This document is a thesis presented by Sebastian Aguiar to the Keck Science Department in partial fulfillment of a Bachelor of Arts degree. It aims to characterize a novel monoclonal antibody targeting the AMPA receptor subunits GluA1, GluA2, and GluA3. The thesis provides background on AMPA receptor structure, function, trafficking, role in synaptic plasticity, involvement in neuropathology, and pharmacology. It then describes the research questions and methods used to visualize the antibody's binding in rat, monkey and human brain tissue using confocal and electron microscopy.
This document discusses several types of protein targets for drugs, including transporters, ion channels, nuclear hormone receptors, G-protein coupled receptors, and enzymes. It covers how these proteins work, the ligands that bind to them, and the downstream effects of ligand binding such as changes in gene expression or intracellular signaling pathways. Key concepts covered include the structures and functions of symporters, antiporters, ion channels, nuclear hormone receptors, GPCRs, the cAMP pathway, PLC pathway, and characteristics of drug-receptor interactions like affinity, efficacy, and types of agonists and antagonists.
The document discusses various techniques for amino acid analysis. It defines amino acids and explains that 20 are encoded by the genetic code. There are essential and non-essential amino acids. Amino acid analysis involves hydrolyzing proteins into individual amino acids, separating them using chromatography, and quantifying amounts. Common techniques discussed include determining amino acid composition, Edman degradation, and mass spectrometry.
1) The study examines the molecular mechanism by which the small molecule distamycin A displaces the HMGA1 protein from DNA. NMR experiments show that distamycin is able to displace an AT hook peptide (DNA binding motif of HMGA1) from DNA.
2) Displaced AT hook peptides weakly interact with DNA but alter distamycin's binding by weakening the distamycin-DNA complex. Distamycin dissociation is also slowed by excess AT hook peptide.
3) Fluorescence anisotropy experiments show that distamycin can effectively displace the full HMGA1a protein from DNA. However, the cooperative binding normally exhibited by distamycin is eliminated by the displaced HMGA1a protein.
- For every molecule of glucose that enters glycolysis, there is an initial investment of 2 ATP molecules before generating subsequent ATP.
- Phosphofructokinase-1 (PFK-1) is a key regulated enzyme in glycolysis. AMP activates PFK-1.
- There are three irreversible reactions in glycolysis catalyzed by hexokinase, phosphofructokinase 1, and pyruvate kinase.
The document reports that monomeric tail domains from kinesin-1 are able to tightly bind and inhibit a dimer of the motor (head) domains, with only one tail peptide binding tightly per head dimer in a "half-site" interaction. This half-site binding of a single tail peptide is sufficient to inhibit both head domains, and occurs rapidly and reversibly. The finding suggests that the second tail peptide in folded kinesin-1 may be available to interact with other molecules while kinesin-1 remains folded.
1. Transcription is the process by which DNA is copied into messenger RNA (mRNA) by RNA polymerase. This involves three phases - initiation, elongation, and termination.
2. Eukaryotic transcription is more complex than prokaryotic transcription due to multiple RNA polymerases, nucleosomes, separation of transcription and translation, and intron-exon structure of genes.
3. Following transcription, eukaryotic mRNA undergoes processing including capping, polyadenylation, and splicing before being translated into protein by ribosomes.
This document compares two methods for sequencing proteins: Edman degradation and mass spectrometry. Edman degradation involves tagging and removing amino acids from the N-terminal end one by one to determine the sequence. Mass spectrometry involves digesting proteins into peptides, using mass to charge ratios to separate peptides, and fragmenting peptides to measure fragment ion masses and deduce sequences. Mass spectrometry is more sensitive, does not require purified samples, and can handle modified proteins better than Edman degradation.
Frederick Sanger developed two important methods for protein sequencing: 1) using fluorodinitrobenzene to determine the N-terminal amino acid, and 2) cleaving proteins into fragments and piecing their sequences together. The standard strategy involves separating chains, identifying terminal residues, cleaving the protein, sequencing fragments, and reconstructing the full sequence. The Edman degradation method allows sequential removal of residues from the N-terminus. Mass spectrometry techniques like peptide mass fingerprinting and tandem mass spectrometry now dominate protein sequencing.
This document discusses several types of protein targets for drugs, including transporters, ion channels, nuclear hormone receptors, G-protein coupled receptors, and enzymes. It covers how these proteins work, the ligands that bind to them, and the downstream effects of ligand binding such as changes in gene expression or intracellular signaling pathways. Key concepts covered include the structures and functions of symporters, antiporters, ion channels, nuclear hormone receptors, GPCRs, the cAMP pathway, PLC pathway, and characteristics of drug-receptor interactions like affinity, efficacy, and types of agonists and antagonists.
The document discusses various techniques for amino acid analysis. It defines amino acids and explains that 20 are encoded by the genetic code. There are essential and non-essential amino acids. Amino acid analysis involves hydrolyzing proteins into individual amino acids, separating them using chromatography, and quantifying amounts. Common techniques discussed include determining amino acid composition, Edman degradation, and mass spectrometry.
1) The study examines the molecular mechanism by which the small molecule distamycin A displaces the HMGA1 protein from DNA. NMR experiments show that distamycin is able to displace an AT hook peptide (DNA binding motif of HMGA1) from DNA.
2) Displaced AT hook peptides weakly interact with DNA but alter distamycin's binding by weakening the distamycin-DNA complex. Distamycin dissociation is also slowed by excess AT hook peptide.
3) Fluorescence anisotropy experiments show that distamycin can effectively displace the full HMGA1a protein from DNA. However, the cooperative binding normally exhibited by distamycin is eliminated by the displaced HMGA1a protein.
- For every molecule of glucose that enters glycolysis, there is an initial investment of 2 ATP molecules before generating subsequent ATP.
- Phosphofructokinase-1 (PFK-1) is a key regulated enzyme in glycolysis. AMP activates PFK-1.
- There are three irreversible reactions in glycolysis catalyzed by hexokinase, phosphofructokinase 1, and pyruvate kinase.
The document reports that monomeric tail domains from kinesin-1 are able to tightly bind and inhibit a dimer of the motor (head) domains, with only one tail peptide binding tightly per head dimer in a "half-site" interaction. This half-site binding of a single tail peptide is sufficient to inhibit both head domains, and occurs rapidly and reversibly. The finding suggests that the second tail peptide in folded kinesin-1 may be available to interact with other molecules while kinesin-1 remains folded.
1. Transcription is the process by which DNA is copied into messenger RNA (mRNA) by RNA polymerase. This involves three phases - initiation, elongation, and termination.
2. Eukaryotic transcription is more complex than prokaryotic transcription due to multiple RNA polymerases, nucleosomes, separation of transcription and translation, and intron-exon structure of genes.
3. Following transcription, eukaryotic mRNA undergoes processing including capping, polyadenylation, and splicing before being translated into protein by ribosomes.
This document compares two methods for sequencing proteins: Edman degradation and mass spectrometry. Edman degradation involves tagging and removing amino acids from the N-terminal end one by one to determine the sequence. Mass spectrometry involves digesting proteins into peptides, using mass to charge ratios to separate peptides, and fragmenting peptides to measure fragment ion masses and deduce sequences. Mass spectrometry is more sensitive, does not require purified samples, and can handle modified proteins better than Edman degradation.
Frederick Sanger developed two important methods for protein sequencing: 1) using fluorodinitrobenzene to determine the N-terminal amino acid, and 2) cleaving proteins into fragments and piecing their sequences together. The standard strategy involves separating chains, identifying terminal residues, cleaving the protein, sequencing fragments, and reconstructing the full sequence. The Edman degradation method allows sequential removal of residues from the N-terminus. Mass spectrometry techniques like peptide mass fingerprinting and tandem mass spectrometry now dominate protein sequencing.
There are several key steps to sequencing a protein, including:
1) Separating individual protein chains and breaking disulfide bonds. This can involve reagents like mercaptoethanol or performic acid.
2) Purifying individual protein chains using techniques like electrophoresis or chromatography.
3) Cleaving the purified protein into fragments using enzymes like trypsin or chemicals like cyanogen bromide.
4) Determining the amino acid sequence of each fragment, then combining the overlapping sequences to deduce the full protein sequence.
Protein sequencing and its applications in bioinformatics. The document discusses the history of protein sequencing including early work by Fred Sanger in 1951. It describes methods of protein sequencing such as N-terminal sequencing using Edman degradation. Mass spectrometry and DNA sequencing are also covered. Bioinformatics tools for sequence alignment are discussed, including BLAST and multiple sequence alignment using CLUSTAL. Protein sequencing provides important information for understanding protein structure and function and has applications in drug development, recombinant protein synthesis, and studying genetic diseases.
Amino acid sequencing determines the order of amino acids in a protein. Frederick Sanger determined the first protein sequence in 1953 using N-terminal analysis methods like Edman degradation. Large proteins are sequenced by first breaking them into smaller fragments using enzymes or chemicals, then determining the sequence of individual fragments and combining sequences to deduce the full protein sequence. Modern techniques like mass spectrometry have made sequencing faster and applicable to modified proteins.
This document describes research into developing an RNA aptamer antagonist of NMDA receptors for potential therapeutic applications. Key points:
- Researchers used SELEX (Systematic Evolution of Ligands by EXponential enrichment) to isolate an RNA aptamer (clone C26) that selectively binds to GluN2A-containing NMDA receptors with high affinity (Kd = 120 ± 15 nM), while having little effect on closely related AMPA and kainate receptors.
