Non-cell autonomous choroid plexus-derived sAPPα regulates adult hippocampus proliferation and plasticity

While Aβ peptides derived from amyloid precursor protein (APP) play a key role in Alzheimer disease pathogenesis, the extracellular sAPPα soluble ectodomain has been shown to stimulate adult neurogenesis and synaptic plasticity. Elevated expression of App in the choroid plexus has been recently reported, suggesting an important role for APP in cerebrospinal fluid. We conditionally knocked down App expression specifically in the adult mouse choroid plexus either by genetic deletion or shRNA expression, which led to reduced proliferation in both subventricular zone and hippocampus dentate gyrus neurogenic niches. Conversely, proliferation in both niches was increased either by viral expression of App in choroid plexus or by infusion of sAPPα but not sAPPβ in cerebrospinal fluid. To test the hypothesis that favoring the production of Aβ specifically in choroid plexus could negatively affect niche functions, we used AAV5 mediated expression of human mutated APP specifically in the choroid plexus of adult wild type mice. These mice showed reduced niche proliferation and, after one year, exhibited behavioral defects in reversal learning. Consistent with impaired memory, electrophysiological analysis revealed impaired synaptic plasticity as evidenced by deficits in hippocampal LTP. Our findings highlight the unique role played by the choroid plexus in regulating brain function, and suggest that targeting APP in choroid plexus may provide a means to improve hippocampus function and alleviate disease-related burdens.

Introduction neurogenesis and to explore the possibility that the ChPl might constitute an important actor and translational target in healthy and pathological aging. We report that sAPPa produced specifically from the ChPl positively affects proliferation in both neurogenic niches. Conversely, we used serotype 5 adeno-associated virus (AAV5) vectors specifically targeting the ChPl to express human APP bearing the Swedish-Indiana (SwInd) mutations, thus favoring Aβ production, which resulted in reduced proliferation in both niches, caused defects in reversal learning and impaired synaptic plasticity mechanisms in the hippocampus.

Materials and Methods Animals and ethics
C57Bl/6J mice were purchased from Janvier (France) and App flox/flox mice were described previously [22]. All colonies were maintained under a 12:12 light/dark cycle with free access to food and water. In environmental enrichment experiments, App flox/flox mice were placed in cages with running wheels (2 mice per cage) 7 days before surgery and kept for 15 days in these cages before analysis. All animal procedures were carried out in accordance with the guidelines of the European Economic Community (2010/63/UE) and the French National Committee (2013/118). For surgical procedures, animals were anesthetized with xylazine (Rompun 2%, 5 mg/kg) and ketamine (Imalgene 1000, 80 mg/kg) by intraperitoneal injection. This project (no. 00702.01) obtained approval from Ethics committee no. 59 of the French Ministry for Research and Higher Education.

Reversal learning
Spatial memory was assessed by the Morris water maze test [6] in which mice use visual cues to locate an escape platform (9 cm in diameter) in an open circular swimming arena (150 cm in diameter, 40 cm deep) filled with opaque water (Acusol, 20 ± 1 °C). The escape platform was hidden 1 cm below the water surface. Room temperature was kept constant at 24°C and both arena placement and surrounding visual cues were kept fixed during all experiments. Data was acquired by the SMART recording system and tracking software (Panlab). Data processing was automated with NAT (Navigation Analysis Tool), an in-house tool rooted in MATLAB [18]. Mice underwent a two-phase training protocol (Fig. 4e). The first phase consisted of 5 training days, 1 day of rest followed by probe test 1 (initial learning). For the second phase, the platform was moved to a different quadrant, and again training lasted 5 days followed by 1 day of rest before probe test 2 (reversal learning). Training sessions consisted of 4 daily swimming trials (1 h interval between trials) starting randomly from different positions, with each quadrant sampled once per day. For each trial, mice were released at a starting point facing the inner wall and given a maximum of 90 s to locate and climb onto the escape platform. Mice that failed to locate the platform within 90 s were guided to it. In either case, mice were allowed to stay on the platform for 30 s. To assess spatial memory, probe tests were performed 24 h after the last training session by tracking mice for 60 s with the platform absent. Measured parameters during the training phases were the average time taken to reach the platform (i.e., mean escape latency) and the average speed. Measured parameters during probe tests were the mean distance traveled by the mouse before arriving at the platform location, % time spent in the target quadrant, and the number of platform location crossings.

