Neuroprotection as a Potential Therapeutic Perspective in Neurodegenerative Diseases: Focus on Antiepileptic Drugs.

Neuroprotection is conceived as one of the potential tool to prevent or slow neuronal death and hence a therapeutic hope to treat neurodegenerative diseases, like Parkinson's and Alzheimer's diseases. Increase of oxidative stress, mitochondrial dysfunction, excitotoxicity, inflammatory changes, iron accumulation, and protein aggregation have been identified as main causes of neuronal death and adopted as targets to test experimentally the putative neuroprotective effects of various classes of drugs. Among these agents, antiepileptic drugs (AEDs), both the old and the newer generations, have shown to exert protective effects in different experimental models. Their mechanism of action is mediated mainly by modulating the activity of sodium, calcium and potassium channels as well as the glutamatergic and GABAergic (gamma-aminobutyric acid) synapses. Neurological pathologies in which a neuroprotective action of AEDs has been demonstrated in specific experimental models include: cerebral ischemia, Parkinson's disease, and Alzheimer's disease. Although the whole of experimental data indicating that neuroprotection can be achieved is remarkable and encouraging, no firm data have been produced in humans so far and, at the present time, neuroprotection still remains a challenge for the future.

Association of VDR gene polymorphisms with heart disease in chronic kidney disease patients.

OBJECTIVES: It has been postulated that VDR polymorphisms influence mortality in CKD by directly modifying VDR protein levels or VDR sensitivity in target organs. Here we aimed at evaluating the possible association of VDR FokI and BsmI gene polymorphisms with co-morbid conditions of CKD at different stages. DESIGN AND METHODS: The patients included in this study were a Sicilian cohort of 171 subjects, at CKD stage 1-2 (n=49), stage 3 (n=34), stage 4-5 (n=34), and hemodialysis (HD) (n=54). Almost 70% of patients were also suffering from heart disease, with/without diabetes and/or hypertension, and 40% were also suffering of hypertension, with/without diabetes and/or heart disease; only around 20% had no co-morbid conditions. RESULTS: A highly significant association was found between the BsmI B minor allele and heart disease in all CKD stages. Indeed, the odds ratio calculation showed that patients bearing either the bB or BB genotype had, respectively, a seven-fold and around twelve-fold increased risk for heart disease. Instead, the presence of bb wild-type genotype was associated with a fifty-fold reduced risk for heart disease, suggesting that the b allele may display a protective effect. No association was found for FokI genotypes with the different co-morbid conditions. CONCLUSIONS: We first demonstrated that the VDR BsmI B allele may be considered as a genetic determinant for heart disease and hypertension in CKD, independently from disease stage. Thus, the screening for VDR variants should be regarded as a way to better address preventive strategies and improving the management of CKD co-morbid conditions.

Association of the COMT synonymous polymorphism Leu136Leu and missense variant Val158Met with mood disorders.

BACKGROUND: Major depressive disorder (MDD) and bipolar disorder (BD) are the two most common mood disorders. Given the recognized involvement of catecholamines in depression, genetic research focused on the evaluation of polymorphisms in genes coding for proteins that regulate neurotransmitter release, transport and degradation. Here we aimed at evaluating the distribution of two genetic variants of catechol-O-methyltransferase (COMT), namely the well characterized missense polymorphism G1947A (Val158Met) and the recently reported synonymous polymorphism C1886G (Leu136Leu), in MDD and BD patients compared with healthy subjects. METHODS: Genotyping for COMT polymorphisms was carried out by DNA direct sequencing in 112 patients (54 MDD and 58 BD) and 58 healthy subjects. RESULTS: We did not find significant differences in the Val158Met variant distribution between patients and controls. Instead, we found that the C1886 major allele and the CC1886 wild-type genotype frequencies were significantly higher in controls than in both groups of patients. On the contrary, the G1886 minor allele and the heterozygous CG1886 genotype were significantly more present in both MDD and BD patients than in healthy subjects. When looking at combined polymorphisms, we found a significantly higher frequency of the double heterozygous diplotype CG/GAVal/Met158 in both MDD and BD patients than in controls. Instead, the diplotype CC/GAVal/Met158 showed a significantly higher frequency in controls than in BD patients. LIMITATIONS: The small size of our study cohort may limit the generalizability of the present findings. CONCLUSIONS: This work first showed the association of combined Leu136Leu and Val158Met variants of COMT gene with MDD and BD.

