In Huntington’s disease (HD), expansion of CAG codons within the huntingtin gene (HTT) leads to the aberrant formation of protein aggregates and the differential degeneration of striatal medium spiny neurons (MSNs). Modeling HD using patient-specific MSNs has been challenging, as neurons differentiated from induced pluripotent stem cells are free of aggregates and lack an overt cell death phenotype. Here we generated MSNs from HD patient fibroblasts through microRNA-based neuronal conversion, previously shown to bypass the induction of pluripotency and retain age signatures of original fibroblasts. We found that patient MSNs consistently exhibited mutant HTT (mHTT) aggregates, mHTT-dependent DNA damage, mitochondrial dysfunction, and spontaneous degeneration over time in culture. We further provide evidence that erasure of age stored in starting fibroblasts and neuronal conversion of pre-symptomatic HD patient fibroblasts resulted in differential manifestation of cellular phenotypes associated with HD, highlighting the importance of age in modeling late-onset neurological disorders.

SUMMARY Directed reprogramming of human fibroblasts into fully-differentiated neurons requires massive changes in epigenetic and transcriptional states. Induction of a chromatin environment permissive to acquiring neuronal subtype identity is therefore a major barrier to fate conversion. Here we show that the brain-enriched miRNAs miR-9/9* and miR-124 (miR-9/9*-124) trigger reconfiguration of chromatin accessibility, DNA methylation, and mRNA expression to induce a default neuronal state. MiR-9/9*-124-induced neurons (miNs) are functionally excitable and are uncommitted towards specific subtypes yet possess open chromatin at neuronal subtype-specific loci, suggesting such identity can be imparted by additional lineage-specific transcription factors. Consistently, we show ISL1 and LHX3 selectively drive conversion to a highly homogenous population of human spinal cord motor neurons. Taken together, this study shows modular synergism between miRNAs and neuronal subtype-specific transcription factors can drive lineage-specific neuronal reprogramming, thereby providing a general platform for high-efficiency generation of distinct subtypes of human neurons.

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The ability to convert human somatic cells efficiently to neurons facilitates the utility of patient-derived neurons for studying neurological disorders. As such, ectopic expression of neuronal microRNAs (miRNAs), miR-9/9 and miR-124 (miR-9/9-124) in adult human fibroblasts has been found to evoke extensive reconfigurations of the chromatin and direct the fate conversion to neurons. However, how miR-9/9-124 break the cell fate barrier to activate the neuronal program remains to be defined. Here, we identified an anti-neurogenic function of EZH2 in fibroblasts that acts outside its role as a subunit of Polycomb Repressive Complex 2 to directly methylate and stabilize REST, a transcriptional repressor of neuronal genes. During neuronal conversion, miR-9/9-124 induced the repression of the EZH2-REST axis by downregulating USP14, accounting for the opening of chromatin regions harboring REST binding sites. Our findings underscore the interplay between miRNAs and protein stability cascade underlying the activation of neuronal program.

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