Philip M. Beart

Mice with a Homozygous Null Mutation for the Most Abundant Glutathione Peroxidase, Gpx1, Show Increased Susceptibility to the Oxidative Stress-inducing Agents Paraquat and Hydrogen Peroxide*

Glutathione peroxidases have been thought to function in cellular antioxidant defense. However, some recent studies on Gpx1 knockout (−/−) mice have failed to show a role for Gpx1 under conditions of oxidative stress such as hyperbaric oxygen and the exposure of eye lenses to high levels of H2O2. These findings have, unexpectedly, raised the issue of the role of Gpx1, especially under conditions of oxidative stress. Here we demonstrate a role for Gpx1 in protection against oxidative stress by showing that Gpx1 (−/−) mice are highly sensitive to the oxidant paraquat. Lethality was already detected within 24 h in mice exposed to paraquat at 10 mg·kg−1 (approximately 1 7 the LD50of wild-type controls). The effects of paraquat were dose-related. In the 30 mg·kg−1-treated group, 100% of mice died within 5 h, whereas the controls showed no evidence of toxicity. We further demonstrate that paraquat transcriptionally up-regulatesGpx1 in normal cells, reinforcing a role forGpx1 in protection against paraquat toxicity. Finally, we show that cortical neurons from Gpx1 (−/−) mice are more susceptible to H2O2; 30% of neurons fromGpx1 (−/−) mice were killed when exposed to 65 μm H2O2, whereas the wild-type controls were unaffected. These data establish a function for Gpx1 in protection against some oxidative stressors and in protection of neurons against H2O2. Further, they emphasize the need to elucidate the role of Gpx1 in protection against different oxidative stressors and in different disease states and suggest thatGpx1 (−/−) mice may be valuable for studying the role of H2O2 in neurodegenerative disorders.

Transporters for L-glutamate: An update on their molecular pharmacology and pathological involvement

L‐Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian CNS and five types of high‐affinity Glu transporters (EAAT1–5) have been identified. The transporters EAAT1 and EAAT2 in glial cells are responsible for the majority of Glu uptake while neuronal EAATs appear to have specialized roles at particular types of synapses. Dysfunction of EAATs is specifically implicated in the pathology of neurodegenerative conditions such as amyotrophic lateral sclerosis, epilepsy, Huntingtons disease, Alzheimers disease and ischemic stroke injury, and thus treatments that can modulate EAAT function may prove beneficial in these conditions. Recent advances have been made in our understanding of the regulation of EAATs, including their trafficking, splicing and post‐translational modification. This article summarises some recent developments that improve our understanding of the roles and regulation of EAATs.

Excitatory amino acid projections to the nucleus accumbens septi in the rat: a retrograde transport study utilizing D[3H]aspartate and [3H]GABA.

Afferents to the nucleus accumbens septi utilizing glutamate or aspartate have been investigated in the rat by autoradiography following injection and retrograde transport of D[3H]aspartate. Parallel experiments with the intra-accumbal injection of [3H]GABA were employed to establish the transmitter-selective nature of the retrograde labelling found with D[3H]aspartate. The topography of cortical and thalamic perikarya labelled by D[3H]aspartate was extremely precise. D[3H]Aspartate labelled perikarya were found in layer V of agranular insular cortex; bilaterally within prelimbic and infralimbic subareas perikarya, but predominantly ipsilaterally. Ipsilateral labelling was observed in dorsal, ventral and posterior agranular insular cortices, and in perirhinal cortex. Injections into ventral accumbens labelled perikarya in ipsilateral entorhinal cortex, while infusion of D[3H]aspartate into anterior caudate-putamen resulted in labelling of perikarya in ipsilateral cingulate and lateral precentral cortices. Following infusion of D[3H]aspartate, ipsilateral midline thalamic nuclei contained the highest density of labelled perikarya; infusions centred on nucleus accumbens resulted in heavy retrograde labelling of the parataenial nucleus, but labelling was sparse from a lateral site and not observed after injection into anterior caudate-putamen. Less prominent labelling of perikarya was seen in other thalamic nuclei (mediodorsal, central medial, rhomboid, reuniens and centrolateral), mostly near the midline. Perikaryal labelling was also found in the ipsilateral amygdaloid complex, particularly in basolateral and lateral nuclei. Only weak labelling resulted in ventral subiculum. Numerous labelled cells were present bilaterally in anterior olfactory nucleus, although perikarya were more prominent ipsilaterally. Labelled perikarya were not consistently observed in other regions (ventral tegmental area, medial substantia nigra, raphe nuclei and locus coeruleus) known to innervate nucleus accumbens. Presumptive anterograde labelling was detected in ventral pallidum/substantia innominata, ventral tegmental area and medial substantia nigra. [3H]GABA was generally not retrogradely transported to the same regions labelled by D[3H]aspartate; an exception being the anterior olfactory nucleus, where large numbers of labelled perikarya were found. [3H]GABA failed to label perikarya in thalamus and amygdala, and a topographic distribution of label was absent in neocortex.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of the Unfolded Protein Response in Familial Amyotrophic Lateral Sclerosis and Association of Protein-disulfide Isomerase with Superoxide Dismutase 1

