Identification of PTPN12 Phosphatase as a Novel Negative Regulator of Hippo Pathway Effectors YAP/TAZ in Breast Cancer.

The Hippo pathway plays crucial roles in governing various biological processes during tumorigenesis and metastasis. Within this pathway, upstream signaling stimuli activate a core kinase cascade, involving MST1/2 and LATS1/2, that subsequently phosphorylates and inhibits the transcriptional co-activators YAP and its paralog TAZ. This inhibition modulates the transcriptional regulation of downstream target genes, impacting cell proliferation, migration, and death. Despite the acknowledged significance of protein kinases in the Hippo pathway, the regulatory influence of protein phosphatases remains largely unexplored. In this study, we conducted the first gain-of-functional screen for protein tyrosine phosphatases (PTPs) regulating the Hippo pathway. Utilizing a LATS kinase biosensor (LATS-BS), a YAP/TAZ activity reporter (STBS-Luc), and a comprehensive PTP library, we identified numerous novel PTPs that play regulatory roles in the Hippo pathway. Subsequent experiments validated PTPN12, a master regulator of oncogenic receptor tyrosine kinases (RTKs), as a previously unrecognized negative regulator of the Hippo pathway effectors, oncogenic YAP/TAZ, influencing breast cancer cell proliferation and migration. In summary, our findings offer valuable insights into the roles of PTPs in the Hippo signaling pathway, significantly contributing to our understanding of breast cancer biology and potential therapeutic strategies.

Gold Nanoparticles in Neurological Diseases: A Review of Neuroprotection.

This review explores the diverse applications of gold nanoparticles (AuNPs) in neurological diseases, with a specific focus on Alzheimer's disease (AD), Parkinson's disease (PD), and stroke. The introduction highlights the pivotal role of neuroinflammation in these disorders and introduces the unique properties of AuNPs. The review's core examines the mechanisms by which AuNPs exert neuroprotection and anti-neuro-inflammatory effects, elucidating various pathways through which they manifest these properties. The potential therapeutic applications of AuNPs in AD are discussed, shedding light on promising avenues for therapy. This review also explores the prospects of utilizing AuNPs in PD interventions, presenting a hopeful outlook for future treatments. Additionally, the review delves into the potential of AuNPs in providing neuroprotection after strokes, emphasizing their significance in mitigating cerebrovascular accidents' aftermath. Experimental findings from cellular and animal models are consolidated to provide a comprehensive overview of AuNPs' effectiveness, offering insights into their impact at both the cellular and in vivo levels. This review enhances our understanding of AuNPs' applications in neurological diseases and lays the groundwork for innovative therapeutic strategies in neurology.

Molecular characterization of human peripheral nerves using desorption electrospray ionization mass spectrometry imaging.

Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is a molecular imaging method that can be used to elucidate the small-molecule composition of tissues and map their spatial information using two-dimensional ion images. This technique has been used to investigate the molecular profiles of variety of tissues, including within the central nervous system, specifically the brain and spinal cord. To our knowledge, this technique has yet to be applied to tissues of the peripheral nervous system (PNS). Data generated from such analyses are expected to advance the characterization of these structures. The study aimed to: (i) establish whether DESI-MSI can discriminate the molecular characteristics of peripheral nerves and distinguish them from surrounding tissues and (ii) assess whether different peripheral nerve subtypes are characterized by unique molecular profiles. Four different nerves for which are known to carry various nerve fiber types were harvested from a fresh cadaveric donor: mixed, motor and sensory (sciatic and femoral); cutaneous, sensory (sural); and autonomic (vagus). Tissue samples were harvested to include the nerve bundles in addition to surrounding connective tissue. Samples were flash-frozen, embedded in optimal cutting temperature compound in cross-section, and sectioned at 14 µm. Following DESI-MSI analysis, identical tissue sections were stained with hematoxylin and eosin. In this proof-of-concept study, a combination of multivariate and univariate statistical methods was used to evaluate molecular differences between the nerve and adjacent tissue and between nerve subtypes. The acquired mass spectral profiles of the peripheral nerve samples presented trends in ion abundances that seemed to be characteristic of nerve tissue and spatially corresponded to the associated histology of the tissue sections. Principal component analysis (PCA) supported the separation of the samples into distinct nerve and adjacent tissue classes. This classification was further supported by the K-means clustering analysis, which showed separation of the nerve and background ions. Differences in ion expression were confirmed using ANOVA which identified statistically significant differences in ion expression between the nerve subtypes. The PCA plot suggested some separation of the nerve subtypes into four classes which corresponded with the nerve types. This was supported by the K-means clustering. Some overlap in classes was noted in these two clustering analyses. This study provides emerging evidence that DESI-MSI is an effective tool for metabolomic profiling of peripheral nerves. Our results suggest that peripheral nerves have molecular profiles that are distinct from the surrounding connective tissues and that DESI-MSI may be able to discriminate between nerve subtypes. DESI-MSI of peripheral nerves may be a valuable technique that could be used to improve our understanding of peripheral nerve anatomy and physiology. The ability to utilize ambient mass spectrometry techniques in real time could also provide an unprecedented advantage for surgical decision making, including in nerve-sparing procedures in the future.

