Intermittent hypoxia treatments cause cellular priming in human microglia.

Obstructive sleep apnoea syndrome (OSAS) is a sleep-disordered breathing characterized by nocturnal collapses of the upper airway resulting in cycles of blood oxygen partial pressure oscillations, which lead to tissue and cell damage due to intermittent hypoxia (IH) episodes. Since OSAS-derived IH may lead to cognitive impairment through not fully cleared mechanisms, herein we developed a new in vitro model mimicking IH conditions to shed light on its molecular effects on microglial cells, with particular attention to the inflammatory response. The in vitro model was set-up and validated by measuring the hypoxic state, HIF-1α levels, oxidative stress by ROS production and mitochondrial activity by MTS assay. Then, the mRNA and protein levels of certain inflammatory markers (NF-κB and interleukin 6 (IL-6)) after different IH treatment protocols were investigated. The IH treatments followed by a normoxic period were not able to produce a high inflammatory state in human microglial cells. Nevertheless, microglia appeared to be in a state characterized by increased expression of NF-κB and markers related to a primed phenotype. The microglia exposed to IH cycles and stimulated with exogenous IL-1ß resulted in an exaggerated inflammatory response with increased NF-κB and IL-6 expression, suggesting a role for primed microglia in OSAS-driven neuroinflammation.

Carbonic anhydrase activation profile of indole-based derivatives.

Carbonic Anhydrase Activators (CAAs) could represent a novel approach for the treatment of Alzheimer's disease, ageing, and other conditions that require remedial achievement of spatial learning and memory therapy. Within a research project aimed at developing novel CAAs selective for certain isoforms, three series of indole-based derivatives were investigated. Enzyme activation assay on human CA I, II, VA, and VII isoforms revealed several effective micromolar activators, with promising selectivity profiles towards the brain-associated cytosolic isoform hCA VII. Molecular modelling studies suggested a theoretical model of the complex between hCA VII and the new activators and provide a possible explanation for their modulating as well as selectivity properties. Preliminary biological evaluations demonstrated that one of the most potent CAA 7 is not cytotoxic and is able to increase the release of the brain-derived neurotrophic factor (BDNF) from human microglial cells, highlighting its possible application in the treatment of CNS-related disorders.

De novo Neurosteroidogenesis in Human Microglia: Involvement of the 18 kDa Translocator Protein.

Neuroactive steroids are potent modulators of microglial functions and are capable of counteracting their excessive reactivity. This action has mainly been ascribed to neuroactive steroids released from other sources, as microglia have been defined unable to produce neurosteroids de novo. Unexpectedly, immortalized murine microglia recently exhibited this de novo biosynthesis; herein, de novo neurosteroidogenesis was characterized in immortalized human microglia. The results demonstrated that C20 and HMC3 microglial cells constitutively express members of the neurosteroidogenesis multiprotein machinery-in particular, the transduceosome members StAR and TSPO, and the enzyme CYP11A1. Moreover, both cell lines produce pregnenolone and transcriptionally express the enzymes involved in neurosteroidogenesis. The high TSPO expression levels observed in microglia prompted us to assess its role in de novo neurosteroidogenesis. TSPO siRNA and TSPO synthetic ligand treatments were used to reduce and prompt TSPO function, respectively. The TSPO expression downregulation compromised the de novo neurosteroidogenesis and led to an increase in StAR expression, probably as a compensatory mechanism. The pharmacological TSPO stimulation the de novo neurosteroidogenesis improved in turn the neurosteroid-mediated release of Brain-Derived Neurotrophic Factor. In conclusion, these results demonstrated that de novo neurosteroidogenesis occurs in human microglia, unravelling a new mechanism potentially useful for future therapeutic purposes.

The Citrus flavanone naringenin prolongs the lifespan in C. elegans and slows signs of brain aging in mice.

Aging is one of the main risk factors for neurodegenerative disorders, which represent a global burden on healthcare systems. Therefore, identifying new strategies to slow the progression of brain aging is a compelling challenge. In this article, we first assessed the potential anti-aging effects of the Citrus flavanone naringenin (NAR), an activator of the enzyme sirtuin-1 (SIRT1), in a 3R-compliant and short-lived aging model (i.e., the nematode C. elegans). Then, we investigated the preventive effects of a 6-month treatment with NAR (100 mg/kg, orally) against brain aging and studied its mechanism of action in middle-aged mice. We demonstrated that NAR (100 µM) extends lifespan and improves healthspan in C. elegans. In the brain of middle-aged mice, NAR promotes the activity of metabolic enzymes (citrate synthase, cytochrome C oxidase) and increases the expression of the SIRT1 enzyme. Consistently, NAR up-regulates the expression of downstream antioxidant (Foxo3, Nrf2, Ho-1), anti-senescence (p16), and anti-inflammatory (Il-6, Il-18) markers. Our findings support NAR supplementation to slow the signs of brain aging.

