In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Phosphoinositide 3 kinase enhancer (PIKE) is a recently identified nuclear GTPase that activates nuclear phosphoinositide 3-kinase (PI3 kinase). We have identified, cloned and characterized a new form of PIKE, designated PIKE-L, which, unlike the nuclear PIKE-S, localizes to both the cytoplasm and the nucleus. We demonstrate physiologic binding of PIKE-L to Homer, an adaptor protein known to link metabotropic glutamate receptors to multiple intracellular targets including the inositol 1,4,5-trisphosphate receptor (IP3R). We show that activation of group I metabotropic glutamate receptors (mGluRIs) enhances formation of an mGluRI-Homer-PIKE-L complex, leading to activation of PI3 kinase activity and prevention of neuronal apoptosis. Our findings indicate that this complex mediates the well-known ability of agonists of mGluRI to prevent neuronal apoptosis.

The inositol 1,4,5-trisphosphate (InsP3) receptor acts as an InsP3-gated Ca2+ release channel in a variety of cell types. Type 1 InsP3 receptor (IP3R1) is the major neuronal member of the IP3R family in the central nervous system, predominantly enriched in cerebellar Purkinje cells but also concentrated in neurons in the hippocampal CA1 region, caudate-putamen, and cerebral cortex. Here we report that most IP3R1-deficient mice generated by gene targeting die in utero, and born animals have severe ataxia and tonic or tonic-clonic seizures and die by the weaning period. An electroencephalogram showed that they suffer from epilepsy, indicating that IP3R1 is essential for proper brain function. However, observation by light microscope of the haematoxylin-eosin staining of the brain and peripheral tissues of IP3R1-deficient mice showed no abnormality, and the unique electrophysiological properties of the cerebellar Purkinje cells of IP3R1-deficient mice were not severely impaired.

Elevating Akt activation is an obvious clinical strategy to prevent progressive neuronal death in neurological diseases. However, this endeavor has been hindered because of the lack of specific Akt activators. Here, from a cell-based high-throughput chemical genetic screening, we identified a small molecule SC79 that inhibits Akt membrane translocation, but paradoxically activates Akt in the cytosol. SC79 specifically binds to the PH domain of Akt. SC79-bound Akt adopts a conformation favorable for phosphorylation by upstream protein kinases. In a hippocampal neuronal culture system and a mouse model for ischemic stroke, the cytosolic activation of Akt by SC79 is sufficient to recapitulate the primary cellular function of Akt signaling, resulting in augmented neuronal survival. Thus, SC79 is a unique specific Akt activator that may be used to enhance Akt activity in various physiological and pathological conditions.

Inositol phosphates are well-known signaling molecules, whereas the inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (InsP7/IP7) and bis-diphosphoinositol tetrakisphosphate (InsP8/IP8), are less well characterized. We demonstrate physiologic regulation of Dictyostelium chemotaxis by InsP7 mediated by its competition with PtdIns(3,4,5)P3 for binding pleckstrin homology (PH) domain-containing proteins. Chemoattractant stimulation triggers rapid and sustained elevations in InsP7/InsP8 levels. Depletion of InsP7 and InsP8 by deleting the gene for InsP6 kinase (InsP6K/IP6K), which converts inositol hexakisphosphate (InsP6/IP6) to InsP7, causes rapid aggregation of mutant cells and increased sensitivity to cAMP. Chemotaxis is mediated by membrane translocation of certain PH domain-containing proteins via specific binding to PtdIns(3,4,5)P3. InsP7 competes for PH domain binding with PtdIns(3,4,5)P3 both in vitro and in vivo. InsP7 depletion enhances PH domain membrane translocation and augments downstream chemotactic signaling activity.

