Lewy Bodies in the Amygdala Increase of ␣-Synuclein Aggregates in Neurodegenerative Diseases with Tau-Based Inclusions

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ORIGINAL CONTRIBUTION Lewy Bodies in the Amygdala Increase of ␣-Synuclein Aggregates in Neurodegenerative Diseases With Tau-Based Inclusions

Anca Popescu, MD; Carol F. Lippa, MD; Virginia M.-Y. Lee, PhD; John Q. Trojanowski, MD, PhD

Background: Increased attention has been given to Results: Lewy bodies were often abundant in classic Pick ␣-synuclein aggregation in nonsynucleinopathies be- disease, argyrophilic grain disease, Alzheimer disease, and cause ␣-synuclein–containing Lewy bodies (LBs) influ- dementia with LBs but not in cases with amygdala de- ence symptoms. However, the spectrum of disorders in generation lacking tau-based inclusions, control cases, which secondary inclusions are likely to occur has not preclinical disease carriers, or degenerative diseases lack- been defined. Amygdala neurons commonly develop large ing pathologic involvement of the amygdala. The ex- numbers of secondary LBs, making it a practical region posed ␣-synuclein epitopes were similar in all cases con- for studying this phenomenon. taining LBs.

Objective: To characterize the spectrum of diseases as- Conclusions: Abnormal ␣-synuclein aggregation in the sociated with LB formation in the amygdala of neurode- amygdala is disease selective, but not restricted to dis- generative disease and control cases. orders of ␣-synuclein and ␤-amyloid. Our data are com- patible with the notion that tau aggregates predispose neu- Design: An autopsy series of 101 neurodegenerative disease and 34 aged control cases. Using immunohisto- rons to develop secondary LBs. chemistry studies, we examined the amygdala for ␣-synuclein aggregates. Arch Neurol. 2004;61:1915-1919

GGREGATION OF ␣-SY- of these subjects.7-9 It is unknown whether nuclein has a primary this curious finding is restricted to AD, or pathogenic role in spo- whether it is a more universal phenom- radic and familial autoso- enon. In the present study, we compared mal dominant Parkinson ␣-synuclein immunoreactivity in the amyg- disease, multiple system atrophy (MSA), and dala in a variety of diseases, including dis- A 1-6 dementia with Lewy bodies (DLB). In the orders of tau, ␣-synuclein, and ␤-amy- past few years, increased ␣-synuclein ag- loid, and in control cases to determine how gregation in the form of Lewy bodies (LBs) widespread this phenomenon is. We also has been reported in neurodegenerative dis- examined epitope exposure in LBs to screen eases that are not synucleinopathies.7-9 In for obvious differences in LB conforma- this context, LBs may be considered a section in different diseases. ondary phenomenon that reflects the fibrillation of ␣-synuclein induced directly by the formation of fibrillary tau lesions or indi- METHODS rectly by cell stress resulting from the formation of these tau inclusions. Regulatory We examined 135 amygdala specimens from factors involved in ␣-synuclein expression pathologically confirmed cases meeting con- Author Affiliations: and the biological changes leading to pri- sensus pathological criteria for DLB10 (n=9; LBs Department of Neurology, ␣ were common in regions outside the amyg- mary or secondary -synuclein aggrega- 11 Drexel University College of tion in neurodegenerative diseases are not dala), sporadic AD (n=20; lacking LBs out- Medicine (Drs Popescu and well understood. side the amygdala), DS (n=9; with or without Lippa), and Center for evidence of cognitive and/or functional de- Neurodegenerative Disease Secondary LBs are numerous in the cline), early-onset familial AD with preseni- Research, University of amygdala and adjacent entorhinal cortex in lin-1 mutations (n=6), preclinical AD with a Pennsylvania School of cases of Alzheimer disease (AD), includ- presenilin-1 mutation (n=1), classic Pick dis- Medicine (Drs Lee and ing sporadic and familial AD, and Down ease12 (PiD) (n=6; frontotemporal dementia Trojanowski), Philadelphia. syndrome (DS). They occur in at least half with numerous Pick bodies; not meeting con-

