Learning-induced and stathmin-dependent changes in microtubule stability are critical for memory and disrupted in ageing

Abstract

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.

Figure 1. Learning induces biphasic changes in stathmin activity and microtubule stability

(a) Immunoblot estimation of stathmin phosphorylation at Ser16 (pS16), Ser25 (pS25), and Ser38 (pS38) 0.5, 1, 2, 8, or 24 h following contextual fear conditioning. N, naïve. n = 6 per group (pooled tissues from 3–4 mice per sample). *p < 0.05 versus naïve mice (post hoc comparison). (b) Analysis of stathmin-tubulin protein interactions using co-immunoprecipitation. Stathmin-tubulin complexes are formed at 0.5 h and dissociate at 8 h after contextual fear conditioning. n = 4 per group (pooled tissues from 4–5 mice per sample). *p < 0.05 (post hoc comparison). (c) Immunoblot estimation of detyrosinated (Detyr-) and tyrosinated (Tyr-) α-tubulin levels after contextual fear conditioning. n = 6 per group (pooled tissues from 3–4 mice per sample). *p < 0.05 versus naïve mice (post hoc comparison). (d) Experimental design for drug administration 8 h following contextual fear conditioning. Memory was tested 24 h after training. (e) Mice injected with nocodazole 8 h following training show reduced freezing. vehicle, n = 11; nocodazole, n = 12. *p < 0.05 (Student’s t test). (f) Mice injected with paclitaxel 8 h following training show increased freezing. vehicle, n = 11; nocodazole, n = 12. *p < 0.05 (Student’s t test). Data are expressed as mean ± s.e.m.

Figure 2. Stathmin phosphorylation is essential for learning-dependent changes in microtubule stability

(a) Diagram illustrating transgenic design and control of transgene expression by doxycycline (dox) in Stat4A bi-transgenic mice. (b) Immunohistochemistry shows that Stat4A:GFP is expressed in the DG, but not CA1 area of the hippocampus. Scale bar, 200 μm. (c) Immunoblot using anti-GFP antibody demonstrates that transgene expression is repressed by dox. (d) Western blots from the synaptosomal fraction of the DG of Stat4A mice show a deficit in microtubule hyperstability at 8 h after contextual fear conditioning. Tyr, tyrosinated tubulin; Detyr, detyrosinated tubulin. N, naïve. n = 6 per group (pooled tissues from 3–4 mice per sample). *p < 0.05 versus naïve in corresponding genotype (post hoc comparison). Data are expressed as mean ± s.e.m.

Figure 3. Stathmin phosphorylation is essential for synaptic plasticity

(a) Input-output relationship of perforant path to dentate gyrus synaptic responses from wildtype (WT) and mutant mice (n = 4, 6 slices). (b) Deficient perforant path to dentate gyrus LTP in Stat4A mice (n = 4, 6 slices). (c) Input-output relationship of Schaffer Collateral to CA1 (SC-CA1) synaptic responses from WT and Stat4A mice. No effect is found on the relationship between stimulus strength and the size of postsynaptic response (input-output relationship). (d) Stat4A mice show normal LTP at SC-CA1 synapses. Averaged field EPSP data (n = 4, 5 slices). *p < 0.05 versus wildtype mice. Data are expressed as mean ± s.e.m.

Figure 4. Stathmin phosphorylation is essential for hippocampus-dependent memory

(a) Deficient long-term (24 h) contextual fear memory in Stat4A mice is rescued by dox. WT, n = 14; Stat4A, n = 15, Stat4A on dox, n = 14. *p < 0.05 versus wildtype mice (post hoc comparison). (b) Normal short-term (0.5 h) memory in Stat4A mice. n = 10 per group. (c) Cued fear memory is normal in Stat4A mice. n = 14 per group. (d–g) Morris water maze. There was no difference in the visible platform task between the genotypes (d). In hidden platform, the latencies of Stat4A mice were slower than those of wild-type (WT) mice (e). In the reversal learning (transfer task), Stat4A mice showed longer latency to find the platform (f). The probe trial (g) shows normal spatial memory recall in Stat4A mice. n = 10 for each group. *p < 0.05. (h) Reduced spatial learning in Stat4A mice assessed in Barnes maze. n = 11 for each group. * p < 0.05. Data are expressed as mean ± s.e.m.

Figure 5. Stathmin control of microtubule stability in the dentate gyrus is critical for contextual fear conditioning

(a) AAV-mediated overexpression of Stat4A in the dentate gyrus (DG) detected by immunohistochemistry with anti-GFP antibody. Scale bar, 200 μm. (b,c) Immunoblot estimation of detyrosinated α-tubulin levels 0.5 h after training in the DG synaptosomal fraction of mice injected with AAV-GFP or AAV-Stat4A-IRES-GFP. N, naïve. n = 5–7 per group (pooled tissues from 3–4 mice per sample). *p < 0.05 versus naïve mice (Student’s t test). (d) AAV-mediated overexpression of Stat4A reduces contextual fear memory. n = 11–12 per group. *p < 0.01 (Student’s t test). Data are expressed as mean ± s.e.m.

