Ilaria Tamburini

Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma.

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by α-tocopherol. We show here that α-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, α-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3′-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by α-tocopherol. However, α-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-γ (PPARγ; troglitazone), indicating that this pathway is susceptible to α-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and α-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARγ element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARγ and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARγ signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.

Ageing and oxidative stress: A role for dolichol in the antioxidant machinery of cell membranes?

Dolichol is a polyprenol compound broadly distributed in membranes, biosynthetized by the general isoprenoid pathway from acetate via mevalonate and farnesyl pyrophosphate. Dolichol lays inside the membrane between the two leaflets of the lipid bilayer very close to the tail of phospholipid fatty acids. No definite catabolic pathways for this molecule have yet been identified. Evidence is produced that dolichol levels increase dramatically with increasing age; that anti-ageing caloric restriction retards this age-associated change; that dolichol may act as a radical scavenger of peroxidized lipids belonging to the cell membranes. In view of the polyunsaturated fatty acids (PUFA), dolichol and Vitamin E location and stechiometry, it is proposed that molecules might interact each-other to form a highly matched free-radical-transfer chain, whose malfunctioning might be involved in statin toxicity and neurodegenerative diseases.

Effects of dietary restriction on age-related changes in the phospholipid fatty acid composition of various rat tissues

Background and aims: Polyunsaturated fatty acids (PUFAs) are essential components of the cell lipid bilayer and are involved in membrane fluidify and normal functioning, but they are vulnerable to free radical attack. Given the role of oxidative stress in the aging process, age-related changes in phospholipid fatty acid (PLFA) composition in rat liver, kidney and heart were assessed in 3-, 12- and 24-month-old rats fed either ad libitum but only every other day, or daily but only 60% of the quantity normally consumed by age-matched controls. Methods: Lipids were extracted and phospholipids (PLs) were separated using the solid phase extraction technique, then transesterified and assayed by gas-liquid chromatography. Results: Saturated fatty acids (FAs) did not change significantly with age; mono- and bi-unsaturated FAs decreased in the liver and heart, and the ratio of the former to the latter increased in the liver, kidney and heart. PUFAs increased in the liver and heart. As regards individual FAs, 20:1(n-9) decreased in all organs, 14:1 and 18:1(n-7) increased in the kidney and heart, 18:1(n-9) increased in the kidney, 20:2(n-6), 18:2(n-6) and 22:5(n-3) decreased in the liver and heart, 20:3(n-6) decreased in the kidney and increased in the heart. The most abundant PUFAs, 20:4(n-6) and 22:6(n-3), either remained the same or increased with age. The N-9 family increased in the kidney, the N-7 family increased in the kidney and heart, the N-6 family decreased in all three organs, and the N-3 family increased in the liver and kidney. Dietary restriction (DR) significantly counteracted most of these changes, but changes in some FAs [20:2(n-6) in the heart] were magnified by DR and may not be age-related. Conclusions: Most age-related changes (that occurred in the rat liver, kidney and heart and were counteracted by the two different types of DR) may be involved in the mechanism of aging.

MDMA Induces Caspase‐3 Activation in the Limbic System but not in Striatum

Abstract:  Several studies, carried out in chronic (+/−) 3,4‐methylenedioxymethamphetamine (MDMA) abusers, have shown memory loss and cognitive impairment, as well as persistent electroencephalographic changes. This suggests that, at least in humans, forebrain areas, including the limbic system, might be altered by MDMA. Consistently, recent experimental evidences suggest that, in rodents, MDMA, besides effects on the basal ganglia, produces alterations in the hippocampus. Therefore, the aim of the present article was to investigate whether treatment with MDMA produces activation of the caspase‐3 enzyme, which is part of an enzymatic pathway involved in cell death, within limbic areas (i.e., hippocampus, amygdala, and piriform cortex) and striatum. A marked induction of caspase‐3 activity was demonstrated in the amygdala and hippocampus, although MDMA did not affect caspase‐3 activity neither in the striatum nor in the frontal cortex. These data indicate that limbic structures possess a high sensitivity to MDMA with respect to the activation of at least one step in the apoptotic pathway. Potential implications and pitfalls of such an experimental observation are reported.

