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Optogenetic acidification of synaptic vesicles and lysosomes

Nature Neuroscience volume 18, pages 1845–1852 (2015)Cite this article

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Abstract

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.

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Figure 1: Design and localization of the light-driven vesicular proton pump pHoenix.
Figure 2: pHoenix can substitute for V-ATPase function.
Figure 3: Activation of pHoenix in untreated neurons increases the quantal size.
Figure 4: Neurons with residual glutamate release after Baf-treatment have smaller mEPSCs and a high paired-pulse ratio.
Figure 5: Partially filled vesicles have a lower release probability.
Figure 6: Optogenetic acidification of lysosomes.

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Acknowledgements

We thank K. Rösler, B. Brokowski, A. Felies, B. Söhl-Kielczynski, C. Schweynoch, J. Pustogowa and A. Schönherr for excellent technical support, the viral core facility of the Charité for virus production, and M. Camacho-Perez, S. Watanabe and A. Liebkowsky for critical comments on the manuscript. The work was supported by the German Research Council grants Cluster of Excellence Neurocure (EXC 257), Collaborative Research Center SFB958 (D.S., C.R.), Collaborative Research Center SFB1078 (F.S., P.H.), and the Integrative Research Institute for the Life Sciences (D.S., P.H., C.R.), a European Research Council grant (249939 SYNVGLUT; C.R.), a Grass Foundation Fellowship (B.R.R.) and the Louis-Jeantet Foundation (F.S., P.H.).

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Author notes

  1. Franziska Schneider

    Present address: Present address: National Heart and Lung Institute, Imperial College London, London, UK.,

  2. Benjamin R Rost and Franziska Schneider: These authors contributed equally to this work.

Authors and Affiliations

  1. Neuroscience Research Center, Charité – Universitätsmedizin Berlin, Berlin, Germany

    Benjamin R Rost, M Katharina Grauel, Christian Wozny, Claudia G Bentz, Tanja Rosenmund, Dietmar Schmitz & Christian Rosenmund

  2. German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany

    Benjamin R Rost & Dietmar Schmitz

  3. Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany

    Franziska Schneider & Peter Hegemann

  4. Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK

    Christian Wozny

  5. Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany

    Anja Blessing & Thomas J Jentsch

  6. Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany

    Anja Blessing & Thomas J Jentsch

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Contributions

B.R.R. and F.S. developed the concepts for the pHoenix constructs. F.S. performed the molecular biology. B.R.R., F.S., M.K.G., C.W., C.G.B., A.B. and T.R. performed the experiments and analyzed the data. All authors designed the experiments and discussed the results. B.R.R. and F.S. prepared the manuscript, and all authors contributed to editing the paper.

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Correspondence to Benjamin R Rost.

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Integrated supplementary information

Supplementary Figure 1 Molecular design of pHoenix and quantification of surface-resident molecules.

(a) Gene-fusion strategy to generate pHoenix, including the amino acid positions of fusion sites and peptide sequences of linker segments (orange). (b) Live-cell images of a pHoenix expressing neuron. The pHluorin signal was acquired in the presence of 50 mM NH4Cl. Scale bar, 50 µm. (c) Heat-colored pHluorin images (averages of 3), acquired during application of pH 5.5 extracellular solution (1), standard extracellular solution (2), and extracellular solution with 50 mM NH4Cl (3). Scale bar, 25 µm. (d) Time-course of pHluorin fluorescence intensities, excited with 100 ms light flashes of a 490-nm LED. Images were acquired at 2 Hz. Signals are baseline corrected. The surface fraction was determined as ratio of fluorescence quenched by the pH 5.5 application (1) to the total range of fluorescence from the pH 5.5 and NH4Cl applications (1 + 3).

Supplementary Figure 2 Expression of pHoenix does not affect synaptic transmitter release.

(a) APs triggered EPSCs of 2.766 ± 0.3 nA in control and 3.3 ± 0.5 nA in pHoenix-expressing neurons. (P = 0.6, U = 681). (b) The size of the readily-releasable pool (RRP) was 0.4 ± 0.05 nC in control and 0.49 ± 0.08 nC in pHoenix-expressing cells (P = 0.9, U = 726). (c) Vesicular release probability (Pvr) was similar for both groups (Pvr(control) = 4.8 ± 0.5 %, Pvr(pHoenix) = 4.9 ± 0.4 %; P = 0.8, U = 715). For (a) – (c): n(control) = 40, n(pHoenix) = 46, N = 4. (d) mEPSC frequency and (e) mEPSC amplitude were not different in both groups: Control: 6.6 ± 1 Hz, 22 ± 1.4 pA (n = 36, N = 4); pHoenix: 5.2 ± 0.8 Hz, 25.5 ± 1.6 pA (n = 43, N = 4); P(frequency) = 0.5, U = 589; P(amplitude) = 0.06, U = 583. (f) Short-term plasticity assessed by train stimulation (50 APs at 10 Hz) was not affected by expression of pHoenix (Control: n = 31, N = 4; pHoenix: n = 40, N = 4). Statistical significance assessed by two-tailed Mann-Whitney U test. Norm., normalized; ns, not significant.

