The molecules that mediate death of selective neurons in Alzheimer's disease (AD) are mostly unknown. The Forkhead transcription factor FoxO3a has emerged as an important mediator of cell fate including apoptosis. When phosphorylated by Akt, it is localized in the cytosol as an inactive complex bound with 14-3-3 protein. For activation and localization of FoxO3a in the nucleus, further modifications are required, such as phosphorylation by mammalian sterile 20-like kinase 1 (MST1) and arginine methylation by protein arginine methyltransferase1. We report here that Akt-mediated phosphorylation of FoxO3a is diminished in neurons exposed to oligomeric β-amyloid (Aβ), in vitro and in vivo. We also find that oligomeric Aβ activates FoxO3a by MST1 phosphorylation and arginine methylation in primary cultures of hippocampal and cortical neurons. Moreover, FoxO3a translocates from the cytosol to nucleus in cultured neurons in response to Aβ. Most importantly, the nuclear redistribution of FoxO3a is significantly increased in Aβ-overexpressing AβPPswe-PS1dE9 mice and Aβ-infused rat brains. We further find that FoxO3a is essential for loss of neurons and neural networks in response to Aβ. Recent reports implicate Bim, a pro-apoptotic member of Bcl-2 family, in neuron death in AD, as a key target of this transcription factor. We show that Bim is a direct target of FoxO3a in Aβ-treated neurons. Our findings thus indicate that FoxO3a is activated, translocated to the nucleus and mediates neuron death via Bim in response to Aβ toxicity.
Neuron death during development and in Alzheimer’s disease (AD) is associated with aberrant regulation/induction of cell cycle proteins. However, the proximal events in this process are unknown. Cell cycle initiation requires dephosphorylation of cyclin-dependent kinases by cell division cycle 25A (Cdc25A). Here, we show that Cdc25A is essential for neuronal death in response to NGF deprivation or β-amyloid (Aβ) treatment and describe the mechanisms by which it is regulated in these paradigms. Cdc25A mRNA, protein and Cdc25A phosphatase activity were induced by NGF deprivation and Aβ treatment. Enhanced Cdc25A expression was also observed in rat brains infused with Aβ and in Aβ-overexpressing AβPPswe-PS1dE9 mice. In cultured neurons Cdc25A inhibition by chemical inhibitors or shRNA prevented cell death and neurite degeneration caused by NGF deprivation or Aβ. Additionally, Cdc25A inhibition diminished distal signaling events including Cdk-dependent elevation of phospho-pRb and subsequent caspase-3 activation. Mechanism studies revealed that Cdc25A induction by NGF deprivation and Aβ is mediated by activation of Forkhead transcription factors that in turn suppress miR-21, a negative regulator of Cdc25A. Our studies thus identify Cdc25A as a required upstream element of the apoptotic cell cycle pathway that is required for neuron death in response to trophic factor deprivation and to Aβ exposure and therefore as a potential target to suppress pathologic neuron death.
Alzheimer’s disease (AD) is a progressive neurodegenerative disease with no cure till today. Aberrant activation of cell cycle regulatory proteins is implicated in neurodegenerative diseases including AD. We and others have shown that Cyclin dependent kinase 4 (Cdk4) is activated in AD brain and is required for neuron death. In this study, we tested the efficiency of commercially available Cdk4 specific inhibitors as well as a small library of synthetic molecule inhibitors targeting Cdk4 as neuroprotective agents in cellular models of neuron death. We found that several of these inhibitors significantly protected neuronal cells against death induced by nerve growth factor (NGF) deprivation and oligomeric beta amyloid (Aβ) that are implicated in AD. These neuroprotective agents inhibit specifically Cdk4 kinase activity, loss of mitochondrial integrity, induction of pro-apoptotic protein Bim and caspase3 activation in response to NGF deprivation. The efficacies of commercial and synthesized inhibitors are comparable. The synthesized molecules are either phenanthrene based or naphthalene based and they are synthesized by using Pschorr reaction and Buchwald coupling respectively as one of the key steps. A number of molecules of both kinds block neurodegeneration effectively. Therefore, we propose that Cdk4 inhibition would be a therapeutic choice for ameliorating neurodegeneration in AD and these synthetic Cdk4 inhibitors could lead to development of effective drugs for AD.
Parkinson's disease (PD) results from the selective loss of dopaminergic neurons of substantia nigra pars compacta region of the midbrain. It has been reported that the transcription factor forkhead Box O3a (FoxO3a) is activated and induces pro‐apoptotic protein such as Bcl‐2‐interacting mediator of cell death (BIM) and p53 up‐regulated modulator of apoptosis (PUMA) in variety of neuron death paradigms. Activity of FoxO3a is governed by its post‐translational modifications which control its subcellular localization. Aim of this study was to determine whether FoxO3a is activated and up‐regulates its pro‐apoptotic genes to induce neuron death in PD. We exposed neuronal PC12 cells or primary cultures of dopaminergic neurons to 6‐hydroxy dopamine (6‐OHDA) and infused 6‐OHDA in rat brain to develop PD models. We found that FoxO3a undergoes multiple post‐translational modifications which render its nuclear localization in dopaminergic neuronal cells in response to 6‐OHDA. The nuclear redistribution of FoxO3a is significantly increased in dopaminergic neurons of 6‐OHDA infused rat brains as well. Moreover, FoxO3a is required for dopaminergic neurodegeneration in response to 6‐OHDA as RNAi‐mediated silencing of FoxO3a protects these cells from 6‐OHDA toxicity. In a search of the downstream targets we identified PUMA as a direct target of FoxO3a. By knocking down FoxO3a we could successfully block the up‐regulation of the pro‐apoptotic protein PUMA in this model. Recently, it has been reported that chromatin remodeler SWItch/sucrose non‐fermentable binds to FOXO and activates transcription. We found that Brg‐associated factor 57 (BAF57), a subclass of SWItch/sucrose non‐fermentable is up‐regulated and play a necessary role in neuron death induced by 6‐OHDA. Moreover, it is required for induction of PUMA by FoxO3a in this cellular model of PD. Taken together, our study suggest that FoxO3a is activated, translocates to nucleus, induces its pro‐apoptotic target PUMA in the presence of chromatin remodeler BAF57 to execute neuron death in cellular models of PD.
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Cancer continues to be one of the biggest threats to the human civilization because there is no cure of it. Small heterocyclic molecule with low molecular weight and novel structural feature is therapeutically highly demanding. These molecules have the capability to disrupt signaling pathways leading to anticancer activities. Therefore, the search for new anticancer agents continues to draw attention to the research community. In this study, a small triazolo-benzoxazepine scaffolds was synthesized using a one-pot four-step synthetic methodology involving click reaction. Small libraries of 12 compounds were successfully synthesized and screened them against different cancer cell lines. Low micromolar anticancer activity was recorded using MTT assay, and further confirmation of cell death was obtained by phase contrast, fluorescent, and confocal images.
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