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α-Tocopheryl succinate (α-TOS) inhibits oxidative phosphorylation at the level of mitochondrial complex I and II, thus promoting cancer cell death through mitochondrial reactive oxygen species (ROS) generation. Redox imbalance activates NF-E2 p45-related factor 2 (Nrf2), a transcription factor involved in cell protection and detoxification responses. Here we examined the involvement of heme oxygenase-1 (HO-1) in the regulation of nuclear factor κB (NF-κB) signaling by short exposure to α-TOS in prostate cancer cells. A short-term (4 h) exposure to α-TOS causes a significant reduction in cell viability (76%±9%) and a moderate rise in ROS production (113%±8%). α-TOS alters glutathione (GSH) homeostasis by inducing a biphasic effect, i.e., an early (1 h) decrease in intracellular GSH content (56%±20%) followed by a threefold rise at 4 h. α-TOS increases nuclear translocation and electrophile-responsive/antioxidant-responsive elements binding activity of Nrf2, resulting in up-regulation of downstream genes cystine-glutamic acid exchange transporter and HO-1, while decreasing NF-κB nuclear translocation. This effect is suppressed by the pharmacological inhibition of HO-1 and mimicked by the end-products of HO activity, i.e., bilirubin and carbon monoxide. Results suggest a little understood mechanism for α-TOS-induced inhibition of NF-κB nuclear translocation due to HO-1 up-regulation. [ABSTRACT FROM AUTHOR]

Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) accounts for the catabolism of the endogenous inhibitors of nitric oxide (NO) synthases, namely, ADMA ( N ω , N ω -dimethyl-l-arginine) and NMMA ( N ω -monomethyl-l-arginine). Inhibition of DDAH-1 may prove a therapeutic benefit in diseases associated with elevated nitric oxide (NO) levels by providing a tissue-specific increase of ADMA and NMMA. In this work, we have used molecular dynamics to generate a pool of DDAH-1 conformations in the apo and holo forms. Ensemble docking has been instrumental in screening an in-house fragment-based library of 824 compounds. Resulting virtual hits have been validated for their binding activity to recombinant human DDAH-1 using microscale thermophoresis (MST). As a key result, three non-amino acidic ligands of DDAH-1 (VIS212, VIS268, VIS726) are identified with higher binding efficiency index than ADMA. Amid these compounds, purpurogallin (VIS726) proves a potent ligand of DDAH-1, showing a mixed behavior of enzymatic inhibition in a biochemical assay. This finding widens the panel of known molecular targets of purpurogallin and provides clues into the molecular mechanisms of its cellular NO inhibition activity as well as its anti-inflammatory and neuroprotective effects.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cardiovascular disease by metabolising the risk factor asymmetric dimethylarginine (ADMA). However, the question whether the second DDAH isoform, DDAH2, directly metabolises ADMA has remained unanswered. Consequently, it is still unclear if DDAH2 may be a potential target for ADMA-lowering therapies or if drug development efforts should focus on DDAH2's known physiological functions in mitochondrial fission, angiogenesis, vascular remodelling, insulin secretion, and immune responses. Here, an international consortium of research groups set out to address this question using in silico, in vitro, cell culture, and murine models. The findings uniformly demonstrate that DDAH2 is incapable of metabolising ADMA, thus resolving a 20-year controversy and providing a starting point for the investigation of alternative, ADMA-independent functions of DDAH2., (© 2023. The Author(s).)

Fusarium mycotoxins commonly contaminate agricultural products resulting in a serious threat to both animal and human health. The co-occurrence of different mycotoxins in the same cereal field is very common, so the risks as well as the functional and ecological effects of mycotoxins cannot always be predicted by focusing only on the effect of the single contaminants. Enniatins (ENNs) are among the most frequently detected emerging mycotoxins, while deoxynivalenol (DON) is probably the most common contaminant of cereal grains worldwide. The purpose of this review is to provide an overview of the simultaneous exposure to these mycotoxins, with emphasis on the combined effects in multiple organisms. Our literature analysis shows that just a few studies on ENN-DON toxicity are available, suggesting the complexity of mycotoxin interactions, which include synergistic, antagonistic, and additive effects. Both ENNs and DON modulate drug efflux transporters, therefore this specific ability deserves to be explored to better understand their complex biological role. Additionally, future studies should investigate the interaction mechanisms of mycotoxin co-occurrence on different model organisms, using concentrations closer to real exposures.