Blood oxygenation state in COVID-19 patients: Unexplored role of 2,3-bisphosphoglycerate.

BACKGROUND: COVID-19 reduces lung functionality causing a decrease of blood oxygen levels (hypoxemia) often related to a decreased cellular oxygenation (hypoxia). Besides lung injury, other factors are implicated in the regulation of oxygen availability such as pH, partial arterial carbon dioxide tension (PaCO2), temperature, and erythrocytic 2,3-bisphosphoglycerate (2,3-BPG) levels, all factors affecting hemoglobin saturation curve. However, few data are currently available regarding the 2,3-BPG modulation in SARS-CoV-2 affected patients at the hospital admission. MATERIAL AND METHODS: Sixty-eight COVID-19 patients were enrolled at hospital admission. The lung involvement was quantified using chest-Computer Tomography (CT) analysed with automatic software (CALIPER). Haemoglobin concentrations, glycemia, and routine analysis were evaluated in the whole blood, while partial arterial oxygen tension (PaO2), PaCO2, pH, and HCO3- were assessed by arterial blood gas analysis. 2,3-BPG levels were assessed by specific immunoenzymatic assays in RBCs. RESULTS: A higher percentage of interstitial lung disease (ILD) and vascular pulmonary-related structure (VRS) volume on chest-CT quantified with CALIPER had been found in COVID-19 patients with a worse disease outcome (R = 0.4342; and R = 0.3641, respectively). Furthermore, patients with lower PaO2 showed an imbalanced acid-base equilibrium (pH, p = 0.0208; PaCO2, p = 0.0496) and a higher 2,3-BPG levels (p = 0.0221). The 2,3-BPG levels were also lower in patients with metabolic alkalosis (p = 0.0012 vs. no alkalosis; and p = 0.0383 vs. respiratory alkalosis). CONCLUSIONS: Overall, the data reveal a different pattern of activation of blood oxygenation compensatory mechanisms reflecting a different course of the COVID-19 disease specifically focusing on 2,3-BPG modulation.

Human oligodendrocyte-like cell differentiation is promoted by TSPO-mediated endogenous steroidogenesis.

Mature oligodendrocytes (OLs) arise from oligodendrocyte precursor cells that, in case of demyelination, are recruited at the lesion site to remyelinate the axons and therefore restore the transmission of nerve impulses. It has been widely documented that exogenously administered steroid molecules are potent inducers of myelination. However, little is known about how neurosteroids produced de novo by OLs can impact this process. Here, we employed a human OL precursor cell line to investigate the role of de novo neurosteroidogenesis in the regulation of OLs differentiation, paying particular attention to the 18 kDa Translocator Protein (TSPO) which controls the rate-limiting step of the neurosteroidogenic process. Our results showed that, over the time of OL maturation, the availability of cholesterol, which is the neurosteroidogenesis initial substrate, and key members of the neurosteroidogenic machinery, including TSPO, were upregulated. In addition, OLs differentiation was impaired following neurosteroidogenesis inhibition and TSPO silencing. On the contrary, TSPO pharmacological stimulation promoted neurosteroidogenic function and positively impacted differentiation. Collectively, our results suggest that de novo neurosteroidogenesis is actively involved in the autocrine and paracrine regulation of human OL differentiation. Moreover, since TSPO was able to promote OL differentiation through a positive modulation of the neurosteroid biosynthetic process, it could be exploited as a promising target to tackle demyelinating diseases.

The human microglial surveillant phenotype is preserved by de novo neurosteroidogenesis through the control of cholesterol homeostasis: Crucial role of 18 kDa Translocator Protein.

Neurodegenerative disease-associated microglia commonly exhibit harmful cholesterol accumulation that impairs their ability to resolve the neuroinflammatory response, contributing to disease onset and progression. Neurosteroids, whose levels have been often found significantly altered in brain diseases, are the most potent endogenous anti-inflammatory molecules exerting beneficial effects on activities of brain cells, including microglia. For the first time, the impact of neurosteroidogenesis on cholesterol homeostasis for the immune surveillance phenotype maintenance was investigated in a human microglia in vitro model. To enhance and inhibit neurosteroidogenesis, pharmacological stimulation and knock-down of 18 kDa Translocator Protein (TSPO), which is involved in the neurosteroidogenesis rate-limiting step, were used as experimental approaches, respectively. The obtained results point to an essential autocrine control of neurosteroidogenesis in orchestrating cholesterol trafficking in human microglia. TSPO pharmacological stimulation ensured cholesterol turnover by strengthening cholesterol efflux systems and preserving healthy immune surveillant phenotype. Conversely, TSPO knock-down induced an impairment of the controlled interplay among cholesterol synthesis, efflux, and metabolism mechanisms, leading to an excessive cholesterol accumulation and acquisition of a chronically activated dysfunctional phenotype. In this model, the exogenous neurosteroid administration restored proper the cholesterol clearance. The TSPO ability in promoting native neurosteroidogenesis opens the way to restore cholesterol homeostasis, and thus to maintain microglia proper functionality for the treatment of neuroinflammation-related brain diseases.

