Signal transduction in astrocytes: Localization and release of inorganic polyphosphate

Abstract Inorganic polyphosphate (polyP) is present in every cell and is highly conserved from primeval times. In the mammalian cells, polyP plays multiple roles including control of cell bioenergetics and signal transduction. In the brain, polyP mediates signaling between astrocytes via activation of purinergic receptors, however, the mechanisms of polyP release remain unknown. Here we report identification of polyP‐containing vesicles in cortical astrocytes and the main triggers that evoke vesicular polyP release. In cultured astrocytes, polyP was localized predominantly within the intracellular vesicular compartments which express vesicular nucleotide transporter VNUT (putative ATP‐containing vesicles), but not within the compartments expressing vesicular glutamate transporter 2 (VGLUT2). The number of lysosomes which contain polyP was dependent on the conditions of astrocytes. Release of polyP from a proportion of lysosomes could be induced by calcium ionophores. In contrast, polyP release from the VNUT‐containing vesicles could be triggered by various physiological stimuli, such as pH changes, polyP induced polyP release and other stimuli which increase [Ca2+]i. These data suggest that astrocytes release polyP predominantly via exocytosis from the VNUT‐containing vesicles. © 2018 Wiley Periodicals, Inc.

We have previously demonstrated that polyP might act as one of the gliotransmitters as the majority of astrocytes and a proportion (3%) of neurons respond to polyP with increases in cytosolic calcium.
This effect is mediated through activation of P2Y 1 receptors and stimulation of phospholipase C activity (Holmstrom et al., 2013). The concentration of polyP in the mammalian brain (50 μM) is much higher than the concentrations sufficient (10 nM-10 μM) to trigger Ca 2+ signals in astrocytes (Kumble & Kornberg, 1995). This indicates that in the brain polyP is contained inside the cells, likely to be compartmentalized and released in a controlled manner. Previously polyP was found to be present in astroglial lysosomes and neuronal synaptosomes (Holmstrom et al., 2013;Stotz et al., 2014). However, in astrocytes only a small number of polyP containing lysosomes fused with the plasma membrane upon stimulation.
Recently we have developed novel highly specific probes to visualize polyP in living cells: JC-D7 and JC-D8, which track localization of polyP in living cells with high affinity . In this study we used specific polyP indicators and novel molecular tools to determine localization of free polyP in specific cellular compartments of astrocytes and to study the mechanisms of polyP release in response to various stimuli.

| Cell culture
Primary cell cocultures of neurons and astrocytes were prepared as described in detail previously Turovsky et al., 2016) with modifications, from the midbrains and cerebral cortices of Sprague-Dawley P3 rat pups or wildtype and LRRK2 knockout C57BL/6 mice (UCL breeding colony). Experimental procedures were performed in compliance with the United Kingdom Animals (Scientific Procedures) Act of 1986. After trypsinization of the tissue, the cells were plated on poly-d-lysine-coated coverslips, according to the protocols described in (Deas et al., 2016) for 12 DIV.

| Transduction
sGFAP is a transcriptionally enhanced, bidirectional, shortened glial fibrillary acidic protein promoter which drives transgene expression in astrocytes (Figueiredo et al., 2011;Gourine et al., 2010;Liu, Paton, & Kasparov, 2008). Fusions with EGFP or mKate2.7 allow detection of transgene expression via green or red fluorescence, respectively. The VGLUT2 (vesicular glutamate transporter; NM0534271; kind gift from P. Bezzi, Lausanne (Bezzi et al., 2004) is targeted to glutamatergic vesicles in astrocytes, while the mVNUT (vesicular nucleotide transporter; kind gift from T. Miyaji, Japan) delineates putative ATP-containing vesicles (Sawada et al., 2008). CD63 is a lysosomal membrane protein which localizes to lysosome-derived exocytotic vesicle-like structures in astrocytes (CD63 clone BC063173 obtained from ImaGenes, Berlin (Metzelaar et al., 1991). LVV-EF1a-TMPAP-EGFP drives expression of a fluorescently tagged plasma-membrane-anchored phosphatase (transmembrane prostatic acid phosphatase, TMPAP) which is designed to break down ATP within vesicles and extracellularly Wells et al., 2015).  (Aschar-Sobbi et al., 2008). Mitochondrial localization was identified using potential sensitive indicator tetramethylrhodamine (TMRM; Angelova et al., 2018). Cells were loaded for 40 min at room temperature and superfused with 20 nM TMRM, excited at 565 nm and imaged with a 580 nm emission filter as previously described. Measurements of fluorescence in astrocytes determined using different z-tacks. Illumination intensity was kept to a minimum (at 0.1-0.2% of laser output) to avoid phototoxicity and the pinhole set to give an optical slice of 2 μm.

