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 dis- ease 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 sec- tion in different diseases. ondary phenomenon that reflects the fibril- lation of ␣-synuclein induced directly by the formation of fibrillary tau lesions or indi- METHODS rectly by cell stress resulting from the for- mation 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- (REPRINTED) ARCH NEUROL / VOL 61, DEC 2004 WWW.ARCHNEUROL.COM 1915 ©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 oc- curred 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 cog- nitively 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).