**6. Toxic change in microglia**

338 Etiology and Pathophysiology of Parkinson's Disease

LPS-activated neonatal microglia showed the neurotoxic phenotype in an ethanol-induced brain injury model produced by the stereotaxic injection of ethanol into the mouse striatum (Takeuchi et al., 1998; Toyama et al., 2008; Fig. 4). In this ethanol-injected model produced a large and round or oval shaped brain lesion without hemorrhage, infection or other unexpected effects that might affect the cytokine networks in the brain. Neonatal mice were pretreated with systemic LPS or saline injection (i.p.) daily for 5 days from postnatal day 3 (P3) to P7. Local injection of 100% ethanol (2.0 μl) produced more severe neuronal damage than seen in the MPTP-induced PD model. A large lesion with a necrotic core was observed in the ethanol-injected striatum; and activated microglia had migrated to the outside of this necrotic mass, where Fluoro-Jade B (FJB)-positive degenerative neurons were observed (Fig. 4). After the ethanol-induced damage, activated microglia accumulated in the FJB-positive regions and eliminated damaged neurons by causing delayed neuronal death (Fig. 4). By previous treatment with systemic LPS or saline treatment as a control, the volumes of necrotic and degenerative areas in the striatum were further increased along with an increase in the number of activated microglia by LPS (Table 1). The number of iNOS-

positive microglia also tended to be increased by the LPS treatment.

Fig. 4. Morphological changes due to ethanol injection into the striatum of neonatal (P8) mice; detection of neuronal injury by Fluoro-Jade B (FJB) staining and activated microglia by

CD11b immunostaining. A large diameter of lesion was observed in ethanol-injected striatum 24 hr after the injection with the core of necrotic mass. Degenerative cells were observed outside the necrotic mass. **A:** In FJB staining, many FJB-positive cells were seen in the degenerative region. **B:** In CD11b immunostaining, the large number of activated microglia was found in the degenerative region. n; necrotic region, d; degenerative region. FJB-positive degenerative neurons are shown in saline-treated (**C**), and LPS-treated (**D**) mice. The number of FJB-positive degenerative cells was increased in LPS-treated mice. CD11b-positive microglia in ethanol-injected ipsilateral striatum are shown in saline-treated (**E**), and LPS-treated (**F**) mice. Increase in the number of activated microglia was observed

most markedly in LPS-treated mice.

Sawada M. et al. (1998, 1999, 2006) proposed a hypothesis of toxic change of microglia. He with collaborators separated two subsets of microglia with neuroprotective or neurotoxic phenotypes from mouse brain by cell sorting and established a cell line for each. The 6-3 cell line of one subset produced a greater amount of ROS stimulated by phorbol myristate acetate (PMA) than did the other, the Ra2 cell line. Both clones were dependent on granulocyte macrophage colony-stimulating factor (GM-CSF). When both microglia cell lines were co-cultured with N18 neuronal cells, which are sensitive to oxidative stress by hydrogen peroxide to produce dose-dependent cell death, and are stimulated with PMA, the viability of the N18 cells was increased when the cell were co-cultured with Ra2 cells and decreased in the presence of 6-3 cells. These results indicate that 6-3 cells were of the neurotoxic phenotype, and that Ra2 cells were of neuroprotective one.

A toxic change in microglia phenotypes from neuroprotection to neurotoxicity was observed by transfecting the cells with cDNA encoding HIV-1 Nef protein, indicating the conversion of microglia from a neurotrophic to a neurotoxic subtype (Vilhardt et al., 2002). When Ra2 cells were transfected with Nef protein by using lenti virus, these normally neuroprotective cells produced ROS with activation of NADPH oxidase, in contrast to the non-transfected Ra2 cells, which did not produce ROS. When Nef-Ra2 cells were co-cultured with N-18 neuronal cells, the viability of N-18 cells was decreased; whereas that of N-18 cells cocultured with Ra2 cells was not lowered. These results suggest a toxic change in neftransfected microglia accompanied by ROS production.

According to a report on conditional ALS transgenic mice, the disease progression was determined by the expression of mutant superoxide dismutase (SOD)1 protein not in motor neurons but in microglia (Boillée et al., 2006). Microglial activation was observed from the disease onset to the progression in the spinal cord in these animals. The disease progression accelerated in the transgenic mice that expressed mutant SOD1 in all systemic cells including microglia, whereas the viability was improved in the transgenic mice that expressed normal SOD1 only in their microglia. However, the early phase of disease progression showed no difference between above the two types of transgenic mice. This report concluded that microglia had little effect on the early disease phase but participated on the later disease progression in ALS. According to the hypothesis of toxic change in microglia by Sawada M., microglial toxic changes might appear during the progression phase of ALS.

Role of Microglia in Inflammatory Process in Parkinson's Disease 341

Thereafter, ATP at low-to-moderate concentrations (maximal secretion at 1 mM) stimulated TNF-α secretion from microglia, beginning at 2-3 hr after the stimulation (Hide et al., 2000). About 6 hr after the stimulation with ATP, the microglia showed increased release of IL-6, which secretion was ATP concentration dependent (10-1,000 μM; Shigemoto-Mogami et al., 2001). ATP also induced an increase in Ca2+ influx in a concentration-dependent manner. Furthermore, after the stimulation at high concentrations of ATP (10-1,000 μM) about 12 hr later microglial iNOS synthesis was induced; and this iNOS produced NO, then killed neuronal cells (Ohtani et al., 2000). Therefore, initial weaker and shorter stimulation of microglia by extracellular ATP may lead to neuroprotection by secretion of plasminogen, TNF-α, and IL-6; whereas stronger and longer exposures to ATP may be neurotoxic due to NO production. A recent report demonstrated that delayed neural damage was induced by iNOS expression by microglia in brain injured mouse model (Ono et al., 2010). Microglia themselves also release ATP

