John J. P. Brevé

Brain region-specific gene expression profiles in freshly isolated rat microglia

Microglia are important cells in the brain that can acquire different morphological and functional phenotypes dependent on the local situation they encounter. Knowledge on the region-specific gene signature of microglia may hold valuable clues for microglial functioning in health and disease, e.g., Parkinson’s disease (PD) in which microglial phenotypes differ between affected brain regions. Therefore, we here investigated whether regional differences exist in gene expression profiles of microglia that are isolated from healthy rat brain regions relevant for PD. We used an optimized isolation protocol based on a rapid isolation of microglia from discrete rat gray matter regions using density gradients and fluorescent-activated cell sorting. Application of the present protocol followed by gene expression analysis enabled us to identify subtle differences in region-specific microglial expression profiles and show that the genetic profile of microglia already differs between different brain regions when studied under control conditions. As such, these novel findings imply that brain region-specific microglial gene expression profiles exist that may contribute to the region-specific differences in microglia responsivity during disease conditions, such as seen in, e.g., PD.

Neuroinflammation in Parkinson's patients and MPTP-treated mice is not restricted to the nigrostriatal system: Microgliosis and differential expression of interleukin-1 receptors in the olfactory bulb

Neuroinflammation may play a role in the pathogenesis of Parkinsons disease (PD). The present study questioned whether this neuroinflammatory response differs between the olfactory bulb, as an early affected region and the nigrostriatal system. Indeed, increased microgliosis was shown in post-mortem olfactory bulb of PD patients. Also in olfactory bulb of MPTP-treated mice, microgliosis and increased expression of IL-1alpha, IL-1beta and IL-1ra mRNA was observed early after treatment. These observations implicate that neuroinflammation is not restricted to the nigrostriatal system. MPTP-induced microgliosis in striatum and olfactory bulb was reduced in IL-1alpha/beta knockout mice, indicating that IL-1 affects microglia activation. Importantly, MPTP induced differential regulation of IL-1 receptors. mRNA levels of IL-1RI and, to a lesser extent, IL-1RII were increased in striatum. Interestingly, in the olfactory bulb only IL-1RII mRNA was enhanced. We suggest that differential regulation of IL-1 signaling can serve as an important mechanism to modulate neuroinflammatory activity after MPTP treatment and possibly during PD.

Astrocyte-Derived Tissue Transglutaminase Interacts with Fibronectin: A Role in Astrocyte Adhesion and Migration?

An important neuropathological feature of neuroinflammatory processes that occur during e.g. Multiple Sclerosis (MS) is the formation of an astroglial scar. Astroglial scar formation is facilitated by the interaction between astrocytes and extracellular matrix proteins (ECM) such as fibronectin. Since there is evidence indicating that glial scars strongly inhibit both axon growth and (re)myelination in brain lesions, it is important to understand the factors that contribute to the interaction between astrocytes and ECM proteins. Tissue Transglutaminase (TG2) is a multifunctional enzyme with an ubiquitous tissue distribution, being clearly present within the brain. It has been shown that inflammatory cytokines can enhance TG2 activity. In addition, TG2 can mediate cell adhesion and migration and it binds fibronectin with high affinity. We therefore hypothesized that TG2 is involved in astrocyte-fibronectin interactions. Our studies using primary rat astrocytes show that intracellular and cell surface expression and activity of TG2 is increased after treatment with pro-inflammatory cytokines. Astrocyte-derived TG2 interacts with fibronectin and is involved in astrocyte adhesion onto and migration across fibronectin. TG2 is involved in stimulating focal adhesion formation which is necessary for the interaction of astrocytes with ECM proteins. We conclude that astrocyte-derived TG2 contributes to the interaction between astrocytes and fibronectin. It might thereby regulate ECM remodeling and possibly glial scarring.

The Proteome of the Locus Ceruleus in Parkinson's Disease: Relevance to Pathogenesis

The locus ceruleus is among the earliest affected brain regions in Parkinsons disease (PD) showing Lewy body pathology and neuronal loss. To improve our understanding of the pathogenesis of PD, we performed the first proteomic analysis ever of post‐mortem locus ceruleus tissue of six pathologically confirmed PD patients, and six age‐ and gender‐matched non‐neurological controls. In total 2495 proteins were identified, of which 87 proteins were differentially expressed in the locus ceruleus of PD patients compared with controls. The majority of these differentially expressed proteins are known to be involved in processes that have been implicated in the pathogenesis of PD previously, including mitochondrial dysfunction, oxidative stress, protein misfolding, cytoskeleton dysregulation and inflammation. Several individual proteins were identified that have hitherto not been associated with PD, such as regucalcin, which plays a role in maintaining intracellular calcium homeostasis, and isoform 1 of kinectin, which is involved in transport of cellular components along microtubules. In addition, pathway analysis suggests a pathogenetic role for aminoacyl‐tRNA‐biosynthesis. These findings indicate that the proteome of the locus ceruleus of PD patients and non‐neurological controls provides data that are relevant to the pathogenesis of PD, reflecting both known and potentially novel pathogenetic pathways.

