Elias K. Michaelis

Selective Neuronal Vulnerability to Oxidative Stress in the Brain

Oxidative stress (OS), caused by the imbalance between the generation and detoxification of reactive oxygen and nitrogen species (ROS/RNS), plays an important role in brain aging, neurodegenerative diseases, and other related adverse conditions, such as ischemia. While ROS/RNS serve as signaling molecules at physiological levels, an excessive amount of these molecules leads to oxidative modification and, therefore, dysfunction of proteins, nucleic acids, and lipids. The response of neurons to this pervasive stress, however, is not uniform in the brain. While many brain neurons can cope with a rise in OS, there are select populations of neurons in the brain that are vulnerable. Because of their selective vulnerability, these neurons are usually the first to exhibit functional decline and cell death during normal aging, or in age-associated neurodegenerative diseases, such as Alzheimers disease. Understanding the molecular and cellular mechanisms of selective neuronal vulnerability (SNV) to OS is important in the development of future intervention approaches to protect such vulnerable neurons from the stresses of the aging process and the pathological states that lead to neurodegeneration. In this review, the currently known molecular and cellular factors that contribute to SNV to OS are summarized. Included among the major underlying factors are high intrinsic OS, high demand for ROS/RNS-based signaling, low ATP production, mitochondrial dysfunction, and high inflammatory response in vulnerable neurons. The contribution to the selective vulnerability of neurons to OS by other intrinsic or extrinsic factors, such as deficient DNA damage repair, low calcium-buffering capacity, and glutamate excitotoxicity, are also discussed.

Direct measurement of glutamate release in the brain using a dual enzyme-based electrochemical sensor

The in vivo measurement of the rapid changes in the extracellular concentrations of L-glutamic acid in the mammalian brain during normal neuronal activity or following excessive release due to episodes of anoxia or ischemia has not been possible to this date. Current techniques for the measurement of the release of endogenous glutamate into the extracellular space of the central nervous system are relatively slow and do not measure the actual concentration of free glutamate in the extracellular space. An enzyme-based electrode with rapid response times (about 1 s) and high degree of sensitivity (less than 2 microM) and selectivity for L-glutamic acid is described in this paper. This electrode has both L-glutamate and ascorbate oxidase immobilized on its surface. The latter enzyme removes almost completely any interferences produced by the high levels of extracellular ascorbate present in brain tissue. The response of the electrode to glutamate and other potentially interfering substances was fully characterized in vitro and its selectivity, sensitivity and rapidity in responding to a rise in extracellular glutamate concentrations was also demonstrated in vivo. Placement of the electrode in the dentate gyrus of the hippocampus led to the detection of both KCl-induced release of L-glutamic acid and the release induced by stimulation of the axons in the perforant pathway. The development of this selective, sensitive and rapidly responding glutamate sensor should make it now possible to measure the dynamic events associated with glutamate neurotransmission in the central nervous system.

Adenosine modulation of synaptosomal dopamine release.

Abstract The effects of adenosine and its analog 2-chloroadenosine on release of preloaded [ 3 H]-dopamine from striatal synaptosomes was explored. Both adenosine and 2-chloroadenosine were found to decrease the amount of dopamine released both by depolarization (with KCl) and by amphetamine. Addition of exogenous adenosine deaminase enhanced dopamine release above controls, and blockade of the endogenous adenosine deaminase activity with deoxycoformycin resulted in a decrease in dopamine release. The methylxanthines, believed to be adenosine antagonists, inhibited dopamine release by an unknown mechanism, and hence it was impossible to evaluate antagonism of the inhibitory effects of adenosine by these agents. The depolarization-induced release of dopamine appeared to be more sensitive to the actions of adenosine than was the amphetamine-induced release. The data obtained so far seem to indicate that adenosine is capable of modulating the release of transmitter substances in brain tissue in a manner analogous to that which has been observed in the peripheral nervous system.

High-affinity glutamic acid binding to brain synaptic membranes

Abstract Rat brain homogenate preparations exhibited two types of glutamine binding, one a high-affinity ( K 1 = 0.2 μ M ) and the other a low-affinity type ( K 2 = 4.4 μ M ). The high-affinity binding was primarily associated with the plasma membrane subcellular fractions and in particular with the synaptic membrane subfraction. This l -glutamate binding was found to be strongly stereospecific for the l -form and was almost totally reversible. The synaptic membrane glutamate binding was partialy inhibited by neuro-excitatory and neuro-inhibitory amino acids but was not affected by amino acids lacking in neuropharmacologic activity. The membrane-associated l -glutamate binding system could be solubilized by Triton X-100 without loss of its high-affinity binding activity. The chemical nature of this glutamate binding component was found to be that of a glycolipoprotein. It is proposed that this glutamate binding system represents the physiologic receptor on neuronal membranes of this amino acid.

