clusterin/apojexpression is associated with neuronal ...assess the contingent expression of...

1635Journal of Cell Science 110, 1635-1645 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS9526

Clusterin/ApoJ expression is associated with neuronal apoptosis in the

olfactory mucosa of the adult mouse

Denis Michel1,*, Emmanuel Moyse2, Alain Trembleau1,†, François Jourdan2 and Gilbert Brun1

1Laboratoire de Biologie Moléculaire et Cellulaire, UMR49 CNRS-Ecole Normale Supérieure de Lyon, 46 Allée d’Italie, 69364 Lyoncedex 07, France2Laboratoire de Physiologie Neurosensorielle, CNRS ERS 5643, Université Claude Bernard-Lyon I, F-69622, Villeurbanne Cedex,France

*Author for correspondence†Present address: Ecole Normale Supérieure, Développement et Evolution du Système Nerveux, URA CNRS 1414, 46 rue d’Ulm, 75230 Paris cedex 05, France

The molecular events orchestrating neuronal degenerationand regeneration remain poorly understood. Attempts atidentifying genes specifically expressed during theseprocesses, have constantly led to the (re)isolation of theclusterin/ApoJ gene, whose expression is highly reactive toinjury in a wide variety of tissues. To get insight into thefunction of clusterin in neuron loss, we have assessed indetail the clusterin gene expression in an experimentalmodel of neurodegeneration, using the peripheral olfactorysystem of adult mouse. The sensory neurons of olfactorynasal mucosa can be massively induced to degenerate invivo, by surgical removal of their only synaptic target: theolfactory bulb. We have previously shown that this neuronloss results from a near-synchronized induction ofapoptosis genetic programs. We present here evidence thatclusterin gene expression is tightly correlated to the onsetof neuronal apoptoses in lesioned olfactory mucosae. Thesimultaneous preparation of DNA and RNA from the same

tissue samples reveals that a strong clusterin mRNA accu-mulation coincides with the wave of nucleosome-sized DNAfragmentation. However, double detection of apoptoticnuclei by the TUNEL method and of clusterin messengersby in situ hybridization revealed that the clusterin geneexpression is not induced in dying neurons, but in the glialsheath surrounding the axon bundles of degeneratingolfactory neurons. Clusterin immunocytochemistry revealsthat the clusterin protein accumulates not only in theseproducing cells, but also in the olfactory epithelium, sug-gesting the possibility of clusterin internalization by cellslocated at a distance from the synthesis loci. In view of thislocalization and of the activities of the clusterin proteinreported so far, possible functions of clusterin in nervousplasticity are discussed.

Key words: Clusterin/ApoJ, Neuronal apoptosis, Olfactory system,Glial cell, Surgical synaptic target ablation

SUMMARY

INTRODUCTION

The clusterin mRNA has been isolated on the basis of its accu-mulation in a classical model of tissue involution throughapoptotic cell deaths: the ventral prostate regression afterandrogen deprivation (Leger et al., 1987). Clusterin geneexpression has then been associated with numerous other casesof programmed cell death, such as those occurring duringnormal regression of temporary embryonic structures (Buttyanet al., 1989), in the mammary gland involution early afterlactation (Strange et al., 1992; Guenette et al., 1994a; Lund etal., 1996), in the remodeling uterus (Brown et al., 1995) or inkidney after experimental obstruction (Buttyan et al., 1989;Pearse et al., 1992). Such striking coincidences betweenclusterin expression and the onset of programmed cell deathshave logically raised the hypothesis that clusterin could play adirect role in apoptosis (Buttyan et al., 1989; Guenette et al.,1994b; Norman et al., 1995).

In the nervous system, a close relationship between neu-rodegeneration and clusterin gene expression has been estab-

lished (Michel et al., 1992; May and Finch, 1992). Althoughseveral normal populations of neuronal and glial cells containclusterin mRNA (Michel et al., 1992; Danik et al., 1993),higher clusterin mRNA levels are observed after deleteriousexperimental treatments, like surgical (Lampertetchells et al.,1991; Pasinetti et al., 1993), or excitotoxic (Michel et al., 1992;Danik et al., 1993) brain injuries, but also in pathologicalbrains from Alzheimer’s diseased human patients (May et al.,1990), scrapie infected hamsters (Duguid et al., 1989), or atepileptic foci (Danik et al., 1991). Clusterin mRNA accumu-lation has also been observed in retinitis-pigmentosa (Jones etal., 1992), and shown to coincide with the time of photo-receptor cell deaths in mouse models of this disease (Wong etal., 1994). Purkinje cells whose apoptosis is induced by thelurcher gene, were shown to contain high levels of clusterinmRNAs prior to their death (Norman et al., 1995). Besides, theclusterin protein has been found associated with dystrophicneurites (McGeer et al., 1992), amyloid plaques (Choi-Miuraet al., 1992) and the soluble form of beta amyloid protein(Matsubara et al., 1995) from Alzheimer’s diseased human

1636 D. Michel and others

brains. The ischaemic, but not the normal, human Purkinjecells are also intensely immunostained with anti-clusterin anti-bodies (Yasuhara et al., 1994).

