Kenneth H. Fischbeck - US grants

freeze etching; muscular dystrophy; membrane structure; cell membrane; muscle cells; erythrocytes; tissue /cell culture; electron microscopy; human tissue; stainings;

A first step towards the goal of the Human Genome Initiative is to generate clones that contain overlapping inserts covering a region of interest. This proposal is aimed at generating a complete, overlapping set of human DNA cloned in yeast artificial chromosomes (YACs) covering Xcen to Xq21.3 by starting with a set of markers that are ordered physically and genetically and filling in between the markers with contiguous, overlapping YAC clones (contigs). The specific aims are: 1. Construction of a YAC library with 300 kb inserts from the human X chromosome. 2. Isolation of three types of cloned DNA from Xcen-Xq21.3 to serve as the starting points for building YAC contigs: (a) Genomic DNAs containing exons; (b) HpaII tiny fragment linking clones containing NotI restriction endonuclease sites; and (c) polymorphic genomic sequences consisting of variable number poly(TG)n. These markers will join 17 markers already available in this region whose order relative to each other is indisputably known. 3. Acquisition of sequence information within each marker clone to allow it to serve as a "sequence tagged site" or STS. 4. Mapping of these Xcen-q21.3 STS markers. Physical mapping will be accomplished by (a) a panel of breakpoints in cell lines with X;autosome translocations and interstitial deletions; (b) long-range restriction maps generated by pulsed field gel electrophoresis. 5. Confirmation of physical order of polymorphic STS markers using multipoint linkage analysis of previously documented phase-known recombinant X chromosomes. 6. Isolation of contiguous YAC clones anchored at each STS marker. Probes prepared from the ends of these anchoring YACs will be used to isolate additional YACs to build sets of contigs that extend out from each STS marker until overlap is found between neighboring contigs. YAC contigs spanning Xcen-q21.3 will provide the raw material for complete sequencing of the region. The strategy of anchoring contigs at exons and HTF islands targets DNA segments that are likely to contain genes and therefore to have biological and medical importance.

Effective treatment for Duchenne muscular dystrophy depends on replacement of dystrophin in skeletal muscle (and other tissues) with protein of sufficient quality and quantity to prevent the disease manifestations. The objective of this project is to test dystrophin replacement in transgenic mdx mice, where problems of vector capacity, immunogenicity, and delivery are obviated by germline transmission of the dystrophin transgene. We plan to use this system to determine the minimum expression levels and the minimum structural requirements of the dystrophin protein needed to correct the dystrophic phenotype. We will produce transgenic mice with full length dystsrophin cDNA under the control of a series of promoters designed to give expression either systemically or specifically in skeletal and cardiac muscle or the central nervous system. We also will produce transgenic mice with different versions of the dystrophin cDNA, including "minidystrophin" and other constructs with different deletions of the central rod domain. Dystrophin expression will determined in the transgenic mice by immunohistochemistry and western blot analysis. The transgenic mice will then be bred with mdx mice, and the effects of the dystrophin transgene on muscle histochemistry and stretch-induced muscle injury will be assessed. We expect that this project will identify the best dystrophin constructs for use in experimental gene therapy in mdx mice, and eventually in patients with Duchenne muscular dystrophy.

X-linked spinal and bulbar muscular atrophy (SBMA), a form of motor neuron disease, is one of a growing list of disorders caused by expanded trinucleotide repeats. The mutation in SBMA is enlargement of a CAG repeat in the first exon of the androgen receptor gene. This CAG repeat encodes a polyglutamine tract near the amino end of the receptor protein, which is similar to repeats found in other proteins involved in control of transcription and development. A very similar repeat alteration has recently been found in Huntington's disease. Enlargement of CAG/polyglutamine tracts may thus be an important cause of neurodegenerative disease. We plan to characterize the causal connection between the androgen receptor mutations and the motor neuron degeneration of SBMA by studying the effects of the mutant androgen receptor in cultured neurons and transgenic mice. Constructs with normal and expanded versions of the androgen receptor will be assayed for neurotoxicity in vitro and in vivo. Since the normal function of the androgen receptor protein is as a transcription factor, and the disease is likely caused by a toxic gain of function of the receptor protein, the probable mechanism of neurotoxicity is through altered transcriptional regulation of one or more target genes. We plan to identify target genes that are aberrantly regulated by the expanded androgen receptor of SBMA and to look for specific effects of the altered receptor on genes known to play a role in motor neuron survival. We expect that our results will increase understanding of the cell biology of motor neurons and steroid hormone receptors. This project should also elucidate the pathogenesis of SBMA and may lead to effective treatment for this and other hereditary degenerative neurological disorders.

