Two NOTCH1 O-fucose sites have opposing functions in mouse retinal angiogenesis.

Previous in vitro studies demonstrated that Fringe glycosylation of the NOTCH1 extracellular domain at O-fucose residues in Epidermal Growth Factor-like Repeats (EGFs) 6 and 8 is a significant contributor to suppression of NOTCH1 activation by JAG1 or enhancement of NOTCH1 activation by DLL1, respectively. In this study, we sought to evaluate the significance of these glycosylation sites in a mammalian model by generating 2 C57BL/6J mouse lines carrying NOTCH1 point mutations, which eliminate O-fucosylation and Fringe activity at EGFs 6 (T232V) or 8 (T311V). We assessed changes to morphology during retinal angiogenesis, a process in which expression of Notch1, Jag1, Dll4, Lfng, Mfng, and Rfng genes coordinate cell-fate decisions to grow vessel networks. In the EGF6 O-fucose mutant (6f/6f) retinas, we observed reduced vessel density and branching, suggesting that this mutant is a Notch1 hypermorph. This finding agrees with prior cell-based studies showing that the 6f mutation increased JAG1 activation of NOTCH1 during co-expression with inhibitory Fringes. Although we predicted that the EGF8 O-fucose mutant (8f/8f) would not complete embryonic development due to the direct involvement of the O-fucose in engaging ligand, the 8f/8f mice were viable and fertile. In the 8f/8f retina, we measured increased vessel density consistent with established Notch1 hypomorphs. Overall, our data support the importance of NOTCH1 O-fucose residues for pathway function and confirms that single O-glycan sites are rich in signaling instructions for mammalian development.

Mitochondria and Endoplasmic Reticulum Stress in Retinal Organoids from Patients with Vision Loss.

Activating transcription factor 6 (ATF6), a key regulator of the unfolded protein response, plays a key role in endoplasmic reticulum function and protein homeostasis. Variants of ATF6 that abrogate transcriptional activity cause morphologic and molecular defects in cones, clinically manifesting as the human vision loss disease achromatopsia (ACHM). ATF6 is expressed in all retinal cells. However, the effect of disease-associated ATF6 variants on other retinal cell types remains unclear. Herein, this was investigated by analyzing bulk RNA-sequencing transcriptomes from retinal organoids generated from patients with ACHM, carrying homozygous loss-of-function ATF6 variants. Marked dysregulation in mitochondrial respiratory complex gene expression and disrupted mitochondrial morphology in ACHM retinal organoids were identified. This indicated that loss of ATF6 leads to previously unappreciated mitochondrial defects in the retina. Next, gene expression from control and ACHM retinal organoids were compared with transcriptome profiles of seven major retinal cell types generated from recent single-cell transcriptomic maps of nondiseased human retina. This indicated pronounced down-regulation of cone genes and up-regulation in Müller glia genes, with no significant effects on other retinal cells. Overall, the current analysis of ACHM patient retinal organoids identified new cellular and molecular phenotypes in addition to cone dysfunction: activation of Müller cells, increased endoplasmic reticulum stress, disrupted mitochondrial structure, and elevated respiratory chain activity gene expression.

ATF6 is essential for human cone photoreceptor development.

Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling promote the pathology of many human diseases. Loss-of-function variants of the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital vision loss diseases such as achromatopsia by unclear pathomechanisms. To investigate this, we generated retinal organoids from achromatopsia patient induced pluripotent stem cells carrying ATF6 disease variants and from gene-edited ATF6 null hESCs. We found that achromatopsia patient and ATF6 null retinal organoids failed to form cone structures concomitant with loss of cone phototransduction gene expression, while rod photoreceptors developed normally. Adaptive optics retinal imaging of achromatopsia patients carrying ATF6 variants also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the defect in cone formation observed in our retinal organoids. These results establish that ATF6 is essential for human cone development. Interestingly, we find that a selective small molecule ATF6 signaling agonist restores the transcriptional activity of some ATF6 disease-causing variants and stimulates cone growth and gene expression in patient retinal organoids carrying these variants. These findings support that pharmacologic targeting of the ATF6 pathway can promote human cone development and should be further explored for blinding retinal diseases.

