Daniele Nosi

The Neuron-Astrocyte-Microglia Triad in Normal Brain Ageing and in a Model of Neuroinflammation in the Rat Hippocampus

Ageing is accompanied by a decline in cognitive functions; along with a variety of neurobiological changes. The association between inflammation and ageing is based on complex molecular and cellular changes that we are only just beginning to understand. The hippocampus is one of the structures more closely related to electrophysiological, structural and morphological changes during ageing. In the present study we examined the effect of normal ageing and LPS-induced inflammation on astroglia-neuron interaction in the rat hippocampus of adult, normal aged and LPS-treated adult rats. Astrocytes were smaller, with thicker and shorter branches and less numerous in CA1 Str. radiatum of aged rats in comparison to adult and LPS-treated rats. Astrocyte branches infiltrated apoptotic neurons of aged and LPS-treated rats. Cellular debris, which were more numerous in CA1 of aged and LPS-treated rats, could be found apposed to astrocytes processes and were phagocytated by reactive microglia. Reactive microglia were present in the CA1 Str. Radiatum, often in association with apoptotic cells. Significant differences were found in the fraction of reactive microglia which was 40% of total in adult, 33% in aged and 50% in LPS-treated rats. Fractalkine (CX3CL1) increased significantly in hippocampus homogenates of aged and LPS-treated rats. The number of CA1 neurons decreased in aged rats. In the hippocampus of aged and LPS-treated rats astrocytes and microglia may help clearing apoptotic cellular debris possibly through CX3CL1 signalling. Our results indicate that astrocytes and microglia in the hippocampus of aged and LPS-infused rats possibly participate in the clearance of cellular debris associated with programmed cell death. The actions of astrocytes may represent either protective mechanisms to control inflammatory processes and the spread of further cellular damage to neighboring tissue, or they may contribute to neuronal damage in pathological conditions.

Toxic effects of amyloid fibrils on cell membranes: the importance of ganglioside GM1

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 Ca2+. 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.—Bucciantini, M., Nosi, D., Forzan, M., Russo, E., Calamai, M., Pieri, L., Formigli, L., Quercioli, F., Soria, S., Pavone, F., Savistchenko, J., Melki, R., Stefani, M. Toxic effects of amyloid fibrils on cell membranes: the importance of ganglioside GM1. FASEB J. 26, 818–831 (2012). www.fasebj.org

Insights into the molecular basis of the differing susceptibility of varying cell types to the toxicity of amyloid aggregates

It has been reported that different tissue or cultured cell types are variously affected by the exposure to toxic protein aggregates, however a substantial lack of information exists about the biochemical basis of cell resistance or susceptibility to the aggregates. We investigated the extent of the cytotoxic effects elicited by supplementing the media of a panel of cultured cell lines with aggregates of HypF-N, a prokaryotic domain not associated with any amyloid disease. The cell types exposed to early, pre-fibrillar aggregates (not mature fibrils) displayed variable susceptibility to damage and to apoptotic death with a significant inverse relation to membrane content in cholesterol. Susceptibility to damage by the aggregates was also found to be significantly related to the ability of cells to counteract early modifications of the intracellular free Ca2+ and redox status. Accordingly, cell resistance appeared related to the efficiency of the biochemical equipment leading any cell line to sustain the activity of Ca2+ pumps while maintaining under control the oxidative stress associated with the increased metabolic rate. Our data depict membrane destabilization and the subsequent early derangement of ion balance and intracellular redox status as key events in targeting exposed cells to apoptotic death.

Relaxin prevents cardiac fibroblast-myofibroblast transition via notch-1-mediated inhibition of TGF-β/Smad3 signaling.

The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-β1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-β1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-β1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-β1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-β1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-β1 signaling, since the addition of RLX to TGF-β1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-β1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-β1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion, the results of the present study beside supporting the role of RLX in the field of cardiac fibrosis, provide novel experimental evidence on the molecular mechanisms underlying its effects.

