Mitochondrial metabolism is essential for invariant natural killer T cell development and function

Conventional T cell fate and function are determined by coordination between cellular signaling and mitochondrial metabolism. Invariant natural killer T (iNKT) cells are an important subset of “innate-like” T cells that exist in a preactivated effector state, and their dependence on mitochondrial metabolism has not been previously defined genetically or in vivo. Here, we show that mature iNKT cells have reduced mitochondrial respiratory reserve and iNKT cell development was highly sensitive to perturbation of mitochondrial function. Mice with T cell-specific ablation of Rieske iron-sulfur protein (RISP; T-Uqcrfs1^−/−), an essential subunit of mitochondrial complex III, had a dramatic reduction of iNKT cells in the thymus and periphery, but no significant perturbation on the development of conventional T cells. The impaired development observed in T-Uqcrfs1^−/− mice stems from a cell-autonomous defect in iNKT cells, resulting in a differentiation block at the early stages of iNKT cell development. Residual iNKT cells in T-Uqcrfs1^−/− mice displayed increased apoptosis but retained the ability to proliferate in vivo, suggesting that their bioenergetic and biosynthetic demands were not compromised. However, they exhibited reduced expression of activation markers, decreased T cell receptor (TCR) signaling and impaired responses to TCR and interleukin-15 stimulation. Furthermore, knocking down RISP in mature iNKT cells diminished their cytokine production, correlating with reduced NFATc2 activity. Collectively, our data provide evidence for a critical role of mitochondrial metabolism in iNKT cell development and activation outside of its traditional role in supporting cellular bioenergetic demands.

Cellular metabolic pathways are interwoven with traditional signaling pathways to regulate the function and differentiation of T cells (1–3). Upon activation, effector T cells display a marked increase in glycolytic metabolism even in the presence of ample oxygen, termed aerobic glycolysis (4). We have previously shown that despite increased aerobic glycolysis, T cell activation depends on mitochondrial metabolism for generation of reactive oxygen species (ROS) for signaling (5). As activated T cells progress to a memory or regulatory phenotype, they preferentially oxidize fatty acids to support mitochondrial metabolism, and enhanced fatty acid oxidation (FAO) and spare respiratory capacity (SRC) are essential to maintenance of their phenotype (6, 7).CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of lymphocytes that exhibit a preactivated phenotype with rapid effector responses (8, 9). iNKT cells are capable of producing large amount of proinflammatory and antiinflammatory cytokines thus have broad immunomodulatory roles (8–10). Given that these cells are poised for rapid proliferation and cytokine production, we hypothesized that coordination of cellular signaling with cellular metabolism will be especially critical for optimal iNKT function. In support of this hypothesis, several studies suggest that modulation of cellular metabolism affects iNKT cell development and function. iNKT cell development is diminished upon deletion of the miR-181 a1b1 cluster, which regulates phosphoinositide 3-kinase signaling and decreases aerobic glycolysis (11, 12). In addition, T cell-specific deletion of Raptor (a component of mTORC1), a metabolic regulator, leads to defects in iNKT cell development and function (13, 14). Loss of folliculin-interacting protein 1 (Fnip1), an adaptor protein that physically interacts with AMP-activated protein kinase, also results in defective NKT cell development, and interestingly conventional T cells develop normally (15). Furthermore, a number of studies targeting bioenergetics processes or related molecules, like alteration of glucose metabolism, mitochondrial-targeted antioxidant treatment, and receptor-interacting protein kinase 3-dependent activation of mitochondrial phosphatase, showed significant effects on iNKT cell ratio and function (16–19). A recent study showed that iNKT cells are less efficient in glucose uptake than CD4⁺ T cells. Furthermore, activated iNKT cells preferentially metabolize glucose by the pentose phosphate pathway and mitochondria, instead of converting into lactate, since high lactate environment is detrimental to their homeostasis and effector function (20).In conventional lymphocytes, mitochondria clearly play a role in coordination of cell signaling and cell fate decisions outside of production of energy (5, 21–23). During T cell activation mitochondria localize at immune synapses that T cells form with antigen-presenting cells (22). T cell receptor (TCR) stimulation triggers mitochondrial ROS (mROS) production as well as mitochondrial ATP production that are released at the immune synapses and are critical for Ca²⁺ homeostasis and modulation of TCR-induced downstream signaling pathways (22). We previously showed that mice with T-cell–specific deletion of Rieske iron sulfur protein (RISP), a component of mitochondrial complex III of the mitochondrial electron transport chain (ETC), are defective in antigen-specific T cell activation due to deficiency of mROS required for cellular signaling (5). Several recent studies showed that ROS or factors that affect ROS production are also important in iNKT cell development and effector functions (24–27). In addition, inhibition of mitochondrial oxidative phosphorylation (OXPHOS) by oligomycin has been shown to decreased survival and cytokine production by splenic iNKT cells (20). However, the requirement of mitochondrial metabolism for iNKT cell development and function has not been previously defined genetically or in vivo.Here we showed that iNKT cells have comparable basal mitochondrial oxygen consumption to conventional T cells but displayed lower SRC and FAO, which are thought to impart cells with mitochondrial reserve under stress. Using Uqcrfs1^fl/fl;CD4-Cre⁺ (hereafter referred as T-Uqcrfs1^−/−) mice, we showed that abrogation of mitochondrial metabolism resulted in a cell-autonomous defect in iNKT cell development in thymus and periphery. The iNKT cells were able to proliferate but exhibited impaired activation, suggesting that they were not lacking bioenergetically but rather had aberrant TCR signaling in vivo, leading to altered expression of downstream factors required for their terminal maturation. Accordingly, T-Uqcrfs1^−/− iNKT cells displayed lower T-bet and CD122 levels and did not respond to interleukin (IL)-15 stimulation. Knockdown of RISP in mature iNKT cells also limited NFATc2 translocation to the nucleus. Collectively, our data highlighted an important role of mitochondrial metabolism in modulating TCR signaling in vivo and regulating iNKT cell development and function.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

Daily mitochondrial dynamics in cone photoreceptors

Cone photoreceptors in the retina are exposed to intense daylight and have higher energy demands in darkness. Cones produce energy using a large cluster of mitochondria. Mitochondria are susceptible to oxidative damage, and healthy mitochondrial populations are maintained by regular turnover. Daily cycles of light exposure and energy consumption suggest that mitochondrial turnover is important for cone health. We investigated the three-dimensional (3D) ultrastructure and metabolic function of zebrafish cone mitochondria throughout the day. At night retinas undergo a mitochondrial biogenesis event, corresponding to an increase in the number of smaller, simpler mitochondria and increased metabolic activity in cones. In the daytime, endoplasmic reticula (ER) and autophagosomes associate more with mitochondria, and mitochondrial size distribution across the cluster changes. We also report dense material shared between cone mitochondria that is extruded from the cell at night, sometimes forming extracellular structures. Our findings reveal an elaborate set of daily changes to cone mitochondrial structure and function.

Photoreceptor cells in the retina are highly metabolically active. Their energy demands change throughout the day to support phototransduction (1) and regeneration of outer segment (OS) disks (2). Photoreceptors consume more energy as ATP in darkness than in light (1) and some additional ATP comes from mitochondrial metabolism (3).Energy production can be influenced by mitochondrial fission, fusion, and new growth (mitogenesis). Smaller, fragmented mitochondria typically consume less oxygen (4). Mitogenesis is influenced by many factors, including circadian rhythms (5). In neurons mitogenesis can occur far away from the cell body (6). Mitochondria can form networks (7) and folds of cristae within mitochondria can be remodeled (8). These dynamic processes contribute to cell health; mitochondrial dysfunction is associated with neurodegenerative diseases (9–11), including retinal degeneration (12, 13).Over 90% of glucose taken up by photoreceptors is used for aerobic glycolysis (14, 15). Nevertheless, they have a large cluster of mitochondria in the apical portion of the inner segment, the ellipsoid, just below the OS. The density and organization of mitochondrial clusters vary among species, but they are present in photoreceptors of all vertebrates examined, including fish (16), ground squirrels (17), mice (18), and humans (19). In photoreceptors of some species, all mitochondria reside within the cluster, while in mammals with vascularized inner retinas mitochondria are also present at synaptic terminals (20, 21). In cultured chicken retinas, mitochondrial dynamics are circadian (22), but diurnal changes in mitochondrial structure and function in photoreceptors in intact eyes have not been explored. Daily exposure to sometimes intense light and high rates of energy production suggest that mitochondrial turnover may be important for photoreceptor health. Structural and functional changes to mitochondria could enable photoreceptors to meet increased ATP requirements in darkness.In this report we describe daily changes that occur in zebrafish cone photoreceptor mitochondria. Zebrafish provide a useful model to dissect mitochondrial dynamics in specific photoreceptor subclasses. Zebrafish undergo typical vertebrate behavioral and biological circadian rhythms (23, 24), and their retinas have four cone subtypes (red, green, blue, and ultraviolet [UV]) organized in a tiered, mosaic pattern. Each cone type can be identified by its unique morphology and position in the outer retina (25), and each maintains a large cluster of mitochondria just below the OS (16). Our results indicate that mitochondrial clusters in cones undergo diurnal remodeling consistent with enhanced energy production in darkness.     