Electrophysiology
Acute transverse hippocampal slices (400 µm) were prepared as previously described [29]. Briefly, mice were culled by cervical dislocation and decapitation. Brains were rapidly removed and placed in chilled (1-4 °C) artificial cerebrospinal fluid (aCSF) composed of (in mM) 119 NaCl, 2.5 KCl, 0.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3 and 11 glucose. After sectioning, hippocampal slices were maintained at room temperature in a storage chamber containing aCSF saturated with 95% O2 and 5% CO2 for at least 1 h before the experiments. Slices were then transferred to a submerged recording chamber mounted on a Scientifica SliceScope Pro 6000 microscope equipped for infrared-differential interference (IR-DIC) microscopy and were perfused with aCSF (2 ml/min) at RT. All experiments were performed in CA1 stratum radiatum region of the hippocampus. Field excitatory postsynaptic potentials (fEPSPs) were recorded with glass pipettes (2-5 MΩ) filled with 1 M NaCl. Postsynaptic responses were evoked by stimulating Schaffer collaterals (0.033 Hz) in CA1 stratum radiatum with aCSF-filled glass pipettes. Input/output relationships of evoked excitatory postsynaptic potentials (EPSPs) were assayed by incrementing stimulation strength (5 to 50 µA, 100 µs). The test-shock used in subsequent experiments was chosen to elicit 50% of the maximal slope. Paired-pulse experiments consisted of 2 identical stimuli with increasing inter-pulse intervals (50 to 250 ms). Paired-pulse ratios were generated by plotting the maximum slope of the second fEPSP as a percentage of the first. Long-term potentiation (LTP) was induced by highfrequency stimulation (HFS: 2 trains of 100 pulses at 100 Hz, 30 s inter-train interval).

PCR and Western blots
Mice were anesthetized for intracardiac perfusion with PBS. Subsequently, choroid plexus, hippocampus, cortex, retina and/or SVZ samples were extracted in ice-cold PBS, frozen on dry ice, and stored at -20 °C. Genomic DNA, total RNA and proteins were recovered by using the Allprep DNA/RNA/Protein Mini Kit (Qiagen 80004). The efficiency of Cre-induced recombination in App flox/flox mice was verified by PCR with primers F, C and D previously described [22]. For quantitative RT-PCR, cDNA was synthetized from 13 ng total RNA with QuantiTect Reverse Transciption Kit (Qiagen 205313). Quantitative PCR reactions were carried out in triplicate with SYBR Green I Master Mix (Roche S-7563) on a LightCycler 480 system (Roche). Expression was calculated by using the 2 −ΔΔCt method with Gapdh as a reference. For Western blot analysis, proteins samples were separated on NuPAGE 4-12% Bis-Tris pre-cast gels (Invitrogen NP0321) at 200 V for 1 h and transferred onto a methanolactivated PVDF membrane at 400 mV for 1 h. Primary antibody anti-APP (22C11 mouse 1:500, Millipore MAB348) was incubated overnight at 4 °C and anti-mouse-HRP-coupled secondary antibody (1:3000, Life Technologies) was incubated for 1.5 h at RT. Signal was detected by SuperSignal West Femto Substrate (Thermo Scientific 34095) with a LAS-4000 gel imager (Fujifilm) and quantified by densitometry with ImageJ.

Statistical Analysis
Morris water maze training data (escape latency and speed) were analyzed with two-way repeated measures ANOVA with Statview 5.0.1 (SAS). Probe test data were analyzed for normality (D'Agostino & Pearson omnibus normality test) and unpaired t test was used for group comparisons with Statview 5.0.1 (SAS). Electrophysiological data were analyzed by ANOVA with Statistica 6.1 and Statview 5.0.1. Histological and biochemical data were analyzed by unpaired t test or ANOVA (as described in Figure legends) with Prism v6 (GraphPad). All data are given as mean ± standard error of the mean (SEM).

App is highly expressed in the adult choroid plexus
Given that ventricular infusion of recombinant sAPPα has been shown to affect SVZ proliferation [4,8], and that the ChPl has been shown to express APP [21], we hypothesized that the ChPl is a potential endogenous source for CSF-borne sAPPα. Indeed, App is one of the most highly expressed ChPl genes [2]. We confirmed high App expression levels in the ChPl by qPCR. In comparison to the hippocampus (that has been shown previously to strongly express APP) and the SVZ, we found higher and elevated levels in ChPl in lateral and 4th ventricles of 4-month-old adult mice (Fig. 1a). Interestingly, Transthyretin and Apoe, two genes involved in App functions [7,40] are also very highly expressed in the ChPl [2,39].

