Role of autophagy in the regulation of epithelial cell junctions

ABSTRACT

Autophagy is a cell survival mechanism by which bulk cytoplasmic material, including soluble macromolecules and organelles, is targeted for lysosomal degradation. The role of autophagy in diverse cellular processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases is being increasingly recognized. Epithelial cell junctions play an integral role in the cell homeostasis via physical binding, regulating paracellular pathways, integrating extracellular cues into intracellular signaling, and cell-cell communication. Recent data indicates that cell junction composition is very dynamic. The junctional protein complexes are actively regulated in response to various intra- and extra-cellular clues by intracellular trafficking and degradation pathways. This review discusses the recent and emerging information on how autophagy regulates various epithelial cell junctions. The knowledge of autophagy regulation of epithelial junctions will provide further rationale for targeting autophagy in a wide variety of human disease conditions.

KEYWORDS:

• adherens junction
• autophagy
• endocytosis
• gap junction
• inflammatory bowel disease
• tight junction

Epithelial junctions

Cell junctions occur at points of cell-cell and cell-matrix contact in all tissues, particularly in epithelia. In vertebrates, 3 major types of cell junctions occur in the epithelia; tight junctions (TJ), adherens junctions (AJ), and gap junctions (GJ).¹ The most apically located inter-cellular TJs polarize the epithelial cell membrane into apical and basolateral regions (fence function) and regulate passive diffusion of solutes and macromolecules in between adjacent cells (gate function).² TJs consist of an array of membrane-spanning proteins such as occludin and claudins, linked by cytoplasmic plaque protein zona occludin to the cytoskeleton. The TJs act as a paracellular barrier and serve as a first line of defense against paracellular permeation of noxious antigens into the epithelia and subepithelial host tissue.^3,4 The TJs consists of structural barrier forming proteins such as occludin, and claudin−1, −4, −8 etc. as well as pore forming claudins such as claudin−2 and −15. Disturbances in this TJ composition play an important role in variety of diseases including inflammatory bowel diseases, kidney diseases such as hypomagnesemia and hypercalciuria, autoimmune diseases, dermal or retinal disease etc. Just below the TJs, AJs encircle interacting adjoining cells. In AJs, special proteins called cadherins form homodimers in the plasma membrane of opposite cell.^5,6 The intracellular domain of the cadherins binds to intracellular actin filaments via catenin anchor family proteins. The cadherin–β-catenin–α-catenin complex has been shown to interact with actin filaments via several actin binding proteins such as vinculin,⁷ formin,⁸ and EPLIN (epithelial protein lost in neoplasm; also known as Lima-1).⁹ Besides actin filaments, microtubules^10,11 and nectins, a family of immunoglobulin-like molecules¹² are known to regulate the assembly of AJs. In addition to being a mechanical linkage of neighboring cells to each other, AJ protein complexes are important regulators of morphogenesis, contact inhibition, proliferation, and are key sensors and transmitters of the extracellular cues converging into intracellular signaling.¹³ Below the AJs, the adjoining cells communicate via GJs that are made up of intercellular channels formed by 6 subunits of connexins that are bundled together in homomeric or heteromeric fashion. GJs allow exchange of small metabolites, ions, and second messengers between adjacent cells that is essential for physiological functions such as cell synchronization, differentiation, cell growth, and metabolic coordination of avascular organs including epidermis and lens.^14,15

Junctional homeostasis through constant protein turnover

The dynamic nature of cell junctions necessitates continuous endocytosis and recycling of junctional proteins to-and-from the membrane. The endocytosed junctional proteins, under various physiological and pathological conditions, may be diverted to the degradation pathway. Emerging evidence shows that TJs are highly interactive and dynamic in nature and intracellular vesicular membrane transport is a key process in the formation of TJ domains.^16,17 The TJ complex undergoes constant remodeling and intracellular trafficking where TJ proteins are continuously inserted and internalized from the membrane.¹⁸ AJs, too, are regulated by intracellular trafficking in addition to transcriptional and posttranscriptional regulation. Following disruption of AJs, such as after extra cellular calcium depletion, E-cadherins are rapidly internalized.^19,20 Interactions with β-catenin, p120-catenin, and nectins are important for stabilizing E-cadherins at the AJs.^21,22 In GJs, the connexin hemichannels called connexons are carried from ER to the cell surface via microtubule aided vesicles. The hemichannels fuse in homotypic or heterotypic fashion to form intercellular channels. From the GJ plaque of intercellular channels, old channels are removed from the center and new channels are added to the periphery in highly dynamic events. This rapid and constant synthesis and degradation of connexins at the GJs may offer quick adaptation of tissues to the changing environmental conditions.²³

