MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

Inositol polyphosphates are vital metabolic and secondary messengers, involved in diverse cellular functions. Therefore, tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, we describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in the multiple inositol polyphosphate phosphatase 1 gene (MINPP1). Patients were found to have a distinct type of Pontocerebellar Hypoplasia with typical basal ganglia involvement on neuroimaging. We found that patient-derived and genome edited MINPP1-/- induced pluripotent stem cells (iPSCs) are not able to differentiate efficiently into neurons. MINPP1 deficiency results in an intracellular imbalance of the inositol polyphosphate metabolism. This metabolic defect is characterized by an accumulation of highly phosphorylated inositols, mostly inositol hexakiphosphate (IP6), detected in HEK293, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP6 level is expected to be associated with an increased chelation of intracellular cations, such as iron or calcium, resulting in decreased levels of available ions. These data suggest the involvement of IP6-mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis.


INTRODUCTION
Inositol polyphosphate (IPs) comprise an ubiquitous family of small molecule messengers controlling every aspect of cell physiology 1. The most characterized is the calcium release factor inositol trisphosphate (I(1,4,5)P3 or simply IP3), a classical example of second messenger 2, generated after receptor activation by the action of phospholipase C on the lipid phosphoinositide PIP2. Each of the six hydroxyl groups of the inositol ring can be phosphorylated, and the combination of these phosphorylations generates multiple derivatives 3. Among them, inositol hexakisphosphate (IP6, or phytic acid) is the most abundant in nature. In plants, IP6 accumulates in seeds, within storage vacuoles, where it could represent 1-2 % of their dry weight 4. Plant seed IP6 is used as the main source of phosphate and mineral nutrients (e.g. Ca2+, K+, Fe2+) during germination. In mammalian cells, IP6 is the most abundant inositol polyphosphate species, reaching cellular concentrations of ~15-100 M 5. IP6 is synthesized from inositol monophosphate (IP) or from IP3 by the action of several inositol phosphate kinases: IPMK (Inositol Polyphosphate Multikinase, also known as IPK2), IP3-3K (Inositol -1,4,5-trisphophate 3-Kinase), ITPK1 (Inositol Tetrakisphosphate 1-Kinase) and IPPK (Inositol-Pentakisphosphate 2-Kinase, also known as IPK1)1 6. Subsequently, the fully phosphorylated ring of IP6 can be further phosphorylated to generate the more polar inositol pyrophosphates such as IP7 7. While IP6 anabolism is well studied, its catabolism has been less characterized. Mammalian cells dephosphorylate IP6 through the action of the MINPP1 (Multiple Inositol-Polyphosphate Phosphatase 1) enzyme 8 that is able to degrade IP6 to IP3 9. The analysis of mouse knockouts for the inositol kinases responsible for IP6 synthesis have highlighted an important role for this pathway in controlling central nervous system development, since knockout of Itpk1 or Ipmk is embryonically lethal due to improper neural tube development 10 11. In mammals, IP6 has been directly associated with a pleiotropy of functions, including ion channel regulation, control of mRNA export, DNA repair, and membrane dynamics 1. Furthermore, IP6 is considered as a natural antioxidant since its iron-chelating property enables it to inhibit iron-catalyzed radical formation12.
Although not yet thoroughly studied, some of the physiological roles of IP6 could be related to its high affinity for polyvalent cations 13 14. To investigate the role of IP6 in mammalian physiology, many studies use IP6 exogenously added to cell lines in culture, often observing antiproliferative properties 15. These studies give little attention to the chelating property of IP6: cations-IP6 precipitation depletes the medium of essential ions such as calcium or iron. Additionally, the physiological relevance of extracellular IP6 in mammals is not established. Extracellular pools of IP6 have only been demonstrated in a cestode intestinal parasite 16, and several studies suggest that dietary IP6 cannot be absorbed as such through the digestive system and is absent from body fluids 17 18. Instead, de novo synthesis of IP6 occurs in all mammalian cells, including in the brain with high levels in regions such as the brainstem and striatum 17 19. The existence of several cellular pools of IP6 has been suggested 19 20 6. However, the dynamic regulation of the endogenous intracellular pools of IP6 is not fully understood, since its high cellular concentration precludes the determination of IP6 pool specific fluctuations. Therefore, the exact function(s) of IP6 in cell homeostasis and mammalian development remain an area of intense investigation.
Several human diseases have been genetically associated with alterations in phosphoinositide (the lipid derivatives of inositol) metabolism 21. However, so far, no Mendelian disorder has been shown to be caused by an imbalance in the cytosolic inositol polyphosphate pathway, with the exception of a single variant in a gene involved in the conversion of the pyrophosphates forms of inositol and associated with hearing impairment 22. Pontocerebellar hypoplasia (PCH) is a group of early-onset neurodegenerative disorders that includes at least 13 subtypes, based on neuropathological, clinical and MRI criteria 23 24. PCH is usually associated with a combination of degeneration and lack of development of the pons and the cerebellum, suggesting a prenatal onset. The genetic basis is not known for all of the cases, and preliminary data from different PCH cohorts suggest that many subtypes remain to be identified.
Based on the known molecular causes, PCH often results from a defect in apparently ubiquitous cellular processes such as RNA metabolism regulation and especially tRNA synthesis (i.e. mutations in EXOSC3, TSEN54, TSEN2, TSEN34, CLP1 and RARS2). Multiple additional conditions show neurological symptoms and imaging comparable to typical PCH syndromes and are caused by defects in diverse pathways involved in mitochondrial, glycosylation or purine nucleotide metabolisms. This observation further supports the disruption of ubiquitous pathways as the unexplained basis of these neurological conditions23.
In this study, we identify MINPP1 as necessary for the dephosphorylation of intracellular IP6, and describe a new syndrome of PCH caused by a defect in this process that directly regulates cytosolic cation (e.g. Ca2+, Fe3+) homeostasis.

