Publications by authors named "Fabrizio Giuliani"

44 Publications

Cross-sectional analysis of peripheral blood mononuclear cells in lymphopenic and non-lymphopenic relapsing-remitting multiple sclerosis patients treated with dimethyl fumarate.

Mult Scler Relat Disord 2021 Jul 7;52:103003. Epub 2021 May 7.

Division of Neurology, Department of Medicine, University of Alberta, Alberta Canada T6G 2M8, Edmonton, Canada. Electronic address:

Background: Relapsing-remitting multiple sclerosis (RRMS) is an autoimmune disorder of the central nervous system. Dimethyl Fumarate is a disease-modifying medication used to treat RRMS patients that can induce lymphopenia. We aimed to immunophenotype peripheral blood mononuclear cells (PBMC) in RRMS patients cross-sectionally and examine the characteristics and modifications of lymphopenia over time.

Methods: Characterization of PBMC was done by multiparametric flow cytometry. Patients had been on treatment for up to 4 years and were grouped into lymphopenic (DMF-L) and non-lymphopenic (DMF-N) patients.

Results: Lymphopenia affected the cell population changes over time, with other patient characteristics (gender, age, and previous treatment status) also having significant effects. In both lymphopenic and non-lymphopenic patients, PBMC percentages were reduced over time. While overall T and B cells frequencies were not affected, males, older patients and untreated patients had significant changes in B cell subpopulations over time. CD4 to CD8T cell ratio increased significantly in lymphopenic patients over time. CD4CD8T cell population was similarly reduced in both lymphopenic and non-lymphopenic patients, over time. While the monocyte and NK overall populations were not changed, non-classical monocyte subpopulation decreased over time in lymphopenic patients. We also found CD56CD16 and CD56CD16 NK cells frequencies changed over time in lymphopenic patients. Immune populations showed correlations with clinical outcomes measured by EDSS and relapse rate. Analysis of the overall immunophenotype showed that, while groups divided by other patient characteristics showed differences, the lymphopenia status overrode these differences, resulting in similar immunophenotype within DMF-L.

Conclusions: Our data provide evidence that under the same therapy, lymphopenia affects how the immunophenotype changes over time and can override the differences associated with other patient characteristics and possibly mask other significant changes in the immune profile of patients.
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http://dx.doi.org/10.1016/j.msard.2021.103003DOI Listing
July 2021

Multimodal peripheral fluid biomarker analysis in clinically isolated syndrome and early multiple sclerosis.

Mult Scler Relat Disord 2021 May 3;50:102809. Epub 2021 Feb 3.

Departments of Clinical Neurosciences, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.

Background: Increasing evidence suggests that various inflammatory, immunological and metabolic pathways are altered in the clinically isolated syndrome (CIS) of multiple sclerosis (MS). Moreover, recent diagnostic criteria have made possible the very early diagnosis of MS. We evaluated multiple fluid biomarkers in people with early MS and CIS.

Methods: We measured blood levels of cytokines, matrix metalloproteinases (MMPs), serum metabolomics and immune cell immunophenotyping in participants in the Trial of Minocycline in a Clinically Isolated Syndrome of Multiple Sclerosis.

Results: When compared with healthy controls, people with early MS/CIS had higher levels of eotaxin, MCP-3, IL-1 receptor antagonist, IL-1β, IL-9 and IP-10, as well as MMPs 1, 8 and 9. In metabolomics analysis, the alanine, aspartate and glutamate metabolism and the synthesis and degradation of ketone bodies pathways were altered compared to healthy controls. There were no differences in lymphocyte subpopulation numbers. Out of all these biomarkers, only MMP-1 was able to differentiate between early MS and CIS, and was found to correlate with lesion volume and gadolinium enhancing lesions on MRI.

Conclusion: The immunological and metabolic profile of CIS and early MS is remarkably similar, supporting that these are a continuum of a common underlying pathophysiological process.
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http://dx.doi.org/10.1016/j.msard.2021.102809DOI Listing
May 2021

Treatment Optimization in Multiple Sclerosis: Canadian MS Working Group Recommendations.

Can J Neurol Sci 2020 07 6;47(4):437-455. Epub 2020 Apr 6.

The Hospital for Sick Children, Toronto, Ontario, Canada.

