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Leukocyte elastase (LE) is a substrate for SerpinA3N, and it promotes neuropathic allodynia. (a,b) LE activity (a) or MMP9 activity (b), as assessed by in vitro fluorometric assays. AFU, arbitrary fluorescence units; n = 3, *P < 0.05 compared to protease alone, †P < 0.05 compared to BSA control. (c–f) Paw withdrawal thresholds (c,e) and AUC of responses to von Frey filaments (d,f) in LE-null (Elane−/−) and WT mice (c,d) or in WT mice after injection of the LE inhibitor Sivelestat or vehicle 8 d after SNI (e,f); n = 8–9 per group; *P < 0.05 compared to basal (c,d) or vehicle (e,f), †P < 0.05 between genotypes (c,d) or compared to vehicle (e,f). (g) Elane mRNA expression in different cell types in vitro or tissues from WT mice. (h,i) Quantification (h) and staining (i) of LE activity in L4 DRGs of Elane−/− and WT mice; n = 4–5 experiments, 4 mice per data point; *P < 0.05 compared to sham, †P < 0.05 between genotypes, one-way ANOVA, post hoc Tukey's test. Scale bars, 100 μm. (j) Mechanical allodynia in Rag2−/− and WT mice after adoptive transfer of WT- or LE-deficient T cells 6 d before SNI; n = 10 mice per group; *P < 0.05 compared to basal values, †P < 0.05 compared to Rag2−/− mice without T cell transfer. Unless otherwise indicated, we used two-way ANOVA of repeated measures, Tukey's post hoc test. Error bars are means ± s.e.m.

Leukocyte elastase (LE) is a substrate for SerpinA3N, and it promotes neuropathic allodynia. (a,b) LE activity (a) or MMP9 activity (b), as assessed by in vitro fluorometric assays. AFU, arbitrary fluorescence units; n = 3, *P < 0.05 compared to protease alone, †P < 0.05 compared to BSA control. (c–f) Paw withdrawal thresholds (c,e) and AUC of responses to von Frey filaments (d,f) in LE-null (Elane−/−) and WT mice (c,d) or in WT mice after injection of the LE inhibitor Sivelestat or vehicle 8 d after SNI (e,f); n = 8–9 per group; *P < 0.05 compared to basal (c,d) or vehicle (e,f), †P < 0.05 between genotypes (c,d) or compared to vehicle (e,f). (g) Elane mRNA expression in different cell types in vitro or tissues from WT mice. (h,i) Quantification (h) and staining (i) of LE activity in L4 DRGs of Elane−/− and WT mice; n = 4–5 experiments, 4 mice per data point; *P < 0.05 compared to sham, †P < 0.05 between genotypes, one-way ANOVA, post hoc Tukey's test. Scale bars, 100 μm. (j) Mechanical allodynia in Rag2−/− and WT mice after adoptive transfer of WT- or LE-deficient T cells 6 d before SNI; n = 10 mice per group; *P < 0.05 compared to basal values, †P < 0.05 compared to Rag2−/− mice without T cell transfer. Unless otherwise indicated, we used two-way ANOVA of repeated measures, Tukey's post hoc test. Error bars are means ± s.e.m.

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Neuropathic pain is a major, intractable clinical problem and its pathophysiology is not well understood. Although recent gene expression profiling studies have enabled the identification of novel targets for pain therapy, classical study designs provide unclear results owing to the differential expression of hundreds of genes across sham and nerve...

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... representative images shown in the manuscript were created by transformation to the LUT range. Values in Figure 4h were calculated as the percentage change over values derived from sham-treated WT mice, and negative values in Elane −/− mice are indicative of basal LE activity in WT sham-treated mice. The experimenter was blinded to the identity of the mice from which the DRGs were derived for measurement of LE activity. ...

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... HNE is also secreted by a variety of other immune cells, namely, MDSCs, macrophages, and lymphocytes. 31,32 As a matter of fact, HNE is upregulated in numerous cancer types. The HNE levels appear significantly elevated in the setting of cancer, even when compared to non-malignant inflammatory diseases with the exception of cystic fibrosis. ...
