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.

Discovery of pyrrole derivatives for the treatment of glioblastoma and chronic myeloid leukemia.

Long-term survivors of glioblastoma multiforme (GBM) are at high risk of developing second primary neoplasms, including leukemia. For these patients, the use of classic tyrosine kinase inhibitors (TKIs), such as imatinib mesylate, is strongly discouraged, since this treatment causes a tremendous increase of tumor and stem cell migration and invasion. We aimed to develop agents useful for the treatment of patients with GBM and chronic myeloid leukemia (CML) using an alternative mechanism of action from the TKIs, specifically based on the inhibition of tubulin polymerization. Compounds 7 and 25, as planned, not only inhibited tubulin polymerization, but also inhibited the proliferation of both GMB and CML cells, including those expressing the T315I mutation, at nanomolar concentrations. In in vivo experiments in BALB/cnu/nu mice injected subcutaneously with U87MG cells, in vivo, 7 significantly inhibited GBM cancer cell proliferation, in vivo tumorigenesis, and tumor growth, tumorigenesis and angiogenesis. Compound 7 was found to block human topoisomerase II (hTopoII) selectively and completely, at a concentration of 100 µM.

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.

Microglial Pro-Inflammatory and Anti-Inflammatory Phenotypes Are Modulated by Translocator Protein Activation.

A key role of the mitochondrial Translocator Protein 18 KDa (TSPO) in neuroinflammation has been recently proposed. However, little is known about TSPO-activated pathways underlying the modulation of reactive microglia. In the present work, the TSPO activation was explored in an in vitro human primary microglia model (immortalized C20 cells) under inflammatory stimulus. Two different approaches were used with the aim to (i) pharmacologically amplify or (ii) silence, by the lentiviral short hairpin RNA, the TSPO physiological function. In the TSPO pharmacological stimulation model, the synthetic steroidogenic selective ligand XBD-173 attenuated the activation of microglia. Indeed, it reduces and increases the release of pro-inflammatory and anti-inflammatory cytokines, respectively. Such ligand-induced effects were abolished when C20 cells were treated with the steroidogenesis inhibitor aminoglutethimide. This suggests a role for neurosteroids in modulating the interleukin production. The highly steroidogenic ligand XBD-173 attenuated the neuroinflammatory response more effectively than the poorly steroidogenic ones, which suggests that the observed modulation on the cytokine release may be influenced by the levels of produced neurosteroids. In the TSPO silencing model, the reduction of TSPO caused a more inflamed phenotype with respect to scrambled cells. Similarly, during the inflammatory response, the TSPO silencing increased and reduced the release of pro-inflammatory and anti-inflammatory cytokines, respectively. In conclusion, the obtained results are in favor of a homeostatic role for TSPO in the context of dynamic balance between anti-inflammatory and pro-inflammatory mediators in the human microglia-mediated inflammatory response. Interestingly, our preliminary results propose that the TSPO expression could be stimulated by NF-κB during activation of the inflammatory response.

Nanocarriers for Protein Delivery to the Cytosol: Assessing the Endosomal Escape of Poly(Lactide-co-Glycolide)-Poly(Ethylene Imine) Nanoparticles.

Therapeutic proteins and enzymes are a group of interesting candidates for the treatment of numerous diseases, but they often require a carrier to avoid degradation and rapid clearance in vivo. To this end, organic nanoparticles (NPs) represent an excellent choice due to their biocompatibility, and cross-linked enzyme aggregates (CLEAs)-loaded poly (lactide-co-glycolide) (PLGA) NPs have recently attracted attention as versatile tools for targeted enzyme delivery. However, PLGA NPs are taken up by cells via endocytosis and are typically trafficked into lysosomes, while many therapeutic proteins and enzymes should reach the cellular cytosol to perform their activity. Here, we designed a CLEAs-based system implemented with a cationic endosomal escape agent (poly(ethylene imine), PEI) to extend the use of CLEA NPs also to cytosolic enzymes. We demonstrated that our system can deliver protein payloads at cytoplasm level by two different mechanisms: Endosomal escape and direct translocation. Finally, we applied this system to the cytoplasmic delivery of a therapeutically relevant enzyme (superoxide dismutase, SOD) in vitro.