neuroserpin , or proteinase inhibitor 12 ( gene symbol serpini1 ) , was first found in cultured chicken neuronal axons and is predominantly expressed in human neurons of both the central and peripheral nervous systems .
sequence analysis has shown that neuroserpin is a new member of an old proteinase family , the serine proteinase inhibitor ( serpin ) superfamily .
a high neuroserpin expression level is detected during the late stage of neuronal development throughout the central nervous system ( cns ) ; however , it is only detected in some regions of the adult brain , such as the hippocampus , hypothalamus , cerebellum , amygdala and sympathetic nerves , where tissue - type plasminogen activator ( tpa ) mrna and/or protein has also been found , during the early stage of neurogenesis .
the spatial and temporal analysis of neuroserpin and tpa expression suggests a role for neuroserpin in maintaining the proteolytic balance in the cns , which is crucial for axonogenesis , synaptic connection , and synaptic plasticity .
the co - expression and preferential interaction with tpa indicates that neuroserpin is a selective inhibitor of tpa in the cns .
the active balance and interaction between these two proteinases are very important for both physiological and pathological events . under normal conditions , neuroserpin and tpa
both participate in processes such as learning , memory , behavior and the regulation of permeability between vascular compartments and the nervous system . during pathological courses , such as neurodegeneration , cerebral ischemia and seizures , neuroserpin displays neuroprotective effects by eliminating tpa deleterious proteolytic activity .
however , many members of the serpin superfamily have shown nonproteinase - inhibitory roles in many processes , such as hormone transport , protein folding , tumor progression , chromatin condensation , and blood pressure regulation .
neuroserpin also has roles that are independent or partly independent of its tpa inhibitory capacity . in this review
, we aim to clarify the tpa - independent roles of neuroserpin in the cns .
the serpin superfamily , which contains inhibitory and non - inhibitory members , is one of the earliest defined and largest protein superfamilies .
serpins are ubiquitously expressed in all branches of life , and are important regulators in many critical physiological processes , including coagulation , fibrinolysis , inflammation , metastasis and apoptosis .
the typical molecular structure of this family is very conserved and has been well defined , containing at least 30% amino acid sequence homology with the archetype protein , 1-antitrypsin . this protein is composed of eight or nine -helices and three -sheets , and characterized by an exposed peptide loop .
this loop , named the reactive center loop , is composed of 20 residues and works as a pseudosubstrate for the target enzyme .
the structural transformation between the reactive center loop and -sheet is critical for the proteinase inhibition capacity .
neuroserpin is a fully functional inhibitor of trypsin - type proteinases , preferentially reacting with tpa and to a minor extent with the urokinase - type plasminogen activator ( upa ) plasmin , but has no inhibitory activity towards thrombin .
ischemic stroke comprises approximately 88% of the total stroke , which is the leading cause of disability and the second largest contributor to mortality in the world .
it is often caused by an occlusion of a certain cerebral artery , and directly results in an absence of blood flow to this artery and the supplied brain tissue .
lack of oxygen and glucose could induce an energy metabolism disorder , which in turn triggers a collapse of ion gradients and an excessive release of excitotoxic neurotransmitters , such as glutamate and dopamine , ultimately leading to neuronal death and development of infarction .
one effective treatment for acute ischemic stroke is early thrombolytic therapy , and tpa is the only agent approved by the food and drug administration for thrombolytic treatment .
meanwhile , tpa is capable of activating matrix metalloproteinase , degrading laminin , interacting with the n - methyl - d - aspartate receptor , and converting plasminogen to plasmin , which also contributes to blood - brain barrier degradation .
these extravascular deleterious effects of tpa aggravate brain parenchyma injury when administrating tpa beyond its therapeutic window .
this damaging side effect of tpa has also been shown by experiments carried out in the presence of tpa deficiency ; these showed a significant reduction in ischemic lesion volume , an increase in neuronal survival , and a better long - term outcome .
moreover , neuroserpin over - expressing mice and adjuvant treatment with neuroserpin both provided neuroprotective effects and increased the therapeutic window of tpa administration .
based on these findings , it seems that the neuroprotective effect of neuroserpin following cerebral ischemia is associated with tpa , by balancing the proteolytic activity of tpa , or by regulating tpa mediated seizure spreading .
however , tpa has also been shown to rescue neurons from apoptosis induced by serum deprivation , and the elevation of tpa expression and activity induced ischemic tolerance and promoted neuronal survival in the murine hippocampus . in these models ,
additionally , a recent study showed that neuroserpin provides prominent neuroprotective effects in both wild - type and tpa - deficient mice during ischemic - hypoxic injury .
these findings suggest the existence of a tpa - independent neuroprotective role of neuroserpin during cerebral ischemia .
in fact , tpa is just one of the substrates of neuroserpin , which also reacts with upa and plasmin .
