Yun-Li Ma

Enrichment enhances the expression of sgk, a glucocorticoid-induced gene, and facilitates spatial learning through glutamate AMPA receptor mediation.

We have previously demonstrated that the serum and glucocorticoid‐inducible kinase (sgk) gene plays a causal role in facilitating memory performance in rats. Environment enrichment is known to facilitate spatial learning. We therefore examined the effect of enrichment on sgk expression. We also examined the role of sgk in spatial and nonspatial learning and the regulation of sgk expression by activation of different glutamate receptors. Both real‐time polymerase chain reaction and Western blot analyses revealed that enrichment training preferentially increased sgk mRNA and protein levels in the hippocampus. Transfection of sgk mutant DNA to the hippocampal CA1 area markedly impaired spatial learning, fear‐conditioning learning and novel object‐recognition learning in rats, but enrichment training effectively reversed these learning deficits. Meanwhile, S422A mutant DNA transfection prevented enrichment‐induced spatial learning facilitation. In studying glutamate receptor regulation of sgk expression, we found that blockade of N‐methyl‐d‐aspartate (NMDA) receptors in general, and the NR2B subunit in particular both effectively blocked enrichment‐induced spatial learning facilitation, but they did not block enrichment‐induced sgk expression. Upon various glutamate agonist infusions, only α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) increased sgk mRNA levels significantly in the hippocampus. Furthermore, blockade of AMPA receptors effectively blocked both enrichment‐induced spatial learning facilitation and sgk expression. These results indicate that there is a dissociation between NMDA receptor activation and sgk expression. Enrichment enhanced spatial learning through both NMDA and AMPA receptor activation, whereas enrichment‐induced sgk expression is specifically mediated through AMPA receptors. These results suggest that sgk could serve as a novel molecular mechanism, in addition to the NMDA receptor NR2B, underlying enrichment‐induced learning facilitation.

SGK1 Phosphorylation of IκB Kinase α and p300 Up-regulates NF-κB Activity and Increases N-Methyl-D-aspartate Receptor NR2A and NR2B Expression

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a downstream target of phosphatidylinositol 3-kinase signaling, and it regulates various cellular and physiological functions, but the SGK1 substrate proteins and genes regulated by SGK1 are less known. Here we have identified IκB kinase α (IKKα) as a novel substrate of SGK1 by using biochemical and bioinformatic approaches. SGK1 directly phosphorylates IKKα at Thr-23 and indirectly activates IKKα at Ser-180. Furthermore, SGK1 enhanced nuclear factor κB (NF-κB) activity and up-regulated N-methyl-d-aspartate receptor NR2A and NR2B expression through activation of IKKα at Thr-23 and Ser-180, and these two residues play an equally important role in mediating these effects of SGK1. Although SGK1 does not phosphorylate IKKβ, IKKβ activity is still required for IKK complex activation and for SGK1 phosphorylation and activation of NF-κB. In addition, SGK1 increased the acetylation of NF-κB through phosphorylation of p300 at Ser-1834, and this also leads to NF-κB activation and NR2A and NR2B expression. Moreover, an endogenous stimulus of SGK1, insulin, increased IKKα and NF-κB phosphorylation as well as NF-κB acetylation and NF-κB activity, but SGK1 small interfering RNA transfection blocked these effects of insulin. In examination of the functional significance of the SGK1-IKKα-NF-κB signaling pathway, we found that transfection of the IKKα double mutant (IKKαT23A/S180A) to rat hippocampus antagonized SGK-1-mediated spatial memory facilitation. Our results together demonstrated novel substrate proteins of SGK1 and novel SGK1 signaling pathways. Activation of these signaling pathways enhances NR2A and NR2B expression that is implicated in neuronal plasticity.

Impaired retention by angiotensin II mediated by the AT1 receptor

We demonstrated previously that hippocampal dentate gyrus neurons were sensitive to angiotensin II (AII) and recently discovered that AII applied directly to the dentate gyrus inhibited granule cell long-term potentiation induction and that the inhibition is mediated by the AT1 receptor and can be blocked by losartan, a specific AT1 antagonist. The purpose of the present study was to examine the effects of AII administered directly to the dentate gyrus, 1, 5, 50, 150, and 300 ng, on the retention of an inhibitory shock avoidance response and to determine if the resultant impairment of retention can be blocked by losartan. A total of 12 groups of rats in three experiments were studied. Three independent repetitions of 5 ng AII administered bilaterally to the dentate gyrus demonstrate a clear impairment of retention under these experimental conditions and that the impairment can be effectively prevented by pretreatment with 20 mg/kg of losartan IP.

