Differential distribution of the G protein γ3 subunit in the developing zebrafish nervous system

G proteins play an essential role in the transduction and propagation of extracellular signals across the plasma membrane. It was once thought that the G protein α subunit was the sole regulator of intracellular molecules. The G protein βγ complex is now recognized as participating in many signaling events. While screening a zebrafish cDNA library to identify members of the protein 4.1 superfamily (Kelly, G.M., Reversade, B., Biochem. Cell Biol. 75 (1997), 623), we fortuitously identified a clone that encodes a zebrafish G protein γ subunit. The 666 nucleotides of the zebrafish G protein γ subunit cDNA encodes a polypeptide of 75 amino acids with high degree of homology to human, bovine, rat and mouse γ subunits. BLAST search analysis of GenBank revealed that the zebrafish γ subunit is 93% identical and 97% similar to the mammalian γ3 subunit. The γ3 gene was mapped to the zebrafish linkage group 21, approximately 10.76 cRays from bf, a gene with sequence homology to the human properdin factor gene. RT-PCR and in situ hybridization analyses first detected γ3 mRNA during late somitogenesis, where it was expressed preferentially in the Vth cranial nerve, the forebrain and in ventrolateral regions of the mid- and hindbrain including the spinal cord. The ability of the zebrafish γ3 subunit to form a signaling heterodimeric complex with a β subunit was tested using a human β2 subunit. The γ3 formed a heterodimer with β2 and the complex was capable of binding calmodulin in a calcium-dependent manner. Overexpression of the β2γ3 complex in zebrafish embryos lead to the loss of dorsoanterior structures and heart defects, possibly owing to an up-regulation of mitogen-activated protein kinase activity and/or decline in protein kinase A signaling. Together, these data imply that a βγ heterodimer plays a role in signal transduction events involving G protein coupled receptors and that these events occur in specific regions in the nervous system of the developing zebrafish.     

The subunit composition of cerebellar tubulin: evidence for multiple beta tubulin messenger RNAs

Cytoplasmic proteins were isolated from adult rat forebrain and cerebellum and analyzed by two-dimensional gel electrophoresis under conditions in which the major subunits of tubulin were separated. Forebrain cytoplasmic tubulin consisted of two groups of α subunits (α₁ and α₂) and a minimum of two β subunits (β₁ and β₂). However, the rat cerebellar cytoplasmic proteins contained greatly decreased amounts of the β₁ tubulin subunit relative to the analysis of forebrain proteins. Messenger RNA (mRNA) was purified from cerebellum and forebrain and translated in wheat germ homogenate. Analysis of the translation products of cerebellar mRNA indicated only a trace amount of the β₁ subunit, whereas the expected amount of β₁ was found among the translation products of forebrain mRNA. This data is consistent with the conclusion that the β₁ and β₂ subunits of tubulin are synthesized from different mRNAs. A decrease in β₁ mRNA relative to other tubulin mRNAs may be one of the factors responsible for the low steady state amounts of β₁ tubulin in the cerebellum.     

Immunohistochemical localization of interleukin-2 and its receptor subunits α, β and γ in the main olfactory bulb of the rat

Endogenous interleukin-2 (IL-2) was found in the adult rat brain, however, it has not been reported whether this cytokine is present in the olfactory bulb. Immunohistochemical techniques were used to examine the cellular localization of IL-2 and its receptor subunits in the main olfactory bulb of the rat. Strong IL-2 immunoreactivity was localized in glial cells, specifically in the olfactory nerve layer, glomerular layer and external plexiform layer. IL-2 mRNA was detected in the olfactory bulb by RT–PCR. All three IL-2 receptor subunits also showed distinct laminar distributions. The IL-2Rα and IL-2Rβ immunoreactivity was found both in neurons and glial cells, whereas IL-2Rγ imunoreactivity was found in glial cells, and thus resembled IL-2 immunostaining. The present results demonstrated a wide distribution of IL-2 and its receptor subunits in the main olfactory bulb of the rat, suggesting that IL-2 might play a role in the olfactory function through autocrine or paracrine pathways. The exclusive high expression of IL-2 in glial cells in distinct laminar structures, where neuron–glia interactions are closely associated with olfactory nerve regeneration, imply that IL-2 might be involved in the process of nerve regeneration in the olfactory bulb.     

