Daniela Caccamo

Tissue transglutaminase and the stress response

Summary.The expression of the protein crosslinking enzyme tissue transglutaminase (TG2, tTG), the ubiquitous member of transglutaminase family, can be regulated by multiple factors. Although it has been suggested that TG2 can be involved in apoptotic cell death, high levels of enzyme have also been associated with cell survival in response to different stimuli. Furthermore, evidence indicates that increases in TG2 production cause enzyme translocation to cell membrane. Cell stress can also lead to TG2 accumulation on the cell surface and in the extracellular matrix resulting in changes in cell-matrix interactions.Here, we discuss the underlying mechanisms of TG2 up-regulation induced by various stimuli including glutamate exposure, calcium influx, oxidative stress, UV, and inflammatory cytokines.These findings agree with a postulated role for transglutaminases in molecular mechanisms involved in several diseases suggesting that cross-linking reactions could be a relevant part of the biochemical changes observed in pathological conditions.

Hyperhomocysteinemia in epileptic patients on new antiepileptic drugs.

Purpose:  Older enzyme‐inducing antiepileptic drugs (AEDs) may induce supraphysiologic plasma concentrations of total (t) homocysteine (Hcy). The aim of the present study was to investigate the effect of new AEDs on plasma tHcy levels.

Flavonoid Fraction of Bergamot Juice Reduces LPS-Induced Inflammatory Response through SIRT1-Mediated NF-κB Inhibition in THP-1 Monocytes

Plant polyphenols exert anti-inflammatory activity through both anti-oxidant effects and modulation of pivotal pro-inflammatory genes. Recently, Citrus bergamia has been studied as a natural source of bioactive molecules with antioxidant activity, but few studies have focused on molecular mechanisms underlying their potential beneficial effects. Several findings have suggested that polyphenols could influence cellular function by acting as activators of SIRT1, a nuclear histone deacetylase, involved in the inhibition of NF-κB signaling. On the basis of these observations we studied the anti-inflammatory effects produced by the flavonoid fraction of the bergamot juice (BJe) in a model of LPS-stimulated THP-1 cell line, focusing on SIRT1-mediated NF-κB inhibition. We demonstrated that BJe inhibited both gene expression and secretion of LPS-induced pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) by a mechanism involving the inhibition of NF-κB activation. In addition, we showed that BJe treatment reversed the LPS-enhanced acetylation of p65 in THP-1 cells. Interestingly, increasing concentrations of Sirtinol were able to suppress the inhibitory effect of BJe via p65 acetylation, underscoring that NF-κB–mediated inflammatory cytokine production may be directly linked to SIRT1 activity. These results suggest that BJe may be useful for the development of alternative pharmacological strategies aimed at reducing the inflammatory process.

Agmatine effects on mitochondrial membrane potential andNF‐κB activation protect against rotenone‐induced cell damage in human neuronal‐like SH‐SY5Y cells

J. Neurochem. (2011) 116, 67–75.

Cystamine inhibits transglutaminase and caspase-3 cleavage in glutamate-exposed astroglial cells.

Although the precise role of transglutaminase in cell death is unknown, several findings demonstrate that tissue transglutaminase selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Calcium‐dependent transglutaminase reactions are also implicated in several neurodegenerative diseases, including alterations in the release of excitatory amino acids. One prevalent theme in cell damage induced by excitotoxic stimuli in different regions of the CNS is that apoptosis may be executed by intracellular caspase proteases. Furthermore, the presence of functional ion channel‐gated receptors in glial cells suggests that also astrocytes can be susceptible to glutamates toxic effects. In this study, we demonstrated that prolonged exposure to glutamate (100 μM) of cultured astrocytes caused an increase in the expression of tissue transglutaminase (tTG). This effect was prevented by preincubation with GYKI 52466, an antagonist of AMPA/KA receptors. Glutamate exposure also promoted an increase in caspase‐3 compared with control cultures. Confocal laser microscopy analysis demonstrated the presence of activated caspase‐3 in the cytoplasm as well as in the nucleus. The inhibition of TG‐catalyzed reactions by cystamine (1 mM) blocked the activation pathway of caspase‐3, with an evident reduction of enzyme cleavage. These results suggest that glutamate increased both TG and caspase‐3 in astroglial cells early in the excitotoxin‐induced events.

Glutamate-evoked redox state alterations are involved in tissue transglutaminase upregulation in primary astrocyte cultures

The aim of this study was to evaluate the involvement of oxidative stress in glutamate‐evoked transglutaminase (TGase) upregulation in astrocyte cultures (14 DIV). A 24 h exposure to glutamate caused a dose‐dependent depletion of glutathione intracellular content and increased the ROS production in cell cultures. These effects were receptor‐mediated, as demonstrated by inhibition with GYKI 52466. The pre‐incubation with glutathione ethyl ester or cysteamine recovered oxidative status and was effective in significantly reducing glutamate‐increased tissue TGase. These data suggest that tissue TGase upregulation may be part of a biochemical response to oxidative stress induced by a prolonged exposure of astrocyte cultures to glutamate.

