Limits...
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.

Show MeSH

Related in: MedlinePlus

Lipid metabolism in ostes mice. (A) Lipid profiling in ostes/ostes mice. Blood lipids were analysed from fasted ostes/ostes (n = 9 female, 11 male) and wild-type (n = 12 female, 12 male) mice for triglyceride (TG) and free fatty acid (FFA). Both male and female ostes/ostes mice showed significantly decreased levels of both TG and FFA (TG: female: P = 3.107E − 05; male: P = 0.0001; FFA: female: P = 0.0005; male: P = 2.221E − 06). Data represented are mean blood plasma levels ± SEM. (B) Total percentage body fat in ostes/ostes mice. Total body fat was analysed by DEXA analysis in ostes/ostes (n = 9 female, 11 male) and wild-type (n = 22 female, 22 male) mice. Total percentage body fat was significantly decreased in both female and male ostes/ostes mice, compared with wild-type (female: P = 1.922E − 03, male: P = 5.023E − 06). Data represented are mean percentage body fat ± SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2742400&req=5

DDP304F8: Lipid metabolism in ostes mice. (A) Lipid profiling in ostes/ostes mice. Blood lipids were analysed from fasted ostes/ostes (n = 9 female, 11 male) and wild-type (n = 12 female, 12 male) mice for triglyceride (TG) and free fatty acid (FFA). Both male and female ostes/ostes mice showed significantly decreased levels of both TG and FFA (TG: female: P = 3.107E − 05; male: P = 0.0001; FFA: female: P = 0.0005; male: P = 2.221E − 06). Data represented are mean blood plasma levels ± SEM. (B) Total percentage body fat in ostes/ostes mice. Total body fat was analysed by DEXA analysis in ostes/ostes (n = 9 female, 11 male) and wild-type (n = 22 female, 22 male) mice. Total percentage body fat was significantly decreased in both female and male ostes/ostes mice, compared with wild-type (female: P = 1.922E − 03, male: P = 5.023E − 06). Data represented are mean percentage body fat ± SEM.

Mentions: We speculated that the overexpression of PKD1L2 in ostes/ostes skeletal muscle could affect the expression or activity of FASN in this tissue. Surprisingly, we found that FASN was overexpressed in ostes/ostes and Tg(Pkd1l2)/Tg(Pkd1l2) skeletal muscle by western blot analysis, mirroring the overexpression of PKD1L2 (n = 12, representative samples shown in Fig. 5). Overexpression of FASN was observed in both soluble and membrane fractions (Fig. 5) and was also confirmed to occur at the transcriptional level (Supplementary Material, Fig. S8A). The growth defect exhibited by the ostes mouse is counterintuitive to an overexpression of FASN, as the latter is generally found in conditions of metabolic overload such as obesity (reviewed in 9). We therefore hypothesized that PKD1L2 acts as a negative regulator of FASN and that the upregulation of PKD1L2 in the ENU and transgenic lines results in decreased FASN activity. In this hypothesis, the increased expression of FASN in skeletal muscle of ostes mice would result from a positive feedback regulatory mechanism. Relative activity of FASN to total amount of FASN protein was measured in soluble fractions of ostes/ostes and wild-type muscle by spectrophotometric analysis of NADPH > NADP+ oxidation. Although there is a clear trend showing reduced relative activity in ostes/ostes mice, FASN activity varied widely in individual samples of the same genotype, making the difference between ostes/ostes and control statistically not significant (P = 0.236) (Supplementary Material, Fig. S8B). Therefore, we looked for other biochemical markers of lipid metabolism to determine whether fatty acid biosynthesis was abnormal in ostes/ostes mice. We assessed the blood plasma lipid profile of ostes/ostes mice (Fig. 8A). Both triglyceride (TG) and free fatty acid (FFA) levels were significantly and markedly decreased in plasma from both male and female ostes/ostes mice, compared with wild-types (Fig. 8A). Furthermore, total body fat content of ostes/ostes mice was analysed by dual-energy X-ray absorptiometry (DEXA) analysis (Fig. 8B). The total fat percentage of body mass was significantly decreased in ostes/ostes mice from a mean wild-type percentage of ∼20 to ∼17% in female ostes/ostes mice and from ∼17 to ∼14% in male ostes/ostes mice (Fig. 8B). These results indicated a reduction of lipogenesis in ostes mice.


