**3. Neuroinflammation and microglia**

Microglia are derived from myeloid cells having the property of high affinity for the brain and comprise about 10% of the total cells in CNS parenchyma (Ono et al., 1999; Sawada M. et al., 1998). Microglia play important physiological roles in the development, differentiation, and maintenance of neural cells in the brain. They also have immunological functions in the brain and serve to remove dead cells by phagocytic activity after brain injury or neurodegeneration. Microglia are normally in the resting stage, but are activated by some brain lesions in neurodegenerative diseases such as PD.

Activated microglia may play neurotoxic roles by producing pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6, as well as, nitric oxide (NO) and ROS (Cassarino et al., 1997; Chao et al., 1992; Hunot et al., 1996; Kim et al., 2000; Koutsilieri et al., 2002; Liu et al., 1998; McGuire et al., 2001). On the other hand, activated microglia may also function neuroprotectively by producing neurotrophic components such as IL-10, TGF-β, plasminogen, glial cell line-derived neurotrophic factor (GDNF), BDNF, and NGF (Batchelor et al., 1999; Elkabes et al., 1996; Miwa et al., 1997; Nagata et al., 1993b; Nakajima et al., 2001; Sawada M. et al., 1995, 1999; Suzumura et al., 1993).

Pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 are pleiotropic, and may produce either neurotoxic or neuroprotective effects (Arai et al., 2004; Barger et al., 1995; Bolin et al., 2002; Fisher et al., 2001; Liu et al., 1998; Mason et al., 2001; McGuire et al., 2001). TNF-α produced by microglia or astrocytes in the CNS is generally considered to be neurotoxic (Sawada M. et al., 1989; Suzumura et al., 1999). Microglial production of TNF-α is increased when the cells are stimulated with lipopolysaccharide (LPS; Sawada M. et al., 1989, 1995). Neurotoxin-mediated damage to dopaminergic neurons is attenuated in mice deficient in TNF-α or TNF receptors compared with the damage seen in wild-type mice (Ferger et al., 2004; Sriram et al., 2002). A recent study demonstrated that inhibition of TNF reduce the delayed and progressive neurodegeneration in the SN in PD rats (Harms et al., 2011). Two weeks after having received intrastriatal administration of 6-OHDA, PD model rats were injected in their SN with lentivirus encoding a dominant-negative TNF gene. The effects of this abnormal TNF included inhibition of the progressive loss of nigral dopaminergic neurons, when the rats were examined 5 weeks after the initial 6-OHDA administration. The lentivirus dominant-negative TNF therapy also attenuated microglial activation in PD rats. However, some other reports suggest opposite neuroprotective effects of TNF-α. For example, auto-immune-mediated demyelination model mice of multiple sclerosis with deficient TNF-α developed more severe neurological impairment than the normal multiple sclerosis model mice (Liu et al., 1998). Two different subtypes of the TNF receptors may act for neuronal death or survival by different signal transduction pathways (Yang et al., 2002).

LPS is a gram-negative bacterial endotoxin and is a microglial activator substance. LPS treatment causes neurotoxic effects on dopaminergic neurons in various cell culture systems (Gao et al., 2003; Gayle et al., 2002; Kim et al., 2000) or by direct injection into the SN (Arai et al., 2004; Castano et al., 2002; Iravani et al., 2005). LPS is recognized as an

(CNS) spreading to higher levels of the neuroaxis. They observed activation of microglia, αsynuclein phosphorylation and aggregation, and dopaminergic neuron loss in the SN after

Microglia are derived from myeloid cells having the property of high affinity for the brain and comprise about 10% of the total cells in CNS parenchyma (Ono et al., 1999; Sawada M. et al., 1998). Microglia play important physiological roles in the development, differentiation, and maintenance of neural cells in the brain. They also have immunological functions in the brain and serve to remove dead cells by phagocytic activity after brain injury or neurodegeneration. Microglia are normally in the resting stage, but are activated

