Abnormal accumulation of autophagic vesicles correlates with axonal and synaptic pathology in young Alzheimer’s mice hippocampus

ORIGINAL PAPER Abnormal accumulation of autophagic vesicles correlates with axonal and synaptic pathology in young Alzheimer’s mice hippocampus Raquel Sanchez-Varo •Laura Trujillo-Estrada •Elisabeth Sanchez-Mejias •Manuel Torres • David Baglietto-Vargas •Ines Moreno-Gonzalez •Vanessa De Castro •Sebastian Jimenez • Diego Ruano •Marisa Vizuete •Jose Carlos Davila •Jose Manuel Garcia-Verdugo • Antonio Jesus Jimenez •Javier Vitorica •Antonia Gutierrez Received: 22 July 2011 / Revised: 27 September 2011 / Accepted: 13 October 2011 / Published online: 22 October 2011 ÓThe Author(s) 2011. This article is published with open access at Springerlink.com Abstract Dystrophic neurites associated with amyloid plaques precede neuronal death and manifest early in Alzheimer’s disease (AD). In this work we have characterized the plaque-associated neuritic pathology in the hippocampus of young (4to 6-month-old) PS1 M146L / APP 751SL mice model, as the initial degenerative process underlying functional disturbance prior to neuronal loss. Neuritic plaques accounted for almost all fibrillar deposits and an axonal origin of the dystrophies was demonstrated. The early induction of autophagy pathology was evidenced by increased protein levels of the autophagosome marker LC3 that was localized in the axonal dystrophies, and by electron microscopic identification of numerous autophagic vesicles filling and causing the axonal swellings. Early neuritic cytoskeletal defects determined by the presence of phosphorylated tau (AT8-positive) and actin–cofilin rods along with decreased levels of kinesin-1 and dynein motor proteins could be responsible for this extensive vesicle accumulation within dystrophic neurites. Although microsomal Aboligomers were identified, the presence of A11-immunopositive Abplaques also suggested a direct role of plaque-associated Aboligomers in defective axonal transport and disease progression. Most importantly, presynaptic terminals morphologically disrupted by abnormal autophagic vesicle buildup were identified ultrastructurally and further supported by synaptosome isolation. Finally, these early abnormalities in axonal and presynaptic structures might represent the morphological substrate of hippocampal dysfunction preceding synaptic and neuronal loss and could significantly contribute to AD pathology in the preclinical stages. Keywords PS1/APP transgenic mice  Dystrophic neurites Electron microscopy LC3  Amyloid plaques Presynaptic terminals Introduction Alzheimer’s disease (AD) gradually damages the function and structure of particularly vulnerable brain areas, as those used for memory and cognition. Accumulation of aggregated proteins at the extracellular (amyloid-beta, Ab) and intracellular (hyperphosphorylated tau) levels is one of the major abnormalities found in the brain of AD patients (revised in [13,26,50]). Another key pathological feature, that defines this disease, is the early appearance of amyloid R. Sanchez-Varo L. Trujillo-Estrada E. Sanchez-Mejias  D. Baglietto-Vargas I. Moreno-Gonzalez V. De Castro  J. C. Davila A. J. Jimenez A. Gutierrez (&) Dpto. Biologı ´a Celular, Gene ´tica y Fisiologı ´a, Facultad de Ciencias, Universidad de Ma ´laga, Campus de Teatinos, 29071 Ma ´laga, Spain e-mail: [email protected] M. Torres S. Jimenez D. Ruano M. Vizuete J. Vitorica Dpto. Bioquı ´mica y Biologı ´a Molecular, Facultad de Farmacia, Universidad de Sevilla, Seville, Spain J. M. Garcia-Verdugo Laboratorio de Morfologı ´a Celular, Unidad Mixta Centro de Investigacio ´n Prı ´ncipe Felipe-UVEG, Valencia, Spain R. Sanchez-Varo L. Trujillo-Estrada E. Sanchez-Mejias  M. Torres D. Baglietto-Vargas I. Moreno-Gonzalez  V. De Castro S. Jimenez D. Ruano M. Vizuete  J. C. Davila J. M. Garcia-Verdugo J. Vitorica A. Gutierrez Centro de Investigacio ´n Biome ´dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain M. Torres S. Jimenez D. Ruano M. Vizuete J. Vitorica Instituto de Biomedicina de Sevilla (IBiS)-Hospital Universitario Virgen del Rocı ´o/CSIC/Universidad de Sevilla, Seville, Spain 123 Acta Neuropathol (2012) 123:53–70 DOI 10.1007/s00401-011-0896-x plaque-associated neuritic changes in the form of dystrophic neurites, together with a selective loss of connections and neuronal groups. Dystrophic neurites, defined as thickened or irregular neuronal processes, are considered to be an expression of a widespread alteration of the neuronal cytoskeleton. In AD, dystrophic axons are particularly abundant in the hippocampal fiber systems originating from the subiculum, CA1, and the entorhinal cortex [57]. Synaptic loss in both the neocortex and the hippocampus is, so far, the best pathological correlate of early cognitive decline [19,41,52–54,59,61]. The initial neuritic degenerative changes may represent an early manifestation of axonal damage that precede the appearance of synaptic loss and, therefore, a promising disease-modifying morphological target for early intervention strategies to reverse the damage and rescue the deteriorating neurons. Supporting the idea that these changes could be potentially reversible, a recent study has reported that dystrophic axons surrounding amyloid plaques remain connected to viable neuronal bodies over a relatively long period of time [1]. The expression of the human amyloid precursor protein (APP) with single or double mutations in transgenic mouse lines leads to the formation of neuritic plaques with clusters of dystrophic neurites and glial recruitment that resembles the amyloid pathology seen in AD brains [4,28]. After plaque formation, neuritic abnormalities progressively develop as shown by in vivo multiphoton imaging in an AD model [42]; however, the cellular dysfunction underlying the neuritic pathology is not well understood. Numerous autophagic vacuoles accumulate within dystrophic neurites in the brains of humans with AD and AD models [46,47,66] and several lines of investigation support the notion that defects in the autophagy process, a cellular catabolic mechanism essential for the degradation of aggregated proteins and organelles, significantly contributes to AD pathogenesis [11,37,46,48]. Interestingly, it has been reported that autophagic compartments participate in APP processing and Abpeptides production [65, 66] suggesting a possible causal relationship between plaque formation and neuritic