Chang, Su‐Youne

AbstractBackgroundConsistent failure of clinical trials for Alzheimer Disease (AD) emphasizes the necessity to identify novel therapeutic targets for this debilitating condition. We recently identified the mitochondrial complex I (MCI) as a small molecule druggable therapeutic target for AD.MethodUsing rational design and extensive structure‐activity relationship studies, we developed novel classes of partial MCI inhibitors with high selectivity, specificity, safety, and drug‐like properties. Target engagement, selectivity and specificity of new compounds were established using multiple in vitro assays, including surface plasmon resonance (SPR), competitive and direct binding, proteomics and pull down experiments. Using these techniques, we developed new lead compound C458 that was interrogated in in vivo efficacy studies in APP/PS1 mice.ResultWe demonstrated that the new lead C458 directly binds to MCI. A single dose administration of C458 to mice or treatment of primary mouse neurons activates AMPK‐a, Akt, autophagy, Sirtuins 1 and 3, and upregulates PGC‐1a leading to enhanced mitochondrial biogenesis. C458 has excellent oral bioavailability, blood–brain‐barrier penetrance, and was remarkably clean in the 250 kinase and CEREP 44 safety panels. Chronic administration of C458 (25 mg/kg/day in drinking water ad lib) to APP/PS1 mice for 6 months starting at pre‐symptomatic stage did not cause detectable side effects, and resulted in cognitive protection, improved long‐term potentiation, and decreased oxidative stress and inflammation. Furthermore, APP/PS1 mice treated with C458 showed a significant increase in glucose uptake and utilization in the brain detected with a translational in vivo biomarker FDG‐PET, as well as an enhancement of ATP production measured in vivo using 31P NMR.ConclusionMechanistic studies confirmed that C458 treatment triggered mitochondrial stress response augmenting mitochondrial biogenesis, anti‐oxidant signaling, and cellular energetics that ultimately restored energy brain homeostasis, leading to cognitive protection in a mouse model of AD. Our studies demonstrate that modulation of MCI activity using novel specific MCI inhibitors improves bioenergetics in the brain and averts cognitive decline representing novel therapeutic approach for AD.

AbstractBackgroundWe have previously demonstrated that partial reduction of mitochondrial complex I (MCI) activity using small molecules is efficacious in decreasing levels of amyloid beta (Aβ), phosphorylated Tau, augmenting mitochondrial bioenergetics, promoting resistance to oxidative stress and restoring mitochondrial axonal trafficking, levels of BDNF and synaptic proteins in pre‐symptomatic APP/PS1 mice. In parallel, these mice demonstrated an improved cognitive and behavioral phenotype compared to their untreated littermates.MethodIn the current study, we tested whether treatment with MCI inhibitors could halt the disease progression when treatment was administered to symptomatic APP/PS1 mice.ResultTreatment improved glucose tolerance, prevented body weight loss, and ameliorated systemic inflammation. Treatment improved glucose uptake in the brain measured using FDG‐PET imaging. Electrophysiological recording in hippocampus indicated that synaptic activity and LTP were restored in treated APP/PS1 mice. Data gathered using 3D electron microscopy reconstruction of dendrites in CA1 region of hippocampus showed that treatment increased the population of mature dendritic spines in APP/PS1 mice, which correlates with restored synaptic activity and improved glucose uptake and utilization in the brain. Immunohistochemical analysis showed that treated APP/PS1 mice had reduced brain level of Aβ plaques, markers of inflammation and reduced degeneration of TH+ neurons in locus coeruleus. The RNA‐seq gene function enrichment analysis showed that pathways down‐regulated by treatment with MCI inhibitors in APP/PS1 mice included the ones involved in oxidative stress and immune system response. Moreover, RNA‐seq data analysis depicted a cluster of genes involved in dendritic morphology, axonal guidance and neuronal development, which expression were reverted by treatment in APP/PS1 mice to the level observed in NTG mice. Western blot analysis in cortico‐hippocampal brain region of APP/PS1 mice confirmed that treatment increased signaling molecules involved in IGF‐1/insulin pathway, mitochondrial biogenesis, oxidative stress protection, autophagy and synaptic function.ConclusionOverall, our data suggest that long‐term treatment with MCI inhibitors is safe and efficacious in restoring cognitive function and preventing degeneration in symptomatic APP/PS1 mice. Therefore, partial MCI inhibitors represent a promising therapeutic approach to ameliorate AD and promote healthy aging.

