Molecular Characterization of Native and Recom­binant Ionotrophic Glutamate Receptors Expressed in Neurons and Heterologous Systems

Kim Ryun Drasbek

Molecular Characterization of Native and Recombinant Ionotrophic Glutamate Receptors Expressed in Neurons and Heterologous Systems

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Abstract

(RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid receptors (AMPARs) are assembled from four subunits, GluR1-GluR4, of which GluR2 greatly influences receptor properties and has been coupled to activity dependent neurodegeneration. The GluR2 subunit plays a major role in receptor trafficking mediating the continuous replacement of synaptic receptors and is important for receptor tetramerization in the endoplasmatic reticulum. Given the many important properties of the GluR2 subunit, it was of great interest to investigate and compare synaptic properties in neuronal populations expressing AMPARs with or without the GluR2 subunits. Earlier findings suggested that neurons cultured from spinal cord were devoid of GluR2 and expressed high amounts of GluR4. In contrast, GluR2 was detected in almost all cells from cortical cultures (Dai et al., 2001). To investigate differences in synaptic currents of receptors from these neuronal preparations, miniature excitatory postsynaptic currents (mEPSCs) were recorded followed by single cell RT-PCR of the same neuron. Unfortunately, no population of GluR2 lacking neurons was detected by single cell RT-PCR, but a higher detection frequency of GluR3 in cortical neurons was found, while GluR4 was more abundant in spinal cord neurons. Developmental changes of AMPA receptor subunit expression was investigated in an attempt to explain these contrasting findings. A decrease in the presence of the GluR4 subunit for older embryos as well as for longer days in vitro(DIV) was found. However, the GluR2 subunit was detected in about half of the neurons regardless of culture type, embryonic age, and DIV. Analysis of the kinetics of detected mEPSCs showed no correlation to GluR1-GluR4 mRNA levels estimated by single cell RT-PCR. To create a pool of GluR2 negative neurons, the newly described method of acute knock down of specific mRNAs, RNA interference (RNAi), was established in the laboratory and shown to work in cultured primary neurons. The effectiveness of seven plasmids expressing short hairpin RNAs (shRNAs), targeting the GluR2 mRNA, was estimated in BHK cells expressing GluR2. Quantitative RT-PCR on purified RNA showed that all of the constructs down regulated GluR2 mRNA two and three days after transfection, while a mutated shRNA did not. Knockdown of GluR2 protein expression was estimated by immunohistochemistry, which showed that all but one construct mediated GluR2 protein knockdown four days after transfection. Due to the low transfection efficiency of cultured primary neurons, GluR2 knockdown was estimated at the single cell level. Rectification properties in transfected neurons were analyzed followed by single cell RTPCR. No inward rectification was seen in control neurons, while several neurons transfected with the same construct showed inward rectification, indicating GluR2 knockdown in surface expressed AMPARs. In addition, because no mEPSCs were observed at positive holding potentials in these neurons, synaptic AMPARs seemingly lacked GluR2. 1-naphthyl acetyl spermine (Naspm) selectively block AMPARs lacking GluR2 and was seen to reduced responses to exogenously applied AMPA in some transfected neurons. However, single cell RT-PCR still detected GluR2 mRNA in some of the neurons transfected with the knockdown constructs. Therefore, a larger number of transfected neurons for each construct have to be investigated by single cell RT-PCR and patch clamp recordings before final conclusions can be drawn. The ability to down regulate GluR2 mRNA enables the study of neuronal responses to the expected sudden loss of GluR2 in regard to synaptic currents and receptor trafficking. The RNA interference technique is effective in primary neuronal cultures and can readily be employed in knockdown of other components of the AMPAR trafficking pathway. To increase the number of affected neurons, lentiviral mediated gene delivery have been shown to be very effective and would facilitate the analysis of effective gene knockdown.

