ELOVL4 Mutations That Cause Spinocerebellar Ataxia-34 Differentially Alter Very Long Chain Fatty Acid Biosynthesis.

The FA Elongase-4 (ELOVL4) enzyme mediates biosynthesis of both very long chain (VLC)-PUFAs and VLC-saturated FA (VLC-SFAs). VLC-PUFAs play critical roles in retina and sperm function, whereas VLC-SFAs are predominantly associated with brain function and maintenance of the skin permeability barrier. While some ELOVL4 mutations cause Autosomal Dominant Stargardt-like Macular Dystrophy (STGD3), other ELOVL4 point mutations, such as L168F and W246G, affect the brain and/or skin, leading to Spinocerebellar Ataxia-34 (SCA34) and Erythrokeratodermia variabilis. The mechanisms by which these ELOVL4 mutations alter VLC-PUFA and VLC-SFA biosynthesis to cause the different tissue-specific pathologies are not well understood. To understand how these mutations alter VLC-PUFA and VLC-SFA biosynthesis, we expressed WT-ELOVL4, L168F, and W246G ELOVL4 variants in cell culture and supplemented the cultures with VLC-PUFA or VLC-SFA precursors. Total lipids were extracted, converted to FA methyl esters, and quantified by gas chromatography. We showed that L168F and W246G mutants were capable of VLC-PUFA biosynthesis. W246G synthesized and accumulated 32:6n3, while L168F exhibited gain of function in VLC-PUFA biosynthesis as it made 38:5n3, which we did not detect in WT-ELOVL4 or W246G-expressing cells. However, compared with WT-ELOVL4, both L168F and W246G mutants were deficient in VLC-SFA biosynthesis, especially the W246G protein, which showed negligible VLC-SFA biosynthesis. These results suggest VLC-PUFA biosynthetic capabilities of L168F and W246G in the retina, which may explain the lack of retinal phenotype in SCA34. Defects in VLC-SFA biosynthesis by these variants may be a contributing factor to the pathogenic mechanism of SCA34 and Erythrokeratodermia variabilis.

Obesity promotes lipid accumulation in mouse cartilage-A potential role of acetyl-CoA carboxylase (ACC) mediated chondrocyte de novo lipogenesis.

Obesity promotes the development of osteoarthritis (OA). It is also well-established that obesity leads to excessive lipid deposition in nonadipose tissues, which often induces lipotoxicity. The objective of this study was to investigate changes in the levels of various lipids in mouse cartilage in the context of obesity and determine if chondrocyte de novo lipogenesis is altered. We used Oil Red O to determine the accumulation of lipid droplets in cartilage from mice fed high-fat diet (HFD) or low-fat diet (LFD). We further used mass spectrometry-based lipidomic analyses to quantify levels of different lipid species. Expression of genes involving in fatty acid (FA) uptake, synthesis, elongation, and desaturation were examined using quantitative polymerase chain reaction. To further study the potential mechanisms, we cultured primary mouse chondrocytes under high-glucose and high-insulin conditions to mimic the local microenvironment associated with obesity and subsequently examined the abundance of cellular lipid droplets. The acetyl-CoA carboxylase (ACC) inhibitor, ND-630, was added to the culture medium to examine the effect of inhibiting de novo lipogenesis on lipid accumulation in chondrocytes. When compared to the mice receiving LFD, the HFD group displayed more chondrocytes with visible intracellular lipid droplets. Significantly higher amounts of total FAs were also detected in the HFD group. Five out of six significantly upregulated FAs were ω-6 FAs, while the two significantly downregulated FAs were ω-3 FAs. Consequently, the HFD group displayed a significantly higher ω-6/ω-3 FA ratio. Ether linked phosphatidylcholine was also found to be higher in the HFD group. Fatty acid desaturase (Fad1-3), fatty acid-binding protein 4 (Fabp4), and fatty acid synthase (Fasn) transcripts were not found to be different between the treatment groups and fatty acid elongase (Elovl1-7) transcripts were undetectable in cartilage. Ceramide synthase 2 (Cers-2), the only transcript found to be changed in these studies, was significantly upregulated in the HFD group. In vitro, chondrocytes upregulated de novo lipogenesis when cultured under high-glucose, high-insulin conditions, and this observation was associated with the activation of ACC, which was attenuated by the addition of ND-630. This study provides the first evidence that lipid deposition is increased in cartilage with obesity and that this is associated with the upregulation of ACC-mediated de novo lipogenesis. This was supported by our observation that ACC inhibition ameliorated lipid accumulation in chondrocytes, thereby suggesting that ACC could potentially be targeted to treat obesity-associated OA.

