Mutations in the gene encoding UbiA prenyltransferase domain-containing protein-1 (UBIAD1) cause Schnyder corneal dystrophy (SCD), a rare autosomal dominant eye disease characterized by opacification of the cornea (Klintworth, 2009; Weiss, 2009; Orr et al., 2007; Weiss et al., 2007). Although apparent early in life, corneal opacification associated with SCD progresses slowly with age and ultimately leads to reduced visual acuity. The severity of visual impairment is underscored by the frequency in which corneal transplant surgery is utilized for treatment of SCD, which rises from 50% in SCD patients > 50 years of age to more than 70% for those 70 years and older (Weiss, 2007). Biochemical analyses of corneas from SCD patients revealed a marked accumulation of cholesterol (McCarthy et al., 1994; Gaynor et al., 1996; Yamada et al., 1998), suggesting that dysregulation of cholesterol metabolism significantly contributes to the pathogenesis of SCD. Systemic hypercholesterolemia has been reported to be associated with some, but not all cases of the disease (Thiel et al., 1977; Brownstein et al., 1991; Crispin, 2002).

UBIAD1 belongs to the UbiA superfamily of integral membrane prenyltransferases that catalyze transfer of isoprenyl groups to aromatic acceptors, generating a wide variety of molecules ranging from ubiquinones, chlorophylls, and hemes to vitamin E and vitamin K (Li, 2016). UBIAD1 mediates transfer of the 20-carbon geranylgeranyl moiety from geranylgeranyl pyrophosphate (GGpp) to menadione (vitamin K₃) released from plant-derived phylloquinone (vitamin K₁), generating the vitamin K₂ subtype menaquinone-4 (MK-4) (Nakagawa et al., 2010; Hirota et al., 2013). To date, 25 missense mutations that alter 21 amino acids in the UBIAD1 protein have been identified in SCD families. Structural analyses of archaeal UbiA prenyltransferases revealed that residues corresponding to SCD-associated mutations in human UBIAD1 cluster around the active site of the enzyme (Cheng and Li, 2014; Huang et al., 2014). Indeed, all SCD-associated variants of UBIAD1 are defective in mediating synthesis of MK-4 (Hirota et al., 2015; Jun, D.-J. and DeBose-Boyd, R.A., unpublished observations) and for some variants, this defect likely results from reduced affinity for GGpp.

The first link between UBIAD1 and cholesterol metabolism was provided by the discovery of its association with the endoplasmic reticulum (ER)-localized enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) (Nickerson et al., 2013). HMGCR catalyzes reduction of HMG CoA to mevalonate, a reaction that constitutes the rate-limiting step in synthesis of cholesterol and the essential nonsterol isoprenoids GGpp and farnesyl pyrophosphate (Fpp) (Goldstein and Brown, 1990; Wang and Casey, 2016). Fpp and GGpp can become transferred to many cellular proteins and are utilized in synthesis of other nonsterol isoprenoids including ubiquinone, heme, dolichol, and MK-4. HMGCR is subjected to tight feedback control through transcriptional and post-transcriptional mechanisms mediated by sterol and nonsterol isoprenoids (Brown and Goldstein, 1980). Sterols mediate the transcriptional effects by inhibiting proteolytic activation of membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs). SREBPs enhance transcription of genes encoding HMGCR and other cholesterol biosynthetic enzymes as well as the low density lipoprotein (LDL) receptor that removes cholesterol-rich LDL from circulation (Horton et al., 2003). Post-transcriptional regulation of HMGCR is mediated by sterol and nonsterol isoprenoids, which combine to accelerate the ER-associated degradation (ERAD) of HMGCR through a reaction that involves the 26S proteasome (Nakanishi et al., 1988; Ravid et al., 2000). Together, these feedback regulatory mechanisms coordinate metabolism of mevalonate to assure cells maintain constant production of nonsterol isoprenoids but avoid overaccumulation of cholesterol and other sterols.

