De novo neurogenesis in adult hypothalamus as a compensatory mechanism to regulate energy balance

Abstract

The ability to develop counter-regulatory mechanisms to maintain energy balance in response to environmental and physiologic insults is essential for survival, but the mechanisms underlying these compensatory regulations are poorly understood. Agouti-related peptide (AGRP) and Neuropeptide Y are potent orexigens and are coexpressed in neurons in the arcuate nucleus of the hypothalamus. Acute ablation of these neurons leads to severe anorexia and weight loss, whereas progressive degeneration of these neurons has minimal impact on food intake and body weight, suggesting that compensatory mechanisms are developed to maintain orexigenic drive. In this study, we show that cell proliferation is increased in the hypothalamus of adult mutant animals in which AgRP neurons undergo progressive neurodegeneration due to deletion of mitochondrial transcription factor A, and that a subset of these newly generated cells differentiate into AgRP neurons along with other resident neuronal subtypes. Furthermore, some of the newly generated cells are capable of responding to leptin, and a central blockade of cell proliferation in adult animals results in decreases in food intake and body adiposity in mutant but not in control animals. Our study indicates that neurons important for energy homeostasis can be regenerated in adult feeding centers under neurodegenerative conditions. It further suggests that de novo neurogenesis might serve as a compensatory mechanism contributing to the plastic control of energy balance in response to environmental and physiologic insults.

Figure 1.

Cell proliferation is increased in the hypothalamus of AgRP-Tfam mutant mice. A–D, Immunohistochemical analyses for endogenous cell proliferation markers Ki-67 (A, B) and PCNA (C, D) were carried out in 2.5- to 4-month-old male control and AgRP-Tfam mutant mice. E, F, A separate cohort of control (E) and mutant (F) mice was infused intracerebroventricularly with BrdU (3.2 μg/μl) via osmotic minipumps (flow rate of 0.15 μl/h for 6 weeks; Alzet model 2006; Durect). Immunofluorescence analysis of BrdU in the hypothalamus of controls and mutants is shown. G, Proportional increase of Ki-67-, PCNA-, or BrdU-positive cells in the mutants was compared with that of the controls. Detailed quantification of positive cells is shown in Table 1. Error bars represent SEM; **p ≤ 0.01. 3V, Third ventricle.

Figure 2.

Newborn hypothalamic cells take on AgRP neuronal identity. A–C, Representative field showing the colocalization of AGRP immunoreactivity (cytoplasmic signals) and PCNA nuclear signals from a colchicine-injected AgRP-Tfam mutant mouse. The arrows show the colocalized cell. DAPI staining revealed all nuclei in the field. The specificity of the AGRP antibody was previously characterized (Xu et al., 2005a,b). D–G, X-gal staining combined with immunohistochemical analysis of PCNA or Ki-67 in hypothalamic sections from the AgRP-Tfam mutant animals. Control and mutant animals carried the Tg.AgRP-Cre transgene and the Cre-activatable R26R-LacZ reporter so that Agrp neurons were identified by LacZ expression (Xu et al., 2005a,b). In this preparation, LacZ expression is characteristically seen as perinuclear blue dots, whereas brown nuclear staining represents the PCNA or Ki-67 signal as indicated. Arrows show a LacZ/PCNA coexpressing neuron and a LacZ/Ki-67 coexpressing neuron. E, Magnified view of the boxed area in D; G, magnified view of the boxed area in F. The specificity of the Tg.AgRP-Cre transgenic mice has been characterized and validated by multiple studies (Gropp et al., 2005; Xu et al., 2005a,b; Kitamura et al., 2006; Konner et al., 2007; van de Wall et al., 2008; Zhang et al., 2008). 3V, Third ventricle.

Figure 3.

Newborn hypothalamic cells take on Pomc neuronal identity. A, Representative field of the ARC showing expression of PCNA (nuclear signal) and ACTH (product of the Pomc neurons; cytoplasmic signal) in an AgRP-Tfam mutant animal. DAPI marked all nuclei in the field. B–D, Magnified views of the boxed area in A, showing a pair of PCNA-positive cells that expressed ACTH. Arrows indicate the two colocalized cells. Fourteen Pomc cells were identified out of 150 PCNA-positive cells from six different mutant mice. The ACTH antibody was previously used and it was highly specific (Piper et al., 2008). 3V, Third ventricle.

Figure 4.

Newly generated hypothalamic cells are responsive to leptin. Four-and-a-half-month-old AgRP-Tfam mutant mice were treated with either vehicle or leptin (5 mg/kg, i.p.), and mice were killed 45 min later. Immunofluorescence analysis was carried out to identify cells that express pSTAT3 and PCNA. A, B, Leptin but not saline treatment activated pSTAT3 in the ARC of the hypothalamus. C, A field in the ARC of the hypothalamus showing expression of PCNA and leptin-induced pSTAT3 expression. Arrow indicates a colocalized cell. D–F, Magnified views of the colocalized cell indicated by the arrow in C showing PCNA, pSTAT3 immunoreactivity, and merged images. Arrow indicates a colocalized cell. Twenty-four of 106 PCNA cells (22.6%) were found to be positive for pSTAT3. Fourteen sections from six mice were analyzed. 3V, Third ventricle.

Figure 5.

Blockade of cell proliferation in the adult brain leads to decreased food intake and body adiposity in AgRP-Tfam mutants but not in control mice. Vehicle or mitotic blocker AraC (10.9 μg/μl) was infused into the lateral ventricle of 3-month-old control and AgRP-Tfam mutant mice via osmotic minipumps (flow rate of 0.15 μl/h). A, B, AraC effectively blocked cell proliferation in the hypothalamus of mutant mice as evident by reduced number of BrdU-positive cells upon AraC treatment. C, Average weekly food intake during a 4 week AraC treatment in control and mutant mice. D, Body weight of mice after 4 weeks of AraC or vehicle treatment was compared with their own pretreatment body weight. E, F, Body composition was measured by DEXA before and after 4 weeks of AraC or vehicle treatment. Four weeks of AraC treatment did not change body lean mass in either controls or mutants, whereas it significantly decreased body fat mass in the mutants but not in the controls. n = 5–6 mice for each group; *p ≤ 0.05; **p ≤ 0.01. 3V, Third ventricle.