Parkinson’s disease (PD) is a neurodegenerative motor disease with motor symptoms such as rigidity, resting tremor and postural instability (Olanow and Tatton, 1999). According to a recent report, PD also has non-motor symptoms such as cognitive impairment, depression and sleep disorder (Barbosa, 2013). Pathologically, PD is caused by the progressive loss of dopaminergic neurons in the substantia nigra (Baba, et al., 1998;Damier, et al., 1999).
However, the factors affecting progression of PD are largely unknown because all cases of PD are of the sporadic form (90-95%). In investigating the progression of PD, many groups focused on the familial form (~10% of total PD) and reported the mutated genes related to familial forms of PD, such as alpha-synuclein, Parkin, PINK1, DJ-1 and LRRK2. These studies elucidated the molecular mechanism underlying neuronal death in PD, such as abnormal protein aggregation, production of reactive oxygen species (ROS), mitochondrial dysfunction, and excessive inflammation (Dawson and Dawson, 2003). However, animal models of PD based on mutation of these genes did not show PD phenotypes such as dopaminergic neuronal death and Lewy body formation (Chen, et al., 2005;Gispert, et al., 2009;Goldberg, et al., 2003;Kim, et al., 2005), although neuronal damage was enhanced in response to ischemic and/or mitochondrial toxin-induced injury. Therefore, it has been suggested that mutation of PD genes cooperate with certain environmental insults to cause PD.
２ B. Genes associated with PD
1. DJ-1 (PARK7)
DJ-1, PARK7, mutations are the cause of early onset autosomal-recessive PD.
(Bonifati, et al., 2003a). DJ-1 has diverse roles in the brain although it has been identified as an oncogene that is linked to prostate cancer along with one member of the family of protein inhibitors of activated STAT (PIAS), PIASxa (Takahashi, et al., 2001;Tillman, et al., 2007).
DJ-1 regulates intracellular ROS directly as an antioxidant (Mitsumoto, et al., 2001), and indirectly by inducing the expression of antioxidant enzymes through the stabilization of a transcriptional factor, Nrf2 (Clements, et al., 2006). DJ-1 inhibits cell death through interactions with apoptosis-associated protein such as Daxx, Bcl-XL and p53, or through regulation of their stability and transcriptional activity (Fan, et al., 2008;Junn, et al., 2005;Ren, et al., 2011). It also promotes the degradation of protein aggregates through the inhibition of the fibrillation of a-synuclein (Shendelman, et al., 2004). In the brain, DJ-1 is expressed in astrocytes as well as in neurons (Bandopadhyay, et al., 2004;Mullett, et al., 2009). DJ-1 mediates the neuroprotective effect of astrocytes (Mullett and Hinkle, 2009), regulates endocytosis (Kim, et al., 2013b) and inflammation through regulating of LPS induced pro-inflammatory mediators, and interaction with SHP-1, a protein of the IFN-g signaling pathway (Kim, et al., 2013a;Waak, et al., 2009).
A 189 amino acid protein is encoded by the DJ-1 gene and is conserved among many species (Bader, et al., 2005;Bandopadhyay, et al., 2004;Kotaria, et al., 2005).
Mutations of this protein were observed in population with early onset autosomal-recessive PD. A large deletion of DJ-1 and an L166P mutation of DJ-1 have been previously reported
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(Bonifati, et al., 2003b). In particular, L166P was reported to lead to DJ-1 protein instability and rapid degradation due to impaired dimerization (Miller, et al., 2003;Moore, et al., 2003;Olzmann, et al., 2004). In addition, other mutants such as M26I, E64D and E163K have also been reported (Abou-Sleiman, et al., 2003;Hering, et al., 2004), but the effects of DJ-1 mutations are still largely unknown. In addition, animal models of PD based on mutation of DJ-1 did not demonstrate PD phenotypes such as dopaminergic neuronal death, and Lewy body formation (Chen, et al., 2005;Kim, et al., 2005;Kitada, et al., 2009). These studies suggest that examination of the functional links between mutated DJ-1 and the pathological condition are needed to better understand the development of PD
2. Other genes
In addition to DJ-1, the major PD related gene examined in this thesis, several genes, such as a-synuclein, PINK1, Parkin and LRRK2 have also been identified as important PD associated genes. First, a-synuclein (PARK1) is related to an early onset autosomal dominant for of PD (Chartier-Harlin, et al., 2004). a-synuclein is located mainly on the pre-synaptic terminals of the neuron and plays a role in their regulation thorough the maintenance of synaptic vesicles supplement (Maroteaux, et al., 1988). Mutation of a-synuclein leads to cellular toxicity as a result of a-synuclein aggregation (Ostrerova-Golts, et al., 2000;Pandey, et al., 2006). In addition, A50T mutation of a-synuclein in transgenic mice led to abnormal accumulation of a-synuclein in the neurons, causing neuronal degeneration (Lee, et al., 2002;Martin, et al., 2006).
