Lysosomal TRPML1 triggers global Ca2+ signals and nitric oxide release in human cerebrovascular endothelial cells

Lysosomal Ca2+ signaling is emerging as a crucial regulator of endothelial Ca2+ dynamics. Ca2+ release from the acidic vesicles in response to extracellular stimulation is usually promoted via Two Pore Channels (TPCs) and is amplified by endoplasmic reticulum (ER)-embedded inositol-1,3,4-trisphosphate (InsP3) receptors and ryanodine receptors. Emerging evidence suggests that sub-cellular Ca2+ signals in vascular endothelial cells can also be generated by the Transient Receptor Potential Mucolipin 1 channel (TRPML1) channel, which controls vesicle trafficking, autophagy and gene expression. Herein, we adopted a multidisciplinary approach, including live cell imaging, pharmacological manipulation, and gene targeting, revealing that TRPML1 protein is expressed and triggers global Ca2+ signals in the human brain microvascular endothelial cell line, hCMEC/D3. The direct stimulation of TRPML1 with both the synthetic agonist, ML-SA1, and the endogenous ligand phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) induced a significant increase in [Ca2+]i, that was reduced by pharmacological blockade and genetic silencing of TRPML1. In addition, TRPML1-mediated lysosomal Ca2+ release was sustained both by lysosomal Ca2+ release and ER Ca2+- release through inositol-1,4,5-trisphophate receptors and store-operated Ca2+ entry. Notably, interfering with TRPML1-mediated lysosomal Ca2+ mobilization led to a decrease in the free ER Ca2+ concentration. Imaging of DAF-FM fluorescence revealed that TRPML1 stimulation could also induce a significant Ca2+-dependent increase in nitric oxide concentration. Finally, the pharmacological and genetic blockade of TRPML1 impaired ATP-induced intracellular Ca2+ release and NO production. These findings, therefore, shed novel light on the mechanisms whereby the lysosomal Ca2+ store can shape endothelial Ca2+ signaling and Ca2+-dependent functions in vascular endothelial cells.

The hCMEC/D3 cell line is the most widely used model of human cerebrovascular endothelial cells (Bintig et al., 2012;Weksler et al., 2013;Helms et al., 2016;Bader et al., 2017;Luo et al., 2019) and provides a predictive model to investigate how endothelial Ca 2+ signals are shaped and regulate the Ca 2+ -dependent production of vasorelaxing mediators, such as nitric oxide (NO), at the human blood-brain barrier (Negri et al., 2021c;Soda et al., 2023).Several studies have shown that neurotransmitters and neuromodulators, such as acetylcholine, glutamate, γ-aminobutyric (GABA), and histamine, evoke NO release from hCMEC/D3 cells through an increase in [Ca 2+ ] i that is patterned by InsP 3 -induced ER Ca 2+ release, NAADP-induces lysosomal Ca 2+ mobilization, and SOCE (Zuccolo et al., 2019b;Berra-Romani et al., 2020;Negri et al., 2020;Negri et al., 2022).Herein, we demonstrate that the lysosomal TRPML1 is also expressed in hCMEC/D3 cells.We also provide the first evidence that TRPML1-mediated endothelial Ca 2+ signals are supported by ER Ca 2+ release through InsP 3 Rs and by SOCE.We further show that the genetic and pharmacological blockade of TRPML1 reduced the ER Ca 2+ load.Moreover, TRPML1mediated global Ca 2+ signals lead to robust NO release that may regulate a variety of processes, including an increase in local cerebral blood flow (CBF), at the neurovascular unit.Finally, we provide the first evidence that TRPML1 support agonist-induced intracellular Ca 2+ release and NO production in vascular endothelial cells.These findings hint at TRPML1 as an additional component of the endothelial Ca 2+ toolkit that may be recruited by extracellular autacoids to regulate the endothelial Ca 2+ -dependent functions.

