Tyrosine phosphatase PTP1B impairs presynaptic NMDA receptor-mediated plasticity in a mouse model of Alzheimer's disease.

Mutations in the beta-amyloid protein (APP) cause familial Alzheimer's disease. In hAPP-J20 mice expressing mutant APP, pharmacological inhibition or genetic ablation of the tyrosine phosphatase PTP1B prevents CA3 hippocampus neuron loss and cognitive decline. However, how targeting PTP1B affects the cellular mechanisms underlying these cognitive deficits remains unknown. Changes in synaptic strength at the hippocampus can affect information processing for learning and memory. While prior studies have focused on post-synaptic mechanisms to account for synaptic deficits in Alzheimer's disease models, presynaptic mechanisms may also be affected. Here, using whole cell patch-clamp recording, coefficient of variation (CV) analysis suggested a profound presynaptic deficit in long-term potentiation (LTP) of CA3:CA1 synapses in hAPP-J20 mice. While the membrane-impermeable ionotropic NMDA receptor (NMDAR) blocker norketamine in the post-synaptic recording electrode had no effect on LTP, additional bath application of the ionotropic NMDAR blockers MK801 could replicate the deficit in LTP in wild type mice. In contrast to LTP, the paired-pulse ratio and short-term facilitation (STF) were aberrantly increased in hAPP-J20 mice. These synaptic deficits in hAPP-J20 mice were associated with reduced phosphorylation of NMDAR GluN2B and the synaptic vesicle recycling protein NSF (N-ethylmaleimide sensitive factor). Phosphorylation of both proteins, together with synaptic plasticity and cognitive function, were restored by PTP1B ablation or inhibition by the PTP1B-selective inhibitor Trodusquemine. Taken together, our results indicate that PTP1B impairs presynaptic NMDAR-mediated synaptic plasticity required for spatial learning in a mouse model of Alzheimer's disease. Since Trodusquemine has undergone phase 1/2 clinical trials to treat obesity, it could be repurposed to treat Alzheimer's disease.

Ketamine's schizophrenia-like effects are prevented by targeting PTP1B.

Subanesthetic doses of ketamine induce schizophrenia-like behaviors in mice including hyperlocomotion and deficits in working memory and sensorimotor gating. Here, we examined the effect of in vivo ketamine administration on neuronal properties and endocannabinoid (eCB)-dependent modulation of synaptic transmission onto layer 2/3 pyramidal neurons in brain slices of the prefrontal cortex, a region tied to the schizophrenia-like behavioral phenotypes of ketamine. Since deficits in working memory and sensorimotor gating are tied to activation of the tyrosine phosphatase PTP1B in glutamatergic neurons, we asked whether PTP1B contributes to these effects of ketamine. Ketamine increased membrane resistance and excitability of pyramidal neurons. Systemic pharmacological inhibition of PTP1B by Trodusquemine restored these neuronal properties and prevented each of the three main ketamine-induced behavior deficits. Ketamine also reduced mobilization of eCB by pyramidal neurons, while unexpectedly reducing their inhibitory inputs, and these effects of ketamine were blocked or occluded by PTP1B ablation in glutamatergic neurons. While ablation of PTP1B in glutamatergic neurons prevented ketamine-induced deficits in memory and sensorimotor gating, it failed to prevent hyperlocomotion (a psychosis-like phenotype). Taken together, these results suggest that PTP1B in glutamatergic neurons mediates ketamine-induced deficits in eCB mobilization, memory and sensorimotor gating whereas PTP1B in other cell types contributes to hyperlocomotion. Our study suggests that the PTP1B inhibitor Trodusquemine may represent a new class of fast-acting antipsychotic drugs to treat schizophrenia-like symptoms.

Neuronal protein-tyrosine phosphatase 1B hinders sensory-motor functional recovery and causes affective disorders in two different focal ischemic stroke models.