App knock-down in the choroid plexus decreases adult proliferation
In order to knock down App expression locally and specifically in ChPl, we performed Cre-Tat intracerebroventricular (icv) injections in 10-month-old App flox/flox mice (Fig. 1b). Cre-mediated deletion of the App locus and App expression were evaluated 15 days later in various structures including retina, hippocampus, cortex and ChPl ( Fig. 1b-f). The specificity of Cre-Tat targeting selectively the ChPl was confirmed by the absence of recombination in other structures (Fig.  1c), as previously demonstrated for another floxed mouse model [41]. Consequently, only in ChPl do we observe a decrease in App mRNA (by 37 ± 7%, Fig. 1d) and APP protein (by 52 ± 3%, Fig. 1e-f). To evaluate the impact of App knock-down on cell proliferation, ~3-month-old App flox/flox mice were injected with vehicle or Cre-Tat and subsequently implanted with 15-day osmotic minipumps for CSF infusion of either vehicle or sAPPα (Fig. 1g). Compared to vehicle injected/infused controls, animals injected with Cre-Tat and infused with vehicle showed a significant reduction in the number of proliferating cells in the SVZ (Fig. 1h). This decrease was rescued by infusion of sAPPα (Fig. 1h), suggesting that knock-down of sAPPα leads to impaired neurogenesis. In contrast, this experimental paradigm did not alter DG proliferation ( Fig. 1i-j), even under enriched environment conditions with free access to running wheels known to stimulate hippocampal proliferation [46]. In order to reduce App levels more robustly in the ChPl, a viral vector expressing shRNA against the mouse βA42 sequence was injected into the ventricles of ~3-month-old wild type mice (Fig. 2a). As previously reported [48], icv injection of AAV5 results in specific ChPl targeting, and indeed we did not detect expression of co-expressed eGFP marker protein in the parenchyma (Fig. 2b). Eight weeks post-injection, App (mRNA) and APP protein decreased nearly 5-fold in ChPl with no change in hippocampus and cortex levels ( Fig. 2c-e). This decrease led to a significant reduction in the number of proliferating cells in both SVZ and DG ( Fig. 2f-i).

sAPPα gain-of-function in either cerebrospinal fluid or choroid plexus increases adult proliferation
To further investigate the impact of sAPP on adult neurogenesis, either sAPPα or sAPPβ was infused for 7 days in the lateral ventricles of ~3-month-old wild type mice (Fig. 3a). APP can be cleaved either along the non-amyloidogenic pathway to give rise to sAPPα or along the amyloidogenic pathway to produce its sAPPβ counterpart, and the icv injection of the two sAPP forms has been previously shown to have opposite effects on neurogenesis [8]. In our paradigm, infusion of sAPPα but not of sAPPβ increased the number of proliferating cells both in the SVZ and DG of adult mice (Fig. 3b-e). To confirm that CSF and choroid plexus sAPPα gain-offunction are correlated, icv injections of AAV5 expressing mouse App were performed for ChPl-specific over-expression (Fig. 3f). After 8 weeks, the relative App expression was increased by approximately 2-fold (Fig. 3g), resulting in a significant increase in the number of proliferating cells in both the SVZ and DG (Fig. 3h-i).