[Ca 2+ ] i and NO imaging
Cells were on glass gelatin-coated coverslips at a density of 5,000 cells/cm 2 for 24-48 h (Mussano et al., 2020).Cells were next loaded the selective Ca 2+ -fluorophore Fura-2 acetoxymethyl ester (2 µM Fura-2/AM; Thermo Fisher Scientific, Waltham, MA, United States) in PSS for 30 min at 37 °C and 5% CO 2 , as described in (Negri et al., 2022;Berra-Romani et al., 2023).After washing in PSS, the coverslip was fixed to the bottom of a Petri dish and the cells were observed by an upright epifluorescence Axiolab microscope (Carl Zeiss, Oberkochen, Germany), usually equipped with a Zeiss ×40 Achroplan objective (water-immersion, 2.0 mm working distance, 0.9 numerical aperture).The cells were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm.A first neutral density filter (1 or 0.3 optical density) reduced the overall intensity of the excitation light, and a second neutral density filter (optical density = 0.3) was coupled to the 380 nm filter to approach the intensity of the 340 nm light.A round diaphragm was used to increase the contrast.The excitation filters were mounted on a filter wheel (Lambda 10, Sutter Instrument, Novato, CA, United States).Custom software, working in the LINUX environment, was used to drive the camera (Extended-ISIS Camera, Photonic Science, Millham, United Kingdom) and the filter wheel, and to measure and plot online the fluorescence from 10 up to 40 rectangular "regions of interest" (ROIs).Each ROI was identified by a number.Since cell borders were not clearly identifiable, a ROI may not include the whole cell or may include part of an adjacent cell.Adjacent ROIs never superimposed.[Ca 2+ ] i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (F 340 /F 380 ).An increase in [Ca 2+ ] i causes an increase in the ratio (Negri et al., 2022;Berra-Romani et al., 2023).Ratio measurements were performed and plotted online every 3 s.The experiments were performed at room temperature (22 °C).The Fe 2+ quench experiments were performed as described in (Kilpatrick et al., 2016) by replacing CaCl 2 with an equimolar amount of FeCl 2 and measuring the quenching of Fura-2 fluorescence at 360 nm, i.e., the isosbestic point for Fura-2.
To evaluate NO release, hCMEC/D3 cells were loaded with 4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate (1 μM, DAF-FM DA) for 60 min in PSS at 22 °C, as illustrated in (Negri et al., 2020;Berra-Romani et al., 2023).DAF-FM fluorescence was measured by using the same imaging setup described above for Ca 2+ measurements but with a different filter set, i.e., excitation at 480 nm and emission at 535 nm wavelength (emission intensity denoted as NO i (F 535 /F 0 )).The changes in DAF-FM fluorescence evoked by extracellular stimulation were recorded and plotted online every 5 s.Measurements of NO were performed at 22 °C.The cellular production of NO was reported as relative fluorescence (F/F 0 ) of DAF-FM DA, where F is the fluorescence intensity obtained during recordings and F 0 is the basal fluorescence intensity.

Immunoblotting
Cells were seeded on a 6-well culture plate, grown to a confluency of 70%-80%, and then silenced or not, according to the protocol described below.For cell lysis, plates were kept on ice and cells were washed twice in ice-cold PBS, scraped with RIPA buffer (Pierce ® RIPA Buffer, Thermo Fisher Scientific, Waltham, MA, United States) and protease inhibitor cocktail (Halt ™ Protease Inhibitor Cocktail, 1:100, Thermo Fisher Scientific, Waltham, MA, United States).Lysates were vortexed and kept on ice for 10 min, then centrifuged at 4 °C for 15 min at 13,000× g.Protein concentrations were determined by using a Bicinchoninic Acid (BCA) kit (Merck KGaA, Darmstadt, Germany) following the manufacturer's instructions.20 μg of lysates were resuspended in SDS loading buffer, heated 30 min at 37 C and then separated on 4%-15% Mini-PROTEAN TGX Precast Protein Gels Bio-Rad (Bio-Rad, Hercules, CA; United States).Then, the proteins were transferred out of the gel on to the PVDF Membrane (Trans-Blot Turbo Transfer Pack, Bio-Rad, Hercules, CA; United States) with the Trans-Blot Turbo Transfer apparatus (BioRad, Hercules, CA; United States).Membranes were blocked by incubation for 1 h at room temperature in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.6) 5% BSA solution and then incubated in agitation overnight at 4 °C with rabbit anti-TRPML1 (#ACC-081, 1:200 in TBST 5% BSA 0.02% sodium azide; Alomone, Jerusalem, Israel) and anti-β-Actin-Peroxidase (#A385416, 1:1,000 in TBST 5% BSA 0.02% sodium azide; Merck KGaA, Darmstadt, Germany) antibodies.Membranes were then washed with TBST and incubated with the appropriate HPR-conjugated antibody (antirabbit HRP #31460, 1:2,000 in TBST 5% BSA; Thermo Fisher Scientific, Waltham, MA, United States).Differences in protein expression was evaluated by using Fiji (ImageJ software).

