Major Scientific Accomplishments of Center Investigators in the following areas:
In the 1970’s Rakic, Sidman and co-workers in this Center defined the mechanisms of neuronal migration during development of the mammalian cerebral cortex (J. Comp. Neurol., 1972; Science, 1974; J. Comp. Neurol., 1973). Radial glial fibers were discovered and shown to be the major guides of neuronal migration. The research indicated that sequential generations of cells originating in the germinative ventricular zones migrated in waves along these fibers, and that ultimately migrating neurons generated earliest in development reside in the deepest layers in cerebral cortex and neurons generated later reside in progressively more superficial layers, resulting in a cortex that is generated in an “inside out” fashion. These observations set the stage for subsequent insights into the genesis of disorders of neuronal migration in the brains of individuals with mental retardation and developmental disabilities. Indeed, MRI studies now have shown that such disorders, largely invisible to CT scanning, are much more common than previously suspected and are associated not only with the major cognitive deficits of mental retardation but with a variety of more restricted forms of developmental disability. The groundbreaking work of Rakic and Sidman ultimately led to the search for the genes determining neuronal migration. Current work on the molecular basis of neuronal migration and how the disruption of this process leads to disorders of cognitive function is the focus of a number of current IDDRC investigators including Corfas, Segal, and Walsh.
The important observation of neuronal retrograde transport of horseradish peroxidase in the central nervous system by the LaVails in 1972 had a profound impact on neuroanatomy and neurobiology in the decades following the description of this phenomenon (Science, 1972). This fundamental observation has had widespread use as a tool to establish anatomically interconnected areas of brain, and provided the seminal insight that nerve cell processes can sample the chemical environment and transport materials great distances back to the cell body. Current IDDRC research that emanates from this historical work have identified signal transduction mechanisms that involve the rapid transport of ligand receptor complexes from nerve endings to the cell body, as demonstrated in the studies of Chen, Greenberg, Pomeroy, Segal, and Stiles.
In the early 1970’s, Cutler, Lorenzo, Barlow and their colleagues (Brain, 1968; Brain, 1970; Arch. Neurol., 1974) carried out fundamental studies of infants with hydrocephalus which provided much of the current knowledge of cerebrospinal fluid (CSF) production and absorption. By adapting the ventricular-lumbar perfusion technique to study of humans these investigators made a series of quantitative studies of CSF formation and absorption. The normal rate of CSF formation was measured and was found to be essentially unchanged in human hydrocephalus, except in choroid plexus papilloma where CSF formation was greatly increased. The absorption of CSF was shown to begin at a critical pressure. In normal individuals, a linear relationship between the rate of CSF absorption and increase in intracranial pressure was shown, and several patterns of deficient absorption were identified with hydrocephalus. This work serves in part as the foundation for current Children’s Hospital IDDRC studies of infantile hydrocephalus using exciting new techniques of near-infrared spectroscopy and diffusion-based magnetic resonance techniques.
The study of human chromosomes was pursued actively by Dr. Samuel Latt over the many years of his tenure in the Children’s Hospital IDDRC (Ann. Rev. Biophys. Bioengr., 1976; Ann. Rev. Gen., 1981; Banbury Report, Cold Spring Laboratory, 1983). Latt was initially recruited as a “new investigator” in the Center and before his untimely death had risen to become the Director of the IDDRC Genetics Program. From early work with DNA binding dyes, which stained different regions of human chromosomes, Latt developed methods that enabled him to follow the replication pattern of DNA within the chromosome. This interest in DNA binding dyes and newly developed means to detect these dyes within cells led to the application of these procedures to fluorescence-activated cell sorting to track the cell cycle more accurately by following the fluorescence intensity of newly-synthesized DNA. A natural extension of this work was the use of DNA binding dyes to separate each of the human chromosomes by DNA content and to prepare these chromosomes in sufficient quantities to make them available for manipulation of their contained DNA. This accomplishment enabled sufficient DNA to be obtained for the establishment of libraries of cloned DNA from each human chromosome. The research of the Genetics Program of this IDDRC then focused on a few specific chromosomes where important human disease genes were known to be located. This work was the model for the development of the governmental resource of libraries for all of the human chromosomes. Indeed, Latt served as an advisor to this project and was instrumental in its implementation. From the libraries, cloned segments could be obtained from specific chromosomes. These cloned segments were then used as a genetic marker for the region from which the segment was derived. These markers became instrumental in the identification of the gene for Duchenne dystrophy by Dr. Kunkel, and ultimately the identification of a large number of different disease genes. This work is being extended in many different ways in the current IDDRC.
