The Role of SUMOylation and Ubiquitination in Brain Ischaemia: Critical Concepts and Clinical Implications.

Brain ischaemia is a severe form of metabolic stress that activates a cascade of pathological events involving many signalling pathways. Modulation of these pathways is largely mediated by post-translational modifications (PTMs). Indeed, PTMs can rapidly modify pre-existing proteins by attaching chemical or polypeptide moieties to selected amino acid residues, altering their functions, stability, subcellular localizations, or interactions with other proteins. Subsequently, related signalling pathways can be substantially affected. Thus, PTMs are widely deployed by cells as an adaptive strategy at the front line to efficiently cope with internal and external stresses. Many types of PTMs have been identified, including phosphorylation, O-GlcNAcylation, small ubiquitin-like modifier (SUMO) modification (SUMOylation), and ubiquitination. All these PTMs have been studied in brain ischaemia to some extent. In particular, a large body of evidence has demonstrated that both global SUMOylation and ubiquitination are massively activated after brain ischaemia, and this activation may play a critical role in defining the fate and function of cells in the post-ischaemic brain. The goal of this review will be to summarize the current findings on SUMOylation and ubiquitination in brain ischaemia and discuss their clinical implications.

Quantitative high-throughput screening identifies cytoprotective molecules that enhance SUMO conjugation via the inhibition of SUMO-specific protease (SENP)2.

The development of novel neuroprotective treatments for acute stroke has been fraught with failures, which supports the view of ischemic brain damage as a highly complex multifactorial process. Post-translational modifications such as small ubiquitin-like modifier (SUMO)ylation have emerged as critical molecular regulatory mechanisms in states of both homeostasis and ischemic stress, as evidenced by our previous work. Accordingly, the clinical significance of the selective control of the global SUMOylation process has become apparent in studies of ischemic pathobiology and pathophysiology. Herein, we describe a process capable of identifying and characterizing small molecules with the potential of targeting the SUMO system through inhibition of SUMO deconjugation in an effort to develop novel stroke therapies.-Bernstock, J. D., Ye, D., Smith, J. A., Lee, Y.-J., Gessler, F. A., Yasgar, A., Kouznetsova, J., Jadhav, A., Wang, Z., Pluchino, S., Zheng, W., Simeonov, A., Hallenbeck, J. M., Yang, W. Quantitative high-throughput screening identifies cytoprotective molecules that enhance SUMO-conjugation via the inhibition of SUMO-specific protease (SENP)2.

A combined banding method that allows the reliable identification of chromosomes as well as differentiation of AT- and GC-rich heterochromatin.

Сonstitutive heterochromatin areas are revealed by differential staining as C-positive chromosomal regions. These C-positive bands may greatly vary by location, size, and nucleotide composition. CBG-banding is the most commonly used method to detect structural heterochromatin in animals. The difficulty in identification of individual chromosomes represents an unresolved problem of this method as the body of the chromosome is stained uniformly and does not have banding pattern beyond C-bands. Here, we present the method that we called CDAG for sequential heterochromatin staining after differential GTG-banding. The method uses G-banding followed by heat denaturation in the presence of formamide with consecutive fluorochrome staining. The new technique is valid for the concurrent revealing of heterochromatin position due to differential banding of chromosomes and heterochromatin composition (AT-/GC-rich) in animal karyotyping.

Ubc9 overexpression and SUMO1 deficiency blunt inflammation after intestinal ischemia/reperfusion.

The intestinal epithelium constitutes a crucial defense to the potentially life-threatening effects of gut microbiota. However, due to a complex underlying vasculature, hypoperfusion and resultant tissue ischemia pose a particular risk to function and integrity of the epithelium. The small ubiquitin-like modifier (SUMO) conjugation pathway critically regulates adaptive responses to metabolic stress and is of particular significance in the gut, as inducible knockout of the SUMO-conjugating enzyme Ubc9 results in rapid intestinal epithelial disintegration. Here we analyzed the pattern of individual SUMO isoforms in intestinal epithelium and investigated their roles in intestinal ischemia/reperfusion (I/R) damage. Immunostaining revealed that epithelial SUMO2/3 expression was almost exclusively limited to crypt epithelial nuclei in unchallenged mice. However, intestinal I/R or overexpression of Ubc9 caused a remarkable enhancement of epithelial SUMO2/3 staining along the crypt-villus axis. Unexpectedly, a similar pattern was found in SUMO1 knockout mice. Ubc9 transgenic mice, but also SUMO1 knockout mice were protected from I/R injury as evidenced by better preserved barrier function and blunted inflammatory responses. PCR array analysis of microdissected villus-tip epithelia revealed a specific epithelial contribution to reduced inflammatory responses in Ubc9 transgenic mice, as key chemotactic signaling molecules such as IL17A were significantly downregulated. Together, our data indicate a critical role particularly of the SUMO2/3 isoforms in modulating responses to I/R and provide the first evidence that SUMO1 deletion activates a compensatory process that protects from ischemic damage.

