Brain Advance Access originally published online on July 14, 2006
Brain 2006 129(11):2992-3005; doi:10.1093/brain/awl176
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Selective vulnerability of different types of commissural neurons for amyloid ß-protein-induced neurodegeneration in APP23 mice correlates with dendritic tree morphology
1 Department of Neuropathology, University of Bonn Bonn 2 Department of Biochemistry, Adolf-Butenandt Institute Ludwig Maximilians University, Munich 3 Department of Neuropathology, Georg-August University Göttingen, Germany 4 Novartis Institutes for Biomedical Research Basel Basel, Switzerland 5 Gunma University School of Health Sciences, Maebashi Gunma, Japan
Correspondence to: Dietmar R. Thal, MD, Department of Neuropathology, University of Bonn, Sigmund Freud Strasse 25, D-53105 Bonn, Germany E-mail: Dietmar.Thal{at}uni-bonn.de
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Summary |
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The amyloid ß-protein (Aß) is the main component of Alzheimer's disease-related senile plaques. Although Aß is associated with the development of Alzheimer's disease, it has not been shown which forms of Aß induce neurodegeneration in vivo and which types of neurons are vulnerable. To address these questions, we implanted DiI crystals into the left frontocentral cortex of APP23 transgenic mice overexpressing mutant human APP (amyloid precursor protein gene) and of littermate controls. Traced commissural neurons in layer III of the right frontocentral cortex were quantified in 3-, 5-, 11- and 15-month-old mice. Three different types of commissural neurons were traced. At 3 months of age no differences in the number of labelled commissural neurons were seen in APP23 mice compared with wild-type mice. A selective reduction of the heavily ramified type of neurons was observed in APP23 mice compared with wild-type animals at 5, 11 and 15 months of age, starting when the first Aß-deposits occurred in the frontocentral cortex at 5 months. The other two types of commissural neurons did not show alterations at 5 and 11 months. At 15 months, the number of traced sparsely ramified pyramidal neurons was reduced in addition to that of the heavily ramified neurons in APP23 mice compared with wild-type mice. At this time Aß-deposits were seen in the neo- and allocortex as well as in the basal ganglia and the thalamus. In summary, our results show that Aß induces progressive degeneration of distinct types of commissural neurons. Degeneration of the most vulnerable neurons starts in parallel with the occurrence of the first fibrillar Aß-deposits in the neocortex, that is, with the detection of aggregated Aß. The involvement of additional neuronal subpopulations is associated with the expansion of Aß-deposition into further brain regions. The vulnerability of different types of neurons to Aß, thereby, is presumably related to the complexity of their dendritic morphology.
Key Words: Alzheimer's disease pathology; animal models; beta-amyloid; neurodegenerative mechanisms; selective vulnerability
Abbreviations: Aß, amyloid ß protein; APP, amyloid precursor protein; BDA, biotinylated dextran amine; ELISA, enzyme-linked immunosorbent assay; NFTs, neurofibrillary tangles; PFA, paraformaldehyde
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Received February 28, 2006. Revised April 20, 2006. Accepted June 7, 2006.
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Introduction |
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Alzheimer's disease is histopathologically characterized by amyloid ß-protein (Aß) deposits and neurofibrillary tangles (NFTs) (Masters et al., 1985
; Braak and Braak, 1991; Dickson, 1997; Esiri et al., 1997). Aß is derived from the amyloid precursor protein (APP) (Kang et al., 1987). Mutations in the APP gene are linked to early onset familial Alzheimer's disease (Tanzi et al., 1987; Bertram and Tanzi, 2003). Transgenic mice overexpressing human APP harbouring these mutations are used as models for studying Alzheimer's disease-related Aß-deposition. Aß-deposits in humans as well as in transgenic mouse models appear predominantly in the neocortex and hippocampus (Alzheimer, 1907; Braak and Braak, 1991; Arriagada et al., 1992; Games et al., 1995; Hsiao et al., 1996; Sturchler-Pierrat et al., 1997) and expand in a similar hierarchical sequence throughout the brain (Thal et al., 2002b; Wiederhold et al., 2004; Thal et al., 2006). Although synaptic changes represent an important feature of Alzheimer's disease pathology (DeKosky and Scheff, 1990; Masliah et al., 1994) and Aß-toxicity (Lacor et al., 2004; Tsai et al., 2004), it is not clear whether increasing levels of Aß as represented by the hierarchical deposition of Aß in different brain regions support the progression of neurodegeneration.
In APP-transgenic mouse models there is only minor neuronal loss (Calhoun et al., 1998; Schmitz et al., 2004) and no tangle formation (Games et al., 1995; Masliah et al., 1996; Sturchler-Pierrat et al., 1997). Recently, Wu et al. (2004) described dendritic alterations in superficial granule cells of the dorsal blade of the dentate gyrus in young PDAPP mice. However, it is not clear whether such alterations of the dendritic tree lead to changes in neuronal connectivity.
In the human brain, pyramidal cells of the layers III and V are vulnerable to Alzheimer's disease (Alzheimer, 1907; Terry et al., 1981; Braak and Braak, 1991; Duyckaerts and Dickson, 2003). Axonal loss in the corpus callosum seen in the early stages (Hampel et al., 1998; Weis et al., 1991; Yamauchi et al., 1993) indicates that commissural neurons in these layers display an early target of Alzheimer's disease-related neurodegeneration. Furthermore, the number of neurons in layers III and V in the frontal neocortex is reduced in Down syndrome cases (Davidoff, 1928). Therefore, commissural neurons of layer III of the frontal neocortex from APP-transgenic mice represent a suitable model for studying Aß-induced changes in neuronal connectivity.
