Nuclear TDP-43 Research Articles

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.

Pathologic TDP43 Associated Tri‐Glial Dysfunction in Amnestic and Non‐amnestic Clinical Dementia Syndromes: A Multiplexed Immunofluorescence Study

AbstractBackgroundAlzheimer’s disease (AD) is a substantial cause of death and disability. In addition to the plaques and tangles classically observed in AD, other co‐existing brain lesions also contribute to the development of cognitive impairment, such as pathologic trans‐active response DNA‐binding protein 43 (pTDP43). TDP43 is ubiquitously expressed in the brain and mechanistic studies suggest potential roles for TDP43 in maintaining normal physiologic conditions in glia (astrocytes, microglia, oligodendrocytes) (Bulgiani, 2022). Effective glial functioning and signaling are crucial to maintaining functional connectivity and pTDP43 is strongly associated with lower cognitive performance (Flanagan, 2018). However, pTDP43’s role in tri‐glial dysfunction remains unclear. We hypothesize that nuclear astrocytic TDP43 loss alters astrocyte‐to‐microglial signaling, and results in tri‐glial dysfunction. Here we examine glial pTDP43 subtypes in amnestic and non‐amnestic clinical dementia syndromes with matched AD pathology.MethodParaffin embedded autopsy samples (hippocampus and middle frontal cortex) were stained using multiplexed immunofluorescence (Pu.1: microglia, GLUL: astrocytes, total TDP43), and imaged. Indica Labs Halo software was used to perform quantitative analyses on whole slide images. Cases were matched for AD pathology (low and high) (Montine, 2012). Students’ t‐tests were performed to compare cell counts between groups. Kolmogorov–Smirnov test was performed to check approximate normality before applying t‐test and all measurements passed it. Clinical phenotypes (AD dementia vs. behavioral variant frontotemporal dementia (bvFTD)) and pTDP43 status were compared (n = 50). Spatial localization plots, NSInC colocalization indices, and Box plot comparisons were used to assess the impact of glial pTDP43 on the microenvironment.ResultHippocampal astrocyte counts are lowest in the AD dementia setting with the highest AD pathology burden, and presence of co‐existing pTDP43. Cases with high AD pathology had lower astrocyte counts when co‐existing pTDP43 was present when comparing both AD dementia with pTDP43 vs. AD dementia without pTDP43 (p = 0.03), and AD dementia with pTDP43 vs. bvFTD without pTDP43 (p = 0.05).ConclusionContradictory to studies showing increased GFAP in mutant TDP43 mice (Yan, 2014), the lowest astrocyte counts were found in individuals with AD dementia, highest burden of AD pathology, and presence of pTDP43. Results support the role of astroglial heterogeneity contributing to white matter dysfunction via selective vulnerability.

Aggregation-prone TDP-43 sequesters and drives pathological transitions of free nuclear TDP-43

Aggregation of the RNA-binding protein, TDP-43, is the unifying hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43-related neurodegeneration involves multiple changes to normal physiological TDP-43, which undergoes nuclear depletion, cytoplasmic mislocalisation, post-translational modification, and aberrant liquid–liquid phase separation, preceding inclusion formation. Along with toxic cytoplasmic aggregation, concurrent depletion and dysfunction of normal nuclear TDP-43 in cells with TDP-43 pathology is likely a key potentiator of neurodegeneration, but is not well understood. To define processes driving TDP-43 dysfunction, we used CRISPR/Cas9-mediated fluorescent tagging to investigate how disease-associated stressors and pathological TDP-43 alter abundance, localisation, self-assembly, aggregation, solubility, and mobility dynamics of normal nuclear TDP-43 over time in live cells. Oxidative stress stimulated liquid–liquid phase separation of endogenous TDP-43 into droplet-like puncta, or spherical shell-like anisosomes. Further, nuclear RNA-binding-ablated or acetylation-mimicking TDP-43 readily sequestered and depleted free normal nuclear TDP-43 into dynamic anisosomes, in which recruited endogenous TDP-43 proteins remained soluble and highly mobile. Large, phosphorylated inclusions formed by nuclear or cytoplasmic aggregation-prone TDP-43 mutants also caused sequestration, but rendered endogenous TDP-43 immobile and insoluble, indicating pathological transition. These findings suggest that RNA-binding deficiency and post-translational modifications including acetylation exacerbate TDP-43 aggregation and dysfunction by driving sequestration, mislocalisation, and depletion of normal nuclear TDP-43 in neurodegenerative diseases.

Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies.

Loss of nuclear TDP-43 is a hallmark of neurodegeneration in TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 mislocalization results in cryptic splicing and polyadenylation of pre-messenger RNAs (pre-mRNAs) encoding stathmin-2 (also known as SCG10), a protein that is required for axonal regeneration. We found that TDP-43 binding to a GU-rich region sterically blocked recognition of the cryptic 3' splice site in STMN2 pre-mRNA. Targeting dCasRx or antisense oligonucleotides (ASOs) suppressed cryptic splicing, which restored axonal regeneration and stathmin-2-dependent lysosome trafficking in TDP-43-deficient human motor neurons. In mice that were gene-edited to contain human STMN2 cryptic splice-polyadenylation sequences, ASO injection into cerebral spinal fluid successfully corrected Stmn2 pre-mRNA misprocessing and restored stathmin-2 expression levels independently of TDP-43 binding.

A quantitative biology approach correlates neuronal toxicity with the largest inclusions of TDP-43.

A number of neurodegenerative conditions are associated with the formation of cytosolic inclusions of TDP-43 within neurons. We expressed full-length TDP-43 in a motoneuron/neuroblastoma hybrid cell line (NSC-34) and exploited the high-resolution power of stimulated emission depletion microscopy to monitor the changes of nuclear and cytoplasmic TDP-43 levels and the formation of various size classes of cytoplasmic TDP-43 aggregates with time. Concomitantly, we monitored oxidative stress and mitochondrial impairment using the MitoSOX and MTT reduction assays, respectively. Using a quantitative biology approach, we attributed neuronal dysfunction associated with cytoplasmic deposition component to the formation of the largest inclusions, independently of stress granules. This is in contrast to other neurodegenerative diseases where toxicity is attributed to small oligomers. Using specific inhibitors, markers, and electron microscopy, the proteasome and autophagy were found to target mainly the largest deleterious inclusions, but their efficiency soon decreases without full recovery of neuronal viability.

Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export.

SUMMARYNuclear clearance of the RNA-binding protein TDP-43 is a hallmark of neurodegeneration and an important therapeutic target. Our current understanding of TDP-43 nucleocytoplasmic transport does not fully explain its predominantly nuclear localization or mislocalization in disease. Here, we show that TDP-43 exits nuclei by passive diffusion, independent of facilitated mRNA export. RNA polymerase II blockade and RNase treatment induce TDP-43 nuclear efflux, suggesting that nuclear RNAs sequester TDP-43 in nuclei and limit its availability for passive export. Induction of TDP-43 nuclear efflux by short, GU-rich oligomers (presumably by outcompeting TDP-43 binding to endogenous nuclear RNAs), and nuclear retention conferred by splicing inhibition, demonstrate that nuclear TDP-43 localization depends on binding to GU-rich nuclear RNAs. Indeed, RNA-binding domain mutations markedly reduce TDP-43 nuclear localization and abolish transcription blockade-induced nuclear efflux. Thus, the nuclear abundance of GU-RNAs, dictated by the balance of transcription, pre-mRNA processing, and RNA export, regulates TDP-43 nuclear localization.

Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.

Optineurin Deficiency and Insufficiency Lead to Higher Microglial TDP-43 Protein Levels.

