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Hans Gerd Nothwang

Publications and source records attributed to Hans Gerd Nothwang.

13 recordsLinked to original sources
PubMed2006

Oligomerization of KCC2 correlates with development of inhibitory neurotransmission.

The neuron-specific K+-Cl- cotransporter KCC2 extrudes Cl- and renders GABA and glycine action hyperpolarizing. Thus, it plays a pivotal role in neuronal inhibition. Development-dependent KCC2 activation is regulated at the transcriptional level and by unknown posttranslational mechanisms. Here, we analyzed KCC2 activation at the protein level in the developing rat lateral superior olive (LSO), a prominent auditory brainstem structure. Electrophysiology demonstrated ineffective KCC2-mediated Cl- extrusion in LSO neurons at postnatal day 3 (P3). Immunohistochemical analyses by confocal and electron microscopy revealed KCC2 signals at the plasma membrane in the somata and dendrites of both immature and mature neurons. Biochemical analysis demonstrated mature glycosylation pattern of KCC2 at both stages. Immunoblot analysis of the immature brainstem demonstrated mainly monomeric KCC2. In contrast, three KCC2 oligomers with molecular masses of approximately 270, approximately 400, and approximately 500 kDa were identified in the mature brainstem. These oligomers were sensitive to sulfhydryl-reducing agents and resistant to SDS, contrary to the situation seen in the related Na+-(K+)-Cl- cotransporter. In HEK-293 cells, coexpressed hemagglutinin-tagged KCC2 assembled with histidine-tagged KCC2, demonstrating formation of homomers. Based on these findings, we conclude that the oligomers represent KCC2 dimers, trimers, and tetramers. Finally, immunoblot analysis identified a development-dependent increase in the oligomer/monomer ratio from embryonic day 18 to P30 throughout the brain that correlates with KCC2 activation. Together, our data indicate that the developmental shift from depolarization to hyperpolarization can be determined by both increased gene expression and KCC2 oligomerization.

Animals↗
PubMed2006

Comparative gene expression analysis reveals a characteristic molecular profile of the superior olivary complex.

The superior olivary complex (SOC) is a very conspicuous structure in the mammalian auditory brainstem. It represents the first binaural processing center and is important for sound localization in the azimuth and in feedback regulation of cochlear function. In order to define molecular determinants of the SOC, which are of potential functional relevance, we have performed a comprehensive analysis of its transcriptome by serial analysis of gene expression in adult rats. Here, we performed a detailed analysis of the SOC's gene expression profile compared to that of two other neural tissues, the striatum and the hippocampus, and with extraocular muscle tissue. This tested the hypothesis that SOC-specific or significantly upregulated transcripts provide candidates for the specific function of auditory neurons. Thirty-three genes were significantly upregulated in the SOC when compared to the two other neural tissues. Thirteen encoded proteins involved in neurotransmission, including action potential propagation, exocytosis, and myelination; five genes are important for the energy metabolism, and five transcripts are unknown or poorly characterized and have yet to be described in the nervous system. The comparison of functional gene classes indicates that the SOC has the highest energy demand of the three neural tissues, yet protein turnover is apparently not increased. This suggests a high energy demand for fueling auditory neurotransmission. Such a demand may have implications on auditory-specific tasks and relate to central auditory processing disorders. Ultimately, these data provide new avenues to foster investigations of auditory function and to advance molecular physiology in the central auditory system.

Animals↗
PubMed2006

Neuroproteomics - the tasks lying ahead.

The brain is unquestionably the most fascinating organ. Despite tremendous progress, current knowledge falls short of being able to explain its function. An emerging approach toward improved understanding of the molecular mechanisms underlying brain function is neuroproteomics. Today's neuroscientists have access to a battery of versatile technologies both in transcriptomics and proteomics. The challenge is to choose the right strategy in order to generate new hypotheses on how the brain works. The goal of this review is therefore two-fold: first we recall the bewildering cellular, molecular, and functional complexity in the brain, as this knowledge is fundamental to any study design. In fact, an impressive complexity on the molecular level has recently re-emerged as a central theme in large-scale analyses. Then we review transcriptomics and proteomics technologies, as both are complementary. Finally, we comment on the most widely used proteomics techniques and their respective strengths and drawbacks. We conclude that for the time being, neuroproteomics should focus on its strengths, namely the identification of posttranslational modifications and protein-protein interactions, as well as the characterization of highly purified subproteomes. For global expression profiling, emphasis should be put on further development to significantly increase coverage.