Activated microglia may play neurotoxic roles by producing pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6, as well as, nitric oxide (NO) and ROS (Cassarino et al., 1997; Chao et al., 1992; Hunot et al., 1996; Kim et al., 2000; Koutsilieri et al., 2002; Liu et al., 1998; McGuire et al., 2001). On the other hand, activated microglia may also function neuroprotectively by producing neurotrophic components such as IL-10, TGF-β, plasminogen, glial cell line-derived neurotrophic factor (GDNF), BDNF, and NGF (Batchelor et al., 1999; Elkabes et al., 1996; Miwa et al., 1997; Nagata et al., 1993b; Nakajima et al., 2001;

Pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 are pleiotropic, and may produce either neurotoxic or neuroprotective effects (Arai et al., 2004; Barger et al., 1995; Bolin et al., 2002; Fisher et al., 2001; Liu et al., 1998; Mason et al., 2001; McGuire et al., 2001). TNF-α produced by microglia or astrocytes in the CNS is generally considered to be neurotoxic (Sawada M. et al., 1989; Suzumura et al., 1999). Microglial production of TNF-α is increased when the cells are stimulated with lipopolysaccharide (LPS; Sawada M. et al., 1989, 1995). Neurotoxin-mediated damage to dopaminergic neurons is attenuated in mice deficient in TNF-α or TNF receptors compared with the damage seen in wild-type mice (Ferger et al., 2004; Sriram et al., 2002). A recent study demonstrated that inhibition of TNF reduce the delayed and progressive neurodegeneration in the SN in PD rats (Harms et al., 2011). Two weeks after having received intrastriatal administration of 6-OHDA, PD model rats were injected in their SN with lentivirus encoding a dominant-negative TNF gene. The effects of this abnormal TNF included inhibition of the progressive loss of nigral dopaminergic neurons, when the rats were examined 5 weeks after the initial 6-OHDA administration. The lentivirus dominant-negative TNF therapy also attenuated microglial activation in PD rats. However, some other reports suggest opposite neuroprotective effects of TNF-α. For example, auto-immune-mediated demyelination model mice of multiple sclerosis with deficient TNF-α developed more severe neurological impairment than the normal multiple sclerosis model mice (Liu et al., 1998). Two different subtypes of the TNF receptors may act for neuronal death or survival by different signal transduction pathways

LPS is a gram-negative bacterial endotoxin and is a microglial activator substance. LPS treatment causes neurotoxic effects on dopaminergic neurons in various cell culture systems (Gao et al., 2003; Gayle et al., 2002; Kim et al., 2000) or by direct injection into the SN (Arai et al., 2004; Castano et al., 2002; Iravani et al., 2005). LPS is recognized as an

long-term progression of the viral infection.

**3. Neuroinflammation and microglia** 

by some brain lesions in neurodegenerative diseases such as PD.

Sawada M. et al., 1995, 1999; Suzumura et al., 1993).

(Yang et al., 2002).

initiator of dopaminergic neuronal loss, and the degree of neuronal damage may depend on the concentration of LPS used for treatment. The neurotoxicity of microglia is increased by the production of TNF-α in response to LPS stimulation (Sawada M. et al., 1989, 1995). On the other hand, the neurotrophic effects of microglial activation induced by LPS have also been found in several cell culture studies (Elkabes et al., 1998; Kramer et al., 2002; Mallat et al., 1989; Miwa et al., 1997; Nakajima et al., 2001). The neurotrophic effects of LPS may be explained by the fact that LPS induces the secretion of not only proinflammatory cytokines but also neurotrophic compounds. Stimulation by LPS increases the microglial secretion of NT-3, NT-4/5, NGF, and BDNF (Elkabes et al., 1998; Miwa et al., 1997; Nakajima et al., 2001). A rat model of spinal cord injury showed improvement in locomotor function by an LPS-elicited increase in the level of neuroprotective GDNF (Hashimoto et al., 2005). Plasminogen produced by LPS-treated microglia was reported to promote the development of dopaminergic neurons (Nagata et al., 1993b; Nakajima et al., 1992).

