The impact of Aβ-plaques on cortical cholinergic and non-cholinergic presynaptic boutons in alzheimer's disease-like transgenic mice

Neuroscience 121 (2003) 421– 432

THE IMPACT OF A␤-PLAQUES ON CORTICAL CHOLINERGIC AND NON-CHOLINERGIC PRESYNAPTIC BOUTONS IN ALZHEIMER’S DISEASE-LIKE TRANSGENIC MICE L. HU,a,1,2 T. P. WONG,a,1 S. K. F. S. BELLa AND A. C. CUELLOa,b,*

L.

ˆ TE´,a CO

dence of cholinergic boutons within the total pre-synaptic bouton population. Confocal and electron microscopic observations confirmed the preferential infiltration of dystrophic cholinergic boutons into fibrillar amyloid aggregates. We therefore hypothesize that extracellular A␤ aggregation preferentially affects cholinergic terminations prior to progression onto other neurotransmitter systems. This is supported by the observable presence of non-cholinergic sprouting, which may be representative of impending neuritic degeneration. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec, Canada H3G 1Y6 b Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada H3G 1Y6

Abstract—A previous study in our laboratory, involving early stage, amyloid pathology in 8-month-old transgenic mice, demonstrated a selective loss of cholinergic terminals in the cerebral and hippocampal cortices of doubly transgenic (APPK670N,M671LⴙPSlM146L) mice, an up-regulation in the single mutant APPK670N,M671L mice and no detectable change in the PSlM146L transgenics [J Neurosci 19 (1999) 2706]. The present study investigates the impact of amyloid plaques on synaptophysin and vesicular acetylcholine transporter (VAChT) immunoreactive bouton numbers in the frontal cortex of the three transgenic mouse models previously described. When compared as a whole, the frontal cortices of transgenic and control mice show no observable differences in the densities of synaptophysin-immunoreactive boutons. An individual comparison of layer V of the frontal cortex, however, shows a significant increase in density in transgenic models. Analysis of the cholinergic system alone shows significant alterations in the VAChT-immunoreactive bouton densities as evidenced by an increased density in the single (APPK670N,M671L) transgenics and a decreased density in the doubly transgenics (APPK670N,M671LⴙPSlM146L). In investigating the impact of plaque proximity on bouton density at early stages of the amyloid pathology in our doubly (APPK670N,M671LⴙPSlM146L) transgenic mouse line, we observed that plaque proximity reduced cholinergic pre-synaptic bouton density by 40%, and yet increased synaptophysinimmunoreactive pre-synaptic bouton density by 9.5%. Distance from plaques (up to 60 ␮m) seemed to have no effect on bouton density; however a significant inverse relationship was visible between plaque size and cholinergic pre-synaptic bouton density. Finally, the number of cholinergic dystrophic neurites surrounding the truly amyloid, Thioflavin-Sⴙ plaque core, was disproportionately large with respect to the inci-

Key words: amyloid precursor protein, amyloid plaque, presynaptic bouton, vesicular acetylcholine transporter, synaptophysin, presenilin 1.

Although senile plaques (SP) are regarded as one of the main pathological hallmarks of Alzheimer’s disease (AD), the results of clinicopathological studies remain controversial as no definite correlation has been established between SP and the severity of dementia (for review, see Giannakopoulos et al., 1997; Cummings et al., 1998). The significant decrease in presynaptic density seen in AD has been well documented (Hamos et al., 1989; Scheff et al., 1990; Honer et al., 1992; Lippa et al., 1992; Masliah, 1995) and is thought to correlate more closely with cognitive decline than any other neuropathological landmark, including SP and/or neurofibrillary tangle number (Terry et al., 1991). Reduced synaptic density precedes overt neuronal loss and is therefore thought to be one of the first indications of disease presence (DeKosky and Scheff, 1990; Terry et al., 1991; Heinonen et al., 1995). The importance of the cholinergic involvement in AD neuropathology has been relatively consistent in the literature (Bowen and Smith, 1976; Davies and Maloney, 1976) due to the correlation between cholinergic deficits and decreased cognitive impairment (Perry et al., 1978; Collerton, 1986; DeKosky et al., 1992). The cholinergic network has also been implicated in higher order functions such as memory development (Winkler et al., 1995; Tang et al., 1997) and functional, post-learning cortical reorganization (Juliano et al., 1991; Kilgard and Merzenich, 1998). The discovery of specific gene mutations in familial AD (for review, see Price and Sisodia, 1998) and their subsequent in vivo expression in transgenic animal models has greatly increased our understanding of the cholinergic network’s role in AD. Mutations in the presenilin 1 (PS1; Sherrington et al., 1995), PS2 (Levy-Lahad et al., 1995) and amyloid precursor protein (APP) genes (for review, see Selkoe, 1994) have all been linked to significant alter-

