Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause late-onset Parkinson’s disease, but its physiological function has remained largely unknown. Here we report that LRRK2 activates a calcium-dependent protein kinase kinase-β (CaMKK-β)/adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway which is followed by a persistent increase in autophagosome formation. Simultaneously, LRKR2 overexpression increases the levels of the autophagy receptor p62 in a protein synthesis-dependent manner, and decreases the number of acidic lysosomes. The LRRK2-mediated effects result in increased sensitivity of cells to stressors associated with abnormal protein degradation. These effects can be mimicked by the lysosomal Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and can be reverted by an NAADP receptor antagonist or expression of dominant-negative receptor constructs. Collectively, our data indicate a molecular mechanism for LRRK2 deregulation of autophagy and reveal previously unidentified therapeutic targets.

SummaryA cytotoxic T lymphocyte (CTL) kills an infected or tumorigenic cell by Ca2+-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. Although inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum activates the store-operated Ca2+-influx pathway that is necessary for exocytosis, it is not a sufficient stimulus [1–4]. Here we identify the Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and its recently identified molecular target, two-pore channels (TPCs) [5–7], as being important for T cell receptor signaling in CTLs. We demonstrate that cytolytic granules are not only reservoirs of cytolytic proteins but are also the acidic Ca2+ stores mobilized by NAADP via TPC channels on the granules themselves, so that TPCs migrate to the immunological synapse upon CTL activation. Moreover, NAADP activates TPCs to drive exocytosis in a way that is not mimicked by global Ca2+ signals induced by IP3 or ionomycin, suggesting that critical, local Ca2+ nanodomains around TPCs stimulate granule exocytosis. Hence, by virtue of the NAADP/TPC pathway, cytolytic granules generate Ca2+ signals that lead to their own exocytosis and to cell killing. This study highlights a selective role for NAADP in stimulating exocytosis crucial for immune cell function and may impact on stimulus-secretion coupling in wider cellular contexts.

Background: The Ca2+-releasing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) acts via lysosomal two-pore channels (TPC2).Results: Tpcn2−/− cardiac myocytes showed reduced acute responses to β-adrenoreceptor stimulation and chronically reduced cardiac hypertrophy and arrhythmogenesis.Conclusion: Acute and chronic effects of cardiac β-adrenoreceptor stimulation depend on NAADP acting via TPC2 in lysosomes.Significance: NAADP/TPC2 signaling pathways offer new strategies for cardiac therapeutics.

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 ...