DISCUSSION

Almost all studies examining the progression of PD were focused on neuronal death, since it has been reported that PD is characterized by dopaminergic neuronal death in the SNpc. However, recently studies suggest that risk factors for PD progression are not only dopaminergic neuronal death but also dysfunction of non-neuronal cells. Based on this, I examined functional change in astrocytes caused by a PD-related gene, DJ-1 and the relationship between dysfunction of astrocytes and progression of PD.

PART A. DJ-1, (PARK7), plays critical roles in astrogliosis by regulating of the pSTAT3 signaling pathway for tissue repair after brain injury

DJ-1, a gene related to an early onset autosomal-recessive form of PD (Bonifati, et al., 2003a). Interestingly, however, animal models of PD based on mutations of DJ-1 did not show PD phenotypes such as dopaminergic neuronal death and Lewy body formation (Chen, et al., 2005;Kim, et al., 2005;Kitada, et al., 2009). For example, it has been reported that the number of dopamine neurons was not decreased in 6 month and 11 month old DJ-1 null mice although striatal dopaminergic neurons were affected by DJ-1 deficiency, leading to increased dopamine reuptake rates and elevated tissue dopamine content (Chen, et al., 2005).

In addition, DJ-1null mice did not demonstrate any anatomical or neuronal abnormalities, reduction in the number of dopaminergic neuron or change in the dopamine levels in the striatum and in the substantia nigra although dopaminergic neuronal loss was increased by 1-methyl-4-phenyl-1,2,4,5-tetrahydrophyridine (MPTP) treatment (Kim, et al., 2005). These

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studies suggest that genetic mutation and environmental insults are necessary for PD progression.

After an environmental insult, the damaged brain tissues begin a process of repair and regeneration, in order to restore brain structure and function (Compston, 1995;Lo, 2010;Okano, et al., 2007). Unfortunately, however, CNS axons do not spontaneously regenerate, unlike peripheral nervous system (PNS) axons which have the capacity to regenerate and recover after injury (Benfey and Aguayo, 1982;David and Aguayo, 1981;Richardson, et al., 1984;Richardson, et al., 1980). However, mature CNS neurons are able to regenerate if provided with sufficient necessary factors. For example, it has been reported that mature CNS neurons have the capacity to regenerate after transplantation of peripheral nerve graft (Richardson, et al., 1980). This study suggests that regulation of brain environment is necessary for CNS repair and regeneration. Recently, it has been reported that PD patients or PD animal models demonstrate altered release of repair-related factors. For example, nerve growth factor (NGF) expression was significantly decreased in the serum of patients with early-stage PD and PD animal models (Lorigados Pedre, et al., 2002), and brain derived neurotrophic factors (BDNF) expression was increased in the serum of patients with PD (Ventriglia, et al., 2013). In addition, angiogenesis marker, integrin a_vb₃ was highly expressed in the SNpc in patients with parkinsonian syndromes and in the putamen in patients with PD (Desai Bradaric, et al., 2012). These studies strongly suggest that a change in the levels of repair-related factors was observed in patients with PD and could be a risk factor for progression of PD. In this study, I found that DJ-1 KO causes defects in the repair and regeneration of TH positive axons (Fig 1, 11). This result is the first direct evidence of a

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possible defect in tissue repair in a familial form of PD and I suggest that it is one of risk factors for progression of PD.

The next question I addressed was how DJ-1 deficiency reduces repair and regeneration after brain damage. Astrogliosis is related to repair-related factors such as the regulation of supporting nutrients, angiogenesis, remyelination, secretion of neurotrophic factors and neurogenesis. Thus many studies have suggested that astrogliosis has important roles in regeneration and repair (do Carmo Cunha, et al., 2007;Liberto, et al., 2004;Triolo, et al., 2006;White, et al., 2008), although, various studies have suggested negative effects of astrogliosis as a result of scar formation. For example, reactive astrocytes induced glycolytic capacity via glucose uptake, pyruvate kinase activity and lactate dehydrogenase (LDH) in hypoxic conditions, in order to support the energy needs of neurons (Marrif and Juurlink, 1999). In addition, astrocytes store glycogen after stimulation by IGF-1 and use it in glucose-limited conditions to provide energy support to neighboring neurons (Dringen and Hamprecht, 1992). Astrocytes also regulate the outgrowth of DRG neurites and axon regeneration in mature white matter through the action of fibronectin. In slice cultures of the P35 rat brain, astrocyte-associated fibronectin was detected. Furthermore, DRG neurites and axon regeneration were dramatically decreased by administration of anti-fibronectin antibody (Tom, et al., 2004). Reactive astrocytes are also associated with neovascularization and angiogenesis. In a traumatic injury model such as a stab wound and neural grafting, VEGF mRNA and protein expression and its receptor, flt-1, was highly expressed in reactive astrocytes, and not endothelium (Krum and Rosenstein, 1998). In hypoxic conditions, reactive astrocytes also regulate angiogenesis via VEGF. For example, co-culture of

