Design and synthesis of P021

The peptidergic compound P021 (Ac-DGGLAG-NH2; molecular weight: 578.3) used in this work derived from the laboratory of Prof. Khalid Iqbal (New York State Institute for Basic Research in Developmental Disabilities, USA). This compound corresponds with the biologically active region of human ciliary neurotrophic factor (CNTF; amino acid residues 148–151) to which adamantylated glycine was added to increase its stability and lipophilicity [40, 45]. The peptide was synthesized and purified by reverse phase HPLC to ~ 96% purity, as previously described [41].

Cell Lines, Treatments and Measurements

Human neuroblastoma cell line SH-SY5Y, obtained from The European Collection of Authenticated Cell Cultures (Sigma-Aldrich, Saint Louis, MO, USA) and the CDKL5 knockout (KO) SH-SY5Y neuroblastoma cell line (SH-CDKL5-KO; [20]) were maintained in Dulbecco modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated FBS, 2 mM of L-glutamine, and antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml; Thermo Fisher Scientific, Waltham, MA, USA), in a humidified atmosphere of 5% of CO2 at 37 °C. Cell medium was replaced every 3 days and the cells were sub-cultured once they reached 90% confluence.

P021 in vitro Treatment

Cells were plated onto poly-D-lysine-coated slides in a 6-well plate at a density of 2.5 × 105 cells per well in culture medium. The day after, cells were exposed to P021 (1 μM or 10 μM; stock solution 1 mM in deionized water) or vehicle (deionized water) for 24 h.

Retinoic Acid Induced Differentiation

For differentiation analyses, cells were plated onto poly-D-lysine-coated slides in a 6-well plate at a density of 1 × 105 cells per well in culture medium. Two hours after cell plating, retinoic acid (RA; Sigma-Aldrich, Saint Louis, MO, USA) was added to the medium at 10 μM final concentration each day for 5 days. Cells were co-treated with P021 (10 μM) or vehicle (deionized water) every day.

Image Acquisition

Phase-contrast or fluorescence images were taken with an Eclipse TE 2000-S microscope equipped with a DS-Qi2 digital SLR camera (Nikon Instruments, Tokyo, Japan). Images were taken from random microscopic fields (10 for each coverslip).

Mitotic and Apoptotic index

To assess the number of mitotic and apoptotic cells, cultures were fixed in a 4% paraformaldehyde solution at 37 °C for 30 min and nuclei were stained with Hoechst 33342 (Sigma-Aldrich, Saint Louis, MO, USA). The number of mitotic cells was assessed by manually counting the cells in prophase (chromosomes are condensed and visible), metaphase (chromosomes are lined up at the metaphase plate), and anaphase/telophase (chromosomes are pulled toward and arrive at opposite poles) and expressed as a percentage of the total number of cells. Apoptotic cell death was assessed by manually counting the number of pyknotic nuclei and apoptotic bodies and was expressed as a percentage of the total number of cells.

Analysis of Neurite Outgrowth

Neurite outgrowth was measured using the image analysis system Image Pro Plus software version 4.5 (Media Cybernetics, Silver Spring, MD, USA). Only cells with neurites that were longer than one cell body diameter were considered as neurite-bearing cells. In each experiment, a total of 900 cells was analyzed. All experiments were performed at least three times. The total length of neurites was divided by the total number of cells counted in the areas.

Colony

The mice used in this work were the Cdkl5 KO strain in the C57BL/6N background developed in [9] and backcrossed in C57BL/6 J for three generations. For the present study, mice were produced by crossing Cdkl5 KO heterozygous females (+ / −) with wild-type (+ /Y) males and they were genotyped using PCR of genomic DNA as previously described [9]. Littermate controls were used for all experiments. The day of birth was designated as postnatal day zero (P0), and animals with 24 h of age were considered as 1-day-old animals (P1). After weaning (P21-23), mice were housed 3 to 5 per cage and maintained in a temperature- (23 °C) and humidity-controlled environment with a standard 12 h light/dark cycle, and provided with mouse chow and water ad libitum. The animals’ health and comfort were controlled by the veterinary service. All the experiments were conducted in accordance with the Italian and European Community law for the use of experimental animals and with the approval of the National Bioethical Committee (approval number: n° 184/2022-PR). All efforts were made to minimize animal suffering and to reduce the number of animals used. Experiments were carried out on a total of 39 Cdkl5 KO male mice (Cdkl5 + /Y, n = 17; Cdkl5 − /Y, n = 22).

