孕烯醇酮（P5）是所有類固醇的前體，在性腺、腎上腺和大腦中產生。P5可以改善認知功能並減輕精神分裂症的壓力和負性癥狀。P5控制斑馬魚的胚胎細胞遷移。由P5活化的CLIP1 (CLIP-170) 可以加速微管的聚合。CLIP1是一種微管+TIP蛋白，可促進神經元的極化和F-肌動蛋白的延長。
P5和CLIP1協同調節斑馬魚胚胎中的外包運動。本篇論文研究了依賴P5激活的CLIP1對神經元和大腦發育的作用。依賴P5激活的CLIP1影響微管蛋白PTM在發育軸突中的平衡，促進微管-肌動蛋白的交互作用，進而造成神經突和軸突生長，軸突加速特化和大腦的早期發育。
P5-CLIP1作用機制在斑馬魚和小鼠之間是保守的。P5及其不可代謝衍生物以濃度依賴性方式作用，並在不同的神經元中產生相似的效果。P5及其不可代謝衍生物為神經發育和神經退行性疾病的擬議藥物。目前的結果描述了P5神經類固醇功能的基本機制之一是它能夠通過CLIP1重組發育神經元中的細胞骨架。

Pregnenolone (P5) is a precursor of all steroids, that is produced in gonads, adrenal glands and brain. P5 improves cognitive functions, reduces stress and negative symptoms of schizophrenia. P5 is required for embryonic cell movement in zebrafish. CLIP1(CLIP-170) activated by P5 accelerates microtubule polymerization.
CLIP1 is a microtubule +TIP protein, that promotes neuronal polarization, affects dendrites morphology and cell migration. P5 and CLIP1 synergistically regulate epiboly movement in zebrafish embryos. This thesis examines the role of P5-dependent CLIP1 activation on neurons and brain development.
The mechanism of P5-dependent CLIP1 activation affects the balance of tubulin post translational modification (PTM) in developing axon, promotes microtubule-actin crosstalk resulting in increased neurites and axon outgrowth, acceleration of axon specification and early stages of brain development. P5-CLIP1 mechanism is conservative between zebrafish and mice. P5 and its non-metabolized derivatives work in concentration-dependent manner and demonstrate similar effects in different neurons. P5 and its non-metabolized derivatives are proposed drugs for neurodevelopmental and neurodegenerative diseases. Current results characterize one of the basic mechanisms of P5 neurosteroidal function which relies on P5’s ability to reorganize the cytoskeleton in developing neurons via CLIP1.

Table of Contents
Acknowledgments…...…………………………………………….…………..….. II
Abstract (Chinese)………………………………………………….……………… III
Abstract (English)………………………………………………………………….. IV
Table of contents……………………………………………………….………….. V
List of tables …………………………………………..……………………….…... IX
List of figures……………………………………………………………………….. X
Abbreviations………………………………………………………….…………… XIII
1. Introduction……….……………................................................................... 1
1.1. Pregnenolone and its functions…………………………………….……….. 1
1.1.1. Pregnenolone functions in steroidogenesis……………………….…….. 1
1.1.2. Pregnenolone is a functional neurosteroid………………………………. 1
1.1.3. Pregnenolone is required for normal embryonic development………… 2
1.2. Mechanism of pregnenolone action………………………………………… 2
1.2.1. The molecular targets of pregnenolone………………………………….. 2
1.2.2. P5 promotes microtubule polymerization via CLIP1………………...….. 3
1.2.3. Mechanism of P5-CLIP1 binding…………………………………………. 4
1.2.4. P5 requires CLIP1 to regulate microtubule assembly in vivo…….……. 4
1.3. The structure and functions of CLIP1………………………………………. 5
1.3.1. The protein structure of CLIP1……………………………………………. 5
1.3.2. Regulation of CLIP1 activities…………………………………...………… 6
1.3.3. Biological functions of CLIP1…………………………………...…………. 6
1.4. Neuronal development……………………………………………………….. 7
1.5. Cytoskeleton reorganization in developing neurons………………………. 8
1.6. The tubulin post-translational modifications……………………………….. 8
2. Materials and Methods……….……………………………………….………... 12
2.1. Animal models…………………..………………………...………..………… 12
2.2. Reagents, plasmids, antibodies...…….…………………………..………… 12
2.3. Cell transfection, in vitro transcription, mRNA, morpholino oligonucleotide, gRNAs/Cas9 microinjection ………….……………….……….
