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The effects of pinealectomy and of intraperitoneally administered melatonin on the retrograde degeneration of retinal ganglion cells (RGCs) were examined in a novel model of optic nerve (ON) transection in C57BL/16J mice. RGCs were prelabeled with the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (Di-I), and the ON was cut inside the orbital cavity 7 days later. The degree of RGC injury was assessed by counting viable Di-I labeled RGCs in various locations of the retina. In unlesioned control eyes, a mean ganglion cell density of 1,891 +/- 30/mm2 (mean +/- S.E.M.) was determined. The cell density markedly declined at 14 days after axotomy (295 +/- 9 cells/mm2; 15.6% of contralateral). Sham-pinealectomy did not influence the density of RGCs at 14 days after ON transection (382 +/- 37 cells/mm2). In pinealectomized animals, on the other hand, the RGC number was significantly reduced as compared with untreated and sham-pinealectomized animals (91 +/- 33 RGCs/mm2). The effect of pinealectomy was reversed after i.p. administration of melatonin (4 mg/kg bw bolus followed by continuous infusion of 8 mg/kg bw/day) (286 +/- 27 cells/mm2). In nonpinealectomized animals, on the contrary, i.p. melatonin did not influence the RGC density (344 +/- 20 cells/mm2). The present results suggest that endogenous melatonin prevents the delayed degeneration of adult central nervous system (CNS) neurons in vivo, and that exogenous substitution of melatonin may be useful to protect injured neurons against cell death under conditions of melatonin deficiency, e.g. in the aged brain, when melatonin synthesis and secretion have decreased.

Adult rat retinal ganglion cells undergo degeneration after optic nerve transection. Repeated intraocular injection of glial cell-line derived neurotrophic factor (GDNF) has been shown to be efficient in enhancing retinal ganglion cell survival following optic nerve axotomy. In the present study we evaluated the potential survival-promoting effect of adenovirally administered GDNF on axotomized retinal ganglion cells. A single intravitreal injection [7 x 107 plaque-forming units (pfu) or 7 x 108 pfu] of an adenoviral vector expressing the rat GDNF gene from a cytomegalovirus promoter enhanced retinal ganglion cell survival 14 days after axotomy by 67 and 125%, respectively, when compared to control animals. Intraocular administration of the vector rescued 12.6 and 23%, respectively, of the retinal ganglion cells which would otherwise have died after axotomy. An increase in retinal GDNF protein and specific virally transduced GDNF mRNA expression was detected following intraocular vector application. Our data support previous findings showing that adenoviral delivery of neurotrophic factors to the vitreous body is a feasible approach for the prevention of axotomy-induced retinal ganglion cell death in vivo and may constitute a relevant strategy for future treatment in traumatic brain injury and ensuing neurodegeneration.

We examined whether brain damage after focal cortex trauma may be attenuated by adenoviral delivery of the glial cell line-derived neurotrophic factor (GDNF) gene. For this reason, injections of vehicle, of an adenoviral vector deleted in the E1 region (Ad-dE1) or a vector expressing the GDNF gene from a CMV promoter (Ad-GDNF) were stereotactically placed in the rat sensorimotor cortex, and one day later cold lesions of the cerebral cortex were induced. Lesions were associated with pronounced brain swelling one day after injury. The degree of brain swelling was significantly attenuated by Ad-GDNF delivery (Ad-GDNF: 7.4 +/- 2.2%, Ad-dE1: 21.1 +/- 4.9%, vehicle: 20.9 +/- 5.0% of contralateral; mean +/- SEM, P < 0.05). Furthermore, Ad-GDNF treatment resulted in a significant reduction of the lesion volume seven days after lesioning (Ad-GDNF: 21.8 +/- 2.8 mm3, Ad-dE1: 44.1 +/- 1.6 mm3, vehicle 40.9 +/- 8.6 mm3, P < 0.05). The decrease in the lesion size was associated with a reduction in the number of inducible nitric oxide (iNOS)(+), activated caspase-3(+) and DNA fragmented cells in the perilesion rim, as revealed by immunocytochemistry and terminal transferase biotinylated-dUTP nick end labeling (TUNEL). In Ad-GDNF-treated animals, the number of caspase-3(+) and TUNEL(+) cells was also reduced in the lesion-remote thalamus. The present study shows that adenoviral GDNF delivery is protective in focal cortex trauma.

