Age-related appearance of dendritic inclusions in catecholaminergic brainstem neurons

Neurobiology of Aging 34 (2013) 286 –297 www.elsevier.com/locate/neuaging

Age-related appearance of dendritic inclusions in catecholaminergic brainstem neurons Heiko Braaka,*, Dietmar Rudolf Thalb, Jakob Matschkec, Estifanos Ghebremedhinb,d, Kelly Del Tredicia a

b

Clinical Neuroanatomy, Center for Biomedical Research, Department of Neurology, University of Ulm, Ulm, Germany Laboratory for Neuropathology, Institute of Pathology, Center for Biomedical Research, University of Ulm, Ulm, Germany c Institute of Neuropathology, University Medical Center, Hamburg-Eppendorf, Germany d University of Queensland, School of Biomedical Sciences, Brisbane, Australia Received 20 October 2011; received in revised form 27 January 2012; accepted 27 February 2012

Abstract We identified p62-immunoreactive inclusions in dendrites of catecholaminergic brainstem projection neurons using antibodies against p62, ubiquitin, ␣-synuclein, hyperphosphorylated tau, and tyrosine hydroxylase in 100-␮m sections through the brainstem dorsal vagal area, locus coeruleus, and substantia nigra of 149 autopsy cases staged for intraneuronal Alzheimer’s and Parkinson’s disease-associated lesions. The inclusions resembled Marinesco bodies within cell nuclei of catecholaminergic neurons as well as the dot-like structures previously described by Dickson in specific neuropil areas in humans. The p62-positive inclusions were confined to dendrites of catecholaminergic neurons, lacked neuromelanin granules, and were tau- and ␣-synuclein-negative. Their immunoreactivity for ubiquitin varied and their prevalence significantly increased with advancing age. The presence or absence of Alzheimer’s and/or Parkinson’s disease-associated pathology did not influence their existence. There was a strong association between the presence of p62-positive inclusions and Marinesco bodies (p ⬍ 0.0001). Our results reveal a hitherto unknown alteration within specific neuronal types of the human brainstem that may be independent of the sequestosome-ubiquitinproteasomal pathway and unrelated to proteinaceous aggregate-formation of neurodegenerative diseases. © 2013 Elsevier Inc. All rights reserved. Keywords: Aging; Catecholamines; Locus coeruleus; Marinesco bodies; p62; Sequestosome-ubiquitin-proteasomal pathway; Substantia nigra; ␣-synuclein; Tau

1. Introduction Relatively little is known about structural changes within human nerve cells that consistently accompany and characterize normal brain aging (Anderton, 1997, 2002; Dickson et al., 1990, 1991; Hof et al., 1996; Keller et al., 2004; Mrak et al., 1997). Historically, these changes include the appearance of lipofuscin and neuromelanin granules, which, in general, accumulate with advancing age and display a va-

* Corresponding author at: Clinical Neuroanatomy (Dept. of Neurology), Center for Biomedical Research, University of Ulm, Helmholtzstrasse 8/1, 89081 Ulm, Germany. Tel.: ⫹49 731 500 63 111; fax: ⫹49 731 500 63 133. E-mail address: [email protected] (H. Braak). 0197-4580/$ – see front matter © 2013 Elsevier Inc. All rights reserved. 10.1016/j.neurobiolaging.2012.02.031

riety of distribution patterns in specific neuronal types (Benavides et al., 2002; Braak, 1984; Brunk and Terman, 2002; Double et al., 2008; Gray and Woulfe, 2005; Gray et al., 2003; Halliday et al., 2006; Porta, 2002; Terman and Brunk, 2004; Terman et al., 2007, 2010). Additional but less widespread morphological changes have in common immunoreactivity for ubiquitin. Ubiquitin-positive Marinesco bodies occur within the cell nuclei of catecholaminergic nerve cells (Alladi et al., 2010; Beach et al., 2004; Dickson et al., 1990; Kanaan et al., 2007; Schwab et al., 2012; Yuen and Baxter, 1963), whereas dot-like deposits are thought to develop in dystrophic neuronal processes in neuropil areas between the cellular islands of layer II in both the entorhinal and transentorhinal regions (Dickson et al., 1990, 1991; Pappolla et al., 1989).

