Periodic Reporting for period 1 - COST-ATP (The role of intravesicular ATP in excitatory and inhibitory synapses of the central nervous system)
Reporting period: 2021-09-01 to 2023-08-31
What is the problem/issue being addressed by COST-ATP?
With almost no exceptions, nucleotides and specifically ATP are present inside all vesicles from all animal species accompanying all kinds of neurotransmitters and hormones, and often they are present as the sole transmitter in some
neurons. Even more, ATP was the first neurotransmitter found in the most ancient forms of life, like Giardia lamblia, which possess ATP-containing vesicles even having neither mitochondria nor Golgi apparatus. ATP is driven inside the vesicles by a transporter –the vesicular nucleotide transporter (VNUT), which has been identified in the neuroendocrine system and the peripheral and central nervous system. Apart from the well-known function of the ATP as the source of energy for active transport, cell signaling or synthesis of DNA and RNA, a recent report by Estévez-Herrera et al. has demonstrated a role for the ATP within the secretory vesicles. This work suggests that intra-vesicular ATP can control the osmotic pressure to regulate the neurotransmitter content, allowing a high concentration of neurotransmitters within the vesicle. This hypothesis has been demonstrated in vitro and in neuroendocrine chromaffin cells. However, this phenomenon has not been explored in neurons of the Central Nervous System nor with other neurotransmitters, besides catecholamines in large dense core vesicles of chromaffin cells. This project tries to unravel the evidence of the co-release of ATP and NT from the same vesicle and its necessary role in allowing the high accumulation of neurotransmitters in SV of the CNS. The overarching goal of this project is to provide answers to several questions currently unsolved, such as: i) Are ATP-containing SV releasable in inhibitory and excitatory neurons of the CNS and where are their places of release? ii) Are ATP and NT transporters co-release from the same vesicle? and iii) Does ATP impact the storage of neurotransmitters in SV?
Why is it important for society?
ATP is one of the most primitive biological molecules that has been used since the earliest times of evolution as an intracellular energy source and as an extracellular signaling molecule in most cells, including neurons. ATP has been identified as a neurotransmitter in both the peripheral and central nervous systems. The involvement of ATP release in the nervous system has been referred to a variety of biological processes, including maintenance of neuron, astrocyte, and microglia function, development of the cerebellar cortex, neuronal differentiation and neuritogenesis, detection of physiological changes in brain pH, and oxygenation, protection against neurotoxicants, or modulation of cognitive processes among others. In addition, purinergic signaling has been related to pathological processes such as sleep disorders, some forms of epilepsy, depression, and worsening of some neurodegenerative diseases. Due to its exceptionally broad spectrum of signaling functions, there is great interest in purinergic signaling, both under physiological and pathophysiological conditions. A better understanding of the release of ATP will help in the field of biomedicine, to identify new targets with therapeutic potential and to develop new therapies for pathological processes in which purinergic signaling participates. Furthermore, these new and specific targets may generate new lines of research in areas such as the application of new diagnoses or treatments for these pathophysiological conditions.
What are the overall objectives?
This project tries to unravel the evidence of the co-release of ATP and NT from the same vesicle and its necessary role in allowing the high accumulation of neurotransmitters in SV of the CNS, divided into three steps.
I) To locate the ATP-containing vesicles and their release sites in excitatory and inhibitory synapses. To study whether there exists a difference in the neuronal distribution of the ATP-containing vesicles within these neurons and the release of the ATP-containing vesicles location of both cell types.
II) To image VNUT-synaptic vesicles to unveil its vesicular co-release. To visualize whether synaptic vesicles can contain both types of neurotransmitter transporter (VGlut/VGAT) and the ATP transporter (VNUT).
III) To establish a role of intravesicular ATP in neurotransmitter accumulation and release.
With almost no exceptions, nucleotides and specifically ATP are present inside all vesicles from all animal species accompanying all kinds of neurotransmitters and hormones, and often they are present as the sole transmitter in some
neurons. Even more, ATP was the first neurotransmitter found in the most ancient forms of life, like Giardia lamblia, which possess ATP-containing vesicles even having neither mitochondria nor Golgi apparatus. ATP is driven inside the vesicles by a transporter –the vesicular nucleotide transporter (VNUT), which has been identified in the neuroendocrine system and the peripheral and central nervous system. Apart from the well-known function of the ATP as the source of energy for active transport, cell signaling or synthesis of DNA and RNA, a recent report by Estévez-Herrera et al. has demonstrated a role for the ATP within the secretory vesicles. This work suggests that intra-vesicular ATP can control the osmotic pressure to regulate the neurotransmitter content, allowing a high concentration of neurotransmitters within the vesicle. This hypothesis has been demonstrated in vitro and in neuroendocrine chromaffin cells. However, this phenomenon has not been explored in neurons of the Central Nervous System nor with other neurotransmitters, besides catecholamines in large dense core vesicles of chromaffin cells. This project tries to unravel the evidence of the co-release of ATP and NT from the same vesicle and its necessary role in allowing the high accumulation of neurotransmitters in SV of the CNS. The overarching goal of this project is to provide answers to several questions currently unsolved, such as: i) Are ATP-containing SV releasable in inhibitory and excitatory neurons of the CNS and where are their places of release? ii) Are ATP and NT transporters co-release from the same vesicle? and iii) Does ATP impact the storage of neurotransmitters in SV?
