Abstract
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.
Highlights
Amyotrophic lateral sclerosis (ALS), known as motor neuron disease (MND), is the most common adult-onset degenerative disease of motor neurons
Investigating the impact of loss of RNA-binding on TDP43 proteinopathy further, we demonstrate that mutations that leave TDP-43 incapable of binding RNA promote the accumulation of insoluble aggregates in the nucleus and cytoplasm, suggesting the loss of interaction with RNA can be a factor contributing to the escalation of TDP-43 proteinopathy
To investigate the influence of RNA binding on the behaviour of cytoplasmic TDP-43, we introduced the K181E mutation into a TDP-43 construct lacking the nuclear localizing signal (GFP-dNLS TDP-43), which is preferentially retained in the cytoplasm
Summary
Amyotrophic lateral sclerosis (ALS), known as motor neuron disease (MND), is the most common adult-onset degenerative disease of motor neurons. Regardless of the cause, $95% of patients share a common molecular pathology featuring the accumulation of ubiquitinated, hyperphosphorylated and detergent resistant TDP-43 protein aggregates in the cytoplasm of affected neuronal tissues (Neumann et al, 2006; Mackenzie et al, 2007; Tan et al, 2007; Geser et al, 2008; Pamphlett et al, 2009; Brettschneider et al, 2013). In 2006, the accumulation of detergent-resistant, hyperphosphorylated TDP-43 was reported in affected neuronal tissues of ALS and frontotemporal dementia (FTD) cases (Neumann et al, 2006). Mutations in TDP-43 were found in patients of both familial and sporadic ALS (Sreedharan et al, 2008). As TDP-43 cytoplasmic aggregates are observed in $95% of ALS and tau-negative FTD, the understanding of the mechanisms contributing to the build-up of TDP-43 proteinopathy is critical in unravelling disease development and holds the key to developing effective therapeutic strategies
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