Choroid plexus expression of APP(SwInd) impairs behavior
Because altering APP expression selectively in the ChPl impacted neurogenic niches, we hypothesized that favoring the production of Aβ specifically in ChPl could negatively affect niche functions. To explore this possibility, we overexpressed a mutated form of human APP (Swedish K670N/M671L and Indiana V17F mutations) specifically in the ChPl of wild type mice at 3 months of age (Fig. 4a). The consequences of this gain-of-function were evaluated 3 and 12 months after injection (when mice were 6 and 15 months old, respectively). We found strong hAPP(SwInd) mRNA expression in ChPl after 3 months which increased more than 3fold by 12 months (Fig. 4b). Strikingly, proliferation was significantly decreased at 3 months post-injection in both SVZ and DG of hAPP(SwInd) expressing mice. However, no difference was seen at 12 months post-injection ( Fig. 4c-d), although this is likely due to the extremely low levels of proliferation also observed in control mice that may preclude further reduction. Indeed, proliferation already dramatically decreases from the time of injection at 3 months ('No injection' in Fig. 4c-d) to 6 months (3 months post-injection) in both niches, and further decreases in the DG at 15 months (12 months post-injection) as previously reported [9]. This decrease in proliferation was not due to amyloid plaque accumulation, as none was observed in the cortex or hippocampus of mice expressing the human APP(SwInd) in the ChPl (data not shown). We also evaluated spatial memory by using the Morris water maze reversal learning paradigm at either 3 months or 12 months post-injection (Fig. 4e). Mice from either group had not been subjected to previous spatial memory tests. During the learning phases, all groups showed significant improvement in latency to find the hidden platform after 5 days of training, but no differences were observed between groups (control and hAPP(SwInd)) in either latency or swimming speed at both ages (Supp. Fig. 1). During probe tests, platforms were removed and performance was evaluated by the time spent in the target platform quadrant, the number of platform area crossings, and the mean total distance taken to reach the platform area (Fig. 4fh). At either 3 months or 12 months post-injection, hAPP(SwInd) mice showed no difference in performance compared to controls during probe test 1 (initial learning). Performance after probe test 2 (reversal learning) was unchanged in 6-month old mice (3 months post-injection) but showed significant differences for all parameters at 15 months (12 months post-injection). The time spent in the trained target quadrant was decreased, as was the number of platform area crossings, while the mean distance to find the platform area was increased, indicating that reversal learning is impaired after long-term ChPl expression of hAPP(SwInd).

Choroid plexus expression of App(SwInd) impairs synaptic plasticity
Compromised reversal learning may be rooted in plasticity-dependent deficits in hippocampaldependent spatial memory. Interestingly, mouse models of AD show a marked decrease in hippocampal CA1 synaptic plasticity in the form of long-term potentiation (LTP) at 4 months of age [20]. We thus assessed synaptic function at Schaffer collateral CA3 to CA1 synapses in the hippocampus of 15-month old mice expressing hAPP(SwInd) in the ChPl after receiving AAV5 icv injections at 3 months of age (Fig. 5a). We found no change in basal synaptic transmission in CA1 as shown by comparable input/output curves (F>1) (Fig. 5b). However, paired-pulse facilitation, a form of short-term plasticity reflecting presynaptic function, is increased in mice expressing mutated APP thus indicating a decrease in pre-synaptic release probability (Fig. 5c). Consistently, induction of LTP by high-frequency stimulation is significantly impaired in AAV5-hAPP(SwInd) injected mice (Fig. 5d), revealing impaired plasticity which corroborates observed deficits in spatial memory (Fig. 4f-h).