Axotomy of the optic nerve in rodents induces the majority of retinal ganglion cells (RGCs) to undergo apoptosis: Only 10-15% survive 14 days past lesion. The molecular mechanism allowing this survival is not known. To test whether expression of the anti-apoptotic proto-oncogene bcl-2 gene is required in those RGCs, we examined the effect of optic nerve axotomy in bcl-2-/- mice. 7 days and 14 days post-lesion, the same number of surviving RGCs was detected in mutant and wild type retinas. Thus, the bcl-2 gene is not necessary for the survival of the subpopulation of retinal ganglion cells resisting axotomy-induced apoptosis in adult mice, nor does its normal expression delay retinal ganglion cell degeneration.

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., (Copyright 2001 Academic Press.)

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.

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.

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.

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.

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.

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., (Copyright 2000 Academic Press.)

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., (Copyright 1999 Academic Press.)

Axotomy of the rat optic nerve leads to apoptotic cell death of retinal ganglion cells (RGCs). We have used adenoviral vectors to transduce RGCs from the cut optic nerve stump, a paradigm in which only those neurons are transduced which are directly affected by the axonal lesion. Transgenes encoded by the vectors were p35 and CrmA, which are potent intracellular anti-apoptotic proteins. We found that p35, but not CrmA exerted significant rescue effects on RGCs 14 days after axotomy. Expression of the transgenes was driven by the murine CMV (MCMV) promoter. The respective mRNAs were detectable 7 days but not 14 days after transduction. Since surviving RGCs were present beyond the time-point of detectable transcription of the p35 transgene, we conclude that apoptosis has been efficiently inhibited. In addition, we observed that transduction with two control vectors without a transgene in E1 also resulted in a minor but significant RGC rescue, implicating neuroprotective effects due to adenoviral transduction itself. This system will be useful in dissecting the pathways leading to neuronal cell death after axonal lesions and in the evaluation of the important question whether the cellular suicide program can be reverted to survival by therapeutic gene delivery.

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.

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., (Copyright 1999 Academic Press.)

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.

To test the hypothesis whether a failure to express neurotrophins or a neurotrophin receptor might underlie the pathology observed in mutant mice with degeneration of regionally distinct subpopulations of neurons, the expression of BDNF, NT-3, TrkB, TrkC and synaptophysin mRNA was examined in the cerebellum of mutant lurcher (lc/+) and weaver (wv/+)/(wv/wv) mice. To identify the expression patterns of individual neurons, we used in situ hybridization with digoxigenin labeled ribonucleotide probes. RT-PCR of cerebellar mRNA for BDNF, NT-3, TrkB and TrkC (GAPDH as internal standard) was performed in parallel. Although especially in homozygous (wv/wv) weaver mice the normal anatomical order and number of the cerebellar neurons is grossly disturbed, residual Purkinje and granule neurons of both mutants displayed a normal expression pattern of the neurotrophins examined. Thus, the affected animals showed no significant signal decrease compared to healthy littermates or C3H mice. Our results suggest that the loss of specific neuron populations in the cerebellum of either mutant occurs via mechanisms either independent or downstream of the neurotrophins examined in this study., (Copyright 1998 Elsevier Science B.V.)

