1 Department of Cell Biology, University of Alberta, Edmonton T6G 2H7
2 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RNA-mediated interference; peroxin; cyan fluorescent protein; protein targeting; Zellweger syndrome
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Peroxisomes are organelles found in most eukaryotic cells (46, 55, 64). They show remarkable morphological and metabolic plasticity, as their number, size, morphology, protein composition, and biochemical functions vary depending on the organism, cell type, and/or environmental milieu (40, 51, 63, 66). Typically, peroxisomes contain at least one oxidase that produces harmful hydrogen peroxide and catalase, a hydrogen peroxide-degrading enzyme (6). In human and other mammalian cells, peroxisomes compartmentalize a variety of degradative and biosynthetic metabolic pathways. Degradative pathways include the - and ß-oxidation of fatty acids, the catabolism of purines and amino acids, and the breakdown of prostaglandins and polyamines (52, 71). Biosynthetic pathways include the formation of plasmalogens, cholesterol, bile acids, and polyunsaturated fatty acids (30, 52, 70).
Peroxisomes are indispensable for normal human development and physiology, as shown by the severity and lethality of numerous peroxisomal disorders (17, 54, 55, 70). These disorders can be divided into two groups, the peroxisome biogenesis disorders (PBDs) and the peroxisomal single enzyme disorders (9, 13, 17, 42, 6871). The PBDs affect multiple peroxisomal metabolic pathways, as patients fail to assemble functionally intact peroxisomes (13, 17, 46, 55). A hallmark of the Zellweger spectrum of PBDs, including Zellweger syndrome (ZS) itself, neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), is the global developmental delay caused by the incomplete migration of neuroblasts during development (38, 41, 68). Patients with ZS rarely survive their first year, whereas NALD and, especially, IRD are less severe disorders, with some patients surviving into their third and fourth decade (17, 68). Another distinct form of PBD, rhizomelic chondrodysplasia punctata (RCDP) type 1, is characterized clinically by abnormal psychomotor development, mental retardation, and death in early infancy (43). The PBDs are inherited in an autosomal recessive fashion, with an incidence of 1 in 50,000 live births (46).
The second group of disorders, peroxisomal single enzyme disorders, results from mutations in genes encoding single peroxisomal enzymes (45, 68, 70, 71). The most common of these disorders, X-linked adrenoleukodystrophy (X-ALD) (71), occurs in 1 in 15,000 live births (46). X-ALD is caused by defects in ALDP, an integral peroxisomal membrane protein (PMP) that is a member of the ATP-binding cassette (ABC) half-transporter family of proteins (10). Single defects in many other peroxisomal enzymes involved in the
- and ß-oxidation of fatty acids, amino acid catabolism, and the biosynthesis of plasmalogens and bile acids have also been shown to cause peroxisomal disorders (68, 70, 71).
Peroxisome assembly, division, and inheritance are regulated by at least 23 proteins called peroxins that are encoded by the PEX genes, and mutations in 11 of these genes cause the PBDs (13, 17, 55). Peroxisomal proteins are encoded by nuclear genes, synthesized on cytosolic polysomes and imported posttranslationally across the peroxisomal membrane (46, 55, 64). They are sorted to the organelle by specific PTSs. These PTSs are recognized in the cytosol by their cognate receptors, which guide the targeting and docking of proteins to the peroxisomal membrane (21, 57). Most matrix proteins are targeted to the peroxisome by PTS1, a carboxy-terminal tripeptide with the consensus motif (Ser/Ala/Cys)(Lys/Arg/His)(Leu/Met/Ile), and its cytosolic shuttling receptor Pex5p (20, 21, 46, 52, 64). A few matrix proteins are targeted by a distinct PTS2, an amino-terminal nonapeptide with the consensus motif (Arg/ Lys)(Leu/Val/Ile)Xaa5(His/Gln)(Leu/Ala), and its cytosolic shuttling receptor Pex7p (21, 55). As reported above, the PTS2 pathway is absent in C. elegans (39). The initial docking site for both the Pex5p-PTS1 and Pex7p-PTS2 complexes on the cytosolic surface of the peroxisomal membrane consists of three membrane-associated peroxins, Pex13p, Pex14p, and Pex17p (46, 64). After docking, the PTS1 and PTS2 receptors, together with their cargoes, are transferred to other membrane-associated components of the import machinery, including Pex2p, Pex10p, and Pex12p (21, 57). These components function in the translocation of cargo proteins, either alone or together with their receptors, across the peroxisomal membrane into the peroxisomal matrix (13, 55). In human cells, Pex2p, together with two cytosolic peroxins, Pex1p and Pex6p, may act to recycle the PTS1 receptor to the cytosol (13, 17, 46). Many PMPs are sorted to peroxisomes directly from the cytosol by a distinct membrane PTS, called mPTS1, and its putative cytosolic receptor, Pex19p (47, 57), which may also act as a chaperone to assist the assembly of PMPs in the peroxisomal membrane (55, 64).
