Deviant Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Ca2+ Signaling upon Lysosome Proliferation*

Accumulating evidence suggests that the endolysosomal system is a novel intracellular Ca2+ pool mobilized by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). Although lysosomes in neurons are known to proliferate in numerous neurodegenerative diseases and during the normal course of aging, little is known concerning the effect of lysosomal proliferation on Ca2+ homeostasis. Here, we induce proliferation of lysosomes in primary cultures of rat hippocampal neurons and PC12 cells through chronic treatment with the cathepsin inhibitor, Z-Phe-Ala-diazomethylketone. We demonstrate that lysosome proliferation increases the size of the lysosomal Ca2+ pool and enhances Ca2+ signals in response to direct cellular delivery of NAADP and glutamate, an identified NAADP-producing agonist. Our data suggest that deregulated lysosomal Ca2+ signaling through NAADP may contribute to neuronal dysfunction and highlight the usefulness of lysosomal hydrolase inhibition in probing NAADP action.

Accumulating evidence suggests that the endolysosomal system is a novel intracellular Ca 2؉ pool mobilized by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). Although lysosomes in neurons are known to proliferate in numerous neurodegenerative diseases and during the normal course of aging, little is known concerning the effect of lysosomal proliferation on Ca 2؉ homeostasis. Here, we induce proliferation of lysosomes in primary cultures of rat hippocampal neurons and PC12 cells through chronic treatment with the cathepsin inhibitor, Z-Phe-Ala-diazomethylketone. We demonstrate that lysosome proliferation increases the size of the lysosomal Ca 2؉ pool and enhances Ca 2؉ signals in response to direct cellular delivery of NAADP and glutamate, an identified NAADP-producing agonist. Our data suggest that deregulated lysosomal Ca 2؉ signaling through NAADP may contribute to neuronal dysfunction and highlight the usefulness of lysosomal hydrolase inhibition in probing NAADP action.
Changes in the concentration of cytosolic Ca 2ϩ ions form the basis of a ubiquitous signal transduction pathway important for a vast number of cellular processes (1). Cytosolic Ca 2ϩ levels are regulated by a tightly controlled system of Ca 2ϩ channels, pumps, exchangers, and buffers (1). In the central nervous system, Ca 2ϩ is essential for vital processes such as neurotransmitter release and synaptic plasticity (2). Loss of Ca 2ϩ homeostasis has devastating consequences as exemplified by cell death in response to Ca 2ϩ overload during glutamate toxicity (2).
Over the last 15 years, an entirely new Ca 2ϩ -signaling pathway has been described that is controlled by the Ca 2ϩ -mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP) 2 (3)(4)(5)(6). Like the better characterized messengers, inositol 1,4,5-triphosphate and cyclic ADP-ribose, NAADP is produced upon cell stimulation and generates spatio-temporally complex Ca 2ϩ signals deriving from intracellular Ca 2ϩ stores (3,4). NAADP is active in the nervous system, as evidenced by the presence of NAADP binding sites (7), an enzymatic route for NAADP metabolism (8), and the demonstration of changes in cytosolic Ca 2ϩ in response to NAADP in neuronal preparations (9 -14). Indeed, NAADP has been shown to regulate neurotransmission (10,15), neurite outgrowth (11), neuronal differentiation (13), and depolarization (14). Moreover, glutamate has recently been identified as an NAADP-dependent agonist (16) linking, for the first time, the actions of NAADP to a major excitatory neurotransmitter in the brain.
In stark contrast to inositol 1,4,5-triphosphate and cyclic ADP-ribose, in most systems studied, NAADP appears to target novel Ca 2ϩ -permeable channels located not on the endoplasmic reticulum (ER; an established Ca 2ϩ store) but instead on acidic Ca 2ϩ stores (3)(4)(5). These channels have recently been identified as the two-pore channels that localize to the endolysosomal system (17)(18)(19). This location is consistent with the many studies that have shown blockade of NAADP-mediated Ca 2ϩ signals by the lysosomotropic agent, glycyl-L-phenylalanine ␤-naphthylamide (GPN) (12, 13, 16, 18, 20 -24). Acidic stores of Ca 2ϩ are thus a novel determinant of Ca 2ϩ signals highlighting the fundamentally different mode of action of NAADP when compared with its messenger counterparts.
