Transdifferentiating Astrocytes Into Neurons Using ASCL1 Functionalized With a Novel Intracellular Protein Delivery Technology

Abstract

Cellular transdifferentiation changes mature cells from one phenotype into another by altering their gene expression patterns. Manipulating expression of transcription factors, proteins that bind to DNA promoter regions, regulates the levels of key developmental genes. Viral delivery of transcription factors can efficiently reprogram somatic cells, but this method possesses undesirable side effects, including mutations leading to oncogenesis. Using protein transduction domains (PTDs) fused to transcription factors to deliver exogenous transcription factors serves as an alternative strategy that avoids the issues associated with DNA integration into the host genome. However, lysosomal degradation and inefficient nuclear localization pose significant barriers when performing PTD-mediated reprogramming. Here, we investigate a novel PTD by placing a secretion signal sequence next to a cleavage inhibition sequence at the end of the target transcription factor–achaete scute homolog 1 (ASCL1), a powerful regulator of neurogenesis, resulting in superior stability and nuclear localization. A fusion protein consisting of the amino acid sequence of ASCL1 transcription factor with this novel PTD added can transdifferentiate cerebral cortex astrocytes into neurons. Additionally, we show that the synergistic action of certain small molecules improves the efficiency of the transdifferentiation process. This study serves as the first step toward developing a clinically relevant in vivo transdifferentiation strategy for converting astrocytes into neurons.

Induced Neurons for Disease Modeling and Repair: A Focus on Non-fibroblastic Cell Sources in Direct Reprogramming

“…Spontaneous and evoked synaptic responses in these glia-derived NeuroD1-converted neurons suggest that the iN integrated into local neural circuits (Guo et al, 2014). Increased plasticity in reactive astrocytes resulted in higher conversion rates when compared against non-reactive astrocytes (Brulet et al, 2017;Robinson et al, 2018). In a stroke model, in vivo direct reprogramming of astrocytes to iN following stroke resulted in improvements in motor function as soon as 3 weeks after the reprogramming process, with sustained long-term functional recovery (Livingston et al, 2020).…”

Direct Reprogramming of Somatic Cells to Neurons: Pros and Cons of Chemical Approach

“…However, due to their low efficiency, transgene silencing, inflammation and poor nuclear uptake, are less commonly used in transdifferentiation studies [ 45 ]. Lately, the use of neural exosomes [ 46 ] and the protein transduction domains (PTDs) fused to TFs allow the direct delivery of exogenous TFs avoiding the problems associated with DNA integration into the host genome [ 47 ] opening up new strategies for possible clinical applications.…”

“…The inability to produce the neuronal subtypes which are lost in neurodegenerative disorders like Parkinson’s disease, Alzheimer’s disease, Amyotrophic Lateral Sclerosis, Huntingdon’s disease represents a major limitation in current small molecules transdifferentiation field. However, it was showed that a single TF such as ASCL1, using a novel protein intracellular delivery technology, in combination with the small molecules LDN193189, SB431542, DAPT and valproic acid can rapidly reprogram astrocytes into mature GABAergic and glutamatergic interneurons with high efficiency [ 47 ]. Moreover, Chabrat et al developed a novel in vitro model of dopaminergic-like neurons derived from human nasal olfactory stem cells through a six step transdifferentiation protocol based on a specific combination of signaling pathway modulators [ 60 ].…”

Neuronal Reprogramming for Tissue Repair and Neuroregeneration

“…Further work showed that the proneural factor ASCL1 alone was able to guide the conversion of human fibroblasts into a mix of glutamatergic and GABAergic neurons [ 77 ]. Interestingly, ASCL1 promoted reprogramming of astrocytes to mainly GABAergic neurons [ 78 ]. This highlighted that the effect of transcription factor to reprogram cell fate is partly dependent on the starting cell type.…”