Transglutaminase 2 is involved in homocysteine-induced activation of human THP-1 monocytes.

Aberrant transglutaminase 2 (TG2) expression and protein cross-linking activity have been associated with several chronic neurodegenerative disorders in which inflammatory processes triggered by activated microglia and monocytes play a key role, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis. Interestingly, mild-to-moderate hyperhomocysteinemia (HHcy), corresponding to increased plasma homocysteine (Hcy) concentrations in the range 16-60 µM, have recently been associated with the above-cited diseases. Using THP-1 monocytes, here we investigated the role of TG2 in cell response to mildly elevated Hcy concentrations. A five-day incubation with Hcy (∼25 µM) increased reactive oxygen species, peroxide lipids, as well as 8-hydroxyguanosine levels by twofold, and decreased the endogenous cell antioxidant defenses, that is reduced glutathione, by 50% in Hcy-exposed cultures compared with controls (p < 0.01). Hcy-induced oxidative stress was associated with increases in TG2 expression and activity, as well as nuclear factor kappa B activation. Notably, the latter was reduced in the presence of the TG-specific inhibitor R283. Hcy exposure also significantly increased the mRNA levels of tumor necrosis factor alpha, interleukin (IL)-6, and IL-1ß, as well as the level of Hcy-inducible endoplasmic reticulum (ER) stress protein, a marker of ER stress, in Hcy-exposed cultures compared with controls. Notably, these effects were dramatically reduced by R283. These preliminary findings indicate that TG2 plays a key role in Hcy-induced activation of THP-1 monocytes, involving oxidative as well as ER stress and inflammation. This underlines the potential of TG2 inhibition in the therapeutic management of inflammatory processes contributing to neurodegenerative disorders associated with mild HHcy.

Toxic effects of mildly elevated homocysteine concentrations in neuronal-like cells.

Epidemiological and experimental evidence indicated that hyperhomocysteinemia is associated with neurodegeneration. However, homocysteine neurotoxic effects have been so far investigated mostly by employing homocysteine concentrations (≥100 µM) much higher than homocysteine mean plasma levels (20 µM) observed in patients with neurodegenerative disorders. While evaluating the effects of a prolonged exposure to ~20 µM homocysteine in neuronal-like differentiated SH-SY5Y cells, we observed a 35% loss of cell viability and a four-fold increase in reactive oxygen species levels in cells incubated with homocysteine for five days compared with controls. Moreover, homocysteine increased by 30% and around two-fold, respectively, the Comet-positive cell number and DNA damage indexes (tail length, T-DNA, olive tail moment) compared with controls. Cell response to homocysteine-induced DNA damage involved the up-regulation of Bax and, at a greater extent, Bcl-2, but not caspase-3, in association with a p53-independent increase of p21 levels; concomitantly, also p16 levels were increased. When looking at time-dependent changes in cyclin expression, we found that a significant up-regulation of cyclins D1, A1, E1, but not B1, concomitant with p21 down-regulation, occurred in cells incubated with homocysteine for three days. However, in line with the observed increase of p21 and p16 levels, a five days incubation with homocysteine induced cyclin down-regulation accompanied by a strong reduction of phosphorylated pRB amounts. These results suggest that, when prolonged, the exposure of neuronal-like cells to mildly elevated homocysteine concentrations triggers oxidative and genotoxic stress involving an early induction of cyclins, that is late repressed by G1-S check-point regulators.

Differential expression of transglutaminase genes in patients with chronic periodontitis.

OBJECTIVE: Gingival epithelium plays a key role in the protection of oral tissues from microbial challenge, especially during the periodontal disease. This study was aimed to evaluate levels of mRNA transcripts of different forms of transglutaminase in the human gingival tissues from patients with chronic periodontitis and relative controls. SUBJECTS AND METHODS: This study included 22 patients with chronic periodontitis (CP) and 22 healthy controls. For each patient, the values of probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP) were recorded. Gene expression of transglutaminase 1, transglutaminase 2, transglutaminase 3, and metalloprotease 2 was evaluated by real-time PCR, while that of Factor XIIIA and metalloprotease 9 by RT-PCR. RESULTS: The values of all the clinical parameters were significantly higher in the CP group than in the healthy control group (P < 0.05). In the CP group, the mRNA expression of transglutaminase 1 and transglutaminase 3 was significantly decreased in comparison with healthy control group. A slight nonsignificant changes of transglutaminase 2 gene expression were observed in samples from CP patients in comparison with controls. CONCLUSIONS: These observations suggest that transglutaminase gene expression may be modified in response to chronic injury in the damaged gingival and emphasizes the key role of these enzymes in gingival remodelling/healing and adaptive processes.

