Roza Lagoudaki

Variable behavior and complications of autologous bone marrow mesenchymal stem cells transplanted in experimental autoimmune encephalomyelitis

Autologous bone marrow stromal cells (BMSCs) offer significant practical advantages for potential clinical applications in multiple sclerosis (MS). Based on recent experimental data, a number of clinical trials have been designed for the intravenous (IV) and/or intrathecal (ITH) administration of BMSCs in MS patients. Delivery of BMSCs in the cerebrospinal fluid via intracerebroventricular (ICV) transplantation is a useful tool to identify mechanisms underlying the migration and function of these cells. In the current study, BMSCs were ICV administered in severe and mild EAE, as well as naive animals; neural precursor cells (NPCs) served as cellular controls. Our data indicated that ICV-transplanted BMSCs significantly ameliorated mild though not severe EAE. Moreover, BMSCs exerted significant anti-inflammatory effect on spinal cord with concomitant reduced axonopathy only in the mild EAE model. BMSCs migrated into the brain parenchyma and, depending on their cellular density, within brain parenchyma formed cellular masses characterized by focal inflammation, demyelination, axonal loss and increased collagen-fibronectin deposition. These masses were present in 64% of ICV BMASC-transplanted severe EAE animals whereas neither BMSCs transplanted in mild EAE cases nor the NPCs exhibited similar behavior. BMSCs possibly exerted their fibrogenic effect via both paracrine and autocrine manner, at least partly due to up-regulation of connective tissue growth factor (CTGF) under the trigger of TGFb1. Our findings are of substantial relevance for clinical trials in MS, particularly regarding the possibility that ICV transplanted BMSCs entering the inflamed central nervous system may exhibit - under conditions - a local pathology of yet unknown consequences.

Administration of 2-arachidonoylglycerol ameliorates both acute and chronic experimental autoimmune encephalomyelitis.

BACKGROUND AND PURPOSE Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis (MS) and both conditions have been reported to exhibit reduced endocannabinoid activity. The purpose of this study was to address the effect of exogenously administered 2-arachidonoylglycerol (2AG), an endocannabinoid receptor ligand, on acute phase and chronic disability in EAE. EXPERIMENTAL APPROACH Acute and chronic EAE models were induced in susceptible mice and 2AG-treatment was applied for 14 days from day of disease induction. KEY RESULTS 2AG-treatment ameliorated acute phase of disease with delay of disease onset in both EAE models and reduced disease mortality and long-term (70 days post-induction) clinical disability in chronic EAE. Reduced axonal pathology in the chronic EAE- (p<0.0001) and increased activation and ramification of microglia in the 2AG-treated acute EAE- (p<0.05) model were noticed. The latter was accompanied by a 2- to 4-fold increase of the M2-macrophages in the perivascular infiltrations (p<0.001) of the 2AG-treated animals in the acute (day 22), although not the chronic (day 70), EAE model. Expression of cannabinoid receptors 1 (CB1R) and 2 (CB2R) was increased in 2AG-treated animals of acute EAE vs. controls (p<0.05). In addition, ex vivo viability assays exhibited reduced proliferation of activated lymph node cells when extracted from 2AG-treated EAE animals, whereas a dose-dependent response of activated lymphocytes to 2AG-treatment in vitro was noticed. CONCLUSION AND IMPLICATIONS Our data indicate for the first time that 2AG treatment may provide direct (via CBRs) and immune (via M2 macrophages) mediated neuroprotection in EAE.

Davunetide (NAP) as a preventative treatment for central nervous system complications in a diabetes rat model

AIMS Central nervous system complications including cognitive impairment are an early manifestation of diabetes mellitus, also evident in animal models. NAP (generic name, davunetide), a neuroprotective peptide was tested here for its ability to prevent diabetes-related brain pathologies in the streptozotocin injected diabetes rat model. METHODS Diabetes was induced by an intraperitoneal streptozotocin injection (55 mg/kg). Intranasal NAP or vehicle was administered daily starting on the day following streptozotocin injection. Cognitive assessment was performed 12 weeks after diabetes induction, using the Morris water maze paradigm. Brain structural integrity was assessed on the 15th week of diabetes by magnetic resonance T2 scan. Characterization of cellular populations, apoptosis and synaptic density was performed 16 weeks after diabetes induction, using immunohistochemical markers and quantified in the prefrontal cortex, the cerebral cortex and the hippocampus of both hemispheres. RESULTS Impaired spatial memory of the diabetic rats was observed in the water maze by attenuated learning curve and worsened performance in the probe memory test. NAP treatment significantly improved both measurements. T2 magnetic resonance imaging revealed atrophy in the prefrontal cortex of the diabetes rat group, which was prevented by NAP treatment. Immunohistochemical analysis showed that NAP treatment protected against major loss of the synaptic marker synaptophysin and astrocytic apoptosis, resulting from streptozotocin treatment. CONCLUSIONS Our results show for the first time protective effects for NAP (davuentide) in a diabetes rat model at the behavioral and structural levels against one of the most severe complications of diabetes.