Second, Leucine-rich repeat kinase 2 (LRRK2) is gene related to a form of autosomal
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dominant and late-onset familial PD. Interestingly, it has been reported that mutation of LRRK2 is also related with sporadic PD (Rajput, et al., 2006;Ross, et al., 2006;Zimprich, et al., 2004). LRRK2 has multiple domains, including ankyrin-like (ANK), leucin-rich repeat (LRR), Ras of complex (ROC), C-terminal of ROC (COR), kinase and WD40 domains. PD-related mutations have been identified in several domains, for example, R1441C/G.H in the ROC domain, G2019S in the kinase domain, and G2385R in the WD40 domain (Covy, et al., 2009;Paisan-Ruiz, et al., 2004;Wang, et al., 2010;Zheng, et al., 2011;Zimprich, et al., 2004).
LRRK2 regulates filopodia protrusion and neurite outgrowth through interaction with actin regulatory protein, ERM and Rac1, and prevention of the actin polymerization process (Jaleel, et al., 2007;Meixner, et al., 2011;Parisiadou, et al., 2009). In addition, LRRK2 is related to calcium homeostasis (Cherra, et al., 2013;Gomez-Suaga and Hilfiker, 2012), inflammation (Gardet, et al., 2010;Kim, et al., 2012;Moehle, et al., 2012), mitochondria dynamics (Su and Qi, 2013), synaptic vesicle trafficking (Matta, et al., 2012;Piccoli, et al., 2011;Shin, et al., 2008), nuclear envelope integrity (Liu, et al., 2012), autophagy (Gomez-Suaga, et al., 2012;Manzoni, 2012), and microglia motility (Choi, et al., 2015).
Third, PTEN-induced kinase 1 (PINK1) is gene related to an autosomal recessive early-onset form of PD (Valente, et al., 2001). PINK1 has an N-terminal mitochondrial targeting sequence and a serine/threonine kinase domain. PINK1 is widely expressed throughout various brain regions (Blackinton, et al., 2007;Chiba, et al., 2009;Taymans, et al., 2006) and it is in expressed neurons, glial cells and neural stem cells (d'Amora, et al., 2011;Gandhi, et al., 2006). PINK1 has various roles such as the regulation of ATP generation, oxygen consumption (Beilina, et al., 2005;Liu, et al., 2009;Sim, et al., 2006), ROS
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production (Gandhi, et al., 2009) and proliferation (Choi, et al., 2013). In addition, PINK1 deficient cells are more vulnerable to various insults compared with wild-type cells (Deng, et al., 2005;Haque, et al., 2008). Moreover, PINK1 regulates a mitochondrial clearance process called mitophagy by accumulating on the outer membrane of hyperpolarized mitochondria and recruiting another familial PD related protein, Parkin (Kondapalli, et al., 2012;Matsuda, et al., 2010;Narendra, et al., 2010;Vives-Bauza, et al., 2010).
Forth, Parkin is associated with form of autosomal recessive juvenile PD (Kitada, et al., 1998). Parkin has activity as an E3 ubiquitin ligase. Many studies have identified substrates of Parkin such as septins CDC-rel1, CDC-rel2 (Ageta-Ishihara, et al., 2013;Zhang, et al., 2000), synaptotagmin XI (Shimura, et al., 2000), synphilin-1 (Chung, et al., 2001), Paelr1 (Imai, et al., 2001), CHIP (Imai, et al., 2002), cyclinE (Staropoli, et al., 2003), and p38 in the tRNA synthase complex (Corti, et al., 2003).
