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The Unfolded Protein Response Is Activated in Pretangle Neurons in Alzheimer's Disease Hippocampus

      Accumulation of misfolded proteins in the endoplasmic reticulum triggers a cellular stress response called the unfolded protein response (UPR) that protects the cell against the toxic buildup of misfolded proteins. Previously, we reported that UPR activation is increased in Alzheimer's disease (AD) patients. How the UPR relates to the pathological hallmarks of AD is still elusive. In the present study, the involvement of UPR activation in neurofibrillary degeneration in AD was investigated. Immunoreactivity for the phosphorylated UPR activation markers pancreatic ER kinase (pPERK), eukaryotic initiation factor 2α, and inositol-requiring enzyme 1α was observed in hippocampal neurons associated with granulovacuolar degeneration. The percentage of pPERK-immunoreactive neurons was increased in AD cases compared with nondemented control cases and with the Braak stage for neurofibrillary changes. Although absent from neurofibrillary tangles, pPERK immunoreactivity was most abundant in neurons with diffuse localization of phosphorylated tau protein. Additional analyses showed that pPERK immunoreactivity was associated with ubiquitin and the ubiquitin binding protein p62. A strong co-occurrence of immunoreactivity for both pPERK and glycogen synthase kinase 3β in neurons was also observed. Together, these data indicate that UPR activation in AD neurons occurs at an early stage of neurofibrillary degeneration and suggest that the prolonged activation of the UPR is involved in both tau phosphorylation and neurodegeneration in AD pathogenesis.
      Alzheimer's disease (AD), the most common form of dementia, is a chronic neurodegenerative disease causing progressive impairment of memory and other cognitive functions. Apart from the loss of synapses and neurons, AD is characterized at the neuropathological level by neurofibrillary tangles (NFTs), and extracellular aggregates of amyloid β (Aβ) protein. The major component of the intracellular NFTs is hyperphosphorylated tau, a microtubule stabilizing protein. Both Aβ and hyperphosphorylated tau have been shown to interfere with several cellular processes, such as Ca2+ homeostasis and protein trafficking. These disturbances can result in stress in the endoplasmic reticulum (ER) because of accumulation of un- and misfolded proteins in the ER. ER stress triggers a cellular stress response called the unfolded protein response (UPR) intended to protect the cell against the toxic build-up of misfolded proteins.
      • Forman MS
      • Lee VM
      • Trojanowski JQ
      ‘Unfolding’ pathways in neurodegenerative disease.
      • Rutkowski DT
      • Kaufman RJ
      A trip to the ER: coping with stress.
      Under physiological conditions the ER chaperone BiP (or GRP78) is bound to three key proteins in the ER membrane: pancreatic ER kinase (PERK),
      • Harding HP
      • Zhang Y
      • Ron D
      Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase.
      inositol-requiring enzyme 1 (IRE-1), and activating transcription factor 6 (ATF-6). When unfolded proteins accumulate in the ER lumen, BiP is attracted to bind to these unfolded proteins to support correct protein folding and is thereby released from PERK, IRE-1, and ATF-6, which are consequently activated. UPR activation results in an overall decrease in translation, increased protein degradation, and increased levels of ER chaperones, including BiP.
      • Kozutsumi Y
      • Segal M
      • Normington K
      • Gething MJ
      • Sambrook J
      The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins.
      Once sufficient BiP protein is available for binding and inactivation of PERK, IRE1, and ATF6, the UPR will be shut down and ER functioning restored. Although the initial activation of the UPR protects the cell against the toxic build-up of misfolded proteins, prolonged activation of the UPR induces cell death and may therefore be involved in the pathogenesis of protein folding diseases, such as AD.
      Previously, we have shown activation of the UPR in AD brain.
      • Hoozemans JJ
      • Veerhuis R
      • Van Haastert ES
      • Rozemuller JM
      • Baas F
      • Eikelenboom P
      • Scheper W
      The unfolded protein response is activated in Alzheimer's disease.
      Western blot analysis showed that protein levels of BiP are increased in the temporal cortex and the hippocampus of AD cases compared with nondemented control cases. In addition, immunohistochemical detection of BiP and phosphorylated PERK (pPERK) shows activation of the UPR in neurons of AD patients. The expression levels of BiP protein in the different Braak stages suggest that the UPR is activated in an early stage of AD pathology. Recently we showed that oligomeric aggregates of Aβ1–42 peptide induce mild ER stress in neuronal cells and that oligomeric Aβ is more toxic to cells when they are primed for UPR activation.
      • Chafekar SM
      • Hoozemans JJ
      • Zwart R
      • Baas F
      • Scheper W
      Abeta 1–42 induces mild endoplasmic reticulum stress in an aggregation state-dependent manner.
      In addition, our data show that increased intracellular Aβ production, in particular Aβ1–42, enhances UPR induction and ER stress toxicity.
      • Chafekar SM
      • Zwart R
      • Veerhuis R
      • Vanderstichele H
      • Baas F
      • Scheper W
      Increased Abeta1–42 production sensitizes neuroblastoma cells for ER stress toxicity.
      These in vitro data indicate a role for early Aβ forms in the induction of the UPR. How the activation of the UPR in AD pathology relates to the occurrence of tau pathology in AD is unknown yet. Interestingly, in vitro studies indicate a direct connection between ER stress and phosphorylation of tau, where activation of the UPR induces the activity of glycogen synthase kinase 3β (GSK-3β).
      • Kim AJ
      • Shi Y
      • Austin RC
      • Werstuck GH
      Valproate protects cells from ER stress-induced lipid accumulation and apoptosis by inhibiting glycogen synthase kinase-3.
      • Song L
      • De Sarno P
      • Jope RS
      Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation.
      GSK-3β is the major kinase for tau and considered to be involved in the hyperphosphorylation of tau present in NFTs.
      • Jope RS
      • Johnson GV
      The glamour and gloom of glycogen synthase kinase-3.
      In the current study we investigated the role of UPR activation in tau pathology in AD. UPR activation markers pPERK, pIRE1α, and phosphorylated eukaryotic initiation factor 2alpha (peIF2α) were investigated in AD and control hippocampus. The hippocampus has a relative early involvement of tau pathology in AD and neurons with different intermediate stages of tau pathology are present in a single hippocampal section.
      • Braak H
      • Braak E
      Neuropathological stageing of Alzheimer-related changes.
      Also the UPR markers are prominently present in the separate subregions of the hippocampus
      • Hoozemans JJ
      • Veerhuis R
      • Van Haastert ES
      • Rozemuller JM
      • Baas F
      • Eikelenboom P
      • Scheper W
      The unfolded protein response is activated in Alzheimer's disease.
      allowing correlative analysis with tau pathology. pPERK expression was evaluated in different Braak stages to investigate the relation with NFTs. The association of pPERK with tau pathology, ubiquitin, and the ubiquitin-binding protein p62 was analyzed using double immunohistochemistry. In addition, the co-localization between pPERK and GSK-3β was investigated. Our data are in accordance with a model in which activation of the UPR enhances tau phosphorylation and aggregation and precedes tangle formation in the hippocampus of AD patients.

