Subcutaneous Administration of AMD3100 into Mice Models of Alzheimer’s Disease Ameliorated Cognitive Impairment,Reduced Neuroinflammation,and Improved Pathophysiological Markers

Abstract

Background:

Alzheimer’s disease (AD), the prevalent dementia in the elderly, involves many related and interdependent pathologies that manifest simultaneously, leading to cognitive impairment and death. Amyloid-β (Aβ) accumulation in the brain triggers the onset of AD, accompanied by neuroinflammatory response and pathological changes. The CXCR4/CXCL12 (SDF1) axis is one of the major signal transduction cascades involved in the inflammation process and regulation of homing of hematopoietic stem cells (HSCs) within the bone marrow niche. Inhibition of the axis with AMD3100, a reversible antagonist of CXCR4 mobilizes endogenous HSCs from the bone marrow into the periphery, facilitating the recruitment of bone marrow-derived microglia-like cells into the brain, attenuates the neuroinflammation process that involves release of excitotoxic markers such as TNFα, intracellular Ca^2 +, and glutamate and upregulates monocarboxylate transporter 1, the major L-lactate transporter in the brain.

Objective:

Herein, we investigate if administration of a combination of AMD3100 and L-lactate may have beneficial effects in the treatment of AD.

Methods:

We tested the feasibility of the combined treatment for short- and long-term efficacy for inducing endogenous stem cells’ mobilization and attenuation of neuroinflammation in two distinct amyloid-β-induced AD mouse models.

Results:

The combined treatment did not demonstrate any adverse effects on the mice, and resulted in a significant improvement in cognitive/memory functions, attenuated neuroinflammation, and alleviated AD pathologies compared to each treatment alone.

Conclusion:

This study showed AMD3100’s beneficial effect in ameliorating AD pathogenesis, suggesting an alternative to the multistep procedures of transplantation of stem cells in the treatment of AD.

Keywords

Alzheimer’s disease bone marrow-derived macrophages CXCR4/SDF1 axis inflammation hematopoietic stem cells L-lactate MCT1 Plerixafor tumor necrosis factor-alpha tumor suppressor protein p53

INTRODUCTION

The CXCR4/CXCL12 axis is one of the important signal transduction cascades involved in the inflammation process, and plays a crucial role in the quiescence, proliferation, homing, and retention of hematopoietic stem cells (HSCs) and hematopoietic stem progenitor cells (HSPCs) within the bone marrow niche. Inhibition of the CXCR/CXCL12 axis may result in attenuation of the neuroinflammation process, either by directly activating its signaling pathway, and/or by its capacity to recruit bone marrow-derived progenitor and stem cells to insulted regions in the brain. These cells may promote brain repair by inducing processes like neurogenesis, the release of anti-inflammatory cytokines and hematopoietic growth factors that lead to the attraction of bone marrow-derived monocyte precursor to the periphery [1].

The precursor cells transdifferentiate into micro-glia-like cells following infiltration into the brain (so-called bone marrow-derived microglia-like cells, or BMDMLs), which may help the resident microglia population to compensate for its dysfunction by enhancing its phagocytic capacity [2 –5]. In addition to its role in the regulation of HSCs homing in the bone marrow niche, CXCL12/CXCR4 signaling plays a crucial role in activation of the inflammatory cascade, which was found to be significant in several diseases, including psoriasis, Type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, idiopathic inflammatory bowel diseases, and neurodegenerative diseases [6, 7].

In the context of Alzheimer’s disease (AD), bone marrow-derived cells can access the amyloid-β (Aβ)-laden brain in higher numbers, as demonstrated in APP23 transgenic mice versus age-matched non-transgenic control mice. The invading cells exhibit hematopoietic phenotypes heterogeneously scattered throughout the brain [8]. Notably, the damaged brain exhibits high levels of chemokines like CXCL12, also known as stromal cell-derived factor-1 (SDF-1), which attract circulating HSCs due to their surface expression of CXCR4 [9 –11]. Furthermore, increased levels of CXCR4 were found in tissue samples of AD patients [12] and in activated astrocytes in a post-ischemic pain rat model and in a P301L-tau transgenic mouse model, which triggered glutamate release via a mechanism involving TNFα, IL-6, and IL-1β release, a process that is amplified by microglia and that can eventually lead to neurotoxicity [13 –16].

These observations have led a number of researchers to look into the therapeutic potential of stimulating the mobilization of endogenous bone marrow stem cells [17 –19], thus sidestepping the multistep procedures preceding transplantation wherein each step can be accomplished by a wide variety of protocols and methods [20].

Herein, we propose a novel therapeutic approach of inhibiting the CXCL12/CXCR4 axis by AMD3100 (also known as Plerixafor), a specific antagonist of CXCR4. AMD3100 is a small bicyclam molecule (1,10-[1,4-phenylenebis-(methylene)]-bis-1,4,8, tetraazacyclotetradecane) that was originally approved in 2008 by the US-FDA for treating chemotherapy-induced neutropenia and for autologous transplantation in patients with non-Hodgkin’s lymphoma and multiple myeloma [21]. AMD3100 reversibly and selectively blocks CXCL12’s binding to its receptor CXCR4 and induces rapid mobilization of CD34⁺ hematopoietic cells, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells.

Additional studies in healthy volunteers demonstrated that AMD3100 is well tolerated with minimal and reversible side effects such as pain and erythema at injection site, headache, nausea, abdominal distension, and cramping [22]. AMD3100’s half-life after intravenous (i.v) infusion is 3.6 h. Subcutaneous administration of AMD3100 results in rapid absorption and distribution in the liver, kidney, and spleen [22 –25].

AMD3100’s feasibility for short- and long-term efficacy of inducing endogenous stem cell mobilization for AD treatment was investigated here in two different Aβ-induced transgenic and non-transgenic AD mouse models. The acute model was obtained using wild-type mice injected bilaterally intrahippocampi with 10μg Aβ_25–35 fibrils. Based on the previous findings that inhibition of the axis with AMD3100 increases monocarboxylate transporters 1 (MCT1) levels, which is the major L-lactate transporter in the brain [26, 27], we propose a treatment based on the administration of a combination of both AMD3100 and L-lactate in an Aβ-induced acute AD mouse model, which is a proper model that serves to demonstrate that neuroinflammation is stimulated by substantial concentration of pre-made Aβ fibrils [28]. Mice were then treated twice weekly with subcutaneous administrations of AMD3100 (5 mg/Kg mouse weight) and L-lactate (900 mg/Kg mouse weight) for 2 weeks. Alongside, we investigated the chronic administration of AMD3100 in a 3xTg-AD mouse model for 4 months to test the side effects of long-term administration of AMD3100. In both mice models, we observed amelioration in cognitive impairment and reduction in neuroinflammation and other typical AD pathophysiological markers. This study showed AMD3100’s beneficial effect in ameliorating AD pathogenesis, suggesting an alternative to the multistep procedures of transplantation of stem cells in the treatment of AD.

MATERIALS AND METHODS

Ethics statement

All animal experiments were approved and conducted as per the Guidelines of the Institutional Animal Care and Use Committee (licenses no. L-04-16-078, L-12-011). Every effort was made to relieve animal stress and to minimize animal usage.

