Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jan 1:452:37-48.
doi: 10.1016/j.neuroscience.2020.10.021. Epub 2020 Nov 8.

Ring Finger Protein 11 (RNF11) Modulates Dopamine Release in Drosophila

Eve Privman Champaloux  1 , Nathan Donelson  2 , Poojan Pyakurel  3 , Danielle Wolin  3 , Leah Ostendorf  3 , Madelaine Denno  3 , Ryan Borman  3 , Chris Burke  2 , Jonah C Short-Miller  4 , Maria R Yoder  4 , Jeffrey M Copeland  5 , Subhabrata Sanyal  2 , B Jill Venton  6
Affiliations

Ring Finger Protein 11 (RNF11) Modulates Dopamine Release in Drosophila

Eve Privman Champaloux et al. Neuroscience. .

Abstract

Recent work indicates a role for RING finger protein 11 (RNF11) in Parkinson disease (PD) pathology, which involves the loss of dopaminergic neurons. However, the role of RNF11 in regulating dopamine neurotransmission has not been studied. In this work, we tested the effect of RNF11 RNAi knockdown or overexpression on stimulated dopamine release in the larval Drosophila central nervous system. Dopamine release was stimulated using optogenetics and monitored in real-time using fast-scan cyclic voltammetry at an electrode implanted in an isolated ventral nerve cord. RNF11 knockdown doubled dopamine release, but there was no decrease in dopamine from RNF11 overexpression. RNF11 knockdown did not significantly increase stimulated serotonin or octopamine release, indicating the effect is dopamine specific. Dopamine clearance was also changed, as RNF11 RNAi flies had a higher Vmax and RNF11 overexpressing flies had a lower Vmax than control flies. RNF11 RNAi flies had increased mRNA levels of dopamine transporter (DAT) in RNF11, confirming changes in DAT. In RNF11 RNAi flies, release was maintained better for stimulations repeated at short intervals, indicating increases in the recycled releasable pool of dopamine. Nisoxetine, a DAT inhibitor, and flupenthixol, a D2 antagonist, did not affect RNF11 RNAi or overexpressing flies differently than control. Thus, RNF11 knockdown causes early changes in dopamine neurotransmission, and this is the first work to demonstrate that RNF11 affects both dopamine release and uptake. RNF11 expression decreases in human dopaminergic neurons during PD, and that decrease may be protective by increasing dopamine neurotransmission in the surviving dopaminergic neurons.

Keywords: Drosophila; Parkinson disease; RNF11; dopamine; dopamine transporter; voltammetry.

