- Email: [email protected]
BACE1 gene regulation: A novel drug target in Alzheimer's disease
P304
Poster Presentations: P1
and perspective provided by an experienced researcher and investigator involved in a recently completed disease modifying immunotherapy program. Conclusions: It is imperative that methodological issues be identified and appropriately addressed in order to enhance the probability of success as the field moves forward into the long term investigation of earlier therapeutic interventions. We cannot afford to repeat the mistakes of the past.
P1-399
BACE1 GENE REGULATION: A NOVEL DRUG TARGET IN ALZHEIMER’S DISEASE
Baindu Bayon1, Debomoy Lahiri1, Jason Bailey1, 1Indiana University School of Medicine, Indianapolis, Indiana, United States. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is believed to result from the misregulation of the production of amyloid-b (Ab), which forms the plaques seen in AD brains. The rate-limiting step in the production of Ab is the processing of amyloid- b precursor protein (APP) by a b-secretase called b-site APP-cleaving enzyme (BACE1). Current BACE1 inhibiting drugs are unsuccessful due to their numerous side effects. We are focused on not simply globally blocking BACE1 activity, but controlling how the expression of BACE1 is regulated. We seek to demonstrate the activity of Sp1-mediated activation at specific regions of the BACE1 promoter with various doses of Mithramycin A, a selective inhibitor of Sp1. Also, we aim to describe how the latent early-life associated regulation (LEARn) model illustrates a mechanism by which BACE1 is epigenetically regulated. Due to the wide range of application of the LEARn pathway, our work can have translational implications in many psychiatric disorders. Methods: Specific Aim 1: To demonstrate the activity of Sp1 at specific regions of the BACE1 promoter can be manipulated pharmacologically with various doses of Sp1-inhibiting drugs. Specific Aim 2: To describe how the latent early-life associated regulation (LEARn) model illustrates a mechanism by which BACE1 may be epigenetically regulated. Results: Specific Aim 1: We will test several BACE1 promoters by DNA transfection in human neuronal cultures. We will treat SK-N-SH cells with the Sp1-inhibiting drug and observe luciferase reporter activity then protein expression by Western blot. The results of the efficacy of MithA will be analyzed as described. Specific Aim 2: We will treat cells with insults such as lead, Ab itself, and reactive oxygen species and then treat with Sp1 inhibitors. Conclusions: Specific Aim 1: We expect to see levels of BACE1 expression to be reduced in those cells treated with an Sp1 inhibitor. We would expect to see a dose-dependent decrease in the luciferase activity driven by BACE1 promoter activity and a decrease in protein expression seen in Western blot analysis.Specific Aim 2: We expect to see reversal the of damage caused by insults when treated with Sp1 inhibiting drugs.
P1-400
FLUVOXAMINE ATTENUATES TAU PHOSPHORYLATION THROUGH AN UPREGULATION OF SIGMA-1 RECEPTORS (SIG-1RS)
Tsubasa Omi1, Takashi Kudo2, Hitoshi Tanimukai3, Daisuke Kanayama4, Yukako Sakagami5, Hideaki Hara6, Masatoshi Takeda7, 1Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Japan; 2 Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Japan; 3Osaka University Graduate School of Medicine, Suita, Japan; 4Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Japan; 5Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Japan; 6Department of Biofunctional Evaluation Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; 7Osaka University Graduate School of Medicine, Osaka, Japan. Contact e-mail: [email protected] Background: A bnormal tau phosphorylation results in neuronal cell death in Alzheimer’s disease. Recently it has been demonstrated that endoplasmic reticulum (ER) stress increases tau phosphorylation. It suggests that control of ER stress may reduce tau phosphorylation. Our previous study showed that Sig-1Rs are transcriptionally induced via the PERK/eIF2a/ATF4 pathway, one of ER stress responses, and ameliorate cell death consequently. Fluvoxamine behaves as a potent agonist at Sig-1Rs and has the highest affinity of any SSRI. In this study, we investigate effects of fluvoxamine on the Sig-1Rs induction and tau phosphorylation. Methods: We loaded ER stress on neuroblastoma cell lines with or without fluvoxamine for LDH assay and western blotting of Sig-1Rs, phosphor-eIF2a, or ATF4. Middle cerebral artery occlusion (MCAO) mice as models for ER stress were also treated with fluvoxamine for TTC staining to detect infarction and for immunohistostaining with AT8 to monitor the expression of tau phosphorylation. To confirm the Sig-1Rs induction by fluvoxamine, a luciferase reporter system of its promoter was used. RNA interference was used to knockdown ATF4 expression to check its involvement in the fluvoxamine effect. Results: Treatment of neuroblastoma cells with fluvoxamine reduced cell death induced by ER stress. Paroxetine does not have this effect, which is canceled by NE-100, an antagonist for Sig-1Rs. Intraperitoneal treatment with fluvoxamine reduced the area of infarction and AT8 positive cells in the ischemic penumbra region. Fluvoxamine transcriptionally induces Sig-1Rs to yield cellular resistance to ER stress. In the HEK293 cells with knockdown of ATF4 expression, the induction of Sig-1Rs by fluvoxamine is not observed indicating that the effect of fluvoxamine is caused by activation of ATF4. Moreover fluvoxamine does not phosphorylate eIF2a, an overhead of ATF4 expression. Taken together, it is suggested that fluvoxamine directly activates a promoter of ATF4. Conclusions: Fluvoxamine induces Sig1Rs as a result of direct ATF4 induction. The induction of Sig-1Rs induces cellular resistance to ER stress and reduces tau phosphorylation. This result might provide the clue to attenuate tau phosphorylation for treatment of Alzheimer’s disease.
