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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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Intravitreal injection of liposomes loaded with HA-1077 decreases phospho-profilin 1 in mouse retina.One eye of 9-week-old wild-type mice (n = 4) was intravitreally injected with liposomes loaded with 40 µM HA-1077, and the other eye was intravitreally injected with empty liposomes. At 24 hours after injection, animals were sacrificed and whole retina cups were harvested, fixed and stained with anti-phospho profilin1 antibody P3490 (red), mounted on a microscope slide glass and imaged by confocal microscopy. A: The retina of eyes injected with empty liposomes shows many P3490-positive cells with irregularly shaped cytoplasm and processes. B: The retina of eyes injected with liposomes loaded with HA-1077 shows decreased staining density and few stained cells with round shaped cytoplasmic staining. Scale bar: 50 µm.
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pone-0056026-g005: Intravitreal injection of liposomes loaded with HA-1077 decreases phospho-profilin 1 in mouse retina.One eye of 9-week-old wild-type mice (n = 4) was intravitreally injected with liposomes loaded with 40 µM HA-1077, and the other eye was intravitreally injected with empty liposomes. At 24 hours after injection, animals were sacrificed and whole retina cups were harvested, fixed and stained with anti-phospho profilin1 antibody P3490 (red), mounted on a microscope slide glass and imaged by confocal microscopy. A: The retina of eyes injected with empty liposomes shows many P3490-positive cells with irregularly shaped cytoplasm and processes. B: The retina of eyes injected with liposomes loaded with HA-1077 shows decreased staining density and few stained cells with round shaped cytoplasmic staining. Scale bar: 50 µm.

Mentions: We have previously determined that Ser-137 of profilin is a direct target of ROCK, and we have developed a phospho-specific antibody to Ser-137 of profilin (P3490), that reports its phosphorylation [8]. We confirmed that liposome-mediated delivery of HA-1077 would produce the desired bioactive effect by administering the liposomes loaded with 40 µM HA-1077 to wild-type mice and measuring profilin phosphorylation in the retina. We injected 1 µl of liposomes loaded with HA-1077 intravitreally, and 1 µl of empty liposomes into the contralateral eye. After 24 hrs the animals were killed, and whole mounted retinas were stained with anti phospho-profilin antibody P-3490. Phospho-profilin levels were lower in HA-1077 injected eyes than that in empty liposome-injected eyes (Fig. 5). These experiments indicated that we were achieving bioactivity in vivo from the liposome-mediated delivery of HA-1077.


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)

Intravitreal injection of liposomes loaded with HA-1077 decreases phospho-profilin 1 in mouse retina.One eye of 9-week-old wild-type mice (n = 4) was intravitreally injected with liposomes loaded with 40 µM HA-1077, and the other eye was intravitreally injected with empty liposomes. At 24 hours after injection, animals were sacrificed and whole retina cups were harvested, fixed and stained with anti-phospho profilin1 antibody P3490 (red), mounted on a microscope slide glass and imaged by confocal microscopy. A: The retina of eyes injected with empty liposomes shows many P3490-positive cells with irregularly shaped cytoplasm and processes. B: The retina of eyes injected with liposomes loaded with HA-1077 shows decreased staining density and few stained cells with round shaped cytoplasmic staining. Scale bar: 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569418&req=5

pone-0056026-g005: Intravitreal injection of liposomes loaded with HA-1077 decreases phospho-profilin 1 in mouse retina.One eye of 9-week-old wild-type mice (n = 4) was intravitreally injected with liposomes loaded with 40 µM HA-1077, and the other eye was intravitreally injected with empty liposomes. At 24 hours after injection, animals were sacrificed and whole retina cups were harvested, fixed and stained with anti-phospho profilin1 antibody P3490 (red), mounted on a microscope slide glass and imaged by confocal microscopy. A: The retina of eyes injected with empty liposomes shows many P3490-positive cells with irregularly shaped cytoplasm and processes. B: The retina of eyes injected with liposomes loaded with HA-1077 shows decreased staining density and few stained cells with round shaped cytoplasmic staining. Scale bar: 50 µm.
Mentions: We have previously determined that Ser-137 of profilin is a direct target of ROCK, and we have developed a phospho-specific antibody to Ser-137 of profilin (P3490), that reports its phosphorylation [8]. We confirmed that liposome-mediated delivery of HA-1077 would produce the desired bioactive effect by administering the liposomes loaded with 40 µM HA-1077 to wild-type mice and measuring profilin phosphorylation in the retina. We injected 1 µl of liposomes loaded with HA-1077 intravitreally, and 1 µl of empty liposomes into the contralateral eye. After 24 hrs the animals were killed, and whole mounted retinas were stained with anti phospho-profilin antibody P-3490. Phospho-profilin levels were lower in HA-1077 injected eyes than that in empty liposome-injected eyes (Fig. 5). These experiments indicated that we were achieving bioactivity in vivo from the liposome-mediated delivery of HA-1077.

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