In Vivo Experimental ProtocolChronic oral P021 treatment

Starting from postnatal day 21 (P21), Cdkl5 − /Y mice were treated orally with a P021 diet for 70 days. Treatment was administered as 60 nmol peptide/g formulated diet (Research Diets, New Brunswick, NJ, USA). On average, each mouse consumed ~ 2.7 g diet/day. As a control, a group of wild-type (+ /Y) and Cdkl5 − /Y mice received the same diet but without the peptide. On the forty-ninth day of treatment (P70), animals from all the experimental groups received a single intraperitoneal (i.p.) injection (150 µg/g body weight) of BrdU (5-bromo-2-deoxyuridine; Sigma-Aldrich, Saint Louis, MO, USA). Body weight of the mice was monitored every eight days. Animals were behaviorally tested from P74 to P89. The day after completion of the behavioral task (P90), animals were sacrificed and brain tissues were collected for histological and Western blot analyses.

Chronic P021 administration through intraperitoneal injections

Four-month-old Cdkl5 − /Y male mice were intraperitoneally injected with P021 (750 nmol/mouse in saline) daily for 30 days. As a control, a group of wild-type (+ /Y) and Cdkl5 − /Y mice were injected with vehicle (saline). Each animal received four successive i.p. injections of BrdU (5-bromo-2'-deoxyuridine; 150 µg/g body weight) at 2 h intervals (at approximately 10:00, 12:00, 14:00, and 16:00 h) on the first experimental day. Twenty-four hours after the last P021 administration, mice were sacrificed and brain tissues were collected for histological and Western blot analyses. Body weight of the mice was monitored every eight days.

Behavioral Assays

After 53 days of P021 oral treatment, the animals were behaviorally tested with a sequence of tests, arranged to minimize the effect of one test influencing the subsequent evaluation of the next, and mice were allowed to recover for 1 day between different tests. All behavioral studies and analyses were performed blinded to genotype and treatment. Mice were allowed to habituate to the testing room for at least 1 h before the test, and testing was performed at the same time of day.

Marble Burying

The marble burying test was performed by placing animals individually in a home-cage-like environment with 5 cm of unscented standard bedding material and 20 marbles (14.3 mm in diameter) arranged in a 4 × 5 matrix, and were left undisturbed for 30 min. The number of marbles that were at least two-thirds buried at the end of the trial was counted.

Nesting

Nest building ability was evaluated as proposed by Deacon [46]. Animals were placed in individual cages with standard bedding, and a standard piece of paper towel (23 cm × 23 cm) was provided. The nests were independently assessed at 24 h by two operators using the following scoring system: 0—no nest, 1—primitive flat nest (pad-shaped, consisting of a flat paper tissue which slightly elevates a mouse above the bedding), 2—more complex nest (including warping and biting the paper towel), 3—complex accurate cup-shaped nests (with shredded paper interwoven to form the walls of the cup), and 4—complex hooded nest, with walls forming a ceiling so the nest becomes a hollow sphere with one opening.

Hind-Limb Clasping

Animals were suspended by their tail for 2 min and hind-limb clasping time was assessed from video recordings. A clasping event is defined by the retraction of hind-limbs into the body and toward the midline.

Accelerating Rotarod Assay

Before the first test session, animals were briefly trained at a constant speed of 5 rpm on the rotarod apparatus (Ugo Basile, Gemonio, Italy) for 30 s. Thirty minutes later, testing was performed at an accelerating linear speed (5–35 rpm within 270 s + 30 s max speed). Four testing trials, with an intertrial interval of 1 h, were performed. The latency to fall from the rotating rod and the number of passive rotations (rotation in which the mouse does not perform any coordinated movement but is passively transported by the rotating apparatus) were recorded for each trial.

Catalepsy Bar Test

The bar was set at a height of 6 cm. Mice were gently positioned, by placing both fore-limbs on the bar and their hind-limbs on the floor. The time needed for the mice to remove both paws from the bar was measured using a stopwatch.

Open Field

In order to assess locomotion, animals were placed in the center of a square arena (50 × 50 cm) and their behavior was monitored for 20 min using a video camera placed above the center of the arena. Distinct features of locomotor activity, including total distance traveled, average locomotion velocity, and the time spent in the central and peripheral zones, were scored by EthoVision 15XT software (Noldus Information Technology, Wageningen, The Netherlands). The test chambers were cleaned with 70% ethanol between test subjects.

