3. Introduction
● L-glutamate is the major excitatory
neurotransmitter in the mammalian
CNS
● It’s implicated in fundamental brain
functions, such as neuronal
development, learning and memory
3
5. Topology of AMPA Receptors
• AMPA Receptors are responsible for the majority of
fast excitatory synaptic transmission
• Recent findings have implicated AMPA Receptors in
synapse formation and stabilization; (Huganir and
Nicoll 2013)
• AMPA Receptor overactivation is potently
excitotoxic and changes in AMPA Receptor activity
have been described in the pathology of numerous
diseases
5
(Schiffer and Heinemann 1999)
Single Receptor
9. Objectives
• Assaying and identifying potent curcumin derivatives that are selective towards
AMPA Receptors only
• Characterizing the AMPA Receptor through the inhibition effect of curcumin
derivatives
• Promoting the creation of small interfering or mimicking peptides for up or down
regulation of AMPA Receptor activity at the synapse
• Identifying the functional groups accountable for the inhibition effect on AMPA
Receptor
9
11. Significance
One of the most apparent significances for characterization of the action
of Curcumin derivatives on the AMPA receptors is to find the most
potent and selective natural source of AMPA receptors inhibitors and to
use these findings for the purpose of synthesizing newer natural
effective drugs without the chemical side effects
11
12. Specific Aims
• Transfecting GluA2 receptor into Human Embryonic Kidney 293 (HEK293) cells
successfully.
• Characterizing the curcumin derivative A effect on AMPA receptor whole cell
current
• Characterizing the curcumin derivative A effect on AMPA receptor
desensitization
• Characterizing the curcumin derivative A effect on AMPA receptor deactivation
12
20. What can we conclude from this session?
● Curcumin derivative A decreased the peak amplitude of whole cell current, increased
desensitisation rate and extent and increased deactivation rate
● Curcumin derivative A is an inhibitor of AMPA receptor
● Curcumin derivative A might have affected the density of AMPA Receptors in the post
synaptic cleft by altering the trafficking mechanisms of AMPA Receptors
● Curcumin derivative A might have modified the chemical properties of the AMPA
Receptors channel pore, making it less permeable to certain ions
● Curcumin derivative A might have an effect on the conformation of the AMPA
Receptors, destabilizing the desensitization state and reducing the period of time
AMPA Receptors spends in that state
20
21. What can we conclude from this session?
● The modified curcumin derivative A contained an electron donating group which we
suggest plays a key a role in the inhibition effect of the curcumin,it might enhance
the ability of the compound to bind tightly to the AMPA Receptor
● The natural product; Curcumin and its derivatives are a target worth pursuing for
further research
21
22. Acknowledgment
Advisor
Dr. Mohammed Qneibi
Lab Technician:
Mrs. Shuruq Subuh
Chemistry Assistant Professor:
Dr. Othman Hamed
Examination Committee Members
22
This work was supported by MoHE grant.
Good morning everyone. It’s my pleasure to present to you our research today, my name is Hasan Arafat, my colleagues Yasmeen Abu Naba and Remah Yousef, we will be presenting The Effect of Curcumin on AMPA Receptor Kinetics.
So before I start, let me outline what we’re going to talk about in this session. We’ll start off by giving a brief background, the objective and significance of our study, followed by the specific aims and how we conducted this study.our results and Finally a summary and what our research will provide for future studies.
So let me start with the introduction. L-glutamate is the major excitatory neurotransmitter in the mammalian CNS. It’s the most abundant neurotransmitter in the vertebrate CNS. L-Glu is implicated in fundamental brain functions, such as neuronal development, learning and memory. So now, let’s talk briefly about the mechanism by which glutamate conveys its actions.
As action potential reaches the axon terminus, the pre-synaptic membrane depolarizes, opening voltage gated Ca2+, prompting an influx of Ca2+.
Glutamate vesicles fuse with the membrane, releasing glutamate into the cleft.
Glutamate diffuses from the presynaptic membrane through the synaptic cleft in a fraction of millisecond, and binds glutamate receptors at the post synaptic cleft.
This binding changes the permeability of the receptor, ultimately generating a membrane potential at the post-synaptic membrane.
