Human brain samples
Brain samples were obtained from the Institute of Neuropathology, Bellvitge University Hospital. Brain tissues were obtained from the Institute of Neuropathology HUB-ICO-IDIBELL Biobank and the Hospital Clinic-IDIBAPS Biobank following the guidelines of Spanish legislation on this matter (Real Decreto de Biobancos 1716/2011) and approval of the local ethics committees. For autopsy, one hemisphere was rapidly cut in 1 cm thick coronal sections, and selected areas of the encephalon were dissected, frozen on dry ice, and stored at -80ºC in labelled plastic bags until use. The other hemisphere was fixed by immersion in 4% buffered formalin for three weeks for morphologic examination. The neuropathological study was carried out on twenty-five regions of the cerebral cortex, diencephalon, thalamus, brainstem, and cerebellum. De-waxed paraffin sections were stained with haematoxylin and eosin and Klüver-Barrera, then processed for immunohistochemistry of microglia-specific markers, glial fibrillary acidic protein, β-amyloid, phosphorylated tau, α-synuclein, TDP-43, ubiquitin, and p62. Neuropathological diagnosis of AD was carried out following the Braak and Braak stages [28] adapted to paraffin sections 29]. Cases with concomitant pathologies, including Lewy body diseases, tauopathies (particularly argyrophilic grain disease), vascular diseases, TDP-43pathies, and metabolic syndrome were excluded. Control and disease cases were processed in parallel. The anterior hippocampus area was used for further immunohistochemical studies.
Animal model
Considering that amyloid precursor protein (APP) and Presenilin 1 (PS1) are mutated in 78% and 18% of the familial cases of Alzheimer, respectively, the double transgenic APP/PS1 mouse model has been one of the most commonly used models in the study of Alzheimer's disease [30, 31, 32].
APPswe/PS1dE9 with a C57BL/6 background (double transgenic mice expressing a chimeric mouse/human amyloid precursor protein and a mutant human PS1 with deletion in exon 9) were purchased from the Jackson Laboratory and kept in a specific pathogen-free environment under standard animal housing conditions in a 12hrs dark-light cycle with free access to food and water in the animal house facility of the Universitat de Lleida. Heterozygous males were bred with wild-type C57BL/6 females. Animal procedures were conducted according to ethical guidelines (European Communities Council Directive 86/609/EEC) and approved by the local ethics committee of the Universitat de Lleida. For experiments, tail biopsies were taken from P0 offspring for genotyping by PCR according to the PCR conditions suggested by the Jackson Laboratory. Mice not expressing the transgene were used as controls.
Behavioural test: Morris water maze
The experimental animals were subjected to Morris water maze (MWM) test to assess their cognition/memory associated with hippocampal function [33, 34]. All of the behavioural procedures were conducted at the same time of day, in an isolated room every day during 4 days. The mice were trained to find a 50 cm2 platform hidden under the water surface in a water tank of 150 cm diameter, with four different geometrical forms attached to the four sides of the water tank wall. Four trials per day with start positions close to the four geometrical signs were carried out, and latency in reaching the platform was recorded. Cut-off time to find the platform was 90 seconds, and mice failing to find the platform were placed on it and left there for 15 seconds. Each trial for a single animal was 30 minutes apart from the previous [18].
Stereotaxic injection
8-9 months old female and male APPsw695/PS1dE9 mice and control animals were placed under isoflurane anaesthesia for a couple of minutes, after which they were injected intraperitoneally with the analgesia Rompun (2%) and ketamine (10%) in 0.9% NaCl. Then, mice were fixed securely unto the stereotaxic apparatus, and the skull was carefully exposed. The stereotaxic apparatus was then adjusted to the accurate position after which a metal bar driller was slowly used to make a hole in the skull. 5 µl per DG of the anti proNGF antibody or BSA control solution was gradually injected bilaterally into the dentate gyrus according to the stereotaxic coordinates (Bregma: anteroposterior: 2mm, lateral: +/- 1.6 mm, dorsolateral: 2.5 mm from the skull), at a speed of 1µl/min. After the successful injection, the needle was held in place for another minute before it was retracted slowly at the same rate of 1µl/min speed. After this, the incisions were stitched, and the mice were returned to their home cages for a recovery period of 10 days.
Another set of APP/PS1 transgenic mice were stereotaxically injected with enhanced green fluorescent protein (eGFP) expressing the Moloney leukaemia-derived retroviral vector pCMMP-IRES2eGFP-WPRE. This was carried out using the previously described protocol [3].
Immunohistochemistry
A week after the behavioural study, the mice were anesthetized with Rompun (2%, Bayer)/ (Ketamine 0.1mg/g, Merial) and transcardially perfused with NaCl (9%) followed by 4% PFA in PBS. Brains were dissected and fixed overnight in 4% PFA in PBS, then were washed with PBS 3 times for 10 minutes and subsequently cryoprotected in 30% sucrose (Scharlau) in PBSO/N, embedded in cryoprotective tissue-freezing medium (General Data), and stored at -80°C. 30µM serial coronal sections (cryosections) were then made using a cryostat (Leica CM 3000), and collected in Super frost PlusTM slides (ThermoFisher) and stored at -80°C for further use.
