Exosomes induce neurogenesis of pluripotent P19 cells.

Exosomes play a role in tissue/organ development and differentiation. Retinoic acid induces differentiation of P19 cells (UD-P19) to P19 neurons (P19N) that behave like cortical neurons and express characteristic neuronal genes such as NMDA receptor subunits. Here we report P19N exosome-mediated differentiation of UD-P19 to P19N. Both UD-P19 and P19N released exosomes with characteristic exosome morphology, size, and common protein markers. P19N internalized significantly higher number of Dil-P19N exosomes as compared to UD-P19 with accumulation in the perinuclear region. Continuous exposure of UD-P19 to P19N exosomes for six days induced formation of small-sized embryoid bodies that differentiated into MAP2-/GluN2B-positive neurons recapitulating RA-induction of neurogenesis. Incubation with UD-P19 exosomes for six days did not affect UD-P19. Small RNA-seq identified enrichment of P19N exosomes with pro-neurogenic non-coding RNAs (ncRNAs) such as miR-9, let-7, MALAT1 and depleted with ncRNAs involved in maintenance of stem cell characteristics. UD-P19 exosomes were rich with ncRNAs required for maintenance of stemness. P19N exosomes provide an alternative method to genetic modifications for cellular differentiation of neurons. Our novel findings on exosomes-mediated differentiation of UD-P19 to P19 neurons provide tools to study pathways directing neuron development/differentiation and develop novel therapeutic strategies in neuroscience.

Small but Mighty-Exosomes, Novel Intercellular Messengers in Neurodegeneration.

Exosomes of endosomal origin are one class of extracellular vesicles that are important in intercellular communication. Exosomes are released by all cells in our body and their cargo consisting of lipids, proteins and nucleic acids has a footprint reflective of their parental origin. The exosomal cargo has the power to modulate the physiology of recipient cells in the vicinity of the releasing cells or cells at a distance. Harnessing the potential of exosomes relies upon the purity of exosome preparation. Hence, many methods for isolation have been developed and we provide a succinct summary of several methods. In spite of the seclusion imposed by the blood-brain barrier, cells in the CNS are not immune from exosomal intrusive influences. Both neurons and glia release exosomes, often in an activity-dependent manner. A brief description of exosomes released by different cells in the brain and their role in maintaining CNS homeostasis is provided. The hallmark of several neurodegenerative diseases is the accumulation of protein aggregates. Recent studies implicate exosomes' intercellular communicator role in the spread of misfolded proteins aiding the propagation of pathology. In this review, we discuss the potential contributions made by exosomes in progression of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Understanding contributions made by exosomes in pathogenesis of neurodegeneration opens the field for employing exosomes as therapeutic agents for drug delivery to brain since exosomes do cross the blood-brain barrier.

Urea can inhibit efficient reduction and alkylation of protein dimers in solution demonstrated by the beta subunit of alpha glucosidase II.

Protein reduction and alkylation is routinely used for analysis of protein dimers and protein complexes in cell fractions using two dimensional gel electrophoresis and mass spectrometry. To resolve the heterogeneity of a high molecular weight protein band that is highlighted by an antibody to the beta subunit of alpha glucosidase II (GIIß), we performed reduction and alkylation of cytosolic proteins extracted from mouse brain. The presence of urea in the reduction/alkylation buffer inhibited the chemical processes. It is thus recommended that protein reduction/alkylation be performed both in the presence and absence of urea for the separation of mono-/hetero-mers.

Prior alcohol consumption does not impair go/no-go discrimination learning, but causes over-responding on go trials, in rats.

Prior alcohol use is associated with impaired response inhibition in humans, including in laboratory go/no-go discrimination tasks. In two experiments, we determined whether chronic intermittent access to alcohol would alter go/no-go discrimination learning. Rats received 4-6 weeks of chronic intermittent access to 20% alcohol (alone or accompanied by saline or 1.5g/kg alcohol injections) or water. Rats then began discrimination training 4-5days after the end of the alcohol access. Each lever was available for 40s with one lever intermittently reinforced ("active lever") and the other lever non-reinforced ("inactive lever"). The rats given access to alcohol without concurrent alcohol injections drank ∼10g/kg/24-h on average during the last three weeks of alcohol access. The groups given alcohol injections (Alcohol+Injection groups) exhibited suppressed drinking, but the Alcohol+Injection groups exhibited higher blood alcohol spikes than all other alcohol groups (195 vs. 85-90mg/dl, respectively). We found no evidence for impaired go/no-go discrimination learning in either experiment. However, the alcohol access groups with moderate-to-high average alcohol consumption (>3g/kg/24-h) exhibited over-responding to the active lever compared to the water-only groups. One group given alcohol injections (Alcohol+Injection group) that exhibited very low voluntary drinking (<1g/kg/24-h) did not exhibit the over-responding effect, suggesting that the total 24-h alcohol dose matters more than short-lived blood alcohol spikes. Our findings are in accord with previous research showing that repeated alcohol withdrawal causes over-responding for responses that lead to limited reinforcement. Future work will determine the psychological and neurobiological basis of this behavioral change.

