Azad Bonni - US grants

DESCRIPTION (From the Applicant's Abstract): The long-term objectives of the proposed research are to elucidate the mechanisms that regulate cell death and survival in the developing mammalian central nervous system. The neurotrophin comprise a family of secreted proteins that elicit the profound effect of promoting survival of neurons in the developing mammalian nervous system. Although the role of neurotrophins in neuronal survival is firmly established, the mechanisms by which neurotrophins support survival of neurons remains to be elucidated. We propose to investigate the intracellular signaling mechanisms by which the neurotrophin brain-derived neurotrophic factor (BDNF) promotes the survival of granule neurons in the developing rat cerebellum. We have recently found that BDNF enhances the survival of cerebellar granule neurons via the extracellular regulated kinase (ERK) signaling pathway. The ERK activates kinases, the Rsks, cooperate with phosphotidylinositol-3 kinase (PI-3K)-Akt signaling pathway to directly inhibit the apoptotic protein BAD. In addition, preliminary data indicate that the transcription factor myocyte enhancer factor 2 (MEF2) previously implicated in myogenesis mediates BDNF-induced cerebellar granule cell survival. To elucidate the transcription-independent and transcription-dependent mechanisms by which BDNF-induced signal suppress the cell death machinery, we propose the following aims: (1) characterize the mechanisms that underlie the cooperativity of Rsk and Akt in suppressing the apoptotic protein BAD, (2) characterize the intracellular signal transduction pathway by which BDNF induced MEF-2 dependent transcription and neuronal survival, and (3) to determine the role that MEF2 plays in the survival of granule neurons in the intact cerebellar cortex in organotypic culture of the rat cerebellum. Together, the proposed experiments will provide critical insights into the intracellular signaling mechanism by which neurotrophin promote the survival of neurons in the mammalian CNS. Since neruotrophins can remarkably protect neurons against injury in the mature nervous system, our investigation should also provide valuable clues for the development of novel therapies aimed at alleviating neuronal cell death occurring in disorders of the nervous system including the devastating neurodegenerative diseases.

[unreadable] DESCRIPTION (provided by applicant): The long-term goals of the proposed research are to elucidate the mechanisms regulating axonal growth and regeneration in the mammalian brain. We recently discovered that the ubiquitin ligase, the anaphase promoting complex (Cdh1-APC), plays a critical role in the control of axonal growth and patterning in the mammalian cerebellum. Cdh1 knockdown by RNAi in primary rat cerebellar granule neurons robustly promoted axonal growth. In cerebellar slice overlay assays and by in vivo knockdown in the postnatal rat cerebellum, we found that Cdh1 cell-autonomously controls the layer-specific growth of granule neuron axons and parallel fiber patterning. In other experiments, Cdh1 knockdown was found to remarkably override myelin-inhibition of axonal growth. Thus, Cdh1-APC may also contribute to the inability of injured neurons to extend axons in the mammalian central nervous system (CNS). Our findings have raised several fundamental questions. How is Cdh1-APC function regulated in neurons? What are the mechanisms by which Cdh1-APC controls axonal growth? How does Cdh1-APC contribute to the negative influence of myelin inhibitory factors on axonal growth? To address these questions, we propose the following specific aims: (1) characterize mechanisms regulating Cdh1-APC function in neurons. We will perform structure/function analyses of Cdh1 and characterize the role of Cdh1 phosphorylation and Cdh1-interacting proteins in Cdh1-APC's control of axonal growth. (2) Determine the mechanism by which Cdh1-APC controls axonal growth. We will identify the substrates of Cdh1-APC that regulate axonal growth. (3) Characterize cell intrinsic role of Cdh1-APC in limiting axonal growth. We will characterize the activity and role of Cdh1-APC in mammalian CNS neurons beyond the cerebellum, and determine how Cdh1-APC contributes to the myelin-inhibition of axonal growth. The proposed research represents an important set of experiments that should address a major gap in our understanding of the cell-intrinsic mechanisms controlling axonal growth in the mammalian brain. In addition, the proposed research should provide the foundation for the development of drugs that might ultimately be used to stimulate axonal regeneration following injury and disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term goals of the proposed research are to elucidate the signaling and transcriptional mechanisms regulating neuronal differentiation. We recently discovered that the transcription factor myocyte enhancer factor 2A (MEF2A) promotes postsynaptic dendritic morphogenesis in differentiating neurons in the mammalian brain. In the developing mammalian cerebellum, granule neuron differentiation culminates in the generation of dendritic claws upon which mossy fibers and Golgi neuron axons form connections. Genetic knockdown of MEF2A by RNAi in cerebellar slices and in in vivo in the postnatal rat cerebellum revealed an essential function for MEF2A in postsynaptic dendritic claw differentiation. A transcriptional repressive form of MEF2A that is sumoylated at Lys403 promotes the differentiation of dendritic claws. These findings have raised several fundamental questions on how the novel function of sumoylated MEF2A is regulated in neurons and how sumoylated MEF2A orchestrates postsynaptic dendritic differentiation. To address these questions, we propose to identify the enzyme that stimulates the sumoylation of MEF2A and thereby promotes dendritic claw differentiation. Using candidate and non-biased approaches, we will also identify the gene targets of sumoylated MEF2A that mediate its ability to promote postsynaptic differentiation. Finally, we will characterize the developmental role of a calcium/calcineurin-MEF2A signaling pathway that suppresses MEF2A sumoylation and thereby inhibits dendritic claw morphogenesis. The proposed research represents an important set of experiments that should address a major gap in our understanding of the signaling and cell-intrinsic transcriptional mechanisms that underlie neuronal differentiation. In addition, since postsynaptic dendritic pathology is thought to contribute to the pathogenesis of diverse neurologic and psychiatric disorders, including neurodegenerative diseases and mental retardation, the proposed research should provide the foundation for a better understanding of these disorders.