Serum‐ and glucocorticoid‐inducible kinase 1 enhances zif268 expression through the mediation of SRF and CREB1 associated with spatial memory formation

Serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) has been shown to play an important role in spatial memory formation, but the molecular mechanism underlying this effect of SGK1 was not known. zif268 is an immediate early gene that is induced by water maze learning. To investigate the role of SGK1 in the regulation of zif268 expression, the dominant negative mutant of SGK1, SGK1 S422A, was infused to the hippocampal CA1 area of rats, and was found to decrease significantly the mRNA level of zif268 in both naïve animals and trained animals. SGK1 was also found to phosphorylate serum response factor (SRF) at Ser73, Ser75, and Ser99, and phosphorylate CREB1 at Ser133. Inhibition of SGK1 phosphorylation sites on SRF and CREB1 with alanine substitution significantly diminished SGK1‐enhanced zif268 expression in the promoter‐luciferase assay. SGK1 also phosphorylates Elk‐1 and SGK1 phosphorylation of Elk‐1 decreased the transcriptional activity of Elk‐1. But SGK1 phosphorylation of Elk‐1 did not affect SGK1‐enhanced zif268 expression. Moreover, the phosphorylation of SGK1 was increased in rat CA1 area after water maze learning, accompanied by increased phosphorylation of SRF at Ser99 and increased phosphorylation of CREB1 at Ser133. All these effects were antagonized by SGK1 S422A transfection. These results together suggest that SGK1 enhances zif268 expression through the mediation of SRF and CREB1, and these signaling pathways are associated with spatial memory formation in rats.

CREB SUMOylation by the E3 Ligase PIAS1 Enhances Spatial Memory

cAMP-responsive element binding protein (CREB) phosphorylation and signaling plays an important role in long-term memory formation, but other posttranslational modifications of CREB are less known. Here, we found that CREB1Δ, the short isoform of CREB, could be sumoylated by the small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) at Lys271 and Lys290 and PIAS1 SUMOylation of CREB1Δ increased the expression level of CREB1Δ. CREB1Δ could also be sumoylated by other PIAS family proteins, but not by the E3 ligases RanBP2 and Pc2 or by the E2 ligase Ubc9. Furthermore, water maze training increased the level of endogenous CREB SUMOylation in rat CA1 neurons determined by in vitro SUMOylation assay, but this effect was not observed in other brain areas. Moreover, transduction of Lenti-CREBWT to rat CA1 area facilitated, whereas transduction of Lenti-CREB double sumo-mutant (CREBK271RK290R) impaired, spatial learning and memory performance. Transduction of Lenti-CREBWT-SUMO1 fusion vector to rat CA1 area showed a more significant effect in enhancing spatial learning and memory and CREB SUMOylation. Lenti-CREBWT transduction increased, whereas Lenti-CREBK271RK290R transduction decreased, CREB DNA binding to the brain-derived neurotrophic factor (bdnf) promoter and decreased bdnf mRNA expression. Knock-down of PIAS1 expression in CA1 area by PIAS1 siRNA transfection impaired spatial learning and memory and decreased endogenous CREB SUMOylation. In addition, CREB SUMOylation was CREB phosphorylation dependent and lasted longer. Therefore, CREB phosphorylation may be responsible for signal transduction during the early phase of long-term memory formation, whereas CREB SUMOylation sustains long-term memory.