Guardian of Genetic Messenger-RNA-Binding Proteins.

RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

Expression of α-subunit of α-glucosidase II in adult mouse brain regions and selected organs.

α-Glucosidase II (GII), a resident of endoplasmic reticulum (ER) and an important enzyme in the folding of nascent glycoproteins, is heterodimeric, consisting of α (GIIα) and ß (GIIß) subunits. The catalytic GIIα subunit, with the help of mannose 6-phosphate receptor homology domain of GIIß, sequentially hydrolyzes two α1-3-linked glucose residues in the second step of N-linked oligosaccharide-mediated protein folding. The soluble GIIα subunit is retained in the ER through its interaction with the HDEL-containing GIIß subunit. N-glycosylation and correct protein folding are crucial for protein stability and trafficking and cell surface expression of several proteins in the brain. Alterations in N-glycosylation lead to abnormalities in neuronal migration and mental retardation, various neurodegenerative diseases, and invasion of malignant gliomas. Inhibitors of GII are used to inhibit cell proliferation and migration in a variety of different pathologies, such as viral infection, cancer, and diabetes. Despite the widespread use of GIIα inhibitory drugs and the role of GIIα in brain function, little is known about its expression in brain and other tissues. Here, we report generation of a highly specific chicken antibody to the GIIα subunit and its characterization by Western blotting and immunoprecipitation using cerebral cortical extracts. By using this antibody, we showed that the GIIα protein is highly expressed in testis, kidney, and lung, with the lowest amount in heart. GIIα polypeptide levels in whole brain were comparable to those in spleen. However, a higher expression of GIIα protein was detected in the cerebral cortex, reflecting its continuous requirement in correct folding of cell surface proteins.

A cis-acting region in the N-methyl-d-aspartate R1 3'-untranslated region interacts with the novel RNA-binding proteins beta subunit of alpha glucosidase II and annexin A2--effect of chronic ethanol exposure in vivo.

A cis-acting region, Δ4, located in the 3'-untranslated region of N-methyl-d-aspartate R1(NR1) mRNA interacts with several trans-acting proteins present in polysomes purified from fetal cortical neurons. Chronic ethanol exposure of fetal cortical neurons increases Δ4 RNA-protein interactions. This increased interaction is due to an increase in one of the Δ4-binding trans-acting proteins identified as beta subunit of alpha glucosidase II (GIIß). In this study, we examined whether ethanol-mediated regulation of NR1 mRNA in vivo is similar to that in vitro and whether Δ4-trans interactions are important for ethanol-mediated NR1 mRNA stability. Our data show that polysomal proteins from adult mouse cerebral cortex (CC) formed a complex with Δ4 RNA, suggesting the presence of NR1 mRNA-binding trans-acting proteins in CC polysomes. The intensity of the Δ4 RNA-protein complex was increased with polysomes from chronic ethanol-exposed CC. The Δ4 RNA-protein complex harbored GIIß and a second trans-acting protein identified as annexin A2 (AnxA2). Ethanol-sensitive GIIß was upregulated by 70% in ethanol-exposed CC. Heparin, a known binding partner of AnxA2, inhibited Δ4 RNA-protein complex formation. Transient transfection studies using chimeric constructs with and without the Δ4 region revealed that cis-trans interactions are important for ethanol-mediated stability of NR1 mRNA. Furthermore, our data highlight, for the first time, the presence of a binding site on the 3'-untranslated region of NR1 mRNA for AnxA2 and demonstrate the regulation of NR1 mRNA by AnxA2, GIIß and a third NR1 mRNA-binding protein, which is yet to be identified.

Differentiated P19 cells express N-methyl-D-aspartate receptor 1 mRNA binding trans-acting proteins and four N-methyl-D-aspartate receptor 1 splice variants comparable to those in cultured fetal cortical neurons.

