Cerebellar granule cells cultured from adolescent rats express functional NMDA receptors: an in vitro model for studying the developing cerebellum.

In the developing rat cerebellum functional NMDA receptors (NMDARs) expressing the NR2C subunit have been identified on or after postnatal day 19. We obtained primary cultured cells from 19- to 35-day-old rat cerebellum that expressed few oligodendrocytes or astrocytes. Cultured cells were immunoreactive for neuron-specific proteins thus indicating a neuronal population. The primary neuron present was the granule cell as indicated by immunofluorescence for the GABA(A) alpha 6 subunit. Whole-cell patch-clamp experiments indicated that functional NMDARs were present. Functional characteristics of NMDARs expressed in cerebellar granule cells (CGCs) obtained from adolescent animals were similar to those previously reported for NMDARs expressed in CGCs obtained from neonatal rats. Cultured CGCs obtained from older animals contained NMDARs that were inhibited by EtOH and were less sensitive to the NR2B subunit-specific antagonist Ro 25-6981. Furthermore, NMDA-induced currents were smaller than those observed in CGCs. Western blot analysis indicated the presence of the NMDA NR2A and NR2C subunits, but not the NR2B in cultures obtained from the adolescent rats. CGCs obtained from adolescent rats express functional NMDARs consistent with a developmental profile observed in vivo.

In vitro chondrocyte behavior on porous biodegradable poly(e-caprolactone)/polyglycolic acid scaffolds for articular chondrocyte adhesion and proliferation.

In this study, poly(e-caprolactone)/polyglycolic acid (PCL/PGA) scaffolds for repairing articular cartilage were fabricated via solid-state cryomilling along with compression molding and porogen leaching. Four distinct scaffolds were fabricated using this approach by four independent cryomilling times. These scaffolds were assessed for their suitability to promote articular cartilage regeneration with in vitro chondrocyte cell culture studies. The scaffolds were characterized for pore size, porosity, swelling ratio, compressive, and thermal properties. Cryomilling time proved to significantly affect the physical, mechanical, and morphological properties of the scaffolds. In vitro bovine chondrocyte culture was performed dynamically for 1, 7, 14, 28, and 35 days. Chondrocyte viability and adhesion were tested using MTT assay and scanning electron microscopy micrographs. Glycosaminoglycan (GAG) and DNA assays were performed to investigate the extracellular matrix (ECM) formation and cell proliferation, respectively. PCL/PGA scaffolds demonstrated high porosity for all scaffold types. Morphological analysis and poly(ethylene oxide) continuity demonstrated the existence of a co-continuous network of interconnected pores with pore sizes appropriate for tissue engineering and chondrocyte ingrowth. While mean pore size decreased, water uptake and compressive properties increased with increasing cryomilling times. Compressive modulus of 12, 30, and 60 min scaffolds matched the compressive modulus of human articular cartilage. Viable cells increased besides increase in cell proliferation and ECM formation with progress in culture period. Chondrocytes exhibited spherical morphology on all scaffold types. The pore size of the scaffold affected chondrocyte adhesion, proliferation, and GAG secretion. The results indicated that the 12 min scaffolds delivered promising results for applications in articular cartilage repair.

Role of helicase-like transcription factor (hltf) in the G2/m transition and apoptosis in brain.

HLTF participates in transcription, chromatin remodeling, DNA damage repair, and tumor suppression. Aside from being expressed in mouse brain during embryonic and postnatal development, little is known about Hltf's functional importance. Splice variant quantification of wild-type neonatal (6-8 hour postpartum) brain gave a ratio of 5:1 for Hltf isoform 1 (exons 1-25) to isoform 2 (exons 1-21 with exon 21 extended via a partial intron retention event). Western analysis showed a close correlation between mRNA and protein expression. Complete loss of Hltf caused encephalomalacia with increased apoptosis, and reduced viability. Sixty-four percent of Hltf null mice died, 48% within 12-24 hours of birth. An RNA-Seq snapshot of the neonatal brain transcriptome showed 341 of 20,000 transcripts were altered (p < 0.05) - 95 up regulated and 246 down regulated. MetaCore™ enrichment pathway analysis revealed Hltf regulates cell cycle, cell adhesion, and TGF-beta receptor signaling. Hltf's most important role is in the G2/M transition of the cell cycle (p  =  4.672e-7) with an emphasis on transcript availability of major components in chromosome cohesion and condensation. Hltf null brains have reduced transcript levels for Rad21/Scc1, histone H3.3, Cap-E/Smc2, Cap-G/G2, and Aurora B kinase. The loss of Hltf in its yeast Rad5-like role in DNA damage repair is accompanied by down regulation of Cflar, a critical inhibitor of TNFRSF6-mediated apoptosis, and increased (p<0.0001) active caspase-3, an indicator of intrinsic triggering of apoptosis in null brains. Hltf also regulates Smad7/Bambi/Tgf-beta/Bmp5/Wnt10b signaling in brain. ChIP confirmed Hltf binding to consensus sequences in predicted (promoter Scgb3a1 gene) and previously unidentified (P-element on chromosome 7) targets. This study is the first to provide a comprehensive view of Hltf targets in brain. Moreover, it reveals how silencing Hltf disrupts cell cycle progression, and attenuates DNA damage repair.

