Atg5 and Ambra1 differentially modulate neurogenesis in neural stem cells

Neuroepithelial cells undergoing differentiation efficiently remodel their cytoskeleton and shape in an energy-consuming process. The capacity of autophagy to recycle cellular components and provide energy could fulfill these requirements, thus supporting differentiation. However, little is known regarding the role of basal autophagy in neural differentiation. Here we report an increase in the expression of the autophagy genes Atg7, Becn1, Ambra1 and LC3 in vivo in the mouse embryonic olfactory bulb (OB) during the initial period of neuronal differentiation at E15.5, along with a parallel increase in neuronal markers. In addition, we observed an increase in LC3 lipidation and autophagic flux during neuronal differentiation in cultured OB-derived stem/progenitor cells. Pharmacological inhibition of autophagy with 3-MA or wortmannin markedly decreased neurogenesis. These observations were supported by similar findings in two autophagy-deficient genetic models. In Ambra1 loss-of-function homozygous mice (gt/gt) the expression of several neural markers was decreased in the OB at E13.5 in vivo. In vitro, Ambra1 haploinsufficient cells developed as small neurospheres with an impaired capacity for neuronal generation. The addition of methylpyruvate during stem/progenitor cell differentiation in culture largely reversed the inhibition of neurogenesis induced by either 3-MA or Ambra1 haploinsufficiency, suggesting that neural stem/progenitor cells activate autophagy to fulfill their high energy demands. Further supporting the role of autophagy for neuronal differentiation Atg5-null OB cells differentiating in culture displayed decreased TuJ1 levels and lower number of cells with neurites. These results reveal new roles for autophagy-related molecules Atg5 and Ambra1 during early neuronal differentiation of stem/progenitor cells.     

Knockout of Atg5 delays the maturation and reduces the survival of adult-generated neurons in the hippocampus

Autophagy is an evolutionarily conserved lysosomal degradation pathway that plays important roles in cell maintenance, expansion and differentiation. Removal of genes essential for autophagy from embryonic neural stem and precursor cells reduces the survival and inhibits neuronal differentiation of adult-generated neurons. No study has modified autophagy within the adult precursor cells, leaving the cell-autonomous role of autophagy in adult neurogenesis unknown. Here we demonstrate that autophagic flux exists in the adult dividing progenitor cells and their progeny in the dentate gyrus. To investigate the role of autophagy in adult hippocampal neurogenesis, we genetically deleted Autophagy-related gene 5 (Atg5) that reduced autophagic flux and the survival of the progeny of dividing progenitor cells. This significant reduction in survival of adult-generated neurons is accompanied by a delay in neuronal maturation, including a transient reduction in spine density in the absence of a change in differentiation. The delay in cell maturation and loss of progeny of the Atg5-null cells was not present in mice that lacked the essential pro-apoptotic protein Bax (Bcl-2-associated X protein), suggesting that Atg5-deficient cells die through a Bax-dependent mechanism. In addition, there was a loss of Atg5-null cells following exposure to running, suggesting that Atg5 is required for running-induced increases in neurogenesis. These findings highlight the cell-autonomous requirement of Atg5 in the survival of adult-generated neurons.In the adult brain, neurogenesis allows for the continuous development of adult-generated neurons in response to physiological and pathological stimuli. The neural progenitor cells (NPCs) within the neurogenic niche of the subventricular zone (SVZ) and subgranular zone (SGZ) give rise to adult-generated neurons within the olfactory bulb and dentate, respectively.^{1, 2, 3} The ability of the NPCs to proliferate, differentiate and integrate into circuitry to modify behavior makes understanding these cells and the factors that regulate these processes critical to develop new therapies. This is especially important for a number of diseases such as neurodegenerative diseases including Parkinson''s and Huntington''s diseases that are associated with reduced adult neurogenesis, as well as regenerative medicine strategies for recovery after stroke.^{4, 5, 6}Two groups have found that in vivo macroautophagy (hereafter referred to as autophagy) can regulate adult neurogenesis by examining the effect of deleting autophagy-related genes (Atgs). Yazdankhah et al.⁷ found that Ambra1 and Beclin1 heterozygous embryonic knockout mice have less proliferating NPCs in the SVZ and an associated reduction in neurogenesis in the olfactory bulb. Wang et al.⁸ found that conditional removal of FIP200 (focal adhesion kinase (FAK) family interacting protein of M_r 200 K, also known as ULK1, an Atg1 homologue-interacting protein) from embryonic NPCs progressively depletes the number of postnatal NPCs, as well as reduces neurogenesis and increases astrogenesis. In contrast in the embryo, Lv et al.⁹ showed that a specific knockdown of the Autophagy-related gene 5 (Atg5) increases proliferation and inhibits neuronal differentiation of embryonic NPCs during cortical development. These data suggest that embryonic and adult NPCs are altered when autophagy-related genes are deleted in the embryo. However, it remains unknown whether autophagy, independent of effects in the embryo, is directly required for NPCs and their progeny in the adult.Here we tested the functional role of autophagy specifically in the adult brain by removing Atg5 from dividing NPCs. We found that autophagic flux occurs in adult NPCs and that removal of Atg5 is associated with a reduction in autophagic flux. In addition, we find that Atg5-null cells have a significant reduction in survival, as well as a delay in neuronal maturation. The reduction in neurogenesis occurred in the absence of altering proliferation or cell lineage. Furthermore, removal of Bax (Bcl-2-associated X protein) restored neurogenesis in the absence of Atg5, implicating Bax functions downstream of Atg5 to regulate the survival of adult-generated neurons. Finally, we showed that Atg5-dependent signaling is required for running-induced increases in the survival of the adult developing NPCs.     

