Understanding the dynamics of muscle transcriptome during development and between breeds differing in muscle growth is necessary to uncover the complex mechanism underlying muscle development. Herein, we present the first transcriptome-wide longissimus dorsi muscle development research concerning Lantang (LT, obese) and Landrace (LR, lean) pig breeds during 10 time-points from 35 days-post-coitus (dpc) to 180 days-post-natum (dpn) using Solexa/Illumina's Genome Analyzer. The data demonstrated that myogenesis was almost completed before 77 dpc, but the muscle phenotypes were still changed from 77 dpc to 28 dpn. Comparative analysis of the two breeds suggested that myogenesis started earlier but progressed more slowly in LT than in LR, the stages ranging from 49 dpc to 77 dpc are critical for formation of different muscle phenotypes. 595 differentially expressed myogenesis genes were identified, and their roles in myogenesis were discussed. Furthermore, GSK3B, IKBKB, ACVR1, ITGA and STMN1 might contribute to later myogenesis and more muscle fibers in LR than LT. Some myogenesis inhibitors (ID1, ID2, CABIN1, MSTN, SMAD4, CTNNA1, NOTCH2, GPC3 and HMOX1) were higher expressed in LT than in LR, which might contribute to more slow muscle differentiation in LT than in LR. We also identified several genes which might contribute to intramuscular adipose differentiation. Most important, we further proposed a novel model in which MyoD and MEF2A controls the balance between intramuscular adipogenesis and myogenesis by regulating CEBP family; Myf5 and MEF2C are essential during the whole myogenesis process while MEF2D affects muscle growth and maturation. The MRFs and MEF2 families are also critical for the phenotypic differences between the two pig breeds. Overall, this study contributes to elucidating the mechanism underlying muscle development, which could provide valuable information for pig meat quality improvement. The raw data have been submitted to Gene Expression Omnibus (GEO) under series GSE25406.

Polymorphic genes associated with Alzheimer's disease (see ) delineate a clearly defined pathway related to cerebral and peripheral cholesterol and lipoprotein homoeostasis. They include all of the key components of a glia/neurone cholesterol shuttle including cholesterol binding lipoproteins APOA1, APOA4, APOC1, APOC2, APOC3, APOD, APOE and LPA, cholesterol transporters ABCA1, ABCA2, lipoprotein receptors LDLR, LRP1, LRP8 and VLDLR, and the cholesterol metabolising enzymes CYP46A1 and CH25H, whose oxysterol products activate the liver X receptor NR1H2 and are metabolised to esters by SOAT1. LIPA metabolises cholesterol esters, which are transported by the cholesteryl ester transport protein CETP. The transcription factor SREBF1 controls the expression of most enzymes of cholesterol synthesis. APP is involved in this shuttle as it metabolises cholesterol to 7-betahydroxycholesterol, a substrate of SOAT1 and HSD11B1, binds to APOE and is tethered to LRP1 via APPB1, APBB2 and APBB3 at the cytoplasmic domain and via LRPAP1 at the extracellular domain. APP cleavage products are also able to prevent cholesterol binding to APOE. BACE cleaves both APP and LRP1. Gamma-secretase (PSEN1, PSEN2, NCSTN) cleaves LRP1 and LRP8 as well as APP and their degradation products control transcription factor TFCP2, which regulates thymidylate synthase (TS) and GSK3B expression. GSK3B is known to phosphorylate the microtubule protein tau (MAPT). Dysfunction of this cascade, carved out by genes implicated in Alzheimer's disease, may play a major role in its pathology. Many other genes associated with Alzheimer's disease affect cholesterol or lipoprotein function and/or have also been implicated in atherosclerosis, a feature of Alzheimer's disease, and this duality may well explain the close links between vascular and cerebral pathology in Alzheimer's disease. The definition of many of these genes as risk factors is highly contested. However, when polymorphic susceptibility genes belong to the same signaling pathway, the risk associated with multigenic disease is better related to the integrated effects of multiple polymorphisms of genes within the same pathway than to variants in any single gene [Wu, X., Gu, J., Grossman, H.B., Amos, C.I., Etzel, C., Huang, M., Zhang, Q., Millikan, R.E., Lerner, S., Dinney, C.P., Spitz, M.R., 2006. Bladder cancer predisposition: a multigenic approach to DNA-repair and cell-cycle-control genes. Am. J. Hum. Genet. 78, 464-479.]. Thus, the fact that Alzheimer's disease susceptibility genes converge on a clearly defined signaling network has important implications for genetic association studies.

