It takes two to tango: cardiac fibroblast-derived NO-induced cGMP enters cardiac myocytes and increases cAMP by inhibiting PDE3.

The occurrence of NO/cGMP signalling in cardiac cells is a matter of debate. Recent measurements with a FRET-based cGMP indicator in isolated cardiac cells revealed NO-induced cGMP signals in cardiac fibroblasts while cardiomyocytes were devoid of these signals. In a fibroblast/myocyte co-culture model though, cGMP formed in fibroblasts in response to NO entered cardiomyocytes via gap junctions. Here, we demonstrate gap junction-mediated cGMP transfer from cardiac fibroblasts to myocytes in intact tissue. In living cardiac slices of mice with cardiomyocyte-specific expression of a FRET-based cGMP indicator (αMHC/cGi-500), NO-dependent cGMP signals were shown to occur in myocytes, to depend on gap junctions and to be degraded mainly by PDE3. Stimulation of NO-sensitive guanylyl cyclase enhanced Forskolin- and Isoproterenol-induced cAMP and phospholamban phosphorylation. Genetic inactivation of NO-GC in Tcf21-expressing cardiac fibroblasts abrogated the synergistic action of NO-GC stimulation on Iso-induced phospholamban phosphorylation, identifying fibroblasts as cGMP source and substantiating the necessity of cGMP-transfer to myocytes. In sum, NO-stimulated cGMP formed in cardiac fibroblasts enters cardiomyocytes in native tissue where it exerts an inhibitory effect on cAMP degradation by PDE3, thereby increasing cAMP and downstream effects in cardiomyocytes. Hence, enhancing ß-receptor-induced contractile responses appears as one of NO/cGMP's functions in the non-failing heart.

The role of FIBCD1 in response to Aspergillus fumigatus in lung epithelial cells.

Chitin, a polysaccharide, is ubiquitously found in nature and has been known to be an active immunogen in mammals, and interacts with Toll-like, mannose and glucan receptors, to induce cytokine and chemokine secretions. FIBCD1 is a tetrameric type II transmembrane endocytic vertebrate receptor that binds chitin, is found in human lung epithelium and modulates lung epithelial inflammatory responses to A. fumigatus cell wall polysaccharides. We previously reported the detrimental role of FIBCD1 in a murine model of pulmonary invasive aspergillosis. However, the effect that chitin and chitin-containing A. fumigatus conidia exerts on lung epithelium following exposure through FIBCD1 is not yet fully explored. Using both in vitro and in vivo strategies, we examined how lung and lung epithelial gene expression are modified after exposure to fungal conidia or chitin fragments in the presence or absence of FIBCD1. FIBCD1 expression was associated with a decrease in inflammatory cytokines with increasing size of chitin (dimer-oligomer). Thus, our results demonstrate that FIBCD1 expression modulates cytokine and chemokine expression in response to A. fumigatus conidia that is modified by the presence of chitin particles.

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles.

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

Isolation of cancer stem cells by selection for miR-302 expressing cells.

BACKGROUND: Cancer stem cells are believed to be a major reason for long-term therapy failure because they are multi-drug resistant and able to rest mitotically inactive in the hypoxic center of tumors. Due to their variable number and their often low proliferation rate, cancer stem cells are difficult to purify in decent quantities and to grow in cell culture systems, where they are easily outcompeted by faster growing more 'differentiated', i.e., less stem cell-like tumor cells. METHODS: Here we present a proof of principle study based on the idea to select cancer stem cells by means of the expression of a stem cell-specific gene. A selectable egfp-neo coding sequence was inserted in the last exon of the non-coding murine miR-302 host gene. As a stem cell specific regulatory element, 2.1 kb of the genomic region immediately upstream of the miR-302 host gene transcription start site was used. Stable transgenic CJ7 embryonic stem cells were used to induce teratomas. RESULTS: After three weeks, tumors were removed for analysis and primary cultures were established. Stem cell-like cells were selected from these culture based on G418 selection. When the selection was removed, stem cell morphology and miR-302 expression were rapidly lost, indicating that it was not the original ES cells that had been isolated. CONCLUSIONS: We show the possibility to use drug resistance expressed from a regulatory sequence of a stem cell-specific marker, to isolate and propagate cancer stem cells that otherwise might be hidden in the majority of tumor cells.

