EGLN1 Inhibition and Rerouting of α-Ketoglutarate Suffice for Remote Ischemic Protection.

Ischemic preconditioning is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent, more prolonged, ischemic insult. In remote ischemic preconditioning (RIPC), ischemia to one organ protects others organs at a distance. We created mouse models to ask if inhibition of the alpha-ketoglutarate (αKG)-dependent dioxygenase Egln1, which senses oxygen and regulates the hypoxia-inducible factor (HIF) transcription factor, could suffice to mediate local and remote ischemic preconditioning. Using somatic gene deletion and a pharmacological inhibitor, we found that inhibiting Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion (I/R) injury. Parabiosis experiments confirmed that RIPC in this latter model was mediated by a secreted factor. Egln1 loss causes accumulation of circulating αKG, which drives hepatic production and secretion of kynurenic acid (KYNA) that is necessary and sufficient to mediate cardiac ischemic protection in this setting.

Labile glutamate synaptic transmission in the adult CA1 stratum-lacunosum-moleculare region.

The excitatory monosynaptic activation of hippocampal CA1 pyramidal cells is spatially segregated such that the proximal part of the apical dendritic tree in stratum radiatum (SR) receives input from the hippocampal CA3 region while the distal part in the stratum-lacunosum-moleculare (SLM) receives input mainly from the entorhinal cortex. The AMPA receptor-mediated (AMPA) signalling of SLM synapses in slices from neonatal rats was previously found to considerably differ from that of the SR synapses. In the present study, AMPA signalling of SLM synapses in 1-month-old rats has been examined, that is, when the hippocampus is essentially functionally mature. For the SR synapses, this time is characterized by a facilitatory shift in short-term plasticity, in the disappearance of labile postsynaptic AMPA signalling, a property thought to be important for early activity-dependent organization of neural circuits, and the expression of an adult form of long-term potentiation. We found that the SLM synapses alter their short-term plasticity similarly to that of the SR synapses. However, the labile postsynaptic AMPA signalling was not only maintained but substantially enhanced in the SLM synapses. The long-term potentiation observed was not of the adult form but like that of the neonatal SR synapses based on unsilencing of AMPA labile synapses. We propose that these features of the SLM synapses in the mature hippocampus will help to produce a flexible map of the multimodal sensory input reaching the SLM required for its conjunctive operation with the SR input to generate a proper functional output from the CA1 region.

The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells.

Extracellular ATP (eATP) is an ancient 'danger signal' used by eukaryotes to detect cellular damage1. In mice and humans, the release of eATP during inflammation or injury stimulates both innate immune activation and chronic pain through the purinergic receptor P2RX72-4. It is unclear, however, whether this pathway influences the generation of immunological memory, a hallmark of the adaptive immune system that constitutes the basis of vaccines and protective immunity against re-infection5,6. Here we show that P2RX7 is required for the establishment, maintenance and functionality of long-lived central and tissue-resident memory CD8+ T cell populations in mice. By contrast, P2RX7 is not required for the generation of short-lived effector CD8+ T cells. Mechanistically, P2RX7 promotes mitochondrial homeostasis and metabolic function in differentiating memory CD8+ T cells, at least in part by inducing AMP-activated protein kinase. Pharmacological inhibitors of P2RX7 provoked dysregulated metabolism and differentiation of activated mouse and human CD8+ T cells in vitro, and transient P2RX7 blockade in vivo ameliorated neuropathic pain but also compromised production of CD8+ memory T cells. These findings show that activation of P2RX7 by eATP provides a common currency that both alerts the nervous and immune system to tissue damage, and promotes the metabolic fitness and survival of the most durable and functionally relevant memory CD8+ T cell populations.

A Novel Assessment of Metabolic Pathways in Peritoneal Metastases from Low-Grade Appendiceal Mucinous Neoplasms.

