An Intranasal Formulation of Erythropoietin (Neuro-EPO) Prevents Memory Deficits and Amyloid Toxicity in the APP Swe Transgenic Mouse Model of Alzheimer’s Disease

Abstract

Erythropoietin (EPO) is a cytokine known to have effective cytoprotective action in the brain, particularly in ischemic, traumatic, inflammatory, and neurodegenerative conditions. We previously reported the neuroprotective effect of a low sialic form of EPO, Neuro-EPO, applied intranasally in rodent models of stroke or cerebellar ataxia and in a non-transgenic mouse model of Alzheimer’s disease (AD). Here we analyzed the protective effect of Neuro-EPO in APP_Swe mice, a reference transgenic mouse model of AD. Mice were administered 3 times a day, 3 days in the week with Neuro-EPO (125, 250 μg/kg) intranasally, between 12 and 14 months of age. Motor responses, general activity, and memory responses were analyzed during and after treatment. The deficits in spontaneous alternation, place learning in the water-maze, and novel object recognition observed in APP_Swe mice were alleviated by the low dose of Neuro-EPO. Oxidative stress, neuroinflammation, trophic factor levels, and a synaptic marker were analyzed in the hippocampus or cortex of the animals. The increases in lipid peroxidation or in GFAP and Iba-1 contents in APP_Swe mice were significantly reduced after Neuro-EPO. Activation of intrinsic and extrinsic apoptotic pathways was analyzed. The increases in Bax/Bcl-2 ratio, TNFα, or Fas ligand levels observed in APP_Swe mice were reduced by Neuro-EPO. Finally, immunohistochemical and ELISA analyses of Aβ_1–42 levels in the APP_Swe mouse cortex and hippocampus showed a marked reduction in Aβ deposits and in soluble and insoluble Aβ_1–42 forms. This study therefore confirmed the neuroprotective activity of EPO, particularly for an intranasally deliverable formulation, devoid of erythropoietic side effects, in a transgenic mouse model of AD. Neuro-EPO alleviated memory alterations, oxidative stress, neuroinflammation, apoptosis induction, and amyloid load in 14-month-old APP_Swe mice.

Keywords

Alzheimer’s disease erythropoietin intranasal formulation learning and memory Neuro-EPO neuroprotection

Introduction

Erythropoietin (EPO) is a 30-kDa glycoprotein regulating erythropoiesis. It inhibits programmed cell death in erythroid cells, regulates the proliferation and differentiation of erythroid precursor cells and allows the maturation of erythrocytes [1]. EPO is expressed in the kidney and liver, but also in tissues not involved in erythropoiesis, such as reproductive tract, lung, spleen, heart, and brain [2 –6]. EPO interact with specific receptors, mainly the EPO receptor (EPOR), acting as a homodimer belonging to the class 1 cytokine receptors, or through EPOR forming heterodimers with the β-common receptor (βCR, also targeted by the granulocyte macrophage colony-stimulating factor, interleukin 3 and interleukin 5) [7 –9]. The cytoprotective effects of EPO were established in several organs and particularly the central nervous system [10, 11]. EPO and EPOR are upregulated upon neuronal injury and neurodegeneration, such as in the temporal cortex and hippocampus of patients with mild cognitive impairment or Alzheimer’s disease (AD) [12]. Experimental injection of EPO improved neurologic functions and reduce brain damage following cerebral ischemia, intracerebral hemorrhage, traumatic brain injury, spinal cord injury, experimental autoimmune encephalitis, status epilepticus, neonatal hypoxia-ischemia, and amyotrophic lateral sclerosis [13 –18]. Interestingly, both EPOR and βCR are highly expressed in the brain [11] and the [EPOR/βCR] heterodimer has been proposed to be responsible for the majority of the neuroprotective actions of EPO, although some clearly EPOR-independent neuroprotective effects have been described [8]. Indeed, carbamylated EPO (CEPO) and sialic acid-depleted CEPO do not interact with EPOR but are potent neuroprotectants [19, 20]. EPO was considered as a master regulator of injuries and healing in diverse preclinical models of disease and is currently under evaluation in advanced clinical trials as a disease-modifying agent in painful neuropathy and diabetes [8].

In AD models, both in vitro and in vivo evidences for a neuroprotective effect of EPO have been obtained. Recombinant Human EPO (rHu-EPO) protected neurons from neurodegeneration induced by amyloid-β (Aβ) peptides in cell culture models [21 –23]. In vivo, EPO prevented the learning and memory deficits induced in rats injected intracerebroventricularly (ICV) with streptozotocin (STZ), a pharmacological model of acute AD-like toxicity [24]. This effect was accompanied by a marked effect of EPO on newborn cell proliferation in the hippocampus in the STZ-treated animals. In aged APP_Swe (Tg2576) mice, a transgenic model of AD, intraperitoneal (IP) administration of EPO improved contextual memory and enhanced endothelial proliferation, synaptophysin expression, and capillary density in the brain [25]. Finally, in the APP/PS1 mouse model of AD, a 4-week treatment with CEPO or EPO was recently reported to modulate the expression of a variety of genes involved in neurotransmission and neuroregulation [26].

