Protein disulfide isomerase-immunopositive inclusions in patients with Alzheimer disease

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Protein disulfide isomerase-immunopositive inclusions in patients with Alzheimer disease Yasuyuki Honjoa,b,⁎, Hidefumi Itob , Tomohisa Horibea , Ryosuke Takahashib , Koji Kawakamia a

Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Japan Department of Neurology, Facility of Medicine, Kyoto University, Japan

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Alzheimer disease (AD) is the most common neurodegenerative disease, but there is

Accepted 7 June 2010

currently no effective treatment available because the etiology or mechanism of AD is still

Available online 12 June 2010

unclear. Many neurodegenerative diseases feature inclusions, which contain accumulations of misfolded, aggregated proteins. Amyloid plaques and neurofibrillary

Keywords:

tangles (NFTs) are the major pathological hallmarks of AD. NFTs are composed of tubular

endoplasmic reticulum stress

filaments, and paired helical filaments containing polymerized hyperphosphorylated tau

misfolded protein

protein. Another feature is excessive generation of nitric oxide synthetase, reactive

tau

nitrogen species, and reactive oxygen species. Protein disulfide isomerase (PDI) is a member of the thioredoxin (TX) superfamily and is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding in the luminal space of the endoplasmic reticulum (ER). Nitric oxide (NO)-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response in neurodegenerative diseases. In this study, we found NFTs positive for anti-PDI-antibody in the brain of patients with AD. As far as we know, this is the first report of anti-PDI-antibody-immunopositive inclusions in AD. In AD, NO may inhibit PDI by inducing S-nitrosylation, which inhibits its enzymatic activity and thus allows protein misfolding to occur. Consequently, the accumulation of misfolded proteins induces ER stress. The ER stress can cause apoptosis of neuronal cells. These results suggest that PDI could be a therapeutic target to prevent ER stress in neuronal cells in AD. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

The morphology of Alzheimer disease (AD) includes cerebral atrophy, deposition of β-amyloid (senile plaques), and neuritic changes like neurofibrillary tangles (NFTs). Tau, which is normally localized to neurons as a microtubule-associated

protein, is abnormally phosphorylated and deposited in NFTs (Morishima-Kawashima and Ihara, 2002). Furthermore, the contribution of NFTs is a link to the clinical progressive stage of AD (Braak and Braak, 1991). In senile plaques, dystrophic neurites contain tau protein (Cras et al., 1991; Krüger et al., 2000). Ultrastructural studies have shown that NFTs are

⁎ Corresponding author. Yoshida Konoecho, Sakyo-ku, Kyoto 606-8501, Japan. Fax: +81 75 753 4469. E-mail address: [email protected] (Y. Honjo). Abbreviations: AD, Alzheimer disease; NFT, neurofibrillary tangle; PDI, protein disulfide isomerase 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.06.016

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Fig. 1 – Neurons in the frontal lobe of the normal control brain were immunopositive for PDI. Scale bar: 20 μm.

composed of tubular filaments and paired helical filaments (PHFs) containing polymerized hyperphosphorylated tau protein (Morishima-Kawashima and Ihara, 2002). Many neurodegenerative diseases are characterized by the accumulation of aggregated proteins, such as α-synuclein in Parkinson disease (PD) and multiple system atrophy (MSA) and TDP-43 in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (Goedert, 2001; Lenders et al., 1989; Nakamura and Lipton, 2009; Waza et al., 2005). These accumulated proteins contribute to the formation of inclusions and neuronal cell death (Nakamura and Lipton, 2009). Another feature of most neurodegenerative diseases is excessive generation of nitric oxide synthetase (NOS), reactive nitrogen species, and reactive oxygen species due to the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. Physiologically, the NMDA receptor can mediate post-synaptic Ca2+ influx, but if this is excessive it can cause excitotoxicity. NOS can contribute to neuronal cell injury and death in many neurodegenerative diseases (Nakamura and Lipton, 2009). In addition, inhibition of NOS activity ameliorates the progression of disease pathology in animal models of ALS, PD, and AD (Colton et al., 2008; Martin, 2007). The accumulation of misfolded, aggregated proteins and Ca2+ influx can cause endoplasmic reticulum (ER) stress in neurons. ER stress signaling, otherwise known as the unfolded