Appearance of Tissue Transglutaminase in Astrocytes in Multiple Sclerosis Lesions: A Role in Cell Adhesion and Migration?

Multiple Sclerosis (MS) is a neuroinflammatory disease mainly affecting young adults. A major pathological hallmark of MS is the presence of demyelinated lesions in the central nervous system. In the active phase of the disease, astrocytes become activated, migrate and contribute to local tissue remodeling that ultimately can result in an astroglial scar. This process is facilitated by extracellular matrix proteins, including fibronectin. Tissue Transglutaminase (TG2) is a multifunctional enzyme with a ubiquitous tissue distribution and it has been shown that inflammatory cytokines can induce TG2 activity. In addition, TG2 is known to mediate cell adhesion and migration. We therefore hypothesized that TG2 is present in MS lesions and plays a role in cell adhesion and/or migration. Our studies showed that TG2 immunoreactivity appeared in astrocytes in active and chronic active MS lesions. These TG2 positive astrocytes partly co‐localized with fibronectin. Additional in vitro studies showed that TG2 mediated astrocytoma adhesion to and migration on the extracellular matrix protein fibronectin. We therefore speculate that TG2 mediates the enhanced interaction of astrocytes with fibronectin in the extracellular matrix of MS lesions, thereby contributing to astrocyte adhesion and migration, and thus in tissue remodeling and possibly glial scarring.

Transglutaminase 1 and its regulator tazarotene-induced gene 3 localize to neuronal tau inclusions in tauopathies

Alzheimers disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17), and Picks disease (PiD) are commonly known as tauopathies. Neurodegeneration observed in these diseases is linked to neuronal fibrillary hyperphosphorylated tau protein inclusions. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular cross‐links. Both transglutaminase 1 (TG1) and transglutaminase 2 (TG2) are abundantly expressed in the brain and are associated with fibrillary hyperphosphorylated tau protein inclusions in neurons of AD, so‐called neurofibrillary tangles (NFTs). However, other data obtained by our group suggested that tau pathology in the brain may be primarily related to TG1 and not to TG2 activity. To obtain more information on this issue, we set out to investigate the association of TG1, TG2, and TG‐catalysed cross‐links with fibrillary hyperphosphorylated tau inclusions in tauopathies other than AD, using immunohistochemistry. We found strong TG1 and TG‐catalysed cross‐link staining in neuronal tau inclusions characteristic of PSP, FTDP‐17 with mutations in the tau gene (FTDP‐17T), and PiD brain, whereas, in contrast to AD, TG2 was only rarely observed in these inclusions. Furthermore, using a biochemical approach, we demonstrated that tau is a substrate for TG1‐mediated cross‐linking. Interestingly, we found co‐localization of the TG1 activator, tazarotene‐induced gene 3 (TIG3), in the neuronal tau inclusions of PSP, FTDP‐17T, and PiD, but not in NFTs of AD cases, indicating that these tau‐containing protein aggregates are not identical. We conclude that TG1‐catalysed cross‐linking, regulated by TIG3, might play an important role in the formation of neuronal tau inclusions in PSP, FTDP‐17T, and PiD brain. Copyright

Discrepancy in CCL2 and CCR2 expression in white versus grey matter hippocampal lesions of Multiple Sclerosis patients

A remarkable pathological difference between grey matter lesions (GML) and white matter lesions (WML) in Multiple Sclerosis (MS) patients is the paucity of infiltrating leukocytes in GML. To better understand these pathological differences, we hypothesize that the chemokine monocyte chemotactic protein-1 (MCP-1 or CCL2), of importance for leukocyte migration, and its receptor CCR2 are more abundantly expressed in WML than in GML of MS patients. To this end, we analyzed CCL2 and CCR2 expression in the hippocampus, comprising WML and GML, of post-mortem MS patients, and of control subjects.CCL2 and CCR2 mRNA were significantly increased in demyelinated MS hippocampus. Semi-quantification of CCL2 and CCR2 immunoreactivity showed that CCL2 is present in astrocytes only in active WML. CCR2 is upregulated in monocytes/macrophages or amoeboid microglia in active WML, and in ramified microglia in active GML, although to a lesser extent. As a follow-up, we observed a significantly increased CCL2 production by WM-, but not GM-derived astrocytes upon stimulation with bz-ATP in vitro. Finally, upon CCL2 stimulation, GM-derived microglia significantly increased their proliferation rate.We conclude that within hippocampal lesions, CCL2 expression is mainly restricted to WML, whereas the receptor CCR2 is upregulated in both WML and GML. The relative absence of CCL2 in GML may explain the lack of infiltrating immune cells in this type of lesions. We propose that the divergent expression of CCL2 and CCR2 in WML and GML explains or contributes to the differences in WML and GML formation in MS.