AGXT1 encodes alanine:glyoxylate aminotransferase 1 (AGT1), a liver peroxisomal pyridoxal 5'-phosphate dependent-enzyme whose deficit causes Primary Hyperoxaluria Type 1 (PH1). PH1 is a rare disease characterized by overproduction of oxalate, first leading to kidney stones formation, and possibly evolving to life-threatening systemic oxalosis. A minority of PH1 patients is responsive to pyridoxine, while the option for non-responders is liver-kidney transplantation. Therefore, huge efforts are currently focused on the identification of new therapies, including the promising approaches based on RNA silencing recently approved. Many PH1-associated mutations are missense and lead to a variety of kinetic and/or folding defects on AGT1. In this context, the availability of a reliable in vitro disease model would be essential to better understand the phenotype of known or newly-identified pathogenic variants as well as to test novel drug candidates. Here, we took advantage of the CRISPR/Cas9 technology to specifically knock-out AGXT1 in HepG2 cells, a hepatoma-derived cell model exhibiting a conserved glyoxylate metabolism. AGXT1-KO HepG2 displayed null AGT1 expression and significantly reduced transaminase activity leading to an enhanced secretion of oxalate upon glycolate challenge. Known pathogenic AGT1 variants expressed in AGXT1-KO HepG2 cells showed alteration in both protein levels and specific transaminase activity, as well as a partial mitochondrial mistargeting when associated with a common polymorphism. Notably, pyridoxine treatment was able to partially rescue activity and localization of clinically-responsive variants. Overall, our data validate AGXT1-KO HepG2 cells as a novel cellular model to investigate PH1 pathophysiology, and as a platform for drug discovery and development., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Primary hyperoxaluria type I (PH1) is a rare kidney disease due to the deficit of alanine:glyoxylate aminotransferase (AGT), a pyridoxal-5'-phosphate-dependent enzyme responsible for liver glyoxylate detoxification, which in turn prevents oxalate formation and precipitation as kidney stones. Many PH1-associated missense mutations cause AGT misfolding. Therefore, the use of pharmacological chaperones (PCs), small molecules that promote correct folding, represents a useful therapeutic option. To identify ligands acting as PCs for AGT, we first performed a small screening of commercially available compounds. We tested each molecule by a dual approach aimed at defining the inhibition potency on purified proteins and the chaperone activity in cells expressing a misfolded variant associated with PH1. We then performed a chemical optimization campaign and tested the resulting synthetic molecules using the same approach. Overall, the results allowed us to identify a promising hit compound for AGT and draw conclusions about the requirements for optimal PC activity.

The conformational landscape of a protein is constantly expanded by genetic variations that have a minimal impact on the function(s) while causing subtle effects on protein structure. The wider the conformational space sampled by these variants, the higher the probabilities to adapt to changes in environmental conditions. However, the probability that a single mutation may result in a pathogenic phenotype also increases. Here we present a paradigmatic example of how protein evolution balances structural stability and dynamics to maximize protein adaptability and preserve protein fitness. We took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population. By integrating crystallographic studies and molecular dynamics simulations, we show that AGT-Ma is endowed with structurally unstable (frustrated) regions, which become disordered in AGT-Mi. An in-depth biochemical characterization of variants from an anticonsensus library, encompassing the frustrated regions, correlates this plasticity to a fitness window defined by AGT-Ma and AGT-Mi. Finally, co-immunoprecipitation analysis suggests that structural frustration in AGT1 could favor additional functions related to protein-protein interactions. These results expand our understanding of protein structural evolution by establishing that naturally occurring genetic variations tip the balance between stability and frustration to maximize the ensemble of conformations falling within a well-defined fitness window, thus expanding the adaptability potential of the protein., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Cells have evolved sophisticated molecular control systems to maximize the efficiency of the folding process. However, any subtle alteration of the environment or the protein can lead to misfolding or affect the conformational plasticity of the native states. It has been widely demonstrated that misfolding and/or conformational instability are the underlying mechanisms of several rare disorders caused by enzymatic deficits. In fact, disease-causing mutations often lead to the substitution of amino acids that are crucial for the achievement of a folded conformation, or play a role on the equilibrium between native-state conformers. One of the promising approaches to treat conformational disorders is the use of pharmacological chaperones (PCs), small molecules that specifically bind a target protein and stabilize a functional fold, thus increasing the amount of functionally active enzyme. Molecules acting as PCs are usually coenzymes, substrate analogues behaving as competitive inhibitors, or allosteric modulators. In this review, the general features of PCs are described, along with three examples of diseases (Gaucher disease, Phenylketonuria, and Primary Hyperoxaluria) in which this approach is currently under study at preclinical and/or clinical level., (© 2021 The Author(s). Published by BRI.)

