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Aging and neurodegenerative diseases share a condition of neuroinflammation entailing the production of endogenous cell debris in the CNS that must be removed by microglia ( i.e., resident macrophages), to restore tissue homeostasis. In this context, extension of microglial cell branches toward cell debris underlies the mechanisms of microglial migration and phagocytosis. Amoeboid morphology and the consequent loss of microglial branch functionality characterizes dysregulated microglia. Microglial migration is assisted by another glial population, the astroglia, which forms a dense meshwork of cytoplasmic projections. Amoeboid microglia and disrupted astrocyte meshwork are consistent traits in aged CNS. In this study, we assessed a possible correlation between microglia and astroglia morphology in rat models of chronic neuroinflammation and aging, by 3-dimensional confocal analysis implemented with particle analysis. Our findings suggest that a microglia-astroglia interaction occurs in rat hippocampus via cell-cell contacts, mediating microglial cell branching in the presence of inflammation. In aged rats, the impairment of such an interaction correlates with altered distribution, morphology, and inefficient clearance by microglia. These data support the idea that generally accepted functional boundaries between microglia and astrocytes should be re-evaluated to better understand how their functions overlap and interact.-Lana, D., Ugolini, F., Wenk, G. L., Giovannini, M. G., Zecchi-Orlandini, S., Nosi, D. Microglial distribution, branching, and clearance activity in aged rat hippocampus are affected by astrocyte meshwork integrity: evidence of a novel cell-cell interglial interaction.

Modifications in the subunit composition of AMPA receptors (AMPARs) have been linked to the transition from physiological to pathological conditions in a number of contexts, including EtOH-induced neurotoxicity. Previous work from our laboratory showed that EtOH withdrawal causes CA1 pyramidal cell death in organotypic hippocampal slices and changes in the expression of AMPARs. Here, we investigated whether changes in expression and function of AMPARs may be causal for EtOH-induced neurotoxicity. To this aim, we examined the subunit composition, localization and function of AMPARs in hippocampal slices exposed to EtOH by using western blotting, surface expression assay, confocal microscopy and electrophysiology. We found that EtOH withdrawal specifically increases GluA1 protein signal in total homogenates, but not in the post-synaptic density-enriched fraction. This is suggestive of overall increase and redistribution of AMPARs to the extrasynaptic compartment. At functional level, AMPA-induced calcium influx was unexpectedly reduced, whereas AMPA-induced current was enhanced in CA1 pyramidal neurons following EtOH withdrawal, suggesting that increased AMPAR expression may lead to cell death because of elevated excitability, and not for a direct contribution on calcium influx. Finally, the neurotoxicity caused by EtOH withdrawal was attenuated by the non-selective AMPAR antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt as well as by the selective antagonist of GluA2-lacking AMPARs 1-naphthyl acetyl spermine. We conclude that EtOH neurotoxicity involves changes in expression, surface localization and functional properties of AMPARs, and propose GluA2-lacking AMPARs as amenable specific targets for the development of neuroprotective drugs in EtOH-withdrawal syndrome.

We investigated the quantitative and morphofunctional alterations of neuron-astrocyte-microglia triads in CA3 hippocampus, in comparison to CA1, after 2 Vessel Occlusion (2VO) and the protective effect of dipyridamole. We evaluated 3 experimental groups: sham-operated rats (sham, n=15), 2VO-operated rats treated with vehicle (2VO-vehicle, n=15), and 2VO-operated rats treated with dipyridamole from day 0 to day 7 (2VO-dipyridamole, n=15), 90days after 2VO. We analyzed Stratum Pyramidalis (SP), Stratum Lucidum (SL) and Stratum Radiatum (SR) of CA3. 1) ectopic neurons increased in SL and SR of 2VO-vehicle, and 2VO-dipyridamole rats; 2) apoptotic neurons increased in SP of 2VO-vehicle rats and dipyridamole reverted this effect; 3) astrocytes increased in SP, SL and SR of 2VO-vehicle and 2VO-dipyridamole rats; 4) TNF-α expression increased in astrocytes, blocked by dipyridamole, and in dendrites in SR of 2VO-vehicle rats; 5) total microglia increased in SL and SR of 2VO-vehicle and 2VO-dipyridamole rats; 6) triads increased in SR of 2VO-vehicle rats and dipyridamole reverted this effect. Microglia cooperated with astrocytes to phagocytosis of apoptotic neurons and debris, and engulfed ectopic non-fragmented neurons in SL of 2VO-vehicle and 2VO-dipyridamole rats, through a new mechanism called phagoptosis. CA3 showed a better adaptive capacity than CA1 to the ischemic insult, possibly due to the different behaviour of astrocytes and microglial cells. Dipyridamole had neuroprotective effects.

