Paola Palestini

Pin1, a new player in the fate of HIF-1α degradation: an hypothetical mechanism inside vascular damage as Alzheimer’s disease risk factor

Aetiology of neurodegenerative mechanisms underlying Alzheimer’s disease (AD) are still under elucidation. The contribution of cerebrovascular deficiencies (such as cerebral ischemia/stroke) has been strongly endorsed in recent years. Reduction of blood supply leading to hypoxic condition is known to activate cellular responses mainly controlled by hypoxia-inducible transcription factor-1 (HIF-1). Thus alterations of oxygen responsive HIF-1α subunit in the central nervous system may contribute to the cognitive decline, especially influencing mechanisms associated to amyloid precursor protein (APP) amyloidogenic metabolism. Although HIF-1α protein level is known to be regulated by von Hippel-Lindau (VHL) ubiquitin-proteasome system, it has been recently suggested that glycogen synthase kinase-3β (Gsk-3β) promotes a VHL-independent HIF-1α degradation. Here we provide evidences that in rat primary hippocampal cell cultures, HIF-1α degradation might be mediated by a synergic action of Gsk-3β and peptidyl-prolyl cis/trans isomerase (Pin1). In post-ischemic conditions, such as those mimicked with oxygen glucose deprivation (OGD), HIF-1α protein level increases remaining unexpectedly high for long time after normal condition restoration jointly with the increase of lactate dehydrogenase (LDH) and β-secretase 1 (BACE1) protein expression (70 and 140% respectively). Interestingly the Pin1 activity decreases about 40–60% and Pin1S16 inhibitory phosphorylation significantly increases, indicating that Pin1 binding to its substrate and enzymatic activity are reduced by treatment. Co-immunoprecipitation experiments demonstrate that HIF-1α/Pin1 in normoxia are associated, and that in presence of specific Pin1 and Gsk-3β inhibitors their interaction is reduced in parallel to an increase of HIF-1α protein level. Thus we suggest that in post-OGD neurons the high level of HIF-1α might be due to Pin1 binding ability and activity reduction which affects HIF-1α degradation: an event that may highlight the relevance of ischemia/HIF-1α as a risk factor in AD pathogenesis.

A photo-reactive derivative of ganglioside GM1 specifically cross-links VIP21-caveolin on the cell surface

Previous studies have shown that sphingolipids may be enriched in caveolae, plasmalemmal invaginations implicated in endocytosis and signal transduction. We synthesised a radiolabeled derivative of ganglioside GM1 bearing a photo‐reactive cross‐linker at the end of its acyl chain. After insertion in the plasma membrane of cultured A431 or MDCK cells and photoactivation, the main protein cross‐linked by the GM1 derivative was VIP21‐caveolin, an essential structural component of caveolae. This result shows close proximity between GM1 molecules and VIP21‐caveolin in the caveolar membrane and strongly implicates sphingolipid segregation in the biogenesis of caveolae.

Glycolipid-Enriched Caveolae and Caveolae-Like Domains in the Nervous System

Abstract: Recent years have been characterized by a booming interest in research on caveolae and caveolae‐like membrane domains. The interest in this subject grew further, when their involvement in fundamental membrane‐associated events, such as signal transmission and lipid/protein sorting, was postulated. Substantial progress has been reached in understanding the biological role of membrane domains in eukaryotic cells. The neuron, however, which perhaps represents one of the greatest challenges to research on membrane traffic and function, has only been partially investigated. The purpose of the present review is to survey this issue in the nervous system. We confine ourselves to the presence of membrane domains in the nervous system and discuss this in the context of three facts: first, glycolipids are peculiarly enriched in both caveolae and caveolae‐like domains and are particularly abundant in the nervous system; second, the neuron is characterized by a basic dual polarity, similar in this respect to other polarized cells, where the role of glycolipid‐enriched domains for lipid/protein sorting has been better ascertained; and third, neurons evolved from, and are related to, simpler eukaryotic cells, allowing us to find analogies with more investigated nonneuronal cells.

