key: cord-0005035-pgyzluwp authors: nan title: Programmed cell death date: 1994 journal: Experientia DOI: 10.1007/bf02033112 sha: e60ada12648bee985f33cede95b6e82eb9c971ad doc_id: 5035 cord_uid: pgyzluwp nan It is widely held that all developmental cell death is of a single type (apoptosis) and that neuronal death is primarily for adjusting the number of neurons in a population to the size of their target field through competition between equals for target-derived factors. We shall draw on our research and on that of others to criticize these views and replace them by the following. At least three types of neuronal death occur, only one of which resembles apoptosis; a neuron can choose between several self-destruct mechanisms depending on the cause of its death. The purpose of the death is to regulate connectivity, not neuron number. Competitors for trophic factors are unequal, and many losers have made axonal targeting errors. A neuron's survival and differentiation depend on multiple anterograde and retrograde signals. Activity affects retrograde signals and some but not all anterograde ones. The pattern of activity is more important than the overall amount. In rodents, the period of naturally occuring cell death of motoneurons is followed by a period of supersensitivity to axonal injury. Thus, in newborn rodents lesion of the facial nerve leads to a rapid degeneration of the injured motoneurons. We have tested whether overexpression, in rive, of the bcl-2 proto-oncogene was capable of preventing death of axotomized motoneurons. To address this question we used transgenic mice whose motoneurons overexpress the Bcl-2 protein. One of the two facial nerves of newborn mice was transected on the 2nd-3rd post-natal day. Seven days after the lesion, the morphology of the facial nuclei was analyzed. In control mice, and when compared to the intact nucleus, 70 to 80 % of axotomized motoneurons had disappeared. In contrast, in the transgenic animals, the number of motoneurons on the lesioned side remained unchanged when compared to the eontralateral nucleus. Furthermore, their axons remained visible up to the distal lesion site. These experiments show that, in rive, motoneurons overexpressing the Bcl-2 protein survive after axotomy, and suggest that, in rive, Bcl-2 protect neurons from experimentally induced cell death and could be a target for treatment of motoneurons degenerative diseases. Messmer S., Mattenberger L., Sager Y., Blatter-Garin M-C., Pometta D., Kate A., James R.W. Drpt de Mrdeeine, Drpt. de Pharmacologie, Div. de Neurophysiologie Clinique, Facult6 de Mrdecine, Gen~ve. Clusterin is a widely expressed glycoprotein, highly conserved across species. Numerous functions have been postulated for this protein. The most important are roles in lipid transport, as elusterin is associated with apolipoprotein AI in HDL, complement regulation and tissue remodelling, in particular during cell death and differentiation. Using cultures of rat spinal cord neurones (90% neurons and 5-10% non-neuronal cells), we have studied the expression of clusterin and ape E in glutamate-induced neuronal cell death to examine potential roles in lipid management. Up-regulation of the two proteins was observed. Clusterin and ape E appear in the conditioned medium respectively 15h and 7.5h after incubation with glutamate. Control studies, in the presence of a noncompetitive NMDA receptor agonist showed the secretion of clusterin and ape E to be diminished by >60%. No up-regulation of either protein was observed in complementary studies with exclusively non-neuronal cell cultures. The cellular origin of the 2 secreted proteins is presently under investigation. Programmed cell death and tissue remodelling are consequences of hormonally induced restructuring of the rat ventral prostate after castration and the rat mammary gland after weaning. We used the "Differential Display"-method (Liang and Pardee, 1992, Science 257:967) to detect and isolate eDNA fragments whose corresponding RNAs are regulated either coincidentally, or in an organ specific fashion during mammary gland involution and postcastrational prostate regression. Partial sequencing of 12 clones revealed high, but not absolute homology of 5 fragments with sequences, previously characterized in different biological contexts. These five encode functions which could be anticipated to be important for cell growth and/or programmed cell death, We are presently investigating the functions of several of these transcripts in cell culture and in rive. Antisense oligos are being employed in vivo to determine whether these genes contribute to the phenotype of programmed cell death. B epitopes derived from the envelope gp52 glycoprotein (EP3) or from the viral superantigen of MMTV have been incorporated into inert or live