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Loss of function of Slc20a2 associated with familial idiopathic Basal Ganglia calcification in humans causes brain calcifications in mice.

Jensen N, Schrøder HD, Hejbøl EK, Füchtbauer EM, de Oliveira JR, Pedersen L - J. Mol. Neurosci. (2013)

Bottom Line: Familial idiopathic basal ganglia calcification (FIBGC) is a neurodegenerative disorder with neuropsychiatric and motor symptoms.Deleterious mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), were recently linked to FIBGC in almost 50% of the families reported worldwide.Here, we show that knockout of Slc20a2 in mice causes calcifications in the thalamus, basal ganglia, and cortex, demonstrating that reduced PiT2 expression alone can cause brain calcifications.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.

ABSTRACT
Familial idiopathic basal ganglia calcification (FIBGC) is a neurodegenerative disorder with neuropsychiatric and motor symptoms. Deleterious mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), were recently linked to FIBGC in almost 50% of the families reported worldwide. Here, we show that knockout of Slc20a2 in mice causes calcifications in the thalamus, basal ganglia, and cortex, demonstrating that reduced PiT2 expression alone can cause brain calcifications.

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PAS staining of brain sections from homozygous Slc20a2 knockout mouse. Calcifications (purple stains) in close relation to blood vessels, delineated by the PAS-positive basal membrane, are found in the basal ganglia (a), thalamus (b), and cortex (not shown) (scale bars, 30 μm). No calcifications/lesions were observed in wt control (not shown)
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Fig3: PAS staining of brain sections from homozygous Slc20a2 knockout mouse. Calcifications (purple stains) in close relation to blood vessels, delineated by the PAS-positive basal membrane, are found in the basal ganglia (a), thalamus (b), and cortex (not shown) (scale bars, 30 μm). No calcifications/lesions were observed in wt control (not shown)

Mentions: To establish Slc20a2 homozygous knockout mice, we used the Slc20a2tm1a(EUCOMM)Wtsi allele on a C57BL/6NTac background obtained from the European Mouse Mutant Archive. The targeting vector introduced a splice acceptor and SV40 polyadenylation sequences between the second and third coding exons in Slc20a2, which leads to a premature stop of transcription. The truncated RNA lacks eight downstream coding exons and does not code for any functional gene. Breeding of heterozygous Slc20a2tm1a(EUCOMM)Wtsi mice resulted in viable mice homozygous for the mutation (Fig. 1a) in a Mendelian ratio. To test for knockout of Slc20a2, we used primers positioned downstream of the knockout cassette and quantified the expression level by a quantitative reverse transcription (RT)-PCR (qRT-PCR) analysis. As seen in Fig. 1b, the mouse homozygous for the knockout cassette shown in Fig. 2 has no expression of Slc20a2 downstream of the cassette. In sections from the brain, we found extensive, bilateral irregular aggregates of calcified spheres in the thalamus using Von Kossa staining (Fig. 2a). The spheres were about 10 μm in diameter, and the size of the aggregates was about 50–100 μm (Fig. 2c, d). A few calcifications were also found in the basal ganglia (Fig. 2f) and in the cortex (Fig. 2g). There were no calcifications observed in the cerebellum (not shown). No calcifications were detected in wt controls (Fig. 2b). Tissue reaction to the calcification was demonstrated by immunohistochemistry showing F4/80 positive microglia surrounding the individual elements of the calcified aggregates (Fig. 2e). No other lesions were observed in the brain, and there was no additional focal increase in microglia. The calcifications in FIBGC are believed to initiate in or around blood vessels (Sobrido et al. 2004). Interestingly, PAS staining suggest that this is also the case in the homozygous Slc20a2 knockout mouse (Fig. 3). The results show that knockout of Slc20a2 alone is sufficient to cause FIBGC-like calcifications in mice and support the significance of the linkage between mutations in SLC20A2 and FIBGC.Fig. 1


Loss of function of Slc20a2 associated with familial idiopathic Basal Ganglia calcification in humans causes brain calcifications in mice.

