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Pharmacological enhancement of mGlu1 metabotropic glutamate receptors causes a prolonged symptomatic benefit in a mouse model of spinocerebellar ataxia type 1.

Notartomaso S, Zappulla C, Biagioni F, Cannella M, Bucci D, Mascio G, Scarselli P, Fazio F, Weisz F, Lionetto L, Simmaco M, Gradini R, Battaglia G, Signore M, Puliti A, Nicoletti F - Mol Brain (2013)

Bottom Line: Spinocerebellar ataxia type 1 (SCA1) is a genetic disorder characterized by severe ataxia associated with progressive loss of cerebellar Purkinje cells.A single injection of Ro0711401 improved motor symptoms for several days, and no tolerance developed to the drug.The prolonged symptomatic benefit caused by Ro0711401 outlasted the time of drug clearance from the cerebellum, and was associated with neuroadaptive changes in the cerebellum, such as a striking reduction of the ectopically expressed mGlu5 receptors in Purkinje cells, increases in levels of total and Ser880-phosphorylated GluA2 subunit of AMPA receptors, and changes in the length of spines in the distal dendrites of Purkinje cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: I,R,C,C,S, Neuromed, Pozzilli, Italy. ferdinandonicoletti@hotmail.com.

ABSTRACT

Background: Spinocerebellar ataxia type 1 (SCA1) is a genetic disorder characterized by severe ataxia associated with progressive loss of cerebellar Purkinje cells. The mGlu1 metabotropic glutamate receptor plays a key role in mechanisms of activity-dependent synaptic plasticity in the cerebellum, and its dysfunction is linked to the pathophysiology of motor symptoms associated with SCA1. We used SCA1 heterozygous transgenic mice (Q154/Q2) as a model for testing the hypothesis that drugs that enhance mGlu1 receptor function may be good candidates for the medical treatment of SCA1.

Results: Symptomatic 30-week old SCA1 mice showed reduced mGlu1 receptor mRNA and protein levels in the cerebellum. Interestingly, these mice also showed an intense expression of mGlu5 receptors in cerebellar Purkinje cells, which normally lack these receptors. Systemic treatment of SCA1 mice with the mGlu1 receptor positive allosteric modulator (PAM), Ro0711401 (10 mg/kg, s.c.), caused a prolonged improvement of motor performance on the rotarod and the paw-print tests. A single injection of Ro0711401 improved motor symptoms for several days, and no tolerance developed to the drug. In contrast, the mGlu5 receptor PAM, VU0360172 (10 mg/kg, s.c.), caused only a short-lasting improvement of motor symptoms, whereas the mGlu1 receptor antagonist, JNJ16259685 (2.5 mg/kg, i.p.), further impaired motor performance in SCA1 mice. The prolonged symptomatic benefit caused by Ro0711401 outlasted the time of drug clearance from the cerebellum, and was associated with neuroadaptive changes in the cerebellum, such as a striking reduction of the ectopically expressed mGlu5 receptors in Purkinje cells, increases in levels of total and Ser880-phosphorylated GluA2 subunit of AMPA receptors, and changes in the length of spines in the distal dendrites of Purkinje cells.

Conclusions: These data demonstrate that pharmacological enhancement of mGlu1 receptors causes a robust and sustained motor improvement in SCA1 mice, and lay the groundwork for the development of mGlu1 receptor PAMs as novel "cerebellum-specific", effective, and safe symptomatic drugs for the treatment of SCA1 in humans.

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Reduced mGlu1 receptor mRNA and protein levels in the cerebellum of symptomatic SCA1 mice. mGlu1 receptor mRNA and mGlu1α receptor protein levels in the cerebellum of presymptomatic and symptomatic SCA1 mice (and their age-matched wild-type littermates) are shown in (A) and (B), respectively. Values are means ± S.E.M. of 3–4 mice per group. *p < 0.05 (Student’s t test) vs. the corresponding wild-type mice; t values = 5.3 (mRNA values normalized to GAPDH); 6.5 (mRNA values normalized to calbindin); and 7.075 (densitometric analysis of immunoblots). Immunohistochemical analysis of mGlu1α receptors and calbindin in the cerebellum of SCA1 mice and wild-type littermates are shown in (C) and (D).
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Figure 1: Reduced mGlu1 receptor mRNA and protein levels in the cerebellum of symptomatic SCA1 mice. mGlu1 receptor mRNA and mGlu1α receptor protein levels in the cerebellum of presymptomatic and symptomatic SCA1 mice (and their age-matched wild-type littermates) are shown in (A) and (B), respectively. Values are means ± S.E.M. of 3–4 mice per group. *p < 0.05 (Student’s t test) vs. the corresponding wild-type mice; t values = 5.3 (mRNA values normalized to GAPDH); 6.5 (mRNA values normalized to calbindin); and 7.075 (densitometric analysis of immunoblots). Immunohistochemical analysis of mGlu1α receptors and calbindin in the cerebellum of SCA1 mice and wild-type littermates are shown in (C) and (D).