And now let’s talk about glutamate receptors. Glutamatergic receptors can be divided into metabotropic glutamate receptors, a.k.a G-protein coupled receptors, and ionotropic glutamate receptors. For the sack of our discussion, we will focus on ionotropic receptors. Ionotropic receptors are further classified into Kainate, AMPA, and NMDA receptors.
1- Kainate receptors control synaptic integration and spike transmission
2- AMPA receptors mediate fast excitation
3- NMDA receptor activity correlates to a much slower and long-lasting current profile
Each type has its own set of subunits, we will focus on AMPA receptors.
AMPA receptors are composed of four receptor subunits: GluA1 to 4. AMPA receptors can form
homomeric functional channels and heteromeric channel complexes.
GluA1-GluA2 (most abundant in hippocampus) and GluA2-GluA3 combinations make the majority of AMPA Receptors there. On the other hand, GluA4-containing AMPA Receptors are expressed mainly in early postnatal development.
GluA1 forms calcium-permeable (without GluA2), and is associated with LTP, and LTD, which affect the synaptic plasticity.
GluA1 functions in the trafficking and insertion of AMPA Receptors in the synapses, hence its unique role in various nervous disorders
GluA2 has the most impact on the biophysical properties of all heteromeric complexes. GluA2-lacking AMPA Receptors are Ca2+ permeable. In contrast, GluA2-containing AMPA Receptors are impermeable to divalent cations, particularly Ca2+. GluA2 receptors also have a charechterisitc form of kinetics
They are also implicated in receptor plasticity, which is defined as the ability of the brain to change and adapt to new information; Synaptic plasticity is a biological process in which synaptic strength changes in retaliation to a specific pattern of changes in neuronal activity.
Point Two: each subunit is highlighted in different color in this scheme, (point using the flashlight), this is the amintotropic domain, ligand binding domain and the transmembrane domain
Homomeric: all subunits are identical (all GluA1, for example), hetermeric: different subunits.The Synaptic Strength is measured by the degree of changing occurring in the postsynaptic neuron potential in respect to changes in presynaptic stimulation.An essential contributor to the synaptic strength is the fact the AMPA receptors are not a fixed element in the synaptic composure, instead are characteristically dynamic, being moved in and out of a synapse in response to neuronal activity.LTP: Long Term Potentiation:LTD: Long Term Depression:
Trafficking their over-activation is potently excitotoxic triggering either rapid or delayed neurotoxicity. Changes in AMPA Receptor activity and consequently a debilitated regulation of Glutamate have been described in the pathology of several diseases, such as ALS, stroke, epilepsy and Alzheimer's disease.o
So as you can see in this figure, the ligand binding site is represented by the clamshell structure, with D1 representing the upper valve of the shell and D2 representing the lower valve. The gray bars represent the ion channel. In part A here, the receptor is in the resting state, there is no ligand at the LBD, and the ion channel is closed. The current passing through the channel is represented here as a flat trace. In part B, the ligand binds to the LBD, opening the ion channel and allowing ions to pass through. This is represented as an abrupt change in the current trace as you can see. The following step is known as desensitization, in which the ligand remains bound to the LBD, while the ion channel becomes loosely closed, decreasing the passage of ions but allowing a small amount to leak, this is represented as a gradual change in the current trace as you can see. The steady state here is considered a part of the desensitization phase, in this phase, the current reaches a plateau in which the electrical charge leaving the cell is equal to that entering the cell. Then, the receptor returns to the resting state, in which there is no ligand at the LBD, the ion channel is closed.
With this I have explained the 1 important phenomenon that our research has focused on, now since we understand that importance of AMPA Receptor and understand how their overactivation is linked to excitotoxicity and to CNS diseases , to stop this from happening we should explore compounds that stop this over excitation and neuronal death from happening ,so now we'll explain what we used for AMPA Receptor inhibition.
The compounds shown here are AMPA Receptor antagonist or inhibitors and are drugs used for many purposes. As I said, AMPA Receptors are excitotoxic and their overactivation is implicated in many diseases. All of these are chemical compounds that come with many systemic side effects, this matter aspired us to look for natural compounds that work as efficiently but don’t come with the annoying side effects.
This has lead us to curcumin >>> next silde
This lead us to Curcumin, is a well known spice with a scientific history going back two centuries ago when it was first isolated from Turmeric (Curcuma longa) roots.
