Emerging roles of protein kinases in microglia-mediated neuroinflammation

Biochemical Pharmacology 146 (2017) 1–9

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Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

Research update

Emerging roles of protein kinases in microglia-mediated neuroinflammation Sun-Hwa Lee a,⇑, Kyoungho Suk b,⇑ a b

New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Republic of Korea Department of Pharmacology, Brain Science & Engineering Institute, Kyungpook National University School of Medicine, Daegu 41944, Republic of Korea

a r t i c l e

i n f o

Article history: Received 18 May 2017 Accepted 30 June 2017 Available online 4 July 2017 Keywords: Microglia Neuroinflammation Neurodegenerative disease Protein kinase

a b s t r a c t Neuroinflammation is mediated by resident central nervous system glia, neurons, peripherally derived immune cells, blood-brain barrier, and inflammatory mediators secreted from these cells. Neuroinflammation has been implicated in stroke and neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis. Protein kinases have been one of the most exploited therapeutic targets in the current pharmacological research, especially in studies on cancer and inflammation. To date, 32 small-molecule protein kinase inhibitors have been approved by the United States Food and Drug Administration for the treatment of cancer and inflammation. However, there is no drug effectively targeting neuroinflammation and/or neurodegenerative diseases. Recent studies have advanced several protein kinases as important drug targets in neuroinflammation and/or neurodegenerative diseases. Here, we review emerging protein kinases potentially involved in neuroinflammation and subsequent neurodegenerative diseases. Ó 2017 Elsevier Inc. All rights reserved.

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PKs and neuroinflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. PKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Microglia and neuroinflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emerging PKs associated with microglial activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Leucine-rich repeat kinase 2 (LRRK2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. LRRK2 in microglial activation/migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. LRRK2 in microglial phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Cellular Abelson murine leukemia viral oncogene homolog 1 (c-Abl). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Src family protein tyrosine kinases (SFKs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Mammalian Ste20-like kinase 1 (MST1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. TAM family receptor tyrosine kinases (RTKs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 2 2 3 3 3 4 5 5 6 7 7 7 8

1. Introduction

⇑ Corresponding authors at: New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Republic of Korea (S.-H. Lee). Department of Pharmacology, Kyungpook National University School of Medicine, Daegu 41944, Republic of Korea (K. Suk). E-mail addresses: [email protected] (S.-H. Lee), [email protected] (K. Suk). http://dx.doi.org/10.1016/j.bcp.2017.06.137 0006-2952/Ó 2017 Elsevier Inc. All rights reserved.

The definition of neuroinflammation has been recently modified to emphasize its pathological nature. Classically, neuroinflammatory responses were constituted mainly of the activation of central nervous system (CNS)-resident microglia and their release of inflammatory mediators, such as cytokines, chemokines, and

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reactive oxygen/nitrogen species. Recently, however, infiltration of immune and inflammatory cells of blood origin and tissue injury in the CNS have been proposed as major elements of neuroinflammation [1–3]. Regardless of the conceptual differences in the definition of neuroinflammation, glia are undoubtedly the central players in neuroinflammation. In addition to a long-standing role of microglial activation, recent studies indicated an emerging role of astrocytes in neuroinflammation. Although astrocytes are mainly neuroprotective and neuromodulatory in many ways, providing metabolic and structural support to neurons and engaging in synaptic modulation, reactive astrocytes may contribute to neuroinflammation, following microglial activation and neural injury [4]. Nevertheless, microglia are the most important components of neuroinflammation. Furthermore, microglia-mediated neuroinflammation has received increasing attention as a non-cellautonomous mechanism of neurodegeneration and brain injury [5]. Activated microglia display diverse functional phenotypes with M1 and M2 being two extreme states [6]. While microglia with an M1-like phenotype are proinflammatory and often neurotoxic in nature, M2 microglia are associated with beneficial effects toward neurons and the CNS as a whole. The M1/M2 classification of microglial phenotype is, however, considered to be an oversimplified view, and its existence has been recently questioned [7,8]. Seemingly distinct microglial phenotypes are mostly determined by their microenvironment [9] and external stimuli to which they respond. Therefore, to exploit the beneficial properties of activated microglia, it is critical to understand how microglia respond to external stimuli in a given microenvironment, and how to manipulate their responses in order to promote their beneficial characteristics [10]. This is where signal transduction pathways come into play. Signal transduction pathways govern when and how microglia respond to numerous input signals in their microenvironment and determine the functional phenotypes that microglia will ultimately adopt. Given that protein kinases (PKs) are the most important constituent of intracellular signal transduction, it is imperative to appreciate their role in microglia and the associated neuroinflammation, which would ultimately lead to the development of new drugs targeting PKs in microglia.

