Jeffrey A. Golden, MD, University of Pennsylvania - US grants

A hallmark of the vertebrate central nervous system is its extreme complexity. Complexity is exhibited at the level of synaptic connections as well as in the number of cell types. The developmental mechanisms that generate such diversity are just beginning to be explored. Lineage analysis has been a valuable technique in addressing this problem. Studies in the retina and cortex suggest cell fate is determined at least partially by local environmental cues operating near the time when a cell is born. In contrast, lineage analysis in the brain stem suggests that classical developmental compartments, in which the clonally-related progeny are restricted to one rhombomere, may exist. The thalamus, which plays a central role in integrating the hindbrain and forebrain, appears to have developmental and anatomic features which overlap with both the hindbrain and the forebrain. To date, no studies have explored lineal relationships in the thalamus. Given the anatomic similarities to both regions, lineage analysis in the thalamus potentially could provide valuable insights into the strategies determining cell fate in the central nervous system. To determine lineal relationships in the thalamus, embryonic chick brains will be infected with a library of replication incompetent retroviruses modified by inserting the alkaline phosphatase gene or the E. coli lacZ gene and a unique molecular tag. Each brain will be serially sectioned and cells infected by the vector identified by using enzyme histochemistry for the transduced alkaline phosphatase or beta-galactosidase. Positive cells will be 'picked' from the slide and amplification of the unique molecular tag will be accomplished using the polymerase chain reaction (PCR). A statistical analysis will then be conducted to determine whether all cells with identical tags are siblings. This technique will establish the location of cell proliferation, migrational pathways, physical boundaries, and cell type within clones in the thalamus. Finally, we hope to investigate some of the environmental and genetic signals which determine cell fate in the thalamus. Results from this study could eventually lead to a better understanding of development in all parts of the CNS and insight into why certain nuclear groups and cell classes are affected in different diseases.

Epilepsy, mental retardation and structural anomalies of the brain together afflict 3-5 percent of all children, yet the underlying pathogeneses for these disorders is poorly understood in most cases. Cell migration is a central component of normal central nervous system (CNS) development and disruptions in this process have been implicated in the development of these disorders. Two primary patterns of cell migration are recognized during CNS development, radial and non-radial. While the cellular and molecular bases of radial cell migration, long considered the predominant mode of cell migration, have begun to be defined, the mechanisms of guidance for non-radial cell migration remain largely unexplored. Using lineage analysis, we have defined the developmental time and location where non-radial cell migration begins in the chick forebrain. Based on these data we have developed a model to explain the cellular and molecular mechanisms of non-radial cell migration. Our model is based on the hypotheses that cell surface molecules, secreted molecules, and extracellular matrix molecules guide non-radially migrating cells. In this proposal, we intend to develop in vitro assays that will allow us to test our hypothesis and identify new molecules participating in the guidance of non-radial cell migration. We anticipate this system will provide us with a more rapid means of identifying and testing molecules essential for cell migration and therefore normal brain development. We will then be in a position to test the contribution of non-radial cell migration, and these molecules in particular, in the pathogenesis of a variety of conditions that afflict children such as epilepsy, mental retardation and structural malformations of the brain. These data will enhance our understanding of CNS development and may ultimately lead to improvements in the diagnosis, management, and prevention of neurological diseases where abnormal cell migration has a pathogenetic role.