Following intraorbital transection of the optic nerve (ON) in rats, more than 80% of the retinal ganglion cell (RGC) population die by apoptosis within 14 days. Repeated intraocular injection of brain-derived neurotrophic factor (BDNF) has been efficient in enhancing RGC survival following ON axotomy. The present study was designed to define a potential survival-promoting effect of adenovirally administered BDNF on axotomized RGCs. A single injection of an adenoviral vector expressing the human BDNF gene from a CMV promoter/enhancer (Ad-BDNF) enhanced RGC survival 14 days after axotomy by 40.3%. Moreover, a combinatory treatment regimen consisting of intraocular Ad-BDNF administration and systemic application of the free radical scavenger, N-tert-butyl-(2-sulphophenyl)-nitrone (S-PBN), enhanced RGC survival by 63.0%. Our data demonstrate that adenoviral delivery of neurotrophic factors to the vitreous body is a feasible approach for the prevention of axotomy-induced RGC death. Further, as shown for S-PBN, therapeutic regimens that combine local virus-mediated gene delivery with systemic administration of protective compounds, may offer promising strategies for future treatment also in human neurodegenerative conditions.

Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell.

Although the formal pathogenesis of spongiform encephalopathies has been described in detail, it is not known whether the infectious agent targets primarily neurons, glial cells, or both. To address this question, we have transplanted transgenic embryonic neural tissue overexpressing PrP(c) into the forebrain of Prnp -knockout mice, and infected it with scrapie prions. After infection, grafts developed severe spongiform encephalopathy. As the infected hosts were not clinically affected, we were able to prolong the experiment and to assess changes in the graft over periods of time, which vastly exceeded the normal life span of scrapie-infected mice. Sequential contrast-enhanced magnetic resonance imaging (MRI) revealed progressive impairment of blood-brain barrier properties in infected grafts. However, loss of astrocytes was not observed. Subtotal neuronal loss occurred during the progression of the disease in the grafts, reactive astrocytes persisted until the terminal stage of disease. We conclude that scrapie encephalopathy primarily leads to neuronal death, while degeneration of astrocytes does not occur. Functional impairment of the blood-brain barrier suggests involvement of astrocytes and endothelial cells in the pathological process.

Focal ischemia in the parietal cortex of the rat results in massive neuronal death in the infarct zone and penumbra between 12 hours and 6 days after photothrombosis. To examine a possible role of Bcl-2 family proteins in this process of cell death, we investigated their expression by immunoblot assays and immunocytochemistry, and correlated expression patterns with TUNEL as well as morphological signs indicative of apoptosis. In the center of the lesion Bax immunostaining was increased in many degenerating neurons between 4 hours and 3 days after the induction of photothrombosis. At all time points examined, Bcl-2 and Bcl-X protein levels were markedly reduced in injured neurons as compared to the unlesioned side. At the border of the ischemic lesion, two areas were distinguished: 1 - 2 days after induction of photothrombosis, pyknotic cells located immediately adjacent to the lesion core displayed nuclear Bcl-X and Bax immunoreactivity. In contrast, large, morphologically intact neurons located more towards the healthy brain parenchyma displayed an increase in cytoplasmic Bcl-2 and Bcl-X proteins. Double staining for each of the Bcl-2 family proteins and TUNEL revealed that DNA strand breaks and nuclear fragmentation seen in cells located in the lesion core were often associated with increased levels of Bax, but not with elevated Bcl-2 or Bcl-X protein levels, suggesting a role for Bax in the induction of apoptotic death in these cells. The upregulation of Bcl-2 and Bcl-X expression in surviving neurons close to the penumbra might reflect an active survival mechanism that protects these neurons from cell death following a sublethal insult.

The identification of neurotrophic factors ameliorating secondary neuronal death in the mammalian CNS has raised hopes for improved treatment strategies in neurodegenerative diseases, CNS trauma and ischemia. Glial cell-line derived neurotrophic factor (GDNF) has potent neuroprotective properties in both the CNS and PNS. We sought to investigate whether GDNF exerts survival promoting effects on axotomized retinal ganglion cells (RGCs) in the adult rat in vivo. Transection of the optic nerve induces delayed retrograde death of approximately 85% of RGCs within 14 days. Intraocular GDNF rescued 21% of the RGCs which would otherwise have died after axotomy (34% of the normal control population), thereby extending the group of neuronal populations responsive to GDNF.