Homocysteine-induced toxicity increases TG2 expression in Neuro2a cells.

High levels of homocysteine promote cell damage mainly through induction of oxidative stress, endoplasmic reticulum (ER) stress, and activation of pro-inflammatory factors. The effects of homocysteine were here examined in the continuously dividing neuroblastoma cell line Neuro2a. Cell treatment with homocysteine (100-500 microM) for 4 h increased ROS production while reducing cell viability in a dose-dependent manner. Cell exposure to 250 microM homocysteine was able to induce transglutaminase 2 up-regulation and increased in situ transglutaminase activity. These effects were prevented by the incubation with the transglutaminase activity inhibitor cystamine. Homocysteine also induced NF-kappaB activation that seemed associated with transglutaminase 2 up-regulation since the specific NF-kappaB inhibition by SN50 was able to reduce transglutaminase expression and activity levels. In the light of these observations, it may be postulated that TG2 up-regulation is involved in cell response to homocysteine-induced stress, in which NF-kappaB activation plays also a pivotal role.

Effect of acetylcholine precursors on proliferation and differentiation of astroglial cells in primary cultures.

Effects of acetylcholine and of the cholinergic precursors choline, cytidine 5'-diphosphocholine (CDP-choline) and alpha-glyceryl-phosphorylcholine (alpha-GPC) on transglutaminase (TG) and cyclin D1 expression were studied in primary astrocyte cultures by confocal laser microscopy (CLSM) with monodansyl-cadaverine uptake as a marker of enzyme activity and by immunochemistry (Western blotting). CLSM analysis showed an increased cytofluorescence in 0.1 microM choline-treated astrocytes. Treatment with CDP-choline dose-dependently increased TG. A total of 1 microM CDP-choline exposure in 14 days in vitro (DIV) astrocyte cultures increased cytofluorescence. A total of 1 microM alpha-GPC 24 h-treated cultures revealed increased cytofluorescence both in cytosol and nuclei. Western blot analysis showed an increased TG expression in cultures exposed for 24 h to 1 microM choline or alpha-GPC, whereas in 24 h 1 microM CDP-choline and acetylcholine-treated astrocytes TG expression was unaffected. Treatment with 1 microM acetylcholine reduced TG expression at 21 DIV. In cultures at 14 and 35 DIV cholinergic precursor treatment for 24 h induced a marked down-regulation of cyclin D1 expression, with reduced cyclin D1 expression in 1 microM alpha-GPC treated astrocytes. Our data suggest a role of cholinergic precursors investigated independent from acetylcholine on maturation and differentiation of astroglial cells in vitro, rather than on their growth, proliferation and development in culture.

Effect of growth factors and steroids on transglutaminase activity and expression in primary astroglial cell cultures.

Type-2 transglutaminase (TG-2) is a multifunctional enzyme involved in the regulation of cell differentiation and survival that recently has been shown to play an emerging role in astrocytes, where it is involved in both proliferation and differentiation processes. Growth factors (GFs) such as EGF, basic fibroblast growth factor, insulin-like growth factor-I (IGF-I), and insulin (INS) are trophic and mitogenic peptides that participate in neuron-glia interactions and stimulate neuronal and astroglial proliferation and differentiation. Steroid hormones such as glucocorticoids and estrogens also play a pivotal role in neuronal and astroglial proliferation and differentiation and are key hormones in neurodegenerative and neuroprotective processes. We investigated the effects of the interaction of GFs with dexamethasone (DEX) or 17beta-estradiol (E(2)) on TG-2 activity and their expression in cultured astrocytes. We observed a significant increase in TG-2 activity and expression in astroglial cells treated for 24 hr with IGF-I, EGF, or INS. Priming of the cells with DEX or E(2), for 48 hr also led to an increase in TG-2 levels. When growth factors were present in the last 24 hr of the steroid treatment, a reduction in TG-2 expression and activity and a different subcellular TG-2 distribution were found. Our data indicate that steroid hormone-GF interaction may play an important role in astroglial function. The effect on TG-2 could be part of the regulation of intracellular pathways associated with the astrocyte response observed in physiological conditions and, possibly, also in neuropathological diseases.

Effect of MTHFR polymorphisms on hyperhomocysteinemia in levodopa-treated Parkinsonian patients.

