Induction of Autophagy Promotes Clearance of RHOP23H Aggregates and Protects From Retinal Degeneration

Autophagy is a critical metabolic process that acts as a major self-digestion and recycling pathway contributing to maintain cellular homeostasis. An emerging field of research supports the therapeutic modulation of autophagy for treating human neurodegenerative disorders, in which toxic aggregates are accumulated in neurons. Our previous study identified Ezrin protein as an inhibitor of autophagy and lysosomal functions in the retina; thus, in turn, identifying it as a potential pharmacological target for increasing retinal cell clearance to treat inherited retinal dystrophies in which misfolded proteins have accumulated. This study aimed to verify the therapeutic inhibition of Ezrin to induce clearance of toxic aggregates in a mouse model for a dominant form of retinitis pigmentosa (i.e., RHOP23H/+). We found that daily inhibition of Ezrin significantly decreased the accumulation of misfolded RHOP23H aggregates. Remarkably, induction of autophagy, by a drug-mediated pulsatile inhibition of Ezrin, promoted the lysosomal clearance of disease-linked RHOP23H aggregates. This was accompanied with a reduction of endoplasmic reticulum (ER)-stress, robust decrease of photoreceptors' cell death, amelioration in both retinal morphology and function culminating in a better preservation of vision. Our study opens new perspectives for a pulsatile pharmacological induction of autophagy as a mutation-independent therapy paving the way toward a more effective therapeutic strategy to treat these devastating retinal disorders due to an accumulation of intracellular toxic aggregates

Retinal Degeneration in MPS-IIIA Mouse Model

Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo A) is one of the most severe lysosomal storage disorder (LSD) caused by the inherited deficiency of sulfamidase, a lysosomal sulfatase enzyme involved in the stepwise degradation of heparan sulfates (HS). MPS-IIIA patients show multisystemic problems, including a strong impairment of central nervous system (CNS), mild somatic involvement, and ocular manifestations that result in significant visual impairment. Despite the CNS and somatic pathology have been well characterized, studies on visual system and function remain partially explored. Here, we characterized the retina morphology and functionality in MPS-IIIA mouse model and analyzed how the SGSH deficiency affects the autophagic flux. MPS-IIIA mice exhibited a progressive retinal dystrophy characterized by significant alterations in visual function. The photoreceptor degeneration was associated with HS accumulation and a block of autophagy pathway. These events caused a reactive microgliosis, and a development of apoptotic processes in MPS-IIIA mouse retina. Overall, this study provides the first phenotypic spectrum of retinal disorders in MPS-IIIA and significantly contributes for diagnosis, counseling, and potential therapies development

TGF-β signaling regulates RhoA levels via two independent and synergistic cascades.

<p><b>(a)</b> qRT-PCR analysis of erk2 transcripts in total eye RNA derived from St32 control-MOs, miR-181a/b morphants and TGF-β-treated miR-181a/b morphants. The TGF-β-mediated increase of miR-181a/b caused a rescue of miR-181a/b target transcripts, such as <i>prox1</i> and <i>erk2</i>, in miR-181a/b morphants. <b>(b, c)</b> Representative Western blotting on protein from St32 eyes (b) and corresponding quantification (c) show that administration of TGF-β to MO-miR-181a/b embryos leads to restoration of total-, phospho-Erk2 and RhoA protein levels. When MO-miR-181a/b embryos were treated with both TGF-β and the proteasomal inhibitor MG132, total- and phospho-Erk2 protein levels were still rescued, whereas RhoA levels were only partially rescued. Data are means +SEM.* P <0.05; **P <0.01; *** P <0.001 (two-way ANOVA).</p

TGF-β pathway down-regulation from early phases of medaka fish embryo development determines alteration of programmed cell death programs in the retina.

<p><b>(a)</b> MO-<i>tgfβr1</i> is designed to sterically block the fourth intron-exon splice donor site of the <i>tgfβr1</i> transcript, causing a partial retention of the intronic sequence as shown by PCR analysis. <b>(b-g)</b> Control (b), MO-<i>tgfβr1</i> (c), SB43152 (d) and TGF-β- (e) treated control, MO-miR-181a/b (f) and TGF-β treated MO-miR-181a/b (g) medaka fish embryos at stage 32. The MO-<i>tgfβr1</i> injected embryos showed a phenotype characterized by abnormal body and head structures, including microphthalmia. <b>(h-m)</b> Alteration of the TGF-β pathway from the early stages of development caused an increase of retina cell death as shown by TUNEL assay (h, i). Administration of drugs that lead to a TGF-β pathway down-regulation (j, SB43152) or increase (k, TGF-β) from stage 30 onwards did not cause cell death alteration in control medaka retina. Similarly, no significant alterations were found in MO-miR-181a/b- (l) and TGF-β-treated MO-miR-181a/b (m) retinas. <b>(n)</b> Quantification of TUNEL positive cells; N = 12 eyes were analyzed for each treatment.</p

TGF-β signaling modulates miR-181a/b action in the assembly of retinal circuitry.