Viral infection activates transcription factors IRF3 and NF-κB, which collaborate to induce type I interferons (IFNs). Here, we identified glycogen synthase kinase 3β (GSK3β) as an important regulator for virus-triggered IRF3 and NF-κB activation, IFN-β induction, and cellular antiviral response. Overexpression of GSK3β potentiated virus-induced activation of IRF3 and transcription of the IFNB1 gene, whereas reduced expression or deletion of GSK3β impaired virus-induced IRF3 and NF-κB activation, transcription of the IFNB1 gene, as well as cellular antiviral response. GSK3β physically associated with the kinase TBK1 in a viral infection-dependent manner. GSK3β promoted TBK1 self-association and autophosphorylation at Ser172, which is critical for virus-induced IRF3 activation and IFN-β induction. The effect of GSK3β on virus-induced signaling is independent of its kinase activity. Our findings suggest that GSK3β plays important roles in virus-triggered IRF3 activation by promoting TBK1 activation and provide new insights to the molecular mechanisms of cellular antiviral response.

The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. Although DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity also may be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole-transcriptome sequencing of normal, chronic phase, and serially transplantable blast crisis chronic myeloid leukemia (CML) progenitors revealed increased IFN-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the IFN-responsive ADAR1 p150 isoform, and a propensity for increased adenosine-to-inosine RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promotes expression of the myeloid transcription factor PU.1 and induces malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a misspliced form of GSK3β implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of blast crisis CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.

The majority of human colorectal cancers (CRCs) are initiated by mutations arising in the adenomatous polyposis coli (APC) tumour suppressor gene. However, a new class of non-APC mutated CRCs has been defined that have a serrated histopathology and carry the (V600E)BRAF oncogene. Here we have investigated the pathogenesis of serrated CRCs by expressing (V600E)Braf in the proliferative cells of the mouse gastrointestinal tract. We show that the oncogene drives an initial burst of Mek-dependent proliferation, leading to the formation of hyperplastic crypts. This is associated with β-catenin nuclear localization by a mechanism involving Mapk/Erk kinase (Mek)-dependent, Akt-independent phosphorylation of Gsk3β. However, hyperplastic crypts remain dormant for prolonged periods due to the induction of crypt senescence accompanied by upregulation of senescence-associated β-galactosidase and p16(Ink4a). We show that tumour progression is associated with down-regulation of p16(Ink4a) through enhanced CpG methylation of exon 1 and knockout of Cdkn2a confirms this gene is a barrier to tumour progression. Our studies identify (V600E)BRAF as an early genetic driver mutation in serrated CRCs and indicate that, unlike APC-mutated cancers, this subtype arises by the bypassing of a (V600E)Braf driven oncogene-induced senescence programme.

Mutations in triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to increased Alzheimer's disease (AD) risk. Neurobiological functions of TREM2 and its pathophysiological ligands remain elusive. Here we found that TREM2 directly binds to β-amyloid (Aβ) oligomers with nanomolar affinity, whereas AD-associated TREM2 mutations reduce Aβ binding. TREM2 deficiency impairs Aβ degradation in primary microglial culture and mouse brain. Aβ-induced microglial depolarization, K+ inward current induction, cytokine expression and secretion, migration, proliferation, apoptosis, and morphological changes are dependent on TREM2. In addition, TREM2 interaction with its signaling adaptor DAP12 is enhanced by Aβ, regulating downstream phosphorylation of SYK and GSK3β. Our data demonstrate TREM2 as a microglial Aβ receptor transducing physiological and AD-related pathological effects associated with Aβ.

