Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To conf

A Comparative Study of SMN Protein and mRNA in Blood and Fibroblasts in Patients with Spinal Muscular Atrophy and Healthy Controls

BACKGROUND: Clinical trials to test safety and efficacy of drugs for patients with spinal muscular atrophy (SMA) are currently underway. Biomarkers that document treatment-induced effects are needed because disease progression in childhood forms of SMA is slow and clinical outcome measures may lack sensitivity to detect meaningful changes in motor function in the period of 1-2 years of follow-up during randomized clinical trials. OBJECTIVE: To determine and compare SMN protein and mRNA levels in two cell types (i.e. PBMCs and skin-derived fibroblasts) from patients with SMA types 1-4 and healthy controls in relation to clinical characteristics and SMN2 copy numbers. MATERIALS AND METHODS: We determined SMN1, SMN2-full length (SMN2-FL), SMN2-delta7 (SMN2-Δ7), GAPDH and 18S mRNA levels and SMN protein levels in blood and fibroblasts from a total of 150 patients with SMA and 293 healthy controls using qPCR and ELISA. We analyzed the association with clinical characteristics including disease severity and duration, and SMN2 copy number. RESULTS: SMN protein levels in PBMCs and fibroblasts were higher in controls than in patients with SMA (p0.1) or mRNA levels (p>0.05) in either cell type. SMN2 copy number was associated with SMN protein levels in fibroblasts (p = 0.01), but not in PBMCs (p = 0.06). Protein levels in PBMCs declined with age in patients (p<0.01) and controls (p<0.01)(power 1-beta = 0.7). Ratios of SMN2-Δ7/SMN2-FL showed a broad range, primarily explained by the variation in SMN2-Δ7 levels, even in patients with a comparable SMN2 copy number. Levels of SMN2 mRNA did not correlate with SMN2 copy number, SMA type or age in blood (p = 0.7) or fibroblasts (p = 0.09). Paired analysis between blood and fibroblasts did not show a correlation between the two different tissues with respect to the SMN protein or mRNA levels. CONCLUSIONS: SMN protein levels differ considerably between tissues and activity is age dependent in patients and controls. SMN protein levels in fibroblasts correlate with SMN2 copy number and have potential as a biomarker for disease severity

Levels of SMN mRNA in blood and fibroblasts from patients with SMA.

<p>Levels of SMN mRNA in blood and fibroblasts from patients with SMA.</p

Levels of SMN protein in PBMCs and fibroblasts.

<p>Levels of SMN protein in PBMCs and fibroblasts.</p

SMN2 mRNA expression levels in blood and fibroblasts from patients with SMA in relation to SMN2 copy number.

<p>Levels of SMN2-FL and SMN2-Δ7 in relation to <i>SMN2</i> copy number in blood (Panel A; KW SMN2-FL p = 0.7; KW SMN2-Δ7 p = 0.3) and fibroblasts (Panel B; KW SMN2-FL p = 0.3; KW SMN2-Δ7 p = 0.09) from patients with SMA. Data shown are normalized to the geometric mean (= GM) of GAPDH- and 18S-reference genes. Boxplot elements represent: median (line in the middle), 1^st en 3^rd quartile (bottom and top of the box), highest case with 1.5 time inter-quartile range (bottom and top whisker) and outliers (dots and asterisks). SMN2-D7 = SMN2-Δ7.</p

SMN protein levels in PBMCs and fibroblasts from patients and controls and effect of <i>SMN2</i> copy numbers.

<p>Mean SMN protein levels are higher in controls compared to patients. SMN protein levels in PBMCs did not differ significantly between patients with 2, 3, 4 or 5 <i>SMN2</i> copies (p = 0.06). Higher <i>SMN2</i> copy number is associated with higher levels of SMN protein in fibroblasts (p = 0.01). Boxplot elements represent: median (line in the middle), 1^st en 3^rd quartile (bottom and top of the box), highest case with 1.5 time inter-quartile range (bottom and top whisker) and outliers (dots).</p

Baseline characteristics of patients in PBMC study.

<p>Baseline characteristics of patients in PBMC study.</p

Baseline characteristics of patients in fibroblast study.

<p>Baseline characteristics of patients in fibroblast study.</p