Genotyping of fibroblast explants
Four subcutaneous fibroblasts explants, MCH068, MCH070, WG0321 and WG0791, were obtained from the repository for mutant human cell strains at the McGill University Health Center. MCH068 and MCH070 cells were isolated from normal individuals while WG0321 and WG0791 cells were isolated from patients diagnosed with GAN. Both GAN patients experienced difficulty in walking and their electromyography showed diffuse axonal neuropathy. We sequenced the GAN cDNAs prepared from the fibroblast explants. No mutations were detected in MCH068 and MCH070 cells. We obtained two PCR products from WG0791 cells, a major product of ~1.8 kb and a minor product of ~1.7 kb (data not shown). Sequencing of the 1.8-kb product revealed that a missense mutation in exon 3 (c.545T>A). The mutation resulted in the substitution of the isoleucine at amino acid position 182 with an asparagine, I182N (Fig. 1A). The 1.7-kb fragment represented an mRNA product from the other GAN allele because it did not contain the I182N mutation. It was shorter than the wild-type message because it did not contain exon 2 (Fig. 1B). We then sequenced the first three exons and the intron-exon junctions of the GAN gene from WG0791 cells. While confirming the I182N missense mutation, we also discovered an A→C mutation near the exon 2-intron 2 junction (c.282+3A>C), which might account for the misspliced message (data not shown).
Sequencing of the GAN cDNA prepared from WG0321 cells revealed a deletion/insertion in the GAN message: nucleotides 1505–2056 were replaced with a 452-nucleotide-long sequence that was identical to a part of intron 9 of the GAN gene (Fig. 1C). We sequenced the 3' region of the GAN gene from WG0321 cells and discovered that the entire exon 10 and 446 base pairs of the exon 11 5'end were deleted in both alleles. The deletion caused exon 9 to be spliced into intron 9 (data not shown). A schematic representation of the mutated and normal GAN alleles is shown in Fig. 1D.
Because we were unable to screen additional healthy controls, the three novel GAN alleles should be considered putative disease-associated mutations.
Characterization of GAN fibroblasts
Previous studies have shown that vimentin IFs form abnormal aggregates in GAN fibroblasts and that low-serum treatment enhances the aggregation. To determine whether WG0321 and WG0791 cells also contained IF aggregates, we performed immunocytochemical staining with antibodies against various IF proteins. In complete medium, 12% of WG0791 cells and 23% of WG0321 cells displayed compact vimentin aggregates. Upon low-serum treatment for 72 hours, the number of cells containing vimentin aggregates dramatically increased, up to 50% for WG0791 cells and 95% for WG0321 cells (Fig. 2A). Vimentin aggregates were never detected in normal fibroblasts, MCH068 and MCH070. Interestingly, although cytokeratins are usually not expressed in fibroblasts, a small percentage of both normal and GAN cells (~2%) exhibited positive staining for keratins (Fig. 2B–E). In GAN cells, some of the IF aggregates contained both vimentin and cytokeratin (Fig. 2D and 2E).
Microarray analysis of GAN fibroblasts
We studied the expression profiles of GAN and normal fibroblasts grown in low-serum medium by microarray (Affymetrix) analysis. To reduce the background noise, we performed a four-way comparison of MCH068, MCH070, WG0321 and WG0791 cells. We selected genes that showed consistent changes in WG0321 and WG0791 fibroblasts when compared to MCH068 and MCH070 cells. Genes that exhibited more than three-fold differences are grouped in Table 1 in the Supplemental Data according to their proposed functions. Gene products involved in lipid metabolism displayed the most dramatic changes. We confirmed the relative expression levels of these lipid metabolism genes by quantitative RT-PCR (Fig. 3). The results were in agreement with the microarray experiments. We also performed quantitative RT-PCR to determine the expression levels of gigaxonin in GAN fibroblasts (Fig. 3A). Compared to MCH068 and MCH070, GAN mRNA expression was dramatically upregulated in WG0321 (~26 fold) and WG0791 (~7 fold) cells.
We also detected significant changes in members of the ATP-Binding Cassette (ABC) protein family, ABCA6 and ABCB4. ABC transporters are multispan transmembrane proteins that translocate a variety of substrates. ABCA6 has been suggested to play an important role in lipid homeostasis ; it was up-regulated in GAN fibroblasts. ABCB4 is also known as multidrug resistance P-glycoprotein 3 and functions as a translocator of phospholipids. Deficiencies in ABCB4 cause progressive intrahepatic cholestasis type III . ABCB4 was downregulated in GAN fibroblasts. In addition, Fatty Acid Binding Protein 5 (FABP5), Meltrin alpha, Complement C3 and Butyrylcholinesterase (BChE) were upregulated in GAN fibroblasts. FABP5 is involved in intracellular fatty-acid trafficking (reviewed in ). Meltrin alpha is a member of the metalloprotease-disintegrin family and is involved in adipogenesis . C3 is a component of the complement system of innate immunity. It is also the precursor of an acylation-stimulating protein that can increase triglyceride synthesis (reviewed in ). BChE is a serine hydrolase that exhibits increased activity in hyperlipidaemic patients . Acyl-CoA: Cholesterol Acyltransferase (ACAT) and Leptin were downregulated in GAN fibroblasts. ACAT is an enzyme that converts intracellular cholesterol into cholesteryl esters and promotes the storage of excess cholesterol in the form of cholesterol ester droplets . Leptin is a peptide hormone produced predominantly by white adipose cells; however, it is also expressed in non-adipocytes and is important in regulating fatty acid metabolism (reviewed in ).
Cellular studies of lipid droplets in GAN fibroblasts
Because the microarray analysis revealed alterations in the expression of lipid metabolism genes in GAN fibroblasts, we studied the distribution of lipid droplets in the fibroblast explants by cytological staining. We used Oil Red O dye to label neutral lipid droplets of serum-starved GAN and normal fibroblasts. The proportions of cells containing lipid droplets were significantly higher in the mutant explants (52% in WG0321, 21% in WG0791) compared with the normal explants (6% in MCH068, 9% in MCH070; Fig 4A). In addition, GAN fibroblasts contained many more droplets per cell than did normal fibroblasts (Fig. 4B–D). These results were confirmed by Bodipy staining (data not shown).
Previously, it has been shown that vimentin can form cage-like structures around lipid droplets in adipocytes. We wondered whether the vimentin aggregates also surrounded the lipid droplets in GAN cells. We performed fluorescence microscopy on GAN cells co-stained for vimentin and lipid droplets. As shown in Fig. 4F–G, while some of the small vimentin aggregates appeared to encage lipid droplets, most of them did not (~80% in both cell lines).