Neuronal Nsun2 deficiency produces tRNA epitranscriptomic alterations and proteomic shifts impacting synaptic signaling and behavior.

Epitranscriptomic mechanisms linking tRNA function and the brain proteome to cognition and complex behaviors are not well described. Here, we report bi-directional changes in depression-related behaviors after genetic disruption of neuronal tRNA cytosine methylation, including conditional ablation and transgene-derived overexpression of Nsun2 in the mouse prefrontal cortex (PFC). Neuronal Nsun2-deficiency was associated with a decrease in tRNA m5C levels, resulting in deficits in expression of 70% of tRNAGly isodecoders. Altogether, 1488/5820 proteins changed upon neuronal Nsun2-deficiency, in conjunction with glycine codon-specific defects in translational efficiencies. Loss of Gly-rich proteins critical for glutamatergic neurotransmission was associated with impaired synaptic signaling at PFC pyramidal neurons and defective contextual fear memory. Changes in the neuronal translatome were also associated with a 146% increase in glycine biosynthesis. These findings highlight the methylation sensitivity of glycinergic tRNAs in the adult PFC. Furthermore, they link synaptic plasticity and complex behaviors to epitranscriptomic modifications of cognate tRNAs and the proteomic homeostasis associated with specific amino acids.

RNA-seq analysis of amygdala tissue reveals characteristic expression profiles in schizophrenia.

The amygdala brain region has been implicated in the pathophysiology of schizophrenia through emotion processing. However, transcriptome messages in the amygdala of schizophrenia patients have not been well studied. We used RNA sequencing to investigate gene-expression profiling in the amygdala tissues, and identified 569 upregulated and 192 downregulated genes from 22 schizophrenia patients and 24 non-psychiatric controls. Gene functional enrichment analysis demonstrated that the downregulated genes were enriched in pathways such as 'synaptic transmission' and 'behavior', whereas the upregulated genes were significantly over-represented in gene ontology pathways such as 'immune response' and 'blood vessel development'. Co-expression-based gene network analysis identified seven modules including four modules significantly associated with 'synaptic transmission', 'blood vessel development' or 'immune responses'. Taken together, our study provides novel insights into the molecular mechanism of schizophrenia, suggesting that precision-tailored therapeutic approaches aimed at normalizing the expression/function of specific gene networks could be a promising option in schizophrenia.

Delayed emergence of behavioral and electrophysiological effects following juvenile ketamine exposure in mice.

Frequent ketamine abuse in adulthood correlates with increased risk of psychosis, as well as cognitive deficits, including disruption of higher-order executive function and memory formation. Although the primary abusers of ketamine are adolescents and young adults, few studies have evaluated its effects on juvenile cognition. Therefore, the current study analyzes the effect of adolescent ketamine exposure on cognitive development. Juvenile mice (4 weeks of age) were exposed to chronic ketamine (20 mg kg(-1), i.p. daily) for 14 days. Mice were tested immediately after exposure in the juvenile period (7 weeks of age) and again as adults (12 weeks of age). Measures included electroencephalography (EEG) in response to auditory stimulation, the social choice test, and a 6-arm radial water maze task. Outcome measures include low-frequency EEG responses, event-related potential (ERP) amplitudes, indices of social behavior and indices of spatial working memory. Juvenile exposure to ketamine was associated with electrophysiological abnormalities in adulthood, particularly in induced theta power and the P80 ERP. The social choice test revealed that ketamine-exposed mice failed to exhibit the same age-related decrease in social interaction time as controls. Ketamine-exposed mice outperformed control mice as juveniles on the radial water maze task, but did not show the same age-related improvement as adult controls. These data support the hypothesis that juvenile exposure to ketamine produces long-lasting changes in brain function that are characterized by a failure to progress along normal developmental trajectories.

Translational potential of olfactory mucosa for the study of neuropsychiatric illness.

