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Summary Astrocyte-to-neuron conversion is a promising avenue for neuronal replacement therapy. Neurons are particularly dependent on mitochondrial function, but how well mitochondria adapt to the new fate is unknown. Here, we determined the comprehensive mitochondrial proteome of cortical astrocytes and neurons, identifying about 150 significantly enriched mitochondrial proteins for each cell type, including transporters, metabolic enzymes, and cell-type-specific antioxidants. Monitoring their transition during reprogramming revealed late and only partial adaptation to the neuronal identity. Early dCas9-mediated activation of genes encoding mitochondrial proteins significantly improved conversion efficiency, particularly for neuron-enriched but not astrocyte-enriched antioxidant proteins. For example, Sod1 not only improves the survival of the converted neurons but also elicits a faster conversion pace, indicating that mitochondrial proteins act as enablers and drivers in this process. Transcriptional engineering of mitochondrial proteins with other functions improved reprogramming as well, demonstrating a broader role of mitochondrial proteins during fate conversion., Graphical abstract, Highlights • Mitochondrial proteomes of cortical astrocytes and neurons are distinct • Astrocyte-enriched mitochondrial proteins are downregulated late in neuronal conversion • Neuron-enriched mitochondrial proteins are upregulated late in neuronal conversion • Early induction of neuronal mitochondrial proteins improves neuronal reprogramming, Russo et al. identify mitochondrial proteins enriched in neurons or astrocytes. Astrocyte-enriched mitochondrial proteins are often only partially downregulated during astrocyte-to-neuron direct reprogramming. Neuron-enriched ones are upregulated late and mainly in reprogrammed neurons. CRISPRa-mediated early induction of neuron-enriched mitochondrial proteins boosts direct neuronal reprogramming speed and efficiency.

Summary Astrocytes are particularly promising candidates for reprogramming into neurons, as they maintain some of the original patterning information from their radial glial ancestors. However, to which extent the position of astrocytes influences the fate of reprogrammed neurons remains unknown. To elucidate this, we performed stab wound injury covering an entire neocortical column, including the gray matter (GM) and white matter (WM), and targeted local reactive astrocytes via injecting FLEx switch (Cre-On) adeno-associated viral (AAV) vectors into mGFAP-Cre mice. Single proneural factors were not sufficient for adequate reprogramming, although their combination with the nuclear receptor-related 1 protein (Nurr1) improved reprogramming efficiency. Nurr1 and Neurogenin 2 (Ngn2) resulted in high-efficiency reprogramming of targeted astrocytes into neurons that develop lamina-specific hallmarks, including the appropriate long-distance axonal projections. Surprisingly, in the WM, we did not observe any reprogrammed neurons, thereby unveiling a crucial role of region- and layer-specific differences in astrocyte reprogramming., Graphical Abstract, Highlights • AAV can be targeted to reactive astrocytes upon stab wound injury • Expression of Ngn2 and Nurr1 in these astrocytes induces pyramidal neurons • Induced pyramidal neurons acquire correct layer identity and axonal projections • Neurons cannot be induced in the white matter, Neurons dying after brain injury cannot be replaced. Mattugini, Bocchi, et al. show that local astrocytes can be converted into functional neurons acquiring appropriate layer identity and connectivity by expression of neurogenic factors in a mouse model of traumatic brain injury.

Despite the widespread interest in direct neuronal reprogramming, the mechanisms underpinning fate conversion remain largely unknown. Our study revealed a critical time point after which cells either successfully convert into neurons or succumb to cell death. Co-transduction with Bcl-2 greatly improved negotiation of this critical point by faster neuronal differentiation. Surprisingly, mutants with reduced or no affinity for Bax demonstrated that Bcl-2 exerts this effect by an apoptosis-independent mechanism. Consistent with a caspase-independentrole, ferroptosis inhibitors potently increased neuronal reprogramming by inhibiting lipid peroxidation occurring during fate conversion. Genome-wide expression analysis confirmed that treatments promoting neuronal reprogramming elicit an anti-oxidative stress response. Importantly, co-expression of Bcl-2 and anti-oxidative treatments leads to an unprecedented improvement in glial-to-neuron conversion after traumatic brain injury invivo, underscoring the relevance of these pathways in cellular reprograming irrespective of cell type invitro and invivo.

The ability to directly reprogram mature cells to alternative fates challenges concepts of how cell identities are maintained, erased, and acquired. Recent advances in understanding and overcoming hurdles to direct neuronal conversion have provided new insights into mechanisms that maintain cell identity programs and have enabled high efficiency reprogramming invivo. We discuss key cell-intrinsic molecular and metabolic constraints that influence the establishment of a new identity as well as environmental inputs from injured brains that favor or harm the conversion process. Finally, we outline the challenges ahead with a particular focus on direct neuronal reprogramming invivo.

nized with PLP139-151, a clearly defined relapsing–remitting disease course similar to relapsing–remitting multiple sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease course. To prevent the large infiltration of immune cells from skewing resident CNS cell analysis, we incorporated an antibody against the hematopoietic marker CD45 into the panel and gated out those cells in OLC analysis. Flow cytometry results demonstrated a robust loss of oligodendrocytes during onset of EAE and an early expansion of OPCs. By disease peak the early OPCs expressed more mature markers of OLCs but never fully differentiated into mature myelin-forming oligodendrocytes throughout the rest of the disease course. The early expansion of OPCs was further associated with an increase in the chemotactic protein CD44 in OPCs from the brain. In summary we present here quantification of OLCs and functional global analysis of the myelinproducing cells in EAE mice by flow cytometry. Characterization in mouse models of human CNS disease presents a rapid means to evaluate potential therapeutic interventions in vivo for direct and indirect effects on demyelination and myelin regeneration.