Aβ-induced Mitochondrial Dysfunction Research Articles

In vitro ADME, mouse pharmacokinetics of LD14b, and bioanalysis of a novel aβ 17β-HSD10 modulator for the treatment of Alzheimer’s disease

LD14b is an amyloid-β (Aβ) 17β-hydroxysteroid dehydrogenase type 10 (Aβ-17β-HSD10) protein-protein interaction modulator that shows promising in vitro and ex vivo activity to rescue Aβ-induced mitochondrial dysfunction, Aβ-induced toxicity, and Aβ-mediated inhibition of estradiol synthesis. The current study investigated in vitro human S9 fractions metabolic stability, apparent permeability, human and mouse plasma protein binding, in vivo pharmacokinetics, and tissue distribution in Balb/cJ mice. A fast (8-min), sensitive, reliable, and reproducible LC-MS/MS method was developed and validated over the dynamic range of 1-1000 ng/mL for the quantification of LD14b in different biological matrices (plasma, liver, kidney, brain, lungs, heart). LD14b was metabolically stable in human liver S9 fractions with 70% remaining after 90 minutes of incubation, showed intermediate apparent permeability of 3.55 × 10−06 cm/s and 6.16 × 10−06 cm/s for apical-to-basolateral (A-to-B) and basolateral-to-apical (B-to-A), respectively across the Caco-2 monolayer, and was medium/highly bound to human plasma proteins (84.1%), mouse plasma proteins (85.7%), and mouse brain homogenate (95.4%). LD14b showed an in vivo predicted % absorption of 52% in Balb/cJ mice and was well-distributed to the peripheral tissues (liver, kidney, lungs, and heart) including the brain.

N1-methylation of adenosine (m1A) in ND5 mRNA leads to complex I dysfunction in Alzheimer’s disease

One mechanism of particular interest to regulate mRNA fate post-transcriptionally is mRNA modification. Especially the extent of m1A mRNA methylation is highly discussed due to methodological differences. However, one single m1A site in mitochondrial ND5 mRNA was unanimously reported by different groups. ND5 is a subunit of complex I of the respiratory chain. It is considered essential for the coupling of oxidation and proton transport. Here we demonstrate that this m1A site might be involved in the pathophysiology of Alzheimer’s disease (AD). One of the pathological hallmarks of this neurodegenerative disease is mitochondrial dysfunction, mainly induced by Amyloid β (Aβ). Aβ mainly disturbs functions of complex I and IV of the respiratory chain. However, the molecular mechanism of complex I dysfunction is still not fully understood. We found enhanced m1A methylation of ND5 mRNA in an AD cell model as well as in AD patients. Formation of this m1A methylation is catalyzed by increased TRMT10C protein levels, leading to translation repression of ND5. As a consequence, here demonstrated for the first time, TRMT10C induced m1A methylation of ND5 mRNA leads to mitochondrial dysfunction. Our findings suggest that this newly identified mechanism might be involved in Aβ-induced mitochondrial dysfunction.

The neuroprotective N-terminal amyloid-β core hexapeptide reverses reactive gliosis and gliotoxicity in Alzheimer’s disease pathology models

BackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by accumulation of extracellular amyloid beta (Aβ) and intracellular neurofibrillary tangles, leading to chronic activation of astrocytes and microglia and persistent neuroinflammation. Aβ-linked activation of microglia and astrocytes leads to increased intracellular calcium and production of proinflammatory cytokines, impacting the progression of neurodegeneration. An N-terminal Aβ fragment (Aβ1–15) and a shorter hexapeptide core sequence within the N-Aβ fragment (N-Aβcore: Aβ10–15) have previously been shown to protect against Aβ-induced mitochondrial dysfunction, oxidative stress and apoptosis in neurons and rescue synaptic and spatial memory deficits in an APP/PSEN1 mouse model. Here, we hypothesized that the N-Aβ fragment and N-Aβcore are protective against Aβ-induced gliotoxicity, promoting a neuroprotective environment and potentially alleviating the characteristically persistent neuroinflammation present in AD.MethodsWe treated ex vivo organotypic brain slice cultures from an aged familial AD mouse model, 5xFAD, with the N-Aβcore and used immunocytochemistry to assess the impact on astrogliosis and microgliosis and alterations in synaptophysin-positive puncta engulfed by microglia. Isolated neuron/glia cultures, mixed glial cultures or a microglial cell line were treated with oligomeric human Aβ at concentrations mimicking the pathogenic concentrations (μM) observed in AD in the absence or presence of the non-toxic N-terminal Aβ fragments. Resultant changes in synaptic density, gliosis, oxidative stress, mitochondrial dysfunction, apoptosis, and the expression and release of proinflammatory markers were then determined.ResultsWe demonstrate that the N-terminal Aβ fragments mitigated the phenotypic switch leading to astrogliosis and microgliosis induced by pathological concentrations of Aβ in mixed glial cultures and organotypic brain slice cultures from the transgenic 5xFAD mouse model, while protecting against Aβ-induced oxidative stress, mitochondrial dysfunction and apoptosis in isolated astrocytes and microglia. Moreover, the addition of the N-Aβcore attenuated the expression and release of proinflammatory mediators in microglial cells activated by Aβ and rescued microglia-mediated loss of synaptic elements induced by pathological levels of Aβ.ConclusionsTogether, these findings indicate the protective functions of the N-terminal Aβ fragments extend to reactive gliosis and gliotoxicity induced by Aβ, by preventing or reversing glial reactive states indicative of neuroinflammation and synaptic loss central to AD pathogenesis.

Willin/FRMD6 Mediates Mitochondrial Dysfunction Relevant to Neuronal Aβ Toxicity.

Willin/FRMD6 has been reported as a potential Alzheimer’s disease (AD) risk gene in a series of genome-wide association and neuroimaging studies; however, the mechanisms underlying its potential role in AD pathogenesis remain unknown. Here, we demonstrate the direct effects of Aβ on Willin/FRMD6 expression and position mitochondrial oxidative stress as a novel potential mechanism underlying the role of Willin/FRMD6 in AD pathogenesis. Specifically, using mouse hippocampal HT-22 cells and primary mouse neurons, we show that Aβ induces downregulation of Willin/FRMD6 protein expression. Furthermore, we demonstrate that Willin/FRMD6 knockdown leads to mitochondrial dysfunction and fragmentation, as well as upregulation of ERK1/2 signaling, both of which are reported to be key early features of AD pathogenesis. Importantly, increasing Willin/FRMD6 expression was able to rescue Aβ-induced abnormalities in mitochondrial morphology, function, and energetics. Thus, enhancing Willin/FRMD6 expression holds potential as a therapeutic strategy for protecting against Aβ-induced mitochondrial and neuronal dysfunction.

Activation of β2-adrenergic Receptor Ameliorates Amyloid-β-induced Mitophagy Defects and Tau Pathology in Mice

