Zhengui Xia - US grants

Apoptosis is one of the major mechanisms for control of cell death. It plays an important role during neuronal development and in the homeostasis of the nervous systems in adult animals. Abnormal apoptosis may cause or contribute to various neurodegenerative disorders including stroke, epilepsy, Parkinson's disease, Huntington's disease, and Alzheimer's disease. Elucidation of mechanisms that regulate neuronal apoptosis is of fundamental importance for neurobiology and may ultimately lead to the development of pharmacological interventions and clinical strategies for treatment of various neurodegenerative disorders. The overall objective of the proposal is to identify signal transduction pathways for apoptosis in neurons of the central nervous system. Recently, we discovered that three members of the mitogen- activated protein (MAP) kinase family, ERK, JNK, and p38, mediate opposing effects on apoptosis induced by withdrawal of NGF from NGF- differentiated PC-I 2 cells. NGF withdrawal led to a delayed but sustained activation of the JNK and p38 MAP kinases and inhibition of ERKs. The activation of JNK and p38 and concurrent inhibition of ERK were both critical for induction of apoptosis in these cells. While activation of the ERK signaling pathway promoted cell survival, stimulation of the JNK and p38 signaling pathways contributed to cell death. We hypothesize that the dynamic balance between growth factor- activated ERK and stress-activated JNK-p38 pathways determines whether neurons survive or undergo apoptosis. The overall objective of this proposal is to test the generaLity of this hypothesis for neurons from different parts of brain in response to several types of cellular stress. The specific aims of this proposal are to determine if JNK or p38 MAP kinases are activated, while the ERKs are inhibited during apoptosis in primary cultured neurons; determine if direct activation of JNK and/or p38 is sufficient to induce apoptosis in primary cultured neurons; determine if inhibition of JNK or p38 signaling pathways prevents apoptosis in primary cultured neurons; determine if activation of ERKs exerts neural protective effects against apoptosis in primary cultured neurons; and determine if the activation of JNK and p38 is critical for the induction of apoptosis in cultured hippocampal slices.

[unreadable] DESCRIPTION (provided by applicant): Development of the mammalian central nervous system (CNS) requires the production of many types of neurons and glia at the correct numbers and appropriate locations. The controlled proliferation, differentiation and migration of multipotent neural progenitor cells give rise to these diverse cell types and are critical for proper CNS development. Abnormalities in these processes have been implicated in microcephaly and in several forms of mental disorders including mental retardation, depression, and schizophrenia. Furthermore, stem cell-based cell replacement therapy offers enormous potential for the treatment of a variety of developmental, psychiatric, neurodegenerative and aging related diseases for which there are currently no cures. The elucidation of molecular mechanisms that regulate neural progenitor cell differentiation into neurons is important for an understanding of human developmental and neurodegenerative diseases. The multipotent neural progenitor cells can differentiate into neurons or glia depending on developmental cues and environmental signals. Neuron differentiation proceeds by a 2-step process: the initial commitment of the progenitors to a neuronal fate followed by terminal differentiation of the committed precursors into mature neurons. The specification of cortical progenitors to a neuronal fate requires a coordinated expression of the basic-helix-loop-helix (bHLH) family of transcription factors neurogeninl (Ngn1) and neurogenin2 (Ngn2). The transient expression of proneural Ngn1 and Ngn2 induces the expression of the NeuroD family of bHLH differentiation genes (NeuroD, NeuroD2 and Nex), which mediate terminal differentiation of neurons. Despite the known role for Ngn/NeuroD in the development of the CNS, little is known regarding the molecular mechanisms underlying regulation of the commitment of the progenitors to a neuronal fate by the proneural factors Ngn 1 and Ngn2. For example, the signaling pathways and mechanisms that positively regulate the activity of Ngns are completely unknown. In this proposal, we will investigate a potential role for the ERK5 MAP kinase signaling pathway in cell fate specification of cortical progenitor cells. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The c-Jun NH2-terminal protein kinases (JNK) are a family of MAP kinases that are preferentially activated by cell stress-inducing signals, such as trophic-withdrawal, heat shock and UV. JNKs have been implicated in several physiological functions including regulation of apoptosis, inflammatory responses, cell proliferation, differentiation and tissue morphogenesis. For example, JNK may play a role in the induction of apoptosis in neurons during development. However, the mechanism for JNK-mediated neuronal apoptosis is undefined. Using arsenite as a model for toxicant-induced neuronal apoptosis during development, we have begun to elucidate the molecular and cellular mechanisms underlying JNK-induced neuronal cell death. Sodium arsenite is an environmental toxicant that causes developmental defects in the CNS. Our data indicate that JNK isoforms may be differentially regulated during neuronal apoptosis. There are three JNK genes; JNK1, JNK2, and JNK3, cortical neurons express kinase activities for all JNKs. We showed that JNK contributes to arsenite-induced apoptosis in cultured neurons. Furthermore, JNK3 but not JNK1/2 is activated by arsenite. Since JNK3 is the only neural specific JNK isoform, it may provide a neurospecific target for blocking neuronal apoptosis. We propose to test the hypothesis that JNK3 but not JNK1 or 2, may be important for stress-induced neuronal apoptosis both in vivo and in vitro. We propose to address this issue using primary cultured cortical neurons, hippocampal slice cultures and high precision stereotactic delivery of apoptotic agents to the hippocampus and cortex. Moreover, we propose to use JNK3-/- mice as well as transgenic mice over-expressing JBD specifically in neurons in the hippocampus and cortex to elucidate the function of JNK in neuronal apoptosis in vivo. We also propose a novel mechanism for JNK induction of apoptosis in which JNK activation induces post-translational modification that leads to caspase activation, and transcriptional regulation of cell death genes.