To measure the progression of Aß-induced neurodegeneration in commissural neurons in APP transgenic mice, we examined the degree of neuronal connectivity in layer III of the frontocentral neocortex at 3, 5, 11 and 15 months of age. To determine the degree of neuronal connectivity, retrograde tracing—a sensitive method to measure the number of neurons being connected to the tracer application site—was used. A decrease in the number of traced neurons would mean a loss of connectivity caused by several reasons such as neuronal death, axonal injury or decrease of collaterals sent from a given type of neurons.
Using this tracing method we showed that degeneration of neuronal connectivity starts in parallel with the occurrence of fibrillar Aß-deposits, that is, aggregated Aß, in the neocortex of 5-month-old APP23 mice and is restricted to a distinct type of neurons. Expansion of Aß-deposition into further brain regions and the increasing Aß-levels lead to the involvement of a second type of commissural neurons.
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Animals
APP23 mice were generated as described previously (Sturchler-Pierrat et al., 1997
) and continuously back-crossed to C57BL/6. An expression construct containing a murine Thy-1 promoter was used to drive neuron-specific expression of human mutant APP751 with the Swedish double mutation 670/671 KM
NL. Heterozygous female APP23 mice were analysed at 3 months of age (n = 12), 5 months of age (n = 16), 11 months of age (n = 13) and 15 months of age (n = 15). As control, female wild-type littermates were analysed at 3 months of age (n = 8), 5 months of age (n = 13), 11 months of age (n = 14) and 15 months of age (n = 9).
Brain homogenates, immunoprecipitation, western blot analysis and enzyme-linked immunosorbent assay (ELISA)
Forebrain hemispheres (excluding olfactory bulb, cerebellum and brainstem) were weighed and homogenized by sonication in 10 vol. of buffer [20 mM Tris–HCl (pH 7.6), 137 mM sodium chloride, protease inhibitor cocktail (Complete, Roche Molecular Biochemical, Mannheim, Germany)]. For immunoprecipitation, homogenates were supplemented with 1% sodium dodecyl sulphate, heated to 95°C for 3 min, diluted with 9 vol. of homogenization buffer and cleared by centrifugation at 15°C for 15 min at 20 000x g. Aß-peptides were immunoprecipitated using the monoclonal antibody ß1 reacting with the amino-terminus of Aß (Schrader-Fischer and Paganetti, 1996) and protein G-coated magnetic beads (Dynal Biotech, Hamburg, Germany). Precipitates or whole homogenates were separated on 10% Tris–bicine gels with 8 M urea as described (Klafki et al., 1996; Staufenbiel and Paganetti, 1999). In this system, Aß1–40 migrates slower than Aß1–42. Proteins were transferred to Immobilon-P membranes (Millipore, Carrigtwohill, Ireland). Aß-peptides were fixed to the membrane by heating it to 95°C for 3 min in phosphate-buffered saline (PBS; Sigma, Taufkirchen, Germany) (Staufenbiel and Paganetti, 1999). Full-length APP (flAPP) was detected with rabbit antiserum APP-C8 raised against the carboxy-terminal amino acids of APP (Schrader-Fischer and Paganetti, 1996), which are identical in mice and humans. Transgene-derived APP and Aß were detected with the monoclonal antibody 6E10 (Signet, MA, USA). After incubation of the blots with the appropriate peroxidase coupled secondary antibodies (Jackson Immunoresearch, Soham, UK and Sigma, Taufkirchen, Germany), proteins were detected by visualizing chemiluminescence (ECL advance, Amersham Pharmacia Biotech, NJ, USA) on autoradiographic films (Hyperfilm ECL, Amersham Pharmacia Biotech).
For analysis of Aß by ELISA, forebrain homogenates from APP23 mice of each age group (3 months: n = 6; 5 months: n = 6; 11 months: n = 4; 15 months: n = 7) were supplemented with concentrated formic acid to a final concentration of 70%. After 15 min of incubation on ice and mixing every 5 min, the samples were neutralized by addition of 19 vol. 1 M Tris base supplemented with protease inhibitor cocktail. The extracts were cleared by centrifugation (20 000x g for 15 min at 4°C). Supernatants from young (3 or 5 months) APP23 mice were directly loaded on sandwich ELISA plates for quantification of Aß-peptides (Aß1–40: ELISA from IBL, Hamburg, Germany; Aß1–42: ELISA from Innogenetics, Ghent, Belgium). Supernatants from 11- and 15-month-old APP23 mice were diluted as necessary in dilution buffers supplied with the ELISAs. Standard curves were prepared with synthetic peptides Aß1–40 and Aß1–42 purchased from Bachem (Bubendorf, Switzerland) and diluted in extracts of non-transgenic mouse forebrain prepared in parallel as described above. Each sample was analysed in duplicate.
