TDP-43 In Amyotrophic Lateral Sclerosis Research Articles

Colocalization of TDP-43 and stress granules at the early stage of TDP-43 aggregation in amyotrophic lateral sclerosis.

TDP-43 aggregates (skeins and round inclusions [RIs]) are frequent histopathological features of amyotrophic lateral sclerosis (ALS). We have shown that diffuse punctate cytoplasmic staining (DPCS) is the earliest pathologic manifestation of TDP-43 in ALS, corresponding to nonfibrillar TDP-43 located in the rough endoplasmic reticulum. Previous in vitro studies have suggested that TDP-43 inclusions may be derived from stress granules (SGs). Therefore, we investigated the involvement of SGs in the formation of TDP-43 inclusions. Formalin-fixed spinal cords of six ALS patients with a disease duration of less than 1 year (short duration), eight patients with a disease duration of 2-5 years (standard duration), and five normal controls were subjected to histopathological examination using antibodies against an SG marker, HuR. In normal controls, the cytoplasm of anterior horn cells was diffusely HuR-positive. In short-duration and standard-duration ALS, the number of HuR-positive anterior horn cells was significantly decreased relative to the controls. DPCS and RIs were more frequent in short-duration ALS than in standard-duration ALS. The majority of DPCS areas and a small proportion of RIs, but not skeins, were positive for HuR. Immunoelectron microscopy showed that ribosome-like granular structures in DPCS areas and RIs were labeled with anti-HuR, whereas skeins were not. These findings suggest that colocalization of TDP-43 and SGs occurs at the early stage of TDP-43 aggregation.

Low T-cell reactivity to TDP-43 peptides in ALS.

Dysregulation of the immune system in amyotrophic lateral sclerosis (ALS) includes changes in T-cells composition and infiltration of T cells in the brain and spinal cord. Recent studies have shown that cytotoxic T cells can directly induce motor neuron death in a mouse model of ALS and that T cells from ALS patients are cytotoxic to iPSC-derived motor neurons from ALS patients. Furthermore, a clonal expansion to unknown epitope(s) was recently found in familial ALS and increased peripheral and intrathecal activation of cytotoxic CD8+ T cells in sporadic ALS. Here, we show an increased activation of peripheral T cells from patients with sporadic ALS by IL-2 treatment, suggesting an increase of antigen-experienced T cells in ALS blood. However, a putative antigen for T-cell activation in ALS has not yet been identified. Therefore, we investigated if peptides derived from TDP-43, a key protein in ALS pathogenesis, can act as epitopes for antigen-mediated activation of human T cells by ELISPOT and flow cytometry. We found that TDP-43 peptides induced only a weak MHCI or MHCII-restricted activation of both naïve and antigen-experienced T cells from healthy controls and ALS patients. Interestingly, we found less activation in T cells from ALS patients to TDP-43 and control stimuli. Furthermore, we found no change in the levels of naturally occurring auto-antibodies against full-length TDP-43 in ALS. Our data suggests a general increase in antigen-experienced T cells in ALS blood, measured by in-vitro culture with IL-2 for 14 days. Furthermore, it suggests that TDP-43 is a weak autoantigen.

Lattice Light-Sheet Microscopy as a Key Method for the Analysis of the Mislocalization of TDP-43 in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease) is a neurodegenerative disorder that is characterized by the progressive loss of motor neurons. Consequently, patients afflicted with this disease eventually lose the motor function associated with moving, speaking, eating, and even breathing. One neuroscience team identified a common mechanism in nearly all sporadic ALS patients in which transactive response DNA binding protein 43 kDa (TDP-43) was mislocalized from the nucleus to the cytoplasm. This suggests that the mislocalization of TDP-43 into the cytoplasm is central for the onset of ALS in patients, but the underlying factors that result in this process are not fully understood as there are no current laboratory methods to study the progression of TDP-43 mislocalization. However, the development and refinement of lattice light-sheet microscopy (LLSM) as a laboratory technique has significant potential in helping scientists elucidate the unknown processes behind ALS. This review discusses the implications of lattice light-sheet microscopy and its optimization methods as a solution to the issue and identifies LLSM as a key method that could be significant in the field of ALS research.

