Hemisynaptic distribution patterns of presenilins and β‐APP isoforms in the rodent cerebellum and...

Hemisynaptic Distribution Patterns ofPresenilins and b-APP Isoforms in theRodent Cerebellum and Hippocampus

CATHERINE RIBAUT-BARASSIN,1 SALIHA MOUSSAOUI,2 BERNARD BRUGG,3ANNE-MARIE HAEBERLE,1 GERDA HUBER,4 ASSUNTA IMPERATO,2

NICOLE DELHAYE-BOUCHAUD,3 JEAN MARIANI,3 AND YANNICK J. BAILLY1*1Laboratoire de Neurobiologie Cellulaire CNRS UPR 9009, Strasbourg, France

2Rhone-Poulenc Rorer, Centre de Recherche de Vitry-Alfortville, Vitry sur Seine, France3Laboratoire de Neurobiologie du Developpement, Institut des Neurosciences CNRS UMR 7624,

Universite Pierre et Marie Curie, Paris, France4F. Hoffmann-La Roche Ltd., Pharma Division, Preclinical CNS Research, Basel, Switzerland

KEY WORDS synapse; brain; rodent; immunocytochemistry; electron microscopy;Alzheimer’s disease

ABSTRACT Healthy brain neurons co-express Alzheimer’s disease (AD) relatedproteins presenilins (PS) and b-amyloid precursor protein (b-APP). Deposition ofb-amyloid and PS in the senile plaques of AD brain and their ability to interact in vitrosuggest that AD pathology could arise from a defect in the physiological interactionsbetween b-APP and PS within and/or between neurons. The present study compares theimmunocytochemical distribution of PS (1 and 2) and b-APP major isoforms (695 and751/770) in the synapses of the cerebellum and hippocampus of the adult rat and mouse.In the cerebellar cortex of both species, the four molecules are immunodetected in thepresynaptic or the postsynaptic compartments of synapses, suggesting that they are in-volved in interneuronal relationships. In contrast, PS and b-APP are postsynaptic inalmost all the immunoreactive synapses of the hippocampus. The different distributionpatterns of these proteins in cerebellar and hippocampal synapses may reflect specificphysiological differences, responsible for differential vulnerability of neurons to AD synap-tic pathology. Defective interactions between b-APP and PS at the synapses could impedethe synaptic functions of b-APP, inducing the selective loss of synapses that accounts forcognitive impairment in AD. Synapse 35:96–110, 2000. r 2000 Wiley-Liss, Inc.

INTRODUCTION

The precursor protein of the b-amyloid peptide (b-APP) is involved in neurotrophic functions duringsynaptogenesis and synaptic remodeling of mnemoniccircuits in the adult brain (Loffler and Huber, 1992;Moya et al., 1994; Huber et al., 1993, 1997; Mucke et al.,1994). The b-APP isoforms are transported antero-gradely to synaptic terminals (Simons et al., 1995)where they are recruited to the plasma membrane andsecreted (Selkoe, 1994; Bouillot et al., 1996). Oncereleased from the axons terminals, b-APP could reachthe postsynaptic dendrites by a transcytosis process(Simons et al., 1995). Transport, sorting, and traffickingof b-APP by the healthy neurons could require a directinteraction with presenilins (PS) at sites of the smooth

endoplasmic reticulum and Golgi apparatus (Shige-matsu et al., 1992; Kovacs et al., 1996; Lah et al., 1997;Haass and Selkoe, 1998) where the transmembrane

Contract grant sponsors: the ‘‘Fondation de la Recherche Medicale,’’ the‘‘Ministere de la Recherche et de l’Education’’ (ACC-SV4).

*Correspondence to: Yannick J. Bailly, CNRS UPR 9009, 5 rue Blaise Pascal,67084 Strasbourg, France. E-mail: [email protected]

Abbreviations: AD, Alzheimer’s disease; b-APP, b-amyloid precursor protein;APLP, amyloid precursor like protein; BC, basket cell; CF, climbing fiber; DCN,deep cerebellar nucleus; DG, dentate gyrus; EC, entorhinal cortex; FCS, fetal calfserum; FN, fastigial neurons; G, glia; GC, granule cell; GO, Golgi neuron; HRP,horseradish peroxidase; IGL, internal granular layer; ION, inferior olivaryneuron; IN, interneuron; KPI, Kunitz protease inhibitor; MF, mossy fiber; ML,molecular layer; PBS, phosphate buffer saline; PC, Purkinje cell; PCL, Purkinjecells layer; PD, Purkinje cell dendrite; PF, parallel fiber; PFT, perforant fiber; PR,Purkinje cell’s recurrent axon; PS, presenilin (1 and 2); PY, pyramidal neuron;SC, stellate cell; SF, Schaffer’s axon; SL, stratum lucidum; SLM, stratumlacunosum moleculare; SO, stratum oriens; SR, stratum radiatum.

Received 4 December 1998; Accepted 23 March 1999

SYNAPSE 35:96–110 (2000)

r 2000 WILEY-LISS, INC.

