Neuronal Aquaporin 1 Inhibits Amyloidogenesis by Suppressing the Interaction Between Beta-Secretase and Amyloid Precursor Protein.

The accumulation of amyloid-ß (Aß) is a characteristic event in the pathogenesis of Alzheimer's disease (AD). Aquaporin 1 (AQP1) is a membrane water channel protein belonging to the AQP family. AQP1 levels are elevated in the cerebral cortex during the early stages of AD, but the role of AQP1 in AD pathogenesis is unclear. We first determined the expression and distribution of AQP1 in brain tissue samples of AD patients and two AD mouse models (3xTg-AD and 5xFAD). AQP1 accumulation was observed in vulnerable neurons in the cerebral cortex of AD patients, and in neurons affected by the Aß or tau pathology in the 3xTg-AD and 5xFAD mice. AQP1 levels increased in neurons as aging progressed in the AD mouse models. Stress stimuli increased AQP1 in primary cortical neurons. In response to cellular stress, AQP1 appeared to translocate to endocytic compartments of ß- and γ-secretase activities. Ectopic expression of AQP1 in human neuroblastoma cells overexpressing amyloid precussir protein (APP) with the Swedish mutations reduced ß-secretase (BACE1)-mediated cleavage of APP and reduced Aß production without altering the nonamyloidogenic pathway. Conversely, knockdown of AQP1 enhanced BACE1 activity and Aß production. Immunoprecipitation experiments showed that AQP1 decreased the association of BACE1 with APP. Analysis of a human database showed that the amount of Aß decreases as the expression of AQP1 increases. These results suggest that the upregulation of AQP1 is an adaptive response of neurons to stress that reduces Aß production by inhibiting the binding between BACE1 and APP.

The role of adiponectin in obesity-associated female-specific carcinogenesis.

Adipose tissue is a highly vascularized endocrine organ, and its secretion profiles may vary with obesity. Adiponectin is secreted by adipocytes that make up adipose tissue. Worldwide, obesity has been designated a serious health problem among women and is associated with a variety of metabolic disorders and an increased risk of developing cancer of the cervix, ovaries, uterus (uterine/endometrial), and breast. In this review, the potential link between obesity and female-specific malignancies is comprehensively presented by discussing significant features of the intriguing and complex molecule, adiponectin, with a focus on recent findings highlighting its molecular mechanism of action in female-specific carcinogenesis.

Targeting DUSPs in glioblastomas - wielding a double-edged sword?

Several dual-specificity phosphatases (DUSPs) that play key roles in the direct or indirect inactivation of different MAP kinases (MAPKs) have been implicated in human cancers over the past decade. This has led to a growing interest in identifying DUSPs and their specific inhibitors for further testing and validation as therapeutic targets in human cancers. However, the lack of understanding of the complex regulatory mechanisms and cross-talks between MAPK signaling pathways, combined with the fact that DUSPs can act as a double-edged sword in cancer progression, calls for a more careful and thorough investigation. Among the various types of brain cancer, glioblastoma multiforme (GBM) is notorious for its aggressiveness and resistance to current treatment modalities. This has led to the search for new molecular targets, particularly those involving various signaling pathways. DUSPs appear to be a promising target, but much more information on DUSP targets and their effects on GBM is needed before potential therapies can be developed, tested, and validated. This review identifies and summarize the specific roles of DUSP1, DUSP4, DUSP6 and DUSP26 that have been implicated in GBM.

3,6'-dithiothalidomide improves experimental stroke outcome by suppressing neuroinflammation.

Tumor necrosis factor-α (TNF) plays a prominent role in the brain damage and functional deficits that result from ischemic stroke. It was recently reported that the thalidomide analog 3,6'-dithiothalidomide (3,6'-DT) can selectively inhibit the synthesis of TNF in cultured cells. We therefore tested the therapeutic potential of 3,6'-DT in a mouse model of focal ischemic stroke. Administration of 3,6'-DT immediately prior to a stroke or within 3 hr after the stroke reduced infarct volume, neuronal death, and neurological deficits, whereas thalidomide was effective only when administered prior to stroke. Neuroprotection was accompanied by decreased inflammation; 3,6'-DT-treated mice exhibited reduced expression of TNF, interleukin-1ß, and inducible nitric oxide synthase; reduced numbers of activated microglia/macrophages, astrocytes, and neutrophils; and reduced expression of intercellular adhesion molecule-1 in the ischemic brain tissue. 3,6'-DT treatment attenuated stroke-induced disruption of the blood-brain barrier by a mechanism that appears to involve suppression of matrix metalloproteinase-9 and preservation of occludin. Treatment with 3,6'-DT did not reduce ischemic brain damage in mice lacking TNF receptors, consistent with a critical role for suppression of TNF production and TNF signaling in the therapeutic action of 3,6'-DT. These findings suggest that anti-inflammatory mechanisms underlie the therapeutic actions of 3,6-DT in an animal model of stroke.