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)

Lipid metabolism in ostes mice. (A) Lipid profiling in ostes/ostes mice. Blood lipids were analysed from fasted ostes/ostes (n = 9 female, 11 male) and wild-type (n = 12 female, 12 male) mice for triglyceride (TG) and free fatty acid (FFA). Both male and female ostes/ostes mice showed significantly decreased levels of both TG and FFA (TG: female: P = 3.107E − 05; male: P = 0.0001; FFA: female: P = 0.0005; male: P = 2.221E − 06). Data represented are mean blood plasma levels ± SEM. (B) Total percentage body fat in ostes/ostes mice. Total body fat was analysed by DEXA analysis in ostes/ostes (n = 9 female, 11 male) and wild-type (n = 22 female, 22 male) mice. Total percentage body fat was significantly decreased in both female and male ostes/ostes mice, compared with wild-type (female: P = 1.922E − 03, male: P = 5.023E − 06). Data represented are mean percentage body fat ± SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742400&req=5

DDP304F8: Lipid metabolism in ostes mice. (A) Lipid profiling in ostes/ostes mice. Blood lipids were analysed from fasted ostes/ostes (n = 9 female, 11 male) and wild-type (n = 12 female, 12 male) mice for triglyceride (TG) and free fatty acid (FFA). Both male and female ostes/ostes mice showed significantly decreased levels of both TG and FFA (TG: female: P = 3.107E − 05; male: P = 0.0001; FFA: female: P = 0.0005; male: P = 2.221E − 06). Data represented are mean blood plasma levels ± SEM. (B) Total percentage body fat in ostes/ostes mice. Total body fat was analysed by DEXA analysis in ostes/ostes (n = 9 female, 11 male) and wild-type (n = 22 female, 22 male) mice. Total percentage body fat was significantly decreased in both female and male ostes/ostes mice, compared with wild-type (female: P = 1.922E − 03, male: P = 5.023E − 06). Data represented are mean percentage body fat ± SEM.
Mentions: We speculated that the overexpression of PKD1L2 in ostes/ostes skeletal muscle could affect the expression or activity of FASN in this tissue. Surprisingly, we found that FASN was overexpressed in ostes/ostes and Tg(Pkd1l2)/Tg(Pkd1l2) skeletal muscle by western blot analysis, mirroring the overexpression of PKD1L2 (n = 12, representative samples shown in Fig. 5). Overexpression of FASN was observed in both soluble and membrane fractions (Fig. 5) and was also confirmed to occur at the transcriptional level (Supplementary Material, Fig. S8A). The growth defect exhibited by the ostes mouse is counterintuitive to an overexpression of FASN, as the latter is generally found in conditions of metabolic overload such as obesity (reviewed in 9). We therefore hypothesized that PKD1L2 acts as a negative regulator of FASN and that the upregulation of PKD1L2 in the ENU and transgenic lines results in decreased FASN activity. In this hypothesis, the increased expression of FASN in skeletal muscle of ostes mice would result from a positive feedback regulatory mechanism. Relative activity of FASN to total amount of FASN protein was measured in soluble fractions of ostes/ostes and wild-type muscle by spectrophotometric analysis of NADPH > NADP+ oxidation. Although there is a clear trend showing reduced relative activity in ostes/ostes mice, FASN activity varied widely in individual samples of the same genotype, making the difference between ostes/ostes and control statistically not significant (P = 0.236) (Supplementary Material, Fig. S8B). Therefore, we looked for other biochemical markers of lipid metabolism to determine whether fatty acid biosynthesis was abnormal in ostes/ostes mice. We assessed the blood plasma lipid profile of ostes/ostes mice (Fig. 8A). Both triglyceride (TG) and free fatty acid (FFA) levels were significantly and markedly decreased in plasma from both male and female ostes/ostes mice, compared with wild-types (Fig. 8A). Furthermore, total body fat content of ostes/ostes mice was analysed by dual-energy X-ray absorptiometry (DEXA) analysis (Fig. 8B). The total fat percentage of body mass was significantly decreased in ostes/ostes mice from a mean wild-type percentage of ∼20 to ∼17% in female ostes/ostes mice and from ∼17 to ∼14% in male ostes/ostes mice (Fig. 8B). These results indicated a reduction of lipogenesis in ostes mice.

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