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Neuroserpin is a serine protease inhibitor identified in a search for proteins implicated in neuronal axon growth and synapse formation. Since its discovery over 30 years ago, it has been the focus of active research. Many efforts have concentrated in elucidating its neuroprotective role in brain ischemic lesions, the structural bases of neuroserpin conformational change and the effects of neuroserpin polymers that underlie the neurodegenerative disease FENIB (familial encephalopathy with neuroserpin inclusion bodies), but the investigation of the physiological roles of neuroserpin has increased over the last years. In this review, we present an updated and critical revision of the current literature dealing with neuroserpin, covering all aspects of research including the expression and physiological roles of neuroserpin, both inside and outside the nervous system; its inhibitory and non-inhibitory mechanisms of action; the molecular structure of the monomeric and polymeric conformations of neuroserpin, including a detailed description of the polymerisation mechanism; and the involvement of neuroserpin in human disease, with particular emphasis on FENIB. Finally, we briefly discuss the identification by genome-wide screening of novel neuroserpin variants and their possible pathogenicity.

Tissue plasminogen activator (tPA) is commonly used to treat acute ischemic stroke within an appropriate therapeutic window. Its inhibitor, neuroserpin (NSP), is reported to exhibit neuroprotective effects on stroke. This review aims to summarize, from literature, the available evidence, potential mechanisms, and knowledge limitations regarding the neuroprotective role of NSP in stroke. All the available evidence indicates that the regulation of the inflammatory response may play a key role in the mechanisms of NSP, which involve all the constituents of the neuroimmune axis. The neuroinflammatory response triggered by stroke can be reversed by NSP, with complicated mechanisms such as maintenance and reconstruction of the structure and function of the blood-brain barrier (BBB), protection of the cells in the central nervous system, and suppression of cell death in both ischemic and hemorrhagic stroke. Moreover, available evidence strongly suggests a tPA-independent mechanism is involved in NSP. However, there are many important issues that are still unclear and need further investigation, such as the effects of NSP on hemorrhagic stroke, the role of the tPA-independent neuroprotective mechanisms, and the clinical application prospects of NSP. We believe our work will be helpful to further understand the neuroprotective role of NSP.

Neuroserpin is a serine protease inhibitor expressed mainly in the brain and at low levels in other tissues like the kidney, testis, heart, and spinal cord. It is involved in the inhibition of tissue plasminogen activator (tPA), plasmin, and to a lesser extent, urokinase-type plasminogen (uPA). Neuroserpin has also been shown to plays noninhibitory roles in the regulation of N-cadherin-mediated cell adhesion. It is involved in neuroprotection from seizure and stroke through tPA-mediated inhibition and also through its other protease targets. Mutations in critical domains of neuroserpin lead to its polymerization and neuronal death. In this study, a novel truncated isoform of human neuroserpin was identified in the brain and liver, which was confirmed by reverse transcriptase-PCR and DNA sequencing using exon-specific primers. Structural characterization of novel isoform using MD simulations studies indicated that it lacks the reactive center loop (RCL) but largely maintains its secondary structure fold. The novel truncated variant was cloned, expressed, and purified. A comparative intrinsic fluorescence and 4,4'-bis-1-anilino naphthalene 8-sulfonate studies revealed a decrease in fluorescence emission intensity and a more exposed hydrophobic surface as compared to the reported isoform. However, the novel isoform has lost its ability for tPA inhibition and complex formation. The absence of RCL indicates a noninhibitory role for the truncated isoform, prompting a detailed search and identification of two smaller isoforms in the human brain. With indications of the noninhibitory role of neuroserpin, identifying novel isoforms that appear to be without the tPA recognition domain is significant.

Dementia is a clinical syndrome that affects approximately 47 million people worldwide and is characterized by progressive and irreversible decline of cognitive, behavioral and sesorimotor functions. Alzheimer's disease (AD) accounts for approximately 60-80% of all cases of dementia, and neuropathologically is characterized by extracellular deposits of insoluble amyloid-β (Aβ) and intracellular aggregates of hyperphosphorylated tau. Significantly, although for a long time it was believed that the extracellular accumulation of Aβ was the culprit of the symptoms observed in these patients, more recent studies have shown that cognitive decline in people suffering this disease is associated with soluble Aβ-induced synaptic dysfunction instead of the formation of insoluble Aβ-containing extracellular plaques. These observations are translationally relevant because soluble Aβ-induced synaptic dysfunction is an early event in AD that precedes neuronal death, and thus is amenable to therapeutic interventions to prevent cognitive decline before the progression to irreversible brain damage. The plasminogen activating (PA) system is an enzymatic cascade that triggers the degradation of fibrin by catalyzing the conversion of plasminogen into plasmin via two serine proteinases: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Experimental evidence reported over the last three decades has shown that tPA and uPA play a role in the pathogenesis of AD. However, these studies have focused on the ability of these plasminogen activators to trigger plasmin-induced cleavage of insoluble Aβ-containing extracellular plaques. In contrast, recent evidence indicates that activity-dependent release of uPA from the presynaptic terminal of cerebral cortical neurons protects the synapse from the deleterious effects of soluble Aβ via a mechanism that does not require plasmin generation or the cleavage of Aβ fibrils. Below we discuss the role of the PA system in the pathogenesis of AD and the translational relevance of data published to this date.

Altres ajuts: This work was also supported by the National Institutes of Health (R21AG056974 and R01AG061566 to JF); Departament de Salut de la Generalitat de Catalunya, Pla Estratègic de Recerca i Innovació en Salut (SLT002/16/00408 to AL); Fundació La Marató de TV3 (20141210 to JF, 044412 to RB). Fundació Catalana Síndrome de Down and Fundació Víctor Grífols i Lucas partially supported this work. This work was also supported by Generalitat de Catalunya (SLT006/17/00119 to JF) and a grant from the Fundació Bancaria La Caixa to RB. The discovery that nerve growth factor (NGF) metabolism is altered in Down syndrome (DS) and Alzheimer's disease (AD) brains offered a framework for the identification of novel biomarkers signalling NGF deregulation in AD pathology. We examined levels of NGF pathway proteins (proNGF, neuroserpin, tissue plasminogen activator [tPA], and metalloproteases [MMP]) in matched cerebrospinal fluid (CSF)/plasma samples from AD-symptomatic (DSAD) and AD-asymptomatic (aDS) individuals with DS, as well as controls (HC). ProNGF and MMP-3 were elevated while tPA was decreased in plasma from individuals with DS. CSF from individuals with DS showed elevated proNGF, neuroserpin, MMP-3, and MMP-9. ProNGF and MMP-9 in CSF differentiated DSAD from aDS (area under the curve = 0.86, 0.87). NGF pathway markers associated with CSF amyloid beta and tau and differed by sex. Brain NGF metabolism changes can be monitored in plasma and CSF, supporting relevance in AD pathology. These markers could assist staging, subtyping, or precision medicine for AD in DS.

Background Type 2 neurologic complications of coronary artery bypass grafting (CABG) include postoperative cognitive dysfunction and delirium. Although relevant clinically, they are rarely properly recognized outside of dedicated research setting, as their identification is complex and time-consuming. The aim of this study was to examine the diagnostic potential of 4 novel brain-injury biomarkers for predicting these sequelae at the completion of off-pump CABG. Methods A total of 100 consecutive patients scheduled for elective isolated off-pump CABG were enrolled. Control group of patients without neurological complications (n = 48) was compared separately to study groups diagnosed with postoperative cognitive dysfunction (n = 39) and delirium (n = 26). Serum concentrations of glial fibrillary acidic protein, neuroserpin, phosphorylated axonal neurofilament subunit H, and visinin-like protein 1 were evaluated at baseline, end of surgery, as well as on postoperative day 1 and 7. Results Increased end of surgery to baseline ratio of neuroserpin predicted the occurrence of both postoperative cognitive dysfunction (area under the curve = 0.655, 95% confidence interval 0.54-0.77) and delirium (area under curve = 0.643, 95% confidence interval 0.52-0.77). Concentrations of neuroserpin were significantly higher on postoperative day 7 compared with end of surgery and postoperative day 1 in all groups. Conclusions Among novel biochemical markers of brain damage, neuroserpin may be a promising predictor of type 2 neurological complications and may express neuroregeneration after off-pump CABG, whereas glial fibrillary acidic protein, phosphorylated axonal neurofilament subunit H, and visinin-like protein 1 may not be suitable for this clinical setting.

