TAR DNA-binding Protein 43 Aggregation Research Articles

Atomistic Insights into A315E Mutation-Enhanced Pathogenicity of TDP-43 Core Fibrils.

The aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) into fibrillary deposits is implicated in amyotrophic lateral sclerosis (ALS), and some hereditary mutations localized in the low complexity domain (LCD) facilitate the formation of pathogenic TDP-43 fibrils. A recent cryo-EM study reported the atomic-level structures of the A315E TDP-43 LCD (residues 288-319, TDP-43288-319) core fibril in which the protofilaments have R-shaped structures and hypothesized that A315E U-shaped protofilaments can readily convert to R-shaped protofilaments compared to the wild-type (WT) ones. There are no atomic structures of WT protofilaments available yet. Herein, we performed extensive all-atom explicit-solvent molecular dynamics simulations on A315E and WT protofilaments starting from both the cryo-EM-determined R-shaped and our constructed U-shaped structures. Our simulations show that WT protofilaments also adopt the R-shaped structures but are less stable than their A315E counterparts. Except for R293-E315 salt bridges, N312-F316 hydrophobic interactions and F316-F316 π-π stacking interactions are also crucial for the stabilization of the neck region of the R-shaped A315E protofilaments. The loss of R293-E315 salt bridges and the weakened interactions of N312-F316 and F316-F316 result in the reduced stability of the R-shaped WT protofilaments. Simulations starting from U-shaped folds reveal that A315E protofilaments can spontaneously convert to the cryo-EM-derived R-shaped protofilaments, whereas WT protofilaments convert to R-shape-like structures with remodeled neck regions. The R-shape-like WT protofilaments could act as intermediate states slowing down the U-to-R transition. This study reveals that A315E mutation can not only enhance the structural stability of the R-shaped TDP-43288-319 protofilaments but also promote the U-to-R transition, which provides atomistic insights into the A315E mutation-enhanced TDP-43 pathogenicity in ALS.

Novel insights into the structural changes induced by disease-associated mutations in TDP-43: a computational approach

Over the last two decades, the pathogenic aggregation of TAR DNA-binding protein 43 (TDP-43) is found to be strongly associated with several fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTD), etc. While the mutations and truncation in TDP-43 protein have been suggested to be responsible for TDP-43 pathogenesis by accelerating the aggregation process, the effects of these mutations on the bio-mechanism of pathological TDP-43 protein remained poorly understood. Investigating this at the molecular level, we formulized an integrated workflow of molecular dynamic simulation and machine learning models (MD-ML). By performing an extensive structural analysis of three disease-related mutations (i.e., I168A, D169G, and I168A-D169G) in the conserved RNA recognition motifs (RRM1) of TDP-43, we observed that the I168A-D169G double mutant delineates the highest packing of the protein inner core as compared to the other mutations, which may indicate more stability and higher chances of pathogenesis. Moreover, through our MD-ML workflow, we identified the biological descriptors of TDP-43 which includes the interacting residue pairs and individual protein residues that influence the stability of the protein and could be experimentally evaluated to develop potential therapeutic strategies. Communicated by Ramaswamy H. Sarma

Identifying Therapeutic Inhibitors of TDP43 Phase‐Separation

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease for which there has been little improvement in treatment over the past 25 years. ALS is linked to mutations in dozens of genes, but despite its genetic complexity, 97% of patients share a distinct molecular pathology: cytoplasmic inclusions of TAR DNA‐Binding protein 43 (TDP‐43). Further, TDP‐43 aggregates are toxic to healthy neurons, suggesting that TDP‐43 aggregates contribute to neurodegeneration in ALS. Due to the near‐ubiquity of TDP‐43 inclusions in ALS patients and their contribution to ALS pathology, prevention or dissolution of these TDP‐43 inclusions presents a promising therapeutic strategy. In cells, TDP43 can form both solid aggregates or liquid droplets, through liquid‐liquid phase separation (LLPS). Here, we screened a library of small molecules and RNAs to identify inhibitors of TDP‐43 LLPS using an in vitro assay which quantifies TDP‐43 liquid droplet formation. Hits from this screen were further characterized based on their ability to reverse and inhibit both TDP‐43 droplets and aggregates to obtain functional profiles for each promising inhibitor. Pairing functional data of inhibitors with structural information regarding their binding to TDP‐43 enables a close‐dissection of the interactions that drive TDP‐43 self‐assembly. Thus, small molecule screens which target protein self‐assembly present a powerful approach for both developing novel therapeutics and identifying the interactions that govern self‐assembly; these inform each other and may lead to the discovery of novel therapeutic agents which target protein self‐assembly to treat severe diseases, like ALS.