proteins PS 1 and 2 colocalize (Blanchard et al., 1997).At these sites, a physical interaction between PS andthe endoplasmic immature form of b-APP has beenrecently demonstrated (Weideman et al., 1997). Muta-tions in the genes encoding for the presenilins andb-APP interfere with b-APP metabolism, leading toincreased production of amyloid peptide AB1–42, amy-loid deposition, and neurodegeneration in Alzheimer’sdisease (AD) (Selkoe, 1994; Sandbrink and Beyreuther,1996; Beyreuther and Masters, 1997; Tomita et al.,1997). The increased production of amyloid peptide andp3 fragment in cultured PS1-deficient hippocampalneurons indicates that PS1 facilitates the proteolysis ofb-APP C-terminal fragments by g-secretase in normalneurons (De Strooper et al., 1998). As shown recently invitro, PS and b-APP could also interact between theplasma membranes of contacting cells (Dewji and Singer,1998; Weideman et al., 1997). Membrane topology andorientation(s) of neuronal PS are still controversial,although the eight-transmembrane-domain model be-comes more and more credible (Li and Greenwald,1998). Also the significance of direct interactions be-tween plasma membrane-bound b-APPs and full-length PS and/or endoproteolytic PS fragments (Kim etal., 1997) remains to be clarified. The physiologicalmechanism(s) altered by the PS mutations and thesubcellular sites of the active proteins and derivativesseem to be multiple (Duff et al., 1996; Simons et al.,1996; Hartmann et al., 1997; Zhang et al., 1998).Neuronal populations that are differentially vulnerableto AD express both PS (Moussaoui et al., 1996; Ushi-hara et al., 1996; Takami et al., 1997) and b-APP(Ouimet et al., 1994). Whereas cerebellar neurons seemto escape the acute stages of amyloid pathology in mostAD cases (see review in Larner, 1997), except in pa-tients with familial AD-linked PS1 mutation (Lemereet al., 1996), hippocampal neurons display severe pathol-ogy in all AD cases (Braak et al., 1993). In thesewell-known cerebellar (Larramendi and Victor, 1967;Palay and Chan-Palay, 1974) and hippocampal (Brownand Zador, 1990; Bernard and Wheal, 1994) neuronalcircuits, the synaptic location of PS1, PS2, and thedifferent b-APP isoforms (695 and 751/770) were exam-ined at the ultrastructural level in young adult rats andmice. We used detergent-free pre-embedding immuno-cytochemistry which enabled us to recognize the type-specific ultrastructural features of the synapses underinvestigation.

MATERIALS AND METHODSTissue preparation

Long Evans rats (n 5 4) and C57Bl6 mice (n 5 2)were anaesthetized and perfused for 20 min on day 45(P45) with a sodium phosphate (0.1 M) buffered solu-tion (pH 7.4) of paraformaldehyde (4%), glutaraldehyde(0.3%), and CaCl2 (0.1%). All animals were maintained

in accordance with the NIH Guidelines of the US andthe European Communities Council Directive of Novem-ber 24, 1986 (86/609/EEC). After 1 h at 4°C, the brainswere dissected and postfixed for 4 h in the same cold,fixative solution without glutaraldehyde.

Sagittal and transverse sections (40 µm) through thecerebellar vermis, the inferior olivary nucleus, thedentate gyrus and the CA3 field of the hippocampuswere cut with a vibratome (Energy Beam Science,Agawam, MA) and collected in PBS. Some sectionswere also cut through the mouse kidney, where PSexpression has been detected previously (Lee et al.,1996).

Antisera

Seven previously characterized antisera were used:three rabbit polyclonal antisera directed respectively tothe N-terminal and to the C-terminal third cytoplasmicloop of PS1 (Moussaoui et al., 1996) and to the N-terminal of PS2 (Blanchard et al., 1997), a rat monoclo-nal antibody directed to the N-terminal of PS1 (Chemi-con International, Temecula, CA), two mousemonoclonal antibodies directed respectively to N-terminal of b-APP695 (Loffler et al., 1994) and of b-APPholoprotein (22C11, Boehringer Mannheim, Germany),a monospecific rabbit antiserum directed to the Kunitzprotease inhibitor (KPI) domain of b-APP751/770 (Loff-ler and Huber, 1992; Huber et al., 1997). The antibodiesto b-APP751/770 isoforms may cross-react with othersynaptic proteins that contain the KPI domain, such asamyloid precursor-like protein 2 (APLP2) isoformswhich are present in cerebellar and hippocampal syn-apses (Thinakaran et al., 1995). Also, the 22C11 mono-clonal antibody recognizes both APP and APLP iso-forms (Slunt et al., 1994).

Pre-embedding immunocytochemistry

Unspecific sites were blocked by pre-incubating thevibratome sections in 3% fetal calf serum (FCS, SigmaBiosciences, St Louis, MO) in 0.1 M PBS, pH 7.4, for 45min. In addition, a few sections from cerebellum, hippo-campus and kidney were treated with H202 1% for 30min. This suppressed the histochemical staining result-ing from endogenous peroxydase activity in the kidneysections without obvious effect on the light and electronmicroscopic patterns of b-APP and PS immunoreactiv-ity in the brain tissues. The sections were then incu-bated overnight at 4°C in the same buffer with 0.3%FCS to which one of the following had been added: a1:500–1,000 dilution of one of the two PS1 rabbitantisera; a 1:500–1,000 dilution of the PS1 monoclonalantibody; a 1:500 dilution of the PS2 rabbit antiserum;the b-APP695 monoclonal antibody, used as undilutedhybridoma supernatant; a 1:100–400 dilution of theb-APP751/770 rabbit antiserum; a 1:100 dilution of the

97SYNAPTIC PS AND b-APP IN RODENT BRAIN

22C11 b-APP monoclonal antibody. A few sections werepermeabilized with 0.1–0.3% Triton X-100 (Sigma)added to the pre-incubation and incubation media witheach antibody in order to improve the accessibility tothe intracellular antigens. However, modifications ofthe subcellular localization of the immunoreaction prod-uct described below in the nonpermeabilized tissueswere not detected in the permeabilized tissues (notshown). Negative controls were performed by incubat-ing sections in FCS-PBS to which no antiserum or theantiserum preadsorbed with its specific peptide-anti-gen (1 mg/ml PS1, 2 mg/ml PS2) had been added(Moussaoui et al., 1996; Blanchard et al., 1997). Immu-nocytochemical labeling of the tissue-bound primaryantibodies was performed using the ABC method (Hsuet al., 1981). All sections were rinsed in PBS andfurther incubated in species-specific biotinylated goatanti-rabbit (PS1, PS2, b-APP751/770), anti-mouse (b-APP695, b-APP) and anti-rat (PS1) IgG in PBS (1:200dilution, Vector Labs, Burlingame, CA) for 1 h, rinsedagain and incubated with avidin-biotinylated horserad-ish peroxidase (HRP) complexes (Vectastain ABC, Vec-tor Labs) in PBS for 30 min. The enzyme activity ofHRP linked to the specific sites was revealed by incubat-ing the sections in 3,38-diamino-benzidine tetrahydro-chloride (Fast DAB, Sigma) for 45 sec. Over that time,DAB increased the intensity of the immunostainingmasking the ultrastructural content of the stainedprocesses but did not modify remarkably the light andelectron microscopic patterns (not shown). Some of theimmunoreacted sections were briefly rinsed in PBS,slide-mounted, air-dried, dehydrated in graded ethanoland toluene, and finally coverslipped for examinationwith the light microscope equipped for differentialinterference contrast illumination (Axioskop Zeiss, Jena,Germany).