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Current chemotherapies suffer low specificity and sometimes drug resistance. Neutrophil elastase activity in cancer is associated with poor prognosis and metastasis settlement. More generally, tumors harbor various and persistent protease activities unseen in healthy tissues. In an attempt to be more specific, we designed prodrugs that are activatable by neutrophil elastase. Upon activation, these alkoxyamine-based drugs release cytotoxic alkyl radicals that act randomly to prevent drug resistance. As a result, U87 glioblastoma cells displayed high level caspase 3/7 activation during the first hour of exposure in the presence of human neutrophil elastase and the prodrug in vitro. The apoptosis process and cell death occurred between 24 and 48 h after exposure with a half lethal concentration of 150 μM. These prodrugs are versatile and easy to synthetize and can be adapted to many enzymes.
... Differential expression analysis between DOLs and the rest of the oligodendrocytes identified 26 genes significantly upregulated by DOLs (adjusted P < 0.01 and log 2 fold change (log 2 FC) >1; Fig. 2f), encompassing both genes related to immune signaling and non-immune-related genes. Immune-related genes included Serpina3n, a serine protease inhibitor related to immune proteases 21,22 ; the complement component C4b; several major histocompatibility complex I (MHC-I) genes (H2-D1, H2-K1 and B2m); and the cytokine Il33, which was previously shown to be expressed by oligodendrocytes under acute injury 23 . Among the non-immune upregulated genes, several have been previously linked to neuroinflammation, such as Klk6 (ref. ...
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Alzheimer’s disease (AD) is a complex neurodegenerative disease, perturbing neuronal and non-neuronal cell populations. In this study, using single-cell transcriptomics, we mapped all non-immune, non-neuronal cell populations in wild-type and AD model (5xFAD) mouse brains. We identified an oligodendrocyte state that increased in association with brain pathology, which we termed disease-associated oligodendrocytes (DOLs). In a murine model of amyloidosis, DOLs appear long after plaque accumulation, and amyloid-beta (Aβ) alone was not sufficient to induce the DOL signature in vitro. DOLs could be identified in a mouse model of tauopathy and in other murine neurodegenerative and autoimmune inflammatory conditions, suggesting a common response to severe pathological conditions. Using quantitative spatial analysis of mouse and postmortem human brain tissues, we found that oligodendrocytes expressing a key DOL marker (SERPINA3N/SERPINA3 accordingly) are present in the cortex in areas of brain damage and are enriched near Aβ plaques. In postmortem human brain tissue, the expression level of this marker correlated with cognitive decline. Altogether, this study uncovers a shared signature of oligodendrocytes in central nervous system pathologies.
... Interestingly, SerpinA3N regulates neither glial activation nor infiltration of immune cells into DRGs but inhibits LE secretion from T cells infiltrating the DRG after nerve injury. Because LE is a critical modulator in neuropathic pain (see the previous section discussing LE), the findings indicate transient Ser-pinA3N expression might be involved in the chronification of pain via reciprocal interaction between T cells and primary nociceptive neurons [99]. ...
Article
Pain is an unpleasant sensation associated with injury, inflammation, and infection. It has been demonstrated that communication between immune cells and neurons plays a vital role in pain and pain-related diseases (e.g. multiple sclerosis, osteoarthritis, irritable bowel syndrome). Growing data from preclinical and clinical studies have established that the bilateral regulations between peripheral immune cells and nociceptive neurons could be beneficial or detrimental for the development of pain and immune defense. We here review the mechanisms underlying neuroimmune crosstalk between circulating immune cells (e.g. macrophages, T cells, mast cells, neutrophils, monocytes) and nociceptors in the peripheral nervous system and the spinal cord. Deciphering the mechanisms by which neuroimmune interaction integrates neuronal inputs and immune responses helps to understand the pathogenesis of pain-related diseases and develop effective medications.
... TREM2 and the transmembrane immune signaling adaptor (TYRO protein tyrosine kinase-binding protein, TYROBP) complex modulate pro-inflammatory cytokines (e.g., tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and IL-6) and trigger phagocytosis. Microglial stimuli of apoptotic neurons activate this mechanism [44,45]. Our spatial gene expression analysis showed that the expression of Trem2 and Tyrobp, inducing microglial activation, was upregulated specifically in WT mice, but not in SynCAM3 KO mice ( Figure 5). ...
