Akihiro Matsumura

3‐[(2,4‐dimethoxy)benzylidene]‐anabaseine dihydrochloride protects against 6‐hydroxydopamine‐induced parkinsonian neurodegeneration through α7 nicotinic acetylcholine receptor stimulation in rats

To explore a novel therapy against Parkinsons disease through enhancement of α7 nicotinic acetylcholine receptor (nAChR), we evaluated the neuroprotective effects of 3‐[(2,4‐dimethoxy)benzylidene]‐anabaseine dihydrochloride (DMXBA; GTS‐21), a functionally selective α7 nAChR agonist, in a rat 6‐hydroxydopamine (6‐OHDA)‐induced hemiparkinsonian model. Microinjection of 6‐OHDA into the nigrostriatal pathway of rats destroys dopaminergic neurons selectively. DMXBA dose dependently inhibited methamphetamine‐stimulated rotational behavior and dopaminergic neuronal loss induced by 6‐OHDA. The protective effects were abolished by methyllycaconitine citrate salt hydrate, an α7 nAChR antagonist. Immunohistochemical study confirmed abundant α7 nAChR expression in the cytoplasm of dopaminergic neurons. These results indicate that DMXBA prevented 6‐OHDA‐induced dopaminergic neuronal loss through stimulating α7 nAChR in dopaminergic neurons. Injection of 6‐OHDA elevated immunoreactivities to glial markers such as ionized calcium binding adaptor molecule 1, CD68, and glial fibrillary acidic protein in the substantia nigra pars compacta of rats. In contrast, these immunoreactivities were markedly inhibited by comicroinjection of DMXBA. Microglia also expressed α7 nAChR in both resting and activated states. Hence, we hypothesize that DMXBA simultaneously affects microglia and dopaminergic neurons and that both actions lead to dopaminergic neuroprotection. The findings that DMXBA attenuates 6‐OHDA‐induced dopaminergic neurodegeneration and glial activation in a rat model of Parkinsons disease raisethe possibility that DMXBA could be a novel therapeutic compound to prevent Parkinsons disease development.

Evaluation of oxidative stress in the brain of a transgenic mouse model of Alzheimer disease by in vivo electron paramagnetic resonance imaging

Alzheimer disease (AD) is a neurodegenerative disease clinically characterized by progressive cognitive dysfunction. Deposition of amyloid-β (Aβ) peptides is the most important pathophysiological hallmark of AD. Oxidative stress induced by reactive oxygen species is prominent in AD, and several reports suggest the relationship between a change in redox status and AD pathology containing progressive Aβ deposition, the activation of glial cells, and mitochondrial dysfunction. Therefore, we performed immunohistochemical analysis using a transgenic mouse model of AD (APdE9) and evaluated the activity of superoxide dismutase in brain tissue homogenates of APdE9 mice in vitro. Together with those analyses, in vivo changes in redox status with age in both wild-type (WT) and APdE9 mouse brains were measured noninvasively by three-dimensional electron paramagnetic resonance (EPR) imaging using nitroxide (3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy) as a redox-sensitive probe. Both methods found similar changes in redox status with age, and in particular a significant change in redox status in the hippocampus was observed noninvasively by EPR imaging between APdE9 mice and age-matched WT mice from 9 to 18 months of age. EPR imaging clearly visualized the accelerated change in redox status of APdE9 mouse brain compared with WT. The evaluation of the redox status in the brain of AD model rodents by EPR imaging should be useful for diagnostic study of AD.

Intravenous mesenchymal stem cell administration exhibits therapeutic effects against 6-hydroxydopamine-induced dopaminergic neurodegeneration and glial activation in rats.

To explore a novel therapy against Parkinsons disease (PD), we evaluated the therapeutic effects of human bone marrow-derived mesenchymal stem cells (hBM-MSCs), pluripotent stromal cells with secretory potential of various neurotrophic and anti-inflammatory factors, in a hemi-parkinsonian rat model. The unilateral intrastriatal 6-hydroxydopamine (6-OHDA)-lesioned rats were injected hBM-MSCs (1.0 × 10(7)cells) or PBS intravenously 16 days after lesioning. Administration of hBM-MSCs inhibited methamphetamine-stimulated rotational behavior at 7, 14, 21 and 28 days after transplantation. Immunohistochemical analysis also showed that number of TH-positive neurons in the substantia nigra pars compacta was significantly preserved in hBM-MSCs-transplanted rats compared to sham-operated rats, whereas the immunoreactivity of ionized calcium binding adaptor molecule 1 was markedly inhibited. In this study, we demonstrated the therapeutic effects of intravenous hBM-MSCs administration in parkinsonian model rats presenting distinct parkinsonian phenotype at 16 days after 6-OHDA lesioning. The favorable findings raise the possibility that hBM-MSCs could be a novel therapeutic option to promote survival of dopaminergic neurons in PD.