Autophagy functions

Autophagy, an intracellular degradation pathway, refers to the engulfment and processing of cellular proteins, including damaged organelles and long-lived and misfolded proteins. Among the 3 main types of autophagy, ‘microautophagy’ involves the engulfment of cytoplasmic material that is in direct contact with the lysosomal membrane while in ‘chaperone-mediated autophagy’ cargo proteins must be recognized by the Hsc70 chaperone before being translocated into the lysosomal interior. “Macroautophagy” (commonly referred as autophagy) is characterized by sequestration of structures intended for destruction into double-membrane vesicles called autophagosomes²⁴ and is the subject of this review. The kinase mammalian target of rapamycin (mTOR) is a critical regulator of autophagy induction; activated mTOR (presence of growth factors and abundant nutrients, Akt, and MAPK signaling) suppresses autophagy while negative regulation of mTOR (nutrient starvation, stress, AMPK, and p53 signaling) induces autophagy. Downstream of mTOR, several autophagy related ATG proteins participate in ubiquitin-like conjugation reactions to control the autophagosome formation. The sequential events of vesicle nucleation (formation of the isolation membrane/phagophore), vesicle elongation and completion, and fusion of the double-membraned autophagosome with the lysosome to form an autolysosome ultimately culminate into the lysis of the autophagosomal inner membrane and breakdown of its contents inside the autolysosome.^25,26 An essential cell survival mechanism, autophagy plays an important role in diverse processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases.^25-30 The main cellular functions of autophagy includes energy and glucose metabolic adaptation to stress, elimination of excess or unneeded organelles, tumor suppressor function, and elimination of intracellular pathogens.³¹ Besides these conventional functions, autophagy proteins have recently been shown to participate in extracellular secretion of biomolecules and cytokines,^32,33 osteoclastic bone resorption,³⁴ regulation of tumor microenvironment through metabolic coupling,^35,36 and other autophagy-independent functions such as innate viral immune response, mitochondrial homeostasis, and cell death.²⁴ In this review, we examine the potential role autophagy plays in the regulation of cell junctions.