- Electrophysiological characterization found this aptamer (C26-50) selectively inhibits GluN2A-containing NMDA receptors without affecting other receptor subtypes. It acts as a non-competitive antagonist with an IC50 of 1
This document discusses macrolide antibiotics, including their mechanism of action, chemistry, classification, and optimization. Macrolides are a class of antibiotics that contain a macrocyclic lactone ring attached to deoxy sugars. They are bacteriostatic and inhibit bacterial protein synthesis by binding reversibly to the 50S subunit of ribosomes. Examples discussed include erythromycin, clarithromycin, and roxithromycin. Strategies to optimize macrolides focused on improving acid stability, such as modifying the C6 hydroxyl group or adding oxime groups.
Advanced Medicinal Chemistry of GPCR Receptorsaurabh gupta
Contents:-
Introduction
Structure of G-protein
Signal Molecules / Ligands of GPCRs
G- Protein Mediated Pathways
Receptor Site Theories
Forces involved in drug receptor interactions
CPP-115 is a potent mechanism-based inactivator of the enzyme γ-aminobutyric acid aminotransferase (GABA-AT) that was developed to treat seizures and other neurological disorders. This study investigated CPP-115's inactivation mechanism of GABA-AT. It was found that CPP-115 undergoes enzyme-catalyzed hydrolysis of its difluoromethylene group, releasing two fluoride ions. This causes a conformational change in the enzyme, forming a tightly bound complex and inactivating the enzyme. Unexpectedly, CPP-115 does not follow the anticipated Michael addition pathway but instead utilizes a novel catalytic mechanism. This new mechanism of inactivation provides insights for
This study used computational modeling to identify potential inhibitors of LdLIP3, a lipase secreted by Leishmania donovani that is involved in the parasite's energy utilization and virulence. Molecular docking experiments analyzed the binding of natural substrates and competitive inhibitors to a control lipase. Results suggested palmitate and stearate are natural substrates, while THC showed stronger inhibitory binding than citral or menthol. This supports exploring THC as a potential LdLIP3 competitive inhibitor for future Leishmaniasis treatment studies.
The document summarizes research on a photoswitchable DNA complex consisting of a dithienylethene (DET) molecular switch bound electrostatically to double-stranded DNA. The DET switch can undergo reversible photochemical ring-opening and closing, existing in either the open (1o) or closed (1c) form. Both 1o and 1c bind to DNA through electrostatic interactions between their protonated amine groups and the negatively charged phosphate backbone of DNA. This results in an induced circular dichroism signal from the DET, demonstrating it has taken on the chirality of the bound DNA helix. The complex can be photochemically switched between states by irradiating with UV or visible light
TRANSLATION & POST - TRANSLATIONAL MODIFICATIONSYESANNA
The document discusses various aspects of translation - the process by which the sequence of nucleotides in mRNA is used to direct the synthesis of a polypeptide chain. It describes how the genetic code is used to translate mRNA into a protein via tRNA and the ribosome. Key points covered include codon-anticodon interactions, the roles of initiation and elongation factors, and termination of protein synthesis.
Synthesis and application of the first small-molecule radioligand targeting t...lastpook
The poster was presented during International Symposium on Medicinal Chemistry in Berlin, September 2012. It was awarded with one of the Poster Prizes.
Introduction
What is Protein Sequencing?
History
Determination of amino acid composition
Sequencing methods
N terminal sequencing
C terminal sequencing
Mass spectrometer
Application
Reference
The determination of amino acid sequences presentation autumne 2015Richard Trinh
1. The document outlines common strategies used for protein sequencing, including Edman degradation. Edman degradation involves breaking the peptide bonds of a protein one by one to determine the amino acid sequence.
2. Other strategies discussed include peptide mass fingerprinting, tandem mass spectrometry, and de novo sequencing methods. Peptide mass fingerprinting involves breaking a protein into peptides and using mass spectrometry to determine the peptide masses and compare them to databases. Tandem mass spectrometry further breaks peptides into fragment ions for sequencing.
3. The strategies each have advantages and limitations. While Edman degradation was previously standard, mass spectrometry techniques are increasingly used due to their high-throughput capabilities and ability to sequence smaller protein amounts. The
This document discusses lipid composition and analysis. It introduces that lipids are insoluble in water but soluble in organic solvents. They include fatty acids, neutral fats, and waxes. Lipids serve important functions as an energy source and in cell structure. Common methods to analyze lipids include fatty acid methyl ester analysis using gas chromatography and mass spectrometry to characterize bacteria based on their lipid profiles.
This document discusses RNA structure and types. It begins by describing the basic components and functions of RNA, including its role in transcription and as an intermediate molecule in protein synthesis. It then discusses the different forms and structures of RNA, including primary, secondary and tertiary structures. The main types of RNA - mRNA, tRNA, rRNA and others like miRNA and siRNA - are then summarized in terms of their roles and characteristics. Applications of RNA interference are also briefly outlined.
Protein sequencing involves determining the order of amino acids in a protein chain. [1] Edman degradation is commonly used for N-terminal sequencing and involves labeling the N-terminal amino acid, removing it, and identifying it through chromatography and mass spectrometry. [2] The protein must first be purified and digested before Edman degradation can begin. [3] Mass spectrometry is used to analyze the separated amino acid derivatives and identify the sequence.
Protein sequencing is a technique to determine the amino acid sequence of proteins. It involves hydrolyzing proteins into amino acids, separating the amino acids, and using techniques like chromatography to identify the order of the amino acids. There are chemical methods like Edman degradation and physical methods like mass spectrometry to sequentially determine the order of amino acids from the N-terminal to C-terminal end of the protein. Understanding amino acid sequences provides insight into protein structure and function.
1) The first protein sequencing was achieved in 1953 by Frederic Sanger who determined the amino acid sequence of bovine insulin.
2) Common strategies for protein sequencing include determining the number of subunits, disulfide bonds, amino acid composition, and sequencing fragments using Edman degradation or enzymatic cleavage.
3) Techniques like end-group analysis, solid-phase support, exopeptidases, and mass spectrometry help overcome challenges and complete the protein sequence.
This doctoral dissertation discusses molecular dynamics simulations of polyglutamine and insulin aggregation. Two projects are described. The first uses replica-exchange molecular dynamics to simulate one and two polyglutamine peptides. It finds the peptides form helical or coil structures at long distances but β-sheets at short distances. The second simulates insulin and binding peptides LVEALYL and RGFFYT. It discovers both peptides aggregate into β-sheets and bind strongly to insulin.
This document discusses various methods for determining the amino acid sequence of proteins, including:
- Edman degradation, which sequentially removes amino acids from the N-terminus. Up to 60 amino acids can typically be determined.
- Mass spectrometry techniques like MALDI that help determine the mass and sequence of protein fragments.
- Enzymatic cleavage techniques using enzymes like trypsin to break proteins into smaller fragments that can then be sequenced.
(1) The document discusses the history of the discovery of neurotransmitters and the role of Ramón y Cajal and Otto Loewi in determining neurons communicate via chemical messengers rather than electrical signals.
(2) It provides definitions of neurotransmitter and criteria that must be met for a substance to be classified as a neurotransmitter.
(3) Glutamate is described as the major excitatory neurotransmitter in the brain, present at high concentrations in presynaptic terminals and involved in many key pathways.
This document discusses the role of AMPA receptor (AMPAR) surface diffusion in synaptic plasticity and memory. It reports that:
1) AMPAR surface diffusion is important for the establishment and maintenance of long-term potentiation (LTP) through replenishing synaptic AMPARs.
2) Blocking AMPAR surface diffusion through crosslinking attenuates LTP in hippocampal brain slices and impairs hippocampal-dependent memory formation.
3) Postsynaptic AMPAR surface diffusion is a critical trafficking mechanism for the expression of LTP and learning in the hippocampus.
There are several key steps to sequencing a protein, including:
1) Separating individual protein chains and breaking disulfide bonds. This can involve reagents like mercaptoethanol or performic acid.
2) Purifying individual protein chains using techniques like electrophoresis or chromatography.
3) Cleaving the purified protein into fragments using enzymes like trypsin or chemicals like cyanogen bromide.
4) Determining the amino acid sequence of each fragment, then combining the overlapping sequences to deduce the full protein sequence.
Protein sequencing and its applications in bioinformatics. The document discusses the history of protein sequencing including early work by Fred Sanger in 1951. It describes methods of protein sequencing such as N-terminal sequencing using Edman degradation. Mass spectrometry and DNA sequencing are also covered. Bioinformatics tools for sequence alignment are discussed, including BLAST and multiple sequence alignment using CLUSTAL. Protein sequencing provides important information for understanding protein structure and function and has applications in drug development, recombinant protein synthesis, and studying genetic diseases.
Amino acid sequencing determines the order of amino acids in a protein. Frederick Sanger determined the first protein sequence in 1953 using N-terminal analysis methods like Edman degradation. Large proteins are sequenced by first breaking them into smaller fragments using enzymes or chemicals, then determining the sequence of individual fragments and combining sequences to deduce the full protein sequence. Modern techniques like mass spectrometry have made sequencing faster and applicable to modified proteins.
This document describes research into developing an RNA aptamer antagonist of NMDA receptors for potential therapeutic applications. Key points:
- Researchers used SELEX (Systematic Evolution of Ligands by EXponential enrichment) to isolate an RNA aptamer (clone C26) that selectively binds to GluN2A-containing NMDA receptors with high affinity (Kd = 120 ± 15 nM), while having little effect on closely related AMPA and kainate receptors.
- Electrophysiological characterization found this aptamer (C26-50) selectively inhibits GluN2A-containing NMDA receptors without affecting other receptor subtypes. It acts as a non-competitive antagonist with an IC50 of 1
This document discusses macrolide antibiotics, including their mechanism of action, chemistry, classification, and optimization. Macrolides are a class of antibiotics that contain a macrocyclic lactone ring attached to deoxy sugars. They are bacteriostatic and inhibit bacterial protein synthesis by binding reversibly to the 50S subunit of ribosomes. Examples discussed include erythromycin, clarithromycin, and roxithromycin. Strategies to optimize macrolides focused on improving acid stability, such as modifying the C6 hydroxyl group or adding oxime groups.