dystrophy. Remarkably, restoring the intracellular autophagy pathway ameliorates disease progression and cognition deficits in a transgenic model [65] proving the potential therapeutic value of autophagy induction in early stages of the disease for neuronal function recovery. Here, in this work, we have characterized the morphological and subcellular abnormalities associated with dystrophic neurites around plaques in the hippocampus of our PS1/APP model at 4–6 months of age. Such neuritic abnormalities may result in defects in maintaining axonal and synaptic terminals structure and function. As we have reported previously, this bigenic model reproduces major amyloid-induced pathogenic steps seen in humans. The most relevant feature of our model is that, unlike many other transgenic mice in which neuronal loss is not observed, the selective neurodegenerative phenotype with specific subsets of interneurons and pyramidal neurons is affected in hippocampus and entorhinal cortex, following a regional and temporal pattern [5,29,44,51]. This neuronal loss was found to be associated with a neurotoxic inflammatory response induced by soluble oligomeric Abpeptides [29]. A better understanding of the pathological basis of the neuritic changes, prior to neuronal loss in this model, will provide valuable insights into the potential causes of early axonal damage and synaptic dysfunction and will further improve the accuracy of preclinical evaluation of novel therapeutic agents intended to reverse axonal damage. Materials and methods Transgenic mice The generation and characterization of PS1/APP transgenic (tg) mice has been reported previously [5,10,12,29,30, 44,51]. These double transgenic mice (C57BL/6 background) were obtained by crossing homozygotic PS1M146L transgenic mice with heterozygotic Thy1APP751SL (Swedish: K670N, M671L and London: V717I FAD mutations) mice (Charles River, France). Mice represented F6–F10 offspring of heterozygous transgenic mice. Non-transgenic mice of the same genetic background and ages were used as controls. All animal experiments were carried out in accordance with the European Union regulations and approved by the committee of animal use for research at Malaga University. Antibodies The following primary antibodies were used in this study: anti-human amyloid precursor protein (hAPP) rabbit polyclonal (1:20,000, Sigma A8717); anti-Ab(clone 6E10) mouse monoclonal (1:5,000, Sigma A1474); anti-oligomer A11 (recognizes Ab42 oligomers but not monomers or fibrils) rabbit polyclonal (1:500; Millipore AB9234); antiAb 42 rabbit polyclonal (1:40; Biosource 44-344); antiphospho-PHF-tau pSer202/Thr205 mouse monoclonal (clone AT8) (1:500; Pierce MN1020); anti-cofilin rabbit polyclonal (1:2,000, Cytoskeleton ACFLO2); anti-microtubule-associated protein 1 light chain 3 (LC3) goat polyclonal (1:1,000; Santa Cruz Biotechnology Sc16755); anti-neurofilament 150 kDa rabbit polyclonal (1:5,000; Millipore AB1981); anti-microtubule-associated protein 2 (MAP-2); rabbit polyclonal (1:5,000; Chemicon Ab5622); anti-synaptophysin rabbit polyclonal (1:1,000; Abcam 54 Acta Neuropathol (2012) 123:53–70 123 ab14692); anti-vesicular GABA transporter (VGAT) guinea pig polyclonal (1:5,000; Calbiochem 676780); antivesicular glutamate transporter (VGLUT1) guinea pig polyclonal (1:10,000; Millipore AB5905); anti-human Ab, N terminus (clone 82E1) mouse monoclonal (1:1,000, IBL 10323); anti-kinesin heavy chain (clone KN-01) mouse monoclonal (1:1,000, Abcam AB9097); anti-dynein, 74 kDa (clone 74.1) mouse monoclonal (1:1,000, Millipore MAB1618). Tissue preparation After deep anesthesia with sodium pentobarbital (60 mg/ kg), 2-, 4and 6-month-old control (WT), PS1 and PS1/ APP tg mice were perfused transcardially with 0.1 M phosphate buffered saline (PBS), pH 7.4 followed by 4% paraformaldehyde, 75 mM lysine, 10 mM sodium metaperiodate in 0.1 M phosphate buffer (PB), pH 7.4. Brains were then removed, post-fixed overnight in the same fixative at 4°C, cryoprotected in 30% sucrose, sectioned at 40 lm thickness in the coronal plane on a freezing microtome and serially collected in wells containing cold PBS and 0.02% sodium azide. For electronic microscopy, 4.5-month-old PS1/APP tg mice were perfused transcardially with 0.1 M phosphate buffered saline (PBS)/1% heparin, pH 7.4 followed by 2.5% glutaraldehyde–2% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. After being removed, the brains were post-fixed in the same fixative overnight at 4°C, washed several times with PB, sectioned at 50 or 100 lm thickness in the coronal plane on a vibratome (Leica VT1000M) and serially collected in wells containing cold PB and 0.02% sodium azide. Then, the 100-lm sections were fixed in 2% osmium tetroxide in 0.1 M PB and dehydrated, to be finally embedded in Araldite (EMS, USA). Tissue blocks were cut serially into semithin (1.5 lm) with a diamond knife in a Leica ultramicrotome (EM UC6), placed on slides, stained with 1% toluidine blue and explored with the light microscope for amyloid plaques. Then, selected areas from semithins were cut in ultrathin sections. Ultrathin sections were placed on Formvar-coated grids and stained with uranyl acetate and lead citrate before being examined with an electron microscope (FEI Tecnai Spirit, OR, USA). Light microscopy immunohistochemistry Serial sections from control (WT) and both tg mice (PS1 and PS1/APP) were processed in parallel for light microscopy immunostaining using the same batches of solutions to minimize variability in immunohistochemical labeling conditions. Free-floating sections were first treated with 3% H 2 O 2 /10% methanol in PBS, pH 7.4 for 20 min to inhibit endogenous peroxidases, and with avidin–biotin Blocking Kit (Vector Labs, Burlingame, CA, USA) for 30 min to block endogenous avidin, biotin and biotin-binding proteins. Sections were immunoreacted with one or two of the primary antibodies over 24 or 48 h at room temperature. The tissuebound primary antibody was then detected by incubating for 1 h with the corresponding biotinylated secondary antibody (1:500 dilution, Vector Laboratories), and then followed by incubating for 90 min with streptavidin-conjugated horseradish peroxidase (Sigma–Aldrich) diluted 1:2,000. The peroxidase reaction was visualized with 0.05% 3-3-diaminobenzidine tetrahydrochloride (DAB, Sigma–Aldrich), 0.03% nickel ammonium sulphate and 0.01% hydrogen peroxide in PBS. After DAB, sections immunolabeled for APP, MAP-2, neurofilament or synaptophysin were