1

Both authors contributed equally to this work. Present address: Department of Neurology and Neuroscience, First Teaching Hospital, Jilin University, Changchun, Jilin, P.R. China 130021. *Correspondence to: A. C. Cuello, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec H3G 1Y6, Canada. Tel: ⫹1-514-398-3621; fax: ⫹1-514-398-6690. E-mail address: [email protected] (A. C. Cuello). Abbreviations: A␤, amyloid-beta protein; AD, Alzheimer’s disease; APP, amyloid precursor protein; ChAT, choline acetyltransferase; DAB, 3, 3⬘-diaminobenzidine tetrahydrochloride; IR, immunoreactive; PB, phosphate buffer; PBS⫹T, phosphate-buffered saline with 0.2% Triton X-100; PS1, presenilin 1; SP, senile plaques; VAChT, vesicular acetylcholine transporter. 2

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00394-4

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Fig. 1. A schematic representation of the sampling method for quantification of cortical presynaptic boutons in transgenic and non-transgenic mice. In (A) the sampling method used for the random neuropile (dimensions of 104 ␮m⫻81 ␮m) is illustrated. All vascular or plaque occupied areas were excluded from analysis. The sampling method used for the plaque adjacent neuropile is shown in (B), where six successive fields of 300 ␮m2 area were quantified, moving in a successive lateral motion away from the plaque perimeter. Only Thioflavin S⫹ plaques were included in the analysis.

ations in APP metabolism. Transgenic overexpression of any of these gene mutations leads to increased levels of amyloid-␤ protein (A␤; Citron et al., 1992, 1997; Cai et al., 1993; Suzuki et al., 1994; Borchelt et al., 1996; Borchelt et al., 1997; Duff et al., 1996; Scheuner et al., 1996). Certain models display extracellular amyloid deposits, which are highly reminiscent of the SP seen in AD (Hsiao et al., 1996; Games et al., 1995; Masliah et al., 1996; Nalbantoglu et al., 1997; Sturchler-Pierrat et al., 1997). Several of these transgenic models were included in our study to investigate the impact of A␤ plaques on fronto-cortical presynaptic bouton density. Neither of the single mutant transgenic mice generated amyloid plaques by the 8-month time point studied here. Overexpression of the mutated PS1 gene generated a slight increase in A␤ levels; however, it did not lead to plaque formation regardless of survival time (Duff et al., 1996; Citron et al., 1997; Chui et al., 1999). Single transgenic mice overexpressing the APPK670N,M671L mutation (Tg2576; Hsiao et al., 1996) eventually develop plaques; however, no plaque presence was seen by the 8-month time point. Co-expression of the mutated APP and PS1 genes (a result of crossbreeding the F1 of Tg2576⫻PS1M146L; Holcomb et al., 1998) accelerates extracellular amyloid accumulation and deposition, since plaque formation was visible by the 8-month time point studied (Holcomb et al., 1998) and this is accompanied by losses of cholinergic presynaptic boutons in the cerebral and hippocampal cortices (Wong et al., 1999). In this study we analyze the impact of plaque presence and its proximity on cholinergic and synaptophysin immunoreactive (IR) pre-synaptic bouton populations within both the plaque adjacent and random neuropiles. We further investigated the nature of the dystrophic neurites surrounding amyloid plaques in doubly transgenic mice. The results indicate that amyloid aggregates exert a differential

effect on the cholinergic and overall populations of cortical presynaptic boutons.

