5.1 Mitochondria, neurodegenerative diseases and MS
summarizes the involvement of mitochondrial abnormalities in patients with MS and EAE mouse models. As shown, current research revealed that the following mitochondrial abnormalities are involved in the development and progression of multiple sclerosis: 1) mitochondrial DNA defects, 2) abnormal mitochondrial gene expression, 3) defective mitochondrial enzyme activities, 4) deficient mitochondrial DNA repair activity 5) and mitochondrial dysfunction. We propose that abnormal mitochondrial dynamics (increased fission and decreased fusion in neurons affected by MS). Further, we also propose that mitochondrial abnormalities and mitochondrial energy failure may impact other cellular pathways, including increased demyelination and inflammation in neurons and tissues that are affected by multiple sclerosis. The details are given below.
Figure 2 Mitochondrial abnormalities in patients with multiple sclerosis and EAE mouse models. Based on current research, we propose that mitochondrial abnormalities are involved in the development and progression of multiple sclerosis, including mitochondrial (more ...)
Mitochondria contain the respiratory chain where energy in the form of ATP is most efficiently produced. The mitochondrial respiratory chain is located in the inner mitochondrial membrane and consists of five complexes (complexes I-V); the fifth complex is directly involved in ATP synthesis. The complexes of the mitochondrial respiratory chain are made up of multiple subunits, and all contain proteins encoded by nuclear DNA and mtDNA, except for complex II, which is entirely encoded by nuclear DNA. Neurons are highly dependent on oxidative energy metabolism. Axons, in particular, consume significant amounts of ATP, which it uses primarily to fuel the sodium/potassium ATPase, or sodium pump that functions to remove the sodium ions that enter the axon during impulse activity. Mitochondria are not only the energy factory for cells but also the seat of a number of important cellular functions, including essential pathways of intermediate metabolism, amino acid biosynthesis, fatty acid oxidation, steroid metabolism, calcium handling and apoptosis. Of key importance is the role of mitochondria in oxidative energy metabolism. Oxidative phosphorylation generates most of the cell’s ATP, and any impairment of the organelle’s ability to produce energy can have catastrophic consequences, not only due to the primary loss of ATP, but also due to indirect impairment of downstream events. Moreover, the production of superoxide occurs mostly within the mitochondria, mainly in complexes I and III, TCA cycle and conditionally in complex II .
Deficient mitochondrial metabolism may generate more reactive oxygen species (ROS) that can wreak havoc in the cell. Therefore, mitochondrial dysfunction is an attractive candidate for neuronal degeneration. Impairment of mitochondrial energy metabolism is the key pathogenic factor in a number of neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease. Hence, therapeutic approaches targeting mitochondrial dysfunction and oxidative damage in neurodegenerative diseases, including MS have great promise .
Recently, several lines of evidence suggests that mitochondrial dysfunction is present in patients with MS. Mitochondrial DNA alterations, mitochondrial structural changes, defective mitochondrial DNA repair events, abnormal mitochondrial enzyme activities, mitochondrial gene expressions, increased free radical production and oxidative damage have been reported in patients with MS and EAE mouse models ().
5.2 Mitochondrial DNA alternations in MS
Age-related decline of mtDNA copy number is associated with late-onset MS. mtDNA mutations may increase the risk of MS. SNP analysis has shown that genetic variants of complex I genes may influence the response of tissues to inflammation in the CNS. Further, genetic alterations in uncoupling proteins are reported to be implicated in patients with MS. Uncoupling protein 2 (UCP2) is a member of the mitochondrial proton transport family that uncouples proton entry to the mitochondria from ATP synthesis. Vogler and colleagues reported that the UCP2 common -866G/A promoter polymorphism is associated with susceptibility to MS in a German population. In a study of 1,097 MS patients and 462 control subjects, they found the common G allele associated with disease susceptibility (p = 0.0015). The UCP2 -866G allele was correlated with lower levels of UCP2 expression in vitro and in vivo. Thus, UCP2 may contribute to MS susceptibility by regulating the level of UCP2 protein in the CNS and/or in the immune system .
Defects in mtDNA have been associated with late onset MS. Ban and colleagues (2008) sequenced the mtDNA from 159 patients with MS and completed a haplogroup analysis of 835 MS patients and 1,506 controls. They found a trend towards over-representation of super-haplogroup U as the only evidence for association with MS. In a parallel analysis of nuclear-encoded mitochondrial protein genes in the same subjects, they also found a trend towards association with the complex I gene, NDUFS2. Taken together, these studies have contributed to evidence suggesting that variations in mtDNA and nuclear-encoded mitochondrial protein genes may contribute to disease susceptibility in MS.
