Optogenetic Approaches to Restoring Vision.

Severe loss of photoreceptor cells in inherited or acquired retinal degenerative diseases can result in partial loss of sight or complete blindness. The optogenetic strategy for restoration of vision utilizes optogenetic tools to convert surviving inner retinal neurons into photosensitive cells; thus, light sensitivity is imparted to the retina after the death of photoreceptor cells. Proof-of-concept studies, especially those using microbial rhodopsins, have demonstrated restoration of light responses in surviving retinal neurons and visually guided behaviors in animal models. Significant progress has also been made in improving microbial rhodopsin-based optogenetic tools, developing virus-mediated gene delivery, and targeting specific retinal neurons and subcellular compartments of retinal ganglion cells. In this article, we review the current status of the field and outline further directions and challenges to the advancement of this strategy toward clinical application and improvement in the outcomes of restored vision.

Possible formation of mitochondrial-RNA containing chimeric or trimeric RNA implies a post-transcriptional and post-splicing mechanism for RNA fusion.

Human cells are known to express many chimeric RNAs, i.e. RNAs containing two genes' sequences. Wondering whether there also is trimeric RNA, i.e. an RNA containing three genes' sequences, we wrote simple computer code to screen human expression sequence tags (ESTs) deposited in different public databases, and obtained hundreds of putative trimeric ESTs. We then used NCBI Blast and UCSC Blat browsers to further analyze their sequences, and identified 61 trimeric and two tetrameric ESTs (one EST containing four different sequences). We also identified 57 chimeric, trimeric or teterameric ESTs that contained both mitochondrial (mt) RNA and nuclear RNA (nRNA), i.e. were mtRNA-nRNA fusions. In some trimeric ESTs, the downstream partner was fused to the poly-A tail of the upstream partner, which, together with the mtRNA-nRNA fusions, suggests a possible new mechanism for RNA fusion that occurs after both transcription and splicing have been terminated, and possibly outside the nucleus, in contrast to the two current hypothetical mechanisms, trans-splicing and transcriptional-slippage, that occur in the nucleus. The mt-sequences in the mtRNA-nRNA fusions had pseudogenes in the nucleus but, surprisingly, localized mainly in chromosomes 1 and 5. In some mtRNA-nRNA fusions, as well as in some ESTs that were derived only from mtRNA, the mt-sequences might be cis- or trans-spliced. Actually, we cloned a new cis-spliced mtRNA, coined as 16SrRNA-s. Hence, mtDNA may not always be intron-less. Fusion of three or more RNAs to one, fusion of nRNA to mtRNA, and cis- or trans-splicing of mtRNA should all enlarge the cellular RNA repertoire, in turn enlarging the cellular functions. Therefore, future experimental verification of the existence of these novel classes of fusion RNAs and spliced mtRNAs in human cells should significantly advance our understanding of biology and medicine.

Splicing of mouse p53 pre-mRNA does not always follow the "first come, first served" principle and may be influenced by cisplatin treatment and serum starvation.

Transcription of a pre-mRNA in eukaryotic cells elongates from the 5' to the 3' end, but intron removal during a pre-mRNA splicing does not always proceed in this orientation. In this study, we identified eight mouse p53 transcripts that retained one or more of introns 6, 7 and 8. The 5' part of intron 9 was also retained while the 3' part was not studied. These intron-containing transcripts, abbreviated as p53-ICTs, were detected at low abundance in many mouse embryonic fibroblasts (MEF) as well as cancer cell lines and tissues, and the highest ratio of these p53-ICTs to the mature p53 mRNA was seen in the normal pancreas. Serum starvation decreased those p53-ICTs that retained introns 6 and 7 but increased the levels of those lacking these introns while the level of the mature p53 mRNA was unaffected. Treatment of several cancer cell lines with cisplatin increased the mature p53 mRNA level but decreased these p53-ICTs. Transfection of p53(-/-) MEF with the p53 cDNA or several p53-ICT mini-genes slightly increased the cell viability and rendered the cells resistant to cisplatin. These data also suggest that p53 pre-mRNA splicing may have multiple orders of intron removal, some of which may not follow the "first come, first served" principle. It remains possible that these p53-ICTs are splicing intermediates existing as a mechanism for the cell to respond more promptly to a demand for more p53 and that p53 protein may be required for a normal life of MEF.

Complex alternative splicing of the smarca2 gene suggests the importance of smarca2-B variants.