Our group discovered that accumulation of sterols in ER membranes triggers binding of HMGCR to ER membrane proteins called Insigs (Sever et al., 2003a; Sever et al., 2003b). Subsequent ubiquitination of HMGCR by Insig-associated ubiquitin ligases (Song et al., 2005; Jo et al., 2011; Jiang et al., 2018) mark the enzyme for extraction across ER membranes and release into the cytosol for proteasome-mediated ERAD (Elsabrouty et al., 2013; Morris et al., 2014). GGpp augments ERAD of ubiquitinated HMGCR by enhancing its membrane extraction (Elsabrouty et al., 2013). Recently, we discovered that sterols also cause a subset of HMGCR molecules to bind to UBIAD1 (32). This binding protects HMGCR from accelerated ERAD, permitting continued synthesis of nonsterol isoprenoids even when cellular sterols are abundant (Schumacher et al., 2018). GGpp triggers release of UBIAD1 from HMGCR, which allows for maximal ERAD of HMGCR and ER-to-Golgi transport of UBIAD1 (34). Eliminating expression of UBIAD1 relieves the GGpp requirement for HMGCR ERAD, indicating the reaction is inhibited by UBIAD1. Further characterization revealed that despite its steady-state Golgi localization in GGpp-replete cells, UBIAD1 continuously cycles between the ER and Golgi. Upon sensing depletion of GGpp in membranes of the ER, UBIAD1 becomes trapped in the organelle and inhibits ERAD of HMGCR to stimulate synthesis of mevalonate for replenishment of GGpp. The physiologic relevance of UBIAD1-mediated sensing of GGpp is highlighted by the observation that the reaction appears to be disrupted in SCD. SCD-associated UBIAD1 resists GGpp-induced release from HMGCR and becomes sequestered in the ER of GGpp-replete cells (Schumacher et al., 2015; Schumacher et al., 2016). The ensuing inhibition of HMGCR ERAD, which occurs in a dominant-negative fashion, leads to a marked increase in synthesis and intracellular accumulation of cholesterol (Schumacher et al., 2018).

The current studies were designed to confirm the role of UBIAD1 in regulation of HMGCR ERAD and cholesterol metabolism in living animals. For this purpose, we generated mice that harbor a knockin mutation that changes asparagine-100 (N100) in UBIAD1 to a serine residue (N100S) (see Figure 1A). The N100S mutation in mouse UBIAD1 corresponds to the SCD-associated UBIAD1 (N102S) mutation in the human enzyme. We show here that knockin mice homozygous for the N100S mutation (designated as Ubiad1^Ki/Ki mice) accumulate HMGCR protein in several tissues, despite a reduction in the amount of Hmgcr mRNA owing to sterol accumulation and reduced activation of SREBPs. The accumulation of HMGCR protein resulted from sequestration of UBIAD1 (N100S) in the ER and inhibition of HMGCR ERAD at a post-ubiquitination step of the reaction. Aged Ubiad1^Ki/Ki mice exhibited signs of opacification of the cornea, which was accompanied by hallmarks of sterol overaccumulation in the tissue. These findings not only indicate that UBIAD1 modulates ERAD of HMGCR in mice through similar mechanisms previously established in cultured cells, but they also establish Ubiad1^Ki/Ki mice as a model for human SCD.

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Ubiad1^WT/Ki heterozygous male and female mice (C57BL/6 × 129 genetic background) were crossed to obtain wild type (WT) and Ubiad1^Ki/Ki littermates. Mice homozygous for the N100S knockin mutation were born at expected Mendelian ratios. WT and Ubiad1^Ki/Ki littermates were externally indistinguishable and had similar body and liver weights (data not shown). Immunoblot analysis revealed that livers of male Ubiad1^WT/Ki and Ubiad1^Ki/Ki mice consuming chow diet ad libitum exhibited a noticeable increase (1.8- and 5.2-fold, respectively) in the amount of HMGCR protein compared to that in WT littermates (Figure 1B, lanes 1–3). However, the amount of Hmgcr mRNA was reduced approximately 40% in knockin mice (Figure 1—figure supplement 1A). UBIAD1 (N100S) protein also accumulated in livers of heterozygous and homozygous Ubiad1 knockin mice (Figure 1B, lanes 1–3); however, this was not accompanied by an increase in hepatic Ubiad1 mRNA (Figure 1—figure supplement 1A). Levels of nuclear SREBP-1 (Figure 1B, lanes 4–6) and SREBP-2 (lanes 7–9) were reduced in livers of Ubiad1^WT/Ki and Ubiad1^Ki/Ki mice, which coincided with reduced expression of mRNAs encoding SREBP target genes (Figure 1—figure supplement 1A). Cholesterol was slightly, but significantly increased in Ubiad1^Ki/Ki livers; however, plasma cholesterol, triglycerides, and non-esterified fatty acids as well as liver triglycerides were not significantly changed between the groups of animals (Figure 1—figure supplement 1B). Similar results were observed in the analysis of female Ubiad1^Ki/Ki mice (data not shown).