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Morphological changes of this type are considered as being distinct from the structural intraneuronal abnormalities that characterize Alzheimer’s (AD) and Parkinson’s (PD) diseases. Incidental and early (prodromal) lesions related to both disorders do not suffice to produce clinically observable symptoms and, in fact, elderly nonsymptomatic individuals frequently display an admixture of disease- and aging-related structural brain changes (Anderton, 2002; Dickson et al., 1991; Price et al., 2009) (Table 1). The widely expressed and conserved stress-responsive protein p62 consists of 440 amino acids and is thought to contribute to the sequestration of misfolded, aggregationprone proteins and their autophagic or ubiquitin/proteasomal clearance (Gal et al., 2007; Moscat et al., 2007; Olanow and McNaught, 2006; Pan et al., 2008; Seibenhener et al., 2004, 2007; Wooten et al., 2006). In addition, p62 shows a still unexplained propensity to become entrapped in both age- or disease-related protein inclusions (Arai et al., 2003; Kuusisto et al., 2001, 2002, 2003, 2008; Mizuno et al., 2006; Nagaoka et al., 2004; Scott and Lowe, 2007). The present study calls attention to a hitherto unknown age-related structural change in dendrites of human catecholaminergic projection neurons that is distinctly p62and variably ubiquitin-immunopositive. 2. Methods 2.1. Study population A total of 149 autopsy cases were studied (52 females, 97 males; age range 6 –96 years; mean age ⫾ SD: 57 ⫾ 22.4 years). This retrospective study was performed in compliance with university ethics committee guidelines and German federal law governing human tissue usage. Sample demographics and relevant clinical data obtained from medical records of participating university clinics (Hamburg, Bonn, Frankfurt am Main, Mainz) and municipal hospitals (Enschede, The Netherlands; Offenbach am Main, Germany; The Parkinson’s Institute, Sunnyvale, CA, USA) as well as from the autopsy reports appear in Table 1. Of the cases in the study cohort, including controls, 119 belonged to consecutive autopsy series performed between the years 2003 and 2007. Exclusionary criteria for the present study were the presence of (1) schizophrenia, (2) dementia with Lewy bodies (DLB) (McKeith et al., 2005), and (3) severe cerebrovascular disease (Román et al., 1993). Upon admission to hospital, nondemented (“controls”) and demented individuals were examined between 1 and 4 weeks prior to death using standardized protocols, including neurological status. The majority of these protocols included assessment of cognitive function and the ability to perform activities of daily living (ADLs) independently within the hospital setting. Clinical dementia rating (CDR) scores (Hughes et al., 1982) were examined retrospectively for 7 cases (70, 75, 90, 124, 128, 130, and 145) (Table 1). The data were also used to determine whether these patients met the clinical Diag-

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nostic and Statistical Manual of Mental Disorders (DSM)IV criteria for dementia (American Psychiatric Association, 1994), which all fulfilled. AD was diagnosed when dementia was observed and when the degree of AD-related neuropathology indicated the existence of at least a moderate likelihood for AD according to the National Institute on Aging-Reagan criteria (The National Institute on Aging, 1997). PD diagnoses had been made during life using the United Parkinson’s Disease Rating Scale (UPDRS) or Hoehn and Yahr scales by hospital neurologists for 10 cases and were confirmed neuropathologically (Braak et al., 2003a; Dickson et al., 2009; Gelb et al., 1999). At autopsy, the brains of 13 individuals whose brains displayed the presence of Lewy body pathology but failed to fulfill the clinicopathological criteria for PD or dementia with Lewy bodies were diagnosed as having incidental Lewy body disease (ILBD). Of these, 1 case (128) also received the diagnosis AD, and 2 cases (134, 146) received the neuropathological diagnoses of PD and AD (Table 1), whereby clinically the leading diagnosis had been Parkinson’s disease dementia (PDD). The majority of these were from individuals with no history of dementia or movement disorders (127 cases: 41 females, 86 males; age range 6 –96 years; mean ⫾ SD: 54.2 ⫾ 22.4 years). Eleven cases had suffered from clinically documented sporadic PD (3 females, 8 males; age range 68 – 88 years; mean age ⫾ SD: 77.5 ⫾ 6.4 years) and 12 brains were from demented individuals with sporadic AD (9 females, 3 males; age range 62– 89 years; mean age ⫾ SD: 73.7 ⫾ 8.4 years). The postmortem interval (PMI), defined as the time between death and autopsy, ranged from 4 to 312 hours (median: 48 hours). Both hemispheres were examined macroscopically in approximately 1 cm thick coronal slices and macroscopic findings noted. Brains were fixed in a 4% aqueous solution of formaldehyde for 11–20 days prior to standardized neuropathological assessment and dissection. 2.2. Tissue dissection, embedding, sectioning, and storage A set of 3 tissue blocks was dissected, embedded in polyethylene glycol (PEG 1000; Merck, Darmstadt, Germany) (Smithson et al., 1983), and sectioned at 100 ␮m perpendicular to the brainstem axis of Meynert as described previously (Braak et al., 2003a, 2006). Because our primary focus is neuroanatomy rather than molecular biology, we have found that PEG embedding and the unconventional section thickness, which allows for the superimposition of large numbers of biological structures (Braak et al., 2003b), including nerve cells with their entire dendritic tree (Braak and Del Tredici, 2011), provide sufficient epitope preservation for morphological studies (Klosen et al., 1993, Braak et al., 2006, 2007). The first tissue block included the medulla oblongata at the latitude of the dorsal motor nucleus of the vagus nerve, the second block ran through the pontine tegmentum and

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Table 1 Age, sex, and clinical data of the cases examined Case