Axonal lesions to the optic nerve (ON) induce c-Jun expression in retinal ganglion cells (RGCs) of the rat in vivo. Detailed investigations using retrograde tracers, and double labeling studies for c-Jun and regeneration-associated factors, such as the growth-associated protein GAP-43, have suggested that this upregulation of c-Jun is part of a cell body response in an abortive attempt of affected RGCs to survive and regenerate an axon. On the other hand, prolonged expression of c-Jun protein has in several paradigms of neurodegeneration been linked to the induction of apoptotic cell death. In the present study, we examined the time course and subcellular localization of c-Jun protein by immunocytochemistry on retinal sections after optic nerve crush and carried out double labeling for c-Jun protein and DNA strand breaks to detect apoptosis on the same sections. Several days after ON lesion, a subpopulation of RGCs was detected in which c-Jun protein was not confined to the nucleus, but also located in the cytoplasm. In addition, RGCs were seen that displayed morphological signs of apoptosis, DNA strand breaks, and c-Jun immunoreactivity at the same time. Therefore, c-Jun expression is not confined to intact or regenerating ganglion cells, but also occurs in cells that are destined to die. Our results suggest that the decision to undergo either fate depends on additional signaling events that modulate the transcriptional actions of c-Jun.

Retrograde degeneration of retinal ganglion cells as a consequence of optic nerve lesion has been shown to fulfil the criteria of apoptosis. In the present study, we investigated the time course of ganglion cell apoptosis following intraorbital crushing of the optic nerve in adult rats using morphological criteria and applying a terminal transferase technique (TUNEL) for in situ detection of DNA strand breaks. In addition, we examined expression patterns of the anti-apoptotic proteins Bcl-2 and Bcl-X and the cell death-promoting protein Bax in retinae after crushing the optic nerve. Apoptotic nuclei were detected in the ganglion cell layer in the first 3 weeks after optic nerve crush, with a peak after 6 days. Bcl-2 and Bcl-X proteins were expressed in ganglion cells at low levels. Expression of Bcl-2 decreased further during the days following crush. Bcl-X expression was initially increased, followed by a decline over the following days. In contrast, Bax protein, which was expressed in most ganglion cells at moderate baseline levels, was sharply increased as early as 30 min after crush, reached peak levels after 3 days, and remained up-regulated for at least 1 week thereafter. Double labelling for Bax and TUNEL in retinal sections, however, did not reveal colocalization of the two signals in individual retinal ganglion cells, consistent with the idea that increases in Bax precede apoptosis after optic nerve lesion. Thus, retinal ganglion cell death might be prevented by ablation of Bax protein in these cells, or by up-regulation of Bax-antagonists such as Bcl-2 or Bcl-X.

Transgenic and knockout mice have contributed much to our current understanding of the role played by single genes during development and in pathological processes of the CNS, such as neuro-degeneration. However, embryonic lethality resulting from the disruption of important genes has often hindered the interpretation of such experiments. Grafting of immature cells from genetically modified organisms into healthy recipients promises to efficiently bypass this problem. We have used neural transplantation techniques which allow us to keep CNS tissue of knockout and transgenic mice viable for a prolonged period of time in the brain or in the kidney capsule of healthy recipients. We have characterized biological parameters such as growth, proliferation and differentiation and also the formation of an intact blood-brain barrier (BBB) after grafting of wild-type telencephalic anlage in this system. We have also employed this technique to study the longterm properties of neuroepithelial tissue derived from knockout mice. The results of our studies are discussed in the context of neurodegenerative diseases.