Tissue preparation and DiI tracing
For DiI tracing brains of 3-, 5-, 11- and 15-month-old APP23 (3 months: n = 6; 5 months: n = 9; 11 months: n = 8; 15 months: n = 8) and wild-type mice (3 months: n = 6; 5 months: n = 12; 11 months: n = 13; 15 months: n = 9) were studied. Animals were treated in agreement with the German law on the use of laboratory animals. Mice were anaesthetized. Perfusion was performed transcardially with Tris-buffered saline (TBS) with heparin (pH 7.4) followed by the injection of 0.1 M PBS (pH 7.4) containing 2.6% paraformaldehyde (PFA), 0.8% iodoacetic acid, 0.8% sodium periodate and 0.1 M D-L lysine. The brains were removed in total and post-fixed in 2.6% phosphate-buffered PFA (pH 7.4) containing 0.8% iodoacetic acid, 0.8% sodium periodate and 0.1 M D-L lysine (Galuske et al., 2000). Three days later a single crystal (
0.3 mm3) of the carbocyanine dye DiI (Molecular Probes, Eugene, OR, USA) was implanted into the left frontocentral cortex, 1 mm rostrally from the central sulcus, 2 mm laterally from the middle line and 1 mm deep in the cortex (Fig. 1A). This dye allows precise Golgi-like tracing of neurons in post-mortem fixed tissue in a quality similar to in vivo tracing methods using rhodamine tracers even in only weakly traced neurons (Galuske and Singer, 1996). After incubation in 2.6% phosphate-buffered PFA for at least 3 months at 37°C, 100-µm-thick coronal vibratome sections were cut. Sections were mounted in TBS for microscopic analysis.
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In vivo tracing with biotinylated dextrane amine (BDA)
For in vivo tracing with BDA (Molecular Probes), three 3-month-old C57BL/6 wild-type mice were anaesthetized with ketamine/Rompun and placed in a stereotactic frame. After incision of the skin, a fine hole was drilled into the skull 0.1 mm caudal and 0.2 mm lateral to bregma. A finely drawn glass capillary was stereotactically placed into the frontal cortex (approximal depth = 0.5 mm), and 1 µl BDA was slowly injected over a 3-min period. After withdrawal of the capillary, the skin wound was closed by suture, and animals were allowed to survive for 3 days. For tissue sampling, animals were anaesthetized, perfused transcardially with 4% PFA and dissected. Brain slices were embedded in gloop, a mixture of egg albumin, gelatine, sucrose and glutaraldehyde, and 50-µm vibratome sections were cut. BDA was visualized using an avidin–peroxidase-based method (Vectastain, Vector Laboratories, Burlingame, CA, USA) with 3,3-diaminobenzidine (DAB) as chromogen. Sections were dehydrated and coverslipped.
Microscopic and quantitative analysis
In layer III of the frontocentral cortex of the right hemisphere, contralateral to the implantation site of the tracer, the morphology of traced commissural neurons was examined. The traced neurons were assigned to different types according to their morphology. Then the number of traced commissural neurons of each type in wild-type mice was compared with that in APP23 mice. For qualitative and quantitative analysis 10 consecutive sections (100-µm thickness each) representing a tissue block of 1 mm thickness were studied for each mouse. Analysis started at the anterior commissure setting the caudal limit of the investigated tissue block. For each coronal section, the medial boundary of the region investigated was set as the vertical line at the cingulum that separated the cingulate cortex (Cg) from secondary motor cortex (M2) (Fig. 1B). The horizontal boundary was set as the horizontal line separating the primary somatosensory cortex (S1) from the insular cortex (I) (Fig. 1B).
For the qualitative analysis a laser scanning confocal microscope (Leica TCS NT, Leica, Bensheim, Germany) was used. Stacks of 2D images were superimposed digitally using the Image J Image Processing and Analysis software (NIH, Bethesda, MD, USA), and 3D data sets were generated for the visualization of neurons with their entire dendritic tree.
For quantification of the commissural neurons in APP-transgenic and wild-type mice, traced neurons in layer III were counted in the region of interest depicted in Fig. 1 in 10 consecutive sections of the tissue block taken for qualitative and quantitative analysis using a fluorescence microscope (Leica DMLB, Leica). In so doing, we analysed a cortex volume of 5–6 mm3 in each mouse. Mean and median values of the number of traced neurons were calculated and compared between wild-type and APP23 mice. Statistical analysis was performed using the Mann–Whitney U-test to compare wild-type and APP23 mice. Poisson regression was used to identify differences among the different phases of Aß-deposition in the brain. Appropriate corrections for multiple testing were made. SPSS 11.0 (SPSS, Chicago, IL, USA) and LogXact 5.0 (Cytel Software Corporation, Cambridge, MA, USA) software were used to calculate statistical tests.
Immunohistochemistry
Immunohistochemistry was performed for the detection and quantification of Aß-plaques. After formic acid pretreatment free-floating sections were incubated with an anti-Aß antibody [polyclonal rabbit (Wild-Bode et al., 1997), 1/750, 24 h at 22°C]. The polyclonal antibody detected Aß1-40 as well as Aß1-42. To separately detect Aß40 and Aß42 positive material, additional sections were stained with monoclonal antibodies specifically detecting the C-terminus of Aß40 [MBC40 (Yamaguchi et al., 1998), 1/20, 24 h at 22°C, formic acid pretreatment] and Aß42 [MBC42 (Yamaguchi et al., 1998), 1/200, 24 h at 22°C, formic acid pretreatment]. The primary antibody was detected with a biotinylated secondary antibody and the ABC complex, and visualized with DAB (Hsu et al., 1981). Sections were mounted in Corbit® (Hecht, Hamburg, Germany).
