Cytoplasmic TDP-43 Research Articles

Modeling of TDP-43 proteinopathy by chronic oxidative stress identifies rapamycin as beneficial in ALS patient-derived 2D and 3D iPSC models

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized neuropathologically by TDP-43 proteinopathy with loss of TDP-43 nuclear splicing activity and formation of cytoplasmic TDP-43 aggregates. The lack of suitable experimental models of TDP-43 proteinopathy has hampered the discovery of effective therapies. We already showed that chronic and mild oxidative insult by sodium arsenite (ARS) triggered TDP-43 cytoplasmic aggregation and stress granules (SGs) formation in ALS patient-derived fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs). However, whether this insult induces a reduction of TDP-43 splicing activity in the nucleus, thus recapitulating both gain and loss of function pathomechanisms, still remains to be determined.In this study we first showed that chronic ARS in human neuroblastoma cells triggered TDP-43 cytoplasmic mislocalization, SGs formation and defective splicing of TDP-43 target genes UNC13A and POLDIP3 as functional readouts of TDP-43 proteinopathy. Additionally, a dysregulation of autophagy and senescence markers was observed in this condition. In a preliminary drug screening approach with autophagy-promoting drugs, namely rapamycin, lithium carbonate and metformin, only rapamycin prevented ARS-induced loss of TDP-43 splicing activity. We then demonstrated that, in addition to TDP-43 cytoplasmic aggregation, chronic ARS triggered TDP-43 loss of splicing activity also in ALS patient-derived primary fibroblasts and iPSC-MNs and that rapamycin was beneficial to reduce these TDP-43 pathological features. By switching to a neuro-glial 3D in vitro model, we observed that treatment of ALS iPSC-brain organoids with chronic ARS also induced a defective TDP-43 splicing activity which was prevented by rapamycin.Collectively, we established different human cell models of TDP-43 proteinopathy which recapitulate TDP-43 gain and loss of function, prevented by rapamycin administration. Human neuroblastoma cells and patient-derived fibroblasts and 2D- and 3D-iPSC models exposed to chronic oxidative stress represent therefore suitable in vitro platforms for future drug screening approaches in ALS.

Pathogenic TDP-43 accelerates the generation of toxic exon1 HTT in Huntington's disease knock-in mice.

Huntington's disease (HD) is caused by a CAG repeat expansion in exon1 of the HTT gene that encodes a polyglutamine tract in huntingtin protein. The formation of HTT exon1 fragments with an expanded polyglutamine repeat has been implicated as a key step in the pathogenesis of HD. It was reported that the CAG repeat length-dependent aberrant splicing of exon1 HTT results in a short polyadenylated mRNA that is translated into an exon1 HTT protein. Under normal conditions, TDP-43 is predominantly found in the nucleus, where it regulates gene expression. However, in various pathological conditions, TDP-43 is mislocalized in the cytoplasm. By investigating HD knock-in mice, we explore whether the pathogenic TDP-43 in the cytoplasm contributes to HD pathogenesis, through expressing the cytoplasmic TDP-43 without nuclear localization signal. We found that the cytoplasmic TDP-43 is increased in the HD mouse brain and that its mislocalization could deteriorate the motor and gait behavior. Importantly, the cytoplasmic TDP-43, via its binding to the intron1 sequence (GU/UG)n of the mouse Htt pre-mRNA, promotes the transport of exon1-intron1 Htt onto ribosome, resulting in the aberrant generation of exon1 Htt. Our findings suggest that cytoplasmic TDP-43 contributes to HD pathogenesis via its binding to and transport of nuclear un-spliced mRNA to the ribosome for the generation of a toxic protein product.

Clinicopathological analysis of NEK1 variants in amyotrophic lateral sclerosis.

