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Neuroserpin is a member of the serpin superfamily. Point mutations in the neuroserpin gene underlie the autosomal dominant dementia, familial encephalopathy with neuroserpin inclusion bodies. This is characterized by the retention of ordered polymers of neuroserpin within the endoplasmic reticulum of neurons. pH has been shown to affect the propensity of several serpins to form polymers. In particular, low pH favors the formation of polymers of both α1-antitrypsin and antithrombin. We report here opposite effects in neuroserpin, with a striking resistance to polymer formation at acidic pH. Mutation of specific histidine residues showed that this effect is not attributable to the shutter domain histidine as would be predicted by analogy with other serpins. Indeed, mutation of the shutter domain His338 decreased neuroserpin stability but had no effect on the pH dependence of polymerization when compared with the wild-type protein. In contrast, mutation of His119 or His138 reduced the polymerization of neuroserpin at both acidic and neutral pH. These residues are at the lower pole of neuroserpin and provide a novel mechanism to control the opening of β-sheet A and hence polymerization. This mechanism is likely to have evolved to protect neuroserpin from the acidic environment of the secretory granules.

This review is focused on the properties of the most-studied inhibitors of proteolytic enzymes that are found in human cerebrospinal fluid. The possible roles of specific proteinase inhibitors in the pathogenesis of neurological diseases are discussed. We also considered perspectives for the use of assessment of their levels for disease diagnosis and prognosis.

Point mutations cause members of the serine protease inhibitor (serpin) superfamily to undergo a novel conformational transition, forming ordered polymers. These polymers characterize a group of diseases termed the serpinopathies. The formation of polymers underlies the retention of α1-antitrypsin within hepatocytes and of neuroserpin within neurons to cause cirrhosis and dementia, respectively. Point mutations of antithrombin, C1 inhibitor, α1-antichymotrypsin, and heparin cofactor II cause a similar conformational transition, resulting in a plasma deficiency that is associated with thrombosis, angioedema, and emphysema. Polymers of serpins can also form in extracellular tissues where they activate inflammatory cascades. This is best described for the Z variant of α1-antitrypsin in which the proinflammatory properties of polymers provide an explanation for both progressive emphysema and the selective advantage of this mutant allele. Therapeutic strategies are now being developed to block the aberrant conformational transitions and so treat the serpinopathies.

Amyloid-beta plaques are a pathological hallmark of Alzheimer's disease. Several proteases are known to cleave/remove amyloid-beta, including plasmin, the product of tissue plasminogen activator cleavage of the pro-enzyme plasminogen. Although plasmin levels are lower in Alzheimer brain, there has been little analysis of the plasminogen activator/plasmin system in the brains of Alzheimer patients. In this study, zymography, immunocapture, and ELISAs were utilized to show that tissue plasminogen activator activity in frontal cortex tissue of Alzheimer patients is dramatically reduced compared with age-matched controls, while tissue plasminogen activator and plasminogen protein levels are unchanged; suggesting that plasminogen activator activity is inhibited in the Alzheimer brain. Analysis of endogenous plasminogen activator inhibitors shows that while plasminogen activator inhibitor-1 and protease nexin-1 levels are unchanged, the neuroserpin levels are significantly elevated in brains of Alzheimer patients. Furthermore, elevated amounts of tissue plasminogen activator-neuroserpin complexes are seen in the Alzheimer brain, and immunohistochemical studies demonstrate that both tissue plasminogen activator and neuroserpin are associated with amyloid-beta plaques in Alzheimer brain tissue. Thus, neuroserpin inhibition of tissue plasminogen activator activity leads to reduced plasmin and may be responsible for reduced clearance of amyloid-beta in the Alzheimer disease brain. Furthermore, decreased tissue plasminogen activator activity in the Alzheimer brain may directly influence synaptic activity and impair cognitive function.

Mutations in human neuroserpin gene cause an autosomal dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB). We generated and analyzed transgenic mice expressing high levels of either FENIB-type (G392E) or wild-type human neuroserpin in neurons of the central nervous system. G392E neuroserpin accumulated age-dependently in neurons of the neocortex, thalamus, amygdala, pons, and spinal cord of homozygous transgenic mice. Such accumulations were not observed in hemizygous transgenic mice nor in transgenic mice for wild-type neuroserpin. In differential centrifugation of brain homogenates, G392E neuroserpin recovered in the nucleus-rich fraction dramatically increased along with aging, suggesting that the aggregations gradually increase their densities presumably by their conversion into heavier and more compact configurations. In immunoelectron microscopical analyses, immunopositivities for G392E neuroserpin were found not only in endoplasmic reticulum but also in lysosomes. G392E neuroserpin transgenic mice were much more susceptible to seizures induced by kainate administration than nontransgenic mice. Overall, G392E neuroserpin accumulated in the central nervous system neurons of transgenic mice in mutation-, aging-, and gene dosage-dependent manners. The established transgenic mice will be valuable to elucidate not only mechanisms for the formation of G392E neuroserpin aggregations but also pathways for the degradation and/or clearance of the already formed aggregations in neurons.

We have developed a relational database of human SERPINA1 gene mutations, leading to alpha(1)-antitrypsin (AAT) deficiency, called A(1)ATVar, which can be accessed over the World Wide Web at www.goldenhelix.org/A1ATVar. Extensive information has been extracted from the literature and converted into a searchable database, including genotype information, clinical phenotype, allelic frequencies for the commonest AAT variant alleles, methods of detection, and references. Mutation summaries are automatically displayed and user-generated queries can be formulated based on fields in the database. A separate module, linked to the FINDbase database for frequencies of inherited disorders allows the user to access allele frequency information for the three most frequent AAT alleles, namely PiM, PiS, and PiZ. The available experimental protocols to detect AAT variant alleles at the protein and DNA levels have been archived in a searchable format. A visualization tool, called VariVis, has been implemented to combine A(1)ATVar variant information with SERPINA1 sequence and annotation data. A direct data submission tool allows registered users to submit data on novel AAT variant alleles as well as experimental protocols to explore SERPINA1 genetic heterogeneity, via a password-protected interface. Database access is free of charge and there are no registration requirements for querying the data. The A(1)ATVar database is the only integrated database on the Internet offering summarized information on AAT allelic variants and could be useful not only for clinical diagnosis and research on AAT deficiency and the SERPINA1 gene, but could also serve as an example for an all-in-one solution for locus-specific database (LSDB) development and curation.

