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CNNM2 mutations cause impaired brain development and seizures in patients with hypomagnesemia.

Arjona FJ, de Baaij JH, Schlingmann KP, Lameris AL, van Wijk E, Flik G, Regele S, Korenke GC, Neophytou B, Rust S, Reintjes N, Konrad M, Bindels RJ, Hoenderop JG - PLoS Genet. (2014)

Bottom Line: Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg(2+) absorption.We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg(2+) homeostasis.By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for monitoring their effects on the brain and kidney phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.

ABSTRACT
Intellectual disability and seizures are frequently associated with hypomagnesemia and have an important genetic component. However, to find the genetic origin of intellectual disability and seizures often remains challenging because of considerable genetic heterogeneity and clinical variability. In this study, we have identified new mutations in CNNM2 in five families suffering from mental retardation, seizures, and hypomagnesemia. For the first time, a recessive mode of inheritance of CNNM2 mutations was observed. Importantly, patients with recessive CNNM2 mutations suffer from brain malformations and severe intellectual disability. Additionally, three patients with moderate mental disability were shown to carry de novo heterozygous missense mutations in the CNNM2 gene. To elucidate the physiological role of CNNM2 and explain the pathomechanisms of disease, we studied CNNM2 function combining in vitro activity assays and the zebrafish knockdown model system. Using stable Mg(2+) isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg(2+)-permeable cation channel TRPM7. In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg(2+) uptake. Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg(2+) absorption. These phenotypes were rescued by injection of mammalian wild-type Cnnm2 cRNA, whereas mammalian mutant Cnnm2 cRNA did not improve the zebrafish knockdown phenotypes. We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg(2+) homeostasis. By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for monitoring their effects on the brain and kidney phenotype.

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CNNM2 increases Mg2+ uptake in HEK293 cells.(A) Time curve of 25Mg2+ uptake in mock (circles) and CNNM2 (squares) transfected cells. (B) Representation of the normalized Mg2+ uptake after 5 minutes. (C) 25Mg2+ uptake in the presence of inhibitors of ion transporters, black bars represent mock cells and white bars represent CNNM2-transfected cells. (D) Dose-response curve of 25Mg2+ transport inhibition by 2-APB in mock (circles) and CNNM2 (squares) transfected cells. (E) The effect of Na+ and Cl− availability on 25Mg2+ uptake in mock (black bars) and CNNM2 (white bars) transfected cells. (F) 25Mg2+ uptake as a function of extracellular 25Mg2+ availability in mock (circles) and CNNM2 (squares) transfected cells. (G) 25Mg2+ extrusion in mock (circles) and CNNM2 (squares) transfected cells. Each data point represent the mean of 3 independent experiments ± SEM. * indicates significant differences compared to mock (P<0.05).
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pgen-1004267-g002: CNNM2 increases Mg2+ uptake in HEK293 cells.(A) Time curve of 25Mg2+ uptake in mock (circles) and CNNM2 (squares) transfected cells. (B) Representation of the normalized Mg2+ uptake after 5 minutes. (C) 25Mg2+ uptake in the presence of inhibitors of ion transporters, black bars represent mock cells and white bars represent CNNM2-transfected cells. (D) Dose-response curve of 25Mg2+ transport inhibition by 2-APB in mock (circles) and CNNM2 (squares) transfected cells. (E) The effect of Na+ and Cl− availability on 25Mg2+ uptake in mock (black bars) and CNNM2 (white bars) transfected cells. (F) 25Mg2+ uptake as a function of extracellular 25Mg2+ availability in mock (circles) and CNNM2 (squares) transfected cells. (G) 25Mg2+ extrusion in mock (circles) and CNNM2 (squares) transfected cells. Each data point represent the mean of 3 independent experiments ± SEM. * indicates significant differences compared to mock (P<0.05).