To ensure phenotypes associated with the N100S knockin mutation were not influenced by mixed genetic background, we backcrossed BL6/129 Ubiad1^Ki/Ki mice to C57BL/6J mice for at least six generations. For experiments described hereafter, Ubiad1^WT/Ki heterozygous female and male mice on the BL6 background were crossed to obtain WT and Ubiad1^Ki/Ki littermates. The results shown in Figure 2A reveal that male Ubiad1^WT/Ki and Ubiad1^Ki/Ki mice on the BL6 background accumulated hepatic HMGCR and UBIAD1 proteins (lanes 1–3), whereas levels of nuclear SREBP-1 and SREBP-2 were either unchanged (nuclear SREBP-1, lanes 4–6) or reduced (nuclear SREBP-2, lanes 7–9). HMGCR and UBIAD1 proteins accumulated and Hmgcr mRNA was down-regulated to varying degrees in other tissues of the knockin mice (Figure 2B and C). HMGCR and UBIAD1 protein accumulated to a similar extent in livers and eyes of female C57BL/6J Ubiad1^Ki/Ki mice (Figure 2—figure supplement 1).

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Table 1 shows that WT and Ubiad1^Ki/Ki mice had similar body and liver weights. Plasma levels of triglycerides, cholesterol, and non-esterified fatty acids (NEFAs) were slightly reduced in Ubiad1^Ki/Ki mice; however, these reductions were not significant. The knockin mice exhibited a small but significant increase in the amount of cholesterol in the liver (Table 1). We next used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure levels of MK-4 and other nonsterol isoprenoids in livers of WT and Ubiad1^Ki/Ki mice (Figure 3). The results show that levels of MK-4 were reduced 50% relative to those observed in WT animals, despite a 4.2-fold increase in the amount of hepatic UBIAD1 protein. When normalized to the amount of UBIAD1 protein, we estimate the relative level of MK-4 was reduced by more than 80% in livers of Ubiad1^Ki/Ki mice. In contrast to results with MK-4, levels of geranylgeraniol (GGOH; derived from GGpp) and ubiquinone-10, were significantly increased in livers of Ubiad1^Ki/Ki mice. This was accompanied by a small, but significant decrease in plant-derived phylloquinone (vitamin K₁); bacterial-derived vitamin K₂ subtype menaquinone-7 (MK-7) remained unchanged in the knockin livers. MK-4 was reduced, whereas levels of GGOH and/or ubiquinone-10 were increased in kidneys, brains, spleens, and testes of Ubiad1^Ki/Ki mice (Figure 3—figure supplement 1).