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astrocytes and microvascular endothelial cells resulted in in vitro angiogenesis with the formation of tube-like structures. In this model, reactive astrocytes highly expressed VEGF (Ment, et al., 1997). Remyelination is also regulated by reactive astrocyte-released CNTF.

CNTF promotes the proliferation of oligodendrocyte precursors via fibroblast growth factor-2 (FGF-factor-2) (Albrecht, et al., factor-2003). CNTF is located in normal astrocytes and has been found in white matter structures as well (Dallner, et al., 2002). CNTF is located in normal astrocytes and has been found in white matter structures as well. CNTF protein and mRNA expression was highly increased during the remyelination phase and its expression was detected within reactive astrocytes surrounding the injured area. In addition, CNTF increased FGF-2, a factor needed for oligodendrocyte precursor cell (OPC) proliferation, in reactive astrocytes during remyelination in the spinal cord (Albrecht, et al., 2003). These studies suggest that astrogliosis maintains the required microenvironment for brain repair. In this study, I found that DJ-1 KO causes defects in the progression of astrogliosis after ATP-induced injury (Fig 2–8). This suggests the possibility of a defect in astrogliosis is present in a familial form of PD and I suggest that it causes a defect in brain repair.

Insufficient astrogliosis could affect the release of neurotrophic factors that mediate brain repair and regeneration. It has been reported that reactive astrocytes express neurotrophic factors necessary for protection and repair after brain injury. In particular, GDNF is expressed by human astrocytes; it also has been reported that GDNF is expressed in reactive astrocytes (Moretto, et al., 1996;Nakagawa and Schwartz, 2004). GDNF is a well-known glial derived neurotrophic factor and is a major supporting factor for dopaminergic neuronal protection in SNpc. GDNF supports the viability of postnatal nigral dopamine

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neurons by inhibiting apoptotic death (Burke, et al., 1998). In addition, GDNF induces dopaminergic nerve fiber sprouting in the margins of striatal wounds (Batchelor, et al., 1999), which is reduced by inhibition of GDNF via antisense oligonucleotides (Batchelor, et al., 2000). Furthermore, many studies suggest that GDNF regulates the protection and function of dopaminergic neurons. For example, GDNF injection induced significant improvement in the symptomatology and pathophysiological features of MPTP-induced parkinsonism in monkeys (Gash, et al., 1996). Adding GDNF via a lentiviral vector also reversed functional deficits monkeys with in MPTP-induced PD (Kordower, et al., 2000). Interestingly, GDNF induction was observed in patients with severe PD although GDNF receptor molecules, GFRa1 and cRET, were not changed (Backman, et al., 2006). These studies suggest that GDNF expression is necessary for neural protection and repair in the injured brain and PD.

In this study, I found that DJ-1 KO causes defects in GDNF expression in reactive astrocytes after ATP-induced injury (Fig 9, 10). This result represents a functional defect of insufficient astrogliosis. Thus, I suggest that functional defects of astrogliosis lead to delayed brain repair and are one of risk factors for progression of PD.

Intermediate filaments (IFs) play a critical role in the progression of astrogliosis.