incubated 3 min in a solution of 20% of Congo red. Sections were then mounted on gelatine-coated slides, air dried, dehydrated in graded ethanol, cleared in xylene and coverslipped with DPX (BDH) mounting medium. Specificity of the immune reactions was controlled by omitting the primary antisera. For double immunofluorescence labelings, sections were first sequentially incubated with the indicated primaries antibodies followed by the corresponding Alexa488/568 secondary antibodies (1:1,000; Invitrogen). APP-immunolabeled sections were stained with 0.02% thioflavine-S in 50°ethanol for 5 min. Sections processed for immunofluorescence were mounted onto gelatin-coated slides, coverslipped with 0.01 M PBS containing 50% glycerin and 3% triethylenediamine and then examined under a confocal laser microscope (Leica SP5 II). Immunoelectron microscopy Sections of 50 lm from 4.5-month-old PS1/APP mice were first washed with PBS and incubated in a 50 mM glycine solution 10 min in order to increase the antibody-binding efficiency. Following the standard immunohistochemical protocol, the tissue was incubated overnight in primary rabbit polyclonal antibodies anti-hAPP or anti-Ab 42 in a PBS 0.1 M/0.01% Tx-100/1% BSA solution at room temperature. Then, they were washed in PBS, and incubated with 1.4 nm gold-conjugated secondary antibody goat antirabbit IgG (1:100; Nanoprobes) for one night at room temperature. After postfixing with 1% glutaraldehyde and washing with 50 mM sodium citrate, the labeling was enhanced with the HQ Silver TM Kit (Nanoprobes). In negative control experiments, primary antibody was omitted. Then, the slices were processed by the standard fixation, dehydration and embedding steps. Stereological analysis Density and size of 6E10-positive amyloid plaques were obtained by stereology-based quantification in the Acta Neuropathol (2012) 123:53–70 55 123 hippocampal formation of PS1/APP at 6 and 18 months of age (n=4/age; 5 sections per animal) according to the optical fractionator method as previously described [44]. Briefly, an Olympus BX61 microscope and the NewCAST software package (Olympus, Glostrup, Denmark) were used. In order to obtain the plaque density, the number of plaques was quantified in five sections through the anteroposterior extent of the hippocampus and then divided between the sampled areas. CA1 subfields were defined using a 109objective and the number of plaques was counted using a 409objective. The number of counting frames varied with the hippocampal region or subfield layer analyzed. We used a counting frame of 7,154.7 lm 2 with step lengths of 84.58 984.58 lm. Neurite and plaque sizes were estimated by the nucleator application with isotrophic probes (n=5 radii). The number of APP-positive dystrophic neurites per plaque was quantified over Congo red stained Abdeposits. Each analysis was done by a single examiner blinded to sample identities. Total protein extraction and Western blots The protein pellets, obtained using the Tripure TM Isolation Reagent, were resuspended in 4% SDS and 8 M urea in 40 mM Tris–HCl, pH 7.4 and rotated overnight at room temperature. The protein content was evaluated using Lowry. Western blots were performed as described previously [12,29,51]. Briefly, 10–20 lg of protein from the different samples were loaded on 16% SDS-Tris-Tricine-PAGE and transferred to nitrocellulose (Hybond-C Extra, Amersham, Sweden). After blocking, the membranes were incubated overnight, at 4°C, with the appropriate antibody. The membranes were then incubated with anti-mouse horseradish-peroxidase-conjugated secondary antibody (Dako, Denmark) at a dilution of 1/8,000. The blots were developed using the ECL-plus detection method (Amersham, Sweden). For quantification, the scanned (Epson 3200) images were analyzed using PCBAS program. In each experiment, the intensity of bands from WT mice and/or experimental condition were averaged and considered as 1 relative unit. Data were always normalized by the specific signal observed in 6-month-old WT group. Synaptosomes and microsomes preparation, soluble fractions isolation and immunoprecipitation The synaptosomal fractions were obtained basically as described previously [62]. Briefly, the tissue was homogenized (using a Dounce homogenizer) in 0.32 M Sucrose, 10 mM Tris–HCl (pH 7.4) buffer (buffer A) containing complete protease and phosphatase inhibitor cocktails (Sigma). After homogenization, the crude synaptosomal fraction (synaptosomes plus mitochondria) was isolated by two sequential centrifugations (1,5009g, 10 min followed by 12,5009g, 20 min; at 4°C). The crude synaptosomes were resuspended in 13% (final concentration) Ficoll 400 (in buffer A) and layered on the bottom of a discontinuous gradient, composed by buffer A and 7% Ficoll (in buffer A). The gradients were centrifuged at 100,0009g(45 min at 4°C) and the synaptosomes were isolated at the 7.5–13% interface. After washing (twice with buffer A), the protein content of the synaptosomal fractions was quantified by Lowry. The soluble and microsomal fractions (supernatant and pellet, respectively) from PS1/APP and WT mice were obtained after centrifugation at 100,0009g(1 h, 4°C) as described previously [29,30]. The A11 or 6E10 immunoprecipitation experiments were also performed as described in detail previously [29, 30]. Since the epitope recognized by A11 was sensitive to detergents, synaptosomes and microsomes were disturbed by sonication (4 pulses at 100 W, 30 s at 4°C). After sonication, the synaptosomes and microsomes were centrifuged (30,0009g, 30 min at 4°C) and soluble proteins were used for immunoprecipitation. A11 and 6E10 immunoprecipitation was done using 50 lg of soluble protein. Statistical analysis Data was expressed as mean ±SD. The comparison between two mice groups (WT and PS1/APP mice or PS1 and PS1/APP tg mice) was done by two-tailed ttest, and for comparing several groups (WT, PS1 and PS1/APP mice) and ages we used one-way ANOVA, followed by Bonferroni post hoc multiple comparison test (SigmaStat Ò 2.03, SPSS Inc). For both tests, the significance was set at 95% of confidence. Results Amyloid plaque-associated dystrophic neurites display a massive accumulation of autophagic vesicles from early ages This PS1/APP transgenic model exhibited extracellular Ab deposits throughout the hippocampus from a very early age as illustrated in Fig. 1a with Congo red staining at 4 months. The number and size of the amyloid deposits progressively increased with age (Fig. 1b). In young mice (4to 6-month old), the most abundant plaques were those less than 500 lm 2 (70.53 ±9.74%), whereas in older mice (18 months) the vast majority of plaques (69.41 ±11.73%) were medium to large ([500 lm 2 ). 