Electrophysiology of Human Native Receptors in Neurological and Mental Disorders
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Dr. Agenor Limon presents research integrating functional metrics with large anatomical, transcriptomic, and proteomic datasets to evaluate the relationship between synaptic E/I ratio and behavioral abnormalities across postmortem intervals and brain banks. Alterations in synaptic function have been found in transcriptomic, genetic, and proteomic studies of neurological and mental disorders. Clinical and preclinical studies suggest that synaptic dysfunction and behavioral abnormalities in disorders like Alzheimer’s disease and schizophrenia may be mechanistically linked to the emergence of imbalances between excitatory (E) and inhibitory (I) receptors. However, until recently, the electrophysiological E/I synaptic ratio had only been measured in animal models. Using pioneering methods developed in the lab including reactivation and microtransplantation of synaptic receptors from frozen human brains, Dr. Agenor Limon’s research team has obtained electrophysiological metrics of global synaptic E/I ratios in cortical brain regions of subjects that were affected by Alzheimer’s Disease and synaptic measurements in schizophrenia. In this webinar, Dr. Agenor Limon will present recent research integrating functional metrics with large anatomical, transcriptomic, and proteomic datasets to evaluate the relationship between synaptic E/I ratio and behavioral abnormalities across postmortem intervals and brain banks. Key Topics Include: - Understand the global synaptic excitation to inhibition ratio between excitatory and inhibitory synaptic receptors determined from reactivated frozen human brain tissue - Understand the relationship between electrophysiological metrics of receptor function and multi-omic data in neurodegenerative disorders - Understand the role of deviations of the excitation to inhibition ratio with clinical presentation in Alzheimer’s disease
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Electrophysiology of Human Native Receptors in Neurological and Mental Disorders
- 1. Electrophysiology of Human Native Receptors in Neurological and Mental Disorders Agenor Limon, PhD Assistant Professor Department of Neurology Mitchell Center for Neurodegenerative Diseases aglimonr@utmb.edu
- 2. Electrophysiology of Human Native Receptors in Neurological and Mental Disorders Dr. Agenor Limon presents research integrating functional metrics with large anatomical, transcriptomic, and proteomic datasets to evaluate the relationship between synaptic E/I ratio and behavioral abnormalities across postmortem intervals and brain banks.
- 3. Copyright 2021 A. Limon and InsideScientific. All rights reserved. Electrophysiology of Human Native Receptors in Neurological and Mental Disorders Agenor Limon, PhD Assistant Professor Department of Neurology Mitchell Center for Neurodegenerative Diseases aglimonr@utmb.edu
- 4. Overview • The problem of understanding synaptic dysfunction in human disorders. • Microtransplantation of Synaptic Receptors. • Integration of excitatory AMPA receptors with multiomics data in schizophrenia. • Excitation to Inhibition balance in across brain regions in Alzheimer’s disease. 4
- 5. Overview • The problem of understanding synaptic dysfunction in human disorders. • Microtransplantation of Synaptic Receptors. • Integration of excitatory AMPA receptors with multiomics data in schizophrenia. • Excitation to Inhibition balance in across brain regions in Alzheimer’s disease. 5
- 7. Penzes et al., 2008 Synaptic communication across disorders 7
- 8. AD Schizophrenia AD Autism Schizophrenia MDD AD Multifactorial etiology converging into synaptic dysfunction alzforum.org 8
- 9. Healthy twin Twin with schizophrenia Wooley and McGuire, 2005 Healthy brain Advanced AD 9
- 10. * Cognition is preserved despite substantial cortical thinning along life span. * Brain reorganization and plasticity overcomes cognitive damage from acute and extensive brain destruction. From Fjell et al., 2015 From Kliemann et al., 2019 10
- 11. • How to study functional alterations in the neurotransmitter receptors that are fundamental for neuronal activity and the target of toxic oligomers? 11
- 12. 12 Zhao Y. et al. – Science - 2019 Differences: • Arrangement of the subunits to form the receptor • Auxiliary subunits • Subunits ratio abundance (across brain region and ontogeny) • transmembrane AMPA receptor regulatory proteins (TARPs) • cornichon homologues (CNIHs) • cysteine-knot AMPAR modulating proteins 44 (CKAMP44) • germ cell-specific gene 1-like (GSG1L) protein. Shen K. And Zeppillo T. et al –Scientific Report journal Native AMPAr vs Recombinant rGluA2-GluA3 Native Recombinant