Head-to-Head Comparison of Tau-PET Radioligands for Imaging TDP-43 in Post-Mortem ALS Brain.

In vivo detection of transactivation response element DNA binding protein-43kDa (TDP-43) aggregates through positron emission tomography (PET) would impact the ability to successfully develop therapeutic interventions for a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The purpose of the present study is to evaluate the ability of six tau PET radioligands to bind to TDP-43 aggregates in post-mortem brain tissues from ALS patients. Herein, we report the first head-to-head evaluation of six tritium labeled isotopologs of tau-targeting PET radioligands, [3H]MK-6240 (a.k.a. florquinitau), [3H]Genentech Tau Probe-1 (GTP-1), [3H]JNJ-64326067(JNJ-067), [3H]CBD-2115, [3H]flortaucipir, and [3H]APN-1607, and their ability to bind to the β-pleated sheet structures of aggregate TDP-43 in post-mortem ALS brain tissues by autoradiography and immunostaining methods. Post-mortem frontal cortex, motor cortex, and cerebellum tissues were evaluated, and binding intensity was aligned with areas of elevated phosphorylated tau (ptau), pTDP-43, and β-amyloid. Negligible binding was observed with [3H]MK-6240, [3H]JNJ-067, and [3H]GTP-1. While [3H]CBD-2115 displayed marginal specific binding, this binding did not significantly correlate with the distribution of pTDP-43 and AT8 inclusions. Of the remaining ligands, the distribution of [3H]flortaucipir did not significantly correlate to pTDP-43 pathology; however, specific binding trends to a positive relationship with tau. Finally, [3H]APN-1607 relates most strongly to amyloid load and does not indicate pTDP-43 pathology as confirmed by [3H]PiB distribution in sister sections. Our results demonstrate the prominent nature of mixed pathology in ALS, and do not support the application of [3H]MK-6240, [3H]JNJ-067, [3H]GTP-1, [3H]CBD-2115, [3H]flortaucipir, or [3H]APN-1607 for selective imaging TDP-43 in ALS for clinical research with the currently available in vitro data. Identification of potent and selective radiotracers for TDP-43 remains an ongoing challenge.

Biomarkers in Neurodegenerative Diseases: Proteomics Spotlight on ALS and Parkinson's Disease.

Neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) are both characterized by pathogenic protein aggregates that correlate with the progressive degeneration of neurons and the loss of behavioral functions. Both diseases lack biomarkers for diagnosis and treatment efficacy. Proteomics is an unbiased quantitative tool capable of the high throughput quantitation of thousands of proteins from minimal sample volumes. We review recent proteomic studies in human tissues, plasma, cerebrospinal fluid (CSF), and exosomes in ALS and PD that identify proteins with potential utility as biomarkers. Further, we review disease-related post-translational modifications in key proteins TDP43 in ALS and α-synuclein in PD studies, which may serve as biomarkers. We compare relative and absolute quantitative proteomic approaches in key biomarker studies in ALS and PD and discuss recent technological advancements which may identify suitable biomarkers for the early-diagnosis treatment efficacy of these diseases.

Exploiting Caenorhabditis elegans to discover human gut microbiota-mediated intervention strategies in protein conformational diseases.

Exploiting Caenorhabditis elegans to discover human gut microbiota-mediated intervention strategies in protein conformational diseases.

Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins

Mislocalization of the predominantly nuclear RNA/DNA binding protein, TDP-43, occurs in motor neurons of ~95% of amyotrophic lateral sclerosis (ALS) patients, but the contribution of axonal TDP-43 to this neurodegenerative disease is unclear. Here, we show TDP-43 accumulation in intra-muscular nerves from ALS patients and in axons of human iPSC-derived motor neurons of ALS patient, as well as in motor neurons and neuromuscular junctions (NMJs) of a TDP-43 mislocalization mouse model. In axons, TDP-43 is hyper-phosphorylated and promotes G3BP1-positive ribonucleoprotein (RNP) condensate assembly, consequently inhibiting local protein synthesis in distal axons and NMJs. Specifically, the axonal and synaptic levels of nuclear-encoded mitochondrial proteins are reduced. Clearance of axonal TDP-43 or dissociation of G3BP1 condensates restored local translation and resolved TDP-43-derived toxicity in both axons and NMJs. These findings support an axonal gain of function of TDP-43 in ALS, which can be targeted for therapeutic development.

Genoppi is an open-source software for robust and standardized integration of proteomic and genetic data

Combining genetic and cell-type-specific proteomic datasets can generate biological insights and therapeutic hypotheses, but a technical and statistical framework for such analyses is lacking. Here, we present an open-source computational tool called Genoppi (lagelab.org/genoppi) that enables robust, standardized, and intuitive integration of quantitative proteomic results with genetic data. We use Genoppi to analyze 16 cell-type-specific protein interaction datasets of four proteins (BCL2, TDP-43, MDM2, PTEN) involved in cancer and neurological disease. Through systematic quality control of the data and integration with published protein interactions, we show a general pattern of both cell-type-independent and cell-type-specific interactions across three cancer cell types and one human iPSC-derived neuronal cell type. Furthermore, through the integration of proteomic and genetic datasets in Genoppi, our results suggest that the neuron-specific interactions of these proteins are mediating their genetic involvement in neurodegenerative diseases. Importantly, our analyses suggest that human iPSC-derived neurons are a relevant model system for studying the involvement of BCL2 and TDP-43 in amyotrophic lateral sclerosis.

"STRESSED OUT": The role of FUS and TDP-43 in amyotrophic lateral sclerosis.

Mutations in fused-in-sarcoma (FUS) and TAR DNA binding protein-43 (TDP-43; TARDBP) are known to cause the severe adult-onset neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Proteinopathy caused by cellular stresses such as endoplasmic reticulum (ER) stress, oxidative stress, mitochondrial stress and proteasomal stress and the formation of stress granules (SGs), cytoplasmic aggregates and inclusions is a hallmark of ALS. FUS and TDP-43, which are DNA/RNA binding proteins that regulate transcription, RNA homeostasis and protein translation are implicated in ALS proteinopathy. Disease-causing mutations in FUS and TDP-43 cause sequestration of these proteins and their interacting partners in the cytoplasm, which leads to aggregation. This mislocalization and formation of aggregates and SGs is cytotoxic and a contributor to neuronal death. We explore how loss-of-nuclear-function and gain-of-cytoplasmic function mechanisms that affect FUS and TPD-43 localization can generate a 'stressed out' neuronal pathology and proteinopathy that drives ALS progression.

Targeting TDP-43 proteinopathy with drugs and drug-like small molecules.

Following the discovery of the involvement of the ribonucleoprotein TDP-43 in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), a major research focus has been to develop treatments that can prevent or alleviate these disease conditions. One pharmacological approach has been to use TDP-43-based disease models to test small molecules and drugs already known to have some therapeutic effect in a variety of neurodegenerative conditions. In parallel, various disease models have been used to perform high-throughput screens of drugs and small compound libraries. The aim of this review will be to provide a general overview of the compounds that have been described to alter pathological characteristics of TDP-43. These include expression levels, cytoplasmic mis-localization, post-translational modifications, cleavage, stress granule recruitment and aggregation. In parallel, this review will also address the use of compounds that modify the autophagic/proteasome systems that are known to target TDP-43 misfolding and aggregation. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.6/issuetoc.

RNA and Protein Interactors with TDP-43 in Human Spinal-Cord Lysates in Amyotrophic Lateral Sclerosis.

