Peptide Splicing in the Proteasome Creates a Novel Type of Antigen with an Isopeptide Linkage.

The proteasome is able to create spliced Ags, in which two distant parts of a protein are excised and ligated together to form a novel peptide, for presentation by MHC class I molecules. These noncontiguous epitopes are generated via a transpeptidation reaction catalyzed by the proteasomal active sites. Transpeptidation reactions in the proteasome follow explicit rules and occur particularly efficiently when the C-terminal ligation partner contains a lysine or arginine residue at the site of ligation. Lysine contains two amino groups that theoretically may both participate in ligation reactions, implying that potentially not only peptide but also isopeptide linkages could be formed. Using nuclear magnetic resonance spectroscopy, we demonstrate in the present study that the proteasome can use the ε-amino group of an N-terminal lysine residue in transpeptidation reactions to create a novel type of posttranslationally modified epitopes. We show that the overall efficiency of ε ligation is only 10-fold lower as compared with α ligation, suggesting that the proteasome can produce sufficient isopeptide Ag to evoke a T cell response. Additionally, we show that isopeptides are more stable toward further proteasomal processing than are normal peptides, and we demonstrate that isopeptides can bind to HLA-A2.1 and HLA-A3 with high affinity. These properties likely increase the fraction of ε-ligated peptides presented on the cell surface for CD8(+) T cell surveillance. Finally, we show that isopeptide Ags are immunogenic in vivo. We postulate that ε ligation is a genuine posttranslational modification, suggesting that the proteasome can create a novel type of Ag that is likely to play a role in immunity.

Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules.

Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I-restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.

Enhanced uptake of multiple sclerosis-derived myelin by THP-1 macrophages and primary human microglia.

BACKGROUND: The pathological hallmark of multiple sclerosis (MS) is myelin phagocytosis. It remains unclear why microglia and macrophages demyelinate axons in MS, but previously found or yet-unknown changes in the myelin of MS patients could contribute to this process. We therefore studied whether myelin from normal-appearing white matter (NAWM) of MS donors is phagocytosed more efficiently than myelin from control donors. METHODS: Myelin was isolated from 11 MS and 12 control brain donors and labeled with the pH-sensitive fluorescent dye pHrodo to quantify uptake in lysosomes. Phagocytosis by differentiated THP-1 macrophages and by primary human microglia was quantified with flow cytometry. Whereas myelin uptake by THP-1 macrophages reached a plateau after approximately 24 hours, uptake by primary human microglia showed an almost linear increase over a 72-hour period. Data were statistically analyzed with the Mann-Whitney U test. RESULTS: MS-derived myelin was phagocytosed more efficiently by THP-1 macrophages after 6-hour incubation (P = 0.001 for the percentage of myelin-phagocytosing cells and P = 0.0005 for total myelin uptake) and after 24-hour incubation (P = 0.0006 and P = 0.0001, respectively), and by microglia after 24-hour incubation (P = 0.0106 for total myelin uptake). This enhanced uptake was not due to differences in the oxidation status of the myelin. Interestingly, myelin phagocytosis correlated negatively with the age of myelin donors, whereas the age of microglia donors showed a positive trend with myelin phagocytosis. CONCLUSIONS: Myelin isolated from normal-appearing white matter of MS donors was phagocytosed more efficiently than was myelin isolated from control brain donors by both THP-1 macrophages and primary human microglia. These data indicate that changes in MS myelin might precede phagocyte activation and subsequent demyelination in MS. Identifying these myelin changes responsible for enhancing phagocytic ability could be an interesting therapeutic target to prevent or inhibit formation or expansion of MS lesions. Moreover, during aging, microglia enhance their phagocytic capacity for myelin phagocytosis, but myelin reduces its susceptibility for uptake.

Microglia in normal appearing white matter of multiple sclerosis are alerted but immunosuppressed.