The deficit of human ornithine aminotransferase (hOAT) is responsible for gyrate atrophy (GA), a rare recessive inherited disorder. Although more than 60 disease-associated mutations have been identified to date, the molecular mechanisms explaining how each mutation leads to the deficit of OAT are mostly unknown. To fill this gap, we considered six representative missense mutations present in homozygous patients concerning residues spread over the hOAT structure. E. coli expression, spectroscopic, kinetic and bioinformatic analyses, reveal that the R154L and G237D mutations induce a catalytic more than a folding defect, the Q90E and R271K mutations mainly impact folding efficiency, while the E318K and C394Y mutations give rise to both folding and catalytic defects. In a human cellular model of disease folding-defective variants, although at a different extent, display reduced protein levels and/or specific activity, due to increased aggregation and/or degradation propensity. The supplementation with Vitamin B6, to mimic a treatment strategy available for GA patients, does not significantly improve the expression/activity of folding-defective variants, in contrast with the clinical responsiveness of patients bearing the E318K mutation. Thus, we speculate that the action of vitamin B6 could be also independent of hOAT. Overall, these data represent a further effort toward a comprehensive analysis of GA pathogenesis at molecular and cellular level, with important relapses for the improvement of genotype/phenotype correlations and the development of novel treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Montioli, Sgaravizzi, Desbats, Grottelli, Voltattorni, Salviati and Cellini.)

Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5'-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins.

Neurodegenerative diseases are associated with increased levels of nitric oxide (NO) mainly produced by microglial cells through inducible nitric oxide synthase (iNOS) whose expression is induced by inflammatory stimuli. NO can both exert cytotoxic functions and induce a metabolic switch by inhibiting oxidative phosphorylation and upregulating glycolytic flux. Here, we investigated whether two newly synthesized acetamidine based iNOS inhibitors, namely CM292 and CM544, could inhibit lipopolysaccharide (LPS)-induced BV2 microglial cell activation, focusing on both inflammatory and metabolic profiles. We found that CM292 and CM544, without affecting iNOS protein expression, reduced NO production and reverted LPS-induced inflammatory and cytotoxic response. Furthermore, in the presence of the inflammatory stimulus, both the inhibitors increased the expression of glycolytic enzymes. In particular, CM292 significantly reduced nuclear accumulation of pyruvate kinase M2, increased mitochondrial membrane potential and oxygen consumption rate, and augmented the expression of pyruvate dehydrogenase, pointing to a metabolic switch toward oxidative phosphorylation. These data confirm the role played by NO in the connection between cell bioenergetics profile and inflammation, and suggest the potential usefulness of iNOS inhibitors in redirecting microglia from detrimental to pro-regenerative phenotype.

