Hypothalamic Brain-derived Neurotrophic Factor Research Articles

Urinary Bladder Dysfunction in a Novel Model of Neuroendocrine Stress

Chronic stress can lead to urinary bladder dysfunction and pain either through actions within the central nervous system or by mechanisms that are locally induced in the bladder. Prolonged upregulation of brain-derived neurotrophic factor (BDNF) in the paraventricular nucleus of the hypothalamus (PVN) has been shown to induce hypertension and chronically elevate activities of the sympathetic nervous system and hypothalamus-pituitary-adrenal axis. Taking advantage of this mechanism, we created a novel model of chronic neuroendocrine stress by subjecting male Sprague Dawley rats to bilateral PVN injections of AAV2 viral vectors expressing either BDNF or GFP (for control). We tested the hypothesis that model-induced chronic neuroendocrine stress would impair bladder function. Bladder activity was assessed 10 weeks post-injections by monitoring the number of urinary voids and average urinary void volume along with water intake over a 48-hour period. To further characterize changes in the urinary bladder, bladder weights were recorded, and in vitro bladder strip experiments were conducted 14 weeks after viral vector injections. BDNF overexpression in the hypothalamus led to significantly lower daily urine output even though water intake remained unaffected, suggesting that BDNF-treated animals showed a significantly higher non-urine-related fluid compared to GFP controls (daily urine output = GFP: 33.85±4.94, BDNF: 14.82±2.18, p < 0.01. Interestingly, despite a lower total daily urine volume, BDNF-treated animals had a significantly higher number of urinary voids (GFP: 7.50±1.42, BDNF: 15.38±2.76; GFP vs. BDNF: p < 0.001) and lower average void volume (GFP: 1.19±0.15, BDNF: 0.27±0.07; GFP vs. BDNF: p < 0.01), thus emulating the overactive bladder phenotype, a common complication in humans with chronic stress. These findings were complemented by in vitro experiments where bladder strips from BDNF rats showed significantly higher contractility as tested with high K+ concentration and electric field stimulation. Moreover, Carbachol-induced stimulation of muscarinic receptors in BDNF bladder strips also trended towards higher contractility. However, urinary bladder weights were unaffected by BDNF overexpression. In summary, we demonstrated that prolonged hypothalamic BDNF overexpression, a model of chronic neuroendocrine stress, leads to significant alterations in bladder function such that BDNF-treated rats exhibited a higher number of urinary voids and lower average void volume compared to GFP controls, and bladder strip experiments suggest that enhanced smooth muscle contractility may contribute to this overactive bladder phenotype. These findings from our novel experimental model will help elucidate pathways through which neuroendocrine stress mechanisms contribute to the development of the overactive bladder syndrome. Funded by R01HL166464, R03AG072016 and R01DK125543. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neurobehavioral Abnormalities in Offspring of Young Adult Male Rats With a History of Traumatic Brain Injury.

Children of parents with traumatic brain injury (TBI) are more likely to develop psychiatric disorders. This association is usually attributed to TBI-induced changes in parents' personality and families' social environment. We tested the hypothesis that offspring of young adult male rats with TBI develop neurodevelopmental abnormalities in the absence of direct social contact with sires. Male Sprague-Dawley rats (F0 generation) in the TBI group underwent moderate TBI via a midline fluid percussion injury that involved craniectomy under sevoflurane (SEVO) anesthesia for 40 min on post-natal Day 60 (P60), while F0 rats in the control group were placed in a new cage, one per cage, for the equivalent time duration. A subset of F0 rats was sacrificed on P66 to assess acute changes in hypothalamic-pituitary-adrenal (HPA) axis and inflammation markers. The remaining F0 males were mated with naive females on P90 to generate offspring (F1 generation). The F0 males and F1 males and females were sequentially evaluated in the elevated plus maze, for pre-pulse inhibition of acoustic startle, in the Morris water maze, and for resting and stress levels of serum corticosterone starting on ∼P105 (F0) and ∼P60 (F1), followed by tissue collection for further analyses. Acutely, the F0 TBI males had messenger RNA (mRNA) transcripts altered to support an increased hypothalamic and hippocampal Na+-K+-Cl- (Slc12a2) Cl- importer / K+-2Cl- (Slc12a5) Cl- exporter ratio and decreased hippocampal glucocorticoid receptors (Nr3c1), as well as increased serum levels of corticosterone, interleukin-1β (IL-1β), and biomarkers of activated hippocampal microglia and astrocytes. Long-term, F0 TBI rats exhibited increased corticosterone concentrations at rest and under stress, anxiety-like behavior, impaired sensory-motor gating, and impaired spatial memory. These abnormalities were underpinned by reduced mRNA levels of hypothalamic and hippocampal mineralocorticoid receptors (Nr3c2), hippocampal Nr3c1, and hypothalamic brain-derived neurotrophic factor (Bdnf), as well as elevated serum levels of IL-1β, and biomarkers of activated hippocampal microglia and astrocytes. F1 male offspring of TBI sires exhibited abnormalities in all behavioral tests, while their F1 female counterparts had abnormal pre-pulse inhibition responses only. F1 male offspring of TBI sires also had reduced mRNA levels of hippocampal Nr3c1 and Nr3c2, as well as hypothalamic and hippocampal Bdnf, whereas increases in inflammatory markers were more profound in F1 females. These findings suggest that offspring of sires with a history of a moderate TBI that involved craniectomy under SEVO anesthesia for 40 min, develop sex-dependent neurobehavioral abnormalities in the absence of direct social interaction between the sire and the offspring.