Mutations in Cu/Zn superoxide dismutase (SOD1) are linked to motor neuron death in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism, although misfolded SOD1 aggregates are commonly associated with disease. Proteomic analysis of the transgenic SOD1(G93A) ALS rat model revealed significant up-regulation of endoplasmic reticulum (ER)-resident protein-disulfide isomerase (PDI) family members in lumbar spinal cords. Expression of SOD1 mutants (mSOD1) led to an up-regulation of PDI in motor neuron-like NSC-34 cells but not other cell lines. Inhibition of PDI using bacitracin increased aggregate production, even in wild type SOD1 transfectants that do not readily form inclusions, suggesting PDI may protect SOD1 from aggregation. Moreover, PDI co-localized with intracellular aggregates of mSOD1 and bound to both wild type and mSOD1. SOD1 was also found in the microsomal fraction of cells despite being a predominantly cytosolic enzyme, confirming ER-Golgi-dependent secretion. In SOD1(G93A) mice, a significant up-regulation of unfolded protein response entities was also observed during disease, including caspase-12, -9, and -3 cleavage. Our findings therefore implicate unfolded protein response and ER stress-induced apoptosis in the patho-physiology of familial ALS. The possibility that PDI may be a therapeutic target to prevent SOD1 aggregation is also raised by this study.

Excitotoxin lesions suggest an aspartatergic projection from rat medial prefrontal cortex to ventral tegmental area

High affinity D-[3H]aspartate uptake and amino acid concentrations were examined in synaptosome-enriched preparations of microdissected rat ventral tegmental area 6-7 days following N-methyl-D-aspartate lesions confined to medial prefrontal cortex. Specific reductions were observed in the high affinity uptake of D-[3H]aspartate (59% of control, P less than 0.005) and concentrations of L-aspartate (79% of control, P less than 0.05) in the ventral tegmental area suggesting the presence of an aspartatergic projection from medial prefrontal cortex to this area.

Micromolar L-glutamate induces extensive apoptosis in an apoptotic-necrotic continuum of insult-dependent, excitotoxic injury in cultured cortical neurones

Excitotoxicity induced by L-glutamate (Glu), when examined in a pure neuronal cortical culture, involved widespread apoptosis at concentrations of 1-10 microM as part of a continuum of injury, which at its most servere was purely necrotic. Cells, maintained in chemically defined neurobasal/B27 medium, were exposed at d7 for 2 h to Glu (1-500 microM), and cellular injury was analysed 2 and 24 h after insult using morphology (phase-contrast microscopy), a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay, nuclear staining with 4,6-diamidino-2-phenylindole (DAPI), terminal transferase-mediated dUTP nick end-labelling (TUNEL) and DNA fragmentation by gel electrophoresis. Glu-mediated neurotoxicity was prevented by MK-801 (5 microM), whilst CNQX (20 microM) attenuated injury by 20%. Exposure to intensive insults (100 and 500 microM Glu) induced necrosis characterized by rapid cell swelling (< 2 h) and lack of chromatin condensation, confirmed by DAPI nuclear staining. In contrast, mild insults (< 20 microM Glu) failed to produce acute neuronal swelling at < 2 h, but 24 h after injury resulted in a large number of apoptotic nuclei as confirmed by TUNEL and electrophoretic evidence of DNA fragmentation, which was attenuated by cycloheximide (0.1 microg/ml). Our findings indicate for the first time that physiological concentrations of Glu produce neuronal injury across a continuum involving apoptosis (< 20 microM) and increasingly necrosis(> 20 microM), dependent on the severity of the initial insult.

