In vivo diagnosis of TDP-43 proteinopathies: in search of biomarkers of clinical use.

TDP-43 proteinopathies are a heterogeneous group of neurodegenerative disorders that share the presence of aberrant, misfolded and mislocalized deposits of the protein TDP-43, as in the case of amyotrophic lateral sclerosis and some, but not all, pathological variants of frontotemporal dementia. In recent years, many other diseases have been reported to have primary or secondary TDP-43 proteinopathy, such as Alzheimer's disease, Huntington's disease or the recently described limbic-predominant age-related TDP-43 encephalopathy, highlighting the need for new and accurate methods for the early detection of TDP-43 proteinopathy to help on the stratification of patients with overlapping clinical diagnosis. Currently, TDP-43 proteinopathy remains a post-mortem pathologic diagnosis. Although the main aim is to determine the pathologic TDP-43 proteinopathy in the central nervous system (CNS), the ubiquitous expression of TDP-43 in biofluids and cells outside the CNS facilitates the use of other accessible target tissues that might reflect the potential TDP-43 alterations in the brain. In this review, we describe the main developments in the early detection of TDP-43 proteinopathies, and their potential implications on diagnosis and future treatments.

TDP-43 proteinopathy in ALS is triggered by loss of ASRGL1 and associated with HML-2 expression.

TAR DNA-binding protein 43 (TDP-43) proteinopathy in brain cells is the hallmark of amyotrophic lateral sclerosis (ALS) but its cause remains elusive. Asparaginase-like-1 protein (ASRGL1) cleaves isoaspartates, which alter protein folding and susceptibility to proteolysis. ASRGL1 gene harbors a copy of the human endogenous retrovirus HML-2, whose overexpression contributes to ALS pathogenesis. Here we show that ASRGL1 expression was diminished in ALS brain samples by RNA sequencing, immunohistochemistry, and western blotting. TDP-43 and ASRGL1 colocalized in neurons but, in the absence of ASRGL1, TDP-43 aggregated in the cytoplasm. TDP-43 was found to be prone to isoaspartate formation and a substrate for ASRGL1. ASRGL1 silencing triggered accumulation of misfolded, fragmented, phosphorylated and mislocalized TDP-43 in cultured neurons and motor cortex of female mice. Overexpression of ASRGL1 restored neuronal viability. Overexpression of HML-2 led to ASRGL1 silencing. Loss of ASRGL1 leading to TDP-43 aggregation may be a critical mechanism in ALS pathophysiology.

Anterior insula is more vulnerable than posterior insula to TDP-43 pathology in common dementias and ALS.

Based on the anatomic proximity, connectivity, and functional similarities between the anterior insula and amygdala, we tested the hypothesis that the anterior insula is an important focus in the progression of TDP-43 pathology in LATE-NC. Blinded to clinical and neuropathologic data, phospho-TDP (pTDP) inclusion pathology was assessed in paired anterior and posterior insula samples in 105 autopsied patients with Alzheimer disease, Lewy body disease, LATE-NC and hippocampal sclerosis (HS), amyotrophic lateral sclerosis (ALS), and other conditions. Insular pTDP pathology was present in 34.3% of the study cohort, most commonly as neuronal inclusions and/or short neurites in lamina II, and less commonly as subpial processes resembling those described in the amygdala region. Among positive samples, pTDP pathology was limited to the anterior insula (41.7%), or occurred in both anterior and posterior insula (58.3%); inclusion density was greater in anterior insula across all diseases (p < .001). pTDP pathology occurred in 46.7% of ALS samples, typically without a widespread TDP-43 proteinopathy. In LATE-NC, it was seen in 30.4% of samples (mostly LATE-NC stages 2 and 3), often co-occurring with basal forebrain pathology and comorbid HS, suggesting this is an important step in the evolution of this pathology beyond the medial temporal lobe.

Generation and characterization of monoclonal antibodies against pathologically phosphorylated TDP-43.