Mitochondria-dependent synthetic small-molecule vaccine adjuvants for influenza virus infection

Vaccine adjuvants enhance and prolong pathogen-specific protective immune responses. Recent reports indicate that host factors—such as aging, pregnancy, and genetic polymorphisms—influence efficacies of vaccines adjuvanted with Toll-like receptor (TLR) or known pattern-recognition receptor (PRR) agonists. Although PRR independent adjuvants (e.g., oil-in-water emulsion and saponin) are emerging, these adjuvants induce some local and systemic reactogenicity. Hence, new TLR and PRR-independent adjuvants that provide greater potency alone or in combination without compromising safety are highly desired. Previous cell-based high-throughput screenings yielded a small molecule 81 [N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide], which enhanced lipopolysaccharide-induced NF-κB and type I interferon signaling in reporter assays. Here compound 81 activated innate immunity in primary human peripheral blood mononuclear cells and murine bone marrow-derived dendritic cells (BMDCs). The innate immune activation by 81 was independent of TLRs and other PRRs and was significantly reduced in mitochondrial antiviral-signaling protein (MAVS)-deficient BMDCs. Compound 81 activities were mediated by mitochondrial dysfunction as mitophagy inducers and a mitochondria specific antioxidant significantly inhibited cytokine induction by 81. Both compound 81 and a derivative obtained via structure–activity relationship studies, 2F52 [N-benzyl-N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] modestly increased mitochondrial reactive oxygen species and induced the aggregation of MAVS. Neither 81 nor 2F52 injected as adjuvants caused local or systemic toxicity in mice at effective concentrations for vaccination. Furthermore, vaccination with inactivated influenza virus adjuvanted with 2F52 demonstrated protective effects in a murine lethal virus challenge study. As an unconventional and safe adjuvant that does not require known PRRs, compound 2F52 could be a useful addition to vaccines.

Vaccine adjuvants enhance the immunogenic properties of antigens to enable enhanced and prolonged protective immune responses. Defined ligands for the pattern recognition receptors (PRRs)—including Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I like receptors (RLR), and stimulator of interferon genes (STING)—have been developed as adjuvants (1, 2). Although PRR-targeting adjuvants are well-tolerated and show promising protective efficacy, vaccination effectiveness is influenced by host factors causing variance in PRR signaling (e.g., aging, pregnancy, or genetic polymorphisms) (3). In the elderly population, activation of TLR signaling pathways is dysregulated, causing impaired activation or constant low-level activation, thereby reducing immune responses (4). Pregnant women are another vulnerable population where immunologic mechanisms that prevent fetal rejection can also lower the maternal response to vaccination. In addition to physiologic factors, genetic polymorphisms, particularly in TLRs and their adaptor proteins, have been reported to reduce responses to measles, meningococcal, and cytomegalovirus glycoprotein B vaccines (3, 5–7). To overcome variations in vaccine responses by these and other host considerations, adjuvants independent of known PRR should be reevaluated for vaccine development.Saponin and oil emulsions are TLR-independent vaccine adjuvants that have been recently approved by the Food and Drug Administration (FDA) (8, 9). Saponin adjuvants, including QS-21, induce strong humoral and T cell responses, and are FDA-approved for malaria and shingles vaccines (8–11). Saponin can disrupt the cell membrane, causing cytotoxicity, which can be reduced by proper formulation within liposomes or emulsions (12). The vaccines adjuvanted with QS-21 sometimes cause systemic adverse effects, including flu-like symptoms, fever, and malaise (13, 14). Oil-in-water adjuvants, including MF59 and AS03, are efficacious, but are also inflammatory in some recipients, inducing local swelling, pain, and fever (14–16). Therefore, new TLR-independent adjuvants with minimal if any local reactions are highly desirable.To seek additional adjuvants that would act independently or as a coadjuvant to enhance the response to monophosphoryl lipid A (MPLA) (9, 17), an FDA-approved TLR4 agonist, we previously performed a high-throughput screening (HTS) using interferon (IFN)-stimulated response element (ISRE) reporter cells. Compound 81 [N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] that belonged to bis-aryl sulfonamide scaffold was identified as a low molecular weight compound that prolonged activation of ISRE induced by type I IFN (18) and was also found to enhance lipopolysaccharide (LPS)-induced NF-κB responses (19). Further structure–activity relationship (SAR) studies identified the sites critical for immune-stimulatory properties of derivatives of 81. Unlike most PRR ligands, these compounds can be easily synthesized in one to two synthetic steps from readily available commercial reagents. Additionally, compound 81, when used as a coadjuvant with MPLA, showed significant enhancement in antigen-specific immunoglobulin responses compared to MPLA alone (19).This study examines the mechanisms that lead to the downstream signaling pathways and efficacy of the compound 81 scaffold (bis-aryl sulfonamide) as a vaccine adjuvant. We demonstrate that compound 81 and its derivative 2F52 [N-benzyl-N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] increase mitochondrial stress associated with mitochnodrial reactive oxygen species (mtROS) release, enhancing mitochondrial antiviral-signaling protein (MAVS)-dependent cytokine production, independent of known PRRs and inflammasomes. When used as an adjuvant with inactivated influenza A virus (IIAV) in a murine lethal influenza virus challenge model, the lead compound 2F52 protects mice from a lethal infection.     

Inositol hexakisphosphate kinase-2 determines cellular energy dynamics by regulating creatine kinase-B

Inositol hexakisphosphate kinases (IP6Ks) regulate various biological processes. IP6Ks convert IP6 to pyrophosphates such as diphosphoinositol pentakisphosphate (IP7) and bis-diphosphoinositol tetrakisphosphate (IP8). IP7 is produced in mammals by a family of inositol hexakisphosphate kinases, IP6K1, IP6K2, and IP6K3, which have distinct biological functions. The inositol hexakisphosphate kinase 2 (IP6K2) controls cellular apoptosis. To explore roles for IP6K2 in brain function, we elucidated its protein interactome in mouse brain revealing a robust association of IP6K2 with creatine kinase-B (CK-B), a key enzyme in energy homeostasis. Cerebella of IP6K2-deleted mice (IP6K2-knockout [KO]) produced less phosphocreatine and ATP and generated higher levels of reactive oxygen species and protein oxidative damage. In IP6K2-KO mice, mitochondrial dysfunction was associated with impaired expression of the cytochrome-c1 subunit of complex III of the electron transport chain. We reversed some of these effects by combined treatment with N-acetylcysteine and phosphocreatine. These findings establish a role for IP6K2–CK-B interaction in energy homeostasis associated with neuroprotection.