Excitotoxic and post-ischemic neurodegeneration: Involvement of transglutaminases

Summary.Neurodegeneration induced by excitotoxicity is a common feature in various neurological disorders. This pathological condition is caused by prolonged stimulation of glutamate receptor subtypes, followed by both intracellular Ca2+ overload and activation of specific genes, resulting in synthesis of enzymes involved in cell stress response.Using experimental in vitro models of excitotoxicity, we demonstrated that glutamate exposure up-regulated tissue transglutaminase in primary cultures of both cerebellar granule cells and astrocytes. These changes were consequent to receptor-mediated Ca2+ influx, as demonstrated by the inhibition with selective antagonists, MK-801 and GYKI 52466. Early increases in different transglutaminase isoforms were also observed in global cerebral ischemia, which closely resembles neuronal damage caused by NMDA receptor activation.These findings agree with a postulated role for transglutaminases in molecular mechanisms of several neurodegenerative diseases. Indeed, increased cross-linking reactions could be of pathologic relevance, as part of biochemical changes observed in neurological disorders.

Glutamate-induced increases in transglutaminase activity in primary cultures of astroglial cells

Glutamate exposure of astroglial cells caused ligand-gated channel receptor activation, associated with excitotoxic cell response. We investigated the effects of 24 h glutamate exposure on transglutaminase in astrocytes primary cultures at 7, 14, and 21 days in vitro (DIV). Increases in enzyme activity were observed as a function of cell differentiation stage in glutamate-treated cultures. These effects were significantly reduced when GYKI 52466, an AMPA/KA receptors inhibitor, was added to the culture medium prior to incubation with glutamate. Microscopy observation on transglutaminase-mediated, fluorescent dansylcadaverine incorporation in living cells was consistent with these results. Western blotting analysis with monoclonal antibody showed that glutamate also up-regulated tissue transglutaminase expression, which reached the highest values in 14 DIV cultures. Confocal laser scanning microscopy analysis of immunostained astroglial cells showed a mainly cytoplasmic localisation of the enzyme both in control and treated cultures; nevertheless, counterstaining with the nuclear dye acridine orange demonstrated the presence of tissue transglutaminase also into the nucleus of glutamate-exposed and 21 DIV cells. The increases in enzyme expression and localisation in the nucleus of glutamate-treated astroglial cells may be part of biochemical alterations induced by excitotoxic stimulus.

Potential of transglutaminase 2 as a therapeutic target.

Importance of the field: Increased expression and activity of transglutaminase 2 – a calcium-dependent enzyme which catalyzes protein cross-linking, polyamination or deamidation at selective glutamine residues – are involved in the etiopathogenesis of several pathological conditions, such as neurodegenerative disorders, autoimmune diseases and inflammatory diseases. Inhibition of enzyme activity has potential for therapeutic management of these diseases. Areas covered in this review: The major results achieved in the last twelve years of research in the field of inhibition of tranglutaminase activity using cell cultures as well as in vivo models of high-social-impact or widespread diseases, such as CNS neurodegenerative disorders, celiac sprue, cancer and fibrotic diseases. What the reader will gain: Beneficial effects of enzyme activity inhibition have been observed in neurodegeneration and fibrosis in vivo models by delivery of the competitive inhibitor cystamine and more recently designed inhibitors, such as thiomidaziolium or norleucine derivatives, which irreversibly bind the active site cysteine residue. Transglutaminase 2 targeting with specific antibodies has also been shown to be a promising tool for celiac disease treatment. Take home message: New insights from transglutaminase inhibition studies dealing with side effects of in vivo administration of pan-transglutaminase inhibitors will help in design of novel therapeutic approaches to various diseases.

Antiepileptic drugs and MTHFR polymorphisms influence hyper-homocysteinemia recurrence in epileptic patients

Summary:  The influence of antiepileptic drugs (AEDs) and/or common polymorphisms (677C → T, 1298A → C) of the methylene‐tetrahydrofolate‐reductase (MTHFR) gene on the recurrence time of hyper‐total‐homocysteinemia (tHcy > 13 μmol/L) was investigated in 59 hyper‐homocysteinemic patients (34M/25F, 20–49 years). Plasma tHcy and folate were assayed before and after 1‐month folate supplementation (5mg/day), and after 2, 4, and 6 months. Four MTHFR polymorphism groups were identified with the following tHcy (μmol/L) and folate (nmol/L) levels (mean ± SD): (a) MTHFR677TT/1298AA, 24 patients, 36.0 ± 4.8, 4.1 ± 0.7; (b) MTHFR677CT/1298AC 27.1 ± 2.7, 5.3 ± 1.0 (n = 15); (c) MTHFR677CT/1298AA 16.6 ± 3.6, 6.8 ± 1.0 (n = 11), all taking enzyme‐inducing AEDs; and (d) MTHFR677TT/1298AA 24.5 ± 3.2, 5.6 ± 1.1 (n = 9), treated with new AEDs. After folate therapy, plasma t‐Hcy and folate were normal in all patients. At 6 months, 43 patients (72.9%) exhibited hyper‐tHcy, the greater proportion belonging to the EI‐AED‐MTHFR677TT/1298AA (39%). Knowledge of the hyper‐tHcy recurrence time after folate therapy discontinuation may help in optimizing folate supplementation pulses.