Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04

    • In the next set of experiments we

    2021-09-17

    In the next set of experiments, we sought to determine the source of lysosomal p-Cresyl sulfate inhibition by oxidative stress. Lysosomal exocytosis probably involves multiple steps including vesicle positioning, delivery, docking and fusion. The previous evidence and data in Fig. 1C,D show that TRPML1 is critical for this process. However, whether it controls all faucets of lysosomal exocytosis is unclear. Indeed, we have recently shown that Ca2+ influx from the extracellular medium has an important role in lysosomal exocytosis [26] Accordingly, the removal of Ca2+ from the extracellular buffer inhibited the basal rate of lysosomal exocytosis (Fig. 3A). When measured in Ca2+-free buffer, basal lysosomal exocytosis averaged 34% or its levels in regular buffer containing 1mM Ca2+ (3 experiments, 3 individual samples in each experiment, p<0.06). In these experiments, Ca2+ was removed at the onset of the experiment. In our hands, ML-SA1 did not induce ß-hex exocytosis in Ca2+-free buffer (Fig. 3B). The removal of extracellular Ca2+ suppressed the lysosomal exocytosis activation by tBHP as well (Fig. 3C(i). Under these conditions, ionomycin induced some ß-hex exocytosis, although its magnitudes were significantly suppressed compared with the ionomycin effect in normal Ca2+ buffer (Fig. 3C(ii) The nonspecific plasma-membrane Ca2+ channel blocker La3+ (100μM) also suppressed the ß-hex exocytosis activation by tBHP (Fig. 3D). Increasing the extracellular Ca2+ levels to 2mM raised the basal ß-hex exocytosis rates by about 40% (Fig. 4A; 3 experiments, 3 individual samples in each experiment, p<0.06). The magnitude of ß-hex signal under the high-Ca2+ conditions was comparable with that induced by ionomycin or tBHP under normal conditions. Under the high-Ca2+ conditions, the tBHP concentrations that activated ß-hex exocytosis in normal Ca2+ produced little or no increase in ß-hex exocytosis (Fig. 4B; 3 experiments, 3 individual samples in each experiment, p<0.06). However, the tBHP concentrations that inhibited ß-hex exocytosis in normal Ca2+, produced marked decrease in ß-hex exocytosis. Based on this, we propose that extracellular Ca2+ influx is important for the lysosomal exocytosis. While the exocytosis activation by low levels of ROS depends on TRPML1, lysosomal exocytosis inhibition by oxidative stress involves inhibiting of plasma membrane Ca2+ influx.
    Discussion Oxidative stress drives pathological processes in many diseases including ischemic stroke, reperfusion injury, heart failure, Alzheimer’s disease, ALS, genetic and toxic forms of Parkinson's disease, autism and Asperger’s syndrome [20], [53], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65]. A better understanding of the mechanisms of oxidative damage and the recovery from oxidative stress is crucial for better treatments for these diseases. An important component of the cellular defense against oxidative stress is the recovery from oxidative damage. The removal of damaged organelles from the cytoplasm is a function of the autophagic/lysosomal pathway [19], [24]. Beyond the degradation of damaged organelles, the expulsion of damaged cellular components via exocytosis has recently emerged as an important component of the cellular clearance mechanism [21], [31], [66], [67], [68], [69], [70], [71]. Therefore, the lysosomal exocytosis pathway is a potentially important pharmacological target for modulating the effects of oxidative stress. The recent evidence of TRPML1 activation by ROS [41] raised the possibility that TRPML1-dependent processes are regulated by ROS as well. The initial evidence for the role of TRPML1 in lysosomal exocytosis comes from resting unstimulated human skin fibroblasts. TRPML-deficient fibroblasts from mucolipidosis type IV patients were found to have lower exocytosis rates than age-matched heterozygous controls [38]. TRPML1 has been implicated in the exocytosis of Cu-filled lysosomes [28]. Finally, inhibition of lysosomal exocytosis and the resulting membrane deficits were used to explain phagocytosis suppression in TRPML1-deficient cells [32]. Our data on the stimulation of the lysosomal exocytosis by low and moderate levels of the pro-oxidant tBHP in a manner requiring TRPML1 agrees with this evidence.