Researchers from the Institute of Neurosciences (IN), a joint center of the Miguel Hernández University of Elche (UMH) and the Spanish National Research Council (CSIC), who are also part of the Network Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED) and the Alicante Institute of Health and Biomedical Research (ISABIAL) have developed a cellular fractionation protocol. This method allows precise analysis of the proteins located in synaptic membranes and in membranes outside the synapses, known as extrasynaptic membranes, in human postmortem brains.
In this study, recently published in Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association, the authors examined NMDA receptors for their importance in synaptic transmission and their special relevance in Alzheimer’s disease. Compared to healthy individuals, the results show that people with Alzheimer’s disease exhibit reduced NMDA receptors in synapses and increased extrasynaptic membranes.
Alzheimer’s disease is characterized by progressive memory loss and affects communication between neurons. This process is largely dependent on synapses, where NMDA receptors play a crucial role in learning and memory. “Most NMDA receptors are found in synapses, where they strengthen neuronal connections. However, the receptors located outside the synapse are more associated with processes of toxicity and cell death, which can contribute to the progression of the disease,” explains Inmaculada Cuchillo Ibáñez, the researcher at the Altered Molecular Mechanism Laboratory for Alzheimer’s Disease and Dementia, who led the study.
The team analyzed samples of human post-mortem brains, including healthy individuals and patients at various stages of neurodegeneration. The results show a clear distribution of NMDA receptors in the cortex of patients with Alzheimer’s disease, where the number of synaptic NMDA receptors is significantly reduced, while extrasynaptic receptors increase compared to healthy individuals. This imbalance suggests that neuronal toxicity-related activity is favored in Alzheimer’s disease, as opposed to the primary function of synaptic transmission, which likely contributes to disease progression.
A groundbreaking protocol for human postmortem brains
The most significant advance made by the researchers is the optimization of a cellular fractionation protocol that allows the separation of synaptic membranes from extrasynaptic membranes, a feat not previously achieved in frozen human postmortem brains.
Other studies have measured total levels of NMDA receptors in the human brain, but did not distinguish between those in synapses and extrasynapses. We have adapted a protocol designed for fresh mouse brains to human samples, achieving this crucial separation.”
Sergio Escamilla, first author of the article
The method is based on the use of detergents that dissolve the lipids in non-synaptic membranes, while leaving synaptic membranes largely intact due to their high protein content. Centrifugation is then used to separate the two membrane types for analysis.
Towards new therapeutic approaches
The findings of this study could open new avenues for the treatment of Alzheimer’s disease. “This protocol allows us to accurately determine whether specific agents, such as modulators or blockers, have a greater affinity for synaptic or extrasynaptic receptors – and not just NMDA receptors – which has important therapeutic implications,” notes Cuchillo.
The study, which collaborated with the laboratories of José Vicente Sánchez Mut and Isabel Pérez Otaño at IN UMH-CSIC, also used transgenic mice to validate the results obtained in humans. Although similar changes in NMDA receptors were detected, the differences between species underline the need for human tissue research to better understand the disease.
With this groundbreaking protocol, the researchers pave the way to investigate the molecular basis of Alzheimer’s disease and the search for more effective treatments. In this spirit, researcher Javier Sáez Valero, head of the Altered Molecular Mechanism in Alzheimer’s Disease and Dementia laboratory at IN UMH-CSIC, emphasizes the importance of this type of research due to the role of NMDA receptors in current Alzheimer’s treatments, such as memantine. one of the most commonly used medications for the disease is an NMDA receptor blocker.
This work was made possible thanks to the financial support of the Health Research Fund, co-financed by the European Regional Development Fund (ERDF “Investing in your future”); the Network Center for Biomedical Research on Neurodegenerative Diseases (CIBERNED); the Carlos III Health Institute; and the Directorate General for Science and Research of the Generalitat Valenciana.
