A Synthetic Pro-Drug Peptide Reverses Amyloid-β-Induced Toxicity in the Rat Model of Alzheimer’s Disease

Abstract

Background:

Alzheimer’s disease (AD), the most prevalent neurodegenerative disorder, involves the formation of the extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles. The current therapies against AD are symptomatic with limited benefits but associated with major side effects. Inhibition of self-aggregation of Aβ peptides into higher order cross-β structure is one of the potential therapeutic approach which may counter oligomerization of Aβ peptide.

Objective:

The present study aimed to evaluate the neuroprotective and anti-inflammatory potential of a synthetic Pro-Drug type peptide (PDp) against Aβ-induced toxicity in rat model of AD.

Methods:

Intra-hippocampal microinjection of toxic Aβ₄₀ (IHAβ₄₀) by stereotaxic surgery was performed in the male Sprague-Dawley rats to generate an Aβ-induced AD model. Sub-chronic toxicity of synthetic PDp using hematological, biochemical, and histopathological parameters was investigated. Evaluation of PDp on Aβ-induced neurodegeneration and neuroinflammation was performed.

Results:

PDp inhibits plaque formation with increase in Nissl granule staining in the rat hippocampus. Aβ-induced toxicity associated imbalance in reactive oxygen species and antioxidant enzymes activity such as superoxide dismutase and catalase in the rat brain was overcome by PDp treatment. Tau protein hyperphosphorylation was normalized with PDp treatment. Also, the neuroinflammatory response was suppressed with PDp treatment.

Conclusion:

The present study depicts the potential neuroprotective role of PDp against Aβ-induced toxicity in rat. PDp inhibits plaque formation thereby normalizing oxidative stress, inhibiting tau protein hyperphosphorylation, and suppressing neuroinflammatory responses. Future studies done in this direction will pave way for new therapeutic strategies.

Keywords

Alzheimer’s disease neuroinflammation neuroprotection Pro-Drug type peptide rat model of AD

INTRODUCTION

Alzheimer’s disease (AD) is currently the most prevalent form of neurodegenerative disorder which imposes a big health care challenge as clinical symptoms appear much later than the onset of the disease [1]. AD was first described by Alois Alzheimer in 1907, as a condition involving intellectual decline with the histopathological findings that showed the presence of senile plaques and neurofibrillary tangles (NFTs) in the brain [2]. AD slowly and progressively leads to a severe state of dementia [2, 3] involving a group of conditions viz., memory impairment, language problems, defective problem-solving skills, difficulty in walking, swallowing, and other associated cognitive dysfunctions thereby disabling an individual to perform daily routine activities, hence leading to a debilitating condition for an individual [3]. The underlying cause of dementia in AD is linked with the loss of neurons mainly in the hippocampus and cerebral cortex of the brain regions associated with the regulation of cognitive functions [3, 4]. The underlying cause of neuronal loss is not well established. Furthermore, based on our present understanding, it is linked with the hallmark pathologies involving formation of proteinaceous aggregates: the extracellular amyloid-β (Aβ) plaques and the intracellular NFTs [3 –5].

Aβ plaque formation is an outcome of the abnormalities in the processing and secretion of the amyloid-β protein precursor (AβPP), wherein clearance of Aβ peptide is hindered leading to the aggregation of Aβ [4 –6]. This self-aggregation of Aβ is the triggering factor resulting in the formation of senile plaques [4, 5]. Aβ is a peptide consisting of 37–42 amino acids, and the isoforms of the Aβ₄₂ and Aβ₄₀ are more toxic and commonly associated with the plaque formation [7]. The toxicity of Aβ is primarily linked to its high hydrophobicity and the physical properties which results in acquiring β-pleated sheet configuration, thereby resulting in the increased tendency for aggregation and hence forming the core of the plaque [8]. Although the Aβ hypothesis suggests the possible role of senile plaques in AD, etiopathogenesis is not yet clearly understood.

Besides, the formation of NFTs, there seems to be another pathogenic event which contributes to the neurodegeneration in AD [9]. NFTs are primarily found in the hippocampus and cerebral cortex regions of the brain [4, 5]. The formation of NFTs is the outcome of the hyperphosphorylation of the tau protein into tangles wherein the aberrant tau protein tends to aggregates thereby initiating its fibrillization [10, 11]. Tau protein aberrancy resulting in NFT formation is possibly the outcome of the Aβ plaque formation [4 , 13]. The formation of Aβ plaques and NFTs are pathological hallmarks and are linked to synaptic dysfunctions, elevated oxidative stress, neuronal apoptosis, and dementia [4 , 10].

Henceforth, any alteration in Aβ metabolism involving its production and clearance in the brain is one of the critical events which may potentially lead to the development of AD [10]. Studies have shown that alteration in Aβ metabolism results in plaque formation which results in hyperphosphorylation of tau protein, synaptic degeneration, cerebrovascular damage, and microglia/astrocyte activation [14]. Such anomalies result in neurotoxicity that may lead to various defects in cognitive functions [14]. Therefore, identification of the factors leading to aberrancy in Aβ production and clearance could help in deciphering the role of Aβ associated plaques in AD pathology. It has been shown that genetic variations at the apolipoprotein E (ApoE) locus seems critical and inflict pathogenicity associated with Aβ metabolism [15]. Furthermore, studies involving genetic variations which affect the production and clearance of Aβ impose a high risk for the development of AD [10].

The AD pathology involving accumulation of Aβ plaques and NFTs is very much critical for neuronal health and survival. Besides, the role of redox imbalance in neurodegenerative disorder involving AD is also crucial and been shown to be co-associated with the aforementioned pathological hallmarks of AD [16 –18]. The activity of antioxidative enzymes like catalase (CAT) and superoxide dismutase (SOD) has been found to be aberrant in AD patients [19]. The redox disequilibrium, therefore, seems to be an important component in AD pathogenesis. However, the underlying mechanism that inflicts alteration in redox balance still remains elusive and the source of free radical yet remains to be deciphered [4]. It has been shown that Aβ plaque formation may potentially induce the formation of reactive oxygen species (ROS) via activation of N-methyl-D-aspartate (NMDA) receptor [20]. Also, ROS accumulation may initiate Aβ aggregation hence facilitating hyperphosphorylation followed by fibrillization of tau protein. Such vice-versa association of ROS production and Aβ plaque formation may inflict AD progression, although more studies are required to unravel the association [18].

