Authored by Om Gandhi*
Abstract
Because the accumulation of amyloid plaques plays a central role in the pathogenesis of Alzheimer’s disease (AD) and is responsible for many of its neurodegenerative effects, this project aims to evaluate the effectiveness of TRSV phytoalexin infusions as a novel treatment for the disease by comparing three separate agents and determining its impact on the accumulation of amyloid plaques in mouse Neuro2a cells. The experimental model is as follows: Neuro2a cells were first thawed using incubation and centrifugation techniques. The subculturing protocol was then performed to maximize cellular viability using DPBS and TrypLE dissociation reagents. The cells were transfected with pCMV4-ApoE4 bacterial plasmid using Opti-MEM medium and Lipofectamine LTX while also being treated with phytoalexin agents. Bradford’s assay was performed on the samples using CBB G-250 reagent, and they were run through a spectrophotometer and a Python low-pass filter to generate readings. The experimental data showed that natural grape seed phytoalexin extract was the most effective treating agent, followed by curcumin extract and synthetic RDS phytoalexin. All treating agents lessened the accumulation of the amyloid plaques significantly, supporting the novel infusion protocol as a treatment for AD.
Keywords: Amyloid plaques; ApoE4; Trans-resveratrol; Neuro2a cells; Apolipoprotein E (APOE)
Abbreviations: AD: Alzheimer’s Disease; DPBS- Dulbecco’s Phosphate-Buffered Saline; DMSO- Dimethyl Sulfoxide; CBB- Coomassie Brilliant Blue; TRSV- Trans-Resveratrol; RDS- Resveratrol Dietary Supplement; APOE- Apolipoprotein E; Aβ- Beta Amyloid; APP- Amyloid Precursor Protein; ROSReactive Oxygen Species; AICD- APP Intracellular Domain
Introduction
Alzheimer’s disease (AD) is a neurodegenerative condition that results in the continuous decline of one’s cognitive, behavioral, and social capabilities [1]. It is the most common type of dementia that slowly destroys memory along with the ability to carry out both simple and complex tasks [1,2]. In total, AD currently afflicts about an estimated 5.8 million Americans age 65 and older and is the sixth leading cause of death in the United States. It is also the leading cause of mental degeneration in the elderly and accounts for a large percentage of admissions to long-term care facilities [3]. Despite there being no way to stop the progression of the disease completely, advancements in scientific research have allowed for the understanding of AD at the cellular and molecular level.
The biological basis of AD
The healthy human brain consists primarily of tens of billions of neurons that process and transmit information via electrical and chemical signals, regulating everything from muscle movement to energy storage. At first, AD disrupts connections between neurons involved with memory in areas such as the hippocampus, resulting in loss of function and cellular death [4]. It later affects areas in the cerebral cortex responsible for language, reasoning, and social behavior. Eventually, many other regions of the brain are damaged, leading to death [4,5].
What can explain the brain damage commonly associated with AD? Although the definitive causes and mechanisms of the disease have yet to be determined, recent studies point to a series of common factors. A range of research has shown that the neurodegenerative phenomena are initiated and enhanced by oxidative stress, a state characterized by an excessively high ratio of oxidants to antioxidants [6-10]. This imbalance can occur either as a result of decreased oxidation defense or an increase in the concentration of free radicals, commonly defined as compounds with unpaired electrons in their valence shells. Free radicals are well known as highly-reactive health hazards, but they attack some types of molecules more than others [11].
The impact of oxidative stress does not stop with the brain’s physical structure. High concentrations of reactive oxygen species (ROS), such as free radicals, also degrade enzymes and proteins that are crucial to normal neuronal and glial cell function [12]. This is the case for two enzymes, which are especially sensitive to oxidative modification: glutamine synthetase and creatine kinase, both of which are less present in AD brains. Oxidative impairment of creatine kinase may also cause decreased energy metabolism, demonstrating the critical nature of the enzymes that AD-related oxidative stress hurts most [12,13]. AD also results in the abnormal deposition of neurofibrillary tangles, “characterized by the aggregation and hyperphosphorylation of the τ protein into paired helical filaments,” [14]. The pathologic aggregation of the τ protein leads to fibril formation, insolubility, and an inability for affected neurons to send signals to other neurons, leading to neurological degeneration [14,15]. Furthermore, phosphorylation is linked to oxidation through the microtubule-associated protein kinase pathway and activation of the transcription factor nuclear factor- κB (NF-κB), thus linking oxidation to the hyperphosphorylation of τ proteins [14-17]. Perhaps the most significant biological indicator of AD, however, is the presence of amyloid plaques.
