The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool

Missense mutations in the cardiac ryanodine receptor type 2 (RyR2) characteristically cause catecholaminergic arrhythmias. Reminiscent of the phenotype in patients, RyR2-R2474S knockin mice develop exercise-induced ventricular tachyarrhythmias. In cardiomyocytes, increased mitochondrial matrix Ca2+ uptake was recently linked to non-linearly enhanced ATP synthesis with important implications for cardiac redox metabolism. We hypothesize that catecholaminergic stimulation and contractile activity amplify mitochondrial oxidation pathologically in RyR2-R2474S cardiomyocytes. To investigate this question, we generated double transgenic RyR2-R2474S mice expressing a mitochondria-restricted fluorescent biosensor to monitor the glutathione redox potential (EGSH). Electrical field pacing-evoked RyR2-WT and RyR2-R2474S cardiomyocyte contractions resulted in a small but significant baseline EGSH increase. Importantly, β-adrenergic stimulation resulted in excessive EGSH oxidization of the mitochondrial matrix in RyR2-R2474S cardiomyocytes compared to baseline and RyR2-WT control. Physiologically β-adrenergic stimulation significantly increased mitochondrial EGSH further in intact beating RyR2-R2474S but not in RyR2-WT control Langendorff perfused hearts. Finally, this catecholaminergic EGSH increase was significantly attenuated following treatment with the RyR2 channel blocker dantrolene. Together, catecholaminergic stimulation and increased diastolic Ca2+ leak induce a strong, but dantrolene-inhibited mitochondrial EGSH oxidization in RyR2-R2474S cardiomyocytes.

Combinatorial G x G x E CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes1-3. The family is highly redundant and their transport activities are coupled to metabolic state. Here, we introduce a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states — glucose, galactose, OXPHOS inhibition, and absence of pyruvate — designed to unmask the inter-dependence of these genes. In total, we screened 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recovered 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrated that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells were genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. Another GxE interaction hit revealed non-equivalence of the paralogous ATP/ADP exchangers (ANTs) with ANT2 specifically required during OXPHOS inhibition. GxG analysis highlighted a buffering interaction between the iron transporter SLC25A37 and the poorly characterized SLC25A39. Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis suggest SLC25A39 is critical for mitochondrial glutathione (GSH) transport. Our work underscores the importance of systematically investigating family-wide genetic interactions between mitochondrial transporters across many metabolic environments.

Combinatorial G x G x E CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we introduce a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states- glucose, galactose, OXPHOS inhibition, and absence of pyruvate-designed to unmask the inter-dependence of these genes. In total, we screened 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recovered 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrated that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells was genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. Another GxE interaction hit revealed non-equivalence of the paralogous ATP/ADP exchangers (ANTs) with ANT2 specifically required during OXPHOS inhibition. GxG analysis highlighted a buffering interaction between the iron transporter SLC25A37 and the poorly characterized SLC25A39. Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis suggests SLC25A39 is critical for mitochondrial glutathione (GSH) transport. Our work underscores the importance of systemetically investigating family-wide genetic interactions between mitochondrial transporters across many metabolic environments.

SLC25A39 is necessary for mitochondrial glutathione import in mammalian cells

Glutathione (GSH) is a small molecule thiol abundantly present in all eukaryotes with key roles in oxidative metabolism. Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions. GSH is exclusively synthesized in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remain elusive. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, to regulate GSH transport into mitochondria. SLC25A39 loss reduces mitochondrial GSH import and abundance without impacting whole cell GSH levels. Cells lacking both SLC25A39 and its paralog SLC25A40 exhibit defects in the activity and stability of iron-sulfur cluster containing proteins. Moreover, mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Remarkably, the heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enabled mitochondrial GSH production and ameliorated the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH import machinery.

Diabetic Nephropathy and COVID-19: The Potential Role of Immune Actors

Nowadays, type II diabetes mellitus, more specifically ensuing diabetic nephropathy, and severe COVID-19 disease are known to be closely associated. The exact mechanisms behind this association are less known. An implication for the angiotensin-converting enzyme 2 remains controversial. Some researchers have started looking into other potential actors, such as neuropilin-1, mitochondrial glutathione, vitamin D, and DPP4. In particular, neuropilin-1 seems to play an important role in the underlying mechanism linking COVID-19 and diabetic nephropathy. We suggest, based on the findings in this review, that its up-regulation in the diabetic kidney facilitates viral entry in this tissue, and that the engagement of both processes leads to a depletion of neuropilin-1, which was demonstrated to be strongly associated with the pathogenesis of DN. More studies are needed to confirm this hypothesis, and research should be directed towards elucidating the potential roles of all these suggested actors and eventually discovering new therapeutic strategies that could reduce the burden of COVID-19 in patients with diabetic nephropathy.

Ginsenoside Re Protects against Serotonergic Behaviors Evoked by 2,5-Dimethoxy-4-iodo-amphetamine in Mice via Inhibition of PKCδ-Mediated Mitochondrial Dysfunction

It has been recognized that serotonin 2A receptor (5-HT2A) agonist 2,5-dimethoxy-4-iodo-amphetamine (DOI) impairs serotonergic homeostasis. However, the mechanism of DOI-induced serotonergic behaviors remains to be explored. Moreover, little is known about therapeutic interventions against serotonin syndrome, although evidence suggests that ginseng might possess modulating effects on the serotonin system. As ginsenoside Re (GRe) is well-known as a novel antioxidant in the nervous system, we investigated whether GRe modulates 5-HT2A receptor agonist DOI-induced serotonin impairments. We proposed that protein kinase Cδ (PKCδ) mediates serotonergic impairments. Treatment with GRe or 5-HT2A receptor antagonist MDL11939 significantly attenuated DOI-induced serotonergic behaviors (i.e., overall serotonergic syndrome behaviors, head twitch response, hyperthermia) by inhibiting mitochondrial translocation of PKCδ, reducing mitochondrial glutathione peroxidase activity, mitochondrial dysfunction, and mitochondrial oxidative stress in wild-type mice. These attenuations were in line with those observed upon PKCδ inhibition (i.e., pharmacologic inhibitor rottlerin or PKCδ knockout mice). Furthermore, GRe was not further implicated in attenuation mediated by PKCδ knockout in mice. Our results suggest that PKCδ is a therapeutic target for GRe against serotonergic behaviors induced by DOI.