Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences, School of Medicine, "Federico II" University of Naples, Naples, Italy.
Division of Pharmacology, Department of Science and Technology-DST, University of Sannio, Benevento, Italy.
FASEB J. 2021 Feb;35(2):e21277. doi: 10.1096/fj.202001886R.
A robust activity of the lysosomal Ca channel TRPML1 is sufficient to correct cellular defects in neurodegeneration. Importantly, lysosomes are refilled by the endoplasmic reticulum (ER). However, it is unclear how TRPML1 function could be modulated by the ER. Here, we deal with this issue in rat primary cortical neurons exposed to different oxygen conditions affecting neuronal survival. Under normoxic conditions, TRPML1: (1) showed a wide distribution within soma and along neuronal processes; (2) was stimulated by the synthetic agonist ML-SA1 and the analog of its endogenous modulator, PI(3,5)P diC8; (3) its knockdown by siRNA strategy produced an ER Ca accumulation; (4) co-localized and co-immunoprecipitated with the ER-located Ca sensor stromal interacting molecule 1 (STIM1). In cortical neurons lacking STIM1, ML-SA1 and PI(3,5)P diC8 failed to induce Ca release and, more deeply, they induced a negligible Ca passage through the channel in neurons transfected with the genetically encoded Ca indicator GCaMP3-ML1. Moreover, TRPML1/STIM1 interplay changed at low-oxygen conditions: both proteins were downregulated during the ischemic preconditioning (IPC) while during IPC followed by 1 hour of normoxia, at which STIM1 is upregulated, TRPML1 protein was reduced. However, during oxygen and glucose deprivation (OGD) followed by reoxygenation, TRPML1 and STIM1 proteins peaked at 8 hours of reoxygenation, when the proteins were co-immunoprecipitated and reactive oxygen species (ROS) hyperproduction was measured in cortical neurons. This may lead to a persistent TRPML1 Ca release and lysosomal Ca loss. Collectively, we showed a new modulation exerted by STIM1 on TRPML1 activity that may differently intervene during hypoxia to regulate organellar Ca homeostasis.
溶酶体钙通道 TRPML1 的活性增强足以纠正神经退行性变中的细胞缺陷。重要的是，溶酶体是由内质网（ER）填充的。然而，TRPML1 的功能如何被 ER 调节尚不清楚。在这里，我们在暴露于不同氧条件下影响神经元存活的大鼠原代皮质神经元中处理这个问题。在正常氧条件下，TRPML1：（1）在体和神经元突起中表现出广泛的分布；（2）被合成激动剂 ML-SA1 和其内源性调节剂 PI(3,5)P diC8 的类似物刺激；（3）通过 siRNA 策略敲低会导致 ER Ca 积累；（4）与内质网定位的 Ca 传感器基质相互作用分子 1（STIM1）共定位和共免疫沉淀。在缺乏 STIM1 的皮质神经元中，ML-SA1 和 PI(3,5)P diC8 未能诱导 Ca 释放，更深入地说，它们在转染了基因编码 Ca 指示剂 GCaMP3-ML1 的神经元中诱导了微不足道的 Ca 通过通道。此外，TRPML1/STIM1 相互作用在低氧条件下发生变化：两种蛋白在缺血预处理（IPC）期间下调，而在 IPC 后进行 1 小时正常氧时，STIM1 上调，TRPML1 蛋白减少。然而，在氧葡萄糖剥夺（OGD）后再复氧时，TRPML1 和 STIM1 蛋白在再复氧 8 小时时达到峰值，此时在皮质神经元中测量到蛋白质共免疫沉淀和活性氧（ROS）过度产生。这可能导致持久的 TRPML1 Ca 释放和溶酶体 Ca 丢失。总的来说，我们展示了 STIM1 对 TRPML1 活性的新调节，该调节可能在缺氧期间以不同的方式干预以调节细胞器 Ca 稳态。