The TAR DNA-binding protein of 43 kDa (TDP-43) is a dual function RNA- and DNA-binding protein with varied cellular functions. In degenerating motor neurons in amyotrophic lateral sclerosis (ALS), TDP-43 relocalizes from the nucleus to the cytosol, where it is sequestered into inclusions. It is likely that the pathogenic role of TDP-43 in ALS can involve either a gain or a loss of function, depending on the nature of its RNA or protein interactor. However, while TDP-43 binding partners have been identified in a range of model systems and from the human brain, interactors from human spinal-cord tissue have not. In this study, we have characterized both protein and RNA TDP-43 interactors from neuropathologically normal (control) and ALS-affected ventral lumbar spinal cord, including sporadic ALS (sALS) and familial cases harboring either a A4T mutant SOD1 or a 3' UTR *c.41G>A mutant FUS/TLS or expressing pathological c9orf72 expanded repeats. RNA interactors with TDP-43 were similar between the control and ALS spinal cords examined regardless of genotype. In contrast, protein interactors with TDP-43 did demonstrate differences, with the sALS and mtSOD1 harboring cases examined differing from the protein interactors identified in the FUS 3' UTR mutation and c9orf72 repeat-positive cases.

The role of ubiquitinated TDP-43 in amyotrophic lateral sclerosis

The role of ubiquitinated TDP-43 in amyotrophic lateral sclerosis

Sonic Hedgehog and TDP-43 Participate in the Spontaneous Locomotor Recovery in a Mouse Model of Spinal Motoneuron Disease

Several studies have attempted to repair the damaged spinal cord (SC) by stimulating neurogenesis or neuroplasticity, with limited success. Sonic hedgehog (Shh) is involved in neural induction and stem cell functioning, but recent findings also suggest its role in regeneration and functional recovery. Transactive response DNA-binding protein of 43 kDa (TDP-43) is a nuclear DNA/RNA binding protein involved in transcription and RNA processing. Recent findings have reported cytoplasmic inclusions containing TDP-43 in amyotrophic lateral sclerosis. Although substantial attention has been given to the toxic effects of this protein, the functional role of TDP-43 remains largely unclear. We used a mouse model of neurotoxic motoneuron depletion to study the role of the above-described factors in the compensatory changes occurring after the lesion. The injection of cholera toxin-B saporin into the gastrocnemius muscle caused a partial motoneuron death accompanied by an impairment of locomotion. Interestingly, motor activity was significantly restored as soon as one month later. Moreover, we observed an activity-dependent modification of Shh and synaptic proteins: synapsin-I and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Notably, the motor performance of lesioned animals correlated with the expression of synapsin-I and Shh. Conversely, the expression of Shh significantly correlated with the levels of synapsin-I, GluR2, and TDP-43. The results suggest that Shh and TDP-43 are crucial parts of a complex mechanism of neuroplasticity in a mouse model of SC motoneuron disease.

In vitro prion-like behaviour of TDP-43 in ALS

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND), and >95% of familial and sporadic cases involve the deposition of insoluble aggregated, phosphorylated and cleaved TDP-43 protein. Accumulating clinical and biological evidence now indicates that ALS bears a number of similarities to the prion diseases, with TDP-43 acting as a misfolded ‘prion-like’ protein demonstrating similar underlying pathobiology. Here we systematically address the hypothesis that ALS is a prion-like disorder. First we demonstrate that TDP-43 demonstrates seeded polymerisation in vitro directly from both ALS brain and spinal cord. We next show that the seeding of TDP-43 results in the formation of characteristic insoluble, aggregated, and phosphorylated TDP-43 pathology that directly recapitulates the morphological diversity of TDP-43 inclusions detected in ALS patient CNS tissue. We next demonstrate that this reaction can be serially propagated to produce increasing amounts of phosphorylated TDP-43 pathology, and that aggregates can spread from cell to cell in an analogous fashion to that seen in the prion diseases. Finally, we reproduced our findings in a murine motor neuron-like cell line (NSC-34), where the seeding of TDP-43 induces the formation of TDP-43 oligomers and reduced cell viability. These findings may guide therapeutic strategies in this rapidly progressive and invariably fatal disease.

CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS.