56 Acta Neuropathol (2012) 123:53–70 123 Double labeling APP/Congo red (Fig. 1a–c) and APP/ thioflavin-S (Fig. 1d) experiments demonstrated that, at every age examined, almost all (91.61 ±0.14%, the percentage was practically identical at 4, 6 and 18 months of age) fibrillar amyloid deposits were decorated with clusters of APP-positive dystrophic neurites (APP is a well-reported marker for dystrophies) from the time of the appearance of amyloid plaques. The number of dystrophic neurites per plaque increased with age in parallel with the size of the plaque (Fig. 1e). Results showed that, in fact, the number of these dystrophic neurites correlated with the size of the plaque and was independent of the age of the mice. Thus, neuronal pathology in the form of dystrophic neurites occurred very early in this transgenic model. These pathological structures were not found in wild-type (WT) or PS1 transgenic mice of the same age (data not shown). Therefore, plaque-associated abnormal swelling of neuronal processes represented an early indicator of disease development and might compromise neuronal integrity and hippocampal function in young PS1/APP mice. No dystrophic neurites were found in areas remote from Ab plaques or in 2-month-old PS1/APP mice (before the Ab deposition). Transmission electron microscopy analysis of the hippocampus of 4.5-month-old PS1/APP mice revealed a close spatial association between amyloid plaques and neuronal dystrophies (Fig. 2a, b). No dystrophic neurites were found in areas remote from plaques. These abnormal swollen neurites had a round/oval profile and were giantsized, compared to normal neuronal processes in the a b c d APP/Thio-S CA1 CA3 DG CA1 CA3 DG 0 5 10 15 20 25 30 35 Neurites/ plaque < 200 Plaque size ( μ m2) 200-500 500-1000 >1000 e * * * Fig. 1 Early plaque-associated neuritic dystrophy pathology in PS1/APP hippocampus. aand bAPP-immunolabeled sections counterstained with Congo red for fibrillar amyloid deposits at 4(a) and 6 (b) months of age showing the early occurrence of the neuritic pathology. The number of neuritic plaques progressively increases with age. APP-positive dystrophic neurites arise from glutamatergic neurons since the human APP transgene is exclusively expressed by principal neurons as shown in the inset.cA representative neuritic plaque formed by a core of congophilic fibrillar amyloid surrounded by numerous APPpositive dystrophic neurites. dConfocal image showing APP-positive dystrophic neurites (red) around a plaque stained with thioflavin-S (green). eStereological quantification of the dystrophic neurites around plaques. The number of dystrophies/plaque increased with the size of plaque (lm 2 ). Data are expressed as mean ±SD, *p\0.05. Scale bars aand b500 lm, inset 25 lm, cand d10 lm. CA1–CA3 subfields of the hippocampus proper, DG dentate gyrus Acta Neuropathol (2012) 123:53–70 57 123 adjacent neuropil. Ultrastructural morphometric analysis (100 aberrant neurites; n=3) revealed that the predominant size of these dystrophic structures was between 10 and 50 lm 2 (63.74%), followed by those ranging from 50 to 100 lm 2 (20.43%). Notably, 5% of the dystrophic neurites measured over 100 lm 2 , only 9.68% of neurites were in the range 5–10 lm 2 and just 1% under 5 lm 2 . In contrast, normal non-dystrophic neurites had an average size of 1.42 ±0.77 lm 2 . Dystrophic neurites were massively filled with collections of vacuolar structures of putative autophagic nature with different morphologies and heterogeneous intraluminal contents (Fig. 2c, e). The most common morphology corresponded to autophagic vesicles (AVs) consisting of double membrane-bound vesicles with densely compacted amorphous or multilamellar contents named as autophagosomes (Fig. 2c, d). These AVs represent the initial stages of autophagy which contain undigested compacted organellar material. In addition, there were also single or double membrane vesicles with translucent or amorphous electron-dense material in some dystrophic neurites (Fig. 2e, f), and these might presumably represent autophagosomes with partially digested material and/or the mature degradative forms of AVs (autophagolysosomes). Overall, a substantial accumulation of early and, to a less extent, late AVs within hippocampal aberrant neurites, surrounding amyloid plaques, occurred at very early ages in these PS1/APP mice. LC3-positive autophagic vesicles within dystrophic neurites are implicated in the amyloidogenic pathway To corroborate the autophagic nature of the heterogeneous vesicles accumulated within the dystrophic neurites, we immunostained PS1/APP hippocampal sections with the anti-LC3 antibody, a marker of autophagy (Fig. 3a, b). At 4 months (Fig. 3a), LC3 immunoreactivity was mainly found in pyramidal somata and their apical dendrites, as well as in punctate structures resembling dystrophic neurites around plaques (see insets in Fig. 3a). At 6 months (Fig. 3b), the immunoreactivity for LC3 around plaques was markedly increased while, in parallel, the staining of somata and apical dendrites decreased. To more specifically determine the proportion of LC3 that was in the LC3-II form, which migrates faster than LC3-I on SDS-PAGE and is the form associated (by lipidation) with the autophagosomal structures, we performed quantitative immunoblot analysis of LC3-I and LC3-II forms in the hippocampus of 6-month-old PS1/APP and WT mice (Fig. 3c). Significantly higher levels of LC3-II were observed in PS1/APP mice than in age-matched WT mice (2.15 ±0.35 fold, n=6, p\0.05). Confocal imaging of double APP/LC3 immunolabeling (Fig. 3d1–d3) revealed the punctate nature of the LC3 labeling (see inset in Fig. 3d2) and the colocalization of LC3 in both APP-positive (glutamatergic) and non-APP (likely GABAergic or cholinergic) dystrophic neurites (Fig. 3d3). Considering the colocalization of APP and LC3 within dystrophic neurites in our AD model, we next wanted to assess the early implication of AVs in APP processing, and in turn likely involvement in Abproduction. To that end, we performed immunoelectron * * ab cd ef Fig. 2 Extensive accumulation of autophagic vesicles within dystrophic neurites around amyloid plaques. Transmission electron microscopy images of plaque-associated