DESCRIPTION (provided by applicant): The long-term goals of the proposed research are to elucidate the mechanisms that regulate apoptosis of neurons in the developing mammalian brain. Growing evidence suggests that components of the cell cycle are reactivated in dying neurons in the brain. However, the role and mechanisms by which the cell cycle machinery control apoptosis of neurons remain largely to be elucidated. We have found that the mitotic kinase Cdc2 induces apoptosis in primary rat cerebellar granule neurons. These findings raise two major questions: how does Cdc2 trigger apoptosis of postmitotic neurons, and what is the role of Cdc2 function in neuronal apoptosis in the intact developing cerebellar cortex? To address these questions we propose the following specific aims: (1) Elucidate the mechanisms by which Cdc2 induces neuronal apoptosis. Our preliminary studies suggest the hypothesis that Cdc2 triggers apoptosis by phosphorylating protein substrates of the major pro-survival kinase Akt with the net effect of opposing Akt-promotion of neuronal survival. We will take a multi-pronged approach to identify novel substrates of Cdc2 that contribute to neuronal apoptosis. (2) Determine Cdc2 function in the intact cerebellar cortex. We will characterize the expression and activity of Cdc2 in granule neurons in the rat cerebellar cortex in both rat cerebellar slices and in vivo, and we will determine the effect of inhibition of Cdc2 on neuronal apoptosis in the cerebellar cortex. Together, the proposed research should provide fundamental and novel insights into the role and mechanisms of cell cycle reactivation in apoptosis of postmitotic neurons. Since cell cycle reactivation is thought to play an important role in neurodegenerative diseases, our studies should also lead to novel insights into the pathogenesis of these devastating diseases of brain.