      Materials and Methods

      Postmortem Brain Tissue

      Human brain specimens of probable AD, other dementias, and age-matched nondemented control cases were obtained at autopsy with a short postmortem interval (The Netherlands Brain Bank, Amsterdam, The Netherlands). Clinical diagnosis was defined according to DSM-III-R criteria and the severity of dementia was evaluated according to the Global Deterioration Scale of Reisberg.
      • Reisberg B
      • Ferris SH
      • de Leon MJ
      • Crook T
      The Global Deterioration Scale for assessment of primary degenerative dementia.
      Neuropathological evaluation was performed on formalin-fixed, paraffin-embedded tissue from different sites, including the frontal cortex (F2), temporal pole cortex, parietal cortex (superior and inferior lobule), occipital pole cortex, and the hippocampus (essentially CA1 and entorhinal area of the parahippocampal gyrus). The distribution and the density of NFTs was determined using Bodian staining and immunohistochemistry for hyperphosphorylated tau. Senile plaques were stained with the methenamine silver method.
      • Yamaguchi H
      • Haga C
      • Hirai S
      • Nakazato Y
      • Kosaka K
      Distinctive, rapid, and easy labeling of diffuse plaques in the Alzheimer brains by a new methenamine silver stain.
      Staging of AD was evaluated according to Braak and colleagues.
      • Braak H
      • Braak E
      Neuropathological stageing of Alzheimer-related changes.
      • Braak H
      • Alafuzoff I
      • Arzberger T
      • Kretzschmar H
      • Del Tredici K
      Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry.
      Age, sex, clinical diagnosis, and Braak score for NFTs of all cases used in this study are listed in Table 1. Mean postmortem delay of all cases was 6 hours with a range from 4 to 16 hours.
      Table 1Cases Used for Immunohistochemical Analysis in This Study
      Case no.Pathological diagnosisSexAgeBraak stage
      1CONM800
      2CONM800
      3CONF730
      4CONM571
      5CONF771
      6CONM731
      7CONF761
      8CONM902
      9CONF812
      10CONM872
      11CONM852
      12CONM743
      13CONM834
      14CONF944
      15CONF974
      16ADM863
      17ADM864
      18ADM644
      19ADF755
      20ADM875
      21ADF785
      22ADM645
      23ADF625
      24ADF696
      25ADM576
      26ADF726
      27AD/LBVF713
      28AD/LBVF954
      29AD/LBVM754
      30AD/LBVF706
      31AD/LBVM656
      32DEM/VASCF893
      33DEM/VASCM803
      34DEM/VASCF903
      CON, control case; AD, Alzheimer's disease case; AD/LBV, Alzheimer's disease case with Lewy body variant pathology; DEM/VASC, dementia caused by vascular lesions in the brain; F, female; M, male.

      Immunohistochemistry

      Sections (5 μm thick) were mounted on Superfrost plus tissue slides (Menzel-Gläser, Braunschweig, Germany) and dried overnight at 37°C. For all stainings sections were deparaffinized and subsequently immersed in 0.3% H2O2 in methanol for 30 minutes to quench endogenous peroxidase activity. Normal sera and antibodies were dissolved in phosphate-buffered saline (PBS) containing 1% (w/v) bovine serum albumin (Boehringer Mannheim, Mannheim, Germany). Primary antibodies and their sources are listed in Table 2. Negative controls for all single and double immunostainings were generated by omission of primary antibodies. For description of antibody-specificity we refer to previous reports (Table 2).
      • Sokka AL
      • Putkonen N
      • Mudo G
      • Pryazhnikov E
      • Reijonen S
      • Khiroug L
      • Belluardo N
      • Lindholm D
      • Korhonen L
      Endoplasmic reticulum stress inhibition protects against excitotoxic neuronal injury in the rat brain.
      • Luo D
      • He Y
      • Zhang H
      • Yu L
      • Chen H
      • Xu Z
      • Tang S
      • Urano F
      • Min W
      AIP1 is critical in transducing IRE1-mediated endoplasmic reticulum stress response.
      • Hoozemans JJ
      • Van Haastert ES
      • Eikelenboom P
      • de Vos RA
      • Rozemuller JM
      • Scheper W
      Activation of the unfolded protein response in Parkinson's disease.
      • van der Voorn JP
      • van Kollenburg B
      • Bertrand G
      • Van Haren K
      • Scheper GC
      • Powers JM
      • van der Knaap MS
      The unfolded protein response in vanishing white matter disease.
      • Spillantini MG
      • Crowther RA
      • Goedert M
      Comparison of the neurofibrillary pathology in Alzheimer's disease and familial presenile dementia with tangles.
      • Dickson DW
      Required techniques and useful molecular markers in the neuropathologic diagnosis of neurodegenerative diseases.
      • Kuusisto E
      • Salminen A
      • Alafuzoff I
      Ubiquitin-binding protein p62 is present in neuronal and glial inclusions in human tauopathies and synucleinopathies.
      • Kuusisto E
      • Salminen A
      • Alafuzoff I
      Early accumulation of p62 in neurofibrillary tangles in Alzheimer's disease: possible role in tangle formation.
      • Leroy K
      • Boutajangout A
      • Authelet M
      • Woodgett JR
      • Anderton BH
      • Brion JP
      The active form of glycogen synthase kinase-3beta is associated with granulovacuolar degeneration in neurons in Alzheimer's disease.
      Except for the detection of pPERK, sections were treated in 10 mmol/L pH 6.0 sodium citrate buffer heated by autoclave for 10 minutes for antigen retrieval. For detection of pPERK and peIF2α sections were pre-incubated for 10 minutes with normal swine-serum (1:20 dilution; DAKO, Glostrup, Denmark), and for the detection of AT8 sections were pre-incubated for 10 minutes with normal rabbit-serum (1:50 dilution, DAKO). Rabbit anti-pPERK (1:800 dilution), rabbit anti-peIF2α (1:500 dilution), or mouse anti-AT8 (1:200 dilution) was incubated overnight at 4°C. After washing with PBS, sections were incubated for 30 minutes with biotin-conjugated swine anti-rabbit F(ab′)2 (1:300 dilution, DAKO) for the detection of primary rabbit antibodies or biotin-conjugated rabbit anti-mouse F(ab′)2 (1:500 dilution, DAKO) for the detection of primary mouse antibodies. Subsequently, sections were incubated with streptavidin-biotin horseradish peroxidase complex (streptABComplex/HRP, 1:200 dilution; DAKO) for 60 minutes. Color was developed using 3,3′-diaminobenzidine (0.1 mg/ml, 0.02% H2O2, 5 minutes; Sigma, St. Louis, MO) as chromogen. Sections were counterstained with hematoxylin and mounted using Depex (BDH Laboratories Supplies, Poole, UK).
      Table 2Name, Epitope, Source, and References of Primary Antibodies Used in This Study
      Name, typeEpitope (immunogen)SourceReferences
      pPERKPERK phosphorylated at threonine 981Santa Cruz Biotechnology, Santa Cruz, CA
      • Sokka AL
      • Putkonen N
      • Mudo G
      • Pryazhnikov E
      • Reijonen S
      • Khiroug L
      • Belluardo N
      • Lindholm D
      • Korhonen L
      Endoplasmic reticulum stress inhibition protects against excitotoxic neuronal injury in the rat brain.
      ,
      • Luo D
      • He Y
      • Zhang H
      • Yu L
      • Chen H
      • Xu Z
      • Tang S
      • Urano F
      • Min W
      AIP1 is critical in transducing IRE1-mediated endoplasmic reticulum stress response.
      ,
      • Hoozemans JJ
      • Van Haastert ES
      • Eikelenboom P
      • de Vos RA
      • Rozemuller JM
      • Scheper W
      Activation of the unfolded protein response in Parkinson's disease.
      pIRE1αIRE1α phosphorylated at serine 724Novus Biologicals, Littleton, CO
      • Luo D
      • He Y
      • Zhang H
      • Yu L
      • Chen H
      • Xu Z
      • Tang S
      • Urano F
      • Min W
      AIP1 is critical in transducing IRE1-mediated endoplasmic reticulum stress response.
      peIF2αeIF-2α phosphorylated at serine 52Sigma, St. Louis, MO
      • Hoozemans JJ
      • Van Haastert ES
      • Eikelenboom P
      • de Vos RA
      • Rozemuller JM
      • Scheper W
      Activation of the unfolded protein response in Parkinson's disease.
      ,
      • van der Voorn JP
      • van Kollenburg B
      • Bertrand G
      • Van Haren K
      • Scheper GC
      • Powers JM
      • van der Knaap MS
      The unfolded protein response in vanishing white matter disease.
      AT8Tau phosphorylated at serine 202Innogenetics, Ghent, Belgium
      • Spillantini MG
      • Crowther RA
      • Goedert M
      Comparison of the neurofibrillary pathology in Alzheimer's disease and familial presenile dementia with tangles.
      AT100Tau phosphorylated at serine 212 and threonine 214Pierce, Rockford, IL
      • Spillantini MG
      • Crowther RA
      • Goedert M
      Comparison of the neurofibrillary pathology in Alzheimer's disease and familial presenile dementia with tangles.
      AT270Tau phosphorylated at threonine 181Pierce
      • Spillantini MG
      • Crowther RA
      • Goedert M
      Comparison of the neurofibrillary pathology in Alzheimer's disease and familial presenile dementia with tangles.
      UbiquitinUbiquitinChemicon, Temecula, CA
      • Dickson DW
      Required techniques and useful molecular markers in the neuropathologic diagnosis of neurodegenerative diseases.
      p62p62 proteinProgen Biotechnik, Heidelberg, Germany
      • Kuusisto E
      • Salminen A
      • Alafuzoff I
      Ubiquitin-binding protein p62 is present in neuronal and glial inclusions in human tauopathies and synucleinopathies.
      ,
      • Kuusisto E
      • Salminen A
      • Alafuzoff I
      Early accumulation of p62 in neurofibrillary tangles in Alzheimer's disease: possible role in tangle formation.
      GSK-3βGSK-3βBD Biosciences, San Jose, CA
      • Leroy K
      • Boutajangout A
      • Authelet M
      • Woodgett JR
      • Anderton BH
      • Brion JP
      The active form of glycogen synthase kinase-3beta is associated with granulovacuolar degeneration in neurons in Alzheimer's disease.
      GSK-3β pSer9GSK-3β phosphorylated at serine 9Cell Signaling, Danvers, MA
      • Leroy K
      • Boutajangout A
      • Authelet M
      • Woodgett JR
      • Anderton BH
      • Brion JP
      The active form of glycogen synthase kinase-3beta is associated with granulovacuolar degeneration in neurons in Alzheimer's disease.
      For the detection of pIRE1α, GSK-3β, and GSK-3β pSer9, sections were incubated overnight at 4°C with rabbit anti-pIRE1α (1:51,200 dilution), mouse anti-GSK-3β (1:1000 dilution), and rabbit anti-GSK-3β pSer9 (1:50 dilution), respectively. After washing with PBS sections were incubated with EnVision solution (goat anti-mouse/rabbit HRP, undiluted; DAKO). Color was developed using 3,3′-diaminobenzidine (DAB) (EnVision Detection system/HRP, 1:50 dilution, 10 minutes; DAKO) as chromogen. Sections were counterstained with hematoxylin and mounted using Depex.