Enhancement of cerebral blood flow by hypoxia is critical for brain function, but signaling systems underlying its regulation have been unclear. We report a pathway mediating hypoxia-induced cerebral vasodilation in studies monitoring vascular disposition in cerebellar slices and in intact mouse brains using two-photon intravital laser scanning microscopy. In this cascade, hypoxia elicits cerebral vasodilation via the coordinate actions of H 2 S formed by cystathionine β-synthase (CBS) and CO generated by heme oxygenase (HO)-2. Hypoxia diminishes CO generation by HO-2, an oxygen sensor. The constitutive CO physiologically inhibits CBS, and hypoxia leads to increased levels of H 2 S that mediate the vasodilation of precapillary arterioles. Mice with targeted deletion of HO-2 or CBS display impaired vascular responses to hypoxia. Thus, in intact adult brain cerebral cortex of HO-2-null mice, imaging mass spectrometry reveals an impaired ability to maintain ATP levels on hypoxia.gas biology | neurovascular unit | energy metabolism | gasotransmitter T he cerebral circulation is maintained by autoregulation, which prevents marked alterations in response to changes in blood pressure, whereas functional hyperemia links blood flow to neural activity (1). Blood flow regulation in the brain is modulated by O 2 (2), with increased cerebral blood flow in response to hypoxia critical for protecting the brain against diverse insults. Such regulation also participates in functional hyperemia, as demonstrated by functional MRI investigations indicating a transient decrease in O 2 levels preceding activation of blood flow in response to neuronal firing (3).Alterations in cerebral blood flow in response to hypoxia and neural activity are mediated via several neurotransmitter systems, with prominent involvement of the gaseous mediator nitric oxide (NO) (1, 2). In response to glutamate acting on NMDA receptors, neuronal NO synthase (nNOS) is activated by increases in intracellular calcium, with the generated NO stimulating soluble guanylyl cyclase, thereby increasing cGMP levels to dilate blood vessels (4). Functional hyperemia is decreased by ∼50% in rats in response to inhibition of nNOS (5). Another gaseous mediator, CO (6-8), is also vasoactive. In some blood vessel systems (e.g., liver sinusoids), CO causes vasodilation, and inhibition of its biosynthetic enzyme HO-2 leads to vasoconstriction (9-13). However, in the cerebral circulation, CO elicits vasoconstriction. Thus, HO inhibitors cause cerebral vasodilation, an effect reversed by CO (14). This action of CO cannot be readily explained by previously identified CO receptors, such as soluble guanylyl cyclase (6-12, 15) or potassium channels (13, 16), both of which mediate vasodilation. The CO and NO systems interface; thus, the vasodilatory actions of HO inhibitors are partially reversed by inhibitors of NOS (14). A third gaseous mediator, H 2 S, is also vasoactive, eliciting vasodilation in both the peripheral and cerebral circulation (17-21). H 2 S can be physiologically ...

Materials and MethodsNeuronal Cell Cultures and Cytotoxicity. Primary cortical or hippocampal neuronal cultures were prepared as previously described (7). To induce excitotoxicity, the cells were prewashed with Tris-buffered control salt solution (CSS; 120 mM NaCl͞5.4 mM KCl͞1.8 mMCaCl 2 ͞25 mM Tris⅐HCl, pH 7.4͞15 mM glucose) and treated with CSS containing 300 M NMDA for 5 min. Toxicity was assayed 20-24 h after NMDA exposure by microscopic examination with computer-assisted cell counting. Total and dead cells were determined by nuclei staining with 100 ng͞ml 4Ј,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) (10 M), respectively. After a 10-min incubation, the cells were examined under a fluorescence microscope (Zeiss) with excitation at 360 nm. Cell death was determined as the ratio of dead to total cells and quantified by counting 1,000 cells. For staining of dead cells by terminal deoxynucleotidyltransferasemediated dUTP nick end labeling (TUNEL) assay, cells were fixed in 4% paraformaldehyde͞PBS and then stained by using a TUNEL Assay Kit (Molecular Probes) following protocols provided by the manufacturer. The cell death inhibitory effect of various agents was examined essentially as described (7,8).Western blotting was performed essentially as described (9).Focal Cerebral Ischemia Model. C57BL͞6 mice weighing 17-25 g were used for transient focal cerebral ischemia. After a midline neck incision, the left common carotid artery was isolated from the vagus nerve and ligated. The external carotid artery also was ligated, and the internal carotid artery was isolated carefully from the surrounding tissue. An 8-0 nylon filament (Ethicon, Somerville, NJ) was inserted into the common carotid artery through a small incision made in the proximity of the carotid bifurcation and advanced to the proximal part of the anterior cerebral artery to compromise the middle cerebral artery (MCA) flow. The filament was fixed in position by ligature. In sham-operated animals, the above procedures were performed except for the insertion of an intraluminal filament. For histological examinations, mice were perfused transcardially with heparinized PBS followed by 4% paraformaldehyde͞PBS for tissue fixation. Brains were removed and postfixed in 4% paraformaldehyde͞PBS at 4°C overnight. Coronal frozen sections (20 m) were prepared on a cryostat and stored at Ϫ80°C until use. The frozen sections were thawed, washed three times in PBS, permeabilized with 0.1% Triton X-100͞PBS at room temperature for 5 min, and then blocked in 5% skim milk͞3% BSA/PBS for 60 min. Subsequently, they were incubated with primary antibodies (1:200) at 4°C overnight and with secondary antibodies at room temperature for 2 h, and immunoreactivity was visualized by the avidin-biotin complex (ABC) method.Cell Lines and Cell Death Assays. HeLa cells, a human cervical carcinoma-derived cell line, were maintained in DMEM with 10% FBS, 2 mM L-glutamine, and 100 units of penicillin͞ streptomycin at 37°C with a 5% CO 2 atmosphere in a humidified incubator. P...