Many genes have been linked to amyotrophic lateral sclerosis (ALS), including never in mitosis A (NIMA)-related kinase 1 (NEK1), a serine/threonine kinase that plays a key role in several cellular functions, such as DNA damage response and cell cycle regulation. Whole-exome sequencing studies have shown that NEK1 mutations are associated with an increased risk for ALS, where a significant enrichment of NEK1 loss-of-function (LOF) variants were found in individuals with ALS compared to controls. In particular, the p.Arg261His missense variant was associated with significantly increased disease susceptibility. This case series aims to understand the neuropathological phenotypes resulting from NEK1 mutations in ALS. We examined a cohort of three Scottish patients with a mutation in the NEK1 gene and evaluated the distribution and cellular expression of NEK1, as well as the abundance of phosphorylated TDP-43 (pTDP-43) aggregates, in the motor cortex compared to age- and sex-matched control tissue. We show pathological, cytoplasmic TDP-43 aggregates in all three NEK1-ALS cases. NEK1 protein staining revealed no immunoreactivity in two of the NEK1-ALS cases, indicating a LOF and corresponding to a reduction in NEK1 mRNA as detected by in situ hybridisation. However, the p.Arg261His missense mutation resulted in an increase in NEK1 mRNA molecules and abundant NEK1-positive cytoplasmic aggregates, with the same morphologic appearance, and within the same cells as co-occurring TDP-43 aggregates. Here we show the first neuropathological assessment of a series of ALS cases carrying mutations in the NEK1 gene. Specifically, we show that TDP-43 pathology is present in these cases and that potential NEK1 LOF can either be mediated through loss of NEK1 translation or through aggregation of NEK1 protein as in the case with p.Arg261His mutation, a potential novel pathological feature of NEK1-ALS.

Spinal cord neurone loss and foot placement changes in a rat knock-in model of amyotrophic lateral sclerosis type 8

Abstract Amyotrophic Lateral Sclerosis is an age-dependent cell type-selective degenerative disease. Genetic studies indicate that Amyotrophic Lateral Sclerosis is part of a spectrum of disorders ranging from spinal muscular atrophy to frontotemporal dementia that share common pathological mechanisms. Amyotrophic Lateral Sclerosis type 8 is a familial disease caused by mis-sense mutations in VAPB. VAPB is localised to the cytoplasmic surface of the endoplasmic reticulum where it serves as a docking point for cytoplasmic proteins and mediates inter-organelle interactions with the endoplasmic reticulum membrane. A gene knock-in model of Amyotrophic Lateral Sclerosis type 8 based on the VapBP56S mutation and VapB gene deletion have been generated in the rat. These animals display a range of age-dependent phenotypes distinct from those previously reported in mouse models of Amyotrophic Lateral Sclerosis type 8. A loss of motor neurones in VapBP56S/+ and VapBP56S/P56Sanimals is indicated by a reduction in the number of large choline acetyl transferase-staining cells in the spinal cord. VapB-/-animals exhibit a relative increase in cytoplasmic TDP-43 levels compared to the nucleus, but no large protein aggregates. Concomitant with these spinal cord pathologies VapBP56S/+, VapBP56S/P56S and VapB-/-animals exhibit age-dependent changes in paw placement and exerted pressures when traversing a CatWalk apparatus, consistent with a somatosensory dysfunction. Extra motor dysfunction is reported in half the cases of motor neurone disease, and this is the first indication of an associated sensory dysfunction in a rodent model of Amyotrophic Lateral Sclerosis. Different rodent models may offer complementary experimental platforms with which to understand the human disease.

Genotype-phenotype association and functional analysis of hnRNPA1 mutations in amyotrophic lateral sclerosis

Background Pathogenic variants in hnRNPA1 have been reported in amyotrophic lateral sclerosis (ALS) patients. However, studies on hnRNPA1 mutant spectrum and pathogenicity of variants were rare. Methods We performed whole exome sequencing of ALS-associated genes and subsequent verification of rare variants in hnRNPA1 in our ALS patients. The hnRNPA1 mutations reported in literature were reviewed and combined with our results to determine the genotype-phenotype relationship. Functional analysis of the novel variant p.G195A was performed in vitro by transfection of mutant hnRNPA1 into 293T cell. Results Among 207 ALS patients recruited, 3 rare hnRNPA1 variants were identified (mutant frequency 1.45%), including two recurrent mutations (p.P340S and p.G283R), and a novel rare variant p.G195A. In combination with previous reports, there are 27 ALS patients with 15 hnRNPA1 mutations identified. Disease onset age was 47.90 ± 1.52 years with predominant limb onset. The p.P340S mutation caused flail arm syndrome (FAS) in two independent families with extended life expectancy. The newly identified p.G195A mutation, lying at the start of the PrLD (“prion-like” domain)/LCD (low-complexity domain), causes local structural changes in 3D protein prediction. Upon sodium arsenite exposure, mutant hnRNPA1 retained in the nucleus but deficit of cytoplasmic G3BP1-positive stress granule clearance was observed. This is different from the p.P340S mutation which caused both cytoplasmic translocation and stress granule formation. No cytoplasmic TDP-43 translocation was observed. Conclusion Mutations in hnRNPA1 are overall minor in ALS patients. The p.P340S mutation is associated with manifestation of FAS. Mutations in LCD of hnRNPA1 cause stress granule misprocessing.

