Hyperglycemia in rodent models of type 2 diabetes requires insulin-resistant alpha cells

To determine the role of glucagon action in diet-induced and genetic type 2 diabetes (T2D), we studied high-fat-diet–induced obese (DIO) and leptin receptor-defective (LepR^−/−) rodents with and without glucagon receptors (GcgRs). DIO and LepR^−/−,GcgR^+/+ mice both developed hyperinsulinemia, increased liver sterol response element binding protein 1c, and obesity. DIO GcgR^+/+ mice developed mild T2D, whereas LepR^−/−,GcgR^+/+ mice developed severe T2D. High-fat–fed (HFF) glucagon receptor-null mice did not develop hyperinsulinemia, increased liver sterol response element binding protein 1c mRNA, or obesity. Insulin treatment of HFF GcgR^−/− to simulate HFF-induced hyperinsulinemia caused obesity and mild T2D. LepR^−/−,GcgR^−/− did not develop hyperinsulinemia or hyperglycemia. Adenoviral delivery of GcgR to GcgR^−/−,LepR^−/− mice caused the severe hyperinsulinemia and hyperglycemia of LepR^−/− mice to appear. Spontaneous disappearance of the GcgR transgene abolished the hyperinsulinemia and hyperglycemia. In conclusion, T2D hyperglycemia requires unsuppressible hyperglucagonemia from insulin-resistant α cells and is prevented by glucagon suppression or blockade.The prevalence of type 2 diabetes (T2D) in the United States was 29.1 million in 2012, and 37% of adults were identified as prediabetic (1). T2D is now present on every continent (2). Despite the magnitude of this threat to world physical and fiscal health, our understanding of the pathogenic pathway is vague and is based largely on epidemiologic correlations. For example, the correlation between T2D and obesity is so high that most obese Americans can be considered prediabetic, but the precise mechanism of this relationship is unknown. Although the “lipotoxic” effects of ectopic lipids were first suggested in 1994 (3) to link diet-induced obesity to T2D and other components of the metabolic syndrome (3–11), the relationship between IR and T2D is still poorly understood. Proposed hypothetical links range from beta cell “glucotoxicity” (12) to the action of modifier genes (13) to failure of redox control (14).It has recently been shown that glucagon receptor-null mice remain normoglycemic and nonketotic despite total insulin deficiency but that transduction of a glucagon receptor cDNA into their liver makes them severely diabetic (15, 16). This proves that, whether or not insulin action is present, suppression of glucagon action prevents hyperglycemia. It has long been known that insulin suppression of glucagon regulates alpha cell secretion (17, 18). Although the presence of hyperglucagonemia was established unequivocally in type 1 diabetes (T1D) (15, 16), direct evidence that it is essential for the hyperglycemia of T2D is lacking. However, it has long been known that glucagon is elevated in T2D (17, 19, 20) and is resistant to suppression by insulin.     

Hematopoietic RIPK1 deficiency results in bone marrow failure caused by apoptosis and RIPK3-mediated necroptosis

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is recruited to the TNF receptor 1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. RIPK1 deficiency results in postnatal lethality, but precisely why Ripk1^−/− mice die remains unclear. To identify the lineages and cell types that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Tamoxifen administration to adult RosaCreER^T2Ripk1^fl/fl mice results in lethality caused by cell death in the intestinal and hematopoietic lineages. Similarly, Ripk1 deletion in cells of the hematopoietic lineage stimulates proinflammatory cytokine and chemokine production and hematopoietic cell death, resulting in bone marrow failure. The cell death reflected cell-intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, because Vav-iCre Ripk1^fl/fl fetal liver cells failed to reconstitute hematopoiesis in lethally irradiated recipients. We demonstrate that RIPK3 deficiency partially rescues hematopoiesis in Vav-iCre Ripk1^fl/fl mice, showing that RIPK1-deficient hematopoietic cells undergo RIPK3-mediated necroptosis. However, the Vav-iCre Ripk1^fl/fl Ripk3^−/− progenitors remain TNF sensitive in vitro and fail to repopulate irradiated mice. These genetic studies reveal that hematopoietic RIPK1 deficiency triggers both apoptotic and necroptotic death that is partially prevented by RIPK3 deficiency. Therefore, RIPK1 regulates hematopoiesis and prevents inflammation by suppressing RIPK3 activation.The proinflammatory cytokine TNF stimulates receptor-interacting serine/threonine-protein kinase 1 (RIPK1) ubiquitination, NFκB and MAPK activation, and induction of apoptosis or necroptosis (1, 2). TNF signaling via TNF receptor 1 (TNFR1) is highly regulated and results in the recruitment of several adapter proteins including TNFR1-associated death domain (TRADD) protein, the E3 ubiquitin ligases cellular inhibitor of apoptosis protein-1 and -2 (cIAP1/2), and TNFR-associated factor 2 (TRAF2) or 5, and the serine threonine death domain-containing kinase RIPK1 (complex I) (1). We have demonstrated that the kinase activity of RIPK1 is not required for NFκB activation (3); rather, RIPK1 is modified by the addition of Lys63-linked and linear polyubiquitin chains (3–6). Polyubiquitinated RIPK1 then recruits NEMO/IκB kinase-γ (IKKγ) to mediate IKK activation and TAK1/TAB2/3 to mediate MAPK activation, resulting in antiapoptotic and proinflammatory gene expression (7, 8). Deubiquitination of RIPK1 by cylindromatosis (CYLD) results in the formation of a cytosolic complex containing TRADD, Fas-associated death domain protein (FADD), caspase-8, and RIPK1 (complex IIa) (2). Caspase-8 cleaves and inactivates RIPK1 and CYLD and stimulates apoptosis (9–11). In the absence of caspase-8 or the presence of caspase inhibitors, TNF family members and potentially other ligands stimulate the kinase activity of RIPK1 to induce necroptosis (9, 11–16). RIPK1 also is recruited to the Toll-like receptor adapter TRIF via the Rip homotypic interaction motif (RHIM) to mediate NFκB activation (17) and, under conditions of caspase-8 inhibition, initiates necroptosis (14, 16). Necrostatin-1 (Nec-1), an allosteric RIPK1 inhibitor, inhibits necroptosis induced by TNF or the TLR3 ligand poly I:C and abolishes the formation and activation of an RIPK1/3 complex (13–16, 18). Although the molecular details whereby RIPK1 initiates necroptosis are unclear, RIPK3 and the pseudo kinase MLKL appear to be required (2).Genetic studies in mice have revealed cross-regulation between the apoptotic and necroptotic pathways. For example, the FADD/caspase-8/FLICE-like inhibitory protein long form (FLIP_L) complex regulates RIPK1 and RIPK3 activity during development, because the embryonic lethality associated with a caspase-8 deficiency is completely rescued by the absence of RIPK3 (19, 20). Similarly, RIPK1 deficiency rescues FADD-associated embryonic lethality (21). Thus, in the absence of FADD or caspase-8, embryos succumb to RIPK1- and RIPK3-dependent necroptosis. However, Fadd^−/−/Ripk1^−/− mice, die perinatally (21, 22), as do Ripk1^−/− mice, revealing that RIPK1 has prosurvival roles beyond the regulation of the FADD/caspase-8/FLIP_L complex.We have demonstrated that complete RIPK1 deficiency results in increased TNF-induced cell death that can be rescued, in part, by the absence of the TNFR1 (22, 23). However, Ripk1^−/−Tnfr1^−/− animals still succumb (23), indicating that other death ligands/pathways contribute to the RIPK1-associated lethality. Consistent with this hypothesis, RIPK3 deficiency recently has been shown to rescue the perinatal lethality observed in Ripk1^−/−Tnfr1^−/− mice (24, 25). Similarly, combined caspase-8 and RIPK3 deficiency also rescues the RIPK1-associated lethality (24–26). Collectively, these genetic studies in mice reveal that the perinatal death of Ripk1^−/− mice reflects TNF-induced apoptosis and RIPK3-mediated necroptosis. The nature of the ligand(s) or the trigger(s) of RIPK3-mediated necroptosis in vivo remain unclear. However, Ripk1^−/− MEFs are prone to necroptosis induced by poly I:C or by treatment with type I or type II IFN (24, 25), suggesting that these pathways contribute. Although these studies reveal a regulatory role for RIPK1, the multiorgan cell death and inflammation observed in the complete and compound RIPK1-knockout strains have made it difficult to discern the specific tissues that require RIPK1 for survival.     

