College of Science
98 Research Summary: The Role of RAB-37b in Synaptic Autophagy
Lindsey Tamlin; Erik M Jorgensen; and Kevin Kruse
Faculty Mentor: Erik M. Jorgensen (Biological Sciences, University of Utah)
Due to their post-mitotic nature, neurons cannot dilute damaged proteins and organelles through cell division and rely on autophagy to “take out” the molecular trash. Faulty autophagy pathways can lead to devastating neurodegenerative diseases like Alzheimer’s, Parkinson’s, and Amyotrophic Lateral Sclerosis (Ichimiya et al. 2020; Stavoe & Holzbaur 2019). Understanding cellular processes related to autophagy in neurons is crucial to develop and improve technologies used to diagnose and treat neurodegenerative diseases. One such family of proteins involved in autophagy are the Rab GTPases, which act as molecular switches cycling between an “inactive” and “active” state. Rab proteins regulate sorting, fission, transport, tethering, docking and fusion of transport vesicles, including synaptic vesicles (Ao et al. 2014, Mignogna & D’Adamo 2016). Many Rab proteins are ubiquitously expressed, but a few are nearly or completely neuron-specific (Chan et al. 2011).
Mutations in neuronal Rab proteins can cause hereditary neurological diseases such as Griscelli syndrome (Rab27) (Menache et al. 2000) and Charcot-Marie-Tooth type 2B disease (Rab7) (Verhoeven et al. 2003). In D. melanogaster, Rab26 is expressed exclusively in neurons (Chan et al. 2011) and links synaptic vesicles to the autophagy pathway (Binotti et al. 2015). In rat hippocampal cultures, Rab26 is expressed near the autophagosome protein ATG16L1 as well as the synaptic vesicle markers synaptophysin and synaptotagmin-I (Binotti et al. 2015). The closest ortholog to the D. melanogaster Rab26 in C. elegans is RAB-37b. Like Rab26, RAB-37b is highly specific to neurons, but the specific function of this protein remains uncharacterized. The goal of my project is to use cell biological and microscopy techniques to investigate the role of RAB-37b in neuronal function and autophagy using the model organism C. elegans.
To investigate the localization of RAB-37b in C. elegans, I used CRISPR to tag endogenous RAB-37b with a fluorescent protein. Using confocal microscopy, I determined that RAB-37b is primarily localized to synapses. In an unc-104/KIF1A mutant (the primary kinesin involved in anterograde axonal transport of synaptic vesicles) I confirmed that RAB-37b is on synaptic vesicles, as seen in other organisms. Surprisingly, RAB-37b did not colocalize with the synaptic protein SNB- 1/synaptobrevin, which indicates RAB-37b may be primarily on synaptic vesicles that are far away from the active zone and/or may not be involved in exocytosis. RAB-37b also did not co-localize with RAB-7 (part of the endosome-lysosome pathway) indicating that RAB-37b is not part of this pathway. More experiments will be needed to determine the sub-cellular localization of RAB-37b in C. elegans. To investigate the function of RAB-37b, I used a pharmacologic assay (aldicarb induced paralysis) to determine the amount of synaptic transmission (Oh & Kim 2017). Over-expression of RAB-37b in neurons increased the rate of paralysis when worms were exposed to aldicarb, suggesting an increase in neurotransmission. Mutants lacking a functional RAB-37b protein (rab-37b knockout), however, did not show a decrease in neurotransmission. Additionally, I used CRISPR to make single point mutations to make the protein either constitutively active (always “on”) or dominant negative (always “off”). Constitutively active RAB-37b showed a similar increase in neurotransmission to over- expressed RAB-37b. Dominant negative RAB-37b, like the rab-37b knockout, did not show a decrease in neurotransmission.
My results suggest that the role of RAB-37b is to increase neurotransmission. Further experiments will be needed to determine if the change in neurotransmission is related to a change in autophagy, which could impact neurotransmission by either regulating the number of synaptic vesicles available for release or by regulating the number of proteins available at the active zone involved in neurotransmitter release (such as calcium channels and tethering proteins). Additionally, further imaging experiments will help me identify other interacting partners of RAB-37b. These studies will provide valuable insight into the mechanisms by which neurons regulate autophagy and synaptic transmission.
Bibliography
Binotti, Beyenech, Pavlos, Nathan, Riedel, Dietmar, Wenzel, Dirk, Vorbrüggen, Gerd, Schalk, Amanda, Kühnel, Karin, Boyken, Janina, Erck, Christian, Martens, Henrik, Chua, John, & Jahn, Reinhard. (2015). “The GTPase Rab26 links synaptic vesicles to the autophagy pathway.” eLife, 4(2). https://doi.org/10.7554/elife.05597.
Chan, Chih-Chiang, Scoggin, Shane, Wang, Dong, Cherry, Smita, Dembo, Todd, Greenberg, Ben, Eugene, Jennifer Jin, Kuey, Cansu, Lopez, Antonio, Mehta, Sunil Q., Perkins, Theodore J., Brankatschk, Marko, Rothenfluh, Adrian, Buszczak, Michael, & Hiesinger, P Robin. (2011). “Systematic discovery of Rab GTPases with synaptic functions in drosophila.” Current Biology, 21(20), pp. 1704–1715, https://doi.org/10.1016/j.cub.2011.08.058.
