Role of Wdr45b in maintaining neural autophagy and cognitive function

ABSTRACT

Macroautophagy/autophagy functions as a quality control mechanism by degrading misfolded proteins and damaged organelles and plays an essential role in maintaining neural homeostasis. The phosphoinositide phosphatidylinositol-3-phosphate (PtdIns3P) effector Atg18 is essential for autophagosome formation in yeast. Mammalian cells contain four Atg18 homologs, belonging to two subclasses, WIPI1 (WD repeat domain, phosphoinositide interacting 1), WIPI2 and WDR45B/WIPI3 (WD repeat domain 45B), WDR45/WIPI4. The role of Wdr45b in autophagy and in neural homeostasis, however, remains unknown. Recent human genetic studies have revealed a potential causative role of WDR45B in intellectual disability. Here we demonstrated that mice deficient in Wdr45b exhibit motor deficits and learning and memory defects. Histological analysis reveals that wdr45b knockout (KO) mice exhibit a large number of swollen axons and show cerebellar atrophy. SQSTM1- and ubiquitin-positive aggregates, which are autophagy substrates, accumulate in various brain regions in wdr45b KO mice. Double KO mice, wdr45b and wdr45, die within one day after birth and exhibit more severe autophagy defects than either of the single KO mice, suggesting that these two genes act cooperatively in autophagy. Our studies demonstrated that WDR45B is critical for neural homeostasis in mice. The wdr45b KO mice provide a model to study the pathogenesis of intellectual disability.

Abbreviations: ACSF: artificial cerebrospinal fluid; AMC: aminomethylcoumarin; BPAN: beta-propeller protein-associated neurodegeneration; CALB1: calbindin 1; CNS: central nervous system; DCN: deep cerebellar nuclei; fEPSP: field excitatory postsynaptic potential; IC: internal capsule; ID: intellectual disability; ISH: in situ hybridization; KO: knockout; LTP: long-term potentiation; MBP: myelin basic protein; MGP: medial globus pallidus; PtdIns3P: phosphoinositide phosphatidylinositol-3-phosphate; WDR45B: WD repeat domain 45B; WIPI1: WD repeat domain, phosphoinositide interacting 1; WT: wild type.     

B cell autophagy mediates TLR7-dependent autoimmunity and inflammation

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, defined by loss of B cell self-tolerance that results in production of antinuclear antibodies (ANA) and chronic inflammation. While the initiating events in lupus development are not well defined, overexpression of the RNA-recognizing toll-like receptor (TLR)7 has been linked to SLE in humans and mice. We postulated that autophagy plays an essential role in TLR7 activation of B cells for the induction of SLE by delivering RNA ligands to the endosomes, where this innate immune receptor resides. To test this hypothesis, we compared SLE development in Tlr7 transgenic (Tg) mice with or without B cell-specific ablation of autophagy (Cd19-Cre Atg5^f/f). We observed that in the absence of B cell autophagy the 2 hallmarks of SLE, ANA and inflammation, were eliminated, thus curing these mice of lupus. This was also evident in the significantly extended survival of the autophagy-deficient mice compared to Tlr7.1 Tg mice. Furthermore, glomerulonephritis was ameliorated, and the serum levels of inflammatory cytokines in the knockout (KO) mice were indistinguishable from those of control mice. These data provide direct evidence that B cells require TLR7-dependent priming through an autophagy-dependent mechanism before autoimmunity is induced, thereafter involving many cell types. Surprisingly, hyper-IgM production persisted in Tlr7.1 Tg mice in the absence of autophagy, likely involving a different activation pathway than the production of autoantibodies. Furthermore, these mice still presented with anemia, but responded with a striking increase in extramedullary hematopoiesis (EMH), possibly due to the absence of pro-inflammatory cytokines.     