Osteopontin expression by CD103? dendritic cells drives intestinal inflammation

Intestinal CD103⁻ dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103⁻ DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103⁻ DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103⁻ DCs resulted in attenuated colitis in comparison to transfer of WT CD103⁻ DCs, whereas transgenic CD103⁻ DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103⁻ DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103⁻ DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103⁻ DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103⁻ DCs and their function during inflammatory bowel disease.Inflammatory bowel diseases (IBDs), including Crohn disease (CD) and ulcerative colitis (UC), are caused by excessive inflammatory responses to commensal microflora and other antigens present in the intestinal lumen (1). Intestinal dendritic cells (DCs) contribute to these inflammatory responses during human IBD, as well as in murine colitis models (2). DCs that reside in draining mesenteric lymph nodes (MLNs) are also crucial mediators of colitis induction (3) and may be grouped based on their surface CD103 (integrin αE) expression as CD11c^highCD103⁺ (CD103⁺ DCs) and CD11c^highCD103⁻ (CD103⁻ DCs) (4–6). CD103⁺ DCs are considered important mediators of gut homeostasis in steady state (4, 5, 7–9), and their tolerogenic properties are conserved between mice and humans (5). However, their role during intestinal inflammation is not well defined. Instead, CD103⁻ DC function has been described mostly during chronic experimental colitis (10–12). These cells secrete IL-23, IL-6, and IL-12 (10–12), contributing to the development of T helper (Th) 17 and Th1 cells, and are highly inflammatory during CD4⁺ T-cell transfer colitis (12) and during 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis (11). MLN CD103⁻ DCs cultured in the presence of LPS, a Toll-like receptor (TLR) 4 agonist, or R848, a TLR7 agonist, express higher levels of TNF-α and IL-6 (7, 12). In fact, these cells secrete IL-23 and IL-12 even in the absence of TLR stimulation (10). Both MLN CD103⁻ and CD103⁺ DC subsets are present in acute colitis (11, 13); however, their function, as well as their cytokine profile, during this phase of disease, reflecting colitis initiation, remains unknown.Recent studies suggest a proinflammatory role for the cytokine osteopontin (Opn) in TNBS- and dextran sulfate sodium (DSS)-induced colitis (14, 15), which are the models for CD and UC, respectively. Opn is expressed by DCs and other immune cell types, such as lymphocytes, during autoimmune responses (16–22), and its expression by DCs during autoimmunity contributes to disease severity (17–19, 21, 23). In addition, Opn expression is highly up-regulated in intestinal immune and nonimmune cells and in the plasma of patients with CD and UC (24–29), as well as in the colon and plasma of mice with experimental colitis (14, 15, 27, 30). Increased plasma Opn levels are related to the severity of CD inflammation (29), and certain Opn gene (Spp1) haplotypes are modifiers of CD susceptibility (31), indicating that Opn could be used as an IBD biomarker (27). In general, Opn affects DC biology during several inflammatory conditions (17–21, 32–37) and could be a potential therapeutic target in IBD.In this study, we initially asked whether Opn was expressed by MLN CD103⁻ and CD103⁺ DCs during colitis. We found that CD103⁻ DCs express excessive levels of Opn in addition to other proinflammatory cytokines. Conversely, CD103⁺ DCs express profoundly lower levels of Opn and are noninflammatory. Using adoptive transfer of purified specific DC subsets, we determined that MLN CD103⁻ DCs are critical mediators of acute intestinal inflammation and that their Opn expression is essential for their proinflammatory properties in both acute and chronic colitis. Furthermore, Opn-deficient and Opn-neutralized mice developed significantly milder disease. In addition, we constructed transgenic (Tg) mice overexpressing Opn only in DCs. These mice developed exaggerated colitis, and adoptive transfer of their CD103⁻ DCs into recipient mice dramatically exacerbated disease. Because Opn protein contains several domains interacting with various receptors, we defined a specific Opn domain significant for inducing proinflammatory properties in CD103⁻ DCs. Blockade of the interaction of this Opn domain [containing functional Ser-Leu-Ala-Tyr-Gly-Leu-Arg (SLAYGLR) sequence] with integrin α9 expressed on CD103⁻ DCs abrogated their proinflammatory profile and colitogenic effects in vivo.     

Follicle-stimulating hormone regulates expression and activity of epidermal growth factor receptor in the murine ovarian follicle

Fertility depends on the precise coordination of multiple events within the ovarian follicle to ensure ovulation of a fertilizable egg. FSH promotes late follicular development, including expression of luteinizing hormone (LH) receptor by the granulosa cells. Expression of its receptor permits the subsequent LH surge to trigger the release of ligands that activate EGF receptors (EGFR) on the granulosa, thereby initiating the ovulatory events. Here we identify a previously unknown role for FSH in this signaling cascade. We show that follicles of Fshb^−/− mice, which cannot produce FSH, have a severely impaired ability to support two essential EGFR-regulated events: expansion of the cumulus granulosa cell layer that encloses the oocyte and meiotic maturation of the oocyte. These defects are not caused by an inability of Fshb^−/− oocytes to produce essential oocyte-secreted factors or of Fshb^−/− cumulus cells to respond. In contrast, although expression of both Egfr and EGFR increases during late folliculogenesis in Fshb^+/− females, these increases fail to occur in Fshb^−/− females. Remarkably, supplying a single dose of exogenous FSH activity to Fshb^−/− females is sufficient to increase Egfr and EGFR expression and to restore EGFR-dependent cumulus expansion and oocyte maturation. These studies show that FSH induces an increase in EGFR expression during late folliculogenesis and provide evidence that the FSH-dependent increase is necessary for EGFR physiological function. Our results demonstrate an unanticipated role for FSH in establishing the signaling axis that coordinates ovulatory events and may contribute to the diagnosis and treatment of some types of human infertility.Fertility in mammals depends on the coordinated execution of multiple events within the fully grown ovarian follicle at the time of ovulation (1, 2). The oocyte undergoes meiotic maturation, during which it progresses to metaphase II of meiosis and acquires the ability to begin embryonic development (3). Concomitantly, the layer of granulosa cells (GCs) immediately surrounding the oocyte, termed the “cumulus,” undergoes a process termed “expansion,” which is required for sperm to penetrate this layer and reach the oocyte (4–7). At the perimeter of the follicle, an inflammatory response associated with rupture of the follicular wall permits the cumulus–oocyte complex (COC) to escape from the follicle and enter the oviduct where fertilization will occur. These events are triggered by the preovulatory release of luteinizing hormone (LH), which acts on LH receptors (LHCGR) on the mural GCs that line the interior wall of the fully grown follicle (8).Recent studies have identified a key downstream effector of LH activity at ovulation. Binding of LH to LHCGR triggers the release of the EGF-related peptides amphiregulin (AREG, betacellulin (BTC), and epiregulin (EREG) (9–11). These bind to EGF receptors (EGFRs) located on both the mural and cumulus GCs (12–19) and activate MAPK3/1 as well as other signaling networks (20–28). Considerable evidence supports the view that the EGFR signaling mediates many or most ovulatory events. First, the release of the EGFR ligands follows the LH surge but precedes the LH-dependent responses (9–11). Second, EGF and the EGFR ligands can induce cumulus expansion and oocyte maturation in vitro, independently of LH (9, 10, 20, 29). Third, these events are impaired in mice bearing a hypomorphic Egfr allele that reduces EGFR activity by about one-half and in mice in which Egfr has been selectively inactivated in GCs through a targeted mutation (22, 23). Thus, the activation of EGFR signaling in GCs of mature follicles appears to be a major effector of the ovulatory response to LH.FSH binds to receptors located on GCs and induces the expression of numerous genes, including Lhcgr (8, 30). Lhcgr expression is impaired substantially in mice that lack either FSH, because of targeted mutation of the Fshb gene that encodes its β-subunit, or the FSH receptor and in humans bearing spontaneous mutations; these individuals fail to ovulate (31–34). Thus, the ovulatory response to LH depends strictly on the prior FSH-dependent expression of Lhcgr, and in this manner FSH indirectly controls the LHCGR-regulated release of the EGFR ligands. We report here that FSH also drives an increase in EGFR expression during late folliculogenesis and provide evidence that this increase is essential to enable the ovulatory response to EGF. By coordinating the expression of EGFR and the release of its ligands, FSH endows full-grown follicles with the capacity to activate EGFR signaling at ovulation.     

iRhoms 1 and 2 are essential upstream regulators of ADAM17-dependent EGFR signaling

The metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) controls EGF receptor (EGFR) signaling by liberating EGFR ligands from their membrane anchor. Consequently, a patient lacking ADAM17 has skin and intestinal barrier defects that are likely caused by lack of EGFR signaling, and Adam17^−/− mice die perinatally with open eyes, like Egfr^−/− mice. A hallmark feature of ADAM17-dependent EGFR ligand shedding is that it can be rapidly and posttranslationally activated in a manner that requires its transmembrane domain but not its cytoplasmic domain. This suggests that ADAM17 is regulated by other integral membrane proteins, although much remains to be learned about the underlying mechanism. Recently, inactive Rhomboid 2 (iRhom2), which has seven transmembrane domains, emerged as a molecule that controls the maturation and function of ADAM17 in myeloid cells. However, iRhom2^−/− mice appear normal, raising questions about how ADAM17 is regulated in other tissues. Here we report that iRhom1/2^−/− double knockout mice resemble Adam17^−/− and Egfr^−/− mice in that they die perinatally with open eyes, misshapen heart valves, and growth plate defects. Mechanistically, we show lack of mature ADAM17 and strongly reduced EGFR phosphorylation in iRhom1/2^−/− tissues. Finally, we demonstrate that iRhom1 is not essential for mouse development but regulates ADAM17 maturation in the brain, except in microglia, where ADAM17 is controlled by iRhom2. These results provide genetic, cell biological, and biochemical evidence that a principal function of iRhoms1/2 during mouse development is to regulate ADAM17-dependent EGFR signaling, suggesting that iRhoms1/2 could emerge as novel targets for treatment of ADAM17/EGFR-dependent pathologies.ADAM17 (a disintegrin and metalloprotease 17) is a membrane-anchored metalloproteinase that controls two major signaling pathways with important roles in development and disease, the EGF receptor (EGFR) pathway and the proinflammatory tumor necrosis factor α (TNF-α) pathway (1–5). Mice lacking ADAM17 resemble mice with defects in EGFR signaling in that they have open eyes at birth, enlarged semilunar heart valves, and enlarged hypertrophic zones in long bone growth plates, most likely caused by a lack of ADAM17-dependent release of the EGFR ligands transforming growth factor α (TGF-α) and heparin-binding epidermal growth factor (HB-EGF) (3, 6–14). In humans, defects in skin and intestinal barrier protection have been reported in a patient lacking ADAM17 (15) and in patients treated with EGFR inhibitors (16, 17), and similar skin defects were recently identified in a patient with defective EGFR signaling (18). Mouse models of ADAM17/EGFR signaling appear to recapitulate these mechanisms, because defects in skin barrier protection can be observed by inactivating either ADAM17 or the EGFR in keratinocytes (19), as well as in mice expressing very low levels of ADAM17, which also have increased susceptibility to intestinal inflammation (20). A hallmark feature of ADAM17 is its rapid response to various activators of cellular signaling pathways (21–23), which is presumably important to allow a rapid response to injury and to maintain the skin and intestinal barrier. The rapid activation of ADAM17 is controlled by its transmembrane domain whereas the cytoplasmic domain is dispensable in this context (22), suggesting that ADAM17 is regulated by one or more other membrane proteins, yet the underlying mechanism has remained enigmatic.Recent studies have shown that the maturation and function of ADAM17 in myeloid cells depend on inactive Rhomboid 2 (iRhom2), a catalytically inactive member of the Rhomboid family of seven membrane-spanning intramembrane serine proteinases (24–28). Myeloid cells lacking iRhom2 release very little TNF-α in response to activation of Toll-like receptor 4 by lipopolysaccharide (LPS) (24, 26, 28). Therefore, mice lacking iRhom2 are protected from the detrimental effects of TNF-α in mouse models for septic shock and inflammatory arthritis, similar to conditional knockout mice lacking ADAM17 in myeloid cells (11, 26, 29). However, iRhom2^−/− (iR2^−/−) mice are viable with no evident spontaneous pathological phenotypes (26, 29), whereas Adam17^−/− (A17^−/−) mice die shortly after birth (3). A major unresolved question has therefore been whether iRhom2 and the related iRhom1 are the long-sought-after regulators of the function of ADAM17-dependent EGFR signaling in vivo. Here we generate iRhom1^−/− (iR1^−/−) mice, which are viable and healthy, and report that iR1/2^−/− double knockout mice closely resemble mice lacking ADAM17 or the EGFR, providing the first genetic evidence, to our knowledge, that the principal function of iRhoms1/2 during mouse development is to control ADAM17/EGFR signaling.     

PML plays both inimical and beneficial roles in HSV-1 replication

After entry into the nucleus, herpes simplex virus (HSV) DNA is coated with repressive proteins and becomes the site of assembly of nuclear domain 10 (ND10) bodies. These small (0.1–1 μM) nuclear structures contain both constant [e.g., promyelocytic leukemia protein (PML), Sp100, death-domain associated protein (Daxx), and so forth] and variable proteins, depending on the function of the cells or the stress to which they are exposed. The amounts of PML and the number of ND10 structures increase in cells exposed to IFN-β. On initiation of HSV-1 gene expression, ICP0, a viral E3 ligase, degrades both PML and Sp100. The earlier report that IFN-β is significantly more effective in blocking viral replication in murine PML^+/+ cells than in sibling PML^−/− cells, reproduced here with human cells, suggests that PML acts as an effector of antiviral effects of IFN-β. To define more precisely the function of PML in HSV-1 replication, we constructed a PML^−/− human cell line. We report that in PML^−/− cells, Sp100 degradation is delayed, possibly because colocalization and merger of ICP0 with nuclear bodies containing Sp100 and Daxx is ineffective, and that HSV-1 replicates equally well in parental HEp-2 and PML^−/− cells infected at 5 pfu wild-type virus per cell, but poorly in PML^−/− cells exposed to 0.1 pfu per cell. Finally, ICP0 accumulation is reduced in PML^−/− infected at low, but not high, multiplicities of infection. In essence, the very mechanism that serves to degrade an antiviral IFN-β effector is exploited by HSV-1 to establish an efficient replication domain in the nucleus.Several prominent events take place after the entry of herpes simplex virus (HSV) DNA into the nucleus of newly infected cells. Thus, viral DNA becomes coated by repressive proteins, the function of which is to block viral gene expression (1–6); nuclear domain 10 (ND10) bodies colocalize with the viral DNA (7, 8); α or immediate early viral genes are expressed; and one viral protein, ICP0, degrades promyelocytic leukemia protein (PML) and Sp100, two key constituents of ND10 bodies in conjunction with the UbcH5A ubiquitin-conjugating enzyme (9–11). What is left of the ND10 bodies is infiltrated by viral proteins and becomes the viral replication compartment (12–15).ND10 bodies range between 0.1 and 1 μM in diameter. The composition of ND10 bodies varies depending on the cellular function or in response to stress, such as that resulting from virus infection (16–19). Among the constant components of ND10 are PML, Sp100, and death-domain associated protein (Daxx). PML has been reported to be critical for the recruitment of components and for the organization of the ND10 bodies (18–23). The function of ND10 bodies may vary under different cellular conditions and may also depend on their composition.A key question that remains unanswered is the function of ND10 bodies in infection, and in particular, why HSV has evolved a strategy that specifically targets PML and Sp100 for degradation. Two clues that may ultimately shed light on the function of ND10 is that exposure of cells to IFN leads to an increase in the number of ND10 bodies and an increase in PML (16, 24–26). The second clue emerged from the observation reported earlier by this laboratory is that pretreatment of murine PML^+/+ cells with IFN-β led to a drastic reduction in virus yields. In contrast, exposure of PML^−/− cells to IFN-β led to a significantly smaller decrease in virus yields (27). The results suggest PML is an antiviral effector of IFN-β, but many questions regarding the function of PML remain unanswered (28).In this study, we constructed a PML^−/− cell line (1D2) derived from HEp-2 cells. The first part of this report centers on the structure of ND10 bodies bereft of PML and the interaction of these bodies with ICP0. In the second part, we report on the replication of HSV-1 in PML^−/− cells. Here we show that HSV-1 replication and the accumulation of ICP0 are significantly reduced in PML^−/− cells exposed to low ratios of virus per cell. HSV has evolved a strategy to take advantage of PML before its degradation.     

Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala

Stimulating presynaptic terminals can increase the proton concentration in synapses. Potential receptors for protons are acid-sensing ion channels (ASICs), Na⁺- and Ca²⁺-permeable channels that are activated by extracellular acidosis. Those observations suggest that protons might be a neurotransmitter. We found that presynaptic stimulation transiently reduced extracellular pH in the amygdala. The protons activated ASICs in lateral amygdala pyramidal neurons, generating excitatory postsynaptic currents. Moreover, both protons and ASICs were required for synaptic plasticity in lateral amygdala neurons. The results identify protons as a neurotransmitter, and they establish ASICs as the postsynaptic receptor. They also indicate that protons and ASICs are a neurotransmitter/receptor pair critical for amygdala-dependent learning and memory.Although homeostatic mechanisms generally maintain the brain’s extracellular pH within narrow limits, neural activity can induce transient and localized pH fluctuations. For example, acidification may occur when synaptic vesicles, which have a pH of ∼5.2–5.7 (1–3), release their contents into the synapse. Studies of mammalian cone photoreceptors showed that synaptic vesicle exocytosis rapidly reduced synaptic cleft pH by an estimated 0.2–0.6 units (4–6). Transient synaptic cleft acidification also occurred with GABAergic transmission (7). Some, but not all, studies also reported that high-frequency stimulation (HFS) transiently acidified hippocampal brain slices, likely as a result of the release of synaptic vesicle contents (8, 9). Neurotransmission also induces a slower, more prolonged alkalinization (10, 11). In addition to release of synaptic vesicle protons, neuronal and glial H⁺ and HCO₃⁻ transporters, channels, H⁺-ATPases, and metabolism might influence extracellular pH (10–12).ASICs are potential targets of reduced extracellular pH. ASICs are Na⁺-permeable and, to a lesser extent, Ca²⁺-permeable channels that are activated by extracellular acidosis (13–19). In the brain, ASICs consist of homotrimeric and heterotrimeric complexes of ASIC1a, ASIC2a, and ASIC2b. The ASIC1a subunit is required for acid-activation in the physiological range (>pH 5.0) (20, 21). Several observations indicate that ASIC are located postsynaptically. ASICs are located on dendritic spines. Although similar to glutamate receptors, they are also present on dendrites and cell bodies (20, 22–24). ASIC subunits interact with postsynaptic scaffolding proteins, including postsynaptic density protein 95 and protein interacting with C-kinase-1 (20, 24–29). In addition, ASICs are enriched in synaptosome-containing brain fractions (20, 24, 30).Although these observations raised the possibility that protons might be a neurotransmitter, postsynaptic ASIC currents have not been detected in cultured hippocampal neurons (31, 32), and whether localized pH transients might play a signaling role in neuronal communication remains unclear. In previous studies of hippocampal brain slices, extracellular field potential recordings suggested impaired hippocampal long-term potentiation (LTP) in ASIC1a^−/− mice (20), although another study did not detect an effect of ASIC1a (33). Another study using microisland cultures of hippocampal neurons suggested that the probability of neurotransmitter release increased in ASIC1a^−/− mice (32).Here, we tested the hypothesis that protons are a neurotransmitter and that ASICs are the receptor. Criteria to identify substances as neurotransmitters have been proposed (34). Beg and colleagues (35) used these criteria to conclude that protons are a transmitter released from Caenorhabditis elegans intestine to cause muscle contraction. Key questions about whether protons meet criteria for a neurotransmitter are: Does presynaptic stimulation increase the extracellular proton concentration? Do protons activate currents in postsynaptic cells? Can exogenously applied protons reproduce effects of endogenous protons? What is the postsynaptic proton receptor? We studied lateral amygdala brain slices because amygdala-dependent fear-related behavior depends on a pH reduction (36). In addition, ASICs are abundantly expressed there, and ASIC1a^−/− mice have impaired fear-like behavior (36–38).     