Age, y

Sex

NFTa

PDb

c62

cMa

n62

nMa

tre

Remarks

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

6 8 12 14 14 14 15 15 15 16 16 16 17 18 19 21 21 22 23 24 24 24 24 25 25 26 26 26 27 27 28 29 29 29 33 41 46 47 49 49 50 50 50 50 52 52 54 54 55 55 55 56 57 57 57 57 58 58 58 60 61 61 61

M M M F M M F M M M M M M M M F M M F M M M F M F M M F M F F F M F M F F M F F F F M M F F M F F M F M M M F M M M M M M M M

a 0 b b 1a a b 1a a a b c a a c a c a a 1a a 1b 1b b a 1a b 1a b 1b a c 1b 1b a 1b I I 1a 1b 1b I I I I 1b I III I 1b 1b I I I 1b II I II I I I III III

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 0 1 1 1 0 0 2 0 0 1 0 0 NE 1 1 NE 1 0 2 0 NE 0 1 1 1 1 1 1 0 0 0 0 2 0 0 0 1 1 1 0 1 NE 1 2 2 NE 2 1 1 0 NE 1 2 2 2 1 2 2 2 1 1 1

1 0 0 0 0 0 0 1 0 0 0 0 0 NE 0 0 NE 0 0 1 0 NE 0 1 1 0 1 0 1 0 0 0 0 1 0 0 0 0 1 0 0 1 NE 0 0 1 NE 1 1 1 0 NE 1 1 1 0 1 1 0 0 0 0 1

0 0 0 0 1 0 0 0 0 0 0 0 1 1 0 0 2 0 1 0 1 0 0 0 2 0 2 0 NE 0 0 0 0 0 0 0 0 1 1 0 1 2 2 1 1 1 2 2 1 0 1 1 1 2 2 1 1 1 1 2 1 0 2

0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 1 2 0 1 0 NE 0 0 1 0 0 0 0 0 1 1 0 1 1 1 1 0 1 2 2 2 0 1 2 2 2 1 2 2 0 1 2 0 0 1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NE 0 0 0 0 NE 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NE 0 1 1 1 1 1 0 1 1 1 0 0 0 1 0 1 NE NE 1 1 1 1 1 1 1

Sepsis Sepsis Sick sinus syndrome Portal sclerosis (transplant) Duchenne muscular dystrophy Rubella Malignant neoplasm Epilepsy Influenza NA NA ALL, CMV pneumonia Down syndrome Acute head trauma Heart failure Acute head trauma Bronchopneumonia, sepsis Sepsis NA Aplastic anemia Embolism NA Sepsis Renal insufficiency Embolism Malignant non-Hodgkin lymphoma NA AML Trauma Anemia AML Malignant neoplasm Myocardial infarction Malignant neoplasm Traumatic brain injury ALL Malignant neoplasm CML, liver failure Cushing syndrome Septic encephalitis SAH, essential hypertonia CML, ILBD Liver transplant (hepatitis C) Pulmonary embolism CML Malignant neoplasm SAH, essential hypertonia Malignant neoplasm Malignant neoplasm Heart transplantation Plasmocytoma Heart disease Colon perforation Aortic aneurysm NA Peritonitis (ruptured appendix) Heart failure, emphysema Malignant neoplasm Hepatic artery rupture Malignant neoplasm Non-Hodgkin lymphoma Liver cirrhosis Heart disease (continued on next page)

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Table 1 (continued) Case

Age, y

Sex

NFTa

PDb

c62

cMa

n62

nMa

tre

Remarks

64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127

61 61 62 62 62 62 62 63 63 64 64 64 65 65 66 66 66 66 66 68 68 68 68 68 68 68 68 69 69 69 69 69 70 70 70 70 71 71 71 71 71 71 72 72 72 72 72 72 73 73 74 74 74 75 75 75 75 75 75 75 75 76 76 76

F F F M M M F M F M M F F M M M M M M M M F M M M M F M F M M M M F M M M F F M M F F F M M M M M M M M M M M M F F M F M F M M

I I 1b II I I VI II I III III V II I III II I II II II I I II II V I VI III II 1b III II II III I II 1b III III III I III III VI III II III III III I II II I III III III III II III IV V II II 1a

0 0 0 0 0 1 0 0 0 0 0 0 3 0 0 0 1 0 0 2 3 1 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 6 0 0 2 0 0 6 0 0 0 0 0 0 5 0 0 4 0 0 0 2 0 0

0 1 1 2 2 1 0 2 2 NE 1 2 2 1 2 NE 2 1 2 2 NE 2 2 1 2 0 0 1 1 NE 2 2 1 2 2 1 2 2 2 2 1 0 1 0 1 2 2 0 1 1 2 1 2 2 1 1 2 1 2 2 1 1 2 2

0 0 0 1 1 0 0 1 1 NE 0 1 1 1 2 NE 1 1 1 1 NE 1 1 0 1 0 0 1 1 NE 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 1 1 0 0 1 1 1 1 1 0 0 1 0 0 1 0 0 1 0

