We hypothesized increased Nogo-A concentration in ALS patient serum based on findings by another group of increased Nogo-A expression in ALS patient muscle (Dupuis et al. 2002
, Jokic et al. 2005
). Although Nogo-A is a transmembrane protein that is not normally secreted, we reasoned that atrophic changes often associated with ALS could result in release of Nogo-A or fragments of Nogo-A into the bloodstream, in a similar fashion to the release of creatine kinase from dying muscle (Achari & Anderson 1974
). A precedent for detectable Nogo isoforms in human serum exists (Rodriguez-Feo et al. 2007
). Moreover, a precedent exists for the use of decreased rather than increased concentrations of specific CSF peptides such as fragments of cystatin 3 and VGF as biomarkers for ALS (Pasinetti et al. 2006
Although the distribution of values for serum Nogo-A was significantly lower in ALS patients than healthy controls in our study, the mean values were not significantly different. Several possible explanations could underlie these findings. Degenerating muscle may release carboxyl-terminal Nogo-A fragments that would not be detected by our assay, which depends on the presence of Nogo-A residues 1–18 through 623–640 in serum. Alternatively, muscle atrophy may have been so advanced by the time of blood collection in ALS patients that serum Nogo-A levels may have already decreased after potentially increasing earlier during the disease course. A post-hoc analysis demonstrated no correlation between serum Nogo-A values and duration of disease symptoms (not shown). A study to follow Nogo-A levels over time within individual ALS patients could shed light on this possibility – our serum-based assay makes this approach much more feasible than performing multiple muscle biopsies.
Interestingly, a subset of subjects showed highly elevated Nogo-A serum values. This occurred more frequently in the healthy control group than the ALS group. We performed a separate experiment to determine whether these high Nogo-A values could be explained by inadvertent haemolysis of our serum samples. When Nogo-A levels were compared in whole venous blood obtained from five healthy volunteers separated into serum, plasma and cell pellet homogenate, values were 84% higher for the cell homogenate, but still well below the 2 nM cut-off used to delineate ‘highly elevated’ serum Nogo-A values (not shown). Thus, even if there were varying levels of haemolysis among our 344 serum samples, this would not cause a sufficient rise in Nogo-A levels to explain the subset of samples with values higher than 2 nM. We do not have matching muscle biopsy samples to determine whether elevated serum Nogo-A correlates with elevated muscle Nogo-A in healthy controls or ALS patients. However, these findings add to evidence that increased Nogo-A expression may not be specific for ALS (Wojcik et al. 2006
, Teng & Tang 2008
, Magnusson et al. 2003
, Tagerud et al. 2007
). It will be of great interest to determine other potential causes of elevated serum Nogo-A, and whether this elevation has any phenotypic effect.
In summary, unlike muscle Nogo-A, concentrations of serum Nogo-A do not increase in ALS.