Blood‐brain barrier (BBB) integrity injury within the thrombolytic time window is becoming a critical target to reduce haemorrhage transformation (HT). We have previously reported that BBB damage was initially damaged in non‐infarcted striatum after acute ischaemia stroke. However, the underlying mechanism is not clear. Since acute ischaemic stroke could induce a significant increase of dopamine release in striatum, in current study, our aim is to investigate the role of dopamine receptor signal pathway in BBB integrity injury after acute ischaemia using rat middle cerebral artery occlusion model. Our data showed that 2‐h ischaemia induced a significant increase of endogenous tissue plasminogen activator (tPA) in BBB injury area and intra‐striatum infusion of tPA inhibitor neuroserpin, significantly alleviated 2‐h ischaemia‐induced BBB injury. In addition, intra‐striatum infusion of D1 receptor antagonist SCH23390 significantly decreased ischaemia‐induced upregulation of endogenous tPA, accompanied by decrease of BBB injury and occludin degradation. More important, inhibition of hypoxia‐inducible factor‐1 alpha with inhibitor YC‐1 significantly decreased 2‐h ischaemia‐induced endogenous tPA upregulation and BBB injury. Taken together, our data demonstrate that acute ischaemia disrupted BBB through activation of endogenous tPA via HIF‐1α upregulation, thus representing a new therapeutic target for protecting BBB after acute ischaemic stroke.

Funder: Pasteur Institute – Cenci Bolognetti Foundation, Funder: Sapienza University of Rome, Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a progressive neurodegenerative disease caused by point mutations in the gene for neuroserpin, a serine protease inhibitor of the nervous system. Different mutations are known that are responsible for mutant neuroserpin polymerization and accumulation as inclusion bodies in many cortical and subcortical neurons, thereby leading to cell death, dementia and epilepsy. Many efforts have been undertaken to elucidate the molecular pathways responsible for neuronal death. Most investigations have concentrated on analysis of intracellular mechanisms such as endoplasmic reticulum (ER) stress, ER-associated protein degradation (ERAD) and oxidative stress. We have generated a HEK-293 cell model of FENIB by overexpressing G392E-mutant neuroserpin and in this study we examine trafficking and toxicity of this polymerogenic variant. We observed that a small fraction of mutant neuroserpin is secreted via the ER-to-Golgi pathway, and that this release can be pharmacologically regulated. Overexpression of the mutant form of neuroserpin did not stimulate cell death in the HEK-293 cell model. Finally, when treating primary hippocampal neurons with G392E neuroserpin polymers, we did not detect cytotoxicity or synaptotoxicity. Altogether, we report here that a polymerogenic mutant form of neuroserpin is secreted from cells but is not toxic in the extracellular milieu.

Protective strategy against hypoxic-ischemic (H/I) induced injury has been intensively discussed. Neuroserpin, an inhibitor for tissue plasminogen activator (tPA), has been proved a vital neuroprotective agent in cerebral ischemia mouse model and oxygen-glucose deprivation and reoxygenation (OGD/R) cell model. Neuroserpin is a promising therapeutic hint for neonatal hypoxic-ischemia injury. Here, we established a neuroserpin deficient zebrafish to study its role in CoCl2 chemically induced hypoxic injury. CoCl2 exposure was beginning at the embryonic stage. Development defects, neuronal loss, and vascular malformation was assessed by imaging microscopy. Neuroserpin deficient zebrafish showed more development defects, neuronal loss and vascular malformation compared to wide-type. Apoptosis and oxidative stress were evaluated to further identify the possible mechanisms. These findings indicate that neuroserpin could protective against CoCl2 induced hypoxic injury by alleviating oxidative stress.

Neuroserpin is a serine protease inhibitor that regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin is strongly expressed during nervous system development as well as during adulthood, when it is predominantly found in regions eliciting synaptic plasticity. In the hippocampus, neuroserpin regulates developmental neurogenesis, synaptic maturation and in adult mice it modulates synaptic plasticity and controls cognitive and social behavior. High expression levels of neuroserpin in the neocortex starting from prenatal stage and persisting during adulthood suggest an important role for the serpin in the formation of this brain region and in the maintenance of cortical functions. In order to uncover neuroserpin function in the murine neocortex, in this work we performed a comprehensive investigation of its expression pattern during development and in the adulthood. Moreover, we assessed the role of neuroserpin in cortex formation by comparing cortical lamination and neuronal maturation between neuroserpin-deficient and control mice. Finally, we evaluated a possible regulatory role of neuroserpin at cortical synapses in neuroserpin-deficient mice. We observed that neuroserpin is expressed starting from the beginning of corticogenesis until adulthood throughout the neocortex in several classes of glutamatergic projection neurons and GABA-ergic interneurons. However, in the absence of neuroserpin we did not detect any alteration either in cortical layer formation, or in neuronal soma size and dendritic length. Furthermore, no significant quantitative changes were observed in the proteome of cortical synapses upon neuroserpin deficiency. We conclude that, although strongly expressed in the neocortex, absence of neuroserpin does not lead to gross developmental abnormalities, and does not perturb the composition of the cortical synaptic proteome.

Little is known about the involvement of type 2 immune response-promoting intestinal tuft cells in metabolic regulation. We here examined the temporal changes in small intestinal tuft cell number and activity in response to high-fat diet-induced obesity in mice and investigated the relation to whole-body energy metabolism and the immune phenotype of the small intestine and epididymal white adipose tissue. Intake of high fat diet resulted in a reduction in overall numbers of small intestinal epithelial and tuft cells and reduced expression of the intestinal type 2 tuft cell markers Il25 and Tslp. Amongst >1,700 diet-regulated transcripts in tuft cells, we observed an early association between body mass expansion and increased expression of the gene encoding the serine protease inhibitor neuroserpin. By contrast, tuft cell expression of genes encoding gamma aminobutyric acid (GABA)-receptors was coupled to Tslp and Il25 and reduced body mass gain. Combined, our results point to a possible role for small intestinal tuft cells in energy metabolism via coupled regulation of tuft cell type 2 markers and GABA signaling receptors, while being independent of type 2 immune cell involvement. These results pave the way for further studies into interventions that elicit anti-obesogenic circuits via small intestinal tuft cells.

Background The discovery that nerve growth factor (NGF) metabolism is altered in Down syndrome (DS) and Alzheimer's disease (AD) brains offered a framework for the identification of novel biomarkers signalling NGF deregulation in AD pathology. Methods We examined levels of NGF pathway proteins (proNGF, neuroserpin, tissue plasminogen activator [tPA], and metalloproteases [MMP]) in matched cerebrospinal fluid (CSF)/plasma samples from AD-symptomatic (DSAD) and AD-asymptomatic (aDS) individuals with DS, as well as controls (HC). Results ProNGF and MMP-3 were elevated while tPA was decreased in plasma from individuals with DS. CSF from individuals with DS showed elevated proNGF, neuroserpin, MMP-3, and MMP-9. ProNGF and MMP-9 in CSF differentiated DSAD from aDS (area under the curve = 0.86, 0.87). NGF pathway markers associated with CSF amyloid beta and tau and differed by sex. Discussion Brain NGF metabolism changes can be monitored in plasma and CSF, supporting relevance in AD pathology. These markers could assist staging, subtyping, or precision medicine for AD in DS.

