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B H Wainer

Publications and source records attributed to B H Wainer.

At least 19 recordsLinked to original sources
PubMed2006

BLOC-1 complex deficiency alters the targeting of adaptor protein complex-3 cargoes.

Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these genes assemble into five distinct complexes (AP-3, BLOC-1-3, and HOPS) that either sort membrane proteins or interact with SNAREs. Several of these seemingly distinct complexes cause similar phenotypic defects when they are rendered defective by mutation, but the underlying cellular mechanism is not understood. Here, we show that the BLOC-1 complex resides on microvesicles that also contain AP-3 subunits and membrane proteins that are known AP-3 cargoes. Mouse mutants that cause BLOC-1 or AP-3 deficiencies affected the targeting of LAMP1, phosphatidylinositol-4-kinase type II alpha, and VAMP7-TI. VAMP7-TI is an R-SNARE involved in vesicle fusion with late endosomes/lysosomes, and its cellular levels were selectively decreased in cells that were either AP-3- or BLOC-1-deficient. Furthermore, BLOC-1 deficiency selectively altered the subcellular distribution of VAMP7-TI cognate SNAREs. These results indicate that the BLOC-1 and AP-3 protein complexes affect the targeting of SNARE and non-SNARE AP-3 cargoes and suggest a function of the BLOC-1 complex in membrane protein sorting.

Adaptor Protein Complex 3↗
PubMed2004

Genetic analysis of the neuronal and ubiquitous AP-3 adaptor complexes reveals divergent functions in brain.

Neurons express adaptor (AP)-3 complexes assembled with either ubiquitous (beta3A) or neuronal-specific (beta3B) beta3 isoforms. However, it is unknown whether these complexes indeed perform distinct functions in neuronal tissue. Here, we explore this hypothesis by using genetically engineered mouse models lacking either beta3A- or beta3B-containing AP-3 complexes. Somatic and neurological phenotypes were specifically associated with the ubiquitous and neuronal adaptor deficiencies, respectively. At the cellular level, AP-3 isoforms were localized to distinct neuronal domains. beta3B-containing AP-3 complexes were preferentially targeted to neuronal processes. Consistently, beta3B deficiency compromised synaptic zinc stores assessed by Timm's staining and the synaptic vesicle targeting of membrane proteins involved in zinc uptake (ZnT3 and ClC-3). Surprisingly, despite the lack of neurological symptoms, beta3A-deficient mouse brain possessed significantly increased synaptic zinc stores and synaptic vesicle content of ZnT3 and ClC-3. These observations indicate that the functions of beta3A- and beta3B-containing complexes are distinct and divergent. Our results suggest that concerted nonredundant functions of neuronal and ubiquitous AP-3 provide a mechanism to control the levels of selected membrane proteins in synaptic vesicles.

Adaptor Protein Complex 3↗
PubMed2001

Nerve growth factor, but not epidermal growth factor, increases Fra-2 expression and alters Fra-2/JunD binding to AP-1 and CREB binding elements in pheochromocytoma (PC12) cells.

In pheochromocytoma (PC12) cells nerve growth factor (NGF) and epidermal growth factor (EGF) activate similar receptor tyrosine kinase signaling pathways but evoke strikingly different biological outcomes: NGF induces differentiation and EGF acts as a mitogen. A novel approach was developed for identifying transcription factor activities associated with NGF-activated, but not EGF-activated, signaling, using random oligonucleotide clones from a DNA recognition library to isolate specific DNA binding proteins from PC12 nuclear extracts. A protein complex from NGF-treated, but not EGF-treated, cells was identified that exhibits increased mobility and DNA binding activity in gel mobility shift assays. The binding complex was identified in supershift assays as Fra-2/JunD. The clones used as probes contain either AP-1 or cAMP response element binding (CREB) recognition elements. Time course experiments revealed further differences in NGF and EGF signaling in PC12 cells. NGF elicits a more delayed and sustained ERK phosphorylation than EGF, consistent with previous reports. Both growth factors transiently induce c-fos, but NGF evokes a greater response than EGF. NGF specifically increases Fra-1 and Fra-2 levels at 4 and 24 hr. The latter is represented in Western blots by bands in the 40-46 kDa range. NGF, but not EGF, enhances the upper bands, corresponding to phosphorylated Fra-2. These findings suggest that prolonged alterations in Fra-2 and subsequent increases in Fra-2/JunD binding to AP-1 and CREB response elements common among many gene promoters could serve to trigger broadly an NGF-specific program of gene expression.