Ischemic brain injury causes neuronal death and inflammation. Inflammation activates protein-tyrosine phosphatase 1B (PTP1B). Here, we tested the significance of PTP1B activation in glutamatergic projection neurons on functional recovery in two models of stroke: by photothrombosis, focal ischemic lesions were induced in the sensorimotor cortex (SM stroke) or in the peri-prefrontal cortex (peri-PFC stroke). Elevated PTP1B expression was detected at 4 days and up to 6 weeks after stroke. While ablation of PTP1B in neurons of neuronal knockout (NKO) mice had no effect on the volume or resorption of ischemic lesions, markedly different effects on functional recovery were observed. SM stroke caused severe sensory and motor deficits (adhesive removal test) in wild type and NKO mice at 4 days, but NKO mice showed drastically improved sensory and motor functional recovery at 8 days. In addition, peri-PFC stroke caused anxiety-like behaviors (elevated plus maze and open field tests), and depression-like behaviors (forced swimming and tail suspension tests) in wild type mice 9 and 28 days after stroke, respectively, with minimal effect on sensory and motor function. Peri-PFC stroke-induced affective disorders were associated with fewer active (FosB+) neurons in the PFC and nucleus accumbens but more FosB+ neurons in the basolateral amygdala, compared to sham-operated mice. In contrast, mice with neuronal ablation of PTP1B were protected from anxiety-like and depression-like behaviors and showed no change in FosB+ neurons after peri-PFC stroke. Taken together, our study identifies neuronal PTP1B as a key component that hinders sensory and motor functional recovery and also contributes to the development of anxiety-like and depression-like behaviors after stroke. Thus, PTP1B may represent a novel therapeutic target to improve stroke recovery. All procedures for animal use were approved by the Animal Care and Use Committee of the University of Ottawa Animal Care and Veterinary Service (protocol 1806) on July 27, 2018.

Activation of tyrosine phosphatase PTP1B in pyramidal neurons impairs endocannabinoid signaling by tyrosine receptor kinase trkB and causes schizophrenia-like behaviors in mice.

Schizophrenia is a debilitating disorder affecting young adults displaying symptoms of cognitive impairment, anxiety, and early social isolation prior to episodes of auditory hallucinations. Cannabis use has been tied to schizophrenia-like symptoms, indicating that dysregulated endogenous cannabinoid signaling may be causally linked to schizophrenia. Previously, we reported that glutamatergic neuron-selective ablation of Lmo4, an endogenous inhibitor of the tyrosine phosphatase PTP1B, impairs endocannabinoid (eCB) production from the metabotropic glutamate receptor mGluR5. These Lmo4-deficient mice display anxiety-like behaviors that are alleviated by local shRNA knockdown or pharmacological inhibition of PTP1B that restores mGluR5-dependent eCB production in the amygdala. Here, we report that these Lmo4-deficient mice also display schizophrenia-like behaviors: impaired working memory assessed in the Y maze and defective sensory gating by prepulse inhibition of the acoustic startle response. Modulation of inhibitory inputs onto layer 2/3 pyramidal neurons of the prefrontal cortex relies on eCB signaling from the brain-derived neurotrophic factor receptor trkB, rather than mGluR5, and this mechanism was defective in Lmo4-deficient mice. Genetic ablation of PTP1B in the glutamatergic neurons lacking Lmo4 restored tyrosine phosphorylation of trkB, trkB-mediated eCB signaling, and ameliorated schizophrenia-like behaviors. Pharmacological inhibition of PTP1B with trodusquemine also restored trkB phosphorylation and improved schizophrenia-like behaviors by restoring eCB signaling, since the CB1 receptor antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide blocked this effect. Thus, activation of PTP1B in pyramidal neurons contributes to schizophrenia-like behaviors in Lmo4-deficient mice and genetic or pharmacological intervention targeting PTP1B ameliorates schizophrenia-related deficits.

Hyperactivated PTP1B phosphatase in parvalbumin neurons alters anterior cingulate inhibitory circuits and induces autism-like behaviors.

Individuals with autism spectrum disorder (ASD) have social interaction deficits and difficulty filtering information. Inhibitory interneurons filter information at pyramidal neurons of the anterior cingulate cortex (ACC), an integration hub for higher-order thalamic inputs important for social interaction. Humans with deletions including LMO4, an endogenous inhibitor of PTP1B, display intellectual disabilities and occasionally autism. PV-Lmo4KO mice ablate Lmo4 in PV interneurons and display ASD-like repetitive behaviors and social interaction deficits. Surprisingly, increased PV neuron-mediated peri-somatic feedforward inhibition to the pyramidal neurons causes a compensatory reduction in (somatostatin neuron-mediated) dendritic inhibition. These homeostatic changes increase filtering of mediodorsal-thalamocortical inputs but reduce filtering of cortico-cortical inputs and narrow the range of stimuli ACC pyramidal neurons can distinguish. Simultaneous ablation of PTP1B in PV-Lmo4KO neurons prevents these deficits, indicating that PTP1B activation in PV interneurons contributes to ASD-like characteristics and homeostatic maladaptation of inhibitory circuits may contribute to deficient information filtering in ASD.

