Retinal Degeneration Caused by Rod-Specific Dhdds Ablation Occurs without Concomitant Inhibition of ProteinN-Glycosylation

Dehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step indolichol synthesis. Recessive mutations inDHDDScause retinitis pigmentosa(RP59), resulting in blindness. We hypothesized that rod photoreceptor-specificablation ofDhddswould cause retinal degeneration due to diminished dolichol-dependent proteinN-glycosylation.Dhddsflx/flxmice were crossed with rod-spe-cific Cre recombinase-expressing (Rho-iCre75) mice to generate rod-specificDhddsknockout mice (Dhddsflx/flxiCre+).In vivomorphological and electrophys-iological evaluation ofDhddsflx/flxiCre+retinas revealed mild retinal dysfunctionat postnatal (PN) 4 weeks, compared with age-matched controls; however, rapidphotoreceptor degeneration ensued, resulting in almost complete loss of rodsand cones by PN 6 weeks. Retina dolichol levels were markedly decreased byPN 4 weeks inDhddsflx/flxiCre+mice, relative to controls; despite this,N-glycosyl-ation of retinal proteins, including opsin (the dominant rod-specific glycoprotein),persisted inDhddsflx/flxiCre+mice. These findings challenge the conventionalmechanistic view of RP59 as a congenital disorder of glycosylation

Isl1 and Pou4f2 form a complex to regulate target genes in developing retinal ganglion cells.

Precise regulation of gene expression during biological processes, including development, is often achieved by combinatorial action of multiple transcription factors. The mechanisms by which these factors collaborate are largely not known. We have shown previously that Isl1, a Lim-Homeodomain transcription factor, and Pou4f2, a class IV POU domain transcription factor, co-regulate a set of genes required for retinal ganglion cell (RGC) differentiation. Here we further explore how these two factors interact to precisely regulate gene expression during RGC development. By GST pulldown assays, co-immunoprecipitation, and electrophoretic mobility shift assays, we show that Isl1 and Pou4f2 form a complex in vitro and in vivo, and identify the domains within these two proteins that are responsible for this interaction. By luciferase assay, in situ hybridization, and RNA-seq, we further demonstrate that the two factors contribute quantitatively to gene expression in the developing RGCs. Although each factor alone can activate gene expression, both factors are required to achieve optimal expression levels. Finally, we discover that Isl1 and Pou4f2 can interact with other POU and Lim-Homeodomain factors respectively, indicating the interactions between these two classes of transcription factors are prevalent in development and other biological processes

Neuronal p58^IPK Protects Retinal Ganglion Cells Independently of Macrophage/Microglia Activation in Ocular Hypertension

p58IPK is a multifaceted endoplasmic reticulum (ER) chaperone and a regulator of eIF2α kinases involved in a wide range of cellular processes including protein synthesis, ER stress response, and macrophage-mediated inflammation. Systemic deletion of p58IPK leads to age-related loss of retinal ganglion cells (RGC) and exacerbates RGC damage induced by ischemia/reperfusion and increased intraocular pressure (IOP), suggesting a protective role of p58IPK in the retina. However, the mechanisms remain elusive. Herein, we investigated the cellular mechanisms underlying the neuroprotection action of p58IPK using conditional knockout (cKO) mouse lines where p58IPK is deleted in retinal neurons (Chx10-p58IPK cKO) or in myeloid cells (Lyz2-p58IPK cKO). In addition, we overexpressed p58IPK by adeno-associated virus (AAV) in the retina to examine the effect of p58IPK on RGC survival after ocular hypertension (OHT) in wild type (WT) mice. Our results show that overexpression of p58IPK by AAV significantly improved RGC survival after OHT in WT mice, suggesting a protective effect of p58IPK on reducing RGC injury. Conditional knockout of p58IPK in retinal neurons or in myeloid cells did not alter retinal structure or cellular composition. However, a significant reduction in the b wave of light-adapted electroretinogram (ERG) was observed in Chx10-p58IPK cKO mice. Deletion of p58IPK in retinal neurons exacerbates RGC loss at 14 days after OHT. In contrast, deficiency of p58IPK in myeloid cells increased the microglia/macrophage activation but had no effect on RGC loss. We conclude that deletion of p58IPK in macrophages increases their activation, but does not influence RGC survival. These results suggest that the neuroprotective action of p58IPK is mediated by its expression in retinal neurons, but not in macrophages. Therefore, targeting p58IPK specifically in retinal neurons is a promising approach for the treatment of neurodegenerative retinal diseases including glaucoma

Identification of domains within Pou4f2 and Isl1 responsible for their mutual interaction.

