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Aging is a strong risk factor for brain dementia and cognitive decline. Age-related accumulation of metabolites such as advanced glycation end products (AGEs) could serve as danger signals to initiate and accelerate disease process and neurodegeneration. The underlying causes and consequences of cerebral AGEs accumulation remain largely unknown. Here, we comprehensively investigate age-related accumulation of AGEs and dicarbonyls, including methylglyoxal (MG), glyoxal (GO), and 3-deoxyglucosone (3-DG), and the effects of mitochondrial reactive oxygen species (ROS) on cerebral AGEs accumulation, mitochondrial function, and oxidative stress in the aging human and mouse brain. We demonstrate that AGEs, including arginine and lysine derived N(6)-carboxymethyl lysine (CML), Nε-(1-Carboxyethyl)-l-lysine (CEL), and methylglyoxal-derived hydro-imidazolone-1 (MG-H1), were significantly elevated in the cerebral cortex and hippocampus with advanced age in mice. Accordingly, aging mouse and human brains revealed decrease in activities of mitochondrial respiratory chain complexes I & IV and ATP levels, and increased ROS. Notably, administration of mitoTEMPO (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mTEMPO), a scavenger of mitochondrial ROS, not only suppressed ROS production but also reduced aged-induced accumulation of AGEs and dicarbonyls. mTEMPO treatment improved mitochondrial respiratory function and restored ATP levels. Our findings provide evidence linking age-related accumulation of toxic metabolites (AGEs) to mitochondrial oxidative stress. This highlights a novel mechanism by which AGEs-dependent signaling promotes carbonyl stress and sustained mitochondrial dysfunction. Eliminating formation and accumulation of AGEs may represent a new therapeutic avenue for combating cognitive decline and mitochondrial degeneration relevant to aging and neurodegenerative diseases including Alzheimer’s disease.

Tau oligomers (oTau) are thought to precede neurofibrillary tangle formation and likely represent one of the toxic species in disease. This study addresses whether mitochondrial reactive oxygen species (ROS) contribute to tau oligomer accumulation. First, we determined whether elevated oxidative stress correlates with aggregation of tau oligomers in the brain and platelets of human Alzheimer’s disease (AD) patient, tauopathy mice, primary cortical neurons from tau mice and human trans-mitochondrial ‘cybrid’ (cytoplasmic hybrid) neuronal cells, whose mitochondria are derived from platelets of patients with sporadic AD- or mild cognitive impairment (MCI)-derived mitochondria. Increased formation of tau oligomers correlates with elevated ROS levels in the hippocampi of AD patients and tauopathy mice, AD- and MCI-derived mitochondria and AD and MCI cybrid cells. Furthermore, scavenging ROS by application of mito-TEMPO/2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride, a mitochondria-targeted antioxidant, not only inhibits the generation of mitochondrial ROS and rescues mitochondrial respiratory function but also robustly suppresses tau oligomer accumulation in MCI and AD cybrids as well as cortical neurons from tau mice. These studies provide substantial evidence that mitochondria-mediated oxidative stress contributes to tau oligomer formation and accumulation.

Background: Mitochondrial dysfunction, bioenergetic deficit, and extensive oxidative stress underlie neuronal perturbation during the early stage of Alzheimer’s disease (AD). Previously, we demonstrated that decreased PTEN-induced putative kinase 1 (PINK1) expression is associated with AD pathology in AD-affected human brains and AD mice. Objective: In the present study, we highlight the essential role of PINK1 in AD-relevant mitochondrial perturbation and neuronal malfunction. Methods: Using trans-mitochondrial “cybrid” (cytoplasmic hybrid) neuronal cells, whose mitochondria are transferred from platelets of patients with sporadic AD, we observed the effect of PINK1 in neuronal-like differentiation and synaptogenesis and mitochondrial functions. Results: In AD cybrid cells, the downregulation of PINK1 is correlated to the alterations in mitochondrial morphology and function and deficit in neuronal-like differentiation. Restoring/increasing PINK1 by lentivirus transduction of PINK1 robustly attenuates mitochondrial defects and rescues neurite-like outgrowth. Importantly, defective PINK1 kinase activity fails to reverse these detrimental effects. Mechanistically, AD cybrid cells reveal a significant decrease in PINK1-dependent phosphorylated mitofusin (Mfn) 2, a key mitochondrial membrane protein that participates in mitochondrial fusion, and an insufficient autophagic activity for the clearance of dysfunctional mitochondria. Overexpression of PINK1, but not mutant PINK1 elevates phosphorylation of Mfn2 and autophagy signaling LC3-II. Accordingly, PINK1-overexpressed AD cybrids exhibit increases in mitochondrial length and density and suppressed reactive oxygen species. These results imply that activation of PINK1 protects against AD-affected mitochondrial dysfunction and impairment in neuronal maturation and differentiation. Conclusion: PINK1-mediated mitophagy is important for maintaining mitochondrial health by clearance of dysfunctional mitochondria and therefore, improves energy homeostasis in AD.

Mitochondrial dysfunction is one of the early pathological features of Alzheimer's disease (AD). Accumulation of cerebral and mitochondrial Aβ links to mitochondrial and synaptic toxicity. We have previously demonstrated the mechanism by which presequence peptidase (PITRM1)‐mediated clearance of mitochondrial Aβ contributes to mitochondrial and cerebral amyloid pathology and mitochondrial and synaptic stress in adult transgenic AD mice overexpressing Aβ up to 12 months old. Here, we investigate the effect of PITRM1 in an advanced age AD mouse model (up to 19–24 months) to address the fundamental unexplored question of whether restoration/gain of PITRM1 function protects against mitochondrial and synaptic dysfunction associated with Aβ accumulation and whether this protection is maintained even at later ages featuring profound amyloid pathology and synaptic failure. Using newly developed aged PITRM1/Aβ‐producing AD mice, we first uncovered reduction in PITRM1 expression in AD‐affected cortex of AD mice at 19–24 months of age. Increasing neuronal PITRM1 activity/expression re‐established mitochondrial respiration, suppressed reactive oxygen species, improved synaptic function, and reduced loss of synapses even at advanced ages (up to 19–24 months). Notably, loss of PITRM1 proteolytic activity resulted in Aβ accumulation and failure to rescue mitochondrial and synaptic function, suggesting that PITRM1 activity is required for the degradation and clearance of mitochondrial Aβ and Aβ deposition. These data indicate that augmenting PITRM1 function results in persistent life‐long protection against Aβ toxicity in an AD mouse model. Therefore, augmenting PITRM1 function may enhance Aβ clearance in mitochondria, thereby maintaining mitochondrial integrity and ultimately slowing the progression of AD., Restoration/gain of neuronal PITRM1 function by enhancing its proteolytic activity in the aged AD mouse model significantly promotes synaptic mitochondrial and cerebral Aβ degradation and clearance, and thereby improves mitochondrial and synaptic functions, and alleviates loss of synapses.

