Molecular basis of ALS and FTD: implications for translational studies.

A hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic ALS in Taiwan

To assess the amount of phosphorylated and nonphosphorylated TAR DNA-binding protein 43 (TDP-43) in the motor brain regions of cases of amyotrophic lateral sclerosis (ALS) with and without repeat expansions in the ATXN2 or C9ORF72 genes. The 45-kDa phosphorylated form of TDP-43 and 43-kDa nonphosphorylated form of TDP-43 were quantified by immunoblot in postmortem brain tissue from the motor cortex, spinal cord, and cerebellar vermis of 23 cases with ALS with repeat expansions in the ATXN2 or C9ORF72 genes and sporadic disease and 10 controls. Significantly greater levels of phosphorylated TDP-43 were identified in the motor cortex of cases with ALS with C9ORF72 expansions, and significantly greater amounts of phosphorylated TDP-43 were found in the spinal cord of cases with ALS with intermediate ATXN2 expansions. In contrast, however, similar levels of nonphosphorylated TDP-43 were found in all 3 regions between ALS groups. Despite its central role in the pathogenesis of ALS and the emergence of potential targets to modify its aggregation, TDP-43 levels have not been quantified in pathologically confirmed cases with ALS. The present results demonstrating significant differences in phosphorylated but not nonphosphorylated TDP-43 levels suggest that different posttranslational modifications are involved in the generation of greater pathologic TDP-43 levels identified here in our cohort of cases with genetic expansions. These findings are consistent with emerging studies implicating distinct pathomechanisms in the generation of pathologic TDP-43 in cases with ALS with C9ORF72 or ATXN2 expansions and are of relevance to therapeutic research aimed at reducing pathologic TDP-43 in all or a subset of patients with ALS.

Tar DNA-binding protein-43 (TDP-43) regulates axon growth in vitro and in vivo

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is characterized with progressive muscle atrophy. We have attempted to establish the link between angiogenesis and cellular survival in the pathogenesis of ALS by compiling evidence described in various scientific reports. The phenotypes of human ALS have earlier been captured in the mutant SOD1 mice as well as by targeted deletion of the hypoxia response element (HRE) from the promoter of the mouse gene for vascular endothelial growth factor (VEGF). Indirect evidence shows that angiogenesis can help prevent oxidative stress, and hence, enhance cell survival. VEGF and angiogenin chiefly regulate the process of angiogenesis. Transactive response DNA-binding protein 43 (TDP-43) is usually found inside the nucleus, but in large number of cases of ALS, it accumulates in the cytoplasm (TDP-43 proteinopathy). Interestingly, TDP-43 proteinopathy is found to be aggravated in the presence of the OPTN mutation, which is the genetic factor that is responsible for such accumulation. Interaction of TDP-43 with progranulin can further affect the angiogenesis in ALS patients by regulating activity of VEGF receptors, but conclusive evidence is needed to establish its role in pathogenesis of ALS. Certain mutations in UBQLN2 and UBQLN4 indicate that ubiquitination has a role in ALS pathobiology, but its link to angiogenesis has not been adequately studied. Recent studies have shown that several mutations in RNA-binding proteins (RBPs) can also cause ALS. Conclusively, in this review, we have attempted to argue the role of angiogenesis in enhanced ALS survival rate is probably regulated with the activation of NF-κβ. Additionally, interaction between OPTN and TDP-43 can also impact the transcription of various angiogenic molecules. Whether targeting angiogenic substances or TDP-43 can provide clues about extending ALS survival rate, in combination with current treatments, can only be evaluated after additional studies.

Case presentation: An 84-year-old hypertensive patient began experiencing dysarthria and progressive dysphagia in 2020. One year later, notable behavioral changes emerged, characterized by aggressiveness, agitation, and disinhibition. These behaviors included repetitive folding and unfolding of clothes, public lifting of her skirt, and attempts to attack family members with a weapon. Notably, these symptoms manifested without significant memory impairment. Neurological examination revealed motor aphasia, paralysis of the orbicularis oris and right eye muscles, jaw drop, atrophic tongue with fasciculations and immobility, and reduced muscle strength (grade 4+) in the right hemibody and left lower limb, and grade 4 in the left upper limb. Fasciculations were observed in the left deltoid and right quadriceps. Deep tendon reflexes were hyperactive, and there was a questionable plantar response on the right. Electromyography indicated axonal degeneration with chronic reinnervation signs in the C5, C6, C7, C8, T1, L4, L5, and S1 myotomes bilaterally, active denervation in the tongue muscles and left C8/T1 myotomes, and no sensory abnormalities. Discussion: Motor neuron disease may precede or follow the onset of FTD, often presenting as the behavioral variant. In patients with classical amyotrophic lateral sclerosis (ALS), characterized by neuromuscular symptoms, 15 to 20% also exhibit cognitive and behavioral symptoms consistent with FTD, particularly the behavioral subtype. These patients frequently present with bulbar symptoms more prominently than those with classical ALS. A hexanucleotide repeat expansion in the C9orf72 gene is the most commonly identified genetic cause of the combined FTD and ALS presentation, accounting for approximately 5-10% of sporadic ALS cases. Other implicated genes include the autosomal dominant FUS gene, responsible for about 2% of sporadic cases, and the rarely recessive TARDBP gene. In this case, the diagnosis of ALS is confirmed by electromyography according to the El Escorial criteria. The 2011 criteria of the International Behavioral Variant FTD Criteria Consortium (FTDC) suggest possible frontotemporal dementia. Final comments: While most patients with FTD do not initially exhibit significant motor symptoms, these can precede the onset of dementia and may result from frontotemporal lobar degeneration. Patients with concurrent FTD and ALS have a shorter survival than those with isolated FTD. Given the strong association of FTD-ALS with the C9orf72 gene expansion, alongside other known genetic factors, genetic testing is recommended but not mandatory for diagnosis.

