Predictors and moderators of burden of care and emotional distress in first-episode psychosis caregivers: results from the GET UP pragmatic cluster randomised controlled trial.

AIMS: First-episode psychosis (FEP) is a major life event and can have an adverse impact on the diagnosed individual and their families. The importance of intervening early and providing optimal treatments is widely acknowledged. In comparison to patient groups, literature is scarce on identifying treatment predictors and moderators of caregiver outcomes. This study aimed to identify pre-treatment characteristics predicting and/or moderating carer outcomes, based on data from a multi-element psychosocial intervention to FEP patients and carers (GET-UP PIANO trial). METHODS: Carer demography, type of family relationship, patient contact hours, pre-treatment carer burden, patient perceptions of parental caregiving and expressed emotion (EE) were selected, a priori, as potential predictors/moderators of carer burden and emotional distress at 9 months post treatment. Outcomes were analysed separately in mixed-effects random regression models. RESULTS: Analyses were performed on 260 carers. Only patient perceptions of early maternal criticism predicted reports of lower carer burden at follow-up. However, multiple imputation analysis failed to confirm this result. For treatment moderators: higher levels of carer burden at baseline yielded greater reductions in carer emotional distress at follow-up in the experimental group compared with treatment as usual (TAU). Higher levels of perceived EE moderated greater reductions in carer reports of tension in experimental group, compared with TAU, at follow-up. In younger caregivers (<51 years old), there were greater reductions in levels of worry during the baseline to follow-up period, within the experimental group compared with TAU. CONCLUSION: The study failed to identify significant treatment predictors of FEP carer outcomes. However, our preliminary findings suggest that optimal treatment outcomes for carers at first episode might be moderated by younger carer age, and carers reporting higher baseline levels of burden, and where patients perceive higher levels of negative effect from caregivers.

5-Lipoxygenase and epigenetic DNA methylation in aging cultures of cerebellar granule cells.

5-Lipoxygenase (5-Lox), an enzyme involved in the metabolism of arachidonic acid participates in the modulation of the proliferation and differentiation of neural stem cells and cerebellar granule cell (CGC) precursors. Since epigenetic mechanisms including DNA methylation regulate 5-LOX expression and have been suggested as possible modulators of stem cell differentiation and aging, using primary cultures of mouse CGC (1, 5, 10, 14, 30 days in vitro; DIV), we studied DNA methylation patterns of the 5-LOX promoter and 5-LOX mRNA levels. We also measured the mRNA and protein content of the DNA methyltransferases DNMT1 and DNMT3a. 5-LOX, DNMT1, and DNMT3a mRNA levels were measured by real-time PCR. We observed that 5-LOX expression and the expression of maintenance DNMT1 is maximal at 1 DIV (proliferating neuronal precursors), whereas the expression of the de novo DNA methyltransferase DNMT3a mRNA increased in aging cultures. We analyzed the methylation status of the 5-LOX promoter using the methylation-sensitive restriction endonucleases AciI, BstUI, HpaII, and HinP1I, which digest unmethylated CpGs while leaving methylated CpGs intact. The 5-LOX DNA methylation increased with the age of the cells. Taken together, our data show that as cultured CGC mature and age in vitro, a decrease in 5-LOX mRNA content is accompanied by an increase in the methylation of the gene DNA. In addition, an increase in DNMT3a but not DNMT1 expression accompanies an increase of 5-LOX methylation during in vitro maturation.

Dopamine receptor-mediated regulation of neuronal "clock" gene expression.

Using a transgenic mice model (i.e. "clock" knockouts), clock transcription factors have been suggested as critical regulators of dopaminergic behaviors induced by drugs of abuse. Moreover, it has been shown that systemic administration of psychostimulants, such as cocaine and methamphetamine regulates the striatal expression of clock genes. However, it is not known whether dopamine receptors mediate these regulatory effects of psychostimulants at the cellular level. Primary striatal neurons in culture express dopamine receptors as well as clock genes and have been successfully used in studying dopamine receptor functioning. Therefore, we investigated the role of dopamine receptors on neuronal clock gene expression in this model using specific receptor agonists. We found an inhibitory effect on the expression of mClock and mPer1 genes with the D2-class (i.e. D2/D3) receptor agonist quinpirole. We also found a generalized stimulatory effect on the expression of clock genes mPer1, mClock, mNPAS2 (neuronal PAS domain protein 2), and mBmal1 with the D1-class (i.e. D1) receptor agonist SKF38393. Further, we tested whether systemic administration of dopamine receptor agonists causes similar changes in striatal clock gene expression in vivo. We found quinpirole-induced alterations in mPER1 protein levels in the mouse striatum (i.e. rhythm shift). Collectively, our results indicate that the dopamine receptor system may mediate psychostimulant-induced changes in clock gene expression. Using striatal neurons in culture as a model, further research is needed to better understand how dopamine signaling modulates the expression dynamics of clock genes (i.e. intracellular signaling pathways) and thereby influences neuronal gene expression, neuronal transmission, and brain functioning.