Background Advanced glycation end products (AGEs) are an important risk factor for the development of cognitive decline in aging and late-onset neurodegenerative diseases including Alzheimer's disease. However, whether and how dietary AGEs exacerbate cognitive impairment and brain mitochondrial dysfunction in the aging process remains largely unknown. Objective We investigated the direct effects of dietary AGEs on AGE adducts accumulation, mitochondrial function, and cognitive performance in mice. Methods Mice were fed the AGE+ diet or AGE- diet. We examined levels of AGE adducts in serum and cerebral cortexes by immunodetection and immunohistochemistry, determined levels of reactive oxygen species by biochemical analysis, detected enzyme activity associated with mitochondrial respiratory chain complexes I & IV and ATP levels, and assessed learning and memory ability by Morris Water Maze and nesting behavior. Results Levels of AGE adducts (MG-H1 and CEL) were robustly increased in the serum and brain of AGE+ diet fed mice compared to the AGE- group. Furthermore, greatly elevated levels of reactive oxygen species, decreased activities of mitochondrial respiratory chain complexes I & IV, reduced ATP levels, and impaired learning and memory were evident in AGE+ diet fed mice compared to the AGE- group. Conclusion These results indicate that dietary AGEs are important sources of AGE accumulation in vivo, resulting in mitochondrial dysfunction, impairment of energy metabolism, and subsequent cognitive impairment. Thus, reducing AGEs intake to lower accumulation of AGEs could hold therapeutic potential for the prevention and treatment of AGEs-induced mitochondrial dysfunction linked to cognitive decline.

Altered mitochondrial fitness is a potential triggering factor in Alzheimer’s disease (AD). Mitochondrial quality control pathways are dysfunctional and mitochondrially-derived reactive oxygen species (mROS) levels are increased in AD patient brains. However, the pathways responsible for dysregulated mROS accumulation have remained relatively unclear. In this study, we demonstrate that levels of ECSIT, a mitochondrial oxidative phosphorylation (OxPhos) complex I (CI)-associated protein, are reduced in AD-affected brains. Neuronal ECSIT downregulation increased mROS generation and impaired mitophagy of defective mitochondria. Consequently, decreasing neuronal ECSIT caused AD-like changes, including memory loss and neuropathology. In contrast, augmented neuronal expression of ECSIT protected against the development of an AD-like phenotype. Decreased levels of ECSIT in AD patient brains therefore likely contribute to oxidative stress, neuroinflammation and AD pathogenesis.

Receptor for Advanced Glycation End products (RAGE) has been implicated in amyloid β-peptide (Aβ)-induced perturbation relevant to the pathogenesis of Alzheimer's disease (AD). However, whether and how RAGE regulates Aβ metabolism remains largely unknown. Aβ formation arises from aberrant cleavage of amyloid pre-cursor protein (APP) by β- and γ-secretase. To investigate whether RAGE modulates β- and γ-secretase activity potentiating Aβ formation, we generated mAPP mice with genetic deletion of RAGE (mAPP/RO). These mice displayed reduced cerebral amyloid pathology, inhibited aberrant APP-Aβ metabolism by reducing β- and γ-secretases activity, and attenuated impairment of learning and memory compared with mAPP mice. Similarly, RAGE signal transduction deficient mAPP mice (mAPP/DN-RAGE) exhibited the reduction in Aβ40 and Aβ42 production and decreased β-and γ-secretase activity compared with mAPP mice. Furthermore, RAGE-deficient mAPP brain revealed suppression of activation of p38 MAP kinase and glycogen synthase kinase 3β (GSK3β). Finally, RAGE siRNA-mediated gene silencing or DN-RAGE-mediated signaling deficiency in the enriched human APP neuronal cells demonstrated suppression of activation of GSK3β, accompanied with reduction in Aβ levels and decrease in β- and γ-secretases activity. Our findings highlight that RAGE-dependent signaling pathway regulates β- and γ-secretase cleavage of APP to generate Aβ, at least in part through activation of GSK3β and p38 MAP kinase. RAGE is a potential therapeutic target to limit aberrant APP-Aβ metabolism in halting progression of AD.

Summary Astrocytes, the most populous glial cell type in the brain, are critical for regulating the brain microenvironment. In various neurodegenerative diseases, astrocytes determine the progression and outcome of the neuropathological process. We have recently revealed the direct involvement of mitochondrial function in human pluripotent stem cell (hiPSC)-derived dopaminergic (DA) neuronal differentiation. Using the astroglial-neuronal co-culture system, we show here that astrocytes effectively rescue defects in neurogenesis of DA neurons with mitochondrial respiratory chain disruption. Co-culture of astrocytes with defective DA neurons completely restored mitochondrial functions and dynamics insulted by mitochondrial toxins. These results suggest the significance of astroglia in maintaining mitochondrial development and bioenergetics during differentiation of hiPSC-derived DA neurons. Our study also provides an active astroglial-neuronal interaction model for future investigation of mitochondrial involvement in neurogenesis and neurodegenerative diseases., Highlights • Role of astrocyte on the development of hiPSC-derived dopaminergic neuron • Astrocyte protects dopaminergic neurogenesis by powering up mitochondrial respiration • Astrocyte restores mitochondrial function and dynamics in dopaminergic neuron • Evidence of astroglial and neuronal interaction during dopaminergic neurogenesis, In this article, Shirley ShiDu Yan and colleagues show that human pluripotent stem cell-derived astrocytes effectively rescue defects in neurogenesis of dopaminergic neurons with mitochondrial respiratory chain disruption. Co-culture with astrocytes restored mitochondrial functions and dynamics in dopaminergic neurons insulted by mitochondrial toxins. These results provide evidence of astroglia in maintaining mitochondrial development and bioenergetics during dopaminergic neuronal differentiation.

