METHODS AND PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF ALZHEIMER'S DISEASE

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of Alzheimer's disease. In particular the present invention relates to a method of treating Alzheimer's disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a vector which comprises a nucleic acid molecule encoding for a polypeptide which is a soluble member of the APP (amyloid precursor protein) family.

Claims

1. A method of treating Alzheimer's disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a vector which comprise a nucleic acid molecule encoding for a polypeptide which is a soluble member of the APP (amyloid precursor protein) family.

2. The method of claim 1 wherein the vector comprise a nucleic acid molecule that encodes for an APPs, APLP1s or APLP2s polypeptide.

3. The method of claim 1 wherein the vector or the cell comprise a nucleic acid encoding for an APPs polypeptide.

4. The method of claim 1 wherein the nucleic acid molecule encoding for an APPs polypeptide comprising an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO:1 or 2.

5. The method of claim 1 wherein the nucleic acid molecule comprises a sequence having at least 70% of identity with the nucleic acid sequence as set forth in SEQ ID NO:3, or SEQ ID NO:4.

6. The method of claim 1 wherein the vector is a viral vector.

7. The method of claim 1 wherein the vector is an adeno-associated virus (AAV) vector.

8. The method of claim 7 wherein the AAV vector is selected from vectors derived from AAV serotypes having tropism for and high transduction efficiencies in cells of the mammalian central and peripheral nervous system, particularly neurons, neuronal progenitors, astrocytes, oligodendrocytes and glial cells.

9. The method of claim 7 wherein the AAV vector is an AAV4, AAV9 or an AAV10.

10. The method of claim 1 wherein the nucleic acid molecule is operatively linked to a promoter sequence.

11. The method of claim 1 wherein the vector comprises a secretory signal sequence.

12. The method of claim 1 wherein the vector comprises the nucleic acid sequence set forth in SED ID NO:5 or 6.

13. The method of claim 1 wherein the vector is delivered by intrathecal delivery.

14. A method of treating Alzheimer's disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of cells transduced with a vector which comprises a nucleic acid molecule encoding for a polypeptide which is a soluble member of the APP (amyloid precursor protein) family.

15. The method according to claim 14 wherein the cells administrated are autologous hematopoietic stem cell or hematopoietic progenitors.

Description

FIGURES

[0041] FIG. 1: APPs overexpression enhances Morris water maze performance in WT mice and rescues the spatial memory deficit of APP/PS1E9 mice. Transgenic APP/PS1E9 mice (n=8 per group) or littermate (LM) controls (n=3-4 per group) were either injected with AAV-Venus or AAV-APPs vectors at 12 months of age and tested 2 months later at 14 months of age. (A) Escape latency and (B) swim speed of littermate controls or APP/PS1E9 mice injected either with AAV-Venus or AAV-APPs. Swim speed was similar between the different groups (2-way ANOVA: Group effect: F.sub.3,100=2.40; ns; Time effect: F.sub.4,100=1.41; ns; GroupTime interaction: F.sub.12,100<1; ns). Littermates injected with AAV-APPs showed improved performance, as indicated by reduced escape latency (2-way ANOVA: Time effect: F.sub.4,100=7.138; p<0.0001; Group effect: F.sub.3,100=7.247; p=0.0002, followed by Tukey post-hoc test: APP/PS1E9 mice injected with AAV-APPs versus each of the other groups, p<0.013). (C) Probe trial performance at 72 h. 2-way ANOVA, Group effect: F.sub.3,17=3.356; p<0.04; quadrant effect: F.sub.1,17=23.54; p<0.007; Groupquadrant interaction effect: F.sub.3,17=3.356; p<0.04. APP/PS1E9 mice injected with AAV-Venus were impaired in comparison to littermate mice injected with AAV-Venus (Tukey post-hoc test: p=0.023) confirmed by no preference for the trained target quadrant. Strikingly, AAV-APPs treated APP/PS1E9 mice spent more time in the target quadrant compared to APP/PS1E9 mice injected with AAV-Venus (Tukey post-hoc test: p=0.017). Statistics: 2-way ANOVA (genotype and group as factors) with repeated measures followed by Tukey post-hoc test: *<0.05. Values represent meansSEM.

[0042] FIG. 2: APP/PS1E9 mice reveal structural and functional synaptic impairments that are ameliorated by APPs expression. (A) LTP was induced by TBS at hippocampal CA3-CA1 synapses after 20 min baseline recordings. Acute slices of AAV-Venus injected APP/PS1E9 animals exhibited significant lower induction and maintenance of LTP compared to littermate controls (LM) showing similar expression of Venus (averaged potentiation minutes t50-80: 148.476.04% vs. 178.018.98%, p=0.021). Viral expression of APPs restored potentiation after TBS (171.486.29%) in transgenic animals and resulted in an LTP curve progression comparable to that of LM controls. The LTP induction rate is shown as percentage % of mean baseline slope, n=number of slices, N=number of mice. (B, C) Input-Output strength of all AAV-injected mice showed no alterations between genotypes at any fiber volley (FV) amplitude or stimulus intensity tested. (D) Altered PPF at the 10 ms ISI revealed a significant impairment in the pre-synapse of APP/PS1E9 mice injected with AAV-Venus in comparison to LM controls (*p=0.030) that was restored after AAV-APPs injection (#p=0.047). (E) No differences in spine density at CA1 apical neurons between groups, but significantly less spines at basal dendrites of APP/PS1E9 Venus injected mice (p=0.040). APPs overexpression partially restored the spine density deficit. (F) Reduced spine density at CA3 apical (p=0.014) and basal (p=0.011) dendritic segments of APP/PS1E9 AAV-Venus injected mice that is partially rescued at apical and completely at basal dendrites (p=0.039) by APPs. N=number of neurons, N=number of animals. Data represent meanSEM and were analyzed by one-way ANOVA followed by Bonferroni's post-hoc test.

[0043] FIG. 3: AAV-APPs injection decreases A and plaques load. (A) ELISA quantification of -CTF in hippocampus (H) and cortex (Cx) of APP/PS1E9 mice. No difference was detectable between AAV-Venus and AAV-APPs injected animals. (B-D) Quantification (MSD immunoassay) of TBS soluble A38 (Group effect: F.sub.1,14=3.879, p=0.07 (36%), A40 (Group effect: F.sub.1,14=3.094, p=0.10 (32%)) and A42 (Group effect: F.sub.1,14=5.211, p=0.04 (33%); region effect: F.sub.1,14<1, ns; Groupregion interaction effect: F.sub.1,14<1, ns) in hippocampus and cortex of APP/PS1E9 mice. Note that AAV-APPs injected animals show reduced levels of A in both anatomical regions analyzed (hippocampus and cortex). (E) Quantification of 4G8 immunolabeled area in hippocampus and cortex (2-way ANOVA: Treatment effect: F.sub.1,13=5.50, p=0.04 (24%); region effect: F.sub.1,13=22.89, p=0.0004; Treatmentregion interaction effect: F.sub.1,13<1, ns). Note that 4G8 immunoreactive plaque area is significantly reduced in AAV-APPs treated animals. Number of animals n=8/group. 2-way ANOVA (Genotype, treatment and region as factors) followed by Tukey post-hoc test: *p<0.05. Values represent meansSEM.

[0044] FIG. 4: AAV-APPs promotes microglia recruitment around plaques in APP/PS1E9 mice. (A) Quantification signal intensity from western blot analysis showing the expression of GFAP and Iba1 in the hippocampus of AAV-Venus or AAV-APPs injected APP/PS1E9 mice (n=8/group). Quantification signal intensities were normalized to GAPDH used as a loading control. AAV-APPs treatment specifically increased Iba1 (microglial marker) expression (t-test: t.sub.14=3.586; p=0.003), whereas the astrocyte marker GFAP was not affected. (B) Whereas the distribution of GFAP positive astrocytes is unaltered, increased recruitment of Iba1 positive microglia is observed in the vicinity of amyloid plaques (t-test: t.sub.16=5.441; p<0.0001). (C) Western blot analysis showing the expression of IDE and TREM2 in the hippocampus of AAV-Venus or AAV-APPs injected APP/PS1E9 mice. Both A clearance related proteins are significantly upregulated (IDE: t-test: t.sub.14=3.984; p=0.0014; TREM2: t-test: t.sub.14=2.947; p=0.010) following AAV-APPs treatment. Values represent meansSEM. ***P<0.001, **P<0.01, *P<0.05.