Neuronal Protein Tyrosine Phosphatase 1B Hastens Amyloid ß-Associated Alzheimer's Disease in Mice.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, resulting in the progressive decline of cognitive function in patients. Familial forms of AD are tied to mutations in the amyloid precursor protein, but the cellular mechanisms that cause AD remain unclear. Inflammation and amyloidosis from amyloid ß (Aß) aggregates are implicated in neuron loss and cognitive decline. Inflammation activates the protein-tyrosine phosphatase 1B (PTP1B), and this could suppress many signaling pathways that activate glycogen synthase kinase 3ß (GSK3ß) implicated in neurodegeneration. However, the significance of PTP1B in AD pathology remains unclear. Here, we show that pharmacological inhibition of PTP1B with trodusquemine or selective ablation of PTP1B in neurons prevents hippocampal neuron loss and spatial memory deficits in a transgenic AD mouse model with Aß pathology (hAPP-J20 mice of both sexes). Intriguingly, while systemic inhibition of PTP1B reduced inflammation in the hippocampus, neuronal PTP1B ablation did not. These results dissociate inflammation from neuronal loss and cognitive decline and demonstrate that neuronal PTP1B hastens neurodegeneration and cognitive decline in this model of AD. The protective effect of PTP1B inhibition or ablation coincides with the restoration of GSK3ß inhibition. Neuronal ablation of PTP1B did not affect cerebral amyloid levels or plaque numbers, but reduced Aß plaque size in the hippocampus. In summary, our preclinical study suggests that targeting PTP1B may be a new strategy to intervene in the progression of AD.SIGNIFICANCE STATEMENT Familial forms of Alzheimer's disease (AD) are tied to mutations in the amyloid precursor protein, but the cellular mechanisms that cause AD remain unclear. Here, we used a mouse model expressing human amyloid precursor protein bearing two familial mutations and asked whether activation of a phosphatase PTP1B participates in the disease process. Systemic inhibition of this phosphatase using a selective inhibitor prevented cognitive decline, neuron loss in the hippocampus, and attenuated inflammation. Importantly, neuron-targeted ablation of PTP1B also prevented cognitive decline and neuron loss but did not reduce inflammation. Therefore, neuronal loss rather than inflammation was critical for AD progression in this mouse model, and that disease progression could be ameliorated by inhibition of PTP1B.

Dabrafenib, an inhibitor of RIP3 kinase-dependent necroptosis, reduces ischemic brain injury.

Ischemic brain injury triggers neuronal cell death by apoptosis via caspase activation and by necroptosis through activation of the receptor-interacting protein kinases (RIPK) associated with the tumor necrosis factor-alpha (TNF-α)/death receptor. Recent evidence shows RIPK inhibitors are neuroprotective and alleviate ischemic brain injury in a number of animal models, however, most have not yet undergone clinical trials and safety in humans remains in question. Dabrafenib, originally identified as a B-raf inhibitor that is currently used to treat melanoma, was later revealed to be a potent RIPK3 inhibitor at micromolar concentrations. Here, we investigated whether Dabrafenib would show a similar neuroprotective effect in mice subjected to ischemic brain injury by photothrombosis. Dabrafenib administered intraperitoneally at 10 mg/kg one hour after photothrombosis-induced focal ischemic injury significantly reduced infarct lesion size in C57BL6 mice the following day, accompanied by a markedly attenuated upregulation of TNF-α. However, subsequent lower doses (5 mg/kg/day) failed to sustain this neuroprotective effect after 4 days. Dabrafenib blocked lipopolysaccharides-induced activation of TNF-α in bone marrow-derived macrophages, suggesting that Dabrafenib may attenuate TNF-α-induced necroptotic pathway after ischemic brain injury. Since Dabrafenib is already in clinical use for the treatment of melanoma, it might be repurposed for stroke therapy.

