Enhancing NeuroD1 Expression to Convert Lineage-Traced Astrocytes into Neurons

Abstract

Regenerating functional new neurons in adult mammalian brains has been proven a difficult task for decades. Recent advancement in direct glia-to-neuron conversion in vivo opens a new field for neural regeneration and repair. However, this emerging new field is facing serious challenges from misuse of viral vectors to misinterpretation of conversion data. Here, we employ a variety of AAV vectors with different promoters and enhancers to demonstrate that astrocytes can be converted into neurons in a NeuroD1 dose-dependent manner in both wildtype (WT) and transgenic mice. Notably, astrocytes in WT mice were relatively easy to convert with higher conversion efficiency, whereas lineage-traced astrocytes in Aldh1l1-CreERT2 mice showed high resistance to reprogramming but were still converted into neurons after enhancing NeuroD1 expression with CMV enhancer. Furthermore, under two-photon microscope, we observed direct astrocyte-to-neuron conversion within 3 weeks of serial live imaging in the mouse cortex. We also demonstrated that high titre AAV reaching 1013 GC/ml caused severe neuronal leakage using a variety of AAV GFAP::GFP vectors, highlighting the necessity to inject low titre AAV into healthy brains to avoid artifactual results. Together, our studies suggest that lineage-traced astrocytes can be converted into neurons but require stronger conversion force such as enhanced NeuroD1 expression. Failure to recognize the difference between WT astrocytes and lineage-traced astrocytes in terms of conversion barrier will lead to misinterpretation of data.

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

“…One potential solution to correct AAV- GFAP -mediated neuronal leaky expression is to reduce AAV doses, as suggested in recent studies. 40 , 41 We tested this possibility by diluting AAV titers from e13 vg/ml to the suggested dose of e11–e12 vg/ml for the 4 reprogramming factors. We first analyzed the neuronal leaky patterns after reducing AAV titers 10-fold to the dose of e12 vg/ml for the 4 reprogramming factors in Sun1-GFP fate mapping mice at 3 weeks post-injection ( Fig.…”

Efficient Dlx2-mediated astrocyte-to-neuron conversion and inhibition of neuroinflammation by NeuroD1

“…This has also been nicely illustrated in our dose-finding studies using control AAV9 GFAP::GFP, where high titre at 1E13 GC/ml results in severe activation of microglia. In our most recent study, we show that injection of high titre 1E13 GC/ml AAV9 GFAP::GFP (1 μl) results in 50-60% neuronal leakage 37 , highlighting the necessity to use low titre AAV for conversion studies. Based on this study and our previous studies, we recommend that AAV titre should be kept in a safe range of ~E11 to E12 GC/ml when conducting AtN conversion studies.…”

“…On NeuroD1-mediated astrocyte-to-neuron conversion, following our initial study and a series of continuous studies 9,20,23,24,26,31 , many other labs have successfully repeated our NeuroD1 conversion results 32–35 . Moreover, our lab and Davidson lab have used the same astrocytic lineage-tracing mice (Aldh1|1-CreER T2 ) to demonstrate that NeuroD1 can convert lineage-traced astrocytes into neurons, although conversion efficiency varies in different studies 14,36,37 . Importantly, we show that lineage-traced astrocytes are more difficult to convert and require enhancer to induce higher expression level of NeuroD1 to overcome the conversion barrier 37 .…”

“…Moreover, our lab and Davidson lab have used the same astrocytic lineage-tracing mice (Aldh1|1-CreER T2 ) to demonstrate that NeuroD1 can convert lineage-traced astrocytes into neurons, although conversion efficiency varies in different studies 14,36,37 . Importantly, we show that lineage-traced astrocytes are more difficult to convert and require enhancer to induce higher expression level of NeuroD1 to overcome the conversion barrier 37 .…”

Cell Reprogramming for Regeneration and Repair of the Nervous System

“…While the type of promoter and virus can greatly affect the cells targeted, the concentration or titer of the viral injection can also cause misleading results [ 159 ]. Viral loads that are too high can cause leakage of the transcription factors from transduced cells.…”