Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable 1 . This is thought to be due to the release of find-me signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages, and dendritic cells, leading to the prompt clearance of the dying cells 2 . However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or ectopic CD39 expression)Correspondence and requests for materials should be addressed to K.S.R. (ravi@virginia.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing interests.Author Contributions M.R.E. designed, performed and analyzed most of the experiments in this study with input from K.S.R. F.B.C. performed ATP quantitation experiments. P.T.C. helped with in vivo thymic apoptosis experiments. E.R.L. carried out HPLC analysis of supernatants. S.F.W. generated the CD39 expression plasmid and stable Jurkat cell lines. D.P. conducted phagocytosis experiments. A.K. and N.L. carried out the MS analysis and provided critical support in establishing the air-pouch model system. R.I.W. and J.J.L. carried out immunohistochemical detection of apoptotic cells in the thymus. M.O. and P.S. assisted with the BMDM generation and macrophage chemotaxis experiments. T.K.H. provided critical intellectual input in the preparation of the manuscript. K.S.R. provided overall coordination with respect to conception, design and supervision of the study. K.S.R. and M.R.E. wrote the manuscript with comments from co-authors. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 April 8. Most developing thymocytes (95%) undergo apoptosis; yet in steady-state only 1-2% are detectable as apoptotic 4,5 . It is hypothesized that dying thymocytes secrete soluble factors that attract resident phagocytes to promote prompt clearance 2,6 . To determine if apoptotic thymocytes release such factors, cell-free supernatants after apoptosis induction (by antiFas/CD95 crosslinking) were assessed for their ability to attract THP-1 monocytesor primary human monocytes in a transwell migration assay ( Figure 1a and Supplemental Figure S2). Apoptotic supernatants caused a 3-fold increase in monocyte migration compared to supernatants of live thymocytes. Such release of chemotactic factors was also seen with Jurkat cells (...

Apoptotic cells release ‘find-me’ signals at the earliest stages of death to recruit phagocytes1. The nucleotides ATP and UTP represent one class of find-me signals2, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 over-expression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the ‘selective’ plasma membrane permeability of early apoptotic cells to specific dyes3. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.

Huntington's disease (HD) is a fatal, dominantly inherited disorder caused by polyglutamine repeat expansion in the huntingtin (htt) gene. Here, we observe that HD mice develop hypothermia associated with impaired activation of brown adipose tissue (BAT). Although sympathetic stimulation of PPARgamma coactivator 1alpha (PGC-1alpha) was intact in BAT of HD mice, uncoupling protein 1 (UCP-1) induction was blunted. In cultured cells, expression of mutant htt suppressed UCP-1 promoter activity; this was reversed by PGC-1alpha expression. HD mice showed reduced food intake and increased energy expenditure, with dysfunctional BAT mitochondria. PGC-1alpha is a known regulator of mitochondrial function; here, we document reduced expression of PGC-1alpha target genes in HD patient and mouse striatum. Mitochondria of HD mouse brain show reduced oxygen consumption rates. Finally, HD striatal neurons expressing exogenous PGC-1alpha were resistant to 3-nitropropionic acid treatment. Altered PGC-1alpha function may thus link transcription dysregulation and mitochondrial dysfunction in HD.

No abstract

Huntington’s disease (HD) is caused by CAG / polyglutamine repeat expansions in the huntingtin (htt) gene, yielding proteins that misfold and resist degradation. HD belongs to a large class of neurodegenerative proteinopathies including Alzheimer’s disease, Parkinson’s disease, and tauopathies. Previous studies demonstrated that mutant htt interferes with transcriptional programs coordinated by PPARγ co-activator 1α (PGC-1α), a regulator of mitochondrial biogenesis and oxidative stress. To test if restoration of PGC-1α could treat HD, we attempted an in vivo genetic rescue in mice. We found that PGC-1α induction ameliorates HD neurodegeneration and virtually eliminates htt protein aggregation, in part by attenuating oxidative stress. Further studies revealed that PGC-1α promotes htt turnover and aggregate elimination by transactivation of TFEB, a master regulator of the autophagy-lysosome pathway, and that TFEB alone is capable of reducing htt aggregation and neurotoxicity, placing PGC-1α upstream of TFEB. PGC-1α and TFEB thus hold great promise as therapies for HD and other neurodegenerative proteinopathies.

Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca 2؉ -and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A 2b adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N 6 -ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolayers that endogenously express a luminal A 2b adenosine receptor, we found that basal as well as forskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A 2b receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/ nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis.Airways continuously remove noxious materials through a mucus clearance process that requires a complex regulation of airway surface liquid (ASL) 1 volume by active ion transport and coordinated ciliary beating. In normal airways, the cystic fibrosis transmembrane conductance regulator (CFTR) protein mediates liquid balance on airway surfaces by regulating Na ϩ absorption and by acting as a cyclic AMP-regulated Cl Ϫ channel. Proper regulation of salt and water movement across epithelial cell surfaces is required in both ASL compartments: (i) sufficient volume in the periciliary liquid ensures efficient cilia movement; and (ii) sufficient volume optimizes the viscoelastic properties of the mucus layer for transport (1). Defective CFTR activity causes the syndrome of cystic fibrosis (CF), which is characterized by accelerated Na ϩ -dependent volume absorption and failure to secrete Cl Ϫ , leading to ASL volume depletion (2). An alternative Ca 2ϩ -regulated Cl Ϫ channel that is functional in CF airway epithelial cells is also present on the mucosal surface but is relatively inactive in nonstimulated cells (3).The expression of purinergic receptors in airway epithelia that couple via different mechanisms to salt (and water) transport suggests a role of puriner...

Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPK␣␤␥ complex or HEK 293 cells to H 2 O 2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPK␣ and AMPK␤ subunits. In experiments using C-terminal truncation mutants of AMPK␣ (amino acids 1-312), we found that mutation of cysteine 299 to alanine diminished the ability of H 2 O 2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H 2 O 2 on kinase activity. Similar to the results obtained with H 2 O 2 -treated HEK 293 cells, activation and S-glutathionylation of the AMPK␣ subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H 2 O 2 are increased. These results demonstrate that physiologically relevant concentrations of H 2 O 2 can activate AMPK through oxidative modification of the AMPK␣ subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status. AMPK3 is a serine/threonine kinase that consists of three subunits, of which the ␣ subunit has inducible kinase activity and the ␤ and ␥ subunits have regulatory function. Formation of the ␣␤␥ complex is required for optimal allosteric activation of AMPK, which is induced by binding of AMP to the ␥ subunit (1-4). In addition to activation by AMP, phosphorylation of the Thr 172 residue of the ␣ subunit enhances kinase activity (5, 6). Recent studies have shown that the autoinhibitory domain (AID), located between amino acids 312 and 335 of the AMPK␣ subunit, is responsible for the lack of kinase activity under basal conditions (7-9), whereas AMP-induced conformational changes within the ␣␤␥ complex diminish function of the AID and lead to kinase activation.The regulation of AMPK activity is primarily thought to result from alterations in the intracellular AMP/ATP ratio, arising from diminished ATP generation due to hypoxia, glucose deprivation, heat shock, or reduction in mitochondrial oxidative phosphorylation or from increased ATP consumption, such as occurs during strenuous exercise (2, 10 -12). Once activated, AMPK can phosphorylate and modulate the function of essential metabolic pathways participating in the regulation of glucose and lipid homeostasis (13-15). A major effect of AMPK activation is in preserving energy for use under conditions where ATP is limiting (4,16). AMPK activation appears to prevent or diminish inflammation-associated organ injury, including the development of a...

Extracellular nucleotides regulate a broad range of cellular responses such as platelet aggregation, vascular tone, cell proliferation, mucociliary clearance, cardiac and skeletal muscle contraction, and neurotransmission (1-4). The effects of extracellular nucleotides are mediated by two large subfamilies of receptors, the ligand-gated channel P2X receptors (P2X 1-7 ) and the G protein-coupled P2Y receptors (P2Y 1, 2, 4, 6, 11 ) (4 -6). ADP, and less potently ATP, activates the P2Y 1 receptor, while ATP and UTP are the most potent agonists at the P2Y 2 receptor. In addition, ATP selectively activates the P2Y 11 as well as all P2X receptors. UTP is the selective agonist for the P2Y 4 receptor, 1 and UDP acts potently and selectively on the P2Y 6 receptor (7-10). The molecular identification and the wide tissue distribution of these nucleotide target proteins confirm that both adenosine and uridine di-and triphosphates subserve important extracellular signaling roles.ATP is known to be released in a Ca 2ϩ -dependent manner from storage compartments in nerve terminals, chromaffin cells, mast cells, and circulating platelets (1, 4, 11). Nucleotide release also occurs from non-excitatory tissues, and an autocrine/paracrine function for extracellular adenine nucleotides has been proposed (2, 3, 14 -22). Recent studies indicate that relatively large amounts of ATP and UTP are released by mechanical stimulation (e.g. shear stress, hypotonic swelling, or stretch) of epithelial and endothelial cells, smooth muscle, glial cells, fibroblasts, and hepatocytes (14 -23), and these nucleotides in the extracellular medium promote a robust activation of P2 receptors (16,21,(23)(24)(25).Ecto-nucleotidases and other ecto-enzymes metabolize extracellular ATP and UTP (26). However, the relative functional importance of these different enzymatic activities has not been defined for a given cell type, and little understanding is available of how these enzymes work in concert to produce the final pattern of metabolism of nucleotides. This is an important question given the aforementioned different selectivities of activation of P2Y receptors by both di-and triphosphate adenine and uridine nucleotides. As such, we have studied the kinetics of accumulation and metabolism of endogenous ATP in the extracellular medium of several different cell types. Our results illustrate for the first time a "constitutive" release of nucleotide that balances nucleotide hydrolysis and accounts for resting levels of extracellular nucleotides. We establish that the exchange of the ␥-phosphate between adenine and uridine nucleotides at steady state markedly exceeds the ecto-ATPase activity. Moreover, our results highlight the importance of a nucleotide pyrophosphatase activity and reveal that this activity approaches or exceeds that of the ecto-ATPase.