Fabrizio Giuliani

Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation

TH17 lymphocytes appear to be essential in the pathogenesis of numerous inflammatory diseases. We demonstrate here the expression of IL-17 and IL-22 receptors on blood-brain barrier endothelial cells (BBB-ECs) in multiple sclerosis lesions, and show that IL-17 and IL-22 disrupt BBB tight junctions in vitro and in vivo. Furthermore, TH17 lymphocytes transmigrate efficiently across BBB-ECs, highly express granzyme B, kill human neurons and promote central nervous system inflammation through CD4+ lymphocyte recruitment.

Granule-Derived Granzyme B Mediates the Vulnerability of Human Neurons to T Cell-Induced Neurotoxicity

Multiple sclerosis (MS) is considered an autoimmune disease of the CNS and is characterized by inflammatory cells infiltrating the CNS and inducing demyelination, axonal loss, and neuronal death. Recent evidence strongly suggests that axonal and neuronal degeneration underlie the progression of permanent disability in MS. In this study, we report that human neurons are selectively susceptible to the serine-protease granzyme B (GrB) isolated from cytotoxic T cell granules. In vitro, purified human GrB induced neuronal death to the same extent as the whole activated T cell population. On the contrary, activated T cells isolated from GrB knockout mice failed to induce neuronal injury. We found that following internalization through various parts of neurons, GrB accumulated in the neuronal soma. Within the cell body, GrB diffused out of endosomes possibly through a perforin-independent mechanism and induced subsequent activation of caspases and cleavage of α-tubulin. Inhibition of caspase-3, a well-known substrate for GrB, significantly reduced GrB-mediated neurotoxicity. We demonstrated that treatment of neurons with mannose-6-phosphate prevented GrB entry and inhibited GrB-mediated neuronal death, suggesting mannose-6-phosphate receptor-dependent endocytosis. Together, our data unveil a novel mechanism by which GrB induces selective neuronal injury and suggest potential new targets for the treatment of inflammatory-mediated neurodegeneration in diseases such as MS.

Innexin 3, a New Gene Required for Dorsal Closure in Drosophila Embryo

BACKGROUND Dorsal closure is a morphogenetic event that occurs during mid-embryogenesis in many insects including Drosophila, during which the ectoderm migrates on the extraembryonic amnioserosa to seal the embryo dorsally. The contribution of the ectoderm in this event has been known for a long time. However, amnioserosa tension and contractibility have recently been shown also to be instrumental to the closure. A critical pre-requisite for dorsal closure is integrity of these tissues that in part is mediated by cell-cell junctions and cell adhesion. In this regard, mutations impairing junction formation and/or adhesion lead to dorsal closure. However, no role for the gap junction proteins Innexins has so far been described. RESULTS AND DISCUSSION Here, we show that Innexin 1, 2 and 3, are present in the ectoderm but also in the amnioserosa in plaques consistent with gap junctions. However, only the loss of Inx3 leads to dorsal closure defects that are completely rescued by overexpression of inx3::GFP in the whole embryo. Loss of Inx3 leads to the destabilisation of Inx1, Inx2 and DE-cadherin at the plasma membrane, suggesting that these four proteins form a complex. Accordingly, in addition to the known interaction of Inx2 with DE-cadherin, we show that Inx3 can bind to DE-cadherin. Furthermore, Inx3-GFP overexpression recruits DE-cadherin from its wildtype plasma membrane domain to typical Innexin plaques, strengthening the notion that they form a complex. Finally, we show that Inx3 stability is directly dependent on tissue tension. Taken together, we propose that Inx3 is a critical factor for dorsal closure and that it mediates the stability of Inx1, 2 and DE-cadherin by forming a complex.