Metabolically Active Zones Involving Fatty Acid Elongation Delineated by DESI-MSI Correlate with Pathological and Prognostic Features of Colorectal Cancer.

Colorectal cancer (CRC) is the second leading cause of cancer deaths. Despite recent advances, five-year survival rates remain largely unchanged. Desorption electrospray ionization mass spectrometry imaging (DESI) is an emerging nondestructive metabolomics-based method that retains the spatial orientation of small-molecule profiles on tissue sections, which may be validated by 'gold standard' histopathology. In this study, CRC samples were analyzed by DESI from 10 patients undergoing surgery at Kingston Health Sciences Center. The spatial correlation of the mass spectral profiles was compared with histopathological annotations and prognostic biomarkers. Fresh frozen sections of representative colorectal cross sections and simulated endoscopic biopsy samples containing tumour and non-neoplastic mucosa for each patient were generated and analyzed by DESI in a blinded fashion. Sections were then hematoxylin and eosin (H and E) stained, annotated by two independent pathologists, and analyzed. Using PCA/LDA-based models, DESI profiles of the cross sections and biopsies achieved 97% and 75% accuracies in identifying the presence of adenocarcinoma, using leave-one-patient-out cross validation. Among the m/z ratios exhibiting the greatest differential abundance in adenocarcinoma were a series of eight long-chain or very-long-chain fatty acids, consistent with molecular and targeted metabolomics indicators of de novo lipogenesis in CRC tissue. Sample stratification based on the presence of lympovascular invasion (LVI), a poor CRC prognostic indicator, revealed the abundance of oxidized phospholipids, suggestive of pro-apoptotic mechanisms, was increased in LVI-negative compared to LVI-positive patients. This study provides evidence of the potential clinical utility of spatially-resolved DESI profiles to enhance the information available to clinicians for CRC diagnosis and prognosis.

Resveratrol Mitigates Oxygen and Glucose Deprivation-Induced Inflammation, NLRP3 Inflammasome, and Oxidative Stress in 3D Neuronal Culture.

Oxygen glucose deprivation (OGD) can produce hypoxia-induced neurotoxicity and is a mature in vitro model of hypoxic cell damage. Activated AMP-activated protein kinase (AMPK) regulates a downstream pathway that substantially increases bioenergy production, which may be a key player in physiological energy and has also been shown to play a role in regulating neuroprotective processes. Resveratrol is an effective activator of AMPK, indicating that it may have therapeutic potential as a neuroprotective agent. However, the mechanism by which resveratrol achieves these beneficial effects in SH-SY5Y cells exposed to OGD-induced inflammation and oxidative stress in a 3D gelatin scaffold remains unclear. Therefore, in the present study, we investigated the effect of resveratrol in 3D gelatin scaffold cells to understand its neuroprotective effects on NF-κB signaling, NLRP3 inflammasome, and oxidative stress under OGD conditions. Here, we show that resveratrol improves the expression levels of cell viability, inflammatory cytokines (TNF-α, IL-1ß, and IL-18), NF-κB signaling, and NLRP3 inflammasome, that OGD increases. In addition, resveratrol rescued oxidative stress, nuclear factor-erythroid 2 related factor 2 (Nrf2), and Nrf2 downstream antioxidant target genes (e.g., SOD, Gpx GSH, catalase, and HO-1). Treatment with resveratrol can significantly normalize OGD-induced changes in SH-SY5Y cell inflammation, oxidative stress, and oxidative defense gene expression; however, these resveratrol protective effects are affected by AMPK antagonists (Compounds C) blocking. These findings improve our understanding of the mechanism of the AMPK-dependent protective effect of resveratrol under 3D OGD-induced inflammation and oxidative stress-mediated cerebral ischemic stroke conditions.