The Canadian Multiple Sclerosis Working Group has updated its treatment optimization recommendations (TORs) on the optimal use of disease-modifying therapies for patients with all forms of multiple sclerosis (MS). Recommendations provide guidance on initiating effective treatment early in the course of disease, monitoring response to therapy, and modifying or switching therapies to optimize disease control. The current TORs also address the treatment of pediatric MS, progressive MS and the identification and treatment of aggressive forms of the disease. Newer therapies offer improved efficacy, but also have potential safety concerns that must be adequately balanced, notably when treatment sequencing is considered. There are added discussions regarding the management of pregnancy, the future potential of biomarkers and consideration as to when it may be prudent to stop therapy. These TORs are meant to be used and interpreted by all neurologists with a special interest in the management of MS.
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http://dx.doi.org/10.1017/cjn.2020.66DOI Listing
July 2020

Diffusion tensor imaging tractography reveals altered fornix in all diagnostic subtypes of multiple sclerosis.

Brain Behav 2020 01 19;10(1):e01514. Epub 2019 Dec 19.

Department of Biomedical Engineering, University of Alberta, Edmonton, AB, Canada.

Introduction: Diffusion tensor imaging (DTI) has shown abnormalities of the fornix and other limbic white matter tracts in multiple sclerosis (MS), mainly focusing on relapsing-remitting MS.

Methods: The goal here was to evaluate the fornix, cingulum, and uncinate fasciculus with DTI tractography at 1.7 mm isotropic resolution in three MS subgroups (11 relapsing-remitting (RRMS), nine secondary progressive (SPMS), eight primary progressive (PPMS)) versus 11 controls, and assess correlations with cognitive and clinical scores.

Results: The MS group overall showed extensive diffusion abnormalities of the fornix with less volume, lower fractional anisotropy (FA), and higher mean and radial diffusivities, which were similarly affected in all three MS subgroups. The uncinate fasciculus had lower FA only in the secondary progressive subgroup, and the cingulum had no DTI differences in any MS subgroup. The FA and/or volumes of these tracts correlated negatively with larger total lesion volume. The only DTI-cognitive correlation was lower right cingulum FA and greater depression over the entire MS cohort.

Conclusions: Diffusion tractography identified abnormalities in the fornix that appears to be affected early and consistently across all three primary MS phenotypes of RRMS, SPMS, and PPMS regardless of Expanded Disability Status Scale, time since diagnosis, or cognitive scores.
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http://dx.doi.org/10.1002/brb3.1514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955822PMC
January 2020

Repression of phagocytosis by human CD33 is not conserved with mouse CD33.

Commun Biol 2019 3;2:450. Epub 2019 Dec 3.

1Department of Chemistry, University of Alberta, Alberta, Canada.

CD33 is an immunomodulatory receptor linked to Alzheimer's disease (AD) susceptibility via regulation of phagocytosis in microglia. Divergent features between human CD33 (hCD33) and murine CD33 (mCD33) include a unique transmembrane lysine in mCD33 and cytoplasmic tyrosine in hCD33. The functional consequences of these differences in restraining phagocytosis remains poorly understood. Using a new αmCD33 monoclonal antibody, we show that mCD33 is expressed at high levels on neutrophils and low levels on microglia. Notably, cell surface expression of mCD33 is entirely dependent on Dap12 due to an interaction with the transmembrane lysine in mCD33. In RAW264.7 cultured macrophages, BV-2 cultured microglia, primary neonatal and adult microglia, uptake of cargo - including aggregated Aβ - is not altered upon genetic ablation of mCD33. Alternatively, deletion of hCD33 in monocytic cell lines increased cargo uptake. Moreover, transgenic mice expressing hCD33 in the microglial cell lineage showed repressed cargo uptake in primary microglia. Therefore, mCD33 and hCD33 have divergent roles in regulating phagocytosis, highlighting the importance of studying hCD33 in AD susceptibility.
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http://dx.doi.org/10.1038/s42003-019-0698-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890642PMC
July 2020

RNA-Sequencing Highlights Inflammation and Impaired Integrity of the Vascular Wall in Brain Arteriovenous Malformations.

Stroke 2020 01 4;51(1):268-274. Epub 2019 Dec 4.

Department of Neurology, Donders Institute of Brain Cognition & Behaviour, Center for Neuroscience, Radboud University Medical Center, Nijmegen, the Netherlands (C.J.M.K.).

