pharmaceutics Article Cognitive Impairment in the 3xTg-AD Mouse Model of Alzheimer’s Disease is Affected by Aβ-ImmunoTherapy and Cognitive Stimulation Alejandro R. Roda 1,† , Gisela Esquerda-Canals 1,2,† , Joaquim Martí-Clúa 2 and Sandra Villegas 1, * 1 Protein Folding and Stability Group, Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain; Alejandro.ramos@uab.cat (A.R.R.); Gisela.esquerda@uab.cat (G.E.-C.) 2 Departament de Biologia Cel·lular, de Fisiologia i d’Immunologia, Unidad de Citologia i d’Histologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain; Joaquim.marti.clua@uab.cat * Correspondence: sandra.villegas@uab.cat; Tel.: +34-935-814-258; Fax: +34-935-811-264 † These authors contributed equally to this work.  Received: 11 August 2020; Accepted: 30 September 2020; Published: 2 October 2020  Abstract: Clinical symptoms of Alzheimer’s Disease (AD) include behavioral alterations and cognitive impairment. These functional phenotypes early occur in triple-transgenic (3xTg-AD) mice. Specifically, behavioral alterations are first detected when mice are at around 2.5 months old and cognitive impairment in between 3- and 5-month-old mice. In this work, the effect of chronic Aβ-immunotherapy on behavioral and cognitive abilities was tested by monthly administering the antibody fragment scFv-h3D6 to 3xTg-AD female mice from 5 to 9 months of age. An untreated group was used as a reference, as well as to attain some information on the effect of training during the longitudinal study. Behavioral and psychological symptoms of dementia (BPSD)-like symptoms were already evident in 5-month-old mice, in the form of neophobia and anxious-like behavior. The exploratory activity decreased over the longitudinal study, not only for 3xTgAD mice but also for the corresponding non-transgenic mice (NTg). Learning abilities of 3xTg-AD mice were not seriously compromised but an impairment in long-term spatial memory was evident at 5 months of age. Interestingly, scFv-h3D6-treatment affected the cognitive impairment displayed by 5-month-old 3xTg-AD mice. It is worth noting that training also reduced cognitive impairment of 3xTg-AD mice over the longitudinal study, suggesting that to properly quantify the isolated therapeutic potential of any drug on cognition using this model it is convenient to perform a prompt, age-matched study rather than a longitudinal study. In addition, a combination of both training and Aβ-immunotherapy could constitute a possible approach to treat Alzheimer’s disease. Keywords: Aβ; tau; immunotherapy; Alzheimer; scFv; 3xTg-AD; training 1. Introduction Alzheimer’s disease (AD) is the most prevalent cause of dementia worldwide [1]. Accumulation of the amyloid-β (Aβ) peptide in the neural tissue, either because of its overproduction or impaired clearance [2], is considered a key factor in the progression of this neurodegenerative disorder [3,4]. Furthermore, other age-related, protective or disease-promoting factors have been shown to be involved in the disease progression [5]. Clinical symptoms include both cognitive and behavioral alterations. The first consist mainly of memory loss and learning deficiencies, as well as impairments in other cognitive abilities interfering with Pharmaceutics 2020, 12, 944; doi:10.3390/pharmaceutics12100944 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 944 2 of 21 mood, reason, judgment, and language [6,7]. The second comprises a wide range of neuropsychiatric symptoms clustered in psychotic (as delusion, hallucinations, and aberrant motor activity) and emotional symptoms (as agitation, dysphoria, anxiety, irritability, and apathy), and which are commonly referred to as behavioral and psychological symptoms of dementia (BPSD) [8–10]. The modeling of human AD in animals is being quite a challenging process. Neuropathological hallmarks were rather easy to reproduce in mice by introducing those mutations found in the familial form of the same, or related, diseases [11,12]. However, due to the heterogeneity of clinical symptoms [13] and the complexity of its evaluation [14–16], mimicking the cognitive and behavioral AD-phenotypes has become a rather difficult goal [17,18]. An overview of the phenotypes achieved in several mice models for AD is detailed by Webster et al. [19]. In brief, cognitive decline is generally detected early as impairments in spatial working memory [20], followed by disturbances