Horst Bluethmann

Benzodiazepine actions mediated by specific gamma-aminobutyric acidA receptor subtypes

GABAA (γ-aminobutyric acidA) receptors are molecular substrates for the regulation of vigilance, anxiety, muscle tension, epileptogenic activity and memory functions, which is evident from the spectrum of actions elicited by clinically effective drugs acting at their modulatory benzodiazepine-binding site. Here we show, by introducing a histidine-to-arginine point mutation at position 101 of the murine α1-subunit gene, that α1-type GABAA receptors, which are mainly expressed in cortical areas and thalamus, are rendered insensitive to allosteric modulation by benzodiazepine-site ligands, whilst regulation by the physiological neurotransmitter γ-aminobutyric acid is preserved. α1(H101R) mice failed to show the sedative, amnesic and partly the anticonvulsant action of diazepam. In contrast, the anxiolytic-like, myorelaxant, motor-impairing and ethanol-potentiating effects were fully retained, and are attributed to the nonmutated GABAA receptors found in the limbic system (α2, α5), in monoaminergic neurons (α3) and in motoneurons (α2, α5). Thus, benzodiazepine-induced behavioural responses are mediated by specific GABAA receptor subtypes in distinct neuronal circuits, which is of interest for drug design.

Attenuation of allergic airway inflammation in IL‐4 deficient mice

To investigate the role of IL‐4 in vivo in allergic asthma, we developed a murine model of allergen‐induced airway inflammation. Repealed daily exposures of actively immunised C57BL/6 mice to aerosolized ovalbumin (OVA) induced a peribronchial inflammation and an increase in eosinophils and lymphocytes in bronchoalveolar‐lavage(BAL) fluid. In IL‐4 deficient (IL4−/−) mice, treated in the same way, there were substantially fewer eosinophils in BAL and much less peribronchial inflammation compared with wild type mice. In this model, mast cell deficient (W/Wv) mice developed a similar degree of BAL eosinophilia and peribronchial inflammation as wild type mice, demonstrating that the mast cell is not required for the induction of chronic airway inflammation. In contrast, BAL eosinophilia and airway inflammation were absent in OVA‐treated MHC ClassII deficient (B6.Aa−/−) mice which lack mature CD4+ T lymphocytes. In conclusion, these results indicate that IL‐4 is a central mediator of allergic airway inflammation, regulating antigen‐induced eosinophil recruitment into the airways by a T cell dependent mechanism.

Amyloid-β and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer's disease mice

Alzheimers disease (AD) is characterized by amyloid-beta (Aβ)-containing plaques, neurofibrillary tangles, and neuron and synapse loss. Tangle formation has been reproduced in P301L tau transgenic pR5 mice, whereas APPswPS2N141I double-transgenic APP152 mice develop Aβ plaques. Cross-breeding generates triple transgenic (tripleAD) mice that combine both pathologies in one model. To determine functional consequences of the combined Aβ and tau pathologies, we performed a proteomic analysis followed by functional validation. Specifically, we obtained vesicular preparations from tripleAD mice, the parental strains, and nontransgenic mice, followed by the quantitative mass-tag labeling proteomic technique iTRAQ and mass spectrometry. Within 1,275 quantified proteins, we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Notably, deregulation of complex I was tau dependent, whereas deregulation of complex IV was Aβ dependent, both at the protein and activity levels. Synergistic effects of Aβ and tau were evident in 8-month-old tripleAD mice as only they showed a reduction of the mitochondrial membrane potential at this early age. At the age of 12 months, the strongest defects on OXPHOS, synthesis of ATP, and reactive oxygen species were exhibited in the tripleAD mice, again emphasizing synergistic, age-associated effects of Aβ and tau in perishing mitochondria. Our study establishes a molecular link between Aβ and tau protein in AD pathology in vivo, illustrating the potential of quantitative proteomics.