Discussion
The present study takes its origin in the growing interest for the ChPl, a structure increasingly recognized for its physiological importance beyond its classical "kidney of the brain" functions as it secretes CSF containing a plethora of trophic compounds [13]. Among these compounds are growth factors, guidance cues and morphogens, some of which gain access to the SVZ and participate in the regulation of neuroblast migration [11,31,39]. sAPPα infused into the CSF has been shown to increase proliferation and progenitor cell numbers in the SVZ [4,8], with binding sites on type C and type A progenitor cells [4]. Given the very high expression of APP by the ChPl, we hypothesized that sAPPα could be secreted by the ChPl into the CSF and participate in adult neurogenesis. By conditionally knocking down App expression specifically in the ChPl through either genetic deletion or shRNA expression, we establish that ChPl APP has neurogenic activity not only in the SVZ but surprisingly also in the DG. Furthermore, this reduced proliferation can be reversed by the infusion of sAPPα into the CSF. Thus, sAPPα secreted by the ChPl can not only gain access to cells within the "superficial" SVZ, which contact lateral ventricles, but also to cells within the "deeper" DG within the parenchyma. Such transport has been reported for transcription factors secreted into the CSF by the ChPl [41]. Together, these results strongly suggest that sAPPα secreted by the ChPl functions as a neurogenic factor, further confirming ChPl as a major actor in regulating adult neurogenesis. The full range of functions of the APP family have yet to be fully identified, but several studies strongly suggest important developmental and physiological roles [28]. Some functions involve cis or trans dimer interactions between transmembrane APP molecules, and others involve heterodimers between transmembrane APP and secreted sAPPα [23]. These interactions modulate important processes including cell adhesion, synapse formation, synapse stabilization and cell survival [28]. Full-length transmembrane APP or sAPPα also interact with a large number of other trans and cis interactors, thus acting as ligands or receptors in a variety of signaling pathways [28]. Some of these properties are attributed to the extracellular C-terminal 16 amino acids of sAPPα, as compared to sAPPβ. For example, acute in vitro or in vivo application of sAPPα but not sAPPβ rescues hippocampal LTP in the adult brain of conditional double knockout mice lacking APP and the related protein APLP2 [17,33]. This C-terminal sequence facilitates synaptic plasticity in the hippocampus through binding to functional nicotinic α7-nAChRs [26,33]. In keeping with previous results [8], we also find that in vivo icv infusion of sAPPα, but not sAPPβ, positively impacts neurogenic niche cell proliferation in the adult mouse brain. While the reason for this functional divergence between sAPPα and sAPPβ for proliferation remains unknown, a shift in APP processing towards the amyloidogenic pathway would clearly impair both synaptic plasticity and neurogenesis. Mouse models developed to analyze the role of specific human APP mutations typically employ mutated genes often expressed throughout the brain and even the body. Our study is unique in that it explores whether expressing mutated APP only in the ChPl of adult mice (3 months old) could affect neurogenic niche proliferation and trigger learning defects. We found that ChPl expression of the human APP(SwInd) mutant, which favors sAPPβ and Aβ peptide formation, decreases proliferation at 3 months after infection (6 months old) with no change at 12 months after infection (15 months old), probably due to the fact that neurogenesis is naturally low at 15 months and cannot be further reduced. However, a reversal learning test showed a significant decrease in the cognitive performance of these mice at 15 months. Accordingly, electrophysiological analysis at 15 months revealed decreased pre-synaptic release probability and impaired synaptic plasticity. No amyloid plaques were observed in these mice, leading us to speculate that the functional outcome of increased sAPPβ and Aβ accumulation is rooted in either synaptic deficits, reduced neurogenesis, and/or neuronal aging [42]. Given that sAPPβ icv infusion does not affect proliferation in the neurogenic niches, it is more likely that the observed deficits are due to increased Aβ (soluble or oligomers). Finally, while we observe no change in proliferation at 15 months, we cannot discount a negative long-term effect of ChPl human APP(SwInd) on the shaping of hippocampal synaptic circuits due to impaired proliferation during aging. Our findings strengthen the unique role played by the ChPl in regulating adult neurogenesis, and it is important to consider the morphological and transcriptomic alterations of the ChPl during normal aging and in late-onset AD that can impact brain homeostasis [35][36][37]39]. These alterations can lead to changes in blood-CSF barrier properties, in ChPl function regulation, as well as in CSF composition and turnover. Furthermore, adult neurogenesis failure clearly plays a role in the development of AD in familial and possibly sporadic AD patients [14,15,25,27,34,45] and could be in part responsible for the decrease in neurogenesis observed in AD patients [25]. From a translational viewpoint, the fact that expressing a mutated APP gene exclusively in the ChPl can alter the cognitive abilities of mice raises the possibility that modifying the expression of APP or targeting APP mutations specifically in the ChPl, a structure accessible from the venous compartment [41], may represent a novel means to alleviate the burden associated with AD.   b An icv injection of AAV5 leads to specific choroid plexus expression. Note the absence of eGFP in brain parenchyma. Scale bar, 80 µm. c Quantitative PCR analysis of App expression in the ChPl, Hc, and Cx after ChPl App knockdown (shRNA-mAβ42 n=4, shRNA-scrambled n=4). d-e Western blot analysis (d) of various brain structures after ChPl App knock-down for quantification (e) of APP protein levels normalized to actin (shRNA-mAβ42 n=4, shRNAscrambled n=4). f-i Analysis of cell proliferation in SVZ (f,g) and DG (h,i) by quantification of Ki67 positive cells after ChPl App knock-down (shRNA-mAβ42 n=7, shRNA-scrambled n=5). Arrows (f) highlight regional differences in SVZ cells. Scale bars, 100 µm. **p<0.01; ***p<0.001; t test; all values, mean ± SEM. ChPl, choroid plexus; Hc, hippocampus; Cx, cortex; SVZ, subventricular zone; DG, dentate gyrus; icv, intracerebroventricular.