Loss of autophagy in hypothalamic POMC neurons impairs lipolysis

Autophagy degrades cytoplasmic contents to achieve cellular homeostasis. We show that selective loss of autophagy in hypothalamic proopiomelanocortin (POMC) neurons decreases α-melanocyte-stimulating hormone (MSH) levels, promoting adiposity, impairing lipolysis and altering glucose homeostasis. Ageing reduces hypothalamic autophagy and α-MSH levels, and aged-mice phenocopy, the adiposity and lipolytic defect observed in POMC neuron autophagy-null mice. Intraperitoneal isoproterenol restores lipolysis in both models, demonstrating normal adipocyte catecholamine responsiveness. We propose that an unconventional, autophagosome-mediated form of secretion in POMC neurons controls energy balance by regulating α-MSH production. Modulating hypothalamic autophagy might have implications for preventing obesity and metabolic syndrome of ageing.     

Weighing on autophagy: A novel mechanism for the CNS regulation of energy balance

Comment on: Coupé B, et al. Cell Metab 2012; 15:247–55, Kaushik S, et al. EMBO Rep 2012; 13:258-65 and Quan W, et al. Endocrinology 2012; 153: In pressAutophagy has received considerable attention over the past decade owing to the fact that alteration of this cellular process, which degrades cytoplasmic materials, including organelles and misfolded proteins, contributes to a variety of diseases, such as cancer, muscular disorders and neurodegeneration.¹ Recent studies using conditional, cell-specific gene-targeting methods have also revealed the importance of autophagy in the regulation of energy balance. For example, the deletion of essential autophagy genes, such as the autophagy-related gene (Atg) 7, in the liver, pancreas or adipose tissue produces alterations in body weight, adiposity and glucose homeostasis.^2-6 Appetite, energy expenditure and metabolism are also carefully regulated by the central nervous system (CNS), particularly the pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus. These neurons act as major negative regulators of energy balance by reducing food intake and increasing energy expenditure. However, the role of CNS autophagy in the regulation of energy balance is largely unknown.Three recent studies, including one from our laboratory, have implicated CNS autophagy in the pathogenesis of obesity (Fig. 1).^7-9 These studies used a conditional cre-loxP approach to specifically delete Atg7 from POMC neurons. Pomc-Cre; Atg7^loxploxp mice display higher body weights, hyperphagia and impaired glucose tolerance.^7-9 These mice also exhibit an increased adiposity that is associated with leptin resistance.^7-9 Mice lacking autophagy in POMC neurons develop an increased sensitivity to weight gain when they are fed a high-fat/high-energy diet.^8,9Open in a separate windowFigure 1. Schematic diagram illustrating the metabolic and structural effects of autophagy deletion in hypothalamic POMC neurons. POMC, pro-opiomelanocortin; Atg, autophagy-related gene.Autophagy plays a particularly important role in biological processes that involve massive cellular elimination, such as neural development.¹⁰ Autophagy is constitutively present in the hypothalamus during important periods of development, and the loss of Atg7 in POMC neurons produces marked structural alterations during the first weeks of postnatal life and prior to the development of obesity(Fig. 1).⁷ Pomc-Cre; Atg7^loxPloxP mice exhibit a reduced density of POMC-containing projections to each of their target nuclei, including the paraventricular nucleus of the hypothalamus, as early as postnatal day 14. These abnormalities in POMC neural projections persist throughout adult life and appear to be the result of diminished capacity of POMC neurons to extend axons.⁷ However, all developmental processes are not affected by autophagy deficiency. No changes in POMC cell numbers between Pomc-Cre; Atg7^loxploxp and control mice are observed,^7-9 which suggests that autophagy does not influence neurogenesis or programmed cell death, but that it specifically affects axonal growth. However, autophagy may be involved in hypothalamic neurodegeneration, because aging is associated with a decline in hypothalamic autophagy and the accumulation of p62 (a polyubiquitin-binding protein that is normally degraded by autophagy) specifically in POMC neurons.⁸ In addition, axonal swelling, which is a hallmark of neurodegeneration, is observed in the hypothalamus of mice that lack autophagy in POMC neurons (Coupé and Bouret, unpublished data).Together, these recent studies suggest that autophagy is required for the proper development and function of POMC neurons, and that autophagy deficiency in POMC neurons causes marked metabolic and structural alterations. Autophagy is highly regulated by nutrient availability,¹¹ including during perinatal life, and further studies may provide novel mechanistic insights on the influence of perinatal dietary changes on the susceptibility to metabolic diseases.     