６ C. Function of astrocytes in the normal brain
Astrocytes are the most abundant cells in the brain and play diverse roles in the well-being and function of the brain. Astrocytes maintain homeostasis of the brain’s microenvironment including neuronal metabolism, neurotransmitter synthesis, formation of the blood brain barrier (BBB), maintenance of the extracellular environment, and regulation of cerebral blood flow (Ransom and Ransom, 2012).
Typically, it has been reported that astrocytes provide nutrients and growth factors for neurons, (Banker, 1980;Walz and Mukerji, 1988). These cells also provide an energy source to adjacent neurons through lactate shuttling for metabolic regulation (Chih and Roberts Jr, 2003). Processes of astrocytes extended to the pre- and post- synaptic terminals, constructing a physical barrier that limit neurotransmitter diffusion (Araque, et al., 1999a;Araque, et al., 1999b). In addition, accumulated K+, a result of neuronal activity, is rapidly removed and glutamate is converted to glutamine by the uptake of astrocytes to maintain the extracellular concentration of neurotransmitters (Verkhratsky and Kirchhoff, 2007). Astrocytes also have neurotransmitter receptors such as glutamatergic, GABAergic, adrenergic, and serotonergic receptors (Larsson, et al., 1980;Lee, et al., 2010;Perea, et al., 2009;Perez-Alvarez and Araque, 2013;Porter and McCarthy, 1997). In order to modulate homeostasis in brain regions, astrocytes have a cellular network where they couple with other astrocytes via gap junctions (Rouach, et al., 2000), where metabolic substrates and ion for cell-to-cell communication are exchanged (Gardner-Medwin, 1983). In addition, the gap junction between astrocytes plays a role in removing extracellular glutamate and potassium in order to regulate synaptic activity (Rouach, et al., 2008). Astrocytes also play a role in the
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regulation of vessel permeability. Endfeet of astrocyte processes extend to capillaries and surround the endothelial cells of capillaries to form the BBB (Abbott, 2002;Mathiisen, et al., 2010). The BBB is an important regulatory factor between the brain’s bllod supply and the CNS. The movement of molecules, ions and cells between the brain and blood is tightly regulated by the BBB (Abbott and Friedman, 2012;Daneman, 2012;Luissint, et al., 2012;Wong, et al., 2013). Therefore, astrocytes are an important cell type in the maintenance of the brain’s environment and activation.
D. Function of astrocytes in the injured brain
Astrocytes are activated by various insults to the brain, such as trauma, ischemia and neurodegenerative disease, and respond via the process of astrogliosis. In the damaged brain, reactive astrocytes show different morphological features and expression of genes compared with normal astrocytes in order to protect and repair the injured brain (Eddleston and Mucke, 1993;Eng and Ghirnikar, 1994;Hernandez, et al., 2002;Pekny and Nilsson, 2005).
1. Reactive astrocytes: intermediate filaments (IFs) and morphological features
In the injured brain, astrocytes show high expression of intermediate filaments (IFs) such as glial fibrillary acidic protein (GFAP), nestin and vimentin. In particular, GFAP is the primary intermediate filament of astrocytes and has been used as a hallmark of reactive astrocytes in the injured brain (Correa-Cerro and Mandell, 2007;Eddleston and Mucke, 1993;Eng, et al., 2000). GFAP and the other IFs of reactive astrocytes are seen in a wide range of brain insults such as trauma, ischemic or hemorrhagic damage, epilepsy,
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Alzheimer's disease, PD and multiple sclerosis (Burda and Sofroniew, 2014). Interestingly, a lack of GFAP and other IFs leads to the induction of neuronal damage by traumatic cerebrospinal injury, cerebral ischemia and traumatic or kainate excitotoxicity (Li, et al., 2008;Nawashiro, et al., 2000;Nawashiro, et al., 1998;Otani, et al., 2006).
Upregulated IFs cause morphological changes in reactive astrocytes such as hypertrophy of the cell body, and thickening and extension of the cellular processes (Wilhelmsson, et al., 2004). In a GFAP and vimentin dual KO mouse model, reactive astrocytes demonstrated less hypertrophy, process shortening and a reduction of glial scar formation when compared with WT.