A new EPO formulation with a low content in sialic acid, and therefore devoid of hematopoietic effects, Neuro-EPO, is able to rapidly reach the brain after intranasal (IN) delivery [27 –29]. The formulation has the required composition to remain an appropriate time in the nasal grave, avoiding natural elimination or cleaning of the silial mucus. pH, volume, and concentration were optimized to allow the Neuro-EPO release through the smell road into the cerebrospinal fluid and brain. We previously reported, in the acute AD mouse model induced by ICV injection of Aβ_25–35 oligomers [30, 31], that either rHu-EPO, administered intraperitoneally (IP), or Neuro-EPO, administered IN, showed potent neuroprotective activity [32]. rHu-EPO or Neuro-EPO was repeatedly administered during 4 days after the Aβ_25–35 peptide injection. The two EPO formulations efficiently prevented the behavioral deficits, analyzed using the spontaneous alternation, passive avoidance, place learning in the water-maze, and novel object recognition, and the biochemical and morphological hallmarks of Aβ toxicity, including induction of pro-apoptotic proteins, oxidative stress, induction of pro-inflammatory cytokines, and cell loss in the hippocampal pyramidal cell layers. Neuro-EPO was effective at a lower dose than rHu-EPO, i.e., 125 μg/kg.

In the present study, we analyzed the neuroprotec-tive activity of Neuro-EPO, following a chronic IN administration regimen, in a transgenic APP_Swe mouse line, the Tg2576 line established by Hsiaoet al. [33] and overexpressing hAPP⁶⁹⁵ with thefamilial Swedish double mutation (Lys670Asn,Met671Leu), under the control of a prion promoter.These mice present numerous parenchymal Aβ pla-ques by 11–13 months of age with some vascularamyloidopathy [33]. They show oxidative stressbut no evidence of hyperphosphorylated tau or neuro-fibrillary tangles. Dendritic spine loss and declinein long-term potentiation in the dentate gyrusafter perforant path stimulation has been reported as early as by 4-5 months, in the CA1 region of the hippocampus [34 –37]. Moreover, APP_Swe mice showed impaired learning at spatial tasks, working memory, and contextual fear conditioning at less than 6 months, although most of the studies report more progressive impairments until 12 months of age [38, 39]. Both wild-type (WT) and APP_Swe mice were treated IN with 0, 125, or 250 μg/kg Neuro-EPO 3 times a week for 2 months, starting at 12 months of age, so at a period when the pathology is progressing. Behavioral, biochemical, and immunohistochemical analyses were performed during and after treatment and showed a clear neuroprotective effect of the lowest dose tested on almost all aspects of the toxicity, including a substantial decrease of amyloid load in these animals.

Material and Methods

Animals

Tg2576 mice overexpressing APP_Swe, and C57BL/6xSJL WT controls were purchased from Taconic (Lelystad, Netherlands). Animals were received at 2 months of age and housed in the animal facility of the University of Montpellier. They were kept in groups, with free access to food and water. Temperature, humidity, and light were controlled (22±1°C; 55±5%; 12 h/12 h light/dark cycle, respectively). Animals were used between 12 to 14 months of age. Behavioral experiments were carried out between 10 : 00 h and 16 : 00 h, in the experimental room after 30 min habituation period. All animal procedures were conducted in adherence to the European Union directive 2010/63.

Drugs, injections, and experimental series

Erythropoietin (EPO) modified to display lowsialic acid content (Neuro-EPO, patents PCT/cu2006/000001 and 20050138 to CIDEM, Havana, Cuba) were supplied by the Center of Molecular Immunology (Havana, Cuba) through CIMAB SA (Havana, Cuba) and diluted in phosphate buffer saline (pH 7.0) at 0.15 mM. Neuro-EPO was applied intranasally (IN) three times a day (at 08 : 00 h, 12 : 00 h and 16 : 00 h) and 3 days a week, as summarized in Fig. 1a. Treatment lasted 2 months and animals were 12 months old at the beginning of the treatment (Fig. 1b). Neuro-EPO was administered at 125 and 250 μg/kg IN [32, 40]. Control groups received phosphate buffer saline solution (IN). IN injections were performed by an experienced experimenter and the physical and behavioral status of the animals checked daily. Housing parameters (state of fur, behavior in home cage, absence of prostration, level of aggressivity, etc.) did not change during the treatment. Only weight loss and attrition where therefore quantified.

Animals were tested in the spontaneous alternation test after one month of treatment (Fig. 1b). They were then subjected to spontaneous alternation, place learning in the water-maze and novel object recognition, and other behavioral procedures after 2 months of treatment (Fig. 1b). At the end of the behavioral sessions, animals were sacrificed and their hippocampus and frontal cortex dissected out on ice for biochemical analyses or animals were transcardiallyperfused with paraformaldehyde for histology and immunohistochemistry.

Clasping and escape responses

Clasping was analyzed as the loss of abduction reflex when the mouse was suspended by the tail. Animals were suspended by the tail, fixed using tape on a horizontal rod and their behavior videotaped during a during a 5-min observation session. The clasping response was coded as: 0, stable abduction posture; 1, difficulty to maintain the posture; 2, no abduction observed. The escape response was analyzed as the rapidity the animal showed to escape from unrestraint contention. The animal was kept under the experimenter’s hand, and the hand was opened after 10 s leaving free to the mouse. Escape was coded as: 0, no escape; 1, escape after more than 5 s; 2, escape within 0–5 s. Results were then averaged and presented as arbitrary units.

Spontaneous alternation in the Y maze

Animals were tested for spontaneous alternation performance in the Y-maze, an index of spatial working memory. The Y-maze is made of grey PVC. Each arm is 40 cm long, 13 cm high, 3 cm wide at the bottom, 10 cm wide at the top, and converged at an equal angle. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an 8 min session. The series of arm entries, including possible returns into the same arm, were checked visually. An alternation was defined as entries into all three arms on consecutive occasions. The number of maximum alternations was therefore the total number of arm entries minus two and the percentage of alternation was calculated as (actual alternations / maximum alternations)×100. Parameters included the percentage of alternation (memory index) and total number of arm entries (exploration index). Animals that performed less than 8 arm entries in 8 min or showed alternation percentage <20% or >90% were discarded from the calculations. Attrition was 11.3% in this study.