Loss of Atg12, but not Atg5, in pro-opiomelanocortin neurons exacerbates diet-induced obesity

The autophagy-related proteins ATG12 and ATG5 form a covalent complex essential for autophagy. Here, we demonstrate that ATG12 has distinct functions from ATG5 in pro-opiomelanocortin (POMC)-expressing neurons. Upon high-fat diet (HFD) consumption, mice lacking Atg12 in POMC-positive neurons exhibit accelerated weight gain, adiposity, and glucose intolerance, which is associated with increased food intake, reduced ambulation, and decreased LEP/leptin sensitivity. Importantly, although genetic deletion of either Atg12 or Atg5 renders POMC neurons autophagy-deficient, mice lacking Atg5 in POMC neurons do not exhibit these phenotypes. Hence, we propose nonautophagic functions for ATG12 in POMC neurons that counteract excessive weight gain in response to HFD consumption.     

Defective hypothalamic autophagy directs the central pathogenesis of obesity via the IkappaB kinase beta (IKKbeta)/NF-kappaB pathway

Autophagy has been recently demonstrated to control cell and tissue homeostasis, including the functions of various metabolic tissues. However, it remains unclear whether autophagy is critical for the central nervous system and particularly the hypothalamus for exerting metabolic regulation. Using autophagy-related protein 7 (Atg7) as an autophagic marker, this work showed that autophagy was highly active in the mediobasal hypothalamus of normal mice. In contrast, chronic development of dietary obesity was associated with autophagic decline in the mediobasal hypothalamus. To investigate the potential role of autophagy in the hypothalamic control of metabolic physiology, a mouse model was developed with autophagic inhibition in the mediobasal hypothalamus using site-specific delivery of lentiviral shRNA against Atg7. This model revealed that hypothalamic inhibition of autophagy increased energy intake and reduced energy expenditure. These metabolic changes were sufficient to increase body weight gain under normal chow feeding and exacerbate the progression of obesity and whole-body insulin resistance under high-fat diet feeding. To explore the underlying mechanism, this study found that defective hypothalamic autophagy led to hypothalamic inflammation, including the activation of proinflammatory IκB kinase β pathway. Using brain-specific IκB kinase β knockout mice, it was found that the effects of defective hypothalamic autophagy in promoting obesity were reversed by IκB kinase β inhibition in the brain. In conclusion, hypothalamic autophagy is crucial for the central control of feeding, energy, and body weight balance. Conversely, decline of hypothalamic autophagy under conditions of chronic caloric excess promotes hypothalamic inflammation and thus impairs hypothalamic control of energy balance, leading to accelerated development of obesity and comorbidities.     

Telemetric control of peripheral lipophagy by hypothalamic autophagy

Autophagy maintains cellular quality control by degrading organelles, and cytosolic proteins and their aggregates in lysosomes. Autophagy also degrades lipid droplets (LD) through a process termed lipophagy. During lipophagy, LD are sequestered within autophagosomes and degraded by lysosomal acid lipases to generate free fatty acids that are β-oxidized for energy. Lipophagy was discovered in hepatocytes, and since then has been shown to function in diverse cell types. Whether lipophagy degrades LD in the major fat storing cell—the adipocyte—remained unclear. We have found that blocking autophagy in brown adipose tissues (BAT) by deleting the autophagy gene Atg7 in BAT MYF5 (myogenic factor 5)-positive progenitors increases basal lipid content in BAT and decreases lipid utilization during cold exposure—indicating that lipophagy contributes to lipohomeostasis in the adipose tissue. Surprisingly, knocking out Atg7 in hypothalamic proopiomelanocortin (POMC) neurons also blocks lipophagy in BAT and liver suggesting that specific neurons within the central nervous system (CNS) exert telemetric control over lipophagy in BAT and liver.     

