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Intravitreal administration of HA-1077, a ROCK inhibitor, improves retinal function in a mouse model of huntington disease.

Li M, Yasumura D, Ma AA, Matthes MT, Yang H, Nielson G, Huang Y, Szoka FC, Lavail MM, Diamond MI - PLoS ONE (2013)

Bottom Line: It is caused by an expanded polyglutamine tract in huntingtin (Htt).ROCK is thus a therapeutic target in HD.By targeting ROCK with a new inhibitor, and testing its effects in a novel in vivo model, these results validate the in vivo efficacy of a therapeutic candidate, and establish the feasibility of using the retina as a readout for CNS function in models of neurodegenerative disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Washington University in St. Louis, St. Louis, Missouri, USA.

ABSTRACT
Huntington disease (HD) is an inherited neurodegenerative disease that affects multiple brain regions. It is caused by an expanded polyglutamine tract in huntingtin (Htt). The development of therapies for HD and other neurodegenerative diseases has been hampered by multiple factors, including the lack of clear therapeutic targets, and the cost and complexity of testing lead compounds in vivo. The R6/2 HD mouse model is widely used for pre-clinical trials because of its progressive and robust neural dysfunction, which includes retinal degeneration. Profilin-1 is a Htt binding protein that inhibits Htt aggregation. Its binding to Htt is regulated by the rho-associated kinase (ROCK), which phosphorylates profilin at Ser-137. ROCK is thus a therapeutic target in HD. The ROCK inhibitor Y-27632 reduces Htt toxicity in fly and mouse models. Here we characterized the progressive retinopathy of R6/2 mice between 6-19 weeks of age to determine an optimal treatment window. We then tested a clinically approved ROCK inhibitor, HA-1077, administered intravitreally via liposome-mediated drug delivery. HA-1077 increased photopic and flicker ERG response amplitudes in R6/2 mice, but not in wild-type littermate controls. By targeting ROCK with a new inhibitor, and testing its effects in a novel in vivo model, these results validate the in vivo efficacy of a therapeutic candidate, and establish the feasibility of using the retina as a readout for CNS function in models of neurodegenerative disease.

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Related in: MedlinePlus

Expression of human huntingtin exon1 and morphological changes of R6/2 mouse retina.A,B: Frozen sections of 8-week-old R6/2 retina were co-immuno-stained with anti human huntingtin exon1 antibody EM48 (red) and anti phospho profilin1 antibody P3490 (green), and counterstained with DAPI (blue). Confocal microscopy was performed. A: EM48-positive inclusions can be seen in the retinal ganglion cell layer (GCL) and, B: in retinal ganglion cell (GC) nuclei at higher magnification with EM 48 positive (red) nuclear inclusions. C: 13-week-old R6/2 whole retina was stained with EM48 (red) and mounted on a microscope slide glass, then imaged by confocal microscopy. EM48-positive nuclear inclusions can be seen in many ganglion cells. D–G: plastic sections of the posterior retina of R6/2 mice of different ages. D: 8-week-old R6/2 retina that is indistinguishable from wild-type retinas of this age. Most photoreceptors in the outer nuclear layer (ONL) are rods, but cone nuclei are shown at the arrows. E: 11-week-old R6/2 retina showing small focal irregularities in the inner and outer borders of the ONL (arrowheads). F: 18-week-old R6/2 retina illustrating single, large fold in the ONL, giving the typical “wavy” appearance of the ONL in many regions that comprise 3–18% of the retinal length. G: 19-week-old R6/2 retina showing normal-appearing ONL, which is typical of the vast majority of retinal length at 18 to 19 weeks of age, but the photoreceptor outer segments (OS) are significantly shorter and more disrupted and vacuolated than normal. Scale bars: All except B = 25 µm; B = 10 µm. INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segments; OPL, outer plexiform layer; RPE, retinal pigment epithelium.
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pone-0056026-g001: Expression of human huntingtin exon1 and morphological changes of R6/2 mouse retina.A,B: Frozen sections of 8-week-old R6/2 retina were co-immuno-stained with anti human huntingtin exon1 antibody EM48 (red) and anti phospho profilin1 antibody P3490 (green), and counterstained with DAPI (blue). Confocal microscopy was performed. A: EM48-positive inclusions can be seen in the retinal ganglion cell layer (GCL) and, B: in retinal ganglion cell (GC) nuclei at higher magnification with EM 48 positive (red) nuclear inclusions. C: 13-week-old R6/2 whole retina was stained with EM48 (red) and mounted on a microscope slide glass, then imaged by confocal microscopy. EM48-positive nuclear inclusions can be seen in many ganglion cells. D–G: plastic sections of the posterior retina of R6/2 mice of different ages. D: 8-week-old R6/2 retina that is indistinguishable from wild-type retinas of this age. Most photoreceptors in the outer nuclear layer (ONL) are rods, but cone nuclei are shown at the arrows. E: 11-week-old R6/2 retina showing small focal irregularities in the inner and outer borders of the ONL (arrowheads). F: 18-week-old R6/2 retina illustrating single, large fold in the ONL, giving the typical “wavy” appearance of the ONL in many regions that comprise 3–18% of the retinal length. G: 19-week-old R6/2 retina showing normal-appearing ONL, which is typical of the vast majority of retinal length at 18 to 19 weeks of age, but the photoreceptor outer segments (OS) are significantly shorter and more disrupted and vacuolated than normal. Scale bars: All except B = 25 µm; B = 10 µm. INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segments; OPL, outer plexiform layer; RPE, retinal pigment epithelium.

