Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death.

Glutamate is the dominant excitatory neurotransmitter in the brain, but under conditions of metabolic stress it can accumulate to excitotoxic levels. Although pharmacologic modulation of excitatory amino acid receptors is well studied, minimal consideration has been given to targeting mitochondrial glutamate metabolism to control neurotransmitter levels. Here we demonstrate that chemical inhibition of the mitochondrial pyruvate carrier (MPC) protects primary cortical neurons from excitotoxic death. Reductions in mitochondrial pyruvate uptake do not compromise cellular energy metabolism, suggesting neuronal metabolic flexibility. Rather, MPC inhibition rewires mitochondrial substrate metabolism to preferentially increase reliance on glutamate to fuel energetics and anaplerosis. Mobilizing the neuronal glutamate pool for oxidation decreases the quantity of glutamate released upon depolarization and, in turn, limits the positive-feedback cascade of excitotoxic neuronal injury. The finding links mitochondrial pyruvate metabolism to glutamatergic neurotransmission and establishes the MPC as a therapeutic target to treat neurodegenerative diseases characterized by excitotoxicity

Wolfram Syndrome protein, Miner1, regulates sulphydryl redox status, the unfolded protein response, and Ca2+ homeostasis.

Miner1 is a redox-active 2Fe2S cluster protein. Mutations in Miner1 result in Wolfram Syndrome, a metabolic disease associated with diabetes, blindness, deafness, and a shortened lifespan. Embryonic fibroblasts from Miner1(-/-) mice displayed ER stress and showed hallmarks of the unfolded protein response. In addition, loss of Miner1 caused a depletion of ER Ca(2+) stores, a dramatic increase in mitochondrial Ca(2+) load, increased reactive oxygen and nitrogen species, an increase in the GSSG/GSH and NAD(+)/NADH ratios, and an increase in the ADP/ATP ratio consistent with enhanced ATP utilization. Furthermore, mitochondria in fibroblasts lacking Miner1 displayed ultrastructural alterations, such as increased cristae density and punctate morphology, and an increase in O2 consumption. Treatment with the sulphydryl anti-oxidant N-acetylcysteine reversed the abnormalities in the Miner1 deficient cells, suggesting that sulphydryl reducing agents should be explored as a treatment for this rare genetic disease

Colorectal carcinomas in MUTYH-associated polyposis display histopathological similarities to microsatellite unstable carcinomas

<p>Abstract</p> <p>Background</p> <p>MUTYH-associated polyposis (MAP) is a recessively inherited disorder which predisposes biallelic carriers for a high risk of polyposis and colorectal carcinoma (CRC). Since about one third of the biallelic MAP patients in population based CRC series has no adenomas, this study aimed to identify specific clinicopathological characteristics of MAP CRCs and compare these with reported data on sporadic and Lynch CRCs.</p> <p>Methods</p> <p>From 44 MAP patients who developed ≥ 1 CRCs, 42 of 58 tumours were analyzed histologically and 35 immunohistochemically for p53 and beta-catenin. Cell densities of CD3, CD8, CD57, and granzyme B positive lymphocytes were determined. <it>KRAS2</it>, the mutation cluster region (MCR) of <it>APC, p53</it>, and <it>SMAD4 </it>were analyzed for somatic mutations.</p> <p>Results</p> <p>MAP CRCs frequently localized to the proximal colon (69%, 40/58), were mucinous in 21% (9/42), and had a conspicuous Crohn's like infiltrate reaction in 33% (13/40); all of these parameters occurred at a higher rate than reported for sporadic CRCs. Tumour infiltrating lymphocytes (TILs) were also highly prevalent in MAP CRCs. Somatic <it>APC </it>MCR mutations occurred in 14% (5/36) while 64% (23/36) had <it>KRAS2 </it>mutations (22/23 c.34G>T). G>T tranversions were found in <it>p53 </it>and <it>SMAD4</it>, although the relative frequency compared to other mutations was low.</p> <p>Conclusion</p> <p>MAP CRCs show some similarities to micro-satellite unstable cancers, with a preferential proximal location, a high rate of mucinous histotype and increased presence of TILs. These features should direct the practicing pathologist towards a MAP aetiology of CRC as an alternative for a mismatch repair deficient cause. High frequent G>T transversions in <it>APC </it>and <it>KRAS2 </it>(mutated in early tumour development) but not in <it>P53 </it>and <it>SMAD4 </it>(implicated in tumour progression) might indicate a predominant MUTYH effect in <it>early </it>carcinogenesis.</p