Mentions: To clarify the function of CNNM2, Human Embryonic Kidney (HEK293) cells were transiently transfected with mouse Cnnm2 or mock constructs and examined for Mg2+ transport capacity using the stable 25Mg2+ isotope. At baseline, approximately 10% of the total intracellular Mg2+ content consists of 25Mg2+, which is the natural abundance of 25Mg2+[21]. By incubating the cells in a physiological buffer containing pure 25Mg2+, the intracellular 25Mg2+ concentration increases over time. Interestingly, Cnnm2 expressing cells displayed a higher 25Mg2+ uptake compared to mock cells (Figure 2A). After 5 minutes, Cnnm2-expressing cells had approximately 2 times more 25Mg2+ uptake than mock-transfected cells (Figure 2B). All further experiments were performed at the 5 minutes time point to cover the exponential phase of the uptake. To reduce the background in 25Mg2+ uptake, inhibitors of known Mg2+ channels and transporters were added during the uptake process; 2-APB to inhibit TRPM7 [22], Ouabain to block the Na+-K+-ATPase [23], Quinidin for SLC41A1 [24] and Nitrendipin [25] for silencing MagT1 activity (Figure 2C). Only 2-APB was capable of significantly inhibiting 25Mg2+ uptake in HEK293 cells. Moreover, 2-APB inhibition also abolished the CNNM2-dependent increase in 25Mg2+ uptake. Dose-response experiments confirmed that the IC50 of 2-APB inhibition is 22 µM (Figure 2D). CNNM2-dependent 25Mg2+ uptake was found to be independent of Na+ and Cl− availability, when uptakes were performed in N-methyl-d-glucamine (NMDG) or Gluconate buffers (Figure 2E). Interestingly, the highest CNNM2-dependent 25Mg2+ uptake was measured between 1–2 mM, suggesting a Km in the physiological range of approximately 0.5 mM (Figure 2F). At high Mg2+ concentrations (5 mM), 25Mg2+ uptake was inhibited. When subjected to 24 hours 25Mg2+ loading, Cnnm2-expressing cells showed a significantly higher 25Mg2+ content baseline. Subsequently, 15 minutes extrusion of Cnnm2-expressing cells demonstrated no difference in Mg2+ extrusion rate, compared to mock-transfected cells (Figure 2G).


CNNM2 mutations cause impaired brain development and seizures in patients with hypomagnesemia.

Arjona FJ, de Baaij JH, Schlingmann KP, Lameris AL, van Wijk E, Flik G, Regele S, Korenke GC, Neophytou B, Rust S, Reintjes N, Konrad M, Bindels RJ, Hoenderop JG - PLoS Genet. (2014)