Periventricular white matter injury is the most important neuropathological substrate for the subsequent neurological disability, i.e., cerebral palsy and cognitive deficits, observed in premature infants. In 1973 Leviton and Gilles began a classic series of neuroepidemiologic and neuropathologic studies linking bacteriemia and endotoxin to the cause of this type of injury (J. Neurol. Sci., 1973; Ann. Neurol., 1984; J. Neurol. Sci., 1976; The Developing Brain: Growth and Epidemiologic Neuropathology, 1983). Subsequent work showed that ischemia also is crucial in pathogenesis of this injury. The work of Leviton and Gilles led to the later discovery of a link between periventricular white matter injury of prematurity and maternal infection, a very important cause of prematurity. These neuroepidemiologic and neuropathological investigations have proved to be the foundation for current cellular and molecular studies of the role of cytokines, reactive oxygen and nitrogen species and glutamate in oligodendroglial cell death, carried out especially in cell culture and in animal models (Volpe, Rosenberg, Jensen, Follett, Kinney and Vartanian). Leviton now leads a major multi-center, NIH-funded grant, based in this IDDRC that addresses the clinical and epidemiological correlates of periventricular white matter injury identified in the living premature infant by cranial ultrasonography. Landmark work of Dr. Hannah Kinney has shown that PVL is characterized by evidence for oxidative and nitrosative stress and by a remarkable infiltration with activated microglia. This research interfaces with a considerable amount of work in the current IDDRC in both the Clinical/Translational Neuroscience Program (Kinney, Volpe, and Jensen) and the Basic Neuroscience Program (Rosenberg, Volpe, Jensen and Vartanian).
As an early effort at what might be regarded as “genetic engineering” in the mid 70’s, Richard Mullen of the Neuroscience Program of the IDDRC developed the skill of combining eight-cell-stage rodent embryos from genetically normal and abnormal individuals to produce surviving chimeras (Science, 1976; Nature, 1977). This approach was applied to the study of animals with neurological abnormalities, one with retinal degeneration and visual disturbance, and the other, with cerebellar Purkinje cell degeneration and clinical ataxia. In both instances, the clinical disorder was ameliorated in the chimera. The principal value of the experiment was the resulting ability to analyze the cellular abnormality.
An early effective effort at gene therapy evolved as a collaborative project between investigators of this IDDRC and the California Institute of Technology. Thus, Readhead et al. (Cell, 1987) described the first successful use of gene transfer to treat a neurological disorder, i.e., the Shiverer mouse, which has an inherited deficiency of myelin basic protein causing tremor and death. Gene transfer was accomplished by inserting the normal myelin basic protein gene into mouse embryos. Crossbreeding of mice with the incorporated gene with Shiverer mice was successful in introducing the myelin basic protein gene into the offspring. This gene repaired the myelin deficiency, and those animals who had two copies of the gene were asymptomatic, while those with one copy showed partial benefit. This work forms some of the roots of the existing studies in gene therapy using immortalized cells and viral vectors to correct both developmental deficits and neurogenetic and neuroendocrine disorders (Kunkel, Mulligan, Macklis, Geller and Snyder).
Several investigators of the IDDRC (Duffy, Snodgrass, Burchfiel, Nature, 1976) showed that the GABA antagonist, bicuculline, can reverse the inhibition of a significant percentage of the inactive nerve cells in the visual cortex in an experimental model of amblyopia. This study revealed that these inactive cells were undergoing active inhibition that could be reversed. The ability to reverse this inhibition provided avenues for developing therapies for treating amblyopia and possibly other neurologic disorders. These finding may be relevant to the understanding of “critical periods” of neuronal plasticity. Related studies of neurophysiological development and plasticity continue in the current IDDRC both in the Basic Neuroscience Program (Benowitz and Macklis) and in the Clinical/Translational Neuroscience Program (Als). The work of a newly recruited member of the Children’s Hospital Neurobiology Program, Dr. Takao Hensch, focuses on the cellular and molecular mechanisms that regulate critical periods in the visual cortex. As a future IDDRC investigator, we anticipate that Dr. Hensch will make substantial contributions to this research area in the future.
In the late 1970’s and early 1980’s the mutant mouse, Spastic, was studied to determine the origin of its neurological syndrome of tremors and abnormal righting behavior. An electrophysiological analysis suggested a deficit in inhibition at the level of the spinal cord, and treatment of normal mice with strychnine, which blocks glycinergic inhibition, caused these mice to mimic the behavior of the Spastic mice. Chemical studies then demonstrated that the Spastic mouse appears to have an absolute deficit in glycine receptors in the spinal cord and brainstem, despite normal receptor numbers for several other neurotransmitters. Thus, the work of White and Heller with this mutant was the first demonstration of a single-gene mutation causing a specific deficit in a central nervous system neurotransmitter receptor (Nature, 1982). It now appears likely that many neurological diseases without specific anatomic lesions reflect a similar pathophysiological mechanism. Hereditary transmission of certain epilepsies in the human has been known for many years. The discovery and characterization of a mouse model with a single recessive mutant gene on chromosome 8 by Noebels and Sidman provided the opportunity to study an inherited epilepsy in this animal, which in adolescence exhibits absence and clonic seizures (clinically and electrographically) (Science, 1979). Spontaneous electrographic and clinical seizures of this type previously had only been recognized in humans. This work defined the first example of an inherited epilepsy model with a single recessive gene on an identified chromosome with clinical and EEG features similar to those in inherited human epilepsy.
