TRANSYNAPTIC DELIVERY OF ADENOVIRAL GENES FOLLOWING UNILATERAL SCIATIC NERVE VECTOR INJECTION
Boulis N.M., Turner D.E., Dice J.A., Feldman E.L. Dept. of Neurology, Univ. of Michigan, Ann Arbor, MI, USA
The retrograde axonal transport and transynaptic movement of
several neurotropic viruses is a well recognized mechanism of
viral spread throughout the nervous system. Recent work suggests
that adenovirus, a virus without intrinsic neurotropism, may spread
by axonal transport. The current experiment suggests that the
transynaptic movement of adenovirus may also occur. Sciatic nerve
injection of the adenoviral vector Ad5RSVntlacZ results in gene
transfer to primary neurons in the spinal cord's ipsilateral ventral
horn as well as secondary neurons in the dorsal horn and contralateral
ventral horn. Injection of the right sciatic nerve with the retrograde
tracer Fluorogold 3 d after injection with Ad5RSVntLacZ results
in distinct patterns of staining for each. In all animals, gene
expression was detected in all 4 quadrants, but predominantly
in the ipsilateral ventral horn. Fluorogold was only detected
in the ipsilateral ventral horn. In order to test that delivery
was the result of a nerve specific pathway, 5 animals received
Ad5RSVntLacZ delivery to the distal stump of the right sciatic
nerve transected 2 d before injection. These animals showed robust
X-gal staining in the distal stump but no staining in the spinal
cord. Taken together, these results suggest that adenovirus injected
into the sciatic nerve is taken by a nerve specific mechanism
to spinal cord and DRG neurons, then transported across synapses
to secondary neurons. Supported by NIH T32 NS07222 (N.M.B.),
R01 NS38849 and grants from the ADA and the JDFI (E.L.F.)
CHARACTERIZATION OF ADENOVIRAL GENE EXPRESSION IN SPINAL CORD FOLLOWING REMOTE VECTOR DELIVERY
Turner D.E., Boulis N.M., Dice J.A. Noordmans A., Feldman E.L. Dept. of Neurology, Univ. of Michigan, Ann Arbor, MI, USA
Recent work has established that the remote injection of attenuated
adenoviral vectors may result in central nervous system (CNS)
gene expression. These studies suggest that virus passes through
peripheral nerves into the CNS. The present experiment attempts
to systematically characterize this phenomenon. Spinal cord cells
staining for the reporter gene b-galactosidase
were histologically quantified following microinjection of the
viral vector, Ad5RSVntlacZ, into rat footpad, muscle, or sciatic
nerve. The effects of injection location, titer, and time, as
well as nerve crush and dexamethasone were examined. Sciatic nerve
viral vector injection results in significantly higher CNS uptake
than intramuscular and subcutaneous injections (P<0.05). Nerve
crush injury caused a time dependent reduction in spinal cord
gene uptake following sciatic nerve adenoviral injection (P<0.05).
Neuronal staining reaches its peak at 6 d following injection
(P<0.002). Peripheral nerve delivery to the CNS increases with
augmented titers (P<0.03). Lastly, gene expression is both
prolonged and augmented by administration of dexamethasone (P<0.0001).
Remote adenoviral vector injection represents an potential method
or spinal cord gene therapy that avoids any manipulation of CNS
tissue. Supported by NIH T32 NS07222 (N.M.B.), R01 NS38849
and grants from the ADA and the JDFI (E.L.F.)
MICE LACKING COMPLEX GANGLIOSIDES HAVE FIBER LOSS AND MYELINATION DEFECTS
Sheikh K.A., Sun J., Crawford T.O., Proia R.L., Griffin J.W., Schnaar R.L. Johns Hopkins University School of Medicine, Baltimore, MD 21287 U.S.A.
Gangliosides are a family of sialic acid-containing glycosphingolipids highly enriched in the mammalian nervous system. The major nervous system gangliosides GM1, GDla, GDlb, GTlb, and GQlb ("complex gangliosides") are closely related structures containing a ceramide lipid, a neutral tetrasaccharide chain, and one or more sialic acids. Their neurobiological functions remain poorly defined. Complex ganglioside biosynthesis occurs via the sequential actions of specific glycosyltransferases. By disrupting the gene for a key enzyme in complex ganglioside biosynthesis (GM2/GD2 synthase; EC 2.4.1.92) we generated mice which express only simple gangliosides (GM3/GD3), and examined their peripheral and central nervous systems. The SE mice display axonal degeneration in both the peripheral and central nervous systems, demyelination in peripheral nerves, and decreased central myelination. The pathological features of their nervous systems closely resemble those reported in mice with a disrupted gene for myelin-associated glycoprotein (MAG), a myelin receptor which binds to complex brain gangliosides in vitro. Furthermore, GM2/GD2 knockout mice have reduced MAG expression in the central nervous system. These results indicate that complex gangliosides are required in maintaining the integrity of both peripheral and central axons and peripheral myelin and for normal central myelination. They also support the theory that complex gangliosides are endogenous ligands for MAG.
TNFa INDUCED PHOSPHORYLATION OF STRESS ACTIVATED KINASES AND APOPTOSIS IN SCHWANN CELLS ARE INHIBITED BY PROSAPTIDE TX14(A).
