Expression of chromogranin A in lesions in the central nervous system from patients with neurological diseases

Expression of chromogranin A in lesions in the central nervous system from patients with neurological diseases

ELSEVIER Neuroscience Letters 170 (1994) 13 16 NEUROSCiENC[ LETT[RS Expression of chromogranin A in lesions in the central nervous system from pati...

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Neuroscience Letters 170 (1994) 13 16


Expression of chromogranin A in lesions in the central nervous system from patients with neurological diseases Osamu Yasuhara, Toshio Kawamata, Yoshinari Aimi, Edith G. McGeer, Patrick L. McGeer* Kinsmen hlhoratory o/' Neurological Research and The Neurodegenerative Disorders Centre, University q/' British Cohmd~ia. 2255 l~'shrm)k Mall, VancouveJ; BC V6T 1Z3. Canada Received 13 December 1993: Revised version received 21 January 1994: Accepted 21 January 1994

Abstract Expression of chromogranin A in various neurological diseases was examined immunohistochemically using purified anti-human chromogranin A antiserum. The antibody stained dystrophic neurites in senile plaques in Alzheimer disease brain, Pick bodies and ballooned neurons in Pick's disease brain, some Lewy bodies in the substantia nigra of Parkinson's disease, and axonal swellings in various neurological conditions including Parkinson's disease, striatonigral degeneration, Shy-Drager syndrome, amyotrophic lateral sclerosis and cerebral infarction. The present study shows that expression of chromogranin A is not an exclusive feature of Alzheimer disease or Pick's disease, and indicates that it could be a useful marker for various neurological diseases. Key words. Chromogranin A: Alzheimer's disease: Pick's disease: Amyotrophic lateral screlosis: Multiple system atrophy: Dystrophic neurite; Pick body: Spheroid body: Torpedo formation

Chromogranin A (CgA), which was initially isolated as the major soluble protein of adrenal medullary chromaffin granules, is a member of a family of acidic proteins present in secretory vesicles of endocrine and neuronal cells [3,6]. Its function is still unclear. However, it has been proposed that CgA is associated with granule packaging, and calcium binding [12], or is a precursor of biologically active peptides [1 I]. Recent investigations have demonstrated increased levels of CgA in Alzheimer's disease (AD) [18], with localization in senile plaque dystrophic neurites [1,4,9,18]. An analogous finding has been reported for Pick's disease by Weiler et al. [18], who demonstrated that Pick's disease brain also contains an augmented level of CgA and that almost all Pick bodies are immunoreactive for CgA. CgA is a substrate of acetylcholinesterase [15], which has been implicated in the pathogenesis of AD [17]. In addition, the human CgA gene is located on chromosome 14q [5], a region implicated in processing of amyloid precursor protein (APP), and linked to early onset, familial AD [14].

*Corresponding author. Fax: (1) 604-822-7086. 0304-3940/94/$7.00 :~:~1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00084-N

The aim of the present study was to determine whether increased expression of CgA is an exclusive characteristic of these two dementing diseases or whether it also occurs in other degenerative neurological diseases. We carried out immunohistochemical studies in brains with various neurological conditions, including AD, Pick's disease, Parkinson's disease, striatonigral degeneration (SND), Shy-Drager syndrome (SDS), amyotrophic lateral sclerosis (ALS), and cerebral cortical infarction. Brains from 4 cases of AD (aged 67 82), 2 cases of Pick's disease (aged 64 and 75), 4 cases of Parkinson's disease (aged 69-80), 1 case of SND (age 55), 1 case of SDS (age 55), 1 case with cerebral infarction (age, 40), and the spinal cord from 2 cases of ALS (aged, 48 and 75) were obtained from 2 to 24 h ariel" death. Brains and spinal cords from six cases without neurological disorder (aged, 58 82) were used as controls. The diagnosis was based in every case on both clinical and pathological criteria. Small blocks of tissue were dissected from the hippocampus and neocortex of AD, the hippocampus and middle temporal gyrus of Pick's disease, the substantia nigra of Parkinson's disease, the cerebellum of SND and SDS, the lumbar spinal cord of ALS, and the parahippocampal gyrus having a small cortical infarc-


O. Yasuhara et ai./:\eto'oscien~e Letters 17(I ~ 1994) 1 3 16


Fig. 1. Chromogranin A (CgA)-immunoreactive structures in various neurological diseases. A: dystrophic neurites in senile plaques in the dentate gyrus of AD brain. B: Pick bodies in neurons in the dentate fascia in Pick's disease brain. C: ballooned neurons in the Vth layer of the parahippocampal gyms of Pick"s disease brain, showing diffuse staining. D: a Lewy body in the substantia nigra of Parkinson's disease. E -H: axonal swellings intensely stained for CgA in the substantia nigra of Parkinson's disease (E), in the granule cell layer of the cerebellum of multiple system atrophy (torpedo formation) (F), in the anterior horn of amyotrophic lateral sclerosis (GI and in the crebral infarction (H). Bars = 50 lma. t i o n f r o m a 4 0 - y e a r - o l d p a t i e n t . C o r r e s p o n d i n g tissues f r o m all these r e g i o n s w e r e t a k e n f r o m c o n t r o l s . T h e

b l o c k s w e r e fixed in 4 % p a r a f o r m a l d e h y d e in 0.1 M p h o s p h a t e buffer, p H 7.4, for 2 - 3 d a y s a n d t h e n t r a n s -