2. PKs and neuroinflammation 2.1. PKs Kinases constitute a major part of the phosphotransferases in the human genome. They transfer a c-phosphate group of adenosine triphosphate (ATP) to various substrates including lipids, sugars, and proteins. The human genome has been so far confirmed to comprise 20 lipid kinases, 518 protein kinases (PKs), and over 900 genes encoding proteins with kinase activity [11–13]. Therefore, PKs form the largest subcategory among phosphotransferases. Human PKs are largely grouped into two classes: the protein tyrosine kinases (PTKs) and the serine- and threonine-specific kinases (SPKs). The former phosphorylates only the tyrosine residues, whereas the latter phosphorylates the serine and/or threonine residues on the protein substrates. Although there is only one protein histidine kinase that phosphorylates histidine residues and a few dual specificity PKs that phosphorylate both tyrosine and serine/ threonine (S/T) residues on target proteins, SPKs constitute the majority of PKs, with an estimated ratio of cellular protein phosphorylation in serine:threonine:tyrosine of 1000:100:1 [14]. Since kinases are involved in virtually all cellular activities, including cell growth, survival, proliferation, differentiation, and metabolism, and since dysregulation of their activity has been associated with numerous diseases, including cancer, CNS

disorders, and vascular and chronic inflammatory conditions, kinases have become attractive drug targets most intensively pursued by both pharmaceutical industries and academia in the past two decades. Although only a minor number of PTKs’ substrates have been identified, many PTKs gain and/or loss of function mutations have been linked to various diseases, suggesting PTKs as important drug targets. To date, over 40 kinase inhibitors including 32 small-molecule inhibitors and six antibodies, have been approved by the United States Food and Drug Administration [13]. Except for only one lipid kinase inhibitor (Idelalisib) and a few SPK inhibitors, most of the approved small molecule inhibitors are PTK inhibitors. Surprisingly, most of these small molecule kinase inhibitors are approved for cancer indications, while only two for non-cancer indications (tofacitinib for rheumatoid arthritis and nintedanib for idiopathic pulmonary fibrosis) and none for CNS indications [13]. Recently, several review papers have highlighted the importance of PKs’ drug targets in CNS disorders, with an emphasis on the role of PKs in neurons [13,15–19]. It is currently believed that microglia-mediated chronic neuroinflammation is closely linked to various CNS diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and stroke [20–23]. Thus, this review briefly addresses the microgliamediated neuroinflammation and the emerging roles of PKs in CNS diseases with emphasis on the functions of PKs in microglia. 2.2. Microglia and neuroinflammation Neuroinflammation is a complex inflammatory reaction in the peripheral nervous system and the CNS in response to tissue injury and pathogen infection. All the CNS-resident cells, including microglia, astrocytes, and neurons, are involved in neuroinflammation; however, microglia and astrocytes particularly play key roles in the regulation of neuroinflammation [5,22]. Initially viewed as passive contributors, neurons have been recently suggested to contribute to neuroinflammation by providing various neuropeptides, neurotransmitters, and cell surface proteins, in addition to modulating microglia and astrocytes. Astrocytes are the most abundant glial cells in the adult human brain, accounting to approximately 40% of the total glial cell population. They are activated upon traumatic and neurodegenerative injuries, releasing an array of inflammatory cytokines and chemokines, which contribute to neuroinflammation. In contrast, microglia account for 5–20% of the total glial cell population in the brain, and are a foremost important cellular component contributing to neuroinflammation. Under normal physiological conditions, microglia are ramified with multiple branches and long processes. By continuously protruding and retracting their processes, microglia interact with neurons, astrocytes, and blood vessels and constantly monitor the CNS and synapses for the presence of tissue injury and pathogen infections. However, depending on the stimuli they receive, microglia become activated and undergo distinct morphological changes, whereby they switch from a ramified to a round amoeboid form with shortened processes and a full phagocytic activity [4]. These morphological changes are also accompanied by functional phenotypic changes resulting either in an M1-like ‘‘classical” proinflammatory phenotype or in an M2-like ‘‘alternative” antiinflammatory phenotype. The M1 phenotype can be induced by various stimuli such as pathogen-associated molecular patterns (PAMPS; bacterial lipopolysaccharide [LPS]), damage-associated molecular patterns (DAMPs; ATP, DNA, protein aggregates such as amyloid-beta (Ab peptides and a-synuclein [a-SYN]) and cytokines (tumor necrosis factor- [TNF] and interferon-gamma [IFN-c]), leading to release of proinflammatory cytokines, chemokines, and reactive oxygen/nitrogen species (ROS/RNS) that are often neurotoxic in nature (Fig. 1). However, glucocorticoids and certain cytokines, including, interleukins (IL-4 and IL-10), and transform-

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Fig. 1. Microglia-mediated neuroinflammation and protein kinases. Microgliamediated neuroinflammation is closely linked to many neurodegenerative diseases including Parkinson’s disease (PD), Alzheimer’s disease (AD), and stroke. Following protein kinases have been associated with microglia-mediated neuroinflammation: leucine-rich repeat kinase 2 (LRRK2), and mammalian Ste20-like kinase 1 (MST1), Src family protein tyrosine kinases (SFKs), cellular homolog of the Abelson murine leukemia virus oncogene (c-Abl) and TAM family receptor tyrosine kinases. These protein kinases are known to play critical roles in microglial activation by relaying the signals from various exogenous inducers through cell surface receptors such as the toll-like receptors (TLRs), tumor necrosis factor receptor (TNFR), CD11b, and P2Y12. One of the common pathological features of PD and AD is the deposition of protein aggregates (a-synuclein [a-SYN] in PD and Ab peptides in AD). These protein aggregates, released from neuronal death or other propagation mechanisms, bind with either TLR, CD11b, or other receptors on microglia and subsequently activate diverse intracellular signaling pathways. Other stimuli such as bacterial lipopolysaccharide (LPS), adenosine diphosphate (ADP), tumor necrosis factor (TNF), and RNA virus can also activate microglia by engaging in TLRs, P2Y12, and TNFR. TAM receptor tyrosine kinases such as Axl and Mer are involved in many features of microglia-medicated neuroinflammatory pathology in PD. Activated microglia ultimately release a wide range of proinflammatory cytokines, chemokines, and reactive oxygen/nitrogen species (ROS/RNS). Depending on stimuli and intracellular protein kinases, activated microglia also exhibit enhanced migration and phagocytic activity.

