Expression and glucocorticoid-dependent regulation of the stressinducible protein DRR1 in the mouse adult brain

Identifying molecular targets that are able to buffer the consequences of stress and therefore restore brain homeostasis is essential to develop treatments for stress-related disorders. Down-regulated in renal cell carcinoma 1 (DRR1) is a unique stress-induced protein in the brain and has been recently proposed to modulate stress resilience. Interestingly, DRR1 shows a prominent expression in the limbic system of the adult mouse. Here, we analyzed the neuroanatomical and cellular expression patterns of DRR1 in the adult mouse brain using in situ hybridization, immunofluorescence and Western blot. Abundant expression of DRR1 mRNA and protein was confirmed in the adult mouse brain with pronounced differences between distinct brain regions. The strongest DRR1 signal was detected in the neocortex, the CA3 region of the hippocampus, the lateral septum and the cerebellum. DRR1 was also present in circumventricular organs and its connecting regions. Additionally, DRR1 was present in non-neuronal tissues like the choroid plexus and ependyma. Within cells, DRR1 protein was distributed in a punctate pattern in several subcellular compartments including cytosol, nucleus as well as some pre- and postsynaptic specializations. Glucocorticoid receptor activation (dexamethasone 10 mg/kg s.c.) induced DRR1 expression throughout the brain, with particularly strong induction in white matter and fiber tracts and in membrane-rich structures. This specific expression pattern and stress modulation of DRR1 point to a role of DRR1 in regulating how cells sense and integrate signals from the environment and thus in restoring brain homeostasis after stressful challenges

The Kynurenine Pathway Is Upregulated by Methyl-deficient Diet and Changes Are Averted by Probiotics

Scope Probiotics exert immunomodulatory effects and may influence tryptophan metabolism in the host. Deficiency of nutrients related to C1 metabolism might stimulate inflammation by enhancing the kynurenine pathway. This study used Sprague Dawley rats to investigate whether a methyl-deficient diet (MDD) may influence tryptophan/kynurenine pathways and cytokines and whether probiotics can mitigate these effects. Methods and Results Rats are fed a control or MDD diet. Animals on the MDD diet received vehicle, probiotics (L. helveticus R0052 and B. longum R0175), choline, or probiotics + choline for 10 weeks (n = 10 per group). Concentrations of plasma kynurenine metabolites and the methylation and inflammatory markers in plasma and liver are measured. Results MDD animals (vs controls) show upregulation of plasma kynurenine, kynurenic acid, xanthurenic acid, 3-hydroxyxanthranilic acid, quinolinic acid, nicotinic acid, and nicotinamide (all p < 0.05). In the MDD rats, the probiotics (vs vehicle) cause lower anthranilic acid and a trend towards lower kynurenic acid and picolinic acid. Compared to probiotics alone, probiotics + choline is associated with a reduced enrichment of the bacterial strains in cecum. The interventions have no effect on inflammatory markers. Conclusions Probiotics counterbalance the effect of MDD diet and downregulate downstream metabolites of the kynurenine pathway.publishedVersio

The Kynurenine Pathway Is Upregulated by Methyl-deficient Diet and Changes Are Averted by Probiotics

Scope Probiotics exert immunomodulatory effects and may influence tryptophan metabolism in the host. Deficiency of nutrients related to C1 metabolism might stimulate inflammation by enhancing the kynurenine pathway. This study used Sprague Dawley rats to investigate whether a methyl‐deficient diet (MDD) may influence tryptophan/kynurenine pathways and cytokines and whether probiotics can mitigate these effects. Methods and Results Rats are fed a control or MDD diet. Animals on the MDD diet received vehicle, probiotics (L. helveticus R0052 and B. longum R0175), choline, or probiotics + choline for 10 weeks (n = 10 per group). Concentrations of plasma kynurenine metabolites and the methylation and inflammatory markers in plasma and liver are measured. Results MDD animals (vs controls) show upregulation of plasma kynurenine, kynurenic acid, xanthurenic acid, 3‐hydroxyxanthranilic acid, quinolinic acid, nicotinic acid, and nicotinamide (all p < 0.05). In the MDD rats, the probiotics (vs vehicle) cause lower anthranilic acid and a trend towards lower kynurenic acid and picolinic acid. Compared to probiotics alone, probiotics + choline is associated with a reduced enrichment of the bacterial strains in cecum. The interventions have no effect on inflammatory markers. Conclusions Probiotics counterbalance the effect of MDD diet and downregulate downstream metabolites of the kynurenine pathway