MHC Class II Molecules Are Not Required for Survival of Newly Generated CD4+ T Cells, but Affect Their Long-Term Life Span

We grafted fetal thymi from wild-type mice into immunodeficient RAG-2-/- or class II-/-RAG-2-/- (class II MHC-) recipients and followed the fate of naive CD4+ T cells derived from the grafts. In both types of recipients, newly generated CD4+ T cells proliferated to the same extent in the periphery and rapidly filled the empty T cell compartment. However, CD4+ T cells in class II- recipients gradually decreased in number over 6 months. These results show that interactions between the TCR and class II molecules are not required for newly generated CD4+ T cells to survive and proliferate, but are necessary to maintain the size of the peripheral T cell pool for extended periods.

A crucial role for B cells in neuroinvasive scrapie

Although prion proteins are most efficiently propagated through intracerebral inoculation, peripheral administration has caused the diseases kuru, iatrogenic Creutzfeldt–Jakob disease (CJD), bovine spongiform encephalopathy (BSE) and new-variant CJD,. The development of neurological disease after peripheral inoculation depends on prion expansion within cells of the lymphoreticular system,. Here we investigate the identity of these cells by using a panel of immune-deficient mice inoculated with prions intraperitoneally: we found that defects affecting only T lymphocytes had no apparent effect, but that all mutations that disrupted the differentiation and response of B lymphocytes prevented the development of clinical scrapie. As an absence of B cells and of antibodies correlates with severe defects in follicular dendritic cells, a lack of any of these three components may prevent the development of clinical scrapie. However, we found that scrapie developed after peripheral inoculation in mice expressing immunoglobulins that were exclusively of the M subclass and without detectable specificity for the normal form of the prion PrPc, and in mice which had differentiated B cells but no functional follicular dendritic cells. We conclude that differentiated B cells are crucial for neuroinvasion by scrapie, regardless of the specificity of their receptors.

Impaired neuroglial activation in interleukin-6 deficient mice.

Astrocyte activation is a ubiquitous hallmark of the damaged brain and has been suggested to play an important regulatory role in the activation, survival, and regeneration of adjacent neurons, microglia, and oligodendrocytes. Little is known, however, about the endogenous signals that lead to this activation of astrocytes. Here we examined the regulation of interleukin 6 (IL6), a proinflammatory cytokine, its receptors, and the effects of IL6‐deficiency in a model of traumatic central nervous system injury in the axotomized mouse facial motor nucleus. Facial nerve transection led to a massive but transient upregulation of IL6 mRNA in the disconnected motor nucleus, while IL6‐receptor subunits were constitutively expressed on motoneurons and astrocytes. Absence of IL6 in genetically IL6‐deficient mice led to massive reduction in the number of activated GFAP‐positive astrocytes, a more moderate decrease in microglial activation and proliferation, and an increase in the late neuronal response to axotomy. These results emphasize the role of IL6 in the global regulation of neurons, astrocytes, and microglia and their activation in the injured nervous system. GLIA 19:227–233, 1997.

Microglia Protect Neurons against Ischemia by Synthesis of Tumor Necrosis Factor

Microglia and infiltrating leukocytes are considered major producers of tumor necrosis factor (TNF), which is a crucial player in cerebral ischemia and brain inflammation. We have identified a neuroprotective role for microglial-derived TNF in cerebral ischemia in mice. We show that cortical infarction and behavioral deficit are significantly exacerbated in TNF-knock-out (KO) mice compared with wild-type mice. By using in situ hybridization, immunohistochemistry, and green fluorescent protein bone marrow (BM)-chimeric mice, TNF was shown to be produced by microglia and infiltrating leukocytes. Additional analysis demonstrating that BM-chimeric TNF-KO mice grafted with wild-type BM cells developed larger infarcts than BM-chimeric wild-type mice grafted with TNF-KO BM cells provided evidence that the neuroprotective effect of TNF was attributable to microglial- not leukocyte-derived TNF. In addition, observation of increased infarction in TNF-p55 receptor (TNF-p55R)-KO mice compared with TNF-p75R and wild-type mice suggested that microglial-derived TNF exerts neuroprotective effects through TNF-p55R. We finally report that TNF deficiency is associated with reduced microglial population size and Toll-like receptor 2 expression in unmanipulated brain, which might also influence the neuronal response to injury. Our results identify microglia and microglial-derived TNF as playing a key role in determining the survival of endangered neurons in cerebral ischemia.

