Gianluca Miglio

Characterization of ionotropic glutamate receptors in human lymphocytes.

The effect of L‐glutamate (Glu) on human lymphocyte function was studied by measuring anti‐CD3 monoclonal antibody (mAb) or phytohaemagglutinin (PHA)‐induced intracellular Ca2+ ([Ca2+]i) rise (Fura‐2 method), and cell proliferation (MTT assay). Glu (0.001 – 100 μM) did not modify basal lymphocyte [Ca2+]i, but significantly potentiated the effects of anti‐CD3 mAb or PHA. Maximal [Ca2+]i rises over resting cells were: 165±8 and 247±10 nM at 3.0×10−2 mg ml−1 anti‐CD3 mAb; 201±4 and 266±9 nM at 5.0×10−2 mg ml−1 PHA, in the absence or presence of 1 μM Glu, respectively. The Glu effect showed a bell‐shape concentration‐dependent relationship, with a maximum (+90±3% for anti‐CD3 mAb and +57±2% for PHA over Glu‐untreated cells) at 1 μM. Non‐NMDA receptor agonists (1 μM) showed a greater efficacy (+76±2% for (S)‐AMPA; +78±4% for KA), if compared to NMDA (+46±2%), or Glu itself. Ionotropic Glu receptor antagonists completely inhibited the effects of the corresponding specific receptor agonists (1 μM). The IC50 values calculated were: 0.9 μM for D‐AP5; 0.6 μM for (+)‐MK801; 0.3 μM for NBQX. Both NBQX and KYNA were able to abolish Glu effect. The IC50s calculated were: 3.4 μM for NBQX; 0.4 μM for KYNA. Glu (0.1 – 1 mM) did not change the resting cell proliferation, whereas Glu (1 mM) significant inhibited (−27±4%) PHA (1.0×10−2 mg ml−1)‐induced lymphocyte proliferation at 72 h. In conclusion, human lymphocytes express ionotropic Glu receptors functionally operating as modulators of cell activation.

Clovamide and rosmarinic acid induce neuroprotective effects in in vitro models of neuronal death

Background and purpose:  Phenolic compounds exert cytoprotective effects; our purpose was to investigate whether the isosteric polyphenolic compounds clovamide and rosmarinic acid are neuroprotective.

PPARγ stimulation promotes mitochondrial biogenesis and prevents glucose deprivation-induced neuronal cell loss

Peroxisome proliferator-activated receptor (PPAR)gamma stimulation provides protection in several models of neurological disorders, but the mechanisms underlying these effects remain to be fully elucidated. Here we have studied whether two PPARgamma agonists, pioglitazone and rosiglitazone, prevent loss of differentiated SH-SY5Y cells transiently exposed to glucose deprivation (GD). Nanomolar drug concentrations prevented GD-induced cell loss in a concentration- and time-dependent manner. These effects were abolished by malonate, a reversible mitochondrial Complex II inhibitor, while significantly potentiated by pyruvate, thus suggesting that they are related to mitochondrial function. During cell pretreatment, PPARgamma agonists promoted biogenesis of functional mitochondria, as indicated by the up-regulation of PPARgamma coactivator (PGC)-1alpha, NRF1, TFAM, cytochrome c oxidase subunit (CO) I and CO IV, and the increased level of mtDNA, while did not significantly change mitochondrial membrane potential. In addition, the analysis of the concentration-response and time-course curves for the protective effects and the up-regulation of mitochondrial biogenesis markers suggests that mitochondrial biogenesis and cell loss prevention are related effects. In conclusion our data indicate that a prolonged PPARgamma stimulation, by repeated administration of nanomolar pioglitazone or rosiglitazone concentrations, decreases GD-induced loss of differentiated SH-SY5Y cells. In addition, they suggest that mitochondrial biogenesis may contribute to these effects.

