Herpes Simplex Virus Vector-Mediated Expression of Bcl-2 Protects Spinal Motor Neurons from Degeneration Following Root Avulsion Masanobu Yamada,* ,1 Atsushi Natsume,* ,1 Marina Mata,* , ‡ Thomas Oligino,† James Goss,* , ‡ Joseph Glorioso,† and David J. Fink* , † , ‡ ,2 *Department of Neurology and †Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; and ‡GRECC, VA Pittsburgh Healthcare System, Pittsburgh, Pennsylvania 15240 Received January 19, 2000; accepted October 31, 2000; published online February 16, 2001 Proximal axotomy in adult animals results in de- layed death of motor neurons. Features characteristic of both necrosis and apoptosis have been described in motor neurons of the spinal cord following proximal avulsion of the ventral roots. We have previously dem- onstrated that a genomic herpes simplex virus (HSV)- based vector expressing the anti-apoptotic peptide Bcl-2 protects dopaminergic neurons of the substantia nigra from neurotoxin-induced apoptotic cell death and preserves the neurotransmitter phenotype of those cells. In this study we examined whether the same vector could protect adult rat lumbar motor neu- rons from cell death following proximal ventral root avulsion. Injection of the Bcl-2-expressing vector 1 week prior to root avulsion increased the survival of lesioned motor neurons, determined by retrograde Fluorogold labeling, by 50%. The Bcl-2-expressing vec- tor did not preserve choline acetyltransferase neuro- transmitter phenotype of the lesioned cells. These re- sults shed light on the mechanism of cell death follow- ing axonal injury, and have implications for developing an effective treatment for the clinical problem of proximal root avulsion. © 2001 Academic Press Key Words: gene therapy; herpes simplex; apoptosis; motor neuron; trauma. INTRODUCTION In response to axonal injury neurons undergo a ste- reotyped retrograde reaction (20); for neurons of the peripheral nervous system recovery from injury is fol- lowed by axonal regeneration. However, a proximal injury near to the cell body, particularly when com- bined with some degree of mechanical traction, results in the death of a substantial fraction of the axotomized neurons (14), forestalling the possibility of axonal re- generation and functional recovery, even in those cases where fiber continuity is reestablished. This accounts for the devastating paralysis that follows traumatic injuries of the brachial plexus (29), for instance. The mechanism of neuronal cell death following proximal root avulsion has not been satisfactorily es- tablished. Morphologically, early chromatolysis is fol- lowed by loss of transmitter phenotype and accumula- tion of phosphorylated neurofilaments in the perikaryon (14). Motor neuron cell death after root avulsion in adult animals exhibits characteristics of both apoptosis and necrosis with some reports empha- sizing the necrotic features (17), and others describing the close resemblance to apoptosis according to ultra- structural criteria (22). An increase in expression of the pro-apoptotic peptide bax with reciprocal decrease in expression of the anti-apoptotic peptide Bcl-2 has been found after hypoglossal axotomy (2), although the response in that nucleus is not as clear as that de- scribed in the sciatic model (11). The distinction has potential therapeutic implications, because a number of strategies are available to interrupt the apoptotic cascade and rescue cells that are destined to die by apoptosis. We have previously demonstrated that a nonrepli- cating genomic herpes simplex virus-based vector car- rying the coding sequence for Bcl-2 under the tran- scriptional control of the simian cytomegalovirus im- mediate early promoter (SCMV IEp) injected in the striatum 1 week before lesioning with 6-hydroxydopa- mine (6-OHDA) protects the dopaminergic neurons of the substantia nigra from toxin-induced cell death and preserves the neurotransmitter phenotype of those cells (32). Bcl-2, acting through several pathways, blocks activation of the caspase cascade, prevents the opening of the mitochondrial permeability transition complex and the release of cytochrome C (16), and has been demonstrated to be capable of protecting neurons from death resulting from a variety of insults (25). 1 The first two authors contributed equally to the work in this report. 2 To whom correspondence should be addressed at S-520 Biomed- ical Science Tower, 200 Lothrop St., Pittsburgh, PA 15213. Fax: (412) 648-8081. Experimental Neurology 168, 225–230 (2001) doi:10.1006/exnr.2000.7597, available online at http://www.idealibrary.com on 225 0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.