Defective mitophagy and mitochondrial dysfunction have been linked to aging and Alzheimer’s disease (AD). β2-Adrenergic receptor (ADRB2) is critical for mitochondrial and cognitive function. However, researchers have not clearly determined whether ADRB2 activation ameliorates defective mitophagy and cognitive deficits in individuals with AD. Here, we observed that the activation of ADRB2 by clenbuterol (Clen, ADRB2 agonist, 2 mg/kg/day) ameliorated amyloid-β-induced (Aβ1-42 bilateral intracerebral infusion, 2 μl, 5 μg/μl) memory deficits. Activation of ADRB2 also attenuated Aβ-induced mitochondrial dysfunction, as revealed by increased ATP levels, mitochondrial membrane potential (MMP/Δψm) and complex I activity. Further studies revealed that ADRB2 activation restored mitophagy deficits, as revealed by the increased light chain 3 (LC3)-II/LC3-I ratio, Atg5 levels, and Atg7 levels and decreased p62 levels, along with the upregulation of PTEN-induced putative kinase 1 (PINK1), Parkin and NAD+ levels. Activation of ADRB2 rescued Aβ-induced oxidative stress and neuronal death. ADRB2 activation also attenuated Aβ-induced tau hyperphosphorylation by regulating glycogen synthase kinase-3β expression in the hippocampus. Finally, we established that Clen improved mitophagy and attenuated mitochondrial dysfunction, and tau pathology in mice by activating the ADRB2/Akt/PINK1 signaling pathway. Conversely, the inhibition of ADRB2 by propranolol (βAR antagonist, 10 μM) blocked the Clen-mediated improvements in pathological changes in N2a cells. The results from the present study indicate that ADRB2 activation may be a therapeutic strategy for AD.

Aβ-induced mitochondrial dysfunction in neural progenitors controls KDM5A to influence neuronal differentiation

Mitochondria in neural progenitors play a crucial role in adult hippocampal neurogenesis by being involved in fate decisions for differentiation. However, the molecular mechanisms by which mitochondria are related to the genetic regulation of neuronal differentiation in neural progenitors are poorly understood. Here, we show that mitochondrial dysfunction induced by amyloid-beta (Aβ) in neural progenitors inhibits neuronal differentiation but has no effect on the neural progenitor stage. In line with the phenotypes shown in Alzheimer’s disease (AD) model mice, Aβ-induced mitochondrial damage in neural progenitors results in deficits in adult hippocampal neurogenesis and cognitive function. Based on hippocampal proteome changes after mitochondrial damage in neural progenitors identified through proteomic analysis, we found that lysine demethylase 5A (KDM5A) in neural progenitors epigenetically suppresses differentiation in response to mitochondrial damage. Mitochondrial damage characteristically causes KDM5A degradation in neural progenitors. Since KDM5A also binds to and activates neuronal genes involved in the early stage of differentiation, functional inhibition of KDM5A consequently inhibits adult hippocampal neurogenesis. We suggest that mitochondria in neural progenitors serve as the checkpoint for neuronal differentiation via KDM5A. Our findings not only reveal a cell-type-specific role of mitochondria but also suggest a new role of KDM5A in neural progenitors as a mediator of retrograde signaling from mitochondria to the nucleus, reflecting the mitochondrial status.

Functionalized Allopurinols Targeting Amyloid-Binding Alcohol Dehydrogenase Rescue Aβ-Induced Mitochondrial Dysfunction.

Alzheimer's disease (AD) is the most common dementia affecting one in nine people over 65. Only a handful of small-molecule drugs and the anti-β amyloid (Aβ) antibody aducanumab are approved to treat AD. However, they only serve to reduce symptoms of advanced disease. Novel treatments administered early in disease progression before the accumulation of Aβ and tau reaches the threshold where neuroinflammation is triggered and irreversible neuronal damage occurs are more likely to provide effective therapy. There is a growing body of evidence implying that mitochondrial dysfunction occurs at an early stage of AD pathology. The mitochondrial enzyme amyloid-binding alcohol dehydrogenase (ABAD) binds to Aβ potentiating toxicity. Moreover, ABAD has been shown to be overexpressed in the same areas of the brain most affected by AD. Inhibiting the Aβ-ABAD protein-protein interaction without adversely affecting normal enzyme turnover is hypothesized to be a potential treatment strategy for AD. Herein, we conduct structure-activity relationship studies across a series of functionalized allopurinol derivatives to determine their ability to inhibit Aβ-mediated reduction of estradiol production from ABAD. The lead compound resulting from these studies possesses potent activity with no toxicity up to 100 μM, and demonstrates an ability to rescue defective mitochondrial metabolism in human SH-SY5Y cells and rescue both defective mitochondrial metabolism and morphology ex vivo in primary 5XFAD AD mouse model neurons.