Inclusions containing TAR DNA binding protein 43 (TDP-43) are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). One of the disease-specific features of TDP-43 inclusions is the aberrant phosphorylation of TDP-43 at serines 409/410 (pS409/410). Here, we developed rabbit monoclonal antibodies (mAbs) that specifically detect pS409/410-TDP-43 in multiple model systems and FTD/ALS patient samples. Specifically, we identified three mAbs (26H10, 2E9 and 23A1) from spleen B cell clones that exhibit high specificity and sensitivity to pS409/410-TDP-43 peptides in an ELISA assay. Biochemical analyses revealed that pS409/410 of recombinant TDP-43 and of exogenous 25 kDa TDP-43 C-terminal fragments in cultured HEK293T cells are detected by all three mAbs. Moreover, the mAbs detect pS409/410-positive TDP-43 inclusions in the brains of FTD/ALS patients and mouse models of TDP-43 proteinopathy by immunohistochemistry. Our findings indicate that these mAbs are a valuable resource for investigating TDP-43 pathology both in vitro and in vivo.

14-3-3θ, a novel player in TDP-43 pathophysiology: Implications for ALS/FTD.

In this issue of Neuron, Ke et al.1 report a novel non-canonical interaction between 14-3-3θ and TDP-43 that impacts loss-of-function and gain-of-toxic pathology in TDP-43 proteinopathies. The authors further provide proof of principle for a 14-3-3θ-targeted gene therapy to reduce TDP-43-induced deficits in transgenic TDP-43 mutant mice.

Limbic-predominant age-related TDP-43 encephalopathy (LATE-NC): Co-pathologies and genetic risk factors provide clues about pathogenesis.

Limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC) is detectable at autopsy in more than one-third of people beyond age 85 years and is robustly associated with dementia independent of other pathologies. Although LATE-NC has a large impact on public health, there remain uncertainties about the underlying biologic mechanisms. Here, we review the literature from human studies that may shed light on pathogenetic mechanisms. It is increasingly clear that certain combinations of pathologic changes tend to coexist in aging brains. Although "pure" LATE-NC is not rare, LATE-NC often coexists in the same brains with Alzheimer disease neuropathologic change, brain arteriolosclerosis, hippocampal sclerosis of aging, and/or age-related tau astrogliopathy (ARTAG). The patterns of pathologic comorbidities provide circumstantial evidence of mechanistic interactions ("synergies") between the pathologies, and also suggest common upstream influences. As to primary mediators of vulnerability to neuropathologic changes, genetics may play key roles. Genes associated with LATE-NC include TMEM106B, GRN, APOE, SORL1, ABCC9, and others. Although the anatomic distribution of TDP-43 pathology defines the condition, important cofactors for LATE-NC may include Tau pathology, endolysosomal pathways, and blood-brain barrier dysfunction. A review of the human phenomenology offers insights into disease-driving mechanisms, and may provide clues for diagnostic and therapeutic targets.

Loss and microglia phagocytosis of synaptic proteins in frontotemporal lobar degeneration with TDP-43 proteinopathy.

Cortical synaptic loss has emerged as an early abnormality in Alzheimer's disease (AD) with a strong relationship to cognitive performance. However, the status of synapses in frontotemporal lobar degeneration (FTLD) has received meager experimental attention. The purpose of this study was to investigate changes in cortical synaptic proteins in FTLD with tar DNA binding protein-43 (TDP-43) proteinopathy. A second aim was to study phagocytosis of synaptic proteins by microglia as a surrogate for synaptic pruning. Western blot analysis in frozen tissue from the middle frontal gyrus revealed decreased levels of the presynaptic protein synaptophysin, but slightly increased levels of the postsynaptic density protein 95 (PSD95) in FTLD-TDP. Levels of the dendritic spine protein spinophilin displayed the largest decrease. Double immunofluorescent staining visualized aggregate or punctate synaptic protein immunoreactivity in microglia. Overall, the proportion of microglia containing synaptic proteins was larger in FTLD-TDP when compared with normal controls. The increase in PSD95 levels may represent reactive upregulation of this protein, as suggested in AD. While greater numbers of microglia containing synaptic proteins is consistent with loss of synapses in FTLD-TDP, it may also be an indication of abnormal synaptic pruning by microglia.

Disentangling and quantifying the relative cognitive impact of concurrent mixed neurodegenerative pathologies.