Electron microscopy

Two immunoreacted sections per antiserum and con-trol were selected from each animal for electron micros-copy and postfixed in glutaraldehyde (2% in PBS).Small pieces were sampled from sagittal and transver-sal sections of the cerebellar cortex to disclose thetransversal and the longitudinal geometry of immuno-staining in the cerebellar circuits. Samples were cutmainly in sagittal sections of the fastigial nucleus, thedentate gyrus, and the CA3 field of the hippocampusand in transversal sections of the inferior olivarynucleus. All samples including PS-immunoreacted kid-ney were postfixed in osmium tetroxide (1% in PBS) for3 h. After dehydration in graded ethanols and propyl-ene oxide, the samples were flat-embedded in Araldite(Fluka, Buchs, Switzerland) between two glass slides.Ultrathin sections were collected from the surface ofthe tissue section using an ultramicrotome (Ultracut,Reichert-Jung, Wien, Austria). These sections were

counterstained with 1% uranyl acetate and examinedwith a Siemens Elmiskop 101 transmission electronmicroscope. The present study enables the identifica-tion of the synaptic profiles with several criteria: fre-quency, localization, size, shape, aspect of the synapticmembranes, and content in presynaptic vesicles andpostsynaptic organelles.

The synaptic expression of PS and b-APP was esti-mated in percent of immunoreactive asymmetric syn-apses counted in the cerebellum and the hippocampusof the rat. Similar immunoreactivities were detected atthe synapses by the polyclonal (Fig. 2A–D) and themonoclonal (Fig. 2E,F) antibodies. The percentages oflabeled parallel fiber-Purkinje cell (PC) synapses withimmunoreactive postsynaptic spines or presynaptic axonterminals were estimated in ultrathin sections of themolecular layer sampled randomly in two vibratomesections immunoreacted with the monoclonal and thepolyclonal antibodies (1 of 5 molecular layer samplesper section). The sections were also selected randomlyfor each antiserum in each animal studied (2 of 10sections/antiserum/animal). Parallel fiber-PC synapseswere counted blind by two investigators on electronmicrographs (40,000x final magnification) takenthroughout the immunoreactive area of each ultrathinsection. Based on current ultrastructural criteria (Pa-lay and Chan-Palay, 1974; Altman and Bayer, 1997),presynaptic parallel fiber boutons were identified bytheir small size and the concentration of pleomorphicsmall vesicles at the synapse with the dense postsynap-tic membrane of the PC branchlet spine. Climbingfiber-PC synapses were recognized as large varicositiesfilled with dense accumulation of rounded vesiclesmaking asymmetric synapses on large spines of the PCdendritic shafts. The same method was used to countthe immunoreactive asymmetric synapses of the pyra-

Fig. 1. Light microscopic localization pattern of presenilins (PS1,PS2) and b-APP isoforms in the cerebellar cortex and the hippocampalCA3 field of the adult rat. In the molecular layer (ML) of the cerebellarcortex (A,B), parts of Purkinje cell dendrites (arrows) and of processesof radial glia (white arrowheads) as well as somata of interneurons(arrowheads) display similar heterogeneous b-APP751/770 (A) andPS1 (B) immunoreactivity with polyclonal antibodies as with monoclo-nal antibodies to b-APP holoprotein (E) and PS1 (F). The neuropilbetween these profiles displays immunolabeled fine structures. ThePurkinje cell somata (PC) are surrounded by immunoreactive profilesand display generally less intense b-APP staining than PS. Weakerimmunoreactivity stains the granule cells and the Golgi neurons (openarrowheads) in the internal granular layer (IGL). In the CA3 field ofthe hippocampus (C,D), immunoreactive apical and basal dendrites(arrows) of the pyramidal neurons (PY) display b-APP695 (C) and PS2(D) in the strata radiatum (SR), lucidum (SL), and oriens (SO). In thecerebellum (B,F) and the hippocampus (D), most of the immunoreac-tive cells display PS nuclear staining (white arrows) but not b-APP(A,E). The specific immunostainings are absent in the sections ofcerebellar cortex (G,H) and of hippocampal CA3 field (I,J), which havebeen processed without PS or b-APP antibodies (G,I) and in sectionsincubated with the PS1 N-terminal polyclonal antibodies (1:1,000)complexed with 1 mg of PS1 antigen peptide (H,J). A–F: 460x,G–J: 230x; bar 5 500 µm.

98 C. RIBAUT-BARASSIN ET AL.

Fig. 1


midal dendrites in the molecular layer of the CA3 fieldof the rat hippocampus sampled in the same vibratomesections used for counts of PC synapses.

RESULTSHistological pattern of PS and b-APP

immunostaining in the cerebellum andhippocampus (CA3) of rat and mouse

Presenilins 1 and 2 and b-APP isoforms displayedvery similar immunohistochemical staining patterns ofneurons and glial cells in the cerebellum as well as inthe hippocampus of the rat (Fig. 1) and the mouse (notshown) using either the polyclonal (Fig. 1A–D) or themonoclonal (Fig. 1E,F) antibodies. In both regions, theonly difference was present in the nucleus of the labeledcells which displayed immunoreactivity for PS 1 and 2(Fig. 1B,D,F) but not for b-APP (Fig. 1A,C,E).