Mitochondrial abnormalities are well known to cause cognitive decline. However, the underlying molecular basis of mitochondria-associated neuronal and synaptic dysfunction in the diabetic brain remains unclear. Here, using a mitochondrial single-channel patch clamp and cyclophilin D (CypD)-deficient mice (Ppif −/−) with streptozotocin-induced diabetes, we observed an increase in the probability of Ca2+-induced mitochondrial permeability transition pore (mPTP) opening in brain mitochondria of diabetic mice, which was further confirmed by mitochondrial swelling and cytochrome c release induced by Ca2+ overload. Diabetes-induced elevation of CypD triggers enhancement of F1F0 ATP synthase–CypD interaction, which in turn leads to mPTP opening. Indeed, in patients with diabetes, brain cypD protein levels were increased. Notably, blockade of the F1F0 ATP synthase–CypD interaction by CypD ablation protected against diabetes-induced mPTP opening, ATP synthesis deficits, oxidative stress, and mitochondria dysfunction. Furthermore, the absence of CypD alleviated deficits in synaptic plasticity, learning, and memory in diabetic mice. Thus, blockade of ATP synthase interaction with CypD provides a promising new target for therapeutic intervention in diabetic encephalopathy.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized clinically by cognitive decline and memory loss. The pathological features are amyloid-β peptide (Aβ) plaques and intracellular neurofibrillary tangles. Many studies have suggested that oxidative damage induced by reactive oxygen species (ROS) is an important mechanism for AD progression. Our recent study demonstrated that oxidative stress could further impair mitochondrial function. In the present study, we adopted a transgenic mouse model of AD (mAPP, overexpressing AβPP/Aβ in neurons) and performed redox measurements using in vivo electron paramagnetic resonance (EPR) imaging with methoxycarbamyl-proxyl (MCP) as a redox-sensitive probe for studying oxidative stress in an early stage of pathology in a transgenic AD mouse model. Through assessing oxidative stress, mitochondrial function and cognitive behaviors of mAPP mice at the age of 8-9 months, we found that oxidative stress and mitochondrial dysfunction appeared in the early onset of AD. Increased ROS levels were associated with defects of mitochondrial and cognitive dysfunction. Notably, the in vivo EPR method offers a unique way of assessing tissue oxidative stress in living animals under noninvasive conditions, and thus holds a potential for early diagnosis and monitoring the progression of AD.

N-methyl d-aspartate receptor (NMDA) subunit 2B (NR2B)-containing NMDA receptors and mitochondrial protein cyclophilin D (CypD) are well characterized in mediating neuronal death after ischemia, respectively. However, whether and how NR2B and CypD work together in mediating synaptic injury after ischemia remains elusive. Using an ex vivo ischemia model of oxygen–glucose deprivation (OGD) in hippocampal slices, we identified a NR2B-dependent mechanism for CypD translocation onto the mitochondrial inner membrane. CypD depletion (CypD null mice) prevented OGD-induced impairment in synaptic transmission recovery. Overexpression of neuronal CypD mice (CypD+) exacerbated OGD-induced loss of synaptic transmission. Inhibition of CypD-dependent mitochondrial permeability transition pore (mPTP) opening by cyclosporine A (CSA) attenuated ischemia-induced synaptic perturbation in CypD+ and non-transgenic (non-Tg) mice. The treatment of antioxidant EUK134 to suppress mitochondrial oxidative stress rescued CypD-mediated synaptic dysfunction following OGD in CypD+ slices. Furthermore, OGD provoked the interaction of CypD with P53, which was enhanced in slices overexpressing CypD but was diminished in CypD-null slices. Inhibition of p53 using a specific inhibitor of p53 (pifithrin-μ) attenuated the CypD/p53 interaction following OGD, along with a restored synaptic transmission in both non-Tg and CypD+ hippocampal slices. Our results indicate that OGD-induced CypD translocation potentiates CypD/P53 interaction in a NR2B dependent manner, promoting oxidative stress and loss of synaptic transmission. We also evaluate a new ex vivo chronic OGD-induced ischemia model for studying the effect of oxidative stress on synaptic damage.

The mitochondrial permeability transition pore (mPTP) has been considered a key contributor to cell death, inducing the process in several major neurodegenerative diseases. To date, the molecular nature of the mPTP remains confounding but its significance is universally acknowledged. Several targets have been screened and inhibition of mPTP has emerged as an attractive field for researchers. Nowadays, in silico-directed studies help to explore new small molecules targeting the mPTP to improve their drug-like properties and bioactivity. Here, we briefly summarize the role of mPTP in neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson disease (PD), and Huntington's disease (HD), and discusses current and future potential therapeutic targets.

Mitochondrial and synaptic dysfunction is an early pathological feature of Alzheimer's disease (AD). Accumulation of amyloid beta-peptide (Aβ) in mitochondria, particularly in synaptic mitochondria, potentiates and amplifies synaptic injury and disruption of synaptic transmission, leading to synaptic dysfunction and ultimately to synaptic failure. Thus, determination of the presence and levels of Aβ in synaptic mitochondria associated with amyloid pathology is important for studying mitochondrial amyloid pathology. Here, we present a detailed methodology for the isolation of synaptic mitochondria from brain tissues and the determination of Aβ levels in the isolated mitochondria as well as ultrastructural localization of synaptic mitochondrial Aβ. These methods have been used successfully for the identification and characterization of Aβ accumulation in synaptic mitochondria from mouse brains derived from transgenic AD mouse model. Additionally, we comprehensively discuss the sample preparation, experimental details, our unique procedures, optimization of parameters, and troubleshooting.

