Inhibition of FKBP51 induces stress resilience and alters hippocampal neurogenesis.

Stress-related psychiatric disorders such as depression are among the leading causes of morbidity and mortality. Considering that many individuals fail to respond to currently available antidepressant drugs, there is a need for antidepressants with novel mechanisms. Polymorphisms in the gene encoding FK506-binding protein 51 (FKBP51), a co-chaperone of the glucocorticoid receptor, have been linked to susceptibility to stress-related psychiatric disorders. Whether this protein can be targeted for their treatment remains largely unexplored. The aim of this work was to investigate whether inhibition of FKBP51 with SAFit2, a novel selective inhibitor, promotes hippocampal neuron outgrowth and neurogenesis in vitro and stress resilience in vivo in a mouse model of chronic psychosocial stress. Primary hippocampal neuronal cultures or hippocampal neural progenitor cells (NPCs) were treated with SAFit2 and neuronal differentiation and cell proliferation were analyzed. Male C57BL/6 mice were administered SAFit2 while concurrently undergoing a chronic stress paradigm comprising of intermittent social defeat and overcrowding, and anxiety and depressive -related behaviors were evaluated. SAFit2 increased neurite outgrowth and number of branch points to a greater extent than brain derived neurotrophic factor (BDNF) in primary hippocampal neuronal cultures. SAFit2 increased hippocampal NPC neurogenesis and increased neurite complexity and length of these differentiated neurons. In vivo, chronic SAFit2 administration prevented stress-induced social avoidance, decreased anxiety in the novelty-induced hypophagia test, and prevented stress-induced anxiety in the open field but did not alter adult hippocampal neurogenesis in stressed animals. These data warrant further exploration of inhibition of FKBP51 as a strategy to treat stress-related disorders.

The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.

Adult-born neurons from the dorsal, intermediate, and ventral regions of the longitudinal axis of the hippocampus exhibit differential sensitivity to glucocorticoids.

Hippocampal neurogenesis has been shown to play roles in learning, memory, and stress responses. These diverse roles may be related to a functional segregation of the hippocampus along its longitudinal axis. Indeed, the dorsal hippocampus (dHi) plays a predominant role in spatial learning and memory, while the ventral hippocampus (vHi) is predominantly involved in the regulation of anxiety, a behaviour impacted by stress. Recent studies suggest that the area between them, the intermediate hippocampus (iHi) may also be functionally independent. In parallel, it has been reported that chronic stress reduces neurogenesis preferentially in the vHi rather the dHi. We thus aimed to determine whether such stress-induced changes in neurogenesis could be related to differential intrinsic sensitivity of neural progenitor cells (NPCs) from the dHi, iHi, or vHi to the stress hormone, corticosterone, or the glucocorticoid receptor (GR) agonist, dexamethasone. Long-term exposure of rat NPCs to corticosterone or dexamethasone decreased neuronal differentiation in the vHi but not the dHi, while iHi cultures showed an intermediate response. A similar gradient-like response on neuronal differentiation and maturation was observed with dexamethasone treatment. This gradient-like effect was also observed on GR nuclear translocation in response to corticosterone or dexamethasone. Long-term exposure to corticosterone or dexamethasone treatment also tended to induce a greater downregulation of GR-associated genes in vHi-derived neurons compared to those from the dHi and iHi. These data suggest that increased intrinsic sensitivity of vHi NPC-derived neurons to chronic glucocorticoid exposure may underlie the increased vulnerability of the vHi to chronic stress-induced reductions in neurogenesis.

miRNA-mediated inhibition of an actomyosin network in hippocampal pyramidal neurons restricts sociability in adult male mice.

Social deficits are frequently observed in patients suffering from neurodevelopmental disorders, but the molecular mechanisms regulating sociability are still poorly understood. We recently reported that the loss of the microRNA (miRNA) cluster miR-379-410 leads to hypersocial behavior and anxiety in mice. Here, we show that ablating miR-379-410 in excitatory neurons of the postnatal mouse hippocampus recapitulates hypersociability, but not anxiety. At the cellular level, miR-379-410 loss in excitatory neurons leads to larger dendritic spines, increased excitatory synaptic transmission, and upregulation of an actomyosin gene network. Re-expression of three cluster miRNAs, as well as pharmacological inhibition of the actomyosin activator ROCK, is sufficient to reinstate normal sociability in miR-379-410 knockout mice. Several actomyosin genes and miR-379-410 family members are reciprocally dysregulated in isogenic human induced pluripotent stem cell (iPSC)-derived neurons harboring a deletion present in patients with Williams-Beuren syndrome, characterized by hypersocial behavior. Together, our results show an miRNA-actomyosin pathway involved in social behavior regulation.