DESCRIPTION (provided by applicant): Environmental toxicants including pesticides may contribute to the development of various neurodegenerative disorders including Parkinson's disease (PD). One of the mechanisms implicated in various neurodegeneration is neuronal apoptosis. We discovered that chlorpyrifos and rotenone both stimulate apoptosis in neuronal cells, suggesting that pesticide-induced apoptosis may play a role in neurodegeneration. Consequently, it is important to elucidate molecular mechanisms for induction of apoptosis by pesticides in neurons. The overall objective of this project is to elucidate apoptotic mechanisms for chlorpyrifos- and rotenone-induced apoptosis in neurons. Chlorpyrifos, an organophosphate pesticide, is one of the most commonly used pesticides. Its primary target of toxicity is the CNS. Treatment of rats with rotenone, a common insecticide, causes all PD symptoms. Therefore, chlorpyrifos and rotenone, two distinct classes of pesticides, are chosen as models to study pesticide-induced neuronal apoptosis. Our preliminary data suggest that both chlorpyrifos and rotenone induce apoptosis in primary cultured cortical neurons and SH-SY5Y cells. They also activate the stress-activated MAP kinases, JNK and p38. We hypothesize that activation of these kinases is important for chlorpyrifos- and rotenone-induced apoptosis in neurons. We will test this hypothesis and elucidate downstream mechanisms by which chlorpyrifos and rotenone stimulation of JNK and p38 causes neuronal apoptosis. This study should provide valuable new information concerning the molecular basis of pesticide-induced apoptosis in neurons and new insights concerning the role of environmental toxicants in neurodegeneration.

DESCRIPTION (provided by applicant): Although the etiology of Parkinson's disease (PD) has not been defined, epidemiological studies have indicated a correlation between increased risk for PD and occupational exposure to pesticides including paraquat, a widely used herbicide. Interestingly, treatment of mice with paraquat produces many key features of PD including dopaminergic neuron degeneration in the substantia nigra pars compacta (SNpc) of the brain and formation of 1-synuclein containing inclusion bodies. Therefore, studies of paraquat-induced dopaminergic neuron death may provide important new information concerning mechanisms governing the death and survival of dopaminergic neurons and thereby provide important new insights concerning the molecular basis of PD. Recently, we discovered that paraquat selectively kills dopaminergic neurons in primary cultures by a mechanism that requires activation of the c-Jun NH2-terminal protein kinase (JNK) and JNK-induced gene expression. Furthermore, paraquat-induced dopaminergic neuron death is inhibited by bFGF. This proposal is based upon the hypothesis that JNK, specifically the neurospecific JNK3 isoform, plays an important role in paraquat-induced death of dopaminergic neurons, and that this cell death may be mediated by BimEL and antagonized by bFGF. These mechanistic studies should provide critical information concerning the molecular basis of dopaminergic neuron death in the paraquat model of PD. Furthermore, our proposed research meets the goals of NIEHS strategic plan, especially to "support research that improves our understanding of signal transduction pathways and their influence on disease" under goal #2, which is to "use environmental toxicants to understand basic mechanisms in human biology". PUBLIC HEALTH RELEVANCE Parkinson's disease is the second most common aging-related neurodegenerative disorder. We propose to elucidate molecular mechanisms underlying paraquat-induced dopaminergic neuron death in vitro and in vivo. These mechanistic studies should provide critical information concerning the molecular basis of dopaminergic neuron death in the paraquat model of Parkinson's disease, and may provide important new insights concerning the molecular basis Parkinson's disease.