Autophagy--alias self-eating--appetite and ageing

Two articles—one published online in January and in the March issue EMBO reports—implicate autophagy in the control of appetite by regulating neuropeptide production in hypothalamic neurons. Autophagy decline with age in POMC neurons induces obesity and metabolic syndrome.Kaushik et al. EMBO reports, this issue doi:10.1038/embor.2011.260Macroautophagy, which I will call autophagy, is a critical process that degrades bulk cytoplasm, including organelles, protein oligomers and a range of selective substrates. It has been linked with diverse physiological and disease-associated functions, including the removal of certain bacteria, protein oligomers associated with neurodegenerative diseases and dysfunctional mitochondria [1]. However, the primordial role of autophagy—conserved from yeast to mammals—appears to be its ability to provide nutrients to starving cells by releasing building blocks, such as amino acids and free fatty acids, obtained from macromolecular degradation. In yeast, autophagy deficiency enhances death in starvation conditions [2], and in mice it causes death from starvation in the early neonatal period [3,4]. Two recent articles from the Singh group—one of them in this issue of EMBO reports—also implicate autophagy in central appetite regulation [5,6].Autophagy seems to decline with age in the liver [7], and it has thus been assumed that autophagy declines with age in all tissues, but this has not been tested rigorously in organs such as the brain. Conversely, specific autophagy upregulation in Caenorhabditis elegans and Drosophila extends lifespan, and drugs that induce autophagy—but also perturb unrelated processes, such as rapamycin—promote longevity in rodents [8].Autophagy literally means self-eating, and it is therefore interesting to see that this cellular ‘self-eating'' has systemic roles in mammalian appetite control. The control of appetite is influenced by central regulators, including various hormones and neurotransmitters, and peripheral regulators, including hormones, glucose and free fatty acids [9]. Autophagy probably has peripheral roles in appetite and energy balance, as it regulates lipolysis and free fatty acid release [10]. Furthermore, Singh and colleagues have recently implicated autophagy in central appetite regulation [5,6].The arcuate nucleus in the hypothalamus has received extensive attention as an integrator and regulator of energy homeostasis and appetite. Through its proximity to the median eminence, which is characterized by an incomplete blood–brain barrier, these neurons rapidly sense metabolic fluctuations in the blood. There are two different neuronal populations in the arcuate nucleus, which appear to have complementary effects on appetite (Fig 1). The proopiomelanocortin (POMC) neurons produce the neuropeptide precursor POMC, which is cleaved to form α-melanocyte stimulating hormone (α-MSH), among several other products. The α-MSH secreted from these neurons activates melanocortin 4 receptors on target neurons in the paraventricular nucleus of the hypothalamus, which ultimately reduce food intake. The second group of neurons contain neuropeptide Y (NPY) and Agouti-related peptide (AgRP). Secreted NPY binds to downstream neuronal receptors and stimulates appetite. AgRP blocks the ability of α-MSH to activate melanocortin 4 receptors [11]. Furthermore, AgRP neurons inhibit POMC neurons [9].Open in a separate windowFigure 1Schematic diagram illustrating the complementary roles of POMC and NPY/AgRP neurons in appetite control. AgRP, Agouti-related peptide; MC4R, melanocortin 4 receptor; α-MSH, α-melanocyte stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin.The first study from Singh''s group started by showing that starvation induces autophagy in the hypothalamus [5]. This finding alone merits some comment. Autophagy is frequently assessed by using phosphatidylethanolamine-conjugated Atg8/LC3 (LC3-II), which is specifically associated with autophagosomes and autolysosomes. LC3-II levels on western blot and the number of LC3-positive vesicles strongly correlate with the number of autophagosomes [1]. To assess whether LC3-II formation is altered by a perturbation, its level can be assessed in the presence of lysosomal inhibitors, which inhibit LC3-II degradation by blocking autophagosome–lysosome fusion [12]. Therefore, differences in LC3-II levels in response to a particular perturbation in the presence of lysosomal inhibitors reflect changes in autophagosome synthesis. An earlier study using GFP-LC3 suggested that autophagy was not upregulated in the brains of starved mice, compared with other tissues where this did occur [13]. However, this study only measured steady state levels of autophagosomes and was performed before the need for lysosomal inhibitors was appreciated. Subsequent work has shown rapid flux of autophagosomes to lysosomes in primary neurons, which might confound analyses without lysosomal inhibitors [14]. Thus, the data of the Singh group—showing that autophagy is upregulated in the brain by a range of methods including lysosomal inhibitors [5]—address an important issue in the field and corroborate another recent study that examined this question by using sophisticated imaging methods [15].