The present study not only enhances our understanding of the functioning of the C. elegans peroxisomal protein import machinery but also sheds new light on the recently discovered role for peroxisomes in the process of development. Peroxisomes do more than just burn fat and dump toxic waste; they appear to be dynamically integrated into specific cell and organismal developmental programs, with their biogenesis being tightly coordinated with the biogenesis of other cellular compartments (1, 28, 32, 36, 49, 50, 57, 6062). Here, we report the use of RNAi for the creation of an extensive collection of nematode functional gene knockout mutants deficient in a wide range of peroxisome-related functions. The availability of such a collection is a prerequisite for the use of C. elegans as an advantageous model system with which to study the molecular and physiological defects underlying the human peroxisomal disorders. We confirm the findings of Motley and coworkers (39) of the requirement for the enzyme alkyldihydroxyacetonephosphate synthase (ADHAPS) for C. elegans development, and, in addition, we define subsets of other peroxisomal enzymes and of peroxins essential for this program.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
dsRNA Interference
The template for double-stranded RNA (dsRNA) synthesis was generated by PCR of C. elegans genomic DNA with oligonucleotide flankers encoding the T7 promoter and a specific sequence complementary to the gene of interest (Fig. 1). RNA was produced using the MEGAscript kit for RNA synthesis (Ambion, Austin, TX). After synthesis, both strands of RNA were annealed by incubation at 65°C for 10 min followed by slow cooling. dsRNA at a concentration of 0.51.0 mg/ml in 2% PEG-6000, 20 mM potassium phosphate, 3 mM potassium citrate, pH 7.5, was injected into the gonads of one or both gonad arms of young adult hermaphrodites (2030 animals), as described (35). Injected animals were transferred to nematode growth medium (NGM) agar plates (4) containing the OP50 strain of Escherichia coli. After 12 h, the surviving animals were transferred to fresh culture plates, on which they were allowed to lay eggs. The percentage of adult F1 progeny was evaluated 3 days after injection. In RNAi experiments, the percentage of adults in the offspring of worms injected with control dsRNA was set at 100% in each independent experiment.
|
Preparation of Anti-Thiolase Antibodies and Immunoblot Analysis
The last exon of the gene Y57A10C.6 encoding the 242 carboxyl-terminal amino acids of the protein P-44 was fused in-frame and downstream of the open reading frame (ORF) encoding E. coli maltose binding protein (MBP) in the vector pMAL-p2X (New England BioLabs, Beverly, MA). The recombinant protein product was purified by affinity chromatography on amylose resin and cleaved with factor Xa. The thiolase peptide was separated from MBP by SDS-PAGE, excised from the gel, and isolated by electroelution. Antibodies to the peptide were raised in rabbit and guinea pig. Immunoblot analysis and detection of antigen-antibody complexes by enhanced chemiluminescence were performed as described (60).
Immunofluorescence Microscopy
Double-labeling, immunofluorescence microscopy was performed as described (37). Rabbit and guinea pig anti-thiolase antibodies were used at a dilution of 1:100. Affinity-purified rabbit antibodies to green fluorescent protein (GFP) (a gift from Dr. L. Berthiaume, Department of Cell Biology, University of Alberta) were used at a dilution of 1:500. Primary antibodies were detected with rhodamine- or FITC-conjugated donkey anti-rabbit IgG antibodies or with rhodamine- or FITC-conjugated donkey anti-guinea pig IgG antibodies (Jackson, West Grove, PA). Samples were viewed with an Olympus BX50 microscope.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The following candidate C. elegans orthologs of human peroxisomal enzymes implicated in lipid metabolism and peroxins were identified.
Orthologs of human thiolases.
