Understanding In Vitro Pathways to Drug Discovery for TDP-43 Proteinopathies

The use of cellular models is a common means to investigate the potency of therapeutics in pre-clinical drug discovery. However, there is currently no consensus on which model most accurately replicates key aspects of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) pathology, such as accumulation of insoluble, cytoplasmic transactive response DNA-binding protein (TDP-43) and the formation of insoluble stress granules. Given this, we characterised two TDP-43 proteinopathy cellular models that were based on different aetiologies of disease. The first was a sodium arsenite-induced chronic oxidative stress model and the second expressed a disease-relevant TDP-43 mutation (TDP-43 M337V). The sodium arsenite model displayed most aspects of TDP-43, stress granule and ubiquitin pathology seen in human ALS/FTD donor tissue, whereas the mutant cell line only modelled some aspects. When these two cellular models were exposed to small molecule chemical probes, different effects were observed across the two models. For example, a previously disclosed sulfonamide compound decreased cytoplasmic TDP-43 and increased soluble levels of stress granule marker TIA-1 in the cellular stress model without impacting these levels in the mutant cell line. This study highlights the challenges of using cellular models in lead development during drug discovery for ALS and FTD and reinforces the need to perform assessments of novel therapeutics across a variety of cell lines and aetiological models.     

Disease Animal Models of TDP-43 Proteinopathy and Their Pre-Clinical Applications

Frontotemperal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are two common neurodegenerative diseases. TDP-43 is considered to be a major disease protein in FTLD/ALS, but it’s exact role in the pathogenesis and the effective treatments remains unknown. To address this question and to determine a potential treatment for FTLD/ALS, the disease animal models of TDP-43 proteinopathy have been established. TDP-43 proteinopathy is the histologic feature of FTLD/ALS and is associated with disease progression. Studies on the disease animal models with TDP-43 proteinopathy and their pre-clinical applications are reviewed and summarized. Through these disease animal models, parts of TDP-43 functions in physiological and pathological conditions will be better understood and possible treatments for FTLD/ALS with TDP-43 proteinopathy may be identified for possible clinical applications in the future.     

Mechanisms of TDP-43 Proteinopathy Onset and Propagation

TDP-43 is an RNA-binding protein that has been robustly linked to the pathogenesis of a number of neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal dementia. While mutations in the TARDBP gene that codes for the protein have been identified as causing disease in a small subset of patients, TDP-43 proteinopathy is present in the majority of cases regardless of mutation status. This raises key questions regarding the mechanisms by which TDP-43 proteinopathy arises and spreads throughout the central nervous system. Numerous studies have explored the role of a variety of cellular functions on the disease process, and nucleocytoplasmic transport, protein homeostasis, RNA interactions and cellular stress have all risen to the forefront as possible contributors to the initiation of TDP-43 pathogenesis. There is also a small but growing body of evidence suggesting that aggregation-prone TDP-43 can recruit physiological TDP-43, and be transmitted intercellularly, providing a mechanism whereby small-scale proteinopathy spreads from cell to cell, reflecting the spread of clinical symptoms observed in patients. This review will discuss the potential role of the aforementioned cellular functions in TDP-43 pathogenesis, and explore how aberrant pathology may spread, and result in a feed-forward cascade effect, leading to robust TDP-43 proteinopathy and disease.     

Dysregulated Expression of Transposable Elements in TDP-43M337V Human Motor Neurons That Recapitulate Amyotrophic Lateral Sclerosis In Vitro