A study of MS patients in Europe showed that a potentially functional mtDNA SNP, nt13708 G/A, was significantly associated with an increased risk of MS (P = 0.0002). The study identified the nt13708A variant as a allele susceptible to MS, which may suggest a role in MS pathogenesis. Recently, Vyshkina et al. discovered an association among common variants of the mitochondrial ND2 and ATP6 genes with both MS and systemic lupus erythematosus. This finding raises the possibility of a shared mitochondrial genetic background between these two autoimmune diseases. On the other hand, an increasing number of case reports on Leber’s hereditary optic neuropathy (LHON) associated mtDNA point mutations, and some patients with MS and LHON share the same mtDNA mutation, suggesting that mitochondrial determinants may contribute to genetic susceptibility in MS and LHON .
In fact, only a very small subgroup of MS patients, usually with prominent optic neuritis, may carry pathogenic LHON mutations. This overlap between the two diseases may be related to the association of MS with an mtDNA haplotype (a set of mtDNA polymorphisms) within which pathogenic LHON mutations preferentially occur. In a recent study, 58 unrelated Bulgarian patients with RRMS and 104 randomly selected healthy individuals were analyzed for the presence of 14 mtDNA polymorphisms determining major European haplogroups as well as three (4216, 14 798, 13 708) secondary LHON mutations. Restriction enzyme analysis, used to screen patients and controls for common haplogroup-associated polymorphisms, showed that each of these changes which occurred in MS patients at a similar rate to control subjects. However, 21 of the 58 patients (36.2%) were positive for the T4 216C mutation, while only 11.3% of the controls carried this mutation (P < 0.01; OR = 4.38), suggesting that the 4216C base substitution may be a predisposing marker for MS. These findings also supported the hypothesis that particular mtDNA variants may contribute to the genetic susceptibility of some people with MS. To further study the relationship between LHON and MS, Hwang et al. tested 20 Korean MS patients for the presence of mtDNA mutations at nucleotide (nt) 11778, and nt 14484, 3460, and 15257. However, none of the MS patients exhibited any pathogenic LHON mtDNA mutations. This result is in agreement with that of Japanese MS patients. It may be the case that racial characteristics may influence the association.
5.3 Mitochondrial dysfunction in MS
Increasing evidence suggests that mitochondrial dysfunction is involved in MS. Ultrastructural analysis of demyelinated spinal cord lesions showed dramatically reduced numbers of mitochondria and microtubules, and demonstrated Ca2+-mediated destruction of chronically demyelinated axons and axonal swelling. Further, the gene expression study showed an unbalanced gene expression in MS patients. As reduced energy production is a major contributor to Ca2+-mediated axonal degeneration, authors focused on changes in oxidative phosphorylation and inhibitory neurotransmission. Compared with controls, 488 transcripts were decreased and 67 were increased in the MS cortex. Twenty-six nuclear-encoded mitochondrial genes and the functional activities of mitochondrial respiratory chain complexes I and III were decreased in the MS motor cortex. Reduced mitochondrial gene expression was specific for neurons. In addition, synaptic components of GABAergic neurotransmission and the density of inhibitory interneuron processes also were decreased in the MS cortex.
In addition, recently a number of mitochondrial respiratory chain proteins in active lesions from acute MS was analyzed using immunohistochemistry. Functionally important defects of mitochondrial respiratory chain complex IV [cytochrome c oxidase (COX)] including its catalytic component (COX-I) are present in some active MS lesions (Pattern III). The lack of immunohistochemically detected COX-I is apparent in oligodendrocytes, hypertrophied astrocytes and axons, but not in microglia. These findings suggest that hypoxia-like tissue injury in Pattern III MS lesions may be initiated from mitochondrial impairment. On the other hand, in inactive areas of chronic MS lesions the complex IV activity and mitochondrial mass, judged by porin immunoreactivity, are increased within approximately half of large chronically demyelinated axons compared with large myelinated axons in the brain and spinal cord. The axon-specific mitochondrial docking protein (syntaphilin) and phosphorylated neurofilament-H were increased in chronic lesions. These results clearly indicate an adaptive change of mitochondrial function and morphology in chronic MS.
Recently, Regenold and colleagues investigated the relationship between disturbed CNS mitochondrial energy metabolism and MS disease progression by measuring cerebrospinal fluid (CSF) concentrations of sorbitol, fructose, and lactate, all metabolites of extra-mitochondrial glucose metabolism. They found that concentrations of all three metabolites, but not concentrations of glucose or myoinositol, were significantly increased in CSF from secondary progressive and, to a lesser degree, relapsing-remitting patients, compared to healthy controls. Furthermore, CSF concentrations of sorbitol and fructose (polyol pathway metabolites), but not lactate (anaerobic glycolysis metabolite), correlated positively and significantly with Expanded Disability Status Scale (EDSS) score, an index of neurologic disability in MS patients. These findings suggest that abnormal mitochondrial glucose metabolism is increased in MS patients and is associated with disease progression .
