Adaptive immune changes associate with clinical progression of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most frequent cause of dementia. Recent evidence suggests the involvement of peripheral immune cells in the disease, but the underlying mechanisms remain unclear. METHODS: We comprehensively mapped peripheral immune changes in AD patients with mild cognitive impairment (MCI) or dementia compared to controls, using cytometry by time-of-flight (CyTOF). RESULTS: We found an adaptive immune signature in AD, and specifically highlight the accumulation of PD1+ CD57+ CD8+ T effector memory cells re-expressing CD45RA in the MCI stage of AD. In addition, several innate and adaptive immune cell subsets correlated to cerebrospinal fluid (CSF) biomarkers of AD neuropathology and measures for cognitive decline. Intriguingly, subsets of memory T and B cells were negatively associated with CSF biomarkers for tau pathology, neurodegeneration and neuroinflammation in AD patients. Lastly, we established the influence of the APOE ε4 allele on peripheral immunity. CONCLUSIONS: Our findings illustrate significant peripheral immune alterations associated with both early and late clinical stages of AD, emphasizing the necessity for further investigation into how these changes influence underlying brain pathology.

Inflammation-induced TRPV4 channels exacerbate blood-brain barrier dysfunction in multiple sclerosis.

BACKGROUND: Blood-brain barrier (BBB) dysfunction and immune cell migration into the central nervous system (CNS) are pathogenic drivers of multiple sclerosis (MS). Ways to reinstate BBB function and subsequently limit neuroinflammation present promising strategies to restrict disease progression. However, to date, the molecular players directing BBB impairment in MS remain poorly understood. One suggested candidate to impact BBB function is the transient receptor potential vanilloid-type 4 ion channel (TRPV4), but its specific role in MS pathogenesis remains unclear. Here, we investigated the role of TRPV4 in BBB dysfunction in MS. MAIN TEXT: In human post-mortem MS brain tissue, we observed a region-specific increase in endothelial TRPV4 expression around mixed active/inactive lesions, which coincided with perivascular microglia enrichment in the same area. Using in vitro models, we identified that microglia-derived tumor necrosis factor-α (TNFα) induced brain endothelial TRPV4 expression. Also, we found that TRPV4 levels influenced brain endothelial barrier formation via expression of the brain endothelial tight junction molecule claudin-5. In contrast, during an inflammatory insult, TRPV4 promoted a pathological endothelial molecular signature, as evidenced by enhanced expression of inflammatory mediators and cell adhesion molecules. Moreover, TRPV4 activity mediated T cell extravasation across the brain endothelium. CONCLUSION: Collectively, our findings suggest a novel role for endothelial TRPV4 in MS, in which enhanced expression contributes to MS pathogenesis by driving BBB dysfunction and immune cell migration.

Extended interval dosing of ocrelizumab modifies the repopulation of B cells without altering the clinical efficacy in multiple sclerosis.

BACKGROUND: Recent studies suggest that extended interval dosing of ocrelizumab, an anti-B cell therapy, does not affect its clinical effectiveness in most patients with multiple sclerosis (MS). However, it remains to be established whether certain B cell subsets are differentially repopulated after different dosing intervals and whether these subsets relate to clinical efficacy. METHODS: We performed high-dimensional single-cell characterization of the peripheral immune landscape of patients with MS after standard (SID; n = 43) or extended interval dosing (EID; n = 37) of ocrelizumab and in non-ocrelizumab-treated (control group, CG; n = 28) patients with MS, using mass cytometry by time of flight (CyTOF). RESULTS: The first B cells that repopulate after both ocrelizumab dosing schemes were immature, transitional and regulatory CD1d+ CD5+ B cells. In addition, we observed a higher percentage of transitional, naïve and regulatory B cells after EID in comparison with SID, but not of memory B cells or plasmablasts. The majority of repopulated B cell subsets showed an increased migratory phenotype, characterized by higher expression of CD49d, CD11a, CD54 and CD162. Interestingly, after EID, repopulated B cells expressed increased CD20 levels compared to B cells in CG and after SID, which was associated with a delayed repopulation of B cells after a subsequent ocrelizumab infusion. Finally, the number of/changes in B cell subsets after both dosing schemes did not correlate with any relapses nor progression of the disease. CONCLUSIONS: Taken together, our data highlight that extending the dosing interval of ocrelizumab does not lead to increased repopulation of effector B cells. We show that the increase of CD20 expression on B cell subsets in EID might lead to longer depletion or less repopulation of B cells after the next infusion of ocrelizumab. Lastly, even though extending the ocrelizumab interval dosing alters B cell repopulation, it does not affect the clinical efficacy of ocrelizumab in our cohort of patients with MS.