ing growth factor (TGF-b) can induce the M2 phenotype, leading to the production of various anti-inflammatory cytokines that are beneficial to the neurons and CNS. It has been reported that the balance between the M1 and M2 phenotypes has an impact on neuroinflammation and on regenerative and reparative mechanisms. Indeed, an imbalance in M1/M2 populations, with a predominance of the M1 phenotype, has been often observed in many neurodegenerative diseases at late stages [6,24]. 3. Emerging PKs associated with microglial activation 3.1. Leucine-rich repeat kinase 2 (LRRK2) LRRK2 is a large protein of 286 kDa belonging to the mammalian Ras of Complex (ROCO) protein family and is characterized by the presence of the core enzymatic domain comprising Ras of complex proteins (ROC)/guanosine triphosphate hydrolases (GTPase), C-terminal ROC proteins (COR), S/T kinases, and multiple distinct repeats (ankyrin, LRR, and WD40) at the N- and C-terminus, which are probably involved in protein-protein interactions [25].

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LRRK2 is the most commonly mutated gene in both familial and idiopathic PD, with over 50 mutations, identified to date since its discovery [18,25–27]. Most of the LRRK2 mutations are clustered within the core enzymatic domains; R1447C/G/H mutations are found in the ROC/GTPase domain, Y1669C mutation in the COR domain, and I2020T and G2019S mutations in the S/T kinase domain. The majority of LRRK2 mutations in its core enzymatic domains show increased kinase activity [18]. The G2019S mutation is the most common form associated with neurotoxicity [18,28], suggesting a link between enhanced activity of either GTPase and/or S/T kinase with the pathogenic effects of LRRK2 [29]. LRRK2 mutation-associated PD is essentially identical to the idiopathic PD [30]. Thus, it is one of the most ‘‘hot” kinase drug targets pursued by pharmaceutical industries and academia with a therapeutic potential. Interestingly, a number of studies have shown that there are molecular and cellular connections between proteins involved in AD pathology and LRRK2 [31]. However, LRRK2 mutations do not appear to be common among AD patients [32], and potential role of LRRK2 particularly in microglial activation has not been reported in AD yet. Several previous studies have investigated the physiological functions of LRRK2 in neurons, which are mostly affected in PD (Fig. 2). In neurons, LRRK2 has been shown to be involved in various cellular activities including vesicular trafficking, cytoskeletal dynamics, mitochondrial function, apoptosis, and regulation of the autophagy pathway [25,33]. Furthermore, LRRK2 is involved in several signaling pathways including the mitogen-activated protein kinase (MAPK), Ras-related C3 botulinum toxin (Rac)/p21activated kinase (PAK), Wnt, Akt, toll-like receptor (TLR), and cAMP-dependent protein kinase A (cAMP-PKA) pathways [34– 39]. However, despite intensive research, the exact physiological functions and downstream substrates of LRRK2 still remain elusive. In addition to neurons, glia such as microglia and astrocytes are known to express LRRK2 in the normal human brain [34]. Moreover, higher basal levels of LRRk2 expression have been reported in microglia than in neurons [40]. In glial cell cultures, IFN-c and bacterial LPS have been reported to increase the expression of LRRK2 [41,42]. Accordingly, several recent studies have elucidated the functions of LRRK2 in cell types other than neurons, suggesting in particular potential contributing roles of LRRK2 in regulating microglia activation, migration, and phagocytosis [42–48]. 3.1.1. LRRK2 in microglial activation/migration A recent study demonstrated that LRRK2 knockdown in murine microglia results in a decrease of LPS-induced expression of proinflammatory mediators including TNF, IL-1b, IL-6, and inducible nitric oxide synthases (iNOS) as well as a reduced activation of p38 MAPK and nuclear factor j-light-chain-enhancer of activated B cells (NF-jB), the latter due to increased binding of the inhibitory NF-jB homodimer, p50/p50, to DNA [48]. By investigating LPSstimulated rat primary microglia, Moehle et al. [42] showed that inflammation drastically increases activity and expression of LRRKs, while the inhibition of LRRK2 kinase activity or LRRK2 knockdown attenuates TNF secretion and iNOS induction. In addition, LRRK2 inhibition was shown to block the TLR4-stimulated microglia outgrowth and to impair microglial chemotaxis induced by treatment with adenosine diphosphate (ADP), which is a potent microglial chemoattractant [42]. Upon ADP recognition, microglia rapidly form lamellipodia and become highly motile. It has been suggested that LRRK2 regulates microglial migration in response to ADP via the phosphorylation of focal adhesion kinase (FAK) at T474 [46]. Upon binding to the P2Y12 receptor, ADP induces P2Y12 receptor-mediated activation of phospholipase C beta (PLCb) and intracellular Ca2+ release, resulting in the induction of FAK phosphorylation at Y397, indicating FAK activation, in microglia. Subsequently, activated FAK localizes to the leading edge of the

