Raymonde Porchet 1 Alphonse Probst 2 Constantin Bouras 3 Eduarda Dráberová 4 Pavel Dráber 4 Beat M. Riederer 1, 5 1 Institut de Biologie Cellulaire et de Morphologie, Lausanne, Switzerland 2 Institute of Pathology, University Hospital Basel, Switzerland 3 Department of Psychiatry, University of Geneva School of Medicine, Geneva, Switzerland 4 Institute of Molecular Genetics, Academy of Sciences, Prague, Czech Republic 5 Centre des Neurosciences Psychiatriques, Hôpital Psychiatrique, Prilly, Switzerland Analysis of gial acidic fibrillary protein in the human entorhinal cortex during aging and in Alzheimer’s disease Glial fibrillary acidic protein, GFAP, is a major intermediate filament protein of glial cells and major cytoskeletal structure in astrocytes. The entorhinal cortex has a key role in memory function and is one of the first brain areas to reveal hallmark structures of Alzheimer’s disease and therefore provides an ideal tissue to investigate incipient neu- rodegenerative changes. Here we have analyzed age- and disease-related occurrence and composition of GFAP in the human entorhinal cortex by using one- and two- dimensional electrophoresis, Western blots and immunocytochemistry combined with confocal microscopy. A novel monoclonal antibody, GF-02, was characterized that mainly reacted with intact GFAP molecules and indicated that more acidic and soluble GFAP forms were also more susceptible to degradation. GFAP and vimentin increased with aging and in Alzheimer’s disease (AD). Two-dimensional electrophore- sis and Western blots revealed a complex GFAP pattern, both in aging and AD with different modification and degradation forms. Immunohistochemistry indicated that reactive astrocytes mainly accumulated in relation to neurofibrillary tangles and senile plaques in deeper entorhinal cortex layers. GFAP may be used as an additional but not exclusive diagnostic tool in the evaluation of neurodegenerative diseases because its levels change with age and respond to senile plaque and tangle formation. Keywords: Astrocytes / Cytoskeleton / Tangles / Two-dimensional gel electrophoresis / Vimentin / Western blots PRO 0456 1 Introduction Astrocytes have a variety of functions and form develop- mental scaffolds, provide boundaries and guidance for neuronal migration, and a structural, metabolic and trophic support to neurons [1]. Most importantly, they are closely linked to neuron energy metabolism and synaptic activity [2, 3]. Furthermore, any type of central nervous system injury may stimulate astrocytes to proliferate, to hypertrophy and to build glial filaments. Astrogliosis is also well known to occur in Alzheimer’s disease (AD) [4– 6]. AD is a dementing illness which clinically is character- ized by intellectual decline and morphologically by senile plaques, dystrophic neurites and neurofibrillary tangles (NFT). Presently, the pathological diagnosis of AD is mainly based on the distribution and density of NFT [7], and both, senile plaques and NFT, have been shown to be correlated with the severity of dementia in AD [8–10]. Braak and colleagues [7] also have identified the ento- rhinal cortex as a pivotal area among affected brain regions in AD, since the very first neurofibrillary lesions are detect- able here and together with the temporobasal isocortex belong to regions where the first senile plaques are formed [10]. The pathophysiological mechanisms respon- sible for plaque formation and progression are emerging. Glial cells, and among them astrocytes and microglia are likely to play a key role in the formation of senile plaques [11]. Astrocytes were found to be associated with many but not all senile plaques [12]. In addition to the classical lesions, astrocytosis is a well known feature of AD and the association of astrocytes with plaques is well established [13, 14]. Astrocytes colocalize with a dense amyloid core and an attendant microglial reaction via interleukin (IL)1b hypertrophy of astrocytes [12]. Confocal microscopy and 3-D reconstruction have demonstrated that astrocytes surround plaques and many astrocytic processes pene- trate into the plaque core [15]. Nevertheless, such astro- cytic reactions may occur during the normal aging pro- cess, independently of the formation of senile plaques. The GFAP is the building block of glial intermediate fila- ments and is the major cytoskeletal structure in astro- cytes [16]. Human GFAP is composed of 432 amino acids Correspondence: Dr. B. M. Riederer, Institut de Biologie Cellu- laire et de Morphologie, Rue du Bugnon 9, 1005 Lausanne, Switzerland E-mail: beatmichel.riederer@ibcm.unil.ch Fax: +41-21-692-5105 Abbreviations: AD, Alzheimer’s disease; APP , amyeloid precur- sor protein; GFAP , glial acidic fibrillary protein; NFT neurofibrillary tangle; PHF , paired helical filament; pmd, post mortem delay 1476 Proteomics 2003, 3, 1476–1485 DOI 10.1002/pmic.200300456  2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim