Haploinsufficiency of the 22q11.2 microdeletion gene Mrpl40 disrupts short-term synaptic plasticity and working memory through dysregulation of mitochondrial calcium.

Hemizygous deletion of a 1.5- to 3-megabase region on chromosome 22 causes 22q11.2 deletion syndrome (22q11DS), which constitutes one of the strongest genetic risks for schizophrenia. Mouse models of 22q11DS have abnormal short-term synaptic plasticity that contributes to working-memory deficiencies similar to those in schizophrenia. We screened mutant mice carrying hemizygous deletions of 22q11DS genes and identified haploinsufficiency of Mrpl40 (mitochondrial large ribosomal subunit protein 40) as a contributor to abnormal short-term potentiation (STP), a major form of short-term synaptic plasticity. Two-photon imaging of the genetically encoded fluorescent calcium indicator GCaMP6, expressed in presynaptic cytosol or mitochondria, showed that Mrpl40 haploinsufficiency deregulates STP via impaired calcium extrusion from the mitochondrial matrix through the mitochondrial permeability transition pore. This led to abnormally high cytosolic calcium transients in presynaptic terminals and deficient working memory but did not affect long-term spatial memory. Thus, we propose that mitochondrial calcium deregulation is a novel pathogenic mechanism of cognitive deficiencies in schizophrenia.

The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant.

Release of cytochrome c from mitochondria triggers activation of caspase proteases and death of a cell by apoptosis. However, the mechanism and kinetics of cytochrome c release remain unknown. Here we study this event by using green fluorescent protein (GFP)-tagged cytochrome c, and find that the release of cytochrome-c-GFP always precedes exposure of phosphatidylserine and the loss of plasma-membrane integrity - characteristics of apoptotic cells. Once initiated, the release of cytochrome- c-GFP continues until all of the protein is released from all mitochondria in individual cells, within about 5 minutes, regardless of the type or strength of stimulus or the time elapsed since the stimulus was applied. Temperatures ranging from 24 degrees C to 37 degrees C do not change the duration of release, and nor does the addition of caspase inhibitors. Further, we find that the electron-transport chain can maintain the mitochondrial transmembrane potential even after cytochrome c has been released.

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome.

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

T cells are just dying to accept grafts.

Two papers suggest that apoptosis of alloreactive T cells is required for induction of peripheral transplantation tolerance. These findings raise new questions about how lymphocytes reach from beyond the grave to influence the immune response (pages 1298-1302 and 1303-1307).

HIV-1 Vpr suppresses immune activation and apoptosis through regulation of nuclear factor kappa B.

The HIV-1 accessory gene product Vpr can influence viral pathogenesis by affecting viral replication as well as host cell transcription and proliferation. We have investigated the effects of Vpr on host cell activation and confirm that it influences cellular proliferation. However, we have also found that Vpr modulates T-cell receptor (TCR)-triggered apoptosis in a manner similar to that of glucocorticoids. In the absence of TCR-mediated activation, Vpr induces apoptosis whereas in its presence, Vpr interrupts the expected induction of apoptosis. This regulation of apoptosis is linked to Vpr suppression of NF-kappa B activity via the induction of I kappa B, an inhibitor of NF-kappa B. Further, Vpr suppresses expression of IL-2, IL-10, IL-12, TNF alpha and IL-4, all of which are NF-kappa B-dependent. The effects of Vpr could be reversed by RU486. Our finding that Vpr can regulate NF-kappa B supports the hypothesis that some aspects of viral pathogenesis are the consequence of cell dysregulation by Vpr.

Role of contrasuppression in the adoptive transfer of immunity.

The data presented in this paper show that the population of cells that adoptively transfer contact hypersensitivity are Lyt-1+ 2-, I-J- and nonadherent to V. villosa lectin. However, the adoptive transfer of immunity by this population of cells is successful only when the recipient has been treated in such a way as to impair the host immunosuppression mechanism. This population cannot, on its own, transfer immunity to adult, untreated naive recipients unless an additional population of immunoregulatory cells is present. This immunoregulatory population does not itself adoptively transfer immunity. This latter population is differentiated from the immune cells in that they are Lyt-1+ 2-, I-J+ and are adherent to V. villosa lectin. Both populations are required to adoptively transfer immunity to adult untreated naive recipients.