AbstractBackgroundAlzheimer’s Disease (AD) has no effective treatments, and recent clinical trials focused on prevention of amyloid beta (Ab) production have consistently failed. Alternative approaches are urgently needed. We developed small molecules partial mitochondrial complex I (MCI) inhibitors and demonstrated that their application prevents the development of cognitive and behavior phenotype, improves LTP, reduces oxidative stress and inflammation, and restores glucose uptake and utilization in the brain in mouse models of familial AD. Efficacy could be monitored in vivo using translational biomarkers FDG‐PET, 31P NMR and blood biomarkers.MethodWe conducted extensive structure‐activity relationship (SAR) studies to characterize novel classes of compounds partial MCI inhibitors including a new lead compound C458. Target engagement, selectivity and specificity of new compounds were established using multiple in vitro and cellular assays including SPR, competitive and direct binding, proteomics and pull down experiments.ResultWe demonstrated that the lead C458 directly binds and competes with the FMN for binding to complex I. In primary neurons and mouse brain tissue, single dose administration of C458 activates AMPK‐a, autophagy, Sirtuins 1 and 3, and upregulate PGC‐1a leading to enhanced mitochondrial biogenesis. In vivo, chronic administration of C458 had no detectable toxicity at therapeutic doses. C458 has excellent oral bioavailability and blood–brain‐barrier penetration. C458 was remarkably clean in 250 kinase panel. Finally, in pre‐symptomatic APP/PS1 mice, treatment with C458 resulted in cognitive protection and improved long‐term potentiation after 6 months of chronic oral administration (25 mg/kg/day), decreased oxidative stress and inflammation. Furthermore, APP/PS1 mice treated with C458 showed a significant increase in metabolic rates and glucose brain uptake and utilization detected with translational FDG‐PET imaging as well as an enhancement of ATP production in the brain measured in vivo with 31P NMR.ConclusionMechanistic studies demonstrate that MCI inhibitors induce protective stress response increasing mitochondrial biogenesis and turnover, augmenting cellular energetics and the protection against oxidative stress, and engage other mechanisms that intersect with the pathways involved in longevity and health extension. Our studies demonstrate that modulation of MCI activity using novel FMN MCI inhibitors improves bioenergetics in the brain and averts cognitive decline in Alzheimer’s Disease representing novel therapeutic approach.

AbstractProhormone convertase 2 (PC2) is essential for the biosynthesis of many neuropeptides, including several of them in hippocampus. In mouse brain, lacking an enzymatically active PC2 (PC2‐null) causes accumulation of many neuropeptides in their precursor or intermediate forms. Little is known about how a PC2‐null state may affect the function of the hippocampus. In this study, adult PC2‐null mice and their wildtype (WT) littermates were subjected to three analyses to determine possible changes associated with PC2‐null at physiological, behavioral, and molecular levels, respectively, under normal and stressed conditions. Electrophysiological recordings of hippocampal slices were performed to measure evoked field‐excitatory postsynaptic potentials (EPSP), long‐term potentiation (LTP), and paired‐pulse facilitation (PPF). Morris water maze (MWM) testing was conducted to examine behavioral changes that are indicative of hippocampal integrity. Quantitative mass spectrometry analysis was used to determine changes in the hippocampal proteome in response to a focal cerebral ischemic insult. We found that there were no significant differences in the threshold of evoked EPSPs between PC2‐null and WT animals. However, an increase in LTP in both triggering rate and amplitude was observed in PC2‐null mice, suggesting that PC2 may be involved in regulating synaptic strength. The PPF, on the other hand, showed a decrease in PC2‐null mice, suggesting a presynaptic mechanism. Consistent with changes in LTP, PC2‐null mice displayed decreased latencies in finding the escape platform in the MWM test. Further, after distal focal cerebral ischemia, the hippocampal proteomes incurred changes in both WT and PC2‐null mice, with a prominent change in proteins associated with neurotransmission, exocytosis, and transport processes seen in the PC2‐null but not WT mice. Taken together, our results suggest that PC2 is involved in regulating hippocampal synaptic plasticity, learning, and memory behaviors, as well as the hippocampal response to stresses originating in other regions of the brain.

SummaryObjectiveSeizures are currently defined by their electrographic features. However, neuronal networks are intrinsically dependent on neurotransmitters of which little is known regarding their periictal dynamics. Evidence supports adenosine as having a prominent role in seizure termination, as its administration can terminate and reduce seizures in animal models. Furthermore, microdialysis studies in humans suggest that adenosine is elevated periictally, but the relationship to the seizure is obscured by its temporal measurement limitations. Because electrochemical techniques can provide vastly superior temporal resolution, we test the hypothesis that extracellular adenosine concentrations rise during seizure termination in an animal model and humans using electrochemistry.MethodsWhite farm swine (n = 45) were used in an acute cortical model of epilepsy, and 10 human epilepsy patients were studied during intraoperative electrocorticography (ECoG). Wireless Instantaneous Neurotransmitter Concentration Sensor (WINCS)–based fast scan cyclic voltammetry (FSCV) and fixed potential amperometry were obtained utilizing an adenosine‐specific triangular waveform or biosensors, respectively.ResultsSimultaneousECoG and electrochemistry demonstrated an average adenosine increase of 260% compared to baseline, at 7.5 ± 16.9 s with amperometry (n = 75 events) and 2.6 ± 11.2 s withFSCV(n = 15 events) prior to electrographic seizure termination. In agreement with these animal data, adenosine elevation prior to seizure termination in a human patient utilizingFSCVwas also seen.SignificanceSimultaneousECoG and electrochemical recording supports the hypothesis that adenosine rises prior to seizure termination, suggesting that adenosine itself may be responsible for seizure termination. Future work using intraoperativeWINCS–basedFSCVrecording may help to elucidate the precise relationship between adenosine and seizure termination.