IRE1α-XBP1 Affects the Mitochondrial Function of Aβ25-35-Treated SH-SY5Y Cells by Regulating Mitochondria-Associated Endoplasmic Reticulum Membranes.

Background: Neurotoxicity induced by the amyloid beta (Aβ) peptide is one of the most important pathological mechanisms of Alzheimer's disease (AD). Activation of the adaptive IRE1α-XBP1 pathway contributes to the pathogenesis of AD, making it a potential target for AD therapeutics. However, the mechanism of IRE1α-XBP1 pathway involvement in AD is unclear. We, therefore, investigated the effect of the IRE1α-XBP1 axis in an in vitro AD model and explored its potential mechanism.Methods: The human neuroblastoma cell line, SH-SY5Y, was used. Cells were treated with Aβ25–35, with or without 4μ8c, an inhibitor of IRE1α. Cells were collected and analyzed by Western blotting, quantitative real-time PCR, electron microscopy, fluorescence microscopy, calcium imaging, and other biochemical assays.Results: Aβ-exposed SH-SY5Y cells showed an increased expression of XBP1s and p-IRE1α. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and calcium imaging analysis showed that the IRE1α inhibitor, 4μ8c, reduced Aβ-induced cytotoxicity. Increased levels of ATP, restoration of mitochondrial membrane potential, and decreased production of mitochondrial reactive oxygen species after Aβ treatment in the presence of 4μ8c showed that inhibiting the IRE1α-XBP1 axis effectively mitigated Aβ-induced mitochondrial dysfunction in SH-SY5Y cells. Furthermore, Aβ treatment increased the expression and interaction of IP3R, Grp75, and vdac1 and led to an increased endoplasmic reticulum (ER)–mitochondria association, malfunction of mitochondria-associated ER-membranes (MAMs), and mitochondrial dysfunction. These deficits were rescued by inhibiting the IRE1α-XBP1 axis.Conclusion: These findings demonstrate that Aβ peptide induces the activation of the IRE1α-XBP1 axis, which may aggravate cytotoxicity and mitochondrial impairment in SH-SY5Y cells by targeting MAMs. Inhibition of the IRE1α-XBP1 axis provides the protection against Aβ-induced injury in SH-SY5Y cells and may, therefore, be a new treatment strategy.

Effect of AmyTrap, an amyloid-β binding drug, on Aβ induced mitochondrial dysfunction and tau phosphorylation in cultured neuroblastoma cells.

Alzheimer's Disease (AD) is the most common cause of dementia, affecting 25 million people worldwide. Accumulation of Amyloid-β (Aβ) in the mitochondria has been shown to adversely affect key enzymes including pyruvate dehydrogenase (PDH), succinate dehydrogenase (SDH), oxoglutarate dehydrogenase (OGDH). Accumulation of Aβ is also believed to increase Tau expression and pathology. Tau, in its toxic state, results in synaptic damage causing memory and cognitive dysfunction. We are developing a drug to treat AD namely AmyTrap. The active pharmacological ingredient is a retro inverso, Aβ-binding peptide which sequesters Aβ. We wanted to examine the effect of AmyTrap peptide on Aβ-induced mitochondrial dysfunction and Tau phosphorylation. Therefore, we treated SH-SY5Y neuroblastoma cells with wild-type Aβ, a mutant AβY10A, AmyTrap peptide (RI-peptide), or Aβ and RI-peptide for 72h. The mutant AβY10A is known to impact the self-aggregating property of Aβ as this Tyr10 is essential for self-aggregation. As expected, AβY10A reversed PDH, OGDH and SDH dysfunction to near normal levels. Further, AβY10A successfully reversed Tau phosphorylation, suggesting that Tyr10 is also associated with Aβ-induced cytotoxicity. RI-peptide was able to significantly reverse SDH dysfunction with limited effect on PDH and Tau phosphorylation. The findings are suggestive that the Tyr10 on Aβ plays a critical role in the self-aggregation. Further studies are warranted to expand these findings.

