Alterations in lipid metabolism gene expression and abnormal lipid accumulation in fibroblast explants from giant axonal neuropathy patients
© Leung et al; licensee BioMed Central Ltd. 2007
Received: 17 August 2006
Accepted: 01 March 2007
Published: 01 March 2007
Giant axonal neuropathy (GAN) is a hereditary neurological disorder that affects both central and peripheral nerves. The main pathological hallmark of the disease is abnormal accumulations of intermediate filaments (IFs) in giant axons and other cell types. Mutations in the GAN gene, encoding gigaxonin, cause the disease. Gigaxonin is important in controlling protein degradation via the ubiquitin-proteasome system. The goal of this study was to examine global alterations in gene expression in fibroblasts derived from newly identified GAN families compared with normal cells.
We report the characterization of fibroblast explants obtained from two unrelated GAN patients. We identify three novel putative mutant GAN alleles and show aggregation of vimentin IFs in these fibroblasts. By microarray analysis, we also demonstrate that the expression of lipid metabolism genes of the GAN fibroblasts is disrupted, which may account for the abnormal accumulations of lipid droplets in these cells.
Our findings suggest that aberrant lipid metabolism in GAN patients may contribute to the progression of the disease.
Giant Axonal Neuropathy (GAN) is a severe autosomal recessive disorder that affects both the central and peripheral nervous systems. The most prominent pathological feature of GAN is the large, focal accumulations of neuronal intermediate filaments (IFs) in distended axons . Abnormal aggregations of IFs have also been found in astrocytes, endothelial cells, Schwann cells and cultured skin fibroblasts. Many GAN patients have frizzy hair that is distinctive from their parents. Chemical analysis of the hair has revealed a disruption of disulfide-bond formation in hair keratins . Hence, a generalized disorganization of IFs has been proposed to be responsible for GAN .
Skin fibroblast explants collected from GAN patients have been used as a model to study the disease. Under normal culture conditions, a low percentage of GAN fibroblasts exhibit abnormal aggregation and bundling of vimentin IFs [4–7]. Upon various stimuli, such as low serum  or low doses of trypsin , the vimentin networks of GAN fibroblasts collapse and form aggregates. Moreover, the microtubule (MT)-depolymerizing agent nocodazole exerts different effects on normal and GAN fibroblasts. Although the IF networks of both types of fibroblasts collapse under nocodazole treatment, the aggregates formed in GAN cells are significantly more compact and dense . Together, these data suggest that dysfunction of the GAN gene product might cause IFs to form aggregates that are harmful to cells.
A GAN gene has been identified and its product named gigaxonin, with twenty-three different mutations reported to date [8–10]. Gigaxonin is a member of the kelch repeat superfamily. It contains an N-terminal BTB/POZ (Broad-Complex, Tramtrack and Bric-a-brac/Poxvirus and Zinc-finger) domain and six C-terminal kelch motifs. MT-Associated Protein 1B (MAP1B), Tubulin Cofactor B (TBCB), and MT-Associated Protein 8 (MAP8 or MAP1S) have been identified as binding partners of gigaxonin in yeast two-hybrid screens [11–14]. Gigaxonin interacted with these proteins via the kelch repeats. The N-terminal BTB of gigaxonin could bind ubiquitin-activating enzyme E1, suggesting that gigaxonin functions as a scaffold protein in the ubiquitin-proteasome complex and mediates the degradation of MAP1B, TBCB and MAP8 . Mutations in the GAN gene result in accumulation of these cytoskeletal proteins and eventual neurodegeneration.
Here, we report the characterization of two primary lines of cultured GAN fibroblasts carrying a total of three putative disease-linked GAN alleles. We compared the gene expression profiles of the GAN fibroblasts to those of normal fibroblasts. We found that the expression of lipid metabolism genes was perturbed in GAN fibroblasts most dramatically. In addition to changes in the expression levels of lipid metabolism genes, we also discovered an increase in the number of neutral lipid droplets in GAN cells. These data suggest that defects in lipid metabolism may contribute to the pathogenesis of GAN.
Genotyping of fibroblast explants
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
Microarray analysis of GAN fibroblasts
Differentially expressed genes in GAN vs. normal fibroblasts as analyzed by oligonucleotide microarrays. Genes selected displayed at least a three-fold difference in expression level.
Genes involved in lipid metabolism and adipogensis
Complement component 3 precursor
Fatty acid binding protein 5
ATP-binding cassette A6
ATP-binding cassette B4
Acyl coenzyme A:cholesterol acyltransferase
Integral membrane proteins and receptors
Integrin, beta 3
GABA-B receptor R2
GABA-B receptor splice variant 1
Orphan G protein-coupled receptor
Integral membrane serine protease
Hyaluronan-mediated motility receptor
Death receptor 6
Membrane glycoprotein M6
Potassium channel beta subunit
Endothelin receptor type B
Potassium channel beta 1a subunit
Dickkopf homolog 1
Kinetochore associated 2
Cell division cycle 2
Tumor necrosis factor-related protein
EGF-like-domain, multiple 6
Transcription factors and nuclear proteins
Transcription factor AP-2 alpha
High mobility group AT-hook 1
Nuclear factor IB
Interferon-inducible protein p78
Pregnancy specific beta-1-glycoprotein
Pregnancy specific beta-1-glycoprotein 7
Pregnancy specific beta-1-glycoprotein 4
Pregnancy specific beta-1-glycoprotein 1
Hypothetical protein PRO02730
Uncharacterized bone marrow protein
Hypothetical protein FLJ10517
KIAA0101 gene product
KIAA0008 gene product
Doublecortin and CaM kinase-like 1
KIAA1547 gene product
Hypothetical protein DKFZp762E1312
Hypothetical protein FLJ10829
Clone HQ0310 PRO0310p1
Hypothetical protein DKFZp564H1916
KIAA0042 gene product
Hypothetical protein FLJ22009
Hypothetical protein FLJ23468
Hypothetical protein DKFZp564N1116
Hypothetical protein FLJ10781
Hypothetical protein DKFZp564B052
KIAA0008 gene product
KIAA0865 gene product
Matrix metalloproteinase 1
Carbonic anhydrase XII
Topoisomerase II alpha
Step II splicing factor SLU7
Monocyte chemotactic protein
Ribonucleotide reductase M2
Plasminogen activator, urokinase
Atrophin-1 interacting protein 1
Monoamine oxidase A
Type II iodothyronine deiodinase
Scrapie responsive protein 1
Natural killer cell transcript 4
CD24 signal transducer
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
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).