The long-term goals of the proposed research are to elucidate the mechanisms that regulate dendrite morphogenesis in the mammalian brain. We recently discovered that the ubiquitin ligase Cdc20-anaphase promoting complex (Cdc20-APC) promotes the generation and elaboration of dendrites in postmitotic mammalian neurons. Genetic knockdown of Cdc20 by RNAi in cerebellar slices and in in vivo in the postnatal rat cerebellum revealed an essential function for Cdc20 in dendrite growth and arborization. Remarkably, Cdc20 is concentrated at the centrosome in neurons, and the centrosomal localization is required for neuronal Cdc20- APC to drive dendrite development. These findings have raised several fundamental questions on how the novel function of Cdc20-APC is regulated in neurons and how Cdc20-APC orchestrates dendrite development. To address these questions, we propose to identify the key domains and posttranslational modifications within Cdc20 that contribute to Cdc20-APC function in dendrite morphogenesis. We will also identify the substrates of neuronal Cdc20-APC that control dendrite growth and elaboration. Finally, based on preliminary evidence, we will characterize the developmental role of a Cdc20-APC in dendrite remodeling and patterning in the cerebellar cortex. The proposed research represents an important set of experiments that will address a major gap in our understanding of the cell-intrinsic mechanisms that underlie neuronal morphogenesis and connectivity. In addition, since abnormalities of dendrite morphology are thought to contribute to the pathogenesis of diverse neurological and psychiatric disorders, including neurodegenerative diseases and mental retardation, the proposed research should provide the foundation for a better understanding of these disorders.

The long-term objectives of the proposal are to elucidate the molecular mechanisms that drive the pathogenesis of glioblastoma, the most common and aggressive primary brain tumor. We are taking a novel neurodevelopmental perspective to the study of glioblastoma pathogenesis. Glioblastoma tumors are thought to arise from the transformation of astrocytes or their precursor cells, the neural stem cells. During brain development, the transcription factor STAT3 plays a critical role in the regulation of neural stem cell fate specification including their differentiation into astrocytes. The central hypothesis of the project is that deregulation of STAT3 signaling contributes to glioblastoma pathogenesis. Using a rigorous mouse genetics approach, we have discovered that STAT3 plays opposing oncogenic and tumor suppressive roles in astrocytes depending on the mutational profile of the tumor. The major genetic alterations of deficiency of the tumor suppressor PTEN and expression of the oncogenic protein EGFRvIII may mark distinct sets of glioblastoma tumors. Remarkably, we have found that STAT3 suppresses cell transformation downstream of PTEN deficiency, whereas STAT3 behaves in an oncogenic manner downstream of EGFRvIII in astrocytes. We have also found that STAT3 represses IL8 transcription in PTEN-deficient glioblastoma cells and thereby inhibits their proliferation and invasiveness. Our findings raise fundamental questions on STAT3's role and mechanisms in glial malignancy. What is the mechanism by which STAT3 mediates the ability of EGFRvIII to induce astrocyte transformation? How does STAT3 repress IL8 transcription in PTEN-deficient glioblastoma cells? What is the role of STAT3 signaling in the biology of human glioblastoma cancer stem cells? We propose to address these questions by achieving the following three specific aims, (1) determine the mechanism by which STAT3 mediates EGFRvIII-induced astrocyte transformation by identifying the genes that operate downstream of STAT3 in this pathobiological response, (2) identify the transcriptional regulators that couple the STAT3 signal to IL8 repression in glioblastoma cells, and (3) determine the function of STAT3 in the malignant potential of glioblastoma cancer stem cells in vitro and in vivo. The proposed experiments represent an important set of experiments that will significantly improve our understanding of the transcriptional mechanisms that govern glioblastoma pathogenesis. The proposed studies should also lay the foundation for potential identification of novel therapeutic strategies in patient-tailored treatment of glioblastoma.

