Immunothrombosis versus thrombo-inflammation: platelets in cerebrovascular complications.

A State-of-the Art lecture titled "Thrombo-Neuroinflammatory Disease" was presented at the International Society on Thrombosis and Haemostasis Congress in 2023. First, we would like to advocate for discrimination between immunothrombosis and thrombo-inflammation, as immunothrombosis describes an overshooting inflammatory reaction that results in detrimental thrombotic activity. In contrast, thrombo-inflammation describes the interplay of platelets and coagulation with the immunovascular system, resulting in the recruitment of immune cells and loss of barrier function (hence, hallmarks of inflammation). Both processes can be observed in the brain, with cerebral venous thrombosis being a prime example of immunothrombosis, while infarct progression in response to ischemic stroke is a paradigmatic example of thrombo-inflammation. Here, we review the pathomechanisms underlying cerebral venous thrombosis and ischemic stroke from a platelet-centric perspective and discuss translational implications. Finally, we summarize relevant new data on this topic presented during the 2023 International Society on Thrombosis and Haemostasis Congress.

The inducible prostaglandin E synthase (mPGES-1) in neuroinflammatory disorders.

The cyclooxygenase (COX)/prostaglandin E2 (PGE2) signaling pathway has emerged as a critical target for anti-inflammatory therapeutic development in neurological diseases. However, medical use of COX inhibitors in the treatment of various neurological disorders has been limited due to well-documented cardiovascular and cerebrovascular complications. It has been widely proposed that modulation of downstream microsomal prostaglandin E synthase-1 (mPGES-1) enzyme may provide more specificity for inhibiting PGE2-elicited neuroinflammation. Heightened levels of mPGES-1 have been detected in a variety of brain diseases such as epilepsy, stroke, glioma, and neurodegenerative diseases. Subsequently, elevated levels of PGE2, the enzymatic product of mPGES-1, have been demonstrated to modulate a multitude of deleterious effects. In epilepsy, PGE2 participates in retrograde signaling to augment glutamate release at the synapse leading to neuronal death. The excitotoxic demise of neurons incites the activation of microglia, which can become overactive upon further stimulation by PGE2. A selective mPGES-1 inhibitor was able to reduce gliosis and the expression of proinflammatory cytokines in the hippocampus following status epilepticus. A similar mechanism has also been observed in stroke, where the overactivation of microglia by PGE2 upregulated the expression and secretion of proinflammatory cytokines. This intense activation of neuroinflammatory processes triggered the secondary injury commonly observed in stroke, and blockade of mPGES-1 reduced infarction size and edema, suppressed induction of proinflammatory cytokines, and improved post-stroke well-being and cognition. Furthermore, elevated levels of PGE2 have been shown to intensify the proliferation of glioma cells, mediate P-glycoprotein expression at the blood-brain barrier (BBB) and facilitate breakdown of the BBB. For these reasons, targeting mPGES-1, the central and inducible enzyme of the COX cascade, may provide a more specific therapeutic strategy for treating neuroinflammatory diseases.

Transient inhibition of microsomal prostaglandin E synthase-1 after status epilepticus blunts brain inflammation and is neuroprotective.

Status epilepticus (SE) in humans is characterized by prolonged convulsive seizures that are generalized and often difficult to control. The current antiseizure drugs (ASDs) aim to stop seizures quickly enough to prevent the SE-induced brain inflammation, injury, and long-term sequelae. However, sole reliance on acute therapies is imprudent because prompt treatment may not always be possible under certain circumstances. The pathophysiological mechanisms underlying the devastating consequences of SE are presumably associated with neuroinflammatory reactions, where prostaglandin E2 (PGE2) plays a pivotal role. As the terminal synthase for pathogenic PGE2, the microsomal prostaglandin E synthase-1 (mPGES-1) is rapidly and robustly induced by prolonged seizures. Congenital deletion of mPGES-1 in mice is neuroprotective and blunts gliosis following chemoconvulsant seizures, suggesting the feasibility of mPGES-1 as a potential antiepileptic target. Herein, we investigated the effects of a dual species mPGES-1 inhibitor in a mouse pilocarpine model of SE. Treatment with the mPGES-1 inhibitor in mice after SE that was terminated by diazepam, a fast-acting benzodiazepine, time-dependently abolished the SE-induced PGE2 within the brain. Its negligible effects on cyclooxygenases, the enzymes responsible for the initial step of PGE2 biosynthesis, validated its specificity to mPGES-1. Post-SE inhibition of mPGES-1 also blunted proinflammatory cytokines and reactive gliosis in the hippocampus and broadly prevented neuronal damage in a number of brain areas. Thus, pharmacological inhibition of mPGES-1 by small-molecule inhibitors might provide an adjunctive strategy that can be implemented hours after SE, together with first-line ASDs, to reduce SE-provoked brain inflammation and injury.