Laser microirradiation as a tool to investigate the role of liquid-liquid phase separation in DNA damage repair.

Here we describe a protocol for the generation of site-specific DNA damage, including double and single strand breaks, using the 405 nm laser of a confocal microscope in cells pre-sensitized with Hoechst. This is a simple approach, particularly useful to assess the involvement of proteins and the roles of liquid-liquid phase separation in DNA damage repair. Examples of transfection protocol, drug concentrations, and microscopy are provided, although optimization may be needed for specific experimental setups and cell lines used. For complete details on the use and execution of this protocol, please refer to Levone et al. (2021).

Specific sub-regions of the longitudinal axis of the hippocampus mediate behavioural responses to chronic psychosocial stress.

Accumulating evidence suggests that the hippocampus is functionally segregated along its longitudinal axis into a dorsal (dHi) sub-region, shown to play roles in learning & memory and a ventral sub-region (vHi), involved in anxiety and antidepressant action. Recent studies also suggest that the intermediate hippocampus (iHi) might be functionally independent, but it has received relatively little attention. We recently found that the iHi is involved in the behavioural effects of chronic treatment with the antidepressant fluoxetine in the forced swim test. However, the roles of specific sub-regions of the longitudinal axis of the hippocampus in the response to chronic stress, a risk factor for depression and anxiety disorders, has not yet been investigated. Therefore, we used excitotoxic lesions of the dHi, iHi or vHi in male C57BL/6 mice to investigate the roles of these sub-regions in the behavioural (anxiety, anhedonia, depression) responses to chronic psychosocial stress. We found that stress-induced increases in anxiety in the novelty-induced hypophagia and marble burying tests were prevented by each of the sub-region lesions, but only vHi lesions attenuated stress-induced anxiety in the open field test. Stress-induced anhedonia was reduced in dHi- and vHi- but not iHi-lesioned mice. In stressed mice, only vHi lesions induced an antidepressant-like effect in the forced swim test and prolonged latency to adopt a defeat posture during social defeat, suggesting an increase in stress resilience. Interestingly, iHi lesions increased stress-induced social avoidance in the social interaction test. In summary, we found that all hippocampal sub-regions are involved in the anxiogenic effects of chronic stress but that the iHi plays a predominant role in stress-induced social avoidance and the vHi has a predominant role in active coping behaviours and antidepressant-like behaviour following chronic stress.

Specific sub-regions along the longitudinal axis of the hippocampus mediate antidepressant-like behavioral effects.

Current antidepressants are suboptimal due incomplete understanding of the neurobiology underlying their behavioral effects. However, imaging studies suggest the hippocampus is a key brain region underpinning antidepressant action. There is increasing attention on the functional segregation of the hippocampus into a dorsal region (dHi) predominantly involved in spatial learning and memory, and a ventral region (vHi) which regulates anxiety, a symptom often co-morbid with depression. However, little is known about the roles of these hippocampal sub-regions in the antidepressant response. Moreover, the area between them, the intermediate hippocampus (iHi), has received little attention. Here, we investigated the impact of dHi, iHi or vHi lesions on anxiety- and depressive-like behaviors under baseline or antidepressant treatment conditions in male C57BL/6 mice (n = 8-10). We found that in the absence of fluoxetine, vHi lesions reduced anxiety-like behavior, while none of the lesions affected other antidepressant-sensitive behaviors. vHi lesions prevented the acute antidepressant-like behavioral effects of fluoxetine in the tail suspension test and its anxiolytic effects in the novelty-induced hypophagia test. Intriguingly, only iHi lesions prevented the antidepressant effects of chronic fluoxetine treatment in the forced swim test. dHi lesions did not impact any behaviors either in the absence or presence of fluoxetine. In summary, we found that vHi plays a key role in anxiety-like behavior and its modulation by fluoxetine, while both iHi and vHi play distinct roles in fluoxetine-induced antidepressant-like behaviors.

Enduring effects of muscarinic receptor activation on adult hippocampal neurogenesis, microRNA expression and behaviour.

