Francesca Somma

Engagement of CD4 Before TCR Triggering Regulates Both Bax- and Fas (CD95)-Mediated Apoptosis

In the present study, we have aimed at clarifying the CD4-dependent molecular mechanisms that regulate human memory T cell susceptibility to both Fas (CD95)-dependent and Bcl-2-dependent apoptotic pathways following antigenic challenge. To address this issue, we used an experimental system of viral and alloantigen-specific T cell lines and clones and two ligands of CD4 molecules, Leu-3a mAb and HIV gp120. We demonstrate that CD4 engagement before TCR triggering suppresses the TCR-mediated neosynthesis of the Flice-like inhibitory protein and transforms memory T cells from a CD95-resistant to a CD95-susceptible phenotype. Moreover, evidence that the apoptotic programs were executed while Fas ligand mRNA expression was inhibited led us to analyze Bcl-2-dependent pathways. The data show that the engagement of CD4 separately from TCR influences the expression of the proapoptotic protein Bax independently of the anti-apoptotic protein Bcl-2, whereas Ag activation coordinately modulates both Bax and Bcl-2. The increased expression of Bax and the consequent dissipation of the mitochondrial transmembrane potential (ΔΨm) suggest a novel immunoregulatory function of CD4 and demonstrate that both passive cell death and activation-induced cell death are operative in CD4+ memory T cells. Furthermore, analysis of the mechanisms by which IL-2 and IL-4 cytokines exert their protective function on CD4+ T cells in the presence of soluble CD4 ligands shows that they were able to revert susceptibility to Bax-mediated but not to CD95-dependent apoptotic pathways.

Gene activating and proapoptotic potential are independent properties of different CD4 epitopes

CD4 engagement triggers an early signaling cascade which initiates late events such as transcription factor activation. The outcome of CD4 engagement is T-cell commitment to alternative, dramatically different fates, such as activation and apoptosis. We have tested a panel of anti-CD4 mAbs specific for different CD4 epitopes, as well as HIV-1 gp120, for the capacity to activate crucial early events such as enhancement of p56(lck) kinase activity and Shc phosphorylation. The same CD4 epitopes were characterized for their capacity both to deliver a gene activating signal and to program T-cells to activation dependent death. No correlation could be found between capacity of specific CD4 epitopes to deliver a gene activating signal and capacity to prime T-cells to apoptosis, suggesting that gene activating and proapoptotic potential are independent functions of CD4 epitopes. Furthermore, while triggering of the calcium pathway appears critical in NF-AT activation, optimal p56(lck) activation and Shc phosphorylation might be required for initiation of the apoptotic pathway.

Lithium Improves Survival of PC12 Pheochromocytoma Cells in High-Density Cultures and after Exposure to Toxic Compounds

Autophagy is an evolutionary conserved mechanism that allows for the degradation of long-lived proteins and entire organelles which are driven to lysosomes for digestion. Different kinds of stressful conditions such as starvation are able to induce autophagy. Lithium and rapamycin are potent autophagy inducers with different molecular targets. Lithium stimulates autophagy by decreasing the intracellular myo-inositol-1,4,5-triphosphate levels, while rapamycin acts through the inhibition of the mammalian target of rapamycin (mTOR). The correlation between autophagy and cell death is still a matter of debate especially in transformed cells. In fact, the execution of autophagy can protect cells from death by promptly removing damaged organelles such as mitochondria. Nevertheless, an excessive use of the autophagic machinery can drive cells to death via a sort of self-cannibalism. Our data show that lithium (used within its therapeutic window) stimulates the overgrowth of the rat Pheochromocytoma cell line PC12. Besides, lithium and rapamycin protect PC12 cells from toxic compounds such as thapsigargin and trimethyltin. Taken together these data indicate that pharmacological activation of autophagy allows for the survival of Pheochromocytoma cells in stressful conditions such as high-density cultures and exposure to toxins.

Lithium limits trimethyltin-induced cytotoxicity and proinflammatory response in microglia without affecting the concurrent autophagy impairment.

