Catherine M. Drerup, Ph.D. - US grants

DESCRIPTION (provided by applicant): In neurons, axonal transport of proteins and organelles to and from synapses is essential for formation and maintenance of neural connectivity. Impaired axon transport is thought to contribute to numerous neurodevelopmental and neurodegenerative disorders, including Alzheimer's Disease, Amyotrophic Lateral Sclerosis, and Charcot-Marie Tooth Disease. Despite the pervasiveness of these disorders, their underlying causes are still poorly understood, which has hindered the development of effective therapies This is at least partially due to the lack of a vertebrate model system in which to study this process in vivo and test potential therapeutics. I have developed zebrafish as an in vivo model for studying axon transport by 1) developing a novel imaging approach to visualize movement of fluorescently labeled cargo in an intact animal;and 2) participating in a forward genetic screen to isolate mutants with axonal transport defects. One of these mutant strains (rogue) has a phenotype typical of disruptions in axon transport, i.e. nerve truncation, nerve thinning and distal axonal swellings in long sensory axons. Live imaging revealed that rogue has reduced density and altered transport parameters of some actively transported cargos. Positional cloning identified the underlying genetic lesion in the gene encoding jnk interacting protein 3 (jip3). Previous studies in vitro revealed that Jip3 binds both the microtubule motor Kinesin-1 and axonal cargo. Jip3 has also been shown to modulate cJun N- terminal kinase (Jnk) activity in cell culture, which could potentially have downstream effects on the microtubule cytoskeleton and cargo-motor binding. However, which axonal cargos, if any, are directly Jip3- dependent and which of these Jip3-dependent molecular interactions regulate axonal transport during axon extension and synapse formation is not known. To address these questions, I will first use the live embryo imaging approach I developed to determine if microtubule dynamics and axon transport of specific cargos are disrupted in rogue. Second, I will determine if Jip3 interaction with Jnk and/or Kinesin-1 are necessary for proper regulation of the microtubule cytoskeleton or axonal transport of specific cargos, thus promoting axon extension and synapse formation. The proposed experiments will define the Jip3-dependent cellular and molecular processes which mediate axon transport in vivo. Long-term, the system I have developed can be used to analyze axon transport in vivo to fully understand how abnormalities in this process disrupt nervous system formation and function in normal and disease states. PUBLIC HEALTH RELEVANCE: The transport of proteins and organelles from the neuronal cell body to axon terminals and vice versa is critical both to maintain the health of the cell body and support the formation of functional nervous system connections. Defects in this process are associated with numerous developmental and neurodegenerative diseases such as Spinal Muscular Atrophy, Alzheimer's Disease, and Amyotrophic Lateral Sclerosis. The knowledge gained from these studies will advance our understanding of the basic mechanisms required for axonal transport in vivo. Additionally, they will establish zebrafish as a model system that can be used to investigate the function of genes associated with axonal diseases to determine if disease etiology lies in interruptions of this basic cellular process.

DESCRIPTION (provided by applicant): Axonal transport of proteins and organelles between the neuronal cell body and axon terminals is essential for axon outgrowth, formation of functional synapses, and neuronal survival. While anterograde transport (cell body to axon terminal) relies on the large family of kinesin motor proteins, retrograde cargo transport (from axon terminals towards the cell body) primarily utilizes one motor complex, cytoplasmic dynein. How cargo binds to this single motor selectively and is transported to the proper location is largely unknown. It has been postulated that this process involves adaptor proteins, which bind cargo to either the core dynein motor complex or its accessory complex, dynactin. My long-term goals are to identify mediators of specific retrograde cargo transport, define their function and determine how disruption of this process impacts circuit formation and activity. Because of their unique genetic tools and imaging accessibility, zebrafish are the ideal system to study retrograde axonal transport and the functional consequences of its disruption in an intact vertebrate. Importantly, most cellular processes that regulate axonal transport are highly conserved between mammals and zebrafish. To begin addressing my goals, I used a forward genetic screen to identify four mutant strains that display phenotypes indicative of interrupted retrograde cargo transport, including axon terminal swellings. One of these strains carries a mutation in JNK-interacting protein 3 (Jip3). Preliminary analyses revealed that jip3 mutants exhibit truncation of long axons and accumulation of activated Ret (GDNF responsive receptor tyrosine kinase) in mutant axon growth cones. In Aim 1, I will address the hypothesis that Jip3 serves as an adaptor protein required for retrograde transport of Ret signaling endosomes, which is necessary for axon extension. The second mutant identified in my screen displays accumulation of mitochondria in axon terminal swellings due to interrupted retrograde transport of this organelle. Anterograde mitochondrial transport and retrograde transport of other cargos are normal. The phenotype in this mutant is due to loss of Actr10, a known member of the dynein accessory complex, dynactin. In Aim 2, I will determine whether Actr10 functions as an adaptor mediating retrograde transport of mitochondria using in vivo imaging and biochemical dissection of interaction domains in the Actr10 protein. In Aim 3, I will use my established protocols and new techniques to determine if retrograde transport of specific cargos is disrupted in my additional novel mutants and how these defects affect function of the circuit. My preliminary data show that these strains have mutations in known dynein interactors, all with unknown functions in axonal transport. Finally, in Aim 4, I will engineer transgenic zebrafish strains which will be used to identify the Actr10 interactome and further dissect the molecular mechanisms that govern retrograde axonal transport of specific cargos. With the data, skill sets, and tools acquired from the proposed experiments, I will be poised to decipher the modulation of retrograde axonal transport of various cargos as an independent investigator.