in associative learning and reference memory [21,22], and eventually concluding with deficits in recognition memory [20,23,24]. Regarding emotional alterations, although delusion or hallucinations are human symptoms rather difficult to identify in mice, other disturbances such as neophobia, exploratory impairment, or anxiety have been characterized [25]. Despite the first conception about the late-onset of BPSD, these symptoms are currently demonstrated to already appear in the prodromal phases of AD [10]. The triple-transgenic mouse model of AD (3xTg-AD) reproduces human amyloid and tau pathologies through similar regional and temporal patterns [26,27]. In this model, cognitive impairment starts at young ages. Associative learning deficits are first detected at around 3–5 months of age, impairments in spatial working memory in the Morris water maze (MWM) paradigm at 6 months, deficits in recognition memory at around 9–11 months, and reference memory impairment in the Barnes maze task at 12 months [19]. In addition, behavioral alterations in 3xTg-AD mice occur at early ages. Alterations in exploratory activity are first detected in the open-field test (OFT) at 2.5 months of age (and at older ages in the corner test, CT), lower habituation to the novelty and uninhibited behavior at 6 months, and neophobia at 12 months, whereas increased emotionality is observed in most of the tests at the adulthood and elderly stages [25]. Taking into account the amyloid cascade hypothesis [28], enhancing Aβ clearance has become the main pharmacological approach tested for AD. Among different monoclonal antibodies (mAbs) that have been developed, bapineuzumab was the first one to reach phase III [29]. However, clinical trials were discontinued because of the occurrence of amyloid-related imaging abnormalities (ARIAs) and the absence of clinical benefit [30]. The latter has been attributed to the fact that the treatment started when the pathology was at an advanced stage. Because the occurrence of ARIAs is related to an immune over-activation via the Fc-receptor, the use of single-chain variable fragments (scFv), which lack the Fc fragment, has emerged as an alternative to immunization with full-length mAbs. In addition, we have learned from clinical trials that treatments for AD must be started at the prodromal phase, when the symptoms are not yet evident. The anti-Aβ scFv-h3D6, derived from bapineuzumab, has been effective in the prevention of Aβ-induced toxicity in SH-SY5Y neuroblastoma cell-cultures [31] and in ameliorating AD histologically hallmarks, without eliciting any detectable inflammatory response in young 3xTgAD mice [32–35]. Interestingly, a longitudinal study using magnetic resonance imaging and spectroscopy showed the efficacy of scFv-h3D6 treatment in preventing loss of brain volume occurring in 3xTg-AD mice [36]. However, cognitive improvement through a chronic treatment has not ever been assessed. Longitudinal studies are relevant for defining the progress of the disease, in both animal models and humans [37]. Additionally, the monitoring of biomarkers by biochemical analyses, image-based techniques, and/or functional and cognitive testing not only enables the further definition of the chronological evolution of the pathology but it is also crucial for early diagnosis and for evaluating therapies in both clinical and preclinical research [4,38]. In the present work, behavioral and cognitive impairments in 3xTg-AD female mice were longitudinally assessed, from 5 (young adulthood) to 9 (middle adulthood) months of age, and compared to the non-pathological ageing in the corresponding non-transgenic age and gender-matched mice (NTg, Pharmaceutics 2020, 12, 944 3 of 21 B6129SF2). In addition, the effects on cognitive functions after chronic administration of scFv-h3D6 to 3xTg-AD mice were tested. Interestingly, a positive effect of cognitive stimulation derived from the longitudinal training, which consisted in repeated MWM testing, was observed, not only in 3xTg-AD mice but also in NTg mice. Indeed, this result concurs with previous studies showing that periodic cognitive enrichment throughout aging improves memory performance in 3xTg-AD mice [39]. 