Yck3 casein kinase-mediated phosphorylation determines Ivy1 localization and function at endosomes and the vacuole.

The Saccharomyces cerevisiae casein kinase protein Yck3 is a central regulator at the vacuole that phosphorylates several proteins involved in membrane trafficking. Here, we set out to identify novel substrates of this protein. We found that endogenously tagged Yck3 localized not only at the vacuole, but also on endosomes. To disable Yck3 function, we generated a kinase-deficient mutant and thus identified the I-BAR-protein Ivy1 as a novel Yck3 substrate. Ivy1 localized to both endosomes and vacuoles, and Yck3 controlled this localization. A phosphomimetic Ivy1-SD mutant was found primarily on vacuoles, whereas its non-phosphorylatable SA variant strongly localized to endosomes, similar to what was observed upon deletion of Yck3. In vitro analysis revealed that Yck3-mediated phosphorylation strongly promoted Ivy1 recruitment to liposomes carrying the Rab7-like protein Ypt7. Modeling of Ivy1 with Ypt7 identified binding sites for Ypt7 and a positively charged patch, which were both required for Ivy1 localization. Strikingly, Ivy1 mutations in either site resulted in more cells with multilobed vacuoles, suggesting a partial defect in its membrane biogenesis. Our data thus indicate that Yck3-mediated phosphorylation controls both localization and function of Ivy1 in endolysosomal biogenesis.

The HOPS tethering complex is required to maintain signaling endosome identity and TORC1 activity.

The endomembrane system of eukaryotic cells is essential for cellular homeostasis during growth and proliferation. Previous work showed that a central regulator of growth, namely the target of rapamycin complex 1 (TORC1), binds both membranes of vacuoles and signaling endosomes (SEs) that are distinct from multivesicular bodies (MVBs). Interestingly, the endosomal TORC1, which binds membranes in part via the EGO complex, critically defines vacuole integrity. Here, we demonstrate that SEs form at a branch point of the biosynthetic and endocytic pathways toward the vacuole and depend on MVB biogenesis. Importantly, function of the HOPS tethering complex is essential to maintain the identity of SEs and proper endosomal and vacuolar TORC1 activities. In HOPS mutants, the EGO complex redistributed to the Golgi, which resulted in a partial mislocalization of TORC1. Our study uncovers that SE function requires a functional HOPS complex and MVBs, suggesting a tight link between trafficking and signaling along the endolysosomal pathway.

TORC1 Determines Fab1 Lipid Kinase Function at Signaling Endosomes and Vacuoles.

Organelles of the endomembrane system maintain their identity and integrity during growth or stress conditions by homeostatic mechanisms that regulate membrane flux and biogenesis. At lysosomes and endosomes, the Fab1 lipid kinase complex and the nutrient-regulated target of rapamycin complex 1 (TORC1) control the integrity of the endolysosomal homeostasis and cellular metabolism. Both complexes are functionally connected as Fab1-dependent generation of PI(3,5)P2 supports TORC1 activity. Here, we identify Fab1 as a target of TORC1 on signaling endosomes, which are distinct from multivesicular bodies, and provide mechanistic insight into their crosstalk. Accordingly, TORC1 can phosphorylate Fab1 proximal to its PI3P-interacting FYVE domain, which causes Fab1 to shift to signaling endosomes, where it generates PI(3,5)P2. This, in turn, regulates (1) vacuole morphology, (2) recruitment of TORC1 and the TORC1-regulatory Rag GTPase-containing EGO complex to signaling endosomes, and (3) TORC1 activity. Thus, our study unravels a regulatory feedback loop between TORC1 and the Fab1 complex that controls signaling at endolysosomes.

Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex.

The mechanism and regulation of fusion between autophagosomes and lysosomes/vacuoles are still only partially understood in both yeast and mammals. In yeast, this fusion step requires SNARE proteins, the homotypic vacuole fusion and protein sorting (HOPS) tethering complex, the RAB7 GTPase Ypt7, and its guanine nucleotide exchange factor (GEF) Mon1-Ccz1. We and others recently identified Ykt6 as the autophagosomal SNARE protein. However, it has not been resolved when and how lipid-anchored Ykt6 is recruited onto autophagosomes. Here, we show that Ykt6 is recruited at an early stage of the formation of these carriers through a mechanism that depends on endoplasmic reticulum (ER)-resident Dsl1 complex and COPII-coated vesicles. Importantly, Ykt6 activity on autophagosomes is regulated by the Atg1 kinase complex, which inhibits Ykt6 through direct phosphorylation. Thus, our findings indicate that the Ykt6 pool on autophagosomal membranes is kept inactive by Atg1 phosphorylation, and once an autophagosome is ready to fuse with vacuole, Ykt6 dephosphorylation allows its engagement in the fusion event.

