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Upregulation of PKD1L2 provokes a complex neuromuscular disease in the mouse.

Mackenzie FE, Romero R, Williams D, Gillingwater T, Hilton H, Dick J, Riddoch-Contreras J, Wong F, Ireson L, Powles-Glover N, Riley G, Underhill P, Hough T, Arkell R, Greensmith L, Ribchester RR, Blanco G - Hum. Mol. Genet. (2009)

Bottom Line: Genetic and biochemical evidence shows that upregulation of the novel, uncharacterized transient receptor potential polycystic (TRPP) channel PKD1L2 (polycystic kidney disease gene 1-like 2) underlies this disease.We show that, in vivo, PKD1L2 primarily associates with endogenous fatty acid synthase in normal skeletal muscle, and these proteins co-localize to costameric regions of the muscle fibre.This work shows the first role for a TRPP channel in neuromuscular integrity and disease.

View Article: PubMed Central - PubMed

Affiliation: MRC Mammalian Genetics Unit, Harwell OX11 0RD, UK.

ABSTRACT
Following a screen for neuromuscular mouse mutants, we identified ostes, a novel N-ethyl N-nitrosourea-induced mouse mutant with muscle atrophy. Genetic and biochemical evidence shows that upregulation of the novel, uncharacterized transient receptor potential polycystic (TRPP) channel PKD1L2 (polycystic kidney disease gene 1-like 2) underlies this disease. Ostes mice suffer from chronic neuromuscular impairments including neuromuscular junction degeneration, polyneuronal innervation and myopathy. Ectopic expression of PKD1L2 in transgenic mice reproduced the ostes myopathic changes and, indeed, caused severe muscle atrophy in Tg(Pkd1l2)/Tg(Pkd1l2) mice. Moreover, double-heterozygous mice (ostes/+, Tg(Pkd1l2)/0) suffer from myopathic changes more profound than each heterozygote, indicating positive correlation between PKD1L2 levels and disease severity. We show that, in vivo, PKD1L2 primarily associates with endogenous fatty acid synthase in normal skeletal muscle, and these proteins co-localize to costameric regions of the muscle fibre. In diseased ostes/ostes muscle, both proteins are upregulated, and ostes/ostes mice show signs of abnormal lipid metabolism. This work shows the first role for a TRPP channel in neuromuscular integrity and disease.

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Western blot analysis of skeletal muscle extracts from wild-type, ostes/ostes and Tg(Pkd1l2)/Tg(Pkd1l2) mice. (A) PKD1L2 and FASN show increased levels in the membrane fraction of ostes/ostes and Tg(Pkd1l2)/Tg(Pkd1l2) skeletal muscle. Samples shown are from individual mice (n = 2 per genotype). (B) PKD1L2 and FASN show increased levels in the soluble fraction of ostes/ostes skeletal muscle. Samples shown are from individual mice (n = 2 per genotype). PKD1L2 and FASN were detected, respectively, with antibodies APKD1L2_1 and mAFASN. +/+, wild-type; ost, ostes/ostes; Tg, Tg(Pkd1l2)/Tg(Pkd1l2).
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DDP304F5: Western blot analysis of skeletal muscle extracts from wild-type, ostes/ostes and Tg(Pkd1l2)/Tg(Pkd1l2) mice. (A) PKD1L2 and FASN show increased levels in the membrane fraction of ostes/ostes and Tg(Pkd1l2)/Tg(Pkd1l2) skeletal muscle. Samples shown are from individual mice (n = 2 per genotype). (B) PKD1L2 and FASN show increased levels in the soluble fraction of ostes/ostes skeletal muscle. Samples shown are from individual mice (n = 2 per genotype). PKD1L2 and FASN were detected, respectively, with antibodies APKD1L2_1 and mAFASN. +/+, wild-type; ost, ostes/ostes; Tg, Tg(Pkd1l2)/Tg(Pkd1l2).

Mentions: We assessed Pkd1l2 as a candidate gene for ostes by generating PKD1L2-specific antibodies and examining PKD1L2 protein in ostes/ostes muscle. Two rabbit polyclonal antibodies, APKD1L2_1 and APKD1L2_2, were generated and thoroughly characterized (Fig. 4). Western blot analysis of soluble skeletal muscle extracts from wild-type mice showed that both antibodies recognized a band migrating at ∼268 kDa (Fig. 4B), consistent with the PKD1L2 expected size (272 kDa). Western blot analysis of multiple tissues showed that this band was the largest protein recognized in several tissues (Supplementary Material, Fig. S6A), suggesting that this protein is likely to be full-length PKD1L2. We used immunoprecipitation to confirm the specificity of these antibodies. Antibody APKD1L2_1 specifically and efficiently immunoprecipitated a protein migrating at ∼268 kDa from wild-type skeletal muscle lysates (Fig. 4C). Analysis of the corresponding band by peptide mass fingerprinting identified PKD1L2 among other proteins (see below and Fig. 4C). In addition, the immunoprecipitated protein was also recognized by the second antibody, APKD1L2_2 (Fig. 4C). We then assessed PKD1L2 protein expression in ostes/ostes skeletal muscle. Since PKD1L2 is a predicted transmembrane protein, PKD1L2 expression was assessed in both soluble and membrane fractions. The results showed that PKD1L2 is strongly accumulated in ostes/ostes skeletal muscle compared with wild-type (n = 8, representative samples shown in Fig. 5). Accumulation was observed in both soluble and membrane fractions (Fig. 5). These results suggested that the upregulation of PKD1L2 underlies the ostes phenotype.


