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

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

Dopamine receptors and transporters sensitivity to trimethyltin in rat hippocampus and facial nucleus

Trimethyltin (TMT) is considered a useful tool to obtain an experimental model of neurodegeneration. TMT is known to cause neurotoxicant effects especially marked in the hippocampus. Despite many studies are published, there are poor literature on the interaction of this xenobiotic with dopaminergic system. In the present work, we investigate in rat brain, after 21 days following TMT intraperitoneal administration, the cells viability (N-NEU) and the animal behaviour in association with the immunohystochemical expression of dopamine receptors (D1- and D2-like) and transporters membrane (DAT) and vesicular monoamine trasporters (VMAT-1 and -2) in rat hippocampus and facial nucleus. The animal behaviour shows a significant reduction of spatial reference memory in a Morris water maze task according with the reduction (70% Vs control) of hippocampus dopaminergic system expression, despite the cell viability is maintained at about 50%. In the facial nucleus, a different reduction of dopamine receptors and trasporters (30% against 60%) was observed while the N-NEU reduction was 40%. These results suggest that the toxic interaction of TMT with the dopaminergic system in rat hippocampus may be responsible for learning and memory deficits. Data obtained in facial nucleus demonstrate different sensitivity of dopamine receptors and dopamine transporters to xenobiotic. Supported by PRIN 2008 - prot. 20089MANHH_00

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

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

Charcot-Marie-Tooth Type 2B: A New Phenotype Associated with a Novel RAB7A Mutation and Inhibited EGFR Degradation

The rare autosomal dominant Charcot-Marie-Tooth type 2B (CMT2B) is associated with mutations in the RAB7A gene, involved in the late endocytic pathway. CMT2B is characterized by predominant sensory loss, ulceromutilating features, with lesser-to-absent motor deficits. We characterized clinically and genetically a family harboring a novel pathogenic RAB7A variant and performed structural and functional analysis of the mutant protein. A 39-year-old woman presented with early-onset walking diculties, progressive distal muscle wasting and weakness in lower limbs and only mild sensory signs. Electrophysiology demonstrated an axonal sensorimotor neuropathy. Nerve biopsy showed a chronic axonal neuropathy with moderate loss of all caliber myelinated fibers. Next-generation sequencing (NGS) technology revealed in the proband and in her similarly affected father the novel c.377A>G (p.K126R) heterozygous variant predicted to be deleterious. The mutation affects the biochemical properties of RAB7 GTPase, causes altered interaction with peripherin, and inhibition of neurite outgrowth, as for previously reported CMT2B mutants. However, it also shows differences, particularly in the epidermal growth factor receptor degradation process. Altogether, our findings indicate that this RAB7A variant is pathogenic and widens the phenotypic spectrum of CMT2B to include predominantly motor CMT2. Alteration of the receptor degradation process might explain the different clinical presentations in this family

Prescription appropriateness of anti-diabetes drugs in elderly patients hospitalized in a clinical setting: evidence from the REPOSI Register

Diabetes is an increasing global health burden with the highest prevalence (24.0%) observed in elderly people. Older diabetic adults have a greater risk of hospitalization and several geriatric syndromes than older nondiabetic adults. For these conditions, special care is required in prescribing therapies including anti- diabetes drugs. Aim of this study was to evaluate the appropriateness and the adherence to safety recommendations in the prescriptions of glucose-lowering drugs in hospitalized elderly patients with diabetes. Data for this cross-sectional study were obtained from the REgistro POliterapie-Società Italiana Medicina Interna (REPOSI) that collected clinical information on patients aged ≥ 65 years acutely admitted to Italian internal medicine and geriatric non-intensive care units (ICU) from 2010 up to 2019. Prescription appropriateness was assessed according to the 2019 AGS Beers Criteria and anti-diabetes drug data sheets.Among 5349 patients, 1624 (30.3%) had diagnosis of type 2 diabetes. At admission, 37.7% of diabetic patients received treatment with metformin, 37.3% insulin therapy, 16.4% sulfonylureas, and 11.4% glinides. Surprisingly, only 3.1% of diabetic patients were treated with new classes of anti- diabetes drugs. According to prescription criteria, at admission 15.4% of patients treated with metformin and 2.6% with sulfonylureas received inappropriately these treatments. At discharge, the inappropriateness of metformin therapy decreased (10.2%, P < 0.0001). According to Beers criteria, the inappropriate prescriptions of sulfonylureas raised to 29% both at admission and at discharge. This study shows a poor adherence to current guidelines on diabetes management in hospitalized elderly people with a high prevalence of inappropriate use of sulfonylureas according to the Beers criteria