Disrupted in Schizophrenia-1 (DISC1) is a candidate gene for psychiatric disorders and has many roles during brain development. Common DISC1 polymorphisms (variants) are associated with neuropsychiatric phenotypes including altered cognition, brain structure, and function; however, it is unknown how this occurs. Here, we demonstrate using mouse, zebrafish, and human model systems that DISC1 variants are loss of function in Wnt/GSK3β signaling and disrupt brain development. The DISC1 variants A83V, R264Q, and L607F, but not S704C, do not activate Wnt signaling compared with wild-type DISC1 resulting in decreased neural progenitor proliferation. In zebrafish, R264Q and L607F could not rescue DISC1 knockdown-mediated aberrant brain development. Furthermore, human lymphoblast cell lines endogenously expressing R264Q displayed impaired Wnt signaling. Interestingly, S704C inhibited the migration of neurons in the developing neocortex. Our data demonstrate DISC1 variants impair Wnt signaling and brain development and elucidate a possible mechanism for their role in neuropsychiatric phenotypes.

TNF-α is a cytokine with antitumorigenic property. In contrast, low dose, chronic TNF-α production by tumor cells or stromal cells may promote tumor growth and metastasis. Serum levels of TNF-α are significantly elevated in renal cell carcinoma (RCC) patients. Here, we showed that TNF-α induced epithelial-mesenchymal transition (EMT) and promoted tumorigenicity of RCC by repressing E-cadherin, upregulating vimentin, activating MMP9, and invasion activities. In addition, TNF-α treatment inhibited glycogen synthase kinase 3β (GSK-3β) activity through serine-9 phosphorylation mediated by the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway in RCC cells. Inhibition of PI3K/AKT by LY294002 reactivated GSK-3β and suppressed the TNF-α-induced EMT of RCC cells. Inactivation of GSK-3β by LiCl significantly increased MMP9 activity and EMT of RCC cells. Activation of GSK-3β by transduction of constitutively active GSK-3β into RCC cells suppressed TNF-α-mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient GSK-3β, in contrast, potentiated EMT, anchorage-independent growth and drastically enhanced tumorigenicity in vivo. Most importantly, a 15-fold inactivation of GSK-3β activity, 3-fold decrease of E-cadherin, and 2-fold increase of vimentin were observed in human RCC tumor tissues. These results indicated that inactivation of GSK-3β plays a pivotal role in the TNF-α-mediated tumorigenesis of RCC.

Lysosomes respond to environmental cues by controlling their own biogenesis, but the underlying mechanisms are poorly understood. Here we describe a protein kinase C (PKC)-dependent and mTORC1-independent mechanism for regulating lysosome biogenesis, which provides insights into previously reported effects of PKC on lysosomes. By identifying lysosome-inducing compounds we show that PKC couples activation of the TFEB transcription factor with inactivation of the ZKSCAN3 transcriptional repressor through two parallel signalling cascades. Activated PKC inactivates GSK3β, leading to reduced phosphorylation, nuclear translocation and activation of TFEB, while PKC activates JNK and p38 MAPK, which phosphorylate ZKSCAN3, leading to its inactivation by translocation out of the nucleus. PKC activation may therefore mediate lysosomal adaptation to many extracellular cues. PKC activators facilitate clearance of aggregated proteins and lipid droplets in cell models and ameliorate amyloid β plaque formation in APP/PS1 mouse brains. Thus, PKC activators are viable treatment options for lysosome-related disorders.

