An Update of Nucleic Acids Aptamers Theranostic Integration with CRISPR/Cas Technology.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system is best known for its role in genomic editing. It has also demonstrated great potential in nucleic acid biosensing. However, the specificity limitation in CRISPR/Cas has created a hurdle for its advancement. More recently, nucleic acid aptamers known for their high affinity and specificity properties for their targets have been integrated into CRISPR/Cas systems. This review article gives a brief overview of the aptamer and CRISPR/Cas technology and provides an updated summary and discussion on how the two distinctive nucleic acid technologies are being integrated into modern diagnostic and therapeutic applications.

Social isolation alters hypothalamic pituitary adrenal axis activity after chronic variable stress in male C57BL/6 mice.

The chronic variable stress (CVS) paradigm is frequently used to model the changes in hypothalamic pituitary adrenal (HPA) axis function characteristic of many stress-related diseases. However, male C57BL/6 mice are typically resistant to CVS's effects, making it difficult to determine how chronic stress exposure may alter acute HPA function and regulation in these mice. As social support in rodents can profoundly influence physiological and behavioral processes, including the HPA axis, we sought to characterize the effects of CVS exposure on basal and acute stress-induced HPA axis function in pair- and single-housed adult male mice. Despite all subjects exhibiting decreased body weight gain after six weeks of CVS, the corticosterone response to a novel, acute restraint stressor was enhanced by CVS exclusively in single-housed males. CVS also significantly increased arginine vasopressin (AVP) mRNA in the hypothalamic paraventricular nucleus (PVN) in single-housed males only. Moreover, in single-, but not pair-housed mice, CVS attenuated decreases in circulating OT found following acute restraint. Only the effect of CVS to elevate PVN corticotropin releasing hormone (CRH) mRNA levels after an acute stressor was restricted to pair-housed mice. Collectively, our findings suggest that social isolation reveals effects of CVS on the HPA axis in male C57BL/6 mice.

Maternal immune activation with toll-like receptor 7 agonist during mid-gestation alters juvenile and adult developmental milestones and behavior.

Infections during pregnancy are associated with increased risk for adult neuropsychiatric disease, such as major depressive disorder, schizophrenia, and autism spectrum disorder. In mouse models of maternal immune activation (MIA), different toll-like receptors (TLRs) are stimulated to initiate inflammatory responses in mother and fetus. The goal of this study was to determine sex-dependent aspects of MIA using a TLR7/8 agonist, Resiquimod (RQ), on neurodevelopment. RQ was administered to timed-pregnant mice on embryonic day (E) 12.5. At E15, maternal/fetal plasma cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Maternal cytokines interleukin (IL)-6 and IL-10 were higher while tumor necrosis factor (TNF)-α and IL-17 were lower in pregnant dams exposed to RQ. Fetal cytokines (E15) were altered at the same timepoint with fetal plasma IL-6 and IL-17 greater after RQ compared to vehicle, while IL-10 and TNF-α were higher in male fetuses but not female. Other timed-pregnant dams were allowed to give birth. MIA with RQ did not alter the female to male ratio of offspring born per litter. Body weights were reduced significantly in both sexes at birth, and over the next 5 weeks. Offspring from RQ-injected mothers opened their eyes 5 days later than controls. Similarly, female offspring from RQ-injected mothers exhibited pubertal delay based on vaginal opening 2-3 days later than control females. On the behavioral side, juvenile and adult male and female MIA offspring exhibited less social-like behavior in a social interaction test. Anhedonia-like behavior was greater in MIA adult female mice. This study provides support for sex-dependent influences of fetal antecedents for altered brain development and behavioral outputs that could be indicative of increased susceptibility for adult disorders through immune mechanisms. Future studies are needed to determine neural cellular and molecular mechanisms for such programming effects.

Evaluating different models of maternal stress on stress-responsive systems in prepubertal mice.

