Chemically Functionalized Water-Soluble Single-Walled Carbon Nanotubes Obstruct Vesicular/Plasmalemmal Recycling in Astrocytes Down-Stream of Calcium Ions

Abstract

We used single-walled carbon nanotubes chemically functionalized with polyethylene glycol (SWCNT-PEG) to assess the effects of this nanomaterial on astrocytic endocytosis and exocytosis. We observed that the SWCNT-PEG do not affect the adenosine triphosphate (ATP)-evoked Ca²⁺ elevations in astrocytes but significantly reduce the Ca²⁺-dependent glutamate release. There was a significant decrease in the endocytic load of the recycling dye during constitutive and ATP-evoked recycling. Furthermore, SWCNT-PEG hampered ATP-evoked exocytotic release of the loaded recycling dye. Thus, by functionally obstructing evoked vesicular recycling, SWCNT-PEG reduced glutamate release from astrocytes via regulated exocytosis. These effects implicate SWCNT-PEG as a modulator of Ca²⁺-dependent exocytosis in astrocytes downstream of Ca²⁺, likely at the level of vesicle fusion with/pinching off the plasma membrane.

Keywords: Ca2+ dynamics; astrocytes; carbon nanotubes; glutamate release; membrane recycling.

Figure 1

Single-walled carbon nanotubes chemically functionalized with polyethylene glycol (SWCNT-PEG) do not affect the ATP-induced intracellular Ca²⁺ elevations in cultured mouse cortical astrocytes. (A) Individual frames from the time-lapse imaging of RCaMP1h (intracellular Ca²⁺ sensor) in a control astrocyte showing the changes in RCaMP1h fluorescence at selected time points (indicated with triangles in (B)). Scale bar, 20 µm. Gray scale is a linear representation of the fluorescence intensities of the pixels in the images, expressed in fluorescence intensity units (iu). (B) Time-lapse imaging of RCaMP1h fluorescence, reporting on the average intracellular Ca²⁺ levels in astrocytes in the absence and the presence of SWCNT-PEG solute (5 µg/mL). ATP (100 µM) was bath applied to elicit an increase in intracellular Ca²⁺ levels and the Ca²⁺ ionophore 4-Bromo-A23187 (4-Br; 20 µM) was bath applied to elicit maximal Ca²⁺ response in astrocytes. The horizontal double-headed arrows indicate the times of addition of ATP and 4-Br containing external solutions. Changes in RCaMP1h fluorescence are expressed as dF/F₀ (%) after background subtraction. Number of astrocytes studied in each condition is shown in parentheses. (C) Changes in the RCaMP1h fluorescence of astrocytes shown in B, normalized to their maximal Ca²⁺ response after the application of 4-Br, expressed as the normalized change in RCaMP1h fluorescence, dF. Traces in (B) and (C) show means + SEMs. (D,E) Summary graphs showing the average peak (D) and cumulative (E) normalized dF with SEMs.

Figure 2

SWCNT-PEG solute inhibits the ATP-induced glutamate release from cultured mouse cortical astrocytes. (A) Individual frames from the time-lapse imaging of iGluSnFR (extracellular glutamate sensor) in a control astrocyte showing the changes in iGluSnFR fluorescence at selected time points (indicated with triangles in (B)). Scale bar, 20 µm. Gray scale is a linear representation of the fluorescence intensities of the pixels in the images, expressed in fluorescence intensity units (iu). (B) Time-lapse imaging of iGluSnFR fluorescence, reporting on the average extracellular glutamate levels at the plasma membrane of astrocytes in the absence and the presence of SWCNT-PEG solute (5 µg/mL). ATP (100 µM) was bath applied to stimulate Ca²⁺-dependent exocytotic glutamate release from astrocytes. Exogenous glutamate (Glut; 100 µM) was bath applied to saturate the iGluSnFR fluorescence at the plasma membranes of astrocytes. The horizontal double-headed arrows indicate the times of addition of ATP and glutamate containing external solutions. Changes in iGluSnFR fluorescence are expressed as dF/F₀ (%) after background subtraction. Number of astrocytes studied in each condition is shown in parentheses. (C) Changes in the iGluSnFR fluorescence of astrocytes shown in B, normalized to their saturated glutamate response after the application of glutamate, expressed as the normalized change in iGluSnFR fluorescence, dF. Traces in B and C show means + SEMs. (D–F) Summary graphs showing the average trough (D), peak (E) and cumulative (F) normalized dF with SEMs. Asterisks indicate a statistical difference compared to the control. Student’s t-test (pooled variances); * p < 0.05, ** p < 0.01.