Islet-Derived eATP Fuels Autoreactive CD8+ T Cells and Facilitates the Onset of Type 1 Diabetes.

Extracellular ATP (eATP) activates T cells by engaging the P2X7R receptor. We identified two loss-of-function P2X7R mutations that are protective against type 1 diabetes (T1D) and thus hypothesized that eATP/P2X7R signaling may represent an early step in T1D onset. Specifically, we observed that in patients with newly diagnosed T1D, P2X7R is upregulated on CD8+ effector T cells in comparison with healthy control subjects. eATP is released at high levels by human/murine islets in vitro in high-glucose/inflammatory conditions, thus upregulating P2X7R on CD8+ T cells in vitro. P2X7R blockade with oxidized ATP reduces the CD8+ T cell-mediated autoimmune response in vitro and delays diabetes onset in NOD mice. Autoreactive CD8+ T-cell activation is highly dependent upon eATP/P2X7R-mediated priming, while a novel sP2X7R recombinant protein abrogates changes in metabolism and the autoimmune response associated with CD8+ T cells. eATP/P2X7R signaling facilitates the onset of autoimmune T1D by fueling autoreactive CD8+ cells and therefore represents a novel targeted therapeutic for the disorder.

Mice Knocked Out for the Primary Brain Calcification-Associated Gene Slc20a2 Show Unimpaired Prenatal Survival but Retarded Growth and Nodules in the Brain that Grow and Calcify Over Time.

Brain calcification of especially the basal ganglia characterizes primary familial brain calcification (PFBC). PFBC is a rare neurodegenerative disorder with neuropsychiatric and motor symptoms, and only symptomatic treatment is available. Four PFBC-associated genes are known; approximately 40% of patients carry mutations in the gene SLC20A2, which encodes the type III sodium-dependent inorganic phosphate transporter PiT2. To investigate the role of PiT2 in PFBC development, we studied Slc20a2-knockout (KO) mice using histology, microcomputed tomography, electron microscopy, and energy-dispersive X-ray spectroscopy. Slc20a2-KO mice showed histologically detectable nodules in the brain already at 8 weeks of age, which contained organic material and were weakly calcified. In 15-week-old mice, the nodules were increased in size and number and were markedly more calcified. The major minerals in overt calcifications were Ca and P, but Fe, Zn, and Al were also generally present. Electron microscopy suggested that the calcifications initiate intracellularly, mainly in pericytes and astrocytes. As the calcification grew, they incorporated organic material. Furthermore, endogenous IgG was detected around nodules, suggesting local increased blood-brain barrier permeabilities. Nodules were found in all 8-week-old Slc20a2-KO mice, but no prenatal or marked postnatal lethality was observed. Thus, besides allowing for the study of PFBC development, the Slc20a2-KO mouse is a potential solid preclinical model for evaluation of PFBC treatments.

Constitutive transgene expression of Stem Cell Antigen-1 in the hair follicle alters the sensitivity to tumor formation and progression.

The cell surface protein Stem Cell Antigen-1 (Sca-1) marks stem or progenitor cells in several murine tissues and is normally upregulated during cancer development. Although the specific function of Sca-1 remains unknown, Sca-1 seems to play a role in proliferation, differentiation and cell migration in a number of tissues. In the skin epithelium, Sca-1 is highly expressed in the interfollicular epidermis but is absent in most compartments of the hair follicle; however, the function of Sca-1 in the skin has not been investigated. To explore the role of Sca-1 in normal and malignant skin development we generated transgenic mice that express Sca-1 in the hair follicle stem cells that are normally Sca-1 negative. Development of hair follicles and interfollicular epidermis appeared normal in Sca-1 mutant mice; however, follicular induction of Sca-1 expression in bulge region and isthmus stem cells reduced the overall yield of papillomas in a chemical carcinogenesis protocol. Despite that fewer papillomas developed in transgenic mice a higher proportion of the papillomas underwent malignant conversion. These findings suggest that overexpression of Sca-1 in the hair follicle stem cells contributes at different stages of tumour development. In early stages, overexpression of Sca-1 decreases tumour formation while at later stages overexpression of Sca-1 seems to drive tumours towards malignant progression.

