Antisense oligonucleotides enhance SLC20A2 expression and suppress brain calcification in a humanized mouse model.

Primary familial brain calcification (PFBC) is a genetic neurological disease, yet no effective treatment is currently available. Here, we identified five novel intronic variants in SLC20A2 gene from six PFBC families. Three of these variants increased aberrant SLC20A2 pre-mRNA splicing by altering the binding affinity of splicing machineries to newly characterized cryptic exons, ultimately causing premature termination of SLC20A2 translation. Inhibiting the cryptic-exon incorporation with splice-switching ASOs increased the expression levels of functional SLC20A2 in cells carrying SLC20A2 mutations. Moreover, by knocking in a humanized SLC20A2 intron 2 sequence carrying a PFBC-associated intronic variant, the SLC20A2-KI mice exhibited increased inorganic phosphate (Pi) levels in cerebrospinal fluid (CSF) and progressive brain calcification. Intracerebroventricular administration of ASOs to these SLC20A2-KI mice reduced CSF Pi levels and suppressed brain calcification. Together, our findings expand the genetic etiology of PFBC and demonstrate ASO-mediated splice modulation as a potential therapy for PFBC patients with SLC20A2 haploinsufficiency.

Astrocytes modulate brain phosphate homeostasis via polarized distribution of phosphate uptake transporter PiT2 and exporter XPR1.

Aberrant inorganic phosphate (Pi) homeostasis causes brain calcification and aggravates neurodegeneration, but the underlying mechanism remains unclear. Here, we found that primary familial brain calcification (PFBC)-associated Pi transporter genes Pit2 and Xpr1 were highly expressed in astrocytes, with importer PiT2 distributed over the entire astrocyte processes and exporter XPR1 localized to astrocyte end-feet on blood vessels. This polarized PiT2 and XPR1 distribution endowed astrocyte with Pi transport capacity competent for brain Pi homeostasis, which was disrupted in mice with astrocyte-specific knockout (KO) of either Pit2 or Xpr1. Moreover, we found that Pi uptake by PiT2, and its facilitation by PFBC-associated galactosidase MYORG, were required for the high Pi transport capacity of astrocytes. Finally, brain calcification was suppressed by astrocyte-specific PiT2 re-expression in Pit2-KO mice. Thus, astrocyte-mediated Pi transport is pivotal for brain Pi homeostasis, and elevating astrocytic Pi transporter function represents a potential therapeutic strategy for reducing brain calcification.

A phosphate transporter in VIPergic neurons of the suprachiasmatic nucleus gates locomotor activity during the light/dark transition in mice.

The suprachiasmatic nucleus (SCN) encodes time of day through changes in daily firing; however, the molecular mechanisms by which the SCN times behavior are not fully understood. To identify factors that could encode day/night differences in activity, we combine patch-clamp recordings and single-cell sequencing of individual SCN neurons in mice. We identify PiT2, a phosphate transporter, as being upregulated in a population of Vip+Nms+ SCN neurons at night. Although nocturnal and typically showing a peak of activity at lights off, mice lacking PiT2 (PiT2-/-) do not reach the activity level seen in wild-type mice during the light/dark transition. PiT2 loss leads to increased SCN neuronal firing and broad changes in SCN protein phosphorylation. PiT2-/- mice display a deficit in seasonal entrainment when moving from a simulated short summer to longer winter nights. This suggests that PiT2 is responsible for timing activity and is a driver of SCN plasticity allowing seasonal entrainment.

Mechanisms of PiT2-loop7 Missense Mutations Induced Pi Dyshomeostasis / 神经科学通报·英文版

PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.

Mechanisms of PiT2-loop7 Missense Mutations Induced Pi Dyshomeostasis.

PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.

Golgi damage caused by dysfunction of PiT-2 in primary familial brain calcification.

