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.     

Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.     

Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis

Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G₂/M is associated with interstitial fibrosis. However, the role of autophagy in renal fibrosis is elusive. Here, we hypothesized that autophagy activity in tubular epithelial cells is pivotal for inhibition of cell cycle G₂/M arrest and subsequent fibrogenic response. In both renal epithelial cells stimulated by angiotensin II (AGT II) and the murine kidney after unilateral ureteral obstruction (UUO), we observed that occurrence of autophagy preceded increased production of COL1 (collagen, type I). Pharmacological enhancement of autophagy by rapamycin suppressed COL1 accumulation and renal fibrosis. In contrast, genetic ablation of autophagy by proximal tubular epithelial cell-specific deletion of Atg5, with reduction of the LC3-II protein level and degradation of SQSTM1/p62, showed marked cell cycle arrest at the G₂/M phase, robust COL1 deposition, and severe interstitial fibrosis in a UUO model, as compared with wild-type mice. In vitro, AGT II exposure triggered autophagy preferentially in the G₁/S phase, and increased COL1 expression in the G₂/M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G₂/M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G₂/M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G₂/M arrest, suggesting the regulation of cell cycle progression by ATG5 is autophagy dependent. In conclusion, Atg5-mediated autophagy in proximal epithelial cells is a critical host-defense mechanism that prevents renal fibrosis by blocking G₂/M arrest.     

TNFAIP3 promotes survival of CD4 T cells by restricting MTOR and promoting autophagy

Autophagy plays important roles in metabolism, differentiation, and survival in T cells. TNFAIP3/A20 is a ubiquitin-editing enzyme that is thought to be a negative regulator of autophagy in cell lines. However, the role of TNFAIP3 in autophagy remains unclear. To determine whether TNFAIP3 regulates autophagy in CD4 T cells, we first analyzed Tnfaip3-deficient naïve CD4 T cells in vitro. We demonstrated that Tnfaip3-deficient CD4 T cells exhibited reduced MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) puncta formation, increased mitochondrial content, and exaggerated reactive oxygen species (ROS) production. These results indicate that TNFAIP3 promotes autophagy after T cell receptor (TCR) stimulation in CD4 T cells. We then investigated the mechanism by which TNFAIP3 promotes autophagy signaling. We found that TNFAIP3 bound to the MTOR (mechanistic target of rapamycin) complex and that Tnfaip3-deficient cells displayed enhanced ubiquitination of the MTOR complex and MTOR activity. To confirm the effects of enhanced MTOR activity in Tnfaip3-deficient cells, we analyzed cell survival following treatment with Torin1, an MTOR inhibitor. Tnfaip3-deficient CD4 T cells exhibited fewer cell numbers than the control cells in vitro and in vivo. In addition, the impaired survival of Tnfaip3-deficient cells was ameliorated with Torin1 treatment in vitro and in vivo. The effect of Torin1 was abolished by Atg5 deficiency. Thus, enhanced MTOR activity regulates the survival of Tnfaip3-deficient CD4 T cells. Taken together, our findings illustrate that TNFAIP3 restricts MTOR signaling and promotes autophagy, providing new insight into the manner in which MTOR and autophagy regulate survival in CD4 T cells.     

Temporal regulation of intracellular organelle homeostasis in T lymphocytes by autophagy

The highly conserved self-degradation pathway known as autophagy plays important roles in regulating T lymphocyte homeostasis. Recently, we found that T lymphocytes lacking the autophagy-related gene Atg5 or Atg7 have defective survival and contain expanded mitochondria and endoplasmic reticulum (ER); however, whether these defects are caused by impaired autophagy or by defects in their autophagy-independent signaling capacity of Atg5 or Atg7 in T lymphocytes remains unknown. Furthermore, the function of the microtubule-associated protein L chain 3 (LC3) conjugation system in T lymphocytes remains unclear. To address these questions, we generated conditional knockout mice with specific deletion of Atg3, a ubiquitin enzyme E2-like molecule involved in the LC3 conjugation system, in T lymphocytes. Atg3-deficient T lymphocytes displayed a phenotype similar to those of Atg7- and Atg5-deficient T cells. The survival of Atg3-deficient naive CD4(+) and CD8(+) T cells was defective. Furthermore, the mitochondria and ER were expanded in Atg3-deficient T cells. Interestingly, mitochondrial and ER content did not change instantly upon inducible deletion of Atg3 in mature T lymphocytes in vitro. Instead, it began to expand 10 d after inducible deletion of Atg3 in mature T lymphocytes, and mitochondrial content continued to increase on day 18. Cell death began to increase 24 d after inducible deletion of Atg3. These data show that the LC3 conjugation system is essential for autophagy in T lymphocytes. Our data suggest that autophagy promotes T lymphocyte survival by regulating organelle homeostasis and that the decreased survival of autophagy-deficient T cells is due to the temporal accumulation of these autophagy-related defects.     