Smith-Magenis syndrome protein RAI1 regulates body weight homeostasis through hypothalamic BDNF-producing neurons and neurotrophin downstream signalling.

Retinoic acid-induced 1 (RAI1) haploinsufficiency causes Smith-Magenis syndrome (SMS), a genetic disorder with symptoms including hyperphagia, hyperlipidemia, severe obesity, and autism phenotypes. RAI1 is a transcriptional regulator with a pan-neural expression pattern and hundreds of downstream targets. The mechanisms linking neural Rai1 to body weight regulation remain unclear. Here we find that hypothalamic brain-derived neurotrophic factor (BDNF) and its downstream signalling are disrupted in SMS (Rai1+/-) mice. Selective Rai1 loss from all BDNF-producing cells or from BDNF-producing neurons in the paraventricular nucleus of the hypothalamus (PVH) induced obesity in mice. Electrophysiological recordings revealed that Rai1 ablation decreased the intrinsic excitability of PVHBDNF neurons. Chronic treatment of SMS mice with LM22A-4 engages neurotrophin downstream signalling and delayed obesity onset. This treatment also partially rescued disrupted lipid profiles, insulin intolerance, and stereotypical repetitive behaviour in SMS mice. These data argue that RAI1 regulates body weight and metabolic function through hypothalamic BDNF-producing neurons and that targeting neurotrophin downstream signalling might improve associated SMS phenotypes.

Smith–Magenis syndrome protein RAI1 regulates body weight homeostasis through hypothalamic BDNF-producing neurons and neurotrophin downstream signalling

Retinoic acid-induced 1 (RAI1) haploinsufficiency causes Smith–Magenis syndrome (SMS), a genetic disorder with symptoms including hyperphagia, hyperlipidemia, severe obesity, and autism phenotypes. RAI1 is a transcriptional regulator with a pan-neural expression pattern and hundreds of downstream targets. The mechanisms linking neural Rai1 to body weight regulation remain unclear. Here we find that hypothalamic brain-derived neurotrophic factor (BDNF) and its downstream signalling are disrupted in SMS (Rai1+/-) mice. Selective Rai1 loss from all BDNF-producing cells or from BDNF-producing neurons in the paraventricular nucleus of the hypothalamus (PVH) induced obesity in mice. Electrophysiological recordings revealed that Rai1 ablation decreased the intrinsic excitability of PVHBDNF neurons. Chronic treatment of SMS mice with LM22A-4 engages neurotrophin downstream signalling and delayed obesity onset. This treatment also partially rescued disrupted lipid profiles, insulin intolerance, and stereotypical repetitive behaviour in SMS mice. These data argue that RAI1 regulates body weight and metabolic function through hypothalamic BDNF-producing neurons and that targeting neurotrophin downstream signalling might improve associated SMS phenotypes.

Complementary role of peripheral and central autonomic nervous system on insulin-like growth factor-1 activation to prevent fatty liver disease.