The olfactory mucosa (OM) is a unique source of regenerative neural tissue that is readily obtainable from living human subjects and thus affords opportunities for the study of psychiatric illnesses. OM tissues can be used, either as ex vivo OM tissue or in vitro OM-derived neural cells, to explore parameters that have been difficult to assess in the brain of living individuals with psychiatric illness. As OM tissues are distinct from brain tissues, an understanding of the neurobiology of the OM is needed to relate findings in these tissues to those of the brain as well as to design and interpret ex vivo or in vitro OM studies. To that end, we discuss the molecular, cellular and functional characteristics of cell types within the olfactory mucosa, describe the organization of the OM and highlight its role in the olfactory neurocircuitry. In addition, we discuss various approaches to in vitro culture of OM-derived cells and their characterization, focusing on the extent to which they reflect the in vivo neurobiology of the OM. Finally, we review studies of ex vivo OM tissues and in vitro OM-derived cells from individuals with psychiatric, neurodegenerative and neurodevelopmental disorders. In particular, we discuss the concordance of this work with postmortem brain studies and highlight possible future approaches, which may offer distinct strengths in comparison to in vitro paradigms based on genomic reprogramming.

Src kinase as a mediator of convergent molecular abnormalities leading to NMDAR hypoactivity in schizophrenia.

Numerous investigations support decreased glutamatergic signaling as a pathogenic mechanism of schizophrenia, yet the molecular underpinnings for such dysregulation are largely unknown. In the post-mortem dorsolateral prefrontal cortex (DLPFC), we found striking decreases in tyrosine phosphorylation of N-methyl-D aspartate (NMDA) receptor subunit 2 (GluN2) that is critical for neuroplasticity. The decreased GluN2 activity in schizophrenia may not be because of downregulation of NMDA receptors as MK-801 binding and NMDA receptor complexes in postsynaptic density (PSD) were in fact increased in schizophrenia cases. At the postreceptor level, however, we found striking reductions in the protein kinase C, Pyk 2 and Src kinase activity that in tandem can decrease GluN2 activation. Given that Src serves as a hub of various signaling mechanisms affecting GluN2 phosphorylation, we postulated that Src hypoactivity may result from convergent alterations of various schizophrenia susceptibility pathways and thus mediate their effects on NMDA receptor signaling. Indeed, the DLPFC of schizophrenia cases exhibit increased PSD-95 and erbB4 and decreased receptor-type tyrosine-protein phosphatase-α (RPTPα) and dysbindin-1, each of which reduces Src activity via protein interaction with Src. To test genomic underpinnings for Src hypoactivity, we examined genome-wide association study results, incorporating 13 394 cases and 34 676 controls. We found no significant association of individual variants of Src and its direct regulators with schizophrenia. However, a protein-protein interaction-based network centered on Src showed significant enrichment of gene-level associations with schizophrenia compared with other psychiatric illnesses. Our results together demonstrate striking decreases in NMDA receptor signaling at the postreceptor level and propose Src as a nodal point of convergent dysregulations affecting NMDA receptor pathway via protein-protein associations.

Juvenile exposure to ketamine causes delayed emergence of EEG abnormalities during adulthood in mice.

BACKGROUND: Increased susceptibility to cognitive impairment or psychosis in adulthood is associated with adolescent drug abuse. Studies in adults have identified impairments in attention and memory, and changes in EEG, as common consequences of ketamine abuse. In contrast, the effects of ketamine on the juvenile brain have not been extensively tested. This is a significant omission, since abuse of ketamine is often observed within this age group. OBJECTIVES: Juvenile mice (4-6 weeks of age) were administered ketamine (20mg/kg) for 14 days. EEG was assessed in response to auditory stimulation both at one week following ketamine exposure at 7 weeks of age (juvenile) and again at 12 weeks of age (adult). EEG was analyzed for baseline activity, event-related power and event-related potentials (ERPs). RESULTS: While no effects of ketamine exposure were observed during the juvenile period, significant reductions in amplitude of the P20 ERP component and event-related gamma power were seen following ketamine when re-tested as adults. In contrast, reductions in event-related theta were seen in ketamine-exposed mice at both time points. CONCLUSIONS: Age related deficits in electrophysiological components such as P20 or event-related gamma may be due to an interruption of normal neural maturation. Reduction of NMDAR signaling during adolescence leads to delayed-onset disruption of gamma oscillations and the P20 component of the ERP. Further, delayed onset of impairment following adolescent ketamine abuse suggests that methods could be developed to detect and treat the early effects of drug exposure prior to the onset of disability.