Mitochondrial and synaptic dysfunction is an early pathological feature of Alzheimer's disease (AD). Accumulation of amyloid beta-peptide (Aβ) in mitochondria, particularly in synaptic mitochondria, potentiates and amplifies synaptic injury and disruption of synaptic transmission, leading to synaptic dysfunction and ultimately to synaptic failure. Thus, determination of the presence and levels of Aβ in synaptic mitochondria associated with amyloid pathology is important for studying mitochondrial amyloid pathology. Here, we present a detailed methodology for the isolation of synaptic mitochondria from brain tissues and the determination of Aβ levels in the isolated mitochondria as well as ultrastructural localization of synaptic mitochondrial Aβ. These methods have been used successfully for the identification and characterization of Aβ accumulation in synaptic mitochondria from mouse brains derived from transgenic AD mouse model. Additionally, we comprehensively discuss the sample preparation, experimental details, our unique procedures, optimization of parameters, and troubleshooting.

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., Endophilin A1 protein is known to be elevated in Alzheimer’s disease (AD). Here the authors show that endophilin A1 overexpression exacerbates synaptic deficits in a mouse model of AD.

Accumulation of amyloid-β (Aβ) in synaptic mitochondria is associated with mitochondrial and synaptic injury. The underlying mechanisms and strategies to eliminate Aβ and rescue mitochondrial and synaptic defects remain elusive. Presequence protease (PreP), a mitochondrial peptidasome, is a novel mitochondrial Aβ degrading enzyme. Here, we demonstrate for the first time that increased expression of active human PreP in cortical neurons attenuates Alzheimer disease's (AD)-like mitochondrial amyloid pathology and synaptic mitochondrial dysfunction, and suppresses mitochondrial oxidative stress. Notably, PreP-overexpressed AD mice show significant reduction in the production of proinflammatory mediators. Accordingly, increased neuronal PreP expression improves learning and memory and synaptic function in vivo AD mice, and alleviates Aβ-mediated reduction of long-term potentiation (LTP). Our results provide in vivo evidence that PreP may play an important role in maintaining mitochondrial integrity and function by clearance and degradation of mitochondrial Aβ along with the improvement in synaptic and behavioral function in AD mouse model. Thus, enhancing PreP activity/expression may be a new therapeutic avenue for treatment of AD.

The receptor for advanced glycation end products (RAGE)-mediated signaling pathway is related to Aβ-induced pathogenic responses. Geniposide, a pharmacologically active component purified from gardenia fruit, could attenuate the oligomeric Aβ(1-42)-induced inflammatory response by blocking the ligation of Aβ to RAGE and suppressing the RAGE-mediated signaling in vitro. Here, we investigated whether geniposide can exert protective effects on the neuroinflammation and memory deficits in an Alzheimer's disease (AD) mouse model. The results indicate that geniposide treatment significantly suppresses RAGE-dependent signaling (activation of ERK and IκB/NF-κB), the production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and cerebral Aβ accumulation in vivo. Furthermore, we demonstrate that geniposide augments synaptic plasticity by attenuating the Aβ-induced reduction of long-term potentiation and increasing the miniature excitatory postsynaptic current (mEPSC) amplitude and frequency in hippocampal neurons. In addition, the intragastric administration of geniposide improves learning and memory in APP/PS1 mice. Taken together, these studies indicate that geniposide has profound multifaceted neuroprotective effects in an AD mouse model. Geniposide demonstrates its neuroprotection by inhibiting inflammation, ameliorating amyloid pathology and improving cognition. Thus, geniposide may be a potential therapeutic agent for halting and preventing AD progression.

The coexistence of neuronal mitochondrial pathology and synaptic dysfunction is an early pathological feature of Alzheimer's disease (AD). Cyclophilin D (CypD), an integral part of mitochondrial permeability transition pore (mPTP), is involved in amyloid beta (Aβ)-instigated mitochondrial dysfunction. Blockade of CypD prevents Aβ-induced mitochondrial malfunction and the consequent cognitive impairments. Here, we showed the elimination of reactive oxygen species (ROS) by antioxidants probucol or superoxide dismutase (SOD)/catalase blocks Aβ-mediated inactivation of protein kinase A (PKA)/cAMP regulatory-element-binding (CREB) signal transduction pathway and loss of synapse, suggesting the detrimental effects of oxidative stress on neuronal PKA/CREB activity. Notably, neurons lacking CypD significantly attenuate Aβ-induced ROS. Consequently, CypD-deficient neurons are resistant to Aβ-disrupted PKA/CREB signaling by increased PKA activity, phosphorylation of PKA catalytic subunit (PKA C), and CREB. In parallel, lack of CypD protects neurons from Aβ-induced loss of synapses and synaptic dysfunction. Furthermore, compared to the mAPP mice, CypD-deficient mAPP mice reveal less inactivation of PKA–CREB activity and increased synaptic density, attenuate abnormalities in dendritic spine maturation, and improve spontaneous synaptic activity. These findings provide new insights into a mechanism in the crosstalk between the CypD-dependent mitochondrial oxidative stress and signaling cascade, leading to synaptic injury, functioning through the PKA/CREB signal transduction pathway.

Diabetes is considered to be a risk factor in Alzheimer’s disease (AD) pathogenesis. Although recent evidence indicates that diabetes exaggerates pathologic features of AD, the underlying mechanisms are not well understood. To determine whether mitochondrial perturbation is associated with the contribution of diabetes to AD progression, we characterized mouse models of streptozotocin (STZ)-induced type 1 diabetes and transgenic AD mouse models with diabetes. Brains from mice with STZ-induced diabetes revealed a significant increase of cyclophilin D (CypD) expression, reduced respiratory function, and decreased hippocampal long-term potentiation (LTP); these animals had impaired spatial learning and memory. Hyperglycemia exacerbated the upregulation of CypD, mitochondrial defects, synaptic injury, and cognitive dysfunction in the brains of transgenic AD mice overexpressing amyloid-β as shown by decreased mitochondrial respiratory complex I and IV enzyme activity and greatly decreased mitochondrial respiratory rate. Concomitantly, hippocampal LTP reduction and spatial learning and memory decline, two early pathologic indicators of AD, were enhanced in the brains of diabetic AD mice. Our results suggest that the synergistic interaction between effects of diabetes and AD on mitochondria may be responsible for brain dysfunction that is in common in both diabetes and AD.

