ADAM-10 over-expression increases cortical synaptogenesis

Neurobiology of Aging 29 (2008) 554–565

ADAM-10 over-expression increases cortical synaptogenesis Karen F.S. Bell a , Luyu Zheng b , Falk Fahrenholz d , A. Claudio Cuello a,b,c,∗ a

Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada b Department of Anatomy and Cell Biology, McGill University, Montreal, Canada c Department of Neurology and Neurosurgery, McGill University, Montreal, Canada d Institute of Biochemistry, Johannes Gutenberg-University Mainz, Germany

Received 20 June 2006; received in revised form 25 October 2006; accepted 7 November 2006 Available online 21 December 2006

Abstract Cortical cholinergic, glutamatergic and GABAergic terminals become upregulated during early stages of the transgenic amyloid pathology. Abundant evidence suggests that sAPP␣, the product of the non-amyloidogenic ␣-secretase pathway, is neurotrophic both in vitro and when exogenously applied in vivo. The disintegrin metalloprotease ADAM-10 has been shown to have ␣-secretase activity in vivo. To determine whether sAPP␣ has an endogenous biological influence on cortical presynaptic boutons in vivo, we quantified cortical cholinergic, glutamatergic and GABAergic presynaptic bouton densities in either ADAM-10 moderate expressing (ADAM-10 mo) transgenic mice, which moderately overexpress ADAM-10, or age-matched non-transgenic controls. Both early and late ontogenic time points were investigated. ADAM-10 mo transgenic mice display significantly elevated cortical cholinergic, glutamatergic and GABAergic presynaptic bouton densities at the early time point (8 months). Only the cholinergic presynaptic bouton density remains significantly elevated in late-staged ADAM-10 mo transgenic animals (18 months). To confirm that the observed elevations were due to increased levels of endogenous murine sAPP␣, exogenous human sAPP␣ was infused into the cortex of non-transgenic control animals for 1 week. Exogenous infusion of sAPP␣ led to significant elevations in the cholinergic, glutamatergic and GABAergic cortical presynaptic bouton populations. These results are the first to demonstrate an in vivo influence of ADAM-10 on neurotransmitter-specific cortical synaptic plasticity and further confirm the neurotrophic influence of sAPP␣ on cortical synaptogenesis. © 2006 Elsevier Inc. All rights reserved. Keywords: ADAM-10; sAPP␣; Neurotrophic; Cholinergic; Glutamatergic; GABAergic

1. Introduction Previous studies by our lab utilizing transgenic mouse models of the amyloid pathology, detected an increased presynaptic bouton density in the overall cortical presynaptic bouton population, as seen by increased synaptophysinimmunoreactivity (IR, Hu et al., 2003), as well as increases in the cholinergic (Wong et al., 1999), glutamatergic and GABAergic (Bell et al., 2003) presynaptic bouton populations specifically. Our observed upregulation in transmitter-specific markers, agrees with the findings of ∗ Corresponding author at: Room 1210, 3655 Sir William Osler Promenade, Montreal, Que. H3G 1Y6, Canada. Tel.: +1 514 398 3618; fax: +1 514 398 8317. E-mail address: [email protected] (A.C. Cuello).

0197-4580/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2006.11.004

others in transgenic mouse models of the amyloid pathology (Hernandez et al., 2001; Mucke et al., 1994) and in human patients displaying a slight decrease in cognitive performance classified as mild cognitive impairment (MCI), in both the cholinergic (DeKosky et al., 2002) and glutamatergic (Bell et al., 2005) systems. The upregulation of neurotransmitter-specific markers in patients with MCI brings about an interesting conundrum since in contrast with what might be expected, MCI patients display more cognitive impairment not less, despite the apparent elevation of specific transmitter systems. In this regard, a better understanding of the potential source of the upregulation is relevant. Mounting evidence suggests that sAPP␣ is involved in normal physiological aspects of neurotrophic support and neuroprotection. In vitro application of sAPP␣ causes neu-