      Evaluation of Immunohistochemical Stainings and Statistics

      A quantitative analysis of the pPERK-, AT8-, GSK-3β-, and GSK-3β pSer9-immunoreactive neurons was performed in the different regions of the hippocampus. The total number of neurons, assessed by morphology and size of nucleus, were counted, as well as neurons containing pPERK, AT8, GSK-3β, and GSK-3β pSer9 immunoreactivity. Data are expressed as mean ± SEM. SPSS 14.0 for Windows (SPSS Inc., Chicago, IL) was used for statistical analysis of the data. One-way analysis of variance followed by Bonferroni's test for multiple comparisons was used to test for differences between groups. A P value of <0.05 was taken as significant. Correlation analysis was done using the Pearson parametric test.

      Double Immunohistochemistry: pPERK with AT8, AT100, and AT270

      To determine co-localization of pPERK with AT8 and AT100 sections were pre-incubated with serum-free protein blocking (SFPB, DAKO) for 10 minutes and subsequently incubated with rabbit anti-pPERK (1:800 dilution) for 60 minutes. After washing with PBS sections were incubated with EnVision solution (goat anti-rabbit HRP, undiluted; DAKO) for 30 minutes. Color was developed using DAB as chromogen. Sections were treated in 10 mmol/L pH 6.0 sodium citrate buffer heated by autoclave for 10 minutes. After pre-incubation with SFPB for 10 minutes, sections were incubated overnight at 4°C with AT8 (1:200 dilution), AT100 (1:1600 dilution), or AT270 (1:1600 dilution). Sections were washed with PBS and incubated with biotin-conjugated rabbit anti-mouse F(ab′)2 (1:500 dilution, DAKO) for 30 minutes and subsequently with alkaline-conjugated streptavidin (1:100 dilution, DAKO) for 60 minutes. Color was developed using Liquid Permanent Red (LPR, DAKO) as chromogen. Sections were counterstained with hematoxylin and mounted using Aquamount (BDH Laboratories Supplies).

      Double Immunohistochemistry: pPERK with Ubiquitin and p62

      To determine co-localization of pPERK with ubiquitin and p62 sections were pre-incubated with SFPB for 10 minutes and subsequently incubated with rabbit anti-pPERK (1:400 dilution) for 60 minutes. After washing with PBS sections were incubated with biotin-conjugated swine anti-rabbit F(ab′)2 (1:300 dilution, DAKO) for 30 minutes and subsequently with streptavidin-biotin horseradish peroxidase complex (streptABComplex/HRP, 1:200 dilution; DAKO) for 60 minutes. Color was developed using DAB as chromogen. Subsequently, sections were treated in 10 mmol/L pH 6.0 sodium citrate buffer heated by autoclave for 10 minutes for antigen retrieval and in addition to remove all bound antibodies. After pre-incubation with SFPB for 10 minutes, sections were incubated overnight at 4°C with mouse anti-ubiquitin (1:1600 dilution) or mouse anti-p62 (1:1000 dilution). Sections were washed with PBS and incubated with biotin-conjugated rabbit anti-mouse F(ab′)2 (1:500 dilution, DAKO) for 30 minutes and subsequently with alkaline-conjugated streptavidin (1:100 dilution, DAKO) for 60 minutes. Color was developed using LPR as chromogen. Sections were counterstained with hematoxylin and mounted using Aquamount.