“…decreasing autophagy with ageing in POMC neurons could contribute to the metabolic problems associated with age”Singh and colleagues then analysed mice that have a specific knockout of the autophagy gene Atg7 in AgRP neurons [5]. Although fasting increases AgRP mRNA and protein levels in normal mice, these changes were not seen in the knockout mice. AgRP neurons provide inhibitory signals to POMC neurons, and Kaushik and colleagues found that the AgRP-specific Atg7 knockout mice had higher levels of POMC and α-MSH, compared with the normal mice. This indicated that starvation regulates appetite in a manner that is partly dependent on autophagy. The authors suggested that the peripheral free fatty acids released during starvation induce autophagy by activating AMP-activated protein kinase (AMPK), a known positive regulator of autophagy. This, in turn, enhances degradation of hypothalamic lipids and increases endogenous intracellular free fatty acid concentrations. The increased intracellular free fatty acids upregulate AgRP mRNA and protein expression. As AgRP normally inhibits POMC/α-MSH production in target neurons, a defect in AgRP responses in the autophagy-null AgRP neurons results in higher α-MSH levels, which could account for the decreased mouse bodyweight.In follow-up work, Singh''s group have now studied the effects of inhibiting autophagy in POMC neurons, again using Atg7 deletion [6]. These mice, in contrast to the AgRP autophagy knockouts, are obese. This might be accounted for, in part, by an increase in POMC preprotein levels and its cleavage product adrenocorticotropic hormone in the knockout POMC neurons, which is associated with a failure to generate α-MSH. Interestingly, these POMC autophagy knockout mice have impaired peripheral lipolysis in response to starvation, which the authors suggest might be due to reduced central sympathetic tone to the periphery from the POMC neurons. In addition, POMC-neuron-specific Atg7 knockout mice have impaired glucose tolerance.This new study raises several interesting issues. How does the autophagy defect in the POMC neurons alter the cleavage pattern of POMC? Is this modulated within the physiological range of autophagy activity fluctuations in response to diet and starvation? Importantly, in vivo, autophagy might fluctuate similarly (or possibly differently) in POMC and AgRP neurons in response to diet and/or starvation. Given the tight interrelation of these neurons, how does this affect their overall response to appetite regulation in wild-type animals?Finally, the study also shows that hypothalamic autophagosome formation is decreased in older mice. To my knowledge, this is the first such demonstration of this phenomenon in the brain. The older mice phenocopied aspects of the POMC-neuron autophagy null mice—increased hypothalamic POMC preprotein and ACTH and decreased α-MSH, along with similar adiposity and lipolytic defects, compared with young mice. These data are provocative from several perspectives. In the context of metabolism, it is tantalizing to consider that decreasing autophagy with ageing in POMC neurons could contribute to the metabolic problems associated with ageing. Again, this model considers the POMC neurons in isolation, and it would be important to understand how reduced autophagy in aged AgRP neurons counterbalances this situation. In a more general sense, the data strongly support the concept that neuronal autophagy might decline with age.Autophagy is a major clearance route for many mutant, aggregate-prone intracytoplasmic proteins that cause neurodegenerative disease, such as tau (Alzheimer disease), α-synuclein (Parkinson disease), and huntingtin (Huntington disease), and the risk of these diseases is age-dependent [1]. Thus, it is tempting to suggest that the dramatic age-related risks for these diseases could be largely due to decreased neuronal capacity of degrading these toxic proteins. Neurodegenerative pathology and age-related metabolic abonormalities might be related—some of the metabolic disturbances that occur in humans with age could be due to the accumulation of such toxic proteins. High levels of these proteins are seen in many people who do not have, or who have not yet developed, neurodegenerative diseases, as many of them start to accumulate decades before any sign of disease. These proteins might alter metabolism and appetite either directly by affecting target neurons, or by influencing hormonal and neurotransmitter inputs into such neurons.     