The Golgi apparatus is vital for protein modification and molecular trafficking. It is essential for nerve development and activity, and damage thereof is implicated in many neurological diseases. Primary familial brain calcification (PFBC) is a rare inherited neurodegenerative disease characterized by multiple brain calcifications. SLC20A2, which encodes the inorganic phosphate transporter 2 (PiT-2) protein, is the main pathogenic gene in PFBC. The PiT-2 protein is a sodium-dependent phosphate type III transporter, and dysfunction leads to a deficit in the cellular intake of inorganic phosphate (Pi) and calcium deposits. Whether the impaired Golgi apparatus is involved in the PFBC procession requires elucidation. In this study, we constructed induced pluripotent stem cells (iPSCs) derived from two PFBC patients with different SLC20A2 gene mutations (c.613G > A or del exon10) and two healthy volunteers as dependable cell models for research on pathogenic mechanism. To study the mechanism, we differentiated iPSCs into neurons and astrocytes in vitro. Our study found disruptive Golgi structure and damaged autophagy in PFBC neurons with increased activity of mTOR. We also found damaged mitochondria and increased apoptosis in the PFBC dopaminergic neurons and astrocytes. In this study, we prove that dysfunctional PiT-2 leads to an imbalance of cellular Pi, which may disrupt the Golgi apparatus with impaired autophagy, mitochondria and apoptosis in PFBC. Our study provides a new avenue for understanding nerve damage and pathogenic mechanism in brain calcifications.

PiT2 deficiency prevents increase of bone marrow adipose tissue during skeletal maturation but not in OVX-induced osteoporosis.

The common cellular origin between bone marrow adipocytes (BMAds) and osteoblasts contributes to the intimate link between bone marrow adipose tissue (BMAT) and skeletal health. An imbalance between the differentiation ability of BMSCs towards one of the two lineages occurs in conditions like aging or osteoporosis, where bone mass is decreased. Recently, we showed that the sodium-phosphate co-transporter PiT2/SLC20A2 is an important determinant for bone mineralization, strength and quality. Since bone mass is reduced in homozygous mutant mice, we investigated in this study whether the BMAT was also affected in PiT2-/- mice by assessing the effect of the absence of PiT2 on BMAT volume between 3 and 16 weeks, as well as in an ovariectomy-induced bone loss model. Here we show that the absence of PiT2 in juveniles leads to an increase in the BMAT that does not originate from an increased adipogenic differentiation of bone marrow stromal cells. We show that although PiT2-/- mice have higher BMAT volume than control PiT2+/+ mice at 3 weeks of age, BMAT volume do not increase from 3 to 16 weeks of age, leading to a lower BMAT volume in 16-week-old PiT2-/- compared to PiT2+/+ mice. In contrast, the absence of PiT2 does not prevent the increase in BMAT volume in a model of ovariectomy-induced bone loss. Our data identify SLC20a2/PiT2 as a novel gene essential for the maintenance of the BMAd pool in adult mice, involving mechanisms of action that remain to be elucidated, but which appear to be independent of the balance between osteoblastic and adipogenic differentiation of BMSCs.

Phosphate-Sensing.

The blood level of phosphate is tightly regulated in a narrow range. Hyperphosphatemia and hypophosphatemia both lead to the development of diseases, such as hyperphosphatemic tumoral calcinosis and rickets/osteomalacia, respectively. Although several humoral factors have been known to affect blood phosphate levels, fibroblast growth factor 23 (FGF23) is the principal hormone involved in the regulation of blood phosphate. This hormone is produced by bone, particularly by osteocytes and osteoblasts, and has the effect of lowering the blood level of phosphate in the renal proximal tubules. Therefore, some phosphate-sensing mechanism should exist, at least in the bone. However, the mechanisms through which bone senses changes in the blood level of phosphate, and through which the bone regulates FGF23 production remain to be fully elucidated. Our recent findings demonstrate that high extracellular phosphate phosphorylates FGF receptor 1c (FGFR1c). Its downstream extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling pathway regulates the expression of several transcription factors and the GALNT3 gene, which encodes GalNAc-T3, which plays a role in the regulation of posttranslational modification of FGF23 protein, which in turn enhances FGF23 production. The FGFR1c-GALNT3 gene axis is considered to be the most important mechanism for regulating the production of FGF23 in bone in the response to a high phosphate diet. Thus-in the regulation of FGF23 production and blood phosphate levels-FGFR1c may be considered to function as a phosphate-sensing molecule. A feedback mechanism, in which FGFR1c and FGF23 are involved, is present in blood phosphate regulation. In addition, other reports indicate that PiT1 and PiT2 (type III sodium-phosphate cotransporters), and calcium-sensing receptor are also involved in the phosphate-sensing mechanism. In the present chapter, we summarize new insights on phosphate-sensing mechanisms.

Familial idiopathic basal ganglia calcification with a heterozygous missense variant (c.902C>T/p.P307L) in SLC20A2 showing widespread cerebrovascular lesions.

