Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11–positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.

Amyotrophic lateral sclerosis (ALS) presents with focal muscle weakness due to motor neuron degeneration that becomes generalized, leading to death from respiratory failure within 3–5 years from symptom onset. Despite the heterogeneity of aetiology, TDP-43 proteinopathy is a common pathological feature that is observed in >95% of ALS and tau-negative frontotemporal dementia (FTD) cases. TDP-43 is a DNA/RNA-binding protein that in ALS and FTD translocates from being predominantly nuclear to form detergent-resistant, hyperphosphorylated aggregates in the cytoplasm of affected neurons and glia. Mutations in TARDBP account for 1–4% of all ALS cases and almost all arise in the low complexity C-terminal domain that does not affect RNA binding and processing. Here we report an ALS/FTD kindred with a novel K181E TDP-43 mutation that is located in close proximity to the RRM1 domain. To offer predictive gene testing to at-risk family members, we undertook a series of functional studies to characterize the properties of the mutation. Spectroscopy studies of the K181E protein revealed no evidence of significant misfolding. Although it is unable to bind to or splice RNA, it forms abundant aggregates in transfected cells. We extended our study to include other ALS-linked mutations adjacent to the RRM domains that also disrupt RNA binding and greatly enhance TDP-43 aggregation, forming detergent-resistant and hyperphosphorylated inclusions. Lastly, we demonstrate that K181E binds to, and sequesters, wild-type TDP-43 within nuclear and cytoplasmic inclusions. Thus, we demonstrate that TDP-43 mutations that disrupt RNA binding greatly enhance aggregation and are likely to be pathogenic as they promote wild-type TDP-43 to mislocalize and aggregate acting in a dominant-negative manner. This study highlights the importance of RNA binding to maintain TDP-43 solubility and the role of TDP-43 aggregation in disease pathogenesis.

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

A pathological hallmark of neurodegenerative disease is the accumulation of aberrant protein aggregates which contribute to the cytotoxicity and are therefore a target for therapy development. One key mechanism to manage cellular protein homeostasis is heat shock proteins (HSPs), protein chaperones which are known to target aberrant protein accumulation. Activation of HSPs target aberrant TDP-43, tau and amyloid to rescue neurodegenerative disease. As an attempt to target HSP activation for neurodegeneration therapy, we here develop a drug screening assay to identify compounds that will activate the master regulator of HSPs, the transcription factor heat shock factor 1 (HSF1). As HSF1 is bound by HSP90 which prevents its activation, we developed a NanoBRET assay, which allows us to monitor and quantify the HSF1-HSP90 interaction in living cells to screen for compounds disrupting this interaction and thereby releasing HSF1 for activation. After the optimisation and validation of the assay, a two thousand compound library was screened which produced 10 hits including two known HSP90 inhibitors. Follow-up functional study showed that one of the hits oxyphenbutazone (OPB) significantly reduces the accumulation of insoluble TDP-43 in a cell model, eliciting no signs of stress or toxicity. Overall, this study demonstrates a viable strategy for new drug discovery in targeting aberrant proteins and identifies potential candidates for translation into neurodegenerative disease treatment.

TDP-43 is an RNA-binding protein constituting the pathological inclusions observed in ∼95% of ALS and ∼50% of FTD patients. In ALS and FTD, TDP-43 mislocalises to the cytoplasm and forms insoluble, hyperphosphorylated and ubiquitinated aggregates that enhance cytotoxicity and contribute to neurodegeneration. Despite its primary role as an RNA/DNA-binding protein, how RNA-binding deficiencies contribute to disease onset and progression are little understood. Among many identified familial mutations in TDP-43 causing ALS/FTD, only two mutations cause an RNA-binding deficiency, K181E and K263E. In this study, we used CRISPR/Cas9 to knock-in the two disease-linked RNA-binding deficient mutations in SH-SY5Y cells, generating both homozygous and heterozygous versions of the mutant TDP-43 to investigate TDP-43-mediated neuronal disruption. Significant changes were identified in the transcriptomic profiles of these cells, in particular, between K181E homozygous and heterozygous cells, with the most affected genes involved in neuronal differentiation and synaptic pathways. This result was validated in cell studies where the neuronal differentiation efficiency and neurite morphology were compromised in TDP-43 cells compared to unmodified control. Interestingly, divergent neuronal regulation was observed in K181E-TDP-43 homozygous and heterozygous cells, suggesting a more complex signalling network associated with TDP-43 genotypes and expression level which warrants further study. Overall, our data using cell models expressing the ALS/FTD disease-causing RNA-binding deficient TDP-43 mutations at endogenous levels show a robust impact on transcriptomic profiles at the whole gene and transcript isoform level that compromise neuronal differentiation and processing, providing further insights on TDP-43-mediated neurodegeneration.