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Solute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP-binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 ( IP6K1-2 ) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC., Competing Interests: Conflict of interest—J.-L. B. and M. S. are inventors on patents describing the use of RBD ligands; M. S. is the co-founder of METAFORA-biosystems, a start-up company that focuses on metabolite transporters under physiological and pathological conditions., (© 2020 López-Sánchez et al.)

Primary familial brain calcification (PFBC) is a rare neurological disease characterized by deposits of calcium phosphate in the basal ganglia and other regions of the brain. Pathogenic variants in the XPR1/SLC53A1 gene, which encodes the only known inorganic phosphate exporter, cause an autosomal dominant form of PFBC. These variants are typically located in the SPX N-terminal domain of the protein. Here, we characterize three XPR1 variants outside of SPX in three PFBC patients with an apparently sporadic presentation: c.1375C > T p.(R459C), c.1855A > G p.(N619D) and c.1886T > G p.(I629S), with the latter identified as the first XPR1/SLC53A1 de novo mutation to occur in a PFBC proband. When tested in an in vitro physiological complementation assay, the three XPR1 variants were impaired in phosphate export function, although they were normally expressed at the cell surface and could serve as functional receptors for retrovirus entry. Moreover, peripheral blood cells from the p.N619D patient could be assayed ex vivo and displayed significantly impaired phosphate export. Our results establish for the first time the clinical and molecular characteristics of XPR1 variants located outside the SPX domain and assert a direct link between these variants, deficient phosphate export, and PFBC. Moreover, we unveiled new structural features in XPR1 C-terminal domain that play a role in phosphate export and disease.

Mutations in XPR1, a gene encoding an inorganic phosphate exporter, have recently been identified in patients with primary familial brain calcification (PFBC). Using Sanger sequencing, we screened XPR1 in 18 unrelated patients with PFBC and no SLC20A2, PDGFB, or PDGFRB mutation. XPR1 variants were tested in an in vitro physiological complementation assay and patient blood cells were assessed ex vivo for phosphate export. We identified a novel c.260T > C, p.(Leu87Pro) XPR1 variant in a 41-year-old man complaining of micrographia and dysarthria and demonstrating mild parkinsonism, cerebellar ataxia and executive dysfunction. Brain (123)I-Ioflupane scintigraphy showed marked dopaminergic neuron loss. Peripheral blood cells from the patient exhibited decreased phosphate export. XPR1 in which we introduced the mutation was not detectable at the cell surface and did not lead to phosphate export. These results confirm that loss of XPR1-mediated phosphate export function causes PFBC, occurring in less than 8 % of cases negative for the other genes, and may be responsible for parkinsonism.

Primary familial brain calcification (PFBC) is a neurological disease characterized by calcium phosphate deposits in the basal ganglia and other brain regions and has thus far been associated with SLC20A2, PDGFB or PDGFRB mutations. We identified in multiple families with PFBC mutations in XPR1, a gene encoding a retroviral receptor with phosphate export function. These mutations alter phosphate export, implicating XPR1 and phosphate homeostasis in PFBC.