Disruption of retrograde transport is sufficient to cause motor neuron disease in mice and has been linked to neurodegenerative disease in patients expressing a mutation in the dynein/dynactin motor complex. The mechanism by which disruption of retrograde transport causes neurodegeneration is unclear. Several studies implicate mitochondria! dysfunction in motor neuron pathogenesis. We propose that deficits in retrograde transport result in altered mitochondrial transport and/or morphology leading to mitochondrial dysfunction and neuronal cell death. We will analyze mitochondrial distribution and morphology using cellular and murine models of neurodegenerative disease expressing mutations in components of the dynein/dynactin transport complex. We will determine through biochemical analyses which proteins and domains mediate interactions between mitochondria and dynein/dynactin. Live cell imaging will be used to examine mitochondrial movement in wild type cells, as well as cells with mutations in dynein/dynactin. Mitochondrial distribution and morphology will be analyzed using immunofluorescence in both cell and mouse models and temporal correlation between these changes and disease progression in mutant mice will be established. Finally, mitochondrial function will be assessed through measurements of membrane potential, Ca++ buffering capacity, ATP production, and induction of apoptosis using mitochondria from cells as well as mitochondria isolated from mouse neuronal tissue. We believe that understanding the interaction between mitochondria and axonal transport will help to elucidate the molecular cause of neuronal dysfunction and death and suggest pathways that can be manipulated for therapeutic purposes. Lay summary: The molecular mechanism or mechanisms responsible for causing motor neuron diseases, such as ALS are unclear. Motor neurons are very long cell; one possible mechanism for their dysfunction is the loss of active transport of important cargoes along the length of the cell. Mitochondria are organelles that supply energy, maintain ion balance, and can initiate cell death under stressful conditions; they are actively moved throughout the neuron through a process called axonal transport. We will disrupt axonal transport in neuronal cells and in mice and study the effects on mitochondrial distribution and how changes in mitochondrial location affect neuronal function. ? ?