Commentary: Autophagy promotes Braf V600E-driven lung tumorigenesis by preserving mitochondrial metabolism

The role of autophagy in cancer is complex and context-dependent. Here we describe work with genetically engineered mouse models of non-small cell lung cancer (NSCLC) in which the tumor-suppressive and tumor-promoting function of autophagy can be visualized in the same system. We discovered that early tumorigenesis in Braf ^V600E -driven lung cancer is accelerated by autophagy ablation due to unmitigated oxidative stress, as observed with loss of Nfe2l2/Nrf2-mediated antioxidant defense. However, this growth advantage is eventually overshadowed by progressive mitochondrial dysfunction and metabolic insufficiency, and is associated with increased survival of mice bearing autophagy-deficient tumors. Atg7 deficiency alters progression of Braf ^V600E-driven tumors from adenomas (Braf ^V600E ; atg7^−/−) and adenocarcinomas (trp53^−/−; Braf ^V600E ; atg7^−/−) to benign oncocytomas that accumulated morphologically and functionally defective mitochondria, suggesting that defects in mitochondrial metabolism may compromise continued tumor growth. Analysis of tumor-derived cell lines (TDCLs) revealed that Atg7-deficient cells are significantly more sensitive to starvation than Atg7–wild-type counterparts, and are impaired in their ability to respire, phenotypes that are rescued by the addition of exogenous glutamine. Taken together, these data suggest that Braf ^V600E -driven tumors become addicted to autophagy as a means to preserve mitochondrial function and glutamine metabolism, and that inhibiting autophagy may be a powerful strategy for Braf ^V600E -driven malignancies.     

Proper macrophagic differentiation requires both autophagy and caspase activation

Autophagy allows the elimination of superfluous or damaged macromolecules or organelles. Genetic evidence indicates that autophagy plays essential functions during differentiation. The differentiation of human blood monocytes into macrophages is a caspase-dependent process triggered by colony stimulating factor1 (CSF1/CSF-1). We have established, using pharmacological inhibitors, siRNA approaches and Atg7^−/− mice, that autophagy is required for proper CSF1/CSF-1-driven differentiation of human and murine monocytes and acquisition of phagocytic functions. Collectively, these findings highlight an essential role of autophagy during monocyte differentiation and acquisition of macrophage functions. Deciphering the complex interplay between caspase and autophagy that occurs during this process will undoubtedly bring new insights in our understanding of monocyte differentiation.     

Research Article: NOD2 is dispensable for ATG16L1 deficiency-mediated resistance to urinary tract infection

NOD2 (nucleotide-binding oligomerization domain containing 2) functions as a pathogen sensor and is involved in development of Crohn disease, a form of inflammatory bowel disease. NOD2 functions in concert with the autophagy protein ATG16L1, which is also implicated in Crohn disease. Recently, we identified a novel protective role of ATG16L1 deficiency in uropathogenic Escherichia coli-induced urinary tract infections (UTIs), which are common infectious diseases in humans. Given the known roles of NOD2 in recruiting ATG16L1 to the bacterial entry site, autophagy induction, and Crohn disease, we hypothesized that NOD2 may also play an important role in UTI pathogenesis. Instead, we found evidence that NOD2 is dispensable in the pathogenesis of UTIs in mice and humans. First, loss of Nod2 did not affect the clearance of bacteriuria and the recruitment of innate immune cells to the bladder. Second, we showed that, although nod2 ^−/− mice display increased kidney abscesses in the upper urinary tract, there were no increased bacterial loads or persistence in this niche. Third, although a previous study indicates that loss of Nod2 reverses the protection from intestinal infection afforded by loss of ATG16L1 in mice, we found NOD2 deficiency did not reverse the ATG16L1-deficiency-induced protection from UTI. Finally, a population-based study of a cohort of 1819 patients did not reveal any association of NOD2 polymorphisms with UTI incidence. Together, our data indicated that NOD2 is dispensable for UTI pathogenesis in both mice and humans and does not contribute to ATG16L1-deficiency-induced resistance to UTI in mice.     

Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages.     

ERBB2 overexpression suppresses stress-induced autophagy and renders ERBB2-induced mammary tumorigenesis independent of monoallelic Becn1 loss