Animals

Like most acute AD rodent model studies that used adult male [29 –32], we used C57BL/6JOlaHsd inbred mice (#2BL/606, Envigo RMS, Israel) aged 8–9 weeks and weighing 27.7 + 3.0 g at the beginning of the experiment.

The 3xTg-AD mouse model, transgenic 8 month-old male mice (kindly donated by Prof. Frank M. LaFerla), manifests age-dependent neuropathology of AD in the form of both Aβ plaques and neurofibrillary tangles, along with age-dependent learning and memory deficits. These mice express concomitantly the PS1M146V, APPSwe, and tau P301L transgenes, as described previously [33].

All mice were housed 4–5 mice/cage under standard laboratory conditions (12/12-h light/dark cycle with lights on at 7 : 00 a.m. and kept at 22°C±1°C). The mice received food and water ad libitum. All procedures were conducted during the light phase of the cycle.

Acute treatment

Preparation of Aβ_25–35 peptide

Aβ_25–35 (Sigma A4559) peptide was dissolved in sterile saline at a concentration of 1μg/μL (0.94 mM) and stored at –20°C until use. “Aged” Aβ_25–35 was obtained by incubating the stock solution at 37°C in a shaking water bath for 4 days. Ascertainment of the presence of fibrils in the solution was obtained by THT fluorescence assay and transmission electron microscopy.

Characterization of Aβ_25–35 aggregation

Thioflavin T (ThT) fluorescence assay for Aβ fibril detection. The ThT assay measures changes in fluorescence intensity of ThT correlated with its binding to Aβ fibrils. The enhanced fluorescence can be observed by fluorimeter (Horiba Jobin Yvon FL3-11 spectrofluorometer). The spectroscopic assay is commonly used to monitor fibrilization over time. The samples were diluted to 20μM with double-distilled water 1 : 40. The ThT solution for detection was prepared by diluting 2.5μL of the ThT stock (4mM ThT in PBS stock solution obtained by dissolving 64 mg ThT in 50 mL PBS covered with aluminum foil and stored at 4°C) in 1 mL 50 mM tris pH 8. 70μL of the ThT solution was added to 40μL sample in cuvette. Excitation was at 450 nm and readout emission was at 470–520 nm.

Transmission electron microscopy (TEM). The approval for the formation of fibrils was also confirmed by TEM (Jeol 1400 plus). 10μL of either t = 0 (1μg/μL Aβ_25–35) or t = 4 (days of incubation at 37°C) were placed on a copper Formvar carbon-coated grid 400 mesh (Electron Microscopy Sciences). After 2 min of adsorption at room temperature (RT), samples were dried with Whatman filter paper and stained with 4% Uranyl Acetate (Thomas Scientific CAS # 541-09-3) for 3 min at RT. Then the samples were examined under Jeol 1400 plus transmission electron microscopy.

Acute AD mouse model: Experimental procedures

The animals were divided into six groups. One group was considered a control group (C) and left without Aβ_25–35 distribution (1st group). The other five groups were bilaterally gradually injected (1μL/min, Hamilton^® syringe, #701) using a stereotactic device (Stoelting Co. IL, USA). 5μL/hemisphere of saline (2nd group –sham) or aged 0.94 nM Aβ_25–35 (3rd, 4th, 5th, 6th groups) were injected into each hemisphere hippocampal region X (L):+2 mm, Y (AP): –2 mm, Z (DV): –2 mm. The coordinates of injections were previously confirmed by injection of Evans Blue instead of Aβ_25–35. Prior to intrahippocampal administration, all mice were anesthetized using 100 mg/Kg mouse weight Ketamine (Fort Dodge^®, USA) and 20 mg/Kg mouse weight Xylazine (Merck, Germany). At the first stage, we used the first three groups to follow the behavioral effect in correlation with the intrahippocampal injection of Aβ_25–35. At the second stage, we assessed the combined treatment of AMD3100 and L-lactate’s effects on cognition, pathophysiology, and neuroinflammation of the Aβ_25–35-injected mice.

Treatments administration

The mice were divided into groups as shown in Table 1. The time chart of the experimental procedure is shown in Fig. 1. The mice were treated (s.c) 2 h post-surgery and twice in the subsequent 11 days with 0.2 mL of: PBS (2nd, 3rd control groups, BI cat #02-023-1A); L-lactate dissolved in PBS (4th group 900 mg/Kg mouse, sodium lactate, Sigma Cat #71718); AMD3100 dissolved in PBS (5th group, AMD3100 Octa-hydrochloride hydrate Sigma #A5602); and a combination of AMD3100 and L-lactate dissolved in PBS (6th group).

Table 1

Experimental groups of mice

Group	Abbreviation	Description
1	C	WT mouse not treated
2	S∖Sham	Saline-intrahippocampal-injected mice treated with PBS
3	Aβ_25–35∖PBS∖P	Mice were intra hippocampal-injected with Aβ_25–35 and treated subcutaneously with PBS
4	L-lactate∖L	Mice were intra hippocampal-injected with Aβ_25–35 and treated subcutaneously with 900 mg/kg L-lactate
5	AMD3100∖A	Mice were intra hippocampal-injected with Aβ_25–35 and treated subcutaneously with 5 mg/kg AMD3100
6	AMD300 + L-lactate/A + L	Mice were intra hippocampal-injected with Aβ_25–35 and treated subcutaneously with 5 mg/kg AMD3100 and 900 mg/kg L-lactate

Fig. 1

Experimental timeline scheme and table of groups. Mice received single injection of Aβ_25–35 followed by 0.2 ml of PBS (Groups 2, 3), 900 mg/Kg l-lactate (Group 4), 5 mg/Kg AMD3100 (Group 5), or the combination of AMD3100 and l-lactate (Group 6) 2 times/week. Mice were subjected to behavioral tests at days 8–10 and sacrificed on the next day for biochemical analysis.

Cognitive tests

The mice were transferred to a behavioral test room for habituation two days prior to the cognitive tests for 3 h each time. To track the correlation between Aβ_25–35 administration and cognition, at t = 8 days, mice were subjected to 1 day of T-maze followed by 2 days of fear conditioning cognitive tests.

T-maze

The T-maze that was used is an enclosed horizontal T-shape Plexiglas apparatus. The T-Maze is based on two short arms (30 cm long×10 cm wide×15 cm high): “old”, and “novel”. There are two sessions: In the first trial, animals are placed at the base of the T-maze and allowed to explore and choose one of the available arms for 5 min (the 2nd arm is closed by a removal gate). In the second trial, following 5-min waiting period, the gate is open and both arms are available for the mouse to explore. Choosing the “novel” arm indicates a healthy, functioning brain. To avoid any arm preference, the “old” and “novel” arms were switched in between mice. The mice’s activity was recorded and analyzed using EthoVision^® XT (Noldus) video tracking software that tracks and analyzes relapse time and length. Ratio of time spent/frequency of visits to the novel arm was calculated as time/frequency in the novel arm divided by the time/frequency in the old arm. Results are presented in percentages and normalized to the Aβ_25–35-injected and PBS treatment control group (3rd group).