Figure 6. Role of learning-induced microtubule instability in memory

(a) Mice injected with paclitaxel immediately following training show reduced freezing. Vehicle, n = 11; Paclitaxel, n = 12. *p < 0.05 (Student’s t test). (b) stathmin^−/− mice show deficit in microtubule destability and stability at 0.5 and 8 h after training. n = 6 per group (pooled tissues from 3–4 mice per sample). N, naïve. *p < 0.05 versus naïve in corresponding genotype (post hoc comparison). (c) stathmin^−/− mice show reduced long-term (24 h) contextual fear memory. n = 12 per group. *p < 0.01 (Student’s t test).

Figure 7. Stathmin regulates learning-dependent dendritic transport of GluA2

(a–c) Immunoblot estimation of the level of GluA2 in synaptosomal (a, n = 8 per group, pooled tissues from 3–4 mice per sample whole cell extract (b, n = 4 per group) and microtubule (c, n = 4 per group, pooled tissues from 3–4 mice per sample) fractions of the DG in mice injected with AAV-GFP or AAV-Stat4A-IRES-GFP in naïve and 8 h after training conditions. *p < 0.05 (post hoc comparison). (d–f) Reduced GluA2 synaptic transport in stathmin^−/− mice, as analyzed by immunoblotting, in the synaptosomal (d, n = 4 per group, pooled tissues from 3–4 mice per sample), whole cell extract (e, n = 4 per group) and microtubule (f, n = 4 per group, pooled tissues from 3–4 mice per sample) fractions in naïve and trained mice. *p < 0.05 (post hoc comparison). Data are expressed as mean ± s.e.m.

Figure 8. Stathmin controls learning-induced dendritic transport of GluA2 by modulating binding of motor protein KIF5 to microtubules

Co-immunoprecipitation reveals deficiency in protein binding between GluA2 and α-tubulin (a), KIF5 and α-tubulin (b), and GluA2 and KIF5 (c) in stathmin^−/− mice. N, naïve. n = 4 per group (pooled tissues from 2–3 mice per sample). *p < 0.05 (post hoc comparison). (d) Scheme of the experimental design and TAT-GluA2_3Y peptide (TAT-Pep.) injection schedule is shown. (e) Blocking GluA2 endocytosis by intra-hippocampal injection of TAT-GluA2_3Y peptide has no effect in wild-type mice, but rescues contextual fear memory in stathmin^−/− mice. n = 12–13. *p < 0.05 versus mice given TAT-control peptide (post hoc comparison). (f) Rescue of contextual fear memory by TAT-GluA2_3Y peptide in wild-type mice injected with AAV-Stat4A-IRES-GFP. n = 10–12. *p < 0.05 (post hoc comparison). Data are expressed as mean ± s.e.m.

Figure 9. Decreased contextual fear memory in aged wild-type mice

Aged (16–20 month-old) and young adult (2–4 month-old) wild-type mice were subjected to one-footshock or three-footshock contextual fear conditioning. Memory was tested 24 h following training. Aged mice showed reduced contextual fear memory. n = 11–13 per group. p < 0.05 (post hoc comparison). Data are expressed as mean ± s.e.m.

Figure 10. Age-dependent memory loss is associated with microtubule-dependent dendritic transport of GluA2

(a) Immunoblot shows reduced level of total stathmin protein in the dentate gyrus (DG) of aged wild-type mice. n = 5 per group. *p < 0.05, **p < 0.01 versus young adult mice (post hoc comparison). (b) Immunoblot shows deficiency in learning-dependent microtubule destability and hyperstability in aged mice. Detyr, detyrosinated. N, naïve. n = 5 per group (pooled tissues from 3–4 mice per sample). *p < 0.05 versus naïve young adult mice (post hoc comparison). (c–e) Immunoblot estimation of GluA2 levels in aged mice in synaptosomal (c, n = 5 per group, pooled tissues from 3–4 mice per sample), whole cell extract (d, n = 5 per group), and microtubule (e, n = 5 per group, pooled tissues from 3–4 mice per sample) fractions of the DG of naïve and trained (single or three foot-shock pairings) aged and young adult mice. N, naïve; #FS, number of foot-shocks; 1, one foot-shock; 3, three foot-shocks. *p < 0.05, **p < 0.01 versus naive mice in corresponding group (post hoc comparison). (f) Intra-hippocampal injection of the TAT-GluA2_3Y peptide rescued decreased contextual fear memory in aged mice. n = 12–13. *p < 0.05 (post hoc comparison). Data are expressed as mean ± s.e.m.