Dysfunctions of the neuronal-glial crosstalk and/or impaired signaling of neurotrophic factors represent key features of the maladaptive changes in the central nervous system (CNS) in neuroinflammatory as neurodegenerative disorders. Tissue plasminogen activator (tPA)/plasminogen (PA)/plasmin system has been involved in either process of maturation and degradation of nerve growth factor (NGF), highlighting multiple potential targets for new therapeutic strategies. We here investigated the role of intrathecal (i.t.) delivery of neuroserpin (NS), an endogenous inhibitor of plasminogen activators, on neuropathic behavior and maladaptive synaptic plasticity in the rat spinal cord following spared nerve injury (SNI) of the sciatic nerve. We demonstrated that SNI reduced spinal NGF expression, induced spinal reactive gliosis, altering the expression of glial and neuronal glutamate and GABA transporters, reduced glutathione (GSH) levels and is associated to neuropathic behavior. Beside the increase of NGF expression, i.t. NS administration reduced reactive gliosis, restored synaptic homeostasis, GSH levels and reduced neuropathic behavior. Our results hereby highlight the essential role of tPA/PA system in the synaptic homeostasis and mechanisms of maladaptive plasticity, sustaining the beneficial effects of NGF-based approach in neurological disorders.

Serpini1, which encodes neuroserpin, has been implicated in the development and maintenance of the nervous system. In this study, an inducible neuroserpin overexpression transgenic zebrafish was generated to investigate its role in different developmental stages. Neuroserpin overexpression was induced by doxycycline in larval and adult zebrafish respectively. Locomotion and thigmotaxis were recorded and analyzed using the ZebraBox high-throughput monitoring equipment and the ZebraLab software system. We find that Tg (serpini1) (+DOX) zebrafish larvae are more hypoactive than their wild-type counterparts at 7 day-postfertilization and anxiety-like behavior is observed in Tg (serpini1) (+DOX) adult zebrafish at 3 month-postfertilization. Furthermore, RNA-sequencing analysis reveals that neuroserpin overexpression affects neurodegeneration-related gene expression. In summary, we report that neuroserpin overexpression in larval and adult zebrafish shows different behavioral phenotypes.

Serpini1, which encodes neuroserpin, has been implicated in the development and normal function of the nervous system. Mutations in serpini1 cause familial encephalopathy, a rare neurodegenerative disorder characterized with neuroserpin inclusion bodies. However, function of neuroserpin in the nervous system is not fully understood. In this study, we generated a novel serpini1 mutant zebrafish model to investigate the loss of function of neuroserpin. Serpini1- deficient mutation was created with the CRISPR/Cas9 technique. No severe morphological characteristics were found in serpini1- deficient zebrafish. Serpini1-/- zebrafish larvae did not cause locomotor defects but displayed anxiety-like behavior. Extension of motoneurons axon defect was observed in serpini1-/- zebrafish. Furthermore, RNA-sequencing analysis revealed that loss of serpini1 resulted in affected expression of neurodegeneration-related genes.

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a severe and lethal neurodegenerative disease. Upon specific point mutations in the SERPINI1gene-coding for the human protein neuroserpin (NS) the resulting pathologic NS variants polymerize and accumulate within the endoplasmic reticulum of neurons in the central nervous system. To date, embelin (EMB) is the only known inhibitor of NS polymerization in vitro. This molecule is capable of preventing NS polymerization and dissolving preformed polymers. Here, we show that lowering EMB concentration results in increasing size of NS oligomers in vitro. Moreover, we observe that in cells expressing NS, the polymerization of G392E NS is reduced, but this effect is mediated by an increased proteasomal degradation rather than polymerization impairment. For these reasons we designed a systematic chemical evolution of the EMB scaffold aimed to improve its anti-polymerization properties. The effect of EMB analogs against NS polymerization was assessed in vitro. None of the EMB analogs displayed an anti-polymerization activity better than the one reported for EMB, indicating that the EMB&ndash, NS interaction surface is very specific and highly optimized. Thus, our results indicate that EMB is, to date, still the best candidate for developing a treatment against NS polymerization.

The present research aimed at evaluating the protective role of betalain on the in vitro glaucoma model using PC12 neuronal cells. The cultured neuronal cells in a customized pressurized chamber were analyzed for the onset of glutathione, myeloperoxidase (MPO), cathepsin, expression of inflammatory enzymes such as cyclooxygenase (COX-1), lipooxygenase (5- LOX), sPLA2 caveolin-1, glaucoma markers and other inflammatory cytokines in the presence and absence of betalain. The results have shown that a significant increase in the expression of oxidative stress with increased activity of cathepsin B and D. On the other hand, the activity of inflammatory enzymes such as COX-1, 5- LOX, sPLA2 were significantly increased in pressure exposed cells. In addition, glaucoma simulated cells demonstrated a significant increase in the VEGF, TGF-β, BDGF, and neuroserpin compared to control. Moreover, cells predisposed to hydrostatic pressure demonstrated an increase in (p

The NGF metabolic pathway entails the proteins that mature pro-nerve growth factor (proNGF) to NGF and those that degrade NGF. Basal forebrain cholinergic neurons require NGF for maintenance of cholinergic phenotype, are critical for cognition, and degenerate early in Alzheimer’s disease (AD). In AD, NGF metabolism is altered, but it is not known whether this is an early phenomenon, nor how it relates to AD pathology and symptomology. We acquired dorsolateral/medial prefrontal cortex samples from individuals with Alzheimer’s dementia, Mild Cognitive Impairment (MCI), or no cognitive impairment with high (HA-NCI) and low (LA-NCI) brain Aβ from the Religious Orders Study. Cortical proNGF protein, but not mRNA, was higher in AD, MCI, and HA-NCI, while mature NGF was lower. Plasminogen protein was higher in MCI and AD brain tissue, with plasminogen mRNA not likewise elevated, suggesting diminished activation of the proNGF convertase, plasmin. The plasminogen activator tPA was lower in HA-NCI while neuroserpin, the CNS tPA inhibitor, was higher in AD and MCI cortical samples. Matrix metalloproteinase 9 (MMP9), which degrades NGF, was overactive in MCI and AD. Transcription of the MMP9 inhibitor TIMP1 was lower in HA-NCI. ProNGF levels correlated with plasminogen, neuroserpin, and VAChT while NGF correlated with MMP9 activity. In NCI, proNGF correlated with cerebral Aβ and tau deposition and to cognitive performance. In summary, proNGF maturation is impaired in preclinical and clinical AD while mature NGF degradation is enhanced. These differences correlate with cognition, pathology, and cholinergic tone, and may suggest novel biomarkers and therapeutic targets.

Purpose: To investigate the regulation mechanism of long non-coding RNA (lncRNA) plasmocytoma variant translocation 1 (PVT1) in ovarian cancer (OC). Methods: The levels of PVT1, microRNA (miR)-543, serpin peptidase inhibitor-clade I (neuroserpin)-member 1 (SERPINI1) in OC tissues and OVCAR-3, A2780, TOV-112D of OC cell lines were detected by quantitative real-time PCR (qRT-PCR) and Western Blot (WB). Cell proliferation, migration, invasion, apoptosis and the regulatory relationship between genes and target genes were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Transwell, flow cytometry and dual luciferase reporter (DLR). The OC patients were followed up for 5 years to analyze the relationship between PVT1 and 5-year overall survival (OS). Results: In contrast with miR-543, PVT1 and SERPINI1 were highly expressed in OC tissues and cell lines, and high levels of PVT1 were significantly associated with lower 5-year OS of patients. Down-regulating PVT1 not only inhibited the malignant proliferation, migration and invasion of OC cells, but promoted cell apoptosis. PVT1 regulated miR-543 in a targeted manner, and its overexpression could attenuate the anticancer effect of miR-543 on OC cells. In addition, miR-543 also directly targeted SERPINI1, and miR-543 knockdown weakened the inhibitory effect of down-regulated SERPINI1 on OC progression. Furthermore, we found that PVT1 acted as a competitive endogenous RNA to sponge miR-543, thereby regulating the expression of SERPINI1. Conclusion: PVT1 can mediate the molecular mechanism of OC by miR-543/SERPINI1 axis regulatory network, which is a new therapeutic direction for OC.