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To directly study the regulation of HMGCR in the presence of UBIAD1 (N100S), we established lines of mouse embryonic fibroblasts (MEFs) from WT and Ubiad1^Ki/Ki littermates. A low level of HMGCR and UBIAD1 protein was detected in membrane fractions isolated from WT MEFs cultured in sterol and nonsterol isoprenoid-replete medium containing fetal calf serum (Figure 4A, lane 1). Both proteins markedly accumulated in MEFs derived from Ubiad1^Ki/Ki mice (lane 2). In contrast, levels of nuclear SREBP-1 and SREBP-2 were reduced in Ubiad1^Ki/Ki MEFs (Figure 4A, compare lanes 3 and 4), which is consistent with reduced levels of Hmgcr mRNA and increased levels of intracellular cholesterol (Figure 4B). We next compared sterol-accelerated ERAD of HMGCR in Ubiad1^Ki/Ki MEFs to that in MEFs derived from Hmgcr^Ki/Ki mice, which harbor knockin mutations in HMGCR that prevent its sterol-induced ubiquitination (Hwang et al., 2016). Cells were first depleted of isoprenoids through incubation in medium containing lipoprotein-deficient serum and the HMGCR inhibitor compactin to enhance expression of HMGCR. The cells were subsequently treated in the absence or presence of the oxysterol 25-hydroxycholesterol (25-HC) prior to harvest, subcellular fractionation, and immunoblot analysis. The results show that 25-HC caused the disappearance of HMGCR from membranes of WT MEFs as expected (Figure 4C, lanes 1 and 2; 5 and 6). However, this disappearance was blunted in membranes from either Ubiad1^Ki/Ki or Hmgcr^Ki/Ki MEFs (lanes 3 and 4; 7 and 8). Despite resistance of HMGCR to sterol-accelerated ERAD, the experiment of Figure 4D shows that sterols continued to stimulate HMGCR ubiquitination in Ubiad1^Ki/Ki MEFs. Isoprenoid-depleted cells were treated with the proteasome inhibitor MG-132 (to block degradation of ubiquitinated HMGCR) in the absence or presence of 25-HC. Cells were then harvested for preparation of detergent lysates that were immunoprecipitated with polyclonal anti-HMGCR. 25-HC caused HMGCR to become ubiquitinated in WT and Ubiad1^Ki/Ki MEFs, as indicated by smears of reactivity in anti-ubiquitin immunoblots of the HMGCR immunoprecipitates (Figure 4D, lanes 1–6). As expected, HMGCR resisted 25-HC-induced ubiquitination in MEFs derived from Hmgcr^Ki/Ki mice (compare lanes 7 and 8 with lanes 9–12).

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Figure 5A compares expression of HMGCR in WT and Ubiad1^Ki/Ki mice fed a chow diet supplemented with 1% cholesterol. The results show that cholesterol feeding led to reduced expression of HMGCR protein in membranes isolated from livers of WT mice (Figure 5A, lane 2); however, a significant amount of HMGCR protein remained in hepatic membranes of cholesterol-fed Ubiad1^Ki/Ki mice (lane 4). The feeding regimen reduced the amount of Insig-1 protein (lanes 2 and 4) and nuclear SREBP-2 (lanes 6 and 8) in both WT and Ubiad1^Ki/Ki livers. Dietary cholesterol also reduced mRNAs encoding HMGCR and other SREBP targets in livers of WT and Ubiad1^Ki/Ki mice (Figure 5—figure supplement 1). The membrane-bound precursor and nuclear forms of SREBP-1 were induced by cholesterol feeding in livers of both lines of mice (lanes 6 and 8). This induction can be attributed to sterol-mediated activation of liver x receptors (LXRs) that modulate expression of SREBP-1c, the major SREBP-1 isoform in the mouse liver (Repa et al., 2000; Liang et al., 2002). The mRNAs encoding SREBP-1c and two other LXR targets, ABCG5 and ABCG8, were enhanced in WT and Ubiad1^Ki/Ki mice fed cholesterol (Figure 5—figure supplement 1). In contrast to results in the liver, cholesterol-feeding failed to down-regulate levels of HMGCR protein in eyes of Ubiad1^Ki/Ki mice (Figure 5B, lanes 3 and 4); mRNAs encoding both SREBPs and their targets were also unchanged in eyes of cholesterol-fed knockin animals (data not shown).

