Brain Advance Access originally published online on May 4, 2005
Brain 2005 128(7):1686-1706; doi:10.1093/brain/awh503
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Mutant SOD1 alters the motor neuronal transcriptome: implications for familial ALS
1 Academic Neurology Unit, University of Sheffield, School of Medicine and Biomedical Sciences, Sheffield and 2 Genome Instability Group, Department of Cancer Studies and Molecular Medicine, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester, UK
Correspondence: Professor Pamela J. Shaw, Academic Neurology Unit, University of Sheffield, Medical School, Beech Hill Road, Sheffield S10 2RX, UK E-mail: Pamela.Shaw{at}sheffield.ac.uk
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Familial amyotrophic lateral sclerosis (FALS) is caused, in 20% of cases, by mutations in the Cu/Zn superoxide dismutase gene (SOD1). Although motor neuron injury occurs through a toxic gain of function, the precise mechanism(s) remains unclear. Using an established NSC34 cellular model for SOD1-associated FALS, we investigated the effects of mutant SOD1 specifically in cells modelling the vulnerable cell population, the motor neurons, without contamination from non-neuronal cells present in CNS. Using gene expression profiling, 268 transcripts were differentially expressed in the presence of mutant human G93A SOD1. Of these, 197 were decreased, demonstrating that the presence of mutant SOD1 leads to a marked degree of transcriptional repression. Amongst these were a group of antioxidant response element (ARE) genes encoding phase II detoxifying enzymes and antioxidant response proteins (so-called ‘programmed cell life’ genes), the expression of which is regulated by the transcription factor NRF2. We provide evidence that dysregulation of Nrf2 and the ARE, coupled with reduced pentose phosphate pathway activity and decreased generation of NADPH, represent significant and hitherto unrecognized components of the toxic gain of function of mutant SOD1. Other genes of interest significantly altered in the presence of mutant SOD1 include several previously implicated in neurodegeneration, as well as genes involved in protein degradation, the immune response, cell death/survival and the heat shock response. Preliminary studies on isolated motor neurons from SOD1-associated motor neuron disease cases suggest key genes are also differently expressed in the human disease.
Key Words: amyotrophic lateral sclerosis; Nrf2; programmed cell life genes; SOD1
Abbreviations:
Actb = ß-actin; Akr1c13 = aldo-keto reductase family 1, member 13; ALS = amyotrophic lateral sclerosis; AP1 = activator protein 1; ARE = antioxidant response element; Bag3 = Bcl2-associated athanogene 3; Bnip3 = E1B 19 kDa/Bcl2 binding protein Nip3; B2m = ß2-microglobulin; Ccl2 = chemokine (C-C motif) ligand 2; Cox4A = cytochrome c oxidase subunit 4; Ddc = dopa decarboxylase; Erk = extracellular signal-regulated kinase; c-Fos = FBJ osteosarcoma oncogene; Gadd45a = growth arrest and DNA damage inducible 45 a; Gsn = gelsolin; GST = glutathione S-transferase; Gsta3 = glutathione S-transferase alpha 3; Gstm1/2 = glutathione S-transferase mu 1/2; G6pd = glucose-6-phosphate dehydrogenase; Hspa1b/4 = heat shock protein 1b/4; Idb2 = inhibitor of DNA binding 2; Jun = v-jun avian sarcoma virus 17 oncogene homologue; Lmp7 = 20s proteasome ß5 inducible subunit; Ltb4dh = leukotriene B4 12-hydroxydehydrogenase; c-Myc = myelocytomatosis oncogene; ß-NF = ß-napthoflavone; Nrf2 = nuclear factor erythroid 2-like 2; PA28
/ß = proteasome activator 28 /ß subunits; PA200 = proteasome activator 200 kDa; Pdcd6ip = programmed cell death 6 interacting protein; PDTC = pyrrolidinedithiocarbamate; 6Pgd = 6-phosphogluconate dehydrogenase; Prdx3 = peroxiredoxin 3; Prdx4 = peroxiredoxin 4; Rgs2 = regulator of G-protein signalling 2; Scg2 = secretogranin II; Smn = survival motor neuron; SOD1 = Cu/Zn superoxide dismutase; S100a6 = calcyclin; t-BHQ = t-butylhydroquinone; Vegf = vascular endothelial growth factor
Received May 26, 2004. Revised March 9, 2005. Accepted March 15, 2005.
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Introduction |
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Amyotrophic lateral sclerosis (ALS) is one of the most common adult onset neurodegenerative diseases, caused by progressive degeneration of the upper and lower motor neurons in the motor cortex, brainstem and spinal cord. Current evidence suggests that multiple interacting factors contribute to motor neuron injury in ALS. The four key pathogenetic hypotheses comprise genetic factors (Hand and Rouleau, 2002
), oxidative stress (Cookson and Shaw, 1999), glutamatergic toxicity (Heath and Shaw, 2002) and protein misfolding/aggregation (Wood et al., 2003). ALS is sporadic in 90–95% and familial in 5–10% of cases. Mutations in the ubiquitiously expressed free radical scavenging enzyme, Cu/Zn superoxide dismutase (SOD1), are causative in 20% of familial cases (Rosen et al., 1993) and current insights into the pathogenesis of ALS have arisen predominantly from studying the effects of SOD1 mutations. Mutant SOD1 produces motor neuron injury by a toxic gain of function and although the exact mechanism of action is unclear, several hypotheses exist, including aberrant free radical handling, abnormal protein aggregation and increased susceptibility to excitotoxicity (Hand and Rouleau, 2002).
The availability of powerful genomics technologies provides the opportunity to unravel complex regulatory and interactive pathways that govern neuronal phenotype and changes in disease states. Several recent studies have attempted to investigate differential gene expression in relation to motor neuron degeneration. One study of RNA extracted from whole spinal cord homogenates from G93A mutant SOD1 mice used Affymetrix Gene Chips to monitor differential gene expression (Olsen et al., 2001), whilst a similar study used cDNA membrane arrays (Yoshihara et al., 2002). Pooled RNA extracts from whole human spinal cord have also been used to analyse gene expression changes in ALS cases compared with controls (Malaspina et al., 2001). While these studies confirmed that microglial and astrocytic activation, and a neuro-inflammatory response all occur in SOD1-related motor neuron injury, their limitation is that they have not profiled gene expression specifically in the vulnerable cell population: the motor neurons. Since motor neurons are only a minority cell population within the spinal cord, any changes in gene expression will have been diluted and potentially masked by changes occurring in other cell types.
