Janice R. Naegele - US grants

In mammals, neurons destined for the adult cerebral cortex are generated only after the period when two transient neuronal populations are born. These populations are called the subplate and the marginal zone cells, and they differentiate into many chemically and morphologically distinct cell types before undergoing preprogrammed cell death. Subplate neurons send axons into the thalamus, midbrain and contralateral hemisphere early in fetal life, supporting the idea that they function as pioneer neurons. They are also the first cortical cell types to receive functional thalamo-cortical synapses. Most subplate cells eventually die, but only after the adult pattern of cortico-cortical and thalamo-cortical connections become established. Together, these and other observations indicate that subplate neurons have a number of important functions in establishing cortical circuits. Little, if anything is known about molecular mechanisms underlying these functions. An immunological approach to search for subplate specific molecules has been used to produce a new monoclonal antibody which stains neurons in the cortical subplate (Naegele et. al.'89). This new antibody selectively stains the cortical subplate when neurons in this region are undergoing preprogrammed cell death. The goal of the proposed pilot studies is to further characterize this new mAb antibody, called Subplate-1, and the cells that express this antigen. In these pilot studies,Subplate-1 will be used in combination with 3H-thymidine birth-dating autoradiography to determine whether the earliest generated neurons of the subplate are stained. The morphology and neurotransmitter phenotypes of the stained neurons will be determined by using Subplate-1 in combination with intracellular staining and double-label immunocytochemistry. Finally, biochemical approaches will be used to characterize the antigen itself. These studies will require neonatal cats and ferrets principally because nearly all previous work on the times of subplate cell genesis, morphology, connectivity and neurochemistry have been studied in these species. However, preliminary results indicate that mAb Subplate-1 also stains fetal monkey and human subplate, so it is likely that any new findings can be related to the development of the human cerebral cortex. Eventually this information may further our understanding of the role of the subplate in normal cortical development and in congenital defects of this brain region.

The cellular and molecular substrates associated with the death of massive numbers of neurons in the developing visual system remain poorly characterized. One of the more distinctive neuronal populations to undergo developmental cell death is found in the subplate zone, a transient layer of the immature cerebral cortex. This population differentiates into a number of neurochemically and morphologically distinct types which form the initial intra-cortical and subcortical projections of the visual cortex and receive the first functional synaptic contacts from the dorsal lateral geniculate nucleus. Although the formation and the elimination of the subplate and its connections are likely to be of major biological significance for understanding mechanisms of cortical development, the events controlling cell death in this neuronal population are largely unknown. The first objective of the proposed studies is to provide a detailed description of the cellular and molecular interactions between the subplate neurons and geniculocortical and corticocortical axons before and during the period of cell death, using subplate-specific molecular markers and antisera against neurotrophic factors and receptors, in conjunction with anatomical techniques at the light- and electron microscopic levels. The second objective is to biochemically characterize a newly-identified antigen (SP1) expressed by the subplate cells during the period of cell death and its regulation, using monoclonal antibodies, in combination with biochemical approaches. The third objective is to define the types of cell death in the subplate. This objective will be achieved by comparing molecular properties of neurons undergoing naturally-occurring versus experimentally-induced cell death. These studies will further our understanding of the role of the subplate in formation of the visual cortex and the causes of cell death in this structure. They represent important first steps in identifying the early and specific markers for cell death in both normal development and in genetic mutations. The new information gained could also provide a rational strategy for the discovery of clinical interventions to prevent vision loss associated with acute conditions causing neuronal degeneration, such as ischemia, stroke and trauma.

Abstract Naegele NSF 0070352


A Confocal Microscope for Research and Teaching in Biology and Neuroscience

A Zeiss 510 Confocal Microscope will be used for research and training in developmental biology and neuroscience at Wesleyan University. This new confocal microscope will enable the three primary faculty users and members of their research laboratories to study the dynamic movements of cells and proteins in living embryos and to identify intercellular junctions between cells in thick sections of brain tissue. In addition, training on the confocal microscope will be an important component of a Biology department graduate-level course in advanced microscopy, including confocal, immunofluorescence, and electron microscopy. The confocal microscope facility is part of an Advanced Instrumentation Facility in the Science Center at Wesleyan University. This facility already contains scanning and transmission electron microscopes, an adjacent wet laboratory, a room for tissue sectioning, and a computer room with associated digital scanners and other image processing equipment.

