Santosh D'Mello, Ph.D. - US grants

9511013 D'Mello During the development of the vertebrate nervous system, approximately half of the neurons generated die around the time of birth by a process known as programmed cell death (PCD or apoptosis). Although leading to the massive death of neuronal populations, this process is necessary for the normal development of the nervous system. It is now well established that PCD is not restricted to the nervous system, but occurs in virtually all tissue and cell types and represents a fundamental biological process serving to eliminate unwanted or superfluous cells. A question of significant importance is whether the inappropriate activation of the apoptotic program underlies abnormal neuronal loss. The objective of this proposal is to understand the mechanisms controlling PCD in neurons. More specifically, genes that induce or suppress the death process will be identified. A cell culture model system using neurons from the vertebrate cerebellum has previously been established will be used to identify genes that regulate neuronal death. In the long term, these investigations will provide fundamental information concerning the processes underlying neuronal death during development.

DESCRIPTION (provided by applicant): Histone deacetylases (HDACs) are proteins originally identified on the basis of their ability to deacetylate histones resulting in transcriptionally repression. HDACs also deacetylate a large number of other proteins in the nucleus, cytoplasm and mitochondria thereby regulating diverse cellular events. Compelling evidence indicates that HDACs regulate the survival and death of neurons. Indeed, several laboratories have demonstrated that chemical inhibitors of HDACs are strongly protective in many different experimental invertebrate and rodent models of neurodegenerative disease. The focus of this application is on HDAC3, an HDAC that we discovered has strong neurotoxic activity and representing a likely target of HDAC inhibitors in their neuroprotective effect. We found that neurons are selectively sensitive to HDAC3 toxicity and that HDAC3-induced neuronal death requires its phosphorylation by GSK3¿, a kinase implicated in several neurodegenerative disorders. The current application follows up on these findings to examine in more detail the mechanism by which HDAC3 promotes neurodegeneration. In addition to using paradigms of neuronal death unrelated to disease states, we will study the role of HDAC3 in Huntington disease (HD) pathogenesis. We have observed that HDAC3 interacts with huntingtin protein (Htt), mutation of which causes HD. We hypothesize that the HDAC3 plays a pivotal role in the neurotoxic effect of mutant-Htt and that mutant-Htt stimulates the release of HDAC3 thereby de-repressing its neurotoxic activity. Based on recently acquired data, we propose that HDAC3 neurotoxicity requires the participation of HDAC1, another Class I HDAC with which HDAC3 interacts. The specific goals of our application are: (1) To study the contribution of HDAC3 to Htt-mediated neuronal survival and to mutant-huntingtin-induced neuronal death, (2) To investigate the contribution of HDAC1 in HDAC3 and mutant-Htt toxicity, (3) To identify downstream targets of HDAC3- mediated neurotoxicity, and (4) To study the effect of HDAC3 deficiency on neuropathology in the R6/2 and BACHD mouse models of HD by breeding these mice to forebrain-specific HDAC3 conditional knockout mice that we have just generated. The studies we propose will shed new insight into the fundamental mechanisms regulating neuronal survival and death, as well as how these mechanisms relate to HD.