We have previously established a robust cellular model to examine the molecular pathophysiology of motor neuron injury associated with mutations in SOD1. NSC34 cells, which are a hybrid mouse motor neuron/neuroblastoma cell line, retain the ability to proliferate whilst exhibiting many motor neuron characteristics (Cashman et al., 1992; Durham et al., 1993). NSC34 cells have been stably transfected with one of several mutant forms of human SOD1, wild-type SOD1 or vector only and single cell clones derived by limiting dilution (Menzies et al., 2002a). This cell line provides a good model to investigate the effects of mutant SOD1 specifically in cells with a motor neuronal phenotype. Using this model we have previously demonstrated several important insights into the toxicity produced by the mutant SOD1 protein, including: (i) biochemical changes reflecting an increased tendency to apoptosis with increased expression of cleaved caspase 9 and annexin V staining on the cell surface under basal culture conditions (Cookson et al., 2002; Sathasivam et al., 2004); (ii) the development of morphologically abnormal mitochondria, with impaired activity of complexes II and IV of the respiratory chain and impaired cellular bioenergetic status (Menzies et al., 2002a); and (iii) alterations in the cytosolic proteome with specific changes in expression and function of proteins involved in nitric oxide metabolism, antioxidant defence and protein folding and degradation (Allen et al., 2003). The likely importance of these changes is reinforced by studies of apoptotic pathways, mitochondrial function and expression of cellular proteins in murine SOD1 transgenic models and in human CNS tissue (Wong et al., 1995; Martin, 1999; Phul et al., 2000).
In the present study we aimed to elucidate further the pathophysiological alterations induced in motor neurons by the presence of mutant SOD1 protein, using gene expression profiling. Although previously we have used cDNA membrane arrays to screen 588 genes for changes in gene expression (Kirby et al., 2002), by employing the Affymetrix microarray system with the U74Av2 Murine GeneChip we can perform simultaneous analysis of 6000 well characterized genes and a further 6000 expressed sequence tag (EST) transcripts. This provides a more powerful screening protocol with the potential to identify not only individual genes that are altered in response to mutant SOD1, but also specific intracellular pathways. We wished initially to examine changes occurring solely within motor neuronal cells both uncontaminated by the presence of other cell types and without the effects of interactions between the cells, while accepting the role that non-neuronal astrocytes and microglia may play in the generation and/or propagation of motor neuron injury (Pramatarova et al., 2001; Lino et al., 2002).
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Reagents
Tissue culture reagents were purchased from Invitrogen, as were reagents for DNase treatment of RNA and cDNA synthesis. SYBR Green PCR Master Mix was obtained from Applied Biosystems, PCR primers from MWG Biotech and assay substrates from Sigma. Reagents used for the microarray experiments were those recommended by Affymetrix. The rabbit anti-mouse nuclear factor erythroid 2-like 2 (NRF2) polylclonal antibody was a gift from Professor John Hayes, University of Dundee, the rabbit anti-human glucose-6-phosphate dehydrogenase (G6PD) polyclonal antibody was from Jose Bautista, University of Madrid and the rabbit anti-peroxiredoxin 3 (PRDX3) polyclonal antibody was from Professor Chi Dang, John Hopkins University School of Medicine. The sheep anti-mouse SOD1 polyclonal antibody was purchased from Calbiochem, the mouse anti-cytochrome c oxidase subunit 4 (COX4A) monoclonal antibody from Molecular Probes, mouse anti-survival motor neuron (SMN) antibody from BD Transduction Laboratories and mouse anti-ß-actin (ACTB) monoclonal antibody from Sigma (clone AC-40). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Dako, except for the anti-sheep HRP, which was purchased from Sigma.
Cell culture and RNA preparation
The NSC34 cell lines stably transfected with pCEP4 (pCEP), normal human SOD1 (pCN) or G93A mutant human SOD1 (pC93) have been described previously (Menzies et al., 2002a). Three replicate sets of cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS) to 80% confluency before being harvested. Following a wash in Hank's buffered saline solution (HBSS), the cells were resuspended in RNAlater (Ambion), according to the manufacturer's instructions. Total RNA was isolated from 5–10 million NSC34 cells using TRI Reagent (Sigma), following the manufacturer's instructions. Following quantitation, 4 µg total RNA was run on a formaldehyde denaturing agarose gel to assess the RNA quality.
Microarray hybridization and analysis
The mRNA fraction of the total RNA was converted to cDNA by reverse transcription (RT) using the SuperScript kit (Invitrogen) in combination with a T7-(dT)24 oligomer [5'-GCCAGTGAATTGTAATCCGACTCACTATAGGGAGGCGG-(dT)24-3'] (Genset Oligos), according to the manufacturer's instructions. The cDNA was used as template for T7 RNA polymerase in vitro transcription (IVT), using the BioArray High Yield RNA Transcript Labelling Kit (Enzo) to incorporate fluorescent label, according to the manufacturer's protocol. cRNA (10 µg) was applied to the GeneChip U74Av2A array, according to Affymetrix protocols. This contains 6000 functionally characterized genes and a further 6000 EST clusters. Three chips were used for each of the triplicate NSC34 cell lines (pCEP, pCN and pC93). Array washing and staining was performed in the Fluidics Station 400 according to the Affymetrix protocol. Arrays were scanned twice using the GeneArray laser scanner (Agilent Technologies). The Microarray Analysis Suite (MAS v5.0) software (Affymetrix) was used to monitor scanning and to convert the raw image file into a cell intensity file (‘.CEL’).
Data analysis
Quality control of each chip was carried out by confirming the detection of spiked controls, low ratios of actin and GAPDH signals to ensure detection of full length transcripts (mean actin 3–5' ratio 1.101 ± 0.201; Gapdh 3–5' ratio 1.001 ± 0.142) and that scaling factor (mean 1.671 ± 0.514), RawQ (mean 3.754 ± 0.756), average background (mean 113.5 ± 39.28) and percentage of genes expressed (mean 36.57 ± 2.333), were similar for each chip.
The nine .CEL files generated by MAS 5.0 were converted into ‘.DCP’ files using dCHIP version 1.2 (www.dCHIP.org) (Li and Hung Wong, 2001). The .DCP files were normalized, and the raw gene expression data generated was then normalized using the dCHIP system of model-based analysis (PM-MM). Comparative analysis of global gene expression profiles was performed using the dCHIP software. The three replicate murine NSC34 cell lines transfected with the empty vector (pCEP) were designated as ‘baseline’ (B), and the three murine NSC34 cell lines transfected with the normal human SOD1 (pCN) and the 3 murine NSC34 cell lines transfected with mutant human SOD1 (pC93) were designated as ‘experiment’ (E). Genes differentially expressed two-fold or higher in the two cell lines transfected with human normal and mutant SOD1 versus control vector alone were then identified by defining the appropriate filtering criteria in the dCHIP software (E/B-2; E-B > 50, B-E > 50).