1) Regulation of retrograde mitochondrial transport in axons One of the most crucial organelles in axons are mitochondria. Mitochondria perform many functions important for local microenvironments including: 1) generate the energy necessary for cellular metabolism; 2) buffer calcium ion levels; and 3) supply ATP for the proper functioning of ion transporters that regulate neural excitability. In addition, the location of mitochondria has been shown to regulate axon branching. Not only do mitochondria need to be properly localized in axons to maintain axon health and function, mitochondria also need to move in order for them to maintain their own health: Mitochondria undergo fission-fusion dynamics which allow the exchange of proteins, lipids, and mitochondrial DNA. If these dynamics are disrupted, mitochondria rapidly undergo degradation. Consequently, mitochondrial transport is of the utmost importance for axon function and health. Our lab is working to identify the factors that regulate mitochondrial transport by the retrograde motor protein complex. Towards the end of my post-doctoral training, I discovered a mutant which lacks almost all retrograde mitochondrial movement. The causative mutation in this line results in depletion of Actr10 (actin related protein 10) a known component of the dynein-associated complex, dynactin. In vivo analyses of mitochondrial movement in actr10 mutants revealed a lack of retrograde mitochondrial movement but normal anterograde (non-dynein related) transport. Transport of other cargos assayed, including lysosomes and the dynein motor itself, was not altered in actr10 mutants. To determine if Actr10 was in fact necessary to link mitochondria to the retrograde motor, we performed mitochondrial fractionation experiments from actr10 mutants and wildtype siblings. These experiments confirmed that Actr10 is necessary for the dynein motor to interact with mitochondria. This linkage is likely not direct, however, as Actr10 does not have known membrane-associated domains. To identify the proteins which make up this link between Actr10 and mitochondria, we performed an immunoprecipitation experiment followed by mass spectrometry analysis. These experiments yielded a number of interesting candidates which we are currently testing for their role in retrograde mitochondrial transport in axons. Together, our work will define the mechanism of dynein-mitochondrial attachment for retrograde movement of this organelle in axons. 2) Identifying novel regulators of retrograde cargo transport in axons Forward genetics is an ideal and unbiased way to identify proteins with critical functions in cellular processes. We have initiated a forward genetic screen in zebrafish to identify proteins important for the retrograde transport of specific cargos in axons. For this screen, we are using a transgenic line that marks both the sensory and motor neuron axons in zebrafish with cytoplasmic GFP (Green Fluorescent Protein). Because cargos that fail to undergo retrograde transport accumulate over time in axon terminals, we can screen our mutagenized families for axon terminal size using the GFP fluorescent indicator to identify strains with disruptions in retrograde axonal transport. In addition to being an efficient screening procedure, the ability to screen live at various developmental stages also gives us the flexibility necessary to study multiple types of axons that develop at different time-points in the same animals. Additionally, our transgenic line contains a second transgene to label mitochondria with the red fluorescent protein TagRFP. Consequently, our screen will also allow us to identify mutant strains with defect in mitochondrial positioning as well as more general markers of retrograde transport disruption. Together, this screen will identify novel regulators of retrograde cargo transport and regulators of mitochondrial localization and motility in axons. This will advance our goal of defining the mechanisms of cargo-specific retrograde transport in axons.