[0045] FIG. 5: Unlike APPs, APPs overexpression does not rescue spatial memory deficit of APP/PS1E9 mice in the Morris water maze. Littermate (LM) controls injected with Venus (n=3) or transgenic APP/PS1E9 mice (n=7-8 per group) either injected with AAV-Venus, AAV-APPs or AAV-APPs vectors at 12 months of age were tested 2 months later at 14 months of age. Graphic showing the probe trial performance at 72 h. 2-way ANOVA, groupquadrant interaction effect: F3,19=4.04; p=0.02. APP/PS1E9 mice injected with AAV-Venus were impaired in comparison to littermate mice injected with AAV-Venus (Tukey post hoc test: p=0.04) confirmed by no preference for the trained target quadrant. Unlike AAV-APPs treatment in APP/PS1E9 mice which restored time spent in the target quadrant (Tukey post hoc test: p=0.011), AAV-APPs did not improve their performances compared to APP/PS1E9 mice injected with AAV-Venus (Tukey post hoc test: p>0.99). Data represent meanSEM and were analyzed by 2-way ANOVA (genotype and group as factors) with repeated measures followed by Tukey post hoc test. *p<0.05.

[0046] FIG. 6: AAV-APPs injection do not restore long term potentiation in the hippocampus of aged APP/PS1E9 mice. LTP was induced by TBS at hippocampal CA3-CA1 synapses after 20 min baseline recordings. Acute slices of AAV-Venus injected APP/PS1E9 animals exhibited significant lower induction and maintenance of LTP compared to littermate controls (LM) indicating a significant impairment of the transgenic mice. Viral expression of APPs restored potentiation after TBS in transgenic animals and resulted in an LTP comparable to that of LM controls. However, AAV-APPs injection did not restore APP/PS1E9 mice which show a similar level compared to AAV-Venus transgenic mice. The LTP induction rate is shown as percentage % of mean baseline slope, n=number of slices, N=number of mice.

[0047] FIG. 7: AAV-APPs injection does not activate microglia. A Western blot analysis showing the expression of IBA1 (microglial marker) in the hippocampus of AAV-Venus, AAVAPPs or AAV-APPs injected APP/PS1E9 mice (n=7/8 per group). For quantification signal intensity was normalized to GAPDH used as a loading control (One-way ANOVA: group effect: F2,19=14.38, p=0.0002). AAV-APPs treatment specifically increased IBA1 expression (p=0.0002) whereas AAV-APPs did not (p=0.82). B Quantification of IBA1 signal in the hippocampus following immunohistochemistry in APP/PS1E9 mice injected either with AAV-Venus, AAV-APPs or AAV-APPs (One-way ANOVA: group effect: F2,41=14.12, p<0.0001). As seen in western blot, AAV-APPs injection is unable to rise IBA1 levels compared to AAV-Venus treated mice. Values represent meanSEM. ***p<0.001, **p<0.01.

EXAMPLE

[0048] Material & Methods

[0049] AAV Plasmid Design and Vector Production

[0050] The mouse APPs coding sequence (derived from Uniprot: P12023-2) was codon optimized (Geneart, Regensburg) and then cloned under control of the synapsin promoter into the single stranded, rAAV2-based transfer vector pAAVSynMCS-2A-Venus (Tang et al, 2009) via NheI-HindIII restriction sites. For easy detection, an N-terminal double HA-tag was inserted downstream of the APP signal peptide at the N-terminus of APPs. The control vector (pAAV-Venus) encodes the yellow fluorescent protein Venus fused to a C-terminal farnesylation signal for membrane anchoring. All constructs were packaged into AAV9 by the MIRCen viral production platform as described (Berger et al, 2015).

[0051] Animals

[0052] Sixteen APPswe/PS1E9 mice (referred as APP/PS1E9; Jackson Laboratories) and seven age-matched littermate control mice were used for behavior, pathology and biochemistry. Eleven APP/PS1E9 and five littermates were used for electrophysiology and spine density analysis. APP/PS1E9 mice express the human APP gene carrying the Swedish double mutation (K595N/M596L). In addition, they express the human PS1E9 variant lacking exon 9 (Borchelt et al, 1997; Jankowsky et al, 2004; Xiong et al, 2011). Only male mice were used throughout the study. For age at AAV injection and age at analysis/sacrifice see results section. All experiments were conducted in accordance with the ethical standards of French, German and European regulations (European Communities Council Directive of 24 Nov. 1986).

[0053] Stereotactic Injection of AAV

[0054] Mice were anesthetized by intraperitoneal injection of ketamine/xylazine (0.1/0.05 g/kg body weight) and positioned on a stereotactic frame (Stoelting, Wood Dale, USA). Vectors (either AAV-Venus or AAV-APPs) were bilaterally injected into the hippocampus using 2 l of viral preparation (10.sup.10 vg/site) at a rate of 0.2 l/minute. Two injections sites per hippocampus were used to optimize virus spreading. Stereotactic coordinates of injection sites from bregma were: anteroposterior 2 mm; mediolateral+/1 mm; dorsoventral 2.25 mm and anteroposterior 2 mm; mediolateral+/1 mm; dorsoventral 1.75 mm.

[0055] Brain Samples

[0056] APP/PS1E9 mice were sacrificed 5 months post-injection at 17 months of age. Following anesthesia, mice were transcardially perfused with 0.1 M phosphate buffered saline (PBS) before dissection. For immunohistochemistry, the left cerebral hemisphere was dissected and post-fixed in 4% paraformaldehyde (PFA) for 1 week and cryoprotected in 30% sucrose for 24 hours. 40 m sections were cut using a freezing microtome (Leica, Wetzlar, Germany), collected in a cryoprotective solution and stored at 20 C. The right hemisphere was dissected to segregate hippocampus and cortex for biochemical analysis. Samples were then homogenized in lysis buffer (TBS, NaCl 150 mM and Triton 1%) containing phosphatase and protease inhibitors. After centrifugation (20 min, 13 000 rpm, 4 C.), the supernatant was collected and the protein concentration was quantified by BCA Assay (Thermo Fisher Scientific, Waltham, USA). Lysate aliquots (3 mg of protein/ml) were stored at 80 C.

[0057] Immunostaining

[0058] Slices were washed with 0.1 M PBS and permeabilized in 0.25% PBS-Triton before blocking in PBS-Triton 0.25% containing 5% goat serum for 60 minutes. For vector encoded HA-APPs immuno labeling, slices were incubated with an anti-HA antibody (Covance, Princeton, USA, 1/250) overnight at 4 C. After successive washes (PBS-Triton 0.25%, PBS and PB 0.1 M), incubation with a biotinylated anti-mouse antibody was performed for one hour at room temperature. For signal amplification, samples were incubated using the ABC kit (Vector laboratories, Burlingame, USA) for one hour at room temperature. Finally, slices were incubated in Cy3-coupled streptavidine. HA-APPs was co-immunostained overnight with the following primary antibodies: Rabbit anti-Iba1, 1/500, Wako, Richmond, USA; Mouse-GFAP Cy3 conjugate, 1/500, Sigma-Aldrich, Saint-Louis, USA. For immunofluorescent staining of plaques, slices were stained using a 30 min incubation in 1% thioflavin-S solution, rinsed twice (1 min each) in 50% EtOH and mounted in Vectashield fluorescent mounting media (Vector laboratories). Images were taken with a Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan) and a Leica SP8 confocal microscope (Leica). For plaque quantification, slices were incubated in 88% formic acid solution for 15 min (antigen retrieval). To inactivate endogenous peroxidase, samples were incubated in hydrogen peroxide (30 min) before blocking and incubation with the primary antibody (4G8, Covance, 1/1000). Incubation with a horseradish coupled secondary antibody was done at RT, developed using the DAB kit (Vector laboratories) and mounted in Eukitt mounting media (Sigma-Aldrich, Saint-Louis, USA). Images were taken with a Z6 APO macroscope (Leica). Plaques, GFAP and Iba1 immunoreactivity were quantified using ImageJ (NIH, Bethesda, USA) or Icy (Institut Pasteur, Paris, France). Laserpower, numeric gain and magnification were kept constant between animals to avoid potential technical artefacts. Images were first converted to 8-bit gray scale and binary thresholded to highlight a positive staining. At least 2 sections per mouse (between 1.7 mm to 2.3 mm caudal to bregma) were quantified for either hippocampus or cortex. The average value per structure was calculated for each mouse. For quantification of Iba1 and GFAP immunoreactivity around plaques, a region of interest (ROI) was drawn around the center of the plaque. The diameter of the circular ROI was set as 3 times the diameter of the plaque. Mean fluorescence intensity values were measured for either Iba1 or GFAP immunoreactivity and were processed via Icy software (Institut Pasteur, Paris, France). Experimentators and data managers were blind with respect to treatments and genotypes.