0 0 1 2 1 1 2 0 2 1 0 2 2 1 2 2 2 1 1 2 1 2 1 0 2 1 2 2 2 0 1 2 1 2 2 2 2 2 2 2 2 NE 1 1 0 NE 2 2 1 1 2 1 2 1 1 0 2 0 1 2 2 1 1 1

0 1 0 2 2 1 2 0 2 2 0 1 2 2 2 2 1 2 1 2 0 2 0 1 2 1 2 1 2 0 1 2 1 2 2 2 1 1 2 2 2 NE 1 2 1 NE 2 1 1 1 2 1 1 2 1 0 2 0 0 2 2 2 0 0

0 0 1 1 1 0 1 2 1 0 1 NE 1 1 1 2 1 0 0 1 1 0 0 0 0 2 1 1 NE 1 1 1 0 1 NE 2 2 2 1 1 2 2 2 0 1 1 1 2 2 1 2 2 2 0 0 2 1 NE 2 1 2 NE 2 0

Malignant neoplasm Malignant neoplasm Malignant neoplasm Cushing syndrome Abscessed spondylodiscitis Malignant neoplasm, ILBD AD Heart failure Malignant neoplasm Liver cirrhosis Pulmonary embolism AD Liver cirrhosis, ILBD Ileus Malignant neoplasm CML Malignant neoplasm, ILBD Malignant neoplasm Ruptured aortic aneurysm colon perforation, ILBD Malignant neoplasm, ILBD Malignant neoplasm, ILBD Malignant neoplasm Aortic aneurysm AD, malignant neoplasm PD AD Plasmocytoma Essential hypertonia Pulmonary embolism Acute head trauma Malignant neoplasm Malignant neoplasm Emphysema, heart disease Heart disease Malignant neoplasm Cardiac disease Malignant neoplasm Malignant neoplasm Malignant neoplasm, ILBD Malignant neoplasm PD Pulmonary embolism AD, heart disease Subdural hematoma, ILBD Malignant neoplasm Malignant neoplasm, HIV PD Malignant neoplasm Myocardial infarction Malignant neoplasm Malignant neoplasm CLL, heart disease Malignant neoplasm PD, aortic dissection Pneumococcus sepsis Sclerosing cholangitis PD, myocardial infarction Malignant neoplasm Type A aortic dissection AD Malignant non-Hodgkin lymphoma, ILBD Malignant neoplasm Myocardial infarction (continued on next page)

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Table 1 (continued) Case

Age, y

128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149

76 76 76 77 77 78 80 81 81 81 81 81 82 85 85 86 86 87 88 89 91 96

Sex

NFTa

F M F M M M F F M F F M M M M F M F M F M M

V III IV III II II IV III II III III I II II I V I III IV VI II III

PDb

c62

cMa

n62

nMa

tre

Remarks

3 2 0 4 5 0 5 0 0 0 0 2 4 4 0 0 0 0 5 0 0 0

1 1 2 1 0 2 1 1 2 2 2 1 1 1 2 2 2 1 1 1 0 1

0 0 1 1 0 1 1 1 1 1 1 1 1 0 1 2 1 1 1 0 0 1

1 NE 1 2 1 2 NE 2 2 2 2 1 1 1 0 2 2 0 1 2 1 1

1 NE 1 1 1 1 NE 2 2 2 2 0 1 1 1 2 1 0 1 2 1 2

2 1 0 1 0 2 1 1 2 2 2 1 2 2 0 1 2 1 2 0 1 1

AD, ILBD Colitis, sepsis, ILBD AD PD, malignant neoplasm PD Malignant neoplasm PD, aortic dissection SAH Emphysema, heart disease AML Acute head trauma (fall) Myocardial infarction, ILBD PD PD Pancytopenia AD, colon cancer Cholecystitis Malignant neoplasm PD AD, pulmonary embolism Pneumonia Malignant neoplasm

Stages of intraneuronal AD- and PD-related lesions are given followed by the frequency of occurrence of dendritic p62-ir inclusions and Marinesco bodies in the locus coeruleus and substantia nigra as well as of ubiquitin-ir dots in the entorhinal region. A 3-point rating scale is used to assess the density of dendritic inclusions as follows: absent or not detectable (0), a few structures (1), and many structures (2). The same scale applies for the absence and presence (few, many) of Marinesco bodies. Key: AD, Alzheimer’s disease; ALL, acute lymphatic leukemia; AML, acute myeloic leukemia; c62, p62-ir dendrites in the locus coeruleus; CLL, chronic lymphatic leukemia; cMa, Marinesco bodies in the locus coeruleus; CML, chronic myeloic leukemia; CMV, cytomegalia virus; F, female; ILBD, incidental Lewy body disease; ir, immunoreactive; M, male; n62, p62-ir dendrites in the substantia nigra; NA, not available; NE, not evaluated; NFT, neurofibrillary tangle; nMa, Marinesco bodies in the substantia nigra, pars compacta; PD, Parkinson’s disease; SAH, subarachnoid hemorrhage; tre, transentorhinal cortex; y, years. a Stages a–VI. b Neuropathological stages 1– 6.