Animals↗
PubMed2001

Complications of gamma knife surgery for Parkinson disease.

BACKGROUND: Many medical centers throughout the world offer radiosurgery with the gamma knife (GK) for pallidotomy and thalamotomy as a safe and effective alternative to radiofrequency ablative surgery and deep brain stimulation for Parkinson disease (PD). The reported incidence of significant complications varies considerably, and the long-term complication rate remains unknown. DESIGN: We describe 8 patients seen during an 8-month period referred for complications of GK surgery for PD. RESULTS: Of the 8 patients, 1 died as a result of complications, including dysphagia and aspiration pneumonia. Other complications included hemiplegia, homonymous visual field deficit, hand weakness, dysarthria, hypophonia, aphasia, arm and face numbness, and pseudobulbar laughter. In all patients, lesions were significantly off target. CONCLUSIONS: The 8 patients with PD seen in referral at our center for complications of GK surgery highlight a spectrum of potential problems associated with this procedure. These include lesion accuracy and size and the delayed development of neurological complications secondary to radiation necrosis. Gamma knife surgery may have a higher complication rate than has been previously appreciated due to delayed onset and underreporting. We believe that the risk-benefit ratio of the GK will require further scrutiny when considering pallidotomy or thalamotomy in patients with PD. Physicians using this technique should carefully follow up patients postoperatively for delayed complications, and fully inform patients of these potential risks.

Aged↗
PubMed2001

Coupling of cAMP/PKA and MAPK signaling in neuronal cells is dependent on developmental stage.

Neurite formation, an essential feature of neuronal development, is believed to involve participation of the ras-mitogen-activated protein kinase (MAPK) and cAMP-dependent protein kinase A (cAMP/PKA)-mediated signaling pathways. These pathways have been studied extensively in the rat pheochromocytoma cell line PC12, and current hypotheses suggest a single effector mechanism resulting from the convergence of cAMP/PKA and MAPK signaling. However, based on observations using a central neuronal progenitor cell line, AS583-8, there also exists evidence that the two signaling pathways may act independently resulting in neurites with differing dynamic features. In the present study, the upstream components of cAMP/PKA signaling were examined in AS583-8 cells as well as possible interactions with the MAPK pathway. We found that activation of PKA is both necessary and sufficient for the elaboration of rapidly forming processes, typical of the cAMP response. In addition, blockade of the MAPK pathway has no effect on the cAMP response, suggesting that activation of the cAMP/PKA pathway can stimulate neurite formation independent of the MAPK pathway. In order to evaluate which cell line model, PC12 vs AS583-8, best reflects the signaling features of developing central neurons, we examined interactions between cAMP/PKA and MAPK signaling in primary neuronal cultures from several brain regions. In immature cultures (1-day-old), at a point where the initiation of neurite formation is maximal, no interaction was observed. In more mature cultures (7 days old), where synaptic contacts have been established, we found a weak but reproducible activation of MAPK following stimulation of the cAMP/PKA pathway. These results suggest that cAMP/PKA and MAPK signaling act independently at the initiation of neuritogenesis but become coupled during later stages of neuronal development. Therefore, the interaction of the two pathways may be cell stage (younger vs older) specific and may participate in cellular functions that take place after initial neurite formation.

Animals↗
PubMed2001

Tau-associated neuropathology in ganglion cell tumours increases with patient age but appears unrelated to ApoE genotype.

Ganglion cell tumours, including gangliogliomas and gangliocytomas, are low grade neoplasms with a mature neuronal component. Ganglion cells within these lesions occasionally exhibit neurodegenerative changes including neurofibrillary tangles (NFT) similar to those in Alzheimer's disease. The frequency and spectrum of degenerative pathology in these lesions have not been defined, nor has their relation to patient age or factors such as apolipoprotein E (ApoE) genotype that predispose to Alzheimer's disease. We studied 72 ganglion cell tumours (61 gangliogliomas, 11 gangliocytomas) from patients 7 months to 72-years-old. Haematoxylin and eosin (H&E), silver stains (Hirano method) and immunohistochemistry for tau, alpha-synuclein and beta-amyloid were performed on formalin-fixed, paraffin-embedded tissue from surgical specimens. Tau-and silver-positive NFT and neuropil threads (NPT) were present in four of 26 ganglion cell tumours from patients over 30-years-old (ages 31, 38, 50, and 58 years). Neuronal granulovacuolar degeneration (GVD) was noted in five of 26 tumours from patients over 30-years-old (mean, 48 years). NFT, NPT, and GVD were not seen in ganglion cell tumours from patients under 30-years-old[0/46]. Cytoplasmic argentophilic bodies distinct from NFT were present in five of 26 tumours from patients over 30-years-old and in two of 46 under 30 years. Neither alpha-synuclein positive neuronal inclusions nor beta-amyloid immunoreactivity was noted in ganglion cell tumours from any age group. The distribution of ApoE genotypes was similar among those tumours that contained tau-associated neuropathology and those that did not. Neurodegenerative changes are uncommon in ganglion cell tumours, but increase in frequency with patient age. GVD, tau-positive NFT and NPT, and argentophilic bodies occur more often in ganglion cell tumours from patients over 30-yrs-old, but do not appear to be associated with a specific ApoE genotype.