<p><b>(a-e)</b> Retinal frontal sections of St40 DMSO-treated (a), TGF-β-treated (b) control-MO medaka fish embryos, miR-181a/b morphant embryos (c), SB432542-treated embryos (d) and TGF-β-treated (e) miR-181a/b morphant medaka fish embryos processed for Richardson-Romeis staining. Red bars, IPL thickness. Scale bars: 20μm. <b>(f)</b> Quantitative analysis of IPL thickness indicated as the ratio in the central retina between the IPL area and total retinal area. Data are means ± SEM. ***, P <0.001 (one-way ANOVA). <b>(g-j)</b> Representative images of amacrine cells from St40 retinal sections of control-MOs (g), miR-181a/b morphant (h), SB432542-treated control (i) and miR-181a/b morphant treated with TGF-β (j) Six3:eGFP transgenic medaka fish embryos. Cell nuclei are stained with DAPI (blue). GFP (green signal) stains amacrine cell soma and neurites; red arrows, Six3 axon-like structure of amacrine cells; red bars, thickness of the IPL. SB432542 treatment (i) phenocopied the amacrine cell neuritogenesis defects observed in miR-181a/b morphants (h). Addition of TGF-β (from St30 to St40) to miR-181a/b morphants (j) was sufficient to rescue neuritogenesis defects of miR-181a/b morphant transgenic embryos. INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: 20μm. <b>(k-n)</b> Representative 2-D reconstruction of confocal images of St32 control-MOs (k), miR-181a/b morphant (l), SB432542-treated (m) and TGF-β-treated miR-181a/b morphant (n) Ath5:eGFP transgenic whole-heads. Dotted white lines mark optic nerve routes. Treatment of control-MOs embryos with 80μM SB432542 (m) phenocopied the miR-181a/b-morphant optic nerve length decrease (l). Addition of TGF-β for 24 h (from St30 to St32) to miR-181a/b morphants (n) was sufficient to rescue correct optic nerve growth in Ath5:eGFP morphant embryos. Scale bars: 50μm. OT, optic tectum.</p

TGF-β signaling regulates mature miR-181a/b expression levels.

<p><b>(a-b)</b> TGF-β pathway inhibition leads to a decrease in the expression levels of mature miR-181a and miR-181b. <b>(a)</b> Administration of SB432542, a TGF-β receptor inhibitor, induced a decrease of miR-181a and miR-181b mature forms in St32 eyes with respect to DMSO treatment, as detected by Taqman assays. The miR-181a and miR-181b reduction was comparable with that observed in the morpholino-mediated inhibition of the TGF-β pathway (MO-<i>tgfβr1</i>) at St32 <b>(b)</b>. Data are means +SEM. ***, P <0.001 (t-tests). <b>(c-d)</b> TGF-β treatment (10ng/ml) leads to increased levels of miR-181a and miR-181b mature forms in medaka fish St32 eyes in a transcription-independent manner. <b>(c)</b> Administration of TGF-β (10ng/ml) for 24 h (from St30 to St32) led to the increase of mature miR-181a and miR-181b in St32 eyes, as assessed by Taqman assays. Co-treatment with TFG-β and actinomycin D for 24 h (from St30 to St32) did not alter the TGF-β effect on mature miR-181a/b levels. These results indicate that the TGF-β effect on miR-181a/b expression is not transcription-dependent. Data are means +SEM. ***, P <0.001 (two-way ANOVA). <b>(d)</b> qRT-PCR on RNA extracted from DMSO- and TGF-β-treated St32 medaka fish eyes for all the pri-miR-181a and pri-miR-181b transcripts derived from the different genomic loci present in the medaka fish genome. After 24 h (from St30 to St32) of TGF-β treatment (10ng/ml), there were no significant changes in pri-miR-181a/b levels with respect to DMSO treatment. <b>(e)</b> qRT-PCR on RNA extracted from DMSO-, SB432542- and TGF-β-treated St32 medaka fish eyes. In the SB432542-treated eyes the decrease of miR-181a/b levels led to increased <i>prox1</i> and <i>erk2</i> transcript levels, whereas in TGF-β-treated eyes the miR-181a/b increase was accompanied by reduced transcript levels of both <i>prox1</i> and <i>erk2</i>. Data are means +SEM. **P <0.01; ***, P <0.001 (Two-way ANOVA). <b>(f-g)</b> Representative Western blotting (f) and corresponding quantification (g), showing a decrease of total-, phospho-Erk2 proteins and of its downstream target RhoA in TGF-β-treated St32 medaka fish eyes. Data are means +SEM. **P <0.01 (t-tests).</p

TGF-β signaling regulates <i>erk2</i> expression by modulating miR-181a/b levels.