Autophagy regulation of tight junction

In a recent study, we have shown that autophagy profoundly enhances TJ barrier function.³⁷ The TJs are known to have 2 pathways, a small-size, cation-selective, high-capacity “pore” pathway and a large-size, non-charge-selective “leak” pathway. The pore pathway is largely regulated by the TJ membrane proteins claudins, particularly claudin-2, which is a cation-selective, pore-forming protein while leak pathway is regulated by barrier forming occludin.³⁸ Induction of autophagy in human intestinal Caco-2 cells by starvation or mTOR inhibitors rapamycin and PP242 treatment caused reduction in ion permeability as reflected by increased transepithelial resistance (TER) and reduced small-size urea permeability, indicating selective autophagy targeting of the pore pathway (Fig. 1). Furthermore, the levels of the pore forming claudin-2 were reduced markedly after starvation or rapamycin treatment and claudin-2 was increasingly targeted for the lysosomal degradation. Pharmacologic inhibition of autophagy with bafilomycin A (V-ATPase inhibitor that impedes autolysosome formation),³⁹chloroquine (inhibitor of lysosomal acidification),⁴⁰ and wortmannin (inhibitor of class III PI3K required to initiate autophagy)⁴¹ as well as genetic inhibition of autophagy with ATG16L1 and ATG7 siRNA prevented the starvation or rapamycin induced increase in TER, reduction in urea flux, and degradation of claudin-2.³⁷ These findings demonstrated that autophagy induction led to an enhancement of TJ barrier function via claudin-2 degradation. Similarly, in primary porcine intestinal epithelial cells also, autophagy played a critical role in TJ barrier function. Non-essential amino acid (NEAA) deprivation induced TJ barrier dysfunction via reduction in expression of TJ protein claudin-1 and ZO-1. Inhibition of autophagy enhanced Non-essential amino acid (NEAA) deprivation-induced TJ barrier dysfunction whereas activation of autophagy by rapamycin was protective against NEAA deprivation-induced TJ barrier dysfunction.⁴² In mice stomach, the oxidative stress induced by hydrogen peroxide (H₂O₂) disrupted gastric TJ barrier, reduced the levels of TJ protein occludin and ZO-1, and inhibited autophagy. However, treatment with reactive oxygen species scavenger PDTC (Ammonium pyrrolidinedithiocarbamate) restored the H₂O₂-induced disruption of gastric TJ barrier and also increased the conversion of LC3I into LC3II, suggesting synergistic association between autophagy and TJ barrier enhancement.⁴³ Autophagy also plays a role in the TJ function in blood-brain barrier. In an ischemia-reperfusion (I/R) model, oxygen-glucose deprivation/reoxygenation (OGD/R) injury in brain microvascular endothelial cell (BMVEC) caused apoptosis, high levels of reactive oxygen species, and reduction in the level and redistribution of ZO-1. Autophagy induction by rapamycin and lithium carbonate attenuated these adverse effects induced by OGD/R. Rapamycin and lithium carbonate pretreatments also prevented evans blue extravasation and brain water content induced by OGD/R in the ischemic hemisphere of the rat.⁴⁴ In contrast, Macrophage migration inhibitory factor (MIF)-induced autophagy caused a degradation and disorganization of endothelial tight junctions resulting into vascular leakage in human microvascular endothelial cell line (HMEC-1).⁴⁵ Also, under prolonged ischemic conditions (OGD/R injury) in brain endothelial cells, endothelial barrier was disrupted in part by nitric oxide-induced caveolin-1-mediated trafficking of claudin-5 to the autophagosome for autophagy-lysosome-dependent degradation.⁴⁶ However, these effects of autophagy on the endothelial TJ barrier appear to be influenced by activation of different intracellular signaling pathways⁴⁵ and dependent on the cell type and the extent of injury. Synergistic association between autophagy and improvement of epithelial TJ barrier has been demonstrated in a diabetic retinopathy model where hyperglycemia and hypoxia caused disruption of claudin-1 and ZO-1 localization in retinal pigment epithelial cells. Fenofibric acid treatment which is known to reduce the progression of diabetic retinopathy in clinical trials, downregulated stress-mediated signaling, induced autophagy, and prevented TJ barrier disruption induced by hyperglycemia and hypoxia.⁴⁷ Overall, autophagy appears to be critical for homeostasis of the epithelial TJ barrier.

Figure 1. Autophagy enhances the epithelial barrier function. Filter-grown intestinal Caco-2 monolayers were incubated in starvation medium (Earle's balanced salt solution) to induce autophagy. Starvation significantly increased transepithelial resistance (TER) and progressively reduced urea flux (^#, *, p < 0.01 vs. time 0). Adapted from Nighot et. al., 2015.

Autophagy regulation of adherens junction

The interplay between autophagy and AJs is exemplified by the classical Wnt-induced β-catenin signaling. The complex of β-catenin-E-cadherin present normally at the plasma membrane is destabilized during aberrant proliferative signaling events such as Wnt-induced β-catenin signaling in undifferentiated acute myeloid leukemia (AML) cells. The independent and constitutively active β-catenin blocked autophagy and promoted proliferation.⁴⁸ However, treatment with lectin LecB was found to degrade β-catenin, allowing induction of autophagy and suppress deregulated proliferation of leukemia THP-1 cells.⁴⁸ Autophagy is also known to contribute to therapy-induced degradation of the PML/RARA oncoprotein, allowing terminal differentiation of immature precursor cells and suppress deregulated proliferation of myeloid cells.⁴⁹ Moreover, the reduced levels of β-catenin also sensitized leukemia THP-1 cells to mTOR inhibitors, suggesting a potential solution for the low clinical benefit of mTOR inhibitors in AML patients.⁵⁰ The autophagy protein LC3 also directly interacts with β‐catenin via W/YXXI/L motif and LC3‐interacting region (LIR) in β‐catenin, and targets β‐catenin for autophagic degradation.⁵¹ Autophagy is furthermore known to down-regulate SNAIL and SLUG, 2 key transcription repressor factors that regulate cadherin expression during epithelial-mesenchymal transition (EMT) process. This autophagy-mediated upregulation of N- and R-cadherins transcription and translation reduces migration and invasion abilities of glioblastoma cells by reversing EMT process.⁵² Conversely, autophagy deficiency promotes cell migration, invasion, and proliferation via reduction in E-cadherin expression. Under autophagic deficiency, autophagosomal degradation of E-cadherin transcriptional repressor Twist1 was inhibited; further the accumulated autophagy substrate p62 (SQSTM1) stabilizes Twist1 via binding to its ubiquitin-associated domain and causes reduction in E-cadherin expression.⁵³ Thus while autophagy helps maintain E-cadherin expression and thus optimum cellular growth, β-catenin acts as a converging point between proliferative β-catenin/Wnt signaling and autophagy.⁵¹