Place learning in the water maze

The water-maze was a circular pool (diameter 140 cm, height 40 cm). The water temperature, 23±1°C, light intensity, external cues in the room, and water opacity were rigorously reproduced. A transparent Plexiglas non-slippery platform (diameter 10 cm) was immersed under the water surface during acquisition. Swimming could be recorded using Videotrack^® software (Viewpoint, Lissieu, France), with trajectories being analyzed as latencies and distances. The software divided the pool into four quadrants, with the platform set in the middle of one quadrant.

Acquisition. Training consisted in 3 swims per day for 5 days, with 20 min intertrial time interval. Start positions, set at each limit between quadrants, were randomly selected and each animal was allowed a 90 s swim to find the platform. Swimming latency was measured using a stopwatch. Animals were left on the platform during 20 s. Animals that did not find the platform after 90 s had elapsed were placed on it manually and left for 20 s. The median latency was calculated for each training day and expressed for the experimental group as mean±S.E.M.

Retention. A probe test was performed 24 h after the last acquisition session. The platform was removed and each animal was allowed a free 60 s swim. The start position for each mouse was one of the two positions remote from the platform location in counterbalanced order. The swimming was videotracked and the time spent in the training (T) quadrant was analyzed. No exclusion criterion has been set in this test.

Novel object recognition memory

The apparatus consisted in four squared open-fields (50 cm×50 cm×50 cm high) made in white Plexiglas and placed on a floor equipped with infrared (IR) light emitting diodes. The locomotor activity of the animal and position of their nose could captured through an IR-sensitive camera and analyzed using the Videotrack^® and Nosetrack^® softwares (Viewpoint).

Session 1: animals were allowed to acclimate during 10 min to the open-field.

Session 2: two identical objects (50 ml plastic vials with caps) were placed at defined positions, at 1/4 and 3/4 of one diagonal of the arena. Each mouse was placed in the open-field and the exploratory activity and nose position was recorded during 10 min. The activity was analyzed in terms of number of contact with the objects and duration of contacts.

Session 3: the object in position 2 was replaced by a novel one (a soft plastic chair feet protection) differing in color shape and texture from the familiar object. Each mouse was placed again in the open-field and the exploratory activity recorded during 10 min. The activity was analyzed similarly. The preferential exploration index was calculated as the ratio of the number (or duration) of contacts with the object in position 2 over the total number (or duration) of contacts with the two objects. Animals showing less than 10 contacts with objects or no contact with one of the objects during the session 2 or 3 were discarded from the calculations. Sixteen animals were discarded accordingly in the present study (16.3% attrition).

Lipid peroxidation measures

Mice were sacrificed by decapitation and their brains rapidly removed, the hippocampus dissected out, weighed, frozen in liquid nitrogen, and stored at –80°C until assayed. After thawing, the hippocampus was homogenized in cold methanol (1/10 w/v), centrifuged at 1,000 g during 5 min, and the supernatant collected. Homogenate was added to a solution containing FeSO₄ 1 mM, H₂SO₄ 0.25 M, and xylenol orange 1 mM and incubated for 30 min at room temperature. Absorbance was measured at 580 nm (A₅₈₀1), and 10 μl of cumene hydroperoxide (CHP) 1 mM was added to the sample and incubated for 30 min at room temperature, to determine the maximal oxidation level. Absorbance was measured at 580 nm (A₅₈₀2). The level of lipid peroxidation was determined as CHP equivalents according to: CHP eq. = A₅₈₀1/A₅₈₀2×[CHP (nmol)]×dilution, calculated as CHP eq. per wet tissue weight, and presented as percentage of control group value.

ELISA assays

The hippocampus and cortex were dissected out on ice, weighed, frozen in liquid nitrogen, and stored at –80°C until analyses. After thawing, brain tissue was homogenized in 50 mM Tris-150 mM NaCl buffer, pH 7.5, and sonicated for 20 s. After centrifugation at 16,100 g for 15 min at 4°C, supernatants were used for ELISA assays according to the manufacturer’s instructions. The commercial kits used are summarized in Table 1. For each assay, absorbance was read at 450 nm and sample concentration was calculated using the standard curve (except for caspase-3 activity). All samples were assayed in triplicates or duplicates. Results were calculated as pg or ng of marker per mg of protein and then presented as % of V-treated WT control data.

Immunohistology

Each mouse was anesthetized using an IP injection of ketamine, 80 mg/kg, and xylazine, 10 mg/kg, and quickly transcardially perfused with 50 ml of saline solution followed by 50 ml of paraformaldehyde 4%. Brains were removed and kept overnight in the fixative solution. Brains were processed and included in paraffin. They were cut in coronal sections (3–5 μm thickness) using a microtome (Histo Line laboratories, Milano, Italy). Serial sections were selected to include the hippocampus formation and placed in glass strips. Sections were dewaxed in xylene and rehydrated through graded alcohols to PBS. The slides were placed in HercepTest K5207 (Dako, Les Ulis, France) for 5 min to eliminate endogenous peroxidase activity. Antigens were retrieved for 20 min at 97°C. After several rinses in phosphate buffer saline (PBS) pH 7.4, tissues were incubated for 30 min in the biotinylated goat anti-rabbit/mouse immunoglobulin solution. Sections were rinsed and incubated in avidin-biotin peroxidase complex solution for 30 min. The avidin-biotin-complex reaction was visualized by incubation in PBS containing 0.05% diaminobenzidine (DAB, Sigma-Aldrich) and 0.005% hydrogen peroxide for 5 min and then stopped by several washings in PBS. Antibodies were diluted in Da Vinci Green PBS pH 7.0 (Biocare Medical, Concord, CA, USA). The primary and secondary antibodies used are detailed in Table 1. Examination of the CA1 and dendate gyrus areas was performed on fields located between antero-posterior coordinates – 1.90 and – 2.30 from Bregma andlateral coordinates±1.50 to±1.75 [41], using a light microscope (Primo Star, Zeiss, Oberkochen, Germany). Slices were digitalized through a CCD camera (XC-77CE Sony, Japan) with the ImageJ^® software (NIH, Bethesda, MD, USA), to easily process visualization of astroglial cells and counting amyloid deposits. Data were calculated as average of 3-4 slices per animal and 3–5 animals per group, and expressed as number of amyloid plaques per squared millimeter.

