Astrocytes rescue neuronal health after cisplatin treatment through mitochondrial transfer

Timed-pregnant Long Evans rats (Charles River, Wilmington, MA, USA) were sacrificed and E18 fetuses of both sexes were collected in accordance with Institutional Animal Care and Use Committee-approved protocols.

Cortices were dissected and incubated in 10 ml of dissociation media (81.8 mM Na₂So₄, 30 mM K₂SO₄, 5.8 mM MgCl₂, 0.25 mM CaCl₂, 1 mM Hepes, 20 mM glucose, 0.0001% Phenol Red, 0.16 mM NaOH pH = 7.4) that contained 10 U/mL papain and 5 mg of L-cysteine in total (Worthington, Lakewood, NJ, USA) for 10 min at 37 °C, followed by incubation with 150 mg of trypsin inhibitor (Millipore-Sigma, St. Louis, MO) in 10 ml of dissociation media for 10 min at 37 °C. The cortical tissue was mechanically dissociation in Opti-mem (GIBCO, Carlsbad, CA, USA) with 2.5 M Glucose (GIBCO). Cells were cultured on plates coated with 0.05 mg/ml poly-D-lysine (PDL; Millipore-Sigma) in neurobasal medium (NBM) with 100 U/mL penicillin and 1x B-27 supplement (Invitrogen Carlsbad, CA) at 37 °C and 5% CO₂. Neuronal cultures were maintained in NBM with B-27 supplement and media was replaced every 3 days. Cortical astrocytes were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum and 5% of 10,000 units/ ml of penicillin and 10,0000 μg/ml of streptomycin (GIBCO) at 5% CO₂ and 37 °C.

Neuronal cells were used for experiments after 12–15 days of culturing in vitro (DIV). To confirm neuronal enrichment, cells were fixed with 4% paraformaldehyde in PBS, treated with 0.25% Triton X-100, blocked in 2% BSA in PBS and stained with anti-Map2 antibody (1:2000); Sigma-Aldrich); anti-GFAP (1:200, Acris, Rockville, MD) and anti-Olig2 (1:400, Abcam, Cambridge, UK) antibody. On DIV12 > 98% of cells were Map2+ and Olig2 and GFAP-negative. Astrocytes were used until the third passage and were > 99% GFAP+.

Astrocytes were plated in a 6-well plate at 1.5 × 10⁵ cells/well 24 h before transfection. For labeling mitochondria, astrocytes were transfected with 2 μg of pLYS1-FLAG-MitoGFP-HA (Addgene plasmid # 50057) which contains the pore-forming subunit of the mitochondrial calcium uniporter coupled to GFP or a mito-mCherry construct generated by subcloning the targeting sequence of the pLYS1-FLAG-MitoGFP-HA plasmid into the mcherry2-N1 vector (Addgene plasmid # 54517). For Miro-1 knockdown, 5 nmol of Rho-1 siRNA (Qiagen, Germany #SI01401743) was diluted in rnase-free water (provided in kit) to make a 20 μM stock, andAllStars Negative Control Scrambled siRNA (Qiagen #SI03650318) was performed similarly to make a 20 μM stock instructions provided. Astrocytes were transfected with 80 nM of Rho-1 siRNA from a 20 μM stock (Qiagen, Germany #SI01401743) or 80 nM AllStars Negative Control Scrambled siRNA from a 20 μM stock (Qiagen #SI03650318). All transfections were performed using the Astrocyte Transfection kit (Altogen Biosystems, Las Vegas, NV, USA) according to manufacturer’s instructions. Miro-1 knockdown was confirmed by Western blot with anti-Rho1 antibody (Novus Biologicals, Centennial, CO, USA) with GAPDH as control (Abcam) followed detection of bands with enhanced chemoluminescence (GE Healthcare Bio-Sciences, Pittsburgh, PA). Blots were captured in the LAS system using Image Quant software (GE Healthcare Bio-Sciences) for quantification of bands.

Neurons were plated in 96 well plates coated with 0.05 mg/ml PDL at 5 × 10⁴ neurons/well. Viability after exposure to cisplatin (Teva, Petah Tikva, Israel) was quantified using the colorimetric cell viability reagent WST-1 (Millipore-Sigma, #11644807001). To assess the effect of astrocytes on neuronal survival, separate cultures of neurons (1.5 × 10⁵ cells/well in a 6-well plate) and astrocytes (5 × 10⁴ /well in a 6-well plate) were treated with cisplatin for 24 h. Neurons were then labeled with 20 μM CellTracker Blue (CTB; Invitrogen) for 45 min at 37 °C, and washed in serum-free media. Astrocytes (5 × 10⁴ cells/well) were added to the neuronal culture and survival of CTB+ neurons was quantified 17 h later using a Countess II FL automated cell counter (Invitrogen).