We describe a postmortem case of familial idiopathic basal ganglia calcification (FIBGC) in a 72-year-old Japanese man. The patient showed progressive cognitive impairment with a seven-year clinical course and calcification of the basal ganglia, thalami, and cerebellar dentate nuclei. A novel heterozygous missense variant in SLC20A2 (c.920C>T/p.P307L), a type III sodium-dependent phosphate transporter (PiT-2), was subsequently identified, in addition to typical neuropathological findings of FIBGC, such as capillary calcification of the occipital gray matter, confluent calcification of the basal ganglia and cerebellar white matter, widespread occurrence of vasculopathic changes, cerebrovascular lesions, and vascular smooth muscle cell depletion. Immunohistochemistry for PiT-2 protein revealed no apparent staining in endothelial cells in the basal ganglia and insular cortex; however, the immunoreactivity in endothelial cells of the cerebellum was preserved. Moreover, Western blot analysis identified preserved PiT-2 immunoreactivity signals in the frontal cortex and cerebellum. The variant identified in the present patient could be associated with development of FIBGC and is known to be located at the large intracytoplasmic part of the PiT-2 protein, which has potential phosphorylation sites with importance in the regulation of inorganic phosphate transport activity. The present case is an important example to prove that FIGBC could stem from a missense variant in the large intracytoplasmic loop of the PiT-2 protein. Abnormal clearance of inorganic phosphate in the brain could be related to the development of vascular smooth muscle damage, the formation of cerebrovascular lesions, and subsequent brain calcification in patients with FIBGC with SLC20A2 variants.

Characteristics and therapeutic potential of sodium-dependent phosphate cotransporters in relation to idiopathic basal ganglia calcification.

Type-III sodium-dependent phosphate transporters 1 and 2 (PiT 1 and PiT 2, respectively) are proteins encoded by SLC20A1 and SLC20A2, respectively. The ubiquitous distribution of SLC20A1 and SLC20A2 mRNAs in mammalian tissues supports the housekeeping maintenance and homeostasis of intracellular inorganic phosphate (Pi), which is absorbed from interstitial fluid for normal cellular functions. SLC20A2 variants have been found in patients with idiopathic basal ganglia calcification (IBGC), also known as Fahr's disease or primary familial brain calcification (PFBC). Thus, disrupted Pi homeostasis is considered one of the major factors in the pathogenic mechanism of IBGC. In this paper, among the causative genes of IBGC, we focused specifically on PiT2, and its potential for a therapeutic target of IBGC.

The impact of type III sodium-dependent phosphate transporters (Pit 1 and Pit 2) on podocyte and kidney function.

The sodium-dependent phosphate transporters Pit 1 and Pit 2 belong to the solute carrier 20 (SLC20) family of membrane proteins. They are ubiquitously distributed in the human body. Their crucial function is the intracellular transport of inorganic phosphate (Pi) in the form of H2 PO4- . They are one of the main elements in maintaining physiological phosphate homeostasis. Recent data have emerged that indicate novel roles of Pit 1 and Pit 2 proteins besides the well-known function of Pi transporters. These membrane proteins are believed to be precise phosphate sensors that mediate Pi-dependent intracellular signaling. They are also involved in insulin signaling and influence cellular insulin sensitivity. In diseases that are associated with hyperphosphatemia, such as diabetes and chronic kidney disease (CKD), disturbances in the function of Pit 1 and Pit 2 are observed. Phosphate transporters from the SLC20 family participate in the calcification of soft tissues, mainly blood vessels, during the course of CKD. The glomerulus and podocytes therein can also be a target of pathological calcification that damages these structures. A few studies have demonstrated the development of Pi-dependent podocyte injury that is mediated by Pit 1 and Pit 2. This paper discusses the role of Pit 1 and Pit 2 proteins in podocyte function, mainly in the context of the development of pathological calcification that disrupts permeability of the renal filtration barrier. We also describe the mechanisms that may contribute to podocyte damage by Pit 1 and Pit 2.

Modulation of Intestinal Phosphate Transport in Young Goats Fed a Low Phosphorus Diet.

