Central nervous system injury and neurodegenerative diseases cause irreversible loss of neurons. Overexpression of exogenous specific transcription factors can reprogram somatic cells into functional neurons for regeneration and functional reconstruction. However, these practices are potentially problematic due to the integration of vectors into the host genome. Here, we showed that the activation of endogenous genes Ngn2 and Isl1 by CRISPRa enabled reprogramming of mouse spinal astrocytes and embryonic fibroblasts to motor neurons. These induced neurons showed motor neuronal morphology and exhibited electrophysiological activities. Furthermore, astrocytes in the spinal cord of the adult mouse can be converted into motor neurons by this approach with high efficiency. These results demonstrate that the activation of endogenous genes is sufficient to induce astrocytes into functional motor neurons in vitro and in vivo. This direct neuronal reprogramming approach may provide a novel potential therapeutic strategy for treating neurodegenerative diseases and spinal cord injury.

No abstract

The development of the neuromuscular junction depends on signaling processes that involve protein phosphorylation. Motor neuron releases agrin to activate muscle protein Dok‐7, a key tyrosine kinase essential for the formation of a mature and functional neuromuscular junction. However, the signaling cascade downstream of Dok‐7 remains poorly understood. In this study, we combined the clustered regularly interspaced short palindromic repeats/Cas9 technique and quantitative phosphoproteomics analysis to study the tyrosine phosphorylation events triggered by agrin/Dok‐7. We found tyrosine phosphorylation level of 36 proteins increased specifically by agrin stimulation. In Dok‐7 mutant myotubes, however, 13 of the 36 proteins failed to be enhanced by agrin stimulation, suggesting that these 13 proteins are Dok‐7‐dependent tyrosine‐phosphorylated proteins, could work as downstream molecules of agrin/Dok‐7 signaling. We validated one of the proteins, Anxa3, by in vitro and in vivo assays. Knocking down of Anxa3 in the cultured myotubes inhibited agrin‐induced AChR clustering, whereas reduction of Anxa3 in mouse muscles induced abnormal postsynaptic development. Collectively, our phosphoproteomics analysis provides novel insights into the complicated signaling network downstream of agrin/Dok‐7.

Background Skeletal muscle‐secreted myokines widely participate in lipids metabolism through autocrine, paracrine and endocrine actions. The myokines represented by FGF21 and Irisin can promote the browning of adipocytes and serve as promising targets for treating obesity. Although recombinant myokines replacement therapy and AAV (adeno‐associated virus)‐based myokines overexpression have shown a definite effect in ameliorating obesity, novel myokine activation strategies with higher efficacy and safety are still in pressing need. This study aimed to evaluate the therapeutic potential of a novel CRISPR‐based myokines activation strategy in obesity treatments. Methods In this study, we used lentivirus and a single AAV vector containing dCas9‐VP64 with a single‐guide RNA to selectively activate Fgf21 and Fndc5 expression in skeletal muscles both in vitro and in vivo. The activation efficacy of the CRISPRa system was determined by qRT‐PCR, Western blotting and ELISA. The treatment effect of CRISPR‐based myokines activation was tested in 3T3‐L1‐derived adipocytes and diet‐induced obese (DIO) mice (male C57BL/6 mice, induced at 6‐week‐old for 10 weeks). Results The virus upregulates myokines expression in both mRNA and protein levels of muscle cells in vitro and in vivo. Myokines secreted by muscle cells promoted browning of 3T3‐L1‐derived adipocytes. In vivo activation of myokines by AAVs can reduce body weight and fat mass, increase the adipocytes browning and improve glucose tolerance and insulin sensitivity in DIO mice. Conclusions Our study provides a novel CRISPR‐based myokines activation strategy that can ameliorate obesity by promoting adipocytes browning.