The Unfolded Protein Response Is Activated in Pretangle Neurons in Alzheimer's Disease Hippocampus
2009; Elsevier BV; Volume: 174; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.080814
ISSN1525-2191
AutoresJeroen J.M. Hoozemans, Elise S. van Haastert, Diana A.T. Nijholt, Annemieke J.M. Rozemüller, Piet Eikelenboom, Wiep Scheper,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoAccumulation 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. 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.1Forman MS Lee VM Trojanowski JQ ‘Unfolding’ pathways in neurodegenerative disease.Trends Neurosci. 2003; 26: 407-410Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 2Rutkowski DT Kaufman RJ A trip to the ER: coping with stress.Trends Cell Biol. 2004; 14: 20-28Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar Under physiological conditions the ER chaperone BiP (or GRP78) is bound to three key proteins in the ER membrane: pancreatic ER kinase (PERK),3Harding HP Zhang Y Ron D Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase.Nature. 1999; 397: 271-274Crossref PubMed Scopus (2533) Google Scholar 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.4Kozutsumi 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.Nature. 1988; 332: 462-464Crossref PubMed Scopus (988) Google Scholar 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.5Hoozemans JJ Veerhuis R Van Haastert ES Rozemuller JM Baas F Eikelenboom P Scheper W The unfolded protein response is activated in Alzheimer's disease.Acta Neuropathol (Berl). 2005; 110: 165-172Crossref PubMed Scopus (434) Google Scholar 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.6Chafekar SM Hoozemans JJ Zwart R Baas F Scheper W Abeta 1–42 induces mild endoplasmic reticulum stress in an aggregation state-dependent manner.Antioxid Redox Signal. 2007; 9: 2245-2254Crossref PubMed Scopus (81) Google Scholar In addition, our data show that increased intracellular Aβ production, in particular Aβ1–42, enhances UPR induction and ER stress toxicity.7Chafekar SM Zwart R Veerhuis R Vanderstichele H Baas F Scheper W Increased Abeta1–42 production sensitizes neuroblastoma cells for ER stress toxicity.Curr Alzheimer Res. 2008; 5: 469-474Crossref PubMed Scopus (36) Google Scholar 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).8Kim AJ Shi Y Austin RC Werstuck GH Valproate protects cells from ER stress-induced lipid accumulation and apoptosis by inhibiting glycogen synthase kinase-3.J Cell Sci. 2005; 118: 89-99Crossref PubMed Scopus (235) Google Scholar, 9Song L De Sarno P Jope RS Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation.J Biol Chem. 2002; 277: 44701-44708Crossref PubMed Scopus (252) Google Scholar GSK-3β is the major kinase for tau and considered to be involved in the hyperphosphorylation of tau present in NFTs.10Jope RS Johnson GV The glamour and gloom of glycogen synthase kinase-3.Trends Biochem Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1331) Google Scholar 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.11Braak H Braak E Neuropathological stageing of Alzheimer-related changes.Acta Neuropathol (Berl). 1991; 82: 239-259Crossref PubMed Scopus (11641) Google Scholar Also the UPR markers are prominently present in the separate subregions of the hippocampus5Hoozemans JJ Veerhuis R Van Haastert ES Rozemuller JM Baas F Eikelenboom P Scheper W The unfolded protein response is activated in Alzheimer's disease.Acta Neuropathol (Berl). 2005; 110: 165-172Crossref PubMed Scopus (434) Google Scholar 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. 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.12Reisberg B Ferris SH de Leon MJ Crook T The Global Deterioration Scale for assessment of primary degenerative dementia.Am J Psychiatry. 1982; 139: 1136-1139PubMed Google Scholar 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.13Yamaguchi 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.Acta Neuropathol (Berl). 