Loss of function mutations of the MINPP1 gene are associated with a distinct subtype of Pontocerebellar Hypoplasia
To identify new etiological diagnoses of patients with PCH, we explored a group of 15 probands previously screened negative with a custom gene panel approach 25. Whole exome sequencing (WES) was then performed through trio sequencing (i.e. both parents and the proband). Among the new candidate genes that were identified, the MINPP1 gene was recurrent and the most obvious candidate (Table 1 and Supplementary Note). The MINPP1 gene has not been previously associated with any Mendelian disorders. To assess how frequently MINPP1 mutations could be involved in PCH, we explored two other cohorts of pediatric cases with neurological disorders. The presence of MINPP1 mutations was investigated using a custom gene panel or WES. Three additional families with MINPP1 bi-allelic variants were identified, all the affected being diagnosed with PCH.
In total, bi-allelic variants in MINPP1 were identified in eight affected children from six unrelated families ( Fig.1, Table 1, Supplementary Fig.1). These variants include homozygous early-truncating mutations in the families CerID-30 and PCH-2712, compound heterozygous missense and frameshift variants in family CerID-11, a homozygous missense variant in the endoplasmic reticulum (ER) retention domain of the protein in the family CerID-09 and homozygous missense variants in the histidine phosphatase domain of the protein in the families TR-PCH-01 and PCH-2456 (Fig.1B, D).
These four missense variants are predicted to be disease-causing using MutationTaster and SIFT 26, and involve amino acids fully conserved across evolution (Table 1, Fig.1C). To predict the impact of the variants on protein structure, we utilized a crystal structure of D. castellii phytase and evaluated the consequences of the missense variants involving amino-acids included in the model ( Supplementary   Fig.1B). Tyr53Asp variant introduces a buried charge and disrupts a hydrogen bond with the donor amino-acid Ser299. The Phe228Leu substitution breaks a buried hydrogen bond with Lys241, both amino-acid positions are close to the IP6 binding site. The Arg401Gln substitution replaces a buried charged residue with an uncharged residue and disrupts a salt bridge formed with the amino-acid Asp318. Thus, all the missense variants tested are predicted to cause structural damages with potential consequences on the enzyme activity.
The eight patients presented with almost complete absence of motor and cognitive development, progressive or congenital microcephaly, spastic tetraplegia or dystonia, and vision impairments ( Table   2). For most of the patients, the first symptoms included neonatal severe axial hypotonia and epilepsy that started during the first months or years of life. Pre-natal symptoms of microcephaly associated with increased thalami echogenicity were detected for the individual CerID-11, while the seven other patients presented with progressive microcephaly. For patients from the families CerID-09 and 11, the phenotype appeared to be progressive and the affected children died in their infancy or middlechildhood. Strikingly, all the affected children harbor a unique brain MRI showing a mild to severe PCH, fluid-filled posterior fossa, with dilated lateral ventricles. Additionally, severe atrophy at the level of the basal ganglia or thalami often associated with typical T2 hypersignal were identified in all the patients MRI (    The MINPP1 enzyme is predominantly localized in the ER lumen27. It removes phosphate groups from inositol polyphosphate substrates starting at position 3 28,29, with high affinity for IP5 and IP6 30. Indeed, it has been described as the main mammalian phytase, or enzyme involved in IP6 degradation ( Fig. 2A). Despite its name, this gene does not have any paralog in the human or mouse genome. In order to determine the effect of the patient mutations on the endogenous enzyme, we obtained skin fibroblast from patients of the CerID-30 family. MINPP1 protein was undetectable in patients' cells  Supplementary Fig.2D). Contrastingly, overexpression of two of the MINPP1 missense variants (i.e. Y53D and E486K) did not rescue growth, suggesting that these variants have a major impact on the protein function. MINPP1 has two predicted N-glycosylation sites (Fig.1D). In order to evaluate the glycosylation status of the endogenous and over-expressed WT MINPP1 as well as the Y53D and E486K missense variants, we treated the protein extracts with the PNGase enzyme 31

