Xenbase may experience sporadic downtime from September 1st to September 3rd due to scheduled IT maintenance work.
We apologize for the inconvenience.
Click on this message to dismiss it.
Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Proc Natl Acad Sci U S A
2016 Mar 15;11311:3060-5. doi: 10.1073/pnas.1600251113.
Show Gene links
Show Anatomy links
Acetylcholine receptors from human muscle as pharmacological targets for ALS therapy.
Palma E
,
Reyes-Ruiz JM
,
Lopergolo D
,
Roseti C
,
Bertollini C
,
Ruffolo G
,
Cifelli P
,
Onesti E
,
Limatola C
,
Miledi R
,
Inghilleri M
.
???displayArticle.abstract???
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons that leads to progressive paralysis of skeletal muscle. Studies of ALS have revealed defects in expression of acetylcholine receptors (AChRs) in skeletal muscle that occur even in the absence of motor neuron anomalies. The endocannabinoid palmitoylethanolamide (PEA) modified the clinical conditions in one ALS patient, improving muscle force and respiratory efficacy. By microtransplanting muscle membranes from selected ALS patients into Xenopus oocytes, we show that PEA reduces the desensitization of acetylcholine-evoked currents after repetitive neurotransmitter application (i.e., rundown). The same effect was observed using muscle samples from denervated (non-ALS) control patients. The expression of human recombinant α1β1γδ (γ-AChRs) and α1β1εδ AChRs (ε-AChRs) in Xenopus oocytes revealed that PEA selectively affected the rundown of ACh currents in ε-AChRs. A clear up-regulation of the α1 subunit in muscle from ALS patients compared with that from non-ALS patients was found by quantitative PCR, but no differential expression was found for other subunits. Clinically, ALS patients treated with PEA showed a lower decrease in their forced vital capacity (FVC) over time as compared with untreated ALS patients, suggesting that PEA can enhance pulmonary function in ALS. In the present work, data were collected from a cohort of 76 ALS patients and 17 denervated patients. Our results strengthen the evidence for the role of skeletal muscle in ALS pathogenesis and pave the way for the development of new drugs to hamper the clinical effects of the disease.
Fig. 1. PEA affects ACh currents in oocytes injected with ALS muscle membranes. Bar graphs show the percentage of ACh amplitude remaining before and after 10 μM PEA treatment (10 min, 50 oocytes), as indicated. (Inset) Sample current traces elicited during prolonged ACh application (500 μM for 60 s) in one oocyte (representative of 10) injected with ALS muscle membranes before and after PEA treatment. In this and subsequent figures, horizontal filled bars indicate the timing of ACh applications, and horizontal open bars indicate the drug applications. *P < 0.05. Holding potential, â60 mV.
Fig 2. Effect of PEA on IACh rundown. (A) Bar graphs of residual IACh (i.e., the ratio between sixth and first IACh amplitude) before and after treatment with 10 µM PEA (10 min) in oocytes injected with ALS muscle membranes. (Inset) Representative currents elicited by the first and sixth ACh application (500 µM; horizontal filled bar) in one oocyte injected with ALS muscle membranes before and after PEA (open bar), as indicated. (B) Bar graphs represent IACh remaining in oocytes injected with non-ALS denervated muscle membranes before and after PEA, as in A. (Inset) Representative currents elicited in one oocyte injected with non-ALS denervated muscle membranes, as in A. Data are the means ± SEM of 40 oocytes (ALS patients #1â4,14, 20, and 39â42) (Table S1) and 35 oocytes (non-ALS patients #79, 80, 82â84, and 90â93) (Table S1). *P < 0.05. Holding potential, â60 mV.
Fig 3. Effect of PEA on oocytes expressing ε-AChRs or γ-AChRs. Bar graphs of residual IACh (i.e., the ratio between sixth and first IACh amplitude) before and after 10 µM PEA treatment (10 min) in oocytes expressing ε-AChRs (Left) or γ-AChRs (Right), as indicated. (Inset) Representative superimposed currents elicited by the first and sixth ACh application (500 µM; horizontal filled bar) in oocytes expressing ε-AChRs or γ-AChRs before and after PEA (open bar), as indicated. *P < 0.05. Holding potential, â60 mV.
Fig 4. The α1 AChR subunit is increased in ALS patients. A statistically significant difference (P = 0.0058) was found in the relative expression level of the α1 ACh subunit in ALS (gray bars) compared with non-ALS (white bars) patients (twelve for each group; Table S1). In contrast, no significant change was found in the expression of the β1 subunit. (Inset) Amplitude of currents evoked by various ACh concentrations, expressed as a percentage of the maximal current evoked by 1 mM ACh, plotted as mean ± SEM and best fitted with Hill curves. Averaged EC50 values and nH in oocytes injected with α1β1εδ cDNAs (1:1:1:1 ratio) were 18.4 ± 3.8 μM and 1.2 ± 0.01 (â); in oocytes injected with α1β1εδ cDNA (5:1:1:1 ratio), the values were 56 ± 7 μM and 1 ± 0.01 (â).
Fig 5. Effects of PEA treatment on pulmonary function in ALS patients. Predicted FVC% of untreated (â, Table S2) and PEAâtreated (â, Table S2) patients at baseline (0) and after 12 and 24 wk of treatment. Data are normalized to the baseline value in each patient; values are expressed as mean ± SEM. The decrease in FVC% was higher in untreated patients than in PEA-treated patients (repeated measures ANOVA, F = 4.46, P = 0.0154 for time à treatment effect), indicating that the disease progressed more slowly during the 24-wk follow-up in patients receiving PEA.