At present, several hypotheses other than the Amyloid Cascade Hypothesis have been proposed, e.g., the mitochondrial cascade hypothesis, the dual pathway hypothesis, the metabolism hypothesis, the cell cycle re-entry hypothesis, the vascular hypothesis, and Aβ soluble protofibril hypothesis [21]. However, most of them are directly or indirectly related to the aggregation of the Aβ peptide. AD pathogenesis mainly revolves around Aβ plaques, tangles, and redox imbalance; therefore, understanding this association will shed light on the underlying mechanism thereby strategizing the therapeutic approach against such a debilitating disease. There is no available specific therapy that restricts or halts the progression of the AD. Current therapeutic only provides limited benefits by slowing down the rate of cognitive decline associated with AD [22]. Some of the currently available drugs used are antagonists of NMDA receptor or inhibitors of acetylcholinesterase [10, 23] having hepatotoxicity and gastrointestinal symptoms as major side effects. The therapeutic approaches which target pathways involved in Aβ production and clearance may provide potential relief in combating AD. The activators of α-secretase, inhibitors of β- and γ-secretase, non-steroidal anti-inflammatory drugs (NSAIDs), metal chelators, anti-aggregate compounds, and immunotherapy are some of the approaches which may help against Aβ plaque-associated AD pathogenesis [10, 22].

One of the potential therapeutic approaches against AD may involve inhibition of protein aggregation (Aβ and NFTs). Several synthetic and natural compounds are being studied using in vitro and in vivo approaches [22 , 25]. In this milieu, one of the strategies involves a β-sheet breaker approach which may inhibit amyloidosis [24 , 27]. The successful utilization of such approach may counter oligomerization of Aβ peptide. In this context, a synthetic Pro-Drug type peptide (PDp, sequence in one letter code: Ac-LD(OBzl)FFD-NH₂) has been shown to inhibit amyloidogenesis [28] and disrupt preformed fibril [27] of Aβ₄₀ as evident by the results of the biophysical experiments. PDps are different than other β-sheet breaker peptides as they are able to form β-sheet initially, therefore can co-aggregate with the amyloid forming agents (e.g., Aβ peptide) easily, but at physiological pH and temperature, they undergo a cascade of chemical transformations, first to form a cyclic aspartimide, that further undergo racemization and hydrolysis to form β-aspartyl residues. All these transformation products can not fit in a planer β-sheet topology, thus finally fibril and amyloid structures collapse. Thus, PDp makes a robust strategy for amyloid disruption. It also provides a neuroprotective effect against Aβ₄₀-induced cytotoxicity in human neuroblastoma SH-SY5Y cells [24]. We have also demonstrated its cell penetrating capability as well. Inspired by the positive results obtained using in vitro approach in our earlier published reports, we were interested to test the potential of PDp in an Aβ-induced rat model of AD. Therefore, the present study was undertaken to evaluate the potential of PDp as a neuroprotective agent against neurodegeneration and neuroinflammation associated with an Aβ-induced rat model of AD. Based on our finding in the present study, it was observed that PDp significantly helps in retaining the cognitive functions in rats, caused by the alleviation of neuronal degeneration, and exhibited anti-inflammatory responses. Further, it was also found that PDp tends to downregulate AD-associated marker proteins p-Tau, GSK-3β, and PP-2A. The present study confirms the potential of PDp as an anti-amyloidosis agent. However, future studies involving characterization of PDp such as half-life, its stability and blood brain barrier breaching property will shed more light on its potential role as anti-amyloidosis drug for AD.

MATERIALS AND METHODS

Ethical statement

The experiments were conducted on the male Sprague-Dawley rats approved by the Institutional Animal Ethics Committee (IAEC), Raja Peary Mohan College, Uttarpara, Hooghly (W.B.). Reg No. 1148/PO/ac/CPCSEA. Standard protocols were used for animal handling and experimentation as per guidelines and regulations of CPCSEA, New Delhi.

Peptides

PDp was synthesized, purified, and characterized by previously described methods [24, 27]. In brief peptides were synthesized using solid phase peptide synthesis technique on Rink amide MBHA resin following Fmoc/^tBut orthogonal protection strategy. Then peptides were purified by RP-HPLC.

Animals

Adult Sprague-Dawley rats weighing 250–300 grams were used for the present study. They were housed in polypropylene cages in the Animal House, with free access to food and water ad libitum. The animals were kept in the rooms with temperature maintained constantly at 21±2°C and humidity at 55%, under a constant 12 h light/dark cycle. Whole experiment was divided into three parts to achieve targeted objectives.

Intra-hippocampal microinjection of toxic Aβ₄₀ by stereotaxic surgery

A total of 18 rats were used (n = 6/group). This was followed by the random assignment of rats to the control, sham, and IHAβ₄₀ groups for the purpose of further experiments. Rats were trained for behavioral test for 5 days. Surgery was performed on the 6th day and prior to that rats were anesthetized intraperitoneally with ketamine (100 mg/kg) and xylazine (10 mg/kg) and placed in a stereotaxic instrument (Stoelting, USA). The scalp was incised and drilled to allow the insertion of a Hamilton micro-syringe (Hamilton, USA). The coordinates were measured from bregma (antero-posterior: –3.8 mm; lateral: ±2.2 mm; dorsal/ventral: –2.7 mm). Aβ₄₀ dissolved in artificial cerebrospinal fluid (ACSF) at concentration 20 μM/ml was administered bilaterally to the IHAβ₄₀ group, sham group was injected with ACSF and the control group was left undisturbed. After surgery, rats were left for 7 days for recovery. Two behavioral tests, passive avoidance test (PAT) and elevated plus maze (EPM) were used to validate successful establishment of a neurobehavioral model of AD in rat, while cresyl violet staining used for estimating neuronal insult caused by intra-hippocampal microinjection of toxic Aβ₄₀. The PAT was performed on the day 14, 21, and 28, while EPM was performed on the day 14 and 28. Rats were sacrificed after the end of the behavioral experiments by decapitation and brains were rapidly removed on ice. Hippocampus was dissected and was processed for cresyl violet staining for locating Nissl granules of the hippocampal neurons.

Evaluation of sub-chronic toxicity of synthetic PDp in rat

A total of 36 rats were used (n = 12/group). Sub-chronic toxicity of synthetic PDp was performed using male Sprague-Dawley rats (n = 36) by random assignment of rats to three groups: control, PDpG1 (n = 12, receiving 100 μg/kg of PDp), and PDpG2 (n = 12, receiving 200 μg/kg of PDp) injection into the tail vein for 42 days. To evaluate sub-chronic toxicity of PDp, the weights of the rats were recorded on 0, 21, and 42 days. After 42 days, rats were anesthetized by intravenous injection of Thiopentone sodium (100 mg/kg b.w.) (Pentothal sodium, Abbott Laboratories) and were sacrificed. Blood was collected by cardiac puncture in a vacutainer tube with a clot activator. The serum was obtained by centrifugation at 3000 rpm for 15 min and analyzed for various hematological and biochemical parameters, while liver and kidney was collected for histopathological evaluations using hematoxylin and eosin (H&E) staining.

