A number of gene expression studies have demonstrated the utility of PBMC as a source of biomarkers in neurological disorders 
. This is the first study that describes a highly feasible strategy to identify and validate multiprotein biomarkers in PBMC, potentially applicable to several neurological/neurodegenerative diseases. Protein profiles are directly connected to changes in molecular pathways related to health and disease, therefore have the potential to accurately monitor the progression of the disease or the response to a treatment.
We have previously shown that PBMC undergo immunophenotypic changes in sALS patients 
. We have now identified a panel of protein biomarkers in these cells that are associated with sALS with high discriminatory power. These PBMC proteins are easily measurable in large-scale immunoassays aimed at developing diagnostic/prognostic tests for clinical use. The great advantages of such an in vitro
test is low invasiveness for the patient compared to CSF tests, the consequent greater availability of samples for large clinical studies, including longitudinal ones, and the simple laboratory procedures involved.
The ideal diagnostic marker should detect disease before clinical diagnosis, which is highly challenging for a rare and sporadic disease. ALS patients very often see the specialized neurologist only months after the first symptoms, when they are unquestionably ill. It is therefore very difficult to test and validate the applicability of biomarkers in preclinical diagnosis. Our PBMC protein biomarkers seems to be promising to support prompt clinical diagnosis, since all the 14 validated proteins can distinguish with high significance ALS patients with low disability from healthy controls (Table S6
). PBMC biomarkers could be measured on patients after as early as 5 months up to 108 months from symptom onset, and with high (from 45) and low (up to 10) ALSFRS-R scores (Table S5
). Now large-scale validations are needed, in which data will be verified in a prospective cohort with analysis of the biomarkers at the time of symptom presentation. In the G93A SOD1 rat model of ALS, alterations of the level of CypA, GSTO1, FUBP1, CLIC1 and actinNT
are detected before disease onset. It is possible that some of these biomarkers may predict the onset of SOD1-linked familial ALS. This potential application will be also considered in future analysis.
Up to 10% of patients initially diagnosed as having ALS are false positive 
. A similar percentage is false negative and undergoes inappropriate medical or surgical procedures 
. Thus, there is a need for biomarkers that distinguish ALS with high accuracy from neurological disorders that in some cases may resemble it, e.g. some peripheral neuropathies that are treatable and do not have a fatal prognosis. We found that there are PBMC protein biomarkers that significantly distinguish ALS from the group of neurological disorders considered in our study. These are chaperones (CALR, CypA), proteins involved in redox homeostasis (GSTO1, CLIC1) and immune responses (IRAK4). IRAK4, which has no previous association with ALS, has a central function in innate immunity 
Finally, multivariate analysis helped us in defining the most convenient combination of protein biomarkers that could be potentially used in clinics (i) to support diagnosis (CLIC1, actinNT and ROA2), (ii) to contribute to differential diagnosis of ALS from other neurological conditions (CypA and IRAK4), and (iii) to determine disease severity (ERp57).
There are no precise measures of ALS disease progression that allow for short-term monitoring of the disease and assessment of treatment efficacy. In clinical trials survival time is therefore used as the primary measure of outcome. This requires large number of patients followed over a long period of time making ALS clinical trials very expensive. A panel of biomarkers that can reliably assess disease progression would enable a substantial reduction of the costs of the clinical trial and accelerate therapy development in ALS. ERp57, CypA and TDP-43, that were able to discriminate between patients with high and low disease severity, were selected for a pilot longitudinal study and proved to be good candidates for such applications. Large longitudinal studies are now needed to further validate the use of these proteins in clinical practice.
The pathogenesis of sALS is largely unknown. The concept of PBMC as a window into the CNS has been already proposed for several neurological disease states 
. CNS and immune cells communicate through multiple mechanisms and have several similarities in receptor expression and transduction processes 
. We therefore hypothesized that PBMC protein profiles could help to elucidate pathways underlying ALS etiology. Indeed, some of the protein biomarkers identified in PBMC of sALS patients were previously found as hallmarks of disease in CNS. Studies in spinal cord tissues of sALS patients showed that PDI and ERp57 were up-regulated 
, CypA and HSC70 were accumulated in the detergent-insoluble fraction 
, HSC70 was present in hyaline inclusions 
, and TDP-43 was identified as the major component of the ubiquitinated inclusions 
The protein profile changes detected in PBMC of ALS patients are suggestive of possible pathogenic mechanisms. For instance, up-regulation of endoplasmic reticulum (ER) chaperones (PDI, ERp57, CALR) is a typical cellular response to ER stress that triggers the unfolded protein response leading eventually to cell death 
. The increased level of a nitrotyrosine-linked protein, actinNT
, is indicative of nitrative stress 
. Alterations in CypA, GSTO1, PRDX2 suggest disturbances in cellular redox regulation 
. It is important to note that all these pathogenic alterations were previously reported in the spinal cord of sALS patients and mutant SOD1 animal models 
. ROA2 belongs to the family of heterogeneous nuclear ribonucleoproteins that participates to several RNA-related biological processes such as transcription, pre-mRNA processing, mRNA transport to the cytoplasm and translation 
. It is also a binding partner of TDP-43 and seems to be crucial for at least one of its putative functions 
. The up-regulation of ROA2 and TDP-43 in PBMC of both ALS>24 and ALS≤24 patients may underline aberrant RNA processing events that are now emerging as central in ALS and other neurological disorders 
. These specific intracellular alterations would not be detectable from the protein profiles of CSF or plasma. Neuroinflammation, which is characterized by activated microglia and infiltrating peripheral blood immune cells, is a prominent pathological feature in ALS 
. It is possible to speculate that such consistent protein profile changes in immune cells may influence their infiltration and/or interaction with microglia and neurons, thus upsetting the delicate balance between neuroprotection and neurotoxicity. In summary, all these data endorse the use of PBMC for further in vitro
mechanistic studies. Experimental models for the sporadic form are not available and all mechanistic studies are done with transgenic cells and animals expressing one of the mutant genes linked to familial ALS. Studies with PBMC would have the advantage to consider the influence of the genetic background of the patient.
Translational biomarkers, that link responses between human and animal model, are of particular interest because their role in the pathogenesis can be investigated in detail in the animal model where they can also offer important preliminary information for clinical trials. We found that CypA, GSTO1, FUBP1, CLIC1 and actinNT are translational biomarkers. Moreover, all of them except for CLIC1 are altered in the ventral horn spinal cord of SOD1 G93A rats before disease onset, suggesting a possible involvement in pathways that trigger the disease. Further mechanistic studies in the animal models with these proteins are now warranted.
In conclusion, we identified and verified a panel of highly promising protein biomarkers of ALS in PBMC that may be useful in clinical studies, helping elucidate pathogenic mechanisms and pin-pointing pathways to tackle for future therapeutic interventions. The fact that our protein biomarkers are easily measurable in accessible clinical samples using straightforward immunoassays makes them attractive candidates for true multi-centric large-scale validations and eventually introduction into clinical practice.