Neurodegenerative pathologies such as Alzheimer disease neuropathologic change (ADNC), Lewy body disease (LBD), limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC), and cerebrovascular disease (CVD) frequently coexist, but little is known about the exact contribution of each pathology to cognitive decline and dementia in subjects with mixed pathologies. We explored the relative cognitive impact of concurrent common and rare neurodegenerative pathologies employing multivariate logistic regression analysis adjusted for age, gender, and level of education. We analyzed a cohort of 6,262 subjects from the National Alzheimer's Coordinating Center database, ranging from 0 to 6 comorbid neuropathologic findings per individual, where 95.7% of individuals had at least 1 neurodegenerative finding at autopsy and 75.5% had at least 2 neurodegenerative findings. We identified which neuropathologic entities correlate most frequently with one another and demonstrated that the total number of pathologies per individual was directly correlated with cognitive performance as assessed by Clinical Dementia Rating (CDR®) and Mini-Mental State Examination (MMSE). We show that ADNC, LBD, LATE-NC, CVD, hippocampal sclerosis, Pick disease, and FTLD-TDP significantly impact overall cognition as independent variables. More specifically, ADNC significantly affected all assessed cognitive domains, LBD affected attention, processing speed, and language, LATE-NC primarily affected tests related to logical memory and language, while CVD and other less common pathologies (including Pick disease, progressive supranuclear palsy, and corticobasal degeneration) had more variable neurocognitive effects. Additionally, ADNC, LBD, and higher numbers of comorbid neuropathologies were associated with the presence of at least one APOE ε4 allele, and ADNC and higher numbers of neuropathologies were inversely correlated with APOE ε2 alleles. Understanding the mechanisms by which individual and concomitant neuropathologies affect cognition and the degree to which each contributes is an imperative step in the development of biomarkers and disease-modifying therapeutics, particularly as these medical interventions become more targeted and personalized.

Genetic associations with dementia-related proteinopathy: Application of item response theory.

INTRODUCTION: Although dementia-related proteinopathy has a strong negative impact on public health, and is highly heritable, understanding of the related genetic architecture is incomplete. METHODS: We applied multidimensional generalized partial credit modeling (GPCM) to test genetic associations with dementia-related proteinopathies. Data were analyzed to identify candidate single nucleotide variants for the following proteinopathies: Aß, tau, α-synuclein, and TDP-43. RESULTS: Final included data comprised 966 participants with neuropathologic and WGS data. Three continuous latent outcomes were constructed, corresponding to TDP-43-, Aß/Tau-, and α-synuclein-related neuropathology endophenotype scores. This approach helped validate known genotype/phenotype associations: for example, TMEM106B and GRN were risk alleles for TDP-43 pathology; and GBA for α-synuclein/Lewy bodies. Novel suggestive proteinopathy-linked alleles were also discovered, including several (SDHAF1, TMEM68, and ARHGEF28) with colocalization analyses and/or high degrees of biologic credibility. DISCUSSION: A novel methodology using GPCM enabled insights into gene candidates for driving misfolded proteinopathies. HIGHLIGHTS: Latent factor scores for proteinopathies were estimated using a generalized partial credit model. The three latent continuous scores corresponded well with proteinopathy severity. Novel genes associated with proteinopathies were identified. Several genes had high degrees of biologic credibility for dementia risk factors.

TDP-43-M323K causes abnormal brain development and progressive cognitive and motor deficits associated with mislocalised and increased levels of TDP-43.

TDP-43 pathology is found in several neurodegenerative disorders, collectively referred to as "TDP-43 proteinopathies". Aggregates of TDP-43 are present in the brains and spinal cords of >97% of amyotrophic lateral sclerosis (ALS), and in brains of ∼50% of frontotemporal dementia (FTD) patients. While mutations in the TDP-43 gene (TARDBP) are usually associated with ALS, many clinical reports have linked these mutations to cognitive impairments and/or FTD, but also to other neurodegenerative disorders including Parkinsonism (PD) or progressive supranuclear palsy (PSP). TDP-43 is a ubiquitously expressed, highly conserved RNA-binding protein that is involved in many cellular processes, mainly RNA metabolism. To investigate systemic pathological mechanisms in TDP-43 proteinopathies, aiming to capture the pleiotropic effects of TDP-43 mutations, we have further characterised a mouse model carrying a point mutation (M323K) within the endogenous Tardbp gene. Homozygous mutant mice developed cognitive and behavioural deficits as early as 3 months of age. This was coupled with significant brain structural abnormalities, mainly in the cortex, hippocampus, and white matter fibres, together with progressive cortical interneuron degeneration and neuroinflammation. At the motor level, progressive phenotypes appeared around 6 months of age. Thus, cognitive phenotypes appeared to be of a developmental origin with a mild associated progressive neurodegeneration, while the motor and neuromuscular phenotypes seemed neurodegenerative, underlined by a progressive loss of upper and lower motor neurons as well as distal denervation. This is accompanied by progressive elevated TDP-43 protein and mRNA levels in cortex and spinal cord of homozygous mutant mice from 3 months of age, together with increased cytoplasmic TDP-43 mislocalisation in cortex, hippocampus, hypothalamus, and spinal cord at 12 months of age. In conclusion, we find that Tardbp M323K homozygous mutant mice model many aspects of human TDP-43 proteinopathies, evidencing a dual role for TDP-43 in brain morphogenesis as well as in the maintenance of the motor system, making them an ideal in vivo model system to study the complex biology of TDP-43.