PubMed Disclaimer

Figures

Figure 1 –
Figure 1 –. RNF11 affects dopamine release.
(A) Schematic of FSCV recording from larval Drosophila VNC. (B) Peak oxidation current of stimulated dopamine in isolated larval ventral nerve cords. The 2 s stimulation is marked by the red line (60 Hz, 120 pulses, 4 ms red light each pulse). The three genotypes plotted are control (black, TH-GAL4; UAS-CsChrimson), RNF11 knockdown (red, TH- GAL4; UAS-RNF11JF11021-RNAi, UAS-CsChrimson), and RNF11 overexpression (blue, TH- GAL4; UAS-RNF11, UAS-CsChrimson). (C) Cyclic voltammograms showing characteristic oxidation peaks for dopamine at 0.6 V and reduction peaks at −0.2 V (colors and genotypes same as B). (D) Average peak stimulated dopamine concentration changes with genotype (one-way ANOVA, p<0.001). RNF11 RNAi flies show greater DA release than control or RNF11 overexpressing flies (Dunnett’s post-test), while RNF11 overexpression does not affect peak dopamine concentration. Data shown are box plots, with the box showing 25–75%, the line marking the median, and the whiskers denoting the range (n=10 flies). Asterisks denote significant differences between groups: *p<0.05, **p<0.01.
Figure 2.
Figure 2.. Increased dopamine release is not specific to the RNF11 RNAi allele.
(A) The RNAi lines RNF11JF11021 and RNF11HMJ22085 target the 3’ coding region (shaded) of the RNF11 transcript. RNF11JF11021 targets a 300 nt region while RNF11HMJ22085 targets a 21 nt region. The two RNAi alleles overlap only in 3 nt. (B) Average peak dopamine release is significantly higher in both RNF11 alleles (one-way ANOVA, p<0.05, Dunnett’s post-test, p<0.05), though difference between alleles is insignificant. The three genotypes plotted are control (black, TH-GAL4; UAS-CsChrimson), RNF11 RNAiJF11021 (red, TH-GAL4; UAS-RNF11JF11021-RNAi, UAS-CsChrimson), and RNF11 RNAiHMJ22085 (maroon, TH-GAL4; UAS-RNF11HMJ22085-RNAi, UAS-CsChrimson). (C) Capacity of RNF11 knockdown and overexpression were measured by RT-qPCR. The Act5C-GAL4 ubiquitous driver was used to activate RNAi in the RNF11JF11021 and the RNF11HMJ22085 lines. RNF11JF11021 shows a 75% knockdown and the RNF11HMJ22085 shows an 89% knockdown in RNF11 mRNA levels. The UAS-RNF11 overexpression (OE) displays 366% increase in RNF11 levels.
Figure 3 –
Figure 3 –. Knocking down RNF11 with RNAi does not change peak serotonin or octopamine levels.
RNF11JF11021 RNAi expressed in (A) serotonergic (Tph-GAL4 driver) or (B) octopaminergic cells (Tdc2-GAL4 driver) did not significantly change peak stimulated neurotransmitter levels (unpaired t-tests, p>0.05, n=4–6 flies per group). Stimulation was 2 s, 60 Hz, 120 pulses (4 ms per pulse).
Figure 4 –
Figure 4 –. RNF11 knockdown increases dopamine release and clearance.
Dopamine release was modeled and release and uptake parameters determined. In A-C, RNF11 is driven by the TH-GAL4 driver. (A) Dopamine released per light stimulation pulse ([DA]p, 120 pulse, 60 Hz, 4 ms pulse width stimulation) is affected by genotype (one-way ANOVA, n=10 flies, p=0.0006). [DA]p is greater in RNF11JF11021 RNAi flies than control or RNF11 overexpressing flies (**, Tukey post-test, p<0.01). (B) Vmax is significantly affected by genotype (one-way ANOVA, n=10 flies, p<0.0001). RNF11 knockdown significantly increases Vmax compared to control (*, Tukey post-test p<0.05) and RNF11 overexpression (****, p<0.0001). RNF11 overexpression significantly decreases Vmax compared to control (*, p<0.05). (C) Km is not significantly affected by genotype (one-way ANOVA, n=10 flies, p>0.5). (D) Dopamine transporter (DAT) mRNA levels by qPCR in adults. RNF11 is driven by the Act5C-GAL4 driver. There is an effect of genotype (one-way ANOVA, p=0.0022, n=4 flies) and RNF11 RNAi flies have significantly higher DAT mRNA than control flies and RNF11 overexpression flies (Tukey post-test, **, p<0.01).
Figure 5 –
Figure 5 –. RNF11 knockdown larval CNS have more dopamine tissue content than RNF11 overexpressing flies.
Capillary electrophoresis coupled to FSCV was used to measure the tissue content of dopamine. The dopamine content was significantly affected by the genotype of the flies (Kruskal-Wallis, p=0.0004, n=7–9 flies). Post hoc tests showed that the RNF11 RNAi larval CNS had significantly more dopamine than the RNF11 overexpressing CNS (p=0.0019). However, RNF11JF11021 RNAi or RNF11 overexpressing did not differ from control.
Figure 6 –
Figure 6 –. Blocking DAT with nisoxetine causes slowed uptake in all three genotypes.
(A) Peak oxidation current for dopamine was monitored over time before (orange) and 15 minutes after (purple) application of 20 μM nisoxetine. This example data shows an RNF11 RNAi VNC. (B) Nisoxetine caused an increase in t50. A two-way ANOVA showed a significant main effect of genotype (p=0.0002, n=4 flies) and nisoxetine (p=0.0012, n=4 flies), but no interaction. (C) The fold increase from baseline upon application of nisoxetine was not significantly different among the different genotypes (Kruskal-Wallis, p=0.3697).
Figure 7 –
Figure 7 –. Blocking the D2 autoreceptor with flupenthixol causes increased dopamine release in all three genotypes.
Peak oxidation current for dopamine was monitored before and 15 minutes after the application of 5 μM flupenthixol. (A) Example plot of the effect of flupenthixol on dopamine release in an RNF11 JF11021 RNAi larval CNS. (B) Flupenthixol caused an increase in peak oxidation current of dopamine in all genotypes. Two way ANOVA revealed a main effect of genotype (p=0.011) and flupenthixol (p=0.0007), and a significat interaction (p=0.0206). (C) There was no significant difference among the genotypes in fold increase of dopamine release from baseline after flupenthixol (one way ANOVA, p=0.0812, n=5–7 flies).
Figure 8 –
Figure 8 –. Dopamine turnover is faster in RNF11 RNAi flies.
Larval CNS was stimulated for 2 seconds every 30 seconds, and the peak oxidation current for dopamine was monitored. There was a significant main effect of both stimulation number (p<0.0001) and genotype (p<0.0001), but no interaction (p=0.1468, two-way ANOVA, n=4 flies). Post hoc-tests showed that RNF11JF11021 RNAi flies have a higher plateau than both control and RNF11 overexpressing flies. The data for each genotype was fit to a single-phase exponential decay, which were significantly different (comparison of fits, p<0.0001).

  1. RNF11 knockdown increases dopamine release in Drosophila larvae.

  2. RNF11 knockdown also increases the rate of dopamine clearance.

  3. RNF11 directly affects dopamine release and clearance.

  4. RNF11 is knocked down during Parkinson disease

  5. Increasing dopamine may be a compensatory mechanism to maintain dopaminergic signaling

See this image and copyright information in PMC

References

    1. Anderson LR, Betarbet R, Gearing M, Gulcher J, Hicks A a, Stefánsson K, Lah JJ, Levey AI (2007) PARK10 candidate RNF11 is expressed by vulnerable neurons and localizes to Lewy bodies in Parkinson disease brain. J Neuropathol Exp Neurol 66:955–964. - PubMed
    1. Bellucci A, Bubacco L, Longhena F, Parrella E, Faustini G, Porrini V, Bono F, Missale C, Pizzi M (2020) Nuclear Factor-κB Dysregulation and α-Synuclein Pathology: Critical Interplay in the Pathogenesis of Parkinson’s Disease. Front Aging Neurosci 12:68. - PMC - PubMed
    1. Borue X, Cooper S, Hirsh J, Condron B, Venton BJ (2009) Quantitative evaluation of serotonin release and clearance in Drosophila. J Neurosci Methods 179:300–308. - PMC - PubMed
    1. Bosse KE, Maina FK, Birbeck JA, France MM, Joseph JP, Colombo ML, Mathews TA (2013) Aberrant striatal dopamine transmitter dynamics in brain- derived neurotrophic factor-deficient mice. J Neurochem 120:385–395. - PMC - PubMed
    1. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415. - PubMed

Publication types

LinkOut - more resources