Maria Teresa Cencioni

Inflammation Triggers Synaptic Alteration and Degeneration in Experimental Autoimmune Encephalomyelitis

Neurodegeneration is the irremediable pathological event occurring during chronic inflammatory diseases of the CNS. Here we show that, in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, inflammation is capable in enhancing glutamate transmission in the striatum and in promoting synaptic degeneration and dendritic spine loss. These alterations occur early in the disease course, are independent of demyelination, and are strongly associated with massive release of tumor necrosis factor-α from activated microglia. CNS invasion by myelin-specific blood-borne immune cells is the triggering event, and the downregulation of the early gene Arc/Arg3.1, leading to the abnormal expression and phosphorylation of AMPA receptors, represents a culminating step in this cascade of neurodegenerative events. Accordingly, EAE-induced synaptopathy subsided during pharmacological blockade of AMPA receptors. Our data establish a link between neuroinflammation and synaptic degeneration and calls for early neuroprotective therapies in chronic inflammatory diseases of the CNS.

Anandamide Suppresses Proliferation and Cytokine Release from Primary Human T-Lymphocytes Mainly via CB2 Receptors

Background Anandamide (AEA) is an endogenous lipid mediator that exerts several effects in the brain as well as in peripheral tissues. These effects are mediated mainly by two types of cannabinoid receptors, named CB1R and CB2R, making AEA a prominent member of the “endocannabinoid” family. Also immune cells express CB1 and CB2 receptors, and possess the whole machinery responsible for endocannabinoid metabolism. Not surprisingly, evidence has been accumulated showing manifold roles of endocannabinoids in the modulation of the immune system. However, details of such a modulation have not yet been disclosed in primary human T-cells. Methodology/Significance In this investigation we used flow cytometry and ELISA tests, in order to show that AEA suppresses proliferation and release of cytokines like IL-2, TNF-α and INF-γ from activated human peripheral T-lymphocytes. However, AEA did not exert any cytotoxic effect on T-cells. The immunosuppression induced by AEA was mainly dependent on CB2R, since it could be mimicked by the CB2R selective agonist JWH-015, and could be blocked by the specific CB2R antagonist SR144528. Instead the selective CB1R agonist ACEA, or the selective CB1R antagonist SR141716, were ineffective. Furthermore, we demonstrated an unprecedented immunosuppressive effect of AEA on IL-17 production, a typical cytokine that is released from the unique CD4+ T-cell subset T-helper 17. Conclusions/Significance Overall, our study investigates for the first time the effects of the endocannabinoid AEA on primary human T-lymphocytes, demonstrating that it is a powerful modulator of immune cell functions. In particular, not only we clarify that CB2R mediates the immunosuppressive activity of AEA, but we are the first to describe such an immunosuppressive effect on the newly identified Th-17 cells. These findings might be of crucial importance for the rational design of new endocannabinoid-based immunotherapeutic approaches.

Distinct Modulation of Human Myeloid and Plasmacytoid Dendritic Cells by Anandamide in Multiple Sclerosis

The immunopathogenesis of multiple sclerosis (MS) has always been thought to be driven by chronically activated and autoreactive Th‐1 and Th‐17 cells. Recently, dendritic cells (DCs) have also been thought to significantly contribute to antigenic spread and to maturation of adaptive immunity, and have been linked with disease progression and exacerbation. However, the role of DCs in MS pathogenesis remains poorly understood.

Apoptosome-deficient Cells Lose Cytochrome c through Proteasomal Degradation but Survive by Autophagy-dependent Glycolysis

Cytochrome c release from mitochondria promotes apoptosome formation and caspase activation. The question as to whether mitochondrial permeabilization kills cells via a caspase-independent pathway when caspase activation is prevented is still open. Here we report that proneural cells of embryonic origin, when induced to die but rescued by apoptosome inactivation are deprived of cytosolic cytochrome c through proteasomal degradation. We also show that, in this context, those cells keep generating ATP by glycolysis for a long period of time and that they keep their mitochondria in a depolarized state that can be reverted. Moreover, under these conditions, such apoptosome-deficient cells activate a Beclin 1-dependent autophagy pathway to sustain glycolytic-dependent ATP production. Our findings contribute to elucidating what the point-of-no-return in apoptosis is. They also help in clarifying the issue of survival of apoptosome-deficient proneural cells under stress conditions. Unraveling this issue could be highly relevant for pharmacological intervention and for therapies based on neural stem cell transfer in the treatment of neurological disorders.