contained portions of the locus coeruleus, and the third block was removed at the level of the inferior colliculus including posterior portions of the substantia nigra. Additional hemisphere blocks served for diagnosis of AD- and PD-associated pathology according to published staging criteria (Braak and Braak, 1991; Braak et al., 2003a, 2006; Dickson et al., 2008, 2009). After sectioning on a giant microtome, free-floating sections of all blocks from all cases were immediately processed for (1) overview purposes using a pigmentNissl stain for lipofuscin and basophilic material (Braak, 1984), (2) demonstration of catecholaminergic nerve cells, including their dendritic arbor, using tyrosine hydroxylase immunoreactions, (3) assessment of tauopathies and synucleinopathies using immunoreactions against hyperphosphorylated tau and ␣-synuclein, and (4) detection of amyloid-␤ deposition using the antibody 4G8. Additional 100-␮m serial tissue sections were then stored in a 4% aqueous solution of formaldehyde at 8°C–20°C. Age-related changes were evaluated using immunoreactions for ubiquitin and p62 (Dwork et al., 1998; Kuusisto et al., 2001, 2002, 2003, 2008) (Table 1). All 5 antibodies consistently produce robust and reliable results in our stored tissue sections

(Braak et al., 2006, 2007, 2008, 2011; Pikkarainen et al., 2010). Antibodies routinely used in our laboratory undergo testing on paraffin sections of various thicknesses (3– 80 ␮m) and on PEG-embedded thick sections of various thicknesses (30 –200 ␮m) at different dilutions and concentrations to determine whether adequate antibody penetration takes place. Thus, antibodies that do not reliably penetrate the tissue with repeated testing are not utilized. 2.3. Staining and immunocytochemistry Tissue sections were first treated in a mixture of 10 mL methanol plus 10 mL concentrated (30%) H2O2 and 80 mL Tris buffer for 30 minutes to inhibit endogenous peroxidase before blocking with bovine serum to prevent nonspecific binding. After pretreatment with 100% formic acid for 3 minutes to facilitate epitope retrieval, each of the sets of free-floating sections was incubated for 18 hours at 20 °C using the following 4 primary antibodies: (1) a monoclonal antibody against human p62 (anti-p62 LCK ligand; 1:500, 250 ␮g/mL; Clone 3/P62 LCK Ligand; BD Biosciences, Mountain View, CA, USA), (2) polyclonal antibody antiubiquitin (1:1000, 0.76 g/L; Dako, Glostrup, Denmark), (3)

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a monoclonal mouse antibody against ␣-synuclein (antisyn-1; 1:2000, 250 ␮m/mL; Clone number 42, BD Biosciences) as a marker of PD-related intraneuronal synucleinopathy, (4) a monoclonal antibody against phosphorylated tau (PHF-Tau; 1:2000, 200 mg/mL; Clone AT8; Pierce Biotechnology, Rockford, IL, USA) to detect Alzheimerassociated tau pathology (pretangle material and neurofibrillary changes of the Alzheimer type) as well as lesions associated with other tauopathies, and (5) a polyclonal antibody anti-TH (1:2000, 150 ␮g/mL; AB152, Chemicon/ Millipore, Schwalbach, Germany) as a marker for tyrosine hydroxylase-containing nerve cells and cellular processes, and (6) a monoclonal antibody anti-beta-amyloid (1:5000, 1 mg/mL; Clone 4G8; Covance, Dedham, MA, USA) for detection of amyloid-␤ deposition. Amyloid-␤ plaque pathology was assessed semiquantitatively as previously published (Thal et al., 2000). Subsequent to processing with a corresponding secondary biotinylated antibody (anti-mouse IgG, 1:200; Linaris/ Vector Laboratories, Burlingame, CA, USA) for 1.5 hours, all immunoreactions were visualized with the avidin-biotin complex (ABC, Vectastain, Vector Laboratories, Burlingame, CA, USA) for 2 hours and the chromogen 3,3’diaminobenzidine tetrahydrochloride (DAB, D5637; Sigma, Taufkirchen, Germany). Omission of the primary antiserum resulted in nonstaining. Positive as well as negative control sections were routinely included to confirm specificity of the immunostaining. The tissue sections were cleared and mounted in a synthetic resin (Permount; Fisher, Fair Lawn, NJ, USA). Additional sets of 100 ␮m tissue sections underwent double-immunostaining for p62 (DAB) and TH (red chromogen Nova red; Zytomed, Berlin, Germany). Other sections underwent a first immunoreaction for p62 visualized with a red chromogen that could be washed off in ethanol (Fast red; Zytomed). After cover-slipping in a water-soluble medium (Aquatex; Merck) and photographic documentation of p62-immunoreactive (ir) dendritic inclusions, the chromogen was removed in 70% ethanol, followed by a second immunoreaction this time using antibodies against ubiquitin, or ␣-synuclein, or hyperphosphorylated tau, or followed by silver staining for neuromelanin granules. For this purpose we used the Campbell-Switzer silver method, which exploits the physical development of nucleation sites (Braak and Braak, 1991; Sandmann-Keil et al., 1999). Tissue sections were cleared, mounted, and cover-slipped in a synthetic resin (Permount; Fisher). The degree of p62-immunolabeling in cellular processes was assessed according to a 3-point rating scale as follows: absent or not detectable (0), a few positive structures (1), many positive structures (2) (Table 1, Figs. 1 and 2). Sections were viewed with a Vanox AHB53 research microscope (Olympus, Optical Co., Tokyo, Japan), and digital micrographs were taken using a digital camera (Olympus, Optical Co., Münster, Germany) to-