Contextual fear conditioning

The test is based on a hippocampal-dependent task, as hippocampal lesions lead to cognitive deficit in contextual fear tests [34]. The test was conducted as described [35]: Briefly, mice were subjected to an unconditioned electric stimulus (one foot shock; 1 s/1 mA) 3 min after being placed in a conditioning chamber, and left 1 min after the shock to recover. Conditioning was tested after 24 h in the same conditioning chamber by scoring freezing behavior, e.g., the absence of all but respiratory movement, for 180 s using EthoVision^® XT (Noldus) video tracking software.

Chronic treatment

Transgenic mice and treatment

The 3xTg mice were treated subcutaneously with PBS, 5 mg or 500μg/Kg mouse weight AMD3100, once a week for four months, starting at 8 months of age.

Cognitive tests

After four months of treatment, mice were subjected to Y-maze and fear conditioning cognitive tests as described below.

Y-maze

The test was conducted in a symmetrical black Plexiglas Y-Maze with three arms (30 cm long×10 cm wide×15 cm high) at 120° angles, designated A, old, and novel. The mice were placed in the distal end of arm A and allowed to explore the maze for 5 min with only the old arm available to exploration. After 5-min waiting period, mice were reintroduced to the maze with both arms (old and new) available to explore, and documented for 5 min. Percentage of time spent/frequency of visits to the novel arm was calculated as time/frequency in the novel arm divided by time/frequency in the old arm.

Contextual fear conditioning

The test was conducted as described [35, 36]. Briefly, mice were placed in a conditioning chamber and subjected to an unconditioned electric stimulus (one foot shock; 1 s/1 mA). Conditioning was tested after 24 h in the same conditioning chamber by scoring freezing behavior, e.g., the absence of all but respiratory movement for 180 s using a Freeze Frame automated scoring system (Coulbourn Instruments).

Brain homogenates preparation

On Day 11, mice were anaesthetized (i.p) with ketamine/xylazine (100 mg/kg and 10 mg/kg mouse weight, respectively) and perfused with saline. Brain tissues were cut sagittally and the right hemispheres were frozen in liquid nitrogen and stored for 2 days at –70°C until homogenization. Brains were then thawed and the right hemispheres placed on petri dishes. Cerebellum and olfactory bulb regions were dissected, and hippocampal regions were immediately transferred into homogenizer tubes. Hippocampi were fractionated into soluble, membrane, and insoluble fractions. Soluble fractions were obtained by adding 700μL soluble buffer-0.1% Triton X-100 in TBSx1 to the homogenizer tubes that contained the hippocampal region on ice, followed by homogenization (30 s), sonication (50% frequency, 30 s total, and 50% power), ultra-centrifugation (100,000×g, 60 min, 4°C). The supernatant obtained was collected and assigned as soluble fraction. Membrane fractions were obtained by suspending the precipitation in 600μL of membrane buffer-TBSX1, 1% NP-40, 0.5% Na-Deoxycholate, 0.1% SDS (50μL) on ice and repeating homogenization (30 s), sonication (50% frequency, 15 s total, 50% power), ultra-centrifugation (100,000×g, 60 min, 4°C), and collecting the supernatant. Protein Inhibitor (cOmplete™, Roche) Phosphatase Inhibitor (PhosSTOP™, Roche) was added to the membrane and soluble solutions immediately before use. The pellets were re-suspended in 50 mM Tris HCl pH 8, 6 M guanidine-HCl overnight at 4°C and centrifuged at 20,000×g for 30 min at 4°C. The resulting supernatants constitute the insoluble fraction. Protein concentrations were determined using the BCA protein assay kit (Thermo Scientific, USA).

Western immunoblot analysis

Equal amounts of mice brain homogenates protein (35μg) were resolved on 10–15% SDS-PAGE, transferred to nitrocellulose membrane (400 mA, 90 min), and blocked overnight with 5% skim milk in TBS-T (0.1% Tween-20). Blots were probed with the following primary antibodies: mouse anti-N-terminal β cytoplasmic actin (1 : 10,000; A5316-Sigma-Aldrich), mouse anti-C-terminal β tubulin (1 : 25,000; T6199-Sigma-Aldrich), rabbit anti-Ly6A/E antibody [EPR3355] (1 : 1,000; ab109211-abcam), mouse anti-N-terminus of mouse iNOS (1 : 500; MABN236-EMD-Millipore), rabbit anti-TYR703 human c-Kit (1 : 1,000; CS3074-Cell Signaling), rabbit anti-C-terminus of IL-1β (1 : 1,000; AB1413-I-EMD-Millipore), goat anti-human Iba1 aa 28–42 (1 : 500; Ab48004-abcam), rabbit anti- KLH-conjugated linear peptide corresponding to human CX3CL1 (1 : 1,000; ABN467-EMD-Millipore), chicken anti-full-length recombinant protein corresponding to rat Arginase-1 (1 : 1,000; ABS535-EMD-Millipore), goat anti-synthetic peptide corresponding to a.a 154–165 of mouse TREM2 (1 : 1,000; Ab95470-abcam), mouse anti-a.a 66–81 of APP (N-terminus) clone 22C11 (1 : 2,000; MAB348-EMD-Millipore), rabbit anti-A 10 a.a corresponding to the amino terminal of Brain-derived neurotrophic factor (BDNF) (1 : 2,000; AB15234SP -EMD-Millipore), rabbit anti-Recombinant Mouse TNFα (1 : 2,000; AB2148P-EMD-Millipore), mouse anti-MBP (myelin basic protein) CLONE SKB3 (1 : 1,000; 05-675-EMD-Millipore), rabbit anti-E.coli-derived recombinant murine IL-6 (1 : 1,000; 500-P56-PeproTech), rabbit anti-Residues a.a 350–450 of human occludin (1 : 1,000; Ab-31721-abcam), rabbit anti-C-terminal cytoplasmic domain (a.a 441–485) of MCT1 of mouse origin (1 : 1,000; SC-50325-Santa Cruz), a mouse anti-synthetic linear peptide BACE1, Clone 61-3E7 (1 : 1,000; Mab5308-EMD-Millipore), rabbit anti-Glial Fibrillary Acidic Protein (1 : 10,000; Z0334-Dako), mouse anti-Peptide with sequence C-RNDGEDSYRTQ of CD11b (1 : 1,000; SAB 2500547-Sigma-Aldrich), mouse anti-bovine brain S-100B (1 : 500; S-100B-Novus Biologicals), rabbit anti-synthetic peptide corresponding to C-terminal a.a 751–770 of human APP (1 : 1,000; 171610-Calbiochem), rabbit anti-Phospho-APP (Thr668) (1 : 1,000; CS-3823-Cell Signaling), rabbit anti-GSKβ [3D10] (1 : 1,000; ab93926-abcam), rabbit anti-p53 d-200 (1 : 1,000; SC-25767-Santa Cruz), mouse anti-Phospho-Tau (Ser202, Thr205) monoclonal antibody (AT8) (1 : 1,000; MN1020-Thermo Fisher Scientific), mouse anti-Phospho-Tau (Ser396/404) Paired Helical Filaments (PHF1) (1 : 1,000; generously provided by Dr. Peter Davies, Albert Einstein University, New York), mouse anti-epitope maps Tau-5 (1 : 1,000; 577801-Calbiochem^®), mouse anti-C-terminal fragment of Human GSK3β expressed in E. coli. Tyr216, Tyr279 (1 : 1,000; 44604G-Thermo Fisher Scientific), mouse anti-Recombinant Murine JE/MCP-1 (CCL2) (1 : 1,000; 250-10-PeproTech), and the corresponding secondary antibodies conjugated to peroxidase: donkey anti-goat IgG H&L (HRP) (1 : 10,000; abcam), HRP-conjugated goat anti-mouse IgG (Fc Fragment Specific) (1 : 10,000; Jackson ImmunoResearch Laboratories), HRP-conjugated AffiniPure Goat anti-rabbit IgG (H + L) (1 : 10,000; Jackson ImmunoResearch Laboratories), and Rabbit Anti-Chicken IgG-HRP conjugated (1 : 10,000; Merck). Immunoblots were developed with EZ-ECL detection kit (Biological Industries, Israel) and Imager 600 (Amersham). Quantitative analysis was performed using ImageStudio Lite (version 2.0) software.

