AgRP and Npy neurons are key first-order neurons in the arcuate nucleus of the hypothalamus that sense and integrate diverse metabolic signals. Paradoxically, chronic and acute disruptions of AgRP gene or AgRP neurons have produced very different outcomes. While acute reduction of AgRP gene expression by 50% using RNA interference results in significant weight loss days after siRNA administration (Makimura et al., 2002
), germline mutation of AgRP gene has minimal consequence on body weight (Qian et al., 2002
; Wortley et al., 2005
). Similarly, acute ablation of AgRP neurons leads to severe anorexia and weight loss, while progressive degeneration of AgRP neurons in the AgRP-Tfam mutants has minimal impact on feeding and body weight even though the same Tg.AgRP-Cre
line was used in both studies (Gropp et al., 2005
; Xu et al., 2005b
). These results clearly demonstrate that compensatory mechanisms develop during the gradual loss of AgRP neuronal function, which allow the mice to escape the deleterious effects of weight loss. In this study, we provide evidence that de novo
cell proliferation increases in the hypothalamus of adult AgRP-Tfam mutant mice in response to degeneration of the AgRP neurons, and that some of the newly generated cells take on AgRP neuronal cell fate. We further show that a subset of the newly proliferative cells are capable of responding to leptin and that central blockade of cell proliferation leads to decreased feeding and body adiposity in the mutants but not in the controls. Thus, our study suggests that de novo
neurogenesis provides a new level of neural plasticity in reshaping the hypothalamic feeding circuits.
It should be noted that there are methodological limitations in the detection of neurogenesis in the adult brain (Emsley et al., 2005
), and the most relevant to this study is the potential incorporation of BrdU into AgRP neurons that are undergoing DNA repair. However, evidence of mitotic divisions indicated by closely situated cell doublets, the generation of non-AgRP neurons such as Pomc neurons, and alteration of energy balance upon inhibition of CNS cell proliferation strongly argue that cell proliferation is indeed increased in the hypothalamus of AgRP-Tfam mutant mice. The nuclear localization of BrdU signals also excludes mitochondrial labeling. The basal levels of BrdU positive cells in our control hypothalamus appear to be lower than previously reported by Kokoeva and colleagues (Kokoeva et al., 2005
). There are several possible reasons for this discrepancy. First, there are differences in experimental procedures. For example, Kokoeva and colleagues prepared hypothalamic sections at 25 μm and we prepared ours at 10 μm, so our sections were thinner, which may contribute to the lower cell counts. Also, different types of osmotic minipumps were used. Second, genetic background may influence the rate of adult neurogenesis. While Kokoeva and colleagues used C57BL/6 mice, our AgRP-Tfam mice were on mixed background with contributions from 129/SvJ, C57BL/6 and FVB. It has been shown that C57BL/6 mice exhibit higher degree of adult neurogenesis in the hippocampus region compared with other mouse strains including 129/SvJ (Kempermann et al., 1997
; Kempermann and Gage, 2002
). Thus, it is possible that genetic background variation may contribute to the discrepant cell counts in these studies.
AGRP and NPY are two of the most potent orexigens identified to date. Disruption of AgRP/Npy neuronal function due to mutations, injuries or neurodegeneration can lead to severe anorexia and weight loss, which is life-threatening. Thus, evolution may have favored the development of multiple mechanisms to ensure that the orexigenic drive is not lost. The fact that AgRP and Npy are co-expressed in the same neurons in the arcuate nucleus suggests that one such safeguarding mechanism is by coexpression of genes that perform similar functions. Another level of compensation could be provided by the redundant wirings of AgRP/Npy neurons on their targets. AgRP/Npy neurons are known to project to MC4R neurons and also to their upstream Pomc neurons that express the ligand for MC4R (Bagnol et al., 1999
; Cowley et al., 1999
; Cowley et al., 2001
; Roseberry et al., 2004
). Thus, AgRP/Npy neurons exert their orexigenic functions by inhibiting two different anorexigenic neurons, the Pomc and MC4R neurons, via regulation of neuronal activities and receptor signaling. The redundant wiring and differential regulation of Pomc and MC4R neurons provide another safeguarding mechanism to ensure sufficient inhibition of anorexigenic drive. The current study provides an additional compensatory mechanism in that de novo cell proliferation functions to replenish the diminishing AgRP neuronal cell pool. Previous studies indicate that neurons can survive and remain functional for a few months following Tfam deletion (Sorensen et al., 2001
). Thus, the ability to regenerate neurons in the AgRP-Tfam mutants would allow other compensatory mechanisms to develop, which would act in concert to provide full functional compensation. Some of these potential compensatory mechanisms include modulation of synaptic densities on MC4R, Pomc or other downstream neurons, change in MC4R and NPY receptor sensitivity or alterations of gene expression in downstream neurons.
