Western blot analysis of <i>PINK1^B9</i> heads shows a reduced expression of the bruchpilot protein.

<p><b>A:</b> Western blot analysis of adult head homogenate from WT and <i>PINK1^B9</i> flies showing the nc82-labeled Bruchpilot protein band (BRP, top) and the loading control Tubulin (bottom). The amount of Bruchpilot protein was quantified by analyzing the intensity of the bands on the autoradiogram with a densitometer. Data were normalized by dividing optical density of the bands corresponding to Bruchpilot protein by that of the band for α-Tubulin. <b>B:</b> Statistical evaluation of the densitometric data shows that the expression of Bruchpilot protein in <i>PINK1^B9</i> is significantly lower compared to WT (*significantly lower than its matching value; <i>P</i><0.05).</p

The antennal lobes reveal abnormalities in <i>PINK1^B9</i> mutants.

<p>ALs were stained for the expression of Bruchpilot protein. Panel A: Confocal micrographs of a frontal view of a couple of ALs (indicated by the arrow heads), with their well-defined glomeruli in WT and the mutant (Scale bar = 50 µm). <b>B,C:</b> The volume rendering for the monoclonal nc82 antibody-stained AL as seen from a frontal view in a WT (B) and in a <i>PINK1^B9</i> mutant (C) (scale bars = 30 µm). Ten specimens in the age range of 3–10 days for each strain were analyzed; the volumes measured were averaged and the statistical differences evaluated. Mean values ± S.E. are reported in C: no significant difference was detected (<i>P</i>>0.05). E,F: Higher magnification photomicrographs of a single AL in WT and <i>PINK1^B9</i> mutant respectively (scale bars = 20 µm). The latter reproducibly displays a less intensive staining and the glomeruli are not as clearly defined as in the WT. The gray scale value, taken as the index of intensity, was measured on ALs from binarized stacks of images (n = 10 for each strain). Mean values ± S.E. for both WT and <i>PINK1^B9</i> mutants are shown in G. Statistical evaluation of the data shows that the staining intensity in <i>PINK1^B9</i> is significantly lower compared to WT; (*significantly different from its matching value; <i>P</i><0.05).</p

Mitochondria within the presynaptic boutons in the antennal lobes present structural alterations in <i>PINK1^B9</i>mutants.

<p>Transmission electron microscopy (TEM) images of mitochondria within the olfactory bulbs of WT and <i>PINK1^B9 Drosophila</i>. A: Two mitochondria in a presynaptic bouton of a WT (scale bar = 200 µm). The regular array of mitochondrial cristae (arrowheads) is surrounded by an electron-dense matrix. B: A mitochondrion in a presynaptic bouton of a <i>PINK1^B9</i> mutant (scale bar = 100 µm). The mitochondrion presents a swelling on its external membrane (arrow) and the mitochondrial cristae are highly degenerated (asterisk). C: a conventional mitochondrion in a WT (scale bar = 200 µm). The regular electron-dense matrix surrounds the mitochondrial cristae (asterisk). D: The abnormal mitochondrial morphology in a <i>PINK1^B9</i> mutant. The mitochondrial cristae are fragmented (arrowhead). E: The abnormal mitochondrial morphology in a <i>PINK1^B9</i> mutant (scale bar = 100 µm). Remnants of the mitochondrial cristae are visible within the mitochondrial matrix (scale bar = 100 µm).</p

Electroantennogram responses in wild type and <i>PINK1^B9</i> mutants.

<p>Dose-response relationships for olfactory stimulations in WT and <i>PINK1^B9</i> adult flies and their differences in signal amplitude. Histograms in A and D show the EAG results in flies from Group I (age range from 3 to 6 days), from Group II (age range from 15 to 20 days) in B and E and from Group III (age range from 27 to 30) in C and F. Values shown are mean ± S.E.M. of the EAG amplitude. Stimuli are dilutions of essential oils (rosemary, RM; lentisk, LT; myrtle, MT) and synthetic compounds (α-pinene, αp; hexanol, hex; isoamyl acetate, iso; ethyl 3-hydroxybutyrate, e3OH) administered in a 3-step dose from 0.1 to 10% in hexane. EAGs obtained in WT and <i>PINK1^B9</i> displayed strong similarities in the dose-response to stimuli; statistically significant differences were observed between WT and the mutant strains (^* significantly different from its previous concentration; ^** significantly different from its matching stimulus; <i>P</i><0.05). In G, representative EAG tracings recorded in WT (upper) and <i>PINK1^B9</i> (lower) in response to olfactory stimulation with rosemary oil (RM) administered in increasing concentrations. The sample tracings show that the amplitude of the depolarization in the baseline is clearly higher in WT than in <i>PINK1^B9</i>.</p

Lifespan in wild type and <i>PINK^B9</i> mutant adults.

<p>The graph shows the survival rate observed in wild type and <i>PINK1^B9</i> mutants. <i>PINK1^B9</i> had a reduced lifespan compared to WT.<i>PINK1^B9</i> flies started to die dramatically at the 15^th days after eclosion with 50% of flies being dead after 30 days (P<0.0001).</p

Effect of LRRK2 gene mutation and treatment with <i>Wse</i> on the “frequency of following” recorded in <i>Drosophila</i> DLM.