Autophagy regulation of gap junction

Autophagy plays an important role in the degradation of gap junctions during their rapid turnover. In a complex process, internalization of GJ occurs in form of a peculiar double membrane structure called annular gap junction (AGJ) which are constitutively degraded via autophagy.^54,55 Autophagy proteins associated with autophagosome formation and maturation, ATG5 and LC3, has been shown to be localized to AGJ⁵⁶ and the autophagy receptor p62 has been found to be co-localized with connexin isotypes, Cx50 and Cx43.⁵⁷ Following traumatic brain injury (TBI), autophagy inhibition caused increased levels of phosphorylated connexin 43 (pCx43) in hippocampal astrocytes, indicating autophagy based degradation of GJs.⁵⁸ A single or several AGJs can be engulfed simultaneously within an individual autophagosome.⁵⁴ Furthermore, plasma-membrane bound connexin proteins have been shown to be involved in early biogenesis of autophagosome. Connexins form a constitutive complex with autophagy-related proteins involved in the initial steps of autophagosome formation, such as ATG16 and components of the PI(3)K autophagy initiation complex (Vps34, Beclin-1 and Vps15). Following autophagy stimulus, ATG14 joins the connexin–ATG complex leading to its internalization. The connexin containing pre-autophagosomal complex further matures into autophagosomes permitting sustained activation of autophagy.⁵⁹ Over all, this involvement of connexins in the early biogenesis of autophagosomes complements cell's efficiency to counteract the stressful conditions by modulation of cell communication via autophagy-mediated degradation of GJ and possibly the larger hazardous molecules entrapped in the internalized AGJ vesicle lumen.⁶⁰

Importance of the context for autophagy regulation of cell junctions

Whether autophagy is beneficial or harmful for the host is a highly debated question. Critical evaluation of the role of autophagy in the organization and function of cell junctions is needed. This is warranted due to the fact that autophagy is very dynamic, multistep, cell specific process, the outcome of which is dependent on the substrate that is being degraded and the context of autophagic flux.⁶¹ The effect of autophagy on cell junctions may have untoward outcomes. For instance, in hypoxic melanoma cells, autophagy induction destabilizes immune synapses by increasing degradation of gap-junctional connexin Cx43, thus impairing NK cell-mediated killing. Conversely, genetic or pharmacological autophagy inhibition stabilizes immune synapses by increasing the presence of Cx43 within the immune synapses and improves NK cell-mediated lysis of hypoxic melanoma cells.⁶² In another instance, autophagy is rapidly up-regulated in necrotizing enterocolitis (NEC) mice model. Under this situation, erythropoietin, a breast milk component that downregulates autophagy via the Akt/mTOR signaling pathway has been shown to preserve the intestinal barrier function and decrease the incidence of NEC.⁶³ The extent of autophagy induction is also a deciding factor for the outcome of autophagy induction. Among the 2 mTOR complexes that regulate autophagy, mTORC1 and mTORC2, sustained AMP-activated protein kinase (AMPK) activation of ERK MAPK has been shown to cause disassembly of mTORC2 via marked increase in beclin 1 expression, leading to cytodestructive autophagy.⁶⁴ In this context, mTORC2 assembly is known to regulate actin cytoskeleton and localization of TJ proteins occludin and ZO-1 via PKC-α and small GTPase Rac1.^65,66 Further, the substrate specific outcome of autophagy induction is illustrated by our recent study in Madin-Darby Canine Kidney MDCK cells. MDCK cells have 2 distinct strains based in part on the claudin-2 expression. MDCK I cells are claudin-2 deficient and therefore have very high TER whereas MDCK II cells have high claudin-2 expression and have low TER.^67,68 Autophagy induction by starvation or mTOR inhibitors rapamycin and PP242 treatment enhanced TJ barrier in MDCK II cells by markedly increasing TER, decreasing urea flux, and reducing claudin-2 protein levels. This autophagy-induced enhancement of TJ barrier was however not evident in claudin-2 deficient MDCK I cells.³⁷ Autophagy regulators may also have autophagy-independent role in TJ barrier function. For instance, AMPK is involved in cytokine-induced TJ barrier modulation, independent of intracellular energy levels.^69,70