BackgroundHuntington’s disease (HD) and the amyotrophic lateral sclerosis (ALS)-frontotemporal dementia (FTD) disease spectrum share early features including metabolic, behavioral and sleep disturbances. Studies in both HD and ALS cases show...

BackgroundNeuronal cytoplasmic inclusions containing TAR DNA-binding protein 43 (TDP-43) are a neuropathological feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s Disease (AD). Emerging evidence also indicates that systemic inflammation may be a contributor to the pathology progression of these neurodegenerative diseases.MethodsTo investigate the role of systemic inflammation in the progression of neuronal TDP-43 pathology, AAV9 particles driven by the UCHL1 promoter were delivered to the frontal cortex of wild-type aged mice via intracranial injections to overexpress TDP-43 or green fluorescent protein (GFP) in corticospinal motor neurons. Animals were then subjected to a low-dose (500 μg/kg) intraperitoneal E. coli lipopolysaccharide (LPS) administration challenge for 2 weeks to mimic a chronically altered low-grade systemic inflammatory state. Mice were then subjected to neurobehavioral studies, followed by biochemical and immunohistochemical analyses of the brain tissue.ResultsIn the present study, we report that elevated neuronal TDP-43 levels induced microglial and astrocytic activation in the cortex of injected mice followed by increased RANTES signaling. Moreover, overexpression of TDP-43 exerted abundant mouse immunoglobulin G (IgG), CD3, and CD4+ T cell infiltration as well as endothelial and pericyte activation suggesting increased blood-brain barrier permeability. The BBB permeability in TDP-43 overexpressing brains yielded the frontal cortex vulnerable to the systemic inflammatory response following LPS treatment, leading to marked neutrophil infiltration, neuronal loss, reduced synaptosome-associated protein 25 (SNAP-25) levels, and behavioral impairments in the radial arm water maze (RAWM) task.ConclusionsThese results reveal a novel role for TDP-43 in BBB permeability and leukocyte recruitment, indicating complex intermolecular interactions between an altered systemic inflammatory state and pathologically prone TDP-43 protein to promote disease progression.

TAR DNA binding protein 43 (TDP-43) is a nuclear protein with multiple RNA/DNA binding and editing functions. It is a key protein that causes Amyotrophic lateral sclerosis (ALS), frontotemporal lobe degeneration (FTLD), and other neurodegenerative diseases (NDDs). It is present in the patient’s tissue in the form of abnormal amyloid deposits and has been identified as the cause of the disease. The mutation site of TDP-43 is known to be associated with possible abnormal folding, and it has been determined that the cause of its formation of precipitation is the disruption of the phase separation state. The TDP-43 protein itself is well studied, but its interaction with other genes to cause ALS is relatively scarce. In this paper, the interaction of TDP-43 protein with HSPB1 and UNC13A will be introduced, which indirectly leads to the increased risk of ALS. And whether the association triggered by CE of UNC13A can specifically select and detect TDP-43 lesions at the early stage of the disease. By summarizing part of the pathogenic mechanism of TDP-43, this paper provides a reference for future further studies.

BackgroundAbnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP-43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Researchers have identified 44 mutations in the TARDBP gene that encode TDP-43 as causative for cases of sporadic and familial ALS http://www.molgen.ua.ac.be/FTDMutations/. Certain mutant forms of TDP-43, such as M337V, are associated with increased low molecular weight (LMW) fragments compared to wild-type (WT) TDP-43 and cause neuronal apoptosis and developmental delay in chick embryos. Such findings support a direct link between altered TDP-43 function and neurodegeneration.ResultsTo explore the pathogenic properties of the M337V mutation, we generated and characterized two mouse lines expressing human TDP-43 (hTDP-43M337V) carrying this mutation. hTDP-43M337V was expressed primarily in the nuclei of neurons in the brain and spinal cord, and intranuclear and cytoplasmic phosphorylated TDP-43 aggregates were frequently detected. The levels of TDP-43 LMW products of ~25 kDa and ~35 kDa species were also increased in the transgenic mice. Moreover, overexpression of hTDP-43M337V dramatically down regulated the levels of mouse TDP-43 (mTDP-43) protein and RNA, indicating TDP-43 levels are tightly controlled in mammalian systems. TDP-43M337V mice displayed reactive gliosis, widespread ubiquitination, chromatolysis, gait abnormalities, and early lethality. Abnormal cytoplasmic mitochondrial aggregates and abnormal phosphorylated tau were also detected in the mice.ConclusionOur novel TDP-43M337V mouse model indicates that overexpression of hTDP-43M337V alone is toxic in vivo. Because overexpression of hTDP-43 in wild-type TDP-43 and TDP-43M337V mouse models produces similar phenotypes, the mechanisms causing pathogenesis in the mutant model remain unknown. However, our results suggest that overexpression of the hTDP-43M337V can cause neuronal dysfunction due to its effect on a number of cell organelles and proteins, such as mitochondria and TDP-43, that are critical for neuronal activity. The mutant model will serve as a valuable tool in the development of future studies designed to uncover pathways associated with TDP-43 neurotoxicity and the precise roles TDP-43 RNA targets play in neurodegeneration.

TAR DNA-binding protein 43 (TDP-43) inclusions are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), including cases caused by G4C2 repeat expansions in the C9orf72 gene (c9FTD/ALS). Providing mechanistic insight into the link between C9orf72 mutations and TDP-43 pathology, we demonstrated that a glycine-arginine repeat protein [poly(GR)] translated from expanded G4C2 repeats was sufficient to promote aggregation of endogenous TDP-43. In particular, toxic poly(GR) proteins mediated sequestration of full-length TDP-43 in an RNA-independent manner to induce cytoplasmic TDP-43 inclusion formation. Moreover, in GFP-(GR)200 mice, poly(GR) caused the mislocalization of nucleocytoplasmic transport factors and nuclear pore complex proteins. These mislocalization events resulted in the aberrant accumulation of endogenous TDP-43 in the cytoplasm where it co-aggregated with poly(GR). Last, we demonstrated that treating G4C2 repeat-expressing mice with repeat-targeting antisense oligonucleotides lowered poly(GR) burden, which was accompanied by reduced TDP-43 pathology and neurodegeneration, including lowering of plasma neurofilament light (NFL) concentration. These results contribute to clarification of the mechanism by which poly(GR) drives TDP-43 proteinopathy, confirm that G4C2-targeted therapeutics reduce TDP-43 pathology in vivo, and demonstrate that alterations in plasma NFL provide insight into the therapeutic efficacy of disease-modifying treatments.

The molecular pathogenesis of amyotrophic lateral sclerosis (ALS) has been studied through analysis of the function of the protein produced by the causative genes of familial ALS. The products of these genes are classified as RNA binding proteins, or proteins related to proteolytic systems. However, most case of familial ALS, and sporadic ALS show TAR DNA binding protein-43 (TDP-43) immune-positive cytoplasmic inclusions. Therefore, the molecular mechanism of formation of TDP-43 inclusions and dysfunction caused by TDP-43 inclusions has been studied. As for the mechanism of inclusion formation, non-membrane organelle formation by liquid-liquid phase separation (LLPS) is important. The ubiquitin-proteasome and autophagy systems are important for the degradation of these inclusions. Several genes associated with these systems have been identified as causative genes for ALS. The formation of cytoplasmic inclusions results in the loss of TDP-43 from the nucleus, resulting in abnormalities in RNA metabolism, through the alteration of spliceosomes and Gemini of coiled bodies. Furthermore, in ALS, the regulation of TDP-43 mRNA/protein expression levels has failed. Failure of the autoregulation system facilitates TDP-43 inclusion formation. Development of treatments for ALS based on these elucidated molecular mechanisms is desirable.

