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

Michael Vanlandewijck,David Kenkel,Maarja Andaloussi Mäe,Khayrun Nahar,Annika Keller,Simone Hornemann,Thibaud Lebouvier,Johan Lennartsson,Sara I Cunha,Carl-Henrik Heldin,Andreas Boss,Christer Betsholtz

doi:10.1371/journal.pone.0143407

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.

Highlights

The role of the platelet-derived growth factors (PDGFs) and their tyrosine kinase receptors (PDGFRs) has been extensively studied in the developing organism [1,2]
The simultaneous discovery that mutations in PDGFB and PDGFRB can cause primary familial brain calcification (PFBC) demonstrates new functions of PDGF-Rβ signaling that could not have been predicted based on existing knowledge about PDGF-B/ PDGF-Rβ functions, demonstrating that there is still much to be learnt about PDGF biology [14,15]
To study the functional consequences of PDGFB mutations described in PFBC (Fig 1A and 1B), cDNA sequences carrying the different mutations identified in six PFBC families were introduced into the pcDNA3.1 vector, transfected into human embryonic kidney (HEK) 293 cells, and after G-418 selection, stably expressing clones were generated

Summary

Introduction

The role of the platelet-derived growth factors (PDGFs) and their tyrosine kinase receptors (PDGFRs) has been extensively studied in the developing organism [1,2]. Homozygous knockouts of either receptor or ligand display widespread vascular alterations leading to perinatal death [1,5,6]. Regarding PDGF-B, increased PDGF-Rβ signaling has been demonstrated in diseases like cancer, vascular inflammation, and tissue fibrosis [1]. Focal uncontrolled PDGF-Rβ signaling due to somatic genetic aberrations has severe consequences in humans, playing a causative role in diseases such as dermatofibrosarcoma protuberans [9], gastric cancer and leukemia [10]. The simultaneous discovery that mutations in PDGFB and PDGFRB can cause primary familial brain calcification (PFBC) demonstrates new functions of PDGF-Rβ signaling that could not have been predicted based on existing knowledge about PDGF-B/ PDGF-Rβ functions, demonstrating that there is still much to be learnt about PDGF biology [14,15]

Methods

Results

Conclusion