1990; 79: 569-572Crossref PubMed Scopus (86) Google Scholar Staging of AD was evaluated according to Braak and colleagues.11Braak H Braak E Neuropathological stageing of Alzheimer-related changes.Acta Neuropathol (Berl). 1991; 82: 239-259Crossref PubMed Scopus (11641) Google Scholar, 14Braak H Alafuzoff I Arzberger T Kretzschmar H Del Tredici K Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry.Acta Neuropathol. 2006; 112: 389-404Crossref PubMed Scopus (1849) Google Scholar 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 StudyCase no.Pathological diagnosisSexAgeBraak stage1CONM8002CONM8003CONF7304CONM5715CONF7716CONM7317CONF7618CONM9029CONF81210CONM87211CONM85212CONM74313CONM83414CONF94415CONF97416ADM86317ADM86418ADM64419ADF75520ADM87521ADF78522ADM64523ADF62524ADF69625ADM57626ADF72627AD/LBVF71328AD/LBVF95429AD/LBVM75430AD/LBVF70631AD/LBVM65632DEM/VASCF89333DEM/VASCM80334DEM/VASCF903CON, 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. Open table in a new tab 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. 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).15Sokka 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.J Neurosci. 2007; 27: 901-908Crossref PubMed Scopus (266) Google Scholar, 16Luo 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.J Biol Chem. 2008; 283: 11905-11912Crossref PubMed Scopus (99) Google Scholar, 17Hoozemans JJ Van Haastert ES Eikelenboom P de Vos RA Rozemuller JM Scheper W Activation of the unfolded protein response in Parkinson's disease.Biochem Biophys Res Commun. 2007; 354: 707-711Crossref PubMed Scopus (370) Google Scholar, 18van 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.J Neuropathol Exp Neurol. 2005; 64: 770-775Crossref PubMed Scopus (85) Google Scholar, 19Spillantini MG Crowther RA Goedert M Comparison of the neurofibrillary pathology in Alzheimer's disease and familial presenile dementia with tangles.Acta Neuropathol. 1996; 92: 42-48Crossref PubMed Scopus (149) Google Scholar, 20Dickson DW Required techniques and useful molecular markers in the neuropathologic diagnosis of neurodegenerative diseases.Acta Neuropathol. 2005; 109: 14-24Crossref PubMed Scopus (59) Google Scholar, 21Kuusisto E Salminen A Alafuzoff I Ubiquitin-binding protein p62 is present in neuronal and glial inclusions in human tauopathies and synucleinopathies.Neuroreport. 2001; 12: 2085-2090Crossref PubMed Scopus (292) Google Scholar, 22Kuusisto E Salminen A Alafuzoff I Early accumulation of p62 in neurofibrillary tangles in Alzheimer's disease: possible role in tangle formation.Neuropathol Appl Neurobiol. 2002; 28: 228-237Crossref PubMed Scopus (165) Google Scholar, 23Leroy 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.Acta Neuropathol (Berl). 2002; 103: 91-99Crossref PubMed Scopus (171) Google Scholar 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 StudyName, typeEpitope (immunogen)SourceReferencespPERKPERK phosphorylated at threonine 981Santa Cruz Biotechnology, Santa Cruz, CA15Sokka 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.J Neurosci. 2007; 27: 901-908Crossref PubMed Scopus (266) Google Scholar, 16Luo 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.J Biol Chem. 2008; 283: 11905-11912Crossref PubMed Scopus (99) Google Scholar, 17Hoozemans JJ Van Haastert ES Eikelenboom P de Vos RA Rozemuller JM Scheper W Activation of the unfolded protein response in Parkinson's disease.Biochem Biophys Res Commun. 2007; 354: 707-711Crossref PubMed Scopus (370) Google ScholarpIRE1αIRE1α phosphorylated at serine 724Novus Biologicals, Littleton, CO16Luo 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.J Biol Chem. 2008; 283: 11905-11912Crossref PubMed Scopus (99) Google ScholarpeIF2αeIF-2α phosphorylated at serine 52Sigma, St. Louis, MO17Hoozemans JJ Van Haastert ES Eikelenboom P de Vos RA Rozemuller JM Scheper W Activation of the unfolded protein response in Parkinson's disease.Biochem Biophys Res Commun. 2007; 354: 707-711Crossref PubMed Scopus (370) Google Scholar, 18van 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.J Neuropathol Exp Neurol. 