Sandwich ELISA for IL-10 measurement

The procedures were calibrated and performed by R&D system protocol (DY41705-Fisher Scientific). The kit is based on a Solid Phase Sandwich ELISA that quantifies mouse IL-10 coated in a 96-well microplate. Results were obtained by dilutions of 1 : 1 of the homogenate samples with kit diluent. The capture antibody was applied overnight and incubated at RT. The next day, blocking buffer was added and incubated at RT for 1 h, followed by incubation of the samples and standards at RT for 2 h. Detection was obtained by incubating with the secondary antibody at RT for 2 h, followed by the addition of Streptavidin-HRP and incubation for 20 min and substrate solution-HRP for 20 min. Readings were obtained at 450 nm and calculated by the subtraction of the 540 nm readings.

TNFα measurement

Soluble fractions of spinal cord homogenates were subjected to Mouse TNF alpha ELISA Ready-SET-Go! kit, as per the manufacturer’s instructions (eBioscience, USA).

Sandwich ELISA for Aβ_1–42 measurement

Microtiter plates (Maxisorp, Nunc) were coated with 0.12μg/well antibody (ELAN, Ireland), recognizing Aβ_17–28, in 0.1 M Na₂CO₃ (pH 9.6) at 37°C for 3 h and blocked with 3% BSA in PBS at 37°C for 3 h. Brain homogenates were added and incubated overnight at 4°C. Biotinylated 21F12 (ELAN, Ireland) diluted 1 : 2,500 in 1% BSA was added to each well and incubated at 37°C for 3 h. Streptavidin conjugated peroxidase was added at 37°C for 1 h. TMB was used as a substrate to monitor Aβ_1–42 levels by absorption at 450 nm.

Histology sections

The right hemisphere of the brain was fixed in 4% formaldehyde in PBS for 48 h. After fixation, brains were immersed in 30% sucrose-PBS overnight. Brains were frozen on dry ice and kept at –70°C. 25μm free-floating coronal sections were obtained using a cryostat (LEICA CM 1900, Germany) and stored in cryo-protectant buffer.

Luxol blue staining

Floating coronal sections of the left hemisphere (from the region of Aβ_25–35 injection X (L): + 2 mm) were washed by incubation in distilled water for 10 min×3 to remove the cryoprotectant, mounted on positively charged slides, then dried by incubation at 37°C for 30 min. Sections were de-fatted for 30 min by incubation in xylene for 10 min, a step that we repeated three times. Sections were incubated in 100% ethanol for 15 min×3 and were stained with 0.1% (w/v) Luxol blue for 12 h at 56°C. Excess staining was rinsed first in distilled water and then in 95% ethanol for 5 s and differentiated in 0.05% lithium carbonate for 30 s following rinsing in distilled water and 70% ethanol. Differentiation steps were repeated until myelinated and unmyelinated regions were defined. Sections were dehydrated in graded alcohol, cleared in xylene, and cover slipped with Entellan. Images were captured by a CCD camera (ProgRes C14, Jenoptic, Jena, Germany) attached to a Leica MZ6 binocular (Leica, Germany) for Luxol fast blue staining. Image-J Software (NIH, freeware) was used for all analysis.

Statistical analysis

Data was analyzed by one-way ANOVA (where applicable by GrapPhad Prism 8.4.0) followed by post-hoc Tukey’s test or nonparametric tests where non-normal distribution was displayed followed by Dunn’s test. All data are presented as means±SEM; p < 0.05 was considered statistically significant.

RESULTS

Acute AD mouse model

Aβ_25–35 peptide’s ability to form fibrils was tested after 4 days of incubation at 37°C (Supplementary Figure 1). Validation of the Aβ_25–35 acute model was performed prior to administration of the combined treatment by subjecting three groups of C57BL/6JOlaHsd mice (C-non-injected control group, sham group-intrahippocampal injection of vehicle, Aβ_25–35 group) to a spatial short-term memory T-maze as well as to long-term contextual fear conditioning tests. T-maze-analysis of ratios’ variance of the time spent in the novel arm versus the old arm and the number of visits to the novel arm versus the old arm is presented in Supplementary Figure 2. Other typical AD pathophysiology effects were also exhibited by this model (Supplementary Figure 3).

AMD3100 increases MCT1 and EAAT2 transporters levels

Mice were administered subcutaneously (s.c) twice a week, starting at 2 h post-surgery, with PBS and AMD3100. As shown in Fig. 2A, mice were injected with Aβ_25–35 and treated with AMD3100 exhibited elevated levels (+70%) of MCT1 in their hippocampi and Excitatory Amino Acid Transporters (EAAT2), the predominant glutamate transporter in the brain mainly expressed in astrocytes, neurons, and oligodendrocytes; and distributed predominantly in the hippocampus. High levels of glutamates, which correlate with low levels or dysfunction of EAAT2, are well demonstrated in AD patients and AD mouse models [37 –40]. As demonstrated in Fig. 2B, AMD3100 treatment resulted in upregulated levels of EAAT2 (+60%) within the hippocampi of Aβ_25–35-injected mice.

Fig. 2

The treatments effects on Aβ_25–35-injected mice A) AMD3100 regulation of MCT, L-lactate main transporter monocarboxylate transporter-1 (PBS, n = 4; AMD3100, n = 4) and B) EAAT2-Glutamate transporter Excitatory Amino Acid Transporter 2 (PBS, n = 3; AMD3100, n = 3). The levels were evaluated by western blot analysis (Image Studio. 5.2 Li-Core) after twice/week s.c administration of AMD3100 (5 mg/Kg mouse) for two weeks. Markers were normalized to their actin levels. Results are presented as % of Aβ_25–35-injected and PBS-treated mice values (set as 100%). Bars represent the mean±SEM. ^*p < 0.05; ^***p < 0.001.

