Figure 6.
TPC2 disrupts dopaminergic function in vivo. (a) Schematic depicting the UAS/GAL4 system to drive the expression of TPCN2 and/or LRRK2 G2019S under the control of the TH promoter in Drosophila. (b) Confocal micrograph of an adult brain showing the expression of TPC2-mCherry in dopaminergic neurons that drive vision (PPL) and movement (VUM). (c) Schematic depicting in vivo vision assay to assess dopaminergic function. SSVEP analyses in the retina following mild visual stress induced by repetitive light stimulation resembling a disco. (d) Visual responses in lamina neurons (mean ± SEM) from transgenic flies expressing empty vector (n = 13), LRRK2 G2019S (n = 10), TPC2-mRFP (n = 14/11), or both (n = 10/10) following disco treatment (mean ± SEM). Flies reared in the dark were used as controls (n = 12, 14, 10/11, 11/6 for EV, GS, TPC2, and TPC2 GS, respectively). Each point represents the response from an individual animal. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA, Sidak’s test). (e) Schematic depicting the PER in response to a sucrose solution to assess dopaminergic-mediated movement in vivo. (f) PER in transgenic flies expressing TPC2-mCherry, LRRK2 G2019S, or both (mean ± SEM). Flies were analyzed according to age after enclosure where 1 day was designated “young,” 4 days designated “middle aged,” and 7 days designated “aged.” Each point represents the proportion of responsive animals from an independent trial (n = 3). **P < 0.01, ***P < 0.001 (two-way ANOVA, Tukey’s test). (g) Schematic depicting crawling of larvae to assess dopaminergic-mediated movement in vivo. (h) Exemplar crawling tracks recorded over a 60-s period from lines expressing TPC2-GCaMP6s and/or LRRK2 G2019S. Scale bar: 1 mm. (i) Summary data (mean ± SEM) quantifying movement of the indicated single- and double-transgenic larvae (n = 20–42). Each point represents the distance traveled by an individual animal. ***P < 0.001, ****P < 0.0001 (one-way ANOVA, Tukey’s test). (j) Confocal micrographs of larval brain showing the expression of TPC2-mCherry and LAMP1-GFP in dopaminergic neurons. Scale bar: 10 µm. PER, proboscis extension reflex; TH, tyrosine hydroxylase; EV, empty vector.

TPC2 disrupts dopaminergic function in vivo. (a) Schematic depicting the UAS/GAL4 system to drive the expression of TPCN2 and/or LRRK2 G2019S under the control of the TH promoter in Drosophila. (b) Confocal micrograph of an adult brain showing the expression of TPC2-mCherry in dopaminergic neurons that drive vision (PPL) and movement (VUM). (c) Schematic depicting in vivo vision assay to assess dopaminergic function. SSVEP analyses in the retina following mild visual stress induced by repetitive light stimulation resembling a disco. (d) Visual responses in lamina neurons (mean ± SEM) from transgenic flies expressing empty vector (n = 13), LRRK2 G2019S (n = 10), TPC2-mRFP (n = 14/11), or both (n = 10/10) following disco treatment (mean ± SEM). Flies reared in the dark were used as controls (n = 12, 14, 10/11, 11/6 for EV, GS, TPC2, and TPC2 GS, respectively). Each point represents the response from an individual animal. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA, Sidak’s test). (e) Schematic depicting the PER in response to a sucrose solution to assess dopaminergic-mediated movement in vivo. (f) PER in transgenic flies expressing TPC2-mCherry, LRRK2 G2019S, or both (mean ± SEM). Flies were analyzed according to age after enclosure where 1 day was designated “young,” 4 days designated “middle aged,” and 7 days designated “aged.” Each point represents the proportion of responsive animals from an independent trial (n = 3). **P < 0.01, ***P < 0.001 (two-way ANOVA, Tukey’s test). (g) Schematic depicting crawling of larvae to assess dopaminergic-mediated movement in vivo. (h) Exemplar crawling tracks recorded over a 60-s period from lines expressing TPC2-GCaMP6s and/or LRRK2 G2019S. Scale bar: 1 mm. (i) Summary data (mean ± SEM) quantifying movement of the indicated single- and double-transgenic larvae (n = 20–42). Each point represents the distance traveled by an individual animal. ***P < 0.001, ****P < 0.0001 (one-way ANOVA, Tukey’s test). (j) Confocal micrographs of larval brain showing the expression of TPC2-mCherry and LAMP1-GFP in dopaminergic neurons. Scale bar: 10 µm. PER, proboscis extension reflex; TH, tyrosine hydroxylase; EV, empty vector.

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