BRM is an ATPase component of the SWI/SNF complex that regulates chromatin remodeling and cell proliferation and is considered a tumor suppressor. In this study we characterized transcripts from the Smarca2 gene that encodes the BRM protein. We found that the human Smarca2 gene (hSmarca2), like its mouse counterpart (mSmarca2), also initiated a short transcript from intron 27 of the long transcript. We name the long and short transcripts as Smarca2-a and Smarca2-b, respectively. Like its human counterpart, mSmarca2-a also underwent alternative splicing at the 54-bp exon 29. The hSmarca2-b had two alternative initiation sites and underwent alternative splicing at three different 3' sites of exon 1 and at exons 2, 3 and/or 5. We identified nine hSmarca2-b mRNA variants that might produce five different proteins. mSmarca2-b also underwent alternative splicing at exon 3 and/or exon 5, besides alternatively retaining part of intron 1 in exon 1. Smarca2-b was expressed more abundantly than Smarca2-a in many cell lines and was more sensitive to serum starvation. Moreover, cyclin D1 also regulated the expression of both Smarca2-a and Smarca2-b in a complex manner. These data suggest that the functions of the Smarca2 gene may be very complex, not just simply inhibiting cell proliferation, and in certain situations may be elicited mainly by expressing the much less known Smarca2-b, not the better studied Smarca2-a and its products BRM proteins.

Ectopic expression of multiple microbial rhodopsins restores ON and OFF light responses in retinas with photoreceptor degeneration.

By expressing channel rhodopsin-2 (ChR2) in inner retinal neurons, previous studies have demonstrated restoration of ON responses in the retina after the death of rod and cone photoreceptors. In this study, we report that the expression of halorhodopsin (HaloR), a light-driven chloride pump, can effectively restore OFF responses in inner retinal neurons of mice with retinal degeneration. We show that HaloR-expressing retinal ganglion cells respond to light with rapid hyperpolarization and suppression of spike activity. After termination of the light stimulus, their membrane potential exhibits a rapid rebound overshoot with robust sustained or transient spike firing. Furthermore, we show that coexpression of ChR2/HaloR in retinal ganglion cells can produce ON, OFF, and even ON-OFF responses, depending on the wavelength of the light stimulus. Our results suggest that the expression of multiple microbial rhodopsins such as ChR2 and HaloR is a possible strategy to restore both ON and OFF light responses in the retina after the death of rod and cone photoreceptors.

Differential expression of three T-type calcium channels in retinal bipolar cells in rats.

Retinal bipolar cells convey visual information from photoreceptors to retinal third-order neurons, amacrine and ganglion cells, with graded potentials through diversified cell types. To understand the possible role of voltage-dependent T-type Ca2+ currents in retinal bipolar cells, we investigated the pharmacological and biophysical properties of T-type Ca2+ currents in acutely dissociated retinal cone bipolar cells from rats using whole-cell patch-clamp recordings. We observed a broad group of cone bipolar cells with prominent T-type Ca2+ currents (T-rich) and another group with prominent L-type Ca2+ currents (L-rich). Based on the pharmacological and biophysical properties of the T-type Ca2+ currents, T-rich cone bipolar cells could be divided into three subgroups. Each subgroup appeared to express a single dominant T-type Ca2+ channel subunit. The T-type calcium currents could generate low-threshold regenerative potentials or spikes. Our results suggest that T-type Ca2+ channels may play an active and distinct signaling role in second-order neurons of the visual system, in contrast to the common signaling by L-rich bipolar cells.

Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration.

The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.

Isolation and Cloning of 17 Novel C(2)H(2) Zinc Finger Gene Fragments.

A pair of degenerate primers were designed according to the DNA sequences of the C(2)H(2) zinc finger genes conservative domain and then homologious PCR was performed with the human genomic total DNA as template. The zinc finger fragments thus obtained were used as probes to screen cDNA libraries of human fetal kidney, muscle and marrow and 22 C(2)H(2) zinc finger gene fragments were selected. Among these fragments, 17 were confirmed to be novel zinc finger gene fragments by literature searches in the National Center Biotechnology Information (NCBI) database. Expression patterns of the clones K3-4 and K5-12 selected from kidney cDNA library were analyzed. The results showed that the expression level of K3-4 in kidney is obviously higher than in other tissues and K5-12 is expressed at different levels in 8 tissues.