Christopher M. Gomez - US grants

The formation of a mature neuromuscular junction (NMJ) requires changes in expression of several muscle genes that are regulated by activation of the nicotinic acetylcholine receptor (ChR). During development, after its insertion into the NMJ the AChR undergoes a series of physiological and structural changes that have been ascribed to the replacement of the gamma (gamma) subunit by the epsilon (epsilon) subunit. These changes may have a role in protecting muscle from potentially excitotoxic effects of excessive stimulation by acetylcholine (ACh) since they lead to a diminution of the ACh-sensitivity of muscle fibers just as the nerve has arrived to establish cholinergic transmission. Failure to make this subunit switch might be the molecular basis for the slow channel syndrome, a hereditary muscle disease. The goal of this study is threefold: To investigate 1) the the mode of regulation of the gamma to epsilon AChR subunit switch and its role in the developmental changes of the AChR of the NMJ; 2) the influence these changes may have on regulation of cholinergic genes and other proteins involved in synaptic transmission; 3) the role of these changes in maintaining the integrity and long-term viability of neuromuscular transmission. This latter goal will test the hypothesis that the phenomenon of excitotoxicity can arise as a result of an abnormal response on the part of an excitatory neurotransmitter receptor. For this study several lines of transgenic mice have been generated that constitutively express the mRNA for the embryonic (gamma) subunit of AChR in muscle. By studying the transcription and stability of the mRNA for gamma, the synthesis of the gamma subunit protein, and its assembly into mature AChRs, the control system for regulation of the embryonic phenotype will be clarified. By studying the properties of the gamma-containing AChRs incorporated into adult motor endplates, the role of the subunit switch in the developmental changes of AChR will be determined. The effect of this persistence of fetal properties on both muscle viability and expression of other genes involved in cholinergic transmission will be evaluated. Further understanding of the molecular basis for the developmentally related phenotypic changes in AChRs and the NMJ will broaden insights into the basis for such changes in other neurotransmitter systems and in neural control in general. Pathologic changes demonstrated in the muscle of animals with such abnormal AChRs would implicate genes for excitatory neurotransmitter receptors in the pathogenesis of hereditary neurodegenerative diseases. It may also provide a model of excitotoxicity for investigation of the second messenger systems participating in its pathogenesis and for development of possible drug therapies.

DESCRIPTION: (adapted from Applicant's Abstract) The slow channel congenital myasthenic syndrome (SCCMS) is a hereditary, progressive muscle disease characterized by weakness, atrophy, degenerative changes confined to the neuromuscular junction (NMJ) and electrophysiological evidence of abnormal acetylcholine receptor (AChR) ion channel function. It may be the first degenerative disease recognized to result from an inherited abnormality of synaptic receptor function. The goal of this project is to demonstrate that the SCCMS is due to a mutation in the protein-coding region of one of four AChR subunit genes. This will be accomplished by first screening for mutations in these four candidate genes using a two-armed analysis, followed by functional confirmation of the phenotype of mutations: 1) The investigator will systematically screen for coding-region mutations along the entire length of each subunit in patients with SCCMS in parallel using the technique of single-stranded conformation polymorphism (SSCP) analysis. 2) They will determine the nucleotide sequence of any regions that show single-strand polymorphisms, as well as the sequences of the four transmembrane domains of each AChR subunit in cases where no polymorphism is found. 3) In order to be able to screen each exon completely, they will determine intron sequences and design intron-specific primers for each subunit. 4) Finally, the phenotype of any mutation identified in the SCCMS will be tested by generating the corresponding mouse AChR subunit cDNA mutation for co-expression along with the other mouse wild- type subunits in vitro. In this way they will directly correlate the clinical AChR phenotype with an AChR mutation and a molecular phenotype in vitro. The overall goal of this research is to characterize the role that abnormal receptors in excitatory synapses might play in degenerative disease using the NMJ and the SCCMS as models. In a related project, a transgenic mouse model for the SCCMS has been generated using mutant AChR subunits. This model and additional transgenic mice expressing the mutations in the SCCMS will be used to investigate the electro- physiological conditions and biochemical pathways responsible for the degenerative changes in muscle. This approach may lead to identification of potential targets for therapeutic intervention relevant to excitotoxicity.