Additionally, Sidman, Rakic and co-workers utilized mutant models of cerebral and cerebellar cortical development, e.g., “Reeler” and “Weaver” mice, to elucidate fundamental mechanisms of neuronal migration. This approach laid the groundwork for the explosion in the use of mutant mice in neuroscience and genetics to elucidate fundamental mechanisms of neuronal development and later to define the molecular genetics of neurogenetic disorders. Work with related animal models is very widespread in the current IDDRC (Kunkel, Corfas, Greenberg, Walsh and Pomeroy).
Between approximately 1980 and 1990, major insights into the relationships between the sex chromosomes and behavior were established by interactions in the Children’s Hospital IDDRC between investigators in the Genetics Program and in the Clinical/Translational Neuroscience Program. Three major groups of patients were studied, i.e., XXY and XYY males, fragile X males, XO females. The behavioral profile in XXY and XYY males was established by studies of Dr. Stanley Walzer (Department of Psychiatry) and Dr. Gerald Parks (Division of Genetics, Department of Medicine) in this IDDRC in the late 1980’s (Birth Defects Original Article Series, 1990). The 47 XXY boys evidenced a continuum of language learning disability over many years. Deficits in language production and processing in the pre-school years were associated with severe and chronic reading and writing disabilities during the school years. The pattern of language deficits was relatively distinct for these boys. The temperamental style of XXY boys, also distinctive, was characterized by low activity levels, low energy content of responsiveness, high pliancy and withdrawal from novel situations and experiences. Interestingly, the boys with 47 XYY chromosomal abnormality evidenced a greater clinical diversity of communication deficits than those noted in the 47 XXY boys. Moreover, in contrast to the 47 XXY boys, the temperamental style of the 47 XYY boys was characterized by high activity level, high energy content of responsiveness, non-pliancy and distractibility. Dr. Peter Wolff and his colleagues found that mentally retarded males with the fragile site on the X chromosome exhibited a highly idiosyncratic but well coordinated stereotypic form of gaze avoidance during greeting ceremonies that involved the whole upper body (Am. J. Ment. Retard., 1989). A comparison group of persons with other etiologically defined syndromes of mental retardation did not exhibit this abnormality. Results suggested that the aberrant greeting behavior was uniquely associated with this defined genetic variety of retardation. Dr. Deborah Waber (Dev. Med. Child Neurol., 1979) studied in depth 11 patients with Turner syndrome (XO) and showed that affected patients performed less well than controls on word fluency, perception of left and right, visual-motor coordination, visual memory and motor learning.
These chromosomal-phenotypic correlations have been of particular importance in subsequent research focused on the molecular genetics of these disorders. Dr. Christopher Walsh has defined the importance of gene defects on the X-chromosome in the genesis of neuronal migration disorders (e.g., periventricular nodular heterotopia, X-linked lissencephaly/double cortex disorder).
Retinal disease that causes deficits of central and peripheral vision in infants and young children is a recognized accompaniment of several disorders in which mental retardation and developmental disability are also prominent and is an independent cause of developmental disturbances. Young age and limited abilities had interfered with quantitative assessment of visual acuity, retinal sensitivity and retinal adaptor processes in these patients. Dr. Ann Fulton developed procedures that depend on modifying the child’s looking behavior with visual stimuli and used these methods to begin evaluation of visual function in children with recognized retinal and brain disorders. Using these procedures and complementary electrophysiological techniques, Fulton also discovered retinal malfunction in several newly described groups of children with progressive neurological degeneration (Arch Ophthalmol 2002, Arch Ophthalmol 2005). The results indicated that retinal malfunction as well as central nervous system abnormalities compromises the visual abilities of these children. This work has been developed considerably over the past 10 years by Fulton in this IDDRC, particularly re: retinopathy of prematurity (Invest Opthalmolo Vis Sci 2002, OptomVis Sci 2005).
Beginning in the mid-1970’s, the IDDRC at Children’s Hospital became the site of a multidisciplinary longitudinal study of the adverse effects of concentrations of lead below previously accepted exposure levels. Drs. Alan Leviton and David Bellinger were the key figures in that work. The results indicated that lead has an adverse biological effect at much lower levels than those previously thought to be acceptable and that the adverse effects do not improve with time (Genetics Resource, 1988; Research in Infant Assessment, 1989; N. Engl. J. Med., 1990). These and other studies resulted in a national effort to reduce environmental lead, and over the years the lowering of blood lead levels in the population has been achieved. This work has led to expanded interest by Dr. Bellinger in the roles of exogenous environmental toxins as well as endogenous insults in the genesis of developmental disabilities. His recent work has focused on exposure to mercury in dental amalgams and in deleterious neurocognitive effects of low levels of arsenic and manganese.
