Sebastian Kügler

Cascade of caspase activation in potassium-deprived cerebellar granule neurons: targets for treatment with peptide and protein inhibitors of apoptosis.

Cerebellar granule neurons (CGN) cultured in the presence of serum and depolarizing potassium concentrations undergo apoptosis when switched to serum-free medium containing physiological potassium concentrations. Here we show that processing of the key protease, caspase-3, depends on the activation of caspase-9, but not of caspase-8. Selective peptide inhibitors of caspase-9 block processing of caspase-3 and caspase-8 and inhibit apoptosis, whereas a selective inhibitor of caspase-8 blocks neither processing of caspase-3 nor cell death. The data obtained with peptide inhibitors were confirmed by adenovirally mediated ectopic expression of the cytokine response modifier A (crmA), the baculovirus protein p35, and the X chromosome-linked inhibitor of apoptosis (XIAP). Further, caspase-8-activating death receptors do not mediate apoptosis in CGN and potassium withdrawal-induced apoptosis evolves unaltered in gld or lpr mice, which harbor mutations in the CD95/CD95 ligand system. Thus, neuronal apoptosis triggered by potassium deprivation is death receptor-independent but involves the mitochondrial pathway of caspase activation.

Dose-dependent rescue of axotomized rat retinal ganglion cells by adenovirus-mediated expression of glial cell-line derived neurotrophic factor in vivo.

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 × 107 plaque‐forming units (pfu) or 7 × 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.

The human vigilin gene: identification, chromosomal localization and expression pattern

Chick vigilin cRNA clones were used to isolate the cognate human gene, by screening a pWE15 genomic library. Three independent cosmid clones were isolated and characterized by restriction mapping. The gene was identified by sequencing an internal EcoRI fragment containing two exons homologous to exon 24 and 25 of the chicken vigilin gene and corresponding to nucleotides 1973–2104 of the human HBP-cDNA. The homology between the chicken and human sequences was 77% and 82% at the cDNA level, and 91% and 100% at the amino acid level. In addition, the analyzed intron/exon boundaries were invariantly conserved. The 5′ and 3′ regions of the human gene were mapped by Southern analysis of the respective clones with synthetic oligonucleotides. The entire vigilin gene spans a region of about 50 kb and has been assigned to chromosome 2q36–q37.2 (FL-pter value of 0.96 ± 0.03) by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes. The vigilin gene is localized in a chromosomal region comprising a cluster of collagen genes (COLIVA3, COLVIA3) and the locus of the Waardenburg syndrome I. Only one mRNA species of 4.4 kb is transcribed from the human vigilin gene. In accordance with previous observations on chicken mRNA, the expression of the human vigilin mRNA depends on the stage of cytodifferentiation both in vitro and in situ.

Adenovirus-Mediated Glial Cell Line-Derived Neurotrophic Factor (GDNF) Expression Protects against Subsequent Cortical Cold Injury in Rats

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.

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.

Ehlers-Danlos syndrome type VI: lysyl hydroxylase deficiency due to a novel point mutation (W612C)

Abstract Ehlers-Danlos syndrome type VI (EDS VI) is a rare autosomal recessively inherited disease of connective tissue. The characteristic symptoms are hyperflexibility of joints and hyperelasticity of skin together with marked scoliosis, ocular manifestations and involvement of the vascular system. The underlying biochemical defect in EDS VI is a deficiency in lysyl hydroxylase (PLOD) activity resulting from mutations in the PLOD gene causing a low hydroxylysine content in various tissues. We found that two out of three patients showed a recently described duplication of about 800 bp in their LH mRNA. In the third patient we identified a new point mutation (2036 G→C) resulting in a substitution of tryptophan by cysteine in the highly conserved C-terminal region of the enzyme (W612C). In addition, this mutation destroys a restriction site of MwoI. Restriction analysis of the patient’s cDNA with MwoI showed the sole occurrence of the mutated transcript, while one allele in his genomic DNA contained the MwoI restriction site. Restriction analysis of the genomic DNA of the unaffected parents displayed a heterozygous loss of the restriction site for MwoI in the mother while the DNA of the father appeared normal. Our study demonstrates that the new point mutation (W612C) in conjunction with a functionless allele, most probably a null allele, for the LH gene may explain the functional deficiencies seen in this patient.

Identification of a new heterozygous point mutation in the COL1A2 gene leading to skipping of exon 9 in a patient with joint laxity, hyperextensibility of skin and blue sclerae

A heterozygous deletion of exon 9 in the COL1A2 ‐ mRNA of a patient with symptoms of both the Ehlers ‐ Danlos ‐ Syndrome and the Osteogenesis Imperfecta is described. In the genomic DNA of the patient, exon 9 is homozygously present. We identified a novel heterozygous point mutation in the splice donor site of intron 9, leading to a G→A substitution in position +5. This mutation leads to heterozygous skipping of exon 9 in the COL1A2 ‐ mRNA of this patient. The deletion results in a shortened (by 18 amino acids) but in frame l2(1) chain, which probably leads to the formation of abberantly processed triple helices. Hum Mutat 12:138, 1998.

Identification of a new heterozygous point mutation in the COL1A2 gene leading to skipping of exon 9 in a patient with joint laxity, hyperextensibility of skin and blue sclerae. Mutations in brief no. 166. Online.

A heterozygous deletion of exon 9 in the COL1A2 ‐ mRNA of a patient with symptoms of both the Ehlers ‐ Danlos ‐ Syndrome and the Osteogenesis Imperfecta is described. In the genomic DNA of the patient, exon 9 is homozygously present. We identified a novel heterozygous point mutation in the splice donor site of intron 9, leading to a G→A substitution in position +5. This mutation leads to heterozygous skipping of exon 9 in the COL1A2 ‐ mRNA of this patient. The deletion results in a shortened (by 18 amino acids) but in frame l2(1) chain, which probably leads to the formation of abberantly processed triple helices. Hum Mutat 12:138, 1998.