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....................... Deleterious effects of amyloid beta peptide in the neuromuscular junction: consequences in ALS disease. Maud Combes , Philippe Poindron , Jean Mariani and Noelle Callizot #1 # # 1 Neuro-Sys SAS, 410 Chemin Départemental 60, 13120 Gardanne, France ● 1 #2 UPMC, UFR 927, 4 place Jussieu, 75252 Paris, France ● 2 Corresponding author : [email protected] Methods Introduction Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neurodegenerative disease of adults which preferentially attacks the neuromotor system. It has been shown that Amyloid-beta (Aβ) levels are elevated in spinal cords of late-stage superoxide dismutase 1 (SOD1) G93A mice (model of familial amyotrophic lateral sclerosis [ALS]) and that Aβ peptide(s) were localized predominantly within affected motor neurons (MN) and surrounding glial cells. Moreover, neuromuscular junction (NMJ) loss and MN degeneration were reduced in SOD1 mice when APP was genetically ablated, suggesting that endogenous APP actively contributes to the pathophysiology of this form of ALS. Additionally, Aβ and glutamate have been physiologically found in NMJs. Previous work done in our lab, showed the tight relationship between glutamate and Aβ in the NMJ. We showed that an interconnection between glutamate and Aβ peptide, as demonstrated in cortical and hippocampal neurons, is also operating in nerve– muscle co-cultures (Combes et al., 2015). Here, using a nerve–muscle co-culture system, we studied the toxicity of Aβ and the mechanisms involved in the process of NMJ death. The aim of this study was to investigated the role and the mechanism of Aβ on an in vitro model of functional NMJ. Culture: The nerve/muscle co-cultures were cultured according to Combes et al., 2015 (Askanas et al., 1987; Braun et al., 1996). Briefly, human muscle cells (SkMC) were plated onto gelatin-coated wells in 48-wells plates and grown in a proliferation medium. The medium was changed every 2 days. Five days after the beginning of the culture, immediately after satellite cell fusion, whole transverse slices of 13-day-old rat Wistar embryos spinal cord with dorsal root ganglia (DRG) were placed onto the muscle cell monolayer. After 24 h of co-culture, neurites were observed growing out of spinal cord explants. These neurites made contacts with myotubes and induce the first contractions after ~ 8 days. Pharmacological treatments: On day 33 following innervation, the co-cultures were injured with Aβ solution (Callizot et al., 2013) at 1.25, 2.5, 5 and 10 µmol/L for 4, 8, 16, 24 and 48 hours. Untreated cultures served as controls. MK801 at 20 µmol/L, Ifenprodil at 1 µmol/L, Memantine at 5 µmol/L, and Riluzole at 5 µmol/L were diluted in culture medium and pre-incubated 1 hour before Aβ intoxication. Staining of NMJs and immunostaining of axons: After 4, 8, 16, 24 and 48 h, co-cultures were incubated with 500 nmol/L α-bungarotoxin (αBgt) coupled with Alexa 488 to detect NMJs. The mean size of NMJs was measured to assess the quality of innervation, (the larger the NMJs, the stronger the innervation). The immunolabelled cultures were examined with MetaXpress (Molecular Devices, USA) at X 20 magnification. For each condition, 63 fields per well were observed (representing the total surface of the well), and six wells per conditions were analyzed. Quantification of glutamate in co-culture supernatants: After 16 h, glutamate was dosed in supernatant using Amplex red Glutamic acid assay kit (Molecular probes) and following manufacturer’s recommendations. Caspase 3 evaluation (Western-blotting [WB]): After 4 h of Aβ treatment, cells were lysed with Cellytic and immediately frozen at -80°C. All reagents were prepared and used according to manufacturer’s recommendations (Simon™ - ProteinSimple - www. proteinsimple.com). Anti-Caspase 3, primary antibody was used for WB analysis. Results Representative pictures Effect of different concentrations of β amyloid applied for different timing on the NMJ Glutamate release in co-culture supernatant after β amyloid application Control (green: [α-bungarotoxin, NMJ]) Muscle fiber Aβ (10 µmol/L) Release of glutamate after 16 h of Aβ application Effect of Aβ on NMJ mean size: Only a minor effect was observed on the NMJ area when Aβ was applied at 1,25 µmol/L (except for 48 h). At 2,5 µmol/L of Aβ the toxic effect was significant at 8, 16, 24 and 48 h of exposure. The toxic effect on the mean size of NMJs was significant at 5 and 10 µmol/L after all times of exposure. The extent of injury was dependent on the time of exposure and concentrations of Aβ. Caspase 3 evaluation: An increase of caspase-3 level was observed after 4 h of Aβ (1,25 µmol/L) application. Aβ (10 µmol/L) + MK801 (20 µmol/L) NMJ Aβ (10 µmol/L) + Ifenprodil (1 µmol/L) AβO treated .................................................................................................................................... Effect of Riluzole on NMJ mean size in nerve/muscle co-culture injured with β amyloid Effect of different antagonists on NMJ mean size after β amyloid injury (24 h) Aβ (10 µmol/L) + Memantine (5 µmol/L) Aβ (10 µmol/L) + Riluzole (5 µmol/L) Conclusions Protective effect of MK801, Ifenprodil and Memantine on NMJ mean size injured with Aβ: MK801 (20 µmol/L, a non competitive N-methylD-aspartate receptor [NMDAR]) and Memantine (5 µmol/L, NMDAR antagonist with extrasynaptic localization selectivity) exerted a fully protective effect, Ifenprodil (1 µmol/L, Non-competitive NMDAR antagonist displays GluN2B [formerly NR2B] subunit selectivity) exerted a partially protective effect against Aβ injury (10 µmol/L) as seen by measurement of NMJ mean size. © Neuro-Sys SAS - JSFM 2015 - November 2015 Protective effect of Riluzole on NMJ mean size injured with Aβ: Pre-treatment of 1 h with Riluzole (5 µmol/L) displayed a significant protective effect on the mean size of NMJ after 24 h and 48 h of exposure to Aβ (2,5 µmol/L). Riluzole, a Na+ channel blocker, fully protected NMJ from Aβ injuries. Riluzole is known to reduce intracellular increases of Na+ and to reverse the sodium calcium exchangers. In addition, Riluzole acts as an anti-glutamatergic agent via the increase of glutamate uptake by activating glutamate transporters and reducing the release of glutamate (Nagoshi et al., 2015). INNOVATIVE RESEARCH www.neuro-sys.fr The following points were shown in this study: ● Aβ application on functional co-culture induced a large toxic effect on NMJ. ● Aβ application induced a large glutamate release. ● Aβ application induced an increase of caspase-3 preceding the NMJ loss. ● MK801 or Memantine fully protected co-culture from injuries. ● Ifenprodil showed a partial protection. ● Riluzole, the only approved molecule for ALS treatment, significantly protected co-culture from Aβ injuries. Altogether these results suggest that Aβ could be an important element triggering excitotoxicity events found in ALS. Further investigations are ongoing in our lab to unravel the respective role of each cellular compartment of the co-culture in the pathological role of Aβ. References: Combes et al., (2015) J Neurosci Res. 2015 Apr;93(4):633-43 ● Callizot et al., (2013) J Neurosci Res. 2013;91(5):70616 ● Braun, S. et al., (1996) J Neurol Sci 136: 17-23 ● Askanas V., et al., (1987). J Neurocytol., 16(4):523-37 ● Bryson et al., (2012) Hum Mol Genet. 1;21(17):3871-82 ● Nagoshi et al., (2015) Molecules. 2015, 20, 7775–7789