High plasma homocysteine levels have been observed in Parkinson's disease (PD) patients treated with levodopa. In this study, we investigated the effects of C677T and A1298C MTHFR polymorphisms, in association with L-DOPA daily dose and vitamin status, on hyperhomocysteinemia development in PD patients. Plasma homocysteine and folate/vitamin B12 levels were assayed in 49 L-DOPA-treated PD patients, and compared with those of 86 healthy subjects. Genotyping for MTHFR polymorphisms was carried out by DG-DGGE. Homocysteine levels were significantly higher in patients than in controls (16.3 +/- 5.7 vs. 11.7 +/- 2.7 micromol/l, P < 0.01). No significant differences were found between patients and controls with regard to folate/vitamin B12 levels, and MTHFR allele distribution. The TT+AA genotype was significantly more frequent in PD patients than in controls (32.5% vs. 17.4%, P < 0.05), but not associated with an increased risk for PD (OR = 2.3, CI = 1.0-5.2). Further, patients carrier of this genotype exhibited a mild hyperhomocysteinemia (22.1 +/- 4.9 micromol/l), while a protective effect was observed in patients having the CC+AA genotype (11.2 +/- 1.6 micromol/l; OR = 0.19, CI = 0.06-0.59). Interestingly, homocysteine levels were also moderately increased in patients with CT heterozygous genotype, in the context of either AA or AC (14.5 +/- 3.6 micromol/l), in comparison to subjects with the CC + AA genotype. Finally, we did not find any significant association of combined C677T and A1298C MTHFR polymorphisms with an increased risk for hyperhomocysteinemia in PD patients. A better understanding of the role of homocysteine and MTHFR genotypes in PD is needed to reveal novel approaches for disease management.

Tissue transglutaminase and the stress response.

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.

Increased transglutaminase activity was associated with IL-6 release in cultured human gingival fibroblasts exposed to dental cast alloys.

Molecular mechanisms underlying gingival and periodontal inflammation caused by dental alloys are still poorly understood. Recently, it has been demonstrated that tissue transglutaminase can be involved in inflammatory cell response. The aim of this study was to evaluate effects of exposure to orthodontic materials on transglutaminase in cultured human gingival fibroblasts. The incubation with Ni-Ti heat-activated (T3) or Ni-Ti super-elastic (T4), and with Ni-Cr-Co (T2) alloys produced respectively 2.5-fold and 8-fold increases in IL-6 release compared with control cultures. Transglutaminase activity was significantly increased in cells exposed to T3 and T4 alloys (about 170% of control; p < 0.05), where it was mainly localized close to inner part of cell membrane. The exposure to T3 and T4 specimens significantly up-regulated also tTG expression compared with control cultures. These data first show an association between IL-6 release and tissue transglutaminase increases, suggesting that TGase-mediated reactions may play a major role in periodontal inflammation.

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.

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.

Tissue transglutaminase was up-regulated by EGF-retinoid interplay in epithelial carcinoma cells.

Retinoids have been shown the most powerful inducers of transglutaminase activity, a well known marker of differentiation. In this work, we tested the effects of all-trans retinoic acid and EGF, used alone or in combination, on transglutaminase activity in a squamous, epithelial carcinoma cell line, HEp-2. We demonstrated that nanomolar EGF further enhances transglutaminase activity previously induced by all-trans retinoic acid. Confocal laser scanning microscopy revealed functional changes in transglutaminase activity localisation, at first restricted to the outermost region of cytosol, then diffused both in the membrane region and extracellular space. RT-PCR showed the presence of mRNA transcripts of different transglutaminases (1, 2, 3). Transglutaminase 2 expression was increased by either all-trans retinoic acid or EGF, and further up-regulated by the simultaneous addition of both substances. These effects were confirmed by Western blotting with transglutaminase 2 specific antibody. The results obtained by combined use of retinoic acid and EGF suggest that transglutaminase activity and expression are differently regulated, and that EGF-signalling can be involved in differentiation of epithelial carcinoma cells induced by retinoids.

Excitotoxin-induced changes in transglutaminase during differentiation of cerebellar granule cells.