Gene silencing
Genetic deletion of MCOLN1, which encodes for TRPML1, was carried by using a similar approach to that described in (Mussano et al., 2020;Negri et al., 2020).Cells were transiently transfected with the esiRNA targeting TRPML1 (EHU062561, MISSION ® esiRNA, 100 nM final concentration) purchased from Merck (Merck KGaA, Darmstadt, Germany) by using the Lipofectamine ™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, United States) protocol in Opti-MEM ™ I Reduced Serum

Medium
(Thermo Fisher Scientific, Waltham, MA, United States), according to manufacturer's instructions.4 h after transfection, the esiRNA-Lipofectamin complex was eliminated and fresh culture media containing 5% FBS was added to the cells.Cells were then kept in incubator at 37 °C and 5% CO 2 and allowed to grow according to the protocol to be used.The effectiveness of silencing was determined by immunoblotting and the silenced hCMEC/ D3 cells were used 48 h after transfection.The Trypan blue exclusion assay confirmed that the genetic silencing of TRPML1 did not affect hCMEC/D3 cell viability (Supplementary Figure S1).

Statistics
All the data have been obtained from at least three independent experiments on hCMEC/D3 cells.The peak amplitude of agonistevoked Ca 2+ signals was measured as the difference between the ratio (F 340 /F 380 ) at the Ca 2+ peak and the mean ratio of 30 s baseline before the peak.
Data were analyzed with GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA, United States).Preliminary Shapiro-Wilk test was performed to check the normal distributions of each dataset: accordingly, statistical analysis was performed by using either the non-parametric tests (Mann-Whitney test or Kruskal-Wallis test) or parametric tests (Student's t-test or oneway ANOVA test).A p-value of <0.05 was considered significant.

Lysosomal TRPML1 is expressed in hCMEC/D3 cells
The synthetic agonist, ML-SAI, has been widely used to evaluate the functional expression of functional TRPML1 channels in a variety of cell lines (Kilpatrick et al., 2016;Thakore et al., 2020;Tedeschi et al., 2021;Boretto et al., 2023;Scorza et al., 2023).In hCMEC/D3 cells loaded with the ratiometric Ca 2+ -sensitive fluorophore, Fura-2, ML-SAI induced a dose-dependent increase in [Ca 2+ ] i that was evident at concentrations ≥10 µM (Figure 1A).At 10-50 μM, ML-SA1 caused a slow but transient increase in [Ca 2+ ] i that started with some delay after the application of the agonist (Figure 1A).Higher concentrations of ML-SA1 (100 μM and 200 µM) induced a biphasic Ca 2+ signal following the slow increase in [Ca 2+ ] i and representing a rapid Ca 2+ peak followed by a sustained plateau level (Figure 1A).The amplitude of the Ca 2+ response to increasing concentrations of ML-SA1 has been reported in Figure 1B.Immunofluorescence showed that punctate vesicular structures could be detected in hCMEC/D3 cells stained with the lysosomal marker LAMP-1 (Supplementary Figure S2A) and Lysotracker Red (Supplementary Figure S2B), a red fluorescent weak base that is selective for acidic organelles (Faris et al., 2019;Scorza et al., 2023).Consistent with this, nigericin (50 µM), a H + /K + ionophore that alkalinizes the lysosomal pH (Morgan et al., 2011;Riva et al., 2018;Morgan and Galione, 2021), erased Lysotracker Red fluorescence (Supplementary Figure S2C), confirming that it selectively labels lysosomal vesicles.Co-immunofluorescence analysis showed that both Lysotracker Red and a TRPML1 specific antibody exhibited a similar punctate