Fluorescence microscopy imaging of mitochondrial metabolism in cancer cells.

Mitochondrial metabolism is an important contributor to cancer cell survival and proliferation that coexists with enhanced glycolytic activity. Measuring mitochondrial activity is useful to characterize cancer metabolism patterns, to identify metabolic vulnerabilities and to identify new drug targets. Optical imaging, especially fluorescent microscopy, is one of the most valuable tools for studying mitochondrial bioenergetics because it provides semiquantitative and quantitative readouts as well as spatiotemporal resolution of mitochondrial metabolism. This review aims to acquaint the reader with microscopy imaging techniques currently used to determine mitochondrial membrane potential (ΔΨm), nicotinamide adenine dinucleotide (NADH), ATP and reactive oxygen species (ROS) that are major readouts of mitochondrial metabolism. We describe features, advantages, and limitations of the most used fluorescence imaging modalities: widefield, confocal and multiphoton microscopy, and fluorescent lifetime imaging (FLIM). We also discus relevant aspects of image processing. We briefly describe the role and production of NADH, NADHP, flavins and various ROS including superoxide and hydrogen peroxide and discuss how these parameters can be analyzed by fluorescent microscopy. We also explain the importance, value, and limitations of label-free autofluorescence imaging of NAD(P)H and FAD. Practical hints for the use of fluorescent probes and newly developed sensors for imaging ΔΨm, ATP and ROS are described. Overall, we provide updated information about the use of microscopy to study cancer metabolism that will be of interest to all investigators regardless of their level of expertise in the field.

Fast Determination of Mitochondrial Metabolism and Respiratory Complex Activity in Permeabilized and Intact Cells.

Assessment of mitochondrial metabolism is multidimensional and time consuming, usually requiring specific training. Respiration, NADH generation, and mitochondrial membrane potential (ΔΨm) are dynamic readouts of the metabolism and bioenergetics of mitochondria. Methodologies available to determine functional parameters in isolated mitochondria and permeabilized cells are sometimes of limited use or inapplicable to studies in live cells. In particular, the sequential assessment of the activity of each complex in the electron transport chain has not been reported in intact cells. Here, we describe a novel approach to sequentially assess electron flow through all respiratory complexes in permeabilized and intact cells by respirometry. We also describe a highly sensitive and fast method to assess ΔΨm and NADH generation in live cells using plate reader assays. Thus, our combined method allows a relatively inexpensive and fast determination of three major readouts of mitochondrial function in a few hours, using equipment that is frequently available in many laboratories worldwide.

Polyploid giant cancer cells are dependent on cholesterol for progeny formation through amitotic division.

Polyploid Giant Cancer Cells (PGCC) are increasingly being recognized as drivers of cancer recurrence. Therapy stress promotes the formation of these cells, which upon stress cessation often successfully generate more aggressive progeny that repopulate the tumor. Therefore, identification of potential PGCC vulnerabilities is key to preventing therapy failure. We have previously demonstrated that PGCC progeny formation depends on the lysosomal enzyme acid ceramidase (ASAH1). In this study, we compared transcriptomes of parental cancer cells and PGCC in the absence or presence of the ASAH1 inhibitor LCL521. Results show that PGCC express less INSIG1, which downregulates cholesterol metabolism and that inhibition of ASAH1 increased HMGCR which is the rate limiting enzyme in cholesterol synthesis. Confocal microscopy revealed that ceramide and cholesterol do not colocalize. Treatment with LCL521 or simvastatin to inhibit ASAH1 or HMGCR, respectively, resulted in accumulation of ceramide at the cell surface of PGCC and prevented PGCC progeny formation. Our results suggest that similarly to inhibition of ASAH1, disruption of cholesterol signaling is a potential strategy to interfere with PGCC progeny formation.

Small molecules targeting the NADH-binding pocket of VDAC modulate mitochondrial metabolism in hepatocarcinoma cells.

