Gordon P. Meares

Resolution of the nuclear localization mechanism of glycogen synthase kinase-3: functional effects in apoptosis.

Mechanisms regulating the nuclear localization of glycogen synthase kinase-3β (GSK3β) remained enigmatic despite the crucial regulation by nuclear GSK3β of important cellular functions. These include regulation of gene expression, cell cycle progression, and apoptosis, achieved by the phosphorylation by GSK3 of nuclear substrates (e.g. numerous transcription factors). We resolved this mechanism by identifying a bipartite nuclear localization sequence (NLS) that is necessary for the nuclear accumulation of GSK3β and is sufficient to drive yellow fluorescent protein into the nucleus. Despite the NLS, most GSK3β is cytosolic, sequestered in protein complexes that, although still mobile in the cytosol, block the NLS. Conditions promoting nuclear translocation of GSK3β release it from cytosolic complexes, allowing the NLS to direct nuclear import. Using this information to prepare a nucleus-excluded active GSK3 construct, we found that the antiapoptotic effect of GSK3β in tumor necrosis factor-induced apoptosis is mediated by cytosolic, not nuclear, GSK3β. Identification of a GSK3β NLS allows new strategies to decipher and manipulate its subcellular actions regulating gene expression and apoptosis and its involvement in diseases.

Glycogen synthase kinase-3 regulates endoplasmic reticulum (ER) stress-induced CHOP expression in neuronal cells.

Endoplasmic reticulum (ER) stress, often resulting from cellular accumulation of misfolded proteins, occurs in many neurodegenerative disorders, in part because of the relatively long lifetime of neurons. Excessive accumulation of misfolded proteins activates the unfolded protein response (UPR) that dampens protein synthesis and promotes removal of misfolded proteins to support survival of ER-stressed cells. However, the UPR also initiates apoptotic signaling to kill cells if recovery is not achieved. Thus, there is much interest in identifying determinants of the life-death switch and interventions that promote recovery and survival. One intervention that has consistently been shown to protect cells from ER stress-induced apoptosis is application of inhibitors of glycogen synthase kinase-3 (GSK3). Therefore, we examined where in the UPR pathway GSK3 inhibitors intercede to impede signaling towards apoptosis. Apoptosis following UPR activation can be mediated by activation of two transcription factors, ATF4 and ATF6, that activate expression of the death-inducing transcription factor C/EBP homologous protein (CHOP/GADD153) following ER stress. We found that ER stress activated ATF6 and ATF4, but these responses were not inhibited by pretreatment with GSK3 inhibitors. However, inhibition of GSK3 effectively reduced the expression of CHOP, and this was apparent in several types of neural-related cells and was evident after application of several structurally diverse GSK3 inhibitors. Therefore, reduction of CHOP activation provides one mechanism by which inhibitors of GSK3 are capable of shifting cell fate towards survival instead of apoptosis following ER stress.

Regulation of CCL20 expression in astrocytes by IL-6 and IL-17

Astrocytes have an important role in the regulation of inflammation within the central nervous system (CNS). In neuroinflammatory conditions such as multiple sclerosis, numerous cytokines and chemokines are elevated including IL‐6, IL‐17, and CCL20. IL‐17 enhances IL‐6 signaling and subsequent IL‐6 expression in astrocytes. CCL20 is a CC motif chemokine that functions as a chemoattractant to facilitate the recruitment of CCR6‐expressing cells, including Th17 cells. In this study, we examined the role of IL‐6 and IL‐17 on CCL20 production in primary murine astrocytes. IL‐6 in combination with the IL‐6 soluble receptor (sIL‐6R) stimulated CCL20 expression in part through STAT3 activation, whereas IL‐17 alone had no effect. However, the combination of IL‐6, sIL‐6R, and IL‐17 led to a robust increase in CCL20 production. IL‐17 increased the activation‐associated phosphorylation of NF‐κB, and inhibition of the NF‐κB pathway significantly inhibited the enhancement of CCL20 expression by IL‐17. In addition, chromatin immunoprecipitation revealed that stimulation of primary astrocytes with IL‐6 plus the sIL‐6R induced STAT3 binding to the CCL20 promoter. Combined stimulation with IL‐6, sIL‐6R, and IL‐17 increased the recruitment of phosphorylated NF‐κB to the CCL20 promoter, increased binding of coactivators such as p300 and CBP, and enhanced H3 and H4 histone acetylation, consistent with a transcriptionally active gene. The astrocyte‐produced CCL20 increased T cell migration as determined by transwell migration assay. Collectively, these results suggest that astrocytes, in response to IL‐6, sIL‐6R, and IL‐17, may shift chemokine production to that favoring T cell recruitment to the CNS.