Changes in the Ceramide Composition of Rat Forebrain Gangliosides with Age

Abstract: Five major gangliosides (GM1, GD1a, GDlb, GTlb, and GQlb) were extracted and isolated by normal‐phase HPLC from the forebrain of Sprague‐Dawley rats of ages ranging from 3 days to 24 months. Each ganglioside was fractionated by reverse‐phase HPLC into the molecular species carrying a single long‐chain base moiety. At all ages, the C18:1 and C20:l long‐chain base species predominated, whereas the C18:0 and C20:0 ones represented 1–3% of the total. The C18:l long‐chain base species, predominant at 3 days (91–96%), diminished with age and reached, at 2 years, 73%, 65%, 61%, 59%, and 45% of the total for GDla, GM1, GTlb, GDlb, and GQlb, respectively. The content of the C20:1 long‐chain base species, low at birth (4–9%), increased with age in all gangliosides and reached, at 2 years, 27–55% of the total. The developmental behavior of the ganglioside species containing the C18:1 long‐chain base was characterized by the following: (a) a biphasic profile with a maximum around 15 days for GD1a, the most abundant ganglioside at all ages; (b) an increase until 6 months for GM1; (c) a sharp decrease until 30 days, followed by leveling for GTlb; and (d) a low, constant level for GDlb and GQlb. All the ganglioside species containing the C20:1 long‐chain base showed a constant increase during development, the increase being more marked in the first 30 days. The molecular species of all gangliosides carrying the C18:1 long‐chain base were virtually devoid of 20:0 fatty acids and carried a higher content of 18:0 fatty acids than the corresponding C20:l long‐chain base species (average 80 versus 57%). Moreover, in the C18:1 long‐chain base species, the 18:0 fatty acid content diminished with age from 89 to 72%, with a concurrent increase of 16:0 and 18:1 fatty acids, whereas the C20:l long‐chain base species had an age‐constant fatty acid composition.

Comparative acute lung inflammation induced by atmospheric PM and size-fractionated tire particles.

A comparison of the effects produced by size-fractionated tire particles (TP10 and TP2.5) and similar-sized urban particulate matter (PM10 and PM2.5), collected in Milan in 2007, on the lungs of mice has been performed. The focus is on early acute lung responses following intratracheal instillation of aerosolized particles at a 3-h recovery period. Together with bronchoalveolar lavage (BAL) conventional endpoints like total and differential cell counts, total protein, alkaline phosphatase, lactate dehydrogenase and pro-inflammatory cytokines (TNF-alpha, MIP-2), the expression of different stress protein markers (caspase8, Hsp70, H0-1, NF-kB) was evaluated 3h after particle instillation into Balb/c mice. The TP2.5 fraction reached the alveolar spaces and produced an acute inflammatory response as evidenced by increased LDH and AP activities, total protein and Hsp70 content. TNF-alpha and MIP-2 production was significantly increased and polymorphonuclear neutrophils (PMN) recruitment was apparent. The TP10 fraction distributed mainly in the bronchial district and the only modified BAL parameter was the expression of MIP-2. PM2.5 induced an inflammatory response lesser in magnitude than that produced by PM10 fraction. The TNF-alpha increase was not significant, and HO-1, though significantly increased with respect to the control, was unable to reduce NF-kB activation, suggesting a role of the endotoxin component of PM in stimulating a pro-inflammatory limited response. This response was maximized by the PM10 that induced a significant increase in MIP-2, TNF-alpha, and HO-1. Lung immunohistochemistry showed fine particles, TPs in particular, being able to deeply penetrate and rapidly induce inflammatory events in the parenchyma, even involving endothelial cells, while PM10 produced a strong pro-inflammatory response mediated by the bronchiolar cells and residential macrophages of the proximal alveolar sacs, likely as a consequence of its larger dimension and endotoxin content. These results provide evidence of variable inflammatory mechanisms in mouse lungs in response to both urban PM and tire particles.