Mammalian glycogen synthase kinase-3 (GSK3) is generated from two genes, GSK3α and <em>GSK3β</em>, while a splice variant of <em>GSK3β</em> (<em>GSK3β</em>2), containing a 13 amino acid insert, is enriched in neurons. GSK3α and <em>GSK3β</em> deletions generate distinct phenotypes. Here, we show that phosphorylation of CRMP2, CRMP4, β-catenin, c-Myc, c-Jun and some residues on tau associated with Alzheimer's disease, is altered in cortical tissue lacking both isoforms of GSK3. This confirms that they are physiological targets for GSK3. However, deletion of each GSK3 isoform produces distinct substrate phosphorylation, indicating that each has a different spectrum of substrates (e.g. phosphorylation of Thr509, Thr514 and Ser518 of CRMP is not detectable in cortex lacking <em>GSK3β</em>, yet normal in cortex lacking GSK3α). Furthermore, the neuron-enriched <em>GSK3β</em>2 variant phosphorylates phospho-glycogen synthase 2 peptide, CRMP2 (Thr509/514), CRMP4 (Thr509), Inhibitor-2 (Thr72) and tau (Ser396), at a lower rate than <em>GSK3β</em>1. In contrast phosphorylation of c-Myc and c-Jun is equivalent for each <em>GSK3β</em> isoform, providing evidence that differential substrate phosphorylation is achieved through alterations in expression and splicing of the GSK3 gene. Finally, each <em>GSK3β</em> splice variant is phosphorylated to a similar extent at the regulatory sites, Ser9 and Tyr216, and exhibit identical sensitivities to the ATP competitive inhibitor CT99021, suggesting upstream regulation and ATP binding properties of <em>GSK3β</em>1 and <em>GSK3β</em>2 are similar.

BACKGROUND

Identifying feasible therapeutic interventions is crucial for ameliorating the intellectual disability and other afflictions of fragile X syndrome (FXS), the most common inherited cause of intellectual disability and autism. Hippocampal glycogen synthase kinase-3 (GSK3) is hyperactive in the mouse model of FXS (FX mice), and hyperactive GSK3 promotes locomotor hyperactivity and audiogenic seizure susceptibility in FX mice, raising the possibility that specific GSK3 inhibitors may improve cognitive processes.

METHODS

We tested if specific GSK3 inhibitors improve deficits in N-methyl-D-aspartate receptor-dependent long-term potentiation at medial perforant path synapses onto dentate granule cells and dentate gyrus-dependent cognitive behavioral tasks.

RESULTS

GSK3 inhibitors completely rescued deficits in long-term potentiation at medial perforant path-dentate granule cells synapses in FX mice. Furthermore, synaptosomes from the dentate gyrus of FX mice displayed decreased inhibitory serine-phosphorylation of GSK3β compared with wild-type littermates. The potential therapeutic utility of GSK3 inhibitors was further tested on dentate gyrus-dependent cognitive behaviors. In vivo administration of GSK3 inhibitors completely reversed impairments in several cognitive tasks in FX mice, including novel object detection, coordinate and categorical spatial processing, and temporal ordering for visual objects.

CONCLUSIONS

These findings establish that synaptic plasticity and cognitive deficits in FX mice can be improved by intervention with inhibitors of GSK3, which may prove therapeutically beneficial in FXS.

Hyperphosphorylation of microtubule-associated protein tau is one of the major pathological events in Alzheimer's disease (AD) and other related neurodegenerative diseases, including frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Mutations in the tau gene MAPT are a cause of FTDP-17, and the mutated tau proteins are hyperphosphorylated in patient brains. Thus, it is important to determine the molecular mechanism of hyperphosphorylation of tau to understand the pathology of these diseases collectively called tauopathy. Tau is phosphorylated at many sites via several protein kinases, and a characteristic is phosphorylation at Ser/Thr residues in Ser/Thr-Pro sequences, which are targeted by proline-directed protein kinases such as ERK, GSK3β, and Cdk5. Among these kinases, Cdk5 is particularly interesting because it could be abnormally activated in AD. Cdk5 is a member of the cyclin-dependent kinases (Cdks), but in contrast to the major Cdks, which promote cell cycle progression in proliferating cells, Cdk5 is activated in post-mitotic neurons via the neuron-specific activator p35. Cdk5-p35 plays a critical role in brain development and physiological synaptic activity. In contrast, in disease brains, Cdk5 is thought to be hyperactivated by p25, which is the N-terminal truncated form of p35 and is generated by cleavage with calpain. Several reports have indicated that tau is hyperphosphorylated by Cdk5-p25. However, normal and abnormal phosphorylation of tau by Cdk5 is still not completely understood. In this article, we summarize the physiological and pathological phosphorylation of tau via Cdk5.

BACKGROUND

Lung cancer is the most common cause of cancer-related deaths. Tobacco smoke exposure is the strongest aetiological factor associated with lung cancer. In this study, using serial analysis of gene expression (SAGE), we comprehensively examined the effect of active smoking by comparing the transcriptomes of clinical specimens obtained from current, former and never smokers, and identified genes showing both reversible and irreversible expression changes upon smoking cessation.

