Purification of cells from fresh human brain tissue: primary human glial cells.

In order to translate the findings obtained from postmortem brain tissue samples to functional biologic mechanisms of central nervous system disease, it will be necessary to understand how these findings affect the different cell populations in the brain. The acute isolation and analysis of pure glial cell populations are common practice in animal models for neurologic diseases, but are not yet regularly applied to human postmortem brain material. The development of novel cell isolation techniques and methods for transcriptomic and proteomic analysis have made it possible to isolate and phenotype primary human cell populations from the central nervous system. The psychiatric program of the Netherlands Brain Bank has considerable experience with the purification of glial cells. This chapter will review the rapid isolation and phenotyping procedures for two major glia cell populations in the human brain, microglia and astrocytes, and will also discuss the potential for biobanking these cells, as well as the possible alternatives to cell isolations. The acute isolation of glial cells without culture-based adherence steps allows the analysis of glial alterations that underlie, or are the result of, disease neuropathology of the donor

Immune cell trafficking across the barriers of the central nervous system in multiple sclerosis and stroke

Each year about 650,000 Europeans die from stroke and a similar number lives with the sequelae of multiple sclerosis (MS). Stroke and MS differ in their etiology. Although cause and likewise clinical presentation set the two diseases apart, they share common downstream mechanisms that lead to damage and recovery. Demyelination and axonal injury are characteristics of MS but are also observed in stroke. Conversely, hallmarks of stroke, such as vascular impairment and neurodegeneration, are found in MS. However, the most conspicuous common feature is the marked neuroinflammatory response, marked by glia cell activation and immune cell influx. In MS and stroke the blood-brain barrier is disrupted allowing bone marrow-derived macrophages to invade the brain in support of the resident microglia. In addition, there is a massive invasion of auto-reactive T-cells into the brain of patients with MS. Though less pronounced a similar phenomenon is also found in ischemic lesions. Not surprisingly, the two diseases also resemble each other at the level of gene expression and the biosynthesis of other proinflammatory mediators. While MS has traditionally been considered to be an autoimmune neuroinflammatory disorder, the role of inflammation for cerebral ischemia has only been recognized later. In the case of MS the long track record as neuroinflammatory disease has paid off with respect to treatment options. There are now about a dozen of approved drugs for the treatment of MS that specifically target neuroinflammation by modulating the immune system. Interestingly, experimental work demonstrated that drugs that are in routine use to mitigate neuroinflammation in MS may also work in stroke models. Examples include Fingolimod, glatiramer acetate, and antibodies blocking the leukocyte integrin VLA-4. Moreover, therapeutic strategies that were discovered in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, turned out to be also effective in experimental stroke models. This suggests that previous achievements in MS research may be relevant for stroke. Interestingly, the converse is equally true. Concepts on the neurovascular unit that were developed in a stroke context turned out to be applicable to neuroinflammatory research in MS. Examples include work on the important role of the vascular basement membrane and the BBB for the invasion of immune cells into the brain. Furthermore, tissue plasminogen activator (tPA), the only established drug treatment in acute stroke, modulates the pathogenesis of MS. Endogenous tPA is released from endothelium and astroglia and acts on the BBB, microglia and other neuroinflammatory cells. Thus, the vascular perspective of stroke research provides important input into the mechanisms on how endothelial cells and the BBB regulate inflammation in MS, particularly the invasion of immune cells into the CNS. In the current review we will first discuss pathogenesis of both diseases and current treatment regimens and will provide a detailed overview on pathways of immune cell migration across the barriers of the CNS and the role of activated astrocytes in this process. This article is part of a Special Issue entitled: Neuro inflammation: A common denominator for stroke, multiple sclerosis and Alzheimer's disease, guest edited by Helga de Vries and Markus Swaninger

Post-mortem multiple sclerosis lesion pathology is influenced by single nucleotide polymorphisms