DESCRIPTION (provided by applicant): Neurological diseases disrupt the quality of the lives of patients, puts a tremendous burden on family caregivers, and cost society billions of dollars annually. A common feature of neurological diseases is the degeneration of neurons by apoptosis. Drugs that inhibit neuronal apoptosis could thus be candidates for therapeutic intervention in neurodegenerative disorders. Moreover, identifying the molecular targets of such neuroprotective drugs and understanding the signal transduction pathways that are utilized in their action would lead to the development of more effective therapeutic strategies. Working with a cell culture paradigm of neuronal apoptosis that uses rat cerebellar granule neurons we have identified a drug, GW5074 {5-Iodo-3-[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-indolinone} that completely inhibits neuronal apoptosis. GW5074 is a specific and potent inhibitor of c-Raf when tested in vitro. Paradoxically, however, treatment of cultured neurons with GW5074 leads to the accumulation of activating modifications on c-Raf. Moreover, GW5074 treatment stimulates B-Raf activity. Among the molecules affected downstream of c-Raf in neurons treated with GW5074 are the antiapoptotic molecule NF-kappa b. GW5074 also inhibits the proapoptotic transcription factor, c-jun. Although GW5074 is the most effective, neuroprotection is also observed by two other chemical inhibitors of c-Raf. The utility of small molecule inhibitors of c-Raf as neuroprotective agents has not been described previously. The overall goal of this proposal is to use GW5074 to understand the molecular mechanism by which inhibitors of c-Raf exert their antiapoptotic effect and to more thoroughly investigate the potential of GW5074 as a neurotherapeutic agent. Our specific goals are to: (1) better understand the effect of GW5074 on c-Raf and B-Raf understand the effect of GW5074 on c-Raf, (2) analyze the downstream mechanisms by which GW5074 exerts its neuroprotective effect, and (3) examine whether the molecular effects of GW5074 in cultured cerebellar granule neurons are also observed in an animal model of neurodegeneration. It is our hope that GW5074 (or other c-Raf inhibitors like it) will emerge as a highly effective and versatile therapeutic agent that could proceed towards clinical trials for the treatment of neurological diseases in the near future.

DESCRIPTION (provided by applicant): Neurological diseases disrupt the quality of the lives of patients, puts a tremendous burden on family caregivers, and cost society billions of dollars annually. A common feature of neurological diseases is the degeneration of neurons by apoptosis. Drugs that inhibit neuronal apoptosis could thus be candidates for therapeutic intervention in neurodegenerative disorders. Moreover, identifying the molecular targets of such neuroprotective drugs and understanding the signal transduction pathways that are utilized in their action would lead to the development of more effective therapeutic strategies. Working with a cell culture paradigm of neuronal apoptosis that uses rat cerebellar granule neurons we have identified a drug, GW5074 {5-Iodo-3-[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-indolinone} that completely inhibits neuronal apoptosis. GW5074 is a specific and potent inhibitor of c-Raf when tested in vitro. Paradoxically, however, treatment of cultured neurons with GW5074 leads to the accumulation of activating modifications on c-Raf. Moreover, GW5074 treatment stimulates B-Raf activity. Among the molecules affected downstream of c-Raf in neurons treated with GW5074 are the antiapoptotic molecule NF-kappa b. GW5074 also inhibits the proapoptotic transcription factor, c-jun. Although GW5074 is the most effective, neuroprotection is also observed by two other chemical inhibitors of c-Raf. The utility of small molecule inhibitors of c-Raf as neuroprotective agents has not been described previously. The overall goal of this proposal is to use GW5074 to understand the molecular mechanism by which inhibitors of c-Raf exert their antiapoptotic effect and to more thoroughly investigate the potential of GW5074 as a neurotherapeutic agent. Our specific goals are to: (1) better understand the effect of GW5074 on c-Raf and B-Raf understand the effect of GW5074 on c-Raf, (2) analyze the downstream mechanisms by which GW5074 exerts its neuroprotective effect, and (3) examine whether the molecular effects of GW5074 in cultured cerebellar granule neurons are also observed in an animal model of neurodegeneration. It is our hope that GW5074 (or other c-Raf inhibitors like it) will emerge as a highly effective and versatile therapeutic agent that could proceed towards clinical trials for the treatment of neurological diseases in the near future.

