Sunil Sahdeo

Dyclonine rescues frataxin deficiency in animal models and buccal cells of patients with Friedreich's ataxia

Inherited deficiency in the mitochondrial protein frataxin (FXN) causes the rare disease Friedreichs ataxia (FA), for which there is no successful treatment. We identified a redox deficiency in FA cells and used this to model the disease. We screened a 1600-compound library to identify existing drugs, which could be of therapeutic benefit. We identified the topical anesthetic dyclonine as protective. Dyclonine increased FXN transcript and FXN protein dose-dependently in FA cells and brains of animal models. Dyclonine also rescued FXN-dependent enzyme deficiencies in the iron–sulfur enzymes, aconitase and succinate dehydrogenase. Dyclonine induces the Nrf2 [nuclear factor (erythroid-derived 2)-like 2] transcription factor, which we show binds an upstream response element in the FXN locus. Additionally, dyclonine also inhibited the activity of histone methyltransferase G9a, known to methylate histone H3K9 to silence FA chromatin. Chronic dosing in a FA mouse model prevented a performance decline in balance beam studies. A human clinical proof-of-concept study was completed in eight FA patients dosed twice daily using a 1% dyclonine rinse for 1 week. Six of the eight patients showed an increase in buccal cell FXN levels, and fold induction was significantly correlated with disease severity. Dyclonine represents a novel therapeutic strategy that can potentially be repurposed for the treatment of FA.

Facilitatory interplay in α1A and β2 adrenoceptor function reveals a non-Gq signaling mode: implications for diversification of intracellular signal transduction

Agonist occupied α1-adrenoceptors (α1-ARs) engage several signaling pathways, including phosphatidylinositol hydrolysis, calcium mobilization, arachidonic acid release, mitogen-activated protein (MAP) kinase activation, and cAMP accumulation. The natural agonist norepinephrine (NE) activates with variable affinity and intrinsic efficacy all adrenoceptors, and in cells that coexpress α1- and β-AR subtypes, such as cardiomyocytes, this leads to coactivation of multiple downstream pathways. This may result in pathway cross-talk with significant consequences to heart physiology and pathologic state. To dissect signaling components involved specifically in α1A- and β2-AR signal interplay, we have developed a recombinant model system that mimics the levels of receptor expression observed in native cells. We followed intracellular Ca2+ mobilization to monitor in real time the activation of both Gq and Gs pathways. We found that coactivation of α1A- and β2-AR by the nonselective agonist NE or via a combination of the highly selective α1A-AR agonist A61603 and the β-selective agonist isoproterenol led to increases in Ca2+ influx from the extracellular compartment relative to stimulation with A61603 alone, with no effect on the associated transient release of Ca2+ from intracellular stores. This effect became more evident upon examination of an α1A-AR variant exhibiting a partial defect in coupling to Gq, and we attribute it to potentiation of a non Gq-pathway, uncovered by application of a combination of xestospongin C, an endoplasmic reticulum inositol 1,4,5-triphosphate receptor blocker, and 2-aminoethoxydiphenyl borate, a nonselective storeoperated Ca2+ entry channel blocker. We also found that stimulation with A61603 of a second α1A-AR variant entirely unable to signal induced no Ca2+ unless β2-AR was concomitantly activated. These results may be accounted for by the presence of α1A/β2-AR heterodimers or alternatively by specific adrenoceptor signal cross-talk resulting in distinct pharmacological behavior. Finally, our findings provide a new conceptual framework to rationalize outcomes from clinical studies targeting α- and β-adrenoceptors.

Characterization of RO5126946, a novel α7 nicotinic acetylcholine receptor positive allosteric modulator

Both preclinical evidence and clinical evidence suggest that α7 nicotinic acetylcholine receptor activation (α7nAChR) improves cognitive function, the decline of which is associated with conditions such as Alzheimer’s disease and schizophrenia. Moreover, allosteric modulation of α7nAChR is an emerging therapeutic strategy in an attempt to avoid the rapid desensitization properties associated with the α7nAChR after orthosteric activation. We used a calcium assay to screen for positive allosteric modulators (PAMs) of α7nAChR and report on the pharmacologic characterization of the novel compound RO5126946 (5-chloro-N-[(1S,3R)-2,2-dimethyl-3-(4-sulfamoyl-phenyl)-cyclopropyl]-2-methoxy-benzamide), which allosterically modulates α7nAChR activity. RO5126946 increased acetylcholine-evoked peak current and delayed current decay but did not affect the recovery of α7nAChRs from desensitization. In addition, RO5126946’s effects were absent when nicotine-evoked currents were completely blocked by coapplication of the α7nAChR-selective antagonist methyl-lycaconitine. RO5126946 enhanced α7nAChR synaptic transmission and positively modulated GABAergic responses. The absence of RO5126946 effects at human α4β2nAChR and 5-hydroxytryptamine 3 receptors, among others, indicated selectivity for α7nAChRs. In vivo, RO5126946 is orally bioavailable and brain-penetrant and improves associative learning in a scopolamine-induced deficit model of fear conditioning in rats. In addition, procognitive effects of RO5126946 were investigated in the presence of nicotine to address potential pharmacologic interactions on behavior. RO5126946 potentiated nicotine’s effects on fear memory when both compounds were administered at subthreshold doses and did not interfere with procognitive effects observed when both compounds were administered at effective doses. Overall, RO5126946 is a novel α7nAChR PAM with cognitive-enhancing properties.