We describe a 47-year-old patient with intracerebral tuberculoma of the falx cerebri and fronto-parietal cerebral cortex. The patient presented after a single episode of epileptic seizures. Computed tomography and MRI scans were suggestive of meningioma. At surgery a granulomatous tumor was removed that adhered to the cerebral cortex. The diagnosis of tuberculoma was verified by histology and growth in culture of mycobacterium tuberculosis. The patient recovered after surgery and chemotherapy. Three years later we confirmed the diagnosis using polymerase chain reaction for mycobacterial DNA sequences on the formalin-fixed, paraffin-embedded tissue.

So far, it has not been possible to treat many neurological conditions causally. However, in the past few years underlying genetic defects have been characterized for a substantial number of neurodegenerative disorders. Experimental methods have been developed that allow for efficient gene transfer into defined regions of the mammalian CNS. Such techniques can be applied to deliver genes into target cells of a recipient organism or to transfer genetically modified cells into defined regions of the CNS. Candidate genes for gene therapy are those encoding for neurotrophins and neurotransmitters for symptomatic therapy and, in the case of neurodegenerative disorders with localized gene defects, the wild-type allele as a causal treatment approach. In this review article, we describe some of the most widely used strategies for gene transfer to the CNS. We also report on the results obtained with animal models for human disease, and discuss both the chances and problems of gene therapy approaches in clinical medicine.

Accumulation of the prion protein PrPSc, a pathological and protease-resistant isoform of the normal host protein PrPC, is a feature of prion disease such as scrapie. It is still unknown whether scrapie pathology comes about by neurotoxicity of PrPSc, acute depletion of PrPC, or some other mechanism. Here we investigate this question by grafting neural tissue overexpressing PrPC into the brain of PrP-deficient mice which are scrapie-resistant and do not propagate infectivity. After intracerebral inoculation with scrapie prions, the grafts accumulated high levels of PrPSc and infectivity and developed the severe histopathological changes characteristic of scrapie. Moreover, substantial amounts of graft-derived PrPSc migrated into the host brain. Even 16 months after inoculation no pathological changes were seen in PrP-deficient tissue, not even in the immediate vicinity of the grafts. Therefore, in addition to being resistant to scrapie infection, brain tissue devoid of PrPC is not damaged by exogenous PrPSc.

The pregnant uterus of humans and rodents contains a population of granulated lymphoid cells, which, in the mouse, are called granular metrial gland (GMG) cells and have been described to express high levels of perforin. Since there is evidence for cytolytic activity of these cells and since perforin is a crucial effector molecule for the lytic action of cytotoxic T cells and natural killer cells, we evaluated the function of perforin in the pregnant uterus by using perforin-deficient mice. Perforin-deficient female mice were found to reproduce as efficiently as normal control females when bred either with syngeneic or allogeneic males. However, perforin-deficient mice differed from normal mice in that the frequency of GMG cells was significantly higher within maternal blood spaces and within several compartments of the feto-maternal interface. Proliferating GMG cells, identified by [3H] thymidine incorporation, were observed during more advanced stages of pregnancy when compared to normal controls. In contrast to normal mice, perforin-deficient mice did not display GMG cells attached to degenerating trophoblasts; instead perforin-deficient GMG cells were often observed in association with small maternal lymphocytes. In addition, the lack of transmission of lymphocytic choriomeningitis virus from infected pregnant perforin-deficient mice to the fetuses argued against a role of perforin expression by GMG cells in prevention of virus transmission from the mother to the fetus. Our data indicate that functional perforin is not necessary for successful pregnancies. The morphological changes in the pregnant uterus of perforin-deficient mice might, however, point to a certain, as-yet undefined function of perforin in the uterus of pregnant normal mice, which is functionally compensated in perforin-deficient mice.