Why is it important for society?
ATP is one of the most primitive biological molecules that has been used since the earliest times of evolution as an intracellular energy source and as an extracellular signaling molecule in most cells, including neurons. ATP has been identified as a neurotransmitter in both the peripheral and central nervous systems. The involvement of ATP release in the nervous system has been referred to a variety of biological processes, including maintenance of neuron, astrocyte, and microglia function, development of the cerebellar cortex, neuronal differentiation and neuritogenesis, detection of physiological changes in brain pH, and oxygenation, protection against neurotoxicants, or modulation of cognitive processes among others. In addition, purinergic signaling has been related to pathological processes such as sleep disorders, some forms of epilepsy, depression, and worsening of some neurodegenerative diseases. Due to its exceptionally broad spectrum of signaling functions, there is great interest in purinergic signaling, both under physiological and pathophysiological conditions. A better understanding of the release of ATP will help in the field of biomedicine, to identify new targets with therapeutic potential and to develop new therapies for pathological processes in which purinergic signaling participates. Furthermore, these new and specific targets may generate new lines of research in areas such as the application of new diagnoses or treatments for these pathophysiological conditions.
What are the overall objectives?
This project tries to unravel the evidence of the co-release of ATP and NT from the same vesicle and its necessary role in allowing the high accumulation of neurotransmitters in SV of the CNS, divided into three steps.
I) To locate the ATP-containing vesicles and their release sites in excitatory and inhibitory synapses. To study whether there exists a difference in the neuronal distribution of the ATP-containing vesicles within these neurons and the release of the ATP-containing vesicles location of both cell types.
II) To image VNUT-synaptic vesicles to unveil its vesicular co-release. To visualize whether synaptic vesicles can contain both types of neurotransmitter transporter (VGlut/VGAT) and the ATP transporter (VNUT).
III) To establish a role of intravesicular ATP in neurotransmitter accumulation and release.
This MSC Action has been diligently focused on achieving its objectives, aiming to shed light on the intriguing possibility of co-releasing ATP and neurotransmitters from synaptic vesicles, as well as exploring their localization and release dynamics within neurons. Here are the key findings:
I) Within the Banker cultures, we observed a significant concentration of VNUT-associated fluorescence in specific neuronal regions, namely dendrites and soma. Additionally, it's essential to note that the presence of VNUT in other neuronal regions, like axons and presynaptic terminals, while less pronounced, has not been definitively ruled out. These findings provide a clear demonstration of VNUT's distribution within neuronal cultures, with a particular focus on the "Sandwich culture" as our reference model.
II) Our investigations revealed positive immunostaining not only for VNUT but also for VGAT and VGlut in the SH-SY5Y neuroblastoma cell line. Through immunocytochemistry and western blot, we observed a notable increase in VNUT expression during the differentiation process, which will aid in the study of ATP-containing vesicle release using TIRF microscopy. Currently, VNUT has been observed in both the soma and neuritic compartment, and we are actively characterizing this localization fro the final transfection of tolls to accoplish the TIRFM experiments.
III) We have recently collected data related to mEPSC and explored its variations through silencing and overexpression, which did not reveal significant differences.
We are currently in the process of drafting a manuscript that will be submitted in 2024 to a high-impact, open-access journal. Once the paper is submitted, we will share the announcement on our Twitter account.
I) Within the Banker cultures, we observed a significant concentration of VNUT-associated fluorescence in specific neuronal regions, namely dendrites and soma. Additionally, it's essential to note that the presence of VNUT in other neuronal regions, like axons and presynaptic terminals, while less pronounced, has not been definitively ruled out. These findings provide a clear demonstration of VNUT's distribution within neuronal cultures, with a particular focus on the "Sandwich culture" as our reference model.
II) Our investigations revealed positive immunostaining not only for VNUT but also for VGAT and VGlut in the SH-SY5Y neuroblastoma cell line. Through immunocytochemistry and western blot, we observed a notable increase in VNUT expression during the differentiation process, which will aid in the study of ATP-containing vesicle release using TIRF microscopy. Currently, VNUT has been observed in both the soma and neuritic compartment, and we are actively characterizing this localization fro the final transfection of tolls to accoplish the TIRFM experiments.
III) We have recently collected data related to mEPSC and explored its variations through silencing and overexpression, which did not reveal significant differences.
We are currently in the process of drafting a manuscript that will be submitted in 2024 to a high-impact, open-access journal. Once the paper is submitted, we will share the announcement on our Twitter account.
The storage of ATP in vesicles and its release must play an essential role in purinergic chemical transmission. Although much progress has been made in recent years, multiple aspects of ATP release via exocytosis are still unknown. For example, it has not been described which neuronal types show vesicular ATP release, nor any of the molecular components for vesicular release mediated by this pathway, which is essential to advance our understanding of purinergic transmission in both its physiological and pathophysiological context. This project's objective is to delve deeper into and advance the understanding of purinergic transmission, a subject of significant relevance within the realm of neuroscience, yet one that continues to pose numerous unanswered questions. This pursuit is driven by the profound significance of ATP release in the nervous system, as it plays a pivotal role in a wide spectrum of biological processes. Therefore, the new information on purinergic signaling that this project will provide will be useful for both basic scientists and clinicians and may lead to the commercialization of new products and services and, ultimately, generate a social and economic impact that will have an impact on improving the health of the population.