Astrocytic deletion of protein kinase R-like ER kinase (PERK) does not affect learning and memory in aged mice but worsens outcome from experimental stroke.

Aging is associated with cognitive decline and is the main risk factor for a myriad of conditions including neurodegeneration and stroke. Concomitant with aging is the progressive accumulation of misfolded proteins and loss of proteostasis. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress and activation of the unfolded protein response (UPR). The UPR is mediated, in part, by the eukaryotic initiation factor 2α (eIF2α) kinase protein kinase R-like ER kinase (PERK). Phosphorylation of eIF2α reduces protein translation as an adaptive mechanism but this also opposes synaptic plasticity. PERK, and other eIF2α kinases, have been widely studied in neurons where they modulate both cognitive function and response to injury. The impact of astrocytic PERK signaling in cognitive processes was previously unknown. To examine this, we deleted PERK from astrocytes (AstroPERKKO ) and examined the impact on cognitive functions in middle-aged and old mice of both sexes. Additionally, we tested the outcome following experimental stroke using the transient middle cerebral artery occlusion (MCAO) model. Tests of short-term and long-term learning and memory as well as of cognitive flexibility in middle-aged and old mice revealed that astrocytic PERK does not regulate these processes. Following MCAO, AstroPERKKO had increased morbidity and mortality. Collectively, our data demonstrate that astrocytic PERK has limited impact on cognitive function and has a more prominent role in the response to neural injury.

The role of endoplasmic reticulum stress in astrocytes.

Astrocytes are glial cells that support neurological function in the central nervous system (CNS), in part, by providing structural support for neuronal synapses and blood vessels, participating in electrical and chemical transmission, and providing trophic support via soluble factors. Dysregulation of astrocyte function contributes to neurological decline in CNS diseases. Neurological diseases are highly heterogeneous but share common features of cellular stress including the accumulation of misfolded proteins. Endoplasmic reticulum (ER) stress has been reported in nearly all neurological and neurodegenerative diseases. ER stress occurs when there is an accumulation of misfolded proteins in the ER lumen and the protein folding demand of the ER is overwhelmed. ER stress initiates the unfolded protein response (UPR) to restore homeostasis by abating protein translation and, if the cell is irreparably damaged, initiating apoptosis. Although protein aggregation and misfolding in neurological disease has been well described, cell-specific contributions of ER stress and the UPR in physiological and disease states are poorly understood. Recent work has revealed a role for active UPR signaling that may drive astrocytes toward a maladaptive phenotype in various model systems. In response to ER stress, astrocytes produce inflammatory mediators, have reduced trophic support, and can transmit ER stress to other cells. This review will discuss the current known contributions and consequences of activated UPR signaling in astrocytes.

Liver kinase B1 depletion from astrocytes worsens disease in a mouse model of multiple sclerosis.

Liver kinase B1 (LKB1) is a ubiquitously expressed kinase involved in the regulation of cell metabolism, growth, and inflammatory activation. We previously reported that a single nucleotide polymorphism in the gene encoding LKB1 is a risk factor for multiple sclerosis (MS). Since astrocyte activation and metabolic function have important roles in regulating neuroinflammation and neuropathology, we examined the serine/threonine kinase LKB1 in astrocytes in a chronic experimental autoimmune encephalomyelitis mouse model of MS. To reduce LKB1, a heterozygous astrocyte-selective conditional knockout (het-cKO) model was used. While disease incidence was similar, disease severity was worsened in het-cKO mice. RNAseq analysis identified Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in het-cKO mice relating to mitochondrial function, confirmed by alterations in mitochondrial complex proteins and reductions in mRNAs related to astrocyte metabolism. Enriched pathways included major histocompatibility class II genes, confirmed by increases in MHCII protein in spinal cord and cerebellum of het-cKO mice. We observed increased numbers of CD4+ Th17 cells and increased neuronal damage in spinal cords of het-cKO mice, associated with reduced expression of choline acetyltransferase, accumulation of immunoglobulin-γ, and reduced expression of factors involved in motor neuron survival. In vitro, LKB1-deficient astrocytes showed reduced metabolic function and increased inflammatory activation. These data suggest that metabolic dysfunction in astrocytes, in this case due to LKB1 deficiency, can exacerbate demyelinating disease by loss of metabolic support and increase in the inflammatory environment.

