Axonal Endoplasmic Reticulum Ca2+ Content Controls Release Probability in CNS Nerve Terminals

Abstract

Although the endoplasmic reticulum (ER) extends throughout axons and axonal ER dysfunction is implicated in numerous neurological diseases, its role at nerve terminals is poorly understood. We developed novel genetically encoded ER-targeted low-affinity Ca²⁺ indicators optimized for examining axonal ER Ca²⁺. Our experiments revealed that presynaptic function is tightly controlled by ER Ca²⁺ content. We found that neuronal activity drives net Ca²⁺ uptake into presynaptic ER although this activity does not contribute significantly to shaping cytosolic Ca²⁺ except during prolonged repetitive firing. In contrast, we found that axonal ER acts as an actuator of plasma membrane (PM) function: [Ca²⁺]_ER controls STIM1 activation in presynaptic terminals, which results in the local modulation of presynaptic function, impacting activity-driven Ca²⁺ entry and release probability. These experiments reveal a critical role of presynaptic ER in the control of neurotransmitter release and will help frame future investigations into the molecular basis of ER-driven neuronal disease states.

Keywords: Ca(2+) imaging; ER-GCaMP; GCaMP6; STIM1; low affinity Ca(2+) indicator; presynaptic endoplasmic reticulum.

Figure 1. ER Ca²⁺ handling is critical for presynaptic function at physiological temperature

(A-D) Representative traces of single AP presynaptic cytosolic Ca²⁺ responses measured using Fluo-5F AM (A, C) and vesicular exocytosis measured using vG-pH (B, D) before and after CPA treatment. Experiments were performed at 37°C (A, B, shown in red) and 26°C (C, D, shown in blue). Fluo-5F AM traces were normalized to the pre-CPA ΔF response (black trace). vG-pH responses are shown as percentage of their maximum fluorescence, obtained by brief perfusion of NH₄Cl buffered at pH 7.4. Differential effects observed are summarized by showing the remaining response after CPA treatment in each of the conditions (E, F): Fluo-5F AM, (26°C) n =10, (37°C) n =14; ***p=1.44·10⁻⁷. vG-pH, (26°C) n =7, (37°C) n =10; ***p=2.75·10⁻⁴.

Figure 2. A new generation of ultrasensitive ER Ca²⁺ indicators

(A) Predicted relative fluorescence vs Ca²⁺ curves based on binding parameters obtained in vitro (Table S1) of low affinity calcium indicators GCaMP3-44, GCaMP6-150, GCaMP6-210 and GCaMP3-373 (light to dark green, high to low affinity) and high affinity cytosolic indicators GCaMP3 (grey) and GCaMP6f (black). Approximate ranges of Ca²⁺ concentration in the cytosol and ER are indicated by grey gradient boxes. (B) top: targeting scheme for expression in the neuronal ER by adding the N-terminal signal peptide of calreticulin (CALR sig peptide) and the C-terminal KDEL retention motif. Bottom: high resolution image of somatic ER-GCaMP6-150 shows neuronal ER structure. Scale bar 2 µM. (C) Montage of several fields from a neuron expressing ER-GCaMP6-150, which localizes in the ER network in the soma, dendrites and axons. Scale bar 40 µM. (D-F) Neurons were treated with 500 µM ionomycin to induce indicator saturation for calibration. (D) Example pseudocolor images of somatic ER-GCaMP6-150 before and after ionomycin treatment. Pseudocolor scale shows low to high intensity. Scale bar 8 µM. (E) Average peak fold-change in fluorescence (F_ionomycin/F₀) during ionomycin application for each ER-GCaMP. ER-GCaMP3-44, n=14; ER-GCaMP6-150, n=8; ER-GCaMP6-210, n=9; ER-GCaMP3-373, n=8; errors are ± S.E.M. (F) pH-corrected estimate of resting ER calcium concentration based on each indicator saturation response. Dashed line indicates the average of the estimates from each of the indicators.

