Hypothalamic dysfunction of the thrombospondin receptor α2δ-1 underlies the overeating and obesity triggered by brain-derived neurotrophic factor deficiency

Abstract

Brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, are critical components of the neural circuitry controlling appetite and body weight. Diminished BDNF signaling in mice results in severe hyperphagia and obesity. In humans, BDNF haploinsufficiency and the functional Bdnf Val66Met polymorphism have been linked to elevated food intake and body weight. The mechanisms underlying this dysfunction are poorly defined. We demonstrate a chief role of α2δ-1, a calcium channel subunit and thrombospondin receptor, in triggering overeating in mice with central BDNF depletion. We show reduced α2δ-1 cell-surface expression in the BDNF mutant ventromedial hypothalamus (VMH), an energy balance-regulating center. This deficit contributes to the hyperphagia exhibited by BDNF mutant mice because selective inhibition of α2δ-1 by gabapentin infusion into wild-type VMH significantly increases feeding and body weight gain. Importantly, viral-mediated α2δ-1 rescue in BDNF mutant VMH significantly mitigates their hyperphagia, obesity, and liver steatosis and normalizes deficits in glucose homeostasis. Whole-cell recordings in BDNF mutant VMH neurons revealed normal calcium currents but reduced frequency of EPSCs. These results suggest calcium channel-independent effects of α2δ-1 on feeding and implicate α2δ-1-thrombospondin interactions known to facilitate excitatory synapse assembly. Our findings identify a central mechanism mediating the inhibitory effects of BDNF on feeding. They also demonstrate a novel and critical role for α2δ-1 in appetite control and suggest a mechanism underlying weight gain in humans treated with gabapentinoid drugs.

Figure 1.

Cell-surface expression of α2δ-1 is reduced in the VMH of BDNF^2L/2LCk-cre mice. A, Representative in situ hybridization showing high α2δ-1 mRNA expression in wild-type VMH (arrows). B, Representative immunolabeling of α2δ-1 protein in wild-type hypothalamus showing high expression in the VMH and minimal expression in the DMH. C, Western blot analysis of total, cell-surface (Surf.), and cytosolic (Cyt.) α2δ-1 protein content in the VMH of BDNF^2L/2LCk-cre CM mice compared with WT controls (n = 6). *p = 0.002; **p = 0.02. β-tub, β-Tubulin. D, Representative confocal images of WT VMH coimmunolabeled with anti-α2δ-1 (green) and anti-TrkB (red) showing a high level of colocalization of these two markers.

Figure 2.

Selective inhibition of α2δ-1 in the VMH induces hyperphagia and excessive weight gain. Food intake (A) and relative body weight changes (B) in WT mice with chronic delivery of saline (Sal; n = 5) or GP (n = 6) to the VMH are shown. *p = 0.02; **p = 0.04.

Figure 3.

Viral-mediated delivery of α2δ-1 to the VMH of wild-type and BDNF mutant mice. A, Representative image of GFP⁺ cells in the hypothalamus of a mouse delivered AAV-GFP bilaterally showing accurate targeting of the VMH at a coronal level corresponding to bregma −1.70. B, C, Images of coronal levels from the same animal corresponding to bregma −1.06 mm (B) and −2.60 mm (C) and lacking GFP⁺ cell bodies indicate the restricted rostrocaudal span of the viral injections. D, Representative images of brain sections before and after being subjected to laser capture microdissection (LCM) to selectively dissect VMH cells. E, Quantitative RT-PCR post hoc analysis of α2δ-1 mRNA expression in cells laser captured from the VMH of WT and BDNF^2L/2LCk-cre (CM) mice delivered AAV-GFP or AAV-α2δ-1 to the VMH via stereotaxic surgery. F, Measurements of cell-surface and cytosol content of α2δ-1 in BDNF^2L/2LCk-cre mice treated with AAV-α2δ-1 (CM-α2δ-1) relative to control mutants that received AAV-GFP (CM-GFP). Minimal detection of β-tubulin (β-tub), a cytoskeletal protein, in the cell-surface fraction confirms the success of the biotinylation assay. Arc, Arcuate nucleus; 3V, third ventricle; PVN, paraventricular nucleus; AHP, anterior hypothalamic area; MT, mammillotegmental tract; PMN, premammillary nucleus; LH, lateral hypothalamic area. *p < 0.05.

Figure 4.