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protein response (UPR), is triggered by an increased load of misfolded proteins in the organelle. ER stress is linked with many neurodegenerative diseases, such as ALS and PD. ALS is a motor neuron disease and a mutation in superoxide dismutase-1 (SOD-1) causes familial ALS. In a SOD-1-mutant mouse model, the mutant SOD-1 protein was localized to the ER of motor neurons (Kikuchi et al., 2006). In addition, aggregated α-synuclein blocks ER-Golgi traffic in a mouse model of PD (Cooper et al., 2006). Protein disulfide isomerase (PDI) is a member of the thioredoxin (TX) superfamily and is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding in the luminal space of the ER (Noiva and Lennarz, 1992). Such exchange reactions can occur intramolecularly, leading to rearrangement of disulfide bonds in a single protein. Oxidized PDI can catalyze the formation of a disulfide bridge (Noiva and Lennarz, 1992). The second major function of PDI is as a chaperone to unfold cholera toxin. This is independent of its first function in disulfide exchange (Tsai et al., 2001). In sporadic AD and PD, nitric oxide (NO)-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to the accumulation of polyubiquitinated proteins (Uehara et al., 2006). Thus, PDI prevents the neurotoxicity associated with ER stress and misfolding (Uehara et al., 2006). Furthermore, nitrosative stress leads to S-nitrosylation of wild-type Parkin and regulates its E3 ubiqutin ligase activity (Yao et al., 2004). In addition, PDI protects against protein aggregation and is S-nitrosylated in ALS (Walker et al., 2010). Therefore, PDI could be inhibited by NO and this inhibition cause ER stress in neuronal cells in the brain of patients with AD. In this study, we identified anti-PDI-antibodyimmunopositive NFTs in the brain of patients with AD.

2.

Results

2.1. PDI-immunopositive neuronal cells in normal control brains In the control specimens, a lot of neurons were immunopositive for the anti-PDI antibody. PDI-immunoreactivity was typically observed in the neuronal bodies and dendrites, but nuclei were not stained. The neurons in the frontal lobe were

Fig. 2 – (A), (B) Anti-PDI-antibody-immunopositive NFTs (arrows) in the hippocampus of the AD brain. (C) The dystrophic neurites of senile plaque (arrow) were also PDI-immunopositive in the hippocampus of the AD. Scale bars: A = 50 μm, B and C = 20 μm.

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Fig. 3 – Double immunostaining of NFTs (arrow) in the hippocampus of the normal control brain. Green: anti-PDI antibody immunostaining (A). Red: anti-AT8 antibody immunostaining (B). Yellow: merged immunostaining (C). Scale bars: A–C = 10 μm.

immunostained by the anti-PDI antibody (Fig. 1). Moderately PDI-immunopositive small neurons were found in the caudate, putamen, globus pallidus, hypothalamus, pons, and neocortex layers II–V. In addition, the glial cells were PDI-immunopositive, but they were stained only weakly. In the tissue sections from the patients with AD, we detected numerous PDI-immunopositive NFTs (Fig. 2A, B). Furthermore, dystrophic neurites of senile plaques were also PDI-immunopositive in the brain of patients with AD (Fig. 2C). These PDI-immunopositive NFTs were observed in all patients with AD and were found in the

hippocampus and frontal lobe. In double staining of anti-PDI and anti-AT8 antibodies, we found the small number of PDIimmunopositive NFTs in normal control brains (Fig. 3). These PDI-immunopositive NFTs were scattered in normal control brains and the distribution of NFTs was consistent with previous findings (Guillozet et al., 2003; Braak and Braak, 1991). The proportion of PDI-immunopositive NFTs compared with AT8immunopositive NFTs was not markedly different between normal control and AD brains. Other immunoreactivity was not markedly different between the normal and AD brains.

Fig. 4 – Double immunostaining of NFTs (arrows) in the hippocampus from a patient with AD. Green: anti-PDI antibody immunostaining (A, D). Red: anti-AT8 antibody immunostaining (B, E). Yellow: merged immunostaining (C, F). Scale bars: A–C = 50 μm; D–F = 10 μm.

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Fig. 5 – Double immunostaining of NFTs in the hippocampus from a patient with AD. Green: anti-PDI antibody immunostaining (A). Red: anti-Alzheimer Tau antibody immunostaining (B). Yellow: merged immunostaining (C). Scale bars: A–C = 50 μm.

