|Role of postsynaptic density proteins in central nervous system disease.|
|Our work has established the first proteome of the human postsynaptic density (PSD) from cortex, showing it presents a very high complexity with over 1400 proteins identified. This work has helped understand its role in nervous system disease. We have primarily focused on brain diseases with well-known genetic basis and found that PSD genes cause more than 130 of them. These diseases, which we have classified according to the World Health Organisation Classification System (ICD-10), cover a wide range of types including common neurodegenerative diseases (Alzheimer’s, Parkinson’s, Huntington’s), adult and childhood mental and behavioural disorders such as intellectual disabilities, motor disorders such as ataxia or dystonia, epilepsies and many rare diseases. Our findings have proven that the human PSD is a neuronal structure with a disproportionately high neural disease susceptibility; presenting it as a more central structure in brain disease. Furthermore, we have also shown that the PSD is particularly relevant to some types of diseases, predominantly for cognitive and motor disorders. Our published data strongly points towards a very important role of the PSD in intellectual disabilities. We have also contributed to a seminal paper on copy number variation (CNV) in schizophrenic patients (Kirov G et al. 2012 Molecular Psychiatry), which has given evidence for PSD molecules having a primary pathogenic role in this important disorder. Our work indicates that we will need a better understanding of the structure and function of the PSD in order to comprehend and treat many brain disorders particularly mental and behavioural ones.We have also worked to adapt well-known synapse biochemical methods to human post-mortem samples (PMS).
|Figure Legend. Classification of Nervous System diseases caused by human PSD proteins.
a. Distribution and relative abundance of monogenic Nervous System diseases caused by hPSD proteins. Central nervous system diseases were classified using the International Classification of Disease (ICD-10) from the World Health Organisation (WHO) and are shown in coloured sections. The proportion of Peripheral Nervous System (PNS) diseases is also shown. b. Distribution and relative abundance of CNS diseases caused by hPSD proteins within Diseases of the Nervous System (Chapter VI, ICD-10).
|Glutamate Receptor Mutations in Psychiatric and Neurodevelopmental Disorders.
David Soto, Xavier Altafaj, Carlos Sindreu, Àlex Bayés.
Communicative and Integrative Biology. 2014, e27887. Download pdf
|De novo CNV analysis implicates specific abnormalities of postsynaptic signalling complexes in the pathogenesis of schizophrenia.
Kirov G, et. al.
Molecular Psychiatry. 2012 Feb;17(2):142-53.
|Characterization of the proteome, diseases and evolution of the human postsynaptic density.
Bayés A, van de Lagemaat LN, Collins MO, Croning MD, Whittle IR, Choudhary JS, Grant SG.
Nat Neurosci. 2011 Jan;14(1):19-21. Download pdf
|Postsynaptic proteins play a major role in neurological and psychiatric diseases.
Alex Bayés & Seth Grant.
Advances in Clinical Neurosciences and Rehabilitation 2011 Jul/Aug; 11(3): 13- 14. Download pdf
|Evolution of the postsynaptic proteome.|
In the past several years the systematic study of the synaptic proteome has identified its components, exposing a much larger complexity than originally anticipated. This is particularly the case for the postsynaptic density (PSD), which could contain up to a few thousand different proteins.
In this context we realised that it was important to understand how did this unexpected complexity arouse through evolution. Our studies in this direction have focused in the PSD, showing that its composition has not grown gradually through animal and mammalian evolution but rather formed in a two-step fashion; from organisms without nervous system to invertebrates and from invertebrates to vertebrates. In this general framework invertebrates share a similar PSD proteome size that is generally half in size to that observed in vertebrates, showing the highest complexity. Furthermore, our work on human, mouse and zebrafish has proven that the composition of the human PSD established very early in vertebrate evolution and has remained highly conserved since then. We have demonstrated that this conservation is also maintained at the quantitative level, that is, PSD protein abundance between species is usually similar. Finally, we have also observed that genes expressed at the PSD have experienced a much slower mutation rate than the average gene expressed in neurons. Altogether our evolutionary work on vertebrates has proven that this molecular machine key to cognition has experienced a very strong evolutionary conservation in vertebrates.
|Figure Legend. Human PSD sequence conservation.|
|a. Cumulative frequency plot of dN/dS values for human PSD and non-PSD genes expressed in cortical neurons and human genome. Human PSD neuronal genes are more constrained than non-PSD neuronal genes (P < 10−11). b. Median mouse-human dN/dS shown for PSD and subcellular structures expressed in cortical neurons14. *P < 0.05, ***P < 0.001. c. Box plots of dN/dS distribution in hPSD hub proteins (>15 interactions, n = 23), non-hubs (≤15 interactions, n = 725) and the tandem affinity purification of PSD-95 complex.|
|Evolution of complexity in the zebrafish synapse proteome.