Differentiated P19 cells naturally express N-methyl-D-aspartate (NMDA) receptors and serve as a good in vitro model system with which to study NMDA receptor regulation. Here we examined expression of NR1 mRNA binding trans-acting proteins and NR1 splice variants in P19 cells. After exposure to retinoic acid, P19 cells were differentiated for 2, 4, 6, and 8 days in vitro (DIV). Total RNA and protein extracts from differentiated P19 cells were utilized to examine NR1 and NR2B expression. A steady increase in NR1 and NR2B mRNA and protein levels was observed with respect to days of differentiation. NR2B mRNA was detected within 2 DIV. However, NR2B protein appeared only at 4 DIV. By contrast, minimal expression of NR1 mRNA could be detected in undifferentiated P19 cells, whereas NR1 protein was detected at 4 DIV. RT-PCR analysis identified expression of four of eight full-length NR1 splice variants, similar to the expression pattern seen in fetal cortical neurons (FCN). These data were confirmed by ribonuclease protection assays. RNA gel shift assays and Northwestern analysis revealed the expression of NR1 mRNA binding trans-acting proteins in P19 neurons comparable to those expressed in FCN. RNA super gel shift assays confirmed the presence of the NR1 mRNA binding trans-acting protein GIIbeta in the NR1-3'UTR-P19 protein complex. Levels of GIIbeta polypeptide increased with increase in days of differentiation. Taken together, our data demonstrate that differentiated P19 cells are comparable to FCN and hence provide an excellent in vitro model for studying NR1 mRNA regulation at the posttranscriptional level.

Supplementing the liquid alcohol diet with chow enhances alcohol intake in C57 BL/6 mice.

BACKGROUND: Administration of alcohol-containing liquid diet is associated with body weight loss in rodents. AIM: The aim of this study was to modify the alcohol-containing liquid diet paradigm to reduce the body weight loss in mice during the alcohol treatment period. METHODS: Two sets of animals (Chow and No Chow groups) were exposed to chronic alcohol with a step-wise increase of alcohol in the diet. One set of control and alcohol exposed animals (Chow group) received chow during alcohol treatment. Food intake and body weight was measured every 24 h. Level of intoxication was determined by measuring blood alcohol levels. Alcohol dependence of mice was determined by handling-induced convulsions (HICs) scoring. Chronic alcohol-mediated effects on brain and liver were examined. RESULTS: Body weight loss was attenuated in chronic alcohol exposed mice in Chow group as compared to No Chow group. Chow group mice consumed higher amounts of alcohol diet resulting in higher blood alcohol levels. Brain NMDA R1 protein levels and liver Cyp 2E1 levels were significantly enhanced in chronic alcohol exposed mice in Chow and No Chow groups suggesting that known medical consequences of alcohol are not interfered with in our modified alcohol treatment paradigm. HIC in Chow and No Chow group mice peaked between 3 and 5 h after alcohol withdrawal. However, the severity of alcohol withdrawal was greater in Chow group mice. CONCLUSIONS: Supplementing alcohol diet with chow not only attenuated body weight loss associated with alcohol intake in mice but also resulted in higher consumption of alcohol diet and higher blood alcohol levels.

A novel RNA binding protein that interacts with NMDA R1 mRNA: regulation by ethanol.

Excitatory NMDA receptors are an important target of ethanol. Chronic ethanol exposure, in vivo and in vitro, increases polypeptide levels of NR1 subunit, the key subunit of functional NMDA receptors. In vitro, chronic ethanol treatment increases the half-life of NR1 mRNA and this observation is dependent on new protein synthesis. The present study was undertaken to locate cis-acting region(s) within the NR1 3'-untranslated region (UTR) and identify NR1 3'-UTR binding trans-acting proteins expressed in mouse fetal cortical neurons. Utilizing RNA gel shift assays we identified a 156-nt cis-acting region that binds to polysomal trans-acting proteins. This binding was highly specific as inclusion of cyclophilin RNA or tRNA did not interfere with cis-trans interactions. Importantly, the 3'-UTR binding activity was significantly up-regulated in the presence of ethanol. UV cross-link analysis detected three NR1 3'-UTR binding proteins and their molecular mass calculated by Northwestern analysis was approximately 88, 60 and 47 kDa, respectively. Northwestern analysis showed a significant up-regulation of the 88-kDa protein after chronic ethanol treatment. The 88-kDa protein was purified and identified by tandem mass spectrometry as the beta subunit of alpha glucosidase II (GIIbeta). That GIIbeta is indeed a trans-acting protein and binds specifically to 3'-UTR of NR1 mRNA was confirmed by RNA gel mobility supershift assays and immuno RT-PCR. Western blotting data established a significant increase of GIIbeta polypeptide in chronic ethanol-exposed fetal cortical neurons. We hypothesize that the identified cis-acting region and the associated RNA-binding proteins are important regulators of NR1 subunit gene expression.