Quantification of the Aß-plaque load was performed in an area of the frontocentral neocortex in one selected section with plaques using a Zeiss Stemi2000C microscope and Axiovision AC 4.2 image analysis software (Carl Zeiss Lichtmikroskopie, Göttingen, Germany). The Aß-plaque load was measured for plaques stained with the polyclonal antibody raised against Aß1–42 (Wild-Bode et al., 1997) according to the following determination:
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Mean values of the Aß-plaque load were compared between 3-, 5-, 11- and 15-month-old APP23 mice with ANOVA (analysis of variance).
To characterize the expansion of Aß-deposition in the mouse brain we determined the phases of Aß-deposition valid for human and APP-transgenic mouse brain (Thal et al., 2002b; Wiederhold et al., 2004; Thal et al., 2006) (Table 1).
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To identify axonal sprouting and axonal damage, sections of the area of interest of each mouse were stained with antibodies directed against APP (22C11, monoclonal mouse, Chemicon (Temecula, CA, USA), 1/75, 24 h at 22°C) and against growth-associated protein GAP43 (polyclonal rabbit, Chemicon, 1/75, 24 h at 22°C) (Masliah et al., 1992a
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Subpopulations of commissural neurons in layer III of the frontocentral cortex
Using retrograde tracing with DiI, three different types of commissural neurons were identified in layer III of the frontocentral cortex in wild-type and APP23 mice at the age of 3, 5, 11 and 15 months (Fig. 2A–F). The first type of commissural neurons, referred to as type I, showed a DiI-labelled perikaryon of pyramidal shape, an apical dendrite and multiple basal dendrites with a heavily ramified and spiny dendritic tree. Ramification of the dendrites was characterized by branching of apical and basal dendrites into primary branches exhibiting secondary and tertiary ramifications within layer III (Fig. 2A and D). The basal dendrites were restricted to layers II/III. The apical dendrite showed further ramification within the molecular layer. These type I commissural neurons were predominantly located in the upper part of layer III (Fig. 2D). The second type of commissural neurons, referred to as type II, exhibited a DiI-labelled perikaryon of pyramidal shape. The apical and basal dendrites were detectable, but further ramification and dendritic spines were less frequently observed and the basal dendrites were thicker than those of type I commissural neurons (Fig. 2B, E and F). 3D reconstruction revealed that the basal dendrites of type II commissural neurons start branching distant from the perikaryon (Fig. 2F). Commissural neurons of type II showed dendritic ramification and spines in the molecular layer as detected by following the apical dendrite in consecutive sections and, thus, represent the classical pyramidal cell morphology. These neurons were most frequently located in the lower part of layer III. Branches of their basal dendrites were also seen in the upper part of layer IV (Fig. 2E and F). The third type of commissural neurons, referred to as type III, did not exhibit clearly distinguishable apical and basal dendrites within layer III. Only the cell body with a circular shape was clearly labelled with the tracer. Single, very thin dendrites without spines were labelled in layer III (Fig. 2C and F). These neurons did not show a predilection site in layer III. They occurred in the upper as well as in the lower parts of this cell layer. In all types of commissural neurons the axon left the neuron at the base and was part of the callosal commissural fibre system. In vivo tracing of wild-type mice with BDA confirmed the staining pattern and the existence of three different types of commissural neurons seen in the DiI-traced sections (Fig. 2G–I).
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Alteration of commissural neurons in APP23 mice
At 3 months of age there were no differences between wild-type and APP23 mice in the morphology of the commissural neurons (Fig. 3A and B). In contrast, type I commissural neurons were morphologically altered in APP23 mice compared with wild-type mice at 5, 11 and 15 months of age (Fig. 3C and D). In APP23 mice, the apical dendrite was shorter than that in wild-type animals (Fig. 3C and D). The basal dendrites exhibited a non-symmetric pattern and short secondary or tertiary branches, and their diameter was often reduced (Fig. 3D). In comparison, in wild-type mice this type of neurons showed a symmetrical architecture of the basal dendrites and secondary and tertiary branches were longer than those in APP23 transgenic mice (Fig. 3C and D). Altered type I commissural neurons in APP23 mice were often located distant from Aß-plaques identified by subsequent immunostaining with anti-Aß1–42 or by Aß-plaque-induced autofluorescence (Thal et al., 2002a
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There were no apparent differences in the morphological patterns of type II and type III commissural neurons among APP23 and wild-type mice.
Selective reduction of type I commissural neurons in APP23 mice
Quantitative analysis of traced commissural neurons in 5- to 15-month-old wild-type mice showed that type II commissural neurons were predominant (60–80%), while type I (10–20%) and type III neurons (10–20%) were less abundant. The number of traced type I neurons appeared to decrease with age, but this trend failed significance (trend test: P = 0.108) (Fig. 4A). In 3-month-old animals a similar percentage of type I neurons was found (10–20%) while type II neurons were less predominant (40–45%). In these animals the number of type III neurons was higher than that in older animals (P < 0.01; ANOVA corrected for multiple testing by using the Tamhane T2 post hoc test) and covered 40–45% of all traced commissural neurons (Fig. 4C).