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 tients with DLB and AD. Moreover, we made the novel Table. Summary Table Showing the Incidence of LBs and observation that LBs were also often present in the amyg- the Average Severity of LB Pathology Across Diseases dala of PiD cases. Three of the 6 PiD cases studied showed numerous LBs in the amygdala (Figure). Rare diffuse No. of No. (%) of Mean Diagnosis Cases Cases With LBs LB Score amyloid plaques were present in the amygdala of 1 PiD case; the other 2 cases lacked plaques. Lewy bodies were DLB 9 8 (89) 2.9 also numerous in 1 case of AGD, another tau disorder PS-1 AD 7 4 (57) 1.7 PiD 6 3 (50) 1.3 involving the amygdala. Lewy bodies were rare in neu- DS 9 4 (44) 1.3 rodegenerative diseases where the amygdala is not heavily AGD 2 1 (50) 1.5 involved by the disease process (ALS and PSP). In MSA, SAD 20 7 (35) 0.7 occasional glial cytoplasmic inclusions in the surround- DLDH 40 2 (5) 0.1 ing white matter were immunoreactive for ␣-synuclein, Aged normal controls* 34 1 (3) 0.1 but intraneuronal aggregates were not seen. Lewy bod- ALS 4 0 0.0 MSA 3 0 0.0 ies and Lewy threads were present in only 2 of 40 cases PSP 1 0 0.0 of DLDH, although severe neuronal loss was seen in most amygdalae. Amygdala LBs were also absent in cases with Abbreviations: AD, Alzheimer disease; AGD, argyrophilic grain disease; amyloid plaques but no neurofibrillary tangles (the ALS, amyotrophic lateral sclerosis; DLB, dementia with LBs; DLDH, dementia younger patients with DS and our patient with a pre- lacking distinctive histopathological features; DS, Down syndrome; LB, Lewy body; MSA, multiple system atrophy; PiD, Pick disease; PS-1 AD, AD related clinical presenilin-1 mutation). to presenilin-1 mutations; PSP, progressive supranuclear palsy; In cases where LBs are present, the LB density is typi- SAD, sporadic AD. cally higher than that described in other brain regions.11 *Indicates elderly subjects with no significant neurological disease. Our semiquantitative grading (reported as mean±SD grade) indicated that the highest number of LBs occurred in DLB (2.9±1.1; 8 of 9 cases), followed by fa- current criteria for AD11), dementia lacking distinctive histo- milial AD (1.7±1.9; 4 of 7 cases), AGD (1.5+2.1; 1 of 2 12 pathological features (DLDH) (n=40), amyotrophic lateral scle- cases), DS (1.3±1.6; 4 of 9 cases), PiD (1.3±1.6; 3 of 6 rosis (ALS) (n=4), MSA (n=3), argyrophilic grain disease (AGD) cases), sporadic AD (0.7±1.1; 7 of 20 cases), and DLDH (n=2), progressive supranuclear palsy (PSP) (n=1), and cognitively normal elderly control cases lacking significant neu- (0.08±0.4; 2 of 40 cases). Lewy bodies were also almost rological diagnoses (n=34) (Table). Control cases had docu- never observed in the amygdalae of aged, cognitively nor- mentation of a normal mental status during life, lacked other mal control cases (0.1±0.1; 1 of 34 cases). When pres- neurological diagnoses before death, and did not meet patho- ent in AD, DLB, AGD, and PiD, LBs were almost always logical criteria for AD or other disorders. All cases, except DLB graded 3 or 4. When grade 3 changes were present, the cases, lacked ␣-synuclein pathology in other regions, includ- superficial regions (central and medial nuclei) tended to ing brainstem structures. be more heavily involved. However, the atrophy and glio- Formalin-fixed, paraffin-embedded tissue was sectioned at 6 sis were too severe to allow us to distinguish exact bor- µm at the level of the midamygdala and stained with a well- 1 ders between subregions, so we did not attempt to com- characterized monoclonal antibody (LB509) to purified full- pare the subregions. In cases where grade 4 changes were length recombinant ␣-synuclein. The antibody LB509 was gen- erated with partially purified LBs as the antigen and was present, LBs occurred throughout the amygdala. Lewy subsequently proved to recognize ␣-synuclein. Some tissues re- body density was low in DLDH, and the 2 cases with LBs active for LB509 were then immunostained using a panel of mono- were graded 1 and 2. clonal antibodies (SNL-4, 204, LB509, 211, and 202) described We usually found ␣-synuclein–positive threads in cases previously13 that are directed at epitopes throughout the with an LB burden of 2 or greater. They were most com- ␣-synuclein protein. Formic acid pretreatment was used with all mon in DLB (mean±SD grade, 1.3±0.9), but were also antibodies.13 The ␣-synuclein epitopes were detected using avidin- present in familial AD (1±1.3), DS (0.8±1.3), PiD biotin complex kits (Vector Laboratories, Burlingame, Calif) and (0.4±0.9), AGD (0.5±0.7), and sporadic AD (0.18±0.38). Ј 3,3 -diaminobenzidine. Positive control tissue consisted of af- Lewy threads were not present in cases lacking LBs. fected regions of a DLB case. Consecutive sections where the su- Because LBs in amyloidopathies are often restricted pernatant from unfused SP2/0-Ag14 mouse myeloma cells re- placed the primary antibodies were used as negative control tissue. to the amygdala, we characterized the distribution of LBs A previously defined semiquantitative scale8,9 for rating of in the PiD cases by screening other brain regions con- LBs was used, where 0 indicates no LBs in the amygdala; 1, taining Pick bodies, including the frontal cortex (middle 1 to 5; 2, 6 to 20; and 3, more than 20 per amygdala. Since LBs frontal and cingulate gyri), temporal lobe (entorhinal cor- were more numerous than envisioned when the scale was de- tex and hippocampus), and brainstem (locus coeruleus, veloped, we added grade 4 to indicate an average of more than midbrain, and medulla). In PiD cases where amygdala 20 LBs per high-power field. We rated ␣-synuclein–positive LBs were abundant, we observed occasional LBs in the threads (Lewy neurites) on a 0- to 3-point scale. All grading periamygdaloid entorhinal cortex. One PiD case lack- was performed by 2 observers (A.P. and C.F.L.), who were ing LBs in the amygdala had an isolated LB in the locus blinded to diagnosis. coeruleus. Lewy bodies were absent in other areas. Because focal LBs are not commonly described in high RESULTS densities in tauopathies, we further examined the PiD cases using double-label immunohistochemistry stud- Using the LB509 antibody, we confirmed our previous ies to determine how frequently LBs coexisted with Pick finding that LBs are common in the amygdala of pa- bodies. Double-label studies with ␣-synuclein and tau

