Cyclin M2 (CNNM2) knockout mice show mild hypomagnesaemia and developmental defects

Patients with mutations in Cyclin M2 (CNNM2) suffer from hypomagnesaemia, seizures, and intellectual disability. Although the molecular function of CNNM2 is under debate, the protein is considered essential for renal Mg2+ reabsorption. Here, we used a Cnnm2 knock out mouse model, generated by CRISPR/Cas9 technology, to assess the role of CNNM2 in Mg2+ homeostasis. Breeding Cnnm2+/− mice resulted in a Mendelian distribution at embryonic day 18. Nevertheless, only four Cnnm2−/− pups were born alive. The Cnnm2−/− pups had a significantly lower serum Mg2+ concentration compared to wildtype littermates. Subsequently, adult Cnnm2+/− mice were fed with low, control, or high Mg2+ diets for two weeks. Adult Cnnm2+/− mice showed mild hypomagnesaemia compared to Cnnm2+/+ mice and increased serum Ca2+ levels, independent of dietary Mg2+ intake. Faecal analysis displayed increased Mg2+ and Ca2+ excretion in the Cnnm2+/− mice. Transcriptional profiling of Trpm6, Trpm7, and Slc41a1 in kidneys and colon did not reveal effects based on genotype. Microcomputed tomography analysis of the femurs demonstrated equal bone morphology and density. In conclusion, CNNM2 is vital for embryonic development and Mg2+ homeostasis. Our data suggest a previously undescribed role of CNNM2 in the intestine, which may contribute to the Mg2+ deficiency in mice and patients.

www.nature.com/scientificreports/ Dounce homogeniser. Samples were centrifuged 10 min 1000 g at 4 °C to remove nuclei. Membranes were pelleted by centrifugation at 100,000 g at 4 °C for 30 min and resuspended in lysis buffer (50 mmol/L Tris-HCl pH 6.8, 5 mmol/L EDTA, 2% (w/v) SDS, with protease inhibitors). For SDS-PAGE, 25 µg protein was loaded, followed by transfer to a polyvinylidene fluoride membrane. Membranes were blocked with 5% (w/v) non-fat dry milk in phosphate buffered solution (PBS) for 1 h at room temperature (RT) and incubated overnight with blocking buffer supplemented with primary antibody at 4 °C. The next day, membranes were washed with PBS and incubated with appropriate peroxidase-conjugated secondary antibodies (Roche, Mannheim, Germany) and visualized by enhanced chemiluminescence.
Histology. Paraffin-embedded brains from adult mice and E17.5 embryos were processed into 5 µm-thick sections and mounted on slides for Nissl staining. Briefly, slides were deparaffinized in xylene for 10 min followed by 5 min incubation in the graded ethanol series (100, 95, 75, 50%). Slides were incubated in 0,1% (w/v) cresyl violet solution prepared in distilled water for 8 mi,n followed by a 2 min differentiation step in 95% ethanol and dehydrated in 100% ethanol for 2 min. Slides were then cleared in xylene and mounted with permanent mounting medium.

RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR
(v/v) formalin for 24 h and subsequently kept in 70% (v/v) ethanol. Femurs were scanned using the Skyscan 1076 in vivo X-ray computed tomography (Bruker microCT, Kontich, Belgium) with a voxel size of 8.88 μm and 2300 ms exposure time in line with the guidelines for the assessment of bone microstructure 23 . The following settings were used: X-ray power of 40 kV and tube current of 250 mA. Beam hardening (20%) was reduced using a 1 mm aluminum filter, ring-artefacts was set at 5 and an average of three photos (frame averaging) at each angle (0.8°) were taken to generate the final images. For the analysis of trabecular bone parameters, the distal metaphysis was scanned (a scan area of 1.35 mm from the distal growth plate towards femoral center). Analysis of the cortical bone parameters was performed in the diaphyseal cortex, which comprised a scan area of 0.45 mm in the femoral center. 3D reconstruction and data analysis were performed with using manufacturer-provided software (NRecon, Data viewer, CT analyzer; Bruker microCT).
Statistics. Data is represented as mean ± SEM. Comparisons were made by a One-or-Two-way ANOVA, followed by a Tukey's post-hoc test. An alpha of P < 0.05 was considered statistically significant. Statistical analysis is described in detail in the figure legends.