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

Tissue-type plasminogen activator (tPA) is characterized as an effective drug for early thrombolytic therapy in acute cerebral infarction (ACI). However, tPA will increase the risk of hemorrhage if it is used beyond the treatment time window. The study aims to explore the effects of neuroserpin (NSP) on the time window of tPA thrombolysis in ACI and the underlying mechanism. The middle cerebral artery occlusion (MCAO) model was constructed in rats, which were randomly divided into six groups: sham operation group, infarction group, 1-hour thrombolysis group, 1-hour thrombolytic + NSP intervention group, 4-hour thrombolytic group, and 4-hour thrombolysis + NSP intervention group. The neurological changes in rats were evaluated by modified neurological severity scores and rota-rod test. The brain edema and cerebral infarction area were evaluated by dry-wet method and triphenyl tetrazolium chloride staining. The blood-brain barrier (BBB) integrity was examined by Evans blue method. The expressions of malondialdehyde, superoxide dismutase, and glutathione peroxidase in brain were also investigated. The expression of caspase-3 and Bcl-2 in brain tissue and apoptosis of neurons were examined by Western blot analysis and toluidine blue staining. tPA thrombolysis significantly attenuated the neurological impairment in rats with MCAO at 1 hour. Conversely, the effect of tPA thrombolysis at 4 hours after MCAO did not significantly help the recovery of neurological function. However, a combination of tPA treatment and NSP treatment at 4 hours after MCAO markedly ameliorated the neurological impairment, cerebral edema, cerebral infarction volume, BBB injury, oxidative stress products, and neuron apoptosis. NSP can probably expand the time window for tPA treatment to reduce neurological impairment in ACI.

In utero alcohol exposure can cause fetal alcohol spectrum disorders (FASD), characterized by structural brain abnormalities and long-lasting behavioral and cognitive dysfunction. Neuronal plasticity is affected by in utero alcohol exposure and can be modulated by extracellular proteolysis. Plasmin is a major extracellular serine-protease whose activation is tightly regulated by the plasminogen activator (PA) system. In the present study we explored the effect of ethanol on the expression of the main components of the brain PA system in sex-specific cortical astrocyte primary cultures in vitro and in the cortex and hippocampus of post-natal day (PD) 9 male and female rats. We find that ethanol alters the PA system in astrocytes and in the developing brain. In particular, the expression of tissue-type PA (tPA), encoded by the gene Plat, is consistently upregulated by ethanol in astrocytes in vitro and in the cortex and hippocampus in vivo. Astrocytes exhibit endogenous plasmin activity that is increased by ethanol and recombinant tPA and inhibited by tPA silencing. We also find that tPA is expressed by astrocytes of the developing cortex and hippocampus in vivo. All components of the PA system investigated, with the exception of Neuroserpin/Serpini1, are expressed at higher levels in astrocyte cultures than in the developing brain, suggesting that astrocytes are major producers of these proteins in the brain. In conclusion, astrocyte PA system may play a major role in the modulation of neuronal plasticity; ethanol-induced upregulation of tPA levels and plasmin activity may be responsible for altered neuronal plasticity in FASD.

1,3,7-Trihydroxyxanthone is a compound isolated from Polygalae Radix, a medicinal herb frequently applied for treatment of psychiatric disordres with symptoms of forgetfulness and depression in ancient China. In current research, this compound was applied onto rat astrocyte primary cultures in exploring the action mechanisms of 1,3,7-trihydroxyxanthone on regulating synthesis of neurotrophic factors. It was found that 1,3,7-trihydroxyxanthone could significantly stimulate the expression of NGF and BDNF in dose-dependent manners: the stimulation was both in mRNA and protein levels. Furthermore, 1,3,7-trihydroxyxanthone might fulfill this effect by regulating critical enzymes, such as plasminogen, tissue plasminogen activator, neuroserpin and tissue inhibitor of metalloproteinases in metabolic pathway of neurotrophic factors. Besides, inhibitors of cAMP- and ERK-dependent pathways, which implied the possible signaling pathway, could reverse this inducing effect. These results might support the potentiality of 1,3,7-trihydroxyxanthone in drug development in treating psychiatric disorders.

FENIB (familial encephalopathy with neuroserpin inclusion bodies) is a human monogenic disease caused by point mutations in the SERPINI1 gene, characterized by the intracellular deposition of polymers of neuroserpin (NS), which leads to proteotoxicity and cell death. Despite the different cell and animal models developed thus far, the exact mechanism of cell toxicity elicited by NS polymers remains unclear. Here, we report that human wild-type NS and the polymerogenic variant G392E NS form protein aggregates mainly localized within the endoplasmic reticulum (ER) when expressed in the yeast S. cerevisiae. The expression of NS in yeast delayed the exit from the lag phase, suggesting that NS inclusions cause cellular stress. The cells also showed a higher resistance following mild oxidative stress treatments when compared to control cells. Furthermore, the expression of NS in a pro-apoptotic mutant strain-induced cell death during aging. Overall, these data recapitulate phenotypes observed in mammalian cells, thereby validating S. cerevisiae as a model for FENIB. ispartof: MICROORGANISMS vol:9 issue:7 ispartof: location:Switzerland status: published

Postoperative recovery can be impaired by many conditions, some of which are difficult to diagnose clinically. These include type 2 neurological complications such as hypoactive subtype of postoperative delirium (PD) and early postoperative cognitive dysfunction (ePOCD). Hope for their timely detection may lie with novel biomarkers. Plasma concentrations of microRNA-1-3p, microRNA-21-5p, glial fibrillary acidic protein (GFAP), neuroserpin (NSP), phosphorylated axonal neurofilament subunit H (pNfH) and visinin-like protein 1 (VILIP-1) were investigated in 30 patients undergoing elective off-pump coronary artery bypass grafting. Blood samples were collected at the start and end of a surgery as well as 24 h postoperatively. Associations between the studied biomarkers’ perioperative expression and type 2 neurological complications were analyzed. PD was associated with postoperative expression of GFAP; ePOCD was associated with postoperative expression of microRNA-21-5p and GFAP as well as intraoperative expression of NSP. The predictive accuracy of these molecules was found acceptable, with all their areas under the curve (AUC) values above 0.7. Multivariable regression indicated that microRNA-21-5p, GFAP and NSP were the only significant predictors of ePOCD. Evaluation of a multi-marker model including these three molecules revealed its outstanding predictive accuracy for ePOCD (AUC = 0.95). The use of microRNA-21-5p, GFAP and NSP for monitoring postoperative recovery warrants further research considering their potential to predict PD and ePOCD.

The serine protease inhibitor neuroserpin regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin expression is particularly prominent at late stages of neuronal development in most regions of the central nervous system (CNS), whereas it is restricted to regions related to learning and memory in the adult brain. The physiological expression pattern of neuroserpin, its high degree of colocalization with tPA within the CNS, together with its dysregulation in neuropsychiatric disorders, suggest a role in formation and refinement of synapses. In fact, studies in cell culture and mice point to a role for neuroserpin in dendritic branching, spine morphology, and modulation of behavior. In this study, we investigated the physiological role of neuroserpin in the regulation of synaptic density, synaptic plasticity, and behavior in neuroserpin-deficient mice. In the absence of neuroserpin, mice show a significant decrease in spine-synapse density in the CA1 region of the hippocampus, while expression of the key postsynaptic scaffold protein PSD-95 is increased in this region. Neuroserpin-deficient mice show decreased synaptic potentiation, as indicated by reduced long-term potentiation (LTP), whereas presynaptic paired-pulse facilitation (PPF) is unaffected. Consistent with altered synaptic plasticity, neuroserpin-deficient mice exhibit cognitive and sociability deficits in behavioral assays. However, although synaptic dysfunction is implicated in neuropsychiatric disorders, we do not detect alterations in expression of neuroserpin in fusiform gyrus of autism patients or in dorsolateral prefrontal cortex of schizophrenia patients. Our results identify neuroserpin as a modulator of synaptic plasticity, and point to a role for neuroserpin in learning and memory.