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2^−/− mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2′-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory disease that arises through unknown genetic, environmental, and bacterial origins (1, 2). Ulcerative colitis (UC) and Crohn disease (CD) are the two main forms of IBD, and their incidence is increasing in industrialized countries (3). Furthermore, IBD is a risk factor for the development of colon cancer (4). Although the specific determinants remain elusive, persistent inflammation is believed to play a significant role in colon cancer development (5).Neutrophil recruitment and activation are key steps in the intestinal innate immune response observed in IBD (6–8), and studies with animal models of colitis highlight the relationship between neutrophil infiltration and disease severity (9–11). We recently reported results of a comprehensive analysis of histopathology, changes in gene expression, and nucleic acid damage occurring during progression of lower bowel disease in Rag2^−/− mice infected with Helicobacter hepaticus (Hh) (10). This mouse model emulates many aspects of human IBD, and infected mice develop severe colitis that progress into colon carcinoma, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages (12, 13).Phagocytes produce strong oxidants and radicals that damage cellular macromolecules and promote tissue damage at sites of inflammation (14–16). Myeloperoxidase (MPO) is an abundant enzyme in neutrophils that produces hypochlorous acid (HOCl) from hydrogen peroxide (H₂O₂) and chloride ion (17, 18). HOCl can oxidize and chlorinate DNA, proteins, and lipids (19, 20). A prominent target of HOCl is tyrosine, which leads to the formation of the stable aromatic residue, 3-chlorotyrosine (Cl-Tyr) (21, 22). MPO also produces chlorinating species that react with DNA to form chlorinated adducts such as 5-chloro-2′-deoxycytidine (5-Cl-dC) (23), the presence of which was identified in colon tissue of H. hepaticus-infected Rag2^−/− mice (10). This modification of DNA may provide a mechanistic link between neutrophil activity and colitis-associated carcinoma (10, 24, 25).Macrophages also contribute to the array of oxidants and radicals at sites of inflammation through release of nitric oxide (NO) generated by the inducible NO synthase (iNOS) enzyme. NO reacts with superoxide anion (O₂^−•) at diffusion-controlled rates to yield highly reactive peroxynitrite (ONOO⁻) (26, 27). MPO also reacts H₂O₂ with nitrite (NO₂⁻, the endpoint of cellular NO oxidation) to produce the strong nitrating agent, nitrogen dioxide radical (NO₂^•) (28). Both NO₂^• and ONOO⁻ can react with tyrosine residues to generate the stable tyrosine nitration product, 3-nitrotyrosine (Nitro-Tyr) (29, 30).Multiple MS methods have been applied for determination of Cl-Tyr and Nitro-Tyr levels in biological systems (10, 31–38), and both have been detected in inflamed tissues from animals and humans (11, 39). The presence of Nitro-Tyr has been demonstrated in colon tissue of IBD patients by immunohistochemistry, and levels were reported to correlate with disease activity (40, 41). We undertook the present study to test the null hypothesis that the H. hepaticus-infected mouse model of colitis and colitis-associated carcinoma represents a useful surrogate of human IBD. To examine this hypothesis, we first quantified levels of Nitro-Tyr and Cl-Tyr in proteins and 5-Cl-dC in DNA of colon tissues of IBD patients. Comparison of these data with our previous findings (10) further assessed the validity of this animal model. We then tested the hypothesis that inflammation-induced damage in the colon would be reflected in changes in serum constituents, and would therefore serve as a noninvasive measure of IBD activity. For this purpose, we determined levels of protein chlorination and nitration products, acute-phase proteins, cytokines, and chemokines in human and mouse sera. In addition, gene expression of several inflammatory signaling molecules was monitored in mice colons to determine whether colonic inflammation was directly associated with serum cytokine levels. We then used multivariate analysis to determine which systemic inflammatory markers in serum were most closely associated with disease activity and were also common to human IBD and H. hepaticus-associated colitis in Rag2^−/− mice.     

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO₃⁻); it thereby serves as a cellular sensor for HCO₃⁻, carbon dioxide (CO₂), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO₃⁻ binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO₃⁻ activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO₃⁻ sensing and a basis for targeting this system with drugs.The ubiquitous second messenger cAMP regulates diverse physiological processes, from fungal virulence to mammalian brain function (1, 2). In mammals, cAMP can be generated by any of 10 differently expressed and regulated adenylyl cyclases (ACs): nine transmembrane enzymes (tmACs) and one soluble AC (sAC) (3). TmACs reside in the cell membrane, where they mediate cellular responses to hormones acting through G protein-coupled receptors (4). In contrast, sAC functions in various intracellular locations, providing cell-specific spatial and temporal patterns of cAMP (5–7) in response to intracellular signals, including calcium, ATP, and bicarbonate (HCO₃⁻) (3, 8–10). HCO₃⁻ regulation of sAC enzymes is a direct effect on their catalytic domains and is conserved across bacterial, fungal, and animal kingdoms (1, 11–13). Via modulation of sAC, and sAC-like cyclase activities, HCO₃⁻ serves as an evolutionarily conserved signaling molecule mediating cellular responses to HCO₃⁻, CO₂, and pH (3, 14). In mammals, sAC acts as a CO₂/HCO₃⁻/pH sensor in processes such as sperm activation (15), acid-base homeostasis (16), and various metabolic responses (10, 17, 18). sAC has also been implicated in skin and prostate cancer and as a target for male contraceptives (19–21).All mammalian ACs are class III nucleotidyl cyclases sharing homologous catalytic domains. Their catalytic cores are formed through symmetrical or pseudosymmetrical association of two identical or highly similar catalytic domains, C₁ and C₂ (22–24); in mammalian ACs, both domains reside on a single polypeptide chain. Such C₁C₂ pseudoheterodimers form two pseudosymmetrical sites at the dimer interface: the active site and a degenerated, inactive pocket (3, 23). A conserved Lys and an Asp/Thr in the active site recognize the base of the substrate ATP, and two conserved Asp residues bind two divalent cations, normally Mg²⁺ (23). The ions, called ion A and ion B, coordinate the substrate phosphates and support the intramolecular 3′-hydroxyl (3′-OH) attack at the α-phosphorous to form cAMP and pyrophosphate (PP_i) (3). In tmACs, the degenerate site binds forskolin (24), a plant diterpene that activates tmACs but has no effect on sAC (25). The forskolin activation mechanism and the existence of physiological ligands for this site in tmACs or in sAC remain unclear.There are two sAC isoforms known to be generated by alternative splicing (26). Full-length sAC comprises N-terminal catalytic domains along with ∼1,100 residues with a little understood function except for an autoinhibitory motif and a heme-binding domain (3, 27, 28). Exclusion of exon 12 (26) generates a truncated isoform, sAC_t (residues 1–490), which comprises just the two sAC catalytic domains (sAC-cat) (25). sAC_t is widely expressed, and it is the isoform most extensively biochemically characterized (3, 8, 11). It is directly activated by Ca²⁺ and HCO₃⁻; Ca²⁺ supports substrate binding, and HCO₃⁻ increases turnover and relieves substrate inhibition (8), and this regulation is conserved in sAC-like enzymes from Cyanobacteria to humans (3, 13, 29). In a homodimeric, HCO₃⁻-regulated sAC homolog from Spirulina platensis, adenylyl cyclase C (CyaC), HCO₃⁻ appeared to facilitate an active site closure required for catalysis (13), but the HCO₃⁻ binding site and its mechanism of activation remained unknown.Here, we present crystal structures of the human sAC-cat in apo form and in complex with substrate, products, bicarbonate, and a pharmacological inhibitor. The structures reveal insights into binding sites and mechanisms for sAC catalysis and for its regulation by physiological and pharmacological small molecules.     