Patient and genome-edited MINPP1 mutant iPSCs show an impaired neuronal differentiation
To investigate the mechanism at the origin of the neurological symptoms of MINPP1 patients, we derived induced pluripotent stem cells (iPSCs) from patient CerID-30-2 ( Supplementary Fig.3A, B).
In order to assess a contribution of the genetic background or other factors to the phenotype, we also generated MINPP1-/-iPSCs in isogenic background ( Supplementary Fig. 3A, B). Surprisingly, a dual SMAD inhibition-based neural induction protocol32 33, did not allow the generation of viable neural progenitor cells for both MINPP1 mutant lines (data not shown). Differentiation of patient-derived iPSCs systematically generated mixed cell populations with undefined HNK1 negative cells ( Supplementary Fig. 3C). These observations suggest a critical role for MINPP1 during neuroectodermal induction, and led us to use a different protocol that preserved neural rosette environment, using only noggin as a SMAD inhibitor 34. In these conditions, control cells efficiently differentiated toward TUJ1+ neurons after 14 days (Fig. 3A). In contrast, both MINPP1 mutant lines showed significant 42% and 65% decreases in TUJ1+ post-mitotic cells mirrored by significant 2.6 and 3.1-fold increases in the number of PAX6+ neural progenitors in MINPP1-/-and CerID-30-2 derived cells respectively (Fig. 3A, B). These changes reflect the inability of neural progenitors to efficiently differentiate into post-mitotic neurons. Altogether, these observations show that MINPP1 plays a direct role during human neuronal differentiation, and suggest that a differentiation defect could be involved in the neuronal vulnerability underlying this early-onset neurodegenerative disorder.