2. Material and Methods 2.1. ScFv-h3D6 Production ScFv-h3D6 was recombinantly expressed in Escherichia coli Origami 2 (DE3)/pETtrx-1a and purified as previously described [31]. Briefly, after refolding by dilution, the Trx-tag was removed from the precursor construct by TEV (Tobacco Etch Virus) proteolysis followed by Immobilized Metal Affinity Chromatography (IMAC), and then, the scFv-h3D6 was purified by CEX cation-exchange chromatography ((CEX) Resource S column). Lipopolysaccharides were detached from the protein by using Detoxi-Gel Endotoxin Removing columns (ThermoFisher Scientific, Waltham, MA, USA). 2.2. Animals The triple-transgenic mouse model of Alzheimer’s disease (3xTg-AD) was engineered at the University of California, Irvine, by introducing APPSwe and MAPTP301L transgenes into a PS1M146V homozygous knock-in single-cell embryo [26]. Female mice were selected because they conserve the phenotype as originally described [40] and exhibit greater Aβ burden and larger behavioral deficits than age-matched male mice [41]. Animals used in the present work belong to the 3xTg-AD colony, and the corresponding non-transgenic (NTg) mice (B6129SF2), stablished by our group in the Animal Facility (Servei d’Estabulari) at the Universitat Autònoma de Barcelona (UAB). Founder animals were provided by The Jackson Laboratory, Bar Harbor, ME, USA. Animals were maintained under standard laboratory conditions (temperature of 22 ± 2 ◦ C and relative humidity of 55 ± 5%, a 12 h light:dark cycle starting at 08:00 a.m., food and water provided ad libitum). All the experiments were approved by the UAB Animal Research Committee and the Government of Catalonia (CEEAH 0661) and performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. 2.3. Experimental Design The experimental design is schematized in Figure 1. Twenty 5-month-old 3xTg-AD female mice and 10 gender- and age-matched NTg mice were used in this study. The 3xTg-AD mice were randomly distributed into 2 groups, PBS-treated 3xTg-AD mice and scFv-h3D6-treated 3xTg-AD mice (n = 10 each group). NTg mice (n = 10) were PBS-treated to obtain references for non-pathological condition and natural aging. Once a month, starting at 5 months of age, mice were intraperitoneally administered with 100 µg of scFv-h3D6 diluted in 200 µL of PBS or with the vehicle (PBS). Behavioral and cognitive testing was performed at 5, 7, and 9 months of age and started 24 h after the corresponding administration (see Figure 1). Two PBS-treated 3xTg-AD mice died at 8 months of age and, therefore, n = 8 was used for this group at 9 months of age. All the experiments and analyses were carried out in a blind fashion. Statistical analyses were performed using Graphpad Prism v6 software. Results are expressed as means ± SD. To check sample normality, the D’Agostino Pearson omnibus K2 test was performed. The effects of the factors group (NTg, PBS-treated 3xTg-AD or scFv-h3D6-treated 3xTg-AD) and time (t) were analyzed with repeated measures (for time factor) two-way ANOVA. Tukey’s multiple comparisons test was performed to compare groups between one another. To evaluate differences among all groups in immunohistochemical analysis, non-repeated measures one-way ANOVA with Tukey’s multiple comparisons test was used. A p-value ≤ 0.05 was considered statistically significant. A p-value over 0.05 and minor or equal to 0.1 (0.05 < p ≤ 0.1) was considered as a low, but not significant Pharmaceutics 2020, 12, 944 4 of 21 value. These low values were considered since the real relevance of p values is being questioned by Pharmaceutics 2020, 12, x several researchers [42]. 4 of 22 Figure Figure1.1.Experimental ExperimentalDesign. Design.(A)(A)Mice Micewere weredivided dividedinto into33experimental experimentalgroups groups(n(n==1010each eachgroup): group): PBS-treated PBS-treated Non-Transgenic Non-Transgenic micemice (NTg); (NTg);PBS-treated PBS-treated3xTg-AD 3xTg-ADmice,mice,and and scFv-h3D6 scFv-h3D6 treated treated 3xTg- 3xTg-AD AD mice. mice. (B) (B) OnceOnce a month, a month, starting starting at 5 at 5 months months of age, of age, micemice werewere intraperitoneally intraperitoneally administered administered with with the treatment the treatment (100ofμg (100 µg of h3D6) scFv scFv h3D6) or with or the with the vehicle vehicle (PBS). (PBS). (C) Behavioral (C) Behavioral and cognitive and cognitive testing testing was longitudinally was longitudinally assessedassessed at 95,months at 5, 7, and 7, and of9 age months of age 24 h after the24 h after the corresponding corresponding administration. administration. The sorting of theThe sorting tests of the tests based was performed was performed based on the degree of on thein stress degree of stress each test, with in theeach mosttest, with stressful the onemost at thestressful end. Theone at the corner end. test (CT)The andcorner test (CT) open-field test and (OFT)open-field test out were carried (OFT) on were day 1 carried and theout on Morris day 1 and water mazethe(MWM) Morris onwater daysmaze 3–14(MWM) (3–8 MWM, on days 9–143–14 (3–8 MWM, reversal MWM).9–14 reversal MWM). 