Insulin-like growth factor-1 (IGF-1) is involved in the pathology of non-alcoholic fatty liver disease (NAFLD) and ameliorates fatty infiltration in the liver. It is activated by growth hormone (GH); however, the role of GH-IGF-1 axis in NAFLD developmental phase has not been well identified. Therefore, in this study, we focused on the effect of IGF-1 in NAFLD pathology and GH excretion activation from the pituitary gland by peripheral autonomic neural pathways relaying liver-brain-gut pathway and by central neuropeptides. GH and IGF-1 levels were assessed in wild-type and melanocortin-4 receptor knockout mice upon the development of diet-induced NAFLD. The contribution of the peripheral autonomic nervous system connecting the liver-brain-gut axis was assessed by its blockade using capsaicin and that of the central nervous system was assessed by the expression of hypothalamic brain-derived neurotrophic factor (BDNF) and corticotropin-releasing factor (CRH), which activates GH release from the pituitary gland. In the NAFLD mouse models, the levels of GH and IGF-1 increased (p < .05). Further, hepatic fatty infiltration was suppressed even under peripheral autonomic nervous system blockade (p < .001), which inhibited gastric ghrelin expression. In mice with peripheral autonomic nervous blockade, hypothalamic BDNF and CRH were inhibited (p < .05), resulting in GH and IGF-1 excretion, whereas other neuropeptides of somatostatin and cortistatin showed no changes. These complementary effects were canceled in melanocortin-4 receptor knockout mice, which diminished BDNF and CRH release control. Our study demonstrates that the release of IGF-1 by the nervous system is a key factor in maintaining the pathological homeostasis of NAFLD, suggesting its therapeutic potential.

Hypothalamic AAV-BDNF gene therapy improves metabolic function and behavior in the Magel2-null mouse model of Prader-Willi syndrome

Individuals with Prader-Willi syndrome (PWS) display developmental delays, cognitive impairment, excessive hunger, obesity, and various behavioral abnormalities. Current PWS treatments are limited to strict supervision of food intake and growth hormone therapy, highlighting the need for new therapeutic strategies. Brain-derived neurotrophic factor (BDNF) functions downstream of hypothalamic feeding circuitry and has roles in energy homeostasis and behavior. In this preclinical study, we assessed the translational potential of hypothalamic adeno-associated virus (AAV)-BDNF gene therapy as a therapeutic for metabolic dysfunction in the Magel2-null mouse model of PWS. To facilitate clinical translation, our BDNF vector included an autoregulatory element allowing for transgene titration in response to the host’s physiological needs. Hypothalamic BDNF gene transfer prevented weight gain, decreased fat mass, increased lean mass, and increased relative energy expenditure in female Magel2-null mice. Moreover, BDNF gene therapy improved glucose metabolism, insulin sensitivity, and circulating adipokine levels. Metabolic improvements were maintained through 23 weeks with no adverse behavioral effects, indicating high levels of efficacy and safety. Male Magel2-null mice also responded positively to BDNF gene therapy, displaying improved body composition, insulin sensitivity, and glucose metabolism. Together, these data suggest that regulating hypothalamic BDNF could be effective in the treatment of PWS-related metabolic abnormalities.

Network Pharmacology and ­Experimental Analyses of the Mechanism of Analgesic and Glucose Intolerance Through Glucocorticoid Signaling in C57 Mice Treated with Water Extract of Prunella vulgaris L. Spica

The aim of this study was to confirm the anti-inflammatory effect and explore the adverse effects and underlying mechanisms of Prunella vulgaris L., which has been extensively used for hundreds of years in East Asia. Network pharmacology studies predicted that glucocorticoids (GCs), GC-targeting molecules, and brain-derived neurotrophic factor (BDNF) were intensively involved in the anti-inflammation and glucose intolerance. To attest the effects and underlying mechanisms, C57 male mice were randomly divided into 5 groups, control (C), dexamethasone (Dex), water extract of P. vulgaris (PE 35 or 70 mg), and PE (70 mg) + mifepristone (PEM). After a 3-week treatment, acetic acid-induced writhing and hot plate tests confirmed the peripheral and central analgesic effects, respectively. Plasma GCs and BDNF were significantly increased. Coincidently, plasma pro-inflammatory cytokines, including IL1β, IL6, and IL10, were decreased by PE treatment, which were blocked by the application of mifepristone ( P &lt; 0.5). Western blots confirmed GC receptor (GR) translocation, and decreased cyclooxygenase 2 in the lumber spine by PE treatment. Food intake was impeded after a 4-week PE treatment, but the ratio of bodyweight gain to food intake was increased in a time-dependent manner. An intraperitoneal glucose tolerance test disclosed that PE treatment impaired glucose disposal in mice. Quantitative polymerase chain reaction (PCR) showed that hepatic GC-responsive genes such as GC-induced leucine zipper protein and glucose-6-phosphatase catalytic subunit 1 were up-regulated, and hypothalamic neuropeptide Y and agouti-related protein expressions were decreased by PE treatment. Hypothalamic BDNF was up-regulated, whereas hepatic BDNF was down-regulated. The regulation of these genes by PE was reversed by mifepristone administration. In conclusion, PE treatment plays analgesic and glucose regulation roles simultaneously through GC-induced signaling pathways, and P. vulgaris may provide a natural ligand of GR for the treatment of inflammation with glucose dysregulation.