Inositol polyphosphate metabolism is altered in HEK293, iPSCs and induced neurons mutated for MINPP1
The localization of human MINPP1 into the ER, and the demonstration that its drosophila homolog (i.e. mipp1) is anchored to the plasma membrane outside of the cell35, prompted us to investigate the presence of phytase activity in conditioned media from control and MINPP1 mutant HEK293 cells. In conditioned medium from control cells, exogenously added IP6 was substantially processed after two hours, and completely degraded after four hours ( Supplementary Fig.4A). Conversely, although partially degraded, IP6 is still detectable after six hours of incubation in MINPP1 mutant conditioned media. This result suggests that MINPP1 accounts for the main secreted phytase activity of HEK293 cells.
To explore precisely a disruption in inositol phosphate metabolism, and to better address the role of MINPP1 in this metabolic pathway, we used tritium inositol (myo-[3H]-inositol) metabolic labeling of cultured cells, and analyzed inositol derivatives with SAX-HPLC (strong anion-exchange highperformance liquid chromatography) as previously described 36 37. We applied this method to HEK293, skin fibroblasts, and iPSCs before or during neuronal differentiation at day 10 (referred to as Day 10 differentiating neurons) from control and MINPP1 mutant cell lines. Exogenously added [3H]inositol is imported into the cytosol and converted into phosphoinositide lipids before processing into inositol phosphates (IPs) ( Fig. 2A). As expected, after 3 days of [3H]-inositol labeling, IP6 was detected as the most, or the second most abundant intracellular inositol derivative in control cell lines (hollow and filled bars in Fig.4), but was absent in the cell culture media (Fig.4, Supplementary   Fig.4B). In all the cell models studied, the disruption of MINPP1 enzyme activity had a strong impact on intracellular IPs profile when compared with their respective controls. The investigation of MINPP1-/-HEK293 cells revealed a 3-fold significant increase in IP6 level, as well as an increase in IP5 levels, and surprisingly a severe decrease in IP and IP2 levels (Fig.4A). Trends in the same direction, although not significant, were detected in patient fibroblasts (Fig.4B) suggesting cell-type specific differences. Indeed, in iPSCs, IP6 levels showed a significant 1.6-fold increase in both patientderived and MINPP1 KO iPSCs, also associated with an increase in IP5 levels ( Fig.4C, D). Finally, the study of Day 10 differentiating neurons revealed significant 1.9-fold and 1.6-fold increases in IP6 levels in patient-derived and MINPP1 KO differentiating cells respectively, with a trend or significant decrease in IP2 levels (Fig.4E, F) In all the cell models tested, including HEK293 cells, patients' fibroblasts, and undifferentiated and differentiated iPSCs, a comparable imbalance of IPs levels were observed, where increase in the amounts of higher inositol polyphosphate derivatives IP5 and IP6 were associated with a decrease in lower-phosphorylated IP2 and IP species (Fig. 4). Differences observed between the various cell models tested are likely to be caused by cell type differences and potentially also by the genetic background. Nevertheless, these observations clearly demonstrate the critical role played by MINPP1 in cellular inositol polyphosphate homeostasis, with the conversion of higher to lower IPs. The most robust finding was that IP6 is systematically increased in MINPP1 mutant cells compared to controls.
Altogether, these observations exclude a major contribution of extracellular higher-phosphorylated IPs to this metabolic defect, but highlight an unappreciated role for MINPP1 in the regulation of the intracellular pool of de novo synthesized IP6, the most abundant inositol derivative.

IP6 accumulation can deplete free iron in presence of high iron condition
Considering the strong impact of MINPP1 mutations on cellular IP6 levels, and the known chelator properties of this molecule, we hypothesized that MINPP1 defects can have consequences for intracellular cations homeostasis. An intracellular accumulation of IP6 could theoretically lead to the accumulation of chelated cations inside the cell, potentially reducing the pool of free cations. At physiological pH, IP6 has a strong binding affinity to iron14, therefore we evaluated the ability of HEK293 cells to store iron, in low iron (-FAC) or high iron (provided with ferric ammonium citrate; +FAC) conditions. Then, we used a colorimetric ferrozine-based assay with a HCl/KMnO4 pretreatment step that separates iron from its binding molecules to measure total intracellular iron 38 39. After two days of incubation with FAC, we observed a significant 1.5-fold increase in the total iron content in MINPP1-/-HEK293, under high iron conditions compared to control (Fig. 5A). Although based on non-physiological iron conditions, this observation suggests that IP6 could play a role in the regulation of metal ion cellular storage such as iron. To investigate a potential increase of iron chelation affecting the free iron cellular pool, we measured the cellular free Fe2+/3+ content using standard cell lysis and colorimetric assay. We detected a 58% depletion in total free iron levels in MINPP1 mutant cells under high iron conditions (Fig.5B). Interestingly, this depletion was mainly contributed by a decrease in Fe3+ levels (Fig.5C, D). IP6, the major IPs accumulating in mutant HEK293, is known to have higher affinity for Fe3+ versus Fe2+ 40,41. These data are consistent with a massive accumulation of complexed iron in the absence of the MINPP1 enzyme, in the presence of high iron, and suggest the potential involvement of IP6-mediated abnormal cation homeostasis as the underlying disease mechanism.

