Precise and long-term tracking of mitochondria in neurons using a bioconjugatable and photostable AIE luminogen

Tracking mitochondrial movement in neurons is an attractive but challenging research field as dysregulation of mitochondrial motion is associated with multiple neurological diseases. To realize accurate and long-term tracking of mitochondria in neurons, we elaborately designed a novel aggregation-induced emission (AIE)-active luminogen, TPAP-C5-yne, where we selected a cationic pyridinium moiety to target mitochondria and employed an activated alkyne terminus to achieve long-term tracking through bioconjugation with amines on mitochondria. For the first time, we successfully achieved the accurate analysis of the motion of a single mitochondrion in live primary hippocampal neurons and the long-term tracking of mitochondria for up to a week in live neurons. Therefore, this new AIEgen can be used as a potential tool to study the transport of mitochondria in live neurons.


General Information
All chemicals were purchased from J&K Chemical Co. and Sigma-Aldrich and used as received without further purification. Anhydrous tetrahydrofuran (THF) and toluene were distilled from sodium benzophenone ketyl under dry nitrogen right before fluorescence property investigation. Deionized water was used throughout this study. All aqueous solutions were freshly prepared with deionized water. 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were recorded on a Bruker ARX 400 spectrometer using tetramethylsilane (TMS) as internal standard. High-resolution mass spectra (HRMS) were recorded on a GCT premier CAB048 mass spectrometer operated in a MALDI-TOF mode. Absorption spectra were measured on a PerkinElmer Lambda 365 UV/Vis spectrophotometer. Steady-state photoluminescence (PL) spectra were measured on a Perkin-Elmer spectrofluorometer LS55. Absolute fluorescence quantum yield was measured by a Hamamatsu Quantaurus-QY Plus UV-NIR Absolute PL quantum yield spectrometer. Hydrated diameter of aggregates was measured at room temperature by NanoBrook 90 Plus Zeta Particle Size Analyzer.

Sodium Dedecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
0.5 mg/mL bovine serum albumin (BSA) stock solution was obtained by diluting in phosphatebuffered saline (PBS, pH 7.4). 10 µl of 1 mM TPAP-C5-yne prepared in anhydrous dimethyl sulfoxide (DMSO) was added to 1 mL of BSA stock solution and reacted for 30 min at the room temperature. 5 µg of protein was loaded to each well of 12 % polyacrylamide gel. Fluorescence and Coomassie blue SDS-PAGE gels were visualized using Bio-rad Laboratories ChemiDoc Touch Imaging System.

HeLa and Primary Neuronal Cultures
HeLa cells were cultured and regularly passaged in the culture medium (Dulbecco's modified Eagle medium (DMEM), supplemented with 10 % fetal bovine serum (FBS) and 1% penicillin and streptomycin) at 5 % CO 2 /air at 37 ℃ in a humidified incubator. A day prior to the experiment, the cells were split, and approximately 8 × 10 5 cells were seeded onto each coverslip in 35 mm petri dish. Hippocampal neurons were isolated from postnatal day 0 (P0) Sprague Dawley rat pups obtained from the Animal and Plant Care Facility at the Hong Kong University of Science and Technology (HKUST) and cultured using a similar protocol as previously described. [1] The hippocampal tissues were dissected and digested briefly with papain containing DNase I. After digestion, the density of dissociated neurons was determined. About 5 × 10 4 cells were plated onto 35 mm clear cover glass-bottom confocal petri dish coated with poly-D-lysine. Neurobasal medium supplemented with 2 % B-27 supplement, 0.5 mM Glutamax supplement, 10 % fetal bovine serum (FBS) and 1% penicillin and streptomycin), was used as a culture medium. After 50 h, 20 μM 5-fluoro-2′-deoxyuridine was added to inhibit the proliferation of glial cells. The neurons were incubated at 5% CO 2 /air at 37 ℃ in a humidified incubator and imaging experiments were performed at day in vitro (DIV) 6-21. These experiments were performed in compliance with the relevant laws and intuitional guidelines. The institutional committee had approved the experiment.

Transfection of HeLa and Primary Neuronal Cultures
Mito-BFP (a gift from G. Voeltz, Addgene plasmid 49151) was extracted from DH5α Escherichia coli with PureLink® HiPure Plasmid Maxiprep Kit (Thermo Fisher Scientific). Transfection of plasmid encoding Mito-BFP was performed using lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions in Opti-MEM Reduced-Serum Medium (Thermo Fisher Scientific). For primary neuronal cultures, transfection reagent was added at DIV 7 and incubated for 4 h prior to changing to fresh culture medium. Confocal fluorescence imaging was performed 24 to 48 h after transfection.

Cell Viability Test
The cytotoxicity of TPAP-C5-yne, TPAP-C8 and MTDR were investigated by the standard tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay using a similar protocol as previously described. [4] 5 × 10 3 cells were seeded onto each well of 96-well plate and allowed to adhere for 24 h for HeLa cells and 6 days for hippocampal neuron cells. 200 µL of culture medium containing one of: TPAP-C5-yne, TPAP-C8 or MTDR was added into each well (concentration range: 0.1 ~ 3 µM) and culture medium with only DMSO was added into wells to serve as a negative control group. The cells were incubated for 24 h at 5 % CO 2 /air at 37 °C in a humidified incubator. MTT solution (5 mg/mL in culture medium) was added into each well and cells were incubated for another 4 h. The MTT solution was replaced with 100 µL of DMSO for each well to lysis cells and dissolve water insoluble formazan. 96-well plate was shaken on the shaker for 3 min and the optical density (OD) readings at 570 nm were measured using a plate reader.

Synthesis of compound 3.
In a two-neck round-bottomed flask, a solution of compound 1 (3.24 g, 10 mmol), compound 2 (1.48 g, 12 mmol), Pd(PPh 3 ) 4 (578 mg, 0.5 mmol) and K 2 CO 3 (5.53 g, 40 mmol) were refluxed under nitrogen in THF and MeOH (1:1, v/v, 100 mL) at 80 o C for 24 h. After cooling to room temperature, the solvent was removed under reduced pressure and the residue was extracted with dichloromethane (DCM) and water. The crude product was purified by a silica gel chromatography using hexane/DCM (3:1 v/v) as the eluent to yield compound 3 as a white solid (2.90 g, 90% yield). 1

Synthesis of TPAP-C5-yne.
In a 100 mL round-bottom flask equipped with a Dean-Stark apparatus were added compound 5 (488 mg, 1 mmol), propiolic acid 6 (210 mg, 3 mmol) and p-TSA (19 mg, 0.1 mmol) and 50 mL of dry toluene. The mixture was allowed to reflux overnight with constant removal of yielded water. After cooling to room temperature, the solvent was removed under reduced pressure, the residue was purified by silica gel chromatography using DCM/MeOH (from 95:5 to 85:15, v/v) as the eluent to yield compound TPAP-C5-yne as a yellow powder (270 mg, 50% yield). 1