Adi Neufeld-Cohen

Resilience to social stress coincides with functional DNA methylation of the Crf gene in adult mice

DNA methylation regulates gene transcription and has been suggested to encode psychopathologies derived from early life stress. We found that methylation regulated the expression of the Crf (also known as Crh) gene and that chronic social stress in adult mice induced long-term demethylation of this genomic region. Demethylation was observed only in the subset of defeated mice that displayed social avoidance and site-specific knockdown of Crf attenuated the stress-induced social avoidance.

Circadian Clocks and Feeding Time Regulate the Oscillations and Levels of Hepatic Triglycerides

Circadian clocks play a major role in orchestrating daily physiology, and their disruption can evoke metabolic diseases such as fatty liver and obesity. To study the role of circadian clocks in lipid homeostasis, we performed an extensive lipidomic analysis of liver tissues from wild-type and clock-disrupted mice either fed ad libitum or night fed. To our surprise, a similar fraction of lipids (∼17%) oscillated in both mouse strains, most notably triglycerides, but with completely different phases. Moreover, several master lipid regulators (e.g., PPARα) and enzymes involved in triglyceride metabolism retained their circadian expression in clock-disrupted mice. Nighttime restricted feeding shifted the phase of triglyceride accumulation and resulted in ∼50% decrease in hepatic triglyceride levels in wild-type mice. Our findings suggest that circadian clocks and feeding time dictate the phase and levels of hepatic triglyceride accumulation; however, oscillations in triglycerides can persist in the absence of a functional clock.

Prolonged and site-specific over-expression of corticotropin-releasing factor reveals differential roles for extended amygdala nuclei in emotional regulation

Corticotropin-releasing factor (CRF) has a key role in the central stress response, and altered levels of this neuropeptide are linked to stress-related psychopathologies such as anxiety and depression. These disorders are associated with the inability to properly regulate stress response, specifically following exposure to prolonged stressful stimuli. Therefore, the current study assessed the effects of prolonged and site-specific over-expression of CRF, which mimics the state of chronic production, in extended amygdala nuclei that are known to be involved in mediating anxiety-like states. We first constructed and generated lentiviruses that overexpress (OE) CRF in a robust and stable manner, and then generated two male mouse models continuously over-expressing CRF, either at the central nucleus of the amygdala (CeA), or at the dorsolateral subdivision of the bed nucleus of the stria terminalis (BNSTdl). After 4 months, behavioral assessments were conducted for anxiety and depressive indices on these mice. Surprisingly, prolonged CRF OE at the CeA attenuated stress-induced anxiety-like behaviors, whereas prolonged CRF OE in the BNSTdl increased depressive-like behaviors, without affecting anxiety levels. These results show possible differential roles for CRF expressed by distinct loci of the extended amygdala, in mediating stress-induced emotional behaviors.

A triple urocortin knockout mouse model reveals an essential role for urocortins in stress recovery

Responding to stressful events requires numerous adaptive actions involving integrated changes in the central nervous and neuroendocrine systems. Numerous studies have implicated dysregulation of stress-response mechanisms in the etiology of stress-induced psychopathophysiologies. The urocortin neuropeptides are members of the corticotropin-releasing factor family and are associated with the central stress response. In the current study, a triple-knockout (tKO) mouse model lacking all three urocortin genes was generated. Intriguingly, these urocortin tKO mice exhibit increased anxiety-like behaviors 24 h following stress exposure but not under unstressed conditions or immediately following exposure to acute stress. The inability of these mutants to recover properly from the exposure to an acute stress was associated with robust alterations in the expression profile of amygdalar genes and with dysregulated serotonergic function in stress-related neurocircuits. These findings position the urocortins as essential factors in the stress-recovery process and suggest the tKO mouse line as a useful stress-sensitive mouse model.