White matter damage as a consequence of vascular dysfunction in a spontaneous mouse model of chronic mild chronic hypoperfusion with eNOS deficiency.

Vascular cognitive impairment and dementia (VCID) is the second most common form of dementia after Alzheimer's disease (AD). Currently, the mechanistic insights into the evolution and progression of VCID remain elusive. White matter change represents an invariant feature. Compelling clinical neuroimaging and pathological evidence suggest a link between white matter changes and neurodegeneration. Our prior study detected hypoperfused lesions in mice with partial deficiency of endothelial nitric oxide (eNOS) at very young age, precisely matching to those hypoperfused areas identified in preclinical AD patients. White matter tracts are particularly susceptible to the vascular damage induced by chronic hypoperfusion. Using immunohistochemistry, we detected severe demyelination in the middle-aged eNOS-deficient mice. The demyelinated areas were confined to cortical and subcortical areas including the corpus callosum and hippocampus. The intensity of demyelination correlated with behavioral deficits of gait and associative recognition memory performances. By Evans blue angiography, we detected blood-brain barrier (BBB) leakage as another early pathological change affecting frontal and parietal cortex in eNOS-deficient mice. Sodium nitrate fortified drinking water provided to young and middle-aged eNOS-deficient mice completely prevented non-perfusion, BBB leakage, and white matter pathology, indicating that impaired endothelium-derived NO signaling may have caused these pathological events. Furthermore, genome-wide transcriptomic analysis revealed altered gene clusters most related to mitochondrial respiratory pathways selectively in the white matter of young eNOS-deficient mice. Using eNOS-deficient mice, we identified BBB breakdown and hypoperfusion as the two earliest pathological events, resulting from insufficient vascular NO signaling. We speculate that the compromised BBB and mild chronic hypoperfusion trigger vascular damage, along with oxidative stress and astrogliosis, accounting for the white matter pathological changes in the eNOS-deficient mouse model. We conclude that eNOS-deficient mice represent an ideal spontaneous evolving model for studying the earliest events leading to white matter changes, which will be instrumental to future therapeutic testing of drug candidates and for targeting novel/specific vascular mechanisms contributing to VCID and AD.

Ablation of Siglec-E augments brain inflammation and ischemic injury.

Sialic acid immunoglobulin-like lectin E (Siglec-E) is a subtype of pattern recognition receptors found on the surface of myeloid cells and functions as a key immunosuppressive checkpoint molecule. The engagement between Siglec-E and the ligand α2,8-linked disialyl glycans activates the immunoreceptor tyrosine-based inhibitory motif (ITIM) in its intracellular domain, mitigating the potential risk of autoimmunity amid innate immune attacks on parasites, bacteria, and carcinoma. Recent studies suggest that Siglec-E is also expressed in the CNS, particularly microglia, the brain-resident immune cells. However, the functions of Siglec-E in brain inflammation and injuries under many neurological conditions largely remain elusive. In this study, we first revealed an anti-inflammatory role for Siglec-E in lipopolysaccharide (LPS)-triggered microglial activation. We then found that Siglec-E was induced within the brain by systemic treatment with LPS in mice in a dose-dependent manner, while its ablation exacerbated hippocampal reactive microgliosis in LPS-treated animals. The genetic deficiency of Siglec-E also aggravated oxygen-glucose deprivation (OGD)-induced neuronal death in mouse primary cortical cultures containing both neurons and glial cells. Moreover, Siglec-E expression in ipsilateral brain tissues was substantially induced following middle cerebral artery occlusion (MCAO). Lastly, the neurological deficits and brain infarcts were augmented in Siglec-E knockout mice after moderate MCAO when compared to wild-type animals. Collectively, our findings suggest that the endogenous inducible Siglec-E plays crucial anti-inflammatory and neuroprotective roles following ischemic stroke, and thus might underlie an intrinsic mechanism of resolution of inflammation and self-repair in the brain.

