Sex differences play a role in pain sensitivity, efficacy of analgesic drugs and prevalence of neuropathic pain, even if the underlying mechanisms are far from being understood. We demonstrate that male and female mice react differently to structural and functional changes induced by sciatic nerve ligature, used as model of neuropathic pain. Male mice show a gradual decrease of allodynia and a complete recovery while, in females, allodynia and gliosis are still present four months after neuropathy induction. Administration of 17β-estradiol is able to significantly attenuate this difference, reducing allodynia and inducing a complete recovery also in female mice. Parallel to pain attenuation, 17β-estradiol treated-mice show a functional improvement of the injured limb, a faster regenerative process of the peripheral nerve and a decreased neuropathy-induced gliosis. These results indicate beneficial effects of 17β-estradiol on neuropathic pain and neuronal regeneration and focuses on the importance of considering gonadal hormones also in clinical studies.

Ethyl acetate (EA), methanol (MeOH), and aqueous extracts of aerial parts of Anthemis tinctoria var. pallida (ATP) and A. cretica subsp. tenuiloba (ACT) were investigated for their phenol and flavonoid content, antioxidant, and key enzyme inhibitory potentials. All extracts displayed antiradical effects, with MeOH and aqueous extracts being a superior source of antioxidants. On the other hand, EA and MeOH extracts were potent against AChE and BChE. Enzyme inhibitory effects against tyrosinase and α-glucosidase were observed, as well. We also studied Anthemis extracts in an ex vivo experimental neurotoxicity paradigm. We assayed extract influence on oxidative stress and neurotransmission biomarkers, including lactate dehydrogenase (LDH) and serotonin (5-HT), in isolated rat cortex challenged with K+ 60 mM Krebs-Ringer buffer (excitotoxicity stimulus). An untargeted proteomic analysis was finally performed in order to explore the putative mechanism in the brain. The pharmacological study highlighted the capability of ACT water extract to blunt K+ 60 mM increase in LDH level and 5-HT turnover, and restore physiological activity of specific proteins involved in neuron morphology and neurotransmission, including NEFMs, VAMP-2, and PKCγ, thus further supporting the neuroprotective role of ACT water extract.

Type 2 diabetes is characterized by progressive β cell dysfunction, with lipotoxicity playing a possible pathogenetic role. Palmitate is often used to examine the direct effects of lipotoxicity and it may cause mitochondrial alterations by activating protein acetylation. However, it is unknown whether palmitate influences protein acetylation in β cells. We investigated lysine acetylation in mitochondrial proteins from INS-1E β cells (INS-1E) and in proteins from human pancreatic islets (HPI) after 24 h palmitate exposure. First, we confirmed that palmitate damages β cells and demonstrated that chemical inhibition of deacetylation also impairs INS-1E function and survival. Then, by 2-D gel electrophoresis, Western Blot and Liquid Chromatography-Mass Spectrometry we evaluated the effects of palmitate on protein acetylation. In mitochondrial preparations from palmitate-treated INS-1E, 32 acetylated spots were detected, with 13 proteins resulting over-acetylated. In HPI, 136 acetylated proteins were found, of which 11 were over-acetylated upon culture with palmitate. Interestingly, three proteins, glutamate dehydrogenase, mitochondrial superoxide dismutase, and SREBP-1, were over-acetylated in both INS-1E and HPI. Therefore, prolonged exposure to palmitate induces changes in β cell protein lysine acetylation and this modification could play a role in causing β cell damage. Dysregulated acetylation may be a target to counteract palmitate-induced β cell lipotoxicity.

