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A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity.

Yao Y, Cui X, Al-Ramahi I, Sun X, Li B, Hou J, Difiglia M, Palacino J, Wu ZY, Ma L, Botas J, Lu B - Elife (2015)

Bottom Line: Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum.Finally, reducing Gpr52 suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models.Thus, our discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, Department of Biophysics, School of Life Sciences, Fudan University, Shanghai, China.

ABSTRACT
Huntington's disease (HD) represents an important model for neurodegenerative disorders and proteinopathies. It is mainly caused by cytotoxicity of the mutant huntingtin protein (Htt) with an expanded polyQ stretch. While Htt is ubiquitously expressed, HD is characterized by selective neurodegeneration of the striatum. Here we report a striatal-enriched orphan G protein-coupled receptor(GPCR) Gpr52 as a stabilizer of Htt in vitro and in vivo. Gpr52 modulates Htt via cAMP-dependent but PKA independent mechanisms. Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum. Finally, reducing Gpr52 suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models. Thus, our discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies.

No MeSH data available.


Related in: MedlinePlus

Gpr52's effect is not mediated by Rap1 or Rap2.Left: Representative western-blots of STHdhQ7/Q111 cells transfected with the Gpr52 siRNA (Gpr52_si2) versus control siRNA, and then with constitutively active or dominant negative Rap1 (Rap1CA or Rap1DN) or Rap2 (Rap2CA or Rap2DN). The Bars plot: Htt level reduction (%) measured by 2B7/2166 HTRF of the total lysates of cells with same transfections as in the western-blots.DOI:http://dx.doi.org/10.7554/eLife.05449.009
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fig3s3: Gpr52's effect is not mediated by Rap1 or Rap2.Left: Representative western-blots of STHdhQ7/Q111 cells transfected with the Gpr52 siRNA (Gpr52_si2) versus control siRNA, and then with constitutively active or dominant negative Rap1 (Rap1CA or Rap1DN) or Rap2 (Rap2CA or Rap2DN). The Bars plot: Htt level reduction (%) measured by 2B7/2166 HTRF of the total lysates of cells with same transfections as in the western-blots.DOI:http://dx.doi.org/10.7554/eLife.05449.009

Mentions: Other than PKA, the guanine exchange factor (GEF) Epac, Rapgef2 and potentially other GEFs have been reported as cAMP sensors that mediate downstream signals (de Rooij et al., 1998; Emery and Eiden, 2012; Emery et al., 2013). Thus, Gpr52 may function via a PKA-independent and cAMP-dependent signaling mechanism by activating GEFs. The reported small GTPases downstream of these GEFs are Rap1 and Rap2, and thus we tested their potential involvement by dominant negative and constitutively active Rap proteins (Fu et al., 2007). None of these showed obvious effects on the Htt lowering by Gpr52 knock-down (Figure 3—figure supplement 3), suggesting novel mechanisms potentially involving other GEFs and/or small GTPases, which is suggested in other models as well (Emery and Eiden, 2012; Kuwayama et al., 2013). Given that the major function of small GTPases is trafficking, we tested potential Htt translocation events upon treatments of PKA-insensitive analogs Rp-cAMP or 8-pCPT-2′-O-Me cAMP, and found that they lead to Htt enrichment at the perinuclear regions and co-localization with the endoplasmic reticulumn (ER) marker (Figure 3E). Translocation of Htt to the ER may prevent its proteasomal degradation due to lack of proteasomes in the ER (Plemper et al., 1997).


A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity.

Yao Y, Cui X, Al-Ramahi I, Sun X, Li B, Hou J, Difiglia M, Palacino J, Wu ZY, Ma L, Botas J, Lu B - Elife (2015)

Gpr52's effect is not mediated by Rap1 or Rap2.Left: Representative western-blots of STHdhQ7/Q111 cells transfected with the Gpr52 siRNA (Gpr52_si2) versus control siRNA, and then with constitutively active or dominant negative Rap1 (Rap1CA or Rap1DN) or Rap2 (Rap2CA or Rap2DN). The Bars plot: Htt level reduction (%) measured by 2B7/2166 HTRF of the total lysates of cells with same transfections as in the western-blots.DOI:http://dx.doi.org/10.7554/eLife.05449.009
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372774&req=5

fig3s3: Gpr52's effect is not mediated by Rap1 or Rap2.Left: Representative western-blots of STHdhQ7/Q111 cells transfected with the Gpr52 siRNA (Gpr52_si2) versus control siRNA, and then with constitutively active or dominant negative Rap1 (Rap1CA or Rap1DN) or Rap2 (Rap2CA or Rap2DN). The Bars plot: Htt level reduction (%) measured by 2B7/2166 HTRF of the total lysates of cells with same transfections as in the western-blots.DOI:http://dx.doi.org/10.7554/eLife.05449.009
Mentions: Other than PKA, the guanine exchange factor (GEF) Epac, Rapgef2 and potentially other GEFs have been reported as cAMP sensors that mediate downstream signals (de Rooij et al., 1998; Emery and Eiden, 2012; Emery et al., 2013). Thus, Gpr52 may function via a PKA-independent and cAMP-dependent signaling mechanism by activating GEFs. The reported small GTPases downstream of these GEFs are Rap1 and Rap2, and thus we tested their potential involvement by dominant negative and constitutively active Rap proteins (Fu et al., 2007). None of these showed obvious effects on the Htt lowering by Gpr52 knock-down (Figure 3—figure supplement 3), suggesting novel mechanisms potentially involving other GEFs and/or small GTPases, which is suggested in other models as well (Emery and Eiden, 2012; Kuwayama et al., 2013). Given that the major function of small GTPases is trafficking, we tested potential Htt translocation events upon treatments of PKA-insensitive analogs Rp-cAMP or 8-pCPT-2′-O-Me cAMP, and found that they lead to Htt enrichment at the perinuclear regions and co-localization with the endoplasmic reticulumn (ER) marker (Figure 3E). Translocation of Htt to the ER may prevent its proteasomal degradation due to lack of proteasomes in the ER (Plemper et al., 1997).

Bottom Line: Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum.Finally, reducing Gpr52 suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models.Thus, our discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, Department of Biophysics, School of Life Sciences, Fudan University, Shanghai, China.

ABSTRACT
Huntington's disease (HD) represents an important model for neurodegenerative disorders and proteinopathies. It is mainly caused by cytotoxicity of the mutant huntingtin protein (Htt) with an expanded polyQ stretch. While Htt is ubiquitously expressed, HD is characterized by selective neurodegeneration of the striatum. Here we report a striatal-enriched orphan G protein-coupled receptor(GPCR) Gpr52 as a stabilizer of Htt in vitro and in vivo. Gpr52 modulates Htt via cAMP-dependent but PKA independent mechanisms. Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum. Finally, reducing Gpr52 suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models. Thus, our discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies.

No MeSH data available.


Related in: MedlinePlus