Targeting EP2 receptor with multifaceted mechanisms for high-risk neuroblastoma.

Prostaglandin E2 (PGE2) promotes tumor cell proliferation, migration, and invasion, fostering an inflammation-enriched microenvironment that facilitates angiogenesis and immune evasion. However, the PGE2 receptor subtype (EP1-EP4) involved in neuroblastoma (NB) growth remains elusive. Herein, we show that the EP2 receptor highly correlates with NB aggressiveness and acts as a predominant Gαs-coupled receptor mediating PGE2-initiated cyclic AMP (cAMP) signaling in NB cells with high-risk factors, including 11q deletion and MYCN amplification. Knockout of EP2 in NB cells blocks the development of xenografts, and its conditional knockdown prevents established tumors from progressing. Pharmacological inhibition of EP2 by our recently developed antagonist TG6-129 suppresses the growth of NB xenografts in nude mice and syngeneic allografts in immunocompetent hosts, accompanied by anti-inflammatory, antiangiogenic, and apoptotic effects. This proof-of-concept study suggests that the PGE2/EP2 signaling pathway contributes to NB malignancy and that EP2 inhibition by our drug-like compounds provides a promising strategy to treat this deadly pediatric cancer.

Inhibition of TRPC3 channels by a novel pyrazole compound confers antiseizure effects.

OBJECTIVE: As a key member of the transient receptor potential (TRP) superfamily, TRP canonical 3 (TRPC3) regulates calcium homeostasis and contributes to neuronal excitability. Ablation of TRPC3 lessens pilocarpine-induced seizures in mice, suggesting that TRPC3 inhibition might represent a novel antiseizure strategy. Among current TRPC3 inhibitors, pyrazole 3 (Pyr3) is most selective and potent. However, Pyr3 only provides limited benefits in pilocarpine-treated mice, likely due to its low metabolic stability and potential toxicity. We recently reported a modified pyrazole compound 20 (or JW-65) that has improved stability and safety. The objective of this study was to explore the effects of TRPC3 inhibition by our current lead compound JW-65 on seizure susceptibility. METHODS: We first examined the pharmacokinetic properties including plasma half-life and brain to plasma ratio of JW-65 after systemic administration in mice. We then investigated the effects of TRPC3 inhibition by JW-65 on behavioral and electrographic seizures in mice treated with pilocarpine. To ensure our findings are not model specific, we assessed the susceptibility of JW-65-treated mice to pentylenetetrazole (PTZ)-induced seizures with phenytoin as a comparator. RESULTS: JW-65 showed adequate half-life and brain penetration in mice, justifying its use for central nervous system conditions. Systemic treatment with JW-65 before pilocarpine injection in mice markedly impaired the initiation of behavioral seizures. This antiseizure action was recapitulated when JW-65 was administered after pilocarpine-induced behavioral seizures were well established and was confirmed by time-locked electroencephalographic monitoring and synchronized video. Moreover, JW-65-treated mice showed substantially decreased susceptibility to PTZ-induced seizures in a dose-dependent manner. SIGNIFICANCE: These results suggest that pharmacological inhibition of the TRPC3 channels by our novel compound JW-65 might represent a new antiseizure strategy engaging a previously undrugged mechanism of action. Hence, this proof-of-concept study establishes TRPC3 as a novel feasible therapeutic target for the treatment of some forms of epilepsy.

Inducible Prostaglandin E Synthase as a Pharmacological Target for Ischemic Stroke.

