A4 amyloid protein precursor derivatives in human cerebrospinal fluid

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Molecular Brain Research 27 (1994) 320-322

Short communication

Developmental changes of sialylation of soluble fl/A4 amyloid protein precursor derivatives in human cerebrospinal fluid Nobuyuki Sodeyama a, Fumiaki Saito a, Kayoko Saito b, Tadashi Miyatake ~, Katsuhiko Yanagisawa a,, a

Department of Neurology, School of Medicine, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113, Japan b Department of Pediatrics, Tokyo Women's Medical College, Kawata-cho 8-1, Shinjuku-ku, Tokyo 162, Japan

Accepted 30 August 1994

Abstract

Soluble f l / A 4 amyloid protein precursor derivatives (APPs) in cerebrospinal fluid from infants, children, adults and aged individuals were treated with neuraminidase. In the samples from infants, reduction of molecular weight of APPs following neuraminidase treatment was significantly less than those from adults or aged individuals. Hyposialylation of fl/A4 amyloid protein precursor in infants may be relevant to a physiological role of this molecule in the development of the nervous system. Keywords: Alzheimer's disease; f l / A 4 amyloid protein precursor; Sialic acid; Neuraminidase; Developmental change; Cerehrospinal fluid; Western blot analysis

Senile plaques are one of the neuropathological hallmarks of Alzheimer's disease (AD). f l / A 4 amyloid protein, a major component of senile plaques, is a cleaved product of a larger f l / A 4 amyloid protein precursor (APP). To date, aberrant processing of APP in AD has not been determined, and little is known of its physiological role. Recently we and another group reported the preferential localization of APP on the synaptic membrane [12,13]. We also found that the soluble APP derivatives (APPs) in cerebrospinal fluid (CSF) were sialylated [11]. Sialic acid has been reported to play a pivotal role in the regulation of m e m b r a n e - m e m b r a n e interaction [10]. The purpose of the present study is to determine the extent of sialylation of APP at different stages of human development so as to investigate the role of APP molecule in brain. CSF samples were obtained from 4 groups classified according to age: 6 infants with various diseases as follows: viral meningitis (1), bacterial meningitis (1), gastroenterocolitis (1), hydrocephalus (1), and convul-

* Corresponding author. Present address: Department of Neuropathology, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan. Fax: (81) 3-5800-6852. 0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0169-328X(94)00201-0

sions (2), (1, 3, 4, 5, 7, 8 months old); 9 children: somatization disorder (2), spinocerebellar degeneration (1), acute cerebellar ataxia (1), spastic paraplegia (1), carnitine deficiency (1), carnitine palmityl transferase deficiency (1), neuronal ceroid lipofuscinosis (1), and adrenoleukodystrophy (1), (7, 7, 8, 8, 9, 13, 14, 14, 15 years old); 11 adults: alcoholic neuropathy (1), epilepsy (1), cerebral infarction (1), consciousness disturbance with unknown etiology (1), astrocytoma (1), Parkinson's disease (1), pyelonephritis (l), somatization disorder (1), transient ischemic attack (1), primary lateral sclerosis (1), and brachial plexus neuropathy (1), (43, 43, 44, 46, 50, 52, 52, 55, 56, 56, 59 years old); and 11 aged individuals: cerebral infarction (3), Parkinson's disease (3), cervical spongylosis (1), polymyalgia rheumatica (2), lung cancer (1), and vertigo (1), (71, 73, 73, 74, 76, 76, 80, 80, 81, 81, 89 years old). Five hundred microliters of each sample was desalted by a NAP-5 column (Pharmacia) and lyophilized. Samples containing 25 mg of CSF protein were solubilized in 20 /.d of buffer A (20 mM sodium phosphate pH 7.0, containing 0.1% sodium dodecyl sulfate (SDS)) and denatured by boiling for 3 min. Samples were then incubated in buffer B (10 mM calcium acetate, 20 mM sodium phosphate, and 17 mU of neuraminidase (Sigma)), for 1 hour at 37°C. Proteins ifi the samples were separated by 8%

N. Sodeyama et aL / Molecular Brain Research 27 (1994) 320-322

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Fig. 1. The specificity of immunoreactivity with anti-APP antibody (NAPP). A major band of soluble APP derivatives was observed with an apparent molecular weight of 103 kDa. Twenty-five/zg of protein from CSF of a 68-year-old woman was electrophoresed and blotted. Blots were immunolabeled with NAPP (A) and antigen-absorbed NAPP (B).