GSH-AuNP anti-oxidative stress, ER stress and mitochondrial dysfunction in amyloid-beta peptide-treated human neural stem cells.

Amyloid-beta (Aß) peptides have a role in the pathogenesis of Alzheimer's disease (AD) and are thought to promote oxidative stress, endoplasmic reticulum (ER) stress and mitochondrial deficiency, causing neuronal loss in the AD brain. The potential applications of glutathione conjugated gold nanoparticles (GSH-AuNPs) suggest they might have therapeutic value. Several studies have demonstrated that the effects of nanoparticles could provide protective roles in AD. Here, we showed that GSH-AuNPs mediate the viability of human neural stem cells (hNSCs) with Aß, which was correlated with decreased caspase 3 and caspase 9. Importantly, hNSCs co-treated with GSH-AuNPs were significantly protected from Aß-induced oxidative stress, as detected using the DCFH-DA, DHE, and MitoSOX staining assays. Furthermore, hNSCs co-treated with GSH-AuNPs were significantly protected from the Aß-induced reduction in the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2 downstream antioxidant target genes (SOD-1, SOD-2, Gpx, Catalase, and HO-1). In addition, GSH-AuNPs rescued the expression levels of ER stress-associated genes (Bip, CHOP, and ASK1) in Aß-treated hNSCs. GSH-AuNPs normalized ER calcium and mitochondrial cytochrome c homeostasis in Aß-treated hNSCs. Furthermore, treatment with GSH-AuNPs restored the levels of ATP, D-loop, mitochondrial mass, basal respiration, ATP-linked reparation, maximal respiration capacity, COX activity, mitochondrial membrane potential, and mitochondrial genes (PGC1α, NRF-1 and Tfam) in Aß-treated hNSCs. Taken together, these findings extend our understanding of the protective effects of GSH-AuNPs against oxidative stress, ER stress and mitochondrial dysfunction in hNSCs with Aß.

Novel MicroRNA-Regulated Transcript Networks Are Associated with Chemotherapy Response in Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is a highly lethal gynecologic cancer, in part due to resistance to platinum-based chemotherapy reported among 20% of patients. This study aims to generate novel hypotheses of the biological mechanisms underlying chemotherapy resistance, which remain poorly understood. Differential expression analyses of mRNA- and microRNA-sequencing data from HGSOC patients of The Cancer Genome Atlas identified 21 microRNAs associated with angiogenesis and 196 mRNAs enriched for adaptive immunity and translation. Coexpression network analysis identified three microRNA networks associated with chemotherapy response enriched for lipoprotein transport and oncogenic pathways, as well as two mRNA networks enriched for ubiquitination and lipid metabolism. These network modules were replicated in two independent ovarian cancer cohorts. Moreover, integrative analyses of the mRNA/microRNA sequencing and single-nucleotide polymorphisms (SNPs) revealed potential regulation of significant mRNA transcripts by microRNAs and SNPs (expression quantitative trait loci). Thus, we report novel transcriptional networks and biological pathways associated with resistance to platinum-based chemotherapy in HGSOC patients. These results expand our understanding of the effector networks and regulators of chemotherapy response, which will help to improve the management of ovarian cancer.

Targeting the Ezrin Adaptor Protein Sensitizes Metastatic Breast Cancer Cells to Chemotherapy and Reduces Neoadjuvant Therapy-induced Metastasis.