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.

TDP-43 pathology: From noxious assembly to therapeutic removal.

Our understanding of amyotrophic lateral sclerosis and frontotemporal dementia has advanced dramatically since the discovery of cytoplasmic TAR DNA-binding protein 43 (TDP-43) inclusions as the hallmark pathology of these neurodegenerative diseases. Recent studies have provided insights into the physiological function of TDP-43 as an essential DNA-/RNA-modulating protein, and the triggers and consequences of TDP-43 dysfunction and aggregation. The formation of TDP-43 pathology is a progressive process, involving the generation of multiple distinct protein species, each with varying biophysical properties and roles in neurodegeneration. Here, we explore how the pathogenic changes to TDP-43, including mislocalisation, misfolding, aberrant liquid-liquid phase separation, stress granule assembly, oligomerisation, and post-translational modification, drive disease-associated aggregation in TDP-43 proteinopathies. We highlight how pathological TDP-43 species are formed and contribute to cellular dysfunction and toxicity, via both loss-of-function and gain-of-function mechanisms. We also review the role of protein homeostasis mechanisms, namely the ubiquitin proteasome system, autophagy-lysosome pathway, heat-shock response, and chaperone-mediated autophagy, in combating TDP-43 aggregation and discuss how their dysfunction likely promotes disease pathogenesis and progression. Finally, we evaluate pre-clinical studies aimed at enhancing TDP-43 protein clearance via these mechanisms and provide insight on promising strategies for future therapeutic advances. Harnessing the mechanisms that protect against or ameliorate TDP-43 pathology presents promising opportunities for developing disease-modifying treatments for these neurodegenerative diseases.

USP10 Inhibits Aberrant Cytoplasmic Aggregation of TDP-43 by Promoting Stress Granule Clearance.

TAR DNA-binding protein 43 (TDP-43) is a causative factor of amyotrophic lateral sclerosis (ALS). Cytoplasmic TDP-43 aggregates in neurons are a hallmark pathology of ALS. Under various stress conditions, TDP-43 localizes sequentially to two cytoplasmic protein aggregates, namely, stress granules (SGs) first and then aggresomes. Accumulating evidence suggests that delayed clearance of TDP-43-positive SGs is associated with pathological TDP-43 aggregates in ALS. We found that ubiquitin-specific protease 10 (USP10) promotes the clearance of TDP-43-positive SGs in cells treated with proteasome inhibitor, thereby promoting the formation of TDP-43-positive aggresomes, and the depletion of USP10 increases the amount of insoluble TDP-35, a cleaved product of TDP-43, in the cytoplasm. TDP-35 interacted with USP10 in an RNA-binding-dependent manner; however, impaired RNA binding of TDP-35 reduced the localization in SGs and aggresomes and induced USP10-negative TDP-35 aggregates. Immunohistochemistry showed that most of the cytoplasmic TDP-43/TDP-35 aggregates in the neurons of ALS patients were USP10 negative. Our findings suggest that USP10 inhibits aberrant aggregation of TDP-43/TDP-35 in the cytoplasm of neuronal cells by promoting the clearance of TDP-43/TDP-35-positive SGs and facilitating the formation of TDP-43/TDP-35-positive aggresomes.

PiRNA/PIWI Protein Complex as a Potential Biomarker in Sporadic Amyotrophic Lateral Sclerosis

The pathological hallmark of the majority of amyotrophic lateral sclerosis (ALS) cases is the mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43), an RNA-binding protein. Several studies have attributed disease processes of ALS to abnormal RNA metabolism. However, dysregulated biogenesis of RNA, especially non-coding RNA (ncRNA), is poorly understood. To resolve it, RNA-Seq, biochemical, and immunohistochemical analyses were performed on the pyramidal tract of the medulla oblongata of sporadic ALS (sALS) and control postmortem brain samples. Here, we report perturbation of ncRNA biogenesis in PIWI-interacting RNA (piRNA) in several sALS brain samples associated with TDP-43 pathology. In addition, we confirmed the dysregulation of two PIWI homologs, PIWI-like-mediated gene silencing 1 (PIWIL1) and PIWIL4, which bind to piRNAs to regulate their expression. PIWIL1 was mislocalized and co-localized with TDP-43 in motor neurons of sporadic ALS lumbar cords. Our results imply that dysregulation of piRNA, PIWIL1, and PIWIL4 is linked to pathogenesis of ALS. Based on these results, piRNAs and PIWI proteins are potential diagnostic biomarkers and therapeutic targets of ALS.

