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. 2015 Nov;130(5):633-42.
doi: 10.1007/s00401-015-1487-z. Epub 2015 Oct 8.

PERK inhibition prevents tau-mediated neurodegeneration in a mouse model of frontotemporal dementia

Helois Radford  1 , Julie A Moreno  1   2 , Nicholas Verity  1 , Mark Halliday  1 , Giovanna R Mallucci  3   4
Affiliations

PERK inhibition prevents tau-mediated neurodegeneration in a mouse model of frontotemporal dementia

Helois Radford et al. Acta Neuropathol. 2015 Nov.

Abstract

The PERK-eIF2α branch of the Unfolded Protein Response (UPR) mediates the transient shutdown of translation in response to rising levels of misfolded proteins in the endoplasmic reticulum. PERK and eIF2α activation are increasingly recognised in postmortem analyses of patients with neurodegenerative disorders, including Alzheimer's disease, the tauopathies and prion disorders. These are all characterised by the accumulation of misfolded disease-specific proteins in the brain in association with specific patterns of neuronal loss, but the role of UPR activation in their pathogenesis is unclear. In prion-diseased mice, overactivation of PERK-P/eIF2α-P signalling results in the sustained reduction in global protein synthesis, leading to synaptic failure, neuronal loss and clinical disease. Critically, restoring vital neuronal protein synthesis rates by inhibiting the PERK-eIF2α pathway, both genetically and pharmacologically, prevents prion neurodegeneration downstream of misfolded prion protein accumulation. Here we show that PERK-eIF2α-mediated translational failure is a key process leading to neuronal loss in a mouse model of frontotemporal dementia, where the misfolded protein is a form of mutant tau. rTg4510 mice, which overexpress the P301L tau mutation, show dysregulated PERK signalling and sustained repression of protein synthesis by 6 months of age, associated with onset of neurodegeneration. Treatment with the PERK inhibitor, GSK2606414, from this time point in mutant tau-expressing mice restores protein synthesis rates, protecting against further neuronal loss, reducing brain atrophy and abrogating the appearance of clinical signs. Further, we show that PERK-eIF2α activation also contributes to the pathological phosphorylation of tau in rTg4510 mice, and that levels of phospho-tau are lowered by PERK inhibitor treatment, providing a second mechanism of protection. The data support UPR-mediated translational failure as a generic pathogenic mechanism in protein-misfolding disorders, including tauopathies, that can be successfully targeted for prevention of neurodegeneration.

Keywords: Dementia; Neurodegeneration; PERK; Tau; Unfolded Protein Response.