Evaluation of the PDp on neurodegeneration and neuroinflammation associated with rat model of AD

We have already established an Aβ-induced rat model of AD. A total of 30 rats were used (n = 6/group). Now, to evaluate the effects of PDp on neurodegeneration and neuroinflammation associated with AD pathology on the Aβ₄₀-injected rat model of AD, animals were grouped as the control, sham, Aβ₄₀, PDp (50 μM) + Aβ₄₀, and PDp (100 μM) + Aβ₄₀. For this, the infusion of PDp and Aβ₄₀ was done on both sides of the hippocampus for two doses, i.e., 50 μM and 100 μM, using a Hamilton microsyringe. Coordinates were measured from bregma (antero-posterior: –3.8 mm; lateral: ±2.2 mm; dorsal/ventral: –2.7 mm). Rats were allowed to recover for 7 days and were sacrificed after 28 days by decapitation, and brains were rapidly removed on ice. To evaluate the effects of PDp on neurodegeneration and neuroinflammation associated with AD pathology, brain was dissected out and processed for Congo red and cresyl violet staining. Hippocampus was dissected out, frozen in liquid nitrogen, and then stored at –80°C for further estimating ROS, nitrite level, activity of antioxidative enzymes SOD, CAT for analysis of neuroinflammatory markers [inducible nitric oxide synthase (iNOS), Interleukin-1β (IL-1β), Tumor necrosis factor-α (TNF-α)], and AD pathology associated markers (p-Tau, GSK-3β, PP-2A) using western blot.

Passive avoidance test by shuttle box apparatus

The passive avoidance test was conducted as described previously [29], started 1 week after the Aβ₄₀ injection using shuttle box apparatus. It consisted of two boxes of the same size (20×20×30 cm). There was a guillotine door in the middle of a dividing wall. The walls and floor of one compartment consisted of white opaque resin and the other one was dark. Intermittent electric shocks (50 Hz, 3 s, 1.5 mA intensity) were delivered to the grid floor of the dark compartment by an isolated stimulator. Each animal was gently placed in the white compartment and after 5 s the guillotine door was opened and the animal was allowed to enter the dark module. Once the animal entered with all four paws to the next chamber, the guillotine door was closed and the rat was immediately withdrawn from the compartment. This trial was repeated after 2 min. As in the acquisition trial, when the animal entered the dark (shock) compartment the door was closed, and a foot shock (50 Hz, 1.5 mA, 3 s) was immediately delivered to the grid floor of the dark room. After 20 s, the rat was removed from the apparatus and placed temporarily into its home cage. Two minutes later, the animal was retested in the same way as in the previous trials; if the rat did not enter the dark compartment during 300 s, a successful acquisition of inhibitory avoidance response was recorded. Otherwise, when the rat entered the dark compartment (before 300 s) a second time, the door was closed and the animal received the shock again. Twenty-four hours later, each rat was again placed in the light chamber (retention trial) and after 5s, the door was opened and the latency with which the animal entered the dark chamber and the total time spent in dark compartment was recorded in the absence of electric foot shocks, as an indicator of inhibitory avoidance behavior.

Elevated plus maze

Each rat was placed in the center of the EPM facing one of the open arms and the number of entries, time spent (s) in open and closed arms were recorded during a 3 min test period. The EPM apparatus was cleaned with 75% ethanol before introduction of each animal. Data for EPM was quantified and presented as percent retention.

Extraction of hippocampus from the rat brain

Thiopentone sodium (100 mg/kg) (Pentothal sodium, Abbott Laboratories) was used to anesthetize the rats. The rats were decapitated before the brains were extracted from the skull, which was then kept on ice. The hippocampus was dissected out using the bregma as a reference landmark [30]. The isolated hippocampus samples were then subjected to flash freezing in liquid nitrogen and were stored in –80°C until further analysis.

Cresyl violet staining of hippocampal neurons to assess neurodegeneration and neuroprotection

The brain tissues were prepared as described previously [31]. The hippocampal paraffin sections were coronally cut at 5 μm thickness using Microtome (Leica Microsystems, Bensheim, Germany) and mounted on gelatin coated glass slides, air-dried. The tissue slides were rehydrated in a graded ethanol series. The brain tissues were briefly stained with 0.125% cresyl violet, dehydrated again and immersed in xylene for 20 min, and then cover slipped. Intensity of Nissl granules over the area of 3000 μm² (100 μm length×30 μm width) in the hippocampus was estimated under a light microscope (Nikon 90i, Tokyo, Japan) under 40× objective. Eight hippocampal sections in each rat were counted (total 48 sections in each group, n = 6).

Analysis of blood for hematological and biochemical parameters in respect to PDp toxicity

The blood sample was analyzed for glucose, serum urea, creatinine, total bilirubin, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, and alkaline phosphatase using commercially available diagnostic test kits (Crest Biosystems kits, India). Blood parameters including red blood cells, white blood cells, hemoglobin, neutrophil, lymphocyte, eosinophil, monocyte, and basophil were estimated using standard kits by following the manufacturer’s manual.

H&E staining of liver and kidney samples for sub-chronic toxicity

To verify the effect of PDp on histopathological studies on the liver and kidney for sub-chronic toxicity in rat, histopathological assessment was done using H&E staining protocol. Brain samples were fixed in formalin solution for 48–72 h. Then dehydration and paraffin embedding were performed via an automated processor. The liver and kidney were sectioned (5 μm) and stained by H&E as described [32]. Slides were observed under the light microscope (Nikon 80i) and images were taken at 40× magnification and examined for histopathological alterations.

Congo red staining for Aβ deposition

The brain tissues were prepared and stained as described previously [33]. To confirm the formation of Aβ plaques in the brain, the rats were decapitated and hippocampus was removed for histopathological observation. The samples were immersed in a fixative formalin solution for 48–72 h. Then dehydration and paraffin embedding were performed via an automated processor. The brain was sectioned and stained with Congo red. The staining verified the formation of Aβ plaques in the hippocampal area of the brain in the Aβ-treated animals. The images were observed and captured under 40× objective and examined for histopathological alterations.

Activity of tissue antioxidant enzymes

Tissue homogenization and preparation of supernatant

Tissue homogenate was prepared by homogenizing the hippocampus in 0.1 M phosphate buffer saline (PBS) at pH 7.4 to obtain 10% sample homogenate. A Potter-Elvehjem homogenizer was utilized for the procedure with 6–8 strokes at medium speed while the sample was kept continuously on the ice. The homogenate was then centrifuged at 10000 rpm at 4°C for 20 min. 10% supernatant was obtained and stored in –20°C for further analysis. The amount of protein in the samples was estimated by utilizing the method used previously [34]. Bovine serum albumin was used as reference standard.

Superoxide dismutase activity

Determination of activity levels of SOD was done as described previously [35, 36]. The assay mixture comprised of 0.8 ml glycine buffer (50 mM, pH 10.4), 0.2 ml of supernatant, and 2 μl of 2% epinephrine. The final volume of the assay mixture was 1.02 ml. SOD activity was determined kinetically at 480 nm by measuring the level of oxidized product of epinephrine, i.e., adrenochrome. The activity of SOD is expressed in nmol of epinephrine that is protected from oxidation by the sample by comparing it with the corresponding reading in the blank. The calculation was done using a molar coefficient (ɛ) of 4.02×10³ M^–1 cm^–1and expressed in nmoles of epinephrine protected from oxidation/min/mg of protein.