Peroxisomal alanine:glyoxylate aminotransferase (AGT) is responsible for glyoxylate detoxification in human liver and utilizes pyridoxal 5'-phosphate (PLP) as coenzyme. The deficit of AGT leads to Primary Hyperoxaluria Type I (PH1), a rare disease characterized by calcium oxalate stones deposition in the urinary tract as a consequence of glyoxylate accumulation. Most missense mutations cause AGT misfolding, as in the case of the G41R, which induces aggregation and proteolytic degradation. We have investigated the interaction of wild-type AGT and the pathogenic G41R variant with d-cycloserine (DCS, commercialized as Seromycin), a natural product used as a second-line treatment of multidrug-resistant tuberculosis, and its synthetic enantiomer l-cycloserine (LCS). In contrast with evidences previously reported on other PLP-enzymes, both ligands are AGT reversible inhibitors showing inhibition constants in the micromolar range. While LCS undergoes half-transamination generating a ketimine intermediate and behaves as a classical competitive inhibitor, DCS displays a time-dependent binding mainly generating an oxime intermediate. Using a mammalian cellular model, we found that DCS, but not LCS, is able to promote the correct folding of the G41R variant, as revealed by its increased specific activity and expression as a soluble protein. This effect also translates into an increased glyoxylate detoxification ability of cells expressing the variant upon treatment with DCS. Overall, our findings establish that DCS could play a role as pharmacological chaperone, thus suggesting a new line of intervention against PH1 based on a drug repositioning approach. To a widest extent, this strategy could be applied to other disease-causing mutations leading to AGT misfolding., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Neuroinflammation, i.e. self-propelling progressive cycle of microglial activation and neuron damage, as well as improper protein folding, are recognized as major culprits of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). Mutations in several proteins have been linked to ALS pathogenesis, including the G93A mutation in the superoxide dismutase 1 (SOD1) enzyme. SOD1(G93A) mutant is prone to aggregate thus inducing both oxidative stress and neuroinflammation. In this study we used hSOD1(G93A) microglial cells to investigate the effects of the antioxidant and anti-inflammatory cyclic dipeptide (His-Pro) on LPS-induced inflammasome activation. We found that cyclo(His-Pro) inhibits NLRP3 inflammasome activation by reducing protein nitration via reduction in NO and ROS levels, indicative of lower peroxynitrite generation by LPS. Low levels in peroxynitrite are related to NF-κB inhibition responsible for iNOS down-regulation and NO dampening. On the other hand, cyclo(His-Pro)-mediated ROS attenuation, not linked to Nrf2 activation in this cellular model, is ascribed to increased soluble SOD1 activity due to the up-regulation of Hsp70 and Hsp27 expression. Conclusively, our results, besides corroborating the anti-inflammatory properties of cyclo(His-Pro), highlight a novel role of the cyclic dipeptide as a proteostasis regulator, and therefore a good candidate for the treatment of ALS and other misfolding diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Most pathogenic missense mutations cause specific molecular phenotypes through protein destabilization. However, how protein destabilization is manifested as a given molecular phenotype is not well understood. We develop here a structural and energetic approach to describe mutational effects on specific traits such as function, regulation, stability, subcellular targeting or aggregation propensity. This approach is tested using large-scale experimental and structural perturbation analyses in over thirty mutations in three different proteins (cancer-associated NQO1, transthyretin related with amyloidosis and AGT linked to primary hyperoxaluria type I) and comprising five very common pathogenic mechanisms (loss-of-function and gain-of-toxic function aggregation, enzyme inactivation, protein mistargeting and accelerated degradation). Our results revealed that the magnitude of destabilizing effects and, particularly, their propagation through the structure to promote disease-associated conformational states largely determine the severity and molecular mechanisms of disease-associated missense mutations. Modulation of the structural perturbation at a mutated site is also shown to cause switches between different molecular phenotypes. When very common disease-associated missense mutations were investigated, we also found that they were not among the most deleterious possible missense mutations at those sites, and required additional contributions from codon bias and effects of CpG sites to explain their high frequency in patients. Our work sheds light on the molecular basis of pathogenic mechanisms and genotype-phenotype relationships, with implications for discriminating between pathogenic and neutral changes within human genome variability from whole genome sequencing studies.

