Parkinson’s disease-implicated kinases in the brain; insights into disease pathogenesis

POLO-LIKE KINASES (PLKs)

Polo-like kinases (PLKs) comprise a serine/threonine kinase family containing an N-terminal kinase catalytic domain and a C-terminal polo-box domain (PBD) that is involved in substrate binding and regulation of kinase activity. Five mammalian PLK family members from three subfamilies have been identified, including the PLK1 subfamily, the PLK4 subfamily, and the PLK2 subfamily (containing PLK2, PLK3, and PLK5; de Carcer et al., 2011b). The study of PLKs has focused primarily on their critical roles in the cell cycle (Winkles and Alberts, 2005); however, recent studies suggest PLKs also have important roles in terminally differentiated cells of the nervous system (Seeburg et al., 2005).

In particular, PLKs 1–3 are capable of phosphorylating α-synuclein (de Carcer et al., 2011a, b). Comparative studies suggest that PLK2 and PLK3 directly phosphorylate α-synuclein at Ser129 in vitro with high stoichiometry, whilst PLK4 is unable to phosphorylate α-synuclein at this residue (Anderson et al., 2006; Inglis et al., 2009). The low kinase activity of PLK4 against α-synuclein, and other substrates, is partially explained by its unique structure, with only a single polo-box in the PBD, resulting in a much-reduced electropositive environment in its substrate-binding site (Mbefo et al., 2010). Human PLK5 lacks a functional kinase domain due to a premature stop codon in exon 6 and is therefore unable to phosphorylate α-synuclein.

Increasing PLK2 or PLK3 significantly up-regulates α-synuclein Ser129 phosphorylation (Mbefo et al., 2010; Waxman and Giasson, 2011), whilst their inhibition or reduction remarkably decreases α-synuclein phosphorylation in both cell and animal models (Inglis et al., 2009; Waxman and Giasson, 2011). This has led to efforts to generate small molecule PLK inhibitors for potential therapeutic use (Bowers et al., 2013; Fitzgerald et al., 2013; Bergeron et al., 2014). The utility of such compounds, however, has been questioned by a recent study showing that Ser129 phosphorylation by PLK2 is required for autophagic degradation of α-synuclein (Oueslati et al., 2013). In this study overexpression of PLK2, as opposed to inhibition, prevented the toxic accumulation of α-synuclein in rodent models, suggesting more work is required to delineate the exact role of PLKs in α-synuclein pathology.

In addition, studies investigating the association of PLKs with α-synuclein pathology / phosphorylation in human brain are lacking. The central nervous system has relatively high levels of PLK2, 3, and 5, low levels of PLK1 and seems to lack PLK4 (Winkles and Alberts, 2005; de Carcer et al., 2011a, b). PLK2 and PLK3 are expressed in most regions of the brain, but surprisingly there is almost no expression of either PLK2 or PLK3 in the cerebellum (Winkles and Alberts, 2005; de Carcer et al., 2011b). Whether PLK2 or PLK3, the main family members that can phosphorylate α-synuclein are increased, or indeed more active, in PD brain remains to be determined. The recent identification of autophosphorylation sites on PLK2 (Rozeboom and Pak, 2012) and other potential selective substrates (Salvi et al., 2012) could allow readouts of PLK2 activity to be examined in PD brain. It would be of interest to determine if the phosphorylation of such substrates correlated to levels of α-synuclein phosphorylation in pathology rich brain regions in PD cases.

CASEIN KINASES (CKs)

Casein kinases (CKs) comprise a ubiquitously expressed serine/threonine kinase family (Peters et al., 1999) containing two members, CK1 and CK2, which differ substantially in terms of structure, localization and function (Perez et al., 2011). CK1 consists of a small N-terminal lobe, a large C-terminal lobe and a catalytic cleft where ATP and substrates bind (Cheong and Virshup, 2011). To date, at least seven CK1 isoforms (α, β, γ1–3, δ, and ∊) and their various splice variants, ranging from 22 to 55 kDa, have been localized within the membrane, nucleus, and cytoplasm of eukaryote cells, and additionally in the mitotic spindles of mammalian cells (Fish et al., 1995). All CK1 isoforms are highly homologous in their kinase domains (Knippschild et al., 2005), presenting a strong preference for “primed,” pre-phosphorylated substrate. However, they can also phosphorylate related unprimed sites under certain conditions (Cheong and Virshup, 2011).