Inhibition of Immune Synapse by Altered Dendritic Cell Actin Distribution: A New Pathway of Mesenchymal Stem Cell Immune Regulation

Immune synapse formation between dendritic cells (DCs) and T cells is one of the key events in immune reaction. In immunogenic synapses, the presence of fully mature DCs is mandatory; consequently, the modulation of DC maturation may promote tolerance and represents a valuable therapeutic approach in autoimmune diseases. In the field of cell therapy, bone marrow mesenchymal stem cells (MSCs) have been extensively studied for their immunoregulatory properties, such as inhibiting DC immunogenicity during in vitro differentiation and ameliorating in vivo models of autoimmune diseases (e.g., experimental allergic encephalomyelitis). MSCs seem to play different roles with regard to DCs, depending on cell concentration, mechanism of stimulation, and accompanying immune cells. The aim of this work was to elucidate the immunogenic effects of MSC/DC interactions during DC activation (LPS stimulation or Ag loading). Human monocyte-derived DCs, bone marrow-derived MSCs, and circulating lymphocytes obtained from healthy donors, as well as the laboratory-generated influenza virus hemagglutinin-derived peptide, aa 306–318 peptide-specific T cell line were used for this study. We demonstrate that MSCs mediate inhibition of DC function only upon cell–cell contact. Despite no modification observed in cell phenotype or cytokine production, MSC-treated DCs were unable to form active immune synapses; they retained endocytic activity and podosome-like structures, typical of immature DCs. The transcriptional program induced by MSC–DC direct interaction supports at the molecular pathway level the phenotypical features observed, indicating the genes involved into contact-induced rearrangement of DC cytoskeleton.

Selective adenosine A2a receptor antagonism reduces JNK activation in oligodendrocytes after cerebral ischaemia

Adenosine is a potent biological mediator, the concentration of which increases dramatically following brain ischaemia. During ischaemia, adenosine is in a concentration range (muM) that stimulates all four adenosine receptor subtypes (A(1), A(2A), A(2B) and A(3)). In recent years, evidence has indicated that the A(2A) receptor subtype is of critical importance in stroke. We have previously shown that 24 h after medial cerebral artery occlusion (MCAo), A(2A) receptors up-regulate on neurons and microglia of ischaemic striatum and cortex and that subchronically administered adenosine A(2A) receptor antagonists protect against brain damage and neurological deficit and reduce activation of p38 mitogen-activated protein kinase (MAPK) in microglial cells. The mechanisms by which A(2A) receptors are noxious during ischaemia still remain elusive. The objective of the present study was to investigate whether the adenosine A(2A) antagonist SCH58261 affects JNK and MEK1/ERK MAPK activation. A further aim was to investigate cell types expressing activated JNK and MEK1/ERK MAPK after ischaemia. We hereby report that the selective adenosine A(2A) receptor antagonist, SCH58261, administered subchronically (0.01 mg/kg i.p) 5 min, 6 and 20 h after MCAo in male Wistar rats, reduced JNK MAPK activation (immunoblot analysis: phospho-JNK54 isoform by 81% and phospho-JNK46 isoform by 60%) in the ischaemic striatum. Twenty-four hours after MCAo, the Olig2 transcription factor of oligodendroglial progenitor cells and mature oligodendrocytes was highly expressed in cell bodies in the ischaemic striatum. Immunofluorescence staining showed that JNK MAPK is maximally expressed in Olig2-stained oligodendrocytes and in a few NeuN stained neurons. Striatal cell fractioning into nuclear and extra-nuclear fractions demonstrated the presence of Olig2 transcription factor and JNK MAPK in both fractions. The A(2A) antagonist reduced striatal Olig 2 transcription factor (immunoblot analysis: by 55%) and prevented myelin disorganization, assessed by myelin-associated glycoprotein staining. Twenty-four hours after MCAo, ERK1/2 MAPK was highly activated in the ischaemic striatum, mostly in microglia, while it was reduced in the ischaemic cortex. The A(2A) antagonist did not affect activation of the ERK1/2 pathway. The efficacy of A(2A) receptor antagonism in reducing activation of JNK MAPK in oligodendrocytes suggests a mechanism of protection consisting of scarring oligodendrocyte inhibitory molecules that can hinder myelin reconstitution and neuron functionality.