2.4.Sample 2.4. SampleCollection Collectionand andProcessing Processing Animals were Animals were anesthetized anesthetized using using 1%1%isoflurane isoflurane (Esteve) (Esteve) (Barcelona, (Barcelona, Spain) Spain) and andeuthanized euthanized by by decapitation [43]. decapitation [43]. Brains Brainswere wereimmediately immediatelyremoved removedfromfromthe theskulls, skulls,rinsed rinsedinincold coldPBS, PBS,weighed, weighed, and dissected on ice. Brains were rapidly fixed by immersion in 4% paraformaldehyde (PFA) and dissected on ice. Brains were rapidly fixed by immersion in 4% paraformaldehyde (PFA) diluted diluted in PBS for 48 h. Samples were embedded in paraffin following common procedures, serially sectioned in PBS for 48 h. Samples were embedded in paraffin following common procedures, serially sectioned in the coronal plane in the (10-µm coronalthick), plane and mounted (10-µm thick),onand SuperfrostTM mounted on Plus microscope slides SuperfrostTM (Thermo Fisher Plus microscope Scientific). slides (Thermo Fisher Scientific). 2.5. Immunohistochemistry 2.5. Immunohistochemistry Sections were deparaffinized by immersion in xylene and rehydrated in serial dilutions of alcohol (n = 8, each group, to avoid unbalanced groups). Endogenous peroxidase activity was blocked by Sections were deparaffinized by immersion in xylene and rehydrated in serial dilutions of immersion in 3% H2 O2 in pure methanol for 10 min. Antigen retrieval was achieved by immersion alcohol (n = 8, each group, to avoid unbalanced groups). Endogenous peroxidase activity was blocked in 0.01M citrate buffer (pH 6.0) supplemented with 0.1% Tween-20 (Sigma-Aldrich Química SL, by immersion in 3% H2O2 in pure methanol for 10 min. Antigen retrieval was achieved by immersion Saint Louis, MO, USA) at 96 ◦ C for 20 min. Then, tissues were cooled at room temperature (RT) in in 0.01M citrate buffer (pH 6.0) supplemented with 0.1% Tween-20 (Sigma-Aldrich Química SL, Saint PBS-T (PBS (pH 7.6), 0.1% Tween-20) and incubated in blocking buffer (5% normal goat serum (NGS, Louis, MO, USA) at 96 °C for 20 min. Then, tissues were cooled at room temperature (RT) in PBS-T Sigma-Aldrich Química SL) and 5% bovine serum albumin (BSA, Sigma-Aldrich Química SL) in PBS-T (PBS (pH 7.6), 0.1% Tween-20) and incubated in blocking buffer (5%◦normal goat serum (NGS, Sigma- to avoid non-specific binding. Slides were incubated overnight at 4 C with the corresponding primary Aldrich Química SL) and 5% bovine serum albumin (BSA, Sigma-Aldrich Química SL) in PBS-T to antibody (mouse anti-Aβ 6E10 mAb, 1:200, Covance Signet, ref. SIG-39320-200, lot. D13BF00601; or avoid non-specific binding. Slides were incubated overnight at 4 °C with the corresponding primary mouse monoclonal anti-human tau mAb, HT7, 1:100 Thermo Scientific, ref. MN1000, lot. TA2494341). antibody (mouse anti-Aβ 6E10 mAb, 1:200, Covance Signet, ref. SIG-39320-200, lot. D13BF00601; or After incubation with the secondary antibody and extravidin (Mouse ExtrAvidin Peroxidase Staining mouse monoclonal anti-human tau mAb, HT7, 1:100 Thermo Scientific, ref. MN1000, lot. TA2494341). After incubation with the secondary antibody and extravidin (Mouse ExtrAvidin Peroxidase Staining Kit antibody, Sigma-Aldrich Química SL, ref. EXTRA2-1KT), the slides were visualized using 3-3′ Diaminobenzidine (DAB, Sigma-Aldrich Química SL). Pharmaceutics 2020, 12, 944 5 of 21 Kit antibody, Sigma-Aldrich Química SL, ref. EXTRA2-1KT), the slides were visualized using 3-30 Diaminobenzidine (DAB, Sigma-Aldrich Química SL). 