Stimulated by the original observations of Mabry and his colleagues in the early 1960’s, Dr. Harvey Levy in this IDDRC had a central role in the study of the effect of untreated maternal PKU during pregnancy on the offspring (Transplacental Effects on Fetal Health, 1988). An international survey found that when mothers had classic PKU and their pregnancies were untreated, 92% of the offspring were mentally retarded. In a further followup study of 435,000 women screened in Massachusetts, Levy identified 22 with persistent hyperphenylalaninemia; two of these women had classic PKU and all four of their children were adversely affected. Of the remaining 20 with atypical PKU or non-PKU, mild hyperphenylalaninemia, it was found that in those with concentrations between 10 and 20 mg/ml, there was a roughly linear correlation with mental retardation. It was also learned that if dietary treatment is instituted prior to pregnancy, the offspring are normal, and that if treatment is instituted in the middle of the first trimester, mentation is preserved but there is a greater than expected incidence of cardiac malformations. An additional finding of great interest was the fact that the human placenta concentrates phenylalanine, and thereby umbilical cord blood concentrations are higher than maternal blood concentrations, thus exposing the fetus to a higher concentration during pregnancy. Dr. Susan Waisbren has pursued this work in the current IDDRC with great vigor and has delineated the importance of timing of intrauterine exposure to hyperphenylalaninemia in the pathogenesis of the adverse effects in offspring.
Observations reported in the mid-1980’s described the occurrence of cognitive impairments subsequent to prophylactic cranial radiation in children with leukemia. Waber and her collaborators in this IDDRC followed up on this initial observation (Dev. Med. Child Neurol., 1990). Cognitive function and physical growth were measured in 51 children who had been treated for acute lymphoblastic leukemia with the combination of chemotherapy, cranial radiation and intrathecal methotrexate and were continuously disease-free for 5 to 12 years. A comparison group of children treated for a solid tumor, Wilms tumor, also was studied. The rates of cognitive impairment and growth retardation were found to be clearly higher among the acute lymphoblastic leukemia group. Of particular significance was the finding that females were at greater risk for central nervous system toxicity from therapy than males. Both cognitive impairment and short stature were more prevalent among females. In addition, the younger the child at diagnosis and treatment, the greater the risk. These observations are important because the combined therapeutic approach to acute lymphoblastic leukemia has resulted in very high cure rates for this once fatal disease so that prevention of subsequent cognitive deficits in the survivors has become a critical future challenge. This work spawned a study of the sex- dependent relationship between chemotherapy and cognitive impairment in an animal model and led to an expanded investigation of neuro-oncological issues in our IDDRC, including recent work that describes stroke and other vascular disorders as late effects of cranial irradiation ( Pomeroy and Waber ).
The two major forms of brain injury in premature infants are periventricular leukomalacia (PVL) and periventricular hemorrhagic infarction (PHI), a form of severe germinal matrix/intraventricular hemorrhage (GMH-IVH). Research in this IDDRC from the early 1990’s to the present has addressed the pathogenesis of this injury in living infants and the mechanisms of cell death of the cellular target in white matter, the developing oligodendrocyte. The research was carried out primarily by Drs. Volpe, du Plessis and Rosenberg and represents a stunning example of how the IDDRC has fostered research that ranges all the way from the bedside to the laboratory bench (for key initial work see N. Engl. J. Med., 1991; J. Neurosci., 1993; Pediatrics, 1994; Ann. Neurol., 1995). The major results of this work were: application of near-infrared spectroscopy to the study in vivo of cerebral hemodynamics in the critically ill premature infant at high risk for the development of brain injury; demonstration of parallel changes in cerebral blood volume and arterial blood pressure, consistent with impaired cerebrovascular autoregulation, in the premature infant; and demonstration of a relation between impaired cerebrovascular autoregulation and subsequent occurrence of ultrasonographically identified PVL and GMH-IVH. In parallel with this clinical research, basic neuroscientific findings were: development of a system of primary cultures of differentiating oligodendroglia to model the cell that is the target in periventricular white matter injury of the premature infant.; discovery that glutamate is highly toxic to differentiating oligodendroglia in culture and that the toxicity is caused by activation of a glutamate-cystine exchange system, leading to cystine and thereby glutathione depletion, and ultimately to apoptotic death induced by oxidative stress; and demonstration that differentiating oligodendroglia are highly vulnerable to oxidative stress, in part because of the acquisition of iron during differentiation, and that the toxicity with oxidative stress is totally preventable by exposure to such clinically safe agents as vitamin E. This work has led to a remarkable confluence of basic and clinical research in this IDDRC. The research has involved the study of living infants by advanced near-infrared spectroscopic and magnetic resonance techniques, the characterization of postmortem human brain by modern immunocytochemical approaches, and studies of cellular and whole animal systems by sophisticated cellular and molecular biological approaches. The research has defined the molecular basis for the vulnerability to oxidative and nitrosative stress and the importance of both excitotoxic and inflammatory (microglia-mediated) injury in the white matter injury. Novel preventative therapies based on this research are currently under investigation (Volpe, Kinney, Rosenberg, Jensen and Vartanian).