a previous study showed that upa was upregulated following ischemia , and that this could be inhibited by amiloride providing a better outcome .
a prior study indicated that plasmin was capable of activating protease - activated receptor-1 ( par-1 ) , and the activation of par-1 increased infarct volume by approximately 68% compared with par-1-null mice .
neuroserpin had already been showed to abrogate the excitotoxic neuronal death induced by plasmin and kainic acid .
there are 20 methionine residues in neuroserpin , and these contribute to the oxidative tolerance produced by this serpin protein .
this molecular structural character of neuroserpin explains a part of its neuroprotective effects against oxidative stress .
it was reported that neuroserpin possessed anti - inflammatory activity in systemic arteries , modifying th cell responses and reducing plaque growth .
since systemic inflammatory stimuli are detrimental to stroke outcomes , we assume that the anti - inflammatory activity of neuroserpin also plays a neuroprotective role in cerebral ischemic conditions .
the human neuroserpin gene is localized to chromosome 3q26 . despite its predominant expression in neurons ,
neuroserpin mrna and/or protein has also been detected in pancreas , heart , kidney , testis , placenta , liver , pituitary and adrenal glands .
the role of neuroserpin in these tissues is still unclear , and recent research has shown that neuroserpin participates in tumorigenesis and tumor migration , including in brain tumor tissue .
the genomic locus of the programmed cell death gene pdcd10 is close to that of serpini1 ( the gene encoding neuroserpin ) and the two genes are oriented in a head - to - head configuration .
serpini1-pdcd10 had been found to be regulated by the oncogenic transcription factor c - myc , and is possibly involved in cns diseases such as brain tumors .
moreover , there is evidence showing that the neuroserpin gene is downregulated in the brain tumor tissues and even absent in two brain cancer cell lines : u-87 mg and h4 .
these findings suggest that the neuroserpin gene is a cancer - associated gene , and that neuroserpin functions as a tissue - specific tumor - suppressor gene in the brain .
however , this tissue - specific tumor - suppressive character is not only observed with neuroserpin , but also with some other members of the serpin family , such as pancpin and maspin .
mapsin was downregulated in breast cancer , like neuroserpin , while the transgenic high - expression of mapsin resulted in a reduction of tumor progression . based on this
the currently poor understanding of how neuroserpin produces its tumor - suppressive effect needs to be addressed .
contrarily , high neuroserpin gene expression was detected in high - grade prostate cancer and hepatocellular carcinoma , where it was associated with a poorer outcome .
this result suggests a need for suspicion towards the potential use of neuroserpin as a chemotherapeutic agent .
tpa converts inactive plasminogen into active plasmin , which is capable of degrading most of the extracellular matrix , and tpa itself degrades laminin and activates matrix metalloproteinase contributing to the degradation of extracellular matrix .
sequentially , the breakdown of the extracellular matrix facilitates the invasion of cancer cells and enables tumor migration .
the absence of neuroserpin , a crucial inhibitor of tpa in the cns , may promote brain tumorigenesis .
the tpa - inhibitory function of neuroserpin is necessary in this tumor - suppressive process .
however , neuroserpin is not only a protease inhibitor : its expression has also been detected in pituitary and adrenal glands , suggesting a possible role for neuroserpin in the endocrine system .
both the att-20 cell line and pc12 cells , two endocrine cell lines , responded to altered neuroserpin expression levels by extending neurite - like outgrowths .
interestingly , compared with the parent and neuroserpin- overexpressing cell lines , there was only a small activation of tpa expression and no accumulation of tpa - neuroserpin complex was observed .
these results suggest an effect of neuroserpin in mediating neurite outgrowth , and that this process is tpa independent .
lee et al , using pc12 cells treated with nerve growth factor , showed that the high expression of neuroserpin induced an increased level of cell - cell adhesion and higher n - cadherin expression .
one mutation was in the reactive site of neuroserpin , blocking recognition by tpa ; the other mutation was at the hinge p14 serine residue , preventing reactive center loop insertion into -sheet a , which resulted in a noninhibitory neuroserpin .
this finding implied that neuroserpin could mediate cell - cell adhesion and alter the expression of n - cadherin , and this function was independent of its enzyme inhibitory activity .
these tpa - independent capabilities of neuroserpin , neurite outgrowth mediation and cell adhesion encouragement possibly result in the promotion of cancer cell metastasis and enhancement of the connection between tumor cells and local normal cells , promoting a secondary lesion .
although the innate protease inhibitory activity of neuroserpin provides us with a potential target for brain tumor treatment , the multiple tpa - independent roles of neuroserpin may lead an opposite result during tumorigenesis .
these processes all require neuronal plasticity , but the mechanisms underlying their effects remain undefined .
explorative behavior analysis revealed that these mice had a selective reduction of locomotor activity in a new environment , an anxiety - like response in the o - maze , and a neophobic response to novel objects .