Bayés À, Collins MO, Reig-Viader R, Gou G, Goulding D, Izquierdo A, Choudhary JS, Emes RD, Grant SG.
Nat Commun. 2017 Mar 2;8:14613. doi: 10.1038/ncomms14613. Download pdf.
|Comparative analysis of human and mouse postsynaptic proteomes identifies high compositional conservation and abundance differences for key synaptic proteins.
Alex Bayés Mark O Collins, Mike DR Croning, Louie N van de Lagemaat, Jyoti S Choudhary and Seth GN Grant.
PLoS ONE. 2012;7(10):e46683. Download pdf
|Evolutionary expansion and anatomical specialization of synapse proteome complexity.
Richard D. Emes*, Andrew J. Pocklington*, Christopher N.G. Anderson*, Alex Bayés, Mark O. Collins, Catherine A. Vickers, Mike D.R. Croning, Bilal R. Malik, Jyoti S. Choudhary, J. Douglas Armstrong, Seth G.N. Grant. (*) These authors equally contributed to the work.
Nature Neuroscience 2008 Jul;11(7): 799-806.
|Development of methods to evaluate stability and quality of synaptic protein complexes from human postmortem brain tissue.|
|The study of human brain disease can be approached using animal models, but at some point most research projects will need to use human samples; this will be particularly relevant for complex psychiatric diseases such as schizophrenia. Post-mortem tissue provides a unique opportunity to perform in-depth molecular and cellular analyses that are not otherwise possible. Nevertheless, post-mortem tissue has its own limitations, particularly those concerning the integrity of the tissue. There are no methods to asses the integrity of synaptic structures in post-mortem tissue.For this reason we have developed a method to predict the integrity in post-mortem tissue of the postsynaptic density (PSD) and the protein complexes within it. This method is based on the stability of the NMDA receptor subunit NR2B, a component of the PSD. We have seen that different brain samples have different NR2B degradation patterns and that those that have not gone over a certain level of NR2B proteolysis have intact postsynaptic supramolecular complexes. This method is very valuable to chose those brains that can be used in a given experiment, not to pool samples with different levels of PSD integrity. Using this method we have seen that the traditional subfractionation methods, used in rodents and other mammals, to isolate synaptic structures, can also be applied to human post-mortem tissue with good protein recovery yields. All this work is in process of publication. Using the developed methods we have characterised from human post-mortem and biopsy brain the complex of proteins associated to the NMDA receptor, a central structure in synapse function extensively studied in rodents.|
|Figure Legend. Method for rapid isolation of synaptic-enriched fractions from PM human cortex. GluN2B degradation and correlation with number of intact PSD proteins.|
|a. Schematic representation of the subcellular fractionation method used to obtain postsynaptic protein enriched fractions (P2). Procedure time is indicated. H and S, homogenized cortex; P1, nucleus/cell debris; S1, cytosolic fraction; P2, triton insoluble fraction; S2, triton soluble fraction. b. Immunoblot showing protein enrichment or depletion between sample S and P2 from isolation protocol described in A. Postsynaptic markers: PSD95/DLG4, GluN2B and SAP102/DLG3. Pre-synaptic-markers: SYP and VGluT-1. A marker of mitochondria is also included: COXIV-1. c. Mean fold enrichment of proteins in final P2 fraction compared to starting S fraction analyzed by immunoblotting (n = 5). Postsynaptic proteins (PSD-95/DLG4, GluN2B and SAP102/DLG3) were enriched in P2, while presynaptic (SYP and VGluT-1) and mitochondrial markers (COX IV-1) were depleted (S/P2 < 1). d. GluN2B immunoblot from control NSB and 28 PM samples (Additional File 2). PM samples shows three main bands, band 1 corresponding with the full-length protein. The ratio of band1 over band2 provides HUSPIR ratio. The antibody used was designed against the C-terminal region of GluN2B (BD Bioscience ref. 610416). e. For each PM sample the HUSPIR ratio (intensity between GluN2B bands 1 and 2) is plotted against the number of intact PSD proteins. Significant positive Spearman’s coefficient of correlation (r) and p-value (p) are indicated. f. HUSPIR ratio for the set of 28 unselected samples and the set of 9 samples selected. Median and interquartile range shown. g. Comparison between percentage of PSD components observed in the set of 28 unselected samples and the prospective set of 9 selected samples. Median and interquartile range shown.|
|Human post-mortem synapse proteome integrity screening for proteomic studies of postsynaptic complexes.
Àlex Bayés, Mark O. Collins, Clare M. Galtrey, Clémence Simonnet, Marcia Roy, Mike D.R. Croning, Gemma Gou, Louie N. van de Lagemaat, David Milward, Ian R. Whittle, Colin Smith, Jyoti S. Choudhary & Seth G.N. Grant.
Molecular Brain 2014, 7:88. Download pdf