Gains or losses: molecular mechanisms of TDP43-mediated neurodegeneration

Abstract

RNA-binding proteins, and in particular TAR DNA-binding protein 43 (TDP43), are central to the pathogenesis of motor neuron diseases and related neurodegenerative disorders. Studies on human tissue have implicated several possible mechanisms of disease and experimental studies are now attempting to determine whether TDP43-mediated neurodegeneration results from a gain or a loss of function of the protein. In addition, the distinct possibility of pleotropic or combined effects - in which gains of toxic properties and losses of normal TDP43 functions act together - needs to be considered.

Figure 1. The genetics, pathology and biochemistry of TDP43 proteinopathies

a | TAR DNA-binding protein 43 (TDP43) protein contains two RNA-recognition motifs (RNA-recognition motif 1 (RRM1) and RRM2), a carboxy-terminal glycine-rich domain, a bipartite nuclear localization signal (NLS) and a nuclear export signal (NES). Numerous mutations (shown by short vertical lines) have been linked to sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). These are almost exclusively found within or immediately adjacent to the glycine-rich domain with the exception of an Asp169Gly mutation within exon 4 (the site at which TDP43 cleavage putatively occurs is shown by an arrow). Known TDP43 phosphorylation sites (Ser 379, Ser403 + Ser404, and Ser409 + Ser 410), when heavily phosphorylated, also contribute to the disease-specific TDP43 biochemical signature (these sites are indicated by asterisks). b | TDP43 immunohistochemistry of human FTLD brain reveals intracytoplasmic inclusions affecting neurons. Dystrophic neurites and glia also exhibit TDP43 inclusions (not shown). An image of the dentate gyrus stained with an anti-TDP43 antibody shows cells without inclusions with normal nuclear immunoreactivity, whereas inclusion-bearing cells show a loss of normal nuclear staining that leads to a presumptive loss of TDP43 function in the nucleus. TDP43 immunofluorescence (shown in green) of an intranuclear inclusion in FTLD-TDP43 is shown. TDP43 immunostaining using a phospho-specific anti-TDP43 antibody of human ALS spinal cord shows characteristic round inclusions (shown by brown immunohistochemistry) and skeins (shown by red immunofluorescence) in lower motor neurons. c | Biochemical analysis demonstrates the distinct biochemical disease-specific TDP43 signature characterized by the accumulation of sarkosyl-insoluble TDP43 species comprised of phosphorylated TDP43, ubiquitylated high molecular weight TDP43 and truncated C-terminal fragments.

Figure 2. Normal functions of TDP43

TAR DNA-binding protein 43 (TDP43) exhibits multiple normal biological functions, predominantly those that regulate RNA pathways. a | TDP43 is a component of heterogenous nuclear ribonucleoprotein (hnRNP) particles, which regulate splicing of pre-mRNA species. b | TDP43 also binds to mRNA sequences, particularly within the 3′ untranslated region, and affects mRNA stability and turnover. c | TDP43 is thought to play a part in mRNA trafficking, as TDP43 undergoes rapid nucleo–cytoplasmic shuttling and is localized within dendritic RNA granules. d | TDP43 is also a component of the Drosha complex, which functions to process primary microRNAs. e | TDP43 can act as a transcriptional repressor by binding to single stranded DNA (ssDNA) promoter sequences. f | TDP43 also colocalizes with stress granules that are thought to sequester and protect mRNAs under conditions of stress.

Figure 3. TDP43 modification, stability and turnover

Both proteosomal and autophagasomal degradation of TAR DNA-binding protein 43 (TDP43) protein has been described. We found that full-length TDP43 is a long-lived protein with a half-life of greater than 34 hours, although other studies have reported that it has a half-life ranging from 4 to 12 hours depending on the cell type^,. Truncation of TDP43 results in the production of carboxy-terminal fragments (CTFs) that are rapidly translocated to the cytoplasm and degraded. TDP43 aggregates can form under various conditions in which CTFs and full-length TDP43 protein seem to co-aggregate, and TDP43 aggregation is tightly linked with the presence of phosphorylated TDP43. Ubiquitylation of TDP43 aggregates seems to occur late in the lifecycle of an inclusion. Given that TDP43 aggregates are resistant to degradation, different TDP43 isoforms and conformers exhibit different turnover rates, ranging from the labile soluble CTFs to stable insoluble aggregates. t_1/2, half-life.