Human Motor Neurons Elicit Pathological Hallmarks of ALS and Reveal Potential Biomarkers of the Disease in Response to Prolonged IFNγ Exposure.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder marked by progressive motor neuron degeneration and muscle denervation. A recent transcriptomic study integrating a wide range of human ALS samples revealed that the upregulation of p53, a downstream target of inflammatory stress, is commonly detected in familial and sporadic ALS cases by a mechanism linked to a transactive response DNA-binding protein 43 (TDP-43) dysfunction. In this study, we show that prolonged interferon-gamma (IFNγ) treatment of human induced pluripotent stem cell-derived spinal motor neurons results in a severe cytoplasmic aggregation of TDP-43. TDP-43 dysfunction resulting from either IFNγ exposure or an ALS-associated TDP-43 mutation was associated with the activation of the p53 pathway. This was accompanied by the hyperactivation of neuronal firing, followed by the complete loss of their electrophysiological function. Through a comparative single-cell transcriptome analysis, we have identified significant alterations in ALS-associated genes in motor neurons exposed to IFNγ, implicating their direct involvement in ALS pathology. Interestingly, IFNγ was found to induce significant levels of programmed death-ligand 1 (PD-L1) expression in motor neurons without affecting the levels of any other immune checkpoint proteins. This finding suggests a potential role of excessive PD-L1 expression in ALS development, given that PD-L1 was recently reported to impair neuronal firing ability in mice. Our findings suggest that exposing motor neurons to IFNγ could directly derive ALS pathogenesis, even without the presence of the inherent genetic mutation or functional glia component. Furthermore, this study provides a comprehensive list of potential candidate genes for future immunotherapeutic targets with which to treat sporadic forms of ALS, which account for 90% of all reported cases.

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

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

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

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

IκB kinase thwarts aggregation: Phosphorylating TDP-43 for degradation.

TDP-43 aggregation is a hallmark of neurodegeneration. In this issue, Iguchi et al. (https://doi.org/10.1083/jcb.202302048) report that IκB kinase (IKK), an important mediator of inflammation, phosphorylates cytoplasmic TDP-43 to promote proteasomal degradation, revealing an unexpected link between inflammation and TDP-43 homeostasis.

IκB kinase phosphorylates cytoplasmic TDP-43 and promotes its proteasome degradation.

Cytoplasmic aggregation of TDP-43 in neurons is a pathological feature common to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We demonstrate that the IκB kinase (IKK) complex promotes the degradation of cytoplasmic TDP-43 through proteasomes. While IKKβ is a major factor in TDP-43 degradation, IKKα acts as a cofactor, and NEMO functions as a scaffold for the recruitment of TDP-43 to the IKK complex. Furthermore, we identified IKKβ-induced phosphorylation sites of TDP-43 and found that phosphorylation at Thr8 and Ser92 is important for the reduction of TDP-43 by IKK. TDP-43 phosphorylation at Ser92 was detected in a pattern different from that of C-terminal phosphorylation in the pathological inclusion of ALS. IKKβ was also found to significantly reduce the expression level and toxicity of the disease-causing TDP-43 mutation. Finally, the favorable effect of IKKβ on TDP-43 aggregation was confirmed in the hippocampus of mice. IKK and the N-terminal phosphorylation of TDP-43 are potential therapeutic targets for ALS and FTLD.

The new missense G376V-TDP-43 variant induces late-onset distal myopathy but not amyotrophic lateral sclerosis.