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rite outgrowth in cultured fibroblasts (Bhasin et al., 1991; Saitoh et al., 1989), PC12 cells (Milward et al., 1992), cortical and hippocampal neuronal cells (Araki et al., 1991; Ohsawa et al., 1997; Qiu et al., 1995) and human neuroblastoma cell lines (Wang et al., 2004), as well as elicits protection against hypoglycaemia-induced cytotoxicity (Mattson et al., 1993) and oxidative impairment (Mattson et al., 1999). Ex vivo application of sAPP␣ to organotypic hippocampal slices increases expression of transthyretin, a neuroprotective protein which binds A␤ (Stein et al., 2004). In vivo application of sAPP␣ increases synaptic density and memory retention (Roch et al., 1994), improves performance in tasks involved in short and long term memory, reverses scopolamine-induced learning deficits (Meziane et al., 1998) and causes proliferation of progenitor cells in the adult subventricular zone (Caille et al., 2004). The present study aims to determine whether the in vivo over-expression of endogenous ␣-secretase, would cause an increase in cortical cholinergic, glutamatergic and GABAergic presynaptic bouton densities. To achieve this we utilized the recently created ADAM-10 mo transgenic mouse model (for full model description please see Postina et al., 2004), where “mo” indicates a moderate level of over-expression of ADAM10, a putative ␣-secretase candidate (Lammich et al., 1999). Quantitative analysis revealed a 30% increase in mature ADAM-10 in ADAM-10 mo mice, as compared to nontransgenic FVB/N mice, while an additional line named ADAM-10 hi showed a 70% increase in mature ADAM10, as compared to non-transgenic FVB/N mice. In mice not over-expressing human APP, it is nearly impossible to determine endogenous mouse sAPP␣ levels and a potential increase via over-expression of ADAM-10. However, in mice which are doubly transgenic for both ADAM-10 and human APP (resulting from the crossing of ADAM-10 mo with APP[V717I] mice), Western blot analysis shows a 2.5fold increase in sAPP␣, as compared to mice which do not overexpress ADAM-10, as was previously reported (Postina et al., 2004). It is important however, to mention that the levels of murine A␤-40 and A␤-42 are decreased by 42% and 28%, respectively, in ADAM-10 mo singly transgenic animals (F. Fahrenholz, unpublished observations), suggesting that sAPP␣ would indeed be expected to be elevated as compared to non-transgenic controls. Neurotransmitter-specific presynaptic bouton densities were investigated at early (8 months old) and late (18 months old) ontogenic time points to assess the influence of ADAM-10 over-expression throughout the aging process. Since ADAM-10 has also been shown to cleave other potentially relevant proteins (such as Ephrin, Janes et al., 2005; E-Cadherin Maretzky et al., 2005; TNF-␣, Rosendahl et al., 1997; Notch, Hartmann et al., 2002), sAPP␣ was exogenously infused into the brains of non-transgenic control animals in order to confirm findings from the first portion of the study. The results confirm that both endogenously overexpressed ADAM-10 and exogenously applied sAPP␣ have neurotrophic effects on cortical synaptogenesis.

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2. Methods All animal experimentation was performed according to guidelines approved by the McGill University Animal Care Committee and the Canadian Council on Animal Care. Efforts were made to minimize both the number of animals used and their suffering. 2.1. ADAM-10 mo characterization Full characterization of the ADAM-10 transgenic mouse model is available in (Postina et al., 2004). Briefly, two transgenic mouse lines were created which overexpress bovine ADAM-10, which has a 97% and 95% sequence identity with the human and mouse forms of ADAM-10, respectively. In both models, the transgene is under the control of the neuron-specific post-natally active mouse Thy1 promoter, which agrees with the subsequent finding that ADAM-10 expression within these models is restricted to neurons, where the highest expression levels are found in layers of the neocortex and in hippocampal large pyramidal neurons. Previous experimentation shows that ADAM-10 is processed normally to yield both the pro-and the mature forms. Indeed, immunocytochemical detection, via a C-terminal hemaglutanin-tag in cultured cells, demonstrates that HA-tagged ADAM-10 maintains its alpha-secretase activity and is trafficked normally to the cell surface and trans-Golgi (Lammich et al., 1999). The ADAM-10 moderate and high transgenic mouse models show a 30% and 70% elevation in mature ADAM10, respectively, as compared to non-transgenic FVB/N mice. The abbreviation FVB/N indicates an imbred mouse strain which is preferable for transgenic analysis, as is described in detail by Taketo et al. (1991). Of the two lines the ADAM10 mo line offers a closer representation of a physiologically relevant scenario and was thus the most appropriate line to use in our study. We then investigated the impact of moderate over-expression of ADAM-10 on synaptic structure in the ADAM-10 mo transgenic mouse line (Postina et al., 2004) at both an early (8 months, n = 8 ADAM-10 mo, n = 7 age-matched FVB/N controls) and late (18 months, n = 5 ADAM-10 mo, n = 8 age-matched FVB/N controls) ontogenic time point. 2.2. Perfusions Animals were anaesthetized and perfused as previously described (Wong et al., 1999). Briefly, Equithesin (2.5 ml/kg), and heparin (4 USP/kg) were injected i.p. prior to intra-cardial perfusion first with 0.1 M perfusion buffer [for composition, see Cˆot´e et al., 1993] containing 0.1% sodium nitrite (BDH Inc., Toronto, Ont.), followed by 4% paraformaldehyde (BDH Inc.) fixative solution in phosphate buffer. Following a 3 h post-fixation in the same fixative, brains were cryo-protected with 10% sucrose in PBS and cut into 40 ␮m coronal sections with a Leica sledge freezing microtome at −20 ◦ C from bregma 1.40 mm to bregma