Diabetes has adverse effects on the brain, especially the hippocampus, which is particularly susceptible to synaptic injury and cognitive dysfunction. The underlying mechanisms and strategies to rescue such injury and dysfunction are not well understood. Using a mouse model of type 2 diabetes (db/db mice) and a human neuronal cell line treated with high concentration of glucose, we demonstrate aberrant mitochondrial morphology, reduced ATP production, and impaired activity of complex I. These mitochondrial abnormalities are induced by imbalanced mitochondrial fusion and fission via a glycogen synthase kinase 3β (GSK3β)/dynamin-related protein-1 (Drp1)-dependent mechanism. Modulation of the Drp1 pathway or inhibition of GSK3β activity restores hippocampal long-term potentiation that is impaired in db/db mice. Our results point to a novel role for mitochondria in diabetes-induced synaptic impairment. Exploration of the mechanisms behind diabetes-induced synaptic deficit may provide a novel treatment for mitochondrial and synaptic injury in patients with diabetes.

Mild cognitive impairment (MCI) occurs during the predementia stage of Alzheimer disease (AD) and is characterized by a decline in cognitive abilities that frequently represents a transition between normal cognition and AD dementia. Its pathogenesis is not well understood. Here, we demonstrate the direct consequences and potential mechanisms of oxidative stress and mitochondrial dynamic and functional defects in MCI-derived mitochondria. Using a cytoplasmic hybrid (cybrid) cell model in which mitochondria from MCI or age-matched non-MCI subjects were incorporated into a human neuronal cell line depleted of endogenous mitochondrial DNA, we evaluated the mitochondrial dynamics and functions, as well as the role of oxidative stress in the resultant cybrid lines. We demonstrated that increased expression levels of mitofusin 2 (Mfn2) are markedly induced by oxidative stress in MCI-derived mitochondria along with aberrant mitochondrial functions. Inhibition of oxidative stress rescues MCI-impaired mitochondrial fusion/fission balance as shown by the suppression of Mfn2 expression, attenuation of abnormal mitochondrial morphology and distribution, and improvement in mitochondrial function. Furthermore, blockade of MCI-related stress-mediated activation of extracellular signal-regulated kinase (ERK) signaling not only attenuates aberrant mitochondrial morphology and function but also restores mitochondrial fission and fusion balance, in particular inhibition of overexpressed Mfn2. Our results provide new insights into the role of the oxidative stress-ERK-Mfn2 signal axis in MCI-related mitochondrial abnormalities, indicating that the MCI phase may be targetable for the development of new therapeutic approaches that improve mitochondrial function in age-related neurodegeneration.

Loss of synapse and synaptic dysfunction contribute importantly to cognitive impairment in Alzheimer's disease (AD). Mitochondrial dysfunction and oxidative stress are early pathological features in AD-affected brain. However, the effect of AD mitochondria on synaptogenesis remains to be determined. Using human trans-mitochondrial "cybrid" (cytoplasmic hybrid) neuronal cells whose mitochondria were transferred from platelets of patients with sporadic AD or age-matched non-AD subjects with relatively normal cognition, we provide the first evidence of mitochondrial dysfunction compromises synaptic development and formation of synapse in AD cybrid cells in response to chemical-induced neuronal differentiation. Compared to non-AD control cybrids, AD cybrid cells showed synaptic loss which was evidenced by a significant reduction in expression of two synaptic marker proteins: synaptophysin (presynaptic marker) and postsynaptic density protein-95, and neuronal proteins (MAP-2 and NeuN) upon neuronal differentiation. In parallel, AD-mediated synaptic deficits correlate to mitochondrial dysfunction and oxidative stress as well as activation of p38 MAP kinase. Notably, inhibition of p38 MAP kinase by pharmacological specific p38 inhibitor significantly increased synaptic density, improved mitochondrial function, and reduced oxidative stress. These results suggest that activation of p38 MAP kinase signaling pathway contributes to AD-mediated impairment in neurogenesis, possibly by inhibiting the neuronal differentiation. Our results provide new insight into the crosstalk of dysfunctional AD mitochondria to synaptic formation and maturation via activation of p38 MAP kinase. Therefore, blockade of p38 MAP kinase signal transduction could be a potential therapeutic strategy for AD by alleviating loss of synapses.

The Entorhinal cortex (EC) has been implicated in the early stages of Alzheimer’s disease (AD). In particular, spreading of neuronal dysfunction within the EC-Hippocampal network has been suggested. We have investigated the time course of EC dysfunction in the AD mouse model carrying human mutation of amyloid precursor protein (mhAPP) expressing human Aβ. We found that in mhAPP mice plasticity impairment is first observed in EC superficial layer and further affected with time. A selective impairment of LTP was observed in layer II horizontal connections of EC slices from 2 month old mhAPP mice, whereas at later stage of neurodegeneration (6 month) basal synaptic transmission and LTD were also affected. Accordingly, early synaptic deficit in the mhAPP mice were associated with a selective impairment in EC-dependent associative memory tasks. The introduction of the dominant-negative form of RAGE lacking RAGE signalling targeted to microglia (DNMSR) in mhAPP mice prevented synaptic and behavioural deficit, reducing the activation of stress related kinases (p38MAPK and JNK). Our results support the involvement of the EC in the development and progression of the synaptic and behavioural deficit during amyloid-dependent neurodegeneration and demonstrate that microglial RAGE activation in presence of Aβ-enriched environment contributes to the EC vulnerability.

A major obstacle to the development of effective treatment of Alzheimer's disease (AD) is successfully delivery of drugs to the brain. We have previously identified a series of benzothiazole phosphonate compounds that block the interaction of amyloid-β peptide with amyloid-β binding alcohol dehydrogenase (ABAD). A selective and sensitive method for the presence of three new benzothiazole ABAD inhibitors in mouse plasma, brain, and artificial cerebrospinal fluid has been developed and validated based on high performance liquid chromatography tandem mass spectrometry. Mass spectra were generated using Micromass Quattro Ultima "triple" quadrupole mass spectrometer equipped with an Electrospray Ionization interface. Good linearity was obtained over a concentration range of 0.05-2.5 μg/ml. The lowest limit of quantification and detection was found to be 0.05 μg/ml. All inter-day accuracies and precisions were within ± 15% of the nominal value and ± 20%, respectively, at the lower limit of quantitation. The tested compounds were stable at various conditions with recoveries90.0% (RSD10%). The method used for pharmacokinetic studies of compounds in mouse cerebrospinal fluid, plasma, and brain is accurate, precise, and specific with no matrix effect. Pharmacokinetic data showed that these compounds penetrate the blood-brain barrier (BBB) yielding 4-50 ng/ml peak brain concentrations and 2 μg/ml peak plasma concentrations from a 10 mg/kg dose. These results indicate that our newly synthesized small molecule ABAD inhibitors have a good drug properties with the ability to cross the blood-brain barrier, which holds a great potential for AD therapy.