ARCAM-1 Facilitates Fluorescence Detection of Amyloid-Containing Deposits in the Retina.

Purpose: To investigate the use of an amyloid-targeting fluorescent probe, ARCAM-1, to identify amyloid-containing deposits in the retina of a transgenic mouse model of Alzheimer's disease (AD) and in human postmortem AD patients. Methods: Aged APP/PS1 transgenic AD and wild-type (WT) mice were given an intraperitoneal (IP) injection of ARCAM-1 and their retinas imaged in vivo using a fluorescence ophthalmoscope. Eyes were enucleated and dissected for ex vivo inspection of retinal amyloid deposits. Additionally, formalin-fixed eyes from human AD and control patients were dissected, and the retinas were stained using ARCAM-1 or with an anti-amyloid-ß antibody. Confocal microscopy was used to image amyloid-containing deposits stained with ARCAM-1 or with immunostaining. Results: Four out of eight APP/PS1 mice showed the presence of amyloid aggregates in the retina during antemortem imaging. Retinas from three human AD patients stained with ARCAM-1 showed an apparent increased density of fluorescently labeled amyloid-containing deposits compared to the retinas from two healthy, cognitively normal (CN) patients. Immunolabeling confirmed the presence of amyloid deposits in both the retinal neuronal layers and in retinal vasculature. Conclusions: ARCAM-1 facilitates antemortem detection of amyloid aggregates in the retina of a mouse model for AD, and postmortem detection of amyloid-containing deposits in human retinal tissues from AD patients. These results support the hypothesis of AD pathology manifesting in the eye and highlight a novel area for fluorophore development for the optical detection of retinal amyloid in AD patients. Translational Relevance: This paper represents an initial examination for potential translation of an amyloid-targeting fluorescent probe to a retinal imaging agent for aiding in the diagnosis of Alzheimer's disease.

Multiexon deletion alleles of ATF6 linked to achromatopsia.

Achromatopsia (ACHM) is an autosomal recessive disease that results in severe visual loss. Symptoms of ACHM include impaired visual acuity, nystagmus, and photoaversion starting from infancy; furthermore, ACHM is associated with bilateral foveal hypoplasia and absent or severely reduced cone photoreceptor function on electroretinography. Here, we performed genetic sequencing in 3 patients from 2 families with ACHM, identifying and functionally characterizing 2 mutations in the activating transcription factor 6 (ATF6) gene. We identified a homozygous deletion covering exons 8-14 of the ATF6 gene from 2 siblings from the same family. In another patient from a different family, we identified a heterozygous deletion covering exons 2 and 3 of the ATF6 gene found in trans with a previously identified ATF6 c.970C>T (p.Arg324Cys) ACHM disease allele. Recombinant ATF6 proteins bearing these exon deletions showed markedly impaired transcriptional activity by qPCR and RNA-Seq analysis compared with WT-ATF6. Finally, RNAscope revealed that ATF6 and the related ATF6B transcripts were expressed in cones as well as in all retinal layers in normal human retina. Overall, our data identify loss-of-function ATF6 disease alleles that cause human foveal disease.

Pathomechanisms of ATF6-Associated Cone Photoreceptor Diseases.

Activating transcription factor 6 (ATF6) is a key regulator of the unfolded protein response (UPR). In response to endoplasmic reticulum (ER) stress, ATF6 is transported from the ER to the Golgi apparatus where it is cleaved by intramembrane proteolysis, releasing its cytosolic fragment. The cleaved ATF6 fragment, which is a basic leucine zipper (bZip) transcription factor, translocates to the nucleus and upregulates the expression of ER protein-folding chaperones and enzymes. Mutations in ATF6 cause heritable forms of cone photoreceptor dysfunction diseases. These mutations include missense, nonsense, splice site, and deletion or duplication changes found across the entire ATF6. To date, there are 11 ATF6 mutations reported, and we classified them into three classes based on their functional defects that interrupt distinct steps in the ATF6 signaling pathway.

ER stress and unfolded protein response in ocular health and disease.