Advanced Medicinal Chemistry of GPCR Receptorsaurabh gupta
Contents:-
Introduction
Structure of G-protein
Signal Molecules / Ligands of GPCRs
G- Protein Mediated Pathways
Receptor Site Theories
Forces involved in drug receptor interactions
CPP-115 is a potent mechanism-based inactivator of the enzyme γ-aminobutyric acid aminotransferase (GABA-AT) that was developed to treat seizures and other neurological disorders. This study investigated CPP-115's inactivation mechanism of GABA-AT. It was found that CPP-115 undergoes enzyme-catalyzed hydrolysis of its difluoromethylene group, releasing two fluoride ions. This causes a conformational change in the enzyme, forming a tightly bound complex and inactivating the enzyme. Unexpectedly, CPP-115 does not follow the anticipated Michael addition pathway but instead utilizes a novel catalytic mechanism. This new mechanism of inactivation provides insights for
This study used computational modeling to identify potential inhibitors of LdLIP3, a lipase secreted by Leishmania donovani that is involved in the parasite's energy utilization and virulence. Molecular docking experiments analyzed the binding of natural substrates and competitive inhibitors to a control lipase. Results suggested palmitate and stearate are natural substrates, while THC showed stronger inhibitory binding than citral or menthol. This supports exploring THC as a potential LdLIP3 competitive inhibitor for future Leishmaniasis treatment studies.
The document summarizes research on a photoswitchable DNA complex consisting of a dithienylethene (DET) molecular switch bound electrostatically to double-stranded DNA. The DET switch can undergo reversible photochemical ring-opening and closing, existing in either the open (1o) or closed (1c) form. Both 1o and 1c bind to DNA through electrostatic interactions between their protonated amine groups and the negatively charged phosphate backbone of DNA. This results in an induced circular dichroism signal from the DET, demonstrating it has taken on the chirality of the bound DNA helix. The complex can be photochemically switched between states by irradiating with UV or visible light
TRANSLATION & POST - TRANSLATIONAL MODIFICATIONSYESANNA
The document discusses various aspects of translation - the process by which the sequence of nucleotides in mRNA is used to direct the synthesis of a polypeptide chain. It describes how the genetic code is used to translate mRNA into a protein via tRNA and the ribosome. Key points covered include codon-anticodon interactions, the roles of initiation and elongation factors, and termination of protein synthesis.
Synthesis and application of the first small-molecule radioligand targeting t...lastpook
The poster was presented during International Symposium on Medicinal Chemistry in Berlin, September 2012. It was awarded with one of the Poster Prizes.
Introduction
What is Protein Sequencing?
History
Determination of amino acid composition
Sequencing methods
N terminal sequencing
C terminal sequencing
Mass spectrometer
Application
Reference
The determination of amino acid sequences presentation autumne 2015Richard Trinh
1. The document outlines common strategies used for protein sequencing, including Edman degradation. Edman degradation involves breaking the peptide bonds of a protein one by one to determine the amino acid sequence.
2. Other strategies discussed include peptide mass fingerprinting, tandem mass spectrometry, and de novo sequencing methods. Peptide mass fingerprinting involves breaking a protein into peptides and using mass spectrometry to determine the peptide masses and compare them to databases. Tandem mass spectrometry further breaks peptides into fragment ions for sequencing.
3. The strategies each have advantages and limitations. While Edman degradation was previously standard, mass spectrometry techniques are increasingly used due to their high-throughput capabilities and ability to sequence smaller protein amounts. The
This document discusses lipid composition and analysis. It introduces that lipids are insoluble in water but soluble in organic solvents. They include fatty acids, neutral fats, and waxes. Lipids serve important functions as an energy source and in cell structure. Common methods to analyze lipids include fatty acid methyl ester analysis using gas chromatography and mass spectrometry to characterize bacteria based on their lipid profiles.
This document discusses RNA structure and types. It begins by describing the basic components and functions of RNA, including its role in transcription and as an intermediate molecule in protein synthesis. It then discusses the different forms and structures of RNA, including primary, secondary and tertiary structures. The main types of RNA - mRNA, tRNA, rRNA and others like miRNA and siRNA - are then summarized in terms of their roles and characteristics. Applications of RNA interference are also briefly outlined.
Protein sequencing involves determining the order of amino acids in a protein chain. [1] Edman degradation is commonly used for N-terminal sequencing and involves labeling the N-terminal amino acid, removing it, and identifying it through chromatography and mass spectrometry. [2] The protein must first be purified and digested before Edman degradation can begin. [3] Mass spectrometry is used to analyze the separated amino acid derivatives and identify the sequence.
Protein sequencing is a technique to determine the amino acid sequence of proteins. It involves hydrolyzing proteins into amino acids, separating the amino acids, and using techniques like chromatography to identify the order of the amino acids. There are chemical methods like Edman degradation and physical methods like mass spectrometry to sequentially determine the order of amino acids from the N-terminal to C-terminal end of the protein. Understanding amino acid sequences provides insight into protein structure and function.
1) The first protein sequencing was achieved in 1953 by Frederic Sanger who determined the amino acid sequence of bovine insulin.
2) Common strategies for protein sequencing include determining the number of subunits, disulfide bonds, amino acid composition, and sequencing fragments using Edman degradation or enzymatic cleavage.
3) Techniques like end-group analysis, solid-phase support, exopeptidases, and mass spectrometry help overcome challenges and complete the protein sequence.
This doctoral dissertation discusses molecular dynamics simulations of polyglutamine and insulin aggregation. Two projects are described. The first uses replica-exchange molecular dynamics to simulate one and two polyglutamine peptides. It finds the peptides form helical or coil structures at long distances but β-sheets at short distances. The second simulates insulin and binding peptides LVEALYL and RGFFYT. It discovers both peptides aggregate into β-sheets and bind strongly to insulin.
This document discusses various methods for determining the amino acid sequence of proteins, including:
- Edman degradation, which sequentially removes amino acids from the N-terminus. Up to 60 amino acids can typically be determined.
- Mass spectrometry techniques like MALDI that help determine the mass and sequence of protein fragments.
- Enzymatic cleavage techniques using enzymes like trypsin to break proteins into smaller fragments that can then be sequenced.
(1) The document discusses the history of the discovery of neurotransmitters and the role of Ramón y Cajal and Otto Loewi in determining neurons communicate via chemical messengers rather than electrical signals.
(2) It provides definitions of neurotransmitter and criteria that must be met for a substance to be classified as a neurotransmitter.
(3) Glutamate is described as the major excitatory neurotransmitter in the brain, present at high concentrations in presynaptic terminals and involved in many key pathways.
This document discusses the role of AMPA receptor (AMPAR) surface diffusion in synaptic plasticity and memory. It reports that:
1) AMPAR surface diffusion is important for the establishment and maintenance of long-term potentiation (LTP) through replenishing synaptic AMPARs.
2) Blocking AMPAR surface diffusion through crosslinking attenuates LTP in hippocampal brain slices and impairs hippocampal-dependent memory formation.
3) Postsynaptic AMPAR surface diffusion is a critical trafficking mechanism for the expression of LTP and learning in the hippocampus.
This document describes the discovery of a novel clinical AMPA receptor positive modulator called N-[(2S)-5-(6-Fluoro-3-pyridinyl)-2,3-dihydro-1H-inden-2-yl]-2-propanesulfonamide (17i). A series of indane analogs were synthesized and tested for their ability to potentiate AMPA receptor activity. Compound 17i was found to be a potent, efficacious modulator with excellent pharmacokinetic properties across preclinical species. It was well tolerated and orally bioavailable in humans, making it a promising candidate for further clinical development.
The document discusses nerve cell membranes and their role in communication between nerve cells. It describes how nerve cell membranes are composed of lipids and proteins, including integral membrane proteins and peripheral membrane proteins. It also discusses several key proteins involved in postsynaptic densities, including PSD-95, Homer, and Shank, and their role in forming a structural framework at neuronal synapses.
The document presents a computational model of episodic memory encoding in the dentate gyrus region of the hippocampus using an ART neural network. The dentate gyrus is proposed to encode episodes in binary patterns in a way that achieves high levels of pattern encoding and separation. The model incorporates a method for encoding episodes as binary patterns that are stored sparsely. Simulation results show that the model achieves good separation of different episodes depending on the vigilance parameter, which is assumed to correlate with attention during episode perception.
This document summarizes the discovery and optimization of a new class of positive allosteric modulators of AMPA receptors. Key points:
- A novel series was identified from a high throughput screen and optimized from an initial hit to a clinical candidate.
- Unusually for an ion channel target, optimization was integrated with regular generation of ligand-bound crystal structures, which uncovered a novel chemotype with a conserved trifluoromethyl interaction site.
- The hit was optimized through various modifications including changing substituents on aromatic rings, modifying amide linkages, adding or removing fluorine atoms, and altering fused ring systems to improve properties like developability, tolerability, and efficacy.
- Crystal
1) The active zone is composed of an evolutionarily conserved protein complex containing RIM, Munc13, RIM-BP, α-liprin, and ELKS proteins as core constituents. This complex docks and primes synaptic vesicles for exocytosis.
2) In addition to transmitting information, synapses transform information encoded in bursts of action potentials through short-term and long-term plasticity mediated by the active zone protein complex.
3) The five core proteins work together to recruit voltage-gated calcium channels to the active zone, position the active zone opposite postsynaptic specializations, and mediate both short-term and long-term presynaptic plasticity.