      Double Immunohistochemistry: pPERK with GSK-3β and pGSK-3β

      To determine co-localization of pPERK with GSK-3β and pGSK-3β sections were pre-incubated with SFPB for 10 minutes and subsequently incubated with rabbit anti-pPERK (1:800 dilution) for 60 minutes. After washing with PBS sections were incubated with biotin-conjugated swine anti-rabbit F(ab′)2 (1:300 dilution, DAKO) for 30 minutes and subsequently with alkaline-conjugated streptavidin (1:100 dilution, DAKO) for 60 minutes. Color was developed using LPR as chromogen. Sections were treated in 10 mmol/L pH 6.0 sodium citrate buffer heated by autoclave for 10 minutes. After pre-incubation with SFPB for 10 minutes, sections were incubated overnight at 4°C with mouse anti-GSK-3β (1:50 dilution) or rabbit anti-GSK-3β pSer9 (1:10 dilution). Sections were washed with PBS and incubated with EnVision solution (goat anti-mouse HRP (or goat anti-rabbit HRP, undiluted; DAKO) for 30 minutes. Color was developed using DAB as chromogen. Sections were counterstained with hematoxylin and mounted using Aquamount.

      Analysis of Double-Immunohistochemical Stainings

      In this study we used the Nuance spectral imaging system (CRi, Woburn, MA) for the analysis of double-stained specimens. Spectral imaging unmixes colors based on their spectral characteristics, enabling visualization of the different colored reaction products.
      • Levenson RM
      • Mansfield JR
      Multispectral imaging in biology and medicine: slices of life.
      Spectral imaging data cubes were taken from 460 to 660 nm at 10-nm intervals and analyzed with the Nuance software. Spectral libraries of single-brown (DAB), single-red (LPR), and hematoxylin were obtained from control slides. The resulting library was applied to the double-stained slides and the different reaction products were then spectrally unmixed into individual black and white images, representing the localization of each of the reaction products, and reverted to fluorescence-like images composed of pseudo-colors using the Nuance software.

      Results

      UPR Markers pPERK, peIF2α, and pIRE1α Are Associated with GVD in AD Hippocampus

      The immunohistochemical localization of pPERK, peIF2α, and pIRE1α was investigated in the hippocampus of AD and nondemented controls. As described previously,
      • Hoozemans JJ
      • Veerhuis R
      • Van Haastert ES
      • Rozemuller JM
      • Baas F
      • Eikelenboom P
      • Scheper W
      The unfolded protein response is activated in Alzheimer's disease.
      pPERK immunoreactivity was observed as granules in pyramidal neurons (Figure 1A). In this study a similar granular staining pattern was detected for peIF2α and pIRE1α (Figure 1, B and C). pPERK-, peIF2α-, and pIRE1α-positive granules were surrounded by a clear halo and were morphologically similar to the classic granules of granulovacuolar degeneration (GVD). GVD is characterized by basophilic granules surrounded by a clear zone measuring 1 to 5 μm in diameter, occurring predominantly in hippocampal neurons in AD.
      • Okamoto K
      • Hirai S
      • Iizuka T
      • Yanagisawa T
      • Watanabe M
      Reexamination of granulovacuolar degeneration.
      In addition to granular structures that can be defined as GVD also smaller granules (<1 μm in diameter) positive for pPERK, peIF2α, and pIRE1α could be observed.
      Figure thumbnail gr1
      Figure 1Immunohistochemical localization of pPERK, peIF2α, and pIRE1α in AD hippocampus. A: pPERK is detected by immunohistochemistry in pyramidal neurons in the hippocampus. In this case a part of the subiculum of an AD case is shown. The inset shows the cellular localization of pPERK, which is present in granules that can be defined as GVD. B: peIF2α immunohistochemistry on the same AD case and area as shown in A. peIF2α can also be detected in GVD granules (inset). C: pIRE1α can also be detected in pyramidal neurons in the hippocampus of AD cases (shown is subiculum). Similar granular structures were detected as observed with pPERK and peIF2α immunohistochemistry (inset). Scale bars: 200 μm (A–C); 20 μm (insets).

      UPR Activation Is Associated with AD Pathology in the Hippocampus

      In AD cases, a marked increase in the number of neurons immunoreactive for pPERK, peIF2α, and pIRE1α was noted. To investigate this in more depth, the percentages of neurons immunoreactive for pPERK throughout the different regions of the hippocampus were quantified in nondemented control cases and AD cases as well as AD cases with mixed Lewy body pathology and cases with vascular dementia (Table 1). In the CA4, CA1, and subiculum (SUB) a significant increase in pPERK-positive neurons was observed in AD cases compared with nondemented control cases (Figure 2). Several clinically nondemented cases have been staged with Braak stage 4 for neurofibrillary changes. Analyses of the data with these cases as a separate group showed intermediate numbers of pPERK-positive neurons in relation to control and AD cases (data not shown).
      Figure thumbnail gr2
      Figure 2Quantitative analyses of pPERK immunoreactivity. A: Neurons immunoreactive for pPERK were quantified in different regions of the hippocampus. Arrows indicate the borders between different hippocampal regions. B: The percentages of neurons immunoreactive for pPERK were quantified in nondemented control cases, AD cases, AD cases with Lewy body variant (AD/LBV), and dementia cases with a vascular insufficiency (DEM/VASC). Data are expressed as mean ± SEM. *P < 0.05 compared with control levels in same region.
      Previously, we reported that UPR activation is increased in Parkinson's disease with α-synuclein pathology in the substantia nigra.
      • Hoozemans JJ
      • Van Haastert ES
      • Eikelenboom P
      • de Vos RA
      • Rozemuller JM
      • Scheper W
      Activation of the unfolded protein response in Parkinson's disease.
      No significant increase in pPERK immunoreactivity was observed in the hippocampus of AD cases with Lewy body pathology (AD/LBV) as compared with nondemented control cases. In these cases no Lewy bodies or α-synuclein immunoreactive neurons were observed in the hippocampal areas investigated in this study (results not shown). Although a possible contribution of α-synuclein in UPR activation in the mixed AD/LBV cases examined in this study cannot be excluded, our data indicate that UPR activation is associated with AD pathology. In addition, in cases with vascular dementia no significant difference was detected in pPERK immunoreactivity in the hippocampus compared with nondemented control cases (Figure 2).