Brain↗
PubMed2006

Aqueous polymer two-phase systems: effective tools for plasma membrane proteomics.

Plasma membranes (PMs) are of particular importance for all living cells. They form a selectively permeable barrier to the environment. Many essential tasks of PMs are carried out by their proteinaceous components, including molecular transport, cell-cell interactions, and signal transduction. Due to the key role of these proteins for cellular function, they take center-stage in basic and applied research. A major problem towards in-depth identification and characterization of PM proteins by modern proteomic approaches is their low abundance and immense heterogeneity in different cells. Highly selective and efficient purification protocols are hence essential to any PM proteome analysis. An effective tool for preparative isolation of PMs is partitioning in aqueous polymer two-phase systems. In two-phase systems, membranes are separated according to differences in surface properties rather than size and density. Despite their rare application to the fractionation of animal tissues and cells, they represent an attractive alternative to conventional fractionation protocols. Here, we review the principles of partitioning using aqueous polymer two-phase systems and compare aqueous polymer two-phase systems with other methods currently used for the isolation of PMs.

Animals↗
PubMed2005

Proteomic analysis of brain plasma membranes isolated by affinity two-phase partitioning.

A comprehensive analysis of plasma membrane proteins is essential to in-depth understanding of brain development, function, and diseases. Proteomics offers the potential to perform such a comprehensive analysis, yet it requires efficient protocols for the purification of the plasma membrane compartment. Here, we present a novel and efficient protocol for the separation and enrichment of brain plasma membrane proteins. It lasts only 4 h and is easy to perform. It highly enriches plasma membrane proteins and can be applied to small amounts of brain tissue, such as the cerebellum of a single rat, which was used in the present study. The protocol is based on affinity partitioning of microsomes in an aqueous two-phase system. Marker enzyme assays demonstrated a more than 12-fold enrichment of plasma membranes and a strong reduction of other compartments, such as mitochondria and the endoplasmic reticulum. 506 different proteins were identified when the enriched proteins underwent LC-MS/MS analysis subsequent to protein separation by SDS-PAGE. Using gene ontology, 146 proteins were assigned to a subcellular compartment. Ninety-three of those (64%) were membrane proteins, and 49 (34%) were plasma membrane proteins. A combined literature and database search for all 506 identified proteins revealed subcellular information on 472 proteins, of which 197 (42%) were plasma membrane proteins. These comprised numerous transporters, channels, and neurotransmitter receptors, e.g. the inward rectifying potassium channel Kir7.1 and the cerebellum-specific gamma-aminobutyric acid receptor GABRA6. Surface proteins involved in cell-cell contact and disease-related proteins were also identified. Six of the 146 assigned proteins were derived from mitochondrial membranes and 5 from membranes of the endoplasmic reticulum. Taken together, our protocol represents a simple, rapid, and reproducible tool for the proteomic characterization of brain plasma membranes. Because it conserves membrane structure and protein interactions, it is also suitable to enrich multimeric protein complexes from the plasma membrane for subsequent analysis.

Alkaline Phosphatase↗
PubMed2005

Developmental pattern of three vesicular glutamate transporters in the rat superior olivary complex.