Introduction: Maternal adversity during pregnancy influences neurodevelopment in human and model animal offspring. Adversity can result from stressors coming from many different directions ranging from environmental to nutritional and physiological to immune (e.g., infection). Most stressors result in fetal overexposure to glucocorticoids that have been directly linked to long- and short-term negative impacts on neurological health of offspring. Neuropsychiatric diseases postulated to have fetal origins are diverse and include such things cardiovascular disease, obesity, affective disorders, and metabolic and immune disorders. Methods: The experiments in the current study compare 3 stressors: prenatal exposure to dexamethasone (DEX), maternal high fat diet (HFD), and maternal caloric restriction (CR). Offspring of mothers with these treatments were examined prepubertally to evaluate stress responsiveness and stress-related behaviors in in male and female mice. Results: Prenatal exposure to synthetic glucocorticoid, DEX, resulted in decreased neonatal body weights, reduced social interaction behavior, and hypoactive stress response offspring exposed to maternal DEX. Maternal CR resulted in decreased body weights and social interaction behavior in males and females and increased anxiety-like behavior and acute stress response only in males. HFD resulted in altered body weight gain in both sex offspring with decreased anxiety-like behavior in a female-biased manner. Discussion: The idea that glucocorticoid responses to different stressors might serve as a common stimulus across stress paradigms is insufficient, given that different modes of prenatal stress produced differential effects. Opposite nutritional stressors produced similar outcomes for anxiety-like behavior in both sexes, social-like behavior in females, and a hyperactive adrenal stress response in males. One common theme among the three models of maternal stress (DEX, CR, and HFD) was consistent data showing their role in activating the maternal and fetal immune response. By tuning in on the more immediate immunological aspect on the developing fetus (e.g., hormones, cytokines), additional studies may tease out more direct outcomes of maternal stress in rodents and increase their translational value to human studies.

Sex Differences in Acute Neuroendocrine Responses to Stressors in Rodents and Humans.

Sex differences in the neuroendocrine response to acute stress occur in both animals and humans. In rodents, stressors such as restraint and novelty induce a greater activation of the hypothalamic-pituitary-adrenal axis (HPA) in females compared to males. The nature of this difference arises from steroid actions during development (organizational effects) and adulthood (activational effects). Androgens decrease HPA stress responsivity to acute stress, while estradiol increases it. Androgenic down-regulation of HPA responsiveness is mediated by the binding of testosterone (T) and dihydrotestosterone (DHT) to the androgen receptor, as well as the binding of the DHT metabolite, 3ß-diol, to the ß form of the estrogen receptor (ERß). Estradiol binding to the α form of the estrogen receptor (ERα) increases HPA responsivity. Studies of human sex differences are relatively few and generally employ a psychosocial paradigm to measure stress-related HPA activation. Men consistently show greater HPA reactivity than women when being evaluated for achievement. Some studies have found greater reactivity in women when being evaluated for social performance. The pattern is inconsistent with rodent studies but may involve the differential nature of the stressors employed. Psychosocial stress is nonphysical and invokes a significant degree of top-down processing that is not easily comparable to the types of stressors employed in rodents. Gender identity may also be a factor based on recent work showing that it influences the neural processing of positive and negative emotional stimuli independent of genetic sex. Comparing different types of stressors and how they interact with gender identity and genetic sex will provide a better understanding of sex steroid influences on stress-related HPA reactivity.

Glucocorticoid regulation of diurnal spine plasticity in the murine ventromedial prefrontal cortex.

The ventromedial prefrontal cortex (vmPFC) regulates fear acquisition, fear extinction, mood, and HPA axis function. Multiple brain regions exhibit time-of-day dependent variations in learning, long term potentiation (LTP), and dendritic morphology. Glucocorticoids have been implicated in the regulation of dendritic structure in the context of stress. Glucocorticoids are also known to regulate molecular clock entrainment via upregulation of Per1 transcription. In the present study, C57BL/6 N mice were sacrificed at three distinct times of day (ZT3, ZT12, and ZT16, lights off at ZT12) and Per1 mRNA expression was measured in the infralimbic and prelimbic vmPFC subregions using droplet digital (dd) PCR after recovering from adrenalectomy or sham surgery for 10 days. Sham mice showed Per1 rhythmicity in both infralimbic (IL) and prelimbic (PL) cortex, with peak expression occurring at ZT12. Adrenalectomized mice showed reductions in Per1 amplitude at ZT12 in both IL and PL, suggesting that the vmPFC molecular clock is entrained by diurnal glucocorticoid oscillations. Thy1-eGFP mice were used to visualize and quantify dendritic spine density on deep layer pyramidal dendrites at ZT 3, 12, and 16. Spine density in both PL and IL exhibited changes between the light (inactive) and dark (active) phases, with peak spine density observed at ZT16 and trough spine density observed at ZT3. These changes in spine density were restricted to changes in long thin and stubby type spines. To determine if changes in spine density is regulated by glucocorticoid oscillations, the 11ß-hydroxylase inhibitor metyrapone was administered 2 h prior to the onset of the active phase (ZT10) daily for 7 days. Metyrapone administration blocked both the diurnal peak of plasma corticosterone and peak spine densities in the IL and PL at ZT16. These results suggest that vmPFC molecular clock gene and dendritic spine diurnal rhythms depend on intact diurnal glucocorticoid oscillations.