Figure 3. Neuronal activity drives ER Ca²⁺ uptake

(A-C) Somatic ER Ca²⁺ responses to 20AP (20Hz) stimulation using different indicators. (A) Representative pseudocolor images of somatic ER-GCaMP6-150 before and after 20AP (20Hz) stimulation and (B) the corresponding fluorescence intensity over time (pseudocolor scale below showing low to high intensity). Scale bar 8 µM. (C) Average of 20AP peak somatic ER-GCaMP responses: G-CEPIA1er, n=5; ER-GCaMP3-44, n=11; ER-GCaMP6-150, n=12; ER-GCaMP6-210, n=6; ER-GCaMP3-373, n=11; errors are ± S.E.M. (D) Diagram showing axonal ER crossing nerve terminals where ER Ca²⁺ responses were measured. (E) ER-GCaMP-150 signals in presynaptic boutons identified by VAMP-mCherry expression (left, colored in green; scale bar 4 µM) at rest (middle image) and during a 20 AP stimulus (displayed as a kymograph, right; pseudocolor scale shows low to high intensity). Scale bar 3 s. (F) Representative 20AP presynaptic response measured with ER-GCaMP6-150 or cytosolic GCaMP6-150 (same variant without ER-targeting sequences).

Figure 4. SERCA function is necessary for activity-driven ER Ca²⁺ uptake

(A) Representative presynaptic responses of ER-GCaMP6-150 to 20AP (20Hz) or (B) ER-GCaMP6-210 to a single AP stimulus before and after 5 min of CPA (50 µM) application (black and red, respectively). Average of single AP Δ[Ca²⁺]_ER responses before CPA was 5.9 ± 1.3 µM (n=5), which was reduced to 0.2 ± 0.2 µM after CPA treatment (n=3). (C) Box plots showing average and single-cell calibrated peak responses of neurons stimulated with 20AP (20Hz) before and after 5 min of treatment with CPA (n=10), thapsigargin (TG, 1 µM, n=9) or 1,4-dihydroxy-2,5-di-tert-butylbenzene (BHQ, 50 µM, n=9).

Figure 5. Presynaptic inhibition is slower than ER Ca²⁺ depletion following SERCA block

(A-C) Average axonal ER Ca²⁺ dynamics were measured using ER-GCaMP6-150 at 26°C, (A, blue) or 37°C (B, red). Grey traces are individual experiments, although some individual responses overshot the scale used here and their peaks were not included in the graph. Neurons were stimulated with 20 AP (20 Hz) and then treated with CPA to induce ER Ca²⁺ depletion. After 3 min of CPA treatment the responses to a second stimulus were abolished (indicated by second arrow, 20AP 20Hz). (C) Estimate of Δ[Ca²⁺]_ER assuming an average resting [Ca²⁺]_ER based on ionomycin responses; n(26°C)=8, n(37°C)=15; n.s. p=0.36. (D) Single-AP cytosolic Ca²⁺ responses (Fluo-5F AM, normalized to average pre-CPA ΔF response) every 60s measured before (black traces) and after CPA treatment at 26°C (D, G, blue traces) or 37°C (E, H, red traces). (G, H) Comparison of the kinetics of presynaptic inhibition and axonal ER Ca²⁺ depletion. Curves were fit to single exponential decays where possible and time constants (τ) were obtained for comparison. τ_ER (26°C)= 47.5 ± 3.4 s, n=8; τ_ER (37°C)= 26 ± 0.9 s, n=15; τ_{cyto Ca} ²⁺ (37°C)= 148 ± 18 s, n=7.

Figure 6. Presynaptic Ca²⁺ influx is correlated with ER Ca²⁺ content in wild type but not in STIM1 KD neurons

(A-B) Neurons expressing ER-GCaMP6-150 and cytosolic jRCaMP1b were co-transfected with or without an shRNA targeting STIM1 and were used to examine single AP-driven Ca²⁺ influx (jRCaMP1b) and axonal [Ca²⁺]_ER (ER-GCaMP6-150) in the same cells. Recordings from wild type (n=15) and STIM1 KD (n=12) neurons were grouped using a binning size of [Ca²⁺]_ER=25µM to better estimate fitting parameters (see methods; see Fig. S6 for unbinned data). These two variables are correlated only in wild type cells (A) and are well described by a generalized Hill equation (red line) with a K_1/2 of 105 µM ± 5 µM and a Hill coefficient of 5.4 ± 2.7(Adjusted R-square = 0.87). Fitting in the case of STIM1 KD was not possible (B, see methods). Grey and brown lines show the predicted impact of CPA on Ca²⁺ influx when the average value of [Ca²⁺]_ER (152 µM) decreases by 48 µM, as measured during SERCA block.