The cholinergic system is one of the most important neurotransmitter systems in the brain with key roles in autonomic control and the regulation of cognitive and emotional responses. However, the precise mechanism by which the cholinergic system influences behaviour is unclear. Adult hippocampal neurogenesis (AHN) is a potential mediator in this context based on evidence, which has identified this process as putative mechanism of antidepressant action. More recently, post-transcriptional regulation by microRNAs is another candidate mechanism based on its involvement in the regulation of AHN and neurotransmission. Taking into account this background, we evaluated the behavioural effects of a non-convulsant dose of pilocarpine - a non-selective muscarinic receptor (mAChR) agonist - in adult Wistar rats. Furthermore, we quantified the expression of different microRNAs implicated in the regulation of AHN. Our results suggests that pilocarpine treatment increases AHN in the granular cell layer but also induced ectopic neurogenesis. Pilocarpine treatment reduced immobility time in forced swimming test but did not affect fear conditioning response, sucrose preference or novelty supressed feeding behaviour. In addition, treatment with pilocarpine down-regulated the expression of 6 microRNAs implicated in the regulation of neurotrophin signalling and axon guidance pathways. Therefore, we suggest that the low-dose stimulation of the cholinergic system is sufficient to alter AHN of rats through post-transcriptional mechanisms, which might contribute to long-lasting behavioural effects.

Ingestive and locomotor behaviours induced by pharmacological manipulation of -adrenoceptors into the median raphe nucleus.

The present study evaluated the involvement of α-adrenoceptors of the median raphe nucleus (MRN) in satiated rats, in food and water intake and motor behaviour. Control groups were treated with saline (SAL) or adrenaline (ADR), injected into the MRN seven minutes after injection of the vehicle used to solubilize the antagonists, propylene glycol (PLG) or SAL. Experimental groups were treated with an α-adrenoceptor antagonist, prazosin (α1, 20 or 40 nmol) or yohimbine (α2, 20 or 40 nmol) or phentolamine (non-selective α, 20 or 40 nmol), followed (later) by injection of ADR or SAL. Behaviour was recorded for 30 min. The injection of ADR and the blockade of α1 receptors resulted in hyperphagia whereas blocking α2 or α1 and α2 simultaneously did not change feeding behaviour. Pre-treatment with prazosin, followed by injection of ADR was not able to cause an increase in the amount of food ingested, while the higher dose of the α1 antagonist reduced the latency to start feeding. Pre-treatment with prazosin also caused hyperactivity. However, pre-treatment with phentolamine or yohimbine was able to block ADR-induced feeding. The present study supports the hypothesis that there is a tonic activation of α1-adrenoceptors in the MRN in satiated rats, which activates an inhibitory influence in areas that control food intake. Injection of ADR seems to activate α2 receptors, resulting in a decrease in the availability of endogenous catecholamines, which reduces the release of the signal that inhibits food intake, leading to hyperphagia.

The microinjection of a cannabinoid agonist into the accumbens shell induces anxiogenesis in the elevated plus-maze.

This study investigated the effect of a cannabinoid agonist injected into the shell region of the nucleus accumbens (nAcb shell) on anxiety-related behaviors. The animals (male Wistar rats) were unilaterally microinjected with either ACEA (arachidonyl-2'-chloroethylamide a CB1 receptor agonist) at doses of 0.005, 0.05 or 0.5 pmol, or vehicle (ethanol 0.04% in saline 0.9%) and submitted to the elevated plus-maze (EPM), a pre-clinical test of anxiety. The data showed that rats microinjected with ACEA (0.05 pmol/0.2 µl) into the nAcb shell exhibited decreased % open arm time and open arm entries in comparison with the control group, which is compatible with an anxiogenic-like effect. To rule out the hypothesis that spread of the drug into the ventricle was responsible for the observed anxiogenic effect, 0.05 pmol ACEA was injected into the lateral ventricle and shown not to alter the responses representative of fear/anxiety and locomotion. The locomotor activity was not changed at the dose of 0.05 pmol ACEA microinjected into the nAcb shell. The present data suggest that activation of cannabinoid receptors in the nAcb shell may modulate fear/anxiety in the EPM.

Blockade of median raphe nucleus α1-adrenoceptor subtypes increases food intake in rats.