12
2.4. Preparation mouse and zebrafish primary neuronal cultures…..……….. 14
2.5. Live-cell microtubule comet tracking…………………………...…………… 16
2.6. Live-cell protein colocalization analysis………………………..…………… 16
2.7. Immunocytochemistry ……………………………….……..….…...…….….. 16
2.8. Immunoblotting ………………………….……..….………………….………. 17
2.9. Immunohistochemistry …………………………..………..…..…...………… 18
2.10. Drug treatment………………………………………………….…………… 19
2.11. Data acquisition and analysis…………………………………..………….. 20
3. Results…...…..……………………………………..…………….....….….…… 22
3.1. Pregnenolone accelerates microtubule comets dynamic in developing neurons………………………………………………………………………………
22
3.2. Pregnenolone increases early neurite outgrowth in different types of neurons……………..…………………………………………………..…….….….
22
3.3. Pregnenolone accelerates axon specification and increases axon outgrowth………………………………………………………………….…..…….
23
3.4. Pregnenolone increases dynamic microtubules pool and accelerates microtubule stability along developing neurites and nascent axons…………..
24
3.5. CLIP1 mediates the pregnenolone effect to promote neurite outgrowth... 25
3.6. The effect of pregnenolone on dynamic and stable microtubule pools is CLIP1-dependent……………………………………………………………….….
26
3.7. Pregnenolone regulates cytoskeleton reorganization in growth cone of developing neurons via CLIP1.…………………………………………...………
27
3.7.1. Pregnenolone decreases growth cone area in developing neurons via CLIP1……………………………………………………………………….……….
27
3.7.2. Pregnenolone increases microtubule penetration into filopodia of developing neurons……………………………………………………….….…….
28
3.7.3. Pregnenolone effect on microtubule penetration into filopodia is CLIP1-dependent……………………………………………………………….….
28
3.8. Pregnenolone promotes CLIP1-Diaph1 colocalization in developing neurites…………………………………………………………………….…….….
29
3.9. Pregnenolone accelerates zebrafish cerebellum development in CLIP1-dependent manner…………………………………………………………..……..
29
3.9.1. Pregnenolone accelerates formation of stable microtubule tracks in developing axon via CLIP1……………….…………………………………….....
29
3.9.2. Pregnenolone promotes the functional axons connections via CLIP1... 30
3.9.3. P5-CLIP1 mechanism is effective in early developmental stages…..... 30
3.10. Endogenous pregnenolone is required for normal development of zebrafish cerebellum.………………………………………………………….…..
31
3.11. Non-metabolized P5 analog promotes neurite and axon development in vitro similarly to pregnenolone………………………………………..…..……
31
3.12. Non-metabolized P5 analog accelerates zebrafish cerebellum development……………………………………………………………….…..…...
32
3.13. Complex regulation of zebrafish epiboly movement by cooperative action of P5 and CLIP1. ………………………………………………….…..…...
33
4. Conclusion………..…...……………………………………………….….…….. 35
5. Discussion…...….………………………………………………...……….……. 36
5.1. Conserved effects of P5 on neurites and axon outgrowth in different types of neurons …………………………………………………….……..….…..
36
5.2. The response to P5 is stage- and concentration-dependent in developing neurons………………………………………………………….…..…
36
5.3. P5-CLIP1 accelerates microtubule network maturation in developing neurons………………………………………………………………………..….…
37
5.3.1. P5 affects dynamic microtubule pool in early neurites………….…..….. 37
5.3.2. P5-CLIP1 effects on tubulin PTM is compartment-specific……..……... 38
5.4. P5 reorganizes cytoskeleton, promoting embryogenesis and brain development……………………………………………………………….…..……
38
5.5. Synthetic P5 derivatives with microtubule polymerization activity similar to P5 accelerate an early stage of brain development……………....…...........
39
5.6. The complexity of analysis of Cyp11a1, Clip1a genes functions in zebrafish due to presence of paralogous genes………………………...……...
39
6. Future directions.………………………………………………...…...………… 40
7. The questions from thesis committee members…………………..….…….. 42
8. Bibliographies……………..…………………………………………………….. 48
9. Tables ……….…………………….…………….………………………….…… 63
10. Figures and legends.……………….…………….………………..…….….... 68
 