IP6 accumulation causes cytosolic calcium depletion in mutant HEK293 and primary mouse neural progenitors
To further explore the involvement of the disruption of cellular cations homeostasis in PCH, we evaluated the intracellular free Ca2+ levels in the absence of MINPP1, using the FLUO-4-AM calcium binding indicator. Strikingly, we found a significant, close to 50% depletion of free basal Ca2+ levels in MINPP1-/-HEK293, compared to control cell line (Fig. 5E). To further validate the involvement of such calcium depletion in the neurological phenotype, we generated a CRISPR/Cas9-mediated Minpp1-/-mouse model ( Supplementary Fig. 5A-E). Minpp1 KO mice were fertile and are born at Mendelian ratio (data not shown) as described in a previously generated Minpp1 KO mouse model30.
Brain histology did not identify major differences in cerebellar ( Supplementary Fig.5C) or cerebral cortex architecture (not shown). However, we identified a mild but significant ~10% decrease in the brain weight associated with a reduced cortical thickness in homozygous mutant mice at P21 ( Supplementary Fig.5D, E). This observation suggests the presence of an evolutionarily conserved requirement for MINPP1 activity in mammalian brain development. To identify a potential cause for this cortical phenotype, we isolated neural progenitors at E14.5 and measured the intracellular free Ca2+ levels. Surprisingly, we observed a significant 33.6% decrease in intracellular free Ca2+ levels in the Minpp1-/-mouse cells when compared to wild type neural progenitors (p=0.007; Fig. 5F).
To assess the effects of IP6 accumulation on calcium signaling, we studied the caffeine sensitive ER calcium release in the MINPP1-/-HEK293 cells42. In response to 10 mM caffeine, we observed a significant peak in the cytosolic Ca2+ levels within a minute in the control cells. However, we could only detect a slight increase with a sustained plateau in MINPP1-/-HEK293 cells, indicating an altered response potentially caused by a decrease of Ca2+ in intracellular ER stores (Fig.5G). To further study the Ca2+ mobilization in MINPP1-/-HEK293 cells, we treated the cells with ionomycin as it is known to initially increase the cytoplasmic calcium levels, which in turn activates calcium induced calcium response and eventually causes Ca2+ depletion in the ER43. In response to ionomycin, the control and MINPP1-/-HEK293 cells exhibited an initial Ca2+ peak with a sustained plateau. However, the relative response to ionomycin stimulation was again significantly decreased in MINPP1-/-cells (Fig.5H).
These calcium signaling defects were specifically rescued in a MINPP1-/-HEK293 line with stable expression of MINPP1 ( Fig.5G and H). Interestingly, we observed similar results in the absence of extracellular calcium (Supplementary Fig. 5F and G), for both caffeine and ionomycin, indicating that the defect is not due to the inhibition of calcium entry. Therefore, these results clearly suggest that MINPP1 absence affects the calcium levels in the cytosol as well as in the intracellular stores such as the ER. Altogether, these data support the critical role played by MINPP1 in the regulation of the intracellular IPs and available cations with strong implications for neural cell signaling and homeostasis.