Translocator Protein 18-kDa: A Promising Target to Treat Neuroinflammation- related Degenerative Diseases.

In the nervous system, inflammatory responses physiologically occur as defense mechanisms triggered by damaging events. If improperly regulated, neuroinflammation can contribute to the development of chronically activated states of glial cells, with the perpetuation of inflammation and neuronal damage, thus leading to neurological and neurodegenerative disorders. Interestingly, neuroinflammation is associated with the overexpression of the mitochondrial translocator protein (TSPO) in activated glia. Despite the precise role of TSPO in the immunomodulatory mechanisms during active disease states is still unclear, it has emerged as a promising target to promote neuroprotection. Indeed, TSPO ligands have been shown to exert beneficial effects in counteracting neuroinflammation and neuronal damage in several in vitro and in vivo models of neurodegenerative diseases. In particular, the regulation of neurosteroids' production, cytokine release, metabolism of radical oxidative species, and cellular bioenergetics appear to be the main cellular events that underlie the observed effects. The present review aims to illustrate and summarize recent findings on the potential effect of TSPO ligands against neuroinflammation and related neurodegenerative mechanisms, taking into consideration some pathologies of the nervous system in which inflammatory events are crucial for the onset and progression of the disease and attempting to shed light onto the immunomodulatory effects of TSPO.

Translocator Protein Ligand PIGA1138 Reduces Disease Symptoms and Severity in Experimental Autoimmune Encephalomyelitis Model of Primary Progressive Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune and demyelinating disease of the central nervous system (CNS) caused by CNS infiltration of peripheral immune cells, immune-mediated attack of the myelin sheath, neuroinflammation, and/or axonal/neuronal dysfunctions. Some drugs are available to cope with relapsing-remitting MS (RRMS) but there is no therapy for the primary progressive MS (PPMS). Because growing evidence supports a regulatory role of the translocator protein (TSPO) in neuroinflammatory, demyelinating, and neurodegenerative processes, we investigated the therapeutic potential of phenylindolyilglyoxylamydes (PIGAs) TSPO ligands in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) mice mimicking the human PPMS. MOG-EAE C57Bl/6-mice were treated by TSPO ligands PIGA839, PIGA1138, or the vehicle. Several methods were combined to evaluate PIGAs-TSPO ligand effects on MOG-EAE symptoms, CNS infiltration by immune cells, demyelination, and axonal damages. PIGA1138 (15 mg/kg) drastically reduced MOG-EAE mice clinical scores, ameliorated motor dysfunctions assessed with the Catwalk device, and counteracted MOG-EAE-induced demyelination by preserving Myelin basic protein (MBP) expression in the CNS. Furthermore, PIGA1138-treatment prevented EAE-evoked decreased neurofilament-200 expression in spinal and cerebellar axons. Moreover, PIGA1138 inhibited peripheral immune-CD45 + cell infiltration in the CNS, suggesting that it may control inflammatory mechanisms involved in PPMS. Concordantly, PIGA1138 enhanced anti-inflammatory interleukin-10 serum level in MOG-EAE mice. PIGA1138-treatment, which increased neurosteroid allopregnanolone production, ameliorated all pathological biomarkers, while PIGA839, unable to activate neurosteroidogenesis in vivo, exerted only moderate/partial effects in MOG-EAE mice. Altogether, our results suggest that PIGA1138-based treatment may represent an interesting possibility to be explored for the innovation of effective therapies against PPMS.

Antitumoral effects of attenuated Listeria monocytogenes in a genetically engineered mouse model of melanoma.

Attenuated Listeria monocytogenes (Lmat-LLO) represents a valuable anticancer vaccine and drug delivery platform. Here we show that in vitro Lmat-LLO causes ROS production and, in turn, apoptotic killing of a wide variety of melanoma cells, irrespectively of their stage, mutational status, sensitivity to BRAF inhibitors or degree of stemness. We also show that, when administered in the therapeutic setting to Braf/Pten genetically engineered mice, Lmat-LLO causes a strong decrease in the size and volume of primary melanoma tumors, as well as a reduction of the metastatic burden. At the molecular level, we confirm that the anti-melanoma activity exerted in vivo by Lmat-LLO depends also on its ability to potentiate the immune response of the organism against the infected tumor. Our data pave the way to the preclinical testing of listeria-based immunotherapeutic strategies against metastatic melanoma, using a genetically engineered mouse rather than xenograft models.