The human eye is the organ that is able to react to light in order to provide sharp three-dimensional and colored images. Unfortunately, the health of the eye can be impacted by various stimuli that can lead to vision loss, such as environmental changes, genetic mutations, or aging. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) signaling have been detected in many diverse ocular diseases, and chemical and genetic approaches to modulate ER stress and specific UPR regulatory molecules have shown beneficial effects in animal models of eye disease. This review highlights specific eye diseases associated with ER stress and UPR activity, based on a recent symposia exploring this theme.

The unfolded protein response regulator ATF6 promotes mesodermal differentiation.

ATF6 encodes a transcription factor that is anchored in the endoplasmic reticulum (ER) and activated during the unfolded protein response (UPR) to protect cells from ER stress. Deletion of the isoform activating transcription factor 6α (ATF6α) and its paralog ATF6ß results in embryonic lethality and notochord dysgenesis in nonhuman vertebrates, and loss-of-function mutations in ATF6α are associated with malformed neuroretina and congenital vision loss in humans. These phenotypes implicate an essential role for ATF6 during vertebrate development. We investigated this hypothesis using human stem cells undergoing differentiation into multipotent germ layers, nascent tissues, and organs. We artificially activated ATF6 in stem cells with a small-molecule ATF6 agonist and, conversely, inhibited ATF6 using induced pluripotent stem cells from patients with ATF6 mutations. We found that ATF6 suppressed pluripotency, enhanced differentiation, and unexpectedly directed mesodermal cell fate. Our findings reveal a role for ATF6 during differentiation and identify a new strategy to generate mesodermal tissues through the modulation of the ATF6 arm of the UPR.

In Vivo Visualization of Endoplasmic Reticulum Stress in the Retina Using the ERAI Reporter Mouse.

PURPOSE: Endoplasmic reticulum (ER) stress activates inositol requiring enzyme 1 (IRE1), a key regulator of the unfolded protein response. The ER stress activated indicator (ERAI) transgenic mouse expresses a yellow fluorescent GFP variant (Venus) when IRE1 is activated by ER stress. We tested whether ERAI mice would allow for real-time longitudinal studies of ER stress in living mouse eyes. METHODS: We chemically and genetically induced ER stress, and qualitatively and quantitatively studied the Venus signal by fluorescence ophthalmoscopy. We determined retinal cell types that contribute to the signal by immunohistology, and we performed molecular and biochemical assays using whole retinal lysates to assess activity of the IRE1 pathway. RESULTS: We found qualitative increase in vivo in fluorescence signal at sites of intravitreal tunicamycin injection in ERAI eyes, and quantitative increase in ERAI mice mated to RhoP23H mice expressing ER stress-inducing misfolded rhodopsin protein. As expected, we found that increased Venus signal arose primarily from photoreceptors in RhoP23H/+;ERAI mice. We found increased Xbp1S and XBP1s transcriptional target mRNA levels in RhoP23H/+;ERAI retinas compared to Rho+/+;ERAI retinas, and that Venus signal increased in ERAI retinas as a function of age. CONCLUSIONS: Fluorescence ophthalmoscopy of ERAI mice enables in vivo visualization of retinas undergoing ER stress. ER stress activated indicator mice enable identification of individual retinal cells undergoing ER stress by immunohistochemistry. ER stress activated indicator mice show higher Venus signal at older ages, likely arising from amplification of basal retinal ER stress levels by GFP's inherent stability.

The unfolded protein response is shaped by the NMD pathway.

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD's ability to regulate normal mRNAs.

Robust Endoplasmic Reticulum-Associated Degradation of Rhodopsin Precedes Retinal Degeneration.