Endoplasmic reticulum stress is transmissible in vitro between cells of the central nervous system.

Improper protein folding and trafficking are common pathological events in neurodegenerative diseases that result in the toxic accumulation of misfolded proteins within the lumen of the endoplasmic reticulum (ER). While low-level stimulation of the unfolded protein response (UPR) is protective, sustained UPR activation resulting from prolonged ER stress can promote neurotoxicity. The cell-autonomous mechanisms of the UPR have been extensively characterized. However, the cell-extrinsic role of the UPR under physiological and pathological states in the CNS remains to be elucidated. To begin to address this, we evaluated if transferring conditioned media between ER-stressed astrocytes and neurons could modulate their functional characteristics. Our results indicate that ER-stressed astrocytes and neurons secrete a molecule(s) with lipid characteristics which regulates both inflammatory and ER stress responses in other astrocytes, neurons, and microglia in vitro. Initial exposure to this stress factor(s) confers resistance against subsequent ER stress to neurons. However, persistent exposure to this unidentified mediator(s) suppresses the initial protective effect and becomes cytotoxic. Overall, these findings provide insight into the cell non-autonomous influence of ER stress on cells of the central nervous system. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Glucagon and Insulin Cooperatively Stimulate Fibroblast Growth Factor 21 Gene Transcription by Increasing the Expression of Activating Transcription Factor 4.

Previous studies have shown that glucagon cooperatively interacts with insulin to stimulate hepatic FGF21 gene expression. Here we investigated the mechanism by which glucagon and insulin increased FGF21 gene transcription in primary hepatocyte cultures. Transfection analyses demonstrated that glucagon plus insulin induction of FGF21 transcription was conferred by two activating transcription factor 4 (ATF4) binding sites in the FGF21 gene. Glucagon plus insulin stimulated a 5-fold increase in ATF4 protein abundance, and knockdown of ATF4 expression suppressed the ability of glucagon plus insulin to increase FGF21 expression. In hepatocytes incubated in the presence of insulin, treatment with a PKA-selective agonist mimicked the ability of glucagon to stimulate ATF4 and FGF21 expression. Inhibition of PKA, PI3K, Akt, and mammalian target of rapamycin complex 1 (mTORC1) suppressed the ability of glucagon plus insulin to stimulate ATF4 and FGF21 expression. Additional analyses demonstrated that chenodeoxycholic acid (CDCA) induced a 6-fold increase in ATF4 expression and that knockdown of ATF4 expression suppressed the ability of CDCA to increase FGF21 gene expression. CDCA increased the phosphorylation of eIF2α, and inhibition of eIF2α signaling activity suppressed CDCA regulation of ATF4 and FGF21 expression. These results demonstrate that glucagon plus insulin increases FGF21 transcription by stimulating ATF4 expression and that activation of cAMP/PKA and PI3K/Akt/mTORC1 mediates the effect of glucagon plus insulin on ATF4 expression. These results also demonstrate that CDCA regulation of FGF21 transcription is mediated at least partially by an eIF2α-dependent increase in ATF4 expression.

MicroRNA-31 is required for astrocyte specification.

Previously, we determined microRNA-31 (miR-31) is a noncoding tumor suppressive gene frequently deleted in glioblastoma (GBM); miR-31 suppresses tumor growth, in part, by limiting the activity of NF-κB. Herein, we expand our previous studies by characterizing the role of miR-31 during neural precursor cell (NPC) to astrocyte differentiation. We demonstrate that miR-31 expression and activity is suppressed in NPCs by stem cell factors such as Lin28, c-Myc, SOX2 and Oct4. However, during astrocytogenesis, miR-31 is induced by STAT3 and SMAD1/5/8, which mediate astrocyte differentiation. We determined miR-31 is required for terminal astrocyte differentiation, and that the loss of miR-31 impairs this process and/or prevents astrocyte maturation. We demonstrate that miR-31 promotes astrocyte development, in part, by reducing the levels of Lin28, a stem cell factor implicated in NPC renewal. These data suggest that miR-31 deletions may disrupt astrocyte development and/or homeostasis.