After brain injury, IFs are highly expressed in reactive astrocytes and promote numerous features of morphological change such as hypertrophy of the cell body, and thickening and extension of cellular processes. For example, GFAP and vimentin double-KO showed insufficient features of reactive astrocytes such as low hypertrophy, process shortening, and reduction of glial scar formation, when compared with WT astrocytes after brain injury (Wilhelmsson, et al., 2004). Interestingly, it has been reported that brain damage was

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increased by a lack of GFAP and other IFs. For example, more GFAP-null mice died than WT mice following percussive head injury induced by mechanical stress (Nawashiro, et al., 1998). In addition, GFAP-null mice showed larger cortical infarct volume than WT because of defects in process formation and the formation, regulation, and induction of the BBB, in permanent middle cerebral artery occlusion (pMCAO) and transient carotid artery occlusion (tCAO) (Nawashiro, et al., 2000), although GFAP-null astrocytes did not differ compared with WT astrocytes in the intact brain (Gomi, et al., 1995). These studies suggest that IFs are key molecules in regulating the morphology and function of reactive astrocytes after brain injury. In patients with PD, interestingly, it has been reported that astrocytes did not show reactive morphology such as hypertrophic cell body, and thickening and extension of cellular processes, which are seen in other neurodegenerative disease (Mirza, et al., 2000;Song, et al., 2009). These studies suggest that insufficient astrogliosis is related to PD. In this study, I found that DJ-1 KO causes defects in features of reactive astrocytes in the penumbral region and GFAP expression after ATP-induced injury (Fig 3). In addition, this result demonstrates a defect of reactive astrocytes due to insufficient expression of IFs (Fig 2–4). Therefore, I suggest that DJ-1 regulates expression of IFs during astrocyte activation.

Interestingly, it has been reported that reactive astrocytes have regional morphological heterogeneity in the penumbra of the injured area. Astrocytes, next to damaged regions, have long processes that are directed toward the damage sites while astrocytes close to damaged area become hypertrophic without any polarity in the cell (Ding, 2014;Shimada, et al., 2012). In this study, I found that DJ-1 KO caused defects in morphological features of reactive astrocytes next to the damaged region. The

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processed reactive astrocytes expressed not only GFAP, but also nestin (Fig. 6). Nestin also strongly induced IFs in reactive astrocytes (Duan, et al., 2015;Lin, et al., 1995;Sirko, et al., 2013;Wiese, et al., 2004). It has been reported that nestin regulates the migration and invasion of cells. For example, nestin knock-down cells exhibit attenuated migration and invasion by the induction of filamentous F-actin and E-cadherin expression in pancreatic cancer cells (Matsuda, et al., 2011). Nestin has also been shown to regulate migration and invasion in other cell lines (Akiyama, et al., 2013;Narita, et al., 2014;Zhao, et al., 2014). In addition, nestin regulates angiogenesis. Nestin knock-down endothelial cells displayed suppressed cell growth and tumor formation ability of pancreatic cancers in vivo. (Matsuda, et al., 2013). In addition, it has been suggested that nestin-positive astrocytes may have potential to dedifferentiate (Heinrich, et al., 2010), since nestin is a marker of neural stem cells (Doetsch, et al., 1997;Lendahl, et al., 1990). In this study, although nestin protein expression level was not detected by western blot, I found that DJ-1 KO caused defects in process extension and invasion in GFAP and nestin-positive reactive astrocytes (Fig. 7, 8) In addition, it is closely related with the repair of TH-positive processes (Fig. 3, 11). Therefore, I speculate that long-process baring GFAP-, and nestin-positive astrocytes may participate in the repair and regeneration of damaged brain regions, and that DJ-1 KO astrocytes may not be able to support regeneration of damaged brain as much as WT astrocytes because of attenuated expression of GFAP and nestin.

The Janus kinase/signal transducer and antivator of transcription3 (JAK/STAT3) pathway is known to central player in the activation of astrocytes (Levy and Darnell, 2002).

Activation of JAK/STAT3 is demonstrated in reactive astrocytes in several conditions. For

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example, STAT3 translocation was strongly detected in reactive astrocytes that surrounded the infarct from 3 to 7 days after transient focal ischemia (Justicia, et al., 2000). Interestingly, with astrocyte-specific inhibition of the STAT3 pathway, reactive astrocytes did not show morphological change (Herrmann, et al., 2008) and STAT3 KO mice showed severe motor deficits with demyelination and neural disruption (Okada, et al., 2006) after spinal cord injury. In a slice culture system, I found that STAT3 phosphorylation could be regulated by DJ-1 expression (Fig. 14). In addition, I detected that GFAP expression was decreased by inhibition of STAT3 phosphorylation (Fig. 14). Therefore, I suggest that DJ-1 regulates astrocyte activation by controlling STAT3 phosphorylation.