The serpins are a family of proteins that can multimerize via β-sheet linkages. Accumulation of such multimers can give rise to diseases such as thrombosis, cirrhosis and dementia. While the structures of many serpins are known, the structure of the linkage between monomers was unclear. In this work, Huntington and colleagues have solved the crystal structure of an antithrombin dimer. They find that the high stability of the serpin polymer is due to a large domain swap between beta sheets of the neighbouring monomers. In addition, the structure explains the how certain pathogenic mutations stabilize a polymerogenic folding intermediate. Repeating intermolecular protein association by means of β-sheet expansion is the mechanism underlying a multitude of diseases including Alzheimer’s, Huntington’s and Parkinson’s and the prion encephalopathies1. A family of proteins, known as the serpins, also forms large stable multimers by ordered β-sheet linkages leading to intracellular accretion and disease2. These ‘serpinopathies’ include early-onset dementia caused by mutations in neuroserpin, liver cirrhosis and emphysema caused by mutations in α1-antitrypsin (α1AT), and thrombosis caused by mutations in antithrombin3. Serpin structure and function are quite well understood, and the family has therefore become a model system for understanding the β-sheet expansion disorders collectively known as the conformational diseases4. To develop strategies to prevent and reverse these disorders, it is necessary to determine the structural basis of the intermolecular linkage and of the pathogenic monomeric state. Here we report the crystallographic structure of a stable serpin dimer which reveals a domain swap of more than 50 residues, including two long antiparallel β-strands inserting in the centre of the principal β-sheet of the neighbouring monomer. This structure explains the extreme stability of serpin polymers, the molecular basis of their rapid propagation, and provides critical new insights into the structural changes which initiate irreversible β-sheet expansion.

Otoliths and the homologous otoconia in the inner ear are essential for balance. Their morphogenesis is less understood than that of other biominerals, such as bone, and only a small number of their constituent proteins have been characterized. As a novel approach to identify unknown otolith proteins, we employed shotgun proteomics to analyze crude extracts from trout and catfish otoliths. We found three proteins that had not been associated previously with otolith or otoconia formation: 'Secreted acidic cysteine rich glycoprotein' (Sparc), an important bone protein that binds collagen and Ca(2+); precerebellin-like protein, which contains a C1q domain and may associate with the collagenous otolin-1 during its assembly into a framework; and neuroserpin, a serine protease inhibitor that may regulate local protease activity during framework assembly. We then used the zebrafish to investigate whether Sparc plays a role in otolith morphogenesis. Immunodetection demonstrated that Sparc is a true constituent of otoliths. Knockdown of Sparc expression in morphant zebrafish resulted in four principal types of defective otoliths: smaller, extra and ectopic, missing and fused, or completely absent. Smaller size was the predominant phenotype and independent of the severity of otic-vesicle defects. These results suggested that Sparc is directly required for normal otolith growth.

Our laboratory has shown in numerous experiments that the neurosteroids progesterone (PROG) and allopregnanolone (ALLO) improve molecular and functional outcomes after traumatic brain injury (TBI). As coagulopathy is an important contributor to the secondary destruction of nervous tissue, we hypothesized that PROG and ALLO administration may also have a beneficial effect on coagulation protein expression after TBI. Adult male Sprague—Dawley rats were given bilateral contusions of the medial frontal cortex followed by treatments with PROG (16 mg/kg), ALLO (8 mg/kg), or vehicle (22.5% hydroxypropyl-β-cyclodextrin). Controls received no injury or injections. Progesterone generally maintained procoagulant (thrombin, fibrinogen, and coagulation factor XIII), whereas ALLO increased anticoagulant protein expression (tissue-type plasminogen activator, tPA). In addition, PROG significantly increased the ratio of tPA bound to neuroserpin, a serine protease inhibitor that can reduce the activity of tPA. Our findings suggest that in a model of TBI, where blood loss may exacerbate injury, it may be preferable to treat patients with PROG, whereas it might be more appropriate to use ALLO as a treatment for thrombotic stroke, where a reduction in coagulation would be more beneficial.

Members of the serine proteinase inhibitor or serpin superfamily inhibit their target proteinases by a conformational transition that involves the enzyme being translocated from the upper to the lower pole of the protein. This sophisticated mechanism is subverted by point mutations to form ordered polymers that are retained within the endoplasmic reticulum of the cell of synthesis. These polymers activate NF-kappaB and cause cytotoxicity by a pathway that is independent of the unfolded protein response. As diverse conditions can be explained the same mechanism of polymerisation we have grouped them together as a new class of disease, the serpinopathies. We review here the structural basis of the serpinopathies and discuss how the ordered accumulation of polymers causes cell death.

An understanding of the diabetes-induced alterations in vitreous protein composition in the absence and in the presence of proliferative diabetic retinopathy (PDR) may provide insights into factors and mechanisms responsible for this disease. We have performed a comprehensive proteomic analysis and comparison of vitreous samples from individuals with diabetes but without diabetic retinopathy (noDR) or with PDR and nondiabetic individuals (NDM). Using preparative one-dimensional SDS-PAGE and nano-LC/MS/MS of 17 independent vitreous samples, we identified 252 proteins from human vitreous. Fifty-six proteins were differentially abundant in noDR and PDR vitreous compared with NDM vitreous, including 32 proteins increased and 10 proteins decreased in PDR vitreous compared with NDM vitreous. Comparison of noDR and PDR groups revealed increased levels of angiotensinogen and decreased levels of calsyntenin-1, interphotoreceptor retinoid-binding protein, and neuroserpin in PDR vitreous. Biological pathway analysis revealed that vitreous contains 30 proteins associated with the kallikrein-kinin, coagulation, and complement systems. Five of them (complement C3, complement factor I, prothrombin, alpha-1-antitrypsin, and antithrombin III) were increased in PDR vitreous compared with NDM vitreous. Factor XII was detected in PDR vitreous but not observed in either NDM or noDR vitreous. PDR vitreous also had increased levels of peroxiredoxin-1 and decreased levels of extracellular superoxide dismutase, compared with noDR or NDM vitreous. These data provide an in depth analysis of the human vitreous proteome and reveal protein alterations that are associated with PDR.