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

UNLABELLED: Parkinson's 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.

Attenuation of PKR-like ER Kinase (PERK) Signaling Selectively Controls Endoplasmic Reticulum Stress-induced Inflammation Without Compromising Immunological Responses.

Inflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. ER stress is brought on by the accumulation of misfolded proteins in the ER, which leads to activation of the unfolded protein response (UPR), a conserved pathway that transmits signals to restore homeostasis or eliminate the irreparably damaged cell. We provide evidence that inhibition or genetic haploinsufficiency of protein kinase R-like endoplasmic reticulum kinase (PERK) can selectively control inflammation brought on by ER stress without impinging on UPR-dependent survival and adaptive responses or normal immune responses. Using astrocytes lacking one or both alleles of PERK or the PERK inhibitor GSK2606414, we demonstrate that PERK haploinsufficiency or partial inhibition led to reduced ER stress-induced inflammation (IL-6, CCL2, and CCL20 expression) without compromising prosurvival responses. In contrast, complete loss of PERK blocked canonical PERK-dependent UPR genes and promoted apoptosis. Reversal of eIF2α-mediated translational repression using ISRIB potently suppressed PERK-dependent inflammatory gene expression, indicating that the selective modulation of inflammatory gene expression by PERK inhibition may be linked to attenuation of eIF2α phosphorylation and reveals a previously unknown link between translational repression and transcription of inflammatory genes. Additionally, ER-stressed astrocytes can drive an inflammatory M1-like phenotype in microglia, and this can be attenuated with inhibition of PERK. Importantly, targeting PERK neither disrupted normal cytokine signaling in astrocytes or microglia nor impaired macrophage phagocytosis or T cell polarization. Collectively, this work suggests that targeting PERK may provide a means for selective immunoregulation in the context of ER stress without disrupting normal immune function.

Protein kinase CK2 is important for the function of glioblastoma brain tumor initiating cells.

Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase composed of two catalytic subunits (α) and/or (α') and two regulatory (ß) subunits. The expression and kinase activity of CK2 is elevated in many different cancers, including glioblastoma (GBM). Brain tumor initiating cells (BTICs) are a subset of cells that are highly tumorigenic and promote the resistance of GBM to current therapies. We previously reported that CK2 activity promotes prosurvival signaling in GBM. In this study, the role of CK2 signaling in BTIC function was examined. We found that expression of CK2α was increased in CD133+ BTICs compared to CD133- cells within the same GBM xenolines. Treatment with CX-4945, an ATP-competitive inhibitor of CK2, led to reduced expression of Sox2 and Nestin, transcription factors important for the maintenance of stem cells. Similarly, inhibition of CK2 also reduced the frequency of CD133+ BTICs over the course of 7 days, indicating a role for CK2 in BTIC persistence and survival. Importantly, using an in vitro limiting dilution assay, we found that inhibition of CK2 kinase activity with CX-4945 or siRNA knockdown of the CK2 catalytic subunits reduced neurosphere formation in GBM xenolines of different molecular subtypes. Lastly, we found that inhibition of CK2 led to decreased EGFR levels in some xenolines, and combination treatment with CX-4945 and Gefitinib to inhibit CK2 and EGFR, respectively, provided optimal inhibition of viability of cells. Therefore, due to the integration of CK2 in multiple signaling pathways important for BTIC survival, CK2 is a promising target in GBM.

Preferential Recruitment of Neutrophils into the Cerebellum and Brainstem Contributes to the Atypical Experimental Autoimmune Encephalomyelitis Phenotype.