High folic acid diet enhances tumour growth in PyMT-induced breast cancer.

BACKGROUND: The B-vitamin folate is among the most studied bioactive food compound, and a dietary intake meeting the daily requirements has been found to reduce the risk of cancer and cardiovascular diseases as well as preventing neural tube defects during fetal development. Several countries have therefore introduced dietary fortification with folic acid. However, clinical and animal studies suggest that folic acid has a dual role in cancer development. METHODS: During the period of initial tumour progression, MMTV-PyMT (MMTV-polyoma virus middle T) transgenic mice were fed with normal diet and high folic acid diet. RESULTS: We found that PyMT-induced breast tumours highly express the cancer-specific folate receptor (FR), a feature they share with several human epithelial cancers in which expression of FRα correlates with tumour grade. Mice receiving a high folic acid diet displayed a significantly increased tumour volume compared with mice receiving normal diet. In the largest tumours, only found in mice on high folic acid diet, STAT3 was activated. In primary cells from PyMT tumours, STAT3 was activated upon treatment with folic acid in culture. CONCLUSIONS: Our results offer a novel molecular explanation for folic acid-induced growth of existing tumours.

The Schizophrenia-Associated BRD1 Gene Regulates Behavior, Neurotransmission, and Expression of Schizophrenia Risk Enriched Gene Sets in Mice.

BACKGROUND: The schizophrenia-associated BRD1 gene encodes a transcriptional regulator whose comprehensive chromatin interactome is enriched with schizophrenia risk genes. However, the biology underlying the disease association of BRD1 remains speculative. METHODS: This study assessed the transcriptional drive of a schizophrenia-associated BRD1 risk variant in vitro. Accordingly, to examine the effects of reduced Brd1 expression, we generated a genetically modified Brd1+/- mouse and subjected it to behavioral, electrophysiological, molecular, and integrative genomic analyses with focus on schizophrenia-relevant parameters. RESULTS: Brd1+/- mice displayed cerebral histone H3K14 hypoacetylation and a broad range of behavioral changes with translational relevance to schizophrenia. These behaviors were accompanied by striatal dopamine/serotonin abnormalities and cortical excitation-inhibition imbalances involving loss of parvalbumin immunoreactive interneurons. RNA-sequencing analyses of cortical and striatal micropunches from Brd1+/- and wild-type mice revealed differential expression of genes enriched for schizophrenia risk, including several schizophrenia genome-wide association study risk genes (e.g., calcium channel subunits [Cacna1c and Cacnb2], cholinergic muscarinic receptor 4 [Chrm4)], dopamine receptor D2 [Drd2], and transcription factor 4 [Tcf4]). Integrative analyses further found differentially expressed genes to cluster in functional networks and canonical pathways associated with mental illness and molecular signaling processes (e.g., glutamatergic, monoaminergic, calcium, cyclic adenosine monophosphate [cAMP], dopamine- and cAMP-regulated neuronal phosphoprotein 32 kDa [DARPP-32], and cAMP responsive element binding protein signaling [CREB]). CONCLUSIONS: Our study bridges the gap between genetic association and pathogenic effects and yields novel insights into the unfolding molecular changes in the brain of a new schizophrenia model that incorporates genetic risk at three levels: allelic, chromatin interactomic, and brain transcriptomic.