aberrant neurites in young PS1/APP hippocampus. aLow magnification image of an amyloid plaque (asterisk) surrounded by dystrophic neurites (discontinuous white circles). bA diversity of dystrophic neurites based on their subcellular content can be identified. cand dDystrophic neurite filled with heterogeneous electrodense double-membrane vesicles (white square magnified in d) belonging to the early degrading autophagylysosomal pathway (autophagosomes); these AVs had a dense compacted amorphous (filled white arrows) or multilamellar content (empty white arrows). eand fDystrophic neurite filled with autophagic vesicles (white square magnified in f) of distinct morphologies showing translucent (filled white arrows) or amorphous electrodense material (empty white arrows) which might represent late autophagic vesicles (autophagolysosomes). Scale bars a10 lm, b2lm, cand e1lm, dand f500 nm 58 Acta Neuropathol (2012) 123:53–70 123 microscopy for APP and Ab 42 in 4.5-month-old PS1/APP hippocampus. Silver-enhanced immunogold labeling revealed that APP localized preferentially to the AVs within plaque-associated dystrophic neurites (Fig. 3e–g), as well as to the Golgi and endoplasmic reticulum (ER) membranes in the neuronal cell somata (Fig. 3h). No APPlabeling was found in other organelles or plasma membrane. Immunoelectron microscopy detection of Abwas much less stronger than of APP, since optimal intracellular labeling with the antibodies for Abforms in our model requires pre-treatment with formic acid which is not compatible with EM processing. Nevertheless, as expected and in contrast to APP immunolabeling, Ab 42 label was mainly associated with plaques (asterisk in Fig. 3i). Interestingly, some autophagic vesicles within dystrophic neurites were also positive for the Ab 42 antibody (inset in Fig. 3i). Dystrophic neurites represent axonal structures with cytoskeletal abnormalities To determine the dendritic and/or axonal nature of the plaque-associated dystrophic neurites in our PS1/APP model, we have performed light and electron microscopy studies in 4to 6-month-old mice. Immunolabeling for the MAP-2 protein (a marker of dendritic processes) (Fig. 4a, b) and for the postsynaptic marker a1GABA A R (not shown) revealed no positive dystrophic neurites around plaques at the early ages investigated. Moreover, confocal double MAP-2/APP immunofluorescence labeling confirmed the lack of colocalization for the dendritic marker in APP-positive dystrophic neurites (Fig. 4c1–c3). On the other hand, the close spatial relationship between amyloid plaques and axonal fibers tracts in the hippocampus, as revealed by neurofilament (NF) immunolabeling and Congo red staining (Fig. 4d), along with the presence of swollen NF-positive neurites (insets in Fig. 4d) indicated a possible axonal/synaptic origin of these dystrophic structures. In fact, the labeling of the dystrophic neurites was very patent with the presynaptic marker synaptophysin (Fig. 4e). Numerous synaptophysin-positive punctated structures were observed around amyloid plaques. To determine the neurochemical nature of these synaptophysin-positive dystrophic neurites, we performed double immunofluorescence labeling for APP and the two major neurotransmitter vesicular transporters, VGLUT1 for glutamate (Fig. 4f1–f3) and VGAT for GABA (Fig. 4g1–g3). As shown in Fig. 4f3 most APP-positive dystrophic neurites contained VGLUT1 indicating the glutamatergic nature of the abnormal axons surrounding amyloid plaques. Consistent with the exclusive expression of the human mutated transgene for APP by principal cells, many enlarged inhibitory GABAergic dystrophic neurites, immunonegative for APP, were also identified around the plaques (Fig. 4g3). In addition, electron microscopy in the hippocampus of 4.5-month-old PS1/APP mice confirmed the presence of some dystrophic myelinated axons around/near plaques (Fig. 4h–j). These axonal dystrophies had a severe (Fig. 4h, i) to moderately (not shown) pathological number of autophagic vesicles. The enlarged size of an aberrant axon (110.22 lm 2 ) compared to adjacent normal ones (1.57 ±0.63 lm 2 ) is shown in Fig. 4j. In order to identify possible early microtubule-associated axonal transport deficits in the PS1/APP hippocampus, which might lead to vesicle accumulation (autophagic, synaptic, etc.) along axons and the consequent development of dystrophy, we first assessed tau abnormalities by quantitative Western blots experiments with the AT8 antibody (which detects tau phosphorylated at both serine 202 and threonine 205 residues, one of the first to be phosphorylated) (Fig. 5a). Immunoblotting revealed a significantly higher level of expression in young PS1/APP mice (1.75 ±0.15 fold) compared to age-matched controls. We have also confirmed by AT8 immunohistochemistry the presence of phospho-tau positive neurites surrounding amyloid plaques in 4to 6-month-old transgenic animals (Fig. 5b). To determine whether phospho-tau was present within APPpositive dystrophic neurites, we performed double APP/AT8 immunofluorescence labeling (Fig. 5c1–c4 and d1–d4). The presence of AT8 was found in some, but not all, APP-positive dystrophic neurites (Fig. 5c3 and detail in c4; Fig. 5d3 and detail in d4). Tau could also induce changes in the organization and stability of neuronal actin filaments, and it is known that the formation of cofilin/actin pathological bundles occludes neurites and vesicle transport. To assess possible early alterations of the actin cystoskeleton in our AD model we analyzed actin and cofilin immunolabeling in the hippocampus of young PS1/APP mice. Results showed numerous rod-like inclusions around amyloid plaques as shown for cofilin in Fig. 5e. Moreover, double APP/cofilin labeling (Fig. 5f1–f3) showed colocalization of both markers in some, particularly small, APP-positive neurites. Finally, to further explore whether microtubule vesicular transport was compromised in young PS1/APP mice we measured the levels of kinesin-1 and dynein, two microtubule-associated motor proteins, in hippocampal protein extracts prepared from 6-month-old PS1/APP and WT mice (Fig. 5g). We found significantly lower levels of both kinesin-1 heavy chain (-24.71 ±11.80%, n=8) and dynein (-38.06 ±14.40%, n=8) in PS1/APP than agematched WT animals. These microtubule, actin cytoskeletal and molecular motor defects are early pathogenic events in our AD model Acta Neuropathol (2012) 123:53–70 59 123 LC3-I LC3-II β-actin WT PS1/APP WT