf72 C4G2 repeat RNA.

The expanded hexanucleotide GGGGCC repeat mutation in the C9orf72 gene is the main genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Under one disease mechanism, sense and antisense transcripts of the repeat are predicted to bind various RNA-binding proteins, compromise their function and cause cytotoxicity. Here we identify phenylalanine-tRNA synthetase (FARS) subunit alpha (FARSA) as the main interactor of the CCCCGG antisense repeat RNA in cytosol. The aminoacylation of tRNAPhe by FARS is inhibited by antisense RNA, leading to decreased levels of charged tRNAPhe. Remarkably, this is associated with global reduction of phenylalanine incorporation in the proteome and decrease in expression of phenylalanine-rich proteins in cellular models and patient tissues. In conclusion, this study reveals functional inhibition of FARSA in the presence of antisense RNA repeats. Compromised aminoacylation of tRNA could lead to impairments in protein synthesis and further contribute to C9orf72 mutation-associated pathology.

Psychotic symptoms in frontotemporal dementia with TDP-43 tend to be associated with type B pathology.

AIMS: Psychotic symptoms are increasingly recognized as a distinguishing clinical feature in patients with dementia due to frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP). Within this group, carriers of the C9orf72 repeat expansion are particularly prone to develop delusions and hallucinations. METHODS: The present retrospective study sought to provide novel details about the relationship between FTLD-TDP pathology and the presence of psychotic symptoms during life. RESULTS: We found that FTLD-TDP subtype B was more frequent in patients with psychotic symptoms than in those without. This relationship was present even when corrected for the presence of C9orf72 mutation, suggesting that pathophysiological processes leading to the development of subtype B pathology may increase the risk of psychotic symptoms. Within the group of FTLD-TDP cases with subtype B pathology, psychotic symptoms tended to be associated with a greater burden of TDP-43 pathology in the white matter and a lower burden in lower motor neurons. When present, pathological involvement of motor neurons was more likely to be asymptomatic in patients with psychosis. CONCLUSIONS: This work suggests that psychotic symptoms in patients with FTLD-TDP tend to be associated with subtype B pathology. This relationship is not completely explained by the effects of the C9orf72 mutation and raises the possibility of a direct link between psychotic symptoms and this particular pattern of TDP-43 pathology.

AAV-mediated expression of a new conformational anti-aggregated α-synuclein antibody prolongs survival in a genetic model of α-synucleinopathies.

Prion-like transmission of pathology in α-synucleinopathies like Parkinson's disease or multiple system atrophy is increasingly recognized as one potential mechanism to address disease progression. Active and passive immunotherapies targeting insoluble, aggregated α-synuclein are already being actively explored in the clinic with mixed outcomes so far. Here, we report the identification of 306C7B3, a highly selective, aggregate-specific α-synuclein antibody with picomolar affinity devoid of binding to the monomeric, physiologic protein. 306C7B3 binding is Ser129-phosphorylation independent and shows high affinity to several different aggregated α-synuclein polymorphs, increasing the likelihood that it can also bind to the pathological seeds assumed to drive disease progression in patients. In support of this, highly selective binding to pathological aggregates in postmortem brains of MSA patients was demonstrated, with no staining in samples from other human neurodegenerative diseases. To achieve CNS exposure of 306C7B3, an adeno-associated virus (AAV) based approach driving expression of the secreted antibody within the brain of (Thy-1)-[A30P]-hα-synuclein mice was used. Widespread central transduction after intrastriatal inoculation was ensured by using the AAV2HBKO serotype, with transduction being spread to areas far away from the inoculation site. Treatment of (Thy-1)-[A30P]-hα-synuclein mice at the age of 12 months demonstrated significantly increased survival, with 306C7B3 concentration reaching 3.9 nM in the cerebrospinal fluid. These results suggest that AAV-mediated expression of 306C7B3, targeting extracellular, presumably disease-propagating aggregates of α-synuclein, has great potential as a disease-modifying therapy for α-synucleinopathies as it ensures CNS exposure of the antibody, thereby mitigating the selective permeability of the blood-brain barrier.

GAA-FGF14 ataxia (SCA27B): phenotypic profile, natural history progression and 4-aminopyridine treatment response.