The JAK/STAT pathway is critical for development, regulation, and termination of immune responses, and dysregulation of the JAK/STAT pathway, that is, hyperactivation, has pathological implications in autoimmune and neuroinflammatory diseases. Suppressor of cytokine signaling 3 (SOCS3) regulates STAT3 activation in response to cytokines that play important roles in the pathogenesis of neuroinflammatory diseases, including IL-6 and IL-23. We previously demonstrated that myeloid lineage-specific deletion of SOCS3 resulted in a severe, nonresolving atypical form of experimental autoimmune encephalomyelitis (EAE), characterized by lesions, inflammatory infiltrates, elevated STAT activation, and elevated cytokine and chemokine expression in the cerebellum. Clinically, these mice exhibit ataxia and tremors. In this study, we provide a detailed analysis of this model, demonstrating that the atypical EAE observed in LysMCre-SOCS3(fl/fl) mice is characterized by extensive neutrophil infiltration into the cerebellum and brainstem, increased inducible NO synthase levels in the cerebellum and brainstem, and prominent axonal damage. Importantly, infiltrating SOCS3-deficient neutrophils produce high levels of CXCL2, CCL2, CXCL10, NO, TNF-α, and IL-1ß. Kinetic studies demonstrate that neutrophil infiltration into the cerebellum and brainstem of LysMCre-SOCS3(fl/fl) mice closely correlates with atypical EAE clinical symptoms. Ab-mediated depletion of neutrophils converts the atypical phenotype to the classical EAE phenotype and, in some cases, a mixed atypical/classical phenotype. Blocking CXCR2 signaling ameliorates atypical EAE development by reducing neutrophil infiltration into the cerebellum/brainstem. Thus, neutrophils lacking SOCS3 display elevated STAT3 activation and expression of proinflammatory mediators and play a critical role in the development of atypical EAE.

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.

Myelin oligodendrocyte glycoprotein reactive Th17 cells drive Janus Kinase 1 dependent transcriptional reprogramming in astrocytes and alter cell surface cytokine receptor profiles during experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune demyelinating disease affecting the central nervous system (CNS). T helper (Th) 17 cells are involved in the pathogenesis of MS and its animal model of experimental autoimmune encephalomyelitis (EAE) by infiltrating the CNS and producing effector molecules that engage resident glial cells. Among these glial cells, astrocytes have a central role in coordinating inflammatory processes by responding to cytokines and chemokines released by Th17 cells. In this study, we examined the impact of pathogenic Th17 cells on astrocytes in vitro and in vivo. We identified that Th17 cells reprogram astrocytes by driving transcriptomic changes partly through a Janus Kinase (JAK)1-dependent mechanism, which included increased chemokines, interferon-inducible genes, and cytokine receptors. In vivo, we observed a region-specific heterogeneity in the expression of cell surface cytokine receptors on astrocytes, including those for IFN-γ, IL-1, TNF-α, IL-17, TGFß, and IL-10. Additionally, these receptors were dynamically regulated during EAE induced by adoptive transfer of myelin-reactive Th17 cells. This study overall provides evidence of Th17 cell reprogramming of astrocytes, which may drive changes in the astrocytic responsiveness to cytokines during autoimmune neuroinflammation.

Dim light at night shifts microglia to a pro-inflammatory state after cerebral ischemia, altering stroke outcome in mice.