Maintaining T Lymphocyte Homeostasis: Another Duty of Autophagy

First identified as a pathway for nutrient recovery during periods of starvation, the role of autophagy has expanded to the clearance of “toxic” intracellular material including ubiquitin-positive protein aggregates, damaged organelles as well as microbial pathogens in various cell types. We have examined the role of autophagy in the development and function of the adaptive immune system. Genes encoding autophagy machinery are expressed in T lymphocytes, and autophagy occurs in primary CD4⁺ and CD8⁺ T cells. By generating fetal liver chimeric mice, we found that thymocyte development is largely normal but the mature T cell compartment is severely reduced in the absence of the essential autophagy gene Atg5. Consistent with a critical role for autophagy in promoting T cell survival, Atg5^-/- CD8⁺ T cells display high levels of apoptosis. Surprisingly, Atg5-deficient T cells were also unable to efficiently proliferate after T-cell receptor (TCR) stimulation. These findings suggest that autophagy regulates T lymphocyte homeostasis by promoting both survival and proliferation. In addition, T cells offer a new, physiologically relevant system to study the regulation and function of autophagy pathways in vivo.

Addendum to:

A Critical Role for the Autophagy Gene Atg5 in T Cell Survival and Proliferation

H.H. Pua, I. Dzhagalov, M. Chuck, N. Mizushima and Y.W. He

J Exp Med 2007; 204:25-31     

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.     

Atg1 allows second-signaled autophagic cell death in Dictyostelium

We investigated the role of Atg1 in autophagic cell death (ACD) in a Dictyostelium monolayer model. The model is especially propitious, not only because of genetic tractability and absence of apoptosis machinery, but also because induction of ACD requires two successive exogenous signals, first the combination of starvation and cAMP, second the differentiation factor DIF-1. This enables one to analyze separately first-signal-induced autophagy and subsequent second-signal-induced ACD. We used mutants of atg1, a gene that plays an essential role in the initiation of autophagy. Upon starvation/cAMP, in contrast to parental cells, atg1 mutant cells showed irreversible lesions, clearly establishing a protective role for Atg1. Upon subsequent exposure to DIF-1 or to more ACD-specific second signals, starved parental cells progressed to ACD, but starved atg1 mutant cells did not, showing that Atg1 was required for ACD. Thus, in the same cells Atg1 was required in two apparently opposite ways, upon first-signaling for cell survival and upon second-signaling for ACD. Our findings strongly suggest that Atg1, thus presumably autophagy, protects the cells from starvation-induced cell death, allowing subsequent induction of ACD by the second signal. ACD is therefore not only “with” autophagy (since

it showed signs of autophagy throughout), but is also “allowed by” autophagy. This does not exclude a role for autophagy also after second signaling. These results may account for discrepancies reported in the literature, encourage searches for second signals in different developmental models of ACD, and incite caution in autophagy-related therapeutic attempts.     

Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila

Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. Here we characterized Atg17/FIP200, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and we showed that it binds to the other subunits including Atg1, Atg13, and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, we found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. We propose a model according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila.     

