ABSTRACT
To ensure efficient energy supply to the high demanding brain, nutrients are transported into brain cells via specific glucose (GLUT) and monocarboxylate transporters (MCT). Mitochondrial dysfunction and altered glucose metabolism are thought to play an important role in the progression of neurodegenerative diseases, including multiple sclerosis (MS). Here, we investigated the cellular localization of key GLUT and MCT proteins in human brain tissue of non-neurological controls and MS patients. We show that in control brain tissue GLUT and MCT proteins were abundantly expressed in a variety of central nervous system cells, particularly in microglia and endothelial cells. In active MS lesions, GLUTs and MCTs were highly expressed in infiltrating leukocytes and reactive astrocytes. Astrocytes manifest increased MCT1 staining and maintain GLUT expression in inactive lesions, whereas demyelinated axons exhibit significantly reduced GLUT3 and MCT2 immunoreactivity in inactive lesions. Finally, we demonstrated that the co-transcription factor peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), an important protein involved in energy metabolism, is highly expressed in reactive astrocytes in active MS lesions. Overexpression of PGC-1α in astrocyte-like cells resulted in increased production of several GLUT and MCT proteins. In conclusion, we provide for the first time a comprehensive overview of key nutrient transporters in white matter brain samples. Moreover, our data demonstrate an altered expression of these nutrient transporters in MS brain tissue, including a marked reduction of axonal GLUT3 and MCT2 expression in chronic lesions, which may impede efficient nutrient supply to the hypoxic demyelinated axons thereby contributing to the ongoing neurodegeneration in MS.
Subject(s)
Brain/metabolism , Glutamate Plasma Membrane Transport Proteins/metabolism , Monocarboxylic Acid Transporters/metabolism , Multiple Sclerosis/metabolism , White Matter/metabolism , Adult , Aged , Aged, 80 and over , Astrocytes/metabolism , Astrocytes/pathology , Axons/metabolism , Axons/pathology , Brain/blood supply , Brain/pathology , Cell Line , Endothelial Cells/metabolism , Endothelial Cells/pathology , Female , Glucose Transporter Type 3/metabolism , Humans , Leukocytes/metabolism , Leukocytes/pathology , Male , Microglia/metabolism , Microglia/pathology , Middle Aged , Multiple Sclerosis/pathology , Multiple Sclerosis, Chronic Progressive/metabolism , Multiple Sclerosis, Chronic Progressive/pathology , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Transcription Factors/metabolism , White Matter/blood supply , White Matter/pathologyABSTRACT
Multiple sclerosis (MS) lesions are characterized by the presence of activated astrocytes, which are thought to actively take part in propagating lesion progression by secreting pro-inflammatory mediators. Conversely, reactive astrocytes may exert disease-dampening effects through the production of trophic factors and anti-inflammatory mediators. Astrocytic control of the blood-brain barrier (BBB) is crucial for normal brain homeostasis and BBB disruption is a well-established early event in MS lesion development. Here, we set out to unravel potential protective effects of reactive astrocytes on BBB function under neuroinflammatory conditions as seen in MS, where we focus on the role of the brain morphogen retinoic acid (RA). Immunohistochemical analysis revealed that retinaldehyde dehydrogenase 2 (RALDH2), a key enzyme for RA synthesis, is highly expressed by reactive astrocytes throughout white matter lesions compared to control and normal appearing white matter. In vitro modeling of reactive astrocytes resulted in increased expression of RALDH2, enhanced RA synthesis, and a protective role for astrocyte-derived RA on BBB function during inflammation-induced barrier loss. Furthermore, RA induces endothelial immune quiescence and decreases monocyte adhesion under inflammatory conditions. Finally, we demonstrated that RA attenuated oxidative stress in inflamed endothelial cells, through activation of the antioxidant transcription factor nuclear factor E2 related factor 2. In summary, RA synthesis by reactive astrocytes represents an endogenous protective response to neuroinflammation, possibly aimed at protecting the BBB against inflammatory insult. A better understanding of RA signaling in MS pathophysiology may lead to the discovery of novel targets to halt disease progression.
