Standardized phytotherapic extracts rescue anomalous locomotion and electrophysiological responses of TDP-43 Drosophila melanogaster model of ALS

Abstract

Findings from studies using animal models expressing amyotrophic lateral sclerosis (ALS) mutations in RNA-binding proteins, such as Transactive Response DNA-binding protein-43 (TDP-43), indicate that this protein, which is involved in multiple functions, including transcriptional regulation and pre-mRNA splicing, represents a key candidate in ALS development. This study focuses on characterizing, in a Drosophila genetic model of ALS (TDP-43), the effects of Mucuna pruriens (Mpe) and Withania somnifera (Wse). Electrophysiological and behavioural data in TDP-43 mutant flies revealed anomalous locomotion (i.e. impaired climbing with unexpected hyperactivity) and sleep dysregulation. These features, in agreement with previous findings with a different ALS model, were at least partially, rescued by treatment with Mpe and Wse. In addition, electrophysiological recordings from dorsal longitudinal muscle fibers and behavioral observations of TDP-43 flies exposed to the volatile anaesthetics, diethyl ether or chloroform, showed paradoxical responses, which were normalized upon Mpe or Wse treatment. Hence, given the involvement of some potassium channels in the effects of anaesthetics, our results also hint toward a possible dysregulation of some potassium channels in the ALS-TDP-43 Drosophila model, that might shed new light on future therapeutic strategies pertaining to ALS.

Figure 1

Mpe or Wse treatment ameliorates the climbing and locomotion behaviour of TDP-43 mutant flies. Effects of Mucuna pruriens (Mpe) or Withania somnifera (Wse) extracts on the climbing (A) and walking (B) activity of adult GAL4 flies, untreated mutants (TDP-43) and Mpe- or Wse-treated TDP-43. The treatment was administered during the adult stage and its effect was assayed in two age groups (Group I: 3–6 day old flies, Group II: 10–15 day old flies). In each set of data in (A) the green horizontal line indicates the mean of the corresponding distribution. * and ^# indicate p < 0.05, one-way ANOVA (non parametric) followed by Dunn’s multiple comparisons test, compared to GAL4 and TDP-43, respectively. The histograms in (B) display the walking activity during the 12-h daytime (white) and 12-h nighttime (black) periods. * and ^# indicate p < 0.05, one-way ANOVA followed by HSD post-hoc test, compared to GAL4 and TDP-43, respectively.

Figure 2

Effect of TDP-43 loss-of-function mutation and treatment with Mpe or Wse on electrophysiological parameters recorded from DLM. (A) Representative traces obtained from different experimental conditions, in which ePSP latency is calculated as the time (ms) from stimulus application to the peak of post-synaptic potential (PSP) (red scattered lines), and PSP peak is calculated by measuring the maximal amplitude of the response starting from the baseline. Scale bar, 10 mV/1 ms. (B) Ámplitude (mV) and (C) latency (ms) recorded in flies from different experimental groups. Bar graphs represent the mean  ± standard error of the mean (SEM) of ePSPs. * indicates p < 0.05 compared to GAL4, one-way ANOVA, followed by Bonferroni post-hoc test. N = 22 for all experimental groups. (D) Representative traces obtained from different experimental groups in which PSPs were evoked in response to 10 stimuli delivered at 100 Hz. Scale bar 10 mV/20 ms. * indicates the presence of the event. (E) Scatter plot graphs showing the changes in ePSPs amplitude following stimulation at increasing frequency (from 10 to 200 Hz) of a train of 10 consecutive stimuli (the effect at 100 Hz is highlighted in yellow). All values are expressed as the mean ± SEM of the % of failure observed in every train. * indicates p < 0.05 vs GAL4. (F) Bar graph representing the averaged % of failure that was plotted in all experimental groups. “Strike” is the % of recordings where failure was not greater that 10%, while “failure” is the number of recordings where the % of failure was greater than 20%. Data corresponds to the % of failures evaluated @ 100 Hz. * indicates p < 0.05 compared to GAL4, unpaired t-test.

Figure 3

Mpe or Wse treatment modulates sleep parameters inTDP-43 mutant flies. Effects of Mucuna pruriens (Mpe) or Withania somnifera (Wse) extracts on total sleep (A,B), the number of sleep episodes (C,D) during the 12-h daytime (white) and 12-h nighttime (gray) periods and on the average of longest sleep episodes (AvLo)/24 h (E,F) of adult GAL4 flies, untreated mutants (TDP-43) and Mpe- and Wse-treated TDP-43 flies. The treatment was administered during the adult stage and its effect assessed in two groups (Group I: 7 days and Group II: 14 days) of the flies’ life-span. The top and bottom of the box and whisker plots show the upper and lower quartiles, respectively. The horizontal line in the middle indicates the median of the corresponding distribution, while the minimum and maximum observed values are indicated by the bars connected to the box. * and ^# indicate p < 0.05, one-way ANOVA followed by HSD post-hoc test, compared to GAL4 and TDP-43, respectively.

Figure 4

Mpe or Wse treatment prevents the effect of ether and chloroform administration on spontaneous PSPs in TDP-43 mutant flies. Treatment with Mpe or Wse rescues the effect of TDP-43 loss-of-function mutation to the enhancement of frequency of spike potentials due to Drosophila DLMs spontaneous contractions induced by 30 s of diethyl ether (Et) or chloroform (Chl) exposure. (A–H) Representative traces and plots obtained from GAL4 flies, TPD-43 treated and untreated mutants in which spontaneous PSPs were recorded in the absence, after 30 s and after 4 min wash-out of anaesthetic exposure. Arrows in graphs indicate when vapours of volatile anaesthetics delivery was begun. Scale bars: left panels, 20 mV/2 s, right panels 10 mV/2 s.