Rottlerin Enhances the Autophagic Degradation of Phosphorylated Tau in Neuronal Cells.

Intra-neuronal accumulation of hyper-phosphorylated tau as neurofibrillary tangles (NFT) is a hallmark of Alzheimer's disease (AD). To prevent the aggregation of phosphorylated tau in neurons, decreasing the phosphorylated tau protein levels is important. Here, we examined the biological effects of rottlerin, a phytochemical compound extracted from the Kamala tree, Mallotus philippinensis, on phosphorylated tau levels. Notably, rottlerin decreased the levels of intracellular phosphorylated and total tau. A marked increase in the LC3-II, a hallmark of autophagy, was observed in these cells, indicating that rottlerin strongly induced autophagy. Interestingly, rottlerin induced the phosphorylation of Raptor at S792 through the activation of adenosine-monophosphate activated-protein kinase (AMPK), which likely inhibits the mammalian target of rapamycin complex 1 (mTORC1), thus resulting in the activation of transcription factor EB (TFEB), a master regulator of autophagy. In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) activity increased in the presence of rottlerin. The decrease of phosphorylated tau levels in the presence of rottlerin was ameliorated by the knockdown of TFEB and partially attenuated by the knockout of the Nrf2 gene. Taken together, rottlerin likely enhances the degradation of phosphorylated tau through autophagy activated by TFEB and Nrf2. Thus, our results suggest that a natural compound rottlerin could be used as a preventive and therapeutic drug for AD.

Peroxiredoxin 2 Ameliorates AßO-Mediated Autophagy by Inhibiting ROS via the ROS-NRF2-p62 Pathway in N2a-APP Swedish Cells.

In Alzheimer's disease, reactive oxygen species (ROS) are generated by the deposition of amyloid-beta oligomers (AßOs), which represent one of the important causes of neuronal cell death. Additionally, AßOs are known to induce autophagy via ROS induction. Previous studies have shown that autophagy upregulation aggravates neuronal cell death. In this study, the effects of peroxiredoxin 2 (Prx2), a member of the peroxidase family of antioxidant enzymes, on regulating AßO-mediated autophagy were investigated. Prx2 decreased AßO-mediated oxidative stress and autophagy in N2a-APPswe cells. Further, we examined the relationship between the neuronal protective effect of Prx2 and a decrease in autophagy. Similar to the effects of N-acetyl cysteine, Prx2 decreased AßO-induced ROS and inhibited p62 protein expression levels by downregulating the activation of NRF2 and its translocation to the nucleus. In addition, treatment with 3-methyladenine, an autophagy inhibitor, ameliorates neuronal cell death. Overall, these results demonstrate that the Prx2-induced decrease in autophagy was associated with the inhibition of ROS via the ROS-NRF2-p62 pathway in N2a-APPswe cells. Therefore, our results revealed that Prx2 is a potential therapeutic target in anti-Alzheimer therapy.

Amyloid beta oligomers-induced parkin aggravates ER stress-mediated cell death through a positive feedback loop.

Recently, Parkin has been reported to induce endoplasmic reticulum (ER) stress. In addition, amyloid beta oligomers (AßO), hallmarks of Alzheimer's disease (AD), also increase ER stress in neurons. Because a mutation in the Parkin gene is a well-known predominant cause of familial Parkinson's disease (PD), Parkin has been well studied in PD but has not been well researched in AD. In this study, we investigated the role of AßO-mediated Parkin associated with ER stress in AD. For AD-based research, we used AßO treatments in mouse hippocampus-derived HT-22 cells. We stably expressed Parkin in HT-22 cells to confirm the hypothesis and used siParkin for downregulation of Parkin expression. Moreover, using hippocampi from amyloid precursor protein/presenilin 1/Tau triple transgenic mice (3xTg-AD mice), which are used for AD models, we confirmed the relationship between ER stress and Parkin in vivo. We observed that ATF4 upregulated AßO-increases in Parkin. Parkin overexpression aggravated ER stress in AßO-treated HT-22 cells and the hippocampi of 3xTg-AD mice. Parkin downregulation led to no significant change when compared to AßO-treated cells. Moreover, Parkin-mediated ER stress was not related to oxidative stress. Our study indicates that AßO-induced ATF4 upregulated Parkin levels and that Parkin increases ER stress as a positive feedback loop. Through this study, our findings provide a foundation for future studies on the specific mechanisms related to the role of Parkin in AD.

Amyloid-beta oligomers induce Parkin-mediated mitophagy by reducing Miro1.