Little is known about the functional phenotype of microglia in normal appearing white matter (NAWM) of multiple sclerosis (MS), although it may hold valuable clues about mechanisms for lesion development. Therefore, we studied microglia from NAWM obtained post-mortem from controls (n = 25) and MS patients (n = 21) for their phenotype ex vivo and their immune responsiveness in vitro, using a microglia isolation method that omits culture and adherence. By flow cytometry, microglia in MS NAWM displayed elevated CD45 levels and increased size and granularity but were distinct from autologous choroid plexus macrophages by absent or low expression of additional markers, in particular CD206. Flow cytometric analysis of microglia from NAWM of three controls and four MS patients showed alterations in levels of Fc-gamma receptors in MS. In primary microglia from a bigger sample of subjects, analysis of Fc-gamma receptors by quantitative PCR indicated a significant increase in mRNA levels of the inhibitory CD32b isoform in MS NAWM. Despite their changed activation status, microglia from MS NAWM were unresponsive to lipopolysaccharide in vitro. Notably, culture with dexamethasone led to an impaired induction of the inflammation-limiting cytokine CCL18 in microglia from MS NAWM compared with those from control NAWM. Together, these data demonstrate that microglia in MS NAWM are in an alerted state, but display features of immunosuppression. Thus, the activation status of microglia in NAWM of MS patients likely reflects a response to ongoing neuroinflammation, which coincides with upregulation of immunoregulatory molecules to prevent full activation and damage to the vulnerable milieu.

Characteristics of differentiated CD8(+) and CD4 (+) T cells present in the human brain.

Immune surveillance of the central nervous system (CNS) by T cells is important to keep CNS-trophic viruses in a latent state, yet our knowledge of the characteristics of CNS-populating T cells is incomplete. We performed a comprehensive, multi-color flow-cytometric analysis of isolated T cells from paired corpus callosum (CC) and peripheral blood (PB) samples of 20 brain donors. Compared to PB, CC T cells, which were mostly located in the perivascular space and sporadically in the parenchyma, were enriched for cells expressing CD8. Both CD4(+) and CD8(+) T cells in the CC had a late-differentiated phenotype, as indicated by lack of expression of CD27 and CD28. The CC contained high numbers of T cells expressing chemokine receptor CX3CR1 and CXCR3 that allow for homing to inflamed endothelium and tissue, but hardly cells expressing the lymph node-homing receptor CCR7. Despite the late-differentiated phenotype, CC T cells had high expression of the IL-7 receptor α-chain CD127 and did not contain the neurotoxic cytolytic enzymes perforin, granzyme A, and granzyme B. We postulate that CNS T cells make up a population of tissue-adapted differentiated cells, which use CX3CR1 and CXCR3 to home into the perivascular space, use IL-7 for maintenance, and lack immediate cytolytic activity, thereby preventing immunopathology in response to low or non-specific stimuli. The presence of these cells in this tightly regulated environment likely enables a fast response to local threats. Our results will enable future detailed exploration of T-cell subsets in the brain involved in neurological diseases.

Phenotyping primary human microglia: tight regulation of LPS responsiveness.

Much is still unknown about mechanisms underlying the phenotypical and functional versatility of human microglia. Therefore, we developed a rapid procedure to isolate pure microglia from postmortem human brain tissue and studied their immediate ex vivo phenotype and responses to key inflammatory mediators. Microglia were isolated, along with macrophages from the choroid plexus by tissue dissociation, density gradient separation, and selection with magnetic microbeads. By flow cytometry, microglia were identified by a CD11b(+) CD45(dim) phenotype and a smaller size compared with CD11b(+) CD45(high) macrophages. Interestingly, white matter microglia from donors with peripheral inflammation displayed elevated CD45 levels and increased size and granularity, but were still distinct from macrophages. The phenotype of isolated microglia was further specified by absent surface expression of CD14, CD200 receptor, and mannose receptor (MR, CD206), all of which were markedly expressed by macrophages. Microglia stimulated immediately after isolation with LPS and IFNγ failed to upregulate TNFα or CCR7. Notably, responsiveness to LPS and IFNγ was clearly instigated in microglia after overnight preculture, which coincided with a strong upregulation of CD14. Culture of microglia with IL-4 resulted in the induction of HLA-DR and CCL18 but not MR, whereas culture with dexamethasone did induce MR, in addition to CD163 and CCL18. In conclusion, this study demonstrates phenotypic changes of microglia associated with peripheral inflammation, and reveals tight regulation of responses to LPS and IFNγ as well as distinct microglial responses to IL-4 and glucocorticoids. These findings are of high relevance to studies on human microglia functioning in health and disease.

