James I. Morgan - US grants

DESCRIPTION (Taken from the Investigator's Abstract) Parkinson's disease (PD) is the most prevalent movement disorder in the United States. Despite recent advances in treatment there remain pressing needs for novel therapies that halt disease progression, means for early diagnosis and methods to identify environmental and genetic risk factors. PD is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) accompanied by gliosis and evidence of oxidative stress, mitochondrial dysfunction and excessive iron deposition. Although dopamine metabolism, environmental and genetic factors have been implicated in the etiology of idiopathic PD, the precise cause is unknown. In this application, the investigators will exploit the observation that MPTP toxicity is genetically determined in mice to characterize a novel molecular signaling pathway that links this xenobiotic to neuronal death and several of the pathological hallmarks of PD. MPTP is a xenobiotic neurotoxin that produces many of the features of PD in animal models and man. Furthermore, susceptibility to MPTP is species- dependent and is inherited as a dominant trait in mice. Therefore, the murine MPTP model represents a unique opportunity to dissect the interaction between a prototypic exogenous neurotoxin and genetic risk factors that may contribute to the pathogenesis of PD. Using a novel chimeric primary culture paradigm where isolated astrocytes or neurons from resistant or sensitive strains of mice are co-cultured in different combinations, the genetic susceptibility to MPTP is conferred by astrocytes. This led the investigators to uncover elements of a previously undescribed response pathway to MPTP in vivo that includes alterations in gene expression in astrocytes. As this response involves dopamine metabolism, oxidative stress and the local production of endogenous neurotoxins, such as iron, they hypothesize that it is capable both of linking MPTP to the selective loss of SNpc dopaminergic neurons and accounting for some of the ancillary pathological findings in PD. Furthermore, as dopamine but not other catecholamines triggers this pathway it can account for the neuronal specificity of MPTP killing.

DESCRIPTION (provided by applicant): Programmed cell death (PCD) is a strictly regulated process and its disruption results in myriad developmental deficits and pathological sequelae. PCD is especially critical in the mammalian nervous system where its perturbation results in aberrant neural development and contributes to many neural disorders. There has been much research into the role of caspases in cell death. However, there is growing evidence for the importance of caspase-independent cell killing in the mammalian nervous system and other tissues. It is difficult to identify the components of the latter pathway or establish its contribution to cell elimination in vivo as it is intimately interwoven with, and masked by, the ubiquitous caspase cascades. We have developed a paradigm that can circumventthis limitation. CED-4S is a pro-apoptotic protein from C. elegans that is lethal when expressed in Saccharomyces cerevisiae. CED-4S lethality in yeast shows physiological specificity as it is blocked by its natural antagonist, CED-9 and is not mimicked by its anti-apoptotic splice variant, CED-4L. However, CED-4S toxicity in yeast does not require a caspase. Given the high degree of structural conservation amongst components of the cell suicide machinery, we propose to use CED-4S lethality in yeast as a paradigm to isolate molecules involved in caspase-independent killing. Subsequently, we will identify the mammalian counterparts of these molecules and investigate their function in the vertebrate nervous system. Using a CED-4S suppresser screen, we isolated 2 yeast AAA-ATPases, Cdc48 and yAPO-1 that have homologs in higher eukaryotes that have been implicated in neuronal death. Cdc48 binds to CED-4 whereas yAPO-l does not. This suggests a scenario in which CED-4S complexes with Cdc48 and alters its function, thereby leading to death. yAPO-l may have a redundant function with Cdc48 or it may lie downstream in the caspase-independent death pathway. Based upon these findings, we will use yeast and mammalian models to characterize the caspase-independent death pathway and determine the role that these and other CED-4 suppressers play in neuronal death in mice. In Specific Aim 1, we will determine the composition and functional domains of CED-4-containing complexes in yeast. In Specific Aim 2, downstream targets of CED-4 will be identified in yeast using a CED-4S suppresser screen. In Specific Aim 3, we will determine the expression and function of the mammalian homologs of the CED-4 suppressers in developing and adult brain and assess their contribution to normal and pathological cell death in the nervous system.