Results
Cnnm2 −/− mice are embryonically lethal and display exencephaly. Cnnm2 −/− mice were developed using CRISPR/Cas9 technology. In a first approach using Cas9 and a single guideRNA (sgRNA), deletions were introduced in exon 1 of the Cnnm2 locus. Two independent lines were established with either a deletion of 55 bp resulting in a frameshift at position 28 (p.A28D fsX9) or a deletion of 37 bp resulting in a frameshift at position 40 (p.V40H fsX3) (Fig. 1A). In a second approach using Cas9, two sgRNAs and a donor plasmid, LoxP sites were introduced upstream and downstream of exon 1, which was removed by cre mediated excision after crossing the mice with the floxed allele to a cre deleter strain (Fig. 1B). At E18, embryos were present at Mendelian ratio (Cnnm2 +/+ : 27%, Cnnm2 +/− : 49% Cnnm2 −/− : 24%) (Fig. 1C). However, the Cnnm2 −/− offspring all died within the first day after birth in the three investigated lines. Consequently, Cnnm2 −/− animals were analysed at embryonic day 18 (E18). 36% of Cnnm2 −/− embryos displayed exencephaly, which may contribute to the mortality of these mice (Fig. 1D,E). The Cnnm2 −/− embryos (E17.5) that did not show exencephaly also did not show obvious morphological neurological differences, as demonstrated with a Nissl staining (Fig. 1F). Both lines were used to generate mice homozygous for the deleted Cnnm2 allele and resulted in early postnatal lethality and a similar penetrance of exencephaly. Absence of CNNM2 protein was confirmed by Western blot analysis of embryonic brains and specificity of the antibody was confirmed using CNNM2 overexpressing Madin-Darby canine kidney (MDCK) cells (Fig. 1G)  www.nature.com/scientificreports/ addition, immunohistochemistry verified that CNNM2 expression in the DCT, defined by Na + /Cl − co-transporter (NCC) positive tubules, was absent in kidney sections of Cnnm2 −/− pups (Fig. 1H).
As Cnnm2 −/− mice died shortly after birth, further studies to determine the role of CNNM2 in Mg 2+ homeostasis were performed in the adult Cnnm2 +/− mice. Adult Cnnm2 +/− had comparable brain morphology with wild type mice ( Supplementary Fig. 2).
Cnnm2 deficiency leads to mild urinary Mg 2+ wasting. To pinpoint the cause of the low serum Mg2 + levels in the Cnnm2 +/− mice, the 24 h urinary Mg 2+ excretion and kidney mRNA expression of genes involved in Mg 2+ reabsorption were assessed. Morphology of the kidneys was similar in both genotypes (Fig. 3A). Cnnm2 +/− deficient mice did not show a significant decrease in urinary Mg 2+ excretion (38 ± 3.2 versus 37 ± 3.0 mmol/L/24hrs, respectively) in the urine compared to wild type mice across the different diets (Fig. 3B,C), despite having decreased serum Mg 2+ concentration, suggestive for a small renal reabsorption defect. Interestingly, Cnnm2 +/− displayed Ca 2+ retention at the baseline urine measurement (1.0 ± 0.1 vs 1.7 ± 0.1 μmol/24hrs, respectively, P < 0.0001) (Fig. 3D), although this effect was not observed at the final day of the diets (Fig. 3E). Gene expression analysis showed significantly decreased renal expression of Cnnm2 in Cnnm2 +/− mice on the control and high Mg 2+ diet (Fig. 3F). Expression of the family member Cnnm4 was decreased 3.6-fold in Cnnm2 +/− mice fed on the normal Mg 2+ -diet (Fig. 3G). Levels of magnesiotropic genes Trpm6, Trpm7, Slc41a1 were measured in the kidney, but did not show any genotype-specific effect (Fig. 3H-J). The expression of the epithelial Ca 2+ channel Trpv5 showed an overall significant increase due the diets, but no differences were observed between the Cnnm2 +/− compared to wild type mice (Fig. 3K).
Cnnm2 +/− mice have perturbed intestinal Mg 2+ absorption. Before the start of the diets Cnnm2 +/− mice displayed a 17.5% higher faecal Mg 2+ content compared to Cnnm2 +/+ mice (Fig. 4A,B). This was similar to Ca 2+ levels, which were also significantly increased (Fig. 4C,D). Furthermore, we investigated the mRNA expression of key magnesiotropic genes present in the distal part of the colon, where transcellular Mg 2+ absorption takes place. Similar to the observation in the kidney, Cnnm2 transcript levels were significantly reduced in the Cnnm2 +/− mice compared to control mice. Yet there was an overall increase of Trpm6 expression and Cnnm4 expression based on diet or genotype, respectively (Two-Way ANOVA). No diet or genotype dependent changes were observed for other magnesiotropic genes ( Fig. 4E-I). The expression of the epithelial Ca 2+ channel Trpv6 was not significantly different among all mice groups (Fig. 4J).