Seladin‐1/DHCR24 protects neuroblastoma cells against Aβ toxicity by increasing membrane cholesterol content

The role of brain cholesterol in Alzheimers 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.

Cytoskeleton/stretch-activated ion channel interaction regulates myogenic differentiation of skeletal myoblasts.

In the present study, we investigated the functional interaction between stress fibers (SFs) and stretch‐activated channels (SACs) and its possible role in the regulation of myoblast differentiation induced by switch to differentiation culture in the presence or absence of sphingosine 1‐phosphate. It was found that there was a clear temporal correlation between SF formation and SAC activation in differentiating C2C12 myoblasts. Inhibition of actin polymerization with the specific Rho kinase inhibitor Y‐27632, significantly decreased SAC sensitivity in these cells, suggesting a role for Rho‐dependent actin remodeling in the regulation of the channel opening. The alteration of cytoskeletal/SAC functional correlation had also deleterious effects on myogenic differentiation of C2C12 cells as judged by combined confocal immunofluorescence, biochemical and electrophysiological analyses. Indeed, the treatment with Y‐27632 or with DHCB, an actin disrupting agent, inhibited the expression of the myogenic markers (myogenin and sarcomeric proteins) and myoblast‐myotube transition. The treatment with the channel blocker, GdCl3, also affected myogenesis in these cells. It impaired, in fact, myoblast phenotypic maturation (i.e., reduced the expression of α‐sarcomeric actin and skeletal myosin and the activity of creatine kinase) but did not modify promoter activity and protein expression levels of myogenin. The results of this study, together with being in agreement with the general idea that cytoskeletal remodeling is essential for muscle differentiation, describe a novel pathway whereby the formation of SFs and their contraction, generate a mechanical tension to the plasma membrane, activate SACs and trigger Ca2+‐dependent signals, thus influencing the phenotypic maturation of myoblasts. J. Cell. Physiol. 211: 296–306, 2007.

Protein N-homocysteinylation induces the formation of toxic amyloid-like protofibrils.

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 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.

Heterogeneity of histaminergic neurons in the tuberomammillary nucleus of the rat

Histaminergic neurons of the hypothalamic tuberomammillary nuclei (TMN) send projections to the whole brain. Early anatomical studies described histaminergic neurons as a homogeneous cell group, but recent evidence indicates that histaminergic neurons are heterogeneous and organized into distinct circuits. We addressed this issue using the double‐probe microdialysis in freely moving rats to investigate if two compounds acting directly onto histaminergic neurons to augment cell firing [thioperamide and bicuculline, histamine H3‐ and γ‐aminobutyric acid (GABA)A‐receptor (R) antagonists, respectively] may discriminate groups of histaminergic neurons impinging on different brain regions. Intra‐hypothalamic perfusion of either drug increased histamine release from the TMN and cortex, but not from the striatum. Thioperamide, but not bicuculline, increased histamine release from the nucleus basalis magnocellularis (NBM), bicuculline but not thioperamide increased histamine release from the nucleus accumbens (NAcc). Intra‐hypothalamic perfusion with thioperamide increased the time spent in wakefulness. To explore the local effects of H3‐R blockade in the histaminergic projection areas, each rat was implanted with a single probe to simultaneously administer thioperamide and monitor local changes in histamine release. Thioperamide increased histamine release from the NBM and cortex significantly, but not from the NAcc or striatum. The presence of H3‐Rs on histaminergic neurons was assessed using double‐immunofluorescence with anti‐histidine decarboxylase antibodies to identify histaminergic cells and anti‐H3‐R antibodies. Confocal analysis revealed that all histaminergic somata were immunopositive for the H3‐R. This is the first evidence that histaminergic neurons are organized into functionally distinct circuits that influence different brain regions, and display selective control mechanisms.