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The pro-inflammatory cytokine interleukin-1β (IL-1β), whose levels are elevated in the brain in Alzheimer's and other neurodegenerative diseases, has been shown to have both detrimental and beneficial effects on disease progression. In this article, we demonstrate that incubation of mouse primary cortical neurons (mPCNs) with IL-1β increases the expression of the P2Y2 nucleotide receptor (P2Y2R) and that activation of the up-regulated receptor with UTP, a relatively selective agonist of the P2Y2R, increases neurite outgrowth. Consistent with the accepted role of cofilin in the regulation of neurite extension, results indicate that incubation of IL-1β-treated mPCNs with UTP increases the phosphorylation of cofilin, a response absent in PCNs isolated from P2Y2R−/− mice. Other findings indicate that function-blocking anti-αvβ3/5 integrin antibodies prevent UTP-induced cofilin activation in IL-1β-treated mPCNs, suggesting that established P2Y2R/αvβ3/5 interactions that promote G12-dependent Rho activation lead to cofilin phosphorylation involved in neurite extension. Cofilin phosphorylation induced by UTP in IL-1β-treated mPCNs is also decreased by inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII), suggesting a role for P2Y2R-mediated and Gq-dependent calcium mobilization in neurite outgrowth. Taken together, these studies indicate that up-regulation of P2Y2Rs in mPCNs under pro-inflammatory conditions can promote cofilin-dependent neurite outgrowth, a neuroprotective response that may be a novel pharmacological target in the treatment of neurodegenerative diseases.
Neurodegenerative diseases are a serious cause of mortality in the United States with more than 5 million people currently afflicted (Duncan 2011; Thies and Bleiler 2011). Alzheimer's disease (AD) is the most prevalent of these conditions, and it is predicted that AD will affect 80 million people worldwide within 30 years (Blennow et al. 2006; Thies and Bleiler 2011). There are currently no effective treatments to prevent the onset or delay the progression of neurological deficits that degrade the quality of life of AD patients for many years prior to death (Thies and Bleiler 2011). It is now widely accepted that chronic inflammation plays a role in the progression of neurological changes observed in the AD brain, including neuronal loss and degeneration of neurological functions (Zilka et al. 2006; Lee et al. 2010; Obulesu et al. 2011; Wyss-Coray and Rogers 2012). Nevertheless, the initiating factors in AD remain obscure and whether neuroinflammation is primarily a neurodegenerative or a neuroprotective response in AD is an area of intense investigation (Zilka et al. 2006; Lee et al. 2010; Broussard et al. 2012).
Chronic inflammation in the central nervous system (CNS) is a conspicuous feature of many neurodegenerative diseases, including AD, Parkinson's disease and multiple sclerosis (Akiyama et al. 2000; Rothwell and Luheshi 2000; Broussard et al. 2012). A key cytokine associated with the neuroinflammatory phenotype is IL-1β, a pro-inflammatory cytokine produced by microglial cells and macrophages that regulates the production of other pro-inflammatory cytokines (e.g., TNF-α, IL-6, and interferons) and chemokines (e.g., CXCL1 and CXCL2) (Rothwell and Luheshi 2000; Shaftel et al. 2008). Although studies have investigated the neurodegenerative roles of IL-1β in AD progression (Rothwell and Luheshi 2000; Shaftel et al. 2008), observations in a mouse model of AD indicate that over-expression of IL-1β in the hippocampus can promote phagocyte recruitment and the clearance of β-amyloid plaques (Shaftel et al. 2007b). This suggests that IL-1β can also serve a neuroprotective role in the CNS that requires further investigation.
