Journal of Neurochemistry

Ng, Gordon Y. K.; O'Dowd, Brian F.; Caron, Mirelle; Dennis, Michael; Brann, Mark R.; George, Susan R.

doi: 10.1046/j.1471-4159.1994.63051589.xpmid: 7931316

Abstract: We have expressed and biochemically characterized the human D2long (D2L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2L receptors expressed in mammalian cell lines. Dopamine binding to D2L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2L receptor‐specific antibody identified two major protein species at ∼44 kDa and at ∼93 kDa in immunoblots, suggesting the presence of D2L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin‐solubilized preparation of cells expressing D2L receptor prelabeled with 32Pi or [3H]‐palmitate. These results constitute the first direct evidence for D2L receptor phosphorylation and palmitoylation.

Paschen, Wulf; Hedreen, John C.; Ross, Christopher A.

doi: 10.1046/j.1471-4159.1994.63051596.xpmid: 7523595

Abstract: Editing of mRNA in the coding region of the second transmembrane domain of glutamate receptor subunits GluR2, GluR5, and GluR6 involves a change of the base A in genomic DNA to the base G in mRNA as described in rat brain. To determine whether this reaction occurs in humans as well as rats, we studied RNA editing of GluR2 and GluR6 in human brain. We compared the extent of editing in controls and cases with Huntington's disease. To assay the extent of editing in brain RNA, first strand cDNA was amplified using the polymerase chain reaction yielding a product across the region of the second transmembrane spanning segment in which editing takes place in rats. The PCR product was incubated with the restriction enzyme BbvI, which recognizes the sequence GCAGC present in the nonedited sequence of the mRNA in subunits GluR2 and GluR6. Thus, BbvI cuts the nonedited version but leaves the edited version intact. As in the rat, the GluR2 subunit mRNA was completely edited in human brain. The GluR6 subunit was nearly completely edited in all gray matter structures investigated including cortex, striatum, thalamus, hippocampus, amygdala, and cerebellum with extent of editing ranging from 89% in the cerebellum to 95% in the cortex and striatum. No significant differences in the extent of RNA editing were apparent in control versus Huntington's disease brains. To compare the extent of editing in neurons and glia in the brain, editing in cerebral cortex (predominantly gray matter and thus neurons) was compared with editing in corpus callosum (white matter and thus nearly completely glial cells). In white matter, GluR2 was completely edited, whereas GluR6 was only ∼10% edited compared with ∼90% edited in gray matter. Thus, these studies indicate that RNA editing is seen in human brain as well as rat brain and that the extent of editing is similar in Huntington's disease compared with controls. The differences in editing in white matter for GluR6, but not for GluR2, suggest that different templates could be subject to different editing activities that undergo tissue‐specific regulation.

Suh, Yoo‐Hun; Chun, Yhang‐Sook; Lee, In Sik; Kim, Sung‐Soo; Choi, Woong; Chong, Young Hae; Hong, Lyhna; Kim, Seong‐Hun; Park, Chan‐Woong; Kim, Chong‐Gook

doi: 10.1046/j.1471-4159.1994.63051603.xpmid:

Sato, Shoko; Burgess, S. B.; McIlwain, D. L.

doi: 10.1046/j.1471-4159.1994.63051609.xpmid: 7523596

Abstract: Nuclear size and total RNA synthesis were compared in single lumbar motoneurons isolated from the grass frog. Transcription was found to correlate significantly, but not exclusively, with nuclear area or volume over a wide range of nuclear size, the largest nuclei having the highest mean transcriptional activity. Flow cytometric analysis of propidium iodide‐stained nuclei excluded polyploidy or polyteny as an explanation for the increased transcription, but left open the possibility of a small increase in DNA with increasing nuclear size. Alternatively, motoneurons may increase transcription and nuclear size without increasing their DNA content, possibly by increasing the proportion of dispersed chromatin (euchromatin). These two mechanisms for size‐related changes in RNA synthesis in motoneurons present an interesting contrast to mechanisms used by many other large animal cells.

Bennett, Ellen; Stenvers, Kaye L.; Lund, P. Kay; Popko, Brian

doi: 10.1046/j.1471-4159.1994.63051616.xpmid: 7931318

Abstract: We have adopted a polymerase chain reaction approach to identify and clone a cDNA that contains the complete coding sequence of a novel fatty acid binding protein (FABP) from a rat brain λgt10 library. Sequencing of the brain FABP (B‐FABP) cDNA revealed an open reading frame coding for a protein with 132 amino acids and a predicted size of ∼15,000 Da. This putative protein shares extensive sequence homology with other members of the FABP family. Northern blot analysis using the B‐FABP cDNA as a probe established the presence of an abundant mRNA ∼0.8 kb long in rat brain and in the MOCH‐1 oligodendrocyte cell line. This transcript was also present in rat liver but not in other tissues examined. A developmental profile of this mRNA in rat brain demonstrated detectable expression in 15‐day‐old embryos with levels peaking in 1‐day postnatal neonates and declining thereafter, reaching a low steady‐state level at 3 weeks of age. In situ hybridization histochemistry revealed B‐FABP mRNA in various brain regions, with the highest levels in fiber tracts. The B‐FABP message was also detected at a lower level in several gray matter regions. The cloning approach used in this study would likely be useful in the identification and isolation of FABP‐encoding genes from other tissues and species.