[0059] Western Blot Analysis

[0060] Proteins were separated by electrophoresis using 4-12% SDS-PAGE (NuPAGE, Life Technologies, Carlsbad, USA) in MOPS buffer (NuPAGE, Life Technologies) and transferred to nitrocellulose membranes (iBlot, Life Technologies). After blocking in 5% milk-PBS 0.1M for 60 minutes, membranes were incubated with the primary antibodies overnight at 4 C. (HA, 1/2000, Covance, Princeton, USA; Venus (GFP), 1/1000, Vector laboratories Burlingame, USA; GAPDH, 1/4000 Abcam, Cambridge, UK; Iba1, 1/2000, Wako, Richmond, USA; GFAP, 1/4000 Dako, Glostrup, Denmark; IDE, 1/200, Santa Cruz Biotechnology, Dallas, USA; TREM2, 1/500, R&D Systems, Minneapolis, USA). Membranes were then washed with TBS-T (with 0.1% Tween), incubated with a horseradish peroxidase coupled secondary antibody and developed using enhanced chemiluminescence (ECL, GE Healthcare, Little Chalfont, UK and Super Signal, Thermo Fisher Scientific). Signals were detected with Fusion FX7 (Vilber Lourmat, Marne-la-Valle, France) and analyzed and quantified using ImageJ.

[0061] ELISA

[0062] APPs, -CTF, and A were quantified using the sAPP kit (Meso Scale Discovery, Rockville, USA), Human APP -CTF Assay Kit (IBL, Hamburg, Germany), V-PLEX Plus A Peptide Panel 1 (6E10) Kit (Meso Scale Discovery). The procedures were performed according to the respective supplier instructions.

[0063] Morris Water Maze

[0064] Experiments were performed in a 120-cm diameter, 50 cm deep tank filled with opacified water kept at 21 C. and equipped with a 10 cm diameter platform submerged 1 cm under the water surface. Visual clues were disposed around the pool as spatial landmarks for the mouse and luminosity was kept at 430 lux. Training consisted of daily sessions (three trials per session) during 5 consecutive days. Start positions varied pseudo-randomly among the four cardinal points. Mean inter-trial interval was 15 min. Each trial ended when the animal reached the platform. A 60 second cut-off was used, after which mice were gently guided to the platform. Once on the platform, animals were given a 30-second rest before being returned to their cage. 72 hours after the last training trial (day 8), retention was assessed during probe trial in which the platform was no longer present. Animals were video tracked using Ethovision software (Noldus, Wageningen, Netherlands) and behavioral parameters (swim speed, travelled distance, latency, percentage of time spend in each quadrant) were automatically calculated. Experiments and statistical evaluation of data were performed by an experimentator blind to genotype and treatment group.

[0065] Statistics

[0066] Statistical analyses were performed as indicated for the respective experiments. Outliers were detected and rejected using maximum normed residual test (Grubbs' test). In most cases, data were analyzed using non-parametric Mann-Whitney U tests excepted for behavioral experiments. Two-way ANOVA with repeated measures were carried out when required by the experimental plan to assess statistical effects. Correlation matrices were generated using non-parametric Spearman rank correlation coefficient. For all analysis statistical significance was set to a p-value <0.05. All analyses were performed using Statistica (StatSoft Inc., Tulsa, USA) or Prism (GraphPad Software, La Jolla, USA).

[0067] Electrophysiology

[0068] In vitro extracellular recordings were performed on acute hippocampal slices of WT littermates stereotactically injected with the AAV-Venus (N=5), APP/PS1E9 mice injected either with AAV-Venus (N=4) or AAV-APPs virus (N=6) at 8 months of age. Electrophysiological recordings were performed 4-5 months later at an age of 12-13 months. In-between animals were housed in a temperature- and humidity-controlled room with a 12 h light-dark cycle and had access to food and water ad libitum.

[0069] Slice Preparation

[0070] Acute hippocampal transversal slices were prepared from individuals at an age of 12 to 13 months. Mice were anesthetized with isoflurane and decapitated. The brain was removed and quickly transferred into ice-cold carbogenated (95% O.sub.2, 5% CO.sub.2) artificial cerebrospinal fluid (ACSF) containing 125 mM NaCl, 2 mM KCl, 1.25 mM NaH.sub.2PO.sub.4, 2 mM MgCl.sub.2, 26 mM NaHCO.sub.3, 25 mM glucose. After dissection of the two hemispheres one was used for Golgi-Cox staining and the other for electrophysiology. The hippocampus was sectioned into 400 m thick transversal slices with a vibrating microtome (Leica, VT1200S). Slices were maintained in carbogenated ACSF (125 mM NaCl, 2.mM KCl, 1.25 mM NaH.sub.2PO.sub.4, 2 mM MgCl.sub.2, 26 mM NaHCO.sub.3, 2 mM CaCl.sub.2, 25 mM glucose) at room temperature for at least 1.5 h before transferred into a submerged recording chamber. Before recording, each slice of the AAV-Venus injected animals was proofed for fluorescence expression of Venus in area CA1 and CA3 (Zeiss, Axiovert 35). Slices absent of the fluorescence protein in the recording areas were excluded from further analysis.

[0071] Extracellular Field Recordings

[0072] Slices were placed in a submerged recording chamber and perfused with carbogenated ACSF (32 C.; 125 mM NaCl, 2 mM KCl, 1.25 mM NaH.sub.2PO.sub.4, 1 mM MgCl.sub.2, 26 mM NaHCO.sub.3, 2 mM CaCl.sub.2, 25 mM glucose) at a rate of 1.2 to 1.5 ml/min. Field excitatory postsynaptic potentials (fEPSPs) were recorded in stratum radiatum of CA1 region with a borosilicate glass micropipette (resistance 2-4 M) filled with 3 M NaCl at a depth of 150-200 m. Monopolar tungsten electrodes were used for stimulating the Schaffer collaterals at a frequency of 0.1 Hz. Stimulation intensity was adjusted to 40% of maximum fEPSP slope for 20 minutes baseline recording. LTP was induced by applying theta-burst stimulation (TBS: 10 trains of 4 pulses at 100 Hz in an 200 ms interval, repeated 3 times). Basal synaptic transmission properties were analyzed via input-output-(IO) measurements and short-term plasticity was examined via paired pulse facilitation (PPF). The IO-measurements were performed either by application of a defined current values (25-175 A) or by adjusting the stimulus intensity to certain fiber volley (FV) amplitudes (0.1-0.7 mV). PPF was performed by applying a pair of two closely spaced stimuli in different inter-stimulus-intervals (ISI) ranging from 10 to 160 ms.

[0073] Dendrite and Spine Analysis

[0074] Golgi-Cox Staining

[0075] Golgi staining was done using the FD Rapid GolgiStain Kit according to the manufacturer's instructions. All procedures were performed under dark conditions. One hemisphere of each mouse was used for electrophysiology and the other one for Golgi-Cox staining. Hemispheres were immersed in 2 ml mixtures of equal parts of kit solutions A and B and stored at RT for 2 weeks. Afterwards brain tissues were stored in solution C at 4 C. for at least 48 h and up to 7 days before sectioning. Solutions AB and C were renewed within the first 24 h. Coronal sections of 200 m were cut with a vibrating microtome (Leica, VT1200S) while embedded in 2% Agar in 0.1 M PBS. Each section was mounted with Solution C on an adhesive microscope slide pre-coated with 1% gelatin/0.1% chromalaun on both sides and stained according to the manufacturer's protocol with the exception that AppliClear (AppliChem) was used instead of xylene. Finally slices were cover-slipped with Permount (Fisher Scientific).

[0076] Imaging and Analysis of Spine Density in Golgi-Cox Stained Slices

[0077] Imaging of 2.sup.d or 3.sup.rd order dendritic branches of hippocampal pyramidal neurons of area CA3 and CA1 was done with an Axioplan 2 imaging microscope (Zeiss) using a 63 oil objective and a z-stack thickness of 0.5 m under reflected light. The number of spines was determined per micrometer of dendritic length (in total 100 m) at apical and basal compartments using ImageJ (1.48v, National Instruments of Health, USA). At minimum 4 animals per genotype and 4 neurons per animal were analyzed blinded to genotype and injected virus.

