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Neuroscience Letters, 129 (1991) 119-122 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS030439409100411K NSL 07927

Relationship between non-fibrillary amyloid precursors and cell processes in the cortical neuropil of Alzheimer patients Khalid H a m i d E1 Hachimi 1, Laura Verga 2, Giorgio Giaccone 2, Fabrizio Tagliavini 2, Bias Frangione 3, Orso Bugiani 2 and Jean-Francois Foncin 1 1Laboratoite de Neurohistologie EPHE and U.106 INSERM, La Salpdtri~re, Paris (France), 2lstituto Neurologico Carlo Besta, Milan (Italy) and aNew York University School of Medicine, New York, N Y (U.S.A.). (Received 13 February 1991; Revised version received 15 April 1991; Accepted 29 April 1991)

Key words: Alzheimer's disease; Amyloid; lmmunoelectronmicroscopy; Preamyloid deposit; Postsynaptic element; Golgi We examined the ultrastructural localization of amyloid p-protein in 8 Alzheimer neocortical biopsies. Intense immunoreactivity was located extracellularly on amyloid fibrils and amorphous material. Amorphous labelled material was also found in cell processes. No ultrastructural cell marker, such as glial fibrils, glycogen, tubules, paired helical filaments (PFHs) or synaptic vesicles could be seen in these processes that could allow their identification as glial processes, neurites or presynaptic terminals, respectively; occasional membrane stacks were observed. These findings suggest that preamyloid deposits are related to cell processes and, by elimination, that postsynaptic terminals may be involved in abnormal metabolism of the amyloid fibril precursors.

Antibodies to amyloid r-protein [12] isolated from the wall of congophilic cerebral vessels and senile plaque (SP) cores, and antibodies to relevant synthetic ]/-protein fragments, recognize cerebral amyloid of SP and congophilic angiopathy (CA) in patients with Alzheimer's disease (AD) or Down's syndrome (DS) [1, 17, 23]. Besides SP and CA, anti-r-protein antibodies label extracellular preamyloid deposits that differ from plaques in that they are non-birefringent, unstained with thioflavine S, and unrelated to neurites bearing abnormal filaments [2, 3, 7, 11, 19]. According to the immunohistochemical and optical properties of preamyloid deposits, the hypothesis has been put forward that these deposits are due to the accumulation of cleavage products of the amyloid precursor protein (APP) that include the r-protein region [2, 3, 11, 19]. Substantial evidence favouring this view was obtained from further light and electronmicroscopical (EM) immunohistochemical studies showing that preamyloid deposits are made up of electrondense, flaky and irregularly distributed material, and do not react with antibodies to the glycoprotein P-component which is bound to amyloid fibrils [20]. In the above-mentioned studies, tissue samples from autopsy specimens were used. In such material, plasma membranes are discontinCorrespondence: K.H. E1 Hachimi, Laboratoire de Neurohistologie EPHE and U.106 INSERM, Hrpital de la Salp~tri~re, 75651 Paris Cedex 13, France.

uous; therefore, the precise relationship between amorphous labelled material and neuropil structures could not be determined; preamyloid deposits were generally described as extracellular. Knowledge of this relationship would be helpful in identifying elements involved in amyloid deposition. This problem was addressed by means of an immunoEM study of biopsy specimens obtained from the second frontal gyrus of the right hemisphere of 8 Alzheimer patients aged 52-71 years, neuropathologicaUy confirmed by the presence of numerous plaques and tangles on routine light microscopy sections. Most patients were at an early stage of evolution: most biopsies were done before CTscan made easier, by elimination of focal lesions, a diagnosis of degenerative dementia, and restricted diagnostic biopsy indications. Tissue specimens, fixed in Karnovsky's solution, postfixed in osmium tetroxyde and embedded in Araldite, were processed for immuno-EM following a postembedding procedure described elsewhere [6]. Briefly, ultrathin sections were mounted on gold grids, incubated with normal goat serum, anti SP28 (a rabbit polyclonal antibody to the 28 residue synthetic peptide homologous to the NH2-terminal region of r-protein [4]) 1:40 overnight at 4°C, reincubated with goat anti-rabbit serum conjugated to colloidal gold (Auroprobe GARG 15 Janssen, 1:60, 2 h at room temperature), stained with uranyl acetate and lead citrate, and finally observed with a Siemens 102 electronmicroscope. The specificity of immunostaining was con-

