Pathomechanisms of ATF6-Associated Cone Photoreceptor Diseases

Abstract

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.

50.1. Introduction

In eukaryotic cells, the endoplasmic reticulum (ER) is an essential organelle, responsible for folding and assembling secretory and membrane proteins before delivering them to other cellular compartments or the extracellular environment. Pathological insults, such as hypoxia or infection or carrying genetic mutations, can cause protein misfolding in the ER, leading to ER stress. To cope with ER stress, eukaryotic cells use a unique mechanism, termed the unfolded protein response (UPR), to ensure that the ER can adapt to the stress and maintain ER homeostasis (Walter and Ron 2011). One of the key UPR regulators, ATF6, is a glycosylated transmembrane protein localized in the ER (Haze et al. 1999) and normally forms monomer, dimer, and oligomer through intra- and intermolecular disulfide bonds in the unstressed cell (Nadanaka et al. 2007). In response to ER stress, ATF6 is reduced to a monomer and transported from the ER to the Golgi apparatus, where it is cleaved by the site 1 protease (S1P) and site 2 protease (S2P) to release the cytosolic fragment of ATF6 (Haze et al. 1999; Ye et al. 2000; Shen et al. 2002). Upon release from the Golgi membrane, ATF6 cytosolic fragment, a potent transcription factor containing a basic leucine zipper (bZip) domain, migrates to the nucleus and upregulates the expression of its downstream target genes including ER protein-folding chaperones and enzymes (Yamamoto et al. 2007) (Fig. 50.1). Through this signal transduction pathway, ATF6 functions to restore ER protein-folding homeostasis and alleviate ER stress (Nadanaka et al. 2004).

Fig. 50.1. ATF6 signaling pathway.

In response to ER stress, ATF6 is reduced to a monomer and traffics from the ER to the Golgi compartment, where it is cleaved by site 1 protease (S1P) and site 2 protease (S2P) to release the cytosolic fragment of ATF6. Cleaved ATF6 contains a basic leucine zipper (bZip) domain essential for the upregulation of its target genes in the nucleus

Recently, we discovered autosomal recessive mutations in ATF6 in patients with cone photoreceptor dysfunction diseases, such as achromatopsia and cone-rod dystrophy (Kohl et al. 2015; Skorczyk-Werner et al. 2017). Patients carrying these ATF6 mutant alleles suffer from foveal hypoplasia and the loss of cone photoreceptor function, indicating that ATF6 signaling is essential for human foveal development and is required for cone photoreceptor function. To date, there are 11 distinct ATF6 mutations associated with achromatopsia and cone-rod dystrophy (Ansar et al. 2015; Kohl et al. 2015; Xu et al. 2015; Skorczyk-Werner et al. 2017). These mutations span the entire coding region of ATF6 and include missense, nonsense, splice site, and single-nucleotide deletion and duplication changes. Based on the functional and molecular defects, we have classified them into three classes (Chiang et al. 2017).

50.2. Class 1 ATF6 Mutations

Class 1 cone photoreceptor disease associated-ATF6 mutations include point mutations in the ATF6 luminal domain or splicing defects resulting in partial deletion of the ATF6 luminal domain. Using human fibroblast cells from patients carrying class 1 mutant ATF6 alleles, we showed that the homozygous mutant fibroblasts produce significantly less amount of cleaved ATF6 fragment under ER stress conditions, compared to the control fibroblasts (Chiang et al. 2017; Skorczyk-Werner et al. 2017). In addition, we demonstrated that the control fibroblasts upregulated the expression levels of ATF6 downstream target genes with ER stress, whereas the ability of homozygous mutant fibroblasts to upregulate ATF6 target genes was significantly reduced. Furthermore, using cells expressing recombinant class 1 ATF6 mutant protein, we showed that mutant ATF6 was retained in the ER when the cells were treated with ER stress, whereas the wild-type ATF6 molecule was rapidly transported to Golgi apparatus under ER stress (Chiang et al. 2017). These studies indicated that class 1 ATF6 mutations that introduce point mutations in ATF6 luminal domain or partial deletion of ATF6 luminal domain impair ER to Golgi trafficking of ATF6 leading to impairment of ATF6 activation and signaling (Fig. 50.2).