As the inducible terminal enzyme for prostaglandin E2 (PGE2) synthesis, microsomal PGE synthase-1 (mPGES-1) contributes to neuroinflammation and secondary brain injury after cerebral ischemia via producing excessive PGE2. However, a proof of concept that mPGES-1 is a therapeutic target for ischemic stroke has not been established by a pharmacological strategy mainly due to the lack of drug-like mPGES-1 inhibitors that can be used in relevant rodent models. To this end, we recently developed a series of novel small-molecule compounds that can inhibit both human and rodent mPGES-1. In this study, blockade of mPGES-1 by our several novel compounds abolished the lipopolysaccharide (LPS)-induced PGE2 and pro-inflammatory cytokines interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α) in mouse primary brain microglia. Inhibition of mPGES-1 also decreased PGE2 produced by neuronal cells under oxygen-glucose deprivation (OGD) stress. Among the five enzymes for PGE2 biosynthesis, mPGES-1 was the most induced one in cerebral ischemic lesions. Systemic treatment with our lead compound MPO-0063 (5 or 10 mg/kg, i.p.) in mice after transient middle cerebral artery occlusion (MCAO) improved post-stroke well-being, decreased infarction and edema, suppressed induction of brain cytokines (IL-1ß, IL-6, and TNF-α), alleviated locomotor dysfunction and anxiety-like behavior, and reduced the long-term cognitive impairments. The therapeutic effects of MPO-0063 in this proof-of-concept study provide the first pharmacological evidence that mPGES-1 represents a feasible target for delayed, adjunct treatment - along with reperfusion therapies - for acute brain ischemia.

Acute Hyperglycemia Exacerbates Hemorrhagic Transformation after Embolic Stroke and Reperfusion with tPA: A Possible Role of TXNIP-NLRP3 Inflammasome.

OBJECTIVES: Acute hyperglycemia (HG) exacerbates reperfusion injury after stroke. Our recent studies showed that acute HG upregulates thioredoxin-interacting protein (TXNIP) expression, which in turn induces inflammation and neurovascular damage in a suture model of ischemic stroke. The aim of the present study was to investigate the effect of acute HG on TXNIP-associated neurovascular damage, in a more clinically relevant murine model of embolic stroke and intravenous tissue plasminogen activator (IV-tPA) reperfusion. MATERIALS AND METHODS: HG was induced in adult male mice, by intraperitoneal injection of 20% glucose. This was followed by embolic middle cerebral artery occlusion (eMCAO), with or without IV-tPA (10 mg/kg) given 3 h post embolization. Brain infarction, edema, hemoglobin content, expression of matrix metalloproteinase (MMP-9), vascular endothelial growth factor A (VEGFA), tight junction proteins (claudin-5, occluding, and zonula occludens-1), TXNIP, and NOD-like receptor protein3 (NLRP3)-inflammasome activation were evaluated at 24 h after eMCAO. RESULTS: HG alone significantly increased TXNIP in the brain after eMCAO, and this was associated with exacerbated hemorrhagic transformation (HT; as measured by hemoglobin content). IV-tPA in HG conditions showed a trend to decrease infarct volume, but worsened HT after eMCAO, suggesting that HG reduces the therapeutic efficacy of IV-tPA. Further, HG and tPA-reperfusion did not show significant differences in expression of MMP-9, VEGFA, junction proteins, and NLRP3 inflammasome activation between the groups. CONCLUSION: The current findings suggest a potential role for TXNIP in the occurrence of HT in hyperglycemic conditions following eMCAO. Further studies are needed to understand the precise role of vascular TXNIP on HG/tPA-induced neurovascular damage after stroke.

Endothelial Thioredoxin-Interacting Protein Depletion Reduces Hemorrhagic Transformation in Hyperglycemic Mice after Embolic Stroke and Thrombolytic Therapy.