Prostaglandin E2-induced anorexia involves hypothalamic brain-derived neurotrophic factor and ghrelin in chicks

Central administration of prostaglandin E2 (PGE2) is associated with potent anorexia in rodents and chicks, although hypothalamic mechanisms are not fully understood. The objective of the present study was to identify hypothalamic nuclei and appetite-related factors that are involved in this anorexigenic effect, using chickens as a model. Intracerebroventricular injection of 2.5, 5, and 10 nmol of PGE2 suppressed food and water intake in broiler chicks in a dose-dependent manner. c-Fos immunoreactivity was increased in the paraventricular nucleus (PVN) at 60 min post injection of 5 nmol of PGE2. Under the same treatment condition, hypothalamic expression of melanocortin receptor 3 and ghrelin mRNAs increased, whereas neuropeptide Y receptor sub-type 5 and tropomyosin receptor kinase B (TrkB) mRNAs decreased in PGE2-treated chicks. In the PVN, chicks injected with PGE2 had more brain-derived neurotrophic factor (BDNF), ghrelin, and c-Fos mRNA but less corticotrophin-releasing factor receptor 1 (CRFR1), CRFR2, and TrkB mRNA expression. In conclusion, PGE2 injection resulted in decreased food and water intake that likely involves BDNF and ghrelin originating in the PVN. Because the anorexigenic effect is so potent and hypothalamic mechanisms are similar in chickens and rodents, a greater understanding of the role of PGE2 in acute appetite regulation may have implications for treating eating and metabolic disorders in humans.

Environmental activation of a hypothalamic BDNF-adipocyte IL-15 axis regulates adipose-natural killer cells

Physical and social environments influence immune homeostasis within adipose tissue, yet the mechanisms remain poorly defined. We report that an enriched environment (EE) housing modulates the immune cell population in white adipose tissue of mice including an increase in the abundance of natural killer (NK) cells. EE upregulates the expression of IL-15 and its receptor IL-15Rα specifically within mature adipocytes. Mechanistically, we show that hypothalamic brain-derived neurotrophic factor (BDNF) upregulates IL-15 production in adipocytes via sympathetic β-adrenergic signaling. Overexpressing BDNF mediated by recombinant adeno-associated virus (rAAV) vector in the hypothalamus expands adipose NK cells. Conversely, inhibition of hypothalamic BDNF signaling via gene transfer of a dominant negative TrkB receptor suppresses adipose NK cells. In white adipose tissue, overexpression of IL-15 using an adipocyte-specific rAAV vector stimulates adipose NK cells and inhibits the progression of subcutaneous melanoma, whereas local IL-15 knockdown blocks the EE effect. These results suggest that bio-behavioral factors regulate adipose NK cells via a hypothalamic BDNF-sympathoneural-adipocyte IL-15 axis. Targeting this pathway may have therapeutic significance for cancer.

Enriched environment enhances NK cell maturation through hypothalamic BDNF in male mice.

Macroenvironmental factors, including a patient's physical and social environment, play a role in cancer risk and progression. Our previous preclinical studies have shown that the enriched environment (EE) confers anti-obesity and anti-cancer phenotypes that are associated with enhanced adaptive immunity and are mediated by brain-derived neurotrophic factor (BDNF). Natural killer (NK) cells have anti-cancer and anti-viral properties, and their absence or depletion is associated with inferior clinical outcomes. In this study, we investigated the effects of EE on NK cell maturation following their depletion. Mice living in EE displayed a higher proportion of NK cells in the spleen, bone marrow, and blood, compared to those living in the standard environment (SE). EE enhanced NK cell maturation in the spleen and was associated with upregulation of BDNF expression in the hypothalamus. Hypothalamic BDNF overexpression reproduced the EE effects on NK cell maturation in secondary lymphoid tissues. Conversely, hypothalamic BDNF knockdown blocked the EE modulation on NK cell maturation. Our results demonstrate that a bio-behavior intervention enhanced NK cell maturation and was mediated at least in part by hypothalamic BDNF.