Catalase activity

Estimation of CAT activity was done as previously used [37] and modified [38]. In a 3 ml cuvette, 1.95 ml of 0.1 M phosphate buffer (pH 7.4) was added along with 1 ml of 0.05 M hydrogen peroxide and 50 μl PMS. The total volume of the assay mixture amounted to 3 ml. The OD was then measured at 240 nm using spectrophotometer. The activity of CAT was then calculated by using a molar coefficient (ɛ) of 39.6 M^–1 cm^–1. The result was expressed in μmoles of H₂O₂ consumed/min/mg of protein.

Assessment of reactive oxygen species generation

ROS generation was estimated through a method done previously [39]. ROS levels were measured using Dichlorofluorescein diacetate (DCF DA), a nonfluorescent lipophilic dye that passively diffuses through cellular membranes, is cleaved into 2,7-dichlorofluorescein by intracellular esterase enzymes in the presence of intracellular ROS and produces fluorescence. 10 μl Dichlorofluorescein diacetate (10 μM) was added to 150 μL of hippocampus tissue homogenate (10% w/v in radio immunoprecipitation assay (RIPA) buffer) and incubated in amber tubes in the dark for 40 min at 37°C. Fluorescence was measured at 488 nm excitation and 525 nm emission (LS 45 Luminescence Spectrometer, Perkin Elmer, Waltham, MA, USA) and converted to arbitrary unit (AU)/mg of protein and shown in % of the control in the bar graph.

Assessment of nitrite level

Nitrite level was estimated in the hippocampus of the rat brain through Nitrite Assay Kit (Griess Reagent) (Biovision, CA, USA) following manufacturer’s protocol. The results obtained in nmol/μl and shown in % of control in the bar graph.

Western blot analysis for neuroinflammatory (iNOS, IL-1β, TNF-α) and AD pathology associated (p-Tau, GSK-3β, PP-2A) markers

Western blotting was performed on 12% SDS-PAGE following previous method [40]. 50 μg proteins were resolved and transferred on PVDF membrane and blot was placed in the blocking solution (5% non-fat milk powder (w/v) in wash buffer) for 1 h at room temperature, rinsed briefly with wash buffer. Blot was incubated with the primary antibodies (iNOS, IL-1β, TNF-α, p-Tau, GSK-3β, PP-2A, β-actin; 1 : 1000 dilutions) at 4°C for overnight. Blot was washed extensively with wash buffer (3×5 min) with gentle agitation. Anti-rabbit HRP-conjugated secondary antibody (CST, 1 : 1500 dilutions) was added and incubated for 1 h at room temperature with gentle agitation. The membrane was washed with gentle agitation as follows: 3×5 min in wash buffer. Protein bands were visualized by ECL methods for developing in X-ray film to detect protein signals. The expression was quantified by densitometry using ImageJ software and expression was normalized using β-actin as internal control. Data is represented as relative expression of target protein in the treatment groups to the control group.

Statistical analysis

The results are expressed as mean±SEM. Statistical analysis was performed using GraphPad Prism software (version 8.0). One-way ANOVA followed by Tukey test was performed to compare significant difference between the groups. Significant difference is indicated by ^*p < 0.05; ^**p < 0.01 when compared to the control and sham, ^# #p < 0.05 as compared to Aβ₄₀ injected rats.

RESULTS

Intra-hippocampal microinjection of toxic Aβ induces neurobehavioral deficits in rat model of AD

Passive avoidance test

Figure 1 displays PAT in the control, sham, and IHAβ₄₀ groups at the training period on day 14, 21, and 28 of the study. Results revealed no significant change for PAT in the sham group. The IHAβ₄₀ (AD) group indicated significant damage (p < 0.05) in retention in PAT for day 14, 21, and 28 as compared to the control and sham group.

Fig.1

Illustration of the step through passive avoidance test in the control and AD group of rats shown by initial and step-through latency. The AD group rats indicated significant damage in retention in the passive avoidance test as compared to the control and sham group. Values are represented as Mean±SEM (n = 6). ^*p < 0.05, compared to the control and sham group of rats.

Elevated plus maze

Table 1 shows the effect of intra-hippocampal microinjection of toxic Aβ₄₀ on cognitive retention in EPM of the AD group when compared to the control and sham group for the 14th and 28th day of the study period.

Table 1

Effect of intra-hippocampal microinjection of toxic Aβ on cognitive dysfunction in

Groups	Elevated Plus Maze (% retention)
	Training Period	14th Day	28th Day
Control	53.42±5.26	57.32±3.64	60.28±2.92
Sham (ACSF 3 μl)	53.85±5.42	60.97±1.86	63.27±3.20
IHAB₄₀ (20 μM/ml)	54.20±3.67	40.92±4.20^*	19.18±2.15^*

Values are mean±SEM; n = 6 animals/group. ^*p < 0.05 compared to the control and sham animals. ACSF, artificial cerebrospinal fluid; IHAβ, intra-hippocampal microinjection of Aβ.

Aβ induces neuronal loss in the hippocampus of rat brain

The effect of intra-hippocampal microinjection of toxic Aβ₄₀ on the hippocampus was determined by calculating the intensity of Nissl granules in the hippocampus of the rat brain stained with cresyl violet. It was found that a significant decrease (p < 0.05) in the intensity of the Nissl granules in IHAβ₄₀ rat brain were seen as compared to the control and sham operated rats (Fig 2).

Fig.2

Intensity of Nissl granules in the hippocampus of rat brain. A significant decrease in intensity of Nissl granules in IHAβ₄₀ rats compared to the control and sham operated rats. Arrow indicates Nissl granules of neuronal cells. Values are Mean±SEM (n = 6). ^*p < 0.05.

PDp does not affect body weight in sub-chronic toxicity study in rats

Table 2 shows result for the effect of PDp on the body weight of rats in the sub-chronic toxicity study. Result showed that there were no marked changes in the body weight of rats tested for sub-chronic toxicity of PDp at two different doses (100 μg/kg, 200 μg/kg) when compared to their respective control groups on day 0, 21, and 42 of the study. This indicates that there was no toxic effect of PDp administration on PDpG1 and PDpG2 groups on the body weight of the rats.

Table 2

Effect of PDp on body weight (Mean±SEM) of rats in sub-chronic toxicity study

Treatment	Dose (μg/kg)	Body weight (gm)
	0 Day	21 Day	42 Day
Control	–	160±3.65	185±2.21	230±3.15
PDpG1	100	165±2.62	189±3.82	225±4.23
PDpG2	200	170±3.84	192±1.23	220±5.16

Effect of PDp on hematological and biochemical parameters in terms of toxicity in rats

The hematological and biochemical parameters of the rats were determined to analyze the effect of PDp treatment. It was observed that there were no significant changes observed in the hematological and biochemical parameters in both the dosage of PDp administered to the rats (Table 3). Therefore, the two dosage of PDp seems to be safe in the rats for further studies.