Exendin-4 ameliorates motor neuron degeneration in cellular and animal models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by a progressive loss of lower motor neurons in the spinal cord. The incretin hormone, glucagon-like peptide-1 (GLP-1), facilitates insulin signaling, and the long acting GLP-1 receptor agonist exendin-4 (Ex-4) is currently used as an anti-diabetic drug. GLP-1 receptors are widely expressed in the brain and spinal cord, and our prior studies have shown that Ex-4 is neuroprotective in several neurodegenerative disease rodent models, including stroke, Parkinson's disease and Alzheimer's disease. Here we hypothesized that Ex-4 may provide neuroprotective activity in ALS, and hence characterized Ex-4 actions in both cell culture (NSC-19 neuroblastoma cells) and in vivo (SOD1 G93A mutant mice) models of ALS. Ex-4 proved to be neurotrophic in NSC-19 cells, elevating choline acetyltransferase (ChAT) activity, as well as neuroprotective, protecting cells from hydrogen peroxide-induced oxidative stress and staurosporine-induced apoptosis. Additionally, in both wild-type SOD1 and mutant SOD1 (G37R) stably transfected NSC-19 cell lines, Ex-4 protected against trophic factor withdrawal-induced toxicity. To assess in vivo translation, SOD1 mutant mice were administered vehicle or Ex-4 at 6-weeks of age onwards to end-stage disease via subcutaneous osmotic pump to provide steady-state infusion. ALS mice treated with Ex-4 showed improved glucose tolerance and normalization of behavior, as assessed by running wheel, compared to control ALS mice. Furthermore, Ex-4 treatment attenuated neuronal cell death in the lumbar spinal cord; immunohistochemical analysis demonstrated the rescue of neuronal markers, such as ChAT, associated with motor neurons. Together, our results suggest that GLP-1 receptor agonists warrant further evaluation to assess whether their neuroprotective potential is of therapeutic relevance in ALS.

Chronic nicotine administration impairs activation of cyclic AMP-response element binding protein and survival of newborn cells in the dentate gyrus.

Chronic intake of nicotine can impair hippocampal plasticity, but the underlying mechanism is poorly understood. Here, we demonstrate that chronic nicotine administration in adult rats inactivates the cyclic AMP-response element binding protein (CREB), a transcription factor that regulates neurogenesis and other plasticity-related processes necessary for learning and memory. Consequently, we showed that impaired CREB signaling is associated with a significant decline in the production of new neurons in the dentate gyrus. Combining retrovirus labeling with gene expression approaches, we found that chronic nicotine administration reduces the number of adult-generated granule neurons by decreasing the survival of newborn cells but not the proliferation of progenitor cells. Additionally, we found that retroviral-mediated expression of a constitutively active CREB in the dentate gyrus rescues survival of newborn cells and reverses the nicotine-induced decline in the number of mature granule neurons. Prolonged nicotine exposure also compromises CREB activation and reduces the viability of progenitor cells in vitro, thereby suggesting that nicotine may exert its adverse effects directly on immature cells in vivo. Taken together, these data demonstrate that inhibition of CREB activation is responsible for the nicotine-induced impairment of hippocampal plasticity.

Electroconvulsive shock ameliorates disease processes and extends survival in huntingtin mutant mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by expanded polyglutamine repeats in the huntingtin (Htt) protein. Mutant Htt may damage and kill striatal neurons by a mechanism involving reduced production of brain-derived neurotrophic factor (BDNF) and increased oxidative and metabolic stress. Because electroconvulsive shock (ECS) can stimulate the production of BDNF and protect neurons against stress, we determined whether ECS treatment would modify the disease process and provide a therapeutic benefit in a mouse model of HD. ECS (50 mA for 0.2 s) or sham treatment was administered once weekly to male N171-82Q Htt mutant mice beginning at 2 months of age. Endpoints measured included motor function, striatal and cortical pathology, and levels of protein chaperones and BDNF. ECS treatment delayed the onset of motor symptoms and body weight loss and extended the survival of HD mice. Striatal neurodegeneration was attenuated and levels of protein chaperones (Hsp70 and Hsp40) and BDNF were elevated in striatal neurons of ECS-treated compared with sham-treated HD mice. Our findings demonstrate that ECS can increase the resistance of neurons to mutant Htt resulting in improved functional outcome and extended survival. The potential of ECS as an intervention in subjects that inherit the mutant Htt gene merits further consideration.