Interleukin-1β-induced brain injury in the neonatal rat can be ameliorated by α-phenyl-n-tert-butyl-nitrone

To examine the possible role of inflammatory cytokines in mediating perinatal brain injury, we investigated effects of intracerebral injection of interleukin-1beta (IL-1β) on brain injury in the neonatal rat and the mechanisms involved. Intracerebral administration of IL-1β (1 μg/kg) resulted in acute brain injury, as indicated by enlargement of ventricles bilaterally, apoptotic death of oligodendrocytes (OLs) and loss of OL immunoreactivity in the neonatal rat brain. IL-1β also induced axonal and neuronal injury in the cerebral cortex as indicated by elevated expression of β-amyloid precursor protein, short beaded axons and dendrites, and loss of tyrosine hydroxylase-positive neurons in the substantia nigra and the ventral tegmental areas. Administration of α-phenyl-n-tert-butyl-nitrone (PBN, 100 mg/kg i.p.) immediately after the IL-1β injection protected the brain from IL-1β-induced injury. Protection of PBN was linked with the attenuated oxidative stress induced by IL-1β, as indicated by decreased elevation of 8-isoprostane content and by the reduced number of 4-hydroxynonenal or malondialdehyde or nitrotyrosine-positive cells following IL-1β exposure. PBN also attenuated IL-1β-stimulated inflammatory responses as indicated by the reduced activation of microglia. The finding that IL-1β induced perinatal brain injury was very similar to that induced by lipopolysaccharide (LPS), as we previously reported and that PBN was capable to attenuate the injury induced by either LPS or IL-1β suggests that IL-1β may play a critical role in mediating brain injury associated with perinatal infection/inflammation.     

The GABAA receptor γ1-subunit in seizure prone (DBA/2) and resistant (C57BL/6) mice

γ-Aminobutyric acid (GABA)_A receptors are the sites of action for many antiepileptic drugs such as benzodiazepines and barbiturates. We report the results of molecular cloning of the γ₁-subunit from seizure prone DBA/2J and resistant C57BL/6J inbred mice, and analyses of nucleotide sequences and expression of the γ₁-subunit messenger RNA (mRNA) in DBA/2 and C57BL/6 inbred mice. The mouse γ₁-subunit complementary DNA (cDNA) shares 98% similarity with that of the rat at the level of amino acid sequence. Northern blot hybridization indicates that the γ₁-subunit mRNA is expressed predominantly in areas other than the cerebral cortex and cerebellum and shows little change with postnatal development. No differences have been found for the subunit between DBA/2 and C57BL/6 mice either for nucleotide sequence or for level of expression of the subunit’s mRNA in whole brain by Northern blots at 3 weeks of age.     

Lymphocyte-specific inducible expression of potassium channel beta subunits

Many studies have shown that voltage-gated potassium (Kv) channel activity is essential for T-lymphocyte proliferation. The IL-2-inducible neuroimmune gene, I2rf5 is the mouse homologue of the rat Kvβ2 subunit. In this study we show that in addition to constitutive expression in adult murine brain, expression of Kv channel subunits β1.1 and β2.1 is inducible in a cloned T-helper cell line stimulated with IL-2 and in normal murine splenocytes stimulated with Con A or LPS. This expression pattern appears to be lymphocyte specific, because stimulated fibroblasts and vascular smooth muscle cells do not express Kvβ channel subunit mRNA. These observations suggest that Kvβ subunit expression is tissue specific and inducible in stimulated lymphocytes. Because Kvβ subunits modulate K⁺ channel activity, the inducible and variable expression of these subunits in lymphocytes may represent an additional regulatory mechanism for lymphocyte proliferation.     

Memory impairment induced by IL-1β is reversed by α-MSH through central melanocortin-4 receptors

Interleukin-1beta (IL-1β) significantly influences memory consolidation. Treatments that raise the level of IL-1β in the brain, given after training, impair contextual fear conditioning. The melanocortin α-MSH exerts potent anti-inflammatory actions by physiologically antagonizing the effect of pro-inflammatory cytokines. Five subtypes of melanocortin receptors (MC1R–MC5R) have been identified, with MC3R and MC4R predominating in the central nervous system. The present experiments show that injection of IL-1β (5 ng/0.25 μl) in dorsal hippocampus up to 15 min after training decreased freezing during the contextual fear test. The treatment with IL-1β (5 ng/0.25 μl) 12 h after conditioning cause amnesia when animals were tested 7 days post training. Thus, our results also demonstrated that IL-1β can influence persistence of long-term memory. We determined that animals previously injected with IL-1β can acquire a new contextual fear memory, demonstrating that the hippocampus was not damaged. Treatment with α-MSH (0.05 μg/0.25 μl) blocked the effect of IL-1β on contextual fear memory. Administration of the MC4 receptor antagonist HS014 (0.5 μg/0.25 μl) reversed the effect of α-MSH. However, treatment with γ-MSH (0.5 μg/0.25 μl), an MC3 agonist, did not affect IL-1β-induced impairment of memory consolidation. These results suggest that α-MSH, through central MC4R can inhibit the effect of IL-1β on memory consolidation.     