EXPERIMENTAL PROCEDURES Experimental animals and tissue processing All animal experimentation was approved by the McGill University Animal Care Committee and was conducted in accordance with the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. Every effort was made to minimize the number of animals required to render statistically significant results in these experiments and no unnecessary suffering was caused. Tissue samples were obtained in order to study the synaptic pattern in the neuropile at “random” (i.e. the neuropile largely devoid of plaques) as well as plaque-adjacent areas. The sampling method is represented in Fig. 1. Our study investigated the synaptic patterns within the random neuropile of 22, 8-month-old transgenic mice [six APP mutants (APPK670N,M671L; derived from line Tg2576), six PS1 mutants (PS1M146L), five doubly mutants APP and PS1 (F1 of Tg2576⫻PS1M146L) and five non-transgenic littermates, resulting from the crossing of the two single transgenic lines]. All animals were coded to ensure unbiased processing and analysis. Animals were anesthetized with Equithesin (2.5 ml/kg, i.p.) and injected with heparin (4 USP/kg, i.p.) prior to perfusion through the heart with perfusion buffer containing 0.1% sodium nitrite, and fixative containing 3% paraformaldehyde (BDH, Toronto, ON, Canada), 0.1% glutaraldehyde (MECA Lte´e, Montreal, QC, Canada) and 15% picric acid (BDH) in 0.1 M phosphate buffer (PB; pH 7.4; for composition see Coˆte´ et al., 1993). After a 3-h post-fixation in the same fixative, brains were cryoprotected with 10% sucrose in PB and cut into 50 ␮m coronal sections with a Leica sledge freezing microtome at ⫺20 °C from bregma 1.40 mm to bregma ⫺3.40 mm (Franklin and Paxinos, 1997). Sections from the same brain were separated into groups for Nissl staining (0.3% Cresyl Violet; Sigma, Oakville, ON, Canada) to identify different cortical laminae. Alternating sections were immunohistochemically stained for synaptophysin and vesicular acetylcholine transporter (VAChT).

L. Hu et al. / Neuroscience 121 (2003) 421– 432 Studies involving the synaptic patterns of the plaque adjacent neuropile used four 8-month-old doubly transgenic F1 hybrids of the APP (APPK67ON,M671L, Tg2576) and PS1 (PSlM146L) mutants as well as four non-transgenic littermates. Mice were anesthetized, perfused, fixed and stained as described above. Immunostainings were combined with Thioflavin-S histochemical staining.

Immunohistochemical staining for light and electron microscopy Free-floating immunohistochemical staining was performed as previously described (Coˆte´ et al., 1993). Briefly, 0.01 M phosphate-buffered saline with 0.2% Triton X-100 (PBS⫹T) was used for all washings and dilutions throughout the experiments; two PBS⫹T washes were performed in between all antibody incubations. Sections from diverse animal groups were processed simultaneously. VAChT immunohistochemical staining was performed using the avidin– biotin complex method. Brain sections were treated with 0.1% sodium borohydride (Sigma) in 0.01 M PBS for 30 min, then incubated with 5% normal goat serum (Sigma) at 37 °C, for 30 min. Normal goat serum (2.5%) was added to solutions containing immunoreagents to further reduce background staining. A polyclonal antiserum against VAChT (1:8000; a gift from Dr. R. H. Edwards, University of California, San Francisco, USA) was used to identify the presynaptic cholinergic sites. Sections were incubated with the antiserum solution for 48 h at 4 °C, followed by two, 2-h subsequent incubations with biotinylated goat anti-rabbit antibody (1:500; Vector Laboratories, Burlingame, CA, USA) and avidin– biotin complex (1:250; Vector) at room temperature. After treating with 0.6% 3, 3⬘-diaminobenzidine tetrahydrochloride (DAB; Sigma), H2O2 was added for a development time of 4 min. Synaptophysin immunohistochemical staining was performed using a monoclonal mouse anti-synaptophysin antibody (Roche, Laval, QC, Canada; 1:40, 4 °C, overnight). After washing, the tissue was immersed in a goat anti-mouse IgG solution (1:160; American Qualex, San Clemente, CA, USA) for 1 h at room temperature, followed by a monoclonal mouse anti-peroxidase antibody (1:60; Medicorp, Montreal, QC, Canada; Semenenko et al., 1985) with 5 g/ml horseradish peroxidase (Sigma; type IV) for 1 h at room temperature. After several washes, the tissue was incubated in 0.6% DAB in PBS⫹T for 15 min at room temperature. Subsequently, H2O2 was added to the DAB solution and the reaction was continued for 6 min. A mouse monoclonal antibody against the human A␤ peptide (Grant et al., 2000) was used to demonstrate the A␤ aggregates in the transgenic mice. Prior to immunostaining, brain sections were incubated in 0.5% H2O2 for 30 min and treated with 3% bovine serum albumin (Sigma) for 60 min at room temperature. Following this, sections were incubated in the mouse anti-A␤ antibody (1:1500; 4 °C) solution overnight. Sections were then incubated for 1 h with goat anti-mouse IgG (1:50; ICN Biochemicals, Costa Mesa, CA, USA) and for another hour with monoclonal mouse anti-peroxidase antibody (1:30; Medicorp; Semenenko et al., 1985) in the presence of 5 ␮g/ml horseradish peroxidase (Sigma; type IV) at room temperature. After 15 min of DAB treatment, H2O2 was added and the reaction was developed for 4 min. After immunostaining, all sections were mounted on gelatincoated glass slides, air-dried, dehydrated in ascending concentrations of ethanol, cleared with xylene, and cover-slipped with Entellan (EM Sciences, Gibbstown, NJ, USA). To determine amyloid plaque boundaries in VAChT- and synaptophysin-immunostained sections, Thioflavin-S histochemical staining was also performed. Sections were rehydrated in descending concentrations of ethanol, rinsed in distilled water, stained in Thioflavin-S solution (1% in 50% ethanol) for 5 min, rinsed in distilled water, washed twice in PBS-T for 15 min and mounted with Vector mounting medium.

