The low abundance signaling lipid, phosphatidylinositol (3,5)-bis phosphate (PI3,5P2) is present in all eukaryotes, and is postulated to be involved in multiple trafficking pathways from late endosomes. The machinery that synthesizes this lipid includes the PI3P 5'-kinase Fab1/PIKfyve and a regulatory complex composed of Vac14 and Fig4. In mammals, these proteins are expressed in all tissues. We recently discovered that mice that lack either Vac14 or Fig4 have reduced PI3,5P2 levels in their cells and die prematurely. Unexpectedly, the main defect is massive neurodegeneration. Affected neurons in both the brain and the peripheral nervous system develop large vacuoles that arise from endosomes. Consistent with the importance of PI3,5P2 in the nervous system, we identified patients with Charcot-Marie Tooth syndrome (CMT4J) whose disease corresponds to a single point mutation in Fig4. Little is known about PI3,5P2 and virtually nothing is known about its role in the nervous system. The overall goals of this proposal are to determine the mechanisms that regulate PI3,5P2 levels in neurons and to conduct studies to determine why loss of PI3,5P2 causes neurological defects. Our three specific aims are: 1) Determine whether any or all WIPI family proteins regulate Fab1/PIKfyve activity. Based on homology with yeast Atg18, we predict that one or more WIPI family members negatively regulate Fab1/PIKfyve. We will directly test this hypothesis and will also screen for additional negative and positive regulators of Fab1/PIKfyve. 2) Determine whether PI3,5P2 in neurons solely regulates general membrane trafficking, or whether it also regulates neuronal- specific membrane trafficking pathways. Why are neurons particularly affected by loss of PI3,5P2? To address this, the following questions will be tested. Are neurons, by virtue of their long processes especially vulnerable to defects in membrane trafficking pathways regulated by PI3,5P2? Are there neuronal-specific organelles either in presynaptic and/or postsynaptic termini that require PI3,5P2? We will measure the effects of loss of PI3,5P2 on pathways specific to neurons, as well as general pathways. We will also develop methods to elevate PI3,5P2 levels in cultured cells. Based on a dominant active yeast Fab1 mutant that produces 17-fold higher levels of PI3,5P2, we will test candidate mammalian Fab1/PIKfyve mutants that we predict will be dominant active. 3) Determine whether PI3,5P2 can be generated in the absence of Fab1/PIKfyve. In yeast, all PI3,5P2 is generated through Fab1. However, phosphoinositide metabolism in mammals is more complex. We will test whether PI3,5P2 in mice can be generated in the absence of Fab1/PIKfyve. Achievement of these aims will provide insights into the pathophysiology of neurodegenerative disorders and may ultimately lead to novel approaches for treatments for a variety of neurodegenerative diseases.

Public Health Relevance

Common neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, are complex conditions that arise from defects in a variety of pathways. Our laboratory has discovered a new pathway that when disrupted unexpectedly causes neurodegeneration. The overall goal of this application is to determine how defects in this pathway lead to neurodegeneration.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS064015-02
Application #
7751295
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Gubitz, Amelie
Project Start
2009-01-01
Project End
2013-11-30
Budget Start
2009-12-01
Budget End
2010-11-30
Support Year
2
Fiscal Year
2010
Total Cost
$334,590
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Al-Ramahi, Ismael; Panapakkam Giridharan, Sai Srinivas; Chen, Yu-Chi et al. (2017) Inhibition of PIP4K? ameliorates the pathological effects of mutant huntingtin protein. Elife 6:
Lang, Michael J; Strunk, Bethany S; Azad, Nadia et al. (2017) An intramolecular interaction within the lipid kinase Fab1 regulates cellular phosphatidylinositol 3,5-bisphosphate lipid levels. Mol Biol Cell 28:858-864
Jin, Natsuko; Jin, Yui; Weisman, Lois S (2017) Early protection to stress mediated by CDK-dependent PI3,5P2 signaling from the vacuole/lysosome. J Cell Biol 216:2075-2090
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Jin, Natsuko; Lang, Michael J; Weisman, Lois S (2016) Phosphatidylinositol 3,5-bisphosphate: regulation of cellular events in space and time. Biochem Soc Trans 44:177-84
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Jin, Yui; Strunk, Bethany S; Weisman, Lois S (2015) Close encounters of the lysosome-peroxisome kind. Cell 161:197-8
McCartney, Amber J; Zolov, Sergey N; Kauffman, Emily J et al. (2014) Activity-dependent PI(3,5)P2 synthesis controls AMPA receptor trafficking during synaptic depression. Proc Natl Acad Sci U S A 111:E4896-905
McCartney, Amber J; Zhang, Yanling; Weisman, Lois S (2014) Phosphatidylinositol 3,5-bisphosphate: low abundance, high significance. Bioessays 36:52-64
Samie, Mohammad; Wang, Xiang; Zhang, Xiaoli et al. (2013) A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis. Dev Cell 26:511-24

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