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

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Review

Physiological and pathological functions of neuroserpin: Regulation of cellular responses through multiple mechanisms Tet Woo Lee a,∗ , Vicky W.K. Tsang a , Evert Jan Loef a,b , Nigel P. Birch a,c,∗ a b c

School of Biological Sciences and Centre for Brain Research, University of Auckland, Auckland, New Zealand Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand Brain Research New Zealand, Rangahau Roro Aotearoa, Auckland, New Zealand

a r t i c l e

i n f o

Article history: Received 14 March 2016 Received in revised form 9 September 2016 Accepted 12 September 2016 Available online xxx Keywords: Protease inhibitor Tissue plasminogen activator Synaptic plasticity Neuroprotection Alzheimer’s disease Brain cancer

a b s t r a c t 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. © 2016 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Neuroserpin as an inhibitory serpin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Neuroserpin expression in neural, endocrine and immune cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Physiological functions of neuroserpin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1. Involvement of neuroserpin in behaviour, synaptic plasticity and neuronal function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2. Contributions of neuroserpin to neuronal survival and effects on the neurovascular unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Neuroserpin in disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.1. Familial encephalopathy with neuroserpin inclusion bodies: a neurological disease caused by neuroserpin mutations . . . . . . . . . . . . . . . . . . . . 00 5.2. Neuroserpin in Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.3. Neuroserpin in brain cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The distinct inhibitory mechanism of neuroserpin – formation of a short-lived acyl-enzyme complex with tPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Structural features of neuroserpin that may contribute to its instability and unusual inhibitory kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Neuroserpin – a temporal inhibitor to regulate pericellular proteolytic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Correspondence authors at: School of Biological Sciences, Level 2, Thomas Building, University of Auckland, 3a Symonds Street, Auckland 1010, New Zealand. E-mail addresses: [email protected] (T.W. Lee), [email protected] (N.P. Birch). http://dx.doi.org/10.1016/j.semcdb.2016.09.007 1084-9521/© 2016 Elsevier Ltd. All rights reserved.

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1. Introduction Neuroserpin (serpin I1) was originally identified as an axonallysecreted protein from cultured chicken dorsal root ganglion neurons grown in a compartmentalised system, and was later identified as a member of the serpin superfamily [1]. In this review, we will summarise the literature surrounding the physiological roles of neuroserpin, its contribution to neurological disease and highlight its unusual inhibitory characteristics.

2. Neuroserpin as an inhibitory serpin Neuroserpin (serpin I1) is a member of the serine protease inhibitor or serpin superfamily, a large family of proteins that share a universal conformation, the ‘serpin fold’ – a core of three ␤-sheets that are surrounded by a number of ␣-helices (8–9 being the most common) [2]. While not all serpins function as protease inhibitors, those that do so share a unique inhibitory mechanism. A defining feature of serpins, which also leads to their name, is their ability to inhibit serine proteases through a unique inhibitory mechanism [2]. This involves a conformational change in the structure of the molecule and the formation of a binary covalent complex between the serpin and its target protease. A prominent exposed loop called the reactive centre loop (RCL) in the serpin acts as ‘bait’ for a target protease and is the main determinant of its inhibitory specificity. Initial indications that neuroserpin was an inhibitory serpin came from identification of an inhibitory ‘hinge’ sequence in the neuroserpin RCL, with the presence of an arginine residue at the P1 site of the RCL suggesting that neuroserpin was likely to inhibit trypsin-like proteases [1]. Biochemical evidence subsequently showed that neuroserpin strongly inhibited the protease tissue plasminogen activator (tPA) in both the single-chain and two-chain forms, although the two-chain form is inhibited more strongly [3,4]. Urokinase plasminogen activator (uPA), trypsin and NGF-␥ were also inhibited but at efficiencies 1–2 orders of magnitude less and there was weak inhibition of plasmin and thrombin [3,4]. As analyses of expression patterns in the central nervous system indicated that neuroserpin and tPA are largely co-expressed [3,5,6], the physiological target of neuroserpin is considered to be tPA [3,4,7]. This is supported by in vivo data showing that tPA activity levels are decreased in the brains of transgenic mice engineered to over-express neuroserpin [8].