Human olfactory epithelial cells generated in vitro express diverse neuronal characteristics.

The olfactory epithelium constitutes the sole source of regenerating neural cells that can be obtained from a living human. As such, primary cultures derived from human olfactory epithelial biopsies can be utilized to study neurobiological characteristics of individuals under different conditions and disease states. Here, using such human cultures, we report in vitro generation of cells that exhibit a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. Using in situ hybridization, we demonstrate for the first time the native expression of olfactory receptors in cultured cells derived from human olfactory epithelial tissue. We further establish the presence and function of olfactory transduction molecules in these cells using immunocytochemistry, calcium imaging and molecular methods. Western blot analysis revealed the expression of neurotransmitter receptors for dopamine (D2R), 5-HT (5HT2C) and NMDA subtypes 1 and 2A/2B. Stimulation with dopamine or 5-HT enhanced receptor G protein activation in a subtype specific manner, based on 35S-guanosine triphosphate incorporation assay. Functional characteristics of the cultured cells are demonstrated through enhanced tyrosine phosphorylation of NMDAR 2A/2B and recruitment of signaling partners in response to NMDA stimulation. The array of neuronal characteristics observed here establishes that proliferating cells derived from the human olfactory epithelium differentiate in vitro to express functional and molecular attributes of mature olfactory neurons. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders. Their ready availability from living humans thus provides a new tool to link functional and molecular features of neural cells with clinical characteristics of individual living patients.

Dysregulation of olfactory receptor neuron lineage in schizophrenia.

BACKGROUND: Growing evidence implicates abnormal neurodevelopment in schizophrenia. While neuron birth and differentiation is largely completed by the end of gestation, the olfactory epithelium (OE) is a unique part of the central nervous system that undergoes regeneration throughout life, thus offering an opportunity to investigate cellular and molecular events of neurogenesis and development postmortem. We hypothesized that OE neurons exhibit deviant progress through neurodevelopment in schizophrenia characterized by an increase in immature neurons. METHODS: Olfactory epithelium was removed at autopsy from 13 prospectively assessed elderly subjects who had schizophrenia and 10 nonpsychiatric control subjects. Sections were immunolabeled with antibodies that distinguish OE neurons in different stages of development, including basal cells (low-affinity nerve growth factor receptor, p75NGFR), postmitotic immature neurons (growth-associated protein 43 [GAP43]), and mature olfactory receptor neurons (olfactory marker protein). Absolute and relative densities of each cell type were determined. RESULTS: We observed a significantly lower density of p75NGFR basal cells (37%) in schizophrenia and increases in GAP43 + postmitotic immature neurons (316%) and ratios of GAP43 + postmitotic immature neurons to p75NGFR + cells (665%) and olfactory marker protein + mature neurons to p75NGFR + basal cells (328%). Neuroleptic-free schizophrenia subjects exhibited the highest GAP43 + postmitotic immature neuron values. CONCLUSIONS: Abnormal densities and ratios of OE neurons at different stages of development indicate dysregulation of OE neuronal lineage in schizophrenia. This could be because of intrinsic factors controlling differentiation or an inability to gain trophic support from axonal targets in the olfactory bulb. While caution is necessary in extrapolating developmental findings in mature OE to early brain development, similarities in molecular events suggest that such studies may be instructive.

Characteristics of odorant elicited calcium changes in cultured human olfactory neurons.