Background and Purpose- Interventional treatment of unruptured brain arteriovenous malformations (BAVMs) has become increasingly controversial. Because medical therapy is still lacking, we aimed to obtain insight into the disease mechanisms implicated in BAVMs and to identify potential targets for medical treatment to prevent rupture of a BAVM. Methods- We used next-generation RNA sequencing to identify differential expression on a transcriptome-wide level comparing tissue samples of 12 BAVMs to 16 intracranial control arteries. We identified differentially expressed genes by negative binominal generalized log-linear regression (false discovery rate corrected <0.05). We selected 10 genes for validation using droplet digital polymerase chain reaction. We performed functional pathway analysis accounting for potential gene-length bias, to establish enhancement of biological pathways involved in BAVMs. We further assessed which Gene Ontology terms were enriched. Results- We found 736 upregulated genes in BAVMs including genes implicated in the cytoskeletal machinery and cell-migration and genes encoding for inflammatory cytokines and secretory products of neutrophils and macrophages. Furthermore, we found 498 genes downregulated including genes implicated in extracellular matrix composition, the binary angiopoietin-TIE system, and TGF (transforming growth factor)-β signaling. We confirmed the differential expression of top 10 ranked genes. Functional pathway analysis showed enrichment of the protein digestion and absorption pathway (false discovery rate-adjusted =1.70×10). We identified 47 enriched Gene Ontology terms (false discovery rate-adjusted <0.05) implicated in cytoskeleton network, cell-migration, endoplasmic reticulum, transmembrane transport, and extracellular matrix composition. Conclusions- Our genome-wide RNA-sequencing study points to involvement of inflammatory mediators, loss of cerebrovascular quiescence, and impaired integrity of the vascular wall in the pathophysiology of BAVMs. Our study may lend support to potential receptivity of BAVMs to medical therapeutics, including those promoting vessel maturation, and anti-inflammatory and immune-modifying drugs.
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http://dx.doi.org/10.1161/STROKEAHA.119.025657DOI Listing
January 2020

Diagnosis and management of secondary-progressive multiple sclerosis: time for change.

Neurodegener Dis Manag 2019 12 26;9(6):301-317. Epub 2019 Nov 26.

University of Montreal Research Centre, Montreal, QC H9S 1A9, Canada.

Identifying the transition of relapsing-remitting multiple sclerosis (MS) to the secondary-progressive MS form remains a clinical challenge due to the gradual nature of the transition, superimposed relapses, the heterogeneous course of disease among patients and the absence of validated biomarkers and diagnostic tools. The uncertainty associated with the transition makes clinical care challenging for both patients and physicians. The emergence of new disease-modifying treatments for progressive MS and the increasing emphasis of nonpharmacological strategies mark a new era in the treatment of progressive MS. This article summarizes challenges in diagnosis and management, discusses novel treatment strategies and highlights the importance of establishing a clear diagnosis and instituting an interdisciplinary management plan in the care of patients with progressive MS.
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http://dx.doi.org/10.2217/nmt-2019-0024DOI Listing
December 2019

Coding and Non-Coding RNA Abnormalities in Bipolar Disorder.

Genes (Basel) 2019 11 19;10(11). Epub 2019 Nov 19.

Department of Translational Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht University, 3584 CX Utrecht, The Netherlands.

The molecular mechanisms underlying bipolar disorder (BPD) have remained largely unknown. Postmortem brain tissue studies comparing BPD patients with healthy controls have produced a heterogeneous array of potentially implicated protein-coding RNAs. We hypothesized that dysregulation of not only coding, but multiple classes of RNA (coding RNA, long non-coding (lnc) RNA, circular (circ) RNA, and/or alternative splicing) underlie the pathogenesis of BPD. Using non-polyadenylated libraries we performed RNA sequencing in postmortem human medial frontal gyrus tissue from BPD patients and healthy controls. Twenty genes, some of which not previously implicated in BPD, were differentially expressed (DE). PCR validation and replication confirmed the implication of these DE genes. Functional in silico analyses identified enrichment of angiogenesis, vascular system development and histone H3-K4 demethylation. In addition, ten lncRNA transcripts were differentially expressed. Furthermore, an overall increased number of alternative splicing events in BPD was detected, as well as an increase in the number of genes carrying alternative splicing events. Finally, a large reservoir of circRNAs populating brain tissue not affected by BPD is described, while in BPD altered levels of two circular transcripts, cNEBL and cEPHA3, are reported. cEPHA3, hitherto unlinked to BPD, is implicated in developmental processes in the central nervous system. Although we did not perform replication analyses of non-coding RNA findings, our findings hint that RNA dysregulation in BPD is not limited to coding regions, opening avenues for future pharmacological investigations and biomarker research.
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http://dx.doi.org/10.3390/genes10110946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895892PMC
November 2019

The Role of Inflammation in Depression and Fatigue.