This study investigated the relationship between matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), and sialic acid (NANA) in human glial cells. Treatment with NANA upregulated MMP-9 and TIMP-1 mRNA expression, indicating NANA involvement in signaling pathways regulating these genes. At lower NANA concentrations, MMP-9 and TIMP-1 expression increased similarly, but at the highest concentration MMP-9 increased more than TIMP-1, resembling an imbalance seen in multiple sclerosis. This suggests NANA affects MMP-9 and TIMP-1 expression and their balance, which may influence inflammatory demyelination.
Lydia Yeshitla, Research Scholar at the Neurobiology Section of UCSDLydia Yeshitla
1) The document describes an experiment cloning a pH-sensitive fluorescent protein (pHRed) onto the GLUA1 AMPA receptor subunit to track intracellular trafficking and degradation of AMPA receptors by lysosomes.
2) Restriction enzymes (AGE1 and BSRG1) were used to cut the DNA in order to ligate pHRed onto GLUA1 using PCR. This would allow detection of AMPA receptors in the acidic lysosome lumen.
3) Bacteria were transformed with the ligated pHRed-GluA1 DNA. Colonies were selected and the DNA was sequenced to validate that the cloning procedure was done correctly.
This document provides an overview of post-transcriptional gene control mechanisms. It discusses processing of eukaryotic pre-mRNA including 5' capping, polyadenylation, and splicing. Splicing involves spliceosome complexes containing small nuclear RNAs. The document also covers regulation of alternative splicing and mechanisms of gene repression by microRNAs and short interfering RNAs.
This document provides an overview of post-transcriptional gene control mechanisms. It discusses processing of eukaryotic pre-mRNA including 5' capping, polyadenylation, and splicing. Splicing involves spliceosome complexes containing small nuclear RNAs. The document also covers regulation of alternative splicing and mechanisms of gene repression by microRNAs and short interfering RNAs.
The N-methyl-D-aspartate receptor (also known as the NMDA receptor or NMDAR), a glutamate receptor, is the predominant molecular device for controlling synaptic plasticity and memory function...
The second large class of proteins are membrane proteins, which make up 20-30% of genes. They are the targets of over 50% of modern drugs. Membrane proteins perform vital functions like transport, signaling, and enzymatic activity. They have hydrophobic residues that allow them to span or attach to cell membranes. Their structures generally involve alpha helical bundles or beta barrels. Their folding and estimation of molecular weight differs from soluble proteins due to their hydrophobic and membrane-bound nature. They are an important class of drug targets.
1. The study used high-resolution fluorescence recovery after photobleaching (FRAP) to examine the dynamics and organization of AMPA receptors (AMPARs) within the postsynaptic density (PSD) of single dendritic spines in cultured hippocampal neurons.
2. They found that under basal conditions, AMPARs showed limited lateral diffusion within the PSD, but clustered together in a matrix that continuously reshaped in an actin-dependent manner.
3. Application of glutamate increased the intrasynaptic mobility of AMPARs, suggesting activated synapses promote exchange of receptors among subdomains. This supports the idea that the PSD regulates subsynaptic receptor distribution.
The document discusses membrane structure and ligand-gated ion channels. It covers topics like lipid structure and properties, membrane structure and fluidity, membrane proteins and their interactions with lipids, and different classes of ligand-gated ion channels including ATP-sensitive potassium channels, G protein-gated potassium channels, cyclic nucleotide-gated channels involved in phototransduction, nicotinic acetylcholine receptors, and glutamate receptors. Experimental techniques like fluorescence recovery after photobleaching are also mentioned for studying membrane component diffusion.
This document discusses synaptic transmission and neurotransmitters. It begins by defining a synapse as the contact point between neurons. It then differentiates between electrical and chemical synapses. Chemical synapses use neurotransmitters to transmit signals across the synaptic cleft in one direction, while electrical synapses allow direct ion flow between neurons. The document outlines the process of neurotransmitter release via calcium-triggered exocytosis and postsynaptic receptor activation. It discusses both ionotropic and metabotropic receptors and how they contribute to postsynaptic potentials. Finally, it covers specific neurotransmitters like glutamate and GABA, the neuromuscular junction, and clostridial toxins that impact synaptic function.
NMDA Receptor Physiological Activators and Inhibitors A Three-fold Molecular ...Laurensius Mainsiouw
1) NMDA receptors demonstrate slow kinetics, are highly permeable to calcium ions, and require binding of glutamate and glycine for activation. Their function is important for processes like long-term potentiation that underlie memory and learning.
2) The receptor consists of four subunits that can vary, leading to differences in properties like agonist binding affinity. Single channel recordings have provided insight into the kinetic schemes of receptor interactions.
3) Binding of agonists is believed to cause conformational changes in the receptor subunits, bringing the transmembrane domains closer together and opening the ion channel. Mutational studies support models where agonist binding causes lobes of the ligand binding domains to move, transmitting the signal to open
This document provides scientific background on the 2009 Nobel Prize in Chemistry, which was awarded for studies of the structure and function of the ribosome. It summarizes the key contributions of Venkatraman Ramakrishnan, Thomas Steitz, and Ada Yonath, including obtaining the first high resolution crystal structures of the ribosomal subunits, solving the long-standing mysteries of the ribosome's catalytic mechanisms and role in protein synthesis, and advancing understanding of its accuracy and interactions with antibiotic drugs. Their work over decades was instrumental in revealing the ribosome's structure and function at the atomic level.
Regulation of atp7 a gene expression by the grx1 as an inducer in menkes d...
Thesis Final Draft_SA
1. Characterizing a novel monoclonal AMPA
receptor 1/2/3 antibody in the hippocampus and
prefrontal cortex of rat, monkey, and human
A Thesis Presented
by
Sebastian Aguiar
To the Keck Science Department
Of Claremont McKenna, Pitzer, and Scripps Colleges
In partial fulfillment of
The degree of Bachelor of Arts
Senior Thesis in Human Biology
December 2013
3. Characterizing a novel monoclonal AMPA receptor 1/2/3 antibody
in the hippocampus and prefrontal cortex of rat, monkey, and human
Sebastian Aguiar
Pitzer College!, W.M. Keck Science Department
!
John Morrison, PhD, Mentor
Dean of Basic Sciences and the Graduate School of Biomedical Sciences
Professor of Neuroscience, Geriatrics and Palliative Medicine
Friedman Brain Institute, Icahn School of Medicine at Mount Sinai
!
Alan Jones, PhD, First Reader
Professor of Neuroscience and Psychology, Pitzer College
Abstract
The excitatory, ionotropic glutamatergic AMPA receptor is the most common
membrane-bound receptor in the central nervous system. AMPARs and the
NMDA receptors are central to synaptic plasticity, memory, and mechanisms of
neurodegeneration. The AMPAR is an obligate heterotetramer, composed of
subunits GluA1-4. Subunit permutation determines ion conductance, trafficking
and other functional characteristics. Few available antibodies are subunit-specific,
disabling researchers from accurately visualizing differential AMPAR subunit
distribution in the nervous system. This study sought to visualize a novel
monoclonal GluA1/2/3 antibody with functional avidity for three of four receptor
subunits and to characterize the ultrastructural localization of these receptors using
confocal and electron microscopy.
4. Table of Contents
Acknowledgements
Introduction
Receptor Structure and Mechanics 1
Receptor Trafficking 3
Biogenesis 3
Synaptic Targeting 4
Exocytosis and Endocytosis 5
Synaptic Plasticity 7
Long-Term Potentiation 8
Long-Term Depression 10
Homeostatic Scaling 11
Neuropathology 11
Pharmacology 13
Allosteric Modulators 14
AMPAR Subunit Characterization 19
Research Questions 20
Methods and Materials
GluA1 Transformation 22
Transcardial Perfusion 23
Immunohistochemistry 23
Confocal and Electron Microscopy 24
Results
Anti-GluA Monoclonal Antibody 25
Single / Double Label Confocal Microscopy 28
PSD95, pAb AMPA, mAb Colocalization 30
Colocalization Plot 31
Immunogold Electron Microscopy 32
Discussion 33
References 34
Appendix 42
5. Table of Figures
Introduction
Figure 1: AMPAR Heterotetramer and Generic Subunit
Structure
Fig 2: Electron micrograph of GluA2 tetramer showing
dual N-terminal domains, ligand binding domains, and
the transmembrane domain
Fig 3: Proteins that associate with the C-terminus of the
AMPAR
Figure 4: Preliminary activity state models of the
AMPAR
Figure 5: TARP-mediated AMPAR trafficking from
endoplasmic reticulum to dendrite, followed by PSD95
binding
Figure 6: Schematic of AMPAR associated trafficking
proteins
Figure 7: LTP Recording Setup and Electrophysiology
Figure 8: Phosphorylation and Receptor Dynamics
Figure 9: Suspected Pathological Molecules in
Alzheimer’s Dementia
Fig 10: Perampanel and tezampanel
Figure 11: Locations of ligand binding for both AMPA
and kainate receptors
Figure 12: Effect of CX717 on Sleep Deprivation and
Cognition in DMS Task
Figure 13: Dose-Response Data
Fig 14: Glucose consumption PET scan
Figure 15: Dimer of dimer subunit ratios in rat Shaffer-
CA1 pyramidal neuron synapses
6. Results
Fig 1. Western blot
Figure 2. Non-antigen retrieved rat prefrontal cortex
stained with monoclonal GluA1/2/3 antibody
Figure 3: Non-antigen retrieved hippocampus stained
with monoclonal GluA1/2/3 antibody
Figure 4: Human Prefrontal Cortex with antigen
retrieval
Figure 5. Colocalization: antigen retrieved human
prefrontal cortex stain of DAPI, GluA1/2/3 and Post
Synaptic Density-95.
Figure 6. Double labeling with GluA1/2/3 and PSD-95
in human, monkey and rat hippocampus
Figure 7. Double labeling of rat CA1 hippocampus with
the GluA1/2/3 monoclonal antibody colocalized with the
polyclonal GluA2/3 antibody.