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For electron microscopical observations we utilized three doubly transgenic mice, intracardially fixed and perfused as above. Fifty micrometer thick sections were immunostained for VAChT as free-floating sections as described above and processed for electron microscopy as previously described (Ribeiro-da-Silva et al., 1993). Lamina V sections were trimmed from flat-embedded samples of cerebral cortex F1/F2 and re-embedded in epoxy resin blocks. Ultrathin sections were cut with a dimatome diamond knife to 66 nm thickness and immunostained for A␤ immunoreactivity with the monoclonal antibody McSA1 (Grant et al., 2000) followed by a post-embedding immunostaining procedure as previously described (Pickel, 1981). The immunoreaction was developed using a rabbit anti-mouse immunoglobulin conjugated with 10 nm gold particles (McLeod et al., 1998, 2000).

Quantification of immunohistochemical staining Quantification of the density of VAChT-IR terminals and synaptophysin-IR presynaptic boutons was performed essentially as previously described (Wong et al., 1998, 1999). A BH-2 Olympus microscope equipped with a 100⫻ oil immersion plan achromatic objective and a 10⫻ projection lens was used. The microscope was equipped with a CDD video camera and was connected to the MCID-M4 image analysis system (Imaging Research Inc., St. Catherines, ON, Canada). Immunopositive punctae (varicosities of cholinergic terminals and presynaptic synaptophysin boutons) were detected by the image analysis system using software designed for silver grain counting. Areas occupied by vessels or plaques (in the case of the doubly transgenic) were excluded from quantification. Studies involving the “random” neuropile quantified the densities of presynaptic elements within all six layers of the F1 region as well as from laminae V and VI individually (see Fig. 1A). The F1 area of the frontal cortex was determined according to Franklin and Paxinos, 1997. Studies involving the plaque proximal neuropile areas were performed in fields close to relatively isolated amyloid plaques in lamina V of the F1 region of the frontal cortex, as facilitated by adjacent Nissl-stained sections. Plaques were considered “relatively isolated” if no other plaques were present within a radial distance of 100 ␮m of the plaque border. Plaque size and perimeter were revealed by both Thioflavin S-staining and A␤ immunostaining. Those plaques which met the criteria of being “relatively isolated” were digitized in six successive 300 ␮m2 fields (30 ␮m in height and 10 ␮m in width), starting at the plaque perimeter and moving laterally in a horizontal manner (as displayed in Fig. 1B). The background staining of all sections was uniformly and individually normalized by the M3D module of the M4 system, enabling us to use a single detection threshold to measure the numbers of VAChT-IR terminals and synaptophysin-IR presynaptic boutons. Segmentation values were selected by a trial and error method, namely by keeping values which provided the most accurate measurements when compared with direct visual counting of punctae within a selected area of the computer screen. Once ideal detection thresholds for VAChT-IR terminals and synaptophysin-IR presynaptic boutons were determined, values were saved in the computer program and applied to all subsequent quantifications. In quantifying synaptophysin-IR boutons, all cell bodies, blood vessels, and unfocused cortical areas were excluded. Results are expressed as the number of varicosities of cholinergic terminals and the number of presynaptic boutons per 1000 ␮m2.

Statistics Mouse identification was unveiled post-quantification and results are displayed as mean⫾S.E.M. One-way ANOVA was used to compare the size and density of cholinergic terminals and presynaptic boutons among different groups of plaques. A post hoc

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Table 1. Pre-synaptic bouton density in the random neuropile of control versus transgenic mice (number of synapses/1000/␮m2) Control Frontal cortex Lamina I–VI Frontal cortex Lamina V⫹VI only

PS1M146L

Cholinergic

35.2⫾0.7

34.4⫾1.3

Overall Cholinergic

355.8⫾21.0 34.6⫾1.2

395.0⫾32.2 34.3⫾1.1

Overall

354.0⫾13.7

394.7⫾20.9

APPK670N,M671L

APPK670N,M671L⫹PS1M146L

39.8⫾1.7* (112.9%) 414.7⫾33.6 40.0⫾1.5** (115.8%) 412.1⫾21.1* (116.4%)

30.2⫾1.3** (214.3%) 403.5⫾13.1 30.6⫾1.2* (211.6%) 400.6⫾10.1

Percentages in brackets indicate differences with respect to controls. * P⬍0.05; ** P⬍0.01.

