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.

IRF2BP2 Reduces Macrophage Inflammation and Susceptibility to Atherosclerosis.

RATIONALE: Inflammation impairs macrophage cholesterol clearance from vascular tissues and promotes atherosclerosis. Inflammatory macrophages suppress expression of the transcription cofactor interferon regulatory factor 2-binding protein 2 (IRF2BP2), and genetic variants near IRF2BP2 associate with ischemic heart disease progression in humans. OBJECTIVES: To test whether IRF2BP2 in macrophages affects atherosclerosis in mice and humans. METHODS AND RESULTS: We generated mice that delete IRF2BP2 in macrophages. IRF2BP2-deficient macrophages worsened atherosclerosis in irradiated low-density lipoprotein receptor null-recipient mice and in apolipoprotein E null mice. IRF2BP2-deficient macrophages were inflammatory and had impaired cholesterol efflux because of their inability to activate the cholesterol transporter ABCA1 in response to cholesterol loading. Their expression of the anti-inflammatory transcription factor Krüppel-like factor 2 was markedly reduced. Promoter studies revealed that IRF2BP2 is required for MEF2-dependent activation of Krüppel-like factor 2. Importantly, restoring Krüppel-like factor 2 in IRF2BP2-deficient macrophages attenuated M1 inflammatory and rescued M2 anti-inflammatory gene activation and improved the cholesterol efflux deficit by restoring ABCA1 activation in response to cholesterol loading. In a cohort of 1066 angiographic cases and 1011 controls, homozygous carriers of a deletion polymorphism (rs3045215) in the 3' untranslated region sequence of human IRF2BP2 mRNA had a higher risk of coronary artery disease (recessive model, odds ratio [95% confidence interval]=1.560 [1.179-2.065], P=1.73E-03) and had lower IRF2BP2 (and Krüppel-like factor 2) protein levels in peripheral blood mononuclear cells. The effect of this deletion polymorphism to suppress protein expression was confirmed in luciferase reporter studies. CONCLUSION: Ablation of IRF2BP2 in macrophages worsens atherosclerosis in mice, and a deletion variant that lowers IRF2BP2 expression predisposes to coronary artery disease in humans.

Stanniocalcin-1 controls ion regulation functions of ion-transporting epithelium other than calcium balance.

Stanniocalcin-1 (STC-1) was first identified to involve in Ca(2+) homeostasis in teleosts, and was thought to act as a hypocalcemic hormone in vertebrate. Recent studies suggested that STC-1 exhibits broad effects on ion balance, not confines to Ca(2+), but the mechanism of this regulation process remains largely unknown. Here, we used zebrafish embryos as an alternative in vivo model to investigate how STC-1 regulates transepithelial ion transport function in ion-transporting epithelium. Expression of stc-1 mRNA in zebrafish embryos was increased in high-Ca(2+) environments but decreased by acidic and ion-deficient treatments while overexpression of stc-1 impaired the hypotonic acclimation by decreasing whole body Ca(2+), Na(+), and Cl(-) contents and H(+) secretion ability. Injection of STC-1 mRNA also down-regulated mRNA expressions of epithelial Ca(2+) channel, H(+)-ATPase, and Na(+)-Cl(-) cotransporter, suggesting the roles of STC-1 in regulation of ions other than Ca(2+). Knockdown of STC-1 caused an increase in ionocyte progenitors (foxi3a as the marker) and mature ionocytes (ion transporters as the markers), but did not affect epithelium stem cells (p63 as the marker) in the embryonic skin. Overexpression of STC-1 had the corresponding opposite effect on ionocyte progenitors, mature ionocytes in the embryonic skin. Taken together, STC-1 negatively regulates the number of ionocytes to reduce ionocyte functions. This process is important for body fluid ionic homeostasis, which is achieved by the regulation of ion transport functions in ionocytes. The present findings provide new insights into the broader functions of STC-1, a hypocalcemic hormone.

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.

Anion exchanger 1b, but not sodium-bicarbonate cotransporter 1b, plays a role in transport functions of zebrafish H+-ATPase-rich cells.

Similar to mammalian proximal tubular cells, H(+)-ATPase rich (HR) cells in zebrafish skin and gills are also responsible for Na(+) uptake and acid secretion functions. However, the basolateral transport pathways in HR cells are still unclear. In the present study, we tested the hypothesis if there are specific slc4 members involved in basolateral ion transport pathways in HR cells. Fourteen isoforms were identified in the zebrafish(z) slc4 family, and the full-length cDNAs of two novel isoforms, zslc4a1b (anion exchanger, zAE1b) and zslc4a4b (Na(+)/HCO(3)(-) cotransporter, zNBCe1b), were sequenced. mRNA signals of zslc4a1b and zslc4a4b were mainly detected in certain groups of ionocytes in zebrafish skin/gills. Further double immunocytochemistry or in situ hybridization demonstrated that zAE1b, but not zNBCe1b, was localized to basolateral membranes of HR cells. Acclimation to low-Na(+) or acidic environments stimulated the mRNA expression of zslc4a1b in zebrafish gills, and loss-of-function of zslc4a1b with specific morpholinos caused significant decreases in both the whole body Na(+) content and the skin H(+) activity in the morphants. On the basis of these results, it was concluded that zAE1b, but not zNBCe1b, is involved in the basolateral transport pathways in Na(+) uptake/acid secretion mechanisms in zebrafish HR cells.

Ghrelin affects carbohydrate-glycogen metabolism via insulin inhibition and glucagon stimulation in the zebrafish (Danio rerio) brain.

Carbohydrate-glycogen metabolism (CGM) is critical for emergency energy supplies in the central nervous system (CNS). Ghrelin (GHRL) in pancreas is known to significantly regulate a dominant player in CGM, insulin (INS). However, its regulatory effect on extrapancreatic INS synthesis is yet unknown. In this study, we used adult zebrafish to elucidate the expression and role of zebrafish GHRL (zGHRL) in genes primarily involved in the brain's CGM. Results showed that zebrafish brain expressed zghrl and its receptor, growth hormone secretagogue-receptor (GHS-R: zghs-r1a and zghs-r2a), according to RT-PCR and in situ hybridization. Protein localization coupled with mRNA spatial expression further verified zGHRL's presence in the brain. For the in vivo study, significant increases in zghs-r1a and zghs-r2a synthesis were observed after injection of synthetic peptide goldfish GHRL-12 (gGHRL) using brain templates analyzed by quantitative real-time PCR (qPCR). Ligand-receptor synthesis of INS (zinsa; zins-r1 and zins-r2) significantly decreased, while glucagon (GCG) (zgcgb1 and zgcgb2; zgcg-r1 and zgcg-r2) exhibited a significant transient increase. In CGM, subsequent processes indicate urgent glucose-sensing response that will balance glycogen degradation and energy storage. Taken together, these findings suggest that GHRL regulates INS synthesis by mediating its action on GHS-R in the CNS and partly involved in CGM.