Trushin, Sergey

Abstract Weak inhibition of mitochondrial complex I (mtCI) has been shown to have neuroprotective effects in cellular and animal models of Alzheimer’s and Huntington’s diseases, at least in part by enhancing mitochondrial biogenesis and function. Mitochondrial dysfunction has also been demonstrated in schizophrenia patients and mouse models of schizophrenia. We tested whether weak inhibition of mtCI would ameliorate mitochondrial and behavioral phenotypes in a mouse model of schizophrenia. In mice with four copies of the Gldc gene, 8 weeks of treatment with the weak mtCI inhibitor, the small-molecule tricyclic pyrone compound CP2, reversed spontaneous alternation deficits in the Y maze, startle habituation deficits, and social novelty deficits in the three-chamber social interaction test. Consistent with the mechanism of action, Western blots revealed that CP2 reverses the reduced expression of PGC-1α, a master regulator of mitochondrial biogenesis, and of the VDAC1, a primary gatekeeper for the exchange of metabolites, ions, and ATP between mitochondria and the cytosol. These findings suggest that the improvement of mitochondrial function may represent a novel strategy to reverse pathophysiological and behavioral deficits in schizophrenia.

Abstract Alzheimer’s disease (AD) is characterized by diminished capacity to mount adaptive cellular stress responses required to maintain energy homeostasis and proteostasis. An emerging therapeutic strategy is to restore adaptive stress responses by inducing mild energetic stress through inhibition of mitochondrial complex I (mtCI). However, pharmacological inhibition of the respiratory chain has remained challenging, as it can induce bioenergetic failure rather than beneficial signaling. Here, we describe C273, a brain-penetrant small molecule that delivers controlled, weak attenuation of mtCI activity to therapeutically restore endogenous adaptive stress pathways. This work establishes a first-in-class mechanism in which calibrated activation of multifaceted adaptive mechanisms enhances cellular resilience, rather than impairing mitochondrial function. Structure-activity relationship optimization yielded a compound with high potency against Aβ-induced cellular toxicity, strong selectivity for mtCI, and favorable drug-like properties. C273 demonstrated excellent oral bioavailability, metabolic stability in mouse, rat, and human microsomes, minimal CYP liabilities, and a clean ancillary pharmacology profile in the Eurofins CEREP44 panel. In vivo , C273 readily crosses the blood-brain barrier and activates AMP-activated protein kinase (AMPK), initiating a coordinated hormetic response characterized by enhanced antioxidant defenses, suppression of inflammatory signaling, induction of autophagy, and increased mitochondrial biogenesis and turnover. Genetic deletion of AMPKα1/α2 abolished these responses, establishing AMPK as a critical mediator of C273 activity. Pharmacological competition experiments further confirmed the target, as pretreatment with non-toxic concentrations of rotenone blocked C273 interaction with the quinone-binding site of mtCI and eliminated its neuroprotective effects. Repeated oral administration of C273 (20-80 mg/kg/day) to wild-type mice for one month produced no detectable cardiac or hepatic toxicity, indicating a favorable in vivo safety margin. Importantly, C273 activated these mechanisms and reduced Aβ and p-Tau levels in induced pluripotent stem cell-derived cerebral organoids from patients with sporadic AD. Collectively, these results establish controlled mtCI modulation as a therapeutic strategy and position C273 as a promising disease-modifying candidate for AD.