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The overall number of commissural neurons did not show significant differences between wild-type and transgenic mice at a given age (Mann–Whitney U-test, corrected for multiple testing: 3 months: P = 0.9917; 5 months: P = 0.2126; 11 months: P = 0.9579; 15 months: P = 0.3232). However, with increasing expansion of Aß-deposition throughout the brain the total number of neurons decreased (Poisson regression analysis controlled for age: risk-ratio = 0.967, 95% confidence interval (CI) = 0.941–0.9938, P < 0.05; trend-test: P < 0.05).
In detail, the number of traced type I commissural neurons in APP23 mice was reduced by >90% compared with that of wild-type mice at 5, 11 and 15 months of age (Mann–Whitney U-test, corrected for multiple testing: 5 months APP23 versus wild-type: P < 0.05; 11 months APP23 versus wild-type: P < 0.005; 15 months APP23 versus wild-type: P < 0.01) but not at 3 months of age (Mann–Whitney U-test, corrected for multiple testing: 3 months APP23 versus wild-type: P = 0.9917) (Fig. 4A). With the expansion of Aß-deposition the number of traced type I neurons decreased significantly (Poisson regression analysis controlled for age: risk-ratio = 0.3417, CI = 0.174 – 0.6712, P < 0.005).
The number of type II commissural neurons did not show significant differences between wild-type and APP23 mice at 3, 5 and 11 months of age (Mann–Whitney U-test, corrected for multiple testing: 3 months: P = 1; 5 months: P = 0.182; 11 months: P = 0.9375) (Fig. 4B). However, at 15 months of age a significant reduction of traced type II neurons was observed in APP23 mice compared with wild-type (Mann–Whitney U-test, P-values corrected for multiple testing: 15 months: P < 0.05). Over all ages the number of traced type II neurons decreased significantly with the expansion of Aß-deposition (Poisson regression analysis controlled for age: risk-ratio = 0.9432, CI = 0.9033–0.9849, P < 0.01).
The number of type III commissural neurons did not significantly differ among APP23 and wild-type mice, neither at 3 nor at 5, 11 or 15 months of age (Mann–Whitney U-test, corrected for multiple testing: APP23 versus wild-type: 3 months: P = 0.8651; 5 months: P = 0.9749; 11 months: P = 0.453; 15 months: P = 0.3232) (Fig. 4C). In addition, there was no significant change in the number of traced type III neurons throughout the phases of Aß-deposition (Poisson regression analysis controlled for age: risk-ratio = 1.0409, P = 0.1533).
Aß-production and Aß-deposition in APP23 mice
Western blot analysis confirmed overexpression of human APP and production of Aß1–40 and Aß1–42 in APP23 mice (Fig. 5). Quantification of total Aß levels by ELISA in APP23 mice at 3, 5, 11 and 15 months of age showed a significant exponential increase of Aß with the age (Table 2) (ANOVA corrected for multiple testing by using the Tamhane T2 post hoc test: Aß1–40 P < 0.001; Aß1–42 P < 0.001). Total Aß1–40 levels, thereby, were higher than total Aß1–42 levels (sign test: P < 0.001). Between 3 and 5 months of age total Aß levels did not differ significantly (ANOVA corrected for multiple testing by using the Tamhane T2 post hoc test: Aß1-40 P = 0.786; Aß1-42 P = 0.963). However, between 5 and 11 months of age an increase of both Aß1-40 and Aß1-42 was seen (ANOVA corrected for multiple testing by using the Tamhane T2 post hoc test: Aß1-40 P < 0.05; Aß1-42 P < 0.01), as well as between 11 and 15 months of age (ANOVA corrected for multiple testing by using the Tamhane T2 post hoc test: Aß1-40 P < 0.01; Aß1-42 P < 0.001).
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Immunohistochemistry with a polyclonal antibody detecting Aß1-40 as well as Aß1-42 revealed no Aß-deposits in wild-type mice at 3, 5, 11 or 15 months. APP23 mice did not show plaques at 3 months of age. Single plaques in the frontocentral neocortex were observed in 67% of the APP23 mice at 5 months of age representing Phase 1 of Aß-deposition (Table 3) (ANOVA corrected for multiple testing by using the Tamhane T2 post hoc test: P < 0.05). However, differences in the Aß-plaque load did not reach significance between 3- and 5-month-old APP23 mice (ANOVA corrected for multiple testing by using the Tamhane T2 post hoc test: P = 0.096) (Fig. 6A). Between 5- and 11-month-old APP23 mice the Aß-plaque load in the frontocentral cortex increased (ANOVA corrected for multiple testing by using the Tamhane T2 post hoc test: P < 0.005). Additionally, 11-month-old mice often exhibited an involvement of allocortical brain regions, that is, the hippocampal formation and the cingulate gyrus in Aß-deposition representing Phase 2 of Aß-deposition (Fig. 6B, Table 3) (ANOVA corrected for multiple testing by using the Tamhane T2 post hoc test: P < 0.005). Between the ages of 11 and 15 months there was a further increase in the Aß-plaque load within the frontocentral cortex (ANOVA corrected for multiple testing by using the Tamhane T2 post hoc test: P < 0.001) (Fig. 6A), and additional Aß-plaques occurred in the basal ganglia as well as in the thalamus, indicating Phase 3 of Aß-deposition (ANOVA corrected for multiple testing by using the Tamhane T2 post hoc test: P < 0.001) (Fig. 6B, Table 3). Aß-deposits in APP23 mice exhibited equal staining patterns with anti-Aß42 and anti-Aß40 antibodies. Aß-deposition according to the pattern of Phase 4 or Phase 5 has not been observed in 3-, 5-, 11- and 15-month-old APP23 mice.