0-11 years old; 1,3-Naphthalenedisulfonic acid, 6,6'-((3,3'-dimethyl(1,1'-biphenyl)-4,4'-diyl)bis(azo))bis(4-amino-5-hydroxy)-, tetrasodium salt; Adrenal Cortex Hormones; Adverse effects; Affect; Antiproteases; Area; Azovan Blue; Biopsy; Bowman-Birk inhibitor; Cell Nucleus; Child; Child Youth; Children (0-21); Clinical Research; Clinical Study; Clinical Trials, Other; Coloring Agents; Corticoids; Corticosteroids; Disease; Disorder; Dose; Duchene; Duchenne; Duchenne de Boulogne muscular dystrophy; Duchenne disease; Duchenne dystrophy; Duchenne muscular dystrophy; Duchenne muscular dystrophy (DMD); Duchenne myodystrophy; Duchenne pseudohypertrophic muscular dystrophy; Duchenne syndrome; Duchenne-Griesinger syndrome; Dyes; Dystrophin; ENPT; Ellis-van Creveld (EvC) syndrome; End Point; EndPointCode; Endopeptidase Inhibitors; Endopeptidases; Endpoints; Enzymes; Esteroproteases; Evans Blue; Evans blue stain; Fibrosis; Human, Child; Inbred mdx Mice; Injury; Investigation; Laboratories; Lipids; Measures; Mice, Inbred mdx; Mouse, mdx; Muscle; Muscle Fibers; Muscle Tissue; Muscle Weakness; Muscular Dystrophies; Muscular Dystrophy, Duchenne; Muscular Dystrophy, Pseudohypertrophic; Muscular Weakness; Myodystrophica; Myodystrophy; Myotubes; Nucleus; Patients; Peptidase Inhibitors; Peptidases; Peptide Hydrolase Inhibitors; Peptide Hydrolases; Peptide Peptidohydrolase Inhibitors; Peptide Peptidohydrolases; Phase; Play; Progressive Muscular Dystrophy, Duchenne Type; Protease Antagonists; Protease Inhibitor; Proteases; Proteinase Inhibitors; Proteinases; Proteolytic Enzyme; Proteolytic Enzymes; Pseudohypertrophic Muscular Dystrophy, Childhood; Reporting; Research; Rhabdomyocyte; Role; Skeletal Fiber; Skeletal Muscle Cell; Skeletal Muscle Fiber; Skeletal Myocytes; Testing; Therapeutic Agents; Therapeutic Corticosteroid; Toxic effect; Toxicities; Treatment Side Effects; X-linked dilated cardiomyopathy; X-linked dilated cardiomyopathy (XLCM); X-linked muscular dystrophy; X-linked recessive muscular dystrophy; base; benign X-linked recessive muscular dystrophy; childhood pseudohypertrophic muscular dystrophy; children; classic X-linked recessive muscular dystrophy; disease/disorder; mild X-linked recessive muscular dystrophy; muscle necrosis; muscle strength; myostatin; new therapeutics; next generation therapeutics; novel therapeutics; placebo controlled study; placebo controlled trial; progressive muscular dystrophy of childhood; protease; proteinase; pseudohypertrophic adult muscular dystrophy; pseudohypertrophic muscular paralysis; pseudohypertrophic progressive muscular dystrophy, Duchenne type; restoration; side effect; social role; therapy adverse effect; treatment adverse effect; uptake; youngster

The purpose of this research program is to develop safe and effective treatments for hereditary neurological disorders. Specific research accomplishments in the past year include the following: (1) completion of a phase 2 study of high dose idebenone treatment for Friedreich's ataxia, and (3) implementation of a phase 2 clinical trial to test the feasibility of dutasteride treatment for spinal and bulbar muscular atrophy (Kennedy's disease). The idebenone trial showed evidence of neurological efficacy with intermediate and high dose treatment in ambulatory subjects. Analysis of other outcome measures is in progress.

