Understanding the regulation of molecular chaperones in motor neurons

Cells are constantly challenged by acute and chronic stresses that must be counteracted by upregulation of protective pathways. The premise of this thesis is that motor neurons have an impaired ability to trigger these protective mechanisms, which may contribute to their preferential vulnerability in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). The first objective was to study the involvement of metallochaperones in motor neuronal stress response, including their potential for rescuing motor neurons from toxicity conferred by a mutant Cu/Zn-superoxide dismutase (SOD1G93A ) that causes a form of familial ALS. Motor neurons in dissociated spinal cord cultures failed to induce the metallochaperone, metallothionein (MT), in response to classical MT inducers, although overexpression of MT in motor neurons failed to protect them from SOD1G93A. A second response system, involving protein chaperones called heat shock proteins (Hsp), was more therapeutically promising, but was also impaired in motor neurons due to an inability to activate the regulatory protein heat shock transcription factor 1 (Hsf1). The remaining objectives were to examine if activation of Hsf1 in motor neurons would protect against SOD1G93A and to understand the mechanisms responsible for its impaired activation. A constitutively active form of Hsf1 induced multiple Hsps in motor neurons and nearly eliminated SOD1G93A toxicity and aggregation. Experiments also demonstrated that failure of stressed motor neurons to activate endogenous Hsf1 is not a result of inappropriate or insufficient activity of kinases that phosphorylate key residues of Hsf1 in nonneuronal cell lines with a competent heat shock response. Disruption of inhibitory Hsp90/multichaperone complexes is another important step in Hsf1 activation. Four different pharmacological inhibitors of Hsp90 induced multiple Hsps in motor neurons, although failure to observe the same response by targeting inhibitory complexes with activator of Hsp90 ATPase 1 (Aha1) or Daxx suggested other mechanisms were involved. A constitutively active form of calcium/calmodulin-dependent kinase N induced Hsp70 in motor neurons, but not in fibroblasts and likely through an Hsf1-independent mechanism. These results provide further evidence for disparity between the stress response of motor neurons and other cells and suggest the possibility of a unique Hsp regulatory system in neurons