Aillaud, C., Bosc, C., Peris, L., Bosson, A., Heemeryck, P., Van Dijk, J., Le Friec, J., Boulan, B., Vossier, F., Sanman, L. E., Syed, S., Amara, N., Couté, Y., Lafanechère, L., Denarier, E., Delphin, C., Pelletier, L., Humbert, S., Bogyo, M., Andrieux, A., Rogowski, K., & Moutin, M.-J. (2017). Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation. Science, 358(6369), 1448-1453. https://doi.org/doi:10.1126/science.aao4165
Akhmanova, A., Hoogenraad, C. C., Drabek, K., Stepanova, T., Dortland, B., Verkerk, T., Vermeulen, W., Burgering, B. M., De Zeeuw, C. I., Grosveld, F., & Galjart, N. (2001). Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts. Cell, 104(6), 923-935. https://doi.org/10.1016/s0092-8674(01)00288-4
Akhmanova, A., & Maiato, H. (2017). Closing the tubulin detyrosination cycle. Science, 358(6369), 1381-1382. https://doi.org/doi:10.1126/science.aar3895
Akwa, Y., Young, J., Kabbadj, K., Sancho, M. J., Zucman, D., Vourc'h, C., Jung-Testas, I., Hu, Z. Y., Le Goascogne, C., Jo, D. H., & et al. (1991). Neurosteroids: biosynthesis, metabolism and function of pregnenolone and dehydroepiandrosterone in the brain. J Steroid Biochem Mol Biol, 40(1-3), 71-81. https://doi.org/10.1016/0960-0760(91)90169-6
al Kandari, H., Katsumata, N., Alexander, S., & Rasoul, M. A. (2006). Homozygous mutation of P450 side-chain cleavage enzyme gene (CYP11A1) in 46, XY patient with adrenal insufficiency, complete sex reversal, and agenesis of corpus callosum. J Clin Endocrinol Metab, 91(8), 2821-2826. https://doi.org/10.1210/jc.2005-2230
Amin, M. A., Itoh, G., Iemura, K., Ikeda, M., & Tanaka, K. (2014). CLIP-170 recruits PLK1 to kinetochores during early mitosis for chromosome alignment. J Cell Sci, 127(Pt 13), 2818-2824. https://doi.org/10.1242/jcs.150755
Amin, M. A., Kobayashi, K., & Tanaka, K. (2015). CLIP-170 tethers kinetochores to microtubule plus ends against poleward force by dynein for stable kinetochore-microtubule attachment. FEBS Lett, 589(19 Pt B), 2739-2746. https://doi.org/10.1016/j.febslet.2015.07.036
Arakawa, Y., Bito, H., Furuyashiki, T., Tsuji, T., Takemoto-Kimura, S., Kimura, K., Nozaki, K., Hashimoto, N., & Narumiya, S. (2003). Control of axon elongation via an SDF-1??/Rho/mDia pathway in cultured cerebellar granule neurons. The Journal of cell biology, 161, 381-391. https://doi.org/10.1083/jcb.200210149
Bae, Y. K., Kani, S., Shimizu, T., Tanabe, K., Nojima, H., Kimura, Y., Higashijima, S., & Hibi, M. (2009). Anatomy of zebrafish cerebellum and screen for mutations affecting its development. Dev Biol, 330(2), 406-426. https://doi.org/10.1016/j.ydbio.2009.04.013
Barbiero, I., Peroni, D., Siniscalchi, P., Rusconi, L., Tramarin, M., De Rosa, R., Motta, P., Bianchi, M., & Kilstrup-Nielsen, C. (2020). Pregnenolone and pregnenolone-methyl-ether rescue neuronal defects caused by dysfunctional CLIP170 in a neuronal model of CDKL5 Deficiency Disorder. Neuropharmacology, 164, 107897. https://doi.org/10.1016/j.neuropharm.2019.107897
Barbiero, I., Zamberletti, E., Tramarin, M., Gabaglio, M., Peroni, D., De Rosa, R., Baldin, S., Bianchi, M., Rubino, T., & Kilstrup-Nielsen, C. (2022). Pregnenolone-methyl-ether enhances CLIP170 and microtubule functions improving spine maturation and hippocampal deficits related to CDKL5 deficiency. Hum Mol Genet, 31(16), 2738-2750. https://doi.org/10.1093/hmg/ddac067
Beaudoin, G. M., 3rd, Lee, S. H., Singh, D., Yuan, Y., Ng, Y. G., Reichardt, L. F., & Arikkath, J. (2012). Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc, 7(9), 1741-1754. https://doi.org/10.1038/nprot.2012.099
Beltramo, D. M., Arce, C. A., & Barra, H. S. (1987). Tubulin, but not microtubules, is the substrate for tubulin:tyrosine ligase in mature avian erythrocytes. Journal of Biological Chemistry, 262(32), 15673-15677. https://doi.org/https://doi.org/10.1016/S0021-9258(18)47779-4
Bieling, P., Kandels-Lewis, S., Telley, I. A., van Dijk, J., Janke, C., & Surrey, T. (2008). CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites. J Cell Biol, 183(7), 1223-1233. https://doi.org/10.1083/jcb.200809190