TDP-43 (43-kDa TAR DNA-binding domain protein) is a major constituent of ubiquitin-positive cytoplasmic aggregates present in neurons of patients with fronto-temporal lobular dementia and amyotrophic lateral sclerosis (ALS). The pathologic significance of TDP-43 aggregation is not known; however, dominant mutations in TDP-43 cause a subset of ALS cases, suggesting that misfolding and/or altered trafficking of TDP-43 is relevant to the disease process. Here, we show that the presenilin-binding protein ubiquilin 1 (UBQLN) plays a role in TDP-43 aggregation. TDP-43 interacted with UBQLN both in yeast and in vitro, and the carboxyl-terminal ubiquitin-associated domain of UBQLN was both necessary and sufficient for binding to polyubiquitylated forms of TDP-43. Overexpression of UBQLN recruited TDP-43 to detergent-resistant cytoplasmic aggregates that colocalized with the autophagosomal marker, LC3. UBQLN-dependent aggregation required the UBQLN UBA domain, was mediated by non-overlapping regions of TDP-43, and was abrogated by a mutation in UBQLN previously linked to Alzheimer disease. Four ALS-associated alleles of TDP-43 also coaggregated with UBQLN, and the extent of aggregation correlated with in vitro UBQLN binding affinity. Our findings suggest that UBQLN is a polyubiquitin-TDP-43 cochaperone that mediates the autophagosomal delivery and/or proteasome targeting of TDP-43 aggregates.

Abnormal accumulation of TAR DNA-binding protein 43 (TDP-43), a DNA/RNA binding protein, is a pathological signature of amyotrophic lateral sclerosis (ALS). Missense mutations in the TARDBP gene are also found in inherited and sporadic ALS, indicating that dysfunction in TDP-43 is causative for ALS. To model TDP-43-linked ALS in rodents, we generated TDP-43 knock-in mice with inherited ALS patient-derived TDP-43M337V mutation. Homozygous TDP-43M337V mice developed normally without exhibiting detectable motor dysfunction and neurodegeneration. However, splicing of mRNAs regulated by TDP-43 was deregulated in the spinal cords of TDP-43M337V mice. Together with the recently reported TDP-43 knock-in mice with ALS-linked mutations, our finding indicates that ALS patient-derived mutations in the TARDBP gene at a carboxyl-terminal domain of TDP-43 may cause a gain of splicing function by TDP-43, however, were insufficient to induce robust neurodegeneration in mice.

A key pathological feature of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) is the loss of nuclear localization and accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43). TDP-43 is a nucleic acid-binding protein involved in transcriptional repression, mRNA splicing, and the regulation of retrotransposable elements (RTEs) and endogenous retroviruses (ERVs). RTEs/ERVs are mobile virus-like genetic elements that constitute about 45% of our genome and encode the capacity to replicate through an RNA intermediate and insert cDNA copies at de novo chromosomal locations. A causal role of RTEs/ERVs has been demonstrated in Drosophila in mediating both intracellular toxicity of TDP-43 and the intercellular spread of toxicity from glia to neurons. RTEs/ERVs are inappropriately expressed in postmortem tissues from ALS, FTD, and Alzheimer’s Disease (AD) patients, but the role of RTEs/ERVs has not yet been examined in a vertebrate model of TDP-43 pathology. We utilized established transgenic mouse models that overexpress moderate levels of human wild-type TDP-43 or a mutant version with a specific ALS-causal Q331K amino acid substitution, together with a LINE-1-EGFP retrotransposon indicator line. We found that TDP-43 animals exhibit broad expression of RTEs/ERVs with LINE-1 retrotransposition in glia and neurons in the motor cortex. Expression begins with onset of neurological phenotypes, earlier in hTDP-43-Q331K animals and later in hTDP-43-WT. The LINE-1-EGFP retrotransposition reporter transiently labels spatially clustered groups of neurons and glia at the time of onset of motor symptoms, while EGFP-labeled neurons undergo cell death and are therefore lost over time. Unlabeled cells also die as a function of distance from the clusters of LINE-1-EGFP labeled neurons and glial cells. Together, these findings support the hypothesis that TDP-43 pathology triggers RTE/ERV expression in the motor cortex, that such expression marks cells for programmed cell death, with cell non-autonomous effects on nearby neurons and glial cells.

Aggregates of the RNA-binding protein TDP-43 (TAR DNA-binding protein) are a hallmark of the overlapping neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The process of TDP-43 aggregation remains poorly understood, and whether it includes formation of intermediate complexes is unknown. Here, we analyzed aggregates derived from purified TDP-43 under semidenaturing conditions, identifying distinct oligomeric complexes at the initial time points before the formation of large aggregates. We found that this early oligomerization stage is primarily driven by TDP-43's RNA-binding region. Specific binding to GU-rich RNA strongly inhibited both TDP-43 oligomerization and aggregation, suggesting that RNA interactions are critical for maintaining TDP-43 solubility. Moreover, we analyzed TDP-43 liquid-liquid phase separation and detected similar detergent-resistant oligomers upon maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, the ALS-linked TDP-43 mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that aggregates generated from purified TDP-43 seed intracellular aggregation detected by established TDP-43 pathology markers. Remarkably, cytoplasmic aggregate seeding was detected earlier for the A315T and M337V variants and was 50% more widespread than for WT TDP-43 aggregates. We provide evidence for an initial step of TDP-43 self-assembly into intermediate oligomeric complexes, whereby these complexes may provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.