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−3.40 mm (Franklin and Paxinos, 1997). Animals were coded to ensure unbiased processing and analysis. 2.3. Immunohistochemistry Free floating immunohistochemistry was performed on alternate sections from both transgenic and non-transgenic brains simultaneously, as previously described (Bell et al., 2006; Wong et al., 1999). Following extensive washing in phosphate buffered saline solution plus triton (2%, PBST) sections were blocked for 30 min in 5% normal goat serum (NGS, Sigma, St. Louis, MO). Sections were then transferred without washing, to primary antibody solutions; cholinergic labeling: rabbit anti-vesicular acetyl choline transporter (VAChT), a specific marker of cholinergic presynaptic boutons (1:10,000, a kind gift from Dr. R.H. Edwards, for characterization, see Roghani et al., 1996), glutamatergic labeling: in-house generated monoclonal mouse-anti-vesicular glutamate transporter (VGluT) 1 antibody, coded McKA1 and which specifically labels glutamatergic presynaptic boutons (1:50, for full characterization, see Bell et al., 2006), and GABAergic labeling: rabbit anti-glutamic acid decarboxylase (GAD) 65 (1:2000, Chemicon, Temecula, CA). The 65 isoform of the GAD enzyme was selected as opposed to the 67 isoform due to the fact that GAD65 is localized within the axonal boutons of GABAergic neurons whereas GAD67 is found within the cell body (Esclapez et al., 1994; Kaufman et al., 1991). Following overnight incubation at 4 ◦ C, sections were washed and transferred to secondary antibody solutions for 1 h at room temperature; cholinergic and GABAergic labelings: biotinylated goat anti-rabbit (Vector Laboratories, Burlingame, CA) and glutamatergic labeling: goat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA). Subsequent development utilized an ABC complex kit (Vector), or MAP/HRP incubation [monoclonal mouse anti-peroxidase antibody (1:30; Medicorp, Montreal, Que.) containing 5 ␮g/ml horseradish peroxidase (Sigma, type IV)]. Section were then washed, incubated in 0.6% 3,3 -diaminobenzedine (Sigma) for 15 min and developed using 0.03% H2 O2 , as previously described (Wong et al., 1999). Following staining development, sections were mounted, dehydrated and cover slipped as previously described (Wong et al., 1999). 2.4. Digital imaging and quantification Bright field digital images were captured using a Zeiss Axioplan 2 imaging Microscope, equipped with motorized stage and focus, and Zeiss AxioCamHRC digital camera coupled to the AxioVision 4 software program (Zeiss Canada, Montreal, Que.). Digital images from Lamina V and VI of the Frontal 1 and Frontal 2 (F1/F2) anatomical range were included as indicated in Franklin and Paxinos (1997). Starting just laterally from the dorsal horn within Lamina VI of the F1 region, four consecutively juxtaposed images spanning an 8200 ␮m2 area were captured moving laterally. Follow-

