Peter J. Hollenbeck - US grants

Although the rapid transport of neuronal organelles has been intensively studied using in vitro cell models, virtually nothing is known about how this phenomenon is controlled and coordinated in intact cells. One major aim of this study is to determine whether rapid organelle transport is modulated to deliver specific classes of organelles to regions of the neuron where they are needed. Using primary cultured chick neurons maintained in a culture system in which their elongation can be controlled, quantitative measurements of organelle transport will made to determine movement is coordinated with growth. The second major aim is to address how the direction of organelle movement is controlled, using conditions that influence the direction of transport in non-neuronal cells to probe intact and permeabilized neurons in culture. The third major aim of this study is to determine what role the mechanochemical ATPase kinesin plays in bidirectional rapid organelle transport. Specific antibodies directed against this enzyme will be introduced into cultured neurons in an effort to disrupt movement of a class of bidirectionally-transported organelles. Because it provides a metabolic link between the periphery of neurons and the site of synthesis in the cell body, rapid axonal transport is essential for the development and maintenance of all nerves; thus, the insight into its mechanism gained by the proposed research can be expected to contribute to our understanding of the development, defects, and diseases of the nervous system. In addition, since neuronal organelle transport must share many features in common with organelle traffic in other cell types, information acquired in this system should provide insight into many other systems of intracellular motility.

ABSTRACT The goalofthis research is to understand how mitochondrialtransport,distribution,and metabolism areregulatedinneurons. Mostneurodegenerativediseasesinvolvemitochondrialdysfunction,and many result directly from specific failures of mitochondrial traffic, distribution, or metabolism. This is probably because the size and asymmetry of neurons result in a non-uniform distribution of demand for mitochondrial functions such as ATP synthesis. As a result, neurons must extensively redistribute theirmitochondria in responseto localphysiologicalconditions,both in vivo and in vitro. Mitochondria are transported and redistributed within the axon by several motor proteins that translocate along microtubule and actin tracks, as well as by docking interactions. But how movement, docking, and mitochondrial metabolism are regulated and coordinated to deliver the right amount of function to the right location at the right time remains unclear. Our efforts to understand these events are focused on both the specific proteins involved in transport and docking, and on larger scale processes in the healthy and diseased nervous system. In the first two aims, we will test the hypotheses that mitochondrial distribution is regulated by myosin-based disruptions of protracted movements, along with anchorage of motor proteins to the organelle by specific linker proteins. We will use double-stranded RNA inhibition to knock down expression of myosins V, VI and II, and three putative motor-organelle linker proteins in isolated Drosophila neurons and quantify the resulting transport phenotypes. We will also use observation of mitochondrial traffic in segmental nerve axons of intact larvae to assess the transport phenotype of myosin and linker protein mutations. In the third aim, we will use Drosophila models of human mitochondrial diseases to test the hypothesis that the proximal cause of neuropathology in mitochondrial neurodegenerative disease is oxidative damage rather than defects in mitochondrial transport or metabolism. Using quantitative fluorescence microscopy methods, we will determine the relationships among mitochondrial traffic, metabolism and reactive oxygen species production throughout the nervous system and across development in models for Friedreich ataxia, Barth syndrome and other disorders.

? DESCRIPTION (provided by applicant): Intracellular bacterial pathogens such as Legionella pneumophila induce a complex set of changes in the eukaryotic cells they infect. Some of these underlie the cell's defensive innate immune response, while other changes are subversions of the cell's own processes to benefit the bacterial replication cycle. The latter are induced by the secretion into the cell by L. pneumophila of diverse effector proteins. Understanding the range of cellular responses produced by these proteins, and how, remain major challenges in bacterial pathogenesis. Anecdotal reports indicate that mitochondria are affected by L. pneumophila infection but this has not been addressed clearly or quantitatively, and which aspects of mitochondrial behavior and function are modulated by L. pneumophila infection and effector protein secretion remain unknown. We seek to elucidate the full nature of the response of macrophage mitochondria to L. pneumophila infection, quantitatively and with good spatial and temporal resolution, and to identify the effectors that produce it. This study represents a new interdisciplinary collaboration between an investigator who studies the cell biology of mitochondria and another who is a specialist in L. pneumophila effector proteins. Together, our goal is to accrue detailed data that will allow us to frame and test informed and mechanistic hypotheses about the role or subversion of specific mitochondrial functions during infection of macrophages with L. pneumophila. Our specific aims are: (1) to identify and characterize the responses of macrophage mitochondria to infection; (2) to determine which among the ~300 effector proteins are targeted to mitochondria, and which produce the mitochondrial phenotypes we observe.