Of the 30,000 infants born annually in the United States with congenital heart disease, more than one-third will require cardiac surgery in the neonatal period. Dramatic reductions in surgical mortality for such deep hypothermic cardiac surgery with cardiopulmonary bypass have been accompanied by the recognition that the survivors frequently experience adverse neurological sequelae, including cognitive deficits and other developmental disabilities. The majority of such brain injury appears to be attributable to operative events, particularly the cardiopulmonary support systems used to protect vital organs during cardiac repair. From the early 1990’s a major program in this IDDRC has focused on identifying the causes of cognitive impairment during cardiac surgery. The clinical research of Newburger, du Plessis and Bellinger began to focus on the relations between deep hypothermic circulatory arrest and the subsequent functional deficits (for key initial work see Pediatrics, 1991; N. Engl. J. Med., 1993; J. Thorac. Cardiovasc. Surg., 1993; N. Engl. J. Med., 1995; Ann. Neurol., 1995). The principal findings were: 1) the demonstration that deep hypothermic cardiac surgery using a predominantly total circulatory arrest strategy is associated with a greater risk of acute neurological complications, delayed motor development and neurological abnormalities than is surgery using a predominantly low-flow cardiopulmonary bypass strategy; and 2) the discovery, in an intraoperative study of intravascular and cellular oxygenation by near-infrared spectroscopy, of a paradoxical dissociation of changes in intravascular and mitochondrial oxygenation during hypothermic cardiopulmonary bypass, suggesting a previously unexpected impairment of mitochondrial function or of delivery of oxygen to the mitochondrion. Later work has demonstrated that the deleterious effect of circulatory arrest is nonlinear, with relatively little impact after shorter duration and steadily worsening outcomes after longer durations. The results have led to major and beneficial changes in cardiac surgery in infants. (Newburger and Bellinger).
Medulloblastomas and other embryonal brain tumors, the most common malignant brain tumors of childhood, have a 40-50% overall mortality. Survivors typically live with profound developmental and neurological disabilities, largely due to toxic therapies currently in use. In 1994, the Pomeroy lab was the first to show that a molecular marker, the neurotrophin-3 receptor TrkC, can predict overall survival of embryonal tumors with significantly greater accuracy than clinical or histological features. The group, then, was the first to apply genomic methods to show that favorable prognosis tumors have substantially different gene expression profiles than poor prognosis tumors, identifying a molecular signature that can distinguish tumors with different biological properties but identical histological features. Outcome prediction models developed by the Pomeroy lab based on gene expression profiles are by far the most accurate predictors of embryonal tumor outcome currently available. They are being tested at a national level in Children’s Oncology Group clinical trials focused on improving cognitive outcome by lowering radiation doses in good prognosis patients.
This research, which developed most actively in the mid-1980’s and continues vigorously to the present, has been led in a most remarkable way by Dr. Louis Kunkel, Director of the Genetics Program and Associate Director of this IDDRC. Duchenne muscular dystrophy is a severe X-linked dystrophic myopathy; Becker muscular dystrophy is a phenotypically less severe disorder. In Duchenne muscular dystrophy a cognitive disturbance is present in all patients, such that as a group, IQ is distributed in a bell-shaped fashion but with a distinct shift of the curve to the left. The accomplishments of Kunkel and co-workers in the years leading to the 1995 renewal of this IDDRC were of profound importance (Adv. Hum. Gen., 1988; N. Engl. J. Med., 1988; Neuron, 1989; Nature, 1989; Nature, 1990; Am. J. Hum. Gen., 1991; J. Cell Biol., 1992; Nature Genetics, 1993; J. Cell Biol., 1995). Of particular note, Kunkel and his colleagues were the first to use restriction length polymorphisms to clone a disease gene. This work represents a landmark in the field of human genetics and sparked a revolution in genetics that has led to the positional cloning of dozens of human disease genes. The accomplishments of Kunkel and his colleagues that are relevant to muscular dystrophy include: cloning of the gene for Duchenne/Becker muscular dystrophy, discovery and characterization of the gene product dystrophin, demonstration that this protein is a cytoskeletal component in muscle, utilization of molecular analysis of specific defects in the dystrophin gene to establish molecular-phenotypic correlations in Duchenne muscular dystrophy, correction of the dystrophin defect in the MDX mouse by myoblast transfer, demonstration of dystrophin in postsynaptic elements of cerebral cortical neurons, and discovery of dystrophin-associated proteins. This groundbreaking work has provoked worldwide research which has focused on possible approaches to gene therapy in this devastating disease. The work continues in depth in new directions in this IDDRC.
In 1984 and 1985, Kurnit and coworkers reported that Down’s syndrome cells from developing lungs and hearts are more adhesive to each other than are the cells of normal controls (Am. J. Med. Gen., 1985; Proc. Natl. Acad. Sci., 1984). They also showed by computer simulations how such increased adhesiveness could result in the congenital lung and heart defects that are characteristically seen in Down’s syndrome. Subsequent work in this IDDRC has discovered families of cell adhesion proteins important in neural development. An important relationship between Alzheimer’s disease and Down’s syndrome was established with the demonstrations that patients with Down’s syndrome suffer a progressive dementing illness in adult life and that the pathological characteristics of this illness are similar to those that occur in Alzheimer’s disease. The core of the characteristic pathological change is the formation of plaques composed of the Beta-amyloid peptide. This similarity between Down syndrome and Alzheimer’s disease focused attention on chromosome 21. Dr. Rachel Neve et al., formerly in the Genetics Program of this IDDRC, reported the isolation and location of the Beta-amyloid gene on chromosome 21 in a paper in Science in 1987. In later experiments, reported in Science in 1989, Dr. Bruce Yankner of this Center showed that a fragment of the amyloid precursor protein (Beta?-amyloid) is toxic to neurons grown in tissue culture. This polypeptide appears to have a major role in the pathogenesis of the Alzheimer’s disease process. Dr. Yankner subsequently discovered that the fibrillar form of Beta-amyloid, as found in mature plaques in Alzheimer’s disease and in Down’s syndrome, and not the amorphous form is neurotoxic, and that microinjection of the fibrillar form into monkey brain results in Alzheimer-type neuronal injury. Dr. Yankner’s research then delineated the mechanism of amyloid toxicity and has raised the possibility of a rational therapeutic approach to therapy. In recent years Dr. Yankner has amplified these observations, establishing a crucial link between Beta?-amyloid neurotoxicity and the toxicity of the related compound amylin to the Beta-cells of pancreatic islets and therefore diabetes, and has extended his studies in important ways to provide insight into the dementia of Down syndrome.