Neurons were plated at 1.5 × 10⁵ cells/well in a 6-well plate, treated with cisplatin for 24 h, and labeled with 20 μM CellTracker Green (Thermo Fisher). Astrocytes (5 × 10⁴ cells/well) were added and co-cultured with the neurons for 17 h. The co-cultures were stained with tetramethylrhodamine methyl ester (TMRM, Invitrogen; 250 nM) for 45 min at 37 °C, or Mitotracker (50 nM, Thermo Fisher, Waltham, MA, USA) for 30 min at 37 °C. As a positive control, neurons were treated with 10 μM carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP, Sigma-Aldrich), a mitochondrial uncoupler, for 15 min. Cells were collected and TMRM fluorescence intensity of the cell tracker green positive cells was quantified using an Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA USA). For confocal microscopy, neurons were plated in cell culture imaging dishes (ibidi, Fitchburg, WI, USA), treated with cisplatin, stained with CTB and cultured with or without mito-GFP and cell tracker deep red-labeled astrocytes for 17 h followed by staining with TMRM or Mitotracker. The TMRM was used in sub-quench mode as described previously [34].

For analysis of mitochondrial transfer, neurons exposed to cisplatin or vehicle were labeled with 20 μM CTB (Thermo Fisher) prior to co-culture with astrocytes. Astrocytes were transfected with either mito-GFP or mito-mCherry prior to exposure to cisplatin followed by labeling with 20 μM Celltracker DeepRed (Thermo Fisher) and co-culture with neurons. Neurons containing an area positive for the mito-GFP or mito-mCherry signal that was larger than 8 pixels were scored as containing astrocyte-derived mitohcondria. The co-cultures were imaged on an SPE Leica Confocal Microscope (Leica Microsystems, Buffalo Grove, IL, USA) with a 63 X or 40 X objective and images were analyzed with LAS X software.

To assess mitochondrial bioenergetics, astrocytes (5 × 10⁴ cells/well) were plated in a Seahorse XFe 24 microplate (Seahorse Biosciences/Agilent Technologies, Santa Clara, CA, USA) coated with 0.05 mg/ml PDL and treated with 1 μM cisplatin or vehicle for 24 h. Cells were washed and incubated for 1 h at 37 °C in XF base media (Seahorse Biosciences) supplemented with 11 mM glucose (Sigma-Aldrich), 2 mM glutamine (Sigma Aldrich), and 1 mM pyruvate (Sigma-Aldrich), 2 mM Oligomycin (Sigma-Alrich), 4 mM FCCP, and rotenone/antimycin A (Sigma-Aldrich, 2 mM each) were used with a 3-time repeat of a 2-min mix, 3-min wait, and 2-min measure assay cycle. Oxygen consumption rates were normalized to the total protein content of each well. Basal respiration, maximal respiratory capacity, and spare respiratory capacity were determined as described previously [20].

Functional Ca²⁺ imaging on cortical neurons was performed as described previously [35]. 12 mm circular glass coverslips containing cells were incubated at room temperature for 20 min with the Ca²⁺-sensitive dye Fura-2-AM (Invitrogen, 2 μM), dissolved in standard extracellular HEPES-buffered HBSS (known hereafter as extracellular imaging buffer) containing the following (in mM): 140 NaCl, 5 KCl, 1.3 CaCl₂, 0.4 MgSO₄, 0.5 MgCl₂, 0.4 KH₂PO₄, 0.6 NaHPO₄, 3 NaHCO₃, 10 glucose, and 10 HEPES adjusted to pH 7.4 with NaOH and 310 mOsm with sucrose. The coverslip was placed in the recording chamber (ALA scientific Instruments, Farmingdale, NY, USA) mounted on the stage of an inverted Nikon Ti2 microscope and continuously superfused for 5 min at room temperature with extracellular imaging buffer. Fura-2 fluorescence was alternately excited at 340 and 380 nm (12 nm band-pass, 50 ms exposure) at 1 Hz using a Lambda LS Xenon lamp (Sutter Instruments, Novato, CA, USA) and a 10x/NA 0.5 objective or 40x/NA 0.6. The CFI Super Fluor 10X was used to measure the ratio of Fura-2 fluorescence. Emitted fluorescence was collected at 510 nm using a sCMOS pco.edge camera for the entire experimental duration, including the first 5 min wash duration, and the ratio of fluorescence (F340:F380) was calculated. The shift in the ratio of Fura-2 fluorescence between excitation at 340 nm versus 380 nm is used as a readout of changes in intracellular calcium concentration [Ca²⁺_i]. Baseline recording of F340:F380 ratio without stimulus was completed to obtain an indication of resting [Ca²⁺_i]. Neurons were stimulated with 20 mM of KCl in the extracellular imaging buffer with continuous superfusion at room temperature. Imaging was analyzed with the NIS Elements software. To identify neurons that did or did not receive astrocytic mitochondria, the Nikon inverted Ti2 microscope with associated A1Rsi-HD confocal system and NIS Elements software was used with the CFI S Plan Fluor ELWD 40XC objective. A single exposure at 576 nm excitation was used to quantify fluorescence within neurons before beginning measurement of F340:F380 ratio.

