The Effect of of Mangifera indica on N-methyl-D-aspartate (NMDA) Receptor Activity

Neurological disorders stemming from oxidative stress and excitotoxicity are significant health concerns, with implications ranging from neurodegenerative diseases to acute injuries like stroke. This project investigates the potential neuroprotective role of mangiferin, an antioxidant compound, by observing its effect on NMDA receptor currents. Using Xenopus laevis oocytes expressing NMDA receptors, a two-electrode voltage clamp will record current responses at -60 mV while exposing the receptors to varying concentrations of mangiferin under conditions. The hypothesis proposes that mangiferin will weaken current activity by negatively modulating NMDA receptor activity, indicating a potential reduction in neuronal damage. This study bridges gaps in understanding mangiferin’s direct effects on neurotransmitter receptors, particularly NMDA receptors, offering insights into its therapeutic potential for neurological disorders. In order to measure currents from NMDA receptors, we developed or improved the following techniques. The oocyte digestion technique was improved to efficiently separate oocytes, involving the development of collagenase solution, precise oocyte cutting methods, determination of optimal digestion time, and the formulation of an incubation solution to maintain oocyte viability. Injection techniques were also improved, focusing on setting up the microinjection rig, preparing micropipettes, and identifying healthy oocytes suitable for injection based on specific criteria. Additionally, recording skills were enhanced through rigorous preparation and troubleshooting of the rig, electrodes, and perfusion system, along with improved data analysis capabilities to differentiate between signals and noise accurately.

Introduction:
Hello everyone, I am Diborah Gutema and this my project: The effect of The Effect of of Mangifera indica on N-methyl-D-aspartate (NMDA) Receptor Activity
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The Brain’s Challenges:
Oxidative stress is the imbalance between the production of reactive oxygen species (ROS) and the body’s ability to counteract their harmful effects. In the central nervous system, buildup of oxidative damage can compromise the integrity of proteins, lipids, and DNA. This can lead to various cellular dysregulations, including neuroinflammation and neurotransmitter imbalance. Oxidative stress is also closely connected with excitotoxicity, a process where excessive activation of glutamate receptors, such as NMDA receptors, leads to neuronal damage. A positive feedback look is formed between excitotoxicity and oxidative stress as excitotoxicity contributes to increased ROS production which then leads to further oxidative damage. The resulting damage from this loop is implicated in the pathogenesis of neurodegenerative disorders like Alzheimer‘s and Parkinson‘s and acute disorders like stroke and traumatic brain injury
The Role of NMDA Receptors:
NMDA (N-methyl-D-aspartate) receptors are glutamate receptors that are found throughout the central nervous system. When glutamate, the primary excitatory neurotransmitter in the brain, is released from the presynaptic neuron, they can bind onto NMDA receptors on the postsynaptic neuron. This binding opens ion channels within the receptor, allowing an influx of calcium and sodium ions. This leads to depolarization of the membrane and, if the threshold potential is met, action potentials are generated and propagated. The excitatory neurotransmission from NMDA receptors plays vital roles in facilitating learning and memory, motor functions, sensory processing, and neurological health. However, excessive activation of NMDA receptors can lead to excitotoxicity (Purves).

Mangiferin’s Potential:
Mangiferin is a natural xanthone glycoside compound found in mangos and is a novel research interest due to its antioxidant and anti-inflammatory properties. In the context of neurological health, Mangiferin has shown potential neuroprotective effects. One study by Zhou et al. showed mangiferin to be a potential therapy for Parkinson‘s through its ability to influence cellular pathways involved in protecting against oxidative stress (Zhou). Additionally, other research indicates that mangiferin has been shown to reduce damage caused by strokes by modulating inflammatory responses, enhancing antioxidant enzyme activity, and regulating signaling pathways related to cell survival and protection against ischemic brain injury. This versatile compound may offer a promising avenue for neuroprotection in various neurological conditions.

