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A role for Alström syndrome protein, alms1, in kidney ciliogenesis and cellular quiescence.

Li G, Vega R, Nelms K, Gekakis N, Goodnow C, McNamara P, Wu H, Hong NA, Glynne R - PLoS Genet. (2006)

Bottom Line: ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia.The stunted-cilium phenotype can be rescued with a 5' fragment of the Alms1 cDNA, which resembles disease-associated alleles.As renal failure is a common cause of mortality in Alström syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alström syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia.

View Article: PubMed Central - PubMed

Affiliation: Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.

ABSTRACT
Premature truncation alleles in the ALMS1 gene are a frequent cause of human Alström syndrome. Alström syndrome is a rare disorder characterized by early obesity and sensory impairment, symptoms shared with other genetic diseases affecting proteins of the primary cilium. ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia. Here, we show that in vitro knockdown of Alms1 in mice causes stunted cilia on kidney epithelial cells and prevents these cells from increasing calcium influx in response to mechanical stimuli. The stunted-cilium phenotype can be rescued with a 5' fragment of the Alms1 cDNA, which resembles disease-associated alleles. In a mouse model of Alström syndrome, Alms1 protein can be stably expressed from the mutant allele and is required for cilia formation in primary cells. Aged mice developed specific loss of cilia from the kidney proximal tubules, which is associated with foci of apoptosis or proliferation. As renal failure is a common cause of mortality in Alström syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alström syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia.

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Suppression of Alms1 Expression Alters Primary Cilium Formation in Kidney Epithelial Cells(A) Elongated cilia, visualized with staining of acetylated tubulin (green), form normally in mIMCD3 cells after mock-transfection, transfection with a negative control siRNA, or transfection with two inactive siRNAs directed against Alms1 (Alms1c and Alms1d). Focal staining of acetylated tubulin without axoneme extension is seen after transfection with two active siRNAs targeting Alms1 (Alms1a and Alms1b).(B) Real-time PCR analysis with two mouse Alms1 probes recognizing the junctions of exons 1 and 2 and exons 12 and 13, respectively: Alms1a and Alms1b siRNAs both cause 70%–80% knockdown of Alms1 mRNA; no effect on Alms1 mRNA was seen with the three siRNAs that were inactive in the ciliogenesis assay.(C) Alms1a siRNA-treated cells lose endogenous Alms1 protein expression. Acet, acetylated. Scale bars, 10 μm.
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pgen-0030008-g001: Suppression of Alms1 Expression Alters Primary Cilium Formation in Kidney Epithelial Cells(A) Elongated cilia, visualized with staining of acetylated tubulin (green), form normally in mIMCD3 cells after mock-transfection, transfection with a negative control siRNA, or transfection with two inactive siRNAs directed against Alms1 (Alms1c and Alms1d). Focal staining of acetylated tubulin without axoneme extension is seen after transfection with two active siRNAs targeting Alms1 (Alms1a and Alms1b).(B) Real-time PCR analysis with two mouse Alms1 probes recognizing the junctions of exons 1 and 2 and exons 12 and 13, respectively: Alms1a and Alms1b siRNAs both cause 70%–80% knockdown of Alms1 mRNA; no effect on Alms1 mRNA was seen with the three siRNAs that were inactive in the ciliogenesis assay.(C) Alms1a siRNA-treated cells lose endogenous Alms1 protein expression. Acet, acetylated. Scale bars, 10 μm.

Mentions: To determine whether Alms1 was necessary for cilium formation and/or function, we used an in vitro model of kidney cell ciliogenesis and signaling. Mouse inner medullary collecting duct (mIMCD3) cells form cilia 5 d after confluency [23]. The cilia can be visualized as long protrusions from the cell surface using an antibody raised against acetylated tubulin (Figure 1A). We tested several short interfering RNA (siRNA) molecules designed against the mouse Alms1 sequence for their effects on formation of cilia in this model. Cilia were formed normally in the presence of a negative control siRNA. However, transfection with two siRNA sequences against Alms1, Alms1a and Alms1b, led to a markedly different phenotype. The acetylated tubulin staining on each cell manifested as a single ball of fluorescence, and very few cells showed the elongated staining typical of ciliary axoneme (Figure 1A). Both of these siRNA species also caused a decrease in the Alms1 mRNA level (Figure 1B). In contrast, two additional siRNAs (Alms1c and Alms1d) did not affect the pattern of acetylated tubulin staining in the ciliogenesis assay, nor did they affect the Alms1 mRNA level (Figure 1A and 1B). To demonstrate that the active siRNA species also decreased levels of Alms1 protein, we raised an antiserum in rabbits against the predicted N-terminal 13 amino acids of the open reading frame of Alms1. Using this antiserum, we detected positive staining at the base of cilia, consistent with previous reports on the subcellular localization of ALMS1 [21]. The antibody signal was reduced below detection in the presence of the active siRNA molecules (Figure 1C).


