TGF-ß-induced PI3K/AKT/mTOR pathway controls myofibroblast differentiation and secretory phenotype of valvular interstitial cells through the modulation of cellular senescence in a naturally occurring in vitro canine model of myxomatous mitral valve disease.

PI3K/AKT/mTOR signalling contributes to several cardiovascular disorders. The aim of this study was to examine the PI3K/AKT/mTOR pathway in myxomatous mitral valve disease (MMVD). Double-immunofluorescence examined expression of PI3K and TGF-ß1 in canine valves. Valve interstitial cells (VICs) from healthy or MMVD dogs were isolated and characterized. Healthy quiescent VICs (qVICs) were treated with TGF-ß1 and SC-79 to induce activated myofibroblast phenotypes (aVICs). Diseased valve-derived aVICs were treated with PI3K antagonists and expression of RPS6KB1 (encoding p70 S6K) was modulated using siRNA and gene overexpression. SA-ß-gal and TUNEL staining were used to identify cell senescence and apoptosis, and qPCR and ELISA to examine for senescence-associated secretory phenotype. Protein immunoblotting was used to examine expression of phosphorylated and total proteins. TGF-ß1 and PI3K are highly expressed in mitral valve tissues. Activation of PI3K/AKT/mTOR and increased expression of TGF-ß are found in aVICs. TGF-ß transitions qVICs to aVICs by upregulation of PI3K/AKT/mTOR. Antagonism of PI3K/AKT/mTOR reverses aVIC myofibroblast transition by inhibiting senescence and promoting autophagy. Upregulation of mTOR/S6K induces transformation of senescent aVICs, with reduced capacity for apoptosis and autophagy. Selective knockdown of p70 S6K reverses cell transition by attenuating cell senescence, inhibiting apoptosis and improving autophagy. TGF-ß-induced PI3K/AKT/mTOR signalling contributes to MMVD pathogenesis and plays crucial roles in the regulation of myofibroblast differentiation, apoptosis, autophagy and senescence in MMVD.

p53 Regulates Mitochondrial Dynamics in Vascular Smooth Muscle Cell Calcification.

Arterial calcification is an important characteristic of cardiovascular disease. It has key parallels with skeletal mineralization; however, the underlying cellular mechanisms responsible are not fully understood. Mitochondrial dynamics regulate both bone and vascular function. In this study, we therefore examined mitochondrial function in vascular smooth muscle cell (VSMC) calcification. Phosphate (Pi)-induced VSMC calcification was associated with elongated mitochondria (1.6-fold increase, p < 0.001), increased mitochondrial reactive oxygen species (ROS) production (1.83-fold increase, p < 0.001) and reduced mitophagy (9.6-fold decrease, p < 0.01). An increase in protein expression of optic atrophy protein 1 (OPA1; 2.1-fold increase, p < 0.05) and a converse decrease in expression of dynamin-related protein 1 (DRP1; 1.5-fold decrease, p < 0.05), two crucial proteins required for the mitochondrial fusion and fission process, respectively, were noted. Furthermore, the phosphorylation of DRP1 Ser637 was increased in the cytoplasm of calcified VSMCs (5.50-fold increase), suppressing mitochondrial translocation of DRP1. Additionally, calcified VSMCs showed enhanced expression of p53 (2.5-fold increase, p < 0.05) and ß-galactosidase activity (1.8-fold increase, p < 0.001), the cellular senescence markers. siRNA-mediated p53 knockdown reduced calcium deposition (8.1-fold decrease, p < 0.01), mitochondrial length (3.0-fold decrease, p < 0.001) and ß-galactosidase activity (2.6-fold decrease, p < 0.001), with concomitant mitophagy induction (3.1-fold increase, p < 0.05). Reduced OPA1 (4.1-fold decrease, p < 0.05) and increased DRP1 protein expression (2.6-fold increase, p < 0.05) with decreased phosphorylation of DRP1 Ser637 (3.20-fold decrease, p < 0.001) was also observed upon p53 knockdown in calcifying VSMCs. In summary, we demonstrate that VSMC calcification promotes notable mitochondrial elongation and cellular senescence via DRP1 phosphorylation. Furthermore, our work indicates that p53-induced mitochondrial fusion underpins cellular senescence by reducing mitochondrial function.

Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor.