Cognitive deficits caused by a disease-mutation in the α3 Na(+)/K(+)-ATPase isoform.

The Na(+)/K(+)-ATPases maintain Na(+) and K(+) electrochemical gradients across the plasma membrane, a prerequisite for electrical excitability and secondary transport in neurons. Autosomal dominant mutations in the human ATP1A3 gene encoding the neuron-specific Na(+)/K(+)-ATPase α3 isoform cause different neurological diseases, including rapid-onset dystonia-parkinsonism (RDP) and alternating hemiplegia of childhood (AHC) with overlapping symptoms, including hemiplegia, dystonia, ataxia, hyperactivity, epileptic seizures, and cognitive deficits. Position D801 in the α3 isoform is a mutational hotspot, with the D801N, D801E and D801V mutations causing AHC and the D801Y mutation causing RDP or mild AHC. Despite intensive research, mechanisms underlying these disorders remain largely unknown. To study the genotype-to-phenotype relationship, a heterozygous knock-in mouse harboring the D801Y mutation (α3(+/D801Y)) was generated. The α3(+/D801Y) mice displayed hyperactivity, increased sensitivity to chemically induced epileptic seizures and cognitive deficits. Interestingly, no change in the excitability of CA1 pyramidal neurons in the α3(+/D801Y) mice was observed. The cognitive deficits were rescued by administration of the benzodiazepine, clonazepam, a GABA positive allosteric modulator. Our findings reveal the functional significance of the Na(+)/K(+)-ATPase α3 isoform in the control of spatial learning and memory and suggest a link to GABA transmission.

C4.4A gene ablation is compatible with normal epidermal development and causes modest overt phenotypes.

C4.4A is a modular glycolipid-anchored Ly6/uPAR/α-neurotoxin multidomain protein that exhibits a prominent membrane-associated expression in stratified squamous epithelia. C4.4A is also expressed in various solid cancer lesions, where high expression levels often are correlated to poor prognosis. Circumstantial evidence suggests a role for C4.4A in cell adhesion, migration, and invasion, but a well-defined biological function is currently unknown. In the present study, we have generated and characterized the first C4.4A-deficient mouse line to gain insight into the functional significance of C4.4A in normal physiology and cancer progression. The unchallenged C4.4A-deficient mice were viable, fertile, born in a normal Mendelian distribution and, surprisingly, displayed normal development of squamous epithelia. The C4.4A-deficient mice were, nonetheless, significantly lighter than littermate controls predominantly due to differences in fat mass. Congenital C4.4A deficiency delayed migration of keratinocytes enclosing incisional skin wounds in male mice. In chemically induced bladder carcinomas, C4.4A deficiency attenuated the incidence of invasive lesions despite having no effect on total tumour burden. This new C4.4A-deficient mouse line provides a useful platform for future studies on functional aspects of C4.4A in tumour cell invasion in vivo.

Prior renovascular hypertension does not predispose to atherosclerosis in mice.

BACKGROUND: Hypertension is a major risk factor for development of atherosclerotic cardiovascular disease (ASCVD). Although lowering blood pressure with antihypertensive drugs reduces the increased risk of ASCVD, residual increased risk still remains, suggesting that hypertension may cause chronic changes that promote atherosclerosis. Thus, we tested the hypothesis that hypertension increases the susceptibility to atherosclerosis in mice even after a period of re-established normotension. METHODS: We used the 2-kidney, 1-clip (2K1C) technique to induce angiotensin-driven renovascular hypertension, and overexpression of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene to cause severe hypercholesterolemia and atherosclerosis. RESULTS: First, we performed 2K1C (n = 8) or sham surgery (n = 9) in PCSK9 transgenic mice before they were fed a high fat diet for 14 weeks. As expected, 2K1C did not affect cholesterol levels, but induced cardiac hypertrophy and significantly increased the atherosclerotic lesion area compared to sham mice (1.8 fold, p < 0.01). Next, we performed 2K1C (n = 13) or sham surgery (n = 14) in wild-type mice but removed the clipped/sham-operated kidney after 10 weeks to eliminate hypertension, and subsequently induced hypercholesterolemia by way of adeno-associated virus-mediated hepatic gene transfer of PCSK9 combined with high-fat diet. After 14 weeks of hypercholesterolemia, atherosclerotic lesion areas were not significantly different in mice with or without prior 2K1C hypertension (0.95 fold, p = 0.35). CONCLUSION: Renovascular hypertension in mice does not induce pro-atherogenic changes that persist beyond the hypertensive phase. These results indicate that hypertension only promotes atherogenesis when coinciding temporally with hypercholesterolemia.