TAR DNA binding protein of 43 kDa (TDP-43)-positive inclusions in neurons are a hallmark of several neurodegenerative diseases including familial amyotrophic lateral sclerosis (fALS) caused by pathogenic TARDBP variants as well as more common non-Mendelian sporadic ALS (sALS). Here we report a G376V-TDP-43 missense variant in the C-terminal prion-like domain of the protein in two French families affected by an autosomal dominant myopathy but not fulfilling diagnostic criteria for ALS. Patients from both families presented with progressive weakness and atrophy of distal muscles, starting in their fifth to seventh decade. Muscle biopsies revealed a degenerative myopathy characterized by accumulation of rimmed (autophagic) vacuoles, disruption of sarcomere integrity and severe myofibrillar disorganization. The G376V variant altered a highly conserved amino acid residue and was absent in databases on human genome variation. Variant pathogenicity was supported by in silico analyses and functional studies. The G376V mutant increased the formation of cytoplasmic TDP-43 condensates in cell culture models, promoted assembly into high molecular weight oligomers and aggregates in vitro, and altered morphology of TDP-43 condensates arising from phase separation. Moreover, the variant led to the formation of cytoplasmic TDP-43 condensates in patient-derived myoblasts and induced abnormal mRNA splicing in patient muscle tissue. The identification of individuals with TDP-43-related myopathy, but not ALS, implies that TARDBP missense variants may have more pleiotropic effects than previously anticipated and support a primary role for TDP-43 in skeletal muscle pathophysiology. We propose to include TARDBP screening in the genetic work-up of patients with late-onset distal myopathy. Further research is warranted to examine the precise pathogenic mechanisms of TARDBP variants causing either a neurodegenerative or myopathic phenotype.

Modelling TDP-43 proteinopathy in Drosophila uncovers shared and neuron-specific targets across ALS and FTD relevant circuits

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comprise a spectrum of neurodegenerative diseases linked to TDP-43 proteinopathy, which at the cellular level, is characterized by loss of nuclear TDP-43 and accumulation of cytoplasmic TDP-43 inclusions that ultimately cause RNA processing defects including dysregulation of splicing, mRNA transport and translation. Complementing our previous work in motor neurons, here we report a novel model of TDP-43 proteinopathy based on overexpression of TDP-43 in a subset of Drosophila Kenyon cells of the mushroom body (MB), a circuit with structural characteristics reminiscent of vertebrate cortical networks. This model recapitulates several aspects of dementia-relevant pathological features including age-dependent neuronal loss, nuclear depletion and cytoplasmic accumulation of TDP-43, and behavioral deficits in working memory and sleep that occur prior to axonal degeneration. RNA immunoprecipitations identify several candidate mRNA targets of TDP-43 in MBs, some of which are unique to the MB circuit and others that are shared with motor neurons. Among the latter is the glypican Dally-like-protein (Dlp), which exhibits significant TDP-43 associated reduction in expression during aging. Using genetic interactions we show that overexpression of Dlp in MBs mitigates TDP-43 dependent working memory deficits, conistent with Dlp acting as a mediator of TDP-43 toxicity. Substantiating our findings in the fly model, we find that the expression of GPC6 mRNA, a human ortholog of dlp, is specifically altered in neurons exhibiting the molecular signature of TDP-43 pathology in FTD patient brains. These findings suggest that circuit-specific Drosophila models provide a platform for uncovering shared or disease-specific molecular mechanisms and vulnerabilities across the spectrum of TDP-43 proteinopathies.

Multiscale simulations reveal TDP-43 molecular-level interactions driving condensation