Adolescent↗
PubMed1999

Cellular aspects of trophic actions in the nervous system.

During the past three decades the number of molecules exhibiting trophic actions in the brain has increased drastically. These molecules promote and/or control proliferation, differentiation, migration, and survival (sometimes even the death) of their target cells. In this review a comprehensive overview of small diffusible factors showing trophic actions in the central nervous system (CNS) is given. The factors discussed are neurotrophins, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors, ciliary neurotrophic factor and related molecules, glial-derived growth factor and related molecules, transforming growth factor-beta and related molecules, neurotransmitters, and hormones. All factors are discussed with respect to their trophic actions, their expression patterns in the brain, and molecular aspects of their receptors and intracellular signaling pathways. It becomes evident that there does not exist "the" trophic factor in the CNS but rather a multitude of them interacting with each other in a complicated network of trophic actions forming and maintaining the adult nervous system.

Animals↗
PubMed1999

Tau pathology in a family with dementia and a P301L mutation in tau.

Familial forms of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) have recently been associated with coding region and intronic mutations in the tau gene. Here we report our findings on 2 affected siblings from a family with early-onset dementia, characterized by extensive tau pathology and a Pro to Leu mutation at codon 301 of tau. The proband, a 55-year-old woman, and her 63-year-old brother died after a progressive dementing illness clinically diagnosed as Alzheimer disease. Their mother, 2 sisters, maternal aunt and uncle, and several cousins were also affected. Autopsy in both cases revealed frontotemporal atrophy and degeneration of basal ganglia and substantia nigra. Sequencing of exon 10 of the tau gene revealed a C to T transition at codon 301, resulting in a Pro to Leu substitution. Widespread neuronal and glial inclusions, neuropil threads, and astrocytic plaques similar to those seen in corticobasal degeneration were labeled with a battery of antibodies to phosphorylation-dependent and phosphorylation-independent epitopes spanning the entire tau sequence. Isolated tau filaments had the morphology of narrow twisted ribbons. Sarkosyl-insoluble tau exhibited 2 major bands of 64 and 68 kDa and a minor 72 kDa band, similar to the pattern seen in a familial tauopathy associated with an intronic tau mutation. These pathological tau bands predominantly contained the subset of tau isoforms with 4 microtubule-binding repeats selectively affected by the P301L missense mutation. Our findings emphasize the phenotypic and genetic heterogeneity of tauopathies and highlight intriguing links between FTDP-17 and other neurodegenerative diseases.

Atrophy↗
PubMed1998

Beta-adrenergic and fibroblast growth factor receptors induce neuronal process outgrowth through different mechanisms.