Table 3

Effect of PDp on hematological and biochemical parameters in rats

	Control Rats	PDp Treated Rats
		100 μg/kg	200 μg/kg
Hematological Parameters
RBC (10⁶/mm³)	7.05±0.34	6.95±0.52	7.12±0.12
WBC (10³/mm³)	7.24±0.99	7.12±0.45	7.09±0.55
Hb (%)	13.25±0.35	14.52±0.94	13.82±1.12
Neutrophil (%)	66.50±2.20	64.62±2.56	68.40±2.15
Lymphocyte (%)	46.50±2.91	42.66±3.63	41.60±2.84
Eosinophil (%)	2.6±0.55	0.92±0.41	1.42±0.52
Monocyte (%)	4.12±0.81	3.72±0.46	3.94±0.56
Basophil (%)	0.40±0.52	0.00±0.0	0.00±0.0
Biochemical Parameters
Glucose (mg/dl)	90±1.2	94±1.28	97±1.42
Serum Urea (mg/dl)	29.5±1.16	26.65±2.0	24.95±3.2
Creatinine (mg/dl)	0.86±0.15	0.89±0.09	0.94±0.19
Total Bilirubin (mg/dl)	0.24±0.03	0.21±0.04	0.23±0.03
SGOT (IU/L)	62.20±5.2	38.8±4.10	32.6±4.3
SGPT (IU/L)	36.5±1.51	64.20±2.85	59.8±3.13
ALP (IU/L)	59.27±3.02	63.72±7.80	62.52±2.64

RBC, red blood cells; WBC, white blood cells; Hb, hemoglobin; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase; ALP, alkaline phosphatase.

Histopathological evaluation depicted no sub-chronic toxicity for PDp in the liver and kidney of rats

The effect of PDp on the histopathological studies of liver and kidney for sub-chronic toxicity in rats were determined. Figure 3a shows histopathology of the liver and results indicated that there were no marked histopathological changes in the basic histology of liver at both the doses of the PDp (100 μg/kg, 200 μg/kg) as compared to the control, evaluated for sub chronic toxicity of PDp in rats. Similarly, no significant changes in the histopathology of kidney found at both doses of PDp (100 μg/kg, 200 μg/kg) as compared to the control (Fig. 3 panel b), indicating no sub-chronic toxicity of PDp in rats.

Fig.3

Effect of PDp on histopathological studies on the liver and kidney for sub-chronic toxicity in the rat. a) Histological section of the liver. b) Histological section of the kidney.

PDp rescues plaque formation and neurodegeneration in the brain of Aβ-induced AD rats

The Aβ aggregation results in plaque formation, hence deposition of Aβ in the brain sections of different experimental groups (control, sham, Aβ₄₀-, PDp (50 μM) + Aβ₄₀, and PDp (100 μM)+Aβ₄₀-) were validated through Congo red staining. Our results showed that the orange colored amyloid plaques were present in the brain section of the Aβ₄₀-injected rat, unlike their control and sham control (Fig. 4a). Following treatment of PDp at two different concentration (50 and 100 μM), there was a reduction in amyloid plaque formation (Fig. 4a). The reduction in amyloid plaque formation in the treated groups suggests the positive role of PDp in inhibiting Aβ-induced plaque formation in the brain section of Aβ-induced AD rats. Furthermore, the intra-hippocampal microinjection of toxic Aβ on hippocampal neurons showed a decrease in the intensity of Nissl granules (Fig. 4b) as compared to the control and sham group. However, PDp treatment at both the dosages to Aβ₄₀-injected rat blocks the conditions toward normal, thereby suggesting its neuroprotective function (Fig. 4b). Hence, PDp treatment exhibits neuroprotective function against Aβ-induced toxicity.

Fig.4

PDp provides protection against Aβ₄₀-induced toxicity. a) Congo red staining of plaques in the hippocampus of different experimental groups of rats shown after 28 days of study. Number of plaques in the hippocampus: a) significant increase in the number of plaques in Aβ₄₀-injected rats. PDp treated rats showed a significant decrease in the number of plaques. Solid arrows indicate plaques. (n = 6 in each group). b) Nissl granules (cresyl violet staining) of hippocampal neurons in the different experimental groups of rats. There was significant decrease in the intensity of Nissl granules in Aβ₄₀-infused rats. PDp injected rats had a significant increase in the intensity of Nissl granules in the hippocampus when compared to only Aβ₄₀-infused rats.

PDp alleviates activity of antioxidant enzymes and oxidative stress against Aβ-induced toxicity in the rat model of AD

Aβ aggregation leads to the formation of free radicals and redox imbalance thereby causing adverse effect on neuronal health and survival. The study was therefore undertaken to analyze the activity of antioxidant enzymes in the hippocampus of Aβ-injected rats. It was found that SOD activity was elevated in the hippocampus of Aβ-injected rats (Fig. 5a). Following treatment of PDp to the Aβ-injected AD rat showed decrease in SOD activity toward normal (Fig. 5a). Similarly, CAT activity was also found to be elevated in the hippocampus of the Aβ-injected rats (Fig. 5b). Following treatment of PDp to the Aβ-injected AD rat, a decrease in CAT activity toward normal was seen (Fig. 5b). The total ROS generation in the hippocampus of Aβ-injected rat was found to be elevated (Fig. 5c). Following treatment of PDp to the Aβ-injected rat, a decrease in total ROS level toward normal was found (Fig. 5c). Further, nitrite level was also found to be elevated in the hippocampus of Aβ-injected AD rats (Fig. 5d). Following treatment of PDp to Aβ-injected AD rats, a decrease in nitrite formation and tendency toward normal was seen (Fig. 5d), thereby suggesting its neuroprotective effect against ROS imbalance due to Aβ-induced toxicity.

Fig.5

Illustration of SOD and CAT activity, total ROS and nitrite level in the different experimental groups as labeled. a) SOD activity in different group. b) CAT activity in different group. c) Total ROS level in the different groups. d) Nitrite level in different groups. Values are Mean±SEM (n = 6). ^**p < 0.01 as compared to the control and sham.

Neuroprotective effect of PDp against Aβ-induced neurotoxicity via optimization of expression of proteins (iNOS, IL-1β, TNF-α, p-Tau, PP-2A, and GSK-3 β) involved in neuroinflammation and AD pathology

The hallmark pathologies of AD are also associated with elevated neuroinflammatory responses. Therefore, the expression profile of proteins linked to AD pathology (p-Tau, PP-2A, and GSK-3β) and neuroinflammatory responses (iNOS, IL-1β, and TNF-α) were analyzed through western blotting. It was found that the neuroinflammatory marker proteins (iNOS, IL-1β, and TNF-α) were found to be upregulated in the hippocampal tissue of the Aβ-injected rats (Fig. 6a). However, the expression levels were optimized toward normal in the hippocampal tissue of the treated groups [PDp (50 μM) + Aβ₄₀ and PDp (100 μM) + Aβ₄₀] (Fig. 6a). Furthermore, proteins linked to Aβ-induced AD pathology involved in hyperphosphorylation of tau protein were also analyzed, revealing levels that were altered in the hippocampal tissue of the Aβ-injected rats (Fig. 6b). However, the expression levels were optimized toward normal in the hippocampal tissue of the treated groups [PDp (50 μM) + Aβ₄₀ and PDp (100 μM) + Aβ₄₀] (Fig. 6b). Therefore, it is evident that PDp tends to provide neuroprotection against Aβ-induced neuropathology in the rat model of AD.