Mutations in optineurin, a ubiquitin-binding adaptor protein, cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease of motor neurons linked to chronic inflammation and protein aggregation. The majority of ALS patients, including those carrying the optineurin mutations, exhibit cytoplasmic mislocalization, ubiquitination, and aggregation of nuclear TAR DNA-binding protein 43 kDa (TDP-43). To address the crosstalk between optineurin and TDP-43, we generated optineurin knockout (KO) neuronal and microglial cell lines using the CRISPR/Cas9 approach. Interestingly, we observed that loss of optineurin resulted in elevated TDP-43 protein expression in microglial BV2 but not neuronal Neuro 2a and NSC-34 cell lines. No changes were observed at the mRNA level, suggesting that this increase was post-translationally regulated. To confirm this observation in primary cells, we then used microglia and macrophages from an optineurin loss-of-function mouse model that lacks the C-terminal ubiquitin-binding region (Optn470T), mimicking optineurin truncations in ALS patients. As observed in the BV2 cells, we also found elevated basal levels of TDP-43 protein in Optn470T microglia and bone marrow-derived macrophages. To test if inflammation could further enhance TDP-43 accumulation in cells lacking functional optineurin, we stimulated them with lipopolysaccharide (LPS), and we observed a significant increase in TDP-43 expression following LPS treatment of WT cells. However, this was absent in both BV2 Optn KO and primary Optn470T microglia, which exhibited the same elevated TDP-43 levels as in basal conditions. Furthermore, we did not observe nuclear TDP-43 depletion or cytoplasmic aggregate formation in either Optn470T microglia or LPS-treated WT or Optn470T microglia. Taken together, our results show that optineurin deficiency and insufficiency post-translationally upregulate microglial TDP-43 protein levels and that elevated TDP-43 levels in cells lacking functional optineurin could not be further increased by an inflammatory stimulus, suggesting the presence of a plateau.

Importin-Mediated Pathological Tau Nuclear Translocation Causes Disruption of the Nuclear Lamina, TDP-43 Mislocalization and Cell Death.

Tau is a cytosolic protein that has also been observed in the nucleus, where it has multiple proposed functions that are regulated by phosphorylation. However, the mechanism underlying the nuclear import of tau is unclear, as is the contribution of nuclear tau to the pathology of tauopathies. We have previously generated a pathological form of tau, PH-tau (pseudophosphorylation mutants S199E, T212E, T231E, and S262E) that mimics AD pathological behavior in cells, Drosophila, and a mouse model. Here, we demonstrated that PH-tau translocates into the nucleus of transiently transfected HEK-293 cells, but wildtype tau does not. We identified a putative importin binding site in the tau sequence, and showed that disruption of this site prevents tau from entering the nucleus. We further showed that this nuclear translocation is prevented by inhibitors of both importin-α and importin-β. In addition, expression of PH-tau resulted in an enlarged population of dying cells, which is prevented by blocking its entry into the nucleus. PH-tau-expressing cells also exhibited disruption of the nuclear lamina and mislocalization of TDP-43 to the cytoplasm. We found that PH-tau does not bundle microtubules, and this effect is independent of nuclear translocation. These results demonstrate that tau translocates into the nucleus through the importin-α/β pathway, and that PH-tau exhibits toxicity after its nuclear translocation. We propose a model where hyperphosphorylated tau not only disrupts the microtubule network, but also translocates into the nucleus and interferes with cellular functions, such as nucleocytoplasmic transport, inducing mislocalization of proteins like TDP-43 and, ultimately, cell death.

Gel‐like inclusions of C‐terminal fragments of TDP‐43 sequester stalled proteasomes in neurons

Aggregation of the multifunctional RNA‐binding protein TDP‐43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both diseases. In disease, characteristic C‐terminal fragments of ~25 kDa ("TDP‐25") accumulate in cytoplasmic inclusions. Here, we analyze gain‐of‐function mechanisms of TDP‐25 combining cryo‐electron tomography, proteomics, and functional assays. In neurons, cytoplasmic TDP‐25 inclusions are amorphous, and photobleaching experiments reveal gel‐like biophysical properties that are less dynamic than nuclear TDP‐43. Compared with full‐length TDP‐43, the TDP‐25 interactome is depleted of low‐complexity domain proteins. TDP‐25 inclusions are enriched in 26S proteasomes adopting exclusively substrate‐processing conformations, suggesting that inclusions sequester proteasomes, which are largely stalled and no longer undergo the cyclic conformational changes required for proteolytic activity. Reporter assays confirm that TDP‐25 impairs proteostasis, and this inhibitory function is enhanced by ALS‐causing TDP‐43 mutations. These findings support a patho‐physiological relevance of proteasome dysfunction in ALS/FTD.

TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1–3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies.

Rapid and automated quantification of TDP-43 and FUS mislocalisation for screening of frontotemporal dementia and amyotrophic lateral sclerosis gene variants.

Identified genetic mutations account for ∼50% of familial frontotemporal dementia (FTD) and ∼70% of familial amyotrophic lateral sclerosis (ALS) cases, however, for the remainder of patients the origin of the disease is uncertain. Post-mortem, affected neurons from 97% of ALS and ∼50% of FTD patients show cytoplasmic mislocalisation of the typically nuclear proteins, TDP-43 and/or FUS. We exploited this predominant neuropathological feature to develop a high-throughput, automated method for the quantification of cytoplasmic TDP-43 and FUS in human cell lines. Utilising fluorescently tagged cDNA constructs to identify cells of interest, fluorescence intensity of TDP-43 or FUS in the nucleus and cytoplasm of HEK293 and SH-SY5Y cells was measured from confocal microscope images using the freely available analysis software, CellProfiler. The assay was validated using known ALS-causative mutations in the genes encoding TDP-43 (TARDBP) and FUS, and pharmacological interventions known to cause TDP-43 and FUS mislocalisation. The ability of the assay to detect effects of a secondary gene on endogenous TDP-43 was tested using CYLD wild-type and mutant constructs. Expression of known TARDBP and FUS mutations showed significantly higher cytoplasmic to nuclear ratios when compared to wild-type protein. Treatment with apoptosis inducers MG132 and staurosporine induced a similar effect. Lastly, we recapitulated the effect of the CYLD FTD-ALS mutation p.M719V on TDP-43 mislocalisation, as observed previously by manual counting of primary mouse neurons [Dobson-Stone et al 2020, Brain 143:783-799]. The current study validates our methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used to rapidly and systematically assess the pathogenicity of predicted FTD/ALS gene variants. This methodology can be employed to aid genetic diagnosis of patients carrying novel variants in known FTD/ALS genes, and in the prioritisation of novel candidate genes for further functional analysis.

The role of the RNA binding protein HnRNP K in the pathogenesis of frontotemporal lobar degeneration.

HnRNP K is an RNA-binding protein belonging to the family of heterogeneous nuclear ribonucleoproteins (hnRNPs). Like other hnRNPs such as the better known TDP-43, hnRNP K is predominantly nuclear where it plays an important role in pre-mRNA splicing. HnRNPs can also shuttle to the cytoplasm and we find that hnRNP K is predominantly cytoplasmic in pyramidal neurons in Frontotemporal Lobar Degeneration (FTLD). The loss of nuclear hnRNPs in disease states is directly linked to splicing misregulation and aberrant RNA processing. Indeed, loss of nuclear TDP-43 leads to cryptic exon inclusion in gene targets and the generation of truncated proteins. We aimed to investigate changes in RNA processing and presence of cryptic events upon hnRNP K mislocalisation in FTLD. Bioinformatic analysis of a neuronal cell line with hnRNP K knockdown identified a set of cryptic events. We have validated these events using PCR amplification and gel electrophoresis in both SH-SY5Y cells with hnRNP K knockdown and in post-mortem human brain tissue. The loss of nuclear hnRNP K results in the inclusion of cryptic exons in multiple gene targets in both SH-SHY5Y cells with hnRNP K knockdown and FTLD brain regions exhibiting hnRNP K nuclear loss. The mislocalisation of hnRNP K from the nucleus to the cytoplasm in FTLD is associated with the inclusion of cryptic exons in multiple gene targets. This suggests that similarly to TDP-43, hnRNP K plays a crucial role in RNA processing and splicing regulation which when impaired could lead to transcriptional dysregulation and disease pathogenesis.