Ischemia is known to increase the deleterious effect of β-amyloid (Aβ), contributing to early cognitive impairment in Alzheimer's disease. Here, we investigated whether transient ischemia may function as a trigger for Aβ-dependent synaptic impairment in the entorhinal cortex (EC), acting through specific cellular signaling. We found that synaptic depression induced by oxygen glucose deprivation (OGD) was enhanced in EC slices either in presence of synthetic oligomeric Aβ or in slices from mutant human amyloid precursor protein transgenic mice (mhAPP J20). OGD-induced synaptic depression was ameliorated by functional suppression of RAGE. In particular, overexpression of the dominant-negative form of RAGE targeted to microglia (DNMSR) protects against OGD-induced synaptic impairment in an amyloid-enriched environment, reducing the activation of stress-related kinases (p38MAPK and JNK) and the release of IL-1β. Our results demonstrate a prominent role for the RAGE-dependent neuroinflammatory pathway in the synaptic failure induced by Aβ and triggered by transient ischemia.

The neuroinflammation induced by amyloid-β (Aβ ) is one of the key events in Alzheimer’s disease (AD) progress in which microglia are the main cells involved. Receptor for advanced glycation end products (RAGE) mediates and enhances A β-induced microglial activation and leads to induction of proinflammatory mediators, such as tumor necrosis factor- α (TNF-α ) and interleukin-1β (IL-1 β). Geniposide, a pharmacologically active component purified from gardenia fruit, exhibits a broad spectrum anti-inflammatory effect as well as neurotrophic and neuroprotective properties. However, the effects of geniposide on A β-mediated microglial pathways have not been fully discovered. Here, we demonstrate that geniposide treatment significantly blocks Aβ -induced RAGE-dependent signaling (activation of ERK and NF-κ B) along with the production of TNF- α and IL-1β in cultured BV2 microglia cells. Notably, based on the data from coimmunoprecipitation assay, we infer that geniposide exerts protective effects on A β-induced inflammatroy response through blocking A β binding to RAGE and suppressing the RAGE-mediated signaling pathway. Taken together, these findings indicate that geniposide is a potent suppressor of neuroflammation through inhibiting RAGE-dependent signaling pathway. Thus, geniposide may be a potential therapeutic agent for the treatment of neuroinflammation that is involved in neurological diseases such as AD.

Amyloid beta (Aβ ) binding alcohol dehydrogenase (ABAD) is a cellular cofactor for promoting (Aβ )-mediated mitochondrial and neuronal dysfunction, and cognitive decline in transgenic Alzheimer’s disease (AD) mouse models. Targeting mitochondrial ABAD may represent a novel therapeutic strategy against AD. Here, we report the biological activity of small molecule ABAD inhibitors. Using in vitro surface plasmon resonance (SPR) studies, we synthesized compounds with strong binding affinities for ABAD. Further, these ABAD inhibitors (ABAD-4a and 4b) reduced ABAD enzyme activity and administration of phosphonate derivatives of ABAD inhibitors antagonized calcium-mediated mitochondrial swelling. Importantly, these compounds also abolished A β-induced mitochondrial dysfunction as shown by increased cytochrome c oxidase activity and adenosine-5'-triphosphate levels, suggesting protective mitochondrial function effects of these synthesized compounds. Thus, these compounds are potential candidates for further pharmacologic development to target ABAD to improve mitochondrial function.

Cyclophilin D (CypD) is a peptidyl prolyl isomerase F that resides in the mitochondrial matrix and associates with the inner mitochondrial membrane during the mitochondrial membrane permeability transition. CypD plays a central role in opening the mitochondrial membrane permeability transition pore (mPTP) leading to cell death and has been linked to Alzheimer’s disease (AD). Because CypD interacts with amyloid beta (Aβ) to exacerbate mitochondrial and neuronal stress, it is a potential target for drugs to treat AD. Since appropriately designed small organic molecules might bind to CypD and block its interaction with Aβ, 20 trial compounds were designed using known procedures that started with fundamental pyrimidine and sulfonamide scaffolds know to have useful therapeutic effects. Two-dimensional (2D) quantitative structure–activity relationship (QSAR) methods were applied to 40 compounds with known IC50 values. These formed a training set and were followed by a trial set of 20 designed compounds. A correlation analysis was carried out comparing the statistics of the measured IC50 with predicted values for both sets. Selectivity-determining descriptors were interpreted graphically in terms of principle component analyses. These descriptors can be very useful for predicting activity enhancement for lead compounds. A 3D pharmacophore model was also created. Molecular dynamics simulations were carried out for the 20 trial compounds with known IC50 values, and molecular descriptors were determined by 2D QSAR studies using the Lipinski rule-of-five. Fifteen of the 20 molecules satisfied all 5 Lipinski rules, and the remaining 5 satisfied 4 of the 5 Lipinski criteria and nearly satisfied the fifth. Our previous use of 2D QSAR, 3D pharmacophore models, and molecular docking experiments to successfully predict activity indicates that this can be a very powerful technique for screening large numbers of new compounds as active drug candidates. These studies will hopefully provide a basis for efficiently designing and screening large numbers of more potent and selective inhibitors for CypD treatment of AD.