Rapamycin Ameliorates Age-Dependent Obesity Associated with Increased mTOR Signaling in Hypothalamic POMC Neurons

The prevalence of obesity in older people is the leading cause of metabolic syndromes. Central neurons serving as homeostatic sensors for body-weight control include hypothalamic neurons that express pro-opiomelanocortin (POMC) or neuropeptide-Y (NPY) and agouti-related protein (AgRP). Here, we report an age-dependent increase of mammalian target of rapamycin (mTOR) signaling in POMC neurons that elevates the ATP-sensitive potassium (K(ATP)) channel activity cell-autonomously to silence POMC neurons. Systemic or intracerebral administration of the mTOR inhibitor rapamycin causes weight loss in old mice. Intracerebral rapamycin infusion into old mice enhances the excitability and neurite projection of POMC neurons, thereby causing?a reduction of food intake and body weight. Conversely, young mice lacking the mTOR-negative regulator TSC1 in POMC neurons, but not those lacking TSC1 in NPY/AgRP neurons, were obese. Our study reveals that an increase in mTOR signaling in hypothalamic POMC neurons contributes to age-dependent obesity. VIDEO ABSTRACT:     

Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance

Macroautophagy is a lysosomal degradative pathway that maintains cellular homeostasis by turning over cellular components. Here we demonstrate a role for autophagy in hypothalamic agouti-related peptide (AgRP) neurons in the regulation of food intake and energy balance. We show that starvation-induced hypothalamic autophagy mobilizes neuron-intrinsic lipids to generate endogenous free fatty acids, which in turn regulate AgRP levels. The functional consequences of inhibiting autophagy are the failure to upregulate AgRP in response to starvation, and constitutive increases in hypothalamic levels of pro-opiomelanocortin and its cleavage product α-melanocyte-stimulating hormone that typically contribute to a lean phenotype. We propose a conceptual framework for considering how autophagy-regulated lipid metabolism within hypothalamic neurons may modulate neuropeptide levels to have immediate effects on food intake, as well as long-term effects on energy homeostasis. Regulation of hypothalamic autophagy could become an effective intervention in conditions such as obesity and the metabolic syndrome.     

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.     

Autophagy–physiology and pathophysiology

“Autophagy” is a highly conserved pathway for degradation, by which wasted intracellular macromolecules are delivered to lysosomes, where they are degraded into biologically active monomers such as amino acids that are subsequently re-used to maintain cellular metabolic turnover and homeostasis. Recent genetic studies have shown that mice lacking an autophagy-related gene (Atg5 or Atg7) cannot survive longer than 12 h after birth because of nutrient shortage. Moreover, tissue-specific impairment of autophagy in central nervous system tissue causes massive loss of neurons, resulting in neurodegeneration, while impaired autophagy in liver tissue causes accumulation of wasted organelles, leading to hepatomegaly. Although autophagy generally prevents cell death, our recent study using conditional Atg7-deficient mice in CNS tissue has demonstrated the presence of autophagic neuron death in the hippocampus after neonatal hypoxic/ischemic brain injury. Thus, recent genetic studies have shown that autophagy is involved in various cellular functions. In this review, we introduce physiological and pathophysiological roles of autophagy.     

Neuronal autophagy: Going the distance to the axon

Autophagy, a regulated cellular degradation process responsible for the turnover of long-lived proteins and organelles, has been increasingly implicated in neurological disorders. Although autophagy is mostly viewed as a stress-induced process, recent studies have indicated that it is constitutively active in central nervous system (CNS) neurons and is protective against neurodegeneration. Neurons are highly specialized, post-mitotic cells that are typically composed of a soma (cell body), a dendritic tree and an axon. The detailed process of autophagy in such a highly differentiated cell type remains to be characterized. To elucidate the physiological role of neuronal autophagy, we generated mutant mice containing a neural cell type-specific deletion of Atg7, an essential gene for autophagy. Establishment of these mutant mice allowed us to examine cell-autonomous events in cerebellar Purkinje cells deficient in autophagy. Our data reveal the indispensability of autophagy in the maintenance of axonal homeostasis and the prevention of axonal dystrophy and degeneration. Furthermore, our study implicates dysfunction of axonal autophagy as a potential mechanism underlying axonopathy, which is linked to neurodegeneration associated with numerous human neurological disorders. Finally, our study has raised a possibility that “constitutive autophagy” in neurons involves processes that are not typical of autophagy in other cell types, but rather is highly adapted to local physiological function in the axon, which is projected in a distance from one neuron to another for transducing neural signals.