3. Neuroserpin expression in neural, endocrine and immune cells Analyses of expression in chicken, mouse and human tissues indicate that neuroserpin transcripts and protein are largely restricted to the nervous system, both during development and in the adult [1,3,5]. Expression was detected from an early stage in neuronal development of the mouse (E10), increasing to a maximal level perinatally before declining to a moderate level in the adult [5]. Developmentally, this corresponds to neuroserpin first being detected during the period of neuronal migration, expression continuing during axonal outgrowth and peaking during late development processes such as the synapse formation and remodelling. In the brain, neuroserpin expression is present in cells that were identified as neurons [3,5,6]. Neuroserpin expression is not, however, restricted to a particular neuronal type [5,6]. In the dentate gyrus subgranular zone of the adult rat, which is a site of adult neurogenesis, neuroserpin expression was detected in post-mitotic immature neurons but not in mature neurons [9], consistent with a role in the maturation in these new neurons prior to them becoming integrated into existing circuits. Expression has also been noted

in a few populations of non-neuronal cells such as the ependyma and epithelial cells of choroid plexus [3,6]. Low levels of neuroserpin expression has been reported in a number of other tissues [3,5]. Of particular interest are recent reports of neuroserpin expression in the endocrine and immune systems. The presence of neuroserpin in endocrine tissue [10,11] may reflect a common function with the nervous system, as some endocrine cells including those in the adrenal medulla and parts of the pituitary have a neuronal origin, while even non-neural endocrine cells share many properties of neurons [12]. Neuroserpin has also been shown to be present in cells of the immune system, including the phagocytotic macrophages, the neuronal counterparts the microglia, T cells and antigen-presenting dendritic cells [13,14]. Modifying roles for exogenous neuroserpin and neuroserpin peptides have been described on inflammatory responses and plaque growth [15,16] and macrophage infiltration in pancreatic tumours [17]. Regulated expression of neuroserpin mRNA and secretion by T cells and/or antigen-presenting cells following activation [18] may also parallel neuronal function through regulation of pericellular proteolysis at the immunological synapse formed between antigen presenting cells and T cells [19]. Secretion of neuroserpin from a vesicular compartment [10,20,21] in both neuronal and immune cells would be consistent with an activity-dependent regulatory function at neuronal and immunological synapses. While functional data on the role of neuroserpin in the immune system is limited, a recent study showed that neuroserpin can regulate the tPA/plasmin-mediated cleavage of the chemokine CCL21 to release soluble CCL21 that can then act as a chemotactic agent [22].

4. Physiological functions of neuroserpin 4.1. Involvement of neuroserpin in behaviour, synaptic plasticity and neuronal function Insight into the function of neuroserpin in vivo has come from the study of ThycNS transgenic mice, which over-express neuroserpin postnatally in neurons several fold higher than wild-type mice, and neuroserpin-knockout mice [23]. Detailed behavioural analysis of ThycNS and neuroserpin-knockout mice revealed changes in the exploratory behaviour of these mice, with both types of mice exhibiting increased phobic and anxiety-like responses, suggesting that neuroserpin may regulate emotional behaviour [23]. It is noteworthy that the behavioural changes followed a U-shaped response curve, with both increases and decreases in neuroserpin levels leading to similar responses. In addition, while the mechanism leading to the behavioural changes could not be determined, the lack of any change in tPA activity levels in the neuroserpinknockout mice led the researchers to suggest that it may not involve regulation of tPA proteolytic activity. These behavioural effects suggest neuroserpin may play a role in synaptic plasticity. A number of pieces of indirect evidence support this possibility. Firstly, there are high levels of neuroserpin expression late in neuronal development, and in adult brain areas associated with plasticity [3,5,6]. In addition, the expression and secretion of neuroserpin are regulated by neuronal activity [20,21]. Altered neuroserpin expression has also been found to occur in the activity-dependent remodelling processes that occur in the developing visual cortex [24]. In another study, localized overexpression of neuroserpin in the adult rat hippocampus did not cause any changes in learning and memory, although it did result in changes in expression levels of the postsynaptic scaffolding protein PSD-95 [25]. There is also considerable evidence that tPA, neuroserpin’s inhibitory target, carries such functions [26,27]. Neuroserpin has also been identified as one