The mechanisms that initiate and direct neuronal process formation remain poorly understood. We have recently described a neuronal progenitor cell line, AS583-8.E4.22 (AS583-8) which undergoes neurite formation in response to beta2-adrenergic and basic fibroblast growth factor (bFGF) receptor activation [Kwon, J.H. et al., (1996) Eur. J. Neurosci., 8, 2042-2055]. In the present study, a comparison of these responses revealed that isoproterenol (ISO), a beta-adrenergic receptor agonist, induces multiple, highly branched processes within 30 min while bFGF induces fewer, unbranched processes within 24 h. In contrast to the ISO response, bFGF induces mitogen-activated protein kinase activation and c-fos expression in the cell line and results in neurite outgrowth that is dependent on new mRNA and protein synthesis. Two-dimensional isoelectric focusing-sodium dodecyl sulphate-polyacrylamide gel electrophoresis of cytoskeletal preparations revealed different patterns following ISO vs. bFGF exposure suggesting selective changes in protein expression and/or post-translational modifications. Immunoblot analysis of these preparations for beta-tubulin, tyrosinated alpha-tubulin and acetylated alpha-tubulin also revealed different patterns following each type of treatment. Follow-up confocal microscopy revealed that following ISO, the distribution of tyrosinated tubulin extends to the distal ends of processes whereas acetylated alpha-tubulin is diminished within distal ends. This pattern has been reported to be associated with enhanced microtubule dynamics, a state in which process outgrowth is facilitated. In contrast, following bFGF treatment the distributions of tyrosinated and acetylated alpha-tubulin were identical, a state associated with a diminution of microtubule dynamics. These results, a different time course of neurite formation, dependency on new gene expression and differential expression and cellular distribution of major cytoskeleton proteins suggest that neurite outgrowth induced by ISO vs. bFGF is mediated by two distinct intracellular effector mechanisms in AS583-8 cells. In addition, studies, using the differential distribution of post-translational modified alpha-tubulins in neurites of primary neuronal cultures as marker for the two distinct processes of neurite formation suggest, that similar mechanisms are present in vivo. Therefore, the AS583-8 cell line provides a useful model to study these signalling mechanisms that couple neurotransmitter and growth factor receptor activation to the cytoskeletal changes that mediate neurite formation.

Animals↗
PubMed1997

Ultrastructural study of cholinergic and noncholinergic neurons in the pars compacta of the rat pedunculopontine tegmental nucleus.

A group of medium-to-large cholinergic neurons situated in the dorsolateral mesopontine tegmentum comprises the pedunculopontine tegmental nucleus (PPT). The PPT pars compacta (PPT-pc), which occupies the lateral part of the caudal two-thirds of the nucleus, contains a dense aggregation of cholinergic neurons. In the present study, we have employed immunohistochemistry for choline acetyltransferase (ChAT) and electron microscopy to investigate the ultrastructure and synaptic organization of neuronal elements in the PPT-pc. Our results demonstrate that: (1) ChAT-immunoreactive (i.e., cholinergic) PPT-pc neurons are characterized by abundant cytoplasm and organelles, and have few axosomatic synapses (both asymmetric and symmetric); (2) ChAT-immunoreactive dendrites comprise 6-15% of total dendritic elements in the neuropil; the mean percentage of dendritic membrane covered by synaptic terminals is approximately 15%, and nearly all synapses with ChAT-immunoreactive dendrites are asymmetric; (3) within the boundaries described by cholinergic PPT-pc, there are noncholinergic neurons which, in contrast, exhibit a lucent cytoplasm and a higher frequency of axosomatic synapses (10.5% versus 3.7% for cholinergic neurons); and (4) noncholinergic neurons are morphologically heterogeneous with one subpopulation exhibiting a mean diameter that approximates that of cholinergic cells (i.e., > 15 microns and < 20 microns) and a very high frequency of axosomatic synapses (> 20%). Only 0.2-0.7% of terminal elements in the neuropil were ChAT-immunoreactive and these were not observed to synapse with cholinergic dendrites or somata. This relative paucity of terminal labeling and lack of cholinergic-cholinergic interactions seems inconsistent with the recognized and prominent physiological actions of acetylcholine on cholinergic PPT-pc neurons, and suggests a methodological limitation and/or a potential paracrine-like action of nonsynaptically released acetylcholine in the PPT region.

Acetylcholine↗
PubMed1997

Serotonergic dorsal raphe nucleus projections to the cholinergic and noncholinergic neurons of the pedunculopontine tegmental region: a light and electron microscopic anterograde tracing and immunohistochemical study.

The serotonergic dorsal raphe nucleus is considered an important modulator of state-dependent neural activity via projections to cholinergic neurons of the pedunculopontine tegmental nucleus (PPT). Light and electron microscopic analysis of anterogradely transported biotinylated dextran, combined with choline acetyltransferase (ChAT) immunohistochemistry, were employed to describe the synaptic organization of mesopontine projections from the dorsal raphe to the PPT. In a separate set of experiments, we utilized immunohistochemistry for the serotonin transporter (SERT), combined with ChAT immunohistochemistry at the light and electron microscopic levels, to determine whether PPT neurons receive serotonergic innervation. The results of these studies indicate that: (1) anterogradely labeled and SERT-immunoreactive axons and presumptive boutons invest the PPT at the light microscopic level; (2) at the ultrastructural level, dorsal raphe terminals in the PPT pars compacta synapse mainly with dendrites and axosomatic contacts were not observed; (3) approximately 12% of dorsal raphe terminals synapse with ChAT-immunoreactive dendrites; and (4) at least 2-4% of the total synaptic input to ChAT-immunoreactive dendrites is of dorsal raphe and/or serotonergic origin. This serotonergic dorsal raphe innervation may modulate cholinergic PPT neurons during alterations in behavioral state. The role of these projections in the initiation of rapid eye movement (REM) sleep and the ponto-geniculo-occipital waves that precede and accompany REM sleep is discussed.