Autophagy and neurodegenerative disorders

Accumulation of aberrant proteins and inclusion bodies are hallmarks in most neurodegenerative diseases. Consequently, these aggregates within neurons lead to toxic effects, overproduction of reactive oxygen species and oxidative stress. Autophagy is a significant intracellular mechanism that removes damaged organelles and misfolded proteins in order to maintain cell homeostasis. Excessive or insufficient autophagic activity in neurons leads to altered homeostasis and influences their survival rate, causing neurodegeneration. The review article provides an update of the role of autophagic process in representative chronic and acute neurodegenerative disorders.

Time Course and Spatial Profile of Nogo-A Expression in Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice

Abstract Inhibition of the myelin-associated neurite outgrowth inhibitor Nogo-A has been found to be beneficial in experimental autoimmune encephalomyelitis (EAE), but there are little data on its expression dynamics during the disease course. We analyzed Nogo-A mRNA and protein during the course of EAE in 27 C57BL/6 mice and in 8 controls. Histopathologic and molecular analyses were performed on Day 0 (naive), preclinical (Day 10), acute (Days 18–22) and chronic (Day 50) time points. In situ hybridization and real-time polymerase chain reaction analyses revealed reduced Nogo-A mRNA expression at preclinical (p < 0.0001) and acute phases (p < 0.0001), followed by upregulation during the chronic phase (p < 0.0001). Nogo-A mRNA was expressed in neurons and oligodendrocytes. By immunohistochemistry and Western blot, there was increased Nogo-A protein expression (p < 0.001) in the chronic phase. Moreover, spatial differences were observed within EAE lesions. The pattern of Nogo-A protein expression inversely correlated with axonal regeneration growth-associated protein 43–positive axons (60% of which were Nogo-A contact–free during the acute phase) and axonal injury (&bgr;-amyloid precursor protein–positive axons). Cortical Nogo-66 receptor protein and mRNA levels increased during the chronic phase. The results indicate that Nogo-A and Nogo receptor are actively regulated in EAE lesions; this may indicate a specific time window for localized axonal regeneration in the acute phase of EAE.

New horizons in schizophrenia treatment: autophagy protection is coupled with behavioral improvements in a mouse model of schizophrenia

Autophagy plays a key role in the pathophysiology of schizophrenia as manifested by a 40% decrease in BECN1/Beclin 1 mRNA in postmortem hippocampal tissues relative to controls. This decrease was coupled with the deregulation of the essential ADNP (activity-dependent neuroprotector homeobox), a binding partner of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) another major constituent of autophagy. The drug candidate NAP (davunetide), a peptide fragment from ADNP, enhanced the ADNP-LC3B interaction. Parallel genetic studies have linked allelic variation in the gene encoding MAP6/STOP (microtubule-associated protein 6) to schizophrenia, along with altered MAP6/STOP protein expression in the schizophrenic brain and schizophrenic-like behaviors in Map6-deficient mice. In this study, for the first time, we reveal significant decreases in hippocampal Becn1 mRNA and reversal by NAP but not by the antipsychotic clozapine (CLZ) in Map6-deficient (Map6+/−) mice. Normalization of Becn1 expression by NAP was coupled with behavioral protection against hyperlocomotion and cognitive deficits measured in the object recognition test. CLZ reduced hyperlocomotion below control levels and did not significantly affect object recognition. The combination of CLZ and NAP resulted in normalized outcome behaviors. Phase II clinical studies have shown NAP-dependent augmentation of functional activities of daily living coupled with brain protection. The current studies provide a new mechanistic pathway and a novel avenue for drug development.