Functional Myc-Max heterodimer is required for activation-induced apoptosis in T cell hybridomas.

T cell hybridomas respond to activation signals by undergoing apoptotic cell death, and this is likely to represent comparable events related to tolerance induction in immature and mature T cells in vivo. Previous studies using antisense oligonucleotides implicated the c-Myc protein in the phenomenon of activation-induced apoptosis. This role for c-Myc in apoptosis is now confirmed in studies using a dominant negative form of its heterodimeric binding partner, Max, which we show here inhibits activation-induced apoptosis. Further, coexpression of a reciprocally mutant Myc protein capable of forming functional heterodimers with the mutant Max can compensate for the dominant negative activity and restore activation-induced apoptosis. These results imply that Myc promotes activation-induced apoptosis by obligatory heterodimerization with Max, and therefore, by regulating gene transcription.

Transforming growth factor beta1 inhibits Fas ligand expression and subsequent activation-induced cell death in T cells via downregulation of c-Myc.

Activation-induced cell death (AICD) is a process that regulates the size and the duration of the primary immune T cell response. In this report, we investigated the mechanisms involved in the regulation of AICD by transforming growth factor beta1 (TGF-beta1). We found that TGF-beta1 decreased apoptosis of human T cells or T cell hybridomas after activation by anti-CD3. This decrease was associated with inhibition of Fas (Apo-1/CD95) ligand (FasL) expression, whereas Fas signaling was not affected by TGF-beta1. In parallel, TGF-beta1 inhibited c-Myc expression in T cell hybridomas, and ectopic expression of a chimeric molecule composed of c-Myc and the steroid binding domain of the estrogen receptor (Myc-ER) blocked both the inhibition of FasL and the decrease of AICD induced by TGF-beta1, providing that 4-hydroxytamoxifen was present. These results identify one mechanism by which TGF-beta1 blocks AICD to allow the clonal expansion of effector T cells and the generation of memory T cells during immune responses.

Selective cleavage of nuclear autoantigens during CD95 (Fas/APO-1)-mediated T cell apoptosis.

Intracellular proteases appear to be important mediators of apoptosis. Substrates cleaved by proteases during apoptosis include nuclear autoantigens targeted in systemic autoimmune diseases. Using human autoantibodies as probes, we demonstrate here that T cell apoptosis mediated by CD95 (Fas/APO-1) is associated with substantial cleavage of a subset of nuclear autoantigens (7 of 33 examined). This subset included poly (ADP-ribose) polymerase, the 70-kD protein of the U1 small nuclear ribonucleoprotein particle, lamin B, the nuclear mitotic apparatus protein NuMA, DNA topoisomerases I and II, and the RNA polymerase I upstream binding factor UBF. Several of the cleaved autoantigens are involved in ensuring the integrity and proper conformation of DNA in the nucleus through interactions with the nuclear matrix, suggesting the possibility that their cleavage may contribute to the collapse of nuclear structure during apoptosis. The relative cleavage kinetics indicated that the autoantigens were targeted at various times after induction of apoptosis, suggesting either differential accessibility or activation of distinct proteases during the cell death process. These data reinforce the hypothesis that apoptosis is accompanied by selective cleavage of key substrates and not by a generalized degradation of intracellular material.

Immunoregulatory circuits that modulate responsiveness to suppressor cell signal. Failure of B10 mice to respond to suppressor factors can be overcome by quenching the contrasuppressor circuit.