[0078] Data Analysis

[0079] Data of electrophysiological recordings were collected, stored and analyzed with LABVIEW software (National Instruments, Austin, Tex.). The initial slope of fEPSPs elicited by stimulation of the Schaffer collaterals was measured over time, normalized to baseline and plotted as averageSEM. Analysis of the PPF data was performed by calculating the ratio of the slope of the second fEPSP divided by the slope of the first one and multiplied by 100. Data of Golgi-Cox staining were analyzed using GraphPad Prism (Version, 5.01) software. Spine density is expressed as meanSEM. Differences between genotypes were detected with one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test using IBM SPSS Statistics 21.

[0080] Results

[0081] AAV-APPs Injection Mediates Efficient and Long Lasting Neuronal Expression of APPs in the Hippocampus of APP/PS1E9 Mice

[0082] To assess the therapeutic potential of APPs we used AAV-mediated overexpression of APPs in the brain of aged (12 month-old) APP/PS1E9 mice. APP/PS1E9 mice show progressive plaque deposition starting at about 5-6 months of age and highly abundant plaques are observed at 12 months of age (Jankowsky et al, 2004; Xiong et al, 2011). AAV9 vectors expressing either Venus or codon optimized HA-tagged murine APPs (HA-APPs) under the control of the neuronal synapsin promoter (further referred to as AAV-Venus and AAV-APPs, data not shown) were bilaterally injected into the stratum lacunosum moleculare region of the dorsal hippocampus and into the dentate gyrus (data not shown) of 12 month-old male APP/PSAE9 mice.

[0083] To monitor vector-mediated Venus and APPs expression, mice were sacrificed 5 months after injection. Immunohistochemistry using an HA-tag specific antibody revealed widespread expression of HA-APPs not only in the hippocampus, but also in the cortical layers above the injected hippocampus (data not shown). Analysis of serial anteroposterior coronal sections demonstrated widespread HA-APPs immunoreactivity (over 3.5 mm) in the hippocampus from 2.6 mm posterior to +0.9 mm anterior from the injection site (data not shown) and in the adjacent cortex. More detailed analysis showed prominent expression of vector-mediated HA-APPs in the pyramidal cells of the subiculum, in the CA1, CA2 regions and in granular neurons of dentate gyrus (data not shown). Within the CA3 subfield HA-APPs expression was detectable but considerably lower. As APPs expression was driven by the neuron-specific synapsin promotor, HA-APPs expression was restricted to neuronal cells as revealed by double immunostaining against NeuN (data not shown). Consistently, no expression was detectable in microglia (Iba1, data not shown) or in astrocytes (GFAP, data not shown). The AAV-Venus expression pattern was largely similar to that of AAV-APPs.

[0084] Western blot analysis of hippocampal extracts confirmed vector-mediated HA-APPs protein expression in all injected animals. Comparable levels of either HA-APPs or Venus were detected in injected APP/PS1E9 mice or nontransgenic littermates, respectively (data not shown). Altogether we demonstrate that our AAV based approach leads to efficient and long lasting APPs expression in the hippocampus and adjacent cortex.

[0085] AAV-APPs Treatment Rescues the Spatial Memory Impairment of APP/PS1E9 Mice

[0086] To analyze the consequences of AAV-APPs or AAV-Venus injection for spatial learning and memory, mice were tested in the Morris water maze place navigation task (FIG. 1). To this end, transgenic APP/PS1E9 mice (n=8 per group) or nontransgenic littermate controls (n=3-4 per group) were either injected with AAV-Venus or AAV-APPs vectors at 12 months of age and tested 2 months later at 14 months of age. Swim speed was comparable in all groups of animals (FIG. 1A) over the 5 days of training, thus excluding impairments in motor performances. While all 4 groups of mice did show learning, as evidenced by reduced latency to reach the platform over the 5 days of training, we observed a group effect resulting from an overall significantly increased performance in nontransgenic littermates that had received AAV-APPs (FIG. 1B). Injection of AAV-APPs did not, however, improve the performance of APP/PS1E9 mice (FIG. 1B). Similar results were obtained when analyzing the path length to reach the platform (data not shown). During the probe trial that assesses spatial reference memory and was conducted 72 hours after the last trial of training APP/PS1E9 mice injected with AAV-Venus were strongly impaired (FIG. 1C) in comparison to littermate mice injected with AAV-Venus and showed no preference for the trained target quadrant (FIG. 1C; paired t-test: t.sub.7=0.96; p=0.37). Strikingly, APP/PS1E9 mice that had been injected with AAV-APPs showed a clear preference for the trained target quadrant (FIG. 1C; paired t-test: t.sub.7=2.516; p=0.045), that was statistically indistinguishable from the performance of littermate controls (FIG. 1C; p>0.84, 2-way ANOVA followed by Tukey's post-hoc test). Thus, vector mediated APPs expression rescued the spatial memory impairment in aged APP/PS1E9 mice despite established plaque deposition.

[0087] Impaired Synaptic Plasticity and Reduced Spine Density of APP/PS1E9 Mice are Rescued by AAV-APPs Expression

[0088] Having established that AAV-APPs expression restored the spatial memory deficits of APP/PS1E9 mice we evaluated whether these improvements were also reflected at the functional neuronal network level. We analyzed synaptic plasticity which is considered to represent the basis of newly formed declarative memory, 4-5 months after AAV injection at an age of 12-13 months. To this end, we induced long term potentiation (LTP) at the Schaffer collateral to CA1 pathway by theta-burst stimulation (TBS) after baseline recording (data not shown). Consistent with our previous results in noninjected APP/PS1E9 mice (Heneka et al, 2013) AAV-Venus injected APP/PS1E9 mice exhibited significantly lower induction and maintenance of LTP (n=22 slices), as compared to AAV-Venus injected littermate controls (n=22, data not shown). Nontransgenic control slices showed at the stable phase of LTP (t50-80 min after TBS) a potentiation of 178.018.98%, that was significantly reduced to only 148.476.04% in AAV-Venus injected APP/PS1E9 mice (FIG. 2A; p=0.021, 1-way ANOVA followed by Bonferroni's post-hoc test). In contrast, the LTP curve recorded from AAV-APPs injected APP/PS1E9 slices (n=26) closely overlapped with and was statistically indistinguishable (1-Way ANOVA for t50-80, p>1) from that of nontransgenic littermate controls (data not shown). AAV mediated expression of APPs largely ameliorated LTP deficits of APP/PS1E9 mice as evidenced by nearly identical average potentiation at t50-80 in AAV-APPs treated APP/PS1E9 mice (171.486.29%) and littermate controls (178.018.98%) receiving AAV-Venus control virus (FIG. 2A). While basal synaptic transmission was comparable in all groups (FIGS. 2B and C), short-term synaptic plasticity evaluated by paired pulse facilitation (PPF, FIG. 2D) was significantly impaired in APP/PS1E9 mice. Transgenic animals injected with AAV-Venus showed an overall lowered response towards the second stimulus in the PPF paradigm, reaching significance at an inter-stimulus interval (ISI) of 10 ms compared to littermate controls (p=0.03; 1-way ANOVA followed by Bonferroni's post-hoc test). Strikingly, AAV-APPs treatment completely rescued presynaptic functionality in APP/PS1E9 animals, as evidenced by PPF values statistically indistinguishable from littermate controls and significantly different from that of AAV-Venus injected transgenic animals (p(ISI.sub.20ms)=0.047; FIG. 2D).

[0089] Next we evaluated spine density as a correlate of excitatory synapses in the same set of animals as used for electrophysiology. Previous studies had indicated reduced spine density in various AD mouse models, presumably due to A mediated toxic effects (reviewed in (Spires-Jones & Knafo, 2012). Spine density of basal and mid-apical dendritic segments of hippocampal CA1 and CA3 pyramidal cells was assessed using Golgi staining (data not shown). Apical dendrites of CA1 neurons showed comparable spine density between experimental groups, whereas significantly reduced spine density was observed in the basal dendrites of CA1 neurons from APP/PS1E9 mice (n=16 neurons) as compared to littermates controls (n=24 neurons, both treated with AAV-Venus, FIG. 2E). Analysis of CA3 neurons revealed significantly fewer spines in both basal (t-test, p=0.01) and apical (p=0.014) dendritic segments when comparing AAV-Venus expressing APP/PS1E9 mice and nontransgenic littermates controls. Importantly, AAV-APPs overexpression partially restored spine density in CA3 apical segments (n=24) and completely rescued the spine density deficit in basal dendrites of CA3 neurons from APP/PS1E9 mice (p=0.031; FIG. 2F). Together, these data indicate that APPs expression substantially ameliorates both structural and functional synaptic impairments of aged AD model mice.

