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Proc Natl Acad Sci U S A
2016 Mar 15;11311:3060-5. doi: 10.1073/pnas.1600251113.
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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
.
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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, −60 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 #1–4,14, 20, and 39–42) (Table S1) and 35 oocytes (non-ALS patients #79, 80, 82–84, and 90–93) (Table S1). *P < 0.05. Holding potential, −60 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, −60 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 (●); in oocytes injected with α1β1εδ cDNA (5:1:1:1 ratio), the values were 56 ± 7 μM and 1 ± 0.01 (○).
Fig 5. Effects of PEA treatment on pulmonary function in ALS patients. Predicted FVC% of untreated (●, Table S2) and PEA–treated (○, 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 × 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).
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