distribution throughout the cells (Figure 1C).Finally, immunoblotting with a TRPML1-specific antibody detected a major band of ~75 kDa (Figure 1D), which is the predictive molecular weight for the TRPML1 protein (Tedeschi et al., 2021).Taken together, these findings show that the lysosomal TRPML1 is expressed and triggers global Ca 2+ signals in hCMEC/D3 cells.Recent studies have shown that the global Ca 2+ response to TRPML1 activation involves both lysosomal Ca 2+ release and extracellular Ca 2+ entry across the plasma membrane (Kilpatrick et al., 2016;Tedeschi et al., 2021).The long-lasting plateau phase of the Ca 2+ responses illustrated in Figure 1A suggests that ML-SA1 at concentrations higher than 50 μM may also activate a Ca 2+ entry pathway in hCMEC/D3 cells.Consistent with this hypothesis, in the absence of extracellular Ca 2+ (0Ca 2+ ), 100 μM ML-SA1 induced a transient increase in [Ca 2+ ] i whose amplitude was significantly (p < 0.05) smaller as compared to the amplitude of the Ca 2+ response recorded in the presence of extracellular Ca 2+ (Figures 2A,B).Restoration of extracellular Ca 2+ 300 s after the removal of ML-SA1 induced a prompt increase in [Ca 2+ ] i that was obviously independent of the presence of the agonist in the bath (Figure 2C).As discussed in (Morgan et al., 2011;Garrity et al., 2016;Faris et al., 2019;Lloyd-Evans and Waller-Evans, 2019;Moccia et al., 2021b), depletion of the lysosomal Ca 2+ pool with nigericin has long been used as a pharmacological approach to confirm that ML-SA1 and NAADP mobilize the lysosomal Ca 2+ store.Consistent with this, the application of nigericin (50 µM) under 0Ca 2+ conditions induced a transient increase in [Ca 2+ ] i reflecting the depletion of the lysosomal Ca 2+ content (Figure 2D).The subsequent application of 100 μM ML-SA1 failed to induce a discernible Ca 2+ response in hCMEC/D3 cells (Figures 2D,F).In addition, ML-SA1-induced intracellular Ca 2+ release was abolished by ML-SI3 (10 µM) (Figures 2E,F), a specific TRPML1 antagonist (Wang et al., 2015;Kilpatrick et al., 2016;Boretto et al., 2023), and by the genetic deletion of TRPML1 with a selective small interfering RNA (siTRPML1) (Figures 2E,F).The efficacy of the siTRPML1-mediated reduction in TRPML1 protein expression has been illustrated in Figures 2G,H.Taken together, these findings show that TRPML1-mediated global Ca 2+ signals are triggered by lysosomal Ca 2+ release and sustained by extracellular Ca 2+ entry.

Discussion
In the present investigation, we showed that the lysosomal TRPML1 can trigger global Ca 2+ signals, involving both Ca 2+ release and Ca 2+ entry, in human cerebrovascular endothelial cells, as recently shown for HeLa cells and primary cultured human skin fibroblasts (Kilpatrick et al., 2016), rat primary cortical neurons (Tedeschi et al., 2021), and MDA-MB-231 breast cancer cells (Boretto et al., 2023).We further showed that TRPML1-mediated global Ca 2+ signals lead to robust NO production, which is not only a recognized proxy for endothelial Ca 2+ signaling, but also a critical vasorelaxing pathway in brain microcirculation.Therefore, the endothelial TRPML1 channel stands out as a novel component of the Ca 2+ toolkit at the neurovascular unit that could be involved in the regulation of CBF during neuronal activity (Negri et al., 2021c;Longden et al., 2021;Moccia et al., 2022;Mughal et al., 2024).In agreement with this hypothesis, we showed that TRPML1 supports ATP-induced intracellular Ca 2+ release and NO production in hCMEC/D3 cells.