Original language	English

Place of publication	Det Sundhedsvidenskabelige Fakultet, Aarhus universitet
Number of pages	97
Publication status	Published - 2005

Keywords

Ionotropic glutamate receptor
GluR2
AMPA receptor
Single cell RT-PCR
RNA interference

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Cite this

@phdthesis{75e62b301d7511dcbee902004c4f4f50,

title = "Molecular Characterization of Native and Recombinant Ionotrophic Glutamate Receptors Expressed in Neurons and Heterologous Systems",

abstract = "(RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid receptors (AMPARs) are assembled from four subunits, GluR1-GluR4, of which GluR2 greatly influences receptor properties and has been coupled to activity dependent neurodegeneration. The GluR2 subunit plays a major role in receptor trafficking mediating the continuous replacement of synaptic receptors and is important for receptor tetramerization in the endoplasmatic reticulum. Given the many important properties of the GluR2 subunit, it was of great interest to investigate and compare synaptic properties in neuronal populations expressing AMPARs with or without the GluR2 subunits. Earlier findings suggested that neurons cultured from spinal cord were devoid of GluR2 and expressed high amounts of GluR4. In contrast, GluR2 was detected in almost all cells from cortical cultures (Dai et al., 2001). To investigate differences in synaptic currents of receptors from these neuronal preparations, miniature excitatory postsynaptic currents (mEPSCs) were recorded followed by single cell RT-PCR of the same neuron. Unfortunately, no population of GluR2 lacking neurons was detected by single cell RT-PCR, but a higher detection frequency of GluR3 in cortical neurons was found, while GluR4 was more abundant in spinal cord neurons. Developmental changes of AMPA receptor subunit expression was investigated in an attempt to explain these contrasting findings. A decrease in the presence of the GluR4 subunit for older embryos as well as for longer days in vitro(DIV) was found. However, the GluR2 subunit was detected in about half of the neurons regardless of culture type, embryonic age, and DIV. Analysis of the kinetics of detected mEPSCs showed no correlation to GluR1-GluR4 mRNA levels estimated by single cell RT-PCR. To create a pool of GluR2 negative neurons, the newly described method of acute knock down of specific mRNAs, RNA interference (RNAi), was established in the laboratory and shown to work in cultured primary neurons. The effectiveness of seven plasmids expressing short hairpin RNAs (shRNAs), targeting the GluR2 mRNA, was estimated in BHK cells expressing GluR2. Quantitative RT-PCR on purified RNA showed that all of the constructs down regulated GluR2 mRNA two and three days after transfection, while a mutated shRNA did not. Knockdown of GluR2 protein expression was estimated by immunohistochemistry, which showed that all but one construct mediated GluR2 protein knockdown four days after transfection. Due to the low transfection efficiency of cultured primary neurons, GluR2 knockdown was estimated at the single cell level. Rectification properties in transfected neurons were analyzed followed by single cell RTPCR. No inward rectification was seen in control neurons, while several neurons transfected with the same construct showed inward rectification, indicating GluR2 knockdown in surface expressed AMPARs. In addition, because no mEPSCs were observed at positive holding potentials in these neurons, synaptic AMPARs seemingly lacked GluR2. 1-naphthyl acetyl spermine (Naspm) selectively block AMPARs lacking GluR2 and was seen to reduced responses to exogenously applied AMPA in some transfected neurons. However, single cell RT-PCR still detected GluR2 mRNA in some of the neurons transfected with the knockdown constructs. Therefore, a larger number of transfected neurons for each construct have to be investigated by single cell RT-PCR and patch clamp recordings before final conclusions can be drawn. The ability to down regulate GluR2 mRNA enables the study of neuronal responses to the expected sudden loss of GluR2 in regard to synaptic currents and receptor trafficking. The RNA interference technique is effective in primary neuronal cultures and can readily be employed in knockdown of other components of the AMPAR trafficking pathway. To increase the number of affected neurons, lentiviral mediated gene delivery have been shown to be very effective and would facilitate the analysis of effective gene knockdown.",

keywords = "Ionotrof glutamat receptor, GluR2, AMPA receptor, Enkel celle RT-PCR, RNA interferens, Ionotropic glutamate receptor, GluR2, AMPA receptor, Single cell RT-PCR, RNA interference",

author = "Drasbek, {Kim Ryun}",

year = "2005",

language = "English",

}

TY - BOOK

T1 - Molecular Characterization of Native and Recombinant Ionotrophic Glutamate Receptors Expressed in Neurons and Heterologous Systems