Oxidation and interaction of DJ-1 with 20S proteasome in the erythrocytes of early stage Parkinson’s disease patients

Parkinson’s disease (PD) is a progressive, age-related, neurodegenerative disorder, and oxidative stress is an important mediator in its pathogenesis. DJ-1, the product of the causative gene of a familial form of PD, plays a significant role in anti-oxidative defence to protect cells from oxidative stress. DJ-1 undergoes preferential oxidation at the cysteine residue at position 106 (Cys-106) under oxidative stress. Here, using specific antibodies against Cys-106-oxidized DJ-1 (oxDJ-1), it was found that the levels of oxDJ-1 in the erythrocytes of unmedicated PD patients (n = 88) were higher than in those of medicated PD patients (n = 62) and healthy control subjects (n = 33). Elevated oxDJ-1 levels were also observed in a non-human primate PD model. Biochemical analysis of oxDJ-1 in erythrocyte lysates showed that oxDJ-1 formed dimer and polymer forms, and that the latter interacts with 20S proteasome. These results clearly indicate a biochemical alteration in the blood of PD patients, which could be utilized as an early diagnosis marker for PD.

Role of Suppressor of Cytokine Signaling 3 (SOCS3) in Altering Activated Microglia Phenotype in APPswe/PS1dE9 Mice

In response to changes of the central nervous system environment, microglia are capable of acquiring diverse phenotypes for cytotoxic or immune regulation and resolution of injury. Alzheimers disease (AD) pathology also induces several microglial activations, resulting in production of pro-inflammatory cytokines and reactive oxygen species or clearance of amyloid-β (Aβ) through phagocytosis. We previously demonstrated that microglial activation and increase in oxidative stress started from the middle age in APPswe/PS1dE9 mice, and hypothesized that M1 activation occurs in middle-aged AD mice by Aβ stimulation. In the present study, we analyzed in vivo expressions of pro-inflammatory cytokines (M1 microglial markers), M2 microglial markers, and suppressor of cytokine signaling (SOCS) family, and examined the microglial phenotypic profile in APPswe/PS1dE9 mice. Then we compared the in vitro gene expression patterns of Aβ- and lipopolysaccharide (LPS)-stimulated primary-cultured microglia. Microglia in APPswe/PS1dE9 mice exhibited an M1-like phenotype, expressing tumor necrosis factor α (TNFα) but not interleukin 6 (IL6). Aβ-stimulated primary-cultured microglia also expressed TNFα but not IL6, whereas LPS-stimulated primary-cultured microglia expressed both pro-inflammatory cytokines. Furthermore, both microglia in APPswe/PS1dE9 mice and Aβ-stimulated primary-cultured microglia expressed SOCS3. Reduction of SOCS3 expression in Aβ-challenged primary-cultured microglia resulted in upregulation of IL6 expression. Our findings indicate that SOCS3 suppresses complete polarization to M1 phenotype through blocking IL6 production, and Aβ-challenged primary-cultured microglia replicate the in vivo gene expression pattern of microglia in APPswe/PS1dE9 mice. Aβ may induce the M1-like phenotype through blocking of IL6 by SOCS3.

Temporal Changes of CD68 and α7 Nicotinic Acetylcholine Receptor Expression in Microglia in Alzheimer's Disease-Like Mouse Models

We previously reported that activated microglia are involved in amyloid-β (Aβ) clearance and that stimulation of α7 nicotinic acetylcholine receptors (nAChR) in microglia enhances Aβ clearance. Nevertheless, how microglia and α7 nAChR in microglia are affected in Alzheimers disease (AD) remains unknown. The present study aimed to collect fundamental data for considering whether microglia are potential targets for AD treatment and the appropriate timing of therapeutic intervention, by evaluating the temporal changes of Aβ, microglia, neurons, presynapses, and α7 nAChR by immunohistochemical studies in mouse models of AD. In an Aβ-injected AD mouse model, we observed early accumulation of CD68-positive microglia at Aβ deposition sites and gradual reduction of Aβ. Microglia were closely associated with Aβ deposits, and were confirmed to participate in clearing Aβ. In a transgenic mouse model of AD, we observed an increase in Aβ deposition from 6 months of age, followed by a gradual increase in microglial accumulation at Aβ deposit sites. Activated microglia in APdE9 mice showed two-step transition: a CD68-negative activated form at 6-9 months and a CD68-positive form from 12 months of age. In addition, α7 nAChR in microglia increased markedly at 6 months of age when activated microglia appeared for the first time, and decreased gradually coinciding with the increase of Aβ deposition. These findings suggest that early microglial activation is associated with α7 nAChR upregulation in microglia in APdE9 mice. These novel findings are important for the development of new therapeutic strategy for AD.