miR-483-5p offsets functional and behavioural effects of stress in male mice through synapse-targeted repression of Pgap2 in the basolateral amygdala.

Severe psychological trauma triggers genetic, biochemical and morphological changes in amygdala neurons, which underpin the development of stress-induced behavioural abnormalities, such as high levels of anxiety. miRNAs are small, non-coding RNA fragments that orchestrate complex neuronal responses by simultaneous transcriptional/translational repression of multiple target genes. Here we show that miR-483-5p in the amygdala of male mice counterbalances the structural, functional and behavioural consequences of stress to promote a reduction in anxiety-like behaviour. Upon stress, miR-483-5p is upregulated in the synaptic compartment of amygdala neurons and directly represses three stress-associated genes: Pgap2, Gpx3 and Macf1. Upregulation of miR-483-5p leads to selective contraction of distal parts of the dendritic arbour and conversion of immature filopodia into mature, mushroom-like dendritic spines. Consistent with its role in reducing the stress response, upregulation of miR-483-5p in the basolateral amygdala produces a reduction in anxiety-like behaviour. Stress-induced neuromorphological and behavioural effects of miR-483-5p can be recapitulated by shRNA mediated suppression of Pgap2 and prevented by simultaneous overexpression of miR-483-5p-resistant Pgap2. Our results demonstrate that miR-483-5p is sufficient to confer a reduction in anxiety-like behaviour and point to miR-483-5p-mediated repression of Pgap2 as a critical cellular event offsetting the functional and behavioural consequences of psychological stress.

Myelin-associated proteins are potential diagnostic markers in patients with primary brain tumour.

INTRODUCTION: Taking into account the possibility of myelin-associated proteins having a role in brain tumour development, the study aimed to evaluate the diagnostic usefulness of myelin-associated proteins (Nogo-A, MAG, OMgp) released into extracellular space in patients with brain tumours. PATIENTS AND METHODS: Protein concentration in primary brain tumour (n = 49) and non-tumoural subjects (n = 24) was measured in cerebrospinal fluid (CSF) and serum by means of ELISA. Immunohistochemistry for IDH1-R132H was done on 5-µm thick formalin-fixed, paraffin-embedded tumour sections with the use of an antibody specific for the mutant IDH1-R132H protein. RESULTS: The receiver operator characteristic curve analysis showed that CSF Nogo-A and serum MAG were useful in differentiating patients with primary brain tumour from non-tumoural individuals. This was also true in the case of the separate analysis of the astrocytic tumour versus non-tumoural groups and the meningeal tumour versus non-tumoural groups. Neither Nogo-A nor MAG or OMgp concentrations were significantly different, in serum or CSF, between IDH1 wild-type astrocytic brain tumour patients compared to IDH1 mutant patients. CONCLUSIONS: Our results indicated the potential usefulness of CSF Nogo-A and serum MAG evaluation as circulating biomarkers of primary brain tumours. Because blood is relatively easy to obtain, future research should be conducted to explicitly indicate the value of serum MAG concentration evaluation as a brain tumour biomarker.Key messagesMyelin-associated proteins may be circulating brain tumour biomarkers.Nogo-A and MAG proteins seem to be the most useful in brain tumour diagnosis.Decreased CSF Nogo-A concentration is an adverse prognostic factor for patients' survival.

Modulation of brain cation-Cl- cotransport via the SPAK kinase inhibitor ZT-1a.

The SLC12A cation-Cl- cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide ("ZT-1a"). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.

Comparison between cuff-based and radial tonometry exercise-induced central blood pressure.

PURPOSE: Non-invasive central blood pressure assessed during exercise may provide better cardiovascular prognostic than measurements taken at rest. Radial tonometry is the only technique validated to perform this type of assessment; however, it relies on the experience of the tester. Cuff-based devices have been developed to avoid operator dependency, although these systems have yet to be validated during exercise. The purpose of this study was to compare exercise-induced central blood pressure estimations between a cuff-based device and radial tonometry. METHODS: Twenty young healthy subjects were recruited to perform a three-workload steady-state exercise test at blood lactate levels of < 2, 2-4, and > 4 mmol/L, respectively. Central systolic and diastolic blood pressure (cSBP and cDBP, respectively), central pulse pressure (cPP), and augmentation index (AIx) were assessed at rest and during each workload with a cuff-based device and radial tonometry. Statistical analysis included Bland-Altman analysis for agreement between techniques. Agreement was considered when 95% of the data set for each central blood pressure parameter was within 1.96 standard deviations from the mean difference. Significance was considered at α = 0.05. RESULTS: Central blood pressure measurements with the cuff device were obtained only at rest and during low-intensity exercise. During low-intensity exercise, all measurements showed agreement between both devices (cSBP 95% CI [- 6.0 to 10.7], cDBP 95% CI [- 4.5 to 6.3], cPP 95% CI [- 4.7 to 8.3], and AIx (95% CI [- 20.1 to 22.2]). CONCLUSION: A cuff-based device can estimate central blood pressure at low-intensity exercise, without operator dependency, and showing agreement to radial tonometry.