Androgens and Their Role in Regulating Sex Differences in the Hypothalamic/Pituitary/Adrenal Axis Stress Response and Stress-Related Behaviors.

Androgens play a pivotal role during development. These gonadal hormones and their receptors exert organizational actions that shape brain morphology in regions controlling the stress regulatory systems in a male-specific manner. Specifically, androgens drive sex differences in the hypothalamic/pituitary/adrenal (HPA) axis and corresponding hypothalamic neuropeptides. While studies have examined the role of estradiol and its receptors in sex differences in the HPA axis and associated behaviors, the role of androgens remains far less studied. Androgens are generally thought to modulate the HPA axis through the activation of androgen receptors (ARs). They can also impact the HPA axis through reduction to estrogenic metabolites that can bind estrogen receptors in the brain and periphery. Such regulation of the HPA axis stress response by androgens can often result in sex-biased risk factors for stress-related disorders, such as anxiety and depression. This review focuses on the biosynthesis pathways and molecular actions of androgens and their nuclear receptors. The impact of androgens on hypothalamic neuropeptide systems (corticotropin-releasing hormone, arginine vasopressin, oxytocin, dopamine, and serotonin) that control the stress response and stress-related disorders is discussed. Finally, this review discusses potential therapeutics involving androgens (androgen replacement therapies, selective AR modulator therapies) and ongoing clinical trials.

The Hypothalamic-Pituitary-Adrenal Axis: Development, Programming Actions of Hormones, and Maternal-Fetal Interactions.

The hypothalamic-pituitary-adrenal axis is a complex system of neuroendocrine pathways and feedback loops that function to maintain physiological homeostasis. Abnormal development of the hypothalamic-pituitary-adrenal (HPA) axis can further result in long-term alterations in neuropeptide and neurotransmitter synthesis in the central nervous system, as well as glucocorticoid hormone synthesis in the periphery. Together, these changes can potentially lead to a disruption in neuroendocrine, behavioral, autonomic, and metabolic functions in adulthood. In this review, we will discuss the regulation of the HPA axis and its development. We will also examine the maternal-fetal hypothalamic-pituitary-adrenal axis and disruption of the normal fetal environment which becomes a major risk factor for many neurodevelopmental pathologies in adulthood, such as major depressive disorder, anxiety, schizophrenia, and others.

Chronic variable stress alters hypothalamic-pituitary-adrenal axis function in the female mouse.

Chronic stress is often associated with a dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, which can greatly increase risk for a number of stress-related diseases, including neuropsychiatric disorders. Despite a striking sex-bias in the prevalence of many of these disorders, few preclinical studies have examined female subjects. Hence, the present study aimed to explore the effects of chronic stress on the basal and acute stress-induced activity of the HPA axis in the female C57BL/6 mouse. We used a chronic variable stress (CVS) paradigm in these studies, which successfully induces physiological and behavioral changes that are similar to those reported for some patients with mood disorders. Using this model, we found pronounced, time-dependent effects of chronic stress on the HPA axis. CVS-treated females exhibited adrenal hypertrophy, yet their pattern of glucocorticoid secretion in the morning resembled that of controls. CVS-treated and control females had similar morning basal corticosterone (CORT) levels, which were both significantly elevated following a restraint stressor. Although morning basal gene expression of the key HPA-controlling neuropeptides corticotropin releasing hormone (CRH), arginine vasopressin (AVP) and oxytocin (OT) was unaltered within the paraventricular nucleus (PVN) by CVS, CVS altered the PVN OT and AVP mRNA responses to acute restraint. In control females, acute stress decreased AVP, but not OT mRNA; whereas, in CVS females, it decreased OT, but not, AVP mRNA. Unlike the morning pattern of HPA activity, in the evening, CVS-treated females showed increased basal CORT with hypoactive responses of CORT and PVN c-Fos immunoreactivity to restraint stress. Furthermore, CVS elevated evening PVN CRH and OT mRNAs in the PVN, but it did not influence anxiety- or depressive-like behavior after a light/dark box or tail suspension test. Taken together, these findings indicate that CVS is an effective model for HPA axis dysregulation in the female mouse and may be relevant for stress-related diseases.