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In Figure 5C, we compared cholesterol-mediated regulation of hepatic HMGCR in Ubiad1^Ki/Ki and Hmgcr^Ki/Ki mice. As little as 0.1% cholesterol caused a significant decrease in the amount of HMGCR in hepatic membranes from WT controls (Figure 5C, lanes b and j). HMGCR was partially resistant to 0.1% cholesterol in livers of Hmgcr^Ki/Ki mice (compare lanes e and f); however, higher concentrations of cholesterol (0.3% and 1%) caused complete disappearance of HMGCR from membranes (lanes g and h). The resistance of HMGCR to cholesterol feeding was more pronounced in Ubiad1^Ki/Ki mice; levels of the protein persisted when the animals were fed 0.1–1% cholesterol (Figure 5C, compare lane m with lanes n-p). Importantly, cholesterol-feeding continued to suppress levels of nuclear SREBP-2 in livers of Hmgcr^Ki/Ki and Ubiad1^Ki/Ki mice as well their WT littermates (compare lanes a-d with e-h and i-l with m-p). The mRNAs encoding SREBP-2 target genes were also reduced in livers of the cholesterol-fed mice (Figure 5D).

We next evaluated the effect of cholesterol depletion on HMGCR levels in WT and Ubiad1^Ki/Ki mice. Cholesterol depletion using lovastatin, a competitive inhibitor of HMGCR, led to the dose-dependent accumulation of HMGCR protein in livers of WT animals (Figure 6A, lanes a-d). Lovastatin also caused HMGCR to accumulate in livers of Ubiad1^Ki/Ki mice (lanes e-h) (see Figure 6B for quantification). The precursor and nuclear forms of SREBP-2 were induced by lovastatin, whereas those of SREBP-1 were reduced by the treatment in both WT and Ubiad1^Ki/Ki mice (lanes i-p). The mRNAs for SREBP-2 and its target genes (including HMGCR) were elevated in livers of lovastatin-treated animals; mRNA for SREBP-1c was reduced by the inhibitor (Figure 6—figure supplement 1). HMGCR protein was also increased in the eyes of lovastatin-treated WT mice; however, this increase required the highest concentration (0.2%) of the drug (Figure 6C, lanes a-d).

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In the experiment of Figure 6D, we analyzed the subcellular localization of UBIAD1 in WT and Ubiad1^Ki/Ki mice using a fractionation scheme previously utilized to isolate ER membranes from Chinese hamster ovary-K1 cells (Radhakrishnan et al., 2008). Liver homogenates (lysates) were first subjected to centrifugation at 3,000 X g to eliminate unbroken cells and nuclei. The resulting post-nuclear supernatants (PNS) were then applied to discontinuous sucrose gradients and centrifuged at 100,000 X g, generating two distinct membrane layers: a light membrane fraction enriched in Golgi and a heavy membrane fraction enriched in ER. Immunoblot analysis revealed the presence of UBIAD1 and the Golgi membrane protein GM-130 in the light, Golgi-enriched membrane fraction obtained from livers of WT mice fed a chow diet (Figure 6D, lane 4). ER-localized calnexin was observed in the ER-enriched fraction as expected (lane 5). When the mice were fed the chow diet supplemented with lovastatin (0.2%), we observed a shift in the localization of UBIAD1 from the Golgi-enriched fraction to the ER (Figure 6D, compare lanes 9 and 10). GM-130 remained in the Golgi-enriched fraction (lane 9) and calnexin continued to localize to the ER (lane 10) of livers from lovastatin-treated mice. In contrast to results with WT mice, UBIAD1 was concentrated in ER-enriched hepatic membranes of chow-fed Ubiad1^Ki/Ki mice (Figure 6D, compare lanes 4 and 5 with lanes 14 and 15). The ER localization of UBIAD1 did not change when the knockin mice were challenged with lovastatin (lanes 19 and 20). Importantly, calnexin and GM-130 were localized to ER- and Golgi-enriched membranes, respectively, regardless of feeding regimen (compare lanes 14 and 15 with lanes 19 and 20).