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gether with the Analysis Soft Imaging Solutions GmbH (Münster, Germany). Statistical analysis was performed using independent samples t test for comparison of mean values for normally distributed continuous outcomes among 2 groups. The Mann-Whitney U test was used for ordered variables, whereas the Spearman’s ␳ statistics was used for measuring rank correlations. Partial correlation analysis was carried out to examine the relationship between different pathological variables after adjusting for the effects of age. Multiple logistic regression analyses were performed to investigate whether pathological and demographic variables, including PMI, predicted the presence or absence of p62-immunoreactive intradendritic inclusions. Multicollinearity was assessed by calculating variance inflation factors (VIF) for independent variables. Statistical tests were mostly 2-sided, with significance declared at p ⬍ 0.05. Computations were performed with the aid of IBM SPSS, Release 20, 2011 (SPSS, Inc., an IBM company).

3. Results Inclusions immunoreactive for p62 were readily visualized within neuronal processes of catecholaminergic neuromelanin-containing cells in the locus coeruleus (Fig. 1A and B) and nigral pars compacta of the human brainstem (Fig. 1C and D) (Bogerts, 1981; Saper and Petito, 1982; Zecca et al., 2001, 2003, 2006). These inclusions were not visible in pigment-Nissl-stained overview sections (data not shown) but were seen in immunoreactions against p62 in the neuropil between the cell bodies of melanized neurons as well as in the immediate vicinity of the catecholaminergic nuclei. They were also visible in the vicinity of smaller groups of melanized neurons elsewhere in the lower brainstem, e.g., within the group of catecholaminergic nerve cells in the medullary dorsal vagal area. The autopsy cases examined contained cases with a low burden of lesions related to AD and/or PD but also included 23 clinically diagnosed AD and PD cases showing severe pathologies related to these diseases (Table 1). Inclusions positive for p62 could be observed in all of these cases. The presence or absence of AD-and/or PD-associated pathology did not appear to influence the distribution pattern and density of the inclusions. There were significant correlations between p62 inclusions, Marinesco bodies, and neurofibrillary tangle stage (NFT) stage (all p ⬍ 0.0001) but not PD-associated pathology. The correlation with NFT stage, however, was diminished after controlling for age (all p ⬎ 0.1). The presence of p62 inclusions was significantly associated with advanced age (p ⬍ 0.0001), with aged individuals showing higher prevalence of p62 inclusions than their younger counterparts. Most of the p62-positive structures resembled short dowel-like or spindle-like structures with lightly rippled surfaces (Fig. 1E–G). Sometimes, their aspect gave the impres-

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Fig. 1. Dendritic changes in the locus coeruleus and substantia nigra as seen in immunoreactions for p62 (100 ␮m, polyethylene glycol [PEG] sections). (A and B) Locus coeruleus. Twenty-four-year-old male (case 20). Framed area in (A) is seen in greater detail in (B). (C and D) Substantia nigra with pars compacta in the left half of the micrograph, reticulate portion in the middle, and crura cerebri at right. Eighty-one-year-old female (case 137). Framed area in (C) again is seen at higher magnification in (D). (E) The p62-positive structures resemble short dowels or spindles, sometimes giving the impression of multiple units of varying length resembling segments of bamboo (50-year-old female, case 42). (F and G) Arrows point to p62-positive dendritic changes in the vicinity of nigral melanized neurons (70-year-old male, case 99). Cells in (F) and (G) additionally show presence of intranuclear Marinesco bodies. Scale bar in (A) applies to (C), bar in (B) is also valid for (D), bar in (E) applies to (F).

sion that small multiple units, resembling segments of bamboo that varied in length, had formed a single large entity (Fig. 1E). The diameters of the inclusions were less variable than their lengths and failed to considerably exceed those of dendritic processes, yet clearly surpassed the di-

ameters of axons of catecholaminergic nerve cells. The strongly p62-positive inclusions were encountered only near the cell bodies of melanized neurons. Occasionally, the somatodendritic domain of catecholaminergic neurons displayed an additional diffuse and weak p62-immunolabeling

