Synaptic mitochondria regulate hair-cell synapse size and function

Abstract

Sensory hair cells in the ear utilize specialized ribbon synapses. These synapses are defined by electron-dense presynaptic structures called ribbons, composed primarily of the structural protein Ribeye. Previous work has shown that voltage-gated influx of Ca²⁺ through Ca_V1.3 channels is critical for hair-cell synapse function and can impede ribbon formation. We show that in mature zebrafish hair cells, evoked presynaptic-Ca²⁺ influx through Ca_V1.3 channels initiates mitochondrial-Ca²⁺ (mito-Ca²⁺) uptake adjacent to ribbons. Block of mito-Ca²⁺ uptake in mature cells depresses presynaptic-Ca²⁺ influx and impacts synapse integrity. In developing zebrafish hair cells, mito-Ca²⁺ uptake coincides with spontaneous rises in presynaptic-Ca²⁺ influx. Spontaneous mito-Ca²⁺ loading lowers cellular NAD⁺/NADH redox and downregulates ribbon size. Direct application of NAD⁺ or NADH increases or decreases ribbon size respectively, possibly acting through the NAD(H)-binding domain on Ribeye. Our results present a mechanism where presynaptic- and mito-Ca²⁺ couple to confer proper presynaptic function and formation.

Keywords: developmental biology; metabolism; mitochondrial calcium; neuroscience; ribbon synapse; sensory cell; zebrafish.

Figure 1.. Mito-Ca²⁺ uptake initiates adjacent to ribbons.

(A) Cartoon illustration of a lateral-line hair cell containing: an apical mechanosensory bundle (blue), mitochondria (green), presynaptic ribbons (magenta), Ca_V1.3 channels (orange) and postsynaptic densities (purple). (B) Airyscan confocal image of 6 live hair cells (1 cell outlined in white) expressing MitoGCaMP3 (mitochondria) and Ribeye a-tagRFP (ribbons) in a developing neuromast at 2 dpf. Also see Figure 1—figure supplement 1. (C) A representative TEM showing a mitochondrion (m) in close proximity to a ribbon (R) at 4 dpf. (D) Quantification of mitochondrion to ribbon distance in TEM sections (n = 17 ribbons). (E) Side-view of a hair cell (outlined in white) shows the spatio-temporal dynamics of evoked mito-Ca²⁺ signals during a 2 s stimulation at 6 dpf. The change in MitoGCaMP3 signal (∆F) from baseline is indicated by the heatmap and are overlaid onto the pre-stimulus grayscale image. (E’-E’’) Circles 1–3 (1.3 μm diameter) denote regions used to generate the normalized (∆F/F₀) temporal traces of mito-Ca²⁺ signals in E’’: adjacent to the presynapse (‘1’), and midbody (‘2’ and ‘3’) in the same cell as E. (F) Average evoked mito-Ca²⁺ response before (solid black) and after 30 min treatment with 10 μM Ru360 (dashed green), 2 μM Ru360 (solid green), or 10 μM isradipine (gray) (3–5 dpf, n ≥ 9 cells per treatment). Error bars in D are min and max; in F the shaded area denotes SEM. Scale bar = 500 nm in C, 5 µm in B and 2 µm in E and E’.

Figure 1—figure supplement 1.. The time course of mechanically-evoked mito-Ca²⁺ signals are longer-lasting than cyto-Ca²⁺ signals.