Vesicular glutamate transporters (VGLUTs) mediate the packaging of the excitatory neurotransmitter glutamate into synaptic vesicles. Three VGLUT subtypes have been identified so far, which are differentially expressed in the brain. Here, we have investigated the spatiotemporal distribution of the three VGLUTs in the rat superior olivary complex (SOC), a prominent processing center, which receives strong glutamatergic inputs and which lies within the auditory brainstem. Immunoreactivity (ir) against all three VGLUTs was found in the SOC nuclei throughout development (postnatal days P0-P60). It was predominantly seen in axon terminals, although cytoplasmic labeling also occurred. Each transporter displayed a characteristic expression pattern. In the adult SOC, VGLUT1 labeling varied from strong in the medial nucleus of the trapezoid body, lateral superior olive, and medial superior olive (MSO) to moderate (ventral and lateral nuclei of the trapezoid body) to faint (superior paraolivary nucleus). VGLUT2-ir was moderate to strong throughout the SOC, whereas VGLUT3 was only weakly expressed. These results extend previous reports on co-localization of VGLUTs in the auditory brainstem. As in the adult, specific features were seen during development for all three transporters. Intensity increases and decreases occurred with both VGLUT1 and VGLUT3, whereas VGLUT2-ir remained moderately high throughout development. A striking result was obtained with VGLUT3, which was only transiently expressed in the different SOC nuclei between P0 and P12. A transient occurrence of VGLUT1-immunoreactive terminals on somata of MSO neurons was another striking finding. Our results imply a considerable amount of synaptic reorganization in the glutamatergic inputs to the SOC and suggest differential roles of VGLUTs during maturation and in adulthood.

Amino Acid Transport Systems, Acidic↗
PubMed2005

A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue.

Subcellular localization represents an essential, albeit often neglected, aspect of proteome analysis. Generally, the subcellular location of proteins determines the function of cells and tissues. Here we present a robust and versatile prefractionation protocol for mammalian cells and tissues which is appropriate for minute sample amounts. The protocol yields three fractions: a nuclear, a cytoplasmic, and a combined membrane and organelle fraction. The subcellular specificity and the composition of the fractions were demonstrated by immunoblot analysis of five marker proteins and analysis of 43 proteins by two-dimensional gel electrophoresis and mass spectrometry. To cover all protein species, both conventional two-dimensional and benzyldimethyl-n-hexadecyl ammonium chloride-sodium dodecyl sulfate (16-BAC-SDS) gel electrophoresis were performed. Integral membrane proteins and strongly basic nuclear histones were detected only in the 16-BAC-SDS gel electrophoresis system, confirming its usefulness for proteome analysis. All but one protein complied to the respective subcellular composition of the analyzed fractions. Taken together, the data make our subcellular prefractionation protocol an attractive alternative to other prefractionation methods which are based on less physiological protein properties.

Animals↗
PubMed2004

Gene expression profiling of the rat superior olivary complex using serial analysis of gene expression.

The superior olivary complex (SOC) is an auditory brainstem region that represents a favourable system to study rapid neurotransmission and the maturation of neuronal circuits. Here we performed serial analysis of gene expression (SAGE) on the SOC in 60-day-old Sprague-Dawley rats to identify genes specifically important for its function and to create a transcriptome reference for the subsequent identification of age-related or disease-related changes. Sequencing of 31 035 tags identified 10 473 different transcripts. Fifty-seven per cent of the unique tags with a count greater than four were statistically more highly represented in the SOC than in the hippocampus. Among them were genes encoding proteins involved in energy supply, the glutamate/glutamine shuttle, and myelination. Approximately 80 plasma membrane transporters, receptors, channels, and vesicular transporters were identified, and 25% of them displayed a significantly higher expression level in the SOC than in the hippocampus. Some of the plasma membrane proteins were not previously characterized in the SOC, e.g. the purinergic receptor subunit P2X(6) and the metabotropic GABA receptor Gpr51. Differential gene expression between SOC and hippocampus was confirmed using RNA in situ hybridization or immunohistochemistry. The extensive gene inventory presented here will alleviate the dissection of the molecular mechanisms underlying specific SOC functions and the comparison with other SAGE libraries from brain will ease the identification of promoters to generate region-specific transgenic animals. The analysis will be part of the publicly available database ID-GRAB.

Animals↗
PubMed2003

Protein analysis in the rat auditory brainstem by two-dimensional gel electrophoresis and mass spectrometry.