Mentions: We used the anti-Htt antibody EM48 to stain for Htt accumulation. We observed that EM48-positive intranuclear inclusions first appeared in the retinal ganglion cells of R6/2 mice at approximately 7 weeks of age (Fig. 1A–C), extending to all 3 layers of the retina at approximately 11 weeks of age (data not shown). This roughly parallels the development of such inclusions elsewhere in the brain [2].


Intravitreal administration of HA-1077, a ROCK inhibitor, improves retinal function in a mouse model of huntington disease.

Li M, Yasumura D, Ma AA, Matthes MT, Yang H, Nielson G, Huang Y, Szoka FC, Lavail MM, Diamond MI - PLoS ONE (2013)

Expression of human huntingtin exon1 and morphological changes of R6/2 mouse retina.A,B: Frozen sections of 8-week-old R6/2 retina were co-immuno-stained with anti human huntingtin exon1 antibody EM48 (red) and anti phospho profilin1 antibody P3490 (green), and counterstained with DAPI (blue). Confocal microscopy was performed. A: EM48-positive inclusions can be seen in the retinal ganglion cell layer (GCL) and, B: in retinal ganglion cell (GC) nuclei at higher magnification with EM 48 positive (red) nuclear inclusions. C: 13-week-old R6/2 whole retina was stained with EM48 (red) and mounted on a microscope slide glass, then imaged by confocal microscopy. EM48-positive nuclear inclusions can be seen in many ganglion cells. D–G: plastic sections of the posterior retina of R6/2 mice of different ages. D: 8-week-old R6/2 retina that is indistinguishable from wild-type retinas of this age. Most photoreceptors in the outer nuclear layer (ONL) are rods, but cone nuclei are shown at the arrows. E: 11-week-old R6/2 retina showing small focal irregularities in the inner and outer borders of the ONL (arrowheads). F: 18-week-old R6/2 retina illustrating single, large fold in the ONL, giving the typical “wavy” appearance of the ONL in many regions that comprise 3–18% of the retinal length. G: 19-week-old R6/2 retina showing normal-appearing ONL, which is typical of the vast majority of retinal length at 18 to 19 weeks of age, but the photoreceptor outer segments (OS) are significantly shorter and more disrupted and vacuolated than normal. Scale bars: All except B = 25 µm; B = 10 µm. INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segments; OPL, outer plexiform layer; RPE, retinal pigment epithelium.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569418&req=5