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E F G H

I J K L M

Figure. Photomicrographs of ␣-synuclein expression in neurodegenerative disease cases. Lewy bodies (LBs) positive for ␣-synuclein are absent in the amygdalae of cognitively normal cases using the LB509 antibody (A). In contrast, LBs (identified with the LB509 antibody) are numerous in the amygdala in Alzheimer disease (B), dementia with LB (C), and classic Pick disease (D). Lewy bodies are absent in amyotrophic lateral sclerosis (E) and the negative control slide (H, primary antibody omitted). Double-label immunohistochemistry studies demonstrate that in Pick disease, the LBs (␣-synuclein, fast red/red) and Pick bodies (tau-2, diaminobenzidine/brown) may commix (F) or LBs may be more discrete (G). The LB grading scale is as follows: 0 indicates no LBs in the amygdala (I); 1, 1-5 (J); 2, 6-20 (K); 3, more than 20 per amygdala (L); and 4, more than 20 per high-power field (M). Cases with an LB grade of 4 also contain numerous Lewy threads. Scale bar indicates 20 µm in parts A through E and I through M, 10 µm in parts F and G, and 60 µm in part H.

demonstrated that LBs in PiD usually colocalized with showing less consistent immunoreactivity. This finding is tau-positive Pick bodies. Here, both proteins could be consistent with epitope mapping in other MSA studies.13 commixed, although sometimes the inclusions re- mained discrete, with a Pick body adjacent to the LB. When we obtained counts in a band of adjacent fields COMMENT across the medial-to-lateral amygdala in PiD cases, tau- positive immunoreactivity was present in 82% of neu- We report for the first time, to our knowledge, the pres- rons containing ␣-synuclein aggregates. Only 18% of PiD ence of intraneuronal ␣-synuclein aggregates in the PiD neurons with LBs lacked clear-cut tau aggregates in the amygdala, a region heavily affected with Pick bodies. De- same section. This is similar to our AD and DS cases where spite the small PiD sample size, it was clear that LBs were LBs typically colocalize with tau-positive neurofibril- abundant when present, sometimes reaching densities of lary tangles.8,9 greater than 20 per high-power field. They usually co- To address the question of whether secondary localized with Pick bodies. In our AD cases, LBs colo- ␣-synuclein exposure was similar in all diseases, we ex- calized with tau-positive neurofibrillary tangles. One of amined our cases containing LBs with the additional epi- our AGD cases also had amygdala LBs, supporting the tope-specific ␣-synuclein antibodies described in the “Meth- notion that tau is important in the process of LB forma- ods” section. When we compared the epitope-mapping tion. Because AGD is a 4-repeat (4R) tau disorder, PiD properties of LBs in PiD with those in familial and spo- is a 3R tau disorder, and AD has equal tau ratios, our data radic AD, DS, and DLB, we found epitope mapping to be suggest that amygdala LBs occur regardless of the 3R:4R identical with strong exposure of all epitopes. In con- tau ratio. Progressive supranuclear palsy, a tauopathy lack- trast, our MSA cases showed nonuniform epitope map- ing amygdala LBs, also lacked tau aggregates in the amyg- ping in glial aggregates, with amino-terminal epitopes dala. Secondary LBs developed less commonly in con-