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Neurons

Microglia

GSK3 DYRK1A CDK4/5/6 ROCK PLK PINK1

CK PI3K

STK39

LRRK2 MST1 c-ABL SFKs MAPKs

AXL/MER

PKB (AKT) PAK DAPK1 Fig. 2. Protein kinases identified as potential therapeutic targets in neuronal cells and microglia. A number of studies have suggested important roles of protein kinases in neuronal toxicity and microglial activation. Compared to neurons, only a handful of protein kinases (PKs) have emerged as important signaling components modulating microglial activation. Some kinases such as Leucine-rich repeat kinase 2 (LRRK2), mammalian Ste20-like kinase 1 (MST1), tyrosine kinases, and mitogenactivated protein kinases (MAPKs) appear to play crucial roles in both neuronal toxicity and microglial activation, whereas TAM receptor tyrosine kinases (RTKs) such as Axl and Mer have recently emerged as potential therapeutic targets for microglia-mediated inflammation. It is expected that more kinases playing critical roles in microglia activation will appear in the near future.

cell, inducing stable lamellipodia formation for proper cell migration. LRRK2 was shown to interact directly with FAK and phosphorylate it at T474, thus inhibiting its phosphorylation at Y397. The GS2019S mutant form of LRRK2 induced further inhibition of FAK-Y397 phosphorylation, resulting in unstable lamellipodia and improper microglial migration [46] (Table 1). In a separate study [44], microglia isolated from LPS-treated transgenic (TG) mice overexpressing R1441G mutation exhibited higher levels of proinflammatory cytokine expression compared with those isolated from LPS-treated wild-type (WT) mice. This study further showed that conditioned medium from LPS-stimulated R1441G TG microglia can induce significant cell death when added to neuronal cultures [44]. Taken together, these studies suggest that LRRK2 is involved in microglial activation upon various stimulations and that a change in LRRK2 kinase activity may contribute to microglia-mediated neuroinflammation in PD. 3.1.2. LRRK2 in microglial phagocytosis The neuropathological hallmarks of PD include the presence of intracellular inclusions containing a-SYN called Lewy bodies and the loss of dopaminergic neurons in the substantia nigra of the midbrain and in other brain regions [20]. A number of earlier in vitro and in vivo studies demonstrated that a-SYN species released from neurons into the extracellular space can activate microglia through TLRs, followed by the initiation of neuroinflammation and progressive neuronal damage in PD [43,49]. Activated microglia also exhibit a profile of full activity of phagocytes [6]. By phagocytosing these extracellular a-SYN species, microglia can exert a protective role in PD [29,43,50,51].

Table 1 Summary of protein kinases (PKs) and their inducers, receptors, signaling events, and amplifiers/effectors under different disease conditions. Disease

PKs

Inducers

Receptors

Signaling

Amplifiers/effectors

References

PD

LRRK2

LPS TNF

TLR TNFR

LRRK2 p38 " NF-jB "

[48]

ADP

P2Y12

a-SYN

TLR

a-SYN

CD11b

LPS TNF a-SYN

TLR TNFR

PLC-b" Ca2+ " LRRK2 G2019S FAKpY397; FAKpT474" LRRK2 G2019S Nox2 " (p47phox) O 2 " H2O2 " Lyn " pCortactin " Fyn " PKCd pY311 " MAPK " NF-jB "

TNF IL-1b IL-6 iNOS Migration /Motility Phagocytosis

TAM

a-SYN

Axl/Mer

Abl/Src

RNA virus MPTP Ab

TLR3

LPS/OGD

TLR

SFKs

PD AD Stroke

MST1

[42,46]

[47] [74]

Cytokines Chemokines iNOS (NO) ROS TNF IL-1b IL-6 CD11b Nox2

[73]

[83]

c-Abl " pMAVS " c-Abl/Src

TNF IFN-b Phagocytosis iNOS

[64]

c-Abl/Src MST1 pY433 " IjB p32/p36 " NF-jB "

TNF IL-1b IL-6 MCP1

[12,78]

[59]

