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

Impairment of the autophagic flux in astrocytes intoxicated by trimethyltin

Autophagy is a lysosomal catabolic route for protein aggregates and damaged organelles which in different stress conditions, such as starvation, generally improves cell survival. An impairment of this degradation pathway has been reported to occur in many neurodegenerative processes. Trimethyltin (TMT) is a potent neurotoxin present as an environmental contaminant causing tremors, seizures and learning impairment in intoxicated subjects. The present data show that in rat primary astrocytes autophagic vesicles (AVs) appeared after few hours of TMT treatment. The analysis of the autophagic flux in TMT-treated astrocytes was consistent with a block of the late stages of autophagy and was accompanied by a progressive accumulation of the microtubule associated protein light chain 3 (LC3) and of p62/SQSTM1. Interestingly, an increased immunoreactivity for p62/SQSTM1 was also observed in hippocampal astrocytes detected in brain slices of TMT-intoxicated rats. The time-lapse recordings of AVs in EGFP-mCherry-LC3B transfected astrocytes demonstrated a reduced mobility of autophagosomes after TMT exposure respect to control cells. The observed block of the autophagic flux cannot be overcome by known autophagy inducers such as rapamycin or 0.5mM lithium. Although ineffective when used at 0.5mM, lithium at higher concentrations (2mM) was able to protect astrocyte cultures from TMT toxicity. This effect correlated well with its ability to determine the phosphorylation/inactivation of glycogen kinase synthase-3β (GSK-3β)

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

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

Measuring source properties and quasi-normal-mode frequencies of heavy massive black-hole binaries with LISA

The laser-interferometer space antenna (LISA) will be launched in the mid 2030s. It promises to observe the coalescence of massive black-hole (BH) binaries with signal-to-noise ratios (SNRs) reaching thousands. Crucially, it will detect some of these binaries with high SNR both in the inspiral and the merger-ringdown stages. Such signals are ideal for tests of General Relativity (GR) using information from the whole waveform. Here, we consider astrophysically motivated binary systems at the high-mass end of the population observable by LISA, and simulate their LISA signals using the newly developed parametrised, multipolar, aligned-spin effective-one-body model: pSEOBNRv5HM. The merger-ringdown signal in this model depends on the binary properties (masses and spins), and also on parameters that describe fractional deviations from the GR quasi-normal-mode frequencies of the remnant BH. Performing full Bayesian analyses, we assess to which accuracy LISA will be able to constrain deviations from GR in the ringdown signal when using information from the whole signal. We find that these deviations can typically be constrained to within $10\%$ and in the best cases to within $1\%$. We also show that we can measure the binary masses and spins with great accuracy even for very massive BH systems with low SNR in the inspiral: individual source-frame masses can typically be constrained to within $10\%$ and as precisely as $1\%$, and individual spins can typically be constrained to within $0.1$ and as precisely as $0.001$. Finally, we probe the accuracy of the SEOBNRv5HM waveform family by performing synthetic injections of GR numerical-relativity waveforms. For the source parameters considered, we measure erroneous deviations from GR due to systematics in the waveform model. These results confirm the need for improving waveform models to perform tests of GR with binary BHs at high SNR with LISA.Comment: 15 pages, 18 with appendices, 19 figure

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 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

Theoretical groundwork supporting the precessing-spin two-body dynamics of the effective-one-body waveform models SEOBNRv5

Waveform models are essential for gravitational-wave (GW) detection and parameter estimation of coalescing compact-object binaries. More accurate models are required for the increasing sensitivity of current and future GW detectors. The effective-one-body (EOB) formalism combines the post-Newtonian (PN) and small mass-ratio approximations with numerical-relativity results, and produces highly accurate inspiral-merger-ringdown waveforms. In this paper, we derive the analytical precessing-spin two-body dynamics for the \texttt{SEOBNRv5} waveform model, which has been developed for the upcoming LIGO-Virgo-KAGRA observing run. We obtain an EOB Hamiltonian that reduces to the exact Kerr Hamiltonian in the test-mass limit. It includes the full 4PN precessing-spin information, and is valid for generic compact objects (i.e., for black holes or neutron stars). We also build an efficient and accurate EOB Hamiltonian that includes partial precessional effects, notably orbit-averaged in-plane spin effects for circular orbits, and derive 4PN-expanded precessing-spin equations of motion, consistent with such an EOB Hamiltonian. The results were used to build the computationally-efficient precessing-spin multipolar \texttt{SEOBNRv5PHM} waveform model.Comment: 35 page

pySEOBNR: a software package for the next generation of effective-one-body multipolar waveform models

We present pySEOBNR, a Python package for gravitational-wave (GW) modeling developed within the effective-one-body (EOB) formalism. The package contains an extensive framework to generate state-of-the-art inspiral-merger-ringdown waveform models for compact-object binaries composed of black holes and neutron stars. We document and demonstrate how to use the built-in quasi-circular precessing-spin model SEOBNRv5PHM, whose aligned-spin limit (SEOBNRv5HM) has been calibrated to numerical-relativity simulations and the nonspinning sector to gravitational self-force data using pySEOBNR. Furthermore, pySEOBNR contains the infrastructure necessary to construct, calibrate, test, and profile new waveform models in the EOB approach. The efficiency and flexibility of pySEOBNR will be crucial to overcome the data-analysis challenges posed by upcoming and next-generation GW detectors on the ground and in space, which will afford the possibility to observe all compact-object binaries in our Universe.Comment: 21 pages, 4 figure

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