Internal validation of an automated system for brachial and femoral flow mediated dilation.

BACKGROUND: Flow Mediated Dilation (FMD) has immense potential to become a clinical, non-invasive biomarker of endothelial function and nitric oxide bioavailability, which regulate vasomotor activity. Unfortunately, FMD analysis techniques could deviate significantly in different laboratories if a validation process is not involved. The purpose of this study was to provide validation to the assessment of FMD analysis in our laboratory and to standardize this process before reporting results of FMD. METHODS: Brachial and femoral arteries FMD were performed on 28 apparently healthy participants (15 male and 13 female, ages 18-35 years). For the intratester reliability study, nine subjects were asked to come to the lab for a second brachial FMD within 48 h. All FMD procedures were performed by the same investigator, while the FMD analyses were performed by 2 independent testers who were blind to each other's analyses. FMD analyses included baseline artery diameter measurements, peak artery diameter after 5 min of ischemia, and FMD. Analysis was completed via an automated edge detection system by both testers after training of the methodical process of analysis to minimize variability. Intratester and intertester reliability were determined by using coefficient of variation (CV) between first and second visit (intratester) and between results obtained by both testers (intertester). RESULTS: The intratester CVs for tester 1 and 2 were 3.28 and 2.62%, 3.74 and 3.27%, and 4.95 and 2.38% for brachial baseline artery diameter, brachial peak artery dilation, and brachial FMD, respectively. In the intertester CVs were 2.40, 3.16, and 3.37% for brachial baseline artery diameter, peak artery dilation, and FMD, respectively and 4.52, 5.50, and 3.46% for femoral baseline artery diameter, peak artery dilation, and FMD, respectively. CONCLUSION: All CVs were under or around 5%, confirming a strong reliability of the method. Our laboratory has shown that the FMD protocol is reproducible due to the significantly low coefficient of variation. This is one step closer to use FMD as a biomarker for endothelial function in our laboratory.

M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss.

The current frontline symptomatic treatment for Alzheimer's disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR-selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.

Novel Genetic, Clinical, and Pathomechanistic Insights into TFG-Associated Hereditary Spastic Paraplegia.

Hereditary spastic paraplegias (HSPs) are genetically and clinically heterogeneous axonopathies primarily affecting upper motor neurons and, in complex forms, additional neurons. Here, we report two families with distinct recessive mutations in TFG, previously suggested to cause HSP based on findings in a single small family with complex HSP. The first carried a homozygous c.317G>A (p.R106H) variant and presented with pure HSP. The second carried the same homozygous c.316C>T (p.R106C) variant previously reported and displayed a similarly complex phenotype including optic atrophy. Haplotyping and bisulfate sequencing revealed evidence for a c.316C>T founder allele, as well as for a c.316_317 mutation hotspot. Expression of mutant TFG proteins in cultured neurons revealed mitochondrial fragmentation, the extent of which correlated with clinical severity. Our findings confirm the causal nature of bi-allelic TFG mutations for HSP, broaden the clinical and mutational spectra, and suggest mitochondrial impairment to represent a pathomechanistic link to other neurodegenerative conditions.

Localization and regulation of PML bodies in the adult mouse brain.

PML is a tumor suppressor protein involved in the pathogenesis of promyelocytic leukemia. In non-neuronal cells, PML is a principal component of characteristic nuclear bodies. In the brain, PML has been implicated in the control of embryonic neurogenesis, and in certain physiological and pathological phenomena in the adult brain. Yet, the cellular and subcellular localization of the PML protein in the brain, including its presence in the nuclear bodies, has not been investigated comprehensively. Because the formation of PML bodies appears to be a key aspect in the function of the PML protein, we investigated the presence of these structures and their anatomical distribution, throughout the adult mouse brain. We found that PML is broadly expressed across the gray matter, with the highest levels in the cerebral and cerebellar cortices. In the cerebral cortex PML is present exclusively in neurons, in which it forms well-defined nuclear inclusions containing SUMO-1, SUMO 2/3, but not Daxx. At the ultrastructural level, the appearance of neuronal PML bodies differs from the classic one, i.e., the solitary structure with more or less distinctive capsule. Rather, neuronal PML bodies have the form of small PML protein aggregates located in the close vicinity of chromatin threads. The number, size, and signal intensity of neuronal PML bodies are dynamically influenced by immobilization stress and seizures. Our study indicates that PML bodies are broadly involved in activity-dependent nuclear phenomena in adult neurons.