Gyrate atrophy (GA) is a rare recessive disorder characterized by progressive blindness, chorioretinal degeneration and systemic hyperornithinemia. GA is caused by point mutations in the gene encoding ornithine δ-aminotransferase (OAT), a tetrameric pyridoxal 5'-phosphate-dependent enzyme catalysing the transamination of l-ornithine and α-ketoglutarate to glutamic-γ-semialdehyde and l-glutamate in mitochondria. More than 50 OAT variants have been identified, but their molecular and cellular properties are mostly unknown. A subset of patients is responsive to pyridoxine administration, although the mechanisms underlying responsiveness have not been clarified. Herein, we studied the effects of the V332M mutation identified in pyridoxine-responsive patients. The Val332-to-Met substitution does not significantly affect the spectroscopic and kinetic properties of OAT, but during catalysis it makes the protein prone to convert into the apo-form, which undergoes unfolding and aggregation under physiological conditions. By using the CRISPR/Cas9 technology we generated a new cellular model of GA based on HEK293 cells knock-out for the OAT gene (HEK-OAT_KO). When overexpressed in HEK-OAT_KO cells, the V332M variant is present in an inactive apodimeric form, but partly shifts to the catalytically-competent holotetrameric form in the presence of exogenous PLP, thus explaining the responsiveness of these patients to pyridoxine administration. Overall, our data represent the first integrated molecular and cellular analysis of the effects of a pathogenic mutation in OAT. In addition, we validated a novel cellular model for the disease that could prove instrumental to define the molecular defect of other GA-causing variants, as well as their responsiveness to pyridoxine and other putative drugs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neuroinflammation, characterized by the appearance of reactive microglial and astroglial cells, is one of the several pathogenic mechanisms of amyotrophic lateral sclerosis (ALS), a fast-progressing and fatal neurodegenerative disease. Cerebrospinal fluid and spinal cord of ALS patients and SOD1 mutant mice show high concentrations of IL-1β. This interleukin, expressed as an inactive precursor, undergoes a proteolytic maturation by caspase1, whose activation, in turn, depends on inflammasomes. Whether and how inflammasome is activated in ALS models is still to be clarified. The mechanism of inflammasome activation was studied in murine microglial cells overexpressing hSOD1(G93A) and verified in the spinal cord of hSOD1(G93A) mice. Murine microglial hSOD1(G93A) cells express all the inflammasome components and LPS activates caspase1 leading to an increase in the secretion of IL-1β. By activating NF-κB, LPS increases ROS and NO levels that spontaneously react to form peroxynitrite, thus leading to protein nitration. Reduction in peroxynitrite levels results in a decrease in caspase1 activity. Protein nitration and caspase1 activity are concomitantly increased in the spinal cord of pre-symptomatic SOD1(G93A) mice. Oxidative/nitrosative stress induces peroxynitrite formation that may be a key trigger of caspase1/inflammasome activation. Peroxynitrite formation may play a critical role in inflammasome activation and might be exploited as potential therapeutic target for ALS.

Protein function is dependent on assumption of the correct three-dimensional structure, achieved through the folding process. As a central element in ensuring cellular homeostasis, proteostasis i.e. the control of correct protein folding, trafficking and degradation, is a highly regulated process ensured by three integrated molecular pathways: i) the unfolded protein response (UPR) which is activated by the engulfment of misfolded proteins and results in protein re-folding through the expression of chaperones; ii) the ubiquitin-proteasome system (UPS) which 'flags' misfolded proteins with ubiquitin, directing them to the 26S proteasome for proteolytic degradation; iii) autophagy that, through lysosomes, removes misfolded or aggregated proteins. All three of these proteostatic controls can be impaired by the aging process and by pathological mutations highlighting the potential role of proteostasis in conditions associated with aging such as neurodegeneration, type 2 diabetes and cancer. Indeed, neurodegenerative diseases are characterised by an interconnected triumvirate of deregulated proteostasis, neuroinflammation (i.e. the uncontrolled activation of microglial cells), and oxidative stress (i.e. the unbuffered increase in reactive oxygen species). The transcription factor Nrf2, classically associated with protection against oxidative stress, can also modulate the UPR, UPS and autophagy, while inhibiting the activation of NF-kB, the key transcription factor of the inflammatory response. In this review we focus on recent data from our laboratory and others demonstrating that the protective Nrf2 pathway can be activated by the endogenous cyclic dipeptide (His-Pro), thereby driving neuroprotective effects in different pathological settings. In this context we discuss the possible utility of clyclo (His-Pro) as a promising future therapeutic option for protein misfolding disorders., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

In prostate cancer, oxidative stress and the subsequent Nrf2 activation promote the survival of cancer cells and acquired chemoresistance. Nrf2 links prostate cancer to endoplasmic reticulum stress, an event that triggers the unfolded protein response, aiming to restore cellular homeostasis as well as an adaptive survival mechanism. Glucose-regulated protein of 78 kD /immunoglobulin heavy chain binding protein (GRP78/BiP) is a key molecular chaperone in the endoplasmic reticulum that, when expressed at the cell surface, acts as a receptor for several signaling pathways enhancing antiapoptotic and proliferative signals. We showed GRP78/BiP translocation to PC3 cell surface in the presence of tunicamycin, an ER stress inductor, and demonstrated the existence of a GRP78/BiP-dependent non-canonical Nrf2 activation, responsible for increased resistance to ER-stress induced apoptosis. We found that, even in the absence of ROS production, tunicamycin causes Nrf2 activation, and activates Akt signaling, events bulnted by anti-GRP78/BiP antibody treatment. The presence of GRP78/BiP at the cell surface might be exploited for the immunotherapeutic strategy of prostate cancer since its blockage by anti-GRP78/BiP antibodies might promote cancer death by suppressing some of the several molecular protective mechanisms found in aggressive cancer cells., Competing Interests: CONFLICTS OF INTEREST The Authors declare no competing interest.