Lysosomal Ca 2+ signaling is emerging as an additional regulator of endothelial Ca 2+ dynamics (Moccia et al., 2021a;Negri et al., 2021b).The lysosomal agonist, nicotinic acid adenine dinucleotide phosphate (NAADP), has been shown to gate endothelial TPCs to regulate mean arterial pressure via NO release (Brailoiu et al., 2010), to stimulate angiogenesis (Favia et al., 2014), von Willebrand factor release (Esposito et al., 2011), andvasculogenesis (Di Nezza et al., 2017;Moccia et al., 2021b).In addition, NAADP-induced lysosomal Ca 2+ release may interact with InsP 3 -induced ER Ca 2+ mobilization to shape the Ca 2+ signal and thereby increase eNOS activity in the human cerebrovascular endothelial cell line, hCMEC/D3, employed in the present investigation (Berra-Romani et al., 2020;Negri et al., 2020;Negri et al., 2021a;Negri et al., 2022).The endothelial role of TRPML1 is less known.A recent study has shown that TRPML1 can increase the interaction between lysosomes and multivesicular bodies in mouse coronary artery endothelial cells, which results in reduced exosome release (Li et al., 2022).The application of low concentrations of ML-SA1 (10 μM), a TRPML1 synthetic agonist, induced a spatially-restricted lysosomal Ca 2+ signal, as revealed by a GCaMP3-ML1 construct that was designed by expressing the genetic Ca 2+ -indicator, GCaMP3, on the cytoplasmic NH 2 -tail of TRPML1 (Li et al., 2022).Herein, we assessed whether TRPML1 was expressed and able to induce global Ca 2+ signals in the human cerebrovascular endothelial cell line, hCMEC/D3, as cytosolic Ca 2+ signals are believed to play a crucial role in endotheliumdependent NO release and CBF regulation at the neurovascular unit (Negri et al., 2021c;Kuppusamy et al., 2021;Longden et al., 2021;Thakore et al., 2021;Moccia et al., 2022;Peters et al., 2022;Mughal et al., 2024).ML-SA1 induced a dose-dependent global Ca 2+ signal consisting of a slow rise in [Ca 2+ ] i that leads to rapid Ca 2+ upstroke followed by a plateau-like phase slightly above the baseline.This long-lasting increase in [Ca 2+ ] i was evident at concentrations of ML-SA1 ≥ 50 µM.Immunofluorescence and immunoblotting confirmed that the TRPML1 protein was expressed in acidic lysosomal vesicles that were widely distributed throughout the cytosol.These findings demonstrate that the endothelial TRPML1 channel is not only expressed in coronary, but also in brain circulation.Lysosomalderived Ca 2+ signals can be amplified into a global increase in [Ca 2+ ] i by the recruitment of ER-embedded InsP 3 Rs via the CICR process (Kilpatrick et al., 2013;Penny et al., 2014;Kilpatrick et al., 2016;Galione et al., 2023).An additional pathway that could be activated upon the NAADP-triggered depletion of the InsP 3 -sensitive ER Ca 2+ store is SOCE, as reported in human cardiac mesenchymal stromal cells (Faris et al., 2022) and MDA-MB-231 cells (Boretto et al., 2023), but not ECFCs (Moccia et al., 2021b) and metastatic colorectal cancer cells (Faris et al., 2019).Furthermore, extracellular Ca 2+ entry through TRPML1 channels that are located on the plasma membrane can support lysosomal Ca 2+ mobilization (Kilpatrick et al., 2016).Consistently, in the absence of extracellular Ca 2+ , ML-SA1 evoked a tiny and transient increase in [Ca 2+ ] i in hCMEC/D3 cells, which is similar to that recorded under the same conditions in HeLa cells (Kilpatrick et al., 2016).This finding supports the emerging notion that ML-SA1 can induce both intracellular Ca 2+ release and extracellular Ca 2+ entry (Kilpatrick et al., 2016;Tedeschi et al., 2021).The intracellular Ca 2+ response to ML-SA1 was abolished by depleting the lysosomal Ca 2+ pool and by the pharmacological or genetic blockade of TRPML1.These findings confirm that TRPML1 activation can also result in lysosomal Ca 2+ mobilization in human cerebrovascular endothelial cells.We must point out that the tiny Ca 2+ response evoked by ML-SA1 under 0Ca 2+ conditions was almost suppressed despite the 60% reduction in TRPML1 protein expression achieved by the specific siTRPML1 used in the present study.The following mechanisms could explain these seemingly contradictory results.First, the remaining TRPML1 protein (≈40%) is not expressed in the acidic vesicle membranes and, therefore, cannot contribute to the Ca 2+ signal.Second, our epifluorescence imaging system does not detect subcellular Ca 2+ signals, as already reported in (Moccia et al., 2019).Thus, we cannot rule out that some TRPML1-mediated local Ca 2+ release events still occurred but were not detected.This hypothesis would be further consistent with the evidence that, although lysosomes contain ≈500 µM free Ca 2+ , they occupy ≈3% of the total endothelial cell volume and are sparse throughout the cytoplasm (Lloyd-Evans and Waller-Evans, 2019).Thus, reducing TRPML1 protein expression by 60% could result in discrete subcellular Ca 2+ signals that can be recorded only with confocal or 2-photon microscopy (Boittin et al., 2002;Kinnear et al., 2008), especially if they are uncoupled from the ER, as further discussed below.For instance, Kinnear et al. revealed that NAADP elicited subcellular Ca 2+ signals that did not significantly increase Fura-2 fluorescence until they led to ER Ca 2+ release in rat pulmonary artery vascular smooth muscle cells (Kinnear et al., 2008).In agreement with these hypotheses, we have recently shown that the lysosomal Ca 2+ response to NAADP is virtually abolished in circulating endothelial colony forming cells transfected with a siRNA selectively targeting TPC1 despite the fact the TPC1 protein expression is downregulated by ≈ 60% (Moccia et al., 2021b).Therefore, we are confident that the overall evidence based upon the pharmacological and genetic blockade of TRPML1 supports its primary role in the Ca 2+ -response to ML-SA1.
ML-SA1-induced intracellular Ca 2+ mobilization was also attenuated or virtually abolished by depleting the ER Ca 2+ store and by blocking InsP 3 Rs.These findings are consistent with those reported in HeLa cells (Kilpatrick et al., 2016) and indicate that InsP 3 Rs sustain the lysosomal Ca 2+ signal in hCMEC/D3 cells.Therefore, the absence of a regenerative Ca 2+ response at low concentrations of ML-SA1 (<50 µM) can be explained by the failure of spatially-restricted lysosomal Ca 2+ nanodomains to fully recruit the juxtaposed InsP 3 Rs (Medina et al., 2015;Davis et al., 2023).On the other hand, an increase in the amount of lysosomal Ca 2+ mobilized by higher concentrations of ML-SA1 (>50 µM) could successfully trigger ER Ca 2+ release through InsP 3 Rs.Lysosomal Ca 2+ release could mobilize the ER Ca 2+ pool by either triggering CICR through RyRs/InsP 3 Rs (Yuan et al., 2024;Yuan et al., 2024) or by refilling the ER in a SERCA-dependent manner, thereby leading to luminal Ca 2+ overload and InsP 3 R/RyR opening (Macgregor et al., 2007).A recent investigation revealed that TRPML1-mediated lysosomal Ca 2+ release tonically regulates the ER Ca 2+ content in primary rat cortical neurons (Tedeschi et al., 2021).Herein, we found that CPA-evoked ER Ca 2+ release, which can be used as a proxy for the free ER Ca 2+ concentration (Brandman et al., 2007;Pierro et al., 2014;Lodola et al., 2017), was significantly reduced upon the pharmacological (with ML-SI3) or genetic (with the specific siTRPML1) blockade of TRPML1.This finding strongly suggests that local TRPML1-mediated Ca 2+ signals are rerouted into the ER in a SERCA-dependent manner, thereby controlling the free ER Ca 2+ concentration that is available to be released through the ER leakage channels and/or the InsP 3 R.Therefore, it is reasonable to conclude that ML-SA1 gates TRPML1, which is likely to lead to ER Ca 2+ overload and InsP 3 R activation from the luminal side.Consistent with this hypothesis, the pharmacological