Helicase-like transcription factor (Hltf) regulates G2/M transition, Wt1/Gata4/Hif-1a cardiac transcription networks, and collagen biogenesis.

HLTF/Hltf regulates transcription, remodels chromatin, and coordinates DNA damage repair. Hltf is expressed in mouse brain and heart during embryonic and postnatal development. Silencing Hltf is semilethal. Seventy-four percent of congenic C57BL/6J Hltf knockout mice died, 75% within 12-24 hours of birth. Previous studies in neonatal (6-8 hour postpartum) brain revealed silencing Hltf disrupted cell cycle progression, and attenuated DNA damage repair. An RNA-Seq snapshot of neonatal heart transcriptome showed 1,536 of 20,000 total transcripts were altered (p < 0.05) - 10 up- and 1,526 downregulated. Pathway enrichment analysis with MetaCore™ showed Hltf's regulation of the G2/M transition (p=9.726E(-15)) of the cell cycle in heart is nearly identical to its role in brain. In addition, Brca1 and 12 members of the Brca1 associated genome surveillance complex are also downregulated. Activation of caspase 3 coincides with transcriptional repression of Bcl-2. Hltf loss caused downregulation of Wt1/Gata4/Hif-1a signaling cascades as well as Myh7b/miR499 transcription. Hltf-specific binding to promoters and/or regulatory regions of these genes was authenticated by ChIP-PCR. Hif-1a targets for prolyl (P4ha1, P4ha2) and lysyl (Plod2) collagen hydroxylation, PPIase enzymes (Ppid, Ppif, Ppil3) for collagen trimerization, and lysyl oxidase (Loxl2) for collagen-elastin crosslinking were downregulated. However, transcription of genes for collagens, fibronectin, Mmps and their inhibitors (Timps) was unaffected. The collective downregulation of genes whose protein products control collagen biogenesis caused disorganization of the interstitial and perivascular myocardial collagen fibrillar network as viewed with picrosirius red-staining, and authenticated with spectral imaging. Wavy collagen bundles in control hearts contrasted with collagen fibers that were thin, short and disorganized in Hltf null hearts. Collagen bundles in Hltf null hearts were tangled and fragmented. Thus, silencing Hltf during heart organogenesis compromised DNA double-strand break repair, and caused aberrant collagen biogenesis altering the structural network that transmits cardiomyocyte force into muscle contraction.

An in vitro model for studying the effects of continuous ethanol exposure on N-methyl-D-aspartate receptor function.

Long-term ethanol exposure has deleterious effects on both glial and neuronal function. We assessed alterations in both astrocytic and neuronal viability, and alterations in N-methyl-d-aspartate receptor (NMDAR) function, in cocultures of rat cerebellar granule cells (CGCs) and astrocytes after continuous ethanol exposure (CEE). Treatment of cells with 100 mM EtOH once every 24 h for 4 days resulted in a mean ethanol concentration of 57.3 ± 2.1 mM. Comparisons between control and post-ethanol-treated cells were made 4 days after the last ethanol treatment. CEE did not alter glial cell viability, as indicated by the absence of either changes in astrocytic morphology, actin depolymerization, or disruption of astrocytic intracellular mitochondrial distribution at any day postethanol treatment. The CGCs were healthy and viable after CEE, as indicated by phase-contrast microscopy and the trypan-blue exclusion method. Whole-cell patch-clamp experiments indicated that NMDA-induced currents (I(NMDA)) were altered by CEE treatment. Similar to previous results obtained during the withdrawal phase from chronic ethanol exposure, I(NMDA) from CEE-treated cells were significantly larger than I(NMDA) from NMDARs in control CGCs, but returned to control values by the fourth day post-CEE. However, after the last ethanol dosing and during a time when ethanol concentrations remained high, I(NMDA) were significantly smaller than control values. Identical results were observed in CGCs expressing the NR2A or NR2B subunit. In summary, both neurons and astrocytes remained healthy following exposure to CEE with no signs of neurotoxicity at the cellular level, and modulation of NMDAR function is consistent with findings from prior experiments. Thus, we conclude that the CEE paradigm in glial-neuronal cocultures readily lends itself to long-term in vitro studies of ethanol effects that include glial-neuronal interactions and the ability to study ethanol withdrawal-induced neurotoxicity.