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 trol cases, asymptomatic carriers (those with early DS and be viewed as an area susceptible to primary and second- the preclinical case with the presenilin-1 mutation), and ary ␣-synuclein inclusions, although the reasons for this degenerative disease cases lacking tau aggregates (DLDH). remain elusive. Indeed, Marui et al20 recently reported Preexisting amygdala pathology appears to be an impor- that Lewy pathology in the cerebrum begins in the amyg- tant factor in LB formation. The frequent colocalization dala in LB disease. This differs from the staging system of tau and ␣-synuclein in the same neuron in diseases for PD, where pathology starts in the medulla.21 We ex- with tau aggregates and the rare occurrence of LBs in dis- amined other brain regions in our cases, including the eases without tau pathology (normal controls and DLDH) medulla, and found little subcortical ␣-synuclein pathol- speak for an association between tau and ␣-synuclein ag- ogy. This is in keeping with the findings of Jellinger,22 gregation that could directly or indirectly be mediated who also noted that ␣-synuclein aggregates in AD do not by tau inclusions that promote the fibrillation of follow the regional pattern of distribution described by ␣-synuclein to form LBs in regions with abundant fibril- Braak et al21 for PD. lary tau lesions. One interesting question is whether the formation of Immunoreactivity of ␣-synuclein has previously been LBs in the amygdala influences the clinical course. Lewy reported in tauopathies. Although nigral LB counts did bodies influence symptoms in AD cases, even when they not differ between control subjects and those with PSP, are present in much lower densities.23 Results of retro- Tsuboi and colleagues14 found LBs and Lewy neurites in spective medical chart reviews in cases where history was 18% of PSP cases, with amygdala LBs being one of the available showed only occasional cases with classic fea- most involved regions. Using epitope specific antibod- tures of DLB (spontaneous parkinsonism or visual hal- ies, Takeda et al15 found that a C-terminus fragment lucinations). However, PD cases with amygdala LBs have ␣-synuclein antibody stained tau-positive aggregates in more visual hallucinations than comparable PD cases lack- AD, PSP, corticobasal degeneration, and PiD. These in- ing amygdala LBs.24 Although the preexisting tau pathol- teresting findings support the notion that ␣-synuclein frag- ogy and neuronal loss makes it likely that the clinical pre- ments commonly co-occur with pathologic tau inclu- sentations would have features of the primary disorder, sions. Evidence of an interaction between tau and prospective studies are needed to more fully determine ␣-synuclein at a biochemical level has also been found. to what extent, if any, amygdala LBs have an impact on Microtubule-associated, soluble axonal tau protein binds symptoms. It is possible that these subjects would expe- to the C-terminus of ␣-synuclein, which in turn modu- rience differential involvement of autonomic, olfactory, lates tau phosphorylation.16 visceral, endocrine, affective, and mnemonic activities It could be argued that ␣-synuclein aggregates are not (which are subserved by intra-amygdala circuits). true LBs if they do not have a fibrillary nature. A previ- Disease duration did not correlate strongly with the ous study from our group demonstrated the fibrillary presence of LBs. The mean duration of illness in PiD cases structure of amygdala LBs by means of electron micros- with LBs in amygdala was 12.7 years (range, 3-17 years), copy.8 Thioflavine stains have also demonstrated the fi- whereas in non–LB-containing PiD cases, it was 17.8 years brillary nature of these lesions. (range, 14-19 years). The Braak AD stage was VI25 in al- The common coexistence of LBs and tau aggregates most all of our postmortem symptomatic AD cases, re- (neurofibrillary tangles, Pick bodies, or 4R grains) could gardless of whether amygdala LBs were present. Our be related to failure of tau function (including microtu- symptomatic cases all had end-stage symptoms at death, bule assembly and/or axonal transport) or could be a di- requiring assistance for all activities of daily living. This rect effect of the tau aggregate. Tau hyperphosphoryla- made it impossible to determine whether LB formation tion may lead to structural changes in ␣-synuclein that is related to symptom severity. However, LBs did not oc- accelerate its aggregation. Another possibility is that im- cur in young patients with DS or in the case with pre- paired axonal transport leads to backup and concentra- clinical, presenilin-related AD, suggesting that amyg- tion of ␣-synuclein in the cytoplasm, which predisposes dala LBs are not an early finding. to fibrillogenesis and aggregation. However, since AGD The reason why central nervous system proteins co- grains most commonly occur in dendrites, we do not think precipitate remains fertile ground for future research. A the process is triggered exclusively within the axon. Al- growing body of evidence suggests that one pathologic ternatively, the LBs may form as more of a general com- protein promotes fibrillation of other abnormal pro- pensatory response to cell stress or other perturbations teins.26 Herein, we focused on cases with tau as a pri- resulting from the accumulation of filamentous tau ag- mary aggregate. This study does not address mecha- gregates.17 nisms involved in secondary (tau) inclusion formation The reason why the amygdala is susceptible to LB for- in cases where ␣-synuclein is the primary aggregate, such mation is unknown. Tsuchiya et al18 studied 8 autop- as the Contursi kindred.27 The present study also does sied classic PiD cases and found that the amygdala showed not completely address whether ␤-amyloid deposition in- the most severe lesions of all basal ganglia regions. In our fluences amygdala LB formation. However, ␤-amyloid 3 cases with PiD and LBs in the amygdala, none had LBs deposition is not crucial for secondary LB formation in in the basal ganglia or brainstem (except for 1 case with the amygdala in PiD, because our affected PiD cases had LBs in the locus coeruleus). Yamazaki et al19 reported 5 little ␤-amyloid deposition. Future studies are also needed cases of Guam dementia complex with ␣-synuclein– to explain why the 2 types of pathology overlap only in positive inclusions in the amygdala reminiscent of cor- certain brain regions. It is feasible that all 3 pathologic tical LBs. These coexisted with tau-positive pretangles proteins (tau, ␣-synuclein, and ␤-amyloid) can pro- and/or neurofibrillary tangles. Overall, the amygdala can mote secondary inclusion formation under different cir-

