Sergei Musatov

Silencing of estrogen receptor α in the ventromedial nucleus of hypothalamus leads to metabolic syndrome

Estrogen receptor α (ERα) plays a pivotal role in the regulation of food intake and energy expenditure by estrogens. Although it is well documented that a disruption of ERα signaling in ERα knockout (ERKO) mice leads to an obese phenotype, the sites of estrogen action and mechanisms underlying this phenomenon are still largely unknown. In the present study, we exploited RNA interference mediated by adeno-associated viral vectors to achieve focused silencing of ERα in the ventromedial nucleus of the hypothalamus, a key center of energy homeostasis. After suppression of ERα expression in this nucleus, female mice and rats developed a phenotype characteristic for metabolic syndrome and marked by obesity, hyperphagia, impaired tolerance to glucose, and reduced energy expenditure. This phenotype persisted despite normal ERα levels elsewhere in the brain. Although an increase in food intake preceded weight gain, our data suggest that a leading factor of obesity in this model is likely a decline in energy expenditure with all three major constituents being affected, including voluntary activity, basal metabolic rate, and diet-induced thermogenesis. Together, these findings indicate that ERα in the ventromedial nucleus of the hypothalamus neurons plays an essential role in the control of energy balance and the maintenance of normal body weight.

RNAi-mediated silencing of estrogen receptor α in the ventromedial nucleus of hypothalamus abolishes female sexual behaviors

Estrogen receptor α (ERα) plays a major role in the regulation of neuroendocrine functions and behaviors by estrogens. Although the generation of ERα knockout mice advanced our knowledge of ERα functions, gene deletion using this method is global and potentially confounded by developmental consequences. To achieve a site-specific knockdown of ERα in the normally developed adult brain, we have generated an adeno-associated virus vector expressing a small hairpin RNA targeting ERα. After bilateral injection of this vector into the hypothalamic ventromedial nucleus in ovariectomized female mice, expression levels of ERα as well as the estrogen-inducible progesterone receptor were profoundly reduced despite the continued presence of this receptor elsewhere in the brain. Functionally, silencing of ERα in the ventromedial nucleus abolished female proceptive and receptive sexual behaviors while enhancing rejection behavior. These results provide evidence that adeno-associated virus-mediated long-term knockdown of genes can be used to delineate their effects on complex behaviors in discrete brain regions.

Reversal of Depressed Behaviors in Mice by p11 Gene Therapy in the Nucleus Accumbens

Reduction of the 5-HT1B receptor–binding protein p11 in the mouse nucleus accumbens induces depression-like behaviors, and gene therapy to restore p11 expression in this region reverses depression-like behaviors. Dialing Down Depression Despite much progress in the study and treatment of depression, the mechanisms underlying this debilitating disease are still unclear. Altered activity of several major neurotransmitters in the brain including serotonin is involved, but pinpointing the parts of the brain affected in depression has proved challenging. Alexander and colleagues now implicate a brain region called the nucleus accumbens and a protein called p11 expressed in this region as important mediators of depression in humans and mice. The authors were alerted to the potential importance of p11 in depression because mice that lack this protein show depressive-like behavior. This protein is involved in the activation of two receptors for serotonin, 5-HT1B and 5-HT4. Alexander et al. decided to down-regulate expression of p11 specifically in the nucleus accumbens by injecting a viral vector containing a small interfering RNA against p11 directly into this brain region in healthy mice. They then tested the treated mice to see if they exhibited depressive-like behaviors in response to two stress tests (suspension by the tail and swimming in a water tank). In both tests, treated mice showed greater immobility compared with control animals, a sign of depressive-like behavior. To show that these depressive symptoms were indeed caused by loss of p11 in the nucleus accumbens, the investigators overexpressed p11 in the nucleus accumbens of mice that completely lacked this protein. They demonstrated restoration of normal immobility times on the two stress tests and an increased desire to sip sucrose solution (a treat that rodents normally enjoy but depressed animals do not). They also showed increased activity of 5-HT1B serotonin receptors expressed by striatal neurons in the nucleus accumbens of mice overexpressing p11. But do these results have any relevance to depression in humans? Alexander and colleagues tackled this question by comparing postmortem nucleus accumbens brain tissue from individuals with and without depression at the time of death. They discovered that expression of p11 was much lower in the nucleus accumbens of depressed individuals compared with healthy persons. These new findings pinpoint the nucleus accumbens and the p11 protein as important mediators of depression and provide new therapeutic targets for drug development. The etiology of major depression remains unknown, but dysfunction of serotonergic signaling has long been implicated in the pathophysiology of this disorder. p11 is an S100 family member recently identified as a serotonin 1B [5-hydroxytryptamine 1B (5-HT1B)] and serotonin 4 (5-HT4) receptor–binding protein. Mutant mice in which p11 is deleted show depression-like behaviors, suggesting that p11 may be a mediator of affective disorder pathophysiology. Using somatic gene transfer, we have now identified the nucleus accumbens as a key site of p11 action. Reduction of p11 with adeno-associated virus (AAV)–mediated RNA interference in the nucleus accumbens, but not in the anterior cingulate, of normal adult mice resulted in depression-like behaviors nearly identical to those seen in p11 knockout mice. Restoration of p11 expression specifically in the nucleus accumbens of p11 knockout mice normalized depression-like behaviors. Human nucleus accumbens tissue shows a significant reduction of p11 protein in depressed patients when compared to matched healthy controls. These results suggest that p11 loss in rodent and human nucleus accumbens may contribute to the pathophysiology of depression. Normalization of p11 expression within this brain region with AAV-mediated gene therapy may be of therapeutic value.