Lack of effect of cannabis-based treatment on clinical and laboratory measures in multiple sclerosis

The endocannabinoid system (ECS) is involved in the pathophysiology of multiple sclerosis (MS), and relief from pain and spasticity has been reported in MS patients self-medicating with marijuana. A cannabis-based medication containing Δ9-tetrahydrocannabinol and cannabidiol (Sativex®) has been approved in some countries for the treatment of MS-associated pain. The effects of this pharmaceutical preparation on other clinically relevant aspects of MS pathophysiology, however, are still unclear. In 20 MS patients, we measured the effects of Sativex® on clinically measured spasticity and on neurophysiological and laboratory parameters that correlate with spasticity severity or with the modulation of the ECS. Sativex® failed to affect spasticity and stretch reflex excitability. This compound also failed to affect the synthesis and the degradation of the endocannabinoid anandamide, as well as the expression of both CB1 and CB2 cannabinoid receptors in various subpopulations of peripheral lymphocytes.

Transcriptional Profiling of γδ T Cells Identifies a Role for Vitamin D in the Immunoregulation of the Vγ9Vδ2 Response to Phosphate-Containing Ligands

Vitamin D is a steroid hormone that, in addition to its well-characterized role in calcium/phosphate metabolism, has been found to have regulatory properties for immune system function. The nuclear vitamin D receptor is widely expressed in tissues, but has also been shown to be regulated by hormones, growth factors, and cytokines. In this study we show that activation of human Vδ2Vγ9 T cells by nonpeptidic monoalkyl phosphates such as isopentenyl pyrophosphate leads to the up-regulation of the vitamin D receptor via a pathway that involves the classical isoforms of protein kinase C. We further show that this receptor is active by demonstrating that the ligand 1α,25-dihydroxyvitamin D3 (vitD3) significantly inhibits in a dose-dependent fashion phospholigand-induced γδ T cell expansion, IFN-γ production, and CD25 expression. We also show that vitD3 negatively regulates signaling via Akt and ERK and, at high concentrations, potentiates Ag-induced cell death. As such, these data provide further support for the immunoregulatory properties of vitamin D, and suggest that the ability of vitD3 to negatively regulate the proinflammatory activity of γδ T cells may contribute to the protection this vitamin affords against inflammatory and autoimmune disorders dependent upon Th1-type responses.

Apaf1 plays a pro-survival role by regulating centrosome morphology and function

The apoptotic protease activating factor 1 (Apaf1) is the main component of the apoptosome, and a crucial factor in the mitochondria-dependent death pathway. Here we show that Apaf1 plays a role in regulating centrosome maturation. By analyzing Apaf1-depleted cells, we have found that Apaf1 loss induces centrosome defects that impair centrosomal microtubule nucleation and cytoskeleton organization. This, in turn, affects several cellular processes such as mitotic spindle formation, cell migration and mitochondrial network regulation. As a consequence, Apaf1-depleted cells are more fragile and have a lower threshold to stress than wild-type cells. In fact, we found that they exhibit low Bcl-2 and Bcl-XL expression and, under apoptotic treatment, rapidly release cytochrome c. We also show that Apaf1 acts by regulating the recruitment of HCA66, with which it interacts, to the centrosome. This function of Apaf1 is carried out during the cell life and is not related to its apoptotic role. Therefore, Apaf1 might also be considered a pro-survival molecule, whose absence impairs cell performance and causes a higher responsiveness to stressful conditions.

