Thrombin in peripheral nerves: friend or foe?

Differently from the central nervous system (CNS), the peripheral nervous system (PNS) exhibits a high regenerative capacity. This ability is related to the remarkable plasticity of Schwann cells (SCs) which after nerve injury convert to a repair-promoting phenotype to a large extent. Nerve injury is accompanied by a rapid rise of thrombin levels (Bushi et al., 2016; Gera et al., 2016). Thrombin is the key effector protease of the coagulation cascade which elicits hormonelike actions by the activation of G-protein coupled receptors known as proteaseactivated receptors (PARs). Inflammation and coagulation are two complex and interconnected pathways whose mutual interactions have been only partially elucidated

Membrane protein remodeling in microglia exposed to amyloid peptides

Infection, neurodegeneration, and other conditions associated with loss of brain homeostasis, induce changes in microglial morphology, gene expression and function, generally referred to as “activation”. Alzheimer’s disease (AD) is the most common dementia and is characterized by neuroinfammatory changes, including alterations in the morphology and distribution of microglia and astrocytes, and deposition of complement and other infammatory mediators. Our previous observations show that microglial cells challenged in vitro with amyloid peptides clustered and rounded up, dramatically changing their morphology. Besides, in these cells we observed the early acetylation and then the phosphorylation of STAT3 which is required for the expression of the epsilon isoform of 14-3-3, a marker of Abeta-activated microglia (1, 2). We applied afnity partitioning approach combined with high throughput mass spectrometric analysis in order to identify variation of proteins on plasma membrane of BV2 immortalized microglia upon treatment with amyloid peptides. By this method several proteins up- or down-regulated by amyloid treatment were identifed in microglial plasma membrane. Among them annexins (5 and 7), IFITM3 and MARK3. These data have been confrmed in primary microglial cultures. In microglia, plasma membrane plays a relevant role in the cross-talking with the external neuronal environment and in the resulting trophic or infammatory response of these sentinel cells. As such, knowledge of the microglia responsiveness to beta amyloids in term of changes in its plasma membrane proteome is imperative for unveiling the molecular landscape in which AD occurs

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 bloc

Autophagy is differently regulated in astrocytes and microglia exposed to environmental toxic molecules

Autophagy is generally considered a degradation pathway involved in many neurodegenerative processes. It can be observed in different stress conditions such as starvation generally improving cell survival. Our previous results described the occurrence of autophagy in neuronal cultures exposed to the toxic compound trimethyltin (TMT) (1). TMT belongs to a family of organotin compounds with wide industrial and agricultural applications, especially as heat stabilizers in PVC production and as biocides. In the nervous system TMT determines the selective destruction of neurons in specific brain regions such as the olfactory bulb and the hippocampus. When this toxic molecule was administered to glial cells we observed in astrocytes a rapid block of the autophagic flux and a consequent increased expression of LC3 and p62 which can be observed both in cultured astrocytes and in the brain of intoxicated animals. Conversely, in microglia autophagy was not impaired in the same conditions and p62 accumulation was not observed neither in vitro primary cultures, nor in brain sections of TMT-treated rats. The protein p62 (also known as SQTM1) is known to be selectively degraded through autophagy and its accumulation activates the transcription factor Nrf2 by sequestering Keap1 (2). To note the block of autophagy has been reported to exert an immunosopressive effect in macrophages (3). Thus, the impairment of autophagy in astrocytes could be related to their limited production of pro-inflammatory cytokines and nitric oxide respect to microglia observed after TMT treatment. This work was supported by grant from Ricerca Scientifica 2013 to L.F

Thrombin regulates the ability of Schwann cells to support neuritogenesis and to maintain the integrity of the nodes of Ranvier

Schwann cells (SC) are characterized by a remarkable plasticity that enables them to promptly respond to nerve injury promoting axonal regeneration. In peripheral nerves after damage SC convert to a repair-promoting phenotype activating a sequence of supportive functions that drive myelin clearance, prevent neuronal death, and help axon growth and guidance. Regeneration of peripheral nerves after damage correlates inversely with thrombin levels. Thrombin is not only the key regulator of the coagulation cascade but also a protease with hormone-like activities that affects various cells of the central and peripheral nervous system mainly through the protease-activated receptor 1 (PAR1). Aim of the present study was to investigate if and how thrombin could affect the axon supportive functions of SC. In particular, our results show that the activation of PAR1 in rat SC cultures with low levels of thrombin or PAR1 agonist peptides induces the release of molecules, which favor neuronal survival and neurite elongation. Conversely, the stimulation of SC with high levels of thrombin or PAR1 agonist peptides drives an opposite effect inducing SC to release factors that inhibit the extension of neurites. Moreover, high levels of thrombin administered to sciatic nerve ex vivo explants induce a dramatic change in SC morphology causing disappearance of the Cajal bands, enlargement of the Schmidt-Lanterman incisures and calcium-mediated demyelination of the paranodes. Our results indicate thrombin as a novel modulator of SC plasticity potentially able to favor or inhibit SC pro-regenerative properties according to its level at the site of lesion

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

The thrombin/PAR1 axis as regulator of Schwann cell functions in health and disease

Thrombin, the key eff ector protease of the coagulation cascade, mediates hemostasis, throm bosis, and infl ammatory responses to vascular injury predominantly acting through its main receptor, protease-activated receptor 1 (PAR1). PAR1 is a member of a family of four G-protein coupled receptors which are activated by proteolytic cleavage of their N-terminal extracellular domains. The expression and role of PAR1 in peripheral nervous system is still poorly inves tigated, although high PAR1 expression was found in the dorsal root ganglia and in the non compacted Schwann cell myelin microvilli at the nodes of Ranvier. Our previous results indicate that rat Schwann cell plasticity can be widely modulated by thrombin acting through PAR1 (Pompili et al., Mol and Cell Neurosci 2017; Pompili et al., Eur J Histochem 2020). Here we extend those previous data showing that thrombin regulates prolifer ation and survival of human Schwann cells increasing the expression of factors, such as matrix metalloprotease 2 (MMP2) and macrophage migration inhibitory factor (MIF), which are key in modulating nerve regeneratio