The role of the estrogen receptor α in the medial amygdala and ventromedial nucleus of the hypothalamus in social recognition, anxiety and aggression.

Social recognition manifests itself in decreased investigation of a previously encountered individual. Estrogen receptor alpha (ERalpha) knock out mice show deficient social recognition and anxiety. These data show that the ERalpha is involved in these effects, but they do not say anything about the brain sites important for these effects. In this study, an shRNA encoded within an AAV viral vector directed against the ERalpha receptor gene (or containing luciferase control), was injected bilaterally into the posterodorsal amygdala (MePDA) or the ventromedial nucleus of the hypothalamus (VMN) of female rats. An 81% reduction of ERalpha expression in the MePDA eliminated social recognition. Moreover, this diminution of ERalpha in the MePDA reduced anxiety in the light/dark choice test. In contrast, social recognition was unaffected after ERalpha knockdown in the VMN while aggressiveness against the juvenile was enhanced. In conclusion, social recognition and anxiety in female rats are modulated by the ERalpha in the amygdala. Moreover, aggression against juveniles but not against adults could, at least partly, depend on the ERalpha in the VMN.

Viral Vector-Mediated Overexpression of Estrogen Receptor-α in Striatum Enhances the Estradiol-Induced Motor Activity in Female Rats and Estradiol-Modulated GABA Release

Classical estrogen receptor-signaling mechanisms involve estradiol binding to intracellular nuclear receptors [estrogen receptor-α (ERα) and estrogen receptor-β (ERβ)] to promote changes in protein expression. Estradiol can also exert effects within seconds to minutes, however, a timescale incongruent with genomic signaling. In the brain, estradiol rapidly potentiates stimulated dopamine release in the striatum of female rats and enhances spontaneous rotational behavior. Furthermore, estradiol rapidly attenuates the K+-evoked increase of GABA in dialysate. We hypothesize that these rapid effects of estradiol in the striatum are mediated by ERα located on the membrane of medium spiny GABAergic neurons. This experiment examined whether overexpression of ERα in the striatum would enhance the effect of estradiol on rotational behavior and the K+-evoked increase in GABA in dialysate. Ovariectomized female rats were tested for rotational behavior or underwent microdialysis experiments after unilateral intrastriatal injections of a recombinant adeno-associated virus (AAV) containing the human ERα cDNA (AAV.ERα) into the striatum; controls received either the same vector into areas outside the striatum or an AAV containing the human alkaline phosphatase gene into the striatum (AAV.ALP). Animals that received AAV.ERα in the striatum exhibited significantly greater estradiol-induced contralateral rotations compared with controls and exhibited behavioral sensitization of contralateral rotations induced by a low-dose of amphetamine. ERα overexpression also enhanced the inhibitory effect of estradiol on K+-evoked GABA release suggesting that disinhibition of dopamine release from terminals in the striatum resulted in the enhanced rotational behavior.