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by the retention of polymers of neuroserpin as inclusions within the endoplasmic reticulum (ER) of neurons. We have developed monoclonal antibodies that detect polymerized neuroserpin and have used COS-7 cells, stably transfected PC12 cell lines and transgenic Drosophila melanogaster to characterize the cellular handling of all four mutant forms of neuroserpin that cause FENIB. We show a direct correlation between the severity of the disease-causing mutation and the accumulation of neuroserpin polymers in cell and fly models of the disease. Moreover, mutant neuroserpin causes locomotor deficits in the fly allowing us to demonstrate a direct link between polymer accumulation and neuronal toxicity.

Ischemic stroke is a leading cause of morbidity and mortality worldwide and recombinant human tissue-type plasminogen activator (tPA) is the prominent therapeutic among very few therapeutics used in its treatment. Due to complications attributed to the drug, most notably transformation of ischemia to hemorrhage, tPA is only used in a small number of ischemic stroke cases, albeit significantly more often in specialized stroke centers.What are the mechanisms of tPA action and side effects in ischemic stroke, and can the knowledge about these mechanisms aid in making tPA a more efficacious and safe therapeutic or in developing alternative therapeutics?tPA use and alternative/combination therapies in acute ischemic stroke treatment are summarized. The review focuses on literature concerning tPA neurotoxicity and its implications for further development of tPA as a stroke therapeutic.Exogenously administered recombinant tPA and endogenous tPA have both turned into promising therapeutic targets for the stroke patient.

The balance between tissue-type plasminogen activator (tPA) and one of its inhibitors, neuroserpin, has crucial roles in the central nervous system, including the control of neuronal migration, neuronal plasticity and neuronal death. In the present study, we demonstrate that the activation of the transforming growth factor-β (TGFβ)-related BMPR-IB (also known as BMPR1B and Alk6)- and Smad5-dependent signalling pathways controls neuroserpin transcription. Accordingly, we demonstrate for the first time that anti-Mullerian hormone (AMH), a member of the TGFβ family, promotes the expression of neuroserpin in cultured neurons but not in astrocytes. The relevance of these findings is confirmed by the presence of both AMH and AMH type-II receptor (AMHR-II) in brain tissues, and is supported by the observation of reduced levels of neuroserpin in the brain of AMHR-II-deficient mice. Interestingly, as previously demonstrated for neuroserpin, AMH protects neurons against N-methyl-D-aspartate (NMDA)-mediated excitotoxicity both in vitro and in vivo. This study demonstrates the existence of an AMH-dependent signalling pathway in the brain leading to an overexpression of the serine-protease inhibitor, neuroserpin, and neuronal survival.

Objective: Serine protease inhibitors (serpins), the acute phase reactants and regulators of the proteolytic processing of proteins, have been recognized as potential contributors to the pathogenesis of Alzheimer disease (AD). We measured plasma and CSF levels of serpins in controls and patients with dementia. Methods: Using rocket immunoelectrophoresis, ELISA, and Luminex xMAP technology, we analyzed plasma levels of α 1 -antichymotrypsin and α 1 -antitrypsin, and CSF levels of α 1 -antichymotrypsin, α 1 -antitrypsin, and neuroserpin along with three standard biomarkers (total tau, tau phosphorylated at threonine-181, and the Aβ 1-42 ) in patients with AD (n = 258), patients with dementia with Lewy bodies (DLB; n = 38), and age-matched controls (n = 37). Results: The level of CSF neuroserpin was significantly higher in AD compared with controls and DLB, whereas CSF α 1 -antichymotrypsin and α 1 -antitrypsin were significantly higher in both AD and DLB groups than in controls. Results from logistic regression analyses demonstrate a relationship between higher CSF levels of α 1 -antichymotrypsin and neuroserpin and increased predicted probability and odds ratios (ORs) of AD (OR 5.3, 95% CI 1.3 to 20.8 and OR 3.3, CI 1.3 to 8.8). Furthermore, a logistic regression model based on CSF α 1 -antichymotrypsin, neuroserpin, and Aβ 1-42 enabled us to discriminate between AD patients and controls with a sensitivity of 94.7% and a specificity of 77.8%. Conclusions: Higher CSF levels of neuroserpin and α 1 -antichymotrypsin were associated with the clinical diagnosis of Alzheimer disease (AD) and facilitated the diagnostic classification of AD vs controls. CSF serpin levels did not improve the diagnostic classification of AD vs dementia with Lewy bodies. GLOSSARY: AAT = α 1 -antitrypsin; ACT = α 1 -antichymotrypsin; AD = Alzheimer disease; ApoE = apolipoprotein E; AUC = area under the curve; BBB = blood-brain barrier; COPD = chronic obstructive pulmonary disease; %CV = coefficients of variation percentage; DLB = dementia with Lewy bodies; IL = interleukin; MMSE = Mini-Mental State Examination; NSAIDs = nonsteroidal anti-inflammatory drugs; OR = odds ratio; P-tau = tau phosphorylated at threonine-181; ROC = receiver operating characteristic; T-tau = total tau.

Familial encephalopathy with neuroserpin inclusion bodies is a recently described neurodegenerative disease that is responsible for progressive myoclonic epilepsy or presenile dementia. In a French family with the S52R mutation of the neuroserpin gene, progressive myoclonic epilepsy was associated with a frontal syndrome. The typical cerebral inclusions (Collins bodies) were abundant in the frontal cortex and in the head of the caudate nucleus but spared the cerebellum.

Background and Objective: Molecular gene markers, which can distinguish human bone marrow mesenchymal stem cells from human fibroblasts, have recently been reported. Messenger RNA levels of tissue factor pathway inhibitor-2, major histocompatibility complex-DR-α, major histocompatibility complex-DR-β, and neuroserpin are higher in human bone marrow mesenchymal stem cells than in human fibroblasts. However, human bone marrow mesenchymal stem cells express less apolipoprotein D mRNA than human fibroblasts. Periodontal ligament cells are a heterogeneous cell population including fibroblasts, mesenchymal stem cells, and progenitor cells of osteoblasts or cementoblasts. The use of molecular markers that distinguish human bone marrow mesenchymal stem cells from human fibroblasts may provide insight into the characteristics of human periodontal ligament cells. In this study, we compared the molecular markers of human periodontal ligament cells with those of human bone marrow mesenchymal stem cells and human gingival fibroblasts. Material and Methods: The mRNA expression of the molecular gene markers was analyzed using real-time polymerase chain reaction. Statistical differences were determined with the two-sided Mann–Whitney U-test. Results: Messenger RNA levels of major histocompatibility complex-DR-α and major histocompatibility complex-DR-β were lower and higher, respectively, in human periodontal ligament cells than in human bone marrow mesenchymal stem cells or human gingival fibroblasts. Human periodontal ligament cells showed the lowest apolipoprotein D mRNA levels among the three types of cells. Conclusion: Human periodontal ligament cells may be distinguished from human bone marrow mesenchymal stem cells and human gingival fibroblasts by the genes for apolipoprotein D, major histocompatibility complex-DR-α, and major histocompatibility complex-DR-β.

