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Background It is unclear to what extent pre-clinical studies in genetically homogeneous animal models of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder, can be informative of human pathology. The disease modifying effects in animal models of most therapeutic compounds have not been reproduced in patients. To advance therapeutics in ALS, we need easily accessible disease biomarkers which can discriminate across the phenotypic variants observed in ALS patients and can bridge animal and human pathology. Peripheral blood mononuclear cells alterations reflect the rate of progression of the disease representing an ideal biological substrate for biomarkers discovery. Methods We have applied TMTcalibrator™, a novel tissue-enhanced bio fluid mass spectrometry technique, to study the plasma proteome in ALS, using peripheral blood mononuclear cells as tissue calibrator. We have tested slow and fast progressing SOD1G93A mouse models of ALS at a pre-symptomatic and symptomatic stage in parallel with fast and slow progressing ALS patients at an early and late stage of the disease. Immunoassays were used to retest the expression of relevant protein candidates. Results The biological features differentiating fast from slow progressing mouse model plasma proteomes were different from those identified in human pathology, with only processes encompassing membrane trafficking with translocation of GLUT4, innate immunity, acute phase response and cytoskeleton organization showing enrichment in both species. Biological processes associated with senescence, RNA processing, cell stress and metabolism, major histocompatibility complex-II linked immune-reactivity and apoptosis (early stage) were enriched specifically in fast progressing ALS patients. Immunodetection confirmed regulation of the immunosenescence markers Galectin-3, Integrin beta 3 and Transforming growth factor beta-1 in plasma from pre-symptomatic and symptomatic transgenic animals while Apolipoprotein E differential plasma expression provided a good separation between fast and slow progressing ALS patients. Conclusions These findings implicate immunosenescence and metabolism as novel targets for biomarkers and therapeutic discovery and suggest immunomodulation as an early intervention. The variance observed in the plasma proteomes may depend on different biological patterns of disease progression in human and animal model. Electronic supplementary material The online version of this article (10.1186/s13024-018-0292-2) contains supplementary material, which is available to authorized users.

Plasma proteome composition reflects the inflammatory and metabolic state of the organism and can be predictive of system-level and organ-specific pathologies. Circulating protein aggregates are enriched with neurofilament heavy chain—axonal proteins involved in brain aggregate formation and recently identified as biomarkers of the fatal neuromuscular disorder amyotrophic lateral sclerosis. Using unbiased proteomic methods, we have fully characterized the content in neuronal proteins of circulating protein aggregates from amyotrophic lateral sclerosis patients and healthy controls, with reference to brain protein aggregate composition. We also investigated circulating protein aggregate protein aggregation propensity, stability to proteolytic digestion and toxicity for neuronal and endothelial cell lines. Circulating protein aggregates separated by ultracentrifugation are visible as electron-dense macromolecular particles appearing as either large globular or as small filamentous formations. Analysis by mass spectrometry revealed that circulating protein aggregates obtained from patients are enriched with proteins involved in the proteasome system, possibly reflecting the underlying basis of dysregulated proteostasis seen in the disease, while those from healthy controls show enrichment of proteins involved in metabolism. Compared to the whole human proteome, proteins within circulating protein aggregates and brain aggregates show distinct chemical features of aggregation propensity, which appear dependent on the tissue or fluid of origin and not on the health status. Neurofilaments’ two high-mass isoforms (460 and 268 kDa) showed a strong differential expression in amyotrophic lateral sclerosis compared to healthy control circulating protein aggregates, while aggregated neurofilament heavy chain was also partially resistant to enterokinase proteolysis in patients, demonstrated by immunoreactive bands at 171 and 31 kDa fragments not seen in digested healthy controls samples. Unbiased proteomics revealed that a total of 4973 proteins were commonly detected in circulating protein aggregates and brain, including 24 expressed from genes associated with amyotrophic lateral sclerosis. Interestingly, 285 circulating protein aggregate proteins (5.7%) were regulated (P, Circulating protein aggregates in blood contain 4973 brain peptides including heavy chain neurofilaments and 24 amyotrophic lateral sclerosis-associated proteins. In amyotrophic lateral sclerosis, circulating protein aggregates express proteins involved in metabolism and proteosome and may be biologically active on endothelial cells., Graphical Abstract Graphical Abstract

Background: plasma proteins composition reflects the inflammatory and metabolic state of an organism and can be predictive of system-level and organ-specific pathologies. Circulating protein aggregates (CPA) are enriched with heavy chain neurofilaments (NfH), axonal proteins involved in brain protein aggregates (BPA) formation and recently identified as biomarkers of the fatal neuromuscular disorder amyotrophic lateral sclerosis (ALS). Methods: here we use mass spectrometry and brain-enhanced TMTcalibrator™-based proteomics to evaluate the composition and the brain-derived protein component of CPA extracted from ALS and healthy controls (HC) plasma samples using high-performance ultracentrifugation. We also test CPA and BPA proteins aggregation propensity and the resistance to proteases digestion by trypsin, chymotrypsin, calpain and enterokinase of NFH within aggregates. Finally, we study CPA biological effects on neuronal and endothelial cell lines. Results: electron microscopy confirms the presence in CPA of electron-dense macromolecular particles appearing as either large globular or as small filamentous formations. CPA from ALS are enriched with proteasome system proteins while HC CPA show a prominent expression of proteins involved in metabolism. CPA enterokinase digestion in ALS generates 171 and 31 KDa NfH fragments not seen in HC samples. Compared to the whole human proteome, proteins within CPA and BPA show distinct chemical features of aggregation propensity, which appear dependent on the tissue or fluid of origin and not on the healthy or pathological source of plasma. The use of a TMTcalibrator™ proteomics workflow reveals 4973 brain-derived low-abundance proteins in CPA, including products of translation of 24 ALS risk genes. 285 (5.7%) are regulated in ALS (p < 0.05) and belong to biochemical pathways previously linked to ALS pathogenesis and aggregates formation. CPA from both ALS and HC have a higher effect on hCMEC/D3 endothelial and PC12 neuronal cells viability than immunoglobulins extracted from the same plasma samples. Compared to HC, CPA from ALS plasma samples exert a higher toxic effect on both cell lines at lower concentrations.Conclusions: this study demonstrates that CPA are significantly enriched with brain proteins which are representative of ALS pathology and a potential source of biomarkers and therapeutic targets for this incurable disorder.

