Do Corticosteroid Receptor mRNA Levels Predict the Expression of Their Target Genes?

The glucocorticoid stress hormones affect brain function via high-affinity mineralocorticoid receptors (MRs) and lower-affinity glucocorticoid receptors (GRs). MR and GR not only differ in affinity for ligands, but also have distinct, sometimes opposite, actions on neuronal excitability and other cellular and higher-order parameters related to cerebral function. GR and MR messenger RNA (mRNA) levels are often used as a proxy for the responsiveness to glucocorticoids, assuming proportionality between mRNA and protein levels. This may be especially relevant for the MR, which because of its high affinity is already largely occupied at low basal (trough) hormone levels. Here we explore how GR and MR mRNA levels are associated with the expression of a shared target gene, glucocorticoid-induced leucine zipper (GILZ, coded by Tsc22d3) with basal and elevated levels of corticosterone in male mice, using in situ hybridization. Depending on the hippocampal subfield and the corticosterone levels, mRNA levels of MR rather than GR mostly correlated with GILZ mRNA in the hippocampus and hypothalamus at the bulk tissue level. At the individual cell level, these correlations were much weaker. Using publicly available single-cell RNA sequencing data, we again observed that MR and GR mRNA levels were only weakly correlated with target gene expression in glutamatergic and GABAergic neurons. We conclude that MR mRNA levels can be limiting for receptor action, but many other cell-specific and region-specific factors ultimately determine corticosteroid receptor action. Altogether, our results argue for caution while interpreting the consequences of changed receptor expression for the response to glucocorticoids.

Biallelic PAX5 mutations cause hypogammaglobulinemia, sensorimotor deficits, and autism spectrum disorder.

The genetic causes of primary antibody deficiencies and autism spectrum disorder (ASD) are largely unknown. Here, we report a patient with hypogammaglobulinemia and ASD who carries biallelic mutations in the transcription factor PAX5. A patient-specific Pax5 mutant mouse revealed an early B cell developmental block and impaired immune responses as the cause of hypogammaglobulinemia. Pax5 mutant mice displayed behavioral deficits in all ASD domains. The patient and the mouse model showed aberrant cerebellar foliation and severely impaired sensorimotor learning. PAX5 deficiency also caused profound hypoplasia of the substantia nigra and ventral tegmental area due to loss of GABAergic neurons, thus affecting two midbrain hubs, controlling motor function and reward processing, respectively. Heterozygous Pax5 mutant mice exhibited similar anatomic and behavioral abnormalities. Lineage tracing identified Pax5 as a crucial regulator of cerebellar morphogenesis and midbrain GABAergic neurogenesis. These findings reveal new roles of Pax5 in brain development and unravel the underlying mechanism of a novel immunological and neurodevelopmental syndrome.

A Novel Automated Approach for Improving Standardization of the Marble Burying Test Enables Quantification of Burying Bouts and Activity Characteristics.

The marble burying test is a commonly used paradigm to describe phenotypes in mouse models of neurodevelopmental and psychiatric disorders. The current methodological approach relies predominantly on reporting the number of buried marbles at the end of the test. By measuring the proxy of the behavior (buried marbles), many important characteristics regarding the temporal aspect of this assay are lost. Here, we introduce a novel, automated method to quantify mouse behavior during the marble burying test with the focus on the burying bouts and movement dynamics. Using open-source software packages, we trained a supervised machine learning algorithm (the "classifier") to distinguish burying behavior in freely moving mice. In order to confirm the classifier's accuracy and characterize burying events in high detail, we performed the marble burying test in three mouse models: Ube3am-/p+ [Angelman syndrome (AS) model], Shank2-/- (autism model), and Sapap3-/- [obsessive-compulsive disorder (OCD) model] mice. The classifier scored burying behavior accurately and consistent with the previously reported phenotype of the Ube3am-/p+ mice, which showed decreased levels of burying compared with controls. Shank2-/- mice showed a similar pattern of decreased burying behavior, which was not found in Sapap3-/- mice. Tracking mouse behavior throughout the test revealed hypoactivity in Ube3am-/p+ and hyperactivity in the Shank2-/- mice, indicating that mouse activity is unrelated to burying behavior. Reducing activity with midazolam in Shank2-/- mice did not alter the burying behavior. Together, we demonstrate that our classifier is an accurate method for the analysis of the marble burying test, providing more information than currently used methods.