Addendum to: Komatsu M, Wang QJ, Holstein GR, Friedrich Jr. VL, Iwata J, Kominami E, Chait BT, Tanaka K, Yue Z. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci USA 2007; 104:14489-94.     

Intracellular Quality Control by Autophagy: How Does Autophagy Prevent Neurodegeneration?

Autophagy is an intracellular bulk degradation process, through which a portion of cytoplasm is delivered to lysosomes to be degraded. In many organisms, the primary role of autophagy is adaptation to starvation. However, we have found that autophagy is also important for intracellular protein quality control. Atg5^-/- mice die shortly after birth due, at least in part, to nutrient deficiency. These mice also exhibit an intracellular accumulation of protein aggregates in neurons and hepatocytes. We now report the generation of neural cell-specific Atg5-deficient mice. Atg5^flox/flox;Nestin-Cre mice show progressive deficits in motor function and degeneration of some neural cells. In autophagy-deficient cells, diffuse accumulation of abnormal proteins occurs, followed by the generation of aggregates and inclusions. This study emphasizes the point that basal autophagy is important even in individuals who do not express neurodegenerative disease-associated mutant proteins. Furthermore, the primary targets of autophagy are diffuse cytosolic proteins, not protein aggregates themselves.     

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.     

Atg5- and Atg7-dependent autophagy in dopaminergic neurons regulates cellular and behavioral responses to morphine

The molecular basis of chronic morphine exposure remains unknown. In this study, we hypothesized that macroautophagy/autophagy of dopaminergic neurons would mediate the alterations of neuronal dendritic morphology and behavioral responses induced by morphine. Chronic morphine exposure caused Atg5 (autophagy-related 5)- and Atg7 (autophagy-related 7)-dependent and dopaminergic neuron-specific autophagy resulting in decreased neuron dendritic spines and the onset of addictive behaviors. In cultured primary midbrain neurons, morphine treatment significantly reduced total dendritic length and complexity, and this effect could be reversed by knockdown of Atg5 or Atg7. Mice deficient for Atg5 or Atg7 specifically in the dopaminergic neurons were less sensitive to developing a morphine reward response, behavioral sensitization, analgesic tolerance and physical dependence compared to wild-type mice. Taken together, our findings suggested that the Atg5- and Atg7-dependent autophagy of dopaminergic neurons contributed to cellular and behavioral responses to morphine and may have implications for the future treatment of drug addiction.     

Intracellular quality control by autophagy: how does autophagy prevent neurodegeneration?

Autophagy is an intracellular bulk degradation process, through which a portion of cytoplasm is delivered to lysosomes to be degraded. In many organisms, the primary role of autophagy is adaptation to starvation. However, we have found that autophagy is also important for intracellular protein quality control. Atg5(-/-) mice die shortly after birth due, at least in part, to nutrient deficiency. These mice also exhibit an intracellular accumulation of protein aggregates in neurons and hepatocytes. We now report the generation of neural cell-specific Atg5-deficient mice. Atg5( flox/flox);Nestin-Cre mice show progressive deficits in motor function and degeneration of some neural cells. In autophagy-deficient cells, diffuse accumulation of abnormal proteins occurs, followed by the generation of aggregates and inclusions. This study emphasizes the point that basal autophagy is important even in individuals who do not express neurodegenerative disease-associated mutant proteins. Furthermore, the primary targets of autophagy are diffuse cytosolic proteins, not protein aggregates themselves.     

