Peter A. Boxer

Assessment of the Role of the Glutathione and Pentose Phosphate Pathways in the Protection of Primary Cerebrocortical Cultures from Oxidative Stress

Abstract: Reactive oxygen species have been implicated in neuronal injury associated with various neuropathological disorders. However, little is known regarding the relationship between antioxidant enzyme capacity and resultant toxicity. The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coupled to glutathione peroxidase (GPx) detoxification of hydrogen peroxide (H2O2). PPP activity was quantified from data obtained by gas chromatography/mass spectrometry analysis of released labeled lactate following metabolic degradation of [1,6‐13C2,6,6‐2H2]glucose by cerebrocortical cultures. The antioxidant capacity of these cultures was systematically evaluated using H2O2, and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H2O2 caused stimulation of PPP activity in a concentration‐dependent fashion from 0.25 to 22.2% and from 6.9 to 66.7% of glucose metabolized to lactate through the PPP, respectively. In the mixed cultures, chelation of iron before H2O2 exposure was protective and resulted in a correlation between PPP saturation and toxicity. Conversely, addition of iron, inhibition of GPx, or depletion of glutathione decreased H2O2‐induced PPP stimulation and increased toxicity. These results implicate the Fenton reaction, reflect the pivotal role of GPx in H2O2 detoxification, and contribute to our understanding of the etiological role of free radicals in neuropathological conditions.

Mechanisms of neuronal cell injury/death and targets for drug intervention

The loss of neurons is responsible for many acute neurological disorders, as well as many chronic neurodegenerative diseases. Drug discovery research has concentrated on blocking necrosis produced by activation of one or more excitatory amino acid (EAA) receptors. Both receptor antagonists and compounds reducing EAA release are currently in clinical trials. With the recent advances in the understanding of apoptosis new strategies for neuroprotection are also emerging. It is anticipated that clinically useful neuroprotective drugs will soon be available for treatment of a range of CNS disorders. This article reviews some of the mechanisms responsible for neuronal cell death and outlines strategies to block calcium overload and free radical production.

Inhibitors of V-Type ATPases, Bafilomycin A1 and Concanamycin A, Protect Against β-Amyloid-Mediated Effects on 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction

Abstract: The functional viability of cells can be evaluated using a number of different assay determinants. One common assay involves exposing cells to 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), which is converted intracellularly to a colored formazan precipitate and often used to assess amyloid peptide‐induced cytotoxic effects. The MTT assay was employed to evaluate the role of endosomal uptake and lysbsomal acidification in amyloid peptide‐treated differentiated PC12 cell cultures using selective vacuolar‐type (N‐type) ATPase inhibitors. The macrolides bafilomycin A1 (BAF) and concanamycin A (CON) block lysosomal acidification through selective inhibition of the V‐type ATPase. Treating nerve growth factor‐differentiated PC12 cells with nanomolar concentrations of BAF or CON provides complete protection against the effects of β‐amyloid peptides Aβ(1‐42), Aβ(1‐40), and Aβ(25‐35) and of amylin on MTT dye conversion. These macrolides do not inhibit peptide aggregation, act as antioxidants, or inhibit Aβ uptake by cells. Measurements of lysosomal acidification reveal that the concentrations of BAF and CON effective in reversing Aβ‐mediated MTT dye conversion also reverse lysosomal pH. These results suggest that lysosomal acidification is necessary for Aβ effects on MTT dye conversion.

Noninvasive assessment of the relative roles of cerebral antioxidant enzymes by quantitation of pentose phosphate pathway activity

Cerebral pentose phosphate pathway (PPP) plays a role in the biosynthesis of macromolecules, antioxidant defense and neurotransmitter metabolism. Studies on this potentially important pathway have been hampered by the inability to easily quantitate its activity, particularly in vivo. In this study we review the use of [1,6-13C2,6,6-2H2]glucose for measuring the relative activities of the PPP and glycolysis in a single incubation in cultured neurons and in vivo, when combined with microdialysis techniques. PPP activity in primary cerebrocortical cultures and in the caudate putamem of the rat in vivo was quantitated from data obtained by GC/MS analysis of released labeled lactate following metabolic degradation of [1,6-13C2,6,6-2H2]glucose. Exposure of cultures to H2O2 resulted in stimulation of PPP activity in a concentration-dependent fashion and subsequent cell death. Chelation of iron during H2O2 exposure exerted a protective effect thus implicating the participation of the Fenton reaction in mediating damage caused by the oxidative insult. Partial inhibition of glutathione peroxidase, but not catalase, was extremely toxic to the cultures reflecting the pivotal role of GPx in H2O2 detoxification. These results demonstrate the ability to dynamically monitor PPP activity and its response to oxidative challenges and should assist in facilitating our understanding of antioxidant pathways in the CNS.