Morris Water Maze

Hippocampal-dependent spatial learning and memory was assessed using the Morris water maze (MWM). Mice were trained to locate a hidden escape platform in a circular pool. The apparatus consisted of a circular water tank (1 m in diameter, 50 cm high) with a transparent round escape platform (10 cm2) placed in a fixed position. The tank was filled with tap water at a temperature of 22 °C up to 0.5 cm above the top of the platform, and the water was made opaque with milk. In the experimental room, intra-maze and extra-maze visual cues were placed to enable spatial orientation. Mouse behavior was automatically videotracked (EthoVision 3.1; Noldus Information Technology, Wageningen, The Netherlands). During training, each mouse was subjected to either 1 swimming session of 4 trials (day 1) or 2 sessions of 4 trials per day (days 2–5), with an intersession interval of 1 h (acquisition phase). Mice were allowed to search for the platform for up to 60 s. If a mouse did not find the platform, it was gently guided to it and allowed to remain there for 15 s. During the intertrial interval (15 s), mice were placed in an empty cage. The latency to find the hidden platform was used as a measure of learning. Twenty-four hours after the last acquisition trial, on day 6, the platform was removed and a probe test was run. Animals were allowed to search for the platform for up to 60 s. The latency of the first entrance into the former platform area was employed as measures of retention of acquired spatial preference. During the learning phase, the average and maximum swim speeds were also analyzed.

Passive Avoidance

For the passive avoidance task, a memory task that involves contributions from both the hippocampus and amygdala, the equipment consisted of a tilting-floor box (47 × 18 × 26 cm) divided into 2 compartments (lit and dark) by a sliding door, and a control unit that incorporated a shocker (Ugo Basile, Gemonio, Italy). Upon entering the dark compartment, mice received a brief mild foot shock (0.4 mA for 3 s) and were removed from the chamber after a 15-s delay. After a 24-h retention period, mice were returned to the illuminated compartment, and the latency to re-enter the dark chamber was measured, up to 360 s. The chambers were cleaned with 70% ethanol between testing of one subject and another.

Histological and Immunohistochemistry Procedures

Animals were anesthetized with isoflurane (2% in pure oxygen) and sacrificed through cervical dislocation. Brains were quickly removed and cut along the midline. Left hemispheres were Golgi-stained or quickly frozen and used for Western blot analyses. Right hemispheres were fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 48 h, kept in 15–20% sucrose for an additional 24 h, frozen with dry ice, and stored at -80 °C. Right hemispheres were then cut with a freezing microtome (Microm GmbH, Walldorf, Germany) into 30-μm-thick coronal sections, which were serially collected in 96-well plates containing a solution composed of 30% glycerol, 30% ethylene glycol, 0.02% sodium azide in 0.1 M PBS, and then processed for immunohistochemistry procedures.

Immunofluorescence staining

One out of every eight free-floating sections from the hippocampal formation was incubated overnight at 4 °C with one of the following primary antibodies: rabbit polyclonal anti-AIF-1 antibody (1:300; Thermo Fisher Scientific, Waltham, MA, USA), mouse monoclonal anti-NeuN antibody (1:250; Merck Millipore, Burlington, MA, USA), or rabbit polyclonal anti-DCX antibody (1:300; Thermo Fisher Scientific, Waltham, MA, USA). The following day, the sections were incubated for 2 h at room temperature with a Cy3-conjugated anti-rabbit IgG secondary antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for AIF-1 and DCX immunohistochemistry, and with a Cy3-conjugated anti-mouse IgG secondary antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for NeuN immunohistochemistry. Nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich, Saint Louis, MO, USA).

For BrdU immunofluorescence, one out of every eight free-floating sections from the hippocampal formation was denatured in 2 N HCl for 30 min at 37 °C, and then incubated overnight at 4 °C with a rat monoclonal anti-BrdU antibody (1:200; Abcam, Cambridge, UK). The following day, the sections were incubated for 2 h at room temperature with a Cy3-conjugated anti-rat IgG secondary antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich, Saint Louis, MO, USA).

Fluorescent images were acquired using an Eclipse TE 2000-S microscope equipped with a DS-Qi2 digital SLR camera (Nikon Instruments, Tokyo, Japan).

Golgi staining

Left hemispheres were Golgi-stained using the FD Rapid Golgi StainTM Kit (FD Neuro Technologies, Columbia, MD, USA). Briefly, hemispheres were immersed in the impregnation solution containing mercuric chloride, potassium dichromate, and potassium chromate, and stored at room temperature in the dark for 3 weeks. Hemispheres were then cut with a cryostat (Histo-Line Laboratories, Pantigliate, Italy) into 100-µm-thick coronal sections, which were directly mounted onto Superfrost® Plus Microscope Slides (Thermo Fisher Scientific, Waltham, MA, USA) and air-dried at room temperature for 1 day. After drying, sections were rinsed with distilled water, stained in the developing solution of FD Rapid Golgi StainTM Kit (FD NeuroTechnologies, Columbia, MD, USA), and coverslipped with DPX mounting medium (Sigma-Aldrich, Saint Louis, MO, USA). A light microscope (Leica Mycrosystems, Wetzlar, Germany) equipped with motorized stage, focus control system, and color digital camera (Coolsnap-Pro; Media Cybernetics, Rockville, MD, USA) were used to acquire bright field images.