RNA-mediated ribonucleoprotein assembly controls TDP-43 nuclear retention.

TDP-43 is an essential RNA-binding protein strongly implicated in the pathogenesis of neurodegenerative disorders characterized by cytoplasmic aggregates and loss of nuclear TDP-43. The protein shuttles between nucleus and cytoplasm, yet maintaining predominantly nuclear TDP-43 localization is important for TDP-43 function and for inhibiting cytoplasmic aggregation. We previously demonstrated that specific RNA binding mediates TDP-43 self-assembly and biomolecular condensation, requiring multivalent interactions via N- and C-terminal domains. Here, we show that these complexes play a key role in TDP-43 nuclear retention. TDP-43 forms macromolecular complexes with a wide range of size distribution in cells and we find that defects in RNA binding or inter-domain interactions, including phase separation, impair the assembly of the largest species. Our findings suggest that recruitment into these macromolecular complexes prevents cytoplasmic egress of TDP-43 in a size-dependent manner. Our observations uncover fundamental mechanisms controlling TDP-43 cellular homeostasis, whereby regulation of RNA-mediated self-assembly modulates TDP-43 nucleocytoplasmic distribution. Moreover, these findings highlight pathways that may be implicated in TDP-43 proteinopathies and identify potential therapeutic targets.

Cryptic exon inclusion is a molecular signature of LATE-NC in aging brains.

The aggregation, mislocalization, and phosphorylation of TDP-43 are pathologic hallmarks of several neurodegenerative diseases and provide a defining criterion for the neuropathologic diagnosis of Limbic-predominant Age-related TDP-43 Encephalopathy (LATE). LATE neuropathologic changes (LATE-NC) are often comorbid with other neurodegenerative pathologies including Alzheimer's disease neuropathologic changes (ADNC). We examined whether TDP-43 regulated cryptic exons accumulate in the hippocampus of neuropathologically confirmed LATE-NC cases. We found that several cryptic RNAs are robustly expressed in LATE-NC cases with or without comorbid ADNC and correlate with pTDP-43 abundance; however, the accumulation of cryptic RNAs is more robust in LATE-NC with comorbid ADNC. Additionally, cryptic RNAs can robustly distinguish LATE-NC from healthy controls and AD cases. These findings expand our current understanding and provide novel potential biomarkers for LATE pathogenesis.

A transient protein folding response targets aggregation in the early phase of TDP-43-mediated neurodegeneration.

Understanding the mechanisms that drive TDP-43 pathology is integral to combating amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and other neurodegenerative diseases. Here we generated a longitudinal quantitative proteomic map of the cortex from the cytoplasmic TDP-43 rNLS8 mouse model of ALS and FTLD, and developed a complementary open-access webtool, TDP-map ( https://shiny.rcc.uq.edu.au/TDP-map/ ). We identified distinct protein subsets enriched for diverse biological pathways with temporal alterations in protein abundance, including increases in protein folding factors prior to disease onset. This included increased levels of DnaJ homolog subfamily B member 5, DNAJB5, which also co-localized with TDP-43 pathology in diseased human motor cortex. DNAJB5 over-expression decreased TDP-43 aggregation in cell and cortical neuron cultures, and knockout of Dnajb5 exacerbated motor impairments caused by AAV-mediated cytoplasmic TDP-43 expression in mice. Together, these findings reveal molecular mechanisms at distinct stages of ALS and FTLD progression and suggest that protein folding factors could be protective in neurodegenerative diseases.