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In a recent study, primary microglia isolated from LRRK2 knockout mice were shown to exhibit a more effective phagocytosis and clearance of a-SYN and an increased early endosomes activity compared with those isolated from LRRK2-WT mice [47] (Table 1). Therefore, this study suggested that LRRK2 may function as a negative regulator of a-SYN clearance by downregulating the endocytosis pathway and that LRRK2 inhibition would serve as a potential therapeutic approach for PD. All these studies collectively suggest that LRRK2 regulates various microglia activities and that LRRK2 mutations with increased kinase activity would be one of the possible mechanisms for microglia-mediated neuroinflammation. Thus, it is plausible that the inhibition of LRRK2 kinase activity would be an attractive therapeutic approach for PD [47]. However, it has been reported that LRRK2 kinase activity is not explored in the brain of patients with PD and that it remains unclear whether neuronal toxicity associated with LRRK2 mutation is kinase-dependent or not [18]. Moreover, potential contributing roles of several distinct repeats in LRRK2 functions remain largely unknown, suggesting a more complicated function of LRRKs. Thus, further studies are required to identify more detailed signaling pathways particularly mediated by LRRK2 mutations in microglia. 3.2. Cellular Abelson murine leukemia viral oncogene homolog 1 (c-Abl) c-Abl (ABL1), the cellular homolog of the Abelson murine leukemia virus oncogene, belongs to the Abl family of nonreceptor tyrosine kinase, which also includes the Abl related gene (Arg). Similar to the other nonreceptor sarcoma (Src) family-protein tyrosine kinases (SFKs), it is composed of N-terminal myristoylation domain, followed by sequential Src-homology domains SH3 and SH2 and a core catalytic domain with tyrosine-kinase activity. However, unlike SFKs, the N-terminal myristoylation domain is known to regulate its kinase activity negatively. Localized in the cytoplasm and nucleus of most cells, c-Abl is involved in a variety of cellular functions including the regulation of cell growth and motility, cytoskeleton dynamics, receptor endocytosis, DNA repair, cell survival, and autophagy [52]. Furthermore, c-Abl is also known to impact the development of the CNS by affecting neurogenesis, neurite outgrowth, and neuronal plasticity [19]. Earlier studies have demonstrated that activated c-Abl is one of the underlying mechanisms of neuronal cell death in human neurodegenerative diseases including PD and AD [52–56] (Fig. 2). In AD, Tau phosphorylation was mediated by c-Abl or Arg in neurons [57,58]. Upon oxidative stress, c-Abl was shown to be activated in neuronal cells, resulting in increased expression of c-Abl and p73. In addition, Ab fibrils in primary neurons induced c-Abl/p73 proapoptotic signaling, while STI571, a pharmacological c-Abl inhibitor, prevented Ab-dependent toxicity [55]. In patients with PD, it was reported that c-Abl protein level was elevated postmortem in the striatum [56] and that c-Abl phosphorylation at Y413 was detected in the substantia nigra [53,56] and striatum [53]. At the molecular level, c-Abl was known to phosphorylate Parkins and a-SYN. Phosphorylation of parkins by c-Abl inhibited E3 ligase activity of parkins, leading to the loss of dopaminergic neurons in the substantia nigra [53]. Additionally, c-Abl negatively regulated a-SYN degradation by phosphorylating a-SYN at Y37 [52]. In terms of the functions of c-Abl in microglia, Dhawan et al. [59] indicated a link between enhanced c-Abl activity and microglial activation. This study showed that primary murine microglia stimulated with Ab upregulate the level of tyrosine phosphorylation of cellular proteins and TNF secretion. In addition, intracerebroventricular infusion of Ab increases microgliosis and the levels of tyrosine phosphorylated proteins in mice. By showing that in vitro and in vivo treatment with dasatinib, an Src/Abl inhibitor,

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attenuates these Ab-induced tyrosine phosphorylation of cellular proteins and TNF secretion, this study suggested that tyrosine kinase activation is involved in Ab-mediated microglial activation and that the use of tyrosine kinase inhibitors may attenuate the microglia-mediated neuroinflammatory process in AD [59] (Table 1). Previously, naked poly(I:C), a synthetic double-stranded RNA recognized by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), such as RIG-I and melanoma differentiation-associated gene 5 (MDA-5), has been shown to induce inflammation through TLR3 [60,61]. Moreover, it has been demonstrated that the RNA virus infection is associated with the development of PD [62]. Upon RNA virus infection, RLRs, such as RIG-I and MDA-5, recognize the viral RNA and interact with the mitochondrial activating signaling (MAVS), leading to subsequent activation of NF-jB and Type I IFN signaling, respectively [63]. In a recent study, Cheng et al. [64] showed that, compared with the MAVS/ mice brain, poly(I:C) injection into the substantia nigra significantly enhanced the production of inflammatory cytokines including IFN-b and TNF. Treatment of microglia with poly(I:C) resulted in the increase in phosphorylated interferon regulatory factor 3 (pIRF3) and light chain 3 (LC3II) and degradation of p62. This study demonstrated that LC3 knockdown or autophagy-related gene (ATG5) deficiency further induced poly(I:C)-mediated increase in pIRF3 and proinflammatory cytokine levels, suggesting that autophagy negatively regulates MAVS-mediated inflammatory response in microglia. Furthermore, MAVS was shown to interact directly with LC3, resulting in MAVS degradation, while c-Abl/ primary microglia or pretreatment of BV-2 cells with STI571 increased MAVS stability. Upon Sendai virus infection, BV-2 cells exhibited enhanced tyrosine phosphorylation of both c-Abl and MAVS. The phosphorylation of MAVS by c-Abl was validated in 293T cells. In addition, the interaction of Abl with MAVS was detected in both 293T and BV-2 cells, while both c-Abl knockdown and Abl inhibition significantly reduced the poly(I:C)-mediated induction of p-IRF3 and IFN-b in BV-2 cells. Upon Sendai viral infection, c-Abl/ primary microglia exhibited a significant reduction of both p-IRF3 and IFN-b expression compared with WT primary microglia. In vivo, this study also reported that the deficiencies of either c-Abl or MAVS prevented microglial activation as well as 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neuron loss in the MPTP-induced PD mouse model [64] (Table 1). Taken together, the findings in this study implied that c-Abl is required for the induction of MAVS-dependent microglial activation, which may contribute to the development of MPTP-induced dopaminergic neurotoxicity. 3.3. Src family protein tyrosine kinases (SFKs) The SFKs are the major nonreceptor tyrosine kinases expressed in the cytoplasm of many different types of animal cells and include c-Src, c-Yamaguchi (Y73 virus) sarcoma oncogene (Yes), c-feline Gardner-Rasheed sarcoma virus (Fgr), Fgr/Yes related novel protein (Fyn), Lyn, Hck, Lck, and Blk. Several of the SFKs such as c-Src, c-Yes, and c-Fgr were originally discovered as the cellular homologs of the avian retroviral oncogenes (v-Src, v-Yes, and v-Fgr) [65]. The SFKs are composed of N-terminal lipid modification signals, which are myristoylation (in all SFKs) or palmitoylation (in all SFKs, except c-Src and Blk), the SH3 and SH2 domains, the catalytic domain, and the C-terminal regulatory tail. These Nterminal lipid modification signals are required for membrane localization of SFKs. Once activated by the cognate ligands, the transmembrane receptors, including the epidermal growth factor (EGF) receptor, T cell receptor and integrins, become activated and subsequently involved in the regulation of a wide range of cellular activities, including cell growth, division, differentiation,