AMD3100 treatment upregulated bone marrow-derived hematopoietic stem cell markers (Sca1, c-Kit)

Sca1 (Ly6A/E) and c-kit (CD117) are markers of murine hematopoietic stem and progenitor cells [41, 42]. Accordingly, their expression in mice hippocampi may suggest that AMD3100 induced the mobilization of hematopoietic stem and progenitor cells, and may imply that bone marrow-derived microglia cells may be present due to the recruitment of HSCs. Following administration of AMD3100, 170% elevation in Sca1 (Fig. 3A) and 24% (Fig. 3B) elevation in c-Kit levels in the hippocampus of Aβ_25–35-injected mice were observed (Fig. 3).

Fig. 3

Upregulation in markers of HSPC following the AMD3100 treatment. A) Sca-1 (stem cell antigen 1). B) c-Kit. Both markers are expressed on the surface of mice HSC. Markers were evaluated by western blot analysis (Image Studio™. 5.2 Li-Core). Markers were normalized to their actin levels. Results are presented as % of Aβ_25–35 injected and PBS-treated mice values (set as 100%). Bars represent the mean±SEM. ^*p < 0.05.

Remyelination

The correlation between the effect of AMD3100 on MCT1 level and remyelination was analyzed by MBP and Luxol Fast Blue staining assay. Demyelination is a well-known pathology manifesting in AD [43]. MBP is a component of the myelin sheath and is known to be downregulated in AD patients [44] and in the 3xTg [45] and APP/PS1 [46] AD mouse models. Several isoforms of classic MBP are formed by differential splicing of a single mRNA transcript [47]. It has been found that MBP downregulation is correlated with MCT1 downregulation in an APP/PS1 mouse model, as both are associated with oligodendrocytes [45, 46]. In this study, MBP protein levels were significantly elevated (by 40–60%, Fig. 4A). In addition, Luxol Fast Blue staining coincided with the protein level increase, showing a 130% increase of total myelin in the AMD3100 + L-lactate treated group (Fig. 4B). The observed elevated levels of myelin may be attributed to the ability of AMD3100 and L-lactate to nourish oligodendrocytes, which is characterized by elevated MBP levels and remyelination.

Fig. 4

The effects of treatment on Aβ_25–35-injected mice. Remyelination in treated Aβ_25–35-injected mice (PBS, n = 4; Lactate, n = 3; AMD3100, n = 3; AMD3100 + lactate, n = 3) demonstrated by A) MBP isoforms (15, 16, 18, 21 KDa) in results observed from western blot analysis of mice’s brain lysates. Markers were normalized to their actin levels. The levels of MBP’s derivatives were evaluated using Image Studio Lite (Li-Core). Bars represent the mean±SEM. B) Luxol Fast Blue Staining assay. Display of the % of staining area of representative coronal section –25 u of the left hemisphere from the hippocampus close to the coordinates of Aβ_25–35 injection of the brain of mice injected with Aβ_25–35 and treated with PBS (n = 3, 6 sections/mouse) or the combination (n = 3, 6 sections/mouse). Quantification analysis of stained sections was obtained by ImageJ software. Results are presented as % of Aβ_25–35 injected and PBS-treated mice values (set as 100%). Bars represent the mean±SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Neuroinflammation

The combined treatment of AMD3100 + L-lactate resulted in the alteration of the levels of several markers, which may be correlated with microglial polarization shift from the neurotoxic M1 profile to presenting neuroprotective M2 phenotypes (Fig. 5A). Increase in TREM2 and CX3CL1 (fractalkine) levels by 250% and 130%, respectively, may indicate an increase in microglia activity. Significant elevation in TREM2 levels may correlate with the elevation of phagocytic activity of further microglia-like cells recruited by AMD3100 and by the contribution of lactate as an energy source. Interestingly, our data show downregulation of IL-6 (–40%), TNFα (–30%), and MCP-1 (–80%), with upregulated expression of IL-4 (+55%) and IL-10 (+35%). This observation may be indicative of an anti-inflammatory effect of the treatment, and might support the polarization shifting of microglia toward the M2 state. Moreover, the M2 state was further confirmed by upregulation of Arg1 by 40%. Based on many studies focusing on microglia’s role in neuroinflammation, we must keep in mind that microglia in various neurodegenerative diseases exhibit mixed profiles expressing elevated levels of both pro- and anti-inflammatory molecules. This variation may be explained by the relapsed onset time and the disease stage [48 –51].

Neuroplasticity

Synaptophysin is an integral membrane glycoprotein presenting in presynaptic vesicles of neurons. Loss of the presynaptic vesicle protein synaptophysin in the hippocampus correlates with cognitive decline in AD [52]. BDNF is an essential neurotrophin for the development, maintenance, and survival of the central and peripheral nervous system [53], and accordingly is crucial to plasticity and cognitive function. AMD3100 + L-lactate showed significant increase in both BDNF (+30%) (Fig. 5B.1) and synaptophysin (+60%) (Fig. 5B.2).

Fig. 5

Effect of the treatment combination on common AD pathologies. A) Microglia activity evaluated by TREM2 (1) and CX3CL1(neurons) (2). TNFα (3), IL-6 (4), and MCP1 (5) anti-inflammatory effect was also evaluated by IL-4 (6), IL-10 (7), and Arg1 (8). B) Synaptic plasticity. (1) Secreted brain-derived neurotrophic factor (BDNF) combination treatment (n = 3) compared to the untreated group (PBS, n = 4), AMD3100 (n = 3), and Lactate (n = 3). (2) Synaptophysin regulation in the combination treatment group (n = 3) compared to AMD3100 (n = 3), treatment group lactate (n = 3), and the PBS-treated group (n = 3). C) (1) AβPP processing in Aβ_25–35-injected mice’s hippocampi. Processing of AβPP is presented as ratio of pAβPP/AβPP (C-term). Both AβPP and pAβPP were normalized to their actin levels. (PBS n = 4, lactate n = 3, AMD3100 n = 3, combination n = 3). (2) BACE1-beta-site AβPP cleaving enzyme 1: AMD3100 + lactate (n = 3), AMD3100 (n = 3), lactate (n = 3) versus PBS (n = 4). D) (1) Phosphorylation of GSK obtained by the ratio pGSK/GSK3_α/β combination treatment (n = 3), PBS (n = 4), AMD3100 (n = 3), lactate (n = 3). (2) Tumor suppressor p53: The combination treatment (n = 3) compared to AMD3100 (n = 3), lactate (n = 3), PBS (n = 4). E) Hyperphosphorylation of Tau in Aβ_25–35 injected mice: AT-8 (1) and PHF1 (2) antibodies were used to evaluate phosphorylation of tau. Total tau was evaluated by tau-5 (3) antibody. Tubulin (4) was also evaluated. (PBS n = 4, Lactate n = 3, AMD3100 n = 3, combination n = 3). F) BBB integration was evaluated by: (1) S100B the combination treatment (n = 3), AMD3100 (n = 3) compared to the PBS-treated group (n = 3), and (2) occludin levels. The combination (n = 4) and AMD3100 (n = 4) compared to PBS-treated group (n = 4). Markers were normalized to their actin levels. Results are presented as % of Aβ_25–35-injected and PBS-treated mice (set as 100%). Bars represent the mean±SEM. ^*p < 0.05; ^**p < 0.01.