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Synaptic cell adhesion molecules (SynCAMs) play an important role in the formation and maintenance of synapses and the regulation of synaptic plasticity. SynCAM3 is expressed in the synaptic cleft of the central nervous system (CNS) and is involved in the connection between axons and astrocytes. We hypothesized that SynCAM3 may be related to the astrocytic scar (glial scar, the most important factor of CNS injury treatment) through extracellular matrix (ECM) reconstitution. Thus, we investigated the influence of the selective removal of SynCAM3 on the outcomes of spinal cord injury (SCI). SynCAM3 knock-out (KO) mice were subjected to moderate compression injury of the lower thoracic spinal cord using wild-type (WT) (C57BL/6JJc1) mice as controls. Single-cell RNA sequencing analysis over time, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and immunohistochemistry (IHC) showed reduced scar formation in SynCAM3 KO mice compared to WT mice. SynCAM3 KO mice showed improved functional recovery from SCI by preventing the transformation of reactive astrocytes into scar-forming astrocytes, resulting in improved ECM reconstitution at four weeks after injury. Our findings suggest that SynCAM3 could be a novel therapeutic target for SCI.
... Specie-specific Serpina3n/SERPINA3 performs important physiological roles during both quiescent and pathological states by inhibiting attributed proteases [15]. Reported functions of Serpina3n/SERPINA3 during various pathologies especially during CNS calamities have made them emerging aspirations for research studies [16,17]. Upregulated expression of Serpina3n (by reactive astrocytes [18] or neurons [19]) during CNS (central nervous system) insults reduces neuronal damage by protease inhibition [17] and has been proposed as a biomarker of astrogliosis [18], while SERPINA3 still needs validation for such scenario. ...
... Analysis of infarct volume illustrated that SERPINA3 was lacking behind in curative effect. The prime reason behind pronounced therapeutical efficacy of Serpina3n can be its aptness to inhibit a wide range of serine proteases, i.e., MMP-2, MMP-9, leukocyte elastase, and granzyme B which contribute to the neuronal damage [11,16,19,38,39]. Postischemic-elevated granzyme B levels pro-mote neuronal death not only through activation of cellular caspases, i.e., caspase-3, PARP, lamins, HSP-70, and Bid [10,11], but also through a noikis [11,40,41]. ...
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Ischemic stroke is a devastating CNS insult with few clinical cures. Poor understanding of underlying mechanistic network is the primary limitation to develop novel curative therapies. Extracellular accumulation of granzyme B subsequent ischemia promotes neurodegeneration. Inhibition of granzyme B can be one of the potent strategies to mitigate neuronal damage. In present study, we investigated the effect of murine Serpina3n and human (homolog) SERPINA3 against cerebral ischemia through granzyme B inactivation. Recombinant Serpina3n/SERPINA3 were expressed by transfected 293 T cells, and eluted proteins were examined for postischemic influence both in vitro and in vivo. During in vitro test, Serpina3n was found effective enough to inhibit granzyme B, while SERPINA3 was ineffectual to counter cytotoxic protease. Treatment of hypoxic culture with recombinant Serpina3n/SERPINA3 significantly increased cell viability in dosage-dependent manner, recorded maximum at the highest concentration (4 mM). Infarct volume analysis confirmed that 50 mg/kg dosage of exogenous Serpina3n was adequate to reduce disease severity, while SERPINA3 lacked behind in analeptic effect. Immunohistochemical test, western blot analysis, and protease activity assay’s results illustrated successful diffusion of applied protein to the ischemic lesion and reactivity with the target protease. Taken together, our findings demonstrate therapeutic potential of Serpina3n by interfering granzyme B-mediated neuronal death subsequent cerebral ischemia.
... These genes included Scgb1a1 (secretoglobin family 1A member 1, fold change = 41.4), Pdk4 (Pyruvate dehydrogenase lipoamide kinase isozyme 4, fold change = 2.8) and Serpina3n (serine protease inhibitor A3N, fold change = 2.7), etc. [28][29][30] In total, RNA-Seq identified 63 genes (40 up-and 23 down-regulated), which showed over 10-fold expression changes over 10 fold; 15 genes (4 up-and 11 down-regulated) had expression changes of 5-10 fold. We further summarize the detailed information regarding the top 20 up-or down-regulated genes in Tables 3 and 4, respectively. ...