Ataxia due to an autosomal dominant intronic GAA repeat expansion in FGF14 [GAA-FGF14 ataxia, spinocerebellar ataxia 27B (SCA27B)] has recently been identified as one of the most common genetic late-onset ataxias. We here aimed to characterize its phenotypic profile, natural history progression, and 4-aminopyridine (4-AP) treatment response. We conducted a multi-modal cohort study of 50 GAA-FGF14 patients, comprising in-depth phenotyping, cross-sectional and longitudinal progression data (up to 7 years), MRI findings, serum neurofilament light (sNfL) levels, neuropathology, and 4-AP treatment response data, including a series of n-of-1 treatment studies. GAA-FGF14 ataxia consistently presented as late-onset [60.0 years (53.5-68.5), median (interquartile range)] pancerebellar syndrome, partly combined with afferent sensory deficits (55%) and dysautonomia (28%). Dysautonomia increased with duration while cognitive impairment remained infrequent, even in advanced stages. Cross-sectional and longitudinal assessments consistently indicated mild progression of ataxia [0.29 Scale for the Assessment and Rating of Ataxia (SARA) points/year], not exceeding a moderate disease severity even in advanced stages (maximum SARA score: 18 points). Functional impairment increased relatively slowly (unilateral mobility aids after 8 years in 50% of patients). Corresponding to slow progression and low extra-cerebellar involvement, sNfL was not increased relative to controls. Concurrent second diseases (including progressive supranuclear palsy neuropathology) represented major individual aggravators of disease severity, constituting important caveats for planning future GAA-FGF14 trials. A treatment response to 4-AP with relevance for everyday living was reported by 86% of treated patients. A series of three prospective n-of-1 treatment experiences with on/off design showed marked reduction in daily symptomatic time and symptom severity on 4-AP. Our study characterizes the phenotypic profile, natural history progression, and 4-AP treatment response of GAA-FGF14 ataxia. It paves the way towards large-scale natural history studies and 4-AP treatment trials in this newly discovered, possibly most frequent, and treatable late-onset ataxia.

C9ORF72 knockdown triggers FTD-like symptoms and cell pathology in mice.

The GGGGCC intronic repeat expansion within C9ORF72 is the most common genetic cause of ALS and FTD. This mutation results in toxic gain of function through accumulation of expanded RNA foci and aggregation of abnormally translated dipeptide repeat proteins, as well as loss of function due to impaired transcription of C9ORF72. A number of in vivo and in vitro models of gain and loss of function effects have suggested that both mechanisms synergize to cause the disease. However, the contribution of the loss of function mechanism remains poorly understood. We have generated C9ORF72 knockdown mice to mimic C9-FTD/ALS patients haploinsufficiency and investigate the role of this loss of function in the pathogenesis. We found that decreasing C9ORF72 leads to anomalies of the autophagy/lysosomal pathway, cytoplasmic accumulation of TDP-43 and decreased synaptic density in the cortex. Knockdown mice also developed FTD-like behavioral deficits and mild motor phenotypes at a later stage. These findings show that C9ORF72 partial loss of function contributes to the damaging events leading to C9-FTD/ALS.

Update on quality assurance in neuropathology: Summary of the round robin trials on TERT promoter mutation, H3-3A mutation, 1p/19q codeletion, and KIAA1549::BRAF fusion testing in Germany in 2020 and 2021.

We previously reported on the first neuropathological round robin trials operated together with Quality in Pathology (QuIP) GmbH in 2018 and 2019 in Germany, i.e., the trials on IDH mutational testing and MGMT promoter methylation analysis [1]. For 2020 and 2021, the spectrum of round robin trials has been expanded to cover the most commonly used assays in neuropathological institutions. In addition to IDH mutation and MGMT promoter methylation testing, there is a long tradition for 1p/19q codeletion testing relevant in the context of the diagnosis of oligodendroglioma. With the 5th edition of the World Health Organization (WHO) classification of the central nervous system tumors, additional molecular markers came into focus: TERT promoter mutation is often assessed as a molecular diagnostic criterion for IDH-wildtype glioblastoma. Moreover, several molecular diagnostic markers have been introduced for pediatric brain tumors. Here, trials on KIAA1549::BRAF fusions (common in pilocytic astrocytomas) and H3-3A mutations (in diffuse midline gliomas, H3-K27-altered and diffuse hemispheric gliomas, H3-G34-mutant) were most desired by the neuropathological community. In this update, we report on these novel round robin trials. In summary, success rates in all four trials ranged from 75 to 96%, arguing for an overall high quality level in the field of molecular neuropathological diagnostics.

Clinical outcome of biomarker-guided therapies in adult patients with tumors of the nervous system.

Background: The clinical utility of molecular profiling and targeted therapies for neuro-oncology patients outside of clinical trials is not established. We aimed at investigating feasibility and clinical utility of molecular profiling and targeted therapy in adult patients with advanced tumors in the nervous system within a prospective observational study. Methods: molecular tumor board (MTB)@ZPM (NCT03503149) is a prospective observational precision medicine study for patients with advanced tumors. After inclusion of patients, we performed comprehensive molecular profiling, formulated ranked biomarker-guided therapy recommendations based on consensus by the MTB, and collected prospective clinical outcome data. Results: Here, we present initial data of 661 adult patients with tumors of the nervous system enrolled by December 31, 2021. Of these, 408 patients were presented at the MTB. Molecular-instructed therapy recommendations could be made in 380/408 (93.1%) cases and were prioritized by evidence levels. Therapies were initiated in 86/380 (22.6%) cases until data cutoff. We observed a progression-free survival ratio >1.3 in 31.3% of patients. Conclusions: Our study supports the clinical utility of biomarker-guided therapies for neuro-oncology patients and indicates clinical benefit in a subset of patients. Our data might inform future clinical trials, translational studies, and even clinical care.