Circadian rhythms are endogenous biological cycles that regulate physiology and behavior and are set to precisely 24-h by light exposure. Light at night (LAN) dysregulates physiology and function including immune response; a critical component that contributes to stroke pathophysiological progression of neuronal injury and may impair recovery from injury. The goal of this study is to explore the effects of dim LAN (dLAN) in a murine model of ischemic stroke to assess how nighttime lighting from hospital settings can affect stroke outcome. Further, this study sought to identify mechanisms underlying pathophysiological changes to immune response after circadian disruption. Male and female adult Swiss Webster (CFW) mice were subjected to transient or permanent focal cerebral ischemia, then were subsequently placed into either dark night conditions (LD) or one night of dLAN (5 lx). 24 h post-stroke, sensorimotor impairments and infarct sizes were quantified. A single night of dLAN following MCAO increased infarct size and sensorimotor deficits across both sexes and reduced survival in males after 24 h. Flow cytometry was performed to assess microglial phenotypes after MCAO, and revealed that dLAN altered the percentage of microglia that express pro-inflammatory markers (MHC II+ and IL-6) and microglia that express CD206 and IL-10 that likely contributed to poor ischemic outcomes. Following these results, microglia were reduced in the brain using Plexxikon 5622 (PLX 5622) a CSFR1 inhibitor, then the mice received an MCAO and were exposed to LD or dLAN conditions for 24 h. Microglial depletion by PLX5622 resulted in infarct sizes that were comparable between lighting conditions. This study provides supporting evidence that environmental lighting exacerbates ischemic injury and post-stroke mortality by a biological mechanism that exposure to dLAN causes a fundamental shift of activated microglial phenotypes from beneficial to detrimental at an early time point after stroke, resulting in irreversible neuronal death.

Lesion-remote astrocytes govern microglia-mediated white matter repair.

Spared regions of the damaged central nervous system undergo dynamic remodeling and exhibit a remarkable potential for therapeutic exploitation. Here, lesion-remote astrocytes (LRAs), which interact with viable neurons, glia and neural circuitry, undergo reactive transformations whose molecular and functional properties are poorly understood. Using multiple transcriptional profiling methods, we interrogated LRAs from spared regions of mouse spinal cord following traumatic spinal cord injury (SCI). We show that LRAs acquire a spectrum of molecularly distinct, neuroanatomically restricted reactivity states that evolve after SCI. We identify transcriptionally unique reactive LRAs in degenerating white matter that direct the specification and function of local microglia that clear lipid-rich myelin debris to promote tissue repair. Fueling this LRA functional adaptation is Ccn1 , which encodes for a secreted matricellular protein. Loss of astrocyte CCN1 leads to excessive, aberrant activation of local microglia with (i) abnormal molecular specification, (ii) dysfunctional myelin debris processing, and (iii) impaired lipid metabolism, culminating in blunted debris clearance and attenuated neurological recovery from SCI. Ccn1 -expressing white matter astrocytes are specifically induced by local myelin damage and generated in diverse demyelinating disorders in mouse and human, pointing to their fundamental, evolutionarily conserved role in white matter repair. Our findings show that LRAs assume regionally divergent reactivity states with functional adaptations that are induced by local context-specific triggers and influence disorder outcome. Astrocytes tile the central nervous system (CNS) where they serve vital roles that uphold healthy nervous system function, including regulation of synapse development, buffering of neurotransmitters and ions, and provision of metabolic substrates 1 . In response to diverse CNS insults, astrocytes exhibit disorder-context specific transformations that are collectively referred to as reactivity 2-5 . The characteristics of regionally and molecularly distinct reactivity states are incompletely understood. The mechanisms through which distinct reactivity states arise, how they evolve or resolve over time, and their consequences for local cell function and CNS disorder progression remain enigmatic. Immediately adjacent to CNS lesions, border-forming astrocytes (BFAs) undergo transcriptional reprogramming and proliferation to form a neuroprotective barrier that restricts inflammation and supports axon regeneration 6-9 . Beyond the lesion, spared but dynamic regions of the injured CNS exhibit varying degrees of synaptic circuit remodeling and progressive cellular responses to secondary damage that have profound consequences for neural repair and recovery 10,11 . Throughout these cytoarchitecturally intact, but injury-reactive regions, lesion-remote astrocytes (LRAs) intermingle with neurons and glia, undergo little to no proliferation, and exhibit varying degrees of cellular hypertrophy 7,12,13 . The molecular and functional properties of LRAs remain grossly undefined. Therapeutically harnessing spared regions of the injured CNS will require a clearer understanding of the accompanying cellular and molecular landscape. Here, we leveraged integrative transcriptional profiling methodologies to identify multiple spatiotemporally resolved, molecularly distinct states of LRA reactivity within the injured spinal cord. Computational modeling of LRA-mediated heterotypic cell interactions, astrocyte-specific conditional gene deletion, and multiple mouse models of acute and chronic CNS white matter degeneration were used to interrogate a newly identified white matter degeneration-reactive astrocyte subtype. We define how this reactivity state is induced and its role in governing the molecular and functional specification of local microglia that clear myelin debris from the degenerating white matter to promote repair.