Genes were grouped functionally using the Gene Ontology (GO) system available through NetAffx (www.affymetrix.com/analysis/index.affx), taking into consideration the biological process, cellular component and molecular function listed for each gene.
Quantitative RT–PCR
Three sets of NSC34 cells (pCEP, pCN and pC93) were harvested when flasks reached 80% confluency, with each set from a separate passage. As an additional control for the specificity of the gene changes, a further set of cells containing the G37R mutation were also screened. Following a wash in HBSS, RNA was extracted using TRIzol (Invitrogen), according to the manufacturer's instructions. RNA (2 µg) was DNase I treated and the sample divided into two. cDNA synthesis was performed, with and without Superscript II reverse transcriptase, following the manufacturer's protocol. Primers were designed to genes in key pathways to confirm their differential expression, and where possible, crossed intron/exon boundaries. Primer concentrations were optimized using 0.5 µl pCEP cDNA. Quantitative RT–PCR (Q-PCR) was performed using 0.5 µl cDNA, 1x SYBR Green PCR Master Mix (Applied Biosystems) and optimized concentrations of forward and reverse primers, to a total volume of 20 µl. Primer sequences and optimized final primer concentrations are given in the figure legends. Following an initial denaturation of 95°C for 10 min, products were amplified by 40 cycles of 95°C for 15 s and 60°C for 1 min, on an MX3000P Real Time PCR System (Stratagene). Finally, a dissociation curve was performed to ensure amplification of a single product and absence of primer dimers. For each of the genes, a standard curve using 1, 0.5, 0.25, 0.125 and 0.0625 µl of cDNA was carried out to determine the efficiency of the PCR was 100% ± 10%, such that values of pCEP, pCN, pC37 and pC93, normalized to Actb expression, could be determined using the ddCt calculation (SYBR Green PCR mix and RT–PCR protocol; Applied Biosystems). The levels of expression of each gene in pCN, pC37 and pC93 cells are shown as a percentage of the gene expression seen in pCEP control cells. Paired t-tests were used to analyse the data and determine the statistical significance of any differences in gene expression.
Isolation of human motor neurons and amplification of RNA
An Arcturus Pixcell II laser capture microdissector was used to isolate individual motor neurons from snap-frozen samples of spinal cord of two neuropathologically normal control subjects (one male and one female, mean age 50 ± 3 years, post mortem delay 16.5 ± 2.5 h) and two subjects with familial ALS, carrying an I113T mutation in the SOD1 gene (one male and one female, mean age 62 ± 2 years, post mortem delay 23.3 ± 3.5 h), as previously described (Heath et al., 2002). Following extraction of RNA using PicoPure RNA Isolation Kit (Arcturus), a double round of RNA amplification was performed using the RiboAmp RNA Amplification kit (Arcturus), according to the manufacturer's protocol. First strand cDNA synthesis was then performed on 1 µg of cRNA, and semi-quantitative RT–PCR was performed. Primers were designed to cross intron/exon boundaries. Aliquots of PCR product were removed at 25, 30, 35 and 40 cycles to ensure that densitometric analyses were carried out during the logorithmic range of amplification. Densitometry was carried out using the AlphaImager system and Alpha Innotech Spot Densitometry software (Alpha Innotech, San Leandro, CA, USA). Relative level of expression for each of the genes was determined by normalizing to levels of ACTB expression in each of the cDNA samples, and results combined to compare normals against FALS cases. Statistical analysis was not performed, as data were only available from two cases in each group.
Enzyme and metabolite assays
For all enzyme and metabolite assays, the NSC34 cells were grown for 96 h, harvested at 80% confluency, and S1 cytosolic fractions prepared as described previously (Allen et al., 2003). All statistical analyses were done using paired t-tests. G6PD assay reaction mixtures (Lee, 1982) consisted of 100 mM Tris/HCl pH 8.0 containing 1 mM glucose-6-phosphate and 1 mM NADP. 6-Phosphogluconate dehydrogenase (6PGD) assay reaction mixtures consisted of 100 mM Tris/HCl pH8.0 containing 1 mM 6-phosphogluconate and 1 mM NADP (Lee, 1982). Malic enzyme assay reaction mixtures consisted of 67 mM triethanolamine pH 7.4 containing 4 mM MnCl2·4H2O, 1 mM L-malate and 1 mM NADP (Hsu and Lardy, 1969). All reactions were initiated by adding 200 mg of NSC34 post-nuclear S1 protein per ml assay reaction (Allen et al., 2003). The increase in absorbance at 340 nm was measured at 1 min intervals for 5 min at room temperature.
Total intracellular NADP(H) concentrations were determined with modification to the method of Zerez et al. (1987). NSC34 cell monolayers previously seeded at a density of 3 x 105 cells per T75 flask and incubated at 37°C for 96 h were resuspended in PBS pH 7.4. The cells were centrifuged at 400 g for 5 min then resuspended in 1 ml per flask of an ice-cold solution of 10 mM nicotinamide, 20 mM NaHCO3 and 100 mM Na2CO3. The cells were immediately snap-frozen in liquid nitrogen, rapidly thawed in a room temperature water bath and then centrifuged at 13 000 g at 4°C for 5 min to obtain supernatants. Total NADP(H) assay reaction mixtures consisted of 100 mM Tris/HCl pH 8.0 containing 5 mM EDTA, 2 mM phenazine ethosulphate, 0.5 mM MTT, 1.3 U/ml G6PD, 1 mM glucose-6-phosphate and either 20–200 nM standard NADP or 100 ml lysate supernatant per ml assay reaction. Reactions were initiated by addition of glucose-6-phosphate and the increase in absorbance at 570 nm was measured as above.
Protein extraction and western blotting
Total cellular protein and the S1 cytosolic fraction were prepared according to protocols described previously (Cookson et al., 1998; Allen et al., 2003). Ten micrograms of protein was run on 14% SDS–PAGE gels, and electroblotted onto Immobilon-P membranes. Membranes were then blocked in TBS-Tween [20 mM Tris–HCl pH 7.6, 137 mM NaCl, 0.1% (v/v) Tween-20] with 5% (w/v) dried skimmed milk. The membranes were probed either with 1 in 5000 dilution of sheep anti-mouse polyclonal SOD1, 1 in 4000 dilution of rabbit anti-mouse polyclonal NRF2, 1 in 500 of rabbit anti-human G6PD polyclonal or 1 in 10 000 of mouse anti-human SMN, followed by peroxide-conjugated secondary antibody of 1 in 5000 for anti-sheep antibody, 1 in 2000 for anti-rabbit antibodies and 1 in 1000 for the anti-mouse antibody. To control for variation in protein concentration of the samples, membranes were also probed with 1 in 2000 of mouse anti-ACTB monoclonal, followed by 1 in 1000 of secondary antibody of rabbit anti-mouse.