The formation of amyloid plaques requires two specific proteins: beta-amyloid protein and apolipoprotein E (APOE) [18]. While the beta-amyloid protein is the primary protein involved, APOE promotes the accumulation of beta-amyloid proteins into plaques. These insoluble plaques have toxic properties, including synaptic dysfunction, τ phosphorylation, and neurodegeneration, that contribute significantly to the progression of AD [18,19].
Beta-amyloid protein
The beta-amyloid (Aβ) 4-κDa peptide has a length that varies from 38 to 43 amino acids. The protein itself usually is present in a variety of somatic cells, but its normal biological function remains unknown [20]. “Under physiological conditions, Aβ40 constitutes about 90 percent of the total amount of Aβ,” [20]. Of the two significant species of Aβ (Aβ40 and Aβ42), the latter is more aggregation-prone due to two additional hydrophobic amino acids [20,21]. This accounts for Aβ42’s position as the predominant compound accumulating in plaques in the AD brain.
Abnormal deposition of the Aβ protein is caused by aggregation of the transmembrane amyloid precursor protein (APP), which is synthesized by the sequential action of β-secretase (BACE1) and γ-secretase [22-25]. “Amyloid precursor protein (APP) is an integral membrane protein, with a large N-terminal extracellular domain and a short C-terminal cytoplasmic domain,” [22]. It is synthesized in the endoplasmic reticulum (ER), post-transcriptionally modified in the Golgi apparatus, and then transported to the cell surface, where it is attached to the plasma membrane. APP is expressed throughout the body, and Aβ is a typical product of APP metabolism. “The APP gene is spliced to yield isoforms of various lengths; the 751- and 770- isoforms predominate in non-brain or nerve cells, whereas the 695-amino acid form (APP695) is by far the most predominant form in neurons,” [22]. APP and APP-like protein have orthologues across nearly all animals, indicating that they may be necessary for a variety of biological processes. However, their exact functions are not currently known. “Possible roles for APP and its proteolytic products range from axonal transport to transcriptional control and from cell adhesion to apoptosis,” [24].
Though comparatively little may be known about the cellular function of APP, much is known about its genetics and proteolytic processing, particularly concerning AD. APP is first cleaved by either α-or β-secretase at the respective α- or β-sites [20,25-27]. These two sites are near the extracellular side of APP’s transmembrane domain, allowing for more efficient cleavage. α- and β-secretase proteases compete for APP cleavage to give two products: a soluble APP and a membrane-anchored C-terminal stub [20,25- 29]. This stub is the site of the crucial AD indicated cleavage, where γ-secretase acts.
“While the division of C83 generates the 6-κDa APP intracellular domain (AICD) and releases the N-terminal 3κDa peptide (p3) into the extracellular space, cleavage of C99 generates AICD and the Aβ peptide,” [20]. The processing of APP by beta-secretase and gammasecretase and the corresponding formation of beta-amyloid protein is known as the amyloidogenic pathway. In the alternative path of APP processing termed as the non-amyloidogenic pathway, no Aβ is produced as α-secretase cleaves APP with an Aβ sequence [27-30]. The second type of protein required for the formation of amyloid plaques is the Apolipoprotein E.
Apolipoprotein E (APOE)
The ApoE gene has demonstrated to code for Apolipoprotein E, a fat-binding protein. Although everyone inherits a copy of some form of the APOE gene from each parent, it is estimated that between 40 and 65 percent who are diagnosed with AD have the ApoE4 gene, implying a genetic basis for the disease [31].
