GFAP-Driven GFP Expression in Activated Mouse Muller Glial Cells Aligning Retinal Blood Vessels Following Intravitreal Injection of AAV2/6 Vectors

Background: Muller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Muller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.Methodology/Principal Findings: We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Muller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Muller glial cells, several other inner retinal cell types were transduced. To obtain Muller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1(-/-) retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Muller glial cells aligning retinal blood vessels.Conclusions/Significance: Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells

Chemorepellent axon guidance molecules in spinal cord injury.

peer reviewedRegenerating axons stop growing when they reach the border of the glial-fibrotic scar, presumably because they encounter a potent molecular barrier inhibiting growth cone advance. Chemorepulsive axon guidance molecules provide a non-permissive environment restricting and channeling axon growth in the developing nervous system. These molecules could also act as growth-inhibitory molecules in the regenerating nervous system. The receptors for repulsive guidance cues are expressed in the mature nervous system, suggesting that adult neurons are sensitive to the activity of developmentally active repulsive proteins. In this review, we summarize recent observations on semaphorins, ephrins, and slits in the injured brain and spinal cord, providing evidence that these proteins are major players in inhibiting axonal regeneration and establishing the glial-fibrotic scar

Genetic Deletion of the Transcriptional Repressor NFIL3 Enhances Axon Growth <i>In Vitro</i> but Not Axonal Repair <i>In Vivo</i>

<div><p>Axonal regeneration after injury requires the coordinated expression of genes in injured neurons. We previously showed that either reducing expression or blocking function of the transcriptional repressor NFIL3 activates transcription of regeneration-associated genes <i>Arg1</i> and <i>Gap43</i> and strongly promotes axon outgrowth <i>in vitro</i>. Here we tested whether genetic deletion or dominant-negative inhibition of NFIL3 could promote axon regeneration and functional recovery after peripheral nerve lesion <i>in vivo</i>. Contrary to our expectations, we observed no changes in the expression of regeneration-associated genes and a significant delay in functional recovery following genetic deletion of <i>Nfil3</i>. When NFIL3 function was inhibited specifically in dorsal root ganglia prior to sciatic nerve injury, we observed a decrease in regenerative axon growth into the distal nerve segment rather than an increase. Finally, we show that deletion of <i>Nfil3</i> changes sciatic nerve lesion-induced expression in dorsal root ganglia of genes that are not typically involved in regeneration, including several olfactory receptors and developmental transcription factors. Together our findings show that removal of NFIL3 <i>in vivo</i> does not recapitulate the regeneration-promoting effects that were previously observed <i>in vitro</i>, indicating that <i>in vivo</i> transcriptional control of regeneration is probably more complex and more robust against perturbation than <i>in vitro</i> data may suggest.</p></div

<i>Nfil3</i> deletion impairs functional recovery from peripheral nerve injury <i>in vivo</i>.

<p>(a) <i>Nfil3</i> KO mice show no differences in the total distance moved in the open field task (n = 12, <i>t</i>₍₂₂₎ = -0.27, <i>p</i> = 0.98). (b) The latency to fall off an accelerating rotarod was not affected in <i>Nfil3</i> KO mice (n = 12, F_(1,22) = 1.02, <i>p</i> = 0.32). (c) <i>Nfil3</i> KO mice have a significantly longer beam crossing latency than wildtype mice (main effect genotype, n = 11/10, F_(1,19) = 8.893, ^#<i>p</i> = 0.008). Post-hoc t-tests indicated indicate significant differences in performance at post-lesion days 5, 13, 15 and 17 (**<i>p</i> < 0.01, *<i>p</i> < 0.05). (d) <i>Nfil3</i> KO mice also make significantly more errors when crossing the beam (main effect genotype, n = 11/10, F_(1,19) = 7.145, ^#<i>p</i> = 0.015; interaction genotype*time, F_(12,228) = 2.131, ^$<i>p</i> = 0.016). Post-hoc t-tests indicate significant differences in performance at post-lesion days 9, 12, 13, 15 and 19 (**<i>p</i> < 0.01, *<i>p</i> < 0.05).</p

Gene ontology terms overrepresented within genes differentially regulated in <i>Nfil3</i> KO mice.

<p>Gene ontology terms overrepresented within genes differentially regulated in <i>Nfil3</i> KO mice.</p

Generation and validation of <i>Nfil3</i> KO mice.

