Dan Sunnemark

Pivotal Advance: HMGB1 expression in active lesions of human and experimental multiple sclerosis

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the CNS, most frequently starting with a series of bouts, each followed by complete remission and then a secondary, progressive phase during which the neurological deficit increases steadily. The underlying molecular mechanisms responsible for disease progression are still unclear. Herein, we demonstrate that high mobility group box chromosomal protein 1 (HMGB1), a DNA‐binding protein with proinflammatory properties, is evident in active lesions of MS and experimental autoimmune encephalomyelitis (EAE) and that HMGB1 levels correlate with active inflammation. Furthermore, the expression of the innate HMGB1 receptors—receptor for advanced glycation end products, TLR2, and TLR4—was also highly increased in MS and rodent EAE. Additionally, in vitro activation of rodent CNS‐derived microglia and bone marrow‐derived macrophages demonstrated that microglia were equally as capable as macrophages of translocating HMGB1 following LPS/IFN‐γ stimulation. Significant expression of HMGB1 and its receptors on accumulating activated macrophages and resident microglia may thus provide a positive feedback loop that amplifies the inflammatory response during MS and EAE pathogenesis.

Characterization of AZD4694, a novel fluorinated Abeta plaque neuroimaging PET radioligand

J. Neurochem. (2010) 114, 784–794.

CX3CL1 (fractalkine) and CX3CR1 expression in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis: kinetics and cellular origin

BackgroundMultiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). It is associated with local activation of microglia and astroglia, infiltration of activated macrophages and T cells, active degradation of myelin and damage to axons and neurons. The proposed role for CX3CL1 (fractalkine) in the control of microglia activation and leukocyte infiltration places this chemokine and its receptor CX3CR1 in a potentially strategic position to control key aspects in the pathological events that are associated with development of brain lesions in MS. In this study, we examine this hypothesis by analyzing the distribution, kinetics, regulation and cellular origin of CX3CL1 and CX3CR1 mRNA expression in the CNS of rats with an experimentally induced MS-like disease, myelin oligodendrocyte glycoprotein (MOG)-induced autoimmune encephalomyelitis (EAE).MethodsThe expression of CX3CL1 and its receptor CX3CR1 was studied with in situ hybridization histochemical detection of their mRNA with radio labeled cRNA probes in combination with immunohistochemical staining of phenotypic cell markers. Both healthy rat brains and brains from rats with MOG EAE were analyzed. In defined lesional stages of MOG EAE, the number of CX3CR1 mRNA-expressing cells and the intensity of the in situ hybridization signal were determined by image analysis. Data were statistically evaluated by ANOVA, followed by Tukey\primes multiple comparison test.ResultsExpression of CX3CL1 mRNA was present within neuronal-like cells located throughout the neuraxis of the healthy rat. Expression of CX3CL1 remained unaltered in the CNS of rats with MOG-induced EAE, with the exception of an induced expression in astrocytes within inflammatory lesions. Notably, the brain vasculature of healthy and encephalitic animals did not exhibit signs of CX3CL1 mRNA expression. The receptor, CX3CR1, was expressed by microglial cells in all regions of the healthy brain. Induction of MOG-induced EAE was associated with a distinct accumulation of CX3CR1 mRNA expressing cells within the inflammatory brain lesions, the great majority of which stained positive for markers of the microglia-macrophage lineage. Analysis in time-staged brain lesions revealed elevated levels of CX3CR1 mRNA in microglia in the periplaque zone, as well as a dramatically enhanced accumulation of CX3CR1 expressing cells within the early-active, late-active and inactive, demyelinated lesions.ConclusionOur data demonstrate constitutive and regulated expression of the chemokine CX3CL1 and its receptor CX3CR1 by neurons/astrocytes and microglia, respectively, within the normal and inflamed rat brain. Our findings propose a mechanism by which neurons and reactive astrocytes may control migration and function of the surrounding microglia. In addition, the accumulation of CX3CR1 expressing cells other than microglia within the inflammatory brain lesions indicate a possible role for CX3CL1 in controlling invasion of peripheral leucocytes to the brain.

Age related changes in brain metabolites observed by 1H MRS in APP/PS1 mice.

Translational biomarkers in Alzheimers disease based on non-invasive in vivo methods are highly warranted. (1)H magnetic resonance spectroscopy (MRS) is non-invasive and applicable in vivo in both humans and experimental animals. In vivo(1)H MRS and 3D MRI were performed on brains of double transgenic (tg) mice expressing a double mutant human beta-amyloid precursor protein APP(K670N,M671L) and human mutated presenilin gene PS1M146L, and wild-type (wt) littermates at 2.5, 6.5 and 9 months of age using a 9.4T magnet. For quantification, LCModel was used, and the data were analyzed using multivariate data analysis (MVDA). MVDA evidenced a significant separation, which became more pronounced with age, between tg and wt mice at all time points. While myo-inositol and guanidoacetate were important for group separation in young mice, N-acetylaspartate, glutamate and macrolipids were important for separation of aged tg and wt mice. Volume segmentation revealed that brain and hippocampus were readily smaller in tg as compared to wt mice at the age of 2.5 months. Amyloid plaques were seen in 6.5 and 9 months, but not in 2.5 months old animals. In conclusion, differences in brain metabolites could be accurately depicted in tg and wt mice in vivo by combining MRS with MVDA. First differences in metabolite content were readily seen at 2.5 months, when volume defects in tg mice were present, but no amyloid plaques.