Diphosphoinositol pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate contain pyrophosphate bonds. InsP7 is formed from inositol hexakisphosphate (InsP6) by a family of three inositol hexakisphosphate kinases (InsP6K). In this study we establish one of the InsP6Ks, InsP6K2, as a physiologic mediator of cell death. Overexpression of wild-type InsP6K2 augments the cytotoxic actions of multiple cell stressors in diverse cell lines, whereas transfection with a dominant negative InsP6K2 decreases cell death. During cell death, InsP6 kinase activity is enhanced, and intracellular InsP7 level is augmented. Deletion of InsP6K2 but not the other forms of InsP6K diminishes cell death, suggesting that InsP6K2 is the major InsP6 kinase involved in cell death. Cytotoxicity is associated with a translocation of InsP6K2 from nuclei to mitochondria, whereas the intracellular localization of the other isoforms of the enzyme does not change. The present study provides compelling evidence that endogenous InsP6K2, by generating InsP7, provides physiologic regulation of the apoptotic process.Inositol phosphates are major intracellular signaling molecules with the best known of these, inositol 1,4,5-trisphosphate, releasing intracellular calcium (1, 2). Inositol pyrophosphates occur physiologically, the most prominent being diphosphoinositol pentakisphosphate (InsP7) 1 and bis-diphosphoinositol tetrakisphosphate (InsP8) (3, 4). Inositol pyrophosphates have been implicated in diverse functions including vesicular trafficking (5-7), DNA recombination and repair (8, 9), and chemotaxis (10). Molecular mechanisms whereby they influence these functions are not well established, although there is evidence that InsP7 can compete for PH domain binding with PtdIns(3,4,5)P3 (10) and serve as a phosphate donor to proteins. 2The formation of InsP7 and InsP8 is mediated by a family of three inositol hexakisphosphate kinases (InsP6Ks) (11)(12)(13)(14). Differential functions of the enzymes are implied by their varying intracellular localizations with InsP6K2 being exclusively nuclear, whereas the other two enzymes are both cytosolic and nuclear (12). A role for InsP6K2 in cell death is suggested by the findings of Lindner and co-workers (15, 16) that deletion of InsP6K2 prevents apoptotic actions of interferon ␤ (15, 16) and ␥-irradiation (15, 16) in ovarian carcinoma cells, whereas transfection of InsP6K2 augments cell death, and interferon ␤ treatment leads to enhanced InsP6K2 activity.In the present study we show that transfection of InsP6K2 in multiple cell lines augments apoptotic actions of several cell stressors, accompanied by major increases in InsP7 formation. Although transfection of all three subtypes of InsP6K increases cell death, deleting InsP6K2, but not InsP6K1 or InsPK3, prevents apoptosis. Additionally, apoptotic stimuli elicit translocation of InsP6K2 from nuclei to damaged mitochondria, whereas no alteration in the intracellular localizations of InsP6K1 or InsP6K3 is demonstrable. EXPERIMENTAL PROCEDURESCell L...