blockade of SOCE with two distinct pyrazole derivatives, Pyr6 and BTP-2, converted the global increase in [Ca 2+ ] i induced by ML-SA1 into a tiny Ca 2+ transient that strongly resembled that measured under 0Ca 2+ conditions.In addition, the "Ca 2+ add-back" protocol revealed that TRPML1-dependent Ca 2+ entry did not require the presence of the extracellular agonist to occur, while it was only associated with the previous depletion of the intracellular Ca 2+ pool.This feature is a hallmark of SOCE activation, which confirms that TRPML1 leads to ER Ca 2+ depletion (Murata et al., 2007;Bird et al., 2008;Moccia et al., 2023a).Conversely, the Fe 2+ quench assay revealed that TRPML1 does not directly contribute to extracellular Ca 2+ entry in hCMEC/D3 cells.A recent study showed that TRPML1 may physically interact with STIM1 (Tedeschi et al., 2021), which could facilitate the assembly of the SOCE machinery.Collectively, these findings strongly suggest that TRPML1-mediated lysosomal Ca 2+ release can trigger ER Ca 2+ release through the Ca 2+ -dependent luminal recruitment of InsP 3 Rs, thereby leading to SOCE activation.Nevertheless, a direct measurement of the ER Ca 2+ concentration is necessary to provide the clear-cut evidence that TRPML1 contributes to ER Ca 2+ refilling.
The endogenous agonist of TRPML1 is still debated, but several lines of evidence indicate that the lysosomal associated PI(3,5)P 2 could fulfil this role (Dong et al., 2010;Zhang et al., 2012;Isobe et al., 2019;Li et al., 2019;Negri et al., 2021b).Consistent with this, the membrane permeable analogue of PI(3,5)P 2 induced a global increase in [Ca 2+ ] i that was similar, although slightly slower, to that induced by ML-SA1.Furthermore, the Ca 2+ response to PI(3,5) P 2 was inhibited by the depletion of the lysosomal Ca 2+ store and by the pharmacological or genetic blockade of TRPML1.Therefore, PI(3,5)P 2 could also serve as an endogenous agonist of TRPML1 in human cerebrovascular endothelial cells, where it could elicit weak or strong Ca 2+ signals depending on the strength of TRPML1 activation and/or its functional coupling with InsP 3 Rs.A further hint at the physiological role played by TRPML1-mediated global Ca 2+ signals arises from NO measurements.Single-cell imaging of DAF-FM DA fluorescence showed that ML-SA1 induced robust NO release via the Ca 2+ -dependent recruitment of eNOS.As reported by single-cell Fura-2 imaging, ML-SA1-induced NO production was abolished by preventing lysosomal Ca 2+ release through TRPML1 and the Ca 2+ -dependent eNOS activation.These findings provide the first evidence that, in addition to NAADP-gated TPCs (Brailoiu et al., 2010;Negri et al., 2021b), the lysosomal Ca 2+ pool could also lead to endothelium-dependent NO release via TRPML1.A recent study has shown that TRPML1-mediated Ca 2+ release can trigger Ca 2+ sparks via CICR through juxtaposed RyRs in vascular smooth muscle cells (VSMCs) from cerebral arteries (Thakore et al., 2020).Ca 2+ sparks, in turn, induce vasodilation and increase local CBF by activating big conductance Ca 2+ -dependent K + channels (Thakore et al., 2020).However, these data collectively suggest that TRPML1 could be critical to regulate vascular reactivity in brain microcirculation, in both VSMCs and endothelial cells.In addition, TRPML1 could also regulate other functions in human cerebrovascular endothelial cells, including lysosome size and trafficking (Yang et al., 2019), autophagy (Yang et al., 2019), and membrane repair (Morabito et al., 2017;Li et al., 2019).TRPML1 could also be physiologically regulated by reactive oxygen species (ROS) (Zhang et al., 2016), which are emerging as crucial regulators of the endothelial Ca 2+ signals at the neurovascular unit (Thakore et al., 2021;Berra-Romani et al., 2023).Future work might assess whether ROS modulate TRPML1 activation in human cerebrovascular endothelial cells.