In contrast to CK1, CK2 is a tetrameric enzyme assembled from two catalytic subunits (CK2α and CK2α′) and a regulatory subunit (CK2β dimer). The two catalytic subunits α and α′ share 90% sequence homology in their N-terminal region, but the regulatory subunit β does not have any similarity to the other two subunits. CK2 is found in many organisms and tissues and nearly every subcellular compartment. It can phosphorylate more than 300 substrate proteins (Meggio and Pinna, 2003) involved in diverse cellular processes including cell division, proliferation, apoptosis, and DNA repair.

Both CK1 and CK2 can constitutively phosphorylate α-synuclein at Ser129 in vitro (Okochi et al., 2000; Waxman and Giasson, 2008), and inhibition of CK1 or CK2 reduces α-synuclein Ser129 phosphorylation in vivo (Okochi et al., 2000; Ishii et al., 2007; Waxman and Giasson, 2008), with CK2 inhibition seemingly more efficient at reducing phosphorylation (Ishii et al., 2007). However, at least one study failed to find an effect of CK1 inhibition on α-synuclein Ser129 phosphorylation in a cellular model (Waxman and Giasson, 2011). This discrepancy could result from, at least partially, the specificity of CK1 inhibitors and more studies are needed to define the relationship between CKs and α-synuclein.

In pathological human brain CK1δ co-localizes predominantly with tau-containing inclusions such as neurofibrillary tangles, and does not co-localize with α-synuclein in Lewy bodies in PD (Schwab et al., 2000). In contrast, CK2β regulatory subunits are present in the halo region of Lewy bodies in PD substantia nigra (Ryu et al., 2008), suggesting that CK2 may be more pathologically relevant to PD. More work is required to determine any correlations between CK isoforms and the pathological accumulation of phosphorylated α-synuclein in PD.

G PROTEIN COUPLED RECEPTOR KINASES (GRKs)

G Protein coupled receptor kinases comprise a serine/threonine kinase family that regulate G protein-coupled receptors (GPCRs) by phosphorylating their intracellular domains after their associated G proteins have been released and activated (Gurevich et al., 2012). Structurally, GRKs contain a central catalytic domain flanked by an N-terminus containing a regulator of G protein signaling homology domain and a variable length C-terminal end. Based on sequence homology and tissue expression, GRKs are further classified into three subfamilies: the rhodopsin kinase or visual GRK subfamily (GRK1 and GRK7), the β-adrenergic receptor kinases subfamily (GRK2 and GRK3), and the GRK4 subfamily (GRK4, GRK5, and GRK6; Gurevich et al., 2012; Kamal et al., 2012).

Exactly which GRK isoforms phosphorylate α-synuclein under pathological conditions is unclear. In vitro GRK2 preferentially phosphorylates α and β synuclein isoforms while GRK5 prefers α-synuclein as a substrate (Pronin et al., 2000). However, knockdown of either GRK5 or GRK2 failed to diminish the phosphorylation of α-synuclein in cell models (Sakamoto et al., 2009; Liu et al., 2010). In contrast, knockdown of GRK3 or GRK6 significantly decreased α-synuclein Ser129 phosphorylation levels (Sakamoto et al., 2009), suggesting further work is required to verify the role of GRK isoforms in phosphorylating α-synuclein.

G protein coupled receptor kinase isoforms, 2, 3, 5, and 6, are highly expressed in the human brain. In PD brain, however, GRK protein levels tend to be lower than controls (Bychkov et al., 2008) with conflicting reports regarding the co-localization of GRK5 in Lewy bodies (Arawaka et al., 2006; Takahashi et al., 2006).