ing the fourth image the user then scanned down ventrally into Lamina V, whereupon the fifth image was captured. The sixth, seventh and eighth pictures were sequentially taken in juxtaposition by traveling medially backward toward the midline. This process was repeated for each brain hemisphere within a given section. Given that three sections were included per animal, this meant a total of 48 images per neurotransmitter, per animal or more clearly, 1344 images per neurotransmitter or a total of 4032 images. The anatomical area of interest was always identical across all animals and neurotransmitter systems. Following digital image capturing, photos were uploaded into the MCID Elite Image analysis system (Imaging Research Inc., St. Catharine’s, Ont.), coded to unsure non-biased processing and then quantified by a blind user, essentially as previously described (Bell et al., 2003, 2006). For all quantification analyses original bright field tagged-image files were transformed into digital files, which increase the computer’s detection accuracy (see Fig. 1B, E and H). The computer’s detection level and related ability to accurately isolate specific areas of interest from the background is dependent on the establishment of specific detection parameters including color (the detection of brown colored pixels representative of the specific DAB immunohistochemical coloration), pixel saturation and intensity, form factor (roundness) and size. These parameters were adjusted by the user until the best possible level of accuracy was visually obtained between the true immunohistochemical staining of presynaptic boutons and the area picked up by the computer based on the employed parameters. Once the best parameters were identified, the specific profile was saved within the MCID image analysis program and reapplied to all subsequent images within the same neurotransmitter-specific staining (see Fig. 1C, F and I). For consistency sake K.F.S.B. performed all of the quantitative analyses. Neurotransmitterspecific presynaptic bouton densities were then quantified by the program across the entire digital image (an area of 8200 ␮m2 ), yielding a specific number of presynaptic boutons per defined area, which was later converted to a per 1000 ␮m2 area for ease of presentation. 2.5. Cerebral infusion of sAPPα Eight-month old FVBN mice were anesthetized with the McGill Animal Resource Center “rodent cocktail”, composed of Ketamine (Bioniche, Belleville, Ont.), Xylazine (Novopharm, Toronto, Ont.) and Acepromazine (WyethAyerst, Guelph, Ont.) with a drug dose ratio of 50:5:1 mg/kg via intra-muscular injection with total dose equivalent to 1 ␮l/g of body weight. Following placement into a Kopf stereotaxic apparatus, the frontal cortex of the animals were implanted with an Alzet cannula (brain infusion kit # 3, Alzet, Palo Alto, CA and as done previously, Caille et al., 2004; Meziane et al., 1998; Roch et al., 1994) according to the following coordinates from bregma—A/P: −0.82 mm; L/M: 1.25 mm; D/V: 0.5 mm (Franklin and Paxinos, 1997). See Fig. 2 for needle insertion sites and qualitative upregulation

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Fig. 1. Illustration of the image analysis quantification method used to determine cortical presynaptic bouton density in the cholinergic (A), glutamatergic (D) and GABAergic (G) populations. Panels A, D and G show the original images displaying neurotransmitter-specific immunohistochemical stainings. Panels B, E and H show the transformed digital images which facilitate quantification for the MCID Image analysis program. Finally, panels C, F and I show the close level of overlap between light immunohistochemical staining of presynaptic boutons and the computer’s detection level as shown in blue. The image analysis software then adds the total number of detected elements and divides it by the entire scanned area to yield a density per 1000 ␮m2 area. Scale bar for (A)–(I) = 10 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 2. (A–D) Illustration of the anatomical region in which the cannulae were stereotaxically inserted (black arrow depicts insertion site). Black squares represent the proximal (A and B) and distal (C and D) neuropile areas investigated. (E–H) show a higher magnification representative sample of the respective neuropile areas. Note the apparent upregulation in presynaptic bouton number (in this case cholinergic) following infusion of sAPP␣ (F and H) in both the proximal (F vs. E) and distal (H vs. G) neuropile areas, as compared to infusion of vehicle alone (E and G). For quantitative data, see Fig. 6. Scale bar in D (for A–D) is 500 ␮m and in H (for E–H) is 20 ␮m.

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of presynaptic boutons in response to sAPP␣ infusion. Cannulas were connected to sterile coiled polyethylene tubing filled with either human sAPP␣ in PBS (0.25 mg/ml, Sigma) or PBS vehicle alone as control. The sAPP␣ concentration was determined based on pilot study efficacy, as well as previous studies (Caille et al., 2004). At the opposite end, the tubing was connected to a heat-activated dye-filled (0.1% methylene blue) Alzet 1007D osmotic mini-pump (7 day, 0.5 ␮l/h, Alzet), which had been pre-tested to confirm activation and delivery rate and which was subcutaneously placed into the animal’s neck and shoulder area. At the end of the 7-day period (total volume of 84 ␮l infused) the pumps were removed and weighed to confirm the total volume infused. Animals (n = 5 sAPP␣ infused, n = 6 controls) were anesthetized, perfused and processed immunohistochemically as described above. Digital imaging and quantification were performed as described above. In an effort to determine whether a dose-dependent type of effect was present in response to sAPP␣, two distinct anatomical regions were selected based on their relative proximity to the cannula. The proximal region was identical to that used previously for the ADAM10 mo portion of the study, namely Lamina V and VI of the F1/F2 cortical regions which in this case was adjacent to the injection site. The distal site was located within Lamina V and VI of the Parietal regions 1 and 2 or P1/P2 as indicated in Franklin and Paxinos (1997). Pictures within the

