Klotho Regulated by Estrogen Plays a Key Role in Sex Differences in Stress Resilience in Rats.

Klotho (KL) is a glycosyl hydrolase and aging-suppressor gene. Stress is a risk factor for depression and anxiety, which are highly comorbid with each other. The aim of this study is to determine whether KL is regulated by estrogen and plays an important role in sex differences in stress resilience. Our results showed that KL is regulated by estrogen in rat hippocampal neurons in vivo and in vitro and is essential for the estrogen-mediated increase in the number of presynaptic vesicular glutamate transporter 1 (Vglut1)-positive clusters on the dendrites of hippocampal neurons. The role of KL in sex differences in stress response was examined in rats using 3-week chronic unpredictable mild stress (CUMS). CUMS produced a deficit in spatial learning and memory, anhedonic-like behaviors, and anxiety-like behaviors in male but not female rats, which was accompanied by a reduction in KL protein levels in the hippocampus of male but not female rats. This demonstrated the resilience of female rats to CUMS. Interestingly, the knockdown of KL protein levels in the rat hippocampus of both sexes caused a decrease in stress resilience in both sexes, especially in female rats. These results suggest that the regulation of KL by estrogen plays an important role in estrogen-mediated synapse formation and that KL plays a critical role in the sex differences in cognitive deficit, anhedonic-like behaviors, and anxiety-like behaviors induced by chronic stress in rats, highlighting an important role of KL in sex differences in stress resilience.

A balance of outward and linear inward ionic currents is required for generation of slow-wave oscillations.

Regenerative inward currents help produce slow oscillations through a negative-slope conductance region of their current-voltage relationship that is well approximated by a linear negative conductance. We used dynamic-clamp injections of a linear current with such conductance, INL, to explore why some neurons can generate intrinsic slow oscillations whereas others cannot. We addressed this question in synaptically isolated neurons of the crab Cancer borealis after blocking action potentials. The pyloric network consists of a distinct pacemaker and follower neurons, all of which express the same complement of ionic currents. When the pyloric dilator (PD) neuron, a member of the pacemaker group, was injected with INL with dynamic clamp, it consistently produced slow oscillations. In contrast, all follower neurons failed to oscillate with INL To understand these distinct behaviors, we compared outward current levels of PD with those of follower lateral pyloric (LP) and ventral pyloric (VD) neurons. We found that LP and VD neurons had significantly larger high-threshold potassium currents (IHTK) than PD and LP had lower-transient potassium current (IA). Reducing IHTK pharmacologically enabled both LP and VD neurons to produce INL-induced oscillations, whereas modifying IA levels did not affect INL-induced oscillations. Using phase-plane and bifurcation analysis of a simplified model cell, we demonstrate that large levels of IHTK can block INL-induced oscillatory activity whereas generation of oscillations is almost independent of IA levels. These results demonstrate the general importance of a balance between inward pacemaking currents and high-threshold K+ current levels in determining slow oscillatory activity.NEW & NOTEWORTHY Pacemaker neuron-generated rhythmic activity requires the activation of at least one inward and one outward current. We have previously shown that the inward current can be a linear current (with negative conductance). Using this simple mechanism, here we demonstrate that the inward current conductance must be in relative balance with the outward current conductances to generate oscillatory activity. Surprisingly, an excess of outward conductances completely precludes the possibility of achieving such a balance.

The role of linear and voltage-dependent ionic currents in the generation of slow wave oscillations.

Neuronal oscillatory activity is generated by a combination of ionic currents, including at least one inward regenerative current that brings the cell towards depolarized voltages and one outward current that repolarizes the cell. Such currents have traditionally been assumed to require voltage-dependence. Here we test the hypothesis that the voltage dependence of the regenerative inward current is not necessary for generating oscillations. Instead, a current I NL that is linear in the biological voltage range and has negative conductance is sufficient to produce regenerative activity. The current I NL can be considered a linear approximation to the negative-conductance region of the current-voltage relationship of a regenerative inward current. Using a simple conductance-based model, we show that I NL , in conjunction with a voltage-gated, non-inactivating outward current, can generate oscillatory activity. We use phase-plane and bifurcation analyses to uncover a rich variety of behaviors as the conductance of I NL is varied, and show that oscillations emerge as a result of destabilization of the resting state of the model neuron. The model shows the need for well-defined relationships between the inward and outward current conductances, as well as their reversal potentials, in order to produce stable oscillatory activity. Our analysis predicts that a hyperpolarization-activated inward current can play a role in stabilizing oscillatory activity by preventing swings to very negative voltages, which is consistent with what is recorded in biological neurons in general. We confirm this prediction of the model experimentally in neurons from the crab stomatogastric ganglion.