Development of a systems-based in situ multiplex biomarker screening approach for the assessment of immunopathology and neural tissue plasticity in male rats after traumatic brain injury.

Traumatic brain injuries (TBIs) pose a massive burden of disease and continue to be a leading cause of morbidity and mortality throughout the world. A major obstacle in developing effective treatments is the lack of comprehensive understanding of the underlying mechanisms that mediate tissue damage and recovery after TBI. As such, our work aims to highlight the development of a novel experimental platform capable of fully characterizing the underlying pathobiology that unfolds after TBI. This platform encompasses an empirically optimized multiplex immunohistochemistry staining and imaging system customized to screen for a myriad of biomarkers required to comprehensively evaluate the extent of neuroinflammation, neural tissue damage, and repair in response to TBI. Herein, we demonstrate that our multiplex biomarker screening platform is capable of evaluating changes in both the topographical location and functional states of resident and infiltrating cell types that play a role in neuropathology after controlled cortical impact injury to the brain in male Sprague-Dawley rats. Our results demonstrate that our multiplex biomarker screening platform lays the groundwork for the comprehensive characterization of changes that occur within the brain after TBI. Such work may ultimately lead to the understanding of the governing pathobiology of TBI, thereby fostering the development of novel therapeutic interventions tailored to produce optimal tissue protection, repair, and/or regeneration with minimal side effects, and may ultimately find utility in a wide variety of other neurological injuries, diseases, and disorders that share components of TBI pathobiology.

Global SUMOylation facilitates the multimodal neuroprotection afforded by quercetin against the deleterious effects of oxygen/glucose deprivation and the restoration of oxygen/glucose.

The putative neuroprotective properties of various flavonoids have long been reported. Among this class of chemicals, quercetin, a major flavone/flavonol naturally occurring in plants, deserves focused attention because of the myriad of beneficial effects observed in various in vitro and in vivo models of central nervous system damage/degeneration. However, the mechanisms governing the beneficial outcomes mediated by quercetin remain to be elucidated. In an effort to define the underlying molecular mechanisms, our study employed human/rat neuroblastoma cell lines (SHSY5Y and B35, respectively) and E18-derived rat primary cortical neurons upon which the effects of various flavonoids were examined. Of note, increases in the levels of global SUMOylation, a post-translational modification with the Small Ubiquitin-like MOdifier (SUMO) were pronounced. Quercetin treatment increased SUMOylation levels in both SHSY5Y cells and rat cortical neurons in a dose and time-dependent manner, possibly via the direct inactivation of certain SENPs (SUMO-specific isopeptidases). Of particular interest, cells treated with quercetin displayed increased tolerance to oxygen/glucose deprivation exposure, an in vitro model of ischemia. SHSY5Y cells treated with quercetin also increased the expression of Nrf2 (via a decrease in the levels of Keap1), heme oxygenase-1 (HO-1), and nitric oxide synthase 1 (NOS1), which provide further protection from oxidative stress. In addition, the increased SUMOylation of HIF-1α was noted and deemed to be significant. We hypothesize that SUMOylated HIF-1α plays a fundamental role in the protection afforded and may underlie some of quercetin's ability to protect cells from oxygen/glucose deprivation-induced cell death, via an up-regulation of HO-1 and NOS1, which ultimately leads to the induction of pro-life NOS1/protein kinase G signaling. Quercetin acts to increase survival in the face of ischemia via an increase of SENP3 expression, the possible inactivation of SENPs 1/2, and via a decrease in KEAP1 levels (thereby increasing Nrf2 stability). These changes may then lead to increase in HIF-1α SUMOylation and HO-1 activation, followed by an up-regulation of NOS1/PKG signaling. Pathways altered via quercetin treatment within our experimental system are represented by blue arrowheads. Solid black arrows represent relationships that have been explored while a dotted arrow represents a relationship that has yet to be confirmed.