The projects to be carried out by major users of this confocal microscope, Drs. Devoto, Kirn, and Naegele, and members of their laboratories, each focus on vertebrate development. The Devoto laboratory will focus on the genetic and molecular guidance cues used by migrating muscle cells in living zebrafish embryos. The Kirn laboratory will address the mechanisms of neuronal replacement and neurogenesis in the brains of adult birds. The Naegele laboratory will study cellular and molecular signals regulating programmed cell death and engulfment of dying neurons in the rodent cerebral cortex and visual system. Occasional use of the confocal is also planned by five additional faculty in the Biology and Molecular Biology and Biochemistry Departments who study a variety of biological problems ranging from the role of transcription factors in embryo development to the cell cycle regulation in yeast. Additional minor use by one extramural group Pfizer, Inc. is planned for 2 days/month. Funds from this extramural group will be a significant source of revenue for long-term maintenance of the confocal, including service contracts.

We anticipate that this confocal microscope and the associated research programs will have a significant impact on research and training at Wesleyan University in the fields of developmental biology and neurobiology. The objective of our science training programs at Wesleyan University is to provide high-quality research experiences for undergraduate and graduate students, as well as postdoctoral fellows and visiting scientists. This hands-on training is of fundamental importance for careers in scientific research, technology, and education. Special initiatives at Wesleyan University advance women and minorities in science, as well as providing access for students with disabilities. It is anticipated that the new confocal microscope will contribute significantly to a basic understanding of how cells in the developing embryo migrate, form connections, and how some undergo programmed cell death as part of their normal developmental plan. These studies will ultimately lead to a better understanding of how the vertebrate brain and body are constructed during development and maintained throughout the life of the organism.

[unreadable] DESCRIPTION (provided by applicant): Our long-term objective is to understand how programmed cell death and DNA repair guide development and maintenance of the nervous system. This ROl focuses a specific form of DNA repair called Non-Homologous End Joining (NHEJ), which is required for proper development of the CNS. Enzymes involved in NHEJ and other DNA repairs serve critical roles in genomic stability and neuronal survival in neurodegenerative disorders such as Alzheimer's and ALS. In the developing brain, programmed cell death is regulated process acting in concert with neurogenesis and differentiation. Knockout mice deficient NHEJ or other DNA repair processes typically have increased apoptosis in the embryonic CNS and perinatal lethality. We will utilize NHEJ deficient mice in combination with cellular and molecular approaches to study the impact of genomic instability on neuronal survival and differentiation. To accomplish this broad goal, we have four Specific aims. The first proposes to compare the molecular phenotypes of neural progenitors and postmitotic neurons in the cerebral cortex of normal and NHEJ-deficient mouse embryos. We will monitor cell death and examine the expression of cell cycle and other molecular markers. We will also generate CNS-specific conditional knockouts of NHEJ genes using cre-recombinase technology to evaluate whether failed NHEJ in neural progenitors and neurons is responsible for the differences in apoptosis. The second aim will determine if NHEJ deficient embryonic CNS cultures exhibit increased apoptotic responses to hypoxia, free radical damage, or excitotoxic injury. The third aim will examine whether NHEJ deficiency and accumulated DNA breaks coupled with hypoxia or excitotoxic injury activate p53 and downstream apoptotic effects such as mitochondrial dysfunction. We will generate compound knockout mice lacking NHEJ genes and either p53 or Bax to test this hypothesis. Our fourth aim will extend these studies to ask whether exogenous growth factors or free radical scavengers can delay or prevents downstream apoptotic signaling in NHEJ deficient mice.