We recently identified nitroxoline as a repurposed drug candidate in pancreatic cancer (PC) showing a dose-dependent antiproliferative activity in different PC cell lines. This antibiotic is effective in several in vitro and animal cancer models. To date, the mechanisms of nitroxoline anticancer action are largely unknown. Using shotgun proteomics we identified 363 proteins affected by nitroxoline treatment in AsPC-1 pancreatic cancer cells, including 81 consistently deregulated at both 24- and 48-hour treatment. These proteins previously unknown to be affected by nitroxoline were mostly downregulated and interconnected in a single highly-enriched network of protein-protein interactions. Integrative proteomic and functional analyses revealed nitroxoline-induced downregulation of Na/K-ATPase pump and β-catenin, which associated with drastic impairment in cell growth, migration, invasion, increased ROS production and induction of DNA damage response. Remarkably, nitroxoline induced a previously unknown deregulation of molecules with a critical role in cell bioenergetics, which resulted in mitochondrial depolarization. Our study also suggests that deregulation of cytosolic iron homeostasis and of co-translational targeting to membrane contribute to nitroxoline anticancer action. This study broadens our understanding of the mechanisms of nitroxoline action, showing that the drug modulates multiple proteins crucial in cancer biology and previously unknown to be affected by nitroxoline.

RNA-binding proteins (RBPs) play important roles in modulating miRNA-mediated mRNA target repression. Argonaute2 (Ago2) is an essential component of the RNA-induced silencing complex (RISC) that plays a central role in silencing mechanisms via small non-coding RNA molecules known as siRNAs and miRNAs. Small RNAs loaded into Argonaute proteins catalyze endoribonucleolytic cleavage of target RNAs or recruit factors responsible for translational silencing and mRNA target destabilization. In previous studies we have shown that KCC2, a neuronal Cl (-) extruding K (+) Cl (-) co-transporter 2, is regulated by miR-92 in neuronal cells. Searching for Ago2 partners by immunoprecipitation and LC-MS/MS analysis, we isolated among other proteins the Serpine mRNA binding protein 1 (SERBP1) from SH-SY5Y neuroblastoma cells. Exploring the role of SERBP1 in miRNA-mediated gene silencing in SH-SY5Y cells and primary hippocampal neurons, we demonstrated that SERBP1 silencing regulates KCC2 expression through the 3' untranslated region (UTR). In addition, we found that SERBP1 as well as Ago2/miR-92 complex bind to KCC2 3'UTR. Finally, we demonstrated the attenuation of miR-92-mediated repression of KCC2 3'UTR by SERBP1 silencing. These findings advance our knowledge regarding the miR-92-mediated modulation of KCC2 translation in neuronal cells and highlight SERBP1 as a key component of this gene regulation.

Neuropathic pain (NeP) in humans is often a life-long condition with no effective therapy available. The higher incidence of female gender in NeP onset is worldwide reported, and although the cause is generally attributed to sex hormones, the actual mechanisms and the players involved are still unclear. Glial and immune cells take part in NeP development, and orchestrate the neuroimmune and inflammatory response, releasing pro-inflammatory factors with chemoattractant properties that activate resident immune cells and recruit immune cells from circulation. The neuro-immune crosstalk is a key contributor to pain hypersensitivity following peripheral nervous system injury. Our previous works showed that in spite of the fact that female mice had an earlier analgesic response than males following nerve lesion, the recovery from NeP was never complete, suggesting that this difference could occur in the very early stages after injury. To further investigate gender differences in immune and neuroimmune responses to NeP, we studied the main immune cells and mediators elicited both in plasma and sciatic nerves by peripheral nerve lesion. After injury, we found a different pattern of distribution of immune cell populations showing either a higher infiltration of T cells in nerves from females or a higher infiltration of macrophages in nerves from males. Moreover, in comparison to male mice, the levels of cytokines and chemokines were differently up- and down-regulated in blood and nerve lysates from female mice. Our study provides some novel insights for the understanding of gender-associated differences in the generation and perseveration of NeP as well as for the isolation of specific neurodegenerative mechanisms underlying NeP. The identification of gender-associated inflammatory profiles in neuropathy is of key importance for the development of differential biomarkers and gender-specific personalized medicine.