BACKGROUND: There is a paucity of targeted therapies for patients with pseudomyxoma peritonei (PMP) secondary to low-grade appendiceal mucinous neoplasms (LAMNs). Dysregulated metabolism has emerged as a hallmark of cancer, and the relationship of metabolomics and cancer is an area of active scientific exploration. We sought to characterize phenotypic differences found in peritoneal metastases (PM) derived from LAMN versus adenocarcinoma. METHODS: Tumors were washed with phosphate-buffered saline (PBS), microdissected, then dissociated in ice-cold methanol dried and reconstituted in pyridine. Samples were derivatized in tert-butyldimethylsilyl (TBDMS) and subjected to gas chromatography-coupled mass spectrometry. Metabolites were assessed based on a standard library. RNA sequencing was performed, with pathway and network analyses on differentially expressed genes. RESULTS: Eight peritoneal tumor samples were obtained and analyzed: LAMNs (4), and moderate to poorly differentiated adenocarcinoma (colon [1], appendix [3]). Decreases in pyroglutamate, fumarate, and cysteine in PM from LAMNs were found compared with adenocarcinoma. Analyses showed the differential gene expression was dominated by the prevalence of metabolic pathways, particularly lipid metabolism. The gene retinol saturase (RETSAT), downregulated by LAMN, was involved in the multiple metabolic pathways that involve lipids. Using network mapping, we found IL1B signaling to be a potential top-level modulation candidate. CONCLUSIONS: Distinct metabolic signatures may exist for PM from LAMN versus adenocarcinoma. A multitude of genes are differentially regulated, many of which are involved in metabolic pathways. Additional research is needed to identify the significance and applicability of targeting metabolic pathways in the potential development of novel therapeutics for these challenging tumors.

The B56α subunit of PP2A is necessary for mesenchymal stem cell commitment to adipocyte.

Adipose tissue plays a major role in maintaining organismal metabolic equilibrium. Control over the fate decision from mesenchymal stem cells (MSCs) to adipocyte differentiation involves coordinated command of phosphorylation. Protein phosphatase 2A plays an important role in Wnt pathway and adipocyte development, yet how PP2A complexes actively respond to adipocyte differentiation signals and acquire specificity in the face of the promiscuous activity of its catalytic subunit remains unknown. Here, we report the PP2A phosphatase B subunit B56α is specifically induced during adipocyte differentiation and mediates PP2A to dephosphorylate GSK3ß, thereby blocking Wnt activity and driving adipocyte differentiation. Using an inducible B56α knock-out mouse, we further demonstrate that B56α is essential for gonadal adipose tissue development in vivo and required for the fate decision of adipocytes over osteoblasts. Moreover, we show B56α expression is driven by the adipocyte transcription factor PPARγ thereby establishing a novel link between PPARγ signaling and Wnt blockade. Overall, our results reveal B56α is a necessary part of the machinery dictating the transition from pre-adipocyte to mature adipocyte and provide fundamental insights into how PP2A complex specifically and actively regulates unique signaling pathway in biology.

Ion concentrations in cerebrospinal fluid in wakefulness, sleep and sleep deprivation in healthy humans.

Sleep is controlled by a circadian rhythmicity, via a reduction of arousal-promoting neuromodulatory activity, and by accumulation of somnogenic factors in the interstitial fluid of the brain. Recent experiments in mice suggest that a reduced neuronal excitability caused by a reduced concentration of potassium in the brain, concomitant with an increased concentration of calcium and magnesium, constitutes an important mediator of sleep. In the present study, we examined whether such changes in ion concentrations could be detected in the cerebrospinal fluid of healthy humans. Each subject underwent cerebrospinal fluid collection at three occasions in a randomized order: at 15:00 hours-17:00 hours during waking, at 06:00 hours-07:00 hours immediately following 1 night of sleep, and at 06:00 hours-07:00 hours following 1 night of sleep deprivation. When compared with wakefulness, both sleep and sleep deprivation produced the same effect of a small (0.1 mm, about 3%), but robust and highly significant, reduction in potassium concentration. Calcium and magnesium concentrations were unchanged. Our results support a circadian modulation of neuronal excitability in the brain mediated via changes of the interstitial potassium concentration.