2.2. Double staining of PDI and NFT indicators in AD brains Immunohistochemical double staining for PDI and AT8 showed that the number of NFTs labeled by antibodies to AT8 was greater than the number of PDI-immunopositive NFTs (Fig. 4). To confirm the NFTs, we also used monoclonal mouse anti-Alzheimer's disease Tau clone DC11 (SigmaAldrich, Saint Louis, USA). Immunohistochemical double staining for PDI and Alzheimer Tau was not markedly different compare with double staining for PDI and AT8 (Fig. 5). A quantitative examination revealed that approximately 78 ± 11.0% of the AT8-immunopositive NFTs were also PDI-immunoreactive. The proportion of PDI-immunopositive NFTs compared with AT8-immunopositive NFTs was not markedly different among the brain regions assayed, or among patients.

3.

Discussion

AD is the most common neurodegenerative disease, but an effective treatment is not available currently because the etiology or mechanism of AD is still unclear. Many neurodegenerative diseases have cytoplasmic inclusions such as Lewy bodies in PD, glial cytoplasmic inclusions (GCIs) in MSA, Lewy body-like hyaline inclusions in ALS, and NFTs in AD. These inclusions contain aberrant accumulations of aggregated proteins (Braak and Braak, 1991; Goedert, 2001; Lenders et al., 1989; Nakamura and Lipton, 2009; Wang et al., 2008). These inclusions could be the key to elucidating the mechanism of neurodegenerative diseases. Amyloid plaques and NFTs are the major pathological hallmarks of AD. The Amyloid plaques are the pathological end-point of aberrant β-amyloid production (Braak and Braak, 1991). In this study, we found anti-PDI antibody immunopositive dystrophic neurites in amyloid plaques. Genetic causes of AD include mutations in β-amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2) (Kovacs et al., 1996). APP transgenic (APP-tg) mouse is commonly used as a model mouse for AD. This APP-tg mouse shows memory disturbance and accumulation of β-amyloid in the brain (Dodart et al.,

1999; Masliah et al., 1996). In addition, APP and PS1 doubletransgenic mice showed accelerated deposition of β-amyloid in the brain and memory disturbance (Holcomb et al., 1998). However, these mouse models have their drawbacks with respect to their use as models for the human disease. For instance, AD model mice generally show no remarkable neuronal loss in the hippocampus and cerebral cortex. Furthermore, NFTs are not clearly found in these mice. Another important pathological hallmark of AD is the NFTs. NFTs are composed of tubular filaments, and PHFs containing polymerized hyperphosphorylated tau protein (Braak and Braak, 1991). The distribution pattern and packing density of amyloid deposits turned out to be of limited significance for differentiation of neuropathological stages (Braak and Braak, 1991). In contrast, NFTs and neuropil threads exhibited a characteristic distribution pattern permitting the differentiation of six stages (called Braak stages) (Braak and Braak, 1991). Furthermore, there is a linear correlation between the Braak stage and clinical mental state of AD (Bancher et al., 1996). The accumulation of tau protein may be toxic and cause neuronal death in AD. But the presence of tangles in aged patients without dementia symptoms was reported (Guillozet et al., 2003). In this study, we found anti-PDI-antibody-immunopositive NFTs in normal control brains (Fig. 3). In normal ageing brains, chaperons may be reduced and misfolded proteins could be accumulated. As PDI is an ER-specific chaperone, PDI may be included in NFTs of normal aging brain. In these days, a repressible mutant tau transgenic mouse was reported (Santacruz et al., 2005). The mouse, which expresses a human tau variant that is repressible using tetracycline, develops progressive age-related NFTs, neuronal loss, and behavioral impairments. After the suppression of transgenic tau, memory function recovers, and neuron numbers stabilized but NFTs continue to accumulate. Thus, accumulated tau may be neuronally toxic, but NFTs are not sufficient to cause neuronal death in this model of tauopathy. The abnormally phosphorylated tau is not only inactive in promoting microtubule assembly but also causes disassembly of microtubules assembled with tau, MAP1, and MAP2 (Alonso et al., 1997). Thus, neurofibrillary degeneration might be prevented by inhibiting abnormal hyperphosphorylation of tau. In addition, polymerization of hyperphosphorylated tau

