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. 2012;7(10):e47884.
doi: 10.1371/journal.pone.0047884. Epub 2012 Oct 29.

Usp14 deficiency increases tau phosphorylation without altering tau degradation or causing tau-dependent deficits

Youngnam N Jin  1 , Ping-Chung Chen, Jennifer A Watson, Brandon J Walters, Scott E Phillips, Karen Green, Robert Schmidt, Julie A Wilson, Gail V Johnson, Erik D Roberson, Lynn E Dobrunz, Scott M Wilson
Affiliations

Usp14 deficiency increases tau phosphorylation without altering tau degradation or causing tau-dependent deficits

Youngnam N Jin et al. PLoS One. 2012.

Abstract

Regulated protein degradation by the proteasome plays an essential role in the enhancement and suppression of signaling pathways in the nervous system. Proteasome-associated factors are pivotal in ensuring appropriate protein degradation, and we have previously demonstrated that alterations in one of these factors, the proteasomal deubiquitinating enzyme ubiquitin-specific protease 14 (Usp14), can lead to proteasome dysfunction and neurological disease. Recent studies in cell culture have shown that Usp14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3. Using Usp14-deficient ax(J) mice, we investigated if loss of Usp14 results in decreased levels of endogenous tau and ataxin-3 in the nervous system of mice. Although loss of Usp14 did not alter the overall neuronal levels of tau and ataxin-3, we found increased levels of phosphorylated tau that correlated with the onset of axonal varicosities in the Usp14-deficient mice. These changes in tau phosphorylation were accompanied by increased levels of activated phospho-Akt, phosphorylated MAPKs, and inactivated phospho-GSK3β. However, genetic ablation of tau did not alter any of the neurological deficits in the Usp14-deficient mice, demonstrating that increased levels of phosphorylated tau do not necessarily lead to neurological disease. Due to the widespread activation of intracellular signaling pathways induced by the loss of Usp14, a better understanding of the cellular pathways regulated by the proteasome is required before effective proteasomal-based therapies can be used to treat chronic neurological diseases.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Steady-state levels of aggregate-prone tau and ataxin-3 proteins in the cerebellum of wt and Usp14-deficient axJ mice.
(A) Representative immunoblot of ataxin-3, tau, α- and β-tubulin, and Usp14 from the cerebellum of two different 4-week-old wt and axJ mice. The tau5 antibody was used to determine total tau levels, and the PHF-1 antibody was used to identify phosphorylated tau. (B) Quantitation of total and PHF-1-reactive tau. n = 4 to 5 animals per genotype (C) Immunohistochemistry of PHF-1-reactive tau in the cerebellum of wt and axJ mice. Cryosections from each genotype were stained with the PHF-1 antibody and visualized using DAB. The boxed area in a and b is enlarged in c and d, respectively. Arrows indicate localized regions of intense staining with PHF-1 in Purkinje cell dendrites. ML, molecular layer; PCL, Purkinje cell layer; and GC, granule cell layer. Scale bars 50 µm.
Figure 2
Figure 2. Structural analysis of the cerebellum from wt and axJ mice.
(A) Indirect immunofluorescence of cerebellum from wt and axJ mice using calbindin (red), β-tubulin (green) and DAPI (blue). (B) Electron micrographs of cerebellar sections from wt and axJ mice. Black line indicates the outline of a Purkinje cell axon. Magnification at 2,500X. n = 3 to 4 animals per genotype.
Figure 3
Figure 3. Immunoblot analysis of tau levels in specific brain regions.
(A) Cortical and (B) hippocampal extracts were prepared from two different 3 and 6-week-old wt and axJ mice. The PHF-1 antibody was used to determine levels of phosphorylated tau, the tau5 antibody was used to detect total tau, and Gapdh was used as a loading control. n = 4 to 5 animals per genotype.
Figure 4
Figure 4. Analysis of phosphorylated tau-epitopes in wt and axJ mice.
Hippocampal extracts from two different 3 and 6-week-old mice were probed with either the 12E8 antibody (pSer262), AT180 antibody (pThr231), AT100 antibody (pSer212/pThr214), tau1 antibody (unphosphorylated tau), or tau5 antibody (total tau). Gapdh was used as a loading control. n = 3 to 4 mice per genotype.
Figure 5
Figure 5. Immunoblot analysis of putative tau kinases in axJ and wt mice.
(A) Steady-state levels of total and inactive (pSer9) GSK3β, total and activated (pSer473 and pThr308) Akt, Cdk5, p35 and β-actin in the hippocampus of two different wt and axJ mice. (B) Total and phosphorylated MAPKs MEK1, JNK1/2 and ERK1/2 were examined in hippocampal extracts from 4-week-old wt and axJ mice. (C) Graph depicts the quantitation of proteins from blots in A and B. n = 3 to 4 mice per genotype.
Figure 6
Figure 6. Effect of tau reduction on development and survival of axJ mice.
(A) Four-month survival curves of wt, axJ, tauKO and axJtauKO mice. n = 4 mice per genotype. (B) Body weights of 4 and 8-week-old wt, axJ, tauKO and axJtauKO mice. n = 4 mice per genotype. (C) Purkinje cell axonal swellings were quantitated in wt, axJ, tauKO and axJtauKO mice. n = 4 mice per genotype. ***indicates p≤10−7 (D) Paired-pulse facilitation was measured at hippocampal CA3-CA1 synapses. Facilitation was measured at 5 interpulse intervals ranging from 50 to 500 ms. n = 4 mice per genotype.
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