Treadmill Exercise Attenuates Aβ-Induced Mitochondrial Dysfunction and Enhances Mitophagy Activity in APP/PS1 Transgenic Mice.

Mitochondrial dysfunction is a hallmark of Alzheimer's disease (AD), which may be related to mitophagy failure. Previous reports suggest that treadmill exercise protects against mitochondrial dysfunction in AD. However, few studies have investigated the relationship between mitophagy and mitochondrial adaptation caused by treadmill exercise in AD. The current study aimed to investigate whether exercise-ameliorated AD is associated with changes in mitophagy activity. Both Wild-type and APP/PS1 transgenic mice were divided into sedentary (WTC and ADC) and exercise (WTE and ADE) groups (n = 9 for each group). WTE and ADE mice were subjected to treadmill exercise for 12weeks, followed by evaluating the effect of treadmill exercise on learning and memory ability, Aβ plaques, mitochondrial Aβ peptide level, synaptic activity and mitochondrial function. Meanwhile, mitophagy-related proteins PINK1, Parkin, LC3II and P62 were measured in the hippocampal mitochondrial fractions. The results indicated that exercise not only restored learning and memory ability, but also reduced Aβ plaque area, mitochondrial Aβ peptide level, and increased levels of synaptic markers SYN and GAP43, as well as reversed mitochondrial dysfunction (defective mitochondrial ultrastructure, decreased PGC-1α, TFAM and ATP levels) in APP/PS1 transgenic mice. Moreover, exercise increased mitophagy activity as evidenced by a significant decrease in levels of P62 and PINK1 as well as an increase in levels of LC3II and Parkin in ADE mice. These findings suggest that treadmill exercise can enhance mitophagy activity in the hippocampus, which is efficient in ameliorating pathological phenotypes of APP/PS1 transgenic mice.

Parkin overexpression attenuates Aβ-induced mitochondrial dysfunction in HEK293 cells by restoring impaired mitophagy

AimsMitochondrial dysfunction is an early prominent feature of Alzheimer's disease (AD). In the present study, we sought to investigate whether defective mitophagy is tightly related to amyloid-β (Aβ)-induced mitochondrial dysfunction. Main methodsImmunofluorescence, western blot and transmission electron microscopy were used to examine mitophagy. Mitochondrial membrane potential was assessed using the JC-1 dye. Mitochondrial ROS was detected using MitoSOX™ Red staining. Key findingsAβ induced mitochondrial dysfunction in HEK293 cells. Moreover, Aβ induced an increase in parkin translocation to mitochondria and led to a drastic reduction in cytosolic parkin. Furthermore, Aβ-treated cells displayed a microtubule-associated protein 1 light chain 3 (LC3) punctate pattern and elevated mitochondrial LC3-II levels, suggesting the upregulation of mitophagy. Notably, Aβ induced the accumulation of mitochondrial p62, which was associated with impaired mitophagy. In addition, Aβ-treated cells exhibited fragmented or swollen mitochondria with severely decreased cristae. We then investigated whether overexpression of parkin could protect cells against Aβ-induced mitochondrial dysfunction. Interestingly, parkin overexpression inhibited Aβ-induced mitochondrial dysfunction. Besides, parkin overexpression increased cytosolic and mitochondrial parkin levels as well as mitochondrial LC3-II levels in Aβ-treated cells. Additionally, parkin overexpression reversed the accumulation of p62 in mitochondria, indicating that parkin overexpression restored impaired mitophagy in Aβ-treated cells. Importantly, parkin overexpression remarkably reversed Aβ-induced mitochondrial fragmentation. SignificanceOur data demonstrate that overexpression of parkin ameliorates impaired mitophagy and promotes the removal of damaged mitochondria in Aβ-treated cells, indicating that upregulation of parkin-mediated mitophagy may be a potential strategy for the therapy of AD.

