Apoptotic stimuli have been shown to trigger lysosomal membrane permeability (LMP), leading to the release of cathepsins, which activate death signaling pathways in the cytosol. However, it is unknown whether this process is an initiating or amplifying event in apoptosis. In this study, we used fibroblasts and monocytes exposed to etoposide, ultraviolet light, FasL or deprived of interleukin-3 (IL-3) to show that LMP and the cytosolic release of cathepsins B, L and D consistently depends on Bax/Bak and components of the apoptosome. Neither Bax nor Bak resided on the lysosomes, indicating that lysosomes were not directly perforated by Bax/Bak but by effectors downstream of the apoptosome. Detailed kinetic analysis of cells lacking cathepsin B or L or treated with the cysteine protease inhibitor, E64d, revealed a delay in these cells in etoposide-and IL-3 deprivation-induced caspase-3 activation and apoptosis induction but not clonogenic survival, indicating that cathepsins amplify rather than initiate apoptosis.

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, have been implicated in the development of the retinotectal projection. Here, glycosylphosphatidylinositol-anchored A-ephrins are not only expressed in the tectum but also on retinal axons, raising the possibility that they function in this context as receptors. We now show that activation of ephrin-A2 or ephrin-A5 by one of their receptors, ephA3, results in a ␤1-integrin-dependent increased adhesion of ephrin-A-expressingcellstolaminin.Inthesearchforanephrin-Adependent signaling pathway controlling integrin activation, we identified a 120-kDa raft membrane protein that is tyrosine-phosphorylated specifically after ephrin-A activation. Tyrosine phosphorylation of this protein is not seen after stimulating ephrin-A2-expressing cells with basic fibroblast growth factor, epidermal growth factor, insulin growth factor, or fetal calf serum containing a large set of different growth factors. The role of p120 as a mediator of an ephrin-A-integrin coupling is supported by the finding that inhibiting tyrosine phosphorylation of p120 correlates with an abolishment of the ␤1-dependent cell adhesion.During development of the retinotectal projection, members of the Eph family of receptor tyrosine kinases and their "ligands", the ephrins, are strongly involved in guiding retinal axons to, and in, the tectum (for review see Refs. 1-3). Besides the graded expression of Eph receptors on retinal axons and of ephrins in the tectum, the ephrin-As are also differentially expressed on retinal axons themselves (4, 5). Gain of function and loss of function analyses suggest that here the ephrin-As modulate the function of the coexpressed receptors in that coexpression of ligands and receptors runs in parallel to a decrease in sensitivity for the repellent activity of the tectally expressed ephrins (4, 5). These findings fit to subsequent results of in vivo analyses of ephrin-A5 single and ephrin-

Endoplasmic reticulum-associated aminopeptidase 1 (ERAP1) is involved in the final processing of endogenous peptides presented by MHC class I molecules to CTLs. We generated ERAP1-deficient mice and analyzed cytotoxic responses upon infection with three viruses, including lymphocytic choriomeningitis virus, which causes vigorous T cell activation and is controlled by CTLs. Despite pronounced effects on the presentation of selected epitopes, the in vivo cytotoxic response was altered for only one of several epitopes tested. Moreover, control of lymphocytic choriomeningitis virus was not impaired in the knockout mice. Thus, we conclude that lack of ERAP1 has little influence on antiviral immunohierarchies and antiviral immunity in the infections studied. We also focused on the role of ERAP1 in cross-presentation. We demonstrate that ERAP1 is required for efficient cross-presentation of cell-associated Ag and of OVA/anti-OVA immunocomplexes. Surprisingly, however, ERAP1 deficiency has no effect on cross-presentation of soluble OVA, suggesting that for soluble exogenous proteins, final processing may not take place in an environment containing active ERAP1.

Loss of plasticity‐related gene 1 (PRG‐1), which regulates synaptic phospholipid signaling, leads to hyperexcitability via increased glutamate release altering excitation/inhibition (E/I) balance in cortical networks. A recently reported SNP in prg‐1 (R345T/mutPRG‐1) affects ~5 million European and US citizens in a monoallelic variant. Our studies show that this mutation leads to a loss‐of‐PRG‐1 function at the synapse due to its inability to control lysophosphatidic acid (LPA) levels via a cellular uptake mechanism which appears to depend on proper glycosylation altered by this SNP. PRG‐1+/− mice, which are animal correlates of human PRG‐1+/mut carriers, showed an altered cortical network function and stress‐related behavioral changes indicating altered resilience against psychiatric disorders. These could be reversed by modulation of phospholipid signaling via pharmacological inhibition of the LPA‐synthesizing molecule autotaxin. In line, EEG recordings in a human population‐based cohort revealed an E/I balance shift in monoallelic mutPRG‐1 carriers and an impaired sensory gating, which is regarded as an endophenotype of stress‐related mental disorders. Intervention into bioactive lipid signaling is thus a promising strategy to interfere with glutamate‐dependent symptoms in psychiatric diseases.

Alterations in dendritic spine numbers are linked to deficits in learning and memory. While we previously revealed that postsynaptic plasticity-related gene 1 (PRG-1) controls lysophosphatidic acid (LPA) signaling at glutamatergic synapses via presynaptic LPA receptors, we now show that PRG-1 also affects spine density and synaptic plasticity in a cell-autonomous fashion via protein phosphatase 2A (PP2A)/β1-integrin activation. PRG-1 deficiency reduces spine numbers and β1-integrin activation, alters long-term potentiation (LTP), and impairs spatial memory. The intracellular PRG-1 C terminus interacts in an LPA-dependent fashion with PP2A, thus modulating its phosphatase activity at the postsynaptic density. This results in recruitment of adhesome components src, paxillin, and talin to lipid rafts and ultimately in activation of β1-integrins. Consistent with these findings, activation of PP2A with FTY720 rescues defects in spine density and LTP of PRG-1-deficient animals. These results disclose a mechanism by which bioactive lipid signaling via PRG-1 could affect synaptic plasticity and memory formation.