Figure 4. Lifecycle of TDP43 pathology

Normal neurons show robust intranuclear TAR DNA-binding protein 43 (TDP43) immunoreactivity (shown in red) with little cytoplasmic TDP43. So-called ‘pre-inclusions’ have been described, and these consist of granular cytoplasmic aggregates that are positive for phospho-TDP43 epitopes (p409 and p410) but that are often negative for ubiquitin. Neurons with pre-inclusions show characteristic loss of normal nuclear staining. Bona fide inclusions exhibit a variety of morphologies ranging from dense round inclusions and skeins in motor neurons to dystrophic neurites, cytoplasmic inclusions or intranuclear ‘cat eye’ inclusions in other neurons. Neuronophagia can rarely be seen in amyotrophic lateral sclerosis, and neuronophagic cell clusters have been reported to be associated with TDP43 inclusions.

Figure 5. Subcellular localization of TDP43 protein

TAR DNA-binding protein 43 (TDP43) shows a predominantly nuclear localization, although nucleo–cytoplasmic shuttling of TDP43 has been shown and low levels of cytoplasmic TDP43 can be demonstrated. TDP43 accumulation in the cytoplasm can be induced by a variety of cellular stressors that result in the formation of TDP43-positive stress granules. TDP43 protein has also been found in RNA transport granules within neuronal processes. TDP43 protein is thought to undergo proteolytic cleavage by caspase 3 to generate a carboxy-terminal fragment (CTF). As the CTF no longer contains the bipartite nuclear localization signal (NLS, shown by a red rectangle), CTFs translocate into the cytoplasm, where they may participate in aggregate formation. Experimentally generated mutations of the NLS and disease-associated mutations (shown by a yellow lightning bolt) have been found to increase the amount of cytoplasmic versus nuclear TDP43. MG132 is a proteasome inhibitor.

Figure 6. Autoregulation of TDP43 and models of TDP43 toxicity

a | Normal TAR DNA-binding protein 43 (TDP43) autoregulation. TDP43 expression is tightly regulated under normal conditions, and overexpression of exogenous TDP43 results in a reduction of endogenous TDP43 expression. This autoregulation of TDP43 expression by itself is mediated at the level of mRNA stability. Within the 3′ untranslated region (UTR) of TDP43 mRNA is a binding site for TDP43 protein that is critical for autoregulation. TDP43 binding to this 3′ UTR site promotes degradation of TDP43 mRNA, at least partly through the exosome. There are conflicting reports about the mechanism of autoregulation, in particular whether alternative splicing leading to nonsense-mediated decay (NMD) is the mechanism of TDP43 autoregulation. b | Loss of autoregulation. According to this model, the exposure of neurons to as-yet-unidentified stressors can lead to cytoplasmic mislocalization of TDP43 (1), and this is perhaps related to the stress granule response. Given TDP43’s propensity to aggregate, TDP43 forms phosphorylated pre-inclusions within the cytoplasm that sequester free TDP43 protein. This cytoplasmic sequestration leads to a loss of normal nuclear TDP43. If autoregulation occurs within the nucleus, a loss of TDP43 autoregulation ensues (2), which results in increased TDP43 mRNA and protein (3). This further exacerbates TDP43 aggregation (4). This vicious cycle leads to cell death (5) possibly through a variety of gain and loss of functions. c | Gain of autoregulation. In this model, neurons that are exposed to unknown stressors can undergo cytoplasmic mislocalization of TDP43 protein (1), and this is perhaps related to the stress granule response. Given the propensity of TDP43 to aggregate, TDP43 forms phosphorylated pre-inclusions within the cytoplasm which are resistant to degradation. If autoregulation occurs within the cytoplasm, an increase in cytoplasmic TDP43 may result in an increase in TDP43 autoregulation (2) that would decrease TDP43 mRNA and therefore decrease synthesis of new TDP43 protein (3). This reduction in TDP43 protein synthesis leads to a loss of normal nuclear TDP43 protein. Given the plethora of normal nuclear TDP43 functions, the absence of nuclear TDP43 is detrimental to neuronal viability, increasing the stress response (4) and leading to cell death (5). Again, this model allows for the possibly of a variety of gain and loss of functions that coordinately result in toxicity.