Josephine Nalbantoglu

Expression of the Primary Coxsackie and Adenovirus Receptor Is Downregulated during Skeletal Muscle Maturation and Limits the Efficacy of Adenovirus-Mediated Gene Delivery to Muscle Cells

Skeletal muscle fibers are infected efficiently by adenoviral vectors only in neonatal animals. This lack of tropism for mature skeletal muscle may be partly due to inefficient binding of adenoviral particles to the cell surface. We evaluated in developing mouse muscle the expression levels of two high-affinity receptors for adenovirus, MHC class I and the coxsackie and adenovirus receptor (CAR). The moderate levels of MHC class I transcripts that were detected in quadriceps, gastrocnemius, and heart muscle did not vary between postnatal day 3 and day 60 adult tissue. A low level of CAR expression was detected on postnatal day 3 in quadriceps and gastrocnemius muscles, but CAR expression was barely detectable in adult skeletal muscle even by reverse transcriptase-polymerase chain reaction. In contrast, CAR transcripts were moderately abundant at all stages of heart muscle development. Ectopic expression of CAR in C2C12 mouse myoblast cells increased their transducibility by adenovirus at all multiplicities of infection (MOIs) tested as measured by lacZ reporter gene activity following AVCMVlacZ infection, with an 80-fold difference between CAR-expressing cells and control C2C12 cells at an MOI of 50. Primary myoblasts ectopically expressing CAR were injected into muscles of syngeneic hosts; following incorporation of the exogenous myoblasts into host myofibers, an increased transducibility of adult muscle fibers by AVCMVlacZ was observed in the host. Expression of the lacZ reporter gene in host myofibers coincided with CAR immunoreactivity. Furthermore, sarcolemmal CAR expression was markedly increased in regenerating muscle fibers of the dystrophic mdx mouse, fibers that are susceptible to adenovirus transduction. These analyses show that CAR expression by skeletal muscle correlates with its susceptibility to adenovirus transduction, and that forced CAR expression in mature myofibers dramatically increases their susceptibility to adenovirus transduction.

Oligodendrocyte injury in multiple sclerosis: a role for p53

Multiple sclerosis (MS) is a neurological disorder characterized by myelin destruction and a variable degree of oligodendrocyte death. We have previously shown that overexpression of the transcription factor p53 can induce oligodendrocyte apoptosis. We investigated the mechanism of p53‐induced apoptosis using primary cultures of central nervous system‐derived adult human oligodendrocytes. Adenovirus‐mediated p53 overexpression resulted in up‐regulation of the death receptors Fas, DR4 and DR5 with subsequent caspase‐mediated apoptosis of the oligodendrocytes. The oligodendrocytes were protected from p53‐induced cell death by blocking signaling through Fas and/or tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) receptors. Although lower levels of p53 did not induce apoptosis, the increase in death receptor expression was sufficient to render the oligodendrocytes susceptible to apoptosis in the presence of exogenous Fas ligand and TRAIL. These ligands are present in the inflammatory milieu of active MS lesions. In situ analysis of active MS lesions revealed increased p53 expression in oligodendrocytes in lesions that featured oligodendrocyte apoptosis and cell loss. Our data provide evidence for a novel role for p53 in the pathogenesis of MS.

Muscle-specific overexpression of the adenovirus primary receptor CAR overcomes low efficiency of gene transfer to mature skeletal muscle.

ABSTRACT Significant levels of adenovirus (Ad)-mediated gene transfer occur only in immature muscle or in regenerating muscle, indicating that a developmentally regulated event plays a major role in limiting transgene expression in mature skeletal muscle. We have previously shown that in developing mouse muscle, expression of the primary Ad receptor CAR is severely downregulated during muscle maturation. To evaluate how global expression of CAR throughout muscle affects Ad vector (AdV)-mediated gene transfer into mature skeletal muscle, we produced transgenic mice that express the CAR cDNA under the control of the muscle-specific creatine kinase promoter. Five-month-old transgenic mice were compared to their nontransgenic littermates for their susceptibility to AdV transduction. In CAR transgenics that had been injected in the tibialis anterior muscle with AdVCMVlacZ, increased gene transfer was demonstrated by the increase in the number of transduced muscle fibers (433 ± 121 in transgenic mice versus 8 ± 4 in nontransgenic littermates) as well as the 25-fold increase in overall β-galactosidase activity. Even when the reporter gene was driven by a more efficient promoter (the cytomegalovirus enhancer–chicken β-actin gene promoter), differential transducibility was still evident (893 ± 149 versus 153 ± 30 fibers; P < 0.001). Furthermore, a fivefold decrease in the titer of injected AdV still resulted in significant transduction of muscle (253 ± 130 versus 14 ± 4 fibers). The dramatic enhancement in AdV-mediated gene transfer to mature skeletal muscle that is observed in the CAR transgenics indicates that prior modulation of the level of CAR expression can overcome the poor AdV transducibility of mature skeletal muscle and significant transduction can be obtained at low titers of AdV.