Stimulating VAPB-PTPIP51 ER-mitochondria tethering corrects FTD/ALS mutant TDP43 linked Ca2+ and synaptic defects.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are clinically linked major neurodegenerative diseases. Notably, TAR DNA-binding protein-43 (TDP43) accumulations are hallmark pathologies of FTD/ALS and mutations in the gene encoding TDP43 cause familial FTD/ALS. There are no cures for FTD/ALS. FTD/ALS display damage to a broad range of physiological functions, many of which are regulated by signaling between the endoplasmic reticulum (ER) and mitochondria. This signaling is mediated by the VAPB-PTPIP51 tethering proteins that serve to recruit regions of ER to the mitochondrial surface so as to facilitate inter-organelle communications. Several studies have now shown that disrupted ER-mitochondria signaling including breaking of the VAPB-PTPIP51 tethers are features of FTD/ALS and that for TDP43 and other familial genetic FTD/ALS insults, this involves activation of glycogen kinase-3ß (GSK3ß). Such findings have prompted suggestions that correcting damage to ER-mitochondria signaling and the VAPB-PTPIP51 interaction may be broadly therapeutic. Here we provide evidence to support this notion. We show that overexpression of VAPB or PTPIP51 to enhance ER-mitochondria signaling corrects mutant TDP43 induced damage to inositol 1,4,5-trisphosphate (IP3) receptor delivery of Ca2+ to mitochondria which is a primary function of the VAPB-PTPIP51 tethers, and to synaptic function. Moreover, we show that ursodeoxycholic acid (UDCA), an FDA approved drug linked to FTD/ALS and other neurodegenerative diseases therapy and whose precise therapeutic target is unclear, corrects TDP43 linked damage to the VAPB-PTPIP51 interaction. We also show that this effect involves inhibition of TDP43 mediated activation of GSK3ß. Thus, correcting damage to the VAPB-PTPIP51 tethers may have therapeutic value for FTD/ALS and other age-related neurodegenerative diseases.

Persistence of Kii amyotrophic lateral sclerosis after the 2000s and its characteristic aging-related tau astrogliopathy.

Kii amyotrophic lateral sclerosis (ALS) is a unique disease that occurs in the southern portion of the Kii Peninsula and exhibits a dual pathology of TAR DNA-binding protein of 43 kDa (TDP-43) proteinopathy and tauopathy. The incidence of ALS in this region was very high in the 1960s, briefly decreased through the 1980s, but began increasing again after 2000 with a change of high-concentration geographic foci. It is unclear, however, whether the unique pathological features have changed along with the incidence changes. This study analyzed postmortem specimens from neuropathologically confirmed Kii ALS cases from the 1970s (n = 4) and those after 1999 (n = 12) from the southern Kii Peninsula or outside of the area. Our results confirm the continued occurrence of Kii ALS after 2000 in the southern Kii Peninsula and the preservation of disease-specific neuronal tau pathology, including the widespread occurrence throughout the brain and spinal cord, sparse neuropil threads, and predominance in superficial layers. Furthermore, we assessed the glial tau pathology of Kii and non-Kii ALS in accordance with the aging-related tau astrogliopathy classification method for the first time and detected a unique brainstem predominant appearance of gray matter aging-related tau astrogliopathy in Kii ALS cases, which may provide clues to pathogenetic mechanisms.

Pathologic correlates of aging-related tau astrogliopathy: ARTAG is associated with LATE-NC and cerebrovascular pathologies, but not with ADNC.