Inositol pyrophosphates are versatile messenger molecules that mediate a variety of cellular functions, including cell growth, apoptosis, endocytosis, and cell differentiation. The most extensively studied inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP7), displays a 5′-diphosphate (1, 2). IP7 is generated in mammals by a family of inositol hexakisphosphate kinases (IP6Ks) (3, 4). IP6Ks exists in three isoforms: IP6K1, IP6K2, and IP6K3. Inositol hexakisphosphate kinase-2 (IP6K2) sensitizes cells to apoptosis (5, 6). Mice with targeted deletion of IP6K2 display an increased incidence of aero-digestive tract carcinoma (7). Cell survival associated with heat shock protein 90 also involves IP6K2 (8, 9).We previously reported a major role for IP6K2 in the disposition of cerebellar granule cells as well as Purkinje cell morphology and motor coordination. The influence of IP6K2 upon cerebellar disposition involved protein 4.1N, both of which were highly expressed in cerebellar granule cells (10).To further assess the functions of IP6K2 in the brain, we explored its binding partners using coimmunoprecipitation and tandem liquid chromatography mass spectrometry (LC-MS/MS). Here, we report that IP6K2 robustly interacts with creatine kinase-B (CK-B), which regulates energy homeostasis of cells and exists in two forms, brain type (CK-B) and muscle type (CK-M). CK catalyzes the reversible transfer of the phosphate group of phosphocreatine to ADP to yield ATP (11, 12). A functional interplay between mitochondrial and cytosolic isoforms of CK regulates cellular energy homeostasis. Cytosolic CK rephosphorylates locally produced free ADP and increases creatine globally, while the mitochondrial enzyme catalyzes the conversion of creatine to phosphocreatine utilizing mitochondrial ATP (13–15).Here, we show that IP6K2 loss leads to decreased CK-B expression, reduced ATP levels, and diminished mitochondrial activity associated with increased oxidative stress. About 80 to 90% of ATP is generated in the mitochondria by oxidative phosphorylation, and diminished ATP levels are the immediate effect of mitochondrial dysfunction. Loss of IP6K2 and CK-B reflects the suppression of the mitochondrial cytochrome c1 expression, a component of complex III of the mitochondrial electron transport chain. In the present study, we report a physiologic association of CK-B and IP6K2, whose disruption impacts mitochondrial functions.Dendritic morphogenesis was reduced in IP6K2-deficient neurons and was rescued by restoring normal levels of ATP. These observations reveal an essential role of IP6K2 in the energy production of the brain. Our findings indicate that IP6K2 is a key regulator of mitochondrial homeostasis which promotes neuroprotection.     

Selective autophagy of AKAP11 activates cAMP/PKA to fuel mitochondrial metabolism and tumor cell growth

Autophagy is a catabolic pathway that provides self-nourishment and maintenance of cellular homeostasis. Autophagy is a fundamental cell protection pathway through metabolic recycling of various intracellular cargos and supplying the breakdown products. Here, we report an autophagy function in governing cell protection during cellular response to energy crisis through cell metabolic rewiring. We observe a role of selective type of autophagy in direct activation of cyclic AMP protein kinase A (PKA) and rejuvenation of mitochondrial function. Mechanistically, autophagy selectively degrades the inhibitory subunit RI of PKA holoenzyme through A-kinase–anchoring protein (AKAP) 11. AKAP11 acts as an autophagy receptor that recruits RI to autophagosomes via LC3. Glucose starvation induces AKAP11-dependent degradation of RI, resulting in PKA activation that potentiates PKA-cAMP response element-binding signaling, mitochondria respiration, and ATP production in accordance with mitochondrial elongation. AKAP11 deficiency inhibits PKA activation and impairs cell survival upon glucose starvation. Our results thus expand the view of autophagy cytoprotection mechanism by demonstrating selective autophagy in RI degradation and PKA activation that fuels the mitochondrial metabolism and confers cell resistance to glucose deprivation implicated in tumor growth.

Macroautophagy (henceforth autophagy) is a catabolic process that degrades various cellular cargos through lysosomes. The autophagy process includes the formation and trafficking of autophagosomes, which sequester the cellular cargos destined for the clearance. Autophagy is activated in response to nutrient deprivation or cellular injuries and serves as a recycling mechanism that maintains cellular homeostasis through degradation of cytoplasmic components. Autophagy provides cell self-nourishment and supports cellular metabolism by supplying breakdown products (1, 2); therefore, autophagy is a fundamental cell protection mechanism. Whether autophagy has a direct function beyond recycling of the breakdown molecules to maintain metabolic homeostasis and cell survival, however, is poorly understood. In certain cancer types, autophagy plays an important role in sustaining the aggressive growth of the tumor cells by enhancing cell metabolism. Although our group and others have previously shown an inhibitory function of Beclin 1–mediated autophagy in tumorigenesis (3, 4), the current view is that tumors, once established, rely heavily on autophagy to survive due to high metabolic demand. One potential mechanism is that the metabolic products generated by autophagy provide tumor cells with metabolic rewiring that enables them to survive even under nutrient-poor conditions (5, 6). However, it remains unclear whether autophagy plays a role beyond the production of metabolic fuel sources to maintain metabolic plasticity and tumor cell growth.Available evidence has demonstrated the selectivity of autophagy in the digestion of certain cellular cargoes mediated by autophagy adaptors/receptors. Characterization of the autophagy adaptors has shed light on the versatile physiological function of autophagy in the maintenance of the homeostasis for large molecules and cellular organelles (7, 8). These adaptors recognize and recruit selective cargos to autophagy machinery for degradation through direct interaction with yeast autophagy gene Atg8 homologs of mammalian LC3/GABARAP/Gate16 proteins. While a few autophagy receptors have been reported, it is clear that many more are yet to be identified (7, 8).The best-known signaling pathways that control the metabolic stress-induced autophagy are mediated by mTOR and AMPK kinases, both of which are the master regulators for cellular metabolism (9, 10). Cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is also a key kinase of cell metabolism that governs diverse cellular pathways, including cellular glucose metabolism and bioenergetic processes (11–13). Surprisingly, whether and how cAMP/PKA regulates autophagy or vice versa is poorly understood in mammals (14–16). cAMP/PKA signaling has emerged over recent years as a key regulator for mitochondrial functions, highlighting the mechanism of cAMP/PKA in cellular metabolism control (17, 18). Despite an established role for PKA in the regulation of mitochondrial metabolism, whether autophagy and PKA converge to regulate metabolic reprogramming and cell survival remains unknown.The PKA holoenzyme consists of two regulatory subunits (R) and two catalytic subunits (C). The R subunits are inhibitory of catalytic kinase activity; upon binding to cAMP, the R subunit dissociates from C subunit, resulting in activation of PKA (19). Furthermore, the specific cellular functions of PKA are controlled by a number of A-kinase–anchoring proteins (AKAPs). The AKAPs bind the R subunits and restrict the PKA holoenzyme to various intracellular compartments, providing spatiotemporal regulation of PKA activity (20). However, the functions of many AKAPs are poorly characterized.Here, we report a role of autophagy that controls cellular metabolism beyond the production of metabolic sources—it activates cAMP/PKA kinase activity by selective degradation of the inhibitory subunit of R1α through autophagy receptor AKAP11 in response to glucose starvation. AKAP11-mediated cAMP/PKA activation leads to elevation of mitochondrial metabolism and cell protection. Our study reveals a previously unrecognized function of autophagy in metabolic rewiring of cells that promote cell survival under energy crisis. Our study thus suggests that selective autophagy induced RI degradation and PKA activation may contribute to the resistance of tumor cells to metabolic stress.     

Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus–induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

Myopia (nearsightedness) in school-age children is generally axial myopia, which is the consequence of elongation of the eyeball along the visual axis. This shape change results in blurred vision but can also lead to severe complications including cataract, retinal detachment, myopic choroidal neovascularization, glaucoma, and even blindness (1–3). Despite the current worldwide pandemic of myopia, the mechanism of myopia onset is still not understood (4–8). One hypothesis that has earned a current consensus is the suggestion that a change in the lighting environment of modern society is the cause of myopia (9, 10). Consistent with this, outdoor activity has a protective effect on myopia development (9, 11, 12), though the main reason for this effect is still under debate (7, 12, 13). One explanation is that bright outdoor light can promote the synthesis and release of dopamine in the eye, a myopia-protective neuromodulator (14–16). Another suggestion is that the distinct wavelength composition of sunlight compared with fluorescent or LED (light-emitting diode) artificial lighting may influence myopia progression (9, 10). Animal studies have shown that different wavelengths of light can affect the development of myopia independent of intensity (17, 18). The effects appear to be distinct in different species: for chicks and guinea pigs, blue light showed a protective effect on experimentally induced myopia, while red light had the opposite effect (18–22). For tree shrews and rhesus monkeys, red light is protective, and blue light causes dysregulation of eye growth (23–25).It has been shown that visible violet light (VL) has a protective effect on myopia development in mice, in chick, and in human (10, 26, 27). According to Commission Internationale de l’Eclairage (International Commission on Illumination), VL has the shortest wavelength of visible light (360 to 400 nm). These wavelengths are abundant in outside sunlight but can only rarely be detected inside buildings. This is because the ultraviolet (UV)-protective coating on windows blocks all light below 400 nm and because almost no VL is emitted by artificial light sources (10). Thus, we hypothesized that the lack of VL in modern society is one reason for the myopia boom (9, 10, 26).In this study, we combine a newly developed lens-induced myopia (LIM) model with genetic manipulations to investigate myopia pathways in mice (28, 29). Our data confirm (10, 26) that visible VL is protective but further show that delivery of VL only in the evening is sufficient for the protective effect. In addition, we show that the protective effect of VL on myopia induction requires OPN5 (neuropsin) within the retina. The absence of retinal Opn5 prevents lens-induced, VL-dependent thickening of the choroid, a response thought to play a key role in adjusting the size of the eyeball in both human and animal myopia models (30–33). This report thus identifies a cell type, the Opn5 retinal ganglion cell (RGC), as playing a key role in emmetropization. The requirement for OPN5 also explains why VL has a protective effect on myopia development.     

