Prostate cancer may trigger paraneoplastic limbic encephalitis: a case report and a review of the literature.

We present a case of a previously healthy and active 64-year-old man who experienced a rapid neuropsychiatric decline. All tests for metabolic causes, neuroinfection, intracranial infarction or tumor were negative. By the means of magnetic resonance imaging, electroencephalography and the anti-Hu antibody test the patient was diagnosed with paraneoplastic limbic encephalitis related to prostate cancer. The patient died within 6 months. We review the literature on prostate cancer-related paraneoplastic limbic encephalitis. High-risk prostate cancer can trigger paraneoplastic limbic encephalitis, a rapidly progressive neurological syndrome with a bad prognosis.

The two different effects of the potential neuroprotective compound minocycline on AMPA-type glutamate receptors.

BACKGROUND: Minocycline has demonstrated neuroprotective effects in experimental neurodegenerative diseases. The aim of this study was to investigate if there is any direct interaction between minocycline and the AMPA-type receptor channels, and to elucidate the underlying molecular pharmacological mechanisms. METHODS: The patch-clamp technique was used combined with an ultrafast solution exchange system to investigate the interaction of minocycline with recombinant AMPA-type glutamate receptor channels (homomeric GluR2flipGQ or nondesensitizing GluR2L504Y). RESULTS: Dose-dependent decreases in the relative peak current amplitude (rAmp) and the relative steady-state current (rC(des)) were found in coapplication experiments with GluR2L504Y receptors, but not in preincubation experiments. Furthermore, coapplication of 1 or 3 mmol/l minocycline showed a decrease in the fast time constant of current decay, and reopening currents were observed. But in the test with GluR2flipGQ receptors, rAmp, relative area under the curve and rC(des) increased with increasing concentrations of minocycline, and the steady-state time constant also increased when 3 µmol/l glutamate were used as agonist. CONCLUSION: Minocycline modulates AMPA-type receptor channels in a combination of a weaker open-channel block effect and a stronger potentiation effect, and the latter effect arises mainly from attenuating the extent of receptor desensitization.

The interaction of the neuroprotective compounds riluzole and phenobarbital with AMPA-type glutamate receptors: a patch-clamp study.

BACKGROUND: Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors is a promising pharmacological strategy in the treatment of neurodegenerative diseases. The aim of the study is to elucidate if there are direct interactions of riluzole and phenobarbital with AMPA-type receptor channels and to determinethe molecular pharmacological mechanisms. METHODS: The patch-clamp technique was used combining an ultrafast solution exchange system to investigate the interaction of riluzole and phenobarbital with recombinant AMPA-type glutamate receptor channels (homomeric GluR2flipGQ or nondesensitizing GluR2L504Y). RESULTS: A dose-dependent decrease in the relative peak current amplitude (rAmp) and the relative area-under-the-current curve (rAUC) were found after preincubation with 0.1 mmol/l or higher concentrations of riluzole. Furthermore, in coapplication experiments with GluR2L504Y, the application of 1 or 3 mmol/l riluzole showed a decrease in the current decay time constant, and a reopening current was observed at 3 mmol/l riluzole. Phenobarbital blocks AMPA receptor channels dose-dependently in the coapplication experiments, and reopening currents after removing glutamate and blocker were observed. A slight block effect after preincubation should indicate an additional competitive block effect. CONCLUSION: Riluzole and phenobarbital modulate AMPA-type receptor channels separately, which could be both characterized as a combination of open-channel block and competitive-block mechanism.

Molecular analysis of the interaction of the pyrazine derivatives RPR119990 and RPR117824 with human AMPA-type glutamate receptor channels.

Antagonizing glutamatergic neurotransmission by blockade of AMPA-type glutamate receptors is a promising pharmacological strategy in the treatment of neurodegenerative diseases. We investigated the interaction of two new pyrazine derivatives (RPR119990 and RPR117824) with recombinant AMPA-type glutamate receptors. Recombinant homooligomeric GluR1flop, GluR2flip, GluR2flop, GluR6, non-desensitizing GluR2 L504Y channels and heterooligomeric GluR1/2 channels were expressed in HEK293 cells. AMPA-type channels were competitively inhibited by RPR119990 or RPR117824 with an IC(50) around 10 nM, at GluR6 channels the dose-response relation of the inhibition was shifted to higher concentrations. Non-desensitizing GluR2 L504Y channels were used to further characterize the inhibition. After equilibration with the agonist a marked dose-dependent current decay upon coapplication of glutamate and RPR119990 and a dose-independent time course of recovery from block was observed. The extents of current inhibition as well as the time constant of current decay upon addition of the blocker to the test solution were dependent on agonist concentration, pointing to a competitive inhibition. Quantitative analysis of the experimental data using computerized simulations are compatible with a competitive block mechanism and provides hints to binding sites at unliganded and liganded closed states of the receptor.

