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Axon regeneration is hindered by a decline of intrinsic axon growth capability in mature neurons. Reversing this decline is associated with the induction of a large repertoire of regeneration-associated genes (RAGs), but the underlying regulatory mechanisms of the transcriptional changes are largely unknown. Here, we establish a correlation between diminished axon growth potential and histone 4 (H4) hypoacetylation. When neurons are triggered into a growth state, as in the conditioning lesion paradigm, H4 acetylation is restored, and RAG transcription is initiated. We have identified a set of target genes of Smad1, a proregenerative transcription factor, in conditioned DRG neurons. We also show that, during the epigenetic reprogramming process, histone-modifying enzymes work together with Smad1 to facilitate transcriptional regulation of RAGs. Importantly, targeted pharmacological modulation of the activity of histone-modifying enzymes, such as histone deacetylases, leads to induction of multiple RAGs and promotion of sensory axon regeneration in a mouse model of spinal cord injury. Our findings suggest epigenetic modulation as a potential therapeutic strategy to enhance axon regeneration.

The embryonic formation of midbrain dopaminergic (mDA) neuronsin vivoprovides critical guidelines for thein vitrodifferentiation of mDA neurons from stem cells, which are currently being developed for Parkinson's disease cell replacement therapy. Bone morphogenetic protein (BMP)/SMAD inhibition is routinely used during early steps of stem cell differentiation protocols, including for the generation of mDA neurons. However, the function of the BMP/SMAD pathway forin vivospecification of mammalian mDA neurons is virtually unknown. Here, we report that BMP5/7-deficient mice (Bmp5−/−;Bmp7−/−) lack mDA neurons due to reduced neurogenesis in the mDA progenitor domain. As molecular mechanisms accounting for these alterations inBmp5−/−;Bmp7−/−mutants, we have identified expression changes of the BMP/SMAD target genes MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog). Conditionally inactivating SMAD1 in neural stem cells of micein vivo(Smad1Nes) hampered the differentiation of progenitor cells into mDA neurons by preventing cell cycle exit, especially of TH+SOX6+(tyrosine hydroxylase, SRY-box 6) and TH+GIRK2+(potassium voltage-gated channel subfamily-J member-6) substantia nigra neurons. BMP5/7 robustly increased thein vitrodifferentiation of human induced pluripotent stem cells and induced neural stem cells to mDA neurons by up to threefold. In conclusion, we have identified BMP/SMAD signaling as a novel critical pathway orchestrating essential steps of mammalian mDA neurogenesisin vivothat balances progenitor proliferation and differentiation. Moreover, we demonstrate the potential of BMPs to improve the generation of stem-cell-derived mDA neuronsin vitro, highlighting the importance of sequential BMP/SMAD inhibition and activation in this process.SIGNIFICANCE STATEMENTWe identify bone morphogenetic protein (BMP)/SMAD signaling as a novel essential pathway regulating the development of mammalian midbrain dopaminergic (mDA) neuronsin vivoand provide insights into the molecular mechanisms of this process. BMP5/7 regulate MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog) expression to direct mDA neurogenesis. Moreover, the BMP signaling component SMAD1 controls the differentiation of mDA progenitors, particularly to substantia nigra neurons, by directing their cell cycle exit. Importantly, BMP5/7 increase robustly the differentiation of human induced pluripotent and induced neural stem cells to mDA neurons. BMP/SMAD are routinely inhibited in initial stages of stem cell differentiation protocols currently being developed for Parkinson's disease cell replacement therapies. Therefore, our findings on opposing roles of the BMP/SMAD pathway duringin vitromDA neurogenesis might improve these procedures significantly.

Visual Abstract, Neural stem cells (NSCs) hold great promise for neural repair in cases of CNS injury and neurodegeneration; however, conventional cell-based transplant methods face the challenges of poor survival and inadequate neuronal differentiation. Here, we report an alternative, tissue-based transplantation strategy whereby cerebral organoids derived from human pluripotent stem cells (PSCs) were grafted into lesioned mouse cortex. Cerebral organoid transplants exhibited enhanced survival and robust vascularization from host brain as compared to transplants of dissociated neural progenitor cells (NPCs). Engrafted cerebral organoids harbored a large NSC pool and displayed multilineage neurodifferentiation at two and four weeks after grafting. Cerebral organoids therefore represent a promising alternative source to NSCs or fetal tissues for transplantation, as they contain a large set of neuroprogenitors and differentiated neurons in a structured organization. Engrafted cerebral organoids may also offer a unique experimental paradigm for modeling human neurodevelopment and CNS diseases in the context of vascularized cortical tissue.