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of several genes associated with female mate preference behaviour in poeciliid fish using a behavioural genomics approach [28,29]. Studies in cell culture models suggest cellular mechanism that may underpin potential effects on synaptic plasticity. Roles for neuroserpin have been described for neurite growth in AtT20 cells [10] and PC12 cells [20,30], with both increased and decreased neurite outgrowth triggered by neuroserpin in different experimental settings, as well as for cell–cell adhesion in PC12 cells [31]. Overexpression of neuroserpin in cultures of dissociated hippocampal neurons has led to changes in the number and morphology of dendritic spines [32]. In all cases, little is known about the cellular mechanisms underlying these effects. The cellular effects of neuroserpin on both neurite outgrowth and cell–cell adhesion do not require the inhibition of tPA leading to the suggestion that neuroserpin may act to regulate cell–cell and cell-matrix interactions by signalling through a cell surface receptor [31]. A possible candidate is the LRP1 receptor, which is known to bind and internalize neuroserpin [33]. It has been shown that binding of ligands including tPA to LRP1 receptor leads to transactivation of neurotrophin TrkA receptors and induces neurite outgrowth in PC12 cells [34]. Notwithstanding possible non-inhibitory roles, neuroserpin has been shown to regulate tPA/plasmin-mediated proteolytic maturation of the neurotrophin proNGF to NGF in vitro [35]. In support of such a role in vivo, neuroserpin is co-expressed and co-secreted with these other players [35] and an association between increased proNGF levels and increased in neuroserpin expression (together with decreased plasmin and tPA expression) has been shown in Down’s syndrome brains [36].

4.2. Contributions of neuroserpin to neuronal survival and effects on the neurovascular unit The effects of tPA on neuronal survival have been studied in some detail, and a complex picture is emerging where tPA can act as either a neurotoxic or a neuroprotective factor depending on factors such as the level or form of tPA and/or downstream targets triggered [27,37]. As an inhibitor of tPA, neuroserpin has been shown to protect against neurotoxic effects of tPA in both in vivo and in vitro studies. In some of the earliest work in this area, Yepes and colleagues [38] found that administration of recombinant neuroserpin into the brain reduced infarct size in a rat MCA occlusion model of cerebral ischaemia, while cleaved neuroserpin lacking tPA-inhibitory activity had no effect. Further experimentation suggested that neuroserpin might act to protect against ECM degradation mediated by the tPA/plasmin system and thereby promote neuronal survival. In the same ischaemic model, intracisternal administration of neuroserpin prior to intravenous administration of tPA outside its normal therapeutic window was also found to reduce infarct size, with the results attributed to neuroserpin inhibiting the deleterious extravascular effects of the administered tPA [39]. In addition, mice with neuron-specific over-expression of neuroserpin (ThycNS mice) had smaller infarcts following MCA occlusion compared to wild-type mice [8]. Again, tPA and uPA activity were found to be reduced in the neuroserpin-over-expressors. This was attributed both to direct inhibition of tPA by neuroserpin, as well as a reduction in microglia activation. Lack of microglia activation prevented the further release of tPA and uPA by microglia and may in itself be beneficial for neuronal survival. Completing these results is a study on MCA occlusion in neuroserpin-knockout mice [40]. These mice were found to have increased infarct size and worse neurological scores, which were concomitant with an increase in microglia activation. While these protective effects are generally considered to be due to inhibition of tPA, exogenous neuroserpin has also been shown to reduce ischaemic damage in tPA-knockout mice [41].