[0090] AAV-APPs Expression Decreases A Levels and Plaque Deposition in Aged APP/PS1dE9 Mice

[0091] APPs had previously been reported to bind to BACE-1 and thereby reduce A production (Obregon et al, 2012). We therefore evaluated if beneficial effects of AAV-APPs overexpression on synaptic plasticity and cognitive function were associated with reduced amyloidogenic processing of APP. Employing a sensitive electrochemiluminescence ELISA we quantified products of amyloidogenic metabolism (A and -CTF) in the cortex (Cx) and hippocampus (H) of 17 months old APP/PS1E9 mice (n=8/group), 5 months after viral vector injection. No significant difference in -CTF levels were detectable in APP/PS1E9 mice injected with AAV-APPs vector, as compared to mice injected with AAV-Venus control vector (FIG. 3A). In contrast, we observed a significant decrease in soluble A42 (reduced by about 33% vs control, FIG. 3D) in both cortex and hippocampus of APP/PS1E9 mice injected with AAV-APPs vector, as compared to AAV-Venus control injections. Similarly, we found a trend towards decreased amounts of A38 and A40 that did, however, not reach statistical significance (FIG. 3B, C).

[0092] In order to assess the impact of APPs overexpression on amyloid deposition, we used 4G8 immunostaining to quantify the area covered by plaques both in the hippocampus and cortex of 17 months old APP/PS1E9 mice injected with viral vectors (data not shown). Interestingly, AAV-APPs injection (n=8) resulted in a significantly reduced plaque area both in cortex and hippocampus as compared to AAV-Venus injected controls (FIG. 3E). Together, these results indicate that AAV-mediated APPs overexpression moderately reduces both A generation and amyloid plaque load in APP/PS1E9 mice not only in the AAV injected hippocampus but also in distant cortical areas.

[0093] AAV-APPs Induces Microglia Recruitment and Activation in the Vicinity of Amyloid Plaques

[0094] Accumulation of amyloid plaques in APP/PS1E9 mice has previously been shown to be accompanied by microgliosis and astrocytosis notably at advanced stages of plaque pathology (Kamphuis et al, 2012; Prokop et al, 2013). Here we evaluated the expression of GFAP (as an astrocyte-specific marker) and Iba1 (as a microglial marker) by Western blot analysis (FIG. 4A) and IHC (FIG. 4B) in the hippocampus of 17 month old APP/PS1E9 mice treated either with AAV-APPs or control vector. While no significant difference was detectable for the astroglial marker GFAP, AAV-APPs treatment lead to a significant increase in Iba1 expression (about +44%; t-test, p=0.003; FIG. 4A), as compared to AAV-Venus control injections. Staining of brain sections further confirmed these data (data not shown) at the cellular level. We went on and quantified GFAP and Iba1 immunoreactivity around amyloid plaques in the hippocampus. Consistent with Western blot analysis, GFAP immunoreactivity was not affected by AAV-APPs injection (FIG. 4B). In contrast, the reduction of amyloid deposits observed after injection of the AAV-APPs vector in APP/PS1E9 mice was accompanied by a 2.3-fold increase in Iba1 immunoreactivity in the vicinity of plaques (FIG. 4B). Moreover, we observed an altered morphology of microglia in AAV-APPs treated mice characterized by increased ramifications in AAV-APPs versus control vector injected APP/PS1E9 mice (data not shown). Microglia contribute to A clearance and are thought to play a protective role at least during early stages of AD (Prokop et al, 2013). Indeed, plaque associated microglia (from both AAV-APPs and AAV-Venus treated mice) were also engaged in A uptake as evidenced by Iba1/4G8 double staining (data not shown). Recently, genetic variants of TREM2 (Triggering Receptor Expressed on Myeloid cells) have been associated with an increased risk for AD (Guerreiro et al, 2013; Jonsson et al, 2013). Although the precise role of TREM2 for AD pathogenesis and A pathology is still controversial (Jay et al, 2015; Wang et al, 2015) TREM2 expression has been consistently detected in plaque associated Iba1.sup.+ cells in AD model mice (Frank et al, 2008; Jay et al, 2015). Consistent with an increase in plaque associated microglia we detected a significant increase of TREM2 expression (about 60% of control, t-test, p<0.05) by Western blot analysis in hippocampi of APP/PS1E9 mice injected with AAV-APPs versus controls (FIG. 4C, n=8 per group). We also determined the expression of neprilysin (NEP) and insulin-degrading enzyme (IDE) that are proteases produced by microglia that contribute to A clearance (Tang 2008). Expression of NEP was identical in APP/PS1E9 mice injected with AAV-APPs versus control (not shown). However a significant increase (of about +20%, t-test, p<0.001) in IDE expression was observed after AAV-HA-APPs vector injection (FIG. 4C). Together these data suggest that AAV mediated APPs expression induces microglia recruitment, activation and possibly also phagocytic function which may lead to enhanced A and plaque clearance.

[0095] AAV-APPs Injection Induce an Efficient and Durable Expression of Hippocampal Neurons in APP/PS1E9 Mice

[0096] In order to assess the consequences of APPs neuronal overexpression in APP/PS1E9 mice, AAV9-Venus or AAV9-APPs and AAV9-APPs both hemaglutinine tagged (thereafter referred as AAV-Venus, AAV-APPs and AAV-APPs) (data not shown) were bilaterally injected into the stratum lacunosum moleculare and the dentate gyrus regions of the hippocampus of aged APP/PS1E9 mice (12 months) (data not shown). Mice were sacrificed at 17 months of age, (5 months post-injection) to evaluate the expression of both APPs and APPs. Efficient transduction of the hippocampus (especially in the CA1, CA2 and dentate gyrus) was evidenced. The pattern of expression in the hippocampus was similar in AAV-APPs injected mice and diffusion of APPs expression into the peri-hippocampal cortex was observed (data not shown). The APPs expression showed also a nice diffusion from rostral to caudal coordinates in both cortex and hippocampus (data not shown). Further cellular analysis of the CA1 layer confirmed that the synapsin promoter allowed specific and efficient neuronal transduction without any transduction of neither astrocytes nor microglia (data not shown). Levels of expression of APPs-HA and APPs-HA in hippocampus analyzed by western blot were very close and consistent in every single animal injected. AAV-Venus control animals also displayed a similar level of Venus (data not shown).

[0097] AAV-APPs does not Improve Spatial Reference Memory of Aged APP/PS1E9

[0098] We used the Morris water maze to evaluate the spatial reference of the APP/PS1E9 mice. Transgenic mice (AAV-Venus (n=8), AAV-APPs (n=8) or AAV-APPs (n=7)) or littermate controls (AAV-Venus (n=3)) were injected at 12 months old and assessed 2 months later at 14 months of age. During the five days of the training phase (TQ, data not shown), every group of animals showed an efficient learning of the platform position highlighted by a decreased distance to find it over the five days (data not shown). 72 hours after the last training session, the platform was removed in order to assess spatial reference memory evidenced by the distance spent in the TQ (FIG. 5). APP/PS1E9 control mice injected with AAV-Venus showed an impaired memory as compared to AAV-Venus injected littermate mice (Tukey post-hoc test: p=0.04). AAV-APPs injection in transgenic mice improved spatial reference memory as previously shown (p=0.01). In contrast, however, AAV-APPs treatment did rescue spatial reference memory as the time spent in the TQ was equivalent to AAV-Venus mice (p=0.99) and significantly different compared to AAV-APPs mice (p=0.02). The lack of efficiency of AAV-APPs to restore an efficient search strategy is highlighted by the occupancy plots (data not shown). Together, these results demonstrate that, in contrast to AAV-APPs, AAV-APP injection is not able to restore memory deficits of APP/PS1E9.

[0099] AAV-APPs Injection does not Restore Long-Term Potentiation in the Hippocampus of Aged APP/PS1E9 Mice.

