Abstract
Recombinant adeno-associated viral (rAAV) vectors can be used to introduce neurotrophic genes into injured CNS neurons, promoting survival and axonal regeneration. Gene therapy holds much promise for the treatment of neurotrauma and neurodegenerative diseases; however, neurotrophic factors are known to alter dendritic architecture, and thus we set out to determine whether such transgenes also change the morphology of transduced neurons. We compared changes in dendritic morphology of regenerating adult rat retinal ganglion cells (RGCs) after long-term transduction with rAAV2 encoding: (i) green fluorescent protein (GFP), or (ii) bi-cistronic vectors encoding GFP and ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43). To enhance regeneration, rats received an autologous peripheral nerve graft onto the cut optic nerve of each rAAV2 injected eye. After 5–8 months, RGCs with regenerated axons were retrogradely labeled with fluorogold (FG). Live retinal wholemounts were prepared and GFP positive (transduced) or GFP negative (non-transduced) RGCs injected iontophoretically with 2% lucifer yellow. Dendritic morphology was analyzed using Neurolucida software. Significant changes in dendritic architecture were found, in both transduced and non-transduced populations. Multivariate analysis revealed that transgenic BDNF increased dendritic field area whereas GAP43 increased dendritic complexity. CNTF decreased complexity but only in a subset of RGCs. Sholl analysis showed changes in dendritic branching in rAAV2-BDNF-GFP and rAAV2-CNTF-GFP groups and the proportion of FG positive RGCs with aberrant morphology tripled in these groups compared to controls. RGCs in all transgene groups displayed abnormal stratification. Thus in addition to promoting cell survival and axonal regeneration, vector-mediated expression of neurotrophic factors has measurable, gene-specific effects on the morphology of injured adult neurons. Such changes will likely alter the functional properties of neurons and may need to be considered when designing vector-based protocols for the treatment of neurotrauma and neurodegeneration.
Highlights
Replication-deficient viral vectors such as recombinant adenoassociated virus are increasingly being used to introduce ‘therapeutic’ genes into neural cells, a method that allows targeted supply of neuroprotective and/or growth-promoting molecules to the injured or degenerating CNS [1,2,3,4,5]
Grafts were identified and photographed under UV light (Fig. 1A, G) and at 488 nm to determine whether the retinal ganglion cells (RGCs) were transduced (GFP+) or non-transduced (GFP2; Fig. 1B, K, L)
Throughout the text for each of the 4 vectors used in this study we will denote transduced and nontransduced FG+ RGCs as green fluorescent protein (GFP)/ntGFP, brain-derived neurotrophic factor (BDNF)/ntBDNF, ciliary neurotrophic factor (CNTF)/ntCNTF or growth-associated protein-43 (GAP43)/ntGAP43 respectively
Summary
Replication-deficient viral vectors such as recombinant adenoassociated virus (rAAV) are increasingly being used to introduce ‘therapeutic’ genes into neural cells, a method that allows targeted supply of neuroprotective and/or growth-promoting molecules to the injured or degenerating CNS [1,2,3,4,5]. Vitreal injection of rAAV serotype 2 (rAAV2) or other viral vectors encoding growth factors increases retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) injury [6,7,8,9]. What is not yet clear is the extent to which longterm constitutive expression of transgenes changes the structure and function of transduced neurons. This is especially relevant when using genes that encode, for example, secretable neurotrophic factors because these peptides are known to alter dendritic architecture, synaptic density and plasticity, cause down-regulation of cognate receptors and modulate activity of signaling molecules [15,16,17,18,19]. Persistent over-expression of some transgenes may alter local circuitry and neuronal responsiveness to endogenous neuroactive factors [1,20,21]
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