      pPERK Correlates with Early Ubiquitin Pathology in AD Hippocampus

      Because GVD inclusions have been reported to be immunoreactive for ubiquitin,
      • Okamoto K
      • Hirai S
      • Iizuka T
      • Yanagisawa T
      • Watanabe M
      Reexamination of granulovacuolar degeneration.
      co-localization of pPERK with ubiquitin and p62 was investigated in AD hippocampus. Both proteins are part of the ubiquitin proteasome system (UPS) and are involved in the accumulation of misfolded proteins.
      • Taylor JP
      • Hardy J
      • Fischbeck KH
      Toxic proteins in neurodegenerative disease.
      Ubiquitin co-localizes with NFTs, neuritic plaques, and neuropil threads in AD brain.
      • Perry G
      • Friedman R
      • Shaw G
      • Chau V
      Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains.
      In this study, ubiquitin immunoreactivity was detected in extra- and intracellular inclusions with a diameter between 1 and 5 μm or smaller, however these could not be defined as GVD granules because of the absence of a clear zone surrounding the immunoreactive inclusion. Therefore, although most neurons with pPERK-immunoreactive granules contain ubiquitin-positive inclusions, these were different from GVD granules (Figure 3A). No co-localization was observed between pPERK and ubiquitin-immunoreactive NFTs (Figure 3B). P62 is a ubiquitin-binding protein, involved in the accumulation of ubiquitin-conjugated proteins into cytoplasmic inclusions.
      • Kuusisto E
      • Salminen A
      • Alafuzoff I
      Ubiquitin-binding protein p62 is present in neuronal and glial inclusions in human tauopathies and synucleinopathies.
      • Kuusisto E
      • Salminen A
      • Alafuzoff I
      Early accumulation of p62 in neurofibrillary tangles in Alzheimer's disease: possible role in tangle formation.
      As previously reported,
      • Kuusisto E
      • Salminen A
      • Alafuzoff I
      Early accumulation of p62 in neurofibrillary tangles in Alzheimer's disease: possible role in tangle formation.
      p62 immunoreactivity was present in NFTs and weakly observed in neuropil threads and almost absent in neuritic plaques (Figure 3C). No pPERK immunoreactivity was observed in neurons containing p62-positive NFTs (Figure 3D). Most of pPERK-positive neurons were free of p62 immunoreactivity, although a small part of the pPERK-positive neurons (10 to 20%) contained some p62 immunoreactivity, which appeared diffuse in the cytoplasm.
      Figure thumbnail gr3
      Figure 3Co-localization of pPERK with markers of the ubiquitin proteasome system. A: Double immunohistochemistry for pPERK (red) and ubiquitin (brown). B: Magnification of inset indicated in A. No co-localization is present between pPERK with ubiquitin inclusions. There is an increased association of pPERK-positive neurons with intra- and extracellular ubiquitin immunoreactive granules. C: Double immunohistochemistry for pPERK (red) and p62 (brown). There is no co-localization between pPERK- and p62-immunoreactive inclusions. D: Magnification of inset as indicated in C. Scale bars: 100 μm (A, C); 50 μm (B, D).

      pPERK Is Associated with Phosphorylated Tau

      To further investigate the association of pPERK with tau pathology, the percentage of pPERK-positive neurons in the different subregions of the hippocampus in controls and AD cases was determined. The mean levels were determined for the different Braak stages for neurofibrillary changes (Figure 4A). A significant increase compared with Braak stage 0 was observed in Braak stages 3 to 4 and 5 to 6 in the CA1 and subiculum. The average levels of pPERK-immunoreactive neurons show a similar distribution along the Braak stage as neurons immunoreactive for AT8, which specifically detects tau phosphorylated at serine 202 (Figure 4B). Analysis of the correlation between the number of pPERK- and AT8-positive neurons shows that these markers significantly correlate in the hippocampal areas investigated (Table 3). The subiculum, CA1, and CA4 show the highest correlation coefficient.
      Figure thumbnail gr4
      Figure 4pPERK and AT8 immunoreactivity in different stages of pathology. A: pPERK immunoreactivity in different regions of the hippocampus increases with the Braak score for NFT pathology. B: Similar results are observed with AT8 immunoreactivity. AT8-immunoreactive neurons included immunoreactive NFT as well as diffuse appearance of AT8 immunoreactivity in neurons. Data are expressed as mean ± SEM. *P < 0.05 compared with Braak stage 0 in the same region.
      Table 3Correlation Analyses of pPERK, GSK-3β, and AT8 Immunoreactivity
      Pearson correlation coefficient (P value)
      AreapPERK and AT8pPERK and GSK-3βpPERK and GSK-3β pSer9
      Subiculum0.771 (<0.001)0.883 (<0.001)0.916 (<0.001)
      CA10.859 (<0.001)0.882 (<0.001)0.858 (<0.001)
      CA20.539 (0.001)0.454 (0.009)0.406 (0.021)
      CA30.642 (<0.001)0.728 (<0.001)0.866 (<0.001)
      CA40.922 (<0.001)0.852 (<0.001)0.904 (<0.001)
      The correlation between pPERK and phosphorylated tau was investigated in more detail at the immunohistochemical level. Previously we showed that AT8 partly co-localizes with pPERK but that pPERK does not co-localize with NFTs.
      • Hoozemans JJ
      • Veerhuis R
      • Van Haastert ES
      • Rozemuller JM
      • Baas F
      • Eikelenboom P
      • Scheper W
      The unfolded protein response is activated in Alzheimer's disease.
      In this study we confirm these data using double immunohistochemistry and spectral image analysis (Figure 5, A–D). Approximately 50% of the neurons that were immunoreactive for pPERK showed moderate to intense immunostaining for AT8. Although pPERK could hardly be detected in tangle-bearing neurons (Figure 5D, arrow), neurons with diffuse AT8 immunoreactivity did show pPERK immunoreactivity. Corresponding results were observed with AT100 and AT270 (Figure 5, E–L). AT100 detects tau phosphorylated at serine 212 and threonine 214, whereas AT270 detects tau phosphorylated at threonine 181. No or very few pPERK-immunoreactive granules were observed in neurons with NFTs immunoreactive for AT100 or AT270 (Figure 5, H and L, respectively; arrows). In contrast, in neurons containing pPERK-positive granules a diffuse staining for AT100 or AT270 could be detected. These data indicate that UPR activation increases with tau-associated pathology. However, pPERK can hardly be detected in association with NFTs, suggesting an inverse correlation between UPR activation and the presence of NFTs. This indicates that the UPR is activated at a pretangle stage in pyramidal neurons in the hippocampus.
      Figure thumbnail gr5
      Figure 5Double immunohistochemistry and spectral imaging analyses for pPERK and phosphorylated tau protein. A: Double immunolabeling for pPERK (brown) and AT8 (red). B and C: Immunohistochemical signals were spectrally unmixed and shown as red for AT8 and green for pPERK. D: Merge of these signals indicates no spatial co-localization. Arrow indicates the absence of pPERK in tangle-bearing neuron. E: Double immunohistochemistry for pPERK (brown) and AT100 (red). F and G: Immunohistochemical signals for AT8 (red) and pPERK (green) were spectrally unmixed. H: Merge of signals for pPERK and AT100 indicate no co-occurrence of pPERK and AT100 in tangle-bearing neuron (arrow). I: Double immunolabeling for pPERK (brown) and AT270 (red). J and K: Immunohistochemical signals for AT270 (red) and pPERK (green) were spectrally unmixed. L: Merge of signal for AT270 and pPERK. Arrow indicates the low amount of pPERK in tangle-bearing neuron. Sections were counterstained with hematoxylin indicated as blue color in merged pictures.

