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Exp Neurobiol 2016; 25(4): 147-155
Published online August 31, 2016
© The Korean Society for Brain and Neural Sciences
Eunju Leem1,2#, Kyoung Hoon Jeong1,2#, So-Yoon Won4, Won-Ho Shin5* and Sang Ryong Kim1,2,3,6*
1School of Life Sciences & Biotechnology, 2BK21 plus KNU Creative BioResearch Group, 3Institute of Life Science & Biotechnology, Kyungpook National University, Daegu 41566, 4Department of Biochemistry and Signaling Disorder Research Center, College of Medicine, Chungbuk National University, Cheongju 28644, 5Predictive Research Center, Korea Institute of Toxicology, Daejeon 34114, 6Brain Science and Engineering Institute, Kyungpook National University, Daegu 41944, Korea
Correspondence to: *To whom correspondence should be addressed.
Sang Ryong Kim, TEL: 82-53-950-7362, FAX: 82-53-943-2762
Won-Ho Shin, TEL: 82-42-610-8088, FAX: 82-42-610-8157
#These authors contributed equally to this work.
Although accumulating evidence suggests that microglia-mediated neuroinflammation may be crucial for the initiation and progression of Parkinson's disease (PD), and that the control of neuroinflammation may be a useful strategy for preventing the degeneration of nigrostriatal dopaminergic (DA) projections in the adult brain, it is still unclear what kinds of endogenous biomolecules initiate microglial activation, consequently resulting in neurodegeneration. Recently, we reported that the increase in the levels of prothrombin kringle-2 (pKr-2), which is a domain of prothrombin that is generated by active thrombin, can lead to disruption of the nigrostriatal DA projection. This disruption is mediated by neurotoxic inflammatory events
Keywords: Prothrombin kringle-2, Parkinson’s disease, Microglia, Toll-like receptor 4
Parkinson's disease (PD) is the second-most common neurodegenerative disorder and is characterized by the progressive degeneration of dopaminergic (DA) neurons and a decrease in striatal dopamine. PD is associated with clinical movement disorders, including a tremor at rest, rigidity of the limbs, bradykinesia (slowness and paucity of voluntary movement), and postural instability (a tendency to fall even in the absence of weakness or cerebellar balance disturbance) [1,2,3]. Although we do not fully understand the etiology of PD, accumulating evidence suggests that microglia, which are the resident immune cells of the brain, are crucial mediators of the brain inflammatory processes that lead to neurotoxicity, and that excessive microglial activation contributes to the initiation and progression of PD [3,4,5]. However, it is largely unknown what endogenous biomolecules initiate and stimulate microglial activation, even though the control of microglial activators, which stimulate neurotoxic inflammation, may be a useful strategy for the prevention of the degeneration of the nigrostriatal DA projection in the adult brain.
Toll-like receptors (TLRs) are pattern recognition receptors that recognize specific pathogen-associated molecular signatures and subsequently initiate inflammatory and immune responses [3,6]. TLR4 recognizes various ligands, such as lipopolysaccharide (LPS), envelope proteins, heat-shock proteins, fibrinogen, and hyaluronan [3,6]. The activation of TLR4 in immune cells induces increases in the levels of inflammatory cytokines [3,7]. Although the pattern of TLR expression in the brain is controversial, there are many reports suggesting that microglia are important cells for TLR4-mediated immune responses, which may be involved in neurodegenerative diseases such as Alzheimer's disease (AD) and PD [8,9,10]. Moreover, increases in TLR4 expression have been observed in α-synuclein-overexpressing transgenic mice and in patients with multiple system atrophy , although alterations in TLR4 expression in patients with PD are still unclear. These results suggest that an increase in microglial TLR4 may be crucial for the pathogenesis of PD, and that the discovery of endogenous molecules involved in the induction of microglial TLR4 may be useful in guiding the development of knowledge-based targeted therapeutics for PD.
We previously reported that prothrombin kringle-2 (pKr-2), which is a domain of prothrombin that is generated by active thrombin, is able to induce the death of DA neurons in the rat SN through microglial activation, even though pKr-2 itself was not directly toxic to neurons . Moreover, we recently found that patients with PD have increased pKr-2 expression in the SN, and that nigrostriatal DA projections might degenerate due to neurotoxic inflammation following pKr-2 upregulation-induced production of microglial TLR4 in the SN of adult murine brain . These results suggest that pKr-2 might be a potential pathogenic factor in PD, and that limiting pKr-2-induced microglial activation may be an effective therapeutic strategy for protecting DA neurons in the adult brain.