Defective autophagy has been implicated in mammary tumorigenesis, as the gene encoding the essential autophagy regulator BECN1 is deleted in human breast cancers and Becn1^+/− mice develop mammary hyperplasias. In agreement with a recent study, which reports concurrent allelic BECN1 loss and ERBB2 amplification in a small number of human breast tumors, we found that low BECN1 mRNA correlates with ERBB2-overexpression in breast cancers, suggesting that BECN1 loss and ERBB2 overexpression may functionally interact in mammary tumorigenesis. We now report that ERBB2 overexpression suppressed autophagic response to stress in mouse mammary and human breast cancer cells. ERBB2-overexpressing Becn1^+/+ and Becn1^+/− immortalized mouse mammary epithelial cells (iMMECs) formed mammary tumors in nude mice with similar kinetics, and monoallelic Becn1 loss did not alter ERBB2- and PyMT-driven mammary tumorigenesis. In human breast cancer databases, ERBB2-expressing tumors exhibit a low autophagy gene signature, independent of BECN1 mRNA expression, and have similar gene expression profiles with non-ERBB2-expressing breast tumors with low BECN1 levels. We also found that ERBB2-expressing BT474 breast cancer cells, despite being partially autophagy-deficient under stress, can be sensitized to the anti-ERBB2 antibody trastuzumab (tzb) by further pharmacological or genetic autophagy inhibition. Our results indicate that ERBB2-driven mammary tumorigenesis is associated with functional autophagy suppression and ERBB2-positive breast cancers are partially autophagy-deficient even in a wild-type BECN1 background. Furthermore and extending earlier findings using tzb-resistant cells, exogenously imposed autophagy inhibition increases the anticancer effect of trastuzumab on tzb-sensitive ERBB2-expressing breast tumor cells, indicating that pharmacological autophagy suppression has a wider role in the treatment of ERBB2-positive breast cancer.     

A new genetic model for calcium induced autophagy and ER-stress in Drosophila photoreceptor cells

Cytoplasmic Ca²⁺ overload is known to trigger autophagy and ER-stress. Furthermore, ER-stress and autophagy are commonly associated with degenerative pathologies, but their role in disease progression is still a matter of debate, in part, owing to limitations of existing animal model systems. The Drosophila eye is a widely used model system for studying neurodegenerative pathologies. Recently, we characterized the Drosophila protein, Calphotin, as a cytosolic immobile Ca²⁺ buffer, which participates in Ca²⁺ homeostasis in Drosophila photoreceptor cells. Exposure of calphotin hypomorph flies to continuous illumination, which induces Ca²⁺ influx into photoreceptor cells, resulted in severe Ca²⁺-dependent degeneration. Here we show that this degeneration is autophagy and ER-stress related. Our studies thus provide a new model in which genetic manipulations trigger changes in cellular Ca²⁺ distribution. This model constitutes a framework for further investigations into the link between cytosolic Ca²⁺, ER-stress and autophagy in human disorders and diseases.     

Analysis of a lung defect in autophagy-deficient mouse strains

Yeast Atg1 initiates autophagy in response to nutrient limitation. The Ulk gene family encompasses the mammalian orthologs of yeast ATG1. We created mice deficient for both Ulk1 and Ulk2 and found that the mice die within 24 h of birth. When found alive, pups exhibited signs of respiratory distress. Histological sections of lungs of the Ulk1/2 DKO pups showed reduced airspaces with thickened septae. A similar defect was seen in Atg5-deficient pups as both Ulk1/2 DKO and Atg5 KO lungs show numerous glycogen-laden alveolar type II cells by electron microscopy, PAS staining, and increased levels of glycogen in lung homogenates. No abnormalities were noted in expression of genes encoding surfactant proteins but the ability to incorporate exogenous choline into phosphatidylcholine, the major phospholipid component of surfactant, was increased in comparison to controls. Despite this, there was a trend for total phospholipid levels in lung tissue to be lower in Ulk1/2 DKO and Atg5 KO compared with controls. Autophagy was abundant in lung epithelial cells from wild-type mice, but lacking in Atg5 KO and Ulk1/2 DKO mice at P1. Analysis of the autophagy signaling pathway showed the existence of a negative feedback loop between the ULK1 and 2 and MTORC1 and 2, in lung tissue. In the absence of autophagy, alveolar epithelial cells are unable to mobilize internal glycogen stores independently of surfactant maturation. Together, the data suggested that autophagy plays a vital role in lung structural maturation in support of perinatal adaptation to air breathing.     

Autophagy Modulates Borrelia burgdorferi-induced Production of Interleukin-1β (IL-1β)

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease. Recent studies have shown that recognition of the spirochete is mediated by TLR2 and NOD2. The latter receptor has been associated with the induction of the intracellular degradation process called autophagy. The present study demonstrated for the first time the induction of autophagy by exposure to B. burgdorferi and that autophagy modulates the B. burgdorferi-dependent cytokine production. Human peripheral blood mononuclear cells treated with autophagy inhibitors showed an increased IL-1β and IL-6 production in response to the exposure of the spirochete, whereas TNFα production was unchanged. Autophagy induction against B. burgdorferi was dependent on reactive oxygen species (ROS) because cells from patients with chronic granulomatous disease, which are defective in ROS production, also produced elevated IL-1β. Further, the enhanced production of the proinflammatory cytokines was because of the elevated mRNA expression in the absence of autophagy. Our results thus demonstrate the induction of autophagy, which, in turn, modulates cytokine production by B. burgdorferi for the first time.     