The present study, employing a comparative proteomic approach, analyzes the protein profile of pig claustrum (CLA), putamen (PU), and insula (IN). Pig brain is an interesting model whose key translational features are its similarities with cortical and subcortical structures of human brain. A greater difference in protein spot expression was observed in CLA vs PU as compared to CLA vs IN. The deregulated proteins identified in CLA resulted to be deeply implicated in neurodegenerative (i.e., sirtuin 2, protein disulfide-isomerase 3, transketolase) and psychiatric (i.e., copine 3 and myelin basic protein) disorders in humans. Metascape analysis of differentially expressed proteins in CLA vs PU comparison suggested activation of the α-synuclein pathway and L1 recycling pathway corroborating the involvement of these anatomical structures in neurodegenerative diseases. The expression of calcium/calmodulin-dependent protein kinase and dihydropyrimidinase like 2, which are linked to these pathways, was validated using western blot analysis. Moreover, the protein data set of CLA vs PU comparison was analyzed by Ingenuity Pathways Analysis to obtain a prediction of most significant canonical pathways, upstream regulators, human diseases, and biological functions. Interestingly, inhibition of presenilin 1 (PSEN1) upstream regulator and activation of endocannabinoid neuronal synapse pathway were observed. In conclusion, this is the first study presenting an extensive proteomic analysis of pig CLA in comparison with adjacent areas, IN and PUT. These results reinforce the common origin of CLA and IN and suggest an interesting involvement of CLA in endocannabinoid circuitry, neurodegenerative, and psychiatric disorders in humans.

The genome of Helicobacter pylori encodes for carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α- and β-CA classes, which together with urease, have a pivotal role in the acid acclimation of the microorganism within the human stomach. Recently, in the exoproteome of H. pylori, a CA with no indication of the corresponding class was identified. Here, using the protonography and the mass spectrometry, a CA belonging to the α-class was detected in the outer membrane vesicles (OMVs) generated by planktonic and biofilm phenotypes of four H. pylori strains. The amount of this metalloenzyme was higher in the planktonic OMVs (pOMVs) than in the biofilm OMVs (bOMVs). Furthermore, the content of α-CA increases over time in the pOMVs. The identification of the α-CA in pOMVs and bOMVs might shed new light on the role of this enzyme in the colonization, survival, persistence, and pathogenesis of H. pylori.

Pseudoexfoliation (PEX) syndrome is an age-related progressive disease of the extracellular matrix with ocular manifestations. PEX is clinically diagnosed by the presence of extracellular exfoliative deposits on the anterior surface of the ocular lens. PEX syndrome is a major risk factor for developing glaucoma, the leading cause of irreversible blindness in the world, and is often associated with the development of cataract. PEX reportedly coexists with Alzheimer disease and increases the risk of heart disease and stroke. PEX material deposited on the anterior surface of the ocular lens is highly proteinaceous, complex, and insoluble, making deciphering the protein composition of the material challenging. Thus, to date, only a small proportion of the protein composition of PEX material is known. The aim of this study was to decipher the protein composition of pathological PEX material deposited on the ocular lens in patients and advance the understanding of pathophysiology of PEX syndrome.

The injection of safe doses of botulinum neurotoxin A (BoNT/A) have been reported to be useful for the treatment of neuropathic pain, but it is still unknown how functional recovery is induced after peripheral nerve injury. We evaluated the effects of intranerve application of BoNT/A, on regeneration and sensorimotor functional recovery in partial and complete peripheral nerve injuries in the mouse. After sciatic nerve crush (SNC) and intranerve delivery of BoNT/A (15pg), axonal regeneration was measured by nerve pinch test at different days. Regeneration of myelinated and unmyelinated fibers was assessed by immunohistochemical double labeling for NF200/GAP43 and CGRP/GAP43. S100 was used as Schwann cells marker. Medial footpad skin reinnervation was assessed by PGP staining. Motor functions were assessed by means of nerve conduction tests. In other mice groups, nerve conduction tests were performed also after chronic constriction injury (CCI) of the sciatic nerve and intraplantar injection of BoNT/A (15pg). In SNC mice, BoNT/A increased the rate of axonal regeneration. The advantage of regrowing myelinated axons after BoNT/A injection was evidenced by longer NF200+ nerve profiles and confirmed by nerve histology. We observed also a higher expression of S100 in the distal portion of BoNT/A-injected regenerated nerves. In CCI mice, BoNT/A induced an increase in reinnervation of gastrocnemius and plantar muscles. These results show that a low dose of BoNT/A, insufficient to produce muscular dysfunction, conversely speeds up sensorimotor recovery by stimulating myelinated axonal regeneration, and points out its application as a multipotent treatment for peripheral neuropathies.

