Laura Botto

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.

Role of lipid rafts and GM1 in the segregation and processing of prion protein.

The prion protein (PrPC) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrPC is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrPC raft association, together with raft lipid composition, appears essential for the conversion of PrPC into the scrapie isoform PrPSc, and the development of prion disease. Controversial findings were reported on the nature of PrPC-containing rafts, as well as on the distribution of PrPC between rafts and non-raft membranes. We investigated PrPC/ganglioside relationships and their influence on PrPC localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrPC conformations coexist: in lipid rafts PrPC is present in the native folding (α-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrPSc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrPC conformation and interaction with lipid bilayers, without modifying PrPC distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.

Hypoxia-Induced Modifications in Plasma Membranes and Lipid Microdomains in A549 Cells and Primary Human Alveolar Cells

We evaluated the response to mild hypoxia exposure of A549 alveolar human cells and of a continuous alveolar cell line from human excised lungs (A30) exposed to 5% O2 for 5 and 24 h. No signs of increased peroxidation and of early apoptosis were detected. After 24 h of hypoxia total cell proteins/DNA ratio decreased significantly by about 20%. Similarly, we found a decrease in membrane phospholipid and cholesterol content. The membrane fluidity assessed by fluorescence anisotropy measurements was unchanged. We also prepared the detergent resistant membrane fraction (DRM) to analyze the distribution of the two types of lipid microdomains, caveolae and lipid rafts. The DRM content of Cav‐1, marker of caveolae, was decreased, while CD55, marker of lipid rafts, increased in both cell lines. Total content of these markers in the membranes was unchanged indicating remodelling of their distribution between detergent‐resistant and detergent‐soluble fraction of the cellular membrane. The changes in protein markers distribution did not imply changes in the corresponding mRNA, except in the case of Cav‐1 for A30 line. In the latter case we found a parallel decrease in Cav‐1 and in the corresponding mRNA. We conclude that an exposure to a mild degree of hypoxia triggers a significant remodelling of the lipid microdomains expression, confirming that they are highly dynamic structures providing a prompt signalling platform to changes of the pericellular microenvironment. J. Cell. Biochem. 105: 503–513, 2008.

Changes in the composition of detergent-resistant membrane domains of cultured neurons following protein kinase C activation

Changes in the composition of cell fractions, and in particular of detergent‐resistant membranes (DRM) isolated from cultured rat cerebellar granule cells, were taken as possible changes in lipid raft composition during a signal transduction event. After activation of protein kinase C (PKC) with phorbol esters (PMA) or glutamate, the content of PKC and of proteins highly enriched (GAP43, Fyn, and PrPc) or not (MARCKS) in DRM was followed. PKC activation strongly increased its association with membranes (from 2% to 75%), causing its enrichment within DRM; the substrate GAP43, enriched in DRM, remained membrane associated, but its proportion in DRM dramatically decreased (from about 40% to 2.5%), suggesting its shift from raft to nonraft membranes, possibly as a consequence of phosphorylation by PKC. The distribution of Fyn and PrPc (DRM‐enriched) and of MARCKS (present mainly outside DRM) did not change. PKC activation was followed by an increase of GAP43 and MARCKS phosphorylation (about 7‐ and 8‐fold, respectively). Noteworthy was that, after cell treatment with the lipid raft‐disrupting drug methyl‐β‐cyclodextrin, PKC activation occurred normally, followed by MARCKS phosphorylation, but GAP43 phosphorylation did not occur. Taken altogether, these data suggest that the integrity of lipid rafts is necessary for PKC to affect GAP43 and catalyze its phosphorylation.