The HIV-1 external glycoprotein, gp120, has been implicated in the development of AIDS dementia complex. The goal of this project is to investigate the neurotoxicity of this molecule, as summarized in two specific aims: 1) Is gp120 toxic to neurons in vivo? 2) Is the secreted form of gp120 neurotoxic in vitro and, if so, does neurotoxicity vary with the HIV strain of origin? The hypothesis that gp120 is neurotoxic in vivo will be tested using transgenic mice that secrete gp120 from one of the following central nervous system (CNS) cells: 1) all neurons; 2) oligodendrocytes; 3) cerebellar Purkinje cells. tissue-specific promoters will be used to expose neurons to gp120 arising from several distinct cellular sources in vivo. These studies will also test whether the effect of gp120 secretion is independent of its site of production. gp120-producing transgenic mice will be systematically analyzed for neuropathological alterations similar to those of ADC. In parallel to the transgenic experiments, gp120-induced neurotoxicity will be determined in primary neuronal cultures allowing direct testing of the hypothesis that gp120-induced neurotoxicity in vitro accurately reflects gp120 actions in vivo. The possibility will be explored that neurotoxicity varies with differences in sequence and cell tropism by using gp120 subunits from different isolates. The subunits of macrophage-tropic and lymphotropic isolates will be expressed in cultured cell lines using recombinant expression vectors. Cells will be co-cultured with primary neuronal cells and the viability of the neurons assessed. In addition, the culture medium of the infected cells will be used as the source of gp120 for the neurotoxicity studies in vitro. The studies proposed in this application are aimed at understanding the role of the HIV-1 envelope glycoprotein in the AIDS dementia complex. The information obtained from these experiments may provide an important model of retrovirus-induced neurodegeneration and contribute to the development of improved therapeutic modalities for this AIDS complication.

DESCRIPTION (from applicant's abstract) The autosomal dominant spinocerebellar ataxias (SCAS) and episodic ataxias (EAs) are a group of adult- and juvenile-onset neurodegenerative diseases characterized by progressive or intermittent dysarthria and incoordination due to degeneration of the cerebellum and brainstem. Advances in the genetic understanding of these diseases have established that, despite similar clinical presentations, there are at least 9 genetically distinct subtypes, SCAI-SCA7, EA-1 and EA-2. Clinical observations suggest that eye movements and postural stability are universally but differentially impaired in the SCAs, presumably due to regional differences in brainstem and cerebellar involvement in the disease. The voluntary and reflexive control of oculomotor and vestibular function rely heavily on the normal function of the cerebellum and its interaction with brainstem neurons. A precise understanding of extraocular movements and vestibular dependent reflexes in SCA may identify both common abnormalities useful for comparative scoring among kindreds, and abnormalities that are unique to a given SCA subtype. In this project the applicants propose to take advantage of a large database of SCA patients, and recent developments in both the genetics of autosomal dominant ataxia, and in the technology for recording and analyzing eye movements and the dynamic control of posture to address the question of whether specific patterns of eye movement abnormalities and postural instability characterize genetically-defined SCAs. These studies will allow them to assess the functional integrity of widespread areas of the brainstem and cerebellum. These measurements are non-invasive, more sensitive than static magnetic resonance imaging, and can be applied to a larger number of genetically defined ataxia patients than could be possible using pathological studies. They propose to: 1) Determine the genetic status of all SCA and EA (episodic ataxia) patients in the University of Minnesota Ataxia Database. 2) Determine whether genetically homogeneous forms of SCA manifest unique patterns of oculomotor and vestibular abnomalities. 3) Determine whether the length of CAG repeat expansions in SCA 1, 2, 3 and 6 correlate with the profile of oculomotor and vestibular abnormalities. 4) Define the progression of oculomotor, vestibular and postural abnormalities as a function of disease duration for SCA 1-7. These studies may identify diagnostic features for some SCA types and provide valuable information about selective vulnerability of CNS neurons and the pathogenesis of CAG repeat diseases. They also may identify traits common to all patients with ataxia that will be useful quantitative measures for therapeutic trials. These studies will test the hypothesis that sensitive measures of eye movements and balance can be used to detect and quantify ataxia from its earliest stages.