The main cause of cancer-associated deaths is the spread of cancer cells to distant organs. Despite its success in the primary tumor setting, modern chemotherapeutic strategies are rendered ineffective at treating metastatic disease, largely due to the development of resistance. The adaptor protein ezrin has been shown to promote cancer metastasis in multiple preclinical models and is associated with poor prognosis in several cancer types, including breast cancer. Ezrin promotes pro-survival signaling, particularly in disseminated cancer cells, to facilitate metastatic outgrowth. However, the role of ezrin in breast cancer chemoresistance is not fully known. In this study, we show that upregulating or downregulating ezrin expression modifies the sensitivity of breast cancer cells to doxorubicin and docetaxel treatment in vitro and is associated with changes in PI3K/Akt and NFκB pathway activation. In addition, we tested the effects of systemic treatment with a small-molecule ezrin inhibitor, NSC668394, on lung metastatic burden in vivo as a monotherapy, or in combination with anthracycline- or taxane-based chemotherapy treatment. We show that anti-ezrin treatment alone reduces metastatic burden and markedly sensitizes metastases to doxorubicin or docetaxel in neoadjuvant as well as neoadjuvant plus adjuvant treatment models. Taken together, our findings demonstrate the impact of anti-ezrin treatment in modulating response to chemotherapy in breast cancer cells as well as the efficacy of anti-ezrin treatment in combination with chemotherapy at reducing metastatic burden. Significance: This work provides preclinical evidence for combining anti-ezrin treatment with chemotherapy as a novel strategy for effectively targeting metastasis, particularly in a neoadjuvant treatment setting.

Exposure to PM2.5 induces neurotoxicity, mitochondrial dysfunction, oxidative stress and inflammation in human SH-SY5Y neuronal cells.

Ambient air pollution is a global public health issue. Recent evidence suggests that exposure to fine aerosolized particulate matter (PM) as small as ≤2.5 microns (PM2.5) is neurotoxic to brain structures. Many studies also suggest exposure to PM2.5 may cause neurotoxicity and affect brain function. However, the molecular mechanisms by which PM2.5 exerts these effects are not fully understood. Thus, we evaluated the hypothesis that PM2.5 exposure exerts its neurotoxic effects via increased oxidative and inflammatory cellular damage and mitochondrial dysfunction using human SH-SY5Y neuronal cells. Here, we show PM2.5 exposure significantly decreases viability, and increases caspase 3 and 9 protein expression and activity in SH-SY5Y cells. In addition, PM2.5 exposure decreases SH-SY5Y survival, disrupts cell and mitochondrial morphology, and significantly decreases ATP levels, D-loop levels, and mitochondrial mass and function (maximal respiratory function, COX activity, and mitochondrial membrane potential) in SH-SY5Y cells. Moreover, SH-SY5Y cells exposed to PM2.5 have significantly decreased mRNA and protein expression levels of survival genes (CREB and Bcl-2) and neuroprotective genes (PPARγ and AMPK). We further show SH-SY5Y cells exposure to PM2.5 induces significant increases in the levels of oxidative stress, and expression levels of the inflammatory mediator's TNF-α, IL-1ß, and NF-κB. Taken together, these results provide the first evidence of the biochemical, molecular and morphological effects of PM2.5 on human neuronal SH-SY5Y cells, and support our hypothesis that increased mitochondrial disruption, oxidative stress and inflammation are critical mediators of its neurotoxic effects. These findings further improve our understanding of the neuronal cell impact of PM2.5 exposure, and may be useful in the design of strategies for the treatment and prevention of human neurodegenerative disorders.

TO901317 activation of LXR-dependent pathways mitigate amyloid-beta peptide-induced neurotoxicity in 3D human neural stem cell culture scaffolds and AD mice.