Dunnett test was used for pair-wise comparisons between control littermates and transgenic mice, while remaining pair-wise comparisons were performed using post hoc Tukey tests. To compare synaptic terminal density in doubly transgenic mice and control littermates, unpaired Student’s t-test was applied. Significance level for all analyses was set as P⬍0.05.

RESULTS Cholinergic and total presynaptic bouton densities in the random neuropile of transgenic and nontransgenic mice Based on our previous observations that presynaptic boutons in lamina V and VI are more sensitive to both the A␤ burden (Wong et al., 1999) and the ageing process (Wong et al., 1997), we felt that further investigation on cholinergic and non-cholinergic presynaptic elements in these individual layers was needed. Thus, an analysis of both Lamina V and VI alone, as well as all six layers of the F1 and F2 regions were included in our study. Quantification of the random neuropile (absence of proximal plaque presence)

of doubly transgenic mice (APPK670N,M671L⫹PSlM146L) revealed significantly decreased cholinergic presynaptic bouton densities of 30.6⫾1.2/1000 ␮m2 (versus 34.6⫾1.2 boutons/1000 ␮m2 in controls) in Lamina V and VI individually, and 30.2⫾1.3/1000 ␮m2 (versus 35.2⫾0.75 boutons/1000 ␮m2 in controls) in all layers of the F1/F2 regions (see Table 1). Analysis of the random neuropile in the PS1 mutants revealed no significant changes in cholinergic presynaptic bouton density, regardless of the lamina in which quantification occurred (see Table 1). The APP mutants however, showed a significant up-regulation in cholinergic presynaptic bouton density in both laminar areas (see Table 1), which confirms our previous findings (Wong et al., 1999). Synaptophysin-IR presynaptic boutons were quantified to determine whether the visibly altered bouton densities were exclusive to the cholinergic system. This was achieved by comparing the densities of the total (synaptophysin-IR) and cholinergic (VAChT-IR) pre-synaptic bouton populations within transgenic and control mice. All three of the transgenic mouse models displayed a similar

Fig. 2. Light microscopic representation of the synaptophysin-IR (A–D) and VAChT-IR boutons (E–H) in the random neuropile of the lamina V region of the frontal cortex in different transgenic mouse models. Note the higher density of synaptophysin-IR boutons in the PS1M146L transgenic mice (B), APPK670N,M671L transgenic mice (C) and doubly transgenic mice (D) as compared with non-transgenic controls (A). Comparing VAChT-IR bouton density in control mice (E) with transgenic mice revealed no change in VAChT bouton density in the PS1 transgenic mice (F), an up-regulation of VAChT bouton density in the APP transgenic mice (G) and a decrease in the number of VAChT boutons in the doubly transgenic mice (H). Scale bar⫽20 ␮m.

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trend of increased levels of synaptophysin-IR boutons in the random neuropile areas of Laminae I–VI and V and VI alone (see Table 1) as compared with non-transgenic controls. Notably, the APP mutants displayed a significantly elevated density of synaptophysin-IR presynaptic boutons in the neuropile areas of Lamina V and VI (412.1⫾21.1 versus 354.0⫾13.7 boutons/1000 ␮m2 in control mice; see Table 1). Fig. 2 displays the typical appearance of the cholinergic and synaptophysin-IR neuropile in lamina V of the F1 regions of the transgenic and non-transgenic mice. Amyloid plaque presence in the doubly transgenic APPK670N,M671LⴙPSlM146L mice Doubly transgenic mice displayed obvious extracellular amyloid plaques by the 8-month time point. Mature plaques were revealed by both Thioflavin-S⫹ and A␤-immunostainings; thus diffuse plaques remained undetected. A␤-IR areas typically covered by a larger portion of the plaque than Thioflavin-S⫹ areas, which was likely indicative of the corolla of diffuse, fibrillar A␤ material surrounding the amyloid core. Plaque distribution throughout the cortex was uneven as were the proportional areas, which accounted for 4.49%, 5.58%, 4.18%, 5.2 1%, 3.42%, and 1.63% of the total cingulate, frontal I, frontal II, parietal I, parietal II and entorhinal cortical areas respectively. Plaque size was also variable: layer V of the F1/F2 region showed a range in size of 344 ␮m2 to 6124 ␮m2. Cholinergic and total presynaptic bouton patterns in the plaque adjacent neuropile of doubly transgenic mice (APPK670N,M671LⴙPSlM146L) The density of the VAChT-IR presynaptic bouton population in the plaque adjacent neuropile areas of lamina V and VI of the F1/F2 regions was remarkably lower in the APP⫹PS1 doubly transgenic mice. This decrease in cholinergic bouton density remained relatively consistent throughout all six successive plaque adjacent neuropile fields and did not appear to be significantly influenced by plaque proximity. However, a statistically significant negative influence of plaque size on cholinergic pre-synaptic bouton density in the plaque adjacent neuropile was observed (Fig. 3A). No correlation between plaque size or proximity, and bouton density in the synaptophysin-IR presynaptic bouton population was observed (Fig. 3B). However, the synaptophysin-IR presynaptic bouton population did appear to be affected by the presence of amyloid plaques as an increased density of 9.5% was visible in the plaque adjacent neuropile areas of lamina V and VI. The morphological impact of amyloid burden on the cholinergic and synaptophysin-IR presynaptic bouton populations and presence of dystrophic elements is illustrated in Fig. 4. Synaptophysin-IR and VAChT-IR dystrophic neurites of the plaque perimeter Despite the relatively low proportion of cholinergic network fibers in the cortex (5–10%) in relation to the total population of presynaptic boutons, the number of cholinergic

