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

Co-localization of PKD1L2 and FASN in skeletal muscle. (A) Confocal microscopic analysis of fresh-frozen skeletal muscle sections from wild-type mice immunostained with APKD1L2_1 and gAFASN antibodies. Top panel, a cross-section of muscle showing PKD1L2 and FASN co-localization at the membrane. Middle panel: a view of the surface of longitudinally sectioned fibres showing PKD1L2 and FASN with overlapping localizations to striations. Bottom panel: a deeper level of the same fibres showing PKD1L2 and FASN with overlapping localizations at the membrane. (B) For comparison, a view of the mid-level of a longitudinal fibre stained with dystrophin and α-actinin using the same conditions. Note that, in contrast to the bottom panel shown in (A), the Z-discs are stained here. (C) Top panel: superficial (‘Top’) and middle views of longitudinal fibres showing PKD1L2 co-localization with the costameric protein β-dystroglycan. Bottom panel: a close-up view of top and middle levels of a single fibre. Scale bar: 25 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DDP304F6: Co-localization of PKD1L2 and FASN in skeletal muscle. (A) Confocal microscopic analysis of fresh-frozen skeletal muscle sections from wild-type mice immunostained with APKD1L2_1 and gAFASN antibodies. Top panel, a cross-section of muscle showing PKD1L2 and FASN co-localization at the membrane. Middle panel: a view of the surface of longitudinally sectioned fibres showing PKD1L2 and FASN with overlapping localizations to striations. Bottom panel: a deeper level of the same fibres showing PKD1L2 and FASN with overlapping localizations at the membrane. (B) For comparison, a view of the mid-level of a longitudinal fibre stained with dystrophin and α-actinin using the same conditions. Note that, in contrast to the bottom panel shown in (A), the Z-discs are stained here. (C) Top panel: superficial (‘Top’) and middle views of longitudinal fibres showing PKD1L2 co-localization with the costameric protein β-dystroglycan. Bottom panel: a close-up view of top and middle levels of a single fibre. Scale bar: 25 μm.

Mentions: Despite PKD1L2 being a predicted transmembrane protein and FASN a soluble cytoplasmic enzyme, their association as revealed by immunoprecipitation of endogenous proteins suggested that these proteins must have overlapping subcellular localizations. To assess this, immunostainings on cross- and longitudinal sections of fresh-frozen skeletal muscle samples were performed with APKD1L2_1 and a goat polyclonal antibody against FASN (gAFASN). PKD1L2 and FASN were found localized to the membrane on cross-sections (Fig. 6A). On longitudinal sections, both antibodies produced overlapping localizations at striations overlying the Z-discs on the surface of the fibre (Fig. 6A, middle panel) but not at the centre (Fig. 6A, bottom panel). A similar result was obtained with mAFASN antibody (Supplementary Material, Fig. S7), suggesting a costameric localization of both proteins. Costameric distribution was supported by co-localization of PKD1L2 with the costameric transmembrane protein β-dystroglycan (8) on serial views of longitudinal sections (Fig. 6C). An identical pattern was observed with antibody APKD1L2_2, and for both PKD1L2 antibodies, the fluorescent signal was effectively blocked upon pre-incubation with their respective antigenic peptides (data not shown). On the other hand, expression of PKD1L2 was also detected at slightly higher levels at the postsynaptic membrane of the NMJs (Fig. 7A and B).


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)

Co-localization of PKD1L2 and FASN in skeletal muscle. (A) Confocal microscopic analysis of fresh-frozen skeletal muscle sections from wild-type mice immunostained with APKD1L2_1 and gAFASN antibodies. Top panel, a cross-section of muscle showing PKD1L2 and FASN co-localization at the membrane. Middle panel: a view of the surface of longitudinally sectioned fibres showing PKD1L2 and FASN with overlapping localizations to striations. Bottom panel: a deeper level of the same fibres showing PKD1L2 and FASN with overlapping localizations at the membrane. (B) For comparison, a view of the mid-level of a longitudinal fibre stained with dystrophin and α-actinin using the same conditions. Note that, in contrast to the bottom panel shown in (A), the Z-discs are stained here. (C) Top panel: superficial (‘Top’) and middle views of longitudinal fibres showing PKD1L2 co-localization with the costameric protein β-dystroglycan. Bottom panel: a close-up view of top and middle levels of a single fibre. Scale bar: 25 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DDP304F6: Co-localization of PKD1L2 and FASN in skeletal muscle. (A) Confocal microscopic analysis of fresh-frozen skeletal muscle sections from wild-type mice immunostained with APKD1L2_1 and gAFASN antibodies. Top panel, a cross-section of muscle showing PKD1L2 and FASN co-localization at the membrane. Middle panel: a view of the surface of longitudinally sectioned fibres showing PKD1L2 and FASN with overlapping localizations to striations. Bottom panel: a deeper level of the same fibres showing PKD1L2 and FASN with overlapping localizations at the membrane. (B) For comparison, a view of the mid-level of a longitudinal fibre stained with dystrophin and α-actinin using the same conditions. Note that, in contrast to the bottom panel shown in (A), the Z-discs are stained here. (C) Top panel: superficial (‘Top’) and middle views of longitudinal fibres showing PKD1L2 co-localization with the costameric protein β-dystroglycan. Bottom panel: a close-up view of top and middle levels of a single fibre. Scale bar: 25 μm.
Mentions: Despite PKD1L2 being a predicted transmembrane protein and FASN a soluble cytoplasmic enzyme, their association as revealed by immunoprecipitation of endogenous proteins suggested that these proteins must have overlapping subcellular localizations. To assess this, immunostainings on cross- and longitudinal sections of fresh-frozen skeletal muscle samples were performed with APKD1L2_1 and a goat polyclonal antibody against FASN (gAFASN). PKD1L2 and FASN were found localized to the membrane on cross-sections (Fig. 6A). On longitudinal sections, both antibodies produced overlapping localizations at striations overlying the Z-discs on the surface of the fibre (Fig. 6A, middle panel) but not at the centre (Fig. 6A, bottom panel). A similar result was obtained with mAFASN antibody (Supplementary Material, Fig. S7), suggesting a costameric localization of both proteins. Costameric distribution was supported by co-localization of PKD1L2 with the costameric transmembrane protein β-dystroglycan (8) on serial views of longitudinal sections (Fig. 6C). An identical pattern was observed with antibody APKD1L2_2, and for both PKD1L2 antibodies, the fluorescent signal was effectively blocked upon pre-incubation with their respective antigenic peptides (data not shown). On the other hand, expression of PKD1L2 was also detected at slightly higher levels at the postsynaptic membrane of the NMJs (Fig. 7A and B).

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