Main Research Findings

1) Pinealon has the potential to elicit potent neuroprotective effects in the early stages of Alzheimer’s disease.

2) Administration of pinealon to pregnant rats has been shown to improve learning ability and spatial orientation of the offspring, as well as decrease the accumulation of reactive oxygen species and necrotic cells.

Selected Data

1) Alzheimer’s disease is considered the most common neurodegenerative disease that frequently arises in older adults. Pathogenesis of the disease is related to damage to the limbic system, more specifically the hippocampus, as it is responsible for emotional regulation and memory consolidation. Manifestation of the disease is typically marked by mutations in the beta-amyloid precursor protein genes leading to accumulation of beta-amyloid plaque, as well as the presenilin 1 and 2 genes. While there are currently no preventative measures for Alzheimer’s disease, short peptide chains rich in arginine have been found to elicit neuroprotective properties by reducing excitotoxicity, diminishing mitochondrial dysfunction, and increasing neurovascular viability [1].

Pinealon is considered an arginine containing tripeptide that has been linked to correction of cerebral dysfunctions following oral administration. The neuroprotective effects of Pinealon were related to improved memory and cognitive functioning, a reduction of neuronal apoptosis, increased perceptual-motor response times, and decreased aging rate of the central nervous system. The research team of Khavinson et al reviewed the various molecular aspects of the EDR tripeptide, Pinealon, in an attempt to further define the mechanism of action behind the neuroprotective effects of the compound in cases of Alzheimer’s disease. This analysis was conducted by examining the role of MAPK/ERK, caspase-3, p53, DOS2, GPX1, PPARA, PPARG, serotonin, and calmodulin and how they relate to the pathogenesis of the disease [1].

MAPKs are considered conserved multifunctional signaling molecules that facilitate the conversion of extracellular signals into intracellular responses. The ERK1/2 cascade is one of the most commonly studied MAPK pathways as it plays a crucial role in the transmission of signals. These receptors are typically activated through mechanisms involving Ras proteins that recruit components from the MAPK cascade to the plasma membrane in order to activate the appropriate receptors. Following stimulation of ERK1/2, substrates responsible for proliferation, differentiation, morphology and plasticity of neurons, stress response control, and apoptotic regulation undergo phosphorylation. When the signaling cascade is impaired neurodegenerative diseases tend to develop; if the inhibition of the MAPK/ERK pathways continues, mitochondrial dysfunction, proinflammatory cytokine secretion, and cell death will occur [1].

Research has shown that neurons in the brain consume oxygen and have various defense mechanisms against oxidative stress. That being said, Khavinson et al assessed the ability of the compound superoxide dismutase (SOD) to compensate for oxidative changes throughout neurons, as well as how these mechanisms are altered with the administration of Pinealon. Based on an animal model including Tg2576 mice, it was determined that SOD is found in the mitochondria and concentration levels tend to increase throughout the cerebral cortex in response to aging as an attempt to reduce oxidative stress. The animals without this compensatory mechanism were found to have higher levels of neuronal apoptosis as a result of mitochondrial oxidative stress. That being said, when SOD expression is diminished neuropathology begins to develop as a result of mitochondrial oxidative stress and neuronal apoptosis [1].

Additionally, the p53 transcription factor was evaluated due to the crucial role it plays in DNA damage, maintenance of genome integrity, and suppression of tumor development. Previous research has shown that disruption of p53 typically leads to the development of neurological deficits, cancers, and metabolic syndrome while low levels of the transcription factor has the potential to decrease DNA damage as well as oxidative stress. Under normal conditions, p53 responds to DNA damage by moving to the mitochondrial matrix to form a gene complex that releases cytochrome C and activates various caspases. When the main effector caspase-3 is activated neurodegenerative processes related to cerebral circulation disorders begin to develop as neuronal apoptosis occurs [1].