RESULTS

Twenty-four SAGE profiles of the bronchial epithelium of eight current, twelve former and four never smokers were generated and analyzed. In total, 3,111,471 SAGE tags representing over 110 thousand potentially unique transcripts were generated, comprising the largest human SAGE study to date. We identified 1,733 constitutively expressed genes in current, former and never smoker transcriptomes. We have also identified both reversible and irreversible gene expression changes upon cessation of smoking; reversible changes were frequently associated with either xenobiotic metabolism, nucleotide metabolism or mucus secretion. Increased expression of TFF3, CABYR, and ENTPD8 were found to be reversible upon smoking cessation. Expression of GSK3B, which regulates COX2 expression, was irreversibly decreased. MUC5AC expression was only partially reversed. Validation of select genes was performed using quantitative RT-PCR on a secondary cohort of nine current smokers, seven former smokers and six never smokers.

CONCLUSIONS

Expression levels of some of the genes related to tobacco smoking return to levels similar to never smokers upon cessation of smoking, while expression of others appears to be permanently altered despite prolonged smoking cessation. These irreversible changes may account for the persistent lung cancer risk despite smoking cessation.

BACKGROUND

Hepatocellular carcinoma (HCC) is a malignant cancer with an observable heterogeneity and microRNAs are functionally associated with the tumorigenesis of HCC. We recently identified that EpCAM (CD326)-positive cells isolated from alpha-fetoprotein (AFP)-positive HCC samples are hepatic cancer stem cells (HepCSCs). EpCAM+AFP+ HepCSCs have an activated Wnt/β-catenin signaling with a parallel increased expression of all four microRNA-181 family members. We hypothesized that Wnt/β-catenin signaling transcriptionally activates microRNA-181s in HCC.

RESULTS

Using both western blot and quantitative reverse transcriptase-PCR analyses, we found that the expression of all four microRNA-181 family members was positively correlated with β-catenin expression in HCC cell lines. MicroRNA-181 expression could be directly induced upon an activation of Wnt/β-catenin signaling, which includes Wnt10B overexpression, inhibition of GSK3β signaling by LiCl, or forced expression of β-catenin/Tcf4. Moreover, microRNA-181 expression was inhibited upon an inactivation of Wnt/β-catenin signaling by an induction of adenomatosis polyposis coli (APC) expression or silencing β-catenin via RNA interference. In addition, seven putative β-catenin/Tcf4 binding sites were identified in the promoter region of the microRNA-181a-2 and microRNA-181b-2 transcripts. Consistently, we found that Tcf4 interacted with these regions in vivo using chromatin immunoprecipitation assay.

CONCLUSIONS

Taken together, our results demonstrate that microRNA-181s are transcriptionally activated by the Wnt/beta-catenin signaling pathway in HCC.

OBJECTIVE

Glycogen synthase kinase 3β (GSK-3β) is an enzyme that is suppressed by insulin and when elevated results in insulin resistance in skeletal muscle and diabetes. Its role in beta cell development and function is little known. Because of the enzyme's anti-proliferative and pro-apoptotic properties, the hypothesis to be tested here was that beta cell specific deficiency of GSK-3β in mice would result in enhanced beta cell mass and function.

METHODS

Mice with beta cell deficiency of GSK-3β (β-Gsk-3β [also known as Gsk3b](-/-)) were generated by breeding Gsk-3β (flox/flox) mice with mice overexpressing the Cre recombinase gene under the control of the rat insulin 2 gene promoter (RIP-Cre mice), and glucose tolerance, insulin secretion, islet mass, proliferation and apoptosis were measured. Changes in islet proteins were investigated by western blotting.