Homosynaptic frequency-dependent depression by release site inactivation at neonatal hippocampal synapses in the stratum lacunosum-moleculare.

When activated at low frequencies (0.1-1 Hz), second postnatal week synapses onto the most distal part of the apical dendritic tree (stratum lacunosum-moleculare) of rat hippocampal CA1 pyramidal cells display a frequency-dependent synaptic depression not observed for the more proximal (stratum radiatum) synapses. Depression in this frequency range is thought of as a possible contributor to behavioural habituation. In fact, in contrast to the proximal synapses, the distal synapses provide more direct sensory information from the entorhinal cortex as well as from thalamic nuclei. The use of antagonists showed that the activation of GABAA , GABAB , NMDA, mGlu, kainate, adenosine, or endocannabinoid receptors was not directly involved in the depression, indicating it to be intrinsic to the synapses themselves. While the depression affected paired-pulse plasticity in a manner indicating a decrease in vesicle release probability, the depression could not be explained by a stimulus-dependent decrease in calcium influx. Despite affecting the synaptic response evoked by brief high-frequency stimulation (10 impulses, 20 Hz) in a manner indicating vesicle depletion, the depression was unaffected by large variations in release probability. The depression was found not only to affect the synaptic transmission at low frequencies (0.1-1 Hz) but also to contribute to the depression evolving during brief high-frequency stimulation (10 impulses, 20 Hz). We propose that a release-independent process directly inactivating release sites with a fast onset (ms) and long duration (up to 20 s) underlies this synaptic depression.

Human cerebrospinal fluid promotes spontaneous gamma oscillations in the hippocampus in vitro.

Gamma oscillations (30-80 Hz) are fast network activity patterns frequently linked to cognition. They are commonly studied in hippocampal brain slices in vitro, where they can be evoked via pharmacological activation of various receptor families. One limitation of this approach is that neuronal activity is studied in a highly artificial extracellular fluid environment, as provided by artificial cerebrospinal fluid (aCSF). Here, we examine the influence of human cerebrospinal fluid (hCSF) on kainate-evoked and spontaneous gamma oscillations in mouse hippocampus. We show that hCSF, as compared to aCSF of matched electrolyte and glucose composition, increases the power of kainate-evoked gamma oscillations and induces spontaneous gamma activity in areas CA3 and CA1 that is reversed by washout. Bath application of atropine entirely abolished hCSF-induced gamma oscillations, indicating critical contribution from muscarinic acetylcholine receptor-mediated signaling. In separate whole-cell patch clamp recordings from rat hippocampus, hCSF increased theta resonance frequency and strength in pyramidal cells along with enhancement of h-current (Ih ) amplitude. We found no evidence of intrinsic gamma frequency resonance at baseline (aCSF) among fast-spiking interneurons, and this was not altered by hCSF. However, hCSF increased the excitability of fast-spiking interneurons, which likely contributed to gamma rhythmogenesis. Our findings show that hCSF promotes network gamma oscillations in the hippocampus in vitro and suggest that neuromodulators distributed in CSF could have significant influence on neuronal network activity in vivo.

Ionized calcium in human cerebrospinal fluid and its influence on intrinsic and synaptic excitability of hippocampal pyramidal neurons in the rat.