Metabolic stress is a primary pathogenic event in transgenic Caenorhabditis elegans expressing pan-neuronal human amyloid beta.

Alzheimer's disease (AD) is the most common neurodegenerative disease affecting the elderly worldwide. Mitochondrial dysfunction has been proposed as a key event in the etiology of AD. We have previously modeled amyloid-beta (Aβ)-induced mitochondrial dysfunction in a transgenic Caenorhabditis elegans strain by expressing human Aβ peptide specifically in neurons (GRU102). Here, we focus on the deeper metabolic changes associated with this Aβ-induced mitochondrial dysfunction. Integrating metabolomics, transcriptomics and computational modeling, we identify alterations in Tricarboxylic Acid (TCA) cycle metabolism following even low-level Aβ expression. In particular, GRU102 showed reduced activity of a rate-limiting TCA cycle enzyme, alpha-ketoglutarate dehydrogenase. These defects were associated with elevation of protein carbonyl content specifically in mitochondria. Importantly, metabolic failure occurred before any significant increase in global protein aggregate was detectable. Treatment with an anti-diabetes drug, Metformin, reversed Aβ-induced metabolic defects, reduced protein aggregation and normalized lifespan of GRU102. Our results point to metabolic dysfunction as an early and causative event in Aβ-induced pathology and a promising target for intervention.

Red Ginseng Attenuates Aβ-Induced Mitochondrial Dysfunction and Aβ-mediated Pathology in an Animal Model of Alzheimer's Disease.

Alzheimer’s disease (AD) is the most common neurodegenerative disease and is characterized by neurodegeneration and cognitive deficits. Amyloid beta (Aβ) peptide is known to be a major cause of AD pathogenesis. However, recent studies have clarified that mitochondrial deficiency is also a mediator or trigger for AD development. Interestingly, red ginseng (RG) has been demonstrated to have beneficial effects on AD pathology. However, there is no evidence showing whether RG extract (RGE) can inhibit the mitochondrial deficit-mediated pathology in the experimental models of AD. The effects of RGE on Aβ-mediated mitochondrial deficiency were investigated in both HT22 mouse hippocampal neuronal cells and the brains of 5XFAD Aβ-overexpressing transgenic mice. To examine whether RGE can affect mitochondria-related pathology, we used immunohistostaining to study the effects of RGE on Aβ accumulation, neuroinflammation, neurodegeneration, and impaired adult hippocampal neurogenesis in hippocampal formation of 5XFAD mice. In vitro and in vivo findings indicated that RGE significantly improves Aβ-induced mitochondrial pathology. In addition, RGE significantly ameliorated AD-related pathology, such as Aβ deposition, gliosis, and neuronal loss, and deficits in adult hippocampal neurogenesis in brains with AD. Our results suggest that RGE may be a mitochondria-targeting agent for the treatment of AD.

Overexpression of endophilin A1 exacerbates synaptic alterations in a mouse model of Alzheimer\u2019s disease

Endophilin A1 (EP) is a protein enriched in synaptic terminals that has been linked to Alzheimer’s disease (AD). Previous in vitro studies have shown that EP can bind to a variety of proteins, which elicit changes in synaptic transmission of neurotransmitters and spine formation. Additionally, we previously showed that EP protein levels are elevated in AD patients and AD transgenic animal models. Here, we establish the in vivo consequences of upregulation of EP expression in amyloid-β peptide (Aβ)-rich environments, leading to changes in both long-term potentiation and learning and memory of transgenic animals. Specifically, increasing EP augmented cerebral Aβ accumulation. EP-mediated signal transduction via reactive oxygen species (ROS)/p38 mitogen-activated protein (MAP) kinase contributes to Aβ-induced mitochondrial dysfunction, synaptic injury, and cognitive decline, which could be rescued by blocking either ROS or p38 MAP kinase activity.