The mitochondrially enriched preparations of NSC34 cell lines were generated by differential centrifugation as previously described (Menzies et al., 2002a). Ten micrograms of protein was run on 14% SDS–PAGE gels, and electroblotted onto membranes. These were probed with 1 in 1000 dilution of rabbit anti-PRDX3 and as a control, 1 in 10 000 of mouse anti-COX4A. Both secondary antibodies were used at 1 in 1000.
All proteins were visualized using ECL western blotting detection reagents kit (Amersham), and densitometry carried out using the AlphaImager system and Alpha Innotech Spot Densitometry software (Alpha Innotech). Following normalization to either ACTB for cytosolic and COX4A for mitochondrial fractions, protein levels of each gene in pC37 and pC93 cells were calculated as a percentage of the gene expression seen in pCEP control cells. Paired t-tests were used to analyse the data and determine the statistical significance of any differences in gene expression.
Pharmacological manipulation of antioxidant response element (ARE)-driven gene expression
NSC34 cells expressing human G93A SOD1 (pC93) or vector only (pCEP) were seeded into T175 flasks at a density of 6 x 105 cells and then grown for 96 h. The cells were then incubated for 24 h with fresh medium containing either 10 mM pyrrolidinedithiocarbamate (PDTC), 10 mM t-butylhydroquinone (t-BHQ), 10 mM ß-naphthoflavone (ß-NF), 15 mM sulforaphane or DMSO vehicle solvent only at a 1:1000 dilution. S1 cytosolic fractions were then prepared as described previously and relative specific activities for both G6PD and total glutathione S-transferase (GST) were determined as above.
For MTT assays, NSC34 cells were seeded into 96-well plates at a density of 2000 cells/cm2 and grown for 5 days in DMEM + 10% FCS. Medium was replaced with fresh DMEM ± serum ± PDTC (1–10 mM) for 48 h. MTT assays were performed as described previously (Cookson et al., 1998). Statistical analysis was performed using the Wilcoxon matched pairs test.
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Results |
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Microarray analysis
The transcription profiles of the motor neuron-like NSC34 cell line transfected with vector, normal human SOD1 or mutant G93A SOD1 were generated using the murine GeneChip U74Av2. Between 32% and 39% of the 12 000 transcripts represented on the GeneChip were detected as present in the three groups of NSC34 cells. To identify genes differentially expressed in the presence of the mutant SOD1 protein, comparisons were initially made between the transcription profiles obtained from the vector and the mutant SOD1 protein transfected cell lines using the analysis software dChip version 1.2 (Li and Hung Wong, 2001
). Transcripts that showed an increase/decrease of at least two-fold, and a difference in signal intensity between the baseline and experimental arrays of at least 50 units of fluorescence were selected for further evaluation. Comparisons between the vector and the normal SOD1 protein transfected cell line transcription profiles were also obtained and transcripts identified according to the same selection criteria. The analyses identified 268 transcripts that were consistently differentially expressed in the presence of G93A mutant SOD1 protein: 197 transcripts were decreased and 71 transcripts were increased (Tables 1 and 2). Thus, 268 of the 12 000 transcripts expressed on the array (2.2%), or 6–7% of genes expressed within the NSC34 cells, are differentially expressed in the presence of mutant SOD1. The genes were categorized according to their molecular function, and included genes involved in antioxidant and stress responses, apoptosis, immunity and protein degradation. Unlike previous studies, where heterogeneous cell populations were used, there were a far greater number of genes showing decreases in expression than increases. It appears that expression of mutant SOD1 within motor neuronal cells leads to a marked degree of transcriptional repression.
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In contrast to the numerous changes observed in the presence of mutant SOD1, only 16 transcripts were differentially expressed specifically in the presence of normal SOD1, 10 increased and six decreased (Table 3).
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Expression levels of endogenous mouse and transfected human SOD1 in the NSC34 cell lines
Q-PCR demonstrated that there was no significant change in the gene expression level of endogenous murine SOD1 in the four groups of cell lines (Fig. 1A). Western blotting demonstrated similar levels of murine SOD1 protein in all the cell lines, and that normal human SOD1 or the G37R and G93A mutant forms of the SOD1 protein were expressed at comparable levels (Fig. 1B). The relative expression levels of human SOD1 to endogenous murine SOD1 are comparable to the levels of mutant SOD1 expression expected in the human disease.
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Verification of microarray data by RT–PCR, western blotting and functional assays
Out of 268 genes diffentially expressed in the presence of mutant G93A SOD1, 23 genes were selected for verification based upon several criteria: (i) presence in a pathway of specific interest, particularly those related to the antioxidant response; (ii) a potential involvement in ALS or neurodegeneration; and (iii) a high fold change. Initially, semi-quantitative RT–PCR was used to verify the changes in the NSC34 cell lines (data not shown) and in isolated motor neurons from human SOD1-associated cases (see below). This was followed up by Q-PCR, western blotting and functional assays in the NSC34 cell lines to validate the changes observed by microarray analysis, and to determine whether the alterations correlated with protein and enzyme activity levels. The additional G37R SOD1 mutant-containing cell line was also included in the verification studies to provide further confirmation of the importance of key genes in SOD1-mediated neurodegeneration. A further five of the differentially expressed genes had previously been identified as altered in the presence of mutant SOD1 using proteomic approaches [leukotriene B4 12-hydroxydehydrogenase (Ltb4dh), 20s proteasome beta 5 inducible subunit (Lmp7), glutathione S-transferase mu 1 (Gstm1), arginosuccinate synthase (Ass1), neuronal nitric oxide synthase (nNos)]. These have been validated in the NSC34 cell line and human SOD1-associated familial ALS cases (Allen et al., 2003
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Antioxidant response proteins
Oxidative stress is known to play a role in ALS (Cookson and Shaw, 1999), and cellular oxidative stress results in the binding of the transcription factor NRF2 to ARE, located in the promoters of phase II detoxifying enzymes (Chan et al., 1996). In the presence of mutant SOD1, an unexpected three-fold decrease in the level of Nrf2 was detected. This was associated with decreases in multiple downstream phase II detoxifying enzymes and antioxidant enzymes, including Gst enzymes [Gstm1, 7.5-fold; Gstm2, two-fold; Gst omega 1 (Gsto1), three-fold; Gst alpha 3, (Gsta3) four-fold], a 2.4-fold decrease in G6pdx, a three-fold decrease in aldoketoreductase family 1, member C13 (Akr1c13) and a 125-fold decrease of Ltb4dh.