Pioglitazone improves lipid and insulin levels in overweight rats on a high cholesterol and fructose diet by decreasing hepatic inflammation

Background and purpose:  Nutrient overload leads to obesity and insulin resistance. Pioglitazone, a selective peroxisome proliferator‐activated receptor (PPAR)γ agonist, is currently used to manage insulin resistance, but the specific molecular mechanisms activated by PPARγ are not yet fully understood. Recent studies suggest the involvement of suppressor of cytokine signalling (SOCS)‐3 in the pathogenesis of insulin resistance. This study aimed to investigate the hepatic signalling pathway activated by PPARγ activation in a non‐genetic insulin‐resistant animal model.

TREATMENT WITH THE GLYCOGEN SYNTHASE KINASE-3β INHIBITOR, TDZD-8, AFFECTS TRANSIENT CEREBRAL ISCHEMIA/REPERFUSION INJURY IN THE RAT HIPPOCAMPUS

The serine/threonine glycogen synthase kinase 3&bgr; (GSK-3&bgr;) is abundant in the central nervous system, particularly in the hippocampus, and plays a pivotal role in the pathophysiology of a number of diseases, including neurodegeneration. This study was designed to investigate the effects of GSK-3&bgr; inhibition against I/R injury in the rat hippocampus. Transient cerebral ischemia (30 min) followed by 1 h of reperfusion significantly increased generation of reactive oxygen species and modulated superoxide dismutase activity; 24 h of reperfusion evoked apoptosis (determined as mitochondrial cytochrome c release and Bcl-2 and caspase-9 expression), resulted in high plasma levels of TNF-&agr; and increased expression of cyclooxygenase-2, inducible nitric oxide synthase, and intercellular adhesion molecule-1. The selective GSK-3&bgr; inhibitor, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), was administered before and after ischemia or during reperfusion alone to assess its potential as prophylactic or therapeutic strategy. Prophylactic or therapeutic administration of TDZD-8 caused the phosphorylation (Ser9) and hence inactivation of GSK-3&bgr;. Infarct volume and levels of S100B protein, a marker of cerebral injury, were reduced by TDZD-8. This was associated with a significant reduction in markers of oxidative stress, apoptosis, and the inflammatory response resulting from cerebral I/R. These beneficial effects were associated with a reduction of I/R-induced activation of the mitogen-activated protein kinases JNK1/2 and p38 and nuclear factor-&kgr;B. The present study demonstrates that TDZD-8 protects the brain against I/R injury by inhibiting GSK-3&bgr; activity. Collectively, our data may contribute to focus the role of GSK-3&bgr; in cerebral I/R.

Peroxisome proliferator-activated receptor β/δ agonism protects the kidney against ischemia/reperfusion injury in diabetic rats

Diabetes is an important risk factor for ischemic acute kidney injury, whose pharmacological treatment remains an unmet medical need. The peroxisome proliferator-activated receptor (PPAR) β/δ is highly expressed in the kidney, although its role has not yet been elucidated. Here, we used an in vivo model of renal ischemia/reperfusion (I/R) in streptozotocin-induced diabetic rats (i) to evaluate whether diabetes increases kidney susceptibility to I/R injury and (ii) to investigate the effects of PPARβ/δ activation. The degree of renal injury (1h ischemia/6h reperfusion) was significantly increased in diabetic rats compared with nondiabetic littermates. PPARβ/δ expression was increased after I/R, with the highest levels in diabetic rats. Administration of the selective PPARβ/δ agonist GW0742 attenuated the renal dysfunction, leukocyte infiltration, and formation of interleukin-6 and tumor necrosis factor-α. These effects were accompanied by an increased expression of the suppressor of cytokine signaling (SOCS)-3, which plays a critical role in the cytokine-activated signaling pathway. The beneficial effects of GW0742 were attenuated by the selective PPARβ/δ antagonist GSK0660. Thus, we report herein that PPARβ/δ activation protects the diabetic kidney against I/R injury by a mechanism that may involve changes in renal expression of SOCS-3 resulting in a reduced local inflammatory response.

