Kim Schuske et al. (2005) International Worm Meeting "Characterizing the role of PIP2 in neuron morphology"

Michael Bastiani, Nullin Divecha, Deepa Joshi, David Weinkove, Tamara Chessa, Kim Schuske

[International Worm Meeting, 2005]

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a membrane lipid that has been shown to be important for multiple cellular processes including as a source of second messengers in cell signaling, as a stimulator of actin polymerization, and as a regulator of vesicular trafficking. We are interested in how these various functions are regulated in neurons. PIP2 is predominantly made by PPK-1, the C. elegans type I PIP kinase. We find that animals overexpressing PPK-1 have defects in axon morphology consistent with a role for PIP2 in actin polymerization and similar to those described in animals in which Rho/ Rac GTPase signaling is disrupted. Using a GFP construct expressed in the DD and VD motor neurons we find that the ventral nerve cord is defasciculated in adult animals overexpressing PPK-1. In addition, ectopic axon branches are observed, primarily in the ventral nerve cord although also occasionally seen in commissural axons. Despite the severe phenotype in adults, axon outgrowth appears mostly normal in larvae suggesting the main defect is a failure to maintain structural integrity of the nervous system. In addition to the neuron morphology defects, these animals are also severely uncoordinated and paralyzed, similar to animals unable to release synaptic vesicles. To determine if animals overexpressing PPK-1 release synaptic vesicle we assayed for resistance to aldicarb, a drug that prevents the breakdown of acetylcholine in the synaptic cleft. Surprisingly, PPK-1 overexpression causes worms to be hypersensitive to aldicarb suggesting that these animals release high levels of acetylcholine. Taken together, the hypersensitivity to aldicarb and the excess axon branching observed in animals overexpressing PPK-1 suggest that these animals may have too many synapses. We are currently testing this hypothesis. Within the cell, localized production and degradation of PIP2 could act as a switch that activates signaling pathways and localizes proteins to their site of action. Misregulation of this switch due to excessive production or an inability to degrade PIP2 may lead to a catastrophic failure in multiple cellular processes. Our eventual goal is to gain a better understanding of how PIP2 regulation affects cellular signaling and membrane trafficking by characterizing mutants that disrupt production and degradation of PIP2.

Kim Schuske et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Overexpression of the Type I PIP Kinase in Neurons Causes Progressive Neuron Overgrowth and Degeneration in C. elegans"

Nullin Divecha, David Weinkove, Deepa Joshi, Maelle Jospin, Tamara Chessa, Michael Bastiani, Kim Schuske

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

During axon outgrowth, dramatic remodeling of the membrane and actin cytoskeleton of a growth cone enables the neuron to migrate and find its correct target tissue. Phosphatidylinositol 4,5 bisphosphate (PIP2) is an important membrane lipid that is required for multiple cell functions including membrane trafficking and actin polymerization. Therefore, we proposed that misregulation of PIP2 synthesis by overexpressing the type 1 PIP kinase (PPK-1) in Caenorhabditis elegans neurons would disrupt axon outgrowth. We have shown that PIP2 levels in L1 larvae are indeed higher in worms overexpressing PPK-1 than in wild type animals. Surprisingly, we have not detected defects in larval neuronal development. However, neurons in adults form extensive projections extending from cell bodies, nerve cord, and commissural axons. As adult animals age, some neurons become progressively overgrown, while others appear to degenerate. These data suggest that neurons in adult animals are susceptible to PIP2 induced actin polymerization and membrane growth. Since many of the signaling pathways activated by PIP2 are not known, we are screening for second site genetic suppressors of the uncoordinated phenotype associated with animals overexpressing PPK-1. One of the suppressors was identified as a mutation in the unc-80 gene. We have cloned unc-80 and confirmed what others had previously shown ("unc-80 is a stupid gene" Hudson, et al, WBG114; 20 1990). It encodes a novel neuronal protein that has a single ortholog in all animals. Interestingly, unc-80 was also identified as a suppressor of synaptojanin mutants. Synaptojanin is a lipid phosphatase that is required to degrade PIP2 during synaptic vesicle recycling. Taken together, our current data suggests UNC-80 functions in PIP2 signaling in neurons.