We examined the effects of inflammaging on memory encoding, and qualitative and quantitative modifications on proinflammatory proteins, apoptosis, neurodegeneration and morphological changes of neuron-astrocyte-microglia triads in CA3 Stratum Pyramidale (SP), Stratum Lucidum (SL) and Stratum Radiatum (SR) of young (3 months) and aged rats (20 months). Aged rats showed short-term memory impairments in the inhibitory avoidance task, increased expression of iNOS and activation of p38MAPK in SP, increase of apoptotic neurons in SP and of ectopic neurons in SL, and decrease of CA3 pyramidal neurons. The number of astrocytes and their branches length decreased in the three CA3 subregions of aged rats, with morphological signs of clasmatodendrosis. Total and activated microglia increased in the three CA3 subregions of aged rats. In aged rats CA3, astrocytes surrounded ectopic degenerating neurons forming “micro scars” around them. Astrocyte branches infiltrated the neuronal cell body, and, together with activated microglia formed “triads”. In the triads, significantly more numerous in CA3 SL and SR of aged rats, astrocytes and microglia cooperated in fragmentation and phagocytosis of ectopic neurons. Inflammaging-induced modifications of astrocytes and microglia in CA3 of aged rats may help clearing neuronal debris derived from low-grade inflammation and apoptosis. These events might be common mechanisms underlying many neurodegenerative processes. The frequency to which they appear might depend upon, or might be the cause of, the burden and severity of neurodegeneration. Targeting the triads may represent a therapeutic strategy which may control inflammatory processes and spread of further cellular damage to neighboring cells.

Histamine, a major mediator in allergic diseases, differentially regulates the polarizing ability of dendritic cells after Toll-like receptor (TLR) stimulation, by not completely explained mechanisms. In this study we investigated the effects of histamine on innate immune reaction during the response of human monocyte-derived DCs (mDCs) to different TLR stimuli: LPS, specific for TLR4, and Pam3Cys, specific for heterodimer molecule TLR1/TLR2. We investigated actin remodeling induced by histamine together with mDCs phenotype, cytokine production, and the stimulatory and polarizing ability of Th0. By confocal microscopy and RT-PCR expression of Rac1/CdC42 Rho GTPases, responsible for actin remodeling, we show that histamine selectively modifies actin cytoskeleton organization induced by TLR4, but not TLR2 and this correlates with increased IL4 production and decreased IFNγ by primed T cells. We also demonstrate that histamine-induced cytoskeleton organization is at least in part mediated by down-regulation of small Rho GTPase CdC42 and the protein target PAK1, but not by down-regulation of Rac1. The presence and relative expression of histamine receptors HR1–4 and TLRs were determined as well. Independently of actin remodeling, histamine down-regulates IL12p70 and CXCL10 production in mDCs after TLR2 and TLR4 stimulation. We also observed a trend of IL10 up-regulation that, despite previous reports, did not reach statistical significance.