The in vitro antibody response of spleen cells from B10 strain mice is not suppressed by factor preparations made by primed Ly-2 T cells, although these preparations can suppress the in vitro antibody response of spleen cells from other mouse strains (1-3)2. The factor preparations from Ly-2 cells contain at least two separable activities: one that acts as a suppressor moiety (Ly-2 T cell suppressor factor [Ly-2 TsF]) and a second factor that acts as an inducer of contrasuppression (Ly-2 TcsiF); the latter initiates a series of cellular interactions that leads to the inhibition of suppression that we refer to as contrasuppression. Removal of components (either cellular or humoral) of the contrasuppressor circuit makes spleen cells from B10 strain mice as easily suppressible as are those of other mouse strains. Thus, removal of the contrasuppressor inducer cell and/or its biologically active product with the use of an anit-J serum, or removal of the functional acceptor of the inducer cell with the same or other (Ly-2; Qa-1) antisera breaks the B10 suppressor barrier. Contrasuppressive activity. but not helper activity can be eluted from anit-I-J immunoabsorbents. The addition of B10 T cells to either B6 or B10 spleen cell culture deprived of acceptor cells for the TcsiF reconstitutes contrasuppression more efficiently than does the addition of C57BL/6 T cells. Ly-2 TcsiF is more cross-reactive than is Ly-2 TsF so that absorption of factor preparations from sheep erythrocyte-primed Ly-2 cells with horse erythrocytes also breaks the B10 suppressor barrier. The hyperresponsiveness of splenic T cells from B10 strains to Ly-2 TcsiF may be an in vitro exaggeration of a normal in vivo process. Thus it is possible that one can take advantage of this unusual situation to help dissect out the cellular and subcellular components of T cell circuits that moldulate sensitivity to immunoregulatory signals.

Lymphoma models for B cell activation and tolerance. X. Anti-mu-mediated growth arrest and apoptosis of murine B cell lymphomas is prevented by the stabilization of myc.

Treatment of the WEHI-2131 or CH31 B cell lymphomas with anti-mu or transforming growth factor (TGF)-beta leads to growth inhibition and subsequent cell death via apoptosis. Since anti-mu stimulates a transient increase in c-myc and c-fos transcription in these lymphomas, we examined the role of these proteins in growth regulation using antisense oligonucleotides. Herein, we demonstrate that antisense oligonucleotides for c-myc prevent both anti-mu- and TGF-beta-mediated growth inhibition in the CH31 and WEHI-231 B cell lymphomas, whereas antisense c-fos has no effect. Furthermore, antisense c-myc promotes the appearance of phosphorylated retinoblastoma protein in the presence of anti-mu and prevents the progression to apoptosis as measured by propidium iodide staining. Northern and Western analyses show that c-myc message and the levels of multiple myc proteins were maintained in the presence of antisense c-myc, results indicating that myc species are critical for the continuation of proliferation and the prevention of apoptosis. These data implicate c-myc in the negative signaling pathway of both TGF-beta and anti-mu.

Isotype-specific immunoregulation. Evidence for a distinct subset of T contrasuppressor cells for IgA responses in murine Peyer's patches.

The ability of murine Peyer's patch (PP) T contrasuppressor cells (Tcs) to reverse oral tolerance to the T cell-dependent (TD) antigen SRBC was studied both in vivo and in vitro. C3H/HeJ mice given SRBC orally for 4 wk are not rendered tolerant to this antigen and were used as a source of PP Tcs cells for adoptive transfer to identically treated, orally tolerized C3H/HeN mice. Transfer of 10(4) or 5 X 10(4) V. villosa-adherent PP T cells resulted in splenic IgM, IgG, and mainly IgA responses in C3H/HeN mice challenged systemically with SRBC. The T cell responsible was Lyt-1+, 2-, L3T4-, I-JK+ and V. villosa lectin-adherent, all characteristics of mature effector Tcs cells. This C3H/HeJ PP Tcs cell subset was also effective when added to in vitro cultures of tolerized spleen cells derived from SRBC-fed, C3H/HeN mice. Interestingly, C3H/HeJ PP Tcs cells restored mainly IgA responses when transferred in vivo or when added to suppressed C3H/HeN splenic cultures. Comparison of the functional activity of Tcs cells derived from spleen or PP of orally immunized C3H/HeJ mice revealed that splenic Tcs cells supported responses of all 3 isotypes; however, PP Tcs cells yielded three-fourfold higher IgA responses, when compared with IgM or IgG anti-SRBC responses. Adherence of C3H/HeJ PP Tcs to an Fc alpha R+ T cell line derived from IgA-specific Th cells resulted in a nonadherent cell fraction that potentiated only IgM and IgG responses, while bound Tcs cells preferentially supported IgA responses. These results suggest that murine PP contain IgA-specific Tcs cells that allow IgA response induction in the presence of Ts cells that mediate oral tolerance.

Granzyme B-mediated cytochrome c release is regulated by the Bcl-2 family members bid and Bax.

