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parkin-induced defects in neurophysiology and locomotion are generated by metabolic dysfunction and not oxidative stress.

Vincent A, Briggs L, Chatwin GF, Emery E, Tomlins R, Oswald M, Middleton CA, Evans GJ, Sweeney ST, Elliott CJ - Hum. Mol. Genet. (2012)

Bottom Line: Rescue experiments confirmed that this phenotype was due to a defect in the nervous system and not in the muscle.Furthermore, recordings of motoneuron activity in parkin larvae revealed reduced bursting and a striking reduction in evoked and miniature excitatory junction potentials, suggesting a neuronal deficit.Surprisingly, scavengers of ROS did not rescue the resting membrane potential and locomotory phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of York, P.O. Box 373, York YO10 5YW, UK.

ABSTRACT
Parkinson's disease (PD) is characterized by movement disorders, including bradykinesia. Analysis of inherited, juvenile PD, identified several genes linked via a common pathway to mitochondrial dysfunction. In this study, we demonstrate that the larva of the Drosophila parkin mutant faithfully models the locomotory and metabolic defects of PD and is an excellent system for investigating their inter-relationship. parkin larvae displayed a marked bradykinesia that was caused by a reduction in both the frequency of peristalsis and speed of muscle contractions. Rescue experiments confirmed that this phenotype was due to a defect in the nervous system and not in the muscle. Furthermore, recordings of motoneuron activity in parkin larvae revealed reduced bursting and a striking reduction in evoked and miniature excitatory junction potentials, suggesting a neuronal deficit. This was supported by our observations in parkin larvae that the resting potential was depolarized, oxygen consumption and ATP concentration were drastically reduced while lactate was increased. These findings suggest that neuronal mitochondrial respiration is severely compromised and there is a compensatory switch to glycolysis for energy production. parkin mutants also possessed overgrown neuromuscular synapses, indicative of oxidative stress, which could be rescued by overexpression of parkin or scavengers of reactive oxygen species (ROS). Surprisingly, scavengers of ROS did not rescue the resting membrane potential and locomotory phenotypes. We therefore propose that mitochondrial dysfunction in parkin mutants induces Parkinsonian bradykinesia via a neuronal energy deficit and resulting synaptic failure, rather than as a consequence of downstream oxidative stress.

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Locomotor contractions are less frequent in parkin larvae. (A) A larval extensometer, constructed from a flexible photobeam and quadrant photodiode. (B) Peristaltic waves recorded by the extensometer show that contractions are fewer in parkin knockouts than in wild-type, but the peak–peak excursion is not affected. (C) The mean force is the same in wild-type and parkin knockout (ANOVA: F1,27 = 2.8, P= 0.10). (D) The frequency of peristaltic contractions is reduced in the parkin knockout (ANOVA, Bonferroni post hoc test, P< 0.001), and completely rescued by neuronal expression of wild-type parkin in nerve (Bonferroni post hoc test versus parkin P= 0.014; versus wild-type P= 0.56). Expression in muscle is ineffective (Bonferroni P= 0.38). Genotypes and number of larvae—wild-type: CS 13; parkin: park25/park25 15; drivers: neuronal: elav3EI 10; muscle: G14 12. Rescues are in park25/park25 background.
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DDR609F2: Locomotor contractions are less frequent in parkin larvae. (A) A larval extensometer, constructed from a flexible photobeam and quadrant photodiode. (B) Peristaltic waves recorded by the extensometer show that contractions are fewer in parkin knockouts than in wild-type, but the peak–peak excursion is not affected. (C) The mean force is the same in wild-type and parkin knockout (ANOVA: F1,27 = 2.8, P= 0.10). (D) The frequency of peristaltic contractions is reduced in the parkin knockout (ANOVA, Bonferroni post hoc test, P< 0.001), and completely rescued by neuronal expression of wild-type parkin in nerve (Bonferroni post hoc test versus parkin P= 0.014; versus wild-type P= 0.56). Expression in muscle is ineffective (Bonferroni P= 0.38). Genotypes and number of larvae—wild-type: CS 13; parkin: park25/park25 15; drivers: neuronal: elav3EI 10; muscle: G14 12. Rescues are in park25/park25 background.

Mentions: We assayed the strength and frequency of peristaltic contractions directly, using a larval length assay, in which a restrained larva pulls on a flexible beam (Fig. 2A). Wild-type larvae pulled regularly on the beam, while the parkin knockouts had a reduced frequency of contractions (32% of wild-type; Fig. 2B and D). However, the peak–peak change in length did not differ between the wild-type and parkin (Fig. 2C). This suggests that the reduced velocity of the larvae was due to less frequent steps, rather than a reduced step size. A particular feature of the parkin larval contractions was the slow rate at which the larva shortens, akin to bradykinesia (Fig. 2B). We also tested for rescue by neuronal or muscle driver. Only the neuronal driver restored the contraction frequency to wild-type levels (Fig. 2D).Figure 2.


parkin-induced defects in neurophysiology and locomotion are generated by metabolic dysfunction and not oxidative stress.