Vitamin B12 Reduces TDP-43 Toxicity by Alleviating Oxidative Stress and Mitochondrial Dysfunction

TAR DNA-binding protein 43 (TDP-43) is a member of an evolutionarily conserved family of heterogeneous nuclear ribonucleoproteins that modulate multiple steps in RNA metabolic processes. Cytoplasmic aggregation of TDP-43 in affected neurons is a pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer’s disease (AD), and limbic predominant age-related TDP-43 encephalopathy (LATE). Mislocalized and accumulated TDP-43 in the cytoplasm induces mitochondrial dysfunction and reactive oxidative species (ROS) production. Here, we show that TDP-43- and rotenone-induced neurotoxicity in the human neuronal cell line SH-SY5Y were attenuated by hydroxocobalamin (Hb, vitamin B12 analog) treatment. Although Hb did not affect the cytoplasmic accumulation of TDP-43, Hb attenuated TDP-43-induced toxicity by reducing oxidative stress and mitochondrial dysfunction. Moreover, a shortened lifespan and motility defects in TDP-43-expressing Drosophila were significantly mitigated by dietary treatment with hydroxocobalamin. Taken together, these findings suggest that oral intake of hydroxocobalamin may be a potential therapeutic intervention for TDP-43-associated proteinopathies.

TDP-43 and ER Stress in Neurodegeneration: Friends or Foes?

Nuclear depletion, abnormal modification, and cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP-43) are linked to a group of fatal neurodegenerative diseases called TDP-43 proteinopathies, which include amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Although our understanding of the physiological function of TDP-43 is rapidly advancing, the molecular mechanisms associated with its pathogenesis remain poorly understood. Accumulating evidence suggests that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are important players in TDP-43 pathology. However, while neurons derived from autopsied ALS and FTLD patients revealed TDP-43 deposits in the ER and displayed UPR activation, data originated from in vitro and in vivo TDP-43 models produced contradictory results. In this review, we will explore the complex interplay between TDP-43 pathology, ER stress, and the UPR by breaking down the evidence available in the literature and addressing the reasons behind these discrepancies. We also highlight underexplored areas and key unanswered questions in the field. A better synchronization and integration of methodologies, models, and mechanistic pathways will be crucial to discover the true nature of the TDP-43 and ER stress relationship and, ultimately, to uncover the full therapeutic potential of the UPR.

DUX4 expression is increased in lymphomonocytes of patients with amyotrophic lateral sclerosis: Possible involvement in TDP-43 aggregation?

TAR DNA binding protein-43 (TDP-43) is the main component of cytoplasmic aggregates in motor neurons of Amyotrophic Lateral Sclerosis (ALS) patients. TDP-43 aggregates have also been found in muscle cells of patients with Facioscapulohumeral dystrophy (FSHD), a disease caused by the aberrant expression of DUX4 protein. Interestingly, the expression of DUX4 has been demonstrated to induce TDP-43 aggregation in human FSHD myoblasts and healthy cells transfected with DUX4. Based on these premises, we decided to verify if DUX4 is expressed in cells of ALS patients and, if expressed, its possible involvement in TDP-43 aggregation.

Establishment of chemically oligomerizable TAR DNA-binding protein-43 which mimics amyotrophic lateral sclerosis pathology in mammalian cells