Type-1 Cannabinoid Receptors Reduce Membrane Fluidity of Capacitated Boar Sperm by Impairing Their Activation by Bicarbonate

Background Mammalian spermatozoa acquire their full fertilizing ability (so called capacitation) within the female genital tract, where they are progressively exposed to inverse gradients of inhibiting and stimulating molecules. Methodology/Principal Findings In the present research, the effect on this process of anandamide, an endocannabinoid that can either activate or inhibit cannabinoid receptors depending on its concentration, and bicarbonate, an oviductal activatory molecule, was assessed, in order to study the role exerted by the type 1 cannabinoid receptor (CB1R) in the process of lipid membrane remodeling crucial to complete capacitation. To this aim, boar sperm were incubated in vitro under capacitating conditions (stimulated by bicarbonate) in the presence or in the absence of methanandamide (Met-AEA), a non-hydrolysable analogue of anandamide. The CB1R involvement was studied by using the specific inhibitor (SR141716) or mimicking its activation by adding a permeable cAMP analogue (8Br-cAMP). By an immunocytochemistry approach it was shown that the Met-AEA inhibits the bicarbonate-dependent translocation of CB1R from the post-equatorial to equatorial region of sperm head. In addition it was found that Met-AEA is able to prevent the bicarbonate-induced increase in membrane disorder and the cholesterol extraction, both preliminary to capacitation, acting through a CB1R-cAMP mediated pathway, as indicated by MC540 and filipin staining, EPR spectroscopy and biochemical analysis on whole membranes (CB1R activity) and on membrane enriched fraction (C/P content and anisotropy). Conclusions/Significance Altogether, these data demonstrate that the endocannabinoid system strongly inhibits the process of sperm capacitation, acting as membrane stabilizing agent, thus increasing the basic knowledge on capacitation-related signaling and potentially opening new perspectives in diagnostics and therapeutics of male infertility.

Characterization of prion protein-enriched domains, isolated from rat cerebellar granule cells in culture

The biological functions of prion protein (PrPC) and its possible interaction with other specific molecular membrane partners remain largely unknown. The aim of this study is to gain information on the molecular environment of PrPC by analyzing the lipid and protein composition of a PrPC‐enriched membrane subfraction, called prion domain, PrD. This domain was obtained by immunoprecipitation of detergent‐resistant microdomains (DRM) of rat cerebellar granule cells under conditions designed to preserve lipid‐mediated membrane organization. The electrophoretic pattern of PrD, after staining with Coomassie blue, showed the enrichment of some protein bands in comparison with DRM. μLiquid cromatography‐electrospray ionization‐mass spectrometry (μLC‐ESI‐MS)/MS analysis showed that Thy‐1 and different types of myosin were strongly enriched in PrD and, in a lesser extent, also OBCAM, LSAMP and tubulin, present altogether in a single band. Experiments using the chemical cross‐linker BS3 suggested the existence of an interaction between PrPC and neural cell adhesion molecule (NCAM). Concerning lipids, the comparison between PrD and DRM showed a similar phospholipid/sphingolipid ratio, a phospholipid/cholesterol ratio doubled, and a strong decrease of plasmenilethanolamine (19.7 ± 3.5% vs. 38.3 ± 1.2%). In conclusion, the peculiar lipid composition and in particular the presence of proteins involved in synaptic plasticity, cell adhesion, cytoskeleton regulation and signalling, suggest an important physiological role in neurons of Prion Domain.

Involvement of MEK-ERK1-2 pathway in the anti-oxidant response in C6 glioma cells after diesel exhaust particles exposure.

Ultrafine particles translocate to the central nervous system and activate oxidative stress-related pathways. The transcription factor Nrf2 activation by ERK1-2 has been suggested as a key regulator of cellular response to oxidative stress. C6 glioma cells have been treated with different doses of diesel exhaust particles (25μg/ml, DEP25, and 50μg/ml, DEP50), for different times. Cells have been screened for oxidative stress and inflammatory markers, and for the activation of the MEK-ERK1-2 pathway. The same markers have been examined after inhibition of MEK, the kinase upstream to ERK1-2. 3h and 24h of DEP25 and DEP50 induced a significant increase in HO-1 levels. After 24h, DEP25 and DEP50 induced an increase in HO-1 and Cyp1b1 levels, while increase in OGG1 level was observed only with DEP25. After 5h of treatment with DEP25, ERK1-2 resulted phosphorylated, concomitantly with a significant increase in HO-1 levels, no changes in iNOS levels, and decreased levels of anti-oxidant enzymes. After treatment with MEK inhibitor U0126, ERK1-2 showed no activation, with a consequent decrease in Nrf2, no increase in HO-1 and a significant increase of iNOS. MEK inhibitor is able to deplete anti-oxidant enzymes. In conclusion, the MEK-ERK1-2 pathway is involved in regulating the anti-oxidant strategies to compensate the oxidative status induced by DEP treatment.

