John G.J.M. Bol

Expression of NAD(P)H:quinone oxidoreductase in the normal and Parkinsonian substantia nigra

Dopamine (DA) autooxidation, and consequent formation of neurotoxic DA-derived quinones and reactive oxygen species, has been implicated in dopaminergic cell death and, hence, in the pathogenesis of Parkinsons disease (PD). Stimulation of pathways involved in the detoxication of DA-quinones in the brain is hypothesized to be an effective means to limit oxidative stress and to confer neuroprotection in PD. In this respect, the inducible flavoprotein NAD(P)H:quinone oxidoreductase (NQO1) is of particular interest as it is directly implicated in the detoxication of DA-quinones and, in addition, has broad spectrum anti-oxidant properties. To study the potential pathophysiological role of NQO1 in PD, the cellular expression of NQO1 was examined in the mesencephalon of PD patients and age-matched controls. In the substantia nigra pars compacta (SNpc), NQO1 was found to be expressed in astroglial and endothelial cells and, albeit less frequently, also in dopaminergic neurons. Moreover, while overt NQO1 immunoreactivity was absent in the surrounding nervous tissue, in the Parkinsonian SNpc a marked increase in the astroglial and neuronal expression of NQO1 was consistently observed.

Transglutaminases and Transglutaminase-Catalyzed Cross-Links Colocalize with the Pathological Lesions in Alzheimer's Disease Brain

Alzheimers disease (AD) is characterized by pathological lesions, in particular senile plaques (SPs), cerebral amyloid angiopathy (CAA) and neurofibrillary tangles (NFTs), predominantly consisting of self‐aggregated proteins amyloid beta (Aβ) and tau, respectively. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular covalent cross‐links. Both Aβ and tau are substrates for TG cross‐linking activity, which links TGs to the aggregation process of both proteins in AD brain. The aim of this study was to investigate the association of transglutaminase 1 (TG1), transglutaminase 2 (TG2) and TG‐catalyzed cross‐links with the pathological lesions of AD using immunohistochemistry. We observed immunoreactivity for TG1, TG2 and TG‐catalyzed cross‐links in NFTs. In addition, both TG2 and TG‐catalyzed cross‐links colocalized with Aβ in SPs. Furthermore, both TG2 and TG‐catalyzed cross‐links were associated with CAA. We conclude that these TGs demonstrate cross‐linking activity in AD lesions, which suggests that both TG1 and TG2 are likely involved in the protein aggregation processes underlying the formation of SPs, CAA and/or NFTs in AD brain.

Pinhole SPECT imaging of dopamine transporters correlates with dopamine transporter immunohistochemical analysis in the MPTP mouse model of Parkinson's disease.

The in vivo analysis of dopaminergic degeneration in animal models of Parkinsons disease (PD), using pinhole single photon emission computed tomography (SPECT), ideally should afford a serial study design, enabling the analysis of the degenerative process as well as the potential neuroprotective and/or restorative properties of drugs over time in living animals. Previously, we demonstrated that striatal dopamine transporter (DAT) levels in rats could be analyzed reproducibly, using pinhole SPECT with the DAT probe [(123)I]N-omega-fluoropropyl-2beta-carbomethoxy-3beta-{4-iodophenyl}nortropane (FP-CIT). However, the capacity of this approach to accurately detect a range of striatal DAT levels in the most widely used animal model of PD, i.e., the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse, remains to be determined. For this purpose, various levels of DAT were induced by treating c57BL/6J mice for 1, 3, or 5 days with MPTP (25 mg/kg ip), respectively. [(123)I]FP-CIT SPECT scans were performed 5 days after the last MPTP injection. Mice were perfused 6 days after the last MPTP injection, and the SPECT data were compared to ex vivo striatal and nigral DAT levels as measured by immunohistochemistry within the same animals. The analysis of striatal DAT levels using SPECT and DAT immunohistochemistry yielded highly comparable results on the percentage of DAT reduction in each MPTP group. The in vivo data showed a decrease of specific striatal to non-specific binding ratios by 59%, 82%, and 76% in mice treated for 1, 3, and 5 days, respectively. Moreover, a strong, positive correlation was observed between the in vivo and ex vivo parameters. The present study provides the first evidence that [(123)I]FP-CIT pinhole SPECT allows the accurate detection of a range of striatal DAT (i.e., losses of approximately 60-80%) levels in mice. Since such large dopaminergic lesions could be detected, this SPECT method may at least be useful for analyzing neuroprotective treatment with a clear-cut positive (i.e., complete protection) or negative (i.e., not any protection) effect. Whether this method is also useful for analyzing more subtle effects of neuroprotective treatment (partial protection) remains to be established, by studying mice with small dopaminergic lesions.