[0100] We then evaluated whether the improvements of spatial memory deficits in APP/PS1E9 mice were also reflected at the functional neuronal network level. Up to know the effects of APPs on synaptic plasticity had not been studied, neither in vitro nor in vivo. To analyze synaptic plasticity we induced long-term potentiation (LTP) at the Schaffer collateral to CA1 pathway by theta-burst stimulation (TBS) after baseline recording (FIG. 6). Acute slices of AAV-Venus injected APP/PS1E9 animals exhibited significant lower induction and maintenance of LTP compared to littermate controls indicating a significant impairment of the transgenic mice. Viral expression of APPs restored potentiation after TBS in transgenic animals and resulted in an LTP curve progression comparable to that of LM controls, confirming previous results (FIG. 2). Injection of AAV-APPs vector in APP/PS1E9 mice did also not correct the impairment of the presynaptic compartment as shown by altered PPF at the 10 ms ISI, This parameter was restored after AAV-APPs injection but not with AAV-APPs (data not shown). Together, these data indicate that APPs expression in contrast to APPsa expression fails to ameliorate functional synaptic impairments of aged AD model mice. This in turn indicates a crucial role for the last 16 aminoacids of APPsa (that are lacking in APPsb) as a domain that mediates the rescue effect on memory and synaptic plasticity.

[0101] AAV-APPs does not Activate Microglia In Vivo in Aged APP/PS1E9 Mice

[0102] In order to explain the discrepancy between AAV-APPs and AAV-APPs regarding amyloid plaques degradation, we assessed microglial activation. We first confirmed that APPs is able activate microglia which in turn internalize A and upregulate the amyloid degrading enzyme IDE and the receptor TREM2. In sharp contrast, the activation of microglia evidenced by upregulation of Iba1 in both western blot and immunohistochemistry was not observed after AAV-APPs or AAV-Venus injection (FIG. 7). This result is unexpected and novel as previous in vitro studies indicated that both APPs and APPs may activate microglia in culture.

[0103] This results might indicate that the AAV-APPs effects on soluble A one hand and amyloid plaques the other hand are mediated by two independent mechanisms. Finally, the lack of activation following APPs injection could explain why plaques levels are not altered.

CONCLUSION

[0104] Despite a recent shift of research efforts towards preventive strategies, there is still an urgent lack of an effective treatment of patients with clinically established AD. So far, many therapeutic approaches targeted the secretases processing APP. However, since all secretases act on many different substrates besides APP (Prox et al, 2012; Vassar et al, 2014), these strategies have major drawbacks for clinical application. -secretase is physiologically essential and current clinical trials to develop -secretase inhibitors have been abrogated due to serious side effects, likely resulting from impaired Notch signaling (Doody et al, 2013). Also systemic upregulation of the major -secretase ADAM10 to boost APPs production is problematic, as this may enhance cleavage of substrates implicated in tumorigenesis and in addition of several hundred substrates expressed in neurons (reviewed by Nhan et al, 2015; Prox et al, 2012, Kuhn et al., 2016). Thus, direct overexpression of APPs in the brain may be more promising than pharmacological upregulation of -secretase.

[0105] Here, the inventors explored a gene therapeutic approach and used AAV-based gene transfer to overexpress APPs in the brain of transgenic APP/PS1E9 mice that have been widely used in experimental studies assessing the efficacy of AD therapies. Bigenic APP.sub.SWE/PS1E9 mice express a chimeric mouse/human APP (with Swedish double mutation) and a mutant human PS1 gene (PS1E9) both associated with familial forms of AD. They produce high amounts of huA leading to amyloid deposition starting at 5-6 months and pronounced progression of plaque pathology with age that is associated with impairments in cognitive behavior (Savonenko et al, 2005). Using bilateral injection of AAV-APPs vector particles the inventors achieved highly efficient and widespread expression of APPs throughout the whole hippocamus and also in adjacent cortical areas.

[0106] In this study, a single bilateral injection of AAV-APPs particles was sufficient to mediate long-lasting APPs expression over five months that was well tolerated without apparent adverse effects. This was a crucial prerequisite to study potential therapeutic efficacy of AAV-APPs overexpression. To this end, they used aged (12 month old) APP/PS1E9 mice with preexisting amyloidosis to mimic the situation in AD patients that are usually clinically diagnosed many years after the onset of pathology (Villemagne et al, 2013).

[0107] Taken together, the inventors provide evidence that APPs as a molecule has beneficial effects on cognition, synaptic density, synaptic function and plasticity, microglia activation and reduces both soluble A and insoluble A deposits in the form of plaques.

[0108] Moreover, they show that APPs and not APPs is responsible for the positive rescue effects in an Alzheimer mouse model. They show that APPs but not APPs rescues: a) cognition (MWM) and b) synaptic plasticity as a molecular correlate to synaptic strength. This are totally novel data not reported so far. In addition they also show that APPs activates microglia and recruits microglia towards plaques. This is an unexpected novel in vivo finding, as previously in vitro both APPs and APPs could activate microglia to secrete inflammatory cytokines. The inventors show for the first time that in vivo both molecules have different properties and that APPs could be very useful in AD and Down syndrome treatment strategy.