Fig.6

PDp modulated expression of the proinflammatory cytokines and molecular markers of AD in the hippocampus of Aβ-injected rats. Rats were treated with 20 μM Aβ₄₀, 50 μM and 100 μM of PDp as labeled. a) Effect of PDp on iNOS, IL-1β, TNF-α, p-Tau, GSK-3β, and PP-2A protein expression by western blotting. b) The expression of relative protein level. Values are Mean±SEM (n = 6). ^**p < 0.01 as compared to the control and sham, ^# #p < 0.01 as compared to the Aβ-injected rats.

DISCUSSION

The formation of senile plaques via Aβ aggregation is one of the pathological hallmarks involved in AD [3 –5]. Studies have shown that plaque formation is an outcome of alteration in Aβ metabolism, which results in tau protein hyperphosphorylation, synaptic degeneration, ROS imbalance, and neurotoxicity, thereby leading to the neuronal apoptosis [4 , 14]. There is no available therapy that restricts or halts the progression of the AD. Current therapy provides only limited benefits by slowing down the rate of cognitive decline associated with AD [22].

Inhibition of protein aggregation seems to be one of the potential strategies against Aβ plaques and NFTs. Several compounds (synthetic or/and natural) are being studied using both the in vitro and in vivo experimental model system [22 , 25]. In this context, PDp, which is a special type of β-sheet breaker peptide, has been shown to exhibit neuroprotective property against Aβ₄₀-induced toxicity in SH-SY5Y cells [24]. Based on the positive result obtained using in vitro approach in our earlier studies [24], we were interested to test the potential of PDp in a rat model of AD. The present study reveals that intra-hippocampal microinjection of toxic Aβ to the rat induces AD phenotypes characterized by defective behavioral functions. The Aβ-injected rats exhibited significant damage in retention in passive avoidance test as compared to the control and sham group. Also, the elevated plus maze test depicted poor memory retention in Aβ-injected rats. Besides, a significant decrease in intensity of Nissl granules in the histological section of the hippocampus of Aβ-injected rats was observed as compared to the control and the sham operated rats. We further tested sub-chronic toxicity of PDp in the rats in order to define the dosage and its role against Aβ-induced toxicity. Based on sub-chronic toxicity, two dosages of PDp (50 and 100 μM) were used for the current experiment. It was found that the Aβ-injected AD rat group showed an increased number of plaques, which was significantly decreased in the PDp treated rat groups [PDp (50 μM) + Aβ₄₀ and PDp (100 μM) + Aβ₄₀]. In addition, there was a significant decrease in intensity of the Nissl granules in Aβ-infused rats thereby suggesting neurotoxicity. However, PDp treated groups [PDp (50 μM) + Aβ₄₀ and PDp (100 μM) + Aβ₄₀] showed significant increase in the intensity of Nissl granules in the hippocampus as compared to the Aβ-induced rat group. PDp seems to provide a neuroprotective effect against Aβ-induced toxicity in the hippocampus. Further, Aβ toxicity is known to induce redox imbalance [16, 41]. The antioxidant enzymes activity, total ROS level, and nitrite level were found to be elevated in the Aβ-induced group, while tissue antioxidant enzymes activity, ROS, and nitrite levels were optimized toward normal in the PDp treated groups [PDp (50 μM) + Aβ₄₀ and PDp (100 μM) + Aβ₄₀]. Hence, PDp seems to alleviate activity of antioxidant enzymes and redox imbalance against Aβ-induced toxicity in the hippocampus of rat brain.

Further, neuroinflammatory responses are also evoked in the AD brain on account of Aβ-induced toxicity and these neuroinflammatory responses play a crucial role in the neurodegeneration process whereby it leads to synapse dysfunction and neuronal apoptosis [42 –44]. It has been shown that the inflammatory response in AD causes transcriptional upregulation of inducible form of nitric oxide synthase (iNOS) which aggravates progression of AD [45]. IL-1β is a proinflammatory cytokine expressed in AD brain and is known to be processed by oligomeric Aβ via increased ROS production [46]. Another pro-inflammatory cytokine, TNF-α, is elevated in AD which may lead to enhanced Aβ production with low clearance thereby leading to neuronal damage and apoptosis [47, 48]. In this context, the above-mentioned neuroinflammatory markers were analyzed in the aforementioned experimental groups and was found that iNOS, IL-1β, and TNF-α expression was elevated in the Aβ injected rat group. Such elevated expression of the markers clearly indicates elevated neuroinflammatory response. However, PDp treated groups [PDp (50 μM) + Aβ₄₀ and PDp (100 μM) + Aβ₄₀] showed reduction in the expression of iNOS, IL-1β, and TNF-α as compared to the Aβ-injected rat group.

Further, the protein expression of p-Tau, GSK-3β, and PP-2A were also studied. It was found that p-Tau and GSK-3β expression were elevated in Aβ injected rat group while their levels were normalized on account of the PDp treatment. This finding supports that PDp tends to inhibit Aβ aggregation induced hyperphosphorylation of tau protein. Protein phosphatase 2A (PP-2A) is known to regulate phosphorylation of tau protein and that its dysfunction induces hyperphosphorylation of tau protein [49]. Therefore, PP-2A expression levels were also studied and it was found that its expression level was diminished in the Aβ-injected rat group. However, the expression level of PP-2A was normalized in the PDp treated rat groups [PDp (50 μM) + Aβ₄₀ and PDp (100 μM) + Aβ₄₀], thereby further corroborating the finding that PDp tends to suppress hyperphosphorylation of tau protein induced by Aβ aggregation and plaque formation.

Therefore, the present study shows that PDp imparts neuroprotective effect against Aβ-induced toxicity. However, more studies are required to further explore the PDp in context to its stability, retention time, blood brain barrier breaching capacity, and any associated side effect. The studies done in this context will help to reveal the true anti-amyloidosis property of PDp and will offer a new therapeutic strategy against Aβ-induced AD pathology.

Conclusions

The present study depicts the potential neuroprotective role of PDp against Aβ-induced toxicity in a rat model of AD. It is clear from the study that PDp inhibits plaque formation thereby normalizing oxidative stress, inhibiting tau protein phosphorylation, and suppressing neuroinflammatory responses. It is also clear from the study that inhibition of Aβ oligomerization via PDp offers a potential therapeutic approach against amyloidogenesis. Figure 7 summarizes the schematic illustration of the role of PDp in alleviating Aβ-induced neurotoxicity. Future studies deciphering the underlying mechanism(s) will pave the way to corroborate its potential as an AD therapeutic.