We hypothesize that endothelial-specific thioredoxin-interacting protein knock-out (EC-TXNIP KO) mice will be more resistant to the neurovascular damage (hemorrhagic-transformation-HT) associated with hyperglycemia (HG) in embolic stroke. Adult-male EC-TXNIP KO and wild-type (WT) littermate mice were injected with-streptozotocin (40 mg/kg, i.p.) for five consecutive days to induce diabetes. Four-weeks after confirming HG, mice were subjected to embolic middle cerebral artery occlusion (eMCAO) followed by tissue plasminogen activator (tPA)-reperfusion (10 mg/kg at 3 h post-eMCAO). After the neurological assessment, animals were sacrificed at 24 h for neurovascular stroke outcomes. There were no differences in cerebrovascular anatomy between the strains. Infarct size, edema, and HT as indicated by hemoglobin (Hb)-the content was significantly higher in HG-WT mice, with or without tPA-reperfusion, compared to normoglycemic WT mice. Hyperglycemic EC-TXNIP KO mice treated with tPA tended to show lower Hb-content, edema, infarct area, and less hemorrhagic score compared to WT hyperglycemic mice. EC-TXNIP KO mice showed decreased expression of inflammatory mediators, apoptosis-associated proteins, and nitrotyrosine levels. Further, vascular endothelial growth factor-A and matrix-metalloproteinases (MMP-9/MMP-3), which degrade junction proteins and increase blood-brain-barrier permeability, were decreased in EC-TXNIP KO mice. Together, these findings suggest that vascular-TXNIP could be a novel therapeutic target for neurovascular damage after stroke.

EP2 Antagonists (2011-2021): A Decade's Journey from Discovery to Therapeutics.

In the wake of health disasters associated with the chronic use of cyclooxygenase-2 (COX-2) inhibitor drugs, it has been widely proposed that modulation of downstream prostanoid synthases or receptors might provide more specificity than simply shutting down the entire COX cascade for anti-inflammatory benefits. The pathogenic actions of COX-2 have long been thought attributable to the prostaglandin E2 (PGE2) signaling through its Gαs-coupled EP2 receptor subtype; however, the truly selective EP2 antagonists did not emerge until 2011. These small molecules provide game-changing tools to better understand the EP2 receptor in inflammation-associated conditions. Their applications in preclinical models also reshape our knowledge of PGE2/EP2 signaling as a node of inflammation in health and disease. As we celebrate the 10-year anniversary of this breakthrough, the exploration of their potential as drug candidates for next-generation anti-inflammatory therapies has just begun. The first decade of EP2 antagonists passes, while their future looks brighter than ever.

Thioredoxin interacting protein regulates age-associated neuroinflammation.

Immune system hypersensitivity is believed to contribute to mental frailty in the elderly. Solid evidence indicates NOD-like receptor pyrin domain containing-3 (NLRP3)-inflammasome activation intimately connects aging-associated chronic inflammation (inflammaging) to senile cognitive decline. Thioredoxin interacting protein (TXNIP), an inducible protein involved in oxidative stress, is essential for NLRP3 inflammasome activity. This study aims to find whether TXNIP/NLRP3 inflammasome pathway is involved in senile dementia. According to our studies on sex-matched mice, TXNIP was significantly upregulated in aged animals, paralleled by the NLRP3-inflammasome over-activity leading to enhanced caspase-1 cleavage and IL-1ß maturation, in both sexes. This was closely associated with depletion of the anti-aging and cognition enhancing protein klotho, in aged males. Txnip knockout reversed age-related NLRP3-hyperactivity and enhanced thioredoxin (TRX) levels. Further, TXNIP inhibition along with verapamil replicated TXNIP/NLRP3-inflammasome downregulation in aged animals, with FOXO-1 and mTOR upregulation. These alterations concurred with substantial improvements in both cognitive and sensorimotor abilities. Together, these findings substantiate the pivotal role of TXNIP to drive inflammaging in parallel with klotho depletion and functional decline, and delineate thioredoxin system as a potential target to decelerate senile dementia.

Prostaglandin E receptors as targets for ischemic stroke: Novel evidence and molecular mechanisms of efficacy.

