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

Abstract

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.

Introduction

Changes in the concentration of cytosolic Ca²⁺ ions form the basis of a ubiquitous signal transduction pathway important for a vast number of cellular processes (1). Cytosolic Ca²⁺ levels are regulated by a tightly controlled system of Ca²⁺ channels, pumps, exchangers, and buffers (1). In the central nervous system, Ca²⁺ is essential for vital processes such as neurotransmitter release and synaptic plasticity (2). Loss of Ca²⁺ homeostasis has devastating consequences as exemplified by cell death in response to Ca²⁺ overload during glutamate toxicity (2).

Over the last 15 years, an entirely new Ca²⁺-signaling pathway has been described that is controlled by the Ca²⁺-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP)² (3–6). 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 Ca²⁺ signals deriving from intracellular Ca²⁺ 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 Ca²⁺ 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 Ca²⁺-permeable channels located not on the endoplasmic reticulum (ER; an established Ca²⁺ store) but instead on acidic Ca²⁺ stores (3–5). These channels have recently been identified as the two-pore channels that localize to the endolysosomal system (17–19). This location is consistent with the many studies that have shown blockade of NAADP-mediated Ca²⁺ signals by the lysosomotropic agent, glycyl-l-phenylalanine β-naphthylamide (GPN) (12, 13, 16, 18, 20–24). Acidic stores of Ca²⁺ are thus a novel determinant of Ca²⁺ 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 ∼0.5 μm 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 Ca²⁺ raises the intriguing possibility that their proliferation may contribute to disturbed Ca²⁺ homeostasis and thus cellular dysfunction. Indeed, recent studies have shown altered lysosomal Ca²⁺ stores in a lysosomal storage disorder (30).

In the present study, we examined the effect of proliferating lysosomes on NAADP-mediated Ca²⁺ 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-Ala-diazomethylketone (ZPAD), results in exaggerated NAADP-mediated Ca²⁺ 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 Ca²⁺/Mg²⁺-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 μg/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 μg/ml poly-l-lysine) by overnight incubation at 37 °C in a humidified atmosphere of 95% air, 5% CO₂. 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 Ca²⁺ imaging by monitoring whether they responded to depolarization (see below).

PC12 cells (ATCC® number CRL-1721^TM) were maintained in RPMI 1640 medium (Sigma) supplemented with 10% (v/v) heat-inactivated horse serum, 5% (v/v) fetal calf serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen). Prior to experimentation, cells were plated onto glass coverslips at a density of 200,000 cells/ml.

Proliferation of Lysosomes

Lysosome proliferation was induced using the method described by Bednarski et al. (31). 10–200 μm 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).

LysoTracker Imaging

Cells were incubated with 1 μm LysoTracker Red (Invitrogen) in HEPES-buffered saline (HBS) composed of 156 mm NaCl, 3 mm KCl, 2 mm MgSO₄, 1.25 mm KH₂PO₄, 2 mm CaCl₂, 10 mm glucose, and 10 mm HEPES (pH 7.4) at 37 °C for 30 min in the dark. Cells were washed with HBS, and fluorescence images captured by either epifluorescence or confocal microscopy.

Epifluorescece microscopy was carried out using an Olympus IX71 inverted microscope fitted with either a 10× 0.40 UPLanApo or a 100× 1.35 NA UPLanApo oil immersion objective. Fluorescence images (emission > 590 nm) were captured with a cooled coupled device (CCD) camera following excitation 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 63× 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% K₄Fe(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.

Quantitative RT-PCR

Total RNA was extracted using the RNeasy mini kit (Qiagen) following the manufacturer's instructions, including the on-column digestion of DNA using the RNase-free DNase set (Qiagen). Quantitative RT-PCR was performed as described previously (17). Briefly, the Improm-II reverse transcription system (Promega) was used to prepare cDNA with oligo(dT) primers and gene-specific oligonucleotide primers corresponding to the nucleotide sequences of rat LAMP-1 (forward, 5′-GTAGCAACTTCAGCAAGG-3′; reverse 5′-GTTCCATTGTCACCAGTC-3′), LAMP-2 (forward, 5′-ACCTGAGTCCTGTTGTTC-3′; reverse, 5′-GGAGTGAGTGTCGTAGTAG-3′), and glyceraldehyde-3-phosphate dehydrogenase (forward, 5′-GTATGACTCTACCCACGGCAAG-3′; reverse, 5′-TTCTCCATGGTGGTGAAGACG-3′). RNA samples were denatured for 2 min at 94 °C and then subjected to 40 cycles of denaturation (15 s at 94 °C), annealing (30 s at 60 °C), and extension (30 s at 72 °C) with the SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich). Expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase following simultaneous amplification.

Measurement of Cytosolic Ca²⁺ Concentration

Cells were loaded with the fluorescent ratiometric Ca²⁺ indicator fura-2 by incubation with 2.5 μm 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 20× 0.75 NA UApo/340 objective. Fura-2 fluorescence (emission > 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⁺-enriched HBS (containing 40 mm K⁺ as an equimolar substitution of Na⁺). Experiments were also performed in nominally Ca²⁺-free HBS, which contained 1 mm EGTA in place of Ca²⁺ 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 (ΔR) 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 ΔR 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 μm 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 μm), there was an ∼10-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 μm 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 μm 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.

FIGURE 1.