In the cerebellar cortex of the rat and the mouse,immunoreactivity for PS1 and 2 and for b-APP isoformsstained parts of the PC dendrites and of the palisades ofthe Bergmann astrocytes in the neuropil of the molecu-lar layer (Fig. 1A,B). Both soma and processes of thesecells and the surrounding neuropil displayed heteroge-neous labeling the geometry of which could not bebetter clarified in the sagittal and transversal sections.The PC somata exhibited weaker b-APP than PScytoplasmic immunoreactivity and were surrounded byintensely positive glial-like cells. Immunoreactive so-mata of basket and stellate interneurons were foundthroughout the molecular layer. In the glial cells of bothspecies, PS2 immunoreactivity was more intense thanthat of b-APP and PS1. Cells in the internal granular

layer displayed a weaker staining with much less PS-and b-APP-positive profiles, than the molecular and thePC layers. No immunoreactivity could be detected inthe white matter of the cerebellar lobules. Finally,neurons and glial cells displayed PS and b-APP immu-nostaining in the deep cerebellar nuclei and outside thecerebellum, in the inferior olive (not shown).

In the hippocampal CA3 field of rat and mouse, PSand b-APP immunoreactivity stained the somata ofpyramidal neurons as well as their dendrites in thestratum oriens, radiatum, and lacunosum moleculare,including the stratum lucidum where the mossy fibersof the immunoreactive dentate granule cells project(Fig. 1C,D). In the hippocampal layers, the fine struc-tures of the neuropil displayed less intense PS andb-APP immunostaining than in the cerebellar molecu-lar layer. A few small somata of interneurons and glialcells were labeled for PS and b-APP throughout alllayers. Immunoreactivity of the four molecules markedthe granule cells and their dendrites in the dentategyrus, PS2 immunoreactivity yielding the weakeststaining. As in the cerebellum, neurons and glial cellsdisplayed nuclear PS1 and PS2 immunoreactivity inthe hippocampus and the dentate gyrus.

Incubating the sections without primary antibodiesor with primary antibodies neutralized by the antigenpeptide abolished all immunoreactivity of the rat andmouse tissue and confirmed the specificity of the stain-ings at both histological (Fig. 1G–J) and ultrastructural(Fig. 2G,H) levels.

Hemisynaptic PS and b-APP immunoreactivityat the asymmetric synapses of the excitatory

circuits of the cerebellum

In the cerebellar cortex, prominent PS and b-APPimmunoreactivity located similarly at asymmetric, exci-tatory synapses made by the parallel fibers of granulecells and by the olivary climbing fibers on the PC (Figs.2, 3). Asymmetric synapses made by the extracerebellarmossy fibers on granule cells were also immunoreactive(Fig. 3). The same protein was seen very rarely in bothpresynaptic and postsynaptic compartments of thesame synapse (,0.5%, not shown) in all the types ofsynapses studied. Immunonegative synapses of all typeswere mixed with immunopositive synapses and alsofeatured areas devoid of synaptic labeling in all layers.The patchy geometry of the immunostaining patternscould not be defined more clearly in either orientation ofthe sections. The intensity of immunoreactivity variedalso within the dendrites and the axons. Both displayedunlabeled parts as observed in PF cut longitudinally inthe transversal sections and in vertical PC dendrites inthe sagittal sections.

TABLE I. Distribution of b-APP and PS immunoreactivity in thecerebellar and hippocampal synapses of the rat and the mouse*

Cerebellum Hippocampus

Asymmetricsynapses PRE POST

Asymmetricsynapses PRE POST

PF/PC

PF/SC, BC

CF/PC

CF, MF/GO, UBC

MF/GC

1212121212

2121212121

PFT/PY

PFT/GC

PY/PY

MF/PY

PY/IN

2

2

2

2

2

1

1

1

1

1

Symmetricsynapses PRE POST

Symmetricsynapses PRE POST

PR, SC, BC/PC

GO/GD; PC/FN

122

211

BC/PY

IN/PY, GC

2

2

1

1

*Presynaptic or postsynaptic immunoreactivity (1) is observed in the asymmet-ric and the symmetric synapses of the cerebellar neurons (cortex and fastigialnucleus). The synapses of the hippocampal neurons (CA3 and dentate gyrus)display only postsynaptic immunoreactivity. Immunonegative synapses of alltypes are observed in all regions (see the list of abbreviations for legend).


Fig. 2. Asymmetric distribution of b-APP and PS in the asymmet-ric synapses (arrowheads) between parallel fibers (PF) and dendriticspines of Purkinje cells (PC) in the molecular layer of the cerebellarcortex of the adult rat (A–D) and mouse (E,F). At the ultrastructurallevel using the pre-embedding immunocytochemical method, thePF-PC synapses display hemisynaptic immunoreactivities for b-APP-KPI (751/770) (A,B), PS1 (C,D), and PS2 (E,F). Electron-opaqueimmunocytochemical precipitates locate similarly for each molecule ineither the presynaptic varicosities of PF innervating unlabeled, post-synaptic PC spines (A,C,E) or in the PC spines innervated byunlabeled PF varicosities (B,D,F). Immunoreactivity labels the axonalmembrane, the outer side of the electron-lucent synaptic vesicles inthe PF boutons and is often concentrated on the postsynaptic mem-brane density of the PC spines. Immunonegative PF-PC synapses(open arrowheads) are seen in close proximity to the labeled PF-PCsynapses (A–F). D: The same unlabeled PF bouton makes asymmetric

synapses with a PS1-immunolabeled PC spine (arrowhead) and anunlabeled PC spine (open arrowheads). E: PS2 in presynaptic PFboutons synapsing (arrowheads) on unstained PC spines. UnlabeledPF boutons also make synapses (open arrowheads) on postsynaptic PCspines. A PS2 PC dendrite (PD) runs perpendicularly to unlabeled PF(asterisk). F: Asymmetric synapses between unlabeled PF boutonsand PS2 postsynaptic PC spines (arrowheads) are surrounded byunlabeled glia (G). Immunostained glia (white G) surrounds synapsesbetween unlabeled PF and PC spines (open arrowhead). Arrow pointsto a symmetric synapse between a stellate cell-like axon terminal anda thin dendritic branch both of which are devoid of immunostaining.(G,H) Unlabeled PF-PC asymmetric synapses (open arrowheads) arefound in the cerebellar molecular layer after control immunocytochem-istry (G) without PS or b-APP primary antibodies and (H) with PS1antibodies neutralized by an excess of antigen peptide (see Materialsand Methods). A–D,G,H: 40,000x; E,F: 27,000x; bar 5 500 nm.