Poster Presentations: P1
and perspective provided by an experienced researcher and investigator involved in a recently completed disease modifying immunotherapy program. Conclusions: It is imperative that methodological issues be identified and appropriately addressed in order to enhance the probability of success as the field moves forward into the long term investigation of earlier therapeutic interventions. We cannot afford to repeat the mistakes of the past.
P1-399
BACE1 GENE REGULATION: A NOVEL DRUG TARGET IN ALZHEIMER’S DISEASE
Baindu Bayon1, Debomoy Lahiri1, Jason Bailey1, 1Indiana University School of Medicine, Indianapolis, Indiana, United States. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is believed to result from the misregulation of the production of amyloid-b (Ab), which forms the plaques seen in AD brains. The rate-limiting step in the production of Ab is the processing of amyloid- b precursor protein (APP) by a b-secretase called b-site APP-cleaving enzyme (BACE1). Current BACE1 inhibiting drugs are unsuccessful due to their numerous side effects. We are focused on not simply globally blocking BACE1 activity, but controlling how the expression of BACE1 is regulated. We seek to demonstrate the activity of Sp1-mediated activation at specific regions of the BACE1 promoter with various doses of Mithramycin A, a selective inhibitor of Sp1. Also, we aim to describe how the latent early-life associated regulation (LEARn) model illustrates a mechanism by which BACE1 is epigenetically regulated. Due to the wide range of application of the LEARn pathway, our work can have translational implications in many psychiatric disorders. Methods: Specific Aim 1: To demonstrate the activity of Sp1 at specific regions of the BACE1 promoter can be manipulated pharmacologically with various doses of Sp1-inhibiting drugs. Specific Aim 2: To describe how the latent early-life associated regulation (LEARn) model illustrates a mechanism by which BACE1 may be epigenetically regulated. Results: Specific Aim 1: We will test several BACE1 promoters by DNA transfection in human neuronal cultures. We will treat SK-N-SH cells with the Sp1-inhibiting drug and observe luciferase reporter activity then protein expression by Western blot. The results of the efficacy of MithA will be analyzed as described. Specific Aim 2: We will treat cells with insults such as lead, Ab itself, and reactive oxygen species and then treat with Sp1 inhibitors. Conclusions: Specific Aim 1: We expect to see levels of BACE1 expression to be reduced in those cells treated with an Sp1 inhibitor. We would expect to see a dose-dependent decrease in the luciferase activity driven by BACE1 promoter activity and a decrease in protein expression seen in Western blot analysis.Specific Aim 2: We expect to see reversal the of damage caused by insults when treated with Sp1 inhibiting drugs.
P1-400
FLUVOXAMINE ATTENUATES TAU PHOSPHORYLATION THROUGH AN UPREGULATION OF SIGMA-1 RECEPTORS (SIG-1RS)
Tsubasa Omi1, Takashi Kudo2, Hitoshi Tanimukai3, Daisuke Kanayama4, Yukako Sakagami5, Hideaki Hara6, Masatoshi Takeda7, 1Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Japan; 2 Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Japan; 3Osaka University Graduate School of Medicine, Suita, Japan; 4Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Japan; 5Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Japan; 6Department of Biofunctional Evaluation Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; 7Osaka University Graduate School of Medicine, Osaka, Japan. Contact e-mail: [email protected] Background: A bnormal tau phosphorylation results in neuronal cell death in Alzheimer’s disease. Recently it has been demonstrated that endoplasmic reticulum (ER) stress increases tau phosphorylation. It suggests that control of ER stress may reduce tau phosphorylation. Our previous study showed that Sig-1Rs are transcriptionally induced via the PERK/eIF2a/ATF4 pathway, one of ER stress responses, and ameliorate cell death consequently. Fluvoxamine behaves as a potent agonist at Sig-1Rs and has the highest affinity of any SSRI. In this study, we investigate effects of fluvoxamine on the Sig-1Rs induction and tau phosphorylation. Methods: We loaded ER stress on neuroblastoma cell lines with or without fluvoxamine for LDH assay and western blotting of Sig-1Rs, phosphor-eIF2a, or ATF4. Middle cerebral artery occlusion (MCAO) mice as models for ER stress were also treated with fluvoxamine for TTC staining to detect infarction and for immunohistostaining with AT8 to monitor the expression of tau phosphorylation. To confirm the Sig-1Rs induction by fluvoxamine, a luciferase reporter system of its promoter was used. RNA interference was used to knockdown ATF4 expression to check its involvement in the fluvoxamine effect. Results: Treatment of neuroblastoma cells with fluvoxamine reduced cell death induced by ER stress. Paroxetine does not have this effect, which is canceled by NE-100, an antagonist for Sig-1Rs. Intraperitoneal treatment with fluvoxamine reduced the area of infarction and AT8 positive cells in the ischemic penumbra region. Fluvoxamine transcriptionally induces Sig-1Rs to yield cellular resistance to ER stress. In the HEK293 cells with knockdown of ATF4 expression, the induction of Sig-1Rs by fluvoxamine is not observed indicating that the effect of fluvoxamine is caused by activation of ATF4. Moreover fluvoxamine does not phosphorylate eIF2a, an overhead of ATF4 expression. Taken together, it is suggested that fluvoxamine directly activates a promoter of ATF4. Conclusions: Fluvoxamine induces Sig1Rs as a result of direct ATF4 induction. The induction of Sig-1Rs induces cellular resistance to ER stress and reduces tau phosphorylation. This result might provide the clue to attenuate tau phosphorylation for treatment of Alzheimer’s disease.