Synthesis and evaluation of 2-(3-arylureido)pyridines and 2-(3-arylureido)pyrazines as potential modulators of Aβ-induced mitochondrial dysfunction in Alzheimer's disease

A series of 2-(3-arylureido)pyridines and 2-(3-benzylureido)pyridines were synthesized and evaluated as potential modulators for amyloid beta (Aβ)-induced mitochondrial dysfunction in Alzheimer's disease (AD). The blocking activities of forty one small molecules against Aβ-induced mitochondrial permeability transition pore (mPTP) opening were evaluated by JC-1 assay which measures the change of mitochondrial membrane potential (ΔΨm). The inhibitory activity of twenty five compounds against Aβ-induced mPTP opening was superior to that of the standard cyclosporin A (CsA). Six hit compounds have been identified as likely safe in regards to mitochondrial and cellular safety and subjected to assessment for their protective effect against Aβ-induced deterioration of ATP production and cytotoxicity. Among them, compound 7fb has been identified as a lead compound protecting neuronal cells against 67% of neurocytotoxicity and 43% of suppression of mitochondrial ATP production induced by 5 μM concentrations of Aβ. Using CDocker algorithm, a molecular docking model presented a plausible binding mode for these compounds with cyclophilin D (CypD) receptor as a major component of mPTP. Hence, this report presents compound 7fb as a new nonpeptidyl mPTP blocker which would be promising for further development of Alzheimer's disease (AD) therapeutics.

Discovery of non-peptidic small molecule inhibitors of cyclophilin D as neuroprotective agents in Aβ-induced mitochondrial dysfunction.

Cyclophilin D (CypD) is a mitochondria-specific cyclophilin that is known to play a pivotal role in the formation of the mitochondrial permeability transition pore (mPTP).The formation and opening of the mPTP disrupt mitochondrial homeostasis, cause mitochondrial dysfunction and eventually lead to cell death. Several recent studies have found that CypD promotes the formation of the mPTP upon binding to β amyloid (Aβ) peptides inside brain mitochondria, suggesting that neuronal CypD has a potential to be a promising therapeutic target for Alzheimer's disease (AD). In this study, we generated an energy-based pharmacophore model by using the crystal structure of CypD-cyclosporine A (CsA) complex and performed virtual screening of ChemDiv database, which yielded forty-five potential hit compounds with novel scaffolds. We further tested those compounds using mitochondrial functional assays in neuronal cells and identified fifteen compounds with excellent protective effects against Aβ-induced mitochondrial dysfunction. To validate whether these effects derived from binding to CypD, we performed surface plasmon resonance (SPR)-based direct binding assays with selected compounds and discovered compound 29 was found to have the equilibrium dissociation constants (KD) value of 88.2nM. This binding affinity value and biological activity correspond well with our predicted binding mode. We believe that this study offers new insights into the rational design of small molecule CypD inhibitors, and provides a promising lead for future therapeutic development.

Cofilin-mediated Neuronal Apoptosis via p53 Translocation and PLD1 Regulation

Amyloid β (Aβ) accumulation is an early event in the pathogenesis of Alzheimer’s disease (AD), leading to mitochondrial and synaptic dysfunction, tau accumulation, and eventual neuronal death. While the p53 apoptotic pathway has clearly been associated with Aβ deposits and neuronal apoptosis, the critical upstream factors contributing to p53 activation in AD are not well understood. We have previously shown that cofilin activation plays a pivotal role in Aβ-induced mitochondrial and synaptic dysfunction. In this study, we show that activated cofilin (S3A) preferentially forms a complex with p53 and promotes its mitochondrial and nuclear localization, resulting in transcription of p53-responsive genes and promotion of apoptosis. Conversely, reduction of endogenous cofilin by knockdown or genetic deficiency inhibits mitochondrial and nuclear translocation of p53 in cultured cells and in APP/PS1 mice. This cofilin-p53 pro-apoptotic pathway is subject to negative regulation by PLD1 thorough cofilin inactivation and inhibition of cofilin/p53 complex formation. Finally, activated cofilin is unable to induce apoptosis in cells genetically lacking p53. These findings taken together indicate that cofilin coopts and requires the nuclear and mitochondrial pro-apoptotic p53 program to induce and execute apoptosis, while PLD1 functions in a regulatory multi-brake capacity in this pathway.