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 cumstances. Future studies are needed to better under- detect Lewy bodies in many Down’s syndrome brains with Alzheimer’s disease. stand mechanisms by which abnormal processing Ann Neurol. 1999;45:353-357. 10. McKeith IG, Galasko D, Kosaka K, et al. Consensus guidelines for the clinical and and/or deposition of one central nervous system protein pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consor- is related to deposition of other central nervous system tium on DLB international workshop. Neurology. 1996;47:1113-1124. proteins. 11. National Institute on Aging and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer’s Disease. Accepted for Publication: June 18, 2004. Consensus recommendations for the postmortem diagnosis of Alzheimer’s disease. Neurobiol Aging. 1997;18(suppl 4):S1-S2. Correspondence: Carol F. Lippa, MD, Department of 12. Lund and Manchester Groups. Clinical and neuropathological criteria for fron- Neurology, Drexel University College of Medicine, 3300 totemporal dementia. J Neurol Neurosurg Psychiatry. 1994;57:416-418. Henry Ave, Philadelphia, PA 19129 (carol.lippa@drexel 13. Duda JE, Giasson BI, Gur TL, et al. Immunohistochemical and biochemical stud- .edu). ies demonstrate a distinct profile of alpha-synuclein permutations in multiple system atrophy. J Neuropathol Exp Neurol. 2000;59:830-841. Author Contributions: Study concept and design: Popescu, 14. Tsuboi Y, Ahlskog JE, Apaydin H, Parisi JE, Dickson DW. Lewy bodies are not Lippa, Lee, and Trojanowski. Acquisition of data: Popescu increased in progressive supranuclear palsy compared with normal controls. and Lippa. Analysis and interpretation of data: Popescu, Neurology. 2001;57:1675-1678. Lippa, Lee, and Trojanowski. Drafting of the manuscript: 15. Takeda A, Hashimoto M, Mallory M, Sundsumo M, Hansen L, Masliah E. Popescu and Lippa. Critical revision of the manuscript for C-terminal alpha-synuclein immunoreactivity in structures other than Lewy bodies in neurodegenerative disorders. Acta Neuropathol (Berl). 2000;99:296- important intellectual content: Popescu, Lippa, Lee, and 304. Trojanowski. Obtained funding: Lippa. Administrative, tech- 16. Jensen PH, Hager H, Nielsen MS, Hojrup P, Gliemann J, Jakes R. Alpha- nical, and material support: Lippa. Study supervision: synuclein binds to tau and stimulates the protein kinase A–catalyzed tau phos- Popescu, Lippa, Lee, and Trojanowski. phorylation of serine residues 262 and 356. J Biol Chem. 1999;274:25481- 25489. 17. Armstrong RA, Cairns NJ, Lantos PL. Are pathological lesions in neurodegen- REFERENCES erative disorders the cause or the effect of the degeneration? Neuropathology. 2002;22:133-146. 1. Baba M, Nakajo S, Tu PH, et al. Aggregation of alpha-synuclein in Lewy bodies 18. 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