Statistical analyses

Data were analyzed using one-way or two-way analysis of variance (ANOVA, F value), with genotype and Neuro-EPO treatment as independent factors. Post-hoc comparisons were done using the Dunnett’s test or one-sample t-test, as specified. Amyloid load were analyzed using a non-parametric Kruskal-Wallis ANOVA (H value), since variances were not equal among groups. Post-hoc comparisons were done using a Dunn’s test. Clasping and escape responses were analyzed using a Kruskal-Wallis ANOVA, followed by a Dunn’s test. Swimming latencies did not show a normal distribution, since a cut-off time was set. Acquisition profiles in the water-maze were analyzed using the non-parametric repeated-measure Friedman’s ANOVA (Fr value), followed by a Dunn’s or Mann-Whitney’s test for post-hoc comparisons. Probe test data were presented as times spent in the quadrants. Presence in the T quadrant was analyzed using a one-sample t-test versus the chance level (15 s). The object preference, calculated from the number of contacts with the two objects, was analyzed using a one-sample t-test versus the chance level (50%). All ANOVA results are detailed in the figure legends, for reading clarity. The level of statistical significance was p < 0.05. When the ANOVA value was <1, it could not reach significance whatever the degrees of freedom. It was therefore indicated as such (F < 1).

Results

Twelve-month-old animals were treated IN, 3 times a week, for two months with Neuro-EPO. Their general health state and body weight were systematically checked before each injection. WT animals did not show significant weight change during the 2-month injection period, but APP_Swe mice showed a very significant 5.2 g weight reduction during the period (Fig. 1c). The Neuro-EPO treatment failed to affect body weight in WT mice or the weight reduction observed in APP_Swe mice (Fig. 1c). Attrition during the treatment period was 17% – 19% in WT and APP_Swe mice (Fig. 1d). In WT mice, the Neuro-EPO treatment at the highest dose completely and significantly prevented mice death (Fig. 1d). In APP_Swe mice, the Neuro-EPO treatment tended to dose-dependently decrease attrition, but no significant effect was detected.

Protective effects of Neuro-EPO in APP_Swe mice: behavioral study

Two simple motor responses were checked in the animals at the end of the treatment. First, the clasping response (Fig. 2a) appeared highly significantly increased in APP_Swe mice as compared to WT. The Neuro-EPO treatment, at 250 μg/kg IN, failed to affect this response. Second, the escape response (Fig. 2b) was not significantly increased in APP_Swe mice. Interestingly, the Neuro-EPO treatment decreased the response in both the WT and transgenic mice, although not significantly in the latter (Fig. 2b).

During the habituation session of the novel object recognition test (see later), open-field parameters can be measured during the 10-min session. The total distance traveled in the arena (Fig. 2c) and the locomotor speed of the animals (Fig. 2d) failed to differ among groups. However, the presence in the center of the open-field, a direct measure of anxiety, was significantly decreased in APP_Swe mice as compared to WT controls (Fig. 2e). The Neuro-EPO treatment dose-dependently decreased this parameter, without effect on WT mice.

The spontaneous alternation performance in the Y-maze, a rapid test assessing spatial working memory and general exploration, was analyzed after 1 and 2 months of treatment (Fig. 3). After 1 month of treatment, APP_Swe mice showed an impairment of alternation as compared to WT animals (Fig. 3a). The Neuro-EPO treatment did not affect the performances of WT animals, but attenuated the deficit in APP_Swe mice, particularly at the 125 μg/kg dose (Fig. 3a). Animals showed a similar exploratory activity with 20–25 arm entries whatever the genotype or Neuro-EPO dose (Fig. 3b).

After 2 months of treatment, WT animals showed a marked decrease in alternation, but their performance remained significantly higher than the 50% hazard level (Fig. 3c). APP_Swe mice showed an impairment of alternation that was attenuated by the Neuro-EPO treatment, at both doses (Fig. 3c). Noteworthy, the highest dose of Neuro-EPO impaired alternation behavior in WT mice. Animals showed a decreased exploratory behavior, with only 15–20 arm entries during the session, but again similar among genotype and treatment groups (Fig. 3d).

At the end of the treatment, mice were submitted to place learning in the water-maze, a test assessing spatial reference memory (Fig. 4). The acquisition profile of WT animals showed that they progressively decreased their latency to reach the platform location, an effect unchanged in Neuro-EPO treated groups (Fig. 4a). APP_Swe mice showed a delayed acquisition profile, latencies measured during trials 2–5 being higher than observed for WT animals, with a very significant difference in trial 3 (Fig. 4b). The Neuro-EPO treatment, at both doses tested tended to decrease this delay. Quality of memory was measured in a probe test, performed with platform 24 h after the last training trial (Fig. 4c). All WT groups showed a significantly preferential exploration of the training (T) quadrant, but not Veh-treated APP_Swe mice. The Neuro-EPO treatment resulted in a restoration of the preferential presence in the T quadrant for both dose, with a significant effect at the 125 μg/kg dose (Fig. 4c). Indeed, typical trajectories presented in Fig. 3d illustrated that while a Veh-treated APP_Swe mouse explore all four quadrants during the 1-min duration swimming, a Neuro-EPO (125)-treated mouse markedly focused exploration in the T quadrant. Note that swimming speed measured during the probe test did not differ among groups (F_(2,82) = 1.72, p > 0.05 for the genotype, F < 1 for the treatment, F_(2,82) = 1.70, p > 0.05 for the interaction; data not shown).