Adhesion molecule on glia (AMOG) represents the beta 2-subunit of murine Na,K-ATPase. Mice carrying a targeted deletion of the AMOG/beta 2 gene exhibit tremor and limb paralysis at postnatal day (P) 15 and die 2 days after the onset of symptoms. The brains of these mice show edema and swelling of astrocytic end feet. However, the cause of death has remained unclear. To identify long-term consequences of AMOG/beta 2 deficiency, we have grafted parts of the embryonic telencephalic anlage of AMOG/beta 2-deficient mice into the caudoputamen of wild-type mice and analyzed the grafts up to 500 days after transplantation. Histological, immunocytochemical, and in situ hybridization techniques were applied to examine histoarchitecture, proliferation, differentiation, and long-term survival of grafts. AMOG/beta 2-deficient telencephalic grafts develop normally and form solid neural tissue that cannot be distinguished from control grafts by morphological features or with immunocytochemical stains for neuronal and glial markers. No signs of degeneration can be found. Expression analysis, however, revealed that no AMOG/beta 2 protein of possible host origin can be detected in AMOG/beta 2-deficient grafts. Graft-borne astrocytes express neither the AMOG/beta 1 nor the AMOG/beta 2 subunit of Na,K-ATPase as examined with immunocytochemistry and in situ hybridization. These findings indicate that AMOG/beta 2 is not necessary for long-term survival of telencephalic graft tissue.

Transgenic technologies have come of age, and the field of carcinogenesis has profited extensively from the availability of these methods. Both the inappropriate expression of dominant oncogenes in specific tissues and the ability to "knock out" tumor suppressor genes in mammalian organisms have enabled substantial advancements of our understanding of development and progression of the neoplastic phenotype. In the first part of this article, we review the most popular techniques for modification of the mammalian genome in vivo, i.e. microinjection of fertilized eggs, retrovirus-mediated gene transfer, and targeted gene deletion through homologous recombination. Subsequently, we attempt a critical evaluation of the available models of neurocarcinogenesis, and discuss their impact and future potential for the study of cancer in the nervous system.

Limb muscles develop from cells that migrate from the somites. The signal that induces migration of myogenic precursor cells to the limb emanates from the mesenchyme of the limb bud. Here we report that the c-met-encoded receptor tyrosine kinase is essential for migration of myogenic precursor cells into the limb anlage and for migration into diaphragm and tip of tongue. In c-met homozygous mutant (-/-) mouse embryos, the limb bud and diaphragm are not colonized by myogenic precursor cells and, as a consequence, skeletal muscles of the limb and diaphragm do not form. In contrast, development of the axial skeletal muscles proceeds in the absence of c-met signalling. The specific ligand of the c-met protein, the motility and growth factor scatter factor/hepatocyte growth factor, is expressed in limb mesenchyme and can thus provide the signal for migration which is received by c-met. We have therefore identified a paracrine signalling system that regulates migration of myogenic precursor cells.

Background: Although infection by HIV-1 is the primary cause of AIDS, cofactorial agents of an infectious nature may be involved in the pathogenesis of the disease. The present work addresses the cofactorial potential of human foamy virus (HFV) in AIDS. It has been suggested that HFV seroprevalence reaches 5% in East Africa, and HFV seroprevalence in East African patients suffering from AIDS and AIDS-related complex may be as high as 20%. Although the pathogenic potential of HFV in humans has not yet been investigated in detail, HFV transgenic mice develop an encephalopathy reminiscent of some of the features of HIV-associated brain diseases., Experimental Design: We set out to investigate the possibility that the regulatory genes of HFV may act as transcriptional cofactors of HIV. To study the effects of bel1, the transcriptional activator of HFV, on the HIV-1 LTR, we generated double transgenic mice for bel1 and for the HIV-1 LTR linked to a lacZ reporter gene. Moreover, to identify the cis-acting elements mediating bel1 action on the HIV LTR, we analyzed the consequences of deletions in the negative regulatory element or in the NF-kappa B binding sites., Results: We demonstrate that bel1 is capable of activating the transcription of HIV-1 LTR in vivo. Such transactivational activity, however, was observed exclusively in a subset of hippocampal neurons, whereas cortical neurons expressing bel1 did not show transactivation. Transactivation was completely abolished by the deletion of the NF-kappa B binding sites. In contrast, deletion of the negative regulatory element region seems to enhance transactivation of bel1 on HIV-1 LTR over a prolonged period of time., Conclusions: Our study indicates that transcriptional transactivation of HIV-1 by HFV can be accomplished in vivo and is dependent on NF-kappa B binding sequences. Therefore, transcriptional transactivation is a potential mechanism of cooperation between HFV and HIV. It is conceivable that this phenomenon attains clinical significance in a situation of coinfection with both viruses.