Lysosomes are dynamic, heterogeneous organelles with an internal pH of 4.5-5 and are best known as the terminal degradative compartments of the endocytic and autophagic pathways (25,26). Depending on cell type, they typically occupy between 0.5 and 5% of the volume of a cell and, in neurons, are ϳ0.5 m in diameter (25,26). The critical role of lysosomes in the brain is highlighted by the numerous neurodegenerative diseases attributable to lysosome dysfunction, including Niemann-Pick disease type C, frontotemporal dementia, neuronal ceroid lipofuscinoses, Down syndrome, Creutzfeldt-Jakob, and Alzheimer, Parkinson, and Huntington disease (27). All of these diseases are characterized by impaired lysosomal function and clearance of waste products, an accumulation of lysosomes and associated autophagic vacuoles, and extensive neurodegeneration of the cortex and hippocampus. Similar lysosomal proliferation has been reported in a variety of aged cells, including neurons (28), and in cells undergoing replicative senescence (29). That lysosomes are now recognized as a readily mobilized pool of Ca 2ϩ raises the intriguing possibility that their proliferation may contribute to disturbed Ca 2ϩ homeostasis and thus cellular dysfunction. Indeed, recent studies have shown altered lysosomal Ca 2ϩ stores in a lysosomal storage disorder (30). * This work was supported by grants from the Alzheimer's Research Trust, Research into Ageing, and the Biotechnology and Biological Sciences Research Council (Grant BB/G013721/1 to S. P. and Grant BB/D012694/1 to G. C. C.). □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3. 1 To whom correspondence should be addressed. E-mail: patel.s@ucl.ac.uk.
In the present study, we examined the effect of proliferating lysosomes on NAADP-mediated Ca 2ϩ signaling in primary cultured rat hippocampal neurons and the rat pheochromocytoma PC12 cell line. We demonstrate, for the first time, that lysosome proliferation, induced by treating cultured cells with the cathepsin B and L inhibitor Z-Phe-Aladiazomethylketone (ZPAD), results in exaggerated NAADPmediated Ca 2ϩ signals in response to both the cell-permeant NAADP analogue NAADP-AM and the NAADP-linked neurotransmitter, glutamate.

MATERIALS AND METHODS
Cell Culture-Hippocampal neurons were isolated from 3-day-old rat pups (Sprague-Dawley, University College London breeding colony) and maintained as mixed cultures of neurons and glia, as described previously (16) with some modifications. Dissected hippocampi from two pups were finely chopped in ice-cold Ca 2ϩ /Mg 2ϩ -free Hanks' balanced salt solution and incubated with 0.25% (w/v) Trypsin-EDTA for 7 min at 37°C. The suspension was then washed with Hanks' balanced salt solution and resuspended in advanced Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. Cells were dissociated by passage through an 18-gauge needle and plated on glass coverslips (VWR International, 25 mm, thickness number 1; coated with 20 g/ml poly-L-lysine) by overnight incubation at 37°C in a humidified atmosphere of 95% air, 5% CO 2 . The cells were then maintained in Neurobasal TM medium supplemented with 2% (v/v) B-27 and 2 mM L-glutamine for 6 -7 days prior to experimentation. All reagents used were from Invitrogen. Neurons were identified by their phase-bright rounded appearance and processes, and their identity was confirmed during Ca 2ϩ imaging by monitoring whether they responded to depolarization (see below).
Proliferation of Lysosomes-Lysosome proliferation was induced using the method described by Bednarski et al. (31). 10 -200 M ZPAD (Bachem; dissolved in DMSO) was added to the culture medium of plated cells for 5-10 days and replenished with each regular medium change. DMSO was added to matched controls to a final concentration of 0.05% (v/v).
Epifluorescece microscopy was carried out using an Olympus IX71 inverted microscope fitted with either a 10ϫ 0.40 UPLanApo or a 100ϫ 1.35 NA UPLanApo oil immersion objective. Fluorescence images (emission Ͼ 590 nm) were captured with a cooled coupled device (CCD) camera following excita-tion from a monochromator light source (TILL Photonics). Confocal microscopy was performed using an Axiovert 200M inverted microscope (Carl Zeiss, Inc.) equipped with a confocal scanner (LSM 510, Carl Zeiss, Inc.), a 63ϫ 1.4 NA Plan Apochromat oil immersion objective, and a long pass 560 emission filter. In both cases, the excitation wavelength was 543 nm.
Transmission Electron Microscopy-Cells were fixed with 2% paraformaldehyde, 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3, and then treated with 1% OSO, 1.5% K 4 Fe(CN) 6 in 0.1 M cacodylate buffer, pH 7.3, dehydrated in a graded ethanol-water series, cleared in propylene oxide, and infiltrated with agar resin. Ultrathin sections were cut using a diamond knife on a Reichert Ultracut E microtome and collected on 300 mesh grids, stained with uranyl acetate and lead citrate. Samples were viewed with a Joel 1010 transition electron microscope, and the images were recorded using a Gatan Orius CCD camera. ImageJ software (32) was used to count and measure the area of lysosomes and mitochondria.