Neural ECM proteases in learning and synaptic plasticity.

Recent studies implicate extracellular proteases in synaptic plasticity, learning, and memory. The data are especially strong for such serine proteases as thrombin, tissue plasminogen activator, neurotrypsin, and neuropsin as well as matrix metalloproteinases, MMP-9 in particular. The role of those enzymes in the aforementioned phenomena is supported by the experimental results on the expression patterns (at the gene expression and protein and enzymatic activity levels) and functional studies, including knockout mice, specific inhibitors, etc. Counterintuitively, the studies have shown that the extracellular proteolysis is not responsible mainly for an overall degradation of the extracellular matrix (ECM) and loosening perisynaptic structures, but rather allows for releasing signaling molecules from the ECM, transsynaptic proteins, and latent form of growth factors. Notably, there are also indications implying those enzymes in the major neuropsychiatric disorders, probably by contributing to synaptic aberrations underlying such diseases as schizophrenia, bipolar, autism spectrum disorders, and drug addiction.

Mutations in KPTN cause macrocephaly, neurodevelopmental delay, and seizures.

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis.

Stress-induced lipocalin-2 controls dendritic spine formation and neuronal activity in the amygdala.

Behavioural adaptation to psychological stress is dependent on neuronal plasticity and dysfunction at this cellular level may underlie the pathogenesis of affective disorders such as depression and post-traumatic stress disorder. Taking advantage of genome-wide microarray assay, we performed detailed studies of stress-affected transcripts in the amygdala - an area which forms part of the innate fear circuit in mammals. Having previously demonstrated the role of lipocalin-2 (Lcn-2) in promoting stress-induced changes in dendritic spine morphology/function and neuronal excitability in the mouse hippocampus, we show here that the Lcn-2 gene is one of the most highly upregulated transcripts detected by microarray analysis in the amygdala after acute restraint-induced psychological stress. This is associated with increased Lcn-2 protein synthesis, which is found on immunohistochemistry to be predominantly localised to neurons. Stress-naïve Lcn-2(-/-) mice show a higher spine density in the basolateral amygdala and a 2-fold higher rate of neuronal firing rate compared to wild-type mice. Unlike their wild-type counterparts, Lcn-2(-/-) mice did not show an increase in dendritic spine density in response to stress but did show a distinct pattern of spine morphology. Thus, amygdala-specific neuronal responses to Lcn-2 may represent a mechanism for behavioural adaptation to psychological stress.

Ephs and ephrins: emerging therapeutic targets in neuropathology.

Eph receptors have been the subject of intense research since their discovery. Their widespread pattern of expression, involvement in a variety of important cellular phenomena and unique mode of action have stimulated interest in their role in health and disease across biological and medical domains. However, the function of Ephs in nervous system development and plasticity remains the best characterised. Recent advances suggest that Ephs play an important role in the development of brain pathologies. This review focuses on their basic structure and function and discusses the latest research on their role in neurological diseases.

Lipocalin-2 controls neuronal excitability and anxiety by regulating dendritic spine formation and maturation.

Psychological stress causes adaptive changes in the nervous system directed toward maintaining homoeostasis. These biochemical and structural mechanisms regulate animal behavior, and their malfunction may result in various forms of affective disorders. Here we found that the lipocalin-2 (Lcn2) gene, encoding a secreted protein of unknown neuronal function, was up-regulated in mouse hippocampus following psychological stress. Addition of lipocalin-2 to cultured hippocampal neurons reduced dendritic spine actin's mobility, caused retraction of mushroom spines, and inhibited spine maturation. These effects were further enhanced by inactivating iron-binding residues of Lcn-2, suggesting that they were facilitated by the iron-free form of Lcn-2. Concurrently, disruption of the Lcn2 gene in mice promoted stress-induced increase in spine density and caused an increase in the proportion of mushroom spines. The above changes correlated with higher excitability of CA1 principal neurons and with elevated stress-induced anxiety in Lcn-2(-/-) mice. Our study demonstrates that lipocalin-2 promotes stress-induced changes in spine morphology and function to regulate neuronal excitability and anxiety.