The important role of glutamate as the mediator of neuronal cell death by receptor-mediated mechanisms that involve the accumulation of cytosolic calcium was established primarily in the 1980’s by work in several laboratories, including those of Drs. Stuart Lipton and Paul Rosenberg in this IDDRC. In 1989, Rosenberg demonstrated the crucial role of astrocytes in the modulation of neuronal vulnerability to glutamate. Thus Rosenberg demonstrated a 100-fold increase in neuronal vulnerability to glutamate when neurons were grown in culture in the absence of astrocytes (Neurosci. Lett., 1989). Rosenberg was able to define the intrinsic vulnerability of neurons to glutamate and demonstrate that the effective toxic concentration, i.e., approximately 5 MM, is significantly lower than previously documented by a large number of studies of glutamate toxicity using conventional astrocyte-rich cultures. The protection afforded by astrocytes was shown to be due to their avid glutamate uptake transport systems. These observations were important because they suggested that disturbances in glutamate transport by astrocytes could accentuate neuronal excitotoxicity under various circumstances. Subsequent research by Dr. Rosenberg has provided substantial support for this hypothesis.
A series of studies led by Dr. Marc Lalande, formerly of this IDDRC, delineated the regions on chromosome 15q11-13 that are mutated in Angelman syndrome (AS) and Prader-Willi syndrome (PWS). Lalande’s research also identified candidate genes in these regions and provided important insights into how an abnormality in genetic imprinting can lead to mental retardation (Am. J. Hum. Genet., 1991; Genomics, 1991; Lancet, 1992; Nature Genetics, 1992; Hum. Molec. Genet., 1993; Am. J. Med. Gen., 1993; Nature Genetics, 1994; Am. J. Med. Gen., 1994). The majority of the AS and PWS patients display a cytogenetic deletion of chromosome 15q11-q13. The deletions of 15q11-q13 in AS occur exclusively on the chromosome inherited from the mother whereas the deleted chromosome 15 in PWS is always of paternal origin. This marked difference in the parental origin of the deletions is consistent with the hypothesis that imprinting is involved in the etiology of AS and PWS. The occurrence of uniparental disomy in some patients with no cytogenetic deletion further supports the hypothesis that chromosome 15q11-q13 is an imprinted region. A cluster of genes encoding ?-aminobutyric acid (GABAA) receptor subunits were mapped to this region and thought to play a role in the genesis of the epilepsy characteristic of this disorder. This work has continued in our IDDRC and led to the identification of the principal gene affected in AS, the ubiquitin ligase ube3a. Current work in the IDDRC by Greenberg and his colleagues has shown that ube3a functions to regulate synapse number during development.
In the last decade investigators in this IDDRC have made exceptional use of human genetics to identify genes that cause neurological disorders. Dr. Christopher Walsh has been a pioneer in identifying genes that control the development and function of the human cerebral cortex whose mutation can cause autism and epilepsy as well as mental retardation and other learning disorders. Using human genetics to study Middle-Eastern families with a high incidence of disorders of cognitive function Dr. Walsh and his colleagues are making rapid progress in identifying new genes that control the development of human cognition. These genes include those whose mutation causes Joubert syndrome, bilateral frontoparietal polymicrogyria, periventricular nodular heterotopia, microcephaly with periventriuclar heterotopia, and double cortin syndrome/X linked lissencephaly to name a few. The identification of each of these genes have provided new insight into mechanisms that regulate human cognitive function, and new knowledge of the pathways that control such processes as cerebral cortical size, neuronal migration, and patterning of the human cortex. Another IDDRC investigator, Dr. Mark Keating has been at the forefront of efforts to use human genetics to understand the biochemical basis of common cardiovascular diseases. This work has led to the identification of mutations in six different ion channel genes that lead to cardiac arrhythmias in humans. Dr. Keating has recently discovered that mutations in an L-type voltage sensitive calcium channel gene are the cause of Timothy syndrome a human disorder that results in significant mental retardation and autistic behavior. Another investigator in our IDDRC, Dr. Elizabeth Engle has combined clinical, genetic, and molecular biological approaches to study congenital strabismus. Dr. Engle has defined several new strabismus syndromes and identified the genes mutated in five of these disorders. The cloning of these genes has revealed that familial forms of strabismus often result from mutations in genes expressed early in gestation that are necessary for the development and connectivity of neurons in the brainstem that normally control the eye muscles.