120 trolled by incubating control sections with Tris buffer or preimmune serum as primary antibody. Control biopsies, two from patients with normal pressure hydrocephalus and two from patients with Creutzfeldt-Jakob disease, processed using the same techniques did not show any immunoreactivity against the anti-fl-amyloid. Background labelling was minimal. Immunogold particles were found to decorate both extra- and intracellular structures. Extracellular structures were fibrillary or amorphous. Fibrils, 4-8 nm in size, were arranged in bundles (Fig. 1A,B) or intermingled with amorphous material also labelled by immunogold (Fig. 1B,D). Fibrillary bundles were often located close to cell processes containing dense or lamellar bodies and/or paired helical filaments (PHFs) (Fig. 1A,B,C). Immunolabelled amorphous material was irregularly distributed within abnormally enlarged extracellular spaces surrounded by anonymous cell processes (Fig. 1C). On some occasions immunogold was found to decorate micellar particles or membrane profiles in cell processes containing dense bodies and mitochondria (Fig. 1E). A few of the labelled processes showed densities at the inner side of their limiting membrane (Fig. 1F). No immunolabelling of neuronal, microglial or astrocytic cell bodies was observed. The following points deserve comment. (1) Non-fibrillary material vs amyloid fibrils. Our EM findings confirm that anti-r-protein antibodies bound to colloidal gold particles label not only amyloid fibrils, but also electrondense, amorphous material. The latter was found intermingled with disarrayed amyloid fibrils, but not with fibril bundles in the plaque cores, and displayed the flaky, highly irregular configuration known to be peculiar to the material that accumulates in preamyloid deposits [20]. Our EM findings in cortical biopsies of Alzheimer patients confirm that amyloid non-fibrillary precursors are abundantly present in neuropil devoid of other major changes: PHFs, in particular, were not seen in these regions. Further, they confirm the colocalization of non-fibrillary amyloid precursors and amyloid fibrils in the cortical neuropil and support the view that in vivo the latter might originate from the former, as it was found to occur in vitro [4, 14]. (2) Intracellular vs extracellular immunoreactive material. Whether mixed or not with disarrayed fibrils, extracellular amorphous material labelled with gold particles was observed within enlarged extracellular spaces outlined by cell processes rather than within cell bodies. This observation provides support for the view that amyloid non-fibritlary precursors derive from APP molecules, the density of which is higher in the cell membrane of neuronal processes than in perikaryonal membranes [8, 15, 18]. More surprisingly, intracellular immunogold labelling was occasionally observed, decorating

linear profiles and micellar particles in cell processes that often also contained dense bodies and mitochondria. Since the APP molecule lies across the cell membrane [8, 13, 21] and the r-protein region does not correspond to the intracellular portion of APP [8, 13, 21], this finding could not be easily explained. However, APP is known to be associated also with intracellular membranes [5, 24]; furthermore, it is known that lamellar and dense bodies in the distal portion of neuronal processes increase in number as neurites involved in the plaque formation degenerate [22]. Therefore, the intracellular antir-protein immunoreactivity found in cortical specimens of Alzheimer patients might have been due to increased intracellular protein turnover. The question then arises of the origin of the cell processes associated with antip-protein immunoreactivity. Our starting hypothesis favoured degenerating presynaptic terminals [10]. In fact, the nature of the involved processes could not be positively assessed. No cell marker at the ultrastructural level, such as glial fibrils, glycogen, tubules and PHFs or synaptic vesicles, was found which could identify glial processes, neurites or presynaptic terminals, respectively, as the site of synthesis and deposition of intracellular amyloid non-fibrillary precursors. In some instances, mitochondria, stacks of membranes, and densities adjoining the plasma membrane were observed in processes containing intracellular immunoreactive material. Thus, negative ultrastructural criteria led us to tentatively identify immunolabelled processes as postsynaptic elements, which is not contradicted by the few positive criteria available. This interpretation may suggest two opposite explanations: postsynaptic elements are selectively involved in APP abnormal metabolism, or they are less vulnerable than other cell structures to the dystrophic or degenerative changes that accompany and follow the deposition of amyloid non-fibrillary precursors and of amyloid fibrils. The former view might be supported by the molecular structure that makes APP resemble receptors on the post-synaptic cell membrane [8, 13]. The latter would be in disagreement with the loss of spines observed in the apical dendrites of cortical pyramidal neurons in Alzheimer patients [9, 16], (Fig. 1G,H). In any case, it is noteworthy that no relationship was observed between amorphous material labelled by immunogold and cell processes bearing PHF. This observation favours the hypothesis, put forward from the results of immunohistochemical studies at the light microscopy level [3], that cytoskeletal changes follow the deposition of amyloid non-fibrillary precursors and amyloid fibrils. In conclusion, the present study showed that, in AD, deposition of amyloid non-fibrillary precursor occurs diffusely in the cortical neuropil. Deposits were related