Fig. 3

Parallel fiber and mossy fiber synapses

Throughout the molecular layer, the immunoreactivesynapses made by parallel fibers on PC displayed PS orb-APP either in parallel fiber boutons presynaptic tounreactive PC dendritic spines (Figs. 2A,C,E) or in PCspines postsynaptic to unreactive parallel fiber varicosi-ties (Fig. 2B,D,F, Table 1). Unlabeled and labeledpostsynaptic PC spines were innervated by the sameunlabeled presynaptic parallel fiber (Fig. 2D). At theultrastructural scale, synapses with presynaptic label-ing were sometimes located near synapses with postsyn-aptic labeling in the molecular layer. All synapses wereunlabeled in control sections (Fig. 2G,H).

The percentages of synapses immunoreactive forproteins PS1 (31% of 419 synapses), PS2 (33.8% of 213),and b-APP holoprotein (29% of 149) and isoforms 695(33.5% of 686) and 751/770 (33.2% of 664) indicate thatthe four molecules are detected at equivalent frequencyat the parallel fiber-PC synapses of the rat. A similarsituation is suggested by our qualitative observationsin the cerebellar cortex of the mouse. In addition, foreach antigen pre- and postsynaptic immunoreactivitylabeled equivalent numbers of synapses (Fig. 4). The inter-varicose segments of parallel fibers were generally labeledin the vicinity of immunoreactive presynaptic parallelfiber boutons (Fig. 2C). Most of these parallel fiber seg-ments were immunonegative near unlabeled parallel fiberterminals synapsing on labeled PC spines (Figs. 2D,F).

As shown in Table 1, similar hemisynaptic labelingwas observed in the synapses established by parallel

fibers on interneurons in the molecular layer (Fig.3A,B) and in the synapses made by mossy fibers onpostsynaptic granule cell dendrites in the glomeruli ofthe granular layer (Fig. 3C,D).

Climbing fiber synapses

In the molecular layer, the climbing fiber synapsesmade by olivary axons directly on spines and shafts ofPC dendrites (Ramon y Cajal, 1911) displayed pre- orpostsynaptic immunoreactivity as the parallel fiber PCsynapses (Fig. 3E,F, Table 1). This distribution was alsoobserved in the asymmetric synapses made by climbingfibers on granule cell dendrites (Fig. 3G), Golgi interneu-rons, and unipolar brush cells in the granular layer(Table 1) but not at the asymmetric synapses madeextracortically by climbing fibers on the fastigial neu-rons, which displayed only postsynaptic immunoreactiv-ity (not shown). In the inferior olive neurons, PS andb-APP immunocytochemical labeling was detected in

Fig. 4. Percentages of asymmetric synapses immunoreactive forb-APP isoforms 695 and 751/770 and for PS1 in the cerebellar cortex(black) and the hippocampal CA3 field (gray) of the 45-day-old LongEvans rat. The mean percentages of immunoreactive synapses areestimated from total synapse numbers counted in the sections ofcerebellar and hippocampal molecular layers of four rats after immu-noreaction with the b-APP isoform-specific antibodies and the PS1polyclonal antibodies (see Materials and Methods). Equivalent percent-ages of asymmetric synapses express b-APP695 and PS1 in bothregions. A similar value for the KPI-containing-b-APP isoforms 751/770 (including APLP2) is estimated in the cerebellum but is doubled inthe hippocampus (asterisk). The percentages of synapses with presyn-aptic labeling (hatched bars) are much higher in the cerebellum thanin the hippocampus.

Fig. 3. Hemisynaptic distribution of b-APP and PS in the asymmet-ric synapses of basket interneurons with PF in the ML (A,B), ofgranule cell dendrites with MF (C,D) and CF (G) in the IGL, of PCspines with CF in the ML (E,F) and of olivary neurons with afferencesin the inferior olivary nucleus (H) of the adult rat. A: Unlabeled (openarrowhead) and b-APP-KPI (arrowhead) presynaptic PF boutonsmake asymmetric synapses on an immunonegative basket cell soma(BC). An immunopositive PF bouton makes an asymmetric synapsewith an unlabeled spine (asterisk) at the left top of the figure. Thearrow points to a multivesicular body in the BC neuroplasm. B: Anunreactive PF bouton makes an asymmetric synapse (arrowhead) on aBC dendrite with PS1-immunoreactive density of the postsynapticmembrane. See also a positive glial process (G) close to a PC dendrite(PD). C: In a cerebellar synaptic glomerulus, a PS1 MF terminalmakes an asymmetric synapse (arrowhead) on a negative GC dendritewhich establishes a symmetric synapse (double arrow) with a negativeGolgi axon terminal (GO). D: Symbols as in C. b-APPKPI-positive MFsynapsing on unlabeled GC dendrites. E: Two PS1-immunoreactiveCF varicosities make asymmetric synapses (arrowheads) on unlabeledpostsynaptic spines (PC). One spine belongs to a proximal PC dendrite(PD), the postsynaptic area of a second spine is shown between twoarrowheads on the left. F: Asymmetric synapses (arrowheads) be-tween PC spines from a b-APP695 PC proximal dendrite (PD) and anegative climbing fiber varicosity (CF). See immunostaining of neuro-plasmic organelles within PD. G: An unlabeled CF-like varicositymakes asymmetric synapses with b-APPKPI-positive (arrowheads)and negative (open arrowhead) GC dendrites. It resembles a small-sized CF varicosity containing synaptic vesicles exclusively. H: PS1dendrites of inferior olivary neurons (IO) making labeled asymmetricsynapses (arrowheads) with negative presynaptic profiles (asterisks).A–H: 40,000x; bar 5 500 nm.