Hypophosphorylation of ribosomal protein S6 is a molecular mechanism underlying ischemic tolerance induced by either hibernation or preconditioning.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) have an extraordinary capacity to withstand prolonged and profound reductions in blood flow and oxygen delivery to the brain without incurring any cellular damage. As such, the hibernation torpor of I. tridecemlineatus provides a valuable model of tolerance to ischemic stress. Herein, we report that during hibernation torpor, a marked reduction in the phosphorylation of the ribosomal protein S6 (rpS6) occurs within the brains of I. tridecemlineatus. Of note, rpS6 phosphorylation was shown to increase in the brains of rats that underwent an occlusion of the middle cerebral artery. However, such an increase was attenuated after the implementation of an ischemic preconditioning paradigm. In addition, cultured cortical neurons treated with the rpS6 kinase (S6K) inhibitors, D-glucosamine or PF4708671, displayed a decrease in rpS6 phosphorylation and a subsequent increase in tolerance to oxygen/glucose deprivation, an in vitro model of ischemic stroke. Collectively, such evidence suggests that the down-regulation of rpS6 signal transduction may account for a substantial part of the observed increase in cellular tolerance to brain ischemia that occurs during hibernation torpor and after ischemic preconditioning. Further identification and characterization of the mechanisms used by hibernating species to increase ischemic tolerance may eventually clarify how the loss of homeostatic control that occurs during and after cerebral ischemia in the clinic can ultimately be minimized and/or prevented. Mammalian hibernation provides a valuable model of tolerance to ischemic stress. Herein, we demonstrate that marked reductions in the phosphorylation of ribosomal protein S6 (rpS6), extracellular signal-regulated kinase family of mitogen-activated protein (MAP) kinase p44/42 (p44/42MAPK) and ribosomal protein S6 kinase (S6K) occur within the brains of both hibernating squirrels and rats, which have undergone an ischemic preconditioning paradigm. We therefore propose that the down-regulation of rpS6 signal transduction may account for a substantial part of the observed increase in cellular tolerance to brain ischemia that occurs during hibernation torpor and after ischemic preconditioning, via a suppression of protein synthesis and/or energy consumption.

Integrative transcriptomic and metabolic analyses of the mammalian hibernating brain identifies a key role for succinate dehydrogenase in ischemic tolerance.

Ischemic stroke results in a loss of tissue homeostasis and integrity, the underlying pathobiology of which stems primarily from the depletion of cellular energy stores and perturbation of available metabolites 1 . Hibernation in thirteen-lined ground squirrels (TLGS), Ictidomys tridecemlineatus , provides a natural model of ischemic tolerance as these mammals undergo prolonged periods of critically low cerebral blood flow without evidence of central nervous system (CNS) damage 2 . Studying the complex interplay of genes and metabolites that unfolds during hibernation may provide novel insights into key regulators of cellular homeostasis during brain ischemia. Herein, we interrogated the molecular profiles of TLGS brains at different time points within the hibernation cycle via RNA sequencing coupled with untargeted metabolomics. We demonstrate that hibernation in TLGS leads to major changes in the expression of genes involved in oxidative phosphorylation and this is correlated with an accumulation of the tricarboxylic acid (TCA) cycle intermediates citrate, cis-aconitate, and α-ketoglutarate-αKG. Integration of the gene expression and metabolomics datasets led to the identification of succinate dehydrogenase (SDH) as the critical enzyme during hibernation, uncovering a break in the TCA cycle at that level. Accordingly, the SDH inhibitor dimethyl malonate (DMM) was able to rescue the effects of hypoxia on human neuronal cells in vitro and in mice subjected to permanent ischemic stroke in vivo . Our findings indicate that studying the regulation of the controlled metabolic depression that occurs in hibernating mammals may lead to novel therapeutic approaches capable of increasing ischemic tolerance in the CNS.

Tracks of a non-main path traveler: 2011 Thomas Willis Lecture.

After an unconventional beginning in stroke research, I veered off the main path repeatedly to view problems from a different perspective. In this lecture summary, I would like to return to several points along the byways that led to research with some continuity.

Blood-brain barrier disruption in humans is independently associated with increased matrix metalloproteinase-9.