Alzheimer's disease (AD) is the major cause of neurodegeneration worldwide and is characterized by the accumulation of amyloid beta (Aß) in the brain, which is associated with neuronal loss and cognitive impairment. Liver X receptor (LXR), a critical nuclear receptor, and major regulator in lipid metabolism and inflammation, is suggested to play a protective role against the mitochondrial dysfunction noted in AD. In our study, our established 3D gelatin scaffold model and a well characterized in vivo (APP/PS1) murine model of AD were used to directly investigate the molecular, biochemical and behavioral effects of neuronal stem cell exposure to Aß to improve understanding of the in vivo etiology of AD. Herein, human neural stem cells (hNSCs) in our 3D model were exposed to Aß, and had significantly decreased cell viability, which correlated with decreased mRNA and protein expression of LXR, Bcl-2, CREB, PGC1α, NRF-1, and Tfam, and increased caspase 3 and 9 activities. Cotreatment with a synthetic agonist of LXR (TO901317) significantly abrogated these Aß-mediated effects in hNSCs. Moreover, TO901317 cotreatment both significantly rescues hNSCs from Aß-mediated decreases in ATP levels and mitochondrial mass, and significantly restores Aß-induced fragmented mitochondria to almost normal morphology. TO901317 cotreatment also decreases tau aggregates in Aß-treated hNSCs. Importantly, TO901317 treatment significantly alleviates the impairment of memory, decreases Aß aggregates and increases proteasome activity in APP/PS1 mice; whereas, these effects were blocked by cotreatment with an LXR antagonist (GSK2033). Together, these novel results improve our mechanistic understanding of the central role of LXR in Aß-mediated hNSC dysfunction. We also provide preclinical data unveiling the protective effects of using an LXR-dependent agonist, TO901317, to block the toxicity observed in Aß-exposed hNSCs, which may guide future treatment strategies to slow or prevent neurodegeneration in some AD patients.

A miR-375/YAP axis regulates neuroendocrine differentiation and tumorigenesis in lung carcinoid cells.

Lung carcinoids are variably aggressive and mechanistically understudied neuroendocrine neoplasms (NENs). Here, we identified and elucidated the function of a miR-375/yes-associated protein (YAP) axis in lung carcinoid (H727) cells. miR-375 and YAP are respectively high and low expressed in wild-type H727 cells. Following lentiviral CRISPR/Cas9-mediated miR-375 depletion, we identified distinct transcriptomic changes including dramatic YAP upregulation. We also observed a significant decrease in neuroendocrine differentiation and substantial reductions in cell proliferation, transformation, and tumor growth in cell culture and xenograft mouse disease models. Similarly, YAP overexpression resulted in distinct and partially overlapping transcriptomic changes, phenocopying the effects of miR-375 depletion in the same models as above. Transient YAP knockdown in miR-375-depleted cells reversed the effects of miR-375 on neuroendocrine differentiation and cell proliferation. Pathways analysis and confirmatory real-time PCR studies of shared dysregulated target genes indicate that this axis controls neuroendocrine related functions such as neural differentiation, exocytosis, and secretion. Taken together, we provide compelling evidence that a miR-375/YAP axis is a critical mediator of neuroendocrine differentiation and tumorigenesis in lung carcinoid cells.

Nanogold induces anti-inflammation against oxidative stress induced in human neural stem cells exposed to amyloid-beta peptide.

Alzheimer's disease (AD) is a neurodegenerative disorder with progressive memory loss resulting in dementia. Amyloid-beta (Aß) peptides play a critical role in the pathogenesis of the disease by promoting inflammation and oxidative stress, leading to neurodegeneration in the brains of AD patients. Numerous in vitro 3D cell culture models are useful mimics for understanding cellular changes that occur during AD under in vivo conditions. The 3D Bioprinter developed at the CELLINK INKREDIBLE was used in this study to directly investigate the influence of 3D conditions on human neural stem cells (hNSCs) exposed to Aß. The development of anti-AD drugs is usually difficult, mainly due to a lack of therapeutic efficacy and enhanced serious side effects. Gold nanoparticles (AuNPs) demonstrate benefits in the treatment of several diseases, including AD, and may provide a novel therapeutic approach for AD patients. However, the neuroprotective mechanisms by which AuNPs exert these beneficial effects in hNSCs treated with Aß are still not well understood. Therefore, we tested the hypothesis that AuNPs protect against Aß-induced inflammation and oxidative stress in hNSCs under 3D conditions. Here, we showed that AuNPs improved the viability of hNSCs exposed to Aß, which was correlated with the reduction in the expression of inflammatory cytokines, such as TNF-α and IL-1ß. In addition, AuNPs rescued the levels of the transcripts of inhibitory kappa B kinase (IKK) in Aß-treated hNSCs. The Aß-mediated increases in mRNA, protein, and nuclear translocation levels of NF-κB (p65), a key transcription factor involved in inflammatory responses, were all significantly abrogated following co-treatment of hNSCs with AuNPs. In addition, treatment with AuNPs significantly restored iNOS and COX-2 levels in Aß-treated hNSCs. Importantly, hNSCs co-treated with AuNPs were significantly protected from Aß-induced oxidative stress, as detected using the DCFH-DA and DHE staining assays. Furthermore, hNSCs co-treated with AuNPs were significantly protected from the Aß-induced reduction in the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2 downstream antioxidant target genes (SOD-1, SOD-2, Gpx1, GSH, Catalase, and HO-1). Moreover, AuNPs reduced the aggregates and increased the proteasome activity and the expression of HSP27 and HSP70 genes in Aß-treated hNSCs. Taken together, these findings provide the first evidence extending our understanding of the molecular mechanisms under 3D scaffold conditions by which AuNPs reverse the inflammation and oxidative stress-induced in hNSCs exposed to Aß. These findings may facilitate the development of novel treatments for AD.