Voltage dependent anion channels (VDAC) control the flux of most anionic respiratory substrates, ATP, ADP, and small cations, crossing the outer mitochondrial membrane. VDAC closure contributes to the partial suppression of mitochondrial metabolism that favors the Warburg phenotype of cancer cells. Recently, it has been shown that NADH binds to a specific pocket in the inner surface of VDAC1, also conserved in VDAC2 and 3, closing the channel. We hypothesized that binding of small molecules to the NADH pocket, maintain VDAC in an open configuration by preventing closure induced by NADH and possible other endogenous regulators. We screened in silico, the South Carolina Compound Collection SC3 (~100,000 proprietary molecules), using shape-based queries of the NADH binding region of VDAC. After molecular docking of selected compounds, we physically screened candidates using mitochondrial membrane potential (ΔΨm), as an overall readout of mitochondrial metabolism. We identified SC18, as the most potent compound. SC18 bound to VDAC1, as assessed by a thermal shift assay. Short-term treatment with SC18 decreased ΔΨm in SNU-449 and HepG2 human hepatocarcinoma cells. Mitochondrial depolarization was similar in wild type, VDAC1/2, 1/3, and 2/3 double KO HepG2 cells indicating that the effect of SC18 was not VDAC isoform-dependent. In addition, SC18 decreased mitochondrial NADH and cellular ATP production; and increased basal respiration. Long-term exposure to SC18, decreased cell proliferation as determined by wound-healing and cell viability assays. In summary, SC18 is a novel VDAC-targeting small molecule that induces mitochondrial dysfunction and inhibits cell proliferation.

Carbon Monoxide Activates PERK-Regulated Autophagy to Induce Immunometabolic Reprogramming and Boost Antitumor T-cell Function.

Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells' fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER-mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory-like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control. SIGNIFICANCE: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.

Mitochondrial depolarization after acute ethanol treatment drives mitophagy in living mice.

Ethanol increases hepatic mitophagy driven by unknown mechanisms. Type 1 mitophagy sequesters polarized mitochondria for nutrient recovery and cytoplasmic remodeling. In Type 2, mitochondrial depolarization (mtDepo) initiates mitophagy to remove the damaged organelles. Previously, we showed that acute ethanol administration produces reversible hepatic mtDepo. Here, we tested the hypothesis that ethanol-induced mtDepo initiates Type 2 mitophagy. GFP-LC3 transgenic mice were gavaged with ethanol (2-6 g/kg) with and without pre-treatment with agents that decrease or increase mtDepo-Alda-1, tacrolimus, or disulfiram. Without ethanol, virtually all hepatocytes contained polarized mitochondria with infrequent autophagic GFP-LC3 puncta visualized by intravital microscopy. At ~4 h after ethanol treatment, mtDepo occurred in an all-or-none fashion within individual hepatocytes, which increased dose dependently. GFP-LC3 puncta increased in parallel, predominantly in hepatocytes with mtDepo. Mitochondrial PINK1 and PRKN/parkin also increased. After covalent labeling of mitochondria with MitoTracker Red (MTR), GFP-LC3 puncta encircled MTR-labeled mitochondria after ethanol treatment, directly demonstrating mitophagy. GFP-LC3 puncta did not associate with fat droplets visualized with BODIPY558/568, indicating that increased autophagy was not due to lipophagy. Before ethanol administration, rhodamine-dextran (RhDex)-labeled lysosomes showed little association with GFP-LC3. After ethanol treatment, TFEB (transcription factor EB) translocated to nuclei, and lysosomal mass increased. Many GFP-LC3 puncta merged with RhDex-labeled lysosomes, showing autophagosomal processing into lysosomes. After ethanol treatment, disulfiram increased, whereas Alda-1 and tacrolimus decreased mtDepo, and mitophagy changed proportionately. In conclusion, mtDepo after acute ethanol treatment induces mitophagic sequestration and subsequent lysosomal processing.Abbreviations : AcAld, acetaldehyde; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ALD, alcoholic liver disease; Alda-1, N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; LMNB1, lamin B1; MAA, malondialdehyde-acetaldehyde adducts; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MPT, mitochondrial permeability transition; mtDAMPS, mitochondrial damage-associated molecular patterns; mtDepo, mitochondrial depolarization; mtDNA, mitochondrial DNA; MTR, MitoTracker Red; PI, propidium iodide; PINK1, PTEN induced putative kinase 1; PRKN, parkin; RhDex, rhodamine dextran; TFEB, transcription factor EB; Tg, transgenic; TMRM, tetramethylrhodamine methylester; TOMM20, translocase of outer mitochondrial membrane 20; VDAC, voltage-dependent anion channel.