Probing the specificity and activity profiles of the proteasome inhibitors bortezomib and delanzomib.

The ubiquitin proteasome system is an attractive pharmacological target for the treatment of cancer. The proteasome inhibitor bortezomib has been approved for the treatment of multiple myeloma and mantle cell lymphoma but is associated with substantial adverse effects and the occurrence of resistance, underscoring the continued need for novel proteasome inhibitors. In this study, bortezomib and the novel proteasome inhibitor delanzomib were compared for their ability to inhibit proteasome activity using both fluorogenic substrates and a recently developed fluorescent proteasome activity probe. Bortezomib and delanzomib were equipotent in inhibiting distinct subunits of the proteasome in a panel of cell lines in vitro. In a preclinical multiple myeloma model, both inhibitors inhibited the proteasome in normal tissues to a similar extent. Tumor proteasome activity was inhibited to a significantly higher extent by delanzomib (60%) compared to bortezomib (32%). In addition, delanzomib was able to overcome bortezomib resistance in vitro. The present findings demonstrate that proteasome activity probes can accurately monitor the effects of proteasome inhibitors on both normal and tumor tissues in preclinical models and can be used as a diagnostic approach to predict resistance against treatment with proteasome inhibitors. Furthermore, the data presented here provide rationale for further clinical development of delanzomib.

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 lesion-derived 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.

Transcriptome analysis of normal-appearing white matter reveals cortisol- and disease-associated gene expression profiles in multiple sclerosis.

Inter-individual differences in cortisol production by the hypothalamus-pituitary-adrenal (HPA) axis are thought to contribute to clinical and pathological heterogeneity of multiple sclerosis (MS). At the same time, accumulating evidence indicates that MS pathogenesis may originate in the normal-appearing white matter (NAWM). Therefore, we performed a genome-wide transcriptional analysis, by Agilent microarray, of post-mortem NAWM of 9 control subjects and 18 MS patients to investigate to what extent gene expression reflects disease heterogeneity and HPA-axis activity. Activity of the HPA axis was determined by cortisol levels in cerebrospinal fluid and by numbers of corticotropin-releasing neurons in the hypothalamus, while duration of MS and time to EDSS6 served as indicator of disease severity. Applying weighted gene co-expression network analysis led to the identification of a range of gene modules with highly similar co-expression patterns that strongly correlated with various indicators of HPA-axis activity and/or severity of MS. Interestingly, molecular profiles associated with relatively mild MS and high HPA-axis activity were characterized by increased expression of genes that actively regulate inflammation and by molecules involved in myelination, anti-oxidative mechanism, and neuroprotection. Additionally, group-wise comparisons of gene expression in white matter from control subjects and NAWM from (subpopulations of) MS patients uncovered disease-associated gene expression as well as strongly up- or downregulated genes in patients with relatively benign MS and/or high HPA-axis activity, with many differentially expressed genes being previously undescribed in the context of MS. Overall, the data suggest that HPA-axis activity strongly impacts on molecular mechanisms in NAWM of MS patients, but partly also independently of disease severity.

Staining of HLA-DR, Iba1 and CD68 in human microglia reveals partially overlapping expression depending on cellular morphology and pathology.