Previous work has suggested that the ARE may be negatively regulated by the activator protein 1 (AP1) component FBJ osteosarcoma oncogene (c-FOS) (Venugopal and Jaiswal, 1996; Wilkinson et al., 1998), which was increased over three-fold in the presence of mutant SOD1. Extracellular signal-regulated kinase (ERK), also known as mitogen-activated protein kinase 3 (MAPK3), reportedly phosphorylates both NRF2, allowing its translocation into the nucleus, and myelocytomatosis oncogene (MYC) transcription factor (Owuor and Kong, 2002). MYC induces many genes including the peroxiredoxins (Prdx) (Wonsey et al., 2002). Both Erk1 and c-Myc were decreased, two- and three-fold, respectively, in the presence of mutant SOD1, as well as the MYC target genes Prdx3 and Prdx4, which decreased four- and two-fold, respectively.
To confirm the differential expression of specific genes involved in the antioxidant response element, Q-PCR was carried out on RNA isolated from the four groups of NSC34 cells: pCEP, pCN, pC37 and pC93. Significant decreases in expression were detected in Nrf2, Gsta3, G6pdx, Akr1c13 and Prdx4, in both mutant containing cell lines compared with vector-only transfected cells (Fig. 2A and B; P values given in figure legends). Significant decreases were also detected in Prdx3 and C-myc along with a significant increase in c-Fos, in the presence of mutant G93A SOD1 compared with vector-only transfected cells, although these changes were not significant in the G37R SOD1 mutant cell line (Fig. 2B). Western blotting was used to investigate the protein levels of the key gene products NRF2, G6PD and PRDX3. Analysis of total cell lysate clearly showed a significant decrease in protein levels of NRF2 and G6PD, in the presence of both mutant G93A and G37R SOD1, compared with both vector only and normal human SOD1-transfected cells (Fig. 3A–C). Since PRDX3 is a mitochondrial protein, a mitochondrial enriched fraction of cell extract was used for western blotting. The results show a low level of PRDX3 protein in both the G37R and G93A SOD1 mutant-containing cells compared with vector-only transfected cells. Whilst this decrease is only significant for the pC93 cell, both mutants are significantly decreased compared with the normal SOD1-transfected cells (Fig. 3D and E). In addition, previous work with this cell model in our laboratory identified decreased protein and mRNA levels of GSTM1 and LTB4DH in the presence of mutant SOD1 (Allen et al., 2003).
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Enzyme activity of NADPH generators and levels of NADPH
G6PD is an NADPH generator, which is required as a cofactor to provide reducing power in numerous enzymatic reactions, and the decrease in G6pdx expression in the presence of mutant SOD1 is reflected in the protein level. 6PGD is also an NADPH generator, and was also found by the microarray analysis to be decreased in the presence of G93A mutant SOD1. To investigate the effect of decreased levels of G6pdx and 6Pgd on enzymatic activity and NADPH levels, functional assays were carried out. Using cytosolic extracts, G6PD and 6PGD activities were significantly decreased to 40% and 60%, respectively, in the presence of G93A mutant SOD1, and to 66% and 76%, respectively, in the presence of G37R mutant SOD1, compared with vector-only transfected cells (Fig. 4A). Total NADP(H) levels were also reduced by 25% in the both mutant transfected cell lines (Fig. 4B). However, the activity of malic enzyme, another NADPH generator, was identical in all three cell lines (Fig. 4A). Although there was no change in gene expression of enzymes requiring NADPH, [e.g. glutathione reductase (Gsr); thioredoxin reductase (Txnrd)], there was a decrease in glutathione peroxidase 4 (Gpx4) (2.3-fold), and the peroxiredoxins Prdx3 (4.8-fold) and Prdx4 (2.4-fold), which use the reduced forms of glutathione and thioredoxin synthesized by glutathione reductase and thioredoxin reductase, respectively. There was also a decrease in several of the Gst enzymes (two- to seven-fold), which eliminate compounds to which reduced glutathione has been attached. Glutathione is synthesized from cysteine and glutamate, and the glutamate is transported into the cell by solute carrier 1, member 4 (Slc1a4). This gene also showed a two-fold decrease in expression in the presence of mutant SOD1.
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Genes previously implicated in ALS and neurodegeneration
Several genes which have previously been implicated in ALS or neurodegeneration were identified as being altered in our cellular model of SOD1 associated ALS. Following deletion of the hypoxia response element in the promoter of the vascular endothelial growth factor (Vegf), transgenic mice developed a late onset, progressive degeneration of the motor neurons in the spinal cord and brainstem (Oosthuyse et al., 2001
). Vegf was increased two-fold in the presence of mutant G93A SOD1, whilst the calcyclin or S100 calcium binding protein A6 (S100a6), which is overexpressed in reactive astrocytes of G93A SOD1 transgenic mice (Hoyaux et al., 2000), is increased 10-fold. Whilst the increase in S100a6 was confirmed by Q-PCR in the presence of both mutations, the changes in VEGF were not (Fig. 5A and B). The Smn gene, mutations in which cause spinal muscular atrophy (Lefebvre et al., 1995), was decreased by two-fold in the presence of mutant G93A SOD1, and this was confirmed at the protein level in both mutant cell lines by western blotting (Fig. 3F).
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Genes altered in other pathways of interest
Other genes of interest that were altered in the presence of G93A SOD1 include those involved in protein degradation, immunity and apoptosis, cell survival, and cell death. Involvement of the proteasome is implicated by the presence of protein aggregates in neuronal cell bodies. The 20s proteasome associates with regulatory proteins that function as proteasome activators, such as the ubiquitin-independent proteasome activator 28 (PA28) complex and the proteasome activator 200 kDa (PA200). The genes encoding both PA28 subunits (PA28
and PA28ß) and PA200 are decreased two-fold in the presence of mutant SOD1, as is the inducible 20s proteasomal subunit Lmp7. LMP7 was previously found to be decreased in the cytosolic proteome of this model (Allen et al., 2003). A large number of MHC encoded genes involved in antigen presentation were decreased in the presence of mutant SOD1. These included the MHC class I heavy chain variants and ß2-microglobulin (B2m). B2m, which was decreased 7.7-fold in the presence of mutant G93A SOD1, was verified by Q-PCR, with both mutants showing significant decreases, compared with vector-only and normal human SOD1-transfected cells (Fig. 5B). Only three genes involved in apoptosis showed alterations in expression of more than two-fold: E1B 19 kDa/BCL2 binding protein Nip3 (Bnip3), a proapoptotic member of the BCL2 family and programmed cell death 6 interacting protein (Pdcd6ip) were increased two-fold. The anti-apoptotic protein, BCL2 associated athanogene 3 (Bag3), which enhances the activity of BCL2 and also interacts with HSC70/HSP70 proteins, was decreased two-fold, as were two heat shock protein (HSP) 70 protein encoding genes Hspa1b and Hspa4, decreased 22- and two-fold, respectively. The decrease in Hspa1b was confirmed in both mutants by Q-PCR (Figure 5B), although the increase in Bnip3 and decreased in Bag3 expression were not (Figure 5C). Another gene involved in cell survival which was decreased in the presence of mutant G93A SOD1 was growth arrest and DNA-damage inducible 45 a (Gadd45a), and the change was confirmed in both mutants by Q-PCR (Fig. 5C).