| The images were analyzed using Zeiss ZEN software
To determine the percentage of lysosomes, mitochondria, VNUT or VGLUT vesicles that contain polyP, and the percentage of polyP localized within organelles or vesicles, colocalization analysis was per-

| TIRF imaging
An Olympus total internal reflection fluorescence (TIRF) microscope was used to detect vesicular fusion events in astrocytes expressing VNUT-eGFP or CD63-mKate, as described in detail previously Kasymov et al., 2013). Fluorescence was excited at 488 nm and collected at 500-530 nm for eGFP and excited at 488 nm and collected at 600-660 nm for mKate. The imaging setup was equipped with a high-NA oil-immersion objective (60×, 1.65 NA), an Olympus IX71 inverted microscope and a Hamamatsu CCD camera. Images were acquired using Olympus Celltool software

| Data analysis and statistics
Data and statistical analysis were performed using OriginPro PolyP in the mitochondria has its functional role; it plays an important role in energy metabolism and calcium handling (Baev, Negoda, & Abramov, 2017;Pavlov et al., 2010). In agreement with our previous report, we found that in cultured astrocytes the specific polyP indicator has higher intensity in the mitochondrial area   (Figure 1a). Simultaneous measurement of mitochondrial (TMRM) and polyP (JC-D8) signals showed that 40% of cellular polyP resides in mitochondria (here and below coefficient of colocalization is a Mander's X; 0.39 ± 0.08, n = 204 cells; Figure 1a,d). As we reported previously, the level of polyP in mitochondria is dependent on the energy state of mitochondria and can be modified within seconds (Pavlov et al., 2010).

| PolyP in lysosomes
We have previously reported that in cultured astrocytes DAPI-polyP staining partially colocalizes with lysosomes labeled with LysoTracker Red (Holmstrom et al., 2013). We next labeled polyP with either DAPI-polyP or JC-D8 and genetically labeled lysosomes using a fusion Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with a familial form of Parkinson's disease. This mutation manifests with defects in the autophagy/lysosomal degradation pathway (Hockey et al., 2015;Manzoni & Lewis, 2013). We found that in LRRK2 knockout mouse cortical astrocytes the percentage of polyPcontaining lysosomes was significantly decreased (Figure 2c,d; 0.19 ± 0.01, n = 51 for wildtype compared to 0.13 ± 0.02, n = 54 for LRRK2 knockout cultures; p < .001).

| PolyP in glutamate-containing vesicles
It is thought that astrocytes may release glutamate via exocytosis of VGLUT2-containing vesicles. Transfection of cortical astrocytes with VGLUT2-eGFP identified VGLUT2-expressing vesicles (Figure 3a-c) but there was no colocalization of eGFP with either DAPI-polyP or JC-D7 (n = 113 and n = 143 cells, respectively; Figure 3a-d). These results indicate that astroglial vesicles which express VGLUT2 do not contain polyP.