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate how inter- and intra-neuronal signaling contributes to neuronal development and function. Interneuronal communication occurs at synapses; disruption of synapse formation and function in developing and adult humans causes myriad neurological and psychiatric disorders, including schizophrenia, bipolar disease, and learning disabilities. The goal of the studies proposed here is to investigate the biological role of the PDZ domain protein NIL-16 in the nervous system. NIL-16 is uniquely bifunctional, serving both as a specific ion channel binding molecule and as the neuron-specific precursor of the cytokine, interleukin (IL)- 16. Until NIL- 16 was cloned, IL16 had been characterized only in the immune system. The identification of NIL-16 revealed an unsuspected parallel between the immune and nervous systems. IL-16 induces a signaling cascade in neurons that leads to upregulation of the transcription factor Fos. Therefore, NIL- 16 represents the first molecular link between cytokine signaling and ion channel function. Remarkably, preliminary studies revealed that NIL-16 can bind CD4, a functional IL- 16 receptor in the immune system. These findings suggest that NIL- 16 may function as both the precursor of IL- 16 and the molecular anchor for its receptor. Moreover, because NIL- 16 also binds ion channels, IL- 16 signaling may ultimately modulate the function of ion channels. These studies have three specific aims: 1) To test the hypothesis that NIL-16 serves as a scaffolding or trafficking protein that influences ion channel function by using electrophysiological recordings, immunoprecipitation, immunofluorescence confocal microscopy, and endocytosis assays; 2) To test the hypothesis that there is a specific IL-16 signaling pathway in neurons by studies in mutant mice, chemical cross-linking studies, kinase and phosphorylation assays, subcellular localization studies, and electrophysiological recordings; and 3) To test the hypothesis that NIL-16 influences long-term potentiation (LTP) by comparative electrophysiological recordings from brain tissue of wild-type and NIL-16-deficient mice.

DESCRIPTION (provided by applicant): The function and viability of neurons is frequently dependent upon secreted growth factors. Although most extensively investigated in the developing nervous system neurotrophic factors are important in the adult brain and have been examined as therapeutic modalities in adult neurodegenerative conditions. Therefore, the isolation of proteins in adult brain with neurotrophic activity could have broad implications both for our understanding of the maintenance of neuronal integrity and function in the mature nervous system and as potential therapeutic agents for a range of neurological and psychiatric disorders. We identified a family of brain-specific proteins (Cbln1-Cbln4), termed synaptotrophins that have properties of adult neurotrophic factors. Cbln1 and Cbln3 are secreted glycoproteins that are co-expressed in mature cerebellar granule cells and form trimeric complexes that are structurally related to tumor necrosis factor-alpha (TNFalpha). Elimination of Cbln1 through homologous recombination in mice causes ataxia, marked structural and physiological defects in granule celI-Purkinje cell synaptic interactions and the progressive degeneration of adult cerebellar granule neurons. Thus Cbln1 is the prototype of a novel class of factor that regulates synaptic stability and function and neuronal survival. Remarkably, loss of the orphan glutamate delta2 receptor (GluRdelta2) in Purkinje cells mimics the phenotype of the cbln1-null mouse. Thus, presynaptic Cbln1 and postsynaptic GluRdelta2 may be components of a novel trophic signaling pathway. This mechanism likely exists elsewhere in brain having implications for neuropsychiatric (disrupted synaptic transmission) and neurodegenerative disorders (neuronal loss and functional impairment) in man. In this application we take advantage of the structural and functional properties of TNFalpha and GluRdelta2 to elucidate the molecular bases of the neural deficits in cbln1-null mice and characterize the components of the Cbln1 signaling pathway.

DESCRIPTION (provided by applicant): The identification of novel mechanisms that contribute to neuronal survival and the ability of neurons to mount regenerative responses have broad implications for a better understanding and treatment of conditions ranging from neural trauma to a host of neurodegenerative disorders. Here, we focus on Nna1, a protein that represents an unexpected and unique mechanistic link between neuronal death and regeneration. Loss of function mutations in Nna1 lead to neurodegeneration in mice that may be mediated by an unprecedented mechanism that has major implications for neurodegenerative disorders of unknown etiology in man. Nna1 was discovered in the applicant's laboratory as a gene induced in spinal motor neurons following surgical axotomy. In a subsequent collaborative study it was established that mutations in Nna1 caused the neurodegenerative phenotype in the classical autosomal recessive mutant mouse, Purkinje cell degeneration (pcd). Thus, enhanced expression of Nna1 is associated with regenerative responses in the CNS whereas loss-of-function mutations in Nna1 result in neurodegeneration. Recently, the applicant's laboratory demonstrated that Nna1 defined a new subfamily of M14 carboxypeptidases with unique aspects of structure and cellular location. This opens the possibility that genetic lesions that affect these genes may cause degeneration of broader categories of neurons and that endogenous or environmental inhibitors of these enzymes will precipitate neurodegeneration. As the function of Nna1 and the pathway in which it acts are unknown their elucidation will provide new insights into mechanisms of neurodegeneration and regeneration. We have identified a molecular event in pcd mice that is the earliest known deficit in these animals and that may directly link Nna1 to neuronal death. This application proposes three specific aims that will define the biochemical function of Nna1 and its role in neuronal death in cerebellum. These aims will exploit our ability to rescue Purkinje cell loss in pcd3J mutants by re-expressing Nna1 with the L7/pcp2 promoter, our demonstration of the activation of a potentially neurotoxic endogenous retroviral like element in pcd mice and structural information obtained from the identification of a subfamily of novel Nna1-related genes to define the biochemical and cell biological properties of Nna1.