Temporal expression and cellular origin of CC chemokine receptors CCR1, CCR2 and CCR5 in the central nervous system: insight into mechanisms of MOG-induced EAE

BackgroundThe CC chemokine receptors CCR1, CCR2 and CCR5 are critical for the recruitment of mononuclear phagocytes to the central nervous system (CNS) in multiple sclerosis (MS) and other neuroinflammatory diseases. Mononuclear phagocytes are effector cells capable of phagocytosing myelin and damaging axons. In this study, we characterize the regional, temporal and cellular expression of CCR1, CCR2 and CCR5 mRNA in the spinal cord of rats with myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE). While resembling human MS, this animal model allows unique access to CNS-tissue from various time-points of relapsing neuroinflammation and from various lesional stages: early active, late active, and inactive completely demyelinated lesions.MethodsThe expression of CCR1, CCR2 and CCR5 mRNA was studied with in situ hybridization using radio labelled cRNA probes in combination with immunohistochemical staining for phenotypic cell markers. Spinal cord sections from healthy rats and rats with MOG-EAE (acute phase, remission phase, relapse phase) were analysed. In defined lesion stages, the number of cells expressing CCR1, CCR2 and CCR5 mRNA was determined. Data were statistically analysed by the nonparametric Mann-Whitney U test.ResultsIn MOG-EAE rats, extensive up-regulation of CCR1 and CCR5 mRNA, and moderate up-regulation of CCR2 mRNA, was found in the spinal cord during episodes of active inflammation and demyelination. Double staining with phenotypic cell markers identified the chemokine receptor mRNA-expressing cells as macrophages/microglia. Expression of all three receptors was substantially reduced during clinical remission, coinciding with diminished inflammation and demyelination in the spinal cord. Healthy control rats did not show any detectable expression of CCR1, CCR2 or CCR5 mRNA in the spinal cord.ConclusionOur results demonstrate that the acute and chronic-relapsing phases of MOG-EAE are associated with distinct expression of CCR1, CCR2, and CCR5 mRNA by cells of the macrophage/microglia lineage within the CNS lesions. These data support the notion that CCR1, CCR2 and CCR5 mediate recruitment of both infiltrating macrophages and resident microglia to sites of CNS inflammation. Detailed knowledge of expression patterns is crucial for the understanding of therapeutic modulation and the validation of CCR1, CCR2 and CCR5 as feasible targets for therapeutic intervention in MS.

Differential Expression of the Chemokine Receptors CX3CR1 and CCR1 by Microglia and Macrophages in Myelin-Oligodendrocyte-Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis

Chemokines are important for the recruitment of immune cells into sites of inflammation. To better understand their functional roles during inflammation we have here studied the in vivo expression of receptors for the chemokines CCL3/CCL5/CCL7 (MIP‐1α/RANTES/MCP‐3) and CX3CL1 (fractalkine), CCR1 and CX3CR1, respectively, in rat myelin oligodendrocyte glycoprotein‐induced experimental autoimmune encephalomyelitis. Combined in situ hybridization and immunohistochemistry demonstrated intensely upregulated CCR1 mRN A expression in early, actively demyelinating plaques, whereas CX3CR1 displayed a more generalized expression pattern. CX3CR1 mRNA expressing cells were identified as microglia on the basis of their cellular morphology and positive GSA/B4 lectin staining. In contrast, CCR1 mRNA was preferentially expressed by ED1+GSA/B4+ macrophages. The notion of differential chemokine receptor expression in microglia and monocyte‐derived macrophages was corroborated at the protein level by extraction and flow cytometric sorting of cells infiltrating the spinal cord using gating for the surface markers CD45, ED‐2 and CD11b. These observations suggest a differential receptor expression between microglia and monocyte‐derived macrophages and that mainly the latter cell type is responsible for active demyelination. This has great relevance for the possibility of therapeutic intervention in demyelinating diseases such as multiple sclerosis, for example by targeting signaling events leading to monocyte recruitment.

Effector stage CC chemokine receptor-1 selective antagonism reduces multiple sclerosis-like rat disease

We have studied the role of the chemokine receptor CCR1 during the effector stage of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in DA rats. In situ hybridization histochemistry revealed local production of the CCR1 ligands CCL3 (MIP-1 alpha) and CCL5 (RANTES), as well as large numbers of CCR1 and CCR5 expressing cells within inflammatory brain lesions. A low-molecular weight CCR1 selective antagonist potently abrogated both clinical and histopathological disease signs during a 5-day treatment period, without signs of peripheral immune compromise. Thus, we demonstrate therapeutic targeting of CCR1-dependent leukocyte recruitment to the central nervous system in a multiple sclerosis (MS)-like rat model.