Estrogen receptors in the medial amygdala inhibit the expression of male prosocial behavior.

Studies using estrogen receptor α (ERα) knock-out mice indicate that ERα masculinizes male behavior. Recent studies of ERα and male prosocial behavior have shown an inverse relationship between ERα expression in regions of the brain that regulate social behavior, including the medial amygdala (MeA), and the expression of male prosocial behavior. These studies have lead to the hypothesis that low levels of ERα are necessary to “permit” the expression of high levels of male prosocial behavior. To test this, viral vectors were used to enhance ERα in male prairie voles (Microtus ochrogaster), which display high levels of prosocial behavior and low levels of MeA ERα. Adult male prairie voles were transfected with ERα in the MeA (MeA-ERα) or the caudate–putamen (ERα control) or luciferase (MeA-site-specific control), and 3 weeks later tested for spontaneous alloparental behavior and partner preference. Enhancing ERα in the MeA altered/reduced male prosocial behavior. Only one-third of MeA-ERα males, compared with all control males, were alloparental. MeA-ERα males also displayed a significant preference for a novel female. This is a critical finding because the manipulations of neuropeptides, oxytocin and vasopressin, can inhibit the formation of a partner preference, but do not lead to the formation of a preference for a novel female. The results support the hypothesis that low levels of ERα are necessary for high levels of male prosocial behavior, and provide the first direct evidence that site-specific ERα expression plays a critical role in the expression of male prosocial behavior.

Differential effects of site-specific knockdown of estrogen receptor α in the medial amygdala, medial pre-optic area, and ventromedial nucleus of the hypothalamus on sexual and aggressive behavior of male mice.

Testosterone is known to play an important role in the regulation of male‐type sexual and aggressive behavior. As an aromatised metabolite of testosterone, estradiol‐induced activation of estrogen receptor α (ERα) may be crucial for the induction of these behaviors in male mice. However, the importance of ERα expressed in different nuclei for this facilitatory action of testosterone has not been determined. To investigate this issue, we generated an adeno‐associated virus vector expressing a small hairpin RNA targeting ERα to site‐specifically knockdown ERα expression. We stereotaxically injected either a control or ERα targeting vector into the medial amygdala, medial pre‐optic area (MPOA), or ventromedial nucleus of the hypothalamus (VMN) in gonadally intact male mice. Two weeks after injection, all mice were tested biweekly for sexual and aggressive behavior, alternating between behavior tests each week. We found that suppressing ERα in the MPOA reduced sexual but not aggressive behavior, whereas in the VMN it reduced both behaviors. Knockdown of ERα in the medial amygdala did not alter either behavior. Additionally, it was found that ERα knockdown in the MPOA caused a parallel reduction in the number of neuronal nitric oxide synthase‐expressing cells. Taken together, these results indicate that the testosterone facilitatory action on male sexual behavior requires the expression of ERα in both the MPOA and VMN, whereas the testosterone facilitatory action on aggression requires the expression of ERα in only the VMN.

AAV-mediated delivery of the caspase inhibitor XIAP protects against cisplatin ototoxicity.

Hypothesis: Delivery of the gene encoding X-linked inhibitor of apoptosis (XIAP) using an adeno-associated viral (AAV) vector can protect against cisplatin-mediated ototoxicity. Background: Cisplatin is a widely used chemotherapeutic agent with significant ototoxic side effects. One possible mechanism of toxicity is apoptotic death of many cochlear cell types. Acute treatment with inhibitors of caspases- enzymes critical for apoptosis- has been shown to prevent hearing loss in vivo, but is too short-acting for therapeutic use. Gene therapy provides a specific and chronic means of delivering potential therapeutic gents. Introducing an anti-apoptotic gene into the cochlea could provide long-term prophylaxis against the ototoxic effects of cisplatin. Method: Two groups of rats were treated with unilateral injection into the round window of AAV harboring a gene encoding either XIAP or green fluorescent protein (GFP). After at least two months of gene expression, auditory-brainstem-response (ABR) threshold shifts and outer-hair-cell (OHC) number were measured in these two groups of animals after 72-hour treatment with cisplatin. Results: Consistent with previous reports, uninjected and AAV.GFP-injected ears displayed profound ABR threshold elevations and OHC loss after cisplatin treatment. Ears that had been injected with AAV encoding XIAP, however, were significantly protected from these effects: cisplatin-induced ABR-threshold shift and hair-cell loss were attenuated by as much as 78% and 45%, respectively, when compared with contralateral (untreated) ears. Conclusion: XIAP delivery to the cochlea can protect against the audiometric changes and hair-cell loss associated with cisplatin ototoxicity. The efficacy, specificity, and duration of the protective effects make this a potentially attractive therapeutic paradigm.

