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S Isenmann

Publications and source records attributed to S Isenmann.

At least 19 recordsLinked to original sources
PubMed2001

Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis.

Decoy receptor 3 (DcR3) is a newly identified soluble protein that binds to CD95 ligand (CD95L) and inhibits its proapoptotic activity. Here we report that DcR3 is expressed by the majority of long-term and ex vivo malignant glioma cell lines as well as in human glioblastoma in vivo. Expression of DcR3 correlates with the grade of malignancy: 15 of 18 (83%) glioblastomas (WHO grade IV) but none of 11 diffuse astrocytomas (WHO grade II) exhibited DcR3 immunoreactivity. We also demonstrate that human malignant glioma cells engineered to release high amounts of DcR3 into the cell culture supernatant are protected from CD95L-induced apoptotic cell death. In contrast, DcR3 does not confer protection from the death ligand Apo2 ligand (TRAIL). Importantly, ectopic expression of DcR3 resulted in substantial differences in immune cell infiltration in the 9L rat gliosarcoma model. Thus, the infiltration of CD4+ and CD8+ T cells as well as microglia/macrophages into glioma was substantially decreased in DcR3-producing tumors compared with control tumors. Chemotaxis assays revealed that DcR3 counteracts the chemotactic activity of CD95L against microglial cells in vitro. These findings suggest that DcR3 may be involved in the progression and immune evasion of malignant gliomas.

Animals↗
PubMed2001

Intravitreal adenoviral gene transfer evokes an immune response in the retina that is directed against the heterologous lacZ transgene product but does not limit transgene expression.

Recombinant E1-deleted adenoviral vectors (DeltaE1-Ad) are promising tools for in vivo gene transfer into the mammalian CNS including the retina. However, the duration of transgene expression is limited, and this limitation has partly been attributed to an immune response directed against vector-derived proteins. Here, we employed immunocytochemistry to assess the immune response to intravitreously injected DeltaE1-Ad encoding the lacZ gene or various neurotrophins (NTs). beta-Galactosidase was expressed by retinal cells for up to 4 weeks. Following intravitreal inoculation of AdCMV-lacZ, microglial and T cells were detected with a panel of antibodies in the retinal cell layers after 2 days (D2). The inflammatory response reached a maximum between D7 and D14. In contrast, no immune response was seen following injection of Ad encoding NTs. Yet, like with Ad-CMV-lacZ, their expression was also limited to approximately 4 weeks. Thus, beta-galactosidase seems to trigger a host immune response following intravitreal adenoviral lacZ gene transfer, but immune responses are not the cause of limited NT transgene expression from the CMV promoter in the inner retina.

Adenoviridae↗
PubMed2001

Neuron-specific expression of therapeutic proteins: evaluation of different cellular promoters in recombinant adenoviral vectors.

In order to achieve neuron-restricted expression of antiapoptotic proteins, cellular promoters were investigated for their expression profiles in the context of adenoviral vectors. Both the synapsin 1 gene and the tubulin alpha1 gene promoters were strictly neuron specific in cocultures of primary neurons with their essential feeder cells. The neuron-specific enolase gene promoter exhibited only weak activity in cultured hippocampal neurons and was not neuron specific in preparations of cerebellar granule cells. By attaining virtually 100% transduction efficiency we were able to generate "quasi-transgenic" primary neuron cultures using both differentiated and completely undifferentiated hippocampal neurons. In a functional assay, we used the synapsin promoter to evaluate the effect of Bcl-X(L) overexpression on potassium-withdrawal-induced apoptosis of cerebellar granule neurons. We found nearly complete inhibition of caspase-9 and -3 activation and apoptosis, indicating a major role for mitochondrial pathways in this paradigm of neuronal cell death. The excellent suitability of the synapsin promoter as a strong panneuronal promoter was further demonstrated by its restricted neuronal activity in various brain regions of adult rats in vivo.

Adenoviridae↗
PubMed2001

Adenovirus-mediated GDNF and CNTF pretreatment protects against striatal injury following transient middle cerebral artery occlusion in mice.