2.6. Image Capture and Processing Bright-field images were captured using a Leica DMRB microscope (Leica, Wetzlar, Germany,) and a Leica DFC 500 camera (Leica) with a Leica PL Fluotar lens. The cerebral sections corresponded to the range of coordinates from Figure 43 (interarual 2.34 mm and Bregma −1.46 mm) to Figure 48 (interaural 1.74 mm and Bregma −2.06 mm) of The Mouse Brain in Stereotaxic Coordinates [44]. To quantify the percentage of immunoreactive 6E10 (Aβ) and HT7 (total tau protein) area, 2–3 sections of the hippocampus were used. This was so because previous studies indicate that scFvs penetrates and accumulates mainly in the periventricular areas, such as the hippocampus [45–47]. Moreover, intraneuronal Aβ and tau pathologies in the hippocampus of 3xTg-AD mice have been directly related with impaired cognitive abilities [48,49]. The percentage of immunoreactive area was calculated from binary images using the ImageJ software 1.8.0 (NIH, Bethesda, USA). 2.7. Behavioral and Cognitive Tests Behavioral and cognitive tests started 24 h after treatment administration at 5, 7, and 9 months of age (Figure 1). The temporal sequence of behavioral testing was performed based on the degree of stress in each test, with the most stressful one at the end: the CT and OFT first, and the MWM on days 3–14. All the tests were recorded by a digital USB camera (The Imaging Source, Bremen, Germany) and processed with the ANY-maze software (v. 5.14, Stoelting Europe, Dublin, Ireland). Rearings, self-groomings, and defecations in the CT and OFT were manually quantified. All the material involved in the behavioral testing was thoroughly cleaned with 20% ethanol between trials to ensure the absence of olfactory cues. Corner test. The CT was performed by placing the mice in the middle of a standard home-cage (makrolon, 22 × 22 × 14.5 cm) for 30 s. Horizontal activity (distance traveled, number of visits to the corner, latency of the first corner visit, and time spent in the corner), vertical activity (number of rearings and latency of the first rearing), and other emotional behaviors (immobility time and number of defecations) were recorded for evaluation. Open-field test. The OFT consisted of an arena (white polyethylene, 42 × 42 × 50 cm) where the animals were placed for 15 min [50]. Horizontal activity (total distance traveled, entries to a virtually delimited central area, time spent and distance traveled in the central area and in the periphery, and ratios of central/peripheral time and distance), vertical activity (number of rearings and latency of the first rearing) and other emotional behaviors (number of self-groomings, latency of the first self-grooming, and total time on self-grooming) were recorded for evaluation. Morris water maze test. The Morris water maze (MWM) paradigm assesses the hippocampal- dependent learning and memory by testing the ability to find a hidden platform in a pool full of opaque water stained with non-toxic white tempera (Ø, 120 cm for the pool, Ø, 11 cm for the platform) [51,52]. The paradigm comprised 3 phases: (1) Visible platform. The first day consisted in the visible platform stage, in which the platform was highlighted by a colored flag. Animals performed 4 trials, with an inter-trial period of 10 min, to ensure that they realized of the existence of the platform. (2) Acquisition phase. During the acquisition phase, the platform was hidden ~5 mm under the water surface and cues were added to make spatial orientation possible. This phase consisted of 4 trials per day, for 5 consecutive days, in which the animals should reach the platform upon being oriented by the cues. Mice were manually guided to the platform when they were unable to find it in 60s. Starting points were randomly ordered to ensure different sequences among stages and days. Since each trial was started from a different virtual cardinal point, values obtained from each trial were normalized by the corresponding starting point-platform distances. (3) Probe. The platform was removed for the final probe. This phase consisted of 60s of navigation in absence of the platform and was performed 24 h after the last acquisition stage to properly evaluate long-term memory. Animals started from the most Pharmaceutics 2020, 12, 944 6 of 21 distant point to the usual platform location. The reversal test consisted of the acquisition and the probe phases but replacing the platform in a different location. Once removed from the water, animals were located under a heating lamp (red-light, 100 W) to prevent hypothermia. Mean swimming speed and distance traveled were measured across trials during the acquisition stages. Latency to the platform, platform crossings and path efficiency, as well as time and distance traveled within a virtual zone centered on the former platform location (normalized by areas), were measured in the final probe. 