Caspase 8 expression and signaling in Fas injury–resistant human fetal astrocytes

Fas (APO‐1/CD95) is a cell surface receptor initially identified in lymphoid cells, but more recently detected in the central nervous system under pathological, usually inflammatory, conditions. In most Fas expressing cells, triggering of Fas by its ligand or by antagonistic antibodies leads to apoptosis. Human fetal astrocytes (HFA) constitutively express Fas yet are resistant to cell death following Fas ligation. In the current study, using dissociated cultures of human fetal central nervous system–derived cells, we attempted to identify a basis for HFA resistance to Fas‐mediated injury. We compared the components of the Fas signaling pathway of HFA to those of two human cell lines susceptible to Fas‐mediated injury, U251 glioma and Jurkat T‐cells. We found that HFA did not express caspase 8 (FLICE), the caspase primarily activated on Fas signaling. Although we could induce caspase 8 in HFA with the inflammatory cytokines IFNγ and TNFα, HFA remained resistant to Fas‐mediated injury. Addition of inflammatory cytokines to the extracellular milieu also increased FLIP mRNA (FLICE inhibitory protein). Furthermore, upon triggering of cytokine‐treated cells with FasL, we observed upregulation of the cleavage product of FLIP (p43‐FLIP) previously shown to associate with the DISC and to block caspase 8 recruitment, thereby inhibiting Fas‐mediated death. Our findings indicate that caspase 8 and its regulators play a central role in determining the response to Fas ligation of HFA and support a role for Fas signaling in the developing central nervous system other than related to cytotoxicity. GLIA 33:217–224, 2001.

Transcriptional Regulation of Amyloid Precursor Protein During Dibutyryl Cyclic AMP‐Induced Differentiation of NG108‐15 Cells

Abstract: Early expression of amyloid precursor protein (APP) during development of the nervous system suggests that this protein may play an important role first in axogenesis and later in synaptogenesis. To study regulation of APP mRNA expression in neuronal cells, NG108‐15 neuroblastoma × glioma cells were induced to differentiate in the presence of dibutyryl cyclic AMP. Steady‐state levels of APP mRNA and APP isoforms increased gradually, concomitantly with the appearance of differentiated phenotype. Northern blot analysis showed a three‐fold increase in APP expression at day 6 of dibutyryl cyclic AMP treatment. Nuclear run‐on assays and transient transfections performed using APP promoter/reporter constructs confirmed a twofold increase in the rate of APP gene transcription. The stability of the mRNA was unchanged, with differentiated and nondifferentiated cells having the same half‐life of about 21 h. These results strongly suggest that APP mRNA induction in the differentiated NG108‐15 cells is due to an increase in the rate of transcription of the gene.

Hypoxia-Induced Loss of Synaptic Transmission Is Exacerbated in Hippocampal Slices of Transgenic Mice Expressing C-Terminal Fragments of Alzheimer Amyloid Precursor Protein

To investigate the possible involvement of β‐amyloid (Aβ) in disrupting neuronal function during ischemia, we examined whether overexpression of C‐terminal fragments of β‐amyloid precursor protein (β‐APP) in transgenic (Tg) mice is capable of altering the capacity of hippocampus slices to recover synaptic transmission after transient hypoxic episodes. Recovery of synaptic transmission was monitored in area CA1 of perfused hippocampal slices prepared from both control and Tg mice. The results obtained indicate that hippocampal slices prepared from Tg mice exhibited a much lower level of recovery in synaptic transmission following reoxygenation. This reduction in the capacity of Tg slices to recover from hypoxia‐induced impairment of synaptic transmission in the hippocampus does not appear to be related to pre‐existing alterations in either functional or biochemical properties of glutamate receptors in Tg mice. The present results provide the first experimental evidence that overexpression of the C‐terminal fragment of APP exacerbates functional damage of hippocampal neurons after hypoxic episodes. Hippocampus 1999;9:201–205.