DM BRAIN · Dissecting the molecular pathways of brain dysfunction in myotonic dystrophy

Final Activity Report Summary - DM BRAIN (Dissecting the molecular pathways of brain dysfunction in myotonic dystrophy)

Increasing the copy number of an existing repetitive deoxyribonucleic acid (DNA) sequence may appear to be a simple process with no major implications. In reality, the pathological consequences are remarkably complex leading to severe diseases. One condition illustrating this great complexity is myotonic dystrophy type 1 (DM1). DM1, the most common form of adult muscular dystrophy, is a complex, autosomal, multisystemic disorder caused by the expansion of a CTG trinucleotide repeat in the 3' UTR of the dystrophia myotonica-protein kinase (DMPK) gene. Although traditionally regarded as a muscle disease, DM1 presents variable degrees of mental dysfunction. Experimental evidence supports a toxic effect of ribonucleic acid (RNA) transcripts containing expanded CUG repeats in disease pathogenesis. Although toxic RNA accumulation has been described in post-mortem DM1 brains, DMPK expression in the central nervous system (CNS) and the extent to which RNA-mediated pathways are responsible for brain-specific disease manifestations have never been studied in detail. To assess this question, DMPK and Dmpk transcripts were quantified by northern blot hybridisation in human and mouse tissues, respectively. Gene expression was detected in brain at lower levels than in heart and skeletal muscle. A detailed study of DMPK expression was subsequently performed on transgenic mice carrying an expanded DMPK gene (circa 400 - 500 CTG) derived from the human DM1 locus, which was previously generated in the laboratory. Real-time quantitative PCR revealed high region-specific DMPK expression in brain, with the highest levels being detected in the brainstem and brain cortex. In situ hybridisation techniques subsequently showed accumulation of expanded DMPK transcripts in intranuclear foci in both neurons and glial cells. Interestingly, regional foci distribution appeared to correlate with the region-specific expression profile of the transgene. Moreover, the trinucleotide DNA repeat was highly unstable towards further expansion in most mouse brain regions. Increased GFAP staining was detected in the brain cortex of mice carrying the DM1 expansion, suggesting previous neuronal cell death and consequent astrogliosis. Further histological studies were ongoing by the time of the project completion. Taken together these results validated the use of this transgenic model to unravel the mechanisms underlying the role of toxic RNA in DM1 brain dysfunction. Moreover, they pointed at the frontal cortex and brainstem as the priority mouse brain areas to look for dysfunctional pathways affected by toxic CUG repeats. The understanding of the cellular and physiological consequences of the DM1 expansion in the CNS was also extended by histological and imaging studies. In order to characterise DM1 dysfunctional pathways, disease targets affected by the toxic RNA were identified, either by a candidate gene approach or by global proteomic analysis. Homozygous mice carrying large repeats (greater than 700 CTG) displayed mis-splicing events in genes that could play a role in neurodegeneration, such as Nmda receptor type 1, Tau and amyloid precursor protein, or in the regulation of alternative splicing, such as muscleblind-like 1 and muscleblind-like 2 genes. Our animals represented the first transgenic DM1 model showing splicing abnormalities in the CNS that resembled those detected in human patients. This important result endorsed the use of these mice to unravel DM1 neuropathogenesis. Novel proteins and pathways targeted by toxic DMPK transcripts were identified by a proteomic approach. The comparison of frontal cortex and brainstem proteomic profiles between mice carrying around 400 - 500 CTG expansion and a short 20 CTG control sequence revealed about 30 candidate proteins which appeared to be affected by the toxic expansion. These proteins seemed to be involved in critical pathways, such as vesicle transport and synaptic transmission, calcium metabolism and response to cellular stress. The relevance of these candidates for disease pathogenesis was subsequently investigated. In the long term, the characterisation of the molecular pathways linking CTG repeat expansion, RNA foci accumulation and neuronal dysfunction would provide great insight into DM1 neuropathogenesis, RNA toxicity and brain biology.

Data: CORDIS, © European Union