Morphogenesis, the process of cellular organization, determines the form and function of the organs. A relatively few cellular behaviors are responsible for all of morphogenesis: these include changes in cell shape, motility, adhesion, proliferation and differentiation. These changes are initiated, controlled and integrated by effectors in a cell’s microenvironment, especially extracellular matrix components, growth factors, cell surface proteins, proteases and anti-proteases. Cell surface receptors mediate the action of these effectors and mutations in these molecules and their receptors can cause abnormalities in morphogenesis. The CNS is the first organ system to undergo morphogenesis during embryogenesis. A series of landmark studies by the late Dr. Merton Bernfield in this IDDRC focused on the role of the syndecans, a family of heparan sulfate proteoglycans, and on the curly-tail mouse, a mouse model of human neural tube defects in morphogenesis (for key initial reports see Develop. Biol., 1991; Development, 1991; Genomics, 1991; J. Biol. Chem., 1993; PNAS, 1994; J. Biol. Chem., 1994; Nature Genomics, 1994).
The major findings of Dr. Bernfield and his colleagues have included: delineation of syndecans as a family of heparan sulfate proteoglycans crucial for “binding together” of components of the cellular microenvironment with the cytoskeleton; definition of syndecan as a “coreceptor” for bFGF and fibronectin and crucial for the action of such external effectors in CNS morphogenesis; determination of the importance of syndecans in reparative as well as developmental events; development of mouse models, e.g., the curly-tail mutant, that mimics human neural tube defects; and definition of the functional and biochemical disturbance in the curly-tail mutant and mapping of the defective gene.
In the 1980s Dr. Greenberg and his colleagues discovered that neuronal activity induces a genetic program that plays a key role in mediating brain development and function (Science 1986). Recent evidence from the laboratory indicates that mutations in components of the signaling network that regulates this gene program can lead to profound disruptions of cognitive function resulting in mental retardation and possibly autism. Over two decades, and under the auspices of the IDDRC since 1994, this laboratory has studied the activity-regulated gene program in considerable detail. Greenberg and his colleagues identified a signaling network that conveys a calcium signal from cell surface calcium channels to the nucleus, where the modification of transcriptional complexes triggers the induction of new gene expression. This characterization of activity-regulated genes has provided insight into an important new function of calcium channels, revealed how calcium signals are conveyed from the plasma membrane to the nucleus, and elucidated a number of the functions of activity-regulated genes (Science 1994, 1999, Cell 2000). Additional accomplishments include the discovery of the role that the transcription factor CREB plays in mediating calcium signaling in the brain and the purification and characterization of CREB kinases that convey the calcium signal to the nucleus (Science 1996).
Greenberg and his colleagues have also contributed significantly to the understanding of the function of this activity-dependent gene program. Through the development of knockout mice that lack specific Fos family members, they demonstrated that activity-regulated genes mediate adaptive neuronal responses that underlie animal behavior – in particular circadian entrainment the response of animals to drugs of abuse, and maternal nurturing. This work established that the activity- dependent gene program is essential for adaptive neuronal responses and provided the first evidence that maternal nurturing responses are genetically encoded. Other related studies provided some of the first mechanistic understanding of how synaptic activity promotes neuronal survival during development.
A critical early contribution of Greenberg and his colleagues was the development of phosphorylation site-specific antibodies to CREB. With these phospho-CREB specific antibodies, they were able to show that synaptic activity within the brain triggers CREB activation. This provided the first evidence that the signal transduction pathways that had been implicated in the induction of activity-regulated genes in cell culture were also activated by neuronal activity in live animals. Perhaps more importantly, this study was among the first to demonstrate the utility of phospho-specific antibodies for studying signal transduction pathways within cells and organisms. Subsequently, the lab has generalized the use of phospho-specific antibodies to over 50 different neuronal signaling proteins. By making these reagents and methods available to hundreds of investigators in the field, these investigators have helped to define neural circuits that mediate a wide range of behavioral responses (e.g. sleep, feeding behavior, circadian rhythms, responses to drugs of abuse).
Wnts are secreted proteins that play critical roles in mammalian brain development and function. During embryogenesis, Wnt genes not only control brain patterning and neuronal progenitor proliferation and differentiation, but also regulate axon pathfinding and synapse formation. Wnt genes also govern the proliferation of adult neural stem cells. Dr. Xi He’s laboratory in this IDDRC has made groundbreaking progress in charactering the mechanisms of Wnt signal transduction. Among their discoveries are: (1) the demonstration that the LDL receptor-related proteins, LRP5 and LRP6, are co-receptors for the canonical Wnt/beta-catenin signaling pathway and (2) the finding that the mammalian head inducer Dkk1 is a ligand for LRP5 and LRP6, and that Dkk1 inhibits Wnt signaling via disrupting Wnt-induced Fz-LRP6 complex formation. These and numerous other findings of the He laboratory have had a major impact on our understanding of the mechanisms of Wnt signaling and the role of these important molecules in nervous system development.