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Apart from ischaemic death, neuroserpin administration has been shown to reduce lesion size following excitotoxic insult with NMDA in mice [42]. Neuroserpin has also been shown to protect against indirect neuronal death caused by seizures that spread throughout the brain following injection of the excitotoxin kainate into the brain [43]. The authors found that kainate injection into the amygdala led to a progressive increase in tPA activity in adjacent neuronal structures that was associated with the spread of seizures. Exogenous neuroserpin, acting to inhibit tPA activity, was found to delay seizure spread, preventing synchronous electrical activity and reducing the concomitant neuronal death. In addition to effects on infarct size, tPA has been shown to contribute to the opening of the blood-brain barrier following ischemia, and exogenously administered neuroserpin could also block this effect of tPA [44]. In a recent study, a tPA-mediated increase in blood-brain barrier permeability was shown to contribute to the progression of kainite-induced seizures [45]. Endogenous neuroserpin inhibits this effect of tPA, as neuroserpin-knockout mice were found to have more rapid seizure progression, while seizure progression was reduced in tPA-knockout and neuroserpin/tPA double knockout mice. These effects on the neurovascular unit were found to be mediated by increased PDGF receptor alpha (PDGFR␣) signalling [45], likely due to proteolytic activation of platelet-derived growth factor-CC (PDGF-CC) by tPA [46]. Another example of a contribution of neuroserpin to neuronal survival in vivo is the study on pmn/pmn mice which exhibit a dying-back motor neuron disease called progressive motor neuronopathy [47]. Neuronal degeneration in these mice is associated with increased plasminogen activator activity (both tPA and uPA). Overexpression of neuroserpin in these mice led to improved clinical symptoms and reduced neuronal degeneration, highlighting a contribution of plasminogen activators to neuronal damage linked to axonal degeneration and a protective function for neuroserpin. Intravitreal administration of exogenous neuroserpin has also been found to improve retinal function and reduced apoptosis in a mouse model of acute retinal ischemic/reperfusion-induced injury [48]. Neuroserpin treatment was linked to inhibition of the caspase-3 cell death signalling pathway. As in the study by Wu et al. [41], neuroserpin produced similar effects in both wild-type and tPA-deficient mice suggesting that neuroserpin was acting independently of tPA. The neuroprotective effects of neuroserpin and its mechanism of action have also been studied in neuronal cultures. For example, recombinant neuroserpin has been shown to reduce NMDA-mediated excitotoxic death in neuronal cultures [42]. This was linked to a reduced NMDA-mediated influx leading to the suggestion that neuroserpin may be acting to prevent the potentiation of NMDA receptor activity [42], which is known to be triggered by single-chain tPA [49,50]. Neuroserpin has also been shown to play a neuroprotective role during oxidative stress, with neuroserpin inhibiting hydrogen peroxide-induced neurotoxicity in cultured neurons by reducing apoptosis through the AKT-BCL-2 signalling pathway [51]. Other research has made use of oxygen and glucose deprivation (OGD) or oxygen-glucose deprivation and reoxygenation (OGD/R) in neuronal cultures to model ischaemic death. In two such studies, neuroserpin treatment was found to have a neuroprotective effect with the data implicating neuroserpinmediated inhibition of tPA as the mechanism of this effect [52,53]. In contrast, Wu et al. [41] found that preconditioning either hippocampal or cortical neuron cultures with neuroserpin resulted in concentration-dependent increases in neuronal survival following OGD but this effect also occurred in neurons from tPA-knockout mice. This study provides clear evidence that neuroserpin can also elicit neuroprotection independently of tPA, possibly through a mechanism involving inhibition of plasmin or uPA [41]. The levels of neuroserpin used in these treatment experiments were rather