Front Immunol 2019 19;10:1696. Epub 2019 Jul 19.

Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada.

Depression and fatigue are conditions responsible for heavy global societal burden, especially in patients already suffering from chronic diseases. These symptoms have been identified by those affected as some of the most disabling symptoms which affect the quality of life and productivity of the individual. While many factors play a role in the development of depression and fatigue, both have been associated with increased inflammatory activation of the immune system affecting both the periphery and the central nervous system (CNS). This is further supported by the well-described association between diseases that involve immune activation and these symptoms in autoimmune disorders, such as multiple sclerosis and immune system activation in response to infections, like sepsis. Treatments for depression also support this immunopsychiatric link. Antidepressants have been shown to decrease inflammation, while higher levels of baseline inflammation predict lower treatment efficacy for most treatments. Those patients with higher initial immune activation may on the other hand be more responsive to treatments targeting immune pathways, which have been found to be effective in treating depression and fatigue in some cases. These results show strong support for the hypothesis that depression and fatigue are associated with an increased activation of the immune system which may serve as a valid target for treatment. Further studies should focus on the pathways involved in these symptoms and the development of treatments that target those pathways will help us to better understand these conditions and devise more targeted treatments.
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http://dx.doi.org/10.3389/fimmu.2019.01696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658985PMC
October 2020

Characterization of lymphopenia in patients with MS treated with dimethyl fumarate and fingolimod.

Neurol Neuroimmunol Neuroinflamm 2018 Mar 28;5(2):e432. Epub 2017 Dec 28.

Division of Neurology, Department of Medicine, University of Alberta, Edmonton, Canada.

Objective: Lymphopenia is a common occurrence of disease-modifying therapies (DMTs) for relapsing-remitting MS (RRMS). The aim of this study was to dissect the prevalence of various lymphocyte subsets in patients with RRMS treated with 2 DMTs commonly associated with lymphopenia, dimethyl fumarate (DMF), and fingolimod (FTY).

Methods: Multicolor flow cytometry and multiplex assays were used to identify up to 50 lymphocyte subpopulations and to examine the expression of multiple cytokines in selected patients. We compared patients untreated (NT) or treated with FTY or DMF who did (DMF-L) or did not (DMF-N) develop lymphopenia.

Results: All FTY patients developed lymphopenia in both T-cell and B-cell compartments. CD41 T cells were more affected by this treatment than CD81 cells. In the B-cell compartment, the CD271IgD2 subpopulation was reduced. T cells but not B cells were significantly reduced in DMF-L. However, within the B cells, CD271 cells were significantly lower. Both CD41 and CD81 subpopulations were reduced in DMF-L. Within the remaining CD41 and CD81 compartments, there was an expansion of the naive subpopulation and a reduction of the effector memory subpopulation. Unactivated lymphocyte from DMF-L patients had significantly higher levels of interferon-γ, interleukin (IL)-12, IL-2, IL-4, IL-6, and IL-1β compared with DMF-N. In plasma, TNFβ was significantly higher in DMF-N and DMF-L compared with NT, whereas CCL17 was significantly higher in DMF-L compared with NT and DMF-N.

Conclusions: This study shows that different treatments can target different lymphocyte compartments and suggests that lymphopenia can induce compensatory mechanisms to maintain immune homeostasis.
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http://dx.doi.org/10.1212/NXI.0000000000000432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746425PMC
March 2018

GFAPδ/GFAPα ratio directs astrocytoma gene expression towards a more malignant profile.

Oncotarget 2017 Oct 4;8(50):88104-88121. Epub 2017 Oct 4.

Netherlands Institute for Neuroscience, an Institute of The Royal Netherlands Academy of Arts and Sciences, 1105 BA Amsterdam, The Netherlands.

