by Lara López de Jesús

Neuroprotective activity of P2Y₂

WHEN A PERSON GOES INTO cardiac arrest, very often the body is partially paralyzed because blood flow decreases and brain tissue suffers due to lack of oxygen and glucose. This phenomenon is known as ischemia, and the brain responds in various ways. One of these responses is known as reactive astrogliosis.

“In reactive astrogliosis, astrocytes—the star-shaped cells that fill the spaces between the neurons in the brain—proliferate and cause lesions, called glial scars, to form on the brain. Some suggest that these scars are a defensive response, a neuroprotective response; however, they also interrupt nerve connections that previously existed, causing paralysis,” says Fernando González, Ph.D., professor of chemistry at the UPR, RP.

Scientists around the world have studied reactive astrogliosis. It is a complex process involving a wide range of molecules, including glutamate and its receptors. In 2004, González and his team of researchers—including postdoctoral associates Cynthia Santos Berríos, and Nataliya Chorna—proposed a controversial hypothesis: the P2Y₂ neuroreceptor is one of the primary molecules that participate in reactive astrogliosis.

“Until that time, nobody had proposed the important role that the P2Y₂ receptor plays in survival situations when neuroprotective mechanisms cause pathological responses. Our most recent research proposes that the P2Y₂ receptor can be considered a new control point for reactive astrogliosis in neurodegenerative diseases. We are now trying to identify the cascade of signals that become active in the nervous system after the receptor is activated with its agonists, molecules that induce the receptor’s response,” says Santos Berríos.

The nervous system: a living tapestry Think of the nervous system as a living tapestry in which the threads form communication centers that control movement, behavior, perception, and memory. The executive center of this network is the brain, which processes, analyzes, and interprets the information it receives, but the execution of orders depends on the ability of its network to intertwine, weave the information together, and communicate it. Almost all the communication centers of the body are stimulated by nerve fibers, which are formed by cells that have the ability to understand thousands of signals, translate millions of messages, and communicate. One hundred million neurons and almost one trillion glial cells make up the nervous system. The neurons are arranged in local circuits, which in turn are arranged in regions that connect to each other by forming netwoks capable of communicating among themselves and with the effector cells, in charge of acting upon nervous stimulus. Photo: Cornell Nanofabrication Facility Neurons receive nervous impulses via dendrites, and generally send information along the prolongation of the axon, which travels to the presynaptic zone, in which the synaptic vesicles are found. The synaptic vesicles are small structures stored by neurotransmitters. The center of the action is the synapses, the small crevice between neurons in which neurotransmitters can act and help in the transmission of messages. The neurotransmitter completes its task when it binds to a specific receptor, activating it to interpret the different biochemical and bioelectric signals and inducing a physiological response. Neurotransmitters and receptors carry out different functions. All cells have different types of neurotransmitters that work with different receptors. In neurons, adenosine triphosphate (ATP) may work as a neurotransmitter and act with two main types of receptors: ionotrophic, which generally mediate rapid, short-term responses, and the metabotrophic, which generally mediate long-term actions. Metabotrophic receptors are quite abundant in both sides of the synaptic zone, and they originate signals coupled with intracellular cascades. These cascades are part of the transformation of a signal and imply a set of processes and biochemical reactions that are interconnected; this allows the cell to respond to external stimuli.

P2Y₂ receptors are proteins that appear on the surface of cells, cross the membrane seven times, and adhere to a protein as part of the machinery of the transduction of signals. The “P” means that these receptors are purinergic, activated by purine and pyramidine nucleotides. Nucleotides—structural units that make up nucleic acids such as RNA and DNA—play important roles in the transportation and transformation of cellular energy and in the regulation of enzymes.

Purinergic receptors have been classified as P1 or P2, depending on their nucleotide preferences. P1 receptors prefer adenosine, whereas P2 receptors respond better to ATP (adenosine triphosphate), UTP (uridine triphosphate), and ADP (adenosine diphosphate). P2Y₂ receptors are found in a variety of cellular tissues, including neurons, where they are activated by their natural agonists—ATP and UTP molecules.

In 1989, González helped discover a ligand, a molecule capable of taking the place of the neurotransmitter by interacting with the P2Y₂ and P2X7 receptors. The molecule BzATP (benzoyl adenosine triphosphate) is the best ligand that currently exists for P2X7 receptors. A synthetic modification introduced the ability to be photoreactive, a very useful characteristic when detecting proteins that join with ATP and when characterizing the places where they join.

P2Y₂ receptors have been involved in various problems related to the secretion of bodily fluids, as in intraocular pressure, dry eyes, and vaginal secretions. Experiments performed by González’ team suggest that P2Y₂ receptors may have important functions in immunological responses to the processes of inflammation in the human body.