Developmental expression, compartmentalization, and possible role in excitotoxicity of a putative NMDA receptor protein in cultured hippocampal neurons

The mechanisms regulating the expression and localization of excitatory amino acid (EAA) neurotransmitter receptors in neurons of the developing mammalian brain, and roles for these receptors in the plasticity and degeneration of neural circuits are not well understood. We previously isolated and characterized a 71 kDa glutamate binding protein (GBP) from rat brain, and have recently obtained evidence that this GBP is a component of a functional N-methyl-D-aspartate (NMDA) receptor-ion channel complex. We have now used antibodies to this putative NMDA receptor protein to examine its expression and localization, and consequences of its activation in cultured embryonic (18 day) rat hippocampal neurons. Immunocytochemistry and Western blots using monoclonal antibodies to the GBP demonstrated an increase in GBP-positive neurons and their staining intensity with time in culture. GBP was localized to the somata and dendrites of pyramidal-like neurons and was sparse or absent in the axons. The expression and compartmentalization of GBP occurred in isolated neurons indicating that direct cell interactions were not required for these processes. Cell surface staining for GBP occurred in patches on the soma and dendrites. The developmental expression of GBP immunoreactivity closely paralleled the expression of sensitivity to NMDA neurotoxicity. There was a direct relationship between GBP immunoreactivity and neuronal vulnerability to glutamate-induced degeneration; vulnerable neurons stained heavily whereas resistant neurons showed either low levels of staining or no staining. Finally, a GBP antiserum greatly reduced NMDA neurotoxicity (but not kainate neurotoxicity). Taken together, these findings demonstrate the expression of presumptive NMDA receptors within a subpopulation of embryonic hippocampal neurons, and their segregation to the soma and dentrites of pyramidal neurons. This spatial distribution of glutamate receptors among and within neurons is likely to play important roles in regulating the structure of neural circuitry during development, and may also be an important determinant of selective neuronal vulnerability in pathological conditions.

Effects of acute and chronic ethanol intake on synaptosomal glutamate binding activity

Abstract The effects of acute and chronic ethanol administration in vivo, on brain synaptosomal glutamate binding activity were explored. In the l -glutamic acid concentration range in which high affinity stereospecific binding to synaptic membranes takes place, both acute and chronic ethanol administration led to progressively greater glutamate binding depending on the chronicity of exposure to ethanol (2 hr to 16 days). These changes appeared to be primarily due to an increase in the maximum binding capacity of these membrane binding sites rather than to changes in their affinity towards the ligand. The withdrawal from ethanol following 16 days of continuous exposure brought about a slow reversal of the increased glutamate-binding activity over a period of 6 days. A brief (2 hr) exposure to ethanol in vitro, produced a small decrease in glutamate binding, whereas prolonged exposure to the alcohol during equilibrium dialysis had a biphasic effect on ligand binding to synaptosomal membranes. These results are suggestive of a possible role for l -glutamic acid in the nervous system during ethanolism and the post-withdrawal reaction.

Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2D21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2D21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2D21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2D21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2D21 to SERCA. In contrast, Bcl-2D21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2D21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2D21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.

Partial purification and characterization of a glutamate-binding membrane glycoprotein from rat brain*

Summary A 200-fold purification of a synaptic membrane glutamate-binding protein was achieved by a combination of affinity batch separation on glutamate-loaded glass fiber and affinity chromatography on concanavalin A sepharose. Analytical gel electrophoresis of this fraction in Triton X-100 revealed a predominant acidic, small-molecular weight protein species exhibiting all of the glutamate binding activity and having a molecular weight of 13,800 as estimated by SDS gel electrophoresis. This purified protein did not show any glutamate dehydrogenase, glutamate decarboxylase, or glutamine synthetase activity, and it differed from the glutamate transport system in many of its characteristics. On the other hand, its interaction with L-glutamate and other neuroexcitatory amino acids resembled that of the physiologic receptor for L-glutamic acid.

Glutamic acid and ethanol dependence.

Glutamate diethyl ester, a specific glutamate antagonist, attenuated the seizures and decreases in behavioral activity that were observed in mice during withdrawal. Prior to withdrawal, ethanol-dependent animals were supersensitive to kainic acid, a potent glutamate agonist, but they were not supersensitive to the convulsant drug pentylenetetrazol. These findings suggest that supersensitivity to glutamate develops during ethanol dependence, and that this phenomenon contributes to the signs of ethanol withdrawl.

Transgenic Expression of Glud1 (Glutamate Dehydrogenase 1) in Neurons: In Vivo Model of Enhanced Glutamate Release, Altered Synaptic Plasticity, and Selective Neuronal Vulnerability

The effects of lifelong, moderate excess release of glutamate (Glu) in the CNS have not been previously characterized. We created a transgenic (Tg) mouse model of lifelong excess synaptic Glu release in the CNS by introducing the gene for glutamate dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. Glud1 is, potentially, an important enzyme in the pathway of Glu synthesis in nerve terminals. Increased levels of GLUD protein and activity in CNS neurons of hemizygous Tg mice were associated with increases in the in vivo release of Glu after neuronal depolarization in striatum and in the frequency and amplitude of miniature EPSCs in the CA1 region of the hippocampus. Despite overexpression of Glud1 in all neurons of the CNS, the Tg mice suffered neuronal losses in select brain regions (e.g., the CA1 but not the CA3 region). In vulnerable regions, Tg mice had decreases in MAP2A labeling of dendrites and in synaptophysin labeling of presynaptic terminals; the decreases in neuronal numbers and dendrite and presynaptic terminal labeling increased with advancing age. In addition, the Tg mice exhibited decreases in long-term potentiation of synaptic activity and in spine density in dendrites of CA1 neurons. Behaviorally, the Tg mice were significantly more resistant than wild-type mice to induction and duration of anesthesia produced by anesthetics that suppress Glu neurotransmission. The Glud1 mouse might be a useful model for the effects of lifelong excess synaptic Glu release on CNS neurons and for age-associated neurodegenerative processes.