Denervation leads to the activation of the catabolic pathways, such as the ubiquitin-proteasome and autophagy, resulting in skeletal muscle atrophy and weakness. Furthermore, denervation induces oxidative stress in skeletal muscle, which is thought to contribute to the induction of skeletal muscle atrophy. Several muscle diseases are characterized by denervation, but the molecular pathways contributing to muscle atrophy have been only partially described. Our study delineates the kinetics of activation of oxidative stress response in skeletal muscle following denervation. Despite the denervation-dependent induction of oxidative stress in skeletal muscle, treatments with anti-oxidant drugs do not prevent the reduction of muscle mass. Our results indicate that, although oxidative stress may contribute to the activation of the response to denervation, it is not responsible by itself of oxidative damage or neurogenic muscle atrophy.

Neurodegenerative diseases may have distinct genetic etiologies and pathological manifestations, yet share common cellular mechanisms underpinning neuronal damage and dysfunction. These cellular mechanisms include excitotoxicity, calcium dysregulation, oxidative damage, ER stress and neuroinflammation. Recent data have identified a dual role in these events for glial cells, such as microglia and astrocytes, which are able both to induce and to protect against damage induced by diverse stresses. Cyclo(His-Pro), a cyclic dipeptide derived from the hydrolytic removal of the amino-terminal pyroglutamic acid residue of the hypothalamic thyrotropin-releasing hormone, may be important in regulating the nature of the glial cell contribution. Cyclo(His-Pro) is ubiquitous in the central nervous system and is a key substrate of organic cation transporters, which are strongly linked to neuroprotection. The cyclic dipeptide can also cross the brain-blood-barrier and, once in the brain, can affect diverse inflammatory and stress responses by modifying the Nrf2-NF-κB signaling axis. For these reasons, cyclo(His-Pro) has striking potential for therapeutic application by both parenteral and oral administration routes and may represent an important new tool in counteracting neuroinflammation-based degenerative pathologies. In this review, we discuss the chemistry and biology of cyclo(His-Pro), how it may interact with the biological mechanisms driving neurodegenerative disease, such as amyotrophic lateral sclerosis, and thereby act to preserve or restore neuronal function.

Many neurological and neurodegenerative diseases are associated with oxidative stress and glial inflammation, all related to endoplasmic reticulum stress. Cyclo(His-Pro) is an endogenous cyclic dipeptide that exerts cytoprotection by interfering with the Nrf2-NF-κB systems, the former presiding the antioxidant and the latter the pro-inflammatory cellular response. Here we investigated whether the cyclic dipeptide inhibits glial inflammation thus reducing the detrimental effect of inflammatory neurotoxins on neurons. We found that systemic administration of cyclo(His-Pro) exerts in vivo anti-inflammatory effects in the central nervous system by down-regulating hepatic and cerebral TNFα expression thereby counteracting LPS-induced gliosis. Mechanistic studies indicated that the cyclic dipeptide-mediated effects are achieved through the activation of Nrf2-driven antioxidant response and the inhibition of the pro-inflammatory NF-κB pathway. Moreover, by up-regulating Bip, cyclo(His-Pro) increases the ER stress sensitivity and triggers the unfolded protein response to alleviate the ER stress. These results unveil a novel potential therapeutic use of cyclo(His-Pro) against neuroinflammatory-related diseases and we might now consider its potential anti-inflammatory role in other neuropathological conditions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