IRF2BP2-deficient microglia block the anxiolytic effect of enhanced postnatal care.

Enhanced postnatal care (EPC) increases resilience to adversity in adulthood. Since microglia participate in shaping neural circuits, we asked how ablation of an inflammation-suppressing factor IRF2BP2 (Interferon Regulatory Factor 2 Binding Protein 2) in microglia would affect the responses to EPC. Mice lacking IRF2BP2 in microglia (KO) and littermate controls (WT) were subjected to EPC during the first 3 weeks after birth. EPC reduced anxiety in WT but not KO mice. This was associated with reduced inflammatory cytokine expression in the hypothalamus. Whole genome RNAseq profiling of the hypothalamus identified 101 genes whose expression was altered by EPC: 95 in WT, 11 in KO, with 5 in common that changed in opposite directions. Proteoglycan 4 (Prg4), prostaglandin D2 synthase (Ptgds) and extracellular matrix protease inhibitor Itih2 were suppressed by EPC in WT but elevated in KO mice. On the other hand, the glutamate transporter VGLUT1 (Slc17a7) was increased by EPC in WT but not KO mice. Prostaglandin D2 (PGD2) is known to enhance microglial inflammation and promote Gfap expression. ELISA confirmed reduced PGD2 in the hypothalamus of WT mice after EPC, associated with reduced Gfap expression. Our study suggests that the anxiety-reducing effect of EPC operates by suppressing microglial inflammation, likely by reducing neuronal prostaglandin D2 production.

Loss of IRF2BP2 in Microglia Increases Inflammation and Functional Deficits after Focal Ischemic Brain Injury.

Ischemic stroke causes neuronal cell death and triggers a cascade of inflammatory signals that contribute to secondary brain damage. Microglia, the brain-resident macrophages that remove dead neurons, play a critical role in the brain's response to ischemic injury. Our previous studies showed that IRF2 binding protein 2 (IRF2BP2) regulates peripheral macrophage polarization, limits their inflammatory response and reduces susceptibility to atherosclerosis. Here, we show that loss of IRF2BP2 in microglia leads to increased inflammatory cytokine expression in response to lipopolysaccharide challenge and impaired activation of anti-inflammatory markers in response to interleukin-4 (IL4) stimulation. Focal ischemic brain injury of the sensorimotor cortex induced by photothrombosis caused more severe functional deficits in mice with IRF2BP2 ablated in macrophages/microglia, associated with elevated expression of inflammatory cytokines in the brain. These mutant mice had larger infarctions 4 days after stroke associated with fewer anti-inflammatory M2 microglia/macrophages recruited to the peri-infarct area, suggesting an impaired clearance of injured tissues. Since IRF2BP2 modulates interferon signaling, and interferon beta (IFNß) has been reported to be anti-inflammatory and reduce ischemic brain injury, we asked whether loss of IRF2BP2 in macrophages/microglia would affect the response to IFNß in our stroke model. IFNß suppressed inflammatory cytokine production of macrophages and reduced infarct volumes at 4 days after photothrombosis in wild type mice. The anti-inflammatory effect of IFNß was lost in IRF2BP2-deficient macrophages and IFNß failed to protect mice lacking IRF2BP2 in macrophages/microglia from ischemic injury. In summary, IRF2BP2 expression in macrophages/microglia is important to limit inflammation and stroke injury, in part by mediating the beneficial effect of IFNß.

Chronic stress induces anxiety via an amygdalar intracellular cascade that impairs endocannabinoid signaling.

Collapse of endocannabinoid (eCB) signaling in the amygdala contributes to stress-induced anxiety, but the mechanisms of this effect remain unclear. eCB production is tied to the function of the glutamate receptor mGluR5, itself dependent on tyrosine phosphorylation. Herein, we identify a novel pathway linking eCB regulation of anxiety through phosphorylation of mGluR5. Mice lacking LMO4, an endogenous inhibitor of the tyrosine phosphatase PTP1B, display reduced mGluR5 phosphorylation, eCB signaling, and profound anxiety that is reversed by genetic or pharmacological suppression of amygdalar PTP1B. Chronically stressed mice exhibited elevated plasma corticosterone, decreased LMO4 palmitoylation, elevated PTP1B activity, reduced amygdalar eCB levels, and anxiety behaviors that were restored by PTP1B inhibition or by glucocorticoid receptor antagonism. Consistently, corticosterone decreased palmitoylation of LMO4 and its inhibition of PTP1B in neuronal cells. Collectively, these data reveal a stress-responsive corticosterone-LMO4-PTP1B-mGluR5 cascade that impairs amygdalar eCB signaling and contributes to the development of anxiety.

