In this study we used label-free proteomic technology to identify two proteins with the potential to act as molecular biomarkers for SMA. The combination of proteomics technology with an established mouse model of SMA (where it is possible to accurately identify and isolate tissue from animals at different stages of disease) revealed that increased levels of GRP75/Mortalin and Calreticulin in skeletal muscle correlated with disease progression. Importantly, these protein biomarkers were also accessible in skin samples from SMA mice, suggesting that they can also be monitored in a peripherally accessible tissue during clinical trials. A preliminary study on a small sample of patient muscle biopsies suggested that GRP75/Mortalin and Calreticulin were detectable and measurable in human tissue, including biopsies from SMA patients.
The use of label-free proteomics in this study provides further evidence that proteomics technologies represent a powerful tool for biomarker discovery . Indeed, proteomics technology has previously been used to screen for potential biomarkers in human SMA patients [19, 21]. Previous studies combining proteomics with animal models of SMA have mainly utilized the technology to uncover molecular pathways disrupted downstream of SMN [7, 30, 35, 36], but the current study demonstrates that similar approaches can be used to identify potential protein biomarkers for future use in the human clinical context. In addition, our ability to identify protein biomarkers conserved between different mouse models of SMA and human SMA patients suggests that common biomarkers can be utilized in both pre-clinical testing of new treatments in animal models as well as in human clinical trials. It should be noted that our proteomics study identified approximately 500 muscle proteins, which is predicted to represent only a fraction of the total muscle proteome. Thus, there are likely to be other proteins in SMA skeletal muscle yet to be identified that have the potential to act as novel biomarkers for the disease alongside GRP75/Mortalin and Calreticulin.
Calreticulin is a multifunctional protein that has previously been identified as a potential biomarker for other diseases. For example, serum levels of Calreticulin have been shown to increase in patients with rheumatoid arthritis  and increased levels of Calreticulin have been reported in breast cancer [38, 39], gastric cancer , and lung cancer . Calreticulin has also been identified as a prognostic factor for neuroblastoma . However, Calreticulin has not previously been linked to SMA, and whether it is actively involved in disease pathogenesis or not remains unclear. Interestingly, Calreticulin has been implicated in regulating motor neuron pathology in a related motor neuron disease (amyotrophic lateral sclerosis; ALS) , suggesting that, alongside its potential to act as a molecular biomarker for SMA, further investigations into its possible contribution to the pathogenesis of SMA are warranted.
GRP75/Mortalin is a member of the Hsp70 family of chaperones, with roles including the regulation of energy generation, stress responses, muscle activity, mitochondrial activity, and cellular viability [44–46]. As with Calreticulin, GRP75/Mortalin has previously been identified as a possible biomarker for cancer and cardiovascular diseases  as well as being a potential prognostic factor for neuroblastoma . GRP75/Mortalin (also known as HSPA9) has also been implicated in the pathogenesis of other neurodegenerative conditions, including Parkinson’s disease  and Alzheimer’s disease , suggesting that it, too, may be contributing directly to SMA pathogenesis. Nevertheless, it is important to note that biomarkers do not need to actively contribute to disease pathogenesis in order to be effective. What is critical is that the levels of a biomarker must alter in a temporally traceable and predictable manner as an accurate measure of the molecular and physiological processes of disease progression. Both GRP75/Mortalin and Calreticulin appear to meet these criteria in SMA.
Our preliminary investigation of GRP75/Mortalin and Calreticulin levels in human skeletal muscle suggests that these proteins may represent viable biomarkers in human SMA patients. However, it represents only an initial demonstration of the ability to detect and measure these proteins in human tissue, and was hampered by a lack of detailed information from the biobank regarding the actual stage of disease progression for each patient at the time of muscle biopsy, as well as the small sample size. As a result, further, large-scale studies on patient cohorts will now be required to validate GRP75/Mortalin and Calreticulin as robust protein biomarkers for SMA in humans. The demonstration in mouse models that increased levels of these proteins correlated with increasing disease severity suggests that such a study is now warranted. Moreover, the finding that these proteins can be tracked in skin samples suggests that the use of skin biopsies might be of more practical use for these studies, reducing the need for repeated invasive muscle biopsies.