An old story with a new twist: do NMDAR1 mRNA binding proteins regulate expression of the NMDAR1 receptor in the presence of alcohol?

NMDA receptors not only play a pivotal role in normal physiological processes in the central nervous system (CNS), but have been identified as an important target of ethanol. Chronic exposure to ethanol induces a number of adaptive processes in the CNS, including an upregulation of NMDA receptor number and function. The increase in NMDA receptor number in response to chronic ethanol exposure both in vivo and in vitro is accompanied by an increase in NMDAR1 and NMDAR2B polypeptide levels. It is widely believed that these adaptive changes play an important role in the development of alcohol dependence and withdrawal syndrome. At the molecular level, chronic ethanol exposure of fetal cortical neurons selectively increases expression of NMDAR1 splice variants lacking exon 5 and exon 22. Chronic ethanol exposure of fetal cortical neurons also increases NMDAR1 mRNA half-life in these neurons. However, when new protein synthesis is inhibited, the half-life of NR1 mRNA in these neurons returns to control values, strongly suggesting that ethanol induces the synthesis of protein(s) that may regulate the decay of NR1 mRNA. In recent years, it has become apparent that regulation of mRNA stability is an important aspect of regulation of gene expression. Changes in mRNA stability can be accomplished by interaction between cis-acting sequences in the 3' untranslated region (3'UTR) of mRNAs and trans-acting proteins expressed in cells. Such interactions may protect RNAs from degradation by ribonucleases, thereby increasing the half-life of mRNAs.

The molecular effects of alcohol: clues to the enigmatic action of alcohol.

Chronic ethanol treatment (50 mM, five days) induces stabilization of NR1 receptor subunit mRNA in cultured fetal cortical neurons. In this paper, we investigate the mechanism(s) by which ethanol mediates its effects on NR1 mRNA. Specifically, we have determined if cellular localization of NR1 mRNA in cortical neurons and/or de novo protein synthesis is essential for ethanol-mediated stabilization of NR1 mRNA. Subcellular fractionation studies show that all detectable NR1 mRNA is associated with rough endoplasmic reticulum, indicating that subcellular distribution of NR1 mRNA in fetal cortical neurons does not play a role in ethanol-mediated NR1 mRNA stabilization. However, inhibition of protein synthesis by cycloheximide abolished the mRNA stabilizing effect of ethanol on NR1 mRNA, thus suggesting de novo protein synthesis is crucial for the action of ethanol on NR1 mRNA stabilization.

Effect of ethanol on lipid-mediated transfection of primary cortical neurons.

Successful introduction of nucleic acids into mammalian neurons has revolutionized the analyses of gene regulation and cellular function. Various methods, including viral infection, have been developed to introduce plasmid DNA into primary neuronal cultures. However, transfection of primary cultures of neurons using the calcium phosphate precipitation method and electroporation have been comparatively inefficient. In this paper, we describe a method to successfully transfect cultured fetal cortical neurons using a cationic lipid reagent, lipofectamine. Cells were cultured in the absence and presence of 50 mM ethanol. To monitor transfection of neurons, we employed three mammalian expression vectors containing Renilla luciferase and/or firefly luciferase, or the beta-galactosidase reporter gene. Fetal cortical neurons were isolated and cultured in the absence or presence of 50 mM ethanol, for two days. On day 3, neurons were washed, fed with serum-free medium, and transfected with the DNA-lipofectamine complex. After two hours, cells were washed, fed complete medium lacking or containing 50 mM ethanol and cultured for two additional days with a change of medium after 24 h. Cultures were terminated 48 h after transfection. Cells were either stained for beta-galactosidase activity using X-gal or lysed to prepare cell extracts to assay for luciferase activity using a luminometer. When neurons were cotransfected, Renilla luciferase was used as an internal control to normalize the expression of the firefly luciferase reporter gene. Analysis of results showed that expression of the reporter gene, firefly luciferase, was approximately 2.5 times greater in ethanol treated neuronal cultures than for neurons cultured in the absence of ethanol. An increased number of neurons expressing beta-galactosidase was also observed in ethanol-treated neurons. These data suggest that perhaps ethanol treatment of fetal cortical neurons improved the DNA uptake and/or increased the expression of the reporter genes.