During the last few years, adenoviral gene transfer techniques have achieved increasing interest in the treatment of neurodegenerative diseases. However, gene therapy requires that delivered genes are translated into proteins. This may pose a problem in focal ischemia where protein synthesis is compromized. The present study was conducted to find out the feasibility of adenoviral GDNF and CNTF delivery in transient focal ischemia, as induced by 30 min of intraluminar middle cerebral artery (MCA) occlusion in mice. Injections of vehicle, of an adenoviral vector deleted in the E1 region (Ad-dE1) and of vectors expressing the GDNF (Ad-GDNF), CNTF (Ad-CNTF), or GFP (Ad-EGFP) gene from a CMV promoter were stereotactically placed in the dorsolateral striatum, i.e., the core of the MCA territory, and focal ischemia was induced seven days later. Thread occlusion resulted in disseminated injury of the striatum, but not the overlying cortex. The number of viable neurons was significantly increased after 1 and 3 days of reperfusion both in Ad-GDNF and Ad-CNTF as compared with vehicle or Ad-dE1-treated animals, whereas the number of injured cells was significantly reduced, as shown by cresyl violet staining, terminal transferase biotinylated-dUTP nick end-labeling (TUNEL), and immunocytochemistry for activated caspase-3. Interestingly, the protective effects of Ad-GDNF were similarly strong in areas of the striatum adjacent and remote of the adenoviral infusion site, while Ad-CNTF showed pronounced rescue effects in the surrounding, but rather little effects distant to the infusion. The present study demonstrates that adenoviral delivery of neurotrophic factors may be a useful tool for the treatment of focal ischemia.

Adenoviridae↗
PubMed2001

Graded expression patterns of ephrin-As in the superior colliculus after lesion of the adult mouse optic nerve.

The idea has been put forward that molecules and mechanisms acting during development are re-used during regeneration in the adult, for example in response to traumatic injury in order to re-establish the functional integrity of neuronal circuits. Members of the Eph family of receptor tyrosine kinases and their 'ligands', the ephrins, play a prominent role during development of the retinocollicular projection in rodents, where EphA receptors and ephrin-As are expressed in gradients in both the retina and the superior colliculi (SC). We were interested in investigating whether EphA family members are also expressed or re-expressed in the adult after optic nerve lesion, since the presence of axon guidance information is an important prerequisite for a topographically appropriate re-connection by retinal ganglion cell (RGC) axons. This analysis was encouraged by results showing that RGC axons do not exert guidance preferences in response to membranes from adult unlesioned SC, but in response to membranes from the adult deafferented SC. We found a graded expression pattern of ephrin-As in the SC both before and after deafferentation, which was remarkably similar to those found during development. EphA receptor levels were reduced in the SC after deafferentation and the expression patterns of the EphB family were not changed. In particular, the presence of a graded ephrin-A expression in the deafferented SC suggests that - if robust regeneration of RGC axons can be achieved - topographic guidance information as a likely requirement for a functionally successful re-establishment of the retinocollicular projection is available.

Animals↗
PubMed2001

Increased expression and activation of poly(ADP-ribose) polymerase (PARP) contribute to retinal ganglion cell death following rat optic nerve transection.

Excessive activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) by free-radical damaged DNA mediates necrotic cell death in injury models of cerebral ischemia-reperfusion and excitotoxicity. We recently reported that secondary retinal ganglion cell (RGC) death following rat optic nerve (ON) transection is mainly apoptotic and can significantly but not entirely be blocked by caspase inhibition. In the present study, we demonstrate transient, RGC-specific PARP activation and increased retinal PARP expression early after ON axotomy. In addition, intravitreal injections of 3-aminobenzamide blocked PARP activation in RGCs and resulted in an increased number of surviving RGCs when compared to control animals 14 days after ON transection. These data indicate that secondary degeneration of a subset of axotomized RGCs results from a necrotic-type cell death mediated by PARP activation and increased PARP expression. Furthermore, PARP inhibition may constitute a relevant strategy for clinical treatment of traumatic brain injury.

Animals↗
PubMed2000

Protection by synergistic effects of adenovirus-mediated X-chromosome-linked inhibitor of apoptosis and glial cell line-derived neurotrophic factor gene transfer in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces clinical, biochemical, and neuropathological changes reminiscent of those occurring in idiopathic Parkinson's disease (PD). Here we show that a peptide caspase inhibitor, N-benzyloxy-carbonyl-val-ala-asp-fluoromethyl ketone, or adenoviral gene transfer (AdV) of a protein caspase inhibitor, X-chromosome-linked inhibitor of apoptosis (XIAP), prevent cell death of dopaminergic substantia nigra pars compacta (SNpc) neurons induced by MPTP or its active metabolite 1-methyl-4-phenylpyridinium in vitro and in vivo. Because the MPTP-induced decrease in striatal concentrations of dopamine and its metabolites does not differ between AdV-XIAP- and control vector-treated mice, this protection is not associated with a preservation of nigrostriatal terminals. In contrast, the combination of adenoviral gene transfer of XIAP and of the glial cell line-derived neurotrophic factor to the striatum provides synergistic effects, rescuing dopaminergic SNpc neurons from cell death and maintaining their nigrostriatal terminals. These data suggest that a combination of a caspase inhibitor, which blocks death, and a neurotrophic factor, which promotes the specific function of the rescued neurons, may be a promising strategy for the treatment of PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2000