Two mechanisms of action of the adamantane derivative IEM-1460 at human AMPA-type glutamate receptors.

1. Antagonizing glutamatergic neurotransmission by blockade of AMPA-type glutamate receptors (GluR) is a promising pharmacological strategy for neuroprotection in neurodegenerative diseases and acute treatment of stroke. 2. We investigated the interaction of the adamantane derivative IEM-1460 with human wild-type and mutant AMPA-type GluR channels. Different recombinant homooligomeric human AMPA-type GluR channels and a rat nondesensitizing mutant GluR (GluR2 L504Y) channel were expressed in HEK293 cells and investigated using the patch-clamp technique in combination with ultrafast agonist application. 3. When IEM-1460 was coapplied with glutamate, an open channel block mechanism was observed at slow desensitizing GluR2 flip (>/=0.1 mM IEM-1460) and nondesensitizing GluR2 L504Y channels (>/=1 microM IEM-1460). 4. A competitive block of AMPA-type channels was observed with IC(50) values for the dose block curves of 0.1 mM IEM-1460 at human unmutated and 10 microM IEM-1460 at mutant GluR channels. 5. Nondesensitizing GluR2 L504Y channels were used to further characterize the block mechanism. After equilibration with the agonist, a current decay upon coapplication of glutamate and IEM-1460 was observed. The recovery from block was independent of the glutamate and IEM-1460 concentration. The extent of current inhibition as well as the time constant of current decay upon addition of the blocker to the test solution were dependent on agonist concentration; this strongly points to an additional competitive-like block mechanism of IEM-1460 at human AMPA-type GluR channels. 6. The data were interpreted in the frame of a molecular scheme with two binding sites of IEM-1460 at the receptor, one at the unliganded resting and the other at the fully liganded open state of the channels.

Effects of propofol on recombinant AMPA receptor channels.

The interaction of the anaesthetic propofol with recombinant human AMPA-type glutamate receptor channels was investigated by a patch-clamp study using fast agonist application techniques. Despite the marked effects of propofol on inhibitory synaptic transmission and voltage gated sodium channels, there is also evidence for a specific pharmacological action on AMPA receptors. In our study, we observed a deceleration of AMPA receptor channel desensitization in the prolonged presence of glutamate and propofol that is likely to account for the enhancement of ion currents through AMPA receptor channels observed in previous studies. While there was an increase in the rate and extent of desensitization at glutamate receptor 1, glutamate receptor 2, and glutamate receptor 3 AMPA receptors, no affection of current rise time, peak current amplitude, and deactivation properties was observed. Thus, our findings point to an isolated interaction with processes that control desensitization of AMPA receptor channels rather than indicating an interaction with channel opening and closing processes due to agonist binding and unbinding. The pharmacological effect described resembles in part that of compounds like cyclothiazide and aniracetam which are known to interact with channel desensitization.

Competitive and open channel block of recombinant nAChR channels by different antibiotics.

Various antibiotics may impair neuromuscular transmission, provoking symptoms of myasthenia in patients with a compromised safety margin of the synaptic transmission, but little is known about the underlying mechanisms at the molecular level. Using a modified patch-clamp technique in combination with an ultrafast system for solution exchange we investigated the functional interaction of gentamicin, penicillin G, tetracycline, erythromycin and ceftriaxone with nAChR transiently transfected into HEK293 cells as a potential molecular target. Gentamicin, penicillin G, tetracycline and erythromycin induced a combination of open channel and competitive block of nAChR channel currents whereas ceftriaxone had no effect. The IC50 for the competitive block was close to or within the range of clinically relevant concentrations. Except for erythromycin the open channel block was observed only at higher concentrations. From our in-vitro results we conclude that competitive inhibition of nAChR channels by antibiotics is an important mechanism underlying the impairment of neuromuscular transmission under clinical conditions.

Synaptic currents and transmitter responses in human NT2 neurons differentiated in aggregate culture.

Postmitotic neurons were generated from the human NT2 teratocarcinoma cell line in a novel cell aggregate differentiation procedure. The NT2 model neurons express punctate immunoreactivity for synapsin and for cell markers related to GABAergic and glutamatergic neurotransmission. Using the outside-out patch-clamp configuration, we characterized the kinetics of currents elicited by a rapid application of the amino acid neurotransmitters. Moreover, we detected spontaneous postsynaptic currents in glia free cell cultures that may result from the firing activity of glutamatergic and GABAergic NT2 neurons. These cultured spontaneously active networks may be a useful tool to analyze factors that modulate the formation and efficacy of synapses between human neurons.