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.
Here we describe three novel mutant GAN alleles, including a missense mutation, an intronic mutation, and a 9.3-kb deletion. We also characterize two GAN fibroblast explants and detect perturbations of lipid metabolism in both of them. Based on the previous studies of vimentin IFs and lipid metabolism, we speculate that the abnormal accumulation of lipid droplets that we observe in GAN fibroblasts is an indirect effect of the GAN mutations and is probably mediated through IF network disruption. These fibroblast explants will be a useful tool to study the physiological functions of gigaxonin.
Genotyping of fibroblasts
Total RNA was isolated from fibroblasts using Trizol reagent (Invitrogen). First-strand cDNA was synthesized with oligo-dT primers and reverse transcriptase (Invitrogen). The procedures were performed according to the manufacturer's protocol. Gigaxonin cDNAs were amplified from the cDNA pool using forward primer, 5'-TTGATGGCTGAGGGCAGTGCCGTGTCTG-3' and reverse primer, 5'-TTCCTCCTCAAGGGGAATGAACACGAAT-3'. After electrophoresis, PCR products were purified by the GeneClean purification system (Q-Biogen) and were sequenced with the BigDye™ sequencing kit (Applied Biosystems). The shorter gigaxonin cDNA products from explant WG0791 were first cloned into pCR2.1-TOPO vector (Invitrogen) before sequencing. The conditions used for genomic PCR and the sequences of the PCR primers have been reported elsewhere .
Cell culture, immunocytochemistry and Oil-Red O staining
Fibroblast explants were obtained from the repository for mutant human cell strains at McGill University. They were maintained at 37°C and 5% CO2 in MEM Eagle medium (Earle's) with 10% fetal bovine serum. For low-serum treatment, cells were incubated in medium containing 0.1% fetal bovine serum for 72 hours. All experiments were performed on cells from passages 13–18. For immunocytochemical analyses, cells seeded on coverslips were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.1% Triton-X100 for 5 minutes. Fixed cells were incubated with primary antibodies at room temperature for one hour, followed by several washes with PBS and incubation with appropriate secondary antibodies for 30 minutes. The coverslips were then washed with PBS and mounted onto slides with Aquamount (Lerner Laboratories) for immunofluorescent microscopy. To co-stain lipid droplets and vimentin filaments, formaldehyde-fixed cells were permeabilized with 0.05% Saponin and incubated with an anti-vimentin antibody overnight. Before mounting onto slides, cells were stained for lipid droplets with 0.5% Oil Red O in propylene glycol (Poly Scientific). To stain the nuclei and the lipid droplets, cells were fixed in 4% paraformaldehyde and incubated with 0.5% Oil Red O in propylene glycol and Gill's Hematoxylin I (Poly Scientific) for 20 minutes. Antibodies used: monoclonal mouse anti-vimentin, clone V9 (Sigma), monoclonal mouse pan anti-keratin (Sigma), and polyclonal anti-vimentin .
Human 133A Genechips from Affymetrix were used to study the expression profiles. Biotin-labeled cRNA probes were prepared according to the manufacturer's protocol. In brief, cells were treated with low-serum medium for 72 hours, and total RNA was extracted. First-strand cDNAs were synthesized with oligo-dT-T7 primers and reverse transcriptase (Invitrogen). After second-strand cDNA synthesis, double-stranded cDNAs were used to produce biotin-labeled cRNA probes by T7 polymerase (Enzo Laboratory). The cRNAs were fragmented before being used for hybridization. Hybridization and scanning were carried out at the GeneChip analysis facility at Columbia University.
GeneChip results were analyzed by Affymetrix Microarray Suite (version 5.0). To reduce background noise, a four-way comparison of MCH068, MCH070, WG0321 and WG0791 cells was performed. Only genes that showed consistent changes in both WG0321 and WG0791 fibroblasts when compared to both MCH068 and MCH070 cells were selected. Genes that exhibited more than three-fold differences were considered as significantly altered.
Quantitative RT-PCR conditions and gene-specific primer sequences.
Quantitative PCR Primers
Fatty acid binding protein 5 (FABP5)
ATP-binding cassette A6 (ABCA6)
ATP-binding cassette B4 (ABCB4)
Acyl coenzyme A: cholesterol acyltransferase
We are grateful to Gee Ying Ching for helpful discussions and advice. This work was supported by National Institutes of Health grants P50 AG08702 for C.L.L. and NIH15182 for R.K.H.L.
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