<p>A. Top: GST-Isl1 could interact with Pou4f2D4 and Pou4f2D5, but not Pou4f2D1, Pou4f2D2 or Pou4f2D3, as detected by anti-His. Full-length Pou4f2 serves as positive control. Middle: Input GST or GST-Isl1 detected by anti-GST. Bottom: Input Pou4f2 and its deletions could all be detected by anti-His. B: Top: GST-Pou4f2 (GST-P), but not GST, could interact with Isl1, Isl1D3, Isl1D4 and Isl1D5, but not with Isl1D1 and Isl1D2. Middle: Input GST or GST-Pou4f2 as detected by anti-GST. Bottom: Input Isl1 or its deletions as detected by a rabbit polyclonal anti-Isl1. Sizes (Kda) of protein markers are indicated on the side. Antibodies used for each blot are indicated at the bottom of each panel.</p

Pou4f2 and Isl1 form a complex in vitro and in vivo.

<p>A. Top: GST-Pou4f2 fusion protein (GST-P), but not GST, can interact with Isl1, as detected by anti-Isl1. Bottom: anti-GST demonstrates the GST and GST-Pou4f2 proteins used in the pull-down assays. Smaller bands in the GST-Pou4f2 lane are degradation products. B. Top: GST-Isl1 fusion protein (GST-I), but not GST alone, can interact with Pou4f2 as detected by anti-Pou4f2. Bottom: anti-GST recognizes both GST and GST-Isl1. Like GST-Pou4f2, there was also degradation of GST-Isl1 as indicated by the smaller bands. C. Co-Immunoprecipitation (IP) of Isl1 and Pou4f2 by a rabbit anti-Isl1. Both proteins were expressed in the HEK293 cells and could be detected in the input lysate by a mouse anti-Isl1 and goat anti-Pou4f2. In the rabbit IgG control, neither Isl1 nor Pou4f2 could be detected. When rabbit anti-Isl1 was used for the IP, both Isl1 and Pou4f2 could be detected.</p

Isl1 and Pou4f2 contribute quantitatively to the full levels of expression of downstream genes.

<p>A. Luciferase assays indicating that whereas Pou4f2 and Isl1 can each activate transcription from the reporter alone, together they promote transcription to a higher level. N = 3 for all transfections. Significance of differences was evaluated by student t test. Double black asterisks indicate p<0.01 when compared with control, double red asterisks indicate p<0.01 when compared with Pou4f2, double purple asterisks indicates p<0.01 when compared with Isl1. B. In situ hybridization of three downstream genes, <i>Gap43</i>, <i>Nefl</i>, and <i>Stmn2</i>, of Isl1 and Pou4f2. Compared to the wild-type retina, although all three genes have significantly reduced levels of expression in the RGCs of both <i>Isl1</i>- and <i>Pou4f2</i>-null retinas, substantial levels of expression remain. In the <i>Isl1</i>/<i>Pou4f2</i> double null retinas, <i>Gap43</i> and <i>Nefl</i> are not detectable, but Stmn2 is still expressed. C. The expression levels of downstream genes of Isl1 and Pou4f2 vary greatly. Shown are the relative expression levels of five downstream genes as revealed by RNA-seq. Y axis is Fragments Per Kilobase of exon per Million fragments mapped (FPKM) based on the number of sequence reads and lengths of the transcripts of individual genes. D. Changes of expression levels of five downstream genes of Isl1 and Pou4f2 in the <i>Isl1</i>- and <i>Pou4f2</i>–null retinas as revealed by RNA-seq. WT: wild-type; P4f2: Pou4f2. The expression levels (Y axis) are calculated based on the FPKM of three replicates and the average values for each gene in the wild-type is set to1. N = 3 for all samples. Significance of differences was evaluated by student's t test. Single asterisk indicates p<0.05, double asterisks indicate p<0.01.</p