Serpins promote cancer cell survival and vascular co-option in brain metastasis.

Brain metastasis is an ominous complication of cancer, yet most cancer cells that infiltrate the brain die of unknown causes. Here, we identify plasmin from the reactive brain stroma as a defense against metastatic invasion, and plasminogen activator (PA) inhibitory serpins in cancer cells as a shield against this defense. Plasmin suppresses brain metastasis in two ways: by converting membrane-bound astrocytic FasL into a paracrine death signal for cancer cells, and by inactivating the axon pathfinding molecule L1CAM, which metastatic cells express for spreading along brain capillaries and for metastatic outgrowth. Brain metastatic cells from lung cancer and breast cancer express high levels of anti-PA serpins, including neuroserpin and serpin B2, to prevent plasmin generation and its metastasis-suppressive effects. By protecting cancer cells from death signals and fostering vascular co-option, anti-PA serpins provide a unifying mechanism for the initiation of brain metastasis in lung and breast cancers.

Frontal deficits and atrophy in a patient with familial encephalopathy with neuroserpin inclusion bodies detected by single-case voxel-based morphometry: a case report.

BACKGROUND: Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a rare genetic disorder characterized by progressive cognitive decline and myoclonic epilepsy, caused by pathogenic variants of SERPINI1. We reported a case of genetically confirmed FENIB with de novo H338R mutation in the SERPINI1, in which frontal deficits including inattention and disinhibition, and relevant atrophy in the vmPFC on brain MRI were observed in the early stage of the disease. CASE PRESENTATION: A 23-year-old Japanese man presented with progressive inattention and disinhibition over 4 years followed by myoclonic epilepsy. The whole-genome sequencing and filtering analysis showed de novo heterozygous H338R mutation in the SERPINI1, confirming the diagnosis of FENIB. Single-case voxel-based morphometry using brain magnetic resonance imaging obtained at the initial visit revealed focal gray matter volume loss in the ventromedial prefrontal cortices, which is presumed to be associated with inattention and disinhibition. CONCLUSION: Frontal deficits including inattention and disinhibition can be the presenting symptoms of patients with FENIB. Single-case voxel-based morphometry may be useful for detecting regional atrophy of the frontal lobe in FENIB. Detecting these abnormalities in the early stage of disease may be key findings for differentiating FENIB from other causes of progressive myoclonic epilepsy.

Neuroserpin gene therapy inhibits retinal ganglion cell apoptosis and promotes functional preservation in glaucoma.

Our research has proven that the inhibitory activity of the serine protease inhibitor neuroserpin (NS) is impaired because of its oxidation deactivation in glaucoma. Using genetic NS knockout (NS-/-) and NS overexpression (NS+/+ Tg) animal models and antibody-based neutralization approaches, we demonstrate that NS loss is detrimental to retinal structure and function. NS ablation was associated with perturbations in autophagy and microglial and synaptic markers, leading to significantly enhanced IBA1, PSD95, beclin-1, and LC3-II/LC3-I ratio and reduced phosphorylated neurofilament heavy chain (pNFH) levels. On the other hand, NS upregulation promoted retinal ganglion cell (RGC) survival in wild-type and NS-/- glaucomatous mice and increased pNFH expression. NS+/+Tg mice demonstrated decreased PSD95, beclin-1, LC3-II/LC3-I ratio, and IBA1 following glaucoma induction, highlighting its protective role. We generated a novel reactive site NS variant (M363R-NS) resistant to oxidative deactivation. Intravitreal administration of M363R-NS was observed to rescue the RGC degenerative phenotype in NS-/- mice. These findings demonstrate that NS dysfunction plays a key role in the glaucoma inner retinal degenerative phenotype and that modulating NS imparts significant protection to the retina. NS upregulation protected RGC function and restored biochemical networks associated with autophagy and microglial and synaptic function in glaucoma.

A Potential Link between Myeloperoxidase Modified LDL, Atherosclerosis and Depression.

Atherosclerosis is a chronic inflammatory disease that involves modified low-density lipoproteins (LDL) which play a pivotal role in the initiation and progression of the disease. Myeloperoxidase oxidized LDL (Mox-LDL) is considered to be the most patho-physiologically relevant type of modified LDL and has been reported to be ubiquitously present in atheroma plaques of patients with atherosclerosis. Besides its involvement in the latter disease state, Mox-LDL has also been shown to be implicated in the pathogenesis of various illnesses including sleep disorders, which are in turn associated with heart disease and depression in many intricate ways. Meanwhile, we have recently shown that lox-1-mediated Mox-LDL signaling modulates neuroserpin activity in endothelial cells, which could have major implications that go beyond the pathophysiology of stroke and cerebrovascular disease (CD). Of note is that tissue plasminogen activator (tPA), which is the main target of neuroserpin in the brain, has a crucial function in the processing of brain-derived neurotrophic factor (BDNF) into its mature form. This factor is known to be involved in major depressive disorder (MDD) development and pathogenesis. Since tPA is more conventionally recognized as being involved in fibrinolytic mechanisms, and its effect on the BDNF system in the context of MDD is still not extensively studied, we speculate that any Mox-LDL-driven change in the activity of tPA in patients with atherosclerosis may lead to a decrease in the production of mature BDNF, resulting in impaired neural plasticity and depression. Deciphering the mechanisms of interaction between those factors could help in better understanding the potentially overlapping pathological mechanisms that regulate disease processes in CD and MDD, supporting the possibility of novel and common therapeutic opportunities for millions of patients worldwide.

