P7C3-A20 Neuroprotection Is Independent of Wallerian
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1544 Cellular, molecular and developmental neuroscience P7C3-A20 neuroprotection is independent of Wallerian degeneration in primary neuronal culture Ciaran S. Hilla,b, David K. Menonb,c and Michael P. Colemana The antiapoptotic, neuroprotective compound P7C3-A20 transection or vincristine administration. Furthermore, there 12/04/2018 on BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3p1zuFA1MWB0krHBsTFhDcZJFJVHasAXOPbtj6FLYIePhYDNS6FsNeg== by https://journals.lww.com/neuroreport from Downloaded reduces neurological deficits when administered to murine was a concentration-dependent neurotoxicity. These in-vivo models of traumatic brain injury. P7C3-A20 is findings are important in understanding the mechanism by Downloaded thought to exert its activity through small-molecule which P7C3-A20 mediates its effects – a key step before activation of the enzyme nicotinamide moving to human clinical trials. NeuroReport 29:1544–1549 from phosphoribosyltransferase. This enzyme converts Copyright © 2018 The Author(s). Published by Wolters https://journals.lww.com/neuroreport nicotinamide to nicotinamide mononucleotide, the Kluwer Health, Inc. precursor to nicotinamide adenine dinucleotide synthesis. NeuroReport 2018, 29:1544–1549 Alterations to this bioenergetic pathway have been shown to induce Wallerian degeneration (WD) of the distal neurite Keywords: axon, P7C3-A20, protection, Wallerian degeneration following injury. This study aimed to establish whether aJohn van Geest Centre for Brain Repair, bDepartment of Medicine, Division of Anaesthesia and cDepartment of Clinical Neurosciences, Wolfson Brain Imaging by P7C3-A20, through induction of nicotinamide BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3p1zuFA1MWB0krHBsTFhDcZJFJVHasAXOPbtj6FLYIePhYDNS6FsNeg== Centre, University of Cambridge, Cambridge, UK phosphoribosyltransferase activity, would affect the rate of WD. The model systems used were dissociated primary Correspondence to Ciaran S. Hill, MD, PhD, University of Cambridge, Cambridge CB2 0PY, UK cortical neurons, dissociated superior cervical ganglion Tel: + 44 122 333 1160; fax: + 44 122 333 1174; e-mail: [email protected] neurons and superior cervical ganglion explants. P7C3-A20 Received 18 June 2018 accepted 21 September 2018 failed to show any protection against WD induced by neurite Introduction ameliorated behavioural deficits, including motor, learning, In 2010 the antiapoptotic, neuroprotective aminopropyl car- memory loss and axon loss in the CA1 stratum radiatum bazole agent P7C3 was discovered using a novel in-vivo region of blast injury exposed mice [5]. Furthermore, screening approach [1]. It has been proposed that the com- P7C3-A20 administration in a rat fluid-percussion injury pound exerts its actions by activating the enzyme nicotina- model showed a reduction in contusion volume and mide phosphoribosyltransferase (NAMPT). NAMPT is the improved sensorimotor and cognitive function [6]. rate-limiting enzyme in the nicotinamide adenine dinucleo- The in-vivo phenotypic screening method by which the tide (NAD) salvage pathway that converts nicotinamide to P7C3 compounds were discovered meant that further nicotinamide mononucleotide (NMN), the precursor to work has been required to elucidate their mode of action. NAD synthesis (Fig. 1). The NAD salvage pathway is Subsequent research has shown that P7C3-related com- important in Wallerian degeneration (WD), the active pounds have some NAMPT-activating activity. NAMPT degeneration of an axon or neurite distal to a site of injury. is an important enzyme in the WD pathway. However, P7C3, and one of its more active fluorinated enantiomers the ability of NAMPT activation to protect against WD is P7C3-A20, has shown promise as a therapeutic agent in a not established and it has not been definitively shown number of conditions that have been associated with WD. that NAMPT activation is the means by which It was capable of blocking 1-methyl-4phenyl-1,2,3,4-tetra- P7C3-based compounds exert their therapeutic action, or on 12/04/2018 hydropyridine-induced death of dopaminergic substantia that they do not have other actions. nigra cells in an adult mouse model of Parkinson’sdisease [2]. Its ability to protect spinal motor neurons from cell death was demonstrated both histologically and functionally in the Materials and methods G93A-SOD1 mouse model of amyotrophic lateral sclerosis Mouse origin and cell culture − / − [3]. The potential for benefit of P7C3 derivatives in trau- C57BL/6 wild-type and SARM1 mice were obtained from Babraham Institute (Babraham, UK). C57BL/ matic brain injury (TBI) was indicated by the findings that s the analogue