Ciliobrevin d

4-Aminopyridine, PTX, poly-L-lysine, D(−)-2-amino-5-phosphonopentanoic acid, Trolox, DAPI nuclear dye, Nocodazole, and Ciliobrevin D were purchased from Sigma. Bicuculline methiodide, MNI-caged-L-glutamate, Anisomycin were purchased from Tocris Bioscience. Leptomycin B (10 nM) was purchased from Beyotime. Peptides used are Tat-APPL1₁₃ (YGRKKRRQRRRRRASEKQKEIERVKEK) and Tat-APPL1_Scr (YGRKKRRQRRRRRASEKQKEIEAAAAA). The following antibodies were used: anti-APPL1 (sc-67402) and anti-Rab5 (sc-46692) from Santa Cruz Biotechnology, anti-pERK (4370S) and anti-GAPDH (2118) from Cell Signaling Techonology, anti-HDAC2 (ab32117), anti-Importin α1 (ab84440), anti-Histone H4K5 (ab51997), anti-Histone H4K12 (ab177793), anti-APPL2 (ab95196), and anti-Histone H4 (ab10158) from Abcam, anti-MAP2 (M9942, M3696), anti-FLAG (F1804), and anti-APPL1 (1409089) from Sigma–Aldrich, and anti-pCREB (06-519 and 04-218) from Millipore. Glutathione sepharose beads and protein A sepharose beads were purchased from GE Healthcare. Phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor cocktails 2 and 3 were purchased from Sigma. Horseradish peroxidase (HRP)-linked goat anti-mouse immunoglobulin G (IgG), goat anti-rabbit IgG, and donkey anti-goat IgG, secondary antibodies conjugated to Dylight (488 or 555), and chemiluminescence kit were purchased from Pierce.

Specific inhibitors were used to reveal the possible role of different cytoskeletal elements in mitochondrial transport via TNTs by selectively blocking the polymerisation of microtubules, as well as the ATPase activity of actin- and microtubule-based motor proteins, preventing their binding to the cytoskeletal polymers and thus hindering their normal functioning. Microtubule polymerisation was blocked with 10 or 20 µM of nocodazole (Thermo Fisher Scientific, Waltham, MA, USA), the activity of dynein was blocked with 20 µM of ciliobrevin D (Sigma Aldrich, St. Louis, MO, USA), and kinesin was inhibited with 15 µM of ispinesib (Selleck Chemicals GmbH, Cologne, Germany). The actin-based myosin II motor protein was blocked with 25 µM of para-nitroblebbistatin (Optopharma Ltd., Budapest, Hungary), myosin V with 30 µM of MyoVin-1 (Merk Millipore, Burlington, MA, USA), and the activity of myosin VI was inhibited with 30 µM of 2,4,6-triiodophenol (TIP) (Alfa Aesar, Haverhill, MA, USA). The optimal concentrations for all inhibitors were determined before each experiment. All dyes, inhibitors, or DMSO (Sigma Aldrich, St. Louis, MO, USA) for control experiments were diluted in culture medium.

The use of all animals was approved by the Institutional Animal Care and Use Committee at the University of California, Irvine. Primary neuronal cultures were obtained from embryonic day 18 (E18) rat embryos. Dissociated cells were cultured on either pre-coated poly-L-Lysine 6-well plates (Biocoat plates, BD Bioscience) or poly-L-Lysine coated glass coverslips affixed to microfluidic chambers at 1 × 10⁶ cells/9.5 cm² and 5 × 10⁶ cells/ml (for microfluidic chamber), respectively. All cells were maintained in Neurobasal (Gibco) supplemented with B27, penicillin/streptomycin, and Glutamax at 37 ^oC, 5% CO₂, 95% humidity. Rat recombinant IL-1β (PeproTech) was used at a final concentration of 10 ng/ml. Human BDNF (PeproTech) was used at a concentration of 50 ng/ml and Ciliobrevin D (Sigma) was dissolved in sterile Me₂SO at 100 μM and used at 1 μM.

The micro-TENNs were treated with various chemical compounds exhibiting well established, specific inhibition of cytoskeleton proteins and enzymes involved in the motility and growth of neurites. All the inhibitors were dissolved in DMSO first at a defined concentration and then were added in the culture media for 24 h. Phase contrast images were taken before and after 24 h of exposure to quantify the contraction rate to test for effects of the drug on this contraction phenomenon. Rho-kinase inhibitor Y27632 (Cayman Chemical 10,005,583) was added at three different concentrations, namely, 50 μM (n = 4), 100 μM (n = 3) and 150 μM (n = 3). The dynein inhibitor ciliobrevin D (Sigma 250,401) and the microtubule-destabilizing agent nocodazole (Sigma M1404) were added at 150 μM (n = 3) and 20 μM (n = 4) respectively. The untreated micro-TENNs acted as a control group (n = 4), whereas a 0.6% DMSO group (n = 3) was included to control for the effects of the vehicle. Between 4–7 DIV, a time interval in which the aggregates have already connected and we could ensure contraction was happening, drugs were administered to determine their effect on contraction by measuring the length change after a 24 h treatment period.