2) The research team of Arutjunyan et al examine the effect of Pinealon on the development of hyperhomocysteinemia during pregnancy. Hyperhomocysteinemia is defined as a metabolic imbalance that causes accumulation of homocysteine in the blood that can lead to toxicosis, nephropathy, preeclampsia, and eclampsia in the mother, and various mental delays in the infant. For the purpose of this study, the research team used pregnant female Wistar rats weighing 180-200 grams, each. Hyperhomocysteinemia was induced by administering methionine to the rats starting in the secondary trimester of pregnancy and continuing until the end of the experiment. The three experimental groups included in the study were a control group that was not administered Pinealon or methionine, a group administered methionine only so the infants would develop under constant oxidative stress, and a group administered both Pinealon and methionine. Pinealon was administered by dissolving the compound in 0.9% NaCl and injected intraperitoneally for 5 days prior to the administration of 10 ug/kg of methionine [2].

When the infants were 10 days old they were registered and weighed in order for cerebellum granule cells to be isolated. The cerebellar slices were treated with 2 mg/ml of collagenase and washed with standard Tyrode’s solution composed of NaCl 148 mM, KCl 5 mM, MgCl2 mM, glucose 10 mM, and HEPES 10 mM at a pH of 7.4, followed by a final filtration. Oxidative stress was induced by incubating the cells in a suspension of 5 mM H2O2 and the resulting intracellular levels of reactive oxygen species was measured using a BD FACSCalibur flow cytometer [2].

At 45 days post-birth the amount of homocysteine in the blood was determined through the immunofluorescent method. At this point the rat pups underwent Morris water maze testing to determine the effects of administration of Pinealon and methionine on overall cognitive functioning. The water maze test included a water pool apparatus measuring 1.7 meters in diameter filled with both water and an extra 2 liters of milk to make the water cloudy. From there a 15 cm platform was placed under the water level and away from the pool edge while the apparatus was monitored via video recording to track the rats’ activity levels [2].

Discussion

1) The results of the review developed by the research team of Khavinson et al found the administration of the EDR peptide was about to reduce the level of synthesis of reactive oxygen species in cerebellar granular cells caused by oxidative stress. This is important to mention considering that reactive oxygen species trigger the MAPK/ERK1/2 pathway. That being said, Pinealon has an inhibitory effect on the ERK1/2 pathway activation indicating that its antiapoptotic and neuroprotective effects are related to its ability to delay pathway activation and change the onset of the cellular phase changes. Additionally, it was determined that when Pinealon was administered at lower concentrations, accumulation of reactive oxygen species and total cell death was significantly reduced. These findings allowed the research team to conclude that the tripeptide elicits neuroprotective and antiapoptotic qualities by manipulating the expression of MAPK/ERK1/2 signaling pathways [1].

When assessing the effects of Pinealon on the expression of SOD, the tripeptide was shown to reduce levels of hydroperoxides resulting in the neutralization of the primary products related to lipid peroxidation. Observations of cerebellar granule cells that were isolated from hypoxia-induced rats treated with Pinealon were found to be resistant to oxidative stress. In addition to reduced oxidative stress the hypoxia-induced rats experienced a decrease in the accumulation of free radicals. That being said, the research team was able to conclude that Pinealon is able to regulate the activity of neuronal antioxidant enzymes, such as SOD. This was indicated by the finding that SOD activity in the brain tissue of hypoxia-resistance rats was significantly higher than animals not treated with Pinealon, indicating that the increased enzymatic activity induced by the tripeptide was neuroprotective against the effects of hypoxia [1].

As it was previously mentioned, the transcription factor p53 travels to the mitochondrial matrix in response to DNA damage, resulting in caspase activation that contributes to neuron death in cases of Alzheimer’s disease. In an animal model of acute hypoxia, administration of Pinealon was found to reduce the expression of caspase-3 in the brain resulting in improved scores in the Morris water maze test in both young and aged animals. Additionally, the tripeptide was shown to activate caspase-3 proteases in the cerebrum and brain stem, as well as prevent an increase in activation of caspase-3. These findings allowed the research team of Khavinson et al to conclude that administration of Pinealon has the potential to elicit neuroprotective effects in cases of Alzheimer’s disease by mediating various apoptotic factors such as p53 and caspase-3 [1].

2) The research team of Arutjunyan et al examine the effects of Pinealon on prenatal hyperhomocysteinemia in rat pups, induced by loading adult rats with methionine while pregnant. Three groups were utilized for the purpose of this study including: a control group, a group administered methionine only, and a group administered both methionine and Pinealon. In order to assess the neuroprotective effects of the peptide, cerebellar granular samples were collected from 10 day old rat pups and subjected to oxidative stress. Additionally, 45 day old rat pups were subjected to a Morris water maze test to observe changes in both learning ability and spatial orientation [2].