TipC and the chorea-acanthocytosis protein VPS13A regulate autophagy in Dictyostelium and human HeLa cells

Deficient autophagy causes a distinct phenotype in Dictyostelium discoideum, characterized by the formation of multitips at the mound stage. This led us to analyze autophagy in a number of multitipped mutants described previously (tipA⁻, tipB⁻, tipC⁻, and tipD⁻). We found a clear autophagic dysfunction in tipC⁻ and tipD⁻ while the others showed no defects. tipD codes for a homolog of Atg16, which confirms the role of this protein in Dictyostelium autophagy and validates our approach. The tipC-encoded protein is highly similar to human VPS13A (also known as chorein), whose mutations cause the chorea-acanthocytosis syndrome. No member of the VPS13 protein family has been previously related to autophagy despite the presence of a region of similarity to Atg2 at the C terminus. This region also contains the conserved domain of unknown function DUF1162. Of interest, the expression of the TipC C-terminal coding sequence containing these 2 motifs largely complemented the mutant phenotype. Dictyostelium cells lacking TipC displayed a reduced number of autophagosomes visualized with the markers GFP-Atg18 and GFP-Atg8 and an impaired autophagic degradation as determined by a proteolytic cleavage assay. Downregulation of human VPS13A in HeLa cells by RNA interference confirmed the participation of the human protein in autophagy. VPS13A-depleted cells showed accumulation of autophagic markers and impaired autophagic flux.     

Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis

Autophagy is an essential process for eliminating ubiquitinated protein aggregates and dysfunctional organelles. Defective autophagy is associated with various degenerative diseases such as Parkinson disease. Through a genetic screening in Drosophila, we identified CG11148, whose product is orthologous to GIGYF1 (GRB10-interacting GYF protein 1) and GIGYF2 in mammals, as a new autophagy regulator; we hereafter refer to this gene as Gyf. Silencing of Gyf completely suppressed the effect of Atg1-Atg13 activation in stimulating autophagic flux and inducing autophagic eye degeneration. Although Gyf silencing did not affect Atg1-induced Atg13 phosphorylation or Atg6-Pi3K59F (class III PtdIns3K)-dependent Fyve puncta formation, it inhibited formation of Atg13 puncta, suggesting that Gyf controls autophagy through regulating subcellular localization of the Atg1-Atg13 complex. Gyf silencing also inhibited Atg1-Atg13-induced formation of Atg9 puncta, which is accumulated upon active membrane trafficking into autophagosomes. Gyf-null mutants also exhibited substantial defects in developmental or starvation-induced accumulation of autophagosomes and autolysosomes in the larval fat body. Furthermore, heads and thoraxes from Gyf-null adults exhibited strongly reduced expression of autophagosome-associated Atg8a-II compared to wild-type (WT) tissues. The decrease in Atg8a-II was directly correlated with an increased accumulation of ubiquitinated proteins and dysfunctional mitochondria in neuron and muscle, which together led to severe locomotor defects and early mortality. These results suggest that Gyf-mediated autophagy regulation is important for maintaining neuromuscular homeostasis and preventing degenerative pathologies of the tissues. Since human mutations in the GIGYF2 locus were reported to be associated with a type of familial Parkinson disease, the homeostatic role of Gyf-family proteins is likely to be evolutionarily conserved.     