<p>(a) A schematic representation of the knockout strategy is indicated. (b) Southern blot analysis confirming correct homologous recombination at the 5’ probe side using AvrII digestion yielding fragments of 11.4 kb (wildtype) and 7.6 kb (mutant), at the 3’ probe side using EcoRV digestion yielding fragments of 12.2 kb (wildtype) and 8.9 kb (mutant), and at the Neo cassette using NheI digestion yielding a band of 12 kb (mutant only). (c) <i>Nfil3</i> mRNA levels in <i>Nfil3</i> KO and wildtype brains as measured by quantitative real-time PCR. Gene expression was normalized against Gapdh expression.</p

NFIL3 deletion does not alter the expression of regeneration-associated genes.

<p>(a) Gene expression was profiled in <i>Nfil3</i> KO mice and wildtype controls at 2 days and at 5 days post-lesion, relative to non-injured control DRGs. Gene regulation values in wildtype animals at post-lesion day 5 show a highly significant correlation (r = 0.48, <i>p</i> < 2.2x10^-16) with previously published data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127163#pone.0127163.ref031" target="_blank">31</a>] describing injury-induced gene expression changes in mouse DRGs at the same time point (GEO datasets GSM827127/8). (b) The expression of well-established regeneration-associated genes and/or NFIL3 target genes is not affected in <i>Nfil3</i> KO animals compared with wildtype controls. Of the 20 genes indicated here, 16 are in the core set of regeneration-associated genes identified in three or more independent microarray studies (bold print) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127163#pone.0127163.ref032" target="_blank">32</a>], and 8 are experimentally validated NFIL3 target genes (underlined) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127163#pone.0127163.ref011" target="_blank">11</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127163#pone.0127163.ref012" target="_blank">12</a>]. No significant differences were observed between expression profiles of <i>Nfil3</i> KO animals and wildtype controls.</p

Gene ontology terms overrepresented within genes differentially regulated at 5 days post-lesion.

<p>Gene ontology terms overrepresented within genes differentially regulated at 5 days post-lesion.</p

Dominant-negative inhibition of NFIL3 impairs regenerative axon growth <i>in vivo</i>.

<p>(a) Overview of the experimental design. At day 0 L4/L5 DRGs were injected with AAV5 virus expressing either DN-NFIL3 and GFP, or GFP only. At day 14 the animals received a unilateral crush of the sciatic nerve. At day 21 we transsected the sciatic nerve 1 cm distal from the crush and treated the proximal stump with the retrograde tracer FastBlue. The distal stump was removed for histological analysis. At day 27 animals were sacrificed, and the DRGs were removed for histological analysis. (b) Examples of control and DN-NFIL3 treated DRG sections stained with anti-βIII-tubulin in red, anti-GFP in green, and showing FastBlue labeling in blue (scale bar: 100 <b>μ</b>m). (c) The total fraction of FastBlue-positive βIII-tubulin expressing neurons was slightly lower in DN-NFIL3-treated animals compared with controls (n = 8, t₍₁₄₎ = 1.180, <i>p</i> = 0.25). (d) When the quantification of FastBlue-positive cells was limited to GFP-positive (i.e. virally transduced) neurons, a significant reduction was observed in DN-NFIL3-treated animals compared with controls (n = 8, t_(9.214) = 2.390, <i>p</i> = 0.040). (e) Examples of control and DN-NFIL3 treated sciatic nerve sections stained with anti-βIII-tubulin (scale bar: 100 <b>μ</b>m). (f) No significant difference was observed in fiber densities in the sciatic nerve at 1 cm distal of the crush (n = 8, t₍₁₄₎ = 0.095, <i>p</i> = 0.925).</p

<i>Nfil3</i> deletion enhances axon growth of DRG neurons in culture.

<p>(a) Example images of cultured embryonic DRG neurons from wildtype mice (top panels) and <i>Nfil3</i> KO mice (bottom panels) at 1, 5 and 8 days <i>in vitro</i> (DIV; scale bar: 500 <b>μ</b>m). (b) Quantification of axon lengths showed that the average axon length of <i>Nfil3</i> KO neurons was significantly higher compared to wildtype neurons at DIV1 (222±10 <b>μ</b>m vs. 178±9 <b>μ</b>m; n = 69/71), at DIV5 (931±101 <b>μ</b>m vs. 595±48 <b>μ</b>m; n = 26/31) and at DIV8 (1134±77 <b>μ</b>m vs. 806±57 <b>μ</b>m; n = 32/34) (Student’s <i>t</i> test; mean ± SEM; **<i>p</i> < 0.01).</p