Transthyretin (TTR) is involved in a subset of familial or sporadic amyloid diseases including senile systemic amyloidosis (SSA), familial amyloid polyneuropathy and cardiomyopathy (FAP/FAC) for which no effective therapy has been found yet. These conditions are characterized by extracellular deposits primarily found in the heart parenchyma and in peripheral nerves whose main component are amyloid fibrils, presently considered the main culprits of cell sufferance. The latter are polymeric assemblies grown from misfolded TTR, either wt or carrying one out of many identified mutations. The recent introduction in the clinical practice of synthetic TTR-stabilizing molecules that reduce protein aggregation provides the rationale to search natural effective molecules able to interfere with TTR amyloid aggregation by hindering the appearance of toxic species or by favoring the growth of harmless aggregates. Here we carried out an in depth biophysical and morphological study on the molecular features of the aggregation of wt- and L55P-TTR involved in SSA or FAP/FAC, respectively, and on the interference with fibril aggregation, stability and toxicity to cardiac HL-1 cells to demonstrate the ability of Oleuropein aglycone (OleA), the main phenolic component of the extra virgin olive oil. We describe the molecular basis of such interference and the resulting reduction of TTR amyloid aggregate cytotoxicity. Our data offer the possibility to validate and optimize the use of OleA or its molecular scaffold to rationally design promising drugs against TTR-related pathologies that could enter a clinical experimental phase.

Alterations of the tightly interwoven neuron/astrocyte interactions are frequent traits of aging, but also favor neurodegenerative diseases, such as Alzheimer disease (AD). These alterations reflect impairments of the innate responses to inflammation-related processes, such as β-amyloid (Aβ) burdening. Multidisciplinary studies, spanning from the tissue to the molecular level, are needed to assess how neuron/astrocyte interactions are influenced by aging. Our study addressed this requirement by joining fluorescence-lifetime imaging microscopy/phasor multiphoton analysis with confocal microscopy, implemented with a novel method to separate spectrally overlapped immunofluorescence and Aβ autofluorescence. By comparing data from young control rats, chronically inflamed rats, and old rats, we identified age-specific alterations of neuron/astrocyte interactions in the hippocampus. We found a correlation between Aβ aggregation (+300 and +800% of aggregated Aβ peptide in chronically inflamed and oldvs.control rats, respectively) and fragmentation (clasmatodendrosis) of astrocyte projections (APJs) (+250 and +1300% of APJ fragments in chronically inflamed and oldvs.control rats, respectively). Clasmatodendrosis, in aged rats, associates with impairment of astrocyte-mediated Aβ clearance (-45% of Aβ deposits on APJs, and +33% of Aβ deposits on neurons in oldvs.chronically inflamed rats). Furthermore, APJ fragments colocalize with Aβ deposits and are involved in novel Aβ-mediated adhesions between neurons. These data define the effects of Aβ deposition on astrocyte/neuron interactions as a key topic in AD biology.-Mercatelli, R., Lana, D., Bucciantini, M., Giovannini, M. G., Cerbai, F., Quercioli, F., Zecchi-Orlandini, S., Delfino, G., Wenk, G. L., Nos, D. Clasmatodendrosis and β-amyloidosis in aging hippocampus.

It is widely reported that the Ca2+ increase following nonspecific cell membrane permeabilization is among the earliest biochemical modifications in cells exposed to toxic amyloid aggregates. However, more recently receptors with Ca2+ channel activity such as α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl d-aspartate (NMDA), ryanodine, and inositol 1,4,5-trisphosphate receptors have been proposed as mediators of the Ca2+ increase in neuronal cells challenged with β-amyloid peptides. We previously showed that prefibrillar aggregates of proteins not associated with amyloid diseases are toxic to exposed cells similarly to comparable aggregates of disease-associated proteins. In particular, prefibrillar aggregates of the prokaryotic HypF-N were shown to be toxic to different cultured cell lines by eliciting Ca2+ and reactive oxygen species increases. This study was aimed at assessing whether NMDA and AMPA receptor activations could be considered a generic feature of cell interaction with amyloid aggregates rather than a specific effect of some aggregated protein. Therefore, we investigated whether NMDA and AMPA receptors were involved in the Ca2+ increase following exposure of rat cerebellar granule cells to HypF-N prefibrillar aggregates. We found that the intracellular Ca2+ increase was associated with the early activation of NMDA and AMPA receptors, although some nonspecific membrane permeabilization was also observed at longer times of exposure. This result matched a significant co-localization of the aggregates with both receptors on the plasma membrane. Our data support the possibility that glutamatergic channels are generic sites of interaction with the cell membrane of prefibrillar aggregates of different peptides and proteins as well as the key structures responsible for the resulting early membrane permeabilization to Ca2+.

