Phosphorylation dynamics regulate Hsp27-mediated rescue of neuronal plasticity deficits in tau transgenic mice

Abstract

Molecular chaperones regulate the aggregation of a number of proteins that pathologically misfold and accumulate in neurodegenerative diseases. Identifying ways to manipulate these proteins in disease models is an area of intense investigation; however, the translation of these results to the mammalian brain has progressed more slowly. In this study, we investigated the ability of one of these chaperones, heat shock protein 27 (Hsp27), to modulate tau dynamics. Recombinant wild-type Hsp27 and a genetically altered version of Hsp27 that is perpetually pseudo-phosphorylated (3×S/D) were generated. Both Hsp27 variants interacted with tau, and atomic force microscopy and dynamic light scattering showed that both variants also prevented tau filament formation. However, extrinsic genetic delivery of these two Hsp27 variants to tau transgenic mice using adeno-associated viral particles showed that wild-type Hsp27 reduced neuronal tau levels, whereas 3×S/D Hsp27 was associated with increased tau levels. Moreover, rapid decay in hippocampal long-term potentiation (LTP) intrinsic to this tau transgenic model was rescued by wild-type Hsp27 overexpression but not by 3×S/D Hsp27. Because the 3×S/D Hsp27 mutant cannot cycle between phosphorylated and dephosphorylated states, we can conclude that Hsp27 must be functionally dynamic to facilitate tau clearance from the brain and rescue LTP; however, when this property is compromised, Hsp27 may actually facilitate accumulation of soluble tau intermediates.

Figure 1.

wt and 3×S/D Hsp27 bind tau despite differences in their oligomerization states. A, SC–WB for Hsp27. Recombinant wt and 3×S/D Hsp27 were independently subjected to SC ultracentrifugation. Hsp27 levels were analyzed in equal volumes of the recovered SC fractions. B, CoIP–WB of His-tagged recombinant tau (rTau) with rabbit anti-tau antibody showed that rTau coprecipitated with both wt and 3×S/D Hsp27 but not BSA. Unlabeled rabbit IgG was used as a negative co-IP control.

Figure 2.

Heparin-induced tau fibril formation is abrogated by wtHsp27 and 3×S/D Hsp27. A, Tau (circles) fibril growth rate in the absence or presence of either wtHsp27 (squares) or 3×S/D Hsp27 (triangles). B, C, Particle size monitoring of tau alone (circles) or in combination with wt (squares) or 3×S/D (triangles) Hsp27 at day 0 (B) and day 5 (C) by DLS. D, AFM images of tau alone or preincubated with either wtHsp27 or 3×S/D Hsp27. Buffer without protein was used as a negative control. Heparin was used to induce tau aggregation for 5 d (A–C), and 15 d (D). Preincubation consisted of 35 mol of recombinant tau per mole of recombinant Hsp27s.

Figure 3.

Hsp27 upregulation by intrahippocampal AAV9 injections has robust distribution throughout the hippocampus, but it does not transduce all neurons. A, Immunohistochemistry detection of Hsp27 in brain sections of mice injected with AAV1, AAV9, or non-injected mice. Brains were harvested 8 weeks after viral injections. Dark staining, which is not present in the non-injected brain section, corresponds to Hsp27. B, Immunofluorescence of the hippocampus neuronal layer using antibodies against Hsp27 and the neuronal marker NeuN shows that not all neurons (blue) expressed Hsp27 (green).

Figure 4.

Neuronal overexpression of Hsp27–AAV9 variants differentially modulates neuronal tau levels. A, Confocal images of immunofluorescently labeled neurons (blue), tau (red), and Hsp27 or GFP (green) in CA1 from rTg4510 mice injected with AAV9-expressing wtHsp27, 3×S/D Hsp27, or GFP (scale bar, 125 μm). B, Higher zoom images of fields from CA1 regions of immunofluorescently labeled sections from AAV9-injected mice (scale bar, 50 μm). C, Quantification of red signal (tau) in blue areas (neurons) sharing positive green signal (Hsp27 or GFP). Statistical significance was determined with Student's t tests. ***p = 0.001.

Figure 5.

wtHsp27 is unable to disrupt preformed tau aggregates. A, DLS scatter plot measuring the particle size of stably formed recombinant tau fibrils before and after addition of wtHsp27. Heparin was used to induce aggregation over the course of 22 d. wtHsp27 was then incubated, and particle size was measured over time. B, Tau can enter 3 protein folding pathways once it loses form and function: aggregation (Kagg), degradation (degrade), or refolding (Krefold). Aggregation and refolding of tau is based on equilibrium, as indicated by the bidirectional arrows. Hsp27 prevents tau aggregation and facilitates the entry of tau into degradation or refolding pathways. Because refolding, or recycling, of tau is in equilibrium with unfolded intermediates, it is possible that Hsp27 may increase the prevalence of these intermediates. Hsp27 is not able to disaggregate tau once fibrillization has already occurred.

Figure 6.

LTP deficits in rTg4510 mice are rescued by wtHsp27 but remain unaffected with 3×S/D Hsp27. After recording baselines for 20 min, LTP was induced with TBS (5 bursts of 200 Hz separated by 200 ms, repeated 6 times with 10 s between the 6 trains), and LTP was recorded for 60 min. Changes in fEPSP slope are expressed as a percentage of baseline. A, Representative fEPSP traces for rTg4510 and age-matched NTG mice injected with either saline or GFP–AAV9. Changes in the slopes of NTG–GFP (n = 6) and NTG–saline (n = 9) were significantly different (*p < 0.01) from that of TG–GFP (n = 5) and TG–saline (n = 9). LTP was not significantly different between mice of the same genotype despite treatments (p > 0.05). B, Representative fEPSP traces for rTg4510 injected with wtHsp27–AAV9 (n = 7), 3×S/D–AAV9 Hsp27 (n = 9), or GFP–AAV9 (n = 7) and age-matched NTG mice injected with GFP–AAV9 (n = 6). Changes in fEPSP slopes between NTG–GFP versus TG–wtHsp27 and TG–3×S/D versus TG–GFP were not significantly different (p > 0.05). NTG–GFP and TG–wtHsp27 fEPSP slopes were significantly different from both TG–3×S/D and TG–GFP (p < 0.001). p values were determined by Student's t tests using data from the first and last 10 min of the LTP curves.

Figure 7.

Proposed mechanism of wtHsp27 and 3×S/D Hsp27 modulation of tau aggregation. During stress-induced client denaturation or loss of function, both wtHsp27 and phospho-Hsp27 (S/DHsp27) bind and prevent client aggregation (in vitro). Client-bound wtHsp27 is then able to facilitate client clearance. Conversely, retention of Hsp27 in a mock-phosphorylated state subverts client clearance, leading to accumulation of soluble intermediates (in vivo).