Over the last few decades, several common single nucleotide polymorphisms (SNPs) have been identified that correlate with clinical outcome in multiple sclerosis (MS), but the pathogenic mechanisms underlying the clinical effects of these SNPs are unknown. This is in part because of the difficulty in the functional translation of genotype into disease-relevant mechanisms. Building on our recent work showing the association of clinical disease course with post-mortem MS lesion characteristics, we hypothesized that SNPs that correlate with clinical disease course would also correlate with specific MS lesion characteristics in autopsy tissue. To test this hypothesis, 179 MS brain donors from the Netherlands Brain Bank MS autopsy cohort were genotyped for 102 SNPs, selected based on their reported associations with clinical outcome or their associations with genes that show differential gene expression in MS lesions. Three SNPs linked to MS clinical severity showed a significant association between the genotype and either the proportion of active lesions (rs2234978/FAS and rs11957313/KCNIP1) or the proportion of mixed active/inactive lesions (rs8056098/CLEC16A). Three SNPs linked to MS pathology-associated genes showed a significant association with either proportion of active lesions (rs3130253/MOG), incidence of cortical gray matter lesions (rs1064395/NCAN) or the proportion of remyelinated lesions (rs5742909/CTLA4). In addition, rs2234978/FAS T-allele carriers showed increased FAS gene expression levels in perivascular T cells and perilesional oligodendrocytes, cell types that have been implicated in MS lesion formation. Thus, by combining pathological characterization of MS brain autopsy tissue with genetics, we now start to translate genotypes linked to clinical outcomes in MS into mechanisms involved in MS lesion pathogenesis

Transcriptional profiling of human microglia reveals grey-white matter heterogeneity and multiple sclerosis-associated changes.

Here we report the transcriptional profile of human microglia, isolated from normal-appearing grey matter (GM) and white matter (WM) of multiple sclerosis (MS) and non-neurological control donors, to find possible early changes related to MS pathology. Microglia show a clear region-specific profile, indicated by higher expression of type-I interferon genes in GM and higher expression of NF-κB pathway genes in WM. Transcriptional changes in MS microglia also differ between GM and WM. MS WM microglia show increased lipid metabolism gene expression, which relates to MS pathology since active MS lesionderived microglial nuclei show similar altered gene expression. Microglia from MS GM show increased expression of genes associated with glycolysis and iron homeostasis, possibly reflecting microglia reacting to iron depositions. Except for ADGRG1/GPR56, expression of homeostatic genes, such as P2RY12 and TMEM119, is unaltered in normal-appearing MS tissue, demonstrating overall preservation of microglia homeostatic functions in the initiation phase of MS

Retinoic acid induces blood-brain barrier development

The blood-brain barrier (BBB) is crucial in the maintenance of a controlled environment within the brain to safeguard optimal neuronal function. The endothelial cells (ECs) of the BBB possess specific properties that restrict the entry of cells and metabolites into the CNS. The specialized BBB endothelial phenotype is induced during neurovascular development by surrounding cells of the CNS. However, the molecular differentiation of the BBB endothelium remains poorly understood. Retinoic acid (RA) plays a crucial role in the brain during embryogenesis. Because radial glial cells supply the brain with RA during the developmental cascade and associate closely with the developing vasculature, we hypothesize that RA is important for the induction of BBB properties in brain ECs. Analysis of human postmortem fetal brain tissue shows that the enzyme mainly responsible for RA synthesis, retinaldehyde dehydrogenase, is expressed by radial glial cells. In addition, the most important receptor for RA-driven signaling in the CNS, RA-receptor β (RARβ), is markedly expressed by the developing brain vasculature. Our findings have been further corroborated by in vitro experiments showing RA- and RARβ-dependent induction of different aspects of the brain EC barrier. Finally, pharmacologic inhibition of RAR activation during the differentiation of the murine BBB resulted in the leakage of a fluorescent tracer as well as serum proteins into the developing brain and reduced the expression levels of important BBB determinants. Together, our results point to an important role for RA in the induction of the BBB during human and mouse developmen

Microglia transcriptional profiling in major depressive disorder shows inhibition of cortical grey matter microglia