Immunotherapy targeting the C-terminal domain of TDP-43 decreases neuropathology and confers neuroprotection in mouse models of ALS/FTD.

Effective therapies are urgently needed to safely target TDP-43 pathology as it is closely associated with the onset and development of devastating diseases such as frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). In addition, TDP-43 pathology is present as a co-pathology in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Our approach is to develop a TDP-43-specific immunotherapy that exploits Fc gamma-mediated removal mechanisms to limit neuronal damage while maintaining physiological TDP-43 function. Thus, using both in vitro mechanistic studies in conjunction with the rNLS8 and CamKIIa inoculation mouse models of TDP-43 proteinopathy, we identified the key targeting domain in TDP-43 to accomplish these therapeutic objectives. Targeting the C-terminal domain of TDP-43 but not the RNA recognition motifs (RRM) reduces TDP-43 pathology and avoids neuronal loss in vivo. We demonstrate that this rescue is dependent on Fc receptor-mediated immune complex uptake by microglia. Furthermore, monoclonal antibody (mAb) treatment enhances phagocytic capacity of ALS patient-derived microglia, providing a mechanism to restore the compromised phagocytic function in ALS and FTD patients. Importantly, these beneficial effects are achieved while preserving physiological TDP-43 activity. Our findings demonstrate that a mAb targeting the C-terminal domain of TDP-43 limits pathology and neurotoxicity, enabling clearance of misfolded TDP-43 through microglia engagement, and supporting the clinical strategy to target TDP-43 by immunotherapy. SIGNIFICANCE STATEMENT: TDP-43 pathology is associated with various devastating neurodegenerative disorders with high unmet medical needs such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Thus, safely and effectively targeting pathological TDP-43 represents a key paradigm for biotechnical research as currently there is little in clinical development. After years of research, we have determined that targeting the C-terminal domain of TDP-43 rescues multiple patho-mechanisms involved in disease progression in two animal models of FTD/ALS. In parallel, importantly, our studies establish that this approach does not alter the physiological functions of this ubiquitously expressed and indispensable protein. Together, our findings substantially contribute to the understanding of TDP-43 pathobiology and support the prioritization for clinical testing of immunotherapy approaches targeting TDP-43.

Accumulation of TMEM106B C-terminal fragments in neurodegenerative disease and aging.

Several studies using cryogenic electron microscopy (cryo-EM) techniques recently reported the isolation and characterization of novel protein filaments, composed of a C-terminal fragment (CTF) of the endolysosomal transmembrane protein 106B (TMEM106B), from human post-mortem brain tissue with various neurodegenerative conditions and normal aging. Genetic variation in TMEM106B is known to influence the risk and presentation of several neurodegenerative diseases, especially frontotemporal dementia (FTD) caused by mutations in the progranulin gene (GRN). To further elucidate the significance of TMEM106B CTF, we performed immunohistochemistry with antibodies directed against epitopes within the filament-forming C-terminal region of TMEM106B. Accumulation of TMEM106B C-terminal immunoreactive (TMEM-ir) material was a common finding in all the conditions evaluated, including frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP), Alzheimer's disease, tauopathies, synucleinopathies and neurologically normal aging. TMEM-ir material was present in a wide range of brain cell types and in a broad neuroanatomical distribution; however, there was no co-localization of TMEM-ir material with other neurodegenerative proteins in cellular inclusions. In most conditions, the presence and abundance of TMEM-ir aggregates correlated strongly with patient age and showed only a weak correlation with the TMEM106B haplotype or the primary pathological diagnosis. However, all patients with FTD caused by GRN mutations were found to have high levels of TMEM-ir material, including several who were relatively young (< 60 years). These findings suggest that the accumulation of TMEM106B CTF is a common age-related phenomenon, which may reflect lysosomal dysfunction. Although its significance in most neurodegenerative conditions remains uncertain, the consistent finding of extensive TMEM-ir material in cases of FTLD-TDP with GRN mutations further supports a pathomechanistic role of TMEM106B and lysosomal dysfunction in this specific disease population.

LATE-NC staging in routine neuropathologic diagnosis: an update.