Over the past two decades the interest has waned in therapeutically targeting cyclooxygenase-2 (COX-2) due to growing concerns over the potential cardiovascular and cerebrovascular toxicities of the long-term use of COX-2 inhibitors. Attention thus has recently been shifted downstream to the prostaglandin signaling pathways for new druggable anti-inflammatory targets aiming for higher therapeutic specificity. Prostaglandin E2 (PGE2) is robustly synthesized in the ischemic cortex by quickly induced COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) following cerebral ischemia. The elevated PGE2, in turn, divergently regulates the excitotoxic injury and neuroinflammation by acting on four membrane-bound G protein-coupled receptors (GPCRs), namely, EP1-EP4. Markedly, all four EP receptors have been implicated in the excitotoxicity-associated brain inflammation and injury in animal models of cerebral ischemia. However promising, these preclinical studies have not yet led to a clinical trial targeting any PGE2 receptor for ischemic stroke. The goal of this article is to review the recent progress in understanding the pathogenic roles of PGE2 in cerebral ischemia as well as to provide new mechanistic insights into the PGE2 signaling via these four GPCRs in neuronal excitotoxicity and inflammation. We also discuss the feasibility of targeting EP1-EP4 receptors as an emerging delayed treatment, together with the first-line reperfusion strategy, to manage acute ischemic stroke with potentially extended window as well as improved specificity.

Inhibiting the PGE2 Receptor EP2 Mitigates Excitotoxicity and Ischemic Injury.

Prostaglandin E2 (PGE2) is elevated in the brain by excitotoxic insults and, in turn, aggravates the neurotoxicity mainly through acting on its Gαs-coupled receptor EP2, inspiring a therapeutic strategy of targeting this key proinflammatory pathway. Herein, we investigated the effects of several highly potent and selective small-molecule antagonists of the EP2 receptor on neuronal excitotoxicity both in vitro and in vivo. EP2 inhibition by these novel compounds largely decreased the neuronal injury in rat primary hippocampal cultures containing both neurons and glia that were treated with N-methyl-d-aspartate and glycine. Using a bioavailable and brain-permeant analogue TG6-10-1 that we recently developed to target the central EP2 receptor, we found that the poststroke EP2 inhibition in mice decreased the neurological deficits and infarct volumes as well as downregulated the prototypic inflammatory cytokines in the brain after a transient ischemia. Our preclinical findings together reinforced the notion that targeting the EP2 receptor represents an emerging therapeutic strategy to prevent the neuronal injury and inflammation following ischemic stroke.

Inverse Agonism of Cannabinoid Receptor Type 2 Confers Anti-inflammatory and Neuroprotective Effects Following Status Epileptics.

Prolonged status epilepticus (SE) in humans causes high mortality and brain inflammation-associated neuronal injury and morbidity in survivors. Currently, the only effective treatment is to terminate the seizures swiftly to prevent brain damage. However, reliance on acute therapies alone would be imprudent due to the required short response time. Follow-on therapies that can be delivered well after the SE onset are in an urgent need. Cannabinoid receptor type 2 (CB2), a G protein-coupled receptor that can be expressed by activated brain microglia, has emerged as an appealing anti-inflammatory target for brain conditions. In the current study, we reported that the CB2 inverse agonism by our current lead compound SMM-189 largely prevented the rat primary microglia-mediated inflammation and showed moderate neuroprotection against N-methyl-D-aspartic acid (NMDA) receptor-mediated excitotoxicity in rat primary hippocampal cultures containing both neurons and glia. Using a classical mouse model of epilepsy, in which SE was induced by systemic administration of kainate (30 mg/kg, i.p.) and proceeded for 1 h, we demonstrated that SE downregulated the CB1 but slightly upregulated CB2 receptor in the hippocampus. Transient treatment with SMM-189 (6 mg/kg, i.p., b.i.d.) after the SE was interrupted by diazepam (10 mg/kg, i.p.) prevented the seizure-induced cytokine surge in the brain, neuronal death, and behavioral impairments 24 h after SE. Our results suggest that CB2 inverse agonism might provide an adjunctive anti-inflammatory therapy that can be delivered hours after SE onset, together with NMDA receptor blockers and first-line anti-convulsants, to reduce brain injury and functional deficits following prolonged seizures.

Thioredoxin-Interacting Protein (TXNIP) Associated NLRP3 Inflammasome Activation in Human Alzheimer's Disease Brain.