Phosphodiesterases coordinate cAMP propagation induced by two stimulatory G protein-coupled receptors in hearts.

Inflammation is a significant player in the progression of heart failure and has detrimental effects on cardiac function. Prostaglandin (PG)E2, a major proinflammatory prostanoid in the cardiovascular system, is a potent stimulus in inducing intracellular cAMP but minimally affects cardiac contractile function. Here, we show that the PGE2 stimulation attenuates the adrenergic-induced cardiac contractile response in animal hearts. Stimulation with PGE2 leads to stimulatory G protein (Gs)-dependent production of cAMP. However, the induced cAMP is spatially restricted because of its degradation by phosphodiesterase (PDE)4 and cannot access the intracellular sarcoplasmic reticulum (SR) for increasing calcium signaling and myocyte contraction. Moreover, pretreatment with PGE2 significantly inhibits PKA activities at the SR induced by a ß-adrenergic agonist, isoproterenol, and subsequently blocks isoproterenol-induced PKA phosphorylation of phospholamban and contractile responses in myocytes. Further analysis reveals that the PGE2-induced cAMP/PKA is sufficient to phosphorylate and activate PDE4D isoforms, which, in turn, spatially inhibits the diffusion of adrenergic-induced cAMP from the plasma membrane to the SR. Inhibition of PDE4 rescues the adrenergic-induced increase in cAMP/PKA activities at the SR, PKA phosphorylation of phospholamban, and contractile responses in PGE2-pretreated myocytes. Thus, this offers an example that one Gs-coupled receptor is able to inhibit the intracellular signaling transduction initiated by another Gs-coupled receptor via controlling the diffusion of cAMP, presenting a paradigm for G protein-coupled receptor (GPCR) signal transduction. It also provides a mechanism for the integration of signaling initiated by different neurohormonal stimuli, as well as long-term effects of chronically circulating proinflammatory factors in myocardium.

Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice.

The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell-based approaches could be beneficial to infants with this often lethal condition.

Enhanced basal contractility but reduced excitation-contraction coupling efficiency and beta-adrenergic reserve of hearts with increased Cav1.2 activity.

Cardiac remodeling during heart failure development induces a significant increase in the activity of the L-type Ca(2+) channel (Cav1.2). However, the effects of enhanced Cav1.2 activity on myocyte excitation-contraction (E-C) coupling, cardiac contractility, and its regulation by the beta-adrenergic system are not clear. To recapitulate the increased Cav1.2 activity, a double transgenic (DTG) mouse model overexpressing the Cavbeta2a subunit in a cardiac-specific and inducible manner was established. We studied cardiac (in vivo) and myocyte (in vitro) contractility at baseline and upon beta-adrenergic stimulation. E-C coupling efficiency was evaluated in isolated myocytes as well. The following results were found: 1) in DTG myocytes, L-type Ca(2+) current (I(Ca,L)) density, myocyte fractional shortening (FS), peak Ca(2+) transients, and sarcoplasmic reticulum (SR) Ca(2+) content (caffeine-induced Ca(2+) transient peak) were significantly increased (by 100.8%, 48.8%, 49.8%, and 46.8%, respectively); and 2) cardiac contractility evaluated with echocardiography [ejection fraction (EF) and (FS)] and invasive intra-left ventricular pressure (maximum dP/dt and -dP/dt) measurements were significantly greater in DTG mice than in control mice. However, 1) the cardiac contractility (EF, FS, dP/dt, and -dP/dt)-enhancing effect of the beta-adrenergic agonist isoproterenol (2 microg/g body wt ip) was significantly reduced in DTG mice, which could be attributed to the loss of beta-adrenergic stimulation on contraction, Ca(2+) transients, I(Ca,L), and SR Ca(2+) content in DTG myocytes; and 2) E-C couplng efficiency was significantly lower in DTG myocytes. In conclusion, increasing Cav1.2 activity by promoting its high-activity mode enhances cardiac contractility but decreases E-C coupling efficiency and the adrenergic reserve of the heart.