- 13. Overview • The problem of understanding synaptic dysfunction in human disorders. • Microtransplantation of Synaptic Receptors. • Integration of excitatory AMPA receptors with multiomics data in schizophrenia. • Excitation to Inhibition balance in across brain regions in Alzheimer’s disease. 13
- 15. FM4-64-labeled rat brain membranes a-Btx on oocyte injected with torpedo membranes 100 µm Visualization of microtransplanted receptors 15
- 16. Microtransplantation of synaptic membranes 16
- 17. Overview • The problem of understanding synaptic dysfunction in human disorders. • Microtransplantation of Synaptic Receptors. • Integration of excitatory AMPA receptors with multiomics data in schizophrenia. • Excitation to Inhibition balance in across brain regions in Alzheimer’s disease. 17
- 18. From Glantz and Lewis (2000) Loss of spine density in schizophrenia 18
- 19. Zeppillo et al., Schiz Res, 2020 Diagnosis Gender Age (years) PMI (hr) pH RIN CTRL 3/7 44 ± 10 22.8 ± 10 6.6 ± 0.4 5.4 ± 1.2 SZ 3/7 48 ± 13 20.5 ± 8 6.2 ± 0.2* 6.6 ± 1.3* *P<0.05 (Student t test),N =10 each group. PMI, postmortem interval AMPA receptors in Schizophrenia Frozen DLPFC Enriched Synaptosome prep. MSM Label free LC- MS/MS RNAseq 19
- 20. Expression profile in DLPFC Schizophrenia 20
- 21. AMPA receptors in Schizophrenia Zeppillo et al., Schiz Res, 2020 * 21
- 22. Proteins positively correlated with AMPA receptor current in human synaptosome (P2) preparations Zeppillo et al., Schiz Res, 2020 Ribosomal proteins External membrane Mitochondrial proteins Inner membrane Mitochondrial proteins AMPA receptor subunits 22
- 23. Proteins negatively correlated with AMPA receptor current in human synaptosome (P2) preparations Traffic, folding and signaling proteins 23
- 24. Zeppillo et al., Schiz Res, 2020 Genes downregulated in SZ. Proteins downregulated in SZ. Proteins (-) r with AMPA responses in SZ. Multifactorial convergence on synaptic dysfunction 24
- 25. Directionality of signaling pathways on synaptic function Zeppillo et al., Schiz Res, 2020 From Glantz and Lewis (2000) 25
- 26. • AMPA receptors from frozen brains can be reactivated and recorded by TEVC. • Amplitude of synaptic receptors’ currents correlate with synaptic elements at the proteomic level. • Function of receptors provide global directionality to signaling pathways that are commonly found altered in brain disorders. 26
- 27. Overview • The problem of understanding synaptic dysfunction in human disorders. • Microtransplantation of Synaptic Receptors. • Integration of excitatory AMPA receptors with multiomics data in schizophrenia. • Excitation to Inhibition balance in across brain regions in Alzheimer’s disease. 27
- 28. From Boros et al., 2017, Barral and Reyes 2016 Glutamatergic and GABAergic synapses Principal postsynaptic receptors A B How is the E/I balance in brain disorders? 28
- 29. • Can we use MSM to measure global excitation to inhibition balance in brain disorders? 29
- 30. Ze Scaduto et al., Sci Rep 2020 Preserved E/I Balance in Human Cx after long PMI 30
- 31. Modified from La Ferla & Oddo, 2005, Alzheimer’s Association 2007, Boros et al., 2017 AD neuropathological change 31
- 32. Sperling et al., 2009 Reduced deactivation of the DMN is observed in old individuals with no Ab burden 32
- 33. Celone et al., 2006, Pihlajamaki et al., 2008 Sperling et al., 2009 Paradoxical increase of the DMN is observed in old individuals with no Ab burden 33
- 34. Determination of global synaptic anatomical and electrophysiological E/I in AD Parietal Cortex Aβ accumulation Sperling et al., 2009 34
- 35. FDT analysis of anatomical E/I ratio Lauterborn et al, Nat Comm, 2021 35
- 36. No changes in total synaptic density but shifts from high to low PSDs intensity in AD and DS Lauterborn et al, Nat Comm, 2021 36
- 37. Increased electrophysiological E/I in AD Lauterborn et al, Nat Comm, 2021 37
- 38. 38 Global E/I balance in AD Frontal Cortex Parietal Cortex Medial Temporal Cortex Hippocampus Aβ accumulation Hyperactivation Reduction of Glu activity First areas affected by the pathology
- 39. Global eE/I balance in Health Frontal Cortex Parietal Cortex Medial Temporal Cortex Hippocampus E/I ratio Singh A. … Scaduto P. et al. accepted - Journal of Alzheimer's Disease 2020 Lauterborn J. and Scaduto P. et al. Nature Communications 2021 Scaduto P. et al. in preparation 39
- 40. eE/I imbalance in AD p=0.039 p=0.0296 Frontal Cortex Parietal Cortex Medial Temporal Cortex Hippocampus E/I ratio Singh A. … Scaduto P. et al. accepted - Journal of Alzheimer's Disease 2020 Lauterborn J. and Scaduto P. et al. Nature Communications 2021 Scaduto P. et al. in preparation p=0.041 40
- 41. Aging Dementia and TBI study 41
- 42. Increased transcriptional E/I in PCx AD Lauterborn et al, Nat Comm, 2021 42
- 43. Increased cellular E/I in PCx AD Lauterborn et al, Nat Comm, 2021 43