Rhodopsin is a G protein-coupled receptor essential for vision and rod photoreceptor viability. Disease-associated rhodopsin mutations, such as P23H rhodopsin, cause rhodopsin protein misfolding and trigger endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR). The pathophysiologic effects of ER stress and UPR activation on photoreceptors are unclear. Here, by examining P23H rhodopsin knock-in mice, we found that the UPR inositol-requiring enzyme 1 (IRE1) signaling pathway is strongly activated in misfolded rhodopsin-expressing photoreceptors. IRE1 significantly upregulated ER-associated protein degradation (ERAD), triggering pronounced P23H rhodopsin degradation. Rhodopsin protein loss occurred as soon as photoreceptors developed, preceding photoreceptor cell death. By contrast, IRE1 activation did not affect JNK signaling or rhodopsin mRNA levels. Interestingly, pro-apoptotic signaling from the PERK UPR pathway was also not induced. Our findings reveal that an early and significant pathophysiologic effect of ER stress in photoreceptors is the highly efficient elimination of misfolded rhodopsin protein. We propose that early disruption of rhodopsin protein homeostasis in photoreceptors could contribute to retinal degeneration.

Endoplasmic reticulum stress in vertebrate mutant rhodopsin models of retinal degeneration.

Rhodopsin mutations cause many types of heritable retinitis pigmentosa (RP). Biochemical and in vitro studies have demonstrated that many RP-linked mutant rhodopsins produce misfolded rhodopsin proteins, which are prone to aggregation and retention within the endoplasmic reticulum, where they cause endoplasmic reticulum stress and activate the Unfolded Protein Response signaling pathways. Many vertebrate models of retinal degeneration have been created through expression of RP-linked rhodopsins in photoreceptors including, but not limited to, VPP/GHL mice, P23H Rhodopsin frogs, P23H rhodopsin rats, S334ter rhodopsin rats, C185R rhodopsin mice, T17M rhodopsin mice, and P23H rhodopsin mice. These models have provided many opportunities to test therapeutic strategies to prevent retinal degeneration and also enabled in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death. Here, we examine and compare the contribution of endoplasmic reticulum stress to retinal degeneration in several vertebrate models of RP generated through expression of mutant rhodopsins.

Induction of endoplasmic reticulum stress genes, BiP and chop, in genetic and environmental models of retinal degeneration.

PURPOSE: Endoplasmic reticulum (ER) stress has been observed in animal models of retinitis pigmentosa expressing P23H rhodopsin. We compared levels of tightly induced ER stress genes, Binding of immunoglobulin protein (BiP) and CCAAT/enhancer-binding protein homologous protein (Chop), in seven additional models of retinal degeneration arising from genetic or environmental causes. METHODS: Retinas from transgenic S334ter rhodopsin (lines 3, 4, and 5) and Royal College of Surgeons (RCS and RCS-p+) rats from postnatal (P) days 10 to 120 were analyzed. In a constant light (CL) model of retinal degeneration, BALB/c mice were exposed to 15,000 lux of CL for 0 to 8 hours. Retinal tissues from three to eight animals per experimental condition were collected for histologic and molecular analyses. RESULTS: S334ter animals revealed significant increases in BiP, S334ter-3 (3.3× at P15), S334ter-4 (4× at P60), and S334ter-5 (2.2× at P90), and Chop, S334ter-3 (1.3× at P15), S334ter-4 (1.5× at P30), and S334ter-5 (no change), compared with controls. P23H-3 rats showed significant increase of BiP at P60 (2.3×) and Chop (1.6×). RCS and RCS-p+ rats showed significant increases in BiP at P60 (2.4×) and P20 (1.8×), respectively, but no statistically significant changes in Chop. BALB/c mice showed increases in BiP (1.5×) and Chop (1.3×) after 4 hours of CL. Increased levels of these ER stress markers correlated with photoreceptor cell loss. CONCLUSIONS: Our study reveals surprising increases in BiP and to a lesser degree Chop in retinal degenerations arising from diverse causes. We propose that manipulation of ER stress responses may be helpful in treating many environmental and heritable forms of retinal degeneration.

Selective activation of ATF6 and PERK endoplasmic reticulum stress signaling pathways prevent mutant rhodopsin accumulation.

