Differential gene expression in LPS/IFNgamma activated microglia and macrophages: in vitro versus in vivo.

Two different macrophage populations contribute to CNS neuroinflammation: CNS-resident microglia and CNS-infiltrating peripheral macrophages. Markers distinguishing these two populations in tissue sections have not been identified. Therefore, we compared gene expression between LPS (lipopolysaccharide)/interferon (IFN)gamma-treated microglia from neonatal mixed glial cultures and similarly treated peritoneal macrophages. Fifteen molecules were identified by quantative PCR (qPCR) as being enriched from 2-fold to 250-fold in cultured neonatal microglia when compared with peritoneal macrophages. Only three of these molecules (C1qA, Trem2, and CXCL14) were found by qPCR to be also enriched in adult microglia isolated from LPS/IFNgamma-injected CNS when compared with infiltrating peripheral macrophages from the same CNS. The discrepancy between the in vitro and in vivo qPCR data sets was primarily because of induced expression of the 'microglial' molecules (such as the tolerance associated transcript, Tmem176b) in CNS-infiltrating macrophages. Bioinformatic analysis of the approximately 19000 mRNAs detected by TOGA gene profiling confirmed that LPS/IFNgamma-activated microglia isolated from adult CNS displayed greater similarity in total gene expression to CNS-infiltrating macrophages than to microglia isolated from unmanipulated healthy adult CNS. In situ hybridization analysis revealed that nearly all microglia expressed high levels of C1qA, while subsets of microglia expressed Trem2 and CXCL14. Expression of C1qA and Trem2 was limited to microglia, while large numbers of GABA+ neurons expressed CXCL14. These data suggest that (i) CNS-resident microglia are heterogeneous and thus a universal microglia-specific marker may not exist; (ii) the CNS micro-environment plays significant roles in determining the phenotypes of both CNS-resident microglia and CNS-infiltrating macrophages; (iii) the CNS microenvironment may contribute to immune privilege by inducing macrophage expression of anti-inflammatory molecules.

Regulation of alpha-synuclein expression in limbic and motor brain regions of morphine-treated mice.

Chronic exposure to opiates produces dependence and addiction, which may result from neuroadaptations in the dopaminergic reward pathway and its target brain regions. The neuronal protein alpha-synuclein has been implicated in neuronal plasticity and proposed to serve as a negative regulator of dopamine neurotransmission. Thus, alpha-synuclein could mediate some effects of opiates in the brain. The present study investigated the influence of acute and chronic morphine administration on alpha-synuclein mRNA and protein expression in the brains of mice. Downregulation of alpha-synuclein mRNA was observed in the basolateral amygdala, dorsal striatum, nucleus accumbens, and ventral tegmental area of mice withdrawn from chronic morphine treatment. The changes were the most pronounced after longer periods of withdrawal (48 h). In contrast, levels of alpha-synuclein protein, as assessed by Western blotting, were significantly increased in the amygdala and striatum/accumbens (but not in the mesencephalon) of morphine-withdrawn mice. In both brain regions, levels of alpha-synuclein were elevated for as long as 2 weeks after treatment cessation. Because alpha-synuclein is a presynaptic protein, the detected opposite changes in its mRNA and protein levels are likely to take place in different populations of projection neurons whose somata are in different brain areas. Axonal localization of alpha-synuclein was confirmed by immunofluorescent labeling. An attempt to identify postsynaptic neurons innervated by alpha-synuclein-containing axon terminals revealed their selective apposition to calbindin D28K-negative projection neurons in the basolateral amygdala. The observed changes in alpha-synuclein levels are discussed in connection with their putative role in mediating suppression of dopaminergic neurotransmission during opiate withdrawal.

Heterogeneous expression of the triggering receptor expressed on myeloid cells-2 on adult murine microglia.

Microglial activation is an early and common feature of almost all neuropathologies, including multiple sclerosis, Alzheimer's disease and mechanical injury. To better understand the relative contributions microglia make toward neurodegeneration and neuroprotection, we used TOGA(R) to identify molecules expressed by microglia and regulated by inflammatory signals. Triggering receptor expressed on myeloid cells-2 (TREM-2) was among the mRNAs identified as being expressed by unactivated microglia, but down-regulated by lipopolysaccharide/interferon gamma. In the healthy CNS, not all microglia expressed TREM-2. Microglial expression of TREM-2 varied not only between brain regions but also within each brain region. Brain regions with an incomplete blood-brain barrier had the lowest percentages of TREM-2- expressing microglia, whereas the lateral entorhinal and cingulate cortex had the highest percentages. A novel form of TREM-2b that lacked a transmembrane domain was detected, perhaps indicating a soluble form of the protein. Taken together, these data suggest that (1) subsets of microglia are specialized to respond to defined extracellular signals; and (2) regional variations in TREM-2 expression may contribute to the varying sensitivities of different brain regions to similar pathological signals.

A novel mRNA expressed along brain ventricles.

The present descriptive study shows the expression pattern of an mRNA, IIIG9, whose most striking expression is seen along the lining of all four ventricles of the adult rat brain. Lower levels of expression are evident in other areas of the brain, notably in cortex, hippocampus, and various thalamic, hypothalamic and brainstem nuclei where it seems to be primarily neuronal, with little or no expression in white matter tracts. However, expression in cells on both sides of the basement membrane of the ependyma suggests expression in other cell types such as astrocytes, neuroblasts and putative precursors (subependyma), as well as ependymal cells and tanycytes. In the periphery, ovary had an expression level similar to the brain, whereas testes had a much higher level of expression.

Localization of olfactomedin-related glycoprotein isoform (BMZ) in the golgi apparatus of glomerular podocytes in rat kidneys.

Agene encoding olfactomedin-related glycoprotein was isolated from rat glomerulus despite its prior identification as a neuron-specific gene. The mRNA expression was remarkably intense in renal glomerulus and brain and faint in the lung and eye among rat systemic organs. Although the brain contained four mRNA variants (AMY, AMZ, BMY, and BMZ) transcribed from a single gene, the glomerulus, lung, and eye expressed only two variants (BMZ and BMY). The glycoprotein was intensely immunolocalized in glomerular podocytes and neurons by using an antibody against synthetic peptide of the M region, but weak in endothelial cells of the kidney and lung. Bronchiolar epithelial cells in the lung, and ciliary, corneal, and iris epithelial cells in the eye were also stained. Immunogold electron microscopy revealed selective localization of olfactomedin-related glycoprotein at the Golgi apparatus in podocytes. In glomerular culture, the staining was also intense at a juxtanuclear region in synaptopodin-positive epithelial cells of irregular shape (phenotypic feature of podocytes), whereas it was weak in synaptopodin-negative ones of cobblestone-like appearance (phenotypic feature of parietal epithelial cells of Bowman's capsule). Interestingly, Western blot analysis identified an intense band corresponding to BMZ isoform and another faint band corresponding to BMY isoform in the glomerulus, whereas the intensity of these two bands were nearly equal in the lung and eye. In the brain, four bands corresponding to four isoforms were observed apparently. Computer sequence analysis predicted coiled-coil structures in the secondary structure of the glycoprotein similar to those in Golgi autoantigens, suggesting significant roles in the unique functions of the Golgi apparatus in rat podocytes and neurons.