Non-enzymatic glycoxidation linked with nutrition enhances the tumorigenic capacity of prostate cancer epithelia through AGE mediated activation of RAGE in cancer associated fibroblasts.

The molecular implications of food consumption on cancer etiology are poorly defined. The rate of nutrition associated non-enzymatic glycoxidation, a reaction that occurs between reactive carbonyl groups on linear sugars and nucleophilic amino, lysyl and arginyl groups on fats and proteins, is rapidly increased by food cooking and manufacturing processes. In this study, we assign nutrition-associated glycoxidation with significant oncogenic potential, promoting prostate tumor growth, progression, and metastasis in vivo. Advanced glycation end products (AGEs) are the final irreversible product of non-enzymatic glycoxidation. Exogenous treatment of prostate tumor cells with a single AGE peptide replicated glycoxidation induced tumor growth in vivo. Mechanistically, receptor for AGE (RAGE) deficiency in the stroma inhibited AGE mediated tumor growth. Functionally, AGE treatment induced RAGE dimerization in activated fibroblasts which sustained and increased the migratory potential of tumor epithelial cells. These data identify a novel nutrition associated pathway that can promote a tissue microenvironment conducive for aggressive tumor growth. Targeted and/or interventional strategies aimed at reducing AGE bioavailability as a consequence of nutrition may be viewed as novel chemoprevention initiatives.

Flavin Adenine Dinucleotide Depletion Caused by electron transfer flavoprotein subunit alpha Haploinsufficiency Leads to Hepatic Steatosis and Injury in Zebrafish.

The electron transfer flavoprotein (ETF) complex, made up of the ETF alpha subunit (ETFA), ETF beta subunit (ETFB), and ETF dehydrogenase (ETFDH), regulates fatty acid ß-oxidation activity while scavenging leaked electrons through flavin adenine dinucleotide (FAD)/reduced form FAD (FADH2) redox reactions in mitochondria. Here, we hypothesized that ETF dysfunction-mediated FAD deficiency may result in increased mitochondrial oxidative stress and steatosis and subsequent liver injury. We report that etfa haploinsufficiency caused hyperlipidemia, hypercholesterolemia, and hepatic steatosis and injury in adult zebrafish. Further, etfa+/ - mutant livers had reduced levels of FAD and glutathione and an increase in reactive oxygen species. Because FAD depletion might be critical in the pathogenesis of the liver lesion identified in etfa+/ - mutants, we used riboflavin to elevate FAD levels in the liver and found that riboflavin supplementation significantly suppressed hepatic steatosis and injury in etfa+/ - mutants through suppression of oxidative stress and de novo lipogenesis in the liver. Additionally, we found that adenosine triphosphate-linked mitochondrial oxygen consumption and mitochondrial membrane potential were reduced in etfa+/ - primary hepatocytes and that riboflavin supplementation corrected these defects. Conclusion: FAD depletion caused by etfa haploinsufficiency plays a key role in hepatic steatosis and oxidative stress-mediated hepatic injury in adult zebrafish. This raises the possibility that people with ETFA haploinsufficiency have a high risk for developing liver disease.

Neuropilin-2b facilitates resistance to tyrosine kinase inhibitors in non-small cell lung cancer.