Nanogold Neuroprotection in Human Neural Stem Cells Against Amyloid-beta-induced Mitochondrial Dysfunction.

Alzheimer's disease (AD) is a neuronal dementia with progressive memory loss. Amyloid-beta (Aß) peptides has major effect in the neurodegenerative disorder, which are thought to promote mitochondrial dysfunction in AD brains. Anti-AD drugs acting upon the brain are generally difficult to develop, often cause serious side effects or lack therapeutic efficacy. Numerous studies have shown the beneficial therapeutic applications of gold nanoparticles (AuNPs), including for neuroprotective events and AD. The aim of this study is to understand how AuNPs could exert their neuroprotective role in AD, for which cell model have chosen human neural stem cells (hNSCs) as the experimental tool. We hypothesize AuNPs protect against Aß-induced cellular impairment and mitochondrial dysfunction in hNSCs. Here, we show AuNPs increase the survival of hNSCs treated with Aß via downregulation of caspase 3 and 9 activities. Moreover, AuNPs abrogated the Aß-mediated decrease neuroprotective (CREB and Bcl-2) and mitochondrial (PGC1α, NRF-1 and Tfam) gene expressions in treated hNSCs. Importantly, co-treatment with AuNPs significantly rescued hNSCs from Aß-mediated mitochondrial function and morphology. AuNPs also significantly normalizes the immunostaining of mitochondrial marker and mass in differentiated hNSCs with Aß. The effects may be exerted by the AuNPs, as supported by its protective reversal of Aß-induced cellular impairment and mitochondrial dysfunction in hNSCs. In fact, the results presented extend our understanding of the mechanisms through which AuNPs could exert their neuroprotective role in hNSCs treated with Aß.

An epigenetic increase in mitochondrial fission by MiD49 and MiD51 regulates the cell cycle in cancer: Diagnostic and therapeutic implications.

Excessive proliferation and apoptosis-resistance are hallmarks of cancer. Increased dynamin-related protein 1 (Drp1)-mediated mitochondrial fission is one of the mediators of this phenotype. Mitochondrial fission that accompanies the nuclear division is called mitotic fission and occurs when activated Drp1 binds partner proteins on the outer mitochondrial membrane. We examine the role of Drp1-binding partners, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51), as drivers of cell proliferation and apoptosis-resistance in non-small cell lung cancer (NSCLC) and invasive breast carcinoma (IBC). We also evaluate whether inhibiting MiDs can be therapeutically exploited to regress cancer. We show that MiD levels are pathologically elevated in NSCLC and IBC by an epigenetic mechanism (decreased microRNA-34a-3p expression). MiDs silencing causes cell cycle arrest through (a) increased expression of cell cycle inhibitors, p27Kip1 and p21Waf1 , (b) inhibition of Drp1, and (c) inhibition of the Akt-mTOR-p70S6K pathway. Silencing MiDs leads to mitochondrial fusion, cell cycle arrest, increased apoptosis, and tumor regression in a xenotransplant NSCLC model. There are positive correlations between MiD expression and tumor size and grade in breast cancer patients and inverse correlations with survival in NSCLC patients. The microRNA-34a-3p-MiDs axis is important to cancer pathogenesis and constitutes a new therapeutic target.

Neuroprotective effects of resveratrol against oxygen glucose deprivation induced mitochondrial dysfunction by activation of AMPK in SH-SY5Y cells with 3D gelatin scaffold.