RESULTS

On a normal diet β-Gsk-3β ( -/- ) mice were found to have mild improvement of glucose tolerance and glucose-induced insulin secretion, and increased beta cell mass accompanied by increased proliferation and decreased apoptosis. On a high-fat diet β-Gsk-3β (-/-) mice exhibited improved glucose tolerance and expanded beta cell mass with increased proliferation relative to that in control mice, resisting fat-fed diabetes. Molecular mechanisms accounting for these phenotypic changes included increased levels of islet IRS1 and IRS2 proteins and phospho-Akt, suggesting enhanced signalling through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and increased islet levels of pancreas/duodenum homeobox protein 1 (PDX1). Inhibition of GSK3 in MIN6 cells in vitro led to increased IRS1 and IRS2 protein levels through inhibition of proteosomal degradation.

CONCLUSIONS

These results are consistent with a mechanism whereby endogenous GSK-3β activity controls islet beta cell growth by feedback inhibition of the insulin receptor/PI3K/Akt signalling pathway.

Insufficient pancreatic β-cell mass or function results in diabetes mellitus. While significant progress has been made in regulating insulin secretion from β-cells in diabetic patients, no pharmacological agents have been described that increase β-cell replication in humans. Here we report aminopyrazine compounds that stimulate robust β-cell proliferation in adult primary islets, most likely as a result of combined inhibition of DYRK1A and GSK3B. Aminopyrazine-treated human islets retain functionality in vitro and after transplantation into diabetic mice. Oral dosing of these compounds in diabetic mice induces β-cell proliferation, increases β-cell mass and insulin content, and improves glycaemic control. Biochemical, genetic and cell biology data point to Dyrk1a as the key molecular target. This study supports the feasibility of treating diabetes with an oral therapy to restore β-cell mass, and highlights a tractable pathway for future drug discovery efforts.

The ST2L receptor for interleukin 33 (IL-33) mediates pulmonary inflammation and immune system-related disorders, such as asthma and rheumatoid arthritis. At present, very little is known about the molecular regulation of ST2L expression. Here we found that FBXL19, an 'orphan' member of the Skp1-Cullin-F-box family of E3 ubiquitin ligases, selectively bound to ST2L to mediate its polyubiquitination and elimination in the proteasome. Degradation of ST2L involved phosphorylation of ST2L at Ser442 catalyzed by the kinase GSK3β. Overexpression of FBXL19 abrogated the proapoptotic and inflammatory effects of IL-33 and lessened the severity of lung injury in mouse models of pneumonia. Our results suggest that modulation of the IL-33-ST2L axis by ubiquitin ligases might serve as a unique strategy for lessening pulmonary inflammation.

Both neuroprotective and neurotoxic roles have previously been described for histone deacetylase-1 (HDAC1). Here we report that HDAC1 expression is elevated in vulnerable brain regions of two mouse models of neurodegeneration, the R6/2 model of Huntington disease and the Ca(2+)/calmodulin-dependent protein kinase (CaMK)/p25 double-transgenic model of tauopathic degeneration, suggesting a role in promoting neuronal death. Indeed, elevating HDAC1 expression by ectopic expression promotes the death of otherwise healthy cerebellar granule neurons and cortical neurons in culture. The neurotoxic effect of HDAC1 requires interaction and cooperation with HDAC3, which has previously been shown to selectively induce the death of neurons. HDAC1-HDAC3 interaction is greatly elevated under conditions of neurodegeneration both in vitro and in vivo. Furthermore, the knockdown of HDAC3 suppresses HDAC1-induced neurotoxicity, and the knockdown of HDAC1 suppresses HDAC3 neurotoxicity. As described previously for HDAC3, the neurotoxic effect of HDAC1 is inhibited by treatment with IGF-1, the expression of Akt, or the inhibition of glycogen synthase kinase 3β (GSK3β). In addition to HDAC3, HDAC1 has been shown to interact with histone deacetylase-related protein (HDRP), a truncated form of HDAC9, whose expression is down-regulated during neuronal death. In contrast to HDAC3, the interaction between HDRP and HDAC1 protects neurons from death, an effect involving acquisition of the deacetylase activity of HDAC1 by HDRP. We find that elevated HDRP inhibits HDAC1-HDAC3 interaction and prevents the neurotoxic effect of either of these two proteins. Together, our results suggest that HDAC1 is a molecular switch between neuronal survival and death. Its interaction with HDRP promotes neuronal survival, whereas interaction with HDAC3 results in neuronal death.

