A team of SMU biological scientists has confirmed that P-glycoprotein (P-gp) has the ability to remove from the brain a toxin that is associated with Alzheimer’s disease.
The finding could lead to new treatments for the disease that affects nearly 6 million Americans. It was that hope that motivated lead researchers James W. McCormick and Lauren Ammerman to pursue the research as SMU graduate students after they both lost a grandmother to the disease while at SMU.
In the Alzheimer’s brain, abnormal levels of amyloid-β proteins clump together to form plaques that collect between neurons and can disrupt cell function.
This is believed to be one of the key factors that triggers memory loss, confusion and other common symptoms from Alzheimer’s disease.
“We were able to demonstrate both computationally and experimentally that P-gp, a critical toxin pump in the body, is able to transport this amyloid-β protein,” said John Wise, associate professor in the SMU Department of Biological Sciences and co-author of the study published in PLOS ONE.
“If you could find a way to induce more P-glycoprotein in the protective blood-brain barrier for people who are susceptible to Alzheimer’s disease, perhaps they could take such a treatment and it would help postpone or prevent the onset of the disease,” he said. Wise stressed that the theory needs more research.
The SMU (Southern Methodist University) study also provides strong evidence for the first time that P-gp may be able to pump out much larger toxins than previously believed.
P-gp is nature’s way of removing toxins from cells. Similar to how a sump pump in your house removes water from the basement, P-gp swallows harmful drugs or toxins within the cell and then spits them back outside the cell.
“You find P-gp wherever the body is looking to protect an organ from toxins, and the brain is no exception,” explained co-author Pia Vogel, SMU professor and director of SMU’s Center for Drug Discovery, Design and Delivery.
Amyloid-β’s large size created questions about whether P-glycoprotein could actually inhale it and pump it back out.
“Amyloid-β is maybe five times bigger than the small, drug-like molecules that P-glycoproteins are well-known to move. It would be like taking New York pizza and trying to stuff that whole slice in your mouth and swallow it,” Wise said.
The fact that P-gp appears to be able to do just that “greatly expands the possible range of things that P-gp can transport, which opens the possibility that it may interact with other factors that were previously thought impossible,” said McCormick, a former SMU graduate student in biological sciences.
The research was personal
SMU researchers might never have investigated the link between P-gp and amyloid-β proteins if not for McCormick’s dogged pursuit of the connection. The Ph.D. student, who graduated in 2017, had seen preliminary work suggesting that P-gp might play a role in pulling amyloid protein out of the brain and asked his faculty advisors, Vogel and Wise, if he could take some time to check it out.
The professors concede they first tried to discourage him because they were more focused on P-gp’s role in creating resistance to chemotherapy in cancer patients. However, McCormick was “passionate,” about figuring out if P-gp might be able to shield someone from getting Alzheimer’s, Vogel said.
He devoted hours of his own time to use a computer-generated model of P-glycoprotein that he and Wise created. The model allows researchers to dock nearly any drug to the P-gp protein and observe how it would behave in P-gp’s “pump.” Vogel, Wise and other SMU scientists have been studying the protein for years to identify compounds that might reverse chemotherapy failure in aggressive cancers.
McCormick completed the computational work with the help of his fiancée, Ammerman, who got her Ph.D in biology from SMU in May.
Together, they ran multiple simulations of the P-gp protein using SMU’s high performance computer, ManeFrame II, and found that each time, P-gp was able to “swallow” amyloid-β proteins and push them out of cells.
“For the scientist in me, it was just absolutely amazing that this pump could consume something that big,” Vogel said. “John [Wise] and I did not predict that would be possible.”
Two in vitro experiments confirmed the computational work
The researchers conducted two experiments in the lab to confirm the computational results.
In one experiment, Ammerman used lab-purchased amyloid-β proteins that had been dyed fluorescent green, allowing them to be easily spotted easily in a microscope. In multiple trials, Ammerman exposed human cells to these amyloid-β proteins.
She used two types of human cells – one where P-gp was strongly expressed and one where P-gp was not. This allowed her to test the difference between the two and see if P-gp was pumping amyloid-β out.
The amyloid proteins were clearly shown to be pushed out of the human cells that had overexpressed P-gp in them, supporting the theory that P-gp removes amyloid proteins on contact.
Another in vitro experiment reached the same conclusion from a different direction. Former graduate student Gang (Mike) Chen worked in SMU’s Center for Drug Discovery, Design and Delivery to show that an Alzheimer’s-linked amyloid-β caused changes in the P-gp’s usage of adenosine triphosphate (ATP), indicating that there was a physical interaction between the two.
ATP hydrolysis produces the energy that P-gp uses to transport toxins or drugs out of the cell. When no toxins are present, P-gp’s rate of ATP stays rather low. When challenged with transporting cargo, P-gp’s ATP hydrolysis activity usually increases quite dramatically.
“Even though our work can’t help our grandparents, I hope that it might help others in the future,” Ammerman said. “The more we know, the more power we have—and researchers after us—to address and target these devastating diseases.”