In spite of these numerous observations, the precise functionof clusterin in damaged nervous system remains to be eluci-dated. In fact, answers to several basic questions would bedeterminant for understanding the biological involvement ofclusterin in neurodegeneration: (i) is clusterin expressionlinked to degeneration itself or to subsequent processes ofnervous tissue reorganization? (ii) is clusterin expressed bydegenerating or surviving cells in the lesioned nervous system?and (iii) where is the clusterin protein located relative to theclusterin-producing cells?

We have addressed simultaneously these different pointswith a favourable in vivo model: the peripheral olfactorysystem of adult mammals. The olfactory turbinates are linedup by an almost pure population of easily accessible sensoryneurons, which belong to a very simple network since theyall connect to a single synaptic target: the mitral cells of theolfactory bulb. Their degeneration can be massively inducedby surgical removal of the olfactory bulb (Monti-Graziadeiand Graziadei, 1979). We have recently shown that this neu-rodegeneration results from the near synchronized inductionof apoptotic genetic programs in vivo (Michel et al., 1994).The olfactory neuroepithelium is also unique in its ability tocompletely regenerate after lesion, even in the adult, due tothe presence of neurogenetic precursor cells, named globosebasal cells, residing in the depths of the epithelium (Monti-Graziadei and Graziadei, 1992; Caggiano et al., 1994). Inaddition, because of the absence of anatomical connectionsbetween the two symmetrical sides of the peripheralolfactory system, it is possible to cause a unilateral lesion byeliminating a single olfactory bulb out of the two, and thusto obtain both control and targetless olfactory neuroepi-thelia in the same transverse tissue section of olfactoryturbinates. Finally, the great number, the synchrony and theanatomical compartmentation of apoptotic neurons at thehistological level render this model highly appropriate forthe study of relationships between clusterin induction andapoptosis in vivo.

MATERIALS AND METHODS

Animals and tissue preparationAdult, three-month-old mice of the C57Bl/6J strain were used in thepresent study. Unilateral or bilateral bulbectomies were performedunder anesthesia with equithesine (0.3 g/0.3 ml per 100 g bodyweight) as follows. A 1 mm wide hole was first dug with a dentaldrill in the dorsal face of the skull 2 mm rostro-laterally to thebregma; a curved glass pipet connected to a vacuum pump was thenintroduced through the hole, the underlying olfactory bulb was totallyremoved by aspiration and the resulting space filled up with gel-foam. Bulbectomized mice were housed and allowed to recover fromanesthesia in individual cages with food and water ad libitum untilsacrifice.

For kinetic assessment of apoptosis and of gene expression, mice werekilled at various intervals, ranging from one hour to 8 days, followingbilateral bulbectomy; the bulk of olfactory turbinates was immediatelydissected out of each mouse head and snap-frozen at −80°C.

For in situ labeling of either clusterin mRNA or apoptotic nuclei,mice were killed by decapitation, and the bulk of the olfactory

turbinates was rapidly dissected out of the heads and immersed undervacuum in histological embedding medium (OCT, Miles, USA). OCT-embedded turbinates were frozen by immersion in isopentane at−50°C, and stored at −80°C until sectioning. Serial 20 µm thick,coronal sections were cut at −20°C on a cryostat (Reichert) and touch-mounted on slides that had been previously coated with 0.5% poly-L-lysine (Sigma) after heat-sterilization. Slide-mounted sections werekept frozen at −20°C until use. From each animal, 15-20 sets of 4 con-secutive, adjacent sections were collected for in situ hybridizationwith sense and antisense clusterin riboprobes and TUNEL labeling.

For clusterin immunocytochemistry, animals taken 48 or 75 hoursafter bulbectomy were deeply anesthetized and perfused transcardiallywith 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS),pH 7.4. The bulk of the olfactory turbinates was dissected out of thehead and rinsed in PBS, then immersed in a 30% sucrose solution inPBS. Following vacuum-embedding in OCT, the nasal bulk wasfrozen into isopentane at −50°C and stored at −80°C until sectioning.Serial 20 µm thick coronal sections were cut in a cryostat andcollected on gelatin-coated slides. Five animals of the same strainwere sham operated and submitted to the same protocol in order toassess the contingent expression of clusterin in the olfactory mucosaof intact animals.