<p>(A) Representative traces obtained from three different flies in which PSPs were evoked in response to 10 stimulations at 100 (top) or 200 Hz (bottom). (B,C) Scatter plot graphs showing the changes in PSP amplitude following stimulation at 100 (B) or 200 Hz (C). All values are expressed as the mean ± SEM of the % relative to the amplitude of the first PSP. *indicates p< 0.05 compared to WT and <i>Wse</i>-untreated LRRK2 (B) and compared to WT and <i>Wse</i>-treated LRRK2 (C), two-way ANOVA.</p

Effect of LRRK2 gene mutation and treatment with <i>Wse</i> 1% (L^-/A⁺) on PSP latency and amplitude recorded from <i>Drosophila</i> DLM.

<p>(A) Representative traces obtained from three different flies in which PSP latency is calculated as the time (ms) from stimulus application to the peak of PSP (black arrows). (B, C) Bar graphs represent the mean ± SEM of PSP latency (ms) and amplitude (mV) recorded from flies of the indicated experimental groups. *indicates p< 0.05 compared to WT, **indicate p<0.05 compared to treated LRRK2; one-way ANOVA, followed by Bonferroni post-hoc test.</p

Effects of <i>Wse</i> on climbing activity.

<p>(A-B) Climbing activity of LRRK2 adult males treated with <i>Wse</i> 1% as compared with WT and untreated LRRK2 (A) and climbing activity of LRRK2 adult males treated with L-Dopa 0.01% (0.5mM) as compared with WT and untreated LRRK2 (B). Values are average ± SEM. * indicates p<0.05 at one-way ANOVA followed by LSD post hoc test as compared to WT; ** indicates p<0.05 at one-way ANOVA followed by LSD post hoc test as compared to LRRK2.</p

<i>Mucuna pruriens</i> (<i>Velvet bean</i>) Rescues Motor, Olfactory, Mitochondrial and Synaptic Impairment in <i>PINK1^B9 Drosophila melanogaster</i> Genetic Model of Parkinson’s Disease

<div><p>The fruit fly <i>Drosophila melanogaster</i> (<i>Dm</i>) mutant for PTEN-induced putative kinase 1 (<i>PINK1^B9</i>) gene is a powerful tool to investigate physiopathology of Parkinson's disease (PD). Using <i>PINK1^B9</i> mutant <i>Dm</i> we sought to explore the effects of <i>Mucuna pruriens</i> methanolic extract (<i>Mpe</i>), a L-Dopa-containing herbal remedy of PD. The effects of <i>Mpe</i> on <i>PINK1^B9</i> mutants, supplied with standard diet to larvae and adults, were assayed on 3–6 (I), 10–15 (II) and 20–25 (III) days old flies. <i>Mpe</i> 0.1% significantly extended lifespan of <i>PINK1^B9</i> and fully rescued olfactory response to 1-hexanol and improved climbing behavior of <i>PINK1^B9</i> of all ages; in contrast, L-Dopa (0.01%, percentage at which it is present in <i>Mpe</i> 0.1%) ameliorated climbing of only <i>PINK1^B9</i> flies of age step II. Transmission electron microscopy analysis of antennal lobes and thoracic ganglia of <i>PINK1^B9</i> revealed that <i>Mpe</i> restored to wild type (WT) levels both T-bars and damaged mitochondria. Western blot analysis of whole brain showed that <i>Mpe</i>, but not L-Dopa on its own, restored bruchpilot (BRP) and tyrosine hydroxylase (TH) expression to age-matched WT control levels. These results highlight multiple sites of action of <i>Mpe</i>, suggesting that its effects cannot only depend upon its L-Dopa content and support the clinical observation of <i>Mpe</i> as an effective medication with intrinsic ability of delaying the onset of chronic L-Dopa-induced long-term motor complications. Overall, this study strengthens the relevance of using <i>PINK1^B9 Dm</i> as a translational model to study the properties of <i>Mucuna pruriens</i> for PD treatment.</p></div

Samples of transmission electron microscopy images of thoracic ganglia and antennal lobes in <i>Drosophila</i> LRRK2 mutant (A) and after treatment with 1% in L^-/A⁺ insects (B, C) and 10% L⁺/A⁺ (D-F) extract of <i>Wse</i>.

<p>(A) abnormal mitochondria in the thoracic ganglia neuropil of <i>Drosophila</i> LRRK2. (B, C) conventional mitochondria in thoracic ganglia of <i>Drosophila</i> LRRK2 after treatment with 1% <i>Wse</i> L^-/A⁺imaged at low (B) and higher magnification (C). (D, E) abnormal mitochondria in <i>Drosophila</i> LRRK2 thoracic ganglia cell bodies after treatment with 10% <i>Wse</i>L⁺/A⁺. Note the irregular electron-dense substance clearly recognizable inside the mitochondria. (F and Inset) numerous endosomes are present inside the antennal lobes neurites of <i>Drosophila</i> LRRK2 after treatment with 10% <i>Wse</i>. Scale bars are 0.5 μm except in B that is 2.5 μm.</p