Combined Tissue-Fluid Proteomics to Unravel Phenotypic Variability in Amyotrophic Lateral Sclerosis.

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.

A motor neuron strategy to save time and energy in neurodegeneration: adaptive protein stoichiometry.

Neurofilament proteins (Nf) are a biomarker of disease progression in amyotrophic lateral sclerosis (ALS). This study investigated whether there are major differences in expression from in vivo measurements of neurofilament isoforms, from the light chain, NfL (68 kDa), compared with larger proteins, the medium chain (NfM, 150 kDa) and the heavy (NfH, 200-210 kDa) chains in ALS patients and healthy controls. New immunological methods were combined with Nf subunit stoichiometry calculations and Monte Carlo simulations of a coarse-grained Nf brush model. Based on a physiological Nf subunit stoichiometry of 7 : 3 : 2 (NfL:NfM:NfH), we found an 'adaptive' Nf subunit stoichiometry of 24 : 2.4 : 1.6 in ALS. Adaptive Nf stoichiometry preserved NfL gyration radius in the Nf brush model. The energy and time requirements for Nf translation were 56 ± 27k ATP (5.6 h) in control subjects compared to 123 ± 102k (12.3 h) in ALS with 'adaptive' (24:2.4:1.6) Nf stoichiometry (not significant) and increased significantly to 355 ± 330k (35.5 h) with 'luxury' (7:3:2) Nf subunit stoichiometry (p < 0.0001 for each comparison). Longitudinal disease progression-related energy consumption was highest with a 'luxury' (7:3:2) Nf stoichiometry. Therefore, an energy and time-saving option for motor neurons is to shift protein expression from larger to smaller (cheaper) subunits, at little or no costs on a protein structural level, to compensate for increased energy demands.

Systemic inflammatory response and neuromuscular involvement in amyotrophic lateral sclerosis.

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)-1ß, 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.

Targeting protein homeostasis in sporadic inclusion body myositis.

Sporadic inclusion body myositis (sIBM) is the commonest severe myopathy in patients more than 50 years of age. Previous therapeutic trials have targeted the inflammatory features of sIBM but all have failed. Because protein dyshomeostasis may also play a role in sIBM, we tested the effects of targeting this feature of the disease. Using rat myoblast cultures, we found that up-regulation of the heat shock response with arimoclomol reduced key pathological markers of sIBM in vitro. Furthermore, in mutant valosin-containing protein (VCP) mice, which develop an inclusion body myopathy, treatment with arimoclomol ameliorated disease pathology and improved muscle function. We therefore evaluated arimoclomol in an investigator-led, randomized, double-blind, placebo-controlled, proof-of-concept trial in sIBM patients and showed that arimoclomol was safe and well tolerated. Although arimoclomol improved some IBM-like pathology in the mutant VCP mouse, we did not see statistically significant evidence of efficacy in the proof-of-concept patient trial.

The role of heat shock proteins in Amyotrophic Lateral Sclerosis: The therapeutic potential of Arimoclomol.

Arimoclomol is a hydroxylamine derivative, a group of compounds which have unique properties as co-inducers of heat shock protein expression, but only under conditions of cellular stress. Arimoclomol has been found to be neuroprotective in a number of neurodegenerative disease models, including Amyotrophic Lateral Sclerosis (ALS), and in mutant Superoxide Dismutase 1 (SOD1) mice that model ALS, Arimoclomol rescues motor neurons, improves neuromuscular function and extends lifespan. The therapeutic potential of Arimoclomol is currently under investigation in a Phase II clinical trial for ALS patients with SOD1 mutations. In this review we summarize the evidence for the neuroprotective effects of enhanced heat shock protein expression by Arimoclomol and other inducers of the Heat Shock Response. ALS is a complex, multifactorial disease affecting a number of cell types and intracellular pathways. Cells and pathways affected by ALS pathology and which may be targeted by a heat shock protein-based therapy are also discussed in this review. For example, protein aggregation is a characteristic pathological feature of neurodegenerative diseases including ALS. Enhanced heat shock protein expression not only affects protein aggregation directly, but can also lead to more effective clearance of protein aggregates via the unfolded protein response, the proteasome-ubiquitin system or by autophagy. However, compounds such as Arimoclomol have effects beyond targeting protein mis-handling and can also affect additional pathological mechanisms such as oxidative stress. Therefore, by targeting multiple pathological mechanisms, compounds such as Arimoclomol may be particularly effective in the development of a disease-modifying therapy for ALS and other neurodegenerative disorders.