Glutamate-system defects behind psychiatric manifestations in a familial hemiplegic migraine type 2 disease-mutation mouse model.

Migraine is a complex brain disorder, and understanding the complexity of this prevalent disease could improve quality of life for millions of people. Familial Hemiplegic Migraine type 2 (FHM2) is a subtype of migraine with aura and co-morbidities like epilepsy/seizures, cognitive impairments and psychiatric manifestations, such as obsessive-compulsive disorder (OCD). FHM2 disease-mutations locate to the ATP1A2 gene encoding the astrocyte-located α2-isoform of the sodium-potassium pump (α2Na(+)/K(+)-ATPase). We show that knock-in mice heterozygous for the FHM2-associated G301R-mutation (α2(+/G301R)) phenocopy several FHM2-relevant disease traits e.g., by mimicking mood depression and OCD. In vitro studies showed impaired glutamate uptake in hippocampal mixed astrocyte-neuron cultures from α2(G301R/G301R) E17 embryonic mice, and moreover, induction of cortical spreading depression (CSD) resulted in reduced recovery in α2(+/G301R) male mice. Moreover, NMDA-type glutamate receptor antagonists or progestin-only treatment reverted specific α2(+/G301R) behavioral phenotypes. Our findings demonstrate that studies of an in vivo relevant FHM2 disease knock-in mouse model provide a link between the female sex hormone cycle and the glutamate system and a link to co-morbid psychiatric manifestations of FHM2.

Stanniocalcin-2 inhibits mammalian growth by proteolytic inhibition of the insulin-like growth factor axis.

Mammalian stanniocalcin-2 (STC2) is a secreted polypeptide widely expressed in developing and adult tissues. However, although transgenic expression in mice is known to cause severe dwarfism, and targeted deletion of STC2 causes increased postnatal growth, its precise biological role is still unknown. We found that STC2 potently inhibits the proteolytic activity of the growth-promoting metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). Proteolytic inhibition requires covalent binding of STC2 to PAPP-A and is mediated by a disulfide bond, which involves Cys-120 of STC2. Binding of STC2 prevents PAPP-A cleavage of insulin-like growth factor-binding protein (IGFBP)-4 and hence release within tissues of bioactive IGF, required for normal growth. Concordantly, we show that STC2 efficiently inhibits PAPP-A-mediated IGF receptor signaling in vitro and that transgenic mice expressing a mutated variant of STC2, STC2(C120A), which is unable to inhibit PAPP-A, grow like wild-type mice. Our work identifies STC2 as a novel proteinase inhibitor and a previously unrecognized extracellular component of the IGF system.

An eEF1A1 truncation encoded by PTI-1 exerts its oncogenic effect inside the nucleus.

