Biomedical subjects

Karl-Heinz Smalla

Publications and source records attributed to Karl-Heinz Smalla.

14 recordsLinked to original sources

PubMed2006

The immunolocalization of the synaptic glycoprotein neuroplastin differs substantially between the human and the rodent brain.

Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that exists in two splice isoforms, np65/np55, and that was reported to play a prominent role in synaptic plasticity processes. The splice isoform np65 associates with synapses in an activity-dependent manner and has been shown to play a role for the induction of hippocampal long-term potentiation in rodents. We have therefore analyzed the distribution of neuroplastins in human brain. Neuroplastin is present in many neuronal cell types of the forebrain and cerebellum and immunoreactive label covers the cell soma, neurites and also puncta in the neuropil were visible. Interestingly, we found some remarkable species differences in the expression patterns of neuroplastins between the human and the rodent brain. In human brain np65 is prominently present in cerebellum while np55 is the predominant isoform in mouse and rat cerebellum. Moreover, the parasagittal stripe-type of staining seen with np55 in mouse cerebellum is not found in human brain. In addition we found no segregation of np65 immunolabel in hippocampal subregions like it was reported previously for the rat. These results might indicate different cellular functions of the molecule in different species.

Animals↗

PubMed2006

Neuronal Ca2+ signaling via caldendrin and calneurons.

The calcium sensor protein caldendrin is abundantly expressed in neurons and is thought to play an important role in different aspects of synapto-dendritic Ca2+ signaling. Caldendrin is highly abundant in the postsynaptic density of a subset of excitatory synapses in brain and its distinct localization raises several decisive questions about its function. Previous work suggests that caldendrin is tightly associated with Ca2+ - and Ca2+ release channels and might be involved in different aspects of the organization of the postsynaptic scaffold as well as with synapse-to-nucleus communication. In this report we introduce two new EF-hand calcium sensor proteins termed calneurons that apart from calmodulin represent the closest homologues of caldendrin in brain. Calneurons have a different EF-hand organization than other calcium sensor proteins, are prominently expressed in neurons and will presumably bind Ca2+ with higher affinity than caldendrin. Despite some significant structural differences it is conceivable that they are involved in similar Ca2+ regulated processes like caldendrin and neuronal calcium sensor proteins.

Amino Acid Sequence↗

PubMed2006

ProSAP-interacting protein 1 (ProSAPiP1), a novel protein of the postsynaptic density that links the spine-associated Rap-Gap (SPAR) to the scaffolding protein ProSAP2/Shank3.

ProSAPs/Shanks are a family of proteins that have a major scaffolding function for components of the postsynaptic density (PSD) of excitatory brain synapses. Members of the family harbor a variety of domains for protein-protein interactions, one of which is a unique PDZ domain that differs significantly from those of other proteins. We have identified a novel binding partner for this PDZ domain, termed ProSAPiP1, that is highly enriched in the PSD and shares significant sequence homology with the PSD protein PSD-Zip70. Both molecules code for a Fez1 domain that can be found in a total of four related proteins. ProSAPiP1 is widely expressed in rat brain and co-localizes with ProSAP2/Shank3 in excitatory spines and synapses. ProSAP2/Shank3 co-immunoprecipitates with ProSAPiP1 but not with PSD-Zip70. Both proteins, however, bind and recruit SPAR to synapses with a central coiled-coil region that harbors a leucine zipper motif. This region is also responsible for homo- and heteromultimerization of ProSAPiP1 and PSD-Zip70. Thus, ProSAPiP1 and PSD-Zip70 are founders of a novel family of scaffolding proteins, the "Fezzins," which adds further complexity to the organization of the PSD protein network.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Brevican-containing perineuronal nets of extracellular matrix in dissociated hippocampal primary cultures.

Perineuronal nets (PNN) are specialized extracellular matrix structures enwrapping CNS neurons, which are important regulators for neuronal and synaptic functions. Brevican, a chondroitin sulfate proteoglycan, is an integral component of PNN. Here, we have investigated the appearance of these structures in hippocampal primary cultures. The expression profile of brevican in mixed cultures resembles the in vivo pattern with a strong upregulation of all isoforms during the second and 3rd weeks in culture. Brevican is primarily synthesized by co-cultured glial fibrillary acidic protein (GFAP-)-positive astrocytes and co-assembles with its interaction partners in PNN-like structures on neuronal somata and neurites as identified by counterstaining with the PNN marker Vicia villosa lectin. Both excitatory and inhibitory synapses are embedded into PNN. Furthermore, axon initial segments are strongly covered by a dense brevican coat. Altogether, we show that mature primary cultures can form PNN, and that basic features of these extracellular matrix structures may be studied in vitro.