Stereomicroscopic examinations revealed that 8–12 week-old Ubiad1^Ki/Ki mice similar to those analyzed in Figures 2, 3, 5 and 6 failed to exhibit significant corneal opacification that characterizes human SCD (data not shown). However, 46% (11/24) of the knockin mice exhibited signs of corneal opacification at 50 weeks of age (Figure 7—figure supplement 1). One of these animals manifested signs of bilateral opacification of the cornea (Figure 7A). None of the aged WT mice developed corneal opacification; heterozygous knockin mice were not examined (data not shown). Immunohistochemical staining with anti-HMGCR revealed a marked increase in the amount of HMGCR protein in corneas of Ubiad1^Ki/Ki mice compared to their WT littermates (Figure 7B). The accumulation of HMGCR protein in Ubiad1^Ki/Ki corneas was accompanied by reduced levels of mRNAs encoding SREBP-2, HMGCR, and other cholesterol biosynthetic enzymes (Figure 7C). In contrast, expression of mRNAs encoding the LXR targets ABCG5, ABCG8, and ABCA1 was enhanced in Ubiad1^Ki/Ki corneas. Although the amount of total cholesterol remained unchanged in corneas of WT and Ubiad1^Ki/Ki mice, we measured a small, but significant increase in free cholesterol in the knockin mice (Figure 7D). Moreover, significant increases in the amount of several sterol intermediates of cholesterol synthesis including lanosterol, follicular fluid meiosis-activating sterol (FFMAS), 7- and 8-dehydrocholesterol, desmosterol, and 7-dehydrodesmosterol were observed in corneas from Ubiad1^Ki/Ki mice (Figure 7E).

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The current studies provide evidence that inhibition of HMGCR ERAD directly contributes to corneal sterol accumulation and opacification that characterizes the human eye disease SCD. This conclusion was drawn from the analysis of Ubiad1^Ki/Ki mice harboring a knockin mutation (N100S) that corresponds to the SCD-associated N102S mutation in human UBIAD1 (Figure 1A). Consistent with our studies of human UBIAD1 (N102S) in cultured cells (Schumacher et al., 2015; Schumacher et al., 2018), mouse UBIAD1 (N100S) inhibited ERAD of HMGCR in vivo as indicated by accumulation of the protein in livers and other tissues of Ubiad1^Ki/Ki mice (Figures 1B, 2 and 3, and Figure 2—figure supplement 1). These increases in HMGCR protein occurred despite reduced levels of its mRNA (Figures 1 and 2), which was attributable to reduced proteolytic activation of SREBP-2 (Figures 1B and 2A) resulting from accumulation of hepatic cholesterol (Figure 1—figure supplement 1B and Table 1). Corneas from 8 to 12 week-old Ubiad1^Ki/Ki mice failed to exhibit opacification of the cornea (data not shown), despite the marked accumulation of HMGCR protein in the eye (Figure 2B and C and Figure 2—figure supplement 1). Conversely, corneal opacification was observed in aged Ubiad1^Ki/Ki mice (50 weeks) (Figure 7A) that was accompanied by a buildup of HMGCR protein in the tissue (Figure 7B). This opacification occurred in the absence of an increase in total cholesterol (Figure 7D and E). However, Ubiad1^Ki/Ki corneas displayed other hallmarks of sterol overaccumulation including an increase in levels of free cholesterol and sterol intermediates of cholesterol biosynthesis (Figure 7D and E), reduced levels of mRNAs for HMGCR and other cholesterol biosynthetic enzymes, and enhanced expression of mRNAs encoding ABCG5, ABCG8, and ABCA1 (Figure 7C). Based on these observations, we conclude that UBIAD1 (N100S)-mediated inhibition of HMGCR ERAD enhances flux through the cholesterol biosynthetic pathway, thereby initiating sterol accumulation and development of corneal opacification. Our current findings suggest the slow progression of corneal opacification in Ubiad1^Ki/Ki mice results from reduced activation of SREBP-2, which leads to reduced expression of genes encoding cholesterol biosynthetic enzymes. Studies are now underway to monitor progression of corneal opacification in Ubiad1^Ki/Ki mice > 50 weeks of age. We anticipate that as the disorder progresses, free cholesterol will continue to accumulate, initiating formation and accumulation of cholesterol esters, which will further exacerbate opacification of the cornea. Additionally, Ubiad1^Ki/Ki mice will be challenged with high cholesterol/Western diets to determine whether dietary cholesterol contributes to progression of corneal opacification in SCD.