Intracellular protein deposition due to aggregation caused by conformational alteration is the hallmark of a number of neurodegenerative disorders, including Parkinson's disease, tauopathies, Huntington's disease, and familial encephalopathy with neuroserpin inclusion bodies. The latter is an autosomal dominant disorder caused by point mutations in neuroserpin resulting in its destabilization. Mutant neuroserpin polymerizes and forms intracellular aggregates that eventually lead to neurodegeneration. We generated genetically modified mice expressing the late-onset S49P-Syracuse or the early-onset S52R-Portland mutation of neuroserpin in central nervous system neurons. Mice exhibited morphological, biochemical, and clinical features resembling those found in the human disease. Analysis of brains revealed large intraneuronal inclusions composed exclusively of mutant neuroserpin, accumulating long before the development of clinical symptoms in a time-dependent manner. Clinical symptoms and amount of neuroserpin inclusions correlated with the predicted instability of the protein. The presence of inclusion bodies in subclinical mice indicates that in humans the prevalence of the disease could be higher than anticipated. In addition to shedding light on the pathophysiology of the human disorder, these mice provide an excellent model to study mechanisms of neurodegeneration or establish novel therapies for familial encephalopathy with neuroserpin inclusion bodies and other neurodegenerative diseases with intracellular protein deposition.

Ns (neuroserpin) is a member of the serpin (serine protease inhibitor) gene family that is primarily expressed within the central nervous system. Its principal target protease is tPA (tissue plasminogen activator), which is thought to contribute to synaptic plasticity and to be secreted in a stimulus-dependent manner. In the present study, we demonstrate in primary neuronal cultures that Ns co-localizes in LDCVs (large dense core vesicles) with the regulated secretory protein chromogranin B. We also show that Ns secretion is regulated and can be specifically induced 4-fold by secretagogue treatment. A novel 13-amino-acid sorting signal located at the C-terminus of Ns is identified that is both necessary and sufficient to target Ns to the regulated secretion pathway. Its deletion renders Ns no longer responsive to secretagogue stimulation, whereas PAI-Ns [Ns (neuroserpin)–PAI-1 (plasminogen activator inhibitor-1) chimaera appending the last 13 residues of Ns sequence to the C-terminus of PAI-1] shifts PAI-1 secretion into a regulated secretory pathway.

Although its roles in the vascular space are most well known, tissue plasminogen activator (tPA) is widely expressed in the developing and adult nervous system, where its activity is believed to be regulated by neuroserpin, a predominantly brain-specific member of the serpin family of protease inhibitors. In the normal physiological state, tPA has been shown to play roles in the development and plasticity of the nervous system. Ischemic damage, however, may lead to excess tPA activity in the brain and this is believed to contribute to neurodegeneration. In this article, we briefly review the physiological and pathological roles of tPA in the nervous system, which includes neuronal migration, axonal growth, synaptic plasticity, neuroprotection and neurodegeneration, as well as a contribution to neurological disease. We summarize the tPA’s multiple mechanisms of action and also highlight the contributions of the inhibitor neuroserpin to these processes.

Neuroserpin (NSP) reportedly exerts neuroprotective effects in cerebral ischemic animal models and patients; however, the mechanism of protection is poorly understood. We thus attempted to confirm neuroprotective effects of NSP on astrocytes in the ischemic state and then explored the relative mechanisms. Astrocytes from neonatal rats were treated with oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/R). To confirm the neuroprotective effects of NSP, we measured the cell survival rate, relative lactate dehydrogenase (LDH) release; we also performed morphological methods, namely Hoechst 33342 staining and Annexin V assay. To explore the potential mechanisms of NSP, the release of nitric oxide (NO) and TNF-α related to NSP administration were measured by enzyme-linked immunosorbent assay. The proteins related to the NF-κB, ERK1/2, and PI3K/Akt pathways were investigated by Western blotting. To verify the cause-and-effect relationship between neuroprotection and the NF-κB pathway, a NF-κB pathway inhibitor sc3060 was employed to observe the effects of NSP-induced neuroprotection. We found that NSP significantly increased the cell survival rate and reduced LDH release in OGD/R-treated astrocytes. It also reduced NO/TNF-α release. Western blotting showed that the protein levels of p-IKKBα/β and P65 were upregulated by the OGD/R treatment and such effects were significantly inhibited by NSP administration. The NSP-induced inhibition could be significantly reversed by administration of the NF-κB pathway inhibitor sc3060, whereas, expressions of p-ERK1, p-ERK2, and p-AKT were upregulated by the OGD/R treatment; however, their levels were unchanged by NSP administration. Our results thus verified the neuroprotective effects of NSP in ischemic astrocytes. The potential mechanisms include inhibition of the release of NO/TNF-α and repression of the NF-κB signaling pathways. Our data also indicated that NSP has little influence on the MAPK and PI3K/Akt pathways.

Subplate cells are among the first generated neurons in the mammalian cerebral cortex and have been implicated in the establishment of cortical wiring. In rodents some subplate neurons persist into adulthood. Here we would like to highlight several converging findings which suggest a novel secretory function of subplate neurons during cortical development. Throughout the postnatal period in rodents, subplate neurons have highly developed rough endoplasmic reticulum (ER) and are under an ER stress condition. By comparing gene expression between subplate and layer 6, we found that several genes encoding secreted proteins are highly expressed in subplate neurons. One of these secreted proteins, neuroserpin, encoded by the serpini1 gene, is localized to the ER in subplate cells. We propose that subplate might influence cortical circuit formation through a transient secretory function.