Characterization of the NT2‐derived neuronal and astrocytic cell lines as alternative in vitro models for primary human neurons and astrocytes

Primary human fetal neurons and astrocytes (HFNs and HFAs, respectively) provide relevant cell types with which to study in vitro the mechanisms involved in various human neurological diseases, such as multiple sclerosis, Parkinsons disease, and Alzheimers disease. However, the limited availability of human fetal cells poses a significant problem for the study of these diseases when a human cell model system is required. Thus, generating a readily available alternative cell source with the essential features of human neurons and astrocytes is necessary. The human teratoma‐derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal and glial cells can be generated. Nevertheless, a direct comparison of NT2 neurons and primary HFNs in terms of their morphology physiological and chemical properties is still missing. This study directly compares NT2‐derived neurons and primary HFNs using immunocytochemistry, confocal calcium imaging, high‐performance liquid chromatography, and high‐content analysis techniques. We investigated the morphological similarities and differences, levels of relevant amino acids, and internal calcium fluctuations in response to certain neurotransmitters/stimuli. We also compared NT2‐derived astrocytes and HFAs. In most of the parameters tested, both neuronal and astrocytic cell types exhibited similarities to primary human fetal neurons and astrocytes. NT2‐derived neurons and astrocytes are reliable in vitro tools and a renewable cell source that can serve as a valid alternative to HFNs/HFAs for mechanistic studies of neurological diseases.

Rab32 connects ER stress to mitochondrial defects in multiple sclerosis

BackgroundEndoplasmic reticulum (ER) stress is a hallmark of neurodegenerative diseases such as multiple sclerosis (MS). However, this physiological mechanism has multiple manifestations that range from impaired clearance of unfolded proteins to altered mitochondrial dynamics and apoptosis. While connections between the triggering of the unfolded protein response (UPR) and downstream mitochondrial dysfunction are poorly understood, the membranous contacts between the ER and mitochondria, called the mitochondria-associated membrane (MAM), could provide a functional link between these two mechanisms. Therefore, we investigated whether the guanosine triphosphatase (GTPase) Rab32, a known regulator of the MAM, mitochondrial dynamics, and apoptosis, could be associated with ER stress as well as mitochondrial dysfunction.MethodsWe assessed Rab32 expression in MS patient and experimental autoimmune encephalomyelitis (EAE) tissue, via observation of mitochondria in primary neurons and via monitoring of survival of neuronal cells upon increased Rab32 expression.ResultsWe found that the induction of Rab32 and other MAM proteins correlates with ER stress proteins in MS brain, as well as in EAE, and occurs in multiple central nervous system (CNS) cell types. We identify Rab32, known to increase in response to acute brain inflammation, as a novel unfolded protein response (UPR) target. High Rab32 expression shortens neurite length, alters mitochondria morphology, and accelerates apoptosis/necroptosis of human primary neurons and cell lines.ConclusionsER stress is strongly associated with Rab32 upregulation in the progression of MS, leading to mitochondrial dysfunction and neuronal death.

Hydroxychloroquine reduces microglial activity and attenuates experimental autoimmune encephalomyelitis

BACKGROUND Microglial activation is thought to be a key pathophysiological mechanism underlying disease activity in all forms of MS. Hydroxychloroquine (HCQ) is an antimalarial drug with immunomodulatory properties that is widely used in the treatment of rheumatological diseases. In this series of experiments, we explore the effect of HCQ on human microglial activation in vitro and on the development of experimental autoimmune encephalitis (EAE) in vivo. METHODS We activated human microglia with lipopolysaccharide (LPS), and measured concentrations of several pro- and anti-inflammatory cytokines in untreated and HCQ pretreated cultures. We investigated the effect of HCQ pretreatment at two doses on the development of EAE and spinal cord histology. RESULTS HCQ pretreatment reduced the production of pro-inflammatory (TNF-alpha, IL-6, and IL-12) and anti-inflammatory (IL-10 and IL-1 receptor antagonist) cytokines in LPS-stimulated human microglia. HCQ pretreatment delayed the onset of EAE, and reduced the number of Iba-1 positive microglia/macrophages and signs of demyelination in the spinal cords of HCQ treated animals. CONCLUSION HCQ treatment reduces the activation of human microglia in vitro, delays the onset of EAE, and decreases the representation of activated macrophages/microglia and demyelination in the spinal cord of treated mice. HCQ is a plausible candidate for further clinical studies in MS.

Reprogramming of HUVECs into Induced Pluripotent Stem Cells (HiPSCs), Generation and Characterization of HiPSC-Derived Neurons and Astrocytes

Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for “patient-matched” cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their treatments.