Hypothalamic gene transfer of BDNF promotes healthy aging.

The aging process and age-related diseases all involve metabolic decline and impaired ability to cope with adversity. Environmental enrichment (EE)-a housing environment which recapitulates aspects of active lifestyle-exerts a wide range of health benefits in laboratory rodents. Brain-derived neurotrophic factor (BDNF) in the hypothalamus orchestrates autonomic and neuroendocrine processes, serving as one key brain mediator of EE-induced resistance to obesity, cancer, and autoimmunity. Recombinant adeno-associated virus (AAV)-mediated hypothalamic BDNF gene transfer alleviates obesity, diabetes, and metabolic syndromes in both diet-induced and genetic models. One recent study by our lab demonstrates the efficacy and safety of a built-in autoregulatory system to control transgene BDNF expression, mimicking the body's natural feedback systems in middle-age mice. Twelve-month old mice were treated with autoregulatory BDNF vector and monitored for 7months. BDNF gene transfer prevented age-associated metabolic decline by: reducing adiposity, preventing the decline of brown fat activity, increasing adiponectin while reducing leptin and insulin in circulation, improving glucose tolerance, increasing energy expenditure, alleviating hepatic steatosis, and suppressing inflammatory genes in the hypothalamus and adipose tissues. Furthermore, BDNF treatment reduced anxiety-like and depression-like behaviors. This chapter summarizes this work and discusses potential roles that hypothalamic BDNF might play in promoting healthy aging.

Adipose PTEN acts as a downstream mediator of a brain-fat axis in environmental enrichment

Background/ObjectivesEnvironmental enrichment (EE) is a physiological model to investigate brain-fat interactions. We previously discovered that EE activates the hypothalamic-sympathoneural adipocyte (HSA) axis via induction of brain-derived neurotrophic factor (BDNF), thus leading to sympathetic stimulation of white adipose tissue (WAT) and an anti-obesity phenotype. Here, we investigate whether PTEN acts as a downstream mediator of the HSA axis in the EE. MethodsMice were housed in EE for 4- and 16-week periods to determine how EE regulates adipose PTEN. Hypothalamic injections of adeno-associated viral (AAV) vectors expressing BDNF and a dominant negative form of its receptor were performed to assess the role of the HSA axis in adipose PTEN upregulation. A β-blocker, propranolol, and a denervation agent, 6-hydroydopamine, were administered to assess sympathetic signaling in the observed EE-PTEN phenotype. To determine whether inducing PTEN is sufficient to reproduce certain EE adipose remodeling, we overexpressed PTEN in WAT using an AAV vector. To determine whether adipose PTEN is necessary for the EE-mediated reduction in adipocyte size, we injected a rAAV vector expressing Cre recombinase to the WAT of adult PTENflox mice and placed the mice in EE. ResultsEE upregulated adipose PTEN expression, which was associated with suppression of AKT and ERK phosphorylation, increased hormone-sensitive lipase (HSL) phosphorylation, and reduced adiposity. PTEN regulation was found to be controlled by the HSA axis—with the hypothalamic BDNF acting as the upstream mediator—and dependent on sympathetic innervation. AAV-mediated adipose PTEN overexpression recapitulated EE-mediated adipose changes including suppression of AKT and ERK phosphorylation, increased HSL phosphorylation, and reduced adipose mass, whereas PTEN knockdown blocked the EE-induced reduction of adipocyte size. ConclusionsThese data suggest that adipose PTEN responds to environmental stimuli and serves as downstream mediator of WAT remodeling in the EE paradigm, resulting in decreased adipose mass and decreased adipocyte size.

BDNF Val66Met polymorphism alters food intake and hypothalamic BDNF expression in mice.

