Periodic Reporting for period 1 - GABARPET (ImmunoPET directed to specific subtypes of GABAA receptors as strategy for molecular mapping of behaviour and mental illness)
Periodo di rendicontazione: 2020-09-09 al 2023-09-08
What is the problem/issue being addressed?
The communication between neurons of the vertebrate’s brain is inhibited by an ion channel known as the GABA-A receptor. GABA-A receptors are formed by five proteins, or subunits, and their different classes are classified according to α subunit (α1–6). The differential expression of GABA-A receptors has been associated with schizophrenia or affective disorders between other pathologies or human conditions. In addition to the brain, GABA-A receptors have been detected in the peripheral nervous system of other organs such as the stomach, lungs, bladder, kidneys, liver and heart.
Schizophrenia is a severe psychiatric disorder that has a profound effect on both the individuals affected and society. Postmortem investigations of GABA-A receptor α subunit expression have found reductions in α1 and increases in α2 expression, but inconsistent results for the α5 subunit. However, the density of GABA-A receptors in schizophrenia does not appear abnormal when it is studied in living patients though positron emission tomography (PET) in contrast to what the postmortem studies have demonstrated. Some researchers and GABARPET researchers believe that these negative findings can be explained because of the lack of PET tracers with high affinity and specificity.
The immuno-positron emission tomography (immunoPET) is a non-invasive imaging technology based on tracking and quantification of radiolabeled monoclonal antibodies, antibody fragments, and peptides in vivo. Antibody imaging provides a specific and sensitive, noninvasive means for molecular detection of the cell surface proteins in vivo (e.g. ion channels), which aid diagnosis, prognosis, therapy selection, and monitoring of treatment for many diseases. Nevertheless, its success in neuroimaging is limited because intact antibodies cannot penetrate the Brain Blood Barrier (BBB) in healthy conditions. In 2019, a new type of bispecific antibody fragment, with one arm binding the neural target of interest and the other arm directed against the transferrin receptor expressed on the endothelial cells of BBB, was successfully created. This antibody fragment was able to cross the BBB, and it was used to visualize in vivo intrabrain amyloid-beta.
Why is it important for society?
Schizophrenia affects approximately 24 million people or 1 in 300 people (0.32%) worldwide. It is frequently associated with significant distress and impairment in personal, family, social, educational, occupational, and other important areas of life. The diagnosis of schizophrenia is clinical, made exclusively after obtaining a full psychiatric history and excluding other causes of psychosis. However, as other psychiatric disorders, schizophrenia no specific marker has been established thus far, and the development of antibody-based PET probes could help find them. Therefore, the high specificity of antibodies could be used to detect each of the 19 subunits of GABA-A receptors. In this way, new immunoPET tools could be developed to look for new biomarkers in neuropsychiatric disorders. In addition, this approach could be applied to other ion channels in the nervous system.
In this research project, GABARPET researchers will utilize a completely novel approach to study the GABA-A receptor in the nervous system, which is expected to yield unprecedented insights into various diseases (e.g. Alzheimer's disease, epilepsy, schizophrenia, anxiety, etc.) and human conditions (Down syndrome, autism, etc..) in the future.
What are the overall objectives?
The goal of GABARPET researchers is to develop novel immunoPET radioligands capable of non-invasively detecting changes in GABA-A receptor subunits linked to neuropsychiatric disorders.
The communication between neurons of the vertebrate’s brain is inhibited by an ion channel known as the GABA-A receptor. GABA-A receptors are formed by five proteins, or subunits, and their different classes are classified according to α subunit (α1–6). The differential expression of GABA-A receptors has been associated with schizophrenia or affective disorders between other pathologies or human conditions. In addition to the brain, GABA-A receptors have been detected in the peripheral nervous system of other organs such as the stomach, lungs, bladder, kidneys, liver and heart.
