Abstract A pathological hallmark of neurodegenerative disease is the accumulation of aberrant protein aggregates which contribute to the cytotoxicity and are therefore a target for new therapy development. One key machinery to manage cellular protein homeostasis is the chaperones proteins, the heat shock proteins (HSPs) which are known to target aberrant proteins including TDP-43, tau and amyloid and rescue neurodegeneration in various disease models. As an attempt to target HSP activation for neurodegeneration therapy, we develop a drug screening assay to search compounds that 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 develop 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 assay was optimised and validated for its robustness and reliability, a two thousand compound library was screened which produced 10 hits including a couple of known HSP90 inhibitors. Follow-up functional study showed that one of the hit oxyphenbutazone (OPB) significantly reduces the accumulation of insoluble TDP-43 in a cell model, displaying no signs of stress or toxicity. Further works are needed to characterise the mode-of-action of OPB in HSF1 activation and aberrant protein reduction, and the physiological impact of OPB on TDP-43 needs to be examined in an in vivo model. However, this study demonstrates a viable strategy for new drug discovery in targeting aberrant proteins and identifies potential candidates for translation into neurodegenerative disease treatment.
TARDBP (TDP-43) is the major pathological protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Large TDP-43 aggregates decorated by degradation adaptor proteins are seen in the cytoplasm of remaining neurons in patients post mortem. TDP-43 accumulation, and ALS-linked mutations within degradation pathways, implicates failed TDP-43 clearance as a primary disease mechanism. Here we report the differential roles of the ubiquitin proteasome system (UPS) and autophagy in the clearance of TDP-43. We have investigated the effects of UPS and autophagy inhibitors on the degradation, localisation and mobility of soluble and insoluble TDP-43. We find that soluble TDP-43 is degraded primarily by the UPS, while aggregated TDP-43 clearance requires autophagy. Cellular macroaggregates, which recapitulate many pathological features of patient aggregates, are reversible when both the UPS and autophagy are competent. Their clearance involves the autophagic removal of oligomeric TDP-43. We speculate that in addition to age-related decline, a second hit in the UPS or autophagy pathways drives the accumulation of TDP-43 in ALS and FTD. Therapies for clearing excess TDP-43 should therefore be targeted to a combination of these pathways.
Background The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [1,2]. The mechanism by which the G4C2 intronic repeats cause neurodegeneration is unknown. Decreased tissue levels of the C9ORF72 transcript implicate a loss of protein function due to haploinsufficiency, intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic [1].
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.
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.
Following the mutation screening of genes known to cause amyotrophic lateral sclerosis (ALS) in index cases from 107 familial ALS (FALS) kindred, a point mutation was identified in vesicle-associated membrane protein-associated protein B (VAPB), or VAMP-associated protein B, causing an amino acid change from threonine to isoleucine at codon 46 (T46I) in one FALS case but not in 257 controls. This is an important finding because it is only the second mutation identified in this gene that causes ALS. In order to investigate the pathogenic effects of this mutation, we have used a motor neuron cell line and tissue-specific expression of the mutant protein in Drosophila. We provide substantial evidence for the pathogenic effects of this mutation in abolishing the effect of wild type VAPB in the unfolded protein response, promoting ubiquitin aggregate formation, and activating neuronal cell death. We also report that expression of the mutant protein in the Drosophila motor system induces aggregate deposition, endoplasmic reticulum disorganization, and chaperone up-regulation both in neurons and in muscles. Our integrated analysis of the pathogenic effect of the T46I mutation and the previously identified P56S mutation indicate extensive commonalities in the disease mechanism for these two mutations. In summary, we show that this newly identified mutation in human FALS has a pathogenic effect, supporting and reinforcing the role of VAPB as a causative gene of ALS. Following the mutation screening of genes known to cause amyotrophic lateral sclerosis (ALS) in index cases from 107 familial ALS (FALS) kindred, a point mutation was identified in vesicle-associated membrane protein-associated protein B (VAPB), or VAMP-associated protein B, causing an amino acid change from threonine to isoleucine at codon 46 (T46I) in one FALS case but not in 257 controls. This is an important finding because it is only the second mutation identified in this gene that causes ALS. In order to investigate the pathogenic effects of this mutation, we have used a motor neuron cell line and tissue-specific expression of the mutant protein in Drosophila. We provide substantial evidence for the pathogenic effects of this mutation in abolishing the effect of wild type VAPB in the unfolded protein response, promoting ubiquitin aggregate formation, and activating neuronal cell death. We also report that expression of the mutant protein in the Drosophila motor system induces aggregate deposition, endoplasmic reticulum disorganization, and chaperone up-regulation both in neurons and in muscles. Our integrated analysis of the pathogenic effect of the T46I mutation and the previously identified P56S mutation indicate extensive commonalities in the disease mechanism for these two mutations. In summary, we show that this newly identified mutation in human FALS has a pathogenic effect, supporting and reinforcing the role of VAPB as a causative gene of ALS.
Detergent-resistant, ubiquitinated and hyperphosphorylated Tar DNA binding protein 43 (TDP-43, encoded by TARDBP ) neuronal cytoplasmic inclusions are the pathological hallmark in ∼95% of amyotrophic lateral sclerosis and ∼60% of frontotemporal lobar degeneration cases. We sought to explore the role for the heat shock response in the clearance of insoluble TDP-43 in a cellular model of disease and to validate our findings in transgenic mice and human amyotrophic lateral sclerosis tissues. The heat shock response is a stress-responsive protective mechanism regulated by the transcription factor heat shock factor 1 (HSF1), which increases the expression of chaperones that refold damaged misfolded proteins or facilitate their degradation. Here we show that manipulation of the heat shock response by expression of dominant active HSF1 results in a dramatic reduction of insoluble and hyperphosphorylated TDP-43 that enhances cell survival, whereas expression of dominant negative HSF1 leads to enhanced TDP-43 aggregation and hyperphosphorylation. To determine which chaperones were mediating TDP-43 clearance we over-expressed a range of heat shock proteins (HSPs) and identified DNAJB2a (encoded by DNAJB2 , and also known as HSJ1a) as a potent anti-aggregation chaperone for TDP-43. DNAJB2a has a J domain, allowing it to interact with HSP70, and ubiquitin interacting motifs, which enable it to engage the degradation of its client proteins. Using functionally deleted DNAJB2a constructs we demonstrated that TDP-43 clearance was J domain-dependent and was not affected by ubiquitin interacting motif deletion or proteasome inhibition. This indicates that TDP-43 is maintained in a soluble state by DNAJB2a, leaving the total levels of TDP-43 unchanged. Additionally, we have demonstrated that the levels of HSF1 and heat shock proteins are significantly reduced in affected neuronal tissues from a TDP-43 transgenic mouse model of amyotrophic lateral sclerosis and patients with sporadic amyotrophic lateral sclerosis. This implies that the HSF1-mediated DNAJB2a/HSP70 heat shock response pathway is compromised in amyotrophic lateral sclerosis. Defective refolding of TDP-43 is predicted to aggravate the TDP-43 proteinopathy. The finding that the pathological accumulation of insoluble TDP-43 can be reduced by the activation of HSF1/HSP pathways presents an exciting opportunity for the development of novel therapeutics. Insoluble TDP-43 inclusions are the pathological hallmark of ALS and tau-negative frontotemporal lobar degeneration. Chen et al. show that the heat shock response (HSR), which regulates chaperone expression, is compromised in an ALS mouse model and in patients. Activation of the HSR clears insoluble TDP-43 and increases cell survival.
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