The long-term objectives of the proposal are to elucidate the molecular mechanisms that drive the pathogenesis of glioblastoma, the most common and aggressive primary brain tumor. We are taking a novel neurodevelopmental perspective to the study of glioblastoma pathogenesis. Glioblastoma tumors are thought to arise from the transformation of astrocytes or their precursor cells, the neural stem cells. During brain development, the transcription factor STAT3 plays a critical role in the regulation of neural stem cell fate specification including their differentiation into astrocytes. The central hypothesis of the project is that deregulation of STAT3 signaling contributes to glioblastoma pathogenesis. Using a rigorous mouse genetics approach, we have discovered that STAT3 plays opposing oncogenic and tumor suppressive roles in astrocytes depending on the mutational profile of the tumor. The major genetic alterations of deficiency of the tumor suppressor PTEN and expression of the oncogenic protein EGFRvIII may mark distinct sets of glioblastoma tumors. Remarkably, we have found that STAT3 suppresses cell transformation downstream of PTEN deficiency, whereas STAT3 behaves in an oncogenic manner downstream of EGFRvIII in astrocytes. We have also found that STAT3 represses IL8 transcription in PTEN-deficient glioblastoma cells and thereby inhibits their proliferation and invasiveness. Our findings raise fundamental questions on STAT3's role and mechanisms in glial malignancy. What is the mechanism by which STAT3 mediates the ability of EGFRvIII to induce astrocyte transformation? How does STAT3 repress IL8 transcription in PTEN-deficient glioblastoma cells? What is the role of STAT3 signaling in the biology of human glioblastoma cancer stem cells? We propose to address these questions by achieving the following three specific aims, (1) determine the mechanism by which STAT3 mediates EGFRvIII-induced astrocyte transformation by identifying the genes that operate downstream of STAT3 in this pathobiological response, (2) identify the transcriptional regulators that couple the STAT3 signal to IL8 repression in glioblastoma cells, and (3) determine the function of STAT3 in the malignant potential of glioblastoma cancer stem cells in vitro and in vivo. The proposed experiments represent an important set of experiments that will significantly improve our understanding of the transcriptional mechanisms that govern glioblastoma pathogenesis. The proposed studies should also lay the foundation for potential identification of novel therapeutic strategies in patient-tailored treatment of glioblastoma.

DESCRIPTION (provided by applicant): The long-term goals of the proposed research are to elucidate the signaling mechanisms regulating neuronal morphogenesis and connectivity in the mammalian brain. The major protein kinase CaMKII predominantly consists of the Alpha and Beta isoforms in the brain. Although CaMKIIBeta functions have been elucidated, the isoform- specific catalytic functions of CaMKIIBeta have remained largely unexplored. Using rigorously controlled knockdown analyses in primary rat neurons and in the rodent cerebellar cortex in vivo, we recently discovered the first unique catalytic function of CaMKIIBeta in the mammalian brain. Remarkably, CaMKIIBeta operates at the centrosome in a CaMKIIAlpha-independent manner to drive dendrite retraction and pruning. In other studies, we found that the TRP channel TRPC5 forms a specific complex with CaMKIIBeta, but not CaMKIIAlpha, and thereby triggers the activation of centrosomal CaMKIIBeta signaling leading to dendrite retraction and pruning in granule neurons and in the cerebellar cortex in vivo. Our findings define a novel TRPC5-regulated centrosomal CaMKIIBeta signaling pathway that controls dendrite patterning in the mammalian brain. Our findings also raise fundamental questions on the molecular basis of TRPC5-regulation of CaMKIIBeta signaling at the centrosome and the mechanisms by which centrosomal CaMKIIBeta regulates dendrite morphogenesis. To address these questions, in structure-function analyses we will test the hypothesis that distinct peptide motifs within TRPC5 and CaMKIIBeta specify the TRPC5/CaMKIIBeta interaction and thereby regulate dendrite patterning in the rodent cerebellar cortex in vivo. Using candidate and innovative unbiased biochemical approaches, we will identify novel substrates of centrosomal CaMKIIBeta and determine their role in the CaMKIIBeta-regulation of dendrite morphogenesis in granule neurons and in the rodent cerebellar cortex in vivo. Finally, in recent exciting studies, we have discovered that proteasomes operate at the centrosome to promote dendrite growth. Based on preliminary data, we will test the hypothesis that centrosomal CaMKIIBeta signaling regulates proteasome activity at the centrosome and thereby controls dendrite morphogenesis. The proposed research represents an important set of experiments that will advance our understanding of the mechanisms that control dendrite patterning and connectivity in the mammalian brain. Since disruption of dendrite connectivity contributes to the pathogenesis of diverse neurological diseases including intellectual disability and autism spectrum disorders, our studies will also advance our understanding of these devastating neurological diseases.