Subject(s)
Astrocytes/drug effects , Blood-Brain Barrier/physiopathology , Brain/pathology , Multiple Sclerosis/pathology , Tretinoin/pharmacology , Adult , Aged , Aged, 80 and over , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase/metabolism , Aldehyde Dehydrogenase 1 Family , Astrocytes/metabolism , Autopsy , Cells, Cultured , Cytokines/metabolism , Endothelial Cells/drug effects , Endothelial Cells/physiology , Female , Glial Fibrillary Acidic Protein/metabolism , HEK293 Cells , Humans , Male , Middle Aged , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Reactive Oxygen Species/metabolism , Retinal Dehydrogenase/genetics , Retinal Dehydrogenase/metabolism , Time FactorsABSTRACT
Oxidative stress has been strongly implicated in both the inflammatory and neurodegenerative pathological mechanisms in multiple sclerosis (MS). In response to oxidative stress, cells increase and activate their cellular antioxidant mechanisms. Glutathione (GSH) is the major antioxidant in the brain, and as such plays a pivotal role in the detoxification of reactive oxidants. Previous research has shown that GSH homeostasis is altered in MS. In this review, we provide a comprehensive overview on GSH metabolism in brain cells, with a focus on its involvement in MS. The potential of GSH as an in vivo biomarker in MS is discussed, along with a short overview of improvements in imaging methods that allow non-invasive quantification of GSH in the brain. These methods might be instrumental in providing real-time measures of GSH, allowing the assessment of the oxidative state in MS patients and the monitoring of disease progression. Finally, the therapeutic potential of GSH in MS is discussed.
Subject(s)
Antioxidants/metabolism , Brain/metabolism , Glutathione/metabolism , Homeostasis/physiology , Multiple Sclerosis/metabolism , Oxidative Stress/physiology , Animals , HumansABSTRACT
There is growing evidence that mitochondrial dysfunction and associated reactive oxygen species (ROS) formation contribute to neurodegenerative processes in multiple sclerosis (MS). Here, we investigated whether alterations in transcriptional regulators of key mitochondrial proteins underlie mitochondrial dysfunction in MS cortex and contribute to neuronal loss. Hereto, we analyzed the expression of mitochondrial transcriptional (co-)factors and proteins involved in mitochondrial redox balance regulation in normal-appearing grey matter (NAGM) samples of cingulate gyrus and/or frontal cortex from 15 MS patients and nine controls matched for age, gender and post-mortem interval. PGC-1α, a transcriptional co-activator and master regulator of mitochondrial function, was consistently and significantly decreased in pyramidal neurons in the deeper layers of MS cortex. Reduced PGC-1α levels coincided with reduced expression of oxidative phosphorylation subunits and a decrease in gene and protein expression of various mitochondrial antioxidants and uncoupling proteins (UCPs) 4 and 5. Short-hairpin RNA-mediated silencing of PGC-1α in a neuronal cell line confirmed that reduced levels of PGC-1α resulted in a decrease in transcription of OxPhos subunits, mitochondrial antioxidants and UCPs. Moreover, PGC-1α silencing resulted in a decreased mitochondrial membrane potential, increased ROS formation and enhanced susceptibility to ROS-induced cell death. Importantly, we found extensive neuronal loss in NAGM from cingulate gyrus and frontal cortex of MS patients, which significantly correlated with the extent of PGC-1α decrease. Taken together, our data indicate that reduced neuronal PGC-1α expression in MS cortex partly underlies mitochondrial dysfunction in MS grey matter and thereby contributes to neurodegeneration in MS cortex.
Subject(s)
Cerebral Cortex/pathology , Heat-Shock Proteins/physiology , Mitochondria/pathology , Multiple Sclerosis/genetics , Multiple Sclerosis/pathology , Neurons/pathology , Transcription Factors/physiology , Adult , Aged , Aged, 80 and over , Blotting, Western , Cell Count , Down-Regulation , Female , Genetic Vectors , Gyrus Cinguli/pathology , Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/genetics , Humans , Immunohistochemistry , Lentivirus/genetics , Male , Middle Aged , Oxidation-Reduction , Oxidative Phosphorylation , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Pyramidal Cells/pathology , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction , Tissue Banks , Transcription Factors/biosynthesis , Transcription Factors/geneticsABSTRACT
Alterations in sphingolipid metabolism are described to contribute to various neurological disorders. We here determined the expression of enzymes involved in the sphingomyelin cycle and their products in postmortem brain tissue of multiple sclerosis (MS) patients. In parallel, we investigated the effect of the sphingosine-1 receptor agonist Fingolimod (Gilenya(®)) on sphingomyelin metabolism in reactive astrocytes and determined its functional consequences for the process of neuro-inflammation. Our results demonstrate that in active MS lesions, marked by large number of infiltrated immune cells, an altered expression of enzymes involved in the sphingomyelin cycle favors enhanced ceramide production. We identified reactive astrocytes as the primary cellular source of enhanced ceramide production in MS brain samples. Astrocytes isolated from MS lesions expressed enhanced mRNA levels of the ceramide-producing enzyme acid sphingomyelinase (ASM) compared to astrocytes isolated from control white matter. In addition, TNF-α treatment induced ASM mRNA and ceramide levels in astrocytes isolated from control white matter. Incubation of astrocytes with Fingolimod prior to TNF-α treatment reduced ceramide production and mRNA expression of ASM to control levels in astrocytes. Importantly, supernatants derived from reactive astrocytes treated with Fingolimod significantly reduced transendothelial monocyte migration. Overall, the present study demonstrates that reactive astrocytes represent a possible additional cellular target for Fingolimod in MS by directly reducing the production of pro-inflammatory lipids and limiting subsequent transendothelial leukocyte migration.