BACKGROUND: Microglia have been implicated in the pathophysiology of major depressive disorder (MDD), but information on biological mechanisms is limited. Therefore, we investigated the gene expression profile of microglial cells in relation to neuronal regulators of microglia activity in well-characterized MDD and control autopsy brains. METHODS: Pure, intact microglia were isolated at brain autopsy from occipital cortex grey matter (GM) and corpus callosum white matter (WM) of 13 MDD and 10 age-matched control donors for RNA sequencing. Top differentially expressed genes were validated using immunohistochemistry (IHC) staining. Since gene expression changes were only detected in GM microglia, neuronal regulators of microglia were investigated in cortical tissue and synaptosomes from the cortex by RT-qPCR and Western blot. RESULTS: Transcriptome analysis revealed 92 genes differentially expressed in microglia isolated from GM, but none in microglia from WM in MDD, compared to controls. Of these, 81 genes were less abundantly expressed in GM MDD, including CD163, MKI67, SPP1, CD14, FCGR1A/C, and C1QA/B/C. Accordingly, pathways related to effector mechanisms, such as the complement system and phagocytosis were differentially regulated in GM microglia in MDD. IHC staining revealed significantly lower expression of CD163 protein in MDD. Whole tissue analysis showed an increase in CD200 (p+0.0009) and CD47 (p=0.068) mRNA, and CD47 protein was significantly elevated (p=0.0396) in synaptic fractions of MDD cases. CONCLUSIONS: Transcriptional profiling indicates an immune-suppressed microglial phenotype in MDD, possibly caused by neuronal regulation

Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables

Microglia are key players in the central nervous system in health and disease. Much pioneering research on microglia function has been carried out in vivo with the use of genetic animal models. However, to fully understand the role of microglia in neurological and psychiatric disorders, it is crucial to study primary human microglia from brain donors. We have developed a rapid procedure for the isolation of pure human microglia from autopsy tissue using density gradient centrifugation followed by CD11b-specific cell selection. The protocol can be completed in 4 h, with an average yield of 450,000 and 145,000 viable cells per gram of white and grey matter tissue respectively. This method allows for the immediate phenotyping of microglia in relation to brain donor clinical variables, and shows the microglia population to be distinguishable from autologous choroid plexus macrophages. This protocol has been applied to samples from over 100 brain donors from the Netherlands Brain Bank, providing a robust dataset to analyze the effects of age, post-mortem delay, brain acidity, and neurological diagnosis on microglia yield and phenotype. Our data show that cerebrospinal fluid pH is positively correlated to microglial cell yield, but donor age and post-mortem delay do not negatively affect viable microglia yield. Analysis of CD45 and CD11b expression showed that changes in microglia phenotype can be attributed to a neurological diagnosis, and are not influenced by variation in ante- and post-mortem parameters. Cryogenic storage of primary microglia was shown to be possible, albeit with variable levels of recovery and effects on phenotype and RNA quality. Microglial gene expression substantially changed due to culture, including the loss of the microglia-specific markers, showing the importance of immediate microglia phenotyping. We conclude that primary microglia can be isolated effectively and rapidly from human post-mortem brain tissue, allowing for the study of the microglial population in light of the neuropathological status of the donor

Single-cell mass cytometry reveals complex myeloid cell composition in active lesions of progressive multiple sclerosis

Myeloid cells contribute to inflammation and demyelination in the early stages of multiple sclerosis (MS), but it is still unclear to what extent these cells are involved in active lesion formation in progressive MS (PMS). Here, we have harnessed the power of single-cell mass cytometry (CyTOF) to compare myeloid cell phenotypes in active lesions of PMS donors with those in normal-appearing white matter from the same donors and control white matter from non-MS donors. CyTOF measurements of a total of 74 targeted proteins revealed a decreased abundance of homeostatic and TNFhi microglia, and an increase in highly phagocytic and activated microglia states in active lesions of PMS donors. Interestingly, in contrast to results obtained from studies of the inflammatory early disease stages of MS, infiltrating monocyte-derived macrophages were scarce in active lesions of PMS, suggesting fundamental differences of myeloid cell composition in advanced stages of PMS