Schizophrenia is a severe psychiatric disorder that has a profound effect on both the individuals affected and society. Postmortem investigations of GABA-A receptor α subunit expression have found reductions in α1 and increases in α2 expression, but inconsistent results for the α5 subunit. However, the density of GABA-A receptors in schizophrenia does not appear abnormal when it is studied in living patients though positron emission tomography (PET) in contrast to what the postmortem studies have demonstrated. Some researchers and GABARPET researchers believe that these negative findings can be explained because of the lack of PET tracers with high affinity and specificity.
The immuno-positron emission tomography (immunoPET) is a non-invasive imaging technology based on tracking and quantification of radiolabeled monoclonal antibodies, antibody fragments, and peptides in vivo. Antibody imaging provides a specific and sensitive, noninvasive means for molecular detection of the cell surface proteins in vivo (e.g. ion channels), which aid diagnosis, prognosis, therapy selection, and monitoring of treatment for many diseases. Nevertheless, its success in neuroimaging is limited because intact antibodies cannot penetrate the Brain Blood Barrier (BBB) in healthy conditions. In 2019, a new type of bispecific antibody fragment, with one arm binding the neural target of interest and the other arm directed against the transferrin receptor expressed on the endothelial cells of BBB, was successfully created. This antibody fragment was able to cross the BBB, and it was used to visualize in vivo intrabrain amyloid-beta.
Why is it important for society?
Schizophrenia affects approximately 24 million people or 1 in 300 people (0.32%) worldwide. It is frequently associated with significant distress and impairment in personal, family, social, educational, occupational, and other important areas of life. The diagnosis of schizophrenia is clinical, made exclusively after obtaining a full psychiatric history and excluding other causes of psychosis. However, as other psychiatric disorders, schizophrenia no specific marker has been established thus far, and the development of antibody-based PET probes could help find them. Therefore, the high specificity of antibodies could be used to detect each of the 19 subunits of GABA-A receptors. In this way, new immunoPET tools could be developed to look for new biomarkers in neuropsychiatric disorders. In addition, this approach could be applied to other ion channels in the nervous system.
In this research project, GABARPET researchers will utilize a completely novel approach to study the GABA-A receptor in the nervous system, which is expected to yield unprecedented insights into various diseases (e.g. Alzheimer's disease, epilepsy, schizophrenia, anxiety, etc.) and human conditions (Down syndrome, autism, etc..) in the future.
What are the overall objectives?
The goal of GABARPET researchers is to develop novel immunoPET radioligands capable of non-invasively detecting changes in GABA-A receptor subunits linked to neuropsychiatric disorders.
GABARPET researchers developed two types of immunoPET probes targeting the α1 subunit of the GABA-A receptor, based on intact antibodies sourced from a database. These immunoPET probes are designed as antibody fragments: single-chain variable fragments (scFv) and di-scFv. First, an scFv targeting the α1 subunit was radiolabeled with fluorine-18 (18F) using a click chemistry approach. Two distinct approaches were employed:
1) Pretargeting Approach: In this method, the scFv was initially intravenously injected into mice. After one or two hours, 18F-tetrazine was injected, followed by PET scans.
2) Direct Targeting: Here, the TCO-scFv + 18F-tetrazine reaction was performed in vitro prior to injections, and PET scans were conducted at various intervals after injection.
Following PET scans, the uptake in selected tissues was quantified using a gamma counter, and autoradiography of the brain, heart, and pancreas was conducted. Secondly, a di-scFv was developed by combining the previously mentioned scFv targeting GABA-A receptors with an scFv against the transferrin receptor in the laboratory. The di-scFv was radiolabeled using zirconium-89 (89Zr) and tetrazine-DFO as a chelator. It was then injected into wild-type mice, and PET scans were conducted at different time points after injection. Subsequently, important tissues were quantified using a gamma counter. As anticipated, the scFv did not penetrate the brain, whereas the di-scFv did. Interestingly, GABA-A receptor PET measurements were taken with the scFv in the peripheral nervous system.