Activated PI3Kδ syndrome, an immunodeficiency disorder, leads to sensorimotor deficits recapitulated in a murine model.

The phosphoinositide-3-kinase (PI3K) family plays a major role in cell signaling and is predominant in leukocytes. Gain-of-function (GOF) mutations in the PIK3CD gene lead to the development of activated PI3Kδ syndrome (APDS), a rare primary immunodeficiency disorder. A subset of APDS patients also displays neurodevelopmental delay symptoms, suggesting a potential role of PIK3CD in cognitive and behavioural function. However, the extent and nature of the neurodevelopmental deficits has not been previously quantified. Here, we assessed the cognitive functions of two APDS patients, and investigated the causal role of the PIK3CD GOF mutation in neurological deficits using a murine model of this disease. We used p110δE1020K knock-in mice, harbouring the most common APDS mutation in patients. We found that APDS patients present with visuomotor deficits, exacerbated by autism spectrum disorder comorbidity, whereas p110δE1020K mice exhibited impairments in motor behaviour, learning and repetitive behaviour patterning. Our data indicate that PIK3CD GOF mutations increase the risk for neurodevelopmental deficits, supporting previous findings on the interplay between the nervous and the immune system. Further, our results validate the knock-in mouse model, and offer an objective assessment tool for patients that could be incorporated in diagnosis and in the evaluation of treatments.

The selective glucocorticoid receptor antagonist CORT125281 has tissue-specific activity.

Glucocorticoids mediate numerous essential processes in the human body via binding to the glucocorticoid receptor (GR). Excessive GR signaling can cause disease, and GR antagonists can be used to treat many symptoms of glucocorticoid-induced pathology. The purpose of this study was to characterize the tissue-specific properties of the selective GR antagonist CORT125281. We evaluated the antagonistic effects of CORT125281 upon acute and subchronic corticosterone exposure in mice. In the acute corticosterone setting, hypothalamus-pituitary-adrenal-axis activity was investigated by measurement of basal- and stress-induced corticosterone levels, adrenocorticotropic hormone levels and pituitary proopiomelanocortin expression. GR signaling was evaluated by RT-PCR analysis of GR-responsive transcripts in liver, muscle, brown adipose tissue (BAT), white adipose tissue (WAT) and hippocampus. Pretreatment with a high dose of CORT125281 antagonized GR activity in a tissue-dependent manner. We observed complete inhibition of GR-induced target gene expression in the liver, partial blockade in muscle and BAT and no antagonism in WAT and hippocampus. Tissue distribution only partially explained the lack of effective antagonism. CORT125281 treatment did not disinhibit the hypothalamus-pituitary-adrenal neuroendocrine axis. In the subchronic corticosterone setting, CORT125281 partially prevented corticosterone-induced hyperinsulinemia, but not hyperlipidemia and immune suppression. In conclusion, CORT125281 antagonizes GR transcriptional activity in a tissue-dependent manner and improves corticosterone-induced hyperinsulinemia. Tailored dosing of CORT125281 may allow tissue-specific inhibition of GR transcriptional activity.

Effectiveness of the Aloe Vera extract in the treatment of fistula-in-ano / Eficácia do extrato de Aloe Vera no tratamento da fístula anal

