Ascl1 phospho-site mutations enhance neuronal conversion of adult cortical astrocytes in vivo

Abstract

Direct neuronal reprogramming, the process whereby a terminally differentiated cell is converted into an induced neuron without traversing a pluripotent state, has tremendous therapeutic potential for a host of neurodegenerative diseases. While there is strong evidence for astrocyte-to-neuron conversion in vitro, in vivo studies in the adult brain are less supportive or controversial. Here, we set out to enhance the efficacy of neuronal conversion of adult astrocytes in vivo by optimizing the neurogenic capacity of a driver transcription factor encoded by the proneural gene Ascl1. Specifically, we mutated six serine phospho-acceptor sites in Ascl1 to alanines (Ascl1SA6) to prevent phosphorylation by proline-directed serine/threonine kinases. Native Ascl1 or Ascl1SA6 were expressed in adult, murine cortical astrocytes under the control of a glial fibrillary acidic protein (GFAP) promoter using adeno-associated viruses (AAVs). When targeted to the cerebral cortex in vivo, mCherry+ cells transduced with AAV8-GFAP-Ascl1SA6-mCherry or AAV8-GFAP-Ascl1-mCherry expressed neuronal markers within 14 days post-transduction, with Ascl1SA6 promoting the formation of more mature dendritic arbors compared to Ascl1. However, mCherry expression disappeared by 2-months post-transduction of the AAV8-GFAP-mCherry control-vector. To circumvent reporter issues, AAV-GFAP-iCre (control) and AAV-GFAP-Ascl1 (or Ascl1SA6)-iCre constructs were generated and injected into the cerebral cortex of Rosa reporter mice. In all comparisons of AAV capsids (AAV5 and AAV8), GFAP promoters (long and short), and reporter mice (Rosa-zsGreen and Rosa-tdtomato), Ascl1SA6 transduced cells more frequently expressed early- (Dcx) and late- (NeuN) neuronal markers. Furthermore, Ascl1SA6 repressed the expression of astrocytic markers Sox9 and GFAP more efficiently than Ascl1. Finally, we co-transduced an AAV expressing ChR2-(H134R)-YFP, an optogenetic actuator. After channelrhodopsin photostimulation, we found that Ascl1SA6 co-transduced astrocytes exhibited a significantly faster decay of evoked potentials to baseline, a neuronal feature, when compared to iCre control cells. Taken together, our findings support an enhanced neuronal conversion efficiency of Ascl1SA6 vs. Ascl1, and position Ascl1SA6 as a critical transcription factor for future studies aimed at converting adult brain astrocytes to mature neurons to treat disease.

NEUROG2regulates a human-specific neurodevelopmental gene regulatory program

“…We and others found that proneural transcription factors are inhibited by phosphorylation in embryonic mouse brain and spinal cord, in Xenopus and fruit fly primary neurogenesis, and in cancer cells (Ali et al, 2011; Ali et al, 2020; Azzarelli et al, 2015; Azzarelli et al, 2022; Ge et al, 2006; Hand et al, 2005; Hindley et al, 2012; Li et al, 2014; Li et al, 2012; Quan et al, 2016; Sun et al, 2001). We used this knowledge to mutate Ascl1 so that it cannot be phosphorylated and remains active, and showed that this designer proneural transcription factor is more efficient at lineage conversion of an astrocyte to an induced neuron than native Ascl1 (Ghazale et al, 2022). In this study, we used a similar strategy to mutate SP and TP sites in Neurog2 to generate Neurog2 SA9TA1 , and we demonstrated that this mutated version has an enhanced capacity to transactivate known Neurog2 target genes, Ppp1r17 and Col3a1.…”

Can pluripotent/multipotent stem cells reverse Parkinson’s disease progression?

“…Among the targets of ASCL1, Klf10 is involved in neuritogenesis of iN cells in the early stage, Myt1 and Myt1l are critical for the electrophysiological maturation of iN cells, and Neurod4 and Chd7 are required for the efficient conversion of astrocytes into neurons ( Rao Z. et al, 2021 ). Another study found that mutating six serine phosphorylation receptor sites in Ascl1 to Ascl1 SA6 could enhance the conversion of astrocytes into neurons in the mouse cerebral cortex ( Ghazale et al, 2022 ). Giehrl-Schwab et al (2022) developed the AAV-based intronic peptide split dcas9 activator system (AAV DCAS) by delivering Ascl1, Lmx1a NeuroD1, Lmx1a, Nr4a2 (ALN)/Ascl1, and miRNA218 (ALNe-218) in various combinations to efficiently reprogram striatal astrocytes into GABAergic neurons and integrate them into neural circuits, thereby improving voluntary motor function in a 6-OHDA-induced PD mouse model.…”

Neuroplasticity and regeneration after spinal cord injury

“…Ghazale et al. mutated proneural gene Asc11 by mutating 6 serine prospho-acceptor sites (Asc11 SA6 ), which was found to enhance reprogramming and downregulate astrocytic markers such as Sox9 and GFAP [124] .…”