“proximal” F1/F2 and “distal” P1/P2 neuropile regions were digitally captured from the ipsilateral brain hemisphere, relative to injection. Five hundred and twenty-eight images per neurotransmitter were captured for a total of 1584 images. 2.6. Statistics Mean presynaptic bouton densities were compared via independent Student’s t-test. All findings were considered significant when p < 0.05.

3. Results 3.1. Increased cholinergic, glutamatergic and GABAergic densities in ADAM-10 over-expressing transgenic animals Qualitative observations of immunohistochemically stained fronto-cortical brain tissue from early staged ADAM-10 mo transgenic animals show upregulations in the cholinergic, glutamatergic and GABAergic presynaptic bouton populations (see Fig. 3B, F and J versus A, E and I). Qualitative observations in late-staged ADAM-10 mo transgenic animals suggest that only the cholinergic terminals remain elevated during aging (see Fig. 3D versus

Fig. 3. Neurotransmitter-specific immunohistochemical stainings in the frontal cortex of early (A, B, E, F, I and J) and late (C, D, G, H, K and L) staged ADAM-10 mo transgenic animals (B, D, F, H, J and L) and age-matched non-transgenic controls (A, C, E, G, I and K). Immunohistochemical stainings are shown for the cholinergic system (A–D), the glutamatergic system (E–H) and the GABAergic system (I–L) using anti-VAChT, anti-VGluT and anti-GAD antibodies, respectively. Note the upregulation in terminal number in early staged ADAM-10 mo animals (B, F, and J vs. A, E, and I). Interestingly, only the cholinergic system appears to remain elevated in late-staged animals (D vs. C). Scale bar for (A)–(L) = 10 ␮m.

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Fig. 4. Graphic illustration of the cholinergic (A and B), glutamatergic (C and D) and GABAergic (E and F) presynaptic bouton densities in the frontal cortex of early (A, C and E) and late-staged (B, D and F) ADAM-10 mo transgenic animals (dark bars) as compared to age-matched non-transgenic controls (open bars). Groups were compared by independent Student’s t-test, where * p < 0.05 and ** p < 0.01, error bars represent S.E.M.

C). Subsequent quantification of cortical presynaptic bouton densities in early staged ADAM-10 mo mice, demonstrates a significant increase in the cholinergic, glutamatergic and GABAergic presynaptic bouton populations, as compared to age-matched controls (see Fig. 4 A, C and E and Table 1A). Interestingly, at the late-staged time point only the cholinergic presynaptic bouton density remains Table 1A Relative changes in neurotransmitter-specific presynaptic bouton densities in early and late-staged ADAM-10 mo transgenic mice Early staged ADAM-10 mo relative to controls (%)

Late-staged ADAM-10 mo relative to controls (%)

+61.3** +14.2** +14.1*

+31.5* +11.3 +8.3

* p < 0.05, ** p < 0.01

by independent Student’s t-test.

VAChT VGluT1 GAD65

significantly elevated (see Fig. 4B), while the glutamatergic and GABAergic presynaptic populations show elevated but non-significantly different densities (see Fig. 4D and F, respectively, and Table 1A). As expected, late-staged control animals display a normal age-associated diminution in terminal number (see Table 1B). Interestingly, Table 1B Relative changes in neurotransmitter-specific presynaptic bouton densities in early vs. late-staged control animals, and late-staged ADAM-10 mo transgenic animals vs. early staged controls

VAChT VGluT1 GAD65 * p < 0.05

Late stage vs. early stage controls (%)

Late-staged ADAM-10 mo vs. early staged controls (%)

−10.7* −4.7 −9.3*

+17.4 +6.1 −1.8

by independent Student’s t-test.

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the cholinergic presynaptic bouton density in late-staged ADAM-10 mo animals is 17.4% higher than early staged non-transgenic controls, despite a 10-month age difference (p = 0.13, see Table 1B). While the trend fails to reach significance it may still be indicative of a potential protective effect of sAPP␣ on the normal aging process of cholinergic terminals.