α-Tocopheryl succinate is one of the most effective analogues of vitamin E for inhibiting cell proliferation and inducing cell death in a variety of cancerous cell lines while sparing normal cells or tissues. αTocopheryl succinate inhibits oxidative phosphorylation at the level of mitochondrial complexes I and II, thus enhancing reactive oxygen species generation which, in turn, induces the expression of Nrf2-driven antioxidant/detoxifying genes. The cytoprotective role of Nrf2 downstream genes/proteins prompted us to investigate whether and how α-tocopheryl succinate increases resistance of PC3 prostate cancer cells to pro-oxidant damage. A 4h α-tocopheryl succinate pre-treatment increases glutathione intracellular content, indicating that the vitamin E derivative is capable of training the cells to react to an oxidative insult. We found that α-tocopheryl succinate pre-treatment does not enhance paraquat-/hydroquinone-induced cytotoxicity whereas it exhibits an additional/synergistic effect on H₂O₂₋/docetaxel-induced cytotoxicity. While glutathione and heme oxygenase-1 are not involved in α-tocopheryl succinate-induced adaptive response to paraquat,, Nad(p)h: quinone oxidoreductase seems to be responsible, at least in part, for the lack of the additional response. Silencing the gene and/or the inhibition of, Nad(p)h: quinone oxidoreductase activity counteracts the α-tocopheryl succinate-induced adaptive response. In conclusion, the adaptive response to α-tocopheryl succinate shows that the activation of Nrf2 can promote the survival of cancer cells in an unfavourable environment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We investigated the in vitro anti-inflammatory activity of 1(10),4-furanodien-6-one, one the most active compounds of the hexane extract of Commiphora erythraea (Ehrenb.) Engl., by exposing microglial BV-2 cells to lipopolysaccharide. We showed that furanodien-6-one pre-treatment restored cell viability and ROS to control levels while halving NO generation. Production of pro-inflammatory IL-6, IL-23, IL-17, TGF-β, and INF-γ, significantly induced by LPS, was also markedly reduced by furanodien-6-one treatment. We further showed that furanodien-6-one protects primary neuronal cultures against the inflammatory/toxic insults of LPS-treated BV-2 conditioned media, indicating that furanodien-6-one exerts anti-inflammatory/cytoprotective effects in neuronal cells. We then investigated whether furanodien-6-one exerts anti-inflammatory properties in an in vivo model of microglial activation. In adult mice ip-injected with LPS we found that furanodien-6-one had strong cerebral anti-inflammatory properties by inhibiting liver and brain TNFα as well as IL-1β expression. Results were not unexpected since FTIR-metabolomic analyses showed that furanodien-6-one-treated mice had a reduced dissimilarity to control animals and that the response to LPS treatment was markedly modified by furanodien-6-one. In conclusion our data provide strong evidence of the anti-inflammatory properties of furanodien-6-one that could be exploited to counteract degenerative pathologies based on neuroinflammation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cyclo(His-Pro) is an endogenous cyclic dipeptide that exerts oxidative damage protection by selectively activating the transcription factor Nrf2 signalling pathway. Given the existence of a tight interplay of the Nrf2/NF-κB systems and that the pro-inflammatory response is governed by transcription factor NF-κB, here we sought to investigate whether and how cyclo(His-Pro) interferes with the cross-talk between the antioxidant Nrf2/heme oxygenase-1 and the pro-inflammatory NF-κB pathways. By knocking down the Nrf2 gene, we confirmed that cyclo(His-Pro) inhibits NF-κB nuclear accumulation induced by paraquat in rat pheochromocytoma PC12 cells via the Nrf2/heme oxygenase-1 pathway. The protection required functional heme oxygenase-1 activity, since zinc protoporphyrin IX, a heme oxygenase-1 inhibitor, prevented NF-κB inhibition, and the presence of exogenous carbon monoxide and bilirubin afforded cytoprotection against paraquat-induced toxicity by preventing NF-κB activation. Cyclooxygenase-2 and matrix metalloproteinase 3, two gene products governed by NF-κB, were down-regulated by cyclo(His-Pro) and up-regulated in heme oxygenase-1 knock-down cells. We validated the general mechanism underlying the anti-inflammatory effects by treating PC12 and murine microglial BV2 cells with different pro-inflammatory agents. Finally, cyclo(His-Pro) reduced 12-otetradecanoylphorbol-13-acetate-induced oedema in mouse ear inflammation model. Results, by showing that cyclo(His-pro) suppresses the pro-inflammatory NF-κB signalling via the Nrf2-mediated heme oxygenase-1 activation, contribute to the understanding of essential cellular pathways and allow the proposal of cyclo(His-Pro) as an in vivo anti-inflammatory compound., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A(1) adenosine receptors are highly expressed in the central nervous system. Mitochondrial function is a major player in adenosine receptors-mediated effects. Here, by using mice with genetic deletion of the A(1) receptor, we addressed the existence of a relationship between mitochondria functions and adenosine A(1) receptor. Mitochondrial functions and effects of MPP(+) in primary mixed cultures are influenced by the presence of the A(1) receptor, demonstrating, for the first time, the mitochondrial localization of the adenosine A(1) receptor and suggesting a role for this receptor as a mitochondrial vulnerability factor., (Copyright 2010 Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.)

