Lipid production in the industrial microalga Nannochloropsis gaditana exceeds that of model algal species and can be maximized by nutrient starvation in batch culture. However, starvation halts growth, thereby decreasing productivity. Efforts to engineer N. gaditana strains that can accumulate biomass and overproduce lipids have previously met with little success. We identified 20 transcription factors as putative negative regulators of lipid production by using RNA-seq analysis of N. gaditana during nitrogen deprivation. Application of a CRISPR-Cas9 reverse-genetics pipeline enabled insertional mutagenesis of 18 of these 20 transcription factors. Knocking out a homolog of fungal Zn(II)Cys-encoding genes improved partitioning of total carbon to lipids from 20% (wild type) to 40-55% (mutant) in nutrient-replete conditions. Knockout mutants grew poorly, but attenuation of Zn(II)Cys expression yielded strains producing twice as much lipid (∼5.0 g m d) as that in the wild type (∼2.5 g m d) under semicontinuous growth conditions and had little effect on growth.
Regulation of the hypothalamic-pituitary-adrenal (HPA) axis is critical for adaptation to environmental changes. The principle regulator of the HPA axis is corticotrophin-releasing hormone (CRH), which is made in the parventricular nucleus and is an important target of negative feedback by glucocorticoids. However, the molecular mechanisms that regulate CRH are not fully understood. Disruption of normal HPA axis activity is a major risk factor of neuropsychiatric disorders in which decreased expression of the glucocorticoid receptor (GR) has been documented. To investigate the role of the GR in CRH neurons, we have targeted the deletion of the GR, specifically in the parventricular nucleus. Impairment of GR function in the parventricular nucleus resulted in an enhancement of CRH expression and an up-regulation of hypothalamic levels of BDNF and disinhibition of the HPA axis. BDNF is a stress and activity-dependent factor involved in many activities modulated by the HPA axis. Significantly, ectopic expression of BDNF in vivo increased CRH, whereas reduced expression of BDNF, or its receptor TrkB, decreased CRH expression and normal HPA functions. We find the differential regulation of CRH relies upon the cAMP response-element binding protein coactivator CRTC2, which serves as a switch for BDNF and glucocorticoids to direct the expression of CRH.knockout | cAMP response-element binding protein | CRTC2 | transcription T he hypothalamic-pituitary-adrenal axis (HPA) axis is regulated by the corticotrophin-releasing hormone (CRH), which controls the secretion of adrenocorticotropin (ACTH) from the anterior pituitary and glucocorticoids from the adrenal gland (1). Stress or threats activate the HPA axis via polysynaptic circuits that converge on the hypothalamic paraventricular nucleus (PVN) to activate CRH-producing neurons (2). Classically, endocrine feedback control is characterized by the down-regulation of CRH and ACTH by glucocorticoids to prevent the escalation of glucocorticoids to toxic levels (1, 3). Maintenance of the HPA axis, therefore, involves a homeostatic equilibrium between the activation and inhibitory feedback. Disruption of normal HPA axis is a major risk factor of neuropsychiatric disorders (4, 5). Elevated CRH levels have been documented in the cerebrospinal fluid of depressed individuals exhibiting hypercortisolism, indicating a close relationship between the production of glucocorticoids and CRH (4). Both activation and feedback regulation require the glucocorticoid receptor (GR), which occurs in brain structures that control HPA axis reactivity (2, 6).We find that genetic disruption of GR in the PVN disinhibited both the HPA axis and the expression of hypothalamic BDNF. Neurotrophic factors, such as BDNF, are involved in regulating many functions carried out by the HPA axis (7). BDNF and its receptor, TrkB, are both expressed in the PVN (8, 9) and many other brain regions, and display dramatic effects upon neuroprotection and synaptic plasticity (10, 11). Despite studies that indicate that BDN...