REFERENCES

[0109] Throughout this application, various references, including United States patents and patent applications, describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference in entirety into the present disclosure. [0110] Ahmed R R, Holler C J, Webb R L, Li F, Beckett T L, Murphy M P (2010) BACE1 and BACE2 enzymatic activities in Alzheimer's disease. J Neurochem 112: 1045-1053 [0111] Austin S A, Combs C K (2008) Mechanisms of Microglial Activation by Amyloid precursor Protein and its Proteolytic Fragments. In Central Nervous System Diseases and Inflammation, Lane T E, Carson M, Bergmann C, Wyss-Coray T (eds) pp 13-32. Springer US [0112] Aydin D, Weyer S W, Muller U C (2012) Functions of the APP gene family in the nervous system: insights from mouse models. Exp Brain Res 217: 423-434 [0113] Bell K F, Zheng L, Fahrenholz F, Cuello A C (2008) ADAM-10 over-expression increases cortical synaptogenesis. Neurobiol Aging 29: 554-565 [0114] Berger A, Lorain S, Josephine C, Desrosiers M, Peccate C, Voit T, Garcia L, Sahel J A, Bemelmans A P (2015) Repair of Rhodopsin mRNA by Spliceosome-Mediated RNA Trans-Splicing: A New Approach for Autosomal Dominant Retinitis Pigmentosa. Mol Ther [0115] Bodles A M, Barger S W (2005) Secreted beta-amyloid precursor protein activates microglia via JNK and p38-MAPK. Neurobiol Aging 26: 9-16 [0116] Borchelt D R, Ratovitski T, van Lare J, Lee M K, Gonzales V, Jenkins N A, Copeland N G, Price D L, Sisodia S S (1997) Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron 19: 939-945 [0117] Bour A, Little S, Dodart J C, Kelche C, Mathis C (2004) A secreted form of the beta-amyloid precursor protein (sAPP695) improves spatial recognition memory in OF1 mice. Neurobiol Learn Mem 81: 27-38 [0118] Caille I, Allinquant B, Dupont E, Bouillot C, Langer A, Muller U, Prochiantz A (2004) Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone. Development 131: 2173-2181 [0119] Casanova F, Carney P R, Sarntinoranont M (2014) Effect of needle insertion speed on tissue injury, stress, and backflow distribution for convection-enhanced delivery in the rat brain. PloS one 9: e94919 [0120] Claasen A M, Guevremont D, Mason-Parker S E, Bourne K, Tate W P, Abraham W C, Williams J M (2009) Secreted amyloid precursor protein-alpha upregulates synaptic protein synthesis by a protein kinase G-dependent mechanism. Neurosci Lett 460: 92-96 [0121] Corrigan F, Vink R, Blumbergs P C, Masters C L, Cappai R, van den Heuvel C (2012) sAPPalpha rescues deficits in amyloid precursor protein knockout mice following focal traumatic brain injury. J Neurochem 122: 208-220 [0122] Cousins S L, Hoey S E, Anne Stephenson F, Perkinton M S (2009) Amyloid precursor protein 695 associates with assembled NR2A- and NR2B-containing NMDA receptors to result in the enhancement of their cell surface delivery. J Neurochem 111: 1501-1513 [0123] Cousins S L, Innocent N, Stephenson F A (2013) Neto1 associates with the NMDA receptor/amyloid precursor protein complex. J Neurochem 126: 554-564 [0124] Dobrowolska J A, Kasten T, Huang Y, Benzinger T L, Sigurdson W, Ovod V, Morris J C, Bateman R J (2014) Diurnal patterns of soluble amyloid precursor protein metabolites in the human central nervous system. PloS one 9: e89998 [0125] Doody R S, Raman R, Farlow M, Iwatsubo T, Vellas B, Joffe S, Kieburtz K, He F, Sun X, Thomas R G et al (2013) A phase 3 trial of semagacestat for treatment of Alzheimer's disease. N Engl J Med 369: 341-350 [0126] Endres K, Fahrenholz F (2012) Regulation of alpha-secretase ADAM10 expression and activity. Exp Brain Res 217: 343-352 [0127] Frank S, Burbach G J, Bonin M, Walter M, Streit W, Bechmann I, Deller T (2008) TREM2 is upregulated in amyloid plaque-associated microglia in aged APP23 transgenic mice. Glia 56: 1438-1447 [0128] Furukawa K, Mattson M P (1998) Secreted amyloid precursor protein alpha selectively suppresses N-methyl-D-aspartate currents in hippocampal neurons: involvement of cyclic GMP. Neuroscience 83: 429-438 [0129] Furukawa K, Sopher B L, Rydel R E, Begley J G, Pham D G, Martin G M, Fox M, Mattson M P (1996) Increased activity-regulating and neuroprotective efficacy of alpha-secretase-derived secreted amyloid precursor protein conferred by a C-terminal heparin-binding domain. J Neurochem 67: 1882-1896 [0130] Goodman Y, Mattson M P (1994) Secreted forms of beta-amyloid precursor protein protect hippocampal neurons against amyloid beta-peptide-induced oxidative injury. Exp Neurol 128: 1-12 [0131] Guerreiro R, Wojtas A, Bras J, Carrasquillo M, Rogaeva E, Majounie E, Cruchaga C, Sassi C, Kauwe J S, Younkin S et al (2013) TREM2 variants in Alzheimer's disease. N Engl J Med 368: 117-127 [0132] Hardy, J. and D. Allsop (1991). Amyloid deposition as the central event in the aetiology of Alzheimer's disease. Trends Pharmacol Sci 12(10): 383-388. [0133] Heneka M T, Kummer M P, Stutz A, Delekate A, Schwartz S, Vieira-Saecker A, Griep A, Axt D, Remus A, Tzeng T C et al (2013) NLRP3 is activated in Alzheimer's disease and contributes to pathology in APP/PS1 mice. Nature 493: 674-678 [0134] Hick M, Herrmann U, Weyer S W, Mallm J P, Tschape J A, Borgers M, Mercken M, Roth F C, Draguhn A, Slomianka L et al (2015) Acute function of secreted amyloid precursor protein fragment APPsalpha in synaptic plasticity. Acta Neuropathol 129: 21-37 [0135] Hick, M., U. Herrmann, S. W. Weyer, J. P. Mallm, J. A. Tschape, M. Borgers, M. Mercken, F. C. Roth, A. Draguhn, L. Slomianka, D. P. Wolfer, M. Korte and U. C. Muller (2015). Acute function of secreted amyloid precursor protein fragment APPsalpha in synaptic plasticity. Acta Neuropathol 129(1): 21-37. [0136] Hoe H S, Lee H K, Pak D T (2012) The upside of APP at synapses. CNS Neurosci Ther 18: 47-56 [0137] Hoey S E, Williams R J, Perkinton M S (2009) Synaptic NMDA receptor activation stimulates alpha-secretase amyloid precursor protein processing and inhibits amyloid-beta production. J Neurosci 29: 4442-4460 [0138] Holsinger R M, McLean C A, Beyreuther K, Masters C L, Evin G (2002) Increased expression of the amyloid precursor beta-secretase in Alzheimer's disease. Annals of neurology 51: 783-786 [0139] Ishida A, Furukawa K, Keller J N, Mattson M P (1997) Secreted form of beta-amyloid precursor protein shifts the frequency dependency for induction of LTD, and enhances LTP in hippocampal slices. Neuroreport 8: 2133-2137 [0140] Jankowsky J L, Fadale D J, Anderson J, Xu G M, Gonzales V, Jenkins N A, Copeland N G, Lee M K, Younkin L H, Wagner S L et al (2004) Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. Hum Mol Genet 13: 159-170 [0141] Jay T R, Miller C M, Cheng P J, Graham L C, Bemiller S, Broihier M L, Xu G, Margevicius D, Karlo J C, Sousa G L et al (2015) TREM2 deficiency eliminates TREM2+ inflammatory macrophages and ameliorates pathology in Alzheimer's disease mouse models. J Exp Med 212: 287-295 [0142] Jonsson T, Stefansson H, Steinberg S, Jonsdottir I, Jonsson P V, Snaedal J, Bjornsson S, Huttenlocher J, Levey A I, Lah J J et al (2013) Variant of TREM2 associated with the risk of Alzheimer's disease. N Engl J Med 368: 107-116 [0143] Jonsson, T., J. K. Atwal, S. Steinberg, J. Snaedal, P. V. Jonsson, S. Bjornsson, H. Stefansson, P. Sulem, D. Gudbjartsson, J. Maloney, K. Hoyte, A. Gustafson, Y. Liu, Y. Lu, T. Bhangale, R. R. Graham, J. Huttenlocher, G. Bjornsdottir, O. A. Andreassen, E. G. Jonsson, A. Palotie, T. W. Behrens, 0. T. Magnusson, A. Kong, U. Thorsteinsdottir, R. J. Watts and K. Stefansson (2012). A mutation in APP protects against Alzheimer's disease and age-related cognitive decline. Nature 488(7409): 96-99. [0144] Kamphuis W, Mamber C, Moeton M, Kooijman L, Sluijs J A, Jansen A H, Verveer M, de Groot L R, Smith V D, Rangarajan S et al (2012) GFAP isoforms in adult mouse brain with a focus on neurogenic astrocytes and reactive astrogliosis in mouse models of Alzheimer's disease. PloS one 7: e42823 [0145] Klevanski M, Saar M, Baumkotter F, Weyer S W, Kins S, Muller U C (2014) Differential role of APP and APLPs for neuromuscular synaptic morphology and function. Mol Cell Neurosci 61C: 201-210 [0146] Kogel D, Deller T, Behl C (2012) Roles of amyloid precursor protein family members in neuroprotection, stress signaling and aging. Exp Brain Res 217: 471-479 [0147] Kuhn P H, Colombo A V, Schusser B, Dreymueller D, Wetzel S, Schepers U, Herber J, Ludwig A, Kremmer E, Montag D, Mller U, Schweizer M, Saftig P, Brase S, Lichtenthaler S F (2016) Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function. eLife: 2016; available online DOI:10.7554/eLife.12748 [0148] Lannfelt L, Basun H, Wahlund L O, Rowe B A, Wagner S L (1995) Decreased alpha-secretase-cleaved amyloid precursor protein as a diagnostic marker for Alzheimer's disease. Nat Med 1: 829-832 [0149] Lassek