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Rilmenidine attenuates toxicity of polyglutamine expansions in a mouse model of Huntington's disease.

Rose C, Menzies FM, Renna M, Acevedo-Arozena A, Corrochano S, Sadiq O, Brown SD, Rubinsztein DC - Hum. Mol. Genet. (2010)

Bottom Line: This screen suggested that rilmenidine, a well tolerated, safe, centrally acting anti-hypertensive drug, could induce autophagy in cell culture via a pathway that was independent of the mammalian target of rapamycin.Rilmenidine administration attenuated the signs of disease in a HD mouse model and reduced levels of the mutant huntingtin fragment.As rilmenidine has a long safety record and is designed for chronic use, our data suggests that it should be considered for the treatment of HD and related conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of Cambridge, Cambridge Institute for Medical Research, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, UK.

ABSTRACT
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a polyglutamine expansion in huntingtin. There are no treatments that are known to slow the neurodegeneration caused by this mutation. Mutant huntingtin causes disease via a toxic gain-of-function mechanism and has the propensity to aggregate and form intraneuronal inclusions. One therapeutic approach for HD is to enhance the degradation of the mutant protein. We have shown that this can be achieved by upregulating autophagy, using the drug rapamycin. In order to find safer ways of inducing autophagy for clinical purposes, we previously screened United States Food and Drug Administration-approved drugs for their autophagy-stimulating potential. This screen suggested that rilmenidine, a well tolerated, safe, centrally acting anti-hypertensive drug, could induce autophagy in cell culture via a pathway that was independent of the mammalian target of rapamycin. Here we have shown that rilmenidine induces autophagy in mice and in primary neuronal culture. Rilmenidine administration attenuated the signs of disease in a HD mouse model and reduced levels of the mutant huntingtin fragment. As rilmenidine has a long safety record and is designed for chronic use, our data suggests that it should be considered for the treatment of HD and related conditions.

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Related in: MedlinePlus

Rilmenidine enhances autophagy in wild-type mice. (A) Endogenous LC3-II levels were measured in stable inducible PC12 cells 48 h after switching on expression of either huntingtin exon 1 with 23 polyglutamine repeats (htt-23Q) or 74 polyglutamine repeats (htt-74Q) in the presence or absence of rilmenidine (for the final 24 h). Actin was used as a loading control. (B) LC3-II levels were measured using fluorescent intensity of the bands by Li-Cor Odyssey. Results are shown as a percentage of control in each individual cell line (n = 5, *P < 0.05 by t-test). The increase in LC3-II levels in rilmenidine treated, htt-74Q is not significantly greater than in htt-23Q treated cells, and possible differences may be due to variations between the clonal cell lines used. (C) LC3-II levels in muscle lysates from rilmenidine-treated and placebo-treated wild-type mice after 24 weeks of treatment. Western blots were also probed for tubulin as a loading control (D) Densitometric analysis of LC3-II-levels relative to tubulin. Control condition is set to 100%. Error bars show SEM (*P = 0.036, t-test, n = 4 for rilmenidine, n = 5 for control). (E) In cultured primary cortical neurons, LC3-II levels were assessed by western blot. Two exposures are shown to allow comparison of weaker bands in non-bafilomycin A1-treated lanes (−Baf A1) and stronger bands in bafilomycin A1-treated lanes without saturation. (F) Densitometric quantification of LC3-II levels relative to actin in triplicate experiments. (*P < 0.05 by t-test). Effect of rilmenidine treatment on phosphorylation of downstream mTOR targets was investigated by western blotting, (G) phosphorylated p70 S6 kinase levels, (H) phosphorylated S6 ribosomal protein and (I) phosphorylated 4EBP1. In these experiments, rapamycin treatment was used as a control for the inactivation of mTOR where the effects of treatment can be clearly seen.
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DDQ093F1: Rilmenidine enhances autophagy in wild-type mice. (A) Endogenous LC3-II levels were measured in stable inducible PC12 cells 48 h after switching on expression of either huntingtin exon 1 with 23 polyglutamine repeats (htt-23Q) or 74 polyglutamine repeats (htt-74Q) in the presence or absence of rilmenidine (for the final 24 h). Actin was used as a loading control. (B) LC3-II levels were measured using fluorescent intensity of the bands by Li-Cor Odyssey. Results are shown as a percentage of control in each individual cell line (n = 5, *P < 0.05 by t-test). The increase in LC3-II levels in rilmenidine treated, htt-74Q is not significantly greater than in htt-23Q treated cells, and possible differences may be due to variations between the clonal cell lines used. (C) LC3-II levels in muscle lysates from rilmenidine-treated and placebo-treated wild-type mice after 24 weeks of treatment. Western blots were also probed for tubulin as a loading control (D) Densitometric analysis of LC3-II-levels relative to tubulin. Control condition is set to 100%. Error bars show SEM (*P = 0.036, t-test, n = 4 for rilmenidine, n = 5 for control). (E) In cultured primary cortical neurons, LC3-II levels were assessed by western blot. Two exposures are shown to allow comparison of weaker bands in non-bafilomycin A1-treated lanes (−Baf A1) and stronger bands in bafilomycin A1-treated lanes without saturation. (F) Densitometric quantification of LC3-II levels relative to actin in triplicate experiments. (*P < 0.05 by t-test). Effect of rilmenidine treatment on phosphorylation of downstream mTOR targets was investigated by western blotting, (G) phosphorylated p70 S6 kinase levels, (H) phosphorylated S6 ribosomal protein and (I) phosphorylated 4EBP1. In these experiments, rapamycin treatment was used as a control for the inactivation of mTOR where the effects of treatment can be clearly seen.