The multi-functional SNARE protein Ykt6 in autophagosomal fusion processes.

Autophagy is a degradative pathway in which cytosolic material is enwrapped within double membrane vesicles, so-called autophagosomes, and delivered to lytic organelles. SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins are key to drive membrane fusion of the autophagosome and the lytic organelles, called lysosomes in higher eukaryotes or vacuoles in plants and yeast. Therefore, the identification of functional SNARE complexes is central for understanding fusion processes and their regulation. The SNARE proteins Syntaxin 17, SNAP29 and Vamp7/VAMP8 are responsible for the fusion of autophagosomes with lysosomes in higher eukaryotes. Recent studies reported that the R-SNARE Ykt6 is an additional SNARE protein involved in autophagosome-lytic organelle fusion in yeast, Drosophila, and mammals. These current findings point to an evolutionarily conserved role of Ykt6 in autophagosome-related fusion events. Here, we briefly summarize the principal mechanisms of autophagosome-lytic organelle fusion, with a special focus on Ykt6 to highlight some intrinsic features of this unusual SNARE protein.

A novel in vitro assay reveals SNARE topology and the role of Ykt6 in autophagosome fusion with vacuoles.

Autophagy is a catabolic pathway that delivers intracellular material to the mammalian lysosomes or the yeast and plant vacuoles. The final step in this process is the fusion of autophagosomes with vacuoles, which requires SNARE proteins, the homotypic vacuole fusion and protein sorting tethering complex, the RAB7-like Ypt7 GTPase, and its guanine nucleotide exchange factor, Mon1-Ccz1. Where these different components are located and function during fusion, however, remains to be fully understood. Here, we present a novel in vitro assay to monitor fusion of intact and functional autophagosomes with vacuoles. This process requires ATP, physiological temperature, and the entire fusion machinery to tether and fuse autophagosomes with vacuoles. Importantly, we uncover Ykt6 as the autophagosomal SNARE. Our assay and findings thus provide the tools to dissect autophagosome completion and fusion in a test tube.

Molecular mechanism to target the endosomal Mon1-Ccz1 GEF complex to the pre-autophagosomal structure.

During autophagy, a newly formed double membrane surrounds its cargo to generate the so-called autophagosome, which then fuses with a lysosome after closure. Previous work implicated that endosomal Rab7/Ypt7 associates to autophagosomes prior to their fusion with lysosomes. Here, we unravel how the Mon1-Ccz1 guanosine exchange factor (GEF) acting upstream of Ypt7 is specifically recruited to the pre-autophagosomal structure under starvation conditions. We find that Mon1-Ccz1 directly binds to Atg8, the yeast homolog of the members of the mammalian LC3 protein family. This requires at least one LIR motif in the Ccz1 C-terminus, which is essential for autophagy but not for endosomal transport. In agreement, only wild-type, but not LIR-mutated Mon1-Ccz1 promotes Atg8-dependent activation of Ypt7. Our data reveal how GEF targeting can specify the fate of a newly formed organelle and provide new insights into the regulation of autophagosome-lysosome fusion.

Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation.

The biogenesis of autophagosomes depends on the conjugation of Atg8-like proteins with phosphatidylethanolamine. Atg8 processing by the cysteine protease Atg4 is required for its covalent linkage to phosphatidylethanolamine, but it is also necessary for Atg8 deconjugation from this lipid to release it from membranes. How these two cleavage steps are coordinated is unknown. Here we show that phosphorylation by Atg1 inhibits Atg4 function, an event that appears to exclusively occur at the site of autophagosome biogenesis. These results are consistent with a model where the Atg8-phosphatidylethanolamine pool essential for autophagosome formation is protected at least in part by Atg4 phosphorylation by Atg1 while newly synthesized cytoplasmic Atg8 remains susceptible to constitutive Atg4 processing.The protease Atg4 mediates Atg8 lipidation, required for autophagosome biogenesis, but also triggers Atg8 release from the membranes, however is unclear how these steps are coordinated. Here the authors show that phosphorylation by Atg1 inhibits Atg4 at autophagosome formation sites.

Multivalent Rab interactions determine tether-mediated membrane fusion.

Membrane fusion at endomembranes requires cross-talk between Rab GTPases and tethers to drive SNARE-mediated lipid bilayer mixing. Several tethers have multiple Rab-binding sites with largely untested function. Here we dissected the lysosomal HOPS complex as a tethering complex with just two binding sites for the Rab7-like Ypt7 protein to determine their relevance for fusion. Using tethering and fusion assays combined with HOPS mutants, we show that HOPS-dependent fusion requires both Rab-binding sites, with Vps39 being the stronger Ypt7 interactor than Vps41. The intrinsic amphipathic lipid packaging sensor (ALPS) motif within HOPS Vps41, a target of the vacuolar kinase Yck3, is dispensable for tethering and fusion but can affect tethering if phosphorylated. In combination, our data demonstrate that a multivalent tethering complex uses its two Rab bindings to determine the place of SNARE assembly and thus fusion at endomembranes.