An international consensus report in 2019 recommended a classification system for limbic-predominant age-related TDP-43 encephalopathy neuropathologic changes (LATE-NC). The suggested neuropathologic staging system and nomenclature have proven useful for autopsy practice and dementia research. However, some issues remain unresolved, such as cases with unusual features that do not fit with current diagnostic categories. The goal of this report is to update the neuropathologic criteria for the diagnosis and staging of LATE-NC, based primarily on published data. We provide practical suggestions about how to integrate available genetic information and comorbid pathologies [e.g., Alzheimer's disease neuropathologic changes (ADNC) and Lewy body disease]. We also describe recent research findings that have enabled more precise guidance on how to differentiate LATE-NC from other subtypes of TDP-43 pathology [e.g., frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS)], and how to render diagnoses in unusual situations in which TDP-43 pathology does not follow the staging scheme proposed in 2019. Specific recommendations are also made on when not to apply this diagnostic term based on current knowledge. Neuroanatomical regions of interest in LATE-NC are described in detail and the implications for TDP-43 immunohistochemical results are specified more precisely. We also highlight questions that remain unresolved and areas needing additional study. In summary, the current work lays out a number of recommendations to improve the precision of LATE-NC staging based on published reports and diagnostic experience.

Germline findings in patients with advanced malignancies screened with paired blood-tumour testing for personalised treatment approaches.

BACKGROUND: Sequencing of tumour tissue with comprehensive gene panels is increasingly used to guide treatment in precision oncology. Analysis of tumour-normal pairs allows in contrast to tumour-only assessment direct discrimination between somatic and germline alterations, which might have important implications not only for the patients but also their families. METHODS: We performed tumour normal sequencing with a large gene panel in 1048 patients with advanced cancer to support treatment decision. Sequencing results were correlated with clinical and family data. RESULTS: We identified 156 likely pathogenic or pathogenic (LP/P) germline variants in cancer predisposition genes (CPGs) in 144 cases (13.7%). Of all patients, 8.8% had a LP/P variant in autosomal-dominant cancer predisposition genes (AD-CPGs), most of them being genes with high or moderate penetrance (ATM, BRCA2, CHEK2 and BRCA1). In 48 cases, the P/LP variant matched the expected tumour spectrum. A second variant in tumour tissue was found in 31 patients with AD-CPG variants. Low frequency mutations in either TP53, ATM or DNMT3A in the normal sample indicated clonal haematopoiesis in five cases. CONCLUSIONS: Tumour-normal testing for personalised treatment identifies germline LP/P variants in a relevant proportion of patients with cancer. The majority of them would not have been referred to genetic counselling based on family history. Indirect functional readouts of tumour-normal sequencing can provide novel links between CPGs and unexpected cancers. The interpretation of increasingly complex datasets in precision oncology is challenging and concepts of interdisciplinary personalised cancer prevention are needed to support patients and their families.

Common Variants Near ZIC1 and ZIC4 in Autopsy-Confirmed Multiple System Atrophy.

BACKGROUND: Multiple System Atrophy is a rare neurodegenerative disease with alpha-synuclein aggregation in glial cytoplasmic inclusions and either predominant olivopontocerebellar atrophy or striatonigral degeneration, leading to dysautonomia, parkinsonism, and cerebellar ataxia. One prior genome-wide association study in mainly clinically diagnosed patients with Multiple System Atrophy failed to identify genetic variants predisposing for the disease. OBJECTIVE: Since the clinical diagnosis of Multiple System Atrophy yields a high rate of misdiagnosis when compared to the neuropathological gold standard, we studied only autopsy-confirmed cases. METHODS: We studied common genetic variations in Multiple System Atrophy cases (N = 731) and controls (N = 2898). RESULTS: The most strongly disease-associated markers were rs16859966 on chromosome 3, rs7013955 on chromosome 8, and rs116607983 on chromosome 4 with P-values below 5 × 10-6 , all of which were supported by at least one additional genotyped and several imputed single nucleotide polymorphisms. The genes closest to the chromosome 3 locus are ZIC1 and ZIC4 encoding the zinc finger proteins of cerebellum 1 and 4 (ZIC1 and ZIC4). INTERPRETATION: Since mutations of ZIC1 and ZIC4 and paraneoplastic autoantibodies directed against ZIC4 are associated with severe cerebellar dysfunction, we conducted immunohistochemical analyses in brain tissue of the frontal cortex and the cerebellum from 24 Multiple System Atrophy patients. Strong immunohistochemical expression of ZIC4 was detected in a subset of neurons of the dentate nucleus in all healthy controls and in patients with striatonigral degeneration, whereas ZIC4-immunoreactive neurons were significantly reduced inpatients with olivopontocerebellar atrophy. These findings point to a potential ZIC4-mediated vulnerability of neurons in Multiple System Atrophy. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

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.