Figure 8. Colocalization plot
Figure 9. Electron microscopy
Figure 10. Dilution series
7. 1
Introduction
AMPA Receptors
The identification of the AMPA-type ionotropic glutamate receptor as the primary
regulator of fast excitatory transmission in the CNS constitutes one of the major
achievements of modern neuroscience. These ubiquitous receptors are critical for
synaptic plasticity, mediating memory-associated changes in dendritic morphology,
signaling the expression of neurotrophins, and are implicated in many neurological
disorders. 1
AMPA receptors (AMPARs) were named for the artificial
selective agonist and glutamate analogue !-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid by Tage Honore and colleagues
at the Royal Danish School of Pharmacy in Copenhagen, and
published in 1982 in the Journal of Neurochemistry. 2
Receptor Structure and Mechanics
AMPARs are obligate heterotetramers, composed of subunits GluA1-4, 3
with
each subunit possessing a binding pocket for the neurotransmitter glutamate. 4
From 1989
to 1992, the genes encoding the four subunits (GRIA1-4) were identified. 5
Different
combinations of subunits determine the pharmacological, functional and trafficking
characteristics of the channel. 6
Diversity can also come in the form of post-translational
modifications and splice variants (such as the Arg607 Q/R residue). 7
Figure 1: AMPAR Heterotetramer and Generic Subunit Structure. 8
AMPA
8. 2
Fig 2: Electron micrograph of GluA2 tetramer showing dual N-terminal domains, ligand binding domains,
and the transmembrane domain. 9
Receptor subunits are composed of an N-terminal extracellular domain (NTD)
with an intermediate ligand binding domain (LBD), four transmembrane domains (TMD),
and an intracellular C-terminal tail that interacts with a multitude of scaffolding proteins
involved in structure and signaling at the post-synaptic density. These include NSF, AP-2,
PICK1, GRIP, ABP, KIF5, PKC, PKA, SAP97, PSD95, and a family of Transmembrane
AMPAR Regulatory Proteins (TARPs) such as stargazin. 10, 11 12
Fig 3: Proteins that associate with the C-terminus of the AMPAR. 11
In adult excitatory hippocampal neurons, most AMPARs are composed of GluA1-
2 or GluA2-3 complexes. 13
All AMPARs are ligand-gated cation channels, allowing
sodium in and potassium ions out, tending toward an equilibrium potential of 0 mV
(about halfway between ENa+ and EK+). GluA2, however, is calcium impermeable. And
GluA2 trumps GluA1 in a heterotetramer. The majority of AMPARs in the hippocampus
(approximately 80%), however, possess GluA2 and are therefore calcium impermeable. 14
9. 3
As a result, AMPARs are “dependent” upon NMDA-mediated calcium influx in the
LTP/LTD response (see the section on Synaptic Plasticity below).
In 1998, Eric Gouaux’s laboratory reported the first structural assessment of the
isolated ligand-binding domain. They identified the N-terminal extracellular structure as
a “clamshell” that is closed by the binding of two glutamate molecules, leading to the
opening of the ion pore in a scissor-like motion. 15
Models of activation and
desensitization states have been developed using computational x-ray crystallography.
Figure 4: Preliminary activity state models of the AMPAR. Desensitization is characterized by reduced
response after prolonged exposure to agonist. This process is mediated by separation of the upper lobes of
the LBD dimer.
16
Receptor Trafficking
Biogenesis
AMPAR subunits are mostly synthesized in the somatic endoplasmic reticulum,
modified and assembled into tetramers in the Golgi apparatus and finally inserted into the
membrane where they laterally diffuse in and around the dendrite. AMPARs are
synthesized in response to neural activity, a process mediated by the metabotropic GluRs
1 and 5. 17
AMPARs are also synthesized within dendrites. The mRNA is packaged and
transported via microtubules in response to glutamatergic stimuli. Translational outposts
in the dendrites consist of polyribosomes and golgi structures. Interestingly, D1/D5
dopaminergic agonism is also capable of augmenting AMPAR synthesis and eliciting a
10. 4
heightened frequency of spontaneous miniature excitatory postsynaptic currents
(mEPSCs) in the hippocampus. 18
Synaptic Targeting
Long-term potentiation, depression, and the attendant memory and learning are
mediated by rates of synthesis, membrane transport, and endocytosis of AMPA receptors.
Vesicular trafficking from the Golgi involves dyneins and kinesins, small molecular
motors that move cargo along microtubules. 19
Trafficking from the soma to the dendrites
is mediated by the TARP family of chaperone-like proteins.
Figure 5: TARP-mediated AMPAR trafficking from endoplasmic reticulum to dendrite, followed by
PSD95 binding. 11
A complex of GluR2, the PSD protein GRIP1, and kinesin can be
immunoprecipitated from brain lysates, and the expression of nonfunctional versions of
kinesin decreases synaptic abundance of AMPARs. 20
Within the dendritic spines, actin
and myosin have been implicated in clathrin-dependent endocytosis and local AMPAR
trafficking. 21
Stargazin, a calcium channel gamma-subunit homolog, plays a major role in
several points of the AMPAR secretory process. 22
Originally identified as the mutant
gene in the Stargazer mouse, which is afflicted with cerebellar ataxia and epilepsy, it has
since been shown to promote transport of AMPARs to the cell surface. Stargazer mouse
cerebellar granule cells consequently show very low AMPAR levels at the synapse. 23
Post-synaptic density protein 95 (PSD 95) is a structural protein critical to
AMPAR stability. Stargazin is complexed with both AMPARs and PSD95, which
anchors the group to the post synaptic density. Overexpression of PSD95 results in
11. 5
increased AMPAR-mediated synaptic currents, 24
and knockdown results in decreased
AMPARs at the synapse. 25
Exocytosis and Endocytosis
AMPARs have a metabolic half-life of approximately 30 hours. 26
Explaining
their synaptic dynamics, especially in light of their role in long-term memory, is a critical
area of research. For example, it remains controversial whether AMPARs are primarily
inserted into the synapse directly or into the adjacent extrasynaptic membrane and then
laterally diffuse to the synapse.
AMPAR insertion is dependent upon subunit composition. Insertion of long-tailed
C-terminus AMPARs GluA1/4 occurs slowly under basal conditions and is stimulated by
NDMA activation and neural activity. In contrast, receptors containing GluA2/3 tend to
be constitutively trafficked to the synapse under basal conditions and are not dependent
upon neural activity. 27
AMPA receptors are held in intracellular reserve pools and are fused with the
membrane as necessary. The administration of tetanus toxin cleaves SNAREs and blocks
AMPAR insertion at the synapse. 28
N-ethylmaleimide-sensitive fusion protein (NSF) has
been shown to bind with the C terminus of the GluA2 subunit, regulating rapid
exocytosis. AMPARs in the reserve pool complex with PICK1, a protein that is thought
to stabilize the reserve pool. When NSF binds with PICK1, the former dissociates from
AMPARs and enables them to fuse with the synapse. 29
Endocytosis of AMPARs is similar to that of G-protein coupled receptors
(GPCRs) because both processes feature clathrin-coated pits and require dynamin. 30
After being internalized, AMPARs are engulfed by early endosomes and sent either to a
specialized recycling endosome compartment that allows quick reinsertion to the surface
or to late endosomes and ultimately lysosomes for degradation. 31
The immediate early gene CPG2 mediates basal and activity-regulated AMPAR
internalization and localizes to the endocytic zone. CPG2 knockdown inhibited AMPAR
and NMDAR endocytosis. 32
Another immediate early gene, Arc, is induced by neural
activity and directly implicated in cognition and long-term memory. Arc mRNA is tightly
regulated and translated at activated synapses. Arc regulates AMPAR trafficking via
12. 6
interactions with the endocytic proteins dynamin and endophilin. 33
Finally, Tumor
Necrosis Factor Alpha (TNF-!) augments AMPAR insertion. 34
Figure 6: Schematic of AMPAR associated trafficking proteins. 1
Recycling and degradation may be coordinated, at least in part, by ubiquitination
signals and the ubiquitin-proteasome system (UPS). Monoubiquitination has been shown
in C. elegans to signal endocytosis, whereas polyubiquitination leads to proteasome
13. 7
degradation. 35
In mammalian neurons, targeted mutation of the ubiquitin peptide at
lysine 48 prevented AMPA-induced receptor internalization. 36
The role of ubiquitination
in AMPAR recycling remains an active area of study.
Synaptic Plasticity
Changes in synaptic strength are believed to underlie memory storage. 37
Hebbian
Learning postulates “neurons that fire together wire together,” and that neurons sensitize
or desensitize in response to repeated stimuli. In the terms of Neural Network Theory,
memory is encoded in the pattern of synaptic strength or “weights” distributed across the
probability space of possible connections. Likewise, forgetting is the loss or decay of this
pattern of synaptic weights.
Long-term potentiation (LTP) and long-term depression (LTD) are the two most
studied cellular models of synaptic plasticity. 38
AMPARs are the metaphorical “currency”
of LTP and LTD, and the NMDA receptor can be understood as the cashier – transacting
in AMPARs under various stimulus conditions. 39
Long-term memory formation
putatively involves cyclic AMP response element binding protein (CREB) and Mitogen-
activated protein kinases (MAPK). 40
Figure 7: A) LTP Recording Setup, B) Augmented AMPAR current and number of AMPARs at the
synapse in response to high frequency vs. low frequency stimulation. 7
14. 8
Long-Term Potentiation
Long-term potentiation can be defined as the long lasting enhancement of signal
transmission that results from synchronous stimulation. The phenomenon was first
demonstrated in the rabbit hippocampus by Terje Lømo and Tim Bliss in 1966, who
stimulated Schaffer collateral (pyramidal CA3) neurons and recorded from CA1
pyramidal cells. The effect has been shown to last for weeks in vivo, and to correlate with
performance on spatial memory tasks. 41
LTP also involves other regions of the brain
such as the limbic system and cortex. 42
Inducing LTP experimentally involves stimulating the presynaptic neuron with a
high frequency (typically 100 Hz for one second) tetanus, resulting in an excitatory post-
synaptic potential. The postsynaptic neuron will be sensitized and more likely to respond
with a higher amplitude output for an extended period of time. A single AMPAR-
mediated excitatory post-synaptic potential (EPSP) has a rise time-to-peak of
approximately 2–5 ms and a mean duration of 30 ms. If stimulated one hundred times a
second, the AMPAR will attempt to open every 10 ms (including the refractory period),
the EPSC will increase, and EPSPs will sum resulting in a moderate local depolarization,
triggering NMDARs and initiating a sequence that leads to increased synaptic weighting.