Excitotoxicity induced by NMDA receptor stimulation is able to increase the activity of many enzymes involved in neuronal cell death. Primary cultures of rat cerebellar granule cells were used to elucidate the role of transglutaminase reaction in the excitotoxic cell response, and to evaluate the role of glutamate receptors in cell survival and degeneration. Granule neurons, maintained in vitro for two weeks, were exposed to NMDA at different stages of differentiation. Following NMDA receptor activation, increases in transglutaminase activity were observed in cell cultures. The levels of enzyme activity were higher in cells at 5 days in vitro than in those at 8-9 or 13-14 days in vitro. Moreover, NMDA exposure up-regulated tTG expression in neurons as young as 5 days in vitro. These cultures also exhibited morphological changes with clear apoptotic features. Results obtained demonstrate that susceptibility of granule cells to excitotoxicity depends on the developmental stage of neurons.

Screening for C677T and A1298C MTHFR polymorphisms in patients with epilepsy and risk of hyperhomocysteinemia.

Hyperhomocysteinemia can result from decreased methylenetetrahydrofolate reductase (MTHFR) enzyme activity, owing to genetic polymorphisms andor inadequate folate intake. This study was aimed at investigating the prevalence of C677T and A1298C MTHFR polymorphisms, and their impact on hyperhomocysteinemia in 95 epileptic patients and 98 controls. Double gradient-denaturing gradient gel electrophoresis screening revealed that the frequency of T677 polymorphic allele was similar between cases and controls (46.3% vs 42.3%), whereas that of C1298 allele was significantly higher in patients (30.5% vs 19.4%, p < 0.05). Significant differences between the two groups were also found for the frequencies of genotypes AA1298 (46.3% in cases vs 67.3% in controls, p < 0.01) and AC1298 (46.3% in cases vs 26.6% in controls, p < 0.01). Other genotype frequencies did not show any statistically significant differences. Haplotype frequencies significantly differed between the two groups. The CT677/AC1298 diplotype was significantly more frequent in epileptic patients than in controls (32.6% vs 18.4%, p < 0.05). Patients treated with enzyme-inducing antiepileptic drugs, having this diplotype and concomitant low folate concentration (i.e., < 3.4 nmol/L), exhibited plasma homocysteine levels significantly higher than normal values (27.1 +/- 2.44 micromol/L, p < 0.001). This increase, however, was lower than that observed in folate-deficient patients with diplotype TT677/AA1298 (41.3 +/- 3.41 micromol/L, p < 0.001). Indeed, these two diplotypes could be regarded as risk factors for hyperhomocysteinemia. Conversely, we found that the CC677/AA1298 diplotype was significantly more frequent in controls (p < 0.01), suggesting a protective role. Our study suggests that both C677T and A1298C MTHFR polymorphisms should be examined when assessing genetic risk factors of hyperhomocysteinemia in epilepsy.

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 glutamate's toxic effects. In this study, we demonstrated that prolonged exposure to glutamate (100 microM) 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-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.

NMDA-evoked excitotoxicity increases tissue transglutaminase in cerebellar granule cells.

In neuronal cells, excessive activation of glutamate receptors causes excitotoxic damage culminating in apoptotic and necrotic cell death. The molecular mechanism of excitotoxicity has been associated with excessive Ca(2+) influx and overload, triggering biochemical events that lead to cell death and tissue degeneration. Following mild insults via NMDA-receptor activation, central neurons undergo several biochemical modifications recognizable as early events in apoptotic machinery.Tissue transglutaminase, the most ubiquitous among cell transglutaminases, catalyzes the Ca(2+)-dependent protein cross-linking probably associated with morphological changes in several neurodegenerative disorders. The possible involvement of this enzyme in excitotoxicity-mediated events was investigated in primary cultures of cerebellar granule cells exposed for 30 min to NMDA (100 microM) in Locke's buffer. Under these conditions time-dependent increases in transglutaminase activity were observed. Tissue transglutaminase expression reached the highest levels within 3-4 h of NMDA exposure. Similarly, high levels of incorporation of fluorescent substrates were observed in living cells. Confocal laser microscopy analysis showed that fluorescein-labelled structures were distributed within the cytoplasm and close to the membranes of NMDA-exposed cells. These effects were dependent on the Ca(2+) influx triggered by the excitotoxic stimulus. Morphological changes in NMDA-treated cells gave evidence of significant cell damage which appeared within 5-6 h of NMDA exposure. These results suggest that increases in tissue transglutaminase may be associated to the effects of NMDA-induced excitotoxicity. Therefore, it is reasonable to hypothesize that if tissue transglutaminase levels and activity are up-regulated under such conditions, the protein cross-linking could be likely involved in excitotoxic response.