Vascular calcification is associated with aging, type 2 diabetes, and atherosclerosis, and increases the risk of cardiovascular morbidity and mortality. It is an active, highly regulated process that resembles physiological bone formation. It has previously been established that pharmacological doses of metformin alleviate arterial calcification through adenosine monophosphate-activated protein kinase (AMPK)-activated autophagy, however the specific pathway remains elusive. In the present study we hypothesized that metformin protects against arterial calcification through the direct autophagic degradation of runt-related transcription factor 2 (Runx2). Calcification was blunted in vascular smooth muscle cells (VSMCs) by metformin in a dose-dependent manner (0.5-1.5 mM) compared to control cells (p < 0.01). VSMCs cultured under high-phosphate (Pi) conditions in the presence of metformin (1 mM) showed a significant increase in LC3 puncta following bafilomycin-A1 (Baf-A; 5 nM) treatment compared to control cells (p < 0.001). Furthermore, reduced expression of Runx2 was observed in the nuclei of metformin-treated calcifying VSMCs (p < 0.0001). Evaluation of the functional role of autophagy through Atg3 knockdown in VSMCs showed aggravated Pi-induced calcification (p < 0.0001), failure to induce autophagy (punctate LC3) (p < 0.001) and increased nuclear Runx2 expression (p < 0.0001) in VSMCs cultured under high Pi conditions in the presence of metformin (1 mM). Mechanistic studies employing three-way coimmunoprecipitation with Runx2, p62, and LC3 revealed that p62 binds to both LC3 and Runx2 upon metformin treatment in VSMCs. Furthermore, immunoblotting with LC3 revealed that Runx2 specifically binds with p62 and LC3-II in metformin-treated calcified VSMCs. Lastly, we investigated the importance of the autophagy pathway in vascular calcification in a clinical setting. Ex vivo clinical analyses of calcified diabetic lower limb artery tissues highlighted a negative association between Runx2 and LC3 in the vascular calcification process. These studies suggest that exploitation of metformin and its analogues may represent a novel therapeutic strategy for clinical intervention through the induction of AMPK/Autophagy Related 3 (Atg3)-dependent autophagy and the subsequent p62-mediated autophagic degradation of Runx2.

The Role of Transforming Growth Factor-ß Signaling in Myxomatous Mitral Valve Degeneration.

Mitral valve prolapse (MVP) due to myxomatous degeneration is one of the most important chronic degenerative cardiovascular diseases in people and dogs. It is a common cause of heart failure leading to significant morbidity and mortality in both species. Human MVP is usually classified into primary or non-syndromic, including Barlow's Disease (BD), fibro-elastic deficiency (FED) and Filamin-A mutation, and secondary or syndromic forms (typically familial), such as Marfan syndrome (MFS), Ehlers-Danlos syndrome, and Loeys-Dietz syndrome. Despite different etiologies the diseased valves share pathological features consistent with myxomatous degeneration. To reflect this common pathology the condition is often called myxomatous mitral valve degeneration (disease) (MMVD) and this term is universally used to describe the analogous condition in the dog. MMVD in both species is characterized by leaflet thickening and deformity, disorganized extracellular matrix, increased transformation of the quiescent valve interstitial cell (qVICs) to an activated state (aVICs), also known as activated myofibroblasts. Significant alterations in these cellular activities contribute to the initiation and progression of MMVD due to the increased expression of transforming growth factor-ß (TGF-ß) superfamily cytokines and the dysregulation of the TGF-ß signaling pathways. Further understanding the molecular mechanisms of MMVD is needed to identify pharmacological manipulation strategies of the signaling pathway that might regulate VIC differentiation and so control the disease onset and development. This review briefly summarizes current understanding of the histopathology, cellular activities, molecular mechanisms and pathogenesis of MMVD in dogs and humans, and in more detail reviews the evidence for the role of TGF-ß.

Mitochondrial Dysfunction: Cause or Consequence of Vascular Calcification?