Obesity, a rising public health burden, is a multifactorial disease with an increased risk for patients to develop several pathological conditions including type 2 diabetes mellitus, hypertension, and cardiovascular disease. Increasing evidence suggests a relationship between the human brain-derived neurotrophic factor (BDNF) Val66Met single-nucleotide polymorphism (SNP) and obesity, although the underlying mechanisms of this connection are still not completely understood. In the present study, we found that homozygous knock-in BDNFMet/Met mice were overweight and hyperphagic compared to wildtype BDNFVal/Val mice. Increased food intake was associated withreduction of total BDNF and BDNF1, BDNF4 and BDNF6 transcripts in the hypothalamus of BDNFMet/Met mice. Incontrast, in the white adipose tissue total BDNF and Glut4 expression levels were augmented, while sirtuin 1 and leptin receptor (Ob-R) expression levels were reduced in BDNFMet/Met mice. Moreover, plasmatic leptin levels were decreased in BDNFMet/Met mice. However, BDNFVal/Val and BDNFMet/Met mice showed a similar response to the insulin tolerance test and glucose tolerance test. Altogether, these results suggest that BDNF Val66Met SNP strongly contributes to adipose tissue pathophysiology, resulting in reduced circulating leptin levels and hypothalamic expression of BDNF, which, in turn, promote increased food intake and overweight in BDNFMet/Met mice.

Hypothalamic gene transfer of BDNF promotes healthy aging in mice.

The aging process and age‐related diseases all involve perturbed energy adaption and impaired ability to cope with adversity. Brain‐derived neurotrophic factor (BDNF) in the hypothalamus plays important role in regulation of energy balance. Our previous studies show that recombinant adeno‐associated virus (AAV)‐mediated hypothalamic BDNF gene transfer alleviates obesity, diabetes, and metabolic syndromes in both diet‐induced and genetic models. Here we examined the efficacy and safety of a built‐in autoregulatory system to control transgene BDNF expression mimicking the body's natural feedback systems in middle‐aged mice. Twelve‐month‐old mice were treated with either autoregulatory BDNF vector or yellow fluorescence protein (YFP) control, maintained on normal diet, and monitored for 28 weeks. BDNF gene transfer prevented the development of aging‐associated metabolic declines characterized by: preventing aging‐associated weight gain, reducing adiposity, reversing the decline of brown fat activity, increasing adiponectin while reducing leptin and insulin in circulation, improving glucose tolerance, increasing energy expenditure, alleviating hepatic steatosis, and suppressing inflammatory genes in the hypothalamus and adipose tissues. Moreover, BDNF treatment reduced anxiety‐like and depression‐like behaviors. These safety and efficacy data provide evidence that hypothalamic BDNF is a target for promoting healthy aging.

The antidepressant fluoxetine acts on energy balance and leptin sensitivity via BDNF

Leptin and Brain Derived Neurotrophic Factor (BDNF) pathways are critical players in body weight homeostasis. Noninvasive treatments like environmental stimulation are able to increase response to leptin and induce BDNF expression in the brain. Emerging evidences point to the antidepressant selective serotonin reuptake inhibitor Fluoxetine (FLX) as a drug with effects similar to environmental stimulation. FLX is known to impact on body weight, with mechanisms yet to be elucidated. We herein asked whether FLX affects energy balance, the leptin system and BDNF function. Adult lean male mice chronically treated with FLX showed reduced weight gain, higher energy expenditure, increased sensitivity to acute leptin, increased hypothalamic BDNF expression, associated to changes in white adipose tissue expression typical of “brownization”. In the Ntrk2tm1Ddg/J model, carrying a mutation in the BDNF receptor Tyrosine kinase B (TrkB), these effects are partially or totally reversed. Wild type obese mice treated with FLX showed reduced weight gain, increased energy output, and differently from untreated obese mice, a preserved acute response to leptin in terms of activation of the intracellular leptin transducer STAT3. In conclusion, FLX impacts on energy balance and induces leptin sensitivity and an intact TrkB function is required for these effects to take place.

Molecular Therapy of Melanocortin-4-Receptor Obesity by an Autoregulatory BDNF Vector