BACKGROUND AND PURPOSE: Matrix metalloproteinases (MMP) may play a role in blood-brain barrier (BBB) disruption after ischemic stroke. We hypothesized that plasma concentrations of MMP-9 are associated with a marker of BBB disruption in patients evaluated for acute stroke. METHODS: Patients underwent MRI on presentation and approximately 24 hours later. The MRI marker, termed hyperintense acute reperfusion injury marker (HARM), is gadolinium enhancement of cerebrospinal fluid on fluid-attenuated inversion recovery MRI. Plasma MMP-9 and tissue inhibitor of matrix metalloproteinase-1 were measured by enzyme-linked immunosorbent assay. Logistic regression models tested for predictors of HARM on 24-hour follow-up scans separately for MMP-9 and the ratio of MMP-9 to TIMP-1. RESULTS: For the 41 patients enrolled, diagnoses were: acute ischemic cerebrovascular syndrome, 33 (80.6%); intracerebral hemorrhage, 6 (14.6%); stroke mimic, 1 (2.4%); and no stroke, 1 (2.4%). HARM was present in 17 (41.5%) patients. In model 1, HARM was associated with baseline plasma MMP-9 concentration (odds ratio [OR], 1.01; 95% confidence interval [CI], 1.001-1.019; P=0.033). In model 2, HARM was associated with the ratio of MMP-9 to tissue inhibitor of matrix metalloproteinase-1 (OR, 4.94; 95% CI, 1.27-19.14; P=0.021). CONCLUSIONS: Baseline MMP-9 was a significant predictor of HARM at 24-hour follow-up, supporting the hypothesis that MMP-9 is associated with BBB disruption. If the association between MMP-9 and BBB disruption is confirmed in future studies, HARM may be a useful imaging marker to evaluate MMP-9 inhibition in ischemic stroke and other populations with BBB disruption.

SUMOylation participates in induction of ischemic tolerance.

Ground squirrels in hibernation torpor have been shown to have striking increases in global SUMOylation on tissue immunoblots. Here, we find evidence that global SUMOylation is also involved in ischemic tolerance in primary cortical neuronal cultures (from rats and mice) and SHSY5Y human neuroblastoma cells. Cultured cortical neurons preconditioned by sublethal oxygen/glucose deprivation (OGD) were less vulnerable to severe OGD than non-preconditioned neurons. Preconditioned neurons maintained elevated SUMO-1 conjugation levels (and, to a lesser extent those of SUMO-2/3) on western blots in contrast to non-preconditioned cells. Further, cortical neurons and SHSY5Y cells in which transfected SUMO-1 or SUMO-2 were over-expressed showed increased survival after severe OGD. In contrast, cell cultures subjected to depletion of endogenous SUMO-1 protein by RNAi had reduced survival after exposure to this form of in vitro ischemia and an attenuated protective response to preconditioning. These findings suggest that maintenance of a globally elevated SUMO-1 (and maybe SUMO-2/3) conjugation level as revealed by immunoblot assays is a component of ischemic tolerance.

Molecular signature of penumbra in acute ischemic stroke: a pilot transcriptomics study.

We aimed to characterize peripheral blood gene expression profile of penumbra defined as MRI perfusion-diffusion mismatch (PD MM) in peripheral blood of patients with acute ischemic stroke. We studied 23 patients. Perfusion-diffusion mismatch volume was observed to be associated and significantly correlated with the expression of 34 genes including those related to inflammation, SUMOylation, and coagulation; while lipopolysaccharide inhibition was identified to be a candidate upstream regulator of these processes (z-score -2.38, P = 0.04). Penumbral volume is correlated with a specific gene expression profile in the peripheral blood characterized by overlap of inflammatory and neuroprotective pathways that are regulated by lipopolysaccharide inhibition.

SUMOylation promotes survival and integration of neural stem cell grafts in ischemic stroke.

BACKGROUND: Neural stem cell (NSC)-based therapies hold great promise for treating diseases of the central nervous system (CNS). However, several fundamental problems still need to be overcome to fully exploit the clinical potential of NSC therapeutics. Chief among them is the limited survival of NSC grafts within hostile microenvironments. METHODS: Herein, we sought to engineer NSCs in an effort to increase graft survival within ischemic brain lesions via upregulation of global SUMOylation, a post-translational modification critically involved in mediating tolerance to ischemia/reperfusion. FINDINGS: NSCs overexpressing the SUMO E2-conjugase Ubc9 displayed resistance to oxygen-glucose-deprivation/restoration of oxygen/glucose (OGD/ROG) and enhanced neuronal differentiation in vitro, as well as increased survival and neuronal differentiation when transplanted in mice with transient middle cerebral artery occlusion in vivo. INTERPRETATION: Our work highlights a critical role for SUMOylation in NSC biology and identifies a biological pathway that can be targeted to increase the effectiveness of exogenous stem cell medicines in ischemic stroke. FUND: Intramural Research Program of the NINDS/NIH, the Italian Multiple Sclerosis Foundation (FISM), the Bascule Charitable Trust, NIH-IRTA-OxCam and Wellcome Trust Research Training Fellowships.