LEUCINE-RICH REPEAT KINASE 2 (LRRK2)

Mutations in the LRRK2 gene were discovered as causal for PD in 2004 (Paisan-Ruiz et al., 2004; Zimprich et al., 2004). Subsequently, some 40 missense mutations have been described across the LRRK2 protein with six of these demonstrated as pathogenic (Paisan-Ruiz, 2009). Collectively these LRRK2 mutations account for the majority of autosomal-dominantly inherited PD (Kett and Dauer, 2012). LRRK2-associated PD is largely clinically and pathologically indistinguishable from sporadic PD (Healy et al., 2008), suggesting that understanding LRRK2 function has implications for all forms of PD. Moreover, large-scale genome-wide association studies show that common variations in non-coding regions of the LRRK2 gene also confer greater risk for sporadic PD (Satake et al., 2009; Simon-Sanchez et al., 2009; Sharma et al., 2012).

Leucine-rich repeat kinase 2 is a 286 kDa multi-domain containing member of the receptor interacting protein kinase (RIPK) family. LRRK2 has N-terminal ankyrin repeats, leucine-rich repeats, a ras of complex (ROC) GTPase domain with adjoining C-terminal of ROC (COR) domain, a serine/threonine protein kinase domain and C-terminal WD40 repeats. Intriguingly, the majority of the pathogenic mutations lie in the catalytic domains of LRRK2. The most common mutation results in the substitution of glycine to serine (G2019S) in the activation loop of the protein kinase domain resulting in a constitutive threefold increase in LRRK2 kinase activity (West et al., 2005; Jaleel et al., 2007). The next most common mutations, substitution of arginine to either histidine (R1141H), cysteine (R1441C), or glycine (R1441G) lie in the GTPase domain. Some evidence suggests that these mutations also increase kinase activity (Sheng et al., 2012), potentially by trapping LRRK2 in a GTP bound active state (Liao et al., 2014). It has previously been shown that GTP binding is required for LRRK2 kinase activity (Taymans et al., 2011) and a complex relationship exists between the two domains (Taymans, 2012). The increase in catalytic kinase activity with LRRK2 mutations has led to the development of LRRK2 kinase inhibitors as potential PD therapeutics (Deng et al., 2012) and much interest has focused on determining the targets of LRRK2 kinase activity (Dzamko and Halliday, 2013). One such robust effect for LRRK2 kinase activity is to mediate the phosphorylation-dependent interaction of LRRK2 with isoforms of the 14-3-3 adaptor protein (Dzamko et al., 2010); however, consensus regarding the PD-relevant physiological functions of LRRK2 has remained largely elusive.

LRRK2 mRNA expression shows a widespread neuronal localization in human brain; however, intriguingly, only weak levels are detected in the substantia nigra (Higashi et al., 2007; Sharma et al., 2011). Moreover, decreased LRRK2 mRNA was found in certain non-nigral regions of PD brain (cerebellum, amygdala, frontal cortex, and cingulate gyrus; Sharma et al., 2011), suggesting a pathogenic role for LRRK2 outside of nigral neurons. This contrasts with the increased levels of LRRK2 protein reported in PD brain regions with pathological accumulation of α-synuclein (Cho et al., 2013; Guerreiro et al., 2013). The exact nature of the relationship between LRRK2 and α-synuclein is somewhat unclear as postmortem localization studies have produced conflicting results. Some studies have demonstrated localization of LRRK2 to α-synuclein pathology (Miklossy et al., 2006; Alegre-Abarrategui et al., 2008; Qing et al., 2009; Sharma et al., 2011) whilst others have not (Giasson et al., 2006; Higashi et al., 2007; Melrose et al., 2007; Waxman et al., 2009). Studies comparing different LRRK2 antibodies have shown that discrepancies in LRRK2 tissue localization likely occurs through use of antibodies unsuitable for immunohistochemistry (Biskup et al., 2007; Melrose et al., 2007; Davies et al., 2013). Indeed, recent data using more rigorous methods shows LRRK2 and α-synuclein co-localize in a small proportion of PD pathologies (Guerreiro et al., 2013). Despite such data, further work is required to define the relationship between LRRK2 expression and protein levels, between LRRK2 and α-synuclein increases and aggregation, and indeed determine if these proteins interact in the same molecular pathway.

