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

Abstract

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.

Astrocyte–microglial association and matrix composition are common events in the natural history of primary familial brain calcification

“…Previously reported anatomical locations of the calcifications and their age-dependent growth in size and number (14,15,17) were largely confirmed in our present study. However, unlike a previous study (40), we did not detect any calcifications in Slc20a2 -/+ heterozygous mice.…”

“…Moreover, the close interaction with astrocytes, microglia and vasculature to calcifications was consistently observed. Astrocyte-microglial reactivity around calcifications also occurs in PFBC patients (28), and the relation between the nodules and astrocytes has been described in mice (15,46).…”

Ossified blood vessels in primary familial brain calcification elicit a neurotoxic astrocyte response

“…This interindividual variation of calcification load has been reported in human PFBC cases, but the clinical significance is not clear (Nicolas et al , 2013 b ). Interindividual variation in calcification load was also recently reported in Slc20a2 -deficent animals; however, no quantification of calcification load was performed (Jensen et al , 2018). This and our data (Fig.…”

“…Additional factors other than a gene mutation contribute to the initiation and/or growth of vessel-associated calcifications. Thus, mouse models of PFBC show similarities in disease pathology, such as variation in calcification load and the time-dependent growth of calcifications (Keller et al , 2013; Jensen et al , 2018).…”

Haploinsufficiency of the Primary Familial Brain Calcification Gene SLC20A2 Mediated by Disruption of a Regulatory Element

“…Loss-offunction variants result in perivascular and cerebrospinal fluid accumulation of Pi that may contribute to vascular mural calcifications in the brain. 20,21 Pathogenic SLC20A2 variants typically introduce premature stop codons (nonsense, canonical splice site single nucleotide variants and frameshift insertions or deletions), leading to haploinsufficiency through nonsensemediated mRNA decay. Missense variants have also been identified, with a loss of Pi import function consequences on PiT2.…”