In conclusion, I provide the first evidence that DJ-1 deficiency negatively regulates STAT3 phosphorylation, which is critical for the progression of astrogliosis. In addition, DJ-1 deficiency could cause delayed tissue repair as a result of insufficient astrogliosis, since reactive astrocytes play a critical role in the repair and regeneration of the injured brain. This evidence suggests that degeneration is not only a problem of neuronal death. Therefore, it should be considered that the regulation of repair and function of reactive astrocytes plays a role in the development and progression of PD and other neurodegenerative diseases.

PART B.DJ-1 exerts the anti-inflammatory effects of astrocytes through stabilization of Sox9, a transcription factor that regulates lipocalin-type prostaglandin D2 synthase (L-PGDS) expression

After brain injury, inflammation has a critical defense mechanism and process to regenerate microenvironment after brain injury. However, uncontrolled brain inflammation

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could accelerate the progression of damage injury (Chao, et al., 1992;Choi, et al., 2003;Kitamura, et al., 1996). Many studies suggest that inflammation is a risk factor for neurodegenerative disease such as AD, PD, and MS (Breitner, 1996;Chen, et al., 2003;Klegeris and McGeer, 2005;Raivich and Banati, 2004;Sheng, et al., 1998), although the affected neuron type and location differs between the diseases. For example, it has been reported that microglia were highly activated in PD patients (McGeer, et al., 1988;Vijitruth, et al., 2006) and inhibition of microglial activation induced neuroprotective effects in PD models (Du, et al., 2001;He, et al., 2001). In addition, it has been reported that exceed pro-inflammatory mediators were observed in PD patients (Knott, et al., 2000;Mogi, et al., 1994).

These studies suggest that regulation of inflammation is an important key factor in the progression of PD. Therefore, I investigated whether the PD related gene, DJ-1, could modulate the anti-inflammatory function of astrocytes.

The anti-inflammatory function of astrocytes is one of a neuroprotective manner, via regulation of the brain’s microenvironment (Sofroniew, 2015). Astrocytes produce several soluble factors to regulate inflammation (Jensen, et al., 2013;John, et al., 2005;Meeuwsen, et al., 2003;Zamanian, et al., 2012). In particular, studies involving ACM provide strong evidence of an anti-inflammatory function for astrocytes (Min KJ et al., 2006).

A previous study suggested that IFN-g-induced nitrite, iNOS, and TNF-a expression were reduced by ACM treatment in BV2 cells or primary microglia. In this study, I found that DJ-1 KO caused defects in the anti-inflammatory function of ACM (Fig. DJ-15). Since DJ-DJ-1 could regulate inflammation via the p38 and JAK/STAT1 signal pathway in astrocytes and microglia (Kim, et al., 2013a;Waak, et al., 2009), this result is the first direct evidence of the possible role that lack of astrocyte-derived anti-inflammatory factors in a familial form of

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ACM contains soluble factors, which induce HO-1 expression via NRF2 translocation and increase the HO-1-reduced pro-inflammatory response in BV2 cells or primary microglia (Min, et al., 2006). Induction of HO-1 has been reported in several neurological conditions. For example, HO-1 induction was observed in microglia in the infarct and penumbral regions after transient MCAO (Koistinaho, et al., 1996) and HO-1-expressing microglia accumulated after traumatic brain traumatic injury (Beschorner, et al., 2000). In addition, it has been reported that HO-1 has anti-apoptotic and anti-inflammatory functions. For example, HO-1-overexpressing human pulmonary epithelial cells are resistant to cell death due to hyperoxia (Lee, et al., 1996) and HO-1 deficient fibroblast cells are more showed weakness to oxidative stress (Dennery, et al., 1997). Moreover, HO-1 deficient mice showed chronic inflammation of the renal tubules (Nath, et al., 2001) induction of a pro-inflammatory response in splenocytes (Kapturczak, et al., 2004). . In this study, I found that HO-1 was insufficiently induced in BV2 cells by DJ-1 deficient ACM (Fig. 16). Therefore, I suggest that uncontrolled inflammation was caused by insufficient induction of HO-1 in BV2 cells, which is regulated by ACM. This result provides evidence that DJ-1 KO astrocyte-derived soluble factors could not induce HO-1 expression in BV2 cells to regulate the anti-inflammatory response.

Previously, our group has been reported that ACM contained factors are lipid characterized molecules which are heat-labile and smaller than 3KD (Min, et al., 2006).