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Fig. 2. Dendritic changes in immunoreactions for p62 and other proteins (100 ␮m, polyethylene glycol [PEG] sections). (A–F) Trials in which dendritic changes were first visualized in immunoreactions against p62 using a red and soluble chromogen. (A1 and F1) Seventy-eight-year-old male, case 133; (B1, C1, D1), 71-year-old male, case 104). After photographic documentation, the chromogen has been washed out and a second immunoreaction was carried out against ubiquitin (A2–D2), hyperphosphorylated tau protein (E2), and ␣-synuclein (F2). Note that the coreactions for ubiquitin varied considerably including intense (A2, B2) as well as weak reactions (C2), but also absence of any reactivity (D2). (G1) Shows again red immunoreaction of a p62-positive dendritic change (arrow, 75-year-old male, case 117). (G2) After washing out of the reaction the section was silver stained for neuromelanin (Switzer technique). Note the intense reaction of neuromelanin granules. The dendritic change (arrow) did not react. The granular material of the spindle does not contain granules of neuromelanin. (H and J) Double immunoreaction for tyrosin hydroxylase (red chromogen) and p62 (brown chromogen) (50-year-old female, case 42). Note that the p62-positive spindle (arrows) is seen in dendrites of tyrosin hydroxylase-positive projection neurons of the substantia nigra, shown in greater detail in (J). (K–O) A few of the neuromelanin-containing projection neurons of the substantia nigra show a subtle diffuse p62-immunoreactivity of the somatodendritic compartment. Such a neuron is shown in (K) including cell body (dashed arrow) and major dendrites. The apical dendrite displays an intensely p62-immunoreactive inclusion (arrow), shown in more detail in (L) (69-year-old male, case 91). (M–O) Other examples of spindle-shaped and strongly immunoreactive inclusions in nigral neurons with diffuse labeling of their somatodendritic compartment (50-year-old female, case 42). Scale bar in (A1) is valid for (A2–G2), bar in (H) applies also to (K), bar in (O) is also valid for (J) and (L–N).

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(Braak et al., 2011; Kuusisto et al., 2008). Such cells (Fig. 2K–O) as well as double immunoreactions with antibodies directed against p62 (brown chromogen) and tyrosine hydroxylase (red chromogen) (Fig. 2H and J) made it possible to identify the strongly immunoreactive inclusions as intradendritic in nature. Because melanized lipofuscin granules (neuromelanin) tend to bunch together in portions of the somata and can protrude from there into the proximal shafts of dendrites, even producing isolated spindle-shaped accumulations within more distal portions of the dendrites, care had to be taken to distinguish the physiologically light-brown neuromelanin granules from the immunoreactive spindle-like structures when visualized with DAB. Thus, we also employed a soluble red chromogen and made micrographs of the p62-positive spindle-like structures (Fig. 2G1) previous to bleaching out the chromogen and counterstaining neuromelanin granules using the Campbell Switzer silver technique (Fig. 2G2, arrow) to show that it was not the neuromelanin pigment granules that constituted the substrate for the p62-immunopositive material seen. Additional tissue sections using a similar procedure confirmed that the p62immunoreactive inclusions (Fig. 2E1 and F1) were neither immunopositive for hyperphosphorylated tau protein (Fig. 2E2) nor for aggregated ␣-synuclein (Fig. 2F2). Some of the p62-positive inclusions (Figs. 2A1, B1, C1, and D1) showed strong reactions with antibodies against ubiquitin (Fig. 2A2 and B2), others were weakly ubiquitin-immunoreactive (Fig. 2C2) and again others were ubiquitin-immunonegative (Fig. 2D2). 3.1. Statistical analyses Males predominated across all age categories (65.1% vs. 34.9%), and there was no significant age difference between females and males. Multiple logistic regression was used to assess the impact of several independent variables (sex, age group, presence of Marinesco bodies, PD pathology, and PMI) upon the likelihood of the presence or absence of p62 inclusions. Inasmuch as NFT pathology and age group were highly correlated (r ⫽ 0.78; p ⬍ 0.0001), NFT pathology was excluded from the regression model to avoid multicollinearity (variance inflation factor ⫽ 2.6). The strongest predictor of the presence of p62 inclusions was the presence of Marinesco bodies (odds ratio, 37.3; p ⫽ 0.001), thereby indicating that individuals with Marinesco bodies are almost 37 times more likely than those without to have p62 inclusions. Age also proved to be a significant predictor (odds ratio, 6.9; p ⫽ 0.02), with older individuals (⬎ 50 years of age) showing 6.9 greater likelihood of having p62 inclusions than their younger counterparts. Sex, PD pathology, and PMI were nonsignificant predictors (sex p ⫽ 0.31; PD pathology p ⫽ 0.45; PMI p ⫽ 0.27).