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high at ∼0.1–1 ␮M and it remains unclear whether the levels of neuroserpin used would normally occur in a physiological context. High levels of neuroserpin would tilt the kinetics in favour of strong protease inhibition, even with the tendency of neuroserpin-tPA complexes to dissociate (see below) or for other proteases (such as plasmin or uPA) that neuroserpin inhibits poorly, and this may result in inhibitory effects of neuroserpin being detected when they otherwise would not occur. One recent study has shown that the survival of cultured astrocytes in an OGD/R model is enhanced by a significantly lower concentration of neuroserpin (∼0.1 nM) [54]. Investigation into the mechanism of this effect revealed that neuroserpin treatment inhibited the release of NO and TNF-␣, as well as inhibition of the NF-␬B. Some of the effects of neuroserpin could be blocked by an NF-␬B inhibitor, suggesting that neuroserpin acts upstream of NF-␬B. The involvement of tPA was not addressed in this study. In addition, it is unclear why such a low concentration of neuroserpin was capable of eliciting a neuroprotective response in this study, but not in the others. Overall, there is a body of evidence that neuroserpin is neuroprotective. Most data suggest that neuroserpin does so through inhibition of tPA, although there is also evidence of tPAindependent mechanisms. In addition, the bulk of the evidence show protective functions of neuroserpin following experimentally increased levels of neuroserpin. A number of these studies report that neuroserpin expression levels are upregulated upon the onset of the neurodegenerative process being studied [8,38,41,43], which argues in favour of neuroserpin being a physiological neuroprotective factor that is induced to protect neurons from degeneration. However, given that neuroserpin secretion and expression has been shown to be activity-dependent [20,21], it remains possible that these increases in neuroserpin levels occur merely as a consequence of uncontrolled neuronal excitation. Nevertheless, the data from neuroserpin-knockout mice do support a physiological importance of neuroserpin in protecting against ischemic damage [40] and maintaining the integrity of the blood-brain barrier [45]. 5. Neuroserpin in disease In addition to possible roles for neuroserpin as a neuroprotective factor, neuroserpin mutations have been shown to lead to a rare form of neurological disease called familial encephalopathy with neuroserpin inclusion bodies (FENIB). Potential links between neuroserpin and Alzheimer’s disease, schizophrenia [55,56] and a role in brain metastasis have also been described. The roles of neuroserpin in FENIB, Alzheimer’s disease and brain metastasis will be considered in more detail. 5.1. Familial encephalopathy with neuroserpin inclusion bodies: a neurological disease caused by neuroserpin mutations The structural flexibility of serpins that is required for their inhibitory mechanism can lead to dysfunction under certain circumstances. In particular, mutations of key residues in the serpin fold, especially those involved in the opening of ␤-sheet A, can make a serpin prone to form loop-sheet polymers [57,58]. Polymerisation results in a loss of function of the serpin, as the levels of the native functional serpin are reduced, while the machinery of the cells producing the serpin can easily be overwhelmed by the accumulation of large amounts of serpin polymers, resulting in cell death [57,58]. A serpinopathy caused by mutations in the neuroserpin gene was discovered not long after neuroserpin was first cloned [59]. This disease was named Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB) and leads to progressive neurodegeneration with symptoms of cognitive decline, epilepsy and dementia that become more severe as the disease progresses

[57–59]. A total of six different mutations causing FENIB are known [57,60]. These mutations are of conserved residues in the ‘shutter’ region of neuroserpin, which is involved in the opening of ␤-sheet A for RCL insertion. They disrupt key interactions in the shutter region and resulting in instability of the native protein [61–63]. Clinical findings and studies in animal and cell culture models indicate that the severity of the mutation (i.e. the degree of instability of the protein) correlates with the accumulation of neuroserpin polymers, as well as decreased age of onset and disease severity [21,57,61]. These studies indicate that polymerisation and inclusion body formation is not only sufficient to cause the disease, but is also the principle factor driving neurodegeneration and the symptoms of the disease. The mutant proteins, however, are also not readily secreted and tend to be poor inhibitors of tPA [21,64]. Given results showing that neuroserpin can block the seizure progression in animal models [43,45], neuroserpin loss-of-function may account for the symptoms of epilepsy that can occur in some individuals during early stages of the disease [45,64]. Several studies have investigated the clearance of the mutant neuroserpin molecules and cellular response to the accumulation of neuroserpin polymers. This research has revealed that the mutant neuroserpin molecules are mainly degraded by ERassociated degradation (ERAD) [65], however, when this pathway is overloaded, the mutant neuroserpin accumulates as ordered polymers in the ER. This does not activate the unfolded protein response (UPR) but instead triggers ER overload response (EOR) in which there is a calcium-dependent activation of NF-␬B signalling that may ultimately trigger cell death [66]. Other studies have revealed a relationship between sterol metabolism and clearance of mutant neuroserpin [67], and molecular players involved in ERAD-mediated degration of neuroserpin [68,69]. Recently, it has also shown that N-linked glycosylation reduces neuroserpin polymerisation [70] and the small molecule embelin can bind to and inhibit the polymerisation of neuroserpin [71].