(A) Airyscan confocal image of a live neuromast expressing MitoGCaMP3 (mitochondria) and Ribeye a-tagRFP (ribbons) at 6 dpf. Insets show the base of 4 individual hair cells from the neuromast in A (dashed white boxes). (B) Average cyto-Ca²⁺ (blue) and mito-Ca²⁺ (green) ∆F/F GCaMP3 signals during the onset of a 2 s stimulus. Mito-Ca²⁺ signals rise with a delay compared to cyto-Ca²⁺ signals, 3–6 dpf, n ≥ 18 cells. (C-C’) Average cyto-Ca²⁺ and mito-Ca²⁺ ∆F/F GCaMP3 signals during and after a 2 s stimulation shows that cyto-Ca²⁺ signals return to baseline shortly after stimulation (C) while mito-Ca²⁺ remains elevated up to 5 min after stimulation (C–C’), 3–6 dpf, n ≥ 7 cells. (D) A series of 3, evoked 2 s stimuli initiated at: t = 0–2, 12–14 and 24–26 s. A rise in MitoGCaMP3 can be detected during each stimulus, prior to MitoGCaMP3 signals returning to baseline. (E) 10 µM of the VDAC inhibitor TRO 19622 partially blocks evoked MitoGCaMP3 signals, 5 dpf, n = 15 cells. (F) A dose response curve indicates that Ru360 blocks evoked MitoGCaMP3 signals with an IC₅₀ value of 1.37 µM at 5 dpf, n ≥ 9 cells per dose. Error in panel (B-C’) E and F represent SEM. Scale bar = 5 µm in A and 2 µm in inset.

Figure 1—figure supplement 2.. Mito-Ca²⁺ uptake occurs in anterior lateral-line hair cells.

(A) A live Image of an anterior-lateral line (ALL) neuromast viewed top-down, expressing the mito-Ca²⁺ sensor MitoGCaMP3 at 5 dpf. (A’) shows the spatio-temporal dynamics of evoked mito-Ca²⁺ signals during a 2 s stimulation. The MitoGCaMP3 signals during the stimulation (∆F) are indicated by the heatmap overlaid onto the baseline grayscale image. (A’’) Temporal traces of evoked mito-Ca²⁺ signals were generated from three regions denoted by three circles in A. Scale bar = 5 µm in A.

Figure 2.. Mito-Ca²⁺ uptake can impact presynaptic-Ca²⁺ signals.

(A) A live Image of a neuromast viewed top-down, expressing the presynaptic-Ca²⁺ sensor GCaMP6sCAAX (green) and mito-Ca²⁺ sensor MitoRGECO1 (magenta) at 5 dpf. A’-A’’, GCaMP6sCAAX (A’) and MitoRGECO1 (A’’) signals (∆F) from baseline during a 2 s stimulation are indicated by the heatmaps and occur in the same cells (white outline). (B) Scatter plot with linear regression of peak presynaptic- and mito-Ca²⁺ response for individual cells at 4–5 dpf, n = 136 cells. Gray background in graph denotes presynaptic-Ca²⁺ signals below 0.25, a threshold used as a cutoff for presynaptic activity (below inactive, above active). (B’) Plot of mito-Ca²⁺responses segregated based on the activity threshold in B. (C-D’) Presynaptic-Ca²⁺ response (example in Figure 2—figure supplement 1C–C’) averaged per cell before (blue) and after a 30 min treatment with 10 μM Ru360 (light green) or 2 μM Ru360 (dark green), n ≥ 10 cells per treatment. C and D show averaged traces while C’ and D’ show before-and-after dot plots of the peak response per cell. (E-F) Representative images of mature neuromasts (5 dpf) immunostained with Ca_V1.3 (white, calcium channels) and MAGUK (green, postsynapses) after a 1 hr incubation in 0.1% DMSO (E) or 2 μM Ru360 (F). G-H, Scatter plots show percentage of postsynapses that pair with Ca_V1.3-channel clusters (Ca_V1.3 + MAGUK) and orphan postsynapses (MAGUK only) (G). The integrated intensity of Ca_V1.3-channel immunolabel at presynapses is lower in control compared to treatment group (H), n ≥ 7 neuromasts per treatment. Whiskers on plots in B’ represent min and max; the shaded area in plots C and D and the error bars in C’, D’ and G-H denotes SEM. Mann-Whitney U test was used in B’; Wilcoxon matched-pairs signed-rank test was used in C’ and D’. Welch’s unequal variance t-test was used in G-H. *p<0.05, ***p<0.001, ****p<0.0001. Scale bar = 5 µm in A and E.

Figure 2—figure supplement 1.. The effects of MCU and VDAC block on mechanotransduction and the effect of VDAC block on presynaptic-Ca²⁺ signals.