Cytotoxic T lymphocytes (CTLs) destroy target cells through a mechanism involving the exocytosis of cytolytic granule components including granzyme B (grB) and perforin, which have been shown to induce apoptosis through caspase activation. However, grB has also been linked with caspase-independent disruption of mitochondrial function. We show here that cytochrome c release requires the direct proteolytic cleavage of Bid by grB to generate a 14-kD grB-truncated product (gtBid) that translocates to mitochondria. In turn, gtBid recruits Bax to mitochondria through a caspase-independent mechanism where it becomes integrated into the membrane and induces cytochrome c release. Our results provide evidence for a new pathway by which CTLs inflict damage and explain the caspase-independent mechanism of mitochondrial dysfunction.

Contrasuppression. A novel immunoregulatory activity.

We have described an interaction between two T cells subsets that results in interference with the expression of Ly-1-, 2+ (Ly-2) T cell-mediated suppression. We refer to this novel immunoregulatory activity as contrasuppression. The T cell responsible for the induction of contrasuppression (inducer cell) expresses the phenotype Ly-1-, 2+;I-J+;Qa-1+. This phenotype distinguishes it from the suppressor effector cells which we find to be I-J-2.3. An I-J+ soluble mediator from the contrasuppressor inducer cell acts on another cell (acceptor cell) that expresses the phenotype Ly-1+, 2+; I-J+; Qa-1+. This phenotype distinguishes it from T helper cells. Both the inducer cell (or its biologically active mediator) and its acceptor cell are required for the expression of contrasuppression. Because contrasuppressor cells can block the suppressive activity of cell-free mediators released by Ly-2 suppressor T cells, the mechanism of contrasuppression is either separated from or in addition to the inactivation of suppressor cells themselves. The potential importance of contrasuppressor activity in the regulation of suppressor T cell activity in allowing immunologic memory to be expressed and in permitting microenvironmental immune regulation is discussed.

Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.

A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.

Antibody profiling of a Borreliella burgdorferi (Lyme disease) C6 antibody positive, symptomatic Rottweiler and her pups.

Lyme disease (LD) is a tick-transmitted disease caused by Borreliella burgdorferi (Bb). Temporal studies of maternal antibody (Ab) profiles in Bb infected pregnant dogs and their pups have not been conducted. In this study, Ab profiles of a client-owned Bb C6 Ab positive Rottweiler and her nine pups were assessed. The dam presented with lameness 12 days prior to parturition and was C6 Ab positive with a Quant C6 Ab concentration of 237U/mL. Treatment with amoxicillin was initiated and 11 days later nine pups were delivered. Screening of the sera from the dam and pups against Bb cell lysates and a panel of antigens revealed similar immunoreactivity profiles. While antigen-specific IgG and IgM reactivity persisted in the dam for at least 7 months, a rapid decline in IgG specific for BBA36, BBK53, BB0238, BBA73 and outer surface protein (Osp) E in the pups occurred between days 29 and 52 post-parturition. In contrast, Ab specific for DbpA and the diagnostic antigens VlsE (C6) and OspF, remained elevated in the pups. Sera from the dam displayed potent complement-dependent bactericidal activity against Bb. Sera from the pups was also bactericidal but primarily through a complement-independent mechanism. Lastly, single dose vaccination of the dam at day 51 post-parturition with a LD subunit vaccine consisting of OspA and an OspC chimeritope triggered a broad anti-OspC Ab response indicative of an anamnestic response. Although this study focused on a single case, these findings add to our knowledge of maternal Ab profiles and will aid the interpretation of serological assays in pups delivered by a Bb C6 Ab positive dog.

Living with death: the evolution of the mitochondrial pathway of apoptosis in animals.

The mitochondrial pathway of cell death, in which apoptosis proceeds following mitochondrial outer membrane permeabilization, release of cytochrome c, and APAF-1 apoptosome-mediated caspase activation, represents the major pathway of physiological apoptosis in vertebrates. However, the well-characterized apoptotic pathways of the invertebrates C. elegans and D. melanogaster indicate that this apoptotic pathway is not universally conserved among animals. This review will compare the role of the mitochondria in the apoptotic programs of mammals, nematodes, and flies, and will survey our knowledge of the apoptotic pathways of other, less familiar model organisms in an effort to explore the evolutionary origins of the mitochondrial pathway of apoptosis.

Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways.

Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.

Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity.

Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.