Fig. 5. Hemisynaptic distribution of b-APP and PS at the PCsymmetric synapses in the cortex and the fastigial nucleus of the ratcerebellum. A: Symmetric synapse (arrow) made by a varicosity of apresumptive PC recurrent axon (PR) on a b-APP-KPI PC dendrite(PD). B: A PS1 PR-like terminal makes a symmetric synapse (arrows)on a PC soma which contains immunoreactive Golgi saccules (doublearrowhead). C: Symmetric synapse (arrow) between a PC dendrite(PD) and a b-APP695 basket cell-like terminal (BC) with numerousneurofilaments and presynaptic clustering of immunoreactive synap-tic vesicles. D: A presynaptic axon terminal resembling the basket

cell-like bouton (BC) shown in C displays intense PS2 immunoreactiv-ity and makes a symmetric synapse (arrow) on a smooth domain of anunstained PC dendrite (PD). Open arrowhead points to a negativeasymmetric synapse between a PF bouton and a PC spine. E: Synapses(arrows) made by presumptive PC axon terminals on the b-APP695dendrites of the neurons of the fastigial nucleus (FN). Open arrowheadpoints to an unlabeled synapse of this type. F: A PS1 dendrite of afastigial neuron (FN) establishes labeled synapses (arrows) withunlabeled PC-like axon terminals. A,B: 45,000x; C,D: 40,000x;E: 30,000x; F: 35,000x; bar 5 500 nm.


the dendrites and the somata (Fig. 3H). Only thepostsynaptic side of the asymmetric synapses made byunlabeled presynaptic boutons displayed PS and b-APPimmunoreactivity.

Hemisynaptic PS and b-APP immunoreactivityin the symmetric synapses of the inhibitory

circuits of the cerebellum

Based on their ultrastructural aspect and localiza-tion, several types of inhibitory symmetric synapseswere recognized in the cerebellar cortex (Larramendiand Victor, 1967; Palay and Chan-Palay, 1974). Un-stained presynaptic boutons of basket and stellateinterneurons as well as of PC recurrent collaterals wereidentified making symmetric synapses with unlabeled(Fig. 2F) or with immunoreactive postsynaptic PCsomata and dendritic shafts (Fig. 5A); presynapticlabeling was found occasionally in these synapses (Fig.5B,C,D, Table 1). The symmetric synapses made byGolgi axons on granule cell dendrites in the granularlayer displayed only postsynaptic PS and b-APP immu-noreactivity (not shown).

In the fastigial nuclei, postsynaptic PS and b-APPimmunoreactivity featured the symmetric synapsesmade by the axons of PC and by intrinsic inhibitoryafferences (Baurle and Grusser-Cornehls, 1997) on thedendrites and the somata of the fastigial neurons (Fig.5E,F, Table 1).

Hemisynaptic PS and b-APP immunoreactivityin the asymmetric synapses of the hippocampus

(CA3) and the dentate gyrus

In all layers of the CA3 field, the distribution of PS1(Fig. 6A,D), PS2 (not shown), and b-APP (Fig. 6B,C,E)immunoreactivity was mainly postsynaptic in the asym-metric synapses of the spines and the dendritic branchesof the pyramidal neurons as well as of interneurons.Less than 10% of the positive hippocampal synapsesdisplayed presynaptic PS or b-APP immunoreactivityin the rat (Fig. 4) and the mouse. Immunostaining wasnot found in the presynaptic varicosities synapsing onspines throughout the hippocampal molecular layer(Fig. 6A–D). The lack of consistent immunolabeling ofaxons in the hippocampus but not in the cerebellarcortex at the ultrastructural level agrees with theweaker immunohistochemical staining of the hippocam-pal neuropil. In the molecular layer of the rat hippocam-pus, including the strata lucidum, radiatum, and lacu-nosum moleculare, the percentages of dendriticasymmetric synapses immunoreactive for PS1 (34.6%of 407 synapses) and b-APP695 (30.5% of 1,040) weresimilar to the percentages of cerebellar parallel fiberPC synapses immunoreactive for PS and b-APP as well.The percentage of synapses displaying b-APP751/770(61% of 1,155 synapses) was higher than any of the

other percentages of synapses labeled for b-APP and PSestimated in the hippocampus and cerebellum (Fig. 4).This increase was not detected by the 22C11 monoclo-nal antibody to b-APP holoprotein (36.2% of 92 syn-apses).

A similar postsynaptic pattern of PS and b-APPimmunoreactivity was also seen in the dentate gyrus,where only the dendrites of the granule cells werelabeled at the asymmetric synapses made by the presyn-aptic varicosities of their own recurrent axons and ofthe entorhinal perforant fibers (Fig. 6F).

