title: 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 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.
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjölds väg 20, Uppsala 75185, Sweden.|Integrated Cardio Metabolic Centre (ICMC), Karolinska Institute, Novum, SE-141 57 Huddinge, Stockholm, Sweden.
authors.forename: Michael
authors.initials: M
authors.lastname: Vanlandewijck
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjölds väg 20, Uppsala 75185, Sweden.
authors.forename: Thibaud
authors.initials: T
authors.lastname: Lebouvier
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjölds väg 20, Uppsala 75185, Sweden.
authors.forename: Maarja
authors.initials: M
authors.lastname: Andaloussi Mäe
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjölds väg 20, Uppsala 75185, Sweden.
authors.forename: Khayrun
authors.initials: K
authors.lastname: Nahar
authors.affiliation: Institute of Neuropathology, University Hospital Zürich, Zürich University, CH-8091 Zürich, Switzerland.
authors.forename: Simone
authors.initials: S
authors.lastname: Hornemann
authors.affiliation: Institute of Diagnostic and Interventional Radiology, University Hospital Zürich, Zürich University, CH-8091 Zürich, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Kenkel
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjölds väg 20, Uppsala 75185, Sweden.|Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Box 595, SE-75124, Uppsala, Sweden.
authors.forename: Sara I
authors.initials: SI
authors.lastname: Cunha
authors.affiliation: Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Box 595, SE-75124, Uppsala, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Lennartsson
authors.affiliation: Institute of Diagnostic and Interventional Radiology, University Hospital Zürich, Zürich University, CH-8091 Zürich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Boss
authors.affiliation: Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Box 595, SE-75124, Uppsala, Sweden.
authors.forename: Carl-Henrik
authors.initials: CH
authors.lastname: Heldin
authors.affiliation: Division of Neurosurgery, University Hospital Zürich, Zürich University, CH-8091 Zürich, Switzerland.
authors.forename: Annika
authors.initials: A
authors.lastname: Keller
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjölds väg 20, Uppsala 75185, Sweden.
authors.forename: Christer
authors.initials: C
authors.lastname: Betsholtz
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000473:pathology; D001927:Brain Diseases / Q000235:genetics*; D002114:Calcinosis / Q000235:genetics*; D022081:Cell Surface Extensions / Q000187:drug effects / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D020022:Genetic Predisposition to Disease; D018095:Germ-Line Mutation / Q000235:genetics*; D057809:HEK293 Cells; D057895:Haploinsufficiency / Q000235:genetics; D006801:Humans; D051379:Mice; D020286:Pericytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D020574:Proto-Oncogene Proteins c-sis / Q000235:genetics*; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014945:Wound Healing / Q000187:drug effects
chemical_list: D017077:Culture Media, Conditioned; D020574:Proto-Oncogene Proteins c-sis; D020797:Receptor, Platelet-Derived Growth Factor beta