Alzheimer's disease (AD) is the most common form of age-associated dementia characterized by amyloid-ß plaques and neurofibrillary tangles. Recent studies have demonstrated that thioredoxin-interacting protein (TXNIP), an endogenous regulator of redox/glucose induced stress and inflammation, is now known to be upregulated in stroke, traumatic brain injury, diabetes and AD. We hypothesized that TXNIP overexpression sustains neurodegeneration through activation of the nucleotide binding and oligomerization domain-like receptor protein 3 in human AD brains. We analyzed TXNIP and the components of the NLRP3 inflammasome in the cortex of postmortem human brain samples by western blotting, real-time PCR, and immunohistochemical techniques in comparison with age-matched non-demented controls. Our results demonstrate that TXNIP protein as well as its mRNA levels in the cortex was significantly upregulated in AD compared to control brains. Moreover, using double immunofluorescence staining, TXNIP and interlukin-1ß (IL-1ß) were co-localized near Aß plaques and p-tau. These results suggest an association between TXNIP overexpression levels and AD pathogenesis. Further, a significant increased expression of cleaved caspase-1 and IL-1ß, the products of inflammasome activation, was detected in the cortex of AD brains. Together, these findings suggest that TXNIP, an upstream promising new therapeutic target, is a molecular link between inflammation and AD. The significant contribution of TXNIP to AD pathology suggests that strategies focusing on specific targeting of the TXNIP-NLRP3 inflammasome may lead to novel therapies for the management of AD and other age-related dementias.

Neuronal HIF-1α and HIF-2α deficiency improves neuronal survival and sensorimotor function in the early acute phase after ischemic stroke.

Hypoxia-inducible factors mediate adaptive responses to ischemia, among others, by induction of anti- and pro-survival genes. Thus, the impact of HIF on neuronal survival upon stroke is controversial. Therefore, neuron-specific knockout mice deficient for Hif1a and Hif2a were exposed to inspiratory hypoxia or ischemia-reperfusion injury. Both Hif1a- and Hif2a-deficient mice showed no altered infarct and edema size, suggesting that both HIF-α subunits might compensate for each other. Accordingly, hypoxic HIF-target gene regulation was marginally affected with exception of anti-survival Bnip3 and pro-survival erythropoietin. In the early acute stage upon stroke, Hif1a/Hif2a double knockout mice exhibited significantly reduced expression of the anti-survival Bnip3, Bnip3L, and Pmaip1 Accordingly, global cell death and edema were significantly reduced upon 24 h but not 72 h reperfusion. Behavioral assessment indicated that Hif1a/Hif2a-deficient mice initially performed better, but became significantly more impaired after 72 h accompanied by increased apoptosis and reduced angiogenesis. Our findings suggest that in neurons HIF-1 and HIF-2 have redundant functions for cellular survival under ischemic conditions. By contrast, lack of anti-survival factors in Hif1a/Hif2a-deficient mice might protect from early acute neuronal cell death and neurological impairment, indicating a benefit of HIF-pathway inhibition in neurons in the very acute phase after ischemic stroke.

Neuronal prolyl-4-hydroxylase 2 deficiency improves cognitive abilities in a murine model of cerebral hypoperfusion.

Episodes of cerebral hypoxia/ischemia increase the risk of dementia, which is associated with impaired learning and memory. Previous studies in rodent models of dementia indicated a favorable effect of the hypoxia-inducible factor (HIF) targets VEGF (vascular endothelial growth factor) and erythropoietin (Epo). In the present study we thus investigated whether activation of the entire adaptive HIF pathway in neurons by cell-specific deletion of the HIF suppressor prolyl-4-hydroxylase 2 (PHD2) improves cognitive abilities in young (3months) and old (18-28months) mice suffering from chronic brain hypoperfusion. Mice underwent permanent occlusion of the left common carotid artery, and cognitive function was assessed using the Morris water navigation task. Under conditions of both normal and decreased brain perfusion, neuronal PHD2 deficiency resulted in improved and faster spatial learning in young mice, which was preserved to some extent also in old animals. The loss of PHD2 in neurons resulted in enhanced hippocampal mRNA and protein levels of Epo and VEGF, but did not alter local microvascular density, dendritic spine morphology, or expression of synaptic plasticity-related genes in the hippocampus. Instead, better cognitive function in PHD2 deficient animals was accompanied by an increased number of neuronal precursor cells along the subgranular zone of the dentate gyrus. Overall, our current pre-clinical findings indicate an important role for the endogenous oxygen sensing machinery, encompassing PHDs, HIFs and HIF target genes, for proper cognitive function. Thus, pharmacological compounds affecting the PHD-HIF axis might well be suited to treat cognitive dysfunction and neurodegenerative processes.