Previous studies have shown that the blockade of α1-adrenoceptors in the median raphe nucleus (MnR) of free-feeding animals increases food intake. Since there is evidence for the presence of α1A-, α1B- and α1D-adrenoceptors in the MnR of rats, this study investigated the involvement of MnR α1-adrenoceptor subtypes in the control of feeding behavior, looking for possible differences on the role of each α1-adrenoceptor in feeding. Male adult rats weighing 280-300 g with guide cannulae chronically implanted above the MnR were injected with antagonists of α1A- (RS100329, 0, 2, 4 or 20 nmol), α1B- (Rec 15/2615, 0, 2, 4 or 20 nmol) or α1D-adrenoceptor (BMY 7378, 0, 2, 4 or 20 nmol). Subsequently, behavioral evaluation of ingestive and non-ingestive parameters was monitored for 1h and the amount of food and water ingested was assessed for 4h. The highest dose (20 nmol) of RS100329 and BMY 7378 increased food intake, feeding duration and frequency, and decreased the latency to start feeding. During the second hour 2 nmol dose of Rec 15/2615 increased food intake and all doses of BMY 7378 decreased water intake. No behavioral alterations were observed during the fourth hour. The results corroborate previous work from our lab in which we describe the involvement of α1-adrenoceptors of MnR on food intake control. Moreover, we show evidence that α1A- and α1D-adrenoceptors mediate feeding responses to adrenaline injections and that the behavioral modifications are of considerable duration, persisting up to 2h after injection of the antagonists.

Qualidade de vida e sintomas depressivos em pacientes renais crônicos submetidos à hemodiálise / Quality of life and depressive symptoms in patients with chronic renal failure undergoing hemodialysis

Introdução: nos períodos iniciais do uso da diálise, todos os esforços eram dedicados para a manutenção da vida; atualmente, com os progressos alcançados neste sentido, observa-se preocupação com os aspectos emocionais dos pacientes renais crônicos e com sua qualidade de vida. Objetivo: Este trabalho objetiva verificar a relação entre qualidade de vida e sintomas depressivos em pacientes renais crônicos submetidos à hemodiálise. Métodos: a amostra foi composta de 30 pacientes, aos quais foram aplicados o Inventário de Depressão Maior (MDI) e o questionário de qualidade de vida WHOQOL- -abreviado. Resultados: a prevalência de depressão foi de 37%. A qualidade de vida foi considerada boa em 89,6% da amostra. Obteve-se fraca associação (Pearson 0,455) entre as variáveis depressão e qualidade de vida. Conclusões: esta amostra apresentou bons índices de qualidade de vida e índices moderados de sintomas depressivos.

Introduction: during the initial periods of the use of dialysis, all effort is devoted to the maintenance of life. Following the progress reached in this regard, there is now concern about the emotional aspects of chronic renal patients and their quality of life. Objective: This study aims to investigate the relationship between quality of life and depressive symptoms in chronic renal failure patients undergoing hemodialysis. Methods: The sample consisted of 30 patients interviewed using the the Major Depression Inventory (MDI) and abbreviated WHOQOL quality of life questionnaires. Results: The prevalence of depression was 37%. Quality of life was considered good for 89.6% of the sample. There was a weak association (Pearson 0.455) between the depression and quality of life variables. Conclusions: This sample showed good levels of quality of life and moderate levels of depressive symptoms.

Desenvolvimento do controle cervical em criança com encefalopatia crônica não-progressiva da infância / Development of neck control in children with chronic non-progressive encephalopathy of childhood

O controle cervical é uma das primeiras aquisições motoras voluntárias da criança. A disfunção motora na Encefalopatia Crônica Não-Progressiva da Infância (ECNPI) pode ocasionar atraso no desenvolvimento desse controle, além da fixação de padrões posturais patológicos. Identifica os recursos disponíveis na literatura para promover a aquisição do controle cervical e relacioná-los com o caso de uma criança que apresenta severo atraso do desenvolvimento motor. Os reflexos primitivos, as reações de retificação e equilíbrio, o grau de espasticidade, a função motora ampla e as habilidades funcionais, foram avaliados. Observou-se presença de reflexos primitivos e espasticidade em todos os membros, deficiência ou ausência das reações de retificação e equilíbrio e limitação funcional muito severa. O severo atraso no desenvolvimento motor, observado neste estudo, determina a estimulação da função motora ampla e habilidades funcionais. O controle cervical deve ser priorizado, já que é um precursor necessário para outras aquisições motoras e posturais.

The cervical control is an early voluntary motor acquisition of child. The motor dysfunction in cerebral palsy may cause delay in this control development and determination of pathological postural patterns. Identifies the available resources in the literature for the acquisition of cervical control and relate them to the case of a child who has severe development delay. The primitive reflexes, postural correction and balance reactions, spasticity rate, motor function and broad functional skills were evaluated. The presence of primitive reflexes and spasticity in all limbs were observed as well as disability or absence of postural correction and equilibrium reactions and a very severe functional limitation. The severe delay in motor development observed in this study determines the broad stimulation of motor function and function abilities. The neck control should be prioritized since it is a necessary precursor for new motor and postural acquisition.