[unreadable] DESCRIPTION (provided by applicant): A common feature of neurodegenerative diseases is the aberrant and excessive loss of neurons by activation of apoptosis. Numerous molecules involved in the promotion or inhibition of neuronal apoptosis have been identified and these are being organized as components of signal transduction pathways. This proposal focuses on histone deacetylases (HDACs), a family of enzymes originally identified on the basis of their ability to deacetylate histones. More recent work has shown that HDACs are involved in a variety of different biological processes and they have therefore emerged as the subject of intense investigation. We have found that one member of the HDAC family of proteins, HDAC4, protects neurons from apoptosis. Neuroprotection does not involve signaling pathways that are commonly used by other survival-promoting genes and biological factors. The objective of the proposal is to use a multi-pronged approach to elucidate the mechanism by which HDAC4 exerts its neuroprotective action with the long-term goal of developing novel and effective strategies to prevent cell death in neurodegenerative pathologies. The specific aims are- (1) To identify the region within HDAC4 that mediates neuroprotection, (2) To identify HDAC4-interacting proteins in neurons using mass-spectrometry, and (3) To identify downstream targets of HDAC4 action in neurons with a focus on cell cycle components. [unreadable] PUBLIC HEALTH RELEVANCE: Neurological diseases disrupt the quality of life for patients and cost society billions of dollars annually. While symptomatic treatments are available for many neurological diseases, a cure is not presently available. Identifying molecules that regulate neuronal survival and understanding the mechanism by which they act would lead to the development of more effective therapeutic strategies. Our proposal focuses on HDAC4, a protein that is neuroprotective. It is our hope that the results from the studies we propose will shed insight into how HDAC4 exerts its neuroprotective effect and thus provide novel strategies to prevent neuronal loss in neurodegenerative conditions. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A common feature of neurodegenerative diseases is the aberrant and excessive loss of neurons by activation of apoptosis. Numerous molecules involved in the promotion or inhibition of neuronal apoptosis have been identified and these are being organized as components of signal transduction pathways. This proposal focuses on sirtuins, a family of deacetylating enzymes that are the subject of intense investigation because of their role in a variety of different biological processes including the regulation of neurodegeneration. Like some other labs, we have found that one of the sirtuin proteins, SIRT1, protects neurons from apoptosis. But contrary to the conclusions of previous studies, we find that neuroprotection by SIRT1 is mediated by a novel, non-catalytic mechanism. The objective of the proposal is to use a multi- pronged approach to elucidate the novel mechanism by which SIRT1 exerts its neuroprotective action. The specific aims are - (1) to identify the region within SIRT1 that mediates neuroprotection, (2) to identify downstream targets and mechanism of SIRT1 action in neurons, and (3) to identify SIRT1-interacting proteins in neurons using mass-spectrometry. The long term goal of this research is to develop novel and effective strategies to prevent cell death in neurodegenerative pathologies. PUBLIC HEALTH RELEVANCE: Neurological diseases disrupt the quality of life for patients and cost society billions of dollars annually. While symptomatic treatments are available for many neurological diseases, a cure is not presently available. Identifying molecules that regulate neuronal survival and understanding the mechanism by which they act would lead to the development of more effective therapeutic strategies. Our proposal focuses on SIRT1, a protein that protects neurons from degeneration. It is our hope that the results from the studies we propose will shed insight into how SIRT1 exerts its neuroprotective effect and thus provide novel strategies to prevent neuronal loss in neurodegenerative conditions.

DESCRIPTION (provided by applicant): Neurodegenerative diseases are characterized by the progressive and relentless loss of neurons. One strategy to prevent or slow down neurodegeneration is to stimulate molecules that have neuroprotective activity. Using tissue culture systems we have demonstrated that FoxG1, a member of the Forkhead family of transcription factors, is necessary for the maintenance of neuronal survival. Furthermore, elevated expression of FoxG1 has strong neuroprotective effects. We propose to extend our studies in vivo and predict that elevated levels of FoxG1 will be also be protective in animal models of neurodegenerative disease. We propose to test this prediction by generating transgenic mice that express elevated levels of FoxG1 selectively in neurons. These mice will be crossed with two different mouse models of neurodegenerative disease and the effect on progression of neuropathology evaluated. The two specific aims of this proposal are - Aim 1: To generate transgenic mice overexpressing wild-type FoxG1, a constitutively-active form of FoxG1, and a dominant-negative form of FoxG1. We will use the mouse prion protein (mPrP) promoter to drive expression of the three forms of FoxG1 in mice. Aim 2: Analysis of the effect of FoxG1 overexpression on the brain and in mouse models of neurodegeneration. We will examine the effect of increasing FoxG1 activity, through expression of WT and CA FoxG1, on neuronal survival in the normal brain. In a second part of this aim we will examine whether elevated FoxG1 activity protects mice against neurodegeneration. We will cross FoxG1-overexpressing transgenic mice with R6/2 mice (a model of HD) and with p25/CDK5 inducible-transgenic mice (a model of AD). The project will result in the development of three new mouse lines expressing wild-type and mutant FoxG1 transgenic mice. FoxG1 transgenic mice do not currently exist. We feel that generating these mice and testing whether elevated FoxG1 can protect against neurodegenerative disease will shed insight into the function of FoxG1 in postmitotic neurons in vivo and could identify it as a target for the development of novel therapeutic strategies for neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: Previous work in our laboratory has demonstrated that elevated activity of the FoxG1 protein protects neurons against death. The goal of our project is to extend this observation in vivo by generating transgenic mice that express higher and lower activity of FoxG1. These mice will then be bred with existing models of neurodegenerative disease.