Abstract Rationale: Considering that anal fistulae are still challenging regarding their treatment due to the risk of fecal incontinence in the most complex cases and the increasing use of phytotherapeutic drugs such as Aloe Vera in medicine, even with proven healing effectiveness, there is interest in researching this drug in the treatment of anal fistulae. Objective: To evaluate the efficacy of Aloe Vera extract in the treatment of anal fistulae in rats. Method: Thirty male Wistar rats weighing 250-300 g were submitted to anal fistula and after 30 days were divided into three groups: Control Group (GCo) (n = 5), Carbopol Group (GCa) (n = 5), and Aloe Vera Group (GAV) (n = 10). In the GCo no treatment was performed, while in the others a daily infusion of 0.3 mL of solution (Carbopol only in GCa and Carbopol plus Aloe Vera extract in the GAV) was performed through the external orifice of the fistula for 30 days. Afterwards, euthanasia was performed and specimens were removed for histological study. It was evaluated the closure of the fistulous tract, the area of the remaining tract, the inflammatory infiltrate and the degree of vascular congestion. The results were submitted to statistical treatment by Kruskall-Wallis test, considering p<0.05. Results: There was no complete closure of the fistulous tract in any of the animals. The mean area of the remaining tract was 847.2 µm in the GCo, 565.6 µm in the GCa and 377.8 µm in the GAV (p<0.05). The mean of the inflammatory infiltrate score was 2.4 in the GCo, 2.4 in the GCa and 2.3 in the GAV (p<0.05), while in the evaluation of vascular congestion, we observed a mean of 1.6 in the GCo, 1.4 in GCa and 1.1 in GAV (p<0.05). Conclusion: The extract of Aloe vera was able to reduce the lumen of the fistulous tract and reduce the degree of vascular congestion; however, it did not allow the complete closure of the fistulous tract nor diminished the inflammatory process.

Resumo Racional: Considerando que o tratamento das fístulas anais tem risco de incontinência fecal e o crescente uso do Aloe Vera na medicina, há interesse em se pesquisar este fármaco. Objetivo: Avaliar a eficácia do extrato de Aloe vera no tratamento das fístulas anais em ratos. Método: Utilizou-se 30 ratos Wistar, os quais foram submetidos à criação de fístula anal e após 30 dias distribuídos em três grupos: Controle (GCo), Carbopol (GCa) e Aloe Vera (GAV). No GCo nenhum tratamento foi realizado, enquanto nos outros realizou-se infusão diária de 0,3 mL de Carbopol GCa e Carbopol mais extrato de Aloe Vera no GAV por 30 dias. Foram retirados os espécimes para estudo histológico, avaliou-se o fechamento do trajeto fistuloso, a área do trajeto remanescente, o infiltrado inflamatório e o grau de congestão vascular. Resultados: Não houve fechamento completo do trajeto fistuloso em nenhum dos animais. A média da área do trajeto remanescente foi 847,2 µm no GCo; 565,6 µm no GCa e 377,8 µm no GAV (p<0,05). A média do escore de infiltrado inflamatório foi 2,4 no GCo; 2,4 no GCa e 2,3 no GAV (p<0.05), enquanto na avaliação da congestão vascular observou-se média 1,6 no GCo; 1,4 no GCa e 1,1 no GAV (p<0,05). Conclusão: O extrato de Aloe Vera foi capaz de diminuir o lumen dos trajetos fistulosos e reduzir o grau de congestão vascular, porém, não permitiu o fechamento completo dos trajetos fistulosos nem diminuiu o processo inflamatório.

Treatment of anal fistula with Baccharis dracunculifolia extract. Experimental study in rats. / Tratamento da fístula anal com extrato de Baccharis dracunculifolia. Estudo experimental em ratos.

OBJECTIVE: to evaluate the efficacy of Baccharis dracunculifolia extract in the treatment of anal fistulas in rats. METHODS: twenty male Wistar rats were submitted to anal fistula and, after 30 days, were divided into three groups: Control Group, with five animals; Carbopol Group, with five animals; and Baccharis dracunculifolia Group, with ten animals. In the Control Group, no treatment was performed. In the Carbopol Group, a daily infusion of Carbopol was performed for 30 days. In the Baccharis dracunculifolia Group, a daily infusion of Carbopol plus Baccharis dracunculifolia extract was performed for 30 days. Specimens were taken for histological analysis after euthanasia. RESULTS: there was no complete closure of the fistulous tract in any of the animals. The mean area of the remaining tract was of 847.2µm2, 565.6µm2 and 372.7µm2, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.001). The mean of the inflammatory process score was of 2.4, 2.4, and 2.1, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.285), while the mean values of vascular congestion were of 1.6, 1.4, and 1.1, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.031). CONCLUSION: Baccharis dracunculifolia extract was able to reduce the lumen of the fistulous tracts and the degree of vascular congestion, without, however, reducing the local inflammatory process or totally closing the fistulous tracts.