Initial results of the study found that the weight of the methionine loaded animals averaged 17.86 +/- 3.05 grams and was shown to be significantly lower than the control group whose weight measured as 23.05 +/- 1.45 grams. There was also a more profound standard deviation of weight changes between the control group and both the methionine loaded animals and the animals treated with both methionine and Pinealon. The researchers noted that this large difference in standard deviation was potentially due to variation in the sensitivity to methionine, however, it was mentioned that the weight of the animals treated with Pinealon did not undergo any significant changes [2].

As it was previously mentioned, cognitive functioning of the animals was assessed through the results of the Morris water maze test. When under the conditions of hyperhomocysteinemia induced by methionine, the rate of swimming was found to be significantly lower than both the control group and the experimental group treated with both methionine and Pinealon. It is important to mention that while the group treated with methionine and Pinealon measured at a higher swimming rate than the control group, the increase was not deemed as significant by the research team [2].

Figure 1: Average swimming rate of the rats measured in meters/second as they learned to find the hidden platform included in the Morris water maze apparatus. The graph displays the changes in rate between the three experimental groups including a control group, a methionine loaded group, and a group treated with both methionine and Pinealon.

In terms of finding the hidden platform in the Morris water maze apparatus, animals from the three different experimental groups scored vastly different from each other. For animals included in the control group the average time to find the platform was 112 seconds; for animals included in the methionine loaded group the average time to find the platform was 170 seconds; and for the animals included in the methionine and Pinealon loaded group the average time to find the platform was 137 seconds. The research team determined that these changes in scores were considered statistically significant. It is important to note that by the 5th experimental trail, any differences in scoring had decreased and all of the animals from each experimental group were able to find the hidden platform in approximately the same amount of time [2].

Figure 2: Time spent searching for the platform by the animals of the three different experimental groups during the Morris water maze test. The graph displays the changes in latency between the three experimental groups including a control group, a methionine loaded group, and a group treated with both methionine and Pinealon.

In addition to the Morris water maze, the researchers also utilized flow cytometry to compare properties of the cerebellar neurons isolated from animals belonging to the methionine loading experimental group and the experimental group treated with both methionine and Pinealon. In the cells obtained from the methionine loaded animals, there was a significant amount of necrotic cells present, as well as increased accumulation of reactive oxygen species. This was compared to the cells obtained from the group treated with methionine and Pinealon which had almost no necrotic cells present and remarkably lower accumulation of reactive oxygen species [2].

Furthermore, it should be mentioned that the research team monitored the levels of homocysteine in the blood of animals included in the methionine loaded experimental group as well as the experimental group treated with methionine and Pinealon. When actively consuming methionine, the pregnant rats experienced a clear increase in homocysteine in the blood; these findings were consistent in the offspring of these rats as well. The research team also reported that the high levels of homocysteine in the blood of the pregnant rat was not attenuated by the administration of Pinealon in addition to methionine loading. The high levels of homocysteine in the blood were also observed in the offspring of the mature rats, however, they were not as dramatically elevated. Based on this data, the researchers were able to conclude that in terms of levels of homocysteine in the blood, Pinealon may not actively metabolize the compound and abolish all negative effects of hyperhomocysteinemia, but rather it may play a neuroprotective role [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Khavinson V, Linkova N, Kozhevnikova E, Trofimova S. EDR Peptide: Possible Mechanism of Gene Expression and Protein Synthesis Regulation Involved in the Pathogenesis of Alzheimer’s Disease. Molecules. 2020 Dec 31;26(1):159. doi: 10.3390/molecules26010159. PMID: 33396470; PMCID: PMC7795577.

[2] Arutjunyan A, Kozina L, Stvolinskiy S, Bulygina Y, Mashkina A, Khavinson V. Pinealon protects the rat offspring from prenatal hyperhomocysteinemia. Int J Clin Exp Med. 2012;5(2):179-85. Epub 2012 Apr 6. PMID: 22567179; PMCID: PMC3342713.

PEPTIDES PREFER THE COLD
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