Subject(s)
Astrocytes/drug effects , Blood-Brain Barrier/drug effects , Ceramides/metabolism , Immunosuppressive Agents/pharmacology , Multiple Sclerosis/physiopathology , Propylene Glycols/pharmacology , Sphingosine/analogs & derivatives , Adult , Aged , Aged, 80 and over , Astrocytes/metabolism , Astrocytes/pathology , Blood-Brain Barrier/pathology , Blood-Brain Barrier/physiopathology , Cell Movement/drug effects , Cells, Cultured , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/pathology , Female , Fingolimod Hydrochloride , Humans , Male , Middle Aged , Monocytes/drug effects , Monocytes/metabolism , Monocytes/pathology , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Sphingomyelins/metabolism , Sphingosine/pharmacologyABSTRACT
OBJECTIVES: To test for structural and functional contribution of mitochondrial dysfunction to neurodegeneration in multiple sclerosis (MS). A visual pathway model void of MS lesions was chosen in order to exclude neurodegeneration secondary to lesion related axonotmesis. METHODS: A single-centre cohort study (230 MS patients, 63 controls). Spectral domain optical coherence tomography of the retina, 3T magnetic resonance imaging of the brain, spectrophotometric assessment of serum lactate levels. Postmortem immunohistochemistry. RESULTS: The visual pathway was void of MS lesions in 31 patients and 31 age-matched controls. Serum lactate was higher in MS compared to controls (P = 0.029). High serum lactate was structurally related to atrophy of the retinal nerve fiber layer at the optic disc (P = 0.041), macula (P = 0.025), and the macular ganglion cell complex (P = 0.041). High serum lactate was functionally related to poor color vision (P < 0.01), Expanded Disability Status Scale score (R = 0.37, P = 0.041), Guy's Neurological disability score (R = 0.38, P = 0.037), MS walking scale (R = 0.50, P = 0.009), upper limb motor function (R = 0.53, P = 0.002). Immunohistochemistry demonstrated increased astrocytic expression of a key lactate generating enzyme in MS lesions as well as profound vascular expression of monocarboxylate transporter-1, which is involved in lactate transport. INTERPRETATION: This study provides structural, functional, and translational evidence for visual pathway neurodegeneration in MS related to mitochondrial dysfunction.
ABSTRACT
INTRODUCTION: Demyelinated axons in multiple sclerosis (MS) lesions have an increased energy demand in order to maintain conduction. However, oxidative stress-induced mitochondrial dysfunction likely alters glucose metabolism and consequently impairs neuronal function in MS. Imaging and pathological studies indicate that glucose metabolism is altered in MS, although the underlying mechanisms and its role in neurodegeneration remain elusive. We investigated expression patterns of key enzymes involved in glycolysis, tricarboxylic acid (TCA) cycle and lactate metabolism in well-characterized MS tissue to establish which regulators of glucose metabolism are involved in MS and to identify underlying mechanisms. RESULTS: Expression levels of glycolytic enzymes were increased in active and inactive MS lesions, whereas expression levels of enzymes involved in the TCA cycle were upregulated in active MS lesions, but not in inactive MS lesions. We observed reduced expression and production capacity of mitochondrial α-ketoglutarate dehydrogenase (αKGDH) in demyelinated axons, which correlated with signs of axonal dysfunction. In inactive lesions, increased expression of lactate-producing enzymes was observed in astrocytes, whereas lactate-catabolising enzymes were mainly detected in axons. Our results demonstrate that the expression of various enzymes involved in glucose metabolism is increased in both astrocytes and axons in active MS lesions. In inactive MS lesions, we provide evidence that astrocytes undergo a glycolytic shift resulting in enhanced astrocyte-axon lactate shuttling, which may be pivotal for the survival of demyelinated axons. CONCLUSION: In conclusion, we show that key enzymes involved in energy metabolism are differentially expressed in active and inactive MS lesions. Our findings imply that, in addition to reduced oxidative phosphorylation activity, other bioenergetic pathways are affected as well, which may contribute to ongoing axonal degeneration in MS.