Alzheimer's disease (AD) is a neurodegenerative disease associated with the accumulation of amyloid-beta oligomers (AßO). Recent studies have demonstrated that mitochondria-specific autophagy (mitophagy) contributes to mitochondrial quality control by selectively eliminating the dysfunctional mitochondria. Mitochondria motility, which is regulated by Miro1, is also associated with neuronal cell functions. However, the role played by Miro1 in the mitophagy mechanism, especially relative to AßO and neurodegenerative disorders, remains unknown. In this study, AßO induced mitochondrial dysfunction, enhanced Parkin-mediated mitophagy, and reduced mitochondrial quantities in hippocampal neuronal cells (HT-22 cells). We demonstrated that AßO-induced mitochondrial fragmentation could be rescued to the elongated mitochondrial form and that mitophagy could be mitigated by the stable overexpression of Miro1 or by pretreatment with N-acetylcysteine (NAC)-a reactive oxygen species (ROS) scavenger-as assessed by immunocytochemistry. Moreover, using time-lapse imaging, under live cell-conditions, we verified that mitochondrial motility was rescued by the Miro1 overexpression. Finally, in hippocampus from amyloid precursor protein (APP)/presenilin 1 (PS1)/Tau triple-transgenic mice, we noted that the co-localization between mitochondria and LC3B puncta was increased. Taken together, these results indicated that up-regulated ROS, induced by AßO, increased the degree of mitophagy and decreased the Miro1 expression levels. In contrast, the Miro1 overexpression ameliorated AßO-mediated mitophagy and increased the mitochondrial motility. In AD model mice, AßO induced mitophagy in the hippocampus. Thus, our results would improve our understanding of the role of mitophagy in AD toward facilitating the development of novel therapeutic agents for the treatment of AßO-mediated diseases.

Streptozotocin Induces Alzheimer's Disease-Like Pathology in Hippocampal Neuronal Cells via CDK5/Drp1-Mediated Mitochondrial Fragmentation.

Aberrant brain insulin signaling plays a critical role in the pathology of Alzheimer's disease (AD). Mitochondrial dysfunction plays a role in the progression of AD, with excessive mitochondrial fission in the hippocampus being one of the pathological mechanisms of AD. However, the molecular mechanisms underlying the progression of AD and mitochondrial fragmentation induced by aberrant brain insulin signaling in the hippocampal neurons are poorly understood. Therefore, we investigated the molecular mechanistic signaling associated with mitochondrial dynamics using streptozotocin (STZ), a diabetogenic compound, in the hippocampus cell line, HT-22 cells. In this metabolic dysfunctional cellular model, hallmarks of AD such as neuronal apoptosis, synaptic loss, and tau hyper-phosphorylation are induced by STZ. We found that in the mitochondrial fission protein Drp1, phosphorylation is increased in STZ-treated HT-22 cells. We also determined that inhibition of mitochondrial fragmentation suppresses STZ-induced AD-like pathology. Furthermore, we found that phosphorylation of Drp1 was induced by CDK5, and inhibition of CDK5 suppresses STZ-induced mitochondrial fragmentation and AD-like pathology. Therefore, these findings indicate that mitochondrial morphology and functional regulation may be a strategy of potential therapeutic for treating abnormal metabolic functions associated with the pathogenesis of AD.

Peroxiredoxin 5 deficiency exacerbates iron overload-induced neuronal death via ER-mediated mitochondrial fission in mouse hippocampus.

Iron is an essential element for cellular functions, including those of neuronal cells. However, an imbalance of iron homeostasis, such as iron overload, has been observed in several neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Iron overload causes neuronal toxicity through mitochondrial fission, dysregulation of Ca2+, ER-stress, and ROS production. Nevertheless, the precise mechanisms between iron-induced oxidative stress and iron toxicity related to mitochondria and endoplasmic reticulum (ER) in vivo are not fully understood. Here, we demonstrate the role of peroxiredoxin 5 (Prx5) in iron overload-induced neurotoxicity using Prx5-deficient mice. Iron concentrations and ROS levels in mice fed a high iron diet were significantly higher in Prx5-/- mice than wildtype (WT) mice. Prx5 deficiency also exacerbated ER-stress and ER-mediated mitochondrial fission via Ca2+/calcineurin-mediated dephosphorylation of Drp1 at Serine 637. Moreover, immunoreactive levels of cleaved caspase3 in the CA3 region of the hippocampus were higher in iron-loaded Prx5-/- mice than WT mice. Furthermore, treatment with N-acetyl-cysteine, a reactive oxygen species (ROS) scavenger, attenuated iron overload-induced hippocampal damage by inhibiting ROS production, ER-stress, and mitochondrial fission in iron-loaded Prx5-/- mice. Therefore, we suggest that iron overload-induced oxidative stress and ER-mediated mitochondrial fission may be essential for understanding iron-mediated neuronal cell death in the hippocampus and that Prx5 may be useful as a novel therapeutic target in the treatment of iron overload-mediated diseases and neurodegenerative diseases.

Peroxiredoxin 4 ameliorates amyloid beta oligomer-mediated apoptosis by inhibiting ER-stress in HT-22 hippocampal neuron cells.

