Edward Giniger - US grants

We seek to answer two questions: how do neurons become connected during development, and why do they become disconnected during neurodegenerative disease? The mechanisms responsible for growth of axons along the longitudinal axis of the central nervous system (CNS) have been mysterious in both vertebrates and invertebrates. This is a crucial question for metazoan neural development, and is also the essential process for repair of catastrophic spinal cord injuries. In light of results we have obtained over the past year, we can now largely account for how this process occurs in the Drosophila central nervous system. First, we have disentangled the mechanism by which the development of four different CNS cell types are coordinated to produce a track along which longitudinal axons can grow between successive segments. This involves spreading a thin layer of neuronal tissue along the surfaces of specialized glial cells, and shaping that layer into a continuous adhesive band that bridges between segments. Second, we have determined what drives growing axons to extend along that track. A receptor on the surface of the growing axon, Notch, suppresses the activity of a major cytoplasmic signaling protein, the Abl tyrosine kinase. This promotes the formation of long filopodia by the growing axons, and suppresses the adhesion of those axons to their substratum. Together, these effects establish a balance among the steps in the cycle of dynamics of the cytoskeleton that is conducive to growth. The view that axon guidance molecules achieve their effects by producing a particular balance in the dynamics of cytoskeleton organization, and not by explicit growth-promoting and growth-retarding activities, is a novel hypothesis that brings into focus a large body of data on neural wiring that has until now been contradictory and confusing. We argue that this alternate perspective will greatly advance our understanding of the mechanisms of neural wiring, and also of cell motility. In the course of this work we have been forced to dissect the signaling network defined by the Abl protein tyrosine kinase. Abl was the first cellular proto-oncogene found to be responsible for a major human cancer. Central components of its signaling pathway have been known for many years, but how they combine to form a pathway has resisted analysis. We found that by analyzing Abl function in epithelia we could order the steps in this signaling pathway. We found, first, that the Disabled protein, long thought to be an effector of Abl, is actually an upstream regulator that localizes the kinase. Moreover, preliminary results suggest that rather than being a single, linear pathway, the Abl signaling system has two branches that are largely separate, though coordinated. This likely accounts for many of the ambiguities and complexities in prior studies of this pathway. Unraveling the mechanism of Abl signaling is central to understanding the regulation and machinery of nerve growth, and also for treatment of hematopoietic malignancies such as chronic myelogenous leukemia.

We seek to answer two questions: how do neurons become connected during development, and why do they become disconnected during neurodegenerative disease? There is a great deal of interest in developing models of neurodegenerative diseases in simple, invertebrate model systems that provide unequaled experimental power for characterizing the cellular events of a complex process and establishing its molecular genetic basis. Use of Drosophila for studies of neurodegeneration have been problematic, however, since in general they have either relied on highly artificial manipulations, such as high-level expression of mutated human genes in the fly, or have identified genes that clearly affect neuronal survival in the fly but are not related to any gene or pathway demonstrated to play a role in neurodegeneration in mammals. We have now identified a natural, adult-onset neurodegenerative syndrome of Drosophila in flies mutant for the ortholog of a gene directly implicated in human diseases including Alzheimer Disease and ALS. The protein kinase Cdk5, together with its regulatory subunit, p35, is one of the major kinases that phosphorylates cytoskeletal proteins to generate the neurofibrillary tangles that are characteristic of the "tauopathy" class of neurodegenerative diseases. Moreover, activated Cdk5 is found concentrated in degenerating tissue in the brains of Alzheimer patients, and experimental activation of Cdk5 induces degenerating lesions in the mouse brain. We have generated a null mutation of the gene encoding the fly homolog of the Cdk5 activating subunit, p35, and find that it causes adult-onset neurodegeneration of a specific portion of the Drosophila brain, the "mushroom bodies" that are the seat of learning and memory. We took advantage of the Drosophila system to ask what are the earliest defects in neurons that are fated to degenerate from loss of Cdk5 activity, and found two completely unexpected phenotypes. First, we found that Cdk5 is essential for the development of the portion of an axon where nerve impulses are initiated. Improper organization of this cellular compartment is expected to cause profound defects in the ability of a nerve cell to act in a neural circuit. It is certainly plausible that such defects play a role in the initiation or progression of Cdk5-associated neurodegeneration, but additional experiments will be necessary to test this hypothesis. Second, we found that Cdk5 controls the machinery responsible for the genetically-programmed disassembly of axons and dendrites at particular developmental stages. Here as well, the potential significance for this in pathological Cdk5-associated disassembly of neurons is clear, but additional experiments will be necessary to fully understand the link.