Receptor channel TRPC6 is a key mediator of Notch-driven glioblastoma growth and invasiveness.

Glioblastoma multiforme (GBM) is the most frequent and incurable type of brain tumor of adults. Hypoxia has been shown to direct GBM toward a more aggressive and malignant state. Here we show that hypoxia increases Notch1 activation, which in turn induces the expression of transient receptor potential 6 (TRPC6) in primary samples and cell lines derived from GBM. TRPC6 is required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces the development of the malignant GBM phenotypes under hypoxia. Clinically, expression of TRPC6 was elevated in GBM specimens in comparison with normal tissues. Collectively, our studies indicate that TRPC6 is a key mediator of tumor growth of GBM in vitro and in vivo and that TRPC6 may be a promising therapeutic target in the treatment of human GBM.

Possible angiogenic roles for claudin-4 in ovarian cancer.

Claudin proteins are frequently overexpressed in various tumors such as breast, prostate and ovarian cancer. While their functions in cancer have not been completely elucidated, roles in survival, adhesion and invasion have been suggested. In order to clarify the roles of claudins in ovarian cancer, we have performed gene expression profiling of ovarian surface epithelial cells overexpressing claudin-4 and compared the expression patterns to the parental, non-expressing cells. Claudin-4 expression leads to the differential expression of several genes, including many that have previously been implicated in angiogenesis. In particular, angiogenic cytokines, such as IL-8, were found elevated while genes of the angiostatic interferon pathway were found downregulated. In vitro assays show that claudin-4-expressing cells produce factors that can stimulate angiogenesis as measured by tube formation and migration in HUVEC cells. In addition, an in vivo mouse dorsal skinfold assay confirms that cells expressing claudin-4 secrete factors that can mediate angiogenesis in the dorsal skin of mice. Our data suggest a novel function for claudin-4 in cancer and provide an additional rationale for its common overexpression in human tumors.

The brain uncoupling protein UCP4 attenuates mitochondrial toxin-induced cell death: role of extracellular signal-regulated kinases in bioenergetics adaptation and cell survival.

Increased bioenergetics demand can stimulate compensatory increases in glucose metabolism. We previously reported that neural cells expressing the brain uncoupling protein UCP4 exhibit enhanced dependency on glucose for support of cellular bioenergetics and survival. The switch from oxidative toward glycolytic metabolism reduces the production of toxic reactive oxygen species (ROS) and increases cellular resistance to toxicity induced by 3-nitropropionic acid, a mitochondrial complex II inhibitor that compromises cellular bioenergetics. In this study we elucidate the underlying mechanism whereby expression of UCP4 promotes bioenergetics adaptation and cell survival. We found that activation of extracellular signal-regulated kinases (ERKs) is necessary and sufficient for the increased dependency on glucose utilization. Pharmacological inhibition of ERKs not only abrogated bioenergetics adaptation but also reduced the activation of cAMP-responsive element-binding (CREB) protein suggesting that CREB protein signaling contributes in part to UCP4-dependent cell death rescue from 3-nitropropionic acid-induced toxicity. We also demonstrated that activation of ERKs by growth factors ameliorated the bioenergetics compromise and reduced cellular toxicity induced by 3-nitropropionic acid. Collectively, our results support the involvement of ERKs in UCP4 dependent bioenergetics adaptation and cell survival.

The homocysteine-inducible endoplasmic reticulum stress protein counteracts calcium store depletion and induction of CCAAT enhancer-binding protein homologous protein in a neurotoxin model of Parkinson disease.

