Pergamon 0361-9230(95)02006-D Brain ResearchBulletin,Vol.38, No. 5, pp. 417-423, 1995 Copyright© 1995Elsevier ScienceInc. Printed in the USA.All rightsreserved 0361-9230/95 $9.50 + .00 fl-Amyloid Precursor Protein (flAPP) in Human Gut With Special Reference to the Enteric Nervous System A. CABAL, V. ALONSO-CORTINA, L. O. GONZALEZ-VAZQUEZ, F. J. NAVES, M. E. DEL VALLE AND J. A. VEGA 1 Departamento de Morfologia y Biolog[a Celular, Facultad de Medicina, Universidad de Oviedo, C~ Julian Claverfa, s/n, E-33006 Oviedo, Spain [Received 31 January 1995; Revised 15 May 1995; Accepted 21 June 1995] ABSTRACT: The distribution of the ~-amyloid precursor protein ~APP) in the human gastrointesUnal tract, from esophagus trough rectum, was studied using immunoblotting, as well as combined immunohistochemical and image analysis (optic mi- croclensitometry) techniques. The study was focused on the en- teric nervous system. ~APP was detected by means of a mono- clonal antibody (22Cll), which recognizes all/3APP isoforms as well as ~APP-like proteins. Immunoblo1L13ng revealed two main protein bends, one corresponding to full-length ~APPs (asti- mated molecular masses of ~ 97-115 kDa); the other corre- sponcled to a protein with estimated molecular masses of 55 kDa. Specific ~APP immunoreactivity (IR) was found in the sub- mucous and myenteric plexuses localized in the supporting glial calls rather than in neurons. Differences were encountered nei- ther in the localization nor in the intensity of immunostaining among different segments of the gastrointestinal tract. More- over, no age-dependent changes were found, jUAPP IR was also regularly observed in blood vessels, primarily labelling endothe- lial cells. Our results provide evidence for the occurrence of ~APP in human gastrointestinal tract of healthy people in both neuronal and nonneuronal tissues. Whether or not these findings have functional or clinical relevance remains to be clarified in future studies. KEY WORDS:/3-Amyloid precursor protein ~APP), Gut, Enteric nervous system, Immunohistochemistry, Human. INTRODUCTION The fl-amyloid precursor protein (/3APP) is regarded as the main source of the/3-amyloid peptide, which typically accumulates in brain and cerebral blood vessels of nondemented elderly subjects, and of patients undergoing several neurodegenerative disorders, especially Alzheimer' s disease [2,6,14,16,17]. Five isoforms of /3APP (/3APP563, /3APP695, /3APP714, /3APP75j, and/3APP770) are derived from a single gene by alter- native splicing, all containing the /3-amyloid amino acidic se- quence [see ref. 20]. However, the mechanisms involved in the /3APP metabolism have not yet been fully elucidated, and both amyloidogenic and nonamyloidogenic pathways exist [7,19-21 ]. /3APP mRNAs and/3APPs are widely distributed in the pe- ripberal nervous system of mammals, including man. They have To whom requests for reprints should be addressed. been found in nerve cell bodies and supporting glial cells of sym- pathetic and dorsal root ganglia, as well as in axons and Schwann cells of peripheral nerves [1,3,11-13,15,18,24]. However, data concerning the presence of/3APP or fl-amyloid peptide in the other main division of the peripheral nervous system, that is, the enteric nervous system (ENS; see for a review [5]) are very scarce. Arai and coworkers [1] observed occurrence of/3APP immunoreactivity (IR) in the enteric ganglia extending from esophagus through transverse colon, whereas the other gastro- intestinal tract tissues were never labelled. On the other hand,/3- amyloid peptide has been localized in the submucosa of the in- testine of Alzheimer's disease patients, primarily around microvessels and perivascular connective tissue [9]. This article reports the immunohistochemical localization of/3APP in the gastrointestinal tract, sampled from the esophagus to the rectum, of adult and old subjects free of neurologic disease. The study was focused on the enteric nervous system. We used a mono- clonal antibody (clone 22C11), which maps an epitope localized between residues 66 and 88 of the N-domain of/3APP [8]. To ascertain the localization of/3APP in enteric ganglia, neurofila- ment proteins and S-100 protein were studied in parallel [3,15]. MATERIALS AND METHODS Tissue Treatment Samples of the gastrointestinal tract from esophagus to rectum were obtained at surgery from patients of different ages without neurological disease. Specimens were from Servicio de Anato- mia Patologica, Hospital Central of Oviedo, Spain. The number of specimens available from the various levels of the gastroin- testinal tract, as well as the age and gender of the examined sub- jects are given in Table 1. Most of the pieces were routinely fixed in buffered 10% formaldehyde, dehydrated, and embedded in paraffin. Moreover, representative specimens of jejune and colon (see Table 1) were fixed in 10% formaldehyde, Bouin's, and Zamboni's fixatives for 12 h and cryosectioned at -20°C to eval- uate the effect of the fixative and of the paraffin-embedding on the expression of/3APP immunoreactivity (IR). Finally, six sam- ples of small intestine were frozen, stored at -80°C until use, and used for immunoblotting analysis. 417