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Direct reprogramming based on genetic factors resembles a promising strategy to replace lost cells in degenerative diseases such as Parkinson's disease. For this, we developed a knock-in mouse line carrying a dual dCas9 transactivator system (dCAM) allowing the conditional in vivo activation of endogenous genes. To enable a translational application, we additionally established an AAV-based strategy carrying intein-split-dCas9 in combination with activators (AAV-dCAS). Both approaches were successful in reprogramming striatal astrocytes into induced GABAergic neurons confirmed by single-cell transcriptome analysis of reprogrammed neurons in vivo. These GABAergic neurons functionally integrate into striatal circuits, alleviating voluntary motor behavior aspects in a 6-OHDA Parkinson's disease model. Our results suggest a novel intervention strategy beyond the restoration of dopamine levels. Thus, the AAV-dCAS approach might enable an alternative route for clinical therapies of Parkinson's disease.

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.