Homozygous Expression of Mutant ELOVL4 Leads to Seizures and Death in a Novel Animal Model of Very Long-Chain Fatty Acid Deficiency.

Lipids are essential components of the nervous system. However, the functions of very long-chain fatty acids (VLC-FA; ≥ 28 carbons) in the brain are unknown. The enzyme ELOngation of Very Long-chain fatty acids-4 (ELOVL4) catalyzes the rate-limiting step in the biosynthesis of VLC-FA (Agbaga et al., Proc Natl Acad Sci USA 105(35): 12843-12848, 2008; Logan et al., J Lipid Res 55(4): 698-708, 2014), which we identified in the brain as saturated fatty acids (VLC-SFA). Homozygous mutations in ELOVL4 cause severe neuropathology in humans (Ozaki et al., JAMA Neurol 72(7): 797-805, 2015; Mir et al., BMC Med Genet 15: 25, 2014; Cadieux-Dion et al., JAMA Neurol 71(4): 470-475, 2014; Bourassa et al., JAMA Neurol 72(8): 942-943, 2015; Aldahmesh et al., Am J Hum Genet 89(6): 745-750, 2011) and are post-natal lethal in mice (Cameron et al., Int J Biol Sci 3(2): 111-119, 2007; Li et al., Int J Biol Sci 3(2): 120-128, 2007; McMahon et al., Molecular Vision 13: 258-272, 2007; Vasireddy et al., Hum Mol Genet 16(5): 471-482, 2007) from dehydration due to loss of VLC-SFA that comprise the skin permeability barrier. Double transgenic mice with homozygous knock-in of the Stargardt-like macular dystrophy (STDG3; 797-801_AACTT) mutation of Elovl4 with skin-specific rescue of wild-type Elovl4 expression (S + Elovl4 mut/mut mice) develop seizures by P19 and die by P21. Electrophysiological analyses of hippocampal slices showed aberrant epileptogenic activity in S + Elovl4 mut/mut mice. FM1-43 dye release studies showed that synapses made by cultured hippocampal neurons from S + Elovl4 mut/mut mice exhibited accelerated synaptic release kinetics. Supplementation of VLC-SFA to cultured hippocampal neurons from mutant mice rescued defective synaptic release to wild-type rates. Together, these studies establish a critical, novel role for ELOVL4 and its VLC-SFA products in regulating synaptic release kinetics and epileptogenesis. Future studies aimed at understanding the molecular mechanisms by which VLC-SFA regulate synaptic function may provide new targets for improved seizure therapies.

Endogenous n-3 polyunsaturated fatty acids delay progression of pancreatic ductal adenocarcinoma in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice.

Preclinical studies suggest that diets rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs) may be beneficial for prevention of pancreatic cancer. Nutritional intervention studies are often complex, and there is no clear evidence, without potential confounding factors, on whether conversion of n-6 PUFAs to n-3 PUFAs in pancreatic tissues would provide protection. Experiments were designed using n-3 fatty acid desaturase (Fat-1) transgenic mice, which can convert n-6 PUFA to n-3 FAs endogenously, to determine the impact of n-3 PUFAs on pancreatic intraepithelial neoplasms (PanINs) and their progression to pancreatic ductal adenocarcinoma (PDAC). Six-week-old female p48(Cre/+)-LSL-Kras(G12D/+) and compound Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice were fed (AIN-76A) diets containing 10% safflower oil for 35 weeks. Pancreata were evaluated histopathologically for PanINs and PDAC. Results showed a dramatic reduction in incidence of PDAC (84%; P < .02) in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice compared to p48(Cre/+)-LSL-Kras(G12D/+) mice. Importantly, significant reductions of pancreatic ducts with carcinoma (90%; P < .0001) and PanIN 3 (~50%; P < .001) lesions were observed in the compound transgenic mice. The levels of n-3 PUFA were much higher (>85%; P < .05-0.01) in pancreas of compound transgenic mice than in those of p48(Cre/+)-LSL-Kras(G12D/+) mice. Molecular analysis of the pancreas showed a significant down-regulation of proliferating cell nuclear antigen, cyclooxygenase-2, 5-lipoxygenase (5-LOX), 5-LOX-activating protein, Bcl-2, and cyclin D1 expression levels in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice compared to p48(Cre/+)-LSL-Kras(G12D/+) mice. These data highlight the promise of dietary n-3 FAs for chemoprevention of pancreatic cancer in high-risk individuals.

Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice.

Delta-6 desaturase-null mice ((-/-)) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The (-/-) males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type ((+/+)) and (-/-) males at weaning until 16 weeks of age (n = 3-5). A breeding success rate of DHA-supplemented (-/-) was comparable to (+/+). DHA-fed (-/-) showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed (-/-), but DPAn6 remained depleted. In AA-fed (-/-), AA was restored at the (+/+) level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of (+/+) and DHA-fed (-/-), whereas it diminished in (-/-) and AA-fed (-/-), suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA.

High levels of retinal membrane docosahexaenoic acid increase susceptibility to stress-induced degeneration.

The fat-1 gene cloned from C. elegans encodes an n-3 fatty acid desaturase that converts n-6 to n-3 PUFA. Mice carrying the fat-1 transgene and wild-type controls were fed an n-3-deficient/n-6-enriched diet [fat-1- safflower oil (SFO) and wt-SFO, respectively]. Fatty acid profiles of rod outer segments (ROS), cerebellum, plasma, and liver demonstrated significantly lower n-6/n-3 ratios and higher docosahexaenoic acid (DHA) levels in fat-1-SFO compared with wt-SFO. When mice were exposed to light stress: 1) the outer nuclear layer (ONL) thickness was reduced; 2) amplitudes of the electroretinogram (ERG) were lower; 3) the number of apoptotic photoreceptor cells was greater; and 4) modification of retinal proteins by 4-hydroxyhexenal (4-HHE), an end-product of n-3 PUFA oxidation was increased in both fat-1-SFO and wt mice fed a regular lab chow diet compared with wt-SFO. The results indicate a positive correlation between the level of DHA, the degree of n-3 PUFA lipid peroxidation, and the vulnerability of the retina to photooxidative stress. In mice not exposed to intense light, the reduction in DHA resulted in reduced efficacy in phototransduction gain steps, while no differences in the retinal morphology or retinal biochemistry. These results highlight the dual roles of DHA in cellular physiology and pathology.

Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.

Stargardt-like macular dystrophy (STGD3) is a dominantly inherited juvenile macular degeneration that eventually leads to loss of vision. Three independent mutations causing STGD3 have been identified in exon six of a gene named Elongation of very long chain fatty acids 4 (ELOVL4). The ELOVL4 protein was predicted to be involved in fatty acid elongation, although evidence for this and the specific step(s) it may catalyze have remained elusive. Here, using a gain-of-function approach, we provide direct and compelling evidence that ELOVL4 is required for the synthesis of C28 and C30 saturated fatty acids (VLC-FA) and of C28-C38 very long chain polyunsaturated fatty acids (VLC-PUFA), the latter being uniquely expressed in retina, sperm, and brain. Rat neonatal cardiomyocytes and a human retinal epithelium cell line (ARPE-19) were transduced with recombinant adenovirus type 5 carrying mouse Elovl4 and supplemented with 24:0, 20:5n3, or 22:5n3. The 24:0 was elongated to 28:0 and 30:0; 20:5n3 and 22:5n3 were elongated to a series of C28-C38 PUFA. Because retinal degeneration is the only known phenotype in STGD3 disease, we propose that reduced VLC-PUFA in the retinas of these patients may be the cause of photoreceptor cell death.

Lipid differences in rod outer segment membranes of rats with P23H and S334ter opsin mutations.