Recently, we demonstrated that the P2Y2 nucleotide receptor (P2Y2R), a G protein-coupled receptor that is activated equally well by ATP and UTP, is up-regulated in rat primary cortical neurons in response to IL-1β (Kong et al. 2009). Subsequent activation of the P2Y2R by extracellular nucleotides promotes the non-amyloidogenic processing of amyloid precursor protein (APP) (Camden et al. 2005; Kong et al. 2009). In mouse primary microglial cells, the P2Y2R is up-regulated by the neurotoxic β-amyloid (Aβ1-42) peptide associated with AD pathogenesis, whereupon activation of the microglial P2Y2R enhances Aβ phagocytosis and degradation (Kim et al. 2012), suggesting that P2Y2R up-regulation and P2Y2R-mediated non-amyloidogenic APP processing are neuroprotective responses that prevent excessive neurotoxic Aβ1-42 accumulation. Other studies have found that activation of ionotropic P2X7 receptors in microglial cells by extracellular ATP, a pathway that induces cell apoptosis, increases both IL-1β and ATP release from microglia (Takenouchi et al. 2009, 2011), thereby providing the agonists for both P2Y2R up-regulation and activation. Other potential neuroprotective responses to P2Y2R activation include the induction of intracellular calcium waves (Halassa et al. 2009), the up-regulation of anti-apoptotic protein expression in astrocytes (Chorna et al. 2004) and the enhancement of neuronal differentiation and survival (Arthur et al. 2005, 2006a, b; Pooler et al. 2005). Thus, P2Y2Rs in neurons, microglial cells, and astrocytes likely coordinately regulate neuroprotective responses to elevated levels of extracellular nucleotides that occur under pro-inflammatory, pro-apoptotic, and necrotic conditions (Peterson et al. 2010; Weisman et al. 2012a, b) and may prevent or delay neurodegeneration. Therefore, P2Y2Rs represent promising pharmacological targets in the treatment of AD and other diseases of the CNS.
This study was undertaken to further evaluate the role of P2Y2Rs in mouse primary cortical neurons (mPCNs), in particular the mechanism underlying the effect of extracellular nucleotides on neurite extension, a neuroprotective pathway that has not been characterized. It has been established that neurite outgrowth in response to activation of other G protein-coupled receptors requires the sequential activation of Rho, ROCK, LIMK, and cofilin (Meng et al. 2002; Bamburg et al. 2010; Bernstein and Bamburg 2010). Furthermore, we have previously demonstrated that activation of the P2Y2R can increase Rho and ROCK activities because of the presence of an Arg-Gly-Asp (RGD) sequence in the first extracellular loop of the P2Y2R that promotes its direct binding to αvβ3/5 integrins, an interaction required for extracellular nucleotides to activate heterotrimeric G12 protein and subsequently the small G protein Rho (Erb et al. 2001; Bagchi et al. 2005; Liao et al. 2007). In this study, we utilized mPCNs to demonstrate that up-regulation of the P2Y2R because of the pro-inflammatory cytokine IL-1β followed by P2Y2R activation with UTP increases both cofilin phosphorylation and neurite outgrowth. Furthermore, UTP-induced cofilin phosphorylation and neurite outgrowth was found to be absent in mPCNs from P2Y2R−/− mice and occurred via a pathway involving αvβ3/5 and CaMKII. These results strongly suggest that by virtue of P2Y2R interactions with αvβ3/5 integrins, nucleotides can activate Rho-dependent cofilin phosphorylation to regulate cytoskeletal rearrangements required for neurite extension and stabilization, which is critical for neuronal survival.
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In this study, we demonstrate that IL-1β up-regulates the P2Y2R in mPCNs, where-upon activation of the P2Y2R promotes neurite extension through inactivation (phosphorylation) of the actin-depolymerizing protein cofilin, a known regulator of neurite extension (Meberg et al. 1998; Meberg and Bamburg 2000; Aizawa et al. 2001; Endo et al. 2003, 2007; Figge et al. 2012). Using an antibody that blocks αv integrin function and selective inhibitors, we also provide evidence that the P2Y2R mediates cofilin inactivation required for neurite outgrowth in mPCNs by a mechanism involving αv integrin, Rho/ROCK/LIMK, and CaMKII/CaMKK/LIMK. The P2Y2R also is known to modulate ADAM activity in neurons (Kong et al. 2009); however, we determined that TAPI-2, a matrix metalloprotease inhibitor, had no effect on UTP-induced cofilin phosphorylation in IL-1β-treated mPCNs (data not shown). Based on these findings, a model of the mechanisms underlying P2Y2R-mediated neurite extension is provided in Fig. 6.