The P2Y₂ receptor is also used in the treatment of cystic fibrosis. This disease causes mucus and bacteria to accumulate in the lungs, and those suffering from it can die from chronic complications such as pneumonia. As a form of treatment, patients inhale solutions with P2Y₂ receptor ligands, which stimulate their bodies to secrete and dilute mucus. The problem is that when the P2Y₂ receptor becomes desensitized, the drug stops working. González hopes to identify and manage the events that cause desensitization, thereby allowing the drug to continue working. Melvin G. Hernández, a graduate student in chemistry at UPR, RP, currently works with González and is trying to determine which kinase proteins participate in the desensitization of P2Y₂.

In their research on reactive astrogliosis, González and his team used the results of the 1989 experiments of Joseph T. Neary, from the School of Medicine at the University of Miami and current collaborator of González. In his lab, González adapted the in vitro mechanical model Neary used in his experiments. “The mechanical model is a convenient system because it eliminates the need to affect a live animal every time an experiment is performed. It also allows many elements to be controlled. However, the response shown by the experiment does not imitate the physiological system completely. It simplifies the organism by representing it with only one type of cell, whereas in reality, tissue has many different types of cells that interact,” says González.

In the mechanical model, cells are cultivated on a stretched membrane, which is subjected to a deformation process to simulate impact or injury to cells from lesions. This in turn initiates neurodegeneration. In his research, Neary discovered that some extracellular nucleotides and their receptors participate in the rapid responses that follow a deformation event. González and his team have focused on the role of P2Y₂ receptors in these events.

They performed a pharmacological analysis using antagonists, substances which stick to cellular receptors and inhibit their biological responses, and attempted to identify which receptors were involved in the cascade of responses related to reactive astrogliosis. The inhibition pattern of the antagonists suggests that P2Y₂ receptors participate in this process.

In other experiments that demonstrate the role of P2Y₂, González has used astrocytomas, a type of tumor that originates in the nervous system and maintains the properties of astrocytes. These cells can easily maintain themselves in a culture and do not respond to nucleotides ATP, UTP, and ADP. González introduced the P2Y₂ gene to create a type of astrocyte that expresses only the P2Y₂ receptor when exposed to ATP, UTP, and ADP nucleotides. He and his team have performed a series of experiments with these cells, revealing important images and data about these receptors, including the intracellular movement of the receptor, which is made possible by the fusion of the P2Y₂ gene to an autofluorescent protein.

Adenosine triphosphate (ATP) as a neurotransmitter ATP, the almost universal currency of cells for management of energy, was discovered in the 1920s. It exists in all living cells, including neurons. This molecule carries out a large number of functions related to metabolic processes. In addition, researchers have attributed the function of neurotransmission to it. Different groups of investigators have demonstrated that ATP—specifically neuronal ATP—is synthesized and stored in the pre-synaptic terminal and is liberated in response to a nervous stimulus, like any neurotransmitter. The first indirect evidence of ATP as a neurotransmitter came from the experiments by Pamela Holton in the 1950s. As early as 1941, Fritz Lipman and Herman Kalkar had introduced the concept of "high-energy phosphate link," which highlighted the importance of ATP in intracellular metabolism. In the1950s, researchers already knew that nerve cells liberated ATP, but it was twenty years later that the British physiologist, Geoffrey Burnstock, proposed the hypothesis that nucleotides (ATP included) had extracellular functions. The purinergic hypothesis, which proposes neurotransmission based on purines within the nervous system, caused a commotion in the scientific community. The Burnstock hypothesis, questioned for many years, is now widely accepted. At the time, it was thought highly unlikely that the molecule—originally implicated in intracellular processes—could be used as an extracellular messenger. At present, ATP's function is admittedly that of a neurotransmitter, both in the central and peripheral nervous systems. A series of possible ATP pathways have been explored. Research on ATP-based neurotransmission has intensified in the past few years, and important data has been collected, although many aspects of this neurotransmission are still unknown. During the 1990s researchers characterized many membrane receptor families for ATP.

The experiments show that P2Y₂ receptor activity induces neuroprotective responses in the astrocytes. These responses, including the activation of transcription factors, help produce proteins important to neuroprotective processes. For example, in his experiments, González has observed how UTP activates the P2Y₂ receptor, causing signals to multiply. In the nucleus, some genes codify factors that encourage the nerve terminals, structures known as neurites, to form and elongate. They have also found that in this cascade of events, proteins (such as brevican and the integrins), enzymes (such as kinase proteins), and transduction factors (such as CREB) cause genes in the nucleus to be transcribed. All of these elements are related to the proliferation of astrocytes that is observed when the cell experiences damage or neurodegeneration.

“Our experiments indicate that the cellular responses that follow the activation of the P2Y₂ receptor are conducive to cellular protection and the proliferation of astrocytes, which may be important in the regeneration of tissue. Some responses associated with the proliferation of astrocytes are responses that promote life.

“Present knowledge about the P2Y₂ receptor and the types of signals it gives off related to neuroprotective responses could have biomedical implications in the treatment of Alzheimer’s disease and cardiac arrest, as well as in treatments in which neurodegenerative events promote the formation of glial scars in the brain,” says González.

fgonzal@upracd.upr.clu.edu
http://web.uprr.pr/faculty/gonzalez