It is well-known that the extracellular concentration of calcium affects neuronal excitability and synaptic transmission. Less is known about the physiological concentration of extracellular calcium in the brain. In electrophysiological brain slice experiments, the artificial cerebrospinal fluid traditionally contains relatively high concentrations of calcium (2-4 mM) to support synaptic transmission and suppress neuronal excitability. Using an ion-selective electrode, we determined the fraction of ionized calcium in healthy human cerebrospinal fluid to 1.0 mM of a total concentration of 1.2 mM (86%). Using patch-clamp and extracellular recordings in the CA1 region in acute slices of rat hippocampus, we then compared the effects of this physiological concentration of calcium with the commonly used 2 mM on neuronal excitability, synaptic transmission, and long-term potentiation (LTP) to examine the magnitude of changes in this range of extracellular calcium. Increasing the total extracellular calcium concentration from 1.2 to 2 mM decreased spontaneous action potential firing, induced a depolarization of the threshold, and increased the rate of both de- and repolarization of the action potential. Evoked synaptic transmission was approximately doubled, with a balanced effect between inhibition and excitation. In 1.2 mM calcium high-frequency stimulation did not result in any LTP, whereas a prominent LTP was observed at 2 or 4 mM calcium. Surprisingly, this inability to induce LTP persisted during blockade of GABAergic inhibition. In conclusion, an increase from the physiological 1.2 mM to 2 mM calcium in the artificial cerebrospinal fluid has striking effects on neuronal excitability, synaptic transmission, and the induction of LTP. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Read the Editorial Highlight for this article on page 435.

Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses.

NMDA-type glutamate receptors (NMDAR) are central actors in the plasticity of excitatory synapses. During adaptive processes, the number and composition of synaptic NMDAR can be rapidly modified, as in neonatal hippocampal synapses where a switch from predominant GluN2B- to GluN2A-containing receptors is observed after the induction of long-term potentiation (LTP). However, the cellular pathways by which surface NMDAR subtypes are dynamically regulated during activity-dependent synaptic adaptations remain poorly understood. Using a combination of high-resolution single nanoparticle imaging and electrophysiology, we show here that GluN2B-NMDAR are dynamically redistributed away from glutamate synapses through increased lateral diffusion during LTP in immature neurons. Strikingly, preventing this activity-dependent GluN2B-NMDAR surface redistribution through cross-linking, either with commercial or with autoimmune anti-NMDA antibodies from patient with neuropsychiatric symptoms, affects the dynamics and spine accumulation of CaMKII and impairs LTP. Interestingly, the same impairments are observed when expressing a mutant of GluN2B-NMDAR unable to bind CaMKII. We thus uncover a non-canonical mechanism by which GluN2B-NMDAR surface dynamics plays a critical role in the plasticity of maturing synapses through a direct interplay with CaMKII.

AMPA-silent synapses in brain development and pathology.

Synapses are constantly generated at a high rate in the developing, prepubescent brain. Newly generated glutamatergic synapses lack functional AMPA receptor-mediated transmission. Most of these 'AMPA-silent' synapses are eliminated during the developmental period, but some are specifically selected for AMPA unsilencing by correlated pre-and postsynaptic activity as the first step in a process that leads to stabilization of the synapse. Premature, or delayed, unsilencing of AMPA-silent synapses has been implicated in neurodevelopmental disorders, and abnormal generation of AMPA-silent synapses is associated with brain trauma, addiction and neurodegenerative disorders, further highlighting the importance of AMPA-silent synapses in brain pathology.

Neuromodulation of fast-spiking and non-fast-spiking hippocampal CA1 interneurons by human cerebrospinal fluid.

KEY POINTS: How the brain extracellular fluid influences the activity of GABAergic interneurons in vivo is not known. This issue is examined in the hippocampal brain slice by comparing GABAergic interneuron activity in human versus artificial cerebrospinal fluid. Human cerebrospinal fluid (hCSF) substantially increases the excitability of fast-spiking and non-fast-spiking CA1 interneurons. CA1 pyramidal cells are even more strongly excited by hCSF. The tonic excitation of pyramidal cells, in combination with an increased responsiveness of interneurons to excitatory input, is likely to promote the generation of synchronized network activity in the hippocampus. ABSTRACT: GABAergic interneurons intricately regulate the activity of hippocampal and neocortical networks. Their function in vivo is likely to be tuned by neuromodulatory substances in the brain extracellular fluid. However, in vitro investigations of GABAergic interneuron function do not account for such effects, as neurons are kept in artificial extracellular fluid. To examine the neuromodulatory influence of brain extracellular fluid on GABAergic activity, we recorded from fast-spiking and non-fast-spiking CA1 interneurons, as well as from pyramidal cells, in the presence of human cerebrospinal fluid (hCSF), using a matched artificial cerebrospinal fluid (aCSF) as control. We found that hCSF increased the frequency of spontaneous firing more than twofold in the two groups of interneurons, and more than fourfold in CA1 pyramidal cells. hCSF did not affect the resting membrane potential of CA1 interneurons but caused depolarization in pyramidal cells. The increased excitability of interneurons and pyramidal cells was accompanied by reductions in after-hyperpolarization amplitudes and a left-shift in the frequency-current relationships. Our results suggest that ambient concentrations of neuromodulators in the brain extracellular fluid powerfully influence the excitability of neuronal networks.