Campana W.M.(1), Darin S.(2), O'Brien J.S.(1) (1)Department of Neurosciences, University of California, San Diego, La Jolla, CA and (2)Myelos Neurosciences, San Diego, CA.
TNFa is a pro-inflammatory cytokine associated with pain following peripheral nerve injury. Recently, the neurotrophic prosaptide, TX14(A), has been demonstrated to alleviate TNFa induced hyperalgesia and reduce TNF receptor 1 expression following injection of TNFa into peripheral nerve. We therefore investigated whether TNFa-induced apoptosis in primary Schwann cell cultures could be prevented by TX14(A). TNFa dose-dependently (3-100 ng/mL) increased the amount of cytoplasmic nucleosomes, an indicator of early stage apoptosis, in primary Schwann cells within 18 hours of exposure. After 48 hours, approximately 30% of Schwann cells treated with TNFa (50 ng/mL) were trypan blue positive, indicating cell death. However, in the presence of TX14(A) at 2 nM and 25 nM, cell death was reduced to 15% and 10%, respectively. To define the signaling pathways associated with these events, we examined the effects of TNFa on stress activated kinases, including p38 kinase and c-Jun N-terminal kinases (JNKs). TNFa dose-depen-dently increased phosphorylation of p38 (up to 4.5±1.1-fold; mean ± SEM) and both 46 and 54 kDa JNKs (up to 8 and 6-fold, respectively) within 5 minutes. TX14(A) (25 nM) attenuated TNFa-induced p38 and JNKs phosphorylation. SB203580, a specific inhibitor of p38 phosphor-ylation, also attenuated TNFa induced cell death, thereby linking activation of p38 to apoptotic pathways in Schwann cells. P38 phosphorylation was also increased by LY294002, a specific PI3K inhibitor, suggesting convergence of these signaling pathways. TX14(A) activated the PI3K/ Akt pathway in Schwann cells, which is directly involved in cell survival. Collectively, these data indicate that prosaptides regulate signaling pathways that antagonize pro-apoptotic signaling events. Supported by Myelos Corporation.
INHIBITION OF THE TRANSCRIPTION FACTOR NFkB PREVENTS CHEMOTHERAPY INDUCED NEURONAL DEATH
Gill J.S., Windebank A.J. Mayo Molecular Neuroscience Program, Rochester, MN 55905.
Sensory neuropathy is a debilitating side effect associated with a number of anti-cancer drugs. Previous studies from our laboratory have revealed drug-induced cell cycle re-entry by post-mitotic dorsal root ganglion neurons leading to apoptosis in cultures exposed to the chemotherapeutic agent suramin. The focus of the present study was to characterize the molecular elements mediating cell cycle re-entry and neuronal death. In suramin treated neuronal cultures, the lipid messenger ceramide was found to be a specific signaling element. Ceramide accumulation mediated activation and nuclear translocation of the transcription factor NFkB with subsequent cyclin D1 protein expression, a G1 phase specific protein. Specific inhibition of NFkB using a novel molecular decoy strategy resulted in increased cell viability accompanied by diminished caspase-3 activity. Inhibition of NFkB activity reduced cyclin D1 expression to control levels. Our findings describe an ordered signaling pathway implicated in suramin induced neuronal death: NFkB activation, cell cycle re-entry, caspase activation, and apoptosis. These studies warrant the development of neuron-specific molecular strategies to inhibit NFkB as potential adjuvant therapy to ameliorate the peripheral neuropathy associated with suramin treatment.
MYELIN ASSOCIATED GLYCOPROTEIN (MAG): A NOVEL METHOD OF ISOLATION AND A MORE CLINICALLY USEFUL ANTI-MAG ELISA?
Lunn M.P.T., Gregson N.A., Hughes R.A.C. Department of Clinical Neurosciences, Guy's King's and St Thomas' School of Medicine, Guy's Hospital, London, UK.
Monoclonal antibodies to MAG are found in the serum of 50% of patients with an IgM paraproteinaemic demyelinating neuropathy. They are implicated in pathogenesis. A sensitive, specific clinical diagnostic test is required. We developed an ELISA using pure intact MAG, isolated by a novel method, as the solid phase. Ovine CNS myelin was extracted by differential centrifugation of homogenised whole brain followed by discontinuous sucrose density gradient centrifugation and osmotic shock. Myelin proteins were extracted in 2% Nonidet NP-40 and 0.4% SDS detergent. A glycoprotein fraction was prepared by lectin affinity chromatography using Con-A. MAG was isolated from this by size exclusion chromatography and low molecular weight centrifuge filtration. The yield of MAG was estimated at 5-10%. The molecule retains its HNK-1 epitopes and complete protein structure. An ELISA was developed using the pure MAG produced. Sera from 64 patients with paraprotein associated neuropathy and 36 controls were tested for antibodies against MAG using the ELISA. Results were compared to those from CFT against human nerve homogenate and western blotting for MAG, and TLC overlay for sulphated glycolipids. 13/16 CFT positive cases were also positive on ELISA. Three clinically atypical cases were ELISA negative. Five clinically typical cases with a negative CFT were identified. Four IgM paraproteinaemic controls (no identified neuropathy) were positive. All other controls were negative. ELISA using intact ovine CNS MAG as the solid phase is a clinically useful, sensitive and specific test for anti-MAG antibodies.