26miR-124 plays a major regulatory role in neurogenesis and neuronal differentiation during 27 brain development through control of its multiple non-neuronal targets and has therefore 28 been employed in direct reprogramming protocols supplementary to neurogenic TFs, and 29 other miRNAs to enhance neurogenic conversion. However, its capacity to instruct 30 neurogenic conversion of astrocytes and its independent mechanism of direct 31 reprogramming action have been poorly investigated. Aim of the study was to investigate 32 whether miR-124 is a master-regulatory reprogramming agent, potent to drive direct 33 reprogramming of astrocytes to induced-neurons (iNs) on its own and to elucidate its 34 mechanism of reprogramming action. To this end we overexpressed miR-124 either alone or 35 in combination with the small neurogenic compound ISX9 both in vitro and in in vivo in a 36 mouse mechanical cortical trauma model and analyzed their mechanism of reprogramming 37 action. Our data indicate that miR-124 and ISX9 exhibit both unique and convergent 38 molecular contributions in the reprogramming process to iNs. miR-124 is a potent driver of 39 the astrocytic reprogramming switch of astrocytes towards an immature neuronal fate by 40 repressing genes regulating astrocytic function, among which we identified the RNA-binding 41 protein Zfp36l1 as a novel miR-124 direct target. We also provide evidence that ISX9 greatly 42 improves both miR-124-induced reprogramming efficiency and functional maturation of iNs. 43 Importantly, miR-124 either alone or along with ISX9 is potent to guide direct neuronal 44 reprogramming of reactive astrocytes to iNs of cortical identity in vivo, a novel finding 45 confirming the robust direct reprogramming action of the two molecules in activated 46 astrocytes in vivo. 47 48 49 50 51 52 53 2006). Its mechanism of action has been studied over the years, where it has been shown to 79 act globally to increase the expression levels of neuronal genes, by repressing components 80 of major neuronal gene repressor complexes, such as the anti-neural transcriptional 81repressor REST complex, targeting Scp1 and Rcor1 (Baudet et al., 2012; Visvanathan et al., 82 2007; Volvert et al., 2014) and the Polycomb Repressive Complex 2 (PRC2), repressing the 83 histone 3 lysine 27 methyltransferase Ezh2 (Lee et al., 2018;Neo et al., 2014), while it also 84 participates at the post-transcriptional regulation of neuronal transcripts by targeting the 85 neuron-specific splicing global repressor Ptbp1 (Makeyev et al., 2007). Besides its roles in 86 transcriptional and post-transcriptional regulation, miR-124 neurogenic action also relates to 87 chromatin dynamics, as it has emerged as one of the key mediators of a chromatin 88 permissive environment for neuronal reprogramming through its involvement in the 89 formation of the neuron specific chromatin remodeling complex nBAF, by targeting BAF53a 90 subunit mediating its exchange with its homologous, neuronal specific BAF53b (Tang et al., 91 2013a;Yoo et al., 2009a...
