Oliver Ullrich

RABAPTIN-5 IS A DIRECT EFFECTOR OF THE SMALL GTPASE RAB5 IN ENDOCYTIC MEMBRANE FUSION

We have identified a novel 100 kDa coiled-coil protein, rabaptin-5, that specifically interacts with the GTP form of the small GTPase Rab5, a potent regulator of endocytic transport. It is mainly cytosolic, but a fraction colocalizes with Rab5 to early endosomes. Expression of a GTPase-deficient Rab5 mutant enhances the binding of rabaptin-5 to enlarged endosomes. Overexpression of rabaptin-5 alone is sufficient to promote expansion of early endosomes. Rab5 recruits rabaptin-5 to purified early endosomes in a GTP-dependent manner, demonstrating functional similarities with other members of the Ras superfamily. Immunodepletion of rabaptin-5 from cytosol strongly inhibits Rab5-dependent early endosome fusion. Rabaptin-5 is thus a Rab effector required for membrane docking and fusion.

The endocannabinoid anandamide protects neurons during CNS inflammation by induction of MKP-1 in microglial cells.

Endocannabinoids are released after brain injury and believed to attenuate neuronal damage by binding to CB(1) receptors and protecting against excitotoxicity. Such excitotoxic brain lesions initially result in primary destruction of brain parenchyma, which attracts macrophages and microglia. These inflammatory cells release toxic cytokines and free radicals, resulting in secondary neuronal damage. In this study, we show that the endocannabinoid system is highly activated during CNS inflammation and that the endocannabinoid anandamide (AEA) protects neurons from inflammatory damage by CB(1/2) receptor-mediated rapid induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) in microglial cells associated with histone H3 phoshorylation of the mkp-1 gene sequence. As a result, AEA-induced rapid MKP-1 expression switches off MAPK signal transduction in microglial cells activated by stimulation of pattern recognition receptors. The release of AEA in injured CNS tissue might therefore represent a new mechanism of neuro-immune communication during CNS injury, which controls and limits immune response after primary CNS damage.

Different requirements for NSF, SNAP, and Rab proteins in apical and basolateral transport in MDCK cells

We used an in vitro system based on streptolysin O-permeabilized MDCK cells to study the involvement of NSF, SNAP, SNAREs, and Rab proteins in polarized membrane transport of epithelial cells. In MDCK cells, transport from the trans-Golgi network (TGN) to the basolateral plasma membrane is inhibited by anti-NSF antibodies and stimulated by alpha-SNAP. In contrast, transport from the TGN to the apical cell surface is not affected by anti-NSF antibodies or alpha-SNAP. Furthermore, apical transport is insensitive to Rab-GDI and tetanus and botulinum neurotoxins, which inhibit basolateral transport. These results provide evidence that the Rab-NSF-SNAP-SNARE mechanism operates in basolateral transport, while other molecules constitute the machinery for vesicular delivery in the apical pathway.

Distinct structural elements of rab5 define its functional specificity

Members of the rab family of small GTPases are localized to distinct cellular compartments and function as specific regulators of vesicle transport between organelles. Overexpression of rab5, which is associated with early endosomes and the plasma membrane, increases the rate of endocytosis [Bucci et al. (1992) Cell, 70, 715‐728]. From sequence alignments and molecular modelling we identified structural elements that might contribute to the definition of the functional specificity of rab5. To test the role of these elements experimentally, we transplanted them onto rab6, which is associated with the Golgi complex. The chimeric proteins were assayed for intracellular localization and stimulation of endocytosis. First, we found that the C‐terminus of rab5 could target rab6 to the plasma membrane and early endosomes but it did not confer rab5‐like stimulation of endocytosis. Further replacement of other regions revealed that the N‐terminus, helix alpha 2/loop 5 and helix alpha 2/loop 7 were all required to functionally convert rab6 into rab5. Reciprocal hybrids of rab5 containing these regions replaced with those of rab6 were inactive, demonstrating that each region is essential for rab5 function. These results indicate that distinct structural elements specify the localization, membrane association and regulatory function of rab5.

Regulation of microglial expression of integrins by poly(ADP-ribose) polymerase-1.

Excitotoxic brain lesions initially result in the primary destruction of brain parenchyma, after which microglial cells migrate towards the sites of injury. At these sites, the cells produce large quantities of oxygen radicals and cause secondary damage that accounts for most of the loss of brain function. Here we show that this microglial migration is strongly controlled in living brain tissue by expression of the integrin CD11a, regulated by the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) through the formation of a nuclear PARP–NF-κB-protein complex. Downregulation of PARP or CD11a by transfection with antisense DNA abrogated microglial migration almost completely and prevented neurons from secondary damage.

Distribution of HLA-DR-positive microglia in schizophrenia reflects impaired cerebral lateralization

Immunological alterations have been demonstrated in peripheral blood and cerebrospinal fluid of patients with schizophrenia, while previous postmortem studies have provided an inconsistent picture as to the role of microglia in the context of schizophrenia. Microglial activation is a sensitive indicator of changes in the CNS microenvironment, such as inflammatory and neurodegenerative processes. The aim of the present postmortem study was to examine HLA class II (HLA-DR) expression on microglia in brain regions which are particularly relevant for schizophrenia, with regard to hemispheric lateralization. Dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex (ACC), hippocampus and mediodorsal thalamus (MD) were studied in 16 cases with schizophrenia and 16 control subjects. Immunostaining was found in all brain regions and was not restricted to macrophage-like ameboid cells, but also appeared in ramified cells. Region-specific HLA-DR-positive cell density was not significantly different between cases with schizophrenia and controls. However, ameboid microglial cells were lateralized towards the right hemisphere in healthy subjects but not in the schizophrenia group (P=0.01). Postmortem interval correlated with ramified cell numbers in ACC/DLPFC (P=0.01/0.04) and ameboid cell density in hippocampus (P=0.03). Age, gender, duration of disease, medication dosage, storage delay and whole brain volume had no effect. Single case analysis revealed highly elevated microglial cell numbers in ACC and MD of two schizophrenic patients who had committed suicide during acute psychosis. In conclusion, the present data suggest the absence of microgliosis but decreased cerebral lateralization of ameboid microglia in schizophrenia.

