Denise A. Figlewicz - US grants

Amyotrophic lateral sclerosis (ALS) is an almost invariably lethal disorder which involves loss of the large motor neurons from the cerebral cortex, brain stem, and spinal cord. During the past three decades, mortality due to ALS has increased consistently. While most cases of ALS are sporadic, 5-10% of cases (which are clinically indistinguishable) are familial. Recently, we have identified the gene responsible for ALS in a subset of the familial cases: Cu/Zn superoxide dismutase (SOD-1), an enzyme which inactivates the highly reactive free radical superoxide anion. The aim of this study is to establish and characterize an in vitro model of ALS, in order to identify the mechanisms by which mutations in the SOD-1 gene lead to the death of motor neurons. We propose to study this by the preparation and characterization of motor neurons which are stably transduced with human SOD-1 cDNA containing one of eleven different mutations identified in our FALS patients. We aim to: 1) Prepare constructs with human SOD-1 cDNAs, both control and mutants. 2) Package mutant SOD-1 constructs into Adeno-Associated Virus (AAV) Vectors. AAV is a human DNA virus which offers a promising alternative to the viral vectors now in frequent use (adenovirus, Herpes, retroviruses). 3) Prepare cultures of rat motor neurons grown under defined conditions which will be characterized for survival, morphology, neurite outgrowth, and expression of neuronal markers. 4) Transduce cultured motor neurons with mutant-human SOD-1 using a replication deficient AAV vector. 5) Measure the activity of enzymes responsible for the detoxification of free-radical metabolites such as superoxide anion and hydrogen peroxide, in control and transduced neurons. And, 6) Use infected motor neurons to test the effectiveness of several therapeutic approaches: trophic factors such as NT-3, BDNF, or NT 4/5, that have motor neurons as targets; pharmacologic agents which are believed to counteract the effects of excessive free radicals; or genes, such as bcl-2, which have been shown to rescue cells from death. Therapeutic agents can be introduced directly into the culture medium (pharmaceutics, trophic factors); secreted by co-cultured, genetically engineered astrocytes (trophic factors); or introduced with the human SOD- 1 cDNA by transduction of a bicistronic AAV construct (trophic factors, cell survival genes). The long-term objective of this project is to identify putative therapeutic agents that will prolong the survival of diseased or injured motor neurons.

9728728 HOLLIDAY There is much evidence indicating that the development of undifferentiated cells into neurons is dependent both upon intrinsic patterns of gene expression as well as on interactions with neighboring cells, including electrical signaling. Early in neuronal differentiation, the elevation of intracellular calcium ions carries much of the electrical signal. Calcium elevation is one of the first steps in the activation of a large variety of cellular signal transduction cascades in all cell types and is therefore very likely to be involved in regulating gene expression and other "experience-dependent" mechanisms that regulate development. There is accumulating evidence that precisely timed changes in the cellular mechanisms that produce transient increases in intracellular calcium may underlie the "critical periods" of neuronal development. This project is designed to determine how the alteration of the level of expression of specific calcium ion regulatory proteins affects transient calcium signaling and subsequent neuronal development. Two calcium regulatory proteins will be separately investigated: a calcium buffering protein (calbindin) and an intracellular calcium induced calcium release channel (CICR). The level of these proteins will be experimentally manipulated by the introduction DNA constructs that either produce additional target protein or specifically reduce endogenous target protein expression. The experiments will be performed in a well- controlled and characterized culture system of rat cerebellar granule neurons. Since intracellular calcium homeostasis is tightly controlled by a variety of mechanisms, alteration of one target may trigger compensatory changes in other regulatory mechanisms. Therefore, the physiological consequences of changing protein expression will be measured with imaging techniques that directly measure intracellular calcium ion concentrations, both at rest and during stimulation. The developmental consequences of changes in target protein expression will be assessed by measuring the acquisition of several developmental markers. The results of these studies will refine our understanding of how calcium transients regulate neuronal development at the single cell level, and develop tools to more definitively test hypotheses about the mechanisms regulating neuronal synaptic organization. The long-range goal of this project is to transiently manipulate calcium regulatory mechanisms during development in vivo to determine whether perturbations of calcium levels, during sensitive defined in vitro, will result in developmental delays or permanent changes in the mature central nervous system.

Facioscapulohumeral muscular dystrophy (FSHD), one of the most common types of muscular dystrophy, is characterized by the development of progressive weakness in the face, proximal arm, and scapular stabilizer muscles. In more severe cases, the pelvic girdle may be involved leading to confinement in a wheelchair. FSHD is inherited as an autosomal dominant trait. The genetic lesion accounting for most cases is the deletion of integral copies of a tandemly repeated 3.3kb unit ("D4Z4 repeat") from chromosome 4q35. Previous studies have failed to identify a gene within the region of the D4Z4 repeats themselves, or distal to the D4Z4 repeat region. Therefore we hypothesize that the gene(s) involved in the development of FSHD lie proximal to the D4Z4 repeat region; the FSHD gene(s) most likely do not themselves contain any mutations, but will display aberrant expression levels in patients compared to their control counterpart(s). Thus, the overall objective of this project is to identify genes lying proximal to the D4Z4 repeat region of chromosome 4q35 and to test each gene for its possible involvement in the pathogenesis of FSHD. In addition, genes whose expression changes downstream of the primary FSHD gene(s) will be identified. Specifically, we aim to: (1) Acquire FSHD patient material--blood samples and muscle biopsy specimens-- for DNA analysis and for gene expression studies. (2) Characterize small-fragment phage libraries of expressed sequences which have been derived from chr. 4q35 yeast artificial chromosomes (YACs) using the direct selection protocol. (3) Characterize mRNAs which are uniquely expressed in FSHD patient skeletal muscle samples vs. control samples, identified via the technique of differential display or gene chip hybridization analysis. (4) Test all appropriate candidate gene fragments for expression in mRNA from control and FSHD skeletal muscle, and from cultured myoblasts of different stages which have been derived from these tissues, using competitive quantitative RT/PCR and Northern Blot techniques. (5) When expression and mapping studies suggest that a given candidate gene might be the FSHD gene, functional studies will be undertaken to characterize the gene, its expression, and the role of its protein product. Our long-term goals are: to identify the gene(s) involved in FSHD; to work closely with our FSHD patient population to characterize the spectrum of genetic effects in muscle biopsy and cultured myoblasts derived from each individual; and to correlate these changes with variations in phenotypic expression of the disease. The ultimate aim is to identify rational therapeutic strategies for FSHD.