Mentions: We measured mGlu1 receptor mRNA and mGlu1α receptor protein levels by real-time PCR and immunoblotting, respectively. The mGlu1α antibody detected a major band at 140 kDa corresponding to the deduced molecular size of receptor monomers. Labeling was highly specific because the band disappeared in the cerebellum of mGlu1-deficient crv4 mice [17] (not shown). mGlu1α mRNA and protein levels in the cerebellum did not differ between 4-week old presymptomatic SCA1 mice and their age-matched wild-type littermates (Figure 1A). In contrast, 30-week old symptomatic SCA1 mice showed large reductions in mGlu1 receptor mRNA levels, which were equally seen when data were normalized to both GAPDH and calbindin mRNA levels (Figure 1B). mGlu1α receptor protein levels were also reduced by about 50% in the cerebellum of symptomatic SCA1 mice, at least when expression data were normalized to β-actin levels (Figure 1B). Immunohistochemical analysis showed a reduced intensity of mGlu1α receptor staining in symptomatic SCA1 mice, which was particularly evident in the dendritic arborization of Purkinje cells (Figure 1C). High magnification analysis showed that mGlu1α receptor protein expression was also reduced at least in some Purkinje cells that appeared morphologically intact and were regularly stained with anti-calbindin antibody (Figure 1D). Taken together, these data indicated that a loss of mGlu1α receptors in Purkinje cells was associated with the pathological phenotype of SCA1 mice.


Pharmacological enhancement of mGlu1 metabotropic glutamate receptors causes a prolonged symptomatic benefit in a mouse model of spinocerebellar ataxia type 1.

Notartomaso S, Zappulla C, Biagioni F, Cannella M, Bucci D, Mascio G, Scarselli P, Fazio F, Weisz F, Lionetto L, Simmaco M, Gradini R, Battaglia G, Signore M, Puliti A, Nicoletti F - Mol Brain (2013)

Reduced mGlu1 receptor mRNA and protein levels in the cerebellum of symptomatic SCA1 mice. mGlu1 receptor mRNA and mGlu1α receptor protein levels in the cerebellum of presymptomatic and symptomatic SCA1 mice (and their age-matched wild-type littermates) are shown in (A) and (B), respectively. Values are means ± S.E.M. of 3–4 mice per group. *p < 0.05 (Student’s t test) vs. the corresponding wild-type mice; t values = 5.3 (mRNA values normalized to GAPDH); 6.5 (mRNA values normalized to calbindin); and 7.075 (densitometric analysis of immunoblots). Immunohistochemical analysis of mGlu1α receptors and calbindin in the cerebellum of SCA1 mice and wild-type littermates are shown in (C) and (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225515&req=5