A novel defense mechanism that is activated on amyloid- β insult to mediate cell survival: role of SGK1-STAT1/STAT2 signaling

Amyloid-β (Aβ) is known to induce apoptotic cell death and its underlying mechanism has been studied extensively, but the endogenous protection mechanism that results from Aβ insult is less known. In this study, we have found that Aβ1−42 produced a dose-dependent decrease in cell viability and dose-dependent increase in apoptotic cell death in PC12 cells. Meanwhile, Aβ1−42 (0.1 μM) increased the phosphorylation of serum- and glucocorticoid-inducible kinase1 (SGK1) at Ser-78 specifically. A parallel increase in ERK1/2, STAT1 and STAT2 phosphorylation and the anti-apoptotic gene Mcl-1 expression was also observed. Transfection of rat siRNAs against ERK1/2, SGK1, STAT1 and STAT2 abolished these effects of Aβ. Transfection of sgkS78D, the constitutively active SGK1, dose-dependently protected against Aβ-induced apoptosis and dose-dependently increased the expression of Mcl-1. SGK1 activation further phosphorylates STAT1 at Tyr-701 and Ser-727 directly, and activates STAT2 at Tyr-690 indirectly. Phosphorylation of STAT1/STAT2 upregulated Mcl-1 expression which in turn protected against Aβ-induced apoptosis. But Mcl-1 siRNA transfection enhanced Aβ-induced apoptosis. Mutation of SGK1 at Ser-78 blocked the effect of Aβ on STAT1/STAT2 phosphorylation and Mcl-1 expression. Further, mutation of STAT1/STAT2 prevented the effect of both Aβ and SGK1 on Mcl-1 expression. These results together showed a novel endogenous protection mechanism that is activated on Aβ insult to mediate cell survival.

STAT1 negatively regulates spatial memory formation and mediates the memory-impairing effect of Aβ

Signal transducer and activator of transcription-1 (STAT1) has an important role in inflammation and the innate immune response, but its role in the central nervous system is less well understood. Here, we examined the role of STAT1 in spatial learning and memory, and assessed the involvement of STAT1 in mediating the memory-impairing effect of amyloid-beta (Aβ). We found that water maze training downregulated STAT1 expression in the rat hippocampal CA1 area, and spatial learning and memory function was enhanced in Stat1-knockout mice. Conversely, overexpression of STAT1 impaired water maze performance. STAT1 strongly upregulated the expression of the extracellular matrix protein laminin β1 (LB1), which also impaired water maze performance in rats. Furthermore, Aβ impaired spatial learning and memory in association with a dose-dependent increase in STAT1 and LB1 expression, but knockdown of STAT1 and LB1 both reversed this effect of Aβ. This Aβ-induced increase in STAT1 and LB1 expression was also associated with a decrease in the expression of the N-methyl-D-aspartate receptor (NMDAR) subunits, NR1, and NR2B. Overexpression of NR1 or NR2B or exogenous application of NMDA reversed Aβ-induced learning and memory deficits as well as Aβ-induced STAT1 and LB1 expression. Our results demonstrate that STAT1 negatively regulates spatial learning and memory through transcriptional regulation of LB1 expression. We also identified a novel mechanism for Aβ pathogenesis through STAT1 induction. Notably, impairment of spatial learning and memory by this STAT1-mediated mechanism is independent of cAMP responsive element-binding protein signaling.

NMDA receptor signaling mediates the expression of protein inhibitor of activated STAT1 (PIAS1) in rat hippocampus.

Protein inhibitor of activated STAT1 (PIAS1) was shown to play an important role in inflammation and innate immune response, but how PIAS1 is regulated is not known. We have recently demonstrated that PIAS1 enhances spatial learning and memory performance in rats. In this study, we examined the signaling pathway and neural mechanism that regulate PIAS1 expression in the brain by using pharmacological and molecular approaches. Our results revealed that pias1 gene expression is rapidly induced upon NMDA receptor activation in rat hippocampus, but this effect is blocked by transfection of sub-threshold concentrations of ERK1 siRNA/ERK2 siRNA or CREB siRNA. Pias1 gene expression is similarly induced by overexpression of the ERK1/ERK2 plasmids in rat hippocampus, and this effect is also blocked by sub-threshold concentration of CREB siRNA transfection. On the other hand, transfection of ERK1 siRNA/ERK2 siRNA or CREB siRNA at a higher concentration is sufficient to down-regulate PIAS1 expression. Inhibition of PI-3 kinase signaling and CaMKII signaling, which both result in CREB inactivation, similarly decreases PIAS1 expression. But NMDA and MK-801 do not affect the expression of IL-6 and TNFα. NMDA also did not affect the expression of PIAS2, PIAS3 and PIAS4. Further, pias1 mRNA has a similar degradation rate to that of the zif268 gene. These results together suggest that pias1 may function as an immediate early gene in an activity-dependent manner and PIAS1 expression is regulated by the NMDA-MAPK/ERK-CREB signaling pathway implicated in neuronal plasticity.