The molecular machinery responsible for cytosolic accumulation of misfolded TDP-43 in amyotrophic lateral sclerosis (ALS) remains elusive. Here we identified a cullin-2 (CUL2) RING complex as a novel ubiquitin ligase for fragmented forms of TDP-43. The von Hippel Lindau protein (VHL), a substrate binding component of the complex, preferentially recognized misfolded TDP-43 at Glu246 in RNA-recognition motif 2. Recombinant full-length TDP-43 was structurally fragile and readily cleaved, suggesting that misfolded TDP-43 is cleared by VHL/CUL2 in a step-wise manner via fragmentation. Surprisingly, excess VHL stabilized and led to inclusion formation of TDP-43, as well as mutant SOD1, at the juxtanuclear protein quality control center. Moreover, TDP-43 knockdown elevated VHL expression in cultured cells, implying an aberrant interaction between VHL and mislocalized TDP-43 in ALS. Finally, cytoplasmic inclusions especially in oligodendrocytes in ALS spinal cords were immunoreactive to both phosphorylated TDP-43 and VHL. Thus, our results suggest that an imbalance in VHL and CUL2 may underlie oligodendrocyte dysfunction in ALS, and highlight CUL2 E3 ligase emerges as a novel therapeutic potential for ALS.

筋萎縮性側索硬化症におけるTDP-43 の機能異常

筋萎縮性側索硬化症におけるTDP-43 の機能異常

TDP-43 in amyotrophic lateral sclerosis - is it a prion disease?

Amyotrophic lateral sclerosis is a devastating disease characterized by rapidly progressive paresis. The neuropathological hallmark of most amyotrophic lateral sclerosis cases are neuronal and glial aggregates of phosphorylated 43-kDa TAR DNA-binding protein (pTDP-43). The accumulation of similar proteins into insoluble aggregates is now recognized as a common pathological hallmark of neurodegenerative diseases in general. Importantly, many of these proteins such as tau and amyloid-β in Alzheimer's disease and α-synuclein in Parkinson's show a stereotypical sequential distribution pattern with progressing disease. In this review, we discuss recent evidence that TDP-43 in ALS may propagate similarly to other neurodegenerative disease proteins. We furthermore delineate similarities and important differences of TDP-43 proteinopathies to prion diseases.

Novel atomic force microscopy based biopanning for isolation of morphology specific reagents against TDP-43 variants in amyotrophic lateral sclerosis.

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson's disease and beta-amyloid and tau in Alzheimer's disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson’s disease and beta-amyloid and tau in Alzheimer’s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

The neurobiology of dysautonomia in Parkinson's disease

Neurodegenerative diseases (NDs) include a large variety of disorders that affects specific areas of the central nervous system, leading to psychiatric and movement pathologies. A common feature that characterizes these disorders is the neuronal formation and accumulation of misfolded protein aggregates that lead to cell death. In particular, different proteinaceous aggregates accumulate to trigger a variety of clinical manifestations: prion protein (PrPSc) in prion diseases, β-amyloid (Aβ) in Alzheimer’s disease (AD), α-synuclein in Parkinson’s disease (PD), huntingtin in Huntington’s disease (HD), superoxide dismutase and TDP-43 in amyotrophic lateral sclerosis (ALS), tau in tauopathies. Non-motor alterations also occur in several viscera, in particular the gastrointestinal tract. These often precede the onset of motor symptoms by several years. For this reason, dysautonomic changes can be predictive of NDs and their correct recognition is being assuming a remarkable importance. This peculiar feature led more and more to the concept that neurodegeneration may initiate in the periphery and propagate retrogradely towards the central nervous system in a prion-like manner. In recent years, a particular attention was dedicated to the clinical assessment of autonomic disorders in patients affected by NDs. In this respect, experimental animal models have been developed to understand the neurobiology underlying these effects as well as to investigate autonomic changes in peripheral organs. This review summarizes experimental studies that have been carried out to understand autonomic symptoms in NDs, with the purpose to provide appropriate tools for comprehensive and integrated studies.