Well-established studies have shown an elevated level of reactive oxygen species (ROS) that induces oxidative stress in the Alzheimer's disease (AD) patient's brain and an animal model of AD. Herein, we investigated the underlying anti-oxidant neuroprotective mechanism of natural dietary supplementation of anthocyanins extracted from Korean black beans in the amyloid precursor protein/presenilin-1 (APP/PS1) mouse model of AD. Both in vivo (APP/PS1 mice) and in vitro (mouse hippocampal HT22 cells) results demonstrated that anthocyanins regulate the phosphorylated-phosphatidylinositol 3-kinase-Akt-glycogen synthase kinase 3 beta (p-PI3K/Akt/GSK3β) pathways and consequently attenuate amyloid beta oligomer (AβO)-induced elevations in ROS level and oxidative stress via stimulating the master endogenous anti-oxidant system of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (Nrf2/HO-1) pathways and prevent apoptosis and neurodegeneration by suppressing the apoptotic and neurodegenerative markers such as activation of caspase-3 and PARP-1 expression as well as the TUNEL and Fluoro-Jade B-positive neuronal cells in the APP/PS1 mice. In vitro ApoTox-Glo™ Triplex assay results also showed that anthocyanins act as a potent anti-oxidant neuroprotective agent and reduce AβO-induced neurotoxicity in the HT22 cells via PI3K/Akt/Nrf2 signaling. Importantly, anthocyanins improve memory-related pre- and postsynaptic protein markers and memory functions in the APP/PS1 mice. In conclusion, our data suggested that consumption and supplementation of natural-derived anti-oxidant neuroprotective agent such as anthocyanins may be beneficial and suggest new dietary-supplement strategies for intervention in and prevention of progressive neurodegenerative diseases, such as AD.

The generation of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β fuel the acute phase response (APR). To maintain body homeostasis, the increase of inflammatory proteins is resolved by acute phase proteins via presently unknown mechanisms. Hepatocyte growth factor (HGF) is transcribed in response to IL-6. Since IL-6 production promotes the generation of HGF and induces the APR, we posited that accumulating HGF might be a likely candidate for quelling excess inflammation under non-pathological conditions. We sought to assess the role of HGF and how it influences the regulation of inflammation utilizing a well-defined model of inflammatory activation, lipopolysaccharide (LPS)-stimulation of bone marrow derived macrophages (BMM). BMM were isolated from C57BL6 mice and were stimulated with LPS in the presence or absence of HGF. When HGF was present, there was a decrease in production of the pro-inflammatory cytokine IL-6, along with an increase in the anti-inflammatory cytokine IL-10. Altered cytokine production correlated with an increase in phosphorylated GSK3β, increased retention of the phosphorylated NFκB p65 subunit in the cytoplasm, and an enhanced interaction between CBP and phospho-CREB. These changes were a direct result of signaling through the HGF receptor, MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274) or when using BMM from macrophage-specific conditional MET knockout mice. Combined, these data provide compelling evidence that under normal circumstances, HGF acts to suppress the inflammatory response.

Progression of colon cancer from microadenoma to macroscopic tumors is coupled to augmentation of canonical Wnt signaling. We recently reported that tumor associated macrophages, through interleukin 1β (IL-1β) dependent phosphorylation of GSK3β, promote Wnt signaling in colon cancer cells, demonstrating that proinflammatory cytokines can enhance TCF4/β-catenin transcriptional activity in tumor cells. Here we investigated the pathway whereby IL-1β inactivates GSK3β and promotes Wnt signaling in colon cancer cells. We showed that normal human monocytes, THP1 macrophages and IL-1 failed to induce Wnt signaling in tumor cells expressing dominant negative IκB (dnIκB), demonstrating that macrophages and IL-1 activate Wnt signaling in a NF-κB-dependent manner. NF-κB activity was required for macrophages and IL-1 to activate PDK1 and AKT in tumor cells and thereby inhibit GSK3β activity. Consistently, dominant negative AKT (dnAKT), or pharmacological inhibition of AKT in tumor cells, prevented macrophage/IL-1 mediated phosphorylation of GSK3β, activation of Wnt signaling, and induction of c-jun and c-myc, confirming that macrophages and IL-1 promote Wnt signaling in an AKT dependent manner. Finally, we showed IL-1 and macrophages failed to promote growth of colon cancer cells with impaired NF-κB or AKT signaling, confirming the requirement for NF-κB and AKT activation for the protumorigenic activity of tumor associated macrophages. Thus, we showed that IL-1 and tumor associated macrophages activate NF-κB-dependent PDK1/AKT signaling in tumor cells, and thereby inactivate GSK3β, enhance Wnt signaling and promote growth of colon cancer cells, establishing a novel molecular link between inflammation and tumor growth.

