Identifying a novel role for cytoplasmic dynein in microtubule acetylation and investigating the multi-hit hypothesis in Motor Neuron Disease
Spinal muscular atrophy with lower extremity predominance (SMA-LED) and amyotrophic lateral sclerosis (ALS) are childhood and adult-onset motor neuron diseases, respectively, that are characterised by loss of motor neurons. Although the underlying molecular mechanisms of these disorders are not fully understood, deficiencies in axonal transport, microtubule dynamics and protein quality control systems have been implicated in the pathogenesis. As such, this thesis investigates consequences of mutations in dynein cytoplasmic 1 heavy chain (DYNC1H1), encoding the heavy chain subunit of cytoplasmic dynein, on MT stability in cellular models of SMA-LED. In addition, it investigates how defective cytoplasmic dynein could make cells more prone to toxic protein aggregation.
Post-translational acetylation of α-tubulin modulates MT dynamics and altered MT dynamics has been linked to neurodegeneration. Analysing fibroblasts derived from an SMA-LED patient and a mouse model of SMA-LED known as Legs at odd angles (Loa), identified a reduced interaction between dynein and alpha-tubulin N-acetyltransferase 1 (ATAT1), the canonical enzyme for acetylation of MTs. The alteration in dynein-ATAT1 interaction suggests a regulatory role of dynein in MT acetylation, which when compromised could contribute to the pathology of SMA-LED.
Furthermore, dynein is involved in autophagic removal of misfolded protein aggregates and damaged organelles. There is a plethora of evidence suggesting decreased autophagic activity with ageing, which could contribute to the cytoplasmic accumulation of misfolded proteins and mislocalisation of TDP-43 as hallmarks of ALS. Here, I utilised cells derived from the Loa mouse model and manipulation of the autophagy associated kinase TANK-binding kinase 1 (TBK1) to reveal a significant increase in the levels of LC3II and p62, as autophagy markers, when TBK1 is genetically or pharmacologically inhibited in cells harbouring the Loa mutation. Importantly, analysis of Loa fibroblasts and hippocampal neurons after an exposure to sequential hits such as TBK1 or proteasome inhibitors led to significant increase in TDP-43 mislocalisation. Moreover, there was a delayed recovery in clearance of stress granules in Loa fibroblasts and neurons, which could mediate further iv formation of protein aggregates, demonstrating the vulnerability of neurons harbouring defective dynein to ageing and cellular stress.
In conclusion, these data suggest regulation of microtubule acetylation through dynein-dependent recruitment of ATAT1 to the microtubule and a potential mechanism involved in the pathogenesis of SMA-LED. Moreover, these data support a multiple-hit hypothesis of neurodegeneration in which the dynein malfunction could establish the primary susceptibility which in combination with variations in another ALS-linked gene such as TBK1 or functional decline in the ubiquitin-proteasome system could exacerbate the formation of aggregates and mislocalisation of TDP-43 that are hallmarks of ALS. Therefore, these data provide further insight into the origin or progression of SMA-LED and ALS, opening the way to find more effective therapeutic targets of the disease.
History
File Version
- Published version
Pages
363.0Department affiliated with
- BSMS Neuroscience Theses
Qualification level
- doctoral
Qualification name
- phd
Language
- eng
Institution
University of SussexFull text available
- Yes