Sebastian Schuchmann

Quantitating protein synthesis, degradation, and endogenous antigen processing.

Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cells 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cells 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate.

Axo-Axonal Coupling: A Novel Mechanism for Ultrafast Neuronal Communication

We provide physiological, pharmacological, and structural evidence that axons of hippocampal principal cells are electrically coupled, with prepotentials or spikelets forming the physiological substrate of electrical coupling as observed in cell somata. Antidromic activation of neighboring axons induced somatic spikelet potentials in neurons of CA3, CA1, and dentate gyrus areas of rat hippocampal slices. Somatic invasion by these spikelets was dependent on the activation of fast Na(+) channels in the postjunctional neuron. Antidromically elicited spikelets were suppressed by gap junction blockers and low intracellular pH. Paired axo-somatic and somato-dendritic recordings revealed that the coupling potentials appeared in the axon before invading the soma and the dendrite. Using confocal laser scanning microscopy we found that putative axons of principal cells were dye coupled. Our data thus suggest that hippocampal neurons are coupled by axo-axonal junctions, providing a novel mechanism for very fast electrical communication.

Experimental febrile seizures are precipitated by a hyperthermia-induced respiratory alkalosis

Febrile seizures are frequent during early childhood, and prolonged (complex) febrile seizures are associated with an increased susceptibility to temporal lobe epilepsy. The pathophysiological consequences of febrile seizures have been extensively studied in rat pups exposed to hyperthermia. The mechanisms that trigger these seizures are unknown, however. A rise in brain pH is known to enhance neuronal excitability. Here we show that hyperthermia causes respiratory alkalosis in the immature brain, with a threshold of 0.2–0.3 pH units for seizure induction. Suppressing alkalosis with 5% ambient CO2 abolished seizures within 20 s. CO2 also prevented two long-term effects of hyperthermic seizures in the hippocampus: the upregulation of the Ih current and the upregulation of CB1 receptor expression. The effects of hyperthermia were closely mimicked by intraperitoneal injection of bicarbonate. Our work indicates a mechanism for triggering hyperthermic seizures and suggests new strategies in the research and therapy of fever-related epileptic syndromes.

Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis

Glucocorticoids mediate their therapeutic actions mostly by genomic effects via cytosolic receptors, but some effects are too rapid to be mediated by changes at the genomic level. The detailed mechanisms of these nongenomic actions are still unclear. Membrane‐bound glucocorticoid receptors (mGCR) have been suggested to be involved, although their physiological existence in humans so far is hypothetical. For the first time we demonstrate the existence of mGCR on monocytes and B cells obtained from healthy blood donors using high‐sensitivity immunofluorescent staining. Immunostimulation with lipopolysaccharide increases the percentage of mGCR‐positive monocytes, which can be prevented by inhibiting the secretory pathway. Overexpression of the human glucocorticoid receptor α alone is not sufficient to enhance mGCR expression. These in vitro findings are consistent with our clinical observation that in patients with rheumatoid arthritis the frequency of mGCR positive monocytes is increased and positively correlated with disease activity. We conclude that mGCR are 1) indeed physiologically present in healthy blood donors, but remained unidentified by conventional techniques due to their small number per cell and 2) actively up‐regulated and transported through the cell after immunostimulation. These receptors may reflect a feedback mechanism of the organism upon immunostimulation and/or play a role in pathogenesis.—Bartholome, B., Spies, C. M., Gaber, T., Schuchmann, S., Berki, T., Kunkel, D., Bienert, M., Radbruch, A., Burmester, G.‐R., Lauster, R., Scheffold, A., Buttgereit, F. Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up‐regulated after in vitro stimulation and in patients with rheumatoid arthritis. FASEB J. 18, 70–80 (2004)

The cation-chloride cotransporter NKCC1 promotes sharp waves in the neonatal rat hippocampus