Group I mGlu receptor stimulation inhibits activation-induced cell death of human T lymphocytes.

1 The effects of L‐glutamate on activation‐induced cell death (AICD) of human activated (1 μg ml−1 phytohemagglutinin plus 2 U ml−1 interleukin‐2; 8 days) T lymphocytes were studied by measuring anti‐CD3 monoclonal antibody (10 μg ml−1; 18 h)‐induced cell apoptosis (Annexin V and propidium iodide staining). 2 L‐Glutamate (1 × 10−8–1 × 10−4 M) significantly (P0.01) inhibited AICD in a concentration‐dependent manner (EC50=6.3 × 10−8 M; maximum inhibition 54.8±6.3% at 1 × 10−6 M). 3 The L‐glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 × 10−7 M for (1S,3R)‐ACPD; 4.5 × 10−8 M for quisqualate; 1.0 × 10−6 M for (S)‐3,5‐DHPG; 2.0 × 10−5 M for CHPG. 4 Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 × 10−6 M. The IC50 values calculated were: 8.7 × 10−5, 4.3 × 10−6 and 6.3 × 10−7 M for AIDA, LY 367385 and MPEP, respectively. 5 L‐Glutamate (1 × 10−6 M; 18 h) significantly (P0.05) inhibited FasL expression (40.8±11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. 6 Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T‐cell lines, as demonstrated by reverse transcriptase–PCR analysis, suggests that L‐glutamate‐mediated inhibition of AICD was exerted on T cells. 7 These data depict a novel role for L‐glutamate in the regulation of the immune response through group I mGlu receptor‐mediated mechanisms.

Cabergoline prevents necrotic neuronal death in an in vitro model of oxidative stress

To study if cabergoline, a long-lasting specific dopamine D2 receptor agonist, has neuroprotective effects against oxidative stress, we exposed (3 h) SH-SY5Y human neuroblastoma cells to tert-butylhydroperoxide (t-BOOH; 500 microM). t-BOOH caused a 42+/-4% neuronal death, which was prevented by cabergoline (2 h before) in a concentration-dependent manner (EC(50): 1.24 microM). This effect was not antagonised by haloperidol (concentration up to 10 microM), and was associated with an increased availability of intracellular GSH contents (+30+/-11%) and a decrease in the membrane lipid peroxidation (-23+/-9%). Our data suggest that cabergoline has neuroprotective effects useful for Parkinsons disease therapy.

The subtypes of peroxisome proliferator‐activated receptors expressed by human podocytes and their role in decreasing podocyte injury

BACKGROUND AND PURPOSE Peroxisome proliferator‐activated receptors (PPARs) are ligand‐activated transcription factors, and three subtypes (α, β and γ) have been identified. PPAR activation has been reported to decrease renal injury and markers of glomerular dysfunction in models of renal ischemia/reperfusion (I/R). However, both the I/R effects and the effects of PPAR agonists on podocytes, an integral cellular part of the glomerular filtration barrier, remain to be established.

PPARγ stimulation promotes neurite outgrowth in SH-SY5Y human neuroblastoma cells

Several evidences indicate that PPARgamma stimulation promotes neuronal differentiation. However, to date, no data describe the effects of PPARgamma agonists on neurite outgrowth. Here we have evaluated the effects of pioglitazone, a synthetic PPARgamma agonist, on differentiation and neurite outgrowth in SH-SY5Y human neuroblastoma cells. Our results show that pioglitazone promotes cell differentiation and the outgrowth of cell processes in a concentration-dependent manner with the maximal effect at 100 nM-1 microM. It significantly increases both the mean process length and the percentage of neurite-bearing cells. In addition, these effects are accompanied by significant activation of p42 and p44 mitogen-activated protein kinases. In conclusion, albeit preliminary, these findings suggest the possibility that PPARgamma stimulation may contribute to the development and maintenance of a proper neuronal connectivity within neuronal networks.