There are three common forms of the APOE gene: ApoE2, ApoE3, and ApoE4. “ApoE has an N-terminal domain and a C-terminal domain, which are the receptor-binding region and fat-binding region, respectively,” [32]. There are three different isoforms, APOE2, APOE3, and APOE4. The structural differences between them are located at amino acid residues 112 and 158. While ApoE2 has cysteine 112 and cysteine 158, ApoE3 has cysteine 112 and arginine 158, and ApoE4 has arginine 112 and arginine
The changes in the R-group of amino acids result in structural and functional (i.e., binding ability) differences between them [32,33]. The exact mechanism connecting APOE isoforms and Aβ metabolism is not yet clear. Recent studies, with evidence collected from both neuron cultures and mice models, have identified that ApoE could activate the DLK-MKK7-ERK1/2 cascade, which stimulates transcription factor AP-1 [33-35]. Increased activity of AP-1 eventually results in a higher rate of APP transcription and, as previously discussed, elevated Aβ production. ApoE4 was shown to have the highest activation of this cascade and, in turn, generated more Aβ than APOE3 and APOE2. Aβ also exhibited accelerated fibrillization when incubated with ApoE4, when compared to ApoE3 in vitro [35,36].
Moreover, extracellular ApoE positively affected the targeting of lipid rafts with Aβ, where it was converted to an oligomer. As previously mentioned, the brain is primarily composed of vulnerable lipid-based substances. Extracellular ApoE can direct Aβ protein to bind to fatty substances, like those found in the brain [37]. Therefore, ApoE4 can assist in the formation of Aβ, playing a crucial role in the accumulation of beta-amyloid proteins that results in the nefarious amyloid plaques. Since these plaques play such a central role in the pathogenesis of AD [18,19], it is vital to investigate how to limit their accumulation.
Trans-resveratrol
Resveratrol is a natural phytoalexin that acts against pathogens and is an integral part of the immune system in more than 70 plant species [38]. Its unique structure as a 3,5,4′ -trihydroxytrans- stilbene possessing two phenyl rings linked to each other by an ethylene bridge allows it to possess a variety of properties and capabilities [38,39]. “As a natural food ingredient, numerous studies have demonstrated that resveratrol possesses a very high antioxidant potential,” [38]. This makes it a prime candidate for the restoration of the correct balance between oxidants and antioxidants, thus limiting oxidative stress, one of the neurodegenerative factors present in AD. Resveratrol also exhibits antitumor activity, and its anti-cancer properties contribute significantly to its ability to inhibit all carcinogenesis stages [38-40].
Other positive bioactive effects have also been reported. These effects stretch from anti-inflammatory action to cardioprotection, and from vasorelaxation to neuroprotection. Although resveratrol and trans-resveratrol are similar in their many characteristics, one defining feature of trans-resveratrol is its ability to act as a free radical scavenger, stabilizing harmful unpaired valence electrons and decreasing damage to body tissues [40]. This makes the compound especially promising as a potential treatment agent for AD.
The primary purpose of the project is to determine whether grape seed phytoalexin extract or curcumin phytoalexin extract serves as the most effective natural trans-resveratrol (TRSV) treating agent by best limiting the accumulation of amyloid plaques. This project also aims to compare the treatment capabilities of the natural TRSV phytoalexins to a chemical phytoalexin supplement: resveratrol dietary supplement (RDS).
Materials and Methods
Preparing the cells
To maximize cellular survival, the procedure was performed under optimal conditions (296K and seeding at 4x10,000 cells cm2 ) and in a sterile environment. A vial of Neuro2a (neuroblastoma) mouse cells was thawed using a water bath, and its contents were transferred to a 15mL centrifuge tube. 10mL of pre-warmed cell culture medium (Eagle Medium) was added to the centrifuge tube in a drop by drop manner to avoid osmotic shock. The cells were centrifuged at 200 x g for five minutes to allow for the removal of the freezing medium that contains dimethyl sulfoxide (DMSO).
The supernatant was discarded, and the pellet was resuspended in 10mL of the complete, pre-warmed medium in a flask. 3mL of 100 units/mL penicillin, 100 μg/mL streptomycin, 0.3 μg/mL G418,
2.0 μg/mL puromycin, and 1.0 μg/mL tetracycline was each added to the flask to minimize the growth of unwanted bacteria. The flask was placed in the incubator with a rocking motion to ensure even distribution of the cells in solution. The cells were incubated at 37C in a humidified atmosphere containing 95 percent air and 5 percent CO2 until the cells have reached about 70-80 percent confluency (approximately 24 hours). This is when the cells are still growing exponentially (near the end of the log phase) and will result in improved overall cell viability, shorter lag time, and less aggregation.
To read more about this article.....Open access Journal of Neurology & Neuroscience
Please follow the URL to access more information about this article
To know more about our Journals...Iris Publishers
No comments:
Post a Comment