In order to confirm that TRPML1 is involved in agonist-evoked Ca 2+ signaling in hCMEC/D3 cells, we assessed whether TRPML1 modulates the Ca 2+ response to ATP, which is one of the most widespread endothelial agonists (Scarpellino et al., 2019;Scarpellino et al., 2022;Lisec et al., 2024).ATP has been shown to elicit InsP 3 -induced ER Ca 2+ release in hCMEC/D3 cells (Bintig et al., 2012;Forcaia et al., 2021).Herein, we found that ATP-induced Ca 2+ signals and NO release were both impaired by blocking TRPML1 with ML-SI3 or the specific siTRPML1 or by inhibiting PI(3,5)P 2 production with apilimod.The most plausible hypothesis to explain these findings is that TRPML1 is gated by PI(3,5)P 2 to maintain ER Ca 2+ levels, thereby enabling the proper Ca 2+ response to ATP.

Conclusion
The present investigation provides the first evidence that the lysosomal TRPML1 channel is expressed and mediates a global increase in [Ca 2+ ] i in human cerebrovascular endothelial cells.Lysosomal Ca 2+ release through TRPML1 is supported by ER Ca 2+ mobilization through InsP 3 Rs and by SOCE.Physiologically, TRPML1 could be gated by the lysosome associated phosphoinositide, PI(3,5)P 2 , and could be involved in the regulation of CBF by promoting endothelium-dependent NO release.

FIGURE 4
FIGURE 4 The inhibition of TRPML1 reduces ER Ca 2+ release.(A) The transient Ca 2+ response induced by CPA (10 µM) under 0 Ca 2+ conditions is strongly reduced in the presence of ML-SI3 (10 μM, 20 min) and in hCMEC/D3 cells transfected with the selective siTRPML1.(B) Mean ± SEM of the amplitude of the Ca 2+ responses in cells under the designated treatments.**** indicates p < 0.0001 (Kruskal-Wallis one-way Anova test followed by the Dunn's post hoc test).

FIGURE 7 TRPML1
FIGURE 7 TRPML1 mediates ATP-induced intracellular Ca 2+ release and NO production.(A) The Ca 2+ response to ATP (100 μM) under 0 Ca 2+ conditions was strongly reduced in the presence of ML-SI3 (10 μM, 20 min) or apilimod (100 nM, 20 min) and in hCMEC/D3 cells transfected with the selective siTRPML1.(B) Mean ± SEM of the amplitude of the Ca 2+ responses in cells under the designated treatments.**** indicates p < 0.0001 (Kruskal-Wallis one-way Anova test followed by Dunn's post hoc test).(C).ATP (100 μM) induced a sustained increase in DAF-DM fluorescence, which reflected NO release and was inhibited in the presence of ML-SI3 (10 μM, 20 min) and in hCMEC/D3 cells transfected with the selective siTRPML1.(D) Mean ± SEM of the amplitude of the Ca 2+ responses in cells under the designated treatments.**** indicates p < 0.0001 (Kruskal-Wallis one-way Anova test followed by the Dunn's post hoc test).