Gamma-protocadherins regulate the functional integrity of hypothalamic feeding circuitry in mice

The hypothalamic neuronal circuits that modulate energy homeostasis become mature and functional during early postnatal life. However, the molecular mechanism underlying this developmental process remains largely unknown. Here we use a mouse genetic approach to investigate the role of gamma-protocadherins (Pcdh-γs) in hypothalamic neuronal circuits. First, we show that rat insulin promoter (RIP)-Cre conditional knockout mice lacking Pcdh-γs in a broad subset of hypothalamic neurons are obese and hyperphagic. Second, specific deletion of Pcdh-γs in anorexigenic proopiomelanocortin (POMC) expressing neurons also leads to obesity. Using cell lineage tracing, we show that POMC and RIP-Cre expressing neurons do not overlap but interact with each other in the hypothalamus. Moreover, excitatory synaptic inputs are reduced in Pcdh-γ deficient POMC neurons. Genetic evidence from both knockout models shows that Pcdh-γs can regulate POMC neuronal function autonomously and non-autonomously through cell-cell interaction. Taken together, our data demonstrate that Pcdh-γs regulate the formation and functional integrity of hypothalamic feeding circuitry in mice.     

Atg9a deficiency causes axon-specific lesions including neuronal circuit dysgenesis

Conditional knockout mice for Atg9a, specifically in brain tissue, were generated to understand the roles of ATG9A in the neural tissue cells. The mice were born normally, but half of them died within one wk, and none lived beyond 4 wk of age. SQSTM1/p62 and NBR1, receptor proteins for selective autophagy, together with ubiquitin, accumulated in Atg9a-deficient neurosoma at postnatal d 15 (P15), indicating an inhibition of autophagy, whereas these proteins were significantly decreased at P28, as evidenced by immunohistochemistry, electron microscopy and western blot. Conversely, degenerative changes such as spongiosis of nerve fiber tracts proceeded in axons and their terminals that were occupied with aberrant membrane structures and amorphous materials at P28, although no clear-cut degenerative change was detected in neuronal cell bodies. Different from autophagy, diffusion tensor magnetic resonance imaging and histological observations revealed Atg9a-deficiency-induced dysgenesis of the corpus callosum and anterior commissure. As for the neurite extensions of primary cultured neurons, the neurite outgrowth after 3 d culturing was significantly impaired in primary neurons from atg9a-KO mouse brains, but not in those from atg7-KO and atg16l1-KO brains. Moreover, this tendency was also confirmed in Atg9a-knockdown neurons under an atg7-KO background, indicating the role of ATG9A in the regulation of neurite outgrowth that is independent of autophagy. These results suggest that Atg9a deficiency causes progressive degeneration in the axons and their terminals, but not in neuronal cell bodies, where the degradations of SQSTM1/p62 and NBR1 were insufficiently suppressed. Moreover, the deletion of Atg9a impaired nerve fiber tract formation.     

Neuronal autophagy: going the distance to the axon

Autophagy, a regulated cellular degradation process responsible for the turnover of long-lived proteins and organelles, has been increasingly implicated in neurological disorders. Although autophagy is mostly viewed as a stress-induced process, recent studies have indicated that it is constitutively active in central nervous system (CNS) neurons and is protective against neurodegeneration. Neurons are highly specialized, post-mitotic cells that are typically composed of a soma (cell body), a dendritic tree and an axon. The detailed process of autophagy in such a highly differentiated cell type remains to be characterized. To elucidate the physiological role of neuronal autophagy, we generated mutant mice containing a neural cell type-specific deletion of Atg7, an essential gene for autophagy. Establishment of these mutant mice allowed us to examine cell-autonomous events in cerebellar Purkinje cells deficient in autophagy. Our data reveal the indispensability of autophagy in the maintenance of axonal homeostasis and the prevention of axonal dystrophy and degeneration. Furthermore, our study implicates dysfunction of axonal autophagy as a potential mechanism underlying axonopathy, which is linked to neurodegeneration associated with numerous human neurological disorders. Finally, our study has raised a possibility that "constitutive autophagy" in neurons involves processes that are not typical of autophagy in other cell types, but rather is highly adapted to local physiological function in the axon, which is projected in a distance from one neuron to another for transducing neural signals.