In the process of designing their own antibody fragments, GABARPET researchers utilized phage display technology to generate scFv targeting α1 and α2 subunits. Phage display libraries were enriched against various α1 and α2 subunit peptides. Additionally, the enriched libraries were transformed from scFv to Fab format. While potential candidates were identified, further characterization work is required to fully assess their suitability.
Regarding the exploitation and dissemination of GABARPET project results, several accomplishments have been achieved. The project's findings were presented at both national (Finland) and international (Spain and Czech Republic) scientific congresses. Furthermore, GABARPET researchers authored a review article published in an international journal. They also have plans to publish a minimum of two additional research papers in international journals. In terms of outreach and scientific dissemination, when the project commenced, the University of Turku issued a press release. Additionally, two web pages were created: one for the Preclinical Turku PET Center unit and another for the Spanish Neuroscience Blog.
1) Pretargeting Approach: In this method, the scFv was initially intravenously injected into mice. After one or two hours, 18F-tetrazine was injected, followed by PET scans.
2) Direct Targeting: Here, the TCO-scFv + 18F-tetrazine reaction was performed in vitro prior to injections, and PET scans were conducted at various intervals after injection.
Following PET scans, the uptake in selected tissues was quantified using a gamma counter, and autoradiography of the brain, heart, and pancreas was conducted. Secondly, a di-scFv was developed by combining the previously mentioned scFv targeting GABA-A receptors with an scFv against the transferrin receptor in the laboratory. The di-scFv was radiolabeled using zirconium-89 (89Zr) and tetrazine-DFO as a chelator. It was then injected into wild-type mice, and PET scans were conducted at different time points after injection. Subsequently, important tissues were quantified using a gamma counter. As anticipated, the scFv did not penetrate the brain, whereas the di-scFv did. Interestingly, GABA-A receptor PET measurements were taken with the scFv in the peripheral nervous system.
In the process of designing their own antibody fragments, GABARPET researchers utilized phage display technology to generate scFv targeting α1 and α2 subunits. Phage display libraries were enriched against various α1 and α2 subunit peptides. Additionally, the enriched libraries were transformed from scFv to Fab format. While potential candidates were identified, further characterization work is required to fully assess their suitability.
Regarding the exploitation and dissemination of GABARPET project results, several accomplishments have been achieved. The project's findings were presented at both national (Finland) and international (Spain and Czech Republic) scientific congresses. Furthermore, GABARPET researchers authored a review article published in an international journal. They also have plans to publish a minimum of two additional research papers in international journals. In terms of outreach and scientific dissemination, when the project commenced, the University of Turku issued a press release. Additionally, two web pages were created: one for the Preclinical Turku PET Center unit and another for the Spanish Neuroscience Blog.
This project marked the first instance in which scientists utilized immunoPET to detect GABA-A receptors in the nervous system. It is likely also the first time that any ion channel inside the brain has been detected using immunoPET. This innovative approach paves the way for the design of more specific PET probes based on antibodies, which can be employed to investigate various neurological and neuropsychiatric disorders.
Another noteworthy aspect is that this marks the inaugural detection of GABA-A receptor PET probes targeting these ion channels in the peripheral nervous system. Consequently, this project has initiated the exploration of GABA-A receptors in vivo in other organs, such as the heart, in addition to the brain. This development is particularly intriguing in light of the advent of whole-body PET scans, as it allows for the examination of GABA-A receptors throughout the body in the context of different illnesses.
Another noteworthy aspect is that this marks the inaugural detection of GABA-A receptor PET probes targeting these ion channels in the peripheral nervous system. Consequently, this project has initiated the exploration of GABA-A receptors in vivo in other organs, such as the heart, in addition to the brain. This development is particularly intriguing in light of the advent of whole-body PET scans, as it allows for the examination of GABA-A receptors throughout the body in the context of different illnesses.