Functional and genetic analyses of HSPB mutations underlying progressive neuronal deficits in Charcot-Marie-Tooth disease

Charcot-Marie-Tooth (CMT) disease is one of the most commonly inherited progressive neurological disorders, characterised by muscle degeneration and neurosensory deficits caused by mutations in neuronal proteins. The majority of CMT disease cases are categorised into type 1 (demyelinating) and type 2 (intra-axonal dysfunction). Patients diagnosed with CMT type 2F (CMT2F) caused by dominant mutations in the small heat shock protein beta-1 (HSPB1) display progressive weakness within the distal muscles of the arms and legs. Genetic mutations within the HSPB1 gene have been identified as specific causative factors in CMT type 2; however, the mechanisms by which these mutations create progressive neuronal defects are largely unknown. HSPB1 is an ATP-independent chaperone that regulates proteostasis through dynamic interactions with client proteins. Structurally, HSPB1 consists of an alpha-crystallin domain flanked by N and C termini. Mutations in the alpha-crystallin domain have been the subject of many studies. In this project we focused on the C-terminal HSPB1 Q175X mutation first identified in a family of CMT-affected patients. Using the C. elegans nematode model, we expressed either wild-type (WT) human HSPB1 or HSPB1 Q175X specifically in neurons. In the behavioural aldicarb assay (a proxy assay for analysing synaptic transmission) we found that expression of the Q175X mutation caused a resistance to the acetylcholinesterase inhibitor aldicarb that changed progressively with C. elegans age. Next, we quantitatively assessed the expression levels of WT and/or Q175X mutant in mammalian cells (human embryonic kidney (HEK) and neuroblastoma-derived cell line (SH-SY5Y). We found that expression of Q175X is unable to form oligomers in the absence of HSPB1 WT. To examine the mechanisms underlying progressive neurotransmission dysfunction in response to the Q175X mutation further, we compared the effects of WT and mutant HSPB1 on stimulus-evoked exocytosis in bovine chromaffin cells using whole-cell patch clamp recording and membrane capacitance measurements. We demonstrated that the Q175X mutation affected asynchronous exocytosis by possibly altering the chromaffin cells' ability to regulate calcium.