The purpose of this research program is to investigate the causes of hereditary neurological diseases, with the goal of developing effective treatments for these disorders. A genetic outreach program allows the identification and characterization of patients and families with hereditary neurological diseases. Specific research accomplishments in the past two years include (1) the evaluation of a candidate gene for familial autoimmune myasthenia gravis, (2) Charcot-Marie-Tooth disease type 2C (CMT2C), and (3) a new form of hereditary spastic paraplegia mapped to chromosome 19. Myasthenia gravis is usually sporadic. However, familial cases suggest a genetic predisposition. We sought to identify a causative mutation in a previously reported Italian-American kindred with parental consanguinity and five out of ten siblings affected by adult-onset autoimmune myasthenia gravis. We performed genome-wide homozygosity mapping, and sequenced all genes in the one region of extended homozygosity. Quantitative and allele specific RT-PCR were performed on a candidate gene to determine the RNA expression level in affected siblings and controls and the relative abundance of the wild-type and mutant alleles. A region of shared homozygosity at chromosome 13q13.3-13q14.11 was found in four affected subjects and one unaffected individual. A homozygous single nucleotide variant was found in the 3'-untranslated region of the ecto-NADH oxidase 1 gene (ENOX1). No other variants likely to be pathogenic were found in genes in this region or elsewhere. The ENOX1 sequence variant was not found in 764 controls. Quantitative RT-PCR showed that the expression of ENOX1 is decreased to about 20% normal levels in lymphoblastoid cells from individuals who are homozygous for the variant and to about 50% in two unaffected heterozygotes. Allele specific RT-PCR showed a 55-60% reduction in the transcript level of the variant in heterozygote cells due to reduced mRNA stability. These results indicate that this sequence variant in ENOX1 may contribute to the late onset myasthenia in these patients. To our knowledge, this is the first single-gene defect to be associated with autoimmune myasthenia. CMT2C is an autosomal dominant neuropathy characterized by limb, diaphragm, and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. All genes in this region were sequenced and heterozygous missense mutations were identified in the TRPV4 gene causing the amino acid substitutions R269C and R269H. TRPV4 is a well known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously associated with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of peripheral nerves. The CMT2C mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this striking phenotypic variability may be due to differential effects on regulatory protein-protein interactions. We subsequently investigated interacting proteins and used exome-sequencing looked for a causative mutation in a CMT2C family in which we had found no TRPV4 mutation. The exome analysis revealed a novel TRPV4 mutation in this family, R186Q, which had been missed by Sanger sequencing due to a rare polymorphism at the primer site. We identified a family in Mali with two sisters affected by spastic paraplegia. In addition to spasticity and weakness of the lower limbs, the patients had marked atrophy of the distal upper extremities. Homozygosity mapping using single nucleotide polymorphism arrays showed that the sisters shared a region of extended homozygosity at chromosome 19p13.11-q12 that was not shared by controls. These findings indicate a clinically and genetically distinct form of hereditary spastic paraplegia with amyotrophy, designated SPG43. We subsequently evaluated candidate genes in the critical region by exome sequencing, and identified one gene, C19orf12, with a sequence variant not found in controls. The variant is associated with aberrant localization of the recombinant protein in transfected cells. Mutation in the protein product of this gene, which we have named senfaganin, may be a novel cause of hereditary spastic paraparesis.

