Functional Characterization of Germline Mutations in PDGFB and PDGFRB in Primary Familial Brain Calcification

Abstract

Primary Familial Brain Calcification (PFBC), a neurodegenerative disease characterized by progressive pericapillary calcifications, has recently been linked to heterozygous mutations in PDGFB and PDGFRB genes. Here, we functionally analyzed several of these mutations in vitro. All six analyzed PDGFB mutations led to complete loss of PDGF-B function either through abolished protein synthesis or through defective binding and/or stimulation of PDGF-Rβ. The three analyzed PDGFRB mutations had more diverse consequences. Whereas PDGF-Rβ autophosphorylation was almost totally abolished in the PDGFRB L658P mutation, the two sporadic PDGFRB mutations R987W and E1071V caused reductions in protein levels and specific changes in the intensity and kinetics of PLCγ activation, respectively. Since at least some of the PDGFB mutations were predicted to act through haploinsufficiency, we explored the consequences of reduced Pdgfb or Pdgfrb transcript and protein levels in mice. Heterozygous Pdgfb or Pdgfrb knockouts, as well as double Pdgfb +/-;Pdgfrb +/- mice did not develop brain calcification, nor did Pdgfrb redeye/redeye mice, which show a 90% reduction of PDGFRβ protein levels. In contrast, Pdgfb ret/ret mice, which have altered tissue distribution of PDGF-B protein due to loss of a proteoglycan binding motif, developed brain calcifications. We also determined pericyte coverage in calcification-prone and non-calcification-prone brain regions in Pdgfb ret/ret mice. Surprisingly and contrary to our hypothesis, we found that the calcification-prone brain regions in Pdgfb ret/ret mice model had a higher pericyte coverage and a more intact blood-brain barrier (BBB) compared to non-calcification-prone brain regions. While our findings provide clear evidence that loss-of-function mutations in PDGFB or PDGFRB cause PFBC, they also demonstrate species differences in the threshold levels of PDGF-B/PDGF-Rβ signaling that protect against small-vessel calcification in the brain. They further implicate region-specific susceptibility factor(s) in PFBC pathogenesis that are distinct from pericyte and BBB deficiency.

Regional early and progressive loss of brain pericytes but not vascular smooth muscle cells in adult mice with disrupted platelet-derived growth factor receptor-β signaling

“…These findings are consistent with a previous report showing that mice with reduced PDGF-BB bioavailability also have regional differences in pericyte coverage with thalamus showing less severe loss, but more severe deposition of calcium, than the cortex and hippocampus showing more severe pericyte loss, but no deposition of calcium [30]. The reasons for these regional differences in vascular phenotype and brain pathology remain currently unclear, but they likely might reflect regional differences in signaling within the respective regional brain neurovascular units, which should be explored by future studies.…”

Identification of partial SLC20A2 deletions in primary brain calcification using whole-exome sequencing

“…Whether the BBB alteration is the direct cause of microvascular calcification or there is other links with inorganic phosphate metabolism remains to be determined. 8 In our French series of PBC patients, 450% of probands or sporadic cases do not exhibit any causative variant in one of these genes after Sanger sequencing (unpublished data).…”

Involvement of platelet‐derived growth factor ligands and receptors in tumorigenesis

“…Knockout of the genes for PDGFB or PDGFR β gave similar phenotypes, including a vascular defect with a reduction in pericytes and a weakening of the vessel wall because of defect smooth muscle cell development, leading to bleedings . The importance of PDGFRβ for proper vascular function is also illustrated by the finding that loss‐of‐function mutations in PDGFB or PDGFR β in humans are associated by brain calcification, probably due to deficiencies in pericytes and the blood‐brain barrier . Moreover, the kidney function was perturbed after knockout of PDGFB or PDGFR β because of defect development of mesangial cells.…”