Chanaday, Natali, Cousin, Michael, Milosevic, Ira, Watanabe, Shigeki, & Morgan, Jennifer. (2019). “The synaptic vesicle cycle revisited: New insights into the modes and mechanisms.” The Journal of Neuroscience, 39(42), pp. 8209–8216, https://doi.org/10.1523/jneurosci.1158-19.2019.
Ichimiya, Tadashi, Yamakawa, Tsukasa, Hirano, Takehiro, Yokoyama, Yoshihiro, Hayashi, Yuki, Hirayama, Daisuke, Wagatsuma, Kohei, Itoi, Takao, & Nakase, Hiroshi. (2020). “Autophagy and autophagy-related diseases: A Review.” International Journal of Molecular Sciences, 21(23), p. 8974, https://doi.org/10.3390/ijms21238974.
Jin, Ramon & Mills, Jason. (2014). “Rab26 coordinates lysosome traffic and mitochondrial localization.” Journal of Cell Science, https://doi.org/10.1242/jcs.138776.
Kohrs, Friederike, Daumann, Ilsa-Maria, Pavlovic, Bojana, Jin, Eugene Jennifer, Kiral, F. Ridvan, Lin, Shih-Ching, Port, Filip, Wolfenberg, Heike, Mathejczyk, Thomas F., Linneweber, Gerit A., Chan, Chih-Chiang, Boutros, Michael & Hiesinger, P. Robin . (2021). “Systematic functional analysis of rab GTPases reveals limits of neuronal robustness to environmental challenges in flies.” eLife, 10, https://doi.org/10.7554/elife.59594.
Lüningschrör, Patrick, Binotti, Beyenech, Dombert, Benjamin, Heimann, Peter, Perez-Lara, Angel, Slotta, Carsten, Thau-Habermann, Nadine, von Collenberg, Cora R., Karl, Franziska, Damme, Markus, Horowitz, Arie, Maystadt, Isabelle, Füchtbauer, Annette, Füchtbauer,Ernst-Martin, Jablonka, Sibylle, Blum, Robert, Üçeyler, Nurcan, Petri, Susanne, Kaltschmidt, Barbara, Jahn, Reinhard, Kaltschmidt, Christian, & Sendtner, Michael. (2017). “PLEKHG5-regulated autophagy of synaptic vesicles reveals a pathogenic mechanism in motoneuron disease.” Nature Communications, 8(1), https://doi.org/10.1038/s41467-017-00689-z.
Ménasché, Gaël, Pastural, Elodie, Feldmann, Jérôme, Certain, Stéphanie, Ersoy, Fügen, Dupuis, Sophie, Wulffraat, Nico, Bianchi, Diana, Fischer, Alain, Le Deist, Françoise, & de Saint Basile, Geneviève. (2000). “Mutations in rab27a cause GRISCELLI syndrome associated with haemophagocytic syndrome.” Nature Genetics, 25(2),pp.173–176, https://doi.org/10.1038/76024.
Menzies, Fiona, Fleming, Angeleen, & Rubinsztein, David. (2015). “Compromised autophagy and neurodegenerative diseases.” Nature Reviews Neuroscience, 16(6), , pp. 345–357, https://doi.org/10.1038/nrn3961.
Lidia Mignogna, Maria & D’Adamo, Patrizia. (2017). “Critical importance of rab proteins for synaptic function.” Small GTPases, 9(1–2), pp. 145–157, https://doi.org/10.1080/21541248.2016.1277001.
Molendijk, Arthur, Ruperti, Benedetto, & Palme, Klaus. (2004) “Small GTPases in vesicle trafficking.” Current Opinion in Plant Biology, 7(6), pp. 694–700, https://doi.org/10.1016/j.pbi.2004.09.014.
Oh, Kelly, & Hongkyun Kim. (2017). “Aldicarb-induced paralysis assay to determine defects in synaptic transmission in Caenorhabditis elegans.” BIO-PROTOCOL, 7(14), https://doi.org/10.21769/bioprotoc.2400.
Stavoe, Andrea K.H., & Holzbaur, Erika L.F. (2019). “Autophagy in neurons.” Annual Review of Cell and Developmental Biology, 35(1), pp. 477–500, https://doi.org/10.1146/annurev-cellbio-100818-125242.
Verhoeven, Kristien, De Jonghe, Peter, Coen, Katrien, Verpoorten, Nathalie, Auer-Grumbach, Michaela, Kwon, Jennifer M., FitzPatrick, David, Schmedding, Eric, De Vriendt, Els, Jacobs, An, Van Gerwen, Veerle, Wagner, Klaus, Hartung, Hans-Peter, & Timmerman, Vincent. (2003). “Mutations in the small GTP-ase late endosomal protein Rab7 cause Charcot-Marie-Tooth type 2B neuropathy.” The American Journal of Human Genetics, 72(3), pp. 722–727, https://doi.org/10.1086/367847.