PERK-Dependent Activation of JAK1 and STAT3 Contributes to Endoplasmic Reticulum Stress-Induced Inflammation

ABSTRACT Neuroinflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. Here, we have examined the interaction between ER stress and JAK/STAT-dependent inflammation in glial cells. We show that ER stress is present in the central nervous system (CNS) concomitant with inflammation and astrogliosis in the multiple sclerosis (MS) mouse model of experimental autoimmune encephalomyelitis (EAE). Astrocytes do not easily succumb to ER stress but rather activate an inflammatory program involving activation of STAT3 in a JAK1-dependent fashion. ER stress-induced activation of the JAK1/STAT3 axis leads to expression of interleukin 6 (IL-6) and several chemokines. Moreover, the activation of STAT3 signaling is dependent on PERK, a central component of the ER stress response, which we show is phosphorylated by JAK1. Disruption of PERK abrogates ER stress-induced activation of STAT3 and subsequent gene expression. Additionally, ER-stressed astrocytes, via paracrine signaling, can stimulate activation of microglia, leading to production of IL-6 and oncostatin M (OSM). These IL-6 cytokines can then synergize with ER stress in astrocytes to drive inflammation. Together, this work describes a new PERK/JAK1/STAT3 signaling pathway that elicits a feed-forward inflammatory loop involving astrocytes and microglia to drive neuroinflammation, which may be relevant in diseases such as MS.

IRE1-Dependent Activation of AMPK in Response to Nitric Oxide

ABSTRACT While there can be detrimental consequences of nitric oxide production at pathological concentrations, eukaryotic cells have evolved protective mechanisms to defend themselves against this damage. The unfolded-protein response (UPR), activated by misfolded proteins and oxidative stress, is one adaptive mechanism that is employed to protect cells from stress. Nitric oxide is a potent activator of AMP-activated protein kinase (AMPK), and AMPK participates in the cellular defense against nitric oxide-mediated damage in pancreatic β-cells. In this study, the mechanism of AMPK activation by nitric oxide was explored. The known AMPK kinases LKB1, CaMKK, and TAK1 are not required for the activation of AMPK by nitric oxide. Instead, this activation is dependent on the endoplasmic reticulum (ER) stress-activated protein IRE1. Nitric oxide-induced AMPK phosphorylation and subsequent signaling to AMPK substrates, including Raptor, acetyl coenzyme A carboxylase, and PGC-1α, is attenuated in IRE1α-deficient cells. The endoribonuclease activity of IRE1 appears to be required for AMPK activation in response to nitric oxide. In addition to nitric oxide, stimulation of IRE1 endoribonuclease activity with the flavonol quercetin leads to IRE1-dependent AMPK activation. These findings indicate that the RNase activity of IRE1 participates in AMPK activation and subsequent signaling through multiple AMPK-dependent pathways in response to nitrosative stress.

FoxO1 and SIRT1 Regulate β-Cell Responses to Nitric Oxide

For many cell types, including pancreatic β-cells, nitric oxide is a mediator of cell death; paradoxically, nitric oxide can also activate pathways that promote the repair of cellular damage. In this report, a role for FoxO1-dependent transcriptional activation and its regulation by SIRT1 in determining the cellular response to nitric oxide is provided. In response to nitric oxide, FoxO1 translocates from the cytoplasm to the nucleus and stimulates the expression of the DNA repair gene GADD45α, resulting in FoxO1-dependent DNA repair. FoxO1-dependent gene expression appears to be regulated by the NAD+-dependent deacetylase SIRT1. In response to SIRT1 inhibitors, the FoxO1-dependent protective actions of nitric oxide (GADD45α expression and DNA repair) are attenuated, and FoxO1 activates a proapoptotic program that includes PUMA (p53-up-regulated mediator of apoptosis) mRNA accumulation and caspase-3 cleavage. These findings support primary roles for FoxO1 and SIRT1 in regulating the cellular responses of β-cells to nitric oxide.

Inhibition of the JAK/STAT Pathway Protects Against α-Synuclein-Induced Neuroinflammation and Dopaminergic Neurodegeneration

Parkinsons Disease (PD) is an age-related, chronic neurodegenerative disorder. At present, there are no disease-modifying therapies to prevent PD progression. Activated microglia and neuroinflammation are associated with the pathogenesis and progression of PD. Accumulation of α-synuclein (α-SYN) in the brain is a core feature of PD and leads to microglial activation, inflammatory cytokine/chemokine production, and ultimately to neurodegeneration. Given the importance of the JAK/STAT pathway in activating microglia and inducing cytokine/chemokine expression, we investigated the therapeutic potential of inhibiting the JAK/STAT pathway using the JAK1/2 inhibitor, AZD1480. In vitro, α-SYN exposure activated the JAK/STAT pathway in microglia and macrophages, and treatment with AZD1480 inhibited α-SYN-induced major histocompatibility complex Class II and inflammatory gene expression in microglia and macrophages by reducing STAT1 and STAT3 activation. For in vivo studies, we used a rat model of PD induced by viral overexpression of α-SYN. AZD1480 treatment inhibited α-SYN-induced neuroinflammation by suppressing microglial activation, macrophage and CD4+ T-cell infiltration and production of proinflammatory cytokines/chemokines. Numerous genes involved in cell–cell signaling, nervous system development and function, inflammatory diseases/processes, and neurological diseases are enhanced in the substantia nigra of rats with α-SYN overexpression, and inhibited upon treatment with AZD1480. Importantly, inhibition of the JAK/STAT pathway prevented the degeneration of dopaminergic neurons in vivo. These results indicate that inhibiting the JAK/STAT pathway can prevent neuroinflammation and neurodegeneration by suppressing activation of innate and adaptive immune responses to α-SYN. Furthermore, this suggests the feasibility of targeting the JAK/STAT pathway as a neuroprotective therapy for neurodegenerative diseases. SIGNIFICANCE STATEMENT α-SYN plays a central role in the pathophysiology of PD through initiation of neuroinflammatory responses. Using an α-SYN overexpression PD model, we demonstrate a beneficial therapeutic effect of AZD1480, a specific inhibitor of JAK1/2, in suppressing neuroinflammation and neurodegeneration. Our findings document that inhibition of the JAK/STAT pathway influences both innate and adaptive immune responses by suppressing α-SYN-induced microglia and macrophage activation and CD4+ T-cell recruitment into the CNS, ultimately suppressing neurodegeneration. These findings are the first documentation that suppression of the JAK/STAT pathway disrupts the circuitry of neuroinflammation and neurodegeneration, thus attenuating PD pathogenesis. JAK inhibitors may be a viable therapeutic option for the treatment of PD patients.