Two human peroxisomal thiolases have been identified. Peroxisomal thiolase 1 (pTH1) acts only on straight-chain substrates and is not reactive with 3-ketoacyl-CoA esters carrying a methyl group at the -carbon (20, 71). pTH1 is therefore not involved in the
-oxidation of very-long-chain fatty acids (VLCFAs) (20, 70). In silico searching revealed three candidate C. elegans orthologs of human pTH1: T02G5.8, T02G5.7, and T02G5.4. The carboxy termini of all three potential C. elegans orthologs of human pTH1 carry tripeptides that closely resemble the PTS1 signal for matrix proteins, namely QKL for T02G5.8, KKL for T02G5.7, and QKL for T02G5.4, all of which have been shown to function in targeting proteins to peroxisomes in other organisms (39). Previous studies have also predicted that C. elegans ORFs T02G5.8, T02G5.7, and T02G5.4 represent orthologs of human pTH1 (18, 39).
Peroxisomal thiolase 2 (pTH2) is the second human peroxisomal thiolase. pTH2, which is alternatively termed SCP2/3-ketothiolase or SCPx (for "sterol carrier protein x"), is required for the metabolism of 3-oxoacyl-CoA esters with a methyl branched chain at the -carbon (20, 71). These CoA esters include intermediates of peroxisomal
-oxidation of 3-methyl branched VLCFAs, such as cerebronic and phytanic acids. The
-oxidation of VLCFAs has been shown to be impaired in ZS patients (27, 48). During
-oxidation, phytanic acid is shortened to pristanic acid, which then enters peroxisomal ß-oxidation, with the last step catalyzed by pTH2 (20, 71). In silico searching revealed the ORF Y57A10C.6 of C. elegans as a potential ortholog of human pTH2. This ORF encodes the C. elegans peroxisomal thiolase, P-44 (34), which is required for the metabolism of 3-oxoacyl-CoA esters with a methyl branched chain at the
-carbon (65).
Orthologs of human catalase.
Catalase converts hydrogen peroxide to water and molecular oxygen. In silico searching revealed two candidate C. elegans orthologs, Y54G11A.6 and Y54G11A.5b, of the only known human catalase. These C. elegans ORFs have been shown to encode two distinct forms of catalase, a cytosolic catalase, ctl-1 (56), and a peroxisomal catalase, ctl-2 (65). Notably, mutations in the ctl-1 gene reduce the adult lifespan (56). Peroxisomal catalase ctl-2 encoded by ORF Y54G11A.5b contains a PTS1 tripeptide at its carboxyl terminus.
Ortholog of human ADHAPS.
ADHAPS catalyzes the second step in the biosynthesis of plasmalogen in peroxisomes (19). In humans, defects in ADHAPS result in a peroxisomal single enzyme disorder, RCDP type 3 (9). ADHAPS, which is targeted to peroxisomes by a PTS2 in mammals (7), contains a PTS1 in C. elegans (8). The C. elegans counterpart of human ADHAPS is encoded by ORF Y50D7A.7 (39).
Orthologs of human ABC half-transporters.
Four human peroxisomal membrane ABC half-transporters have been identified, namely ALDP, ALDRP, PMP70, and PMP69 (70, 71). They are all members of the so-called ABCD subfamily of the ABC transporter superfamily (10). Different combinations of ABC half-transporters to form dimers (peroxisomal half-transporter heterodimers) have been proposed to mediate the transport of distinct subsets of their substrates across the peroxisomal membrane (10). ALDP is implicated in the uptake of VLCFA-CoAs, as a deficiency in this ABC half-transporter leads to the accumulation of C26:0 VLCFA-CoA in the plasma of patients suffering from X-ALD (23). The functions of the other three transporters remain to be established, although, considering their high similarity to each other and to ALDP, they may be involved in fatty acid transport across the peroxisomal membrane (10). Indeed, PMP70 has recently been implicated in the peroxisomal transport of 2-methylacyl-CoA esters (71). In silico searching revealed three candidate C. elegans orthologs of human ALDP, namely, T02D1.5, C44B7.9, and C44B7.8. The proteins encoded by these C. elegans ORFs also show high similarity to two human ALDP-like transporters, ABCD2 and ABCD3. T02D1.5 is also a putative ortholog of human ALDRP. Of note, the ORFs C44B7.8 and C44B7.9, as well as the ORFs T02G5.7 and T02G5.8, are linked, being separated by 380 bp and 220 bp, respectively. Therefore, their levels of expression may be modulated by common regulatory elements.