Background The neuroprotective effects of neuroserpin (NSP) have been well documented in both patients and animal models with cerebral ischemia; however, have never been investigated in hemorrhagic stroke. The aim of this study is to verify the neuroprotection of NSP in the non-tPA-induced intracerebral hemorrhage (ICH) mouse model. Methods C57BL/6J male mice (n = 198) were involved in this study. ICH models were established with infusion of autologous blood into the brain parenchyma. We then detected NSP expression in ICH brains by morphological methods and western blotting analysis. We measured the brain water content and detected blood-brain barrier (BBB) permeability to verify the neuroprotective effects of NSP. Results We found that NSP protein expression was upregulated in ICH models, with a peak at 48 h after ICH induction. NSP local administration reduced the brain edema and the BBB permeability in ICH models. The neurological deficits were also ameliorated. Thus, the neuroprotection of NSP in ICH state was confirmed. Additionally, we also found that the distribution pattern of occludin-expressing cells was obviously changed by the ICH procedure but partly recovered after NSP administration. This finding indicated that protecting and/or repairing the injured vascular endothelial cells may be a potential mechanism involved in NSP neuroprotection, which needs further verification. Conclusions Our results supported the fact that NSP may be considered as a potential therapy for ICH for the neuroprotective effects including amelioration of the edema. Electronic supplementary material The online version of this article (10.1186/s12883-017-0976-1) contains supplementary material, which is available to authorized users.

// Maria Ramnefjell 1 , Christina Aamelfot 2 , Lars Helgeland 3 and Lars A. Akslen 1,3 1 Centre for Cancer Biomarkers CCBIO, Department of Clinical Medicine, Section for Pathology, Haukeland University Hospital, University of Bergen, Bergen, Norway 2 Department of Thoracic Medicine, Haukeland University Hospital, Bergen, Norway 3 Department of Pathology, Haukeland University Hospital, Bergen, Norway Correspondence to: Lars A. Akslen, email: // Keywords : lung cancer, metastases, SerpinB2, neuroserpin, L1CAM, Pathology Section Received : March 16, 2017 Accepted : September 22, 2017 Published : October 03, 2017 Abstract Lung cancer is a leading cause of cancer deaths worldwide and new biomarkers are of utmost importance. Studies have indicated that the anti-plasminogen activators SerpinB2 and Neuroserpin, and the adhesion molecule L1CAM, have a coordinated impact on development of metastasis. Here, we examined whether expression of these markers was associated with clinico-pathologic characteristics and prognosis in resected non-small cell lung cancer (NSCLC). Surgical specimens from 438 NSCLC patients treated at Haukeland University Hospital, Bergen, Norway (1993-2010) were included (median age 68 years; 213 adenocarcinomas, 135 squamous cell carcinomas, 90 others). Representative tumor sections were stained for SerpinB2, Neuroserpin, and L1CAM. Low expression of SerpinB2 was associated with reduced lung cancer specific survival (LCSS) in adenocarcinomas ( p = 0.017), also in stage I ( p = 0.031). In contrast, high SerpinB2 was associated with reduced LCSS in stage I squamous cell carcinomas ( p = 0.022). Although Neuroserpin and L1CAM showed some associations with clinico-pathologic phenotype, there were no associations with survival. In multivariate survival analysis of adenocarcinomas, low SerpinB2 demonstrated independent prognostic value (HR 1.8, p = 0.008). In summary, low expression of SerpinB2 in lung adenocarcinomas was an independent prognostic factor. In contrast to findings by others, we found no impact of L1CAM on survival.

Neuroserpin (NSP) is known for its neuroprotective effects. The aim of this study was to investigate the relationship between NSP level and clinical outcomes and inflammatory markers in Chinese patients with acute ischemic stroke.A total of 133 patients with acute cerebral infarction (ischemia group) and 44 controls were recruited. The modified Rankin Scale (mRS) was used to determine the functional outcome three months after onset. We investigated the relationship between serum NSP levels [on admission and a decrease in NSP levels (10 days after admission versus on admission)] and serum levels of several inflammatory markers.We confirmed that NSP levels on admission in the ischemia group were significantly higher than those in the control group. NSP levels in patients with good outcomes were significantly higher than those in patients with poor outcomes. NSP levels on admission were associated with having a good outcome in these patients. We found that a larger decrease in NSP levels (on admission vs. 10 days after admission) was correlated with lower serum levels of IL-6, IL-1β, and ICAM-1 10 days after admission.A larger decrease in NSP levels related to lower levels of inflammatory marker, while higher NSP levels were associated with lower inflammatory markers and better functional outcomes. Decreasing the infarct size may play a role in this process. These results provide more evidence of the neuroprotective effect of NSP in cerebral ischemic patients. Decrease in the Serum NSP level and NSP level at admission may be considered as potential predictive factors for outcome of acute ischemic stroke.

Progressive myoclonic epilepsies are rare neurodegenerative diseases with a wide spectrum of clinical presentations and genetic heterogeneity that render their diagnosis perplexing. Discovering new imputable genes has been an ongoing process in recent years. We present two pediatric cases of progressive myoclonic epilepsy with SERPINI1 pathogenic variants that lead to a severe presentation; we highlight the importance of including this gene, previously known as causing an adult-onset dementia-epilepsy syndrome, in the genetic work-up of childhood-onset progressive myoclonic epilepsies.

Current understanding of the neurobiology of depression has grown over the past few years beyond the traditional monoamine theory of depression to include chronic stress, inflammation and disrupted synaptic plasticity. Tissue plasminogen activator (tPA) is a key factor that not only promotes fibrinolysis via the activation of plasminogen, but also contributes to regulation of synaptic plasticity and neurogenesis through plasmin-mediated activation of a probrain derived neurotrophic factor (BDNF) to mature BDNF. ProBDNF activation could potentially be supressed by competition with fibrin for plasmin and tPA. High affinity binding of plasmin and tPA to fibrin could result in a decrease of proBDNF activation during brain inflammation leading to fibrosis further perpetuating depressed mood. There is a paucity of data explaining the possible role of the fibrinolytic system or aberrant extravascular fibrin deposition in depression. We propose that within the brain, an imbalance between tPA and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) and neuroserpin favors the inhibitors, resulting in changes in neurogenesis, synaptic plasticity, and neuroinflammation that result in depressive behavior. Our hypothesis is that peripheral inflammation mediates neuroinflammation, and that cytokines such as tumor necrosis factor alpha (TNF-α) can inhibit the fibrinolytic system by up- regulating PAI-1 and potentially neuroserpin. We propose that the decrement of the activity of tPA and uPA occurs with downregulation of uPA in part involving the binding and clearance from the surface of neural cells of uPA/PAI-1 complexes by the urokinase receptor uPAR. We infer that current antidepressants and ketamine mitigate depressive symptoms by restoring the balance of the fibrinolytic system with increased activity of tPA and uPA with down-regulated intracerebral expression of their inhibitors. We lastly hypothesize that psychedelic 5-ht2a receptor agonists, such as psilocybin, can improve mood through anti- inflammatory and pro-fibrinolytic effects that include blockade of TNF-α activity leading to decreased PAI-1 activity and increased clearance. The process involves disinhibition of tPA and uPA with subsequent increased cleavage of proBDNF which promotes neurogenesis, decreased neuroinflammation, decreased fibrin deposition, normalized glial-neuronal cross-talk, and optimally functioning neuro-circuits involved in mood. We propose that psilocybin can alleviate deleterious changes in the brain caused by chronic stress leading to restoration of homeostatic brain fibrinolytic capacity leading to euthymia.