Genes showing highest fold changes
There were 15 genes the expression levels of which were markedly altered (>10-fold) in the presence of mutant SOD1, and 24 whose expression altered between five- and 10-fold. Of these genes, those which were located in pathways of specific interest were: (i) inhibitor of DNA binding 2 (Idb2) increased by eight-fold; (ii) chemokine (C-C motif) ligand 2 (Ccl2) decreased by 120-fold; (iii) regulator of G-protein signalling 2 (Rgs2), which was decreased by 12-fold; (iv) dopa decarboxylase (Ddc), decreased by seven-fold; (v) secretogranin II (Scg2) decreased by 130-fold; and (vi) gelsolin (Gsn) decreased by six-fold. All these changes were confirmed in both SOD1 mutant-containing cell lines by Q-PCR, except Ccl2 and Gsn, where the decreases in G37R SOD1 expressing cells were not significant compared with the vector-only transfected cells but were to the normal human SOD1-transfected cells (Figure 5A and D).
Expression in isolated human motor neurons from SOD1-associated ALS cases
To determine whether the changes observed in our cellular model were applicable to the human disease state, preliminary experiments were undertaken using semi-quantitative RT–PCR on motor neurons isolated from spinal cord sections of two familial cases carrying the I113T SOD1 mutation and two neuropathologically normal control cases, using laser capture microdissection. These initial studies suggest the differential expression of NRF2, B2M and VEGF is also present in the human disease (Figure 6). However, the significant decrease of SCG2 in the presence of mutant SOD1 was not supported. In addition, our previous work has shown significant decreases in the expression of GSTM1, LMP7 and LTB4DH compared with control motor neurons from human SOD1-related ALS cases (Allen et al., 2003).
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Reversal of mutant SOD1 down-regulation of antioxidant enzyme activity
Multiple studies have demonstrated that the expression of genes containing AREs can be promoted using small electrophilic compounds that activate transcription factors such as NRF2 and AP1. To investigate whether the mutant SOD1-dependent downregulation of antioxidant enzymes was reversible, NSC34 expressing either vector only or G93A SOD1 were treated with either 10 mM PDTC, 10 mM t-BHQ, 10 mM ß-NF or 15 mM sulforaphane, then post-nuclear supernatants prepared from the cells were assayed for G6PD and total GST activity. t-BHQ, ß-NF and sulforaphane had no effect upon antioxidant enzyme activity (data not shown). In contrast, PDTC was shown to significantly increase the G6PD and GST activities in cells expressing G93A SOD1 from 44% to 63% and 72% to 88%, respectively (Fig. 7A). Similarly, PDTC also increased G6PD and GST activities in cells expressing vector only from 100% to 123% and 100% to 125%, respectively.
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NSC34 cells expressing G93A SOD1 have previously been shown to be more susceptible than vector-only expressing cells to cell death following oxidative stress induced by serum withdrawal (Cookson et al., 2002
; Menzies et al., 2002a). In this model, PDTC is partly protective and can increase pC93 viability after 48 h serum withdrawal from 46 ± 2.4% to 54 ± 3.2% (Fig. 7B). |
Discussion |
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TopSummaryIntroductionMaterial and methodsResultsDiscussionNote added in proofReferences |
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Mutations in SOD1 were identified as causative for ALS in 1993 (Rosen et al., 1993
), and although there is substantial evidence that motor neuron degeneration occurs through a toxic gain of function of the mutant Cu/Zn SOD protein, the underlying pathophysiological mechanism is still unknown. In order to identify cellular pathways that are altered, specifically in motor neurons, in the presence of mutant SOD1, transcription profiles were obtained from our cellular model of SOD1-associated familial ALS. Comparison of transcription profiles from either mutant G93A SOD1 or normal SOD1 versus vector-only transfected cells resulted in the identification of 268 transcripts altered by more than two-fold in the presence of mutant SOD1; 197 transcripts were decreased and 71 transcripts increased. Although this is a large number of genes, it became apparent that distinct pathways were affected. The genes could be categorized into several groups including antioxidant response and related genes, protein degradation, immunity, apoptosis, and cell survival/cell death genes. There were also changes in several genes that have been previously implicated in neurodegeneration.
A striking feature of the gene expression profile within NSC34 motor neuronal cells in the presence of mutant SOD1 was the marked degree of transcriptional repression. This is in contrast to gene expression changes identified in whole spinal cord homogenates, where an increase in expression of genes reflecting reactive gliosis and inflammatory mechanisms have been observed, most likely arising from numerically dominant non-neuronal cells and interactions between the cell types (Malaspina et al., 2001; Olsen et al., 2001). Recent evidence has highlighted the potential importance of transcriptional repression in other neurodegenerative disorders including Huntington's disease (Zuccato et al., 2003).
The antioxidant response and related genes
NRF2 is a bZIP transcription factor that is a master regulator of ARE-driven gene expression, which includes phase II detoxification enzymes and antioxidant proteins, in a process that has been referred to as ‘programmed cell life’ (Lee et al., 2003b). NRF2 is post-translationally regulated by kelch-like ECH-asssociated protein 1 (KEAP1), a cytosolic actin binding protein localized to the cytoskeleton, which binds NRF2 within the cytosol under basal conditions. Oxidative stress causes the release of NRF2 followed by translocation to the nucleus, where it induces transcription by binding to the ARE sequences in the promoters of specific genes.
Recent studies applying microarray analysis to Nrf2–/– cellular models have identified genes that are either directly or indirectly transcriptionally regulated by NRF2. Although these studies vary with regard to the stimulus [sulforaphane (Thimmulappa et al., 2002); mitochondrial toxins (Lee et al., 2003b); t-BHQ (Lee et al., 2003a); 3H-1,2-dithiole-3-thione (Kwak et al., 2003)], or specific pathways involved [phosphatidylinositol-3 kinase dependent/independent (Li et al., 2002)], the expression of multiple genes, in addition to the phase II detoxifying enzymes, have been shown to be regulated by NRF2. Correlating these data with the results obtained in the present experiments, NRF2 is not only involved in the transcriptional regulation of Gsta3, Gstm1, Gstm2, G6pdx, 6Pgd, Akr1c13, Ltb4dh and Prdx3 (Fig. 8), but also Gadd45a, B2m, Rgs2, Ddc, Scg2 and Gsn, all of which were decreased in the presence of mutant SOD1 in motor neuronal cells.