Effects of alkyl side chain modification of coenzyme Q10 on mitochondrial respiratory chain function and cytoprotection

The effect of the alkyl side chain length of coenzyme Q10 on mitochondrial respiratory chain function has been investigated by the use of synthetic ubiquinone derivatives. Three analogues (3, 4 and 6) were identified that exhibited significantly improved effects on mitochondrial oxygen consumption and mitochondrial membrane potential, and also conferred significant cytoprotection on cultured mammalian cells in which glutathione had been depleted by treatment with diethyl maleate. The analogues also exhibited lesser inhibition of the electron transport chain than idebenone. The results obtained provide guidance for the design of CoQ10 analogues with improved activity compared to that of idebenone (1), the latter of which is undergoing evaluation in the clinic as a therapeutic agent.

Shc depletion stimulates brown fat activity in vivo and in vitro

Adipose tissue is an important metabolic organ that integrates a wide array of homeostatic processes and is crucial for whole‐body insulin sensitivity and energy metabolism. Brown adipose tissue (BAT) is a key thermogenic tissue with a well‐established role in energy expenditure. BAT dissipates energy and protects against both hypothermia and obesity. Thus, BAT stimulation therapy is a rational strategy for the looming pandemic of obesity, whose consequences and comorbidities have a huge impact on the aged. Shc‐deficient mice (ShcKO) were previously shown to be lean, insulin sensitive, and resistant to high‐fat diet and obesity. We investigated the contribution of BAT to this phenotype. Insulin‐dependent BAT glucose uptake was higher in ShcKO mice. Primary ShcKO BAT cells exhibited increased mitochondrial respiration; increased expression of several mitochondrial and lipid‐oxidative enzymes was observed in ShcKO BAT. Levels of brown fat‐specific markers of differentiation, UCP1, PRDM16, ELOVL3, and Cox8b, were higher in ShcKO BAT. In vitro, Shc knockdown in BAT cell line increased insulin sensitivity and metabolic activity. In vivo, pharmacological stimulation of ShcKO BAT resulted in higher energy expenditure. Conversely, pharmacological inhibition of BAT abolished the improved metabolic parameters, that is the increased insulin sensitivity and glucose tolerance of ShcKO mice. Similarly, in vitro Shc knockdown in BAT cell lines increased their expression of UCP1 and metabolic activity. These data suggest increased BAT activity significantly contributes to the improved metabolic phenotype of ShcKO mice.

Dimethyl fumarate mediates Nrf2-dependent mitochondrial biogenesis in mice and humans

The induction of mitochondrial biogenesis could potentially alleviate mitochondrial and muscle disease. We show here that dimethyl fumarate (DMF) dose-dependently induces mitochondrial biogenesis and function dosed to cells in vitro, and also dosed in vivo to mice and humans. The induction of mitochondrial gene expression is more dependent on DMFs target Nrf2 than hydroxycarboxylic acid receptor 2 (HCAR2). Thus, DMF induces mitochondrial biogenesis primarily through its action on Nrf2, and is the first drug demonstrated to increase mitochondrial biogenesis with in vivo human dosing. This is the first demonstration that mitochondrial biogenesis is deficient in Multiple Sclerosis patients, which could have implications for MS pathophysiology and therapy. The observation that DMF stimulates mitochondrial biogenesis, gene expression and function suggests that it could be considered for mitochondrial disease therapy and/or therapy in muscle disease in which mitochondrial function is important.

In Vitro Evaluation of Mitochondrial Function and Estrogen Signaling in Cell Lines Exposed to the Antiseptic Cetylpyridinium Chloride

Background: Quaternary ammonium salts (QUATS), such as cetylpyridinium chloride (CPC) and benzalkonium chloride (BAK), are frequently used in antiseptic formulations, including toothpastes, mouthwashes, lozenges, throat and nasal sprays, and as biocides. Although in a recent ruling, the U.S. Food and Drug Administration (FDA) banned CPC from certain products and requested more data on BAK’s efficacy and safety profile, QUATS, in general, and CPC and BAK, in particular, continue to be used in personal health care, food, and pharmaceutical and cleaning industries. Objectives: We aimed to assess CPCs effects on mitochondrial toxicity and endocrine disruption in vitro. Method: Mitochondrial O2 consumption and adenosine triphosphate (ATP) synthesis rates of osteosarcoma cybrid cells were measured before and after CPC and BAK treatment. Antiestrogenic effects of the compounds were measured by a luciferase-based assay using recombinant human breast carcinoma cells (VM7Luc4E2, ERalpha-positive). Results: CPC inhibited both mitochondrial O2 consumption [half maximal inhibitory concentration (IC50): 3.8μM] and ATP synthesis (IC50: 0.9μM), and additional findings supported inhibition of mitochondrial complex 1 as the underlying mechanism for these effects. In addition, CPC showed concentration-dependent antiestrogenic activity half maximal effective concentration [(EC50): 4.5μM)]. BAK, another antimicrobial QUATS that is structurally similar to CPC, and the pesticide rotenone, a known complex 1 inhibitor, also showed mitochondrial inhibitory and antiestrogenic effects. In all three cases, there was overlap of the antiestrogenic activity with the mitochondrial inhibitory activity. Conclusions: Mitochondrial inhibition in vitro occurred at a CPC concentration that may be relevant to human exposures. The antiestrogenic activity of CPC, BAK, rotenone, and triclosan may be related to their mitochondrial inhibitory activity. Our findings support the need for additional research on the mitochondrial inhibitory and antiestrogenic effects of QUATS, including CPC and BAK. https://doi.org/10.1289/EHP1404