The serpinopathies are human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity. The dementia FENIB is caused by polymerisation of the neuronal serpin neuroserpin (NS) within the endoplasmic reticulum (ER) of neurons. With the aim of understanding the toxicity due to intracellular accumulation of neuroserpin polymers, we have generated transgenic neural progenitor cell (NPC) cultures from mouse foetal cerebral cortex, stably expressing the control protein GFP (green fluorescent protein), or human wild type, G392E or delta NS. We have characterised these cell lines in the proliferative state and after differentiation to neurons. Our results show that G392E NS formed polymers that were mostly retained within the ER, while wild type NS was correctly secreted as a monomeric protein into the culture medium. Delta NS was absent at steady state due to its rapid degradation, but it was easily detected upon proteasomal block. Looking at their intracellular distribution, wild type NS was found in partial co-localisation with ER and Golgi markers, while G392E NS was localised within the ER only. Furthermore, polymers of NS were detected by ELISA and immunofluorescence in neurons expressing the mutant but not the wild type protein. We used control GFP and G392E NPCs differentiated to neurons to investigate which cellular pathways were modulated by intracellular polymers by performing RNA sequencing. We identified 747 genes with a significant upregulation (623) or downregulation (124) in G392E NS-expressing cells, and we focused our attention on several genes involved in the defence against oxidative stress that were up-regulated in cells expressing G392E NS (Aldh1b1, Apoe, Gpx1, Gstm1, Prdx6, Scara3, Sod2). Inhibition of intracellular anti-oxidants by specific pharmacological reagents uncovered the damaging effects of NS polymers. Our results support a role for oxidative stress in the cellular toxicity underlying the neurodegenerative dementia FENIB., Graphical abstract Image 1, Highlights • A cell model system for FENIB was generated using mouse neural progenitor cells. • Cells expressing polymerogenic neuroserpin upregulated several anti-oxidant genes. • Inhibition of the anti-oxidant defences led to apoptosis of these cells. • Oxidative stress and lipid oxidation may contribute to neurodegeneration in FENIB.

Tissue plasminogen activator (tPA) is a serine protease involved in cleavage of neurotrophic factors. In addition, tPA and neuroserpin can also directly bind to low density lipoprotein receptor-related protein 1 (LRP1), promoting neurogenesis and neurite outgrowth. Given both the cleavage and non-cleavage actions of the fibrinolytic system are crucial in neurological functions, the present study, for the first time, systematically detected the changes of fibrinolytic system factors in rats exposed to chronic unpredictable mild stress (CUMS) or lipopolysaccharide (LPS) and patients with depression. In general, our data demonstrated that both CUMS and LPS reduced tPA but elevated plasminogen activator inhibitor-1 (PAI-1; SERPINE1) mRNA expression. Intriguingly, decreased expression of neuroserpin and LRP1 was also observed in rats exposed to CUMS or LPS. The down-regulated neuroserpin and LRP1 signaling were confirmed by western blotting and immunoflurence data. Likewise, elevated PAI-1 but a significant reduction of neuroserpin and LRP1 mRNA expression were observed in the peripheral blood mononuclear cells (PBMCs) of patients with first-episode depression, and the mRNA levels of PAI-1, neuroserpin and LRP1 were correlated with the Beck Depression inventory (BDI) scores, further strengthening the clinical significance and involvement of the fibrinolytic system in depression. Collectively, the present study demonstrated the alterations of fibrinolytic system in stressed and inflamed brain and in patients with first-episode depression, firstly showing that not only the cleavage actions, but also the non-cleavage actions of the system may play an essential role in the development of depression. Keywords: tPA, PAI-1, Neuroserpin, LRP1, Depression

Objective: Neuroserpin is a serine protease inhibitor predominantly expressed in the nervous system functioning mainly in neuronal migration and axonal growth. Neuroprotective effects of neuroserpin were shown in animal models of stroke, brain, and spinal cord injury. Postmortem studies confirmed the involvement of neuroserpin in Alzheimer’s disease. Since altered adult neurogenesis was postulated as an aetiological mechanism for bipolar disorder, the possible effect of neuroserpin gene expression in the disorder was evaluated. Methods: Neuroserpin mRNA expression levels were examined in the peripheral blood of bipolar disorder type I manic and euthymic patients and healthy controls using the polymerase chain reaction method. The sample comprised of 60 physically healthy, middle-aged men as participants who had no substance use disorder. Results: The gene expression levels of neuroserpin were found lower in the bipolar disorder patients than the healthy controls (p=0.000). The neuroserpin levels did not differ between mania and euthymia (both 96% down-regulated compared to the controls). Conclusion: Since we detected differences between the patients and the controls, not the disease states, the dysregulation in the neuroserpin gene could be interpreted as a result of the disease itself.

Neuroserpin is a serine‐protease inhibitor mainly expressed in the CNS and involved in the inhibition of the proteolytic cascade. Animal models confirmed its neuroprotective role in perinatal hypoxia‐ischaemia and adult stroke. Although neuroserpin may be a potential therapeutic target in the treatment of the aforementioned conditions, there is still no information in the literature on its distribution during human brain development. The present study provides a detailed description of the changing spatiotemporal patterns of neuroserpin focusing on physiological human brain development. Five stages were distinguished within our examined age range which spanned from the 7th gestational week until adulthood. In particular, subplate and deep cortical plate neurons were identified as the main sources of neuroserpin production between the 25th gestational week and the first postnatal month. Our immunohistochemical findings were substantiated by single cell RNA sequencing data showing specific neuronal and glial cell types expressing neuroserpin. The characterization of neuroserpin expression during physiological human brain development is essential for forthcoming studies which will explore its involvement in pathological conditions, such as perinatal hypoxia‐ischaemia and adult stroke in human.

Antithrombin (AT3) is one of the most important inhibitors of blood coagulation proteases that belong to the serpin family of protease inhibitors. In this study, a novel alternatively spliced isoform of AT3 was identified, both at transcript and protein level. This novel transcript contains an additional region in the continuation of exon 3b that was included in the transcript due to use of an alternate 5’ splice site. The existence of the novel transcript was confirmed in human brain and liver through RT-PCR. An analysis of the complete transcript indicated that the native reactive centre loop (RCL) of AT3 is maintained; however the novel amino acid sequence projects out as an additional loop as evident from MD simulation studies. A unique amino acid sequence present in the novel isoform was used for the development of polyclonal antibody. The expression of novel isoform was confirmed in human brain and liver tissue using Western blot analysis. Interestingly an alignment of RCL like domain with other inhibitory serpins showed significant similarity with the neuroserpin RCL. To the best of our knowledge, this is the first evidence of alternatively spliced AT3 sequence containing an additional loop and could have physiological relevance.

Neuroserpin is a serine protease inhibitor of the nervous system required for normal synaptic plasticity and regulating cognitive, emotional and social behavior in mice. The high expression level of neuroserpin detected at late stages of nervous system formation in most regions of the brain points to a function in neurodevelopment. In order to evaluate the contribution of neuroserpin to brain development, we investigated developmental neurogenesis and neuronal differentiation in the hippocampus of neuroserpin-deficient mice. Moreover, synaptic reorganization and composition of perineuronal net were studied during maturation and stabilization of hippocampal circuits. We showed that absence of neuroserpin results in early termination of neuronal precursor proliferation and premature neuronal differentiation in the first postnatal weeks. Additionally, at the end of the critical period neuroserpin-deficient mice had changed morphology of dendritic spines towards a more mature phenotype. This was accompanied by increased protein levels and reduced proteolytic cleavage of aggrecan, a perineuronal net core protein. These data suggest a role for neuroserpin in coordinating generation and maturation of the hippocampus, which is essential for establishment of an appropriate neuronal network.

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent edema attacks associated with morbidity and mortality. HAE results from variations in the SERPING1 gene that encodes the C1 inhibitor (C1INH), a serine protease inhibitor (serpin). Reduced plasma levels of C1INH lead to enhanced activation of the contact system, triggering high levels of bradykinin and increased vascular permeability, but the cellular mechanisms leading to low C1INH levels (20%-30% of normal) in heterozygous HAE type I patients remain obscure. Here, we showed that C1INH encoded by a subset of HAE-causing SERPING1 alleles affected secretion of normal C1INH protein in a dominant-negative fashion by triggering formation of protein-protein interactions between normal and mutant C1INH, leading to the creation of larger intracellular C1INH aggregates that were trapped in the endoplasmic reticulum (ER). Notably, intracellular aggregation of C1INH and ER abnormality were observed in fibroblasts from a heterozygous carrier of a dominant-negative SERPING1 gene variant, but the condition was ameliorated by viral delivery of the SERPING1 gene. Collectively, our data link abnormal accumulation of serpins, a hallmark of serpinopathies, with dominant-negative disease mechanisms affecting C1INH plasma levels in HAE type I patients, and may pave the way for new treatments of HAE.