Reverse transcription-mediated PCR (RT-PCR)RNAs were extracted as previously described (Michel et al., 1994),from the same olfactory turbinates which were used to quantify DNAladderization intensity, in order to compare the time-course of thegenomic DNA fragmentation, with that of clusterin mRNA accumu-lation. Reverse transcriptions were done starting with 0.3 µg RNAfrom each sample in the presence of 50 µM random hexamer primersas described (Michel et al., 1994). PCRs were performed using 1/20of total cDNA, 1 mM primers, 1 mM dNTP and 0.1 mCi of [α-32P]dCTP. Forward and reverse primers were defined in differentexons. They were, for clusterin: 5′-TCTCCAGCAGGGAGTCGAT-GCG-3′ and 5′-TGATGGCCCTCTGGGAGGAGTG-3′; for β-actin:5′-TTGCTGATCCACATCTGCTG-3′ and 5′-GACAGGATGCA-GAAGGAGAT-3′. Amplified fragments of clusterin and β-actincDNAs are 177 bp and 146 bp long, respectively. To determine theclusterin/β-actin ratio, we amplified simultaneously the clusterin andβ-actin cDNAs, by adding the four corresponding primers in the PCRtubes. 1/5 of the clusterin/β-actin co-amplifications were elec-trophoresed through a 4% polyacrylamide (38:2; acrylamide:bisacry-lamide) gel. After UV-visualization, the gel was dried and autoradi-ographed for 2 hours with Amersham MP films to quantify the cDNAamplifications by 32P-incorporation.

In situ hybridization Frozen cryostat sections of olfactory turbinates were brought to roomtemperature, fixed for 30 minutes at room temperature by immersionin 4% paraformaldehyde-containing PBS and rinsed three times (5minutes each) in PBS. The sections were then treated with proteinaseK, acetylated according to the method of Simmons et al. (1989). Senseand antisense clusterin riboprobes have been labeled with 33P duringin vitro transcription of the coding region of the rat clusterin cDNA(generously supplied by Dr Michael Griswold, Washington State Uni-versity). The transcription reaction contained 1 µg of linearized DNAtemplate, 100 µCi [33P]UTP (3,000 Ci/mmol, Amersham), ATP, CTP,GTP (10 µM each), 2 µl transcription buffer (Boehringer Mannheim),20 units of SP6 or T7 RNA polymerases (Boehringer Mannheim) and20 units of RNase inhibitor (Boehringer Mannheim), in a final volumeof 20 µl. The labeled probe was digested with DNase I, ethanol pre-cipitated, and resuspended in Tris-EDTA (10 mM-1 mM) buffer.Following dehydration, tissue sections were incubated overnight at52°C in hybridization buffer containing radioactive probe (107

cpm/ml), 50% formamide, 10 mM Tris-HCl, pH 8.0, 0.3 M NaCl,1 mM EDTA, 1× Denhardt’s solution (0.02% BSA, 0.02%polyvinylpyrrolidone, 0.02% tri-sodium citrate), and 0.5 mg/ml

1637Clusterin and neuronal apoptosis in vivo

tRNA. Following hybridization, the sections were washed four times(5 minutes each) in 2× SSC (1× SSC = 0.15 M NaCl, 0.015 M tri-sodium citrate), and RNase A treated (30 minutes at 37°C) in a buffercontaining 10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1 mM EDTA and0.02 mg/ml RNase A (Sigma). Then, the sections were washed atroom temperature as follows: 2× SSC, 2× 5 minutes; 1× SSC, 10minutes; 0.5× SSC, 10 minutes, then 0.2× SSC for 30 minutes at 60°C.Finally, the sections were rinsed in 0.1× SSC at room temperature,dehydrated in graded ethanols and air-dried. Section-hybridized radio-probe molecules were detected by dry and wet autoradiography asfollows. All sections were first autoradiographed simply by apposi-tion onto Amersham MP films in light-proof cassettes for 2 to 4 days;films were developed in a developer apparatus (P2000, 3M). Sectionswere then autoradiographed by dipping into liquid nuclear emulsion(Amersham), exposed for 10-15 days in light-proof, sealed boxes,developed in Kodak D-19 for 3 minutes at 17°C, fixed in Kodak Unifixfor 10 minutes at 4°C and rinsed under tap water and distilled water;sections were then Nissl-counterstained with 0.5% acidified CresylViolet, dehydrated-defatted in graded ethanols and xylene, and cov-erslipped with Depex.

In situ labeling of nuclear DNA fragmentationVisualization of DNA laddering in situ was performed by theTUNEL method (Gavrieli et al., 1992). Frozen, slide-mounted tissuesections were brought to room temperature, fixed for 30 minutes atroom temperature by immersion in 4% paraformaldehyde-contain-ing 0.1 M PBS at 4°C and rinsed 2× 15 minutes in PBS. Slides werethen incubated for 15 minutes at room temperature with proteinaseK at 20 µg/ml PBS and rinsed 2× 5 minutes with PBS. Slides werefurther treated with 2% H2O2 in PBS for 5 minutes at room tem-perature in order to block endogenous peroxidases, and rinsed inPBS. After 5 minutes rinse in 30 mM Tris-HCl, pH 7.5, containing140 mM sodium cacodylate and 1 mM cobalt chloride, slides wereincubated with biotinylated dUTP (Boehringer, 6 nM/ml) andterminal transferase (Boehringer, 300 units/ml) in TdT buffer for 1hour at 37°C. Slides were then rinsed for 15 minutes at room tem-perature in 300 mM sodium chloride-30 mM sodium citrate solution,further rinsed in PBS and incubated for 10 minutes with 2% bovineserum albumin (Eurobio) in PBS. After rinsing in PBS, slides wereincubated for 30 minutes with a horseradish peroxidase-coupledABC system (Vector) prepared in PBS as described in the manu-facturer’s instructions, rinsed 5 minutes in PBS and 5 minutes in 50mM Tris-HCl, pH 7.5. Slides were then reacted with 0.05%diaminobenzidine (Sigma) in 50 mM Tris-HCl, pH 7.5, containing0.6% H2O2 and 0.03% nickel chloride. After 5-7 minutes reactionat room temperature, slides were rinsed in cold buffer, dehydrated-defatted through graded ethanols and xylene and coverslipped withDepex.