PS1/APP Relativeunits 3 2 1 0 LC3-II p<0.05 c 4 months 6 months so so a b sp sp sr sr gf APP LC3 Merge d1 d2 d3 ** * e* * i h A β 42 ** * * 60 Acta Neuropathol (2012) 123:53–70 123 and might lead to transport abnormalities and the accumulation of organelles (synaptic vesicles, autophagosomes, mitochondria, lysosomes) within axonal neurites promoting the dystrophic process. The early axonal pathology includes morphologically disrupted presynaptic terminals The axonal defects in our AD model could also affect presynaptic terminals. Therefore, in order to investigate the possible early synaptic pathology we examined the hippocampus of 4.5-month-old PS1/APP mice using electron microscopy (Fig. 6a–e). Our ultrastructural study showed that, near to amyloid plaques, there were presynaptic elements that displayed pathological changes including large diameter with a considerate number of AVs and, in contrast, fewer synaptic vesicles (Fig. 6a, b; presynaptic terminals outlined with a white line). The presence of presynaptic terminals, at the beginning of the dystrophy process, with few and early stage AVs formation, as well as with synaptic-like vesicles were also detected (Fig. 6c–e; see also the presynaptic terminal outlined with a black line in Fig. 6b). However, these altered presynaptic elements were making synaptic contacts with morphologically normal dendrites or dendritic spines with postsynaptic density (see Fig. 6a–d). These morphologically altered presynaptic terminals may represent the initial stages of synaptic disruption and loss. Moreover, we compared the LC3-II accumulation in synaptosomal and microsomal fractions isolated from 6-month-old WT and PS1/APP animals (Fig. 6f). As expected, a low percentage of LC3-II was observed in isolated synaptosomes from WT mice (15.42 ±2.25%, n=4, of microsomal fractions in WT mice). Further, in agreement with our electron microscopy studies, the amount of LC3-II in PS1/APP mice was higher in both synaptosomal and microsomal fractions. Although the relative abundance of LC3-II presented in PS1/APPderived synaptosomes was still low (22.22 ±3.84% of PS1/APP microsomal LC3-II), the level was consistently higher (2.54 ±0.54, n=5) than in WT synaptosomes. These data demonstrated the existence of an early autophagy-associated axonal/synaptic pathology in the hippocampus of this AD mouse model. Extracellular periplaque oligomeric Abspatially correlates with axonal/synaptic dystrophy Taking into account the close spatial relationship between axonal dystrophies and Abplaques, we next examined the possible intracellular and/or extracellular origin of the pathogenic Abagent. We first investigated the possible intracellular accumulation of oligomeric Ab in isolated synaptosomes and microsomes by immunoprecipitation experiments using the monoclonal antibody 6E10 (Fig. 7a). Results demonstrated the presence of a relatively large accumulation of monomeric Abin synaptosomes, whereas lower levels were detected in microsomes (Fig. 7a1). Furthermore, within the different Abpeptides, the Ab 42 was the major form observed in these synaptosomal fractions (Fig. 7a2). On the other hand, no Aboligomers were observed with this approach. We have also used the anti-oligomeric Abantibody A11 in immunohistochemistry and immunoprecipitation experiments. Our results demonstrated that most Abplaques were immunopositive for the A11 antibody (Fig. 7b), with a preferential immunolabeling at the plaque periphery (see inset). Furthermore, A11-immunoprecipitation using the soluble (S1) fractions demonstrated the presence of minute amount of extracellular oligomeric Ab(Fig. 7c) in the hippocampus of the PS1/APP mice at this early age (6 months). These data were consistent with previous experiments (see also [29,30]). However, in spite of the relative abundance of monomeric Abin synaptosomes, under the present experimental conditions, no Aboligomers Fig. 3 Autophagic compartments within dystrophic neurites are sites of APP processing. aand bImmunohistochemistry for the autophagosome marker LC3 in CA1 subfield of PS1/APP mice hippocampus. At 4 months (a), immunoreactivity was concentrated in pyramidal cell somata (stratum pyramidale) and their apical dendrites (stratum radiatum). LC3-positive dystrophic neurites were surrounded amyloid plaques (upper inset in a) and were punctate in nature (the lower inset shows a high magnification image of the LC3positive dystrophy circled in the upper inset). At 6 months (b), the immunoreaction was mainly associated with dystrophic neurites around plaques (arrows) as shown here for stratum oriens (SO). cQuantitative immunoblot analysis of LC3-II form in the hippocampus of 6-month-old PS1/APP and WT mice. PS1/APP mice had significantly higher LC3-II levels than age-matched WT mice (n=6, p\0.05). d1–d3 Confocal images of double immunofluorescence labeling for APP (green) and LC3 (red); APP-positive dystrophic neurites around an amyloid plaque (asterisk) displayed the LC3 marker (long arrows); some LC3-positive dystrophic neurites were negative for APP (short arrows) indicating the non-glutamatergic origin of these aberrant neurites. eDystrophic neurites (encircled) around amyloid plaques (asterisk) were APP immunopositive as revealed by electron microscopy using silver-enhanced immunogold labeling. fHigher magnification of a dystrophic neurite immunopositive for APP; small open squares indicate mitochondria; inset shows two autophagosomes immunogold-labeled for APP. gHigher magnification image of two autophagic vesicles immunogold-labeled for APP. hIn addition to autophagic vesicles, APP immunogold labeling was associated with the endoplasmic reticulum (ER) and Golgi cisternae in neuronal somata. iImmunogold labeling revealed that Ab42 peptides localize to amyloid plaques (asterisk) and autophagic vesicles (inset). so stratum oriens, sp stratum pyramidale, sr stratum radiatum. Scale bars aand b100 lm(upper inset in a50 lm, lower inset in a10 lm); d1–d3 100 lm(inset in d2 2.5 lm), e–h500 nm, inset in f200 nm, i2lm and inset 400 nm b Acta Neuropathol (2012) 123:53–70 61 123 dystrophies. In any case, further experiments are needed to clarify this point. A further significant result was the identification by electron microscopy of dystrophic axon terminals that were making contact with morphologically normal postsynaptic elements. These abnormal presynaptic boutons contained numerous AVs and were observed to have a low content in synaptic vesicles. These