siRNA silencing of estrogen receptor-α expression specifically in medial preoptic area neurons abolishes maternal care in female mice

The medial preoptic area has been shown to be intricately involved in many behaviors, including locomotion, sexual behavior, maternal care, and aggression. The gene encoding estrogen receptor-α (ERα) protein is expressed in preoptic area neurons, and a very dense immunoreactive field of ERα is found in the preoptic region. ERα knockout animals show deficits in maternal care and sexual behavior and fail to exhibit increases in these behaviors in response to systemic estradiol treatment. In the present study, we used viral-vector mediated RNA interference to silence ERα expression specifically in the preoptic area of female mice and measured a variety of behaviors, including social and sexual aggression, maternal care, and arousal activity. Suppression of ERα in the preoptic area almost completely abolished maternal care, significantly increasing the latency to pup retrieval and significantly reducing the time the moms spent nursing and licking the pups. Strikingly, maternal aggression toward a male intruder was not different between control and preoptic ERα-silenced mice, demonstrating the remarkably specific role of ERα in these neurons. Reduction of ERα expression in preoptic neurons significantly decreased sexual behavior in female mice and increased aggression toward both sexual partners and male intruders in a seminatural environment. Estrogen-dependent increases in arousal, measured by home cage activity, were not mediated by ERα expression in the preoptic neurons we targeted, as ERα-suppressed mice had increases similar to control mice. Thus, we have established that a specific gene in a specific group of neurons is required for a crucially important natural behavior.

The role of the estrogen receptor α in the medial preoptic area in sexual incentive motivation, proceptivity and receptivity, anxiety, and wheel running in female rats

Ovariectomized females were given an infusion in the medial preoptic area (MPOA) of a viral vector carrying either a shRNA directed against the estrogen receptor α (ERα) or luciferase. The females were subjected to a test for sexual incentive motivation immediately followed by a test for receptivity and proceptive behaviors. Two weeks later they were tested in the light/dark choice procedure, and after another 2 weeks they were subjected to a test in a brightly lit open field. Finally, the females were given free access to a running wheel for 88h. The females were treated with estradiol benzoate (EB), 18 or 1.5μg/kg, in randomized order 52h before each test except the running wheel. In that experiment, they were given EB 48h after introduction into the wheel cage. They were given progesterone, 1mg/rat, about 4h before all tests, except the running wheel. The shRNA reduced the number of ERα with 83%. Females with few ERα in the MPOA showed increased lordosis quotient after the 1.5μg/kg dose of EB. There was no effect on proceptive behaviors or on rejections. When given the 18μg/kg EB dose, there was no difference between females with few preoptic ERα and controls. In the test for sexual incentive motivation, females with few preoptic ERα approached the castrated male incentive more than controls, regardless of EB dose. They also moved a shorter distance. In the light/dark choice test as well as in the open field, females with few ERα in the MPOA showed signs of reduced fear/anxiety, since they spent more time in the light part of the dark/light box and in the center of the open field. Finally, the data from the running wheel showed that females with few preoptic ERα failed to show enhanced activity after treatment with EB. These data show that the preoptic ERα inhibits lordosis in females with an intermediate level of receptivity while it fails to do so in fully receptive females. The ERα in the MPOA seems to be necessary for selective approach to a sexual incentive. Finally, activation of this receptor appears to have anxiogenic effects in the procedures employed here. A hypothesis for how all these actions of the preoptic ERα contributes to efficient reproductive behavior is outlined.