Fig. 3. A graphic representation of the correlation between presynaptic bouton density and plaque size. In (A) we see a significant negative correlation between amyloid plaque size and density of presynaptic boutons, stained for the VAChT in the plaque adjacent neuropile of double transgenic PS1M146L⫹APPK670N,M671L mice as compared with non-transgenic controls. In contrast no correlation between plaque size and overall presynaptic bouton population is visible, as revealed by immunohistochemical staining with anti-synaptophysin antibodies (B).

dystrophic elements surrounding Thioflavin-S⫹ mature plaques is disproportionately high. In fact the total area occupied by cholinergic dystrophic neurites was approximately half of the total area occupied by synaptophysin-IR dystrophic neurites. Light microscopy observations suggest that VAChT-IR dystrophic neurites are located more proximally to the plaque core (Fig. 4A and C) than the synaptophysin-IR dystrophic neurites (Fig. 4B and D). This observation was confirmed by confocal microscopy analyses, which showed that dystrophic cholinergic terminals tended to be in direct contact with the Thioflavin-S⫹ amyloid material (Fig. 5). This relationship was further confirmed at the EM level, where cholinergic boutons and dystrophic neurites were often seen in direct contact with A␤-IR fibrillar material, while non-cholinergic (VAChT nonIR) dystrophic processes tended to be positioned further from the amyloid material (Fig. 6).

DISCUSSION AD-related dementia is clearly linked in some manner to a cholinergic deficit since pharmacological studies have shown a symptomatic relief and delay of cognitive decline upon anticholinesterase drug administration (for results and reviews see Gauthier et al., 1989, 1990, 2002; Giacobini, 1991, 2001, 2002; Giannakopoulos et al., 1997; Rockwood et al., 1997; Cummings et al., 1998; Ladner and Lee,

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Fig. 4. Light microscopy staining of the lamina V plaque adjacent neuropile in the F1 region of the cerebral cortex of PS1M146L⫹APPK670N,M671L double transgenic mice, as revealed by immunohistochemistry using anti-VAChT antibodies and anti-synaptophysin antibodies, for the cholinergic and total presynaptic bouton populations respectively. Note the relatively large number of cholinergic boutons surrounding plaques as well as the more proximal location to the plaque core. Scale bars⫽60 ␮m (A, B); C, D⫽20 ␮m

1998; Geula et al., 1998; Burns et al., 1999; Gauthier, 1999; Baladi et al., 2000; Feldman et al., 2001; Lahiri et al., 2003). Despite major advances in the field, anticholinesterases remain the only widely accepted form of symptomatic therapy by prolonging the actions of acetylcholine transmission on muscarinic receptors which appear to be relatively well-preserved in the AD brain (Svensson et al., 1992; Ladner and Lee, 1998; Mulugeta et al., 2003). While the mechanistic pathway leading to cholinergic basal forebrain attrition is relatively unknown, our findings here show that a cortical amyloid burden is sufficient to initiate a cholinergic presynaptic involvement in transgenic mice. Synaptic loss has been suggested to be a crucial component of AD pathology. Several authors have provided evidence that synaptic loss correlates more closely with dementia than other structural lesions (DeKosky and Scheff, 1990; Terry et al., 1991; Lassmann et al., 1992; Giannakopoulos et al., 1997). In fact, in the postmortem AD brain a decreased presynaptic bouton density of up to 45% has been seen (DeKosky and Scheff, 1990; Davies et al., 1987; Hamos et al., 1989; McGeer et al., 1994; Ikonomovic et al., 2002); however, the reasoning behind this “synaptoses” is unknown. An in vitro postmortem tracing method was recently used to compare axonal transport in neuronal tissue of the temporal and prefrontal cortical regions of AD brains, revealing a decreased level of axonal transport in the former region, as would be expected given its marked implication in the AD pathology (Dai et al.,