A preliminary analysis of UBIAD1 (N100S) knockin mice has been recently described (Dong et al., 2018). However, this study lacks the molecular characterization of HMGCR and documentation of age-dependent corneal opacification in the knockin animals. Instead, the authors report evidence for mitochondrial damage and accumulation of mitochondrial-localized glycerophosphoglycerols in knockin corneas. The authors conclude that mitochondrial dysfunction is linked to the abnormal deposition of cholesterol in corneas of SCD patients, constrasting our conclusion the response results from inhibition of HMGCR ERAD. The possibility exists that this mitochondrial dysfunction is secondary to cholesterol accumulation and/or impairment of MK-4 synthesis (see Figure 3) in Ubiad1^Ki/Ki mice. Further investigation is required to resolve this discrepancy.

Examination of Ubiad1^Ki/Ki-derived MEFs cultured under isoprenoid-replete conditions yielded results remarkably similar to those obtained with whole mouse tissues – marked accumulation of HMGCR protein, overproduction of cholesterol, reduced levels of Hmgcr mRNA, and reduced activation of SREBPs (Figure 4A and B). HMGCR was also refractory to accelerated ERAD stimulated by the oxysterol 25-HC in Ubiad1^Ki/Ki MEFs and Hmgcr^Ki/Ki MEFs, which were derived from mice expressing ubiquitination-resistant HMGCR (Hwang et al., 2016) (Figure 4C). However, 25-HC continued to stimulate ubiquitination of HMGCR in Ubiad1^Ki/Ki MEFs, but not in those derived from Hmgcr^Ki/Ki mice (Figure 4D). In addition to providing direct evidence that SCD-associated UBIAD1 inhibits ERAD of HMGCR, these results indicate that the inhibition results from a block in a post-ubiquitination step of the reaction (Schumacher et al., 2015).

Our previous characterization of Hmgcr^Ki/Ki mice indicated that dietary cholesterol reduces levels of HMGCR protein primarily by inhibiting activation of SREBP-2 (35); similar results were obtained in the current study (Figure 5C). In contrast, levels of HMGCR protein persisted in livers of Ubiad1^Ki/Ki mice fed cholesterol, even though the feeding regimen continued to block proteolytic activation of SREBP-2 (Figure 5A and C) and reduce levels of mRNAs for HMGCR and other SREBP-2 targets (Figure 5—figure supplement 1). Previous studies found that HMGCR is subjected to sterol-independent ubiquitination and ERAD in cultured cells (Doolman et al., 2004). Thus, we speculate that in livers of Hmgcr^Ki/Ki mice, ubiquitination-resistant HMGCR continues to become degraded through this sterol-independent ERAD pathway. However, both sterol-independent and sterol-dependent pathways for HMGCR ERAD are blocked in Ubiad1^Ki/Ki mice, owing to the ability of UBIAD1 (N100S) to inhibit post-ubiquitination steps of the reaction (see Figure 4D). As a result of this inhibition, levels of HMGCR protein persist in livers of cholesterol-fed Ubiad1^Ki/Ki mice.

Okano and co-workers previously attempted to generate mice lacking Ubiad1; however, Ubiad1-deficient embryos failed to survive past embryonic day 7.5 (40). Ubiad1^-/- embryonic stem cells failed to synthesize MK-4, suggesting the compound plays a pivotal role in development. Taking this and the observation that human UBIAD1 (N102S) is defective in MK-4 synthetic activity (Hirota et al., 2015), we were somewhat surprised that Ubiad1^Ki/Ki mice were born at normal Mendelian ratios and appear grossly normal. Throughout our studies, we noticed that UBIAD1 (N100S) protein accumulated in all tissues of Ubiad1^Ki/Ki mice (see Figures 1–3). Similar results have been observed for SCD-associated variants of human UBIAD1 in transfected cells; stabilization of SCD-associated UBIAD1 has been attributed to ER sequestration and protection from autophagic degradation from the Golgi (Jun, D.-J. and DeBose-Boyd, R.A., unpublished observations). The level of MK-4 was significantly reduced, but not absent, in various tissues of Ubiad1^Ki/Ki mice (Figure 3 and Figure 3—figure supplement 1). We speculate that despite reduced enzymatic activity, accumulated UBIAD1 (N100S) produces sufficient amounts of MK-4 to support development and survival of Ubiad1^Ki/Ki mice. Accumulation of SCD-associated UBIAD1 in the ER has important implications for the pathology of the disease. SCD is an autosomal dominant disorder and our previous studies revealed that SCD-associated variants of UBIAD1 inhibit HMGCR ERAD in a dominant-negative fashion (Schumacher et al., 2015; Schumacher et al., 2016). Enhanced stability of SCD-associated UBIAD1 owing to its ER sequestration helps to explain how cholesterol accumulation occurs in corneas of SCD patients harboring heterozygous UBIAD1 mutations.