Tau and AβPP

The attenuation in neuroinflammation may be associated with AβPP and tau pathologies. In this study, we evaluated AβPP phosphorylation levels at T668 (pAβPP) versus CT-AβPP levels by western blot. pAβPP levels were significantly reduced (by 40–50%, Fig. 5.1) in all treatments. Furthermore, significant reduction in BACE1 levels (–80%) was observed in the combined-treated group (Fig. 5C.2). Many studies have shown a correlation between AβPP processing and phosphorylation of GSK-3β and tau, together with activation of p53 and even neuroinflammation [54 –57]. In our study, both GSK and p53 were significantly elevated in response to Aβ_25–35 intrahippocampal injection (Supplementary Figure 3). pGSK/GSK ratio in mice hippocampi treated with AMD3100 + L-lactate was significantly downregulated (–50%, Fig. 5D.1).

Hyperphosphorylated tau is known to be neurotoxic and to promote the neuronal injury and cell death observed in AD brains [58]. Total human tau (tau5) was reduced by 25% in response to AMD3100 + L-lactate treatment (Fig. 5E.3). The hyperphosphorylated form of tau, PHF1 (Fig. 5E.2), was significantly downregulated (–49%) in the AMD3100 treatment group and in the combined treatment group (–41%). However, evaluation of hyperphosphorylated tau by AT-8 showed significant downregulation in the AMD3100 group (–46%) and downregulation (–32%), but not significantly, in the combined treatment group (Fig. 5E.1). This result may be explained due to non-specificity of AT-8 antibody known to react with other non-tau proteins that may be generated in response to L-lactate treatment [59]. Tubulin was not affected by any of the treatments (Fig. 5E.4). Here we demonstrated the correlation between several signaling cascades involving the regulation of TNFα, AβPP, BACE1, GSK3β, p53; and tau expression followed the Aβ injection and was restored by the combined treatment.

Blood-brain barrier integrity

The effect of the combined treatment of AMD3100 + L-lactate on blood-brain barrier (BBB) integrity was demonstrated by the protein levels of S100B (Fig. 5F.1) and occludin (Fig. 5F.2). Occludin levels were elevated by 50% in mice treated with the AMD3100 + L-lactate, compared to the control group (PBS-treated). A significant decrease in S100B levels was also demonstrated (∼–40%) compared to the non-treated group. This evidence of improvement in the BBB markers levels can be attributed to HSCs infiltration, microglia state, and the release of growth and neurotrophic factors, as implied by the results that were previously shown.

Cognitive functions

In order to assess the effect on cognitive function following each of the treatments, we used spatial short-term memory, T maze learning, and long-term contextual fear conditioning tests.

According to the analyses of the behavioral tests, the cognitive impairment depicted previously in the Aβ_25–35 acute model was significantly attenuated by the combined AMD3100 + L-lactate treatment as can be concluded by both T-maze and contextual fear conditioning tests (Fig. 6). T-maze analysis of ratio variance of the time spent and the number of visits in the novel arm versus the familiar arm is presented in Fig. 6A. T-maze analysis of ratio variance shows that mice treated with AMD3100 + L-lactate spent 333% more time in and 123% more frequently visited the novel arm than did the PBS-treated mice. Fear conditioning test results show longer freezing time (132%) in the AMD3100 + L-lactate treated group compared to the PBS-treated mice (Fig. 6B).

Fig. 6

AMD3100 + Lactate effect on cognitive impairment alleviation following Aβ_25–35 intrahippocampal injection. Mice were subjected to A) short-term spatial memory test in a T-maze. The results show the time spent in the novel arm and the frequency of visits thereto in terms of the percentages of ratios of novel/familiar arms compared to the PBS-treated group (set as 100%); and to B) long-term spatial memory test-fear conditioning. Bars represent the mean±SEM. Statistical comparisons were performed using student T-test. ^*p < 0.05; ^***p < 0.005 versus no treatment control group. Time and video recorded using Noldus, EthoVision XT 11.5 software.

Chronic administration of AMD3100 in the 3xTg AD model

Although AMD3100 is considered a safe drug with minor side effects [22, 24] and more than 80 completed clinical trials (ClinicalTrials.gov), its original purpose was acute daily course for up to one month. Regarding the beneficial effect of the short treatment and regarding the need for extended usage in clinical trials for AD treatment, we were next interested in whether it can be used for long course treatment with no adverse effect. Taking into account possible side effects, we also tested a lower dosage of 0.5 mg/kg AMD3100 with the original dosage (5 mg/kg) in a chronic treatment of 3xTg-AD mice.

Cognitive functions

3xTg-AD mice were treated with AMD3100 or PBS for four months and then subjected to Y-maze and fear conditioning testing. Mice treated with 5 mg/kg AMD3100 spent nearly 50% of the time in the novel arm in the Y-maze test (which reflects +182% more time spent that the PBS-treated group); in contrast, mice treated with 0.5 mg/kg AMD3100 spent only 25% of their time in the novel arm (Fig. 7A). Consistently, when subjected to fear conditioning testing, 5 mg/kg AMD3100-treated mice also exhibited better cognitive function than did 0.5 mg/kg AMD3100-treated mice (Fig. 7B). More specifically, on the next day of the electric shock, 5 mg AMD3100-treated mice exhibited significantly longer freezing time (+383%) when introduced to the same arena than did the PBS group. The 0.5 mg/kg AMD3100-treated group showed non-significant improvement in this test (+233%) compared to the PBS group.

Fig. 7

Cognitive function following chronic AMD3100 treatment versus PBS-treated mice. 3xtg-AD mice were treated with 5 mg AMD3100 (n = 4); 500μg AMD3100 (n = 5); PBS (n = 5) starting at 8 months of age. After four months of treatment, mice were subjected to cognitive testing. A) Y-maze learning test: Both duration spent in and frequency of visits to each arm were recorded. B) Fear conditioning test using a FreezeFrame automated scoring system (Coulbourn Instruments). Results are presented as % of PBS-treated mice (set as 100%). Bars represent the mean±SEM. ^*p < 0.05.

Neuroinflammation (TNFα, IL-6)

The 3xTg-AD mouse model is known to demonstrate age-dependent TNFα changes in the entorhinal cortex, which are strongly correlated with learning and memory deficits. In this model, elevation in TNFα levels was found to precede the onset of overt extracellular amyloid or tau pathology in the brain [60, 61]. The correlation between TNFα levels and cognitive decline was is well demonstrated by a 6-month study: AD patients with systemic inflammatory events exhibited high levels of TNFα in their serum, which were associated with a two-fold decrease in cognitive function during the study. In the same study, subjects that maintained low baseline levels of TNFα over the same period exhibited no cognitive decline [62]. Our data show that following four months of treatment with AMD3100, 3xTg-AD mice treated with 5 mg/kg exhibited a significant decrease in TNFα levels (–44%) compared to control mice (Fig. 8A.1) with a corresponding significant decrease in IL-6 levels (–24%) in those treated with the same dose (Fig. 8A.2). No significant difference between the lower dose (0.5 mg/kg) and the higher dose of AMD3100 was evident regarding both cytokines. As the level of neuroinflammation markers (TNFα and IL-6) is known to be correlated with both tau and Aβ pathologies, we further evaluated AMD3100’s effect on both phenotypes associated with the 3xTg-AD mouse model.