Breaching Brain Barriers: B Cell Migration in Multiple Sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) known for the manifestation of demyelinated lesions throughout the CNS, leading to neurodegeneration. To date, not all pathological mechanisms that drive disease progression are known, but the clinical benefits of anti-CD20 therapies have put B cells in the spotlight of MS research. Besides their pathological effects in the periphery in MS, B cells gain access to the CNS where they can contribute to disease pathogenesis. Specifically, B cells accumulate in perivascular infiltrates in the brain parenchyma and the subarachnoid spaces of the meninges, but are virtually absent from the choroid plexus. Hence, the possible migration of B cells over the blood-brain-, blood-meningeal-, and blood-cerebrospinal fluid (CSF) barriers appears to be a crucial step to understanding B cell-mediated pathology. To gain more insight into the molecular mechanisms that regulate B cell trafficking into the brain, we here provide a comprehensive overview of the different CNS barriers in health and in MS and how they translate into different routes for B cell migration. In addition, we review the mechanisms of action of diverse therapies that deplete peripheral B cells and/or block B cell migration into the CNS. Importantly, this review shows that studying the different routes of how B cells enter the inflamed CNS should be the next step to understanding this disease.

Meningeal inflammation in multiple sclerosis induces phenotypic changes in cortical microglia that differentially associate with neurodegeneration.

Meningeal inflammation strongly associates with demyelination and neuronal loss in the underlying cortex of progressive MS patients, thereby contributing significantly to clinical disability. However, the pathological mechanisms of meningeal inflammation-induced cortical pathology are still largely elusive. By extensive analysis of cortical microglia in post-mortem progressive MS tissue, we identified cortical areas with two MS-specific microglial populations, termed MS1 and MS2 cortex. The microglial population in MS1 cortex was characterized by a higher density and increased expression of the activation markers HLA class II and CD68, whereas microglia in MS2 cortex showed increased morphological complexity and loss of P2Y12 and TMEM119 expression. Interestingly, both populations associated with inflammation of the overlying meninges and were time-dependently replicated in an in vivo rat model for progressive MS-like chronic meningeal inflammation. In this recently developed animal model, cortical microglia at 1-month post-induction of experimental meningeal inflammation resembled microglia in MS1 cortex, and microglia at 2 months post-induction acquired a MS2-like phenotype. Furthermore, we observed that MS1 microglia in both MS cortex and the animal model were found closely apposing neuronal cell bodies and to mediate pre-synaptic displacement and phagocytosis, which coincided with a relative sparing of neurons. In contrast, microglia in MS2 cortex were not involved in these synaptic alterations, but instead associated with substantial neuronal loss. Taken together, our results show that in response to meningeal inflammation, microglia acquire two distinct phenotypes that differentially associate with neurodegeneration in the progressive MS cortex. Furthermore, our in vivo data suggests that microglia initially protect neurons from meningeal inflammation-induced cell death by removing pre-synapses from the neuronal soma, but eventually lose these protective properties contributing to neuronal loss.

Enhancing mitochondrial activity in neurons protects against neurodegeneration in a mouse model of multiple sclerosis.

While transcripts of neuronal mitochondrial genes are strongly suppressed in central nervous system inflammation, it is unknown whether this results in mitochondrial dysfunction and whether an increase of mitochondrial function can rescue neurodegeneration. Here, we show that predominantly genes of the electron transport chain are suppressed in inflamed mouse neurons, resulting in impaired mitochondrial complex IV activity. This was associated with post-translational inactivation of the transcriptional co-regulator proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). In mice, neuronal overexpression of Ppargc1a, which encodes for PGC-1α, led to increased numbers of mitochondria, complex IV activity, and maximum respiratory capacity. Moreover, Ppargc1a-overexpressing neurons showed a higher mitochondrial membrane potential that related to an improved calcium buffering capacity. Accordingly, neuronal deletion of Ppargc1a aggravated neurodegeneration during experimental autoimmune encephalomyelitis, while neuronal overexpression of Ppargc1a ameliorated it. Our study provides systemic insights into mitochondrial dysfunction in neurons during inflammation and commends elevation of mitochondrial activity as a promising neuroprotective strategy.