The immunohistochemistry protocol followed in this study varied slightly according to the sets of samples analysed: hippocampal slices obtained from blocking α-proNGF stereotaxically injected mice, GFP retroviral-injected mice, or human samples. The procedures are summarized as follows:
Immunostaining protocol for human samples
Human samples were deparaffined by heating them to 62ºC for 30 min and then substituted in xylol 2 times at 10-minute intervals. A series of ethanol washes were then done in the lamina flow hood at varying concentrations as follows: 2 times in ethanol 100%, 2 times in ethanol 95%, and 2 times in ethanol 60%; the interval between each of these washes was 5 minutes. The resulting samples were rinsed in distilled water for 10 min, followed by 3 10-minute washes in PBS 1X and then 1 10-minute wash in 50mM NH4Cl+PBS1X. Lipofusion was done under black conditions to eliminate autofluorescence by blocking in black Sudan suspension for 25 minutes at room temperature before antigen retrieval (10-minutes wash in preheated Tris-HCl buffer 20 mM pH 9.5 at 97oC for 20 minutes), after which it was cooled down. The next step was to wash it in 0.1% PBST 2 times for 15 minutes and block with 5% donkey serum for 1 hour. Primary antibodies were then added at working concentration 1:100 and kept at 4ºC till the next day (rabbit DCX Santa Cruz; goat anti-p75ECD R and D). On the following day, having spent 28 hours in the cold room, the samples were left at room temperature for 2 hours before proceeding with the 3 washes in PBST 0.1% at 15-minute intervals. The samples were then incubated with secondary antibodies (working dilution 1: 500 in blocking solution) for 2 hours. They were then washed 3 times in PBS Tween 0.1% and a last washing in 0.1% PBS before they were finally mounted with the coverslip using the mounting medium Fluoromount G (Southern Biotech 0100-01).
Immunostaining procedure for mouse cryosections.
The sections were consequently subjected to one-hour blocking and permeabilization process with PBST and 1% donkey serum (5%) (Jackson 017-000-121 IR), then incubated with primary antibodies (in blocking solution 1:100) (rabbit DCX Santa Cruz; goat anti-p75ECD R and D), and subsequently kept at 4° C for 5 days. On the 5th day, the samples were removed from the cold room and kept at room temperature for 2 hours. After that, they were washed three times with PBST 0.1% at 10-minute intervals; and then incubated with secondary antibodies in blocking solution (Working dilution 1:500 plus DAPI for 2 hours at room temperature). Finally, the cells were washed three more times with PBST 0.1% for 15 minutes, after which they were mounted with coverslips using Fluoromount G (Southern Biotech 0100-01).
Immunostaining protocol for GFP retroviral injected hippocampal slices
The mouse hippocampal slices were subjected to immunohistochemistry protocol performed in batches in round-bottomed Eppendorf tubes. The slices were first placed at room temperature for 40 minutes, followed by 4 free-floating washes with PBST (0.25% Triton) of 5 minutes each. After that, they were washed 3 times in PBST (0.1%) at 10 min intervals and then in PBST0.1%+NH4Cl 50 mM for 10 minutes. Antigen retrieval was done using pre-heated citrate buffer and the antigens were then placed in 95ºC boiling water for 30 min. The slices were cooled for about 15minutes and washed 2 times in PBST (0.1% Triton) to remove remains of the fixing solution. Then, they were incubated with the blocking solution following the protocol described in the section below but in batches in the Eppendorf tubes. The antibodies were used in blocking solution at 1:100 (goat anti-p75ECD R and D; rabbit anti-DCX Santa Cruz). After that, they were washed three times with PBST 0.1% at 10 min intervals, and then incubated with secondary antibodies in blocking solution (working dilution 1:500 plus DAPI for 2 hours at room temperature). Finally, the slices were carefully transferred from Eppendorf tubes to super frost glass slides and mounted with coverslips using Fluoromount G (Southern Biotech 0100-01). Fluorescence images were acquired on a confocal microscopy setup (Olympus FV1000, 60X PlanApo using software Fluoview v.4.3) or on an inverted fluorescence microscope (OlimpusIX71, 20X LCPlanFl).
All microscopes were set to collect images below saturation and were kept constant for all images taken in one experiment.
Statistical analysis
Statistical analysis of data was performed using SPSS statistical software (SPSS for Windows, v.16, SPSS, Inc., Chicago, IL). Student’s t-test and ANOVA were used to compare quantitative with qualitative variables and χ2 test was used for categorical variables. Values were expressed as mean ± SD, and statistical significance are indicated as follows; p<0.05 (*), p<0.01 (**) or p<0.001 (***).