ATP13A2-mediated endo-lysosomal polyamine export counters mitochondrial oxidative stress

Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson’s disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6. These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.

Loss-of-function mutations in ATP13A2 (PARK9) are causative for a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome (KRS, a juvenile onset parkinsonism with dementia) (1), early-onset Parkinson’s disease (PD) (2, 3), hereditary spastic paraplegia (HSP) (4), neuronal ceroid lipofuscinosis (5), and amyotrophic lateral sclerosis (6), which are commonly hallmarked by lysosomal and mitochondrial dysfunction (4, 6, 7). Also, ATP13A2 deficiency causes lysosomal and mitochondrial impairment in various models, as evidenced by decreased lysosomal functionality (8, 9), reduced mitochondrial clearance capacity (8–10), mitochondrial fragmentation, mitochondrial DNA damage, and increased oxygen consumption (11, 12).We recently discovered that ATP13A2 transports the polyamines spermidine and spermine from the late endo/lysosome to the cytosol (9). Polyamines are ubiquitous polycationic aliphatic amines that stabilize nucleic acids, influence protein folding, regulate ion channels, and modulate cell proliferation and differentiation (13–15). We found that the late endo-lysosomal transporter ATP13A2 strongly contributes to the total cellular polyamine content via a two-step process: Firstly, polyamines enter the cell via endocytosis and subsequently, polyamines are transported by ATP13A2 into the cytosol (9). This process complements polyamine biosynthesis via the ornithine decarboxylase (ODC) pathway (9). Importantly, ATP13A2’s polyamine transport function is crucial for its neuroprotective effect, since it prevents lysosomal polyamine accumulation and subsequent lysosomal rupture, while improving lysosomal health and functionality (9). Moreover, when activated by its two regulatory lipids—phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] and phosphatidic acid (PA)—ATP13A2 exerts a cell protective effect against the mitochondrial neurotoxin rotenone (16), an environmental risk factor for PD (17). Rotenone is a mitochondrial complex I inhibitor, which leads to high levels of reactive oxygen species (ROS), promoting protein aggregation and damaging organelles. However, how ATP13A2’s polyamine transport function exerts a cell protective effect against rotenone, or other mitochondrial neurotoxins, is not yet clear.Interestingly, the transported substrates spermine and spermidine reduce oxidative stress (14, 15). Spermine is a potent free radical scavenger (18) and a biologically important antioxidant (19–23). We therefore hypothesize that ATP13A2-mediated polyamine transport may counteract oxidative stress (16, 24) and preserve mitochondrial health (11, 12). Here, we demonstrate in complementary human cell models and Caenorhabditis elegans that lysosomal polyamine export by ATP13A2 effectively lowers ROS levels and promotes mitochondrial health and functionality, pointing to a lysosomal-dependent cell protective pathway that may be implicated in ATP13A2-related neurodegenerative disorders.     

Adipose tissue is a critical regulator of osteoarthritis

Osteoarthritis (OA), the leading cause of pain and disability worldwide, disproportionally affects individuals with obesity. The mechanisms by which obesity leads to the onset and progression of OA are unclear due to the complex interactions among the metabolic, biomechanical, and inflammatory factors that accompany increased adiposity. We used a murine preclinical model of lipodystrophy (LD) to examine the direct contribution of adipose tissue to OA. Knee joints of LD mice were protected from spontaneous or posttraumatic OA, on either a chow or high-fat diet, despite similar body weight and the presence of systemic inflammation. These findings indicate that adipose tissue itself plays a critical role in the pathophysiology of OA. Susceptibility to posttraumatic OA was reintroduced into LD mice using implantation of a small adipose tissue depot derived from wild-type animals or mouse embryonic fibroblasts that undergo spontaneous adipogenesis, implicating paracrine signaling from fat, rather than body weight, as a mediator of joint degeneration.

Osteoarthritis (OA) is the leading cause of pain and disability worldwide and is associated with increased all-cause mortality and cardiovascular disease (1, 2). OA is strongly associated with obesity, suggesting that either increased biomechanical joint loading or systemic inflammation and metabolic dysfunction related to obesity are responsible for joint degeneration (1, 2). However, increasing evidence is mounting that changes in biomechanical loading due to increased body mass do not account for the severity of obesity-induced knee OA (1–9). These observations suggest that other factors related to the presence of adipose tissue and adipose tissue-derived cytokines—termed adipokines—play critical roles in this process and other musculoskeletal conditions (1, 2, 6, 7, 10). As there are presently no disease-modifying OA drugs available, direct evidence linking adipose tissue and cartilage health could provide important mechanistic insight into the natural history of OA and obesity and therefore guide the development and translation of novel OA therapeutic strategies designed to preserve joint health.The exact contribution of the adipokine-signaling network in OA has been difficult to determine due to the complex interactions among metabolic, biomechanical, and inflammatory factors related to obesity (11). To date, the link between increased adipose tissue mass and OA pathogenesis has largely been correlative (6, 7, 12), and, as such, the direct effect of adipose tissue and the adipokines it releases has been difficult to separate from other factors such as dietary composition or excess body mass in the context of obesity, which is most commonly caused by excessive nutrition (2, 6, 7). In particular, leptin, a proinflammatory adipokine and satiety hormone secreted proportionally to adipose tissue mass is most consistently increased in obesity-induced OA (1), and leptin knockout mice are protected from OA (6, 7). However, it remains to be determined whether leptin directly contributes to OA pathogenesis, independent of its effect on metabolism (and weight). Additional adipokines that have been implicated in the onset and progression of OA include adiponectin, resistin, visfatin, chimerin, and inflammatory cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α) (13). The infrapatellar fat pad represents a local source of adipokines within the knee joint, but several studies indicate strong correlations with visceral adipose tissue, outside of the joint organ system, with OA severity (14). Furthermore, adipokine receptors are found on almost all cells within the joint and, therefore, could directly contribute to OA pathogenesis through synovitis, cartilage damage, and bone remodeling (13). The role of other adipokines (15) in OA pathogenesis remains to be determined, as it has been difficult to separate and directly test the role of adipokines from other biomechanical, inflammatory, and metabolic factors that contribute to OA pathogenesis.To directly investigate the mechanisms by which adipose tissue affects OA, we used a transgenic mouse with lipodystrophy (LD) that completely lacks adipose tissue and, therefore, adipokine signaling. The LD model system affords the unique opportunity to directly examine the effects of adipose tissue and its secretory factors on musculoskeletal pathology without the confounding effect of diet (16, 17). While LD mice completely lack adipose tissue depots, they demonstrate similar body mass to wild-type (WT) controls on a chow diet (12, 16–19). These characteristics provide a unique model that can be used to eliminate the factor of loading due to body mass on joint damage and, thus, to directly test the effects of fat and factors secreted by fat on musculoskeletal tissues. Of particular interest, LD mice also exhibit several characteristics that have been associated with OA, including sclerotic bone (11, 20), metabolic derangement (3, 5, 7–9, 21, 22), and muscle weakness (2). Despite these OA-predisposing features, LD mice are protected from OA and implantation of adipose tissue back into LD mice restores susceptibility to OA—demonstrating a direct relationship between adipose tissue and cartilage health, independent of the effect of obesity on mechanical joint loading.     