The RNA-binding protein TDP-43 is associated with mRNA processing and transport from the nucleus to the cytoplasm. TDP-43 localizes in the nucleus as well as accumulating in cytoplasmic condensates such as stress granules. Aggregation and formation of amyloid-like fibrils of cytoplasmic TDP-43 are hallmarks of numerous neurodegenerative diseases, most strikingly present in >90% of amyotrophic lateral sclerosis (ALS) patients. If excessive accumulation of cytoplasmic TDP-43 causes, or is caused by, neurodegeneration is presently not known. In this work, we use molecular dynamics simulations at multiple resolutions to explore TDP-43 self- and cross-interaction dynamics. A full-length molecular model of TDP-43, all 414 amino acids, was constructed from select structures of the protein functional domains (N-terminal domain, and two RNA recognition motifs, RRM1 and RRM2) and modeling of disordered connecting loops and the low complexity glycine-rich C-terminus domain. All-atom CHARMM36m simulations of single TDP-43 proteins served as guides to construct a coarse-grained Martini 3 model of TDP-43. The Martini model and a coarser implicit solvent C⍺ model, optimized for disordered proteins, were subsequently used to probe TDP-43 interactions; self-interactions from single-chain full-length TDP-43 simulations, cross-interactions from simulations with two proteins and simulations with assemblies of dozens to hundreds of proteins. Our findings illustrate the utility of different modeling scales for accessing TDP-43 molecular-level interactions and suggest that TDP-43 has numerous interaction preferences or patterns, exhibiting an overall strong, but dynamic, association and driving the formation of biomolecular condensates.

Chronic exposure to l-BMAA cyanotoxin induces cytoplasmic TDP-43 accumulation and glial activation, reproducing an amyotrophic lateral sclerosis-like phenotype in mice

BackgroundAmyotrophic lateral sclerosis (ALS) is a progressive and often fatal neurodegenerative disease characterized by the loss of Motor Neurons (MNs) in spinal cord, motor cortex and brainstem. Despite significant efforts in the field, the exact pathogenetic mechanisms underlying both familial and sporadic forms of ALS have not been fully elucidated, and the therapeutic possibilities are still very limited. Here we investigate the molecular mechanisms of neurodegeneration induced by chronic exposure to the environmental cyanotoxin L-BMAA, which causes a form of ALS/Parkinson’s disease (PD) in several populations consuming food and/or water containing high amounts of this compound. MethodsIn this effort, mice were chronically exposed to L-BMAA and analyzed at different time points to evaluate cellular and molecular alterations and behavioral deficits, performing MTT assay, immunoblot, immunofluorescence and immunohistochemistry analysis, and behavioral tests. ResultsWe found that cyanotoxin L-BMAA determines apoptotic cell death and a marked astrogliosis in spinal cord and motor cortex, and induces neurotoxicity by favoring TDP-43 cytoplasmic accumulation. ConclusionsOverall, our results characterize a new versatile neurotoxic animal model of ALS that may be useful for the identification of new druggable targets to develop innovative therapeutic strategies for this disease.

Reduction of Nemo-like kinase increases lysosome biogenesis and ameliorates TDP-43-related neurodegeneration.

Protein aggregation is a hallmark of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although mutations in TARDBP, encoding TDP-43, account for less than 1% of all ALS cases, TDP-43-positive aggregates are present in nearly all ALS patients, including patients with sporadic ALS (sALS) or carrying other familial ALS (fALS)-causing mutations. Interestingly, TDP-43 inclusions are also present in subsets of patients with frontotemporal dementia, Alzheimer's disease, and Parkinson's disease; therefore, methods of activating intracellular protein quality control machinery capable of clearing toxic cytoplasmic TDP-43 species may alleviate disease-related phenotypes. Here, we identify a novel function of Nemo-like kinase (Nlk) as a negative regulator of lysosome biogenesis. Genetic or pharmacological reduction of Nlk increased lysosome formation and improved clearance of aggregated TDP-43. Furthermore, Nlk reduction ameliorated pathological, behavioral, and lifespan deficits in two distinct mouse models of TDP-43 proteinopathy. Because many toxic proteins can be cleared along the autophagy-lysosome axis, targeted reduction of Nlk represents a potential approach to therapy development for multiple neurodegenerative disorders.

Nuclear Expression of TDP-43 Is Linked with Morphology and Ubiquitylation of Cytoplasmic Aggregates in Amyotrophic Lateral Sclerosis.

