Expression level of the reprogramming factor NeuroD1 is critical for neuronal conversion efficiency from different cell types

Abstract

Several transcription factors, including NeuroD1, have been shown to act as neuronal reprogramming factors (RFs) that induce neuronal conversion from somatic cells. However, it remains unexplored whether expression levels of RFs in the original cells affect reprogramming efficiency. Here, we show that the neuronal reprogramming efficiency from two distinct glial cell types, microglia and astrocytes, is substantially dependent on the expression level of NeuroD1: low expression failed to induce neuronal reprogramming, whereas elevated NeuroD1 expression dramatically improved reprogramming efficiency in both cell types. Moreover, even under conditions where NeuroD1 expression was too low to induce effective conversion by itself, combined expression of three RFs (Ascl1, Brn2, and NeuroD1) facilitated the breaking down of cellular barriers, inducing neuronal reprogramming. Thus, our results suggest that a sufficiently high expression level of RFs, or alternatively their combinatorial expression, is the key to achieving efficient neuronal reprogramming from different cells.

New AAV tools fail to detect Neurod1-mediated neuronal conversion of Müller glia and astrocytes in vivo

“…One hypothesis for the failure of glia-to-neuron conversion is likely due to the low expression level of Neurod1. 27 , 47 , 48 Without improved gene delivery to restrict transgene expression in glial cells, however, simply increasing the expression levels of Neurod1 by using higher viral doses 48 or an enhanced GFAP promoter 41 may further increase the leakage into endogenous neurons, a strategy counterproductive to glia-to-neuron reprogramming. In fact, a new study 49 demonstrates that the failure of Neurod1-induced microglia-to-neuron conversion is not due to the low expression level as comparable or even higher expression levels of Neurod1 cannot induce microglia-derived neurogenesis, arguing against the conclusion from the original study.…”

Lineage tracing identifies in vitro microglia‐to‐neuron conversion by NeuroD1 expression

“…Forskolin activates adenylate cyclase to produce cyclic adenosine monophosphate (cAMP), and this cAMP activates protein kinase A, which improves the survivability and maturation after neuronal conversion (Liu et al, 2013). Forskolin is also known to induce Brn2 expression (Liu et al, 2021), which has been shown to promote NeuroD1-mediated neuronal reprogramming (Matsuda-Ito et al, 2022). These facts suggest that VPA and Forskolin enhance neuronal conversion, possibly through the induction of endogenous gene expression associated with reprogramming.…”

“…We prepared primary microglia from mouse using a previously reported protocol (Matsuda et al, 2019; Matsuda‐Ito et al, 2022). We dissected cortexes of BL6 mice at postnatal day 1 (P1) or Hexb‐Cre ERT2 ::Stop‐YFP mice at P4 (Masuda et al, 2020) after peeling of meninges to obtain microglia from glial cell mixtures.…”

“…The vector backbone of the lentiviral plasmid expressing M2rtta is FUW. Plasmids used in this study are similar to those described in our previous report (Matsuda et al, 2019;Matsuda-Ito et al, 2022). For cells to be infected efficiently with the lentivirus, the virus must be added as soon as possible after plating the microglia.…”

“…Microglia and macrophages, which accumulate at tissue damage sites to remove dead cells and debris after a brain injury such as stroke (Fumagalli et al, 2015), can be target cells for neuronal reprogramming. We previously showed that mouse microglia are converted into neurons both in vitro and in vivo by the ectopic expression of lentivirus‐encoded NeuroD1 (Matsuda et al, 2019; Matsuda‐Ito et al, 2022). Microglia and macrophages are known to share similar gene expression patterns in the healthy CNS, although they reside in different locations (Grassivaro et al, 2020).…”