In situ measurements of mitochondrial matrix enzyme activities using plasma and mitochondrial membrane permeabilization agents

Activities of enzymes localized to the mitochondrial matrix of mammalian cells are often critical regulatory steps in cellular metabolism. As such, measurement of matrix enzyme activities in response to genetic modifications or drug interventions is often desired. However, measurements in intact cells are often hampered by the presence of other isozymes in the cytoplasm as well as the inability to deliver enzyme substrates across cellular membranes. Classic approaches to liberate matrix enzymes utilize harsh treatments that disrupt intracellular architecture or require significant starting material to allow mitochondrial isolation prior to sample extraction. We describe a method using permeabilization reagents for both the plasma and mitochondrial membranes to allow in situ measurement of matrix enzyme activities. It is applied to adherent cell monolayers in 96-well plates treated with perfringolysin O to permeabilize the plasma membrane and alamethicin to permeabilize the mitochondrial inner membrane. We present three examples validated with inhibitor sensitivity: (i) Complex I-mediated oxygen consumption driven by NADH, (ii) ATP hydrolysis by the F1FO complex measuring pH changes in an Agilent Seahorse XF Analyzer, and (iii) Mitochondrial glutaminase (GLS1) activity in a coupled reaction monitoring NADH fluorescence in a plate reader

Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

<div><p>Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.</p> </div

Bax-induced pore formation requires heat-labile protein(s) and is inhibited by mdivi-1 analogs.

<p>For heat-induced protein inactivation, OMVs were preincubated at 68°C for 10 min and equilibrated at room temperature. Dextran-fluorescein release induced by 100 nM Bax in the presence of 40 nM cBid was inhibited in heat-treated OMVs (A), but restored at higher (400–800 nM) Bax concentrations (B). OMVs were incubated with mdivi-1 analogs B and H for 5 min prior to the addition of Bax (C–E). Note that at 25°C, compound H (25–50 µM) completely inhibits dextran release (D); at 45°C, however, this compound produces merely a long lag phase without much effect on the rate of pore formation (E). Black lines, control OMVs; red lines, heat-treated OMVs; purple lines, OMVs treated with the mdivi-1 analogs. Arrows indicate additions of Bax; cBid was added 2–3 min prior to Bax. Data shown are representative of at least three independent experiments (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001394#pbio.1001394.s006" target="_blank">Figure S6</a>).</p

The kinetic parameters showed saturation with respect to cBid, but not Bax concentrations, and exhibited an inverse relationship between [cBid] and [Bax].

<p>(A) Dependence of the rate constants on Bax concentration was linear. Some small deviations from linearity at low Bax concentrations probably reflect systematic error due to the need for long incubations under those conditions. (B) Dependence of the rate constants on cBid concentration was saturable and displayed an inverse relationship with the amount of Bax (lower EC₅₀ values at higher Bax concentration). The assay was performed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001394#pbio-1001394-g002" target="_blank">Figure 2A,C</a>. Data shown in (A) and (B) are representative of three independent experiments. Kinetic parameters were determined using <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001394#pbio.1001394.e009" target="_blank">equation 9</a>; cBid dose-response data (B) were fit to hyperbolic curves (solid lines) using GraphPad Prizm software. (C) cBid-dependent dextran-Cascade Blue release induced by 70 nM (blue lines) and 210 nM (red lines) Bax at indicated concentrations of cBid. Bax was added at time zero; cBid was added 2 min prior to Bax. The data were used for quantification of kinetic parameters shown in (B). Dextran content is defined as the fluorescence signal at a given time point relative to the maximal fluorescence and quantified as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001394#s4" target="_blank">Materials and Methods</a>. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001394#pbio.1001394.s005" target="_blank">Figure S5</a> demonstrating theoretical Bax dose-response curves. (D) Molecular scheme, modified from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001394#pbio-1001394-g004" target="_blank">Figure 4B</a> to include the data in (C) confirming that cBid activates Bax nonstoichiometrically, that is catalytically. Also, as k₁ shows the same dependencies on [cBid] and [Bax] as k₂, we infer that activated Bax enters into both tier I and tier II reactions. As k₁ and k2 are linearly related to [Bax], the entire process shows no cooperativity with regard to Bax, and we conclude that Bax promotes the Catalyst Assembly reaction but cannot be the monomer M. Activated Bax could enter into the tier II reaction II, either as a catalyst or a direct participant. Data are representative of three independent experiments.</p