3. Results 3.1. Progression of BPSD-Like Symptoms BPSD-like symptoms were evaluated by the corner test and the open-field test (Figures 2 and 3 and Tables 1 and 2). Both PBS-treated NTg and 3xTg-AD mice exhibited similar performances at 5 months of age in most of the parameters analyzed in the CT (Figure 2, Table 1). However, track plots of the pathway followed by animals showed dramatic differences upon aging (Figure 2A). In both groups, the distance traveled shortened over time (F (2, 52) = 32.32, p ≤ 0.0001) (Figure 2B) concomitantly with a decrease in the number of rearings (F (2, 51) = 7.507, p = 0.0014) (Figure 2C) and in the number of corner visits (F (2, 52) = 63.95, p ≤ 0.0001) (Figure 2D), and an increase in the immobility time (F (2, 52) = 39.36, p ≤ 0.0001) (Figure 2E) and in the latency to the first rearing (F (2, 51) = 35.25, p ≤ 0.0001) (Figure 2F). Altogether this evidences a reduction in the exploratory activity upon aging in both genotypes. However, 3xTg-AD mice exhibited a more pronounced inactive behavior indicating a higher vulnerability to the novelty. This is especially evident since mice are 7-month-old. At 9 months of age and compared to NTg mice, 3xTg-AD mice showed increased immobility time (t = 1.984, p = 0.0064) and latency to the first rearing (t = 3.147, p = 0.0062) and decreased number of rearings (t = 2.354, p = 0.0317) and number of corner visits (t = 2.296, p = 0.0356). These data showed an enhanced anxious-like behavior in the 3xTg-AD female mice that exacerbated over-time. In the OFT (Figure 3, Table 2), 5-month-old 3xTg-AD mice traveled a shorter distance (t = 6.107, p ≤ 0.0001) (Figure 3A,B), displayed a lower number of rearings (t = 7.922, p ≤ 0.0001) (Figure 3C), and an increased latency to the first rearing (t = 3.336, p = 0.0030) (Figure 3D) than the NTg mice. This agrees with the reduced exploratory activity observed in the CT. In consonance, other traits such as the lower number of self-groomings (Figure 3E), even though statistical significance was not reached, and the larger latency to the first one (Table 2) (t = 2.240, p = 0.0388) pointed to the inactivity of the 3xTg-AD mice already at 5 months of age. The representation of the distance travelled during the test shows an evident decrease over time indicative of a habituation process in both groups (F (14, 257) = 7.897, p ≤ 0.0001) (Figure 3F). Similarly to what was observed in the corner test, the exploratory activity was reduced over time. This behavior is shown by the decrease in the total distance travelled (F (2, 54) = 22.96, p ≤ 0.0001) and in the number of rearings (F (2, 52) = 17.73, p ≤ 0.0001) and the increase in the latency to the first rearing (F (2, 52) = 3.016, p = 0.0576). Nevertheless, 9-month-old 3xTg-AD female mice exhibited a much larger decrease in exploratory activity than NTg mice (Figure 3). In summary, BPSD-like symptoms were already evident in 5-month-old 3xTg-AD mice and exacerbated upon aging. Such an early onset and exacerbation of BPSD-like symptoms are triggered by the disease, but these exacerbated symptoms also occur late in the NTg group because of natural aging. Pharmaceutics 2020, 12, 944 7 of 21 Pharmaceutics 2020, 12, x 7 of 22 Figure 2. Behavioral and psychological symptoms of dementia (BPSD)-like symptoms as tested in the Figure corner 2. Behavioral test. and of (A) Track plots psychological the pathwaysymptoms followed by of dementia PBS-treated(BPSD)-like NTg and symptoms as tested in the 3xTg-AD representative corner test. (A) Track plots of the pathway followed by PBS-treated NTg animals. The blue point indicates the animal location at the starting point and the red point and 3xTg-AD representative the final animals. location. The Evolution (B–F) blue pointofindicates NTg (green),the animal 3xTg-AD location (red),atand thescFv-h3D6-treated starting point and3xTg-AD the red point (blue)the final mice location. with (B–F) Evolution age considering (B) theof distance NTg (green), 3xTg-AD travelled (m),(red), and scFv-h3D6-treated (C) number of rearings, (D)3xTg-AD number of (blue) mice corner with(E) visits, age considering immobility time(B) (s),the (F)distance latency to travelled the first(m), (C) (s), rearing number and (G)of latency rearings,to(D) the number of corner first corner visit (s).visits, Values(E) are