O. Yasuhara et al./Neuroscience Letters 170 (1994) 13 16

ferred to a maintenance solution of 15% sucrose in 0.01 M phosphate-buffered saline (PBS), pH 7.4. Sections were cut on a freezing microtome at 30/*m thickness, collected in maintenance solution, and stored until stained. Following pretreatment for 30 min with 0.3% H2Oe solution in PBS containing 0.3% Triton X-100 (PBS-T) to eliminate endogenous peroxidase activity, sections were incubated for 72 h in the cold with antiCgA antibody at a concentration of 2/ag/ml in PBS-T and 0.5% normal goat serum. The rabbit anti-human CgA (Dakopatts) is the purified immunoglobulin fraction of rabbit antiserum raised against the C-terminal half of CgA purified from urine of patients with carcinoid syndrome. The sections were next treated with biotinylated anti-rabbit IgG (Vector Lab., Burlingame, CA; diluted 1:1,000) for 2 h at room temperature, followed by incubation in the avidin-biotinylated HRP complex (ABC Elite, Vector Lab.) for 1 h at room temperature. Peroxidase labeling was visualized by incubating with a solution containing 0.01% 3,3'-diaminobenzidine (DAB, Sigma), 0.6% nickel ammonium sulfate. 0.05 M imidazole, and 0.00015% H202in 0.05 M TrisHCI buffer, pH 7.6. When a dark purple reaction product appeared, the reaction was terminated by transferring the sections to PBS-T. Sections were mounted on glass slides, dried, dehydrated, and coverslipped with Entellan (Merck). As controls, serum from a rabbit hyperimmunized with tobacco mosaic virus was substituted for the primary antibody: no positive staining was seen in any of the cases. In the central nervous system (CNS) of control cases, CgA-like immunoreactivity was observed in widespread. but selective populations of neuronal cell bodies and fiber systems, the distribution of which was compatible with that reported in bovine [10], ovine [16], and human CNS [6 8]. Thus, the present results focused on the pathological lesions immunoreactive for CgA. In keeping with previous reports, the anti-CgA antibody immunostained dystrophic neurites in senile plaques of AD brain, most of which had globular profiles (Fig. 1A). In Pick's disease brain, Pick bodies (Fig. 1B) and ballooned neurons (Fig. IC) were immunopositive for CgA. When immunostained sections were compared with adjacent sections stained by Bielschowsky's method, it was found that the antibody intensely stained Pick bodies in the dentate fascia and layers II, III, and VI of the parahippocampal and middle temporal cortices. However, Pick bodies in hippocampal pyramidal neurons were not, or were only faintly, stained with the antibody. In contrast to the punctate neuronal staining in control brain [4,8], ballooned neurons were stained rather diffusely, but weakly. In the substantia nigra of Parkinson's disease, Lewy bodies (Fig. 1D) and spheroid bodies (Fig. 1E) were immunoreactive for CgA. The staining of Lewy bodies was inconsistent and varied from case to case, while


spheroid bodies were consistently and intensely stained in every case. The presence of CgA immunoreactivity was a prominent feature shared by spheroid bodies in the other neurological coditions examined in the present study. Fig. 1F shows an axonal swelling o f a Purkinje cell (torpedo formation) immunoreactive for CgA. Such swellings were abundantly found in the granule cell layer of the cerebellum in the SND and SDS cases. Many CgA-positive spheroid bodies were also found in the anterior horn of the ALS cases (Fig. 1G) and in the periphery of the infarcted area in the stroke case (Fig. 1H). The present study shows that CgA immunoreactivity is not an exclusive feature of dystrophic neurites in senile plaques and Pick bodies, but is also present in Lewy bodies and axonal swellings in a variety of other conditions including Parkinson's disease, ALS, SND, SDS and cerebral infarction. Thus, CgA could be a usefnl histochemical marker for various neurological diseases. Weiler et al. [18] reported that all Pick bodies were intensely stained with antibodies against CgA. The present results confirm the presence of CgA immunoreactivity in Pick bodies, but differ somewhat from those of Weiler et al. In the present study, Pick bodies in nonpyramidal neurons in the dentate fascia and in the neocortex showed intense staining, while those in hippocampal pyramidal neurons showed little immunoreactivity for CgA. The reason for the discrepant results is unknown. However, the present study suggests that there may exist some antigenic differences between Pick bodies in hippocampal pyramidal neurons and those in nonpyramidal neurons. Disturbances ofaxonal flow and transport may underlie the formation of axonal spheroids [13]. Therefore, accumulation of CgA in axonal swellings is understandable, since CgA is a protein present in dense core vesicles, which undergo fast axonal transport [3]. In addition, it is reasonable to assume that the same mechanism underlies the formation of CgA-positive plaque-associated dystrophic neurites in AD brain. Although the significance of CgA immunoreactivity in Lewy bodies is uncertain, it is interesting to note that Lewy bodies also contain large numbers of vesicles in the periphery of the central core [2]. We thank Ms. J. Sunahara for excellent technical assistance. This research was supported by a grant from the Alzheimer Society of British Columbia, and by donations from individual British Columbians.

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[2] Duffy, RE. and Tennysom V.M., Phase and electron microscopic observations of Lewy bodies and melanin granules in the substan-


O. Yasuhara el aL/Neuroscience Letters 170 (1~94) 13 16

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