Figure 7. STIM1 regulates presynaptic function impairment induced by ER Ca²⁺ depletion

(A-D) Single AP-driven cytosolic Ca²⁺ signals and exocytosis were measured using GCaMP6f or vG-pH, respectively. Single-AP stimulated signals were quantified in wild type neurons, STIM1 KD neurons, STIM1 KD neurons expressing an shRNA-resistant version of STIM1 (STIM1^WT) or STIM1 KD neurons expressing an shRNA-resistant version of STIM1 that is insensitive to ER Ca²⁺ content due to EF-hand mutations (STIM1^EF). Responses were quantified before (black) and after (red) CPA treatment. Black dashed lines represent average response before treatment whereas red dashed lines represent the effect quantified in wild type neurons for ease of comparison in the different conditions. (C-D) Differential effects of CPA are summarized by showing the remaining response after CPA treatment in each of the conditions. Solid black line indicates response before CPA treatment, normalized to 1 in each case. (C) Effects of CPA in single-AP-driven presynaptic Ca²⁺ signals, Control n=16, ***p=0.0016; STIM1 KD n=14, n.s. p=0.60; STIM1 KD + STIM1^WT n=10, *p=0.02; STIM1 KD + STIM1^EF n=10, n.s. p=0.14. (D) Effects of CPA in single-AP vG-pH peak responses, Control n=10, ***p=6.68·10⁻⁵; STIM1 KD n=7, n.s. p=0.22; STIM1 KD + STIM1^WT n=7, *p=0.031; STIM1 KD + STIM1^EF n=7, n.s. p=0.55. Statistics were analyzed using paired sample Student’s t-test.

Figure 8. Local STIM1 enrichment at nerve terminals inhibits Ca²⁺ influx

(A) Representative image of an individual axon expressing STIM1-RFP and the presynaptic marker synapsin-GFP (straightened for ease of visualization). (B-E) Synapsin-GFP and STIM1-RFP were imaged before (B) and after CPA treatment (C). (D-F) Intensity profiles of both proteins were analyzed in 218 individual boutons from 8 neurons and compared before and after 15 min of CPA treatment to obtain 1 dimensional intensity cross-correlation profiles, showing that the presynaptic enrichment of STIM1-RFP increases following CPA treatment (F; control=0.66 ± 0.03, CPA=0.75 ± 0.02; ** p=0.0012) with negligible impact on the distribution of synapsin-GFP (control, black profile in D; CPA, blue profile in E). (G) Average decrease in single AP-Ca²⁺ influx following overexpression of STIM1-RFP. (H) Representative image of a neuron co-transfected with Phy-GCaMP6 (top, left) and STIM1-RFP (bottom left) showing the response to a 10 AP stimulus (middle left). Note that nerve terminals with greater STIM1-RFP abundance (1) have lower Ca²⁺ influx than neighboring synapses (2,3) with lower STIM1-RFP abundance (right) (I) Quantification of 10AP-driven phy-GCaMP6 signals shown in H. (J) Quantification of single AP-driven phy-GCaMP6 signals and their corresponding STIM1-RFP intensities acquired from 564 individual boutons from 10 neurons co-transfected as in H. STIM1-RFP intensities are shown as fluorescence normalized to the auto-fluorescence. Phy-GCaMP6 responses were binned into 4 different STIM1-RFP intensity groups (1–1.5, 1.5–2.5, 2.5–4.5 and >4.5) and reveal a strong inverse correlation between STIM1-RFP abundance and Ca²⁺ influx.