The hereditary spastic paraplegias (HSP) comprise a genetically heterogeneous group of disorders characterized by progressive bilateral weakness and spasticity of the lower limbs. This hallmark clinical symptom of HSP results from progressive dysfunction and degeneration of upper motor neurons in corticospinal tracts and dorsal column fibers. Over fifteen mutations in structurally and functionally unrelated genes have been identified as causative of HSP. Among these, mutations in the SPG4 locus coding for the microtubule-severing protein spastin represent the most common cause of HSP. Despite these major breakthroughs, pathogenic mechanisms underlying HSP pathogenesis remain unknown. Pathological observations from HSP patients and HSP animal models indicate that neurons affected in HSP degenerate following a "dying back" pattern, which is characterized by early abnormalities in synapses and distal axons and progressive degeneration of axons. Significantly, recent genetic data demonstrated that reductions in fast axonal transport (FAT) result in such pattern of neuronal degeneration. Moreover, loss of function mutations in the SPG10 locus coding for a specific subunit of the molecular motor protein conventional kinesin lead to HSP, suggesting that abnormalities in FAT might indeed represent a critical event in HSP pathogenesis. Our recent studies demonstrated that nanomolar levels of specific mutant spastin isoforms inhibit FAT in an axon-autonomous manner. Consistent with a role of kinases in the regulation of FAT, pharmacological studies presented in this application indicate that this effect of pathogenic spastin was mediated by the activity of casein kinase 2 (CK2). Complementing these observations, active CK2 was found to inhibit FAT. Moreover, CK2 was found to directly phosphorylate and inhibit the functionality of the molecular motor protein conventional kinesin. Based on these and additional findings herein, we propose that activation of axonal CK2 and inhibition of FAT induced by pathogenic spastin represent critical pathogenic events in HSP. Experiments in this application will characterize effects of pathogenic spastin on FAT. Biochemical, immunochemical, pharmacological and cell biological methods will be used to identify specific axonal cargoes and molecular motors associated with pathogenic spastin. Based on our previous work, lentiviral approaches will evaluate isoform-specific effects of pathogenic spastin in vivo. Finally, biochemical, and cell biological approaches will identify and characterize CK2 targets associated with pathogenic spastin including molecular motors and cytoskeletal proteins. These studies will help identifying molecular components and mechanisms mediating the inhibition of FAT induced by pathogenic spastin. Studies proposed here will characterize pathogenic mechanisms underlying the axonal defects characteristic of HSP. The ultimate goal of this project is to identify novel therapeutic targets in HSP that help prevent distal axonopathy and degeneration of motor neurons.
Hereditary spastic paraplegias (HSPs) comprise a heterogeneous group of genetic diseases that lead to lower limb spasticity in affected patients. This clinical symptom hallmark results from dysfunction and dying back degeneration of upper motor neurons. Although HSP pathogenesis involves early alterations in axonal and synaptic function, specific mechanisms and molecular components involved in this pathogenic event remain elusive. Mutations in the SPG4 locus coding for the microtubule-severing protein spastin represent the most common cause of HSP. The mammalian spastin gene has two start codons, resulting in the production of two alternatively spliced spastin isoforms. Consistent with genetic data linking alterations in axonal transport to HSP, our recently published studies and data presented here indicate that pathogenic forms of spastin protein inhibit axonal transport through a mechanism involving the activity of casein kinase 2 (CK2). Moreover, the effects of pathogenic spastin are isoform-specific and occur independently of changes in gene transcription. Consistent with these observations, active CK2 dramatically inhibited axonal transport. Together, our observations suggest that HSP pathogenesis might involve reductions in the delivery of axonal and synaptic proteins essential for neuronal function and survival. Experiments in this application will characterize alterations in axonal transport induced by pathogenic spastin, and evaluate underlying molecular mechanisms. The ultimate goal of this project is to define disease mechanisms and identify novel therapeutic targets in HSP.
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