Mitochondria are often involved in the pathogenesis of neurodegenerative diseases. This is not surprising considering the fundamental functions of mitochondria in intracellular Ca2+ homeostasis and ATP synthesis, which are critical for synaptic function. Appropriate distribution and supply of mitochondria to critical neuronal sites are thought to be necessary for the normal maintenance of neuronal architecture and activity, including synaptic plasticity and function. However, the role that abnormalities in mitochondrial transport and localization within neurons play in neurodegeneration remains to be investigated in a relevant in vivo model. Thus, the goal of this proposal is to generate a novel mouse model and make them available to the scientific community to study in vivo the effects of abnormal mitochondrial transport and distribution in neurons. We will genetically alter the normal function of Miro1, a mitochondrial-bound protein, which regulates mitochondrial transport along axons in response to Ca2+ signaling.
In Aim 1, we propose to generate transgenic wild-type Miro1 (Miro1WT) and mutant Miro1 mouse (Miro1V13). The V13 mutation is expected to impair the docking of mitochondria to the anterograde transport molecular machinery, which will result in the reduction of mitochondria trafficking towards the periphery of the neuron. Preliminary results obtained in cultured neurons confirm this expectation. We will use the well-established Rosa26 knock-in approach and Cre-lox technology to activate transgene expression in a conditional and tissue-specific manner. The Miro1 transgene is preceded by a floxed stop-cassette and followed by an IRES element that allows coordinated expression of a mitochondrial fluorescent protein (mitoDendra).
In Aim 2, we will characterize the transgenic mice by crossing them with Cre-Thy1 mouse, which expresses Cre recombinase specifically in neurons. In this pilot project, as a proof of principle, we will study mitochondrial distribution along motor neurons. We will also assess neurodegeneration of motor axons and neuromuscular junctions. Successful completion of this project will provide a new valuable tool for the scientific community to study the effects f altered mitochondria transport and distribution in vivo.
Proper distribution and supply of mitochondria are necessary for the normal maintenance of neuronal architecture and activity, including synaptic plasticity and function. We propose to alter mitochondria trafficking by generating a transgenic mouse that express a mutant protein that disrupts the normal mitochondrial distribution in neurons. Successful completion of this project will provide a valuable tool for the scientific community to study the pathological consequences of altered mitochondrial transport in vivo.