Neurotrophins and glucocorticoids are robust synaptic modifiers, and deregulation of their activities is a risk factor for developing stress-related disorders. Low levels of brain-derived neurotrophic factor (BDNF) increase the desensitization of glucocorticoid receptors (GR) and vulnerability to stress, whereas higher levels of BDNF facilitate GR-mediated signaling and the response to antidepressants. However, the molecular mechanism underlying neurotrophic-priming of GR function is poorly understood. Here we provide evidence that activation of a TrkB-MAPK pathway, when paired with the deactivation of a GR-protein phosphatase 5 pathway, resulted in sustained GR phosphorylation at BDNF-sensitive sites that is essential for the transcription of neuronal plasticity genes. Genetic strategies that disrupted GR phosphorylation or TrkB signaling in vivo impaired the neuroplasticity to chronic stress and the effects of the antidepressant fluoxetine. Our findings reveal that the coordinated actions of BDNF and glucocorticoids promote neuronal plasticity and that disruption in either pathway could set the stage for the development of stress-induced psychiatric diseases.BDNF | glucocorticoid | coincidence detection | stress | antidepressant G lucocorticoids can either facilitate or deteriorate the structure and function of brain circuits involved in perception, cognition, and mood by modulating neurotransmission and remodeling dendritic spines as a function of time at exposure, dose, and duration (1, 2). Neuronal growth defects and cognitive impairment manifest upon disruption of circadian oscillations and of stress-mediated peaks of glucocorticoids, common features of major depression (3). Paradoxically, despite the often high glucocorticoid concentrations in patients with depression, signaling through glucocorticoid receptors (GR) appears defective, as evidenced by the inability of the endocrine stress response to be suppressed by dexamethasone, and reduction in expression of GR-sensitive genes and an inability of GR to suppress inflammation (4, 5). This so called "glucocorticoid-resistant" state is not solely the result of GR down-regulation (6, 7). What accounts for this lack of GR responsiveness during chronic stress is not understood.One hypothesis to account for glucocorticoid resistance is that additional stress-sensitive pathways influence GR function (8). For example, brain-derived neurotrophic factor (BDNF), is essential for the enhancement of contextual fear memories by glucocorticoids (9, 10). In contrast, BDNF-deficiency diminished the complexity of hippocampal dendritic arborization without further atrophy by stress (11). This raises the interesting question of whether BDNF initiates a cellular pathway that could modulate GR activity during stress. Using mass spectrometry, we previously found that BDNF promotes the phosphorylation of GR at two conserved phosphorylation sites (Fig. 1A) involved in the expression of select glucocorticoid-regulated genes when BDNF and glucocorticoid stimulation were paired in ...
Abnormal glucocorticoid and neurotrophin signaling has been implicated in numerous psychiatric disorders. However, the impact of neurotrophic signaling on glucocorticoid receptor (GR)-dependent gene expression is not understood. We therefore examined the impact of brain-derived neurotrophic factor (BDNF) signaling on GR transcriptional regulatory function by gene expression profiling in primary rat cortical neurons stimulated with the selective GR agonist dexamethasone (Dex) and BDNF, alone or in combination. Simultaneous treatment with BDNF and Dex elicited a unique set of GR-responsive genes associated with neuronal growth and differentiation and also enhanced the induction of a large number of Dex-sensitive genes. BDNF via its receptor TrkB enhanced the transcriptional activity of a synthetic GR reporter, suggesting a direct effect of BDNF signaling on GR function. Indeed, BDNF treatment induces the phosphorylation of GR at serine 155 (S155) and serine 287 (S287). Expression of a nonphosphorylatable mutant (GR S155A/S287A) impaired the induction of a subset of BDNF-and Dex-regulated genes. Mechanistically, BDNF-induced GR phosphorylation increased GR occupancy and cofactor recruitment at the promoter of a BDNF-enhanced gene. GR phosphorylation in vivo is sensitive to changes in the levels of BDNF and TrkB as well as stress. Therefore, BDNF signaling specifies and amplifies the GR transcriptome through a coordinated GR phosphorylation-dependent detection mechanism.
The lack of diversity among faculty at universities and medical schools in the United States is a matter of growing concern. However, the factors that influence the career choices of underrepresented minority and female postdoctoral researchers have received relatively little attention. Here we report the results of a survey of 1284 postdocs working in the biomedical sciences in the US. Our findings highlight possible reasons why some underrepresented minority and female postdocs choose not to pursue careers in academic research, and suggest interventions that could be taken in the early stages of postdoctoral training to prevent this attrition of underrepresented groups.
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