Data are presented as mean ± SEM of at least 3 independent experiments performed in duplicate or triplicate. For survival analysis and mitochondrial membrane potential analysis, data were normalized to the control of each experiment and replicates were averaged. We used One-way or Two-way analysis of variance (ANOVA) with or without repeated measure followed by Tukey’s correction for multiple comparisons or Sidak’s correction for multiple comparisons according to experimental set up. For mitochondrial transfer analysis, the percentage of neurons that received mitochondria was calculate for each group. We used Student’s t-test or Two-way analysis of variance (ANOVA) without repeated measures followed by Tukey’s correction for multiple comparisons. For calcium imaging, the ratio of fluorescence (F340:F380) was calculated and the ratios at baseline were subtracted from the maximum peak values upon KCl stimulus to calculate change in [Ca²⁺_i]. To calculate Ca²⁺ clearance, F340:F380 ratios were converted to a 0–1 scale with 1 being the maximum value in response to 20 mM KCl. Then, we quantified the time to return to 20% of baseline by counting the values from 1 (maximum peak) to 20% of the baseline value for each neurons. All analyses were performed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA).

To examine the effect of cisplatin on neuron and astrocyte survival, we treated separate cultures of primary cortical neurons and astrocytes with cisplatin for 24 h and measured survival using the WST assay. Cisplatin dose-dependently reduced neuronal survival as assessed immediately after removal of cisplatin (Fig. 1a) or 17 h later (Fig. 1b). Astrocytes were shown to be resistant to cisplatin since only the highest dose (4 μM) resulted in a modest reduction in astrocyte survival (Fig. 1c and d). For all further experiments we used cisplatin at a dose of 1 μM. Exposure of astrocytes to 1 μM cisplatin did not induce detectable changes in astrocytic mitochondrial respiration; basal respiration, spare respiratory capacity and maximal respiration were similar in control and cisplatin-treated astrocytes (Fig. 1e).

To test the hypothesis that astrocytes improve the survival of neurons treated with cisplatin, primary cortical neurons and astrocytes were cultured separately in the presence or absence of 1 μM cisplatin for 24 h. Next cisplatin was removed, neurons were labeled with celltracker blue (CTB), and co-cultured with astrocytes for an additional 17 h. Co-culture with astrocytes significantly improved survival of cisplatin-treated neurons. Astrocytes did not affect survival of control neurons (Fig. 1f).

To assess the effect of cisplatin on neuronal mitochondrial integrity, we quantified mitochondrial membrane potential by labeling the cells with tetramethylrhodamine (TMRM) and assessed fluorescence intensity by flow cytometry. Figure 2a clearly shows that cisplatin depolarized the neuronal mitochondrial membrane potential as indicated by a reduction in TMRM fluorescence intensity. The cisplatin-induced reduction in TMRM fluorescence intensity was not associated with changes in neuronal mitochondrial content as determined by labeling mitochondria with the membrane potential-independent dye Mitotracker (Fig. 2b). Together these findings indicate that cisplatin reduces neuronal mitochondrial membrane potential without leading to an actual loss of mitochondria under the conditions tested. Co-culture with astrocytes restored the mitochondrial membrane potential of neurons cultured with cisplatin (Fig. 2a). Confocal microscopy confirmed that cisplatin reduces TMRM staining in somata and dendrites of neurons and that co-culture with astrocytes restored TMRM staining (Fig. 2c). The increase in TMRM fluorescence intensity of cisplatin-treated neurons in response to astrocytes was associated with an increase in mitotracker fluorescence intensity in neuronal cells (Fig. 2b), indicating an increase in neuronal mitochondrial content as a result of co-culture with astrocytes.