Amyloid β Peptides Promote Autophagy-Dependent Differentiation of Mouse Neural Stem Cells

Although regarded as neurotoxic, amyloid β (Aβ) peptides may also mediate a wide range of nonpathogenic processes. Autophagy has been implicated in Aβ-mediated effects, although its precise function in neural differentiation remains unknown. Here, we addressed the role of different Aβ fragments in neural stem cell (NSC) proliferation and differentiation, and investigated whether autophagy is involved in Aβ-induced alterations of neural fate. Our results demonstrate that neuronal and glial-specific protein markers are significantly induced by both Aβ_1–40 and Aβ_1–42. However, Aβ_1–40 preferentially enhances neurogenesis of NSCs, as determined by βIII-tubulin, NeuN, and MAP2 neuronal marker immunoreactivity, while Aβ_1–42 appears to favor gliogenesis. In contrast, Aβ_25–35 does not influence NSC fate. The effect of Aβ_1–40 on neurogenesis is partially dependent on its role in NSC self-renewal as both S-phase of the cell cycle and BrdU labeling were markedly increased. Nevertheless, Aβ_1–40 resulted also in increased Tuj1 promoter activity. Autophagy, assessed by conversion of endogenous LC3-I/II, fluorescence of pGFP-LC3-transfected cells, and Atg9 protein levels, was evident in both Aβ_1–40- and Aβ_1–42-treated NSCs, independently of reactive oxygen species production and apoptosis. Finally, inhibition of autophagy by pharmacologic means abrogated Aβ-induced lineage-specific protein markers. These results support distinct roles for different Aβ peptides in NSC fate decision and underline the importance of autophagy control of this process.     

GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells

Hippocampal activity influences neurogenesis in the adult dentate gyrus; however, little is known about the involvement of the hippocampal circuitry in this process. In the subgranular zone of the adult dentate gyrus, neurogenesis involves a series of differentiation steps from radial glia-like stem/progenitor (type-1) cells, to transiently amplifying neuronal progenitor (type-2) cells, to postmitotic neurons. In this study, we conducted GFP-targeted recordings of progenitor cells in fresh hippocampal slices from nestin-GFP mice and found that neuronal progenitor (type-2) cells receive active direct neural inputs from the hippocampal circuitry. This input was GABAergic but not glutamatergic. The GABAergic inputs depolarized type-2 cells because of their elevated [Cl(-)](i). This excitation initiated an increase of [Ca(2+)](i) and the expression of NeuroD. A BrdU-pulse labeling study with GABA(A)-R agonists demonstrated the promotion of neuronal differentiation via this GABAergic excitation. Thus, it appears that GABAergic inputs to hippocampal progenitor cells promote activity-dependent neuronal differentiation.     