Purpose
The primary objective of this experiment is to investigate a potential neuroprotective property of mangiferin, an antioxidant compound. Overactivation of NMDA receptors is known to be a key factor in excitotoxicity, a process linked to oxidative stress and implicated in various neurological disorders. Dysregulation by excitotoxicity has been shown to lead to neuronal damage in neurodegenerative diseases, stroke, and traumatic brain injury. Mangiferin has been shown in a study to decrease oxidative stress by interacting with signaling pathways, but it is unknown if mangiferin works directly on receptors themselves. This experiment will work to bridge this knowledge gap by unveiling the potentially neuroprotective mechanism that targets excitotoxic processes directly. This research holds significant promise for novel clinical approaches to be used for treatment of neuropathology and for broader understanding of mangiferin‘s therapeutic applications
Hypothesis:
The hypothesis for this project is that mangiferin, a negative modulator of NMDA receptors, will reduce the current response elicited by NMDA receptor activation in a dose-dependent manner.
Experimental Approach and Methodology:
We start with the Xenopus laevis oocyte expression system, a widely used model organism in neuroscience research. This model organism is advantageous due to its large size, robust membrane durability, and efficient expression of exogenous proteins.
In this system, we utilize the technique of injecting cRNA (complementary RNA) encoding NMDA receptor subunits. Injecting cRNA allows us to control the composition of NMDA receptors expressed on the oocyte membranes, enabling us to study specific subunit combinations and their functional implications.
After incubation, we will set up the two-electrode voltage clamp rig for recording. technique enables us to measure and manipulate ion currents across cell membranes, providing invaluable insights into the function of ion channels such as NMDA receptors. One electrode, known as the voltage electrode, maintains a fixed membrane potential in the oocyte. The other electrode, called the current electrode, measures the resulting current flow across the membrane when NMDA receptors are activated.
The voltage clamp technique allows us to study the properties of NMDA receptors, such as their ion conductance and gating kinetics, under controlled experimental conditions. By applying our solutions and recording resulting currents, we can analyze NMDA receptor activation, desensitization, and modulation by ligands like glutamate.
Mangiferin Introduction and Concentration Gradient:
We’ll start with baseline recordings in ND96 solution for both control and NMDA-expressing oocytes. Then, we’ll perfuse the oocytes with glutamate to induce excitotoxic conditions and perfuse serial dilutions of mangiferin to analyze its effect on NMDA receptor activity.

Expected Outcomes and Future Implications:
If my hypothesis is supported, I expect to see mangiferin act as a negative modulator of NMDA receptors. Here’s how the negative modulation of NMDA receptors by mangiferin might be reflected in current readings:
1. Reduction in Current Amplitude: Negative modulation of NMDA receptors by mangiferin could lead to a decrease in the peak current amplitude measured during receptor activation. This reduction in current flow indicates that fewer ions are passing through the NMDA receptor channels upon stimulation.
2. Slower Rise and Decay of Currents: Mangiferin’s negative modulation may also result in slower kinetics of current rise and decay during NMDA receptor activation and deactivation, respectively. This change in kinetics can be observed as a prolonged duration of the current response compared to control conditions.
3. Shift in Dose-Response Curve: When testing different concentrations of glutamate or NMDA to activate NMDA receptors, the presence of mangiferin may shift the dose-response curve to the right. This shift indicates that higher concentrations of agonists are required to elicit the same level of current response in the presence of mangiferin compared to control conditions.
4. Attenuation of Potentiation: If NMDA receptors normally undergo potentiation (increased response) under certain conditions, mangiferin’s negative modulation may attenuate or reduce this potentiation. This attenuation can be observed as a blunted increase in current response compared to what would be expected in the absence of mangiferin.
Awknowledgements
Thank you to Dr. Greta Ann Herin for her continuous support and enlightening learning experiences
Thank you to fellow research assistants and technicians of the lab for helping move the progress forward
Thank you to OSCAR for funding this project by of URSP Grant