Emanuel M. DiCicco-Bloom - US grants

Candidate: The author is Chief Resident in pediatric neurology at New York Hospital-Cornell Medical Center with research training in developmental neuro-science. This award will prepare the author for a career in academic medicine as an independent investigator in developmental neurology. Proposal: Recent evidence suggests that neurotransmitter phenotypic expression is remarkably mutable. This study seeks to define the extracellular factors and intracellular processes through which sympathetic neurons regulate multiple transmitter phenotypes. The specific aims are to define mechanisms through which presynaptic neurons, the central nervous system and target organ regulate peptide and catecholamine traits, determine the role of specific messenger RNA (mRNA) in modulation and expression of catecholamine enzyme, tyrosine hydroxylase (TH), and examine the roles of neurite outgrowth and extracellular factors in initial embryonic phenotypic expression. The superior cervical sympathetic ganglion, serving as a model system of the multiple transmitter neuron, will be assayed for markers of catecholamine (TH) and peptide (Substance P, SP) phenotypes in adults and neonates in vivo and embryos in vitro. The effects of long term deafferentation on ganglion TH and SP will be defined to examine chronic regulatory mechanims during maturity and ontogeny. The regulation of transmitter trait development by central synapses and Nerve Growth Factor will also be examined. The specific role of mRNA in TH modulation and expression during maturity and ontogeny, will be defined after altering extracellular regulatory factors. The rigorous conditions of tissue culture will permit analysis of cellular and molecular mechanisms in initial phenotypic expression. Characterization of the extracellular factors regulating phenotypic plasticity may lead to therapeutic interventions which can alter deranged neurologic function and, thus, alleviate symptoms of neural tube dysgenesis, hereditary ataxias and dystonias and cerebral palsy. Elucidation of molecular genetic mechanisms of phenotypic expression may suggest new approaches to degenerative processes such as the spinal muscular atrophies, Huntington's chorea and lysosomal storage disease. Environment: The Laboratory of Developmental Neurology is actively examining classical and peptide neurotransmitter plasticity during development and maturity, in vivo and in vitro.

Generation ofthe correct numbers and types of neural cells, including neurons and astrocytes, is fundamental to normal brain development and function. Conversely, alterations in brain cell composition, especially forebrain, are considered substrate of mature mental disorders with developmental origins, such as schizophrenia, depression and autism spectrum disorder. Previously we defined positive and negative extracellular signals, including FGF, IGF1 and PACAP, and intrinsic cell cycle mechanisms that regulate neurogenesis in cerebral cortex. Using this model, we are defining mechanisms by which the neurotherapeutic valproic acid (VPA), routinely administered to women of childbearing age, affects neuro/gliogenesis, because it is a teratogen that causes malformations and contributes to neuropsychiatric disorders. We hypothesize that VPA disrupts normal brain development by differentially regulating generation of neurons and glia, altering BDNF signaling and disturbing subsequent behavioral function. We find that VPA stimulates neurogenesis in culture and in embryos via cell cycle machinery, differentially regulates gliogenesis and alters BDNF signaling. Our Aims are: 1. Define effects of VPA on prenatal cortical neurogenesis and cell cycle machinery;2. Define VPA effects on proliferation and differentiation of astrocytes;3. Define effects of maternal VPA treatment on behavior of offspring during development and maturity. Studies will examine DNA synthesis, proliferation, differentiation, cell death, cell cycle western/RT- PCR and kinase analyses, cell composition by stereology, in culture and/or in developing pre- and postnatal animals, as well as assessments of exploratory behavior, social and anxiety measures and learning and memory processes. By defining cell type specific effects of VPA on intracellular signaling and cell cycle machinery, and characterizing consequences for brain cell composition and animal behavior during development, we may provide fundamental knowledge to effectively evaluate the benefits and risks of drug therapy, and identify pathways where intervention may counter detrimental effects of drug exposure. RELEVANCE (See instructions): Valproic acid (VPA) is routinely administered to women of childbearing age as therapy for epilepsy, mood disorders and migraine prophylaxis, raising concerns about contributions to developmental brain disorders. By defining effects on intracellular signaling, cell cycle machinery, brain cell composition and behavior, we may provide insights to evaluate VPA risks and benefits and identify pathways to prevent detrimental effects.

