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. 2022 Sep 19:10:914386.
doi: 10.3389/fcell.2022.914386. eCollection 2022.

Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina

Nguyet Le  1 , Haley Appel  1 , Nicole Pannullo  1 , Thanh Hoang  1 , Seth Blackshaw  1   2   3   4   5
Affiliations

Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina

Nguyet Le et al. Front Cell Dev Biol. .

Abstract

Direct reprogramming of retinal Müller glia is a promising avenue for replacing photoreceptors and retinal ganglion cells lost to retinal dystrophies. However, questions have recently been raised about the accuracy of studies claiming efficient glia-to-neuron reprogramming in retina that were conducted using GFAP mini promoter-driven adeno-associated virus (AAV) vectors. In this study, we have addressed these questions using GFAP mini promoter-driven AAV constructs to simultaneously overexpress the mCherry reporter and candidate transcription factors predicted to induce glia-to-neuron conversion, in combination with prospective genetic labeling of retinal Müller glia using inducible Cre-dependent GFP reporters. We find that, while control GFAP-mCherry constructs express faithfully in Müller glia, 5 out of 7 transcription factor overexpression constructs tested are predominantly expressed in amacrine and retinal ganglion cells. These findings demonstrate strong insert-dependent effects on AAV-based GFAP mini promoter specificity that preclude its use in inferring cell lineage relationships when studying glia-to-neuron conversion in retina.

Keywords: adeno associated viral vectors; adeno-associated virus; cell lineage analysis; ectopic; glia; glial fibrillary acidic protein; muller glia; reprogramming.

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Conflict of interest statement

SB is a cofounder and shareholder of CDI Labs, and has received research support from Genentech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Summary of constructs tested in the study. (A) Schematic of the GFAP AAV constructs and (B) the workflow: three weeks old GlastCreERT2;Sun1-GFP mice were induced with 3 daily doses of 4-OHT delivered intraperitoneally, intravitreally injected with GFAP AAV constructs at 2 months of age, and collected 8 days and 21 days following AAV infection for analysis.
FIGURE 2
FIGURE 2
GFAP promoter constructs expressing mCherry, Nrl, and Oct4 show strong Müller glia-specific expression. Representative immunostaining for GFP, mCherry and 3 neuronal markers: (A) Tfap2a, (B) Rbpms, and (C) Otx2 expression in the retinas collected 21 days post GFAP AAV infection. GFAP-mCherry, GFAP-Oct4-mCherry and GFAP-Nrl-mCherry showed almost complete colocalization of construct-derived mCherry and Müller glia-specific Sun1-GFP. White arrowheads indicate co-labeled mCherry+/GFP + cells. Little to no mCherry expression in amacrine, retinal ganglion and bipolar cells is observed in these GFAP AAV constructs. Quantification of transduction efficiency (mCherry+ & GFP + cells/GFP + cells) (D) and transduction specificity (mCherry+ & GFP + cells/mCherry + cells) (E). Quantification of mean percentage ±SD of marker & mCherry+/GFP cells: Tfap2a+ & mCherry+/GFP + cells (in INL and GCL), Rbpms+ & mCherry+/GFP + cells (in GCL) and Otx2+&mCherry+/GFP + cells (in INL) (F). Significance was determined via one-way ANOVA or two-way ANOVA with Dunnett’s test: ***p < 0.001, ****p < 0.0001. Each data point in the bar graphs was calculated from an individual retina (n = 6). INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 40 μm.
FIGURE 3
FIGURE 3
GFAP promoter constructs expressing NeuroD1, Mybl1 and Insm1 show ectopic mCherry expression in amacrine and ganglion cells. Representative immunostaining for GFP, mCherry and 3 neuronal markers: (A) Tfap2a, (B) Rbpms, and (C) Otx2 expression in the retinas collected 21 days post GFAP AAV infection. GFAP-NeuroD1-mCherry, GFAP-Mybl1-mCherry and GFAP-Insm1-mCherry showed little to no colocalization of construct-derived mCherry and Müller glia-specific Sun1-GFP. High levels of mCherry expression in amacrine and ganglion cells, but not in bipolar cells were observed in retinas infected with these GFAP AAV constructs. Yellow arrowheads indicate co-labeled mCherry+ & marker + cells. Quantification of transduction efficiency (mCherry+ & GFP + cells/GFP + cells) (D) and transduction specificity (mCherry+ & GFP + cells/mCherry + cells) (E). Quantification of mean percentage ±SD of marker & mCherry+/GFP cells: Tfap2a+ & mCherry+/GFP + cells (in INL and GCL), Rbpms+ & mCherry+/GFP + cells (in GCL) and Otx2+&mCherry+/GFP + cells (in INL) (F). Significance was determined via one-way ANOVA or two-way ANOVA with Dunnett’s test: ****p < 0.0001. Each data point in the bar graphs was calculated from an individual retina (n = 6). INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 40 μm.
FIGURE 4
FIGURE 4
GFAP promoter constructs tandemly expressing Atoh7-Ascl1 and Atoh7-Brn3b show ectopic mCherry expression in amacrine and ganglion cells. Representative immunostaining for GFP, mCherry and 3 neuronal markers: (A) Tfap2a, (B) Rbpms, and (C) Otx2 expression in the retinas collected 21 days post GFAP AAV infection. GFAP-Atoh7-Ascl1-mCherry and GFAP-Atoh7-Brn3b-mCherry showed little to no colocalization of construct-derived mCherry and Müller glia-specific Sun1-GFP. High levels of mCherry expression in amacrine and ganglion cells, but not in bipolar cells were observed in retinas infected with these GFAP AAV constructs. Yellow arrowheads indicate co-labeled mCherry+ & marker + cells. Quantification of transduction efficiency (mCherry+ & GFP + cells/GFP + cells) (D) and transduction specificity (mCherry+ & GFP + cells/mCherry + cells) (E). Quantification of mean percentage ±SD of marker & mCherry+/GFP cells: Tfap2a+ & mCherry+/GFP + cells (in INL and GCL), Rbpms+ & mCherry+/GFP + cells (in GCL) and Otx2+&mCherry+/GFP + cells (in INL) (F). Significance was determined via one-way ANOVA or two-way ANOVA with Dunnett’s test: ****p < 0.0001. Each data point in the bar graphs was calculated from an individual retina (n = 6). INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 40 μm.
FIGURE 5
FIGURE 5
Inserts that show ectopic neuronal expression when driven by the GFAP promoter are restricted to Müller glia when expressed using GlastCreERT2-dependent EF1a FLEX AAV constructs. (A) A schematic of Cre-dependent FLEX-mCherry control and FLEX-Atoh7-Brn3b-mCherry constructs. (B) A schematic of the experimental workflow: two months old GlastCreERT2;Sun1-GFP mice were intravitreally injected with FLEX AAV constructs and collected 21 days following the 4th dose of 4-OHT i.p injection for analysis. (C) Representative immunostaining for GFP and mCherry expression in FLEX AAV infected retinas collected 21 days post Cre-recombination induction. FLEX-Atoh7-Brn3b-mCherry showed extensive colocalization of GFP and mCherry. White arrowheads indicate mCherry+/GFP + cells. (D) Quantification of mean percentage ±SD of mCherry+/GFP + cells. Significance was determined via unpaired two-tailed t-test and Gaussian distribution: ***p < 0.001. Each data point in the bar graphs was calculated from an individual retina (n = 2). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer Scale bar = 40 μm.
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References