The significance of the HMGCR regulatory system is evidenced by the widespread use of statins to lower plasma LDL-cholesterol and reduce the incidence of atherosclerotic cardiovascular disease (Stossel, 2008). However, statins block synthesis of sterol and nonsterol isoprenoids that mediate feedback regulation of HMGCR, causing the enzyme’s accumulation in livers of humans and animals (Kita et al., 1980; Reihnér et al., 1990) (Figure 6). This accumulation partially overcomes inhibitory effects of statins, which allows for continued synthesis of cholesterol that limits cholesterol-lowering (Schonewille et al., 2016; Goldberg et al., 1990; Engelking et al., 2006). We showed previously that the statin-induced increase in HMGCR was blunted 5-fold in Hmgcr^Ki/Ki versus WT mice, suggesting inhibition of ERAD significantly contributes to the response (Hwang et al., 2016). Our current findings argue that statin-induced accumulation of HMGCR results in part, from depletion of GGpp from ER membranes. This was indicated by accumulation and ER sequestration of UBIAD1 in livers of lovastatin-treated mice, which coincided with accumulation of HMGCR (Figure 6A and C). UBIAD1 (N100S), which resists GGpp-induced release from HMGCR, accumulated and remained sequestered in the ER, regardless of whether Ubiad1^Ki/Ki mice were treated in the absence or presence of lovastatin (Figure 6A and C). The resultant inhibition of HMGCR ERAD led to accumulation of the protein and overproduction of sterol and nonsterol isoprenoids in various tissues of Ubiad1^Ki/Ki mice (see Figures 3 and 7, and Figure 3—figure supplement 1). Thus, GGpp-regulated, ER-to-Golgi transport of UBIAD1 regulates HMGCR ERAD to coordinate synthesis of sterol and nonsterol isoprenoids in mice through similar mechanisms previously described in cultured cells (Schumacher et al., 2018). The current results indicate that modulating ER-to-Golgi transport of UBIAD1 may have therapeutic value. For example, agents that mimic GGpp in stimulating ER-to-Golgi transport of UBIAD1 should relieve inhibition of ERAD and prevent accumulation of HMGCR that limits the effectiveness of statins in lowering plasma levels of LDL-cholesterol. Agents that restore Golgi localization of SCD-associated UBIAD1 or block its interaction with HMGCR may prevent or retard cholesterol accumulation and corneal opacification associated with SCD. The establishment of a mouse model for SCD described here will prove instrumental in the identification and characterization of such molecules.

All data generated in this study are included in the manuscript.

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Author details

1. Youngah Jo

   Departments of Molecular Genetics, Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

2. Jason S Hamilton

   Departments of Molecular Genetics, Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Data curation, Validation, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3291-3125

3. Seonghwan Hwang

   Departments of Molecular Genetics, Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Resources, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Kristina Garland

   Departments of Molecular Genetics, Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Gennipher A Smith

   Departments of Molecular Genetics, Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

6. Shan Su

   Departments of Molecular Genetics, Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9369-1642

7. Iris Fuentes

   Departments of Molecular Genetics, Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

8. Sudha Neelam

   Department of Ophthalmology, Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

9. Bonne M Thompson

   Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

10. Jeffrey G McDonald

    Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States

    Contribution

    Investigation, Methodology

    Competing interests

    No competing interests declared

11. Russell A DeBose-Boyd

    Departments of Molecular Genetics, Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States

    Contribution

    Conceptualization, Supervision, Funding acquisition, Visualization, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    Russell.Debose-Boyd@utsouthwestern.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7452-5227

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