AMPARs are mostly found as GluA2-containing heterotetramers, which are
impermeable to calcium. For LTP to occur, NMDA (named for the selective agonist N-
methyl-D-aspartate) receptors must be activated. NMDARs are dual-ligand (glutamate
and D-serine or glycine) and voltage-gated due to magnesium ion blockade at resting
potential (-70 mV). AMPARs depolarize the membrane potential enough to dislodge the
Mg2+
ion (about 0 mV) and enable the NMDAR Ca2+
current to flow inward.
In short, the role of the AMPAR is to initiate a local membrane depolarization
sufficient to revoke the Mg2+
blockade, leading to the activation of the more “powerful”
NMDARs, which, in turn, instigate a cascade that results in the regulation of AMPAR
membrane insertion. Many excitatory synapses are actually thought to be
postsynaptically 'silent', possessing functional NMDARs but lacking AMPARs. The
acquisition of AMPARs at silent synapses may also be important in synaptic plasticity
15. 9
and neural development. 43
Kinase phosphorylation is crucial in the regulation of neural function, like in most
cell types. 44
AMPAR phosphorylation is also an integral part of LTP. 45
A substantial rise
in calcium concentration within the dendritic spine initiates LTP by activating kinases.
Low calcium influx activates phosphatases leading to long-term depression (LTD).
AMPARs are phosphorylated by Calcium/calmodulin-dependent kinase II
(CaMKII), which was discovered in the lab of Paul Greengard in 1978, another
monumental discovery in the molecular neuroscience of memory and learning. 46
CaMKIIA isoform knockout mice demonstrate a low frequency of LTP and fail to form
persistent, stable place cells in the hippocampus. 47
CaMKII itself is autophosphorylated
in the presence of NMDAR-mediated calcium influx. CaMKII does not appear to be
necessary for recruiting AMPARs to the synapse but phosphorylation Ser831 of GluA1
does indeed augment ion conductance. 48, 49, 50
Figure 8: Phorphorylation of AMPARs influences cation conductance, as
well as playing a role in dendritic spine trafficking and membrane
insertion. 45
16. 10
Protein Kinase A (PKA) phosphorylation of GluA1 at Ser845 is also critical to
LTP. Intracellular perfusion of PKA into HEK 293 cells transfected with GluA1 led to a
40% potentiation of whole cell glutamate current peak amplitude, 51
likely by increasing
open-channel probability. 52
This potentiation effect was lost when Ser845 was mutated to
alanine. Site-directed mutagenesis and pharmacological studies indicate that this
phosphorylation is necessary but not sufficient for GluA1 membrane insertion during
LTP. 53
LTP was diminished in mice with mutated knock-in Ser381 and Ser845 sites that
resist phosphorylation. 54
Protein Kinase C (PKC) has also been shown to phosphorylate
Ser818 of GluA1, a modification that is implicated in synaptic trafficking during LTP. 55
Long-Term Depression
As downtrodden as this molecular process sounds, it is actually critical to synaptic
plasticity and rather something to be upbeat about. LTD is the means by which a
postsynaptic neuron becomes desensitized to input from the presynaptic neuron,
putatively by means of NMDAR-dependent AMPA receptor endocytosis. 56,57
The state is
induced by low frequency stimulus (1 Hz) over an extended period of time. Most studies
take place in hippocampal sections in vitro, so it remains a challenge determining how
the mechanism actually works in vivo. Heynen et al., however, have demonstrated that
hippocampal AMPAR postsynaptic membrane surface expression increased with LTP
and decreased with LTD in vivo. 58
Insulin plays a role in LTD, by activating phosphatidylinositol 3-kinase (PI3K)
and protein kinase B (PKB) via an NMDAR-dependent mechanism, 59
as well as by a
distinct AMPAR sorting pathway to specialized endosomes. 60
LTD is dependent upon
calcium influx and the activation of the phosphatase calcineurin. 61
For hippocampal LTD,
Ser845, the PKA site on GluA1, is dephosphorylated. Mice with this site mutated exhibit
deficits of LTD and NMDAR-induced AMPAR internalization. 62
In the cerebellum,
chemical activation of PKC was sufficient to phosphorylate Ser880 of GluA2 and induce
LTD and AMPAR internalization. 63
Interestingly, PKC does not appear to mediate the
phosphorylation of Ser880 in the hippocampus, indicating that this requirement is
satisfied by other kinases. 64
17. 11
Homeostatic Scaling
Chronic excitation or inhibition leads to compensatory mechanisms in cultured
neurons. Raising activity by blocking inhibitory synaptic transmission markedly
decreased the synaptic AMPAR population and reduced the EPSC. This also applied to
NMDARs. 65
Likewise, the chronic administration of AMPAR antagonists for hours to
days at a time increased the synaptic AMPAR population. 66
Both TNF-! and the immediate early gene Arc have been implicated in this
homeostatic process. By administering tetrodotoxin (TTX), a voltage-gated Na+
channel
blocker, glutamate release can be suppressed. This low level of glutamate causes glia to
release TNF-!, which upregulates AMPARs through an unknown mechanism. 67
Arc acts
as a sensor of neural activity, augmenting endocytosis when activity is elevated.
Conversely, blocking neural activity increases Arc levels and the AMPAR surface
population. Arc overexpression nullifies the gains in AMPARs that normally
accompanies TTX-induced activity reduction, and Arc hippocampal cell culture
knockouts exhibit no compensatory scaling in either direction. 68
This compensatory
mechanism poses an obstacle for the development of sustainable drug therapy. 69
Furthermore, any therapy must be precise enough to distinguish between constitutive and
regulated AMPA trafficking. The activity-independent constitutive pathway, typically of
GluA2/3 bearing receptors, maintains the total number of AMPARs and is thought to
replace damaged receptors and preserve newly formed memories. The regulated pathway
traffics predominantly GluA1-containing AMPARs, is inactive under basal conditions
and is activated by LTP. 70
Neuropathology
AMPARs are putatively involved in many diseases, including X-linked mental
retardation, Alzheimer’s disease, amyotrophic lateral sclerosis, limbic encephalitis,
epilepsy, ischemic brain injury, and Rasmussen’s encephalitis. 71, 72, 73, 74, 75
Given that Alzheimer’s dementia (AD) threatens to exacerbate the looming
demographic-healthcare crisis, the role of AMPAR dysfunction in AD deserves special
attention. An estimated 3.4 million people are affected by dementia in the United States.
18. 12
76
The elderly population (aged over 65) is expected to double by 2030, reaching 72
million, or 20% of the total U.S. population. 77
A similar shift awaits the developed world.
AD is characterized by the progressive loss of neural function, culminating in cell
death and impaired cognition. The fundamental etiology of AD remains controversial,
though there seem to be certain histological hallmarks including "-amyloid (A"42),
neurofibriliary tangles composed of hyperphosphorylated tau, inflammation, oxidative
stress, adrenergic and cholerinergic deficits. 78 79
A novel disease model even implicates
insulin resistance – the “type 3 diabetes” hypothesis. 80
Figure 9: Suspected Pathological Molecules in Alzheimer’s Dementia. A"42 can exist as intercellular
monomers, inhibiting the proteasome, as well as in the more familiar form of intracellular plaques. 81
Whether A" or tau are by-products of another underlying mechanism, or whatever
precise ratio of causal factors, AMPARs are intimately involved. A" secretion adversely
affects LTP and depresses both AMPAR and NMDAR currents in cultured slices. 82, 83
Furthermore, A" has proven capable of phosphorylating AMPARs at Ser880, signaling
LTD and endocytosis, indicating that A" may be responsible for synaptic depression,
AMPAR withdrawal, and dendritic spine loss. 84
The loss of spines and synaptic
19. 13
sensitivity presages cognitive impairment, and safe, effective methods of preserving
AMPAR function and neural integrity are certainly called for.
Pharmacology
Agonists
Each AMPAR has four glutamate binding sites, putatively formed by the
extracellular N-tail and the extracellular loop between transmembrane domains three and
four. The channel opens when two sites are bound, and as more sites are bound, more
current flows through the ionopore. 85
Full agonists are almost exclusively used to induce lesions for ablation
experiments. They include various AMPA analogues, williardiine analogues (found in
Mimosa bark), 86
domoic acid (culprit in amnesic shellfish poisoning and algal bloom
toxicity), 87
and quisqualic acid, an L-glutamic acid analogue found in Combretum
indicum (traditionally used as an antihelmintic). These few compounds that have been
characterized thus far are simply too potent for therapeutic use. (See Appendix for IC50
values).