A substantial lack of information is recognized on the features underlying the variable susceptibility to amyloid aggregate toxicity of cells with different phenotypes. Recently, we showed that different cell types are variously affected by early aggregates of a prokaryotic hydrogenase domain (HypF-N). In the present study we investigated whether differentiation affects cell susceptibility to amyloid injury using a human neurotypic SH-SY5Y cell differentiation model. We found that retinoic acid-differentiated cells were significantly more resistant against Abeta1-40, Abeta1-42 and HypF-N prefibrillar aggregate toxicity respect to undifferentiated cells treated similarly. Earlier and sharper increases in cytosolic Ca(2+) and ROS with marked lipid peroxidation and mitochondrial dysfunction were also detected in exposed undifferentiated cells resulting in apoptosis activation. The reduced vulnerability of differentiated cells matched a more efficient Ca(2+)-ATPase equipment and a higher total antioxidant capacity. Finally, increasing the content of membrane cholesterol resulted in a remarkable reduction of vulnerability and ability to bind the aggregates in either undifferentiated and differentiated cells.

The role of brain cholesterol in Alzheimer's disease (AD) is currently a matter of debate. Experimental evidence suggests that reducing circulating and brain cholesterol protects against AD, however recent data indicate that low membrane cholesterol results in neurode-generation and that the cholesterol synthesis catalyst seladin-1 is down-regulated in AD-affected brain regions. We previously reported a significant correlation between resistance to amyloid toxicity and content of membrane cholesterol in differing cultured cell types. Here we provide evidence that Aβ42 pre-fibrillar aggregates accumulate more slowly and in reduced amount at the plasma membrane of human SH-SY5Y neuroblastoma cells overexpressing seladin-1 or treated with PEG-cholesterol than at the membrane of control cells. The accumulation was significantly increased in cholesterol-depleted cells following treatment with the specific seladin-1 inhibitor 5,22E-cholestadien-3-ol or with methyl-β-cyclodextrin. The resistance to amyloid toxicity and the early cytosolic Ca2+ rise following exposure to Aβ42 aggregates were increased and prevented, respectively, by increasing membrane cholesterol whereas the opposite effects were found in cholesterol-depleted cells. These results suggest that seladin-1-dependent cholesterol synthesis reduces membrane-aggregate interaction and cell damage associated to amyloid-induced imbalance of cytosolic Ca2+. Our findings extend recently reported data indicating that seladin-1 overexpression directly enhances the resistance to Aβ toxicity featuring seladin-1/DHCR 24 as a possible new susceptibility gene for sporadic AD.

Much experimental evidence suggests that an imbalance in cellular redox status is a major factor in the pathogenesis of Alzheimer's disease (AD). Our previous data showed a marked increase in membrane lipoperoxidation in primary fibroblasts from familial AD (FAD) patients. In the present study, we demonstrate that when oligomeric structures of Aβ 1-40 and Aβ 1-42 are added to the culture media, they accumulate quicker near the plasma membrane, and are internalized faster and mostly in APPV717I fibroblasts than in age-matched healthy cells; this results in an earlier and sharper increase in the production of reactive oxygen species (ROS). Higher ROS production leads in turn to an increase in membrane oxidative-injury and significant impairment of cellular antioxidant capacity, giving rise to apoptotic cascade activation and finally to a necrotic outcome. In contrast, healthy fibroblasts appear more resistant to amyloid oxidative-attack, possibly as a result of their plasma membrane integrity and powerful antioxidant capacity. Our data are consistent with increasing evidence that prefibrillar aggregates, compared to mature fibrils, are likely the more toxic species of the peptides. These findings provide compelling evidence that cells bearing increased membrane lipoperoxidation are more susceptible to aggregate toxicity as a result of their reduced ability to counteract amyloid oligomeric attack.