2005; 64: 770-775Crossref PubMed Scopus (85) Google ScholarAT8Tau phosphorylated at serine 202Innogenetics, Ghent, Belgium19Spillantini MG Crowther RA Goedert M Comparison of the neurofibrillary pathology in Alzheimer's disease and familial presenile dementia with tangles.Acta Neuropathol. 1996; 92: 42-48Crossref PubMed Scopus (149) Google ScholarAT100Tau phosphorylated at serine 212 and threonine 214Pierce, Rockford, IL19Spillantini MG Crowther RA Goedert M Comparison of the neurofibrillary pathology in Alzheimer's disease and familial presenile dementia with tangles.Acta Neuropathol. 1996; 92: 42-48Crossref PubMed Scopus (149) Google ScholarAT270Tau phosphorylated at threonine 181Pierce19Spillantini MG Crowther RA Goedert M Comparison of the neurofibrillary pathology in Alzheimer's disease and familial presenile dementia with tangles.Acta Neuropathol. 1996; 92: 42-48Crossref PubMed Scopus (149) Google ScholarUbiquitinUbiquitinChemicon, Temecula, CA20Dickson DW Required techniques and useful molecular markers in the neuropathologic diagnosis of neurodegenerative diseases.Acta Neuropathol. 2005; 109: 14-24Crossref PubMed Scopus (59) Google Scholarp62p62 proteinProgen Biotechnik, Heidelberg, Germany21Kuusisto E Salminen A Alafuzoff I Ubiquitin-binding protein p62 is present in neuronal and glial inclusions in human tauopathies and synucleinopathies.Neuroreport. 2001; 12: 2085-2090Crossref PubMed Scopus (292) Google Scholar, 22Kuusisto E Salminen A Alafuzoff I Early accumulation of p62 in neurofibrillary tangles in Alzheimer's disease: possible role in tangle formation.Neuropathol Appl Neurobiol. 2002; 28: 228-237Crossref PubMed Scopus (165) Google ScholarGSK-3βGSK-3βBD Biosciences, San Jose, CA23Leroy 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.Acta Neuropathol (Berl). 2002; 103: 91-99Crossref PubMed Scopus (171) Google ScholarGSK-3β pSer9GSK-3β phosphorylated at serine 9Cell Signaling, Danvers, MA23Leroy 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.Acta Neuropathol (Berl). 2002; 103: 91-99Crossref PubMed Scopus (171) Google Scholar Open table in a new tab 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. 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. 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). 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. 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. 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.24Levenson RM Mansfield JR Multispectral imaging in biology and medicine: slices of life.Cytometry A. 2006; 69: 748-758Crossref PubMed Scopus (228) Google Scholar 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. The immunohistochemical localization of pPERK, peIF2α, and pIRE1α was investigated in the hippocampus of AD and nondemented controls. As described previously,5Hoozemans JJ Veerhuis R Van Haastert ES Rozemuller JM Baas F Eikelenboom P Scheper W The unfolded protein response is activated in Alzheimer's disease.Acta Neuropathol (Berl). 2005; 110: 165-172Crossref PubMed Scopus (434) Google Scholar 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.25Okamoto K Hirai S Iizuka T Yanagisawa T Watanabe M Reexamination of granulovacuolar degeneration.Acta Neuropathol (Berl). 1991; 82: 340-345Crossref PubMed Scopus (107) Google Scholar 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. 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). Previously, we reported that UPR activation is increased in Parkinson's disease with α-synuclein pathology in the substantia nigra.17Hoozemans JJ Van Haastert ES Eikelenboom P de Vos RA Rozemuller JM Scheper W Activation of the unfolded protein response in Parkinson's disease.Biochem Biophys Res Commun. 2007; 354: 707-711Crossref PubMed Scopus (370) Google Scholar 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). Because GVD inclusions have been reported to be immunoreactive for ubiquitin,25Okamoto K Hirai S Iizuka T Yanagisawa T Watanabe M Reexamination of granulovacuolar degeneration.Acta Neuropathol (Berl). 1991; 82: 340
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