Targeting and insertion of C-terminally anchored proteins to the mitochondrial outer membrane is specific and saturable but does not strictly require ATP or molecular chaperones.

A distinct class of proteins contain a C-terminal membrane anchor and a cytoplasmic functional domain. A subset of these proteins is targeted to the mitochondrial outer membrane. Here, to probe for the involvement of a saturable targeting mechanism for this class of proteins, and to elucidate the roles of chaperone proteins and ATP, we have utilized an in vitro targeting system consisting of in vitro-synthesized proteins and isolated mitochondria. To establish the specificity of targeting we have used a closely related protein pair. VAMP-1A and VAMP-1B are splice variants of the vesicle-associated membrane protein/synaptobrevin-1 (VAMP-1) gene. In intact cells VAMP-1B is targeted to mitochondria whereas VAMP-1A is targeted to membranes of the secretory pathway, yet these isoforms differ by only five amino acids at the extreme C-terminus. Here we demonstrate that, in vitro, VAMP-1B is imported into both intact mitochondria and mitochondrial outer-membrane vesicles with a 15-fold greater efficiency than VAMP-1A. We generated and purified bacterially expressed fusion proteins consisting of the C-terminal two-thirds of VAMP-1A or -1B proteins fused to glutathione S-transferase (GST). Using these fusion proteins we demonstrate that protein targeting and insertion is saturable and specific for the VAMP-1B membrane anchor. To elucidate the role of cytosolic chaperones on VAMP-1B targeting, we also used the purified, Escherichia coli-derived fusion proteins. (33)P-Labelled GST-VAMP-1B(61-116), but not GST-VAMP-1A(61-118), was efficiently targeted to mitochondria in a chaperone-free system. Thus the information required for targeting is contained within the targeted protein itself and not the chaperone or a chaperone-protein complex, although chaperones may be required to maintain a transport-competent conformation. Moreover, ATP was required for transport only in the presence of cytosolic chaperone proteins. Therefore the ATP requirement of transport appears to reflect the participation of chaperones and not any other ATP-dependent step. These data demonstrate that targeting of C-terminally anchored proteins to mitochondria is sequence specific and mediated by a saturable mechanism. Neither ATP nor chaperone proteins are strictly required for either specific targeting or membrane insertion.

Adenosine Triphosphate↗
PubMed2000

Adenovirus-mediated expression of ciliary neurotrophic factor (CNTF) rescues axotomized rat retinal ganglion cells but does not support axonal regeneration in vivo.

Rat optic nerve (ON) transection leads to mainly apoptotic cell death of about 85% of the retinal ganglion cell (RGC) population within 14 days after lesion. In the present study, we tested the effect of adenovirally delivered CNTF (Ad-CNTF) on survival and regeneration of axotomized adult RGCs in vivo. Single intravitreal Ad-CNTF injection led to stable CNTF mRNA and protein expression for at least 18 days and significantly enhanced RGC survival by 155% when compared to control animals 14 days after ON transection. ON stump application of Ad-CNTF also resulted in an increased number of surviving RGCs. Ad-CNTF injection led to better preservation of intraretinal RGC axons but did not support regeneration of axotomized RGCs into a peripheral nerve graft. Thus, adenovirus-mediated neurotrophic factor supply is a suitable approach for reducing axotomy-induced RGC death in vivo and may constitute a relevant strategy for clinical treatment of traumatic brain injury.

Adenoviridae↗
PubMed2000

Apoptosis in the developing visual system.