Programmed cell death 1 inhibits inflammatory helper T-cell development through controlling the innate immune response

Programmed cell death 1 (PD-1) is an inhibitory coreceptor on immune cells and is essential for self-tolerance because mice genetically lacking PD-1 (PD-1^−/−) develop spontaneous autoimmune diseases. PD-1^−/− mice are also susceptible to severe experimental autoimmune encephalomyelitis (EAE), characterized by a massive production of effector/memory T cells against myelin autoantigen, the mechanism of which is not fully understood. We found that an increased primary response of PD-1^−/− mice to heat-killed mycobacteria (HKMTB), an adjuvant for EAE, contributed to the enhanced production of T-helper 17 (Th17) cells. Splenocytes from HKMTB-immunized, lymphocyte-deficient PD-1^−/− recombination activating gene (RAG)2^−/− mice were found to drive antigen-specific Th17 cell differentiation more efficiently than splenocytes from HKMTB-immunized PD-1^+/+ RAG2^−/− mice. This result suggested PD-1’s involvement in the regulation of innate immune responses. Mice reconstituted with PD-1^−/− RAG2^−/− bone marrow and PD-1^+/+ CD4⁺ T cells developed more severe EAE compared with the ones reconstituted with PD-1^+/+ RAG2^−/− bone marrow and PD-1^+/+ CD4⁺ T cells. We found that upon recognition of HKMTB, CD11b⁺ macrophages from PD-1^−/− mice produced very high levels of IL-6, which helped promote naive CD4⁺ T-cell differentiation into IL-17–producing cells. We propose a model in which PD-1 negatively regulates antimycobacterial responses by suppressing innate immune cells, which in turn prevents autoreactive T-cell priming and differentiation to inflammatory effector T cells.Autoimmune disease development is impacted by both genetic and environmental factors. Programmed cell death 1 (PD-1) is a type I membrane protein that delivers inhibitory signals to immune cells upon the binding of its ligand, PD-L1 or PD-L2 (1). PD-1 has been shown to be important for self-tolerance because spontaneous autoimmune diseases develop in PD-1^−/− mice (2–4). A single-nucleotide polymorphism that affects PD-1 expression is associated with autoimmune diseases in humans, such as systemic lupus erythematosus (5), type I diabetes (6), rheumatoid arthritis (7), and multiple sclerosis (MS) (8), suggesting that PD-1 deficiency may be a genetic factor involved in the development of autoimmunity.Experimental autoimmune encephalomyelitis (EAE) is a rodent model of T-cell–mediated inflammatory disease in the central nervous system (CNS), causing demyelination, axonal damage, and paralysis, and is a commonly used model for human MS. Previous reports suggested that PD-1 functions to attenuate EAE. PD-1 and its ligands were found to be strongly expressed on immune infiltrates in the CNS during the peak phase of EAE (9–11). In EAE studies, PD-1–deficient mice or the use of blocking antibodies that inhibit PD-1 engagement by ligands resulted in earlier disease onset, increased inflammatory infiltrates, and increased severity of clinical symptoms compared with normal disease progression (10–16). It has been demonstrated that ligand engagement of PD-1 inhibits T-cell activation, expansion, and cytokine production (17–19). Similarly, in EAE, PD-1 signaling in CNS-specific helper T cells may inhibit their expansion and secretion of inflammatory cytokines (10–12). Recently, T-helper 17 (Th17) cells were shown to be involved in EAE by producing IL-17 and GM-CSF (20, 21). Two reports showed that PD-1^−/− mice mount an augmented Th17 response to EAE induction (14, 16). However, the fundamental mechanisms by which PD-1 regulates antigen-specific Th17 cell differentiation, expansion, and effector function in EAE remain to be understood.To induce EAE, mice are immunized with myelin autoantigens in an emulsion of Mycobacterium tuberculosis (MTB)-derived adjuvants, causing a strong innate inflammatory response, leading to Th skewing (22). Curiously, recent studies showed that PD-1^−/− mice exhibited an altered response to infection with mycobacteria, characterized by uncontrolled bacterial burden; massive production of cytokines, termed “cytokine storm”; and early death (23–25). We wondered if this unique response of PD-1^−/− mice to mycobacteria contributed to their Th response in EAE.In this study, we took a combination of genetic and immunological approaches in which the innate response to MTB-derived adjuvant and antigen-specific T-cell polarization were separately analyzed. The present data suggest that an enhanced innate response of PD-1^−/− mice to MTB contributes to the susceptibility of these mice to severe EAE. We propose a previously undescribed function of PD-1 in controlling the basal state of the innate immune response, the failure of which can cause the activation of adaptive immune responses, provoking autoimmunity.     

Effects of lymphocyte profile on development of EBV-induced lymphoma subtypes in humanized mice

Epstein-Barr virus (EBV) infection causes both Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). The present study reveals that EBV-induced HL and NHL are intriguingly associated with a repopulated immune cell profile in humanized mice. Newborn immunodeficient NSG mice were engrafted with human cord blood CD34⁺ hematopoietic stem cells (HSCs) for a 8- or 15-wk reconstitution period (denoted ^8whN and ^15whN, respectively), resulting in human B-cell and T-cell predominance in peripheral blood cells, respectively. Further, novel humanized mice were established via engraftment of hCD34⁺ HSCs together with nonautologous fetal liver-derived mesenchymal stem cells (MSCs) or MSCs expressing an active notch ligand DLK1, resulting in mice skewed with human B or T cells, respectively. After EBV infection, whereas NHL developed more frequently in B-cell–predominant humanized mice, HL was seen in T-cell–predominant mice (P = 0.0013). Whereas human splenocytes from NHL-bearing mice were positive for EBV-associated NHL markers (hBCL2⁺, hCD20⁺, hKi67⁺, hCD20⁺/EBNA1⁺, and EBER⁺) but negative for HL markers (LMP1⁻, EBNA2⁻, and hCD30⁻), most HL-like tumors were characterized by the presence of malignant Hodgkin’s Reed–Sternberg (HRS)-like cells, lacunar RS (hCD30⁺, hCD15⁺, IgJ⁻, EBER⁺/hCD30⁺, EBNA1⁺/hCD30⁺, LMP⁺/EBNA2⁻, hCD68⁺, hBCL2⁻, hCD20^-/weak, Phospho STAT6⁺), and mummified RS cells. This study reveals that immune cell composition plays an important role in the development of EBV-induced B-cell lymphoma.Epstein Barr virus (EBV) infects human B lymphocytes and epithelial cells in >90% of the human population (1, 2). EBV infection is widely associated with the development of diverse human disorders that include Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphomas (NHL), including diffused large B-cell lymphoma (DLBCL), follicular B-cell lymphoma (FBCL), endemic Burkitt’s lymphoma (BL), and hemophagocytic lymphohistiocytosis (HLH) (3).HL is a malignant lymphoid neoplasm most prevalent in adolescents and young adults (4–6). Hodgkin/Reed–Sternberg (HRS) cells are the sole malignant cells of HL. HRS cells are characterized by CD30⁺/CD15⁺/BCL6⁻/CD20^+/− markers and appear large and multinucleated owing to multiple nuclear divisions without cytokinesis. Although HRS cells are malignant in the body, surrounding inflammatory cells greatly outnumber them. These reactive nonmalignant inflammatory cells, including lymphocytes, histiocytes, eosinophils, fibroblasts, neutrophils, and plasma cells, compose the vast majority of the tumor mass. The presence of HRS cells in the context of this inflammatory cellular background is a critical hallmark of the HL diagnosis (4). Approximately 50% of HL cases are EBV-associated (EBVaHL) (7–11). EBV-positive HRS cells express EBV latent membrane protein (LMP) 1 (LMP1), LMP2A, LMP2B, and EBV nuclear antigen (EBNA) 1 (EBNA1), but lack EBNA2 (latency II marker) (12). LMP1 is consistently expressed in all EBV-associated cases of classical HL (13, 14). LMP1 mimics activated CD40 receptors, induces NF-κB, and allows cells to become malignant while escaping apoptosis (15).The etiologic role of EBV in numerous disorders has been studied in humanized mouse models in diverse experimental conditions. Humanized mouse models recapitulate key characteristics of EBV infection-associated disease pathogenesis (16–24). Different settings have given rise to quite distinct phenotypes, including B-cell type NHL (DLBCL, FBCL, and unspecified B-cell lymphomas), natural killer/T cell lymphoma (NKTCL), nonmalignant lymphoproliferative disorder (LPD), extremely rare HL, HLH, and arthritis (16–24). Despite considerable efforts (16–24), EBVaHL has not been properly produced in the humanized mouse setting model, owing to inappropriate animal models and a lack of in-depth analyses. After an initial report of infected humanized mice, HRS-like cells appeared to be extremely rare in the spleens of infected humanized mice; however, the findings were inconclusive (18). Here we report direct evidence of EBVaHL or HL-like neoplasms in multiple humanized mice in which T cells were predominant over B cells. Our study demonstrates that EBV-infected humanized mice display additional EBV-associated pathogenesis, including DLBCL and hemophagocytic lymphohistiocytosis (16, 17).     

Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability

Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ^−/− mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ^−/− mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ^−/− mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper–tyrosine-phosphorylated in the PTPσ^−/− mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ^−/− mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD.Protein tyrosine phosphatase (PTP)σ, encoded by PTPRS (1), consists of a cell adhesion molecule-like ectodomain containing three immunoglobulin (Ig)-like and three to eight fibronectin type III repeats, a transmembrane domain, and a cytosolic region with two PTPase domains, of which the first (D1) is catalytically active (2). PTPσ expression is developmentally regulated and found primarily in the nervous system and specific epithelia (3, 4). It was previously shown to play a role in axon growth and path finding (5–7), neuroregeneration (5, 8, 9), autophagy (10), and neuroendocrine development (11–13).To investigate the function of PTPσ in vivo, our group (11) and Tremblay and coworkers (12) generated PTPσ^−/− mice. These mice exhibited high neonatal mortality, various neurological and neuroendocrine defects, colitis, and cachexia (5, 11, 13, 14). Analysis of the intestinal tissue in surviving mice by our group revealed the presence of mucosal inflammation, intestinal crypt branching, and villus blunting: all features of colitis similar to the enteropathy associated with human inflammatory bowel disease (IBD) (15). Notably, PTPσ^−/− mice also showed increased susceptibility to chemical and infectious models of murine colitis, specifically treatment with dextran sodium sulfate (DSS) or infection with Citrobacter rodentium (15). The intestinal phenotype in the mice strongly inferred a connection between PTPσ and IBD.IBD is a chronic, idiopathic, relapsing disorder affecting the gastrointestinal tract, where Crohn disease and ulcerative colitis (UC) are the two major forms (16). In IBD pathogenesis, the presence of environmental factors together with polymorphisms in IBD-susceptibility genes cause an abnormal innate and adaptive host immune response to commensal gut bacteria, leading to sustained and deleterious inflammation (17). Chronic infection (18, 19), dysbiosis (19), defective mucosal barrier defense (20), and insufficient microbial clearance (19) have all been implicated as factors contributing to IBD pathogenesis. The disease is known to have a strong genetic component, as evidenced by specific populations exhibiting a disproportionately high incidence (21) and the high disease concordance between monozygotic twins (22). Genome-wide association studies and associated metaanalyses have implicated several genes and pathways in IBD, notably genes associated with intestinal barrier defense [MYO9B (23), PARD3 (24), MAGI2 (24), CDH1 (25, 26)].Through SNP analysis of IBD patients, we showed that PTPRS is genetically associated with UC (15). The identified SNP polymorphism leads to alternative splicing in the extracellular region of the epithelial isoform of PTPσ, causing loss of the third Ig domain (15). This splicing might potentially lead to altered ligand recognition or may affect receptor dimerization (27). In addition, through an interaction-trap assay, we identified the apical junction complex (AJC) proteins E-cadherin (CDH1) and β-catenin (CTNNB1) as colonic substrates of PTPσ (15). Interestingly, recent large-scale genetic studies have identified over 160 loci that affect risk of developing IBD, many of which involved in barrier regulation (24, 28, 29).The AJC confers polarity to epithelial cells and maintains intestinal barrier integrity (30). Defective regulation of AJC proteins creates disrupted epithelial barriers, permeability defects, and aberrant intestinal morphology (30, 31), similar to defects seen in IBD. The connection between the AJC and IBD is further demonstrated by our earlier SNP analysis, which revealed a haplotype polymorphism in CDH1 that is associated with Crohn disease, leading to a truncated E-cadherin protein that fails to localize to the plasma membrane (PM), as also observed in IBD patient biopsy samples (25). Thus, we postulate that PTPσ regulates epithelial barrier integrity through regulation of AJC proteins and that defective PTPσ function may contribute to IBD.In this report, we demonstrate an intestinal epithelial barrier defect in the PTPσ^−/− mice and identify the AJC protein ezrin as an in vivo colonic substrate for PTPσ. We further demonstrate that dephosphorylation of ezrin-Y353 or -Y145 by PTPσ leads to its redistribution from the PM to the cytosol, similar to its localization following induction of IBD in mice.     

Deletion of microRNA-155 reduces autoantibody responses and alleviates lupus-like disease in the Faslpr mouse

MicroRNA-155 (miR-155) regulates antibody responses and subsequent B-cell effector functions to exogenous antigens. However, the role of miR-155 in systemic autoimmunity is not known. Using the death receptor deficient (Fas^lpr) lupus-prone mouse, we show here that ablation of miR-155 reduced autoantibody responses accompanied by a decrease in serum IgG but not IgM anti-dsDNA antibodies and a reduction of kidney inflammation. MiR-155 deletion in Fas^lpr B cells restored the reduced SH2 domain-containing inositol 5′-phosphatase 1 to normal levels. In addition, coaggregation of the Fc γ receptor IIB with the B-cell receptor in miR-155^−/−-Fas^lpr B cells resulted in decreased ERK activation, proliferation, and production of switched antibodies compared with miR-155 sufficient Fas^lpr B cells. Thus, by controlling the levels of SH2 domain-containing inositol 5′-phosphatase 1, miR-155 in part maintains an activation threshold that allows B cells to respond to antigens.MicroRNA-155 (miR-155) plays a critical role in the generation of effective antibody responses to exogenous antigenic challenges in mice (1–3). MiR-155 levels have been reported to be elevated in B but low in T cells from patients with systemic lupus erythamosus (4), yet it is not known whether miR-155 controls autoimmune responses and the expression of related pathology.Mice harboring ubiquitous or B-cell-specific ablation of the death receptor Fas develop a severe lupus-like disease. B-cell-specific deletion of the death receptor (fas^−/−) fas^−/− mice develop an excessive germinal center (GC)-derived IgG autoantibody deposition in their kidneys and succumb to renal failure (5). It has been suggested that loss of tolerance in lpr mice results from the down-regulation of the low-affinity IgG inhibitory receptor FcγRIIB (Fc γ receptor IIB), thereby rendering their B cells incapable of terminating stimulatory signals delivered by autoantigen-containing immune complexes (6–8). However, the mechanisms whereby lack of FcγRIIB engagement would lead to autoimmunity, and whether additional factors contribute to autoimmunity, are still unclear.The SH2 domain-containing inositol 5′-phosphatase 1 (SHIP-1) phosphatase acts downstream of inhibitory cell-surface receptors (9–12), including the FcγRIIB, which is essential in opposing B-cell activation signals in mice and humans (13, 14). FcγRIIB inactivation has been implicated in the development of autoreactive GC B cells and plasma cells (15), as well as in the regulation of the persistence and longevity of bone marrow plasma cells (16). After coligation of the FcγRIIB with the B-cell receptor (BCR), FcγRIIB recruits SHIP-1 to the plasma membrane, where it negatively regulates cell survival, Ca²⁺-dependent effector functions, and ERK activation, thus controlling cell proliferation, anergy, and apoptosis (17–23). As a consequence of these wide-ranging activities, germ-line or B-cell-specific deletion of FcγRIIB or SHIP-1 in mice results in a severe lupus-like disease characterized by high-titer serum IgG antinuclear autoantibodies, lymphadenopathy, splenomegaly, renal failure, and increased mortality (23–27). MiR-155 has been reported to regulate SHIP-1 expression in mammalian myeloid and malignant B cells (28–31). However, it is not known whether SHIP-1 regulation by miR-155 affects GC reactions or peripheral tolerance during a protective immune response or in an autoimmune environment, such as that in Fas^lpr mice.To understand the role of miR-155 in autoimmunity, we crossed Fas^lpr mice with our bic/miR-155^−/− mice to generate miR-155^−/−-Fas^lpr animals. Here we demonstrate that deletion of miR-155 reduced serum IgG but not IgM anti-dsDNA autoantibody levels and kidney damage. Further, we show that the absence of miR-155 derepresses the expression of SHIP-1, thus mitigating B-cell activation, proliferation, and autoimmune responses. We provide evidence that miR-155 could be targeted to control autoimmunity and lupus nephritis.     

