Modeling Myotonic Dystrophy Type 1 and Type 2 (DM1 and DM2) Neuropathology with iPSC-Derived Cortical Organoids

About This Grant

Repeat-expansion disorders myotonic dystrophy 1 and 2 (DM1, DM2) produce disabling cognitive and behavioral deficits, yet the molecular drivers of central-nervous-system (CNS) pathology remain undefined. DM1 is caused by expanded CTG repeats in the dystrophia-myotonica protein kinase (DMPK) gene that produces toxic CUG RNAs, whereas DM2 is caused by expanded CCTG repeats in the cellular nucleic-acid–binding protein (CNBP) gene that yield toxic CCUG RNAs. These expanded RNAs sequester the splicing regulator muscleblind-like protein 1 (MBNL1), disrupt microtubule-associated protein tau (MAPT) processing, and may provoke excitotoxicity via hyperactive CUG-binding protein ELAV-like family 2 (CELF2). The long-term objective is to enable CNS-directed therapies for myotonic dystrophy. The central hypothesis is that repeat-expansion RNA toxicity mis-splices MAPT and activates CELF2-mediated glutamatergic hyperexcitability, jointly driving tauopathy and neuronal loss. This project will delineate convergent and divergent mechanisms of cortical dysfunction in DM1 and DM2 using human induced-pluripotent-stem-cell (iPSC) cortical organoids produced by an optimized protocol validated in more than sixty lines. Organoids derived from this protocol retain physiological DMPK1, CNBP, and MBNL1 expression overcoming the low expression of these genes observed in other organoid models. The optimized protocol will be applied to DM1 iPSC lines (238–1,600 CTG repeats), DM2 lines (8.8–11.9 kb CCTG repeats), and healthy control lines to generate side-by-side disease and reference cortical organoids. Aim 1 will track RNA- foci formation, splice defects, and tau aggregation at 2, 4, and 6 months in DM1, DM2, and control organoids, combining long-read RNA-seq with quantitative neuropathology. Aim 2 will test whether hyper-phosphorylated CELF2 drives glutamatergic mis-splicing, network hyperexcitability, and neuronal loss across DM1 and DM2 organoids, and whether inducible shRNA knock-down of CELF2 restores receptor splicing, electrophysiological balance, and tau status relative to controls. High-density multielectrode-array recordings, long-read transcriptomics, and tau biochemistry will integrate molecular and functional endpoints throughout the project. The study is expected to (i) establish mechanistic links between repeat RNA toxicity and tauopathy, (ii) identify CELF2 as a modifiable driver of excitotoxicity, and (iii) deliver a scalable, biomarker-rich organoid platform for therapeutic screening. By clarifying disease pathways and providing human assay systems, the work will accelerate the development of tau-lowering and synapse-stabilizing strategies, directly advancing NIH priorities to translate mechanistic insight into treatments for rare neurodegenerative diseases.