DESCRIPTION (provided by applicant): Our long-term goal is to advance cell-based treatments for severe temporal lobe epilepsy (TLE). The proposed work examines the mechanisms regulating synaptic integration of transplanted GABAergic interneurons transplanted into the dentate gyrus of the hippocampus in the adult nervous system of mice with pilocarpine-induced TLE. GABAergic interneuron grafts made into the adult dentate gyrus suppress temporal lobe seizures in TLE mice. Our working hypothesis is that the mechanism for seizure suppression is formation of inhibitory synapses by the transplanted neurons onto granule cells (GCs) born into the epileptic brain environment. To test this hypothesis we will compare seizure suppression and inhibitory synapse formation by transplants of GABAergic precursors derived from three sources: 1) the medial ganglionic eminence (MGE) of fetal mouse brain, 2) mouse embryonic stem cells (mESCs), and 3) mouse induced pluripotent stem cells (mIPS cells) in the following two Specific Aims: Aim 1: Identify the functionality and synaptic partners of fetal MGE-derived GABAergic interneurons transplanted into the dentate gyrus of TLE mice. This aim tests the hypothesis that fetal GABAergic interneuron transplants suppress seizures due to formation of inhibitory synapses onto GCs born into the epileptic brain environment. Aim 1.1: Evaluate seizure suppression by EEG in TLE mice with transplants of fetal basal forebrain cells. Aim 1.2: Measure inhibitory post-synaptic currents in retrovirally-labeled populations of GCs by patch-clamp electrophysiological recordings in hippocampal slices from TLE mice with fetal interneuron transplants. Aim 1.3: Compare the extent of axon outgrowth and inhibitory synapse formation by the fetal GABAergic interneuron transplants onto retrovirally-labeled GCs in TLE mice. Aim 2: Identify the functionality and synaptic partners of mouse pluripotent stem cell (mPSC)-derived GABAergic interneurons transplanted into the dentate gyrus of TLE mice. This aim tests the hypothesis that mPSC-derived GABAergic precursor transplants will suppress seizures due to formation of inhibitory synapses onto GCs born into the epileptic brain environment. Aim 2.1: Derive MGE-like basal forebrain cells in vitro from mESCs with an Nkx2.1 BAC reporter construct. Aim 2.2: Derive MGE-like basal forebrain cells in vitro from miPS cells with an Nkx2.1 BAC reporter construct. Aim 2.3: Evaluate seizure suppression by EEG in mice with transplants of mPSC-derived MGE-like basal forebrain cells. Aim 2.4: Measure inhibitory post-synaptic currents in retrovirally-labeled populations of GCs by patch-clamp electrophysiological recordings in hippocampal slices from mice with transplants of mPSC-derived MGE-like basal forebrain cells.

An award is made to Wesleyan University to support the acquisition of a confocal microscope for use by faculty and students across multiple departments and disciplines. The new instrument will profoundly enhance the training environment and be used during many highly interactive laboratory courses taught by Wesleyan faculty to promote STEM career advancement. The proposed microscope will be housed in an advanced imaging facility that includes scanning and transmission electron microscopes. To support dissemination of science to the general public, as exemplified by the large number of articles authored by Wesleyan faculty that are printed in the popular media, data generated by the new system will be used in outreach presentations, science camps, local high-schools, an annual scientific imaging student prize, and similar forums.

Wesleyan boasts an unusually broad portfolio of research topics that will benefit from the new system, including evolution and development of morphology, optogenetic-based control of behavior, cell biology, physiology, developmental neuroscience, and chromosomal dynamics. Each of these areas is experiencing an expansion of research into spatial relationships, driven by advances in microscopy, and by the realization that researchers must study three-dimensional shapes and how those change over time to understand the structure and function of biological systems. Several of the research projects will only be possible because the local availability of the system. Data and analyses from these studies will be published in peer-reviewed scientific journals, presented at scientific meetings, and used in both educational and public outreach activities.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.