The intestinal absorption of phosphate (Pi) takes place transcellularly through the active NaPi-cotransporters type IIb (NaPiIIb) and III (PiT1 and PiT2) and paracellularly by diffusion through tight junction (TJ) proteins. The localisation along the intestines and the regulation of Pi absorption differ between species and are not fully understood. It is known that 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3) and phosphorus (P) depletion modulate intestinal Pi absorption in vertebrates in different ways. In addition to the apical uptake into the enterocytes, there are uncertainties regarding the basolateral excretion of Pi. Functional ex vivo experiments in Ussing chambers and molecular studies of small intestinal epithelia were carried out on P-deficient goats in order to elucidate the transepithelial Pi route in the intestine as well as the underlying mechanisms of its regulation and the proteins, which may be involved. The dietary P reduction had no effect on the duodenal and ileal Pi transport rate in growing goats. The ileal PiT1 and PiT2 mRNA expressions increased significantly, while the ileal PiT1 protein expression, the mid jejunal claudin-2 mRNA expression and the serum 1,25-(OH)2D3 levels were significantly reduced. These results advance the state of knowledge concerning the complex mechanisms of the Pi homeostasis in vertebrates.

EOS789, a novel pan-phosphate transporter inhibitor, is effective for the treatment of chronic kidney disease-mineral bone disorder.

Chronic kidney disease is characterized as impaired renal function along with the imbalance and dysregulation of mineral metabolism; recognized as chronic kidney disease-mineral and bone disorder. Hyperphosphatemia, characterized by altered phosphate homeostasis along with elevated fibroblast growth factor-23 and intact parathyroid hormone, is such an alteration of mineral metabolism. We discovered a novel inhibitor, EOS789, that interacts with several sodium-dependent phosphate transporters (NaPi-IIb, PiT-1, and PiT-2) known to contribute to intestinal phosphate absorption. This inhibitor dose-dependently increased the fecal phosphorus excretion rate and inversely decreased the urinary phosphorus excretion rate in normal rats, suggesting inhibition of intestinal phosphorus absorption. In rats with adenine-induced hyperphosphatemia, EOS789 markedly decreased the serum phosphate, fibroblast growth factor-23, and intact parathyroid hormone below values found in normal control rats. Notably, this pan-phosphate transporter inhibitor exhibited a more potent effect on serum phosphate than a NaPi-IIb-selective inhibitor in rats with hyperphosphatemia indicating that PiT-1 and PiT-2 play important roles in intestinal phosphate absorption. Moreover, in a long-term study, EOS789 sustained the suppression of serum phosphorus in parallel with fibroblast growth factor-23 and intact parathyroid hormone and ameliorated ectopic calcification of the thoracic aorta. Additionally, EOS789 treatment also ameliorated kidney deterioration in rats with progressive kidney injury, probably due to the strict phosphate control. Thus, EOS789 has potent efficacy against hyperphosphatemia and its complications and could provide a significant benefit to patients who are ineffectively treated with phosphate binders.

Significant Species Differences in Intestinal Phosphate Absorption between Dogs, Rats, and Monkeys.

A treatment for hyperphosphatemia would be expected to reduce mortality rates for CKD and dialysis patients. Although rodent studies have suggested sodium-dependent phosphate transporter type IIb (NaPi-IIb) as a potential target for hyperphosphatemia, NaPi-IIb selective inhibitors failed to achieve efficacy in human clinical trials. In this study, we analyzed phosphate metabolism in rats, dogs, and monkeys to confirm the species differences. Factors related to phosphate metabolism were measured and intestinal phosphate absorption rate was calculated from fecal excretion in each species. Phosphate uptake by intestinal brush border membrane vesicles (BBMV) and the mRNA expression of NaPi-IIb, PiT-1, and PiT-2 were analyzed. In addition, alkaline phosphatase (ALP) activity was evaluated. The intestinal phosphate absorption rate, including phosphate uptake by BBMV and NaPi-IIb expression, was the highest in dogs. Notably, urinary phosphate excretion was the lowest in monkeys, and their intestinal phosphate absorption rate was by far the lowest. Dogs and rats showed positive correlations between Vmax/Km of phosphate uptake in BBMV and NaPi-IIb expression. Although phosphate uptake was observed in the BBMV of monkeys, NaPi-IIb expression was not detected and ALP activity was low. This study revealed significant species differences in intestinal phosphate absorption. NaPi-IIb contributes to intestinal phosphate uptake in rats and dogs. However, in monkeys, phosphate is poorly absorbed due to the slight degradation of organic phosphate in the intestine.

PiT-2, a type III sodium-dependent phosphate transporter, protects against vascular calcification in mice with chronic kidney disease fed a high-phosphate diet.