Galectin-3 Negatively Regulates Hippocampus-Dependent Memory Formation through Inhibition of Integrin Signaling and Galectin-3 Phosphorylation

Galectin-3, a member of the galectin protein family, has been found to regulate cell proliferation, inhibit apoptosis and promote inflammatory responses. Galectin-3 is also expressed in the adult rat hippocampus, but its role in learning and memory function is not known. Here, we found that contextual fear-conditioning training, spatial training or injection of NMDA into the rat CA1 area each dramatically decreased the level of endogenous galectin-3 expression. Overexpression of galectin-3 impaired fear memory, whereas galectin-3 knockout (KO) enhanced fear retention, spatial memory and hippocampal long-term potentiation. Galectin-3 was further found to associate with integrin α3, an association that was decreased after fear-conditioning training. Transfection of the rat CA1 area with small interfering RNA against galectin-3 facilitated fear memory and increased phosphorylated focal adhesion kinase (FAK) levels, effects that were blocked by co-transfection of the FAK phosphorylation-defective mutant Flag-FAKY397F. Notably, levels of serine-phosphorylated galectin-3 were decreased by fear conditioning training. In addition, blockade of galectin-3 phosphorylation at Ser-6 facilitated fear memory, whereas constitutive activation of galectin-3 at Ser-6 impaired fear memory. Interestingly galectin-1 plays a role in fear-memory formation similar to that of galectin-3. Collectively, our data provide the first demonstration that galectin-3 is a novel negative regulator of memory formation that exerts its effects through both extracellular and intracellular mechanisms.

HDAC1 SUMOYLATION PROTECTS AGAINST AMYLOID-BETA TOXICITY IN A MOUSE MODEL OF ALZHEIMER'S DISEASE

Background: Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder for which there are no effective treatments. By far the strongest genetic risk factor for AD is possession of the e4 allele of apolipoprotein E (ApoE4), a protein involved in lipid transport. Despite knowing for over a decade that the ApoE e4 allele is somehow contributory to the disease process, the precise role of ApoE4 in the AD pathogenesis remains unclear. We now provide evidence that ApoE4 acts as a transcription factor, binds DNA with high affinity, including the promoter regions of 1700 different genes, several of which have previously been linked to AD pathogenesis. Methods: Through a series of experiment involving neural cells, fibroblasts from AD patients, and hApoEKI-Tg brains and using a combination of techniques including ChiP-Seq, SPR, and IHC, we show that ApoE4 undergoes nuclear translocation, binds double-stranded DNA with high affinity and functions as a transcription factor. Results: Our results indicate that ApoE not only binds to the SirT1 promoter with high affinity but also represses SirT1 promoter activity, suggesting a plausible role for ApoE4 in the nucleus as a transcriptional repressor. ChIP sequencing data indicate that the ApoE4 DNA binding sites include w1700 gene promoter regions that include genes associated with neurotrophins, programmed cell death, synaptic function, sirtuins and aging, and insulin resistance, all processes that have been implicated in AD pathogenesis. Further experiments revealed that ApoE4 reduced transcription of at least two genes besides SirT1 and both of these genes are involved in inflammatory processes. Thus, ApoE4 leads to an entire reprogramming of the cell, with increased inflammatory response and increased AD-related signaling. Conclusions: ApoE4 functions as a transcription factor, binding DNA and modulating the transcription of >3000 genes and approximately half of these did not bind ApoE3. Notably, ApoE4 targets genes associated with sirtuins and aging, neurotrophins and programmed cell death, microtubule disassembly, synaptic function, NFkB and inflammation, and insulin resistance and diabetes, providing a roadmap for what is essentially a ‘unified theory’ of Alzheimer’s disease.