Animals were then tested in the novel object recognition test (Fig. 5). Data were treated in terms of number of contacts (Fig. 5a, b) and duration of contacts (Fig. 5c, d). During session 2, when two similar objects were presented, animals failed to show a preference for the object in either position 1 or 2 (Fig. 5a, c). In session 3, when the object in position 2 was replaced by a novel one, control WT animals showed a strong preference for the novel object, while Veh-treated APP_Swe mice did not, for both parameters (Fig. 5b, d). In APP_Swe mice, the Neuro-EPO treatment at the lowest dose significantly restored the preference for the novel object (Fig. 5b, d), but the highest dose did not. Interestingly this dose also impaired novel object recognition in WT animals.

Protective effects of Neuro-EPO in APP_Swe mice: Biochemical and immunohistochemical analyses

Oxidative stress was assessed by measuring the level of lipid peroxidation in the mouse hippocampus (Fig. 6a). Lipid peroxidation was significantly increased in APP_Swe mice as compared with WT controls and the Neuro-EPO treatment at 125 μg/kg IN significantly prevented this increase. Neuroinflammation was first assessed by measuring by a direct measure of GFAP, marker of reactive astrocytes, and Iba-1, marker of reactive microglia (Fig. 6b, c).Both markers were significantly increased in the hippocampus of APP_Swe mice as compared with WT controls. The Neuro-EPO treatment significantly decreased GFAP expression at 250 μg/kg (Fig. 6b), and Iba-1 expression at both doses tested (Fig. 6c). Interestingly, the active dose of Neuro-EPO also significantly attenuated GFAP expression in WT animals (Fig. 6b). An immunohistochemical analysis of GFAP labeling was performed in the hippocampus of the mice (Fig. 7). GFAP immunolabeling was observed in the hilus, strati molecular, oriens, and radiatum of the hippocampal formation surrounding pyramidal cell layers throughout CA1-3 and in the dentate gyrus. The labeling was dispersed and moderate but present in Veh-treated WT mice (Fig. 7a, b).The Neuro-EPO treatment at both doses did not change or tended to decrease the labeling moderately (Fig. 7c-f). In Veh-treated APP_Swe the labeling, particularly in the hilus and stratum oriens, appeared markedly more intense and dense (Fig. 7g, h). The Neuro-EPO treatment, particularly at the 125 μg/kg dose (Fig. 7i, j), but also at the 250 μg/kg dose (Fig. 7k, l) decreased the number of GFAP-expressing cells and the intensity of the labeling. These qualitative observations appeared highly coherent with the Elisa quantification of global GFAP levels(Fig. 6b).

Two trophic factors were also directly measured: brain-derived neurotrophic factor (BDNF) (Fig. 6d) and nerve growth factor (NGF) (Fig. 6e). BDNF tended to be decreased in APP_Swe mice, but group comparison failed to show a significant difference. The Neuro-EPO treatment dose-dependently increased BDNF expression in the hippocampus of WT animals, but not in APP_Swe mice (Fig. 6d). NGF levels also tended to decrease in APP_Swe mice, but without a significant difference among groups (Fig. 6e). The Neuro-EPO treatment failed to affect NGF levels in WT or APP_Swe mice. A similar result was obtained with a marker of synapse integrity, synaptophysin (Fig. 6f). Synaptophysin levels tended to decrease in APP_Swe mice, but without a significant difference among groups (Fig. 6f). The Neuro-EPO treatment failed to affect synaptophysin levels in WT or APP_Swe mice.

Intrinsic apoptotic pathways were assessed using Bax/Bcl-2 expression in the mouse hippocampus (Fig. 8a-c). A moderate increase in Bax was measured in APP_Swe mice as compared with WT controls (Fig. 8a). The Neuro-EPO treatment prevented Bax increase, with a significant effect at the 125 μg/kg dose (Fig. 8a). Bcl-2 levels appeared highly significantly decreased for all groups as compared with V-treated WT animals (Fig. 8b). Contrary to what was expected in APP_Swe mice, this observation was surprising as concerns the Neuro-EPO treatment. As a result, the Bax/Bcl-2 ratio, calculated for each animal, appeared significantly increased in APP_Swe mice and normalized with the Neuro-EPO treatment (Fig. 8c). The treatment did not affect the ratio in WT mice.

The extrinsic apoptotic pathways were evaluated by measuring TNFα and FasL levels in the mouse hippocampus. An increase in TNFα level was measured in APP_Swe mice as compared with WT controls (Fig. 8d). The Neuro-EPO treatment tended to prevent the increase in TNFα level although no significant effect was noted (Fig. 8d). FasL level was significantly increased in APP_Swe mice (Fig. 8e). The two doses of Neuro-EPO tested decreased FasL level, with a significant difference at the lowest dose tested. Finally, Caspase-3 activity was analyzed using an activity kit. The protease activity was increased in APP_Swe mice (Fig. 8f). Indeed, the lowest dose of Neuro-EPO, but not the highest, significantly decreased caspase-3 activity.