[unreadable] DESCRIPTION (provided by applicant): Cellular composition of the cerebral cortex is critical for normal function. While major deficits accompany extensive loss of tissue, more common problems like attentional/learning disability, schizophrenia and autism reflect more subtle changes undetected by neuroimaging. Specifically, abnormalities in numbers or types of neurons may underlie certain developmental disorders. As a corollary, deficiency in one neuron subpopulation due to disordered regulation at a specific ontogenetic stage may be compensated numerically by later neurogenesis, yielding an apparently normal cortical population size. Thus, by defining molecular regulation of neurogenesis, with regard to stage-specific cell production, we may discover pathways affecting cortical composition. We hypothesize signals stimulating precursor proliferation are balanced by anti-mitogenic regulators of extra- (PACAP) and intracellular (p57) origins, which halt cell cycle progression and impact cortical neurogenesis. We identified PACAP as one such anti-mitogenic agent, acting via G-protein coupled receptor/cAMP to inhibit cell cycle progression via increased CDK inhibitor, p57. We plan to define roles and mechanisms of anti-mitogenic signals in producing cortical populations. Our aims are: 1) Define the changes in cell cycle machinery elicited by intracerebroventricular injections of PACAP in utero, and identify responsive ventricular zone cells. 2) Characterize the roles of PACAP anti-mitogenic pathway components, specifically PAC1 receptor and CDK inhibitor p57, in promoting cell cycle exit and determining cell fate using deletion mutants. 3) Define effects of anti-mitogenic signaling on cell fate of mitotic precursors in culture and in vivo using over-expression vectors. Thus, we may gain insight into fundamental processes generating cellular diversity. [unreadable] [unreadable]

Drs. Dreyfus and DiCicco-Bloom are experienced histologists and have been performing immunohistochemistry for over 25 years, tn addition to its other responsibilities, the Core wfti now provide service in quantitative stereology. We have added Dr Alder who wift assist in the supen/ision of the facility as well as train students in morphological techniques. Dr. Alder is an experienced morphologist, versed in immunocytochemical analysis, autoradiography, radioactive and nonradioactive in situ hybridization, and quantitative stereology. The Core will be staffed by Lauren Lercher and Xiaofeng Zhou who are technicians (RTS) experienced in all aspects of the immunohistochemical procedures

DESCRIPTION (provided by applicant): Methylmercury (MeHg) is a widespread and persistent environmental factor that adversely affects the developing brain, which is far more sensitive than the adult organ. From previous poisonings, we know that exposure of the developing fetus in the mother to MeHg can cause severe consequences, from mental retardation to seizures and malformations, many times without harming the mother. However, there are concerns that more subtle exposures may harm the developing brain, altering learning and memory, for which the hippocampus is critical. In previous work, we found that exposing postnatal rats to moderate levels of MeHg rapidly impaired production of neurons and later produced deficiencies in hippocampal structure and function. We have now discovered that hippocampal neural stem cells, that produce neurons and glia, are exquisitely sensitive to MeHg, which causes them to die within hours. We now plan to define the lowest levels of MeHg that can cause stem cell death, and define its relationship to later loss of neurons and memory functions. In Aim 1 we will define how little MeHg in newborn rats can cause stem cell death, and identify which of several stem cell types are affected. Subsequently, 2 weeks later, we will count total number and types of neurons and glia to see whether killing precursors leads to later losses of specific populations of hippocampal cells, as we see with higher exposures. In Aim 2, we will determine whether lower exposures and losses of cells can impact learning and memory of the animals at 4 weeks of age. When complete, these studies will identify a new, highly sensitive target of MeHg, hippocampal stem cells, and will establish a novel model system. By focusing on the stem cells, rather than simply the whole brain organ as is frequently done, we may detect toxic effects of environmental factors that are otherwise missed. Or we may find that levels of known factors, like MeHg, thought to be safe, may actually have previously unrecognized harmful effects. This model system offers the possibility of defining the temporal sequence of molecular and cellular events that lead to subtle structural and functional changes in the brain during a critical window of developmental vulnerability. This information may shed light on the ways environmental exposures can impair learning and memory.