2002). The proven correlation between axoplasmic transport and the subsequent onset of neuronal degeneration and death (Catsicas et al., 1992), may explain in part the decrease in presynaptic bouton density observed in AD patient autopsies. Over the last 10 years the “Amyloid Hypothesis” (Selkoe, 2000; Hardy and Selkoe, 2002) has been the most commonly accepted hypothesis to explain the physiopathology of AD. In most interpretations of this hypothesis, A␤ deposition within the parenchyma is listed as the initiating factor of dementia. However, if the degeneration of cholinergic nerve terminals and resultant dementia occur due to plaque formation, one would expect a firm correlation between the incidence of SP and severity of dementia to exist. Studies addressing this issue however have found that amyloid plaques can occupy as much as 15% of the total cortical surface area in human AD brains without displaying a significant correlation with duration or severity of dementia (Giannakopoulos et al., 1997; Gomez-Isla et al., 1997). Furthermore, a doubly transgenic mouse model, bearing both APP and PS1 gene mutations, displayed a remarkable reduction in the spontaneous alternation performance in a “Y” maze, prior to substantial amyloid plaque presence (Holcomb et al., 1998). Likewise, Mucke and colleagues (2000) have described synaptic losses preceding plaque formation in a transgenic model expressing wild type human APP. Neuropathological changes have also been detected in the absence of plaque

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Fig. 5. Confocal microscopy displaying the Thioflavin-S⫹ core of mature amyloid plaques as well as the immunoreactivity of the cholinergic nerve terminals (A) of the F1 region of the cerebral cortex of double transgenic PS1M146L⫹APPK670N,M671L mice. Green denotes amyloid staining using Thioflavin-S⫹ staining (B), while the red fluorescence denotes VAChT-IR terminals, as revealed by rhodamine-red immunofluorescence. In (A) numerous VAChT dystrophic neurites can be observed surrounding and overlapping (yellow signals) the amyloid plaque material. Scale bar⫽40 ␮m.

formation in a PS1 mutant transgenic mouse line (Chui et al., 1999), suggesting that certain aspects of AD-like neuropathology likely occur independently of amyloid plaque formation (Holcomb et al., 1998; Chui et al., 1999) and may potentially account for the synaptic dysfunction visible in plaque absent environments (Hsia et al., 1999). Conversely, however, extracellular aggregated A␤ has been shown to radically alter dendritic morphology (Knowles et al., 1999) and in vivo studies have demonstrated that axons filled with a marker for anterograde transport deviate from their course and become morphologically altered in the presence of A␤ plaques in AD transgenic models (Phinney et al., 1999). Thus, an important question within the potential correlation of amyloid pathology, neurotransmission and resultant dementia, is whether A␤-induced neurotoxicity requires the deposition of aggregated A␤ into plaques. Our study investigated this question by analyzing the impact of the A␤ burden on presynaptic bouton density and dystrophic neurite morphology in both the cholinergic and the total (cholinergic and non-cholinergic, together) presynaptic bouton populations of three different transgenic mouse models, carrying APP and/or PS1 gene mutations. Immunocytochemical markers were used to identify and quantify these presynaptic populations. Cholinergic presynaptic boutons were labeled with an antibody specific to VAChT. The VAChT antigenic sites are considered to be reliable presynaptic cholinergic network markers (Gilmor et