E-SELECTIN is an inducible cell-adhesion molecule on endothelial cells, which mediates the binding of neutrophils and functions as a Ca(2+)-dependent lectin. We have recently identified a 150K glycoprotein as the major ligand for E-selectin on myeloid cells, using a recombinant antibody-like form of mouse E-selectin as an affinity probe. Here we report the isolation of a mouse complementary DNA for this E-selectin ligand (ESL-1). The predicted amino-acid sequence of ESL-1 is 94% identical (over 1,078 amino acids) to the recently identified chicken cysteine-rich fibroblast growth-factor receptor, except for a unique 70-amino-acid aminoterminal domain of mature ESL-1. Fucosylation of ESL-1 is imperative for affinity isolation with E-selectin-IgG. A fucosylated, recombinant antibody-like form of ESL-1, but not of L-selectin, supports adhesion of E-selectin-transfected Chinese hamster ovary cells. Antibodies against ESL-1 block the binding of mouse myeloid cells to E-selectin. ESL-1, with a structure essentially identical to that of a receptor, thus functions as a cell adhesion ligand of E-selectin.

Although primary treatment of medulloblastoma is now successful in a high percentage of patients, its secondary manifestations still bear a poor prognosis. Thorough studies of secondary manifestations are therefore pivotal to plan therapeutic approaches for the long-term management of medulloblastoma. Here we describe the incidence of secondary tumour manifestations in 66 patients of a single centre who underwent surgery for medulloblastoma between 1975 and 1990. No patient was excluded due to a poor postoperative course. Thirty-five patients showed evidence of secondary tumour growth. Of these, 17 suffered from local recurrence, and 27 developed metastastatic disease. The median latencies for secondary manifestations were 25 months for local recurrence (n = 17), 11 months for spinal metastases (n = 10), 15 months for supratentorial metastases (n = 8), 8 months for subleptomeningeal dissemination (n = 6), and 23 months for systemic metastases (n = 8). Two patients developed primary metastatic spread to the posterior fossa. Of 8 patients with supratentorial metastases, 6 developed fronto-basal lesions. In our patients, 89% of secondary lesions occurred within less than 3 years after primary diagnosis. 85% of patients with extra-axial tumour spread had been treated with a permanent shunt. Radical tumour resection and radiotherapy with 30 Gy to the neuraxis and 20 Gy boost to the posterior fossa was an important prognostic factor in this series. Patients with additional chemotherapy did not benefit significantly from this treatment. We conclude that optimal management of the primary lesions should aim at (i) total resection, (ii) avoid permanent shunting, and (iii) completion of the radiotherapy with inclusion of the medial frontobasal cisterns in the radiotherapeutic regimen. Our analysis suggests that adequate postoperative screening programmes should consist of 3-monthly scans of the neuraxis in the first three postoperative years and 6-monthly scans thereafter.