Mitochondria are crucial bioenergetics powerhouses and biosynthetic hubs within cells, which can generate and sequester toxic reactive oxygen species (ROS) in response to oxidative stress. Oxidative stress-stimulated ROS production results in ATP depletion and the opening of mitochondrial permeability transition pores, leading to mitochondria dysfunction and cellular apoptosis. Mitochondrial loss of function is also a key driver in the acquisition of a senescence-associated secretory phenotype that drives senescent cells into a pro-inflammatory state. Maintaining mitochondrial homeostasis is crucial for retaining the contractile phenotype of the vascular smooth muscle cells (VSMCs), the most prominent cells of the vasculature. Loss of this contractile phenotype is associated with the loss of mitochondrial function and a metabolic shift to glycolysis. Emerging evidence suggests that mitochondrial dysfunction may play a direct role in vascular calcification and the underlying pathologies including (1) impairment of mitochondrial function by mineral dysregulation i.e., calcium and phosphate overload in patients with end-stage renal disease and (2) presence of increased ROS in patients with calcific aortic valve disease, atherosclerosis, type-II diabetes and chronic kidney disease. In this review, we discuss the cause and consequence of mitochondrial dysfunction in vascular calcification and underlying pathologies; the role of autophagy and mitophagy pathways in preventing mitochondrial dysfunction during vascular calcification and finally we discuss mitochondrial ROS, DRP1, and HIF-1 as potential novel markers and therapeutic targets for maintaining mitochondrial homeostasis in vascular calcification.

Osteoblast-specific deficiency of ectonucleotide pyrophosphatase or phosphodiesterase-1 engenders insulin resistance in high-fat diet fed mice.

Supraphysiological levels of the osteoblast-enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase-1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast-specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6-week-old mice lacking osteoblast NPP1 expression (osteoblast-specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast-specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast-specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin-sensitizing under-carboxylated osteocalcin (195% increase; p < .05). However, following high-fat-diet challenge, osteoblast-specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity.

Osteocalcin Regulates Arterial Calcification Via Altered Wnt Signaling and Glucose Metabolism.

Arterial calcification is an important hallmark of cardiovascular disease and shares many similarities with skeletal mineralization. The bone-specific protein osteocalcin (OCN) is an established marker of vascular smooth muscle cell (VSMC) osteochondrogenic transdifferentiation and a known regulator of glucose metabolism. However, the role of OCN in controlling arterial calcification is unclear. We hypothesized that OCN regulates calcification in VSMCs and sought to identify the underpinning signaling pathways. Immunohistochemistry revealed OCN co-localization with VSMC calcification in human calcified carotid artery plaques. Additionally, 3 mM phosphate treatment stimulated OCN mRNA expression in cultured VSMCs (1.72-fold, p < 0.001). Phosphate-induced calcification was blunted in VSMCs derived from OCN null mice (Ocn -/- ) compared with cells derived from wild-type (WT) mice (0.37-fold, p < 0.001). Ocn -/- VSMCs showed reduced mRNA expression of the osteogenic marker Runx2 (0.51-fold, p < 0.01) and the sodium-dependent phosphate transporter, PiT1 (0.70-fold, p < 0.001), with an increase in the calcification inhibitor Mgp (1.42-fold, p < 0.05) compared with WT. Ocn -/- VSMCs also showed reduced mRNA expression of Axin2 (0.13-fold, p < 0.001) and Cyclin D (0.71 fold, p < 0.01), markers of Wnt signaling. CHIR99021 (GSK3ß inhibitor) treatment increased calcium deposition in WT and Ocn -/- VSMCs (1 µM, p < 0.001). Ocn -/- VSMCs, however, calcified less than WT cells (1 µM; 0.27-fold, p < 0.001). Ocn -/- VSMCs showed reduced mRNA expression of Glut1 (0.78-fold, p < 0.001), Hex1 (0.77-fold, p < 0.01), and Pdk4 (0.47-fold, p < 0.001). This was accompanied by reduced glucose uptake (0.38-fold, p < 0.05). Subsequent mitochondrial function assessment revealed increased ATP-linked respiration (1.29-fold, p < 0.05), spare respiratory capacity (1.59-fold, p < 0.01), and maximal respiration (1.52-fold, p < 0.001) in Ocn -/- versus WT VSMCs. Together these data suggest that OCN plays a crucial role in arterial calcification mediated by Wnt/ß-catenin signaling through reduced maximal respiration. Mitochondrial dynamics may therefore represent a novel therapeutic target for clinical intervention. © 2019 American Society for Bone and Mineral Research.

Autophagy as a novel therapeutic target in vascular calcification.