Contact between T cells and APCs and activation of an effective immune response trigger cellular polarization and the formation of a structured interface known as the immunological synapse. Interactions across the synapse and secretion of T cell and APC-derived factors into the perisynaptic compartment regulate synapse formation and activation of T cells. We report that the serine protease inhibitor neuroserpin, an axonally secreted protein thought to play roles in the formation of the neuronal synapse and refinement of synaptic activity, is expressed in human nai¨ve effector memory and central memory subsets of CD4+ and CD8+ T cells, as well as monocytes, B cells, and NK cells. Neuroserpin partially colocalized with a TGN38/LFA-1-positive vesicle population in T cells and translocates to the immunological synapse upon activation with TCR antibodies or antigen-pulsed APCs. Activation of T cells triggered neuroserpin secretion, a rapid, 8.4-fold up-regulation of the serine protease tissue plasminogen activator, the protease target for neuroserpin, and a delayed, 6.25-fold down-regulation of neuroserpin expression. Evidence of polarization and regulated neuroserpin expression was also seen in ex vivo analyses of human lymph nodes and blood-derived T cells. Increased neuroserpin expression was seen in clusters of T cells in the paracortex of human lymph nodes, with some showing polarization to areas of cell:cell interaction. Our results support a role for neuroserpin and tissue plasminogen activator in activation-controlled proteolytic cleavage of proteins in the synaptic or perisynaptic space to modulate immune cell function.

Serine protease inhibitors, termed serpins, regulate myriad physiological processes in the mammalian body from thrombotic and thrombolytic pathways to inflammation, angiogenesis, hormone transport, and hypertension. The large percentage of serpins among the plasma proteins in the circulating blood as in the case of plasminogen activators, the functional redundancy of serpins, and also the debilitating serpinopathies of antithrombin III (SERPINC1), neuroserpin (SERPINI1), and alpha-1 antitrypsin (SERPINA1) provide evidence of the importance of serpins and their widespread impact in normal physiological homeostasis. Inflammation, also termed innate immunity, interacts closely with and both regulates and is regulated by thrombotic and thrombolytic serine proteases. Activation of the coagulation proteases is, in turn, controlled by serpins. Apoptosis is also modulated by serpins with cross-class inhibitory activity for cysteine and serine proteases. Excessive inflammation and cell death processes are now recognized as interacting with the thrombotic and thrombolytic proteases. Viruses have evolved to communicate and control these processes by encoding their own serpins, which confer on them the ability to evade host immune defenses. This chapter provides an introduction to the viral serpins derived from poxvirus origins that have been shown not only to be essential for successful viral infection but, in some cases, as for Serp-1 and Serp-2, to have the potential to mitigate inflammatory disease in animal models. Serp-1 has in fact been successfully tested in a small phase 2A clinical trial in unstable angina patients with coronary stent implants. A tandem discussion of mammalian serpins with actions similar to those of the viral serpins is also presented to emphasize potential evolutionary relationships between viral and mammalian serpins. The anti-inflammatory serpins hold the potential to be effective in disease states such as atheroma, sepsis, cancer, and wound healing given that these conditions are all associated with aberrant inflammatory responses and with dysregulation of thrombotic, thrombolytic, and apoptotic protease cascades. The capacity of viral serpins to provide antiviral protection by modulating the virome as well as possible therapeutic effects of serpin metabolites in inflammation will also be discussed. In summary, viral serpins have evolved over many millions of years and provide a unique and highly potent reservoir for both the study of serpin modulation of inflammatory responses and for new therapeutic approaches to inflammatory and even infectious diseases.

GİRİŞ VE AMAÇ: Romatoid artrit (R.A), yetişkin nüfusunun% 1 ila % 2'sini etkileyen önemli bir otoimmun hastalıktır. Neuroserpin (N.S) doku plazminojen aktivatör (t-PA) inhibitörü olan serin proteaz inhibitörüdür. tPA matriks metalloproteinazları (MMPler), özellikle MMP-9'u aktifleyebilmektedir. MMP-9'un anormal upregülasyonu kan beyin bariyerini çevreleyen bazal lamina proteinleri, sıkı bağlantı komplekleri ve ekstraselüler matriks degredasyonu ile oluşan kan beyin bariyerinin bozulması ile bağlantılır. Patolojik bazı hastalıklarda, MMP-9 ekspresyonu yükselmekte ve claudin-5 (Cld-5) ekspresyonunu azalmaktadır. Bu çalışmada R.A'lı hastalarda hastalığın klinik aktivitesi ile N.S, Cld-5 ve MMP-9 arasındaki ilişki araştırılmıştır.GEREÇ VE YÖNTEM: Seçilen hastalar daha sonra kendi DAS-28 puanlarına göre dört gruba ayrıldı: remisyon grubu (R.G), 16 hasta (DAS-28 2,6-3,2); orta hastalık aktivitesi grubu (O.H.G), 28 hasta (DAS-28> 3,2-5,1); yüksek hastalık aktivitesi grubu (Y.H.G), 15 hasta (DAS-28> 5.1). On sağlıklı birey (S.B) kontrol olarak belirlendi.BULGULAR: Elde edilen veriler ilk defa Cld-5, MMP-9 ve N.S düzeylerinin sağlıklı bireylere göre R.A hastalarında anlamlı ölçüde farklı olduğunu (sırasıyla p = 0.035, 0.026 ve 0.014, sırasıyla) göstermektedir.SONUÇ: Kısacası çalışmamız R.A'da hastalık aktivitesi ve endotel fonksiyon/disfonksiyon biyobelirteçleri arasında farklı bir ilişki olduğunu göstermektedir. Bu bozukluğun nasıl ve niçin meydana geldiği tam olarak anlaşılmamaktadır. Plazmadaki N.S, MMP-9 ve Cld-5 ekpresyonu ile ilgili daha çok veriye ihtiyaç duyulmaktadır. INTRODUCTION AND AIM: Rheumatoid arthritis (R.A) is a major autoimmune disease affecting 1% to 2% of the adult population. Neuroserpin (N.S) is a serine protease inhibitor and member of the serpin family that acts as an inhibitor of protease tissue plasminogen activator (t-PA). tPA can also activate matrix metalloproteinases (MMPs), specifically MMP-9. Abnormal upregulation of MMP-9 has been linked to blood brain barrier (BBB) disruption by extracellular matrix degradation, basal lamina proteins, and tight junctions surrounding the BBB. In pathological some diseases, MMP-9 expression levels increase and decreased expression of Cld-5. The present study investigated the relationship between N.S and Cld-5, as well as MMP-9, with respect to clinical activity of disease in patients with R.AMATERIAL AND METHOD: Selected patients were then divided into four groups based on their DAS-28 scores: remission group (R.G), 16 patients (DAS-28 < 2.6); low disease activity group (H.H.G), 16 patients (DAS-28 > 2.6–3.2); moderate disease activity (O.H.G), 28 patients (DAS-28 > 3.2–5.1); high disease activity group (H.H.G), 15 patients (DAS-28 > 5.1). Ten healthy subjects (S.B) served as controls.FINDINGS: Our results demonstrate, for the first time, that Cld-5, MMP-9, and N.S levels are significantly different in R.A patients relative to healthy subjects (P = 0.035, 0.026, and 0.014, respectively).RESULT: In brief, our study demonstrates differential associations of endothelial function/dysfunction biomarkers and disease activity in R.A How and why this impairment occurs is not fully understood, and more data regarding N.S, MMP, and Cld-5 expression in plasma are warranted. 69

Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

Serpins are well-known inhibitors of plasminogen activators, key components of fibrinolysis. Within the last decades, tissue-type plasminogen activator (tPA) became more than a fibrinolytic agent and has been demonstrated to be a neuromodulator with physiological and pathological roles within the brain. Indeed, tPA is involved in brain plasticity, learning and memory and development. But in a stroke context, tPA contributes to the increase of intraneuronal calcium, by interacting with the GluN1 subunit of the NMDA receptor, leading to an increase in excitotoxicity. It also promotes blood-brain barrier leakage and inflammation, but in certain conditions tPA can also exert beneficial effects in stroke.

06.03.2018 tarihli ve 30352 sayılı Resmi Gazetede yayımlanan “Yükseköğretim Kanunu İle Bazı Kanun Ve Kanun Hükmünde Kararnamelerde Değişiklik Yapılması Hakkında Kanun” ile 18.06.2018 tarihli “Lisansüstü Tezlerin Elektronik Ortamda Toplanması, Düzenlenmesi ve Erişime Açılmasına İlişkin Yönerge” gereğince tam metin erişime açılmıştır. GİRİŞ VE AMAÇ: Romatoid artrit (R.A), yetişkin nüfusunun% 1 ila % 2'sini etkileyen önemli bir otoimmun hastalıktır. Neuroserpin (N.S) doku plazminojen aktivatör (t-PA) inhibitörü olan serin proteaz inhibitörüdür. tPA matriks metalloproteinazları (MMPler), özellikle MMP-9'u aktifleyebilmektedir. MMP-9'un anormal upregülasyonu kan beyin bariyerini çevreleyen bazal lamina proteinleri, sıkı bağlantı komplekleri ve ekstraselüler matriks degredasyonu ile oluşan kan beyin bariyerinin bozulması ile bağlantılır. Patolojik bazı hastalıklarda, MMP-9 ekspresyonu yükselmekte ve claudin-5 (Cld-5) ekspresyonunu azalmaktadır. Bu çalışmada R.A'lı hastalarda hastalığın klinik aktivitesi ile N.S, Cld-5 ve MMP-9 arasındaki ilişki araştırılmıştır. GEREÇ VE YÖNTEM: Seçilen hastalar daha sonra kendi DAS-28 puanlarına göre dört gruba ayrıldı: remisyon grubu (R.G), 16 hasta (DAS-28 2,6-3,2); orta hastalık aktivitesi grubu (O.H.G), 28 hasta (DAS-28> 3,2-5,1); yüksek hastalık aktivitesi grubu (Y.H.G), 15 hasta (DAS-28> 5.1). On sağlıklı birey (S.B) kontrol olarak belirlendi. BULGULAR: Elde edilen veriler ilk defa Cld-5, MMP-9 ve N.S düzeylerinin sağlıklı bireylere göre R.A hastalarında anlamlı ölçüde farklı olduğunu (sırasıyla p = 0.035, 0.026 ve 0.014, sırasıyla) göstermektedir. SONUÇ: Kısacası çalışmamız R.A'da hastalık aktivitesi ve endotel fonksiyon/disfonksiyon biyobelirteçleri arasında farklı bir ilişki olduğunu göstermektedir. Bu bozukluğun nasıl ve niçin meydana geldiği tam olarak anlaşılmamaktadır. Plazmadaki N.S, MMP-9 ve Cld-5 ekpresyonu ile ilgili daha çok veriye ihtiyaç duyulmaktadır. INTRODUCTION AND AIM: Rheumatoid arthritis (R.A) is a major autoimmune disease affecting 1% to 2% of the adult population. Neuroserpin (N.S) is a serine protease inhibitor and member of the serpin family that acts as an inhibitor of protease tissue plasminogen activator (t-PA). tPA can also activate matrix metalloproteinases (MMPs), specifically MMP-9. Abnormal upregulation of MMP-9 has been linked to blood brain barrier (BBB) disruption by extracellular matrix degradation, basal lamina proteins, and tight junctions surrounding the BBB. In pathological some diseases, MMP-9 expression levels increase and decreased expression of Cld-5. The present study investigated the relationship between N.S and Cld-5, as well as MMP-9, with respect to clinical activity of disease in patients with R.A MATERIAL AND METHOD: Selected patients were then divided into four groups based on their DAS-28 scores: remission group (R.G), 16 patients (DAS-28 < 2.6); low disease activity group (H.H.G), 16 patients (DAS-28 > 2.6–3.2); moderate disease activity (O.H.G), 28 patients (DAS-28 > 3.2–5.1); high disease activity group (H.H.G), 15 patients (DAS-28 > 5.1). Ten healthy subjects (S.B) served as controls. FINDINGS: Our results demonstrate, for the first time, that Cld-5, MMP-9, and N.S levels are significantly different in R.A patients relative to healthy subjects (P = 0.035, 0.026, and 0.014, respectively). RESULT: In brief, our study demonstrates differential associations of endothelial function/dysfunction biomarkers and disease activity in R.A How and why this impairment occurs is not fully understood, and more data regarding N.S, MMP, and Cld-5 expression in plasma are warranted.

The serpin superfamily encompasses hundreds of proteins, spread across all kingdoms of life, linked by a common tertiary fold. This review focuses on five diseases caused by serpin dysfunction: variants of antithrombin III lose their ability to interact with heparin; the α1-antitrypsin Pittsburgh mutation causes a change in target proteinase; the α1-antitrypsin Z mutation and neuroserpin, polymerisation of which lead to cellular cytotoxicity; and a loss of maspin expression resulting in cancer.