PURPOSE: Many rhodopsin mutations that cause retinitis pigmentosa produce misfolded rhodopsin proteins that are retained within the endoplasmic reticulum (ER) and cause photoreceptor cell death. Activating transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) control intracellular signaling pathways that maintain ER homeostasis. The aim of this study was to investigate how ATF6 and PERK signaling affected misfolded rhodopsin in cells, which could identify new molecular therapies to treat retinal diseases associated with ER protein misfolding. METHODS: To examine the effect of ATF6 on rhodopsin, wild-type (WT) or mutant rhodopsins were expressed in cells expressing inducible human ATF6f, the transcriptional activator domain of ATF6. Induction of ATF6f synthesis rapidly activated downstream genes. To examine PERK's effect on rhodopsin, WT or mutant rhodopsins were expressed in cells expressing a genetically altered PERK protein, Fv2E-PERK. Addition of the dimerizing molecule (AP20187) rapidly activated Fv2E-PERK and downstream genes. By use of these strategies, it was examined how selective ATF6 or PERK signaling affected the fate of WT and mutant rhodopsins. RESULTS: ATF6 significantly reduced T17M, P23H, Y178C, C185R, D190G, K296E, and S334ter rhodopsin protein levels in the cells with minimal effects on monomeric WT rhodopsin protein levels. By contrast, the PERK pathway reduced both levels of WT, mutant rhodopsins, and many other proteins in the cell. CONCLUSIONS: This study indicates that selectively activating ATF6 or PERK prevents mutant rhodopsin from accumulating in cells. ATF6 signaling may be especially useful in treating retinal degenerative diseases arising from rhodopsin misfolding by preferentially clearing mutant rhodopsin and abnormal rhodopsin aggregates.

Endoplasmic reticulum-associated degradation (ERAD) and autophagy cooperate to degrade polymerogenic mutant serpins.

The serpinopathies are a family of diseases characterized by the accumulation of ordered polymers of mutant protein within the endoplasmic reticulum. They are a diverse group including alpha(1)-antitrypsin deficiency and the inherited dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. We have used transient transfection of COS7 cells and mouse embryonic fibroblasts, PC12 cell lines that conditionally express wild type and mutant neuroserpin and fly models of FENIB to assess the cellular handling of wild type and mutant serpins. By using a polymer-specific monoclonal antibody, we show that mutant neuroserpin forms polymers after a delay of at least 30 min and that polymers can be cleared in PC12 cell lines and from the brain in a fly model of FENIB. At steady state, the fractions of intracellular polymerogenic G392E mutant neuroserpin in the monomeric and polymeric states are comparable. Inhibition of the proteasome with MG132 reveals that both mutant neuroserpin and alpha(1)-antitrypsin are degraded predominantly by endoplasmic reticulum-associated degradation (ERAD). Pharmacological and genetic inhibitions demonstrate that autophagy is responsible for bulk turnover of wild type and mutant serpins, but can be stimulated by rapamycin to compensate for proteasome inhibition. The significance of these findings to the treatment of serpinopathies is discussed.

Aberrant CpG island methylation in acute myeloid leukemia is accentuated at relapse.

DNA methylation of CpG islands around gene transcription start sites results in gene silencing and plays a role in leukemia pathophysiology. Its impact in leukemia progression is not fully understood. We performed genomewide screening for methylated CpG islands and identified 8 genes frequently methylated in leukemia cell lines and in patients with acute myeloid leukemia (AML): NOR1, CDH13, p15, NPM2, OLIG2, PGR, HIN1, and SLC26A4. We assessed the methylation status of these genes and of the repetitive element LINE-1 in 30 patients with AML, both at diagnosis and relapse. Abnormal methylation was found in 23% to 83% of patients at diagnosis and in 47% to 93% at relapse, with CDH13 being the most frequently methylated. We observed concordance in methylation of several genes, confirming the presence of a hypermethylator pathway in AML. DNA methylation levels increased at relapse in 25 of 30 (83%) patients with AML. These changes represent much larger epigenetic dysregulation, since methylation microarray analysis of 9008 autosomal genes in 4 patients showed hypermethylation ranging from 5.9% to 13.6% (median 8.3%) genes at diagnosis and 8.0% to 15.2% (median 10.6%) genes in relapse (P < .001). Our data suggest that DNA methylation is involved in AML progression and provide a rationale for the use of epigenetic agents in remission maintenance.