Subcellular PS and b-APP immunoreactivity

The PS and b-APP immunoreactivity displayed simi-lar subcellular localizations in rat and mouse. Theplasma membrane and the cytoplasmic face of intracel-lular organelles such as synaptic vesicles were stainedby PS and b-APP immunoreactivity within the labeledpresynaptic boutons and preterminal varicosities in thecerebellum (Figs. 2A,C,E, 3A,C–E, 5B–D). In the den-drites and the spines of the cerebellar, olivary, andhippocampal neurons, the postsynaptic membrane ofboth asymmetric and symmetric synapses and thesubsynaptic organelles were stained by PS and b-APPimmunoreactivity (Figs. 2B,D,F, 3B,F–H, 5A,E,F, 6A–F). Immunolabeling of areas of nonsynaptic plasmamembrane and of the cytoplasmic face of vesicles andergastoplasmic saccules revealed PS and b-APP in theimmunoreactive dendrites and soma of all types ofneurons observed in this study (Figs. 2E, 3B,F,G,5A,E,F, 6). The cis saccules of dictyosomes displayedlumenal immunolabeling in the PC somata (Fig. 5B),but the lumen of most organelles, including multivesicu-lar bodies, remained unlabeled in parallel fibers andPC. Permeabilization with Triton and prolonged chromo-gen reaction did not induce detectable changes in theimmunolocalizations described here in nonpermeabi-lized tissues. Ultrastructural staining of the neuro-plasm often appeared increased at the periphery of thecells between the labeled organelles and the plasmamembrane. Nuclear PS1 and PS2 immunoreactivityfrequently labeled the inner membrane of the nuclearenvelope, nuclear pores, and dense chromatin of thenucleoplasm, except the nucleolus (not shown). A posi-tive control for the specificity of PS immunoreactivity inthe brain tissue was achieved in a peripheral organ: themouse kidney, where intense PS immunoreactivity wasdetected at plasma and subjacent endoplasmic mem-branes of the apical pole of the epithelial cells circlingthe lumen of the renal tubules in the mouse kidney(data not shown).

PS and b-APP immunoreactivity of glial cells

As in neurons, PS and b-APP immunostaining variedwithin labeled glial cell processes and was more fre-


Fig. 6. Postsynaptic immunoreactivity of b-APP and PS at theasymmetric synapses (arrowheads) of neurons in the CA3 field of thehippocampus and of granule cells (GC) in the dentate gyrus of the ratand mouse. Rat (A,B,C,E) and mouse (D) CA3. A: Stratum radiatum.Asymmetric synapses (arrowheads) between PS1-immunoreactivespines (PY) and unreactive presynaptic terminals (asterisks) of pre-sumed Schaffer’s axons of CA3 neurons. B: Stratum lacunosummoleculare. Unreactive, presynaptic boutons (asterisks) of presumedentorhinal perforant fibers and Schaffer’s axons of CA3 neurons makeasymmetric synapses with a b-APP695-immunolabeled (arrowhead)

and two unlabeled (open arrowheads) postsynaptic spines (PY).C,D: Stratum lucidum. Unlabeled mossy fiber varicosities (MF) estab-lish asymmetric synapses (arrowheads) with the thorns and the shaftsof b-APP695 (C) and PS1 (D) PY dendrites. E: Stratum oriens. Some ofthese unlabeled presynaptic boutons (asterisks) originate presumablyfrom CA3 PY neurons and make asymmetric synapses (arrowheads)on a b-APP695 PY basal dendrite. F: Rat dentate gyrus. Asymmetricsynapse (arrowhead) made by an unlabeled axon terminal (asterisk)on a PS1 granule cell dendrite (GC). A: 50,000; B,F: 40,000x; C: 57,000x;D: 27,000x; E: 45,000x; F: 30,000x; bar 5 500 nm.


quent in the cerebellum than in the hippocampus. Inthe neuropil of both regions, the immunoreactive glialprocesses surrounded unlabeled rather than immunola-beled presynaptic terminals and postsynaptic spines(Figs. 2A–F, 3F). Increased immunoreactivity for allantigens was seen at the tip of some thin glial pro-cesses. Immunostaining for PS and b-APP was bound tothe glial cell membrane and to the same cytoplasmicorganelles as in neurons.

DISCUSSION

The light microscopic staining patterns of b-APP andPS immunoreactivity are similar in the cerebellum andhippocampus of rat and mouse. In agreement withprevious immunocytochemical studies (Shigematsu etal., 1992; Selkoe, 1994; Kovacs et al., 1996; Lah et al.,1997; Li et al., 1997), both neurons and glial cellsexpress the four proteins with a similar cytoplasmicdistribution at the membranes of the cytoplasmic organ-elles (i.e., endoplasmic reticulum, saccules and vesiclesof the Golgi dictyosomes) in both regions (Culvenor etal., 1997). The PS immunoreactivity detected at thenucleus of the immunoreactive cells in our preparationsagrees with the current data, suggesting that thepresenilins are involved in cell cycle mechanisms (Li etal., 1997) as well as in regulation of apoptosis (Vito etal., 1997; Zhang et al., 1998).

By contrast, the cerebellar and hippocampal syn-apses displayed different immunostaining patterns.This discrepancy regarding the hemisynaptic localiza-tion of AD molecules is also detected at the ultrastruc-tural level in the triton-permeabilized tissues, indicat-ing that it is not due to variable penetration of theimmunoreagents into these tissues. Immunostaining ofthe surrounding nonsynaptic nerve profiles and glialprocesses as well as the numerous unlabeled synapsesblurred the light microscopic contrast of the synapticlabeling, which thus did not appear as clearly in oursections as the punctate labeling given by specificsynaptic markers such as synaptophysin (Leclerc et al.,1989; Grabs et al., 1994).

Hemisynaptic localization of b-APP and PS

The four molecules are concentrated in the synapsesof both cerebellar and hippocampal neurons, with adistribution restricted to one of the synaptic compart-ments (Ribaut et al., 1998). This suggests that presenil-ins might have synaptic functions, as proposed for theb-APP isoforms (Loffler and Huber, 1992; Huber et al.,1993, 1997; Moya et al., 1994; Mucke et al., 1994) andfor related molecules such as APLP2 (Thinakaran et al.,1995; Von Koch et al., 1997).