2. Reactive astrocytes: function of astrogliosis in neural protection
Reactive astrocytes construct a physical barrier around damaged core regions with microglia/macrophages, extracellular matrix molecules, perivascular fibroblasts and pericytes, to isolate the site of injury (Burda and Sofroniew, 2014;Cregg, et al., 2014;Rolls, et al., 2009). After injury, the number of reactive astrocytes increases and surrounds the damaged core region (Fitch and Silver, 1997;Reier and Houle, 1988). Moreover, reactive astrocytes eliminate increased extracellular glutamate after injury (Rothstein, et al., 1996;Swanson, et al., 2004). Ablation of reactive astrocytes leads to a reduction in glutamate transporter expression and an induction of neuronal degeneration due to the excitotoxic effects of accumulating glutamate (Cui, et al., 2001). In addition, reactive astrocytes regulate oxidative stress after injury (Desagher, et al., 1996). For example, neuronal death due to nitric oxide (NO) or oxidative glutamate toxeicity was increased in a co-culture system with
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glutathione deficient astrocytes (Chen, et al., 2001;Shih, et al., 2003). Reactive astrocytes also provide neuronal protection against ammonia toxicity. Ammonia, CNS dysfunction associated neurotoxin with hepatic encephalopathy, induced extensive degeneration in pure neuron culture. However, extensive degeneration was decreased in co-culture system with astrocytes (Rao, et al., 2005).
3. Reactive astrocytes: function of astrogliosis for regeneration and repair
In the past, various studies have suggested the potential negative effects of astrogliosis scar formation such as the elimination of neural regeneration. Numerous therapeutic studies have reported that the degradation of scar formation through techniques such as enzymes to eliminate scar formation (Moon, et al., 2001;Tester and Howland, 2008), and inhibition of astrocyte proliferation (Tian, et al., 2007), led to beneficial effects for regeneration. However, growing evidence has demonstrated that astrogliosis has important roles in regeneration and repair by regulating the supply of nutrients, angiogenesis, remyelination, neurotrophic factor s and neurogenesis (do Carmo Cunha, et al., 2007;Liberto, et al., 2004;Triolo, et al., 2006;White, et al., 2008). Reactive astrocytes showed increased glucose uptake and lactate release in hypoxic conditions (Marrif and Juurlink, 1999) and stored glycogen following stimulation by insulin-like growth factor-1 (IGF-1) (Dringen and Hamprecht, 1992) to provide energy support for neighboring neurons. In addition, astrocytes are associated with fibronectin, which outgrowth of dorsal root ganglion (DRG) neurites and axon regeneration in mature white matter (Tom, et al., 2004). Reactive astrocytes also correlated with neovascularization and angiogenesis, which provide oxygen and nutrients to
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the injured brain area. Vascular endothelial growth factor (VEGF) is a key molecule in angiogenesis and is induced by platelet-activating factor (PAF) from reactive astrocytes after stab wounds, neural grafting and hypoxia (Krum and Rosenstein, 1998;Yoshida, et al., 2002).
Reactive astrocytes also correlates with remyelination. Ciliary neurotrophic factor (CNTF) has been shown to regulate the induction of oligodendrocyte precursor proliferation through fibroblast growth factor-2 (FGF-2) for remyelination (Albrecht, et al., 2003). Interestingly, the levels of CNTF, which is present in normal astrocytes, were highly increased in reactive astrocytes after brain injury (Dallner, et al., 2002). In addition, CNTF induced FGF-2 expression in reactive astrocytes during remyelination in the spinal cord (Albrecht, et al., 2003). Moreover, it has been reported that reactive astrocytes express neurotrophic factor.
For example, glial cell line-derived neurotrophic factor (GDNF) and its receptors are expressed during development and in the adult brain (Arenas, et al., 1995;Buj-Bello, et al., 1995;Oppenheim, et al., 1995). GDNF reported to effect neuronal survival (Kordower, et al., 2000;Perrelet, et al., 2002) and axonal regeneration (Bjorklund, et al., 1997;Iannotti, et al., 2003;Mills, et al., 2007). It has been reported that reactive astrocytes also express GDNF (Moretto, et al., 1996;Nakagawa and Schwartz, 2004). Recently, detection of reactive astrocyte derived neural stem and progenitor cells suggest that reactive astrocytes have stem cell-like properties after brain injury (Gotz, et al., 2015;Shimada, et al., 2012), since neural stem cell-like cells have been found in cortical tissues after various injuries such as stabbing injuries and stroke (Buffo, et al., 2008;Nakagomi, et al., 2009;Shimada, et al., 2010).