Amyotrophic lateral sclerosis (ALS) is a disease that progressively annihilates spinal cord motor neurons, causing severe motor decline and death. The disease is divided into familial and sporadic ALS. Mutations in the TAR DNA binding protein 43 (TDP-43) have been involved in the pathological emergence and progression of ALS, although the molecular mechanisms eliciting the disease are unknown. Transposable elements (TEs) and DNA sequences capable of transposing within the genome become dysregulated and transcribed in the presence of TDP-43 mutations. We performed RNA-Seq in human motor neurons (iMNs) derived from induced pluripotent stem cells (iPSCs) from TDP-43 wild-type—iMNs-TDP-43^WT—and mutant—iMNs-TDP-43^M337V—genotypes at 7 and 14 DIV, and, with state-of-the-art bioinformatic tools, analyzed whether TDP-43^M337V alters both gene expression and TE activity. Our results show that TDP-43^M337V induced global changes in the gene expression and TEs levels at all in vitro stages studied. Interestingly, many genetic pathways overlapped with that of the TEs activity, suggesting that TEs control the expression of several genes. TEs correlated with genes that played key roles in the extracellular matrix and RNA processing: all the regulatory pathways affected in ALS. Thus, the loss of TE regulation is present in TDP-43 mutations and is a critical determinant of the disease in human motor neurons. Overall, our results support the evidence that indicates TEs are critical regulatory sequences contributing to ALS neurodegeneration.     

Molecular Mechanisms Underlying TDP-43 Pathology in Cellular and Animal Models of ALS and FTLD

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are neurodegenerative disorders that exist on a disease spectrum due to pathological, clinical and genetic overlap. In up to 97% of ALS cases and ~50% of FTLD cases, the primary pathological protein observed in affected tissues is TDP-43, which is hyperphosphorylated, ubiquitinated and cleaved. The TDP-43 is observed in aggregates that are abnormally located in the cytoplasm. The pathogenicity of TDP-43 cytoplasmic aggregates may be linked with both a loss of nuclear function and a gain of toxic functions. The cellular processes involved in ALS and FTLD disease pathogenesis include changes to RNA splicing, abnormal stress granules, mitochondrial dysfunction, impairments to axonal transport and autophagy, abnormal neuromuscular junctions, endoplasmic reticulum stress and the subsequent induction of the unfolded protein response. Here, we review and discuss the evidence for alterations to these processes that have been reported in cellular and animal models of TDP-43 proteinopathy.     

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.     

DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1^G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.     

Regulation of Endoplasmic Reticulum–Mitochondria Tethering and Ca2+ Fluxes by TDP-43 via GSK3β

Mitochondria–ER contacts (MERCs), tightly regulated by numerous tethering proteins that act as molecular and functional connections between the two organelles, are essential to maintain a variety of cellular functions. Such contacts are often compromised in the early stages of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). TDP-43, a nuclear protein mainly involved in RNA metabolism, has been repeatedly associated with ALS pathogenesis and other neurodegenerative diseases. Although TDP-43 neuropathological mechanisms are still unclear, the accumulation of the protein in cytoplasmic inclusions may underlie a protein loss-of-function effect. Accordingly, we investigated the impact of siRNA-mediated TDP-43 silencing on MERCs and the related cellular parameters in HeLa cells using GFP-based probes for MERCs quantification and aequorin-based probes for local Ca²⁺ measurements, combined with targeted protein and mRNA profiling. Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca²⁺ uptake after ER Ca²⁺ release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca²⁺-mediated ER–mitochondria cross-talk, nor alterations of mitochondrial density and morphology were observed by confocal microscopy. Further mechanistic inspections, however, suggested that the observed cellular alterations are correlated to increased expression/activity of GSK3β, previously associated with MERCs disruption.     

Expression of Huntingtin and TDP-43 Derivatives in Fission Yeast Can Cause Both Beneficial and Toxic Effects

Many neurodegenerative disorders display protein aggregation as a hallmark, Huntingtin and TDP-43 aggregates being characteristic of Huntington disease and amyotrophic lateral sclerosis, respectively. However, whether these aggregates cause the diseases, are secondary by-products, or even have protective effects, is a matter of debate. Mutations in both human proteins can modulate the structure, number and type of aggregates, as well as their toxicity. To study the role of protein aggregates in cellular fitness, we have expressed in a highly tractable unicellular model different variants of Huntingtin and TDP-43. They each display specific patterns of aggregation and toxicity, even though in both cases proteins have to be very highly expressed to affect cell fitness. The aggregation properties of Huntingtin, but not of TDP-43, are affected by chaperones such as Hsp104 and the Hsp40 couple Mas5, suggesting that the TDP-43, but not Huntingtin, derivatives have intrinsic aggregation propensity. Importantly, expression of the aggregating form of Huntingtin causes a significant extension of fission yeast lifespan, probably as a consequence of kidnapping chaperones required for maintaining stress responses off. Our study demonstrates that in general these prion-like proteins do not cause toxicity under normal conditions, and in fact they can protect cells through indirect mechanisms which up-regulate cellular defense pathways.     