immobility time by represented (s),means (F) latency and to the bands error first rearing (s), andto(G) correspond SD.latency to theafirst # indicates low,corner visit but not (s). Values significant, are represented p-value (0.05 < p ≤ 0.1)by means and error (Total distance bands correspond travelled (m), 5-mo, NTg to SD. vs. #3xTg-AD-PBS, indicates a low, but not t = 2.022, p =significant, p-value (0.05 0.0583* significant < p ≤ 0.1) differences p ≤(Total 0.05, **distance p ≤ 0.01,travelled (m), 5-mo, *** p ≤ 0.001, **** p ≤NTg vs. 3xTg-AD-PBS, 0.0001. t = 2.022, A refers to NTg mice. p = 0.0583* significant differences p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. A refers to NTg mice. Pharmaceutics 2020, 12, 944 8 of 21 Pharmaceutics 2020, 12, x 8 of 22 Figure 3. BPSD-like symptoms as tested in the open-field test. (A) Track plots of the pathway followed by PBS-treated Figure NTg symptoms 3. BPSD-like and 3xTg-AD representative as tested animals.test. in the open-field The(A) blue point Track indicates plots of the the animalfollowed pathway location at the starting point and the red point the final location. (B–E) Evolution of NTg (green), 3xTg-AD by PBS-treated NTg and 3xTg-AD representative animals. The blue point indicates the animal location (red), and scFv-h3D6-treated 3xTg-AD (blue) mice with age considering B) the distance travelled (m), at the starting point and the red point the final location. (B–E) Evolution of NTg (green), 3xTg-AD (C) number of rearings, (D) latency to the first rearing (s), and (E) number of rearings. (F) Distance (red), and scFv-h3D6-treated 3xTg-AD (blue) mice with age considering B) the distance travelled (m), travelled over-time at different ages. Values are represented by means and error bands correspond to (C) number of rearings, (D) latency to the first rearing (s), and (E) number of rearings. (F) Distance SD. # indicates a low, but not significant, p-value (0.05 < p ≤ 0.1) (Total distance travelled (m), 7-mo, travelled over-time at different ages. Values are represented by means and error bands correspond to NTg vs. 3xTg-AD-PBS, t = 2.022, p = 0.0583). * significant differences p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, SD. # indicates A a low, but not significant, p-value (0.05 < p ≤ 0.1) (Total distance travelled (m), 7-mo, **** p ≤ 0.0001. refers to NTg mice. NTg vs. 3xTg-AD-PBS, t = 2.022, p = 0.0583). * significant differences p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. A refers to NTg mice. Pharmaceutics 2020, 12, 944 9 of 21 Table 1. Corner test data. Values are expressed by means and SD. # indicates a low, but not significant, p-value 0.05 < p ≤ 0.1, * significant differences with p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. A refers to NTg/- mice and B refers to 3xTg/- mice. PBS-Treated ScFv-h3D6-Treated Corner Test NTg 3xTg-AD 3xTg-AD Horizontal activity Distance traveled (m) 5-month-old 1.53 ± 0.14 1.90 ± 0.15 #A 2.29 ± 0.20 **A 7-month-old 0.96 ± 0.06 0.51 ± 0.11 **A 0.56 ± 0.16 *A 9-month-old 0.50 ± 0.13 0.49 ± 0.16 0.49 ± 0.13 Corner visits (n) 5-month-old 11.00 ± 0.68 10.80 ± 0.77 12.10 ± 1.08 7-month-old 6.50 ± 0.37 2.4 ± 0.69 ****A 3.10 ± 0.88 **A 9-month-old 5.20 ± 0.85 2.63 ± 0.58 *A 2.70 ± 0.65 *A Corner latency (s) 5-month-old 3.37 ± 0.63 1.76 ± 0.75 1.15 ± 0.41 **A 7-month-old 1.95 ± 0.43 10.67 ± 3.64 *A 10.67 ± 3.77 *A 9-month-old 1.94 ± 0.67 5.50 ± 2.43 4.88 ± 2.39 Time in the corner (s) 5-month-old 10.56 ± 0.66 10.25 ± 1.12 9.26 ± 1.17 7-month-old 10.52 ± 1.08 5.99 ± 1.78 *A 4.70 ± 1.43 **A 9-month-old 9.08 ± 1.77 8.20 ± 2.93 7.71 ± 1.86 Vertical activity Rearings (n) 5-month-old 4.15 ± 0.49 4.20 ± 0.51 3.70 ± 0.83 7-month-old 4.00 ± 0.45 2.67 ± 1.01 1.00 ± 0.33 ****A 9-month-old 2.80 ± 0.89 0.38 ± 0.24 *A 0.40 ± 0.16 *A 1st rearing latency (s) 5-month-old 0.13 ± 0.02 0.13 ± 0.02 0.12 ±0.03 7-month-old 7.30 ± 1.24 18.00 ± 3.72 *A 23.00 ± 2.53 ****A 9-month-old 12.30 ± 3.18 25.88 ± 2.43 **A 25.40 ± 2.76 **A Other emotional behaviors Immobility time (s) 5-month-old 0.92 ± 0.50 0.65 ± 0.43 0.53 ± 0.35 7-month-old 6.06 ± 1.30 17.18 ± 2.00 ***A 18.54 ± 3.21 **A 9-month-old 12.73 ± 2.75 20.59 ± 2.50 *A 22.06 ± 1.46 **A Defecations (boli) 5-month-old 0.40 ± 0.22 0.30 ± 0.21 0.20 ± 0.13 7-month-old 1.00 ± 0.26 0.56 ± 0.28 0.50 ± 0.22 9-month-old 0.80 ± 0.20 0.25 ± 0.15 #A 0.00 ± 0.00 ***A,#B Table 2. Open-field test data. Values are