Human pluripotent stem cells (hPSCs) constitute a promising resource for use in cell-based therapies and a valuable in vitro model for studying early human development and disease. Despite significant advancements in the derivation of specific fates from hPSCs, the generation of skeletal muscle remains challenging and is mostly dependent on transgene expression. Here, we describe a method based on the use of a small-molecule GSK3β inhibitor to derive skeletal muscle from several hPSC lines. We show that early GSK3β inhibition is sufficient to create the conditions necessary for highly effective derivation of muscle cells. Moreover, we developed a strategy for stringent fluorescence-activated cell sorting-based purification of emerging PAX3+/PAX7+ muscle precursors that are able to differentiate in postsort cultures into mature myocytes. This transgene-free, efficient protocol provides an essential tool for producing myogenic cells for in vivo preclinical studies, in vitro screenings, and disease modeling.

Somatic cells can be reprogrammed to form embryonic stem cell-like induced pluripotent stem cells (iPSCs), but the process suffers from low efficiency and the underlying molecular mechanisms that control reprogramming remain poorly understood. Here we perform an inhibitor screen to identify kinases that enhance, or present a barrier to, reprogramming. In particular, inhibitors of p38, inositol trisphosphate 3-kinase, and Aurora A kinase potently enhance iPSC generation, and iPSCs derived from inhibitor-treated somatic cells are capable of reaching a fully reprogrammed state. Knockdown of target kinases by short interfering RNAs confirms that they function as barrier genes. We show that Aurora A kinase, which functions in centrosome activity and spindle assembly, is highly induced during reprogramming and inhibits Akt-mediated inactivation of GSK3β, resulting in compromised reprogramming efficiency. Together, our results not only identify new compounds that enhance iPSC generation but also shed new light on the function of Aurora A kinase in the reprogramming process.

Experience of mice in a complex environment enhances neurogenesis and synaptic plasticity in the hippocampus of wild type and transgenic mice harboring familial Alzheimer's disease (FAD)-linked APPswe/PS1ΔE9. In FAD mice, this experience also reduces levels of tau hyperphosphorylation and oligomeric β-amyloid. Although environmental enrichment has significant effects on brain plasticity and neuropathology, the molecular mechanisms underlying these effects are unknown. Here we show that environmental enrichment upregulates the Akt pathway, leading to the downregulation of glycogen synthase kinase 3β (GSK3β), in wild type but not FAD mice. Several neurotrophic signaling pathways are activated in the hippocampus of both wild type and FAD mice, including brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and this increase is accompanied by the upregulation of the BDNF receptor, tyrosine kinase B (TrkB). Interestingly, neurotrophin-3 (NT-3) is upregulated in the brains of wild type mice but not FAD mice, while insulin growth factor-1 (IGF-1) is upregulated exclusively in the brains of FAD mice. Upregulation of neurotrophins is accompanied by the increase of N-Methyl-D-aspartic acid (NMDA) receptors in the hippocampus following environmental enrichment. Most importantly, we observed a significant increase in levels of cAMP response element- binding (CREB) transcripts in the hippocampus of wild type and FAD mice following environmental enrichment. However, CREB phosphorylation, a critical step for the initiation of learning and memory-required gene transcription, takes place in the hippocampus of wild type but not of FAD mice. These results suggest that experience of wild type mice in a complex environmental upregulates critical signaling that play a major role in learning and memory in the hippocampus. However, in FAD mice, some of these pathways are impaired and cannot be rescued by environmental enrichment.