Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Regulates Autophagy in Cultured Astrocytes

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2؉ -mobilizing messenger that in many cells releases Ca 2؉ from the endolysosomal system. Recent studies have shown that NAADP-induced Ca 2؉ mobilization is mediated by the two-pore channels (TPCs). Whether NAADP acts as a messenger in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that intracellular delivery of NAADP evokes Ca 2؉ signals from acidic organelles in rat astrocytes and that these signals are potentiated upon overexpression of TPCs. We also show that NAADP increases acidic vesicular organelle formation and levels of the autophagic markers, LC3II and beclin-1. NAADP-mediated increases in LC3II levels were reduced in cells expressing a dominant-negative TPC2 construct. Our data provide evidence that NAADP-evoked Ca 2؉ signals mediated by TPCs regulate autophagy. Increases in cytosolic Ca2ϩ regulate a myriad of cellular functions including information processing in the central nervous system (1). In many cells, these increases can be driven by mobilization of intracellular Ca 2ϩ stores (2). Much attention has focused on the endoplasmic reticulum as a Ca 2ϩ store (3), but accumulating evidence also implicates acidic organelles such as lysosomes in the control of Ca 2ϩ dynamics (4). In particular, NAADP 2 has emerged as a novel intracellular Ca 2ϩ -mobilizing messenger that links cell surface stimulation to the release of Ca 2ϩ from acidic Ca 2ϩ stores (5). Changes in the concentration of cytosolic Ca 2ϩ in glial cells are key for bidirectional control of neuronal activity (1). Previous studies have shown that extracellular application of NAADP can evoke Ca 2ϩ signals in astrocytes, consistent with a messenger role for NAADP in this cell type, following its internalization (6). Interpretation of these results, however, is clouded by the demonstrated lack of specificity with respect to related nucleotides (6) and by the potential activation by NAADP of cell surface purinergic receptors (7). Whether NAADP acts as an intracellular messenger in astrocytes is therefore unclear.Although the role of inositol trisphosphate and ryanodine receptors is established in mediating Ca 2ϩ release from the ER in response to inositol trisphosphate and cyclic ADP-ribose, respectively (2), the molecular basis for Ca 2ϩ release by NAADP from acidic organelles is less certain (8). In a series of recent studies, however, a novel family of Ca 2ϩ channels, known as the two-pore channels (TPCs), have emerged as likely targets (9). Thus, TPCs localize to endosomes and/or lysosomes through an identified targeting motif and enhance NAADPmediated cytosolic Ca 2ϩ signals when overexpressed (10 -12). Inhibition of TPC expression/function using siRNA (10), TPC knock-out mice (11), or a dominant-negative TPC construct (10) reduces NAADP-evoked Ca 2ϩ signals, and biophysical analyses indicate that TPCs are NAADP-gated Ca 2ϩ -permeable channels (13). Moreover, a functional role for TPCs has been identified in events such ...

Effect of aging on calcium signaling in C57Bl6J mouse cerebral arteries

“…The overexpression of calstabin-2 was also implicated in cell hypertrophy and apoptosis [66]. Finally, the increase of TPCN1 could increase the arteriosclerosis risk through the implication of NAADP in autophagy [47] and inflammation [64], two components of arteriosclerosis.…”

Identification of Two-pore Channel 2 as a Novel Regulator of Osteoclastogenesis

“…TPC2 is required for myocyte differentiation (14,15). This channel is also involved in autophagy in astrocytes (16). Considering bone physiology, osteoclasts are regulated by calcium signaling (17).…”

Two-pore channels (TPCs): Novel voltage-gated ion channels with pleiotropic functions

“…38 This process involves a degradation by the autolysosome, which is formed by fusion of an autophagosome and a lysosome, which suggests that NAADP signaling and TPCs could be involved in this process. 79 The first study that established a possible relationship between TPCs and autophagy showed that in rat astrocytes NAADP increased acidic vesicular organelle formation and contributed to increased autophagy markers as LC3II and Beclin1; 35 this data provided an evidence that NAADP-evoked Ca 2C signals mediated by TPCs regulated autophagy in astrocytes. 35 Later, Kayala et al 36 associated the absence of presenilins (which are important for the proteolysis process during autophagy) with abnormalities in lysosomal Ca 2C and changes in expression levels and dimerization of TPCs, which they hypothesized that could lead to an autophagy disruption in these cells.…”

“…79 The first study that established a possible relationship between TPCs and autophagy showed that in rat astrocytes NAADP increased acidic vesicular organelle formation and contributed to increased autophagy markers as LC3II and Beclin1; 35 this data provided an evidence that NAADP-evoked Ca 2C signals mediated by TPCs regulated autophagy in astrocytes. 35 Later, Kayala et al 36 associated the absence of presenilins (which are important for the proteolysis process during autophagy) with abnormalities in lysosomal Ca 2C and changes in expression levels and dimerization of TPCs, which they hypothesized that could lead to an autophagy disruption in these cells. In 2013, Lu et al 80 found that overexpression of TPC2 in HeLa or mouse embryonic stem cells leaded to an acumulattion of LC3II, p62 and autophagosomes, suggesting an inhibition of fusion between the lysosome and the autophagosome.…”

“…Today it is known that TPCs have a role not only in ion signaling but also in intracellular vesicle trafficking, [18][19][20] participating in a wide range of pathophysiological processes; that is why in the last years TPCs have received an increased attention from the main journals in the field, and they have been related to the regulation of many biological functions at different levels, including pancreatic b-cell function, 21,22 thermogenesis, 23 nutrient sensing, 6 endolysosomal transport and functions, 19,20 exocytosis, 24,25 cytokinesis, 26 fertilization and embryogenesis, 27 cell differentiation, [28][29][30][31] angiogenesis, 32 endothelium activation mediated by histamine, 33 smooth muscle contraction, 34 autophagy, [35][36][37][38][39][40] skin pigmentation, 41 or even to the Ebola virus infection mechanism. 42 As well, recent studies have suggested their possible implication in the pathogenesis of Alzheimer disease, 36 46 Thus, TPCs have become attractive therapeutic targets, although it is necessary to go deeper into their main functions and mechanisms of action not only to clinically relevant compounds could be designed but also to understand the pathophysiology of an increased wide range of diseases related to TPCs function/dysfunction.…”