The autophagy pathway is a key regulator of cellular metabolism and homeostasis, and plays a critical role in maintaining normal vascular cell function. It is well recognised that autophagy can regulate endothelial cell homeostasis, vascular smooth muscle cell (VSMC) phenotype transition, and calcium (Ca2+) homeostasis in VSMCs. Emerging evidence has demonstrated that autophagy directly protects against vascular calcification (VC). Crosstalk between endosomes, dysfunctional mitochondria, autophagic vesicles and Ca2+ and phosphate (Pi) enriched matrix vesicles (MVs) may underpin the pathogenesis of VC. In this review, we summarize the current experimental evidence in understanding how autophagy maintains normal vascular cell function and its protective role against vascular calcification. We also discuss the underlying molecular and cellular mechanisms through which autophagy inhibits vascular calcification. Pharmacological modulation of autophagy may offer an exciting new strategy for the treatment of vascular calcification.

Spermine increases acetylation of tubulins and facilitates autophagic degradation of prion aggregates.

Autolysosomal dysfunction and unstable microtubules are hallmarks of chronic neurodegenerative diseases associated with misfolded proteins. Investigation of impaired protein quality control and clearance systems could therefore provide an important avenue for intervention. To investigate this we have used a highly controlled model for protein aggregation, an in vitro prion system. Here we report that prion aggregates traffic via autolysosomes in the cytoplasm. Treatment with the natural polyamine spermine clears aggregates by enhancing autolysosomal flux. We demonstrated this by blocking the formation of mature autophagosomes resulting in accumulation of prion aggregates in the cytoplasm. Further we investigated the mechanism of spermine's mode of action and we demonstrate that spermine increases the acetylation of microtubules, which is known to facilitate retrograde transport of autophagosomes from the cellular periphery to lysosomes located near the nucleus. We further report that spermine facilitates selective autophagic degradation of prion aggregates by binding to microtubule protein Tubb6. This is the first report in which spermine and the pathways regulated by it are applied as a novel approach towards clearance of misfolded prion protein and we suggest that this may have important implication for the broader family of protein misfolding diseases.

Insights into pancreatic ß cell energy metabolism using rodent ß cell models.

Background: Mitochondrial diabetes is primarily caused by ß-cell failure, a cell type whose unique properties are important in pathogenesis. Methods: By reducing glucose, we induced energetic stress in two rodent ß-cell models to assess effects on cellular function. Results: Culturing rat insulin-secreting INS-1 cells in low glucose conditions caused a rapid reduction in whole cell respiration, associated with elevated mitochondrial reactive oxygen species production, and an altered glucose-stimulated insulin secretion profile. Prolonged exposure to reduced glucose directly impaired mitochondrial function and reduced autophagy. Conclusions: Insulinoma cell lines have a very different bioenergetic profile to many other cell lines and provide a useful model of mechanisms affecting ß-cell mitochondrial function.

Dysregulated mitophagy and mitochondrial organization in optic atrophy due to OPA1 mutations.

OBJECTIVE: To investigate mitophagy in 5 patients with severe dominantly inherited optic atrophy (DOA), caused by depletion of OPA1 (a protein that is essential for mitochondrial fusion), compared with healthy controls. METHODS: Patients with severe DOA (DOA plus) had peripheral neuropathy, cognitive regression, and epilepsy in addition to loss of vision. We quantified mitophagy in dermal fibroblasts, using 2 high throughput imaging systems, by visualizing colocalization of mitochondrial fragments with engulfing autophagosomes. RESULTS: Fibroblasts from 3 biallelic OPA1(-/-) patients with severe DOA had increased mitochondrial fragmentation and mitochondrial DNA (mtDNA)-depleted cells due to decreased levels of OPA1 protein. Similarly, in siRNA-treated control fibroblasts, profound OPA1 knockdown caused mitochondrial fragmentation, loss of mtDNA, impaired mitochondrial function, and mitochondrial mislocalization. Compared to controls, basal mitophagy (abundance of autophagosomes colocalizing with mitochondria) was increased in (1) biallelic patients, (2) monoallelic patients with DOA plus, and (3) OPA1 siRNA-treated control cultures. Mitophagic flux was also increased. Genetic knockdown of the mitophagy protein ATG7 confirmed this by eliminating differences between patient and control fibroblasts. CONCLUSIONS: We demonstrated increased mitophagy and excessive mitochondrial fragmentation in primary human cultures associated with DOA plus due to biallelic OPA1 mutations. We previously found that increased mitophagy (mitochondrial recycling) was associated with visual loss in another mitochondrial optic neuropathy, Leber hereditary optic neuropathy (LHON). Combined with our LHON findings, this implicates excessive mitochondrial fragmentation, dysregulated mitophagy, and impaired response to energetic stress in the pathogenesis of mitochondrial optic neuropathies, potentially linked with mitochondrial mislocalization and mtDNA depletion.