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survival, and programmed death, as well as specialized functions such as immune responses, cell adhesion, cell movement, and endocytosis. Dysregulation of SFKs is often associated with various human diseases such as cancers [65]. In terms of CNS disorders, several earlier studies have implied that Fyn is linked to neuronal toxicity and glial cell activation [66–71] (Fig. 2). A recent study further supported potential roles of Fyn and protein kinase Cd (PKCd), a member of the PKC family of SPKs, in microglia-mediated neuroinflammation [72,73]. Panicker et al. [72] showed that both LPS and TNF rapidly induce Fyn activation in microglia. Activated Fyn then phosphorylates Y311 of PKCd, leading to an increase in its activity. Activated PKCd subsequently leads to the activation of the MAPK and NF-jB pathways. By using microglia isolated from Fyn WT (Fyn+/+) and knockout (Fyn/) mice, this study further demonstrated that Fyn is required for the release of proinflammatory cytokines and the activation of iNOS. Compared with WT mice, Fyn/ and PKCd/ mice showed a greater attenuation of MPTP-, LPS-, or 6-hydroxydopamine (OHDA)-induced neuroinflammatory responses. Taken together, this study suggested that Fyn may act as a major signaling molecule in microglia-mediated neuroinflammatory processes in PD. Another study conducted by the same group further elaborated a pivotal role of PKCd activation in microglia-mediated neuroinflammatory responses [73]. They showed that major inflammatory stimuli, including LPS, TNF, and a-SYN, dramatically upregulate PKCd expression with a concomitant increase in its kinase activity and nuclear translocation in BV-2 cells and primary microglia. They confirmed that a marked upregulation of PKCd is detected in the microglia of the ventral midbrain of patients with PD compared with age-matched healthy controls. PKCd knockdown or deficiency in primary microglia dampened proinflammatory responses induced by inflammatory stimuli, resulting in the production of cytokines, chemokines, ROS, nitric oxide (NO), and NF-jB activation. In addition, this study showed that PKCd/ mice exhibit attenuated microglial proinflammatory responses following treatment with MPTP and a reduced susceptibility to the neurotoxininduced dopaminergic neuron degeneration and associated motor impairments [73] (Table 1). Taken together, these studies implied that Fyn acts upstream of PKCd and that both Fyn and PKCd represent potential therapeutic drug targets in PD. Lyn is another member of the SFKs that is potentially involved in microglial activation, in particular microglial motility. Wang et al. [74] recently showed that microglial integrin CD11b detects a-SYN, leading to the translocation of p47phox, a surrogate marker of NADH oxidase (Nox2) activation, to the cell membrane and the  release of the superoxide anion (O 2 ). Increased O2 , in turn, converts to membrane-permeable H2O2, which is known to regulate many cytosolic tyrosine kinases by oxidizing the kinases cysteine residues. In microglia treated with either a-SYN or H2O2, this study demonstrated an enhanced phosphorylation of Lyn and cortactin, an F-actin-associated protein that promotes the polymerization and rearrangement of the actin cytoskeleton. Lyn knockdown or treatment of microglia exposed to a-SYN with protein phosphatase 2 (PP2) resulted in an inhibition of cortactin phosphorylation with a concomitant attenuation of cytoskeleton rearrangement and directional migration of microglia toward sources of a-SYN. Thus, this study suggested that Lyn activation following exposure to aSYN may contribute to neuroinflammation in PD by regulating microglial migration through cortactin phosphorylation [74] (Table 1). 3.4. Mammalian Ste20-like kinase 1 (MST1) MST1 is a multifunctional SPKs belonging to the family of class II germinal center kinases, which also include MST2, MST3, and