DESCRIPTION (Adapted from applicant's abstract): The goal of this research is to understand the processes involved in excitotoxic degeneration of synapses using an animal model. Animal models for human neurodegenerative disease are of great value for exploring the cellular and biochemical mediators and molecular pathogenesis of a slowly progressive disease process. The slow channel congenital myasthenic syndrome (SCCMS) is caused by mutations that result in delayed closure of the ion channel of the acetylcholine receptor (AM) of the neuromuscular (NMJ). The delayed channel closure is associated with calcium overload and degeneration of the NMJ, AChR loss, and progressive muscle weakness. Thus, the SCCMS is a prototype for a hereditary excitotoxic disorder. Using transgenic mice technology and site-directed mutagenesis of AChR subunit coding sequences, we have developed the slow-channel transgenic mouse, an animal model for the SCCS that manifest all the features seen in the human disease. In this proposal, the investigator proposes to: (1) Determine whether slow-channel transgenic mice have reduced expression of neuromuscular synapse-specific genes. This will be accomplished by comparison of mRNA levels for the AChR subunit genes and other NMJ-specific genes between transgenic and control mice and between degenerating NMJ nuclei and remote from the NMJ nuclei; (2) Determine the cause(s) of the organellar damage and endplate myopathy in slow-channel mice. Three likely pathways of intracellular damage: activation of calcium-activated proteases, oxidative damage by free radicals, and apoptosis will be explored using a combination of specific antibody probes and stains to look for damaged proteins and DNA at the NMJ and genetic and pharmacological manipulation of these pathways to alter the course of the disease; and (3). Determine if quinidine can protect the slow-channel transgenic mice from endplate degeneration.

The longterm objective of this study is to define the role of the P/Q-type voltage-gated channel in Purkinje-cell degeneration and hereditary ataxia. P/Q channels are membrane proteins that play a crucial role in membrane excitability and triggering synaptic transmission. The P/Q a subunit (a1A) encoded by the gene, CACNA1A, has been implicated in familial migraine, hereditary episodic ataxia and spinocerebellar ataxia type 6 (SCA6). SCA6, a form of autosomal dominant progressive ataxia associated with nearly selective Purkinje cell degeneration, bears the putative pathogenic mutation in the long C terminus of a novel spliceform of the a1A mRNA. This study proposes to investigate the distribution of this a1A subunit spliceform and the pathogenicity of the CAG repeat expansion to which the disease is attributed. Using antisera specific for the long splice form, fusion proteins composed of the long C terminus and chimeric full length a1A subunits bearing the long tail with and without expanded CAG repeats we propose to: 1) Characterize the distribution and localization of the wild type and SCA6 mutant long splice form of the a1A subunit of the P/Q-type voltage-gated Ca2+ channel in brain and transfected cells and in relation to b subunit subtypes. 2) Characterize the effect of the SCA6 P/Q channel mutation and b subunit subtype on P/Q channel kinetics, and on Ca2+ overload and calcium-coupled pathways. 3) Develop mice that express the a1A subunit with SCA6-associated polyglutamine expansions to investigate the role of these mutations in the development of ataxia and Purkinje cell dysfunction in transgenic mice. These studies will provide new information about the biology of Ca+2 channels and may aid in understanding the basis for selective vulnerability of Purkinje cells in SCA6. Identification of an effect of an elongated polyglutamine tract in the a1A C terminus on specific Ca+2 channel properties or on Purkinje cell viability would help elucidate the pathogenesis of SCA6 and increase our understanding of P/Q-type Ca+2 channel function.

DESCRIPTION (provided by applicant): The long-range goal of this research is to understand the pathophysiology and molecular mechanisms involved in the impairment of neuromuscular transmission in the slow-channel congenital myasthenic syndrome (SCS). Up to 14 distinct missense mutations in the genes coding for the four subunits of the muscle acetylcholine receptor (nAChR) are responsible for a dominantly-inherited syndrome of varied clinical severity and pathological findings. Although the common effect of each mutation identified in the SCS to date is to prolong the duration of the acetylcholine-induced channel bursts and endplate currents, differences in gating kinetics, ion permeability, desensitization rate, activation of cell death pathways, and sensitivity to choline and ethanol may affect pathogenesis. In this project we propose to explore specific, novel aspects of the molecular and cellular phenotype of each SCS mutation and to assess the relative contribution of each to neuromuscular weakness. Specifically, we propose to: 1) Determine whether pathogenicity in SCS correlates with activation of cell death pathways in vitro. This will be accomplished with cultured mammalian cells expressing SCS mutant AChRs using assays of cell lysis and of activation of cellular proteases. 2) Compare the capacity of ethanol as a model "environmental agent" to modulate activity of mutant AChRs in SCS. This will be accomplished by studying the effect of ethanol on channel activity of AChRs bearing SCS mutations. 3) Determine whether pathogenicity in SCS correlates with differential response to serum choline or spontaneous activity. This will be accomplished by direct comparison of transgenic mice expressing choline-sensitive mutations with varied channel properties. 4) Determine whether reduced AChR channel opening rate (() alone can lead to significant weakness and impairment of synaptic responses. This will be accomplished using an AChR mutation that selectively alters rate of channel opening in transgenic mice. These studies extend the focus of the project by exploring newly recognized SCS mutant channel phenotypes in vitro and by using prototypic AChR subunit mutations to study pathogenic mechanisms in vivo. In addition, to extend the study of genetic and molecular mechanisms, we begin to investigate the influence of the environment on disease phenotype, and whether this influence may vary according to SCS mutation.