Earlier studies indicate a crucial role for the interconnected network of intrinsically bursting CA3 pyramidal neurons in the generation of in vivo hippocampal sharp waves (SPWs) and their proposed neonatal in vitro counterparts, the giant depolarizing potentials (GDPs). While mechanisms involving ligand‐ and voltage‐gated channels have received lots of attention in the generation of CA3 network events in the immature hippocampus, the contribution of ion‐transport mechanisms has not been extensively studied. Here, we show that bumetanide, a selective inhibitor of neuronal Cl− uptake mediated by the Na+–K+–2Cl− cotransporter isoform 1 (NKCC1), completely and reversibly blocks SPWs in the neonate (postnatal days 7–9) rat hippocampus in vivo, an action also seen on GDPs in slices (postnatal days 1–8). These findings strengthen the view that GDPs and early SPWs are homologous events. Gramicidin‐perforated patch recordings indicated that NKCC1 accounts for a large (∼10 mV) depolarizing driving force for the GABAA current in the immature CA3 pyramids. Consistent with a reduction in the depolarization mediated by endogenous GABAA‐receptor activation, bumetanide inhibited the spontaneous bursts of individual neonatal CA3 pyramids, but it slightly increased the interneuronal activity as seen in the frequency of spontaneous GABAergic currents. An inhibitory effect of bumetanide was seen on the in vitro population events in the absence of synaptic GABAA receptor‐mediated transmission, provided that a tonic GABAA receptor‐mediated current was present. Our work indicates that NKCC1 expressed in CA3 pyramidal neurons promotes network activity in the developing hippocampus.

Coupling of neuronal activity and mitochondrial metabolism as revealed by nad(p)h fluorescence signals in organotypic hippocampal slice cultures of the rat

During physiological activity neurons may experience localised energy demands which require intracellular signals for stimulation of mitochondrial NADH generation and subsequent delivery of ATP. To elucidate these mechanisms, we applied microfluorimetric monitoring of cytoplasmic (Fluo-3) and mitochondrial (Rhod-2) calcium concentration ([Ca(2+)](c), [Ca(2+)](m)), as well as of mitochondrial oxidative metabolism (NAD(P)H), whilst simultaneously measuring changes in extracellular potassium concentration ([K(+)](o)), as an indicator of neuronal activity in hippocampal slice cultures. Changes in neuronal activity were induced by repetitive stimulation at different frequencies (5, 20, 100 Hz) and intensities. Stimulation parameters were chosen to elicit rises in [K(+)](o) of less than 3 mM which is comparable to physiologically occurring rises in [K(+)](o). The mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) reduced stimulus-induced changes in Rhod-2 fluorescence by 79%, indicating that Rhod-2 signals were primarily of mitochondrial origin. Repetitive stimulation at 20 Hz applied to areas CA1 or CA3 resulted in moderate rises in [K(+)](o) which were associated with stimulus-dependent elevations in [Ca(2+)](c) and [Ca(2+)](m) of up to 15%. The same stimuli also elicited biphasic changes in NAD(P)H fluorescence characterised by an initial decline and a subsequent prolonged elevation of up to 10%. Variation of stimulus parameters revealed close correlations between rises in [K(+)](o), in [Ca(2+)](m) and changes in NAD(P)H fluorescence. To elucidate the role of intracellular Ca(2+) accumulation in induction of NAD(P)H fluorescence signals, the effect of application of Ca(2+)-free solution on these signals evoked by repetitive antidromic stimulation of the alveus during recordings in area CA1 was studied. Lowering extracellular Ca(2+) led to complete blockade of postsynaptic field potential components as well as of rises in [Ca(2+)](c) and [Ca(2+)](m). Amplitudes of NAD(P)H signals were reduced by 59%, though rises in [K(+)](o) were comparable in presence and absence of extracellular Ca(2+). The results suggest i) that mitochondrial oxidative metabolism is fine-tuned to graded physiological activity in neurons and ii) that rapid mitochondrial Ca(2+) signalling represents one of the main determinants for stimulation of oxidative metabolism under physiological conditions.

Synaptic PRG-1 Modulates Excitatory Transmission via Lipid Phosphate-Mediated Signaling

Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.

