Downregulation of protein phosphatase 2A by apolipoprotein E: Implications for Alzheimer's disease

https://doi.org/10.1016/j.mcn.2017.07.002Get rights and content

Highlights

  • ApoE transcriptionally represses PPP2R5E, a regulatory B′ subunit of protein phosphatase 2A.

  • ApoE triggers demethylation of the catalytic subunit (PP2AC) of PP2A.

  • Together, this results in the disruption of the PPP2R5E-PP2AC complex and in PP2A activity.

  • Our work linking ApoE to a reduction in the PP2A activity has implications for Alzheimer's disease.

Abstract

The apolipoprotein E ε4 allele is the single most important genetic risk factor associated with Alzheimer's disease (AD). Tau phosphorylation and hyperphosphorylation is an underlying feature of AD and is regulated by specific kinases and phosphatases. Among phosphatases, protein phosphatase 2A (PP2A) is the principal tau dephosphorylating enzyme in the brain. Several abnormalities of PP2A have been reported in AD, including among others decreased protein levels of PP2A, decreased mRNA and protein levels of the catalytic subunit PP2AC and variable regulatory B subunits and reduced methylation of the catalytic subunit, all of which results in disruption of the PP2A phosphatase activity. In earlier studies we described a novel mechanism for ApoE as a transcription factor that binds regions of double-stranded DNA with high affinity, including the promoter regions of ~ 3000 different genes. The list of genes also included PPP2R5E (B56ε), a regulatory B′ subunit of protein phosphatase 2A. Using a combination of A172 human glioblastoma cells, ApoE3/4 and ApoE−/− NSC and human postmortem tissue, we now demonstrate that ApoE not only binds to the PPP2R5E promoter but also triggers a significant reduction in PP2A activity by two mechanisms: 1) ApoE transcriptionally represses PPP2R5E and reduces protein expression, and 2) ApoE triggers demethylation of the catalytic subunit (PP2AC) of PP2A, resulting in the disruption of the PPP2R5E-PP2AC complex. Our results indicated a significant down-regulation of PPP2R5E gene expression and reduction in PP2A activity by ApoE4 compared with ApoE3. This may also explain an elevated Tau phosphorylation in AD human brains that featured at least one ApoE4 allele. Thus, our present work links ApoE and PPP2R5E expression to a reduction in the PP2A catalytic activity that has implications for Alzheimer's disease.

Introduction

Our recent work on understanding the pathogenesis of Alzheimer's disease (AD) provided a set of mechanisms that links the expression of apolipoprotein E (ApoE) to amyloid precursor protein (APP) processing and Aβ peptide accumulation (Theendakara et al., 2013, Theendakara et al., 2016). Specifically, we showed that the expression of ApoE4 but not ApoE3, significantly (i) reduced the ratio sAPPα/Aβ; (ii) reduced Sirtuin T1 (SirT1) expression, (iii) triggered Tau and APP phosphorylation, and (iv) induced programmed cell death (Theendakara et al., 2013, Theendakara et al., 2016). Using a combination of cell-based studies and chromatin immunoprecipitation experiments, including ChIP-seq, we showed that ApoE binds to the promoter regions of ~ 3000 different genes and included among them PPP2R5E (B56ε), a regulatory B subunit belonging to the PR61/B′ subunit family of protein phosphatase 2A (PP2A) (Theendakara et al., 2016).

PP2A holoenzymes are a family of ubiquitous heterotrimeric proteins consisting of a core dimer, PP2AD, which contains one of the two scaffolding A subunits (PR65α or PR65β; ~ 65 kDa), a ~ 36-kDa catalytic PP2AC subunit (PP2ACα or PP2ACβ) and one of a number of variable regulatory B subunits. There are four multigene families of B-type subunits including B (PR55/B55), B′ (PR61/B56), B″ (PR48/PR72/PR130), and B′′′ (PR93/PR110), all with specific cellular functions (Sangodkar et al., 2016, Sommer et al., 2015). The assembly of the entire PP2A complex with the appropriate B-type subunit is the key to specificity and regulation of PP2A (Iqbal et al., 2016, Janssens et al., 2008, Martin et al., 2013, Sommer et al., 2015, Sontag and Sontag, 2014, Torrent and Ferrer, 2012). The B′ family (PR61/B56) has 5 different isoforms (α, β, γ, δ and ε), that share a central core domain but possess divergent N-and C-termini that provide isoform specificity (McCright et al., 1996, Sangodkar et al., 2016). Although PP2AC is physically responsible for the dephosphorylation process, recent reports indicate that regulatory B subunits that are more diverse than the A and C subunits, influence substrate specificity, rate and extent of dephosphorylation, and subcellular localization of the PP2A holoenzyme (McCright et al., 1996, Sangodkar et al., 2016). Several alterations including phosphorylation and demethylation of the PP2AC catalytic subunit of PP2A, can dissociate the PP2A heterotrimeric subunits in certain situations, resulting in destabilization of the enzyme complex and reduction in the phosphatase activity (Zhou et al., 2008).

While PP2A subunit alterations resulting in cancer have been demonstrated (Perrotti and Neviani, 2013, Sangodkar et al., 2016), the mechanisms that destabilize the PP2A complex leading to reduction in PP2A activity and AD toxicity are still unclear (Sontag and Sontag, 2014). Furthermore, the relationship between ApoE and PP2A, and more specifically PPP2R5E's role in AD pathogenesis is also unclear. Since ApoE bound to the promoter region of PPP2R5E (Theendakara et al., 2016), we chose to investigate what effect ApoE has on the expression of PPP2R5E and utilized a combination of techniques to confirm its potential role in AD.