MeasurementsCell density

The number of BrdU-, and DCX-positive cells were counted in the subgranular and granular zone of the dentate gyrus of the hippocampus and expressed as number of cells/100 µm. The density of Hoechst-positive nuclei and neurons (NeuN-positive cells) in the CA1 field of the hippocampus were manually counted and expressed as cells/mm3.

Morphometric microglial cell analysis

Starting from 20 × magnification images of AIF-1-stained cortical slices, microglial cell body size was manually drawn using the measurement function of the Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA) and expressed in μm2. Approximately 120 microglial cells were analyzed from each sample.

Dendritic Spine Number and Morphology

In Golgi-stained sections, dendritic spines of hippocampal pyramidal neurons were visualized with a 100 × oil immersion objective lens. Dendritic spine density was measured by manually counting the number of dendritic spines on the basal dendrites of hippocampal pyramidal neurons and was expressed as total number of spines per 10 μm. Based on their morphology, dendritic spines can be divided into two different categories that reflect their state of maturation: immature spines (filopodium-like, thin- and stubby-shaped) and mature spines (mushroom- and cup-shaped). The number of mature spines was counted and expressed as a percentage. About 100–150 spines from 15 to 20 dendrites, derived from 10 to 20 neurons, were analyzed per condition.

Western Blotting

To score the amount of phospho-proteins in cell culture we used a rapid protein extraction method [47] that significantly reduces the risk of protein degradation and modifications that may occur during harvesting and cell lysis. SH-SY5Y and SH-CDKL5-KO cells were plated in a 6-well plate at a density of 2.5 × 105 cells per well in culture medium. The following day, cells were exposed to 10 μM P021 or vehicle (deionized water). Six hours after the treatment the culture medium was removed from the plate and adherent cells were washed with ice cold PBS and directly lysed in the plate by adding 100 μl of denaturing Laemmli loading buffer 2x. Samples were collected, boiled in a mixing heating block for 10 min at 99 °C, and equal amounts (20 μl) of each sample were subjected to electrophoresis on a BoltTM 4–12% Bis–Tris Plus gel (Life Technologies Corporation, Carlsbad, CA, USA).

Tissue samples from the hippocampus of vehicle-treated Cdkl5 + /Y and Cdkl5 − /Y mice and of P021-treated Cdkl5 − /Y mice, were lysed in ice-cold RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton-X100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 1 mM PMSF, and with 1% protease and phosphatase inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA). Protein concentration for both cell and tissue extracts was determined using the Bradford method [48]. Equivalent amounts of protein (50 µg) were subjected to electrophoresis on a BoltTM 4–12% Bis–Tris Plus gel and transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare Bio-Science, Piscataway, NJ, USA).

The following primary antibodies were used: rabbit polyclonal anti-BDNF (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-phospho-GSK3β (Ser9; 1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-GSK3β (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-phospho-Akt (Ser473; 1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-Akt (1:1000; Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-Vinculin (7F9; 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit polyclonal anti-GAPDH (1:5000; Sigma-Aldrich, Saint Louis, MO, USA). An HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and an HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used. Densitometric analysis of digitized Western blot images was performed using Chemidoc™ XRS + Imaging System and the Image Lab™ Software (Bio-Rad, Hercules, CA, USA). This software automatically highlights any saturated pixels of Western blot images in red. Images acquired with exposure times that generated protein signals out of a linear range were not considered for quantification. Western blot analyses were performed on protein extracts of multiple samples per experimental group: three to five biological replicates for the cell lines or three to six for animals. Repeated measurements of the same samples were performed by running from two to four independent gels. The signal of one sample (internal control) was used to perform a relative analysis of the antigen expression of each sample on the same gel. We considered the control signal as 100 and assigned a value to the other sample as a percentage of the control. Data analysis was performed by averaging the signals obtained in two to four gels for each individual sample.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 8.0.1 (GraphPad Software, Boston, MA, USA). Values are expressed as means ± standard error (SEM). The significance of results was obtained using Student’s t-test, an ordinary one-way or two-way analysis of variance (ANOVA) or a two-way repeated measurement (RM) ANOVA followed by Fisher’s LSD post hoc test, as specified in the figure legends. A probability level of p < 0.05 was considered statistically significant. The confidence level was taken as 95%.