Cnnm2 +/− mice display normal bone morphology. The femurs of three male mice were subjected to micro-computed tomography analysis. Both the cortical and trabecular bone microarchitecture were not significantly affected by genotype nor diet, except in Cnnm2 +/− mice on a low Mg 2+ diet (Fig. 5A-E).

Discussion
In this study, we used several genetic mouse models to investigate the role of CNNM2 in mice. Cnnm2 −/− mice exhibited maldevelopment of the brain, prominently consisting of exencephaly. The Cnnm2 −/− newborns displayed hypomagnesaemia concomitant with increased serum Ca 2+ levels. Cnnm2 +/− adult mice showed a normal development, but similarly suffered from a low serum Mg 2+ concentration, which could be partially explained by faecal and renal Mg 2+ loss. Our study emphasises the role of CNNM2 in embryonic development and overall Mg 2+ handling, and disclosed a novel role in intestinal Mg 2+ absorption.
Impaired renal Mg 2+ reabsorption and urinary Mg 2+ wasting have been considered as the primary defect of HSMR patients as renal Mg 2+ excretion remains similar despite lowered serum Mg 2+ levels 13,14 . In line with the observation in patients, Cnnm2 +/− mice displayed low serum Mg 2+ levels when compared to Cnnm2 +/+ mice. Yet, no significant increase in urinary Mg 2+ excretion was observed. The Cnnm2 +/− mice displayed lowered urinary Mg 2+ excretion upon exposure to lowered Mg 2+ diet, similarly to wild type mice. This demonstrates the compensatory capacity of Mg 2+ reabsorption in these mice. Yet, Cnnm2 +/− mice showed decreased serum Mg 2+ levels. The fact that this low serum value is not associated with a diminished urinary Mg 2+ excretion can be interpreted as a sign of a renal Mg 2+ leak. This has previously been reported in other mouse models of hypomagnesemia, e.g. Slc41a3 −/− mice and high-fat diet-fed mice 24,25 . The role of CNNM2 in the kidney has been substantiated by studies of kidney-specific knockout mice of Cnnm2 which suffer from hypomagnesemia, demonstrating that CNNM2 regulates renal Mg 2+ handling 19 .  Ca 2+ in newborn Cnnm2 −/− mice is significantly increased compared to Cnnm2 +/+ mice (n = 3-17) (C) Cnnm2 +/+ or Cnnm2 +/− mice were treated with low (0.02%), normal (0.23%), or high (0.48%) (w/w) Mg 2+ diets for two weeks, followed by housing in metabolic cages. (D,F) Cnnm2 +/− mice (black bars) have lower serum Mg 2+ levels and higher compared to wild type mice (white bars), independent of the Mg 2+ diet. (E,G) Cnnm2 +/− mice have lower serum Mg 2+ levels and an overall increase in serum Ca 2+ levels compared to wild type littermates, independent of the Mg 2+ diet. Serum Na + (H) and K + (I) levels in Cnnm2 +/− mice are unchanged (n = 10-12 per group). Data are presented as mean ± SEM. Significance determined with two-tailed Student's T-test or Two-way ANOVA followed by a Tukey posthoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. www.nature.com/scientificreports/ Interestingly, intestinal malabsorption may also play a role in the Mg 2+ deficiency observed in the Cnnm2 +/− mice. At basal conditions, faecal Mg 2+ excretion was increased in Cnnm2 +/− mice, indicative of Mg 2+ malabsorption. Although CNNM2 has been primarily studied in the kidney, our current and previous studies demonstrate that Cnnm2 is expressed in the distal colon 8 . Indeed, several recent single cell RNA sequencing datasets in colon and rectum confirm that CNNM2 is particularly expressed in enterocytes 26,27 . Of interest, CNNM4, a close family member of CNNM2, is present in the basolateral membrane of the colon, where it orchestrates Mg 2+ efflux [28][29][30] . We have previously shown an interaction of CNNM2 with CNNM4 upon expression in HEK293 cells, suggesting that these proteins could form a complex 8 . Indeed, a significant increase of Cnnm4 expression in the colon was observed in Cnnm2 +/− mice, suggesting that CNNM4 compensates for the loss of CNNM2. However, a CNNM4-independent function of CNNM2 cannot be excluded. Although malabsorption of Mg 2+ was not previously reported in Cnnm2 +/− mice or patients 19 , it should be noted that faecal Mg 2+ excretion is often not detected or even determined in mice, although also in intestinal-specific Trmp6 −/− and Trpm7 −/− mice decreased serum Mg 2+ levels and increased faecal Mg 2+ excretion can be observed 6,31 . On average, 30% of dietary Mg 2+ is absorbed and foremost ends in the faeces, making it challenging to detect changes in absorption. However, Mg 2+ supplementation was proven difficult in patients with CNNM2 mutations, which potentially indicates a reduced ability to absorb Mg 2+13,14 . Our findings suggest that oral Mg 2+ supplementation in patient with HSMR syndrome depends on paracellular Mg 2+ transport in the duodenum, as transcellular Mg 2+ absorption in the colon may be reduced by CNNM2 mutations.