The histopathological features of PD are the idiopathic degeneration of DA neurons in the pars compacta of the SN and loss of DA nerve terminals in the striatum . This progressive neurodegeneration, which consequently results in the reduction of dopamine in the nigrostriatal DA system , is generally accompanied by both motor and non-motor symptoms. The non-motor symptoms of PD include olfactory dysfunction, cognitive impairment, psychiatric symptoms, sleep disorders, pain, depression, and rapid eye movement sleep behavior disorders . The motor symptoms of PD include movement disorders such as resting tremor, muscular rigidity, bradykinesia, akinesia, and postural instability . Although the maintenance of the dopamine concentration is considered to be a useful target in the development of therapeutics against PD progression, clinical trials focusing on dopamine production have not been successful [14,15]. Levodopa (L-3,4-dihydroxyphenylalanine), which is a precursor of dopamine, is one of the main drugs used to treat PD symptoms. However, the long-term use of levodopa is associated with complications, such as abnormal involuntary movements called dyskinesias and dystonias [14,15]. Moreover, no treatment has been identified that forestalls deterioration attributable to progressive neurodegeneration [1,2]. These findings indicate that sustained dopamine supplementation alone is unable to protect or restore DA systems during PD progression. Thus, the control of PD pathogenesis using approaches such as inhibition of mitochondrial dysfunction and reduction of activated microglia-derived oxidative stress and/or neuroinflammation, may be more important in treating PD progression than the maintenance of dopamine production [16,17,18,19], even though this is the major therapeutic strategy currently used to treat patients with PD.
Accumulating evidence suggests that microglial activation, which is an important neurotoxic mechanism, plays important roles in the initiation and progression of PD [20,21]. Imamura et al. have previously reported that the accumulation of CR3/43-positive cells (activated microglia) is increased in the SN and putamen in the post-mortem brains of patients with PD . Furthermore, [11C](R)-PK11195, which is a radiotracer used to detect activated microglia, noticeably accumulates in the brains of patients diagnosed with PD . These findings strongly suggest that microglial activation negatively affects neuronal cell survival and consequently leads to neurodegeneration in PD.
Microglia are the resident immune cells in the central nervous system (CNS). In the resting state, microglia have small cell bodies and numerous processes and can support neuronal function and survival . In pathological conditions, microglia stimulated by various activators undergo phagocytic morphological changes, which are characterized by enlarged cell bodies and short processes, and these altered microglia exert beneficial effects that repair tissue by releasing anti-inflammatory cytokines and neurotrophic factors [22,23]. However, the major cause of neurotoxic inflammation in the CNS is the response of microglia to a variety of stimuli, such as infection, trauma, and toxins , and activated microglia produce neurotoxic inflammatory cytokines, such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 [22,24]. In addition, activated microglia can produce reactive oxygen species (ROS), such as O2- and O2--derived oxidants,
Consistent with the observation of increases in NADPH oxidase and neurotoxic cytokines in the brains of patients with PD [3,19,20,28], there are many reports indicating the significance of these factors involved in the degeneration of the nigrostriatal DA system in animal models of PD. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was discovered to be a neurotoxic compound in drug addicts who showed parkinsonian symptoms  and has been widely used in the production of PD animal models . In the SN of adult mice, MPTP induces microglial activation, which is involved in neurotoxicity . The blockade of microglial activation following treatment with minocycline, a broad-spectrum tetracycline antibiotic, inhibits the production of microglial-derived deleterious factors, and exerts neuroprotective effects in the MPTP-lesioned SN . These suggest that microglial activation is involved in the PD pathogenesis related to DA neuronal damage. In addition, the increased expression of gp91phox, which is an NADPH oxidase subunit, is observed in the activated microglia of MPTP-injected mice . In fact, the upregulation of gp91phox within activated microglia is markedly reduced by minocycline treatment, which leads to the protection of DA neurons
6-hydroxydopamine (6-OHDA) is also often used to produce PD models for the testing symptomatic therapies and the study of the mechanisms of DA neuronal death . Since the structure of 6-OHDA is similar to that of dopamine, it is taken up into DA neurons by the dopamine transporter, where it acts as a neuronal toxin by generating ROS . In addition to direct neuronal toxicity, 6-OHDA-induced neurotoxicity can promote microglial activation in nigrostriatal DA projection areas [32,33], resulting in the production of neurotoxic proinflammatory cytokines [34,35]. In addition, microglial activation induced by 6-OHDA treatment parallels the activation of NADPH oxidase, including the p47phox and gp91phox subunits, in nigral microglia . Similar to the effects of MPTP, the 6-OHDA-induced toxic effects in DA neurons are attenuated by minocycline treatment . Taken together, these results suggest that microglial-derived neuroinflammation, which includes the production of neurotoxic cytokines and activation of NADPH oxidase within microglia, may play a crucial role in DA neuronal degeneration in animal models of PD. In addition to MPTP and 6-OHDA, studies using rotenone, which is an odorless, colorless, crystalline isoflavone used as a broad-spectrum insecticide, piscicide, and pesticide, have also indicated that the production of neurotoxic cytokines and activation of NADPH oxidase in microglia play important roles in PD pathogenesis [38,39].
