Maria Loscertales

Experience-dependent Facilitating Effect of Corticosterone on Spatial Memory Formation in the Water Maze

Stress‐related adrenal steroid hormones modulate brain and cognitive function. Electrophysiological studies, including primed burst potentiation and long‐term potentiation, have indicated concentration‐dependent inverted U‐shape effects of corticosterone in hippocampal function and plasticity. Here, we explored the role of corticosterone in the consolidation and long‐term retrieval of spatial learning in the Morris water maze task in rats. We postulated that corticosterone actions might be experience‐dependent with regard to stimulus intensity, such as differential water temperatures. Indeed, rats trained at 19°C showed a quicker rate of acquisition and better long‐term retention than rats trained at 25°C water. In addition, post‐training corticosterone levels, on the first training day, were significantly higher in rats in the 19°C group than in the 25°C group. Performance of rats trained at 25°C, but not at 19°C, water was improved by injecting them i.p. with corticosterone immediately after each training session. Thus, the effect of exogenously administered corticosterone appears to be experience‐dependent, with the experience‐induced corticosterone concentrations as a critical factor determining the cognitive consequences of steroid treatment. Therefore, this work indicates a facilitating corticosterone action, during the post‐training period, on the neural mechanisms determining the strength of information storage under acute, physiological conditions.

Mutations in LRP2 , which encodes the multiligand receptor megalin, cause Donnai-Barrow and facio-oculo-acoustico-renal syndromes

Donnai-Barrow syndrome is associated with agenesis of the corpus callosum, congenital diaphragmatic hernia, facial dysmorphology, ocular anomalies, sensorineural hearing loss and developmental delay. By studying multiplex families, we mapped this disorder to chromosome 2q23.3–31.1 and identified LRP2 mutations in six families with Donnai-Barrow syndrome and one family with facio-oculo-acoustico-renal syndrome. LRP2 encodes megalin, a multiligand uptake receptor that regulates levels of diverse circulating compounds. This work implicates a pathway with potential pharmacological therapeutic targets.

Opposite effects on NCAM expression in the rat frontal cortex induced by acute vs. chronic corticosterone treatments

The temporal pattern of exposure to glucocorticoids has been reported to be a critical factor in determining the outcome of glucocorticoid actions at the brain. In this work, the effects of different regimes of subcutaneous corticosterone administration (acute-single injection-vs. chronic-daily injection for 21 days) on the expression of the neural cell adhesion molecule (NCAM) were evaluated in different rat brain regions (CA1-CA4, dentate gyrus, frontal cortex, striatum, and hypothalamus). The treatments were selected according to previous studies in which we showed biphasic effects of corticosterone on memory formation, with acute corticosterone effects being facilitating and chronic effects being deleterious. In addition, the chronic treatment was shown by others to result in structural alterations at the hippocampus. NCAM was evaluated given its cell-cell recognition and adhesion properties, and the involvement on synaptic stabilisation subserving long-term memory formation. The results showed a biphasic modulation of NCAM levels at the frontal cortex, with acute corticosterone resulting in enhanced NCAM levels at 8 h and 24 h posttraining, and the chronic treatment decreasing its expression. None of the other brain areas examined showed significant changes in NCAM expression with corticosterone treatments, except for the hypothalamus that showed reduced NCAM levels after the chronic corticosterone regime. These results support the view that NCAM regulation at the frontal cortex might be a mechanism by which corticosterone treatments influence memory formation. They also highlight the hypothalamus as a brain area particularly sensitive to NCAM regulation by prolonged exposure to elevated glucocorticoids.