Pharmacological inhibition of the chemokine receptor CX3CR1 attenuates disease in a chronic-relapsing rat model for multiple sclerosis

Significance Multiple sclerosis (MS) is a chronic inflammatory and degenerative disease of the central nervous system (CNS) causing paralysis. The most effective treatments for MS aim to block infiltration of inflammatory cells to the brain. However, severe side effects related to the broad-acting specificity of these treatments exist. AZD8797, a unique inhibitor of the chemokine receptor CX3CR1, provides inhibition of subpopulations of peripheral leukocytes with potential for a beneficial effect: side effect ratio. We provide evidence that blocking this receptor results in reduced paralysis, inflammation, and degeneration in the CNS in a disease model for MS. Furthermore, CX3CR1 expression analysis in the MS brain strengthens the evidence for CX3CR1 as a new target for the treatment of MS. One hallmark of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is infiltration of leukocytes into the CNS, where chemokines and their receptors play a major mediatory role. CX3CR1 is a chemokine receptor involved in leukocyte adhesion and migration and hence a mediator of immune defense reactions. The role of CX3CR1 in MS and EAE pathogenesis however remains to be fully assessed. Here, we demonstrate CX3CR1 mRNA expression on inflammatory cells within active plaque areas in MS brain autopsies. To test whether blocking CNS infiltration of peripheral leukocytes expressing CX3CR1 would be a suitable treatment strategy for MS, we developed a selective, high-affinity inhibitor of CX3CR1 (AZD8797). The compound is active outside the CNS and AZD8797 treatment in Dark Agouti rats with myelin oligodendrocyte glycoprotein-induced EAE resulted in reduced paralysis, CNS pathology, and incidence of relapses. The compound is effective when starting treatment before onset, as well as after the acute phase. This treatment strategy is mechanistically similar to, but more restricted than, current very late antigen-4–directed approaches that have significant side effects. We suggest that blocking CX3CR1 on leukocytes outside the CNS could be an alternative approach to treat MS.

Elevated MARK2-Dependent Phosphorylation of Tau in Alzheimer's Disease

The appearance of neurofibrillary tangles (NFT), one of the major hallmarks of Alzheimers disease (AD), is most likely caused by inappropriate phosphorylation and/or dephosphorylation of tau, eventually leading to the accumulation of NFTs. Enhanced phosphorylation of tau on Ser(262) is detected early in the course of the disease and may have a role in the formation of tangles. Several kinases such as microtubule-affinity regulating kinase (MARK), protein kinase A, calcium calmodulin kinase II, and checkpoint kinase 2 are known to phosphorylate tau on Ser(262) in vitro. In this study, we took advantage of the in situ proximity ligation assay to investigate the role of MARK2, one of the four MARK isoforms, in AD. We demonstrate that MARK2 interacts with tau and phosphorylates tau at Ser(262) in stably transfected NIH/3T3 cells expressing human recombinant tau. Staurosporine, a protein kinase inhibitor, significantly reduced the interaction between MARK2 and tau, and also phosphorylation of tau at Ser(262). Furthermore, we observed elevated interactions between MARK2 and tau in post-mortem human AD brains, compared to samples from non-demented elderly controls. Our results from transfected cells demonstrate a specific interaction between MARK2 and tau, as well as MARK2-dependent phosphorylation of tau at Ser(262). Furthermore, the elevated interactions between MARK2 and tau in AD brain sections suggests that MARK2 may play an important role in early phosphorylation of tau in AD, possibly qualifying as a therapeutic target for intervention to prevent disease progression.

Induction of early atherosclerosis in CBA/J mice by combination of Trypanosoma cruzi infection and a high cholesterol diet

In addition to established factors such as hyperlipidemia, smoking and hypertension, inflammation and infection have recently been implicated as major risk factors for atherosclerotic disease. Proatherogenic effects induced by infection may be related to both systemic inflammation and to direct effects on the vascular wall. We report here that a high fat diet combined with a protozoal infection with known tropism to the heart induced early atherosclerosis and intimal inflammatory infiltrates (CD4+, CD8+ cells and macrophages) in aortas of all (n = 7) CBA/J mice investigated. These mice are normally quite resistant to atherosclerotic development and in the control group (n = 7) receiving only a fatty diet, only one mouse presented a lesion. This lesion was completely devoid of infiltrating CD8+ cells. Parasite-infected mice receiving a normal diet exhibited vasculitis, but no signs of atherosclerosis and control mice receiving normal diet, as expected, exhibited neither signs of vasculitis nor atherosclerosis. Secretion of IL-6, TNF-alpha, and IFN-gamma were demonstrated in all atherosclerotic lesions and IL-6 appeared to be the dominant cytokine, both in the lesions themselves as well as in the intimal-medial junction. There were no traces of parasites present in the artery wall, indicating that atherosclerosis was induced via an indirect route. We conclude that a high fat diet in conjunction with infection and systemic (or localized) inflammation may have a strong proatherogenic effect. Finally, we suggest that CBA/J mice infected with T. cruzi parasites and given a fatty diet could serve as a useful experimental model in the continued analysis of factors contributing to the induction of atherosclerosis.