Figure 3

SWCNT-PEG solute inhibits the endocytotic load of the recycling dye FM4-64 during constitutive and ATP-stimulated recycling in cultured mouse cortical astrocytes. (A) Images (from left to right) showing a control astrocyte loaded with β-Ala-Lys-Nε-AMCA (DAPI filter set) and three subsequent individual frames from the time-lapse imaging showing the changes in FM4-64 fluorescence intensity (TRITC filter set) before the addition of FM4-64 (t = 10 s), immediately after the addition of FM4-64, (peak; t = 400 s), and at steady state (t = 850 s). Scale bar, 50 µm. The t = 10 s image shows astrocyte autofluorescence (TRITC filter set) with the dotted outline representing the cell area traced based on the corresponding β-Ala-Lys-Nε-AMCA image (left). Gray scale is a linear representation of the fluorescence intensities of the pixels in the images, expressed in fluorescence intensity units (iu). (B) Schematics showing FM4-64 labeling and membrane recycling in astrocytes before dye application, after dye application and at steady state after the dye washout, respectively. (C) Time-lapse imaging of FM4-64 fluorescence in the absence and the presence of SWCNT-PEG solute (5 µg/mL) or PEG (1 µg/mL). The arrow indicates the beginning of the 5 min FM4-64 (10 µM) application without or with ATP (100 µM) to study constitutive (triangles) or ATP-stimulated (circles) recycling, respectively (broken abscissa). Number of astrocytes studied in each condition is shown in parentheses. Traces show the background subtracted FM4-64 fluorescence (dF) reported as medians. (D,E) Summary graphs showing the median peak (D) and steady state (E) of FM-4-64 dF with interquartile range. Asterisks indicate a statistical difference compared to the corresponding control group. Other differences are marked by the brackets. KWA followed by Dunn’s test; * p < 0.05, ** p < 0.01.

Figure 4

SWCNT-PEG solute inhibits the ATP-stimulated exocytosis (chase) of the FM4-64 pre-loaded in exocytosis vesicles by ATP-stimulation (pulse) of cultured mouse cortical astrocytes. (A) Time-lapse imaging of FM4-64 fluorescence for astrocyte groups stimulated with ATP once and washed as described in Figure 3 (pulse), followed by second ATP (100 µM) application to stimulate the exocytosis of FM4-64 loaded in the vesicles (chase), the time of addition of which is indicated by the horizontal double-headed arrow. Other annotations as in Figure 3. Number of astrocytes studied in each condition is shown in parentheses and represents a fraction of the astrocytes already reported in Figure 3 but here stimulated again with ATP for the second time. The schematics show FM4-64 labeling and membrane recycling in astrocytes after dye application, at steady state after the dye washout, ATP-stimulated exocytosis and at steady state after ATP-stimulated dye washout, respectively (left to right), the time points of which are marked by the bold arrows. (B) FM4-64 fluorescence normalized to the steady-state level just prior to the second application of ATP. Traces in (A) and (B) show medians. Asterisks and pound signs indicate a statistical difference between the groups indicated on the right at the specific time points. KWA followed by Dunn’s test; * p < 0.05, # p < 0.01.

Figure 5

Scheme showing the possible mechanism of action of SWCNT-PEG solute on astrocytes. SWCNT-PEG obstructs vesicular/plasmalemmal recycling in astrocytes by inhibiting (-) both the endocytic and exocytotic pathways.