Antagonists
Antagonists have demonstrated much more clinical relevance. One excessively
well-characterized compound, ethanol, partially acts as an AMPAR antagonist. 88
Upregulation of AMPARs in alcoholics is implicated in hyperactive excitation during the
withdrawal period. 89
The drug acamprosate (N-acetyl homotaurine) acts as an antagonist
at NMDARs and an agonist at GABAA receptors. The drug is of questionable efficacy, 90
but future compounds may be more effective if they target AMPAR regulation as well. 91
There is important emerging evidence for the involvement of AMPARs in addiction. 92, 93
Other antagonists include L-theanine (found at high levels in tea), a putative
cognitive enhancer 94
and neuroprotective agent; 95
kynurenic acid, an endogenous
tryptophan analogue implicated in schizophrenia 96
and cognitive impairment; 97
as well
as the experimental receptor blockers CNQX and NBQX. There is also reason to believe
that norepinephrine inhibits AMPAR currents in all layers of the rat temporal cortex. 98
20. 14
The AMPAR antagonists perampanel and tezampanel have found clinical
application in the treatment of epilepsy and as neuroprotective agents after stroke,
traumatic brain injury and the attendant excitotoxicity. 99
Furthermore, tezampanel
demonstrates analgesic and anxiolytic activity in animals. 100
Allosteric Modulators
Both positive and negative allosteric modulators (AMs) provide greater precision
of control or “fine tuning” of potential therapies. These drugs are also more selective for
receptor subtypes because they bind less conserved allosteric regions (rather than merely
binding the glutamate site of the LBD or blocking the ion pore with varying affinities). 101
Furthermore, AMs are believed to impact receptor dynamics less, leading to
reduced withdrawal and tolerance formation because these drugs work with pre-existing
receptor populations. AMs generally work by modifying the time course of deactivation
and/or desensitization. These drugs do, however, affect neurotrophin expression – a fact
that even further augments excitement about therapeutic application. 102
Negative AMs (NAMs) have not been studied much, but they include
extracellular protons (via a pH sensing region on the LBD), 103
neurosteroids (like
sulfated pregnenolone analogues), 104
and unsaturated fatty acids like arachidonic acid. 105
More research in this area is certainly warranted.
Positive AMs (PAMs) have been studied more extensively, and they work in at
least two different ways: some slow the rate of desensitization following repeated ligand
binding to the LBD, while others obstruct the exit of the agonist from the LBD.
There are presently three structural classes of PAM:
Fig 10: Perampanel and tezampanel
21. 15
1. Pyrrolidinone and related piperidine compounds (e.g., aniracetam and CX614).
2. Benzothiadiazide compounds (e.g., cyclothiazide, diazoxide)
3. Biarylpropylsulfonamide compounds (e.g., PEPA, LY404,187).
Detailed crystallographic descriptions of these ligand-receptor interactions are
now being correlated with network circuitry and behavioral output. This literature is
quickly evolving and beyond the scope of this thesis. 106
What began with the -racetam family has expanded to encompass a host of other,
highly potent and selective compounds; for example, PEPA has one hundred times the
potency of aniracetam at the AMPAR in vitro. 107
PAMs are promising therapeutics for
dementia, major depression, ADHD, Parkinson’s, Huntington’s, ALS and other cognitive
disorders. Ailments characterized by neuron loss may be particularly amenable to PAM
treatment because several ampakines augment brain-derived neurotrophic factor (BDNF)
22. 16
expression in disease models. 108, 109, 110
Thus far, however, clinical trials for dementia
have been lackluster due to bioavailability and blood-brain barrier issues.
Figure 11: Locations of ligand binding for both AMPA and kainate receptors. 111
The ampakine farampator demonstrated an acute effect on short-term memory in
a preliminary clinical trial of healthy elderly. 112
One concern is that prolonged ampakine
use could downregulate synaptic AMPARs via compensatory homeostatic scaling.
Lauterborn et al found that continuous incubation of cultured hippocampal slices with
CX614 rapidly multiplied BDNF mRNA (over 3–12 h) but this was followed by a decline
to control values over the next 36 hours coupled with a comcomittant decline in
AMPARs at the synapse. 113
This finding portends a “crash.” Lauterborn, Gall, Lynch et
al addressed this concern, finding that CX614 can sustain increases in BDNF protein
without AMPAR downregulation when moderate doses were administered for three hour
intervals rather than continuous (24 hr) incubation of brain slices in the ampakine
solution (see data in appendix). 114
Deadwyler et al. demonstrated dramatic sleep-deprivation alleviating cognitive
effects of CX717 in non-human primates performing a delayed match-to-sample task.
Not only was performance augmented for 30-36 hour sleep-deprived monkeys (to the
point of removing impairment), but also CX717 significantly augmented performance of
well-rested monkeys in a dose dependent manner. Positron emission tomography
measures of regional cerebral metabolic rates for glucose (CMRglc) during the task
I
23. 17
revealed that activity in the PFC, dorsal striatum, and medial temporal lobe (including
hippocampus) was significantly enhanced by CX717. 115
As these data show, correct
matches were highly significantly increased (P < 0.001), and latency of response was
reduced by a similar magnitude.
Figure 12: Normally rested + vehicle vs. Normal + CX717 delayed match-to-
sample task. Animals treated with CX717 outperformed in both accuracy and
latency parameters. Sleep deprived animals administered CX717 outperformed
even those that were normally rested on both parameters. (N = 11) 115
24. 18
Figure 13: A) Dose-Response Data. CX717 is shown administered on consecutive sessions for nine
monkeys over three dose ranges (0.3–0.5 mg/kg, 0.8–1.0 mg/kg, and 1.5 mg/kg, IV). Each CX717 session
(C, arrows) was interspersed with a single normal vehicle (V) session. B) Monkeys were given 0.8 mg/kg
of CX717 midway of their sessions. C) Overall mean dose-effect relationship of CX717 on normal alert
DMS performance across monkeys (n = 9).
Fig 14: Glucose consumption PET scan. Brain Region
Abbreviations: Dorsal Prefrontal Cortex, Dorsal Striatum,
Thalamus, and Medial Temporal Lobe. 115
25. 19
AMPAR Subunit Characterization
Different permutations of subunits of the heterotetrameric AMPAR determine its
functional characteristics. There are efforts underway to develop antibodies that
selectively tag these receptors with subunit specificity with the expectation that novel
therapies will ultimately take advantage of differential subunit localization and activity in
disease states.
GluA2 receptors have been relatively easy to characterize because they exhibit a
signature inward rectifying current and are responsive to an array of polyamines and
toxins. The other three subtypes exhibit more subtle pharmacological and
electrophysiological signatures.
In Neuron, Lu et al in the lab of Roger Nicoll at UCSF published an innovative
single-cell knockout method that has yielded much greater insight into the precise subunit
composition at CA1-Shaffer pyramidal neuron synapses. 116
GluA4 is conspicuously
absent from their data, because although it is found in the brain, it has much lower
expression levels than the other three. 117
According to Lu et al, GluA4 does not play any
role in the AMPAR-mediated transmission in CA1 pyramidal neurons.
Figure 15: Dimer of dimer subunit ratios in rat Shaffer-CA1 pyramidal neuron synapses. GluA1/2
tetramers predominate both at the synapse (80%) and extrasynaptically (>90%), whereas GluA2/3 tetramers
compose only about 16% of synaptic AMPA receptors. 118
Subunit composition is no mere technical curiosity – it is a major mechanism by
which the brain regulates its level of excitation. For example, RNA editing replaces a
80%
26. 20
glutamine with an arginine at the TMD 607 residue of GluA2. This change accounts for
the calcium impermeability of GluA2 and its inwardly rectifying current properties
(letting more current in than out). Transgenic mice with impaired Q/R editing exhibit
epileptic seizures and die within two weeks after birth. 119, 120
Understanding this kind of
subtle change may yield profound dividends in human therapy.
Unaddressed Research Questions
Although great strides have been made, there are many questions that remain to be
answered with regard to AMPARs trafficking and cognition. Two prominent researchers
in the field, Jason Shepherd at the Picower Institute for Learning and Memory at MIT and
Richard Huganir at the Howard Hughes Medical Institute at Johns Hopkins, ask the
following questions in their comprehensive review: 1
“Many basic cell biological questions still remain to be addressed. What is
the role of locally synthesized receptors, and how are local translation and
mRNA trafficking regulated? How do receptors traffic in and out of the
entangled complex of proteins in the PSD? When does lateral diffusion
versus direct insertion/internalization occur at synapses?
In addition, a huge challenge remains to elucidate the role of AMPAR
trafficking in vivo, in terms of precise mechanisms, as well as determine
the role that these processes play in synaptic plasticity and behavior.
Information can be stored in the brain for years, yet AMPARs are highly
dynamic and have a metabolic half-life of only a couple of days. Therefore,
if AMPAR levels do determine synaptic strength, how can synaptic weights
be maintained for weeks, months, or years? Moreover, how do individual
synapses within a neuron know how many receptors it needs to maintain its
potentiated or depressed state?”
Research Question(s)
This experimental thesis set out to address the following questions: Does a newly
developed monoclonal antibody bind to AMPA receptors with subunit specificity and
under what conditions? If the antibody binds to the appropriate sites where AMPARs are
putatively found, and does so for numerous subunit permutations, then the antibody likely
demonstrates avidity for multiple AMPA receptor subtypes.
27. 21
The AMPAR 1/2/3 monoclonal antibody (mAb) functions as a sort of
experimental control – one can compare, for example, a GluA1-specific mAB to the
novel “pan-AMPA” (1/2/3) mAb. If, for whatever reason a researcher must visualize all
of the predominant AMPAR subunits of the CNS, this mAb would also serve that
purpose.
These questions were addressed using double-label immunohistochemistry,
confocal laser scanning microscopy and electron microscopy. These studies were
conducted in accordance with the methods of Morrison et al. 2005. 121
28. 22
Methods and Materials
Expression of GluA1 on a plasmid vector in transformed E. Coli.
GluA1 subunit with an N-terminally fused SUMO protein by the lab of Dr.