The α2δ-1 rescue in the VMH of BDNF^2L/2LCk-cre mutants mitigates their hyperphagia, excessive weight gain, and liver steatosis. A, Weekly food intake of BDNF^2L/2LCk-cre conditional mutant mice with VMH delivery of AAV-α2δ-1 (CM-α2δ-1) or AAV-GFP (CM-GFP) and WT mice delivered AAV-α2δ-1 (WT-α2δ-1) or AAV-GFP (WT-GFP). There was a significant effect of time on food intake (F_(17,25) = 3.175; p = 0.004, two-way ANOVA with repeated measures; n = 9–12). *p < 0.05, CM-α2δ-1 relative to CM-GFP. B, There was also a significant effect on relative body weight gain (F_(18,24) = 53.15; p < 0.00001, two-way ANOVA with repeated measures; n = 9–12). *p < 0.05; ^#p < 0.1; CM-α2δ-1 relative to CM-GFP. C, Measurement of locomotor activity over 3 d expressed as total beam breaks. *p < 0.01. D, There was a significant effect of treatment on liver (F_(3,21) = 70.4; p < 0.001, one-way ANOVA) and total fat (F_(3,21) = 17.0; p < 0.001, one-way ANOVA) tissue weights. *p < 0.05. E, Representative liver tissue sections from WT-GFP, CM-GFP, and CM-α2δ-1 mice stained with hematoxylin and eosin (H&E) or Oil Red O, which stains lipids.

Figure 5.

The α2δ-1 rescue in the VMH of BDNF^2L/2LCk-cre mutants normalizes deficits in glucose homeostasis. A, There was a significant effect on fasted circulating levels of glucose (F_(3,20) = 15.0; p < 0.001), insulin (F_(2,14) = 25.3; p < 0.01), and leptin (F_(3,29) = 20.6; p < 0.000001, one-way ANOVA) in WT-GFP, WT-α2δ-1, CM-GFP, and CM-α2δ-1 mice. *p < 0.04. B, Glucose tolerance test showing compromised responses in CM-GFP mice compared with WT-GFP mice (F_(1,9) = 5.10; p = 0.05, two-way ANOVA with repeated measures) and improved glucose metabolism in CM-α2δ-1 mice compared with the CM-GFP group (F_(1,9) = 5.73; p = 0.04, two-way ANOVA with repeated measures). *p < 0.04, CM-GFP mice compared with CM-α2δ-1 mice. C, There was a significant effect of treatment on area under the curve (AUC) for the glucose tolerance test (F_(3,20) = 3.3; p = 0.04, one-way ANOVA). *p < 0.02.

Figure 6.

Pair-feeding improves but does not normalize glucose homeostasis in BDNF^2L/2LCk-cre conditional mutants. A, There was a significant effect of pair-feeding on liver (F_(2,23) = 8.2; p = 0.002) and total fat (F_(2,23) = 17.4; p = 0.000) tissue weights in naive wild-type (WT Ad Lib) and BDNF^2L/2LCk-cre conditional mutant (CM Ad Lib) mice with ad libitum access to food and in naive BDNF^2L/2LCk-cre conditional mutants pair-fed to BDNF^2L/2LCk-cre mutants treated with AAV α2δ-1 (CM Pair Fed). *p < 0.004. B, Fasted serum levels of glucose (F_(2,31) = 10.2; p = 0.000), insulin (F_(2,13) = 8.1; p = 0.005), and leptin (F_(2,23) = 12.4; p = 0.000) in WT Ad Lib, CM Ad Lib, and CM Pair Fed mice. C, Glucose tolerance test in naive wild-type (WT Ad Lib) and BDNF^2L/2LCk-cre conditional mutant (CM Ad Lib) mice with ad libitum access to food and in naive BDNF^2L/2LCk-cre conditional mutants pair-fed to BDNF^2L/2LCk-cre mutants treated with AAV-α2δ-1. Compromised responses to glucose were observed in CM Ad Lib mice compared with WT Ad Lib mice (F_(1,13) = 97.0; p = 0.000, two-way ANOVA with repeated measures) and in CM Pair Fed mice compared with the WT Ad Lib group (F_(1,13) = 17.9; p = 0.000, two-way ANOVA with repeated measures). *p < 0.02, WT Ad Lib mice compared with CM Pair Fed mice. D, There was a significant effect of treatment on area under the curve (AUC) for the glucose tolerance test (F_(2,19) = 26.6; p = 0.000, one-way ANOVA). *p < 0.01; ^#p < 0.1.

Figure 7.

Normal calcium currents but reduced frequency of EPSCs in VMH cells of BDNF^2L/2LCk-cre mice. A, Representative whole-cell Ca²⁺ currents recorded from VMH cells in WT (n = 14) and BDNF^2L/2LCk-cre (n = 10) mice. The 250 ms test pulses from a holding potential of −70 mV were applied in 10 mV increments. B, I–V relationships resulting from step potentials from −70 to +40 mV in 10 mV increments. C, Amplitude, or kinetics, of Ca²⁺ currents in WT and BDNF^2L/2LCk-cre CM mice. Histograms for peak current (picoamperes per picofarad, p = 0.8), time to peak (milliseconds, p = 0.35), and mean inactivation of the maximal current traces at the end of the 250 ms depolarization (R250%; p = 0.18). D, Representative traces in voltage clamp from wild-type and BDNF^2L/2LCKCre conditional mutant mice show EPSCs under basal conditions and in the presence of bicuculline. E, sEPSC amplitude and frequency data (mean ± SE) for WT (n = 8) and BDNF^2L/2LCKCre mutant (CM; n = 9) VMH cells. *p = 0.002.