Abstract：
Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson\'s disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.
摘要：
已知较低的多巴胺能（DA）神经元群体会增加对PD的易感性，我们早期的研究表明，携带LRRK2-I1371V突变的PD患者iPSCs中DA神经元的产量较低。虽然SHH在底板细胞（FPC）的DA-神经发生中的作用是已知的，LRRK2突变对影响DA-神经元产量的FPC的SHH反应性的影响尚未研究。我们研究了源自LRRK2-I1371VPD患者来源的iPSCs的SHH反应性，涉及SHH受体Patched1（Ptch1）和Smoothened（Smo）的表达，与核Gli1表达结合，细胞内Ca2+上升，和SHH诱导后的胞浆cAMP水平。此外,我们通过评估SH-SY5Y细胞和过表达LRRK2-I1371V的健康对照（HC）-FPC以及FPC中的膜流动性和Rab8A＆Rab10磷酸化，研究了与LRRRK2-I1371V功能获得的机制联系。虽然Ptch1和Smo的总表达具有可比性，LRRK2-I1371VFPCs细胞表面受体表达显著低于HC,下游转录因子Gli1的核表达明显较低。用LRRK2I1371V转染的HC-FPC表现出类似地降低的Ptch1和Smo的细胞表面表达。与SHH刺激后的HC相比，LRRK2-I1371VFPC中的细胞内Ca2响应显着降低，相应的cAMP水平升高。LRRK2-I1371V突变体FPCs和LRRK2-I1371V转染的SH-SY5Y和HC-FPCs均进一步表现出磷酸LRRK2（pLRRK2）serine1292和serine935的更高的自磷酸化，以及Rab8A和Rab10的底物磷酸化。同时增加膜的流动性，伴随着膜胆固醇的减少，在它们中也观察到脂筏标记Caveolin1的较低表达。这些发现表明，LRRK2-I1371VPDFPC的SHH反应性受损确实会导致个体发育过程中DA神经元的产量降低。SHH受体的细胞表面表达减少受到膜流动性改变的影响，这是由于Rab8A的底物磷酸化增加和pRab10引起的膜蛋白运输减少，这两个结果均为LRRK2-I1371V突变。