Approach to discriminate subgroups in multiple sclerosis with cerebrospinal fluid (CSF) basic inflammation indices and TNF-α, IL-1β, IL-6, IL-8

Lumbar CSF and serum pairs of untreated multiple sclerosis patients (MS; n=47) were analyzed on admission. On average, higher CSF leukocyte (lymphocyte and monocyte) counts, IgG index, CSF IgG contents, but not of TNF-α, IL-1β, IL-6, IL-8 in CSF and serum, were revealed in all MS or patients with long disease course (LO-MS) compared with controls. In primary progressive MS (PP-MS) cell counts were low, but IgG contents were high, when compared to relapsing-remitting MS (RR-MS). In clinically probable MS (CP-MS) both contents were low, in clinically definite MS (CD-MS) high. Spearman’s correlation with the four monokines and the basic indices in CSF revealed activation patterns known for microglia/macrophages in the four MS subgroups, for astrocytes in CP-MS and RR-MS, for CSF lymphocytes in CP-MS and PP-MS, for cells of blood–brain barrier (BBB) in CP-MS, for intrathecal IgG synthesis in PP-MS and for lymphocyte transfer in CD-MS. Correlations between CSF and serum parameters indicated CNS disease processes to be associated with systemic processes of inflammation (acute, chronic) in CD-MS, RR-MS, and PP-MS in different ways. CSF IgG content, IgG index and systemic markers of inflammation correlated with overall disability scores in LO-MS; increasing levels may indicate a bad outcome.     

Subunit structure of synaptosomal tubulin

The subunit structure of rat brain synaptosomal tubulin was examined by high resolution two-dimensional gel fractionation. Whole brain cytoplasmic tubulin consists of two groups of α subunits (α₁andα₂), and a minimum of two β subunits (β₁andβ₂). Both α subunits consist of a major relatively acidic form and minor relatively basic forms. In contrast, tubulin purified from synaptoplasm contains an additional subunit, α₃, which has the same isoelectric point but slightly faster electrophoretic mobility than α₁andα₂. All synaptosomal α subunits are the relatively acidic forms and the minor basic forms are absent. The synaptosomal β subunits have electrophoretic properties similar to the corresponding cytoplasmic forms. The α₃ synaptosomal tubulin subunit has affinity for colchicine, has a tryptic peptide map similar to whole brain cytoplasmic α tubulin, and can be purified by a standard tubulin purification method.     

Interleukin-4 Induces the Release of Opioid Peptides from M1 Macrophages in Pathological Pain

Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can be protective in inflammatory and neurologic disorders, and can alleviate pain. Classically, IL-4 diminishes pain by blocking the production of proinflammatory cytokines. Here, we uncovered that IL-4 induces acute antinociception by IL-4 receptor α (IL-4Rα)-dependent release of opioid peptides from M1 macrophages at injured nerves. As a model of pathologic pain, we used a chronic constriction injury (CCI) of the sciatic nerve in male mice. A single application of IL-4 at the injured nerves (14 d following CCI) attenuated mechanical hypersensitivity evaluated by von Frey filaments, which was reversed by co-injected antibody to IL-4Rα, antibodies to opioid peptides such as Met-enkephalin (ENK), β-endorphin and dynorphin A 1–17, and selective antagonists of δ-opioid, µ-opioid, and κ-opioid receptors. Injured nerves were predominately infiltrated by proinflammatory M1 macrophages and IL-4 did not change their numbers or the phenotype, assessed by flow cytometry and qRT-PCR, respectively. Macrophages isolated from damaged nerves by immunomagnetic separation (IMS) and stimulated with IL-4 dose dependently secreted all three opioid peptides measured by immunoassays. The IL-4-induced release of ENK was diminished by IL-4Rα antibody, intracellular Ca²⁺ chelator, and inhibitors of protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), and ryanodine receptors. Together, we identified a new opioid mechanism underlying the IL-4-induced antinociception that involves PKA-mediated, PI3K-mediated, ryanodine receptor-mediated, and intracellular Ca²⁺-mediated release from M1 macrophages of opioid peptides, which activate peripheral opioid receptors in injured tissue.SIGNIFICANCE STATEMENT Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can ameliorate pain. The IL-4-mediated effects are considered to mostly result from the inhibition of the production of proinflammatory mediators (e.g., IL-1β, tumor necrosis factor, prostaglandin E2). Here, we found that IL-4 injected at the injured nerves attenuates pain by releasing opioid peptides from the infiltrating macrophages in mice. The opioids were secreted by IL-4 in the intracellular Ca²⁺-dependent manner and activated local peripheral opioid receptors. These actions represent a novel mode of IL-4 action, since its releasing properties have not been so far reported. Importantly, our findings suggest that the IL-4–opioid system should be targeted in the peripheral damaged tissue, since this can be devoid of central and systemic side effects.     