One of the pathological hallmarks of amyotrophic lateral sclerosis (ALS) is mislocalized, cytosolic aggregation of TAR DNA-Binding Protein-43 (TDP-43). Not only TDP-43 per se is a causative gene of ALS but also mislocalization and aggregation of TDP-43 seems to be a common pathological change in both sporadic and familial ALS. The mechanism how nuclear TDP-43 transforms into cytosolic aggregates remains elusive, but recent studies using optogenetics have proposed that aberrant liquid–liquid phase separation (LLPS) of TDP-43 links to the aggregation process, leading to cytosolic distribution. Although LLPS plays an important role in the aggregate formation, there are still several technical problems in the optogenetic technique to be solved to progress further in vivo study. Here we report a chemically oligomerizable TDP-43 system. Oligomerization of TDP-43 was achieved by a small compound AP20187, and oligomerized TDP-43 underwent aggregate formation, followed by cytosolic mislocalization and induction of cell toxicity. The mislocalized TDP-43 co-aggregated with wt-TDP-43, Fused-in-sarcoma (FUS), TIA1 and sequestosome 1 (SQSTM1)/p62, mimicking ALS pathology. The chemically oligomerizable TDP-43 also revealed the roles of the N-terminal domain, RNA-recognition motif, nuclear export signal and low complexity domain in the aggregate formation and mislocalization of TDP-43. The aggregate-prone properties of TDP-43 were enhanced by a familial ALS-causative mutation. In conclusion, the chemically oligomerizable TDP-43 system could be useful to study the mechanisms underlying the droplet-aggregation phase transition and cytosolic mislocalization of TDP-43 in ALS and further study in vivo.TDP-43 is the primary protein aggregate in amyotrophic lateral sclerosis (ALS), and its mislocalization from nucleus is a hallmark of ALS pathology whose mechanisms remain unclear. In this study, the authors report a novel chemically oligomerizable TDP-43 system. Induction of TDP-43 oligomerization mimics ALS pathology such as mislocalization of TDP-43 and recruitment of critical proteins into the aggregates.

Thioridazine reverts the phenotype in cellular and Drosophila models of amyotrophic lateral sclerosis by enhancing TDP-43 aggregate clearance

Brain inclusions mainly composed of misfolded and aggregated TAR DNA binding protein 43 (TDP-43), are characteristic hallmarks of amyotrophic lateral sclerosis (ALS). Irrespective of the role played by the inclusions, their reduction represents an important therapeutic pathway that is worth exploring. Their removal can either lead to the recovery of TDP-43 function by removing the self-templating conformers that sequester the protein in the inclusions, and/or eliminate any potential intrinsic toxicity of the aggregates. The search for curative therapies has been hampered by the lack of ALS models for use in high-throughput screening. We adapted, optimised, and extensively characterised our previous ALS cellular model for such use. The model demonstrated efficient aggregation of endogenous TDP-43, and concomitant loss of its splicing regulation function. We provided a proof-of-principle for its eventual use in high-throughput screening using compounds of the tricyclic family and showed that recovery of TDP-43 function can be achieved by the enhanced removal of TDP-43 aggregates by these compounds. We observed that the degradation of the aggregates occurs independent of the autophagy pathway beyond autophagosome-lysosome fusion, but requires a functional proteasome pathway. The in vivo translational effect of the cellular model was tested with two of these compounds in a Drosophila model expressing a construct analogous to the cellular model, where thioridazine significantly improved the locomotive defect. Our findings have important implications as thioridazine cleared TDP-43 aggregates and recovered TDP-43 functionality. This study also highlights the importance of a two-stage, in vitro and in vivo model system to cross-check the search for small molecules that can clear TDP-43 aggregates in TDP-43 proteinopathies.

Glial TDP-43 and TDP-43 induced glial pathology, focus on neurodegenerative proteinopathy syndromes.

Since its discovery in 2006, TAR DNA binding protein 43 (TDP-43) has driven rapidly evolving research in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and limbic predominant age-related TDP-43 encephalopathy (LATE). TDP-43 mislocalization or aggregation is the hallmark of TDP-43 proteinopathy and is associated with cognitive impairment that can be mapped to its regional deposition. Studies in human tissue and model systems demonstrate that TDP-43 may potentiate other proteinopathies such as the amyloid or tau pathology seen in Alzheimer's Disease (AD) in the combination of AD+LATE. Despite this growing body of literature, there remain gaps in our understanding of whether there is heterogeneity in TDP-43 driven mechanisms across cell types. The growing observations of correlation between TDP-43 proteinopathy and glial pathology suggest a relationship between the two, including pathogenic glial cell-autonomous dysfunction and dysregulated glial immune responses to neuronal TDP-43. In this review, we discuss the available data on TDP-43 in glia within the context of the neurodegenerative diseases ALS and FTLD and highlight the current lack of information about glial TDP-43 interaction in AD+LATE. TDP-43 has proven to be a significant modulator of cognitive and neuropathological outcomes. A deeper understanding of its role in diverse cell types may provide relevant insights into neurodegenerative syndromes.