Different Intermolecular Interactions Drive Nonpathogenic Liquid–Liquid Phase Separation and Potentially Pathogenic Fibril Formation by TDP-43

The liquid–liquid phase separation (LLPS) of proteins has been found ubiquitously in eukaryotic cells, and is critical in the control of many biological processes by forming a temporary condensed phase with different bimolecular components. TDP-43 is recruited to stress granules in cells and is the main component of TDP-43 granules and proteinaceous amyloid inclusions in patients with amyotrophic lateral sclerosis (ALS). TDP-43 low complexity domain (LCD) is able to de-mix in solution, forming the protein condensed droplets, and amyloid aggregates would form from the droplets after incubation. The molecular interactions regulating TDP-43 LCD LLPS were investigated at the protein fusion equilibrium stage, when the droplets stopped growing after incubation. We found the molecules in the droplet were still liquid-like, but with enhanced intermolecular helix–helix interactions. The protein would only start to aggregate after a lag time and aggregate slower than at the condition when the protein does not phase separately into the droplets, or the molecules have a reduced intermolecular helix–helix interaction. In the protein condensed droplets, a structural transition intermediate toward protein aggregation was discovered involving a decrease in the intermolecular helix–helix interaction and a reduction in the helicity. Our results therefore indicate that different intermolecular interactions drive LLPS and fibril formation. The discovery that TDP-43 LCD aggregation was faster through the pathway without the first protein phase separation supports that LLPS and the intermolecular helical interaction could help maintain the stability of TDP-43 LCD.     

eIF3a Destabilization and TDP-43 Alter Dynamics of Heat-Induced Stress Granules

Stress granules (SGs) are membrane-less assemblies arising upon various stresses in eukaryotic cells. They sequester mRNAs and proteins from stressful conditions and modulate gene expression to enable cells to resume translation and growth after stress relief. SGs containing the translation initiation factor eIF3a/Rpg1 arise in yeast cells upon robust heat shock (HS) at 46 °C only. We demonstrate that the destabilization of Rpg1 within the PCI domain in the Rpg1-3 variant leads to SGs assembly already at moderate HS at 42 °C. These are bona fide SGs arising upon translation arrest containing mRNAs, which are components of the translation machinery, and associating with P-bodies. HS SGs associate with endoplasmatic reticulum and mitochondria and their contact sites ERMES. Although Rpg1-3-labeled SGs arise at a lower temperature, their disassembly is delayed after HS at 46 °C. Remarkably, the delayed disassembly of HS SGs after the robust HS is reversed by TDP-43, which is a human protein connected with amyotrophic lateral sclerosis. TDP-43 colocalizes with HS SGs in yeast cells and facilitates cell regrowth after the stress relief. Based on our results, we propose yeast HS SGs labeled by Rpg1 and its variants as a novel model system to study functions of TDP-43 in stress granules disassembly.     

Molecular Alterations in Sporadic and SOD1-ALS Immortalized Lymphocytes: Towards a Personalized Therapy

Amyotrophic lateral sclerosis (ALS) is a fatal neurological condition where motor neurons (MNs) degenerate. Most of the ALS cases are sporadic (sALS), whereas 10% are hereditarily transmitted (fALS), among which mutations are found in the gene that codes for the enzyme superoxide dismutase 1 (SOD1). A central question in ALS field is whether causative mutations display selective alterations not found in sALS patients, or they converge on shared molecular pathways. To identify specific and common mechanisms for designing appropriate therapeutic interventions, we focused on the SOD1-mutated (SOD1-ALS) versus sALS patients. Since ALS pathology involves different cell types other than MNs, we generated lymphoblastoid cell lines (LCLs) from sALS and SOD1-ALS patients and healthy donors and investigated whether they show changes in oxidative stress, mitochondrial dysfunction, metabolic disturbances, the antioxidant NRF2 pathway, inflammatory profile, and autophagic flux. Both oxidative phosphorylation and glycolysis appear to be upregulated in lymphoblasts from sALS and SOD1-ALS. Our results indicate significant differences in NRF2/ARE pathway between sALS and SOD1-ALS lymphoblasts. Furthermore, levels of inflammatory cytokines and autophagic flux discriminate between sALS and SOD1-ALS lymphoblasts. Overall, different molecular mechanisms are involved in sALS and SOD1-ALS patients and thus, personalized medicine should be developed for each case.     