Acetylcholine↗
PubMed1997

Insulin gene expression in immortalized rat hippocampal and pheochromocytoma-12 cell lines.

Employing reverse transcription-polymerase chain reaction and clonal cell lines derived by retroviral transduction of the temperature sensitive simian virus 40 large T-antigen into dispersed rat embryonic hippocampal cells, we detected the ancestral gene-insulin II mRNA in three progenitor subcloned cell lines. These cell lines upon differentiation are known to express markers indicative of commitment to either neuronal (H19-7; NF + , GFAP -), glial (H19-5; GFAP +, NF -), or bipotential (H583-5, NF +, GFAP + ) lineages. No duplicated, i.e., insulin I gene expression, was observed in any of the three cell lines. Induction of differentiation was associated with the persistence of insulin II mRNA and in the cells expressing a neuronal phenotype (H19-7; NF +, GFAP -) a relative doubling in insulin II mRNA level was present (P < 0.05). Minimal cellular insulin immunoreactivity was detected only in a subpopulation of cells with a differentiated neuronal phenotype. Radioimmunoassayable insulin peptide in the H19-7 cellular conditioned medium revealed a 5-fold increase in the differentiated state. In contrast, peripheral sympathetic PC-12 neuronal cells both in the undifferentiated and nerve growth factor-driven differentiated states, failed to express both insulin I and insulin II genes. We conclude that insulin II is expressed by cultured rat hippocampal clonal cell lines, and not by the peripheral sympathetic PC-12 neuronal cell line.

Animals↗
PubMed1996

Expression of glucocorticoid and mineralocorticoid receptors in an immortalized hippocampal neuronal cell line.

A clonal cell line of rat embryonic hippocampal origin (H19-7) has been examined for the expression of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). H19-7 cells grown at 33 degrees C continue to divide, however when grown at 39 degrees C in reduced levels of serum the cells undergo morphological differentiation and express neuronal properties. Immunocytochemistry demonstrated that H19-7 cells express both MR and GR when grown at either 33 degrees C or 39 degrees C. GR mRNA is readily detected in H19-7 cells by RNase protection assay. MR mRNA levels in H19-7 cells are too low to detect by RNase protection, but can be detected by RT-PCR. RT-PCR also demonstrated that H19-7 cells express more GR mRNA than primary hippocampal neurons. Since previous studies have shown that the level of MR mRNA is higher than that of GR mRNA in hippocampal neurons, these studies suggest that H19-7 cells represent hippocampal neurons immortalized at an early stage when the MR system is not yet fully differentiated.

Animals↗
PubMed1996

Beta-adrenergic receptor activation promotes process outgrowth in an embryonic rat basal forebrain cell line and in primary neurons.

A clonal cell line, AS583-8.E4.22, from the embryonic day 15 rat basal forebrain was established using retrovirus-mediated transduction of a temperature-sensitive mutant of the simian virus 40 (SV40) large tumour antigen. The cell line expresses cytoskeletal and neurotransmitter features indicative of neuronal commitment. In response to agents that increase intracellular cAMP, including forskolin and catecholamines, the cell line exhibits rapid process outgrowth and growth cone formation that does not require new gene expression or protein synthesis. The neurite outgrowth induced by catecholamines is mediated by beta 2-adrenergic receptors and is characterized by a rapid, reversible redistribution of filamentous actin. Neurons from primary cultures of embryonic day 15 basal forebrain were also found to respond to beta-adrenergic receptor agonists by enhancing growth cone formation. These results suggest that catecholamines provide cues that induce cytoskeletal rearrangements leading to neuronal process outgrowth and growth cone formation in the developing basal forebrain and possibly other neuronal progenitor cell populations. The neuronal basal forebrain cell line provides an ideal model to study the signalling mechanisms underlying the catecholamine-induced process outgrowth.

Adrenergic beta-Agonists↗
PubMed1995

BDNF-activated signal transduction in rat cortical glial cells.

