Triplet Repeat Pathogenesis
The spinocerebellar ataxias (SCAs) are dominantly inherited neurodegenerative disorders that belong to a family of diseases caused by dynamic mutations. In the case of SCA1, the mutation is an expansion of a translated CAG repeat that tends to expand further upon transmission to each subsequent generation (hence its description as a "dynamic" mutation). Precisely how ataxin-1 with an expanded glutamine tract causes progressive loss of balance and motor control has been a central question in my lab and that of my collaborator, Harry Orr (University of Minnesota), ever since we codiscovered the genetic basis of SCA1 in 1993.
Genetic studies in mice and fruit flies have yielded tremendous insights into SCA1 pathogenesis. In collaboration with the Orr lab, we found that residue S776 in ataxin-1 is necessary for mutant ataxin-1's toxicity: mice bearing expanded ataxin-1 with an S776A mutation live a normal, disease-free life span. We also found that transcription is disrupted early in pathogenesis, within two weeks of birth for SCA1 mice—long before symptoms begin to appear. In collaboration with Juan Botas (Baylor College of Medicine), we found that high levels of even wild-type ataxin-1 can be toxic to neurons: Drosophila and mice overexpressing wild-type ataxin-1 develop a mild version of the SCA1 phenotype. This led us to propose that the polyglutamine tract might induce ataxin-1 to take on a conformation that resists clearance or alters the protein's interactions with its normal partners. 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 and that the polyglutamine tract modulates the interaction or its consequences. The interactions of 14-3-3 and RBM17 with ataxin-1 require S776, and 14-3-3 augments ataxin-1 levels.
The polyglutamine diseases have long been understood to result from a toxic gain of function, and we did find that the glutamine expansion contributes to the gain-of-function mechanism by intensifying ataxin-1 interactions with RBM17-containing complexes. We also found, however, that the CAG expansion causes loss of function by decreasing interactions with complexes containing Capicua. Studies from labs working on other triplet repeat diseases have provided evidence that loss- and gain-of-function mechanisms also come together in Huntington's disease and spinobulbar muscular atrophy (SBMA).
These and other mechanistic insights are finally providing avenues for potential therapies. The transcriptional down-regulation we observed in early SCA1 pathogenesis led us to test the therapeutic effects of lithium carbonate, a transcriptional enhancer, in our SCA1 knock-in mice. Lithium improved several neurobehavioral and pathological outcomes in these mice. We are also searching for kinases and phosphates that might affect serine 776, in the hope of finding a drug that can target this residue, which seems to be the central key to ataxin-1 toxicity.
Math1 and Neurodevelopment
To understand the neuronal pathways that underlie balance and coordination, we sought and identified the mouse homolog of the Drosophila gene atonal, which controls the development and function of the fly's chordotonal organs (the sensory elements that provide proprioception and hearing sense). Our studies of mouse atonal homolog 1 (Math1) have opened several fascinating and entirely unexpected lines of inquiry in the lab.
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 surprising; even more surprising was our discovery that Math1 is essential for secretory cells in the gut (Paneth, goblet, and enteroendocrine cells), which modulate gut proprioception. Our most recent work tracing the fate of Math1-dependent neurons has allowed us to redefine the rhombic lip and its derivatives and is revealing MATH1's postnatal function in proprioception and regulation of respiration. Other labs have now used Math1 to correct deafness in guinea pigs caused by loss of inner ear hair cells; we hope our current work will shed light on disorders of respiration in infancy.
Rett Syndrome and MeCP2
Rett syndrome, an autism spectrum disorder that causes a broad range of neurological and behavioral disabilities, 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 have since established that MECP2 mutations cause a broad spectrum of phenotypes. 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 hypomorphic mutations can cause mental retardation, motor dysfunction, or psychosis.
MeCP2 is expressed 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—all very reminiscent of the features that characterize classic Rett in humans. 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 would cause postnatal neurologic disorders in human children, which proved to be the case. We have begun to characterize patients with the duplication and found that some have Rett-like features, while 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).
We are studying Mecp2-null and -overexpressing mice to identify the neuron-specific expression and splicing changes that result from MeCP2 dysfunction. Thousands of genes are altered in these mouse models, and the vast majority of the expression changes occur in opposite directions in the Rett and duplication models. This suggests that the MeCP2 duplication syndrome most likely causes disease by a gain-of-function mechanism.
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 have found that deleting Mecp2 in Sim1-expressing neurons leads to hyperphagia, aggression, and an increased stress response. Armed with this new phenotype, we returned to the clinic and have now identified patients with hypomorphic MECP2 mutations and hyperphagia, autism, and aggression. Our mouse studies thus led to the definition of clinical phenotypes not previously appreciated in MeCP2 disorders and revealed an important role for MeCP2 in regulating feeding behavior. We hope that correlating molecular changes with specific behaviors will provide a framework for better understanding of the pathogenesis of Rett and related MeCP2 disorders.