    1. Aartsen W. M., van Cleef K. W. R., Pellissier L. P., Hoek R. M., Vos R. M., Blits B., et al. (2010). GFAP-driven GFP expression in activated mouse Müller glial cells aligning retinal blood vessels following intravitreal injection of AAV2/6 vectors. PLoS One 5, e12387. 10.1371/journal.pone.0012387 - DOI - PMC - PubMed
    1. Blackshaw S., Sanes J. R. (2021). Turning lead into gold: Reprogramming retinal cells to cure blindness. J. Clin. Invest.. 131, 146134. 10.1172/JCI146134 - DOI - PMC - PubMed
    1. Brulet R., Matsuda T., Zhang L., Miranda C., Giacca M., Kaspar B. K., et al. (2017). NEUROD1 instructs neuronal conversion in non-reactive astrocytes. Stem Cell Rep. 8, 1506–1515. 10.1016/j.stemcr.2017.04.013 - DOI - PMC - PubMed
    1. Chen W., Zheng Q., Huang Q., Ma S., Li M. (2021). Repressing PTBP1 is incapable to convert reactive astrocytes to dopaminergic neurons in a mouse model of Parkinson’s disease. bioRxiv. 10.1101/2021.11.12.468309 - DOI - PMC - PubMed
    1. de Melo J., Miki K., Rattner A., Smallwood P., Zibetti C., Hirokawa K., et al. (2012). Injury-independent induction of reactive gliosis in retina by loss of function of the LIM homeodomain transcription factor Lhx2. Proc. Natl. Acad. Sci. U. S. A. 109, 4657–4662. 10.1073/pnas.1107488109 - DOI - PMC - PubMed