Pathological 25 kDa C-Terminal Fragments of TDP-43 Are Present in Lymphoblastoid Cell Lines and Extracellular Vesicles from Patients Affected by Frontotemporal Lobar Degeneration and Neuronal Ceroidolipofuscinosis Carrying a GRN Mutation

Frontotemporal lobar degeneration (FTLD) is a complex disease, characterized by progressive degeneration of frontal and temporal lobes. Mutations in progranulin (GRN) gene have been found in up to 50% of patients with familial FTLD. Abnormal deposits of post-translationally-modified TAR DNA-binding protein of 43 kDa (TDP-43) represent one of the main hallmarks of the brain pathology. To investigate in peripheral cells the presence of the different TDP-43 forms, especially the toxic 25 kDa fragments, we analyzed lymphoblastoid cell lines (LCLs) and the derived extracellular vesicles (EVs) from patients carrying a GRN mutation, together with wild-type (WT) healthy controls. After characterizing EV sizes and concentrations by nanoparticle tracking analysis, we investigated the levels of different forms of the TDP-43 protein in LCLs and respective EVs by Western blot. Our results showed a trend of concentration decreasing in EVs derived from GRN-mutated LCLs, although not reaching statistical significance. A general increase in p-TDP-43 levels in GRN-mutated LCLs and EVs was observed. In particular, the toxic 25 kDa fragments of p-TDP-43 were only present in GRN-mutated LCLs and were absent in the WT controls. Furthermore, these fragments appeared to be more concentrated in EVs than in LCLs, suggesting a relevant role of EVs in spreading pathological molecules between cells.     

Long-Term Effects of Repetitive Mild Traumatic Injury on the Visual System in Wild-Type and TDP-43 Transgenic Mice

Little is known about the impairments and pathological changes in the visual system in mild brain trauma, especially repetitive mild traumatic brain injury (mTBI). The goal of this study was to examine and compare the effects of repeated head impacts on the neurodegeneration, axonal integrity, and glial activity in the optic tract (OT), as well as on neuronal preservation, glial responses, and synaptic organization in the lateral geniculate nucleus (LGN) and superior colliculus (SC), in wild-type mice and transgenic animals with overexpression of human TDP-43 mutant protein (TDP-43^G348C) at 6 months after repeated closed head traumas. Animals were also assessed in the Barnes maze (BM) task. Neurodegeneration, axonal injury, and gliosis were detected in the OT of the injured animals of both genotypes. In the traumatized mice, myelination of surviving axons was mostly preserved, and the expression of neurofilament light chain was unaffected. Repetitive mTBI did not induce changes in the LGN and the SC, nor did it affect the performance of the BM task in the traumatized wild-type and TDP-43 transgenic mice. Differences in neuropathological and behavioral assessments between the injured wild-type and TDP-43^G348C mice were not revealed. Results of the current study suggest that repetitive mTBI was associated with chronic damage and inflammation in the OT in wild-type and TDP-43^G348C mice, which were not accompanied with behavioral problems and were not affected by the TDP-43 genotype, while the LGN and the SC remained preserved in the used experimental conditions.     