Prolactin induces Jak2 phosphorylation of RUSHY195.

Jak2/RUSH-mediated prolactin signaling culminates in RUSH-1α-DNA-binding. Heretofore, Jak2-specific phosphorylation residues in RUSH were unknown. Genpathway's discovery approaches correlated RUSH-DNA binding (-126/-121) in uteroglobin's proximal promoter with recruitment of the transcriptional machinery. NetPhos 2.0 server found a single tyrosine phosphorylation site in RUSH's minimal DNA-binding domain. Y195 had identical context and prediction scores (0.52) for rabbit and human (HLTF) orthologs. The mouse ortholog (Hltf) had a higher prediction score (0.897). Affinity purified RUSHY195ph antibodies recognized native tyrosine phosphorylated RUSH protein immunoprecipitated from nuclear extracts. When R5020-treated HRE-H9 cells±the Jak2 inhibitor, Tyrene CR4, were stimulated with prolactin, confocal immunofluorescence images provided conclusive evidence that Jak2 mediated the availability of phosphorylated RUSHY195 in nucleus and cytoplasm. Catalytically active Jak2 is ipso facto a RUSH site-specific tyrosine kinase. Immunoprecipitation/Western blotting revealed both phosphorylation at Y195 and the physical interaction between p-Jak2/RUSH/HLTF/Hltf are evolutionarily conserved across three mammalian (rabbit, human, mouse) orthologs.

Prolactin-induced Jak2 phosphorylation of RUSH: a key element in Jak/RUSH signaling.

Jak2/Stat-mediated prolactin signaling culminates in Stat5a-DNA-binding. However, not all Jak2-dependent genes have Stat5 sites. Western analysis with inhibitors showed Jak2 is a proximal intermediate in prolactin-induced RUSH phosphorylation. Transfection assays with HRE-H9 cells showed the RUSH-binding site mediated the ability of prolactin to augment progesterone-dependent transcription of the RUSH gene. Jak2 inhibitors or targeted RUSH-site mutation blocked the prolactin effect. RUSH co-immunoprecipitated with phospho-Jak2 from nuclear extracts. Jak2 inhibitors abolished the nuclear pool of phospho-RUSH not the nuclear content of RUSH in HRE-H9 cells. Nucleolar-affiliated partners, e.g. nucleolin, were identified by microLC/MS/MS analysis of nuclear proteins that co-immunoprecipitated with RUSH/GST-RING. RUSH did not exclusively co-localize with fibrillarin to the nucleolus. MG-132 (proteasomal inhibitor) failed to block Tyrene CR4-mediated decrease in phospho-RUSH, and did not promote RUSH accumulation in the nucleolus. These studies authenticate prolactin-dependent Jak2 phosphorylation of RUSH, and provide functional implications on the RUSH network of nuclear interactions.

Actin depolymerization contributes to ethanol inhibition of NMDA receptors in primary cultured cerebellar granule cells.

We have previously reported that a 30s ethanol (10 and 100mM) pre-exposure significantly enhanced EtOH inhibition of N-methyl-d-aspartate (NMDA-induced currents)-induced peak currents in primary cultured cerebellar granule cells (CGCs). The purpose of this study was to determine if intracellular factors play a role in ethanol pre-exposure-enhanced inhibition of NMDA-induced currents and if so, to identify the intracellular target(s) mediating this effect. Ethanol pre-exposure-enhanced inhibition was reduced when ethanol was present intracellularly prior to the initiation of the pretreatment protocol. Similar to results acquired with the whole-cell configuration, ethanol pre-exposure-enhanced inhibition of NMDA-induced currents was also observed in the perforated patch-clamp mode. Collectively, these results suggest an intracellular target not easily dialyzed from the cell. Perturbation of the actin cytoskeleton was responsible for the ethanol pre-exposure-enhanced inhibition of NMDA-induced currents was supported by the observation that the intracellular presence of the actin stabilizer phalloidin prevented ethanol pre-exposure-enhanced inhibition. Similar to the effects of ethanol, the depolymerizing agent latrunculin A inhibited NMDA-induced currents after a 30s pretreatment exposure with full recovery of receptor function after washout of the drug. Furthermore, latrunculin A occluded the enhanced inhibition of NMDA-induced currents by ethanol pre-exposure for both 10 and 100mM ethanol. The microtubule depolymerizing agent taxol had no affect on ethanol pretreatment-enhanced inhibition of NMDA-induced currents. Confocal microscopy with phalloidin-FITC indicated that F-actin filaments in neurites were depolymerized after a 30s treatment of either latrunculin A or 100mM ethanol. Our observations indicate that ethanol inhibition of NMDAR function may involve perturbation of the actin cytoskeleton.