Ischemic stroke arising from the sudden blockage of arteries in the brain, is a common and serious brain damaging problem worldwide, often leading to disability or death. The oxygen glucose deprivation (OGD) model was created to improve understanding of hypoxia- and hypoglycemia-induced neuronal cell injury, and provide an in vitro surrogate to assess novel treatments for cerebral hypoxia-ischemia. AMP-activated protein kinase (AMPK) is a critical neuroprotective regulator of energy homeostasis, metabolism and cell survival. However, the neuroprotective mechanisms by which AMPK achieves these beneficial effects in human SH-SY5Y neural cells exposed to OGD are still not well understood. Resveratrol is a potent activator of AMPK suggesting it may have therapeutic potential as a neuroprotective agent. Therefore, we hypothesized the AMPK activator resveratrol protects against OGD-mediated impairment of human SH-SY5Y neuronal cells. The novelty of the experiment using a 3D gelatin scaffold cell culture assay, we have tested the potential of 3D systems to mimic the endogenous neuronal environment and have applied these systems to study the effect of OGD on neuronal cells with/without resveratrol. Here we show resveratrol reverses, via AMPK-dependent downregulation of caspase 3 and 9 activity, the OGD-mediated decreases in SH-SY5Y cell viability on a 3D gelatin scaffold. In addition, treatment with OGD decreases mRNA levels of AMPK and the neuroprotective genes (Bcl-2 and CREB); however, co-treatment with resveratrol significantly normalizes these effects. Importantly, resveratrol improves the expression of AMPK and p-AMPK in OGD-exposed SH-SY5Y cells. Resveratrol also significantly rescues SH-SY5Y cells from OGD-mediated mitochondrial deficiency (lower D-loop level, mitochondrial mass, maximal respiratory function, COX activity, and mitochondrial membrane potential). Resveratrol also rescues the transcript expression levels of PGC1α and mitochondrial genes (NRF-1 and Tfam) in OGD-treated SH-SY5Y cells. These findings extend our mechanistic understanding of the central role of AMPK in OGD-related neuronal impairment, and may serve as basis for implementing new therapeutic strategies in the treatment of ischemic stroke.

Phosphodiesterase 3B (PDE3B) antagonizes the anti-angiogenic actions of PKA in human and murine endothelial cells.

Recent reports show that protein kinase A (PKA), but not exchange protein activated by cAMP (EPAC), acts in a cell autonomous manner to constitutively reduce the angiogenic sprouting capacity of murine and human endothelial cells. Specificity in the cellular actions of individual cAMP-effectors can be achieved when a cyclic nucleotide phosphodiesterase (PDE) enzyme acts locally to control the "pool" of cAMP that activates the cAMP-effector. Here, we examined whether PDEs coordinate the actions of PKA during endothelial cell sprouting. Inhibiting each of the cAMP-hydrolyzing PDEs expressed in human endothelial cells revealed that phosphodiesterase 3 (PDE3) inhibition with cilostamide reduced angiogenic sprouting in vitro, while inhibitors of PDE2 and PDE4 family enzymes had no such effect. Identifying a critical role for PDE3B in the anti-angiogenic effects of cilostamide, silencing this PDE3 variant, but not PDE3A, markedly impaired sprouting. Importantly, using both in vitro and ex vivo models of angiogenesis, we show the hypo-sprouting phenotype induced by PDE3 inhibition or PDE3B silencing was reversed by PKA inhibition. Examination of the individual cellular events required for sprouting revealed that PDE3B and PKA each regulated angiogenic sprouting by controlling the invasive capacity of endothelial cells, more specifically, by regulating podosome rosette biogenesis and matrix degradation. In support of the idea that PDE3B acts to inhibit angiogenic sprouting by limiting PKA-mediated reductions in active cdc42, the effects of PDE3B and/or PKA on angiogenic sprouting were negated in cells with reduced cdc42 expression or activity. Since PDE3B and PKA were co-localized in a perinuclear region in human ECs, could be co-immunoprecipitated from lysates of these cells, and silencing PDE3B activated the perinuclear pool of PKA in these cells, we conclude that PDE3B-mediated hydrolysis of cAMP acts to limit the anti-angiogenic potential of PKA in ECs.