Adult neurogenesis is readily observed in the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus in the hippocampus, but is limited in the adult hypothalamus under normal conditions (Ming and Song, 2005
). However, resident neural stem cells have been described in the adult hypothalamus, where de novo neurogenesis occurs at a low rate (Kokoeva et al., 2007
). A number of growth factors and neurotrophic factors such as FGF2, BDNF, CNTF, VEGF and TGFα have been shown to regulate neural stem cells and neural progenitor proliferation in the adult rodent brain (Hagg, 2005
; Lledo et al., 2006
). Infusion of BDNF into the lateral ventricle of the adult rat leads to generation of new neurons in the hypothalamus (Pencea et al., 2001
). In addition, central administration of CNTF to mice stimulates neurogenesis in the adult hypothalamus (Kokoeva et al., 2005
). Together, these results suggest that neurogenesis in the adult hypothalamus can be stimulated under certain conditions. It is likely that neurodegeneration in the AgRP-Tfam mutant mice activates this latent pool of neural stem cells, and they differentiate into AgRP neurons along with other resident cell types such as Pomc neurons and glial cells. Since AgRP neurons, but not Pomc or other neurons, are specifically degenerated in the AgRP-Tfam mutant animals, regeneration of AgRP neurons would be expected to have greater impact on the AgRP neuronal cell pool, while the addition of new Pomc neurons may not affect the overall Pomc population. It is difficult to directly compare the relative abundance of new born AgRP neurons with new born Pomc neurons due to differences in antibody sensitivity. However, it is conceivable that the increase in cell proliferation in the mutant hypothalamus would partially restore orexigenic drive while having smaller impact on anorexigenic drive.
Neurogenesis in the adult brain, although at low rates, could be a critical mechanism to replenish degenerated neurons due to aging, gene mutations or environmental toxins, and impairment in this process could lead to disease onset or aggravate disease progression. Impaired adult neurogenesis has been implicated in the etiology and progression of neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease or Amyotrophic Lateral Sclerosis, which are characterized by loss of neurons in particular brain regions (for review, see (Abdipranoto et al., 2008
). Stimulation of neurogenesis has been found to associate with functional recovery of memory and learning in models of Alzheimer’s disease. In addition, it has been shown that neurogenesis increases following ischemia or stroke in both humans and rodents, suggesting that stroke-induced neurogenesis serves as a self-repair mechanism (Okano et al., 2007
; Wiltrout et al., 2007
). Interestingly, recent studies show that consumption of high fat diet in rodents causes hypothalamic ER stress (Zhang et al., 2008
; Ozcan et al., 2009
) and high fat diet induces apoptosis of hypothalamic neurons (Moraes et al., 2009
). Thus, these findings raise the possibility that diet-induced neurodegeneration may play a role in the etiology of obesity. Our current study provides new evidence that AgRP neurons, critical regulators of energy balance, can be regenerated in the adult hypothalamus, suggesting that adult neurogenesis is one adaptive mechanism to reshape the feeding circuits in response to hypothalamic neurodegeneration. Understanding the mechanisms underlying neural stem cell activation would pave the way for future therapeutic intervention to treat feeding disorders that are associated with neurodegeneration.