Jensen N, Schrøder HD, Hejbøl EK, Füchtbauer EM, de Oliveira JR, Pedersen L - J. Mol. Neurosci. (2013)

PAS staining of brain sections from homozygous Slc20a2 knockout mouse. Calcifications (purple stains) in close relation to blood vessels, delineated by the PAS-positive basal membrane, are found in the basal ganglia (a), thalamus (b), and cortex (not shown) (scale bars, 30 μm). No calcifications/lesions were observed in wt control (not shown)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: PAS staining of brain sections from homozygous Slc20a2 knockout mouse. Calcifications (purple stains) in close relation to blood vessels, delineated by the PAS-positive basal membrane, are found in the basal ganglia (a), thalamus (b), and cortex (not shown) (scale bars, 30 μm). No calcifications/lesions were observed in wt control (not shown)
Mentions: To establish Slc20a2 homozygous knockout mice, we used the Slc20a2tm1a(EUCOMM)Wtsi allele on a C57BL/6NTac background obtained from the European Mouse Mutant Archive. The targeting vector introduced a splice acceptor and SV40 polyadenylation sequences between the second and third coding exons in Slc20a2, which leads to a premature stop of transcription. The truncated RNA lacks eight downstream coding exons and does not code for any functional gene. Breeding of heterozygous Slc20a2tm1a(EUCOMM)Wtsi mice resulted in viable mice homozygous for the mutation (Fig. 1a) in a Mendelian ratio. To test for knockout of Slc20a2, we used primers positioned downstream of the knockout cassette and quantified the expression level by a quantitative reverse transcription (RT)-PCR (qRT-PCR) analysis. As seen in Fig. 1b, the mouse homozygous for the knockout cassette shown in Fig. 2 has no expression of Slc20a2 downstream of the cassette. In sections from the brain, we found extensive, bilateral irregular aggregates of calcified spheres in the thalamus using Von Kossa staining (Fig. 2a). The spheres were about 10 μm in diameter, and the size of the aggregates was about 50–100 μm (Fig. 2c, d). A few calcifications were also found in the basal ganglia (Fig. 2f) and in the cortex (Fig. 2g). There were no calcifications observed in the cerebellum (not shown). No calcifications were detected in wt controls (Fig. 2b). Tissue reaction to the calcification was demonstrated by immunohistochemistry showing F4/80 positive microglia surrounding the individual elements of the calcified aggregates (Fig. 2e). No other lesions were observed in the brain, and there was no additional focal increase in microglia. The calcifications in FIBGC are believed to initiate in or around blood vessels (Sobrido et al. 2004). Interestingly, PAS staining suggest that this is also the case in the homozygous Slc20a2 knockout mouse (Fig. 3). The results show that knockout of Slc20a2 alone is sufficient to cause FIBGC-like calcifications in mice and support the significance of the linkage between mutations in SLC20A2 and FIBGC.Fig. 1

Bottom Line: Familial idiopathic basal ganglia calcification (FIBGC) is a neurodegenerative disorder with neuropsychiatric and motor symptoms.Deleterious mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), were recently linked to FIBGC in almost 50% of the families reported worldwide.Here, we show that knockout of Slc20a2 in mice causes calcifications in the thalamus, basal ganglia, and cortex, demonstrating that reduced PiT2 expression alone can cause brain calcifications.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.

ABSTRACT
Familial idiopathic basal ganglia calcification (FIBGC) is a neurodegenerative disorder with neuropsychiatric and motor symptoms. Deleterious mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), were recently linked to FIBGC in almost 50% of the families reported worldwide. Here, we show that knockout of Slc20a2 in mice causes calcifications in the thalamus, basal ganglia, and cortex, demonstrating that reduced PiT2 expression alone can cause brain calcifications.

Show MeSH
Related in: MedlinePlus