A microfluidic approach to rescue ALS motor neuron degeneration using rapamycin

TAR DNA-binding protein-43 (TDP-43) is known to accumulate in ubiquitinated inclusions of amyotrophic lateral sclerosis affected motor neurons, resulting in motor neuron degeneration, loss of motor functions, and eventually death. Rapamycin, an mTOR inhibitor and a commonly used immunosuppressive drug, has been shown to increase the survivability of Amyotrophic Lateral Sclerosis (ALS) affected motor neurons. Here we present a transgenic, TDP-43-A315T, mouse model expressing an ALS phenotype and demonstrate the presence of ubiquitinated cytoplasmic TDP-43 aggregates with > 80% cell death by 28 days post differentiation in vitro. Embryonic stem cells from this mouse model were used to study the onset, progression, and therapeutic remediation of TDP-43 aggregates using a novel microfluidic rapamycin concentration gradient generator. Results using a microfluidic device show that ALS affected motor neuron survival can be increased by 40.44% in a rapamycin dosage range between 0.4-1.0 µM.

Niclosamide affects intracellular TDP-43 distribution in motor neurons, activates mitophagy, and attenuates morphological changes under stress

Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron loss in the brain and spinal cord; however, its etiology is unknown, and no curative treatment exists. TAR DNA-binding protein 43 (TDP-43), encoded by TARDBP, is a genetic mutation observed in 2-5% of familial ALS, and TDP is known to be mislocalized in the cytoplasm. This study aimed to identify compounds that inhibited the nuclear to cytoplasmic localization of TDP-43 in human induced pluripotent stem (iPS) cells-derived neurons. TDP-43 transgenic human iPS cells were constructed, differentiated into motor neurons, and then treated with MG-132 and sodium arsenite (stressors) to induce nuclear to cytoplasmic localization of TDP-43. STAT3 inhibitors, such as niclosamide, prevented TDP-43 mislocalization and degraded TDP-43 aggregates, and attenuated morphological changes under stress. Furthermore, niclosamide activated mitophagy via the PINK1-parkin-ubiquitin pathway. These findings suggest niclosamide may be a therapeutic candidate for ALS.

Cytoplasmic TDP-43 impairs the activity of the ubiquitin-proteasome system

The cytoplasmic inclusions of nuclear TAR DNA-binding protein 43 (TDP-43) are a pathologic hallmark in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTD), and other neurological disorders. We reported that expressing mutant TDP-43(M337V) in rhesus monkeys can mimic the cytoplasmic mislocalization of mutant TDP-43 seen in patient brains. Here we investigated how cytoplasmic mutant TDP-43 mediates neuropathology. We found that C-terminal TDP-43 fragments are primarily localized in the cytoplasm and that the age-dependent elevated UBE2N promotes the accumulation of cytoplasmic C-terminal TDP-43 via K63 ubiquitination. Immunoprecipitation and mass spectrometry revealed that cytoplasmic mutant TDP-43 interacts with proteasome assembly proteins PSMG2 and PSD13, which might lead to the impairment of the proteasomal activity. Our findings suggest that cytoplasmic TDP-43 may participate in age-dependent accumulation of misfolded proteins in the brain by inhibiting the UPS activity.

Phosphorylated TAR DNA-binding protein-43: Aggregation and antibody-based inhibition

TAR DNA-binding protein-43 (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). Hyperphosphorylation and aggregation of TDP-43 contribute to pathology and are viable therapeutic targets for ALS. In vivo inhibition of TDP-43 aggregation was evaluated using anti-TDP-43 antibodies with promising outcomes. However, the exact mechanism of antibody-based inhibition targeting TDP-43 is not well understood but may lead to the identification of viable immunotherapies. Herein, the mechanism of in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Specifically, the aggregation of phosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) spectroscopy. The protein aggregates were insoluble, ThT-positive and characterized with heterogeneous morphologies (fibers, amorphous structures). Antibodies specific to epitopes 178-393 and 256-269, within the RRM2-CTD domain, reduced the formation of β-sheets and insoluble aggregates, at low antibody loading (antibody: protein ratio = 1 μg/mL: 45 μg/mL). Inhibition outcomes were highly dependent on the type and loading of antibodies, indicating dual functionality of such inhibitors, as aggregation inhibitors or aggregation promoters. Anti-SOD1 and anti-tau antibodies were not effective inhibitors against TDP-43 aggregation, indicating selective inhibition.