Paraquat (1,1'-dimethyl-4,4'-bipyridinium), a widely used non-selective herbicide, is a redox cycling agent with adverse effects on dopamine systems. Epidemiological data have shown that exposure to paraquat is one of the several risk factors for Parkinson's disease. We have already shown that cyclo(His-Pro), an endogenous cyclic dipeptide produced by the cleavage of the thyrotropin releasing hormone, has a cytoprotective effect through a mechanism involving Nrf2 activation that decreases production of reactive oxygen species and increases glutathione synthesis. Using primary neuronal cultures and PC12 cells as targets of paraquat neurotoxicity, we addressed whether and how cyclo(His-Pro) causes cellular protective response against paraquat-mediated cell death. We found that cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion by up-regulating Nrf2 gene expression, triggering its nuclear accumulation and activating the expression of heme oxygenase1. These protective effects were abolished by RNA interference-mediated Nrf2 knock down whereas were unaffected by RNA interference-mediated Keap1 knock down. Inhibition of heme oxygenase activity decreased cyclo(His-Pro)-induced neuroprotection. These results suggest that cyclo(His-Pro), acting as a selective activator of the brain modulable Nrf2 pathway, may be a promising candidate as neuroprotective agent that act through induction of phase II genes.

Hystidyl-proline [cyclo(His-Pro)] is an endogenous cyclic dipeptide produced by the cleavage of thyrotropin releasing hormone. Previous studies have shown that cyclo(His-Pro) protects against oxidative stress, although the underlying mechanism has remained elusive. Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells. Cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion caused by glutamate, rotenone, paraquat and beta-amyloid treatment. Moreover, real-time PCR analyses revealed that cyclo(His-Pro) induced the expression of a number of ARE-related genes and protected cells against hydrogen peroxide-mediated apoptotic death. Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown. Finally, pharmacological inhibition of p-38 MAPK partially prevented both cyclo(His-Pro)-mediated Nrf2 activation and cellular protection. These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence.

Adenosine levels are increased in stress and act as anti-oxidant and anti-inflammatory mediators by binding to 4 G-protein-coupled receptors. Using genetically modified mice lacking A(1) and A(3) adenosine receptors, treated with ip buthionine-[S,R]-sulphoximine injections to inhibit gamma-glutamylcysteine ligase, the question was addressed whether these receptors modulate the responses to the stress related to altered glutathione levels. This study determined organ glutathione levels and expression of two sub-units of gamma-glutamylcysteine ligase and the cationic x(c)-transporter and found that deletion of one or both adenosine receptors influenced the responses in an organ-specific manner. The lack of A(1) and A(3) adenosine receptors is related to decreased basal glutathione content and down-regulation of gamma-glutamylcysteine ligase sub-units in several organs. Moreover, responses to buthionine-[S,R]-sulphoximine were different. For example, the lack of A(3) adenosine receptors, or their blockade of A(3) by MRS 1191, caused a marked increase in gene expression, which was not observed in mice lacking both A(1) and A(3) receptors. The results indicate that A(1) and A(3) adenosine receptors play a role in antioxidant responses and their role differs in an organ-specific way.

The effects of dipeptide cyclo-[His-Pro] (CHP), known to participate in the appetite behavior and food intake control, have been investigated using PC12 cells in culture as model system. We found that only in the presence of experimental conditions that cause cellular stress the cyclic dipeptide affect cellular proliferation and protects from apoptosis. It greatly enhances the phosphorylation of hsp27, alpha-B-crystallin, Cdc2, and p-38 MAPK, whereas it decreases the phosphorylation of MEK1, Cav 2, GSK3a, PKB/Akt, PKCdelta, PKCgamma, and Erk2. PKA and PKG are involved in ERK1/2 deactivation via a receptor that appears to be dually coupled to Gs and Gq protein subfamilies.