Autophagy limits proliferation and glycolytic metabolism in acute myeloid leukemia.

Decreased autophagy contributes to malignancies, however it is unclear how autophagy impacts on tumour growth. Acute myeloid leukemia (AML) is an ideal model to address this as (i) patient samples are easily accessible, (ii) the hematopoietic stem and progenitor population (HSPC) where transformation occurs is well characterized, and (iii) loss of the key autophagy gene Atg7 in hematopoietic stem and progenitor cells (HSPCs) leads to a lethal pre-leukemic phenotype in mice. Here we demonstrate that loss of Atg5 results in an identical HSPC phenotype as loss of Atg7, confirming a general role for autophagy in HSPC regulation. Compared to more committed/mature hematopoietic cells, healthy human and mouse HSCs displayed enhanced basal autophagic flux, limiting mitochondrial damage and reactive oxygen species in this long-lived population. Taken together, with our previous findings these data are compatible with autophagy limiting leukemic transformation. In line with this, autophagy gene losses are found within chromosomal regions that are commonly deleted in human AML. Moreover, human AML blasts showed reduced expression of autophagy genes, and displayed decreased autophagic flux with accumulation of unhealthy mitochondria indicating that deficient autophagy may be beneficial to human AML. Crucially, heterozygous loss of autophagy in an MLL-ENL model of AML led to increased proliferation in vitro, a glycolytic shift, and more aggressive leukemias in vivo. With autophagy gene losses also identified in multiple other malignancies, these findings point to low autophagy providing a general advantage for tumour growth.

Analyzing the Colocalization of MAP1LC3 and Lysosomal Markers in Primary Cells.

This technique evaluates the colocalization of the autophagy protein MAP1LC3 (microtubule-associated proteins 1A/1B light chain 3B, here referred to as LC3) with lysosomes (autolysosomes) in primary cells in a high-throughput manner. It uses an imaging fluorescence-activated cell sorting cytometer called the ImageStream to concomitantly detect surface molecules, making possible the identification of cells in mixed cell populations (e.g., in blood or bone marrow). It can be applied to clinical samples and to rare cell populations because only a few cells are needed for detection.

Techniques for the Detection of Autophagy in Primary Mammalian Cells.

Autophagy is a lysosomal catabolic pathway responsible for the degradation of cytoplasmic constituents. Autophagy is primarily a survival pathway for recycling cellular material in times of nutrient starvation, and in response to hypoxia, endoplasmic reticulum stress, and other stresses, regulated through the mammalian target of rapamycin pathway. The proteasomal pathway is responsible for degradation of proteins, whereas autophagy can degrade cytoplasmic material in bulk, including whole organelles such as mitochondria (mitophagy), bacteria (xenophagy), or lipids (lipophagy). Although signs of autophagy can be present during cell death, it remains controversial whether autophagy can execute cell death in vivo. Here, we will introduce protocols for detecting autophagy in mammalian primary cells by using western blots, immunofluorescence, immunohistochemistry, flow cytometry, and imaging flow cytometry.

Autophagy Controls Acquisition of Aging Features in Macrophages.

Macrophages provide a bridge linking innate and adaptive immunity. An increased frequency of macrophages and other myeloid cells paired with excessive cytokine production is commonly seen in the aging immune system, known as 'inflamm-aging'. It is presently unclear how healthy macrophages are maintained throughout life and what connects inflammation with myeloid dysfunction during aging. Autophagy, an intracellular degradation mechanism, has known links with aging and lifespan extension. Here, we show for the first time that autophagy regulates the acquisition of major aging features in macrophages. In the absence of the essential autophagy gene Atg7, macrophage populations are increased and key functions such as phagocytosis and nitrite burst are reduced, while the inflammatory cytokine response is significantly increased - a phenotype also observed in aged macrophages. Furthermore, reduced autophagy decreases surface antigen expression and skews macrophage metabolism toward glycolysis. We show that macrophages from aged mice exhibit significantly reduced autophagic flux compared to young mice. These data demonstrate that autophagy plays a critical role in the maintenance of macrophage homeostasis and function, regulating inflammation and metabolism and thereby preventing immunosenescence. Thus, autophagy modulation may prevent excess inflammation and preserve macrophage function during aging, improving immune responses and reducing the morbidity and mortality associated with inflamm-aging.

