Spinal muscular atrophy (SMA), a lethal hereditary disease caused by mutations of the survival of motor neuron 1 (SMN1) gene, is the leading genetic cause of infant mortality. Its severity directly correlates to the expression level of SMN protein in patients with SMA, but the regulatory mechanisms of SMN protein expression remain incompletely defined. In the present study, we aimed to identify candidate proteins to distinguish SMA fibroblasts from normal fibroblasts.

To identify cellular targets regulating the expression of SMN, we initially utilized a proteomics approach combining 2D electrophoresis and LC-MS/MS, wherein the total proteins extracted from type I SMA patients and normal skin fibroblast cells were compared.

Our initial proteomics analysis discovered significant increase of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in type I SMA fibroblasts when compared to normal fibroblasts. Significantly, UCHL1 proteins directly interacted with SMN protein, as determined by immunoprecipitation and immunofluorscence assays in P19 and NSC34 cells. Over-expression of UCHL1 in P19 and NSC34 cells significantly reduced the level of SMN proteins in vivo, and, in fact, purified UCHL1 was shown to be able to enhance, in a dose-dependent manner, the level of ubiquitinated SMN in vitro. Further, inhibition of UCHL1 activity by UCHL1 inhibitor (LDN-57444) increased cellular SMN protein and gems number in the nucleus in NSC34 and SMA skin fibroblasts. The same results were observed in cells with UCHL1-specific knockdown.

These results suggested that UCHL1 may be a critical regulator in controlling cellular SMN protein turnover, and that it may serve as an attractive therapeutic target for SMA.