Astrocytomas are the most common malignant brain tumours and are to date incurable. It is unclear how astrocytomas progress into higher malignant grades. The intermediate filament cytoskeleton is emerging as an important regulator of malignancy in several tumours. The majority of the astrocytomas express the intermediate filament protein Glial Fibrillary Acidic Protein (GFAP). Several splice variants have been identified and the main variants expressed in human astrocytoma are the α and δ isoforms. Here we show a significant downregulation of α in grade IV astrocytoma compared to grade II and III, resulting in an increased GFAPδ/ ratio. Mimicking this increase in ratio in astrocytoma cell lines and comparing the subsequent transcriptomic changes with the changes in the patient tumours, we have identified a set of ratio-regulated high-malignant and low-malignant genes. These genes are involved in cell proliferation and protein phosphorylation, and their expression correlated with patient survival. We additionally show that changing the ratio of , by targeting expression, affected expression of high-malignant genes. Our data imply that regulating expression and splicing are novel therapeutic targets that need to be considered as a treatment for astrocytoma.
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http://dx.doi.org/10.18632/oncotarget.21540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675697PMC
October 2017

Rab32 connects ER stress to mitochondrial defects in multiple sclerosis.

J Neuroinflammation 2017 01 23;14(1):19. Epub 2017 Jan 23.

Department of Cell Biology, University of Alberta, Edmonton, Canada.

Background: Endoplasmic 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.

Methods: We 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.

Results: We 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.

Conclusions: ER stress is strongly associated with Rab32 upregulation in the progression of MS, leading to mitochondrial dysfunction and neuronal death.
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http://dx.doi.org/10.1186/s12974-016-0788-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5260063PMC
January 2017

RNA Sequencing Analysis of Intracranial Aneurysm Walls Reveals Involvement of Lysosomes and Immunoglobulins in Rupture.

Stroke 2016 05 29;47(5):1286-93. Epub 2016 Mar 29.

From the Department of Neurology and Neurosurgery, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands (R.K., B.H.V., F.G., A.v.d.Z., J.W.B.v.d.S., K.S.H., P.G., P.C.v.R., G.J.E.R., J.H.V., Y.M.R.); Department of Genetics (P.v.d.V., P.D., M.A.S.) and Genomics Coordination Center (P.D., M.A.S.), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Laboratory of Neurobiology, Vesalius Research Center, VIB, Leuven, Belgium (L.d.M., P.V.D.); Department of Neurosurgery, University Hospital Zurich, Zurich, Switzerland (L.R.); and Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands (E.K., J.A.P.).

Background And Purpose: Analyzing genes involved in development and rupture of intracranial aneurysms can enhance knowledge about the pathogenesis of aneurysms, and identify new treatment strategies. We compared gene expression between ruptured and unruptured aneurysms and control intracranial arteries.

Methods: We determined expression levels with RNA sequencing. Applying a multivariate negative binomial model, we identified genes that were differentially expressed between 44 aneurysms and 16 control arteries, and between 22 ruptured and 21 unruptured aneurysms. The differential expression of 8 relevant and highly significant genes was validated using digital polymerase chain reaction. Pathway analysis was used to identify enriched pathways. We also analyzed genes with an extreme pattern of differential expression: only expressed in 1 condition without any expression in the other.

Results: We found 229 differentially expressed genes in aneurysms versus controls and 1489 in ruptured versus unruptured aneurysms. The differential expression of all 8 genes selected for digital polymerase chain reaction validation was confirmed. Extracellular matrix pathways were enriched in aneurysms versus controls, whereas pathways involved in immune response and the lysosome pathway were enriched in ruptured versus unruptured aneurysms. Immunoglobulin genes were expressed in aneurysms, but showed no expression in controls.

Conclusions: For rupture of intracranial aneurysms, we identified the lysosome pathway as a new pathway and found further evidence for the role of the immune response. Our results also point toward a role for immunoglobulins in the pathogenesis of aneurysms. Immune-modifying drugs are, therefore, interesting candidate treatment strategies in the prevention of aneurysm development and rupture.
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http://dx.doi.org/10.1161/STROKEAHA.116.012541DOI Listing
May 2016

Fatigue in Multiple Sclerosis: Assessing Pontine Involvement Using Proton MR Spectroscopic Imaging.

PLoS One 2016 19;11(2):e0149622. Epub 2016 Feb 19.

Department of Biomedical Engineering, University of Alberta, Edmonton, Alberta, Canada.