Animals↗

PubMed2004

Impaired synaptic function in the microglial KARAP/DAP12-deficient mouse.

Several proteins are expressed in both immune and nervous systems. However, their putative nonimmune functions in the brain remain poorly understood. KARAP/DAP12 is a transmembrane polypeptide associated with cell-surface receptors in hematopoeitic cells. Its mutation in humans induces Nasu-Hakola disease, characterized by presenile dementia and demyelinization. However, alteration of white matter occurs months after the onset of neuropsychiatric symptoms, suggesting that other neuronal alterations occur in the early phases of the disease. We hypothesized that KARAP/DAP12 may impact synaptic function. In mice deficient for KARAP/DAP12 function, long-term potentiation was enhanced and was partly NMDA receptor (NMDAR) independent. This effect was accompanied by changes in synaptic glutamate receptor content, as detected by the increased rectification of AMPA receptor EPSCs and increased sensitivity of NMDAR EPSCs to ifenprodil. Biochemical analysis of synaptic proteins confirmed these electrophysiological data. In mutants, the AMPA receptor GluR2 subunit expression was decreased only in the postsynaptic densities but not in the whole membrane fraction, demonstrating specific impairment of synaptic receptor accumulation. Alteration of the BNDF-tyrosine kinase receptor B (TrkB) signaling in the mutant was demonstrated by the dramatic decrease of synaptic TrkB with no change in other regulatory or scaffolding proteins. Finally, KARAP/DAP12 was detected only in microglia but not in neurons, astrocytes, or oligodendrocytes. KARAP/DAP12 may thus alter microglial physiology and subsequently synaptic function and plasticity through a novel microglia-neuron interaction.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Caldendrin but not calmodulin binds to light chain 3 of MAP1A/B: an association with the microtubule cytoskeleton highlighting exclusive binding partners for neuronal Ca(2+)-sensor proteins.

Caldendrin is a neuronal Ca(2+)-sensor protein (NCS), which represents the closest homologue of calmodulin (CaM) in nerve cells. It is tightly associated with the somato-dendritic cytoskeleton of neurons and highly enriched in the postsynaptic cytomatrix. Here, we report that caldendrin specifically associates with the microtubule cytoskeleton via an interaction with light chain 3 (LC3), a microtubule component with sequence homology to the GABAA receptor-associated protein (GABARAP), which is, like LC3, probably involved in cellular transport processes. Interestingly, two binding sites exist in LC3 for caldendrin from which only one exhibits a strict Ca(2+)-dependency for the interaction to take place but both require the presence of the first two EF-hands of caldendrin. CaM, however, is not capable of binding to LC3 at both sites despite its high degree of primary structure similarity with caldendrin. Computer modelling suggests that this might be explained by an altered distribution of surface charges at the first two EF-hands rendering each molecule, in principle, specific for a discrete set of binding partners. These findings provide molecular evidence that NCS can transduce signals to a specific target interaction irrespective of Ca(2+)-concentrations and CaM-levels.

Amino Acid Sequence↗

PubMed2003

Proteomics analysis of rat brain postsynaptic density. Implications of the diverse protein functional groups for the integration of synaptic physiology.

The postsynaptic density contains multiple protein complexes that together relay the presynaptic neurotransmitter input to the activation of the postsynaptic neuron. In the present study we took two independent proteome approaches for the characterization of the protein complement of the postsynaptic density, namely 1) two-dimensional gel electrophoresis separation of proteins in conjunction with mass spectrometry to identify the tryptic peptides of the protein spots and 2) isolation of the trypsin-digested sample that was labeled with isotope-coded affinity tag, followed by liquid chromatography-tandem mass spectrometry for the partial separation and identification of the peptides, respectively. Functional grouping of the identified proteins indicates that the postsynaptic density is a structurally and functionally complex organelle that may be involved in a broad range of synaptic activities. These proteins include the receptors and ion channels for glutamate neurotransmission, proteins for maintenance and modulation of synaptic architecture, sorting and trafficking of membrane proteins, generation of anaerobic energy, scaffolding and signaling, local protein synthesis, and correct protein folding and breakdown of synaptic proteins. Together, these results imply that the postsynaptic density may have the ability to function (semi-) autonomously and may direct various cellular functions in order to integrate synaptic physiology.