Alzheimer disease is characterized by extracellular plaques composed of Abeta peptides. We show here that these plaques also contain the serine protease inhibitor neuroserpin and that neuroserpin forms a 1:1 binary complex with the N-terminal or middle parts of the Abeta(1-42) peptide. This complex inactivates neuroserpin as an inhibitor of tissue plasminogen activator and blocks the loop-sheet polymerization process that is characteristic of members of the serpin superfamily. In contrast neuroserpin accelerates the aggregation of Abeta(1-42) with the resulting species having an appearance that is distinct from the mature amyloid fibril. Neuroserpin reduces the cytotoxicity of Abeta(1-42) when assessed using standard cell assays, and the interaction has been confirmed in vivo in novel Drosophila models of disease. Taken together, these data show that neuroserpin interacts with Abeta(1-42) to form off-pathway non-toxic oligomers and so protects neurons in Alzheimer disease.

金沢大学大学院医学系研究科脳細胞分子学, FENIB (familial encephalopathy with neuroserpin inclusion bodies) is caused by intracellular accumulation/polymerization of mutant neuroserpins in the endoplasmic reticulum (ER). Transgenic rats overexpressing megsin (Tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid Schiff (PAS)-positive inclusions distributed throughout deeper layers of cerebral cortex, CA1 of the hippocampus, and substantia nigra. Hippocampal extracts from Tg meg rats showed increased expression of ER stress proteins, and activation of caspases-12 and -3, associated with decreased neuronal density. Enhanced ER stress was also observed in dopaminergic neurons in the substantia nigra, in parallel with decreased neuronal viability and motor coordination. In each case, PAS-positive inclusions were also positive for megsin. These data suggest that overexpression of megsin results in ER stress, eventuating in the formation of PAS-positive inclusions. Tg meg rats provide a novel model of FENIB, where accumulation of serpins in the ER induces selective dysfunction/loss of specific neuronal populations. © 2006 Elsevier Inc. All rights reserved.

In this report, we provide direct demonstration that the neurotrophin nerve growth factor (NGF) is released in the extracellular space in an activity-dependent manner in its precursor form (proNGF) and that it is in this compartment that its maturation and degradation takes place because of the coordinated release and the action of proenzymes and enzyme regulators. This converting protease cascade and its endogenous regulators (including tissue plasminogen activator, plasminogen, neuroserpin, precursor matrix metalloproteinase 9, and tissue inhibitor metalloproteinase 1) are colocalized in neurons of the cerebral cortex and released upon neuronal stimulation. We also provide evidence that this mechanism operates in in vivo conditions, as the CNS application of inhibitors of converting and degrading enzymes lead to dramatic alterations in the tissue levels of either precursor NGF or mature NGF. Pathological alterations of this cascade in the CNS might cause or contribute to a lack of proper neuronal trophic support in conditions such as cerebral ischemia, seizure and Alzheimer’s disease or, conversely, to excessive local production of neurotrophins as reported in inflammatory arthritis pain.

Mutations in neuroserpin and α1-antitrypsin cause these proteins to form ordered polymers that are retained within the endoplasmic reticulum of neurones and hepatocytes, respectively. The resulting inclusions underlie the dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) and Z α1-antitrypsin-associated cirrhosis. Polymers form by a sequential linkage between the reactive centre loop of one molecule and β-sheet A of another, and strategies that block polymer formation are likely to be successful in treating the associated disease. We show here that glycerol, the sugar alcohol erythritol, the disaccharide trehalose and its breakdown product glucose reduce the rate of polymerization of wild-type neuroserpin and the Ser49Pro mutant that causes dementia. They also attenuate the polymerization of the Z variant of α1-antitrypsin. The effect on polymerization was apparent even when these agents had been removed from the buffer. None of these agents had any detectable effect on the structure or inhibitory activity of neuroserpin or α1-antitrypsin. These data demonstrate that sugar and alcohol molecules can reduce the polymerization of serpin mutants that cause disease, possibly by binding to and stabilizing β-sheet A.

Proteinases and their inhibitors play important roles in neural development, homeostasis and disease. Neuroserpin is a member of the serine proteinase inhibitor (serpin) superfamily that is secreted from the growth cones of neurons and inhibits the enzyme tissue-type plasminogen activator (tPA). The temporal and spatial pattern of neuroserpin expression suggests a role in synaptogenesis and is most prominent in areas of the brain that participate in learning, memory and behaviour. Neuroserpin also provides neuronal protection in pathologies such as cerebral ischaemia and epilepsy by preventing excessive activity of tPA. Point mutations in neuroserpin cause aberrant conformational transitions and the formation of loop-sheet polymers that are retained within the endoplasmic reticulum of neurons, forming inclusion bodies that underlie an autosomal dominant dementia that we have called familial encephalopathy with neuroserpin inclusion bodies or FENIB. We review here the role of neuroserpin and other proteinase inhibitors in brain development, function and disease.

Considering its brain-specific expression, neuroserpin (NS), a potent inhibitor of tissue-type plasminogen activator (tPA), might be a good therapeutic target to limit the pro-excitotoxic effects of tPA within the cerebral parenchyma, without affecting the benefit from thrombolysis in stroke patients. Here, we aimed at determining the mechanisms of action responsible for the previously reported neuroprotective activity of NS in rodent experimental cerebral ischemia. First, we show in vivo that exogenous NS protects the cortex and the striatum against NMDA-induced injury. Then, the cellular mechanisms of this neuroprotection were investigated in primary cultures of cortical neurons. We show that NS fails to prevent serum deprivation-induced apoptotic neuronal death, while it selectively prevents NMDA- but not AMPA-induced excitotoxicity. This beneficial effect is associated to a decrease in NMDA receptor-mediated intracellular calcium influx. Altogether, these data suggest that an overexpression of neuroserpin in the brain parenchyma might limit the deleterious effect of tPA on NMDA receptor-mediated neuronal death, which occurs following experimental ischemia.