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The level of Nrf2 expression was decreased whilst the AP1 complex component c-Fos was increased. It has been reported that FOS negatively regulates ARE-driven expression (Venugopal and Jaiswal, 1996
; Wilkinson et al., 1998), acting by heterodimerizing with another leucine zipper protein and binding the ARE. However, it has also been shown that AP1 activity is repressed during oxidative stress due to direct oxidation of specific cysteine residues in the v-jun avian sarcoma virus 17 oncogene homologue (JUN) and FOS proteins (Abate et al., 1990). Opposing regulatory effects of a single transcriptional factor are not unprecedented, for example regulation of urokinase is achieved by a heterodimer of JUN and ATF-2 positively regulating this gene, whilst a JUN and FOS heterodimer represses transcription (De Cesare et al., 1995). Further work investigating the regulation of ARE transcription is required to elucidate the role of FOS. However, since NRF2 plays a role in basal cellular redox homeostasis and in the mounting of a cellular cytoprotective response to oxidative insults, we suggest that dysregulation of the ‘programmed cell life’ response may represent a key component of the toxicity of mutant SOD1. This is supported by evidence for: (i) the presence of oxidative stress in cellular and animal models of ALS, as well as in human spinal cord tissue (Cookson and Shaw, 1999); (ii) the partial restoration of G6PD and GST activity of cells expressing mutant SOD1 to levels of those expressing normal SOD1, and increased cell viability following serum withdrawal, by PDTC, a compound known to promote binding of both NRF2 and AP-1 (Meyer et al., 1993; Wild et al., 1999); and (iii) the indication of decreased expression of NRF2 in motor neurons from SOD1-associated familial ALS cases. If chronic oxidative stress provides a mechanism to explain the toxicity of mutant SOD1, it also points to potential pharmacological and recombinant approaches aimed at reversing or preventing the deleterious effects of the mutant enzyme.
Dysregulation of the pentose phosphate pathway and NADPH synthesis
The significantly decreased expression and activities of the pentose phosphate pathway enzymes G6PD and 6PGD are likely to result in a significant lowering of the cell's ability to produce reduced NADPH, which is known to be crucial for the regeneration of the antioxidant capacity within the CNS. For example peroxiredoxin, responsible for eliminating hydrogen peroxide, is dependent upon reduced thioredoxin, which is regenerated by thioredoxin reductase at the expense of NADPH. Hydrogen peroxide is also removed by glutathione peroxide, which is dependent upon reduced glutathione, and this is regenerated by glutathione reductase, again at the expense of NADPH. In our cellular model, there is reduced expression and function of NADPH-generating enzymes, total NADP(H) levels are decreased, and, as previously reported, there are decreased levels of reduced glutathione in the presence of mutant SOD1 (Allen et al., 2003). Therefore, in the presence of mutant SOD1, oxidative stress is likely to arise from dysregulation of the pentose phosphate pathway, resulting in reduced availability of NADPH essential to maintain the major intracellular antioxidants glutathione and thioredoxin in their reduced states. Recent work supports a pivotal role for G6pd in the cellular response to oxidative stress (Filosa et al., 2003). Mouse embryonic stem cells carrying an exonic deletion in the G6pd gene, under conditions of oxidative stress, fail to upregulate the activity of the pentose phosphate pathway, resulting in lowered NADPH/NADP ratio, decreased reduced glutathione and ultimately cell death.
Our findings are supported by earlier studies which demonstrated that neurons, but not astrocytes, show mitochondrial dysfunction, glutathione oxidation, decreased NADPH levels and cell death, following glucose deprivation, an effect mediated by the superoxide ion (Almeida et al., 2002). These authors suggest that NADPH generated by the pentose-phosphate pathway prevents oxidative and mitochondrial damage during oxidative stress specifically in neuronal cells. Therefore, dysregulation of Nrf2 coupled with reduced pentose-phosphate activity and decreased generation of NADPH, may represent major and hitherto unrecognized components of the toxic gain of function of mutant SOD1.
Genes implicated in ALS and neurodegeneration
One of the most interesting changes in a gene implicated in ALS was the increase in Vegf expression, and although this was not confirmed by Q-PCR in the cellular model, the expression levels of the human gene were investigated during our preliminary studies of human SOD1 cases using semi-quantitative RT–PCR. These data suggested an increase is also present in the isolated human motor neurons from the two SOD1 cases, compared with neurologically normal controls. VEGF is neuroprotective in cultured primary neurons (Oosthuyse et al., 2001), and therefore, increases of Vegf may represent a neuroprotective cellular response. This effect of mutant SOD1 on Vegf was recently demonstrated in the G93A SOD1 transgenic mice. The basal level of Vegf expression was increased in the mutant SOD1 mice compared with litter mates, but following hypoxic stress, there was impaired Vegf up-regulation (Murakami et al., 2003).
Protein degradation
The 20s proteasome is the catalytic core of the proteasome complex and associates with regulatory proteins that function as proteasome activators. Activation of the ubiquitin-independent pathway is completed by the association of the PA28 complex, which is thought to be the mechanism by which oxidized proteins are degraded (Grune et al., 1997). However, heavily oxidized proteins become extensively crosslinked and aggregate such that they are poor substrates for degradation. In this report we describe a decrease in the expression of both PA28 activator subunits, PA200 and LMP7. We have previously described functional alterations in the proteasome activities in this cell model of ALS (Allen et al., 2003). These changes may impair the motor neuron's ability to remove oxidized proteins and may contribute to the formation of abnormal intracellular protein aggregates.
Genes associated with immunity
Although expression of MHC class I genes in the various subgroups of neurons in the healthy CNS is either absent or very low (Mucke and Oldstone, 1992), motor neurons of the spinal cord and brainstem exhibit significant expression of MHC class I (Linda et al., 1998). Cell surface expression of antigen presenting class I MHC is dependent upon the non-covalent association of B2M with the heavy chain and there are decreases in expression of the B2m and MHC class 1 heavy chain molecules in the presence of mutant SOD1. This corresponds with a previous study where motor neurons from ALS patients did not display any MHC class I or B2M immunoreactivity (Lampson et al., 1990). Our RT–PCR experiments suggest this may be due to reduced gene expression levels, as in both mutant SOD1-transfected cell lines, there was a significant decrease in B2m expression compared with controls, and the preliminary studies in isolated motor neurons also showed decreased B2M expression in the SOD1-associated cases.
In addition to the role LMP7 and PA28 and -ß subunit may play in forming protein aggregates, they are also involved in antigen presentation (Preckel et al., 1999). Previously, it was proposed that the decrease in LMP7 was a neuroprotective response, reducing the antigen presentation function of the proteasome (Allen et al., 2003). However, in the light of our current findings, the down-regulation of the immunoproteasome subunits may be part of a broader survival strategy to reduce the repertoire of MHC-restricted peptides, which could potentially increase both in quantity and variety in cells that are challenged by oxidative stress.
