Abstract
Expression of optogenetic tools in surviving inner retinal neurons to impart retinal light sensitivity has been a new strategy for restoring vision after photoreceptor degeneration. One potential approach for restoring retinal light sensitivity after photoreceptor degeneration is to express optogenetic tools in retinal ganglion cells (RGCs). For this approach, restoration of ON and OFF center-surround receptive fields in RGCs, a key feature of visual information processing, may be important. A possible solution is to differentially express depolarizing and hyperpolarizing optogenetic tools, such as channelrhodopsin-2 and halorhodopsin, to the center and peripheral regions of the RGC dendritic field by using protein targeting motifs. Recombinant adeno-associated virus (rAAV) vectors have proven to be a powerful vehicle for in vitro and in vivo gene delivery, including in the retina. Therefore, the search for protein targeting motifs that can achieve rAAV-mediated subcellular targeted expression would be particularly valuable for developing therapeutic applications. In this study, we identified two protein motifs that are suitable for rAAV-mediated subcellular targeting for generating center-surround receptive fields while reducing the axonal expression in RGCs. Resulting morphological dendritic field and physiological response field by center-targeting were significantly smaller than those produced by surround-targeting. rAAV motif-mediated protein targeting could also be a valuable tool for studying physiological function and clinical applications in other areas of the central nervous system.
Highlights
The severe loss of photoreceptor cells in inherited and acquired retinal degenerative diseases, such as retinitis pigmentosa, could result in partial or complete blindness
The virus vectors were packaged at the Gene Transfer Vector Core of the University of Iowa. rAAV2 constructs carrying an elongation factor I alpha (EF1a) promoter and a double-floxed inverted open-reading frame (DIO) sequence encoding ChR2-YFP or NpHR-YFP were modified from a construct obtained from Dr Karl Deisseroth [25] by replacing YFP with mCherry or inserting motif sequences at the 39 end of YFP
The remaining were potential surround targeting motifs, AMPAR-motif [14], Kv4.2motif [15], MLPH-motif [16,17], nAChR-motif [18], NLG1-motif [19], and TLCN-motif [20], and all were previously reported to target the somatodendritic region of a neuron in vitro or in vivo
Summary
The severe loss of photoreceptor cells in inherited and acquired retinal degenerative diseases, such as retinitis pigmentosa, could result in partial or complete blindness. Expression of optogenetic tools in surviving inner retinal neurons to impart retinal light sensitivity has been a new strategy for restoring vision after photoreceptor degeneration [1,2,3,4,5,6,7]. One approach is to express microbial rhodopsins in retinal ganglion cells (RGCs). ON and OFF light responses can be restored in RGCs by expressing channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively [1,8]. A possible solution to a create centersurround receptive field is to differentially express depolarizing and hyperpolarizing optogenetic tools to the center and peripheral regions of the RGC dendritic field by using protein targeting motifs [10]
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