Orthologs of human acyl-CoA synthetases.
An initial step of fatty acid metabolism in humans is catalyzed by several acyl-CoA synthetases, which all ligate CoA to free fatty acid but differ in their substrate specificities from VLCFAs to long-chain fatty acids (LCFAs) (14, 41, 51). Acyl-CoAs formed in the acyl-CoA synthetase-catalyzed reaction are key intermediates in the biosynthesis of various lipids, including triacylglycerols, phospholipids, cholesterol esters, and sphingomyelin, and in the ß-oxidation of fatty acids (70, 71). C. elegans fatty acid transport protein a (CeFATPa) is a candidate ortholog of two human acyl-CoA synthetases, heart-specific very-long-chain fatty acyl-CoA synthetase-related protein (VLCS-H1) and fatty acid transport protein 4 (FATP4) (22), and is encoded by ORF F28D1.9. Oversynthesis of CeFATPa in COS cells greatly increases their LCFA uptake (22).
The protein encoded by C. elegans ORF Y65B4BL.5 is an ortholog of two Saccharomyces cerevisiae peroxisomal acyl-CoA synthetases, Faa1p and Faa2p, and shows similarity to three human acyl-CoA synthetases, FACL1, FACL2, and FACL5.
Ortholog of human 3,5-
2,4-dienoyl-CoA isomerase.
This enzyme catalyzes the first step of auxiliary reactions that allow polyunsaturated fatty acids to enter the normal pathway for the ß -oxidation of fatty acids (12). A candidate C. elegans ortholog of human peroxisomal 3,5-
2,4-dienoyl-CoA isomerase-like protein ECH1 is encoded by ORF Y25C1A.13.
Orthologs of human peroxins.
Several functional categories of human peroxins have been chosen for the in silico search of their potential C. elegans counterparts. A candidate ortholog of the human PTS1 receptor, Pex5p, is encoded by ORF C34C6.6. An analysis of the interactions between the C. elegans ortholog of human Pex5p and PTS1-containing peroxisomal matrix proteins revealed that the Pex5p-dependent route for the peroxisomal targeting of PTS1 proteins is conserved between humans and nematodes (18). A potential ortholog of human peroxin Pex19p is encoded by ORF F54F2.8. Candidate orthologs of Pex13p and Pex12p are encoded by ORFs F32A5.6 and F08B12.2, respectively, while potential orthologs of human Pex2p, Pex1p, and Pex6p are encoded by ORFs ZK809.7, C11H1.6, and F39G3.7, respectively (15).
Peroxisomal Enzymes and Peroxins Implicated in the Development of C. elegans
We used RNAi for the epigenetic inactivation of genes encoding C. elegans orthologs of human peroxisomal enzymes and peroxins, and the effects of posttranscriptional silencing of the individual genes on normal development of the nematode were evaluated. ORFs were targeted individually in the RNAi experiments, with the exception of those encoding 1) the three ABC half-transporters, 2) the three orthologs of human pTH1, 3) the three orthologs of human pTH1 and the ortholog of human pTH2, and 4) the orthologs of human peroxins Pex1p and Pex6p. These four groups of orthologous proteins were targeted by coinjection of dsRNAs specific for the individual genes coding for the proteins of a particular group.
The offspring of worms injected with dsRNAs specific to ORFs for various peroxisomal enzymes of lipid metabolism can be divided into two distinct phenotypes (Fig. 2). The first phenotype showed significant developmental delay in the worms, with almost no adult worms being found 3 days postinjection. The second phenotype was almost indistinguishable from that of control worms, and, compared with control values, 60100% of the F1 progeny of worms injected with dsRNA reached adult stage.
|
|
Chimera CFP-SKL Shows Differences in Peroxisome Size and Number in Different Parts of the Worm Body
Motley and coworkers (39) constructed fusions of GFP to either a PTS1 or a PTS2 to morphologically define peroxisomes within the cells of C. elegans. We have used a similar approach and have integrated an extrachromosomal array encoding a CFP-SKL fusion protein into the genome of C. elegans to monitor the peroxisomal import of PTS1-targeted matrix proteins and to evaluate the size, number, and morphology of peroxisomes in various dsRNA-injected worms. The integrated construct was expressed under the let-858 promoter, which is active in all tissues and at all stages of C. elegans development (http://www.ciwemb.edu/pages/firelab.html). Expression of this fusion protein in worms produced a punctate pattern of fluorescence characteristic of peroxisomes and which can be visualized in the living organism by fluorescent microscopy (Fig. 4). The peroxisomal targeting of CFP-SKL was confirmed by double-labeling, immunofluorescence microscopy with antibodies to the well-established marker of peroxisomes, P-44 thiolase type 2, and to CFP (Fig. 5). The punctate fluorescence patterns generated by antibodies to thiolase and to CFP were superimposable and characteristic of peroxisomes (Fig. 5).