An important step in establishing and utilizing a cell culture system for the in vitro study of olfaction is assessing whether the cultured cells possess physiological properties similar to those of mature olfactory neurons. Various investigators have successfully established proliferating cell lines from olfactory tissue, but few have demonstrated the characteristics of odor sensitivity of these cells. We successfully established cultured cell lines from adult human olfactory tissue obtained using an olfactory biopsy procedure and measured their ability to respond to odor stimulation using calcium imaging techniques. A subset of the human olfactory cells in culture displayed a distinct morphology and specifically expressed immunocytochemical markers characteristic of mature human olfactory neurons such as OMP, G(olf), NCAM and NST. Under defined growth conditions, these cultured cells responded to odorant mixes that have been previously shown to elicit intracellular calcium changes in acutely-isolated human olfactory neurons. These odorant-elicited calcium responses displayed characteristics similar to those found in mature human olfactory neurons. First, cultured cells responded with either increases or decreases in intracellular calcium. Second, increases in calcium were abolished by removal of extracellular calcium. Third, inhibitors of the olfactory signal transduction cascades reversibly blocked these odorant-elicited intracellular calcium changes. Our results demonstrate that cultures of adult human olfactory cells established from olfactory biopsies retain some of the in vivo odorant response characteristics of acutely isolated cells from the adult olfactory epithelium. This work has important ramifications for investigation of olfactory function and dysfunction using biopsy procedures and in vitro assays of odor sensitivity.

Overview of the mechanism of action of lithium in the brain: fifty-year update.

Since its discovery, lithium has been shown to act upon various neurotransmitter systems at multiple levels of signaling in the brain. Lithium, affecting each neurotransmitter system within complex interactive neuronal networks, is suggested to restore the balance among aberrant signaling pathways in critical regions of the brain. Recent molecular studies have revealed the action of lithium on signal transduction mechanisms, such as phosphoinositide hydrolysis, adenylyl cyclase, G protein, glycogen synthase kinase-3beta, protein kinase C, and its substrate myristoylated alanine-rich C kinase substrate. Such effects are thought to trigger long-term changes in neuronal signaling patterns that account for the prophylactic properties of lithium in the treatment of bipolar disorder. Through its effects on glycogen synthase kinase-3beta and protein kinase C, lithium may alter the level of phosphorylation of cytoskeletal proteins, which leads to neuroplastic changes associated with mood stabilization. Chronic lithium regulates transcriptional factors, which in turn may modulate the expression of a variety of genes that compensate for aberrant signaling associated with the pathophysiology of bipolar disorder. Future studies on long-term neuroplastic changes caused by lithium in the brain will set the stage for new drug-discovery opportunities.

Serotonin transporter gene polymorphism and antidepressant response.

We examined allelic polymorphisms of the serotonin transporter (5-HTT) gene and antidepressant response to 6 weeks' treatment with the selective serotonin reuptake inhibitor (SSRI) drugs fluoxetine or paroxetine. We genotyped 120 patients and 252 normal controls, using polymerase chain reaction of genomic DNA with primers flanking the second intron and promoter regions of the 5-HTT gene. Diagnosis of depression was not associated with 5-HTT polymorphisms. Patients homozygous l/l in intron 2 or homozygous s/s in the promoter region showed better responses than all others (p < 0.0001, p = 0.0074, respectively). Lack of the l/l allele form in intron 2 most powerfully predicted non-response (83.3%). Response to SSRI drugs is related to allelic variation in the 5-HTT gene in depressed Korean patients.

Prevalence of gallstones in a Brazilian population.

The objective of the present study was to determine the prevalence of gallstones in the population of Curitiba, Brazil. A total of 1000 persons was randomly recruited among individuals who were visiting two shopping centers of the city in order to represent the Brazilian population in relation to age and sex. The selected people underwent ultrasonographic examination of the upper abdomen immediately after a medical interview. Of the 1000 persons evaluated, 93 (9.3%) had gallstones (64 persons) or had been subjected to cholecystectomy due to cholelithiasis. The gallstone prevalence increased from 2.4% in persons of 20-29 years of age to 27.5% in persons of more than 70 years (chi2 = 37.29; P <0.001). The prevalence was 2.4 greater in females (12.9%) than in males (5.4%) (chi2 = 16.34; P <0.001). The prevalence increased with the number of pregnancies from 4% in nulliparous women, to 34.6% in persons with a history of six or more pregnancies (chi2 = 200.1; P <0,001). The prevalence also increased according to the body weight (chi2 = 30.08; P <0.001). There was no difference in the prevalence between individuals with diabetes mellitus and controls. It is concluded from this study that the prevalence of gallstones in the city of Curitiba is elevated.