Fig. 8

Chronic AMD3100 effect on AD pathologies A) neuroinflammation (1) TNFα and (2) IL-6 levels in 3xtg-AD mice. 3xtg-AD mice were treated with 5 mg AMD3100; 500μg AMD3100; PBS, starting at 8 months of age. Mice were sacrificed at 12 months of age, and their brains were subjected to biochemical analysis of cytokine levels. B) Aβ pathology. (1) Aβ_1–42 levels measured using sandwich ELISA. (2) pAβPP levels (normalized to total AβPP levels) measured using western blot. C) AMD3100 reduces tau pathology. Western blot analysis of various forms of tau levels. (1) AT-8 levels. (2) PHF1 levels. (3) Tau-5 levels. D) AMD3100 increases BBB integrity-western blot analysis of BBB permeability markers. (1) S100B and (2) occludin levels. All results were normalized to the control (PBS-treated) group, which were set to 100%. Bars represent the mean±SEM. ^*p < 0.05; ^**p < 0.01.

Aβ pathology

Sandwich ELISA of brain homogenates of AMD3100-treated mice was employed to measure Aβ_1–42 levels in soluble and membrane-enriched fractions. A significant change was observed in the membrane fraction, yielding more than 30% reduction in Aβ_1–42 (Fig. 8B.1) in the 5 mg AMD3100-treated mice compared to the PBS-treated group, with no significant effect in the 0.5 mg AMD-treated mice. No significant change in the soluble fraction was observed. Further evaluation of pAβPP at T668 showed significant decrease (35%) in 5 mg AMD3100-treated mice (Fig. 8B.2).

Tau pathology

3XTg mice manifest age-dependent tau pathology. No AT8 immunoreactivity was found in 2-month-old 3xTg mice, while it did appear in 100% of 6-month-old 3xTg mice. Several studies have adopted TNFα inhibition approaches that demonstrated attenuation in tau pathology: 6-week treatment with 3,6’ dithiothalidomide, an immunomodulatory known to lower TNFα protein levels, reduced tau hyperphosphorylation [63]. Knock-out of TNFα receptor 1 (TNFR1) reduced tau pathology in 3xTg-AD mice [64]. Infliximab, a TNFα antibody that was administered intracerebroventricularly to 12-month-old APP/PS1-AD mice, resulted in rapid reduction in brain TNFα, Aβ, and tau phosphorylation levels [65].

3xtg-AD mice treated with both 5 mg and 0.5 mg AMD3100 showed significantly reduced tau pathology as evidenced by the various tau markers (Fig. 8C). AT-8 was reduced by 38% in the 5 mg AMD3100-treated group, and by 34% in the 0.5 mg AMD3100-treated group (Fig. 8C.1). PHF1 was reduced by 36% in the 5 mg AMD3100-treated group, and by 34% in the 0.5 mg AMD3100-treated group (Fig. 8C.2). Tau-5 levels were reduced by 31% in both AMD3100-treated groups (Fig. 8C.3).

Blood-brain barrier integrity

Tight junction protein occludin was significantly upregulated (five-fold) by both AMD3100 doses (Fig. 8D.1), with a significant decrease of 16% in S100B levels in the 5 mg AMD3100-treated mice compared to the PBS-treated mice (Fig. 8D.2). No significant change in S100B levels was observed in 0.5 mg AMD3100-treated mice.

DISCUSSION

Here we showed that administration of a combination of AMD3100 and L-lactate may have beneficial effects in the treatment of AD. We investigated the effects of subcutaneous administration of AMD3100/lactate into two different Aβ-induced AD mouse models following the recovery of their cognitive/memory functions and alleviation of AD related pathologies. The observation that AMD3100 as a result of signaling inhibition the axis, induced overexpression of MCT1 the main L-lactate transporter in the brain [26], motivated us to use the combination of AMD3100 and L-lactate as a potential therapeutic approach to AD. The metabolism of glucose is known to be impaired in AD [66], as well as deficiencies in lactate and MCT1 transporters in the human AD brain and in the mouse APP/PS1 model [67]. Notably, replacement of extracellular glucose with sodium lactate was shown to sustain synaptic transmission after transient glucose depletion [68]. Lactate is a neuroprotectant and significant energy source for the brain under stress conditions [69, 70]. Evidence for L-lactate activation as a signaling molecule suggests that interference with the lactate/pyruvate pathway, ATP production, and activation of a P2Y/KATP cascade lead to neuroprotection [71].

The AD acute rodent model, which is based on the amyloid cascade theory, is one of the main non-transgenic mouse models that has been studied over the past 30 years since its establishment [29]. Glia, activated by Aβ_25–35, can synthesize and release a variety of inflammatory proteins and cytokines, such as cyclooxygenase-2 (COX-2), IL-1, IL-6, and TNFα [72]. Two weeks after Aβ_25–35 intrahippocampal injection, mice displayed cognitive impairment as observed by T-maze and fear conditioning tests (Supplementary Figure 2), similarly to other behavioral tests observed in the literature [73 –77]. Furthermore, AβPP and CTF99 and p53 levels were markedly elevated following intrahippocampal injection of Aβ_25–35 (Supplementary Figure 3). Previous studies showed that p53 can affect GSK3β activity by inducing tau phosphorylation, and under stress, p53 forms a complex with GSK3β [54 , 79], resulting in increased GSK3β activity and consequent phosphorylation of tau. Our data show that alongside GSK3β activity, p53 levels (Fig. 5D.2) were also reduced by 20%, which may suggest amelioration of the neurodegenerative process in response to AMD3100 treatment alone, and even further by the combined treatment of AMD3100 + L-lactate. Tau and GSK3β phosphorylation levels, together with the deposition of neurofibrillary tangles and Aβ plaques, are indicative of successful induction of acute pathology in this mouse model (Supplementary Figure 3). The CXCL12/CXC4 axis-based mechanism in the AD brain suggests that Aβ plaques attract microglia to activate the inflammatory cascade. Binding of CXL12 to CXCR4 enhances activated microglia, and astrocytes trigger several Ca^2 +-dependent pathways, including the activation of kinases (MAPK pathway) and the release of pro-inflammatory cytokines such as TNFα [13], prostagandins, and glutamate. An important support for the migration and brain infiltration is that Aβ_1–40 and Aβ_1–42 induce the migration of BMDMLs to the brain, but not Aβ_1–31 or Aβ_1–57 [80].

Here, we demonstrate the beneficial effect of combined treatment as a potential therapeutic treatment in an acute mouse AD model. The combined treatment was found to recover AD cognitive function and related pathologies better then did each component alone. Notably, the improved cognitive deficit in the acute model may have resulted from attenuated tau and AβPP pathologies and the shift in microglia profile. Downregulation of IL-6, TNFα, and MCP-1 together with upregulation of IL-4 and IL-10 may imply the anti-inflammatory effect that we expected to achieve by changing the lineage of the microglia. This shift may be explained by several mechanisms: Firstly, recruitment of new microglia-like cells from the bone marrow may either change the resident microglial cell population and/or the release factors that may “recover” the resident cells profile. The recruited microglia, as well as neuronal cells, may be supported by the energy supply of lactate, which is more efficiently transported into the brain via elevated MCT1 expression. The second mechanism suggests that AMD3100 acts as a CXCR4 antagonist and therefore inhibits the toxic inflammation process and consequently the general phosphorylation process that eventually leads to attenuation in cognitive impairment. AMD3100 ameliorates AD’s main pathology by increasing both BDNF production and synaptophysin expression. The increase in BDNF and synaptophysin levels suggests that activation of the synaptogenesis process may act as a compensating mechanism for plasticity during neurodegeneration.