A role for Alström syndrome protein, alms1, in kidney ciliogenesis and cellular quiescence.

Li G, Vega R, Nelms K, Gekakis N, Goodnow C, McNamara P, Wu H, Hong NA, Glynne R - PLoS Genet. (2006)

Suppression of Alms1 Expression Alters Primary Cilium Formation in Kidney Epithelial Cells(A) Elongated cilia, visualized with staining of acetylated tubulin (green), form normally in mIMCD3 cells after mock-transfection, transfection with a negative control siRNA, or transfection with two inactive siRNAs directed against Alms1 (Alms1c and Alms1d). Focal staining of acetylated tubulin without axoneme extension is seen after transfection with two active siRNAs targeting Alms1 (Alms1a and Alms1b).(B) Real-time PCR analysis with two mouse Alms1 probes recognizing the junctions of exons 1 and 2 and exons 12 and 13, respectively: Alms1a and Alms1b siRNAs both cause 70%–80% knockdown of Alms1 mRNA; no effect on Alms1 mRNA was seen with the three siRNAs that were inactive in the ciliogenesis assay.(C) Alms1a siRNA-treated cells lose endogenous Alms1 protein expression. Acet, acetylated. Scale bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1761047&req=5

pgen-0030008-g001: Suppression of Alms1 Expression Alters Primary Cilium Formation in Kidney Epithelial Cells(A) Elongated cilia, visualized with staining of acetylated tubulin (green), form normally in mIMCD3 cells after mock-transfection, transfection with a negative control siRNA, or transfection with two inactive siRNAs directed against Alms1 (Alms1c and Alms1d). Focal staining of acetylated tubulin without axoneme extension is seen after transfection with two active siRNAs targeting Alms1 (Alms1a and Alms1b).(B) Real-time PCR analysis with two mouse Alms1 probes recognizing the junctions of exons 1 and 2 and exons 12 and 13, respectively: Alms1a and Alms1b siRNAs both cause 70%–80% knockdown of Alms1 mRNA; no effect on Alms1 mRNA was seen with the three siRNAs that were inactive in the ciliogenesis assay.(C) Alms1a siRNA-treated cells lose endogenous Alms1 protein expression. Acet, acetylated. Scale bars, 10 μm.
Mentions: To determine whether Alms1 was necessary for cilium formation and/or function, we used an in vitro model of kidney cell ciliogenesis and signaling. Mouse inner medullary collecting duct (mIMCD3) cells form cilia 5 d after confluency [23]. The cilia can be visualized as long protrusions from the cell surface using an antibody raised against acetylated tubulin (Figure 1A). We tested several short interfering RNA (siRNA) molecules designed against the mouse Alms1 sequence for their effects on formation of cilia in this model. Cilia were formed normally in the presence of a negative control siRNA. However, transfection with two siRNA sequences against Alms1, Alms1a and Alms1b, led to a markedly different phenotype. The acetylated tubulin staining on each cell manifested as a single ball of fluorescence, and very few cells showed the elongated staining typical of ciliary axoneme (Figure 1A). Both of these siRNA species also caused a decrease in the Alms1 mRNA level (Figure 1B). In contrast, two additional siRNAs (Alms1c and Alms1d) did not affect the pattern of acetylated tubulin staining in the ciliogenesis assay, nor did they affect the Alms1 mRNA level (Figure 1A and 1B). To demonstrate that the active siRNA species also decreased levels of Alms1 protein, we raised an antiserum in rabbits against the predicted N-terminal 13 amino acids of the open reading frame of Alms1. Using this antiserum, we detected positive staining at the base of cilia, consistent with previous reports on the subcellular localization of ALMS1 [21]. The antibody signal was reduced below detection in the presence of the active siRNA molecules (Figure 1C).

Bottom Line: ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia.The stunted-cilium phenotype can be rescued with a 5' fragment of the Alms1 cDNA, which resembles disease-associated alleles.As renal failure is a common cause of mortality in Alström syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alström syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia.

View Article: PubMed Central - PubMed

Affiliation: Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.

ABSTRACT
Premature truncation alleles in the ALMS1 gene are a frequent cause of human Alström syndrome. Alström syndrome is a rare disorder characterized by early obesity and sensory impairment, symptoms shared with other genetic diseases affecting proteins of the primary cilium. ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia. Here, we show that in vitro knockdown of Alms1 in mice causes stunted cilia on kidney epithelial cells and prevents these cells from increasing calcium influx in response to mechanical stimuli. The stunted-cilium phenotype can be rescued with a 5' fragment of the Alms1 cDNA, which resembles disease-associated alleles. In a mouse model of Alström syndrome, Alms1 protein can be stably expressed from the mutant allele and is required for cilia formation in primary cells. Aged mice developed specific loss of cilia from the kidney proximal tubules, which is associated with foci of apoptosis or proliferation. As renal failure is a common cause of mortality in Alström syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alström syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia.

Show MeSH
Related in: MedlinePlus