It’s known for its anti-oxidant , anti-inflammatory, chemotherapeutic and chemopreventive effect.
It’s also known for its neuroprotective effect, esp. Alzheimer disease, ALS and epilepsy.
It’s a potent Reactive Oxygen Species (ROS) scavenger, at least ten times more potent than Vit. C.
Now this compound shown above is native curcumin, the compound shown below is the derivative we chose for our study. Due to time limitation, we chose to study this one compound although we had a variety of other compound, which we will study is the future.
And here we finish with the introduction, we talked about glutamate, glutamate receptors and their types, AMPA receptor, AMPA subunits and kinetics and curcumin. Now I leave you with my colleague Yasmeen Abu Nabaa to continue with the objectives, Yasmeen.
The reason why we chose this particular derivative is because of the amine group it contains, which is an electron donating group as you know. According to a previous study of Dr. Qneibi, BZD, the well-known CNS inhibitors, inhibit AMPA Receptors peak current, this was found to be due to the interaction of the electron-donating group of BDZ with AMPA Receptors. We suggested a similar role for this derivative based on the amine group it contains, and this is why we chose to test it first.
Limitation: We did not have enough time to study all components ..
** to understand the molecular and cellular mechanisms underlying synapse
development and function.
** but due to the limitations ( opening of lab only recently and deadline) we did not have enough time to do this so we started with 1 but will continue on this objective for our future study.
Now this table shows the different derivatives we obtained from curcumin, all of these compounds will be used in our future studies.
As we previously mentioned, AMPA Receptor has been implicated in many CNS diseases , this makes it a target worth pursuing.
Extraction was done by Dr Othman in faculty of chemistry. Curcuma longa powder was placed in a Soxhlet extractor and extracted with methanol for about 4 hr. The methanol solvent was filtered then concentrated under vacuum. The yellow gummy residue containing curcuminoids was subjected to purification by flash chromatography on silica (100-200 mesh).first fraction was eluted with hexane-ethyl acetate and then methanol-ethyl acetate was used to elute the second fraction. It was found that the Second fraction contains the desired curcumin.
DNA preparation:
Cell culture (Human Embryonic Kidney 293) cells were grown in supplemented DMEM (Dulbecco Modified Eagle Medium) supplemented with 10% FBS (fetal bovine serum), 0.1 mg/ml streptomycin, and 1 mM sodium pyruvate.
the picture here shows healthy grown HEK293 under microscope.
Transfection the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. (plasmid containing AMPA Receptor gene into HEK293 cells in our case) .Before the electrophysiology recordings, cells were replated 24 hours after transfection on coverslips coated with Laminin.
The right image here shows transfected HEK293 under fluorescent microscope, the green light represents the fluorescent dye eGFP (enhanced Green Fluorescent Protein) found on the plasmid carrying the gene for the AMPA Receptor. These shiny large spots represent free dye, these irregular shapes are deformed cells that are unsuitable for patch-clamp, circular, well defined cells represent healthy cells that took the plasmid and has mostly expressed the receptor of our interest. All of these cells (point using the laser) represent potential candidates for patch-clamp.
Wash System: made of 5 syringes, the first one contains the wash solution, the second contains glutamate, and the other three contains distilled water.
Glutamate, l
Wash, a buffer solution
As you can see, we attach the pipette to the healthiest cell in the field. We used the whole cell patch clamp, in which only a small part of the membrane is ruptured using the pipette, this allows direct recording of the cell current. The solution flowing through the pipette is known as the internal solution, and it’s carefully prepared to simulate the cytoplasm. As the pipette attaches to the membrane, a GigaOhm seal is applied at the membrane. The whole cell is carried using the pipette outside the field and exposed to the external solution barrel of the theta, this keeps the cell at base current and viable. At command, the theta sweeps to the glutamate barrel in 20 μs second, exposing the cell to glutamate and activating AMPA Receptors, generating an action potential.
At this step, the wash, a.k.a the external solution, a solution prepared to simulate that of the external environment surrounding the cell, is applied to the cell using one of the theta two barrels. When the user is ready for the experiment, glutamate is applied using the other barrel, eliciting a change in the membrane action potential due to the binding of the glutamate to the LBD at AMPA Receptors, the current elicited is measured using the pipette
simultaneously, the wash solution starts flowing through the theta, the wash system contains certain chemicals that keep the cell at base current and keep it healthy and alive while a part
We first studied the effect of curcumin derivative A on glutamate-evoked AMPA receptor-mediated whole cell currents in HEK293 Cells.