Neuronal deficiency of HIF prolyl 4-hydroxylase 2 in mice improves ischemic stroke recovery in an HIF dependent manner.

Hypoxia inducible factors (HIFs) mediate the endogenous adaptive responses to hypoxia. HIF prolyl 4-hydroxylase domain proteins (PHD) are important suppressors of the HIF pathway. Recently, we demonstrated that neuron-specific deletion of Phd2 reduces cerebral tissue damage in the very acute phase of ischemic stroke. In the present study, we investigated whether neuronal Phd2 ablation is likewise beneficial for stroke recovery, and aimed to identify underlying cellular mechanisms. Mice underwent permanent occlusion of the distal middle cerebral artery (pdMCAO) for either 7days (sub-acute stage) or 30days (chronic stage). One week after pdMCAO the infarct size of Phd2-deficient mice was significantly reduced as compared to wild-type (WT) mice. Accordingly, Phd2-deficient animals showed less impaired sensorimotor function. Neuronal loss of Phd2 upregulated vascular endothelial growth factor (VEGF) and significantly increased microvascular density along the infarct border in the sub-acute stage of stroke. Phd2-deficient mice showed reduced expression of pro-inflammatory cytokines and increased numbers of resting microglia/macrophages and reactive astrocytes within peri-infarct regions in comparison to WT littermates. Finally, brain tissue protection and increased angiogenesis upon sub-acute ischemic stroke was completely absent in Phd2 knockout mice that were additionally deficient for both Hif1a and Hif2a. Our findings suggest that lack of PHD2 in neurons improves histological and functional long-term outcome from ischemic stroke at least partly by amplifying endogenous adaptive neovascularization through activation of the HIF-VEGF axis.

Fumaric acid esters promote neuronal survival upon ischemic stress through activation of the Nrf2 but not HIF-1 signaling pathway.

Oxidative stress is a hallmark of ischemic stroke pathogenesis causing neuronal malfunction and cell death. Up-regulation of anti-oxidative genes through activation of the NF-E2-related transcription factor 2 (Nrf2) is one of the key mechanisms in cellular defense against oxidative stress. Fumaric acid esters (FAEs) represent a class of anti-oxidative and anti-inflammatory molecules that are already in clinical use for multiple sclerosis therapy. Purpose of this study was to investigate whether FAEs promote neuronal survival upon ischemia, and analyze putative underlying molecular mechanisms in neurons. Murine organotypic hippocampal slice cultures, and two neuronal cell lines were treated with dimethyl fumarate (DMF) and monomethyl fumarate (MMF). Ischemic conditions were generated by exposing cells and slice cultures to oxygen-glucose deprivation (OGD), and cell death was determined through propidium iodide staining. Treatment with both DMF and MMF immediately after OGD during reoxygenation strongly reduced cell death in hippocampal cultures ex vivo. Both DMF and MMF promoted neuronal survival in HT-22 and SH-SY5Y cell lines exposed to ischemic stress. DMF but not MMF activated the anti-oxidative Nrf2 pathway in neurons. Accordingly, Nrf2 knockdown in murine neurons abrogated the protective effect of DMF but not MMF. Moreover, FAEs did not activate the hypoxia-inducible factor (HIF) pathway suggesting that this pathway may not significantly contribute to FAE mediated neuroprotection. Our results may provide the basis for a new therapeutic approach to treat ischemic pathologies such as stroke with a drug that already has a broad safety record in humans.