Bradke, F., & Dotti, C. G. (1999). The Role of Local Actin Instability in Axon Formation. Science, 283(5409), 1931. https://doi.org/10.1126/science.283.5409.1931
Brown, E. S., Park, J., Marx, C. E., Hynan, L. S., Gardner, C., Davila, D., Nakamura, A., Sunderajan, P., Lo, A., & Holmes, T. (2014). A randomized, double-blind, placebo-controlled trial of pregnenolone for bipolar depression. Neuropsychopharmacology, 39(12), 2867-2873. https://doi.org/10.1038/npp.2014.138
Cai, H., Zhou, X., Dougherty, G. G., Reddy, R. D., Haas, G. L., Montrose, D. M., Keshavan, M., & Yao, J. K. (2018). Pregnenolone-progesterone-allopregnanolone pathway as a potential therapeutic target in first-episode antipsychotic-naïve patients with schizophrenia. Psychoneuroendocrinology, 90, 43-51. https://doi.org/10.1016/j.psyneuen.2018.02.004
Chen, Z., Lee, H., Henle, S. J., Cheever, T. R., Ekker, S. C., & Henley, J. R. (2013). Primary neuron culture for nerve growth and axon guidance studies in zebrafish (Danio rerio). PLoS One, 8(3), e57539. https://doi.org/10.1371/journal.pone.0057539
Coleman, A. K., Joca, H. C., Shi, G., Lederer, W. J., & Ward, C. W. (2021). Tubulin acetylation increases cytoskeletal stiffness to regulate mechanotransduction in striated muscle. J Gen Physiol, 153(7). https://doi.org/10.1085/jgp.202012743
Dixit, R., Barnett, B., Lazarus, J. E., Tokito, M., Goldman, Y. E., & Holzbaur, E. L. (2009). Microtubule plus-end tracking by CLIP-170 requires EB1. Proc Natl Acad Sci U S A, 106(2), 492-497. https://doi.org/10.1073/pnas.0807614106
Don, E. K., Maschirow, A., Radford, R. A. W., Scherer, N. M., Vidal-Itriago, A., Hogan, A., Maurel, C., Formella, I., Stoddart, J. J., Hall, T. E., Lee, A., Shi, B., Cole, N. J., Laird, A. S., Badrock, A. P., Chung, R. S., & Morsch, M. (2021). In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS). Mol Neurobiol, 58(5), 2061-2074. https://doi.org/10.1007/s12035-020-02238-0
Ducharme, N., Banks, W. A., Morley, J. E., Robinson, S. M., Niehoff, M. L., Mattern, C., & Farr, S. A. (2010). Brain distribution and behavioral effects of progesterone and pregnenolone after intranasal or intravenous administration. Eur J Pharmacol, 641(2-3), 128-134. https://doi.org/10.1016/j.ejphar.2010.05.033
Erck, C., Peris, L., Andrieux, A., Meissirel, C., Gruber, A. D., Vernet, M., Schweitzer, A., Saoudi, Y., Pointu, H., Bosc, C., Salin, P. A., Job, D., & Wehland, J. (2005). A vital role of tubulin-tyrosine-ligase for neuronal organization. Proceedings of the National Academy of Sciences, 102(22), 7853-7858. https://doi.org/doi:10.1073/pnas.0409626102
Fernández-Barrera, J., & Alonso, M. A. (2018). Coordination of microtubule acetylation and the actin cytoskeleton by formins. Cell Mol Life Sci, 75(17), 3181-3191. https://doi.org/10.1007/s00018-018-2855-3
Flood, J. F., Morley, J. E., & Roberts, E. (1992). Memory-enhancing effects in male mice of pregnenolone and steroids metabolically derived from it. Proceedings of the National Academy of Sciences, 89(5), 1567-1571. https://doi.org/10.1073/pnas.89.5.1567
Fontaine-Lenoir, V., Chambraud, B., Fellous, A., David, S., Duchossoy, Y., Baulieu, E. E., & Robel, P. (2006). Microtubule-associated protein 2 (MAP2) is a neurosteroid receptor. Proc Natl Acad Sci U S A, 103(12), 4711-4716. https://doi.org/10.1073/pnas.0600113103
Fukata, M., Watanabe, T., Noritake, J., Nakagawa, M., Yamaga, M., Kuroda, S., Matsuura, Y., Iwamatsu, A., Perez, F., & Kaibuchi, K. (2002). Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170. Cell, 109(7), 873-885. http://www.ncbi.nlm.nih.gov/pubmed/12110184
Hahn, I., Voelzmann, A., Liew, Y.-T., Costa-Gomes, B., & Prokop, A. (2019). The model of local axon homeostasis - explaining the role and regulation of microtubule bundles in axon maintenance and pathology. Neural Development, 14(1), 11. https://doi.org/10.1186/s13064-019-0134-0
Hamasaki, M., Matsumura, S., Satou, A., Takahashi, C., Oda, Y., Higashiura, C., Ishihama, Y., & Toyoshima, F. (2014). Pregnenolone functions in centriole cohesion during mitosis. Chem Biol, 21(12), 1707-1721. https://doi.org/10.1016/j.chembiol.2014.11.005