Amyloid load was visualized by 6E10 immunolabeling in the hippocampus and cortex and analyzed by measuring both soluble and insoluble Aβ_1–42 in the mouse cortex using an ELISA kit (Fig. 9). In all APP_Swe animals treated with vehicle, large and dense Aβ deposits were found mainly in the cerebral cortex and hippocampus. Numerous cells were labeled with 6E10 confirming the high level of Aβ expression (Fig. 9a, d, g). Mice treated with 125 μg/kg Neuro-EPO also showed Aβ deposits but less dense and mainly associated with cell soma (Fig. 9b, e, h). Mice treated with Neuro-EPO 250 μg/kg showed clearly fewer smaller deposits with some animals completely devoid of plaques (Fig. 9c, f, i). Counting of the plaques showed a dose-dependent tendency to lower densities in the cortex (Fig. 9j) and at the highest dose in the hippocampus. The quantification of Aβ_1–42 content by ELISA in the cortex showed that Neuro-EPO, at 125 μg/kg, tended to decrease soluble Aβ_1–42 content (–56%; Fig. 9k) and highly significantly decreased insoluble Aβ_1–42 level. A – 83% decrease was measured in the mouse brain at this dose and only a – 37% decrease at 250 μg/kg (Fig. 9l).

Discussion

The impacts of EPO on neurodegenerative pathologies have been extensively reported. In in vitro, in vivo, and transgenic models of AD, particularly, the cytokine allowed not only neuroprotection but also disease modification and regeneration (for reviews, see [1, 42]). EPO reduced tau hyperphosphorylation, oxidative stress, and apoptosis induced by Aβ peptides application in SH-SY5Y neuroblastoma cells, PC12 pheochromocytoma cells, or primary hippocampus neuronal cultures, notably through the NF-κB p65 pathway [21 , 43]. In vivo, rHu-EPO was previously administered to APP_Swe (Tg2576) mice over a short period of 5 days [25]. The treatment was effective in reversing contextual memory deficits in the mice, in enhancing endothelial proliferation, capillary density, and synaptophysin expression, and in decreasing the levels of receptor for advanced glycation endproducts and the amount of amyloid plaques and Aβ species [25]. The short duration of treatment, but with a high dose of EPO (5000 UI/kg/day, IP), the delay taken after administration to evaluate the animals (14 days), and the age of the mice (13 months, i.e., when the pathology is progressing rapidly), suggested a very impressive efficacy of the cytokine. More recently, CEPO, a cytokine form carrying seven carbamylated Lysine residues and able to readily cross the blood-brain barrier, has been compared with rHu-EPO in an APP_Swe/PS1-dE9 mouse line [26]. The study showed that, after a 4-weeks treatment in 5-month-old mice, both CEPO and EPO improved novel object recognition, but only EPO decreased the amount of amyloid plaques and Aβ species. CEPO was devoid of erythropoietic effects and microarray analysis of gene expression revealed that both forms regulated a limited number of common genes, notably genes involved in synaptic transmission [26]. This study confirmed the efficacy of neuroprotective forms of EPO in AD transgenic mouse models, but pointed out mechanistic differences in the neuroprotection induced by EPO and its non-erythropoietic forms.

We previously compared the efficacy of rHu-EPO, administered IP, and Neuro-EPO, administered IN, in a non transgenic mouse model of AD [32]. Both forms prevented the memory deficits induced by Aβ_25-35 injection, the induction of lipid peroxidation, the increases in Bax level, TNFα, and IL-1β production, and decrease in Akt activation, in the hippocampus. Moreover, a significant prevention of the Aβ_25–35-induced cell loss in CA1 was also observed [32]. The study illustrated the efficacy of the low sialic form of EPO and confirmed the pertinence of the IN route in this pathology. Indeed, IN delivery is generally well tolerated by AD patients and effectiveness in humans is outlined with a clinical trial (NCT00438568) recently conducted for an IN insulin therapy [44]. The drug was administered by a specific medical device and no treatment-related adverse effects occurred while a mild therapeutic benefit was shown. The IN route circumvents the blood-brain barrier and offers the advantage of avoiding systemic circulation and associated risks of side effects. The IN-administered agents travel along the olfactory and trigeminal perineural spaces and small substances or macromolecules can overcome the respiratory epithelium transcellularly, through endocytosis, micropinocytosis, or phagocytosis [45, 46].

In the present study, Neuro-EPO was administered IN for 2 months in APP_Swe mice. The animals were used at an age when pathology is progressing rapidly, in order to mimic therapeutic intervention in humans when clinical signs are already present. In this mouse line, tangles and neuronal loss are absent or very limited. But cognitive impairments could appear as early as the age of 6 months, in particular regarding spatial learning and working memory. Memory loss is then progressive and clearly established at ages >10–12 months [33]. Synaptic loss is indeed observed in the CA1 hippocampal area at 4-5 months of age [35] and decline in long-term potentiation also appears at the same age, initially in the dendate gyrus after perforant path stimulation and then in the whole hippocampus formation [34, 36]. The mouse brains present an intense neuroinflammation, with reactive astrocytes and microglia, throughout the hippocampal formation, frontal, entorhinal, and occipital cortex at ages >10 months [47]. Numerous parenchymal Aβ deposits and plaques and accumulation of Aβ species are observed at this age [33]. We therefore chose a clearly different stage of the pathology as compared to Lee et al. [25] and adapted the treatment duration in accordance, i.e., 3 days a week during 2 months instead of once-a-day during 5 days.