Functions of vasopressin and oxytocin in bone mass regulation

Prior studies show that oxytocin (Oxt) and vasopressin (Avp) have opposing actions on the skeleton exerted through high-affinity G protein-coupled receptors. We explored whether Avp and Oxtr can share their receptors in the regulation of bone formation by osteoblasts. We show that the Avp receptor 1α (Avpr1α) and the Oxt receptor (Oxtr) have opposing effects on bone mass: Oxtr^−/− mice have osteopenia, and Avpr1α^−/− mice display a high bone mass phenotype. More notably, this high bone mass phenotype is reversed by the deletion of Oxtr in Oxtr^−/−:Avpr1α^−/− double-mutant mice. However, although Oxtr is not indispensable for Avp action in inhibiting osteoblastogenesis and gene expression, Avp-stimulated gene expression is inhibited when the Oxtr is deleted in Avpr1α^−/− cells. In contrast, Oxt does not interact with Avprs in vivo in a model of lactation-induced bone loss in which Oxt levels are high. Immunofluorescence microscopy of isolated nucleoplasts and Western blotting and MALDI-TOF of nuclear extracts show that Avp triggers Avpr1α localization to the nucleus. Finally, a specific Avpr2 inhibitor, tolvaptan, does not affect bone formation or bone mass, suggesting that Avpr2, which primarily functions in the kidney, does not have a significant role in bone remodeling.Over the past decade, we have described direct actions of anterior and posterior pituitary hormones on the skeleton (1–8). We have shown that these actions are exerted via G protein-coupled receptors resident on both osteoblasts and osteoclasts. We also find that the skeleton is highly sensitive to the action of posterior pituitary hormones; for example, mice haploinsufficient in oxytocin (Oxt) have osteopenic bones, but lactation is normal; lactation is impaired only in Oxt^−/− mice (2). Likewise, Tshr haploinsufficient mice are completely euthyroid with normal thyroid follicles but display significant osteopenia (4). The exquisite sensitivity of the skeleton to pituitary hormones comes as no surprise, considering that the pituitary gland and the skeleton are both evolutionarily more primitive than target endocrine organs (7).Apart from the known actions of growth hormone on the skeleton, Tsh, Fsh, Acth, Oxt, and vasopressin (Avp) have all been shown to regulate the formation and/or function of both osteoblasts and osteoclasts and thus to control bone remodeling in vivo (2–4, 6–8). The two neurohypophyseal hormones Oxt and Avp have opposing functions (2, 3). Oxt stimulates and Avp inhibits osteoblast formation. Consequently, the genetic deletion of the Oxt receptor (Oxtr) and Avp receptor 1α (Avpr1α) yields opposing phenotypes, notably osteopenia in Oxtr^−/− mice and high bone mass in Avpr1α^−/− mice (2, 3). These findings may explain the rapid recovery of bone loss at weaning when plasma Oxt levels are high (9) and also the profound loss of bone noted in chronic hyponatremic states, such as the syndrome of inappropriate antidiuretic hormone secretion (SIADH), in which serum Avp levels are elevated (3).We find high levels of Oxtr expression on both osteoclasts and osteoblasts (2, 10), in addition to their abundant expression in breast and uterine tissue, where they regulate lactation and parturition, respectively (11). Avpr1αs, in contrast, are distributed more ubiquitously, whereas Avpr2s are localized mainly in the kidney, where they regulate free water excretion (12). Osteoblasts express both Avpr1α and Avrpr2 (3). The only other known isoform, Avpr1β, is expressed predominantly in the pancreas and pituitary; it regulates ACTH secretion from pituitary corticotrophs (13). Sequence alignment shows that the binding sites of the Oxtr and Avprs are highly conserved, with specific amino acids within the predicted binding pocket providing ligand selectivity (14–16). The respective ligands Oxt and Avp also are homologous nonapeptides, differing in only two amino acids, and are known to interact with the other’s receptor with different affinities (17).To our knowledge, osteoblasts and osteoclasts are the only cells in which Oxtr, Avpr1α, and Avpr2 are coexpressed. We also have shown that osteoblastic Oxtrs undergo internalization and nuclear translocation upon binding to Oxt and that this action is independent of cytosolic Erk phosphorylation (18). Avpr1α activation by Avp also activates Erk phosphorylation within minutes (3). The homology between the ligands and their respective receptors and converging downstream signals suggest that Avp and Oxtr may share receptors with opposing or convergent signals. Here, we have explored these interactions in the regulation of osteoblastic bone formation by using mice lacking one or both receptors, chemical inhibitors, and physiological models of high bone turnover.     

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme–substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.Since the invention of the Haber–Bosch process, the amount of fixed nitrogen in soils and waters has been increasing, and this trend has significant impact on the global environment (1, 2). Fixed nitrogen is oxidized to nitrite (NO₂⁻) or nitrate (NO₃⁻) by nitrification and then converted to gaseous dinitrogen (N₂) by microbial denitrification, which closes the nitrogen cycle. Microorganisms involved in denitrification couple their respiratory systems to stepwise reduction of nitrogen oxides to N₂ (NO₃⁻ → NO₂⁻ → NO → N₂O → N₂) (3, 4). The reduction of NO₂⁻ to toxic nitric oxide (NO₂⁻ + 2H⁺ + e⁻ → NO + H₂O) is referred to as the key step in denitrification and catalyzed by either cd₁-heme nitrite reductase (cd₁NiR) or copper nitrite reductase (CuNiR) (3, 4). Although the catalytic mechanism of cd₁NiR is well understood (5, 6), that of CuNiR is controversial (7). CuNiR is a homotrimeric protein containing two distinct Cu sites per monomer (SI Appendix, Fig. S1). Type 1 Cu (T1Cu) with a Cys–Met–His₂ ligand set is an electron acceptor incorporated near the molecular surface, whereas type 2 Cu (T2Cu) with a His₃ ligand set is a catalytic center, which is ∼12 Å distant from the molecular surface and located between two adjacent monomers (7, 8). Spaced ∼12.5 Å apart, the two Cu sites are linked by a Cys–His bridge and a sensor loop. Whereas the Cys–His bridge is an electron pathway, the sensor loop is thought to control electron distribution between T1Cu and T2Cu (9).Two conserved residues, Asp98 and His255 (Alcaligenes faecalis numbering), are located above the T2Cu site and bridged by a water molecule called bridging water (SI Appendix, Fig. S1). They are essential to the CuNiR activity because they assist proton transfer (PT) to the substrate (10–12). Although intramolecular electron transfer (ET) from T1Cu to T2Cu can occur in the resting state (RS) (13, 14), the differences in the redox potentials of T2Cu minus T1Cu are small and sometimes negative in the absence of NO₂⁻, meaning that intramolecular ET before NO₂⁻ binding is not energetically favorable (15, 16). By contrast, intramolecular ET is dramatically accelerated in the presence of NO₂⁻ (15, 17). An explanation for this gating-like phenomenon is that substrate binding raises the redox potential of T2Cu and shifts the equilibrium of the ET reaction (16). However, pH dependence of intramolecular ET in the presence of NO₂⁻ cannot be explained by such a change of redox potentials (15). Instead, Kobayashi et al. (15) proposed that reduction-induced structural change of His255 is responsible for the gating-like mechanism. Because it has been recently proven that intramolecular ET in CuNiR is accompanied by PT and hence proton-coupled ET (PCET) (17, 18), one can readily speculate that intramolecular ET contributes PT to NO₂⁻ and that the structural change of His255 is involved in PCET. Crystal structures of CuNiR from Rhodobacter sphaeroides (RhsNiR) implies this possibility because His287 in RhsNiR, which corresponds to His255, seems to show pH- and redox-dependent conformational changes (19, 20). However, presumably because of X-ray radiation damages implied by rerefinement of RhsNiR structures (21), electron density around His287 was so unusual that interpretation of it is difficult (SI Appendix, Fig. S2).Crystal structures determined by synchrotron radiation crystallography (SRX) have provided insights into the enzymatic mechanism of CuNiR (22–25), and these studies are summarized elsewhere (7). High-resolution nitrite complex (NC) structures revealed an O-coordination of NO₂⁻ showing a near face-on binding mode (22, 23), whereas Cu(II)-NO₂⁻ model complexes show a vertical binding mode (7, 26–29). The near face-on coordination manner is thought to facilitate its conversion to side-on NO, which was observed in the crystal structures of CuNiR exposed to NO (22, 23, 25). Skeptical eyes have, however, been cast on these CuNiR structures because SRX data might be affected by some problems connected to the high radiation dose delivered on the crystals. First, strong synchrotron X-rays cause not only radiation damages to amino acid residues but also photoreduction of metalloproteins (30, 31). Although a comparison between oxidized and reduced states is necessary to closely investigate redox reactions, completely oxidized structures are almost impossible to determine by SRX. Indeed, the Cu centers in CuNiR are rapidly reduced by exposure to synchrotron X-rays (21, 32). Second, following the photoreduction of T2Cu, NO₂⁻ is easily reduced and produces NO and water in SRX (21). Consequently, electron density at the catalytic site of an NC structure is derived from the mixture of both substrate and product, making interpretation of data complicated and unreliable. Third, cryogenic manipulations for reducing radiation damages in SRX have also been focused as a factor that changes the population of amino acid residues (33, 34) and enzyme–substrate complexes (35). Crystallographic (36), computational (37), and spectroscopic (38–40) studies actually show that binding modes of NO₂⁻ and NO in CuNiR crystal structures can differ from those in physiological environments.We here ventured to use photoreduction in SRX to initiate a chemical reaction and to trap an enzymatically produced intermediary state (30, 31). Furthermore, to visualize intact CuNiR structures in the resting and NC states, we applied serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) (41), which enables damage-free structural determination of metalloproteins (42, 43) and evaluation of the native conformational population at room temperature (RT) (44). By comparing SRX and SFX data, we discuss PCET and nitrite reduction in CuNiR.     