<p><b>(a)</b> qRT-PCR analysis of <i>prox1</i> and <i>erk2</i> transcripts in RNA derived from St32 control-MOs, MO-protector-<i>erk2</i>–injected and TGF-β-treated MO-protector-<i>erk2</i> medaka fish eyes. The TGF-β rescue on the transcript levels of miR-181a/b targets was mediated by the miR-181a/b increase. Indeed this effect on <i>erk2</i> was completely abolished in the MO-protector-<i>erk2</i> embryos (a), while other miR-181a/b targets, such as <i>prox1</i>, whose miR-181 binding sites are unaffected by the MO-protector, were still sensitive to TGF-β action. Data are means ± SEM. * P <0.05; ** P <0.01 (two-way ANOVA). <b>(b-d)</b> Retinal frontal sections of St40 Control (b), MO-protector-<i>erk2</i> (c) and TGF-β-treated MO-protector-<i>erk2</i> medaka fish embryos (d) processed for Richardson-Romeis staining. Red bars, IPL thickness. Scale bars: 20μm. <b>(e)</b> Quantitative analysis of IPL thickness, indicated as the ratio in the central retina between the IPL area and total retinal area. Data are means ± SEM. *** P <0.0001 (one-way ANOVA). <b>(f-g)</b> Representative Western blotting on protein from St32 eyes (f) and corresponding quantification (g) show that administration of TGF-β to MO-protector-<i>erk2</i> embryos leads to partial rescue of RhoA protein to levels. When MO-protector-<i>erk2</i> embryos were treated with both TGF-β and the proteasomal inhibitor MG132, RhoA levels were not rescued anymore. Data are means +SEM. **P <0.05 (one-way ANOVA).</p

TGF-β administration rescues axon defects in miR-181a/b depleted RGCs.

<p><b>(a-c)</b> Representative images from primary RGC cultures from St30 control-MOs (a), miR-181a/b morphant (b) and TGF-β-treated miR-181a/b morphant medaka fish embryos (c). The RGC axon length defect was rescued by treatment with TGF-β (c). Scale bars: 10μm. <b>(d)</b> Quantification of RGC axonal length. Data are means +SEM (n = 100) from three independent cell culture experiments. ***P <<0.001 (one-way ANOVA).</p

Model of TGF-β cascades in retinal axon specification and growth.

<p>The TGF-β pathway regulates axon growth in the retina via two independent and synergistic pathways: the Par6/Smurf1 and the miR-181/ERK pathways. TGF-β–mediated activation of the Par6/Smurf1 cascade leads to ubiquitination and degradation of RhoA. On the other side, TGF-β also generates increased miR-181a/b levels, enhancing the process of miRNA maturation via activation of the SMAD2/3 protein. In turn, by fine modulation of the MAPK/ERK signaling pathway, miR-181a/b has an inhibitory effect on cofilin and RhoA production.</p

miR-204 Targeting of <i>Ankrd13A</i> Controls Both Mesenchymal Neural Crest and Lens Cell Migration

<div><p>Loss of cell adhesion and enhancement of cell motility contribute to epithelial-to-mesenchymal transition during development. These processes are related to a) rearrangement of cell-cell and cell-substrate adhesion molecules; b) cross talk between extra-cellular matrix and internal cytoskeleton through focal adhesion molecules. Focal adhesions are stringently regulated transient structures implicated in cell adhesion, spreading and motility during tissue development. Importantly, despite the extensive elucidation of the molecular composition of focal adhesions, the complex regulation of their dynamics is largely unclear. Here, we demonstrate, using live-imaging in medaka, that the microRNA miR-204 promotes both mesenchymal neural crest and lens cell migration and elongation. Overexpression of miR-204 results in upregulated cell motility, while morpholino-mediated ablation of miR-204 activity causes abnormal lens morphogenesis and neural crest cell mislocalization. Using a variety of <i>in vivo</i> and <i>in vitro</i> approaches, we demonstrate that these actions are mediated by the direct targeting of the <i>Ankrd13A</i> gene, which in turn controls focal cell adhesion formation and distribution. Significantly, <i>in vivo</i> restoration of abnormally elevated levels of <i>Ankrd13A</i> resulting from miR-204 inactivation rescued the aberrant lens phenotype in medaka fish. These data uncover, for the first time <i>in vivo,</i> the role of a microRNA in developmental control of mesenchymal cell migration and highlight miR-204 as a “master regulator” of the molecular networks that regulate lens morphogenesis in vertebrates.</p></div