Cell migration is fundamental to normal central nervous system (CNS) development and perturbations in this process have been implicated in the pathogenesis of many neurologic disorders including epilepsy, mental retardation and autism. Understanding the pathogenesis of these all to common disorders requires, among other processes, the elucidation of the molecular and cellular mechanisms governing neuronal migration. Two primary pathways of cell migration are recognized during cerebral cortical development, radial and non-radial. At least in rodents, radial cell migration, from the pallial ventricular zone out to the cortical plate, gives rise to the projection neurons of the cerebral cortex, whereas cortical interneurons must travel along non-radial pathways from the subpallial proliferative zones to arrive in the cortical plate. Over the past seven years, supported by this grant, we have made significant contributions to our understanding of interneuron migration. Over the next five years we plan to extend these studies to more clearly define the mechanisms governing non-radial cell migration in mammals. One fundamental questions is how does the leading process respond to guidance cues to direct the migrating interneurons from the ganglionic eminence along their circuitous path and finally into the cerebral cortex. We have hypothesis that the leading process functions like a growth cone by dynamically regulating its cytoskeleton in response to long and short-range cues. Herein we propose to define the cytoskeletal dynamics in the migrating leading processes and establish how guidance cues and intracellular modulators regulate them. Together these data will establish the cellular mechanisms by which the leading process is able to lead the migrating interneuron, an integral component of normal brain development and one that is disrupted in many neurologic disorders.

[unreadable] DESCRIPTION (provided by applicant): The David W. Smith Annual Workshops on Malformations and Morphogenesis have been held for 24 years and bring together a mix of dysmorphologists, embryologists, anatomists, geneticists, and others who work on developing a better understanding of how human malformations occur and there underlying mechanisms of morphogenesis. The workshops are held annually and were initiated in celebration of the career of David W. Smith who was the father of human clinical dysmorphology and first initiated these meetings. Meetings are held over approximately five days typically on an academic campus in the summer and have an attendance of 120-140 individuals who are selected following submissions of abstracts and reviewed by the scientific program committee. The workshop is intended to be an interactive exchange and thus is limited only to those presenting that year. Approximately every five years the meetings have been held internationally but most meetings are in the United States or Canada. Each year three to five central topics are selected by the organizers; the selection of topics takes into account when the topic had last been a focus, timeliness based on new scientific developments, and new fields. As an example, this year will focus on brain development, craniofacial development, urogenital development, and epigenesis, additional sessions are devoted to more general issues of malformation and morphogenesis and based on the abstracts submitted that year. The meetings consist of a workshop format in which short presentations of hypothesis driven findings are then followed by an intense discussion period. The meetings have a long history of wide-ranging and stimulating discussion and have provided an especially important focus for the development and recruitment of fellows and junior faculty into the field of malformation and morphogenesis. This proposal is a request to provide continuing support for these meetings in the form of travel stipends for fellows, junior faculty, and a select number of plenary speakers. These meetings have had a history of continued success based on the dedication of the participants and the goal of this proposal is to provide additional stability with a particular emphasis on the involvement of new members of this research investigative community. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Developmental anomalies of the brain, including mental retardation and malformations, occur in approximately 2% of all live born children and epilepsy in about 0.5%, placing them among the most common known childhood disorders. Despite this high frequency, the molecular and cellular basis for only a very few disorders has been recently elucidated, while the basis of many more remains unknown. Mutations in the transcription factor ARX have been described in several children with early childhood epilepsy and mental retardation, both with and without associated brain malformations. As predicted in our first application, mutations of this gene prove to be a relatively common cause of mental retardation and infantile epilepsy based on the wide spectrum of severity already apparent in this group of children and a recurrent mechanism for mutation in at least two of the four polyalanine tracts found in the gene. The developmental mechanism by which ARX mutations result in this wide spectrum of problems is incompletely understood, although emerging data implicate disturbances in radial and nonradial cell migration, two pathways required for normal brain development. Based on data generated from this grant in humans and mice, the following hypotheses have been generated: (1) the type of ARX mutation predicts the phenotype in both hemizygous males and heterozygous females; (2) the phenotype in affected female humans and mice correlates with X-inactivation; and (3) Arx with an expanded poly-A tract results in defects in transcriptional repression, ultimate resulting in mice with seizures and mental retardation. To test these hypotheses, a series of experiments are proposed that will discover the mutation types in a large series of male and female patients with candidate phenotypes and determine X-inactivation status. In this application we will focus on elucidating the mechanism of a poly-A tract mutation in causing the neurologic phenotype, and in further understanding the downstream targets of Arx and their role in normal and abnormal brain development and function. The phenotypes in humans and mutant mice with different ARX/Arx mutations will be analyzed and compared to each other and to overlapping phenotypes caused by mutations of related genes. These studies are expected to provide a greater understanding of how Arx functions in normal and abnormal development, and will contribute to our understanding of the pathogenesis of such common disorders in children as mental retardation, epilepsy, and structural anomalies of the brain. PUBLIC HEALTH RELEVANCE: Epilepsy and mental retardation co-exist in many children and together extract a significant financial burden on the US health care dollar, an estimated $51.2 billion (in 2003 dollars). Although 3-5% of all children in the United States exhibit epilepsy and/or mental retardation, the underlying pathogeneses for these disorders is poorly understood in most cases. The data from our previous work and from that proposed in this application seeks to understand how one gene, ARX, commonly causes childhood epilepsy and mental retardation. Ultimately we expect these studies will lead to improvements in their diagnosis, treatment, and prevention of these and related neurologic disorders.