SDS-polyacrylamide gel electrophoresis according to the method of Laemmli [7]. After electrophoresis, proteins were electroblotted onto nitrocellulose membranes by the method of Towbin et al. [14]. Membranes were immunolabeled with polyclonal antibody against the amino-terminal portion of APP(45-62) (NAPP) followed by reaction with horseradish peroxidaselabeled anti-rabbit IgG. Detection of the immunoreactive proteins was carried out with an enhanced chemiluminescence system (Amersham). On Western blots of preincubated CSF samples, two bands of APPs with apparent molecular weights of 125 kDa and 103 kDa were observed as our previous report [11]. We adopted only a major band as subject for the present study because a minor band was not consistently observed clearly enough to permit reliable molecular weight analysis. The specificity of immunoreactivity was confirmed by antigen-absorbed NAPP (Fig. 1). The molecular weight of the major band was constant among the samples from 4 groups classified according to age (data not shown). All samples showed a reduction of molecular weight of the major band by desialylation; however, the extent of decrement in infants was much less than those in adults or aged individuals. Fig. 2 shows the results of the samples from infant (A) and aged individual (B). The mean values of the reduction of molecular weight, which were calculated by means of plotting the relative mobility on the regression curve of the molecular weight standard, are as follows: infants, 3.9 + 0.4 kDa; children, 4.5 + 0.4 kDa; adults, 6.1 + 0.3 kDa; and aged individuals, 5.9 + 0.3 kDa (mean + S.E.M.). The reduction of molecular weight in infants compared with those in both adults and aged individuals was found to be statistically significant (Fig. 3).

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Fig. 2. Representative immunoblot of desialylation study. Twenty-five ~g of CSF protein from a 3-month-old infant (A) and an 88-year-old woman (B) were incubated with (2) or without (1) neuraminidase. Reductions of molecular weight were 2.4 kDa (A) and 6.8 kDa (B), respectively.

Since point mutations of the APP gene were discovered in familial AD [2,15], it has been strongly suggested that aberrant processing of APP may be closely linked to the primary cause of the disease. However, little is known of the physiological role of APP in brain. Previous in vitro studies suggested that APP may function in adhesion of membranes in cell to cell interaction [1,16]. We and other groups reported the preferential localization of APP at synaptic sites [8,12,13,17], suggesting that APP may be involved in synaptogenesis or regulation of synaptic activities. Since the main source of APPs in CSF is considered to be neuronal cells because microglias and astroc3,tes generate very small amounts of APPs [3], a developmental study of APPs in CSF is expected to provide information on the roles of APP in the formation of neuronal networks. In the present study, the extent of the reduction of molecular weight of APPs following neuraminidase treatment was found to be significantly

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Fig. 3. Quantitative analysis of the reduction of molecular weight following desialylation. CSF samples from infants (n = 6), children (n = 9), adults (n = 11), and aged individuals (n = 11) were treated with neuraminidase and analyzed by Western blotting with NAPP.

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smaller in the samples from infants than those from adults or aged individuals. This result indicates that APPs molecules in infant brain may be much less sialylated than those in adult or aged individual brains. Rutishauser et al. reported that the removal of sialic acid from neuronal membrane facilitated membranemembrane adhesion via a decrease of electrostatic repulsion [10]. Other investigators also demonstrated the enhanced membrane-membrane interactions by desialylation treatment [9]. One of the characteristics of infant brains, which differentiates these from the brains of adults or aged individuals, is marked synaptogenesis. Huttenlocher et al. reported that synaptic density in human brain increased rapidly from birth to one year of age, and subsequently decreased gradually to about 60% of the maximum between two years of age and adolescence [4-6]. From this line of evidence, one could speculate that hyposialylation of APPs in infant brain may contribute to the synaptogenesis through an adhesion of neuronal membrane. Very recently, Moya et al. demonstrated that expression of APP was developmentally regulated and correlated with synaptogenesis in the primary visual pathways of the hamster [8]. Currently we cannot conclude that APPs in CSF reflect only the metabolism at the synaptic site; however, the present result suggests that a role of APP in the development of the nervous system may be regulated via the extent of sialylation.

We thank Dr. Yasuo Ihara for providing the polyclonal antibody against APP, and Dr. Kenji Nihei for providing cerebrospinal fluid samples from infants and children. This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas, No. 04268104 from the Ministry of Education, Science and Culture, Japan.

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