Influence of endogenous GM1 ganglioside on TrkB activity, in cultured neurons.

We verified the hypothesis that changes in the endogenous GM1 ganglioside density in the environment of TrkB, receptor of brain‐derived neurotrophic factor, can affect receptor activity, and focused on rat cerebellar granule cells expressing both GM1 and TrkB. Changes of the amount of GM1 associated to immunoprecipitated TrkB and of receptor tyrosine phosphorylation were evaluated after treatment with phorbol‐12‐myristate‐13‐acetate (1 μM, 7 min), reported to affect the plasma membrane distribution of endogenous gangliosides in the same cells. After treatment, the amount of GM1 associated to receptor and TrkB phosphorylation decreased by about 40%. The amount of associated GM1 decreased by about 33% also after concomitant treatment with phorbol ester and brain‐derived neurotrophic factor, but in this case the neurotrophin was unable to enhance receptor tyrosine phosphorylation. These results for the first time suggest that changes in the amount of endogenous GM1 in the environment of TrkB can modulate receptor activity, and offer new clues for a better understanding of physiological and pathological events of the nervous system.

Milano Summer Particulate Matter (PM10) Triggers Lung Inflammation and Extra Pulmonary Adverse Events in Mice

Recent studies have suggested a link between particulate matter (PM) exposure and increased mortality and morbidity associated with pulmonary and cardiovascular diseases; accumulating evidences point to a new role for air pollution in CNS diseases. The purpose of our study is to investigate PM10sum effects on lungs and extra pulmonary tissues. Milano PM10sum has been intratracheally instilled into BALB/c mice. Broncho Alveolar Lavage fluid, lung parenchyma, heart and brain were screened for markers of inflammation (cell counts, cytokines, ET-1, HO-1, MPO, iNOS), cytotoxicity (LDH, ALP, Hsp70, Caspase8-p18, Caspase3-p17) for a putative pro-carcinogenic marker (Cyp1B1) and for TLR4 pathway activation. Brain was also investigated for CD68, TNF-α, GFAP. In blood, cell counts were performed while plasma was screened for endothelial activation (sP-selectin, ET-1) and for inflammation markers (TNF-α, MIP-2, IL-1β, MPO). Genes up-regulation (HMOX1, Cyp1B1, IL-1β, MIP-2, MPO) and miR-21 have been investigated in lungs and blood. Inflammation in the respiratory tract of PM10sum-treated mice has been confirmed in BALf and lung parenchyma by increased PMNs percentage, increased ET-1, MPO and cytokines levels. A systemic spreading of lung inflammation in PM10sum-treated mice has been related to the increased blood total cell count and neutrophils percentage, as well as to increased blood MPO. The blood-endothelium interface activation has been confirmed by significant increases of plasma ET-1 and sP-selectin. Furthermore PM10sum induced heart endothelial activation and PAHs metabolism, proved by increased ET-1 and Cyp1B1 levels. Moreover, PM10sum causes an increase in brain HO-1 and ET-1. These results state the translocation of inflammation mediators, ultrafine particles, LPS, metals associated to PM10sum, from lungs to bloodstream, thus triggering a systemic reaction, mainly involving heart and brain. Our results provided additional insight into the toxicity of PM10sum and could facilitate shedding light on mechanisms underlying the development of urban air pollution related diseases.

Lung toxicity induced by intratracheal instillation of size-fractionated tire particles