Next, we tested the hypothesis that astrocytes transfer mitochondria to neurons damaged by cisplatin. We labeled astrocyte mitochondria with mCherry coupled to a mitochondrial localization sequence (mito-mCherry). Neurons and astrocytes were treated separately with cisplatin or control medium for 24 h; neurons were labeled with Celltracker Blue, and co-cultured with Celltracker Deep Red-labeled astrocytes for an additional 17 h. Confocal fluorescence analysis revealed that cisplatin-treated neurons contained astrocyte-derived mCherry+ mitochondria. Only a few astrocyte-derived mitochondria were present in untreated control neurons co-cultured with astrocytes (Fig. 3a-c). These findings indicate that astrocytes transfer mitochondria to neurons damaged by cisplatin. Quantitative assessment of mitochondrial transfer showed that cisplatin induced an approximately 3-fold increase in the percentage of neurons that had received mitochondria from astrocytes (Fig. 3d). 3D reconstruction and orthogonal section analysis confirmed that the mito-mCherry+ mitochondria are localized inside the neurons and were detected in the soma, axons and dendrites of the neuron (Fig. 3e-f).

We next assessed the contribution of Miro-1, a Rho-GTPase mitochondrial adaptor protein, to mitochondrial transfer from astrocytes to damaged neurons. Astrocytes were transfected with short interfering RNA (siRNA) to knock down Miro-1 (astrocytes^{Miro-1 siRNA}) and mitochondria in astrocytes were labeled with mito-GFP. Miro-1 siRNA decreased astrocytic Miro-1 by approximately ~ 50% (Fig. 4 inset). Neurons were treated with cisplatin and co-cultured with astrocytes^{Miro-1 siRNA} or astrocytes^{Scr siRNA} as a control and mitochondrial transfer was quantified. The results in Fig. 4 show that knockdown of Miro-1 in astrocytes decreased mitochondrial transfer to neurons in comparison to astrocytes treated with control scrambled siRNA (Fig. 4). These findings indicate that Miro-1 plays a key role in the transfer of mitochondria from astrocytes to neurons damaged by cisplatin.

We next asked the question whether cisplatin alters neuronal Ca²⁺ levels and whether co-culture with astrocytes affects cisplatin-induced changes in neuronal calcium dynamics. Resting [Ca²⁺_i] levels, as measured using the calcium indicator Fura-2, were significantly higher in cisplatin-treated neurons than in control neurons (Fig. 5a and b). Co-culture with astrocytes normalized resting [Ca²⁺_i] levels in cisplatin-treated neurons (Fig. 5a and b). The increase in neuronal [Ca²⁺_i] level in response to 20 mM KCl was smaller in cisplatin-treated than in control neurons and this was also normalized by co-culture with astrocytes, indicating that astrocytes normalize calcium dynamics in neurons treated with cisplatin. (Fig. 5c and d).

Treatment of neurons with cisplatin increased the time to reach 80% clearance of [Ca²⁺_i] after exposure to 20 mM KCl (Fig. 5e and f), even though maximal calcium levels were significantly lower in cisplatin-treated neurons (Fig. 5a and b). These findings indicate Ca²⁺ clearance is impaired in cisplatin treated neurons. Co-culture with astrocytes increased the rate of calcium clearance in cisplatin-treated neurons to a level that was even higher than that of control neurons (Fig. 5e and f).

To address the question of whether transfer of mitochondria from astrocytes to neurons contributes to the improved neuronal Ca²⁺ dynamics, we compared calcium responses to 20 mM KCl of neurons that did or did not contain mCherry+ mitochondria on the same coverslip. As shown in Fig. 6a neurons that contained astrocyte-derived mitochondria showed a larger increase in 20 mM KCl evoked [Ca²⁺_i] in comparison to neurons in the same culture that did not receive astrocytic mitochondria (Fig. 6a). This finding indicates that transfer of mitochondria from astrocytes to neurons plays a substantial role in restoring the KCl evoked [Ca²⁺_i] increase in neurons damaged by cisplatin.

To further address the contribution of mitochondrial transfer to the normalization of calcium dynamics in neurons in response to co-culture with astrocytes, we assessed the effect of astrocytic Miro-1 knockdown. The results in Fig. 6b and c show that basal calcium levels were lower in cisplatin-damaged neurons co-cultured with control astrocytes^{Scr siRNA} as compared to astrocytes^{Miro-1 siRNA}. This indicates that knockdown of Miro-1 in astrocytes prevented the normalization of baseline calcium levels in cisplatin-treated neurons (Fig. 6b and c). Similarly, co-culture of cisplatin-treated neurons with astrocytes^{Miro-1 siRNA} failed to normalize the 20 mM KCl evoked [Ca²⁺_i] that was observed in the presence of control astrocytes^{Scr siRNA} (Fig. 6d and e). This indicates that the restorative effects of astrocytes on calcium dynamics in cisplatin-treated neurons are abrogated by impairing mitochondrial transfer from astrocytes.