Tau-tubulin kinase 1 phosphorylates TDP-43 at disease-relevant sites and exacerbates TDP-43 pathology

TDP-43 pathology is a hallmark of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal lobar degeneration (FTLD). Namely, both diseases feature aggregated and phosphorylated TDP-43 containing inclusions in the cytoplasm and a loss of nuclear TDP-43 in affected neurons. It has been reported that tau tubulin kinase (TTBK)1/2 phosphorylate TDP-43 and TTBK1/2 overexpression induced neuronal loss and behavioral deficits in a C. elegans model of ALS. Here we aimed to elucidate the molecular mechanisms of TTBK1 in TDP-43 pathology. TTBK1 levels were observed to be elevated in ALS patients' post-mortem motor cortex. Also, TTBK1 was found to phosphorylate TDP-43 at disease-relevant sites in vitro directly, and this phosphorylation accelerated TDP-43 formation of high molecular species. Overexpression of TTBK1 in mammalian cells induced TDP-43 phosphorylation and the construction of high molecular species, concurrent with TDP-43 mis-localization and cytoplasmic inclusions. In addition, when TTBK1 was knocked down or pharmacologically inhibited, TDP-43 phosphorylation and aggregation were significantly alleviated. Functionally, TTBK1 knockdown could rescue TDP-43 overexpression-induced neurite and neuronal loss in iPSC-derived GABAergic neurons. These findings suggest that phosphorylation plays a critical role in the pathogenesis of TDP-43 pathology and that TTBK1 inhibition may have therapeutic potential for the treatment of ALS and FTLD.

Nuclear depletion of RNA-binding protein ELAVL3 (HuC) in sporadic and familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a progressive fatal neurodegenerative disease caused by loss of motor neurons and characterized neuropathologically in almost all cases by nuclear depletion and cytoplasmic aggregation of TDP-43, a nuclear RNA-binding protein (RBP). We identified ELAVL3 as one of the most downregulated genes in our transcriptome profiles of laser captured microdissection of motor neurons from sporadic ALS nervous systems and the most dysregulated of all RBPs. Neuropathological characterizations showed ELAVL3 nuclear depletion in a great percentage of remnant motor neurons, sometimes accompanied by cytoplasmic accumulations. These abnormalities were common in sporadic cases with and without intermediate expansions in ATXN2 and familial cases carrying mutations in C9orf72 and SOD1. Depletion of ELAVL3 occurred at both the RNA and protein levels and a short protein isoform was identified, but it is not related to a TDP-43-dependent cryptic exon in intron 3. Strikingly, ELAVL3 abnormalities were more frequent than TDP-43 abnormalities and occurred in motor neurons still with normal nuclear TDP-43 present, but all neurons with abnormal TDP-43 also had abnormal ELAVL3. In a neuron-like cell culture model using SH-SY5Y cells, ELAVL3 mislocalization occurred weeks before TDP-43 abnormalities were seen. We interrogated genetic databases, but did not identify association of ELAVL3 genetic structure with ALS. Taken together, these findings suggest that ELAVL3 is an important RBP in ALS pathogenesis acquired early and the neuropathological data suggest that it is involved by loss of function rather than cytoplasmic toxicity.

Extensive phenotypic characterisation of a human TDP-43Q331K\xa0transgenic mouse model of amyotrophic lateral sclerosis (ALS)

The majority of preclinical studies in ALS have relied on transgenic models with overexpression of mutant human superoxide dismutase 1 (SOD1), widely regarded to have failed in terms of translation of therapeutic effects. However, there are still no widely accepted models of other genetic subtypes of ALS. The majority of patients show ubiquitinated cytoplasmic inclusions of TAR DNA binding protein of 43 kilodaltons (TDP-43) in spinal motor neurons at the end stage of disease and a small proportion have mutations in TARDBP, the gene encoding TDP-43. TDP-43 transgenic mouse models have been produced, but have not been widely adopted. Here, we characterised one of these models available from the Jackson Laboratory in detail. Compared to TDP-43WT mice, TDP-43Q331K mice had 43% less hindlimb muscle mass at 6 months and a 73% reduction in hindlimb compound muscle action potential at 8 months of age. Rotarod and gait analysis indicated motor system decline with elevated weight gain. At the molecular level, the lack of TDP-43 cellular pathology was confirmed with a surprising increase in nuclear TDP-43 in motor neurons. Power analysis indicated group sizes of 12–14 mice are needed to detect 10–20% changes in measured parameters with a power of 80%, providing valid readouts for preclinical testing. Overall, this model may represent a useful component of multi-model pre-clinical therapeutic studies for ALS.