Tire particles (TP) represent a significant component of urban air pollution (PM), constituting more than 10% of PM10 mass at urban locations with heavy traffic. The purpose of this study was to evaluate the effects of size-fractionated TP in an animal exposure model frequently used to assess the health effects of air pollutants. Potential pro-inflammatory and toxic effects of TP2.5 (<2.5 microm) and TP10 (<10 microm) were investigated through instillation of suspensions of these materials in BALB/c mice. Bronchoalveolar lavage fluid (BALF) was screened for total protein, lactate dehydrogenase (LDH), alkaline phosphatase (AP), and beta-glucuronidase (B-Gluc) as markers of cytotoxicity; glutathione (GSH) and superoxide dismutase (SOD) as markers of oxidative potential; and tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), and inflammatory cells as markers of inflammation. Concomitantly, histological analysis of TP-exposed lungs was performed. A single intratracheal instillation of 10 microg/100 microl, 100 microg/100 microl or 200 microg/100 microl was performed, and after 24h mice were euthanized and BALF examined. Inflammatory cellular profiles showed dose-dependent responses after TP10 exposure, while strong cytotoxic effects, including increases in total protein, LDH and AP, were observed to be associated to TP2.5 exposure. Histologically, TP10-treated lungs mainly showed inflammatory tissue infiltration, in contrast to TP2.5-treated lungs, where lysis of the alveolar barrier appeared to be the most characteristic lesion. Our biochemical, cytological, and histological results indicated differential lung toxicity mechanisms elicited by size-fractionated TP, in agreement with other studies performed in in vivo systems that have shown that lung responses to inhaled or instilled particles are affected by particle size. We conclude that lung toxicity induced by TP10 was primarily due to macrophage-mediated inflammatory events, while toxicity induced by TP2.5 appeared to be related more closely to cytotoxicity.

Immunoseparation of Prion protein-enriched domains from other detergent-resistant membrane fractions, isolated from neuronal cells

The possibility of coexistence of different subtypes of membrane lipid rafts has been investigated in cerebellar granule cells, by submitting detergent‐resistant membrane fractions to immunoprecipitation. Among the proteins and lipids present in detergent‐resistant fractions, almost all Prion protein, GAP43 and PKC were present in the immunoprecipitate obtained with anti‐GAP43 or anti‐Prion protein antibody at 4°C, together with a small fraction of cholesterol and sphingolipids, suggesting that they belong to a distinct subset of membranes. On the contrary, all Fyn and almost all MARCKS remained in the supernatant. Fluorescence microscopy experiments showed that Fyn and Prion protein were mostly not colocalized within a single neuron. Our results suggest that granule cells membranes contains different subtypes of detergent‐resistant fractions, possibly deriving from different lipid rafts.

Health Risk Assessment for Air Pollutants: Alterations in Lung and Cardiac Gene Expression in Mice Exposed to Milano Winter Fine Particulate Matter (PM2.5)

Oxidative stress, pulmonary and systemic inflammation, endothelial cell dysfunction, atherosclerosis and cardiac autonomic dysfunction have been linked to urban particulate matter exposure. The chemical composition of airborne pollutants in Milano is similar to those of other European cities though with a higher PM2.5 fraction. Milano winter fine particles (PM2.5win) are characterized by the presence of nitrate, organic carbon fraction, with high amount of polycyclic aromatic hydrocarbons and elements such as Pb, Al, Zn, V, Fe, Cr and others, with a negligible endotoxin presence. In BALB/c mice, we examined, at biochemical and transcriptomic levels, the adverse effects of repeated Milano PM2.5win exposure in lung and heart. We found that ET-1, Hsp70, Cyp1A1, Cyp1B1 and Hsp-70, HO-1, MPO respectively increased within lung and heart of PM2.5win-treated mice. The PM2.5win exposure had a strong impact on global gene expression of heart tissue (181 up-regulated and 178 down-regulated genes) but a lesser impact on lung tissue (14 up-regulated genes and 43 down-regulated genes). Focusing on modulated genes, in lung we found two- to three-fold changes of those genes related to polycyclic aromatic hydrocarbons exposure and calcium signalling. Within heart the most striking aspect is the twofold to threefold increase in collagen and laminin related genes as well as in genes involved in calcium signaling. The current study extends our previous findings, showing that repeated instillations of PM2.5win trigger systemic adverse effects. PM2.5win thus likely poses an acute threat primarily to susceptible people, such as the elderly and those with unrecognized coronary artery or structural heart disease. The study of genomic responses will improve understanding of disease mechanisms and enable future clinical testing of interventions against the toxic effects of air pollutant.