Our study using A172 human glioblastoma cells, ApoE3/4 and ApoE−/− neural stem cells (NSC), and postmortem hippocampal tissue of normal subjects and AD-individuals, links ApoE to reduced PPP2R5E expression leading to a reduction in PP2A activity. Specifically, we showed that ApoE transcriptionally represses PPP2R5E mRNA expression resulting in a significant reduction in PPP2R5E protein levels. Additionally, ApoE triggers demethylation of PP2AC thereby disrupting the PPP2R5E-PP2AC complex, with ApoE4 effecting a significant change compared with ApoE3. The above events provide an explanation for the reduction in PP2AC activity and an elevation in hyperphosphorylated Tau (p-Tau) as judged by the presence of the paired helical filaments (PHF) in AD human brains. In a pilot study involving screening of small molecules, the ApoE4-mediated reduction in PP2A activity was reversed by a subset of therapeutic candidates. Thus, our study linking ApoE4 expression to reduced PP2A activity through the suppression of PPP2R5E expression offers new insight into the mechanism by which ApoE4 confers risk for the development of AD and provides a format for medium-throughput screening of drug candidates.

Section snippets

Results

Earlier, our genome wide studies using ApoE chromatin immunoprecipitation (ChIP) followed by DNA sequencing revealed that > 3000 genomic sequences interacted with ApoE. In addition to Sirtuin 1 (Theendakara et al., 2013, Theendakara et al., 2016), the list included genes involved in axon guidance, neuronal survival, cell death, energy metabolism, and inflammation, as well as other AD-related genes (Theendakara et al., 2016). Further analysis revealed that ApoE also bound to the promoter for the

Discussion

Accumulation of the amyloid-β (Aβ) peptide, the major component of amyloid plaques, is considered one of the hallmarks of a pathogenic cascade that eventually leads to AD (Hardy and Selkoe, 2002). The sequential proteolytic processing of amyloid-β-precursor protein (APP) by β-secretase and γ-secretase produces several Aβ species, including the most abundant and toxic species-Aβ40 and Aβ42 and a number of other minor species (Selkoe and Hardy, 2016). Given the strong effect of ApoE alleles on

Cell culture conditions and transfections

All experiments were performed in A172 human glioblastoma cells or ApoE3/4 and ApoE−/− iPSC. Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Human ApoE cDNA constructs were generously provided by Dr. Yadong Huang (Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, CA). All DNA constructs were confirmed by sequence analysis. Transient transfection of cells was performed using a

Compliance

This manuscript is not under consideration by another journal, nor has it been published. The research study was conducted to the highest ethical standards.

Competing financial interests

The authors declare no competing financial interests.

Acknowledgements

We thank Dr. Yadong Huang (Gladstone Institute of Neurological Disease, University of California, San Francisco, CA 94158) for providing the ApoE plasmids, Dr. Peter Davies (Albert Einstein College of Medicine, Bronx, NY) for the PHF antibody (tau phosphorylated at Ser396/404; PHF1 mouse IgG), Dr. Xianmin Zeng (Buck Institute for Research on Aging) for the NSC and iPSC, Dr. Varghese John (Easton Center for Alzheimer's Disease Research, University of California, Los Angeles, CA 90025) for the

Author contributions

V.T. and R.V.R. performed the research experiments and analyzed the data, R.V.R designed and supervised the study, D.E.B. and R.V.R wrote the manuscript.

References (48)

  • R.V. Rao

    Molecular components of a cell death pathway activated by endoplasmic reticulum stress

    J. Biol. Chem.

    (2004)
  • A. Shaltouki

    Mitochondrial alterations by PARKIN in dopaminergic neurons using PARK2 patient-specific and PARK2 knockout isogenic iPSC lines

    Stem Cell Reports.

    (2015)
  • S.B. Vafai et al.

    Protein phosphatase 2A methylation: a link between elevated plasma homocysteine and Alzheimer's disease

    FEBS Lett.

    (2002)
  • V. Vogelsberg-Ragaglia

    PP2A mRNA expression is quantitatively decreased in Alzheimer's disease hippocampus

    Exp. Neurol.

    (2001)
  • Y. Xing

    Structural mechanism of demethylation and inactivation of protein phosphatase 2A

    Cell

    (2008)
  • X.Q. Yao

    Glycogen synthase kinase-3beta regulates leucine-309 demethylation of protein phosphatase-2A via PPMT1 and PME-1

    FEBS Lett.

    (2012)
  • C.E. Zhang

    Homocysteine induces tau phosphorylation by inactivating protein phosphatase 2A in rat hippocampus

    Neurobiol. Aging

    (2008)
  • X.W. Zhou

    Tau hyperphosphorylation correlates with reduced methylation of protein phosphatase 2A

    Neurobiol. Dis.

    (2008)
  • D.E. Bredesen

    Metabolic profiling distinguishes three subtypes of Alzheimer's disease

    Aging (Albany NY)

    (2015)
  • G.A. Calin

    Low frequency of alterations of the alpha (PPP2R1A) and beta (PPP2R1B) isoforms of the subunit A of the serine-threonine phosphatase 2A in human neoplasms

    Oncogene

    (2000)
  • D.J. Christensen

    Apolipoprotein E and peptide mimetics modulate inflammation by binding the SET protein and activating protein phosphatase 2A

    J. Immunol.

    (2011)
  • C.E. Finch

    Evolution in health and medicine Sackler colloquium: evolution of the human lifespan and diseases of aging: roles of infection, inflammation, and nutrition

    Proc. Natl. Acad. Sci. U. S. A.

    (2010)
  • C.E. Finch et al.

    Systemic inflammation, infection, ApoE alleles, and Alzheimer disease: a position paper

    Curr. Alzheimer Res.

    (2007)
  • T.F. Gendron et al.

    The role of tau in neurodegeneration

    Mol. Neurodegener.

    (2009)
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