Mechanisms intersecting autophagy and junction protein trafficking

In the autophagy regulation of cell junctions, which are located at the plasma membrane, the crosstalk between autophagy machinery and the endocytic machinery is a crucial step. Autophagy is known to regulate the key modes of endocytosis of junctional proteins. In case of clathrin-mediated endocytosis, various clathrin adaptor protein (AP) complexes (AP-1, −2, and −3) connect cargo proteins to clathrin coat and accessory proteins that regulate coat assembly and disassembly. AP2 complex is involved in membrane protein endocytosis and has been recently shown to function as an LC3 receptor, which shuttles endocytic cargo to autophagosomes.⁷¹ TJ protein claudin-2 has been shown to be endocytosed via clathrin pits that are positive for adaptin α, a key constituent of AP-2 complex⁷² while AJ protein E-cadherin is endocytosed via clathrin in a SMAP1, a clathrin-interacting GTPase-activating protein (GAP) dependent manner.⁷³ Clathrin pits are also implicated in the autophagy mediated connexin Cx31.1 degradation.⁷⁴ Autophagy machinery also regulates caveolae. Under basal conditions, caveolin-1 (Cav-1) binds to ATG5 and ATG12 in the cytosol while formation of ATG5-ATG12 complex during starvation leads to rapid dissociation of Cav-1 from ATG5-ATG12 conjugates.⁷⁵ Moreover, sustained autophagy is known to reduce Cav-1 mRNA level.⁷⁶ Cav-1 deficiency also promotes both basal and inducible autophagy, particularly by increasing lysosomal function and autophagosome-lysosome fusion as a cell survival mechanism under starvation.⁷⁷ TJ protein occludin,^78-80 AJ protein β-catenin,^81,82 and GJ protein connexins⁸³ are known to be associated with caveolins. Macropinocytosis is another mode of vesicular trafficking of junctional proteins⁸⁴ that is regulated by autophagy. LC3 (Light chain 3), a component of autophagosomes, is recruited to macropinosomes via lipidation by ATG5 and ATG7, and the class III phosphatidylinositol-3-kinase VPS34, facilitating lysosomal degradation of internalized cargo.⁸⁵

The autophagy regulation of endocytic processes assumes greater relevance in the context of the larger question about the origin of the autophagosomal membrane. Based upon the location of enrichment of scaffolding ATG proteins with specific lipid-modifying enzymes that promotes autophagosomal membrane formation,⁸⁶ the origin of the autophagosomal membrane has been traced to the endoplasmic reticulum,⁸⁷ mitochondria,⁸⁸ and plasma membrane.⁸⁹ In case of plasma membrane-originated autophagosomal membrane, the contribution of endocytic clathrin machinery for the assembly of the autophagosome-limiting membranes has already been demonstrated.⁸⁹ The key questions that need to be answered include the following: what kind of autophagic stimulus dictates plasma membrane-derived autophagosome formation and how specific junctional proteins are targeted to the autophagosomes assuming they are not moved passively along with the plasma membrane fractions. One such clue has already been revealed regarding the autophagy-mediated degradation of gap junctions. E3 ubiquitin-protein ligase Nedd4-mediated ubiquitinylation of the connexin molecule and recruitment of the adaptor protein Eps15 to the GJ has been shown to be required for the initiation of autophagy-dependent internalization and degradation of connexin 43.⁹⁰