DESCRIPTION (provided by applicant): The autosomal dominant spinocerebellar ataxias (SCA) are a clinically and genetically heterogeneous group of neurodegenerative diseases. It is clear that a diverse of genes and mutational mechanisms can cause SCA, but the molecular process and mechanism for Purkinje cell degeneration that leads to SCA is still unknown. Further insights into SCA pathogenesis may come from more studies that have demonstrated that not all SCAs are due to expanded DNA repeats in novel genes. We recently identified a large family with a novel dominant ataxia, register as SCA26, and mapped the disease locus to 19p13.3. The long-range goal of this research is to expand our understanding of the pathogenesis of the hereditary ataxias, and specifically the basis for the nearly selective Purkinje cell degeneration. We hypothesize that SCA26 is caused by a mutation in a gene that is vital to neuron survival or specific function in the cerebellum, and have identified a few compelling candidate genes. The objective of this project is to refine the locus map by recruiting more family members, and by seeking a different founder haplotype from unrelated families, and to identify the gene and mutational basis by sequencing all coding regions of top candidate genes, and to survey the prevalence of SCA26. This project is significant because: 1) it will directly benefit ataxic patients by providing a new genetic test for diagnosis and genetic consulting; 2) it will provide a new model to study Purkinje cell and cerebellar degeneration in ataxia patients; 3) more broadly, it will establish a new point to inter-connect known factors together, and unveil new insights to delineate the common pathways involved in neurodegeneration or vital to neuron survival and function.

DESCRIPTION (provided by applicant): The Third Ataxia Investigators'Meeting will focus on the multi-disciplinary nature of ataxia research by assembling an international roster of clinical investigators, diagnosticians, pathologists, geneticists and molecular biologists. In addition to focusing on the most recent scientific advances, the goals of the meeting include: 1) To enhance the open exchange of information related to ataxia research;2) To stimulate the initiation of collaborative research between investigators worldwide;3) to improve the understanding of human diseases related to ataxia and establish international protocols for the common investigation and storage of data related to ataxia and its treatment;4) To provide junior investigators with an opportunity to present their work interact with more established scientists in the field, and to provide them with the opportunity to interact with patients and support groups so that they can see the clinical impact and importance of their work. This meeting has been an important stepping stone for collaboration and discussion on ataxia research and therapeutic approaches, and is of great importance now that the field is at the brink of meaningful clinical trials. PUBLIC HEALTH RELEVANCE: The Third Ataxia Investigators'Meeting will focus on the most recent advances in ataxia research, and therapeutic approaches for ataxic disorders. Ataxia, which is defined as the loss of motor control, can affect all aspects of human movement - gait, dexterity, speech, swallowing, eye movements and more - and afflicts approximately 1 in every 2000 individuals worldwide. An expanding genetic understanding of ataxias over the last 15 years has recently led investigators to propose several possible therapeutic approaches, which demand an increase in international communication, collaboration, and standardization of research and clinical methods, all of which will be specifically addressed at the 3rd Ataxia Investigators'Meeting. This meeting will also provide a forum for recruiting new investigators to this field of biomedical research, which is an essential element for achieving the rapid success that appears increasingly possible for treating these diseases.

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type 6 (SCA6) is a dominantly-inherited, untreatable neurodegenerative disease characterized by progressive ataxia and Purkinje cell degeneration associated with CAG repeat expansions in the gene, CACNA1A. Our recent evidence suggests that the disease is attributable to expression of a polyQ repeat expansion within a second CACNA1A gene product, a1ACT, that normally serves as a transcription factor (TF) critical for cerebellar cortical development, and that arises through the action of a cryptic cellular internal ribosomal entry site (IRES) within the coding region. SCA6-sized polyQ expansions in the ?1ACT TF interrupt its cellular and molecular function, cause cell death in vitro, ataxia and cortical thinning. The long-term goal of this project is to understand te pathogenesis of SCA6 by characterizing how a1ACTSCA6 alters gene expression, by confirming the role of the CACNA1A IRES in vivo and by developing a screening strategy for potential IRES-inhibiting compounds as potential therapies. Specifically we propose to ask: 1. Does the SCA6 polyQ expansion in a1ACT in the transcription factor (a1ACTSCA6) changes the gene binding patterns and the expression patterns of Purkinje cell genes? We will utilize chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) to analyze in detail binding profiles of a1ACTWT and a1ACTSCA6 expressed in PC12 cells and isolate RNA from Purkinje cells of mice expressing a1ACTWT or a1ACTSCA6 to generate global gene expression profiles by RNA-seq to correlate a1ACTWT-DNA binding with transcriptional activity and to identify allele-specific changes in gene expression patterns. 2. Does a1ACT expression by CACNA1A IRES and promoter reproduce the normal and pathological functions of ?1ACT? We will generate new lines of a1ACT transgenic mice that will only yield a1ACTWT or a1ACTSCA6 protein by CAP-independent translation, and use the tet-off expression system to drive conditional expression and use a CACNA1A-tTA transgene to generate an endogenous pattern of expression. 3. Can we identify IRES-directed molecules that selectively suppress ?1ACT translation, but not a1A subunit expression? We will use our dual-luciferase bicistronic reporter to determine the optimal RNA sequences involved in IRES function, identify the ITAFs by EMSA and predict the secondary structure of CACNA1A IRES. We will carry out a high throughput screening assay to identify compounds that interfere with ?1ACT IRES function.