Prostaglandins are lipid autacoids and it has been reported that prostaglandins are critical player in the progression of the inflammatory response. Bioactive prostaglandins consist of prostaglandin E2 (PGE2), prostacyclin (PGI2), and prostaglandin D2 (PGD2). These molecules

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have several autocrine and paracrine lipid mediators to maintain tissue homeostasis.

Interestingly, it has been reported that prostaglandin expression level is increased in an inflammatory response (Ricciotti and FitzGerald, 2011). However, our group has reported that PGE₂ and 15d-PGJ₂, which are well-known HO-1 inducible factors, could not increase HO-1 levels in ACM-treated BV2 cells. Therefore, I focused on other prostaglandin families.

PGD₂ is synthesized in the CNS and PNS to regulate inflammation and homeostasis (Jowsey, et al., 2001). It has been reported that PGD₂ is the most abundant prostaglandin in the CNS (Narumiya, et al., 1982) and PGD2 relates with regulation of sleep and pain in the brain (Eguchi, et al., 1999;Urade and Hayaishi, 1999). PGD₂ also has a correlation with inflammatory response. For example, PGD2 and 15d-PGJ2 levels was increased by COX2 and these molecules mediated reduction of inflammation in carrageenin-induced rat model of pleurisy (Gilroy, et al., 1999) and colitis (Ajuebor, et al., 2000). Interestingly, PGD2 is involved in increasing HO-1 mRNA expression via the DP2receptor and phosphorylated AKT signal pathway in retinal pigment epithelium (Kuesap, et al., 2008;Satarug, et al., 2008).

These studies suggest that the most abundant prostaglandin in CNS, PGD2, could be a HO-1 inducible factor in ACM. In this study, I found that PGD2 is found in the ACM and is decreased by DJ-1 deficiency (Fig. 17). In addition, ACM contained PGD₂,which induced HO-1 via DP2 and AKT phosphorylation. DJ-1 deficient ACM did not induce this pathway (Fig. 17). Therefore, I suggest that DJ-1 regulates the production of PGD₂ in astrocytes.

PGD₂ is produced by two types of prostaglandin D₂ synthase: L-PGDS and hematopoietic PGDS (H-PGDS). L-PGDS is expressed in the retina, heart, reproductive organs, cerebrospinal fluid, and CNS. It has been reported that L-PGDS is a glutathione (GSH)-independent enzyme and the major enzyme in the production of PGD₂ in the CNS (Urade and Eguchi, 2002;Urade and Hayaishi, 2000). In this study, I found that DJ-1 KO

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caused a defect in L-PGDS mRNA and protein expression (Fig. 18) and it led to low anti-inflammatory action of the ACM (Fig. 20). Therefore, I suggest that DJ-1 is an important positive transcriptional regulator of L-PGDS.

The next question is how DJ-1 regulates the level of L-PGDS transcription. Recently, it has been reported that Sox9, a well-known transcription factor, regulated L-PGDS mRNA expression (Wilhelm, et al., 2007). In adult brain, interestingly, Sox9 is highly localized to the astrocytes (Pompolo and Harley, 2001). In this study, I found that L-PGDS was regulated by Sox9 expression (Fig. 21) and DJ-1 KO caused a defect in in vitro and in vivo expression of Sox9 protein, which is expressed in astrocytes (Fig 22). It has been reported that Sox9 protein is regulated by ubiquitination and sumorylation (Akiyama, et al., 2005). In this study, I found that DJ-1 KO caused a defect of Sox9 protein stability (Fig. 23, 24). Therefore, I suggest that DJ-1 is an important negative regulator of Sox9 ubiquitination, and the reduction of L-PGDS is caused by excess degradation of Sox9 by the ubiquitination in DJ-1 KO astrocytes.

In conclusion, I provide the first evidence that DJ-1 deficiency positively regulates Sox9 ubiquitination, which is critical for the production of L-PGDS mRNA. In addition, DJ-1 deficiency could cause a lack of PGD2 in the ACM secondary to insufficient L-PGDS production. Finally, a lack of PGD₂ induced low HO-1 expression in BV2 cells.

In conclusion, I provide the first evidence that DJ-1 deficiency positively regulates Sox9 ubiquitination, which is critical for the production of L-PGDS mRNA. In addition, DJ-1 deficiency could cause a lack of PGD2 in the ACM secondary to insufficient L-PGDS production. Finally, a lack of PGD₂ induced low HO-1 expression in BV2 cells.