Functional properties of Claramine: a novel PTP1B inhibitor and insulin-mimetic compound.

Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling, interfering with its control of glucose homeostasis and metabolism. PTP1B activity is elevated in obesity and type 2 diabetes and is a major cause of insulin resistance. Trodusquemine (MSI-1436) is a "first-in-class" highly selective inhibitor of PTP1B that can cross the blood-brain barrier to suppress feeding and promote insulin sensitivity and glycemic control. Trodusquemine is a naturally occurring cholestane that can be purified from the liver of the dogfish shark, Squalus acanthias, but it can also be manufactured synthetically by a fairly laborious process that requires several weeks. Here, we tested a novel easily and rapidly (2 days) synthesized polyaminosteroid derivative (Claramine) containing a spermino group similar to Trodusquemine for its ability to inhibit PTP1B. Like Trodusquemine, Claramine displayed selective inhibition of PTP1B but not its closest related phosphatase TC-PTP. In cultured neuronal cells, Claramine and Trodusquemine both activated key components of insulin signaling, with increased phosphorylation of insulin receptor-ß (IRß), Akt and GSK3ß. Intraperitoneal administration of Claramine or Trodusquemine effectively restored glycemic control in diabetic mice as determined by glucose and insulin tolerance tests. A single intraperitoneal dose of Claramine, like an equivalent dose of Trodusquemine, suppressed feeding and caused weight loss without increasing energy expenditure. In summary, Claramine is an alternative more easily manufactured compound for the treatment of type II diabetes.

LMO4 is required to maintain hypothalamic insulin signaling.

Insulin action at the hypothalamus controls glucose homeostasis by suppressing hepatic glucose production and promoting glucose uptake by muscle. However, the mechanisms that control central insulin signaling have not been fully elucidated. Previously, we showed that LMO4 is highly expressed in hypothalamic nuclei that regulate glucose homeostasis. Here, we determined how loss of LMO4 in the hypothalamus would affect central insulin signaling and glucose homeostasis. In transgenic mice that have LMO4 in ablated in glutamatergic neurons, we found that insulin signaling is impaired in the hypothalamus as well as in peripheral tissues (liver and skeletal muscle). Impaired glucose homeostasis was associated with a markedly elevation in hypothalamic protein tyrosine phosphatase 1B (PTP1B) activity. PTP1B is a key phosphatase that terminates insulin signaling by dephosphorylating its receptor and downstream signaling molecules. Importantly, we found that administration of a selective PTP1B inhibitor Trodusquemine to the hypothalamus restored central insulin signaling and improved the response of peripheral tissues to insulin in these LMO4-deficient mice. Thus, our study reveals an essential requirement for LMO4 to modulate central insulin signaling.

LMO4 is essential for paraventricular hypothalamic neuronal activity and calcium channel expression to prevent hyperphagia.

The dramatic increase in the prevalence of obesity reflects a lack of progress in combating one of the most serious health problems of this century. Recent studies have improved our understanding of the appetitive network by focusing on the paraventricular hypothalamus (PVH), a key region responsible for the homeostatic balance of food intake. Here we show that mice with PVH-specific ablation of LIM domain only 4 (Lmo4) become rapidly obese when fed regular chow due to hyperphagia rather than to reduced energy expenditure. Brain slice recording of LMO4-deficient PVH neurons showed reduced basal cellular excitability together with reduced voltage-activated Ca(2+) currents. Real-time PCR quantification revealed that LMO4 regulates the expression of Ca(2+) channels (Cacna1h, Cacna1e) that underlie neuronal excitability. By increasing neuronal activity using designer receptors exclusively activated by designer drugs technology, we could suppress food intake of PVH-specific LMO4-deficient mice. Together, these results demonstrate that reduced neural activity in LMO4-deficient PVH neurons accounts for hyperphagia. Thus, maintaining PVH activity is important to prevent hyperphagia-induced obesity.