Suppressing thyroid hormone signaling preserves cone photoreceptors in mouse models of retinal degeneration

Cone phototransduction and survival of cones in the human macula is essential for color vision and for visual acuity. Progressive cone degeneration in age-related macular degeneration, Stargardt disease, and recessive cone dystrophies is a major cause of blindness. Thyroid hormone (TH) signaling, which regulates cell proliferation, differentiation, and apoptosis, plays a central role in cone opsin expression and patterning in the retina. Here, we investigated whether TH signaling affects cone viability in inherited retinal degeneration mouse models. Retinol isomerase RPE65-deficient mice [a model of Leber congenital amaurosis (LCA) with rapid cone loss] and cone photoreceptor function loss type 1 mice (severe recessive achromatopsia) were used to determine whether suppressing TH signaling with antithyroid treatment reduces cone death. Further, cone cyclic nucleotide-gated channel B subunit-deficient mice (moderate achromatopsia) and guanylate cyclase 2e-deficient mice (LCA with slower cone loss) were used to determine whether triiodothyronine (T3) treatment (stimulating TH signaling) causes deterioration of cones. We found that cone density in retinol isomerase RPE65-deficient and cone photoreceptor function loss type 1 mice increased about sixfold following antithyroid treatment. Cone density in cone cyclic nucleotide-gated channel B subunit-deficient and guanylate cyclase 2e-deficient mice decreased about 40% following T3 treatment. The effect of TH signaling on cone viability appears to be independent of its regulation on cone opsin expression. This work demonstrates that suppressing TH signaling in retina dystrophy mouse models is protective of cones, providing insights into cone preservation and therapeutic interventions.Rod and cone photoreceptors degenerate under a variety of pathological conditions, including a wide array of hereditary retinal diseases, such as retinitis pigmentosa, macular degeneration, and cone–rod dystrophies. Defects in a large number of genes are linked to inherited retinal degenerative disorders (www.sph.uth.tmc.edu/RetNet/disease.htm), including those encoding enzymes involved in the recycling of 11-cis retinal in the retinal pigment epithelium (RPE), retinoid isomerase (RPE65), and lecithin retinol acyltransferase (LRAT), and the phototransduction-associated proteins (opsins, subunits of transducin, cGMP phosphodiesterase PDE6, guanylate cyclase, and cyclic nucleotide-gated channel). There are currently no treatments for human retinal dystrophies. Despite a high genetic heterogeneity, the degenerating photoreceptors show common cellular disorder features, including oxidative damage (1, 2), endoplasmic reticulum stress (3, 4), and apoptosis (5, 6).Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and apoptosis. The role of TH signaling in retina regarding its regulation of cone opsin expression and patterning has been well documented (7, 8). Most mammals possess dichromatic color vision that is mediated by two opsins with peak sensitivities to medium-long (M, green) and short (S, blue) wavelengths of light (9, 10). In mouse, M- and S-opsins are expressed in opposing gradients such that varying amounts of both opsins are coexpressed in cones in midretinal regions, whereas M-opsin predominates in dorsal (superior) regions and S-opsin predominates in ventral (inferior) regions (10, 11) (Fig. S1). During development and in the adult postmitotic retina, TH signaling via its receptor type β2 (TRβ2) suppresses expression of S-opsin, induces expression of M-opsin, and promotes the dorsal–ventral opsin patterning (7, 8). Importantly, TH signaling has been associated with cone viability. Triiodothyronine (T3) treatment was shown to cause cone death in mice and this effect was reversed by deletion of TRβ2 gene (12). Excessive TH signaling was also shown to induce auditory defects and cochlear degeneration in mice (13). TH signaling has been associated with apoptosis of a variety of human cell lines, including lymphocytes (14), breast cancer cells (15), HeLa cells (16), and pituitary tumor cells (17), and TH signaling has been well documented in apoptotic tissue remodeling during anuran metamorphosis (18, 19). To determine whether TH signaling affects cone viability in inherited retinal degeneration, we investigated cone death/survival in retinal degeneration mouse models following TH signaling suppression and stimulation. Retinol isomerase RPE65-deficient (Rpe65^−/−) (a model of Leber congenital amaurosis, LCA) (20, 21) and cone photoreceptor function loss type 1 (cpfl1) mice (PDE6C mutation, a model of achromatopsia) (22), displaying fast and severe cone degeneration, were used to determine whether suppressing TH signaling with antithyroid treatment reduces cone degeneration. Cone cyclic nucleotide-gated channel B subunit-deficient (Cngb3^−/−) (a model of achromatopsia) (23) and guanylate cyclase 2e-deficient (Gucy2e^−/−) (another model of LCA) mice (24), displaying relatively slow progressive and moderate cone degeneration, were used to determine whether stimulating TH signaling (with T3 treatment) deteriorates cones. We report here that cone survival was greatly improved in Rpe65^−/− and cpfl1 mice following TH signaling suppression, whereas cone degeneration was significantly increased in Cngb3^−/− and Gucy2e^−/− mice following TH signaling stimulation, demonstrating a protective role of suppressing TH signaling in cones.     

Bidirectional homeostatic plasticity induced by interneuron cell death and transplantation in vivo

Chronic changes in excitability and activity can induce homeostatic plasticity. These perturbations may be associated with neurological disorders, particularly those involving loss or dysfunction of GABA interneurons. In distal-less homeobox 1 (Dlx1^−/−) mice with late-onset interneuron loss and reduced inhibition, we observed both excitatory synaptic silencing and decreased intrinsic neuronal excitability. These homeostatic changes do not fully restore normal circuit function, because synaptic silencing results in enhanced potential for long-term potentiation and abnormal gamma oscillations. Transplanting medial ganglionic eminence interneuron progenitors to introduce new GABAergic interneurons, we demonstrate restoration of hippocampal function. Specifically, miniature excitatory postsynaptic currents, input resistance, hippocampal long-term potentiation, and gamma oscillations are all normalized. Thus, in vivo homeostatic plasticity is a highly dynamic and bidirectional process that responds to changes in inhibition.Prolonged changes in activity levels induce bidirectional changes in neuronal excitability and synaptic activity known as homeostatic plasticity (1, 2). This phenomenon has been described well at excitatory synapses and functions to maintain activity within a preferred dynamic range. Maintaining excitatory/inhibitory synaptic balance is critical for neuronal information processing and a potential problem when confronted with aberrant states of excitability, such as those associated with autism, schizophrenia, Alzheimer’s disease, or epilepsy (3–12).Chronic manipulation of synaptic input and/or action potential (AP) output rates in cortical and hippocampal cell cultures induces homeostatic synaptic scaling, in which the amplitude and then the frequency of pyramidal neuron miniature excitatory postsynaptic currents (mEPSCs) increase when activity is lowered or decrease when activity is raised (13–16). Recent studies have begun to reveal the underlying molecular mechanisms of homeostatic synaptic changes, including the AMPA receptor subunits, synapse-associated calcium-binding proteins, and intracellular signaling cascades involved (14, 17, 18). Changes to activity also trigger homeostatic plasticity of inhibitory synaptic transmission (19–23). Homozygous deletion of glutamate decarboxylase 1 (Gad1), the rate-limiting enzyme in the synthesis of GABA, reduced miniature inhibitory postsynaptic current (mIPSC) amplitudes in cultured hippocampal neurons but also blocked further homeostatic changes to mIPSCs. This suggests a key role for regulation of Gad1 expression in inhibitory homeostatic plasticity (23). Intrinsic excitability is also homeostatically regulated by activity. Changes in input resistance (Rin) and voltage-activated K⁺ and Na⁺ channel number (24–27), and in Na⁺ channel compartmentalization (28, 29), have been described following manipulations that chronically alter neuronal activity. Finally, in vivo manipulation of neuronal activity with TTX results in larger mEPSC amplitudes and reduced Rin of CA1 pyramidal neurons (30), suggesting that multiple mechanisms of homeostatic plasticity can occur simultaneously in the intact nervous system.Loss of GABAergic interneurons is common across different neurological disorders. It is unknown whether homeostatic plasticity can be induced by changes in activity related to interneuronopathy or how the combination of interneuron cell death and compensation alters circuit function. To begin to address these issues, we studied synaptic and intrinsic excitability in a hippocampal circuit in which a subpopulation of interneurons is reduced [i.e., distal-less homeobox 1 (Dlx1^−/−) mice] (31–33). At around 30 d of age, these mice lose a subset of somatostatin (Sst)-, calretinin (CR)-, vasoactive intestinal peptide-, and neuropeptide Y (NPY)-positive interneurons; exhibit decreased inhibitory synaptic activity in some brain regions; and subsequently develop epilepsy (31). Our results show that secondary to the in vivo interneuron loss is a homeostatic reduction in mEPSC frequency, decreased AMPA/NMDA ratio, and decreased intrinsic excitability in CA1 pyramidal neurons (that do not express Dlx1). Transplantation of GABA progenitor cells from the medial ganglionic eminence (MGE) (34) causes a reversal of the homeostatic changes in excitatory synaptic activity and Rin. Additionally, we describe unique changes in Dlx1^−/− circuit function that homeostatic compensation does not correct: enhanced long-term potentiation (LTP) and altered gamma frequency oscillations (GFOs). The severity of these phenotypes is reduced by interneuron transplantation. These studies demonstrate the responsiveness of excitatory circuitry to changes in inhibition, using homeostatic plasticity as a mechanism for maintaining excitatory/inhibitory balance.