Granzyme B-inhibitor serpina3n induces neuroprotection in vitro and in vivo

BackgroundMultiple sclerosis (MS) is an autoimmune inflammatory and neurodegenerative disease of the central nervous system (CNS). It is widely accepted that inflammatory cells play major roles in the pathogenesis of MS, possibly through the use of serine protease granzyme B (GrB) secreted from the granules of cytotoxic T cells. We have previously identified GrB as a mediator of axonal injury and neuronal death. In this study, our goal was to evaluate the effect of GrB inhibition in the human system in vitro, and in vivo in EAE using the newly isolated GrB-inhibitor serpina3n.MethodsWe used a well-established in vitro model of neuroinflammation characterized by a co-culture system between human fetal neurons and lymphocytes. In vivo, we induced EAE in 10- to 12-week-old female C57/BL6 mice and treated them intravenously with serpina3n.ResultsIn the in vitro co-culture system, pre-treatment of lymphocytes with serpina3n prevented neuronal killing and cleavage of the cytoskeletal protein alpha-tubulin, a known substrate for GrB. Moreover, in EAE, 50 μg serpina3n substantially reduced the severity of the disease. This dose was administered intravenously twice at days 7 and 20 post EAE induction. serpina3n treatment reduced axonal and neuronal injury compared to the vehicle-treated control group and maintained the integrity of myelin. Interestingly, serpina3n treatment did not seem to reduce the infiltration of immune cells (CD4+ and CD8+ T cells) into the CNS.ConclusionOur data suggest further studies on serpina3n as a potentially novel therapeutic strategy for the treatment of inflammatory-mediated neurodegenerative diseases such as MS.

Canadian Expert Panel Recommendations for MRI Use in MS Diagnosis and Monitoring

Background: A definitive diagnosis of multiple sclerosis (MS), as distinct from a clinically isolated syndrome, requires one of two conditions: a second clinical attack or particular magnetic resonance imaging (MRI) findings as defined by the McDonald criteria. MRI is also important after a diagnosis is made as a means of monitoring subclinical disease activity. While a standardized protocol for diagnostic and follow-up MRI has been developed by the Consortium of Multiple Sclerosis Centres, acceptance and implementation in Canada have been suboptimal. Methods: To improve diagnosis, monitoring, and management of a clinically isolated syndrome and MS, a Canadian expert panel created consensus recommendations about the appropriate application of the 2010 McDonald criteria in routine practice, strategies to improve adherence to the standardized Consortium of Multiple Sclerosis Centres MRI protocol, and methods for ensuring effective communication among health care practitioners, in particular referring physicians, neurologists, and radiologists. Results: This article presents eight consensus statements developed by the expert panel, along with the rationale underlying the recommendations and commentaries on how to prioritize resource use within the Canadian healthcare system. Conclusions: The expert panel calls on neurologists and radiologists in Canada to incorporate the McDonald criteria, the Consortium of Multiple Sclerosis Centres MRI protocol, and other guidance given in this consensus presentation into their practices. By improving communication and general awareness of best practices for MRI use in MS diagnosis and monitoring, we can improve patient care across Canada by providing timely diagnosis, informed management decisions, and better continuity of care.

CD4+CD25+CD127dimFoxp3+ T cells are cytotoxic for human neurons

MS lesions are characterized by destruction of myelin and significant neuronal and axonal loss. Preliminary studies with the use of Tregs in the mouse model of MS have been extremely encouraging. However, recent studies with human cells have shown the presence of different subpopulations of T cells within the CD4+CD25+Foxp3+ T cell phenotype, some of which do not have regulatory functions. These findings suggest a potential difference between mouse and human in the regulatory phenotype. Here, we show that human activated CD4+CD25+Foxp3+ T cells are neurotoxic in vitro. These cells expressed high levels of the cytotoxic molecule GrB and had no suppressive effect. On the contrary, they produced IFN‐γ and low IL‐17, suggesting a shift toward a TH1 phenotype. Thus, our data confirm the presence of a nonregulatory cytotoxic subpopulation within the human CD4+CD25+Foxp3+ T cells and suggest further studies on the human regulatory phenotype prior to any potential therapeutic application.