Edward Giniger - US grants

How is the proper pattern of neural connections established during development? And how is that pattern maintained in the adult nervous system? These are the questions that the Axon Guidance and Neural Connectivity Unit seeks to answer.[unreadable] [unreadable] To understand the mechanisms underlying the establishment of neural connections, we focus on what might be termed the "elementary event" in the process of neural wiring, the mechanism by which a single cell-surface receptor tells a developing neuron where to grow in order to find its synaptic partners. We study a particular cell surface receptor called Notch. Notch is notable because, in addition to directing nerve growth, it also controls the branching of dendrites, the identities of neurons (and many other types of cells), how many neurons are born and whether cells live or die. As such, it controls many aspects of animal development and is responsible for a wide array of human diseases, including some kinds of cancer and stroke. What we learn about Notch in axons, therefore, has implications for biology and health far beyond the particular process we are examining. Previous studies of Notch have focused on a single signaling mechanism for this ubiquitous receptor. We have found, however, that this is only half of the story. About 5% of the Notch protein in the embryo is tyrosine phosphorylated, and this population of molecules associates specifically with an alternate group of downstream effectors, the Abl oncogene and its associated accessory factors, to directly control cell-cell contacts, cell shape and cell migration. We have shown, moreover, that this alternate Notch signaling pathway acts at the plasma membrane (as opposed to the standard signaling pathway, which targets events in the cell nucleus), and that it acts via a protein called Rac that is a direct regulator of the actin cytoskeleton and of cell-adhesion complexes. In growing nerves, the activity of the Notch/Abl/Rac machinery is revealed as regulation of the direction and extent of nerve growth. Current experiments are directed at continuing to elucidate the molecular mechanism of this alternate signaling pathway, and to determine where besides growing axons it may act in biology and disease. We are particularly interested by evidence that the alternate Notch signaling pathway we have discovered may be central to controlling the survival of neural and embryonic stem cells, and that it is key to the mechanism by which activation of Notch can cause cancers, including medulloblastoma, leukemia, rhabdomyosarcoma and breast cancer. [unreadable] [unreadable] 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. We find the mutants are viable and fertile, and are behaviorally normal at birth. However, within a few weeks they show progressive loss of motor coordination, culminating in rigidity and then death, with a significantly shortened lifespan (30% shorter than matched controls). Sectioning the heads of aging p35 mutant flies reveals degenerative lesions in the brain, initially in the neuropil but also around the cell bodies. Remarkably, these lesions are highly localized, being present bilaterally in specific brain nuclei, but not generally distributed through the brain, even though p35 and Cdk5 are present and active throughout the brain. Therefore, these mutants may allow us not only to uncover the genetic pathway leading to Cdk5-associated neurodegeneration, but also to understand how and why disease processes that occur throughout the brain lead to very specific and characteristic structural and behavioral defects in particular brain regions. Moreover, in mutant animals we observe widespread defects in axon patterning, synaptic morphology and protein localization within axons long before we see overt degeneration, raising the possibility that late onset degeneration may actually reflect a delayed response to defective nervous system structure early in development.

We previously developed a potential Drosophila model of mammalian neurodegenerative disease by inactivating the protein kinase Cdk5. This is the fly homolog of one of the two main proteins responsible for phosphorylating tau into the form found in the neurofibrillary tangles that are characteristic of many forms of human neurodegeneration. We showed that there are many phenotypic similarities between the neurodegenerative syndrome in this endogenous process of flies and that observed in humans and mice, thus validating our Drosophila mutant as a model of the mammalian disease process. More recently, we have found that Cdk5 also controls various aspects of developmental disassembly of axons and dendrites, in particular the stability of the microtubule cytoskeleton. Thus, we find that loss of Cdk5 activity delays the onset of developmental remodeling of axons and dendrites of the fly central brain during metamorphosis, while hyperactivation of Cdk5 accelerates remodeling. Genetic interaction and pharmacological experiments reveal that this is mediated partly, but not completely, through Cdk-dependent destabilization of microtubules in these neurons. Given the many anatomic and molecular similarities between developmental remodeling of neurons and their pathological degeneration in disease, it is likely that this function of Cdk5 is directly relevant to the mechanism of neurodegeneration in p35 mutants. We also initiated systematic, genome-wide investigations into the consequences of Cdk5 inactivation. We have used microarrays to perform gene expression profiling of adult flies having, or lacking, Cdk5 activity. We have focused our efforts on early events in degeneration by isolating RNA from young flies, before the onset of overt cellular or organismal defects in the mutants. Preliminary analysis reveals two classes of gene expression changes in mutants lacking Cdk5 activity. We observe alterations in genes directly important for neural function, including sensory transduction, synaptic transmission and ion homeostasis. We also observe changes in the expression of genes associated with a variety of organismal physiological processes we had previously hypothesized to be associated with Cdk5 function, based on our phenotypic analysis. These include proteostasis, redox state, mitochondrial structure and function and stress-sensitivity. The microarray analysis, therefore, serves as a powerful independent validation of the phenotypic analysis. Moreover, as this unbiased, genome-wide array analysis failed to identify major classes of genes not predicted by the earlier studies, these data suggest that we have identified the major functional pathways downstream of Cdk5 that are responsible for its pathological consequences. Remarkably, we find that many of the processes downstream of Cdk5 are also physiological hallmarks of natural aging, and we are currently investigating this connection.