results were confirmed by LC3-II Western blots using synaptosomes isolated from the hippocampus of 6-month-old PS1/APP mice. Although we cannot rule out a direct effect of soluble Abon these presynaptic terminals, their relative distance from Abplaques (between 10 and 30 lm) together with the low soluble Abcontent at this age, suggested that AV accumulation might reflect the axonal transport defects at dystrophies. These altered synaptic terminals may represent one of the initial pathogenic steps of synaptic loss leading to early deficits in synaptic transmission and plasticity. As early memory loss in AD is increasingly attributed to synaptic failure, we can conclude that this PS1/APP transgenic model shows, at young ages and in absence of pyramidal degeneration, a presynaptic pathology progression that may closely resemble the pre-clinical or early stages of human AD. Acknowledgments This work was supported by grants PS09/00099 (to A.G.), PS09/00151 (to J.V.), PS09/00848 (to D.R.) and PS09/ 00376 (to A.J.J.) from Fondo de Investigacio ´n Sanitaria (FIS)-Instituto de Salud Carlos III, Spain. Financial support was also received from Junta de Andalucı ´a grants SAS P09/496 (to A.G.) and CTS-4795 (to J.V.). R.S.V., V.D.C. and S.J. were the recipients of a contract from CIBERNED. E.S.M. and L.T.E. held a PhD fellowship from Spain FPU program. We thank Mercedes Aneiros for her excellent technical assistance and Mario Soriano for his help with the transmission electron microscope. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References 1. Adalbert R, Nogradi A, Babetto E et al (2009) Severely dystrophic axons at amyloid plaques remain continuous and connected to viable cell bodies. Brain 132:402–416 2. Arendt T (2005) Alzheimer’s disease as a disorder of dynamic brain self-organization. Prog Brain Res 147:355–378 3. Arendt T (2009) Synaptic degeneration in Alzheimer’s disease. Acta Neuropathol 118:167–179 4. Ashe KH, Zahs KR (2010) Probing the biology of Alzheimer’s disease in mice. Neuron 66:631–645 5. Baglietto-Vargas D, Moreno-Gonzalez I, Sanchez-Varo R et al (2010) Calretinin interneurons are early targets of extracellular amyloid-beta pathology in PS1/AbetaPP Alzheimer mice hippocampus. J Alzheimers Dis 21:119–132 6. Bamburg JR, Bloom GS (2009) Cytoskeletal pathologies of Alzheimer disease. Cell Motil Cytoskelet 66:635–649 7. Bell KF, Claudio CA (2006) Altered synaptic function in Alzheimer’s disease. Eur J Pharmacol 545:11–21 8. Bell KF, de Kort GJ, Steggerda S et al (2003) Structural involvement of the glutamatergic presynaptic boutons in a transgenic mouse model expressing early onset amyloid pathology. Neurosci Lett 353:143–147 9. Bertoni-Freddari C, Fattoretti P, Solazzi M et al (2003) Neuronal death versus synaptic pathology in Alzheimer’s disease. Ann NY Acad Sci 1010:635–638 10. Blanchard V, Moussaoui S, Czech C et al (2003) Time sequence of maturation of dystrophic neurites associated with Abeta deposits in APP/PS1 transgenic mice. Exp Neurol 184:247– 263 11. Boland B, Kumar A, Lee S et al (2008) Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer’s disease. J Neurosci 28:6926–6937 12. Caballero C, Jimenez S, Moreno-Gonzalez I et al (2007) Interindividual variability in the expression of the mutated form of hPS1M146L determined the production of Abeta peptides in the PS1xAPP transgenic mice. J Neurosci Res 85:787–797 13. Castellani RJ, Rolston RK, Smith MA (2010) Alzheimer disease. Dis Mon 56:484–546 14. Chishti MA, Yang DS, Janus C et al (2001) Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695. J Biol Chem 276:21562–21570 15. Cirrito JR, Kang JE, Lee J et al (2008) Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo. Neuron 58:42–51 16. Cirrito JR, Yamada KA, Finn MB et al (2005) Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo. Neuron 48:913–922 17. De Felice FG, Wu D, Lambert MP et al (2008) Alzheimer’s disease-type neuronal tau hyperphosphorylation induced by Abeta oligomers. Neurobiol Aging 29:1334–1347 18. Decker H, Lo KY, Unger SM, Ferreira ST, Silverman MA (2010) Amyloid-beta peptide oligomers disrupt axonal transport through an NMDA receptor-dependent mechanism that is mediated by glycogen synthase kinase 3beta in primary cultured hippocampal neurons. J Neurosci 30:9166–9171 19. DeKosky ST, Scheff SW (1990) Synapse loss in frontal cortex biopsies in Alzheimer’s disease: correlation with cognitive severity. Ann Neurol 27:457–464 20. Delatour B, Blanchard V, Pradier L, Duyckaerts C (2004) Alzheimer pathology disorganizes cortico-cortical circuitry: direct evidence from a transgenic animal model. Neurobiol Dis 16:41–47 21. Dickson TC, King CE, McCormack GH, Vickers JC (1999) Neurochemical diversity of dystrophic neurites in the early and late stages of Alzheimer’s disease. Exp Neurol 156:100–110 22. Dickson TC, Vickers JC (2001) The morphological phenotype of beta-amyloid plaques and associated neuritic changes in Alzheimer’s disease. Neuroscience 105:99–107 23. Fiala JC (2007) Mechanisms of amyloid plaque pathogenesis. Acta Neuropathol 114:551–571 24. Geddes JW, Anderson KJ, Cotman CW (1986) Senile plaques as aberrant sprout-stimulating structures. Exp Neurol 94:767–776 25. Geddes JW, Monaghan DT, Cotman CW et al (1985) Plasticity of hippocampal circuitry in Alzheimer’s disease. Science 230:1179–1181 26. Gomez-Isla T, Spires T, de Calignon A, Hyman BT (2008) Neuropathology of Alzheimer’s disease. Handb Clin Neurol 89:233–243 68 Acta Neuropathol (2012) 123:53–70 123 27. Gouras GK, Almeida CG, Takahashi RH (2005) Intraneuronal Abeta accumulation and origin of plaques in Alzheimer’s disease. Neurobiol Aging 26:1235–1244 28. Hussain I (2010) APP transgenic mouse models and their use in drug discovery to evaluate amyloid-lowering therapeutics. CNS Neurol Disord Drug Targets 9:395–402 29. Jimenez S, Baglietto-Vargas D, Caballero C et al (2008) Inflammatory response in the hippocampus of PS1M146L/ APP751SL mouse model of Alzheimer’s disease: age-dependent switch in the microglial phenotype from alternative to classic. J Neurosci 28:11650–11661 30. Jimenez S, Torres M, Vizuete M et al (2011) Age-dependent accumulation of soluble Abeta oligomers reverses the neuroprotective effect of sAPPalpha by modulating PI3K/Akt-GSK-3beta pathway in Alzheimer mice model. J Biol Chem 286:18414–18425 31. Jin M, Shepardson N, Yang T et al (2011) Soluble amyloid {beta}-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration. Proc Natl Acad Sci USA 108:5819–5824 32. Kanaan