Therapeutic Treatment of Superoxide Dismutase 1 (G93A) Amyotrophic Lateral Sclerosis Model Mice with Medical Ozone Decelerates Trigeminal Motor Neuron Degeneration,Attenuates Microglial Proliferation,and Preserves Monocyte Levels in Mesenteric Lymph Nodes

Amyotrophic lateral sclerosis (ALS) is an incurable and lethal neurodegenerative disease in which progressive motor neuron loss and associated inflammation represent major pathology hallmarks. Both the prevention of neuronal loss and neuro-destructive inflammation are still unmet challenges. Medical ozone, an ozonized oxygen mixture (O₃/O₂), has been shown to elicit profound immunomodulatory effects in peripheral organs, and beneficial effects in the aging brain. We investigated, in a preclinical drug testing approach, the therapeutic potential of a five-day O₃/O₂ i.p. treatment regime at the beginning of the symptomatic disease phase in the superoxide dismutase (SOD1^G93A) ALS mouse model. Clinical assessment of SOD1^G93A mice revealed no benefit of medical ozone treatment over sham with respect to gross body weight, motor performance, disease duration, or survival. In the brainstem of end stage SOD1^G93A mice, however, neurodegeneration was found decelerated, and SOD1-related vacuolization was reduced in the motor trigeminal nucleus in the O₃/O₂ treatment group when compared to sham-treated mice. In addition, microglia proliferation was less pronounced in the brainstem, while the hypertrophy of astroglia remained largely unaffected. Finally, monocyte numbers were reduced in the blood, spleen, and mesenteric lymph nodes at postnatal day 60 in SOD1^G93A mice. A further decrease in monocyte numbers seen in mesenteric lymph nodes from sham-treated SOD1^G93A mice at an advanced disease stage, however, was prevented by medical ozone treatment. Collectively, our study revealed a select neuroprotective and possibly anti-inflammatory capacity for medical ozone when applied as a therapeutic agent in SOD1^G93A ALS mice.     

Exposure to Environmental Toxicants and Pathogenesis of Amyotrophic Lateral Sclerosis: State of the Art and Research Perspectives

There is a broad scientific consensus that amyotrophic lateral sclerosis (ALS), a fatal neuromuscular disease, is caused by gene-environment interactions. In fact, given that only about 10% of all ALS diagnosis has a genetic basis, gene-environmental interaction may give account for the remaining percentage of cases. However, relatively little attention has been paid to environmental and lifestyle factors that may trigger the cascade of motor neuron degeneration leading to ALS, although exposure to chemicals—including lead and pesticides—agricultural environments, smoking, intense physical activity, trauma and electromagnetic fields have been associated with an increased risk of ALS. This review provides an overview of our current knowledge of potential toxic etiologies of ALS with emphasis on the role of cyanobacteria, heavy metals and pesticides as potential risk factors for developing ALS. We will summarize the most recent evidence from epidemiological studies and experimental findings from animal and cellular models, revealing that potential causal links between environmental toxicants and ALS pathogenesis have not been fully ascertained, thus justifying the need for further research.     

Optimized Protocol for Proportionate CNS Cell Retrieval as a Versatile Platform for Cellular and Molecular Phenomapping in Aging and Neurodegeneration

Efficient purification of viable neural cells from the mature CNS has been historically challenging due to the heterogeneity of the inherent cell populations. Moreover, changes in cellular interconnections, membrane lipid and cholesterol compositions, compartment-specific biophysical properties, and intercellular space constituents demand technical adjustments for cell isolation at different stages of maturation and aging. Though such obstacles are addressed and partially overcome for embryonic premature and mature CNS tissues, procedural adaptations to an aged, progeroid, and degenerative CNS environment are underrepresented. Here, we describe a practical workflow for the acquisition and phenomapping of CNS neural cells at states of health, physiological and precocious aging, and genetically provoked neurodegeneration. Following recent, unprecedented evidence of post-mitotic cellular senescence (PoMiCS), the protocol appears suitable for such de novo characterization and phenotypic opposition to classical senescence. Technically, the protocol is rapid, efficient as for cellular yield and well preserves physiological cell proportions. It is suitable for a variety of downstream applications aiming at cell type-specific interrogations, including cell culture systems, Flow-FISH, flow cytometry/FACS, senescence studies, and retrieval of omic-scale DNA, RNA, and protein profiles. We expect suitability for transfer to other CNS targets and to a broad spectrum of engineered systems addressing aging, neurodegeneration, progeria, and senescence.