The endoplasmic reticulum (ER) is a key organelle regulating intracellular Ca(2+) homeostasis. Oxidants and mitochondria-derived free radicals can target ER-based Ca(2+) regulatory proteins and cause uncontrolled Ca(2+) release that may contribute to protracted ER stress and apoptosis. Several ER stress proteins have been suggested to counteract the deregulation of ER Ca(2+) homeostasis and ER stress. Here we showed that knockdown of Herp, an ubiquitin-like domain containing ER stress protein, renders PC12 and MN9D cells vulnerable to 1-methyl-4-phenylpyridinium-induced cytotoxic cell death by a mechanism involving up-regulation of CHOP expression and ER Ca(2+) depletion. Conversely, Herp overexpression confers protection by blocking 1-methyl-4-phenylpyridinium-induced CHOP up-regulation, ER Ca(2+) store depletion, and mitochondrial Ca(2+) accumulation in a manner dependent on a functional ubiquitin-proteasomal protein degradation pathway. Deletion of the ubiquitin-like domain of Herp or treatment with a proteasomal inhibitor abolished the central function of Herp in ER Ca(2+) homeostasis. Thus, elucidating the underlying molecular mechanism(s) whereby Herp counteracts Ca(2+) disturbances will provide insights into the molecular cascade of cell death in dopaminergic neurons and may uncover novel therapeutic strategies to prevent and ameliorate Parkinson disease progression.

Involvement of notch signaling in wound healing.

The Notch signaling pathway is critically involved in cell fate decisions during development of many tissues and organs. In the present study we employed in vivo and cell culture models to elucidate the role of Notch signaling in wound healing. The healing of full-thickness dermal wounds was significantly delayed in Notch antisense transgenic mice and in normal mice treated with gamma-secretase inhibitors that block proteolytic cleavage and activation of Notch. In contrast, mice treated with a Notch ligand Jagged peptide showed significantly enhanced wound healing compared to controls. Activation or inhibition of Notch signaling altered the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in a scratch wound healing model in ways consistent with roles for Notch signaling in wound healing functions all three cell types. These results suggest that Notch signaling plays important roles in wound healing and tissue repair, and that targeting the Notch pathway might provide a novel strategy for treatment of wounds and for modulation of angiogenesis in other pathological conditions.

Gamma secretase-mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke.

Mice transgenic for antisense Notch and normal mice treated with inhibitors of the Notch-activating enzyme gamma-secretase showed reduced damage to brain cells and improved functional outcome in a model of focal ischemic stroke. Notch endangers neurons by modulating pathways that increase their vulnerability to apoptosis, and by activating microglial cells and stimulating the infiltration of proinflammatory leukocytes. These findings suggest that Notch signaling may be a therapeutic target for treatment of stroke and related neurodegenerative conditions.

Calcitonin increases tumorigenicity of prostate cancer cells: evidence for the role of protein kinase A and urokinase-type plasminogen receptor.

The expression of human (h) calcitonin (CT) and its receptor (CTR) is localized to basal epithelium in benign prostates but is distributed in whole epithelium of malignant prostates. Moreover, the abundance of hCT and CTR mRNA in primary prostate tumors positively correlates with the tumor grade. We tested the hypothesis that the modulation of endogenous hCT expression of prostate cancer (PC) cell lines alters their oncogenicity. The effect of modulation of hCT expression on oncogenic characteristics was examined in LNCaP and PC-3M cell lines. The endogenous hCT expression was modulated using either constitutively active expression vector containing hCT cDNA or anti-hCT hammerhead ribozymes. The changes in the oncogenicity of cell sublines was assessed with cell proliferation assays, invasion assays, colony formation assays, and in vivo growth in athymic nude mice. Up-regulation of hCT in PC-3M cells and or enforced hCT expression in LNCaP cells dramatically enhanced their oncogenic characteristics. In contrast, the down-regulation of hCT in PC-3M cells led to a dramatic decline in their oncogenicity. These results, when combined with our other results, that the expression of hCT in primary PCs increase with tumor grade, suggest an important role for hCT in the progression of PC to a metastatic phenotype.

Calcitonin stimulates the secretion of urokinase-type plasminogen activator from prostate cancer cells: its possible implications on tumor cell invasion.