The LIM domain only 4 protein is a metabolic responsive inhibitor of protein tyrosine phosphatase 1B that controls hypothalamic leptin signaling.

Protein tyrosine phosphatase 1B (PTP1B) counteracts leptin signaling and is a therapeutic target for obesity and diabetes. Here we found that LIM domain only 4 (LMO4) inhibits PTP1B activity by increasing the oxidized inactive form of PTP1B. Mice with neuronal ablation of LMO4 have elevated PTP1B activity and impaired hypothalamic leptin signaling, and a PTP1B inhibitor normalized PTP1B activity and restored leptin control of circulating insulin levels. LMO4 is palmitoylated at its C-terminal cysteine, and deletion of this residue prevented palmitoylation and retention of LMO4 at the endoplasmic reticulum and abolished its inhibitory effect on PTP1B. Importantly, LMO4 palmitoylation is sensitive to metabolic stress; mice challenged with a brief high-fat diet or acute intracerebroventricular infusion of saturated fatty acid had less palmitoylated LMO4, less oxidized PTP1B, and increased PTP1B activity in the hypothalamus. Thus, unleashed PTP1B activity attributable to loss of LMO4 palmitoylation may account for rapid loss of central leptin signaling after acute exposure to saturated fat.

LIM domain only 4 (LMO4) regulates calcium-induced calcium release and synaptic plasticity in the hippocampus.

The LIM domain only 4 (LMO4) transcription cofactor activates gene expression in neurons and regulates key aspects of network formation, but the mechanisms are poorly understood. Here, we show that LMO4 positively regulates ryanodine receptor type 2 (RyR2) expression, thereby suggesting that LMO4 regulates calcium-induced calcium release (CICR) in central neurons. We found that CICR modulation of the afterhyperpolarization in CA3 neurons from mice carrying a forebrain-specific deletion of LMO4 (LMO4 KO) was severely compromised but could be restored by single-cell overexpression of LMO4. In line with these findings, two-photon calcium imaging experiments showed that the potentiation of RyR-mediated calcium release from internal stores by caffeine was absent in LMO4 KO neurons. The overall facilitatory effect of CICR on glutamate release induced during trains of action potentials was likewise defective in LMO4 KO, confirming that CICR machinery is severely compromised in these neurons. Moreover, the magnitude of CA3-CA1 long-term potentiation was reduced in LMO4 KO mice, a defect that appears to be secondary to an overall reduced glutamate release probability. These cellular phenotypes in LMO4 KO mice were accompanied with deficits in hippocampus-dependent spatial learning as determined by the Morris water maze test. Thus, our results establish LMO4 as a key regulator of CICR in central neurons, providing a mechanism for LMO4 to modulate a wide range of neuronal functions and behavior.

Ablation of LMO4 in glutamatergic neurons impairs leptin control of fat metabolism.

The LIM domain only 4 (LMO4) protein is expressed in the hypothalamus, but its function there is not known. Using mice with LMO4 ablated in postnatal glutamatergic neurons, including most neurons of the paraventricular (PVN) and ventromedial (VMH) hypothalamic nuclei where LMO4 is expressed, we asked whether LMO4 is required for metabolic homeostasis. LMO4 mutant mice exhibited early onset adiposity. These mice had reduced energy expenditure and impaired thermogenesis together with reduced sympathetic outflow to adipose tissues. The peptide hormone leptin, produced from adipocytes, activates Jak/Stat3 signaling at the hypothalamus to control food intake, energy expenditure, and fat metabolism. Intracerebroventricular infusion of leptin suppressed feeding similarly in LMO4 mutant and control mice. However, leptin-induced fat loss was impaired and activation of Stat3 in the VMH was blunted in these mice. Thus, our study identifies LMO4 as a novel modulator of leptin function in selective hypothalamic nuclei to regulate fat metabolism.

LIM domain only 4 protein promotes granulocyte colony-stimulating factor-induced signaling in neurons.