New perspectives for (S)-dolichol and (S)-nordolichol synthesis and biological functions

A procedure is described for the preparation of (S)-dolichol and (S)-nordolichol starting from the polyprenyl fraction extracted fromGingko biloba integer or exhausted leaves. The procedure appears extremely valuable in obtaining the two chiral isoprenoid compounds in good chemical yields and retention of a high degree of enantiomeric excess. Also, the (S)-nordolichol represents a good chiral precursor for the preparation of 14C-labelled (S)-dolichol to be used in biological investigations into the (S)-dolichol catabolism in the functional living cell. Furthermore, the possible role of (S)-dolichol as a free radical scavenger in the cell membrane was preliminarily evaluated by means of a 1H-NMR analytical method. Apparently, experimental results substantiate this hypothesis.

The effect of carbon tetrachloride and ultraviolet radiation on dolichol levels in liver cells isolated from 3- and 24-month-old male Sprague-Dawley rats

Dolichol (D) is a long-chain polyprenoid broadly distributed in the cell membranes, possibly endowed with a free-radical scavenging activity, whose concentration in tissues increases with increasing age. No enzyme pathway for D degradation has been discovered. In order to test the hypothesis that D might undergo a non-enzymatic free-radical mediated decomposition the effects of a xenobiotic agent (carbon tetrachloride, CCl4) and ultraviolet-B (UV-B) radiation on D levels were studied in liver cells isolated from male ad libitum fed Sprague-Dawley rats aged 3 or 24 months. Liver cells (90 mg/ml) were incubated in sealed flasks (6 ml cell suspension each) for 0, 5, 10and 20 min after the addition of 25, 50 or 200μl CCl4 in the central well. 50 ml of a 6 mg/ml liver cell suspension were poured in a 120 cm2 Petri dish and the sediment liver cell monolayer was exposed to UV Bradiation for 0, 5, 10, 20 and 40 min. At the given time, cells were taken and D was extracted and assayed by the HPLC procedure. D levels were remarkably higher in older than in younger cells as expected (P < 0.001). Treatment with CCl4 and UVB caused a highly significant decrease in D (P < 0.001) whose percentage was larger in younger than in older cells. The conclusions are that free-radicals generated either by chemical or by physical agents cause a very rapid depletion of D in liver cells, and that the effect of the free radical attack on D decomposition may be lower percentage wise in older than in younger cells, which might account at least in part for the accumulation of D in older tissues.

Autophagy activation in glutamate-induced motor neuron loss

Recent literature demonstrated that exposure to excitatory amino acid in specific experimental conditions might produce a defect in the autophagy pathway. Such an effect was observed in motor neurons exposed chronically to glutamate agonists. On the other hand, it is well known that glutamate induces motor neuron death and this is supposed to play a key role in the physiopathology of motor neuron loss in amyotrophic lateral sclerosis (ALS). Similarly, a defective recruitment of autophagy was recently documented in ALS. In the present study we found that exposure of motor neurons to kainic acid produces intracellular changes associated with defective autophagy. In this experimental conditions, pharmacological activation of autophagy rescues the loss of motor neurons.

Deficiency and supplementation of PUFA in the diet have similar effects on the age-associated changes in rat-plasma cholesterol levels.

Levels of plasma cholesterol, particularly LDL cholesterol, increase with increasing age in humans and rodents. Feeding a fish oil-rich diet may exert hypocholesterolemic effects. The aim of this work was to examine the effects of a life-long administration of a PUFA-enriched diet and of a PUFA-deficient diet in male Sprague-Dawley rats on the age-associated increases in plasma cholesterol and triglycerides. Diet had small effects on body-weight, and had dramatic effects on liver phospholipids-fatty acids. Surprisingly, both diets counteracted the age-associated changes in plasma cholesterol and triglycerides similarly and benefits were already visible in adult rats.