Spine dynamics of PSD-95-deficient neurons in the visual cortex link silent synapses to structural cortical plasticity

Critical periods (CPs) are time windows of heightened brain plasticity during which experience refines synaptic connections to achieve mature functionality. At glutamatergic synapses on dendritic spines of principal cortical neurons, the maturation is largely governed by postsynaptic density protein-95 (PSD-95)-dependent synaptic incorporation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors into nascent AMPA-receptor silent synapses. Consequently, in mouse primary visual cortex (V1), impaired silent synapse maturation in PSD-95-deficient neurons prevents the closure of the CP for juvenile ocular dominance plasticity (jODP). A structural hallmark of jODP is increased spine elimination, induced by brief monocular deprivation (MD). However, it is unknown whether impaired silent synapse maturation facilitates spine elimination and also preserves juvenile structural plasticity. Using two-photon microscopy, we assessed spine dynamics in apical dendrites of layer 2/3 pyramidal neurons (PNs) in binocular V1 during ODP in awake adult mice. Under basal conditions, spine formation and elimination ratios were similar between PSD-95 knockout (KO) and wild-type (WT) mice. However, a brief MD affected spine dynamics only in KO mice, where MD doubled spine elimination, primarily affecting newly formed spines, and caused a net reduction in spine density similar to what has been observed during jODP in WT mice. A similar increase in spine elimination after MD occurred if PSD-95 was knocked down in single PNs of layer 2/3. Thus, structural plasticity is dictated cell autonomously by PSD-95 in vivo in awake mice. Loss of PSD-95 preserves hallmark features of spine dynamics in jODP into adulthood, revealing a functional link of PSD-95 for experience-dependent synapse maturation and stabilization during CPs.

Early life of an animal is characterized by time windows of functionally and structurally enhanced brain plasticity known as critical periods (CPs), which have been described initially in the primary visual cortex (V1) of kittens (1). During CPs, experience refines the connectivity of principal excitatory neurons to establish the mature functionality of neural networks. This refinement is governed by the constant generation and elimination of nascent synapses on dendritic spines that sample favorable connections to be consolidated and unfavorable ones to be eliminated (2–5). A fraction of nascent synapses is or becomes α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor silent, expressing N-methyl-D-aspartate (NMDA) receptors only (6–8). At eye opening, silent synapses are abundant in the primary visual cortex (V1) (9, 10) and mature during CPs by stable AMPA receptor incorporation (11–14). The pace of silent synapse maturation is governed by the opposing yet cooperative function of postsynaptic density protein of 95 kDa (PSD-95) and its paralog PSD-93, two signaling scaffolds of the postsynaptic density of excitatory synapses (12, 13). However, whether silent synapses are preferential substrates for spine elimination during CPs remains to be investigated.In juvenile mice (postnatal days [P] 20 to 35), a brief monocular deprivation (MD) of the dominant contralateral eye results in a shift of the ocular dominance (OD) of binocular neurons in V1 toward the open eye, mediated by a reduction of responses to visual stimulation of the deprived eye (15–17). Structurally, MD induces an increase in spine elimination in apical dendrites of layer (L) 2/3 and L5 pyramidal neurons (PNs) which is only observed during the CP and constitutes a hallmark of juvenile OD plasticity (jODP) (18–20). After CP closure, cortical plasticity declines progressively, and in standard cage-raised mice beyond P40, a 4-d MD no longer induces the functional nor anatomical changes associated with jODP (21–24).At least three different mechanisms involved in experience-dependent maturation of cortical neural networks have been described, but the molecular and cellular mechanisms that cause CP closure remain highly debated (18, 25, 26). First, plasticity of local inhibitory neurons, such as increased inhibitory tone or a reduction of release probability by experience-dependent endocannabinoid receptor 1 (CB1R) activation was reported to close the critical period in rodent V1 (27–29). Second, the expression of so-called “plasticity brakes,” such as extracellular matrix (ECM), Nogo receptor 1 (NgR1), paired immunoglobulin-like receptor B (PirB), and Lynx1 were correlated with the end of critical periods (30–33). Experimentally decreasing the inhibitory tone or absence of plasticity brakes enhanced ODP expression in various knockout (KO) mouse models (32, 34, 35), among which only Lynx1 KO mice were shown to exhibit functional hallmarks of jODP, such as selective deprived eye depression after a short MD (36). Structurally, Lynx1 KO mice exhibited elevated spine dynamics at baseline; however, MD induced a reduction in spine elimination in apical dendrites of L5 PNs, whereas in L2/3 PNs there was no change (37). Thus, the effects of removing plasticity brakes on structural plasticity are variable, and it remains unclear to what extend manipulating the plasticity brakes can reinstate cellular signatures of CP plasticity in the adult wild-type (WT) brain (38). Third, the progressive maturation of AMPAR-silent synapses was correlated with the closure of the CP for jODP (12, 13). Consequently, in PSD-95 KO mice, the maturation of silent synapses is impaired; their fraction remains at the eye opening level, and jODP is preserved lifelong (13). Furthermore, visual cortex-specific knockdown (KD) of PSD-95 in the adult brain reinstated jODP. In contrast, in PSD-93 KO mice, silent synapses mature precociously and the CP for jODP closes precociously (12), correlating the presence of silent synapses with functional plasticity during CPs.While these three mechanisms of CP closure are not mutually exclusive in regulating cortical plasticity (26), it remains elusive whether CP-like structural plasticity can be expressed in the adult brain and whether silent synapses might be substrates for it. Here, we performed chronic two-photon imaging of dendrites of L2/3 pyramidal neurons in binocular V1 of PSD-95 KO (and KD) and WT mice, tracking the same dendritic spines longitudinally before, during, and after a 4-d period of MD. As previous studies have reported anesthesia effects on spine dynamics (39–41), we performed our experiments in awake mice, thoroughly trained for head fixation under the two-photon microscope. Our chronic spine imaging experiments revealed that in adult PSD-95 KO and KD mice, a brief MD indeed increased spine elimination about twofold, while adult WT mice did not display experience-dependent changes in spine elimination or spine formation. Thus, the loss of PSD-95 led to a high number of AMPAR-silent synapses which were correlated with jODP after MD, and with juvenile-like structural plasticity even in the adult brain, underscoring the importance of silent synapses for CP-timing and network maturation and stabilization.     

Circular swimming motility and disordered hyperuniform state in an algae system

Active matter comprises individually driven units that convert locally stored energy into mechanical motion. Interactions between driven units lead to a variety of nonequilibrium collective phenomena in active matter. One of such phenomena is anomalously large density fluctuations, which have been observed in both experiments and theories. Here we show that, on the contrary, density fluctuations in active matter can also be greatly suppressed. Our experiments are carried out with marine algae (Effrenium voratum), which swim in circles at the air–liquid interfaces with two different eukaryotic flagella. Cell swimming generates fluid flow that leads to effective repulsions between cells in the far field. The long-range nature of such repulsive interactions suppresses density fluctuations and generates disordered hyperuniform states under a wide range of density conditions. Emergence of hyperuniformity and associated scaling exponent are quantitatively reproduced in a numerical model whose main ingredients are effective hydrodynamic interactions and uncorrelated random cell motion. Our results demonstrate the existence of disordered hyperuniform states in active matter and suggest the possibility of using hydrodynamic flow for self-assembly in active matter.

Active matter exists over a wide range of spatial and temporal scales (1–6) from animal groups (7, 8) to robot swarms (9–11), to cell colonies and tissues (12–16), to cytoskeletal extracts (17–20), to man-made microswimmers (21–25). Constituent particles in active matter systems are driven out of thermal equilibrium at the individual level; they interact to develop a wealth of intriguing collective phenomena, including clustering (13, 22, 24), flocking (11, 26), swarming (12, 13), spontaneous flow (14, 20), and giant density fluctuations (10, 11). Many of these observed phenomena have been successfully described by particle-based or continuum models (1–6), which highlight the important roles of both individual motility and interparticle interactions in determining system dynamics.Current active matter research focuses primarily on linearly swimming particles which have a symmetric body and self-propel along one of the symmetry axes. However, a perfect alignment between the propulsion direction and body axis is rarely found in reality. Deviation from such a perfect alignment leads to a persistent curvature in the microswimmer trajectories; examples of such circle microswimmers include anisotropic artificial micromotors (27, 28), self-propelled nematic droplets (29, 30), magnetotactic bacteria and Janus particles in rotating external fields (31, 32), Janus particle in viscoelastic medium (33), and sperm and bacteria near interfaces (34, 35). Chiral motility of circle microswimmers, as predicted by theoretical and numerical investigations, can lead to a range of interesting collective phenomena in circular microswimmers, including vortex structures (36, 37), localization in traps (38), enhanced flocking (39), and hyperuniform states (40). However, experimental verifications of these predictions are limited (32, 35), a situation mainly due to the scarcity of suitable experimental systems.Here we address this challenge by investigating marine algae Effrenium voratum (41, 42). At air–liquid interfaces, E. voratum cells swim in circles via two eukaryotic flagella: a transverse flagellum encircling the cellular anteroposterior axis and a longitudinal one running posteriorly. Over a wide range of densities, circling E. voratum cells self-organize into disordered hyperuniform states with suppressed density fluctuations at large length scales. Hyperuniformity (43, 44) has been considered as a new form of material order which leads to novel functionalities (45–49); it has been observed in many systems, including avian photoreceptor patterns (50), amorphous ices (51), amorphous silica (52), ultracold atoms (53), soft matter systems (54–61), and stochastic models (62–64). Our work demonstrates the existence of hyperuniformity in active matter and shows that hydrodynamic interactions can be used to construct hyperuniform states.     