Clusterin immunocytochemistryThe mounted sections were incubated for 30 minutes in PBS con-taining Triton X-100 (0.3%) and normal serum (donkey or rabbit,depending on the secondary antibody used) and immersed for 20hours at 4°C in the same medium containing clusterin antibody(polyclonal, sheep anti-rat clusterin, Quidel, San Diego, CA) at adilution of 1/1,000 or 1/5,000. Following incubation in the primaryantibody, the sections were rinsed several times in PBS and processedfor visualization of the reactive sites. The first staining procedureused a FITC-conjugated secondary antibody (anti-sheep IgG, FITC-conjugate; Sigma). A second set of slides was stained using theavidin-biotin-peroxidase technique (Vectastain kit, Vector Labs,USA). The tissue-bound peroxidase was visualized with diaminoben-zidine (DAB, 25 mg/100 ml). Finally, a few sections adjacent toimmunocytochemically stained ones were counterstained withNeutral Red or Hoechst. Control experiments were carried out by

omitting the primary antibody in the incubation medium of severalslides.

Clusterin/GFAP double immunofluorescenceIn order to assess the contingent glial nature of clusterin immuno-reactive cells, some mucosae were submitted to a double immunoflu-orescence detection of clusterin and GFAP, using FITC and TRITCas fluorescent probes conjugated with respective secondary anti-bodies. The double immunofluorescence procedure was performedeither sequentially on the same sections or separately on 20 µm thickadjacent sections. The procedure for clusterin immunofluorescencehas been described previously. For GFAP immunocytochemistry,sections were pre-incubated in PBS containing Triton X-100 andnormal goat serum (5%) for 30 minutes and then immersed for 20hours at 4°C in the same medium containing the GFAP antibody(Dako) at a concentration of 1/500 or 1/1,000.

Then, the sections were rinsed several times and immersed in PBScontaining a TRITC-conjugated secondary antibody (anti-rabbit IgG,TRITC-conjugated, Sigma) at a concentration of 1/500. Finally, thesections were rinsed in PBS, mounted in Fluoprep (BioMérieux,France) and observed with a Zeiss fluorescence microscope. Controlexperiments were carried out by omitting the primary antibody in theincubation medium of several slides.

RESULTS

Localization of apoptotic nuclei in olfactory mucosainvoluting after bulbectomyThe severe involution of the olfactory mucosa after synaptictarget ablation is evidenced on a Nissl-stained transversesection of olfactory turbinates from a mouse killed 40 hoursafter unilateral bulbectomy (Fig. 1B). This neuroepitheliumdegeneration was previously shown to be preceded by a peakof genomic DNA fragmentation, typical of apoptotic pro-grammed cell deaths (Michel et al., 1994). The cellular site ofthis DNA fragmentation was assessed here in situ, using theTUNEL method (Fig. 2). Comparison of each TUNEL-processed sample with the adjacent Nissl-stained sectionclearly shows that olfactory mucosa, on the side ipsilateral tothe bulbectomy, is strongly enriched in apoptotic nuclei (Fig.2A,B). By contrast, contralateral turbinates are almost devoidof TUNEL staining, except for a few scattered reactive nuclei(Fig. 2C,D). The TUNEL-stained nuclei of target-deprivedmucosa were strictly confined to the neuroepithelium andmore precisely to its inner part containing the nuclei of maturesensory neurons (Fig. 2A,B). No TUNEL reactivity is foundin the superficial sub-layer of the olfactory epithelium con-taining the cell bodies of sustentacular cells, indicating thatthis non-neuronal cell population does not participate in themassive cell loss induced by bulbectomy. No more TUNELreactivity is found in the lamina propria that underlies thebasal lamina of the neuroepithelium and consists of olfactoryaxon bundles, Bowman glandular acini, blood vessels andconnective tissue.

Time-course of clusterin mRNA accumulation afterolfactory bulbectomyOccurrence of clusterin mRNA was then assessed in olfactoryturbinates of mice previously bulbectomized. Since the size ofthe tissue samples was not sufficient to allow northern blotexperiments, we have used RNA minipreparations as


templates for reverse transcription-mediated PCR and testedthe relative variations of clusterin and β-actin mRNAs. β-Actin and clusterin cDNAs from each tissue sample were co-amplified in the same PCR tube, thus allowing us to obtainideally normalized clusterin/β-actin ratios. This co-PCRprocedure applied to olfactory turbinate extracts from micekilled at various post-bulbectomy intervals shows that therelative variations of the two mRNAs are clearly disconnected(Fig. 3). While the β-actin mRNA remains roughly constantbefore and after bulbectomy, the clusterin mRNA, barelydetectable in control turbinates (Fig. 3, first lane), is stronglyoveraccumulated after bulbectomy, reaching transiently a levelsimilar to that of the β-actin mRNA. However, clusterinmRNA induction is only transient and slowly returns tocontrol values 120 hours post-operation and does not increase