Abstract INTRODUCTION Mitochondrial dysfunction is implicated in Alzheimer's disease (AD), but whether it drives AD‐associated changes is unclear. We assessed transcriptomic alterations in the brains of Ndufs4 −/− mice, a model of mitochondrial complex I (mtCI) deficiency, and evaluated the therapeutic effects of the neuroprotective mtCI inhibitor CP2. METHODS Cortico‐hippocampal tissue from Ndufs4 −/− and wild‐type mice was subjected to transcriptomic analysis, followed by cross‐species comparisons to human late‐onset AD and familial AD mouse datasets. RESULTS Knockout of Ndufs4 ‐mediated mtCI deficiency disrupted mitochondrial homeostasis, energy metabolism, and synaptic gene expression, recapitulating transcriptomic signatures of AD. CP2 treatment partially reversed these changes, with female Ndufs4 −/− mice showing greater compensatory adaptations and treatment responses. DISCUSSION Loss of mtCI activity alone is sufficient to induce AD‐like molecular changes in the brain, independent of amyloid beta or phosphorylated tau. CP2‐mediated rescue highlights the potential of targeting mitochondria as a therapeutic strategy for AD. Sex‐specific responses suggest important considerations for personalized therapeutics. Highlights Activity of mitochondrial complex I (mtCI) affects broad mitochondrial and neuronal transcriptional networks. A reduction of mtCI activity is sufficient to induce transcriptomic changes reminiscent of those observed in late‐onset Alsheimer's disease (AD) patients and familial mouse models of AD. Pharmacological targeting of mtCI mediates neuroprotective signaling. Male and female mice have differential responses to the loss of mtCI activity and to the mitochondria‐targeted therapeutics. Mitochondria play a key role in AD development and treatment.

Abstract Despite recent approval of monoclonal antibodies that reduce amyloid (Aβ) accumulation, the development of disease-modifying strategies targeting the underlying mechanisms of Alzheimer’s disease (AD) is urgently needed. We demonstrate that mitochondrial complex I (mtCI) represents a druggable target, where its weak inhibition activates neuroprotective signaling, benefiting AD mouse models with Aβ and p-Tau pathologies. Rational design and structure‒activity relationship studies yielded novel mtCI inhibitors profiled in a drug discovery funnel designed to address their safety, selectivity, and efficacy. The new lead compound C458 is highly protective against Aβ toxicity, has favorable pharmacokinetics, and has minimal off-target effects. C458 exhibited excellent brain penetrance, activating neuroprotective pathways with a single dose. Preclinical studies in APP/PS1 mice were conducted via functional tests, metabolic assessment, in vivo 31 P- NMR spectroscopy, blood cytokine panels, ex vivo electrophysiology, and Western blotting. Chronic oral administration improved long-term potentiation, reduced oxidative stress and inflammation, and enhanced mitochondrial biogenesis, antioxidant signaling, and cellular energetics. These studies provide further evidence that the restoration of mitochondrial function and brain energetics in response to mild energetic stress represents a promising disease- modifying strategy for AD.

AbstractBackgroundAlzheimer’s Disease (AD) has no cure. We showed that partial inhibition of mitochondrial complex I (MCI) with small molecule CP2 induced adaptive stress response activating multiple neuroprotective mechanisms. Chronic treatment reduced inflammation, improved synaptic and mitochondrial functions, and blocked neurodegeneration in symptomatic APP/PS1 mice, a translational model of AD.MethodsAnalysis of mitochondrial morphology and mitochondria‐endoplasmic reticulum (ER) contacts (MERCS) was done in the hippocampal CA1 tissue from APP/PS1 mice treated with CP2 and compared to APP/PS1 and non‐transgenic mice treated with vehicle using serial block‐face scanning electron microscopy (SBFSEM) and three‐dimensional (3D) EM reconstructions combined with western blot analysis and next generation RNA sequencing.ResultsWe demonstrate that CP2 treatment restores mitochondrial morphology and mitochondria‐ ER communication, reducing ER stress in the APP/PS1 mouse brain. Using 3D EM reconstructions, we found that mitochondria in AD dendrites existed primarily as mitochondria‐on‐a‐string (MOAS), a morphological phenotype that is difficult to detect using conventional 2D EM or fluorescence spectroscopy. Compared to other morphological phenotypes, MOAS are extensively enveloped in the ER membranes forming multiple MERCS known to contribute to abnormal lipid and calcium homeostasis, and ER stress in AD. CP2 treatment specifically reduced MOAS formation, consistent with improved energy homeostasis in the brain, with concomitant reduction in MERCS, ER stress, and improved lipid homeostasis.ConclusionsThese data provide novel information on the role MOAS play in AD. Our data demonstrate that in response to energetic stress, mitochondria in dendrites acquire MOAS phenotype with increased interaction with the ER, which may synergistically contribute to the AD development. Since CP2 treatment alleviated the ER stress and improved mitochondrial dynamics and function, these data further support the development of partial MCI inhibitors as disease modifying strategy for AD.