Ambra1 modulates starvation-induced autophagy through AMPK signaling pathway in cardiomyocytes

Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells and cancer cells. However, whether Ambra1 plays an important role in the autophagy pathway in cardiomyocytes is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in cardiomyocytes. To test this hypothesis, we confirmed autophagic activity in serum-starved cardiomyocytes by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes and LC3 protein levels. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in cardiomyocytes. When Ambra1 expression was reduced by siRNA, the cardiomyocytes were more sensitive to staurosporine-induced apoptosis. In addition, co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated cardiomyocytes, suggesting that Ambra1 regulates autophagy in cardiomyocytes by interacting with Beclin1. Finally, we determined that starvation stress-induced activation of Ambra1 contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signaling. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis through AMPK signaling pathway in cardiomyocytes that maintains the balance between autophagy and apoptosis.     

Clock genes regulate neurogenic transcription factors,including NeuroD1, and the neuronal differentiation of adult neural stem/progenitor cells

The circadian clock system plays multiple roles in our bodies, and clock genes are expressed in various brain regions, including the lateral subventricular zone (SVZ) where neural stem/progenitor cells (NSPCs) persist and postnatal neurogenesis continues. However, the functions of clock genes in adult NSPCs are not well understood. Here, we first investigated the expression patterns of Clock and Bmal1 in the SVZ by immunohistochemistry and then verified how the expression levels of 17 clock and clock-related genes changed during differentiation of cultured adult NSPCs using quantitative RT-PCR. Finally, we used RNAi to observe the effects of Clock and Bmal1 on neuronal differentiation. Our results revealed that Clock and Bmal1 were expressed in the SVZ and double-stained with the neural progenitor marker Nestin and neural stem marker GFAP. In cultured adult NSPCs, the clock genes changed their expression patterns during differentiation, and interestingly, Bmal1 started endogenous oscillation. Moreover, gene silencing of Clock or Bmal1 by RNAi decreased the percentages of neuronal marker Map2-positive cells and expression levels of NeuroD1 mRNA. These findings suggest that clock genes are involved in the neuronal differentiation of adult NSPCs and may extend our understanding of various neurological/psychological disorders linked to adult neurogenesis and circadian rhythm.     

The role of autophagy during coxsackievirus infection of neural progenitor and stem cells

Coxsackievirus B3 (CVB3) has previously been shown to utilize autophagy in an advantageous manner during the course of infection of the host cell. However, few studies have determined whether stem cells induce autophagy in a similar fashion, and whether virus-induced autophagy occurs following infection of stem cells. Therefore, we compared the induction of autophagy following CVB3 infection of neural progenitor and stem cells (NPSCs), which we have recently shown to be highly susceptible to CVB3 infection, to HL-1 cells, a transformed cardiomyocyte cell line. As previously demonstrated for other susceptible host cells, HL-1 cells showed an increase in the activity of autophagic signaling following infection with a CVB3 expressing dsRed protein (dsRed-CVB3). Furthermore, viral titers in HL-1 cells increased in the presence of an inducer of autophagy (CCPA), while viral titers decreased in the presence of an inhibitor of autophagy (3-MA). In contrast, no change in autophagic signaling was seen in NPSCs following infection with dsRed-CVB3. Also, basal levels of autophagy in NPSCs were found to be highly elevated in comparison to HL-1 cells. Autophagy could be induced in NPSCs in the presence of rapamycin without altering levels of dsRed-CVB3 replication. In differentiated NPSC precursors, autophagy was activated during the differentiation process, and a decrease in autophagic signaling was observed within all three CNS lineages following dsRed-CVB3 infection. Hence, we conclude that the role of autophagy in modulating CVB3 replication appears cell type-specific, and stem cells may uniquely regulate autophagy in response to infection.     