      Co-Localization of pPERK with GSK-3β in AD Hippocampus

      In vitro studies have shown that activation of the UPR induces the activity of the major tau kinase GSK-3β, providing a connection between ER stress and phosphorylation of tau.
      • Song L
      • De Sarno P
      • Jope RS
      Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation.
      In addition, it has been described previously that GSK-3β and phosphorylated GSK-3β are associated with GVD.
      • Leroy K
      • Boutajangout A
      • Authelet M
      • Woodgett JR
      • Anderton BH
      • Brion JP
      The active form of glycogen synthase kinase-3beta is associated with granulovacuolar degeneration in neurons in Alzheimer's disease.
      With specific antibodies directed against GSK-3β and GSK-3β pSer9 (inactive form) the co-localization with pPERK in neurons was analyzed. Both GSK-3β and GSK-3β pSer9 were present as granules in GVD-containing neurons (Figure 6). Almost all neurons with pPERK-positive GVD granules also contained GSK-3β and GSK-3β pSer9 immunoreactivity (Figure 6, A and B). GSK-3β-positive granules could be identified as GVD granules, however GSK-3β in these granules did not co-localize with pPERK (Figure 6, C–F; arrow). GSK-3β pSer9 was detected in granules in neurons, however these granules could morphologically not be identified as GVD granules (Figure 6, G–J). Both markers occurred as granules in the same neuron. No co-localization was observed between GSK-3β pSer9 and pPERK inside these granules.
      Figure thumbnail gr6
      Figure 6Double immunohistochemistry and spectral imaging analyses for pPERK, GSK-3β, and pGSK-3β. A: Double immunohistochemistry for pPERK (red) and GSK-3β (brown). B: Double immunohistochemistry for pPERK (red) and pGSK-3β (brown). Immunohistochemical signals for pPERK (red) and GSK-3β (brown) (C) were spectrally unmixed in green for GSK-3β (D) and red for pPERK (E). F: Colors were merged with the addition of spectrally unmixed hematoxylin staining indicated in blue. Arrow indicates that GSK-3β in these granules does not co-localize with pPERK. Immunohistochemical signals for pPERK (red) and pGSK-3β (brown) (G) were spectrally unmixed in red for pGSK-3β (H) and green for pPERK (I). J: Colors were merged with the addition of spectrally unmixed hematoxylin staining indicated in blue. Scale bars: 50 μm (A, B); 30 μm (C–J).
      The percentages of GSK-3β- and GSK-3β pSer9-positive neurons in the different subregions of the hippocampus in controls and AD cases were determined. Both markers are significantly increased in Braak stages 3 to 4 and 5 to 6 in the CA1 and subiculum as compared with the levels in these regions in Braak stage 0 (Figure 7, A and B). The percentages of GSK-3β- and GSK-3β pSer9-immunoreactive neurons show a similar distribution along the Braak stages as neurons immunoreactive for pPERK and AT8 (Figure 4, A and B). Both GSK-3β- and GSK-3β pSer9-immunoreactive neurons show a significant correlation with the number of neurons that are pPERK-positive (Table 3).
      Figure thumbnail gr7
      Figure 7GSK-3β and GSK-3β pSer9 immunoreactivity in different stages of pathology. GSK-3β immunoreactivity (A) and GSK-3β pSer9 immunoreactivity (B) in different regions of the hippocampus increases with the Braak score for NFT pathology. Data are expressed as mean ± SEM. *P < 0.05 compared with Braak stage 0 in the same region.