The yeast prion Ure2p assembles in vitro into oligomers and fibrils retaining the alpha-helix content and binding properties of the soluble protein. Here we show that the different forms of Ure2p native-like assemblies (dimers, oligomers, and fibrils) are similarly toxic to murine H-END cells when added to the culture medium. Interestingly, the amyloid fibrils obtained by heat treatment of the toxic native-like fibrils appear harmless. Moreover, the Ure2p C-terminal domain, lacking the N-terminal segment necessary for aggregation but containing the glutathione binding site, is not cytotoxic. This finding strongly supports the idea that Ure2p toxicity depends on the structural properties of the flexible N-terminal prion domain and can therefore be considered as an inherent feature of the protein, unrelated to its aggregation state but rather associated with a basic toxic fold shared by all of the Ure2p native-like assemblies. Indeed, the latter are able to interact with the cell surface, leading to alteration of calcium homeostasis, membrane permeabilization, and oxidative stress, whereas the heat-treated amyloid fibrils do not. Our results support the idea of a general mechanism of toxicity of any protein/peptide aggregate endowed with structural features, making it able to interact with cell membranes and to destabilize them. This evidence extends the widely accepted view that the toxicity by protein aggregates is restricted to amyloid prefibrillar aggregates and provides new insights into the mechanism by which native-like oligomers compromise cell viability.

GM1-gangliosidosis is a lysosomal storage disorder caused by a deficiency of β-galactosidase (GLB1). The GLB1 gene gives rise to the GLB1 lysosomal enzyme and to the elastin binding protein (EBP), involved in elastic fiber deposition. GLB1 forms a complex with protective protein cathepsin A (PPCA), alpha neuraminidase (NEU1), and galactosamine 6-sulphate sulfatase (GALNS) inside lysosomes, while EBP binds to PPCA and NEU1 on the cell surface. We investigated the function of the GLB1 and EBP mutated proteins by analyzing the clinical, genetic, and cellular data of 11 GM1-gangliosidosis patients. Their molecular analysis, followed by expression studies, lead to the identification of four new and 10 known GLB1 mutations. Some common amino acid substitutions [c.1445G>A (p.Arg482H), c.622C>T (p.Arg208His), c.175C>T (p.Arg59Cys) and c.176G>A (p.Arg59His)] were present in the GLB1 enzyme of several patients, all of Mediterranean origin, suggesting a common origin. Western blotting analyses against GLB1, EBP, and PPCA proteins showed that the identified mutations affect GLB1 enzyme activity and/or stability. The c.1445G>A (p.Arg482His), c.175C>T (p.Arg59Cys), c.733+2T>C, c.1736G>A (p.Gly579Asp), and c.1051C>T (p.Arg351X) GLB1 mutations, affect the stabilization of PPCA probably because they hamper the interaction between GLB1/EBP and PPCA within the multiprotein complex. The amount of EBP was normal, but the detection of impaired elastogenesis in such patients suggests an alteration in its function. We conclude that the presence of genetic lesions in both GLB1 and EBP coding region does not directly predict impaired elastogenesis and that elastic fiber assembly has to be evaluated specifically in each case. Nevertheless, the degree of EBP involvement may be linked to specific clinical findings. Hum Mutat 25:285–292, 2005. © 2005 Wiley-Liss, Inc.

Sphingosine-1-phosphate (S1P) is a lipid mediator, which affects many essential processes such as cell proliferation, differentiation and contraction in many cell types. We have previously demonstrated that the lipid mediator elicits Ca2+ transients in a myoblastic cell line (C2C12) by interacting with its specific receptors (S1PRs). In the present study, we wanted to correlate the Ca2+ response with activation of myoblastic cell contractility. C2C12 cells were first investigated for the expression and cellular organization of cytoskeletal proteins by immunoconfocal microscopy. We found that myoblasts exhibited a quite immature cytoskeleton, with filamentous actin dispersed as a web-like structure within the cytoplasm. To evaluate intracellular Ca2+ mobilization, the cells were loaded with a fluorescent Ca2+ indicator (Fluo-3), stimulated with S1P and simultaneously observed with differential interference contrast and fluorescence optics. Exogenous S1P-induced myoblastic cell contraction was temporally unrelated to S1P-induced intracellular Ca2+ increase; cell contraction occurred within 5–8 s from stimulation, whereas intracellular Ca2+ increase was evident only after 15–25 s. To support the Ca2+ independence of myoblastic cell contraction, the cells were pretreated with a Ca2+ chelator, BAPTA/AM, prior to stimulation with S1P. In these experimental conditions, the myoblasts were still able to contract, whereas the S1P-induced Ca2+ transients were completely abolished. On the contrary, when C2C12 cells were induced to differentiate into skeletal myotubes, they responded to S1P with a rapid cell contraction concurrent with an increase in the intracellular Ca2+. These data suggest that Ca2+-independent mechanism of cell contraction may be replaced by Ca2+-dependent ones during skeletal muscle differentiation.