Mitochondrial dysfunction is an early pathological feature of Alzheimer’s disease (AD). The underlying mechanisms and strategies to repair it remain unclear. Here, we demonstrate for the first time the direct consequences and potential mechanisms of mitochondrial functional defects associated with abnormal mitochondrial dynamics in AD. Using cytoplasmic hybrid (cybrid) neurons with incorporated platelet mitochondria from AD and age-matched non-AD human subjects into mitochondrial DNA (mtDNA)-depleted neuronal cells, we observed that AD cybrid cells had significant changes in morphology and function; such changes associate with altered expression and distribution of dynamin-like protein (DLP1) and mitofusin 2 (Mfn2). Treatment with antioxidant protects against AD mitochondria-induced extracellular signal-regulated kinase (ERK) activation and mitochondrial fission-fusion imbalances. Notably, inhibition of ERK activation not only attenuates aberrant mitochondrial morphology and function but also restores the mitochondrial fission and fusion balance. These effects suggest a role of oxidative stress-mediated ERK signal transduction in modulation of mitochondrial fission and fusion events. Further, blockade of the mitochondrial fission protein DLP1 by a genetic manipulation with a dominant negative DLP1 (DLP1K38A), its expression with siRNA-DLP1, or inhibition of mitochondrial division with mdivi-1 attenuates mitochondrial functional defects observed in AD cybrid cells. Our results provide new insights into mitochondrial dysfunction resulting from changes in the ERK-fission/fusion (DLP1) machinery and signaling pathway. The protective effect of mdivi-1 and inhibition of ERK signaling on maintenance of normal mitochondrial structure and function holds promise as a potential novel therapeutic strategy for AD.

Bioenergetic dysfunction occurs in Alzheimer's disease (AD) and mild cognitive impairment (MCI), a clinical syndrome that frequently precedes symptomatic AD. In this study, we modeled AD and MCI bioenergetic dysfunction by transferring mitochondria from MCI, AD and control subject platelets to mtDNA-depleted SH-SY5Y cells. Bioenergetic fluxes and bioenergetics-related infrastructures were characterized in the resulting cytoplasmic hybrid (cybrid) cell lines. Relative to control cybrids, AD and MCI cybrids showed changes in oxygen consumption, respiratory coupling and glucose utilization. AD and MCI cybrids had higher ADP/ATP and lower NAD+/NADH ratios. AD and MCI cybrids exhibited differences in proteins that monitor, respond to or regulate cell bioenergetic fluxes including HIF1α, PGC1α, SIRT1, AMPK, p38 MAPK and mTOR. Several endpoints suggested mitochondrial mass increased in the AD cybrid group and probably to a lesser extent in the MCI cybrid group, and that the mitochondrial fission-fusion balance shifted towards increased fission in the AD and MCI cybrids. As many of the changes we observed in AD and MCI cybrid models are also seen in AD subject brains, we conclude reduced bioenergetic function is present during very early AD, is not brain-limited and induces protean retrograde responses that likely have both adaptive and mal-adaptive consequences.

Mitochondria are well-known cellular organelles widely studied in relation to a variety of disease states, including Alzheimer’s disease. With roles in several metabolic processes, numerous signal transduction pathways, and overall cell maintenance and survival, mitochondria are essential to understanding the inner workings of cells. As mitochondria are able to be utilized by diverse illnesses to increase the likelihood of disease progression, targeting specific processes in these organelles could provide beneficial therapeutic options.

Amyloid binding alcohol dehydrogenase, a mitochondrial protein, is a cofactor facilitating amyloid-β peptide (Aβ) induced cell stress. Antagonizing Aβ-ABAD interaction protects against aberrant mitochondrial and neuronal function and improves learning memory in the Alzheimer's disease mouse model. Therefore, it offers a potential target for Alzheimer's drug design, by identifying potential inhibitors of Aβ-ABAD interaction. 2D QSAR methods were applied to novel compounds with known IC(50) values, which formed a training set. A correlation analysis was carried out comparing the statistics of the measured IC(50) with predicted values. These selectivity-determining descriptors were interpreted graphically in terms of principle component analyses, which are highly informative for the lead optimization process with respect to activity enhancement. A 3D pharmacophore model also was created. The 2D QSAR and 3D pharmacophore models will assist in high-throughput screening. In addition, ADME descriptors were also determined to study their pharmacokinetic properties. Finally, amyloid binding alcohol dehydrogenase molecular docking study of these novel molecules was undertaken to determine whether these compounds exhibit significant binding affinity with the binding site. We have synthesized only the compounds that have shown the best drug-like properties as candidates for further studies.

Mitochondrial dysfunction and synaptic damage are early pathological features of the Alzheimer's disease-affected brain. Memory impairment in Alzheimer's disease is a manifestation of brain pathologies such as accumulation of amyloid-β peptide and mitochondrial damage. The underlying pathogenic mechanisms and effective disease-modifying therapies for Alzheimer's disease remain elusive. Here, we demonstrate for the first time that decreased PTEN-induced putative kinase 1 (PINK1) expression is associated with Alzheimer's disease pathology. Restoring neuronal PINK1 function strikingly reduces amyloid-β levels, amyloid-associated pathology, oxidative stress, as well as mitochondrial and synaptic dysfunction. In contrast, PINK1-deficient mAPP mice augmented cerebral amyloid-β accumulation, mitochondrial abnormalities, impairments in learning and memory, as well as synaptic plasticity at an earlier age than mAPP mice. Notably, gene therapy-mediated PINK1 overexpression promotes the clearance of damaged mitochondria by augmenting autophagy signalling via activation of autophagy receptors (OPTN and NDP52), thereby alleviating amyloid-β-induced loss of synapses and cognitive decline in Alzheimer's disease mice. Loss of PINK1 activity or blockade of PINK1-mediated signalling (OPTN or NDP52) fails to reverse amyloid-β-induced detrimental effects. Our findings highlight a novel mechanism by which PINK1-dependent signalling promotes the rescue of amyloid pathology and amyloid-β-mediated mitochondrial and synaptic dysfunctions in a manner requiring activation of autophagy receptor OPTN or NDP52. Thus, activation of PINK1 may represent a new therapeutic avenue for combating Alzheimer's disease.