Several studies indicate that damaged dopaminergic neurons release various factors that can active microglia. These factors are α-synuclein, matrix metalloproteinase 3 (MMP-3), and neuromelanin, all of which are released from damaged dopaminergic neurons and induce ROS production. α-Synuclein, which is a synaptic vesicle protein and a main component of Lewy bodies, the pathological hallmark of PD, may have an important role in both the onset and progression of PD. Extracellular aggregated α-synuclein induced microglial activation that enhanced neurotoxicity toward dopaminergic neurons, whereas low concentrations of α-synuclein failed to be neurotoxic (Zhang et al., 2005). Microglial enhancement of αsynuclein-mediated neurotoxicity depended on the phagocytosis of α-synuclein and production of ROS by microglia. Nitrated/oxidized α-synuclein was detected in nigral cytoplasmic inclusions, and inhibition of microglial-derived NO and superoxide provided significant neuroprotection to dopaminergic neurons (Gao et al., 2008). MMP3 is a zincdependent proteolytic enzyme that degrades the extracellular matrix; and it is released from damaged neurons, thereby inducing microglial activation with production of inflammatory cytokines such as TNF-α (Kim et al., 2005). MMP-3-deficient mice show reduced MMP-3 induced microglial production of NADPH oxidase-derived superoxide and dopaminergic cell death (Kim et al., 2007).

Microglial activation accompanied by the degeneration of dopaminergic neurons is an early event of neuroinflammation in PD. Purisai et al. (2007) reported that activation of microglia accompanied by the induction of NADPH oxidase was a priming event in paraquatadministered mice, which activation occurred at least 1 or 3 days after the administration. An *in vivo* positron emission tomography (PET) study imaging, microglial activation in nigro-striatal regions indicated that the activation was likely to occur early in the disease process and paralleled the loss of terminals in dopaminergic neurons, as revealed by use of [11C](R)-PK11195, a peripheral benzodiazepine receptor-binding ligand (Gerhard et al., 2006). However, microglial activation by chronic LPS infusion into the SN or single systemic injection in animals caused delayed and progressive neurodegeneration of nigral dopaminergic neurons (Gao et al., 2002; Qin et al., 2007). Using conditional amyotrophic lateral sclerosis (ALS) transgenic mice, Boillée et al. (2006) demonstrated that microglia had a great effect on the later phase of disease progression but little effect on the early phase of the disease.

Role of Microglia in Inflammatory Process in Parkinson's Disease 335

dopaminergic (A9) neurons were observed in the MPTP-treated mice, whereas mice treated with the MPTP and LPS demonstrated marked microglial activation and a tendency toward recovery against cell toxicity, as compared with the MPTP-treated mice (Fig. 3A). These MPTP-LPS-treated mice showed increased levels of pro-inflammatory cytokines of IL-1β and IL-6. LPS-activated microglia in neonatal and aged mice had different phenotypic effects on dopaminergic neurons exposed to MPTP. In contrast, the number of dopaminergic neurons in the SN in aged mice (60 weeks) treated with MPTP was significantly decreased, and an increase in the number of microglia treated with MPTP and LPS produced a further decrease in the number of dopaminergic neurons (Fig. 1 and 2). The relationship between microglial activation and viability of dopaminergic (A9) neurons for the three groups (saline control, MPTP treated, and MPTP-LPS treated mice) of aged mice showed an inverse correlation (Fig. 3B). These results suggest that LPS-activated microglia in aged mice may be

neurotoxic, whereas in neonatal mice they may have neurotrophic potential.

Fig. 2. Analysis of effects of LPS treatment on numbers of dopaminergic (A9) neurons and CD 11b-positive activated microglia in MPTP-treated neonatal and aged mice. **A**: Number of dopaminergic (A9) neurons of the SN for the saline, MPTP, and LPS-MPTP groups in P8 mice. The number of dopaminergic (A9) neurons in the MPTP group was significantly decreased, whereas that for the LPS-MPTP group was recovered. **B**: Number of CD11bimmunopositive microglial cells in the SN in P8 mice. The LPS-MPTP group demonstrated marked microglial activation. **C**: Number of dopaminergic (A9) neurons of the three groups in the SN of 60w mice. The number in the MPTP and LPS-MPTP groups was significantly decreased. **D**: Number of CD11b-immunopositive microglia for the three groups in the SN of 60w mice. Severe microglial activation was observed in the LPS-MPTP group. Values represent the mean±SD. \**p* < 0.05; \*\**p* < 0.01 versus saline group, ##*p* < 0.01 versus MPTP group, by the use of the unpaired Student's *t* test (Sawada H. et al., 2007, J Neurosci Res,

Vol. 85, No. 8, pp. 1752-1761, With permission of John Wiley and Sons).