In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349–357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.

The interaction of amyloid aggregates with the cell plasma membrane is currently considered among the basic mechanisms of neuronal dysfunction in amyloid neurodegeneration. We used amyloid oligomers and fibrils grown from the yeast prion Sup35p, responsible for the specific prion trait [PSI(+)], to investigate how membrane lipids modulate fibril interaction with the membranes of cultured H-END cells and cytotoxicity. Sup35p shares no homology with endogenous mammalian polypeptide chains. Thus, the generic toxicity of amyloids and the molecular events underlying cell degeneration can be investigated without interference with analogous polypeptides encoded by the cell genome. Sup35 fibrils bound to the cell membrane without increasing its permeability to Ca(2+). Fibril binding resulted in structural reorganization and aggregation of membrane rafts, with GM1 clustering and alteration of its mobility. Sup35 fibril binding was affected by GM1 or its sialic acid moiety, but not by cholesterol membrane content, with complete inhibition after treatment with fumonisin B1 or neuraminidase. Finally, cell impairment resulted from caspase-8 activation after Fas receptor translocation on fibril binding to the plasma membrane. Our observations suggest that amyloid fibrils induce abnormal accumulation and overstabilization of raft domains in the cell membrane and provide a reasonable, although not unique, mechanistic and molecular explanation for fibril toxicity.

Previous works reported that a mild increase in homocysteine level is a risk factor for cardiovascular and neurodegenerative diseases in humans. Homocysteine thiolactone is a cyclic thioester, most of which is produced by an error-editing function of methionyl-tRNA synthetase, causing in vivo post-translational protein modifications by reacting with the var epsilon-amino group of lysine residues. In cells, the rate of homocysteine thiolactone synthesis is strictly dependent on the levels of the precursor metabolite, homocysteine. In this work, using bovine serum albumin as a model, we investigated the impact of N-homocysteinylation on protein conformation as well as its cellular actions. Previous works demonstrated that protein N-homocysteinylation causes enzyme inactivation, protein aggregation, and precipitation. In addition, in the last few years, several pieces of evidence have indicated that protein unfolding and aggregation are crucial events leading to the formation of amyloid fibrils associated with a wide range of human pathologies. For the first time, our results reveal how the low level of protein N-homocysteinylation can induce mild conformational changes leading to the formation of native-like aggregates evolving over time, producing amyloid-like structures. Taking into account the fact that in humans about 70% of circulating homocysteine is N-linked to blood proteins such as serum albumin and hemoglobin, the results reported in this article could have pathophysiological relevance and could contribute to clarify the mechanisms underlying some pathological consequences described in patients affected by hyperhomocysteinemia.

Stimulation of histamine H(3) receptors (H(3)R) activates G(i/o)-proteins that inhibit adenylyl cyclase and triggers MAPK and phospholipase A(2). In a previous study, we showed that H(3)R-mediated phosphorylation of Akt at Ser473 occurs in primary cultures of rat cortical neurons, but neither the downstream targets nor the function of such activation were explored. In this report we address these questions. Western blotting experiments showed that H(3)R-mediated activation of Akt in cultured rat cortical neurons was inhibited by LY 294004 and U0126, suggesting that it depends on phosphoinositide-3-kinase and mitogen-activated protein kinase kinase. H(3)R activation phosphorylated, hence inactivated, the Akt downstream effector glycogen synthase kinase-3beta, increased the expression of the antiapoptotic protein Bcl-2 and protected cultured rat and mouse cortical neurons from neurotoxic insults in a dose-dependent manner. All these effects were inhibited by the H(3)R antagonist inverse/agonist thioperamide. Mouse cortical cells expressed H(3)R as revealed by immunostaining experiments, and stimulation of H(3)R phoshorylated Akt and decreased caspase 3 activity. Hence, we uncovered a yet unexplored action of the H(3)R that may help understand the impact of H(3)R signaling in the CNS.