IL-1β is thought to have both detrimental and beneficial effects on the pathogenesis of AD (Mrak and Griffin 2005; Shaftel et al. 2007b). Studies in humans have shown that the IL-1β gene (Colangelo et al. 2002) and IL-1β protein expression (Griffin et al. 1989) are elevated in AD brain tissue and experimental models demonstrate that IL-1β is involved in neuronal injury, degeneration, and loss (Rothwell and Luheshi 2000). Other studies, however, demonstrate that IL-1β promotes remyelination of neurons in the mouse CNS (Mason et al. 2001) and that chronic IL-1β expression causes an increase in blood–brain barrier permeability to leukocytes in mouse brain without causing neurodegeneration (Shaftel et al. 2007a). We show here that a 24 h pre-treatment of mPCNs with IL-1β causes an ~fourfold increase in P2Y2R mRNA expression (Fig. 1), suggesting that signaling cues from the P2Y2R in neurons are intensified under inflammatory conditions. Since P2Y2R activation in IL-1β-treated wild type mPCNs promotes neurite extension, in contrast to P2Y2R−/− mPCNs (Fig. 2), we postulate that P2Y2R up-regulation in the CNS may delay the progression of neurodegeneration that occurs with chronic inflammation. P2Y2R expression levels were found to be lower in post-mortem human AD brain tissue compared to normal tissue (Lai et al. 2008). This may be because of brain atrophy and the degeneration of P2Y2R-expressing cells in the CNS or the down-regulation of the P2Y2R in specific subsets of cells in the CNS during end stage AD, possibilities that we are currently evaluating in a mouse model of AD. A better understanding of the mechanisms of neurite extension may provide insights into the role of P2Y2Rs in the regulation of the transition from acute to chronic inflammation and neurodegeneration and the apparent neuroprotective role of P2Y2Rs during inflammation.
P2Y2R gene expression induced by IL-1β in mPCNs (Fig. 1) and rat cortical neurons (Kong et al. 2009) is inhibited by pre-treatment with Bay 11-7082, an inhibitor of IκB-α phosphorylation (Pierce et al. 1997). This suggests that the IκB/NF-κB signaling pathway, known to be enhanced by pro-inflammatory cytokines (DiDonato et al. 1997; Hacker and Karin 2006), regulates P2Y2R transcription in neurons. Consistent with this hypothesis, the P2Y2R promoter contains a NF-κB binding domain that regulates increased P2Y2R transcription in response to inflammatory agents (Degagne et al. 2009). In vascular smooth muscle cells, IL-1β was shown to up-regulate P2Y2R gene expression by a mechanism involving cyclooxygenase and protein kinase C (PKC), although NF-κB activation was not examined (Hou et al. 2000).
We found that activation of the P2Y2R in wild type mPCNs increases neurite extension, as indicated by an increase in the neurite perimeter of UTP-treated cells (Fig. 2), which is critical for establishing synaptic connections and for neuronal survival (Cline and Haas 2008). Neurite outgrowth in cultured neurons is considered an indication of neuroregenerative potential (Mitchell et al. 2007). Therefore, development of strategies to activate the P2Y2R in vivo may help retard neurodegeneration that occurs in AD and other neuroinflammatory diseases by promoting neurite extension and neuronal survival.
Neurite extension requires molecular signals that promote remodeling of the actin cytoskeleton within the growing neurite. The actin-binding protein cofilin regulates actin dynamics in essentially every type of eukaryotic cell (Maciver and Hussey 2002; Van Troys et al. 2008), and numerous studies indicate that cofilin is important for neurite extension (Meberg et al. 1998; Meberg and Bamburg 2000; Aizawa et al. 2001; Endo et al. 2003, 2007; Figge et al. 2012). Recent studies using Aplysia kurodai neurons found that microinjection of dephosphorylated cofilin led to rod formation, synapse loss (e.g., a decrease in the number of pre-synaptic varicosities) and, distal to the rod, impairment of synaptic plasticity measured by electrophysiological methods (Jang et al. 2005). In addition, phospho-cofilin administration impaired basal synaptic transmission, long-term potentiation (LTP), and the structure and dynamics of post-synaptic dendritic spines (Jang et al. 2005). Other studies indicate that blockade of calcineurin with FK506 or expression of a phosphomimetic mutant of cofilin (cof-S3D) prevented Aβ-induced spine loss in a hippocampal slice model of AD (Shankar et al. 2007). These findings are consistent with previous reports that cofilin signaling is perturbed in AD brain tissue and in neurons treated with synthetic Aβ (Maloney et al. 2005; Zhou et al. 2006). Thus, the ability of the P2Y2R to regulate cofilin phosphorylation in neurons (Figs. 3 and 4) likely has relevance to neurodegenerative disorders, such as AD, where cell damage or apoptosis would be anticipated to increase levels of extracellular nucleotides that activate the P2Y2R.