Hydrogen Peroxide-induced Structural Alterations of RNase A

Proteins exposed to oxidative stress are degraded via proteolytic pathways. In the present study, we undertook a series of in vitro experiments to establish a correlation between the structural changes induced by mild oxidation of the model protein RNase A and the proteolytic rate found upon exposure of the modified protein toward the isolated 20 S proteasome. Fourier transform infrared spectroscopy was used as a structure-sensitive probe. We report here strong experimental evidence for oxidation-induced conformational rearrangements of the model protein RNase A and, at the same time, for covalent modifications of amino acid side chains. Oxidation-related conformational changes, induced by H2O2exposure of the protein may be monitored in the amide I region, which is sensitive to changes in protein secondary structure. A comparison of the time- and H2O2concentration-dependent changes in the amide I region demonstrates a high degree of similarity to spectral alterations typical for temperature-induced unfolding of RNase A. In addition, spectral parameters of amino acid side chain marker bands (Tyr, Asp) revealed evidence for covalent modifications. Proteasome digestion measurements on oxidized RNase A revealed a specific time and H2O2 concentration dependence; at low initial concentration of the oxidant, the RNase A turnover rate increases with incubation time and concentration. Based on these experimental findings, a correlation between structural alterations detected upon RNase A oxidation and proteolytic rates of RNase A is established, and possible mechanisms of the proteasome recognition process of oxidatively damaged proteins are discussed.

Proteasomal degradation of oxidatively damaged endogenous histones in K562 human leukemic cells.

A number of antitumor drugs act via the oxidation of nuclear material in the tumor cell. It is therefore important to know if tumor cells can effectively and precisely cope not only with oxidatively induced DNA damage, but also with nuclear protein oxidation. In this study, we investigated the endogenous degradation of oxidatively damaged histones in K562 human leukemic cells after oxidative challenge and demonstrated a link to the overall cellular stress response pathways by poly-ADP-ribose-polymerase (PARP). After an oxidative challenge, endogenous nuclear protein degradation, as well as histone degradation, was enhanced. Among the histone fractions, histone H1 revealed the highest degradation rate, and more than 85% of the total degraded H1 disappeared in the first 30 min after oxidative challenge. Short-term degradation of histones up to 30 min, as well as long-term degradation up to 48 h after oxidative challenge, was significantly reduced in the presence of the PARP inhibitor 3-aminobenzamide, and nearly completely abrogated by the selective proteasome inhibitor lactacystin. Immunoprecipitation experiments indicated that the proteasome specifically degraded oxidized histones. Thus, we show that the nuclear proteosome system in tumor cells is capable of preventing the accumulation of oxidized proteins in this compartment and may suggest further treatment strategies to effectively interfere with the protein repair and replacement strategies of tumor cells.

Identification of metabolic pathways of the lipid peroxidation product 4‐hydroxynonenal by mitochondria isolated from rat kidney cortex

The cytosolic lipid peroxidation product 4‐hydroxynonenal (HNE) is rapidly metabolized in mitochondria isolated from rat kidney cortex. About 80% of HNE was degraded within 3 min of incubation. Main products of HNE which were identified in mitochondria were the hydroxynonenoic acid, the 1,4‐dihydroxynonene and the glutathione‐HNE‐conjugate. Furthermore, formation of metabolites of the tricarboxylic acid cycle from HNE is suggested. The quantitative share of HNE binding to proteins was high with about 8% of total HNE consumption after 3 min of incubation. Therefore, rapid degradation of HNE by mitochondria might be involved in an intracellular antioxidative defense system.

Turnover of oxidatively damaged nuclear proteins in BV-2 microglial cells is linked to their activation state by poly-ADP-ribose polymerase

During neuroinflammation, activated microglial cells migrate to the sites of neuronal injury, phagocytose neighboring cells, and produce large amounts of oxygen free radicals, which might contribute to severe cell damage and death. It is interesting that microglial cells have withstood this cytotoxic action of free radicals, which indicates that there is an intracellular mechanism that apparently enables microglial cells to cope with such oxidative challenges. In this study, we investigated the capability of BV‐2 murine microglial cells to cope with oxidatively damaged proteins by the proteasomal proteolytic system. To induce a highly activated state, we used the proinflammatory cytokine tumor necrosis factor‐α, which acts as a priming signal for microglial superoxide radical production. We showed that activation of the nuclear enzyme poly(ADP‐ribose)polymerase (PARP) enabled activated microglial cells to resist oxidative damage by an up‐regulation of the nuclear proteasome. Activated microglial cells revealed an efficient recognition and degradation of oxidatively damaged proteins during an enhanced endogenous protein turnover. The impairment of PARP function by inhibitor or antisense experiments resulted in an accumulation of damaged proteins and subsequently cell death. In contrast, this was not the case in resting microglial cells. These findings demonstrate the crucial role of the PARP in microglial cell survival during activation and renders it a potential anti‐inflammatory target.