HLA-DR, Iba1 and CD68 are widely used microglia markers in human tissue. However, due to differences in gene regulation, they may identify different activation stages of microglia. Here, we directly compared the expression of HLA-DR, Iba1 and CD68 in microglia with different phenotypes, ranging from ramified to amoeboid, to foamy phagocytizing macrophages, in adjacent sections immunocytochemically double stained for two of the markers. Material was used from patients diagnosed with multiple sclerosis (MS) and Alzheimer's disease (AD) patients and control subjects because together they contain all the microglia activation stages in an acute and a chronic inflammatory setting. We found a similar, yet not identical, overall expression pattern. All three markers were expressed by ramified/amoeboid microglia around chronic active MS lesions, but overlap between HLA-DR and Iba1 was limited. Foamy macrophages in the demyelinating rims of active MS lesions of MS expressed more HLA-DR and CD68 than Iba1. All markers were expressed by small microglia accumulations (nodules) in MS NAWM. Dense core AD plaques in the hippocampus were mostly associated with microglia expressing HLA-DR. Diffuse AD plaques were not specifically associated with microglia at all. These results indicate that microglia markers have different potential for neuropathological analysis, with HLA-DR and CD68 reflecting immune activation and response to tissue damage, and Iba1 providing a marker more suited for structural studies in the absence of pathology.

Gene Expression Profiling of Multiple Sclerosis Pathology Identifies Early Patterns of Demyelination Surrounding Chronic Active Lesions.

In multiple sclerosis (MS), activated microglia and infiltrating macrophages phagocytose myelin focally in (chronic) active lesions. These demyelinating sites expand in time, but at some point turn inactive into a sclerotic scar. To identify molecular mechanisms underlying lesion activity and halt, we analyzed genome-wide gene expression in rim and peri-lesional regions of chronic active and inactive MS lesions, as well as in control tissue. Gene clustering revealed patterns of gene expression specifically associated with MS and with the presumed, subsequent stages of lesion development. Next to genes involved in immune functions, we found regulation of novel genes in and around the rim of chronic active lesions, such as NPY, KANK4, NCAN, TKTL1, and ANO4. Of note, the presence of many foamy macrophages in active rims was accompanied by a congruent upregulation of genes related to lipid binding, such as MSR1, CD68, CXCL16, and OLR1, and lipid uptake, such as CHIT1, GPNMB, and CCL18. Except CCL18, these genes were already upregulated in regions around active MS lesions, showing that such lesions are indeed expanding. In vitro downregulation of the scavenger receptors MSR1 and CXCL16 reduced myelin uptake. In conclusion, this study provides the gene expression profile of different aspects of MS pathology and indicates that early demyelination, mediated by scavenger receptors, is already present in regions around active MS lesions. Genes involved in early demyelination events in regions surrounding chronic active MS lesions might be promising therapeutic targets to stop lesion expansion.

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.

Expression of vitamin D receptor and metabolizing enzymes in multiple sclerosis-affected brain tissue.

Vitamin D deficiency has been implicated as a risk factor for multiple sclerosis (MS), but how vitamin D metabolism affects MS pathophysiology is not understood. We studied the expression of vitamin D receptor (VDR) and related enzymes, including 1,25(OH)(2)D-24-hydroxylase (24-OHase; CYP24A1) and 25(OH)D-1α-hydroxylase (CYP27B1), in CNS tissues of 39 MS patients and 20 controls and in primary human glial cells in vitro. In control and MS normal-appearing white matter (NAWM), nuclear VDR immunostaining was observed in oligodendrocyte-like cells, human leukocyte antigen (HLA)-positive microglia, and glial fibrillary acidic protein-positive astrocytes. There was a 2-fold increase in VDR transcripts in MS NAWM versus control white matter (p = 0.03). In chronic active MS lesions, HLA-positive microglia/macrophages showed nuclear VDR staining; astrocytes showed nuclear and cytoplasmic VDR staining. Staining for 24-OHase was restricted to astrocytes.VDR and CYP27B1 mRNA expressions were increased in active MS lesions versus NAWM (p < 0.01, p = 0.04, respectively). In primary human astrocytes in vitro, the active form of vitamin D, 1,25(OH)(2)D(3), induced upregulation of VDR and CYP24A1. Tumor necrosis factor and interferon-γ upregulated CYP27B1 mRNA in primary human microglia and astrocytes. Increased VDR expression in MS NAWM and inflammatory cytokine-induced amplified expression of VDR and CYP27B1 in chronic active MS lesions suggest increased sensitivity to vitamin D in NAWM and a possible endogenous role for vitamin D metabolism in the suppression of active MS lesions.