Calcitonin (CT) is synthesized and secreted in prostate epithelium, and its secretion from malignant prostates is several folds higher than that in benign prostates. CT receptor (CTR) is expressed in malignant prostate epithelium, and its activation increases invasiveness of prostate cancer (PC) cells via activation of protein kinase A. Since the role of urokinase-type plasminogen activator (uPA) in invasion of PC has been established, we tested the hypothesis that CT increases invasion of PC cells by stimulating uPA secretion from PC cells. Exogenously added CT stimulated the secretion of uPA from PC-3M cells in a dose-dependent manner, which was blocked by Rp.cAMP, a competitive inhibitor of protein kinase A. CT stimulated the secretion of MMP-2 and MMP-9 from PC-3M cells, and also increased their invasiveness. Both these actions of CT were blocked by uPA-neutralizing antibodies. Immunofluorescence studies with PC-3M cells suggest that CT stimulated redistribution of cellular uPA to focal adhesion sites, which was further confirmed by co-immunoprecipitation of uPA with focal adhesion kinase (FAK) in response to CT. These results suggest that CT increases invasiveness of PC cells by stimulating PKA-mediated uPA secretion and by redirecting the secreted uPA to focal adhesion sites. The results also suggest that uPA may, at least in part, mediate proinvasive actions of CT on PC cells by stimulating the secretion of gelatinases and degradation of focal adhesion sites.

Calcitonin stimulates multiple stages of angiogenesis by directly acting on endothelial cells.

Although a strong correlation between neuroendocrine differentiation and angiogenesis of prostate cancer has been reported, no mechanistic link between the two events has been established. Because neuropeptide calcitonin is secreted by prostate tumors and endothelial cells are known to express calcitonin receptor-like receptor, we examined the potential action of calcitonin on endothelial cells. The presence of calcitonin receptor, calcitonin receptor-like receptor, and receptor activity-modifying proteins in human microvessel endothelial-1 cells was tested by reverse transcriptase-PCR (RT-PCR). The proangiogenic action of calcitonin was examined in several in vitro models of angiogenesis using HMEC-1 cells and also in vivo using dorsal skinfold assays. Calcitonin expression of PC-3M cells was modulated, and its effect on angiogenesis was examined in in vitro as well as in vivo models. The results of RT-PCR and radioligand receptor assays showed the presence of functional calcitonin receptor in HMEC-1 cells. Calcitonin stimulated all phases of angiogenesis through the calcitonin receptor, but its effect on tube morphogenesis by endothelial cells occurred at the concentration of the Kd of calcitonin receptor. Silencing of calcitonin receptor expression in HMEC-1 cells abolished calcitonin-induced tube formation. Vascular endothelial growth factor antibodies attenuated but did not abolish calcitonin-induced tube morphogenesis. PC-3M prostate cancer cells induced angiogenesis in in vivo and in vitro models. Overexpression of calcitonin in PC-3M cells increased their angiogenic activity, whereas the silencing of calcitonin expression abolished it. These results show that prostate tumor-derived calcitonin may play an important role in prostate tumor growth by regulating intratumoral vascularization.

Inhibition of matrix metalloproteinase-9 reduces in vitro invasion and angiogenesis in human microvascular endothelial cells.

The expression of matrix metalloproteinases (MMPs), particularly MMP-9, is significantly increased during tumor progression and is thought to play a major role in mediating angiogenic process. Since microvasculature plays an important role in controlling tumor growth, we investigated the effects of MMP-9 inhibition on endothelial cell migration and tube formation, two determinants of angiogenesis. Adenoviral-mediated MMP-9 downregulation inhibited endothelial cell migration in cell wounding and spheroid migration assays. To determine the effects of MMP-9 reduction in glioblastoma/endothelial co-cultures, we used a three-dimensional co-culture assay of glioblastoma spheroids and endothelial spheroids. Untreated controls showed invasion of both cell populations into each other whereas treatment of the co-cultures with adenoviral antisense MMP-9 particles resulted in reduced invasion. Next, inhibition of MMP-9 by adenoviral vectors in endothelial cells was assessed for in vitro capillary-like structure formation either by co-culture with glioblastoma cells or exposure to glioblastoma-conditioned medium. Addition of conditioned medium from human glioblastoma cells to endothelial cells treated with antisense MMP-9 adenoviral vectors or co-cultures of glioblastoma cell lines with MMP-9-reduced endothelial cells resulted in reduced capillary-like tube formation demonstrating the key role of MMP-9 in endothelial cell network organization. Examination of in vitro capillary-like tube structure formation using Matrigel showed a significant decrease in MMP-9 downregulated endothelial cells as compared to controls. In conclusion, the inhibition of MMP-9 is required for inhibition of endothelial cell migration and tube formation and is likely to be of importance in cerebral angiogenesis for therapeutic targets.