Loss of Long-Term Potentiation at Hippocampal Output Synapses in Experimental Temporal Lobe Epilepsy.

Patients suffering from temporal lobe epilepsy (TLE) show severe problems in hippocampus dependent memory consolidation. Memory consolidation strongly depends on an intact dialog between the hippocampus and neocortical structures. Deficits in hippocampal signal transmission are known to provoke disturbances in memory formation. In the present study, we investigate changes of synaptic plasticity at hippocampal output structures in an experimental animal model of TLE. In pilocarpine-treated rats, we found suppressed long-term potentiation (LTP) in hippocampal and parahippocampal regions such as the subiculum and the entorhinal cortex (EC). Subsequently we focused on the subiculum, serving as the major relay station between the hippocampus proper and downstream structures. In control animals, subicular pyramidal cells express different forms of LTP depending on their intrinsic firing pattern. In line with our extracellular recordings, we could show that LTP could only be induced in a minority of subicular pyramidal neurons. We demonstrate that a well-characterized cAMP-dependent signaling pathway involved in presynaptic forms of LTP is perturbed in pilocarpine-treated animals. Our findings suggest that in TLE, disturbances of synaptic plasticity may influence the information flow between the hippocampus and the neocortex.

Immunoproteasome deficiency alters microglial cytokine response and improves cognitive deficits in Alzheimer's disease-like APPPS1 mice.

The immunoproteasome (iP) represents a specialized type of proteasomes, which plays an important role in the clearance of oxidant-damaged proteins under inflammatory and pathological conditions determining the outcome of various diseases. In Alzheimer's disease (AD)-like APPPS1 mice Aß-deposition is paralleled by iP upregulation, most likely mediated through type I interferon induction. To define the impact of increased iP expression we crossed APPPS1 mice with mice deficient in the iP subunit LMP7 resulting in impaired iP function. While LMP7 deficient APPPS1 mice showed no major change in cerebral Aß-pathology, we observed an altered cytokine response in microglia isolated from LMP7 deficient APPPS1 mice compared to LMP7 expressing APPPS1 control mice. The altered microglial cytokine profile upon iP deficiency in the presence of extracellular Aß-pathology was associated with an improvement of Aß-associated cognitive deficits typically present in APPPS1 mice. Our findings suggest a role for iP in the regulation of the innate immune response towards extracellular Aß-pathology and indicate that inhibition of iP function can modulate the cognitive phenotype upon overexpression of Aß.

TCF11/Nrf1-Mediated Induction of Proteasome Expression Prevents Cytotoxicity by Rotenone.

AIMS: Precise regulation of cellular protein degradation is essential for maintaining protein and redox homeostasis. The ubiquitin proteasome system (UPS) represents one of the major degradation machineries, and UPS disturbances are strongly associated with neurodegeneration. We have previously shown that the transcription factor TCF11/Nrf1 induces antioxidant response element-mediated upregulation of UPS components in response to proteotoxic stress. Knockout of TCF11/Nrf1 is embryonically lethal, and therefore, the present investigation describes the role of oxidative stress in regulating TCF11/Nrf1-dependent proteasome expression in a model system relevant to Parkinson's disease. RESULTS: Using the human dopaminergic neuroblastoma cell line SH-SY5Y and mouse nigrostriatal organotypic slice cultures, gene and protein expression analysis and functional assays revealed oxidative stress is induced by the proteasome inhibitor epoxomicin or the mitochondrial complex I inhibitor rotenone and promotes the upregulation of proteasome expression and function mediated by TCF11/Nrf1 activation. In addition, we show that these stress conditions induce the unfolded protein response. TCF11/Nrf1, thus, has a cytoprotective function in response to oxidative and proteotoxic stress. Innovation and Conclusion: We here demonstrate that adaption of the proteasome system in response to oxidative stress is dependent on TCF11/Nrf1 in this model system. We conclude that TCF11/Nrf1, therefore, plays a vital role in maintaining redox and protein homeostasis. This work provides a vital insight into the molecular mechanisms of neurodegeneration due to oxidative stress by rotenone, and further studies investigating the role of TCF11/Nrf1 in the human condition would be of considerable interest. Antioxid. Redox Signal. 25, 870-885.

Patch clamp combined with voltage/concentration clamp to determine the kinetics and voltage dependency of N-methyl-D-aspartate (NMDA) receptor open channel blockers.

Electrophysiological techniques can be used to great effect to help determine the mechanism of action of a compound. However, many factors can compromise the resulting data and their analysis, such as the speed of solution exchange, expression of additional ion channel populations including other ligand-gated receptors and voltage-gated channels, compounds having multiple binding sites, and current desensitization and rundown. In this chapter, such problems and their solutions are discussed and illustrated using data from experiments involving the uncompetitive NMDA receptor antagonist memantine. Memantine differs from many other NMDA receptor channel blockers in that it is well tolerated and does not cause psychotomimetic effects at therapeutic doses. Various electrophysiological parameters of NMDA-induced current blockade by memantine have been proposed to be important in determining therapeutic tolerability; potency, onset and offset kinetics, and voltage dependency. These were all measured using whole cell patch clamp techniques using hippocampal neurons. Full results are shown here for memantine, and these are summarized and compared to those from similar experiments with other NMDA channel blockers. The interpretation of these results is discussed, as are theories concerning the tolerability of NMDA channel blockers, with the aim of illustrating how electrophysiological data can be used to form and support a physiological hypothesis.

Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds.

Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-ß-cyclodextrine (MßCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

Potency, voltage-dependency, agonist concentration-dependency, blocking kinetics and partial untrapping of the uncompetitive N-methyl-D-aspartate (NMDA) channel blocker memantine at human NMDA (GluN1/GluN2A) receptors.

Both the clinical tolerability and the symptomatic effects of memantine in the treatment of Alzheimer's disease have been attributed to its moderate affinity (IC(50) around 1 microM at -70 mV) for NMDA receptor channels and associated fast, double exponential blocking/unblocking kinetics and strong voltage-dependency. Most of these biophysical data have been obtained from rodent receptors. Some substances show large species-specific differences, so using human rather than rodent receptors and tissue may highlight important differences in the effects of drugs. In the present study we compared the potency of memantine, ketamine and (+)MK-801 in binding to NMDA receptors in post-mortem human cortical tissue and to antagonize intracellular Ca(2+) responses of human GluN1/GluN2A receptors expressed in HEK-293 cells. In addition, the biophysical properties of memantine and ketamine were compared using patch clamp recordings from these cells. Memantine was confirmed to be a moderate affinity (IC(50) at -70 mV of 0.79+/-0.02 microM, Hill=0.92+/-0.02), strongly voltage-dependent (delta=0.90+/-0.09) uncompetitive antagonist of human GluN1/GluN2A receptors. Moreover, the rapid double exponential blocking kinetics (e.g. at 10 microM - onset tau(fast)=273+/-25 ms (weight 69%), onset tau(slow)=2756+/-296 ms, offset tau(fast)=415+/-82 ms (weight 38%) offset tau(slow)=5107+/-1204 ms) and partial untrapping (around 20%) previously reported for memantine on rodent receptors were confirmed for human receptors. Ketamine showed similar potency (IC(50) at -70 mV of 0.71+/-0.03 microM, Hill=0.84+/-0.02) but somewhat less pronounced voltage-dependency (delta=0.79+/-0.04), slower, single exponential kinetics (ketamine: k(on)=0.15+/-0.05 x 10(6)M(-1)s(-1), k(off)=0.22+/-0.05 s(-1)c.f. memantine following normalization k(on)=0.32+/-0.11 x 10(6)M(-1)s(-1), k(off)=0.53+/-0.10s(-1)) and was fully trapped. The present data closely match previously reported data from studies in rodent receptors and suggest that the proposed mechanism of action of memantine in Alzheimer's disease as a fast, voltage-dependent open-channel blocker of NMDA receptors can be confirmed for human NMDA receptors.

Blocking kinetics of memantine on NR1a/2A receptors recorded in inside-out and outside-out patches from Xenopus oocytes.

Previous experiments on primary cultures of hippocampal/cortical neurones revealed that the block and unblock of N-Methyl-D-Aspartate (NMDA) receptor channels by memantine showed double exponential kinetics and that the offset kinetics following a voltage-step were much faster than following a concentration jump. There are, however, two major problems when using such cultured primary neurones for these experiments (1) the almost certain expression of heterogeneous NMDA receptor subunits which could underlie double exponential kinetics due to different potencies at receptor subtypes and (2) slow space- and concentration-clamp due to neuronal morphology which could mask even faster kinetics. Therefore, we performed similar experiments with Xenopus oocytes exclusively expressing one NMDA receptor type (NR1a/2A) at high levels which allowed recordings from membrane patches with large currents. The use of inside-out patches for voltage-step and outside-out patches in combination with a piezo driven fast application system largely negated potential space- and concentration-clamp problems. Block and unblock of the NMDA receptor by memantine after both voltage jump and concentration jumps showed triple exponential kinetics. The fast onset kinetics of NMDA receptor channel block following both concentration-clamp and voltage jumps from +70 to -70 mV were similar. In contrast, offset kinetics after a voltage-step from -70 to +70 mV were much faster than following a concentration jump at the holding potential of -70 mV. These results provide further support for the hypothesis that rapid relief of block via strong synaptic membrane depolarisation underlies the good therapeutic profile of memantine.

Memantine does not show intracellular block of the NMDA receptor channel.

Mg2+ is known to gain access to the NMDA receptor channel from both the extra- and intracellular compartments. Memantine, being an amphiphilic substance, reaches intracellular concentrations of approximately 30 microM, which are much higher than therapeutic extracellular concentrations ( approximately 1 microM). We therefore investigated whether memantine can also block the NMDA receptor channel from the intracellular compartment. NR1a/NR2A receptors were expressed in Xenopus oocytes and in classical two electrode voltage-clamp recordings, voltage-ramps from -100 to +100 mV confirmed moderate inward rectification of NR1a/NR2A receptor control responses at positive membrane potentials above +40 mV. Patch clamp recordings from these same cells (applying 100 microM glutamate and 1 mM Mg2+) revealed similar rectification at positive potentials in cell-attached mode which disappeared after pulling an inside-out patch. Application of 1 mM Mg2+ to the intracellular side of the receptor re-introduced the rectification seen in cell-attached mode, and 5 mM Mg2+ produced much more pronounced block. In contrast, 30 microM memantine was completely unable to block the NMDA receptor from the intracellular compartment. In conclusion, intracellular block of the NMDA receptor, as reported for Mg2+, is not of significance for the therapeutic effects of memantine.