The glutamate hypothesis of depression: the effect of stress and glucocorticoids on glutamate synapse and the action of antidepressants

In the last years, a consistent number of clinical and preclinical studies have demonstrated that glutamatergic transmission has a primary role in the pathophysiology of mood and anxiety disorders (MADI). It has been shown that in depressed patients the levels of glutamate and its metabolites are altered in plasma and in selected brain areas and mRNA and protein levels of glutamate receptors are changed in brain areas. A number of preclinical studies on animal models of MADI have shown that different types of environmental stress and glucocorticoid administration affect glutamate transmission and exert structural brain remodeling in the same areas involved in human pathology. These effects of stress and glucocorticoids have been associated with the onset and exacerbation of neuropsychiatric disorders.In previous studies we found that acute footshock (FS)-stress induces an increase of glutamate release from synaptosomes of prefrontal and frontal cortex (PFC/FC), via glucocorticoid receptors (GR) activation and SNARE complex accumulation in synaptic membranes. Furthermore, we have demonstrated that the increase of glutamate release induced by acute stress is prevented by chronic antidepressants (ADs). Additional studies have also shown that ADs can regulate glutamate transmission through glutamate receptors; reducing the function of NMDA receptors, potentiating the function of AMPA receptor and affecting different subtypes of metabotropic glutamate receptors. Together, these findings have identified the glutamate synapse as a target for novel glutamatergic ADs.Considering the importance of stress-induced alteration of presynaptic glutamate release in the pathophysiology of MADI, we aimed to study whether the enhancement of depolarization-evoked glutamate release induced by acute stress was related to an increase of the readily releasable pool (RRP) of vesicles and whether this effect was mediated by a synaptic non-genomic action of corticosterone (CORT).We found that FS-stress increased glutamate release evoked by hypertonic sucrose (which mobilizes exclusively the RRP), suggesting an increase in the RRP size. Then we found that this synaptic effect of stress was dependent on local CORT action. Indeed, CORT was able to directly affect the RRP size through the activation of GR and mineralcorticoid receptors (MR). The preincubation with RU486, selective GR antagonist, and spironolactone, selective MR antagonist, prevented the CORT-induced increase of RRP. Contrary to acute stress, CORT by itself did not promote vesicle fusion, since CORT application in vitro did not increase glutamate release evoked by depolarization of control synaptosomes, and did not affect excitatory post-synaptic potentials and paired pulse facilitation in mPFC slices. Furthermore, we also found that CORT increased vesicle mobilization towards the RRP via GRs and MRs activation, by using total internal fluorescence microscopy, a technique that allows the study of events occurring in a 100 nm-interval below the plasma membrane. Finally we found that stress and CORT modulated synapsin I, a protein involved in vesicle mobilization and in vesicles docking, fusion and recycling at active zones. We found that both FS-stress and CORT induce an increase of synapsin I phosphorylation in synaptic membranes selectively at site 1. The preincubation with both RU486 and spironolactone, prevented the CORT-induced synapsin I phosphorylation at site I, suggesting that this protein is involved in the pathway downstream of activation of the two receptors. Together these results suggest that the increase of the RRP size induced by stress is promoted by a local action of CORT on synaptic receptors. We speculated that CORT is necessary to promote an increase of the RRP, but not sufficient to increase depolarization-dependent glutamate release, suggesting that additional mediators or neurotransmitters, released during the stress response, are necessary to trigger vesicle releas

Functional and Structural Remodeling of Glutamate Synapses in Prefrontal and Frontal Cortex Induced by Behavioral Stress