P7C3-S243, prevented retinal ganglion cell 6OlaHsd-Wlds (Wld ) mice, originally obtained from loss following experimental blast injury in mice [4]. This Harlan (Bicester, UK), were maintained as a separate colony at the Babraham Institute for 13 years. All animal was followed by a demonstration that P7C3-S243 also work was performed in accordance with the 1986 Animals (Scientific Procedures) Act under project licence PPL This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduc- 70/7620. All superior cervical ganglion explant (SCGe) tion in any medium, provided the original work is properly cited. and dissociated superior cervical ganglion (dSCG) 0959-4965 Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. DOI: 10.1097/WNR.0000000000001146 P7C3-A20 and Wallerian degeneration Hill et al. 1545 Fig. 1 (Olympus, Shinjuku, Tokyo, Japan) using × 20 magnifi- cation and phase-contrast imaging. Neurite degeneration index The neurite degeneration index (NDI) is an established quantitative measure of neurite degeneration (ND) [9]. Images of the same, or similar, the field of neurites are obtained at different time points or treatment conditions at a controlled level of magnification (×20) and illumi- nation. In the case of SCGe the imaged field is typically 5 mm from the main explant mass where the density of the neurites allows clear visualization of morphological change. The NDI is calculated by an Image J (University The nicotinamide adenine dinucleotide (NAD) synthesis pathway. The of Wisconsin, Wisconsin, USA) plug-in algorithm that is de novo NAD synthesis pathway generates NAD from the essential operator independent. The output is a unitless number aromatic amino acid tryptophan. Nicotinic acid mononucleotide (NaMN) is converted to nicotinic acid adenine dinucleotide (NaAD) by from 0 to 1 based on the degree of neurite continuity or nicotinamide mononucleotide adenylyltransferase (NMNAT). NaAD is fragmentation. Most healthy neurites score around then converted to NAD by NAD synthetase. There are three additional – thee salvage pathways. Nicotinamide mononucleotide (NMN) is 0.1 0.3, whereas a profoundly degenerated field would produced by nicotinamide phosphoribosyltransferase (NAMPT) from be in the range of 0.6–0.9. In the case of complete nicotinamide (NAM), and by nicotinamide riboside kinase (NRK) from degeneration, with or without detachment, a score of nicotinamide riboside (NR). NMN is then converted to NAD by NMNAT. Alternatively, NAM is converted to nicotinic acid (NA) by nicotinamidase, 0.9 is assigned [9]. and then NA is converted to NaMN by nicotinic acid phosphoribosyltransferase (NaPRT). P7C3-A20 is reported to acts as a NAD/NADH-Glo assay small-molecule activator of the enzyme NAMPT within the bioenergetic NAD synthesis pathway. Levels of NAD were measured using the NAD/NADH- Glo assay (Promega, Madison, Wisconsin, USA). The assay was performed according to the manufacturer’s protocol. The assay contains a cycling reductase enzyme cultures were obtained from humanely killed postnatal that reduces a proluciferin reductase substrate to luciferin day 0 (P0) to P2 mice and were prepared as described in the presence of NADH. Luminescence signals were in vitro previously [7,8]. Cells were cultured for 7 days measured by PHERAstar plate reader (BMG Labtech, before experimentation. SCGe were plated on 35 mm Ortenberg, Germany). Nunc cell culture dishes (Thermo-Fisher Scientific, Waltham, Massachusetts, USA), and dSCGs were plated Statistics μ on low 35 mm -dishes (ibidi, Martinsried, Germany). Data are expressed as a mean value with error bars Primary cortical neuronal cells were extracted by dis- representing SEM unless otherwise stated. A number of secting out a section of cortical mantle, trypsinizing and repeats and replicates and statistical tests are detailed in dissociating the neurons before separating by cen- each figure legend. A P value of 0.05 or less was con- trifugation. Viability was confirmed to be greater than sidered significant for any data set. Asterisks represent 95%. The cells were then plated and cultured on low the statistical significance as follows: *P ≤ 0.05, μ in vitro 35 mm -dishes (ibidi) for 7 days before experi- **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, P < 0.05 (NS). mentation. In all cases, dishes were coated with poly- All statistical tests and graphs were generated with μ L-lysine (25 g/ml; Sigma, St. Louis, Missouri, USA), GraphPad Prism Software, version 7.0 (GraphPad μ 2 then laminin (2 g/cm ; Sigma). All cultures were main- Software Inc., La Jolla, USA). tained in an incubator at a constant 37°Cwith5%CO2. Results P7C3-A20 To select an appropriate concentration of P7C3-A20 to test The compound P7C3-A20 (cat no. 2850) was sourced in WD, we