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Genetic Studies of Neurodevelopment and Neurodegeneration
Summary: Huda Zoghbi is interested in how an expanded polyglutamine tract renders a protein toxic in neurodegenerative diseases and how to subdue this toxicity. She is also working to understand the roles of Math1 in the development of neurons essential for balance, hearing, and breathing, and how mutations in MECP2 cause Rett syndrome and related autism spectrum disorders.
We use genetic, cell biological, and biochemical approaches to explore the pathogenesis of triplet repeat neurodegenerative disease, the function of Math1 in development, and how MECP2 mutations cause postnatal neurodevelopmental disorders.
Triplet Repeat Pathogenesis
The spinocerebellar ataxias (SCAs) are dominantly inherited neurodegenerative disorders characterized by a progressive and eventually fatal loss of balance and coordination. In collaboration with Harry Orr's group (University of Minnesota), we determined that the mutation responsible for SCA1 is an expansion of a CAG trinucleotide repeat encoding glutamine in the protein ataxin-1. Exactly how the expanded protein causes neuronal degeneration has been the subject of intensive research in our lab.
Genetic studies in mice and fruit flies have shed the most light on SCA1 pathogenesis. In collaboration with Juan Botas (Baylor College of Medicine), we found that high levels of wild-type ataxin-1 produce effects similar to mutant ataxin-1 in Drosophila and mice. This led us to propose that wild-type ataxin-1 might take on a conformation that resists clearance or interacts strongly with other proteins and that such conformation is favored by the expanded polyglutamine tract. This proved to be the case. We found that wild-type and mutant ataxin-1 interact with proteins such as 14-3-3, Gfi-1, and RBM17 through their carboxyl-terminal region and that the polyglutamine tract simply modulates the interaction or its consequences. In collaboration with the Orr lab, we found that residue S776 in ataxin-1 is necessary for ataxin-1's toxicity. We found that 14-3-3 and RBM17 proteins interact with ataxin-1 in a S776-dependent manner and that 14-3-3 augments levels of ataxin-1 when phosphorylated at S776 by Akt kinase.
In biochemical and genetic studies, we determined that disease pathology depends on association of ataxin-1 with its native complexes and that the glutamine expansion contributes to the gain-of-function mechanism by enhancing interactions with RBM17-containing complexes and that it causes some loss of function by decreasing interactions with CAPICUA-containing complexes. These data reveal opposing effects of the glutamine expansion on ataxin-1's function and demonstrate that although SCA1 pathogenesis predominantly results from a gain-of-function mechanism, partial loss of function also contributes to disease.
In addition to the mechanistic insight, these studies provided avenues for investigating potential therapies. Given Ataxin-1's interactions and the finding that transcriptional alterations occur early in SCA1 pathogenesis, we tested lithium carbonate (known to enhance transcription) in our SCA1 knock-in model. We found that lithium improved several neurobehavioral and pathological outcomes in these mice. The finding that S776 phosphorylation is critical for SCA1 pathogenesis is leading us to search for kinases and phosphates that might affect this serine, in the hope of finding a drug that might modulate their activity.
Math1 and Neurodevelopment
In pursuit of genes essential for balance and coordination, we identified and have been studying the mouse atonal homolog 1 (Math1).
Mice lacking Math1 die at birth because they cannot initiate respiration. These mice lack cerebellar granule neurons, pontine neurons, hair cells in the vestibular and auditory systems, and the D1 interneurons of the spinocerebellar tracts. That a single gene controls the genesis and/or differentiation of multiple components of the proprioceptive pathway was a surprise. Math1 is also essential for secretory cells in the gut (Paneth, goblet, and enteroendocrine cells), and the enteroendocrine cells secrete neuropeptides that modulate gut proprioception. Recently we discovered that Math1 also controls multiple components of the auditory and vestibular pathways, and within the cerebellum it controls the genesis of some deep cerebellar neurons in addition to granule neurons. Identification of the Math1-dependent neurons allowed us to propose that Math1 redefines the rhombic lip and its derivatives.
Recently, we determined that TCF4's interaction with MATH1 is critical for specification of pontine nucleus neurons, a finding that provides new insight about the mechanisms underlying transcriptional programs that regulate neuronal differentiation. A conditional allele is allowing us to analyze MATH's postnatal function in the proprioceptive pathway and breathing control.
We are continuing to characterize the Math1-null mice to pinpoint the neurons involved in the respiration phenotype, and we are pursuing the identification of Math1's downstream targets to define the molecular pathways involved in the differentiation of the diverse cell types dependent on Math1.
Rett Syndrome and MeCP2
Girls affected with Rett syndrome appear to develop normally for the first 6 to 18 months of life, then lose the ability to speak and socialize, and develop tremors, ataxia, seizures, and stereotypic hand-wringing movements. I became fascinated with the disorder after seeing my first Rett patient and embarked on a lengthy search for its genetic basis. In 1999, we discovered that Rett syndrome is caused by mutations in the gene encoding methyl-CpG–binding protein 2 (MECP2). Located on the X chromosome, MECP2 encodes a protein that binds methylated cytosines, helping to orchestrate gene silencing via DNA methylation.
We and others discovered that MECP2 mutations cause a broad spectrum of phenotypes in both females and males. Females may present with isolated mental retardation, autism, or milder forms of Rett if they have favorable X-chromosome inactivation. In males, the inactivating mutations cause severe neonatal encephalopathy and death in infancy, whereas milder mutations may cause mental retardation, motor dysfunction, and psychosis.
MeCP2 is in mature neurons, and the number of MeCP2-positive cortical neurons increases postnatally as the brain matures. We generated a mouse model by creating a mutation that truncates the protein past amino acid 308. Male Mecp2308 mice appear normal up to six weeks of age, when they develop tremors, seizures, coordination problems, social behavior abnormalities, and forepaw stereotypies similar to the hand-wringing seen in patients. We also generated transgenic mice that overexpress MeCP2 at twice the normal level in the correct spatiotemporal distribution, and found that they develop a progressive postnatal neurodevelopmental disorder. This led us to propose that duplications of MECP2 might lead to postnatal neurologic disorders, which indeed is proving to be the case. We have begun to characterize patients with the duplication and found that some have Rett-like features and others have autism spectrum phenotypes. In collaboration with Christian Rosenmund (Baylor College of Medicine), we studied hippocampal glutamatergic neurons in both the duplication and loss-of-function mouse models and determined that MECP2 is a key rate-limiting factor in regulating glutamatergic synapse formation in early postnatal development (Figure 1).
In pursuing functional and pathogenesis studies, we found that MeCP2 interacts with the RNA-binding protein YB-1 and that this interaction affects alternative splicing of reporter cassettes regulated by YB-1. We are studying both the Mecp2308 and overexpression mice to identify the neuron-specific expression and splicing changes that result from MeCP2 dysfunction. We found that MeCP2 regulates the expression of corticotropin-releasing hormone (CRH) by binding the promoter of the Crh gene. The elevation of the Crh level in the Mecp2308 mice (Figure 2) could explain the anxiety-like phenotype and the exaggerated poststress corticosterone response in these animals.
To get at the neuroanatomical origins of the Rett phenotypes, we are selectively deleting Mecp2 in different neuronal populations and studying the corresponding gene expression changes. We hope that correlating molecular changes with specific behaviors will provide the framework for better understanding of the pathogenesis of Rett and related autism spectrum disorders. (Grants from the National Institutes of Health, the International Rett Syndrome Research Foundation, and the Simons Foundation provided support for this project.)
Last updated: April 24, 2008
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