Multiple sclerosis is a life-long neurological condition that typically begins when people are in their twenties or thirties. Symptoms vary between individuals, and within a single individual over time, but can include difficulties with vision, balance, movement and thinking. These occur because the immune system of people with multiple sclerosis attacks the brain and spinal cord. This immune assault damages neurons and can eventually cause them to die. But exactly how this happens is unclear, and there are no drugs available that can prevent it. One idea is that the immune attack in multiple sclerosis damages neurons by disrupting structures inside them called mitochondria. These cellular 'organs', or organelles, produce the energy that all cells need to function correctly. If the mitochondria fail to generate enough energy, the cells can die. And because neurons are very active cells with high energy demands, they are particularly vulnerable to the effects of mitochondrial damage. By studying a mouse version of multiple sclerosis, Rosenkranz et al. now show that mitochondria in the neurons of affected animals are less active than those of healthy control mice. This is because the genes inside mitochondria that enable the organelles to produce energy are less active in the multiple sclerosis mice. Most of these genes that determine mitochondrial activity and energy production are under the control of a single master gene called PGC-1alpha. Rosenkranz et al. showed that boosting the activity of this gene ­ by introducing extra copies of it into neurons ­ increases mitochondrial activity in mice. It also makes the animals more resistant to the effects of multiple sclerosis. Boosting the activity of mitochondria in neurons could thus be a worthwhile therapeutic strategy to investigate for multiple sclerosis. Future studies should examine whether drugs that activate PGC-1alpha, for example, could help prevent neuronal death and the resulting symptoms of multiple sclerosis.

Neuron-specific activation of necroptosis signaling in multiple sclerosis cortical grey matter.

Sustained exposure to pro-inflammatory cytokines in the leptomeninges is thought to play a major role in the pathogenetic mechanisms leading to cortical pathology in multiple sclerosis (MS). Although the molecular mechanisms underlying neurodegeneration in the grey matter remain unclear, several lines of evidence suggest a prominent role for tumour necrosis factor (TNF). Using cortical grey matter tissue blocks from post-mortem brains from 28 secondary progressive MS subjects and ten non-neurological controls, we describe an increase in expression of multiple steps in the TNF/TNF receptor 1 signaling pathway leading to necroptosis, including the key proteins TNFR1, FADD, RIPK1, RIPK3 and MLKL. Activation of this pathway was indicated by the phosphorylation of RIPK3 and MLKL and the formation of protein oligomers characteristic of necrosomes. In contrast, caspase-8 dependent apoptotic signaling was decreased. Upregulation of necroptotic signaling occurred predominantly in macroneurons in cortical layers II-III, with little expression in other cell types. The presence of activated necroptotic proteins in neurons was increased in MS cases with prominent meningeal inflammation, with a 30-fold increase in phosphoMLKL+ neurons in layers I-III. The density of phosphoMLKL+ neurons correlated inversely with age at death, age at progression and disease duration. In vivo induction of chronically elevated TNF and INFγ levels in the CSF in a rat model via lentiviral transduction in the meninges, triggered inflammation and neurodegeneration in the underlying cortical grey matter that was associated with increased neuronal expression of TNFR1 and activated necroptotic signaling proteins. Exposure of cultured primary rat cortical neurons to TNF induced necroptosis when apoptosis was inhibited. Our data suggest that neurons in the MS cortex are dying via TNF/TNFR1 stimulated necroptosis rather than apoptosis, possibly initiated in part by chronic meningeal inflammation. Neuronal necroptosis represents a pathogenetic mechanism that is amenable to therapeutic intervention at several points in the signaling pathway.

Correction to: Inflammation of the choroid plexus in progressive multiple sclerosis: accumulation of granulocytes and T cells.

The original publication of this article [1] contained an incorrect author name. The correct and incorrect information is shown in this correction article. The original article has been updated.

Inflammation of the choroid plexus in progressive multiple sclerosis: accumulation of granulocytes and T cells.

The choroid plexus (CP) is strategically located between the peripheral blood and the cerebrospinal fluid, and is involved in the regulation of central nervous system (CNS) homeostasis. In multiple sclerosis (MS), demyelination and inflammation occur in the CNS. While experimental animal models of MS pointed to the CP as a key route for immune cell invasion of the CNS, little is known about the distribution of immune cells in the human CP during progressive phases of MS. Here, we use immunohistochemistry and confocal microscopy to explore the main immune cell populations in the CP of progressive MS patients and non-neuroinflammatory controls, in terms of abundance and location within the distinct CP compartments. We show for the first time that the CP stromal density of granulocytes and CD8+ T cells is higher in progressive MS patients compared to controls. In line with previous studies, the CP of both controls and progressive MS patients contains relatively high numbers of macrophages and dendritic cells. Moreover, we found virtually no B cells or plasma cells in the CP. MHCII+ antigen-presenting cells were often found in close proximity to T cells, suggesting constitutive CNS immune monitoring functions of the CP. Together, our data highlights the role of the CP in immune homeostasis and indicates the occurrence of mild inflammatory processes in the CP of progressive MS patients. However, our findings suggest that the CP is only marginally involved in immune cell migration into the CNS in chronic MS.