NM, Morfini GA, Lapointe NE et al (2011) Pathogenic forms of tau inhibit kinesin-dependent axonal transport through a mechanism involving activation of axonal phosphotransferases. J Neurosci 31:9858–9868 33. Kochl R, Hu XW, Chan EY, Tooze SA (2006) Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes. Traffic 7:129–145 34. Koenigsknecht-Talboo J, Meyer-Luehmann M, Parsadanian M et al (2008) Rapid microglial response around amyloid pathology after systemic anti-Abeta antibody administration in PDAPP mice. J Neurosci 28:14156–14164 35. Koffie RM, Meyer-Luehmann M, Hashimoto T et al (2009) Oligomeric amyloid beta associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques. Proc Natl Acad Sci USA 106:4012–4017 36. Kovacs AL, Reith A, Seglen PO (1982) Accumulation of autophagosomes after inhibition of hepatocytic protein degradation by vinblastine, leupeptin or a lysosomotropic amine. Exp Cell Res 137:191–201 37. Lee JH, Yu WH, Kumar A et al (2010) Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell 141:1146–1158 38. Martins IC, Kuperstein I, Wilkinson H et al (2008) Lipids revert inert Abeta amyloid fibrils to neurotoxic protofibrils that affect learning in mice. EMBO J 27:224–233 39. Masliah E, Alford M, Adame A et al (2003) Abeta1–42 promotes cholinergic sprouting in patients with AD and Lewy body variant of AD. Neurology 61:206–211 40. Masliah E, Hansen L, Albright T, Mallory M, Terry RD (1991) Immunoelectron microscopic study of synaptic pathology in Alzheimer’s disease. Acta Neuropathol 81:428–433 41. Masliah E, Mallory M, Alford M et al (2001) Altered expression of synaptic proteins occurs early during progression of Alzheimer’s disease. Neurology 56:127–129 42. Meyer-Luehmann M, Spires-Jones TL, Prada C et al (2008) Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer’s disease. Nature 451:720– 724 43. Mirra SS, Heyman A, McKeel D et al (1991) The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer’s disease. Neurology 41:479–486 44. Moreno-Gonzalez I, Baglietto-Vargas D, Sanchez-Varo R et al (2009) Extracellular amyloid-beta and cytotoxic glial activation induce significant entorhinal neuron loss in young PS1(M146L)/ APP(751SL) mice. J Alzheimers Dis 18:755–776 45. Nimmrich V, Ebert U (2009) Is Alzheimer’s disease a result of presynaptic failure? Synaptic dysfunctions induced by oligomeric beta-amyloid. Rev Neurosci 20:1–12 46. Nixon RA (2007) Autophagy, amyloidogenesis and Alzheimer disease. J Cell Sci 120:4081–4091 47. Nixon RA, Wegiel J, Kumar A et al (2005) Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study. J Neuropathol Exp Neurol 64:113–122 48. Nixon RA, Yang DS (2011) Autophagy failure in Alzheimer’s disease-locating the primary defect. Neurobiol Dis 43:38–45 49. Noda-Saita K, Terai K, Iwai A et al (2004) Exclusive association and simultaneous appearance of congophilic plaques and AT8positive dystrophic neurites in Tg2576 mice suggest a mechanism of senile plaque formation and progression of neuritic dystrophy in Alzheimer’s disease. Acta Neuropathol 108:435–442 50. Perl DP (2010) Neuropathology of Alzheimer’s disease. Mt Sinai J Med 77:32–42 51. Ramos B, Baglietto-Vargas D, Del Rio JC et al (2006) Early neuropathology of somatostatin/NPY GABAergic cells in the hippocampus of a PS1xAPP transgenic model of Alzheimer’s disease. Neurobiol Aging 27:1658–1672 52. Scheff SW, Price DA, Schmitt FA, DeKosky ST, Mufson EJ (2007) Synaptic alterations in CA1 in mild Alzheimer disease and mild cognitive impairment. Neurology 68:1501–1508 53. Scheff SW, Price DA, Schmitt FA, Mufson EJ (2006) Hippocampal synaptic loss in early Alzheimer’s disease and mild cognitive impairment. Neurobiol Aging 27:1372–1384 54. Selkoe DJ (2002) Alzheimer’s disease is a synaptic failure. Science 298:789–791 55. Sokolow S, Henkins KM, Bilousova T et al (2011) AD synapses contain abundant Abeta monomer and multiple soluble oligomers, including a 56-kDa assembly. Neurobiol Aging. doi: 10.1016/j.neurobiolaging.2011.05.011 56. Stokin GB, Lillo C, Falzone TL et al (2005) Axonopathy and transport deficits early in the pathogenesis of Alzheimer’s disease. Science 307:1282–1288 57. Su JH, Cummings BJ, Cotman CW (1993) Identification and distribution of axonal dystrophic neurites in Alzheimer’s disease. Brain Res 625:228–237 58. Su JH, Cummings BJ, Cotman CW (1998) Plaque biogenesis in brain aging and Alzheimer’s disease. II. Progressive transformation and developmental sequence of dystrophic neurites. Acta Neuropathol 96:463–471 59. Sze CI, Troncoso JC, Kawas C et al (1997) Loss of the presynaptic vesicle protein synaptophysin in hippocampus correlates with cognitive decline in Alzheimer disease. J Neuropathol Exp Neurol 56:933–944 60. Tampellini D, Gouras GK (2010) Synapses, synaptic activity and intraneuronal abeta in Alzheimer’s disease. Front Aging Neurosci 2:13 61. Terry RD, Masliah E, Salmon DP et al (1991) Physical basis of cognitive alterations in Alzheimer’s disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol 30:572–580 62. Vitorica J, Satrustegui J (1986) Involvement of mitochondria in the age-dependent decrease in calcium uptake of rat brain synaptosomes. Brain Res 378:36–48 63. Vossel KA, Zhang K, Brodbeck J et al (2010) Tau reduction prevents Abeta-induced defects in axonal transport. Science 330:198 64. Woodhouse A, Vickers JC, Adlard PA, Dickson TC (2009) Dystrophic neurites in TgCRND8 and Tg2576 mice mimic human pathological brain aging. Neurobiol Aging 30:864–874 65. Yang DS, Stavrides P, Mohan PS et al (2011) Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer’s disease ameliorates amyloid pathologies and memory deficits. Brain 134:258–277 Acta Neuropathol (2012) 123:53–70 69 123 66. Yu WH, Kumar A, Peterhoff C et al (2004) Autophagic vacuoles are enriched in amyloid precursor protein-secretase activities: implications for beta-amyloid peptide over-production and localization in Alzheimer’s disease. Int J Biochem Cell Biol 36:2531–2540 67. Zempel H, Thies E, Mandelkow E, Mandelkow EM (2010) Abeta oligomers cause localized Ca(2?) elevation, missorting of endogenous Tau into dendrites, Tau phosphorylation, and destruction of microtubules and spines. J Neurosci 30:11938–11950 68. Zhang XM, Cai Y, Xiong K et al (2009) Beta-secretase-1 elevation in transgenic mouse models of Alzheimer’s disease is associated with synaptic/axonal pathology and amyloidogenesis: implications for neuritic plaque development. Eur J Neurosci 30:2271–2283 70 Acta Neuropathol (2012) 123:53–70 123