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Axonal sprouting of commissural neurons in APP23 mice
Immunohistochemistry with antibodies directed against APP and GAP43 showed the presence of immunolabelled, thickened axon endings in the frontocentral cortex of 3-month-old wild-type and APP23 mice indicative for axonal sprouting (Fig. 7A–D). In the DiI-traced sections we found collateral sprouting in wild-type and APP23 mice at 3 months of age. Single axons of commissural neurons with growth cones were seen in layers II and III of the frontocentral cortex of the DiI-traced sections (Fig. 7E). In 5-month-old mice thickened axon endings of commissural neurons in DiI-traced sections were no longer observed. However, single APP and GAP43-positive, thickened axon endings were found in APP23 mice but not in wild-type mice at this age (Fig. 7F–I). Sprouting of commissural neurons around plaques was observed in 11- and 15-month-old APP23 mice in DiI-traced sections (Fig. 7L) as well as in APP and GAP43-stained sections (Fig. 7J and K). Wild-type mice did not show sprouting of commissural neurons in this age in DiI-traced sections. Only single APP and GAP43-positive sprouting axons were detected in the frontocentral cortex of 15-month-old wild-type mice.
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Discussion |
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In this study, we show that three types of commissural neurons in layer III of the mouse frontocentral cortex can be identified using tracing methods. These different neuron types are hierarchically affected by Aß-induced neurodegeneration. The deposition of the first Aß-plaques is associated with the onset of neurodegeneration in APP23 mice. Expansion of Aß-deposition into further brain regions goes along with the involvement of a second type of commissural neurons in neurodegeneration. The complexity of the dendritic morphology is, thereby, related to the vulnerability of these different types of neurons.
Characterization of three types of commissural neurons in the mouse frontocentral neocortex
Our study confirms and extends previous reports that describe different types of pyramidal and non-pyramidal neurons projecting through the corpus callosum (Hughes and Peters, 1992; Martinez-Garcia et al., 1994). The distinction of the three types of commissural neurons in the mouse frontocentral cortex is based on their morphological appearance after retrograde tracing with DiI. It is confirmed by using biotinylated dextran in vivo tracing as an additional method.
Type I commissural neurons are characterized by a pyramidally shaped perikaryon and apical and basal dendrites with a heavily ramified and spiny dendritic tree within layer II–III. Type II commissural neurons represent neurons with a pyramidally shaped perikaryon and apical and basal dendrites, which do not show extensive further ramifications in layer III in a given tissue section. However, 3D reconstruction revealed that the basal dendrites of these neurons start branching distant from the perikaryon. Type III commissural neurons are non-pyramidal neurons and have a circular cell body with a few dendrites.
In contrast to the other types of commissural neurons, type III neurons are reduced in number in 5-, 11- and 15-month-old animals in comparison with 3-month-old mice regardless of the genotype. Thus, an effect of Aß or APP-overexpression is not responsible for the reduction of type III neurons because wild-type mice show the same effects as APP23 mice. To explain this finding, further studies are required. Developmental or maturation-related phenomena (Fritzsch et al., 1997) might be involved.
All three types of neurons were found in all mice studied, and their axons projected through the corpus callosum to the contralateral hemisphere. Since tracer-labelled type I, II and III commissural neurons were visible in the same section close to one another and type II neurons showed dendritic ramification in the molecular layer, it seems unlikely that DiI tracing of neurons without a ramified dendritic tree is due to dysfunctional tracing rather than to the exhibition of specific morphological types of neurons. Galuske and Singer (1996) also reported that in the event that a given cell is stained by DiI all cell processes will be labelled. Moreover, a second tracing method based on in vivo transport of the biotinylated dextrane (Reiner et al., 2000) confirmed the presence of three different types of commissural neurons.
The heterogeneous morphological pattern of commissural neurons in the frontocentral cortex of mice confirms and extends the finding of other authors that the visual cortex contains different types of callosal commissural neurons (Voigt et al., 1988; Buhl and Singer, 1989; Martinez-Garcia et al., 1994). They also reported the presence of commissural neurons of pyramidal and non-pyramidal types. Cortical commissural neurons project to different layers of the contralateral hemisphere (Wise and Jones, 1976; Martinez-Garcia et al., 1994). In so doing, the different types of frontocentral commissural neurons may terminate in different layers of the contralateral hemisphere and may have specific functions in commissural information processing.