? DESCRIPTION (provided by applicant): We have discovered that the CACNA1A gene, which encodes the P/Q type voltage gated calcium channel (VGCC) is bicistronic. The CACNA1A mRNA encodes both the a1A subunit and a second protein, a1ACT, a transcription factor, that is translated as a second open reading frame due to a cryptic internal ribosomal entry site (IRES). Simultaneous expression of two proteins in the same cellular mRNA, one of which is a transcription factor, may be a powerful strategy used by calcium channels and other cellular genes to coordinate expression of an ensemble of genes. To understand this new class of IRES and bicistronic genes we will: 1. Determine how the CACNA1A IRES is regulated by RNA-binding proteins and miRNAs; 2. Define the novel properties of bicistronic genes of the VGCC family. 3. Determine whether altered bicistronic gene expression leads to the complex phenotypes of VGCC disorders. This study will help us to understand the biology of bicistronic cellular genes in particular among other VGCCs, the nature of the IRES regulatory mechanisms and proteins they encode, and the possible role in the pathogenesis and treatment of complex human genetic disease.

ABSTRACT Spinocerebellar ataxia type 6 (SCA6) is an incurable hereditary degenerative ataxic disease passed from parent to child. We have developed a novel, microRNA-based gene therapy approach to inhibit expression the disease-causing protein, alpha1ACT made by the gene CACNA1A. This approach, which relies on an adenovirus to deliver the micro RNA, spares the other critical CACNA1A protein, alpha1A calcium channel subunit, although it appears to suppress both the disease-causing and normal alpha1ACT proteins. Here in Aim 1 we propose to expand on our initial study that inhibited a hyper acute neonatal mouse of SCA6 by comparing two different routes (intraventricular and intravenous) of administration of the adenovirus- microRNA, for effectiveness in blocking the hyperacute mouse model and a new BAC-based transgenic mouse model. In Aim 2 we will use a mouse CACNA1A mutant and a novel transgenic mouse model expressing normal alpha1ACT to prove that suppression of normal alpha1ACT is safe in adult mice, paving the way to safely envision a similar approach in patients with SCA6.

Abstract The long term objectives of this project are to explain the basis for the complex genotype-phenotype relationships for a growing number of severe calcium channel gene disorders and develop therapies based on these new insights. To make these advances we will capitalize on our new discovery that at least three of these calcium channel genes are bicistronic i.e. they encode two distinct proteins, the calcium channel proteins and a newly discovered transcription factor. We have discovered that the transcription factor is translated by internal translation by a process resembling an internal ribosomal entry site (IRES). We hypothesize that mutations in these Ca2+ channel genes may have a diversity of outcomes, affecting, in distinct cases, neuronal firing, calcium signaling, regulation of the expression of the transcription factor or directly altering the function of the transcription factor. In this study we will systematically explore the function and biological action of these three novel transcription factors, how their expression is regulated by the IRES, how normal cellular physiology governs their translocation to and from the nucleus, and how different mutations affecting channel gating, IRES function and transcription factor cause impaired neuronal development and or viability in these different disorders. We will study this using recombinant calcium channels expressed in primary neurons and human reprogrammed neurons from normal and patient sources, and in transgenic mice expressing well characterized mutations. We will study gene binding and expression using next generation approaches, nuclear translocation using epitope and fluorescent tags with physiological stimuli, IRES function using dual luciferase reporters and immunoblotting, neuronal development using immunofluorescent microscopy and corrective therapy using antisense oligos, miRNA and AAV viral vectors expressing transcription factors.