Neuropsin cleaves EphB2 in the amygdala to control anxiety.

A minority of individuals experiencing traumatic events develop anxiety disorders. The reason for the lack of correspondence between the prevalence of exposure to psychological trauma and the development of anxiety is unknown. Extracellular proteolysis contributes to fear-associated responses by facilitating neuronal plasticity at the neuron-matrix interface. Here we show in mice that the serine protease neuropsin is critical for stress-related plasticity in the amygdala by regulating the dynamics of the EphB2-NMDA-receptor interaction, the expression of Fkbp5 and anxiety-like behaviour. Stress results in neuropsin-dependent cleavage of EphB2 in the amygdala causing dissociation of EphB2 from the NR1 subunit of the NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2-NR1 interaction enhances NMDA receptor current, induces Fkbp5 gene expression and enhances behavioural signatures of anxiety. On stress, neuropsin-deficient mice do not show EphB2 cleavage and its dissociation from NR1 resulting in a static EphB2-NR1 interaction, attenuated induction of the Fkbp5 gene and low anxiety. The behavioural response to stress can be restored by intra-amygdala injection of neuropsin into neuropsin-deficient mice and disrupted by the injection of either anti-EphB2 antibodies or silencing the Fkbp5 gene in the amygdala of wild-type mice. Our findings establish a novel neuronal pathway linking stress-induced proteolysis of EphB2 in the amygdala to anxiety.

The M3-muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner.

Degeneration of the cholinergic system is considered to be the underlying pathology that results in the cognitive deficit in Alzheimer's disease. This pathology is thought to be linked to a loss of signaling through the cholinergic M(1)-muscarinic receptor subtype. However, recent studies have cast doubt on whether this is the primary receptor mediating cholinergic-hippocampal learning and memory. The current study offers an alternative mechanism involving the M(3)-muscarinic receptor that is expressed in numerous brain regions including the hippocampus. We demonstrate here that M(3)-muscarinic receptor knockout mice show a deficit in fear conditioning learning and memory. The mechanism used by the M(3)-muscarinic receptor in this process involves receptor phosphorylation because a knockin mouse strain expressing a phosphorylation-deficient receptor mutant also shows a deficit in fear conditioning. Consistent with a role for receptor phosphorylation, we demonstrate that the M(3)-muscarinic receptor is phosphorylated in the hippocampus following agonist treatment and following fear conditioning training. Importantly, the phosphorylation-deficient M(3)-muscarinic receptor was coupled normally to G(q/11)-signaling but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. It can, therefore, be concluded that M(3)-muscarinic receptor-dependent learning and memory depends, at least in part, on receptor phosphorylation/arrestin signaling. This study opens the potential for biased M(3)-muscarinic receptor ligands that direct phosphorylation/arrestin-dependent (non-G protein) signaling as being beneficial in cognitive disorders.

Plasmin-mediated degradation of laminin gamma-1 is critical for ethanol-induced neurodegeneration.

BACKGROUND: Alcoholism may result in severe neurological deficits and cognitive impairments. Many of the central effects of ethanol (EtOH) can be explained by upregulation of N-methyl-D-aspartate (NMDA) and downregulation of gamma-aminobutyric acid (GABA) A receptors (GABAA) in response to long-term EtOH consumption. Abrupt ethanol withdrawal (EW) may result in neuronal hyperexcitability leading to hallucinations, seizures, neurodegeneration, and sometimes death. METHODS: Using a multidisciplinary approach in wild-type and genetically modified mice, we examined the contribution of the tissue plasminogen activator (tPA), plasminogen, and laminin to EW-induced cell death. RESULTS: Here we show that EW-induced neurodegeneration is mediated by the tPA/plasmin system. During EW, tPA is upregulated in the hippocampus and converts plasminogen to plasmin, which in turn degrades an extracellular matrix component laminin, leading to caspase-3-dependent cell death. Consequently, mice in which the tPA or plasminogen genes have been deleted do not show EW-induced laminin degradation, mitochondrial dysfunction, and neurodegeneration. Finally, we demonstrated that disruption of the hippocampal laminin gamma-1 renders the mice resistant to neurotoxic effects of EW. CONCLUSIONS: Our data identify laminin gamma-1 as a novel target to combat neurodegeneration.

Proteolytic fragments of laminin promote excitotoxic neurodegeneration by up-regulation of the KA1 subunit of the kainate receptor.