TDP-43 and Inflammation: Implications for Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

The abnormal mislocalisation and ubiquitinated protein aggregation of the TAR DNA binding protein 43 (TDP-43) within the cytoplasm of neurons and glia in the central nervous system (CNS) is a pathological hallmark of early-onset neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The pathomechanisms underlying abnormal mislocalisation and aggregation of TDP-43 remain unknown. However, there is a growing body of evidence implicating neuroinflammation and immune-mediated mechanisms in the pathogenesis of neurodegeneration. Importantly, most of the evidence for an active role of immunity and inflammation in the pathogenesis of ALS and FTD relates specifically to TDP-43, posing the question as to whether immune-mediated mechanisms could hold the key to understanding TDP-43’s underlying role in neurodegeneration in both diseases. Therefore, this review aims to piece together key lines of evidence for the specific association of TDP-43 with key immune and inflammatory pathways to explore the nature of this relationship and the implications for potential pathomechanisms underlying neurodegeneration in ALS and FTD.

Distinct brain-derived TDP-43 strains from FTLD-TDP subtypes induce diverse morphological TDP-43 aggregates and spreading patterns in vitro and in vivo.

The heterogeneity in the distribution and morphological features of TAR DNA-binding protein-43 (TDP-43) pathology in the brains of frontotemporal lobar degeneration (FTLD-TDP) patients and their different clinical manifestations suggest that distinct pathological TDP-43 strains could play a role in this heterogeneity between different FTLD-TDP subtypes (A-E). Our aim was to evaluate the existence of distinct TDP-43 strains in the brains of different FTLD-TDP subtypes and characterise their specific seeding properties in vitro and in vivo. We used an inducible stable cell line expressing a mutant cytoplasmic TDP-43 (iGFP-NLSm) to evaluate the seeding properties of distinct pathological TDP-43 strains. Brain-derived TDP-43 protein extracts from FTLD-TDP types A (n=6) and B (n=3) cases induced the formation of round/spherical phosphorylated TDP-43 aggregates that morphologically differed from the linear and wavy wisps and bigger heterogeneous filamentous (skein-like) aggregates induced by type E (n=3) cases. These morphological differences correlated with distinct biochemical banding patterns of sarkosyl-insoluble TDP-43 protein recovered from the transduced cells. Moreover, brain-derived TDP-43 extracts from type E cases showed higher susceptibility to PK digestion of full-length TDP-43 and the most abundant C-terminal fragments that characterise type E extracts. Finally, we showed that intracerebral injections of different TDP-43 strains induced a distinctive morphological and subcellular distribution of TDP-43 pathology and different spreading patterns in the brains of CamKIIa-hTDP-43NLSm Tg mice. We show the existence of distinct TDP-43 strains in the brain of different FTLD-TDP subtypes with distinctive seeding and spreading properties in the brains of experimental animal models.

Mechanistic and Structural Characterization of Full‐Length, Aggregation‐Prone, TDP‐43 Protein

TAR DNA-binding protein-43 (TDP-43) proteinopathy has been linked to several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) [1]. TDP-43 is a protein that functions in transcriptional repression, the modulation of RNA metabolism, and stress granule formation within cells. As in the case with many other proteins that are partially destructed, characterization of full-length recombinant TDP-43 has proved difficult. This protein is commonly modified post-translationally (PTMs) into aggregated and mutated structures in the cytoplasm and nucleus, including phosphorylation, acetylation, and C-terminal cleavages [2]. Cytoplasmic mislocalization and post-translational modifications of TDP-43 have been linked to aggregate formation, proposedly important for TDP-43-toxicity mechanisms [3, 4]. Since pertinent research has only recently been established on the protein, the pathogenicity and functionality of TDP-43 in ALS has yet to be determined, thus providing us with a critical knowledge gap in TDP-43's functions. Thus, systematic in vitro studies were conducted in our lab to characterize the basic aggregation mechanisms of full-length, native TDP-43 and establish a working model for the in vitro analyses of the protein. Current research on the full-length protein have discovered notable findings regarding the intermolecular properties of TDP-43, providing critical insight into the hydrophobicity of its structure. Along with these intermolecular characteristics and its poor solubility in vitro, the full structure of TDP-43 has not been fully defined. Herein, the in vitro aggregation of TDP-43 protein was achieved and characterized using various spectroscopic methods and electron microscopy. Data indicate that TDP-43 protein is an inherently aggregation-prone protein from the application of several in vitro conditions to induce misfolding of the protein, followed by turbidity and Thioflavin T (ThT) fluorescence assays to monitor β-sheet formation. Transmission electron microscopy (TEM) allowed for visualization of the structural morphologies of these TDP-43 aggregates. As a result, data indicate that full-length TDP-43 protein aggregated under specific environmental conditions and resulted in a high turbidity and ThT intensity. The in vitro analysis of TDP-43 aggregation is a valuable model for future protein studies and provides a preliminary platform for screening therapies towards a viable treatment for ALS.