The role of autophagy during coxsackievirus infection of neural progenitor and stem cells

Coxsackievirus B3 (CVB3) has previously been shown to utilize autophagy in an advantageous manner during the course of infection of the host cell. However, few studies have determined whether stem cells induce autophagy in a similar fashion, and whether virus-induced autophagy occurs following infection of stem cells. Therefore, we compared the induction of autophagy following CVB3 infection of neural progenitor and stem cells (NPSCs), which we have recently shown to be highly susceptible to CVB3 infection, to HL-1 cells, a transformed cardiomyocyte cell line. As previously demonstrated for other susceptible host cells, HL-1 cells showed an increase in the activity of autophagic signaling following infection with a CVB3 expressing dsRed protein (dsRed-CVB3). Furthermore, viral titers in HL-1 cells increased in the presence of an inducer of autophagy (CCPA), while viral titers decreased in the presence of an inhibitor of autophagy (3-MA). In contrast, no change in autophagic signaling was seen in NPSCs following infection with dsRed-CVB3. Also, basal levels of autophagy in NPSCs were found to be highly elevated in comparison to HL-1 cells. Autophagy could be induced in NPSCs in the presence of rapamycin without altering levels of dsRed-CVB3 replication. In differentiated NPSC precursors, autophagy was activated during the differentiation process, and a decrease in autophagic signaling was observed within all three CNS lineages following dsRed-CVB3 infection. Hence, we conclude that the role of autophagy in modulating CVB3 replication appears cell type-specific, and stem cells may uniquely regulate autophagy in response to infection.     

Autophagy: Insights from DEspR-deficiency and haploinsufficiency

We recently showed that DEspR-haploinsufficiency resulted in increased neuronal autophagy and spongiform changes in the adult brain especially the hippocampus, cerebral cortex and basal ganglia, causing cognitive performance deficits. This model demonstrates a causal link between increased autophagy and neurodegenerative changes. This is in contrast with recent observations that decreased autophagy from null mutations of autophagy genes, Atg5 and Atg7, resulted in early neurodegenerative changes. With the observed autophagy phenotype, we then compared the neural tube phenotype of DEspR-deficient mice with knockout mice of genes established to underlie or regulate autophagy. Intriguingly, the hyperproliferative neuroepithelium observed in DEspR-deficient embryos is also detected in null mutants of Ambra1, an autophagy modulator, and two apoptosis genes, Apaf1 and Caspase 9. While all four knockout models exhibited hyperproliferative neuroepithelium, DEspR-deficient mice differed by having greater neural tube cavitation. Additionally, observed DEspR roles in angiogenesis and autophagy recapitulate the association of angiogenesis inhibition and increased autophagy as observed for endostatin and kringle5, thus elucidating an expanding complex network of autophagy, apoptosis and angiogenesis in neuroepithelial development, and an emerging complex spectrum of autophagy effects on neurodegeneration. Nevertheless, DEspR provides a ligand-activated receptor system to modulate autophagy – be it to increase autophagy by inhibition of DEspR-function, or to decrease autophagy by agonist stimulation of DEspR-function.     

Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality

The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.     

Beclin-1-mediated autophagy protects spinal cord neurons against mechanical injury-induced apoptosis

Apoptosis has been widely reported to be involved in the pathogenesis associated with spinal cord injury (SCI). Recently, autophagy has also been implicated in various neuronal damage models. However, the role of autophagy in SCI is still controversial and its interrelationship with apoptosis remains unclear. Here, we used an in vitro SCI model to observe a time-dependent induction of autophagy and apoptosis. Mechanical injury induced autophagy markers such as LC3 lipidation, LC3II/LC3I conversion, and Beclin-1expression. Injured neurons showed decreased cell viability and increased apoptosis. To elucidate the effect of autophagy on apoptosis, the mechanically-injured neurons were treated with the mTOR inhibitor rapamycin and 3-methyl adenine (3-MA), which are known to regulate autophagy positively and negatively, respectively. Rapamycin-treated neurons showed the highest level of cell viability and lowest level of apoptosis among the injured neurons and those treated with 3-MA showed the reciprocal effect. Notably, rapamycin-treated neurons exhibited slightly reduced Bax expression and significantly increasedBcl-2 expression. Furthermore, by plasmid transfection, we showed that Beclin-1-overexpressing neuronal cells responded to mechanical injury with greater LC3II/LC3I conversion and cell viability, lower levels of apoptosis, higher Bcl-2 expression, and unaltered Bax expression as compared to vector control cells. Beclin-1-knockdown neurons showed almost the opposite effects. Taken together, our results suggest that autophagy may serve as a protection against apoptosis in mechanically-injured spinal cord neurons. Targeting mTOR and/or enhancing Beclin-1 expression might be alternative therapeutic strategies for SCI.     