Trimethyltin (TMT) is a highly toxic molecule present as an environmental contaminant causing neurodegeneration particularly of the limbic system both in humans and in rodents. We recently described the occurrence of impairment in the late stages of autophagy in TMT‐intoxicated astrocytes. Here we show that similarly to astrocytes also in microglia, TMT induces the precocious block of autophagy indicated by the accumulation of the autophagosome marker, microtubule associated protein light chain 3. Consistent with autophagy impairment we observe in TMT‐treated microglia the accumulation of p62/SQSTM1, a protein specifically degraded through this pathway. Lithium has been proved effective in limiting neurodegenerations and, in particular, in ameliorating symptoms of TMT intoxication in rodents. In our in vitro model, lithium displays a pro‐survival and anti‐inflammatory action reducing both cell death and the proinflammatory response of TMT‐treated microglia. In particular, lithium exerts these activities without reducing TMT‐induced accumulation of light chain 3 protein. In fact, the autophagic block imposed by TMT is unaffected by lithium administration. These results are of interest as defects in the execution of autophagy are frequently observed in neurodegenerative diseases and lithium is considered a promising therapeutic agent for these pathologies. Thus, it is relevant that this cation can still maintain its pro‐survival and anti‐inflammatory role in conditions of autophagy block. Copyright

PAR1 activation affects the neurotrophic properties of Schwann cells

&NA; Protease‐activated receptor‐1 (PAR1) is the prototypic member of a family of four G‐protein‐coupled receptors that signal in response to extracellular proteases. In the peripheral nervous system, the expression and/or the role of PARs are still poorly investigated. High PAR1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR1 mRNA increased in response to sciatic nerve transection. In the sciatic nerve, functional PAR1 receptor was reported at the level of non‐compacted Schwann cell myelin microvilli of the nodes of Ranvier. Schwann cells are the principal population of glial cells of the peripheral nervous system which myelinate axons playing an important role during axonal regeneration and remyelination. The present study was undertaken in order to determine if the activation of PAR1 affects the neurotrophic properties of Schwann cells. Our results suggest that the stimulation of PAR1 could potentiate the Schwann cell ability to favour nerve regeneration. In fact, the conditioned medium obtained from Schwann cell cultures challenged with a specific PAR1 activating peptide (PAR1 AP) displays increased neuroprotective and neurotrophic properties with respect to the culture medium from untreated Schwann cells. The proteomic analysis of secreted proteins in untreated and PAR1 AP‐treated Schwann cells allowed the identification of factors differentially expressed in the two samples. Some of them (such as macrophage migration inhibitory factor, matrix metalloproteinase‐2, decorin, syndecan 4, complement C1r subcomponent, angiogenic factor with G patch and FHA domains 1) appear to be transcriptionally regulated after PAR1 AP treatment as shown by RT‐PCR. HighlightsSchwann cells (SC) express the protease‐activated receptor PAR1.Activation of this receptor potentiates the neurotrophic properties of SC cultures.Some factors released from SC after PAR1 activation are identified by proteomics.PAR1 activation upregulates Sdc4, Dcn, Akr1b, Mif and Mmp2.PAR1 activation downregulates Aggf1, complement C1r and C1qbp subcomponents.

PAR-1 activation affects the neurotrophic properties of rat Schwann cells

PAR-1 (Protease-activated receptor–1) is a G-protein-coupled receptor that elicits cellular responses to extracellular proteases such as thrombin. In the peripheral nervous system (PNS), the expression and/or the role of PAR-1 are still poorly investigated. Several authors speculated that many functions in PNS, such as motor, secretory, vascular, nociceptive, inflammatory or regenerative processes, may be regulated by PARs (Vergnolle et al. 2003; Shavit et al., 2008; Wang et al., 2013). The present study was aimed to determine if PAR-1 activation affects neurotrophic properties of Schwann cells. By double immunofluorescence experiments we observed a specific staining for PAR-1 in S100β-positive cells of rat sciatic nerve and sciatic teased fibres. Moreover, PAR-1 was highly expressed in Schwann cell cultures obtained from both neonatal and adult rat sciatic nerves. When PAR-1 specific agonists were added to these cultures a higher proliferation rate was observed. Moreover, conditioned medium from primary Schwann cells treated with PAR-1 agonists increased cell survival and neurite outgrowth of PC12 cells. Therefore synthesis and secretion of several factors by Schwann cells treated with PAR-1 agonist peptides were investigated by RT-PCR, western blot and proteomics analysis. By these experiments some molecules, including extracellular matrix components and adhesion molecules, were identified as putative neurotrophic candidates.