Chick pulmonary Wnt5a directs airway and vascular tubulogenesis

Wnt5a is an important factor patterning many aspects of early development, including the lung. We find pulmonary non-canonical Wnt5a uses Ror2 to control patterning of both distal air and vascular tubulogenesis (alveolarization). Lungs with mis/overexpressed Wnt5a develop with severe pulmonary hypoplasia associated with altered expression patterns of Shh, L-CAM, fibronectin, VEGF and Flk1. This hypoplastic phenotype is rescued by either replacement of the Shh protein or inhibition of fibronectin function. We find that the effect of Wnt5a on vascular patterning is likely to be through fibronectin-mediated VEGF signaling. These results demonstrate the pivotal role of Wnt5a in directing the essential coordinated development of pulmonary airway and vasculature, by affecting fibronectin levels directly, and by affecting the fibronectin pattern of expression through its regulation of Shh. Data herein suggest that Wnt5a functions in mid-pulmonary patterning (during alveolarization), and is distinct from the Wnt canonical pathway which is more important in earlier lung development.

Congenital diaphragmatic hernia candidate genes derived from embryonic transcriptomes

Congenital diaphragmatic hernia (CDH) is a common (1 in 3,000 live births) major congenital malformation that results in significant morbidity and mortality. The discovery of CDH loci using standard genetic approaches has been hindered by its genetic heterogeneity. We hypothesized that gene expression profiling of developing embryonic diaphragms would help identify genes likely to be associated with diaphragm defects. We generated a time series of whole-transcriptome expression profiles from laser captured embryonic mouse diaphragms at embryonic day (E)11.5 and E12.5 when experimental perturbations lead to CDH phenotypes, and E16.5 when the diaphragm is fully formed. Gene sets defining biologically relevant pathways and temporal expression trends were identified by using a series of bioinformatic algorithms. These developmental sets were then compared with a manually curated list of genes previously shown to cause diaphragm defects in humans and in mouse models. Our integrative filtering strategy identified 27 candidates for CDH. We examined the diaphragms of knockout mice for one of the candidate genes, pre–B-cell leukemia transcription factor 1 (Pbx1), and identified a range of previously undetected diaphragmatic defects. Our study demonstrates the utility of genetic characterization of normal development as an integral part of a disease gene identification and prioritization strategy for CDH, an approach that can be extended to other diseases and developmental anomalies.

Congenital diaphragmatic hernia interval on chromosome 8p23.1 characterized by genetics and protein interaction networks

Chromosome 8p23.1 is a common hotspot associated with major congenital malformations, including congenital diaphragmatic hernia (CDH) and cardiac defects. We present findings from high‐resolution arrays in patients who carry a loss (n = 18) or a gain (n = 1) of sub‐band 8p23.1. We confirm a region involved in both diaphragmatic and heart malformations. Results from a novel CNVConnect algorithm, prioritizing protein–protein interactions between products of genes in the 8p23.1 hotspot and products of previously known CDH causing genes, implicated GATA4, NEIL2, and SOX7 in diaphragmatic defects. Sequence analysis of these genes in 226 chromosomally normal CDH patients, as well as in a small number of deletion 8p23.1 patients, showed rare unreported variants in the coding region; these may be contributing to the diaphragmatic phenotype. We also demonstrated that two of these three genes were expressed in the E11.5–12.5 primordial mouse diaphragm, the developmental stage at which CDH is thought to occur. This combination of bioinformatics and expression studies can be applied to other chromosomal hotspots, as well as private microdeletions or microduplications, to identify causative genes and their interaction networks.

The corticosteroid synthesis inhibitors metyrapone and aminoglutethimide impair long-term memory for a passive avoidance task in day-old chicks.

Long-term memory for a passive avoidance task in day-old chicks has proved to depend upon an action of the adrenal steroid corticosterone through specific receptors in a brain region, the intermediate medial hyperstriatum ventrale (IMHV), involved in learning the task. In this study, we questioned whether pretraining peripheral administration of drugs described to inhibit either basal levels of corticosterone - aminoglutethimide - or treatment-induced stimulated corticosterone secretion - metyrapone - might interfere with retention for the task at 24 h post-training. The results showed a dose-dependent effect of the inhibitors, with the highest doses tested for both drugs (10 and 50 mg/kg for metyrapone, and 50 mg/kg for aminoglutethimide) being amnestic for the task. Additional experiments, in which we studied possible effects of the inhibitors on concomitant aspects of learning (i.e., reactivity to novelty, and pecking pattern), show that the drugs did not affect general behavioural reactivity. The present results thus support the idea that training-induced corticosterone release plays a key role in the neurobiological processes that determine the establishment of a persistent memory for the aversively motivated avoidance response. In addition, they point to corticosteroid inhibitor drugs as potential tools for the study of the interactions between steroid hormones and cognition-enhancing compounds in this learning task.

