Oxaloacetate treatment preserves motor function in SOD1G93A mice and normalizes select neuroinflammation-related parameters in the spinal cord.

Amyotrophic lateral sclerosis (ALS) remains a devastating motor neuron disease with limited treatment options. Oxaloacetate treatment has a neuroprotective effect in rodent models of seizure and neurodegeneration. Therefore, we treated the ALS model superoxide dismutase 1 (SOD1) G93A mice with oxaloacetate and evaluated their neuromuscular function and lifespan. Treatment with oxaloacetate beginning in the presymptomatic stage significantly improved neuromuscular strength measured during the symptomatic stage in the injected mice compared to the non-treated group. Oxaloacetate treatment starting in the symptomatic stage significantly delayed limb paralysis compared with the non-treated group. For lifespan analysis, oxaloacetate treatment did not show a statistically significant positive effect, but the treatment did not shorten the lifespan. Mechanistically, SOD1G93A mice showed increased levels of tumor necrosis factor-α (TNFα) and peroxisome proliferative activated receptor gamma coactivator 1α (PGC-1α) mRNAs in the spinal cord. However, oxaloacetate treatment reverted these abnormal levels to that of wild-type mice. Similarly, the altered expression level of total NF-κB protein returned to that of wild-type mice with oxaloacetate treatment. These results suggest that the beneficial effects of oxaloacetate treatment in SOD1G93A mice may reflect the effects on neuroinflammation or bioenergetic stress.

Mitochondrial Electron Transfer Cascade Enzyme Activity Assessment in Cultured Neurons and Select Brain Regions.

Measurement of the electron transfer cascade (ETC) enzyme activities and their kinetic profiles is important in assessing mitochondrial function in the nervous system in health and disease or following exposure to toxic agents. The optimization of enzymatic assays for brain tissues and neurons is critical to the development of high-throughput assay formats. This article describes a step-by-step protocol for reliable and reproducible assessment of ETC enzyme kinetics (Complex I-IV) for mitochondria from small quantities of tissue from different brain regions, such as the hippocampus, cerebellum, and frontal cortex, or from neurons in culture. Methods for differential and density gradient centrifugation are detailed for isolating cell body and synaptic mitochondria from brain, as well as measurement of ETC activities in microwell plate or single-cuvette format using spectrophotometric methods. Easy-to follow assay layouts and useful tips are presented, allowing the user to perform these assays in under 3 hr. © 2019 by John Wiley & Sons, Inc.

The Role of Methionine Oxidation/Reduction in the Regulation of Immune Response.

Methionine oxidation by reactive oxygen species and reduction mediated by the methionine sulfoxide reductase (Msr) system may attenuate protein function in signal transduction pathways. This review will focus on two potential protein targets for methionine oxidation involved in signal transduction of the immune response: Ca(2+)/calmodulin-regulated phosphatase calcineurin (Cn) and inhibitor of kappa B-alpha (IkBα). The major known function of Cn is to regulate nuclear localization of the nuclear factor of activated T cells (NFAT), a family of transcription factors during immune stimulus. Like wise, IκBα inhibits the activity of nuclear factor kappa B (NFkB), which is known to regulate the transcription of various genes participating in immunological and oxidative stress response. Modification of Met (45) in IκBα enhances its resistance to protein-degredation; thereby, preventing NFkB from activating transcription in cells of the immune system. Similarly, the human Cn molecule contains several methionine residues that are either located next to a cysteine residue or a methionine residue. Accordingly, it is suggested that oxidation of a specific Cn-methionine may interfere with the proper NFAT nuclear-localization and transcriptional activation in T-cell. Thus, the roles of oxidized-methionine residues and their reduction, by the Msr system, are discussed as potential regulators of cellular immune response.

A rat brain bicistronic gene with an internal ribosome entry site codes for a phencyclidine-binding protein with cytotoxic activity.

The cloning and characterization of the gene for the fourth subunit of a glutamate-binding protein complex in rat brain synaptic membranes are described. The cloned rat brain cDNA contained two open reading frames (ORFs) encoding 8.9- (PRO1) and 9.5-kDa (PRO2) proteins. The cDNA sequence matched contiguous genomic DNA sequences in rat chromosome 17. Both ORFs were expressed within the structure of a single brain mRNA and antibodies against unique sequences in PRO1- and PRO2-labeled brain neurons in situ, indicative of bicistronic gene expression. Dicistronic vectors in which ORF1 and ORF2 were substituted by either two different fluorescent proteins or two luciferases indicated concurrent, yet independent translation of the two ORFs. Transfection with noncapped mRNA led to cap-independent translation of only ORF2 through an internal ribosome entry sequence preceding ORF2. In vitro or cell expression of the cloned cDNA led to the formation of multimeric protein complexes containing both PRO1 and PRO2. These complexes had low affinity (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801)-sensitive phencyclidine-binding sites. Overexpression of PRO1 and PRO2 in CHO cells, but not neuroblastoma cells, caused cell death within 24-48 h. The cytotoxicity was blocked by concurrent treatment with MK-801 or by two tetrahydroisoquinolines that bind to phencyclidine sites in neuronal membranes. Co-expression of two of the other subunits of the protein complex together with PRO1/PRO2 abrogated the cytotoxic effect without altering PRO1/PRO2 protein levels. Thus, this rare mammalian bicistronic gene coded for two tightly interacting brain proteins forming a low affinity phencyclidine-binding entity in a synaptic membrane complex.

Structure and function of an early divergent form of laminin in hydra: a structurally conserved ECM component that is essential for epithelial morphogenesis.

As a major component of the extracellular matrix (ECM), laminin has been found in many vertebrate and invertebrate organisms. Its molecular structure is very similar across species lines and its biological function in the ECM has been extensively studied. In an effort to study ECM structure and function in hydra, we have cloned a partial hydra laminin alpha chain and the full-length hydra laminin beta chain using ECM-enriched cDNA libraries. Analysis of deduced amino acid sequences indicated that both polypeptides have high sequence similarity to a number of invertebrate and vertebrate laminin alpha and beta subunits. Rotary shadow analysis of isolated hydra laminin indicates it has a heterotrimeric organization that is characteristic of vertebrate laminins. A putative integrin-class protein was also identified using a cell-binding peptide sequence from the laminin beta chain as an affinity probe, indicating that integrins are possible cell surface receptors in hydra. In agreement with previous results for the hydra laminin beta chain, in situ hybridization experiments revealed that hydra laminin alpha chain mRNA is restricted to endodermal cells. As with a number of other hydra ECM components, higher levels of laminin alpha chain mRNA are localized to regions where cell migration and differentiation are actively undertaken such as the base of tentacles, the peduncle region, buds, regenerating tentacles, and at the head end during regeneration. The role of laminin in morphogenesis was studied using an antisense approach and the results indicated that translation of the laminin alpha chain is required for head regeneration.