Autophagy, Tight junctions, and inflammatory bowel disease

Defects in intestinal TJ barrier,^4,72,73 AJ,⁹¹ and GJ⁹² are associated with intestinal disorders and inflammatory bowel diseases (IBD) including Crohn disease and ulcerative colitis. The defects in intestinal TJ barrier allows increased antigenic penetration, resulting in amplified inflammatory response in intestinal tissue.^4,93 Genome wide association studies have identified mutations in autophagy related genes ATG16L1 and IRGM as substantiated risk factors for Crohn disease (CD).^94-96 Recent studies have also shown the role of autophagy in dendritic-epithelial cell interactions, NOD2 directed bacterial sensing, and immune mediated clearance which is considered to be important in pathogenesis of IBD.^27-30 In mice, Atg4b-deficiency caused increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with alterations in pro-inflammatory cytokine profiles and Paneth cell abnormalities.⁹⁷ On the other hand, autophagy enhancement ameliorated intestinal inflammation in IL-10 knockout mouse model of spontaneous enterocolitis.^98,99 Clinical data and animal studies show a direct link between defective intestinal TJ barrier and intestinal inflammation in IBD patients and animal models of IBD.^100-102 Previous study from our laboratory showed that starvation-induced autophagy reduced TJ permeability of the intestinal epithelial cells, indicating a role for autophagy in the maintenance of intestinal barrier function.³⁷ Moreover, we have observed that acute deletion of Atg7, which is critical for the autophagy conjugation system and formation of autophagosomes,¹⁰³ in adult mice exacerbate DSS-induced increase in colonic TJ permeability, disease activity, and colonic inflammation (unpublished data). Thus the role of autophagy in the maintenance of intestinal TJ barrier appears to be a critical mechanism in prevention of intestinal inflammation.

Perspective

In summary, various autophagic responses regulate epithelial junctions to maintain cell homeostasis (Fig. 2). Autophagy enhances TJ barrier function and reduces paracellular permeability by modulating TJ protein composition. At AJ, autophagy promotes E-cadherin expression and regulates cell proliferation and migration. Sustained autophagy also regulates cell growth and proliferation via degradation of AJ protein β-catenin. Autophagy-mediated connexin (GJ) degradation helps cell adaptation in the event of stress. These autophagic responses however may have context dependent outcomes and not globally uniform consequences. The role of autophagy in epithelial cell junction regulation has been deduced using pharmacological or genetic modulation of ATG proteins in stress or injury models. Further studies are warranted to identify specific autophagy regulators of epithelial cell junctions. The advances in the understanding of the intricate role of autophagy in the regulation of epithelial junctions will help rationalize and improve therapeutic efforts toward the clinical problems ranging from diabetic retinopathy, edema, jaundice, and diarrhea to autoimmune diseases, cancer metastasis, and infectious diseases.

Figure 2. Summary of the cell homeostatic effects of autophagy on the epithelial cell junctions. Various positive or inhibitory regulators of authophagic effects on cell junctions are listed. The backdrop shows transmission electron micrograph of mouse small intestinal tight junction and adherens junction.

Abbreviations

AGJ	=	annular gap junction
AJ	=	adherens junction
Akt	=	Protein kinase B
AML	=	acute myeloid leukemia
AMPK	=	5′ adenosine monophosphate-activated protein kinase
BMVEC	=	brain microvascular endothelial cell
Cav-1	=	caveolin1
CD	=	Crohn disease
Eps15	=	Epidermal growth factor receptor substrate 15
GJ	=	gap junction
HMEC	=	human microvascular endothelial cell line
IBD	=	inflammatory bowel disease
LC3	=	light chain 3
MAPK	=	Mitogen-activated protein kinases
MIF	=	macrophage migration inhibitory factor
Mtor	=	mammalian target of rapamycin
NEAA	=	non-essential amino acids
NEC	=	necrotizing enterocolitis
OGD/R	=	oxygen-glucose deprivation/reoxygenation
SMAP1	=	Stromal Membrane-Associated Protein 1
SQSTM1	=	Sequestosome 1
TJ	=	tight junction
TER	=	transepithelial resistance

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This research work has been supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants (to T. Ma) and K01-DK-100562-02 (to P. Nighot) and a Veterans Affairs (VA) Merit Review grant from the VA Research Service (to T. Ma).