Setmelanotide, a Novel, Selective Melanocortin Receptor-4 Agonist Exerts Anti-inflammatory Actions in Astrocytes and Promotes an Anti-inflammatory Macrophage Phenotype.

To date, available treatment strategies for multiple sclerosis (MS) are ineffective in preventing or reversing progressive neurologic deterioration, creating a high, and unmet medical need. One potential way to fight MS may be by limiting the detrimental effects of reactive astrocytes, a key pathological hallmark for disease progression. One class of compounds that may exert beneficial effects via astrocytes are melanocortin receptor (MCR) agonists. Among the MCR, MC4R is most abundantly expressed in the CNS and several rodent studies have described that MC4R is-besides neurons-expressed by astrocytes. Activation of MC4R in astrocytes has shown to have potent anti-inflammatory as well as neuroprotective effects in vitro, suggesting that this could be a potential target to ameliorate ongoing inflammation, and neurodegeneration in MS. In this study, we set out to investigate human MC4R expression and analyze its downstream effects. We identified MC4R mRNA and protein to be expressed on astrocytes and observed increased astrocytic MC4R expression in active MS lesions. Furthermore, we show that the novel, highly selective MC4R agonist setmelanotide ameliorates the reactive phenotype in astrocytes in vitro and markedly induced interleukin-6 and -11 production, possibly through enhanced cAMP response element-binding protein (CREB) phosphorylation. Notably, stimulation of human macrophages with medium from astrocytes that were exposed to setmelanotide, skewed macrophages toward an anti-inflammatory phenotype. Taken together, these findings suggest that targeting MC4R on astrocytes might be a novel therapeutic strategy to halt inflammation-associated neurodegeneration in MS.

Calcium Influx through Plasma-Membrane Nanoruptures Drives Axon Degeneration in a Model of Multiple Sclerosis.

Axon loss determines persistent disability in multiple sclerosis patients. Here, we use in vivo calcium imaging in a multiple sclerosis model to show that cytoplasmic calcium levels determine the choice between axon loss and survival. We rule out the endoplasmic reticulum, glutamate excitotoxicity, and the reversal of the sodium-calcium exchanger as sources of intra-axonal calcium accumulation and instead identify nanoscale ruptures of the axonal plasma membrane as the critical path of calcium entry.

Inflammation and mitochondrial dysfunction: A vicious circle in neurodegenerative disorders?

Experimental evidence supports an intricate association between inflammation and mitochondrial dysfunction as main contributors of neurological diseases. Inflammatory mediators produced by activated microglia and infiltrated immune cells trigger intracellular signalling cascades that can alter cellular mitochondrial metabolism. Cytokines, particularly tumor necrosis factor-alpha, impede mitochondrial oxidative phosphorylation and associated ATP production and instigate mitochondrial reactive oxygen species production. This culminates in mitochondrial membrane permeabilization, altered mitochondrial dynamics and might ultimately result in cell death. When severely injured mitochondria are not appropriately removed by mitophagy they can release their contents into the cytosol and extracellular environment and thereby amplify the inflammatory process. Here we provide a comprehensive overview on how inflammatory mediators impair mitochondrial metabolism and discuss how defective mitochondria can elicit and potentiate an inflammatory response.

A Defective Pentose Phosphate Pathway Reduces Inflammatory Macrophage Responses during Hypercholesterolemia.

Metabolic reprogramming has emerged as a crucial regulator of immune cell activation, but how systemic metabolism influences immune cell metabolism and function remains to be investigated. To investigate the effect of dyslipidemia on immune cell metabolism, we performed in-depth transcriptional, metabolic, and functional characterization of macrophages isolated from hypercholesterolemic mice. Systemic metabolic changes in such mice alter cellular macrophage metabolism and attenuate inflammatory macrophage responses. In addition to diminished maximal mitochondrial respiration, hypercholesterolemia reduces the LPS-mediated induction of the pentose phosphate pathway (PPP) and the Nrf2-mediated oxidative stress response. Our observation that suppression of the PPP diminishes LPS-induced cytokine secretion supports the notion that this pathway contributes to inflammatory macrophage responses. Overall, this study reveals that systemic and cellular metabolism are strongly interconnected, together dictating macrophage phenotype and function.