OBJETIVO: avaliar a eficácia do extrato de Baccharis dracunculifolia no tratamento de fístulas anais em ratos. MÉTODOS: vinte ratos Wistar machos foram submetidos à confecção de fístula anal e, após 30 dias, foram distribuídos em três grupos: Grupo Controle, com cinco animais; Grupo Carbopol, com cinco animais; e Grupo Baccharis dracunculifolia, com dez animais. No Grupo Controle, não se realizou nenhum tratamento. No Grupo Carbopol, realizou-se infusão diária de carbopol, e no Grupo Baccharis dracunculifolia, infusão de extrato de Baccharis dracunculifolia com carbopol, ambos por 30 dias. Foram retirados espécimes para análise histológica após a eutanásia. RESULTADOS: não houve fechamento completo do trajeto fistuloso em nenhum dos animais. A média da área do trajeto resultante foi de 847,2µm2, 565,6µm2 e 372,7µm2, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,001). A média do escore de processo inflamatório foi de 2,4, 2,4 e 2,1, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,285), enquanto a média de congestão vascular foi de 1,6, 1,4 e 1,1, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,031). CONCLUSÃO: o extrato de Baccharis dracunculifolia foi capaz de reduzir o lúmen dos trajetos fistulosos e a congestão vascular, sem reduzir, no entanto, o processo inflamatório local ou fechar totalmente os trajetos fistulosos.

Tratamento da fístula anal com extrato de Baccharis dracunculifolia. Estudo experimental em ratos / Treatment of anal fistula with Baccharis dracunculifolia extract. Experimental study in rats

RESUMO Objetivo: avaliar a eficácia do extrato de Baccharis dracunculifolia no tratamento de fístulas anais em ratos. Métodos: vinte ratos Wistar machos foram submetidos à confecção de fístula anal e, após 30 dias, foram distribuídos em três grupos: Grupo Controle, com cinco animais; Grupo Carbopol, com cinco animais; e Grupo Baccharis dracunculifolia, com dez animais. No Grupo Controle, não se realizou nenhum tratamento. No Grupo Carbopol, realizou-se infusão diária de carbopol, e no Grupo Baccharis dracunculifolia, infusão de extrato de Baccharis dracunculifolia com carbopol, ambos por 30 dias. Foram retirados espécimes para análise histológica após a eutanásia. Resultados: não houve fechamento completo do trajeto fistuloso em nenhum dos animais. A média da área do trajeto resultante foi de 847,2µm2, 565,6µm2 e 372,7µm2, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,001). A média do escore de processo inflamatório foi de 2,4, 2,4 e 2,1, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,285), enquanto a média de congestão vascular foi de 1,6, 1,4 e 1,1, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,031). Conclusão: o extrato de Baccharis dracunculifolia foi capaz de reduzir o lúmen dos trajetos fistulosos e a congestão vascular, sem reduzir, no entanto, o processo inflamatório local ou fechar totalmente os trajetos fistulosos.

ABSTRACT Objective: to evaluate the efficacy of Baccharis dracunculifolia extract in the treatment of anal fistulas in rats. Methods: twenty male Wistar rats were submitted to anal fistula and, after 30 days, were divided into three groups: Control Group, with five animals; Carbopol Group, with five animals; and Baccharis dracunculifolia Group, with ten animals. In the Control Group, no treatment was performed. In the Carbopol Group, a daily infusion of Carbopol was performed for 30 days. In the Baccharis dracunculifolia Group, a daily infusion of Carbopol plus Baccharis dracunculifolia extract was performed for 30 days. Specimens were taken for histological analysis after euthanasia. Results: there was no complete closure of the fistulous tract in any of the animals. The mean area of the remaining tract was of 847.2µm2, 565.6µm2 and 372.7µm2, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.001). The mean of the inflammatory process score was of 2.4, 2.4, and 2.1, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.285), while the mean values of vascular congestion were of 1.6, 1.4, and 1.1, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.031). Conclusion: Baccharis dracunculifolia extract was able to reduce the lumen of the fistulous tracts and the degree of vascular congestion, without, however, reducing the local inflammatory process or totally closing the fistulous tracts.