Reactive astrocytes also express several stem cell associated protein such as GFAP, Nestin, RC2 and Sox2 (Buffo, et al., 2008;Pekny and Pekna, 2004;Shimada, et al., 2010).
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Furthermore, glutamate aspartate transporter (GLAST)-positive reactive astrocytes showed de-differentiation and multipotent spheres formation potential (Buffo, et al., 2008).
4. Reactive astrocytes: function of astrogliosis for regulation of inflammation
Brain inflammation is a critical defense mechanism and process to regenerate the microenvironment after brain injury through the action of microglia and infiltrating marcrophages. Microglia and macrophages produce neurotrophic factors, such as transforming growth factor (TGF)-b1, neurotrophin (NT)-3 and brain-derived neurotrophic factor (BDNF). (Batchelor, et al., 1999;Elkabes, et al., 1996;Garg, et al., 2008;Glezer, et al., 2007;Jeong, et al., 2013a;Jeong, et al., 2013b;Lehrmann, et al., 1998;Schwartz, et al., 2006;Streit, 2005;Streit, 2002). However, uncontrolled brain inflammation could accelerate the progression of injury (Chao, et al., 1992;Choi, et al., 2003;Kitamura, et al., 1996). Many studies suggest that brain inflammation is a risk factor for neurodegenerative diseases such as Alzheimer's disease (AD), PD, and multiple sclerosis (MS) (Breitner, 1996;Chen, et al., 2003;Klegeris and McGeer, 2005;Raivich and Banati, 2004;Sheng, et al., 1998). Therefore, brain inflammation is tightly regulated to maintain its beneficial effects.
Following numerous studies, it has been demonstrated that astrocytes or reactive astrocyte have essential anti-inflammatory roles (Sofroniew, 2015). For example, astrocytes release various cytokines such as TGF-b, IL-6, IL-10, IL-11 IL-19, and IL-27 which activate anti-inflammatory signaling and immunosuppressive effects (Jensen, et al., 2013;John, et al., 2005;Meeuwsen, et al., 2003;Zamanian, et al., 2012). Furthermore astrocytes also produce prostaglandins E2 (PGE2) as an anti-inflammatory factor (Molina-Holgado, et al., 2000) and
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astrocyte-conditioned media (ACM) suppresses nitrite, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-a expression in IFN-g treated BV2, primary microglia.
ACM also induced endogenous NRF2 translocation and HO-1 expression. (Min KJ et al., 2006). In addition, several studies have suggested that the inflammatory response is increased in astrogliosis ablated transgenic mice after stroke (Li, et al., 2008;Liu, et al., 2014) and spinal cord injury (Herrmann, et al., 2008;Okada, et al., 2006).
E. Astrocyte dysfunction in neurodegenerative disease
Astrocyte dysfunction has been observed in various neurodegenerative diseases.
Amyotrophic lateral sclerosis (ALS) is an adult motor neuron diseasecharacterized by the progressive degeneration of motor neurons in the cortex, the brainstem and spinal cord.
