ARTICLE Received 11 Dec 2013 | Accepted 13 Jun 2014 | Published 10 Jul 2014 DOI: 10.1038/ncomms5389 Learning-induced and stathmin-dependent changes in microtubule stability are critical for memory and disrupted in ageing Shusaku Uchida1,2,*, Guillaume Martel1,*, Alice Pavlowsky3, Shuichi Takizawa1, Charles Hevi1, Yoshifumi Watanabe2, Eric R. Kandel4, Juan Marcos Alarcon3 & Gleb P. Shumyatsky1 Changes in the stability of microtubules regulate many biological processes, but their role in memory remains unclear. Here we show that learning causes biphasic changes in the microtubule-associated network in the hippocampus. In the early phase, stathmin is dephosphorylated, enhancing its microtubule-destabilizing activity by promoting stathmin- tubulin binding, whereas in the late phase these processes are reversed leading to an increase in microtubule/KIF5-mediated localization of the GluA2 subunit of AMPA receptors at synaptic sites. A microtubule stabilizer paclitaxel decreases or increases memory when applied at the early or late phases, respectively. Stathmin mutations disrupt changes in microtubule stability, GluA2 localization, synaptic plasticity and memory. Aged wild-type mice show impairments in stathmin levels, changes in microtubule stability and GluA2 localization. Blocking GluA2 endocytosis rescues memory deficits in stathmin mutant and aged wild-type mice. These findings demonstrate a role for microtubules in memory in young adult and aged individuals. 1 Department of Genetics, Rutgers University, Room 322 Life Sciences Building,145 Bevier Road, Piscataway, New Jersey 08854, USA. 2 Division of Neuropsychiatry, Department of Neuroscience, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan. 3 Department of Pathology, State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203, USA. 4 Department of Neuroscience, Columbia University, Howard Hughes Medical Institute, Kavli Institute for Brain Science, 1051 Riverside Drive, New York, New York 10032, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to G.P.S. (email: [email protected]). NATURE COMMUNICATIONS | 5:4389 | DOI: 10.1038/ncomms5389 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5389 n the brain, following neuronal activity there is an increase in (Fig. 1a and Supplementary Fig. 2). To confirm that the observed synaptic transport, which results in the strengthening of changes were specific to associative learning, we examined the Isynaptic connections, and ultimately in memory formation1. effects of exposure to the context only (no shock) or the Among the many mechanisms mediating synaptic transport, immediate shock deficit on stathmin phosphorylation cytoskeletal structures—actin filaments and microtubules—play a (Supplementary Fig. 3). In mice exposed to context only or to major role in motor-driven import and export2. Actin filaments the immediate shock, phosphorylation of stathmin at Ser25 and are known to be dynamic and present in dendritic spines, where Ser38 was rapidly decreased 30–60 min following the exposure, they play an essential role in synaptic function and memory but no changes in phospho-stathmin levels were observed at 8 h. formation3. Microtubule dynamics have been described in cell In contrast, there was no effect of either the context only or the division, axonal pathfinding during development and axonal immediate shock on stathmin phosphorylation at Ser16. growth and regeneration. In contrast, in mature neurons, We examined whether the changes in stathmin phosphoryla- microtubules are generally viewed as stable non-dynamic tion following learning regulate stathmin–tubulin binding, which structures present in dendritic shafts but not in dendritic in turn may affect microtubule stability. Using co-immunopre- spines. Intriguingly, recent work in hippocampal primary cipitation on the synaptosomal fractions of the dentate gyrus, we neuronal cultures indicates that microtubules can also be found that stathmin binding to a-tubulin, a major component of dynamically regulated during neuronal activity by moving from microtubules, increased in the first 30 min but decreased the the dendritic shaft to the dendritic spines and affecting synaptic following 8 h after training (Fig. 1b). In mice exposed to context structure and function4–9. Despite this new information, the role only, stathmin binding to a-tubulin was increased 30 min, but not of changes in microtubule stability in memory formation remains 8 h, following the exposure (Supplementary Fig. 4). These data unclear. suggest that during associative learning, stathmin may regulate Microtubule assembly from heterodimers of a-andb-tubulins microtubule stability