Researchers from the Institute of Neurosciences (IN), a joint center of the Miguel Hernández University of Elche (UMH) and the Spanish National Research Council (CSIC), who are also part of the Network Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED) and the Alicante Institute of Health and Biomedical Research (ISABIAL) have developed a cellular fractionation protocol. This method allows precise analysis of the proteins located in synaptic membranes and in membranes outside the synapses, known as extrasynaptic membranes, in human postmortem brains.
In this study, recently published in Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association, the authors examined NMDA receptors for their importance in synaptic transmission and their special relevance in Alzheimer’s disease. Compared to healthy individuals, the results show that people with Alzheimer’s disease exhibit reduced NMDA receptors in synapses and increased extrasynaptic membranes.
Alzheimer’s disease is characterized by progressive memory loss and affects communication between neurons. This process is largely dependent on synapses, where NMDA receptors play a crucial role in learning and memory. “Most NMDA receptors are found in synapses, where they strengthen neuronal connections. However, the receptors located outside the synapse are more associated with processes of toxicity and cell death, which can contribute to the progression of the disease,” explains Inmaculada Cuchillo Ibáñez, the researcher at the Altered Molecular Mechanism Laboratory for Alzheimer’s Disease and Dementia, who led the study.
The team analyzed samples of human post-mortem brains, including healthy individuals and patients at various stages of neurodegeneration. The results show a clear distribution of NMDA receptors in the cortex of patients with Alzheimer’s disease, where the number of synaptic NMDA receptors is significantly reduced, while extrasynaptic receptors increase compared to healthy individuals. This imbalance suggests that neuronal toxicity-related activity is favored in Alzheimer’s disease, as opposed to the primary function of synaptic transmission, which likely contributes to disease progression.
A groundbreaking protocol for human postmortem brains
The most significant advance made by the researchers is the optimization of a cellular fractionation protocol that allows the separation of synaptic membranes from extrasynaptic membranes, a feat not previously achieved in frozen human postmortem brains. “Other studies have measured total levels of NMDA receptors in the human brain, but did not distinguish between the levels in synapses and extrasynapses. We adapted a protocol designed for fresh mouse brains to human samples, achieving this crucial separation “, emphasizes Sergio Escamilla. the first author of the article.
The method is based on the use of detergents that dissolve the lipids in non-synaptic membranes, while leaving synaptic membranes largely intact due to their high protein content. Centrifugation is then used to separate the two membrane types for analysis.
Towards new therapeutic approaches
The findings of this study could open new avenues for the treatment of Alzheimer’s disease. “This protocol allows us to accurately determine whether specific agents, such as modulators or blockers, have a greater affinity for synaptic or extrasynaptic receptors – and not just NMDA receptors – which has important therapeutic implications,” notes Cuchillo.
The study, which collaborated with the laboratories of José Vicente Sánchez Mut and Isabel Pérez Otaño at IN UMH-CSIC, also used transgenic mice to validate the results obtained in humans. Although similar changes in NMDA receptors were detected, the differences between species underline the need for human tissue research to better understand the disease.
With this groundbreaking protocol, the researchers pave the way to investigate the molecular basis of Alzheimer’s disease and the search for more effective treatments. In this spirit, researcher Javier Sáez Valero, head of the Altered Molecular Mechanism in Alzheimer’s Disease and Dementia laboratory at IN UMH-CSIC, emphasizes the importance of this type of research due to the role of NMDA receptors in current Alzheimer’s treatments, such as memantine. one of the most commonly used medications for the disease is an NMDA receptor blocker.
This work was made possible thanks to the financial support of the Health Research Fund, co-financed by the European Regional Development Fund (ERDF “Investing in your future”); the Network Center for Biomedical Research on Neurodegenerative Diseases (CIBERNED); the Carlos III Health Institute; and the Directorate General for Science and Research of the Generalitat Valenciana.
Source:
Magazine reference:
Escamilla, S., et al. (2024). Synaptic and extrasynaptic distribution of NMDA receptors in the cortex of patients with Alzheimer’s disease. Alzheimer’s and dementia. doi.org/10.1002/alz.14125.