Recently our research has focused on three neuromuscular diseases: autosomal recessive spinal muscular atrophy (SMA) due to deficiency of the protein SMN, spinal and bulbar muscular atrophy (SBMA) due to polyglutamine expansion in the androgen receptor, and amyotrophic lateral sclerosis type 4 (ALS4) due to mutation in senataxin. Specific research accomplishments include the following: (1) Polyglutamine expansion in androgen receptor (AR) is responsible for SBMA and leads to selective loss of lower motor neurons. We evaluated the effect of polyglutamine length on AR function in Xenopus oocytes. This allowed us to correlate the nuclear AR concentration to its capacity for specific DNA binding and transcription activation in vivo. AR variants with polyglutamine tracts containing either 25 or 64 residues were expressed in Xenopus oocytes by cytoplasmic injection of the corresponding mRNAs. The intranuclear AR concentration was monitored in isolated nuclei and related to specific DNA binding as well as transcriptional induction from the hormone response element in the mouse mammary tumor virus (MMTV) promoter. The expanded AR with 64 glutamines had increased capacity for specific DNA binding and a reduced capacity for transcriptional induction as related to its DNA binding activity. (2) In SBMA, as in other polyglutamine diseases, a toxic gain of function in the mutant protein is an important factor in the disease mechanism; therefore, therapies aimed at reducing the mutant protein hold promise as an effective treatment strategy. Gene silencing by RNA interference has recently evolved as a viable therapeutic option. We evaluated a microRNA (miRNA) approach to achieve AR repression. We identified and characterized microRNA-298 (miR-298), which binds to the 3'-untranslated region of the human AR transcript, down-regulates AR mRNA and protein levels and counteracts AR toxicity in vitro. Intravenous delivery of miR-298 via adeno-associated virus serotype 9 vector resulted in efficient transduction of muscle and spinal cord and amelioration of the disease phenotype in SBMA mice. Our findings support the development of miRNAs as a therapeutic strategy for SBMA and other polyglutamine diseases. (3) It has been suggested that proteins with expanded polyglutamine tracts impair ubiquitin-dependent proteolysis due to their propensity to aggregate, but recent studies indicate that the overall activity of the ubiquitin-proteasome system is preserved in SBMA models. We found that AR selectively interferes with the function of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), which, together with its substrate adaptor Cdh1, is critical for cell cycle arrest and neuronal architecture. We showed that both wild-type and mutant AR physically interact with the APC/CCdh1 complex in a ligand-dependent fashion without being targeted for proteasomal degradation. Inhibition of APC/CCdh1 by mutant but not wild-type AR in PC12 cells resulted in enhanced neurite outgrowth which is typically followed by rapid neurite retraction and mitotic entry. Our data indicate a role of AR in neuronal differentiation through regulation of APC/CCdh1 and suggest abnormal cell cycle reactivation as a pathogenic mechanism in SBMA. (4) We characterized a novel curcumin analog, ASC-JM17, as an activator of central pathways controlling protein folding, degradation, and oxidative stress resistance. ASC-JM17 acts on Nrf1, Nrf2, and Hsf1, to increase the expression of proteasome subunits, antioxidant enzymes, and molecular chaperones. We showed that ASC-JM17 ameliorates toxicity of the mutant androgen receptor (AR) responsible for SBMA in cell, fly, and mouse models. Knockdown of the Drosophila Nrf1 and Nrf2 ortholog CncC, but not Hsf1, blocked the protective effect of ASC-JM17 on mutant AR-induced eye degeneration in flies. Our observations indicate that activation of the Nrf1/Nrf2 pathway is a viable option for pharmacological intervention in SBMA and potentially other polyglutamine diseases. (5) The development of therapeutics for neurological disorders is constrained by limited access to the central nervous system (CNS). AT-binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), are expressed on the luminal surface of capillaries in the CNS and transport drugs out of the endothelium back into the blood against the concentration gradient. SMN protein, which is deficient in SMA, is a target of the ubiquitin proteasome system. Inhibiting the proteasome in a rodent model of SMA with bortezomib increases SMN protein levels in peripheral tissues but not the CNS, because bortezomib has poor CNS penetrance. We sought to determine if we could inhibit SMN degradation in the CNS of SMA mice with a combination of bortezomib and the ABC transporter inhibitor tariquidar. In cultured cells we showed that bortezomib is a substrate of P-gp. Mass spectrometry analysis demonstrated that intraperitoneal co-administration of tariquidar increased the CNS penetrance of bortezomib, and reduced proteasome activity in the brain and spinal cord. This correlated with increased SMN protein levels and improved survival and motor function of SMA mice. These findings show that CNS penetrance of treatment for this neurological disorder can be improved by inhibiting drug efflux at the blood-brain barrier. (6) Numerous strategies using gene and cell therapy are being developed for the treatment of neurodegenerative disorders. Many of these strategies utilize constitutive expression of therapeutic transgenic proteins. Although functional in animal models of disease, this method is less likely to provide adequate flexibility for regulating the expression of the therapeutic proteins in humans. We described the modification and production of a mifepristone-inducible vector system for regulated expression of transgenes within the central nervous system. Mifepristones ability to cross the blood-brain barrier makes it an especially attractive inducible ligand for transgene expression in the brain and spinal cord. Our inducible system used a lentivirus-based vector platform for the ex vivo production of mifepristone-inducible murine neural progenitor cells that express our transgenes of interest. These cells were processed through a series of selection steps to ensure the cells exhibited appropriate transgene expression in a dose-dependent and temporally controlled manner with minimal background activity. Inducible cells were then transplanted into the brains of rodents, where they exhibited appropriate mifepristone-inducible expression. These studies detail a strategy for regulated expression in the CNS for the potential development of an efficient and safe method for future gene therapies for neurological disorders.