Tonically active NMDA receptors--a signalling mechanism critical for interneuronal excitability in the CA1 stratum radiatum.

In contrast to tonic extrasynaptic γ-aminobutyric acid (GABA)A receptor-mediated signalling, the physiological significance of tonic extrasynaptic N-methyl-D-aspartate (NMDA) receptor (NMDAR)-mediated signalling remains uncertain. In this study, reversible open-channel blockers of NMDARs, memantine and phencyclidine (PCP) were used as tools to examine tonic NMDAR-mediated signalling in rat hippocampal slices. Memantine in concentrations up to 10 µM had no effect on synaptically evoked NMDAR-mediated responses in pyramidal neurons or GABAergic interneurons. On the other hand, 10 µM memantine reduced tonic NMDAR-mediated currents in GABAergic interneurons by approximately 50%. These tonic NMDAR-mediated currents in interneurons contributed significantly to the excitability of the interneurons as 10 µM memantine reduced the disynaptic inhibitory postsynaptic current in pyramidal cells by about 50%. Moreover, 10 µM memantine, but also PCP in concentrations ≤ 1 µM, increased the magnitude of the population spike, likely because of disinhibition. The relatively higher impact of tonic NMDAR-mediated signalling in interneurons was at least partly explained by the expression of GluN2D-containing NMDARs, which was not observed in mature pyramidal cells. The current results are consistent with the idea that low doses of readily reversible NMDAR open-channel blockers preferentially inhibit tonically active extrasynaptic NMDARs, and they suggest that tonically active NMDARs contribute more prominently to the intrinsic excitation in GABAergic interneurons than in pyramidal cells. It is proposed that this specific difference between interneurons and pyramidal cells can explain the disinhibition caused by the Alzheimer's disease medication memantine.

Human cerebrospinal fluid increases the excitability of pyramidal neurons in the in vitro brain slice.

KEY POINTS: The cerebrospinal fluid contains numerous neuromodulators at ambient levels but whether, and how, they affect the activity of central neurons is unknown. This study provides experimental evidence that human cerebrospinal fluid (hCSF) increases the excitability of hippocampal and neocortical pyramidal neurons. Hippocampal CA1 pyramidal neurons in hCSF displayed lowered firing thresholds, depolarized resting membrane potentials and reduced input resistance, mimicking properties of pyramidal neurons recorded in vivo. The excitability-increasing effect of hCSF on CA1 pyramidal neurons was entirely occluded by intracellular application of GTPγS, suggesting that neuromodulatory effects were mediated by G-protein coupled receptors. These results indicate that the CSF promotes spontaneous excitatory neuronal activity, and may help to explain observed differences in the activity of pyramidal neurons recorded in vivo and in vitro. The composition of brain extracellular fluid is shaped by a continuous exchange of substances between the cerebrospinal fluid (CSF) and interstitial fluid. The CSF is known to contain a wide range of endogenous neuromodulatory substances, but their collective influence on neuronal activity has been poorly investigated. We show here that replacing artificial CSF (aCSF), routinely used for perfusion of brain slices in vitro, with human CSF (hCSF) powerfully boosts spontaneous firing of CA1, CA3 and layer 5 pyramidal neurons in the rat brain slice. CA1 pyramidal neurons in hCSF display lowered firing thresholds, more depolarized resting membrane potentials and reduced input resistance, mimicking properties of pyramidal neurons recorded in vivo. The increased excitability of CA1 pyramidal neurons was completely occluded by intracellular application of GTPγS, suggesting that endogenous neuromodulators in hCSF act on G-protein coupled receptors to enhance excitability. We found no increase in spontaneous inhibitory synaptic transmission by hCSF, indicating a differential effect on glutamatergic and GABAergic neurons. Our findings highlight a previously unknown function of the CSF in promoting spontaneous excitatory activity, and may help to explain differences observed in the activity of pyramidal neurons recorded in vivo and in vitro.