Circadian control of oscillations in mitochondrial rate-limiting enzymes and nutrient utilization by PERIOD proteins

Significance Mitochondria are major cellular energy suppliers and have to cope with changes in nutrient supply and energy demand that naturally occur throughout the day. We obtained the first, to our knowledge, comprehensive mitochondrial proteome around the clock and identified extensive oscillations in mitochondrial protein abundance that predominantly peak during the early light phase. Remarkably, several rate-limiting mitochondrial enzymes that process different nutrients accumulate in a diurnal manner and are dependent on the clock proteins PER1/2. Concurrently, we uncovered daily oscillations in mitochondrial respiration that are substrate-specific and peak during different times of the day. We propose that the circadian clock PERIOD proteins regulate the diurnal utilization of different nutrients by the mitochondria and thus, optimize mitochondrial function to daily changes in energy supply/demand. Mitochondria are major suppliers of cellular energy through nutrients oxidation. Little is known about the mechanisms that enable mitochondria to cope with changes in nutrient supply and energy demand that naturally occur throughout the day. To address this question, we applied MS-based quantitative proteomics on isolated mitochondria from mice killed throughout the day and identified extensive oscillations in the mitochondrial proteome. Remarkably, the majority of cycling mitochondrial proteins peaked during the early light phase. We found that rate-limiting mitochondrial enzymes that process lipids and carbohydrates accumulate in a diurnal manner and are dependent on the clock proteins PER1/2. In this conjuncture, we uncovered daily oscillations in mitochondrial respiration that peak during different times of the day in response to different nutrients. Notably, the diurnal regulation of mitochondrial respiration was blunted in mice lacking PER1/2 or on a high-fat diet. We propose that PERIOD proteins optimize mitochondrial metabolism to daily changes in energy supply/demand and thereby, serve as a rheostat for mitochondrial nutrient utilization.

Susceptibility to PTSD-Like Behavior Is Mediated by Corticotropin-Releasing Factor Receptor Type 2 Levels in the Bed Nucleus of the Stria Terminalis

Posttraumatic stress disorder (PTSD) is a debilitating disease, which affects 8–10% of the population exposed to traumatic events. The factors that make certain individuals susceptible to PTSD and others resilient are currently unknown. Corticotropin-releasing factor receptor type 2 (CRFR2) has been implicated in mediating stress coping mechanisms. Here, we use a physiological PTSD-like animal model and an in-depth battery of tests that reflect the symptomology of PTSD to separate mice into subpopulations of “PTSD-like” and “Resilient” phenotypes. PTSD-like mice are hypervigilant, hyperalert, insomniac, have impaired attention and risk assessment, as well as accompanying attenuated corticosterone levels. Intriguingly, PTSD-like mice show long-term robust upregulation of BNST-CRFR2 mRNA levels, and BNST-CRFR2-specific lentiviral knockdown reduces susceptibility to PTSD-like behavior. Additionally, using a BNST mRNA expression array, PTSD-like mice exhibit a general transcriptional attenuation profile, which was associated with upregulation of the BNST-deacetylation enzyme, HDAC5. We suggest PTSD to be a disease of maladaptive coping.

Urocortin-1 and -2 double-deficient mice show robust anxiolytic phenotype and modified serotonergic activity in anxiety circuits

The urocortin (Ucn) family of neuropeptides is suggested to be involved in homeostatic coping mechanisms of the central stress response through the activation of corticotropin-releasing factor receptor type 2 (CRFR2). The neuropeptides, Ucn1 and Ucn2, serve as endogenous ligands for the CRFR2, which is highly expressed by the dorsal raphe serotonergic neurons and is suggested to be involved in regulating major component of the central stress response. Here, we describe genetically modified mice in which both Ucn1 and Ucn2 are developmentally deleted. The double knockout mice showed a robust anxiolytic phenotype and altered hypothalamic–pituitary–adrenal axis activity compared with wild-type mice. The significant reduction in anxiety-like behavior observed in these mice was further enhanced after exposure to acute stress, and was correlated with the levels of serotonin and 5-hydroxyindoleacetic acid measured in brain regions associated with anxiety circuits. Thus, we propose that the Ucn/CRFR2 serotonergic system has an important role in regulating homeostatic equilibrium under challenge conditions.