Table 2 Relative changes in neurotransmitter-specific presynaptic bouton densities following sAPP␣ infusion

VAChT VGluT1 GAD65

Proximal vs. saline (%)

Distal vs. saline (%)

+37** +12.2* +11.7**

+31** +6.2 +4.5

* p < 0.05, ** p < 0.01,

3.2. FVBN control animals receiving exogenously infused sAPPα display significantly increased cholinergic, glutamatergic and GABAergic presynaptic bouton densities In order to confirm that the previous results were due to sAPP␣ itself as opposed to an additional ADAM-10mediated cleavage product, sAPP␣ was infused directly into the cortex of FVBN non-transgenic control animals. Following a 7-day infusion period, animals were perfused and processed as described above. Qualitatively, animals infused with sAPP␣ appeared to display higher numbers of cholinergic, glutamatergic and GABAergic presynaptic boutons in the proximal neuropile (see Fig. 5B, F and J respectively and Fig. 2 for depiction of proximal versus distal neuropile). In the distal neuropile, only the cholinergic terminals appear elevated (see Fig. 5D). Subsequent quantification revealed significantly elevated cholinergic, glutamatergic, and GABAergic presynaptic bouton densities in the proxi-

by independent Student’s t-test.

mal (F1/F2) neuropile area of animals infused with sAPP␣ as compared to saline injected controls (see Fig. 6 and Table 2). Interestingly, only the cholinergic system appeared to respond to sAPP␣ in the distal (P1/P2) neuropile area, as evidenced by a significant increase in cholinergic presynaptic bouton density (see Fig. 6A). The glutamatergic and GABAergic presynaptic bouton densities were slightly elevated in the distal neuropile area of sAPP␣ injected animals, however, the trend failed to reach significance (see Fig. 6B and C). Proportionally the cholinergic system appears to display the most sensitivity to sAPP␣ levels, as can be seen by percentage elevations of 37% and 31% in the proximal and distal neuropile areas, respectively, versus only 12.2% and 6.2% for the glutamatergic terminals and 11.7% and 4.5% for the GABAergic terminals (see Table 2). All three neurotransmitter systems displayed the highest proportional increase in presynaptic bouton density in the proximal neuropile area, potentially indicative of a dose-dependent effect of sAPP␣

Fig. 5. Neurotransmitter-specific immunohistochemical stainings in the proximal (F1/F2, A, B, E, F, I and J) and distal (P1/P2, C, D, G, H, K and L) cortical regions of animals exogenously infused with sAPP␣ (B, D, F, H, J and L) or saline (A, C, E, G, I and K). Anti-VAChT, anti-VGluT and anti-GAD antibodies were used to immunohistochemically label the cholinergic (A–D), glutamatergic (E–H) and GABAergic (I–L) systems, respectively. Note the upregulation in terminal number in animals infused with sAPP␣ in the proximal region (B, F, and J vs. A, E, and I), while only the cholinergic system remains elevated at the distal site (D vs. C). Scale bar for (A)–(L) = 10 ␮m.

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gests the possibility that sAPP␣ is capable of eliciting both a trophic and tropic response from the cholinergic system.

4. Discussion

Fig. 6. Graphic representations of the cortical cholinergic (A), glutamatergic (B) and GABAergic (C) presynaptic bouton densities in non-transgenic control animals stereotaxically infused with sAPP␣ for 1 week. Dark bars represent neurotransmitter-specific densities obtained from animals infused with sAPP␣, while open bars represent animals infused with vehicle alone. Densities were quantified in both the proximal (F1/F2) and distal (P1/P2) neuropile regions relative to the infusion site. Groups were compared by independent Student’s t-test, where * p < 0.05 and ** p < 0.01, error bars represent S.E.M.

on presynaptic bouton density (see Table 2). The proportionally higher recruitment of cholinergic terminals in situations of both elevated endogenous and exogenous sAPP␣ supports the notion that the cholinergic system appears particularly sensitive to APP-mediated changes. Moreover, the detection of cholinergic sprouting in the direction of the sAPP␣ infusion source (data not shown), a phenomena not seen for either the glutamatergic or GABAergic systems, sug-