surprisingly , zymographic analysis of brain extracts revealed unchanged tpa activity in neuroserpin - deficient mice .
this finding suggested that neuroserpin was a novel regulator of behavior and emotion , and that this role of neuroserpin was tpa independent .
it also indicated that other inhibitors , such as pai-1 , which was found to form complex with tpa in demyelinating multiple sclerosis lesions , contributed to the regulation of tpa activity in the brain and compensated for the defect of neuroserpin activity .
as mentioned above , noninhibitory neuroserpin is also capable of mediating n - cadherin expression . through the rho - family gtpases
, n - cadherin plays a key role in synapse formation , controlling dendritic spine maturation , and participating in long - term potentiation .
moreover , a higher level of neuroserpin expression can alter dendritic spine shape and induce an increase in the density of dendritic protrusions .
this suggests a regulatory role of neuroserpin in neuronal structural plasticity . additionally , neuroserpin has a partly tpa - independent activity in promoting neurite outgrowth , while the extension and remodeling of neurites also play critical roles in neuronal development and plasticity .
because neuronal plasticity is an essential composition for emotional and behavioral processes , the roles of neuroserpin in modulating n - cadherin expression , neuronal structural plasticity and neurite outgrowth reveal novel effects of neuroserpin in mediating emotion and behavior .
five point mutations of the neuroserpin gene , ser49pro , ser52arg , his338arg , gly392glu and gly392arg , have been shown to induce deposition of ordered neuroserpin polymers within the neuronal endoplasmic reticulum ( er ) .
fenib patients show clinical features such as cognitive dementia , tremor , seizures and progressive myoclonus . a very clear genotype - phenotype correspondence has been revealed in this disease , which can be explained by the rate of polymerization .
for example , compared with ser49pro , patients with the ser52arg mutation exhibit more rapid polymerization , a greater number of inclusion bodies , an earlier age of onset and more serious clinical symptoms .
although the lack of tpa - inhibitory activity of mutant neuroserpin , resulting in an imbalance of these two important proteins in the cns , underlies the etiology of fenib , the overload of er stress seems to be more harmful . normally , both active neuroserpin and neuroserpin - tpa complexes are recognized and internalized by membrane receptors , typically the ldl receptor - related protein , and degraded predominantly by er - associated degradation ( erad ) and autophagy . however , mutant neuroserpin significantly decelerates secretion and resists erad , resulting in a massive collection of neuroserpin polymers within the er , dramatically increasing er stress .
this er overload damage has also been identified in liver cirrhosis caused by mutant 1-antitrypsin , in which the sequestration of mutant 1-antitrypsin from inclusion bodies is a cell - protective mechanism to maintain er function .
furthermore , the cellular toxicity of mutant neuroserpin was confirmed by a fly fenib model and a rat fenib model . in these two models
the extent of neuroserpin polymer accumulation was directly linked to the severity of locomotor deficits and a selective dysfunction or loss of a specific neuronal population , respectively .
a recent study showed that neuroserpin polymers were capable of activating the calcium - dependent nuclear factor-b pathway .
because chronic activation of nuclear factor-b could induce cell death , the regulation of nuclear factor-b by neuroserpin polymers contributes to neuroserpin - associated cellular toxicity .
this finding reveals a novel mechanism used by neuroserpin to achieve motor and cognitive regulation , and suggests a new strategy for the treatment of fenib .
neuroserpin is an important protein in the cns . through its interaction with its main substrate , tpa , to maintain a proteolytic balance , neuroserpin plays essential roles in both physiological and pathological processes .
importantly , neuroserpin is not only a tpa inhibitor , it also reacts with upa and plasmin , and these interactions underlie neuroserpin 's protective effects during cerebral ischemia .
a better understanding of the roles of neuroserpin in the inflammatory system will widen its use in the treatment of adult stroke as well as neonatal hypoxic - ischemic encephalopathy . additionally , neuroserpin is a tpa - independent mediator of neurite - like outgrowth , cell - cell adhesion , and n - cadherin and nf-b expression .
the tpa - independent regulatory effect of neuroserpin participates in tumorigenesis , as well as emotional and cognitive processes .
further characterization of the multiple roles of neuroserpin and its polymers will be helpful for developing new strategies to treat the associated diseases .
 a number of studies have confirmed the existence of tissue - type plasminogen activator - independent roles of neuroserpin , a member of the serine protease inhibitor superfamily . in this review article 
 , we aim to clarify this role . 
 these unique roles of neuroserpin are involved in its neuroprotective effect during ischemic brain injury , its regulation of tumorigenesis , and the mediation of emotion and cognition through the inhibition of urokinase - type plasminogen activator and fibrinolysin , modification of th cells , reducing plaque formation , promoting process growth and intracellular adhesion , and altering the expression of cadherin and nuclear factor kappa b. 