The presence of HSCs in the brains of treated mice coincided with BDNF production [81] and is also supported by lactate supplementation [70, 82]. Increase in BDNF is linked to the recruitment of the BMDML cells, as these cells benefit the brain by secreting bioactive factors that may, in a paracrine manner, convey intrinsic repair and enhance neurogenesis [1]. The presence of hematopoietic cells in the brain was confirmed by the appearance of markers such as c- KIT and Sca-1. Interestingly, only the combined treatment of AMD3100 and L-lactate increased BDNF and synaptophysin levels, both of which affect cognition directly.

Notably, the observed increase in MCT1 levels is the suggested mechanism by which lactate was transported into the brain in order to enable this cascade of events [83]. Moreover, lactate was shown to be involved in several cellular processes that include interference with the CXCL12/CXCR4 signaling axis, increase in BDNF levels, reduced intracellular Ca^2 + influx, and protection of neurons from glutamate toxicity [71, 82]. Regarding these accumulated data, our study shows that only when combining AMD3100 with lactate as a treatment in both AD mouse models cognitive functions were significantly improved based on two behavioral tests, correlates with the same significance of attenuating AD-like pathologies.

It has been found that downregulation of MBP is correlated with downregulation of MCT1 in the APP/PS1 mouse model, as both are associated with oligodendrocytes [46, 84]. Some evidence suggests that lactate transport by astrocytes may be utilized as an energy substrate by neurons and myelin formation [69]. MCT1 was identified as a target for p53, which leads to downregulation of its expression both in vitro and in vivo [85]. CXCR4 inhibition in acute myeloblastic leukemia (AML), U937 cell line, resulted in downregulation of p53 and p53-dependent genes [86]. These studies corroborate our results that show an increase in MCT1 expression following the inhibition of CXCR4 by AMD3100 accompanied by a decrease in p53 levels in the Aβ_25–35 acute mouse model, as well as increase in EAAT2 levels, which affect glutamate toxicity [87].

In sum, the short treatment with AMD3100 was found to have beneficial effects in the AD acute mouse model, by ameliorating cognitive deficit and reducing tau and AβPP pathologies while affecting microglial polarization, inducing remyelination and affecting BBB associated biomarker levels.

Further, we investigated whether chronic inhibition of the CXCL12/CXCR4 axis by AMD3100 may have clinical potential as a pharmacological therapy for AD, as all of the clinical trials with AMD3100 are based on acute administration for various indications [21]. HSCs’ niche is a specific site in the marrow cavity where HSCs reside and undergo self-renewal, proliferation, and differentiation. Thus, it is very likely that the chronic administration of AMD3100 at intervals of 36–48 h between injections, could enable HSCs to mobilize from “quiescent storage” niches, enter the cell cycle to expand, and then return to their marrow niches and become quiescent again [88]. We previously reported that chronic administration of 5 mg/kg AMD3100 to an SOD1^G93A mice model of amyotrophic lateral sclerosis improved the blood-spinal cord barrier integrity by increasing tight-junction proteins levels, decreasing pro-inflammatory cytokines, promoting remyelination, and increasing EAAT2 levels for the reuptake of glutamate, thus reducing glutamate toxicity. The reported treatment led to significant extension in SOD1^G93A mice lifespans, as well as improved motor function and weight loss [89].

In the present study, we treated a 3xTg-AD mouse model with AMD3100 for four months. The 3×Tg-AD mouse model demonstrates age-dependent changes in TNFα in the entorhinal cortex that strongly correlate with learning and memory deficits. The chronic AMD3100 treatment led to significant decrease in TNFα levels, followed by significant improvement in cognitive function of treated mice, evaluated by two different cognitive tests: Y-maze, and fear conditioning. Furthermore, AMD3100 treatment of 3xtg-AD mice was shown to significantly improve tau pathology as expressed by several markers: AT-8, PHF1, and tau 5. Here, we showed that modulation of TNFα by AMD3100 led to a decrease in Aβ_1–42 levels. Administration of AMD3100, simultaneously with reduction in TNFα levels, might have functioned together to increase mobilization of the HSPC population in the diseased brains.

Taken together, the multifaceted role of inhibition of the CXCL12/CXCR4 signaling pathway may result in prevention of neuroinflammation via mobilization of HSCs from the bone marrow to the bloodstream. As AMD3100 is a direct antagonist of the interaction between CXCL12 and its receptor CXCR4, AMD3100 mobilizes HSCs within hours rather than days. In addition, AMD3100 is known to be well tolerated, with no significant side effects, as supported by various clinical trials conducted to date [22].

Our experimental data supports this notion and suggests AMD3100 as a safe and effective mobilizer of endogenous hematopoietic stem cells. We suggest that AMD3100 can be considered as an alternative approach for the multistep procedures of transplantation of stem cells in the treatment of AD. The therapeutic approach that we presented herein not only opens up an innovative path to intervene in AD, but may also drive clinical trials in shorter time frames, as we focus on repurposing of existing drugs that pharmacokinetics and pharmacodynamics have already established and on which a number of clinical trials are still running [90].

Footnotes

ACKNOWLEDGMENTS

We thank Prof. Frank M. LaFerla for providing the 3xTg mice and Dr. P. Davies for the PHF1 antibody.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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Subcutaneous Administration of AMD3100 into Mice Models of Alzheimer’s Disease Ameliorated Cognitive Impairment,Reduced Neuroinflammation,and Improved Pathophysiological Markers

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Ethics statement

Animals

Acute treatment

Preparation of Aβ25–35 peptide

Characterization of Aβ25–35 aggregation

Acute AD mouse model: Experimental procedures

Treatments administration

Cognitive tests

T-maze

Contextual fear conditioning

Chronic treatment

Transgenic mice and treatment

Cognitive tests

Y-maze

Contextual fear conditioning

Brain homogenates preparation

Western immunoblot analysis

Sandwich ELISA for IL-10 measurement

TNFα measurement

Sandwich ELISA for Aβ1–42 measurement

Histology sections

Luxol blue staining

Statistical analysis

RESULTS

Acute AD mouse model

AMD3100 increases MCT1 and EAAT2 transporters levels

AMD3100 treatment upregulated bone marrow-derived hematopoietic stem cell markers (Sca1, c-Kit)

Remyelination

Neuroinflammation

Neuroplasticity

Tau and AβPP

Blood-brain barrier integrity

Cognitive functions

Chronic administration of AMD3100 in the 3xTg AD model

Cognitive functions

Neuroinflammation (TNFα, IL-6)

Aβ pathology

Tau pathology

Blood-brain barrier integrity

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Preparation of Aβ_25–35 peptide

Characterization of Aβ_25–35 aggregation

Sandwich ELISA for Aβ_1–42 measurement