Alterations in APP expression in transgenic animals have been shown to alter cortical steady state presynaptic bouton levels in vivo. Mucke and collaborators observed an elevation in synaptophysin-IR in mice expressing non-mutated forms of hAPP (Mucke et al., 1994). Interestingly, moderate over-expression of non-mutated hAPP had a higher synaptotrophic effect on cortical presynaptic boutons, than high over-expression (Mucke et al., 1994). Similar elevations in cortical presynaptic bouton density can be seen in pre-plaque staged APPK670N,M671L transgenic mice expressing familial Alzheimer’s disease (FAD) mutated forms of hAPP (Hu et al., 2003). These animals also display an increase in cholinergic presynaptic bouton density (Wong et al., 1999), ChAT mRNA and ChAT activity (Hernandez et al., 2001). Moreover, TgCRND8 transgenic animals display elevated glutamatergic and GABAergic presynaptic bouton densities during early stages of the amyloid pathology (as evidenced by the modest presence of diffuse versus neuritic plaques, Bell et al., 2003), demonstrating that elevations in presynaptic bouton number extend beyond the cholinergic system. Elevations in transmitter-specific presynaptic markers are also visible in the human pathology, since patients with MCI display increased hippocampal and fronto-cortical ChAT activity (DeKosky et al., 2002), as well as increased cortical glutamatergic presynaptic bouton density (Bell et al., 2005). The present finding that mice over-expressing ADAM10, also display elevated cortical cholinergic, glutamatergic and GABAergic presynaptic bouton densities, suggests that the upregulation in terminal number seen in transgenic animal models of the amyloid pathology, as well as in patients with MCI is potentially attributable to an in vivo neurotrophic effect of increased ADAM-10 activity. Our subsequent observation that the exogenous infusion of sAPP␣ into the brains of non-transgenic control mice causes an elevation in cortical cholinergic, glutamatergic and GABAergic presynaptic bouton density, confirms that the in vivo changes visible in the ADAM-10 mo animals are due, at least in part, to increased levels of endogenous sAPP␣. However, an additional possibility not explored in the present study, is that the ADAM-10 over-expression-dependent decrease in the levels of A␤ (murine A␤ 42/40 levels are lowered by 42%/28% in ADAM-10 mo animals, F. Fahrenholz unpublished observations) and presumably C99, would be expected to reduce the levels of A␤ dependent toxicity. This ADAM-10 dependent downregulation in A␤ might contribute to the visibly increased synaptic density, since there is ample evidence of the negative influence of A␤ on steady state presynaptic bouton numbers (for review, see Bell and Claudio, 2006).

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It seems plausible that the upregulation in terminal number could stem from sAPP␣ since the protein’s neurotrophic and neuroprotective capabilities have been consistently demonstrated both in vitro and in vivo (Caille et al., 2004; Jin et al., 1994; Mattson, 1994; Mattson et al., 1993, 1999; Meziane et al., 1998; Roch et al., 1994; Stein et al., 2004; Wang et al., 2004). Indeed, in a previous study similar to our own, ventricular infusion of sAPP␣ led to increased cortical synaptophysin-IR in rats (Roch et al., 1994). Moreover, the offspring of FAD hAPPV7171 mutant transgenic mice crossed with ADAM-10 mo transgenic mice (Postina et al., 2004), display significantly decreased A␤ peptide levels, reduced plaque formation, and most strikingly, an alleviation of the impaired long-term potentiation and cognitive deficits seen in their singly transgenic APPV7171 parents (Postina et al., 2004), the latter of which may be linked to the known influence of sAPP␣ on hippocampal synaptic plasticity (Ishida et al., 1997). The present findings, as well as those of previous studies, clearly support the view that sAPP␣ has a normal physiological neurotrophic and neuroprotective role. In this regard it is interesting to note that the cerebrospinal fluid of AD patients generally contains significantly lower levels of sAPP␣ than age-matched non-demented individuals (Colciaghi et al., 2002; Hock et al., 1998; Lannfelt et al., 1995; Olsson et al., 2003; Palmert et al., 1990; Prior et al., 1991; Sennvik et al., 2000; Van Nostrand et al., 1992). Moreover, carriers of the Swedish Familial Alzheimer’s disease mutation who had yet to show symptoms, showed intermediate sAPP␣ levels, suggesting the possibility of a gradual decline in sAPP␣ with disease progression (Lannfelt et al., 1995). Marcinkiewicz et al. found ADAM-10 mRNA expression was elevated in patients with pre-senile dementia and lowered in patients with moderate to severe AD (Marcinkiewicz and Seidah, 2000), however, the number of cases in the pre-senile group is too low to draw firm conclusions. Interestingly, however, a morphological analysis by Bernstein et al., found an increase in ADAM-10 intraneuronal staining intensity during aging, but a significant reduction in ADAM-10-IR in tissue drawn from AD patients (Bernstein et al., 2003). While it is tempting to draw parallels between the apparent upregulation in ADAM-10 and the increases in transmitter-specific markers in patients with MCI, greater analyses are required to determine whether a correlation indeed exists. In this regard, a direct analysis of ADAM-10 and sAPP␣ levels in patients with MCI would help determine whether a link exists between elevated levels of endogenous sAPP␣ and increased presynaptic bouton density. Assuming such a link does in fact exist (that the upregulation in terminal density stems from a direct action by sAPP␣) it would seem plausible that an increase in synaptic density would equate with improved cognition. Yet no behavioural improvements were detectable in the ADAM-10 mo animals in either the open field, elevated plus-maze or Morris Water Maze hidden platform task and no differences were observed with regard to basal activity and exploration,

