VGF Protein and Its C-Terminal Derived Peptides in Amyotrophic Lateral Sclerosis: Human and Animal Model Studies

RESEARCH ARTICLE VGF Protein and Its C-Terminal Derived Peptides in Amyotrophic Lateral Sclerosis: Human and Animal Model Studies Carla Brancia1*, Barbara Noli1, Marina Boido2, Andrea Boi1, Roberta Puddu3, Giuseppe Borghero3, Francesco Marrosu3, Paolo Bongioanni4, Sandro Orrù5, Barbara Manconi6, Filomena D’Amato1, Irene Messana8, Federica Vincenzoni7, Alessandro Vercelli2, Gian-Luca Ferri1‡, Cristina Cocco1‡ a11111 1 NEF-Laboratory, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy, 2 Neuroscience Institute Cavalieri Ottolenghi, Department of Neuroscience, University of Turin, Turin, Italy, 3 Department of Neurology, Azienda Universitario Ospedaliera di Cagliari and University of Cagliari, Cagliari, Italy, 4 Neurorehabilitation Unit, Neuroscience Department, University of Pisa, Pisa, Italy, 5 Medical Genetics, Department of Medical Sciences, University of Cagliari, Binaghi Hospital, Cagliari, Italy, 6 Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy, 7 Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy, 8 Institute of Chemistry of the Molecular Recognition CNR, Rome, Italy ‡ These authors are co-senior authors on this work. * OPEN ACCESS Citation: Brancia C, Noli B, Boido M, Boi A, Puddu R, Borghero G, et al. (2016) VGF Protein and Its CTerminal Derived Peptides in Amyotrophic Lateral Sclerosis: Human and Animal Model Studies. PLoS ONE 11(10): e0164689. doi:10.1371/journal. pone.0164689 Editor: Anna-Leena Sirén, Julius-MaximiliansUniversitat Wurzburg, GERMANY Received: June 10, 2016 Accepted: September 29, 2016 Published: October 13, 2016 Copyright: © 2016 Brancia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Autonomous Region of Sardinia, Sardinia PO FSE 2007-1013’ funds (the L.R. 7/2007), and NEUROCARE onlus. 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 downregulation 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 NAPPsequence in all tissues studied, while in the mice and fibroblasts, we revealed also AQEEand TLQP- peptides. Taken together, selective VGF fragment depletion may participate in disease onset and/or progression of ALS. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0164689 October 13, 2016 1 / 16 VGF C-Terminal Derived Peptides in ALS Introduction 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 is a neutrophin induced gene that encodes for a single VGF precursor, composed of 617 (rat/mouse) / 615 (human) amino acids [1]. Studies in rats revealed that VGF mRNA is widely expressed in areas that are known to degenerate in ALS, including cerebral cortex, spinal cord, and cranial nerve motor nuclei as trigeminal and hypoglossal nuclei [2]. While the role of VGF in the nervous system is yet to be clarified, in VGF knock-out mice, synaptic plasticity and memory would be affected, in addition to a depressive behaviour [3]. 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 also 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]. In CSF from ALS patients, a VGF-derived 4.8 kDa fragment significantly decreased compared to controls [9], while immunoreactivity of the VGF full-length was reduced in parallel with development of ALS symptoms [8]. Moreover, the density of VGF immunoreactivity was also lower in spinal cords from sporadic ALS patients than in control subjects [10]. Interestingly, increased VGF expression would attenuate excitotoxic injury in primary mixed spinal cord cultures from G93A-SOD1 mice [8]. VGF could be also involved in neuroprotective mechanisms in stress-induced cell death in vitro as well as in vivo [10]. Recent literature data showed as human primary fibroblast cultures from ALS patients reflect some pathophysiological features observed in neuronal cells [11], with altered bioenergetic properties in neurodegenerative diseases, including ALS [12, 13]. Hence, we decided to use both patients’ plasma and fibroblasts to search for evidence of VGF changes. In order to confirm and extend our study, we also analysed the G93A-SOD1 animal model (spinal cord and plasma). Highly 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 pre (...truncated)