Presence of tissue transglutaminase in granular endoplasmic reticulum is characteristic of melanized neurons in Parkinson's disease brain.

Parkinsons disease (PD) is characterized by the accumulation of α‐synuclein aggregates and degeneration of melanized neurons. The tissue transglutaminase (tTG) enzyme catalyzes molecular protein cross‐linking. In PD brain, tTG‐induced cross‐links have been identified in α‐synuclein monomers, oligomers and α‐synuclein aggregates. However, whether tTG and α‐synuclein occur together in PD affected neurons remains to be established. Interestingly, using immunohistochemistry, we observed a granular distribution pattern of tTG, characteristic of melanized neurons in PD brain. Apart from tTG, these granules were also positive for typical endoplasmic reticulum (ER)‐resident chaperones, that is, protein disulphide isomerase, ERp57 and calreticulin, suggesting a direct link to the ER. Additionally, we observed the presence of phosphorylated pancreatic ER kinase (pPERK), a classical ER stress marker, in tTG granule positive neurons in PD brain, although no subcellular colocalization of tTG and pPERK was found. Our data therefore suggest that tTG localization to granular ER compartments is specific for stressed melanized neurons in PD brain. Moreover, as also α‐synuclein aggregates were observed in tTG granule positive neurons, these results provide a clue to the cellular site of interaction between α‐synuclein and tTG.

Regional and temporal expression patterns of interleukin-10, interleukin-10 receptor and adhesion molecules in the rat spinal cord during chronic relapsing EAE

Adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mediate leukocyte infiltration into the CNS, in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). Because exogenous interleukin-10 (IL-10) inhibits ICAM-1 and VCAM-1 expression and clinical EAE, we hypothesize that endogenous IL-10 signaling may suppress expression of adhesion molecules. In a rat model of chronic relapsing EAE, expression levels of IL-10 and its receptor (IL-10R1), ICAM-1 and VCAM-1 mRNA in the spinal cord are markedly increased, whereas levels of IL-10 mRNA remain relatively low. The temporal pattern of mRNA and protein expression showed marked differences between spinal cord levels. During relapse, IL-10, IL-10R1, ICAM-1, VCAM-1 mRNA levels and neurological scores show positive correlations. We conclude that endogenous IL-10 is not a crucial factor inhibiting adhesion molecule expression in this model.

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.

Parkinson's disease-associated parkin colocalizes with Alzheimer's disease and multiple sclerosis brain lesions

Parkin is implicated in the pathogenesis of Parkinsons disease. Furthermore, parkin targets misfolded proteins for degradation and protects cells against various forms of cellular stress, including unfolded-protein and oxidative stress. This points towards a protective role of parkin in neurological disorders in which these stressors are implicated, including Alzheimers disease (AD) and multiple sclerosis (MS). Here, we assessed parkin distribution in AD and MS brain tissue using immunohistochemistry. In AD brains, parkin colocalized with classic senile plaques and amyloid-laden vessels as well as astrocytes associated with both lesions. Similarly, we observed enhanced astrocytic parkin immunoreactivity in MS lesions, particularly in inflammatory lesions. Furthermore, parkin mRNA expression was increased in an astrocytoma cell line after free radical exposure. Our data indicate that parkin is upregulated in AD and MS brain tissue and might represent a defense mechanism to counteract stress-induced damage in AD and MS pathogenesis.

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.

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.

Increased amoeboid microglial density in the olfactory bulb of Parkinson's and Alzheimer's patients.

The olfactory bulb (OB) is affected early in both Parkinsons (PD) and Alzheimers disease (AD), evidenced by the presence of disease‐specific protein aggregates and an early loss of olfaction. Whereas previous studies showed amoeboid microglia in the classically affected brain regions of PD and AD patients, little was known about such changes in the OB. Using a morphometric approach, a significant increase in amoeboid microglia density within the anterior olfactory nucleus (AON) of AD and PD patients was observed. These amoeboid microglia cells were in close apposition to β‐amyloid, hyperphosphorylated tau or α‐synuclein deposits, but no uptake of pathological proteins by microglia could be visualized. Subsequent analysis showed (i) no correlation between microglia and α‐synuclein (PD), (ii) a positive correlation with β‐amyloid (AD), and (iii) a negative correlation with hyperphosphorylated tau (AD). Furthermore, despite the observed pathological alterations in neurite morphology, neuronal loss was not apparent in the AON of both patient groups. Thus, we hypothesize that, in contrast to the classically affected brain regions of AD and PD patients, within the AON rather than neuronal loss, the increased density in amoeboid microglial cells, possibly in combination with neurite pathology, may contribute to functional deficits.