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Fig. 1. A-F: immunogold electronmicroseopy with anti-SP28 antibody (Postembedding procedure, 15 nm colloidal gold particles); G-H: rapid Oolgi method. A ( x 22,600), B (28,300), senile plaque: irnmunogold particles decorate amyloid fibril bundles (bf), note cell processes with synaptic vesicles (sv), degenerating neurites with paired helical filaments (PHF) or without paired helical filament (n) or astroglial processes with glycogen inclusions (g). C ( x 44,200): preamyloid deposit: immunogold particles decorate electrondense amorphous material that is accumulated in enlarged extracellular space outlined by cell process profiles. D ( x 44,200): labelled amorphous material, the nature of the enlarged and burst process cannot be precised. E ( x 22,600): intracellular immunogold labelling of membrane profiles and micellar particles in an enlarged cell process containing dense bodies and mitochondria. F ( x 33,900): diffuse immunogold labelling of a cell process adjacent to a presynaptic terminal provided with unaffected synaptic junction (arrow). G ( x 730): loss of dendritic spines in the apical dendrite of a cortical pyramidal neuron from a patient with Alzheimer's disease, as compared with non-demented, age-matched control (H, x 730).

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to cell processes and were both extra- and intracellular. Extracellular precursors were often mixed with disarrayed amyloid fibrils. Occasional cell processes contained labelled material. By elimination of other hypotheses as well as from a few instances of direct observation of landmarks, these processes were tentatively identified as altered postsynaptic elements. This conclusion is in accordance with observations of dendritic spine loss as demonstrated with Golgi methods. Supported in part by the Italian Ministry of Health, Department of Social Services (to O.B.), the U.S. National Institutes of Health (Grants AG05891 and AG08721 to B.F.), Convention INSERM-CNR, U.F.R. Piti6-Salp~tri6re and La Fondation de France (to J.F.F.). I Allops, D., Landon, M., Kidd, M., Lowe, J.S., Reynolds, G.P. and Gardner, A., Monoclonal antibodies raised against a subsequence of senile plaque core protein react with plaque core, plaque periphery and cerebrovascular amyloid in Alzheimer's disease, Neurosci. Lett., 68 (1986) 252-256. 2 Bugiani, O., Giagcone, G., Frangione, B., Ghetti, B. and Tagliavini, F., Alzheimer patients: preamyloid deposits are more widely distributed than senile plaques throughout the central nervous system, Neurosci. Lett., 103 (1989) 263-268. 3 Bugiani, O., Oia~one, G., Verga, L., Polio, B., Ghetti, B. and Tagliavini, F., Alzheimer patients and Down patients: abnormal presynaptic terminals are related to cerebral preamyloid deposits, Neurosci. Lett., 119 (1990) 56--59. 4 Castafio, E.N., Ghiso, J., Pirelli, F., Gorevic, P.D., Migheli, A. and Frangione, B., In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to Alzheimer's disease/~-protein, Biochem. Biophys. Res. Commun., 114 (1986) 782789. 5 Catteruccia, N., Willingale-Theune, J., Bunke, D., Prior, R., Masters, C.L., Crisanti, A. and Beyreuther, K., Ultrastructural localization of the putative precursors of the A4 amyloid protein associated with Alzheimer's disease, Am. J. Pathol., 137 (1990) 19-26. 6 Defossez, A., E1 Hachimi, K.H., Beauvillain, J.-C., Perre, J., Delacourte, A. and Foncin, J.-F., Etude immunocytochimique l'6chelle ultrastructurale des d6g~n6rescences neurofibrillaires clans la maiadie d'Alzheimer, C.R. Acad. Sc., S6rie III, (1987) 217-222. 7 Deleare, P., Duyckaerts, C., Masters, C., Beyreuther, K., Piette, F. and Hauw, J.J., Large amounts of neocorticai ~8A4deposits without neuritic plaques nor tangles in a psychometrically assessed, non-demented person, Neurosci. Lett., 116 (1990) 87-93. 8 Dyrcks, T., Weidemann, A., Multhaup, G., Saibaum, J.M., Lemaire, H.G., Kang, J., Muller-Hill, B., Masters, C.L. and Beyreuther, K., Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer's disease, EMBO J., 7 (1988) 949-957. 9 El Hachimi, K.H. and Foncin, J.F., Perte des ~pines dendritiques dans la maladie d'Alzheimer, C.R. Acad. Sci., S~rie III, (1990) 397402.

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Relationship between non-fibrillary amyloid precursors and cell processes in the cortical neuropil of Alzheimer patients.

We examined the ultrastructural localization of amyloid beta-protein in 8 Alzheimer neocortical biopsies. Intense immunoreactivity was located extrace...
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