Discovery of subtype-selective NMDA receptor ligands: 4-benzyl-1-piperidinylalkynylpyrroles, pyrazoles and imidazoles as NR1A/2B antagonists.

4-Benzyl-1-[4-(1H-imidazol-4-yl)but-3-ynyl]piperidine (8) has been identified as a potent antagonist of the NR1A/2B subtype of the NMDA receptor. When dosed orally, this compound potentiates the effects of L-DOPA in the 6-hydroxydopamine-lesioned rat, a model of Parkinsons disease.

The voltage-sensitive Ca2+ channel (VSCC) antagonists ω-Aga-IVA and ω-CTX-MVIIC inhibit spontaneous epileptiform dischares in the rat cortical wedge

The ability of VSCC antagonists to modulate excitatory amino acid (EAA) release was evaluated by measuring N-methyl-D-aspartate (NMDA) receptor-dependent spontaneous epileptiform discharges in rat cortical wedges. The N-type channel blocker omega-CTX-GVIA (300 nM) was ineffective. The P-type channel blocker omega-Aga-IVA at 300 nM reduced the frequency of discharges by 63%, while 300 nM omega-CTX-MVIIC reduced the frequency by 35%. These results coupled with the absence of NMDA antagonism by omega-Aga-IVA or omega-CTX-MVIIC in the cortical wedge suggest that the VSCCs blocked by these toxins are primarily responsible for mediating impulse dependent EAA release in the rat neocortex.

N-sulfonyl derivatives of 6,7-dichloro 3,4-dihydro-3-oxo-quinoxalinecarboxylate as glycine-site NMDA and AMPA antagonists

Abstract N-Phenylsulfonyl and N-methylsulfonyl derivatives of 6,7-dichloro-3,4-dihyro-3-oxo-2-quinoxalinecarboxamide have been synthesized. Both compounds have been characterized as antagonists, at the glycine-site of the NMDA receptor and AMPA receptor.

Chapter 2. Neuronal Cell Death and Strategies for Neuroprotection

Publisher Summary Necrotic cell death, prominent in acute neurological conditions, such as stroke, is characterized by swelling of the cell and disruption of the internal and external membranes and cell lysis. Acute neuronal death is easily modeled in primary neuronal cell culture and brain slices. In apoptosis, cells undergo nuclear condensation, fragmentation, and nuclear fragments, along with intracellular organelles. The apoptotic process is energy dependent, and in some instances requires ongoing RNA and protein synthesis. High levels of intracellular calcium [Ca 2+ ] i is thought to be the primary causative event in mediating necrotic neuronal death. Calcium enters the neuron, through a variety of mechanisms, including ligand-gated ion channels, such as the N-methyl-D-aspartate (NMDA) glutamate receptor, voltage-gated calcium channels, leak channels, and reversal of the Na + -Ca 2+ anti-porter. Neurons also have inositol trisphosphate receptors (IPS) on intracellular organelles that can liberate calcium from intracellular stores. Alterations in the mosaic of phosphorylated products, with concomitants, increase and decrease in the activity of cellular enzymes, and ion channels, influence the expression of immediate early genes (IEGs) that is involved in programmed cell death. Sporadic energy supplies disrupt ion homeostasis as in focal ischemia, where depolarization in the penumbra is initially moderate and/or intermittent although adenosine triphosphate (ATP) is produced. Important clues, regarding apoptotic cell death, have come from genetic studies in the nematode, caenorhabditis elegans.

Polyamine modulation of excitatory amino acid responses in the rat cortical wedge

Rat neocortex slices in Mg2+ free buffer show spontaneous discharges which have a constant frequency and are dependent on N-methyl-D-aspartate (NMDA) receptor activation. Spermine increased the frequency of discharges at concentrations below 1 mM, had biphasic effects at 1 mM, and only decreased the frequency of discharges at 3 mM. In contrast, the amplitude of these discharges was only reduced by spermine in a concentration dependent manner (300 microM to 3 mM). Spermidine produced similar effects, but was a less potent inhibitor of discharge frequency and amplitude than spermine. Diethylenetriamine (DET) 300 microM did not significantly inhibit polyamine-induced increases in the discharge frequency. Polyamines inhibited direct depolarizations induced by the glutamate agonists NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in a concentration and time dependent manner. The data are consistent with two sites of action for polyamines, one enhancing the frequency of NMDA-mediated spontaneous epileptiform discharges and the other inhibiting direct glutamate responses in rat cortex. The slow onset (time to maximal enhancement or inhibition by polyamines greater than 30 min) and lack of reversibility with polyamine removal suggest that these sites are intracellular and/or presynaptic.