Persistent paradoxical eff ects on striatal and limbic a-synuclein and tyrosine hydroxylase following methamphetamine withdrawal

Methamphetamine (METH) produces a variety of epigenetic eff ects in the brain, which are seminal to establish long-lasting alterations in neuronal activity. A number of studies were car ried out aimed at rough assessment of the amount of either histone acetylation and methyla tion or direct DNA methylation, without a selective analysis of specifi c genes. In the present study we wish to assess whether METH-induced epigenetic alterations may specifi cally engage the expression of a-synuclein, which is a key protein in neurodegeneration and synaptic plastic ity. In this way, a potential long-term alteration of brain circuitries may produce a variation in the threshold for neurotoxicity, sensitization, addiction and neurodegeneration. Thus, the occur rence of long-term changes in the expression of the protein were analyzed in parallel with per sistent changes in a specifi c marker of integrity of meso-striatal/meso-limbic pathway, which is the expression of tyrosine hydroxylase (TH) both in the mesencephalon and within dorsal striatum. The integrity of dopamine (DA) projection was assessed at the level of the olfactory tubercle, the nucleus accumbens and fundus striati. Prolonged exposure to small doses of METH, produces nigro-striatal toxicity, when assessed at short time intervals following prolonged exposure. However, at prolonged time intervals a paradoxical increase progressively occurred in TH immunostaining within limbic regions. Such an increase exceeds at large the amount of TH expressed in controls. This occurs concomitantly with an overexpression of the primary transcript as well as the protein alpha synuclein within the same brain regions and dorsal striatum. This increase is persistent at prolonged time inter val of METH withdrawal.The increase in the primary a-synuclein transcript is due to hypomethylation of specifi c CPG islands placed in the SNCA gene promoter which ranged roughly ten-fold of controls, it was steady, and it persisted at least 21 days following METH withdrawal. Thus, such an appar ent synucleinopathy induced by METH indeed was associated with increased mesolimbic DA innervation, which equally surpasses several folds the amount which was measured in controls and persists at least for three weeks. The increase in SNCA is not associated with an increase of SNCA copy number. Nonetheless, the amount of the native protein, which is detected by ultra structural stoichiometry, exceeds the increase reported following genetic SNCA multiplications (ten-fold of controls). These fi ndings are discussed in the light of METH-induced phenotype changes which accompany toxicity, sensitization, addiction and neurodegeneration

Protease-activated receptor-1 in Schwann cells and its possible role in the regeneration of peripheral nerves

Protease-activated receptor-1 (PAR-1) 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 PAR-1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR-1 mRNA increased in response to sciatic nerve transection, both in the proximal and in the distal part of the lesioned nerve (1). Other authors revealed that functional PAR-1 receptor exists specifically in the non-compacted Schwann cell myelin microvilli at the nodes of Ranvier in the sciatic nerve (2). 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 (3). The present study was aimed to determine if the activation of PAR-1 affects the neurotrophic properties of Schwann cells. We observed a specific staining for PAR-1 in Schwann cells of rat sciatic nerve and also in primary Schwann cell cultures. To study the role of PAR-1 in Schwann cell cultures, we activated this receptor with a specific activating peptide (PAR-1 AP). Conditioned medium from PAR-1 AP-treated Schwann cells reduced the LDH release of PC12 cells respect to the medium of the untreated cells, suggesting that the stimulation of PAR-1 induces the production of pro-survival molecules. Also an increased neurite outgrowth on PC12 cells was observed using the conditioned medium from Schwann cells treated with PAR-1 AP respect to the control obtained from untreated cells. The synthesis and secretion of several factors produced by Schwann cells treated with PAR-1 AP were investigated by proteomics, western blot and RT-PCR analyses. By these experiments we identified as putative neurotrophic candidates some molecules, such as Macrophage migration inhibitory factor, Syndecan 4 and Annexin A2

The Stimulation of Inducible Nitric-oxide Synthase by the Prion Protein Fragment 106–126 in Human Microglia Is Tumor Necrosis Factor-α-dependent and Involves p38 Mitogen-activated Protein Kinase

A synthetic peptide consisting of amino acid residues 106-126 of the human prion protein (PrP-(106--126)) has been previously demonstrated to be neurotoxic and to induce microglial activation. The present study investigated the expression of the inducible form of the nitric-oxide synthase (NOS-II) in human microglial cells treated with PrP-(106--126). Using reverse transcriptase-polymerase chain reaction, we found that PrP-(106--126) induces NOS-II gene expression after 24 h of treatment and that this effect is accompanied by a peak of nuclear factor kappa B (NF-kappa B) binding at 30 min as evaluated by electrophoretic mobility shift assay. Since our previous data demonstrated tumor necrosis factor-alpha (TNF-alpha) to be a potent inducer of NOS-II in these cells, we analyzed the expression of this cytokine in PrP-(106--126)-treated microglia. PrP-(106--126) caused the release of TNF-alpha as detected by enzyme-linked immunosorbent assay, and a blocking antibody, anti-TNF-alpha, abolished NOS-II induction elicited by this peptide. Moreover, PrP-(106-126) activates p38 mitogen-activated protein kinase, and the inhibition of this pathway determines the ablation of NF-kappa B binding induced by this fragment peptide