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.

No abstract

In the originally published version of our manuscript, values in Table S1 were missing a decimal separator (dot) and some columns were moved due to program incompatibilities when transferring the original data from .csv to .xlsx. To address this, we have now added the ''.'' and corrected the columns so that the Excel file of Table S1 corresponds to the original .csv file. We apologize for the oversight and for any resulting confusion.

Glycosaminoglycans, both cell-surface and exogenous, can interfere with DNA delivery efficiency of nonviral carrier systems. In this work, we report an extensive comparative study to explore the effect of exogenously added chondroitin sulfate on biophysical characteristics, cellular uptake, transfection efficiency, and intracellular trafficking of nanocomplexes formed using primary and secondary amphipathic peptides developed in our laboratory. Our results indicate that the presence of exogenous chondroitin sulfate exhibits differential enhancement in transfection efficiency of the amphipathic peptides depending upon their chemical nature. The enhancement was more pronounced in primary amphipathic peptide-based nanocomplexes as compared to the secondary counterpart. This difference can be attributed to possible alteration of the intracellular entry pathway in addition to increased extracellular stability, less cellular toxicity, and assistance in nuclear accumulation. These results imply potential use of glycosaminoglycans such as chondroitin sulfate to improve the transfection efficiency of primary amphipathic peptides for possible in vivo applications.

The present study is focused on a simple execution in the applicability of non-toxic lower generation poly amidoamine (PA-MAM) dendrimers as effective nano-vectors in targeted gene delivery to the skin. So far the first three lower generation (G1, G2 and G3) PAMAM dendrimers have been overlooked as nucleic acid therapeutics. In this study, we have first carried out a systematic biophysical analysis to analyze their ability for plasmid DNA (pDNA) condensation and subsequent release by ethidium bromide assay to optimize dendrimer to DNA charge ratio for effective pDNA condensation and release. Interestingly, stopped flow fluorescence spectroscopic analysis on pDNAdendrimer binding kinetics revealed the efficiency of generations G2 and G3 in pDNA condensation in comparison with G1 through four steps. Importantly, it validates aggregation and association of the dendrimer in the vicinity of pDNA. Based on these understanding, successful in vitro cellular uptake of dendriplexes followed by their transfection into CHOÀK1 cells was demonstrated at the charge ratio Z + /-5 and 10. The interesting observations of gene transfection with CHOÀK1 cells were extended to HaCaT cell line and efficient pDNA transfections were evidenced with negligible cytotoxicity. In addition to this, the stability of the dendriplexes at the charge ratio Z + /-5 for G3 and Z + /-10 for G2 was found even upto 50 % serum concentration suggesting possible future applicability in in vivo. Overall, the current approach promises the use of lower generation dendrimers for pDNA delivery to the skin using only electrostatic nanocomplex formation without any covalent linkage.