The transactive response DNA-binding protein of 43 kDa (TDP-43) is a pathological protein of amyotrophic lateral sclerosis (ALS). TDP-43 pathology is characterized by a combination of the cytoplasmic aggregation and nuclear clearance of this protein. However, the mechanisms underlying TDP-43 pathology have not been fully clarified. The aim of this study was to evaluate the relationships between the expression level of nuclear TDP-43 and the pathological properties of cytoplasmic aggregates in autopsied ALS cases. We included 22 consecutively autopsied cases with sporadic TDP-43-related ALS. The motor neuron systems were neuropathologically assessed. We identified 790 neurons with cytoplasmic TDP-43 inclusions from the lower motor neuron system of included cases. Nuclear TDP-43 disappeared in 84% (n = 660) and expressed in 16% (n = 130) of neurons with cytoplasmic inclusions; the former was defined as TDP-43 cytoplasmic immunoreactivity (c-ir), and the latter was defined as nuclear and cytoplasmic immunoreactivity (n/c-ir). Morphologically, diffuse cytoplasmic inclusions were significantly more prevalent in TDP-43 n/c-ir neurons than in c-ir neurons, while skein-like and round inclusions were less prevalent in n/c-ir neurons. The cytoplasmic inclusions of TDP-43 n/c-ir neurons were phosphorylated but poorly ubiquitylated when compared with those of c-ir neurons. TDP-43 n/c-ir neurons became less dominant than the c-ir neurons among cases with a prolonged disease duration. The expression level of nuclear TDP-43 was significantly lower in n/c-ir neurons than in normal neurons without cytoplasmic inclusions. Our results indicate that the maturation of cytoplasmic TDP-43 inclusions correlates with the depletion of nuclear TDP-43 in each affected neuron. This finding supports the view that an imbalance between nuclear and cytoplasmic TDP-43 may be an essential pathway to TDP-43 pathology.

Mature iPSC-derived astrocytes of an ALS/FTD patient carrying the TDP43A90V mutation display a mild reactive state and release polyP toxic to motoneurons.

Astrocytes play a critical role in the maintenance of a healthy central nervous system and astrocyte dysfunction has been implicated in various neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). There is compelling evidence that mouse and human ALS and ALS/FTD astrocytes can reduce the number of healthy wild-type motoneurons (MNs) in co-cultures or after treatment with astrocyte conditioned media (ACM), independently of their genotype. A growing number of studies have shown that soluble toxic factor(s) in the ACM cause non-cell autonomous MN death, including our recent identification of inorganic polyphosphate (polyP) that is excessively released from mouse primary astrocytes (SOD1, TARDBP, and C9ORF72) and human induced pluripotent stem cells (iPSC)-derived astrocytes (TARDBP) to kill MNs. However, others have reported that astrocytes carrying mutant TDP43 do not produce detectable MN toxicity. This controversy is likely to arise from the findings that human iPSC-derived astrocytes exhibit a rather immature and/or reactive phenotype in a number of studies. Here, we have succeeded in generating a highly homogenous population of functional quiescent mature astrocytes from control subject iPSCs. Using identical conditions, we also generated mature astrocytes from an ALS/FTD patient carrying the TDP43A90V mutation. These mutant TDP43 patient-derived astrocytes exhibit key pathological hallmarks, including enhanced cytoplasmic TDP-43 and polyP levels. Additionally, mutant TDP43 astrocytes displayed a mild reactive signature and an aberrant function as they were unable to promote synaptogenesis of hippocampal neurons. The polyP-dependent neurotoxic nature of the TDP43A90V mutation was further confirmed as neutralization of polyP in ACM derived from mutant TDP43 astrocytes prevented MN death. Our results establish that human astrocytes carrying the TDP43A90V mutation exhibit a cell-autonomous pathological signature, hence providing an experimental model to decipher the molecular mechanisms underlying the generation of the neurotoxic phenotype.

Regulation of RNG105/caprin1 dynamics by pathogenic cytoplasmic FUS and TDP-43 in neuronal RNA granules modulates synaptic loss

In neurodegenerative diseases, the condensation of FUS and TDP-43 with RNA granules in neurons is linked to pathology, including synaptic disorders. However, the effects of FUS and TDP-43 on RNA granule factors remain unclear. Here, using primary cultured neurons from the mouse cerebral cortex, we show that excess cytoplasmic FUS and TDP-43 accumulated in dendritic RNA granules, where they increased the dynamics of a scaffold protein RNG105/caprin1 and dissociated it from the granules. This coincided with reduced levels of mRNA and translation around the granules and synaptic loss in dendrites. These defects were suppressed by non-dissociable RNG105, suggesting that RNG105 dissociation mediated the defects. In contrast to the model where FUS and TDP-43 co-aggregate with RNA granule factors to repress their activity, our findings provide a novel pathogenic mechanism whereby FUS and TDP-43 dissociate RNA scaffold proteins from RNA granules which are required for local translation that regulates synapse formation.