vaccines. The inert vaccine consists of purified chimeric proteins which contain the B epitopes alone or fused to multimeric promiscuous T helper epitopes from tetanus toxin. Mice were immunized subcutaneously with these chimeric proteins. The live vaccine consists of an avirulent strain of Salmonella typhimurium which expresses the MMTV epitopes in the form of chimeric proteins fused to the nucleocapsid protein of Hepatitis B Virus. This vaccine is given to mice in one oral dose. The level, duration and isotype of the immune response generated by each vaccine have been measured and compared. The level of protection has been investigated by systemically challenging immunized mice with the relzovims. A reduced binding of oxytocin (OT) occurs with aging in some, but not all, areas of the rat brain (Arsenijevic et al., Experientia 1993, 49, A75) . The candate putamen showed the most impressive loss of OT receptors. Two other regions, the hypothalamic ventromedial nucleus (VMH) and the islands of CaUeja (ICj) had also an important deficit of OT binding sites. On the other hand, these two regions were known to be sensitive to sex steroids. In the present work, we treated from 20 month old rats during one month with testosterone propionate (2 #g/kg s.c., once every 3 days) dissolved in oil. Three rats of the same age injected with oil only served as controls. We labelled OT receptors throughout the brain of old rats using a 125I-labelled ligand specific for OT receptors. Analysis of autoradiograms by an image analyzer revealed that the testosterone treatment increased OT binding sites in the VMH, in the ICj, and, to a lesser extent, in the bed nucleus of the stria terminalis, a region also sensitive to sex steroids, By contrast, in the caudate putamen, the disappearance of OT receptors was not compensated. In conclusion, the decrease of OT receptors occurring in VMH and ICj with aging can be reversed by administration of gonadal steroids. In contrast, the loss of OT receptors in the striatum appears to depend on another mecanism. Vasopressin (AVP) receptors are expressed transiently in the facial nucleus during development (Tribollet et el., 1991, Dev. Brain Res., 58, 13-24) . AVP may therefore play a role in the maturation of neuromuscular connexions in the neonate rat, and possibly in the restanration of these connexions after nerve lesion in the adult. In order to investigate the latter proposition, we have sectionned the facial nerve in adult rats and used quantitative autoradiography to look at AVP binding sites in the facial nucleus at various postoperative times. We observed a massive and transient increase of AVP binding sites on the operated side. The number of facial AVP binding sites reaches a maximum about one week after nerve section, remains stable during 2-3 weeks, then begin to decrease towards control level. The induction of AVP receptors is markedly delayed if the proximal stump of the nerve is ligated. To assess whether other motor nuclei would also react to axotomy by up-regulating the expression of AVP receptors, we have sectionned the hypoglossal nerve and the sciatic nerve. In both cases, the binding of AVP receptor ligand increases massively in the respective motor nuclei, with a time-course similar to that found in the facial nucleus. Altogether, our data suggest that central AVP could be involved in the process of nerve regeneration. CYTOTOXIC T-CELL MEDIATED APOPTOSIS Schaerer,E, Karapetian,O.,Adrian,M. and Tschopp,J. Inst.de Biochimie, Univ.de Lausanne, 1066 Epalinges. An apoptotic cell death mechanism is used by cytolytic T cells (CTL) to lyse appropriate target cells. CTL harbor cytoplasmic storage compartments, containing the lytic protein perforin and serineproteases (granzymes), whose content is released upon target cell interaction. We show that these granules are multivesieular bodies and that degranulation releases these intragranular vesicles (IGV) having granzymes, T-cell receptor and yet undefined proteins associated. Isolated IGVs and perforin induce DNA breakdown in target cells within 20 minutes. Microscopic analysis demonstrates that IGV specifically interact with target cell via the T-cell receptor and that their contents is taken up by the target cell. Already 15 min. after interaction, 3 distinct IGV proteins are found in the nucleus of the target cell.One of the molecules has been identified to be granzyme A, previously reported to be involved in apoptosis. We propose that lymphocytes transfer apoptosisinducing proteins to the nucleus of the target cells using vesicles as vehicles for delivery. Cytotoxic T cells kill their targets by a mechanism involving membranolysis and DNA degradation (apoptosis). Recently, two sets of proteins have been proposed as DNA breakdown-inducing molecules in T cells: granzyme