Mutations in the melanocortin-4-receptor (MC4R) comprise the most common monogenic form of severe early-onset obesity, and conventional treatments are either ineffective long-term or contraindicated. Immediately downstream of MC4R—in the pathway for regulating energy balance—is brain-derived neurotrophic factor (BDNF). Our previous studies show that adeno-associated virus (AAV)-mediated hypothalamic BDNF gene transfer alleviates obesity and diabetes in both diet-induced and genetic models. To facilitate clinical translation, we developed a built-in autoregulatory system to control therapeutic gene expression mimicking the body’s natural feedback systems. This autoregulatory approach leads to a sustainable plateau of body weight after substantial weight loss is achieved. Here, we examined the efficacy and safety of autoregulatory BDNF gene therapy in Mc4r heterozygous mice, which best resemble MC4R obese patients. Mc4r heterozygous mice were treated with either autoregulatory BDNF vector or YFP control and monitored for 30 weeks. BDNF gene therapy prevented the development of obesity and metabolic syndromes characterized by decreasing body weight and adiposity, suppressing food intake, alleviating hyperleptinemia and hyperinsulinemia, improving glucose and insulin tolerance, and increasing energy expenditure, without adverse cardiovascular function or behavioral disturbances. These safety and efficacy data provide preclinical evidence that BDNF gene therapy is a compelling treatment option for MC4R-deficient obese patients.

Spontaneous physical activity and mediators of energy homeostasis in the hypothalamus of mice from 4 to 10 months of age.

What is the central question of this study? Is the initial decline of spontaneous physical activity (SPA) in mice related to impaired insulin and leptin signalling or brain-derived neurotrophic factor expression in the hypothalamus? What is the main finding and its importance? We showed that SPA started to decline at an early stage, concomitantly with an impairment of hypothalamic leptin signalling. Consequently, energy expenditure decreased and glucose tolerance worsened. Our results demonstrate the need to counteract the initial decline in SPA to avoid metabolic impairments and indicate the possible involvement of central leptin in the reduction in SPA with age. The biological control of physical activity is poorly understood. Age decreases insulin, leptin and brain-derived neurotrophic factor (BDNF) signalling in the hypothalamus, and all have been shown to modulate spontaneous physical activity (SPA). We investigated the age at which SPA starts to decline and whether this is associated with the emergence of hypothalamic insulin and leptin resistance and reduced BDNF expression. Spontaneous physical activity (and other parameters of locomotion) and energy expenditure were determined monthly in mice from the 4th to the 10thmonth of age. Metabolic and hypothalamic analyses were performed in 4-, 6- and 10-month-old mice. Spontaneous physical activity, distance travelled and speed of locomotion started to decrease in 6-month-old mice. The reduction in SPA became more evident from 8months of age. Energy expenditure decreased from the 8thmonth. Hypothalamic BDNF protein expression and insulin signalling did not change throughout the time span studied. Leptin signalling decreased at 6 and 10months compared with 4months. Also, compared with 4months, 6- and 10-month-old mice were glucose intolerant. In conclusion, SPA begins to decline in parallel with reduced hypothalamic leptin signalling. Metabolic impairment also manifests as SPA decreases, highlighting the need to understand the regulation of SPA in order to combat its decline.

Is There a Role for Bioactive Lipids in the Pathobiology of Diabetes Mellitus?

Inflammation, decreased levels of circulating endothelial nitric oxide (eNO) and brain-derived neurotrophic factor (BDNF), altered activity of hypothalamic neurotransmitters (including serotonin and vagal tone) and gut hormones, increased concentrations of free radicals, and imbalance in the levels of bioactive lipids and their pro- and anti-inflammatory metabolites have been suggested to play a role in diabetes mellitus (DM). Type 1 diabetes mellitus (type 1 DM) is due to autoimmune destruction of pancreatic β cells because of enhanced production of IL-6 and tumor necrosis factor-α (TNF-α) and other pro-inflammatory cytokines released by immunocytes infiltrating the pancreas in response to unknown exogenous and endogenous toxin(s). On the other hand, type 2 DM is due to increased peripheral insulin resistance secondary to enhanced production of IL-6 and TNF-α in response to high-fat and/or calorie-rich diet (rich in saturated and trans fats). Type 2 DM is also associated with significant alterations in the production and action of hypothalamic neurotransmitters, eNO, BDNF, free radicals, gut hormones, and vagus nerve activity. Thus, type 1 DM is because of excess production of pro-inflammatory cytokines close to β cells, whereas type 2 DM is due to excess of pro-inflammatory cytokines in the systemic circulation. Hence, methods designed to suppress excess production of pro-inflammatory cytokines may form a new approach to prevent both type 1 and type 2 DM. Roux-en-Y gastric bypass and similar surgeries ameliorate type 2 DM, partly by restoring to normal: gut hormones, hypothalamic neurotransmitters, eNO, vagal activity, gut microbiota, bioactive lipids, BDNF production in the gut and hypothalamus, concentrations of cytokines and free radicals that results in resetting glucose-stimulated insulin production by pancreatic β cells. Our recent studies suggested that bioactive lipids, such as arachidonic acid, eicosapentaneoic acid, and docosahexaenoic acid (which are unsaturated fatty acids) and their anti-inflammatory metabolites: lipoxin A4, resolvins, protectins, and maresins, may have antidiabetic actions. These bioactive lipids have anti-inflammatory actions, enhance eNO, BDNF production, restore hypothalamic dysfunction, enhance vagal tone, modulate production and action of ghrelin, leptin and adiponectin, and influence gut microbiota that may explain their antidiabetic action. These pieces of evidence suggest that methods designed to selectively deliver bioactive lipids to pancreatic β cells, gut, liver, and muscle may prevent type 1 and type 2 DM.