Perifornical Urocortin-3 mediates the link between stress-induced anxiety and energy homeostasis

In response to physiological or psychological challenges, the brain activates behavioral and neuroendocrine systems linked to both metabolic and emotional outputs designed to adapt to the demand. However, dysregulation of integration of these physiological responses to challenge can have severe psychological and physiological consequences, and inappropriate regulation, disproportional intensity, or chronic or irreversible activation of the stress response is linked to the etiology and pathophysiology of mood and metabolic disorders. Using a transgenic mouse model and lentiviral approach, we demonstrate the involvement of the hypothalamic neuropeptide Urocortin-3, a specific ligand for the type-2 corticotropin-releasing factor receptor, in modulating septal and hypothalamic nuclei responsible for anxiety-like behaviors and metabolic functions, respectively. These results position Urocortin-3 as a neuromodulator linking stress-induced anxiety and energy homeostasis and pave the way toward better understanding of the mechanisms that mediate the reciprocal relationships between stress, mood and metabolic disorders.

Protein Tyrosine Phosphatase Epsilon Affects Body Weight by Downregulating Leptin Signaling in a Phosphorylation-Dependent Manner

Molecular-level understanding of body weight control is essential for combating obesity. We show that female mice lacking tyrosine phosphatase epsilon (RPTPe) are protected from weight gain induced by high-fat food, ovariectomy, or old age and exhibit increased whole-body energy expenditure and decreased adiposity. RPTPe-deficient mice, in particular males, exhibit improved glucose homeostasis. Female nonobese RPTPe-deficient mice are leptin hypersensitive and exhibit reduced circulating leptin concentrations, suggesting that RPTPe inhibits hypothalamic leptin signaling in vivo. Leptin hypersensitivity persists in aged, ovariectomized, and high-fat-fed RPTPe-deficient mice, indicating that RPTPe helps establish obesity-associated leptin resistance. RPTPe associates with and dephosphorylates JAK2, thereby downregulating leptin receptor signaling. Leptin stimulation induces phosphorylation of hypothalamic RPTPe at its C-terminal Y695, which drives RPTPe to downregulate JAK2. RPTPe is therefore an inhibitor of hypothalamic leptin signaling in vivo, and provides controlled negative-feedback regulation of this pathway following its activation.

Topographical distribution of corticotropin-releasing factor type 2 receptor-like immunoreactivity in the rat dorsal raphe nucleus: co-localization with tryptophan hydroxylase

Corticotropin-releasing factor (CRF) and CRF-related neuropeptides are involved in the regulation of stress-related physiology and behavior. Members of the CRF family of neuropeptides bind to two known receptors, the CRF type 1 (CRF₁) receptor, and the CRF type 2 (CRF₂) receptor. Although the distribution of CRF₂ receptor mRNA expression has been extensively studied, the distribution of CRF₂ receptor protein has not been characterized. An area of the brain known to contain high levels of CRF₂ receptor mRNA expression and CRF₂ receptor binding is the dorsal raphe nucleus (DR). In the present study we investigated in detail the distribution of CRF₂ receptor immunoreactivity throughout the rostrocaudal extent of the DR. CRF₂ receptor-immunoreactive perikarya were observed throughout the DR, with the highest number and density in the mid-rostrocaudal DR. Dual immunofluorescence revealed that CRF₂ receptor immunoreactivity was frequently co-localized with tryptophan hydroxylase, a marker of serotonergic neurons. This study provides evidence that CRF₂ receptor protein is expressed in the DR, and that CRF₂ receptors are expressed in topographically organized subpopulations of cells in the DR, including serotonergic neurons. Furthermore, these data are consistent with the hypothesis that CRF₂ receptors play an important role in the regulation of stress-related physiology and behavior through actions on serotonergic and non-serotonergic neurons within the DR.