Cortical glial cells in culture were found to be responsive to the neurotrophin brain-derived neurotrophic factor (BDNF), as evidenced by activation of multiple signal transduction processes. BDNF produced an increase in mitogen-activated protein (MAP) kinase tyrosine phosphorylation, MAP kinase activity, intracellular calcium concentration and c-fos expression in the glial cells. Only a subset of the glial cells responded to BDNF, as reflected in single-cell analysis of calcium transients and c-fos expression. BDNF had no detectable effect on glial mitotic activity, as measured by DNA synthesis. In parallel studies, nerve growth factor and neurotrophin-3 had no effect on signalling in these cultures. BDNF has previously been demonstrated to act via trkB receptors with a cytoplasmic tyrosine kinase domain (gp145trkB). Pretreatment of glial cultures with K252a, which at low concentrations specifically inhibits the trk tyrosine kinases, abolished BDNF effects on MAP kinase stimulation, suggesting that BDNF was acting through gp145trkB. However, subsequent studies showed that gp145trkB was expressed at extremely low levels in the cultures: gp145trkB mRNA transcripts could only be detected using the reverse transcription-polymerase chain reaction, and gp145trkB protein was not detected by either immunoblotting or immunocytochemistry. On the other hand, the glia expressed significantly higher levels of gp95trkB mRNA and protein, which represent truncated forms of trkB receptors lacking the tyrosine kinase domain. The results of these studies demonstrate that a subset of cultured CNS glia respond to BDNF with the activation of conventional signal transduction processes. The mechanism of BDNF-initiated signal transduction in glial cells most likely involves a relatively small number of gp145trkB receptors, but involvement of the more abundant truncated gp95trkB receptors cannot be excluded.

Animals↗
PubMed1995

All-trans- and 9-cis-retinoic acid enhance the cholinergic properties of a murine septal cell line: evidence that the effects are mediated by activation of retinoic acid receptor-alpha.

We investigated the effects of retinoids on the cholinergic properties of a murine septal cell line, SN56. Treatment of the cells with all-trans-retinol (vitamin A), all-trans-retinal, all-trans-retinoic acid (t-RA), 9-cis-retinoic acid (9c-RA), or 13-cis-retinoic acid caused time- and concentration-dependent increases in choline acetyltransferase activity (up to 3.4-fold) and in intracellular acetylcholine levels (up to 2.5-fold, with respective EC50 values of 68, 50, 18, 15, and 56 nM). Furthermore, treatment with either t-RA or 9c-RA at 1 microM for 48 h resulted in an increase in the expression of choline acetyltransferase mRNA by threefold that of controls. These data and the presence of putative retinoic acid response elements in the 5' region of the murine choline acetyltransferase gene indicate that retinoids stimulate choline acetyltransferase transcription in murine cholinergic neurons. No additivity or synergism was observed between the effects of t-RA and 9c-RA on any of these cholinergic properties of SN56 cells, suggesting a common mechanism of action of the two retinoids. However, a combined treatment with t-RA and forskolin, which activates adenylate cyclase, resulted in an additive increase in acetylcholine content. Using an antagonist selective for the retinoic acid receptor-alpha subtype, Ro 41-5253, we found that the effects of t-RA and 9c-RA on acetylcholine levels were abolished. An agonist selective for retinoic acid receptor-alpha, Ro 40-6055, increased acetylcholine levels to a similar extent as t-RA and 9c-RA, and this effect was blocked by the antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1994

Conditional immortalization of neuronal cells from postmitotic cultures and adult CNS.

To determine whether postmitotic neurons can be immortalized by oncogenic transduction, we used two approaches involving conditional expression of a temperature-sensitive SV40 large T antigen (Tts). Initially, Tts was introduced into E17 rat embryonal hippocampal cells that were then cultured at the non-permissive temperature to enrich for postmitotic pyramidal neurons, and subsequently cloned at the permissive temperature. One clonal line (HMR10-3) expressed neuron-specific proteins upon differentiation, was capable of generating action potentials, and formed synapses with primary rat neurons in co-culture. Replating of these postmitotic cells at the permissive temperature resulted in reversible loss of neurofilament expression. Conditionally immortalized cell lines were also generated from the brain of an adult mouse carrying an inducible Tts transgene. These lines proliferated in a T antigen-dependent manner and expressed neuron-specific proteins upon differentiation at the non-permissive temperature. These results suggest that postmitotic neurons can be induced to enter the cell cycle without losing their commitment to a neuronal lineage.

Animals↗