(A) Illustration of a neuromast and the imaging planes used to study the mechanotransduction in hair-bundles and the presynaptic-Ca²⁺ influx at ribbons. Localization of the membrane-localized Ca²⁺ sensor GCaMP6sCAAX shown in green. Inset in A shows an example top-down view of GCaMP6sCAAX bundle plane (6sCAAX) at 5 dpf. (B-B’) Bundle-Ca²⁺ signals before (blue) and after a 30 min treatment with 10 μM Ru360 (green) or 10 μM TRO 19622 (magenta), n ≥ 39 bundles per treatment. Average traces are shown in B while dot plots of the peak response per bundle are shown in B’. (C) Double-transgenic hair cells expressing GCaMP6sCAAX (At presynaptic membranes) and Ribeye a-tagRFP (Labels ribbons) at 5 dpf. Example cells in presynaptic imaging plane are boxed in white and duplicated in right insets. (C’) Example cells show evoked presynaptic-Ca²⁺ signals at ribbons during a 0.2 s stimulation. Circles 1–5 (1.3 μm diameter) in insets in C denote regions at ribbons used to generate the temporal traces of presynaptic-Ca²⁺ signals at each ribbon in C’. Similarly-colored traces of presynaptic-Ca²⁺ signals originate from different presynapses of the same cell. (D-E’) Presynaptic-Ca²⁺ signals averaged per cell before (blue) and after a 30 min 10 μM TRO 19622 (magenta), n ≥ 9 cells per treatment. D and E show averaged traces while D’ and E’ show before-and-after treatment dot plots of the peak response per cell. Error in panel B-B’, D-E’ represent SEM. A Wilcoxon matched-pairs signed-rank test was used in B’,D’ and E’. *p<0.05, **p<0.01, ***p<0.001. Scale bar = 5 µm in A and C and 2 µm in C inset.

Figure 3.. Mito-Ca²⁺ is important for ribbon size and synapse integrity in mature hair cells.

(A-D) Representative images of mature neuromasts (5 dpf) immunostained with Ribeye b (magenta, ribbons) and MAGUK (green, postsynapses) after a 1 hr 0.1% DMSO (A), a 1 hr 2 μM Ru360 (B), a 30 min 10 μM Ru360 (C), or a 1 hr 10 μM Ru360 (D) treatment. Insets show three example synapses (white squares). E-F, Scatter plots show synapse counts (E), and ribbon area (F) in controls and in treatment groups. Ribbon areas, synapse numbers, and hair-cell counts are unaffected after a 1 hr 2 µM Ru360 treatment. Ribbon areas are larger and there are fewer synapses without significant loss of hair cells after a 30 min treatment with 10 µM Ru360 (F). After a 1 hr 10 µM Ru360 treatment there is an increase in ribbon area and a decrease in synapse (E) and hair-cell counts. N ≥ 9 neuromasts per treatment. Error bars in E-F represent SEM. An unpaired t-test was used in E and a Welch’s unequal variance t-test was used in F. ****p<0.0001. Scale bar = 5 µm in A, and 2 µm in inset.

Figure 3—figure supplement 1.. Ribbon and postsynapse size in mature ALL neuromasts.

(A-B) Scatter plots show that ribbon areas (A) and postsynaptic density areas (B) within the same fish are similar between mature anterior lateral-line (ALL) and posterior lateral-line (PLL) neuromasts. (C) Scatter plots show ribbon areas in controls and after a 1 hr treatment with 2 µM Ru360 are similar in mature hair cells within the ALL. Ribbon sizes of untreated anterior lateral-line hair cells are from the same data as A and (C) n ≥ 10 neuromasts per treatment; error bars represent SEM; and a Welch’s unequal variance t-test was used for comparisons.

Figure 3—figure supplement 2.. MCU block does not impact postsynapse size in mature hair cells.

(A) Quantification of postsynapse size assayed by MAGUK immunolabel in mature neuromasts indicate the treatments with 2 µM Ru360 and 10 µM Ru360 do not significantly alter postsynapse size compared to controls, n ≥ 9 neuromasts per treatment. Error bars represent SEM. A Welch’s unequal variance t-test was used for comparisons.