The Load of Amyloid-β Oligomers is Decreased in the Cerebrospinal Fluid of Alzheimer's Disease Patients

Amyloid-β (Aβ) oligomers are heterogeneous and instable compounds of variable molecular weight. Flow cytometry and fluorescence resonance energy transfer (FRET)-based methods allow the simultaneous detection of Aβ oligomers with low and high molecular weight in their native form. We evaluated whether an estimate of different species of Aβ oligomers in the cerebrospinal fluid (CSF) with or without dilution with RIPA buffer could be more useful in the diagnosis of Alzheimers disease (AD) than the measurement of Aβ42 monomers, total tau (t-tau), and phosphorylated tau (p-tau). Increased t-tau (p < 0.01) and p-tau (p < 0.01), and decreased Aβ42 (p < 0.01), were detected in the CSF of patients with AD (n = 46), compared to patients with other dementia (OD) (n = 35) or with other neurological disorders (OND) (n = 56). In native CSF (n = 137), the levels of Aβ oligomers were lower (p < 0.05) in AD than in OD and OND patients; in addition, the ratio Aβ oligomers/p-tau was lower in AD than in OD (p < 0.01) and OND (p < 0.05) patients, yielding a sensitivity of 75% and a specificity of 64%. However, in CSF diluted with RIPA (n = 30), Aβ oligomers appeared higher (p < 0.05) in AD than in OND patients, suggesting they become partially disaggregated and more easily detectable after RIPA. In conclusion, FRET analysis in native CSF is essential to correctly determine the composition of Aβ oligomers. In this experimental setting, the simultaneous estimate of low and high molecular weight Aβ oligomers is as useful as the other biomarkers in the diagnosis of AD. The low amount of Aβ oligomers detected in native CSF of AD may be inversely related to their levels in the brain, as occurs for Aβ monomers, representing a biomarker for the amyloid pathogenic cascade.

FAS-ligand regulates differential activation-induced cell death of human T-helper 1 and 17 cells in healthy donors and multiple sclerosis patients

Functionally distinct T-helper (Th) subsets orchestrate immune responses. Maintenance of homeostasis through the tight control of inflammatory Th cells is crucial to avoid autoimmune inflammation. Activation-Induced Cell Death (AICD) regulates homeostasis of T cells, and it has never been investigated in human Th cells. We generated stable clones of inflammatory Th subsets involved in autoimmune diseases, such as Th1, Th17 and Th1/17 cells, from healthy donors (HD) and multiple sclerosis (MS) patients and we measured AICD. We find that human Th1 cells are sensitive, whereas Th17 and Th1/17 are resistant, to AICD. In particular, Th1 cells express high level of FAS-ligand (FASL), which interacts with FAS and leads to caspases’ cleavage and ultimately to cell death. In contrast, low FASL expression in Th17 and Th1/17 cells blunts caspase 8 activation and thus reduces cell death. Interestingly, Th cells obtained from healthy individuals and MS patients behave similarly, suggesting that this mechanism could explain the persistence of inflammatory IL-17-producing cells in autoimmune diseases, such as MS, where their generation is particularly substantial.

Protein repertoire impact of Ubiquitin-Proteasome System impairment: Insight into the protective role of beta-estradiol

The Ubiquitin-Proteasome System (UPS) and the Autophagy-Lysosome Pathways (ALP) are key mechanisms for cellular homeostasis sustenance and protein clearance. A wide number of Neurodegenerative Diseases (NDs) are tied with UPS impairment and have been also described as proteinopathies caused by aggregate-prone proteins, not efficiently removed by proteasome. Despite the large knowledge on proteasome biological role, molecular mechanisms associated with its impairment are still blur. We have pursued a comprehensive proteomic investigation to evaluate the phenotypic rearrangements in protein repertoires associated with a UPS blockage. Different functional proteomic approaches have been employed to tackle UPS impairment impact on human NeuroBlastoma (NB) cell lines responsive to proteasome inhibition by Epoxomicin. 2-Dimensional Electrophoresis (2-DE) separation combined with Mass Spectrometry and Shotgun Proteomics experiments have been employed to design a thorough picture of protein profile. Unsupervised meta-analysis of the collected proteomic data revealed that all the identified proteins relate each other in a functional network centered on beta-estradiol. Moreover we showed that treatment of cells with beta-estradiol resulted in aggregate removal and increased cell survival due to activation of the autophagic pathway. Our data may provide the molecular basis for the use of beta-estradiol in neurodegenerative disorders by induction of protein aggregate removal.