PURPOSE: Retinal degenerations and diets low in n-3 fatty acids are associated with decreased docosahexaenoic acid (22:6n-3) in retina and plasma and with sterol abnormalities in retina and sperm. Using wild type (WT) and transgenic rats with P23H and S334ter opsin mutations, we evaluated retinal cholesterol levels, cholesterol synthesis, and fatty acid compositions of phospholipid classes in animals fed diets enriched in n-3 or n-6 polyunsaturated fatty acids. METHODS: Pregnant WT and heterozygous P23H and S334ter transgenic (TG) rats were fed safflower (safflower oil [SO], high n-6, trace n-3 fatty acids) or flaxseed oil (flaxseed oil [FO], high n-3, moderate n-6 fatty acids) diets beginning at E15, and pups were continued on the diets after weaning. Rod outer segment (ROS) membranes were prepared from 55-day-old rats, and the ratios of total fatty acid to cholesterol and the fatty acid compositions of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in ROS were determined. Intravitreal injections of [3H]acetate were given to 35-day-old WT and TG rats fed standard chow-diets. Endogenous cholesterol mass and de novo [3H]cholesterol synthesis were measured and normalized to total ROS fatty acid content. Multivariate analysis of variance (ANOVA) with post hoc Newman-Keuls tests were used to determine statistical differences. RESULTS: The relative levels of PC, PE, and PS were similar in all three rat strains independent of diet. Total lipids, PC, PS, and PE of ROS FO fed rats had higher levels of 22:6n-3 and lower levels of 22:5n-6 than those fed SO. Rats fed SO had higher levels of 22:5n-6 than those fed FO. Significant increases in 18:1n-9 were seen in PC and PS of P23H and S334ter rats; arachidonate (20:4n-6) increased only in PE. These changes were independent of diet. ROS membranes of transgenic rats were cholesterol enriched, relative to WT ROS, yet retinal cholesterol synthesis was not altered. Plasma cholesterol levels of transgenic rats were not different from those of WT rats. CONCLUSIONS: Endogenous levels of cholesterol, 18:1n-9, 20:4n-6, 22:5n-6, and 22:6n-3 were altered in ROS membranes of P23H and S334ter compared to WT rats. There appear to be two pools of 22:6n-3 in rat ROS, one that is sensitive to retinal degenerations and one that is not. The stress induced reduction in 22:6n-3 was not specific to any phospholipid class and was not caused by alteration of relative amounts of PC, PS, or PE in the membrane. Elevated retinal cholesterol may be a result of either an increased half life or an increased uptake of cholesterol from the blood.

P23H and S334ter opsin mutations: Increasing photoreceptor outer segment n-3 fatty acid content does not affect the course of retinal degeneration.

PURPOSE: The n-3 polyunsaturated fatty acids (PUFA) facilitate retinal development and function. Rats carrying transgenes with P23H and S334ter rhodopsin mutations lose their photoreceptors and have lower levels of 22:6n-3 in rod photoreceptor outer segments (ROS) than wild type (WT) animals. We tested the hypothesis that the rate of retinal degeneration in these mutant animals could be sensitive to the n-3 fatty acid content of retina. METHODS: Beginning embryonic day 15, WT and heterozygous transgenic rats with P23H and S344ter rhodopsin mutations were fed semi-synthetic diets enriched in n-6 (safflower oil, SO) or n-3 (flaxseed oil, FO) PUFA. At 35 and 55 days of age, electroretinographic (ERG) response, outer nuclear layer (ONL) thickness, and fatty acid composition of plasma and ROS were determined. Student's t-tests and multivariate analysis of variance with post hoc tests determined statistical differences. RESULTS: Rats fed FO or SO diets had different n-6/n-3 PUFA ratios in plasma (1.3 and 62) and ROS (0.2 and 1.1, respectively). Although there were profound effects of the diets on the plasma fatty acid composition, there were only minor differences between WT and transgenic animals within each dietary regime. The ROS of FO fed rats had 70% more 22:6n-3 than those fed SO, and the WT had higher concentrations of 22:6n-3 than the transgenic animals (WT>P23H>S334ter). In contrast, there was no difference in 22:6n-3 levels in ROS of WT and transgenic rats fed the SO diet. At P55, both transgenic lines had diminished ERGs and ONL thickness relative to the WT. There was no detectable effect of ROS fatty acid enrichment on the rate of retinal degeneration in the transgenic animals. However, the FO-diet provided a modest protection of function (b-wave) in S334ter animals. CONCLUSIONS: Feeding n-3 fatty acids to rats with mutant rhodopsin transgenes significantly increased the levels of 22:6n-3 in ROS membranes, but had no effect on the rate of retinal degeneration. Therefore, the degeneration is not the result of low (or high) 22:6n-3 in ROS and supplementation with 18:3n-3 will not rescue dying photoreceptor cells in these animal models of inherited retinal degenerations.