Structure of Atg5.Atg16, a complex essential for autophagy

Atg5 is covalently modified with a ubiquitin-like modifier, Atg12, and the Atg12-Atg5 conjugate further forms a complex with the multimeric protein Atg16. The Atg12-Atg5.Atg16 multimeric complex plays an essential role in autophagy, the bulk degradation system conserved in all eukaryotes. We have reported here the crystal structure of Atg5 complexed with the N-terminal region of Atg16 at 1.97A resolution. Atg5 comprises two ubiquitin-like domains that flank a helix-rich domain. The N-terminal region of Atg16 has a helical structure and is bound to the groove formed by these three domains. In vitro analysis showed that Arg-35 and Phe-46 of Atg16 are crucial for the interaction. Atg16, with a mutation at these residues, failed to localize to the pre-autophagosomal structure and could not restore autophagy in Atg16-deficient yeast strains. Furthermore, these Atg16 mutants could not restore a severe reduction in the formation of the Atg8-phosphatidylethanolamine conjugate, another essential factor for autophagy, in Atg16-deficient strains under starvation conditions. These results taken together suggest that the direct interaction between Atg5 and Atg16 is crucial to the performance of their roles in autophagy.     

Aberrant Membranes and Double-Membrane Structures Accumulate in the Axons of Atg5-Null Purkinje Cells before Neuronal Death

Autophagy (macroautophagy) is an evolutionally conserved process by which cytoplasmic proteins and organelles are surrounded by unique double membranes and are subsequently degraded upon fusion with lysosomes. Many autophagy-related genes (Atg) have been identified in yeast; a ubiquitin-like Atg12-Atg5 system is also essential for the elongation of the isolation membrane in mammalian cells. Nevertheless, the regulation of autophagy in neurons remains largely unknown. In this study, we crossed conditional knockout mice Atg5^flox/flox with pcp2-Cre transgenic mice, which express Cre recombinase through a Purkinje cell–specific promoter, pcp2. In Atg5^flox/flox; pcp2-Cre mice, the Atg5 gene was excised as early as postnatal day 6; Purkinje cells started to degenerate after approximately 8 weeks, and the animals showed an ataxic gait from around 10 months. Initially, however, the Purkinje cells showed axonal swelling around its terminals from as early as 4 weeks after birth. An electron microscopic analysis revealed the accumulation of autophagosome-like double-membrane structures in the swollen regions, together with numerous membranous organelles, such as tubular or sheet-like smooth endoplasmic reticulum and vesicles. These results suggest that Atg5 plays important roles in the maintenance of axon morphology and membrane structures, and its loss of function leads to the swelling of axons, followed by progressive neurodegeneration in mammalian neurons.     

Compensatory activation of ERK1/2 in Atg5-deficient mouse embryo fibroblasts suppresses oxidative stress-induced cell death

Despite of the increasing evidence that oxidative stress may induce non-apoptotic cell death or autophagic cell death, the mechanism of this process is unclear. Here, we report a role and a down-stream molecular event of Atg5 during oxidative stress-induced cell death. Compared to wild type (WT) cells, Atg5-deficient mouse embryo fibroblasts (Atg5^-/- MEFs) and Atg5 knockdown HT22 neuronal cells were more resistant to cell death induced by H₂O₂. On the contrary, Atg5^-/- MEFs were as sensitive to tumor necrosis factor (TNF)-α and cycloheximide as WT cells, and were more sensitive to cell death triggered by amino acid-deprivation than WT MEFs. Treatment with H₂O₂ induced the recruitment of a GFP-LC3 fusion protein and conversion of LC3 I to LC3 II, correlated with the extent of autophagosome formation in WT cells, but much less in Atg5-deficient cells. Among stress kinases, ERK1/2 was markedly activated in Atg5^-/- MEFs and Atg5 knockdown HT22 and SH-SY5Y neuronal cells. The inhibition of ERK1/2 by MEK1 inhibitor (PD98059) or dominant negative ERK2 enhanced the susceptibility of Atg5^-/- MEFs to H₂O₂-induced cell death. Further, reconstitution of Atg5 sensitized Atg5^-/- MEFs to H₂O₂ and suppressed the activation of ERK1/2. These results suggest that the inhibitory effect of Atg5 deficiency on cell death is attributable by the compensatory activation of ERK1/2 in Atg5^-/- MEFs during oxidative stress-induced cell death.     