As LRRK2 is also expressed by glial cells in normal human brain (Miklossy et al., 2006) and in tissue culture, and its expression in glia is increased by interferon gamma (Gardet et al., 2010) and bacterial lipopolysaccharide (LPS; Moehle et al., 2012), the neuroinflammation prevalent in PD affected regions may promote the expression changes observed for LRRK2 specifically in microglia rather than neurons. It will also be important to correlate any changes in LRRK2 expression to cell type. The recent demonstration that there is a primate specific LRRK2 promoter that differentiates primate expression of the protein in the brain from that observed in rodents (West et al., 2014) underlies the requirement for further observations in staged human tissue specimens in order to determine the role of LRRK2 kinase function in PD pathogenesis.

PTEN-INDUCED PUTATIVE KINASE 1 (PINK1)

Homozygous missense mutations in the PINK1 gene were identified as a cause of familial PD in 2004 (Valente et al., 2004). Around 50 missense mutations have subsequently been identified across the PINK1 protein in a number of populations (Kawajiri et al., 2011). Mutations in PINK1 are the second most common cause of recessive PD (following mutations in the ubiquitin ligase Parkin) and are thought to contribute to 1–8% of familial PD (Kawajiri et al., 2011). Unlike LRRK2, PINK1 mutations reduce kinase activity and cause an atypical form of PD characterized by an early age of onset and slower clinical progression (Abou-Sleiman et al., 2006; Woodroof et al., 2011).

The PINK1 protein comprises a serine/threonine protein kinase domain, a N-terminal mitochondrial targeting motif and a trans-membrane domain located between the two. The mitochondrial targeting motif is required for recruitment of PINK1 to mitochondrial membranes. Following recruitment in healthy mitochondria, PINK1 is enzymatically cleaved to produce a shorter fragment, which is degraded by the proteasome (Narendra et al., 2010). In this way, PINK1 is maintained at very low levels. Pharmacological uncoupling of the mitochondrial membrane leading to a loss of membrane potential; however, results in inhibition of PINK1 cleavage and its accumulation on depolarized mitochondrial membranes (Matsuda et al., 2010; Narendra et al., 2010). The kinase activity of PINK1 is also increased under these conditions with PINK1 undergoing autophosphorylation (Kondapalli et al., 2012). Intriguingly, Parkin is then also recruited to depolarized mitochondria where it is phosphorylated and activated by PINK1 (Matsuda et al., 2010; Kondapalli et al., 2012). Parkin then mediates the degradation of dysfunctional mitochondria by mitophagy (mitochondrial autophagy). Therefore, the two proteins responsible for the majority of familial early onset PD appear to function in the same pathway important for the regulation of mitochondrial quality control. Whether this is the pathway ultimately responsible for loss of neurons in PD is still unclear (Grenier et al., 2013) and it should be noted that PINK1 has also been implicated in other biological processes such as neurite maintenance (Dagda et al., 2014) and inflammation (Lee and Chung, 2012; Kim et al., 2013) among others.