Changes in extracellular pH affect glycine receptor channels expressed in HEK 293 cells.

Glycine receptors are expressed throughout the central nervous system working for inhibitory neurotransmission. Since fluctuations of the blood pH value occur under certain physiological and pathological conditions, we investigated the influence of the extracellular pH on glycine homomeric and heteromeric receptor functions using patch clamp in combination with the fast agonist application technique. Our results demonstrated that both alpha1 homomeric and alpha 1 beta heteromeric glycine receptors were remarkably inhibited under acidic extracellular pH. Under alkaline extracellular pH 8.5, there was also a negative functional effect. Desensitization was accelerated depending on pH and a rebound current was observed at an extremely acidic pH value. In double-pulse experiments on alpha1 homomeric receptors, a more rapid recovery of the glycinergic current was shown at pH 4.5 compared to current induced at a physiological pH of 7.2. Our study provided a potential cause for the impaired function of the glycine receptor channels during pH fluctuations occurring in the central nervous system, especially under pathological conditions such as epileptic seizure or ischaemia.

Dopaminergic properties and function after grafting of attached neural precursor cultures.

Generation of dopaminergic (DA) neurons from multipotent embryonic progenitors represents a promising therapeutical strategy for Parkinson's disease (PD). Aim of the present study was the establishment of enhanced cell culture conditions, which optimize the use of midbrain progenitor cells in animal models of PD. In addition, the progenitor cells were characterized during expansion and differentiation according to morphological and electrophysiological criteria and compared to primary tissue. Here, we report that CNS precursors can be expanded in vitro up to 40-fold and afterwards be efficiently differentiated into DA neurons. After 4-5 days under differentiation conditions, more than 70% of the neurons were TH+, equivalent to 30% of the total cell population. Calcium imaging revealed the presence of calcium-permeable AMPA receptors in the differentiated precursors which are capable to contribute to many developmental processes. The overall survival rate, degree of reinnervation and the behavioral performance after transplantation of 4 days in-vitro-differentiated cells were similar to results after direct grafting of E14 ventral mesencephalic cells, whereas after shorter or longer differentiation periods, respectively, less effects were achieved. Compared to the amount of in-vitro-generated DA neurons, the survival rate was only 0.8%, indicating that these cells are very vulnerable. Our results suggest that expanded and differentiated DA precursors from attached cultures can survive microtransplantation and integrate within the striatum in terms of behavioral recovery. However, there is only a short time window during in vitro differentiation, in which enough cells are already differentiated towards a DA phenotype and simultaneously not too mature for implantation. However, additional factors and/or genetical manipulation of these expanded progenitors will be required to increase their in vivo survival in order to improve both the ethical and the technical outlook for the use of fetal tissue in clinical transplantation.

A mutation in the GABA(A) receptor alpha(1)-subunit is associated with absence epilepsy.

OBJECTIVE: To detect mutations in GABRA1 in idiopathic generalized epilepsy. METHODS: GABRA1 was sequenced in 98 unrelated idiopathic generalized epilepsy patients. Patch clamping and confocal imaging was performed in transfected mammalian cells. RESULTS: We identified the first GABRA1 mutation in a patient with childhood absence epilepsy. Functional studies showed no detectable GABA-evoked currents for the mutant, truncated receptor, which was not integrated into the surface membrane. INTERPRETATION: We conclude that this de novo mutation can contribute to the cause of "sporadic" childhood absence epilepsy by a loss of function and haploinsufficiency of the GABA(A) receptor alpha(1)-subunit, and that GABRA1 mutations rarely are associated with idiopathic generalized epilepsy.

Desensitization and resensitization are independently regulated in human recombinant GluR subunit coassemblies.

AMPA-type glutamate receptor (GluR) channels are the most abundant excitatory transmitter receptors of the central nervous system. Four subunits with different posttranscriptional modifications and flip/flop splice variants are known. In vivo they occur as tetrameric heteromeric receptors. In the present study we analyzed the time course of desensitization (tau(D)) and resensitization (tau(rec)) kinetics of different homomeric (coassembly of splice or editing variants of one subunit) and heteromeric (coassembly of different subunits) GluR channels. We found that tau(D) had intermediate values depending on the amount of cDNA of the respective subunit at all heteromeric and homomeric GluR channels tested. The same holds true for tau(rec) except GluR2 flip channels were coexpressed with GluR1 channels. In this case, tau(rec) had values close to that of fast resensitizing GluR2 flip channels, even in the case of an abundance of GluR1 cDNA.

3,4-Methylenedioxymethamphetamine (ecstasy) activates skeletal muscle nicotinic acetylcholine receptors.