Fig.7

Schematic illustration of the role of PDp in alleviating Aβ₄₀-induced neurotoxicity. Aβ intoxication induces amyloid plaque formation, redox imbalance, and neuro-inflammatory response in the hippocampus of rat brain. PDp shows its neuroprotective effects and mitigates Aβ-induced neurotoxicity.

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/18-1273r1).

Footnotes

ACKNOWLEDGMENTS

A.C.M. acknowledges financial support from DBT (BT/347/NE/TBP/2012]) and the partial support from (BT/ PR16164/NER/95/88/2015), DST PURSE-II, UGC RN, UGC-SAP, (UPE-II, JNU- Project Id No. 247). We acknowledge RPM College, Uttarpara, Hooghly, and School of Life Sciences, JNU for providing all the facilities for the commencement of the project. We thank department of Chemistry, IIT Guwahati for providing facilities for peptide synthesis and characterization. The authors are highly thankful to DST PURSE-II (PAC-JNU-DST-PURSE-462), Jawaharlal Nehru University, New Delhi for providing financial assistance for the publication.

References

Scheltens

, Blennow

, Breteler

MMB

, de Strooper

, Frisoni

, Salloway

, Van der Flier

(2016) Alzheimer’s disease. Lancet 388, 505–517.

Selkoe

(2001) Alzheimer’s disease: Genes, proteins, and therapy. Physiol Rev 81, 741–766.

Alzheimer’s Association (2018) 2018 Alzheimer’s disease facts and figures. Alzheimers Dement 14, 367–429.

Sanabria-Castro

, Alvarado-Echeverría

, Monge-Bonilla

(2017) Molecular pathogenesis of Alzheimer’s disease: An update. Ann Neurosci 24, 46–54.

Singh

, Srivastav

, Yadav

, Srikrishna

, Perry

(2016) Overview of Alzheimer’s disease and some therapeutic approaches targeting Aβ by using several synthetic and herbal compounds. Oxid Med Cell Longev 2016, 7361613.

Cummings

, Doody

, Clark

(2007) Disease-modifying therapies for Alzheimer disease: challenges to early intervention. Neurology 69, 1622–1634.

Deane

, Bell

, Sagare

, Zlokovic

(2009) Clearance of amyloid-β peptide across the blood-brain barrier: Implication for therapies in Alzheimer’s disease. CNS Neurol Disord-Drug Targets 8, 16–30.

Jucker

, Walker

(2015) Neurodegeneration: Amyloid-β pathology induced in humans. Nature 525, 193–194.

Roberson

, Scearce-Levie

, Palop

, Yan

, Cheng

, Wu

, Gerstein

, Yu

, Mucke

(2007) Reducing endogenous tau ameliorates amyloid β-induced deficits in an Alzheimer’s disease mouse model. Science 316, 750–754.

10.

Awasthi

, Singh

, Pandey

, Dwivedi

(2016) Alzheimer’s disease: An overview of amyloid beta dependent pathogenesis and its therapeutic implications along with in silico approaches emphasizing the role of natural products. J Neurol Sci 361, 256–271.

11.

Stoothoff

, Johnson

(2005) Tau phosphorylation: Physiological and pathological consequences. Biochim Biophys Acta 1739, 280–297.

12.

Wang

, Li

, Benedetti

, Lee

(2003) α7 nicotinic acetylcholine receptors mediate β-amyloid peptide-induced tau protein phosphorylation. J Biol Chem 278, 31547–53.

13.

Götz

, Chen

, Van Dorpe

, Nitsch

(2001) Formation of neurofibrillary tangles in P301L tau transgenic mice induced by Aβ42 fibrils. Science 293, 1491–1495.

14.

Kumar

, Singh

(2015) A review on Alzheimer’s disease pathophysiology and its management: An update. Pharmacol Rep 67, 195–203.

15.

Hardy

, Selkoe

(2002) The amyloid hypothesis of Alzheimer’s disease: Progress and problems on the road to therapeutics. Science 297,, 353–356.

16.

Cheignon

, Tomas

, Bonnefont-Rousselot

, Faller

, Hureau

, Collin

(2018) Oxidative stress and the amyloid beta peptide in Alzheimer’s disease. Redox Biol 14,, 450–464.

17.

Tönnies

, Trushina

(2017) Oxidative stress, synaptic dysfunction, and Alzheimer’s disease. J Alzheimers Dis 57,, 1105–1121.

18.

Zhao

, Zhao

(2013) Oxidative stress and the pathogenesis of Alzheimer’s disease. Oxid Med Cell Longev 2013, 316523.

19.

Praticò

(2008) Oxidative stress hypothesis in Alzheimer’s disease: A reappraisal. Trends Pharmacol Sci 29, 609–615.

20.

Makhaeva

, Lushchekina

, Boltneva

, Sokolov

, Grigoriev

, Serebryakova

, Vikhareva

, Aksinenko

, Barreto

, Aliev

, Bachurin

(2015) Conjugates of γ-Carbolinesand Phenothiazine as new selective inhibitors of butyrylcholinesterase and blockers of NMDA receptors for Alzheimer disease. Sci Rep 5, 13164.

21.

Karran

, De Strooper

(2016) The amyloid cascade hypothesis: Are we poised for success or failure? J Neurochem 139, 237–252.

22.

Graham

, Bonito-Oliva

, Sakmar

(2017) Update on Alzheimer’s disease therapy and prevention strategies. Annu Rev Med 68, 413–430.

23.

Pohanka

(2011) Cholinesterases, a target of pharmacology and toxicology. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 155, 219–229.

24.

Kumar

, Paul

, Kalita

, Kumar

, Srivastav

, Hazra

, Ghosh

, Mandal

, Mondal

(2017) A peptide based pro-drug ameliorates amyloid-β induced neuronal apoptosis in in vitro SH-SY5Y cells. Curr Alzheimer Res 14, 1293–1304.

25.

Salahuddin

, Fatima

, Abdelhameed

, Nusrat

, Khan

(2016) Structure of amyloid oligomers and their mechanisms of toxicities: Targeting amyloid oligomers using novel therapeutic approaches. Eur J Med Chem 114, 41–58.

26.

Soto

, Sigurdsson

, Morelli

, Kumar

, Castaño

, Frangione

(1998) β-sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: Implications for Alzheimer’s therapy. Nat Med 4, 822–826.

27.

Paul

, Kumar

, Kalita

, Ghosh

, Mondal

, Mandal

(2018) A peptide based pro-drug disrupts Alzheimer’s amyloid into non-toxic species and reduces Aβ induced toxicity in vitro. Int J Pept Res Ther 24, 201–211.

28.