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.     

Coordinated activation of AMP‐activated protein kinase,extracellular signal‐regulated kinase,and autophagy regulates phorbol myristate acetate‐induced differentiation of SH‐SY5Y neuroblastoma cells

We explored the interplay between the intracellular energy sensor AMP‐activated protein kinase (AMPK), extracellular signal‐regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)‐induced neuronal differentiation of SH‐SY5Y human neuroblastoma cells. PMA‐triggered expression of neuronal markers (dopamine transporter, microtubule‐associated protein 2, β‐tubulin) was associated with an autophagic response, measured by the conversion of microtubule‐associated protein light chain 3 (LC3)‐I to autophagosome‐bound LC3‐II, increase in autophagic flux, and expression of autophagy‐related (Atg) proteins Atg7 and beclin‐1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference‐mediated silencing of AMPK suppressed PMA‐induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA‐induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA‐induced differentiation of SH‐SY5Y cells. Therefore, PMA‐induced neuronal differentiation of SH‐SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response.

     

Targeted Deletion of the ERK5 MAP Kinase Impairs Neuronal Differentiation,Migration, and Survival during Adult Neurogenesis in the Olfactory Bulb

Recent studies have led to the exciting idea that adult-born neurons in the olfactory bulb (OB) may be critical for complex forms of olfactory behavior in mice. However, signaling mechanisms regulating adult OB neurogenesis are not well defined. We recently reported that extracellular signal-regulated kinase (ERK) 5, a MAP kinase, is specifically expressed in neurogenic regions within the adult brain. This pattern of expression suggests a role for ERK5 in the regulation of adult OB neurogenesis. Indeed, we previously reported that conditional deletion of erk5 in adult neurogenic regions impairs several forms of olfactory behavior in mice. Thus, it is important to understand how ERK5 regulates adult neurogenesis in the OB. Here we present evidence that shRNA suppression of ERK5 in adult neural stem/progenitor cells isolated from the subventricular zone (SVZ) reduces neurogenesis in culture. By contrast, ectopic activation of endogenous ERK5 signaling via expression of constitutive active MEK5, an upstream activating kinase for ERK5, stimulates neurogenesis. Furthermore, inducible and conditional deletion of erk5 specifically in the neurogenic regions of the adult mouse brain interferes with cell cycle exit of neuroblasts, impairs chain migration along the rostral migratory stream and radial migration into the OB. It also inhibits neuronal differentiation and survival. These data suggest that ERK5 regulates multiple aspects of adult OB neurogenesis and provide new insights concerning signaling mechanisms governing adult neurogenesis in the SVZ-OB axis.     

Extracellular Signal-regulated Kinase 5 (ERK5) Mediates Prolactin-stimulated Adult Neurogenesis in the Subventricular Zone and Olfactory Bulb

Prolactin-stimulated adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) mediates several reproductive behaviors including mating/pregnancy, dominant male pheromone preference in females, and paternal recognition of offspring. However, downstream signaling mechanisms underlying prolactin-induced adult neurogenesis are completely unknown. We report here for the first time that prolactin activates extracellular signal-regulated kinase 5 (ERK5), a MAP kinase that is specifically expressed in the neurogenic regions of the adult mouse brain. Knockdown of ERK5 by retroviral infection of shRNA attenuates prolactin-stimulated neurogenesis in SVZ-derived adult neural stem/progenitor cells (aNPCs). Inducible erk5 deletion in adult neural stem cells of transgenic mice inhibits neurogenesis in the SVZ and OB following prolactin infusion or mating/pregnancy. These results identify ERK5 as a novel and critical signaling mechanism underlying prolactin-induced adult neurogenesis.     