Animals↗

PubMed2003

Kainate-induced epileptic seizures induce a recruitment of caldendrin to the postsynaptic density in rat brain.

Caldendrin defines a novel family of neuronal calcium-sensor proteins, the C-terminal moiety of which displays high similarity to calmodulin. We now report that the protein is recruited to the postsynaptic density (PSD) of cortical and hippocampal neurons in response to kainate-induced epileptic seizures, an animal model of human temporal lobe epilepsy. The translocation of caldendrin to the PSD did not occur in kainate-treated rats that did not develop seizures. The enhanced PSD levels of caldendrin are not due to increased protein synthesis and most likely reflect a recruitment from the soluble caldendrin protein pool. These findings suggest that the transduction of dendritic Ca2+-signals via caldendrin is altered by epileptic seizures and that caldendrin might be involved in the pathophysiology of temporal lobe epilepsy.

Animals↗

PubMed2003

Expression of the immunoglobulin superfamily neuroplastin adhesion molecules in adult and developing mouse cerebellum and their localisation to parasagittal stripes.

Neuroplastin (np) 55 and 65 are immunoglobulin superfamily members that arise by alternative splicing of the same gene and have been implicated in long-term activity-dependent synaptic plasticity. Both biochemical and immunocytochemical data suggest that np55 is the predominant isoform (>95% of total neuroplastin) in cerebellum. Neuroplastin immunoreactivity is concentrated in the molecular layer and synaptic glomeruli in the granule cell layer. Expression in the molecular layer appears to be postsynaptic. First, neuroplastin is associated with Purkinje cell dendrites in two mouse granuloprival cerebellar mutants, disabled and cerebellar deficient folia. Second, in an acid sphingomyelinase knockout mouse with widespread protein trafficking defects, neuroplastin accumulates in the Purkinje cell somata. Finally, primary cerebellar cultures show neuroplastin expression in Purkinje cell dendrites and somata lacking normal histotypic organization and synaptic connections, and high-magnification views indicate a preferential association with dendritic spines. In the molecular layer, differences in neuroplastin expression levels present as a parasagittal array of stripes that alternates with that revealed by the expression of another compartmentation antigen, zebrin II/aldolase c. Neuroplastin immunoreactivity is first detected weakly at postnatal day 3 (P3) in the anterior lobe vermis. By P5, parasagittal stripes are already apparent in the immature molecular layer. At this stage, punctate deposits are also localised at the perimeter of the Purkinje cell perikarya; these are no longer detected by P15. The data suggest a role for neuroplastins in the development and maintenance of normal synaptic connections in the cerebellum.

Animals↗

PubMed2003

Gliap--a novel untypical L-asparaginase localized to rat brain astrocytes.

L-asparaginases catalyse the formation of the neuroactive amino acid L-aspartate by deamination of asparagine. The major pathophysiological significance of L-asparaginase activity is in its clinical use for the treatment of acute lymphatic leukaemia and neoplasias that require asparagine and obtain it from circulating pools. Here we report the identification and characterization of Gliap, a cytosolic L-asparaginase, which is the founding member of a new group of L-asparaginases in mammalia. Structural modelling suggests that Gliap is an atypical mammalian type-I asparaginase inasmuch as it harbours the active centre of a type-I glycosylasparaginase but, like plant-type asparaginases, lacks their auto-proteolytic site and, in addition, exhibits significant type-II L-asparaginase enzymatic activity. Moreover, in contrast to glycosylasparaginases Gliap is enriched in the cytosolic fraction and not in lysosomes. The protein is particularly abundant in liver, testis and brain. In brain Gliap is exclusively expressed in astrocytes and prominently present in structures reminiscent of glial endfeet. These data suggest that Gliap is involved in astroglial production of the neuroactive amino acid L-aspartate.

Amino Acid Sequence↗

PubMed2003

Distribution and cellular localization of caldendrin immunoreactivity in adult human forebrain.

We investigated by immunohistochemistry (IHC) the distribution of caldendrin, the founding member of a novel family of neuronal calcium-binding proteins closely related to calmodulin, in human forebrain. Caldendrin immunoreactivity was unevenly distributed, with prominent staining in the paleo- and neocortex, hippocampus, and hypothalamus. With the exception of the hypothalamus, labeling was restricted to the somato-dendritic compartment of neurons. This distribution completely matches that reported in rat, indicating that the cellular function is most likely conserved among species. Therefore, one prerequisite for functional studies in rodent models aimed at elucidation of mechanisms with relevance for humans can be based on the present findings.