Scientific Reports
In addition to the Mg 2+ disturbances, Cnnm2 +/− mice displayed renal Ca 2+ retention resulting in an increased serum Ca 2+ concentration. Decreased serum Mg 2+ levels could trigger the TAL to increase its paracellular Mg 2+ transport. In addition, this would lead to increased Ca 2+ reabsorption and decreased urinary Ca 2+ levels, which is coordinated via Claudins 10, 14, 16 and 19 [32][33][34][35][36] . This compensatory capacity may be specific for mice, as patients with mutated CNNM2 have significant urinary Mg 2+ wasting. In line with this, 24 h urinary Ca 2+ levels are normal in patients with CNNM2 mutations. Interestingly, a few patients with CNNM2 mutations have been reported with impaired Ca 2+ and phosphate homeostasis. Whether this is a common phenotype in these patients remains to be determined, as the patients are consanguineous or have other genetic defects, such as mutations in the vitamin D receptor (VDR) 12,37 . However, the role of TAL in CNNM2-associated physiology should be interpreted with caution, as direct effects of CNNM2 on Ca 2+ homeostasis cannot be excluded.
Our study demonstrates that CNNM2 is essential for brain development and early life survival. Until embryonic day 18.5, the Cnnm2 −/− embryos were present at Mendelian ratios, but newborn Cnnm2 −/− mice died shortly after birth. Although lethality of Cnnm2 −/− mice was reported earlier 19 , we are the first to report the presence of exencephaly, which suggests that neural tube defects contribute to the brain phenotype. It is known that defects in the closure of the cranial neural tube result in protrusion of the neuroepithelium outside the developing brain, inevitably disturbing normal neurodevelopment 38 . Although previous studies suggested an important role of CNNM2 in brain function, the exact consequences of CNNM2 deficiency may be species-dependent. Patients with recessive CNNM2 mutations exhibit structural brain deformities, such as demyelination, failure of opercularisation, and cerebral cortical atrophy, often concomitant with motor skill defects, epileptic seizures, and intellectual disability 12,13 . Moreover, knockdown of cnnm2 in zebrafish resulted in maldevelopment of the mid-hindbrain boundary 13 .
Interestingly, exencephaly and other neural tube defects have been more often observed in mouse models of hypomagnesemia. Trmp6 knockout mice display embryonic lethality, which is accompanied by the presence of exencephaly and spina bifida 5,39 . Similarly, the absence of Trpm7 expression in mice during embryonic development, a close homologue of TRPM6, has been associated with neural tube defects 40,41 . Although this homology suggests that neural tube defects may be the direct result of hypomagnesaemia, it should be noted that neural tube defects are uncommon in patients with hypomagnesaemia 1 . Consequently, it remains to be determined whether this is a Mg 2+ dependent or independent effect. Of note: as only a subset of Cnnm2 −/− mice displayed exencephaly, neural tube defects may only partially explain the lethality.
The absence of a clear urinary Mg 2+ wasting in Cnnm2 +/− mice shows that renal Mg 2+ reabsorption may be different in mice and men, as HSMR syndrome show clear renal Mg 2+ wasting 13 . In line with this, kidneyspecific Trpm6 knock out mice were reported to have normal serum Mg 2+ levels and without renal Mg 2+ leak, unlike patients with Trpm6 mutations (HOMG1/HSH; MIM# 602014) 31 . Similarly, Kcnj10 and Fxyd2 knock out Table 1. Metabolic parameters of Cnnm2 +/− mice upon exposure to different Mg 2+ diets. Cnnm2 +/+ and Cnnm2 +/− mice were kept in metabolic cages exposed at low (0.02%), normal (0.21%) and high (0.48%) (w/w) Mg 2+ diets for 14 days. The last 48 h, mice were kept in metabolic cages for the collection of urine and faeces. Values represent the latter 24 h (mean ± SEM). M male, F female.