Dual effects of carnosine on energy metabolism of cultured cortical astrocytes under normal and ischemic conditions.

OBJECTIVE: The aim of this study was to investigate the effects of carnosine on the bioenergetic profile of cultured cortical astrocytes under normal and ischemic conditions. METHODS: The Seahorse Bioscience XF96 Extracellular Flux Analyzer was used to measure the oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) of cultured cortical astrocytes treated with and without carnosine under normal and ischemic conditions. RESULTS: Under the normal growth condition, the basal OCRs and ECARs of astrocytes were 21.72±1.59 pmol/min/µg protein and 3.95±0.28 mpH/min/µg protein respectively. Mitochondrial respiration accounted for ~80% of the total cellular respiration and 85% of this coupled to ATP synthesis. Carnosine significantly reduced basal OCRs and ECARs and ATP-linked respiration, but it strikingly increased the spare respiratory capacity of astrocytes. The cellular ATP level in carnosine-treated astrocytes was reduced to ~42% of the control. However, under the ischemic condition, carnosine upregulated the mitochondrial respiratory and cellular ATP content of astrocytes exposed to 8h of oxygen-glucose deprivation (OGD) followed by 24 h of recovery under the normal growth condition. CONCLUSIONS: Carnosine may be an endogenous regulator of astrocyte energy metabolism and a clinically safe therapeutic agent for promoting brain energy metabolism recovery after ischemia/reperfusion injury.

Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes.

Endoplasmic reticulum stress induced by tunicamycin and thapsigargin protects against transient ischemic brain injury: Involvement of PARK2-dependent mitophagy.

Transient cerebral ischemia leads to endoplasmic reticulum (ER) stress. However, the contributions of ER stress to cerebral ischemia are not clear. To address this issue, the ER stress activators tunicamycin (TM) and thapsigargin (TG) were administered to transient middle cerebral artery occluded (tMCAO) mice and oxygen-glucose deprivation-reperfusion (OGD-Rep.)-treated neurons. Both TM and TG showed significant protection against ischemia-induced brain injury, as revealed by reduced brain infarct volume and increased glucose uptake rate in ischemic tissue. In OGD-Rep.-treated neurons, 4-PBA, the ER stress releasing mechanism, counteracted the neuronal protection of TM and TG, which also supports a protective role of ER stress in transient brain ischemia. Knocking down the ER stress sensor Eif2s1, which is further activated by TM and TG, reduced the OGD-Rep.-induced neuronal cell death. In addition, both TM and TG prevented PARK2 loss, promoted its recruitment to mitochondria, and activated mitophagy during reperfusion after ischemia. The neuroprotection of TM and TG was reversed by autophagy inhibition (3-methyladenine and Atg7 knockdown) as well as Park2 silencing. The neuroprotection was also diminished in Park2(+/-) mice. Moreover, Eif2s1 and downstream Atf4 silencing reduced PARK2 expression, impaired mitophagy induction, and counteracted the neuroprotection. Taken together, the present investigation demonstrates that the ER stress induced by TM and TG protects against the transient ischemic brain injury. The PARK2-mediated mitophagy may be underlying the protection of ER stress. These findings may provide a new strategy to rescue ischemic brains by inducing mitophagy through ER stress activation.

Histamine H3 receptors aggravate cerebral ischaemic injury by histamine-independent mechanisms.

The role of the histamine H3 receptor (H3R) in cerebral ischaemia/reperfusion (I/R) injury remains unknown. Here we show that H3R expression is upregulated after I/R in two mouse models. H3R antagonists and H3R knockout attenuate I/R injury, which is reversed by an H3R-selective agonist. Interestingly, H1R and H2R antagonists, a histidine decarboxylase (HDC) inhibitor and HDC knockout all fail to compromise the protection by H3R blockade. H3R blockade inhibits mTOR phosphorylation and reinforces autophagy. The neuroprotection by H3R antagonism is reversed by 3-methyladenine and siRNA for Atg7, and is diminished in Atg5⁻/⁻ mouse embryonic fibroblasts. Furthermore, the peptide Tat-H3R(CT414-436), which blocks CLIC4 binding with H3Rs, or siRNA for CLIC4, further increases I/R-induced autophagy and protects against I/R injury. Therefore, H3R promotes I/R injury while its antagonism protects against ischaemic injury via histamine-independent mechanisms that involve suppressing H3R/CLIC4 binding-activated autophagy, suggesting that H3R inhibition is a therapeutic target for cerebral ischaemia.