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Background: Chronic postsurgical pain (CPSP) is common among patients receiving major surgeries. CPSP produces suffering in patients, both physically and mentally. However, the mechanisms underlying CPSP remain elusive. Here, a genome-wide expression profiling of ipsilateral spinal cord dorsal horn (SCDH) was performed to identify potential genes related with CPSP. Methods: A rat skin/muscle incision and retraction (SMIR) model was established to induce CPSP. Immunostaining was used to study glial cell and neuron activation in ipsilateral SCDH of SMIR model rats. RNA sequencing (RNA-Seq), combined with bioinformatics analysis, was undertaken to explore gene expression profiles. qPCR was applied to validate the expression of some representative genes. Results: The SMIR model rats developed persistent mechanical allodynia in ipsilateral hindpaw for up to 14 days. Ipsilateral SCDH of SMIR rats showed remarkable glial cell and neuron activation. A number of differentially expressed genes (DEGs) were identified in ipsilateral SCDH of SMIR rats by RNA-Seq. qPCR confirmed expression of some representative DEGs. Bioinformatics indicated that chemical synaptic transmission, sensory perception of pain and neuroactive ligand-receptor interaction were predominant functions. We compared our dataset with human pain-related genes and found that several genes exclusively participate in pain modulation and mechanisms. Conclusion: Our study provided novel understandings of the molecular mechanisms possibly contributing to CPSP. These findings may offer new targets for future treatment of CPSP.
... Serine protease inhibitors (SERPINs) are an evolutionary old, structurally conserved [52] superfamily consisting of at least 37 proteins divided into 16 clades (A-P) [53] in humans. They regulate coagulation, inflammation and wound healing by inhibiting protease activity or function as chaperones [54], and their dysregulation is associated with pathologies including inflammation and cancer [55][56][57]. In our studies, several serpin-clade A members-1, 1A, 1C (alpha-1-antitrypsin), 3B (alpha-1-antichymotrypsin), 3C, 3K (kallikrein inhibitor), 3M, 3N, and 10 (protein Z-dependent protease inhibitor)-were elevated at least two-fold in at least one study. ...
... Furthermore, SerpinA3 overexpression decreased cell adhesion to the extracellular matrix and to neighboring cells and protected them from apoptosis [58]. SerpinA-3N was upregulated upon injury [54], including hypoxia [58], and could serve as a marker of the transition of AKI to CKD [62]. Thus, especially SerpinA-3N seems to be a ubiquitous stress-response protein involved both in cancer protection from therapy and renal ischemia as well as AKI-to-CKD transition in the kidney. ...
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The prevailing general view of acute-phase proteins (APPs) is that they are produced by the liver in response to the stress of the body as part of a systemic acute-phase response. We demonstrated a coordinated, local production of these proteins upon cell stress by the stressed cells. The local, stress-induced APP production has been demonstrated in different tissues (kidney, breast cancer) and with different stressors (hypoxia, fibrosis and electromagnetic heat). Thus, this local acute-phase response (APR) seems to be a universal mechanism. APP production is an ancient defense mechanism observed in nematodes and fruit flies as well. Local APP production at the tissue level is also supported by sporadic literature data for single proteins; however, the complex, coordinated, local appearance of this stress response has been first demonstrated only recently. Although a number of literature data are available for the local production of single acute-phase proteins, their interpretation as a local, coordinated stress response is new. A better understanding of the role of APPs in cellular stress response may also be of diagnostic/prognostic and therapeutic significance.
... SERPINA3 upregulation in SZ could be a compensatory response to chronic inflammation [65]. However, SERPINA3 inhibits the catalytic activity of leukocyte elastase [67], a protease that cleaves ICAM1 which in turn favors attachment of leukocytes to endothelium [68]. Hence, elevated SERPINA3 in blood-vesselassociated astrocytes could be cooperating with CD163þ CNS infiltration in the SZ-high-inflammation group [63 && ]. ...