MST4. It is composed of an S/T kinase domain at the N-terminus, an inhibitory domain in the central domain, and a regulatory Salvador/Rassf/Hippo (SARAH) domain at the C-terminus. The SARAH domain is responsible for the homo-dimerization of MST1, contributing to the mechanism underlying MST1 activation [75]. MST1, ubiquitously expressed, is associated with the regulatory mechanisms for many biological events including cell growth, apoptosis, stress response, and senescence. Elevated activity of MST1 kinase was mainly demonstrated in neurons, where it played a critical role in multiple neurodegenerative diseases including AD and ALS [75–77] (Fig. 2). ALS is an adult-onset neurodegenerative disorder characterized by a loss of motor neurons. Although the underlying mechanisms of familial ALS remain unknown, mutations of superoxide dismutase 1 (SOD) gene have been implicated. In an earlier study with mice expressing SOD1 (G93A), the ALS-associated mutant form of human SOD1, Lee et al. [75] showed that SOD1 (G93A) activated MST1 by inducing MST1 dissociation from the redox protein thioredoxin-1. As a result, activated MST1 induced the activation of p38 MAPK and caspases, and impaired autophagy in spinal cord motor neurons of SOD1 (G93A) mice, leading to neuronal toxicity. However, introducing a genetic MST1 deficiency in the SOD1 mice resulted in a delay of disease progression and extension of the mice survival. Thus, this study proposed MST1 as a key determinant of neurodegeneration in ALS [75]. Recent studies indicated that two tyrosine kinases, namely Src and c-Abl, may activate MST1 by its phosphorylating at Y433 in microglia, resulting in microglial activation and inflammation in ischemic conditions [78,79]. Ischemic stroke, accounting for 80% of stroke cases, is a major public concern with high rate of disability and mortality in adults worldwide [80]. The cerebral ischemia is pathophysiologically associated with oxidative stress and inflammation. When stroke occurs, oxidative stress induces neuronal damage, releasing DAMPs and purine (ATP) that subsequently activate TLRs and scavenger receptors in microglia, resulting in the recruitment of active microglia to the injury sites. Subsequently, the recruited microglia clear the dead cells and debris. However, uncontrolled microglial activation could be detrimental, leading to neuroinflammation. By using Lyz2Cre:Mst1f/f, a mouse model in which MST1 is deleted in microglia in the CNS and in monocytes in the periphery, Zhao et al. [78] demonstrated an increased number of CD11b+ microglia in the ischemic striatum of WT or Mst1/ mice subjected to right-sided transient middle cerebral artery occlusion (tMCAO), compared with sham-treated mice. Furthermore, oxygen glucose deprivation (OGD) or LPS-induced production of TNF and IL-6 was significantly decreased in Mst1/ mice. They also reported that the stimulation of microglia by LPS, OGD, or cerebral ischemia resulted in a significant increase in MST1 phosphorylation at Y433, leading to MST1-mediated phosphorylation of IjBa, an inhibitor of NF-jB, at S32 and S36 and subsequent degradation of IjBa. This ultimately led to NF-jB activation, followed by microglial activation and inflammation. By using a siRNA library targeting 32 putative nonreceptor tyrosine kinases, they further suggested that Src was responsible for MST phosphorylation at Y433 in both primary microglia and BV-2 cells. Primary microglia expressing Y433F mutant of MST1 showed an attenuated LPSinduced TNF and IL-6 mRNA expression. Treatment of primary microglia with AZD530, a specific Src kinase inhibitor in PhII/III that is used for chemotherapy in ovarian cancer, resulted in a decreased LPS- or OGD-induced phosphorylation of MST1 at Y433 [78] (Table 1). The same group has recently demonstrated that c-Ablmediated MST1 activation and phosphorylation at Y433 are involved in M1 microglia polarization upon ischemic injury

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[79]. This study also showed that BV-2 cells exposed to OGD exhibit (i) an upregulation of M1 microglia markers including TNF, IL1, and MCP1, (ii) a slight increase in c-Abl expression, and (iii) a striking increase in MST1 phosphorylation at Y433. However, treatment with Malibatol A, a potent scavenger of free radicals, reduced the expression of these M1 microglia markers, but increased M2 microglial markers such as Ym-1 and CD206. Since treatment with both STI571 and Malibatol A inhibited MST1 phosphorylation at Y433, this study suggested that the inhibition of MST1 phosphorylation may be the underlying mechanism for Malibatol A-mediated microglial M2 polarization associated with OGD treatment [79].