Androgens and the androgen receptor (AR) are involved both in early tumorigenesis of prostate cancer (PCa) and in androgen-refractory disease. The role of AR signalling has also been highlighted by the fusion gene TMPRSS2:ERG recently identified in the majority of PCa. Several data indicate that re-expression of AR in PCa cell lines confers a less aggressive phenotype. We observed that re-expression of AR in the AR-negative cells PC3 decreases anchorage-independent growth and Matrigel invasiveness of PC3-AR cells where plasma membrane interaction between AR and EGFR led to an interference with downstream signalling and internalization of activated EGFR. Our data evidenced a shift of EGFR internalization pathway from the clathrin-coated pit one mediating signalling and recycling of EGFR to the lipid raft-mediated one mainly involved in lysosomal degradation of EGFR. These effects involved an altered recruitment to EGFR of the adaptor proteins Grb2 and c-Cbl followed by a reduced ubiquitination of EGFR. Our preliminary results suggest that in PC3-AR cells a pool of classical AR is located within cholesterol-rich membrane microdomains (namely as lipid rafts) and a population of EGFR is within cholesterol-rich membrane microdomains too. However, AR and EGFR membrane interaction that is increased by rapid androgen signalling is not within cholesterol-rich membrane microdomains. Our data enlighten that the crosstalk between genotropic and non-genotropic AR signalling interferes with signalling of EGFR in response to ligand leading to a lower invasive phenotype of AR-positive PCa cells.

A key role of mitotic activation in neuronal cell death in early stages of Alzheimer's disease (AD) has been suggested. Apparently, terminally differentiated neurons are precluded from mitotic division, yet some phenotypic markers of cell cycling are present in AD-vulnerable brain areas. In this paper, we investigated whether dividing human neuroblastoma cells are preferentially vulnerable to amyloid aggregate toxicity in some specific cell cycle stage(s). Our data indicate that Abeta1-40/42 aggregates added to the cell culture media bind to the plasma membrane and are internalized faster in the S than in the G2/M and G1 cells possibly as a result of a lower content in membrane cholesterol in the former. Earlier and sharper increases in reactive oxygen species production triggered a membrane oxidative injury and a significant impairment of antioxidant capacity, eventually culminating with apoptotic activation in S and, to a lesser extent, in G2/M exposed cells. G1 cells appeared more resistant to the amyloid-induced oxidative attack possibly because of their higher antioxidant capacity. The high vulnerability of S cells to aggregate toxicity extends previous data suggesting that neuronal loss in AD could result from mitotic reactivation of terminally differentiated neurons with arrest in the S phase.

Although neurokinin 1 receptor antagonists prevent ethanol (EtOH)-induced gastric lesions, the mechanisms by which EtOH releases substance P (SP) and SP damages the mucosa are unknown. We hypothesized that EtOH activates transient receptor potential vanilloid 1 (TRPV1) on sensory nerves to release SP, which stimulates epithelial neurokinin 1 receptors to generate damaging reactive oxygen species (ROS). SP release was assayed in the mouse stomach, ROS were detected using dichlorofluorescein diacetate, and neurokinin 1 receptors were localized by immunofluorescence. EtOH-induced SP release was prevented by TRPV1 antagonism. High dose EtOH caused lesions, and TRPV1 or neurokinin 1 receptor antagonism and neurokinin 1 receptor deletion inhibited lesion formation. Coadministration of low, innocuous doses of EtOH and SP caused lesions by a TRPV1-independent but neurokinin 1 receptor-dependent process. EtOH, capsaicin, and SP stimulated generation of ROS by superficial gastric epithelial cells expressing neurokinin 1 receptors by a neurokinin 1 receptor-dependent mechanism. ROS scavengers prevented lesions induced by a high EtOH dose or a low EtOH dose plus SP. Gastric lesions are caused by an initial detrimental effect of EtOH, which is damaging only if associated with TRPV1 activation, SP release from sensory nerves, stimulation of neurokinin 1 receptors on epithelial cells, and ROS generation.