Upregulation of PKD1L2 provokes a complex neuromuscular disease in the mouse.

Mackenzie FE, Romero R, Williams D, Gillingwater T, Hilton H, Dick J, Riddoch-Contreras J, Wong F, Ireson L, Powles-Glover N, Riley G, Underhill P, Hough T, Arkell R, Greensmith L, Ribchester RR, Blanco G - Hum. Mol. Genet. (2009)

Western blot analysis of skeletal muscle extracts from wild-type, ostes/ostes and Tg(Pkd1l2)/Tg(Pkd1l2) mice. (A) PKD1L2 and FASN show increased levels in the membrane fraction of ostes/ostes and Tg(Pkd1l2)/Tg(Pkd1l2) skeletal muscle. Samples shown are from individual mice (n = 2 per genotype). (B) PKD1L2 and FASN show increased levels in the soluble fraction of ostes/ostes skeletal muscle. Samples shown are from individual mice (n = 2 per genotype). PKD1L2 and FASN were detected, respectively, with antibodies APKD1L2_1 and mAFASN. +/+, wild-type; ost, ostes/ostes; Tg, Tg(Pkd1l2)/Tg(Pkd1l2).
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DDP304F5: Western blot analysis of skeletal muscle extracts from wild-type, ostes/ostes and Tg(Pkd1l2)/Tg(Pkd1l2) mice. (A) PKD1L2 and FASN show increased levels in the membrane fraction of ostes/ostes and Tg(Pkd1l2)/Tg(Pkd1l2) skeletal muscle. Samples shown are from individual mice (n = 2 per genotype). (B) PKD1L2 and FASN show increased levels in the soluble fraction of ostes/ostes skeletal muscle. Samples shown are from individual mice (n = 2 per genotype). PKD1L2 and FASN were detected, respectively, with antibodies APKD1L2_1 and mAFASN. +/+, wild-type; ost, ostes/ostes; Tg, Tg(Pkd1l2)/Tg(Pkd1l2).
Mentions: We assessed Pkd1l2 as a candidate gene for ostes by generating PKD1L2-specific antibodies and examining PKD1L2 protein in ostes/ostes muscle. Two rabbit polyclonal antibodies, APKD1L2_1 and APKD1L2_2, were generated and thoroughly characterized (Fig. 4). Western blot analysis of soluble skeletal muscle extracts from wild-type mice showed that both antibodies recognized a band migrating at ∼268 kDa (Fig. 4B), consistent with the PKD1L2 expected size (272 kDa). Western blot analysis of multiple tissues showed that this band was the largest protein recognized in several tissues (Supplementary Material, Fig. S6A), suggesting that this protein is likely to be full-length PKD1L2. We used immunoprecipitation to confirm the specificity of these antibodies. Antibody APKD1L2_1 specifically and efficiently immunoprecipitated a protein migrating at ∼268 kDa from wild-type skeletal muscle lysates (Fig. 4C). Analysis of the corresponding band by peptide mass fingerprinting identified PKD1L2 among other proteins (see below and Fig. 4C). In addition, the immunoprecipitated protein was also recognized by the second antibody, APKD1L2_2 (Fig. 4C). We then assessed PKD1L2 protein expression in ostes/ostes skeletal muscle. Since PKD1L2 is a predicted transmembrane protein, PKD1L2 expression was assessed in both soluble and membrane fractions. The results showed that PKD1L2 is strongly accumulated in ostes/ostes skeletal muscle compared with wild-type (n = 8, representative samples shown in Fig. 5). Accumulation was observed in both soluble and membrane fractions (Fig. 5). These results suggested that the upregulation of PKD1L2 underlies the ostes phenotype.

Bottom Line: Genetic and biochemical evidence shows that upregulation of the novel, uncharacterized transient receptor potential polycystic (TRPP) channel PKD1L2 (polycystic kidney disease gene 1-like 2) underlies this disease.We show that, in vivo, PKD1L2 primarily associates with endogenous fatty acid synthase in normal skeletal muscle, and these proteins co-localize to costameric regions of the muscle fibre.This work shows the first role for a TRPP channel in neuromuscular integrity and disease.

View Article: PubMed Central - PubMed

Affiliation: MRC Mammalian Genetics Unit, Harwell OX11 0RD, UK.

ABSTRACT
Following a screen for neuromuscular mouse mutants, we identified ostes, a novel N-ethyl N-nitrosourea-induced mouse mutant with muscle atrophy. Genetic and biochemical evidence shows that upregulation of the novel, uncharacterized transient receptor potential polycystic (TRPP) channel PKD1L2 (polycystic kidney disease gene 1-like 2) underlies this disease. Ostes mice suffer from chronic neuromuscular impairments including neuromuscular junction degeneration, polyneuronal innervation and myopathy. Ectopic expression of PKD1L2 in transgenic mice reproduced the ostes myopathic changes and, indeed, caused severe muscle atrophy in Tg(Pkd1l2)/Tg(Pkd1l2) mice. Moreover, double-heterozygous mice (ostes/+, Tg(Pkd1l2)/0) suffer from myopathic changes more profound than each heterozygote, indicating positive correlation between PKD1L2 levels and disease severity. We show that, in vivo, PKD1L2 primarily associates with endogenous fatty acid synthase in normal skeletal muscle, and these proteins co-localize to costameric regions of the muscle fibre. In diseased ostes/ostes muscle, both proteins are upregulated, and ostes/ostes mice show signs of abnormal lipid metabolism. This work shows the first role for a TRPP channel in neuromuscular integrity and disease.

Show MeSH
Related in: MedlinePlus