OBJECTIVE: Innate and acquired resistance is the principle factor limiting the efficacy of tyrosine kinase inhibitors in lung cancer. We have observed a dramatic upregulation of the cell surface co-receptor neuropilin-2b in lung cancers clinically treated with tyrosine kinase inhibitors correlating with acquired resistance. We hypothesize that neuropilin-2b plays a functional role in acquired tyrosine kinase inhibitor resistance. METHODS: Non-small cell lung cancer proliferation and survival were determined during chronic tyrosine kinase inhibitor exposure in the presence or absence of neuropilin-2b knock-down. Interactions of neuropilin-2a and neuropilin-2b isoforms with PTEN and GSK3ß were assessed by immunoprecipitation. Neuropilin-2a and neuropilin-2b mutants deleted for their cytoplasmic domains were used to identify regions responsible for neuropilin-2b-GSK3ß interaction. Because GSK3ß is known to phosphorylate and degrade PTEN, phospho-PTEN and total PTEN levels were assessed after transfection of neuropilin-2a and neuropilin-2b wild-type and mutant constructs. RESULTS: Non-small cell lung cancer cells chronically treated with gefitinib or osimertinib developed drug resistance and exhibited logarithmic growth in the presence of endothelial growth factor receptor tyrosine kinase inhibitors. However, neuropilin-2b knockdown cells remained sensitive to gefitinib. Likewise, neuropilin-2b knockdown suppressed and neuropilin-2a knockdown enhanced cellular migration. Acquired drug resistance and cell migration correlated with neuropilin-2b-dependent AKT activation with the intermediate step of GSK3ß-dependent PTEN degradation. A specific binding site for GSK3ß on the cytoplasmic domain of neuropilin-2b was identified with truncated protein constructs and computer modeling. CONCLUSIONS: Neuropilin-2b facilitates non-small cell lung cancer resistance to tyrosine kinase inhibitors, and this biological effect relates to AKT activation. Neuropilin-2b GSK3ß interactions appear to be essential for PTEN degradation and AKT activation in lung cancer cells. Disruption of the neuropilin-2b GSK3ß interaction may represent a novel treatment strategy to preserve sensitivity to tyrosine kinase inhibitors in non-small cell lung cancer.

Quantitative analysis of mitochondrial membrane potential heterogeneity in unsynchronized and synchronized cancer cells.

Mitochondrial membrane potential (ΔΨm) is a global indicator of mitochondrial function. Previous reports on heterogeneity of ΔΨm were qualitative or semiquantitative. Here, we quantified intercellular differences in ΔΨm in unsynchronized human cancer cells, cells synchronized in G1, S, and G2, and human fibroblasts. We assessed ΔΨm using a two-pronged microscopy approach to measure relative fluorescence of tetramethylrhodamine methyl ester (TMRM) and absolute values of ΔΨm. We showed that ΔΨm is more heterogeneous in cancer cells compared to fibroblasts, and it is maintained throughout the cell cycle. The effect of chemical inhibition of the respiratory chain and ATP synthesis differed between basal, low and high ΔΨm cells. Overall, our results showed that intercellular heterogeneity of ΔΨm is mainly modulated by intramitochondrial factors, it is independent of the ΔΨm indicator and it is not correlated with intercellular heterogeneity of plasma membrane potential or the phases of the cell cycle.

Differential effects of low-dose sacubitril and/or valsartan on renal disease in salt-sensitive hypertension.

Diuretics and renin-angiotensin system blockers are often insufficient to control the blood pressure (BP) in salt-sensitive (SS) subjects. Abundant data support the proposal that the level of atrial natriuretic peptide may correlate with the pathogenesis of SS hypertension. We hypothesized here that increasing atrial natriuretic peptide levels with sacubitril, combined with renin-angiotensin system blockage by valsartan, can be beneficial for alleviation of renal damage in a model of SS hypertension, the Dahl SS rat. To induce a BP increase, rats were challenged with a high-salt 4% NaCl diet for 21 days, and chronic administration of vehicle or low-dose sacubitril and/or valsartan (75 µg/day each) was performed. Urine flow, Na+ excretion, and water consumption were increased on the high-salt diet compared with the starting point (0.4% NaCl) in all groups but remained similar among the groups at the end of the protocol. Upon salt challenge, we observed a mild decrease in systolic BP and urinary neutrophil gelatinase-associated lipocalin levels (indicative of alleviated tubular damage) in the valsartan-treated groups. Sacubitril, as well as sacubitril/valsartan, attenuated the glomerular filtration rate decline induced by salt. Alleviation of protein cast formation and lower renal medullary fibrosis were observed in the sacubitril/valsartan- and valsartan-treated groups, but not when sacubitril alone was administered. Interestingly, proteinuria was mildly mitigated only in rats that received sacubitril/valsartan. Further studies of the effects of sacubitril/valsartan in the setting of SS hypertension, perhaps involving a higher dose of the drug, are warranted to determine if it can interfere with the progression of the disease.