ATP binding cassette (ABC) transport proteins located at cerebral endothelial cells in the blood-brain barrier (BBB) pump out a wide variety of compounds from the brain to the blood [1, 2], contributing to the maintenance of cerebral homeostasis and the protection of the central nervous system (CNS) [3, 4].
These transporters use ATP hydrolysis to transport substrates from the intracellular to the extracellular compartment [5, 6]. P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 1 (MRP-1) are the most studied ABC transporters, because of their clinical relevance [7, 8].
The P-gp transporter can recognize a large number of structurally diverse exogenous compounds such as anti-cancer, anti-epileptic, and antidepressant drugs and also endogenous compounds. Inflammatory responses, stress, therapeutic drugs, and diet can modify the expression and/or function of the P-gp transporter [9, 10]. An increase in the P-gp function has been related to decreases in drug efficiency (drug resistance) [1, 11].
This phenomenon is especially important in the treatment of brain tumors [12], intractable epilepsy [13], psychiatric diseases [1], and infectious diseases [14]. On the other hand, a decline in the P-gp activity is associated with an increased concentration of neurotoxic compounds inside the CNS, which may be related to the onset of several neurodegenerative diseases [15, 16] or may cause neurological problems [17, 18].
Vogelgesang et al. demonstrated that the β-amyloid deposition inside the brain, which is the pathological hallmark of Alzheimer’s disease [19, 20], is inversely correlated with the P-gp expression in endothelial cells of cerebral blood vessels [21].
Assessment of the P-gp function in vivo may help to diagnose several neurodegenerative diseases and may predict the efficacy of CNS treatments. PET imaging has already been used to study the P-gp function at the BBB in humans [22,23,24,25,26]. Nowadays, (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide are considered “gold standard” tracers for imaging the P-gp function, being the most extensively used in preclinical and clinical research [24, 25, 27].
However, these tracers have been identified as strong P-gp substrates [22, 28, 29]; i.e., the tracers are quickly transported from the brain to the blood. This results in a low tracer concentration inside the brain [25], precluding their use in the assessment of P-gp upregulation, which might occur in treatment-resistant depression [30] and patients with intractable epilepsy [13].
For these reasons, many efforts have been made to develop new P-gp tracers with improved pharmacokinetic properties and lower affinity to the P-gp transporter [25]. [11C]metoclopramide [31], [11C]emopamil [32], [11C]phenytoin [26], and [18F]MC225 [33] were identified as weak substrates of the P-gp transporter, showing higher tracer uptake in the brain than (R)-[11C]verapamil at baseline conditions when P-gp is functioning adequately. [18F]MC225 was selected as the most promising fluorine-18 labeled tracer for in vivo measurement of P-gp function [34]. Recently, the kinetic properties of [18F]MC225 were evaluated, and the results confirmed the ability of this tracer to measure changes in the P-gp function of rats [33] and non-human primates [35].
The present study is the first direct comparison of the characteristics of the weak P-gp substrate [18F]MC225 and the strong P-gp substrate (R)-[11C]verapamil in non-human primates. To this aim, the function of the P-gp transporter was explored in three rhesus monkeys (Macaca mulatta) under normal conditions as well as after the administration of the P-gp inhibitor, tariquidar.
Based on previous publications that analyzed the pharmacokinetics of [18F]MC225 and (R)-[11C]verapamil in non-human primates [35, 36], the 1-Tissue Compartment Model (1-TCM) was fitted to the data of both tracers. Kinetic parameters such as the influx constant K1, the volume of distribution (VT), and the efflux constant k2 were compared between the tracers at baseline and after inhibition. Regional differences between tracers were also analyzed, and simplified methods to quantify the P-gp function were assessed.
Conclusion
This head-to-head comparison between [18F]MC225 and (R)-[11C]verapamil demonstrates that VT calculated using 30-min scan duration may be a more adequate parameter than K1 to measure decreases in the P-gp function with both tracers. Although K1 was selected as the best parameter to measure the P-gp function in the previous publications [35, 36], the present study found that K1 was not different between tracers at baseline conditions, and therefore K1 could not be used to detect differences in affinity of strong and weak substrates towards the P-gp transporter.
Moreover, K1 values of (R)-[11C]verapamil were higher than those from [18F]MC225 in after-inhibition scans, suggesting that (R)-[11C]verapamil K1 values may be affected by other non-specific unknown factors in the brain. The results from both tracers indicate that subcortical regions may present a higher P-gp function than frontal cortex and cerebellum.
The higher baseline VT of [18F]MC225 may allow the quantification of increases in the P-gp function and may facilitate the baseline image registration and fusion to an anatomical image (MRI or CT) (Supplemental Fig. 4). Thereby, [18F]MC225 has the potential to become the first radiofluorinated tracer able to measure both decreases and increases in P-gp function at the BBB. Nevertheless, a first-in-man study is required to verify the properties of [18F]MC225 before the tracer can be clinically applied.
reference link : https://link.springer.com/article/10.1007/s00259-021-05411-2
More information: James W. McCormick et al, Transport of Alzheimer’s associated amyloid-β catalyzed by P-glycoprotein, PLOS ONE (2021). DOI: 10.1371/journal.pone.0250371