A, B and TIA-I. In this study, we cloned and further characterized the TIA-I mouse homologue. Aa sequence comparison with the human TIA-1 showed an overall identity of 93%. Devoid of a signal peptide, TIA is yet localized to cytotoxic granules, probably targeted via a Gly-Tyr-motif. As TIA-I, its mouse homolcgue contains three RNAbinding domains. Expression of TIA during development shows a very strong signal in the brain and weaker signals in thymus, heart and other organs. During embryonic development several structures that contribute to organogenesis form transiently and are later eliminated by apoptosis. This pattern of TIA expression could indicate its involvement in apoptosis. Prostate involution occurs after castration in rats and is associated with the death by apoptosis of a large fraction of the epithelial cells. We have isolated several genes from a prostate involution bacteriophage lambda library using differential screening methods. Among these clones, one d~monstrated an especially strong signal when used as a probe against Northern blots of prostate mlhNA obtained before, and at different times after castration. This gene is down-regulated after castration by 40-fold within 5 days. Intramuscular injection of a testosterone depot resulted in complete restoration of expression within 24 hours. Upon sequencing it became apparent that this clone has a high degree of homology to a known NDAH dehydrogenase encoded in mitochondrial DNA. The clone failed to hybridize to any transcripts from rat organs other than prostate. We are now in the process of isolating the htm~n hc~olog to this gene for use as a biomarker in study of benign hyperplasia and developing carcinoma. This gene is a possible indicator for testosterone-independent cell populations or of cells lacking ftl~ctional testosterone receptor. During the first three postnatal weeks the rat lung undergoes the last two developmental stages, the phase of alveolarization and the phase of microvascular maturation. The latter involves a decrease of the connective tissue mass in the alveolar septa and a merging of the two capillary layers to a single one. Speculating that programmed cell death may play a role during this remodeling, we searched for the presence of apoptotie cells in rat lungs between days 10 and 24. Lung paraffin sections were treated with Y-terminal transferase, digoxigenin-dUTP, and anti-digoxigeninfluorescein-F(ab)-fragments, and the number of fluorescent nuclei was compared between sections at different days. While the number of apoptotie ceils was low until the end of the second week and at day 24, we observed an about eight fold increase of fluorescent nuclei towards the end of the third week. We conclude that programmed cell death is involved in the structural maturation of the lung. Brunner, A., Wallrapp, Ch., Pollack, I, Twardzik, T. and Schneuwly, S. Lehrstuhl Genetik, Biozentrum Universit~t W~rzburg, Mutants in the giant lens (g/l) gene show a strong disturbance in ommatidial development. In the absence of any gene product, additional phetoreceptors, cone cells and pigment cells develop. Opposite effects can be seen in flies in which the gene product of the giant lens gene can be ectopically expressed by heat shock. A second very typical phenotype is the disturbance of photoreceptor axon guidance. Molecular analysis of gil shows that it encodes a secreted protein of 444aa containing three evolutionary conserved cystein-motives very similar to EGF-like repeats. We propose that gil functions as a secreted signal, most likely a lateral inhibitor for the development of specific cell fates and that gil, either directly or indirectly, is involved in targeting photoreceptor axons into the brain. THE DECREASE IN CELLULARITY DURING SCAR ESTABLISHMENT IS MEDIATED THROUGH APOPTOSIS DESMOULIERE, A., REDARD, M., DARBY, I., AND G. GABBIANI Department of Pathology, CMU, 1 rue Michel Server, 1211 Gen~ve 4 Dudng the healing of an open wound, granulation tissue formation is characterized by replication and accumulation of fibroblastic cells, many of which acquire morphological and biochemical features of smooth muscle cells and have been named myofibroblasts (Sch0rch et el., Histology for Pathologists, t992). As the wound evolves into a scar, there is an important decrease in ceUuladty, including disappearance of myofibroblasts. The question adses as to which process is responsible for myofibroblast disappearance. During a previous investigation on the expression of (z-smooth muscle actin in myofibroblasts, we have obsewed that in late phases of wound healing, many of myofibroblasts show signs of apoptosis end suggested that this type of cell death is responsible for the disappearance of myofibroblasts (Darby et al., Lab. Invest. 