Mentions: Our previous studies have demonstrated the ability of rilmenidine to induce autophagy and clear model aggregate-prone proteins such as mutant huntingtin exon 1 (16). In order to further confirm the suitability of rilmenidine as a potential therapeutic for use in HD, we showed that its ability to induce autophagy was not compromised by the presence of mutant huntingtin. We assessed autophagosome numbers using the microtubule-associated protein 1 light chain (LC3) (27). LC3 is processed post-translationally into LC3-I, then converted to LC3-II, the only known protein that specifically associates with autophagosome membranes (28). LC3-positive vesicle numbers or LC3-II levels (versus loading control) correlate with autophagosome numbers (27). Stable inducible PC12 cells expressing either wild-type (23Q) or expanded polyglutamine (74Q) huntingtin both showed a significant increase in LC3-II when treated with rilmenidine (Fig. 1A and B).


Rilmenidine attenuates toxicity of polyglutamine expansions in a mouse model of Huntington's disease.

Rose C, Menzies FM, Renna M, Acevedo-Arozena A, Corrochano S, Sadiq O, Brown SD, Rubinsztein DC - Hum. Mol. Genet. (2010)

Rilmenidine enhances autophagy in wild-type mice. (A) Endogenous LC3-II levels were measured in stable inducible PC12 cells 48 h after switching on expression of either huntingtin exon 1 with 23 polyglutamine repeats (htt-23Q) or 74 polyglutamine repeats (htt-74Q) in the presence or absence of rilmenidine (for the final 24 h). Actin was used as a loading control. (B) LC3-II levels were measured using fluorescent intensity of the bands by Li-Cor Odyssey. Results are shown as a percentage of control in each individual cell line (n = 5, *P < 0.05 by t-test). The increase in LC3-II levels in rilmenidine treated, htt-74Q is not significantly greater than in htt-23Q treated cells, and possible differences may be due to variations between the clonal cell lines used. (C) LC3-II levels in muscle lysates from rilmenidine-treated and placebo-treated wild-type mice after 24 weeks of treatment. Western blots were also probed for tubulin as a loading control (D) Densitometric analysis of LC3-II-levels relative to tubulin. Control condition is set to 100%. Error bars show SEM (*P = 0.036, t-test, n = 4 for rilmenidine, n = 5 for control). (E) In cultured primary cortical neurons, LC3-II levels were assessed by western blot. Two exposures are shown to allow comparison of weaker bands in non-bafilomycin A1-treated lanes (−Baf A1) and stronger bands in bafilomycin A1-treated lanes without saturation. (F) Densitometric quantification of LC3-II levels relative to actin in triplicate experiments. (*P < 0.05 by t-test). Effect of rilmenidine treatment on phosphorylation of downstream mTOR targets was investigated by western blotting, (G) phosphorylated p70 S6 kinase levels, (H) phosphorylated S6 ribosomal protein and (I) phosphorylated 4EBP1. In these experiments, rapamycin treatment was used as a control for the inactivation of mTOR where the effects of treatment can be clearly seen.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2865373&req=5