Numerous disorders, including Alzheimer's, Parkinson's and other late-onset neurodegenerative diseases, arise from the conformationally driven aggregation of individual proteins. Previous focus on just one end-product of such aggregation - extracellular deposits of amyloid - has diverted attention from what is now recognized as being primarily intracellular disease processes. Recent structural findings show how cytotoxicity can result from even minor changes in conformation that do not lead to amyloid formation, as with the accumulation within the endoplasmic reticulum of intact mutant alpha-1-antitrypsin in hepatocytes and of neuroserpin in neurons. Studies in Alzheimer's and other dementias also indicate that the damage occurs at the stage of the initial intermolecular linkages that precede amyloid formation. The challenge now is to determine the detailed mechanisms of this cytotoxicity.

To characterize mesenchymal stem cells (MSC), we compared gene expression profiles in human bone marrow MSC (11 lines) and human fibroblasts (4 lines) by RT-PCR and real time PCR. Messenger RNA levels of MHC-DR-alpha, MHC-DR-beta, MHC-DR-associated protein CD74, tissue factor pathway inhibitor-2, and neuroserpin were much higher in MSC than in fibroblasts, even in the presence of large interindividual variations. Those of adrenomedullin, apolipoprotein D, C-type lectin superfamily member-2, collagen type XV alpha1, CUG triplet repeat RNA-binding protein, matrix metalloproteinase-1, protein tyrosine kinase-7, and Sam68-like phosphotyrosine protein/T-STAR were lower in MSC than in fibroblasts. FACS analysis showed that cell surface expression of MHC-DR was also higher in MSC than in fibroblasts. MHC-DR expression decreased after osteogenic differentiation, whereas the expression of adrenomedullin-a potent stimulator of osteoblast activity-along with collagen XV alpha1 and apolipoprotein D increased after osteogenic differentiation. The marker genes identified in this study should be useful for characterization of MSC both in basic and clinical studies.

Point mutations in members of the serine proteinase inhibitor or serpin superfamily cause them to change shape, polymerise and be deposited in the tissues. This process is best seen in mutants of α 1 -antitrypsin within hepatocytes to cause periodic acid-Schiff (PAS) positive inclu- sions and cirrhosis. An identical process underlies the PAS positive inclusions of mutants of neuro- serpin within neurones to cause a dementia that we have called familial encephalopathy with neuroserpin inclusion bodies (FENIB). In both cases, there is a direct correlation between the molecular instability, the rate of intracellular polymer formation and the severity of disease. This process of polymerisation also explains the failure to secrete mutants of other members of the serpin superfamily - antithrombin, C1 inhibitor and α 1 -antichymotrypsin - to cause thrombosis, angio-oedema and emphysema, respectively. In view of the common mechanism underlying these conditions, we have grouped them together as the serpinopathies.

Serine protease inhibitors form a diverse family of proteins of which most members inhibit target serine proteases. Neuroserpin is a member of this family. Here, we have characterized neuroserpin in the nonmammalian species Xenopus laevis and found a high degree of aminoacid sequence conservation, especially of the reactive center loop, of the Xenopus protein compared to mammalian and chicken neuroserpin sequences, suggesting a conserved target specificity. Neuroserpin mRNA and protein were expressed throughout Xenopus development, while in the adult frog high mRNA expression was found in neuronal and neuroendocrine tissues, and the reproductive organs, and the neuroserpin protein was detected mainly in brain and pituitary. More specifically, in Xenopus pituitary neuroserpin mRNA was expressed higher in the neurointermediate lobe than in the pars distalis. At the protein level, we detected a 55-kDa neuroserpin protein in the pars nervosa, two neuroserpin proteins of 44- and 50-kDa in the melanotrope cells of the pars intermedia, and a 46-kDa product in the pars distalis. On the basis of its relatively high degree of sequence conservation and its expression pattern, we conclude that Xenopus neuroserpin may play an important physiological role, e.g. as a serine protease inhibitor during development, and for proper neuronal and neuroendocrine cell functioning.

Inhibitors of serine proteases (serpins) are important regulators of intracellular and extracellular proteolytic pathways, and they function by forming an irreversible complex with their substrate. Neuroserpin represents a neuroendocrine-specific serpin family member that is expressed in brain regions displaying synaptic plasticity. In this study, we explored the biosynthesis of endogenous neuroserpin in a neuroendocrine model system, namely the melanotrope cells of Xenopus intermediate pituitary. The biosynthetic activity of these cells can be physiologically manipulated (high and low production of the prohormone proopiomelanocortin in black and white animals, respectively), resulting from a synaptic plasticity in innervating hypothalamic neurons. We found that neuroserpin was also differentially expressed in the Xenopus intermediate, but not anterior, pituitary with a 3-fold higher mRNA and more than 30-fold higher protein expression in the active vs. the inactive melanotrope cells. Two newly synthesized glycosylated forms of the neuroserpin protein (47 and 50 kDa) were produced and secreted by the active cells. Intriguingly, neuroserpin was found in an approximately 130-kDa sodium dodecyl sulfate-stable complex in the active, but not in the inactive, melanotrope cells, which correlated with the high and low proopiomelanocortin expression levels, respectively. In conclusion, we report on the biosynthesis of neuroserpin in a physiological context, and we find that the induction of neuroserpin expression and the formation of the 130-kDa neuroserpin-containing complex are linked to neuroendocrine cell activation.

The dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) is caused by point mutations in the neuroserpin gene. We have shown a correlation between the predicted effect of the mutation and the number of intracerebral inclusions, and an inverse relationship with the age of onset of disease. Our previous work has shown that the intraneuronal inclusions in FENIB result from the sequential interaction between the reactive centre loop of one neuroserpin molecule with b-sheet A of the next. We show here that neuroserpin Portland (Ser52Arg), which causes a severe form of FENIB, also forms loop-sheet polymers but at a faster rate, in keeping with the more severe clinical phenotype. The Portland mutant has a normal unfolding transition in urea and a normal melting temperature but is inactive as a proteinase inhibitor. This results in part from the reactive loop being in a less accessible conformation to bind to the target enzyme, tissue plasminogen activator. These results, with those of the CD analysis, are in keeping with the reactive centre loop of neuroserpin Portland being partially inserted into b-sheet A to adopt a conformation similar to an intermediate on the polymerization pathway. Our data provide an explanation for the number of inclusions and the severity of dementia in FENIB associated with neuroserpin Portland. Moreover the inactivity of the mutant may result in uncontrolled activity of tissue plasminogen activator, and so explain the epileptic seizures seen in individuals with more severe forms of the disease.