Fig. S1. Effects of PEA treatment on tracheotomy and death. Cumulative proportion surviving related to tracheotomy and death in untreated (red trace, 36 patients) and PEA-treated (blue trace, 28 patients) patients in the 6-mo follow-up period after 6 mo of PEA treatment. The cumulative proportion surviving is the proportion of ALS patients who survived without tracheotomy (days). Note that death and tracheotomy occurred more frequently in untreated than in PEA-treated patients (P = 0.05).
André,
The endocannabinoid system: its roles in energy balance and potential as a target for obesity treatment.
2010, Pubmed
André,
The endocannabinoid system: its roles in energy balance and potential as a target for obesity treatment.
2010,
Pubmed
Brenner,
On the effect of muscle activity on the end-plate membrane in denervated mouse muscle.
1989,
Pubmed
Brooks,
El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial "Clinical limits of amyotrophic lateral sclerosis" workshop contributors.
1994,
Pubmed
Bustin,
The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.
2009,
Pubmed
Cedarbaum,
The ALSFRS-R: a revised ALS functional rating scale that incorporates assessments of respiratory function. BDNF ALS Study Group (Phase III).
1999,
Pubmed
Clemente,
Amyotrophic lateral sclerosis treatment with ultramicronized palmitoylethanolamide: a case report.
2012,
Pubmed
Deflorio,
Riluzole blocks human muscle acetylcholine receptors.
2012,
Pubmed
,
Xenbase
Dupuis,
Neuromuscular junction destruction during amyotrophic lateral sclerosis: insights from transgenic models.
2009,
Pubmed
Eusebi,
Microtransplantation of ligand-gated receptor-channels from fresh or frozen nervous tissue into Xenopus oocytes: a potent tool for expanding functional information.
2009,
Pubmed
,
Xenbase
Inghilleri,
Clinical neurophysiology in ALS.
2011,
Pubmed
KATZ,
FURTHER OBSERVATIONS ON THE DISTRIBUTION OF ACTYLCHOLINE-REACTIVE SITES IN SKELETAL MUSCLE.
1964,
Pubmed
Mattace Raso,
Palmitoylethanolamide in CNS health and disease.
2014,
Pubmed
Miledi,
Microtransplantation of neurotransmitter receptors from cells to Xenopus oocyte membranes: new procedure for ion channel studies.
2006,
Pubmed
,
Xenbase
MILEDI,
Junctional and extra-junctional acetylcholine receptors in skeletal muscle fibres.
1960,
Pubmed
Miller,
General considerations for lung function testing.
2005,
Pubmed
Miller,
Standardisation of spirometry.
2005,
Pubmed
Moreno-Martet,
Changes in endocannabinoid receptors and enzymes in the spinal cord of SOD1(G93A) transgenic mice and evaluation of a Sativex(®) -like combination of phytocannabinoids: interest for future therapies in amyotrophic lateral sclerosis.
2014,
Pubmed
Musarò,
Understanding ALS: new therapeutic approaches.
2013,
Pubmed
Nakanishi,
Regulation of motoneuron excitability via motor endplate acetylcholine receptor activation.
2005,
Pubmed
NULL,
Standardization of Spirometry, 1994 Update. American Thoracic Society.
1995,
Pubmed
Oppenheim,
The rescue of developing avian motoneurons from programmed cell death by a selective inhibitor of the fetal muscle-specific nicotinic acetylcholine receptor.
2008,
Pubmed
Oppenheim,
Reduction of neuromuscular activity is required for the rescue of motoneurons from naturally occurring cell death by nicotinic-blocking agents.
2000,
Pubmed
Oz,
The endogenous cannabinoid anandamide inhibits alpha7 nicotinic acetylcholine receptor-mediated responses in Xenopus oocytes.
2003,
Pubmed
,
Xenbase
Oz,
Receptor-independent effects of endocannabinoids on ion channels.
2006,
Pubmed
Palma,
Physiological characterization of human muscle acetylcholine receptors from ALS patients.
2011,
Pubmed
,
Xenbase
Pellegrino,
Interpretative strategies for lung function tests.
2005,
Pubmed
Quanjer,
Lung volumes and forced ventilatory flows. Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society.
1993,
Pubmed
Robberecht,
The changing scene of amyotrophic lateral sclerosis.
2013,
Pubmed
Rocha,
Early changes of neuromuscular transmission in the SOD1(G93A) mice model of ALS start long before motor symptoms onset.
2013,
Pubmed
Rossi,
The endocannabinoid system in the inflammatory and neurodegenerative processes of multiple sclerosis and of amyotrophic lateral sclerosis.
2010,
Pubmed
Schroeder,
The RIN: an RNA integrity number for assigning integrity values to RNA measurements.
2006,
Pubmed
Scuderi,
Palmitoylethanolamide exerts neuroprotective effects in mixed neuroglial cultures and organotypic hippocampal slices via peroxisome proliferator-activated receptor-α.
2012,
Pubmed
Silani,
The diagnosis of Amyotrophic lateral sclerosis in 2010.
2011,
Pubmed
Terrado,
Motoneuron survival is enhanced in the absence of neuromuscular junction formation in embryos.
2001,
Pubmed
Thomsen,
Analysis of qPCR data by converting exponentially related Ct values into linearly related X0 values.
2010,
Pubmed
Tsujihata,
The motor end-plate fine structure and ultrastructural localization of acetylcholine receptors in amyotrophic lateral sclerosis.
1984,
Pubmed
Witzemann,
Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses.
1991,
Pubmed