Subject(s)
Brain/enzymology , Glucose/metabolism , Ketoglutarate Dehydrogenase Complex/metabolism , L-Lactate Dehydrogenase/metabolism , Multiple Sclerosis/pathology , Adult , Aged , Aged, 80 and over , Brain/pathology , Cell Line, Tumor , Female , Gene Expression Regulation/drug effects , Glial Fibrillary Acidic Protein/metabolism , Humans , Interferon-gamma/pharmacology , Male , Middle Aged , Neuroblastoma/pathology , Tumor Necrosis Factor-alpha/metabolism , tert-Butylhydroperoxide/pharmacologyABSTRACT
Recent evidence suggests that reactive oxygen species (ROS) produced by inflammatory cells drive axonal degeneration in active multiple sclerosis (MS) lesions by inducing mitochondrial dysfunction. Mitochondria are endowed with a variety of antioxidant enzymes, including peroxiredoxin-3 and thioredoxin-2, which are involved in limiting ROS-induced damage. In this study, we explored the distribution and role of the mitochondrial antioxidants peroxiredoxin-3 and thioredoxin-2 as well as their regulator peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC-1α) in MS pathogenesis. Immunohistochemical analysis of a large cohort of MS patients revealed a striking upregulation of PGC-1α and downstream mitochondrial antioxidants in active demyelinating MS lesions. Enhanced expression was predominantly observed in reactive astrocytes. To elucidate the functional role of astrocytic PGC-1α in MS pathology, we generated human primary astrocytes that genetically overexpressed PGC-1α. Upon an oxidative insult, these cells were shown to produce less ROS and were found to be more resistant to ROS-induced cell death compared to control cells. Intriguingly, also neuronal cells co-cultured with PGC-1α-overexpressing astrocytes were protected against an exogenous oxidative attack compared to neuronal cells co-cultured with control astrocytes. Finally, enhanced astrocytic PGC-1α levels markedly reduced the production and secretion of the pro-inflammatory mediators interleukin-6 and chemokine (C-C motif) ligand 2. Our findings suggest that increased astrocytic PGC-1α in active MS lesions might initially function as an endogenous protective mechanism to dampen oxidative damage and inflammation thereby reducing neurodegeneration. Activation of PGC-1α therefore represents a promising therapeutic strategy to improve mitochondrial function and repress inflammation.
Subject(s)
Astrocytes/metabolism , Brain/metabolism , Encephalitis/etiology , Encephalitis/pathology , Multiple Sclerosis/complications , Transcription Factors/metabolism , Adult , Aged , Antioxidants/metabolism , Case-Control Studies , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Female , Gene Expression Regulation , Humans , Male , Middle Aged , Multiple Sclerosis/pathology , Myelin Proteolipid Protein/metabolism , Oxidative Stress/physiology , Peroxiredoxin III/genetics , Peroxiredoxin III/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Reactive Oxygen Species , Thioredoxins/genetics , Thioredoxins/metabolism , Transcription Factors/genetics , White Matter/metabolism , White Matter/pathologyABSTRACT
BACKGROUND: Foamy macrophages, containing myelin degradation products, are abundantly found in active multiple sclerosis (MS) lesions. Recent studies have described an altered phenotype of macrophages after myelin internalization. However, mechanisms by which myelin affects the phenotype of macrophages and how this phenotype influences lesion progression remain unclear. RESULTS: We demonstrate that myelin as well as phosphatidylserine (PS), a phospholipid found in myelin, reduce nitric oxide production by macrophages through activation of peroxisome proliferator-activated receptor ß/δ (PPARß/δ). Furthermore, uptake of PS by macrophages, after intravenous injection of PS-containing liposomes (PSLs), suppresses the production of inflammatory mediators and ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The protective effect of PSLs in EAE animals is associated with a reduced immune cell infiltration into the central nervous system and decreased splenic cognate antigen specific proliferation. Interestingly, PPARß/δ is activated in foamy macrophages in active MS lesions, indicating that myelin also activates PPARß/δ in macrophages in the human brain. CONCLUSION: Our data show that myelin modulates the phenotype of macrophages by PPAR activation, which may subsequently dampen MS lesion progression. Moreover, our results suggest that myelin-derived PS mediates PPARß/δ activation in macrophages after myelin uptake. The immunoregulatory impact of naturally-occurring myelin lipids may hold promise for future MS therapeutics.
