For this, we treated our primary cortical neurons with jasplakinolide (1 M), a compound that promotes actin polymerization (Lazaro- Dieguez et al., 2008), or with a latrunculin A (at 500 nM), a compound that depolymerizes F-actin into soluble globular actin (Gactin; Coue´ et al., 1987; Fig. 5C). After jasplakinolide application, we observed a large increase in synaptic EGFP-tau fluorescence
Latrunculin treatment produced rapid actin depolymerization and the corresponding disappearance of LifeAct-RFP fluorescence in every spine studied; no synaptic EGFP-tau fluorescence was observed (data not shown).
Abetao exposure induced a translocation of tau into the PSD fraction (***p 0.0002, 2-tailed Student’s t test; control 20.12 1228 vs Abetao 29.74 1.748, N 12 independent culture). There was also an increase of PSD-95 (***p0.0006, 2-tailed Student’s t test; control 19.10 2.557 vs Abetao 33.3 2153, N 9 independent culture), GluA1 (**p 0.0078, 2-tailed Student’s t test; control 18.841.930 vs Abetao 26.221.475,N9 independent culture) and fyn (**p 0.0041, 2-tailed Student’s t test; control 19.42 1.337 vs Abetao 29.67 2.181, N 6 independent cultures; Fig. 6D).
After Abetao treatment, synaptic activation did not trigger any increase in synaptic markers and in fact decreased synaptic actin (***p 0.0009, 2-tailed Student’s t test; Abetao 29.64 1.495, Abetao Bic/4-AP 18.56 2.030, N 7 independent cultures; Fig. 7C), PSD-95 (***p0.0007, 2-tailed Student’s t test; Abetao 33.37 2.153, Abetao Bic/4-AP 19.25 2.550, N 7 independent cultures) and tau levels (**p0.0014, 2-tailed Student’s t test; Abetao 29.74 1.748, Abetao Bic/4-AP 20.68 1.751, N 12 independent cultures).
When we investigated Abetao-driven tau translocation to the synapse, we did not see any change in half-life recovery (4.729 s) from those measured with synaptic activation. However, the plateau value was drastically modified (71.20%), illustrating that, whereas Abetao induced tau translocation and subsequently its interaction with actin filament, the resulting synaptic tau is less stable.
Preceded by 15 min Abetao treatment, synaptic activation disrupted LifeAct-RFP fluorescence, suggesting an alteration of F-actin organization and no additional EGFPtau recruitment at the synapse.
Finally, we performed a phalloidin precipitation assay after a 15 min Abetao treatment on our neuron culture (Fig. 8A) and observed that tau/F-actin content was increased (**,*p 0.05 relative to control, #p 0.05 relative to Abetao, 1-way ANOVA; control 15.45 1.529, Abetao 32.90 3.181, Abetao Bic/ 4-AP 20.182671 for actin; control 16.342.618, Abetao 31.77 1.952, Abetao Bic/4-AP 17.704.080 for tau,N5 independent cultures Fig. 8B). A subsequent synaptic activation did alter tau interaction with F-actin.
Thr-205 phosphorylated tau was only increased under synaptic activation in the PSD fraction (control 24.57 0.9754 vs Bic/4-AP 38.90 1.936; Fig. 9E), whereas it was decreased after Abetao treatment (control 24.57 0.9754 vs Abeta 13.64 2.416). Synaptic activation after Abetao exposure did not produce any significant Thr-205 phosphorylation of tau (Abeta Bic/4-AP 22.892.796 vs Bic/ 4-AP 38.90 1.93 vs Abeta 13.642.50).
Conversely, only Abetao exposure promoted significant tau phosphorylation on Ser 404 (**p0.05, 1-way ANOVA; control 15.672.418 vs Abetao 32.65 3.76 vs Bic/4-AP 26.75 1.17 vs Abetao Bic/4-AP 24.97 4.48, N 4). These results revealed that, although synaptic activation or Abetao promote tau translocation to PSD fractions, the synaptic tau displays a different phosphorylation profile that may be responsible for the conditional tau properties observed.
We observed that synaptic activation promoted EGFP-Tau T205A translocation to the spine but FRAP experiments revealed a shorter tau turnover time in the spine (Fig. 9B), whereas Abetao driven translocation to the spine was no longer observable in EGFP-Tau S404A-transfected neurons (Fig. 9F,G). These experiments highlight the pivotal role of these phosphorylations in tau translocation features to the spine.
The recovery curves indicated 3 pools of tau: a mobile, unbleached fraction comprising 9.72 1.03%, a dynamic fraction at 47.08 3.06%, and a stable, unrecoverable fraction at 42.75 2.78%. This stable fraction suggests that a large portion of tau is anchored in the spine.
We analyzed actin and tau in the PSD-enriched fraction from primary cortical neurons treated with jasplakinolide (Fig. 5E). We observed that increased neuronal F-actin content promotes concurrent tau enrichment (*p0.0150, 2-tailed Student’s t test; control 17.49 0.7755 vs jasplakinolide 27.02 2719, N 4 independent cultures; Fig. 5F). GLUA1, the membrane trafficking of which is known to be actin dependent, was increased (*p 0.0279, 2-tailed Student’s t test; control 16.91 1015 vs jasplakinolide 31.00 4.778, N 4 independent cultures). The amount of Fyn in the PSD was decreased (*p 0.0265, 2-tailed Student’s t test; control 27.25 5.003 vs jasplakinolide 11.71 1.786, N 4 independent cultures).
Therefore, during a long-lasting synaptic activation, we observed an increase in tau, fyn, actin, GluA1, and PSD-95 content in the PSD-positive fraction, which is consistent with the characteristic features of synaptic plasticity (Ehlers, 2003).
These results suggest that tau translocates from the dendritic shaft to the synapse during activation and probably takes part in the activity-driven synaptic reorganization that underlies synaptic plasticity
We observed a similar LTPinduced increase in tau content within the PSD-enriched fraction from CA1 synaptosomes (29.86 +-4.86 to 70.15 +- 4.86, **p = 0.0011; Fig. 3B). As expected, actin and GluA1 were also increased, strengthening the idea that tau is involved in synaptic reorganization processes necessary for synaptic plasticity
These results show that the amount of tau collected is proportional to neuronal F-actin content, suggesting a close link between F-actin and tau.
Together, these results suggest that tau translocation to the synapse depends on the F-actin stabilization that promotes their interaction.
Although tau alone were observed only in the supernatant in both experimental conditions, ruling out a nonspecific coaggregation, we found that tau coprecipitated with the pellet obtained from high- and low-speed centrifugation, illustrating its direct interaction with both F-actin (Fig. 4A) and actin bundles (Fig. 4B).
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If you find BEL Commons useful in your work, please consider citing: Hoyt, C. T., Domingo-Fernández, D., & Hofmann-Apitius, M. (2018). BEL Commons: an environment for exploration and analysis of networks encoded in Biological Expression Language. Database, 2018(3), 1–11.