Developmental changes in the hypothalamic mRNA expression levels of brain-derived neurotrophic factor and serum leptin levels: Their responses to fasting in male and female rats

The actions and responses of hypothalamic appetite regulatory factors change markedly during the neonatal to pre-pubertal period in order to maintain appropriate metabolic and nutritional conditions. In this study, we examined the developmental changes in the hypothalamic mRNA levels of brain-derived neurotrophic factor (BDNF), which is a potent anorectic factor and the changes in the sensitivity of the hypothalamic expression of this factor to fasting during the neonatal to pre-pubertal period. Under fed conditions, hypothalamic BDNF mRNA expression decreased during development in both male and female rats. Similarly, the serum levels of leptin, which is a positive regulator of hypothalamic BDNF expression, also tended to fall during the developmental period. The serum leptin level and the hypothalamic BDNF mRNA level were found to be positively correlated in both sexes under the fed conditions. Hypothalamic BDNF mRNA expression was decreased by 24h fasting (separating the rats from their mothers) in the early neonatal period (postnatal day 10) in both males and females, but no such changes were seen at postnatal day 20. Twenty-four hours’ fasting (food deprivation) did not affect hypothalamic BDNF mRNA expression in the pre-pubertal period (postnatal day 30). On the other hand, the rats’ serum leptin levels were decreased by 24h fasting (separating the rats from their mothers at postnatal day 10 and 20, and food deprivation at postnatal day 30) throughout the early neonatal to pre-pubertal period. The correlation between serum leptin and hypothalamic BDNF mRNA levels was not significant under the fasted conditions. It can be speculated that leptin partially regulates hypothalamic BDNF mRNA levels, but only in fed conditions. Such changes in hypothalamic BDNF expression might play a role in maintaining appropriate metabolic and nutritional conditions and promoting normal physical development. In addition, because maternal separation induces a negative energy balance and short- and long-term stress responses, it is also possible that reductions in hypothalamic BDNF mRNA levels in the early neonatal period (postnatal day 10) may be partially induced by stress responses of the maternal deprivation.

Environmental and Genetic Activation of Hypothalamic BDNF Modulates T-cell Immunity to Exert an Anticancer Phenotype.

Macroenvironmental factors, including a patient's physical and social environment, play a role in cancer risk and progression. Our previous studies show that living in an enriched environment (EE) providing complex stimuli confers an anticancer phenotype in mice mediated, in part by a specific neuroendocrine axis, with brain-derived neurotrophic factor (BDNF) as the key brain mediator. Here, we investigated how an EE modulated T-cell immunity and its role in the EE-induced anticancer effects. Our data demonstrated that CD8 T cells were required to mediate the anticancer effects of an EE in an orthotropic model of melanoma. In secondary lymphoid tissue (SLT), an EE induced early changes in the phenotype of T-cell populations, characterized by a decrease in the ratio of CD4 T helper to CD8 cytotoxic T lymphocytes (CTL). Overexpression of hypothalamic BDNF reproduced EE-induced T-cell phenotypes in SLT, whereas knockdown of hypothalamic BDNF inhibited EE-induced immune modulation in SLT. Both propranolol and mifepristone blocked the EE-associated modulation of CTLs in SLT, suggesting that both the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis were involved. Our results demonstrated that enhanced anticancer effect of an EE was mediated at least in part through modulation of T-cell immunity and provided support to the emerging concept of manipulating a single gene in the brain to improve cancer immunotherapy. Cancer Immunol Res; 4(6); 488-97. ©2016 AACR.