The activity of cofilin in the disassembly of actin filaments (F-actin) is regulated by several mechanisms, including phosphorylation of cofilin at serine3 (Ser3) (van Rheenen et al. 2009). Ser3 phosphorylation of cofilin prevents F-actin binding and severing of actin filaments by cofilin (van Rheenen et al. 2009), and thus it is thought that cofilin phosphorylation/dephosphorylation at Ser3 acts as a switch between actin assembly and disassembly (Huang et al. 2006). Several kinases and phosphatases have been identified that phosphorylate and dephosphorylate cofilin at Ser3, respectively; these include the actin-binding LIM kinases (LIMK1, LIMK2) (Arber et al. 1998; Yang et al. 1998), testicular protein kinases (Toshima et al. 2001a, b), slingshot (SSH) phosphatases (Niwa et al. 2002), and chronophin phosphatase (Niwa et al. 2002; Gohla et al. 2005). Here, we show that activation of the P2Y2R in IL-1β-pre-treated mPCNs causes phosphorylation of cofilin at Ser3 (Figs. 3 and 4) and that the Rho/ROCK/LIMK pathway is involved in this process, since inhibition of ROCK, which is activated by RhoA and controls the activation of LIMK2 (Gungabissoon and Bamburg 2003; Bernstein and Bamburg 2010), prevents P2Y2R-mediated cofilin phosphorylation at Ser3 (Fig. 5a). We also show that P2Y2R-mediated cofilin phosphorylation requires the activities of CaMKII and CaMKK (Fig. 5b). Furthermore, CaMKII controls cofilin phosphorylation and regulates F-actin dynamics through pathways that mainly converge on the Rho family of monomeric GTPases, such as RhoA and Rac1 (Okamoto et al. 2009). The activities of these GTPases are controlled by guanine-nucleotide-exchange factors (GEFs) and GTPase activating proteins (GAPs) (Symons and Settleman 2000; Newey et al. 2005). Although we did not explore which GEFs are activated by the P2Y2R in mPCNs, a likely candidate is kalirin-7, whose activity is reported to be essential for spine enlargement (Penzes and Jones 2008). Phosphorylation of kalirin-7 by CaMKII increases its GEF activity and leads to cofilin phosphorylation through the Rac1/PAK1/LIMK1 pathway (Penzes and Jones 2008). Other GEF candidates that mediate cofilin phosphorylation in dendritic spines and are controlled by CaMKII include βPIX, which signals through the Rac1 pathway, and Lcf, which binds to the actin-binding protein spinophilin and signals through the RhoA/ROCK/LIMK2 pathway (Okamoto et al. 2009).
Ionomycin-induced cofilin phosphorylation and neurite outgrowth are also blocked by KN-93, an inhibitor of Ca2+/calmodulin-dependent protein kinases, and STO-609, an inhibitor of CaMKK (Takemura et al. 2009). Other investigators have previously shown that ROCK and PAK activate LIMK1 by phosphorylation at Thr-508, which is in the activation loop of the kinase domain in LIMK1 (Edwards et al. 1999; Maekawa et al. 1999; Ohashi et al. 2000). Various signaling pathways, including Rac/PAK1 and CaMKIV/CaMKK activate Thr-508 phosphorylation in LIMK1 (Edwards et al. 1999; Maekawa et al. 1999; Ohashi et al. 2000; Takemura et al. 2009), suggesting a similar target for the Rho/ROCK and CaMKII/CaMKK pathways activated by the P2Y2R. This also suggests that LIMK1 activation is a point of convergence that links multiple signaling pathways to the regulation of actin cytoskeletal reorganization in cells (Takemura et al. 2009). Further studies are needed to evaluate cross-talk between the CaMKII/CaMKK, RhoA/ROCK, and other signaling pathways in the regulation of P2Y2R-mediated and LIMK-dependent cofilin phosphorylation and neurite extension.