Fig. 1. (A) Light microscopic structure ofolfactory mucosa in control adult mouse,as observed on a semi-thin (1 µm-thick)section of prefixed olfactory organ. Abasal lamina (bl) separates olfactoryepithelium (above) from underlyinglamina propria that harbors axon bundlesof olfactory nerve (arrows) and Bowmanglands (bg). Note the pseudostratifiedaspect of olfactory neuroepithelium, dueto the laminar distribution of therespective nuclei of horizontal (hbc) andglobose (gbc) basal cells, olfactoryneurons (on) and supporting cells (sc).The upper sub-layer of olfactoryepithelium is made up mainly of olfactoryneuron dendrites at the tip of whichdendrite knobs (dk) protrude into the nasalcavity (N). Bar, 25 µm. (B) Involution ofthe olfactory mucosa following unilateralolfactory. A section of the olfactorymucosa from a unilaterally bulbectomizedmouse was Nissl-stained. This sectionincludes the nasal septum (S) andolfactory turbinates of the two sides of theorgan. Olfactory turbinates and septum arecovered by olfactory neuroepithelium(OE) lying onto lamina propria (LP), asfigures in A. Note the dramatic thicknessreduction of olfactory neuroepithelium onthe side that has been deprived of itssynaptic target, ipsilateral to olfactorybulbectomy (left). N, nasal cavity. Bar,100 µm.

again at longer time periods (data not shown). The time-courseof quantitatively assessed clusterin mRNA accumulation isshown in Fig. 4. It can be compared with that of genomic DNAladderization observed in the same conditions (Michel et al.,1994), prepared from the same tissue samples, dissected outfrom the same animals. Clusterin mRNA content increasessignificantly as soon as 8 hours after bulbectomy and thenparallels the pattern of DNA fragmentation up to 32 hours.Clusterin mRNA decrease is delayed by about 40 hours fromthat of DNA fragmentation.

In situ localization of clusterin mRNA in olfactoryturbinates of unilaterally bulbectomized miceWe further assessed the cellular site of clusterin geneexpression by in situ hybridization of clusterin mRNAs on


sections of olfactory turbinates from mice killed 75 hours afterunilateral olfactory bulbectomy. Clusterin riboprobe hybridiz-ation was higher in the bulbectomized side of olfactoryturbinates than in the control side, as shown by film autoradi-ography (Fig. 5A) and by microscopic autoradiograms (Fig.5B,C). Clusterin gene expression is thus induced by olfactorybulbectomy within the mucosa of olfactory turbinates ipsilat-eral to the lesion, like neuronal apoptoses. Clusterin geneinduction thus seems not only temporally, but also spatiallyrelated to olfactory neurodegeneration.

In order to assess whether bulbectomy-induced apoptoticnuclei and clusterin mRNA accumulation within olfactorymucosa concern the same cells, we have detected these twoparameters in adjacent tissue sections of olfactory mucosafrom mice killed 40 hours after ipsilateral bulbectomy. Insitu hybridization with the clusterin antisense riboproberesulted in dense and heterogeneous labeling within thelamina propria, rows of silver grains conspicuously overlay-ing the glial sheath of olfactory axon bundles (Fig. 6B,C).No specific hybridization signal was found either above thebasal lamina in olfactory epithelium, or in axon bundles per

Fig. 2. Selective presence of apoptotic nuclei at the level of the involutinallows us to specifically reveal nuclei of cells undergoing apoptosis, whiasymmetrical, with a strong enrichment in the lesioned side (A) as compabundance of these apoptotic nuclei in the lesioned side is responsible fohematoxylin-stained sections, B and D). Apoptotic nuclei are specificallneuron body layer. By contrast, the lamina propria, located underneath tnucleus, indicating that apoptoses only concern olfactory neurons. N, na

se (Fig. 6B,C). Conversely, apoptotic nuclei were onlyobserved in the neuroepithelium (Fig. 6A). This experimentdemonstrates that the localizations of apoptotic nuclei and ofclusterin messengers are mutually exclusive, and thus thatclusterin mRNAs do not accumulate in dying neurons, but insurviving, non-neuronal cells located in the underlyinglamina propria.

Clusterin immunocytochemistryThe mere detection of clusterin mRNA is not adequate todetermine the localization of the related protein, sinceclusterin can either remain inside the producing cells (Reddyet al., 1996), or be targeted towards the secretion and even tothe receptor-dependent internalization pathways (Kounnas etal., 1995). Hence, we wanted to visualize directly theclusterin protein. Fig. 7 shows the distribution pattern ofclusterin immunoreactivity 75 hours after bulbectomy, asrevealed by immunofluorescence. Specific labeling has beenfound widely distributed in the olfactory mucosa, but twomain sites of clusterin accumulation could be identified, andobviously confirmed with the most sensitive avidin-biotin-

g neuroepithelium. The DNA end labeling procedure (TUNEL method)ch appear in black. The distribution of apoptotic nuclei is clearlyared to the control side (C) from the same olfactory mucosa. Ther the striking reduction of the neuroepithelium thickness (compare

y located in the inner part of the neuroepithelium, at the level of thehe basal lamina of the neuroepithelium, is devoid of any apoptoticsal cavity; OE, olfactory epithelium. Bar, 50 µm.