Besides providing useful model systems for basic science, studies based on modification of the mammalian germ line are changing our understanding of pathogenetic principles. In this article, we review the most popular techniques for generating specific germ line mutations in vivo and discuss the impact of various transgenic models on the study of neurodegenerative diseases. The "gain of function" approach, i.e., ectopic expression of exogenous genes in neural structures, has deepened our understanding of neurodegeneration resulting from infection with papova viruses, picorna viruses, and human retroviruses. Further, inappropriate expression of mutated cellular molecules in the nervous system of transgenic mice is proving very useful for studying conditions whose pathogenesis is controversial, such as Alzheimer's disease and motor neuron diseases. As a complementary approach, ablation of entire cell lineages by tissue-specific expression of toxins has been useful in defining the role of specific cellular compartments. Modeling of recessive genetic diseases, such as Lesch-Nyhan syndrome, was helped by the development of techniques for targeted gene deletion (colloquially termed "gene knock-out"). Introduction of subtle homozygous mutations in the mouse genome was made possible by the latter approach. Such "loss of function" mutants have been used for clarifying the role of molecules thought to be involved in development and structural maintenance of the nervous system, such as the receptors for nerve growth factor and the P0 protein of peripheral myelin. In addition, these models are showing their assets also in the study of enigmatic diseases such as spongiform encephalopathies.

The adhesion molecule on glia (AMOG) has been reported to function as cell adhesion molecule and also to constitute the beta 2-subunit of the murine Na,K-ATPase. In order to elucidate these functions in vivo, Magyar et al. have generated mice carrying a targeted deletion of the AMOG gene. These mice exhibit behaviourally normal development till postnatal day P16. At this time, they develop muscular weakness, incoordination, and tremor. Death invariably occurs 24-36 hours after onset of the symptoms. Histological and ultrastructural examination of brain sections show enlarged ventricles, brain edema, and swelling of astrocyte end feet. However, no disturbances of the architecture or cell migration in the brain can be detected. In order to identify long-term consequences of AMOG deficiency which might not yet be detectable at the time of death, we have established a CNS grafting model. The embryonal brain anlage (E10.5-E13.5) was grafted into the caudoputamen of wild type mice. The graft recipients are sacrificed up to 7 months after the procedure. Both wild type and AMOG deficient grafts develop and form solid neural tissue with neurons, myelinated axons, glial cells, and ventricular structures, as shown by histological and immunocytochemical analysis. However, no differences in grafts derived from wild type, heterozygous, and AMOG-deficient donors can be detected. Proliferation has been examined by BrdU immunocytochemistry. The blood-brain barrier as examined by repeated magnetic resonance imaging after injection of Gadolinium-DTPA has been shown to be largely reconstituted five weeks after grafting.

The migration of lymphocytes from the blood into various lymphoid organs (lymphocyte homing) occurs in specialized high endothelial venules (HEV). Homing of lymphocytes into mucosa-associated lymph nodes in the mouse is mediated at least in part by the lymphocyte homing receptor LPAM-1, an integrin which has been identified as the heterodimer alpha 4 beta p (alpha 4 beta 7 in humans). The HEV-ligand for this integrin in the homing process has not yet been identified. VCAM-1, a cytokine-inducible, endothelial adhesion molecule for lymphocytes and monocytes, was recently demonstrated in vitro to be a ligand for the alpha 4 beta p integrin. We have tested whether VCAM-1 could be a candidate for an alpha 4 beta p ligand on HEV. Two findings strongly argue against this possibility: First, an anti-VCAM-1 monoclonal antibody (mAb) failed to block the binding of the mouse T-lymphoma TK-1 to HEV in cryostat sections of mesenteric lymph nodes, even though this antibody blocked alpha 4 beta p mediated binding of TK-1 cells to VCAM-1 on TNF-alpha-activated mouse endothelioma cells. Second, expression of VCAM-1 on lymph node endothelium was undetectably low as tested by immunofluorescence staining of mouse tissue sections using four different mAbs against mouse VCAM-1, three of which are described in this report. Surprisingly, expression of VCAM-1 on lymph node endothelium was not found even in mice which had been treated systemically with high doses of TNF-alpha or LPS, although VCAM-1 expression was strongly induced on endothelial of all other organs that were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)