63:21, 1990) . We have tested this hypothesis by means of electron microscopy and morphometry and by in situ end-labeling of fragmented DNA (Wijsman et al., J. Histochem. Cytochem. 41:7, t 993) . Our results show that the number of apoptotic cells increases as the wound closes and suggest that this may be the mechanism for the disappearance of myofibroblasts as well as for the evolution of granulation tissue into a scar. (Supported by the Swiss National Science Foundation, Grant n~ S01-16 R. Jaggl, A. Marti and B. Jehn. Universit~t Bern, AKEF, Tiefenaustr. 120, 3004 Bern At weaning the mammary gland undergoes a reductive remodelling process (involution) which is associated with the cessation of milk protein gene expression and apoptosis of milk-produclng epithelial cells. This process can be reversed by returning the pups to the mother within 1 day. Elevated nuclear protein kinase A (PKA) activity was observed from one day post-lactation, paralleled by increased c-los, junB, ]unD and to a lesser extent c-]un mRNA levels. AP-1 DNA binding activity was transiently induced and the AP-1 complex was shown to consist principally of cFos/JunD. Oct-1 DNA binding activity and Oct-1 protein were gradually lost from the gland over the first four days of involution, whereas Oct-1 m_RNA levels remained unchanged. Comparing nuclear extracts from normal mammary glands with nuclear extracts from glands which had been cleared of all epithelial cells three weeks after birth revealed that PKA activation, AP-1 induction and Oct-1 inactivation are all dependent on the presence of the epithelial compartment. The increased Fos/Jtm expression and the inactivation of Oct-1 may be consequences of the increased PKA activity. When involution is reversed, both, PICA activity and AP-1 DNA binding activity (and fos andjun mRNA levels) are reduced to basal levels. Our data suggests a role for PKA and AP-1 on progranlmed cell death of manlnmry epithelial ceils. Bcl-2~ does not require membrane attachment for its survival activity C. Borner*, I. Martinout, C. Mattmann*, M. Irmler*, E. Sch&rrer*, J.-C. Martinou-j-, and J. Tschopp*. * Institute of Biochemistry, University of Lausanne, 1066 Epalinges, 1 Institute of Molecular Biology, Glaxo Inc.,1228 Plan los Ouates. 8cl-2(z is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. How it exerts this activity is unknown but it is believed that membrane attachment is required. To identify critical regions in bcl-2o~ for subcellular localization and survival activity, we created by site-directed mutagenesis, various mutations in regions which are most conserved between the different bcl-2 species. We show here that membrane attachment is not required for the survival activity of bcl-2o< A truncation mutant of bcl-2(z lacking the last 33 amino acids (T3) including the hydrophobic domain is soluble, yet fully active in blocking apoptosis of sympathetic neurons induced by NGF deprivation or L929 fibroblasts induced by TNFc~ treatment. We further provide evidence for a putative functional region in bcl-2 which lies in the conserved domains 4 and 5 upstream of the hydrophobic COOH terminal tail. The breakdown of nuclear DNA is considered to be a hallmark of apoptosis. We previously identified the perinuclear membrane localized DNase I as the endonuclease involved in the formation of oligonucleosomal-sized fragments (DNA ladder). It is not clear how the nuclease is activated and has access to the DNA. We show that in thymocytes induced to undergo apoptosis, lamin breakdown preceded DNA laddering. By transfeeting HeLa cells with a constitutively active cdc2 mutant, nuclear envelope breakdown and typical apoptotic features (ehromatin condensation) were observed. Moreover, co-transfection with cdc2 mutant and DNase I led to DNA degradation. We propose that apoptosis can be induced by wrongly timed and hence abortive mitosis leading to uncontrolled nuclear membrane disintegration. S02-01 S02-04 Platelet-derived growth factor (PDGF) is thought to play an active role in fibrosing diseases. Bronchiolitis obliterans-organizing pneumonia (BOOP) is a condition characterized by intraluminal proliferation of connective tissue inside distal air spaces. To evaluate PDGF expression in BOOP we performed immunohistoehemistry on lung biopsies from 20 patients and 10 controls free of fibrosis. Sedal sections were stained with an antibody against either PDGF or the monoeyte/macrophage marker CD68, In both groups the PDGF ~9 cells were essentially tissue macrophages. Using point counting to measure volume fraction (Vv) , PDGF-pesitive cells represented 4.65+1.63% (mean+SD) of the volume occupied by lung tissue in the BOOP cases, and 2,12+0.65% in the controls (!0<0,001). Similarily, 10.73+4.69% of the lung tissue was occupied by CD68 E~ macrophages in the BOOP cases, compared to 5.37:~3.73% in the controls (p