Adiponectin inhibits high glucose-induced angiogenesis via inhibiting autophagy in RF/6A cells

Adiponectin, one of the adipose-derived hormone with metabolic activity, has been reported to conversely affect angiogenesis of endothelial cells in vitro. The previous study in animal models has demonstrated that adiponectin has a protective role in retinal vascular injury following pathological stimuli. However, clinical research regarding the relationship between plasma adiponectin level and diabetic retinopathy (DR) are inconclusive. The aim of this study was to investigate the effect of adiponectin on high glucose-induced retinal angiogenesis and its association with autophagy by using rhesus choroid-retinal endothelial (RF-6A) cells as a model. We found that cell vitality decreased and cell migration and tube formation increased in the high-glucose group. Treatment with adiponectin or 3-methyladenine (3-MA, an autophagy inhibitor) increased cell viability and inhibited cell migration and tube formation. In the high-glucose group, the protein expression of Bax and apoptosis rate of cells increased and the expression of Bcl-2 decreased, whereas treatment with adiponectin or 3-MA reversed these results. Autophagy was activated in the high-glucose group to present as more LC3B fluorescent dots and higher expressions of LC3B, Atg5 proteins as well as lower expression of p62. Treatment with adiponectin or 3-MA inhibited autophagy by promoting the expression of p-PI3K, p-AKT, and p-mTOR when compared with the high-glucose group. The results of this study suggested that adiponectin inhibits high glucose-induced angiogenesis of RF/6A cells by inhibiting autophagy, and promotion of the PI3K/AKT/mTOR pathway might be involved in the anti-autophagy activities of adiponectin.     

The autophagy regulators Ambra1 and Beclin 1 are required for adult neurogenesis in the brain subventricular zone

Autophagy is a conserved proteolytic mechanism required for maintaining cellular homeostasis. The role of this process in vertebrate neural development is related to metabolic needs and stress responses, even though the importance of its progression has been observed in a number of circumstances, both in embryonic and in postnatal differentiating tissues. Here we show that the proautophagic proteins Ambra1 and Beclin 1, involved in the initial steps of autophagosome formation, are highly expressed in the adult subventricular zone (SVZ), whereas their downregulation in adult neural stem cells in vitro leads to a decrease in cell proliferation, an increase in basal apoptosis and an augmented sensitivity to DNA-damage-induced death. Further, Beclin 1 heterozygosis in vivo results in a significant reduction of proliferating cells and immature neurons in the SVZ, accompanied by a marked increase in apoptotic cell death. In sum, we propose that Ambra1- and Beclin 1-mediated autophagy plays a crucial role in adult neurogenesis, by controlling the survival of neural precursor cells.In the adult mammalian brain, neural stem cells are localized in two regions: in the subventricular zone (SVZ), a layer extending along the wall of the lateral ventricle, and in the subgranular zone of the dentate gyrus in the hippocampus.¹ SVZ stem cells are strictly controlled under physiological conditions and are believed to replenish dying cells. In addition to their effect in maintaining brain homeostasis, they are also involved in neuronal replacement in response to injury.² Although several factors are known to affect neurogenesis, understanding of the mechanisms that regulate adult neurogenic niches and their metabolism is still incomplete. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved cellular turnover process in which bulk cytoplasmic materials, long-lived proteins or damaged organelles are sequestered and delivered to lysosomes for degradation.³ A complex crosstalk takes place between apoptosis and autophagy that determines the death or life of cells.⁴ Beclin 1 has a key role in autophagy initiation;⁵ it regulates the autophagy-promoting activity of the Class III PI 3-kinase Vps34,⁶ and is involved in the recruitment of membranes to form the key autophagy vesicles, named autophagosomes. Beclin 1 also interacts with Bcl-2,⁷ and plays an important function in the regulation of cell survival.⁸ Ambra1 (activating molecule in Beclin 1-regulated autophagy) is another modulator of autophagy, which is phosphorylated by the upstream autophagy kinase Ulk1 and acts on Ulk1 stability and function.^{9, 10} Ambra1 also interacts with Beclin 1 upon autophagic stimuli, thereby promoting the binding between Beclin 1 and its target kinase, Vps34. The binding between Ambra1 and mitochondrial Bcl-2 is also important for cell survival.¹¹ Moreover, Ambra1 is crucial for nervous system development and is expressed from early neurulation onwards, with a high specificity for the neural plate.¹²In contrast with studies on the pro-survival impact of autophagy in post-mitotic cells and in disease models, the role of autophagy in the maintenance and function of adult neural stem cells (ANSCs) is poorly understood. Here we have found that expression of upstream autophagy-regulating genes in the adult neurogenic region of SVZ, in physiological conditions, plays a crucial role in the regulation of adult neurogenesis.