AMP-Activated Protein Kinase Restricts IFN-γ Signaling

Inflammation in the CNS contributes to neurologic disorders. Neuroinflammation involves the release of inflammatory molecules from glial cells, such as astrocytes and microglia, and can lead to neuronal damage if unabated. In multiple sclerosis, peripheral immune cells, including IFN-γ–producing Th1 cells, infiltrate the CNS and are important in shaping the inflammatory microenvironment, in part through cytokine-mediated interactions with glial cells. Recent evidence suggests that AMP-activated protein kinase (AMPK), a central regulator of energetic metabolism, can regulate inflammatory gene expression. In this study, we identified that IFN-γ induces biphasic AMPK signaling, suggestive of negative-feedback mechanisms. Activation of AMPK suppresses several IFN-γ–induced cytokines and chemokines in primary astrocytes and microglia. IFN-γ regulates gene expression through activation of STAT1, and deletion of AMPK results in a marked increase in basal expression of STAT1. Conversely, activation of AMPK blocks IFN-γ–induced STAT1 expression. Deletion of AMPK leads to increased basal and IFN-γ–induced expression of inflammatory molecules, including TNF-α, CXCL10, and CCL2. AMPK does not affect the phosphorylation of STAT1, but instead attenuates nuclear translocation of STAT1, DNA binding, and subsequent gene expression. In vivo, AMPK signaling during experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, is downregulated in the brain at onset and peak of disease. Diminution of AMPK signaling in vivo correlates with increased expression of IFN-γ and CCL2 in the CNS. Overall, these findings provide the first link between AMPK and STAT1 and may provide important clues about how bioenergetics and inflammation are linked.

Repair of Nitric Oxide-damaged DNA in β-Cells Requires JNK-dependent GADD45α Expression

Proinflammatory cytokines induce nitric oxide-dependent DNA damage and ultimately β-cell death. Not only does nitric oxide cause β-cell damage, it also activates a functional repair process. In this study, the mechanisms activated by nitric oxide that facilitate the repair of damaged β-cell DNA are examined. JNK plays a central regulatory role because inhibition of this kinase attenuates the repair of nitric oxide-induced DNA damage. p53 is a logical target of JNK-dependent DNA repair; however, nitric oxide does not stimulate p53 activation or accumulation in β-cells. Further, knockdown of basal p53 levels does not affect DNA repair. In contrast, expression of growth arrest and DNA damage (GADD) 45α, a DNA repair gene that can be regulated by p53-dependent and p53-independent pathways, is stimulated by nitric oxide in a JNK-dependent manner, and knockdown of GADD45α expression attenuates the repair of nitric oxide-induced β-cell DNA damage. These findings show that β-cells have the ability to repair nitric oxide-damaged DNA and that JNK and GADD45α mediate the p53-independent repair of this DNA damage.

Heat shock protein-90 dampens and directs signaling stimulated by insulin-like growth factor-1 and insulin

Heat shock protein‐90 (Hsp90) buffers cells from genetic mutations and environmental stresses. To test if this capability reflects a normal physiological function of Hsp90 to buffer cellular signals, the effects of Hsp90 inhibition were measured on activation of Akt. Inhibition of Hsp90 with geldanamycin amplified Akt phosphorylation induced by insulin‐like growth factor‐1 (IGF‐1) or insulin, indicating that Hsp90 normally buffers these signals. Furthermore, with IGF‐1 stimulation Hsp90 inhibition increased p38 activation, produced additive activation of p90RSK, and slightly increased the duration of ERK1/2 activation. Hsp90 dampened Akt signaling by facilitating phosphatase‐mediated dephosphorylation of Akt. Thus, Hsp90 not only buffers the cellular effects of mutations and stresses, but also buffers the magnitude and duration of activation of proliferative and survival‐promoting signaling responses.