ZPAD induces lysosome proliferation. A, epifluorescence images of LysoTracker Red-labeled neurons cultured with DMSO (top) or 50 μm ZPAD (bottom) for 10 days. B, epifluorescence images of LysoTracker Red-labeled PC12 cells cultured with the indicated concentration of ZPAD for 5 days. Top panels are wide field images. Bottom panels are higher magnification images of individual cells. C, confocal images of individual LysoTracker Red-labeled neurons (left) and PC12 cells (right) cultured in the presence of DMSO (top) or 50 μm ZPAD (bottom). Scale bar = 5 μm. D and E, electron microscopy images of neurons (D) and PC12 cells (E) cultured in the presence of DMSO (top) or 50 μm ZPAD (bottom). Lysosomes are depicted by the arrowheads. Images were captured at 6,000× magnification in D (scale bar = 2 μm) and 12,000× magnification in E (scale bar = 1 μm). F, effect of the indicated concentration of ZPAD on the number (left) and area (right) of lysosomes (Lyso.) and mitochondria (Mito.) Data (mean ± S.E.) were derived from electron microscopy images of 5–8 cells. G, quantitative RT-PCR of LAMP isoforms from PC12 cells cultured with DMSO or 50 μm ZPAD.

Having successfully induced lysosome proliferation, we examined the effect of lysosome proliferation on the size of the lysosomal Ca²⁺ stores. To achieve this, cytosolic Ca²⁺ 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 Ca²⁺ (to prevent Ca²⁺ influx) and following treatment with thapsigargin (to deplete endoplasmic reticulum Ca²⁺ stores). Although Ca²⁺ signals in response to thapsigargin were not affected by ZPAD, those to GPN were markedly increased (Fig. 2A). Peak responses to GPN were elevated ∼4-fold (Fig. 2F). These data show that ZPAD increases the size of the lysosomal Ca²⁺ store but has no effect on ER Ca²⁺ stores.

FIGURE 2.

Lysosome proliferation selectively potentiates lysosomal Ca²⁺ signals. A–D, cytosolic Ca²⁺ signals from cultures treated with either DMSO (left) or 50 μm ZPAD (right). A, responses from neurons sequentially stimulated with thapsigargin (Tg, 1 μm) and GPN (200 μm) in the absence of extracellular Ca²⁺. B and C, responses from neurons (B) and PC12 cells (C) stimulated with 250 nm and 1 μm NAADP-AM, respectively. Experiments were performed in the presence of extracellular Ca²⁺. D, responses from neurons stimulated with 10 μm glutamate (Glu) in the absence of extracellular Ca²⁺. E, responses from neurons stimulated with 40 mm K⁺ in the presence of extracellular Ca²⁺. All traces are population averages from 3–4 separate cultures. The total number of cells analyzed (n) is indicated. F, summary (mean ± S.E.) showing the magnitude of the responses (ΔR) under the various conditions.

Because NAADP has been proposed to mobilize acidic Ca²⁺ stores, we next examined the effect of lysosomal proliferation on NAADP-mediated Ca²⁺ 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 Ca²⁺ signals, circumventing the more complicated and/or invasive techniques of liposome delivery and microinjection (14, 30, 39). The addition of NAADP-AM evoked robust Ca²⁺ 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 “bell-shaped” concentration-effect relationship for NAADP-AM was observed (supplemental Fig. S3) as reported previously in cortical neurons (38).

We compared NAADP-AM-mediated cytosolic Ca²⁺ changes in cells treated with 50 μm ZPAD or DMSO. As shown in Fig. 2, B and C, ZPAD treatment potentiated Ca²⁺ signals in both neurons and PC12 cells. The mean peak Ca²⁺ response (ΔR) 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 Ca²⁺ pool consistent with the increases in total Ca²⁺ content (Fig. 2A). These data also provide independent evidence that NAADP-sensitive Ca²⁺ 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 NAADP-sensitive Ca²⁺ stores in neurons are recruited through NAADP production by the neurotransmitter glutamate (16). We therefore investigated the impact of lysosome proliferation on Ca²⁺ signaling evoked by this neurotransmitter. As shown in Fig. 2D, ZPAD treatment substantially potentiated Ca²⁺ signals in response to glutamate. Ca²⁺ signals in response to 10 μm ATP and 100 μm carbachol, however, were not significantly affected (data not shown). Finally, we compared Ca²⁺ signals in response to chemical depolarization (with high K⁺) in the presence of extracellular Ca²⁺ to stimulate Ca²⁺ entry through voltage-sensitive Ca²⁺ channels. Ca²⁺ signals were similar in control and ZPAD-treated cultures, further attesting to the specificity of ZPAD in modulating cytosolic Ca²⁺ levels (Fig. 2E). These data, summarized in Fig. 2F, indicate that lysosome proliferation selectively potentiates Ca²⁺ 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 Ca²⁺ store and exaggerated Ca²⁺ 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 Ca²⁺ signaling and other processes associated with neurodegenerative diseases and aging in which lysosomal proliferation features. Indeed, deregulated Ca²⁺ signaling through Ca²⁺ stores has already been associated with neuronal dysfunction, although this is ascribed to changes in the ER (40, 41). Potential changes in lysosomal Ca²⁺ signaling, however, are not mutually exclusive because NAADP is thought to provide a “trigger” release of Ca²⁺, which is then amplified by Ca²⁺-sensitive ER Ca²⁺ channels (3–6). Thus, altered activity of NAADP-sensitive channels is likely to have profound effects on downstream Ca²⁺ signals and Ca²⁺-dependent function. Exaggerated NAADP-mediated signaling as a potential contributor to homeostasis loss therefore represents a novel mechanism underlying deregulated Ca²⁺ 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.