|
|
In worms injected with dsRNAs specific to the ORFs encoding Pex5p (Fig. 4E), Pex13p (Fig. 4H), or Pex19p (Fig. 4I), a diffuse pattern of fluorescence of CFP-SKL, characteristic of the cytosol, was observed. Therefore, these worms are deficient in the peroxisomal import of PTS1-targeted matrix proteins. In contrast, injection of dsRNAs specific to ORFs encoding the other peroxins or the other peroxisomal enzymes did not compromise the punctate fluorescence pattern of CFP-SKL characteristic of peroxisomes (Fig. 4).
It should be noted that the size and morphology of peroxisomes were significantly altered in worms injected with dsRNA to Pex12p, P-44 (type 2 peroxisomal thiolase), or to the three type 1 thiolases. Posttranscriptional silencing of the PEX12 gene in RNAi experiments caused the accumulation of fewer peroxisomes that were significantly larger than those found in worms injected with control dsRNA (compare Fig. 4, K and L). Moreover, whereas peroxisomes in worms injected with control dsRNA were round in shape, of uniform size, and randomly distributed in the organism, the injection of dsRNA to P-44 caused the accumulation of clusters of peroxisomes that were fewer in number, irregular in shape, and of variable size, with most peroxisomes being larger than those found in worms injected with control dsRNA (Fig. 4M). A similar phenomenon has been observed in human, mammalian, and yeast mutant cells deficient in other enzymes of ß-oxidation. In these mutant cells, loss of the enzymatic activity of acyl-CoA oxidase (5, 11, 67), fatty acyl-CoA synthetase (67), and/or another peroxisomal ß-oxidation enzyme, 2-enoyl-CoA hydratase/ D-3-hydroxyacyl-CoA dehydrogenase (5, 49), resulted in significant changes in peroxisome size and/or number. The primary targets for this so-called metabolic control of peroxisome abundance (5) are likely the levels of other peroxisomal ß-oxidation enzymes that are dramatically increased by the loss of acyl-CoA oxidase (11) or 2-enoyl-CoA hydratase/ D-3-hydroxyacyl-CoA dehydrogenase (49) enzymatic activity. The resultant overproduction of abundant matrix proteins leads to a significant change in peroxisome size (5, 49, 67) and/or number (11, 49).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Injection of dsRNAs specific to the ORFs encoding the three C. elegans orthologs of the membrane-embedded ABC half-transporters, ALDP and ALDRP, caused a substantial delay in the development of the nematode. ALDP has been implicated in the transport of VLCFA-CoAs across the peroxisomal membrane (71), and a similar role has been suggested for other ABC half-transporters (10). A defect in ALDP in humans causes developmental delay and severe neurological deficits and leads to the most common peroxisomal disease, X-ALD (46, 70, 71). Another peroxisome-associated metabolic process, which is essential for the normal development of C. elegans, involves an auxiliary metabolic pathway that allows polyunsaturated fatty acids to enter the normal ß-oxidation spiral. In fact, injection of dsRNA specific to the ORF encoding the first enzyme in this auxiliary pathway, 3,5-
2,4-dienoyl-CoA isomerase, caused a substantial delay in development of the nematode. In humans, an inborn error of the auxiliary pathway for the ß-oxidation of fatty acids causes a peroxisomal disorder (45). The third peroxisomal metabolic process, which, according to the data of RNAi analysis presented here (Fig. 2) and elsewhere by Motley and coworkers (39), is required for the normal development of C. elegans, involves the biosynthesis of plasmalogen. RNAi experiments with dsRNA to ADHAPS, which catalyzes the second step in plasmalogen biosynthesis, showed a substantial delay in the development of the nematode. Likewise, in humans, inborn defects in ADHAPS cause abnormal psychomotor development and result in a lethal peroxisomal disorder, RCDP type 3 (7, 9).