What Is the Role of Stathmin-2 in Axonal Biology and Degeneration?

Stathmin-2 (STMN2) is a microtubule-associated protein that has a major role in axonal development and repair. STMN2 promotes microtubule instability necessary for normal axonal outgrowth and regeneration. The expression of STMN2 is strongly regulated by nuclear transactive response DNA-binding protein 43 kDA (TDP-43). This multifunctional nucleic acid binding protein is a primary component of insoluble cytoplasmic aggregates associated with several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) resulting in frontotemporal dementia (FTD). Recent evidence indicates that reduced nuclear TDP-43 function leads to improper splicing of the gene encoding STMN2, leading to the production of a truncated protein.1,2 This finding provides a mechanistic link between TDP-43 proteinopathies and axonal degeneration.1,3

SUMOylation Regulates TDP-43 Splicing Activity and Nucleocytoplasmic Distribution

The nuclear RNA-binding protein TDP-43 forms abnormal cytoplasmic aggregates in the brains of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients and several molecular mechanisms promoting TDP-43 cytoplasmic mislocalization and aggregation have been proposed, including defects in nucleocytoplasmic transport, stress granules (SG) disassembly and post-translational modifications (PTM). SUMOylation is a PTM which regulates a variety of cellular processes and, similarly to ubiquitination, targets lysine residues. To investigate the possible regulatory effects of SUMOylation on TDP-43 activity and trafficking, we first assessed that TDP-43 is SUMO-conjugated in the nuclear compartment both covalently and non-covalently in the RRM1 domain at the predicted lysine 136 and SUMO-interacting motif (SIM, 106–110 residues), respectively. By using the SUMO-mutant TDP-43 K136R protein, we demonstrated that SUMOylation modifies TDP-43 splicing activity, specifically exon skipping, and influences its sub-cellular localization and recruitment to SG after oxidative stress. When promoting deSUMOylation by SENP1 enzyme over-expression or by treatment with the cell-permeable SENP1 peptide TS-1, the cytoplasmic localization of TDP-43 increased, depending on its SUMOylation. Moreover, deSUMOylation by TS-1 peptide favoured the formation of small cytoplasmic aggregates of the C-terminal TDP-43 fragment p35, still containing the SUMO lysine target 136, but had no effect on the already formed p25 aggregates. Our data suggest that TDP-43 can be post-translationally modified by SUMOylation which may regulate its splicing function and trafficking, indicating a novel and druggable mechanism to explore as its dysregulation may lead to TDP-43 pathological aggregation in ALS and FTD.

Cytoplasmic TDP-43 is involved in cell fate during stress recovery.

Transactive response DNA binding protein 43 (TDP-43) is an RNA processing protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Nuclear TDP-43 mislocalizes in patients to the cytoplasm, where it forms ubiquitin-positive inclusions in affected neurons and glia. Physiologically, cytoplasmic TDP-43 is associated with stress granules (SGs). Here, we explored TDP-43 cytoplasmic accumulation and stress granule formation following osmotic and oxidative stress. We show that sorbitol drives TDP-43 redistribution to the cytoplasm, while arsenite induces the recruitment of cytoplasmic TDP-43 to TIA-1 positive SGs. We demonstrate that inducing acute oxidative stress after TDP-43 cytoplasmic relocalization by osmotic shock induces poly (ADP-ribose) polymerase (PARP) cleavage, which triggers cellular toxicity. Recruitment of cytoplasmic TDP-43 to polyribosomes occurs in an SH-SY5Y cellular stress model and is observed in FTD brain lysate. Moreover, the processing body (P-body) marker DCP1a is detected in TDP-43 granules during recovery from stress. Overall, this study supports a central role for cytoplasmic TDP-43 in controlling protein translation in stressed cells.

Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis

Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.