Degeneration of distinct types of commissural neurons in APP23 mice
Two of the three types of commissural neurons were reduced in number when traced in APP23 transgenic mice. Type I commissural neurons were significantly reduced in number in APP23 mice beginning at 5 months of age. The surviving type I commissural neurons in these mice exhibited alterations of the dendritic tree in comparison with wild-type animals. The other types of commissural neurons did not show obvious differences between transgenic and wild-type mouse lines at this point in time. The second type of neurons, type II commissural neurons, was reduced in number at 15 months of age. Thus, the number of traced commissural neurons apparently decreases in APP23-transgenic mice as soon as they start developing Aß-deposits at 5 months of age. With increasing age, Aß-plaque load and Aß-expansion, a second type of neuron becomes involved in Aß-induced neurodegeneration. However, type III commissural neurons were not altered. The degeneration of type I and II commissural neurons in APP23 mice can be interpreted (i) as a result of selective neuronal death; (ii) as the result of dendritic alterations; or (iii) as a consequence of axonal damage. The first possibility of selective neuronal death is supported by the decrease of the total number of neurons in the hippocampus of APP-transgenic mice, indicating that neuronal loss can result from abnormal production of Aß (Calhoun et al., 1998; Schmitz et al., 2004). A second supporting argument is that in Down syndrome patients overexpressing APP (Neve et al., 1988) neuronal loss is accentuated in layer III of the frontocentral cortex (Davidoff, 1928). On the other hand, the second possibility that the reduction of heavily ramified type I commissural neurons is due to dendritic changes is supported by a recent finding indicating that dendritic length is reduced in a specific group of dentate granule cells in PDAPP transgenic mice (Wu et al., 2004). Together with our finding of an asymmetrically ramified dendritic tree in altered type I commissural neurons it is likely that dendritic alterations would at least precede neuronal death. In so doing, it is possible that degenerated type I and II neurons may appear morphologically as type II or III commissural neurons, respectively. The third hypothesis for the reduction of the number of traced commissural neurons is a loss of axonal connectivity between the hemispheres. This hypothesis is supported by axonal sprouting seen in aged APP-transgenic mice (Phinney et al., 1999; Teter and Ashford, 2002) indicating regeneration after axonal injury (Deller and Jucker, 2001) and by the degeneration of myelinated axons (Bartzokis et al., 2003), especially in the corpus callosum of human Alzheimer's disease cases (Weis et al., 1991; Yamauchi et al., 1993; Hampel et al., 1998). However, our tracing study as well as that of Phinney et al. (1999) did not exhibit collateral sprouting of commissural neurons in 5-month-old APP23 mice already exhibiting a decrease in the number of traced type I commissural neurons. Abnormal axonal sprouting of commissural neurons in APP23 mice starts at 11 months of age and, therefore, axonal damage may not be the first step in the degeneration of these neurons.
Single APP and GAP-43 positive neurites occur in the frontocentral cortex of APP23 mice at 5 months, indicative for axonal sprouting (Masliah et al., 1992a, b). Since commissural neurons do not show sprouting at this age, these APP and GAP-43 positive neurites represent sprouting axon terminals from non-commissural neurons. Such a sprouting is not seen in wild-type littermates. Aß-aggregates alone do not explain this selective sprouting. If Aß-aggregates alone would induce such a sprouting, one would expect a contribution of neurites from all types of afferent neurons including commissural neurons. Therefore, it is tempting to speculate that these sprouting neurites in 5-month-old APP23 mice represent reactive sprouting following the degeneration of type I commissural neurons.
Taken together, dendritic and axonal degeneration as well as neuronal death may be involved in the degeneration of type I and II commissural neurons in a distinct sequence. In a first step dendritic processes of the nerve cells may degenerate presumably followed by axonal damage and, finally, nerve cell death. This sequence is similar to that seen in human Alzheimer's disease for neurons accumulating NFTs (Bancher et al., 1989; Braak et al., 1994; Thal et al., 2000a). Here, neurodegeneration starts with dendritic and perikaryal accumulation of abnormal 
-protein, followed by the creation of NFTs, axonal accumulation of abnormal -protein in the target region of affected neurons and, finally, neuronal death (Braak et al., 1994; Sassin et al., 2000; Thal et al., 2000a).
The relationship between neurodegeneration and Aß-pathology
The loss of traceable type I commissural neurons in 5-month-old APP23 mice is associated with the deposition of the first Aß-plaques within the frontal cortex of these animals. However, total Aß-levels as well as the Aß-plaque load do not differ significantly between 3- and 5-month-old APP23 mice, and there is no close spatial relationship between Aß-plaques and degenerating commissural neurons in 5-month-old APP23 mice. These results indicate that Aß-induced degeneration of neurons in the brain is not due to a simple increase in Aß-levels or to Aß-plaques themselves but due to qualitative changes in the status of Aß-aggregation. Our results support other studies showing that the aggregation of Aß to soluble Aß-oligomers and/or fibrils is required for Aß-toxicity (Podlisny et al., 1995; Kayed et al., 2003; Kim et al., 2003; Cleary et al., 2005) and go beyond the findings of other authors that Aß-plaques induce local dendritic or neuritic degeneration (Tsai et al., 2004; Brendza et al., 2005; Spires et al., 2005).
Our finding of a hierarchical involvement of different types of neurons in Aß-induced neurodegeneration further extends the present knowledge of Aß-toxicity and argues in favour of a selective vulnerability of neurons to Aß. Since we have studied only commissural neurons projecting into the same area of the contralateral cortex, all neurons have the same axon lengths and all neurons function as commissural neurons. Axon lengths and the function of the neuron do, therefore, not appear to be responsible for the selective vulnerability of distinct types of neurons. All pyramidal neurons use excitatory amino acids (glutamate or aspartate) as transmitter (Conti et al., 1988; Giuffrida and Rustioni, 1989). Thus, at least type I and II commissural neurons share a similar neurotransmitter but differ in their vulnerability. Therefore, the selective vulnerability of commissural neurons is most likely not due to the neurotransmitter of the different types of commissural neurons examined in this study.