DESCRIPTION (provided by applicant): For the past 29 years the David W. Smith Workshop on Malformations and Morphogenesis has brought together a mix of dysmorphologists, embryologists, anatomists, geneticists, and others who work on developing a better understanding of how human malformations occur and the underlying mechanisms of morphogenesis. The workshops are held annually and were initiated in celebration of the career of Dr. David W. Smith, who was the father of human clinical dysmorphology and first initiated these meetings. Workshops are held over approximately five days, typically on an academic campus in the summer, and have an attendance of 120 to 140 individuals who are selected following submissions of abstracts and reviewed by the scientific program committee. The workshop is intended to provide an interactive exchange and thus is limited only to those presenting that year. Approximately every five years the meetings have been held internationally, but most meetings took place in the United States. Each year three to five central topics are selected by the organizers;which takes into account when the topic had last been a focus, timeliness based on new scientific developments, and new fields. The meetings consist of a workshop format in which short presentations of hypothesis-driven findings are then followed by an intense discussion period. The meetings have a long history of wide-ranging and stimulating discussion, and have provided an especially important focus for the development and recruitment of fellows as well as junior faculty into the field of malformation and morphogenesis. PUBLIC HEALTH RELEVANCE: This application is a request to provide continuing support for the David W. Smith Workshop in the form of travel stipends for fellows, junior faculty, and a select number of plenary speakers. This Workshop has a history for being the primary forum where clinicians and basic scientists exchange information on the pathogenesis of human malformations.

The initial (1999) application for this IDDRC included separate cores in Neuropathology and Neuroscience, with the latter offering expertise in cell culture. As user needs evolved, it became apparent that these facilities should be merged into a single Cellular Neuroscience Core. This was accomplished in 1994 with Dr. Pleasure as the Director. He served in this capacity unfil 2001, when leadership passed to Dr. Jeffrey Golden, a pediatric neuropathologist with a primary interest in developmental disorders. Dr. Golden first joined CHOP in 1997 and immediately became engaged with the IDDRC, both as a user and as an Associate Director of the Cellular Neuroscience Core. Since its inception this Core has provided users with a diverse repertoire of state-of-the-art methods for visualizaion of the distribufions of gene products in normal, developing neural cells and in those undergoing various forms of degeneration and regenerafion. We have confinually and eagerly added new skills, instrumentafion and reagents to better serve our users'needs. In 1995, with the generous assistance of the Children's Hospital, we purchased a Leica confocal microscope to which we added inverted microscopy, stagemounted micromanipulators/microinjectors, and a stage-mounted environmental chamber, thus permitting prolonged observation and manipulation of living cells under physiological conditions. In this manner we offered 8-color capacity fluorescent imaging. Our institution paid for the apparatus and we used IDDRC funding to partially support a technician. We made several other notable technological additions to the Core repertoire, including in situ hybridization in both sections and whole embryos. Our general purpose has been not only to make available a technology, but a consultative sen/ice that facilitates implementation of the method as well as the interpretation of data. We adhered to this policy when we also added video-enhanced microscopy in order to better support IDDRC investigators in analyzing intracellular Ca2+ and Na+ as well as the estimate of mitochondrial potential.