Differential expression of thyroid hormone receptor isoforms by thyroid hormone and lithium in rat GH3 and B103 cells.

BACKGROUND: The interaction between lithium, a mood stabilizer, and the thyroid axis has been extensively studied; however, the regulation of thyroid hormone receptors by lithium is yet to be investigated. METHODS: To test whether lithium affects thyroid hormones at the receptor level, we examined the effects of lithium in combination with triiodothyronine (T3) on gene expression of thyroid hormone receptor isoforms in GH3 and B103 cells. RESULTS: The pattern of expression as well as the magnitude of regulation of the different thyroid hormone receptor isoforms appeared to be cell line specific. Whereas T3 regulated all four isoforms in GH3 cells at both time points, T3 did not alter thyroid hormone receptor TR alpha 1 and TR alpha 2 mRNA in B103 cells. Addition of lithium to thyroid hormone-deficient GH3 cells decreased TR alpha 1, alpha 2, and beta 2 expression without affecting TR beta 1 expression at 2 but not 5 days. Addition of lithium to T3-treated GH3 cells did not further modulate gene expression of TR alpha 1, alpha 2, beta 1, or beta 2 when compared to cells treated with T3 alone. The effects of lithium in B103 cells appeared to be isoform specific as well as time dependent, since TR alpha 1 expression was selectively decreased in B103 cells, when treated with T3 in the presence of lithium. CONCLUSIONS: The present study provides direct evidence that T3 and/or lithium regulate TR gene expression in vitro in a both time-dependent and cell line-specific manner.

Lithium administration affects gene expression of thyroid hormone receptors in rat brain.

Even though lithium has received wide attention in the treatment of manic depressive illness, the mechanisms underlying its mood stabilizing effects are not understood. Lithium is known to interact with the thyroid axis and causes hypothyroidism in a subgroup of patients, which compromises its mood stabilizing effects. Since lithium was recently reported to alter thyroid hormone metabolism in the rat brain, the present study investigated whether these effects were mediated through regulation of thyroid hormone receptor (THR) gene expression. Adult male euthyroid rats were either given a diet containing 0.25% lithium or one without lithium for 14 days. Rats were sacrificed in the evening and RNA was isolated from different brain regions to quantitate the isoform specific mRNAs of THRs. Following 14 days of lithium treatment, THR alpha1 mRNA levels were increased in the cortex and decreased in hypothalamus; THR alpha2 mRNA levels were increased in the cortex and THR beta mRNA levels were decreased in the hypothalamus. No significant difference in the expression of these THR isoforms was observed in the hippocampus or cerebellum. Thus, chronic lithium treatment appeared to regulate THR gene expression in a subtype and region specific manner in the rat brain. It remains to be determined whether the observed effects of lithium on THR gene expression are related to its therapeutic efficacy in the treatment of bipolar disorder.

Abnormalities in protein kinase C signaling and the pathophysiology of bipolar disorder.

Protein kinase C (PKC) is a group of calcium and phospholipid-dependent enzymes, which plays a pivotal role in cell signaling systems. Recently accumulated evidence indicates that alterations in PKC activity play a significant role in the pathophysiology of bipolar disorder. A number of laboratories investigated the effect of mood stabilizers on the regulation of PKC activity in bipolar patients, in animals, and in cultured cells. Following chronic lithium treatment, PKC activation was significantly reduced in rat brains, as measured by the translocation of cytoplasmic PKC to the membrane compartment, or by quantitative binding of the PKC ligand, PDBu. The effect of the therapeutic concentration of lithium in attenuating PKC-dependent intracellular parameters was also demonstrated in cultured cells. More importantly, alterations in platelet PKC was shown in bipolar patients during the manic state of the illness. In comparison to patients with major depressive disorder, schizophrenia, or healthy controls, PKC activity was significantly increased in manic patients, suggesting that changes in PKC may be an illness-specific marker. Interestingly, enhanced PKC activity during mania was suppressed following mood-stabilizer treatment as manic symptoms improved. In parallel to the findings in platelets, postmortem studies demonstrate that membrane-associated PKC and stimulation-induced translocation of cytosolic enzyme to the membrane were also increased in frontal cortex of bipolar patients. Other studies suggest alterations in other signal transduction mechanisms in bipolar disorder. These include alterations in G protein activation, phosphatidylinositol (PI) signaling, cyclic AMP formation, and intracellular calcium homeostasis. The alterations of PKC activity in bipolar disorder may be related to changes in these other intracellular signaling mechanisms. Alternatively, the changes of PKC activity may be the core pathology of the illness. More studies are required to further characterize the association of PKC changes with bipolar disorder, using a proper neuronal model.