Role of mitochondrial depolarization and disrupted mitochondrial homeostasis in non-alcoholic steatohepatitis and fibrosis in mice.

The pathogenesis of non-alcoholic steatohepatitis (NASH) is poorly understood. Here, relationships between mitochondrial depolarization (mtDepo) and mitochondrial homeostasis were studied in a mouse model of NASH. C57BL/6 mice were fed a Western diet (high fat, fructose and cholesterol) for 2 weeks, 2 months and 6 months, and livers were harvested for histology and biochemical analysis. Hepatic mtDepo was evaluated by intravital multiphoton microscopy. After Western diet feeding, mixed hepatic micro- and macrovesicular steatosis and leukocyte infiltration occurred at 2 weeks and continued to increase afterwards. ALT release, mild necrosis, apoptosis, and ballooning degeneration were present at 2 and 6 months. Smooth muscle α-actin expression increased at 2 weeks and longer, and increased collagen-I expression and mild fibrosis occurred at 6 months. After feeding Western diet for 2 weeks and longer, mtDepo appeared in 50-70% hepatocytes, indicating mitochondrial dysfunction at an early stage of NASH. mtDepo can initiate mitophagy, and mitophagic markers increased at 2 and 6 months. Concurrently autophagic processing became impaired. Oxidative phosphorylation proteins, mitochondrial biogenesis signals, and proteins associated with mitochondrial fission and fusion decreased after 2 months and longer of Western diet. Proinflammatory and profibrotic signaling (NLRP3 inflammasome activation, expression of IL-1, osteopontin and TGF-ß1) also increased in association with mitochondrial stress/dysfunction after Western diet feeding. Taken together, we show that hepatic mtDepo occurs early in mice fed a Western diet, followed by increased mitophagic burden, suppressed mitochondrial biogenesis and dynamics, and mitochondrial depletion. These novel mitochondrial alterations in NASH most likely play an important role in promoting steatosis, inflammation, and progression to fibrosis.

Mitochondrial protein import is regulated by p17/PERMIT to mediate lipid metabolism and cellular stress.

How lipid metabolism is regulated at the outer mitochondrial membrane (OMM) for transducing stress signaling remains largely unknown. We show here that this process is controlled by trafficking of ceramide synthase 1 (CerS1) from the endoplasmic reticulum (ER) to the OMM by a previously uncharacterized p17, which is now renamed protein that mediates ER-mitochondria trafficking (PERMIT). Data revealed that p17/PERMIT associates with newly translated CerS1 on the ER surface to mediate its trafficking to the OMM. Cellular stress induces Drp1 nitrosylation/activation, releasing p17/PERMIT to retrieve CerS1 for its OMM trafficking, resulting in mitochondrial ceramide generation, mitophagy and cell death. In vivo, CRISPR-Cas9-dependent genetic ablation of p17/PERMIT prevents acute stress-mediated CerS1 trafficking to OMM, attenuating mitophagy in p17/PERMIT-/- mice, compared to controls, in various metabolically active tissues, including brain, muscle, and pancreas. Thus, these data have implications in diseases associated with accumulation of damaged mitochondria such as cancer and/or neurodegeneration.

Effects of vagus nerve stimulation are mediated in part by TrkB in a parkinson's disease model.