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into filaments inhibits its ability to bind normal tau and as well as the ability to inhibit the assembly of tubulin into microtubules in vitro and in the regenerating microtubule system from cultured cells. The in vitro abnormally hyperphosphorylated recombinant brain normal tau binds normal tau and loses this binding activity on polymerization into filaments. Dissociation of the hyperphosphorylated normal tau filaments by ultrasonication restores its ability to bind normal tau (Alonso et al., 2006). Abnormally phosphorylated tau may be neuronal toxic and NFT may prevent the neuronal death by causing the aggregation into filaments. Recently, granular and fibrillar tau aggregates was reported in atomic-force microscopy study of AD brain tissue and in vitro (Maeda et al., 2007). Circular dichroism spectral analysis and immunostaining with conformation-dependent antibodies indicated that tau may undergo conformational changes during fibril formation. Enriched granules generated filaments, suggesting that granular tau aggregates may be an intermediate form of tau fibrils. The amount of granular tau aggregates was elevated in the prefrontal cortex of Braak stage I cases compared to that of Braak stage 0 cases, suggesting that granular tau aggregation precedes PHF formation (Maeda et al., 2007). Thus, granular tau aggregates may be a relevant marker for the early diagnosis of tauopathy. Reducing the level of these aggregates may be a promising therapy for tauopathies. As NFTs are observed in normal aging brain, reducing tau aggregation may be a therapy for promoting brain aging (Guillozet et al., 2003). PDI is an ER-specific chaperone and is linked to the accumulation of misfolded proteins in many neurodegenerative diseases (Nakamura and Lipton, 2009; Uehara et al., 2006; Walker et al., 2010). In this study, we have shown the localization of PDI in neuronal cells. PDI-immunoreactivity was seen in neuronal bodies and dendrites both for neurons and glial cells. These results were expected, because PDI is normally localized in the ER and not in the nucleus. In a previous report on AD, PDI was localized specifically to neurons, where there is no substantial increase in AD compared to controls (Kim et al., 2000). PDI prevents the neurotoxicity associated with ER stress and protein misfolding, but NO or reactive oxygen species block the enzyme's protective effect through the S-nitrosylation of PDI. This inhibition of PDI leads to ER stress, which can induce apoptosis (Nakamura and Lipton, 2009; Uehara et al., 2006). Recently, some S-nitrosylated proteins have been reported in neurodegenerative diseases. Parkin was S-nitrosylated in familial and sporadic PD, and S-nitrosylated Parkin can regulate E3 ligase activity (Yao et al., 2004). The regulation of E3 ligase activity and its autoubiquitination may result in proteasome dysfunction and protein accumulation. Furthermore, it was recently reported that PDI was S-nitrosylated in sporadic PD and AD (Uehara et al., 2006). The ratio of Snitrosylated-PDI to total PDI was calculated and indicates that pathophysiologically relevant amounts of S-nitrosylated-PDI are present in human brains with PD and AD (Uehara et al., 2006). In addition, PDI protects against protein aggregation and is S-nitrosylated in ALS (Walker et al., 2010). Actually, PDI was present in cerebrospinal fluid and was aggregated in motor neurons of patients with ALS (Atkin et al., 2008). More recently, it was reported that exposure of cerebrocortical neurons in culture to β-amyloid results in the formation of

amounts of S-nitrosylated dynamin-related protein (Drp) 1, which is relatively similar to what happens in human AD brains (Cho et al., 2009). The S-nitrosylation of Drp1 causes dimer formation and increased GTPase activity, thus accelerating the process of mitochondrial fragmentation and contributing to neuronal synaptic damage or cell death. This relationship between nitrosative stress and mitochondrial fragmentation suggests that Drp1 can be a target of S-nitrosylation in AD. Drp1 is nitrosylated via a redox-mediated pathway in response to β-amyloid oligomers, causing mitochondrial fission and synaptic damage. Mitochondrial dysfunction may be linked to the formation of NFTs and the mechanism of AD. In this study, we have revealed anti-PDI-antibody-immunopositive NFTs. Furthermore, PHF-tau and PDI were co-localized in NFTs. As far as we know, this is the first report of anti-PDIantibody-immunopositive inclusions in AD. We assume that NO inhibited PDI and lead to the accumulation of unfolded proteins in AD. Abnormally phosphorylated tau or other proteins may be accumulated in NFTs and cause ER stress in AD. As PDI is working in neurons as a chaperon, PDI may bind to PHF-tau and become to be included in NFTs. The co-localization of PDI and PHF-tau in NFTs may be linked to the formation of these inclusions. In neurodegenerative disease, the neurons are degenerated and the cell structure is markedly changed. The ER is damaged and PDI may come out of the ER. In AD, NO may inhibit PDI by inducing S-nitrosylation, which inhibits its enzymatic activity and thus allows protein misfolding to occur. Consequently, the accumulation of misfolded proteins induces ER stress. The ER stress can cause apoptosis of neuronal cells. These results suggest that PDI could be a therapeutic target to prevent ER stress in neuronal cells in AD.