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Purpose of review: The vascular hypothesis of schizophrenia (SZ) postulates that brain endothelial dysfunction contributes to brain pathophysiology. This review discusses recent evidence for and against this hypothesis, including data related to blood-brain barrier (BBB), brain endothelium, and brain blood supply, to provide a critical weighed update. Recent findings: Different studies report a consistent proportion of SZ patients showing increased BBB permeability, reflected by higher levels of albumin in the cerebral spinal fluid. Of note, this was not a result of antipsychotic medication. The high inflammatory profile observed in some SZ patients is strongly associated with increased BBB permeability to circulating immune cells, and with more severe cognitive deficiencies. Also, sex was found to interact with BBB integrity and permeability in SZ. The strongest independent genetic association with SZ has been identified in FZD1, a hypoxia-response gene that is 600-fold higher expressed in early development endothelium as compared to adult brain endothelium. Regarding brain blood supply, there is evidence to suggest alterations in proper brain perfusion in SZ. Nonetheless, ex-vivo experiments suggested that widely used antipsychotics favor vasoconstriction; thus, alterations in cerebral perfusion might be related to the patients' medication. Summary: In some patients with SZ, a vulnerable brain endothelium may be interacting with environmental stressors, such as inflammation or hypoxia, converging into a more severe SZ symptomatology. Gene expression and performance of human brain endothelium could vary along with development and the establishment of the BBB; therefore, we encourage to investigate its possible contribution to SZ considering this dynamic context.
... These T cells were shown to release leukocyte elastase, which is a serine protease that interacts with and sensitizes sensory neurons. Inhibiting leukocyte elastase with the specific inhibitor sivelestat, which is an approved drug in Southeast Asia for the treatment of acute lung injury, causes a reduction of nerveinjury-induced neuropathic pain in vivo [65]. In another study by the same group, sivelestat was also found to reduce diabetes-induced neuropathic pain and cancer pain in respective rodent models [66]. ...
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Around 20% of the American population have chronic pain and estimates in other Western countries report similar numbers. This represents a major challenge for global health care systems. Additional problems for the treatment of chronic and persistent pain are the comparably low efficacy of existing therapies, the failure to translate effects observed in preclinical pain models to human patients and related setbacks in clinical trials from previous attempts to develop novel analgesics. Drug repurposing offers an alternative approach to identify novel analgesics as it can bypass various steps of classical drug development. In recent years, several approved drugs were attributed analgesic properties. Here, we review available data and discuss recent findings suggesting that the approved drugs minocycline, fingolimod, pioglitazone, nilotinib, telmisartan, and others, which were originally developed for the treatment of different pathologies, can have analgesic, antihyperalgesic, or neuroprotective effects in preclinical and clinical models of inflammatory or neuropathic pain. For our analysis, we subdivide the drugs into substances that can target neuroinflammation or substances that can act on peripheral sensory neurons, and highlight the proposed mechanisms. Finally, we discuss the merits and challenges of drug repurposing for the development of novel analgesics.
... As BQTX demonstrated a strong inhibitory ability against elastase, and inhibitors of elastase display analgesic functions [38], we further investigated the analgesic abilities of BQTX using mouse models. A plantar subcutaneous injection of 5% formalin in the mice induced nociceptive behavior, which occurred in two phases. ...
... Recent research has also shown that secreted modular calcium-binding protein 1 (SMOC1) has a Kazal domain that binds to thrombin and potentiates its activity [52]. Neutrophil elastase is associated with pain and its inhibitors are considered as painkillers [38]. As an elastase inhibitor, BQTX showed a strong ability to inhibit pain-related behaviors in several animal models (Fig. 6B, C). ...
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Multiple representatives of eulipotyphlan mammals such as shrews have oral venom systems. Venom facilitates shrews to hunt and/or hoard preys. However, little is known about their venom composition, and especially the mechanism to hoard prey in comatose states for meeting their extremely high metabolic rates. A toxin (BQTX) was identified from venomous submaxillary glands of the shrew Blarinella quadraticauda. BQTX is specifically distributed and highly concentrated (~ 1% total protein) in the organs. BQTX shares structural and functional similarities to toxins from snakes, wasps and snails, suggesting an evolutional relevancy of venoms from mammalians and non-mammalians. By potentiating thrombin and factor-XIIa and inhibiting plasmin, BQTX induces acute hypertension, blood coagulation and hypokinesia. It also shows strong analgesic function by inhibiting elastase. Notably, the toxin keeps high plasma stability with a 16-h half-life in-vivo, which likely extends intoxication to paralyze or immobilize prey hoarded fresh for later consumption and maximize foraging profit.