Immunofluorescence Labeling of Nuclear Receptor Expression in Formalin-Fixed, Paraffin-Embedded Tissue.

Immunofluorescent staining (IF) uses antigen-antibody complexes tagged with fluorochromes to observe the expression of proteins within a tissue sample. Multiple groups have described optimized methods to visualize several proteins simultaneously within the same tissue section using immunofluorescence in both mouse and human FFPE tissues. Our group routinely uses an optimized protocol described here to examine nuclear receptor expression in experimental samples from conditional knockout in vivo studies.

Examining the Role of Nuclear Receptors During In Vivo Chemical-Mediated Breast Tumorigenesis.

The chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) has been used for many decades to induce skin, mammary, and ovarian tumors in mice. There are however a wide range of doses and treatment regimens in the literature that sometimes confound comparative interpretations of different studies. Here we describe a proven method to generate in vivo DMBA-mediated murine mammary tumors to enable consistent studies of the cell targeted role of genes of interest during this process.

Endothelial Cell-Targeted Deletion of PPARγ Blocks Rosiglitazone-Induced Plasma Volume Expansion and Vascular Remodeling in Adipose Tissue.

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor γ (PPARγ) agonists that represent an effective class of insulin-sensitizing agents; however, clinical use is associated with weight gain and peripheral edema. To elucidate the role of PPARγ expression in endothelial cells (ECs) in these side effects, EC-targeted PPARγ knockout (Pparg ΔEC) mice were placed on a high-fat diet to promote PPARγ agonist-induced plasma volume expansion, and then treated with the TZD rosiglitazone. Compared with Pparg-floxed wild-type control (Pparg f/f) mice, Pparg ΔEC treated with rosiglitazone are resistant to an increase in extracellular fluid, water content in epididymal and inguinal white adipose tissue, and plasma volume expansion. Interestingly, histologic assessment confirmed significant rosiglitazone-mediated capillary dilation within white adipose tissue of Pparg f/f mice, but not Pparg ΔEC mice. Analysis of ECs isolated from untreated mice in both strains suggested the involvement of changes in endothelial junction formation. Specifically, compared with cells from Pparg f/f mice, Pparg ΔEC cells had a 15-fold increase in focal adhesion kinase, critically important in EC focal adhesions, and >3-fold significant increase in vascular endothelial cadherin, the main component of focal adhesions. Together, these results indicate that rosiglitazone has direct effects on the endothelium via PPARγ activation and point toward a critical role for PPARγ in ECs during rosiglitazone-mediated plasma volume expansion.

Rosiglitazone rescues human neural stem cells from amyloid-beta induced ER stress via PPARγ dependent signaling.

Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to a family of ligand-activated nuclear receptors known to regulate many crucial physiological and pathological conditions. Indeed, altered PPARγ transcriptional activity contributes to metabolic syndromes (obesity and hyperglycemia associated with type 2 diabetes mellitus), stroke and neurodegenerative diseases. Various studies suggest that PPARγ agonists influence neuronal deficits in Alzheimer's Disease (AD) patients and rodent models of AD. Expression of amyloid-beta (Aß), a neuropathological marker associated with the pathogenesis of AD neuronal impairment, is inversely correlated with the activation of PPARγ-dependent neuroprotective responses. Nevertheless, molecular mechanisms by which the effects of PPARγ agonists in AD remain to be clarified. Here, we explore the PPARγ signaling pathways and networks that protect against Aß-induced endoplasmic reticulum (ER) stress (e.g., caspase 4, Bip, CHOP, ASK1 and ER calcium), cell death (e.g., viability and cytochrome c) and mitochondrial deficiency (e.g., maximal respiratory function, COX activity, and mitochondrial membrane potential) events in the human neural stem cells (hNSCs) treated with Aß. Co-treatment with GW9662 (an antagonist of PPARγ) effectively blocked these protective effects by rosiglitazone, providing strong evidence that PPARγ-dependent signaling rescues hNSCs from Aß-mediated toxicity. Together, our data suggest activation of PPARγ pathway might be critical to protecting against AD-related ER stress, ER disequilibrium and mitochondrial deficiency. These findings also improve our understanding of the role of PPARγ in hNSCs, and may aid in the development and implementation of new therapeutic strategies for the treatment of AD.