Transcriptional up-regulation of ULK1 by ATF4 contributes to cancer cell survival.

Hypoxia in the microenvironment of many solid tumours is an important determinant of malignant progression. The ISR (integrated stress response) protects cells from the ER (endoplasmic reticulum) stress caused by severe hypoxia. Likewise, autophagy is a mechanism by which cancer cells can evade hypoxic cell death. In the present paper we report that the autophagy-initiating kinase ULK1 (UNC51-like kinase 1) is a direct transcriptional target of ATF4 (activating transcription factor 4), which drives the expression of ULK1 mRNA and protein in severe hypoxia and ER stress. We demonstrate that ULK1 is required for autophagy in severe hypoxia and that ablation of ULK1 causes caspase-3/7-independent cell death. Furthermore, we report that ULK1 expression is associated with a poor prognosis in breast cancer. Collectively, the findings of the present study identify transcriptional up-regulation of ULK1 as a novel arm of the ISR, and suggest ULK1 as a potentially effective target for cancer therapy.

Tightrope act: autophagy in stem cell renewal, differentiation, proliferation, and aging.

Autophagy is a constitutive lysosomal catabolic pathway that degrades damaged organelles and protein aggregates. Stem cells are characterized by self-renewal, pluripotency, and quiescence; their long life span, limited capacity to dilute cellular waste and spent organelles due to quiescence, along with their requirement for remodeling in order to differentiate, all suggest that they require autophagy more than other cell types. Here, we review the current literature on the role of autophagy in embryonic and adult stem cells, including hematopoietic, mesenchymal, and neuronal stem cells, highlighting the diverse and contrasting roles autophagy plays in their biology. Furthermore, we review the few studies on stem cells, lysosomal activity, and autophagy. Novel techniques to detect autophagy in primary cells are required to study autophagy in different stem cell types. These will help to elucidate the importance of autophagy in stem cells during transplantation, a promising therapeutic approach for many diseases.

ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia.

Intratumoral hypoxia is associated with poor prognosis, regardless of the mode of therapy. Cancer cells survive this condition through activating several adaptive signaling pathways, including the integrated stress response (ISR) and autophagy. Activating transcription factor 4 (ATF4) is the major transcriptional mediator of the ISR, which we have shown to be involved in autophagy regulation to protect cells from severe hypoxia. Here we demonstrate that ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia. We find that the BH3-only proteins HRK, PUMA, and NOXA are transcriptionally induced in severe hypoxia and that their expression is abrogated by RNA interference against ATF4. In particular, we show that the BH3-only protein harakiri (HRK) is transactivated by ATF4 in severe hypoxia through direct binding of ATF4 to the promoter region. Furthermore, we demonstrate through siRNA knockdown that HRK induces autophagy and promotes cancer cell survival in severe hypoxia.

Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity.

Most autosomal genetic causes of childhood-onset hypogammaglobulinemia are currently not well understood. Most affected individuals are simplex cases, but both autosomal-dominant and autosomal-recessive inheritance have been described. We performed genetic linkage analysis in consanguineous families affected by hypogammaglobulinemia. Four consanguineous families with childhood-onset humoral immune deficiency and features of autoimmunity shared genotype evidence for a linkage interval on chromosome 4q. Sequencing of positional candidate genes revealed that in each family, affected individuals had a distinct homozygous mutation in LRBA (lipopolysaccharide responsive beige-like anchor protein). All LRBA mutations segregated with the disease because homozygous individuals showed hypogammaglobulinemia and autoimmunity, whereas heterozygous individuals were healthy. These mutations were absent in healthy controls. Individuals with homozygous LRBA mutations had no LRBA, had disturbed B cell development, defective in vitro B cell activation, plasmablast formation, and immunoglobulin secretion, and had low proliferative responses. We conclude that mutations in LRBA cause an immune deficiency characterized by defects in B cell activation and autophagy and by susceptibility to apoptosis, all of which are associated with a clinical phenotype of hypogammaglobulinemia and autoimmunity.

A novel method for autophagy detection in primary cells: impaired levels of macroautophagy in immunosenescent T cells.

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8(+) T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.