DISCUSSION
The direct physiological role(s) played by IP6 in mammals has been difficult to define, due to the technical challenges associated with its measurement, and its complex anabolism. Furthermore, the well described role of IP6 (phytate) and phytase activity in plants and bacteria had led to the thinking that extracellular IP6 degradation also supplies mammalian cells with phosphate and cations. On the contrary, we demonstrate that intracellular, not extracellular, IP6 influences cation homeostasis. An imbalance of IPs derivatives has not been so far directly involved in disease, and the previously investigated Minpp1 KO mouse did not reveal any obvious phenotype 30. Surprisingly, we discovered that the absence of MINPP1 in humans results in a very severe early-onset neurodegenerative disorder with specific features. Patients with loss of function mutations in the MINPP1 gene present with PCH associated with typical basal ganglia or thalami involvement identified by MRI. The prenatal onset of the phenotype is obvious for patient CerID-11, which supports a critical and early role for MINPP1 in neuronal development and survival. In agreement with this, we observed that patient-derived and genome-edited iPSCs mutant for MINPP1 cannot be differentiated toward neural progenitors that efficiently give rise to neurons. Although the exact mechanism underlying this differentiation defect has not been identified yet, the sensitivity of human neural progenitors to the disruption of IPs levels is likely to be involved 44. While a key role for MINPP1 in the regulation of IP6 cellular levels has been investigated before30, we provide the first evidence for its critical importance on cellular physiology and human development.
Our analysis of IPs profiles using metabolic labelling unambiguously identified a typical imbalance resulting from MINPP1 defect. The increase in IP5 and IP6 levels is consistent with a previous mouse model study8, but our more complete assessment of the IPs metabolism imbalance also revealed alterations in lower phosphorylated IPs. Furthermore, the consequences of this metabolic block are associated with cell type-dependent differences in the IPs profile, such as the IP4 depletion in mutant iPSCs and robust IP6 accumulation in day-10 differentiating neurons mutated for MINPP1.
The discrepancy related to the supposed mostly cytosolic localization of IP6 and the ER localization of MINPP1 remains an unsolved problem 19, 26, 30. Hypothetically, the specific subcellular localization of MINPP1 prevents IP6 accumulation in a specific compartment (e.g. the ER) that would have primary consequences on local cation homeostasis. We identified that MINPP1-mediated IP6 regulation impacts free cations availability, as illustrated with the altered iron content of MINPP1-/-HEK293 cells as well as the severe depletion of cytosolic calcium identified in Minpp1-/-mouse primary neural progenitors and HEK293 cells. Interestingly, the absence of MINPP1 also severely disrupts signaling based on ER calcium that could potentially be the place of the primary defect. Calcium signaling has broad functions in neural cell physiology and brain development. Basal calcium levels influence neuronal physiology and cell survival 45-47, and calcium signaling plays a role in neural induction and differentiation 48-51. Consequently, a disruption in calcium homeostasis could be involved in PCH disease pathogenesis. A link between MINPP1 and calcium regulation has been suggested previously but it was through the synthesis of I(1,4,5)P3 52. Hypothetically, coupling the limitation of IP6mediated chelation of calcium with the promotion of IP3 synthesis could be an efficient way for MINPP1 to regulate calcium signaling dynamics and homeostasis.
A mild or absent structural brain defect was also observed in other PCH mouse models. AMPD2 null mutations cause PCH9 but the Ampd2 single KO mouse is not associated with any obvious histological brain defect 53. CLP1 is involved in tRNA processing and mutated in PCH10, however the Clp1 mutant mouse showed only a mild decrease in the brain weight and volume, a phenotype overlooked before the identification of patients with a brain phenotype 54. Differences in the phenotype of human and mouse with MINPP1 loss-of-function mutations could be related to an increased sensitivity of the human brain development to metabolic defects, although the impact of the genetic background cannot be excluded at this point.
Disrupted cation homeostasis, including metal accumulation, is central to multiple degenerative disorders55 such as neurodegeneration with brain iron accumulation56,57, Parkinson's disease (Manganese accumulation)58,59, Wilson's disease (Copper accumulation)60,61. Basal ganglia dysfunction is usually suspected in PCH23, however the severe defects identified in MINPP1 patient MRIs suggest major neurodegeneration at the level of these subcortical nuclei, a feature not typically associated with other PCH subtypes. These structures are well known to be primarily affected by metal ions accumulation and further investigation will be needed to determine how cation chelation could contribute to the disease pathogenesis. Nevertheless, our results reveal an unappreciated basic role for highly phosphorylated IPs in cellular homeostasis which is critical during neurodevelopment.

Patients recruitment and investigation
The  Fig.3A, B).

Generation of Minpp1-/-mice
Minpp1-/-mice were generated with the aid of LEAT platform of Imagine Institute by using a CRISPR/Cas9 system. In this study, animals were used in compliance with the French Animal Care and Use Committee from the Paris Descartes University (APAFIS#961-201506231137361). Guide RNAs (sgRNAs) targeting the first exon of the gene were designed via the CRISPOR (http://crispor.tefor.net/). C57Bl/-J female mice (4 weeks old) were super ovulated by intraperitoneal injection of 5 IU PMSG (SYNCRO-PART® PMSG 600 UI, Ceva) followed by 5 IU hCG (Chorulon 1500 UI, Intervet) at an interval of 46-48 hours and mated with C57BL/6J male mice. The next day, zygotes were collected from the oviducts and exposed to hyaluronidase (H3884, Sigma-Aldrich) to remove the cumulus cells and then placed in M2 medium (M7167, Sigma-Aldrich) into a CO2 incubator (5% CO2, 37 °C). SgRNAs were hybridized with Cas9 (Wild type) protein and injected into the pronucleus of the C57Bl/6J zygotes.
Surviving zygotes were placed in KSOM medium (MR-106-D, Merck-Millipore) and cultured overnight to two-cell stage and then transferred into the oviduct of B6CBAF1 pseudo-pregnant females. The

Cloning of plasmids
The MINPP1 cDNA (transcript NM_004897.5) sequence was isolated by PCR from human brain cDNA. The PCR product was then ligated into the FLAG-HA-pcDNA3.