Supplementary Figure 3 Bleaching does not account for the rapid quenching of pHluorin molecules after activation of pHoenix.

(a) Heat-colored images averaged from three photomicrographs of sypHy-expressing neurons 15 s before and after 15 s of illumination with yellow light. Note the high intensity of pHluorin signals in Baf-treated cells compared to untreated cells, indicating a higher vesicular pH after incubation in Baf. Scale bar, 25 µm. (b) Time-course of normalized pHluorin signals in sypHy- or pHoenix-expressing cells. Blue ticks indicate 100 ms excitation of pHluorin with 490 nm light, and numbers indicate acquisition time-points of the example images in (a). Synaptic fluorescence of sypHy-expressing control neurons was stable in untreated cells (black, n = 6, N = 2), and decreased linearly in Baf-treated cells (blue, n = 8, N = 2). In contrast, activation of the proton pump in pHoenix-expressing neurons caused rapid quenching of pHluorin signals (green, n = 15, N = 2; same data as Fig. 2d). Norm., normalized.

Supplementary Figure 4 Application of pHoenix in hippocampal slice cultures.

(a) Schematic illustrating injection of AAV particles into area CA3. (b) pHluorin signals in the pyramidal layer of CA3 in a pHoenix-expressing slice after Baf-treatment for 16 h. Scale bar, 25 µm. (c) Example traces at indicated time points during illumination with 587-nm light, and time plot of EPSC recorded from a CA3 pyramidal neuron. Activation of presynaptic pHoenix by illumination causes a gradual recovery of glutamatergic transmission. Scale bars, 10 ms and 200 pA. (d) Summary of 5 cells recorded in Baf-treated slices expressing pHoenix.

Supplementary Figure 5 Reversible acidification and refilling by pHoenix.

(a) Experimental outline: Cells were recorded after > 2 h of incubation in Baf, and experiments were performed in the presence of 0.1 µM Baf. Every 20 s a single image of pHluorin signals was acquired, followed by train (40 APs at 25 Hz) or sparse (2 APs at 25 Hz) electrophysiological stimulation. pHoenix activity was stimulated by 120-s illumination with 580-nm light, followed by a 300-s dark interval and a second 120-s illumination. (b) A single trace of EPSCs evoked by 40 APs at the end of the first illumination period. AP artifacts deleted for display. Below, only the 1st EPSCs of the train are shown. Illumination caused an increase of the EPSC amplitude, while EPSCs decreased during the dark period. EPSCs recovered during the 2nd illumination. (c) Trace illustrates the sparse stimulation paradigm with 2 APs every 20 s. EPSCs increased during the first light stimulation, but did not significantly decrease in the dark interval. Time points of EPSCs in (b) and (c): Traces during illuminations depict recordings at 0, 20 and 110 s of light, traces during dark interval at 0, 120 and 260 s of depletion (no averages). Scale bars, 4 nA, 100 and 10 ms, respectively. (d) pHluorin signals of the same cell as in (b) with train stimulation. Note the increase of fluorescence at the end of the dark interval due to ongoing exocytosis and lack of reacidification. (e) pHluorin signals of the same cell as in (c), with sparse stimulation. Images in (d) and (e) are averaged from 3 frames, and processed with rolling ball background subtraction for display. Numbers (1–4) indicate acquisition time-points of the example images. Scale bar, 10 µm. (f) Average time course of the 1st EPSC’s amplitudes during train (open symbols) or sparse (closed symbols) stimulation. Both groups: n = 8; N = 3. (g) Time course of pHluorin fluorescence for both stimulation paradigms from the same cells as in (f). Norm., normalized.

Supplementary Figure 6 Vesicular transmitter uptake in Baf-treated neurons is restored by activation of pHoenix, but not Arch3-eGFP, and reaches fill states comparable to those of untreated neurons.