Figure 1: Reduced mGlu1 receptor mRNA and protein levels in the cerebellum of symptomatic SCA1 mice. mGlu1 receptor mRNA and mGlu1α receptor protein levels in the cerebellum of presymptomatic and symptomatic SCA1 mice (and their age-matched wild-type littermates) are shown in (A) and (B), respectively. Values are means ± S.E.M. of 3–4 mice per group. *p < 0.05 (Student’s t test) vs. the corresponding wild-type mice; t values = 5.3 (mRNA values normalized to GAPDH); 6.5 (mRNA values normalized to calbindin); and 7.075 (densitometric analysis of immunoblots). Immunohistochemical analysis of mGlu1α receptors and calbindin in the cerebellum of SCA1 mice and wild-type littermates are shown in (C) and (D).
Mentions: We measured mGlu1 receptor mRNA and mGlu1α receptor protein levels by real-time PCR and immunoblotting, respectively. The mGlu1α antibody detected a major band at 140 kDa corresponding to the deduced molecular size of receptor monomers. Labeling was highly specific because the band disappeared in the cerebellum of mGlu1-deficient crv4 mice [17] (not shown). mGlu1α mRNA and protein levels in the cerebellum did not differ between 4-week old presymptomatic SCA1 mice and their age-matched wild-type littermates (Figure 1A). In contrast, 30-week old symptomatic SCA1 mice showed large reductions in mGlu1 receptor mRNA levels, which were equally seen when data were normalized to both GAPDH and calbindin mRNA levels (Figure 1B). mGlu1α receptor protein levels were also reduced by about 50% in the cerebellum of symptomatic SCA1 mice, at least when expression data were normalized to β-actin levels (Figure 1B). Immunohistochemical analysis showed a reduced intensity of mGlu1α receptor staining in symptomatic SCA1 mice, which was particularly evident in the dendritic arborization of Purkinje cells (Figure 1C). High magnification analysis showed that mGlu1α receptor protein expression was also reduced at least in some Purkinje cells that appeared morphologically intact and were regularly stained with anti-calbindin antibody (Figure 1D). Taken together, these data indicated that a loss of mGlu1α receptors in Purkinje cells was associated with the pathological phenotype of SCA1 mice.

Bottom Line: Spinocerebellar ataxia type 1 (SCA1) is a genetic disorder characterized by severe ataxia associated with progressive loss of cerebellar Purkinje cells.A single injection of Ro0711401 improved motor symptoms for several days, and no tolerance developed to the drug.The prolonged symptomatic benefit caused by Ro0711401 outlasted the time of drug clearance from the cerebellum, and was associated with neuroadaptive changes in the cerebellum, such as a striking reduction of the ectopically expressed mGlu5 receptors in Purkinje cells, increases in levels of total and Ser880-phosphorylated GluA2 subunit of AMPA receptors, and changes in the length of spines in the distal dendrites of Purkinje cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: I,R,C,C,S, Neuromed, Pozzilli, Italy. ferdinandonicoletti@hotmail.com.

ABSTRACT

Background: Spinocerebellar ataxia type 1 (SCA1) is a genetic disorder characterized by severe ataxia associated with progressive loss of cerebellar Purkinje cells. The mGlu1 metabotropic glutamate receptor plays a key role in mechanisms of activity-dependent synaptic plasticity in the cerebellum, and its dysfunction is linked to the pathophysiology of motor symptoms associated with SCA1. We used SCA1 heterozygous transgenic mice (Q154/Q2) as a model for testing the hypothesis that drugs that enhance mGlu1 receptor function may be good candidates for the medical treatment of SCA1.

Results: Symptomatic 30-week old SCA1 mice showed reduced mGlu1 receptor mRNA and protein levels in the cerebellum. Interestingly, these mice also showed an intense expression of mGlu5 receptors in cerebellar Purkinje cells, which normally lack these receptors. Systemic treatment of SCA1 mice with the mGlu1 receptor positive allosteric modulator (PAM), Ro0711401 (10 mg/kg, s.c.), caused a prolonged improvement of motor performance on the rotarod and the paw-print tests. A single injection of Ro0711401 improved motor symptoms for several days, and no tolerance developed to the drug. In contrast, the mGlu5 receptor PAM, VU0360172 (10 mg/kg, s.c.), caused only a short-lasting improvement of motor symptoms, whereas the mGlu1 receptor antagonist, JNJ16259685 (2.5 mg/kg, i.p.), further impaired motor performance in SCA1 mice. The prolonged symptomatic benefit caused by Ro0711401 outlasted the time of drug clearance from the cerebellum, and was associated with neuroadaptive changes in the cerebellum, such as a striking reduction of the ectopically expressed mGlu5 receptors in Purkinje cells, increases in levels of total and Ser880-phosphorylated GluA2 subunit of AMPA receptors, and changes in the length of spines in the distal dendrites of Purkinje cells.

Conclusions: These data demonstrate that pharmacological enhancement of mGlu1 receptors causes a robust and sustained motor improvement in SCA1 mice, and lay the groundwork for the development of mGlu1 receptor PAMs as novel "cerebellum-specific", effective, and safe symptomatic drugs for the treatment of SCA1 in humans.

Show MeSH
Related in: MedlinePlus