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The goal of this study was to examine global alterations in gene …
Biology Articles » Biochemistry » Lipid Biochemistry » Alterations in lipid metabolism gene expression and abnormal lipid accumulation in fibroblast explants from giant axonal neuropathy patients » Discussion
In this study, we describe two GAN fibroblast explants and identify the underlying mutations in the GAN gene. WG0791 cells contained two different GAN mutant alleles, an intronic mutation near the splice donor site of intron 2 and a missense mutation in exon 3 (I182N). WG0321 cells carried two identical deletion alleles predicted to produce a truncated gigaxonin protein. As revealed by immunocytochemical analysis, both WG0791 and WG0321 cells displayed abnormal vimentin filament aggregation, a phenomenon exacerbated drastically by low-serum treatment. By comparing the expression profiles of these GAN fibroblasts to two normal fibroblasts under low-serum conditions, we found that the GAN cells exhibited defects in lipid metabolism. Unlike normal fibroblasts, which were virtually devoid of lipid droplets under these conditions, GAN fibroblasts accumulated a large number of lipid droplets.
The mechanism that caused lipid defects in GAN fibroblasts is not clear but may involve defects in the vimentin IFs. GAN has long been considered a disease of IFs, and earlier studies have shown that vimentin IFs are closely associated with cytoplasmic lipid droplets in normal cells (reviewed in ). Association of vimentin IFs with lipid droplets is most obvious in adipose cells where the vimentin network forms a cage-like structure surrounding the lipid droplets . Similar interactions of vimentin and lipid droplets have also been observed in steroidogenic cells [25-27], and the interaction is probably direct as indicated by in vitro experiments [28,29]. Although the significance of the vimentin-lipid interactions to cellular functions has not been clearly defined, there is evidence to suggest that vimentin IFs play an important role in cholesterol transport. Using human adrenal carcinoma cells with or without vimentin IFs, Sarria et al. showed that there is a direct correlation between the presence of vimentin IFs and the capacity of the cells to utilize lysosomal cholesterol (Sarria et al., 1992). Their studies also indicated that the intracellular movement of low-density-lipoprotein (LDL)-derived cholesterol from the lysosomes to the site of esterification is dependent on vimentin.
Using 3T3-L1 preadipocytes as a model of adipogenesis, vimentin IFs have been shown to be important for lipid droplet accumulation during adipose development . Perturbation of the vimentin network in 3T3-L1 cells during adipose conversion by nocodazole treatment, anti-IF antibody microinjection, or over-expression of a dominant-negative vimentin mutant protein could abolish the formation of lipid storage droplets in the differentiated adipocytes. The impairment appeared to be the result of an increased turnover rate of triglyceride synthesis. However, the significance of vimentin in adipogenesis has been questioned by the studies of vimentin knockout mice. Vimentin-null mice were viable and exhibited no obvious abnormality in adipose development . Importantly, there was no compensatory increase in the expression of another IF protein. Only some minor pathologies were observed in the null mice. Specifically, the glial fibrillary acidic protein network was disrupted in a subset of astrocytes , and the Bergmann fibers of the cerebellar cortex were hypertrophic . Nonetheless, cultured embryonic fibroblasts from vimentin-null mice displayed a significant decrease in the synthesis of glycosphingolipids . The defect appeared to result from impaired intracellular transport of glycolipids and sphingoid bases between the endosomal/lysosomal pathway and the Golgi apparatus and the endoplasmic reticulum. It is therefore possible that, in GAN fibroblasts, mutations of the GAN gene affect the properties of vimentin IFs, leading to perturbation of lipid metabolism and accumulation of lipid droplets. Our observation that some low-serum-treated GAN cells contained vimentin aggregates but no lipid droplets may be explained by an insufficient sensitivity of Oil Red O staining. Alternatively, the cells could still have been in the process of accumulating oil droplets.
How do GAN mutations lead to defects in IF networks? One possible mechanism is through the disruption of MTs, because gigaxonin can affect the degradation of MAP1B, MAP8 and TBCB [11,12,14]. IFs are closely associated with MTs. Disruption of the MT network by nocodazole can cause IFs to collapse into the perinuclear region, and this MT-mediated effect of IFs is more obvious in GAN fibroblasts than in normal fibroblasts . GAN mutations may therefore affect MTs, leading to IF aggregation and ultimately retention of lipid droplets. However, MT disruption with nocodazole did not have an obvious effect on the number of lipid droplets in either mutant or normal fibroblasts (data not shown). These data suggest that IF aggregation may not be linked mechanistically to lipid droplet accumulation in GAN cells, but rather that the observed defects of lipid metabolism in GAN cells are a compound effect of IF/MT perturbation and other gigaxonin-related functions.
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