A catalogue of the protein repertoire of processing centres in the central auditory system would greatly foster our knowledge on the anatomical and functional properties of this sensory system. Towards this goal, we report on the first mapping study of the protein content in the superior olivary complex (SOC) and the inferior colliculus (IC) of the rat auditory brainstem. The protein content of these two structures was assessed by means of two-dimensional gel electrophoresis (2-DGE) and mass spectrometry. To do so, proteins were first separated into four fractions by differential centrifugation. For comparison, corresponding cerebellar fractions were also analysed. Immunoblot analysis revealed highly enriched microsomal and cytosolic fractions; the other two fractions were mixtures of various subcellular compartments. Separation of the 800 g pellets (enriched for nuclear and plasma membrane proteins) and the 100,000 g supernatants (enriched for cytosolic proteins) by 2-DGE yielded between 456 and 674 distinct protein spots after silver staining. The overall protein pattern of all three tissues was similar for a given fraction. Fifty prominent protein spots of the SOC cytosolic fraction were identified by mass spectrometry and yielded information on thirty different genes with various cellular functions, e.g. primary metabolism, cytoarchitecture, and signal transduction. Sequencing of eleven corresponding spots from the SOC and IC cytosolic fractions confirmed the great similarity between the two samples. The results of this analysis are part of a novel integrated database of the gene repertoire of the auditory brainstem (ID-GRAB), that is publicly available (http://www.id-grab.de).

Animals↗
PubMed2003

Expression and function of chloride transporters during development of inhibitory neurotransmission in the auditory brainstem.

Glycine and GABA, the dominant inhibitory neurotransmitters in the CNS, assume a depolarizing role in early development, leading to increased cytoplasmic Ca2+ levels and action potentials. The effect is thought to be of some significance for maturation. The depolarization is caused by Cl- efflux, and chloride transporters contribute to the phenomenon by raising the intracellular Cl- concentration ([Cl-]i) above equilibrium, thereby generating an outward-directed electrochemical gradient for Cl-. In mature neurons, the [Cl-]i is reduced below equilibrium, thus rendering glycine activity hyperpolarizing. Here, we investigated the temporal expression of the K-Cl cotransporter KCC2 and the Na-K-Cl cotransporter NKCC1 in the lateral superior olive (LSO) of rats and mice. The two cation cotransporters normally extrude and accumulate Cl-, respectively. As evidenced by several methods, KCC2 mRNA was present in LSO neurons during both the depolarizing and hyperpolarizing periods. Western blots confirmed a constant level of KCC2 in the brainstem, and immunohistochemistry showed that the protein is diffusely distributed within neonatal LSO neurons, becoming integrated into the plasma membrane only with increasing age. The glycine reversal potential in KCC2 knock-out mice differed significantly from that determined in wild-type controls at postnatal day 12 (P12) but not at P3, demonstrating that KCC2 is not active in neonates, despite its early presence. NKCC1 mRNA was not detected during the depolarizing phase in the LSO, implying that this transporter does not contribute to the high [Cl-]i. Our results reveal major differences in the development of [Cl-]i regulation mechanisms seen in brainstem versus forebrain regions.

Aging↗
PubMed2003

Differential expression pattern of chloride transporters NCC, NKCC2, KCC1, KCC3, KCC4, and AE3 in the developing rat auditory brainstem.

During development of inhibitory synapses, the action of the two neurotransmitters GABA and glycine shifts from depolarizing to hyperpolarizing. The shift is due to an age-dependent regulation of the intracellular free chloride concentration ([Cl(-)](i)) in postsynaptic neurons. A model system to study this maturation process is a glycinergic projection in the mammalian auditory brainstem. It is formed in the superior olivary complex (SOC) by neurons of the medial nucleus of the trapezoid body, whose axons terminate in the lateral superior olive (LSO). LSO neurons of perinatal rats and mice are depolarized upon glycine application, whereas older cells (>postnatal day (P) 8) are hyperpolarized. Here we examined the expression of six secondary active chloride transporter genes ( NCC, NKCC2, KCC1, KCC3, KCC4, and AE3) in the rat SOC to unravel the molecular mechanisms underlying this change. RT-PCR analysis demonstrated brainstem expression of KCC1, KCC3, KCC4, and AE3, but not of NCC and NKCC2. RNA in situ hybridization showed that only AE3 is highly expressed both at P3 (high [Cl(-)](i)) and P12 (low [Cl(-)](i)) in LSO neurons. KCC1 and KCC4 are weakly expressed in LSO neurons at P3 and P12, respectively. This study completes the expression analysis of all known chloride transporters sensitive to loop diuretic drugs in the SOC and demonstrates differences in the maturation between hippocampal and brainstem inhibitory synapses.

Animals↗