DESCRIPTION (provided by applicant): Deficits in learning and memory are prominent features of many mental disorders. Understanding molecular mechanisms responsible for learning and memory are key to the development of therapies to improve learning and memory in the treatment of mental illness. Recent studies lead to the exciting idea that newly generated neurons in the adult dentate gyrus (DG) of the hippocampus may play a role in neural plasticity including hippocampal-dependent memory formation. Adult neurogenesis occurs in the DG of the mammalian brains including adult human brain under physiological conditions and newly generated neurons functionally integrate to the DG. Many factors, including depression and other mental illness may adversely affect hippocampal adult neurogenesis. In contrast, treatment with anti-depressants and exercise, an effective means to treat depression, enhance adult neurogenesis in the DG. Consequently, elucidation of basic molecular mechanisms regulating adult hippocampal neurogenesis and generation of definitive evidence supporting a role for adult neurogenesis in hippocampal-dependent memory formation is a critical and timely issue for mental health research. Although many studies have assessed the role of adult neurogenesis in hippocampal-dependent learning and memory, the results have been inconsistent, making it still highly controversial whether adult neurogenesis contributes to hippocampus-dependent memory formation. This proposal will test the hypothesis that ERK5 signaling-mediated adult neurogenesis plays a critical role in some but not all forms of hippocampal- dependent learning and memory. PUBLIC HEALTH RELEVANCE: Our proposed studies are likely to provide new insights concerning signal transduction mechanisms regulating adult neurogenesis, and may lead to new insights to the development of therapies to improve learning and memory in the treatment of mental illness.

PROJECT SUMMARY Cadmium (Cd) is a heavy metal of high interest to the Superfund Initiative. It has no known physiological function but is a neurotoxicant. Cd exposure is associated with cognitive and olfactory impairment in humans. However, little is known concerning the underlying molecular and cellular mechanisms. This grant explores the molecular and cellular basis for the deleterious effects of Cd on olfaction and cognition in mouse models, with a focus on its effects on adult neurogenesis and Ca2+signaling critical for hippocampus-dependent memory. We hypothesize that Cd interferes with adult neurogenesis in the dentate gyrus of the hippocampus and in olfactory bulb, and disrupts Ca2+signaling in neurons. We further hypothesize that these adverse cellular and molecular effects may underlie Cd neurotoxicity in cognition and olfaction. We will test these hypotheses both in primary cultured neural stem cells and in vivo in mice. Studies proposed here will provide new insights concerning mechanisms of Cd neurotoxicity, establish animal models to ascertain a causal relationship between Cd exposure and impairment in cognition and olfaction, and elucidate underlying cellular and molecular mechanisms. The proposed studies may provide useful information for Cd risk assessment and designing prevention and intervention strategies against Cd -induced neurotoxicity.

Lead is a heavy metal of great public health concern in the US and globally. In addition to its well- characterized developmental neurotoxicity, cumulative lead exposure is also neurotoxic to adults and can lead to accelerated, persistent cognitive decline in adult humans. The apolipoprotein E gene (ApoE) exists as three polymorphic alleles in humans (?2, ?3, ?4); the ApoE -?3 allele (simplified as ApoE3 hereafter) is the most common allele. The ApoE-?4 allele (simplified as ApoE4 hereafter) is associated with increased risk for Alzheimer?s disease, and accelerated cognitive decline even in the absence of Alzheimer?s disease pathology. The hippocampus is a region of the brain critical for learning and memory, especially spatial learning. Adult hippocampal neurogenesis is the process whereby adult neural stem cells in the dentate gyrus of the hippocampus leads to the generation and functional integration of adult-born neurons in the hippocampus. These adult-born neurons can influence hippocampus-dependent learning and memory. Although adult neurogenesis is modulated by various extracellular stimuli, by the environment, and by neurotoxicants including lead and ethanol, there is a paucity of information regarding the effect of gene-environment interactions (GxE) on adult neurogenesis. This proposal focuses on the effect of lead-genetic background interactions on adult neurogenesis and hippocampus-dependent learning and memory.