Figure 4.. Spontaneous presynaptic- Ca²⁺ influx and mito-Ca²⁺ uptake are linked.

(A-A’) A live Image of an immature neuromast viewed top-down, expressing the presynaptic-Ca²⁺ sensor GCaMP6sCAAX (A) and mito-Ca²⁺ sensor MitoRGECO1 (A’) at 3 dpf. Example GCaMP6sCAAX (A’) and MitoRGECO1 (A’) signals during two 25 s windows within a 900 s acquisition are indicated by the ∆F heatmaps and occur in the same cells. (A’’) A heatmap of Pearson correlation coefficients comparing GCaMP6sCAAX and MitoRGECO1 signals from the cells in A-A’. (A’’’) Example GCaMP6sCAAX (green) MitoRGECO1 (magenta) traces during the 900 s acquisition from the 5 cells numbered in A, also see Video 2. (B) Scatter plot showing the average magnitude of GCaMP6sCAAX signals in developing and mature hair cells, n = 6 neuromasts per age. (C) Scatter plot showing frequency of GCaMP6sCAAX events in developing and mature hair cells, n = 6 neuromasts. Error bars in B-C represent SEM. A Mann-Whitney U test was used in B and C. ****p<0.0001. Scale bar = 5 µm in A’.

Figure 4—figure supplement 1.. Spontaneous presynaptic and mito-Ca²⁺ signals are abolished by Ca_V1.3 channel antagonist isradipine.

(A) A live Image of a neuromast viewed top-down, expressing the presynaptic-Ca²⁺ sensor GCaMP6sCAAX (green) and mito-Ca²⁺ sensor MitoRGECO1 (magenta) at 6 dpf. (B) Representative GCaMP6sCAAX (green) and MitoRGECO1 (magenta) traces from the cells in A during a 900 s continuous image acquisition in the absence of stimuli and 10 µM isradipine. (C) There is no correlation between GCaMP6sCAAX and MitoRGECO1 signals within each cell from B in the presence of isradipine. Scale bar = 5 µm in A.

Figure 5.. Mito-Ca²⁺ regulates ribbon formation.

(A-C) Representative images of immature neuromasts (3 dpf) immunostained with Ribeye b (magenta, ribbons) and MAGUK (green, postsynapses) after a 1 hr 0.1% DMSO (A), 2 μM Ru360 (B) or 10 µM Ru360 (C) treatment. Insets show three representative synapses (white squares) for each treatment. D-E, Scatter plot show quantification of synapse number (D), and ribbon area (E) in controls and in treatment groups. (F) Side-view of 2 hair cells (white outline) shows synaptic ribbon (three magenta asterisks in each cell) and extrasynaptic Ribeye b aggregates after a 1 hr 0.1% DMSO or 10 μM Ru360 treatment. (G) Quantification of extrasynaptic Ribeye puncta. N ≥ 12 neuromasts per treatment. Error bars in D-E and G represent SEM. An unpaired t-test was used in D and a Welch’s unequal variance t-test was used in E and G, *p<0.05, **p<0.01, ****p<0.0001. Scale bar = 5 µm in A, 2 µm in insets and F.

Figure 5—figure supplement 1.. Ribbon and postsynapse size in immature ALL neuromasts.

(A-B) Scatter plots show that ribbon areas (A) and postsynaptic density areas (B) within the same fish are similar between immature anterior lateral-line (ALL) and posterior lateral-line (PLL) neuromasts. (C) Scatter plots show ribbon areas in controls and after a 1 hr treatment with 2 µM Ru360 are larger in immature hair cells within the ALL. Ribbon sizes of untreated anterior lateral-line hair cells are from the same data as A and C, n ≥ 10 neuromasts per treatment; error bars represent SEM; and a Welch’s unequal variance t-test was used for comparisons. *p<0.05.

Figure 5—figure supplement 2.. MCU and Ca_V1.3 block do not impact postsynapse size.