Degradation of the extracellular matrix (ECM) protein laminin contributes to excitotoxic cell death in the hippocampus, but the mechanism of this effect is unknown. To study this process, we disrupted laminin gamma1 (lamgamma1) expression in the hippocampus. Lamgamma1 knockout (KO) and control mice had similar basal expression of kainate (KA) receptors, but the lamgamma1 KO mice were resistant to KA-induced neuronal death. After KA injection, KA1 subunit levels increased in control mice but were unchanged in lamgamma1 KO mice. KA1 levels in tissue plasminogen activator (tPA)-KO mice were also unchanged after KA, indicating that both tPA and laminin were necessary for KA1 up-regulation after KA injection. Infusion of plasmin-digested laminin-1 into the hippocampus of lamgamma1 or tPA KO mice restored KA1 up-regulation and KA-induced neuronal degeneration. Interfering with KA1 function with a specific anti-KA1 antibody protected against KA-induced neuronal death both in vitro and in vivo. These results demonstrate a novel pathway for neurodegeneration involving proteolysis of the ECM and KA1 KA receptor subunit up-regulation.

Tissue plasminogen activator and plasminogen mediate stress-induced decline of neuronal and cognitive functions in the mouse hippocampus.

Repeated stress can impair function in the hippocampus, a brain structure essential for learning and memory. Although behavioral evidence suggests that severe stress triggers cognitive impairment, as seen in major depression or posttraumatic stress disorder, little is known about the molecular mediators of these functional deficits in the hippocampus. We report here both pre- and postsynaptic effects of chronic stress, manifested as a reduction in the number of NMDA receptors, dendritic spines, and expression of growth-associated protein-43 in the cornu ammonis 1 region. Strikingly, the stress-induced decrease in NMDA receptors coincides spatially with sites of plasminogen activation, thereby predicting a role for tissue plasminogen activator (tPA) in this form of stress-induced plasticity. Consistent with this possibility, tPA-/- and plasminogen-/- mice are protected from stress-induced decrease in NMDA receptors and reduction in dendritic spines. At the behavioral level, these synaptic and molecular signatures of stress-induced plasticity are accompanied by impaired acquisition, but not retrieval, of hippocampal-dependent spatial learning, a deficit that is not exhibited by the tPA-/- and plasminogen-/- mice. These findings establish the tPA/plasmin system as an important mediator of the debilitating effects of prolonged stress on hippocampal function at multiple levels of neural organization.

Angiotensin II enhances thrombosis development in renovascular hypertensive rats.

There is an increased number of in vitro evidence that angiotensin II (Ang II) may promote thrombosis. However there are no in vivo experiments exploring the effect of Ang II on thrombus formation. In the present study we have investigated the influence of Ang II on venous thrombosis in renovascular hypertensive rats. Furthermore, we examined the role of AT(1) receptor and Ang II metabolites: angiotensin III (Ang III) and angiotensin IV (Ang IV) in the mechanisms of Ang II action. The contribution of coagulation and fibrinolytic systems in the mode of Ang II action was also determined. Venous thrombosis was induced by ligation of vena cava. Ang II infused into rats developing venous thrombosis caused dose-dependent increase in thrombus weight, which was partially reversed by losartan, selective AT(1) antagonist. Ang III did not influence the thrombus formation in hypertensive rats, while Ang IV caused a marked increase in thrombus weight only in one of the used doses. Our study shows that Ang II via AT(1) receptor enhances thrombosis development. The prothrombotic effect of Ang II may partially depend on enhanced leukocytes adhesion to endothelial cells accompanied by accelerated fibrin formation and increased plasma level of PAI-1. Moreover, Ang II action is partially mediated by one of its metabolites - Ang IV.

Ethanol-withdrawal seizures are controlled by tissue plasminogen activator via modulation of NR2B-containing NMDA receptors.

Chronic ethanol abuse causes up-regulation of NMDA receptors, which underlies seizures and brain damage upon ethanol withdrawal (EW). Here we show that tissue-plasminogen activator (tPA), a protease implicated in neuronal plasticity and seizures, is induced in the limbic system by chronic ethanol consumption, temporally coinciding with up-regulation of NMDA receptors. tPA interacts with NR2B-containing NMDA receptors and is required for up-regulation of the NR2B subunit in response to ethanol. As a consequence, tPA-deficient mice have reduced NR2B, extracellular signal-regulated kinase 1/2 phosphorylation, and seizures after EW. tPA-mediated facilitation of EW seizures is abolished by NR2B-specific NMDA antagonist ifenprodil. These results indicate that tPA mediates the development of physical dependence on ethanol by regulating NR2B-containing NMDA receptors.