In experimental and clinical pain studies, the sex of subjects was rarely taken into account, even if nociceptive inputs appear to be processed and modulated by partially distinct neural mechanisms in each sex. In this study we analysed, in male and female mice, behavioural and neuronal responses in developing, maintaining, and recovering from neuropathic pain. Experiments were carried out in adult CD1 mice by using Chronic Constriction Injury (CCI) as neuropathic pain model. We investigated the temporal trend of mechanical nociceptive threshold together with functional recovery of the injured paw, and the immunofluorescence staining of proteins associated with nerve injury and repair and with spinal gliosis, 7 and 121days after CCI. A proteomic analysis on proteins extracted from sciatic nerves was also performed. Male mice showed a gradual decrease of CCI-induced allodynia, the complete recovery occurring 81days after the sciatic nerve ligation. On the contrary, in female mice, allodynia was still present 121days after CCI. Sex-dependent differences also resulted from immunofluorescence experiments: in sciatic nerve, the expression of P0 and Neu200 is greater in neuropathic males than in neuropathic females, suggesting faster nerve regeneration. Proteomic analysis confirmed sex-related differences of proteins associated with nerve regenerative processes. In addition, the reactive gliosis induced by CCI at day 7, as revealed by colocalization of glial fibrillary acidic protein (astrocytes) and CD11b (microglia) with phosphorylated p38, disappeared 121 days after CCI in male but not in female mice. These results may have important therapeutic implications for the treatment of neuropathic pain.

At birth, contact with external stimuli, such as nutrients derived from food, is necessary to modulate the symbiotic balance between commensal and pathogenic bacteria, protect against bacterial dysbiosis, and initiate the development of the mucosal immune response. Among a variety of different feeding patterns, breastfeeding represents the best modality. In fact, the capacity of breast milk to modulate the composition of infants' gut microbiota leads to beneficial effects on their health. In this study, we used newborn mice as a model to evaluate the effect of parental genetic background (i.e., IgA-producing mice and IgA-deficient mice) and feeding modulation (i.e., maternal feeding and cross-feeding) on the onset and shaping of gut microbiota after birth. To investigate these topics, we used either a culturomic approach that employed Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MS), or bottom-up Liquid Chromatography, with subsequent MSMS shotgun metaproteomic analysis that compared and assembled results of the two techniques. We found that the microbial community was enriched by lactic acid bacteria when pups were breastfed by wild-type (WT) mothers, while IgA-deficient milk led to an increase in the opportunistic bacterial pathogen (OBP) population. Cross-feeding results suggested that IgA supplementation promoted the exclusion of some OBPs and the temporary appearance of beneficial species in pups fed by WT foster mothers. Our results show that both techniques yield a picture of microbiota from different angles and with varying depths. In particular, our metaproteomic pipeline was found to be a reliable tool in the description of microbiota. Data from these studies are available via ProteomeXchange, with identifier PXD004033.

Clinical use of neurotoxins from Clostridium botulinum is well established and is continuously expanding, including in treatment of pain conditions. Background: The serotype A (BoNT/A) has been widely investigated, and current data demonstrate that it induces analgesia and modulates nociceptive processing initiated by inflammation or nerve injury. Given that data concerning the serotype B (BoNT/B) are limited, the aim of the present study was to verify if also BoNT/B is able not only to counteract neuropathic pain, but also to interfere with inflammatory and regenerative processes associated with the nerve injury. Methods: As model of neuropathic pain, chronic constriction injury (CCI) of the sciatic nerve was performed in CD1 male mice. Mice were intraplantarly injected with saline (control) or BoNT/B (5 or 7.5 pg/mouse) into the injured hindpaw. For comparison, another mouse group was injected with BoNT/A (15 pg/mouse). Mechanical allodynia and functional recovery of the injured paw was followed for 101 days. Spinal cords and sciatic nerves were collected at day 7 for immunohistochemistry. Results and Conclusions: The results of this study show that BoNT/B is a powerful biological molecule that, similarly to BoNT/A, can reduce neuropathic pain over a long period of time. However, the analgesic effects are not associated with an improvement in functional recovery, clearly highlighting an important difference between the two serotypes for the treatment of this chronic pain state.