Impairment of the autophagic flux in astrocytes intoxicated by trimethyltin

Autophagy is generally considered a degradation pathway involved in many neurodegenerative processes. It is induced by different stress conditions such as starvation improving cell survival. Conversely, an excess activation of autophagy can drive cells to death by a sort of self-cannibalism. Toxic compounds such as arsenic and lead have been described to affect autophagy in a different way by blocking the correct execution of this pathway. Our previous results show that in hippocampal neuronal cultures the toxic compound trimethyltin (TMT) determines the formation of autophagic vacuoles and that autophagy inducers (lithum, rapamycin) improves neuronal survival (Fabrizi et al., 2012). The present data show that in astrocytes TMT similarly activates the autophagic pathway. Differently from neurons, in astrocytes autophagy inducers are ineffective in modifying cell survival. Moreover, the analysis of the LC3B conversion show in TMT-treated astrocytes a precocious block of the late stages of autophagy which ultimately leads to p62 accumulation, nrf-2 nuclear translocation and induction of ARE-responsive genes.

Analysis of the autophagic flux in astrocytes intoxicated by trimethyltin

Autophagy is an intracellular degradation process that controls the quality of the cytoplasm by eliminating protein aggregates and damaged organelles. In addition to its vital homeostatic role, this degradation pathway is involved in various human disorders, including neurodegenerative diseases. Our previous data show that in hippocampal and cortical neurons the neurotoxic compound trimethyltin (TMT) activates the autophagic pathway (Fabrizi et al., 2012). Recently we extended our analysis to astrocytes, the main population of glia of the central nervous system. As already observed in neurons, in astrocytes autophagy is rapidly induced after TMT administration. LC3-II which is a distinctive marker of autophagy rapidly appeared in TMT-treated astrocytes but then it accumulates indicating a precocious block of the autophagic pathway. The inhibition of autophagy by 3-methyladenine at the level of the autophagosome formation partially rescues astrocytes from TMT-induced cell death. Interestingly, an impairment of autophagy was also observed by other authors following intoxication with arsenic and could represent a common feature of different environmental toxins.

Thrombin receptor PAR-1 is a glial cell receptor involved in the regeneration of peripheral nerves

Thrombin, a multifunctional serine protease, is a key enzyme in the coagulation cascade. Most of its actions are mediated by a G protein-coupled protease activated receptor (PAR-1) which is highly expressed in glial cells especially after injury (Pompili et al., 2006; Pompili et al., 2011) . In the peripheral nerves thrombin and PAR-1 specific agonist peptides produce changes in nerve conduction compatible with a conduction block. Aim of the present study is to determine if the activation of this receptor affects the neurotrophic properties of Schwann cells. In peripheral nerves PAR-1 was predominantly observed by immunofluorescence on non-compacted Schwann cell microvilli at the node of Ranvier. Moreover, PAR- 1 was highly expressed in Schwann cell cultures obtained from both neonatal and adult rat sciatic nerves. When PAR-1 specific peptides were added to these cultures an increased proliferation rate was observed. The synthesis and secretion of several growth factors by Schwann cells treated with PAR-1 agonist peptides were studied by RT-PCR, western blot and proteomics analyses.

Autophagy inhibitors and inducers as modulators of trimethyltin toxicity in neurons and glial cells

Trimethyltin (TMT) is a triorganotin compound which determines neurodegeneration of specific brain areas particularly damaging the limbic system. Organotin compounds are used as heat stabilizers in polyvinyl chloride polymers, industrial and agricultural biocides, and industrial catalysts in chemical reactions. The mechanism underlying TMT selective tissue-specific pattern of cellular damage is not completely known unless a correlation exists between TMT toxicity and the expression of stannin, a highly-conserved protein mainly localized within mitochondria. Ultrastructural studies performed on samples obtained from the brain of humans and rodents intoxicated with TMT describe increased number of lysosomes and big vacuoles suggestive of altered autophagy. Our results show that autophagy inhibitors added to TMT dramatically increased the toxicity of this compound both in hippocampal and cortical neurons. Conversely, autophagy inducers such as rapamycin and lithium prevent the neurotoxicity of TMT. Due to its diverse targets, the action of lithium may be complex and its neuroprotective property is sometimes controversial. Our data show that in acute treatment experiments hippocampal and cortical neurons behave quite differently when challenged with lithium. The neuroprotective effect of lithium acutely administered against TMT in hippocampal but not in cortical neurons can be completely reverted by an excess of inositol and is possibly related to the inactivation of inositol monophosphatase, a key regulator of autophagy. Lithium is also effective in preventing TMT toxicity in astrocyte cell cultures in a range of concentrations of 0.2-4 mM.