Henrie, H., Bakhos-Douaihy, D., Cantaloube, I., Pilon, A., Talantikite, M., Stoppin-Mellet, V., Baillet, A., Pous, C., & Benoit, B. (2020). Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue. J Cell Biol, 219(7). https://doi.org/10.1083/jcb.201909093
Henty-Ridilla, J. L., Rankova, A., Eskin, J. A., Kenny, K., & Goode, B. L. (2016). Accelerated actin filament polymerization from microtubule plus ends. Science, 352(6288), 1004-1009. https://doi.org/10.1126/science.aaf1709
Hsu, H. J., Liang, M. R., Chen, C. T., & Chung, B. C. (2006). Pregnenolone stabilizes microtubules and promotes zebrafish embryonic cell movement. Nature, 439(7075), 480-483. https://doi.org/10.1038/nature04436
Hu, M. C., Hsu, N. C., El Hadj, N. B., Pai, C. I., Chu, H. P., Wang, C. K., & Chung, B. C. (2002). Steroid deficiency syndromes in mice with targeted disruption of Cyp11a1. Mol Endocrinol, 16(8), 1943-1950. https://doi.org/10.1210/me.2002-0055
Janke, C., & Montagnac, G. (2017). Causes and Consequences of Microtubule Acetylation. Current Biology, 27(23), R1287-R1292. https://doi.org/10.1016/j.cub.2017.10.044
Kakeno, M., Matsuzawa, K., Matsui, T., Akita, H., Sugiyama, I., Ishidate, F., Nakano, A., Takashima, S., Goto, H., Inagaki, M., Kaibuchi, K., & Watanabe, T. (2014). Plk1 phosphorylates CLIP-170 and regulates its binding to microtubules for chromosome alignment. Cell Struct Funct, 39(1), 45-59. https://doi.org/10.1247/csf.14001
Kani, S., Bae, Y. K., Shimizu, T., Tanabe, K., Satou, C., Parsons, M. J., Scott, E., Higashijima, S., & Hibi, M. (2010). Proneural gene-linked neurogenesis in zebrafish cerebellum. Dev Biol, 343(1-2), 1-17. https://doi.org/10.1016/j.ydbio.2010.03.024
Kholmanskikh, S. S., Koeller, H. B., Wynshaw-Boris, A., Gomez, T., Letourneau, P. C., & Ross, M. E. (2006). Calcium-dependent interaction of Lis1 with IQGAP1 and Cdc42 promotes neuronal motility. Nat Neurosci, 9(1), 50-57. https://doi.org/10.1038/nn1619
Kim, C. J., Lin, L., Huang, N., Quigley, C. A., AvRuskin, T. W., Achermann, J. C., & Miller, W. L. (2008). Severe combined adrenal and gonadal deficiency caused by novel mutations in the cholesterol side chain cleavage enzyme, P450scc. J Clin Endocrinol Metab, 93(3), 696-702. https://doi.org/10.1210/jc.2007-2330
Kolas, V., Bandonil, J. S. A., Wali, N., Hsia, K. C., Shie, J. J., & Chung, B. C. (2022). A synthetic pregnenolone analog promotes microtubule dynamics and neural development. Cell Biosci, 12(1), 190. https://doi.org/10.1186/s13578-022-00923-2
Kolas, V., Mathieu, G., Wu, Y.-T., & Chung, B.-c. (2022). Evaluation of two gene ablation methods for the analysis of pregnenolone function in zebrafish embryonic development. Biochemical and Biophysical Research Communications, 636, 84-88. https://doi.org/https://doi.org/10.1016/j.bbrc.2022.10.067
Krohmer, A., Brehm, M., Auwarter, V., & Szabo, B. (2017). Pregnenolone does not interfere with the effects of cannabinoids on synaptic transmission in the cerebellum and the nucleus accumbens. Pharmacol Res, 123, 51-61. https://doi.org/10.1016/j.phrs.2017.04.032
Kumar, N., & Flavin, M. (1981). Preferential action of a brain detyrosinolating carboxypeptidase on polymerized tubulin. Journal of Biological Chemistry, 256(14), 7678-7686. https://doi.org/https://doi.org/10.1016/S0021-9258(19)69014-9
Lansbergen, G., Komarova, Y., Modesti, M., Wyman, C., Hoogenraad, C. C., Goodson, H. V., Lemaitre, R. P., Drechsel, D. N., van Munster, E., Gadella, T. W., Jr., Grosveld, F., Galjart, N., Borisy, G. G., & Akhmanova, A. (2004). Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization. J Cell Biol, 166(7), 1003-1014. https://doi.org/10.1083/jcb.200402082
Laurine, E., Lafitte, D., Gregoire, C., Seree, E., Loret, E., Douillard, S., Michel, B., Briand, C., & Verdier, J. M. (2003). Specific binding of dehydroepiandrosterone to the N terminus of the microtubule-associated protein MAP2. J Biol Chem, 278(32), 29979-29986. https://doi.org/10.1074/jbc.M303242200
