Functional Characterization of Germline Mutations in PDGFB and PDGFRB in Primary Familial Brain Calcification.
Functional Characterization of Germline Mutations in PDGFB and PDGFRB in Primary Familial Brain Calcification.
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DOI：
10.1371/journal.pone.0143407
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Betsholtz C
中科院分区：
文献类型：
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作者：
Vanlandewijck M;Lebouvier T;Andaloussi Mäe M;Nahar K;Hornemann S;Kenkel D;Cunha SI;Lennartsson J;Boss A;Heldin CH;Keller A;Betsholtz C
原发性家族性脑钙化（PFBC）是一种以进行性毛细血管周围钙化为特征的神经退行性疾病，最近被认为与PDGFFB和PDGFRB基因的杂合突变有关。在这里，我们在体外功能分析了这些突变中的几个。分析的所有6种PDGFB突变均导致PDGF-B功能完全丧失，这要么是由于蛋白质合成消失，要么是由于PDGF-Rβ结合和/或刺激缺陷。分析的三种PDGFRB突变具有更多样化的后果。尽管PDGFRB L 658 P突变几乎完全消除了PDGF-Rβ自身磷酸化，但两种散发性PDGFRB突变R987 W和E1071 V分别导致蛋白水平降低以及PLCγ激活强度和动力学的特定变化。由于预测至少一些PDGFB突变通过单倍不足起作用，我们探索了小鼠中Pdgfb或Pdgfrb转录物和蛋白质水平降低的后果。杂合Pdgfb或Pdgfrb敲除以及双Pdgfb +/-;Pdgfrb +/-小鼠未发生脑钙化，Pdgfrb红眼/红眼小鼠也未发生脑钙化，其显示PDGFRβ蛋白水平降低90%。相比之下，Pdgfb ret/ret小鼠，由于蛋白聚糖结合基序的丢失而改变了PDGF-B蛋白的组织分布，发生了脑钙化。我们还确定了Pdgfb ret/ret小鼠中钙化倾向和非钙化倾向脑区的周细胞覆盖率。令人惊讶的是，与我们的假设相反，我们发现与非钙化倾向脑区相比，Pdgfb ret/ret小鼠模型中的钙化倾向脑区具有更高的周细胞覆盖率和更完整的血脑屏障（BBB）。虽然我们的研究结果提供了明确的证据，证明PDGFFB或PDGFRB的功能缺失突变导致PFBC，但它们也证明了PDGF-B/PDGF-Rβ信号传导的阈值水平存在物种差异，这些信号传导可以防止大脑中的小血管钙化。他们进一步暗示PFBC发病机制中的区域特异性易感因子，其不同于周细胞和BBB缺陷。
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.