PiT-2, a type III sodium-dependent phosphate transporter, is a causative gene for the brain arteriolar calcification in people with familial basal ganglion calcification. Here we examined the effect of PiT-2 haploinsufficiency on vascular calcification in uremic mice using wild-type and global PiT-2 heterozygous knockout mice. PiT-2 haploinsufficiency enhanced the development of vascular calcification in mice with chronic kidney disease fed a high-phosphate diet. No differences were observed in the serum mineral biomarkers and kidney function between the wild-type and PiT-2 heterozygous knockout groups. Micro computed tomography analyses of femurs showed that haploinsufficiency of PiT-2 decreased trabecular bone mineral density in uremia. In vitro, sodium-dependent phosphate uptake was decreased in cultured vascular smooth muscle cells isolated from PiT-2 heterozygous knockout mice compared with those from wild-type mice. PiT-2 haploinsufficiency increased phosphate-induced calcification of cultured vascular smooth muscle cells compared to the wild-type. Furthermore, compared to wild-type vascular smooth muscle cells, PiT-2 deficient vascular smooth muscle cells had lower osteoprotegerin levels and increased matrix calcification, which was attenuated by osteoprotegerin supplementation. Thus, PiT-2 in vascular smooth muscle cells protects against phosphate-induced vascular calcification and may be a therapeutic target in the chronic kidney disease population.

Phosphorus absorption and gene expression levels of related transporters in the small intestine of broilers.

To investigate the P absorption and gene expression levels of related co-transporters, type IIb sodium-dependent phosphate co-transporter (NaPi-IIb), inorganic phosphate transporter 1 (PiT-1) and inorganic phosphate transporter 2 (PiT-2) in the small intestine of broilers, 450 1-d-old Arbor Acres male broilers were randomly allocated to one of three treatments with ten replicate cages of fifteen birds per cage for each treatment in a completely randomised design. Chickens were fed a diet with no added inorganic P (containing 0·06 % non-phytate P (NPP)) or with either 0·21 or 0·44 % NPP for 21 d. Plasma P concentration in the hepatic portal vein, mRNA and protein expression levels of NaPi-IIb, PiT-1 and PiT-2 were determined at 7, 14 and 21 d of age. The results showed that the concentration of P in plasma in the hepatic portal vein increased as dietary NPP increased (P<0·0001). At 14 and 21 d of age, the increase in dietary NPP inhibited (P<0·003) NaPi-IIb mRNA expression level in the duodenum, as well as PiT-1 mRNA and protein expression levels in the ileum, but promoted NaPi-IIb protein expression level (P<0·002) and PiT-2 mRNA and protein expression levels (P<0·04) in the duodenum. These results suggest that NaPi-IIb, PiT-1 and PiT-2 might be important P transporters in the small intestine of broilers. Higher intestinal P absorption may be achieved by up-regulating the protein expression levels of NaPi-IIb and PiT-2 and down-regulating the protein expression of PiT-1.

Phosphate-dependent FGF23 secretion is modulated by PiT2/Slc20a2.

OBJECTIVE: The canonical role of the bone-derived fibroblast growth factor 23 (Fgf23) is to regulate the serum inorganic phosphate (Pi) level. As part of a feedback loop, serum Pi levels control Fgf23 secretion through undefined mechanisms. We recently showed in vitro that the two high-affinity Na+-Pi co-transporters PiT1/Slc20a1 and PiT2/Slc20a2 were required for mediating Pi-dependent signaling. Here, we addressed the contribution of PiT1 and PiT2 to the regulation of Fgf23 secretion. METHODS: To this aim, we used PiT2 KO and DMP1Cre; PiT1lox/lox fed Pi-modified diets, as well as ex vivo isolated long bone shafts. Fgf23 secretion and expression of Pi homeostasis-related genes were assessed. RESULTS: In vivo, PiT2 KO mice responded inappropriately to low-Pi diets, displaying abnormally normal serum levels of intact Fgf23. Despite the high iFgf23 level, serum Pi levels remained unaffected, an effect that may relate to lower αKlotho expression in the kidney. Moreover, consistent with a role of PiT2 as a possible endocrine Pi sensor, the iFGF23/cFGF23 ratios were suppressed in PiT2 KO mice, irrespective of the Pi loads. While deletion of PiT1 in osteocytes using the DMP1-Cre mice was inefficient, adenovirus-mediated deletion of PiT1 in isolated long bone shafts suggested that PiT1 does not contribute to Pi-dependent regulation of Fgf23 secretion. In contrast, using isolated bone shafts from PiT2 KO mice, we showed that PiT2 was necessary for the appropriate Pi-dependent secretion of Fgf23, independently from possible endocrine regulatory loops. CONCLUSIONS: Our data provide initial mechanistic insights underlying the Pi-dependent regulation of Fgf23 secretion in identifying PiT2 as a potential player in this process, at least in high Pi conditions. Targeting PiT2, therefore, could improve excess FGF23 in hyperphosphatemic conditions such as chronic kidney disease.