Vincent A, Briggs L, Chatwin GF, Emery E, Tomlins R, Oswald M, Middleton CA, Evans GJ, Sweeney ST, Elliott CJ - Hum. Mol. Genet. (2012)

Locomotor contractions are less frequent in parkin larvae. (A) A larval extensometer, constructed from a flexible photobeam and quadrant photodiode. (B) Peristaltic waves recorded by the extensometer show that contractions are fewer in parkin knockouts than in wild-type, but the peak–peak excursion is not affected. (C) The mean force is the same in wild-type and parkin knockout (ANOVA: F1,27 = 2.8, P= 0.10). (D) The frequency of peristaltic contractions is reduced in the parkin knockout (ANOVA, Bonferroni post hoc test, P< 0.001), and completely rescued by neuronal expression of wild-type parkin in nerve (Bonferroni post hoc test versus parkin P= 0.014; versus wild-type P= 0.56). Expression in muscle is ineffective (Bonferroni P= 0.38). Genotypes and number of larvae—wild-type: CS 13; parkin: park25/park25 15; drivers: neuronal: elav3EI 10; muscle: G14 12. Rescues are in park25/park25 background.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3313793&req=5

DDR609F2: Locomotor contractions are less frequent in parkin larvae. (A) A larval extensometer, constructed from a flexible photobeam and quadrant photodiode. (B) Peristaltic waves recorded by the extensometer show that contractions are fewer in parkin knockouts than in wild-type, but the peak–peak excursion is not affected. (C) The mean force is the same in wild-type and parkin knockout (ANOVA: F1,27 = 2.8, P= 0.10). (D) The frequency of peristaltic contractions is reduced in the parkin knockout (ANOVA, Bonferroni post hoc test, P< 0.001), and completely rescued by neuronal expression of wild-type parkin in nerve (Bonferroni post hoc test versus parkin P= 0.014; versus wild-type P= 0.56). Expression in muscle is ineffective (Bonferroni P= 0.38). Genotypes and number of larvae—wild-type: CS 13; parkin: park25/park25 15; drivers: neuronal: elav3EI 10; muscle: G14 12. Rescues are in park25/park25 background.
Mentions: We assayed the strength and frequency of peristaltic contractions directly, using a larval length assay, in which a restrained larva pulls on a flexible beam (Fig. 2A). Wild-type larvae pulled regularly on the beam, while the parkin knockouts had a reduced frequency of contractions (32% of wild-type; Fig. 2B and D). However, the peak–peak change in length did not differ between the wild-type and parkin (Fig. 2C). This suggests that the reduced velocity of the larvae was due to less frequent steps, rather than a reduced step size. A particular feature of the parkin larval contractions was the slow rate at which the larva shortens, akin to bradykinesia (Fig. 2B). We also tested for rescue by neuronal or muscle driver. Only the neuronal driver restored the contraction frequency to wild-type levels (Fig. 2D).Figure 2.

Bottom Line: Rescue experiments confirmed that this phenotype was due to a defect in the nervous system and not in the muscle.Furthermore, recordings of motoneuron activity in parkin larvae revealed reduced bursting and a striking reduction in evoked and miniature excitatory junction potentials, suggesting a neuronal deficit.Surprisingly, scavengers of ROS did not rescue the resting membrane potential and locomotory phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of York, P.O. Box 373, York YO10 5YW, UK.

ABSTRACT
Parkinson's disease (PD) is characterized by movement disorders, including bradykinesia. Analysis of inherited, juvenile PD, identified several genes linked via a common pathway to mitochondrial dysfunction. In this study, we demonstrate that the larva of the Drosophila parkin mutant faithfully models the locomotory and metabolic defects of PD and is an excellent system for investigating their inter-relationship. parkin larvae displayed a marked bradykinesia that was caused by a reduction in both the frequency of peristalsis and speed of muscle contractions. Rescue experiments confirmed that this phenotype was due to a defect in the nervous system and not in the muscle. Furthermore, recordings of motoneuron activity in parkin larvae revealed reduced bursting and a striking reduction in evoked and miniature excitatory junction potentials, suggesting a neuronal deficit. This was supported by our observations in parkin larvae that the resting potential was depolarized, oxygen consumption and ATP concentration were drastically reduced while lactate was increased. These findings suggest that neuronal mitochondrial respiration is severely compromised and there is a compensatory switch to glycolysis for energy production. parkin mutants also possessed overgrown neuromuscular synapses, indicative of oxidative stress, which could be rescued by overexpression of parkin or scavengers of reactive oxygen species (ROS). Surprisingly, scavengers of ROS did not rescue the resting membrane potential and locomotory phenotypes. We therefore propose that mitochondrial dysfunction in parkin mutants induces Parkinsonian bradykinesia via a neuronal energy deficit and resulting synaptic failure, rather than as a consequence of downstream oxidative stress.

Show MeSH
Related in: MedlinePlus