The most significant difference between type I, II and III commissural neurons is the morphology of the dendritic tree (Fig. 8). Type I neurons exhibit a highly ramified dendritic tree with basal dendrites branching exclusively in layer III, whereas type II neurons have a less ramified dendritic tree with dendrites also branching into layer IV. The type III neurons, in contrast, show only single dendrites without significant further ramification into secondary and tertiary dendrites. Thus, it is tempting to speculate that neurons with a highly ramified dendritic tree present a better target for toxic changes by oligomeric and/or fibrillar Aß than neurons with a less ramified dendritic tree. This hypothesis is supported (i) by the finding of Roselli et al. (2005) that soluble Aß-oligomers induce N-methyl-D-aspartate (NMDA) dependent degradation of post-synaptic density-95 at glutamatergic synapses on dendrites; (ii) by the Aß-induced inhibition of long-term potentiation (Wang et al., 2002); and (iii) by the loss of dendritic spines seen in the vicinity of amyloid plaques (Tsai et al., 2004; Spires et al., 2005). Our finding that Aß-induced neurotoxicity induces dendritic alterations of type I commissural neurons also argues in favour of the dendritic tree to be the primary target of Aß-toxicity. The primary deposition of Aß in the neocortical layers III and V (Braak and Braak, 1991; Price et al., 1991; Arriagada et al., 1992; Thal et al., 2000b) may support this hypothesis insofar as neurons with a dendritic tree branching exclusively within layer III and the molecular layer are target of Aß-induced neurodegeneration at an earlier time point compared with neurons with basal dendrites escaping layer III-Aß by branching into layer IV.
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Our results in concert with that of other authors (Wang et al., 2002
; Roselli et al., 2005) support the hypothesis that oligomeric and/or fibrillar Aß-aggregates interact with dendrites and that neurons may degenerate when their dendrites are exposed to Aß-aggregates. The size of the target for Aß, that is, the dendritic tree surface in a given area, presumably contributes to the vulnerability of neurons to Aß. The primary involvement of dendrites in Aß-toxicity has been demonstrated in dentate gyrus neurons as well (Wu et al., 2004). The interaction of Aß-oligomeric diffusible ligands (ADDLs) with the dendrites (Lacor et al., 2004) could explain why Wu et al. (2004) did not detect any Aß-deposits although they found dendritic degeneration and it could also explain why 33% of the 5-month-old APP23 mice in our study did show neurodegeneration in the absence of Aß-deposits. Moreover, it is highly unlikely that the principal function of a neuron (e.g. the function as a commissural neuron), the length of its axon and the neurotransmitter used are the only factors determining selective vulnerability against Aß. Our results show that theories of selective vulnerability in Alzheimer's disease based upon the principal function and axon length of neurons (Braak et al., 2000) need to be supplemented by other factors to explain Aß-induced selective vulnerability. The morphology of the dendritic arbor presumably contributes to this selective vulnerability of neurons against Aß as demonstrated here.
The impact of Aß-induced progressive neurodegeneration in Alzheimer's disease
In this study we demonstrate that Aß alters different types of neurons in a distinct hierarchical sequence in an animal model for Aß-pathology. After onset of degeneration of a distinct type of neurons a further type of neurons became involved with increasing Aß-pathology. Other studies showing degeneration of different types of neurons in animal models for Alzheimer's disease used double or triple transgenic mice (Lewis et al., 2001; Oddo et al., 2003; Schmitz et al., 2004). In these animals neurodegeneration cannot be addressed exclusively to Aß because presenilin 1 (Oddo et al., 2003; Schmitz et al., 2004) and/or mutant -protein (Lewis et al., 2001; Oddo et al., 2003) are co-expressed. In contrast, single APP-transgenic mice used in this study allow the conclusion that Aß alone is capable of inducing progressive neurodegeneration. Since humans show a hierarchical sequence in which different types of neurons develop Alzheimer's disease-related NFTs and neuronal loss (Braak and Braak, 1991) that parallels the step-by-step expansion of Aß-deposits into further brain regions (Thal et al., 2002b) similar to neurodegeneration and Aß-deposition in mice, our present results suggest that Aß is capable of inducing progressive neurodegeneration in Alzheimer's disease. Therefore, our results support Aß to be a major therapeutic target for treatment of Aß-induced neurodegeneration preferentially in pre-clinical sages. However, since Aß supports progression of neurodegeneration, Aß-lowering treatment strategies may also be helpful in later stages to stop the disease progression.
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*Present address: DKFZ-German Cancer Research Center, D-69120 Heidelberg, Germany
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Acknowledgements |
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We thank N. Kolosnjaji, U. Enderlein, C. Kaschke and H.-U. Klatt for the excellent technical assistance. The authors gratefully acknowledge the introduction to tracing techniques by Dr R. Galuske (Max Planck Institute for Brain Research, Frankfurt am Main, Germany). This study was supported by DFG-grant No. TH624/2 (D.R.T.) and BONFOR-grants No. O-154.0041, O-154.0068 (D.R.T.). C.S. is supported by the Gemeinnützige Hertie-Stiftung and the Medical Faculty of Göttingen (junior research group).
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