The amplitude generated by GluA2Q receptors was measured using Integrated Patch Amplifiers (IPA),
Agonist was applied on GluR2Q by using Piezo Fast Exchange solution with 10 mM of glutamate.
Data were analyzed using Igor 7 software.
As shown in figure A, application of Curcumin derivative A in agonist barrels showed significant decrease in the peak glutamate-activated current of GluA2Q.
The amount of peak current has decreased from 4820 pA to 3012 pA (i.e. ~1.6 folds).
The y-axis of figure A was plotted as A/A1, which is a function of inhibitor concentration vs. the concentration of the curcumin derivatives in the x-axis.
From the observed shortening of the activation limb of the AMPA receptor current trace resonating with the decrease in the peak amplitude. This suggest that the curcumin derivative may affect the activation phase time.????
We evaluated the effect of the derivative on desensitization. Following the activation of GluA2, superimposed current traces evoked by 500 ms pulses of 10 mm glutamate in AMPA Receptor alone and AMPA Receptor plus the curcumin derivative were obtained to study its effect on desensitization.
To recall , Desensitization is defined as the characteristic decay in current after a long simulation time following activation. Thus evoking for the period of 500ms allow us to observe this phenomenon and the slow recovery of the receptor.
curcumin derivative affected the desensitization which was markedly reduced following the administration of the curcumin as in figure B.
Comparison of desensitization time constants between AMPA Receptor alone (9.84 ± 1.64 ms, n = 4) and with the addition of curcumin (4.6 ± 1.1 ms, n = 4) shows a significant difference in the desensitization time constant.
The amount of desensitization can be measured by : peak/steady state
the rate of desensitization equals the inverse of time constant obtained by exponential fit to the desensitization (tau) using equation : 1/τ(s-1)
The amount of desensitization increased from an average of 19.08 to that of 30.78 ~1.6 folds, while the average rate of desensitization increased from 0.10 ms-1 to 0.22 ms-1 ~2 folds.
We focused our attention on deactivation.
Following the activation of GluA2, superimposed current traces evoked by 1 ms pulses of 10 mm glutamate in AMPA Receptor alone and AMPA Receptor with curcumin derivative were obtained to study the curcumin derivatives effect on deactivation.
To recall , Deactivation is the characteristic decay in current after a short simulation time following activation. Applying agonist for 1 ms allow us to catch the receptor rapid recovery upon the immediate removal of glutamate.
As shown in figure B the presence of curcumin derivative also affected deactivation, by a distinct decrease of the current peak, which increased the rate of receptor deactivation.
Comparison of deactivation time constants between AMPA Receptor alone (5.88 ± 0.40 ms, n = 5) and with curcumin (4.8 ± 0.6 ms, n = 5) variants (figure A) shows a notable difference in the deactivation time constant.
The decrease in time constant suggest that the curcumin derivative accelerated the rate of deactivation of GluA2.
The amount of deactivation, calculated using :peak/steady state,increased from an average of 895.37 to that of 977.25 ~1.1folds
calculated using equation 1/τ(s-1) : the average rate of deactivation increased from 0.17 ms-1 to 0.2 ms-1 ~1.18 folds
In the presence of curcumin, the time the receptor spent in the desensitized and deactivated phases was reduced, demonstrated by a reduction in the value of tau. This suggests that curcumin might have an effect on the AMPA receptor conformation, destabilizing the desensitized/deactivated conformation and favoring the resting conformation.
Point 2 ; Denoting that curcumin derivative renders AMPA Receptor less sensitive for its agonist glutamate.
Before point 3: We suggest these mechanism for the curcumin effect on AMPA Receptors.
وفي النهاية ،، نتقدم بجزيل الشكر وعظيم الامتنان الأهل الاعزاء الذين كانو مصدر القوة والالهام لنا ،، (وبنتمنى انه نكون رفعنا راسكم وفرحناكم بهاليوم ) وشكرا لجميع الأصدقاء والزملاء الذين كانو بجانبنا منذ بداية الطريق وشكرا لجميع الحضور والمستمعين ،،