During the development of the nervous system there is a critical balance between the survival and death of developing neurons. Neurons that form the proper connections received trophic support from their targets and survive whereas neurons that fail to compete effectively for target derived neurotrophic factors die by a process of programmed cell death termed apoptosis. Research over the last decade had defined the molecular underpinnings of the apoptotic process, but the molecular mechanisms by which target derived neurotrophic factors suppress apoptosis and promote survival were unknown. Investigators in this IDDRC have made outstanding contributions to the understanding of the mechanisms by which neurotrophic factors promote survival in the developing nervous system. These include elucidation of the signaling pathways by which neurotrophins regulate gene transcription, and establishing the importance of CREB as a mediator of neurotrophin-dependent neuronal survival (Cell 1994, Science1996). In addition, it was discovered by investigators in our IDDRC that a primary function of the serine/threonine kinase Akt is to mediate cell survival (Science 1997). This seminal finding opened the flood gates for a huge number of studies implicating Akt in the process of cell survival. Following up on this finding, Children’s Hospital IDDRC investigators identified the first Akt substrates, the Bcl2 family member BAD (Cell 1997), and the forkhead transcription factor FOXO3a (Cell 1999; Science 2004), that mediate Akt-dependent survival. A series of elegant studies established the mechanism by which Akt phosphorylation regulates the activity of BAD and FOXO3a to promote cell survival. These studies provided the first mechanistic insight into how growth factors promote cell survival, identified possible mechanisms to explain the etiology of a variety of neurodegenerative disorders, and have suggested possible therapies for treating these neurodenerative disorders. The papers describing these discoveries stand out as landmarks in the field and are among most highly cited papers in biology in the last five years.
Damage to the developing or mature nervous system can be devastating due to the limiting ability of central nervous system axons to regenerate if they are severed. Two laboratories in the IDDRC (Benowitz and Z. He) have made outstanding contributions to the understanding of the molecular basis of the barriers to axon re-growth after injury and are moving toward the development of potential therapies for promoting the effective re-growth of axons to restore function in the injured nervous system. A focus of this research has been to identify the components in myelin that are inhibitory to re-growth. Work by Zhigang He and his colleagues have identified a new component of myelin that inhibits axon growth and has identified a receptor complex on neurons that mediates the inhibition of axon growth by several myelin proteins. The Benowitz and He laboratories have also each identified small compounds that can reverse the inhibition of axon growth and are therefore candidate therapies for treating spinal cord injuries as well as neurodegenerative disorders that result in substantial neuronal loss (Science 2005).
Beginning in 1991, a major new area of research in this IDDRC has been the study of neural progenitor or “stem cells.” This research has included landmark studies of cell fate determination (i.e., identification of external factors and instrinsic factors that regulate the differentiation of neural progenitor cells into neurons, astrocytes and oligodendrocytes). Dr. Greenberg and his colleagues demonstrated that cilliary neurotrophic factor acting through the Jak/STAT pathway plays a key role in the generation of astrocytes (see Science 1997). Drs. Stiles and Rowitch have carried out seminal work leading to the discovery of a family of transcription factors (Neuron 2000, Cell 2002), the Oligs, that specify the differentiation of neural progenitors into oligodendrocytes. Additional work by Greenberg and his colleagues identified a mechanism by which another transcription factor, neurogenin, supresses the glial fate and promotes neuronal differentiation (Sun et al., Cell 2001). In addition to these fundamental insights into the mechanisms regulating cell fate determination, IDDRC investigators have been pioneers in efforts to harness the potential of neural progenitors for repairing thedamaged nervous system. The work has been pursued principally by Drs. Snyder and Macklis (for key initial reports see Cell, 1992; J. Neurosci., 1993; Nature, 1995; PNAS, 1995; Nature, 1995). The potential impact of this research on prevention and treatment of serious neurological disability could be enormous. The initial work utilized a neuron-specific model of cortical neuron depletion, developed by Dr. Macklis, to study development of transplanted, genetically engineered neural progenitors. Remarkably the transplanted cells differentiated and integrated into the lesioned cortex and sent distant appropriate intercortical projections across the corpus callosum. Dr. Snyder’s work showed that the immortalized donor progenitors, upon transplantation, can engraft throughout the neuraxis and develop into “functional” neurons, oligodendroglia and astrocytes. Dr. Snyder then used neural progenitor transplantation to deliver sustained therapeutic levels of the missing gene product ?-glucuronidase directly to and throughout the brain of the Mucopolysaccharidosis Type VII mouse. Enzyme expression by these donor cells as integral brain components resulted in widespread correction of CNS neuropathology in this model of a human metabolic disease that causes mental retardation and is representative of a class of neurogenetic diseases whose CNS manifestations have heretofore been unresponsive to therapy. Dr. Snyder’s subsequent work has extended this exciting therapy to other neurogenetic disorders. This work in the IDDRC Basic Neuroscience Program was followed by parallel development of the use of stem cells in the treatment of muscular dystrophy by Dr. Kunkel in the Genetics Program (Kunkel, Mulligan and Gussoni).