- 44. 1. The PCx and TCx in AD show strong loss of excitatory and inhibitory synapses 2. The impairment of synapses is unequal leading to increased transcriptional, anatomical and functional E/I synaptic imbalance 3. Pro-excitatory imbalance in AD may explain the hyperexcitability observed in the DMN, which at early stages interferes with the encoding of memory, and promotes activity-dependent release of Ab and Tau oligomers 44
- 46. Thanks! Collaborators: Julie Lauterborn, Adolfo Sequeira, Mark Vawter at UCI David Cotter, Melanie Focking at RCSI Dirk Keene at University of Washington Giulio Taglialatela, Rakez Kayed and Balaji Krishnan at UTMB Limon Lab: Tommaso Zeppillo Pietro Scaduto Kevin Shen https://www.utmb.edu/mitchellcenter/lab-groups/agenor-limon 46
- 47. Thanks for participating! Before you go… • Complete the Survey! We’d love to get your feedback • Still have a question? Use the Ask a Question panel • Want to learn more about Multi Channel Systems’ electrophysiology instrumentation? Visit: www.multichannelsystems.com
Editor's Notes
- During all our life we consistently lose neurons and synaptic complexity with only mild changes in cognitive function. We can lose a third of or cortical thikness and still live a normal life. Every man in purple and women in green across life in cognitively normal people. For example kids or teenagers that undergo complete resection of half the brain, due to intractable epilepsy can see only a mild to moderate loss of cognitively function. Even when the left hemisphere is removed. This is quite remarkable since major areas associated with speech are in the left hemisphere. But reorganization of the right hemisphere allow to remergence of speech. While persistent epilepsy produces cognitive impairment, the loss of half of the brain has only a moderate effect on cognition. Actually people with only half the brain go on to have functional and successful lives.
- Auxiliary subunits play a critical role in the facilitation and regulation
- To gather this information, we used the microtransplantation of synaptic membranes method (MSM). Briefly, synaptosomes are isolated from frozen tissue, the synaptosomes are lised to form small vesicles that containing the human membranes with their receptors and synaptic complexes, and these protoeliposomes are injected into Xenopus oocytes, which are 1mm in diameter and strong enough to resist the process. The human membranes fuse with the membrane of the oocyte and then we can apply agonists of the receptors to observe their responses. Xenopus oocytes do not express endogenous GABA or glutamate receptors, therefore responses elicited by the agonists are mediated by microtransplanted human receptors. 45 s
- For these experiments we used frozen DLPFC of 10 CTRL and 10 SZ matched by Age, PMI, and as closely as possibly by pH and RIN. There was a slight statistically significant difference in pH and RIN between CTRL and SZ. This parameters were included as covariates in the analysis of the data. Enriched synaptosomal preparations from the DLPFC were used for MSM experiments and for label free proteomic analysis. An adjacent dlpfc section was used for RNA Seq. RNA seq
- The severe cognitive impairment and disability observed in patients with AD is a logical consequence of the loss of brain volume at late stages of AD. Postmortem analysis show substantial loss of gray and white matter and the presence of the neuropathological change that define AD: which is he presence of Amyloid plaques and neurofibrillary tangles. Many decades of research have consistently show that the loss of dendritic spines and synaptic density is the best correlate of cognitive impairment. However, it is important to note that loss of neurons or even loss of brain regions does not necessarily mean loss of cognitive function. For example.
- The severe cognitive impairment and disability observed in patients with AD is a logical consequence of the loss of brain volume at late stages of AD. Postmortem analysis show substantial loss of gray and white matter and the presence of the neuropathological change that define AD: which is he presence of Amyloid plaques and neurofibrillary tangles. Many decades of research have consistently show that the loss of dendritic spines and synaptic density is the best correlate of cognitive impairment. However, it is important to note that loss of neurons or even loss of brain regions does not necessarily mean loss of cognitive function. For example.
- M1-selective allosteric agonist, is effective in increasing spontaneous excitatory postsynaptic currents in the medial prefrontal cortex (mPFC) pyramidal cells and improving memory in a transgenic mouse model of AD -Nicotinic Stimulation Produces Multiple Forms of Increased Glutamatergic Synaptic Transmission (nicotine-induced glutamate release in the prefrontal cortex)