We first observed that Neuro-EPO failed to affect the body weight loss of transgenic animals, about -5 g in 2 months, but tended to increase the survival rate, particularly at the highest dose tested. It therefore appears that the peripheral impact of the IN treatment was limited but not completely absent and the mechanism of the effect on survival must be investigated. A likely hypothesis could be a direct impact on stress axis, known to be deregulated in AD mice [48 –50]. Indeed, EPO and EPO receptors are highly expressed in the hippocampus and hypothalamus [51 –53], the two most important structures involved in regulating the hypothalamic-pituitary adrenal axis, controlling the physiological response to stress. Direct evidence that EPO regulates pituitary ACTH secretion has been recently published [54]. Moreover, EPO has been reported to induce BDNF expression in physiological and pathological conditions, an important regulator of the stress response [55, 56]. In our study, we observed a BDNF-enhancing effect of Neuro-EPO in the hippocampus of WT animals but not in APP_Swe mice. We did not measure the trophic factor level in the hypothalamus, and the result could have been different since amyloid toxicity differentially affects trophic systems among brain structures [57]. A precise study regarding the cytokine impact on stress response and its deregulation in AD must therefore be conducted.

The general activity and motor responses of the animals were briefly assessed. Neuro-EPO failed to affect clasping response or locomotor activity in WT and APP_Swe mice. Interestingly, anxiety-related parameters, such as the hand-escape response or anxiety response in the open-field were attenuated by the cytokine treatment, in a dose-related manner. Although more specific behavioral tests assessing anxiety (black-and-white box, elevated plus-maze or novelty-suppression feeding, for instance) were not assessed, these observations are coherent with a global impact on the mouse cognitive status. Indeed, learning and memory was assessed after 1 and 2 months of treatment, using spontaneous alternation response, or at the end, with place learning in the water-maze or object recognition. Results confirmed a beneficial effect of the cytokine on all responses measured. The effect on spatial working memory deficit was significantly observed after only 1 month of treatment and both doses of Neuro-EPO allowed significant effect after 2 months. The spatial reference memory and recognition memory deficits were also alleviated at the end of the treatment but with significant effects observed mainly for the lowest Neuro-EPO dose tested. It appeared that Neuro-EPO exerts its cognitive and neuroprotective effects in a bell-shaped manner and that the most active dose-range depends on the test considered. We here observed that Neuro-EPO at 250 μg/kg was, particularly, not effective in the object recognition test. The ability of WT animals was even diminished. Recognition memory, a highly impacted memory in AD, seems therefore more sensitive to lower doses of the cytokine, contrary to spatial memory in the water-maze.

rHu-EPO has been previously shown to enhance hippocampus dependent memory in young healthy mice. Fear conditioning response was increased during several weeks after a 3-weeks chronic treatment with EPO [58]. This was accompanied by increases in short-term and long-term potentiation and a decrease in short-term depression [58]. Interestingly, these effects on long-term potentiation were also found after acute treatment in rats [59]. EPO also accelerated associative, operant, and discriminant learning in the 5-choice serial reaction time task in mice and improved task adaptation [60]. Finally, the same team described that a 3-weeks chronic treatment with EPO improved a touch-screen visual discrimination task, an effect associated with plastic changes driving the differentiation of oligodendrocyte and neuronal precursor cells in the hippocampus [61]. We did not observe pro-mnesic effects of Neuro-EPO in our middle-aged WT controls, in the water-maze or object recognition tests. Since the highest dose tested even decreased memory ability in WT animals, it seems that the dose range was not optimal to see putative memory enhancing effect of Neuro-EPO. Indeed, cognitive enhancers classically induce bell-shaped dose-dependent effect and lower doses of the cytokine must be tested in the future.

Cognition ability, particularly in neurodegenerative pathologies, is a relevant response to the integrated pathological status. It is notable that several transgenic mouse models of AD, reconciling incomplete description of the pathology, could lead to an absence of memory deficits. The BRI-Aβ₄₂ mouse, overexpressing Aβ_1-42 and showing parenchymal and vascular diffuse amyloid deposits, for instance, did not present memory impairments or cell loss [62]. Similarly, mice overexpressing mutant presenilin-1 showed increased Aβ_1–42 deposits but limited toxicity and no learning deficits [63]. Fourteen-month-old APP_Swe mice showed important learning deficits and the observation that they could be significantly alleviated by Neuro-EPO was coherent with the pleiotropic activity of the cytokine. We observed that although it failed to impact trophic factor or synaptophysin expression, the cytokine alleviated the increases in lipid peroxidation or GFAP content and immunoreactivity or Iba-1 content in the hippocampus of APP_Swe mice. These observations are coherent with previous results and likely to involve direct effects of the cytokine. Indeed, EPO prevented cellular damages, including lipid peroxidation, induced by reactive oxygen or nitrogen species [57 –59]. In amyloid toxicity models, EPO preserved the mitochondrial integrity and therefore directly reduced oxidative stress generation [22]. The cytokine also increased several antioxidant enzyme activities, including catalase [64], superoxide dismutase [65], and glutathione peroxidase [66], in different pathological models. The antioxidant mechanism appears, however, dependent on the model and pathological context. Moreover, some results clearly suggest that EPO may directly scavenge reactive oxygen species [67]. In AD models, no direct antioxidant effect has been shown, but indirect effects could be proposed either through EPO-induced increase in erythrocyte production and related depletion in systemic iron concentration [68], an effect obviously not shared by Neuro-EPO, or through its impact on microglial cells [69]. We confirmed that both astroglial and microglial cells activation in APP_Swe is efficiently alleviated by Neuro-EPO. EPO treatment has previously been shown to decrease microglial proliferation and activation. Notably, EPO decreases phosphatidylserine (PS) exposure on neuronal plasma membrane [21] and the level in PS receptor on microglial plasma membrane [70], thus preventing microglial activation and phagocytosis through the PS binding recognition mechanism. Moreover, EPO regulates EPOR signaling in microglia, altering directly the reactive state of the cell. Through activation of the Wnt1/PI3-K/Akt1/mTOR pathway, EPO translocates the p65 subunit of NK-kB into the nucleus and allow the initiation of anti-apoptotic gene expression [21].