Background/objective: The underlying mechanism of fatigue in multiple sclerosis (MS) remains poorly understood. Our study investigates the involvement of the ascending reticular activating system (ARAS), originating in the pontine brainstem, in MS patients with symptoms of fatigue.

Methods: Female relapsing-remitting MS patients (n = 17) and controls (n = 15) underwent a magnetic resonance spectroscopic imaging protocol at 1.5T. Fatigue was assessed in every subject using the Fatigue Severity Scale (FSS). Using an FSS cut-off of 36, patients were categorized into a low (n = 9, 22 ± 10) or high (n = 10, 52 ± 6) fatigue group. The brain metabolites N-acetylaspartate (NAA) and total creatine (tCr) were measured from sixteen 5x5x10 mm3 spectroscopic imaging voxels in the rostral pons.

Results: MS patients with high fatigue had lower NAA/tCr concentration in the tegmental pons compared to control subjects. By using NAA and Cr values in the cerebellum for comparison, these NAA/tCr changes in the pons were driven by higher tCr concentration, and that these changes were focused in the WM regions.

Discussion/conclusion: Since there were no changes in NAA concentration, the increase in tCr may be suggestive of gliosis, or an imbalanced equilibrium of the creatine and phosphocreatine ratio in the pons of relapsing-remitting MS patients with fatigue.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149622PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760929PMC
July 2016

Hydroxychloroquine reduces microglial activity and attenuates experimental autoimmune encephalomyelitis.

J Neurol Sci 2015 Nov 28;358(1-2):131-7. Epub 2015 Aug 28.

Department of Clinical Neurosciences and Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada.

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.
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http://dx.doi.org/10.1016/j.jns.2015.08.1525DOI Listing
November 2015

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

J Neuroinflammation 2015 Sep 4;12:157. Epub 2015 Sep 4.

Department of Medicine, Division of Neurology, University of Alberta, 4C Kaye Edmonton Clinic, Edmonton, Alberta, T6G 1Z1, Canada.

Background: Multiple 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.

Methods: We 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.

Results: In 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.

Conclusion: Our data suggest further studies on serpina3n as a potentially novel therapeutic strategy for the treatment of inflammatory-mediated neurodegenerative diseases such as MS.
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http://dx.doi.org/10.1186/s12974-015-0376-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4558826PMC
September 2015

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

Can J Neurol Sci 2015 May 21;42(3):159-67. Epub 2015 Apr 21.

19Department of Radiology/Division of Neurology,University of British Columbia,Vancouver,British Columbia,Canada.

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.
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http://dx.doi.org/10.1017/cjn.2015.24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416356PMC
May 2015

Reprogramming of HUVECs into induced pluripotent stem cells (HiPSCs), generation and characterization of HiPSC-derived neurons and astrocytes.

PLoS One 2015 19;10(3):e0119617. Epub 2015 Mar 19.

Department of Medicine, University of Alberta, Edmonton, Canada.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119617PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366250PMC
February 2016

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

J Neurosci Res 2014 Sep 7;92(9):1187-98. Epub 2014 May 7.

Division of Neurology, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada.

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, Parkinson's disease, and Alzheimer's 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.
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http://dx.doi.org/10.1002/jnr.23399DOI Listing
September 2014

The regulation of reactive changes around multiple sclerosis lesions by phosphorylated signal transducer and activator of transcription.

J Neuropathol Exp Neurol 2013 Dec;72(12):1135-44

From the Departments of Laboratory Medicine and Pathology (JQL), and Medicine/Neurology (CP, GB, FG), University of Alberta, Edmonton, Alberta, Canada; and Departments of Clinical Neurosciences and Oncology, University of Calgary, Calgary, Alberta, Canada (VWY).