Fig. 3. Competitive amplification of β-actin and clusterin mRNAs byco-PCR. RNAs were prepared from a control mouse (first lane) orfrom mice previously bulbetomized for the indicated time periods,and reverse-transcribed using random hexamers as primers. Thesingle-stranded cDNAs thus obtained were used as templates forPCR amplification specific of clusterin and β-actin. Bothamplifications were carried out simultaneously in the same PCRtubes and starting from the same reverse-transcription mixtures.Amplification product amounts were quantified by ethidiumintercalation (top panel) or radioactivity incorporation (bottom panel)yielded identical comparative results.

peroxidase method (Fig. 8). As shown in Figs 7 and 8,prominent immunoreactive sites are present in the deep partof the olfactory epithelium, just above the basal lamina (Figs7A, 8A). A weaker and more diffuse labeling has also beenoccasionally observed more superficially in the epithelium.However, the most superficial layer containing supportingcell bodies was always immunoreactive. All these epithelialsites of clusterin accumulation have been found widely dis-tributed in most areas of the olfactory epithelium, at bothpost-operative delays. However, their frequency and intensitywas apparently increasing with time, being higher at thelongest post-operative delay (72 hours). In addition to thisunexpected epithelial labeling, clusterin immunofluorescencewas also observed in the deep olfactory mucosa (laminapropia), in close association with olfactory nerve bundles(Fig. 7). Clusterin accumulation in this area appeared aseither a diffuse labeling within nerve bundles or denseimmunofluorescent patches located around or inside thenerve. Identification of reactive sites at the cellular level usingthe more sensitive ABC staining showed that labeling was

0

MAX

MIN

Fig. 4. Comparative time-courses of clusterin mRNAaccumulation and of genomic DNA degradation.Clusterin/β-actin PCR signal ratios were blotted togetherwith the intensity of apoptotic DNA fragmentation as afunction of time after bulbectomy. The intensity of DNAladderization was determined by densitometricquantification of the two-nucleosome-sized fragments(Michel et al., 1994). For each time point, mRNA andladdering quantifications are done starting from the sameanimals.

concentrated in the unique cell type found in this area, i.e.glial cells which ensheath olfactory axons (Fig. 8B).Immunoreactive glial cell bodies were located either at theperiphery or inside the olfactory nerve bundles whereas theweaker and more diffuse labeling observed inside the nerves(Figs 7A, 8B) was most likely due to clusterin accumulationin glial cell processes. In contrast to clusterin immunoreac-tivity in the epithelium, this deep clusterin accumulation inthe lamina propia appeared more frequently in animals killedearliest (48 hours) after bulbectomy. Control sectionsincubated without primary antibody displayed only a faintdiffuse autofluorescence in the superficial lamina propriawhich could not be mistaken for specific labeling (data notshown).

While there is no doubt about the glial nature of immuno-reactive cells located around or inside the olfactory nerve, theidentity of large immunoreactive sites located in the deepolfactory epithelium cannot be simply inferred from theirlocation. In order to assess their potential glial nature, wehave studied the comparative distribution of clusterin andGFAP in the olfactory mucosa using double immunofluor-escence. As shown in Fig. 9, GFAP immunoreactivity wasconfined to the lamina propia, and was obviously concen-trated in glial cell bodies and processes ensheathing olfactorynerve bundles, in accordance with previous studies (Barberand Dahl, 1987).

Following the clusterin immunofluorescence procedure,clusterin immunoreactivity appears to be of relatively lowintensity in the lamina propria, probably due to the low con-centration of clusterin stored in glial cell bodies and processes.While clusterin and GFAP immunoreactivities are regionallyco-localized in the lamina propia, they are obviously mis-matched in the epithelium. The most intense clusterin accu-mulation took place in large cell bodies located in the deepolfactory epithelium, just above the basal lamina, which provedcompletely devoid of GFAP immunoreactivity, demonstratingthat cells accumulating clusterin in the epithelium were notglial cells.

DISCUSSION

Our work allows us to answer several questions about theinvolvement of clusterin in neuron degeneration, thanks tosome remarkable features of the peripheral olfactory system inadult mammals. The in situ tissue localization of apoptoticcells, of clusterin mRNA and of clusterin protein, was clearly

12011210496888072645648403224168

DNA ladders

clusterine/actine

c-fos/actine

DNA ladders

Hours after bulbectomy

clusterin/β-actin


established, at the light microscopic level, by the histologicalcompartmentation of olfactory turbinates into segregated pop-ulations of sensory neuron cell bodies (85% of all neuroep-ithelial cells) responding massively and quasi-synchronouslyto synaptic target ablation, of bundled olfactory axons and ofnerve sheathing glia. Although the TUNEL method labels allnuclei containing fragmented DNA, including those of necroticcells, in the present case, the apoptotic character of TUNEL-stained nuclei in the olfactory epithelium of bulbectomizedmice is validated by our previous visualization of nucleosome-sized DNA fragmentation, unambigously related to theapoptotic process (Michel et al., 1994).