Studies in human suggested that sporadic and familial ALS showed reduced levels of the astrocyte glutamate transporter, EAAT2 (Bristol and Rothstein, 1996). In a superoxide dismutase (SOD) mutated animal model, glutamate transport and GLT1 expression was decreased in astrocytes before neuronal degeneration (Bruijn, et al., 1997;Howland, et al., 2002). AD is the most common neurodegenerative disease and causes functional loss of cognitive abilities such as memory, language, and calculation (Selkoe, 2001). In human studies, neuron-derived amyloid material was accumulated in astrocytes and human amyloid-b (Aamyloid-b) activated astrocytes. In addition, Aamyloid-b affected the induction of calcium wave signaling in astrocytes (DeWitt, et al., 1998;Haughey and Mattson, 2003;Nagele, et al., 2004). In familial AD, calcium oscillations were founded in astrocytes with mutated presenilin 1 (PSEN1) leading to low concentrations of ATP and glutamate (Johnston, et al., 2006). In
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animal models, expression of tau protein in astrocytes, which is on of the hallmarks of AD, caused a reduction in the expression and function of gluatamate transporters GLT1 and GLAST (Dabir, et al., 2006). PD is the second most common neurodegenerative disease, after AD. In human studies, GFAP immunoreactivity, a marker of reactive astrocytes, was found in the striatum and the substantia nigra pars compacta (SNpc) of postmortem PD cases (Dervan, et al., 2004;Mirza, et al., 2000). Interestingly, morphological changes such as enlarged cell bodies and distorted processed in the reactive astrocytes were different when compared with those in other neurodegenerative disease (Mirza, et al., 2000;Song, et al., 2009). In an animal model, it has been reported that astrocytes that expressed PD-related A53T mutant a-synuclein induced progressive paralysis. In addition, the mutated astrocytes were shown to have down-regulation of the astrocytic glutamate transporters (Gu, et al., 2010).
F. Aim of this study
Since it has been reported that PD is characterized by dopaminergic neuronal death in the SNpc, almost all studies investigating the progression of PD were focused on neuronal death. Unfortunately, little is known about the progression and development of PD. Recently, increasing evidence has suggested that dopaminergic neuronal death could be induced by functional defects in non-neuronal cells necessary for neuronal survival in either the intact brain or pathological conditions. Astrocytes are the most abundant glial cell and have numerous functions in the maintenance of the brain’s environment. In the intact healthy brain, astrocytes regulate brain homeostasis by regulating extracellular ion concentrations,
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neurotransmitters, the BBB, and cerebral blood flow. In pathological conditions, reactive astrocytes perform a number of actins necessary to form a physical barrier to isolating the injury site through morphological change and proliferation, and provide neural protection by regulating of inflammation and oxidative stress. In addition, reactive astrocytes play a critical role in brain repair by providing energy support, releasing neurotrophic and growth factors, and regulating angiogenesis and remyelination. Therefore, dysfunction of astrocytes is intricately linked with the progression of neurodegeneration.
The specific aims of this study were
1. Whether DJ-1 deficiency attenuates endogenous repair and astrogliosis
2. Whether DJ-1 deficiency attenuates the anti-inflammatory function of astrocytes
As a result, I examined how the PD-related gene, DJ-1 affects the functions of astrocytes in pathological conditions.
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II. Materials and methods
1. Ethics statement-
All experiments were performed in accordance with the approved animal protocols and guidelines established by the Ajou University School of Medicine Ethics Review Committee for animal experiments, and all animal work was approved by the Ethical Committee for Animal Research of Ajou University (2014-0029; AMC119).
2. DJ-1 deficient mice
The DJ-1 KO mice used in this study were a generous gift from Dr. UJ Kang (Chicago University, Chicago, IL, USA). DJ-1 KO mice were previously generated by deleting a 9.3- kb region of genomic DNA containing the first five exons and part of the promoter region of the DJ-1 gene (Chen, et al., 2005).
3. Animal MRI
c57/BL6 mouse were anesthetized by Isoflurane. Brain damage was measured by 9.4T MRI (BioSpec 94/30 US/R, BRUKER, USA) and Volume RF coil (Inner diameter 23 mm, BRUKER,USA) CNIR of Sungkyunkwan University. Using parameter was 2D T2 Turbo RARE sequence (TR/TE = 9000/33 ms; Resolution = 78 mM x 78 mM x 250 mM;
Slice thickess = 250 mM; RARE factor = 8; Average = 2; Scan time = 9 m36 s).
１６ 4. Stereotaxic surgery and drug injection
Male c57/BL6 mouse were anesthetized by injection of Tribromethanol (250 mg/kg, i.p) and positioned in a stereotaxic apparatus (David Kopf instruments, USA). ATP (400
Male c57/BL6 mouse were anesthetized by injection of Tribromethanol (250 mg/kg, i.p) and positioned in a stereotaxic apparatus (David Kopf instruments, USA). ATP (400