Microarray analysis of peripheral blood lymphocytes from ALS patients and the SAFE detection of the KEGG ALS pathway

Abstract

Background: Sporadic amyotrophic lateral sclerosis (sALS) is a motor neuron disease with poorly understood etiology. Results of gene expression profiling studies of whole blood from ALS patients have not been validated and are difficult to relate to ALS pathogenesis because gene expression profiles depend on the relative abundance of the different cell types present in whole blood. We conducted microarray analyses using Agilent Human Whole Genome 4 × 44k Arrays on a more homogeneous cell population, namely purified peripheral blood lymphocytes (PBLs), from ALS patients and healthy controls to identify molecular signatures possibly relevant to ALS pathogenesis.

Methods: Differentially expressed genes were determined by LIMMA (Linear Models for MicroArray) and SAM (Significance Analysis of Microarrays) analyses. The SAFE (Significance Analysis of Function and Expression) procedure was used to identify molecular pathway perturbations. Proteasome inhibition assays were conducted on cultured peripheral blood mononuclear cells (PBMCs) from ALS patients to confirm alteration of the Ubiquitin/Proteasome System (UPS).

Results: For the first time, using SAFE in a global gene ontology analysis (gene set size 5-100), we show significant perturbation of the KEGG (Kyoto Encyclopedia of Genes and Genomes) ALS pathway of motor neuron degeneration in PBLs from ALS patients. This was the only KEGG disease pathway significantly upregulated among 25, and contributing genes, including SOD1, represented 54% of the encoded proteins or protein complexes of the KEGG ALS pathway. Further SAFE analysis, including gene set sizes >100, showed that only neurodegenerative diseases (4 out of 34 disease pathways) including ALS were significantly upregulated. Changes in UBR2 expression correlated inversely with time since onset of disease and directly with ALSFRS-R, implying that UBR2 was increased early in the course of ALS. Cultured PBMCs from ALS patients accumulated more ubiquitinated proteins than PBMCs from healthy controls in a serum-dependent manner confirming changes in this pathway.

Conclusions: Our study indicates that PBLs from sALS patients are strong responders to systemic signals or local signals acquired by cell trafficking, representing changes in gene expression similar to those present in brain and spinal cord of sALS patients. PBLs may provide a useful means to study ALS pathogenesis.

Figure 1

Normalization, filtering, SAFE, LIMMA and SAM analyses of lymphocyte-derived microarray data from ALS patients and healthy controls. Microarray analyses were performed on purified PBLs isolated from patients affected by sporadic amyotrophic lateral sclerosis (sALS) (n = 11) and healthy control subjects (HCs) (n = 11). The dual color mode in the common reference design was used to interrogate the expression of ~40000 transcripts (~30000 unique genes) using Agilent Human Whole Genome 4 × 44k Microarrays. Raw expression data were normalized and filtered using the MIDAS pipeline in the TM4 microarray suite (TIGR Genomics, Rockville, MD) to generate the dataset DS7000. SAFE was used for testing enrichment of functional gene ontology (GO) categories related to biological processes, molecular functions, cellular components, protein families (Pfam) and the KEGG databases. DS7000 was subjected to LIMMA and SAM analyses using TM4/TMeV v4.5.1 to determine differentially expressed (DE) genes. The online tool Data Overlapping and Area-Proportional Venn Diagram (http://bioinforx.com/free/bxarrays/overlap.php) was used to generate the Venn diagram.

Figure 2

Genes from lymphocytes of ALS patients contributing to the perturbation of the KEGG ALS pathway per SAFE analysis. The KEGG (Kyoto Encyclopedia of Genes and Genomes) ALS pathway map relates to motor neuron degeneration in the context of a microenvironment represented by glial cells and can be found online at http://www.genome.jp/kegg/pathway/hsa/hsa05014.html. There are 54 unique gene entries (including CASP12 pseudogene) defined by ENTREZ identities. There are 36 protein entities represented on the map that are not all designated by official HUGO gene symbols. Red dashed areas represent subpathway modules affected by differentially expressed genes. Up or down-regulations determined following SAFE for DS7000 are shown by (↑) or (↓), unchanged is shown by (=) (fold changes in expression and HUGO gene symbols are reported in Table 6). HUGO aliases for protein entities represented on the map are as follows: ALS2 [ALS2], Apaf1 [APAF1], ASK1 [MAP3K5], Bad [BAD], Bax [BAX], Bcl2 [BCL2], Bcl-XL [BCL2L1], Bid [BID], CaN [CHP, CHP2, PPP3CA, PPP3CB, PPP3CC, PPP3R1, PPP3R2], CASP1 [CASP1], CASP3 [CASP3], CASP9 [CASP9], CASP12 [CASP12], CAT [CAT], CCS [CCS], CytC [CYCS], Daxx [DAXX], Derlin-1 [DERL1], EAAT2 [SLC1A2], GPX1 [GPX1], GluR [GRIA1, GRIA2, GRIN1, GRIN2A, GRIN2B, GRIN2C, GRIN2D], MKK3 [MAP2K3], MKK6 [MAP2K6], p38 [MAPK11, MAPK12, MAPK13, MAPK14], NEFH [NEFH], NEFL [NEFL], NEFM [NEFM], NOS1 [NOS1], p53 [TP53], PRPH [PRPH, PRPH2], Rab5 [RAB5A], Rac1 [RAC1], SOD1 [SOD1], TNF-α [TNF], TNFR [TNFRSF1A, TNFRSF1B], and Tom [TOMM40, TOMM40L].

Figure 3

Relationship between UBR2 transcriptional expression in lymphocytes from ALS patients and progression of the disease. Expression of UBR2 varies inversely with the length of the disease from onset to lymphocyte gene expression testing, and varies directly with the ALS-FRS-R score. Duration of the disease from onset to sampling (i.e. Disease Duration) (a) (*three close values) or the ALSFRS-R score at the time of sampling (b) are indicated on the x-axis. Log₂ ratios of expression obtained from the dual mode reference design are represented on the y axis. Dot plot (c) shows that with a cut-off of 0.15, discrimination between ALS patients [ALS] and healthy controls [HC] for UBR2 expression is achieved with p = 0.000953 (Fisher's exact test).

Figure 4

Total ubiquitination Western blot (WB) analysis of cultured PBMCs from ALS patients and controls in the presence or absence of added-back serum for 16 hours and treated or not with proteasome inhibitor MG132 for 1.5 hr. Comparison of PBMCs from one healthy control and one ALS patient incubated or not in the presence of added-back matched autologous serum is shown in (a). Semi-quantitative Western blot analysis was performed to measure the accumulation of high molecular weight (HMW) ubiquitinated protein species in PBMCs that were prepared the same day from one healthy control and one ALS patient (WB1). A signal (S) to noise (N) ratio (S/N) was determined with ImageJ program by comparing the integrated density of two areas consistently stained throughout the membrane and visually contrasting the accumulation of HMW ubiquitinated protein species (WB1). ALS patient serum exacerbates the effects of MG132 on total ubiquitination and accumulation of HMW ubiquitinated species, while serum from healthy control mitigates these effects. Comparison of PBMCs from ALS patients (n = 5) and healthy controls (n = 4) incubated in the presence of added-back matched autologous serum is shown in (b). PBMCs obtained at different times from additional ALS patients (n = 5) and healthy controls (n = 4) show similar result (WB2 and WB3).