Co-deleting Pten with Rb in retinal progenitor cells in mice results in fully penetrant bilateral retinoblastomas.

BACKGROUND: Rb1 is the most frequently mutated gene in the pediatric cancer retinoblastoma, and its loss causes E2F transcription factors to induce proliferation related genes. However, high E2F levels following pRB loss also induce apoptosis-promoting genes as a safeguard mechanism to suppress emergent tumors. Although p53 accumulation and apoptosis induction is believed to be a primary mechanism to eliminate cells with excess E2F activity, p53 deletion doesn't suppress RB/E2F induced apoptosis in vivo in the retina. This prompted us to test the PTEN/PI3K/AKT signaling pathway on RB/E2F apoptosis suppression in vivo, to ascertain if the PI3K pathway may provide a potential avenue for retinoblastoma therapy. METHODS: We developed a mouse model in which Rb1 and Pten were conditionally deleted from retinal progenitor cells using Chx10-Cre, whereas Rbl1 (p107) was constitutively deleted. Pathway components were also tested individually by in vivo electroporation into newborn retinas for an effect on apoptosis and tumor initiation. Mouse retinal tissues were analyzed by immunohistochemistry (IHC) for proliferation, apoptosis, and pathway activation. ShRNAs were used in vitro to assess effects on apoptosis and gene expression. RESULTS: Co-deleting Pten with Rb1 and Rbl1 in mouse retinal progenitor cells (RPCs) causes fully penetrant bilateral retinoblastomas by 30 days and strongly suppresses Rb/E2F-induced apoptosis. In vivo electroporation of constitutively active (ca)-Pik3ca, ca-Akt, or dominant-negative (dn)-Foxo1 into apoptosis prone newborn murine retina with deleted Rb/p107 eliminate Rb/E2F induced apoptosis and induce retinoblastoma emergence. Retinal deletion of Pten activates p-AKT and p-FOXO1 signaling in incipient retinoblastoma. An unbiased shRNA screen focusing on Akt phosphorylation targets identified FOXOs as critical mediators of Rb/E2F induced apoptosis and expression of Bim and p73 pro-apoptotic genes. CONCLUSIONS: These data indicate that we defined a key molecular trigger involving E2F/FOXO functioning to control retinal progenitor cell homeostasis and retinoblastoma tumor initiation. We anticipate that our findings could provide contextual understanding of the proliferation of other progenitor cells, considering the high frequency of co-altered signaling from RB/E2F and PTEN/PI3K/AKT pathways in a wide variety of normal and malignant settings.

Irradiation of the Juvenile Brain Provokes a Shift from Long-Term Potentiation to Long-Term Depression.