First-in-class humanized FSH blocking antibody targets bone and fat

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a K_D of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH–FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHβ subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing.

Obesity and osteoporosis affect nearly 650 million and 200 million people worldwide, respectively (1, 2). Yet the armamentarium for preventing and treating these disorders remains limited, particularly when compared with public health epidemics of a similar magnitude. It has also become increasingly clear that obesity and osteoporosis track together clinically. First, body mass does not protect against bone loss; instead, obesity can be permissive to osteoporosis and a high fracture risk (3, 4). Furthermore, the menopausal transition marks the onset not only of rapid bone loss, but also of visceral obesity and dysregulated energy balance (5–9). These physiologic aberrations have been attributed traditionally to a decline in serum estrogen, although, during the perimenopause—2 to 3 y prior to the last menstrual period—serum estrogen is within the normal range, while FSH levels rise to compensate for reduced ovarian reserve (10–12). In our view, therefore, the early skeletal and metabolic derangements cannot conceivably be explained solely by declining estrogen (13, 14).The past decade has shown that pituitary hormones can act directly on the skeleton and other tissues, a paradigm shift that is in stark contrast to previously held views on their sole regulation of endocrine targets (15–25). We and others have shown that FSH can bypass the ovary to act on G_i-coupled FSH receptors (FSHRs) on osteoclasts to stimulate bone resorption and inhibit bone formation (26, 27). This mechanism, which could underscore the bone loss during early menopause, is testified by the strong correlations between serum FSH, bone turnover, and bone mineral density (7–9, 14, 16, 26). Likewise, activating polymorphisms in the FSHR in postmenopausal women are linked to a high bone turnover and reduced bone mass (27). It therefore made biological and clinical sense to inhibit FSH action during this period to prevent bone loss.Toward this goal, we generated murine polyclonal and monoclonal antibodies to a 13-amino-acid–long binding epitope of FSHβ (28–31). The mouse and human FSHβ epitopes differ by just two amino acids; hence, blocking antibodies to the human epitope showed efficacy in mice (28). The antibodies displayed two sets of actions: they attenuated the loss of bone after ovariectomy by inhibiting bone resorption and stimulating bone formation and displayed profound effects on body composition and energy metabolism (28, 29, 31). Most notably, in a series of contemporaneously reproduced experiments, we (M.Z. and C.J.R.) found that FSH blockade reduced body fat, triggered adipocyte beiging, and increased thermogenesis in models of obesity, notably post ovariectomy and after high-fat diet (29). Our findings have been further confirmed independently by two groups who used a FSHβ–GST fusion protein or tandem repeats of the 13-amino-acid–long FSHβ epitope for studies on bone and fat, respectively (32, 33). Consistent with the mouse data, inhibiting FSH secretion using a GnRH agonist in prostate cancer patients resulted in low body fat compared with orchiectomy, wherein FSH levels are high (34). This interventional clinical trial provides evidence for a therapeutic benefit of reducing FSH levels on body fat in people. There is also new evidence that FSH blockade lowers serum cholesterol (35, 36).Thus, both emerging and validated datasets on the antiobesity, osteoprotective, and lipid-lowering actions of FSH blockade in mice and in humans prompted our current attempt to develop and characterize an array of fully humanized FSH-blocking antibodies for future testing in people. Here, we report that our lead first-in-class humanized antibody, Hu6, and two related molecules, Hu26 and Hu28, bind human FSH with a high affinity (K_Ds <10 nM), block the binding of FSH on the human FSHR, and inhibit FSH action in functional cell-based assays.     

Atomic-scale insights into quantum-order parameters in bismuth-doped iron garnet

Bismuth and rare earth elements have been identified as effective substituent elements in the iron garnet structure, allowing an enhancement in magneto-optical response by several orders of magnitude in the visible and near-infrared region. Various mechanisms have been proposed to account for such enhancement, but testing of these ideas is hampered by a lack of suitable experimental data, where information is required not only regarding the lattice sites where substituent atoms are located but also how these atoms affect various order parameters. Here, we show for a Bi-substituted lutetium iron garnet how a suite of advanced electron microscopy techniques, combined with theoretical calculations, can be used to determine the interactions between a range of quantum-order parameters, including lattice, charge, spin, orbital, and crystal field splitting energy. In particular, we determine how the Bi distribution results in lattice distortions that are coupled with changes in electronic structure at certain lattice sites. These results reveal that these lattice distortions result in a decrease in the crystal-field splitting energies at Fe sites and in a lifted orbital degeneracy at octahedral sites, while the antiferromagnetic spin order remains preserved, thereby contributing to enhanced magneto-optical response in bismuth-substituted iron garnet. The combination of subangstrom imaging techniques and atomic-scale spectroscopy opens up possibilities for revealing insights into hidden coupling effects between multiple quantum-order parameters, thereby further guiding research and development for a wide range of complex functional materials.

The element bismuth has been chosen as a substituent, or major element, in a diverse range of functional materials, including multiferroics, superconductors, and catalysts (1–3). On account of the often significantly improved performance and various unique phenomena when bismuth is introduced in functional materials, investigations on the local order parameters underpinning such effects have attracted considerable attention. In the past few years, it has been verified that bismuth doping is also an effective method to enhance the performance of magneto-optical devices (4, 5). Among the iron oxides, ferrimagnetic insulators with the complex iron garnet structure R₃Fe₅O₁₂ (where R is an element with large radius) are already widely utilized in magneto-optic devices owing to their combination of small spin-wave damping, good optical transparency, and a pronounced Faraday effect (6–12). The strength of the Faraday effect, which describes a rotation of the plane of polarization of electromagnetic radiation in a magnetic field, is linearly proportional to the Verdet constant (13, 14) for a given material, which for magneto-optical materials such as substituted garnets depends strongly on the coupling effect of multiple quantum-order parameters (15, 16), including those of lattice, spin, and electronic orbitals (12, 17, 18). In particular, diverse polyhedral sites in the garnet structure are bridged via oxygen atoms with a strong exchange interaction effect, resulting in complex electronic and crystal structures (19–22). Although pure yttrium iron garnet (YIG) has several advantages in terms of magneto-optical response, it has not been widely applied in integrated devices due to its low Verdet constant, resulting in a limited Faraday rotation (23, 24). Due, however, to its chemical flexibility, selective substitution has been established as an effective method to tune various physical properties of iron garnets (7, 12, 25, 26), and it is noteworthy that Bi-substituted lutetium iron garnet films prepared via liquid phase epitaxy (LPE) demonstrate an appreciable enhancement in magneto-optical performance (8). Several models based on diamagnetic transitions have been proposed to explain the effect of Bi substitution on magneto-optical response (4, 12, 17, 19, 21, 27–30), in each case with a strong dependence on the crystal energy levels of the Fe³⁺ ions in differently coordinated lattice sites. There is still, however, a lack of experimental evidence to test these models, as this requires the distribution of substituent atoms to be characterized and related to their effect on the crystal lattice and electronic structure at different lattice sites. In this work, we address this limitation by the use of several advanced electron microscopy techniques (31–40) applied synergistically to a Bi-substituted lutetium iron garnet.     