Detailed in-situ hybridization studies using rodent brain demonstrate that PINK1 mRNA is expressed throughout the brain with the strongest signal in neurons of the olfactory bulb, neocortex, prefrontal cortex, piriform cortex, hippocampus, amygdala, brainstem, and cerebellar Purkinje cells (Taymans et al., 2006). Similarly, in human brain, PINK1 mRNA is widely expressed in neurons with highest signals recorded for the temporal cortex, amygdala, substantia nigra, cerebellar Purkinje cells and the dentate nucleus (Blackinton et al., 2007). PINK1 mRNA is undetectable in glial cells (Taymans et al., 2006; Blackinton et al., 2007) and is not different in the substantia nigra of sporadic PD patients compared to controls (Blackinton et al., 2007). The mRNA distribution of PINK1 has been largely confirmed at the protein level with the exception that PINK1 immunoreactivity was also observed in glia, albeit with weak staining compared to neuronal staining (Gandhi et al., 2006). PINK1 is predominantly localized to mitochondria and does not change in amount or localization in the brain of patients with idiopathic PD, although PINK1 immunoreactivity is detected in ~10% of brainstem Lewy bodies (Gandhi et al., 2006). Interestingly, a case report describing the neuropathology of an early onset PINK1 homozygous mutation patient showed a pattern of Lewy body pathology with atypical Braak Lewy body staging (Samaranch et al., 2010). This was due to the absence of Lewy bodies, and indeed cell loss in the locus coeruleus, potentially helping to explain the longer disease duration of PINK1-associated PD (Samaranch et al., 2010). Collectively these studies suggest that alterations in PINK1 function, rather than protein levels, likely contribute to PD. This is consistent with observations that the majority of described PINK1 mutations result in a loss of kinase activity (Woodroof et al., 2011). Whether PINK1 autophosphorylation or PINK1-induced Parkin phosphorylation are altered in sporadic PD, and how this correlates to mitochondrial health and/or neuronal loss, would be interesting to explore.

CYCLIN G-ASSOCIATED KINASE (GAK)

The Ser/Thr protein kinase GAK was originally identified via its interaction with cyclin G and cyclin-dependent kinase 5 (CDK5; Kanaoka et al., 1997). The kinase domain is located at the N-terminus and a leucine-zipper region is located at the C-terminus. The majority of the protein comprises a TAG domain that has 80% identity to the auxilin protein (Kanaoka et al., 1997). Both auxilin and GAK play key roles in the uncoating of clatherin-coated vesicles and the regulation of clatherin-mediated endocytosis (Eisenberg and Greene, 2007). The latter is completely blocked in GAK deficient mouse embryonic fibroblasts (Lee et al., 2008). GAK also plays a key role in brain development. Conditional deletion of GAK in mouse brain resulted in marked cell loss and morphological changes in new-born pups, potentially due to a lack of proliferation of neural progenitor cells in the subventricular zone of the hippocampus. Conditional GAK knockout mice die soon after birth whilst conventional GAK knockouts are embryonic lethal (Lee et al., 2008). Moreover transgenic mice expressing kinase inactive GAK die within 30 min of birth due to respiratory dysfunction (Tabara et al., 2011). Respiratory problems are also associated with use of gefitinib (Tabara et al., 2011), an anti-cancer epidermal growth factor receptor (EGFR) inhibitor that also inhibits GAK.

Single nucleotide polymorphisms in the GAK locus were first associated with PD susceptibility following genome-wide association analysis of a large number of familial PD patients (Pankratz et al., 2009). The association has since been robustly replicated in different populations (Rhodes et al., 2011; Sharma et al., 2012). One GAK SNP, rs1564282 is associated with higher expression of α-synuclein in PD brain, and when GAK mRNA was knocked down with siRNA, there was accumulation of α-synuclein in cell culture models (Dumitriu et al., 2011). This provides some biochemical evidence for a role for GAK in PD, although the toxic effects of GAK knockdown/inhibition suggest that GAK is unlikely to be dramatically decreased in PD brain. Moreover, an alternative microarray based study has shown that GAK mRNA expression is increased in the substantia nigra of PD patients (Grunblatt et al., 2004). Intriguingly, GAK has also been proposed to interact with LRRK2 and potentially help co-ordinate the clearance of trans-Golgi derived vesicles (Beilina et al., 2014); however, GAK protein expression in PD brain and any association with PD pathology has been poorly explored.

SERINE/THREONINE KINASE 39 (STK39)

Serine/threonine kinase is more commonly referred to in the literature as SPAK. The majority of work on SPAK has focused on the enzymes role as a regulator of the Na⁺/Cl^- and Na⁺/K⁺/2Cl^- ion co-transporters, NCC and NKCC, respectively. In response to osmotic stress SPAK is activated by phosphorylation at T233 in its activation loop by isoforms of WNK (with-no lysine) kinases and in turn phosphorylates NCC/NKCC to promote transporter activity (Richardson and Alessi, 2008). These ion co-transporters are major drug targets of current anti-hypertensive medications and evidence suggests that inhibition of SPAK may also lower blood pressure (Richardson and Alessi, 2008; Glover and O’shaughnessy, 2011). Indeed, variations in the STK39 gene have been implicated in hypertension in the Amish population through genome-wide association, with the resulting non-coding mutations increasing the allelic expression of SPAK (Wang et al., 2009a). This association, however, has failed to reach significance in other populations (Cunnington et al., 2009; Persu and Vikkula, 2011).