Radiotherapy is common in the treatment of brain tumors in children but often causes deleterious, late-appearing sequelae, including cognitive decline. This is thought to be caused, at least partly, by the suppression of hippocampal neurogenesis. However, the changes in neuronal network properties in the dentate gyrus (DG) following the irradiation of the young, growing brain are still poorly understood. We characterized the long-lasting effects of irradiation on the electrophysiological properties of the DG after a single dose of 6-Gy whole-brain irradiation on postnatal day 11 in male Wistar rats. The assessment of the basal excitatory transmission in the medial perforant pathway (MPP) by an examination of the field excitatory postsynaptic potential/volley ratio showed an increase of the synaptic efficacy per axon in irradiated animals compared to sham controls. The paired-pulse ratio at the MPP granule cell synapses was not affected by irradiation, suggesting that the release probability of neurotransmitters was not altered. Surprisingly, the induction of long-term synaptic plasticity in the DG by applying 4 trains of high-frequency stimulation provoked a shift from long-term potentiation (LTP) to long-term depression (LTD) in irradiated animals compared to sham controls. The morphological changes consisted in a virtually complete ablation of neurogenesis following irradiation, as judged by doublecortin immunostaining, while the inhibitory network of parvalbumin interneurons was intact. These data suggest that the irradiation of the juvenile brain caused permanent changes in synaptic plasticity that would seem consistent with an impairment of declarative learning. Unlike in our previous study in mice, lithium treatment did unfortunately not ameliorate any of the studied parameters. For the first time, we show that the effects of cranial irradiation on long-term synaptic plasticity is different in the juvenile compared with the adult brain, such that while irradiation of the adult brain will only cause a reduction in LTP, irradiation of the juvenile brain goes further and causes LTD. Although the mechanisms underlying the synaptic alterations need to be elucidated, these findings provide a better understanding of the effects of irradiation in the developing brain and the cognitive deficits observed in young patients who have been subjected to cranial radiotherapy. © 2015 S. Karger AG, Basel.

Activation of the transcription factor GLI1 by WNT signaling underlies the role of SULFATASE 2 as a regulator of tissue regeneration.

Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known; however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning active multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced ß-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and ß-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway.

Activation of GFRAL+ neurons induces hypothermia and glucoregulatory responses associated with nausea and torpor.

GFRAL-expressing neurons actuate aversion and nausea, are targets for obesity treatment, and may mediate metformin effects by long-term GDF15-GFRAL agonism. Whether GFRAL+ neurons acutely regulate glucose and energy homeostasis is, however, underexplored. Here, we report that cell-specific activation of GFRAL+ neurons using a variety of techniques causes a torpor-like state, including hypothermia, the release of stress hormones, a shift from glucose to lipid oxidation, and impaired insulin sensitivity, glucose tolerance, and skeletal muscle glucose uptake but augmented glucose uptake in visceral fat. Metabolomic analysis of blood and transcriptomics of muscle and fat indicate alterations in ketogenesis, insulin signaling, adipose tissue differentiation and mitogenesis, and energy fluxes. Our findings indicate that acute GFRAL+ neuron activation induces endocrine and gluco- and thermoregulatory responses associated with nausea and torpor. While chronic activation of GFRAL signaling promotes weight loss in obesity, these results show that acute activation of GFRAL+ neurons causes hypothermia and hyperglycemia.

SAPS3 subunit of protein phosphatase 6 is an AMPK inhibitor and controls metabolic homeostasis upon dietary challenge in male mice.

Inhibition of AMPK is tightly associated with metabolic perturbations upon over nutrition, yet the molecular mechanisms underlying are not clear. Here, we demonstrate the serine/threonine-protein phosphatase 6 regulatory subunit 3, SAPS3, is a negative regulator of AMPK. SAPS3 is induced under high fat diet (HFD) and recruits the PP6 catalytic subunit to deactivate phosphorylated-AMPK, thereby inhibiting AMPK-controlled metabolic pathways. Either whole-body or liver-specific deletion of SAPS3 protects male mice against HFD-induced detrimental consequences and reverses HFD-induced metabolic and transcriptional alterations while loss of SAPS3 has no effects on mice under balanced diets. Furthermore, genetic inhibition of AMPK is sufficient to block the protective phenotype in SAPS3 knockout mice under HFD. Together, our results reveal that SAPS3 is a negative regulator of AMPK and suppression of SAPS3 functions as a guardian when metabolism is perturbed and represents a potential therapeutic strategy to treat metabolic syndromes.