      Discussion

      In this study we have shown that UPR activation occurs early during neurofibrillary degeneration. Previously we have shown that the UPR is activated in AD brain. Increased levels of BiP, indicative of UPR activation,
      • Kozutsumi Y
      • Segal M
      • Normington K
      • Gething MJ
      • Sambrook J
      The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins.
      are present in temporal cortex and hippocampus in AD cases compared with nondemented control cases.
      • Hoozemans JJ
      • Veerhuis R
      • Van Haastert ES
      • Rozemuller JM
      • Baas F
      • Eikelenboom P
      • Scheper W
      The unfolded protein response is activated in Alzheimer's disease.
      Because protein expression levels of BiP are increased at an early stage of AD pathology in the temporal cortex, it is suggested that the UPR is involved in AD pathology before the widespread occurrence of neurofibrillary degeneration. pPERK is present in pyramidal neurons in AD hippocampus and almost absent in neurons in neocortical areas.
      • Hoozemans JJ
      • Veerhuis R
      • Van Haastert ES
      • Rozemuller JM
      • Baas F
      • Eikelenboom P
      • Scheper W
      The unfolded protein response is activated in Alzheimer's disease.
      The hippocampus is affected by neurofibrillary degeneration at an earlier pathological stage compared with neocortical areas.
      • Braak H
      • Braak E
      Neuropathological stageing of Alzheimer-related changes.
      These findings support the suggestion that UPR activation in the hippocampus is involved with neurofibrillary degeneration in AD.
      Immunoreactivity for pPERK, peIF2α, and pIRE1α was observed as a granular staining. These granules could morphologically be characterized as GVD. GVD is characterized by basophilic granules surrounded by a clear zone measuring 1 to 5 μm in diameter. GVD is primarily found within pyramidal cells of the hippocampus. It is reported that GVD occurs in adult control brains and increases slightly with increasing age, however the occurrence of GVD in AD brain is increased compared with age-matched control brain.
      • Ball MJ
      • Lo P
      Granulovacuolar degeneration in the ageing brain and in dementia.
      • Xu M
      • Shibayama H
      • Kobayashi H
      • Yamada K
      • Ishihara R
      • Zhao P
      • Takeuchi T
      • Yoshida K
      • Inagaki T
      • Nokura K
      Granulovacuolar degeneration in the hippocampal cortex of aging and demented patients—a quantitative study.
      In line with these reports, we found that the percentage of neurons immunoreactive for pPERK is increased in AD cases as compared with control cases. Although the molecular events in GVD-containing neurons are poorly understood, histochemical and ultrastructural observations suggest that GVD might correspond to a special type of autophagosome.
      • Okamoto K
      • Hirai S
      • Iizuka T
      • Yanagisawa T
      • Watanabe M
      Reexamination of granulovacuolar degeneration.
      The occurrence of UPR activation markers pPERK, pIRE1α, and peIF2α in GVD granules is interesting in the light of reports indicating that ER stress triggers or mediates autophagy.
      • Yorimitsu T
      • Nair U
      • Yang Z
      • Klionsky DJ
      ER stress triggers autophagy.
      • Li J
      • Ni M
      • Lee B
      • Barron E
      • Hinton DR
      • Lee AS
      The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells.
      • Bernales S
      • McDonald KL
      • Walter P
      Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response.
      There are indications that autophagosomes selectively include the ER membrane and that the ER can serve as a membrane source for autophagosome formation.
      • Bernales S
      • McDonald KL
      • Walter P
      Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response.
      • Bernales S
      • Schuck S
      • Walter P
      ER-phagy: selective autophagy of the endoplasmic reticulum.
      This could explain the presence of UPR activation markers in GVD granules. The sequestration of unfolded proteins by autophagy may help to protect the neurons against the toxic build-up of unfolded proteins in the ER.
      Although it is assumed that GVD inclusions are ubiquitin-positive, initial reports on the localization of ubiquitin in GVD granules are contradictory.
      • Okamoto K
      • Hirai S
      • Iizuka T
      • Yanagisawa T
      • Watanabe M
      Reexamination of granulovacuolar degeneration.
      • Manetto V
      • Perry G
      • Tabaton M
      • Mulvihill P
      • Fried VA
      • Smith HT
      • Gambetti P
      • Autilio-Gambetti L
      Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases.
      • Love S
      • Saitoh T
      • Quijada S
      • Cole GM
      • Terry RD
      Alz-50, ubiquitin and tau immunoreactivity of neurofibrillary tangles, Pick bodies and Lewy bodies.
      Also, in the present study no ubiquitin immunoreactivity was observed in GVD granules. However, granules immunoreactive for pPERK were present in neurons with ubiquitin inclusions. In addition we observed that pPERK-immunoreactive neurons were associated with extracellular ubiquitin granules, which have been reported previously as perisomatic granules.
      • Probst A
      • Herzig MC
      • Mistl C
      • Ipsen S
      • Tolnay M
      Perisomatic granules (non-plaque dystrophic dendrites) of hippocampal CA1 neurons in Alzheimer's disease and Pick's disease: a lesion distinct from granulovacuolar degeneration.
      Perisomatic granules are preferentially associated with pretangle neurons and start to occur as from Braak stage 3. It is suggested that perisomatic granules represent degenerating perisomatic neurites and are a pathological entity distinct from GVD.
      • Probst A
      • Herzig MC
      • Mistl C
      • Ipsen S
      • Tolnay M
      Perisomatic granules (non-plaque dystrophic dendrites) of hippocampal CA1 neurons in Alzheimer's disease and Pick's disease: a lesion distinct from granulovacuolar degeneration.
      P62 is a ubiquitin-binding protein and is involved in the accumulation of ubiquitin-conjugated proteins into cytoplasmic inclusions.
      • Kuusisto E
      • Salminen A
      • Alafuzoff I
      Ubiquitin-binding protein p62 is present in neuronal and glial inclusions in human tauopathies and synucleinopathies.
      It has been reported that p62 appears early during neurofibrillary pathogenesis.
      • Kuusisto E
      • Salminen A
      • Alafuzoff I
      Early accumulation of p62 in neurofibrillary tangles in Alzheimer's disease: possible role in tangle formation.
      P62 is found in several types of pathological inclusions composed of phosphorylated tau or α-synuclein, which suggests that p62 might contribute to the aggregation process. Because p62 has been reported to interact with several signaling proteins,
      • Joung I
      • Strominger JL
      • Shin J
      Molecular cloning of a phosphotyrosine-independent ligand of the p56lck SH2 domain.
      • Puls A
      • Schmidt S
      • Grawe F
      • Stabel S
      Interaction of protein kinase C zeta with ZIP, a novel protein kinase C-binding protein.
      • Sudo T
      • Maruyama M
      • Osada H
      p62 functions as a p38 MAP kinase regulator.
      • Wooten MW
      • Seibenhener ML
      • Mamidipudi V
      • Diaz-Meco MT
      • Barker PA
      • Moscat J
      The atypical protein kinase C-interacting protein p62 is a scaffold for NF-kappaB activation by nerve growth factor.
      entrapment of p62 into NFTs could disrupt vital signaling pathways, thereby contributing to dysfunction or death of NFT-containing neurons. In this study we found that pPERK was associated with small ubiquitin-positive inclusions and diffuse p62 immunoreactivity, whereas pPERK was absent from neurons containing ubiquitin or p62-immunoreactive tangle-bearing neurons. These data suggest that activation of the UPR is co-occurring with early UPS activation in pretangle neurons.
      The number of pPERK-positive neurons in the hippocampus is increased in Braak stages 3 to 4 for NFTs and correlates with the number of neurons immunoreactive for phosphorylated tau. pPERK immunoreactivity is observed in neurons containing diffuse phosphorylated tau and is almost absent from tangle-bearing neurons. These data suggest that UPR activation occurs in pretangle neurons. Because postmortem studies are limited in studying the temporal sequence of events, we cannot exclude the possibility that UPR activation occurs in neurons that will not form NFTs. Recent studies on tau transgenic mouse models indicate that diffusible tau contributes to disease pathology.
      • Andorfer C
      • Acker CM
      • Kress Y
      • Hof PR
      • Duff K
      • Davies P
      Cell-cycle reentry and cell death in transgenic mice expressing nonmutant human tau isoforms.
      • Santacruz K
      • Lewis J
      • Spires T
      • Paulson J
      • Kotilinek L
      • Ingelsson M
      • Guimaraes A
      • DeTure M
      • Ramsden M
      • McGowan E
      • Forster C
      • Yue M
      • Orne J
      • Janus C
      • Mariash A
      • Kuskowski M
      • Hyman B
      • Hutton M
      • Ashe KH
      Tau suppression in a neurodegenerative mouse model improves memory function.
      • Yoshiyama Y
      • Higuchi M
      • Zhang B
      • Huang SM
      • Iwata N
      • Saido TC
      • Maeda J
      • Suhara T
      • Trojanowski JQ
      • Lee VM
      Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model.
      Data from these studies imply that NFTs themselves do not lead to synaptic dysfunction and neurotoxicity, but that nonfibrillar tau is responsible for these events.
      • Brunden KR
      • Trojanowski JQ
      • Lee VM
      Evidence that non-fibrillar tau causes pathology linked to neurodegeneration and behavioral impairments.