Vagus nerve stimulation (VNS) is being explored as a potential therapeutic for Parkinson's disease (PD). VNS is less invasive than other surgical treatments and has beneficial effects on behavior and brain pathology. It has been suggested that VNS exerts these effects by increasing brain-derived neurotrophic factor (BDNF) to enhance pro-survival mechanisms of its receptor, tropomyosin receptor kinase-B (TrkB). We have previously shown that striatal BDNF is increased after VNS in a lesion model of PD. By chronically administering ANA-12, a TrkB-specific antagonist, we aimed to determine TrkB's role in beneficial VNS effects for a PD model. In this study, we administered a noradrenergic neurotoxin, DSP-4, intraperitoneally and one week later administered a bilateral intrastriatal dopaminergic neurotoxin, 6-OHDA. At this time, the left vagus nerve was cuffed for stimulation. Eleven days later, rats received VNS twice per day for ten days, with daily locomotor assessment. Daily ANA-12 injections were given one hour prior to the afternoon stimulation and concurrent locomotor session. Following the final VNS session, rats were euthanized, and left striatum, bilateral substantia nigra and locus coeruleus were sectioned for immunohistochemical detection of neurons, α-synuclein, astrocytes, and microglia. While ANA-12 did not avert behavioral improvements of VNS, and only partially prevented VNS-induced attenuation of neuronal loss in the locus coeruleus, it did stop neuronal and anti-inflammatory effects of VNS in the nigrostriatal system, indicating a role for TrkB in mediating VNS efficacy. However, our data also suggest that BDNF-TrkB is not the sole mechanism of action for VNS in PD.

Nkx2-5 Second Heart Field Target Gene Ccdc117 Regulates DNA Metabolism and Proliferation.

The cardiac transcription factor Nkx2-5 is essential for normal outflow tract (OFT) and right ventricle (RV) development. Nkx2-5-/- null mouse embryos display severe OFT and RV hypoplasia and a single ventricle phenotype due to decreased proliferation of Second Heart Field (SHF) cells, a pool of cardiac progenitors present in anterior pharyngeal arch mesoderm at mid-gestation. However, definition of the precise role of Nkx2-5 in facilitating SHF expansion is incomplete. We have found that Nkx2-5 positively and directly regulates a novel target gene, Ccdc117, in cells of the SHF at these stages. The nuclear/mitotic spindle associated protein Ccdc117 interacts with the MIP18/MMS19 cytoplasmic iron-sulfur (FeS) cluster assembly (CIA) complex, which transfers critical FeS clusters to several key enzymes with functions in DNA repair and replication. Loss of cellular Ccdc117 expression results in reduced proliferation rates associated with a delay at the G1-S transition, decreased rates of DNA synthesis, and unresolved DNA damage. These results implicate a novel role for Nkx2-5 in the regulation of cell cycle events in the developing heart, through Ccdc117's interaction with elements of the CIA pathway and the facilitation of DNA replication during SHF expansion.

Baclofen-induced neurotoxicity in patients with compromised renal function: Review.

Baclofen is a centrally-acting γ-amino butyric acid agonist used mainly in the symptomatic management of spasticity originating from the spinal cord. It is absorbed completely from the gastrointestinal tract, metabolized by the liver to a minor degree, and excreted unchanged by the kidneys. Baclofen is moderately lipophilic and can cross the blood-brain barrier easily. At the usual dosage, it acts mainly at the spinal level without central nervous system (CNS) side effects. During renal failure, however, the elimination of the drug will decrease with a prolonged half-life, resulting in a larger area-under-the-curve exposure and disproportionate CNS toxicity. Clinically, these patients with renal failure may present with a variety of toxic symptoms manifesting at therapeutic/sub-therapeutic doses of baclofen. In cases of unexplained mental status changes in a patient receiving baclofen therapy, a careful assessment of renal function and a high suspicion of baclofen-induced encephalopathy will be key to the diagnosis.
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Nitration and Glycation Turn Mature NGF into a Toxic Factor for Motor Neurons: A Role for p75NTR and RAGE Signaling in ALS.

