Loss of autophagy in pro-opiomelanocortin neurons perturbs axon growth and causes metabolic dysregulation

Abstract

The hypothalamic melanocortin system, which includes neurons that produce pro-opiomelanocortin (POMC)-derived peptides, is a major negative regulator of energy balance. POMC neurons begin to acquire their unique properties during neonatal life. The formation of functional neural systems requires massive cytoplasmic remodeling that may involve autophagy, an important intracellular mechanism for the degradation of damaged proteins and organelles. Here we investigated the functional and structural effects of the deletion of an essential autophagy gene, Atg7, in POMC neurons. Lack of Atg7 in POMC neurons caused higher postweaning body weight, increased adiposity, and glucose intolerance. These metabolic impairments were associated with an age-dependent accumulation of ubiquitin/p62-positive aggregates in the hypothalamus and a disruption in the maturation of POMC-containing axonal projections. Together, these data provide direct genetic evidence that Atg7 in POMC neurons is required for normal metabolic regulation and neural development, and they implicate hypothalamic autophagy deficiency in the pathogenesis of obesity.

Figure 1

Identification of autophagy in hypothalamic POMC processes. (A) Representative images and quantification of LC3-GFP puncta in the arcuate nucleus (ARH) and the hypothalamic periventricular zone (PeV) of P10, P14, P21, and adult (8- to 9-week-old) mice (n = 4–5 per group). Dashed boxes in the schematics represent the approximate borders of the areas used for quantification. (B) Immunoblot analysis of LC3 (LC3-I, 18 kDa; LC3-II, 16 kDa) and b-actin (as a loading control) from hypothalami derived from adult mice. (C) Representative electron micrographs showing autophagosomes (arrows) in POMC-immunolabeled perikarya and processes of P24 wild-type mice. D, dendrites, V3, third ventricle. Values are shown as mean ± SEM. *P < 0.05 versus Atg7^loxP/loxP. Scale bar, 15 um (A) and 1 um (C).

Figure 2

Altered metabolism in mice lacking autophagy in POMC neurons. (A) Pre- and (B) post-weaning growth curves of Atg7^loxP/loxP (n ≥ 9) and Pomc-Cre; Atg7^loxP/loxP (n = 15) male mice. (C) Leptin sensitivity of 10-week-old Atg7^loxP/loxP (n = 6) and Pomc-Cre; Atg7^loxP/loxP (n = 8) male mice. Mass of (D) retroperitoneal and (E) epididymal fat of 7- and 15- to 17-week-old Atg7^loxP/loxP (n = 4–5) and Pomc-Cre; Atg7^loxP/loxP (n = 5–11) male mice. (F) Serum leptin and (I) insulin levels in Atg7^loxP/loxP (n = 8) and Pomc-Cre; Atg7^loxP/loxP (n = 6) male mice from 7- and 15–17 weeks of age. (G) Glucose tolerance test (GTT) and (H) area under the GTT curve of 8- to 9-week-old Atg7^loxP/loxP (n = 7) and Pomc-Cre; Atg7^loxP/loxP (n = 12) male mice. Values are shown as mean ± SEM. *P < 0.05 versus Atg7^loxP/loxP.

Figure 3

Lack of autophagy in POMC neurons leads to the gradual accumulation of ubiquitin aggregates in the arcuate nucleus. Quantification of (A) ubiquitin- and (B) p62-immunoreactivity in the arcuate nucleus (ARH) of P14, P22, and adult (15- to 17-week-old) Atg7^loxP/loxP (n = 4) and Pomc-Cre; Atg7^loxP/loxP (n = 4) male mice. (A, B) Confocal images illustrating (A) ubiquitin- and (B) p62-immunoreactivity in the ARH of adult Atg7^loxP/loxP and Pomc-Cre; Atg7^loxP/loxP mice. (C) Confocal images showing the presence of ubiquitin-immunoreactivity (green fluorescence) in aMSH-positive cells (red fluorescence) of an adult Pomc-Cre; Atg7^loxP/loxP mouse. The arrow points to a doubled labeled cell. V3, third ventricle. Values are shown as mean ± SEM. *P < 0.05 versus P14; #P < 0.05 versus P22. Scale bars, 50 um.

Figure 4

Disruption of POMC projections in mice lacking autophagy in POMC neurons. Confocal images and quantification of aMSH-IR fibers in the PVH of (A) P14, (B) P22, and (C) adult (15- to 17-week-old) Atg7^loxP/loxP (n = 4) and Pomc-Cre; Atg7^loxP/loxP (n = 4) male mice. (D) Images and quantification of b-endorphin (a POMC-derived peptide)-immunopositive fibers derived from isolated organotypic cultures of the ARH from P4 Atg7^loxP/loxP and Pomc-Cre; Atg7^loxP/loxP mice. ARH, arcuate nucleus of the hypothalamus, PVH, paraventricular nucleus of the hypothalamus, V3, third ventricle. Values are shown as mean ± SEM. *P < 0.05 versus Atg7^loxP/loxP. Scale bars, 50 um.