| PolyP in ATP-containing vesicles
PolyP may mediate signaling between astrocytes through activation of metabotropic P2Y 1 receptors (Holmstrom et al., 2013). We next identified putative ATP-containing vesicles, by transducing astrocytes to express eGFP-tagged vesicular nucleotide transporter (VNUT) JC-D8 and DAPI specifically label free polyP and have no selectivity towards purines, including ATP or ADP Aschar-Sobbi et al., 2008). To study the functional roles of ATP or ADP as signaling molecules, enzymatic depletion of ATP or ADP by phosphatases has been used in a number of studies (Wells et al., 2015). We have previously demonstrated that transmembrane prostatic acid phosphatase (TMPAP) prevents accumulation of ATP within the intracellular vesicular compartments (Wells et al., 2015). TMPAP should in theory also break down polyP and polyP degradation by TMPAP should be facilitated by the vesicular acidic environment.
Application of GPN triggered release of polyP into the cytosol that was recorded as an increase of polyP-JC-D8 fluorescence (90.5 ± 7.5% n = 244 cells, Figure 5c,d). These data suggest that in Previously we reported that application of polyP to cortical astrocytes induces the release of polyP into the medium (Holmstrom et al., 2013). Here we found that polyP is released by exocytosis of the VNUT-containing vesicles (Figure 6b,e) in response to application of Previously we found that even a short exposure of astrocytes to hypoxia results in an increase in intracellular calcium . In the present study, a short episode of hypoxia induces the release of polyP by fusion of VNUT-containing vesicles (n = 39, Figure 6f ) suggesting that release of polyP from astrocytes may play a certain role in mediating the physiological response to brain hypoxia.

| DISCUSSION
PolyP has been previously shown to be involved in a number of cellular processes (Angelova, Baev, et al., 2016;Morrissey et al., 2012;Schroder, Lorenz, Kurz, & Muller, 1999). Accumulation of polyP in cellular organelles and vesicles can indicate a specific signaling function.
In this study, we found that 39% of the total pool of intracellular polyP in astrocytes is located in mitochondria ( Figure 1) where it plays a role in bioenergetics (Angelova, Baev, et al., 2016;Pavlov et al., 2010) and calcium handling (Abramov et al., 2007;Baev et al., 2017;Elustondo et al., 2016). The level of polyP in mitochondria has been previously shown to be dependent on the metabolic state and the age of the cells (Pavlov et al., 2010). Astrocytes play a role of metabolic sensing and metabolic signaling (Marina et al., 2018) and the level of polyP may play important role in this process as signaling or/and metabolic molecule.
Although polyP is localized in 20-40% of astroglial lysosomes (Figure 2), only a small percentage of these lysosomes undergo exocytosis in response to various stimuli. Lysosomes can elongate the polyP chain in human fibroblasts and granulocytes (Cowling & Birnboim, 1994;Manzoni & Lewis, 2013;Pisoni & Lindley, 1992). PolyP has been shown to play an important role in acidocalcisomes (Docampo & Moreno, 2011). Although the presence of these vesicles is in astrocytes is disputable, eukaryotic acidocalcisomes belong to the group of lysosome-related organelles. They have a variety of functions, from the storage of cations and phosphorus to calcium signaling, autophagy, osmoregulation, blood coagulation, and inflammation Lander, Cordeiro, Huang, & Docampo, 2016)-all processes which can be regulating by lysosomal polyP in astrocytes.
The vesicular nucleotide transporter (VNUT) is a secretory vesicle protein which is responsible for the vesicular packaging of ATP (Sawada et al., 2008). Previously we and others reported that polyP could activate metabotropic P2Y 1 nucleotide receptors (Dinarvand et al., 2014;Holmstrom et al., 2013). Here we detected not only fusion of the VNUT-containing vesicles in response to various stimuli (Ca 2+ , pH, and activation of purinoceptors) but, importantly, release of polyP by exocytosis of these secretory vesicles. PolyP appears to play a role similar to that of ATP (as energy and signaling molecule), and here we found that in astrocytes these molecules could be potentially colocalized within the same intracellular vesicular compartments. At the moment we are not able to separate the functions which are specific to polyP and ATP but on the basis of indicator specificity and the effects induced by application of exogenous polyP can prove polyP induced polyP release-the signaling cascade which is typical for astrocytes. In summary, VNUT-containing vesicles appear to be the prime source of the releasable pool of polyP.
Considering involvement of the interaction of astrocytes and neurons in neurodegenerative, neuropsychiatric, and other neurological disorders Chiou, Lucassen, Sather, Kallianpur, & Connor, 2018;Liu, Teschemacher, & Kasparov, 2017), changes in polyP signaling may potentially be involved in the mechanism of pathology in CNS.