Programmed cellular death is a widespread phenomenon during development of the nervous system. Two classes of molecules are particularly important in the context of apoptosis control in the nervous system: intracellular effectors homologous to the Caenorhabditis elegans Ced-3, -4, and -9 proteins, which in mammals correspond to the proteases of the caspase family, Apaf-1, and the members of the Bcl-2 protein family, and neurotrophic factors. Retinal ganglion cells lend a convenient model system with which to investigate apoptosis in central neurons during development as well as after injury. In this review, we discuss the role of these molecules in the control of programmed cellular death in the retinotectal system. Transgenic animal models and expression studies have shown that caspases, Bcl-2, Bax, and possibly Bcl-X are necessary players for the control of programmed cellular death in retinal ganglion cells. Bax and caspase 3 expression in retinal ganglion cells is upregulated after injury, and inhibition of Bax or caspase 3 increases the survival of injured retinal ganglion cells. Neurotrophins can support the survival of injured retinal ganglion cells, but this effect is transient. The physiological role of neurotrophins in the development of the retinocollicular system seems more related to the topographic refinement of retinocollicular projections, a process that is mediated, at least partially, by selective elimination of retinal ganglion cells making inappropriate topographic projections.

Animals↗
PubMed2000

The X-linked inhibitor of apoptosis (XIAP) prevents cell death in axotomized CNS neurons in vivo.

The inhibition of neuronal apoptosis in acute traumatic and ischemic injuries as well as in long term neurodegenerative disorders like spinal muscular atrophy and possibly Alzheimer's disease is a fundamental requirement for a therapeutic strategy. In this study we used an established in vivo model system of induction of neuronal apoptosis in the CNS to evaluate the properties of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit secondary cell death after axonal lesions. We used adenoviral vectors to transduce retinal ganglion cells after axotomy of the optic nerve of adult rats. Vector application was performed at the optic nerve stump so that only the lesioned retinal neurons could be transduced. We found XIAP to be as effective as the viral broad spectrum caspase inhibitor protein p35. These findings suggest that axotomized RGCs degenerate through class II caspase activity and furthermore offer the possibility of using mammalian XIAP protein to inhibit neuronal apoptosis as a basis for a regenerative therapy in the CNS.

Animals↗
PubMed1999

Locoregional Apo2L/TRAIL eradicates intracranial human malignant glioma xenografts in athymic mice in the absence of neurotoxicity.

Glioblastoma multiforme is a lethal neoplasm refractory to radiochemotherapy. Although glioma cells undergo apoptosis when exposed to the death ligand, CD95 (Fas/APO-1) ligand, the therapeutic use of CD95L is considered impossible because of lethal side effects. Here, we report that the locoregional application of Apo2 ligand (Apo2L) exerts strong antitumor activity on preestablished intracranially growing human U87MG glioma xenografts in athymic mice. Two repetitive intratumoral injections of 2 microg Apo2L resulted in long-term survival of mice (>100 days), whereas the median survival of mock-treated mice was 36 days. The assessment of tumor volumes at 21 and 35 days after inoculation showed complete eradication of glioma xenografts in Apo2L-treated mice. Histology and TUNEL assay confirmed the induction of apoptosis by Apo2L in glioma cells in vivo. Importantly, the intracerebral injection of Apo2L does not result in acute or delayed neurotoxicity. We propose that a phase 1 trial of intralesional Apo2L therapy for human glioblastoma multiforme is warranted.

Animals↗
PubMed1999

Inhibition of experimental rat glioma growth by decorin gene transfer is associated with decreased microglial infiltration.

Decorin gene therapy for experimental malignant glioma is thought to involve antagonism of immunosuppression induced by glioma-derived transforming growth factor-beta (TGF-beta). TGF-beta is chemotactic for cells of the monocyte macrophage lineage but inhibits their functional activity in many in vitro paradigms. Here, we examined changes in the patterns of microglial infiltration of rat C6 gliomas expressing a decorin transgene. We find that the number of OX42/ED-1-positive microglial cells is reduced rather than enhanced in the presence of decorin. Decorin-expressing gliomas contain lower numbers of MHC class II antigen-expressing microglial cells whereas the relative frequency of MHC I immunoreactivity among microglial cells is increased. Interestingly, the reduction of TGF-beta levels in the tumors by decorin is associated with the de novo expression of inducible nitric oxide synthase (iNOS) in a minority of microglial cells. These data suggest that microglial cells do not participate in the regression of decorin-expressing rat C6 gliomas.

Animals↗
PubMed1999

Targeting of tail-anchored proteins to yeast mitochondria in vivo.