As described above, Sawada M. et al. (2006) hypothesized that the microglial activation may occur in two steps in the PD brain (Fig. 5). At the first step, microglia activated by the first stimulation probably by signaling molecules from injured neurons produce neuroprotectants such as neurotrophins, neurotrophic cytokines, and antioxidant. At the secondary step, activated microglia, which are further stimulated by other factors such as a large amount of cytotoxic factors, may undergo toxic changes, thus converting them from the neuroprotective phenotype to the neurotoxic one, with the result being neuronal cell

Neuroinflammation with activated microglia may play important roles in the pathogenesis of various neurodegenerative diseases including PD. Activation of microglia may be produced by some chemical signaling molecules released from the injured neurons such as dopaminergic neurons in the SN of the PD brain. Activated microglia may be neuroprotective, at least in the early phase, but may become neurotoxic later to contribute to the progression of the disease. Sawada M. et al. proposed a hypothesis of a toxic change in activated microglia from the neuroprotective phenotype to the neurotoxic one. Development of drugs to regulate activated microglia could be a promising approach for drug

We thank Dr. R. Hishida, Dr. S. Muramatsu, and Dr. I. Nakano (Jichi Medical University, Department of Medicine, Division of Neurology), Dr. Y. Hirata (Gifu University, Faculty of Engineering, Department of Biomolecular Science), Dr. F. Imai (Fujita Health University, School of Medicine, Department of Neurosurgery), and Dr. M. Mogi (Aichi-Gakuin University, School of Pharmacy, Department of Medical Biochemistry) for their helpful advice and support of this work. Many of our studies described in this review were supported by grants-in-aid from the Ministry of Health, Labor, and Welfare of Japan (MS); Ministry of Education, Culture, Sports, Science, and Technology of Japan (MS); and Japan

depending on the LPS concentration (Ferrari et al., 1997).

development for neurodegenerative diseases such as PD.

death in PD.

**7. Conclusions** 

**8. Acknowledgments** 

Health Sciences Foundation (MS).

Lai and Todd (2008) examined the effect of microglia by changing the severity of hypoxiainduced neuronal injury in a culture study. After exposing neuronal cells to various degrees of hypoxia, i.e., mild, moderate, or severe, the media from the neuronal cell culture were added to microglial cell culture. Neuroprotective phenotype of microglia was observed with the media from neurons with moderate hypoxia, but not with those from the cultured cells under mild or severe hypoxia.

Fig. 5. This scheme shows a hypothesis of toxic change of microglia by two step activation of microglia proposed by Sawada M. et al. At the first step, activated microglia by the first stimulation probably by signaling molecules from injured neurons produce such as neurotrophins, neurotrophic cytokines, antioxidant, and may act for neuroprotection. At the secondary step, activated microglia, which are further stimulated by other factors such as a large amount of cytotoxic factors, may produce toxic change of microglia, which converts neuroprotective phenotypes to neurotoxic ones.

Inoue (2002) hypothesized that the fate of damaged neurons may be regulated in part by ATP through the activation of microglia. Microglia possess the functional P2 receptors, P2X and P2Y, which bind to purines and pyrimidines. Microglial activation is triggered by extracellular ATP or ADP, which is released from damaged neurons under pathological situations. Microglia showed membrane ruffling and enhanced chemotaxis in response to extracellular ATP or ADP produced by neuronal injuries (Honda et al., 2001). A low concentration of extracellular ATP (10-100 μM) stimulated microglial secretion of a neurotrophic substance plasminogen, with a peak response at 5-10 min after the stimulation. This secretion was ATP-concentration dependent (Inoue et al., 1998). Thereafter, ATP at low-to-moderate concentrations (maximal secretion at 1 mM) stimulated TNF-α secretion from microglia, beginning at 2-3 hr after the stimulation (Hide et al., 2000). About 6 hr after the stimulation with ATP, the microglia showed increased release of IL-6, which secretion was ATP concentration dependent (10-1,000 μM; Shigemoto-Mogami et al., 2001). ATP also induced an increase in Ca2+ influx in a concentration-dependent manner. Furthermore, after the stimulation at high concentrations of ATP (10-1,000 μM) about 12 hr later microglial iNOS synthesis was induced; and this iNOS produced NO, then killed neuronal cells (Ohtani et al., 2000). Therefore, initial weaker and shorter stimulation of microglia by extracellular ATP may lead to neuroprotection by secretion of plasminogen, TNF-α, and IL-6; whereas stronger and longer exposures to ATP may be neurotoxic due to NO production. A recent report demonstrated that delayed neural damage was induced by iNOS expression by microglia in brain injured mouse model (Ono et al., 2010). Microglia themselves also release ATP depending on the LPS concentration (Ferrari et al., 1997).

As described above, Sawada M. et al. (2006) hypothesized that the microglial activation may occur in two steps in the PD brain (Fig. 5). At the first step, microglia activated by the first stimulation probably by signaling molecules from injured neurons produce neuroprotectants such as neurotrophins, neurotrophic cytokines, and antioxidant. At the secondary step, activated microglia, which are further stimulated by other factors such as a large amount of cytotoxic factors, may undergo toxic changes, thus converting them from the neuroprotective phenotype to the neurotoxic one, with the result being neuronal cell death in PD.