Polymerogenic neuroserpin causes mitochondrial alterations and activates NFκB but not the UPR in a neuronal model of neurodegeneration FENIB.

The neurodegenerative condition FENIB (familiar encephalopathy with neuroserpin inclusion bodies) is caused by heterozygous expression of polymerogenic mutant neuroserpin (NS), with polymer deposition within the endoplasmic reticulum (ER) of neurons. We generated transgenic neural progenitor cells (NPCs) from mouse fetal cerebral cortex stably expressing either the control protein GFP or human wild type, polymerogenic G392E or truncated (delta) NS. This cellular model makes it possible to study the toxicity of polymerogenic NS in the appropriated cell type by in vitro differentiation to neurons. Our previous work showed that expression of G392E NS in differentiated NPCs induced an adaptive response through the upregulation of several genes involved in the defence against oxidative stress, and that pharmacological reduction of the antioxidant defences by drug treatments rendered G392E NS neurons more susceptible to apoptosis than control neurons. In this study, we assessed mitochondrial distribution and found a higher percentage of perinuclear localisation in G392E NS neurons, particularly in those containing polymers, a phenotype that was enhanced by glutathione chelation and rescued by antioxidant molecules. Mitochondrial membrane potential and contact sites between mitochondria and the ER were reduced in neurons expressing the G392E mutation. These alterations were associated with a pattern of ER stress that involved the ER overload response but not the unfolded protein response. Our results suggest that intracellular accumulation of NS polymers affects the interaction between the ER and mitochondria, causing mitochondrial alterations that contribute to the neuronal degeneration seen in FENIB patients.

Neuroserpin, a crucial regulator for axogenesis, synaptic modelling and cell-cell interactions in the pathophysiology of neurological disease.

Neuroserpin is an axonally secreted serpin that is involved in regulating plasminogen and its enzyme activators, such as tissue plasminogen activator (tPA). The protein has been increasingly shown to play key roles in neuronal development, plasticity, maturation and synaptic refinement. The proteinase inhibitor may function both independently and through tPA-dependent mechanisms. Herein, we discuss the recent evidence regarding the role of neuroserpin in healthy and diseased conditions and highlight the participation of the serpin in various cellular signalling pathways. Several polymorphisms and mutations have also been identified in the protein that may affect the serpin conformation, leading to polymer formation and its intracellular accumulation. The current understanding of the involvement of neuroserpin in Alzheimer's disease, cancer, glaucoma, stroke, neuropsychiatric disorders and familial encephalopathy with neuroserpin inclusion bodies (FENIB) is presented. To truly understand the detrimental consequences of neuroserpin dysfunction and the effective therapeutic targeting of this molecule in pathological conditions, a cross-disciplinary understanding of neuroserpin alterations and its cellular signaling networks is essential.

Neuroserpin: structure, function, physiology and pathology.

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.

Increased anxiety was found in serpini1 knockout zebrafish larval.

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.

Detection of truncated isoforms of human neuroserpin lacking the reactive center loop: Implications in noninhibitory role.

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.

Deficits in developmental neurogenesis and dendritic spine maturation in mice lacking the serine protease inhibitor neuroserpin.

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.

Nerve growth factor (NGF) pathway biomarkers in Down syndrome prior to and after the onset of clinical Alzheimer's disease: A paired CSF and plasma study.

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.

Neuroserpin extends the time window of tPA thrombolysis in a rat model of middle cerebral artery occlusion.

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.

Glycosylation Tunes Neuroserpin Physiological and Pathological Properties.

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.

Physiological and pathological functions of neuroserpin: Regulation of cellular responses through multiple mechanisms.

It is 27 years since neuroserpin was first discovered in the nervous system and identified as a member of the serpin superfamily. Since that time potential roles for this serine protease inhibitor have been identified in neuronal and non-neuronal systems. Many are linked to inhibition of neuroserpin's principal enzyme target, tissue plasminogen activator (tPA), although some have been suggested to involve alternate non-inhibitory mechanisms. This review focuses mainly on the inhibitory roles of neuroserpin and discusses the evidence supporting tPA as the physiological target. While the major sites of neuroserpin expression are neural, endocrine and immune tissues, most progress on characterizing functional roles for neuroserpin have been in the brain. Roles in emotional behaviour, synaptic plasticity and neuroprotection in stroke and excitotoxicity models are discussed. Current knowledge on three neurological diseases associated with neuroserpin mutation or activity, Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), Alzheimer's disease and brain metastasis is presented. Finally, we consider mechanistic studies that have revealed a distinct inhibitory mechanism for neuroserpin and its possible implications for neuroserpin function.

Identification of a novel alternatively spliced isoform of antithrombin containing an additional RCL-like loop.

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 expression during human brain development and in adult brain revealed by immunohistochemistry and single cell RNA sequencing.

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.

The serine protease inhibitor neuroserpin is required for normal synaptic plasticity and regulates learning and social behavior.

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.

Neuroserpin polymers cause oxidative stress in a neuronal model of the dementia FENIB.

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.

SERPINI1 pathogenic variants: An emerging cause of childhood-onset progressive myoclonic epilepsy.

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.

Neuroprotective effect of neuroserpin in non-tPA-induced intracerebral hemorrhage mouse models.

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.