Reperfusion-associated hemorrhagic transformation in SHR rats: evidence of symptomatic parenchymal hematoma.

BACKGROUND AND PURPOSE: Symptomatic hemorrhagic transformation (HT) is the most important complicating factor after treatment with intravenous tissue plasminogen activator. In this study, we used multimodal magnetic resonance imaging to investigate the incidence and severity of reperfusion-based HT in spontaneously hypertensive rats after ischemia/reperfusion. METHODS: Twenty male spontaneously hypertensive rats were subjected to 30 minutes of middle cerebral artery occlusion via the suture model. Diffusion-weighted, T(2)-weighted, and gradient-echo imaging were performed on days 1, 2, 3, 4, and 7 for longitudinal evaluation of lesion evolution, vasogenic edema, and HT, respectively. Findings on gradient-echo images were classified according to the severity of hemorrhage: no HT; punctate or small petechial hemorrhage (HI-1); confluent petechial hemorrhage (HI-2); hematoma with absent/mild space-occupying effect (PH-1, 30% lesion volume). Histopathologic evaluation of HT was performed after final imaging for comparison with magnetic resonance imaging results. RESULTS: Final hemorrhage scores based on severity were as follows: HI-1 23.1%, HI-2 30.8%, PH-1 30.8%, and PH-2 15.4%. Similar to clinical observations, only PH-2 was associated with neurologic deterioration and associated weight loss. CONCLUSIONS: This model has a high incidence of parenchymal hematomas (46.2%) and therefore is appropriate for the evaluation of novel therapeutics targeting blood-brain barrier integrity and the reduction of symptomatic HT events (PH-2), as well as those potentially "at risk" for neurologic deterioration (PH-1).

Mucosal tolerization to E-selectin protects against memory dysfunction and white matter damage in a vascular cognitive impairment model.

Vascular cognitive impairment (VCI) is the second most prevalent type of dementia in the world. The white matter damage that characterizes the common subcortical ischemic form of VCI can be modeled by ligating both common carotid arteries in the Wistar rat to induce protracted cerebral hypoperfusion. In this model, we find that repetitive intranasal administration of recombinant E-selectin to induce mucosal tolerance and to target immunomodulation to activating blood vessels potently suppresses both white matter (and possibly gray matter) damage and markers of vessel activation (tumor necrosis factor and E-selectin); it also preserves behavioral function in T-maze spontaneous alternation, T-maze spatial discrimination memory retention, and object recognition tests. Immunomodulation may be an effective novel strategy to prevent progression of VCI.

Akt Protein Kinase, miR-200/miR-182 Expression and Epithelial-Mesenchymal Transition Proteins in Hibernating Ground Squirrels.

Hibernating 13-lined ground squirrels (Ictidomys tridecemlineatus; TLGS) rank among the most brain hypoperfusion-tolerant mammals known. Herein we provide some evidence of cycling between an epithelial phenotype and a hybrid epithelial/mesenchymal (E/M) phenotype (partial EMT) within the brains of TLGS during each bout of hibernation torpor. During hibernation torpor, expression of the epithelial marker E-cadherin (E-CDH) was reduced, while expression of the well-known mesenchymal markers vimentin and Sox2 were increased. P-cadherin (P-CDH), which has recently been proposed as a marker of intermediate/partial EMT, also increased during torpor, suggesting that a partial EMT may be taking place during hibernation torpor. Members of the miR-200 family and miR-182 cluster and Akt isoforms (Akt1, Akt2), well-known EMT regulators, were also differentially regulated in the TLGS brain during hibernation bouts. Using SHSY5Y cells, we also demonstrate that the Akt1/Akt2 ratio determined the expression levels of miR-200/miR-182 miRNA family members, and that these miRNAs controlled the expression of EMT-related proteins. Accordingly, we propose that such cell state transitions (EMT/MET) may be one of the mechanisms underlying the extraordinary ischemic tolerance of the TLGS brain during hibernation bouts; hibernator brain cells appear to enter reversible states that confer the stress survival characteristics of cancer cells without the risk of neoplastic transformation.

SUMOylation in brain ischemia: Patterns, targets, and translational implications.