Differential responses of pancreatic ß-cells to ROS and RNS.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) direct the activation of distinct signaling pathways that determine cell fate. In this study, the pathways activated and the mechanisms by which ROS and RNS control the viability of pancreatic ß-cells were examined. Although both nitric oxide and hydrogen peroxide (H2O2) induce DNA damage, reduce cell viability, and activate AMPK, the mechanisms of AMPK activation and cell death induction differ between each reactive species. Nitric oxide activates the unfolded protein and heat shock responses and MAPK kinase signaling, whereas H2O2 stimulates p53 stabilization and poly(ADP-ribose) polymerase (PARP) activation but fails to induce the unfolded protein or heat shock responses or MAPK activation. The control of cell fate decisions is selective for the form of stress. H2O2-mediated reduction in ß-cell viability is controlled by PARP, whereas cell death in response to nitric oxide is PARP independent but associated with the nuclear localization of GAPDH. These findings show that both ROS and RNS activate AMPK, induce DNA damage, and reduce cell viability; however, the pathways controlling the responses of ß-cells are selective for the type of reactive species.

FoxO1 and SIRT1 regulate beta-cell responses to nitric oxide.

For many cell types, including pancreatic ß-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 ß-cells to nitric oxide.

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.

Cytokine-mediated ß-cell damage in PARP-1-deficient islets.

Poly(ADP)-ribose polymerase (PARP) is an abundant nuclear protein that is activated by DNA damage; once active, it modifies nuclear proteins through attachment of poly(ADP)-ribose units derived from ß-nicotinamide adenine dinucleotide (NAD(+)). In mice, the deletion of PARP-1 attenuates tissue injury in a number of animal models of human disease, including streptozotocin-induced diabetes. Also, inflammatory cell signaling and inflammatory gene expression are attenuated in macrophages isolated from endotoxin-treated PARP-1-deficient mice. In this study, the effects of PARP-1 deletion on cytokine-mediated ß-cell damage and macrophage activation were evaluated. There are no defects in inflammatory mediator signaling or inflammatory gene expression in macrophages and islets isolated from PARP-1-deficient mice. While PARP-1 deficiency protects islets against cytokine-induced islet cell death as measured by biochemical assays of membrane polarization, the genetic absence of PARP-1 does not effect cytokine-induced inhibition of insulin secretion or cytokine-induced DNA damage in islets. While PARP-1 deficiency appears to provide protection from cell death, it fails to provide protection against the inhibitory actions of cytokines on insulin secretion or the damaging actions on islet DNA integrity.

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.

AMP-activated protein kinase attenuates nitric oxide-induced beta-cell death.

During the initial autoimmune response in type 1 diabetes, islets are exposed to a damaging mix of pro-inflammatory molecules that stimulate the production of nitric oxide by beta-cells. Nitric oxide causes extensive but reversible cellular damage. In response to nitric oxide, the cell activates pathways for functional recovery and adaptation as well as pathways that direct beta-cell death. The molecular events that dictate cellular fate following nitric oxide-induced damage are currently unknown. In this study, we provide evidence that AMPK plays a primary role controlling the response of beta-cells to nitric oxide-induced damage. AMPK is transiently activated by nitric oxide in insulinoma cells and rat islets following IL-1 treatment or by the exogenous addition of nitric oxide. Active AMPK promotes the functional recovery of beta-cell oxidative metabolism and abrogates the induction of pathways that mediate cell death such as caspase-3 activation following exposure to nitric oxide. Overall, these data show that nitric oxide activates AMPK and that active AMPK suppresses apoptotic signaling allowing the beta-cell to recover from nitric oxide-mediated cellular stress.