Interestingly, analysis of mitochondrial enzymes on human muscle showed that in people with MS, there were fewer type I fibers, and that fibers of all types were smaller and had lower succinate dehydrogenase (SDH, component of the respiratory chain complex II) and SDH/alpha-glycerol-phosphate dehydrogenase (GPDH) but not GPDH activities, suggesting that muscle in this disease is smaller and relies more on anaerobic than aerobic-oxidative energy supply than does muscle of healthy individuals. Similar to brain, muscles are also highly dependent on mitochondrial oxidative energy metabolism, so it is reasonable that there is a weaker muscle in MS patients, indicating muscle is also one of the targets of MS. In some rare cases, MS could have a mitochondrial myopathy combination, in which MRI showed widespread white matter lesions, muscle biopsy showed ragged red fibres and COX (complex IV) deficiency, Southern blot analysis revealed a large deletion of mtDNA. Probably the severe mitochondrial genomic deletion is the key cause or initiation factor for this special case.
Another interesting key issue of mitochondria must be discussed below. The mitochondrial permeability transition leads to mitochondrial swelling, outer membrane rupture and the release of apoptotic mediators. The mitochondrial permeability transition pore (PTP) is thought to consist of the adenine nucleotide translocator, a voltage-dependent anion channel, and cyclophilin D (CyPD, the Ppif gene product), a prolyl isomerase located within the mitochondrial matrix. CyPD is a key regulator of the PTP and they are required for mediating Ca2+- and oxidative damage-induced cell death. In experimental animal MS disease model, EAE mice lacking CyPD showed that neurons missing CyPD, are resistant to oxidative agents thought to be the mediators of axonal degeneration observed in both EAE and MS and have mitochondria that are able to more effectively handle elevated Ca2+. Consistent with this neuronal resistance, animals missing CyPD are able to recover, clinically, following the induction of EAE. These results directly implicate pathological activation of the mitochondrial PTP in the axonal damage occurring during MS, in other word, PTP and mitochondria are the critical target of EAE, perhaps multiple sclerosis.
5.4 Oxidative stress in MS
Reactive oxygen species (ROS) are the by-products of cellular metabolism. Excessive ROS or an imbalance between cellular production of ROS and the ability of cells to defend against them is referred to as oxidative stress. Oxidative stress can cause cellular damage and subsequent cell death because the ROS oxidize critical cellular components, such as lipids, proteins, and DNA especially mitochondrial DNA. In neurodegenerative and neuroinflammatory disorders, there is evidence for a primary contribution of oxidative stress in neuronal death, as opposed to other diseases where oxidative stress more likely plays a secondary or by-stander role .
There is increasing evidence that oxidative stress is an important component in the pathogenesis of MS. The inflammatory environment in demyelinating lesions is conducive to the generation of reactive oxygen species. Macrophages and microglia are known to express myeloperoxidase (MPO) and generate ROS during myelin phagocytosis in the white matter. Recent research involving the cerebral cortex in MS indicates that microglial production of ROS is also likely to be involved in cortical demyelination. Protein kinase C (PKC) could induce an increased production of ROS in mononuclear cells of patients with MS compared to those of controls, and it was predominantly or exclusively generated by PKC activated NADPH oxidase. The concentrations of reactive oxygen and/or nitrogen species (e.g. superoxide, nitric oxide and peroxynitrite) can increase dramatically under conditions such as inflammation, and this can overwhelm the inherent antioxidant defences within lesions. Such oxidative and/or nitrative stress can damage the lipids, proteins and nucleic acids of cells and mitochondria, potentially causing cell death. Oligodendrocytes are more sensitive to oxidative and nitrative stress in vitro than are astrocytes and microglia, seemingly due to a diminished capacity for antioxidant defence, and the presence of raised risk factors. Oxidative and nitrative stress might therefore result in selective oligodendrocyte death, and thereby demyelination in vivo. The reactive species may also damage the myelin sheath, promoting its attack by macrophages/microglia.
Evidence for the existence of oxidative and nitrative stress within inflammatory demyelinating lesions includes the presence of both lipid peroxidation and protein peroxides (protein carbonyls), and nitrotyrosine (a marker for peroxynitrite formation). When the ROS/RNS are generated in MS and animal models of MS, products such as superoxide and peroxynitrite are formed that are highly toxic to both glia and neuronal cells. Kalman’s group has determined the level of DNA damage in MS patients using 8-hydroxy-deoxy-guanosine (8-OH-dG) as an oxidative marker, they found that a significant increase in DNA oxidation within plaques compared to NAWM specimens in MS cerebella. A tendency for increase of oxidative markers in normal appearing cortical tissues located in the proximity of MS plaques was also observed when compared to those in control cortical specimens. Also, oxidative damage to mitochondrial DNA and impaired activity of mitochondrial enzyme complexes in MS lesions suggest that inflammation can affect energy metabolism, ATP synthesis, and viability of affected cells .