Subject(s)
Macrophages/immunology , Multiple Sclerosis/immunology , Myelin Sheath/physiology , PPAR delta/metabolism , PPAR-beta/metabolism , Adult , Aged , Animals , Brain/immunology , Brain/pathology , Cell Proliferation/physiology , Cells, Cultured , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Humans , Lysosomes/metabolism , Male , Middle Aged , Multiple Sclerosis/pathology , Nitric Oxide/metabolism , Phosphatidylserines/administration & dosage , Phosphatidylserines/metabolism , Rats , Spleen/immunology , T-Lymphocytes/physiologyABSTRACT
The involvement of parkin, PINK1, and DJ1 in mitochondrial dysfunction, oxidative injury, and impaired functioning of the ubiquitin-proteasome system (UPS) has been intensively investigated in light of Parkinson's disease (PD) pathogenesis. However, these pathological mechanisms are not restricted to PD, but are common denominators of various neurodegenerative and neuroinflammatory disorders. It is therefore conceivable that parkin, PINK1, and DJ1 are also linked to the pathogenesis of other neurological diseases, including Alzheimer's disease (AD) and multiple sclerosis (MS). The importance of these proteins in mechanisms underlying neurodegeneration is reflected by the neuroprotective properties of parkin, DJ1, and PINK1 in counteracting oxidative stress and improvement of mitochondrial and UPS functioning. This review provides a concise overview on the cellular functions of the E3 ubiquitin ligase parkin, the mitochondrial kinase PINK1, and the cytoprotective protein DJ1 and their involvement and interplay in processes underlying neurodegeneration in common neurological disorders.
Subject(s)
Intracellular Signaling Peptides and Proteins/physiology , Multiple Sclerosis/metabolism , Neurodegenerative Diseases/metabolism , Oncogene Proteins/physiology , Protein Kinases/physiology , Ubiquitin-Protein Ligases/physiology , Animals , Humans , Inflammation/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Multiple Sclerosis/pathology , Neurodegenerative Diseases/pathology , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Oxidative Stress , Protein Deglycase DJ-1 , Protein Kinases/genetics , Protein Kinases/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Macrophages are versatile cells, which phenotype is profoundly influenced by their environment. Pro-inflammatory classically activated or M1 macrophages, and anti-inflammatory alternatively-activated or M2 macrophages represent two extremes of a continuum of functional states. Consequently, macrophages that are present in tumors can exert tumor-promoting and tumor-suppressing activity, depending on the tumor milieu. In this study we investigated how human monocytes-the precursors of macrophages-are influenced by carcinoma cells of different origin. We demonstrate that monocytes, stimulated with breast cancer supernatant, showed increased expression of interleukin (IL)-10, IL-8 and chemokines CCL17 and CCL22, which are associated with an alternatively-activated phenotype. By contrast, monocytes that were cultured in supernatants of colon cancer cells produced more pro-inflammatory cytokines (e.g., IL-12 and TNFα) and reactive oxygen species. Secretome analysis revealed differential secretion of proteins by colon and breast cancer cell lines, of which the proteoglycan versican was exclusively secreted by colon carcinoma cell lines. Reducing active versican by blocking with monoclonal antibodies or shRNA diminished pro-inflammatory cytokine production by monocytes. Thus, colon carcinoma cells polarize monocytes toward a more classically-activated anti-tumorigenic phenotype, whereas breast carcinomas predispose monocytes toward an alternatively activated phenotype. Interestingly, presence of macrophages in breast or colon carcinomas correlates with poor or good prognosis in patients, respectively. The observed discrepancy in macrophage activation by either colon or breast carcinoma cells may therefore explain the dichotomy between patient prognosis and macrophage presence in these different tumors. Designing new therapies, directing development of monocytes toward M1 activated tumor macrophages in cancer patients, may have great clinical benefits.