Age-related tau astrogliopathy (ARTAG) is detectable in the brains of over one-third of autopsied persons beyond age 80, but the pathoetiology of ARTAG is poorly understood. Insights can be gained by analyzing risk factors and comorbid pathologies. Here we addressed the question of which prevalent co-pathologies are observed with increased frequency in brains with ARTAG. The study sample was the National Alzheimer's Coordinating Center (NACC) data set, derived from multiple Alzheimer's disease research centers (ADRCs) in the United States. Data from persons with unusual conditions (e.g. frontotemporal dementia) were excluded leaving 504 individual autopsied research participants, clustering from 20 different ADRCs, autopsied since 2020; ARTAG was reported in 222 (44.0%) of included participants. As has been shown previously, ARTAG was increasingly frequent with older age and in males. The presence and severity of other common subtypes of pathology that were previously linked to dementia were analyzed, stratifying for the presence of ARTAG. In logistical regression-based statistical models that included age and sex as covariates, ARTAG was relatively more likely to be found in brains with limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC), and in brains with comorbid cerebrovascular pathology (arteriolosclerosis and/or brain infarcts). However, ARTAG was not associated with severe Alzheimer's disease neuropathologic change (ADNC), or primary age-related tauopathy (PART). In a subset analysis of 167 participants with neurocognitive testing data, there was a marginal trend for ARTAG pathology to be associated with cognitive impairment as assessed with MMSE scores (P = 0.07, adjusting for age, sex, interval between final clinic visit and death, and ADNC severity). A limitation of the study was that there were missing data about ARTAG pathologies, with incomplete operationalization of ARTAG according to anatomic region and pathologic subtypes (e.g., thorn-shaped or granular-fuzzy astrocytes). In summary, ARTAG was not associated with ADNC, whereas prior observations about ARTAG occurring with increased frequency in aging, males, and brains with LATE-NC were replicated. It remains to be determined whether the increased frequency of ARTAG in brains with comorbid cerebrovascular pathology is related to local infarctions or neuroinflammatory signaling, or with some other set of correlated factors including blood-brain barrier dysfunction.

The influence of limbic-predominant age-related TDP-43 encephalopathy on argyrophilic grain disease: A voxel-based morphometry analysis of pathologically confirmed cases.

BACKGROUND: The influence of limbic-predominant age-related TAR DNA-binding protein of 43 kDa encephalopathy neuropathological change (LATE-NC) on structural alterations in argyrophilic grain disease (AGD) have not been documented. This study aimed to investigate the morphological impact of LATE-NC on AGD through voxel-based morphometry (VBM) technique. MATERIALS AND METHODS: Fifteen individuals with pathologically verified AGD, comprising 6 with LATE-NC (comorbid AGD [cAGD]) and 9 without LATE-NC (pure AGD [pAGD]), along with 10 healthy controls (HC) were enrolled. Whole-brain 3D-T1-weighted images were captured and preprocessed utilizing the Computational Anatomy Toolbox 12. VBM was employed to compare gray matter volume among (i) pAGD and HC, (ii) cAGD and HC, and (iii) pAGD and cAGD. RESULTS: In comparison to HC, the pAGD group exhibited slightly asymmetric gray matter volume loss, particularly in the ambient gyrus, amygdala, hippocampus, anterior cingulate gyrus, and insula. Alternatively, the cAGD group exhibited greater gray matter volume loss, with a predominant focus on the inferolateral regions encompassing the ambient gyrus, amygdala, hippocampus, and the inferior temporal area, including the anterior temporal pole. The atrophy of the bilateral anterior temporal pole and right inferior temporal gyrus persisted when contrasting the pAGD and cAGD groups. CONCLUSION: Comorbidity with LATE-NC is linked to different atrophic distribution, particularly affecting the inferolateral regions in AGD. Consequently, the consideration of comorbid LATE-NC is crucial in individuals with AGD exhibiting more widespread temporal atrophy.

Effective Inhibition of TDP-43 Aggregation by Native State Stabilization.

Preventing the misfolding or aggregation of transactive response DNA binding protein with 43 kDa (TDP-43) is the most actively pursued disease-modifying strategy to treat amyotrophic lateral sclerosis and other neurodegenerative diseases. In this work, we provide proof of concept that native state stabilization of TDP-43 is a viable and effective strategy for treating TDP-43 proteinopathies. Firstly, we leveraged the Cryo-EM structures of TDP-43 fibrils to design C-terminal substitutions that disrupt TDP-43 aggregation. Secondly, we showed that these substitutions (S333D/S342D) stabilize monomeric TDP-43 without altering its physiological properties. Thirdly, we demonstrated that binding native oligonucleotide ligands stabilized monomeric TDP-43 and prevented its fibrillization and phase separation in the absence of direct binding to the aggregation-prone C-terminal domain. Fourthly, we showed that the monomeric TDP-43 variant could be induced to aggregate in a controlled manner, which enabled the design and implementation of a high-throughput screening assay to identify native state stabilizers of TDP-43. Altogether, our findings demonstrate that different structural domains in TDP-43 could be exploited and targeted to develop drugs that stabilize the native state of TDP-43 and provide a platform to discover novel drugs to treat TDP-43 proteinopathies.