A recent report showed that oxidative stress occurs in progressive as well as benign MS patients. For example, serum diene conjugate levels (a measure of lipid peroxidation) were significantly elevated in MS patients, especially patients with primary progressive phenotypes. However, serum total antioxidative activity and total antiradical activity were not different between MS patients and healthy controls. On the other hand, the chemical composition of human cerebrospinal fluid is considered to reflect brain metabolism, there is experimental evidence of a decrease in sulfhydryl groups (antioxidants) and increased content of products of lipid peroxidation, such as ultraweak chemiluminescence and liposoluble fluorescence, which was higher in the CSF and plasma of MS patients than in controls, clearly pointing out the role of oxidative stress in the pathogenesis of MS .
Recent insights into the molecular pathogenesis of progression in MS also list oxidative stress as one of main mechanisms. More recently, Van Horssen J et al reported the presence of extensive oxidative damage to proteins, lipids, and nucleotides occurring in active demyelinating MS lesions, predominantly in reactive astrocytes and myelin-laden macrophages. It is reasonable that some of structurally-damaged myelin proteins are the targets of immune system, which are recognized as foreign antigens. Hence we consider that if not all, at lease for some MS patients, oxidative stress (including nitrative stress) may be the primary mechanism in pathogenesis.
On the other hand, antioxidant enzymes, including superoxide dismutase 1 and 2, catalase, and heme oxygenase 1, are markedly upregulated in active demyelinating MS lesions compared to normal-appearing white matter and white matter tissue from nonneurological control brains. Enhanced antioxidant enzyme production in inflammatory MS lesions may reflect an adaptive defense mechanism to reduce ROS-induced cellular damage. These data and observations strongly indicate that antioxidant therapy may be a potential treatment for MS patients.
5.5 Nitric oxide and MS
As described above, human blood macrophages, astrocytes, and microglial cells make NO. NO is present at increased concentrations in acute MS lesions and is known to have a deleterious effect on mitochondria. The relationship between NO and cytochrome c oxidase (mt complex IV) has been investigated at different integration levels of the enzyme, including the in situ state, such as in mouse liver mitochondria or cultured human SY5Y neuroblastoma cells. Micromolar NO rapidly inhibits cytochrome c oxidase in turnover with physiological substrates. The respiratory chain is inhibited by NO, either supplied exogenously or produced endogenously via the NO synthase activation. Inhibition of respiration is reversible, although it remains to be clarified whether reversibility is always full and how it depends on concentration of and time of exposure to NO. At least under hypoxic condition, NO irreversibly inhibits cytochrome oxidase. NO and superoxide redical combine to form peroxynitrate (ONOO-), which breaks down to form the highly reactive radicals hydroxyl radical and nitrogen dioxide. In other words NO can enhance the cellular toxicity of ROS.
Peroxynitrite and other reactive nitrogen oxide species exert a toxic effect on neurons, axons and glia cells and enhance apoptosis. In addition, they increase the blood-brain-barrier (BBB) permeability and can therefore promote invasion of inflammatory cells into the CNS. On the other hand, uric acid, a purine metabolite and peroxynitrite scavenger inhibits blood-CNS-barrier permeability changes, CNS inflammation and tissue damage in EAE and in mice with spinal cord injury. More recently the concentrations of uric acid, purine profile and creatinine in samples of cerebrospinal fluid and serum of MS patients were measured in detail by HPLC. The values of all compounds assayed were significantly higher in both biological fluids of MS patients with respect to values measured in controls. In particular, serum hypoxanthine, xanthine, uric acid and sum of oxypurines were, respectively, 3.17, 3.11, 1.23 and 1.27-fold higher in these patients than corresponding values recorded in controls. Though differently from what previously reported, these data clearly demonstrate that all purine compounds, including uric acid, are elevated in biological fluids of MS patients. Reinforced by the trend observed for creatinine, this corroborates the notion of sustained purine catabolism, possibly due to imbalance in ATP homeostasis, under these pathological conditions. As observed in other pathological states, uric acid, purine compounds and creatinine, can be considered markers of metabolic energy imbalance rather than of reactive oxygen species, even in MS.
Nitric oxide synthases (NOS) also play an important role under physiological as well as pathological conditions. Active iNOS enzyme has been demonstrated in astrocytes in acute and chronic-active MS lesions at the lesion edge where de myelination is occurring. QRT-PCR analysis detected significant upregulation of the neuronal form of NOS (nNOS), in most of the MS normal-appearing white matter tissue samples, this change together with the upregulation of HIF-1 in oligodendrocytes and neurons supports the view of oligodendrocyte and/or neuronal dysfunction in this non-lesion containing tissue as a possible primary cause .