5.2. Neuroserpin in Alzheimer’s disease One of the defining features of Alzheimer’s disease is the presence of extracellular plaques in the brain containing fibrils of amyloid-beta (A␤) protein. It has been hypothesised that these amyloid deposits are neurotoxic and play a major role in the pathogenesis of the disease [72]. Neuroserpin has been found to associate with A␤ in the amyloid plaques of patients with Alzheimer’s disease [73,74] while elevated levels of neuroserpin have been found in the cerebrospinal fluid and brain tissue [74,75], which may be due to activation of the thyroid hormone response system [76]. These findings have led to suggestions that neuroserpin may play a role in the development of the disease, with evidence to suggest that elevated neuroserpin could be either beneficial or detrimental. In terms of the former, biochemical studies showed that neuroserpin forms a binary complex with A␤ peptides, altering the oligomerisation pathway and reducing their toxicity [73]. On the other hand, the increased levels of neuroserpin in Alzheimer’s disease are associated with a concomitant decrease in tPA activity due to neuroserpin’s inhibitory activity, which may reduce plasminmediated clearance of A␤ [74]. Indeed, it has been shown that A␤42 that is injected into the brains of mice is cleared more rapidly in neuroserpin-deficient mice than in wild-type mice, and the knockout of the neuroserpin gene leads to decreased A␤40 and A␤42 levels, fewer and smaller amyloid plaques, as well as reduced memory deficits, in a mouse model of Alzeimer disease [77]. Consistent with the hypothesis that inhibition of tPA by neuroserpin underlies these changes, there was a greater level of tPA accumulation in the plaques of the neuroserpin knockout mice. Given the roles of tPA in synaptic plasticity, there have also been suggestions that changes

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in tPA and neuroserpin levels may have direct consequences on synaptic activity and neuroregeneration [74,75].

5.3. Neuroserpin in brain cancer Inhibitors of plasminogen activators including neuroserpin have recently been shown to play a critical role in brain metastasis [78]. To survive, cancer cells must block killing signals from astrocytes after they traverse the blood-brain barrier and then establish interactions with brain capillaries to undergo metastatic expansion. Neuroserpin has been found to shield metastatic cells from the plasminogen activator (PA)/plasmin system in the brain. Neuroserpin protects cancer cells from Fas-dependent cell death by death by inhibiting PA/plasmin-dependent cleavage of membranebound FasL to release active soluble FasL. Neuroserpin-mediated inhibition of PA/plasmin is also proposed to aid vascular co-option by blocking plasmin-mediated degradation of the cell adhesion molecule L1CAM.