The lack of biomarkers for early diagnosis, clinical stratification and to monitor treatment response has hampered the development of new therapies for amyotrophic lateral sclerosis (ALS), a clinically heterogeneous neurodegenerative disorder with a variable site of disease initiation and rate of progression. To identify new biomarkers and therapeutic targets, two separate proteomic workflows were applied to study the immunological response and the plasma/brain proteome in phenotypic variants of ALS. Conventional multiplex (TMT) proteomic analysis of peripheral blood mononuclear cells (PBMCs) was performed alongside a recently introduced method to profile neuronal-derived proteins in plasma using brain tissue-enhanced isobaric tagging (TMTcalibrator). The combined proteomic analysis allowed the detection of regulated proteins linked to ALS pathogenesis (RNA-binding protein FUS, superoxide dismutase Cu-Zn and neurofilaments light polypeptide) alongside newly identified candidate biomarkers (myosin-9, fructose-bisphosphate aldolase and plectin). In line with the proteomic results, orthogonal immunodetection showed changes in neurofilaments and ApoE in bulbar versus limb onset fast progressing ALS. Functional analysis of significantly regulated features showed enrichment of pathways involved in regulation of the immune response, Rho family GTPases, semaphorin and integrin signalling. Our cross-phenotype investigation of PBMCs and plasma/brain proteins provides a more sensitive biomarker exploratory platform than conventional case-control studies in a single matrix. The reported regulated proteins may represent novel biomarker candidates and potentially druggable targets.

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Increasingly, clinical trials for Alzheimer’s disease (AD) are being conducted earlier in the disease phase and with biomarker confirmation using in vivo amyloid PET imaging or CSF tau and Aβ measures to quantify pathology. However, making such a pre-clinical AD diagnosis is relatively costly and the screening failure rate is likely to be high. Having a blood-based marker that would reduce such costs and accelerate clinical trials through identifying potential participants with likely pre-clinical AD would be a substantial advance. In order to seek such a candidate biomarker, discovery phase proteomic analyses using 2DGE and gel-free LC-MS/MS for high and low molecular weight analytes were conducted on longitudinal plasma samples collected over a 12-year period from non-demented older individuals who exhibited a range of 11C-PiB PET measures of amyloid load. We then sought to extend our discovery findings by investigating whether our candidate biomarkers were also associated with brain amyloid burden in disease, in an independent cohort. Seven plasma proteins, including A2M, Apo-A1, and multiple complement proteins, were identified as pre-clinical biomarkers of amyloid burden and were consistent across three time points (p < 0.05). Five of these proteins also correlated with brain amyloid measures at different stages of the disease (q < 0.1). Here we show that it is possible to detect a plasma based biomarker signature indicative of AD pathology at a stage long before the onset of clinical disease manifestation. As in previous studies, acute phase reactants and inflammatory markers dominate this signature.

OBJECTIVE: To evaluate the combined blood expression of neuromuscular and inflammatory biomarkers as predictors of disease progression and prognosis in amyotrophic lateral sclerosis (ALS). METHODS: Logistic regression adjusted for markers of the systemic inflammatory state and principal component analysis were carried out on plasma levels of creatine kinase (CK), ferritin, and 11 cytokines measured in 95 patients with ALS and 88 healthy controls. Levels of circulating biomarkers were used to study survival by Cox regression analysis and correlated with disease progression and neurofilament light chain (NfL) levels available from a previous study. Cytokines expression was also tested in blood samples longitudinally collected for up to 4 years from 59 patients with ALS. RESULTS: Significantly higher levels of CK, ferritin, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, IL-2, IL-8, IL-12p70, IL-4, IL-5, IL-10, and IL-13 and lower levels of interferon (IFN)-γ were found in plasma samples from patients with ALS compared to controls. IL-6, TNF-α, and IFN-γ were the most highly regulated markers when all explanatory variables were jointly analyzed. High ferritin and IL-2 levels were predictors of poor survival. IL-5 levels were positively correlated with CK, as was TNF-α with NfL. IL-6 was strongly associated with CRP levels and was the only marker showing increasing expression towards end-stage disease in the longitudinal analysis. CONCLUSIONS: Neuromuscular pathology in ALS involves the systemic regulation of inflammatory markers mostly active on T-cell immune responses. Disease stratification based on the prognostic value of circulating inflammatory markers could improve clinical trials design in ALS.