M, Weingarten J, Einsfelder U, Brendel P, Muller U, Volknandt W (2013) Amyloid precursor proteins are constituents of the presynaptic active zone. J Neurochem 127: 48-56 [0150] Lee K J, Moussa C E, Lee Y, Sung Y, Howell B W, Turner R S, Pak D T, Hoe H S (2010) Beta amyloid-independent role of amyloid precursor protein in generation and maintenance of dendritic spines. Neuroscience 169: 344-356 [0151] Leissring M A, Farris W, Chang A Y, Walsh D M, Wu X, Sun X, Frosch M P, Selkoe D J (2003) Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron 40: 1087-1093 [0152] Leyssen M, Ayaz D, Hebert S S, Reeve S, De Strooper B, Hassan B A (2005) Amyloid precursor protein promotes post-developmental neurite arborization in the Drosophila brain. EMBO J 24: 2944-2955 [0153] Lichtenthaler S F, Haass C, Steiner H (2011) Regulated intramembrane proteolysislessons from amyloid precursor protein processing. J Neurochem 117: 779-796 [0154] Lu B, Nagappan G, Guan X, Nathan P J, Wren P (2013) BDNF-based synaptic repair as a disease-modifying strategy for neurodegenerative diseases. Nat Rev Neurosci 14: 401-416 [0155] Marcello E, Gardoni F, Mauceri D, Romorini S, Jeromin A, Epis R, Borroni B, Cattabeni F, Sala C, Padovani A et al (2007) Synapse-associated protein-97 mediates alpha-secretase ADAM10 trafficking and promotes its activity. J Neurosci 27: 1682-1691 [0156] Meziane H, Dodart J C, Mathis C, Little S, Clemens J, Paul S M, Ungerer A (1998) Memory-enhancing effects of secreted forms of the beta-amyloid precursor protein in normal and amnestic mice. Proc Natl Acad Sci USA 95: 12683-12688 [0157] Mileusnic R, Lancashire C L, Rose S P (2004) The peptide sequence Arg-Glu-Arg, present in the amyloid precursor protein, protects against memory loss caused by A beta and acts as a cognitive enhancer. Eur J Neurosci 19: 1933-1938 [0158] Milosch N, Tanriover G, Kundu A, Rami A, Francois J C, Baumkotter F, Weyer S W, Samanta A, Jaschke A, Brod F et al (2014) Holo-APP and G-protein-mediated signaling are required for sAPPalpha-induced activation of the Akt survival pathway. Cell Death Dis 5: e1391 [0159] Mucke L, Abraham C R, Masliah E (1996) Neurotrophic and neuroprotective effects of hAPP in transgenic mice. Ann N Y Acad Sci 777: 82-88 [0160] Murakami N, Yamaki T, Iwamoto Y, Sakakibara T, Kobori N, Fushiki S, Ueda S (1998) Experimental brain injury induces expression of amyloid precursor protein, which may be related to neuronal loss in the hippocampus. J Neurotrauma 15: 993-1003 [0161] Nhan H S, Chiang K, Koo E H (2015) The multifaceted nature of amyloid precursor protein and its proteolytic fragments: friends and foes. Acta Neuropathol 129: 1-19 [0162] Obregon D, Hou H, Deng J, Giunta B, Tian J, Darlington D, Shahaduzzaman M, Zhu Y, Mori T, Mattson M P et al (2012) Soluble amyloid precursor protein-alpha modulates beta-secretase activity and amyloid-beta generation. Nat Commun 3: 777 [0163] Prokop S, Miller K R, Heppner F L (2013) Microglia actions in Alzheimer's disease. Acta Neuropathol 126: 461-477 [0164] Prox J, Rittger A, Saftig P (2012) Physiological functions of the amyloid precursor protein secretases ADAM10, BACE1, and Presenilin. Exp Brain Res 217: 331-341 [0165] Ramirez M J, Heslop K E, Francis P T, Rattray M (2001) Expression of amyloid precursor protein, tau and presenilin RNAs in rat hippocampus following deafferentation lesions. Brain Res 907: 222-232 [0166] Ring S (2007) Phenotypic analysis to define physiolocically essential functional domains of APP family proteins. In Institut fr Phramazie and molekulare BiotechnologieFunktionelle Genomik. Heidelberg: Universitat Heidelberg [0167] Ring S, Weyer S W, Kilian S B, Waldron E, Pietrzik C U, Filippov M A, Herms J, Buchholz C, Eckman C B, Korte M et al (2007) The secreted beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice. J Neurosci 27: 7817-7826 [0168] Roch J M, Masliah E, Roch-Levecq A C, Sundsmo M P, Otero D A, Veinbergs I, Saitoh T (1994) Increase of synaptic density and memory retention by a peptide representing the trophic domain of the amyloid beta/A4 protein precursor. Proc Natl Acad Sci USA 91: 7450-7454 [0169] Savonenko A, Xu G M, Melnikova T, Morton J L, Gonzales V, Wong M P, Price D L, Tang F, Markowska A L, Borchelt D R (2005) Episodic-like memory deficits in the APPswe/PS1dE9 mouse model of Alzheimer's disease: relationships to beta-amyloid deposition and neurotransmitter abnormalities. Neurobiol Dis 18: 602-617 [0170] Selkoe D J (2002) Alzheimer's disease is a synaptic failure. Science 298: 789-791 [0171] Selkoe, D. J. (2001). Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 81(2): 741-766. [0172] Shankar G M, Bloodgood B L, Townsend M, Walsh D M, Selkoe D J, Sabatini B L (2007) Natural oligomers of the Alzheimer amyloid-beta protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway. J Neurosci 27: 2866-2875 [0173] Shankar G M, Li S, Mehta T H, Garcia-Munoz A, Shepardson N E, Smith I, Brett F M, Farrell M A, Rowan M J, Lemere C A et al (2008) Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med 14: 837-842 [0174] Spires-Jones T, Knafo S (2012) Spines, plasticity, and cognition in Alzheimer's model mice. Neural Plast 2012: 319836 [0175] Suh J, Choi S H, Romano D M, Gannon M A, Lesinski A N, Kim D Y, Tanzi R E (2013) ADAM10 missense mutations potentiate beta-amyloid accumulation by impairing prodomain chaperone function. Neuron 80: 385-401 [0176] Tang W, Ehrlich I, Wolff S B, Michalski A M, Wolfl S, Hasan M T, Luthi A, Sprengel R (2009) Faithful expression of multiple proteins via 2A-peptide self-processing: a versatile and reliable method for manipulating brain circuits. J Neurosci 29: 8621-8629 [0177] Taylor C J, Ireland D R, Ballagh I, Bourne K, Marechal N M, Turner P R, Bilkey D K, Tate W P, Abraham W C (2008) Endogenous secreted amyloid precursor protein-alpha regulates hippocampal NMDA receptor function, long-term potentiation and spatial memory. Neurobiol Dis 31: 250-260 [0178] Terry R D, Masliah E, Salmon D P, Butters N, DeTeresa R, Hill R, Hansen L A, Katzman R (1991) Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment. Annals of neurology 30: 572-580 [0179] Thornton E, Vink R, Blumbergs P C, Van Den Heuvel C (2006) Soluble amyloid precursor protein alpha reduces neuronal injury and improves functional outcome following diffuse traumatic brain injury in rats. Brain Res 1094: 38-46 [0180] Van den Heuvel C, Blumbergs P C, Finnie J W, Manavis J, Jones N R, Reilly P L, Pereira R A (1999) Upregulation of amyloid precursor protein messenger RNA in response to traumatic brain injury: an ovine head impact model. Exp Neurol 159: 441-450 [0181] Vassar R, Kuhn P H, Haass C, Kennedy M E, Rajendran L, Wong P C, Lichtenthaler S F (2014) Function, therapeutic potential and cell biology of BACE proteases: current status and future prospects. J Neurochem 130: 4-28 [0182] Villemagne V L, Burnham S, Bourgeat P, Brown B, Ellis K A, Salvado O, Szoeke C, Macaulay S L, Martins R, Maruff P et al (2013) Amyloid beta deposition, neurodegeneration, and cognitive decline in sporadic Alzheimer's disease: a prospective cohort study. Lancet Neurol 12: 357-367 [0183] Wang Y, Cella M, Mallinson K, Ulrich J D, Young K L, Robinette M L, Gilfillan S, Krishnan G M, Sudhakar S, Zinselmeyer B H et al (2015) TREM2 Lipid Sensing Sustains the Microglial Response in an Alzheimer's Disease Model. Cell 160: 1061-1071 [0184] Weyer S W, Klevanski M, Delekate A, Voikar V, Aydin D, Hick M, Filippov M, Drost N, Schaller K L, Saar M et al (2011) APP and APLP2 are essential at PNS and CNS synapses for transmission, spatial learning and LTP. EMBO J 30: 2266-2280 [0185] Weyer S W, Zagrebelsky M, Herrmann U, Hick M, Ganss L, Gobbert J, Gruber M, Altmann C, Korte M, Deller T et al (2014) Comparative analysis of single and combined APP/APLP knockouts reveals reduced spine density in APP-KO mice that is prevented by APPsalpha expression. Acta Neuropathol Commun 2: 36 [0186] Wilhelm B G, Mandad S, Truckenbrodt S, Krohnert K, Schafer C, Rammner B, Koo S J, Classen G A, Krauss M, Haucke V et al (2014) Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344: 1023-1028 [0187] Wolfer D P, Stagljar-Bozicevic M, Errington M L, Lipp H P (1998) Spatial Memory and Learning in Transgenic Mice: Fact or Artifact? News Physiol Sci 13: 118-123 [0188] Xiong H, Callaghan D, Wodzinska J, Xu J, Premyslova M, Liu Q Y, Connelly J, Zhang W (2011) Biochemical and behavioral characterization of the double transgenic mouse model (APPswe/PS1dE9) of Alzheimer's disease. Neurosci Bull 27: 221-232 [0189] Yang L, Wang Z, Wang B, Justice N J, Zheng H (2009) Amyloid precursor protein regulates Cav1.2 L-type calcium channel levels and function to influence GABAergic short-term plasticity. J Neurosci 29: 15660-15668