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

• Published in: PloS one Vol. 10; no. 11; p. e0143407
• Main Authors: Vanlandewijck, Michael, Lebouvier, Thibaud, Andaloussi Mäe, Maarja, Nahar, Khayrun, Hornemann, Simone, Kenkel, David, Cunha, Sara I., Lennartsson, Johan, Boss, Andreas, Heldin, Carl-Henrik, Keller, Annika, Betsholtz, Christer
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 23.11.2015; Public Library of Science (PLoS)
• Subjects: Analysis; Animals; Binding; Blood platelets; Blood-brain barrier; Blood-Brain Barrier - pathology; Brain Diseases - genetics; Brain research; Calcification; Calcification (Physiology); Calcinosis - genetics; Cancer; Care and treatment; Cell Surface Extensions - drug effects; Cell Surface Extensions - metabolism; Chemical bonds; Complications and side effects; Culture Media, Conditioned - pharmacology; Disease; Gene mutation; Genetic aspects; Genetic Predisposition to Disease; Genetics; Germ-Line Mutation - genetics; Growth factors; Haploinsufficiency; Haploinsufficiency - genetics; HEK293 Cells; Humans; Immunology; Influence; Kinases; Kinetics; Laboratories; Ligands; Medical research; Mice; Mutation; Nervous system diseases; Pathogenesis; Pathology; Pericytes - drug effects; Pericytes - metabolism; Pericytes - pathology; Phosphorylation - drug effects; Platelet-derived growth factor; Protein biosynthesis; Protein synthesis; Proteins; Proto-Oncogene Proteins c-sis - genetics; Receptor, Platelet-Derived Growth Factor beta - genetics; Rodents; Signal Transduction - drug effects; Signaling; Smooth muscle; Transcription; Transfection; Wound Healing - drug effects; Switzerland; Sweden
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0143407

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 Pdgfrbredeye/redeye mice, which show a 90% reduction of PDGFRβ protein levels. In contrast, Pdgfbret/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 Pdgfbret/ret mice. Surprisingly and contrary to our hypothesis, we found that the calcification-prone brain regions in Pdgfbret/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.