BACKGROUND: The oncogene PTI-1 was originally isolated from a prostate cancer cell line by its capability to transform rat fibroblasts. The PTI-1 mRNA has a very eccentric structure as the 5'UTR is similar to prokaryotic 23S rRNA, while the major open reading frame and the 3'UTR corresponds to a part of the mRNA encoding human translation elongation factor eEF1A1. Thus, the largest open reading frame encodes a truncated version of eEF1A1 lacking the first 67 amino acids, while having three unique N-terminal amino acids. Previously, the UTRs were shown to be a prerequisite for the transforming capacity of the PTI-1 transcript. In this study, we have investigated the possible role of the UTRs in regulating protein expression and localization. METHODS: The protein expression profiles of a number of PTI-1 mRNA variants were studied in vitro and in vivo. Furthermore, the oncogenic potentials of the same PTI-1 mRNAs were determined by monitoring the capacities of stably transfected cells expressing these mRNAs to induce tumors in nude mice and form foci in cell culture. Finally, the cellular localizations of PTI-1 proteins expressed from these mRNAs were determined by fluorescence microscopy. RESULTS: The PTI-1 mRNA was found to give rise to multiple protein products that potentially originate from translation initiation at downstream, inframe AUGs within the major open reading frame. At least one of the truncated protein variants was also found to be oncogenic. However, the UTRs did not appear to influence the amount and identities of these truncated protein products. In contrast, our localization studies showed that the UTRs of the transcript promote a nuclear localization of the encoded protein(s). CONCLUSIONS: Translation of the PTI-1 mRNA results in multiple protein products of which (a) truncated variant(s) may play a predominant role during cellular transformation. The PTI-1 UTRs did not seem to play a role in translation regulation, but appeared to contribute to a nuclear localization of the PTI-1 protein(s). This indicates that the PTI-1 protein(s) exert(s) its/their oncogenic function inside the nucleus.

SORLA-dependent and -independent functions for PACS1 in control of amyloidogenic processes.

Sorting-related receptor with A-type repeats (SORLA) is a sorting receptor for the amyloid precursor protein (APP) that prevents breakdown of APP into Aß peptides, a hallmark of Alzheimer's disease (AD). Several cytosolic adaptors have been shown to interact with the cytoplasmic domain of SORLA, thereby controlling intracellular routing of SORLA/APP complexes in cell lines. However, the relevance of adaptor-mediated sorting of SORLA for amyloidogenic processes in vivo remained unexplored. We focused on the interaction of SORLA with phosphofurin acidic cluster sorting protein 1 (PACS1), an adaptor that shuttles proteins between the trans-Golgi network (TGN) and endosomes. By studying PACS1 knockdown in neuronal cell lines and investigating transgenic mice expressing a PACS1-binding-defective mutant form of SORLA, we found that disruption of SORLA and PACS1 interaction results in the inability of SORLA/APP complexes to sort to the TGN in neurons and in increased APP processing in the brain. Loss of PACS1 also impairs the proper expression of the cation-independent mannose 6-phosphate receptor and its target cathepsin B, a protease that breaks down Aß. Thus, our data identified the importance of PACS1-dependent protein sorting for amyloidogenic-burden control via both SORLA-dependent and SORLA-independent mechanisms.

The Rag2⁻Il2rb⁻Dmd⁻ mouse: a novel dystrophic and immunodeficient model to assess innovating therapeutic strategies for muscular dystrophies.

The development of innovative therapeutic strategies for muscular dystrophies, particularly cell-based approaches, is still a developing field. Although positive results have been obtained in animal models, they have rarely been confirmed in patients and resulted in very limited clinical improvements, suggesting some specificity in humans. These findings emphasized the need for an appropriate animal model (i.e., immunodeficient and dystrophic) to investigate in vivo the behavior of transplanted human myogenic stem cells. We report a new model, the Rag2(-)Il2rb(-)Dmd(-) mouse, which lacks T, B, and NK cells, and also carries a mutant Dmd allele that prevents the production of any dystrophin isoform. The dystrophic features of this new model are comparable with those of the classically used mdx mouse, but with the total absence of any revertant dystrophin positive fiber. We show that Rag2(-)Il2rb(-)Dmd(-) mice allow long-term xenografts of human myogenic cells. Altogether, our findings indicate that the Rag2(-)Il2rb(-)Dmd(-) mouse represents an ideal model to gain further insights into the behavior of human myogenic stem cells in a dystrophic context, and can be used to assess innovative therapeutic strategies for muscular dystrophies.

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

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.