Liu, Wei; Shafit‐Zagardo, Bridget; Aquino, Dennis A.; Zhao, Meng‐Liang; Dickson, Dennis W.; Brosnan, Celia F.; Lee, Sunhee C.

doi: 10.1046/j.1471-4159.1994.63051625.xpmid: 7931319

Abstract: Previous studies in this and other laboratories have shown that interleukin‐1β (IL‐1β) is a selective and potent activator of human astrocytes with respect to induction of cytokines and hematopoietic growth factors. To study the effect of recombinant human IL‐1β (rhIL‐1β) on astrocyte morphology, glial fibrillary acidic protein (GFAP) and vimentin expression, and actin organization, we conducted a systematic survey using dissociated human fetal astrocyte cultures. Within hours of stimulation with IL‐1β, the majority of astrocytes converted from flat, polygonal cells to small, contracted, highly branched cells. This change in morphology was more striking when serum was eliminated from the medium. Complete dissolution of filamentous actin occurred simultaneously with the change in cell shape, as demonstrated by fluorescein‐phalloidin binding. These “activated” astrocytes displayed intense GFAP and vimentin immunoreactivity in the small perikarya and processes. In contrast, the large, flat astrocytes in control cultures showed diffuse pale immunoreactivity for GFAP and vimentin. To quantify the changes in GFAP and vimentin content with IL‐1β stimulation, densitometric analyses of northern and western blots were performed. Northern blot analysis of IL‐1β‐stimulated astrocytes revealed a transient, marked decrease in steady‐state levels of mRNA for GFAP, vimentin, and microtubule‐associated protein 4. The decrease in mRNA levels was evident by 4–8 h and fell to the lowest level at 16–24 h (80–98% decrease by densitometry) with partial recovery by 72 h. By immunoblotting, a significant decrease in both GFAP and vimentin protein content was observed after IL‐1β stimulation. Furthermore, metabolic labeling studies revealed an almost total loss of GFAP synthesis following stimulation with IL‐1β for 16 h. These observations are consistent with the idea that increases in immunoreactivity were related to factors such as redistribution of epitope, rather than increases in total protein content. We hypothesize that in IL‐1β‐stimulated astrocytes, synthesis of other proteins, e.g., inflammatory cytokines, occurs at the expense of structural proteins and that the decrease in content of cytoskeletal proteins may reflect an “activated” state of astrocytes.

Hosoda, Kohkichi; Feussner, Gretchen K.; Rydelek‐Fitzgerald, Laura; Fishman, Peter H.; Duman, Ronald S.

doi: 10.1046/j.1471-4159.1994.63051635.xpmid: 7931320

Abstract: Exposure of rat C6 glioma cells to either agonists or agents that increase cyclic AMP levels leads to down‐regulation of β1‐adrenergic receptors (β1AR) as measured by loss of radioligand binding sites. The present study examines the influence of isoproterenol and forskolin treatment on levels of β1AR mRNA, mRNA stability, and gene transcription rate. Isoproterenol treatment of C6 cells altered β1AR mRNA levels in a biphasic manner; i.e., short‐term exposure (30–60 min) increased by 50%, whereas longer exposure (2–6 h) decreased by 50% the levels of β1AR mRNA. The extent of both the up‐ and down‐regulation was dependent on agonist concentration. Similar regulation of β1AR mRNA was observed in forskolin‐treated cells. Pretreatment of the cells with Pseudomonas exotoxin A, a potent inhibitor of protein synthesis, completely blocked isoproterenol‐ and forskolin‐mediated down‐regulation of β1AR mRNA, and thereby potentiated the increase in receptor mRNA up to fourfold over the 6‐h time course. The mechanisms underlying β1AR mRNA down‐regulation were examined. The half‐life of β1AR mRNA was slightly increased (from 61 to 77 min) after a 2‐h exposure of the cells to either isoproterenol or forskolin. Nuclear run‐on analysis demonstrated that the rate of β1AR gene transcription was increased after isoproterenol incubation for 60 min, but then decreased after 90–240 min, consistent with the time course for up‐ and down‐regulation of β1AR mRNA. Isoproterenol treatment (120 min) also decreased the level of β1AR nascent transcripts, purified by affinity chromatography of RNA isolated from 4‐thiouridine‐pulsed cells. The results demonstrate that β1AR mRNA has a relatively short half‐life and that agonist regulation of β1AR mRNA is mediated by activation of the cyclic AMP system. Moreover, the results indicate that agonist regulation of β1AR mRNA occurs at the level of β1AR gene transcription, not mRNA stability. Finally, the observed requirement for protein synthesis indicates that β1AR mRNA down‐regulation may be mediated by the induction of a repressor of the β1AR gene.