The relevance of autophagy in the dopaminergic effects of PTEN-induced kinase I (PINK-I)

Autophagy is a main cellular mechanism to remove misfolded proteins and aggregates as well as damaged and dysfunctional organelles, which recently emerged as critical for neuroprotection. In fact, in several disease models suppression of autophagy produces cell death in vitro and in vivo, while autophagy inducers reduce protein aggregates and apoptotic cell death. Recently, PINK1, a kinase mutated in autosomal recessive Parkinson’s disease, was shown to be neuroprotective. Distinct PINK1 isoforms are known to be active outside and within mitochondria to prevent mitochondrial damage and apoptosis and to regulate mitochondria dynamics. Moreover PINK1 also interacts with the pro-autophagic protein Beclin1 and positively regulates basal and starvationinduced autophagy, a key protective mechanism against neuronal cell loss. The aims of this study was to characterize the multiple roles of PINK1 in protecting dopaminergic cells from neurodegeneration. For this reasons this study is articulated in the following steps, to define: (i) whether PINK1 interacts with the pro-autophagic proteins Beclin1and parkin to modulate autophagy; (ii) how PINK1 modulates autophagy/ mitophagy The effect of PINK1 wt, mutant and siRNA on autophagy and apoptotic pathways and on mitochondria dynamics is characterized through a range of complementary techniques using in vitro models of neurodegeneration. Using in vitro models, we showed that PINK1 wild type (wt), but not mutants, strongly interacts with Beclin1. We also found that PINK1wt positively modulates autophagy in baseline conditions through a fairly specific promotion of mitochondrial turn over which keeps mitochondrial function and morphology despite neurotoxic insults. These results highlight a novel role of PINK1 acting as a strategic cellular sensor of mitochondrial damage. This study helps understand the interconnection between mitochondrial dysfunction and DA cell death.

Neuroanatomical substrates for recurrent epileptic limbic seizures

Epilepsy is a neurological disorder characterized by the recurrence of spontaneous, unprovoked epileptic seizures. Mesial temporal lobe epilepsy (more briefly, MTLE) is a very common form of epilepsy which is featured by the occurrence of focal limbic seizures, and associated to a specific neuropathological alteration, the so-called Ammon’s horn sclerosis, whose main features are a selective loss of the CA1 and CA3/4 section of the Ammon’s horn (CA, from Latin Cornu Ammonis, abbreviated as CA), a selective cell loss of inhibitory interneurons in the hilus of the dentate gyrus (DG), and the abnormal sprouting of granule cells mossy fibers (the so called mossy fiber sprouting, MFS). The onset of spontaneous seizures (SRS) is the hallmark of a good model of epilepsy. For temporal lobe epilepsy (TLE), the most used models consist in administering systemically chemoconvulsants inducing limbic status epilepticus (i.e. seizures lasting for more than 30’, SE) and evaluating the occurrence of SRS. However, in these models, the widespread involvement of different structures which complicates the interpretation of experimental findings: limbic seizures and status epilepticus (SE) can be triggered by focal infusion of chemoconvulsants within anterior piriform cortex (abbreviated as APC) This brain region is densely innervated by noradrenergic fibers arising from the locus coeruleus (LC), and we recently showed that a lesion of LC (induced by a selective neurotoxin, DSP-4, i.p.), induces SE. LC plays a critical role in modulating several models of seizures, and it plays a critical role in plastic mechanisms and neuroprotection in the brain. Thus, we compared the group DSP-4+bicuculline and cyclothiazide+bicuculline, to evaluate whether the focal SE evoked from the APC is capable of inducing SRS and AHS, and whether LC plays a significant role in this phenomena. We found that: the loss of LC induced: (i)a higher incidence of SRS; (ii) cell loss in the hippocampal DG hilus and CA3 (iii)the loss of parvalbumin-positive neurons. In conclusion, our study confirms that focal induction of SE from the APC represents a good model of TLE, and that NE released from the fibers originating from the LC plays a significant role both in the hippocampal damage occurring after SE, and in the incidence of SRS.