DESCRIPTION (taken from the application): Fascioscapulohumeral muscular dystrophy (FSHD) is the third most common form of muscular dystrophy. Since the genetic locus has been linked to chromosome 4q35, clinical and genetic research in FSHD has advanced rapidly. Current evidence continues to support a unique genetic mechanism for FSHD. In April of 1997, the second international FSHD symposium was held in Boston; it proved to be a landmark opportunity for FSHD clinicians and research scientists to meet and more importantly to gain an updated comprehensive view of the disease. The present proposal is for a third international FSHD symposium to be held at the National Institutes of Health on May 8, 2000. The purpose of the conference is to once again bring together scientists in the field to summarize current knowledge, to facilitate collaboration, and to establish directions for research strategies.

DESCRIPTION (provided by applicant): Facioscapulohumeral dystrophy (FSHD) is the third most common form of inherited muscle disease, characterized by the development of progressive weakness in the face, proximal arm, and scapular stabilizer muscles. In more severe cases, the pelvic girdle may also be involved leading to confinement in a wheelchair. FSHD is inherited as an autosomal dominant trait. The mutation responsible for >95% of FSHD cases is known-a deletion of integral copies of a tandemly repeated 3.3kb element (D4Z4) on chromosome 4q35. However, the molecular and biochemical mechanisms of FSHD remain elusive. No gene has been identified within the D4Z4 repeats themselves, and the consensus is that the deletion likely affects the expression of genes proximal to the 3.3kb repeat array. Accordingly, multiple primary and secondary pathways are envisaged to be involved in the pathogenesis of FSHD. We have begun cell biology studies of FSHD myoblasts which display an aberrant phenotype in both the undifferentiated and differentiated states. As a result of these studies, we have determined that FSHD myoblasts are more susceptible to oxidative stress compared to myoblasts from control, and other muscle disease. FSHD myoblasts express p21, a cyclin kinase inhibitor and important intermediate in pathways related to intracellular stress, to a greater extent than controls. Their morphology and replicative capacity are reminiscent of senescence in control myoblasts. These are the first biochemical clues about cellular processes which are altered in FSHD. We now aim to: (1) Characterize the redox state of FSHD myoblasts and determine whether they respond to antioxidant agents. (2) Determine the relationship between p21 expression and two factors known to upregulate it-p53 and MyoD. (3) Establish whether FSHD myoblasts have undergone replicative senescence, by quantitation of telomere size and change of telomere size with replication. The overall aim of these experiments is a better understanding of the pathophysiology of FSHD. Moreover, the detailed characterization of the aberrant intracellular processes in FSHD myoblasts will be an important step for the development of intervention strategies based on muscle stem cell or myoblast transfer protocols, where maintenance of a healthy satellite cell population will be crucial to longterm therapeutic efficacy.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is an adult-onset lethal disorder characterized by degeneration of brain and spinal motor neurons. Approximately 10% of ALS cases are the direct result of genetic inheritance; a subset of these is the result of mutations in the Cu-Zn superoxide dismutase (SOD1) gene. For the remaining 90% of cases, the underlying pathogenic mechanisms remain unknown. It is hypothesized that genetic susceptibility combined with specific lifestyle elements including exposure to environmental toxins contribute to the development of sporadic amyotrophic lateral sclerosis. No treatment is currently available to significantly slow or halt the progress of this disease. Transgenic mice overexpressing mutant SOD1 exhibit an autosomal dominant adult onset degeneration of the motor system which bears a striking resemblance to amyotrophic lateral sclerosis, both clinically and pathologically. They provide an excellent opportunity to study the disease process prior to the age when overt symptoms appear, particularly because the phenotype follows a well-characterized and predictable course. The transgenic mice have been utilized for preclinical trials of a large number of putative therapeutic agents. However, these mice do not adequately reflect the heterogeneity both in genetic backgrounds and in environmental exposures of human ALS patients. Extensive phenotypic and genotypic diversity does exist between inbred strains of mice. We now propose to take advantage of this diversity to create models of ALS which represent a greater variation in phenotypic presentation. Specifically, we aim to: 1) Breed the G93A mutant SOD1 trait into 6 distinct genetic strains of mouse; 2) Measure clinical parameters including age at onset of symptoms, motor and sensory functions, and endstage disease; and 3) Examine markers of spinal motor neuron degeneration and glial activation/inflammatory processes at intervals prior to and following the appearance of symptoms. We expect that modifier genes expressed in the various inbred strains will alter the phenotype of the disease, yielding mice with different ages of onset, different disease courses, and at the cellular level, different degrees of involvement of inflammatory processes and pathways of motor neuron death. The overall objective of this R21 is the creation and characterization of models of ALS in which a known disease-causing gene is expressed in the presence of widely varying genetic backgrounds, thus providing a more realistic range of disease phenotypes for testing relative vulnerability to exposure to environmental toxins, and ultimately for identifying genes which contribute to specific vulnerability or resistance to deleterious environmental agents.