Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo Vincent Pernet,* William W. Hauswirth and Adriana Di Polo* *Department of Pathology and Cell Biology, Universite ´ de Montre ´al, Montreal, Quebec, Canada  Departments of Ophthalmology and Powell Gene Therapy Center, University of Florida, Gainesville, Florida, USA Abstract Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal- regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen- activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environ- ment. Keywords: gene therapy, mitogen-activated protein kinase, neuroprotection, neurotrophic factors, regeneration, retinal ganglion cell. J. Neurochem. (2005) 93, 72–83. Neurotrophins mediate a variety of key cellular responses in the developing and mature central nervous system (CNS) including proliferation, differentiation, survival, axon growth, and dendrite formation (Huang and Reichardt 2001; Miller and Kaplan 2003; Lu 2004). There has been great interest in the identification of the intracellular signa- ling pathways that regulate these cellular processes. Upon binding to Trk receptors, neurotrophins stimulate multiple signaling pathways, most notably the extracellular signal- regulated kinase 1/2 (Erk1/2), the phosphatidylinositol 3-kinase (PI3K)/Akt and the phospholipase C pathways (Chao 2003; Huang and Reichardt 2003). The signaling mechanisms that mediate the effects of neurotrophins, particularly as they relate to neuronal survival, have been extensively studied in vitro (Kaplan and Miller 2000; Harper and LoGrasso 2001; Hetman and Gozdz 2004). However, the signaling pathways involved in neurotrophin-induced survival and axon growth of adult central neurons in vivo remain poorly defined. The rat retinocollicular system is ideal to address the role of neurotrophin signaling in the survival and regeneration of adult Received September 14, 2004; revised manuscript received November 16, 2004; accepted November 17, 2004. Address correspondence and reprint requests to Adriana Di Polo, PhD, Department of Pathology and Cell Biology, Universite ´ de Montre ´al, 2900, Boul. Edouard-Montpetit, Pavillon Principal, Room N-535, Montreal, Quebec H3T 1J4, Canada. E-mail: dipoloa@patho.umontreal.ca Abbreviations used: AAV, adeno-associated virus; BDNF, brain-de- rived neurotrophic factor; CTb , cholera toxin b subunit; Erk1/2, extra- cellular signal-regulated kinases 1/2; GFP, green fluorescent protein; HA, hemagglutinin; MEK1, mitogen-activated protein kinase kinase 1; MEK- ca, constitutively active MEK1 mutant; MEK-wt, wild-type MEK1; PI3K, phosphatidylinositol 3-kinase; RGCs, retinal ganglion cells. Journal of Neurochemistry , 2005, 93, 72–83 doi:10.1111/j.1471-4159.2005.03002.x 72 Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 72–83