DESCRIPTION (provided by applicant): Ever since mutations in the methyl-CpG binding protein 2 (MeCP2) gene were found to be the primary cause of Rett syndrome, there has been a tremendous level of interest in understanding the biology and normal function of MeCP2. The MeCP2 gene is alternatively spliced to generated two isoforms commonly referred to as MeCP2-e1 and MeCP2-e2. The significance of these two isoforms is not known and it is widely believed that there is no functional difference. Indeed, most studies in which MeCP2 is ectopically expressed do not specify which isoforms is being used, and expression studies of MeCP2 rarely distinguish between the two. We find that the two isoforms are differentially regulated in postmitotic neurons that are degenerating. Moreover, the enhanced expression of MeCP2-e2 promotes neuronal death while elevated MeCP2-e1 expression has no such effect. In addition to the differential expression pattern and effect on neuronal viability, the two isofors bind the FoxG1 with dramatically different efficiency. FoxG1 is a transcription factor which promotes neuronal survival and whose mutations have also been linked to Rett syndrome. The specific aims of the proposal are (1) to obtain a more detail understanding of MeCP2-e2-induced neurotoxicity, and (2) to define the relationship between MeCP2 and FoxG1 in the regulation of neuronal survival. Although most studies have focused on the role of MeCP2 during brain development, relatively little is known about its function in postmitotic neurons. Our results suggest that MeCP2 is involved in regulating the survival of postmitotic neurons and that the two isoforms regulate this function differently. Results from our proposed experiments will unravel the relationship between two proteins that are linked to Rett syndrome. Furthermore, they will increase existing knowledge on the regulation of neuronal death by focusing on two proteins whose role in the context of neuronal death has not been adequately studied.

SUMMARY Enhanced activity of the heat shock factor-1 (HSF1) transcription factor has protective effects in a variety of tissue culture and in vivo models of protein aggregation-associated neurodegenerative disease. This neuroprotective effect of HSF1 is thought to be mediated through a stimulation of transcription of genes encoding heat shock proteins (HSPs). By acting as molecular chaperones the newly produced HSPs re-fold or help degrade misfolded proteins in cells that suffer thermal or proteotoxic stress. Stimulation of HSP gene transcription by HSF1 requires its homo-trimerization and binding to sequences called heat shock elements (HSEs) located mostly in the promoters of HSP genes. We recently demonstrated that HSF1 can protect neurons from both proteotoxic and non-proteotoxic death by a mechanism does not require its trimerization and is HSP-independent. The goal of this this proposal is to understand this novel HSP-independent mechanism of neuroprotection. We propose that HSF1 acts by binding to non-HSE sequences as a monomer and regulating genes unconnected to HSPs and other chaperones. We propose that neuroprotection by HSF1 through this non-canonical mechanism requires the activity of histone deacetylases (HDACs) and interaction with the Class III HDAC, SIRT1. The specific aims of our project are: The specific aims of our project are - Aim 1: To understand the role of histone deacetylases in HSF1-mediated neuroprotection. Aim 2: To identify genomic sequences bound by monomeric HSF1 using two separate approaches ? ChiP- Seq and Bind-N-Seq. Aim 3: To identify genes regulated by monomeric HSF1 using RNA-Seq. Understanding the mechanism will lead to a better understanding of the process of neurodegeneration and provide the basis for the development of novel therapeutic approaches for neurodegenerative diseases.