(A) Quantification of postsynapse size assayed by MAGUK immunolabel in mature neuromasts indicate the treatments with 10 µM isradipine, 2 µM Ru360 and 10 µM Ru360 do not significantly alter postsynapse size compared to controls, n ≥ 9 neuromasts per treatment. Error bars represent SEM. A Welch’s unequal variance t-test was used for comparisons.

Figure 6.. Cyto-Ca²⁺, mito-Ca²⁺ and NAD⁺/NADH redox baseline measurements.

Live hair cells expressing RGECO1 (A), MitoGCaMP3 (D), or Rex-YFP (G) show resting cyto-Ca²⁺, mito-Ca²⁺ or NAD⁺/NADH levels respectively. (B-C) RGECO1 baseline measurements before and after a 30 min mock treatment (0.1% DMSO) or after a 30 min 10 μM Bay K8644 (BayK), 10 μM isradipine, or 10 μM Ru360 treatment. (E-F) MitoGCaMP3 baseline measurements before and after a 30 min mock treatment (0.1% DMSO) or after a 10 μM BayK, 10 μM isradipine, or 10 μM Ru360 treatment. (H-I) Rex-YFP baseline measurements before and after 30 min mock treatment (0.1% DMSO) or after a 30 min 100 μM NAD⁺, 5 mM NADH, 10 μM isradipine, or 10 μM Ru360 treatment. All plots are box-and-whiskers plot that show median, min and max. N ≥ 9 neuromasts per treatment. A one-way Brown-Forsythe ANOVA with Dunnett’s T3 post hoc was used to calculate the difference in (B-C), (E-F), and a one-way Brown-Forsythe and Welch ANOVA with Holm-Sidak’s post hoc was used in H-I, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Horizontal lines in E, H, and I indicate that both conditions had similar p values compared to mock treatment. Scale bar = 5 μm in A, D and G.

Figure 7.. NAD⁺ and NADH directly influence ribbon formation.

Representative images of immature (A-C, 3 dpf) and mature (G-H, 5 dpf) neuromasts immunostained with Ribeye b (magenta, ribbons) and MAGUK (green, postsynapses) after a 0.1% Tris-HCl (A, F), 100 μM NAD⁺ (B, G) or 5 mM NADH treatment (C, H). Insets show three example synapses (white squares). D-E and I-J, Scatter plots show synapse count (D, I) and ribbon area (E, J) in controls and treatments groups. N ≥ 10 neuromasts per treatment. Error bars in B-C represent SEM. An unpaired t-test was used for comparisons in D and I and a Welch’s unequal variance t-test was used for comparisons in E and J, **p<0.01. Scale bar = 5 µm in A and F, 2 µm in insets.

Figure 7—figure supplement 1.. NAD⁺ and NADH treatment do not impact postsynapse size.

(A-B) Quantification of postsynapse size assayed by MAGUK immunolabel in mature (A) and immature (B) neuromasts indicate the treatment with 100 μM NAD⁺ and 5 mM NADH do not significantly alter postsynapse size compared to controls, n ≥ 9 neuromasts per treatment. Error bars represent SEM. A Welch’s unequal variance t-test was used for comparisons.

Figure 8.. Schematic model of mito-Ca²⁺ in developing and mature hair cells.

(A) In developing hair cells, spontaneous presynaptic-Ca²⁺ influx is linked to mito-Ca²⁺ uptake. Together these Ca²⁺ signals function to regulate ribbon size during ribbon formation. When the Ca_V1.3 or MCU channels are blocked, ribbon formation is increased leading to larger ribbons. These Ca²⁺ signals regulate ribbon formation via NAD(H) redox. MCU block lowers mito-Ca²⁺, increases the NAD⁺/NADH ratio and promotes ribbon formation. (B) In mature hair cells, evoked presynaptic-Ca²⁺ influx is linked to mito-Ca²⁺ uptake. When the MCU is partially blocked there is a reduction in presynaptic-Ca²⁺ influx. When the MCU is completely blocked there are synaptopathic consequences; ribbons are enlarged and synapses are lost.