Dysregulation of Retinal and Photoreceptor Structural Integrity Genes in ATF6 Retinal Organoids.

ATF6 is a key regulator of the unfolded protein response (UPR) pathway that maintains cellular homeostasis during ER stress. In people, loss of ATF6 function causes cone dysfunction, manifesting as achromatopsia (ACHM). Previously, we generated ACHM retinal organoids (ROs) from patient induced pluripotent stem cells (iPSCs) carrying mutant ATF6 variants and gene-edited ATF6-knockout (KO) human embryonic stem cells (hESCs).

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.

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.

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 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 disease variants and from gene-edited null hESCs.

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.

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.

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.

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.

The unfolded protein response regulator ATF6 promotes mesodermal differentiation.

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 ATF6β 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.

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.

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.

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.

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.

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.

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.

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.

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.