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Figures

Fig. 1
Fig. 1
Mutant tau-expressing rTg4510 mice show overactivation of the PERK/eIF2α-P branch of the UPR resulting in a decline in protein synthesis rates by 6 months of age. a Scheme depicting disease progression in rTg4510 tauP301L+ mice from 3 to 8 months (mo). Red circles indicate times of testing. b tauP301L+ mice (black bars) show sustained elevated levels of eIF2α-P and ATF4 by 6 months of age, compared to levels seen in tauP301L− (white bars). These remained elevated at 8 months, eIF2α-P and ATF4 were equivalent in both mutant tau-expressing and transgene-negative mice examined at 4 months. Levels of GADD34 do not change throughout disease progression and remain equivalent to tauP301L− mice. Representative immunoblots of hippocampal lysates and bar charts quantitating relative levels of protein (n = 3) are shown. c RT-PCR of XBP1 transcript shows that there is no splicing in mutant tau-expressing and transgene-negative mice. Control lanes from untreated MEF cells (−Tm), or MEF cells treated with tunicamycin (+Tm) as a positive control for activation of IRE1 d Protein synthesis rates in hippocampal slices, determined by incorporation of 35S-methionine into protein, declined by ~57 % by 6 months of age in tauP301L+ mice in contrast to transgene-negative mice, consistent with eIF2α-P signalling (n = 3–4 mice per time point). e Levels of pre-synaptic protein (SNAP25) and post-synaptic protein (PSD-95) declined in parallel (n = 3, measured relative to GAPDH). f tauP301L+ mice showed neuronal loss in the CA1 region of the hippocampus by 6 months (panel iv) compared to age-matched tauP301L− mice (panel ii). Representative images or hematoxylin and eosin-stained sections, scale bar 50 μm. g Doxycycline treatment from 4 months of age reduced total tau protein levels (green bars) compared to non-treated tauP301L+ mice (black bars) at 8 months of age, with accompanying reduction in eIF2α-P and ATF4 levels in 8-month-old tauP301L+ mice to levels similar to those seen in transgene-negative mice; and in contrast to high levels seen in untreated tauP301L+. Bar charts quantitating levels of total tau, eIF2α-P and ATF4 measured relative to GAPDH (in 3–4 independent samples) are shown. All bar charts show mean ± SEM, *p < 0.05, **p < 0.005, Student’s t test was used except in (c), where one-way ANOVA analysis with Tukey’s post hoc test for multiple comparisons was performed. X-axis represents increasing age in months.
Fig. 2
Fig. 2
PERK inhibitor treatment reduces eIF2α-P and ATF4 protein levels and restores protein synthesis rates in mutant tau-expressing rTg4510 mice. a tauP301L+ mice were treated twice daily by oral gavage from 6 months with either the PERK inhibitor, GSK2606414 50 mg/kg, (blue bars) or vehicle (grey bars) and tested at 8 months of age. b Immunostaining showed a significant reduction in PERK-P (red) and pSer202/Thr205-tau (AT8, green) staining in the hippocampus after GSK2606414 treatment. Graphs show quantification of relative intensity for PERK-P and ptau compared to transgene-negative mice (n = 3–5 mice, scale bar 20 μm). c PERK inhibitor treatment markedly reduced PERK-P, eIF2α-P and ATF4 protein levels in 8-month-old tauP301L+ mice, preventing the decline of global protein synthesis rates as determined by 35S-methionine incorporation into protein (d) in comparison to vehicle-treated animals (n = 3 mice). Representative immunoblots of hippocampal lysates and bar charts quantitating protein levels (in three independent samples). All bar charts show mean ± SEM, *p < 0.05, **p < 0.01, using Student’s t test.
Fig. 3
Fig. 3
PERK inhibitor treatment decreases tau phosphorylation and prevents neurodegeneration and clinical disease in mutant tau-expressing rTg4510 mice. a GSK2606414 treatment prevented clinical signs in 8-month-old tauP301L+ mice, which showed normal grooming, posture and movement compared to vehicle-treated animals and were indistinguishable from transgene-negative animals of the same age (representative images, i–iii). Histologically, PERK inhibitor resulted in marked neuroprotection with preservation of hippocampal volume and CA1-3 neuronal ribbon (iv–vi, hematoxylin and eosin-stained sections), and immunostaining using AT8 showed a significant reduction in pSer202/Thr205-tau staining in the hippocampus after GSK2606414 treatment (vii–ix, representative images of hippocampal sections, scale bar 50 μm). b Average count of the number of CA1 pyramidal neurons in five consecutive slices from tauP301L+ treated with vehicle (grey bars) or GSK2606414 (blue bars) mice relative to control mice (tauP301L−, white bars) shows reduced loss of pyramidal neurons in PERK inhibitor-treated mice (i.e. prevention of neurodegeneration) to numbers similar to 6-month-old mutant tau-expressing mice (black bar) (n = 3–4 mice) (c) GSK2606414 partially prevented brain atrophy in comparison to vehicle-treated mice (n = 3–8 mice). d Total tau levels were not significantly different in PERK inhibitor or vehicle-treated animals (n = 3 mice). e AlphaScreen analysis with AT8 and PHF-1 shows a reduction in soluble phospho-tau at pSer202/Thr205 and pSer396/404 epitopes after PERK inhibitor treatment compared to vehicle-treated animals (n = 4 mice). f Levels of the active form, pTyr216-GSK3β, and total GSK3β levels increased compared to control mice. PERK inhibitor treatment significantly reduced pTyr216-GSK3β and total GSK3β levels in tauP301L+ mice to levels seen in control mice. Representative immunoblots of hippocampal lysates and bar charts quantitating protein levels (in three independent samples). All bar charts show mean ± SEM, *p < 0.05, **p < 0.01 ***p < 0.005, n.s non-significant, using Student’s t test.
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