Lee, H. S., Komarova, Y. A., Nadezhdina, E. S., Anjum, R., Peloquin, J. G., Schober, J. M., Danciu, O., van Haren, J., Galjart, N., Gygi, S. P., Akhmanova, A., & Borisy, G. G. (2010). Phosphorylation controls autoinhibition of cytoplasmic linker protein-170. Mol Biol Cell, 21(15), 2661-2673. https://doi.org/10.1091/mbc.E09-12-1036
Leroux, M. R. (2010). Tubulin acetyltransferase discovered: Ciliary role in the ancestral eukaryote expanded to neurons in metazoans. Proceedings of the National Academy of Sciences, 107(50), 21238-21239. https://doi.org/doi:10.1073/pnas.1016396108
Lim, W. M., Ito, Y., Sakata-Sogawa, K., & Tokunaga, M. (2018). CLIP-170 is essential for MTOC repositioning during T cell activation by regulating dynein localisation on the cell surface. Sci Rep, 8(1), 17447. https://doi.org/10.1038/s41598-018-35593-z
Lomakin, A. J., Semenova, I., Zaliapin, I., Kraikivski, P., Nadezhdina, E., Slepchenko, B. M., Akhmanova, A., & Rodionov, V. (2009). CLIP-170-dependent capture of membrane organelles by microtubules initiates minus-end directed transport. Dev Cell, 17(3), 323-333. https://doi.org/10.1016/j.devcel.2009.07.010
Marchant, C., Malmi-Kakkada, A., Espina, J., & Barriga, E. (2021). Microtubule deacetylation reduces cell stiffness to allow the onset of collective cell migration in vivo. bioRxiv, 2021.2008.2012.456059. https://doi.org/10.1101/2021.08.12.456059
Marx, C. E., Lee, J., Subramaniam, M., Rapisarda, A., Bautista, D. C., Chan, E., Kilts, J. D., Buchanan, R. W., Wai, E. P., Verma, S., Sim, K., Hariram, J., Jacob, R., Keefe, R. S., & Chong, S. A. (2014). Proof-of-concept randomized controlled trial of pregnenolone in schizophrenia. Psychopharmacology (Berl), 231(17), 3647-3662. https://doi.org/10.1007/s00213-014-3673-4
Meijering, E., Dzyubachyk, O., & Smal, I. (2012). Methods for cell and particle tracking. Methods Enzymol, 504, 183-200. https://doi.org/10.1016/b978-0-12-391857-4.00009-4
Melchior, C. L., & Ritzmann, R. F. (1996). Neurosteroids block the memory-impairing effects of ethanol in mice. Pharmacology Biochemistry and Behavior, 53(1), 51-56. https://doi.org/https://doi.org/10.1016/0091-3057(95)00197-2
Mellon, S. H., & Deschepper, C. F. (1993). Neurosteroid biosynthesis: genes for adrenal steroidogenic enzymes are expressed in the brain. Brain Res, 629(2), 283-292. https://doi.org/10.1016/0006-8993(93)91332-m
Milivojevic, V., Charron, L., Fogelman, N., Hermes, G., & Sinha, R. (2022). Pregnenolone Reduces Stress-Induced Craving, Anxiety, and Autonomic Arousal in Individuals with Cocaine Use Disorder. Biomolecules, 12(11). https://doi.org/10.3390/biom12111593
Milivojevic, V., Sullivan, L., Tiber, J., Fogelman, N., Simpson, C., Hermes, G., & Sinha, R. (2022). Pregnenolone effects on provoked alcohol craving, anxiety, HPA axis, and autonomic arousal in individuals with alcohol use disorder. Psychopharmacology (Berl). https://doi.org/10.1007/s00213-022-06278-3
Mizota, K., & Ueda, H. (2008). N-terminus of MAP2C as a neurosteroid-binding site. Neuroreport, 19(15), 1529-1533. https://doi.org/10.1097/WNR.0b013e328310fe97
Naylor, J. C., Kilts, J. D., Shampine, L. J., Parke, G. J., Wagner, H. R., Szabo, S. T., Smith, K. D., Allen, T. B., Telford-Marx, E. G., Dunn, C. E., Cuffe, B. T., O'Loughlin, S. H., & Marx, C. E. (2020). Effect of Pregnenolone vs Placebo on Self-reported Chronic Low Back Pain Among US Military Veterans: A Randomized Clinical Trial. JAMA Netw Open, 3(3), e200287. https://doi.org/10.1001/jamanetworkopen.2020.0287
Neukirchen, D., & Bradke, F. (2011). Cytoplasmic linker proteins regulate neuronal polarization through microtubule and growth cone dynamics. J Neurosci, 31(4), 1528-1538. https://doi.org/10.1523/JNEUROSCI.3983-10.2011
Nieuwenhuis, J., & Brummelkamp, T. R. (2019). The Tubulin Detyrosination Cycle: Function and Enzymes. Trends in Cell Biology, 29(1), 80-92. https://doi.org/10.1016/j.tcb.2018.08.003
Nirschl, J. J., Magiera, M. M., Lazarus, J. E., Janke, C., & Holzbaur, E. L. (2016). alpha-Tubulin Tyrosination and CLIP-170 Phosphorylation Regulate the Initiation of Dynein-Driven Transport in Neurons. Cell Rep, 14(11), 2637-2652. https://doi.org/10.1016/j.celrep.2016.02.046