Prevalence and penetrance of ZFPM2 mutations and deletions causing congenital diaphragmatic hernia.

Zinc finger protein, FOG2 family member 2 (ZFPM2) (previously named FOG2) gene defects result in the highly morbid congenital diaphragmatic hernia (CDH) in humans and animal models. In a cohort of 275 CDH patient exomes, we estimated the prevalence of damaging ZFPM2 mutations to be almost 5%. Genetic analysis of a multigenerational family identified a heritable intragenic ZFPM2 deletion with an estimated penetrance of 37.5%, which has important implications for genetic counseling. Similarly, a low penetrance ZFPM2 frameshift mutation was observed in a second multiplex family. Isolated CDH was the predominant phenotype observed in our ZFPM2 mutation patients. Findings from the patients described herein indicate that ZFPM2 point mutations or deletions are a recurring cause of CDH.

Piracetam facilitates long-term memory for a passive avoidance task in chicks through a mechanism that requires a brain corticosteroid action.

We investigated the effects of piracetam, a nootropic, on learning and memory formation for a passive avoidance task in day‐old chicks. To test for the possible cognitive‐enhancing properties of piracetam, a weak learning version of this task – whereby chicks maintain a memory to avoid pecking at a bead coated in a diluted aversant for up to 10 h – was used. Post‐training (5, 30 or 60 min), but not pretraining, injections of piracetam (10 or 50 mg/kg, i.p.) increased recall for the task when the chicks were tested 24 h later. Because previous studies showed that long‐term memory for the passive avoidance task is dependent upon a brain corticosteroid action, and because the efficacy of piracetam‐like compounds is also modulated by corticosteroids, we tested whether the facilitating effect of piracetam was dependent upon a corticosteroid action through specific brain receptors (mineralocorticoid receptor and glucocorticoid receptor). First, increased plasma levels of corticosterone were found 5 min after piracetam injection. In addition, intracerebral administration of antagonists for each receptor type (RU28318, for mineralocorticoid receptors, and RU38486 for glucocorticoid receptors; i.c.) given before the nootropic inhibited the facilitative effect of piracetam on memory consolidation. These results give further support to a modulatory action of piracetam on the mechanisms involved in long‐term memory formation through a neural action that, in this learning model, requires the activation of the two types of intracellular corticosteroid receptors.

Genome-wide enrichment of damaging de novo variants in patients with isolated and complex congenital diaphragmatic hernia.

Congenital Diaphragmatic Hernia (CDH) is a common and often lethal birth defect characterized by diaphragmatic structural defects and pulmonary hypoplasia. CDH is isolated in 60% of newborns, but may also be part of a complex phenotype with additional anomalies. We performed whole exome sequencing (WES) on 87 individuals with isolated or complex CDH and on their unaffected parents, to assess the contribution of de novo mutations in the etiology of diaphragmatic and pulmonary defects and to identify new candidate genes. A combined analysis with 39 additional trios with complex CDH, previously published, revealed a significant genome-wide burden of de novo variants compared to background mutation rate and 900 control trios. We identified an increased burden of likely gene-disrupting (LGD, i.e. nonsense, frameshift, and canonical splice site) and predicted deleterious missense (D-mis) variants in complex and isolated CDH patients. Overall, an excess of predicted damaging de novo LGD and D-mis variants relative to the expected frequency contributed to 21% of complex cases and 12% of isolated CDH cases. The burden of de novo variants was higher in genes expressed in the developing mouse diaphragm and heart. Some overlap with genes responsible for congenital heart defects and neurodevelopmental disorders was observed in CDH patients within our cohorts. We propose that de novo variants contribute significantly to the development of CDH.