Seipin accumulates and traps diacylglycerols and triglycerides in its ring-like structure

Lipid droplets (LDs) are intracellular organelles responsible for lipid storage, and they emerge from the endoplasmic reticulum (ER) upon the accumulation of neutral lipids, mostly triglycerides (TG), between the two leaflets of the ER membrane. LD biogenesis takes place at ER sites that are marked by the protein seipin, which subsequently recruits additional proteins to catalyze LD formation. Deletion of seipin, however, does not abolish LD biogenesis, and its precise role in controlling LD assembly remains unclear. Here, we use molecular dynamics simulations to investigate the molecular mechanism through which seipin promotes LD formation. We find that seipin clusters TG, as well as its precursor diacylglycerol, inside its unconventional ring-like oligomeric structure and that both its luminal and transmembrane regions contribute to this process. This mechanism is abolished upon mutations of polar residues involved in protein–TG interactions into hydrophobic residues. Our results suggest that seipin remodels the membrane of specific ER sites to prime them for LD biogenesis.

Lipid droplets (LDs) are the intracellular organelles responsible for fat accumulation (1). As such, they play a central role in lipid and cellular metabolism (1–4), and they are crucially involved in metabolic diseases such as lipodystrophy and obesity (5–7).Formation of LDs occurs in the endoplasmic reticulum (ER), where neutral lipids (NLs), namely triglycerides (TG) and cholesteryl esters, constituting the core of LDs are synthesized by acyltransferases that are essential for LD formation (8). The current model of LD formation posits that NLs are stored between the two leaflets of the ER bilayer, where they aggregate in nascent oblate lens-like structures with diameters of 40 to 60 nm (9) before complete maturation and budding toward the cytosol (10–13).Recent experiments suggest that LDs form at specific ER sites marked by the protein seipin (14) upon arrival of its interaction partner protein promethin/LDAF1 (lipid droplet organization [LDO] in yeast) (15–19). These recent observations confirm previous works showing that seipin, in addition to modulating LD budding and growth (14, 19–21) and LD–ER contacts (22, 23), is also a major player in the early stages of LD formation, as deletion of seipin leads to TG accumulation in the ER and a delay in the formation of, possibly aberrant, LDs (20, 24).The role of seipin in LD formation is potentially coupled to its function in regulating lipid metabolism (25, 26) and notably that of phosphatidic acid (PA) (27–31). Recently, seipin-positive ER loci have been shown to be part of a larger protein machinery that also includes membrane and lipid remodeling proteins of the TG synthesis pathway (32), most notably, Lipin (Pah1 in yeast) and FIT proteins (Yft2 and Scs3 in yeast), for which PA is either a known substrate (Lipin/Pah1) (33) or a likely one (FIT/Yft2/Scs3) (34).Despite this thorough characterization of the cellular role of seipin in LD formation, the molecular details of its mechanism remain mostly unclear. Recently, the structure of the luminal part of the seipin oligomer has been solved at 3.7 to 4.0 Å resolution using electron microscopy (27, 35), paving the way for the investigation of the relationship between its three-dimensional structure and its mode of action. These studies revealed that the luminal domain of seipin consists of an eight-stranded beta sandwich, together with a hydrophobic helix (HH), positioned toward the ER bilayer. Notably, the seipin oligomer assembles into a ring-like architecture, an unconventional assembly in lipid bilayers that rather resembles the shape of microbial pore-forming assemblies (36) or GroEL-GroES chaperones (37, 38).From a stochiometric point of view, both fluorescence and electron microscopy data are consistent with the presence of a single seipin oligomer per nascent LD (14, 15). Hence, the structure of the luminal part of seipin is consistent with two proposed modes of action: seipin could mark the sites of LD formation by controlling TG flow in and out of the nascent droplet (14), or, alternatively, seipin could help recognize and stabilize preexisting nascent droplets in the ER membrane (20, 21, 39). In both cases, however, the relationship between the role of seipin in LD formation and its ability to regulate lipid metabolism remains unclear.Here, we use coarse-grain (CG) molecular dynamics (MD) simulations to investigate the mechanism of seipin in molecular detail. We find that seipin is able to cluster TG molecules inside its ring-like structure and that both the transmembrane (TM) helices and the luminal domain contribute to this process. Diacylglycerol (DG), the lipid intermediate between TG and PA in the Kennedy pathway, also accumulates around seipin, further promoting the accumulation of TG at very low TG-to-phospholipids ratios. Our data suggest that by accumulating DG and TG molecules, seipin generates ER sites with a specific lipid composition that in turn could promote the sequential recruitment of additional TG- and DG-sensing proteins involved in LD formation, including promethin/LDOs, FIT/Yft2/Scs3, and perilipins.     

The mitochondrial thioredoxin reductase system (TrxR2) in vascular endothelium controls peroxynitrite levels and tissue integrity

The mitochondrial thioredoxin/peroxiredoxin system encompasses NADPH, thioredoxin reductase 2 (TrxR2), thioredoxin 2, and peroxiredoxins 3 and 5 (Prx3 and Prx5) and is crucial to regulate cell redox homeostasis via the efficient catabolism of peroxides (TrxR2 and Trxrd2 refer to the mitochondrial thioredoxin reductase protein and gene, respectively). Here, we report that endothelial TrxR2 controls both the steady-state concentration of peroxynitrite, the product of the reaction of superoxide radical and nitric oxide, and the integrity of the vascular system. Mice with endothelial deletion of the Trxrd2 gene develop increased vascular stiffness and hypertrophy of the vascular wall. Furthermore, they suffer from renal abnormalities, including thickening of the Bowman’s capsule, glomerulosclerosis, and functional alterations. Mechanistically, we show that loss of Trxrd2 results in enhanced peroxynitrite steady-state levels in both vascular endothelial cells and vessels by using a highly sensitive redox probe, fluorescein-boronate. High steady-state peroxynitrite levels were further found to coincide with elevated protein tyrosine nitration in renal tissue and a substantial change of the redox state of Prx3 toward the oxidized protein, even though glutaredoxin 2 (Grx2) expression increased in parallel. Additional studies using a mitochondria-specific fluorescence probe (MitoPY1) in vessels revealed that enhanced peroxynitrite levels are indeed generated in mitochondria. Treatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin [Mn(III)TMPyP], a peroxynitrite-decomposition catalyst, blunted intravascular formation of peroxynitrite. Our data provide compelling evidence for a yet-unrecognized role of TrxR2 in balancing the nitric oxide/peroxynitrite ratio in endothelial cells in vivo and thus establish a link between enhanced mitochondrial peroxynitrite and disruption of vascular integrity.