The γ2 subunit of GABAA receptors is required for maintenance of receptors at mature synapses

Abstract The γ2 subunit of GABA A receptor chloride channels is required for normal channel function and for postsynaptic clustering of these receptors during synaptogenesis. In addition, GABA A receptor function is thought to contribute to normal postnatal maturation of neurons. Loss of postsynaptic GABA A receptors in γ2-deficient neurons might therefore reflect a deficit in maturation of neurons due to the reduced channel function. Here, we have used the Cre-loxP strategy to examine the clustering function of the γ2 subunit at mature synapses. Deletion of the γ2 subunit in the third postnatal week resulted in loss of benzodiazepine-binding sites and parallel loss of punctate immunoreactivity for postsynaptic GABA A receptors and gephyrin. Thus, the γ2 subunit contributes to postsynaptic localization of GABA A receptors and gephyrin by a mechanism that is operant in mature neurons and not limited to immature neurons, most likely through interaction with proteins involved in trafficking of synaptic GABA A receptors.

Trace Amine-Associated Receptor 1 Modulates Dopaminergic Activity

The recent identification of the trace amine-associated receptor (TAAR)1 provides an opportunity to dissociate the effects of trace amines on the dopamine transporter from receptor-mediated effects. To separate both effects on a physiological level, a Taar1 knockout mouse line was generated. Taar1 knockout mice display increased sensitivity to amphetamine as revealed by enhanced amphetamine-triggered increases in locomotor activity and augmented striatal release of dopamine compared with wild-type animals. Under baseline conditions, locomotion and extracellular striatal dopamine levels were similar between Taar1 knockout and wild-type mice. Electrophysiological recordings revealed an elevated spontaneous firing rate of dopaminergic neurons in the ventral tegmental area of Taar1 knock-out mice. The endogenous TAAR1 agonist p-tyramine specifically decreased the spike frequency of these neurons in wild-type but not in Taar1 knockout mice, consistent with the prominent expression of Taar1 in the ventral tegmental area. Taken together, the data reveal TAAR1 as regulator of dopaminergic neurotransmission.

Strongly compromised inflammatory response to brain injury in interleukin‐6‐deficient mice

Injury to the central nervous system (CNS) elicits an inflammatory response involving activation of microglia, brain macrophages, and astrocytes, processes likely mediated by the release of proinflammatory cytokines. In order to determine the role of interleukin‐6 (IL‐6) during the inflammatory response in the brain following disruption of the blood–brain barrier (BBB), we examined the effects of a focal cryo injury to the fronto‐parietal cortex in interleukin‐6‐deficient (IL‐6−/−) and normal (IL‐6+/+) mice. In IL‐6+/+ mice, brain injury resulted in the appearance of brain macrophages and reactive astrocytes surrounding the lesion site. In addition, expression of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and metallothionein‐I+II (MT‐I+II) were increased in these cells, while the brain‐specific MT‐III was only moderately upregulated. In IL‐6−/− mice, however, the response of brain macrophages and reactive astrocytes was markedly depressed and the number of NSE positive neurons was reduced. Brain damage‐induced GM‐CSF and MT‐I+II expression were also markedly depressed compared to IL‐6+/+ mice. In contrast, MT‐III immunoreactivity was markedly increased in brain macrophages and astrocytes. In situ hybridization analysis indicates that MT‐I+II but not MT‐III immunoreactivity reflect changes in the messenger levels. The number of cell divisions was similar in IL‐6+/+ and IL‐6−/− mice. The present results demonstrate that IL‐6 is crucial for the recruitment of myelo‐monocytes and activation of glial cells following brain injury with disrupted BBB. Furthermore, our results suggest IL‐6 is important for neuroprotection and the induction of GM‐CSF and MT expression. The opposing effect of IL‐6 on MT‐I+II and MT‐III levels in the damaged brain suggests MT isoform‐specific functions. GLIA 25:343–357, 1999.