The above observations indicate that excessive increases in proinflammatory mediators and ROS production following microglial activation lead to severe neurotoxicity, resulting in the deterioration of the DA system in the adult brain. While numerous studies have examined the development of therapeutic and preventive agents against PD, there are currently no cures that stop or slow down the progressive degeneration of DA neurons and PD symptoms. Therefore, efforts to develop therapeutic and preventive agents for PD need to aim at inhibiting microglial-derived neuroinflammation, which would suppress microglial activation and its pathogenic mechanisms.
TLR4 initiates the activation of the innate immune response by recognizing pathogens as a pattern-recognition receptor. LPS is a representative ligand for TLR4 . The TLR4-mediated activation of NF-κB and mitogen-activated protein kinases that occurs
Prothrombin, which is known to be synthesized mainly in the liver and then secreted into the bloodstream , is cleaved to produce fragment 1-2 (kringle regions) and thrombin during activation [53,54]. Activated thrombin, a serine protease which converts soluble fibrinogen into insoluble fibrin for blood coagulation, can cleave pKr-1 and -2 (Fig. 1) . The functions of pKr-2 are well-established in the field of angiogenesis. For instance, pKr-2, which is purified from LPS-treated rabbit serum, acts as an angiogenic inhibitor during bovine capillary endothelial cell proliferation . Treatment with pKr-2 induces the suppression of basic fibroblast growth factor-triggered endothelial cell growth and angiogenesis in the chorioallantoic membrane of chick embryos . Moreover, treatment with pKr-2 inhibits endothelial cell proliferation and angiogenesis by inactivating the cyclin D1/cyclin-dependent kinase 4 (CDK4) complex through the induction of ROS production and upregulation of nuclear CDK inhibitors . It also inhibits fibrin formation and platelet aggregation by binding to thrombin and inducing conformational changes at its active site, resulting in a reduction in the clotting activity of thrombin . In addition, recombinant human pKr-2 reduces the immunoreactivity of matrix metalloproteinases 2 and 9 and the expression of vascular endothelial growth factor, which results in the inhibition of B16F10 melanoma cell metastasis .
Although many studies have investigated the functions of pKr-2 [56,57,58,59,60] and prothrombin is expressed in brain tissues , few reports have examined the roles of pKr-2 in the CNS. The accumulation of prothrombin and thrombin, which might be due to blood-brain barrier leakage, has been shown in the brains of patients with PD and AD [61,62,63], which suggests a possible increase in pKr-2 expression. Moreover, we previously reported that the upregulation of pKr-2 could contribute to microglial activation, resulting in neurodegeneration in the SN of murine brains . Therefore, these results suggest that pKr-2 expression is increased in the lesioned brain and that its upregulation is involved in neurotoxic effects in the adult brain.
pKr-2 can trigger microglial activation, resulting in the production of neurotoxic cytokines such as TNF-α and IL-1β, and inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 [5,64,65]. In addition, pKr-2-activated microglia produce O2- and O2--derived oxidants through the activation of NADPH oxidase, which is also involved in neurotoxic events in the murine cortex . Moreover, we recently suggested that pKr-2 upregulation is involved in the pathogenesis of PD, and that the control of microglial pKr-2 expression and pKr-2-induced microglial TLR4 overexpression might be important for protecting the nigrostriatal DA system against PD . These results help the understanding of the relationships among pKr-2, microglia, and TLR4 (Fig. 2). To ascertain whether pKr-2 is involved in PD
Since inhibiting inflammation is an important strategy for the prevention of neuronal damage in neurodegenerative disease, therapies for the control of the activation of microglia have been proposed. Although the exact mechanisms are unknown, our recent results strongly suggest that TLR4 and pKr-2 are closely associated with neuroinflammation in PD . Similar to our results involved in neurodegeneration in the DA system, previous studies have shown that TLR4 expression is upregulated in the MPTP-treated animal model of PD  and that microglial TLR4 is directly activated by α-synuclein treatment . Taken together, these results suggest that the modulation of microglial TLR4 by controlling pKr-2 production may provide us with important clues regarding the mechanisms responsible for inflammation-associated neurodegeneration in PD and open innovative therapeutic perspectives for the treatment of PD. For instance, treatment with minocycline has neuroprotective effects in preclinical studies of neurodegenerative diseases [2,30,67,68,69,70,71]. In particular, treatment with minocycline protects DA neurons against pKr-2-induced neurotoxicity through the inhibition of inflammatory responses in the brains of adult mice . Moreover, its treatment reduces the expression of proinflammatory cytokines and iNOS, which is significantly increased by activated microglia following pKr-2 upregulation. These results suggest that the development of efficient anti-inflammatory drugs against pKr-2 may be useful for protecting DA neurons in the SN of lesioned adult brain.
The discovery of endogenous biomolecules that initiate and stimulate microglial activation and result in neurodegeneration