Mitochondrial dysfunction and axonal degeneration are early pathological features of Alzheimer's disease (AD)-affected brains. The underlying mechanisms and strategies to rescue it have not been well elucidated. Here, we evaluated axonal mitochondrial transport and function in AD subject-derived mitochondria. We analyzed mitochondrial transport and kinetics in human trans-mitochondrial "cybrid" (cytoplasmic hybrid) neuronal cells whose mitochondria were derived from platelets of patients with sporadic AD and compared these AD cybrid cell lines with cybrid cell lines whose mitochondria were derived from age-matched, cognitively normal subjects. Human AD cybrid cell lines, when induced to differentiate, developed stunted projections. Mitochondrial transport and function within neuronal processes/axons was altered in AD-derived mitochondria. Antioxidants reversed deficits in axonal mitochondrial transport and function. These findings suggest that antioxidants may be able to mitigate the consequences of AD-associated mitochondrial dysfunction. The present study provides evidence of the cause/effect of AD specific mitochondrial defects, which significantly enhances our understanding of the AD pathogenesis and exploring the effective therapeutic strategy for AD.

Mitochondria are essential dynamic organelles for energy production. Mitochondria dynamically change their shapes tightly coupled to fission and fusion. Imbalance of fission and fusion can cause deficits in mitochondrial respiration, morphology and motility. Mfn2 (mitofusin 2), a mitochondrial membrane protein that participates in mitochondrial fusion in mammalian cells, contributes to the maintenance and operation of the mitochondrial network. Due to lack of applicable model systems, the mechanisms and involvement of mitochondria in neurogenesis in human brain cells have not been well explored. Here, by employing the human induced pluripotent stem cells (hiPSCs) differentiation system, we fully characterized mitochondrial development, neurogenesis and synapse formation in hiPSCs-derived cortical neurons. Differentiation of hiPSCs to cortical neurons with extended period demonstrates mature neurophysiology characterization and functional synaptic network formation. Mitochondrial respiration, morphology and motility in the differentiated neurons also exhibit pronounced development during differentiation. Mfn2 knock-down results in deficits in mitochondrial metabolism and network, neurogenesis and synapse formation, while Mfn2 overexpression enhances mitochondrial bioenergetics and functions and promotes the differentiation and maturation of neurons. Together, our data indicate that Mfn2 is essential for human mitochondrial development in neuronal maturation and differentiation, which will enhance our understanding of the role of Mfn2 in neurogenesis.

Accumulation of oxidized proteins, and especially β-amyloid (Aβ), is thought to be one of the common causes of Alzheimer's disease (AD). The current studies determine the effect of an in vivo methionine sulfoxidation of Aβ through ablation of the methionine sulfoxide reductase A (MsrA) in a mouse model of AD, a mouse that overexpresses amyloid precursor protein (APP) and Aβ in neurons. Lack of MsrA fosters the formation of methionine sulfoxide in proteins, and thus its ablation in the AD-mouse model will increase the formation of methionine sulfoxide in Aβ. Indeed, the novel MsrA-deficient APP mice ( APP+/ MsrAKO) exhibited higher levels of soluble Aβ in brain compared with APP+ mice. Furthermore, mitochondrial respiration and the activity of cytochrome c oxidase were compromised in the APP+/ MsrAKO compared with control mice. These results suggest that lower MsrA activity modifies Aβ solubility properties and causes mitochondrial dysfunction, and augmenting its activity may be beneficial in delaying AD progression.

Although epidemiological data associate hypertension with a strong predisposition to develop Alzheimer disease, no mechanistic explanation exists so far. We developed a model of hypertension, obtained by transverse aortic constriction, leading to alterations typical of Alzheimer disease, such as amyloid plaques, neuroinflammation, blood-brain barrier dysfunction, and cognitive impairment, shown here for the first time. The aim of this work was to investigate the mechanisms involved in Alzheimer disease of hypertensive mice. We focused on receptor for advanced glycation end products (RAGE) that critically regulates Aβ transport at the blood-brain barrier and could be influenced by vascular factors. The hypertensive challenge had an early and sustained effect on RAGE upregulation in brain vessels of the cortex and hippocampus. Interestingly, RAGE inhibition protected from hypertension-induced Alzheimer pathology, as showed by rescue from cognitive impairment and parenchymal Aβ deposition. The increased RAGE expression in transverse aortic coarctation mice was induced by increased circulating advanced glycation end products and sustained by their later deposition in brain vessels. Interestingly, a daily treatment with an advanced glycation end product inhibitor or antioxidant prevented the development of Alzheimer traits. So far, Alzheimer pathology in experimental animal models has been recognized using only transgenic mice overexpressing amyloid precursor. This is the first study demonstrating that a chronic vascular insult can activate brain vascular RAGE, favoring parenchymal Aβ deposition and the onset of cognitive deterioration. Overall we demonstrate that RAGE activation in brain vessels is a crucial pathogenetic event in hypertension-induced Alzheimer disease, suggesting that inhibiting this target can limit the onset of vascular-related Alzheimer disease.

Significance: Synaptic degeneration, an early pathological feature in Alzheimer's disease (AD), is closely correlated to impaired cognitive function and memory loss. Recent studies suggest that involvement of amyloid-beta peptide (Aβ) in synaptic mitochondrial alteration underlies these synaptic lesions. Thus, to understand the Aβ-associated synaptic mitochondrial perturbations would fortify our understanding of synaptic stress in the pathogenesis of AD. Recent Advances: Increasing evidence suggests that synaptic mitochondrial dysfunction is strongly associated with synaptic failure in many neurodegenerative diseases including AD. Based on recent findings in human AD subjects, AD animal models, and AD cellular models, synaptic mitochondria undergo multiple malfunctions including Aβ accumulation, increased oxidative stress, decreased respiration, and compromised calcium handling capacity, all of which occur earlier than changes seen in nonsynaptic mitochondria before predominant AD pathology. Of note, the impact of Aβ on mitochondrial motility and dynamics exacerbates synaptic mitochondrial alterations. Critical Issues: Synaptic mitochondria demonstrate early deficits in AD; in combination with the role that synaptic mitochondria play in sustaining synaptic functions, deficits in synaptic mitochondria may be a key factor involved in an early synaptic pathology in AD. Future Directions: The importance of synaptic mitochondria in supporting synapses and the high vulnerability of synaptic mitochondria to Aβ make them a promising target of new therapeutic strategy for AD. Antioxid. Redox Signal. 16, 1467–1475.

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.