Pagnamenta, A. T., Heemeryck, P., Martin, H. C., Bosc, C., Peris, L., Uszynski, I., Gory-Fauré, S., Couly, S., Deshpande, C., Siddiqui, A., Elmonairy, A. A., Jayawant, S., Murthy, S., Walker, I., Loong, L., Bauer, P., Vossier, F., Denarier, E., Maurice, T., Barbier, E. L., Deloulme, J. C., Taylor, J. C., Blair, E. M., Andrieux, A., & Moutin, M. J. (2019). Defective tubulin detyrosination causes structural brain abnormalities with cognitive deficiency in humans and mice. Hum Mol Genet, 28(20), 3391-3405. https://doi.org/10.1093/hmg/ddz186
Perez, F., Diamantopoulos, G. S., Stalder, R., & Kreis, T. E. (1999). CLIP-170 highlights growing microtubule ends in vivo. Cell, 96(4), 517-527. https://doi.org/10.1016/s0092-8674(00)80656-x
Peris, L., Thery, M., Fauré, J., Saoudi, Y., Lafanechère, L., Chilton, J. K., Gordon-Weeks, P., Galjart, N., Bornens, M., Wordeman, L., Wehland, J., Andrieux, A., & Job, D. (2006). Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends. J Cell Biol, 174(6), 839-849. https://doi.org/10.1083/jcb.200512058
Peris , L., Thery , M., Fauré , J., Saoudi , Y., Lafanechère , L., Chilton , J. K., Gordon-Weeks , P., Galjart , N., Bornens , M., Wordeman , L., Wehland , J., Andrieux , A., & Job , D. (2006). Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends. Journal of Cell Biology, 174(6), 839-849. https://doi.org/10.1083/jcb.200512058
Ramírez, S., Haddad-Tóvolli, R., Radosevic, M., Toledo, M., Pané, A., Alcolea, D., Ribas, V., Milà-Guasch, M., Pozo, M., Obri, A., Eyre, E., Gómez-Valadés, A. G., Chivite, I., Van Eeckhout, T., Zalachoras, I., Altirriba, J., Bauder, C., Imbernón, M., Garrabou, G., Garcia-Ruiz, C., Nogueiras, R., Soto, D., Gasull, X., Sandi, C., Brüning, J. C., Fortea, J., Jiménez, A., Fernández-Checa, J. C., & Claret, M. (2022). Hypothalamic pregnenolone mediates recognition memory in the context of metabolic disorders. Cell Metabolism, 34(2), 269-284.e269. https://doi.org/10.1016/j.cmet.2021.12.023
Ran, J., Luo, Y., Zhang, Y., Yang, Y., Chen, M., Liu, M., Li, D., & Zhou, J. (2017). Phosphorylation of EB1 regulates the recruitment of CLIP-170 and p150glued to the plus ends of astral microtubules. Oncotarget, 8(6), 9858-9867. https://doi.org/10.18632/oncotarget.14222
Reddy, D. S. (2010). Neurosteroids: Endogenous role in the human brain and therapeutic potentials. Progress in brain research, 186, 113-137. https://doi.org/10.1016/B978-0-444-53630-3.00008-7
Rohde, L. A., & Heisenberg, C. P. (2007). Zebrafish gastrulation: cell movements, signals, and mechanisms. Int Rev Cytol, 261, 159-192. https://doi.org/10.1016/S0074-7696(07)61004-3
Schumacher, M., Robel, P., & Baulieu, E. E. (1996). Development and regeneration of the nervous system: a role for neurosteroids. Dev Neurosci, 18(1-2), 6-21. https://doi.org/10.1159/000111391
Schwend, T., Loucks, E. J., Snyder, D., & Ahlgren, S. C. (2011). Requirement of Npc1 and availability of cholesterol for early embryonic cell movements in zebrafish. J Lipid Res, 52(7), 1328-1344. https://doi.org/10.1194/jlr.M012377
Shin, Y. Y., Kang, E. J., Jeong, J. S., Kim, M. J., Jung, E. M., Jeung, E. B., & An, B. S. (2019). Pregnenolone as a potential candidate for hormone therapy for female reproductive disorders targeting ERbeta. Mol Reprod Dev, 86(1), 109-117. https://doi.org/10.1002/mrd.23086
Song, Y., & Brady, S. T. (2015). Post-translational modifications of tubulin: pathways to functional diversity of microtubules. Trends in Cell Biology, 25(3), 125-136. https://doi.org/10.1016/j.tcb.2014.10.004
Straiker, A., Mitjavila, J., Yin, D., Gibson, A., & Mackie, K. (2015). Aiming for allosterism: Evaluation of allosteric modulators of CB1 in a neuronal model. Pharmacol Res, 99, 370-376. https://doi.org/10.1016/j.phrs.2015.07.017
Su, H., Wang, B., & Shaik, S. (2019). Quantum-Mechanical/Molecular-Mechanical Studies of CYP11A1-Catalyzed Biosynthesis of Pregnenolone from Cholesterol Reveal a C-C Bond Cleavage Reaction That Occurs by a Compound I-Mediated Electron Transfer. J Am Chem Soc, 141(51), 20079-20088. https://doi.org/10.1021/jacs.9b08561
