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Abstract
Aim: Oxidative stress and NAD+/NADH imbalance caused by alterations in reactive oxygen species (ROS) and NAD(H) metabolism are pathological features associated with normal aging and age-related diseases including Alzheimer’s disease (AD). How abnormalities in ROS and NAD(H) metabolism occur under these pathological conditions is not well understood, nor is it known whether they are mechanistically linked and can be therapeutically targeted together. The aim of this study is to identify the cause of aberrant ROS and NAD(H) metabolism and test its role in the pathogenesis of AD.
Methods: Reverse electron transport (RET) along mitochondrial complex I can occur under certain thermodynamic conditions, leading to excessive ROS generation and NAD+ conversion to NADH, and thus lowered NAD+/NADH ratio. Brain samples from AD patients and mouse AD models were used to assess the status of RET by measuring ROS and NAD+/NADH ratio in brain lysates and purified mitochondria respiring under RET conditions. A small molecule RET inhibitor was used to treat APP(swe)/PS1(deltaE9) and 5xFAD mouse models and human induced pluripotent stem cell (iPSC)-derived neuronal model of AD. Effects on behavior and AD-related neuropathology were examined. The biochemical mechanism underlying RET alteration was examined by protein-protein interaction studies.
Results: RET is aberrantly activated in transgenic AD mouse brains and in individuals with AD. Pharmacological inhibition of RET reduced amyloid burden and neuroinflammation and rescued cognitive and behavioral deficits in the APP(swe)/PS1(deltaE9) and 5xFAD mouse models. In human AD iPSC-derived neurons, RET inhibition reduced amyloid aggregation, tau hyperphosphorylation, and early endosomal defects. Mechanistically, theAD-associatedamyloid precursor protein C-terminal fragment (APP.C99) was found to interact with complex I proteins to promote RET.
Conclusion: RET is aberrantly activated in AD, causing altered ROS and NAD+/NADH metabolism. Pharmacological inhibition of RET is beneficial in mouse and human iPSC models of AD. RET activation represents a key pathological driver and a rational therapeutic target for AD and possibly other age-related neurodegenerative diseases.
Keywords
Mitochondria, reactive oxygen species, NAD+/NADH, reverse electron transport, Alzheimer’s disease, mouse models
INTRODUCTION
Oxidative stress due to increased reactive oxygen species (ROS) production[1–4] and metabolic derailment due to altered NAD+/NADH ratio or lowered NAD+ pool[5–9] are pathological features associated with normal biological aging and age-related diseases. Therapeutic interventions targeting ROS and NAD+/NADH metabolism individually have been tried in the context of aging and age-related diseases, but with mixed results[10–14], highlighting the importance of in-depth understanding of the sources and pathophysiology of ROS and NAD+/NADH metabolism. The mechanisms by which the increased ROS production and decreased NAD+/NADH ratio occur are not well understood, and it is not known if these two processes are connected mechanistically and therefore can be therapeutically targeted together.
Mitochondria have long been recognized as a major source of ROS during aging and age-related diseases[15] and a leading cause of aging based on the free radical theory[16]. Recent studies indicate that the role of ROS in aging and age-related diseases is likely to be complex and multifaceted, with the sites and levels of ROS generation having a significant influence on the outcome[17,18]. It is generally assumed that transient and moderate levels of ROS may serve physiological roles such as stress adaptation and synaptic signaling, whereas prolonged and elevated ROS production can be detrimental. Along the electron transport chain, complex I and complex III have been identified as key sites of ROS generation[19,20]. In certain tissues and under certain respiration conditions, complex I has been shown to be the major site of ROS production[21,22]. Although both beneficial and detrimental effects have been attributed to complex I-generated ROS[23], in general, longevity is associated with a low rate of ROS generation[21].
Mitochondria are also critically involved in NADH/NAD+ metabolism. The central roles of NAD+ and NADH in the TCA cycle and electron transport chain (ETC) underscore the importance of balanced NAD+/NADH to mitochondrial well-being[24]. Beyond this metabolic function, NAD+ critically regulates the activities of NAD+-consuming enzymes, including Sirtuins and poly-ADP-ribose polymerases, which have been implicated in aging and age-related diseases[7,25–27]. NAD+/Sirtuin signaling modulates longevity through the activation of mitochondrial stress responses such as the unfolded protein response (UPRmt) and the nuclear translocation and activation of Foxo[28], linking mitochondrial metabolism and stress signaling with longevity. NAD+ level declines with age, and replenishing NAD+ with precursors offers certain beneficial effects towards aging and age-related disease[6,8], but the long-term health benefits of NAD+ precursor supplementation remain to be tested. Although CD38/157 ectoenzymes have been implicated in NAD+ degradation during aging[24], the causes of NAD+ level decline during age are incompletely delineated and other mechanisms remain to be identified.
Under certain thermodynamic conditions, reverse electron transport (RET) along mitochondrial complex I can occur[29,30], resulting in NAD+ conversion to NADH and thus reduced NAD+/NADH ratio and excessive ROS generation[31–34]. RET is considered a major source of mitochondrial ROS production. While the physiological function of RET remains to be deciphered[35], deregulation of RET has been linked to pathological conditions, e.g., ischemia-reperfusion injury during stroke[32] and cancer[33]. Given that RET influences ROS and NAD+/NADH ratio, two parameters intimately linked to aging and age-related diseases, deregulation of RET is potentially a driver of these pathological conditions. Indeed, simultaneous restoration of tissue ROS and NAD+/NADH homeostasis by inhibition of RET has been explored in the context of aging and age-related diseases and shown to be beneficial in Drosophila models[33,34]. Whether RET is altered in human disease conditions and the in vivo efficacy of RET manipulation in mammalian models of age-related neurodegenerative diseases such as AD are important questions that remain to be tested. CPT2008, 6-chloro-3-(2,4-dichloro-5-methoxyphenyl)-2-mecapto-7-methoxyquinazolin-4(3H)-one (CPT) is a small molecule belonging to the quinazolinone family that was identified as a specific and potent inhibitor of RET[33]. CPT binds to mitochondrial complex I and alters protein-protein interactions within complex I that are involved in RET (e.g., NDUFS3-NDUFV1 interaction). CPT also interferes with the action of certain non-ETC proteins that are recruited to complex I to promote RET (e.g., Notch in cancer settings)[33,34]. In this study, we tested the therapeutic effect of simultaneous restoration of tissue ROS and NAD+/NADH homeostasis through CPT treatment in mouse transgenic models and human iPSC-derived neuron models of AD. We also explored the mechanism of RET deregulation in AD models and tested if RET deregulation occurs in human AD patient samples.