SUMMARY Huntington disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of a CAG repeat in the first exon of the huntingtin gene resulting in a mutant protein with a poly-glutamine expansion. Although mutant huntingtin (mut-Htt) is expressed ubiquitously in the brain, neurodegeneration occurs selectively in the striatum and, to a lesser degree, the cortex. We propose that a key factor in the region-specific vulnerability in HD is a reduction in the expression of FoxP1, a neuroprotective protein that is expressed selectively in medium spiny neurons of the striatum and to a relatively lower level in pyramidal neurons of the cortex. Consistent with our hypothesis, FoxP1 expression is reduced in the striatum of HD patients and HD mouse models. Elevating FoxP1 expression in cultured neurons protects them from mut-Htt toxicity, while knocking down its expression induces death in otherwise healthy neurons. The overall objective of the proposal is to understand the mechanism underlying the reduced expression of FoxP1 in dying neurons and the mechanism by which FoxP1 maintains the survival of neurons normally. Although almost all studies on FoxP1 have focused on a 90 kDa form of the protein called isoform-A, the brain expresses two other major isoforms ? isoforms C and D. The significance of these isoforms as well as other members of the FoxP family that are expressed in the adult striatum will be studied. Finally, we will extend our tissue culture studies to mice and examine whether elevated expression of FoxP1 protects mice from HD and whether the reduced striatal size in brain-specific FoxP1 conditional mice (cKO) is due to neuronal loss. The specific aims of this proposal are: Aim 1: Role of histone deacetylase-3 (HDAC3) and methyl CpG binding protein-2 (MeCP2) in the downregulation of FoxP1 expression in dying neurons. Aim 2: Aim 2: Identify downstream targets of FoxP1 that mediate its protective effect against mut-Htt neurotoxicity. Aim 3: To examine contribution of major FoxP1 isoforms and of FoxP2 and FoxP4 to neuronal survival. Aim 4: Examine the effects of modulating FoxP1 levels on the regulation of neurodegeneration and neuronal survival in vivo. There are currently no effective treatment strategies for the abnormal neuronal loss that occurs in HD. Successful completion of this project has the potential to provide new avenues for the development of a therapy to reduce or stop neurodegeneration in HD.

MeCP2 duplication syndrome is a severe and progressive neurological disorder caused by the duplication (or triplication) of the MeCP2 gene located on the X-chromosome. The disorder is characterized by intellectual disability, motor deficits, ataxia, epilepsy and premature death. Little is known about the molecular mechanisms underlying MeCP2 duplication syndrome and there is no treatment. We are studying this disorder using a transgenic mouse line, MeCP2-Tg, in which human MeCP2 gene locus is inserted on the X- chromosome and in which MeCP2 is expressed at 3 ? 5 times the normal level. As in humans, male but not female MeCP2-Tg mice display cognitive impairment, seizures, motor deficits, ataxia and die at 18 ? 20 weeks of age. We find that male MeCP2-Tg mice display highly elevated expression of glial fibrillary acidic protein (GFAP) and the microtubule-associated protein, Tau, in the hippocampus and cortex, but not appreciably in other brain parts. We propose that MeCP2 duplication syndrome is a neurodegenerative disorder that is triggered by elevated GFAP in astrocytes of the cortex and hippocampus leading to their dysfunction. A consequence of this is the elevation of extracellular glutamate, which increases Tau levels in neurons and promotes their death as a result of excitotoxicity. The goal of our proposal is to investigate the contributions of GFAP and Tau to the motor and cognitive deficits in MeCP2-Tg mice. This will be accomplished using cell culture models and MeCP2-Tg mice deficient in either GFAP or Tau. The specific goals of our proposal are: (1) To understand the mechanisms underlying selective neuronal vulnerability in MeCP2-Tg mice, (2) To investigate why MeCP2 duplication syndrome selectively affects males, and (3)To investigate the contribution of increased GFAP and Tau expression to neuronal death and behavioral deficits. Our research will provide a mechanistic framework for the understanding of the molecular and cellular underpinnings of MeCP2 duplication syndrome and will likely identify two proteins, the deregulation of which likely plays a key role in disease pathogenesis. If our hypothesis is correct, therapeutic approaches to lower GFAP and Tau levels will have benefit to patients with MeCP2 duplication syndrome.