4.

Experimental procedures

4.1.

Tissue preparation

Postmortem brain specimens from four patients with AD (69–91 years old) and four normal control brains (62–94 years old) were utilized in this study. The diagnosis of the patients was defined by pathological study. Specimens from the hippocampus, frontal lobe, midbrain, pons, medulla, basal ganglia and cerebellar hemispheres were obtained from the autopsied brains of the normal control and AD patients. All brains were fixed in 10% neutral formalin at room temperature. Several paraffin-embedded tissue blocks were prepared and cut into 6-μm-thick sections on a microtome. The paraffin-embedded sections were deparaffinized in xylene, followed by rehydration in ethanol solutions of decreasing concentration as previously described (Kawamoto et al., 2007).

4.2.

Immunohistochemistry

Immunohistochemical staining was performed as previously described (Honjyo et al., 2001; Kawamoto et al., 2007). Immunohistochemical staining for PDI and human PHF-tau were performed using polyclonal rabbit anti-PDI antibody that has been previously described before (Kimura et al., 2008, 2004)

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and mouse anti-AT8 antibody purchased from Innogenetics Biologicals (Gent, Belgium). But AT8 immunopositive cells can be free of NFT (Uchihara et al., 2001). To confirm the NFTs, we also used another NFT indicator monoclonal mouse antiAlzheimer's disease Tau clone DC11 (Sigma-Aldrich). The sections were incubated in a microwave oven for a few minutes. And the sections were incubated in 0.3% hydrogen peroxide in 0.1 M PBS at room temperature for 30 min to block the endogenous peroxidase activity. After washing with PBS containing 0.3% Triton X 100 (PBST), the sections were incubated with the primary antibody diluted in PBST for overnight at room temperature. A dilution of 1:100 was used for the primary PDI antibody. After incubation with the primary antibody and washing, the sections were incubated with the secondary antibody (diluted 1:100). The sections were incubated with an avidin–biotin complex, and were allowed to react with a solution containing 0.02% 3,3′-diaminobenzidine tetrahydrochloride (DAB), 0.005% hydrogen peroxide, and 0.6% nickel acetate in 0.05 M Tris/HCl buffer.

4.3. Double staining of PDI and NFT indicators in tissue sections from AD samples To confirm the anatomical relationship between PDI and NFT, we performed double-staining studies using mouse anti-AT8 and rabbit anti-PDI antibody, and mouse anti-Alzheimer Tau antibody and rabbit anti-PDI antibody as described above (Honjyo et al., 2001). Briefly, sections of the hippocampus were incubated in medium containing mouse anti-AT8 (or mouse anti-Alzheimer Tau antibody) and anti-PDI antibodies (each diluted 1:100) in PBST overnight at room temperature. After washing, the sections were reacted with the secondary antibodies consisting of polyclonal goat anti-rabbit immunoglobulins/FITC (The Jackson Laboratory, Bar Harbor, ME, USA) and a polyclonal swine anti-mouse immunoglobulins/TRITC (Cosmo Bio Science, San Diego, CA, USA) for 1 h at room temperature. After rinsing, the slides were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) and photographed using an Olympus Fr1000 (Olympus Corporation, Tokyo, Japan) confocal laser scanning microscope. We selected 100 AT8-immunopositive NFTs in the immunostained sections of the hippocampus from three patients with AD. We then counted the number of PDI-immunopositive NFTs in the selected AT8-immunopositive NFTs from each patient. The statistical analysis was performed on the quantitative data using JMP 8 Introductory Guide (SAS Institute Inc., Cary, NC, USA).

Acknowledgments We thank Kumi Kodama, Nana Kawaguchi (Department of Pharmacoepidemiology, Kyoto University) and Hitomi Nakabayashi (Department of Neurology, Kyoto University) for excellent technical assistance. This work was supported in part by a research grant from the Seijin-kai Medical Group in Japan.

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