Early activation of cellular stress and death pathways caused by cytoplasmic TDP-43 in the rNLS8 mouse model of ALS and FTD.

TAR DNA binding protein 43 (TDP-43) pathology is a key feature of over 95% of amyotrophic lateral sclerosis (ALS) and nearly half of frontotemporal dementia (FTD) cases. The pathogenic mechanisms of TDP-43 dysfunction are poorly understood, however, activation of cell stress pathways may contribute to pathogenesis. We, therefore, sought to identify which cell stress components are critical for driving disease onset and neurodegeneration in ALS and FTD. We studied the rNLS8 transgenic mouse model, which expresses human TDP-43 with a genetically-ablated nuclear localisation sequence within neurons of the brain and spinal cord resulting in cytoplasmic TDP-43 pathology and progressive motor dysfunction. Amongst numerous cell stress-related biological pathways profiled using qPCR arrays, several critical integrated stress response (ISR) effectors, including CCAAT/enhancer-binding homologous protein (Chop/Ddit3) and activating transcription factor 4 (Atf4), were upregulated in the cortex of rNLS8 mice prior to disease onset. This was accompanied by early up-regulation of anti-apoptotic gene Bcl2 and diverse pro-apoptotic genes including BH3-interacting domain death agonist (Bid). However, pro-apoptotic signalling predominated after onset of motor phenotypes. Notably, pro-apoptotic cleaved caspase-3 protein was elevated in the cortex of rNLS8 mice at later disease stages, suggesting that downstream activation of apoptosis drives neurodegeneration following failure of early protective responses. Unexpectedly, suppression of Chop in the brain and spinal cord using antisense oligonucleotide-mediated silencing had no effect on overall TDP-43 pathology or disease phenotypes in rNLS8 mice. Cytoplasmic TDP-43 accumulation therefore causes very early activation of ISR and both anti- and pro-apoptotic signalling that switches to predominant pro-apoptotic activation later in disease. These findings suggest that precise temporal modulation of cell stress and death pathways may be beneficial to protect against neurodegeneration in ALS and FTD.

TDP-43 pathology and functional deficits in wild-type and ALS/FTD mutant cyclin F mouse models.

Amyotrophic lateral sclerosis (ALS) is characterised by a progressive loss of upper and lower motor neurons leading to muscle weakness and eventually death. Frontotemporal dementia (FTD) presents clinically with significant behavioural decline. Approximately 10% of cases have a known family history, and disease-linked mutations in multiple genes have been identified in FTD and ALS. More recently, ALS and FTD-linked variants have been identified in the CCNF gene, which accounts for an estimated 0.6% to over 3% of familial ALS cases. In this study, we developed the first mouse models expressing either wild-type (WT) human CCNF or its mutant pathogenic variant S621G to recapitulate key clinical and neuropathological features of ALS and FTD linked to CCNF disease variants. We expressed human CCNF WT or CCNFS621G throughout the murine brain by intracranial delivery of adeno-associated virus (AAV) to achieve widespread delivery via somatic brain transgenesis. These mice developed behavioural abnormalities, similar to the clinical symptoms of FTD patients, as early as 3months of age, including hyperactivity and disinhibition, which progressively deteriorated to include memory deficits by 8months of age. Brains of mutant CCNF_S621G mice displayed an accumulation of ubiquitinated proteins with elevated levels of phosphorylated TDP-43 present in both CCNF_WT and mutant CCNF_S621G mice. We also investigated the effects of CCNF expression on interaction targets of CCNF and found elevated levels of insoluble splicing factor proline and glutamine-rich (SFPQ). Furthermore, cytoplasmic TDP-43 inclusions were found in both CCNF_WT and mutant CCNF_S621G mice, recapitulating the key hallmark of FTD/ALS pathology. In summary, CCNF expression in mice reproduces clinical presentations of ALS, including functional deficits and TDP-43 neuropathology with altered CCNF-mediated pathways contributing to the pathology observed.