al., 1996; Weihe et al., 1996; Roghani et al., 1996), since VAChT transports acetylcholine into vesicles exclusively within cholinergic nerve terminals of the CNS (for review, see Usdin et al., 1995). For the identification and quantification of the total (cholinergic and non-cholinergic) presynaptic bouton population we applied antibodies against synaptophysin, which is a suitable marker as this protein is located in both the small synaptic, and large dense-core vesicles of almost all presynaptic boutons (Jahn et al., 1985; Wiedenmann and Franke, 1985; Navone et al., 1986; Lowe et al., 1988; Walaas et al., 1988; Sudhof and Jahn, 1991). These markers were used to quantify the VAChT-IR and synaptophysin-IR presynaptic bouton densities within both the plaque adjacent and random neuropile in transgenic mouse models. In line with well-established neurochemical observations (Davies and Maloney, 1976; Bowen et al., 1982; Quirion et al., 1986; Perry et al., 1992) of the human brain, we observed a cholinergic involvement in both the random and plaque adjacent neuropile of APPK670N,M671L⫹ PSlM146L doubly transgenic mice. In the random neuropile, this was a modest compromise (see Table 1). However, the decreased density was much more marked in the plaque adjacent neuropile, reaching a maximum of approximately 40% when in relation to large plaques (see Fig. 3). These results suggest that extracellular aggregated A␤ may be involved in the pathological decline of cholinergic synapses. It is possible that this phenomenon may in fact

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Fig. 6. Electron microscopic micrograph illustrating the close relationship of cholinergic (VAChT-IR) dystrophic presynaptic boutons (immunoenzymatic staining) and the A␤ amyloid-IR fibrillar material (immunogold staining) in the plaque periphery of a doubly transgenic mouse. Note that non-cholinergic dystrophic neurites are positioned further away from the plaque periphery. Electron dense dots overlying the amyloid fibrillar material denote A␤-imunoreactivity. Scale bar⫽1.2 ␮m.

be the substrate for the loss in assayable choline acetyltransferase (ChAT) enzymatic activity in postmortem AD samples (Davies and Maloney, 1976; Bowen et al., 1982; Quirion et al., 1986; Perry et al., 1992). As more recent studies in the human brain seem to indicate (Davis et al., 1999), the cholinergic synaptic loss and dystrophy visible in transgenic models appears in more advanced stages of amyloid pathology. Figs. 5 and 6 illustrate this intimate interaction between degenerating cholinergic fibers and A␤ fibrillar material. It is also important to highlight, however, that a significant elevation of cholinergic boutons was visible in the APP single mutants who showed no plaque presence at the 8-month time point (see Table 1). This observation confirms our previous findings (Wong et al., 1999) and correlates with the elevated level of ChAT activity and mRNA observed by Hernandez and collaborators (Hernandez et al., 2001) at the same time point and in the same

mouse model. Would a similar cholinergic up-regulation occur in the progression of the AD pathology? In this regard it is interesting to note that an increased cholinergic network in the hippocampus of individuals with mild cognitive impairment has been found (DeKosky et al., 2002; Ikonomovic et al., 2002), a condition which is now considered prodromic of AD (Collie and Maruff, 2000; Morris et al., 2001; Thompson and Hodges, 2002; Yesavage et al., 2002). Whether this increased number of cholinergic boutons represents a type of sprouting phenomenon remains to be established. Another issue which also remains unclear at this time is whether a functional or neurochemical counterpart exists. Speculatively, however, it is possible that the observable presynaptic up-regulation could be due to an initial, possibly neurotrophic, influence of APP fragments (Mattson et al., 1993; Wallace et al., 1993, 1995, 1997a,b; Luo et al., 2001), as has been well illustrated in several experimental models.

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Our findings show that synaptic alterations within transgenic mouse models of AD pathology are not entirely A␤ plaque dependent. Alteration of the synaptic pattern within the random neuropile was visible in two different transgenic mouse models in which there was no presence of amyloid plaques. Additionally the consistent up-regulation of synaptophysin-IR presynaptic boutons across all three transgenic models, as compared with the decreased cholinergic presynaptic bouton population, suggests that amyloid burden may have a differential time-related effect on different neurotransmitter systems. In conclusion, we suggest that the dementia observed in AD is not plaque dependent, but rather progresses in accordance with the A␤ burden (soluble, aggregated in the neuropile) in a time-dependent and neurotransmitter specific manner. Acknowledgements—This research was supported by a grant to A.C.C. (CIHR MOP-37996). The authors would like to thank Dr. Karen Duff (Nathan Kline Institute, NY, USA) for the provision of transgenic mice, as well as Dr. R. H. Edwards (UCSF) for his generous gift of anti-vesicular acetylcholine transporter antibodies. We would also like to thank Mr. Alan Forster for assistance in photography, Dr. Paul B. S. Clarke for his suggestions on statistical analyses, Sid Parkinson for editorial assistance, Adriana Ducatenzeiler for expert assistance in genotyping and John LaFrancois for mouse husbandry. T.P.W. was the recipient of a Doctoral award from the Alzheimer Society of Canada.

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(Accepted 7 May 2003)