Toda, Kenichi; Small, Judy A.; Goda, Shuichiro; Quarles, Richard H.

doi: 10.1046/j.1471-4159.1994.63051646.xpmid: 7523597

Abstract: Biochemical and cellular properties of three immortalized Schwann cell lines expressing different levels of the myelin‐associated glycoprotein (MAG) were compared. The S16 line generated by repetitive passaging was described previously and expresses a level of MAG comparable to that in adult sciatic nerve. The S42 line was generated independently by the same procedure, divides more slowly than the S16 line, and expresses an even higher level of MAG. The S16Y line arose spontaneously from a passage of the S16 cells, divides much more rapidly, and does not express MAG. The levels of MAG expression in the three lines are inversely related to their rates of proliferation, and MAG mRNA levels parallel the amounts of MAG. The S16 and S42 lines consist mainly of flat cells at low density and develop many processes at high density, whereas most of the S16Y cells are spindle‐shaped, resembling primary Schwann cells in appearance. Surface immunostaining with the O4 antibody was positive for the S16 and S42 cells and negative for the S16Y cells, but all three lines were negative for surface staining with the O1 antibody. The overall protein compositions of the three lines are very similar, but the S16 and S42 cells express larger amounts of several glycoproteins than the S16Y cells, including the adhesion proteins, neural cell adhesion molecule, L1, and laminin. S16 and S42 cells (but not S16Y cells) also express P0 glycoprotein, galactocerebroside, and sulfatide, but, unlike MAG, these other myelin‐related components were present at much lower levels than in adult nerve. Myelin basic protein and proteolipid protein were not detected in any of the lines, although all three lines contained proteolipid protein mRNA. 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase and glial fibrillary acidic protein were present in all three lines. Conditions have not yet been found in which any of the lines will myelinate dorsal root ganglion neurons in vitro, but the S16 and S42 cells differ from the S16Y cells by clustering around neurons after 1 week in coculture. In many respects, the S16 and S42 cells biochemically resemble Schwann cells at an early stage in their preparation to myelinate and should be useful for investigating the cell biology of MAG and other myelin‐related components.

Hall, Randy A.; Bahr, Ben A.

doi: 10.1046/j.1471-4159.1994.63051658.xpmid: 7931321

Abstract: Telencephalic membranes from rats of different embryonic (E16, E19) and postnatal (P2, P7, P14, adult) ages were assessed for α‐[3H]amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid ([3H]AMPA) binding and for immunoreactivity levels of AMPA receptor subunits (GluR1, GluR2/3, and GluR4). In addition, the synaptic markers synaptophysin and NCAM140 (a neural cell adhesion molecule isoform) were examined by immunoblot. The density of [3H]AMPA binding sites increased steadily with advancing age. This increase was due mainly to the development of the large low‐affinity component (KD = 400 nM) that dominates the [3H]AMPA binding profile of adult rat brain membranes. As resolved by two‐site regression analysis, the high‐affinity component (KD = 15 nM) of the [3H]AMPA binding increased by approximately twofold from E16 to adult, whereas the low‐affinity component increased by 25‐fold. Staining for GluR1 and GluR2/3 increased steadily with increasing age at all time points examined; synaptophysin and NCAM140 exhibited similar ontogenic immunostaining profiles. GluR4 immunoreactivity was first evident at P14 and increased by adulthood. These results indicate that AMPA receptor density increases steadily during development and that this increase is coincident with the ontogenic expression of other synaptic components. Furthermore, there is a shift toward a preponderance of low‐affinity [3H]AMPA binding, which occurs during the period when AMPA receptors are being sorted into postsynaptic regions, suggesting that some element of the postsynaptic membrane environment modulates AMPA receptor properties.

Watson, D. G.; Wainer, B. H.; Lenox, R. H.

doi: 10.1046/j.1471-4159.1994.63051666.xpmid: 7931322

Abstract: The expression of MARCKS, a major protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS protein levels were reduced through an apparent PKC‐dependent mechanism. Exposure to 1 µM phorbol 12‐myristate 13‐acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane‐associated PKC activity. PKC activity was reduced to <20% of control values in both soluble and membrane fractions following 1 h of PMA exposure. Significant reductions in MARCKS protein levels were initially observed in membrane and soluble fractions following PMA exposure for 4 and 8 h, respectively. The reduction in MARCKS protein levels was maximal following 24 h of PMA exposure. MARCKS protein expression was also down‐regulated in a dose‐dependent manner on exposure of HN33 cells to retinoic acid. In cells exposed to 10 µM retinoic acid, the MARCKS protein level was reduced in the membrane fraction within 4 h. Reduction of MARCKS protein levels was maximal (>90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS protein were also observed in the soluble fraction of retinoic acid‐exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down‐regulation of MARCKS protein in PMA‐treated cultures but not in retinoic acid‐treated cells. These findings suggest that the down‐regulation of MARCKS may play an important role in both phorbol ester‐ and retinoic acid‐induced differentiation in cells of neuronal origin.