Research report
Effect of carbamylated erythropoietin Fc fusion protein (CEPO-Fc) on learning and memory impairment and hippocampal apoptosis induced by intracerebroventricular administration of streptozotocin in rats

https://doi.org/10.1016/j.bbr.2020.112554Get rights and content

Abstract

Intracerebroventricular (icv) administration of streptozotocin (STZ) has been used as a metabolic model of sporadic Alzheimer’s disease (AD). Erythropoietin (EPO) possesses neuroprotective and memory-improving effects, which might be advantageous in treating different characteristics of AD. Nevertheless, the hematopoietic effect of EPO has hindered its application as a neuroprotective agent. Previous studies have shown that a new Epo derivative called carbamylated Erythropoietin-Fc (CEPO-Fc), yield noticeable neuroprotective effects without affecting hematopoiesis. In this study, the neuroprotective effects of CEPO-Fc on icv-STZ induced memory impairment and hippocampal apoptosis were examined. Adult male Wistar rats weighing 250−300 g were used. STZ was administered on days 1 and 3 (3 mg/kg in divided doses/icv), and CEPO-Fc was administered at the dose of 5000 IU/ip/daily during days 4–14. The animals were trained in Morris water maze during days 15–17, and the memory retention test was performed on the 18th day. Following behavioral studies, the animals were sacrificed and their hippocampi isolated to determine the amounts of cleaved caspase-3 (the landmark of apoptosis). The results showed that CEPO-Fc treatment at the dose of 5000 IU/kg/ip was able to prevent the learning and memory deficit induced by icv-STZ. Western blot analysis revealed that STZ prompted the cleavage of caspase-3 in the hippocampus while pretreatment with CEPO-Fc significantly reduced the cleavage of this protein. Collectively, our findings suggest that CEPO-Fc could restore STZ-induced learning and memory impairment as well as apoptosis in the hippocampal region in a rat model of sporadic AD induced by icv-STZ.

Introduction

Alzheimer’s disease (AD) is the most common form of dementia, characterized by a gradual loss of cholinergic neurons in brain regions, such as cerebral cortex and hippocampus, which leads to progressive decline in cognitive functions and memory loss [1]. Currently the available treatments, such as memantine, galantamine, rivastigmine and donepezil can produce some functional symptom relief, but do not affect the progression of AD. Furthermore, these treatments have peripheral and central side effects, which warrant for search of other therapeutic agents to manage AD [2].

Despite indistinguishable clinical signs, there are two different forms of AD. Familial AD is caused by mutations in three genes: amyloid-β protein precursor (AβPP), presenilin-1, and presenilin-2 [3]. However, the great majority of AD cases (accounting for 90 % or more of the cases) are sporadic in origin (sporadic AD). The memory impairment and neuropathological alterations observed in AD can be induced by sub-diabetogenic doses of intracerebroventricular streptozotocin (icv-STZ) administration in rats [4], which is considered as an established and reproducible animal model of sporadic AD [5]. Following icv STZ injection, some AD-like changes such as learning and memory impairment as well as neuronal apoptosis [6] occur.

Erythropoietin (EPO) is a glycoprotein hormone, mainly secreted by interstitial fibroblasts in the kidney that regulates erythropoiesis. Furthermore, EPO was reported to act as a neuroprotective factor against various injuries [7,8]. Despite the potential neuroprotective effect of EPO, its low efficiency in crossing the blood brain barrier (BBB), necessitates high doses, which in turn rises the hematocrit and blood viscosity that might induce serious problems such as infarction or stroke [9]. Hence, conventional EPO might not be a suitable neuroprotective agent due to its hematopoietic activity. Recent studies revealed that the hematological and protective effects of EPO are exerted through different receptors [10,11]. Consequently, chemical and genetic modification strategies were used to eliminate its erythropoietic activity while preserving the protective effects [12].

Recently, several types of EPO derivatives that lack hematopoietic effect were generated. Carbamylated form of EPO-Fc fusion protein (CEPO-FC) is derived from the carbamylation of EPO’s lysine residues. This fusion protein holds the constant region of an immunoglobulin which gives it a prolonged half-life. This derivative does not exert erythropoietic effects, but is comparable to EPO in stability, BBB permeability, and pharmacokinetics properties [12].

After proposing carbamylated EPO to remove its erythropoietic effects, some experiments were conducted to evaluate its neuroprotective effects in a wide range of animal models of neurotoxicity including ischemic stroke, spinal cord depression, sciatic nerve compression and peripheral diabetic neuropathy, revealing the neuroprotective effects of this EPO derivative [[12], [13], [14]]. In line, recent studies proved that CEPO exerts neuroprotection in animal models of AD such as in AβPP/PS1 transgenic mice [15] and Aβ 25–35 induced memory deficit [16].

Considering the destructive effects of STZ on learning and memory and the recommended neuroprotective effect of CEPO-FC, this study aimed to investigate if CEPO-FC can protect STZ-induced spatial memory deficit. Moreover, the hippocampal levels of cleaved caspase-3 (an indicator of apoptosis) was assessed following CEPO-FC treatment.

Section snippets

Animals

In-house breeding adult male Wistar rats weighing 250−350 g (n = 8/group) were group housed and had access to water and food ad libitum. They were kept under controlled temperature (20 ± 2 °C) and lighting (07:00 to 19:00 h) status. All experimental protocols were approved by Shiraz University of Medical Sciences Animal Care and Use Committee in accordance with the National Institutes of Health Guidelines (approval number: IR.SUMS.REC.1397.254). The animals were randomly assigned into 4 groups

CEPO-Fc protects against icv-STZ induced spatial learning deficit

A two-way repeated-measures ANOVA of escape latencies showed a significant difference (Fig. 1A, treatment effect, F (3, 28) = 22.30, p < 0.0001; days effect, F (2, 56) = 44.14, p < 0.0001; interaction of treatment vs. day, F (6, 56) = 2.823, P = 0.0180). Since the interaction of treatment vs. day was significant, the escape latency of animals in each day, was analyzed separately by one-way ANOVA which revealed significant differences in all 3 days (day 1: F (3, 28) = 15.4, P < 0.0001; Day 2: F

Discussion

The findings in the present study showed that CEPO-FC protects against icv-STZ induced water maze learning and memory deficit as well as caspase-3 cleavage (activation) as an indicator of apoptosis in the hippocampus. To the best of our knowledge, this is the first study to reveal the attenuation of icv-STZ induced learning and memory impairment in male rats by systemic CEPO-FC administration. In female rats, four stages of estrous cycle (estrus, proestrus, diestrus and metestrus) exist during

Author statement

Maryam Moosavi: Conceptualization, Methodology, Formal analysis, Writing - Original Draft, Funding acquisition. Etrat Hooshmandi: Data curation, Investigation, Software. Pegah Javadpour: Visualization, Investigation. Nader Maghsoudi: Supervision, Writing - Review & Editing. Hermann Katinger: Investigation, Writing - Review & Editing. Rasoul Ghasemi: Validation Methodology Writing - Review & Editing Project administration.

Declaration of Competing Interest

The authors declare that they have no conflict of interests.

Acknowledgments

This work was supported by a grant (No. 97-01-57-16986) from Shiraz University of Medical Sciences, Shiraz, Iran. The authors wish to thank Mr. H. Argasi at the Research Consultation Center (RCC) of Shiraz University of Medical Sciences for his invaluable assistance in editing this manuscript.

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