Primary familial brain calcifications.

Primary familial brain calcification (PFBC) is a neurodegenerative disease with characteristic calcium deposits in the basal ganglia and other brain regions. The disease usually presents as a combination of abnormal movements, cognitive and psychiatric manifestations, clinically indistinguishable from other adult-onset neurodegenerative disorders. The differential diagnosis must be established with genetic and nongenetic disorders that can also lead to calcium deposits in encephalic structures. In the past years PFBC causal mutations have been discovered in genes related to calcium phosphate homeostasis (SLC20A2, XPR1) and in genes involved with endothelial function and integrity (PDGFB, PDGFRB). The most frequently mutated gene is SLC20A2, where mutations can affect any domain of the protein. There is no clearcut relationship between the specific mutation/gene, onset age, neuroimaging pattern, and severity of clinical manifestations. The discovery of the genetic basis of PFBC provides not only a diagnostic tool, but also an insight into the pathomechanisms and potential therapeutic trials for this rare disease.

Primary familial brain calcification with a novel SLC20A2 mutation: Analysis of PiT-2 expression and localization.

Primary familial brain calcification (PFBC) is an autosomal dominant rare disorder characterized by bilateral and symmetric brain calcifications, and neuropsychiatric manifestations. Four genes have been linked to PFBC: SLC20A2, PDGFRB, PDGFB, and XPR1. In this study, we report molecular and clinical data of a PFBC patient carrying a novel SLC20A2 mutation and we investigate the impact of the mutation on PiT-2 expression and function. Sanger sequencing of SLC20A2, PDGFRB, PDGFB, XPR1 led to the identification of a novel duplication of twelve nucleotides (c.1876_1887dup/ p.Trp626_Thr629dup) in SLC20A2 gene. SLC20A2 encodes for a cell membrane transporter (PiT-2) involved in maintenance of inorganic phosphate homeostasis. We performed an analysis of expression and functionality of PiT-2 protein in patient primary cultured fibroblasts. In patient fibroblasts, the mutation does not affect PiT-2 expression but alter sub-cellular localization. The Pi-uptake assay revealed a less Pi depletion in patient than in control fibroblasts, suggesting that SLC20A2 duplication may impair Pi internalization. This is the first study reporting sub-cellular expression analysis of mutant PiT-2 in primary cultured fibroblasts from a PFBC patient, showing that p.Trp626_Thr629dup in SLC20A2 alters PiT-2 sub-cellular localization and reduces Pi-uptake, leading to onset of PFBC in our patient.

Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake.

Extracellular phosphate (Pi) can act as a signaling molecule that directly alters gene expression and cellular physiology. The ability of cells or organisms to detect changes in extracellular Pi levels implies the existence of a Pi-sensing mechanism that signals to the body or individual cell. However, unlike in prokaryotes, yeasts, and plants, the molecular players involved in Pi sensing in mammals remain unknown. In this study, we investigated the involvement of the high-affinity, sodium-dependent Pi transporters PiT1 and PiT2 in mediating Pi signaling in skeletal cells. We found that deletion of PiT1 or PiT2 blunted the Pi-dependent ERK1/2-mediated phosphorylation and subsequent gene up-regulation of the mineralization inhibitors matrix Gla protein and osteopontin. This result suggested that both PiTs are necessary for Pi signaling. Moreover, the ERK1/2 phosphorylation could be rescued by overexpressing Pi transport-deficient PiT mutants. Using cross-linking and bioluminescence resonance energy transfer approaches, we found that PiT1 and PiT2 form high-abundance homodimers and Pi-regulated low-abundance heterodimers. Interestingly, in the absence of sodium-dependent Pi transport activity, the PiT1-PiT2 heterodimerization was still regulated by extracellular Pi levels. Of note, when two putative Pi-binding residues, Ser-128 (in PiT1) and Ser-113 (in PiT2), were substituted with alanine, the PiT1-PiT2 heterodimerization was no longer regulated by extracellular Pi These observations suggested that Pi binding rather than Pi uptake may be the key factor in mediating Pi signaling through the PiT proteins. Taken together, these results demonstrate that Pi-regulated PiT1-PiT2 heterodimerization mediates Pi sensing independently of Pi uptake.