Seroepidemiology of Rickettsia typhi, spotted fever group rickettsiae, and Coxiella burnetti infection in pregnant women from urban Tanzania.

Immunofluorescent antibody (IFA) testing was performed on sera drawn from 150 pregnant women in the port city of Dar es Salaam, Tanzania. Prevalence of antibodies to Rickettsia typhi was 28%, higher than in any of the 12 other African countries in which serosurveys using IFA testing have been performed. Seroprevalence of antibodies to spotted fever group rickettsiae antigens was 25.3%, comparable with that found in other sub-Saharan countries endemic for Amblyomma ticks. Only 4.7% of women were seropositive for Coxiella burnetii.

Inositol 1,4,5-trisphosphate receptor expression in mammalian olfactory tissue.

Two cDNAs encoding inositol 1,4,5-trisphosphate (IP3) receptors were amplified from rat olfactory tissue, and both exhibited 100% sequence identity to the short (Segment II - ) variant of type I IP3 receptor. Type III IP3 receptor was also expressed in olfactory tissue. The distribution of IP3 receptors included the olfactory epithelium, lamina propria, and glandular tissue. These results demonstrate the co-expression of multiple IP3 receptor subtypes in olfactory cells, and suggest multiple functions for IP3 receptors in this tissue.

Neural regulation of N-cadherin gene expression in developing and adult skeletal muscle.

Using monoclonal antibody and cDNA probes, we have studied N-cadherin gene expression in developing and adult chick skeletal muscle. N-cadherin was expressed by developing myotubes during the period of initial nerve-muscle contact but was downregulated within days of innervation. Treatment of embryos with d-tubocurare partially reversed this downregulation. In the adult, there were muscle fiber type differences in N-cadherin expression. N-cadherin was undetectable on normally innervated twitch fibers, while multiply innervated tonic muscle fibers expressed low but readily detectable levels of N-cadherin. Denervation led to the renewed expression of N-cadherin in twitch fibers as well as a marked increase in expression in tonic fibers. Levels of N-cadherin expressed by tonic fibers could also be modulated by animal housing conditions that favored either increased or decreased levels of daily motor activity. Increased motor activity was correlated with decreased levels of N-cadherin, while decreased motor activity correlated with increased levels of muscle N-cadherin. Results of in vitro studies using the calcium channel agonist ryanodine suggest that changes in intracellular calcium may be the initial signal linking neural stimulation with changes in muscle fiber expression of N-cadherin. Together, our results indicate that neural stimulation of chick skeletal muscle fibers dynamically and reversibly downregulates the expression of N-cadherin mRNA and protein. This pattern of regulation may be functionally important in limiting and/or promoting axon growth in innervated versus denervated muscle and may serve as a molecular model system for studies of the activity-dependent regulation of gene expression.

Isolation of transcriptionally active nuclei from striated muscle using Percoll density gradients.

A procedure is described utilizing Percoll density media for the separation of nuclei and myofibrils in homogenates of adult skeletal muscle. Using this method, transcriptionally active nuclei can be readily obtained in relatively high yield (approximately 30%). In vitro RNA polymerase run-on-labeled RNAs isolated from these nuclei can be used in hybridization assays to study the transcriptional activities of specific genes. Percoll density gradient centrifugation should be useful for the isolation of nuclei from a variety of other tissues in which, like skeletal muscle, subcellular or tissue components cosediment with nuclei in conventional sucrose density centrifugation.