Neurodevelopmental disorders (intellectual disability, epilepsy, autism, attention deficit, etc.) are diagnosed in 1 in 6 children in the US according to the report from the Center for Disease Control and Prevention. Despite this high frequency, the molecular and cellular basis for only a few disorders has been elucidated, while the basis of most remains unknown. Interestingly, patients with mutations in ARX exhibit nearly all of these features. However, the developmental mechanism by which ARX mutations result in this wide spectrum of problems is incompletely understood. Our prior work has demonstrated that ARX is expressed in two different neural progenitor populations during forebrain development and that it plays distinct roles in each population by regulating different subsets of genes. Furthermore, we have shown that the loss of Arx from each progenitor population accounts for specific components of the mouse and human phenotypes. In this proposal, building on our data from the past ten years, we seek to understand how ARX regulates different subset of genes in different progenitor populations. The proposed studies will focus on identification of ARX-interacting transcription factors (or co-factors) and their down-stream target genes co-regulated by ARX in each cell population. The combination of proteomics and genomics approach as well as systematic network analysis will identify the key target genes predicted to play crucial roles in each progenitor specific, ARX-mediated, cellular functions that we previously defined. Finally, we will validate these predicted genes with in vivo functional analysis. These studies are expected to provide a greater understanding of how ARX functions in normal and abnormal brain development, and will contribute to our understanding of the pathogenesis of such common disorders in children as intellectual disabilities, epilepsy, autism, and structural anomalies of the brain.

PROJECT SUMMARY/ABSTRACT Nearly 1% of the US population suffers from epilepsy (prevalence 5-8.4/1000), with a slightly higher prevalence in children. Despite this high frequency, the molecular and cellular basis for only a few types of epilepsy have been defined, while the basis for most remains unknown. Mutations in one gene, ARX, are of considerable interest as distinct mutations are associated with a spectrum of neurological disorders with epilepsy representing one of the few consistent features. ARX has 4 poly-alanine (pAla) tracts and expansions in the 1st or 2nd tract are consistently associated with epilepsy. pAla tract expansion mutations are a relatively newly described mutation type and are associated with a growing number of human developmental disorders, epilepsy being a component of several. How this mutation type results in human disorders and epilepsy in particular are not well understood. Our prior work has demonstrated that an expansion in the first pAla tract of ARX results in structural change in the protein and the resulting protein has differential effects on developing cortical interneuron- and projection neuron progenitor cells. In other studies, we have shown that the loss of Arx from each progenitor population accounts for specific components of the mouse and human phenotypes. In this multi-PI R01 proposal, building on our data from the past ten years, we seek to unite human stem cell models with mouse models to elucidate the pathobiology underlying ARX related epilepsy, and specifically the function of pAla tracts along with mutations in these tracts. Aim 1 will evaluate the cellular impact of ARX pAla mutations in patient-derived spheroids. Aim 2 will examine the role of ARX pAla mutations on cortical interneuron migration and network activity. Aim 3 will determine the effects of Arx pAla expansion mutations on brain development and function. This project will utilize human induced pluripotent stem cell (hiPSC) and spheroid models and complement these with mouse embryonic stem cell lines and behavioral and physiological assays in mice. Together, these studies are expected to provide a greater understanding of how pAla tracts function in normal and abnormal brain development, contribute to our understanding of the pathogenesis of epilepsy, and generate valuable resources and mouse models to test potential therapeutic strategies for developmental epilepsies.