Induction of cytokines in glial cells surrounding cortical β-amyloid plaques in transgenic Tg2576 mice with Alzheimer pathology

β-Amyloid plaque deposition observed in brains from Alzheimer patients, might function as immune stimulus for glial/macrophages activation, which is supported by observations of activated microglia expressing interleukin (IL)-1β and elevated IL-6 immunoreactivity in close proximity to amyloid plaques. To elucidate the mechanisms involved in β-amyloid-mediated inflammation, transgenic mice (Tg2576) expressing high levels of the Swedish double mutation of human amyloid precursor protein and progressively developing typical β-amyloid plaques in cortical brain regions including gliosis and astrocytosis, were examined for the expression pattern of a number of cytokines.Using ribonuclease protection assay, interleukin (IL)-1α,-β, IL-1 receptor antagonist, IL-6, IL-10, IL-12, IL-18, interferon-γ, and macrophage migration inhibitory factor (MIF) mRNA were not induced in a number of cortical areas of Tg2576 mice regardless of the postnatal ages studied ranging between 2 and 13 months. Using immunocytochemistry for IL-1α,β, IL-6, tumor necrosis factor (TNF)-α, and macrophage chemotactic protein (MCP)-1, only IL-1β was found to be induced in reactive astrocytes surrounding β-amyloid deposits detected in 14-month-old Tg2576 mice. Using non-radioactive in situ hybridization glial fibrillary acidic protein (GFAP) mRNA was detected to be expressed by reactive astrocytes in close proximity to β-amyloid plaques. The local immune response detected around cortical β-amyloid deposits in transgenic Tg2576 mouse brain is seemingly different to that observed in brains from Alzheimer patients but may represent an initial event of chronic neuroinflammation at later stages of the disease.     

Interleukin-1β System (Ligand, Receptor Type I, Receptor Accessory Protein and Receptor Antagonist), TNF-α, TGF-β1 and Neuropeptide Y mRNAs in Specific Brain Regions During Bacterial LPS-Induced Anorexia

Bacterial lipopolysaccharide (LPS) or endotoxin induces neurological manifestations including anorexia. It is proposed that LPS-induced cytokine production is involved in the generation of neurological manifestations and in neuroinflammatory/immunological responses during Gram-negative infections. For example, LPS-induced effects can be blocked or ameliorated by the interleukin-1 receptor antagonist (IL-1Ra). Here, sensitive and specific RNase protection assays were used to investigate the effects of the intracerebroventricular (i.c.v.) administration of LPS on mRNA levels of interleukin-1β (IL-1β) system components, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and neuropeptide Y (NPY) in the cerebellum, hippocampus, and hypothalamus. The same brain region sample was analyzed with all of the antisense probes. The data show simultaneous local induction of multiple cytokine components messenger ribonucleic acids (mRNAs) within specific brain regions in anorectic rats responding to i.c.v. administered LPS (500 ng/rat). Interleukin-1β and IL-1Ra had a similar mRNA induction profile (hypothalamus > cerebellum > hippocampus). Interleukin-1 receptor type I (IL-1RI) mRNA also increased in all three brain regions examined, and the soluble form of IL-1 receptor accessory protein (IL-1R AcP II) mRNA was induced in the hypothalamus. Tumor necrosis factor-α mRNA levels increased in the hypothalamus > hippocampus > cerebellum. Levels of membrane bound IL-1R AcP, TGF-β1, and NPY mRNAs did not change significantly in any brain region. The results suggest that: (1) endogenous up-regulation of IL-1β and TNF-α in the hypothalamus contribute to LPS-induced anorexia; and (2) the ratio IL-1Ra/IL-1β, and IL-1β ↔ TNF-α interactions may have implications for Gram-negative infections associated with high levels of LPS in the brain-cerebrospinal fluid.