pone-0056026-g001: Expression of human huntingtin exon1 and morphological changes of R6/2 mouse retina.A,B: Frozen sections of 8-week-old R6/2 retina were co-immuno-stained with anti human huntingtin exon1 antibody EM48 (red) and anti phospho profilin1 antibody P3490 (green), and counterstained with DAPI (blue). Confocal microscopy was performed. A: EM48-positive inclusions can be seen in the retinal ganglion cell layer (GCL) and, B: in retinal ganglion cell (GC) nuclei at higher magnification with EM 48 positive (red) nuclear inclusions. C: 13-week-old R6/2 whole retina was stained with EM48 (red) and mounted on a microscope slide glass, then imaged by confocal microscopy. EM48-positive nuclear inclusions can be seen in many ganglion cells. D–G: plastic sections of the posterior retina of R6/2 mice of different ages. D: 8-week-old R6/2 retina that is indistinguishable from wild-type retinas of this age. Most photoreceptors in the outer nuclear layer (ONL) are rods, but cone nuclei are shown at the arrows. E: 11-week-old R6/2 retina showing small focal irregularities in the inner and outer borders of the ONL (arrowheads). F: 18-week-old R6/2 retina illustrating single, large fold in the ONL, giving the typical “wavy” appearance of the ONL in many regions that comprise 3–18% of the retinal length. G: 19-week-old R6/2 retina showing normal-appearing ONL, which is typical of the vast majority of retinal length at 18 to 19 weeks of age, but the photoreceptor outer segments (OS) are significantly shorter and more disrupted and vacuolated than normal. Scale bars: All except B = 25 µm; B = 10 µm. INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segments; OPL, outer plexiform layer; RPE, retinal pigment epithelium.
Mentions: We used the anti-Htt antibody EM48 to stain for Htt accumulation. We observed that EM48-positive intranuclear inclusions first appeared in the retinal ganglion cells of R6/2 mice at approximately 7 weeks of age (Fig. 1A–C), extending to all 3 layers of the retina at approximately 11 weeks of age (data not shown). This roughly parallels the development of such inclusions elsewhere in the brain [2].

Bottom Line: It is caused by an expanded polyglutamine tract in huntingtin (Htt).ROCK is thus a therapeutic target in HD.By targeting ROCK with a new inhibitor, and testing its effects in a novel in vivo model, these results validate the in vivo efficacy of a therapeutic candidate, and establish the feasibility of using the retina as a readout for CNS function in models of neurodegenerative disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Washington University in St. Louis, St. Louis, Missouri, USA.

ABSTRACT
Huntington disease (HD) is an inherited neurodegenerative disease that affects multiple brain regions. It is caused by an expanded polyglutamine tract in huntingtin (Htt). The development of therapies for HD and other neurodegenerative diseases has been hampered by multiple factors, including the lack of clear therapeutic targets, and the cost and complexity of testing lead compounds in vivo. The R6/2 HD mouse model is widely used for pre-clinical trials because of its progressive and robust neural dysfunction, which includes retinal degeneration. Profilin-1 is a Htt binding protein that inhibits Htt aggregation. Its binding to Htt is regulated by the rho-associated kinase (ROCK), which phosphorylates profilin at Ser-137. ROCK is thus a therapeutic target in HD. The ROCK inhibitor Y-27632 reduces Htt toxicity in fly and mouse models. Here we characterized the progressive retinopathy of R6/2 mice between 6-19 weeks of age to determine an optimal treatment window. We then tested a clinically approved ROCK inhibitor, HA-1077, administered intravitreally via liposome-mediated drug delivery. HA-1077 increased photopic and flicker ERG response amplitudes in R6/2 mice, but not in wild-type littermate controls. By targeting ROCK with a new inhibitor, and testing its effects in a novel in vivo model, these results validate the in vivo efficacy of a therapeutic candidate, and establish the feasibility of using the retina as a readout for CNS function in models of neurodegenerative disease.

Show MeSH
Related in: MedlinePlus