Selenocysteine-containing mitochondrial thioredoxin reductase (TrxR2) is the key regulator of the thioredoxin system and essential for mitochondrial redox homeostasis (1–3). This system constitutes a primary defense against peroxides produced in mitochondria, such as hydrogen peroxide and peroxynitrite (4). TrxR2 is necessary for maintaining thioredoxin 2 (Trx2) in its reduced state by using electrons from NADPH. In turn, Trx2 is a cofactor of mitochondrially localized peroxiredoxin 3 (Prx3) and peroxiredoxin 5 (Prx5) that reduce H₂O₂ and peroxynitrite^* generated by mitochondrial metabolism (1, 2, 4–7). The reaction rate constants of H₂O₂ and peroxynitrite with the fast reacting thiols of both Prx3 and Prx5 have been compiled recently (4). Notably, Prx3, the only peroxiredoxin which is exclusively localized in mitochondria (8, 9)^†, was reported to accept electrons also from glutaredoxin 2 (Grx2) (10). Among the biologically recognized functions, TrxR2 plays essential roles in hematopoiesis, heart development, and heart function (11, 12). In a previous study, we could show that loss of TrxR2 in endothelial cells (ECs) attenuated vascular remodeling processes following ischemic events and led to a prothrombotic and proinflammatory endothelium (13). While these studies pointed toward an important role of TrxR2 in the cardiovascular system, the underlying biochemical mechanisms, particularly in vivo, have remained largely unclear. Unlike many other cell types in the body, ECs are unique as they are constantly exposed to changing biochemical and mechanical stimuli. Furthermore, they do not just separate the circulating blood and the vascular smooth muscle cells but also have to fulfill a wide range of physiological tasks, including regulation of vascular tone, cellular adhesion, thromboresistance, smooth muscle cell proliferation, and inflammoresistance (14, 15). One of the most significant biomolecules that is involved in vascular endothelial function is the free radical nitric oxide (·NO) (16, 17). ·NO is not only an important vasodilator but also has antiinflammatory properties by inhibiting the synthesis and expression of cytokines and adhesion molecules that attract inflammatory cells and facilitate their entrance into the vessel wall (18, 19). Furthermore, ·NO suppresses platelet aggregation (20), vascular smooth muscle cell migration, and proliferation (21). Consequentially, a decreased synthesis of ·NO, as well as an enhanced inactivation, can result in endothelial dysfunction (22–24). Oxidative stress contributes to this phenomenon, starting with the diffusion-controlled reaction of ·NO with superoxide radical (O₂^·−), which shortens the biological half-life and compromises the signaling actions of ·NO (25–28). In addition, the oxidative inactivation of ·NO by O₂^·− yields a powerful oxidizing and nitrating species, peroxynitrite anion (28, 29). Moreover, peroxynitrite itself leads to uncoupling of endothelial nitric oxide synthase to become a dysfunctional O₂^·− and peroxynitrite-producing enzyme that additionally contributes to cellular oxidative stress (30, 31). A sustained overload of O₂^·− and, peroxynitrite combined with insufficient levels of ·NO may contribute to a switch of the endothelium from the quiescent stage toward an activated one, setting up a vicious cycle, causing endothelial dysfunction and inflammation. ECs are equipped with a number of antioxidant systems known to be potentially protective against vascular oxidative stress that, however, under persistent pathological stimuli, may become overwhelmed.In this context, it is increasingly recognized that mitochondrial-derived O₂^·− and the disruption of mitochondrial redox homeostasis contribute to alter the signaling actions of ·NO in vascular biology (32–34). Besides the mitochondrial thioredoxin system, a number of professional redox systems, including mitochondrial superoxide dismutase (MnSOD/SOD2), glutathione peroxidase, and glutathione reductase, are involved in maintaining mitochondrial redox homeostasis.However, the specific roles of these enzymes in the context of endothelial dysfunction are far from being understood. The fact that genetically modified mouse models revealed that many of the different antioxidant enzymes are indispensable for murine development (35–38) impedes further insights into their role for vascular homeostasis, and, to our knowledge, only a limited number of EC-specific transgenic mice have yet been described in this context (39–42). Interestingly, Trx2 transgenic mice that overexpress Trx2 specifically in the endothelium demonstrated an increased ·NO bioavailability and EC function, decreased oxidative stress, and reduced propensity to atheroma formation (3, 43).The aim of this study was to analyze the impact of endothelial TrxR2 on vascular homeostasis (in vitro, ex vivo, and in vivo), focusing on its role on mitochondrial peroxynitrite catabolism. The data support that enhanced mitochondrial steady-state levels of peroxynitrite in vascular ECs are connected with disruption of redox homeostasis and vascular structural and functional integrity.     

Astrocyte-secreted IL-33 mediates homeostatic synaptic plasticity in the adult hippocampus

Hippocampal synaptic plasticity is important for learning and memory formation. Homeostatic synaptic plasticity is a specific form of synaptic plasticity that is induced upon prolonged changes in neuronal activity to maintain network homeostasis. While astrocytes are important regulators of synaptic transmission and plasticity, it is largely unclear how they interact with neurons to regulate synaptic plasticity at the circuit level. Here, we show that neuronal activity blockade selectively increases the expression and secretion of IL-33 (interleukin-33) by astrocytes in the hippocampal cornu ammonis 1 (CA1) subregion. This IL-33 stimulates an increase in excitatory synapses and neurotransmission through the activation of neuronal IL-33 receptor complex and synaptic recruitment of the scaffold protein PSD-95. We found that acute administration of tetrodotoxin in hippocampal slices or inhibition of hippocampal CA1 excitatory neurons by optogenetic manipulation increases IL-33 expression in CA1 astrocytes. Furthermore, IL-33 administration in vivo promotes the formation of functional excitatory synapses in hippocampal CA1 neurons, whereas conditional knockout of IL-33 in CA1 astrocytes decreases the number of excitatory synapses therein. Importantly, blockade of IL-33 and its receptor signaling in vivo by intracerebroventricular administration of its decoy receptor inhibits homeostatic synaptic plasticity in CA1 pyramidal neurons and impairs spatial memory formation in mice. These results collectively reveal an important role of astrocytic IL-33 in mediating the negative-feedback signaling mechanism in homeostatic synaptic plasticity, providing insights into how astrocytes maintain hippocampal network homeostasis.

Synaptic plasticity, the ability of neurons to alter the structure and strength of synapses, is important for the refinement of neuronal circuits in response to sensory experience during development (1) as well as learning and memory formation in adults (2, 3). To maintain the stability of neuronal network activity, the synaptic strength of neurons is modified through a negative-feedback mechanism termed homeostatic synaptic plasticity (4–6). Specifically, inhibiting neuronal activity in cultured neuronal cells or hippocampal slices by pharmacological administration of the sodium channel blocker tetrodotoxin (TTX) increases the strength of excitatory synapses to rebalance network activity (5–7).The hippocampus, which comprises the cornu ammonis 1 (CA1), CA2, CA3, and dentate gyrus subregions, is important for memory storage and retrieval. In particular, the CA1 subregion constitutes the primary output of the hippocampus, which is thought to be essential for most hippocampus-dependent memories (8, 9). Moreover, experience-driven synaptic changes in the CA1 microcircuitry impact how information is integrated (10, 11). Accordingly, the induction and expression of synaptic plasticity at hippocampal CA1 excitatory synapses are critically dependent on the structural remodeling and composition of synapses as well as functional modifications of pre- and postsynaptic proteins and neurotransmitter receptors (4–6, 12). As such, structural plasticity is a major regulatory mechanism of homeostatic synaptic plasticity in the hippocampal CA1 region. While most excitatory synapses are located at dendritic spines, morphological changes of dendritic spines likely participate in compensatory adaptations of hippocampal network activity and are therefore involved in learning, memory formation (13), and memory extinction (14).The efficacy of synaptic transmission and the wiring of neuronal circuitry are regulated not only by bidirectional communication between pre- and postsynaptic neurons, but also through the interactions between neurons and their associated glial cells (15–17). Astrocytes, as the most abundant type of glia in the central nervous system, actively regulate synapse formation, function, and maintenance during development and in the adult brain (18–20). However, the molecular basis of astrocyte–neuron communication in synaptic plasticity is largely unknown. Nevertheless, one of the mechanisms by which astrocytes regulate synapses is by secreting factors (21–25); the most well-characterized one is TNFα. Notably, pharmacologically induced deprivation of neuronal activity increases TNFα release from astrocytes, which modulates homeostatic plasticity in both excitatory and inhibitory neurons through regulation of neuronal glutamate and GABA receptor trafficking (24, 26). Further in vivo studies on germline knockout mice support the roles of astrocyte-secreted TNFα in homeostatic adaptations of cortical circuitry during sensory deprivation (27, 28). Another cytokine interleukin-33 (IL-33) is secreted by astrocytes to regulate synapse development in spinal cord and thalamus (29). Nevertheless, it remains largely unknown how astrocytes respond to changes in neuronal activity to regulate homeostatic synaptic plasticity in the hippocampus as well as learning and memory formation.In this study, we identified IL-33 as an astrocyte-secreted factor which mediates homeostatic synaptic plasticity in the CA1 subregion of adult hippocampus. Pharmacological blockade of neuronal activity or in vivo optogenetic inhibition of CA1 pyramidal neurons stimulates a local increase in the expression and release of IL-33 from the astrocytes. In turn, this astrocyte-secreted IL-33 and its ST2/IL-1RAcP receptor complex mediate the increase of excitatory synapses and neurotransmission in homeostatic synaptic plasticity. Two-photon imaging of CA1 pyramidal neurons in vivo reveals that IL-33 promotes dendritic spine formation through the synaptic recruitment of postsynaptic scaffolding protein PSD-95. Importantly, conditional knockout of IL-33 in astrocytes decreases excitatory synapses in the CA1 subregion, and inhibition of IL-33/ST2 signaling in adult mice abolishes the homeostatic synaptic plasticity in CA1 pyramidal neurons, resulting in impaired spatial memory formation. Hence, our findings collectively show that astrocyte-secreted IL-33 plays an important role in homeostatic synaptic plasticity in the adult hippocampus and spatial memory formation.