Diesel exhaust particles (DEP) pre-exposure contributes to the anti-oxidant response impairment in hCMEC/D3 during post-oxygen and glucose deprivation damage

Recently, air pollution has been identified as a significant modifiable risk factor to the increasing stroke burden. Diesel exhaust particles, characterized by high polycyclic aromatic hydrocarbons content, constitute an important component of outdoor air pollution and is known to cause oxidative stress, and could therefore contribute to and exacerbate the effects of ROS in post-ischemic injury. hCMEC/D3 cells have been submitted to 48h treatment with diesel exhaust particles (25μg/ml and 50μg/ml, DEP50) or alternatively to 3h of oxygen and glucose deprivation, followed by 1h of oxygen and glucose restoration. The combined treatment consisted in 48h of diesel exhaust particles (25μg/ml and 50μg/ml, DEP50) followed by 3h of oxygen and glucose deprivation and 1h of restoration. A panel of markers related to oxidative stress and inflammatory responses, such as transcription factors (Nrf2 and HIF-1α), anti-oxidant proteins (HO-1, SOD-1, Hsp70) and proteins potentially inducing further oxidative-stress or inflammation (Cyp1b1, iNOS, COX-2, TNF-α, IL-1α, IL-1β, IL-8, VEGF), have been examined. Data obtained showed that diesel exhaust particles and oxygen and glucose deprivation treatments alone elicited the antioxidants response, each by means of a different transcription factor, while the combined treatment led to a dysregulation of the antioxidant response during ischemic injury reperfusion.

Remodelling of Membrane Rafts Expression in Lung Cells as an Early Sign of Mechanotransduction-Signalling in Pulmonary Edema

Membrane rafts (MRs) are clusters of lipids, organized in a “quasicrystalline” liquid-order phase, organized on the cell surface and whose pattern of molecules and physicochemical properties are distinct from those of the surrounding plasma membrane. MRs may be considered an efficient and fairly rapid cell-activated mechanism to express or mask surface receptors aimed at triggering specific response pathways. This paper reports observations concerning the role of MRs in the control of lung extravascular water that ought to be kept at minimum to assure gas diffusion, supporting the hypothesis that MRs expression is a potential mechanism of sensing minor changes in the volume of extravascular water. We present the evidence that MRs expression specifically relates to signal-transduction processes evoked by mechanical stimuli arising in the interstitial lung compartment when a small increase in extravascular volume occurs. We further hypothesize that a differential expression of MRs might also reflect the damage to precise components of the extracellular matrix caused by the perturbation in water balance and thus can trigger a molecule-oriented specific matrix remodelling.

Early evidence of stress in immortalized neurons exposed to diesel particles: the role of lipid reshaping behind oxidative stress and inflammation

Diesel combustion is the major source of fine particle road emission, whose solid fraction is represented by diesel exhaust particles (DEP). Many studies indicate the contribution of DEP to the onset of different neurological diseases, such as Alzheimers disease (AD), identifying oxidative stress and neuroinflammation as two cardinal processes of brain damage. This study aimed to investigate the effects of different concentrations of DEP (10 μg/ml and 50 μg/ml) on the mouse HT22 cells treated for 3 h or 24 h. Our results demonstrated that DEP contributed to an increased oxidative stress, defined by overexpression of HO-1, Hsp70 and Cyp1b1 protein levels. Moreover, an inflammatory-related processes were also observed, as COX-2 and iNOS levels were higher in treated cells when compared to the control. Furthermore, our investigations highlighted the alteration of fatty acid composition, total cholesterol content in cells and media, and of membrane fluidity, suggesting a lipid reshaping after DEP treatment. Finally, we detected APP and BACE1 increase after 24 h of treatment with 50 μg/ml of DEP. Indeed, our results propose a role of acute exposure in the onset of a deleterious mechanism for AD neurodegeneration, even though no differences were observed in p-APP Thr668 levels, BACE1 activity and APP C-terminal fragment beta amount.