Nadimpally

, Paul

, Mandal

(2014) Reversal of aggregation using β-breaker dipeptide containing peptides: Application to Aβ(1–40) self-assembly and its inhibition. ACS Chem Neurosci 5, 400–408.

29.

Nikkhah

, Ghahremanitamadon

, Zargooshnia

, Shahidi

, Asl

(2014) Effect of amyloid β-peptide on passive avoidance learning in rats: A behavioral study. Avicenna J Neuropsychophysiol 1, 1–4.

30.

Paxinos

, Watson

(1998) The rat brain in stereotaxic coordinates. Academic Press, 6th Edition, San Diego.

31.

Lee

, Brady

, Shapiro

, Dorsa

, Koenig

(2007) Prenatal stress generates deficits in rat social behavior: Reversal by oxytocin. Brain Res 1156, 152–167.

32.

Gray

(1954) The Microtomist’s Formulary and Guide, Blakiston Co., New York.

33.

Skovronsky

, Zhang

, Kung

, Trojanowski

, Lee

(2000) In vivo detection of amyloid plaques in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A 97, 7609–7614.

34.

Lowry

, Rosebrough

, Farr

, Randall

(1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265–275.

35.

Misra

, Fridovich

(1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247, 3170–3175.

36.

Ahmad

, Fatima

, Hossain

, Mondal

(2018) Evaluation of naproxen-induced oxidative stress, hepatotoxicity and in vivo genotoxicity in male Wistar rats. J Pharm Anal 8,, 400–406.

37.

Claiborne

(1985) Catalase activity. In CRC Handbook of Methods for Oxygen Radical Research, Greenwald

, ed. CRC Press, Boca Raton, pp. 283–284.

38.

Fatima

, Usmani

, Firdaus

, Zafeer

, Ahmad

, Akhtar

, Dawar Husain

, Ahmad

, Anis

, Mobarak Hossain

(2015) In vivo induction of antioxidant response and oxidative stress associated with genotoxicity and histopathological alteration in two commercial fish species due to heavy metals exposure in northern India (Kali) river. Comp Biochem Physiol C Toxicol Pharmacol 176-177, 17–30.

39.

Socci

, Bjugstad

, Jones

, Pattisapu

, Arendash

(1999) Evidence that oxidative stress is associated with the pathophysiology of inherited hydrocephalus in the H-Tx rat model. Exp Neurol 155, 109–117.

40.

Fatima

, Srivastav

, Ahmad

, Mondal

(2019) Effects of chronic unpredictable mild stress induced prenatal stress on neurodevelopment of neonates: Role of GSK-3β. Sci Rep 9, 1305.

41.

Swomley

, Förster

, Keeney

, Triplett

, Zhang

, Sultana

, Butterfield

(2014) A beta, oxidative stress in Alzheimer disease: Evidence based on proteomics studies. Biochim Biophys Acta 1842, 1248–1257.

42.

J Fernandez-Perez

, Peters

, G Aguayo

(2016) Membrane damage induced by amyloid beta and a potential link with neuroinflammation. Des 22, 1295–1304.

43.

Heneka

, Carson

, El Khoury

, Landreth

, Brosseron

, Feinstein

, Jacobs

, Wyss-Coray

, Vitorica

, Ransohoff

, Herrup

(2015) Neuroinflammation in Alzheimer’s disease. Lancet Neurol 14, 388–405.

44.

Dorey

, Chang

, Liu

, Yang

, Zhang

(2014) Apolipoprotein E, amyloid-beta, and neuroinflammation in Alzheimer’s disease. Bull 30, 317–330.

45.

Kummer

, Hermes

, Hammerschmidt

, Terwel

, Gorji

, Pape

, Heneka

(2009) iNOS gene deficiency modifies amyloid β deposition, cognitive performance and hippocampal long-term potentiation in an APP transgenic mouse model. Alzheimers Dement 5, P89.

46.

Parajuli

, Sonobe

, Horiuchi

, Takeuchi

, Mizuno

, Suzumura

(2013) Oligomeric amyloid β induces IL-1β processing via production of ROS: Implication in Alzheimer’s disease. Cell Death Dis 4, e975.

47.

Chang

, Yee

, Sumbria

(2017) Tumor necrosis factor α Inhibition for Alzheimer’s Disease. J Cent Nerv Syst Dis 9, 1179573517709278.

48.

Decourt

, Lahiri

, Sabbagh

(2017) Targeting tumor necrosis factor alpha for Alzheimer’s disease. Curr Alzheimer Res 14, 412–425.

49.

Sontag

, Sontag

(2014) Protein phosphatase 2A dysfunction in Alzheimer’s disease. Front Mol Neurosci 7, 16.

A Synthetic Pro-Drug Peptide Reverses Amyloid-β-Induced Toxicity in the Rat Model of Alzheimer’s Disease

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Ethical statement

Peptides

Animals

Intra-hippocampal microinjection of toxic Aβ40 by stereotaxic surgery

Evaluation of sub-chronic toxicity of synthetic PDp in rat

Evaluation of the PDp on neurodegeneration and neuroinflammation associated with rat model of AD

Passive avoidance test by shuttle box apparatus

Elevated plus maze

Extraction of hippocampus from the rat brain

Cresyl violet staining of hippocampal neurons to assess neurodegeneration and neuroprotection

Analysis of blood for hematological and biochemical parameters in respect to PDp toxicity

H&E staining of liver and kidney samples for sub-chronic toxicity

Congo red staining for Aβ deposition

Activity of tissue antioxidant enzymes

Tissue homogenization and preparation of supernatant

Superoxide dismutase activity

Catalase activity

Assessment of reactive oxygen species generation

Assessment of nitrite level

Western blot analysis for neuroinflammatory (iNOS, IL-1β, TNF-α) and AD pathology associated (p-Tau, GSK-3β, PP-2A) markers

Statistical analysis

RESULTS

Intra-hippocampal microinjection of toxic Aβ induces neurobehavioral deficits in rat model of AD

Passive avoidance test

Elevated plus maze

Aβ induces neuronal loss in the hippocampus of rat brain

PDp does not affect body weight in sub-chronic toxicity study in rats

Effect of PDp on hematological and biochemical parameters in terms of toxicity in rats

Histopathological evaluation depicted no sub-chronic toxicity for PDp in the liver and kidney of rats

PDp rescues plaque formation and neurodegeneration in the brain of Aβ-induced AD rats

PDp alleviates activity of antioxidant enzymes and oxidative stress against Aβ-induced toxicity in the rat model of AD

Neuroprotective effect of PDp against Aβ-induced neurotoxicity via optimization of expression of proteins (iNOS, IL-1β, TNF-α, p-Tau, PP-2A, and GSK-3 β) involved in neuroinflammation and AD pathology

DISCUSSION

Conclusions

Footnotes

ACKNOWLEDGMENTS

References

Intra-hippocampal microinjection of toxic Aβ₄₀ by stereotaxic surgery