3.5. TAM family receptor tyrosine kinases (RTKs) The TAM family RTKs are one of the most recently identified RTKs subclasses that include Tyro-3, Axl, and Mer tyrosine kinases [81]. Having been initially discovered in several cancers, the biological functions of TAM family-RTKs have been mostly exploited in oncological studies. Upon activation, these RTKs can regulate several downstream signaling pathways such as the MAPK/ERK and PI3K/AKT signaling pathways, which in turn modulate numerous cellular activities including cell survival, proliferation, migration, and invasion [82]. In normal adult tissues, TAM family RTKs are broadly expressed in the heart, liver, and brain (i.e., hippocampus and cerebellum), as well as in various cell types including the endothelial cells, epithelial cells, and cells derived from hematopoietic lineage (monocytes, platelets, macrophages, dendritic cells, and natural killer cells). The primary physiological function of TAM family RTKs is to regulate inflammation and to remove apoptotic cells and waste material [5,81]. In the CNS, these RTKs are widely expressed in neurons and microglia, where Tyro3 is abundantly expressed in neurons, while both Axl and Mer are prominently expressed in microglia [5]. Recently, Fourgeaud et al. [83] showed that adult mice devoid of microglial Axl and Mer displayed a marked accumulation of apoptotic cells in neurogenic regions of the CNS compared with WT mice. In addition, these Axl/Mer/ microglia exhibit reduced process activity and slower response to injury sites. In a TG mouse model of a hereditary PD, in which the PDassociated mutant form of human a-SYN (SNCAA53T) was expressed in neurons (most prominently in the spinal cord) under the control of the mouse Thy1 promoter, this study further demonstrated that a number of inflammatory marker mRNAs such as TNF, IL-6, IL-1b, Nox2, and CD11b/c, were elevated in the spinal cord and to a lesser extent in the brain, but not in the spleen. This study also demonstrated that both Axl and soluble Axl (sAxl) ectodomain, an inflammatory marker, were upregulated in the spinal cord and to a lesser extent in the brain, but not in the spleen of these aged TG mice. This Axl upregulation was shown to be exclusively associated with Iba1+ microglia in the Thy1-SNCAA53T spinal cord. However, Mer expression was only very modestly increased in the spinal cord, but not in the spleen. Interestingly, a modest expended survival was also observed in Axl/Mer/SNCAA53T mice compared with WT mice. Collectively, this study suggested the importance of TAM RTKs in the regulation of many features of microglial physiology in inflammatory environments associated with neurodegenerative diseases such as PD [60,83] (Table 1). Although further studies are required for a more detailed characterization of TAM-mediated signaling pathways in microglia with respect to other neuroinflammatory and neurodegenerative disease conditions, TAM RTKs could serve as newly identified therapeutic targets for the development of CNS diseases in the near future.

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4. Conclusion and future perspectives It is now believed that neuroinflammation is closely linked to many neurodegenerative diseases, and that sustained chronic activation of microglia is critical for neuroinflammation. Thus, effective therapeutic strategies to slow down or halt the progression of neurodegenerative diseases could be either the suppression of inflammation or the modulation of microglial activation. To date, a wide range of anti-inflammatory drugs have failed to impact the progression of these diseases [84,85]. Moreover, once a disease becomes clinically apparent, the efficacy of anti-inflammatory drugs becomes drastically hindered. Therefore, there is an urgent need to develop drugs that are able to delay or curtail the progression of neurodegenerative diseases effectively. During the past two decades, great advances have been made with kinase-targeting agents in the therapeutic areas of oncology and chronic inflammation. However, progress in the development of kinase inhibitors for CNS disorders appears to be slower and more challenging. This could be largely owing to issues specifically associated with CNS drug discovery [15] and in part due to the lack of protein kinase drug targets to be exploited for CNS disorders. Here, we reviewed selected protein kinases that are currently emerging as potential therapeutic targets in microglia-mediated neuroinflammation (Fig. 1, Table 1). Some of these kinases, including LRRK2, MST1, and tyrosine kinases (c-Abl, Src, and Fyn), have been previously proposed as potential drug targets owing to their critical functions in neuronal toxicity [13,15,18,19,86], while others were presented here as novel protein kinase drug candidates owing to their crucial roles in microglial activation (Fig. 2). While further studies are required for a better understanding of signaling mechanisms mediated by these protein kinases in microglia, it is expected that TAM RTKs, in particular, will be most extensively exploited in the near future as potential drug targets for various neurodegenerative diseases associated with apoptotic cell death. Moreover, the crosstalk between kinases not only in neurons but microglia are also possible under certain circumstances [39,87,88]. It would be an exciting area to be explored in the future. As mentioned above, certain protein kinases are involved in both neurotoxicity and microglial activation (Fig. 2). Thus, targeting these kinases may be an effective therapeutic strategy in neurodegenerative diseases. Alternatively, targeting two or more PK targets (e.g., LRRK2 and c-Abl in PD) may serve as an alternative therapeutic approach for the future treatment of many neurodegenerative diseases. However, the exploration of FDA approved protein kinase inhibitors and whether they can be repurposed or repositioned for neuroinflammation could serve as another approach. Indeed, inhibition of c-Abl has been suggested to be beneficial for patients with AD and PD [19,89] and PhII clinical trials of nilotinib, an Abl inhibitor approved by the U.S. FDA for adults with chronic myeloid leukemia (CML), are currently ongoing for both AD (NCT02947893) and PD (NCT02954978). In summary, the development of kinase-targeted therapeutics for CNS disorders holds great promise in the future. With a growing number of protein kinases emerging as therapeutic targets for CNS disorders and with the rapid advancement and growth in the development of protein kinase inhibitors, it is only a matter of time to witness whether the modulation of the protein kinases presented here may become a valuable option for future treatment of various neurodegenerative diseases with features of microgliamediated neuroinflammation.

Acknowledgments The authors declare no competing financial interests. We apologize to those authors whose works were not cited owing to space

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constraints. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (2015R1A2A1A10051958, 2016M3C7A1904148).

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