Genome-wide association studies have also implicated variations in the STK39 locus with PD. First identified through large-scale meta-analysis (Liu et al., 2011; Nalls et al., 2011), the association of STK39 SNPs with PD has been subsequently replicated in Asian and Caucasian populations (Lill et al., 2012; Sharma et al., 2012). The three reported STK39 SNPs associating with PD differ from the reported SNP for hypertension. Whether these polymorphisms affect SPAK expression is unknown.

Studies using rats show that SPAK is highly expressed in the nervous system, especially brain where it is detected in neurons, Purkinje cells and choroid epithelial cells (Ushiro et al., 1998). Glial cells do not show immunoreactivity for SPAK (Ushiro et al., 1998). In developing brain, SPAK plays a role in the regulation of Cl^- concentration and in-turn release of the neurotransmitter GABA (Delpire and Austin, 2010). SPAK has also been suggested to act as a stress-response kinase with its overexpression or activation leading to increased phosphorylation of p38 MAPK (Yan et al., 2007). Whether levels of SPAK protein, phosphorylation of SPAK T233 or phosphorylation of the SPAK ion transporter substrates are altered in PD brain has not been investigated.

KINASES OF THE MITOGEN ACTIVATED PROTEIN KINASE (MAPK) PATHWAY

The MAPK superfamily of serine/threonine protein kinases consists of three major branches, the JNKs, the p38 kinases and the ERKs (Figure 2; for review see Kyriakis and Avruch, 2012). The three JNK isoforms (JNK1, JNK2, and JNK3) and four p38 isoforms (p38α, p38β, p38γ, and p38δ) are referred to as stress activated protein kinases (SAPKs). In particular, JNK is activated by a number of environmental stresses implicated in PD including, toxins, inflammatory agonists and misfolded protein-induced ER stress. The activation of p38 is more restricted to inflammatory agonists whilst the two ERK isoforms (ERK1 and ERK2) are activated principally in response to mitogens, although a high level of cross-talk exists between the different MAPK branches. Upon activation, JNK, ERK, and p38 phosphorylate a large number of substrates in a proline-directed manner. In some instances substrates can be specific, such as JNK to phosphorylate the AP-1 transcription factor component c-Jun, or ERK to phosphorylate the p90 ribosomal S6 kinase (RSK), or shared, such as ERK and p38 to phosphorylate the mitogen and stress activated kinase (MSK). Biologically, the MAPKs modulate a number of important functions including development, immunity, apoptosis, cell growth and division, autophagy and cell survival.

KINASES OF THE PHOSPHOINOSITIDE 3-KINASE (PI3K) PATHWAY

The PI3K pathway controls cell survival and proliferation and thus has been studied extensively in the context of cancer. PI3K is classically activated by tyrosine kinase receptors following their binding of insulin or insulin like growth factors (e.g., IGF1; Figure 3). Activated PI3K phosphorylates membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2) to produce phosphatidylinositol 2,4,5-triphosphate (PIP3), which results in recruitment of PKB (also known as AKT; Figure 3). PKB is in turn phosphorylated in its activation loop (at Thr308) by phosphoinositide-dependent kinase 1 (PDK1) and at its C-terminal hydrophobic motif (Ser473) by mTOR complex 2 (mTORC2). Activated PKB phosphorylates a number of substrates including mTOR, to promote protein synthesis and inhibit autophagy, glycogen synthase kinase 3β (GSK3β), to induce glycogen and regulate glucose metabolism and fork head box-O class (FOXO), a transcription factor regulating genes essential for cell growth, proliferation, and survival.