as compared to non-transgenic control animals (Schmitt et al., 2006). Furthermore, patients displaying elevated ChAT activity (DeKosky et al., 2002) or increased glutamatergic presynaptic bouton densities (Bell et al., 2005), are clinically considered to be more cognitively impaired not less (hence the diagnosis of MCI). It is conceivable that the visible upregulation is non-functional, and results solely from unorganized aberrant sprouting, potentially due to increasing levels of toxic A␤. Alternatively, however, and perhaps more plausibly, the upregulation could represent a compensatory mechanism designed to counterbalance accumulating levels of synaptotoxic A␤ (see Kar et al., 1996), by increasing the possibility of synaptic contact. In this situation, the compensatory upregulation in terminal number (mediated at least in part by sAPP␣), while functional, might not be discernable if other integral components of synaptic viability were already impaired (e.g. alterations in neurotransmitter or neurotrophic receptor levels (Mufson et al., 2002), intracellular signaling mediators etc.). Several studies have suggested that sAPP␣ is an injury induced neurotrophic factor (Haroutunian et al., 1997; Wallace et al., 1993; Wallace et al., 1995), which potentiates the effects of NGF (Chen et al., 2006; Luo et al., 2001; Wallace et al., 1997). NGF induction appears to shift APP processing toward the non-amyloidogenic route since administration of propentofylline, an NGF synthesis stimulator, to Tg25776 mice caused a 20% increase in NGF-mRNA and a 42% increase in sAPP␣ levels (Chauhan and Siegel, 2003). Interestingly, however, the role of sAPP␣ must encompass more than a potentiation of endogenous NGF alone, since GABAergic neurons, which do not express TrkA (Wu and Yeh, 2005) or respond to NGF application (Hyman et al., 1994), also display an increased presynaptic bouton density in ADAM-10 mo, as shown here, and in FAD mutated hAPP transgenic mice (Bell et al., 2003). While the entirety of sAPP␣’s physiological role remains elusive, its beneficial effect would likely become compromised by increasing A␤ levels, which could explain the subsequent decline in steady state terminal numbers seen in both transgenic animal models (Bell et al., 2006; Wong et al., 1999) and AD patients (Davies et al., 1987; DeKosky and Scheff, 1990; Hamos et al., 1989; Masliah et al., 1989; Scheff et al., 1990). Despite recent advances in elucidating sAPP␣’s effects, a greater understanding of its normal in vivo physiological function is still required. This study is the first to demonstrate that ␣-secretase can have an in vivo neurotrophic effect on cortical cholinergic, glutamatergic and GABAergic presynaptic bouton populations, which is at least partially mediated by sAPP␣.

Disclosure statement The authors disclose that they have no actual or potential conflicts of interest. All animal procedures were done in

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accordance with guidelines set out by the McGill University Animal care Committee and the Canadian Council on Animal Care.

Acknowledgements The authors would like to thank Anja Schr¨oder for mouse husbandry and Vanessa Partridge for assistance with Immunohistochemistry. This work was supported by Canadian Institutes of Health Research grant # MOP62735, to A.C. Cuello, by Deutsche Forschungsgemeinschaft grant Fa122/3 and by Bundesministerium f¨ur Bildung und Forschung to F. Fahrenholz. K.F.S. Bell is the recipient of a CIHR Doctoral Research Award and A.C. Cuello is the holder of the Charles E. Frosst Merck endowed Chair.

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