The present in situ studies have allowed us to discriminatein vivo between dying and clusterin-expressing cells. Theyhave demonstrated that the accumulation of clusterin mRNA isclosely dependent on the presence of apoptotic cells, but doesnot occur at the level of these cells. As revealed by in situhybridization, clusterin signals are completely absent from theolfactory epithelium which contains apoptotic cells. This cleardisconnection demonstrates that clusterin can no longer beconsidered as a gene involved in the apoptosis genetic program,in the olfactory neuron. This conclusion is in line with resultsobtained outside the nervous system, such as the existence ofapparently healthy clusterin-expressing cells (Michel et al.,1992; Aronow et al., 1993) or, conversely, with cases ofapoptosis without clusterin expression, like in leucocytes(Pearse et al., 1992; French et al., 1992), or in cells irradiatedin vitro with ultra violet (French et al., 1994). In turn, thesefindings are in apparent contradiction to recent data demon-strating that in the lurcher mutant mouse strain, Purkinje cells

Fig. 5. In situ hybridization of clusterin mRNA in olfactory mucosa 75 hclusterin mRNA, more important in the ipsilateral side of the lesion, is vapposition onto an X-ray film (A) or of the emulsion microautoradiogramcontrol one (C). B and C derive from the same unilateraly lesioned olfac

strongly accumulate clusterin mRNA prior to their death byapoptosis (Norman et al., 1995). This observation, however,does not imply that clusterin gene expression plays an activerole in the loss of Purkinje cells.

We have shown that clusterin transcripts are exclusivelylocated in the lamina propria of the olfactory mucosa, withthe most intense hybridization signal in olfactory nervebundles. This localization of hybridization signals is highlyconsistent with the assumption that clusterin synthesis occursin glial cells of the olfactory nerve, since glial ensheathingcells are the only cell body type present in this area. Thisstatement is in accordance with previous ones that support thehypothesis that clusterin gene induction in the lesioned brainoccurs mainly, if not exclusively, in glial cells (Danik et al.,1993).

A partial mismatch between clusterin mRNA and proteinhas been observed since immunoreactive sites to anti-clusterin antibody are present not only in the lamina propiabut also in the olfactory epithelium which proved completelydevoid of any hybridization signal for the clusterin mRNA.The possibility that clusterin immunoreactive glial cells havemigrated and penetrated the epithelium might be consideredsince GFAP-containing glial cells of the olfactory nerve havebeen shown to migrate towards the basal membrane liningthe olfactory epithelium during the few days followingolfactory nerve transection (Barber and Dahl, 1987).However, we can rule out this hypothesis since cell bodiesaccumulating clusterin in the olfactory epithelium are clearlyGFAP immunonegative (Fig 9). The alternative, and mostplausible interpretation of our data retains the possibility of

ours after unilateral bulbectomy. Asymmetrical accumulation ofisualized at the level of the global autoradiogram obtained by

s (B,C). More grains are visible in the targetless side (B) than in thetory mucosa, corresponding to that shown in A. Bar, 1 mm.


Fig. 6. Co-detection of clusterin mRNA and apoptotic nuclei.Adjacent sections of an olfactory mucosa from a mousebulbectomized for 40 hours were submitted to the three followingtreatments: (A) TUNEL detection of apoptotic nuclei. (B) In situhybridization for clusterin. (C) Hematoxylin staining. Comparison ofthe three pictures, corresponding to the same tissular region andobserved at identical magnification, shows that clusterin mRNA andapoptotic nuclei are detected in the lamina propria and in theneuroepithelium, respectively. This mutually exclusive distributiondemonstrates that clusterin is not synthesized in the dying neurones,but by lamina propria cells, informed about the physiological state ofolfactory neurones through an unknown mechanism. b, olfactorynerve bundles; N, nasal cavity; OE and arrowheads, olfactoryepithelium. Bar, 250 µm.

a fast secretion of clusterin from glial cells of the olfactorynerve, followed by internalization of the protein by targetcells located mainly, if not exclusively, in the olfactory neu-roepithelium. This hypothesis is highly consistent withprevious demonstrations that clusterin synthesized by glialcells is secreted (Pasinetti et al., 1994) and that extracellularclusterin could be internalized by target cells (Kounnas et al.,1995). Further investigations, using immunocytochemicalstudies at the cellular and ultrastructural levels, are nowneeded for an accurate identification of cells accumulatingclusterin in the olfactory mucosa, and particularly in theolfactory neuroepithelium.