4. Discussion The p62-positive structures in dendrites of human catecholaminergic nerve cells described here are new and their prevalence (frequency of occurrence) shows an age-related increase comparable with that displayed by other age-associated alterations, e.g., Marinesco bodies and dot-like dystrophic neurites (see below) (Table 1). Moreover, the presence or absence of AD-and/or PD-related lesions does not appear to influence their distribution pattern or severity after controlling for age. Marinesco bodies are intensely immunopositive for both p62 and ubiquitin. They develop in cell nuclei of human neuromelanin-containing neuronal types and are thought to develop as a response to cellular stress (Alladi et al., 2010; Beach et al., 2004; Dickson et al., 1990; Kanaan et al., 2007; Schwab et al., 2012; Yuen and Baxter, 1963). Dot-like p62and ubiquitin-positive deposits are discussed as representing local swellings of dystrophic nerve cell processes typically seen in neuropil areas between the cellular islands of layer II in the entorhinal and transentorhinal regions (Dickson et al., 1990, 1991). Immunopositivity for p62 is a feature shared by Marinesco bodies, dot-like dystrophic neurites, and the novel intradendritic inclusions described here. The significant association between the presence of Marinesco bodies and p62-positive inclusions indicates a possible shared pathomechanism. Dysfunctional short-lived proteins generally are subjected to clearance via proteasomal proteolysis (Driscoll, 1994). Unlike the lysosomal pathway, however, protein degradation in proteasomes requires an adenosine triphosphate (ATP)-dependent polyubiquitination of the substrate, a tag that serves as a recognition motif for the 26S proteasome (Layfield and Searle, 2008). Such multiubiquitinated proteins also bind noncovalently to the multifunctional protein p62 to form larger subunits, the sequestosomes, which may help to “shuttle” the substrates to the proteasome (Shin, 1998) but, in so doing, possibly delay their degradation (Korolchuk et al., 2009) or help to direct the substrates into autophagosomes (Komatsu and Ichimura, 2010). Until recently, p62 immunoreactivity has mainly been discussed within the context of clearance via the ubiquitin-proteasomal pathway (Shin, 1998). Inasmuch as several in vitro studies have reported that familial amyotrophic lateral sclerosis mutants of superoxide dismutase (SOD)1 are recognized by the protein p62 in a ubiquitin-independent manner and targeted for the autophagy-lysosome degradation pathway (Gal et al., 2007, 2009), we decided to perform additional immunoreactions on standard paraffin sections from selected cases using a polyclonal superoxide dismutase-1 antibody as directed (Anti-Superoxide Dismutase 1; 1:100, 0.263 mg/mL; ab592950; Abcam, Cambridge, UK). The results (data not shown) were negative.

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In conclusion, although the physiological functions of the p62-immunoreactive structures reported here are currently unknown, our observation that the novel intradendritic alterations frequently are ubiquitin-immunonegative or display only a subtle degree of immunoreactivity indicates that p62 may possess functions relatively independent of the ubiquitin-proteasomal pathway that are capable of influencing intrinsic changes in specific neuronal types of the human brain during normal aging. Alternatively, such p62 inclusions may first become attached to ubiquitin in a later phase of their development. Other possible interactions, including those with cell surface receptors and additional proteins that might contribute to the formation of the p62 aggregates, certainly warrant further study. The intradendritic spindles or dowels seen here cannot be said to markedly impede or to disrupt cellulipetal and cellulifugal transport functions because localized swellings proximal or distal to the inclusions were not present. Disclosure statement The authors have no actual or potential conflicts of interest. This retrospective study was performed in compliance with university ethics committee guidelines and German federal law governing human tissue usage. Acknowledgements This study was supported by the German Research Council (Deutsche Forschungsgemeinschaft, DFG, grant number TR 1000/1-1) and the Michael J. Fox Foundation for Parkinson’s Research (New York City, USA). Technical assistance was provided by Ms. Siegrid Baumann, Ms. Gabriele Ehmke, Ms. Simone Feldengut (immunohistochemistry), and Mr. David Ewert (graphics). The authors thank the Braak Collection (Goethe University, Frankfurt/ Main) and William J. Langston, M.D. (Parkinson’s Institute, Sunnyvale, CA, USA) for providing tissue. References Alladi, P.A., Mahadevan, A., Vijayalakshmi, K., Muthane, U., Shankar, S.K., Raju, T.R., 2010. Ageing enhances alpha-synuclein, ubiquitin and endoplasmic reticular stress protein expression in the nigral neurons of Asian indians. Neurochem. Int. 57, 530 –539. American Psychiatric Association, 1994. Diagnostic and Statistical Manual of Mental Disorders. American Psychiatric Association, Washington, D.C. Anderton, B.H., 1997. Changes in the ageing brain in health and disease. Philos. Trans. R. Soc. Lond. B Biol. Sci. 352, 1781–1792. Anderton, B.H., 2002. Ageing of the brain. Mech. Ageing Dev. 123, 811– 817. Arai, T., Nonaka, T., Hasegawa, M., Akiyama, H., Yoshida, M., Hashizume, Y., Tsuchiya, K., Oda, T., Ikeda, K., 2003. Neuronal and glial inclusions in frontotemporal dementia with or without motor neuron disease are immunopositive for p62. Neurosci. Lett. 342, 41– 44.

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