6. The distinct inhibitory mechanism of neuroserpin – formation of a short-lived acyl-enzyme complex with tPA The preceding discussion highlights roles for neuroserpin in a range of physiological and pathological processes. In many cases these roles require the inhibitory activity of neuroserpin to block the enzymatic activity of the plasminogen activator, tPA. One key tPA substrate is plasminogen, which is activated by tPA and other plasminogen activators to form plasmin. While plasmin is best known for its proteolytic role in thrombolysis through the degradation of fibrin, there is increasing recognition of roles for this enzyme, particularly in its membrane-associated form, in the cleavage of a wide range of other molecules [79]. The two principal inhibitors of tPA are the serpins plasminogen activator inhibitor-1 (serpin E1) and neuroserpin. Why are there two serpins targeting tPA? A major difference between these two serpins is their mechanism of inhibition. Unlike most covalent serpin-protease complexes such as PAI-1-tPA, which are highly stable, neuroserpin-tPA complexes were shown to be unstable and dissociate with a time-scale of minutes (half-life about 10 min) instead of weeks (Fig. 1) [62,80,81]. Complex dissociation occurs by deacylation of the neuroserpin-tPA complex, resulting in the release of cleaved neuroserpin as well as functionally active tPA, and is predicted to occur more rapidly than the complexes can be cleared by internalisation [80]. There was no evidence for cleaved neuroserpin forming prior to complex formation, indicating that neuroserpin does not act as a pure substrate of tPA and instead neuroserpin-tPA interactions proceed through this inhibition/dissociation pathway. This has led to inhibition of tPA by neuroserpin to be described as a kinetic ‘stutter-step’, that is, a detectable intermediate step in the process of the cleavage of neuroserpin that causes a transient inhibition of tPA [80]. This means that neuroserpin neither acts as a pure inhibitor nor as a pure substrate of tPA. Differential inhibition of the different forms of tPA by neuroserpin reveals another level of regulatory complexity. While neuroserpin forms complexes with both single- and two-chain forms of tPA (sctPA and tctPA), tctPA-neuroserpin complexes form more rapidly but are only stable at acidic pH and dissociate quickly at physiological pH, while sctPA-neuroserpin complexes are more stable at physiological pH [80,82]. As cerebral spinal fluid is poorly buffered and decreases in pH in several pathological states, this pHdependence in the inhibitory kinetics of neuroserpin towards tPA may be important in vivo [82]. The finding that sctPA selectively modulates N-methyl-d-aspartate receptor signalling [45] also suggests that the differential inhibition of the two forms of tPA may be functionally relevant.

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These findings indicate that neuroserpin shows unusual kinetics for a serpin in terms of tPA inhibition. It is possible that neuroserpin-tPA complexes could be stabilised in vivo by an unidentified cofactor [80]. It is not uncommon among the serpins for their inhibitory activity to be modulated by a cofactor. For example, the conversion of PAI-1 to an inhibitory inactive latent conformation occurs within hours under physiology conditions unless stabilised by the cofactor vitronectin [83]. It may also be that the specific kinetics of neuroserpin’s inhibition of tPA are important biologically, and neuroserpin may have evolved to be only a transient inhibitor of tPA. In support of this, it has recently been shown that several evolutionarily-conserved residues in neuroserpin regulate the half-life of neuroserpin-tPA complexes [81]. A biological role for (only) short-term inhibition of tPA by neuroserpin has not been directly demonstrated, although one possibility is that this would allow some inhibition of tPA without causing deleterious effects on the neurovascular unit that have been shown to be triggered by tPA-serpin complexes such as tPA-PAI-1 complexes [84]. Alternatively, the contribution of neuroserpin to the physiological regulation of tPA activity levels may be less important than initially suspected. That is, while neuroserpin can function as an inhibitor tPA in vivo, this may not be its main or only function. Neuroserpin may have other protease targets for which it exhibits more traditional inhibitory kinetics. While it has been suggested that neuroserpin’s weak inhibition of plasmin may be physiologically important [41], biochemical data indicate neuroserpin acts as a substrate for plasmin with no evidence for the formation of plasmin-neuroserpin complexes [3,80] arguing against its role as a physiological inhibitor of plasmin. Neuroserpin does form complexes with uPA, thrombin and NGF-␥ [3] but no reports on the stability of these complexes are available. Similarly, while other targets of neuroserpin such as neurotrypsin, neurosin and neuropsin have been suggested [4], there have been no empirical reports indicating neuroserpin inhibits any other protease. Neuroserpin might also possess a function distinct from its inhibitory activity. Not all serpins inhibit proteases and such non-inhibitory serpins perform other functions. Even those that do act as inhibitors may carry additional non-inhibitory functions. If neuroserpin does possess a non-inhibitory function, cleavage by tPA may modulate its biological activity. Indeed, it has been suggested that a function for cleaved neuroserpin could explain its inhibitory kinetics [80].