MTT Proliferation Assay
Cells were cultured with Methylthiazolyldiphenyl-tetrazolium bromide (MTT) (0.5 mg/ml, M5655, Sigma) for 1.5 hours. Then, MTT and the cell culture medium were removed and 100% dimethyl sulfoxide (100 μl/well) was added to dissolve the formazan crystals. Next, the cell culture plate was left to shake for 15-30 minutes in the dark at room temperature, followed by transfer of the samples into flat-bottom 96-well microtiter plate (655101, Greiner). The optical density was read on a microplate reader at 560 nm (1681135, Biorad). or Caffeine (10 mM, C0750, Sigma) were quickly added to the wells in the presence of assay buffer and fluorescence intensities were recorded every 30 seconds for a minimum of 10 minutes. All the Ca2+ stimulation experiments were performed with early batch HEK293 cells4.

Quantification of intracellular total and free iron levels
The total iron content was measured with a colorimetric ferrozine-based assay according to previous studies5,6. Briefly, cells were cultured on 6-well plate (92006, TPP) and incubated with or without ferric ammonium citrate (FAC) (100 μM, RES20400-A702X, Sigma) in serum-free DMEM for 48 hours.
Then, the cells were lysed with NaOH (50 mM, 200μl/well) and 50 μl of the lysate was kept apart for protein quantification. The remaining cell lysate were mixed with equal volumes of 10 mM HCl and iron-releasing reagent (HCl 1.4 M, KMnO4 4.5%) and incubated for 2 hours at 60 °C. Once the samples reached room temperature, 30 μl of iron-detection reagent (6.5 mM ferrozine, 6.5 mM neocuproine, 2.5 M ammonium acetate, and 1 M ascorbic acid) was added to each sample. After 30 minutes of incubation at room temperature, the samples were transferred to flat-bottom 96-well microtiter plate (Greiner, 655101). The optical density was read on a microplate reader at 560 nm (Biorad, 1681135). FAC was used as reference standard.
The free iron levels (Fe2+ and Fe3+) were measured by using the Iron Assay Kit (Abcam, ab83366) according to manufacturer's instructions. In both iron-detection assays, the data are normalized against the protein levels, quantified via Pierce BCA Protein assay kit (23225, Thermo Scientific).

Immunofluorescence
Cells were fixed in cold 4% paraformaldehyde for 10 minutes followed by three washes of PBS and permeabilization in 0.25% PBS-Triton-X-100 for 10 minutes. Cells were blocked for 1 hour in 1% BSA

In vitro phytase Assay
The in vitro phytase assay was performed as previously described9. Briefly, 4 nmol of IP6 was added to 1 ml of conditioned media from Ctrl and MINPP1-/-HEK293 cells and further incubated for 0, 2, 4 and 6 hours at 37 °C. The samples were then mixed with Orange G loading dye and resolved by 35% PAGE followed by toluidine blue staining.

Analysis of extracellular inositol phosphates
For analysis of extracellular inositol phosphates, cells were labelled for 5 days with [3H]-inositol as indicated above. Media were collected and centrifuged at 300xg for 5 minutes to remove cell contaminants. Perchloric acid was added to the supernatant to final 1 M. The acidified medium was incubated on ice for 10 minutes before centrifugation at 18,000 xg to remove precipitates. Inositol phosphates in the supernatant were then purified using titanium dioxide beads10. Briefly, 4 mg beads were added to the media extracts and the samples rotated at 4°C for 15 minutes. Beads were washed twice in 1 M perchloric acid, before elution with ammonium hydroxide. Radiolabeled inositol phosphates in the purified media extracts were separated by SAX-HPLC as before, and radioactivity in each fraction measured with a beta counter.