The plasminogen activation (PA) system is best known for its role in fibrinolysis. However, it has also been shown to regulate many non-fibrinolytic functions in the central nervous system (CNS). In particular, tissue-type plasminogen activator (tPA) is reported to have pleiotropic activities in the CNS, regulating events such as neuronal plasticity, excitotoxicity and cerebrovascular barrier integrity, whereas urokinase-type plasminogen activator (uPA) is mainly associated with tissue remodeling and cell migration. It has been suggested that the role tPA plays in controlling barrier integrity may provide a unifying mechanism for the reported diverse, and often opposing, functions ascribed to tPA in the CNS. Here we will review the possibility that the pleiotropic effects reported for tPA in physiologic and pathologic processes in the CNS may be a consequence of its role in the neurovascular unit in regulation of cerebrovascular responses and subsequently parenchymal homeostasis. We propose that this might offer an explanation for the ongoing debate regarding the neurotoxic versus neuroprotective roles of tPA.

Oxidative stress plays a critical role in neuronal injury and is associated with various neurological diseases. Here, we explored the potential protective effect of neuroserpin against oxidative stress in primary cultured hippocampal neurons. Our results show that neuroserpin inhibits H2O2-induced neurotoxicity in hippocampal cultures as measured by WST, LDH release, and TUNEL assays. We found that neuroserpin enhanced the activation of AKT in cultures subjected to oxidative stress and that the AKT inhibitor Ly294002 blocked this neuroprotective effect. Neuroserpin increased the expression of the anti-apoptotic protein BCL-2 and blocked the activation of caspase-3. Neuroserpin did not increase the level of neuroprotection over levels seen in neurons transduced with a BCL-2 expression vector, and an inhibitor of Trk receptors, K252a, did not block neuroserpin's effect. Taken together, our study demonstrates that neuroserpin protects against oxidative stress-induced dysfunction and death of primary cultured hippocampal neurons through the AKT-BCL-2 signaling pathway through a mechanism that does not involve the Trk receptors and leads to inhibition of caspase-3 activation.

Serpins regulate coagulation and inflammation, binding serine proteases in suicide-inhibitory complexes. Target proteases cleave the serpin reactive center loop scissile P1-P1' bond, resulting in serpin-protease suicide-inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases, whereas mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast, Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon γ-receptor-deficient mice (IFNγR(-/-)). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the reactive center loop produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing Arg or Arg-Asn, not Arg-Met, with a 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking Arg or Arg-Asn with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b(+) monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in plasminogen activator inhibitor-1 serpin-deficient transplants. Plaque reduction correlated with mononuclear cell activation. In a separate study, Serp-1 peptide S-7 improved survival in the MHV68 vasculitis model, whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.

Neuroserpin (NS) is predominantly expressed in brain and inhibits tissue-type plasminogen activator (tPA) with implications in brain development and memory. Nature of conformational change in pathological variants in strand 6B and helix B of NS that cause a relatively mild to severe epilepsy (and/or dementia) remains largely elusive. MD simulation with wild type (WT) NS, strand 6B and helix B variants indicated that substitution in this region affects the conformation of the strands 5B, 5A and reactive centre loop. Therefore, we designed variants of NS in strand 6B (I46D and F48S) and helix B (A54F, L55A and L55P) to investigate their role in tPA inhibition mechanism and propensity to aggregate. An interaction analysis showed disturbance of a hydrophobic patch centered at strands 5B, 6B and helix B in I46D and F48S but not in A54F, L55A, L55P and WT NS. Purified I46D, F48S and L55P variants showed decrease in fluorescence emission intensity but have similar α-helical content, however results of A54F and L55A were comparable to WT NS. Analysis of tPA inhibition showed marginal effect on A54F and L55A variant with tPA-NS complex formation. In contrast, I46D, F48S and L55P variants showed massive decrease in tPA inhibition, with no tPA-NS complex formation. Analysis of native PAGE under under polymerization condition showed prompt conversion of I46D, F48S and L55P to latent conformation but not A54F and L55A variants. Identification of these novel conformational changes will aid in the understanding of variable clinical phenotype of shutter region NS variants and other serpins.

Tissue plasminogen activator is usually used for the treatment of acute ischemic stroke, but the role of endogenous tissue plasminogen activator in traumatic brain injury has been rarely reported. A rat model of traumatic brain injury was established by weight-drop method. The tissue plasminogen activator inhibitor neuroserpin (5 μL, 0.25 mg/mL) was injected into the lateral ventricle. Neurological function was assessed by neurological severity score. Neuronal and axonal injuries were assessed by hematoxylin-eosin staining and Bielschowsky silver staining. Protein level of endogenous tissue plasminogen activator was analyzed by western blot assay. Apoptotic marker cleaved caspase-3, neuronal marker neurofilament light chain, astrocyte marker glial fibrillary acidic protein and microglial marker Iba-1 were analyzed by immunohistochemical staining. Apoptotic cell types were detected by immunofluorescence double labeling. Apoptotic cells in the damaged cortex were detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling staining. Degenerating neurons in the damaged cortex were detected by Fluoro-Jade B staining. Expression of tissue plasminogen activator was increased at 6 hours, and peaked at 3 days after traumatic brain injury. Neuronal apoptosis and axonal injury were detected after traumatic brain injury. Moreover, neuroserpin enhanced neuronal apoptosis, neuronal injury and axonal injury, and activated microglia and astrocytes. Neuroserpin further deteriorated neurobehavioral function in rats with traumatic brain injury. Our findings confirm that inhibition of endogenous tissue plasminogen activator aggravates neuronal apoptosis and axonal injury after traumatic brain injury, and activates microglia and astrocytes. This study was approved by the Biomedical Ethics Committee of Animal Experiments of Shaanxi Province of China in June 2015.

International audience; Human African trypanosomiasis (HAT) is a neglected tropical disease that is endemic in sub-Saharan Africa. Control of the disease has been recently improved by better screening and treatment strategies, and the disease is on the WHO list of possible elimination. However, some physiopathological aspects of the disease transmission and progression remain unclear. We propose a new proteomic approach to identify new targets and thus possible new biomarkers of the disease. We also focused our attention on fluids classically associated with HAT (serum and cerebrospinal fluid (CSF)) and on the more easily accessible biological fluids urine and saliva. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) established the proteomic profile of patients with early and late stage disease. The serum, CSF, urine and saliva of 3 uninfected controls, 3 early stage patients and 4 late stage patients were analyzed. Among proteins identified, in CSF, urine and saliva, respectively, 37, 8 and 24 proteins were differentially expressed and showed particular interest with regards to their function. The most promising proteins (Neogenin, Neuroserpin, secretogranin 2 in CSF; moesin in urine and intelectin 2 in saliva) were quantified by enzyme-linked immunosorbent assay in a confirmatory cohort of 14 uninfected controls, 23 patients with early stage disease and 43 patients with late stage disease. The potential of two proteins, neuroserpin and moesin, with the latter present in urine, were further characterized. Our results showed the potential of proteomic analysis to discover new biomarkers and provide the basis of the establishment of a new proteomic catalogue applied to HAT-infected subjects and controls. SIGNIFICANCE: Sleeping sickness, also called Human African Trypanosomiasis (HAT), is a parasitic infection caused by a parasitic protozoan, Trypanosoma brucei gambiense or T. b. rhodesiense which are transmitted via an infected tsetse fly: Glossina. For both, the haemolymphatic stage (or first stage) signs and symptoms are intermittent fever, lymphadenopathy, hepatosplenomegaly, headaches, pruritus, and for T. b. rhodesiense infection a chancre is often formed at the bite site. Meningoencephalitic stage (or second stage) occurs when parasites invade the CNS, it is characterised by neurological signs and symptoms such as altered gait, tremors, neuropathy, somnolence which can lead to coma and death if untreated. first stage of the disease is characterizing by fevers, headaches, itchiness, and joint pains and progressive lethargy corresponding to the second stage with confusion, poor coordination, numbness and trouble sleeping. Actually, diagnosing HAT requires specialized expertise and significant resources such as well-equipped health centers and qualified staff. Such resources are lacking in many endemic areas that are often in rural locales, so many individuals with HAT die before the diagnosis is established. In this study, we analysed by mass spectrometry the entire proteome of serum, CSF, urine and saliva samples from infected and non-infected Angolan individuals to define new biomarkers of the disease. This work of proteomics analysis is a preliminary stage to the characterization of the whole proteome, of these 4 biological fluids, of HAT patients. We have identified 69 new biomarkers. Five of them have been thoroughly investigated by ELISA quantification. Neuroserpine and Moesin are respectively promising new biomarkers in CSF and urine's patient for a better diagnosis.