      In light of these studies it is interesting to find activation of the UPR, as detected by pPERK immunostaining, co-occurring with diffuse nonfibrillar phosphorylated tau. The presence of diffusible tau or nonfibrillar tau aggregates may present a highly stressful situation for the neuron, whereas the deposition of tau into insoluble aggregates may serve as a mechanism to reduce the levels of the toxic soluble species. This could explain why tangle-bearing neurons show no or low levels of pPERK immunoreactivity.
      Although several reports have shown that the UPR is activated in AD,
      • Hoozemans JJ
      • Veerhuis R
      • Van Haastert ES
      • Rozemuller JM
      • Baas F
      • Eikelenboom P
      • Scheper W
      The unfolded protein response is activated in Alzheimer's disease.
      • Hamos JE
      • Oblas B
      • Pulaski-Salo D
      • Welch WJ
      • Bole DG
      • Drachman DA
      Expression of heat shock proteins in Alzheimer's disease.
      • Unterberger U
      • Hoftberger R
      • Gelpi E
      • Flicker H
      • Budka H
      • Voigtlander T
      Endoplasmic reticulum stress features are prominent in Alzheimer disease but not in prion diseases in vivo.
      it is still elusive what triggers UPR activation in AD neurons. Amyloid plaques are not located in the ER, however in vitro studies have demonstrated that Aβ1–42 can be generated in the ER of neuronal cells.
      • Cook DG
      • Forman MS
      • Sung JC
      • Leight S
      • Kolson DL
      • Iwatsubo T
      • Lee VM
      • Doms RW
      Alzheimer's A beta(1–42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells.
      • Hartmann T
      • Bieger SC
      • Bruhl B
      • Tienari PJ
      • Ida N
      • Allsop D
      • Roberts GW
      • Masters CL
      • Dotti CG
      • Unsicker K
      • Beyreuther K
      Distinct sites of intracellular production for Alzheimer's disease A beta40/42 amyloid peptides.
      Extracellular oligomeric aggregates of Aβ1–42 enhance the ER stress response and intracellular Aβ1–42 sensitizes neuroblastoma cells for ER stress toxicity.
      • Chafekar SM
      • Hoozemans JJ
      • Zwart R
      • Baas F
      • Scheper W
      Abeta 1–42 induces mild endoplasmic reticulum stress in an aggregation state-dependent manner.
      • Chafekar SM
      • Zwart R
      • Veerhuis R
      • Vanderstichele H
      • Baas F
      • Scheper W
      Increased Abeta1–42 production sensitizes neuroblastoma cells for ER stress toxicity.
      Oligomeric aggregates of Aβ1–42 are readily internalized
      • Chafekar SM
      • Baas F
      • Scheper W
      Oligomer-specific Abeta toxicity in cell models is mediated by selective uptake.
      and induce Ca2+ release from the ER.
      • Suen KC
      • Yu MS
      • So KF
      • Chang RC
      • Hugon J
      Upstream signaling pathways leading to the activation of double-stranded RNA-dependent serine/threonine protein kinase in beta-amyloid peptide neurotoxicity.
      Disturbance of Ca2+ homeostasis in the ER is a well known cause of UPR activation. In models for α-synuclein pathology, ER stress is caused by inhibition of protein trafficking from the ER.
      • Cooper AA
      • Gitler AD
      • Cashikar A
      • Haynes CM
      • Hill KJ
      • Bhullar B
      • Liu K
      • Xu K
      • Strathearn KE
      • Liu F
      • Cao S
      • Caldwell KA
      • Caldwell GA
      • Marsischky G
      • Kolodner RD
      • Labaer J
      • Rochet JC
      • Bonini NM
      • Lindquist S
      Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.
      Interestingly, increased levels of the microtubule-binding protein tau inhibits anterograde vesicle transport in neurons.
      • Stamer K
      • Vogel R
      • Thies E
      • Mandelkow E
      • Mandelkow EM
      Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress.
      The resulting accumulation of proteins in the cell body is likely to induce stress in the ER. In addition, disturbances in the degradation machinery may contribute to ER stress and UPR activation. Decreased proteasomal activity is found in specific brain regions of AD patients
      • Keller JN
      • Hanni KB
      • Markesbery WR
      Impaired proteasome function in Alzheimer's disease.
      and neurons affected by neurofibrillary pathology accumulate immature forms of autophagosomes indicating impaired transport and maturation of autophagosomes.
      • Nixon RA
      • Wegiel J
      • Kumar A
      • Yu WH
      • Peterhoff C
      • Cataldo A
      • Cuervo AM
      Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study.
      Beclin 1 has a key role in autophagy and is decreased in affected brain regions of patients with AD,
      • Pickford F
      • Masliah E
      • Britschgi M
      • Lucin K
      • Narasimhan R
      • Jaeger PA
      • Small S
      • Spencer B
      • Rockenstein E
      • Levine B
      • Wyss-Coray T
      The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice.
      which indicates impaired autophagy in AD neurons. In case of proteasome impairment or of increased demand for degradation during ER stress, autophagy is triggered, indicating that the two degradation pathways used by the ER are tightly coupled.
      • Iwata A
      • Riley BE
      • Johnston JA
      • Kopito RR
      HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin.
      • Iwata A
      • Christianson JC
      • Bucci M
      • Ellerby LM
      • Nukina N
      • Forno LS
      • Kopito RR
      Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation.
      • Ding WX
      • Ni HM
      • Gao W
      • Yoshimori T
      • Stolz DB
      • Ron D
      • Yin XM
      Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability.
      More importantly, autophagy protects against ER stress toxicity.
      • Ding WX
      • Ni HM
      • Gao W
      • Yoshimori T
      • Stolz DB
      • Ron D
      • Yin XM
      Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability.
      • Ding WX
      • Ni HM
      • Gao W
      • Hou YF
      • Melan MA
      • Chen X
      • Stolz DB
      • Shao ZM
      • Yin XM
      Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival.
      The overall reduction in protein degradation in AD neurons could induce ER stress and promote UPR activation.
      In vitro studies have shown that activation of the UPR induces the activity of GSK-3β.
      • Kim AJ
      • Shi Y
      • Austin RC
      • Werstuck GH
      Valproate protects cells from ER stress-induced lipid accumulation and apoptosis by inhibiting glycogen synthase kinase-3.
      • Song L
      • De Sarno P
      • Jope RS
      Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation.
      GSK-3β is involved in a remarkably broad spectrum of AD-associated events, especially in the phosphorylation of tau, a prerequisite for the formation of NFTs. This suggests that UPR activation in neurons might directly contribute to tau phosphorylation. In this study we demonstrate a striking co-occurrence of GSK-3β- and pPERK-immunoreactive granules in AD neurons, and in addition, a strong correlation between the occurrence of pPERK-immunoreactive neurons with GSK-3β- or GSK-3β pSer9-immunoreactive neurons was observed. These data support the hypothesis that UPR activation is related to GSK-3β activity as reported in previous in vitro studies. However, a direct co-localization of pPERK and GSK-3β was not observed. This is in agreement with a previous report, which describes the localization of GSK-3β in a part of all GVD granules and the absence of GSK-3β pSer9 in GVD granules.
      • Leroy K
      • Boutajangout A
      • Authelet M
      • Woodgett JR
      • Anderton BH
      • Brion JP
      The active form of glycogen synthase kinase-3beta is associated with granulovacuolar degeneration in neurons in Alzheimer's disease.
      GSK-3β is no direct substrate for pPERK, making co-localization between PERK and GSK-3β not a prerequisite for a functional connection. Although underlying mechanisms remain elusive, there is considerable evidence from in vitro studies that GSK-3β promotes intrinsic apoptotic signaling.
      • Kim AJ
      • Shi Y
      • Austin RC
      • Werstuck GH
      Valproate protects cells from ER stress-induced lipid accumulation and apoptosis by inhibiting glycogen synthase kinase-3.
      • Song L
      • De Sarno P
      • Jope RS
      Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation.
      The presence of GSK-3β in granules could represent an attempt to sequester GSK-3β, to prevent further tau phosphorylation and promote cell survival.
      • Jope RS
      • Johnson GV
      The glamour and gloom of glycogen synthase kinase-3.
      • Leroy K
      • Boutajangout A
      • Authelet M
      • Woodgett JR
      • Anderton BH
      • Brion JP
      The active form of glycogen synthase kinase-3beta is associated with granulovacuolar degeneration in neurons in Alzheimer's disease.
      In conclusion, UPR activation markers pPERK, pIRE1α, and peIF2α are observed in granules associated with GVD in pyramidal neurons in the hippocampus of AD patients. The co-occurrence of pPERK with diffuse phosphorylated tau and markers for pretangles indicates that UPR activation occurs early during neurofibrillary degeneration. The direct connection between UPR activation and GSK-3β activation as previously shown in vitro is supported by data derived from human AD brain tissue. These data support the hypothesis that UPR activation and GVD are involved at the early stages of tau phosphorylation. The initial activation of the UPR in AD neurons might work as neuroprotective to restore homeostasis and increase neuronal survival. Sustained activation of the UPR in AD neurons could accelerate phosphorylation of tau through activation of GSK-3β. Future studies need to address the therapeutic opportunities of this pathway for the treatment of AD and other tauopathies.

      Acknowledgements

      We thank the Netherlands Brain Bank for supplying the human brain tissue, Dr. Wouter Kamphorst for the neuropathological diagnosis of control and AD tissue, Dr. Chris van der Loos for assistance with the spectral imaging system, and Mr. Jaap van Veldhuisen and Mr. Ron Otsen for their assistance with the preparation of the figures.

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