by changing it’s affinity to a-tubulin. involves interaction with microtubule-associated proteins. One of these proteins, stathmin, binds tubulin heterodimers and prevents Changes in microtubule stability following learning.To microtubule assembly, thus controlling microtubule dynamics10,11. examine microtubule stability following learning, we measured When phosphorylated, stathmin releases tubulin dimers allowing the levels of the detyrosinated tubulin, a marker of stable microtubules to be formed12,13. Stathmin has been linked to fear, microtubules, and tyrosinated tubulin, a marker of labile micro- cognition and aging in rodent and human studies14–19,suggesting tubules22–24. We found biphasic changes in tyrosinated and the involvement of microtubule dynamics in memory. detyrosinated tubulin in the synaptosomal fractions of the dentate Therefore, we set out to examine the role of stathmin-regulated gyrus: detyrosinated tubulin levels were decreased 30–60 min and control of microtubule stability in memory formation by focusing increased 8 h following contextual fear conditioning, whereas on learning-induced changes, because activity-dependent signalling tyrosinated tubulin levels were changed in the opposite direction is a hallmark of neuronal function20,21. Our approach reveals that (Fig. 1c and Supplementary Fig. 5). Similar to the changes by regulating microtubule stability and dendritic transport of the in stathmin phosphorylation, exposure to context only GluA2 subunit of AMPA-type glutamate receptors (AMPARs), (Supplementary Fig. 6a) or immediate shock (Supplementary stathmin controls synaptic plasticity and memory consolidation. Fig. 6b) led to a reduction of the detyrosinated tubulin and an Furthermore, we observe that aged wild-type (WT) mice, in increase in tyrosinated tubulin 0.5 h, but not 8 h, following the addition to memory deficits, display deficits in stathmin levels, exposure. These results suggest that the increase in microtubule learning-dependent changes in microtubule stability and GluA2 stability at 8 h is specific to associative learning. These results dendritic transport along microtubules. Our combined approach further suggest that microtubules become unstable 0.5–1 h and demonstrates that learning-induced and stathmin-mediated hyperstable 8 h following learning, and the increase in phospho- changes in microtubule stability control GluA2 dendritic stathmin and subsequent microtubule hyperstability may be transport, synaptic plasticity and memory formation, pointing to involved in memory consolidation. a new learning-dependent intracellular signalling pathway. Memory can be enhanced by increasing microtubule stability. Results We investigated whether pharmacological manipulation of the Stathmin activity is regulated by learning. We first examined increase in microtubule stability following learning affects expression of stathmin protein in adult mice and found it memory. We used paclitaxel or nocodazole to stabilize or desta- to be present in the brain and testis (Supplementary Fig. 1a). bilize microtubules, respectively. The drugs were injected into the In the brain, stathmin is located in the hippocampus, prefrontal dentate gyrus area of WT mice 8 h following fear conditioning, cortex, amygdala, striatum, hypothalamus and cerebellum and memory was tested 24 h after training (Fig. 1d). Nocodazole (Supplementary Fig. 1b). Stathmin is strongly expressed in the injections decreased (Fig. 1e), while paclitaxel injections increased dentate gyrus with lesser expression in the CA3 and CA1 memory (Fig. 1f). To rule out the possibility that nocodazole (Supplementary Fig. 1c). In the dentate gyrus, stathmin was injections disrupted freezing in a non-associative manner, we found not only in the whole-cell extracts but also in the synap- injected nocodazole into the dentate gyrus area of WT mice in tosomal fraction (Supplementary Fig. 1d). Based on this obser- home cage 24 h after they were subjected to contextual fear vation, we focused our work on the dentate gyrus synaptosomes. conditioning (Supplementary Fig. 7). Their freezing responses To examine whether stathmin phosphorylation is regulated by were measured 48 h after training. There were no differences in learning, synaptosomal fractions from the dentate gyrus were freezing between mice injected with nocodazole and vehicle, thus isolated at several time points (5, 15 and 30 min, and 1, 2, 8 or suggesting that the nocodazole treatment alone did not disrupt 24 h) from mice subjected to single-shock contextual fear freezing. These pharmacological experiments demonstrate that conditioning. Compared with naive mice, phosphorylation