(a) Single EPSC traces and time-plot of EPSCs recorded from a Baf-treated autaptic neuron expressing Arch3-EGFP (gray) or pHoenix (green). Diagram shows averages of EPSCs 30 s after illumination. Transmission was rescued in pHoenix- (n = 42; N = 7), but not Arch3-expressing neurons (n = 10, N = 2; P < 0.0001, Mann-Whitney U test, U = 0). Scale bar, 10 ms, 0.5 nA. (b) EPSCs rescued by pHoenix activation in Baf-treated neurons are not significantly different from the EPSCs in untreated neurons expressing pHoenix after illumination (untreated EPSC: 4.0 ± 1.3 nA before, 4.2 ± 1.4 nA after illumination, n = 20, N = 3; Baf-treated EPSC: 0.1 ± 0.02 nA before, 3.5 ± 0.9 nA after illumination, n = 21, N = 3). Multiplicity adjusted P-values for EPSC amplitudes of untreated vs. treated, pre-light: P = 0.02, post-light: P = 0.87. Traces are average of 6 sweeps before and 30 s after illumination. Scale bar, 1 nA, 10 ms. (c) Traces of mEPSCs recorded from an untreated and a Baf-treated neuron before and after illumination. Scale bars, 50 pA, 200 ms. In Baf-treated cells, mEPSCs were small and occurred at a very low frequency (14.5 ± 1.7 pA; at 0.2 ± 0.05 Hz, n = 19, N = 3, 3 cells displayed no mEPSCs before light). Illumination restored both mEPSC amplitude and frequency (to 28 ± 2 pA, at 5.2 ± 1 Hz), which were not significantly different from the untreated group (22.5 ± 1.9 pA, at 7.6 ± 1.6 Hz before illumination; 26.7 ± 2.3 pA, 7 ± 1.6 Hz after illumination, n = 16, N = 3). Events were detected in a 1 min period before and after illumination. Multiplicity adjusted P-values for mEPSC amplitudes, untreated vs. treated, pre-light: P = 0.015, post-light: P = 0.88; and for mEPSC frequencies, untreated vs. treated, pre-light: P < 0.0001, post-light: P = 0.49 (all tests: Repeated-measures two-way ANOVA, with Sidak's multiple comparisons test). ns, not significant.

Supplementary Figure 7 Pharmacological inhibition of AMPA receptor desensitization does not affect the paired-pulse behavior of residual and rescued EPSCs.

(a) Averages of paired EPSCs of a Baf-treated neuron recorded in the presence of 100 µM cyclothiazide (CTZ) before (Baseline) and after (Full) rescue by pHoenix. AP interval was 40 ms. CTZ slowed EPSC decay kinetics and caused summation of the signals during the second EPSC. Horizontal lines indicate the range of EPSC amplitudes. Scale bars, baseline: 50 ms, 200 pA; full: 50 ms, 1 nA. (b) Recovery of transmission by pHoenix activation in CTZ using low intensity light: EPSC amplitudes increased from 0.55 ± 0.1 nA to 2.53 ± 0.5 nA, with τ = 86 s (P < 0.0001, Wilcoxon signed-rank test, W = 435; n = 29, N = 5). (c) Paired EPSCs scaled to the amplitude of the first EPSC. Scale bar 50 ms. (d) Paired-pulse ratios decreased from 1.2 ± 0.05 for residual EPSCs to 0.96 ± 0.03 for recovered EPSCs after the activation of pHoenix, with τ = 47 s (P < 0.0001, two-tailed paired t-test, t28 = 6). Green curves represent monoexponential association or decay fitted to the data.

Supplementary Figure 8 Vesicular acidification and EPSC amplitudes monitored in parallel during the interval recovery protocol.

During the first illumination period activating pHoenix, pHluorin signals (open circles, normalized to baseline) were rapidly quenched. EPSCs (black filled circles) partially recovered and remained stable during the following dark period, while fluorescence slightly increased due to ongoing exocytosis. EPSCs strongly increased during the second, 90 s illumination period, while pHluorin signals further decreased, indicating that constant proton pumping activity is required for glutamate uptake into synaptic vesicles (n = 9, N = 2). Norm., normalized.

Supplementary Figure 9 Colocalization analysis of lyso-pHoenix and different organelle marker proteins.

HeLa cells transfected with lyso-pHoenix were fixed and stained with antibodies against different markers for major cellular organelles, combined with an anti-GFP staining to enhance the pHluorin signal of the lyso-pHoenix construct. Lyso-pHoenix showed strong colabelling with a staining for LAMP-2 (lysosome-associated membrane protein 2), but no overlap with stainings for the cis-Golgi (GM130, Golgi-Matrix protein 130 kD), mitochondria (Hsp60, 60 kDa heat shock protein), the endoplasmic reticulum (PDI, protein disulphide isomerase) or the early endosome (small GTPase Rab5, Ras-associated protein 5). Scale bar, 10 µm.

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Rost, B., Schneider, F., Grauel, M. et al. Optogenetic acidification of synaptic vesicles and lysosomes. Nat Neurosci 18, 1845–1852 (2015). https://doi.org/10.1038/nn.4161

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