INTRODUCTION: Glycating stress can occur together with oxidative stress during neurodegeneration and contribute to the pathogenic mechanism. Nerve growth factor (NGF) accumulates in several neurodegenerative diseases. Besides promoting survival, NGF can paradoxically induce cell death by signaling through the p75 neurotrophin receptor (p75NTR). The ability of NGF to induce cell death is increased by nitration of its tyrosine residues under conditions associated with increased peroxynitrite formation. AIMS: Here we investigated whether glycation also changes the ability of NGF to induce cell death and assessed the ability of post-translational modified NGF to signal through the receptor for advanced glycation end products (RAGEs). We also explored the potential role of RAGE-p75NTR interaction in the motor neuron death occurring in amyotrophic lateral sclerosis (ALS) models. RESULTS: Glycation promoted NGF oligomerization and ultimately allowed the modified neurotrophin to signal through RAGE and p75NTR to induce motor neuron death at low physiological concentrations. A similar mechanism was observed for nitrated NGF. We provide evidence for the interaction of RAGE with p75NTR at the cell surface. Moreover, we observed that post-translational modified NGF was present in the spinal cord of an ALS mouse model. In addition, NGF signaling through RAGE and p75NTR was involved in astrocyte-mediated motor neuron toxicity, a pathogenic feature of ALS. INNOVATION: Oxidative modifications occurring under stress conditions can enhance the ability of mature NGF to induce neuronal death at physiologically relevant concentrations, and RAGE is a new p75NTR coreceptor contributing to this pathway. CONCLUSION: Our results indicate that NGF-RAGE/p75NTR signaling may be a therapeutic target in ALS. Antioxid. Redox Signal. 28, 1587-1602.

Acid sphingomyelinase promotes mitochondrial dysfunction due to glutamate-induced regulated necrosis.

Inhibiting the glutamate/cystine antiporter system xc-, a key antioxidant defense machinery in the CNS, could trigger a novel form of regulated necrotic cell death, ferroptosis. The underlying mechanisms of system xc--dependent cell demise were elucidated using primary oligodendrocytes (OLs) treated with glutamate to block system xc- function. Pharmacological analysis revealed ferroptosis as a major contributing factor to glutamate-initiated OL death. A sphingolipid profile showed elevations of ceramide species and sphingosine that were preventable by inhibiting of an acid sphingomyelinase (ASM) activity. OL survival was enhanced by both downregulating ASM expression and blocking ASM activity. Glutamate-induced ASM activation seems to involve posttranscriptional mechanisms and was associated with a decreased GSH level. Further investigation of the mechanisms of OL response to glutamate revealed enhanced reactive oxygen species production, augmented lipid peroxidation, and opening of the mitochondrial permeability transition pore that were attenuated by hindering ASM. Of note, knocking down sirtuin 3, a deacetylase governing the mitochondrial antioxidant system, reduced OL survival. The data highlight the importance of the mitochondrial compartment in regulated necrotic cell death and accentuate the novel role of ASM in disturbing mitochondrial functions during OL response to glutamate toxicity, which is essential for pathobiology in stroke and traumatic brain injury.

Vagus nerve stimulation improves locomotion and neuronal populations in a model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a progressive, neurodegenerative disorder with no disease-modifying therapies, and symptomatic treatments are often limited by debilitating side effects. In PD, locus coeruleus noradrenergic (LC-NE) neurons degenerate prior to substantia nigra dopaminergic (SN-DA) neurons. Vagus nerve stimulation (VNS) activates LC neurons, and decreases pro-inflammatory markers, allowing improvement of LC targets, making it a potential PD therapeutic. OBJECTIVE: To assess therapeutic potential of VNS in a PD model. METHODS: To mimic the progression of PD degeneration, rats received a systemic injection of noradrenergic neurotoxin DSP-4, followed one week later by bilateral intrastriatal injection of dopaminergic neurotoxin 6-hydroxydopamine. At this time, a subset of rats also had vagus cuffs implanted. After eleven days, rats received a precise VNS regimen twice a day for ten days, and locomotion was measured during each afternoon session. Immediately following final stimulation, rats were euthanized, and left dorsal striatum, bilateral SN and LC were sectioned for immunohistochemical detection of monoaminergic neurons (tyrosine hydroxylase, TH), α-synuclein, astrocytes (GFAP) and microglia (Iba-1). RESULTS: VNS significantly increased locomotion of lesioned rats. VNS also resulted in increased expression of TH in striatum, SN, and LC; decreased SN α-synuclein expression; and decreased expression of glial markers in the SN and LC of lesioned rats. Additionally, saline-treated rats after VNS, had higher LC TH and lower SN Iba-1. CONCLUSIONS: Our findings of increased locomotion, beneficial effects on LC-NE and SN-DA neurons, decreased α-synuclein density in SN TH-positive neurons, and neuroinflammation suggest VNS has potential as a novel PD therapeutic.