ATG16L1 and pathogenesis of urinary tract infections

Autophagy is generally considered to be antipathogenic. The autophagy gene ATG16L1 has a commonly occurring mutation associated with Crohn disease (CD) and intestinal cell abnormalities. Mice hypomorphic for ATG16L1 (ATG16L1^HM) recreate specific features of CD. Our recent study shows that the same ATG16L1^HM mice that are susceptible to intestinal inflammatory disease are protected from urinary tract infections (UTI), a common and important human disease primarily caused by uropathogenic E. coli (UPEC). UPEC colonize the bladder and exhibit both luminal and intra-epithelial stages. The host responds by recruiting innate immune cells and shedding infected epithelial cells to clear infection. Despite these countermeasures, UPEC can persist within the bladder epithelium as membrane-enclosed quiescent intracellular reservoirs (QIRs) that can seed recurrent UTI. The mechanisms of persistence remain unknown. In this study, we show that ATG16L1 deficiency protects the host against acute UTI and UPEC latency. ATG16L1^HM mice clear urinary bacterial loads more rapidly and thoroughly due to ATG16L1-deficient innate immune components. Furthermore, ATG16L1^HM mice exhibit superficial urothelial cell-autonomous architectural aberrations that also result in significantly reduced QIR numbers. Our findings reveal a host-protective effect of ATG16L1 deficiency in vivo against a common pathogen.     

The Wallerian degeneration slow (Wld(s)) gene does not attenuate disease in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a severe neuromuscular disease characterized by loss of spinal α-motor neurons, resulting in the paralysis of skeletal muscle. SMA is caused by deficiency of survival motor neuron (SMN) protein levels. Recent evidence has highlighted an axon-specific role for SMN protein, raising the possibility that axon degeneration may be an early event in SMA pathogenesis. The Wallerian degeneration slow (Wld^s) gene is a spontaneous dominant mutation in mice that delays axon degeneration by approximately 2-3 weeks. We set out to examine the effect of Wld^s on the phenotype of a mouse model of SMA. We found that Wld^s does not alter the SMA phenotype, indicating that Wallerian degeneration does not directly contribute to the pathogenesis of SMA development.     

Inhibition of autophagy attenuates pancreatic cancer growth independent of TP53/TRP53 status

Basal levels of autophagy are elevated in most pancreatic ductal adenocarcinomas (PDAC). Suppressing autophagy pharmacologically using chloroquine (CQ) or genetically with RNAi to essential autophagy genes inhibits human pancreatic cancer growth in vitro and in vivo, which presents possible treatment opportunities for PDAC patients using the CQ-derivative hydroxychloroquine (HCQ). Indeed, such clinical trials are ongoing. However, autophagy is a complex cellular mechanism to maintain cell homeostasis under stress. Based on its biological role, a dual role of autophagy in tumorigenesis has been proposed: at tumor initiation, autophagy helps maintain genomic stability and prevent tumor initiation; while in advanced disease, autophagy degrades and recycles cellular components to meet the metabolic needs for rapid growth. This model was proven to be the case in mouse lung tumor models. However, in contrast to prior work in various PDAC model systems, loss of autophagy in PDAC mouse models with embryonic homozygous Trp53 deletion does not inhibit tumor growth and paradoxically increases progression. This raised concerns whether there may be a genotype-dependent reliance of PDAC on autophagy. In a recent study, our group used a Trp53 heterozygous mouse PDAC model and human PDX xenografts to address the question. Our results demonstrate that autophagy inhibition was effective against PDAC tumors irrespective of TP53/TRP53 status.     

MIR34A regulates autophagy and apoptosis by targeting HMGB1 in the retinoblastoma cell

MIR34A (microRNA 34a) is a tumor suppressor gene, but how it regulates chemotherapy response and resistance is not completely understood. Here, we show that the microRNA MIR34A-dependent high mobility group box 1 (HMGB1) downregulation inhibits autophagy and enhances chemotherapy-induced apoptosis in the retinoblastoma cell. HMGB1 is a multifaceted protein with a key role in autophagy, a self-degradative, homeostatic process with a context-specific role in cancer. MIR34A inhibits HMGB1 expression through a direct MIR34A-binding site within the HMGB1 3′ untranslated region. MIR34A inhibition of HMGB1 leads to a decrease in autophagy under starvation conditions or chemotherapy treatment. Inhibition of autophagy promotes oxidative injury and DNA damage and increases subsequent CASP3 activity, CASP3 cleavage, and PARP1 [poly (ADP-ribose) polymerase 1] cleavage, which are important to the apoptotic process. Finally, upregulation of MIR34A, knockdown of HMGB1, or inhibition of autophagy (e.g., knockdown of ATG5 and BECN1) restores chemosensitivity and enhances tumor cell death in the retinoblastoma cell. These data provide new insights into the mechanisms governing the regulation of HMGB1 expression by microRNA and their possible contribution to autophagy and drug resistance.