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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
Neuro-Sys SAS, 410 Chemin Départemental 60, 13120 Gardanne, France ●
UPMC, UFR 927, 4 place Jussieu, 75252 Paris, France ●
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
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. 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
Control (green: [α-bungarotoxin, NMJ])
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)
Aβ (10 µmol/L) + MK801 (20 µmol/L)
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
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).
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.
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