Post-translational protein modification by small ubiquitin-like modifier (SUMO) regulates a myriad of homeostatic and stress responses. The SUMOylation pathway has been extensively studied in brain ischemia. Convincing evidence is now at hand to support the notion that a major increase in levels of SUMOylated proteins is capable of inducing tolerance to ischemic stress. Therefore, the SUMOylation pathway has emerged as a promising therapeutic target for neuroprotection in the face of brain ischemia. Despite this, it is prudent to acknowledge that there are many key questions still to be addressed in brain ischemia related to SUMOylation. Accordingly, herein, we provide a critical review of literature within the field to summarize current knowledge and in so doing highlight pertinent translational implications of the SUMOylation pathway in brain ischemia.

Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation.

Neural stem cell (NSC) transplantation can influence immune responses and suppress inflammation in the CNS. Metabolites, such as succinate, modulate the phenotype and function of immune cells, but whether and how NSCs are also activated by such immunometabolites to control immunoreactivity and inflammatory responses is unclear. Here, we show that transplanted somatic and directly induced NSCs ameliorate chronic CNS inflammation by reducing succinate levels in the cerebrospinal fluid, thereby decreasing mononuclear phagocyte (MP) infiltration and secondary CNS damage. Inflammatory MPs release succinate, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, leading them to secrete prostaglandin E2 and scavenge extracellular succinate with consequential anti-inflammatory effects. Thus, our work reveals an unexpected role for the succinate-SUCNR1 axis in somatic and directly induced NSCs, which controls the response of stem cells to inflammatory metabolic signals released by type 1 MPs in the chronically inflamed brain.

Protein SUMOylation is massively increased in hibernation torpor and is critical for the cytoprotection provided by ischemic preconditioning and hypothermia in SHSY5Y cells.

Hibernation torpor provides an excellent natural model of tolerance to profound reductions in blood flow to the brain and other organs. Here, we report that during torpor of 13-lined ground squirrels, massive SUMOylation occurs in the brain, liver, and kidney. The level of small ubiquitin-related modifier (SUMO) conjugation coincides with the expression level of Ubc9, the SUMO specific E2-conjugating enzyme. Hypothermia alone also increased SUMO conjugation, but not as markedly as hibernation torpor. Increased SUMO conjugation (induced by Ubc9 overexpression, ischemic preconditioning (PC)+/-hypothermia) was necessary and sufficient for tolerance of SHSY5Y neuroblastoma cells to oxygen/glucose deprivation (OGD) ('in vitro ischemia'); decreased SUMO conjugation (induced by a dominant-negative Ubc9) severely reduced tolerance to OGD in these cells. These data indicate that post-translational modification of proteins by SUMOylation is a prominent feature of hibernation torpor and is critical for cytoprotection by ischemic PC+/-hypothermia in SHSY5Y cells subjected to OGD.

Spatial and temporal activation of brain regions in hibernation: c-fos expression during the hibernation bout in thirteen-lined ground squirrel.

Hibernation results in dramatic changes in body temperature and metabolism; however, the central nervous system remains active during deep torpor. By cloning c-fos cDNA from the 13-lined ground squirrel (Spermophilus tridecemlineatus) and using squirrel c-fos mRNA probe for in situ hybridization histochemistry, we systematically analyzed and identified specific brain regions that were activated during six different phases of the hibernation bout. During entrance into torpor, we detected activation of the ventrolateral subdivision of the medial preoptic area ('thermoregulatory center'), and the reticular thalamic nucleus, which is known to inhibit the somatomotor cortex. During torpor, c-fos expression in the cortex was suppressed while the reticular thalamic nucleus remained uniformly active. Throughout torpor the suprachiasmatic nucleus ('biological clock') showed increasing activity, likely participating in phase-change regulation of the hibernation bout. Interestingly, during torpor very strong c-fos activation was seen in the epithelial cells of the choroid plexus and in tanycytes at the third ventricle, both peaking near the beginning of arousal. In arousal, activity of the suprachiasmatic and reticular thalamic nuclei and choroid epithelial cells diminished, while ependymal cells in the lateral and fourth ventricles showed stronger activity. Increasing body temperature during arousal was driven by the activation of neurons in the medial part of the preoptic area. In interbout awake animals, we demonstrated the activation of hypothalamic neurons located in the arcuate nucleus and the dorsolateral hypothalamus, areas involved in food intake. Our observations indicate that the hibernation bout is closely regulated and orchestrated by specific regions of the central nervous system. J. Comp. Neurol. 505:443-458, 2007. (c) 2007 Wiley-Liss, Inc.