Abstract
Sporadic amyotrophic lateral sclerosis (sALS) is a fatal progressive neurodegenerative disease affecting upper and lower motor neurons. Biomarkers are useful to facilitate the diagnosis and/or prognosis of patients and to reveal possible mechanistic clues about the disease. This study aimed to identify and validate selected putative biomarkers in the cerebrospinal fluid (CSF) of sALS patients at early disease stages compared with age-matched controls and with other neurodegenerative diseases including Alzheimer disease (AD), spinal muscular atrophy type III (SMA), frontotemporal dementia behavioral variant (FTD), and multiple sclerosis (MS). SWATH acquisition on liquid chromatography-tandem mass spectrometry (LC–MS/MS) for protein quantitation, and ELISA for validation, were used in CSF samples of sALS cases at early stages of the disease. Analysis of mRNA and protein expression was carried out in the anterior horn of the lumbar spinal cord in post-mortem tissue of sALS cases (terminal stage) and controls using RTq-PCR, and Western blotting, and immunohistochemistry, respectively. SWATH acquisition on liquid chromatography-tandem mass spectrometry (LC–MS/MS) revealed 51 differentially expressed proteins in the CSF in sALS. Receiver operating characteristic (ROC) curves showed CXCL12 to be the most valuable candidate biomarker. We validated the values of CXCL12 in CSF with ELISA in two different cohorts. Besides sALS, increased CXCL12 levels were found in MS but were not altered in AD, SMA, and FTD. Therefore, increased CXCL12 levels in the CSF can be useful in the diagnoses of MS and sALS in the context of the clinical settings. CXCL12 immunoreactivity was localized in motor neurons in control and sALS, and in a few glial cells in sALS at the terminal stage; CXCR4 was in a subset of oligodendroglial-like cells and axonal ballooning of motor neurons in sALS; and CXCR7 in motor neurons in control and sALS, and reactive astrocytes in the pyramidal tracts in terminal sALS. CXCL12/CXCR4/CXCR7 axis in the spinal cord probably plays a complex role in inflammation, oligodendroglial and astrocyte signaling, and neuronal and axonal preservation in sALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive loss of upper and lower motor neurons [1]
Since YKL40 is expressed in inflammatory astrocytes [4,12], YKL40 in the cerebrospinal fluid (CSF) indicates the final balance of YKL40 metabolism in inflammatory astrocytes, and its delivery to the CSF in ALS
To establish the best candidate for ALS biomarker, we compared the area under the curve (AUC) values of candidates ; the results showed that the most likely was CXCL12 the AUC value of which showed significant differences or tendencies when compared with AAAS (p = 0.08), S100 calcium-binding protein A6 (S100A6) (p = 0.002), proteasomal ATPase associated factor 1 (PAAF1) (p = 0.08) and STX12 (p = 0.08) AUC values (Figure 2F)
Summary
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive loss of upper and lower motor neurons [1]. Based on the statistical significance in the exploratory proteomics study and the putative biological relevance, altered protein CSF levels were assessed in the first cohort of 43 sALS cases and 36 controls using commercial ELISA kits. Higher CXCL12 protein levels were detected in sALS (593.00 ± 33.08 pg/mL) compared with controls (305.00 ± 21.5 pg/mL) (p = 0.000) (Figure 2A). PAAF1 levels were significantly increased in sALS (7.07 ± 0.32 pg/mL) when compared with controls (4.80 ± 0.18 pg/mL) (p = 0.000) (Figure 2D). MRNA levels of CXCL12 (p = 0.002) were significantly increased in sALS when compared with controls. C-X-C motif chemokine ligand 12 (CXCL12/SDF1α), syntaxin-12 (STX12), and proteasomal ATPase associated factor 1 (PAAF1) protein levels are significantly increased, whereas aladin WD repeat nucleoporin (AAAS) and S100 calcium-binding protein A6 (S100A6) are significantly reduced in CSF of sALS. The localization of CXCR4 in axonal spheroids and degenerating motor neurons, and in a subset of oligodendrial cells and/or precursors in the pyramidal tracts, together with the presence of CXCR7 in motor neurons and reactive astrocytes in the pyramidal tracts, suggests a more complex scenario for CXCL12 involving neuron/axon preservation, oligodendroglial activation, and astrocyte signaling in sALS
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