Apoptotic, cell death and cell survival proteins
A body of evidence indicates that apoptosis plays a role in motor neuron degeneration, and previous studies in the NSC34 cellular model have shown characteristic mitochondrial swelling (Menzies et al., 2002a), cell surface annexin V binding (Cookson et al., 2002) and activation of several caspase proteins (Sathasivam et al., 2004). In the presence of mutant G93A SOD1, we identified an increase in the proapoptotic Bnip3 and Pdcd6ip and a decrease in the anti-apoptotic Bag3 genes, although these were not confirmed by Q-PCR. However, although relatively few genes encoding apoptosis regulating protein were altered in our cellular model, it is noteworthy that many of the molecular effectors of apoptosis are regulated by alteration in subcellular localization or by cleavage of a precursor protein, rather than by alterations in gene expression levels.
HspA1B and HspA4, members of the HSP70 multigene family, which are expressed in response to heat shock, oxidative free radicals and toxic metal ions, were both decreased in the presence of G93A mutant SOD1. HSPs are sequestered by mutant SOD1 in the characteristic protein aggregates seen in both the motor neurons of human ALS cases and transgenic mice models of ALS (Okado-Matsumoto and Fridovich, 2002), and overexpression of Hsp70 in a cultured neuronal cell model reduced both aggregate formation and cell death (Takeuchi et al., 2002). Thus, reduced expression of two key Hsp70 protein family members is likely to be detrimental to the ability of the cell to refold and/or eliminate abnormal proteins.
Comparison of microarray studies
In contrast to the microarray studies reported previously using whole spinal cord extracts, the majority of altered genes identified in our motor neuronal cell model were decreased. Since several of the increases in gene expression identified previously were due to reactive gliosis, which occurs in the spinal cord during neurodegeneration, it is suggested that the presence of non-neuronal cells dilutes and potentially masks changes occuring in motor neurons. Comparisons between studies are difficult given variability in starting material and array formats. The cellular model has little biological variation, and does not possess CNS supporting cells, whilst whole spinal cord from transgenic mice show little biological variation, but do possess heterogeneous cell types and human spinal cord sections possess both biological variation and heterogeneous cell types. In array studies using spinal cord from the G93A SOD1 transgenic mice, one used GeneChip microarrays and the other cDNA membrane arrays, resulting in only four common gene changes (Olsen et al., 2001; Yoshihara et al., 2002). In our previous study using cDNA membrane arrays, five genes were identified as differentially expressed (Kirby et al., 2002). However, in this study, none of those genes were identified and subsequentQ-PCR experiments support the current microarray data that they are not differentially expressed in this set of transfected cells. With this in mind, it highlights the importance of validating data obtained by microarray analysis, not only at the RNA and protein level, but also in additional samples to those used to conduct the experiment. In the previous experiment, the RNA template for hybridization to the array and for Q-PCR was from a single source, whereas in the current study the Q-PCR experiments were performed on three further separate samples, distinct from the three used for the original microarray experiments. Discrepancies could also be attributable to the array formats, as highlighted above, as the macroarray sample gene expression levels by hybridization to a duplicate spotted cDNA, whereas the GeneChip samples gene expression by hybridization to 16 probe pairs. Each probe pair is a 25mer oligo consisting of a perfect match probe and a mismatch probe, containing a single nucleotide substitution. The mismatch probes serve as specificity controls for their perfect match probe, and the levels of non-specific hybridization are taken into account by the software during analysis of the data. The GeneChip protocol is also more consistent in stringency washes between the three hybridizations, owing to use of the fluidics station, compared with manual washes. In addition, when using a model of a disease, changes should be correlated back to the human disease. Our preliminary findings suggest the changes identified in NSC34 cells are relevant to human SOD1-related motor neuron degeneration. However, these studies, in collaboration with other brain tissue bank centres, need to be extended in a larger sample size, with different SOD1 mutations, to determine the true relevance to the human disease of the results from the cellular model.
In summary, gene expression profiling in our cellular model of motor neuron degeneration has identified key cellular pathways specifically altered in the presence of mutant SOD1. To our knowledge, this is the first microarray analysis that has attempted to dissect changes in cells with a motor neuron phenotype uncontaminated by the multiple other cellular groups present in spinal cord. We have verified key differentially expressed genes in the cellular model by Q-PCR in both the G37R and G93A SOD1 transfected NSC34 cell lines, and preliminary studies suggest these changes are also present in motor neurons microdissected from human SOD1-associated ALS spinal cord. Previous work from our group and others indicates that motor neurons in the presence of mutant SOD1 are under stress, with abnormal mitochondria, cytoskeletal dysregulation, proteasomal dysfunction, increased sensitivity to oxidative, glutamatergic and nitric oxide insults, as well as basal activation of early components of the apoptotic cascade (Eggett et al., 2000; Cookson et al., 2002; Menzies et al., 2002a, b; Allen et al., 2003; Sathasivam et al., 2004). We have now added significantly to the existing knowledge of the toxic cellular effects of mutant SOD1 by comparative gene expression profiling. Our data suggest that failure to mount a ‘programmed cell life’ or ARE-driven response may play an important role in mutant SOD1-induced motor neuron injury. The pathways showing dysregulated gene expression in the presence of mutant SOD1 are potentially amenable to therapeutic manipulation and may lead to new treatment approaches for human ALS.
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Note added in proof |
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TopSummaryIntroductionMaterial and methodsResultsDiscussionNote added in proofReferences |
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Recently, Jiang and colleagues have published a report detailing the gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis (Jiang et al., 2005
). They compared the gene expression profiles of isolated motor neurons with whole spinal ventral horn. Interestingly, their study showed evidence of transcriptional repression in the gene expression profile of motor neurons. Comparison of the 60 reported genes altered in sporadic ALS motor neurons with our cellular model provided only a few correlates, and this is perhaps not surprising as these are sporadic ALS cases, rather than SOD1-related motor neuron disease, the changes reflect those at end stage of the disease, the genes expressed have been influenced by the presence of astrocytes and other surrounding CNS tissue, and an alternative array platform (glass microarrays with Cy3- and Cy5-labelled probes) has been used. |
Acknowledgements |
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We wish to thank Neil Cashman for supplying the original NSC34 cell line and Denise Figlewicz for the SOD1 constructs. J.K. and P.R.H. are funded by the Motor Neurone Disease Association, S.A., M.J.B., S.C.B., C.A.W.-A. and C.A.L. are funded by the Wellcome Trust, and P.J.S. is supported by both. E.H. and J.L. would like to acknowledge the Food Standards Agency and the Arthritis Research Campaign.
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