A number of studies have illustrated the importanceof interactions between PS and b-APP within theneurons (Dewji and Singer, 1998; Weideman et al.,

1997; Walter et al., 1997; Beyreuther and Masters,1997; De Strooper et al., 1998). Our data extend theirpossible interaction to the key site of interneuronalcommunication, i.e., the synapses. The specific APPimmunoreactivity detected in our sections revealedplasma membrane-bound full-length and intracellularimmature forms of APP as well as its N-terminalderivatives. The predominant PS species detected withinthe synaptic compartments in the present study areprobably N- and C-terminal fragments. They resultfrom the proteolytic cleavage of full-length PS soonafter synthesis (Kim et al., 1997; Beher et al., 1997) andare enriched in synaptic membranes and vesicles ex-tracted from the rat brain (Robakis et al., 1997).Triton-induced permeabilization and over-incubationin DAB increased the intensity of immunostaining onthe plasma membrane and close to the cytoplasmic faceof the synaptic vesicles and the reticular saccules, butneither within them nor on the external surface of thecells. This suggests that most of the antigens detectedat the plasma membranes and at the surface of theorganelles are cytoplasm-oriented. The immunostain-ing detected in the lumen of the Golgi saccules revealsantigens located unequivocally within the lumen ofthese organelles.

The synaptic PS and b-APP proteins are concen-trated at the dendritic pole of neurons in the mainhippocampal circuits, while they are found in bothaxonal and dendritic poles of the cerebellar neurons.This discrepancy suggests that they might interactdifferently in these brain regions. A possibility is thatPS and b-APP colocalize and interact within the samecompartment of asymmetric synapses. In cerebellarsynapses, this could occur in the presynaptic varicosi-ties as well as in the postsynaptic dendrites, suggestingthat the proteins could have distinct presynaptic andpostsynaptic functions. Another possibility is a sepa-rate pre- and postsynaptic localization of PS and b-APPin a given synapse mediating a neuron-to-neuron rela-tionship (Dewji and Singer, 1998) with transport of theproteins (Simons et al., 1995). In the hippocampus,synaptic interactions between PS and b-APP would beconfined to the postsynaptic dendritic compartment, aspresynaptic profiles were rarely immunoreactive. Thedifferent synaptic expression of PS and b-APP betweenhippocampus (mostly postsynaptic) and cerebellum(equally pre- and postsynaptic) might reflect differentsynaptic functions. This difference is detected in themouse and the rat, which are able to develop AD-likeneuropathological features (Sturchler-Pierrat et al.,1997) and could account for the different vulnerabilitiesof the two regions to AD neurodegeneration in thehuman brain.

The immunostaining for b-APP751/770 labeled ap-proximately twice as many asymmetric synapses in themolecular layer of the hippocampus than in the molecu-


lar layer of the cerebellar cortex, while both regionscontained similar proportions of synapses immunoreac-tive for PS1 and for b-APP695. Also, more synapsesdisplayed postsynaptic b-APP751/770 than b-APP695immunoreactivity in the CA1 field of the hippocampusand in the occipital cortex of the rat (Huber et al., 1997).Increased expression of KPI-containing isoforms ofb-APP as well as other APP-like synaptic proteins, suchas APPL2 isoforms (Thinakaran et al., 1995), couldhave a physiological and pathological importance in thehippocampus, since these molecules can modulate pro-teolysis at the surface of the plasma membrane (re-viewed in Selkoe, 1994) and are upregulated duringinflammatory (Brugg et al., 1995) and neurodegenera-tive processes (Johnson et al., 1990; Higgins et al.,1990).

Synaptic PS and b-APP in the cerebellarand hippocampal circuits

Similar numbers of PS/b-APP immunoreactive pre-synaptic parallel fibers and postsynaptic PC indicatethat PS as well as b-APP are present in granule cells atthe presynaptic boutons of a subset of parallel fiber PCsynapses and in PC at the postsynaptic spines ofanother subset of parallel fiber PC synapses. Thissuggests that distinct relationships between PC andgranule cells involve synaptic PS and b-APP. Approxi-mately one-third of these synapses contain PS or b-APPimmunolabeling, while it is well established that more,if not all, granule cells and PC express PS and b-APPmRNA and protein (Gegelashvili et al., 1994; Lee et al.,1996; Blanchard et al., 1997). The heterogeneity of PSand b-APP expression at parallel fiber PC synapsescould reflect a functional subcellular segregation of thesynapses and could be modulated by activity (Mattsonet al., 1993; Nitsch et al., 1992). Studies in vivo (Huberet al., 1993, 1997; Meziane et al., 1998), includingtransgenic mice over- or misexpressing b-APP (Muckeet al., 1994), suggest strong neurotrophic and synapto-genic properties of b-APP isoforms during the establish-ment and the remodeling of synaptic connectivity(Selkoe, 1994; Storey et al., 1996). Enriched environ-ment was recently shown to upregulate the postsynap-tic expression of b-APP isoforms in the occipital cortexand CA1 field of the hippocampus of young rats (Huberet al., 1997). The molecular mechanism(s) which in-volve b-APP in activity-dependent plasticity of centralsynapses (Huber et al., 1993, 1997; Mucke et al., 1994;Roch et al., 1994; Zheng, 1996; Nalbantoglu et al., 1997;Meziane et al., 1998) could require an interaction withsynaptic PS during neuronal differentiation (Lee et al.,1996; Hartmann et al., 1997) and remodeling of adultneuronal circuits (Walter et al., 1997; Brouillet et al.,1999).

The specific loss of synapses that gives rise to demen-tia in AD occurs independently of amyloid deposition in

senile plaques (Masliah and Terry, 1993; Masliah,1995), and the cortical neurons which retain theircapacity for structural plasticity in the adult are thosewhich develop neurofibrillary tangles in the AD brain(Arendt et al., 1998). Accordingly, cell-specific proper-ties seem to modulate AD neuropathology in the centralcircuits. The present ultrastructural analysis suggeststhat distinct cerebellar and hippocampal mechanismscould involve the synaptic functions of PS and b-APP,which might be altered differently by AD in these tworegions, leading or not to synaptolysis.

ACKNOWLEDGMENTS

We thank J. Knobloch (animal care), M. Jamme(immunocytochemistry, ultramicrotomy), F. Scheer, andJ.-C. Barthe (photography) for excellent technical assis-tance.

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