Over the recent years compelling evidence has accumulated indicating that botulinum neurotoxin serotype A (BoNT/A) results in analgesic effects on neuropathic as well as inflammatory pain, both in humans and in animal models. In the present study, the pharmacological interaction of BoNT/A with morphine in fighting inflammatory pain was investigated in mice using the formalin test. Moreover, the effects of BoNT/A on the tolerance-induced by chronic administration of morphine were tested and the behavioral effects were correlated with immunofluorescence staining of glial fibrillary acidic protein, the specific marker of astrocytes, at the spinal cord level. An ineffective dose of BoNT/A (2 pg/paw) combined with an ineffective dose of morphine (1 mg/kg) exerted a significant analgesic action both during the early and the late phases of formalin test. A single intraplantar injection of BoNT/A (15 pg/paw; i.pl.), administered the day before the beginning of chronic morphine treatment (7 days of s.c. injections of 20 mg/kg), was able to counteract the occurrence of tolerance to morphine. Moreover, BoNT/A reduces the enhancement of the expression of astrocytes induced by inflammatory formalin pain. Side effects of opiates, including the development of tolerance during repeated use, may limit their therapeutic use, the possibility of using BoNT/A for lowering the effective dose of morphine and preventing the development of opioid tolerance would have relevant implications in terms of potential therapeutic perspectives.

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease (AD) treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V), which would allow increasing the dose of NGF without triggering pain. We show that "painless" hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( n = 8), hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and anti-amyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of "painless" hNGF variants as a new generation of therapeutics for neurodegenerative diseases.

Cannabidiol (CBD) and cannabigerol (CBG) are Cannabis sativa terpenophenols. Although CBD's effectiveness against neurological diseases has already been demonstrated, nothing is known about CBG. Therefore, a comparison of the effects of these compounds was performed in two experimental models mimicking the oxidative stress and neurotoxicity occurring in neurological diseases. Rat astrocytes were exposed to hydrogen peroxide and cell viability, reactive oxygen species production and apoptosis occurrence were investigated. Cortexes were exposed to K+ 60 mM depolarizing stimulus and serotonin (5-HT) turnover, 3-hydroxykinurenine and kynurenic acid levels were measured. A proteomic analysis and bioinformatics and docking studies were performed. Both compounds exerted antioxidant effects in astrocytes and restored the cortex level of 5-HT depleted by neurotoxic stimuli, whereas sole CBD restored the basal levels of 3-hydroxykinurenine and kynurenic acid. CBG was less effective than CBD in restoring the levels of proteins involved in neurotransmitter exocytosis. Docking analyses predicted the inhibitory effects of these compounds towards the neurokinin B receptor. Conclusion: The results in the in vitro system suggest brain non-neuronal cells as a target in the treatment of oxidative conditions, whereas findings in the ex vivo system and docking analyses imply the potential roles of CBD and CBG as neuroprotective agents.

Prosthetic joint replacement failure has a huge impact on quality of life and hospitalization costs. A leading cause of prosthetic joint infection is bacteria-forming biofilm on the surface of orthopedic devices. Staphylococcus epidermidis is an emergent, low-virulence pathogen implicated in chronic infections, barely indistinguishable from aseptic loosening when embedded in a mature matrix. The literature on the behavior of quiescent S. epidermidis in mature biofilms is scarce. To fill this gap, we performed comparative analysis of the whole proteomic profiles of two methicillin-resistant S. epidermidis strains growing in planktonic and in sessile form to investigate the molecular mechanisms underlying biofilm stability. After 72-h culture of biofilm-forming S. epidermidis, overexpression of proteins involved in the synthesis of nucleoside triphosphate and polysaccharides was observed, whereas planktonic bacteria expressed proteins linked to stress and anaerobic growth. Cytological analysis was performed to determine why planktonic bacteria unexpectedly expressed proteins typical of sessile culture. Images evidenced that prolonged culture under vigorous agitation can create a stressful growing environment that triggers microorganism aggregation in a biofilm-like matrix as a mechanism to survive harsh conditions. The choice of a unique late time point provided an important clue for future investigations into the biofilm-like behavior of planktonic cells. Our preliminary results may inform comparative proteomic strategies in the study of mature bacterial biofilm. Finally, there is an increasing number of studies on the aggregation of free-floating bacteria embedded in an extracellular matrix, prompting the need to gain further insight into this mode of bacterial growth.