Characterization of In Vitro Expanded Bone Marrow-Derived Mesenchymal Stem Cells Isolated from Experimental Autoimmune Encephalomyelitis Mice

Extensive experimental studies indicate that autologous bone marrow mesenchymal stem cells (BMSCs) are able to ameliorate experimental autoimmune encephalomyelitis (EAE) and potentially multiple sclerosis. However, the impact that the inflammatory environment present in EAE may have on the biological properties of BMSCs expanded in vitro for transplantation is yet to be clarified. It was investigated whether BMSCs isolated from EAE-induced C57bl6/J mice and expanded in vitro preserve the properties of BMSCs isolated from healthy donors (BMSCs-control). The mesenchymal origin, the differentiation potential, and the transcriptional expression profile of six histone-modifying genes were studied in both groups of BMSCs. BMSCs-EAE exhibited distinct morphology and larger size compared to BMSCs-control, higher degree of proliferation and apoptosis, differences in the adipogenesis and the osteogenesis induction, and differential expression of stromal markers and markers of progenitor and mature neuronal/glial cells. Moreover, BMSCs-EAE exhibited different expression patterns on a number of histone-modifying genes compared to controls. We recorded manifold differences, both phenotypical and functional, of in vitro expanded BMSCs-EAE in comparison to their healthy donor-derived counterparts that may be attributed to the inflammatory environment they originated from. Whether our findings may be of any clinical relevance needs to be clarified in future studies, in vivo.

Connexin43 and connexin47 alterations after neural precursor cells transplantation in experimental autoimmune encephalomyelitis.

Exogenous transplanted neural precursor cells (NPCs) exhibit miscellaneous immune‐modulatory effects in models of autoimmune demyelination. However, the regional interactions of NPCs with the host brain tissue in remissive inflammatory events have not been adequately studied. In this study we used the chronic MOG‐induced Experimental Autoimmune Encephalomyelitis (EAE) model in C57BL/six mice. Based on previous data, we focused on neuropathology at Day 50 post‐induction (D50) and studied the expression of connexin43 (Cx43) and Cx47, two of the main glial gap junction (GJ) proteins, in relation to the intraventricular transplantation of GFP+NPCs and their integration with the host tissue. By D50, NPCs had migrated intraparenchymally and were found in the corpus callosum at the level of the lateral ventricles and hippocampus. The majority of GFP+ cells differentiated with simple or ramified processes expressing mainly markers of mature GLIA (GFAP and NogoA) and significantly less of precursor glial cells. GFP+NPCs expressed connexins and formed GJs around the hippocampus more than lateral ventricles. The presence of NPCs did not alter the increase in Cx43 GJ plaques at D50 EAE, but prevented the reduction of oligodendrocytic Cx47, increased the number of oligodendrocytes, local Cx47 levels and Cx47 GJ plaques per cell. These findings suggest that transplanted NPCs may have multiple effects in demyelinating pathology, including differentiation and direct integration into the panglial syncytium, as well as amelioration of oligodendrocyte GJ loss, increasing the supply of potent myelinating cells to the demyelinated tissue. GLIA 2015;63:1772–1783

Neuroprotective and anti-inflammatory mechanisms are activated early in Optic Neuritis

The aim of the study was to investigate the expression of different immunological mediators in blood and CSF in patients with acute ON and to estimate whether they were implicated in pro‐ or anti‐inflammatory or even regulatory reactions in comparison with a healthy control group (HC).

Immunophenotype of mouse cerebral hemispheres-derived neural precursor cells

Postnatally isolated neural precursor cells (piNPCs) from mouse cerebral tissue have been studied in cell-based therapeutic approaches for Experimental Autoimmune Encephalomyelitis (EAE). Transplantation experiments in EAE rodents revealed that piNPCs manage to integrate into the host tissue and ameliorate clinical symptoms. When cultured in vitro, mouse cerebral piNPCs form neurospheres consisting of immature cells positive for polysialylated neural adhesion molecule (PSA-NCAM) that differentiate mainly towards glial cells, but also neurons. Herein, we have characterized piNPCs immunophenotype, with flow cytometry. NPCs were positive for CD24, CD44, and CD133 though negative for CD15, CD184 and CD49d. This immunophenotype, determined for the first time, among cells isolated from neonates might be useful for the identification of NPC population aiming at the development of transplantation protocols.