Swiech, L., Blazejczyk, M., Urbanska, M., Pietruszka, P., Dortland, B. R., Malik, A. R., Wulf, P. S., Hoogenraad, C. C., & Jaworski, J. (2011). CLIP-170 and IQGAP1 cooperatively regulate dendrite morphology. J Neurosci, 31(12), 4555-4568. https://doi.org/10.1523/JNEUROSCI.6582-10.2011
Szyk, A., Deaconescu, A. M., Piszczek, G., & Roll-Mecak, A. (2011). Tubulin tyrosine ligase structure reveals adaptation of an ancient fold to bind and modify tubulin. Nature Structural & Molecular Biology, 18(11), 1250-1258. https://doi.org/10.1038/nsmb.2148
Tahirovic, S., & Bradke, F. (2009). Neuronal polarity. Cold Spring Harb Perspect Biol, 1(3), a001644. https://doi.org/10.1101/cshperspect.a001644
Tajima, T., Fujieda, K., Kouda, N., Nakae, J., & Miller, W. L. (2001). Heterozygous mutation in the cholesterol side chain cleavage enzyme (p450scc) gene in a patient with 46,XY sex reversal and adrenal insufficiency. J Clin Endocrinol Metab, 86(8), 3820-3825. https://doi.org/10.1210/jcem.86.8.7748
Tuem, K. B., & Atey, T. M. (2017). Neuroactive Steroids: Receptor Interactions and Responses. Front Neurol, 8, 442. https://doi.org/10.3389/fneur.2017.00442
Vallee, M., Vitiello, S., Bellocchio, L., Hebert-Chatelain, E., Monlezun, S., Martin-Garcia, E., Kasanetz, F., Baillie, G. L., Panin, F., Cathala, A., Roullot-Lacarriere, V., Fabre, S., Hurst, D. P., Lynch, D. L., Shore, D. M., Deroche-Gamonet, V., Spampinato, U., Revest, J. M., Maldonado, R., Reggio, P. H., Ross, R. A., Marsicano, G., & Piazza, P. V. (2014). Pregnenolone can protect the brain from cannabis intoxication. Science, 343(6166), 94-98. https://doi.org/10.1126/science.1243985
Weng, J.-H. (2013). Pregnenolone functions in cytoskeleton, cell migration and zebrafish development [Doctoral dissertation, National Yang-Ming University].
Weng, J. H., Liang, M. R., Chen, C. H., Tong, S. K., Huang, T. C., Lee, S. P., Chen, Y. R., Chen, C. T., & Chung, B. C. (2013). Pregnenolone activates CLIP-170 to promote microtubule growth and cell migration. Nat Chem Biol, 9(10), 636-642. https://doi.org/10.1038/nchembio.1321
Wilkins, A., Majed, H., Layfield, R., Compston, A., & Chandran, S. (2003). Oligodendrocytes Promote Neuronal Survival and Axonal Length by Distinct Intracellular Mechanisms: A Novel Role for Oligodendrocyte-Derived Glial Cell Line-Derived Neurotrophic Factor. The Journal of Neuroscience, 23(12), 4967-4974. https://doi.org/10.1523/jneurosci.23-12-04967.2003
Witte, H., Neukirchen, D., & Bradke, F. (2008). Microtubule stabilization specifies initial neuronal polarization. J Cell Biol, 180(3), 619-632. https://doi.org/10.1083/jcb.200707042
Wu, I.-T. (2016). Probing CLIP-170 in cells and zebrafish [Master thesis, National Yang-Ming University].
Wu, Y.-F. O., Bryant, A. T., Nelson, N. T., Madey, A. G., Fernandes, G. F., & Goodson, H. V. (2021). Overexpression of the microtubule-binding protein CLIP-170 induces a +TIP network superstructure consistent with a biomolecular condensate. PLoS One, 16(12), e0260401. https://doi.org/10.1371/journal.pone.0260401
Yamamoto, H., Demura, T., Morita, M., Banker, G. A., Tanii, T., & Nakamura, S. (2012). Differential neurite outgrowth is required for axon specification by cultured hippocampal neurons. J Neurochem, 123(6), 904-910. https://doi.org/10.1111/jnc.12001
Young, J., Corpéchot, C., Perché, F., Eychenne, B., Haug, M., Baulieu, E. E., & Robel, P. (1996). Neurosteroids in the mouse brain: behavioral and pharmacological effects of a 3 beta-hydroxysteroid dehydrogenase inhibitor. Steroids, 61(3), 144-149. https://doi.org/10.1016/0039-128x(95)00220-k
Zhu, J., Wang, H. T., Chen, Y. R., Yan, L. Y., Han, Y. Y., Liu, L. Y., Cao, Y., Liu, Z. Z., & Xu, H. A. (2020). The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish. Neurosci Bull, 36(9), 1023-1034. https://doi.org/10.1007/s12264-020-00554-y
Zwain, I. H., & Yen, S. S. (1999). Neurosteroidogenesis in astrocytes, oligodendrocytes, and neurons of cerebral cortex of rat brain. Endocrinology, 140(8), 3843-3852. https://doi.org/10.1210/endo.140.8.6907