Parkinson's disease (PD) is the second most frequent neurodegenerative disease worldwide and the availability of early biomarkers and novel biotargets represents an urgent medical need. The main pathogenetic hallmark of PD is the specific loss of nigral dopaminergic neurons, in which mitochondrial dysfunction plays a crucial role. Mitochondrial proteases are central to the maintenance of healthy mitochondria and they have recently emerged as drug targets. However, an exhaustive characterization of these enzymes and their targets is still lacking, due to difficulties in analyzing proteolytic fragments by bottom-up proteomics approaches. Here, we propose the "mitochondrial dimethylation-TAILS" strategy, which combines the isolation of mitochondria with the enrichment of N-terminal peptides to analyze the mitochondrial N-terminome. We applied this method in a cellular model of altered dopamine homeostasis in neuroblastoma SH-SY5Y cells, which recapitulates early steps of PD pathogenesis. The main aim was to identify candidate mitochondrial proteases aberrantly activated by dopamine dysregulation and their cleaved targets. The proposed degradomics workflow was able to improve the identification of mitochondrial proteins if compared to classical shotgun analysis. In detail, 40% coverage of the mitochondrial proteome was obtained, the sequences of the transit peptides of two mitochondrial proteins were unveiled, and a consensus cleavage sequence for proteases involved in the processing of mitochondrial proteins was depicted. Mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD013900. Moreover, sixty-one N-terminal peptides whose levels were affected by dopamine treatment were identified. By an in-depth analysis of the proteolytic peptides included in this list, eleven mitochondrial proteins showed altered proteolytic processing. One of these proteins (i.e., the 39S ribosomal protein L49 - MRPL49) was cleaved by the neprilysin protease, already exploited in clinics as a biotarget. We eventually demonstrated a mitochondrial subcellular localization of neprilysin in human cells for the first time. Collectively, these results shed new light on mitochondrial dysfunction linked to dopamine imbalance in PD and opened up the possibility to explore the mitochondrial targets of neprilysin as candidate biomarkers.

Traumatic spinal cord injury has dramatic consequences and a huge social impact. We propose a new mouse model of spinal trauma that induces a complete paralysis of hindlimbs, still observable 30 days after injury. The contusion, performed without laminectomy and deriving from the pressure exerted directly on the bone, mimics more closely many features of spinal injury in humans. Spinal cord was injured at thoracic level 10 (T10) in adult anesthetized female CD1 mice, mounted on stereotaxic apparatus and connected to a precision impactor device. Following severe injury, we evaluated motor and sensory functions, and histological/morphological features of spinal tissue at different time points. Moreover, we studied the effects of early and subchronic administration of Docosahexaenoic acid, investigating functional responses, structural changes proximal and distal to the lesion in primary and secondary injury phases, proteome modulation in injured spinal cord. Docosahexaenoic acid was able i) to restore behavioural responses and ii) to induce pro-regenerative effects and neuroprotective action against demyelination, apoptosis and neuroinflammation. Considering the urgent health challenge represented by spinal injury, this new and reliable mouse model together with the positive effects of docosahexaenoic acid provide important translational implications for promising therapeutic approaches for spinal cord injuries.

Stevia rebaudiana Bertoni leaf extracts have gained increasing attention for their potential protection against type 2 diabetes. In this study, we have evaluated the possible beneficial effects of Stevia rebaudiana leaf extracts on beta-cells exposed to lipotoxicity and explored some of the possible mechanisms involved.