Functional analysis of motor neuron disease-associated mutations in TAR DNA-binding protein 43 and cytoplasmic dynein heavy chain 1

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy with lower extremity predominance (SMA-LED) are two forms of motor neuron diseases at the opposite ends of the age spectrum, with ALS being mainly an adult-onset progressive and fatal neurodegenerative disease, and SMA-LED being a childhood-onset neuromuscular disease, manifested by muscle weakness, joint contracture, abnormal gait, and in some cases combined with intellectual disability. This thesis represents my research on the role of mutant forms of two proteins, namely Tar-DNA binding protein 43 (TDP-43) and cytoplasmic dynein heavy chain 1 (DYNC1H1) in the pathogenesis of ALS and SMA-LED, respectively. TDP-43 – an RNA/DNA binding protein – has been implicated in ALS. The function of TDP-43 in the nucleus is to regulate RNA processing, including RNA splicing and editing. Abnormal expression of the peripherin splice variant per61 has been found in transgenic mouse models of ALS. In addition, aberrant expression of EAAT2 (excitatory amino acid glutamate transporter 2) protein has been reported in some ALS cases. Thus, I investigated splicing of peripherin and GLT-1 (the murine homologue of EAAT2) RNAs, as potential targets of TDP-43 and examined whether mutations in TDP-43 alter the expression levels of the genes encoding the two proteins. My data show that per61 is expressed in wild type mice at both RNA and protein levels. This suggests a role for this isoform in the assembly of peripherin filaments. Moreover, overexpression of TDP-43A315T increases the expression level of per45 (an alternative translated isoform of peripherin) and leads to the instability of the filament network. Analysis of GLT-1B, the neuronal splice variant of GLT-1, reveals significant down-regulation in TDP-43A315T transgenic mice, indicating impaired RNA processing of GLT-1B. Collectively, these data show that the expression of peripherin and EAAT2 is regulated by TDP-43, and that aberrant expressions of these two genes caused by TDP-43 mutations could have a role in the pathology of ALS. A Phe580Tyr mutation in the mouse gene Dync1h1 impairs growth factor-induced endocytic trafficking in Dync1h1+/F580Y mouse motor neurons, resulting in aberrant activation of extracellular-signal-related kinases 1 and 2 (ERK1/2) and phosphorylation of the immediate early gene c-Fos. My data show that the induction of c-Fos upon serum starvation and/or growth factor stimulation is ERK1/2 dependent and that the mitogen-activated protein kinase p38 is also likely to be involved in c-Fos activation during starvation. Moreover, the activation of autophagy is reduced in Dync1h1+/F580Y motor neurons, suggesting a role for cytoplasmic dynein in autophagy induction/formation. In addition, the Dync1h1F580Y/F580Y mouse embryonic fibroblasts (MEFs) exhibit a defect in cell migration, as manifested by a delayed wound closure and reduced levels of paxillin phosphorylation at Tyr118 (p-paxillin). They also show abnormal and increased number of focal adhesions in spreading assays. Interestingly, human SMA-LED DYNC1H1R399G/R399G fibroblasts show defective lamellipodia formation, as well as reduced levels of p-paxillin. Moreover, Dync1h1+/F580Y mouse motor neurons show a defect in exploratory microtubules in the peripheral domain of their growth cones. As the molecular mechanism of growth cone motility is analogous to that found in fibroblasts, the molecular pathogenesis of SMA-LED caused by mutations in cytoplasmic dynein heavy chain 1, is likely to involve impaired growth cone development and axonal pathfinding, which could be exacerbated by the aberrant endocytic trafficking and signalling in mutant motor neurons.