expressed by means and SD. # indicates a low, but not significant, p-value 0.05 < p ≤ 0.1, * significant differences with p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. A refers to NTg/- mice, B refers to the 3xTg/- mice and C refers to the 3xTg/WT mice. PBS-Treated ScFv-h3D6-Treated Open-Field Test NTg 3xTg-AD 3xTg-AD Horizontal activity Total distance traveled (m) 5-month-old 46.87 ± 3.54 19.36 ± 2.79 ****A 24.30 ± 2.88 ***A 7-month-old 22.49 ± 2.88 12.94 ± 3.75 #A 9.23 ± 1.82 **A 9-month-old 17.54 ± 3.07 10.43 ± 2.05 *A 12.74 ± 2.75 Entries to the center (n) 5-month-old 41.70 ± 5.32 23.50 ± 4.92 *A 26.60 ± 5.73 #A 7-month-old 22.00 ± 2.77 13.20 ± 4.86 12.3 ± 3.81 #A 9-month-old 15.6 ± 2.76 15.25 ± 4.64 14.10 ± 4.29 Pharmaceutics 2020, 12, 944 10 of 21 Table 2. Cont. PBS-Treated ScFv-h3D6-Treated Open-Field Test NTg 3xTg-AD 3xTg-AD Distance in the center (m) 5-month-old 6.61 ± 0.58 3.68 ± 0.73 **A 4.02 ± 0.88 *A 7-month-old 2.85 ± 0.45 2.10 ± 0.82 1.57 ± 0.52 #A 9-month-old 1.93 ± 0.31 1.58 ± 0.45 1.91 ± 0.58 Distance in the periphery (m) 5-month-old 40.26 ± 3.08 15.49 ± 2.20 ****A 20.29 ± 2.34 ****A 7-month-old 19.65 ± 2.52 10.83 ± 3.06 *A 7.66 ± 1.32 **A 9-month-old 15.61 ± 2.82 8.86 ± 1.61 *A 10.84 ± 2.22 Ratio center/periphery (dist) 5-month-old 0.17 ± 0.01 0.24 ± 0.04 0.20 ± 0.03 7-month-old 0.15 ± 0.01 0.23 ± 0.05 0.17 ± 0.03 9-month-old 0.13 ± 0.02 0.15 ± 0.03 0.16 ± 0.03 Time in the center (s) 5-month-old 76.75 ± 9.03 93.07 ± 23.87 75.20 ± 17.30 7-month-old 50.66 ± 7.40 159.5 ± 46.4 *A 52.46 ± 17.91 *B 9-month-old 38.99 ± 7.74 57.98 ± 25.95 68.30 ± 23.00 Time in the periphery (s) 5-month-old 823.3 ± 9.04 723.2 ± 78.2 824.7 ± 17.3 7-month-old 849.3 ± 7.39 740.2 ± 46.34 *A 847.5 ± 17.9 *B 9-month-old 861.01 ± 7.73 841.98 ± 25.94 831.7 ± 23.0 Ratio center/periphery (time) 5-month-old 0.09 ± 0.01 0.15 ± 0.04 0.10 ± 0.02 7-month-old 0.06 ± 0.01 0.27 ± 0.10 #A 0.07 ± 0.02 #B 9-month-old 0.05 ± 0.01 0.08 ± 0.04 0.09 ± 0.03 Vertical activity Rearings (n) 5-month-old 73.20 ± 6.28 14.78 ± 3.22 ****A 14.50 ± 2.25 ****A 7-month-old 35.60 ± 7.26 6.60 ± 1.75 **A 3.80 ± 0.89 ***A 9-month-old 26.50 ± 6.36 3.57 ± 1.09 **A 5.10 ± 1.33 **A 1st rearing latency (s) 5-month-old 18.70 ± 3.10 64.89 ± 13.50 **A 54.40 ± 14.48 ***A 7-month-old 18.20 ± 4.36 360.9 ± 124.8 *A 243.3 ± 57.1 ****A 9-month-old 123.4 ± 87.7 269.1 ± 48.9 314.9 ± 105.1 Other emotional behaviors Immobility time (s) 5-month-old 116.8 ± 38.44 304.95 ± 84.19 #A 215.27 ± 88.67 7-month-old 229.41 ± 80.38 459.06 ± 107.27 310.07 ± 127.14 9-month-old 364.31 ± 108.38 285.86 ± 125.45 525.6 ± 115.84 Self-groomings (n) 5-month-old 4.30 ± 0.34 3.33 ± 0.73 2.90 ± 1.06 7-month-old 3.90 ± 1.14 3.40 ± 0.75 2.40 ± 0.7 9-month-old 5.10 ± 0.74 3.50 ± 0.54 3.10 ± 0.38 *A Self-groomings latency (s) 5-month-old 148.4 ± 39.3 265.4 ± 31.8 *A 343.0 ± 98.8 #A 7-month-old 276.5 ± 71.1 381.7 ± 77.9 402.3 ± 94.1 9-month-old 80.20 ± 12.76 177.4 ± 35.2 *A 201.9 ± 32.0 **A Time on self-grooming (s) 5-month-old 7.20 ± 0.98 5.24 ± 0.95 6.07 ± 2.31 7-month-old 7.35 ± 1.93 6.15 ± 1.63 5.10 ± 1.42 9-month-old 8.77 ± 1.42 7.46 ± 0.94 7.05 ± 0.77 3.2. Effect of Cognitive Stimulation in Spatial Learning and Memory Spatial learning and memory were evaluated by the MWM paradigm (Figure 4). 3xTg-AD mice exhibited a higher swimming speed than NTg mice at all studied ages (t = 3.870 p = 0.0012; Pharmaceutics 2020, 12, 944 11 of 21 t = 3.347 p = 0.0036; t = 5.710 p ≤ 0.0001, respectively) (Figure 4A), supporting the anxious-like behavior observed in the CT and OFT. Pharmaceutics 2020, 12, x 12 of 22 Figure 4. Longitudinally assessed acquisition phase in the MWM paradigm. NTg mice in green, 3xTg-AD Figuremice in red, and assessed 4. Longitudinally scFv-h3D6-treated 3xTg-AD acquisition phase in blue. in the MWM (A) Averaged paradigm. NTg mice swimming in green, 3xTg-speed. (B) Distance AD mice in red, and scFv-h3D6-treated 3xTg-AD in blue. (A) Averaged swimming speed. (B) Distance the traveled in each acquisition stage. (C) Detailed distances traveled in each trial, from traveledvisible-platform preliminary in each acquisition stage.to phase (C) Detailed the distances traveled last acquisition in each are stage. Values trial,represented from the preliminary by the mean visible-platform and error phase to the bands correspond to last SD.acquisition # indicates stage. Values a low, butare notrepresented significant,by p-value and