Thomas Moran, Director Microbiology Department, Icahn School of Medicine at Mount
Sinai. The entire GluR1 receptor subunit has a molecular weight of 106 kD (Milipore) to
101 kD (PhosphoSitePlus). Listed below is a portion of the extracellular N-terminal
domain putatively containing antigenic target portion of the GluR1, fused with a SUMO
peptide in bold. The molecular weight of this 376 peptide subsequence after the SUMO
protein has been cleaved is 43kD. Including the SUMO protein, it weighs 48 kD.
MGSSHHHHHHSSGLVPRGSHMASMSDSEVNQEAKPEVKPEVKPETHINLKV
SDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL
DMEDNDIIEAHREQIGGSNFPNNIQIGGLFPNQQSQEHAAFRFALSQLTEPPKLL
PQIDIVNISDSFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTSFCGALHVCFITPSF
PVDTSNQFVLQLRPELQDALISIIDHYKWQKFVYIYDADRGLSVLQKVLDTAAEK
NWQVTAVNILTTTEEGYRMLFQDLEKKKERLVVVDCESERLNAILGQIIKLEKNG
IGYHYILANLGFMDIDLNKFKESGANVTGFQLVNYTDTIPAKIMQQWKNSDARD
HTRVDWKRPKYTSALTYDGVKVMAEAFQSLRRQRIDISRRGNAGDCLANPAVP
WGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTNYTLHVIEMKHDGIRKIGYWNE
DDKFVPAATD
The clonal line of mAb producing B-cells was expanded with hybridoma
technology in the lab of Dr. Thomas Moran. The avidity of the 5 µg/mL GluA mAb was
verified using a Western blot by sodium dodecyl sulfate polyacrylamide gel
29. 23
electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and visualized
with anti-mouse secondary antibody conjugated with I125
.
Transcardial Perfusion and Brain Acquisition
The brains of six adult Sprague-Dawley rats and one macaque monkey (Macaca
fascicularis) were acquired by transcardial perfusion. The rats were were deeply
anesthetized with a lethal dose of chloral hydrate (300 mg/kg) and the monkeys were
deeply anesthetized with ketamine (25 mg/kg) and Nembutal (30 mg/kg). The animals
were transcardially perfused with cold 1% paraformaldehyde in PBS, followed with cold
4% paraformaldehyde and 0.125% glutaraldehyde in phosphate buffered solution (pH
7.4).
All experiments were performed according to the NIH guidelines for research on
vertebrate animals and the Institutional Animal Care and Use Committee (IACUC) at the
Mount Sinai School of Medicine approved all protocols. The brains were immediately
postfixed in 4% paraformaldehyde for an additional 6 h at 48° C. The brains were then
cut into 50 micron sections on a Vibratome and preserved in 1% sodium azide. The
human brain was accessed via Dr. Patrick Hof’s research program.
Immunohistochemistry
A subset of sections underwent antigen retrieval, a process that breaks aldehyde
crosslinks and renders the target antigens more “visible” to the antibody. The sections
were rinsed in 37 ° C 200 mL dH2O for 5 minutes followed by 6.5 minutes in 100 mL of
0.2M HCl and 1 mL pepsin. This was followed by three washes in RT phosphate
buffered solution.
30. 24
A blocking solution was used in order to reduce non-specific mAb binding. The
solution was made up of 5% bovine serum albumin, 0.2% cold water fish skin gelatin,
and 0.3% Triton-X100 permeabilizing agent in phosphate buffered solution. The same
protocol was followed for the primary (polyclonal GluA2/3 or monoclonal GluA1/2/3)
and secondary Ab (1:400 diluted anti-Mouse Alexa 488 and anti-Rabbit 555) double-
label incubations. The sections then were mounted on microscope slides, stained with
Vectashield and DAPI, dried and cover-slipped.
Confocal and Electron Microscopy
Immunofluorescence of anti-Mouse Alexa 488 and anti-Rabbit 555 secondary
fluorophores were analyzed and imaged using a Zeiss LSM 410 inverted laser scanning
confocal microscope equipped with a ArKr 488/568 laser and Zeiss Plan-Neofluar
objectives (Zeiss, Oberkochen, Germany). A colocalization correlation was generated
using built-in Zeiss analysis software.
Ultrastructural analysis was performed using a Hitachi 7000 (Tokyo, Japan)
electron microscope. We used 10 nanometer immunogold-conjugated secondary
antibodies (preparation credit to Rishi Puri). We also conducted a dilution series in order
to determine the appropriate concentration for secondary antibody, using 2.5, 5, and 10
µg/mL. All images were prepared using Adobe Photoshop 7.
31. 25
Results
Characterization of anti-GluA monoclonal antibody
Fig 1. Western blot analysis demonstrated immunoreactivity to GluA subunits 1, 2 and 3,
but not 4. The antigen binding motif region was recognized at approximately molecular
weight 48 kD. The control lane was using mock-transfected E. coli lysate supernatant.
Single Label Confocal Microscopy
Figure 2. Non-antigen retrieved rat prefrontal cortex stained with monoclonal GluA1/2/3
antibody. Roman numerals denote layers of the neocortex. Ab binding was nonspecific.
32. 26
Figure 3. Non-antigen retrieved rat
hippocampus and stained with
monoclonal GluA1/2/3. On the right
is a magnified image of CA1 and the
dentate gyrus.
26
33. 27
Figure 4. Human Prefrontal Cortex with antigen retrieval. The section was stained with
DAPI and GluA1/2/3. The binding pattern is puntate, indicating greater specificity.
34. 28
Double Label Confocal Microscopy
Figure 5. Antigen retrieved human prefrontal cortex stain of DAPI, GluA1/2/3 and Post
Synaptic Density-95. Yellow punctae denote the sum of GluA1/2/3 and PSD-95,
indicating a high degree of colocalization at the postsynaptic density.
35. Human Hippocampus
GluA1/2/3 + PSD-95
Monkey Hippocampus
GluA1/2/3 + PSD-95
Rat Hippocampus
GluA1/2/3 + PSD-95
Figure 6. Double labeling with GluA1/2/3 and PSD-95 in
human, monkey and rat hippocampus. Yellow denotes
colocalization.
29
37. 31
Figure 8. Colocalization plot of the monoclonal GluA1/2/3 and GluA2/3 showing
moderate colocalization. Generated using Zeiss Zen built-in colocalization correlation
function.
38. Figure 9. Electron microscopy of non-human primate (macaque) hippocampus labeled
with 10 nm immunogold spheres conjugated to GluA1/2/3, which was used at a
concentration of 10 µg/mL. The antibody was successfully localized to the post synaptic
density of the dentritic spines.
Figure 10. Dilution series of 2.5, 5, and 10 µg/mL immunogold nanospheres
conjugated to monoclonal GluA1/2/3 antibody.
32
39. 33
Discussion
The novel monoclonal antibody binds to the ionotropic glutamatergic AMPA
receptor subunits 1, 2, and 3. Confocal laser scanning microscopy showed that the
antibody forms discrete punctae in the neuropil alone and when colocalized with PSD-95,
validating expected synaptic presence. The electron microscopic studies display a precise
localization of gold particles at the postsynaptic density synapse, likely in synthetic pools
within spines as well as stabilized on the membrane. Antigen retrieval is strongly advised
to augment antigen specificity. The fact that the antibody does not bind GluA4 is
acceptable because this subunit does not appear to play any major role in AMPA receptor
trafficking.
The antibody does not colocalize with the polyclonal GluA 2/3 to a convincing
degree, this dysfunction may be due to time-dependent storage degradation of the
GluR2/3 antibody. A follow-up experiment is currently underway.
The monoclonal antibody is functional, particularly with antigen retrieval,
between 5 to 10 µg/mL, pending confirmation with more confocal and EM double-
labeling with different sized nanogold spheres. Upon further validation, this antibody will
be ready for the market and use by neuroscientists all over the world. Hopefully this
novel antibody will provide a higher degree of subunit-specific granularity for
experimentalists and will expedite the development of therapies that attenuate epilepsy,
dementia, amylotrophic lateral sclerosis and other neuropathologies associated with
deranged AMPA receptor dynamics.
40. 34
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Appendix
Appendix Figure 1
Adapted From Lauterborn et al. 114
: “Spaced ampakine treatments sustain elevated BDNF protein content
without down-regulating AMPAR expression.
A) Hippocampal slices were treated with CX614 for varied intervals (1 – 6 hr) and harvested 24 h after
treatment onset.
B) Bar graphs show group mean ± S.E.M. in situ hybridization labeling densities for GluR1 mRNA in str.
granulosum and CA1 str. pyramidale. With CX614 at 50 µM (light bars), labeling was unaffected through 3
h but was lower than control values after 5 h treatments (*p < 0.05, **p < 0.01, ***p < 0.001 vs. con, SNK).
CX614 at 25 µM (dark bars) did not affect GluR1 mRNA levels.
C) Slices were treated with 50 µM CX614 for 3 h on four successive days; slices were collected daily (i) at
the end of treatment for GluR1 mRNA analysis (D) or (ii) at the end of the 24 h period for ELISA measures
of BDNF protein (E).
D) Bar graph shows that GluR1 mRNA levels in str. granulosum (SG) remained at control values through
treatment day 4 [at 50 µM]
E) Plot of protein measures shows that total slice BDNF protein content was elevated at the end of the first
day of treatment and remained elevated through treatment day 4 (**p < 0.01 vs. Con group) whereas GluR1
protein levels were unchanged throughout treatment. Mean ± S.E.M. values shown for n ! 8/group.”
50. 44
The following tables are adapted from Traynelis et al. 111
:
Carboxyl-terminal protein binding partners for AMPAR subunits
Post-Translational Modifications of C-terminal Domains of GluA
51. 45
AMPAR Subunit Agonists at Micromolar Concentrations
The Douglas Adams Equation and Secret to Universe Cube Matrix (Sum 3 x 3).
(Congratulations for Finishing the Paper)