Alzheimer's disease (AD) is a neurodegenerative disorder caused by amyloid beta oligomers (AßO), which induce cell death by triggering oxidative stress and endoplasmic reticulum (ER) stress. Oxidative stress is regulated by antioxidant enzymes, including peroxiredoxins. Peroxiredoxins (Prx) are classified into six subtypes, based on their localization and cysteine residues, and protect cells by scavenging hydrogen peroxide (H2O2). Peroxiredoxin 4 (Prx4) is unique in being localized to the ER; however, whether Prx4 protects neuronal cells from AßO-induced toxicity remains unclear, although Prx4 expression is upregulated in AßO-induced oxidative stress and ER stress. In this study, we established HT-22 cells in which Prx4 was either overexpressed or silenced to investigate its role in AßO-induced toxicity. AßO-stimulation of HT-22 cells with overexpressed Prx4 caused decreases in both AßO-induced ROS and ER stress (followed by ER expansion). In contrast, AßO stimulation caused increases in both ROS and ER stress that were notably higher in HT-22 cells with silenced Prx4 expression than in HT-22 cells. Consequently, Prx4 overexpression decreased apoptotic cell death and ameliorated the AßO-induced increase in intracellular Ca2+. Therefore, we conclude that Prx4 has a protective effect against AßO-mediated oxidative stress, ER stress, and neuronal cell death. Furthermore, these results suggest that Prx4 may be a target for preventing AßO toxicity in AD. Graphical abstract .

Peroxiredoxin 5 prevents iron overload-induced neuronal death by inhibiting mitochondrial fragmentation and endoplasmic reticulum stress in mouse hippocampal HT-22 cells.

Iron is an essential element for neuronal as well as cellular functions. However, Iron overload has been known to cause neuronal toxicity through mitochondrial fission, dysregulation of Ca2+, endoplasmic reticulum (ER) stress, and reactive oxygen species (ROS) production. Nevertheless, the precise mechanisms of iron-induced oxidative stress and mitochondria- and ER-related iron toxicity in neuronal cells are not fully understood. In this study, we demonstrated that iron overload induces ROS production earlier in the ER than in the mitochondria, and peroxiredoxin 5 (Prx5), which is a kind of antioxidant induced by iron overload, prevents iron overload-induced mitochondrial fragmentation mediated by contact with ER and translocation of Drp1, by inhibiting ROS production and calcium/calcineurin pathway in HT-22 mouse hippocampal neuronal cells. Moreover, Prx5 also prevented iron overload-induced ER-stress and cleavage of caspase-3, which consequently attenuated neuronal cell death. Therefore, we suggested that iron overload induces oxidative stress in the ER earlier than in the mitochondria, thereby increasing ER stress and calcium levels, and consequently causing mitochondrial fragmentation and neuronal cell death. So we thought that this study is essential for understanding iron toxicity in neurons, and Prx5 may serve as a new therapeutic target to prevent iron overload-induced diseases and neurodegenerative disorders.

Peroxiredoxin 5 Decreases Beta-Amyloid-Mediated Cyclin-Dependent Kinase 5 Activation Through Regulation of Ca2+-Mediated Calpain Activation.

AIMS: Aberrant Cdk5 (cyclin-dependent kinase 5) and oxidative stress are crucial components of diverse neurodegenerative disorders, including Alzheimer's disease (AD). We previously reported that a change in peroxiredoxin (Prx) expression is associated with protection from neuronal death. The aim of the current study was to analyze the role of Prx in regulating Cdk5 activation in AD. RESULTS: We found that of the six Prx subtypes, Prx5 was increased the most in cellular (N2a-APPswe cells) model of AD. Prx5 in the brain of APP (amyloid precursor protein) transgenic mouse (Tg2576) was more increased than a nontransgenic mouse. We evaluated Prx5 function by using overexpression (Prx5-WT), a mutation in the catalytic residue (Prx5-C48S), and knockdown. Increased neuronal death and Cdk5 activation by amyloid beta oligomer (AßO) were rescued by Prx5-WT expression, but not by Prx5-C48S or Prx5 knockdown. Prx5 plays a role in Cdk5 regulation by inhibiting the conversion of p35 to p25, which is increased by AßO accumulation. Prx5 is also upregulated in both the cytosol and mitochondria and it protects cells from AßO-mediated oxidative stress by eliminating intracellular and mitochondrial reactive oxygen species. Moreover, Prx5 regulates Ca2+ and Ca2+-mediated calpain activation, which are key regulators of p35 cleavage to p25. Innovation and Conclusion: Our study represents the first demonstration that Prx5 induction is a key factor in the suppression of Cdk5-related neuronal death in AD and we show that it functions via regulation of Ca2+-mediated calpain activation. Antioxid. Redox Signal. 27, 715-726.