CNNM2 increases Mg2+ uptake in HEK293 cells.(A) Time curve of 25Mg2+ uptake in mock (circles) and CNNM2 (squares) transfected cells. (B) Representation of the normalized Mg2+ uptake after 5 minutes. (C) 25Mg2+ uptake in the presence of inhibitors of ion transporters, black bars represent mock cells and white bars represent CNNM2-transfected cells. (D) Dose-response curve of 25Mg2+ transport inhibition by 2-APB in mock (circles) and CNNM2 (squares) transfected cells. (E) The effect of Na+ and Cl− availability on 25Mg2+ uptake in mock (black bars) and CNNM2 (white bars) transfected cells. (F) 25Mg2+ uptake as a function of extracellular 25Mg2+ availability in mock (circles) and CNNM2 (squares) transfected cells. (G) 25Mg2+ extrusion in mock (circles) and CNNM2 (squares) transfected cells. Each data point represent the mean of 3 independent experiments ± SEM. * indicates significant differences compared to mock (P<0.05).
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pgen-1004267-g002: CNNM2 increases Mg2+ uptake in HEK293 cells.(A) Time curve of 25Mg2+ uptake in mock (circles) and CNNM2 (squares) transfected cells. (B) Representation of the normalized Mg2+ uptake after 5 minutes. (C) 25Mg2+ uptake in the presence of inhibitors of ion transporters, black bars represent mock cells and white bars represent CNNM2-transfected cells. (D) Dose-response curve of 25Mg2+ transport inhibition by 2-APB in mock (circles) and CNNM2 (squares) transfected cells. (E) The effect of Na+ and Cl− availability on 25Mg2+ uptake in mock (black bars) and CNNM2 (white bars) transfected cells. (F) 25Mg2+ uptake as a function of extracellular 25Mg2+ availability in mock (circles) and CNNM2 (squares) transfected cells. (G) 25Mg2+ extrusion in mock (circles) and CNNM2 (squares) transfected cells. Each data point represent the mean of 3 independent experiments ± SEM. * indicates significant differences compared to mock (P<0.05).
Mentions: To clarify the function of CNNM2, Human Embryonic Kidney (HEK293) cells were transiently transfected with mouse Cnnm2 or mock constructs and examined for Mg2+ transport capacity using the stable 25Mg2+ isotope. At baseline, approximately 10% of the total intracellular Mg2+ content consists of 25Mg2+, which is the natural abundance of 25Mg2+[21]. By incubating the cells in a physiological buffer containing pure 25Mg2+, the intracellular 25Mg2+ concentration increases over time. Interestingly, Cnnm2 expressing cells displayed a higher 25Mg2+ uptake compared to mock cells (Figure 2A). After 5 minutes, Cnnm2-expressing cells had approximately 2 times more 25Mg2+ uptake than mock-transfected cells (Figure 2B). All further experiments were performed at the 5 minutes time point to cover the exponential phase of the uptake. To reduce the background in 25Mg2+ uptake, inhibitors of known Mg2+ channels and transporters were added during the uptake process; 2-APB to inhibit TRPM7 [22], Ouabain to block the Na+-K+-ATPase [23], Quinidin for SLC41A1 [24] and Nitrendipin [25] for silencing MagT1 activity (Figure 2C). Only 2-APB was capable of significantly inhibiting 25Mg2+ uptake in HEK293 cells. Moreover, 2-APB inhibition also abolished the CNNM2-dependent increase in 25Mg2+ uptake. Dose-response experiments confirmed that the IC50 of 2-APB inhibition is 22 µM (Figure 2D). CNNM2-dependent 25Mg2+ uptake was found to be independent of Na+ and Cl− availability, when uptakes were performed in N-methyl-d-glucamine (NMDG) or Gluconate buffers (Figure 2E). Interestingly, the highest CNNM2-dependent 25Mg2+ uptake was measured between 1–2 mM, suggesting a Km in the physiological range of approximately 0.5 mM (Figure 2F). At high Mg2+ concentrations (5 mM), 25Mg2+ uptake was inhibited. When subjected to 24 hours 25Mg2+ loading, Cnnm2-expressing cells showed a significantly higher 25Mg2+ content baseline. Subsequently, 15 minutes extrusion of Cnnm2-expressing cells demonstrated no difference in Mg2+ extrusion rate, compared to mock-transfected cells (Figure 2G).

Bottom Line: Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg(2+) absorption.We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg(2+) homeostasis.By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for monitoring their effects on the brain and kidney phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.

ABSTRACT
Intellectual disability and seizures are frequently associated with hypomagnesemia and have an important genetic component. However, to find the genetic origin of intellectual disability and seizures often remains challenging because of considerable genetic heterogeneity and clinical variability. In this study, we have identified new mutations in CNNM2 in five families suffering from mental retardation, seizures, and hypomagnesemia. For the first time, a recessive mode of inheritance of CNNM2 mutations was observed. Importantly, patients with recessive CNNM2 mutations suffer from brain malformations and severe intellectual disability. Additionally, three patients with moderate mental disability were shown to carry de novo heterozygous missense mutations in the CNNM2 gene. To elucidate the physiological role of CNNM2 and explain the pathomechanisms of disease, we studied CNNM2 function combining in vitro activity assays and the zebrafish knockdown model system. Using stable Mg(2+) isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg(2+)-permeable cation channel TRPM7. In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg(2+) uptake. Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg(2+) absorption. These phenotypes were rescued by injection of mammalian wild-type Cnnm2 cRNA, whereas mammalian mutant Cnnm2 cRNA did not improve the zebrafish knockdown phenotypes. We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg(2+) homeostasis. By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for monitoring their effects on the brain and kidney phenotype.

Show MeSH
Related in: MedlinePlus