Trushin, Sergey

AbstractBackgroundAlzheimer’s disease (AD) is a devastating neuropathological condition where age is the greatest risk factor. There is currently an estimated global prevalence upward of 46.8 million individuals and recent efforts to combat the clinical presentation of AD through late‐stage Amyloid‐beta (Aβ) targeted therapeutic intervention has not been successful, underscoring the need for novel approaches to slow disease progression. Accumulating evidence proposes that mitochondrial and metabolic homoeostasis is an early dysfunction which occurs decades before the presentation of clinical symptoms. Indeed AD has been classified as type‐3 diabetes, suggesting the need to target metabolic modifiers to slow or even abrogate neurodegeneration. We have recently reported that targeting the oxidative phosphorylation (OXPHOS) system through mild inhibition of NADH dehydrogenase (Complex I) with our small molecules partial Complex I inhibitors (MCIi) can halt the development of cognitive and behavioral phenotypes associated with AD. We have also demonstrated that this modulation of mitochondrial complex I, through reversible inhibition at the Flavin Mononucleotide (FMN) domain, serves to enhance mitochondrial bioenergetics and function and protects against oxidative stress. We hypothesize that this mechanism of improved bioenergetics may be due to transformed OXPHOS super‐complex assembly, which can lead to a more efficient bioenergetics system and protect against extrinsic stress.MethodsTo test this hypothesis, we examined bioenergetics and the assembly of mitochondrial OXPHOS complexes and super‐complexes in mitochondria from primary cortical neurons and synaptosomes isolated from the brain tissue of non‐transgenic and 3xTgAD mice treated with MCli using respiratory XF Seahorse analysis, BNPAGE and 2D‐BNPAGE.ResultsOur analyses demonstrates that MCIi can acutely alter the assembly of super‐complexes within the OXPHOS system and protect against stress through enhanced spare respiratory capacity while preserving ATP level, which may aid in the enhancement of mitochondrial bioenergetics and function in AD.ConclusionPartial complex I inhibitors can induce positive bioenergetics through super‐complex assembly

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.

ABSTRACTHuman monocytes and macrophages are persistent reservoirs of human immunodeficiency virus (HIV) type-1. Persistent HIV infection of these cells results in increased levels of NF-κB in the nucleus secondary to increased IκBα, IκBβ, and IκBɛ degradation, a mechanism postulated to regulate viral persistence. To characterize the molecular mechanisms regulating HIV-mediated degradation of IκB, we have sought to identify the regulatory domains of IκBα targeted by HIV infection. Using monocytic cells stably expressing different transdominant molecules of IκBα, we determined that persistent HIV infection of these cells targets the NH2but not the COOH terminus of IκBα. Further analysis demonstrated that phosphorylation at S32and S36is necessary for HIV-dependent IκBα degradation and NF-κB activation. Of the putative N-terminal IκBα kinases, we demonstrated that the Iκκ complex, but not p90rsk, is activated by HIV infection and mediates HIV-dependent NF-κB activation. Analysis of viral replication in cells that constitutively express IκBα negative transdominant molecules demonstrated a lack of correlation between virus-induced NF-κB (p65/p50) nuclear translocation and degree of viral persistence in human monocytes.