Tissue Transglutaminase Activity Is Involved in the Differentiation of Oligodendrocyte Precursor Cells into Myelin-Forming Oligodendrocytes During CNS Remyelination

During normal brain development, axons are myelinated by mature oligodendrocytes (OLGs). Under pathological, demyelinating conditions within the central nervous system (CNS), axonal remyelination is only partially successful because oligodendrocyte precursor cells (OPCs) largely remain in an undifferentiated state resulting in a failure to generate myelinating OLGs. Tissue Transglutaminase (TG2) is a multifunctional enzyme, which amongst other functions, is involved in cell differentiation. Therefore, we hypothesized that TG2 contributes to differentiation of OPCs into OLGs and thereby stimulates remyelination. In vivo studies, using the cuprizone model for de‐ and remyelination in TG2−/− and wild‐type mice, showed that during remyelination expression of proteolipid protein mRNA, as a marker for remyelination, in the corpus callosum lags behind in TG2−/− mice resulting in less myelin formation and, moreover, impaired recovery of motor behavior. Subsequent in vitro studies showed that rat OPCs express TG2 protein and activity which reduces when the cells have matured into OLGs. Furthermore, when TG2 activity is pharmacologically inhibited, the differentiation of OPCs into myelin‐forming OLGs is dramatically reduced. We conclude that TG2 plays a prominent role in remyelination of the CNS, probably through stimulating OPC differentiation into myelin‐forming OLGs. Therefore, manipulating TG2 activity may represent an interesting new target for remyelination in demyelinating diseases.

Validated sandwich ELISA for the quantification of tissue transglutaminase in tissue homogenates and cell lysates of multiple species

Tissue transglutaminase (tTG) is a calcium dependent enzyme that displays diverse functions in various physiological processes. In addition to these physiological functions, there is strong evidence for the implication of tTG in a number of pathologies, including celiac disease, cancer and neurodegeneration. To explore the expression and function of tTG during (patho)physiological conditions, it is of utmost importance to have an assay that specifically measures tTG protein levels in various species and matrices. Therefore, we have developed a sensitive sandwich ELISA to measure tTG protein levels in tissue homogenates and cell lysates of human, rat and mouse origin. The ELISA uses commercially available antibodies, and human recombinant tTG as the standard protein. The limit of detection is 100 pg/ml; the coefficients of intra- and inter-assay variation range from 2.4% to 6.6% and from 12.7% to 15.1%, respectively. Clear detectable levels of tTG protein were measured in human and rat liver and cerebral cortex, as well as in brain-derived neuronal and glial cells. tTG levels in mouse tissues were much lower than observed in human and rat tissues. No cross-reactivity against keratinocyte TG (TG1), epidermal TG (TG3) or blood coagulation factor XIII was observed. The tTG specific sandwich ELISA presented in this paper is a sensitive and reliable tool to accurately measure tTG protein levels in different matrices (cell/tissue) of rat, mouse and human origin. It provides a better alternative for the widely used transglutaminase activity assay with respect to sensitivity and specificity, and may serve as a valuable tool to investigate protein expression levels as part of the approach to unravel the contribution of tTG to health and disease.

Tissue Transglutaminase contributes to experimental multiple sclerosis pathogenesis and clinical outcome by promoting macrophage migration

Multiple sclerosis is a serious neurological disorder, resulting in e.g., sensory, motor and cognitive deficits. A critical pathological aspect of multiple sclerosis (MS) is the influx of immunomodulatory cells into the central nervous system (CNS). Identification of key players that regulate cellular trafficking into the CNS may lead to the development of more selective treatment to halt this process. The multifunctional enzyme tissue Transglutaminase (TG2) can participate in various inflammation-related processes, and is known to be expressed in the CNS. In the present study, we question whether TG2 activity contributes to the pathogenesis of experimental MS, and could be a novel therapeutic target. In human post-mortem material, we showed the appearance of TG2 immunoreactivity in leukocytes in MS lesions, and particular in macrophages in rat chronic-relapsing experimental autoimmune encephalomyelitis (cr-EAE), an experimental MS model. Clinical deficits as observed in mouse EAE were reduced in TG2 knock-out mice compared to littermate wild-type mice, supporting a role of TG2 in EAE pathogenesis. To establish if the enzyme TG2 represents an attractive therapeutic target, cr-EAE rats were treated with TG2 activity inhibitors during ongoing disease. Reduction of TG2 activity in cr-EAE animals dramatically attenuated clinical deficits and demyelination. The mechanism underlying these beneficial effects pointed toward a reduction in macrophage migration into the CNS due to attenuated cytoskeletal flexibility and RhoA GTPase activity. Moreover, iNOS and TNFα levels were selectively reduced in the CNS of cr-EAE rats treated with a TG2 activity inhibitor, whereas other relevant inflammatory mediators were not affected in CNS or spleen by reducing TG2 activity. We conclude that modulating TG2 activity opens new avenues for therapeutic intervention in MS which does not affect peripheral levels of inflammatory mediators.