Measurement of Cytosolic Ca 2ϩ Concentration-Cells were loaded with the fluorescent ratiometric Ca 2ϩ indicator fura-2 by incubation with 2.5 M fura-2 AM and 0.005% (v/v) pluronic acid (Invitrogen) in HBS for 30 min at room temperature in the dark. The cells were washed with HBS and mounted in a viewing chamber on the stage of the Olympus IX71 microscope described above fitted with a 20ϫ 0.75 NA UApo/340 objective. Fura-2 fluorescence (emission Ͼ 440 nm) images were captured every 3 s using a CCD camera (TILL Photonics) following alternate excitation at 340 and 380 nm delivered by a monochromator (TILL Photonics). Recordings were made for 60 s to allow the determination of the basal fluorescence ratio prior to stimulation. Cells were depolarized by the addition of K ϩ -enriched HBS (containing 40 mM K ϩ as an equimolar substitution of Na ϩ ). Experiments were also performed in nominally Ca 2ϩfree HBS, which contained 1 mM EGTA in place of Ca 2ϩ where indicated.
During each experiment, 30 -60 cells were analyzed by calculating the mean 340/380 nm ratio at each time point within user-defined regions of interest using TILLvisION software. The magnitude of each response upon stimulation (⌬R) was determined by subtracting the mean of the basal ratios for 30 -60 s prior to stimulation from the peak fluorescence ratio. Cells were considered responsive to a given agonist if the change in ⌬R was 20% or greater than the basal ratio.

RESULTS AND DISCUSSION
Previous studies have demonstrated that lysosomal proliferation can be induced both in vitro (31,33,34) and in vivo (35,36) by pharmacological inhibition of select lysosomal hydrolases. Here, we treated primary cultured rat hippocampal neurons and PC12 cells with ZPAD for 10 or 5 days, respectively, and examined its effect on lysosome morphology. First, we compared the distribution of the weak florescent base, LysoTracker Red, in live cells. Epifluorescence and confocal imaging indicated that ZPAD treatment had a dramatic effect on LysoTracker staining in both cell types. As shown in the epifluorescence images in Fig. 1A, 50 M ZPAD induced dense perinuclear labeling in neurons consistent with a marked up-regulation of acidic compartments. From wide field epifluorescence images of cultures of PC12 cells, LysoTracker Red staining was increased by ZPAD in a concentration-dependent manner (Fig. 1B, top panels). At the highest concentration tested (200 M), there was an ϳ10-fold increase in fluorescence (supplemental Fig. S1A). Higher magnification images of individual cells indicated that ZPAD increased the number of labeled structures (Fig.  1B, bottom panels). This was confirmed in both cell types by confocal microscopy (Fig. 1C). Second, we examined ZPAD-treated cells by electron microscopy. The effect of 50 M ZPAD on the morphology of a typical neuron and PC12 cell is shown in Fig. 1, D and E. Higher magnification images are shown in supplemental Fig. S2. As evident, ZPAD increased the number and size of electron-dense lysosomes (Fig. 1, D and E). Pooled data from several cells quantifying lysosomal number and area are shown in Fig.  1F. Analysis of frequency distributions of lysosome size indicated that ZPAD treatment induced a concentration-dependent rightward shift (supplemental Fig. S1, B and C). In contrast, ZPAD had little effect on the number, size (Fig. 1, D-F), or morphology (supplemental Fig. S2) of mitochondria. These results, showing selective proliferation of lysosomes, are consistent with previous studies conducted on cultured hippocampal slices treated with ZPAD for 6 days (31). Finally, we examined the effects of ZPAD on transcript levels of the lysosomal proteins LAMP-1 and LAMP-2. As shown in Fig. 1G, 50 M ZPAD induced a 2-3-fold increase in expression of the lysosomal markers. Taken together, the above analysis suggests that lysosome proliferation can be readily induced in culture by inhibition of lysosomal hydrolases.
Having successfully induced lysosome proliferation, we examined the effect of lysosome proliferation on the size of the lysosomal Ca 2ϩ stores. To achieve this, cytosolic Ca 2ϩ levels were measured in fura-2-loaded cells in response to GPN. GPN is a cell-permeable cathepsin C peptide substrate that, when cleaved, causes osmotic lysis of lysosomes (37). Cells were stimulated with GPN in the absence of extracellular Ca 2ϩ (to prevent Ca 2ϩ influx) and following treatment with thapsigargin (to deplete endoplasmic reticulum Ca 2ϩ stores). Although Ca 2ϩ signals in response to thapsigargin were not affected by ZPAD, those to GPN were markedly increased ( Fig. 2A). Peak responses to GPN were elevated ϳ4-fold (Fig. 2F). These data show that ZPAD increases the size of the lysosomal Ca 2ϩ store but has no effect on ER Ca 2ϩ stores.