Surprisingly, the phenotypes of worms treated with dsRNA to inactivate the processes of acyl-CoA synthetase-mediated activation of fatty acids, - and ß-oxidation of fatty acids, and intraperoxisomal decomposition of harmful hydrogen peroxide were close to that of control worms (Fig. 6). These results may suggest that, unlike in humans or yeast, these biochemical functions of peroxisomes may not be of major importance for the development of C. elegans or are duplicated by cytosolic (e.g., the cytosolic form of catalase) or mitochondrial (e.g., mitochondrial forms of thiolase) orthologs. However, as with all RNAi experiments, there is the strong possibility that posttranscriptional silencing may not have reduced the expression of some genes below a threshold necessary to display a phenotype. Immunoblot analysis with antibodies specific to the carboxyl terminus of type 2 thiolase P-44 demonstrated that the level of this protein was decreased more than 10-fold following injection of dsRNA specific for this gene (Fig. 3). Nevertheless, the observed substantial delay in nematode development caused by reductions in the activities of the ABC half-transporters, an auxiliary reaction for the ß-oxidation of polyunsaturated fatty acids, and the biosynthesis of plasmalogen is specific; that is, it is not due to a general defect in the overall metabolic functioning of peroxisomes.
|
C. elegans as a Model System for the Human Peroxisomal Disorders
This study shows that C. elegans can be considered as a valuable model system with which to study the molecular defects underlying the human peroxisomal disorders and to tailor therapeutics for their treatment. In fact, the developmental delay in worms injected with dsRNAs to the ABC half-transporters, 3,5-
2,4-dienoyl-CoA isomerase and ADHAPS, mimics the global developmental delay seen in patients suffering from X-ALD (46, 70, 71), a deficiency in the auxiliary pathway for the ß-oxidation of polyunsaturated fatty acids (45), and RCDP type 3 (7, 9), respectively. Furthermore, the developmental delay observed in Zellweger spectrum patients afflicted with ZS, NALD, and IRD is due to defects in the peroxins Pex5p, Pex12p, Pex13p, and Pex19p (13, 17, 55). Likewise, this study shows that injection of C. elegans with dsRNAs to Pex5p-, Pex12p-, Pex13p-, and Pex19p caused a substantial delay in the development of the nematode.
Although the clinical phenotypes of the various peroxisomal disorders (38) and some aspects of peroxisome biogenesis in cultured fibroblasts of PBD patients (17) are well studied, the precise physiological role of peroxisomes in the normal development of a multicellular organism remains largely unknown. From this perspective, C. elegans provides several important advantages as a model system. First, the nematode is a genetically tractable organism with a sequenced genome and a well-studied lifecycle (2, 25). Second, C. elegans has a relatively simple multicellular organization (24, 31). The pedigrees of cells have been established from egg to adult organism (24, 26). Moreover, the transparent cuticle of the worm permits visualization of all cells, fluorescently tagged proteins and vital dyes within the live worm. Third, since the cell lineage, location, and synaptic connectivity of the C. elegans nervous system have been completely described (2, 59), C. elegans should prove extremely useful to analyze the molecular basis underlying the impaired neuronal development and neurodegeneration observed in patients with peroxisomal disorders (46, 68, 70, 71). Last, although studies with cultured human fibroblasts have opened up the option of pre- or postnatal diagnosis of peroxisomal disorders (69), they have not yet provided any prospect of therapy for these disorders. On the other hand, C. elegans has been extensively used for the testing of various pharmaceutical agents and for drug discovery (44, 59). Therefore, generation of C. elegans knockout mutants carrying deletions in genes that are described in this paper is the next step for unveiling the molecular causes of human peroxisomal disorders and for high-throughput screening of pharmaceuticals for their treatment.
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported by grants from the Canadian Institutes of Health Research (CIHR) to R. A. Rachubinski. R. A. Rachubinski is a CIHR Senior Investigator, Canada Research Chair in Cell Biology, and an International Research Scholar of the Howard Hughes Medical Institute.
Present address for V. I. Titorenko: Dept. of Biology, Concordia Univ., 1455 de Maisonneuve Blvd. W., Montreal, Quebec, Canada H3G 1M8.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: R. A. Rachubinski, Dept. of Cell Biology, Univ. of Alberta, Medical Sciences Bldg. 5-14, Edmonton, Alberta, Canada T6G 2H7 (E-mail: rick.rachubinski{at}ualberta.ca).
10.1152/physiolgenomics.00044.2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|