Astroglial PGC-1alpha increases mitochondrial antioxidant capacity and suppresses inflammation: implications for multiple sclerosis.

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.

Demyelination during multiple sclerosis is associated with combined activation of microglia/macrophages by IFN-γ and alpha B-crystallin.

Activated microglia and macrophages play a key role in driving demyelination during multiple sclerosis (MS), but the factors responsible for their activation remain poorly understood. Here, we present evidence for a dual-trigger role of IFN-γ and alpha B-crystallin (HSPB5) in this context. In MS-affected brain tissue, accumulation of the molecular chaperone HSPB5 by stressed oligodendrocytes is a frequent event. We have shown before that this triggers a TLR2-mediated protective response in surrounding microglia, the molecular signature of which is widespread in normal-appearing brain tissue during MS. Here, we show that IFN-γ, which can be released by infiltrated T cells, changes the protective response of microglia and macrophages to HSPB5 into a robust pro-inflammatory classical response. Exposure of cultured microglia and macrophages to IFN-γ abrogated subsequent IL-10 induction by HSPB5, and strongly promoted HSPB5-triggered release of TNF-α, IL-6, IL-12, IL-1ß and reactive oxygen and nitrogen species. In addition, high levels of CXCL9, CXCL10, CXL11, several guanylate-binding proteins and the ubiquitin-like protein FAT10 were induced by combined activation with IFN-γ and HSPB5. As immunohistochemical markers for microglia and macrophages exposed to both IFN-γ and HSPB5, these latter factors were found to be selectively expressed in inflammatory infiltrates in areas of demyelination during MS. In contrast, they were absent from activated microglia in normal-appearing brain tissue. Together, our data suggest that inflammatory demyelination during MS is selectively associated with IFN-γ-induced re-programming of an otherwise protective response of microglia and macrophages to the endogenous TLR2 agonist HSPB5.

Glutathione in multiple sclerosis: more than just an antioxidant?

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.

Cellular distribution of glucose and monocarboxylate transporters in human brain white matter and multiple sclerosis lesions.

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.

Mitochondrial dysfunction contributes to neurodegeneration in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Current treatments are very effective in reducing the neuroinflammatory attack, but fail to significantly halt disease progression and associated loss of neuronal tissue. In recent years, it has become increasingly clear that dysfunctional mitochondria are important contributors to damage and loss of both axons and neurons. Observations in animal and histopathological studies suggest that infiltrating leukocytes and activated microglia play a central role in neuronal mitochondrial dysfunction. This review provides a comprehensive overview on the current knowledge regarding mitochondrial dysfunction in MS. Importantly, more insight into the cause and consequences of impaired mitochondrial function provide a basis for mitochondrial-targeted medicine to combat progressive MS.

Reduced expression of PGC-1α partly underlies mitochondrial changes and correlates with neuronal loss in multiple sclerosis cortex.

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.

Clusters of activated microglia in normal-appearing white matter show signs of innate immune activation.

BACKGROUND: In brain tissues from multiple sclerosis (MS) patients, clusters of activated HLA-DR-expressing microglia, also referred to as preactive lesions, are located throughout the normal-appearing white matter. The aim of this study was to gain more insight into the frequency, distribution and cellular architecture of preactive lesions using a large cohort of well-characterized MS brain samples. METHODS: Here, we document the frequency of preactive lesions and their association with distinct white matter lesions in a cohort of 21 MS patients. Immunohistochemistry was used to gain further insight into the cellular and molecular composition of preactive lesions. RESULTS: Preactive lesions were observed in a majority of MS patients (67%) irrespective of disease duration, gender or subtype of disease. Microglial clusters were predominantly observed in the vicinity of active demyelinating lesions and are not associated with T cell infiltrates, axonal alterations, activated astrocytes or blood-brain barrier disruption. Microglia in preactive lesions consistently express interleukin-10 and TNF-α, but not interleukin-4, whereas matrix metalloproteases-2 and -9 are virtually absent in microglial nodules. Interestingly, key subunits of the free-radical-generating enzyme NADPH oxidase-2 were abundantly expressed in microglial clusters. CONCLUSIONS: The high frequency of preactive lesions suggests that it is unlikely that most of them will progress into full-blown demyelinating lesions. Preactive lesions are not associated with blood-brain barrier disruption, suggesting that an intrinsic trigger of innate immune activation, rather than extrinsic factors crossing a damaged blood-brain barrier, induces the formation of clusters of activated microglia.