Monitoring NAD(P)H autofluorescence to assess mitochondrial metabolic functions in rat hippocampal–entorhinal cortex slices

Changes in neuronal energy metabolism, mitochondrial functions and homeostasis of reactive oxygen species are often supposed to induce alterations in neuronal activity in hippocampal slice models. In order to investigate the NAD(P)H autofluorescence signal in brain slice models, methods to monitor NAD(P)H signal in isolated mitochondria as described by Chance et al. [J. Biol. Chem. 254 (1979) 4764] and dissociated neurons as described by Duchen [Biochem. J. 283 (1992) 41] were adapted to recording conditions required for brain slices. Considering different experimental questions, we established an approach to monitor NAD(P)H autofluorescence signals from hippocampal slices of 400 microm thickness under either submerged or interface conditions. Therefore the procedure described here allows the measurement of NAD(P)H autofluorescence under conditions typically required in electrophysiological experiments. Depolarization of plasma membrane caused by electrical stimulation or application of glutamate (100 microM) resulted in a characteristic initial decrease followed by a long-lasting increase in the NAD(P)H autofluorescence signal. H(2)O(2) (100 microM) evoked a strong NAD(P)H signal decrease indicating direct oxidation to the nonfluorescencend NAD(P)(+). In contrast, the increase in NAD(P)H signal that followed a brief inhibition of mitochondrial respiratory chain complex I using rotenone (1 microM) indicated an accumulation of NAD(P)H. However, in presence of rotenone (1 microM) electrically evoked long-lasting NAD(P)H signal overshoot decreased progressively, due to a negative feedback of accumulated NAD(P)H to the citrate cycle. A comparable reduction in NAD(P)H signal increase were observed during low-Mg(2+) induced epileptiform activity, indicating a relative energy failure. In conclusion, the method presented here allows to monitor NAD(P)H autofluorescence signals to gain insight into the coupling of neuronal activity, energy metabolism and mitochondrial function in brain slice models.

Altered Ca2+ Signaling and Mitochondrial Deficiencies in Hippocampal Neurons of Trisomy 16 Mice: A Model of Down’s Syndrome

It has been suggested that augmented nerve cell death in neurodegenerative diseases might result from an impairment of mitochondrial function. To test this hypothesis, we investigated age-dependent changes in neuronal survival and glutamate effects on Ca2+ homeostasis and mitochondrial energy metabolism in cultured hippocampal neurons from diploid and trisomy 16 (Ts16) mice, a model of Down’s syndrome. Microfluorometric techniques were used to measure survival rate, [Ca2+]ilevel, mitochondrial membrane potential, and NAD(P)H autofluorescence. We found that Ts16 neurons die more than twice as fast as diploid neurons under otherwise identical culture conditions. Basal [Ca2+]i levels were elevated in Ts16 neurons. Moreover, in comparison to diploid neurons, Ts16 neurons showed a prolonged recovery of [Ca2+]iand mitochondrial membrane potential after brief glutamate application. Glutamate evoked an initial NAD(P)H decrease that was found to be extended in Ts16 neurons in comparison to diploid neurons. Furthermore, for all age groups tested, glutamate failed to cause a subsequent NAD(P)H overshoot in Ts16 cultures in contrast to diploid cultures. In the presence of cyclosporin A, an inhibitor of the mitochondrial membrane permeability transition, NAD(P)H increase was observed in both diploid and Ts16 neurons. The results support the hypothesis that Ca2+ impairs mitochondrial energy metabolism and may play a role in the pathogenesis of neurodegenerative changes in neurons from Ts16 mice.

Increased mitochondrial superoxide generation in neurons from trisomy 16 mice: a model of Down’s syndrome

Increased neuronal cell death in neurodegenerative diseases has been suggested to result from an increased mitochondrial generation of radical oxygen species (ROS). To test this hypothesis, we investigated superoxide formation in cultured hippocampal neurons from diploid and trisomy 16 mice (Ts16), a model of Downs syndrome. Microflurometric techniques were used to measure superoxide-induced oxidation rate of hydroethidine (HEt) to ethidium and reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) autofluorescence signal to monitor changes in neuronal energy metabolism. We found an increase in superoxide formation by more than 50% in Ts16 neurons in comparison with diploid control neurons. In the presence of the mitochondrial respiratory chain complex I inhibitor rotenone superoxide production was blocked in diploid neurons, but the increased superoxide generation in Ts16 neurons remained. Uncoupling of mitochondrial oxidative phosphorylation using carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused irreversible deficiency in the energy metabolism, monitored by NAD(P)H autofluorescence in Ts16 neurons, but not in diploid control neurons. These results suggest an increased basal generation of superoxide in Ts16 neurons, probably caused by a deficient complex I of mitochondrial electron transport chain, which leads to an impaired mitochondrial energy metabolism and finally neuronal cell death.