Modulation of astrocytic glutamine synthetase expression and cell viability by histamine in cultured cortical astrocytes exposed to OGD insults.

Histamine, a neurotransmitter or neuromodulator has been demonstrated to be neuroprotective in cerebral ischemia. However, few reports concern its function on astrocytes during cerebral ischemia. The purpose of this study was to investigate the effects of histamine on astrocytic cell damage and glutamate signaling, especially on glutamine synthetase (GS) expression in primary cultured cortical astrocytes exposed to oxygen-glucose deprivation (OGD) insult. OGD for 6h caused a severe damage of astrocytic mitochondrial function, and decreased GS expression and then increased the extracellular glutamate level. Pretreatment with histamine significantly prevented the cell damage and rescued the expression of GS in a concentration-dependent manner. The protective effect of histamine on astrocytic cell damage could be partly reversed either by H1 receptor antagonist pyrilamine or H2 receptor antagonist cimetidine. However, the regulatory effect of histamine on GS expression was antagonized only by pyrilamine. In addition, bisindolylmaleimide II, a broad-spectrum inhibitor of PKC, reversed the regulatory action of histamine on GS expression. These results indicate that histamine can effectively protect against OGD-induced cell damage in astrocytes through H1 and H2 receptors, and its regulatory effect on astrocytic GS expression may be due to the activation of H1 receptor and PKC pathway. Histamine may be an endogenous protective factor and calls for its further study as a regulator of astrocyte function during ischemic stroke.

Cerebral ischemia-reperfusion-induced autophagy protects against neuronal injury by mitochondrial clearance.

Cerebral ischemia-reperfusion (I-R) is a complex pathological process. Although autophagy can be evoked by ischemia, its involvement in the reperfusion phase after ischemia and its contribution to the fate of neurons remains largely unknown. In the present investigation, we found that autophagy was activated in the reperfusion phase, as revealed in both mice with middle cerebral artery occlusion and oxygen-glucose deprived cortical neurons in culture. Interestingly, in contrast to that in permanent ischemia, inhibition of autophagy (by 3-methyladenine, bafilomycin A 1, Atg7 knockdown or in atg5(-/-) MEF cells) in the reperfusion phase reinforced, rather than reduced, the brain and cell injury induced by I-R. Inhibition of autophagy either with 3-methyladenine or Atg7 knockdown enhanced the I-R-induced release of cytochrome c and the downstream activation of apoptosis. Moreover, MitoTracker Red-labeled neuronal mitochondria increasingly overlapped with GFP-LC3-labeled autophagosomes during reperfusion, suggesting the presence of mitophagy. The mitochondrial clearance in I-R was reversed by 3-methyladenine and Atg7 silencing, further suggesting that mitophagy underlies the neuroprotection by autophagy. In support, administration of the mitophagy inhibitor mdivi-1 in the reperfusion phase aggravated the ischemia-induced neuronal injury both in vivo and in vitro. PARK2 translocated to mitochondria during reperfusion and Park2 knockdown aggravated ischemia-induced neuronal cell death. In conclusion, the results indicated that autophagy plays different roles in cerebral ischemia and subsequent reperfusion. The protective role of autophagy during reperfusion may be attributable to mitophagy-related mitochondrial clearance and inhibition of downstream apoptosis. PARK2 may be involved in the mitophagy process.

Acidic preconditioning protects against ischemia-induced brain injury.

Ischemic preconditioning protects against cerebral ischemia. Recent investigations indicated that acidic preconditioning (APC) protects against ischemia-induced cardiomyocytes injury. However, it is not clear whether APC can protect against cerebral ischemia. To address this issue, C57BL/6 mice were exposed 3 times at 10-min intervals to a normoxic atmosphere containing 20% CO(2) for 5 min before being further subjected to bilateral common carotid artery occlusion. APC reversed the ischemia-induced brain injury as revealed by improved performance in passive avoidance experiments and decreased neuron loss in the hippocampal CA1 region. Consistently, both APC-treated brain slices and primary cultured neurons were more resistant to oxygen-glucose-deprivation (OGD)-induced injury, in a pH- and time-dependent manner, as revealed by reversed cell/tissue viability. In addition, the APC treatment prevented OGD-induced mitochondrial transmembrane potential loss and apoptosis, which was inhibited by the mitochondrial permeability transport pore opener atractyloside. Taken together, these findings indicated that APC protects against ischemia-induced neuronal injury. The beneficial effects may be attributed, at least in part, to decreased mitochondria-dependent neuronal apoptosis.