Melatonin receptor agonist ramelteon activates the extracellular signal-regulated kinase 1/2 in mouse cerebellar granule cells.

The melatonin receptors MT1 and MT2 take part in the regulation of the activity (i.e. phosphorylation) of extracellular-signal-regulated kinase (ERK1/2), an enzyme involved in neuroplasticity. Primary cultures of mouse and rat cerebellar granule cells (CGC), which express both MT1 and MT2 receptors, have been widely used as an in vitro model to study neuronal ERK1/2. A novel MT1/MT2 agonist, ramelteon, has recently become clinically available. In this study, we characterized its action on neuronal ERK1/2. We used CGC cultures prepared from the cerebella of wild-type mice (MT1/MT2 CGC) and MT1- and MT2-knockout (KO) mice (MT1 KO CGC and MT2 KO CGC, respectively), and we employed a Western blot assay to evaluate ERK1/2 phosphorylation. Ramelteon increased ERK1/2 phosphorylation not only in MT1/MT2 CGC but also in CGC expressing only one of the two melatonin receptors. In the MT1 KO CGC, the stimulatory effect of ramelteon was blocked by an MT2 antagonist, 4P-PDOT, whereas in the MT2 KO CGC, this effect of ramelteon was blocked by luzindole. Pertussis toxin treatment did not prevent ramelteon from activating ERK1/2 but pretreatment with a tyrosine kinase (Trk) inhibitor, K252a, did, suggesting that an activation of Trk may mediate melatonin-receptor dependent ERK1/2 activation. In conclusion, we showed for the first time that a clinically used MT1/MT2 agonist, ramelteon, is capable of activating neuronal ERK1/2.

Effect of fluoxetine and cocaine on the expression of clock genes in the mouse hippocampus and striatum.

Long-term drug-induced alterations in CNS gene expression may be responsible for some therapeutic effects, such as antidepressant action, as well as for psychopathological conditions, such as drug addiction and abuse. Transcription factors called "clock" genes can be affected by psychotropic drugs and may modify the expression pattern of other genes. In this study in mice, we investigated the delayed effects of single and repeated (i.e. 14 days) administration of the antidepressant fluoxetine and the psychostimulant cocaine on the brain expression of clock genes Period1, Period2, Period3, Clock, Bmal1, Cryptochrome1, Cryptochrome2, and NPAS2 (neuronal PAS domain protein 2), and their putative target gene, serotonin N-acetyltransferase. Mice were treated at ZT05 (lights on at 5:00 am; ZT00). Brain samples (i.e. hippocampus, striatum, and prefrontal cortex) were processed for a semi-quantitative mRNA assay. Repeated but not single treatment with either drug increased serotonin N-acetyltransferase expression in all areas tested. On the other hand, the expression of clock genes was differentially affected depending on the drug (i.e. fluoxetine and cocaine), treatment schedule (i.e. single and repeated), and brain area (i.e. hippocampus and striatum) tested. More pronounced changes were induced by repeated rather than single administrations of fluoxetine or cocaine. We propose that the effects of psychoactive drugs on clock transcription factors may mediate long-term drug-induced changes, possibly by regulating the expression of a second set of genes (i.e. clock-controlled genes).

Intracellular glutathione levels determine cerebellar granule neuron sensitivity to excitotoxic injury by kainic acid.

Glutathione (GSH) is a key component of the cellular defence cascade against injury caused by reactive oxygen species. Kainic acid (KA) is a potent central nervous system excitotoxin. KA-elicited neuronal death may result from the generation of ROS. The present study was undertaken to characterize the role of GSH in KA-induced neurotoxicity. Cultures of cerebellar granule neurons were prepared from 8-day-old rats, and used at 8, 14 and 20 days in vitro (DIV). Granule neurons displayed a developmental increase in their sensitivity to KA injury, as quantified by an ELISA-based assay with the tetrazolium salt MTT. At DIV 14 and 20, a 30-min challenge with KA (500 microM) reduced cell viability by 45% after 24 h, significantly greater (P<0.01) than the 22% cell loss with DIV 8 cultures. Moreover acute (30 min) KA exposure concentration-dependently reduced intracellular GSH and enhanced reactive oxygen species generation (evaluated by 2', 7'-dichlorofluorescein diacetate). In comparison to control, KA (500 microM) lowered GSH levels in DIV 8 granule neurons by 16% (P=0. 0388), and by 36% (P=0.0001) in both DIV 14 and DIV 20 neurons, after 30 min. Preincubation of granule neurons with the membrane permeant GSH delivery agent, GSH ethyl ester (5 mM), for 30 min significantly increased intracellular GSH content. Importantly, GSH ethyl ester reduced the toxic effects of KA, becoming significant at 1 mM (P=0.007 vs. KA-treated group), and was maximal at >/=2.5 mM (P<0.0001). GSH ethyl ester displayed a similar dose-dependence in its ability to counteract KA-induced depletion of cellular GSH. The data strengthen the notion that cellular GSH levels have a fundamental role in KA-induced neurotoxicity.