Activation of signal transducer and activator of transcription 3 (STAT3) by phosphorylation is thought to mediate anti-inflammatory responses to CNS injury. Several studies have reported an increase in phosphorylated STAT3 (pSTAT3) in peripheral T cells and monocytes from patients with multiple sclerosis (MS) during relapses, suggesting that pSTAT3 might represent an inflammatory marker. Here, we examined immunoreactivity for pSTAT3 in brain tissue samples from MS patients and controls. Phosphorylated STAT3 immunoreactivity was sparse within lesions, with no difference between active and inactive lesions. It was, however, significantly greater in white matter (WM) adjacent to active and inactive lesions; moreover, it was significantly greater in WM adjacent to active versus inactive lesions. Phosphorylated STAT3-positive cells were identified as astrocytes and macrophages/microglia. Phosphorylated STAT3 expression was also detected by Western blotting in WM of patients with MS. In comparison, pSTAT3 immunoreactivity was either rare or found focally in brain tissue samples from patients with other neurologic diseases. Our findings show that pSTAT3 does not correlate with inflammatory activity in MS lesions, but that it may play an important role in regulating reactive changes proximal to MS lesions.
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http://dx.doi.org/10.1097/NEN.0000000000000011DOI Listing
December 2013

The Drosophila RNA-binding protein HOW controls the stability of dgrasp mRNA in the follicular epithelium.

Nucleic Acids Res 2014 Feb 11;42(3):1970-86. Epub 2013 Nov 11.

Hubrecht Institute-KNAW & University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel and The Department of Cell Biology, UMC Utrecht, The Netherlands.

Post-transcriptional regulation of RNA stability and localization underlies a wide array of developmental processes, such as axon guidance and epithelial morphogenesis. In Drosophila, ectopic expression of the classically Golgi peripheral protein dGRASP at the plasma membrane is achieved through its mRNA targeting at key developmental time-points, in a process critical to follicular epithelium integrity. However, the trans-acting factors that tightly regulate the spatio-temporal dynamics of dgrasp are unknown. Using an in silico approach, we identified two putative HOW Response Elements (HRE1 and HRE2) within the dgrasp open reading frame for binding to Held Out Wings (HOW), a member of the Signal Transduction and Activation of RNA family of RNA-binding proteins. Using RNA immunoprecipitations, we confirmed this by showing that the short cytoplasmic isoform of HOW binds directly to dgrasp HRE1. Furthermore, HOW loss of function in vivo leads to a significant decrease in dgrasp mRNA levels. We demonstrate that HRE1 protects dgrasp mRNA from cytoplasmic degradation, but does not mediate its targeting. We propose that this binding event promotes the formation of ribonucleoprotein particles that ensure dgrasp stability during transport to the basal plasma membrane, thus enabling the local translation of dgrasp for its roles at non-Golgi locations.
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http://dx.doi.org/10.1093/nar/gkt1118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919595PMC
February 2014

IL-25 prevents T cell-mediated neurotoxicity by decreasing LFA-1 expression.

J Neuroimmunol 2013 Dec 19;265(1-2):11-9. Epub 2013 Oct 19.

Centre for Neuroscience, Department of Medicine, University of Alberta, Edmonton, Alberta T6G 3G3, Canada.

Autoimmune diseases such as multiple sclerosis (MS) are thought to develop due to a dysregulation in the normal T(H)1-T(H)17/T(H)2 immune system balance, where pro-inflammatory responses with a T(H)1/T(H)17 prevalence develop. Some therapeutic treatments in MS promote a shift toward a TH2-prevalent environment and this has been shown to be protective. However, not all patients respond to current immunomodulatory treatments in MS so that new immunomodulatory drugs that can promote a shift of the immune system into an anti-inflammatory T(H)2 status are needed. IL-25 is a cytokine of the IL-17 family with powerful anti-inflammatory properties. This study demonstrates that IL-25 exerts neuroprotective functions by reducing T cell-mediated killing of human fetal neurons. The mechanism of action of this IL-25-mediated neuroprotective effect appears to be linked to reduction in the expression of the adhesion molecule LFA-1, which is relevant in stabilizing the immune synapse during cytotoxicity.
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http://dx.doi.org/10.1016/j.jneuroim.2013.10.006DOI Listing
December 2013

Region-specific regulation of posterior axial elongation during vertebrate embryogenesis.

Dev Dyn 2014 Jan 6;243(1):88-98. Epub 2013 Sep 6.

Hubrecht Institute and University Medical Center, Utrecht, The Netherlands.

Background: The vertebrate body axis extends sequentially from the posterior tip of the embryo, fueled by the gastrulation process at the primitive streak and its continuation within the tailbud. Anterior structures are generated early, and subsequent nascent tissues emerge from the posterior growth zone and continue to elongate the axis until its completion. The underlying processes have been shown to be disrupted in mouse mutants, some of which were described more than half a century ago.