At all events, we must emphasize the fact that the highestconcentration of clusterin was observed in the area wheredegenerating and regenerating processes (olfactory neurondegeneration and stem cell proliferation) occur concomitantly,

Fig. 7. Immunofluorescence detection of clusterin in the olfactorymucosa of a bulbectomized mouse, 75 hours after surgery (A). Theadjacent section has been stained with Neutral Red (B) in order todisplay the main anatomical subdivisions. In the olfactory epithelium(oe), dense patches of immunofluorescence are visible in the deepestzone, just above the basal lamina. Smaller immunoreactive sites arealso scattered in the intermediate zone of the epithelium. By contrast,the upper layer containing the supporting cell bodies appears ascompletely immunonegative. In the lamina propria (lp), clusterinimmunoreactivity concentrates in the olfactory nerve bundles (on,arrows), as either a diffuse fluorescent signal or small dots of cellularappearance. In the Nissl stained section (B), olfactory nerve bundlescorrespond to the pale circular areas located in the deep mucosa,while the stained areas are Bowman acini (bg). Bar, 50 µm.


Fig. 8. Immunocytochemical detection of clusterin in the olfactorymucosa 75 hours following olfactory bulb ablation, using theavidin/biotin-peroxidase method. In some areas of the olfactoryepithelium (oe), large immunoreactive patches are observed in thebasal area, just above the basal lamina (A, arrows). In the laminapropria (lp), immunoreactive cell bodies are present in olfactorynerve bundles (B, large arrows), either at the periphery, or inside thenerve. A fine network of immunoreactive processes can also bedistinguished inside the olfactory nerve bundle (small arrows). Bars:50 µm (A) and 25 µm (B).

Fig. 9. Regional distribution of clusterin (A) and GFAP (B)immunoreactive sites in two adjacent sections of an olfactoryturbinate, 75 hours after bulbectomy. The pattern of clusterinaccumulation is similar to the one shown in Fig. 7, with the mostprominent reactives sites located in the basal part of the olfactoryepithelium (oe), and a diffuse or patchy fluorescence at the level ofolfactory nerve bundles. GFAP immunoreactivity (B) is exclusivelypresent in the lamina propia (lp). Olfactory nerve bundles containnumerous immunoreactive glial cell bodies and processes. It isnoteworthy that clusterin immunoreactive sites located in theneuroepithelium are obviously not immunoreactive and cannot beinterpreted as migrating glial cells.

in the lower part of the olfactory epithelium (Monti-Graziadeiand Graziadei, 1979, 1992).

Among the different purported activities of the clusterinprotein, it is tempting to retain its possible involvement in alocal lipid homeostasis since clusterin, also namedapolipoprotein J, has been isolated as a component of a minorclass of circulating lipoproteins (de Silva et al., 1990). In thisrespect, it can be involved in the lipid recycling betweendegenerating and regenerating structures, as already proposedfor apolipoprotein E during reactive synaptogenesis (Poirieret al., 1991; Goodrum, 1991). Alternatively, since clusterinhas a potent complement-inhibiting activity (Kirszbaum etal., 1989; Jenne and Tschopp, 1989), it has been proposed tobe upregulated as a defense mechanism against immunologi-cal agressions in wounded brains (McGeer et al., 1992; Zhanet al., 1994). Indeed, the complement pathway has beenimplicated in brains of Alzheimer patients (McGeer et al.,1989). This hypothesis is further supported by observationsthat expression of complement components and clusterin areco-induced after experimental lesioning in the nervoussystem (Pasinetti et al., 1992; Liu et al., 1995; Rozovsky etal., 1994), probably by glial cells (Liu et al., 1995; Gasque etal., 1995).

Finally, taking into account that clusterin does not complexonly with complement molecules but also with other extracel-lular components as the secreted amyloid (Matsubara et al.,1995), one may propose clusterin as a ‘molecular cleaner’

ensuring the solubilization of a variety of compounds poten-tially cytotoxic. Indeed, clusterin is thought to maintainamyloid (Matsubara et al., 1996; Boggs et al., 1996) as well ascomplement membrane attack complexes (Jenne and Tschopp,1992) in soluble forms.

Previous hypotheses assuming that clusterin is expressedby apoptotic cells, have interpreted the decrease of theclusterin mRNA level at the end of the degeneration period,as a mere consequence of the loss of clusterin-producing cells(Guenette et al., 1994a). The demonstration, in the olfactorysystem, that the apoptotic and the clusterin-expressing cellsare clearly separated, means that the clusterin gene is in factsubjected to a stringent regulation in surviving cells, andprobably depends on cell-cell interactions. The way in whichthe ensheathing cells are informed about the physiological


state of the olfactory neurons remains to be elucidated, butour comparative time-course experiments already show thatthis information is needed early on, just before the beginningof DNA fragmentation and when the neuron bodies are stillintact.

In conclusion, the present observations rule out the directinvolvement of clusterin expression in the apoptosis geneticprogram, but at the same time strengthen the existence of a linkbetween clusterin and apoptosis.

This work was supported by the Association pour la Recherche surle Cancer (ARC) and by the Region Rhône-Alpes.

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(Received 22 July 1996 – Accepted 15 May 1997)

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