Calcium-Binding Proteins↗

PubMed2002

Nicotinic acetylcholine receptors of Drosophila: three subunits encoded by genomically linked genes can co-assemble into the same receptor complex.

The second beta-like subunit (SBD) is a putative structural subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here we have produced specific antibodies against SBD to study, which other nAChR subunits can co-assemble with SBD in receptor complexes of the Drosophila nervous system. Immunohistochemical studies in the adult optic lobe revealed that SBD has a distribution similar to that of the alpha-subunit ALS in the synaptic neuropil. The subunits ALS, D(alpha)2 and SBD can be co-purified by alpha-bungarotoxin affinity chromatography. Moreover, anti-SBD antibodies co-precipitate ALS and D(alpha)2 and, vice versa, ALS and D(alpha)2 antibodies co-immunoprecipitate SBD protein. Two-step immunoaffinity chromatography with immobilized antibodies against ALS and D(alpha)2 revealed the existence of nAChR complexes that include ALS, D(alpha)2 and SBD as integral components. Interestingly, the genes encoding these three subunits appear to be directly linked in the Drosophila genome at region 96 A of the third chromosome. In addition, SBD appears to be a component of a different receptor complex, which includes the ARD protein as an additional beta-subunit, but neither ALS nor D(alpha)2 nor the third alpha-subunit D(alpha)3. These findings suggest a considerable complexity of the Drosophila nicotinic receptor system.

Animals↗

PubMed2002

Brevican isoforms associate with neural membranes.

Brevican is a neural-specific proteoglycan of the brain extracellular matrix, which is particularly abundant in the terminally differentiated CNS. It is expressed by neuronal and glial cells, and as a component of the perineuronal nets it decorates the surface of large neuronal somata and primary dendrites. One brevican isoform harbors a glycosylphosphatidylinositol anchor attachment site and, as shown by ethanolamine incorporation studies, is indeed glypiated in stably transfected HEK293 cells as well as in oligodendrocyte precursor Oli-neu cells. The major isoform is secreted into the extracellular space, although a significant amount appears to be tightly attached to the cell membrane, as it floats up in sucrose gradients. Flotation is sensitive to detergent treatment. Brevican is most prominent in the microsomal, light membrane and synaptosomal fractions of rat brain membrane preparations. The association with the particulate fraction is in part sensitive to chondroitinase ABC and phosphatidylinositol-specific phospholipase C treatment. Furthermore, brevican staining on the surface of hippocampal neurons in culture is diminished after hyaluronidase or chondroitinase ABC treatment. Taken together, this could provide a mechanism by which perineuronal nets are anchored on neuronal surfaces.

Animals↗

PubMedDate not supplied

Organelle proteomics of rat synaptic proteins: correlation-profiling by isotope-coded affinity tagging in conjunction with liquid chromatography-tandem mass spectrometry to reveal post-synaptic density specific proteins.

Organelle proteomics is the method of choice for global analysis of cellular proteins. However, it is difficult to isolate organelles to homogeneity. Recently, correlation-profiling has been used to filter off the contaminants ad hoc and to disclose the genuine organelle-specific proteins. In the present study, we further extend the method to include subcellular compartments that contain proteins shared by multiple distinct subcellular domains. We performed correlation profiling of proteins contained in synaptic membrane and postsynaptic density (PSD) fractions isolated from rat brain. Proteins were labeled with isotope-coded affinity-tag reagents, digested with trypsin, and resulting peptides were resolved by cation exchange chromatography followed by reversed phase chromatography. Peptides were then subjected to mass spectrometry for quantification and identification. We confirm that the core PSD proteins were enriched in the PSD preparation. Other functional protein groups such as cytoskeleton-associated proteins, protein kinases and phosphatases, signaling components and regulators, as well as proteins involved in energy production partitioned to multiple organelles. When analyzed as groups, they were shown to accumulate to a lesser extent. Mitochondrial proteins and transporters were generally strongly depleted from the PSD fraction confirming that they were contaminants of the PSD preparation. Finally, immunoelectron microscopy was performed on selected proteins to validate the proteomics results, and confirm that synaptophysin that was highly depleted in the PSD preparation is localized in the presynaptic compartment, whereas LASP-1 that was slightly enriched in the PSD preparation is present in the PSD as well as other subdomains within the synapse.

Affinity Labels↗