DESCRIPTION (provided by applicant): Intellectual disability is a prevalent developmental disorder, affecting 1-3% of the population. Advances in genetics have led to the identification of many intellectual disability proteins. However, how these proteins regulate brain development and the mechanisms by which mutations of these proteins cause intellectual disability remain poorly understood. During the past few years, we have characterized the functions of specific nuclear X-linked intellectual disability (XLID) proteins in brain development. Mutations of the XLID protein PHF6 cause the B¿rjeson-Forssman-Lehmannsyndrome (BFLS), which features intellectual delay and epilepsy. We have discovered that knockdown of PHF6 profoundly impairs neuronal migration in the mouse cerebral cortex in vivo. Remarkably, PHF6 physically associates with the PAF1 transcription elongation complex, and inhibition of PAF1 phenocopies the PHF6 knockdown-induced migration phenotype in vivo. These findings define PHF6 and the PAF1 complex as components of a novel transcriptional pathway that drives neuronal migration in the brain. Our findings have also raised fundamental questions on the mechanisms of the PHF6/PAF1 transcriptional pathway in neuronal migration and on the pathophysiological relevance of this pathway in intellectual disability. To address these questions, we will first perform structure- function analyses of PHF6 in neuronal migration in the mouse cerebral cortex. We will test the effect of BFLS patient-specific mutations of PHF6 on PHF6-dependent transcription and neuronal migration. We will also test the hypothesis that phosphorylation of PHF6 on specific sites regulates PHF6-dependent transcription and neuronal migration. In other studies, we will test the hypothesis that PHF6 regulates transcription elongation of actively transcribed genes in neurons and identify targets of PHF6 that drive neuronal migration. Finally, we will determine the effect of deregulation of the PHF6/PAF1 transcriptional pathway during cortical development on the formation of white matter heterotopias and neuronal excitability in postnatal mice. The proposed research will advance our understanding of the transcriptional mechanisms that govern neuronal positioning in the brain as well as lead to insights into how deregulation of these mechanisms contributes to the pathogenesis of intellectual disability. These studies also hold the potential of laying the foundation for novel therapeutic approaches to the treatment of BFLS and developmental cognitive disorders.

? DESCRIPTION (provided by applicant): The long-term goals of the proposed research are to elucidate the ubiquitin-signaling networks that regulate neuronal connectivity and synaptic plasticity in the brain. We recently discovered that forebrain-specific conditional knockout of Cdh1, the key coactivator of the major E3 ubiquitin ligase Cdh1-APC, profoundly impairs metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at CA1 synapses in the mouse hippocampus. In mechanistic studies, we have identified the fragile X syndrome protein FMRP as a novel substrate of Cdh1-APC in the regulation of mGluR-LTD in the hippocampus. Endogenous Cdh1-APC forms a complex with endogenous FMRP in the hippocampus, and forebrain-specific conditional knockout of Cdh1 impairs mGluR-induced ubiquitination and degradation of FMRP in the hippocampus. In epistatic analyses, knockout of FMRP suppresses the conditional Cdh1 knockout-induced mGluR-LTD phenotype. These findings define the major E3 ubiquitin ligase Cdh1-APC and fragile X syndrome protein FMRP as components of a novel ubiquitin-signaling pathway that regulates mGluR-dependent synaptic plasticity. These findings also raise fundamental questions on the mechanisms and biological implications of Cdh1-APC signaling in synaptic plasticity and disease. To address these questions, in structure-function analyses we will identify distinct domains and motifs within Cdh1 that regulate the ability of Cdh1-APC to drive mGluR-LTD in the hippocampus. We will also determine the role of Cdh1 phosphorylation and Cdh1-interacting proteins in Cdh1-APC function in mGluR-LTD. Using candidate and innovative unbiased genomics approaches, we will determine the mechanism by which Cdh1-APC/FMRP drives mGluR-LTD in the hippocampus. Finally, we will assess the biological role of Cdh1-APC signaling in a premutation model of fragile X syndrome. The proposed research represents an important set of experiments that will advance our understanding of the mechanisms that control synaptic plasticity in the brain as well as neurodevelopmental disorders of cognition.