Fig. 50.2. Class 1 ATF6 mutations.

Under ER stress, class 1 ATF6 mutants show impaired translocation from the ER to the Golgi apparatus. The included table comprises all known class 1 ATF6 mutations

50.3. Class 2 ATF6 Mutations

Class 2 ATF6 mutations introduce a premature stop codon, either by point-nonsense mutation or by frame shift, in the ATF6 gene immediately downstream of the DNA region encoding for the ATF6 bZip domain. Since these mutations introduced a premature stop codon more than 50 nucleotides upstream of the 3′-most exon-exon junction complex of the ATF6 mRNA, these transcripts are likely subjected to nonsense-mediated mRNA decay (NMD) (Fig. 50.3a) (Lewis et al. 2003; Baker and Parker 2004). However, the degradation efficiency of these mutant ATF6 transcripts by NMD is unclear. Any mutant ATF6 mRNA that escapes NMD can still be translated to produce a truncated ATF6 fragment containing the entire cytosolic transcriptional activator domain of ATF6 (Fig. 50.3b). In our previous study, we showed that expressing the cytosolic domain of wild-type ATF6 alone or these class 2 ATF6 mutant proteins in cells strongly upregulated the expression of ATF6 downstream target genes (Chiang et al. 2017), indicating that these class 2 ATF6 mutant proteins are fully and constitutively active, if they are translated.

Fig. 50.3. Class 2 ATF6 mutations.

(a) The mRNA of class 2 ATF6 mutant may undergo nonsense-mediated mRNA decay (NMD). (b) Undegraded mutant ATF6 mRNA is translated, which produces a truncated ATF6 fragment that is constitutively active. The included table comprises all known class 2 ATF6 mutations

50.4. Class 3 ATF6 Mutations

Class 3 ATF6 mutations introduce a missense mutation resulting in an amino acid change in the conserved region of the ATF6 bZip domain (Fig. 50.4a) or frame shift or nonsense mutations resulting in a premature stop codon leading to the deletion of the bZip domain in ATF6 (Fig. 50.4b). Previously, we demonstrated that patient fibroblasts carrying class 3 mutant ATF6 alleles have significantly reduced ability to upregulate ATF6 signaling pathway under ER stress. In addition, we showed that expressing the cytosolic fragment of class 3 mutant ATF6 in cells cannot upregulate the expression of ATF6 downstream targets (Chiang et al. 2017). These studies indicate that class 3 mutant ATF6 is not transcriptionally active due to the defection or absence of bZip DNA-binding domain.

Fig. 50.4. Class 3 ATF6 mutations.

Class 3 ATF6 mutations introduce a point mutation in the bZip domain of ATF6 (a) or produce a truncated ATF6 fragment that does not contain the bZip domain (b). These ATF6 mutants are unable to bind DNA or upregulate ATF6 specific targets. The included table comprises all known class 3 ATF6 mutations

50.5. Concluding Remarks

The cone photoreceptor and foveal pathology caused by these defects in ATF6 activation and signaling is still largely unknown. However, impaired or defective ATF6 signaling could contribute to the increased susceptibility to ER stress during retinal development. Recently Kroeger et al. identified a novel link between ATF6 activation and early stem cell differentiation events (Kroeger et al. 2018). In pluripotent stem cells, it was demonstrated that ATF6 activation, using the small compound AA147 (Plate et al. 2016; Paxman et al. 2018), resulted in accelerated loss of pluripotency and preferential differentiation of an angiogenic pool of cells that were able to undergo in vitro angiogenesis. The results were confirmed using iPS cells derived from the fibroblast cells of patients carrying a class 3 ATF6 mutation. Those data were the first to demonstrate that ATF6 is able to perform separate regulatory functions during development using physiological appropriate conditions that were independent from a comprehensive UPR signaling response. ATF6 is a powerful transcription factor involved in cellular stress signaling pathways and development; hence its potential in targeted drug therapies is substantial. Future research is required to address ATF6’s regulatory role in a wide array of cellular and disease pathologies, concerning ATF6 mutations, to path a way toward its future therapeutical potential.