Cis- and trans-4-aminocrotonic acid as GABA analogues of restricted conformation.

Cis‐4‐aminocrotonic acid, an analogue of GABA in a folded conformation, appears not to act as a GABA analogue with respect to bicuculline‐sensitive postsynaptic receptors, ‘high affinity’ GABA uptake and GABA: 2‐oxoglutarate aminotransferase in the mammalian central nervous system. On the other hand, trans‐4‐aminocrotonic acid, an analogue of GABA in an extended conformation, acts as efficiently as GABA with respect to each of the above systems, indicating that extended rather than folded conformations of GABA are likely to be important in the interaction of GABA with the specific macromolecules concerned.

An Excitant Amino Acid Projection from the Medial Prefrontal Cortex to the Anterior Part of Nucleus Accumbens in the Rat

Abstract: High‐affinity uptake of neurotransmitter substrates in synaptosome‐containing homogenates and tissue concentrations of amino acids were examined in subcortical areas 5–6 days after bilateral N‐methyl‐D‐aspartate lesions confined to rat medial prefrontal cortex. D‐[3H]Aspartate (32% of control) and [3H]γ‐aminobutyric acid ([3H]GABA) (60% of control) uptakes were significantly reduced in medial prefrontal cortex, whereas [3H]choline (110% of control) uptake was unchanged, suggesting the production of axon‐sparing lesions. The uptake of D‐[3H]aspartate (76% of control), but not of [3H]GABA or [3H]choline, was significantly reduced in nucleus accumbens, with no concomitant reduction in amino acid concentrations. When examined in serial coronal sections, reduced D‐[3H]aspartate uptake was confined to the most anterior 500 μm of nucleus accumbens (67% of contralateral sample). No significant reductions of uptake or amino acid concentrations were observed in caudate putamen or ventral tegmental area. These results suggest a role for glutamate or aspartate as neurotransmitters in projections from medial prefrontal cortex to anterior nucleus accumbens. Medial prefrontal cortex may represent the major excitatory cortical input to the nucleus accumbens.

Impairment in T-maze reinforced alternation performance following nucleus basalis magnocellularis lesions in rats

Rats were trained on a reinforced alternation paradigm using an elevated T-maze. After pre-surgical training subjects received either ibotenic acid (4 micrograms/0.4 microliter) or vehicle (pH 7.4, 0.4 microliter) bilaterally into the region of the nucleus basalis magnocellularis--an important source of neocortical acetylcholine projections. Acetylcholinesterase staining of sectioned brains revealed a loss of neocortical, but not hippocampal staining in lesioned animals. On the T-maze task, lesioned rats showed significantly impaired choice performance relative to controls. They also demonstrated significant side biases, the degree of which was correlated with choice performance deficit.

Oxidative Stress: Emerging Mitochondrial and Cellular Themes and Variations in Neuronal Injury

Oxidative stress plays a central role in neuronal injury and cell death in acute and chronic pathological conditions. The cellular responses to oxidative stress embrace changes in mitochondria and other organelles, notably endoplasmic reticulum, and can lead to a number of cell death paradigms, which cover a spectrum from apoptosis to necrosis and include autophagy. In Alzheimers disease, and other pathologies including Parkinsons disease, protein aggregation provides further cellular stresses that can initiate or feed into the pathways to cell death engendered by oxidative stress. Specific attention is paid here to mitochondrial dysfunction and programmed cell death, and the diverse modes of cell death mediated by mitochondria under oxidative stress. Novel insights into cellular responses to neuronal oxidative stress from a range of different stressors can be gained by detailed transcriptomics analyses. Such studies at the cellular level provide the key for understanding the molecular and cellular pathways whereby neurons respond to oxidative stress and undergo injury and death. These considerations underpin the development of detailed knowledge in more complex integrated systems, up to the intact human bearing the neuropathology, facilitating therapeutic advances.