Abstract
VGF mRNA is widely expressed in areas of the nervous system known to degenerate in Amyotrophic Lateral Sclerosis (ALS), including cerebral cortex, brainstem and spinal cord. Despite certain VGF alterations are reported in animal models, little information is available with respect to the ALS patients. We addressed VGF peptide changes in fibroblast cell cultures and in plasma obtained from ALS patients, in parallel with spinal cord and plasma samples from the G93A-SOD1 mouse model. Antisera specific for the C-terminal end of the human and mouse VGF proteins, respectively, were used in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), while gel chromatography and HPLC/ESI-MS/MS were used to identify the VGF peptides present. Immunoreactive VGF C-terminus peptides were reduced in both fibroblast and plasma samples from ALS patients in an advanced stage of the disease. In the G93A-SOD1 mice, the same VGF peptides were also decreased in plasma in the late-symptomatic stage, while showing an earlier down-regulation in the spinal cord. In immunohistochemistry, a large number of gray matter structures were VGF C-terminus immunoreactive in control mice (including nerve terminals, axons and a few perikarya identified as motoneurons), with a striking reduction already in the pre-symptomatic stage. Through gel chromatography and spectrometry analysis, we identified one form likely to be the VGF precursor as well as peptides containing the NAPP- sequence in all tissues studied, while in the mice and fibroblasts, we revealed also AQEE- and TLQP- peptides. Taken together, selective VGF fragment depletion may participate in disease onset and/or progression of ALS.
Highlights
Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disorder characterized by selective degeneration and death of upper and lower motor neurons, respectively, in the cerebral cortex, brainstem and spinal cord
Using plasma samples (Fig 1A), we found that VGF C-terminus peptide levels were decreased in ALS patients at the advanced stage compared to controls and patients at the early stage
We found a VGF C-terminus reduction in fibroblasts (Fig 1B) obtained from ALS patients at the advanced stage, with either TDP-43 mutation or unidentified aetiology, compared to the control cells Raw data from enzyme-linked immunosorbent assay (ELISA) are reported in S2 Dataset
Summary
Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disorder characterized by selective degeneration and death of upper and lower motor neurons, respectively, in the cerebral cortex, brainstem and spinal cord. VGF cleavage can gives rise to a variety of bioactive peptides, of which those derived from its C-terminal portion have so far been more extensively studied. Two peptides from such region, named AQEE-30 and TLQP-62 appear to regulate synaptic function [4], while TLQP-62 can induce neurogenesis [5], or enhance neuronal hippocampal transmission [6], and would be required for hippocampal memory consolidation [7]. In the most commonly used murine model of ALS, i.e G93A-SOD1 transgenic mice overexpressing the mutated human SOD1 gene, VGF immunoreactivity was reported to be reduced in the cerebro-spinal fluid (CSF) and serum, as in the spinal cord in parallel with the progression of muscle weakness [8]. Characterized VGF antisera raised against the human and mouse C-terminal end of the VGF were used in immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA), while gel chromatography and HPLC high-resolution Electron Spray Ionization-MS (HPLC-ESI-MS) and HPLC/ESI-MS/MS (MS/MS) were carried out to identify VGF peptides present
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