Selective upregulation of scavenger receptors in and around demyelinating areas in multiple sclerosis.

Autoantibodies and complement opsonization have been implicated in the process of demyelination in the major human CNS demyelinating disease multiple sclerosis (MS), but scavenger receptors (SRs) may also play pathogenetic roles. We characterized SR mRNA and protein expression in postmortem brain tissue from 13 MS patients in relation to active demyelination. CD68, chemokine (C-X-C motif) ligand 16 (CXCL16), class A macrophage SR (SR-AI/II), LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1), FcγRIII, and LRP-1 (low-density lipoprotein receptor-related protein 1) mRNA were upregulated in the rims of chronic active MS lesions. CD68 and CXCL16 mRNA were also upregulated around chronic active MS lesions. By immunohistochemistry, CD68, CXCL16, and SR-AI/II were expressed by foamy macrophages in the rim and by ramified microglia around chronic active MS lesions. CXCL16 and SR-AI/II were also expressed by astrocytes in MS lesions and by primary human microglia and astrocytes in vitro. These data suggest that SRs are involved in myelin uptake in MS, and that upregulation of CD68, CXCL16, and SR-AI/II is one of the initial events in microglia as they initiate myelin phagocytosis. As demyelination continues, additional upregulation of LOX-1, FcγRIII, and LRP-1 may facilitate this process.

Fluorescence-based proteasome activity profiling.

With the proteasome emerging as a therapeutic target for cancer treatment, accurate tools for monitoring proteasome (inhibitor) activity are in demand. In this chapter, we describe the synthesis and use of a fluorescent proteasome activity probe that allows for accurate profiling of proteasomal activity in cell lysates, intact cells, and murine and human patient-derived material, with high sensitivity using SDS-PAGE. The probe allows for direct scanning of the gel for fluorescent emission of the distinct proteasomal subunits and circumvents the use of Western blot analysis. Due to its suitable biochemical and biophysical properties, the fluorescent probe can also be used for confocal laser scanning microscopy and flow cytometry-based experiments.

Quantifying cross-tissue diversity in proteasome complexes by mass spectrometry.

The composition of 20S mouse proteasome complexes isolated from mice heart, kidney, liver, lung, thymus and spleen was compared using quantitative mass spectrometry. Significant variety was observed in hybrid classes of immunoproteasomes which may have implications for the use of proteasome targeted inhibitors.

Dose-dependent inhibition of proteasome activity by a mutant ubiquitin associated with neurodegenerative disease.

The ubiquitin-proteasome system is the main regulated intracellular proteolytic pathway. Increasing evidence implicates impairment of this system in the pathogenesis of diseases with ubiquitin-positive pathology. A mutant ubiquitin, UBB(+1), accumulates in the pathological hallmarks of tauopathies, including Alzheimer's disease, polyglutamine diseases, liver disease and muscle disease and serves as an endogenous reporter for proteasomal dysfunction in these diseases. UBB(+1) is a substrate for proteasomal degradation, however it can also inhibit the proteasome. Here, we show that UBB(+1) properties shift from substrate to inhibitor in a dose-dependent manner in cell culture using an inducible UBB(+1) expression system. At low expression levels, UBB(+1) was efficiently degraded by the proteasome. At high levels, the proteasome failed to degrade UBB(+1), causing its accumulation, which subsequently induced a reversible functional impairment of the ubiquitin-proteasome system. Also in brain slice cultures, UBB(+1) accumulation and concomitant proteasome inhibition was only induced at high expression levels. Our findings show that by varying UBB(+1) expression levels, the dual proteasome substrate and inhibitory properties can be optimally used to serve as a research tool to study the ubiquitin-proteasome system and to further elucidate the role of aberrations of this pathway in disease.