7. Structural features of neuroserpin that may contribute to its instability and unusual inhibitory kinetics Structures of human neuroserpin in the native and cleaved conformations have been reported [62,63]. As expected, the structure of neuroserpin is the standard serpin fold, with three ␤-sheets and nine ␣-helices. As in other serpins, the RCL in native neuroserpin was found to be exposed and highly flexible [62,63]. The structure of cleaved neuroserpin showed complete insertion of the RCL into ␤-sheet A, similar to that observed for other serpins, and no clear structural reason for the instability of neuroserpin-tPA complexes was apparent [62]. Negative charges on one side of ␤-sheet A and flexibility in the helix C and D region suggests possible sites for intermolecular interactions [62,63]. Compared to other serpins, there is a loss of conserved residues in and adjacent to ␤-sheet A, which contribute the instability of neuroserpin and the propensity of even the wild-type to polymerise and undergo a conversion to a latent form [63]. Increased flexibility in these regions of neuroserpin is supported by biophysical data [85]. Through a combination of sequence analysis and mutagenesis studies, Lee at al. [81] have identified evolutionarily-conserved regions in neuroserpin that are essential for inhibition of tPA (Fig. 2). Interestingly, two distinct effects on tPA inhibition were revealed.

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Fig. 1. Pericellular regulation of the proteases tPA and plasmin by the serpins neuroserpin, PAI-1 and ␣2 -antiplasmin. Neuroserpin (NS) is an extracellular serpin that is secreted by the cell into the pericellular space where it can inhibit tPA activity and block cleavage of a range of substrates including plasminogen to block production of active plasmin. Another serpin, ␣2 -antiplasmin, can directly inhibit plasmin activity. The inhibitory kinetics of tPA inhibition by neuroserpin and PAI-1 are very different. The interaction between tPA and neuroserpin is short-lived, rapidly progressing to complete cleavage of neuroserpin and regeneration of active tPA [80]. In contrast PAI-1 (and ␣2 -antiplasmin) form stable essentially irreversible complexes with tPA (and plasmin). These complexes are typically stable for long periods and are cleared by internalisation and degradation.

neuroserpin-tPA complexes, suggesting that these may function as exosites for tPA binding. Residues in helices CD and E, which are located at the opposite end of the neuroserpin molecule, near the final location of tPA when in a covalent complex with neuroserpin, where found to be important in regulating the rate of complex dissociation. The unique omega loop in neuroserpin between s1B and s2B was also shown important for inhibitory activity of neuroserpin in this and a previous study [63]. Kinetic analysis indicated that deletion of this loop led to increased complex dissociation [81]. 8. Neuroserpin – a temporal inhibitor to regulate pericellular proteolytic activity

Fig. 2. Locations of conserved residues in neuroserpin that regulate inhibitory activity towards tPA. Several evolutionarily conserved residues in neuroserpin that contribute to tPA inhibition have been identified by sequence analysis and mutagenesis experiments [81]. These cluster in two regions of the neuroserpin molecule: (a) residues adjacent to the RCL that when mutated decrease association between neuroserpin and tPA and (b) residues close to the final location of tPA when in a covalent complex with neuroserpin that when mutated cause more rapid complex dissociation. The unique omega loop in neuroserpin that is important for inhibitory activity is shown as (c).

In summary, neuroserpin is a unique member of the serpin superfamily. It is widely expressed in the brain where it is associated with effects on emotional behaviour, synaptic plasticity and neuroprotection in a range of in vivo rodent and cellular models. It has been proposed to play a role in the development of Alzheimer’s disease and more recently in brain metastasis. Many of these functions are likely to be the result of regulating processing of substrates cleaved directly by tPA, or by plasmin following tPA-mediated activation of plasminogen. Future research is likely to identify additional substrates and processes regulated by the interactions of neuroserpin, tPA and plasmin. Perhaps an even greater challenge for the field will be understanding if, and how, the transient inhibition of tPA by neuroserpin regulates biological responses and the interplay between neuroserpin and PAI-1 in those systems where both inhibitors are co-expressed. Acknowledgements

Residues in ␤-sheet C, which is located close to the RCL, were found to be important for acylation of neuroserpin by tPA to form

We apologise in advance to all the investigators whose research could not be appropriately cited owing to space limitations. This

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Please cite this article in press as: T.W. Lee, et al., Physiological and pathological functions of neuroserpin: Regulation of cellular responses through multiple mechanisms, Semin Cell Dev Biol (2016), http://dx.doi.org/10.1016/j.semcdb.2016.09.007