Results: Important progress in elucidating the cellular and genetic events involved in body axis elongation has recently been made on several fronts. Evidence for the residence of self-renewing progenitors, some of which are bipotential for neurectoderm and mesoderm, has been obtained by embryo-grafting techniques and by clonal analyses in the mouse embryo. Transcription factors of several families including homeodomain proteins have proven instrumental for regulating the axial progenitor niche in the growth zone. A complex genetic network linking these transcription factors and signaling molecules is being unraveled that underlies the phenomenon of tissue lengthening from the axial stem cells. The concomitant events of cell fate decision among descendants of these progenitors begin to be better understood at the levels of molecular genetics and cell behavior.

Conclusions: The emerging picture indicates that the ontogenesis of the successive body regions is regulated according to different rules. In addition, parameters controlling vertebrate axial length during evolution have emerged from comparative experimental studies. It is on these issues that this review will focus, mainly addressing the study of axial extension in the mouse embryo with some comparison with studies in chick and zebrafish, aiming at unveiling the recent progress, and pointing at still unanswered questions for a thorough understanding of the process of embryonic axis elongation.
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http://dx.doi.org/10.1002/dvdy.24027DOI Listing
January 2014

Innexin 3, a new gene required for dorsal closure in Drosophila embryo.

PLoS One 2013 24;8(7):e69212. Epub 2013 Jul 24.

Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands ; UMC Utrecht, Utrecht, The Netherlands.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069212PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3722180PMC
February 2014

A reversible cerebral vasoconstriction syndrome.

BMJ Case Rep 2012 Jul 11;2012. Epub 2012 Jul 11.

Division of Neurology, University of Alberta, Edmonton, Canada.

Reversible cerebral vasoconstriction syndrome (RCVS) is typically presented with severe headaches where, vascular imaging demonstrates multiple intracranial arterial narrowing. Variable triggers are related to RCVS, such as serotonin agents and bromocriptine. Thus, a detailed medication history is important. Subarachnoid haemorrhage (SAH) is not uncommon in RCVS. Repeat vascular imaging at 2-3 months with complete reversal of the narrowed vessels confirms the diagnosis of RCVS. The authors present a case where use of triptan along with multiple psychotropic medications, was associated with RVCS. Neuroimaging demonstrated focal SAH and diffuse beaded appearance involving the intracranial vasculature. The patient improved clinically with oral nimodipine treatment. Repeat angiography and a follow-up transcranial Doppler showed complete resolution of vasoconstriction. In the setting of acute severe headache, with any 'red flags', it is important to evaluate the medication use and other precipitating risks for RVCS. Vascular imaging is the key for diagnosis.
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http://dx.doi.org/10.1136/bcr.09.2011.4841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3417010PMC
July 2012

Granule-derived granzyme B mediates the vulnerability of human neurons to T cell-induced neurotoxicity.

J Immunol 2011 Nov 30;187(9):4861-72. Epub 2011 Sep 30.

Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

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.
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http://dx.doi.org/10.4049/jimmunol.1100943DOI Listing
November 2011

Unconventional secretion: a stress on GRASP.

Curr Opin Cell Biol 2011 Aug 14;23(4):498-504. Epub 2011 May 14.

Hubrecht Institute for Developmental Biology and Stem Cell Research, Uppsalalaan 8, Utrecht, The Netherlands.

Most proteins follow the classical secretory pathway from the endoplasmic reticulum, via the Golgi, to the plasma membrane or extracellular medium. However, some proteins reach these final destinations by two alternative routes. One sustains the extracellular delivery of cytoplasmic proteins that lack a signal peptide, the other supports the transport of transmembrane proteins to the plasma membrane in a manner that bypasses the Golgi. Here, we highlight the observation that some unconventional secretion events are triggered by cellular stress. Furthermore, one Golgi protein, Golgi Re-Assembly and Stacking Protein (GRASP), has been shown to be essential to both types of unconventional secretion and we discuss ways in which it may support these events in a Golgi-independent manner.
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http://dx.doi.org/10.1016/j.ceb.2011.04.005DOI Listing
August 2011

Loss of purkinje cells is associated with demyelination in multiple sclerosis.

Can J Neurol Sci 2011 May;38(3):529-31

Department of Medicine (Neurology), University of Alberta, Edmonton, Alberta, Canada.

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http://dx.doi.org/10.1017/s0317167100012014DOI Listing
May 2011