Increasing evidence has shown that the pathophysiology of neuropsychiatric disorders, including mood disorders, is associated with abnormal function and regulation of the glutamatergic system. Consistently, preclinical studies on stress-based animal models of pathology showed that glucocorticoids and stress exert crucial effects on neuronal excitability and function, especially in cortical and limbic areas. In prefrontal and frontal cortex, acute stress was shown to induce enhancement of glutamate release/transmission dependent on activation of corticosterone receptors. Although the mechanisms whereby stress affects glutamate transmission have not yet been fully understood, it was shown that synaptic, non-genomic action of corticosterone is required to increase the readily releasable pool of glutamate vesicles, but is not sufficient to enhance transmission in prefrontal and frontal cortex. Slower, partly genomic mechanisms are probably necessary for the enhancement of glutamate transmission induced by stress. Combined evidence has suggested that the changes in glutamate release and transmission are responsible for the dendritic remodeling and morphological changes induced by stress and it has been argued that sustained alterations of glutamate transmission may play a key role in the long-term structural/functional changes associated with mood disorders in patients. Intriguingly, modifications of the glutamatergic system induced by stress in the prefrontal cortex seem to be biphasic. Indeed, while the fast response to stress suggests an enhancement in the number of excitatory synapses, synaptic transmission and working memory, long-term adaptive changes - including those consequent to chronic stress - induce opposite effects. Better knowledge of the cellular effectors involved in this biphasic effect of stress may be useful to understand the pathophysiology of stress-related disorders, and open new paths for the development of therapeutic approaches

Decoding the Mechanism of Action of Rapid-Acting Antidepressant Treatment Strategies: Does Gender Matter?

Gender differences play a pivotal role in the pathophysiology and treatment of major depressive disorder. This is strongly supported by a mean 2:1 female-male ratio of depression consistently observed throughout studies in developed nations. Considering the urgent need to tailor individualized treatment strategies to fight depression more efficiently, a more precise understanding of gender-specific aspects in the pathophysiology and treatment of depressive disorders is fundamental. However, current treatment guidelines almost entirely neglect gender as a potentially relevant factor. Similarly, the vast majority of animal experiments analysing antidepressant treatment in rodent models exclusively uses male animals and does not consider gender-specific effects. Based on the growing interest in innovative and rapid-acting treatment approaches in depression, such as the administration of ketamine, its metabolites or electroconvulsive therapy, this review article summarizes the evidence supporting the importance of gender in modulating response to rapid acting antidepressant treatment. We provide an overview on the current state of knowledge and propose a framework for rodent experiments to ultimately decode gender-dependent differences in molecular and behavioural mechanisms involved in shaping treatment response

Concordance study on Y-STRs typing between SeqStudio™ genetic analyzer for HID and MiSeq™ FGx forensic genomics system

Background: Massively Parallel Sequencing (MPS) allowed an increased number of information to be retrieved from short tandem repeat (STR) analysis, expanding them not only to the size, as already performed in Capillary Electrophoresis (CE), but also to the sequence. MPS requires constant development and validation of the analytical parameters to ensure that the genotyping results of STRs correspond to those obtained by CE. Given the increased frequency of usage of Y-STRs as supplementary markers to the autosomal STRs analysis, it is urgent to validate the concordance of the typing results between CE and MPS analyses. Methods and results: DNA extracted from 125 saliva samples of unrelated males was genotyped using Yfiler™ Plus PCR Amplification Kit and ForenSeq™ DNA Signature Prep Kit, which were analyzed by SeqStudio™ Genetic Analyzer for HID and MiSeq™ FGx Forensic Genomics System, respectively. For each shared Y-STR, allele designation, number of length- and sequence-based alleles per locus, stutter percentage, and the intra-locus balance of multicopy Y-STRs were screened. Conclusions: Although the number of forensic genetics laboratories that are applying the MPS technique in routine analysis is small and does not allow a global assessment of MPS limitations, this comparative study highlights the ability of MPS to produce reliable profiles despite the generation of large amounts of raw data