AbstractBackgroundAlzheimer Disease (AD) currently has no effective treatments. A consistent failure of recent clinical trials focused on Aβ or pTau necessitates the identification of novel therapeutic targets and strategies. Since abnormal brain energy homeostasis and mitochondria dysfunction underlie early stages of AD, the development of treatments to restore these defects could be beneficial. We synthesized small molecules that selectively and mildly inhibit mitochondrial complex I (MCI) which restored synaptic and cognitive function in APP/PS1 and 3xTgAD mice. One of the most important mechanisms improved by the treatment included restoration of glucose uptake/utilization that resulted in augmented cellular energetics in mouse brains. However, mechanistic relationship between MCI inhibition, glucose uptake/utilization and mitochondrial function remains to be determined.MethodNeuroblastoma SH‐SY5Y cells that express mutant human APP protein (APPswe) and WT cells were treated with MCI inhibitors in a time‐ and dose‐dependent manner. Flow cytometry was used to measure the translocation of glucose transporters to the cell surface. Changes in protein expression in response to treatment were assessed using Western blot analysis. Changes in mitochondrial morphology were monitored using electron microscopy. Cellular energetics was assessed using a Seahorse Extracellular Flux Analyzer. The non‐radioactive Glucose Uptake‐Glo™ assay was utilized for measuring glucose uptake in cells.ResultsTreatment of with SH‐SY5Y‐APPswe cells with MCI inhibitors within an hour increased surface translocation of GLUTs enhancing glucose uptake and utilization. The effect persisted for 48 hrs. The mechanism of enhanced glucose uptake was associated with AMPK‐dependent activation of NRF2 pathway but not AKT. Furthermore, the improvement in the bioenergetic parameters including Spare Respiratory Capacity, an indicator of the mitochondrial ability to produce energy under stress conditions, and ATP levels were found 24 hrs after the compound treatment. Treatment activated mitochondrial morphofunctional pathway (e.g., fission, fusion, biogenesis and turnover) consistent with the improved cellular bioenergetics. Similar mechanisms were confirmed in the brain tissue of 3xTgAD mice treated with one of the compounds.ConclusionsPartial MCI inhibition activates multiple mechanisms essential for improved energy homeostasis and mitochondrial function. These mechanisms could be consistently induced by our compounds in vivo and in human brain cells support translational potential of this therapeutic strategy.

Abstract Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high‐resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.

Alzheimer’s disease (AD) has no cure. Earlier, we showed that partial inhibition of mitochondrial complex I (MCI) with the small molecule CP2 induces an adaptive stress response, activating multiple neuroprotective mechanisms. Chronic treatment reduced inflammation, Aβ and pTau accumulation, improved synaptic and mitochondrial functions, and blocked neurodegeneration in symptomatic APP/PS1 mice, a translational model of AD. Here, using serial block-face scanning electron microscopy (SBFSEM) and three-dimensional (3D) EM reconstructions combined with Western blot analysis and next-generation RNA sequencing, we demonstrate that CP2 treatment also restores mitochondrial morphology and mitochondria-endoplasmic reticulum (ER) communication, reducing ER and unfolded protein response (UPR) stress in the APP/PS1 mouse brain. Using 3D EM volume reconstructions, we show that in the hippocampus of APP/PS1 mice, dendritic mitochondria primarily exist as mitochondria-on-a-string (MOAS). Compared to other morphological phenotypes, MOAS have extensive interaction with the ER membranes, forming multiple mitochondria-ER contact sites (MERCS) known to facilitate abnormal lipid and calcium homeostasis, accumulation of Aβ and pTau, abnormal mitochondrial dynamics, and apoptosis. CP2 treatment reduced MOAS formation, consistent with improved energy homeostasis in the brain, with concomitant reductions in MERCS, ER/UPR stress, and improved lipid homeostasis. These data provide novel information on the MOAS-ER interaction in AD and additional support for the further development of partial MCI inhibitors as a disease-modifying strategy for AD.

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.