The finding that aggregates of disease-unrelated proteins can induce cell death indicates that proteins may act as potential toxins. Cells experiencing toxic protein aggregates often display modifications of the redox status and ion balance, eventually leading to apoptosis; however, in some cases, tissue and cultured cell types die with features of necrosis. To elucidate the pathways leading to such different outcomes, we studied the biochemical features of death in H-END and NIH/3T3 cells exposed to prefibrillar aggregates of a disease-unrelated protein. The two types of cells died by apoptosis and necrosis, respectively. The pattern of caspase and proapoptotic factor activation was investigated together with the extent of mitochondria impairment and the energy load in either cell line. Our data depict a scenario where the events related to the extrinsic pathway of apoptosis are the same in the two cell lines, the difference in the final outcome being related to the extent of mitochondria derangement, possibly due to the different ability of the cells to counteract ion homeostasis impairment.

The intracellular free Ca(2+) concentration and redox status of murine fibroblasts exposed to prefibrillar aggregates of the HypF N-terminal domain have been investigated in vitro and in vivo using a range of fluorescent probes. Aggregate entrance into the cytoplasm is followed by an early rise of reactive oxygen species and free Ca(2+) levels and eventually by cell death. Such changes correlate directly with the viability of the cells and are not observed when cell are cultured in the presence of reducing agents or in Ca(2+)-free media. In addition, moderate cell stress following exposure to the aggregates was found to be fully reversible. The results show that the cytotoxicity of prefibrillar aggregates of HypF-N, a protein not associated with clinical disease, has the same fundamental origin as that produced by similar types of aggregates of proteins linked with specific amyloidoses. These findings suggest that misfolded proteinaceous aggregates stimulate generic cellular responses as a result of the exposure of regions of the structure (such as hydrophobic residues and the polypeptide main chain) that are buried in the normally folded proteins. They also support the idea that a higher number of degenerative pathologies than previously known might be considered as protein deposition diseases.

Sphingosine 1-phosphate (S1P) activates a subset of plasma membrane receptors of the endothelial differentiation gene family (EdgRs) in many cell types. In C2C12 myoblasts, exogenous S1P elicits Ca2+ transients by activating voltage-independent plasma membrane Ca2+ channels and intracellular Ca2+-release channels. In this study, we investigated the effects of exogenous S1P on voltage-dependent L-type Ca2+ channels in skeletal muscle fibers from adult mice. To this end, intramembrane charge movements (ICM) and L-type Ca2+ current (I(Ca)) were measured in single cut fibers using the double Vaseline-gap technique. Our data showed that submicromolar concentrations of S1P (100 nM) caused a approximately 10-mV negative shift of the voltage threshold and transition voltages of q(gamma) and q(h) components of ICM, and of I(Ca) activation and inactivation. Biochemical studies showed that EdgRs are expressed in skeletal muscles. The involvement of EdgRs in the above S1P effects was tested with suramin, a specific inhibitor of Edg-3Rs. Suramin (200 microM) significantly reduced, by approximately 90%, the effects of S1P on ICM and I(Ca), suggesting that most of S1P action occurred via Edg-3Rs. Moreover, SIP at concentration above 10 microM elicited intracellular Ca2+ transients in muscle fibers loaded with the fluorescent Ca2+ dye Fluo-3, as detected by confocal laser scanning microscopy.

a Center of Excellence for the Study of Inflammation, University of Ferrara, Ferrara, Italy b Allergy Center Charite, Berlin, Germany c Department of Critical Care Medicine and Surgery, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy d Departments of Surgery and Physiology, University of California, San Francisco, CA, USA e Department of Human Pathology and Oncology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy f Department of Anatomy, Histology, and Forensic Medicine, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy g Department of Pediatrics, Harvard Medical School, Cambridge, MA, USA h Department of Physiopathology, Experimental Medicine and Public Health, University of Siena, Siena, Italy i Center for Cardiovascular Research, Division of Applied Cachexia Research, Charite, Berlin, Germany

This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca2 + levels and the abnormal Ca2 + signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca2 + responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca2 + response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.