Irwin Levitan, Ph.D. - US grants

It is proposed to continue ongoing studies of neurite regeneration and specificity of synapse formation between nerve cells. A detailed understanding of these and related phenomena will be essential for extending knowledge about normal and abnormal development, and for devising strategies to help patients recover function following injury to the nervous system. The system of large identifiable Aplysia neurons in primary cell culture will be used to examine synaptic specificity and synaptic plasticity. These neurons regenerate neurites and form specific electrical and chemical synapses in culture, and it has been found that the patterns of growth and specificity can be changed by adding the lectin Concanavalin A to the culture medium. Thus, although Concanavalin A itself is unlikely to be involved in neuronal development, it can be used as a tool to investigate fundamental problems in synaptogenesis. It is proposed now to build on these findings and ask questions about the molecular mechanisms of action of Concanavalin A, using a combination of electrophysiological, biochemical and molecular biological approaches. The long term goal of these multidisciplinary studies is to understand the molecular basis of synaptic specificity and synaptic plasticity, phenomena which are essential for the proper functioning of the nervous system.

DESCRIPTION (Adapted from the investigator's application): Ion channels compromise a specialized class of membrane proteins that underlie electrical signalling in nerve, muscle and other cells. Modulation of ion channels properties, particularly by protein phosphorylation, is of fundamental importance for the functioning of many cell types. Although serine/threonine phosphorylation of ion channels has been studied thoroughly, modulation of ion channels by tyrosine phosphorylation is largely unexplored. The investigator proposes to examine modulatory interactions between protein tyrosine kinases and voltage-gated potassium (Kv) channels. The investigators will focus on two representative members of the Kv channel family: Dv1.3, which is prominent in T lymphocytes and some brain neurons, and Kv1.5, which is expressed highly in heart and brain. This proposal will test two hypotheses, each of which is based on extensive preliminary data. The first is that some Kv channels can associate tightly with tyrosine kinases in a regulatory complex, and the second is that there is reciprocal regulation of Kv channels and tyrosine kinases - that is, channels and kinases can regulate each other's activities. The investigator's specific aims are: 1) To investigate the molecular mechanisms and functional consequences of direct association of Kv channels and tyrosine kinases; 2) to investigate the molecular mechanisms by which tyrosine phosphorylation modulates Kv channels current; and 3) to investigate the reciprocal regulation of Kv channels and tyrosine kinsases. The properties of cloned Kv1.3 and Kv1.5 will be examined following their heterologous expression in a human embryonic kidney (HEK 293 cell line, and native Kv channel current will be investigated in cultured olfactory bulb neurons. For each of these specific aims, the investigators will use a combination of biochemical, physiological and molecular approaches to dissect the molecular details and functional consequences of modulatory interactions between Kv channels and tyrosine kinases.

DESCRIPTION (provided by applicant): Deposits of 42 amino acid amyloid-beta peptide (Abeta42) and hyper-phosphorylated microtubule-associated protein tau in the brain are the pathological hallmarks of Alzheimer's disease (AD). Accumulating evidence suggests that Abeta42 lies upstream of tau in a pathological cascade. However, two critical questions remain elusive. First, how does Abeta42 induce pathological consequences in AD? Second, how does Abeta42 induce abnormal phosphorylation and toxicity of tau? Addressing these questions will advance our understanding of complex AD pathogenesis, and lead to the discovery of novel therapeutic interventions. In this study, Drosophila is used as an efficient genetic model system to unravel molecular mechanism underlying Abeta42-induced toxicity in vivo. Using gene expression profiling and a genome-wide screen, the genes/pathways and chromosomal loci that modify Abeta42-induced neuronal dysfunction were identified. Moreover, co-expression of Abeta42 enhanced phosphorylation and toxicity of tau in fly eyes and brains. Based on these data, we designed our specific aims as follows; Specific Aim 1: To examine the role of and mechanisms underlying reduced cAMP/PKA/CREB activity in Abeta42 toxicity. The cAMP/PKA/CREB pathway plays critical roles in the execution and maintenance of complex brain functions such as memory formation and energy metabolism, and the dysregulation of this pathway has been implicated in the pathogenesis of AD. However, it is not fully understood 1) how Abeta42 affects PKA and CREB activity and 2) whether activation of this pathway protects against Abeta42-induced toxicity in vivo. The relationship of Abeta42 toxicity to dysregulation in the cAMP/PKA/CREB pathway will be analyzed in Abeta42 flies. Specific Aim 2: To elucidate molecular mechanisms underlying toxic interactions between microtubule associated protein tau and Abeta42. Our preliminary data showed that co-expression of A242 and tau enhanced tau-induced toxicity in the eyes and brains of flies, which correlates with increased tau phosphorylation at the AD-related sites S202, T231, and S262. Interestingly, tau phosphorylation at T231 and S262 is known to be upregulated in pretangle neurons in AD brains. Moreover, phosphorylation at S262 has been shown to promote both tau phosphorylation at other sites and tau toxicity. The mechanisms underlying the enhancement of tau phosphorylation and toxicity by Abeta42 will be studied using our fly model as a genetic model system, which may recapitulate an initial step in the abnormal metabolism of tau in AD brains. Specific Aim 3: A search for genes that modify the neuronal dysfunction induced by Abeta42. By a genetic screen, ten chromosomal loci whose haploinsufficiency enhances A2beta2-induced behavioral deficits were identified. The modifier genes in these loci will be identified and characterized. This study will provide mechanistic insights into how Abeta42 induces neurotoxicity and tau pathology in vivo, which will facilitate our understanding of AD pathogenesis and may open novel therapeutic avenues for AD.