Background Electrical status epilepticus during slow-wave sleep (ESESS) which is also known as continuous spike-wave of slow sleep (CSWSS) is type of electroencephalographic (EEG) pattern which is seen in ESESS/CSWSS/epilepsy aphasia spectrum. This EEG pattern can occur alone or with other syndromes. Its etiology is not clear, however, brain malformations, immune disorders, and genetic etiologies are suspected to contribute. We aimed to perform a systematic review of all genetic etiologies which have been reported to associate with ESESS/CSWSS/epilepsy-aphasia spectrum. We further aimed to identify the common underlying pathway which can explain it. To our knowledge, there is no available systematic review of genetic etiologies of ESESS/CSWSS/epilepsy-aphasia spectrum. MEDLINE, EMBASE, PubMed and Cochrane review database were searched, using terms specific to electrical status epilepticus during sleep or continuous spike–wave discharges during slow sleep or epilepsy-aphasia spectrum and of studies of genetic etiologies. These included monogenic mutations and copy number variations (CNVs). For each suspected dosage-sensitive gene, further studies were performed through OMIM and PubMed database. Results Twenty-six studies out of the 136 identified studies satisfied our inclusion criteria. I51 cases were identified among those 26 studies. 16 studies reported 11 monogenic mutations: SCN2A (N = 6), NHE6/SLC9A6 (N = 1), DRPLA/ ATN1 (N = 1), Neuroserpin/SRPX2 (N = 1), OPA3 (N = 1), KCNQ2 (N = 2), KCNA2 (N = 5), GRIN2A (N = 34), CNKSR2 (N = 2), SLC6A1 (N = 2) and KCNB1 (N = 5). 10 studies reported 89 CNVs including 9 recurrent ones: Xp22.12 deletion encompassing CNKSR2 (N = 6), 16p13 deletion encompassing GRIN2A (N = 4), 15q11.2–13.1 duplication (N = 15), 3q29 duplication (N = 11), 11p13 duplication (N = 2), 10q21.3 deletion (N = 2), 3q25 deletion (N = 2), 8p23.3 deletion (N = 2) and 9p24.2 (N = 2). 68 of the reported genetic etiologies including monogenic mutations and CNVs were detected in patients with ESESS/CSWSS/epilepsy aphasia spectrum solely. The most common underlying pathway was channelopathy (N = 56). Conclusions Our review suggests that genetic etiologies have a role to play in the occurrence of ESESS/CSWSS/epilepsy-aphasia spectrum. The common underlying pathway is channelopathy. Therefore we propose more genetic studies to be done for more discoveries which can pave a way for proper drug identification. We also suggest development of common cut-off value for spike-wave index to ensure common language among clinicians and researchers. Electronic supplementary material The online version of this article (10.1186/s12863-018-0628-5) contains supplementary material, which is available to authorized users.

Neurodegenerative diseases are disorders characterized by progressive loss of neurons associated with deposition of proteins showing altered physicochemical properties in the brain and in peripheral organs. Molecular classification of neurodegenerative disease is protein-based. This emphasizes the role of protein-processing systems in the pathogenesis. The most frequent proteins involved in the pathogenesis of neurodegenerative diseases are amyloid-β, prion protein, tau, α-synuclein, TAR-DNA-binding protein 43kDa, and fused-in sarcoma protein. There are further proteins associated mostly with hereditary disorders such as proteins encoded by genes linked to trinucleotide repeat disorders, neuroserpin, ferritin, and familial cerebral amyloidoses. The clinical presentations are defined by the distinct involvement of functional systems and do not necessarily indicate the molecular pathologic background. Seeding of pathologic proteins and hierarchic involvement of anatomic regions is commonly seen in neurodegenerative diseases. Overlap of neurodegenerative diseases and combinations of different disorders is frequent. Translation of neuropathologic categories of neurodegenerative diseases into in vivo detectable biomarkers is only partly achieved but intensive research is performed to reach this goal.

As the systematic work on the pathogenesis of the white matter injury in the spinal cord models progresses, it becomes obvious that a severe and extraordinarily protracted, destructive inflammation follows the initial injury. Appropriate anti-inflammatory therapies of sufficient duration should not only inhibit but also lead to the elimination of this destructive inflammation, thus resulting in neuroprotection of the spinal cord tissue and a greater preservation of the neurologic function. While dexamethasone, a powerful, anti-inflammatory steroid analog administered continuously by subdural infusion for 7 days inhibited severe macrophage infiltration in the cavity of injury, the dose used was remarkably toxic. A 2-week-long infusion of lower doses of dexamethasone resulted in dose-dependent inhibition of macrophage infiltration and was better tolerated by the rats, but it became evident that a much longer duration of subdural administration of a powerful anti-inflammatory drug is required to eliminate myelin-rich, necrotic debris from the cavity and synthetic steroids such as dexamethasone, and methylprednisolone may be too toxic for this application. Therefore, nontoxic but powerful anti-inflammatory compounds are required for neuroprotective treatment of the spinal cord injury (SCI) and also brain trauma and stroke where the massive injury to the white matter occurs. Serpins have been associated with neurological damage. The mammalian serpin neuroserpin (SERPINI1) is reported to act in a protective manner after cerebrospinal infarction. The serine protease, tissue-type plasminogen activator (tPA), and the serpin plasminogen activator inhibitor (PAI-1, SERPINE1) are both upregulated at sites of central nervous system damage. In preliminary studies, subdural infusion of the myxomaviral serpin, Serp-1, resulted in the powerful inhibition of the macrophage infiltration of the cavity of injury, comparable to the inhibition by high dose of dexamethasone that has proven to be unduly toxic. Nontoxic, yet powerful neuroprotective, anti-inflammatory effects of Serp-1 may indicate this serpin protein as a potential attractive compound to treat SCI and similar syndromes involving massive injury to the white matter such as brain trauma and stroke. Novel methods of drug delivery, chronic subdural infusion, and novel analytic methods to measure the effectiveness of the neuroprotective serpin treatments are discussed in this chapter.

The fish ear stones (otoliths) consist mainly of calcium carbonate and have lower amounts of a proteinous matrix. This matrix consists of macromolecules, which directly control the biomineralization process. We analyzed the composition of this proteinous matrix by mass spectrometry in a shotgun approach. For this purpose, an enhanced protein purification technique was developed that excludes any potential contamination of proteins from body fluids. Using this method we identified eight proteins in the inner ear of Oreochromis mossambicus. These include the common otolith matrix proteins (OMP-1, otolin-1, neuroserpin, SPARC and otoconin), and three proteins (alpha tectorin, otogelin and transferrin) not previously localized to the otoliths. Moreover, we were able to exclude the occurrence of two matrix proteins (starmaker and pre-cerebellin-like protein) known from other fish species. In further analyses, we show that the absence of the OMP starmaker corresponds to calcitic otoliths and that pre-cerebellin-like protein is not present at any stage during the development of the otoliths of the inner ear. This study shows O. mossambicus does not have all of the known otolith proteins indicating that the matrix proteins in the inner ear of fish are not the same across species. Further functional studies of the novel proteins we identified during otolith development are required.