Granulocyte colony-stimulating factor (GCSF) is currently in clinical trials to treat neurodegenerative diseases and stroke. Here, we tested whether LIM domain only 4 protein (LMO4), a hypoxia-inducible gene that protects neurons from ischemic injury, could modulate the neuroprotective effect of GCSF. We showed that GCSF treatment acetylates and phosphorylates Stat3, activates expression of a Stat3-dependent anti-apoptotic gene, p27, and increases neuron survival from ischemic injury. LMO4 participates in Stat3 signaling in hepatocytes and associates with histone deacetylase 2 (HDAC2) in cancer cells. In the absence of LMO4, GCSF fails to rescue neurons from ischemic insults. In wild-type neurons, inhibition of HDAC promoted Stat3 acetylation and the antiapoptotic effect of GCSF. In LMO4 null cortical neurons, expression of wild-type but not HDAC-interaction-deficient LMO4 restored GCSF-induced Stat3 acetylation and p27 expression. Thus, our results indicate that LMO4 enhances GCSF-induced Stat3 signaling in neurons, in part by sequestering HDAC.

Rescue of neurons from ischemic injury by peroxisome proliferator-activated receptor-gamma requires a novel essential cofactor LMO4.

Activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) signaling after stroke may reduce brain injury, but this effect will depend on the levels of receptor and cofactors. Here, we showed that the direct effect of PPARgamma signaling to protect neurons from ischemic injury requires a novel cofactor LMO4, because this effect was lost in LMO4-null cortical neurons. PPARgamma agonist also failed to reduce cerebral infarction after transient focal ischemia in CaMKIIalphaCre/LMO4loxP mice with LMO4 ablated in neurons of the forebrain. Expressing LMO4 in LMO4-null cortical neurons rescued the PPARgamma-protective effect. PPARgamma signaling activates the promoter of the antioxidant gene SOD2 and this process requires LMO4. Addition of a superoxide dismutase mimetic MnTBAP [manganese(III)tetrakis(4-benzoic acid)porphyrin] bypassed the deficiency in PPARgamma signaling and was able to directly rescue LMO4-null cortical neurons from ischemic injury. Like LMO4, PPARgamma and PGC1alpha (PPARgamma coactivator 1alpha) levels in neurons are elevated by hypoxic stress, and absence of LMO4 impairs their upregulation. Coimmunoprecipitation and mammalian two-hybrid assays revealed that LMO4 interacts in a ligand-dependent manner with PPARgamma. LMO4 augments PPARgamma-dependent gene activation, in part, by promoting RXRalpha (retinoid X receptor-alpha) binding to PPARgamma and by increasing PPARgamma binding to its target DNA sequence. Together, our results identify LMO4 as an essential hypoxia-inducible cofactor required for PPARgamma signaling in neurons. Thus, upregulation of LMO4 expression after stroke is likely to be an important determinant of neuron survival.

Hydrogen sulfide as an oxygen sensor in trout gill chemoreceptors.

O2 chemoreceptors elicit cardiorespiratory reflexes in all vertebrates, but consensus on O2-sensing signal transduction mechanism(s) is lacking. We recently proposed that hydrogen sulfide (H2S) metabolism is involved in O2 sensing in vascular smooth muscle. Here, we examined the possibility that H2S is an O2 sensor in trout chemoreceptors where the first pair of gills is a primary site of aquatic O2 sensing and the homolog of the mammalian carotid body. Intrabuccal injection of H2S in unanesthetized trout produced a dose-dependent bradycardia and increased ventilatory frequency and amplitude similar to the hypoxic response. Removal of the first, but not second, pair of gills significantly inhibited H2S-mediated bradycardia, consistent with the loss of aquatic chemoreceptors. mRNA for H2S-synthesizing enzymes, cystathionine beta-synthase and cystathionine gamma-lyase, was present in branchial tissue. Homogenized gills produced H2S enzymatically, and H2S production was inhibited by O2, whereas mitochondrial H2S consumption was O2 dependent. Ambient hypoxia did not affect plasma H2S in unanesthetized trout, but produced a PO2-dependent increase in a sulfide moiety suggestive of increased H2S production. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, both hypoxia and H2S, produced a similar approximately 10-mV depolarization. These studies are consistent with H2S involvement in O2 sensing/signal transduction pathway(s) in chemoreceptive cells, as previously demonstrated in vascular smooth muscle. This novel mechanism, whereby H2S concentration ([H2S]) is governed by the balance between constitutive production and oxidation, tightly couples tissue [H2S] to PO2 and may provide an exquisitely sensitive, yet simple, O2 sensor in a variety of tissues.