Adverse 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) effects are usually ascribed to neurotransmitter release in the central nervous system. Since clinical features such as fasciculations, muscle cramps, rapidly progressing hyperthermia, hyperkalemia, and rhabdomyolysis point to the skeletal muscle as additional target, we studied the effects of MDMA on native and cultured skeletal muscle. We addressed the question whether malignant hyperthermia (MH)-susceptible (MHS) muscle is predisposed to adverse MDMA reactions. Force measurements on muscle strips showed that 100 microM MDMA, a concentration close to that determined in some MDMA users, regularly enhanced the sensitivity of skeletal muscle to caffeine-induced contractures but did not cause contractures on its own. The left-shift of the dose-response curve induced by MDMA was greater in normal than in MHS muscle. Furthermore, MDMA did not release Ca(2+) from isolated sarcoplasmic reticulum vesicles. These findings do not support the view of an MH-triggering effect on muscle. However, MDMA induced Ca(2+) transients in myotubes and increased their acidification rate. Surprisingly, alpha-bungarotoxin, a specific antagonist of the nicotinic acetylcholine receptor (nAChR), abolished these MDMA effects. The nAChR agonistic action of MDMA was confirmed by patch-clamp measurements of ion currents on human embryonic kidney cells expressing nAChR. We conclude that the neuromuscular junction is a target of MDMA and that an activation of nAChR contributes to the muscle-related symptoms of MDMA users. The drug may be of particular risk in individuals with abundant extrajunctional nAChR such as in generalized denervation or muscle regeneration processes and may act on central nAChR.

Control of kinetic properties of GluR2 flop AMPA-type channels: impact of R/G nuclear editing.

The GluR2 flop subunit of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors greatly determines calcium permeability and kinetic properties of heteromeric AMPA subunit assemblies. Post-transcriptional editing of this subunit at the Q/R/N site controls calcium permeability whereas editing at the R/G site is involved in the regulation of biophysical properties. We used patch-clamp techniques with ultrafast solution exchange to examine the kinetics of recombinant human homomeric GluR2 flop channels transiently expressed in HEK293 cells [edited at the R/G site and Q/R/N site (GR), and unedited (RN) and edited (GN) at the R/G site both with asparagine (N) at the Q/R/N site]. The time constant of desensitization after application of 10 mm glutamate was 1.38 +/- 0.05 ms (n = 10), 5.53 +/- 0.57 ms (n = 7) and 1.33 +/- 0.06 ms (n = 12) for the GluR2 flop GR, RN and GN channels, respectively. The time constant of resensitization was 75 ms for the GluR2 flop RN and 30 ms for the GN channels. The dose-dependence of the peak current amplitude, kinetics of activation and deactivation, and peak open probability did not differ between RN and GN channels. The study shows that desensitization and resensitization kinetics of homomeric GluR2 flop channels are controlled by a single amino acid exchange (glycine by arginine) at the R/G site. Quantitative analysis by computer simulation using a circular kinetic scheme allows the prediction of the main experimental results.

Kinetic properties of human AMPA-type glutamate receptors expressed in HEK293 cells.

AMPA-type glutamate receptors (AMPAR) display a high variability in functional properties, which determine the time course of excitatory postsynaptic potentials. They are assembled as tetramers of GluR subunits 1-4 of different splice variants and nuclear edited isoforms. Presently, the kinetics of activation, desensitization and recovery from desensitization of human AMPARs (GluR1, 3 and 4 flip and flop, and GluR2 flip and flop in R and G edited forms, respectively) transiently expressed in HEK293 cells were studied with patch-clamp techniques and ultra fast agonist application. Activation time constants were identical for all receptors (0.13 ms). The GluR2 flip G variant showed the slowest desensitization (10.8 ms), GluR4 flip the fastest (1.6 ms). Recovery from desensitization varied between 3.1 ms (GluR4 flip) and 178 ms (GluR1 flip). To determine functional interactions between subunits in heteromeric receptors the GluR1 flip and the GluR2 flip R were coexpressed. The time constant of desensitization increased linearly from 2.5 ms (GluR1 flip homomers) to 6.8 ms (GluR2 flip R homomers) with the amount of GluR2 flip R cDNA transfected. Recovery followed a monoexponential time course and had a time constant of 178 ms in GluR1 flip homomeric expression. In all GluR1 flip/GluR2 flip heteromers and in GluR2 flip R homomers desensitization recovered with a time constant of approximately 50 ms. Thus, subunit interaction seems likely during recovery but not desensitization. Both parameters might influence the ability of AMPA receptors to mediate glutamate induced chronic excitotoxicity in neurodegenerative diseases.