Because NAADP has been proposed to mobilize acidic Ca 2ϩ stores, we next examined the effect of lysosomal proliferation on NAADP-mediated Ca 2ϩ signals. As a charged molecule NAADP, like other second messengers, is incapable of passing through the plasma membrane. The recent development of a cell-permeant acetoxymethyl ester of NAADP (NAADP-AM) (38) has provided an excellent pharmacological tool for directly investigating the effect of NAADP on Ca 2ϩ signals, circum-venting the more complicated and/or invasive techniques of liposome delivery and microinjection (14,30,39). The addition of NAADP-AM evoked robust Ca 2ϩ signals in both cell types (Fig. 2, B  and C), whereas the addition of NAADP had no effect (data not shown). In neurons, a typical "bellshaped" concentration-effect relationship for NAADP-AM was observed (supplemental Fig. S3) as reported previously in cortical neurons (38).
We compared NAADP-AM-mediated cytosolic Ca 2ϩ changes in cells treated with 50 M ZPAD or DMSO. As shown in Fig. 2, B and C, ZPAD treatment potentiated Ca 2ϩ signals in both neurons and PC12 cells. The mean peak Ca 2ϩ response (⌬R) of neurons and PC12 cells to NAADP-AM following ZPAD treatment was 1.5-2-fold higher (Fig. 2F). Taken together, the above data indicate that the proliferation of lysosomes by ZPAD results in an increase in the mobilizable NAADP-sensitive intracellular Ca 2ϩ pool consistent with the increases in total Ca 2ϩ content ( Fig.  2A). These data also provide independent evidence that NAADPsensitive Ca 2ϩ channels are expressed on the acidic stores of these cell types (13,16).
Our knowledge concerning the physiological cues that engage the NAADP pathway is currently limited, particularly in the nervous system. We have recently shown that NAADPsensitive Ca 2ϩ stores in neurons are recruited through NAADP production by the neurotransmitter glutamate (16). We therefore investigated the impact of lysosome proliferation on Ca 2ϩ signaling evoked by this neurotransmitter. As shown in Fig. 2D, ZPAD treatment substantially potentiated Ca 2ϩ signals in response to glutamate. Ca 2ϩ signals in response to 10 M ATP and 100 M carbachol, however, were not significantly affected (data not shown). Finally, we compared Ca 2ϩ signals in response to chemical depolarization (with high K ϩ ) in the presence of extracellular Ca 2ϩ to stimulate Ca 2ϩ entry through voltage-sensitive Ca 2ϩ channels. Ca 2ϩ signals were similar in control and ZPAD-treated cultures, further attesting to the specificity of ZPAD in modulating cytosolic Ca 2ϩ levels (Fig.  2E). These data, summarized in Fig. 2F, indicate that lysosome proliferation selectively potentiates Ca 2ϩ signals in response to an identified NAADP-coupled neurotransmitter.
To conclude, we show that pharmacological inhibition of lysosome hydrolases induces marked proliferation of the lysosomal system and that this is associated with an increase in the total size of the acidic Ca 2ϩ store and exaggerated Ca 2ϩ signals in response to direct intracellular delivery of NAADP and to the NAADP-forming neurotransmitter, glutamate. ZPAD-treated cells are likely to provide an extremely tractable system for defining changes in Ca 2ϩ signaling and other processes associated with neurodegenerative diseases and aging in which lysosomal proliferation features. Indeed, deregulated Ca 2ϩ signaling through Ca 2ϩ stores has already been associated with neuronal dysfunction, although this is ascribed to changes in the ER (40,41). Potential changes in lysosomal Ca 2ϩ signaling, however, are not mutually exclusive because NAADP is thought to provide a "trigger" release of Ca 2ϩ , which is then amplified by Ca 2ϩ -sensitive ER Ca 2ϩ channels (3)(4)(5)(6). Thus, altered activity of NAADP-sensitive channels is likely to have profound effects on downstream Ca 2ϩ signals and Ca 2ϩ -dependent function. Exaggerated NAADP-mediated signaling as a potential contributor to homeostasis loss therefore represents a novel mechanism underlying deregulated Ca 2ϩ signals. Additionally, although the use of the lysosomotropic agent, GPN, which decreases lysosomal number, has been widely used to probe NAADP action (12, 13, 16, 18, 20 -24), we suggest that ZPAD, which has the opposite effect on lysosomes, could prove equally useful.