DDQ093F1: Rilmenidine enhances autophagy in wild-type mice. (A) Endogenous LC3-II levels were measured in stable inducible PC12 cells 48 h after switching on expression of either huntingtin exon 1 with 23 polyglutamine repeats (htt-23Q) or 74 polyglutamine repeats (htt-74Q) in the presence or absence of rilmenidine (for the final 24 h). Actin was used as a loading control. (B) LC3-II levels were measured using fluorescent intensity of the bands by Li-Cor Odyssey. Results are shown as a percentage of control in each individual cell line (n = 5, *P < 0.05 by t-test). The increase in LC3-II levels in rilmenidine treated, htt-74Q is not significantly greater than in htt-23Q treated cells, and possible differences may be due to variations between the clonal cell lines used. (C) LC3-II levels in muscle lysates from rilmenidine-treated and placebo-treated wild-type mice after 24 weeks of treatment. Western blots were also probed for tubulin as a loading control (D) Densitometric analysis of LC3-II-levels relative to tubulin. Control condition is set to 100%. Error bars show SEM (*P = 0.036, t-test, n = 4 for rilmenidine, n = 5 for control). (E) In cultured primary cortical neurons, LC3-II levels were assessed by western blot. Two exposures are shown to allow comparison of weaker bands in non-bafilomycin A1-treated lanes (−Baf A1) and stronger bands in bafilomycin A1-treated lanes without saturation. (F) Densitometric quantification of LC3-II levels relative to actin in triplicate experiments. (*P < 0.05 by t-test). Effect of rilmenidine treatment on phosphorylation of downstream mTOR targets was investigated by western blotting, (G) phosphorylated p70 S6 kinase levels, (H) phosphorylated S6 ribosomal protein and (I) phosphorylated 4EBP1. In these experiments, rapamycin treatment was used as a control for the inactivation of mTOR where the effects of treatment can be clearly seen.
Mentions: Our previous studies have demonstrated the ability of rilmenidine to induce autophagy and clear model aggregate-prone proteins such as mutant huntingtin exon 1 (16). In order to further confirm the suitability of rilmenidine as a potential therapeutic for use in HD, we showed that its ability to induce autophagy was not compromised by the presence of mutant huntingtin. We assessed autophagosome numbers using the microtubule-associated protein 1 light chain (LC3) (27). LC3 is processed post-translationally into LC3-I, then converted to LC3-II, the only known protein that specifically associates with autophagosome membranes (28). LC3-positive vesicle numbers or LC3-II levels (versus loading control) correlate with autophagosome numbers (27). Stable inducible PC12 cells expressing either wild-type (23Q) or expanded polyglutamine (74Q) huntingtin both showed a significant increase in LC3-II when treated with rilmenidine (Fig. 1A and B).

Bottom Line: This screen suggested that rilmenidine, a well tolerated, safe, centrally acting anti-hypertensive drug, could induce autophagy in cell culture via a pathway that was independent of the mammalian target of rapamycin.Rilmenidine administration attenuated the signs of disease in a HD mouse model and reduced levels of the mutant huntingtin fragment.As rilmenidine has a long safety record and is designed for chronic use, our data suggests that it should be considered for the treatment of HD and related conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of Cambridge, Cambridge Institute for Medical Research, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, UK.

ABSTRACT
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a polyglutamine expansion in huntingtin. There are no treatments that are known to slow the neurodegeneration caused by this mutation. Mutant huntingtin causes disease via a toxic gain-of-function mechanism and has the propensity to aggregate and form intraneuronal inclusions. One therapeutic approach for HD is to enhance the degradation of the mutant protein. We have shown that this can be achieved by upregulating autophagy, using the drug rapamycin. In order to find safer ways of inducing autophagy for clinical purposes, we previously screened United States Food and Drug Administration-approved drugs for their autophagy-stimulating potential. This screen suggested that rilmenidine, a well tolerated, safe, centrally acting anti-hypertensive drug, could induce autophagy in cell culture via a pathway that was independent of the mammalian target of rapamycin. Here we have shown that rilmenidine induces autophagy in mice and in primary neuronal culture. Rilmenidine administration attenuated the signs of disease in a HD mouse model and reduced levels of the mutant huntingtin fragment. As rilmenidine has a long safety record and is designed for chronic use, our data suggests that it should be considered for the treatment of HD and related conditions.

Show MeSH
Related in: MedlinePlus