Tail-anchored proteins are inserted into intracellular membranes via a C-terminal transmembrane domain. The topology of the protein is such that insertion must occur post-translationally, since the insertion sequence is not available for membrane insertion until after translation of the tail-anchored polypeptide is completed. Here, we show that the targeting information in one such tail-anchored protein, translocase in the outer mitochondrial membrane 22, is contained in a short region flanking the transmembrane domain. An equivalent region is sufficient to specify the localisation of Bcl2 and SNARE proteins to the secretory membranes. We discuss the targeting process for directing members of this protein family to the secretory and mitochondrial membranes in vivo.

Amino Acid Sequence↗
PubMed1999

Excess target-derived brain-derived neurotrophic factor preserves the transient uncrossed retinal projection to the superior colliculus.

During early postnatal development, a widespread ipsilateral projection to the superior colliculus is secondarily restricted to a small topographically defined region by elimination of ipsilaterally projecting retinal ganglion cells. Brain-derived neurotrophic factor (BDNF) has been proposed as the target-derived neurotrophic factor for retinal ganglion cells in several studies. Here we investigated the long-term effects of excess BDNF in the retinal ganglion cell target on naturally occurring retinal ganglion cell (RGC) elimination and on the restriction of the ipsilateral projection. To this end, sustained overexpression of BDNF was achieved in the postnatal superior colliculus using an adenoviral vector. While the total number of retinal ganglion cells in the adenovirus-BDNF treated animals was unchanged, a much higher proportion of RGCs retained a projection to the ipsilateral superior colliculus. We conclude that an excess of target-derived BDNF does not reduce the net amount of naturally occurring cell death in the retino-collicular system, but prevents the negative selection of retinal ganglion cells making inappropriate topographic connections.

Adenoviruses, Human↗
PubMed1999

Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma.

The inhibitor-of-apoptosis (IAP) proteins are a novel family of antiapoptotic proteins that are thought to inhibit cell death via direct inhibition of caspases. Here, we report that human malignant glioma cell lines express XIAP, HIAP-1 and HIAP-2 mRNA and proteins. NAIP was not expressed. IAP proteins were not cleaved during CD95 ligand (CD95L)-induced apoptosis, and loss of IAP protein expression was not responsible for the potentiation of CD95L-induced apoptosis when protein synthesis was inhibited. LN-18 cells are highly sensitive to CD95-mediated apoptosis, whereas LN-229 cells require co-exposure to CD95L and a protein synthesis inhibitor, CHX, to acquire sensitivity to apoptosis. Adenoviral XIAP gene transfer blocked caspase 8 and 3 processing in both cell lines in the absence of CHX. Apoptosis was blocked in the absence and in the presence of CHX. However, XIAP failed to block caspase 8 processing in LN-229 cells in the presence of CHX. There was considerable overlap of the effects of XIAP on caspase processing with those of BCL-2 and the viral caspase inhibitor crm-A. These data define complex regulatory mechanisms for CD95-mediated apoptosis in glioma cells and indicate that there may be a distinct pathway of death receptor-mediated apoptosis that is readily activated when protein synthesis is inhibited. The constitutive expression of natural caspase inhibitors may play a role in the resistance of these cells to apoptotic stimuli that directly target caspases, including radiochemotherapy and immune-mediated tumor cell lysis.

Adenoviridae↗
PubMed1999

Bax antisense oligonucleotides reduce axotomy-induced retinal ganglion cell death in vivo by reduction of Bax protein expression.

Following transection of the optic nerve (ON), retinal ganglion cells (RGCs) upregulate Bax protein expression and undergo apoptosis. The present study aimed at reducing Bax expression in order to test whether Bax plays a causative role in the induction of secondary RGC apoptosis. Following injection into the vitreous, fluoresceinated oligonucleotides transfected RGCs in vivo at the injection site in the temporal superior retina. Following ON lesion, and repeated injections of a partially phosphorothioated Bax antisense oligonucleotide, but not following injection of control oligonucleotides, expression of Bax protein was locally inhibited, and the number of surviving RGCs was increased in Bax antisense treated rats 8 days after axotomy. Our results indicate that Bax induction is a prerequisite for the execution of RGC apoptosis following ON axotomy. While the Bax antisense strategy offers an exciting perspective to inhibit secondary neuronal degeneration in vivo, both limited transfection efficacy, and the temporal restriction of this effect currently limit the use of this approach with respect to clinical applications for the treatment of neurodegeneration.

Animals↗