We addressed both intrinsic and extrinsic apoptosis pathways and observed that the increases in APP_Swe mice of Bax level or Bax/Bcl-2 ratio as well as TNFα or FasL levels are significantly reduced to the level observed in WT control mice by Neuro-EPO, at least for the lowest dose. As a consequence, the activity of the effector caspase triggering apoptotic death, caspase-3, was reduced by the cytokine. Similar direct impacts have been previously reported on in vitro models of microglial cultures [71]. As said above, EPO activated the Wnt1/PI3-K/Akt1/mTOR pathway, which in turn increased phosphorylation and cytosol trafficking of Bad, reduced Bad/Bcl-x_L complex and increased Bcl-x_L/Bax complex. The cytokine therefore protects both mitochondrial related signaling of pro-apoptotic molecules and extrinsic pathway involving death receptors Fas and TNFαR.

Finally, we examined the amyloid load in APP_Swemice, following Neuro-EPO treatment. Using immunolabeling, we observed visually that amyloid deposits decreased in the cortex at both doses of Neuro-EPO and in the hippocampus, at the highest dose tested. Animals presented markedly less large dense deposits as observed in vehicle-treated APP_Swe mice and as expected at this age of the pathology [33]. Deposits appeared limited to cellular labeling and some animals treated with 250 μg/kg did not show any marked labeling. Quantification of the amyloid load using an ELISA kit confirmed that Neuro-EPO treated animals presented less Aβ_1–42 in cortex extracts for both the soluble and insoluble forms. Our data are coherent with the previous report from Armand-Ugón et al. [26], who described that rHu-EPO, at 2500 UI/kg, i.e., 105 μg/kg, decreased plaque burden and soluble Aβ_1–40 in AβPP/PS1 mice. Neuro-EPO therefore appears as effective as rHu-EPO on Aβ pathology. Further treatments with longer duration will be necessary to determine whether EPO could help a complete clearance of Aβ in the brain of APP_Swe mice, but the cytokine appears to be, besides its neuroprotective properties, a major mean to alleviate amyloid load in the brain. Among the strategies used to reduce amyloid burden, especially in transgenic mice, immunization with Aβ and other inflammatory stimuli, inhibitors of Aβ formation, cholesterol lowering agents, β-sheet breaker peptides, antioxidants and various miscellaneous agents have been found to be the most effective means to reduce soluble Aβ and, possibly, deposits in such transgenic mice [72]. Although it has not ever been demonstrated that they would really be an effective treatment for the disease, EPO appears as effective as the most efficient ones. The anti-inflammatory role of EPO must therefore been further investigated, particularly in relation with microglial cells, since (i) they are highly involved in Aβ clearance, (ii) they express the highest density of EPO receptors, and (iii) EPO has been shown to directly activate signaling cascades in microglia, including the Wnt-1, PI-3K, Akt, and mTOR pathways [71 , 74].

In summary, the present study confirmed previous reports based on EPO or other low sialic forms of EPO and using either transgenic mouse models of AD [25, 26] or Aβ-induced toxicity [32, 66], by showing that Neuro-EPO, administered chronically, prevents AD-like pathology and behavioral impairments. The cytokine alleviated oxidative stress, neuroinflammation, and apoptosis in the APP_Swe mouse brain, and an earlier study reporting the presence of EPO in the cerebrospinal fluid of AD patients at a similar level as in control patients [75] suggest that EPO/EPOR system could be considered as a promising endogenous neuroprotection system efficiently targeted by innovative drug strategies. Considering the results found with nasal application of Neuro-EPO in this transgenic AD model, we propose that the cytokine presents a potent neuroprotective effect by blocking the main metabolic pathways that are affected in the neurodegenerative disease. Intranasal Neuro-EPO appears as a potent inducer of endogenous neuroprotection and a novel therapeutic alternative against AD.

Footnotes

ACKNOWLEDGMENTS

We thank Drs. Gaëlle Naert and Catherine Desrumaux (INSERM U1198, Montpellier, France) for helpful advices and CIMAB (Habana, Cuba) for providing Neuro-EPO. This work is a scientific collaboration between the University of Montpellier and the University of Medical Sciences of Havana, Institute of Basic and Preclinical Sciences “Victoria de Girón”. It was funded by a Franco-Cuban scientific cooperation between the French Embassy in Cuba and the Cuban Ministry of Foreign Trade and Foreign Investment (MINCEX), program FSP Cuba 2011-16, 29967RG (Havana, Cuba) and external resources from Inserm (Paris, France) and the University of Montpellier (Montpellier, France). JCGR acknowledges a visiting professor fellowship from the University of Montpellier.

Authors’ disclosures available online ().

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An Intranasal Formulation of Erythropoietin (Neuro-EPO) Prevents Memory Deficits and Amyloid Toxicity in the APP Swe Transgenic Mouse Model of Alzheimer’s Disease

Abstract

Keywords

Introduction

Material and Methods

Animals

Drugs, injections, and experimental series

Clasping and escape responses

Spontaneous alternation in the Y maze

Place learning in the water maze

Novel object recognition memory

Lipid peroxidation measures

ELISA assays

Immunohistology

Statistical analyses

Results

Protective effects of Neuro-EPO in APPSwe mice: behavioral study

Protective effects of Neuro-EPO in APPSwe mice: Biochemical and immunohistochemical analyses

Discussion

Footnotes

ACKNOWLEDGMENTS

References

Protective effects of Neuro-EPO in APP_Swe mice: behavioral study

Protective effects of Neuro-EPO in APP_Swe mice: Biochemical and immunohistochemical analyses