Mitochondrial FOXO3a is involved in amyloid β peptide-induced mitochondrial dysfunction.

Mitochondrial dysfunction is a hallmark of amyloid β peptide (Aβ)-induced neuronal toxicity in Alzheimer's disease (AD). However, the precise mechanism(s) of Aβ-induced mitochondrial dysfunction has not been fully understood. There is evidence that Forkhead box O3a (FOXO3a) is normally present in neuronal mitochondria. Using HT22 murine hippocampal neuronal cells and primary hippocampal neurons, the present study investigated whether mitochondrial FOXO3a was involved in mitochondrial dysfunction induced by Aβ. It was found that Aβ induced dephosphorylation and mitochondrial translocation of FOXO3a. In addition, Aβ enhanced association of FOXO3a with mitochondrial DNA (mtDNA), causing a decrease in the expression of cytochrome c oxidase subunit 1 (COX1) and the activity of COX. In addition, Aβ-induced mitochondrial dysfunction, indicated by the decrease in 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion, mitochondrial adenosine triphosphate (ATP) production and COX activity, could be suppressed by knockdown of FOXO3a (FOXO3a-KD). These results provide new insights into the mechanism underlying Aβ-induced neurotoxicity and open up new therapeutic perspectives for AD.

Identification of a Small Molecule Cyclophilin D Inhibitor for Rescuing Aβ-Mediated Mitochondrial Dysfunction

Cyclophilin D (CypD), a peptidylprolyl isomerase F (PPIase), plays a central role in opening the mitochondrial membrane permeability transition pore leading to cell death. CypD resides in the mitochondrial matrix, associates with the inner mitochondrial membrane, interacts with amyloid beta to exacerbate mitochondrial and neuronal stress and has been linked to Alzheimer's disease (AD). We report the biological activity of a small-molecule CypD inhibitor (C-9), which binds strongly to CypD and attenuates mitochondrial and cellular perturbation insulted by Aβ and calcium stress. Binding affinities for C-9 were determined using in vitro surface plasmon resonance. This compound antagonized calcium-mediated mitochondrial swelling, abolished Aβ-induced mitochondrial dysfunction as shown by increased cytochrome c oxidase activity and adenosine-5'-triphosphate levels, and inhibited CypD PPIase enzymatic activity by real-time fluorescence capture assay using Hamamatsu FDSS 7000. Compound C-9 seems a good candidate for further investigation as an AD drug.

Protective Effects of Ramie (Boehmeria nivea) against Oxidative Stress in C6 Glial Cells

β amyloid protein (Aβ) plays a critical role in the pathogenesis of Alzheimers disease (AD) and possibly in Aβ-induced mitochondrial dysfunction and oxidative stress. Aβ can directly cause reactive oxygen species (ROS) production. Overproduction of ROS is considered to be involved in the pathogenesis of neurodegeneration of AD. Here, we investigated 9 kinds of ramie (Boehmeria nivea, (L.) Gaud., BN; hereafter denoted as BN) for their protective action against oxidative stress in a cellular system using C6 glial cells. We observed loss of cell viability and high levels of ROS generation after treatment with hydrogen peroxide (H2O2) and Aβ25-35. However, treatments with BN extracts led to an increase in cell viability and decrease in ROS production induced by H2O2 and Aβ 25 - 35 . In particular, the extracts of BN-01 (seobang variety from Seocheon) and BN-09 (local variety from Yeonggwang) showed excellent anti-oxidative properties. This indicates that BN extracts could prevent neurodegeneration by reducing oxidative stress in cells.