The contribution of cytoskeleton disruptions in ALS disease pathology



 DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons from the brain and spinal cord. Disease progression is rapid and patients typically die from respiratory failure 3-5 years following disease onset. Numerous ALS causing mutant genes have been identified but the underlying cause of motor neuron degeneration is not known. Recent genome analysis from families that contain a heritable form of ALS have identified multiple mutations in two cytoskeletal genes, profilin 1 and TUBA4A, that cause ALS; linking cytoskeletal dysfunction to ALS pathology. In this proposal I will examine whether an ALS mutant gene disrupts two cytoskeletal-dependent functions that are crucial for motor neuron health. Motor neurons extend structures great distances from the cell body and rely heavily on the microtubule cytoskeleton for the transport of cellular materials to these distal structures. In the first part of the proposl, I will examine whether expression of mutant TUBA4A affects the transport of essential microtubule based cargos to and from the distal structures. I first will use live cell imaging to track the rate of transport of various essential cargos in primary cultured neurons expressing mutant TUBA4A. Next I will examine if developmental or cellular interactions play a contributing role by examining changes in motor protein movement in the sciatic nerve of mutant TUBA4A expressing mice. Finally, I will evaluate weather mutant TUBA4A expression changes the motor neuron tolerance to microtubule-destabilizing neurotoxic insult. Injury to motor neurons causes separation of the motor neuron axon from the neuromuscular junction (NMJ). After the initial denervation of the NMJ the motor neuron can follow one of two paths: axon regrowth and reattachment to the NMJ or continued axon dieback and cell death. The stability of the microtubules in the tip of the injured axon will influence which of these two paths will be the finl outcome. In the second part of the proposal I will examine if mutant TUBA4A expression increases retraction bulb formation and decreases axon regeneration following injury. I will first use live cell imaging to measure the rate of regrowth of severed axons in primary motor neurons expressing mutant TUBA4A. I will next examine whether mutant TUBA4A expression changes the rate of growth cone formation and microtubule organization after sciatic nerve lesion in mutant TUBA4A expressing mice. Finally, I will examine whether mutant TUBA4A affects the reinnervation of muscle tissue following nerve crush in mutant TUBA4A expressing mice. The goal of this proposal is to further knowledge on how cytoskeleton-dependent functions are involved in ALS disease pathology. The results gained from this proposal will offer new insight into the cause of cell death in ALS and will open new areas for drug research and therapy.