Atg9 Trafficking in Yeast Saccharomyces cerevisiae

Autophagy can be divided into selective and non-selective modes. This process is considered selective when a precise cargo is specifically and exclusively incorporated into autophagosomes, the double-membrane vesicles that are the hallmark of autophagy. In contrast, during nonselective, bulk autophagy, cytoplasmic components are randomly enwrapped into autophagosomes. To date, approximately 30 autophagy-related genes called ATG have been identified. Sixteen of them compose the general basic machinery catalyzing the formation of double-membrane vesicles in all eukaryotic cells. The rest of them are often not conserved between species and cooperate with the basic Atg proteins during either selective or nonselective autophagy. Atg9 is the only integral membrane component of the conserved Atg machinery and appears to be a crucial organizational element.5 Recent studies in the S. cerevisiae have shown that Atg9 transport is differentially regulated depending on the autophagy mode. In this addendum, we will review and discuss what has recently been unveiled about yeast S. cerevisiae Atg9 trafficking, its modulators and its potential role in double-membrane vesicle biogenesis.

Addendum to:

Atg9 Sorting from Mitochondria is Impaired in Early Secretion and VFT Complex Mutants in Saccharomyces cerevisiae

F. Reggiori and D.J. Klionsky

J Cell Sci 2006: 119:2903-11     

Evolution of Atg1 function and regulation

The serine/threonine kinase Atg1 plays an essential role downstream of TOR for the regulation of autophagy. In yeast, where Atg1 was first identified, a complex regulatory mechanism has been described that includes at least seven other interacting proteins and a phosphorylation-dependent switch. Recent findings confirm that the mammalian Atg1 homologues ULK1 and 2 have autophagy regulatory roles. However, we and others have also demonstrated mechanistic differences with the yeast model and between these two Atg1 family members. Here, we elaborate on our growing understanding of Atg1 function, incorporating findings from yeast, C. elegans, D. melanogaster and mammalian cells. We propose that through evolution, Atg1 proteins have adopted additional cellular functions and regulatory mechanisms, which could involve multiple gene family isoforms working within multi-functional protein complexes. The gene family expansion observed in higher eukaryotes might reflect an increased functional diversity of Atg1 proteins in cell growth, differentiation and survival.     

Phospholipid methylation controls Atg32‐mediated mitophagy and Atg8 recycling

Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy‐inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria‐anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8–PMME, a mitophagy‐incompetent lipid conjugate of the autophagy‐related ubiquitin‐like modifier. Amelioration of Atg32 expression and attenuation of Atg8–PMME conjugation markedly rescue mitophagy in opi3‐null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32‐mediated mitophagy.     

Bortezomib alleviates antibody-mediated rejection in kidney transplantation by facilitating Atg5 expression

Antibody-mediated rejection (AMR) is one of the most dominant mechanisms responsible for the loss of kidney grafts. Previous researches have shown that donor-specific antibodies (DSAs) are the major mediators of AMR. In order to prolong the survival time of grafts, it is vital to reduce the incidence of AMR and inhibit the generation of DSAs. We established an animal model of AMR by performing kidney transplantation in pre-sensitized rats. Then, we investigated the effect of bortezomib (BTZ) on AMR. We found that BTZ could reduce the serum level of DSAs and alleviate post-transplantation inflammation in peritubular capillaries (PTCs) and glomeruli, which was demonstrated by the reduction of C4d and IgG deposition in PTCs, and the reduced number of B cell and plasma cell in peripheral blood and the transplanted kidney (p < 0.05). Our results also suggested that BTZ increased the number of regulatory T cell (Treg) and significantly reduced the proportion of T helper (Th17) cell (p < 0.05). Besides, BTZ induced the significant upregulation of anti-inflammatory cytokines but downregulated pro-inflammatory cytokines (p < 0.05). After dealing with Atg5 siRNA-lentivirus, the effect of BTZ alleviating AMR was reversed and Th17/Treg proportions were also significantly modulated. Collectively, these findings show that BTZ slows down the process of AMR and Atg5 may be the key mechanism. Furthermore, Atg5 silencing results may be demonstrated that Atg5 alleviated AMR by modulating the ratio of Th17/Treg.