AU - Drasbek, Kim Ryun

PY - 2005

Y1 - 2005

N2 - (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid receptors (AMPARs) are assembled from four subunits, GluR1-GluR4, of which GluR2 greatly influences receptor properties and has been coupled to activity dependent neurodegeneration. The GluR2 subunit plays a major role in receptor trafficking mediating the continuous replacement of synaptic receptors and is important for receptor tetramerization in the endoplasmatic reticulum. Given the many important properties of the GluR2 subunit, it was of great interest to investigate and compare synaptic properties in neuronal populations expressing AMPARs with or without the GluR2 subunits. Earlier findings suggested that neurons cultured from spinal cord were devoid of GluR2 and expressed high amounts of GluR4. In contrast, GluR2 was detected in almost all cells from cortical cultures (Dai et al., 2001). To investigate differences in synaptic currents of receptors from these neuronal preparations, miniature excitatory postsynaptic currents (mEPSCs) were recorded followed by single cell RT-PCR of the same neuron. Unfortunately, no population of GluR2 lacking neurons was detected by single cell RT-PCR, but a higher detection frequency of GluR3 in cortical neurons was found, while GluR4 was more abundant in spinal cord neurons. Developmental changes of AMPA receptor subunit expression was investigated in an attempt to explain these contrasting findings. A decrease in the presence of the GluR4 subunit for older embryos as well as for longer days in vitro(DIV) was found. However, the GluR2 subunit was detected in about half of the neurons regardless of culture type, embryonic age, and DIV. Analysis of the kinetics of detected mEPSCs showed no correlation to GluR1-GluR4 mRNA levels estimated by single cell RT-PCR. To create a pool of GluR2 negative neurons, the newly described method of acute knock down of specific mRNAs, RNA interference (RNAi), was established in the laboratory and shown to work in cultured primary neurons. The effectiveness of seven plasmids expressing short hairpin RNAs (shRNAs), targeting the GluR2 mRNA, was estimated in BHK cells expressing GluR2. Quantitative RT-PCR on purified RNA showed that all of the constructs down regulated GluR2 mRNA two and three days after transfection, while a mutated shRNA did not. Knockdown of GluR2 protein expression was estimated by immunohistochemistry, which showed that all but one construct mediated GluR2 protein knockdown four days after transfection. Due to the low transfection efficiency of cultured primary neurons, GluR2 knockdown was estimated at the single cell level. Rectification properties in transfected neurons were analyzed followed by single cell RTPCR. No inward rectification was seen in control neurons, while several neurons transfected with the same construct showed inward rectification, indicating GluR2 knockdown in surface expressed AMPARs. In addition, because no mEPSCs were observed at positive holding potentials in these neurons, synaptic AMPARs seemingly lacked GluR2. 1-naphthyl acetyl spermine (Naspm) selectively block AMPARs lacking GluR2 and was seen to reduced responses to exogenously applied AMPA in some transfected neurons. However, single cell RT-PCR still detected GluR2 mRNA in some of the neurons transfected with the knockdown constructs. Therefore, a larger number of transfected neurons for each construct have to be investigated by single cell RT-PCR and patch clamp recordings before final conclusions can be drawn. The ability to down regulate GluR2 mRNA enables the study of neuronal responses to the expected sudden loss of GluR2 in regard to synaptic currents and receptor trafficking. The RNA interference technique is effective in primary neuronal cultures and can readily be employed in knockdown of other components of the AMPAR trafficking pathway. To increase the number of affected neurons, lentiviral mediated gene delivery have been shown to be very effective and would facilitate the analysis of effective gene knockdown.

AB - (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid receptors (AMPARs) are assembled from four subunits, GluR1-GluR4, of which GluR2 greatly influences receptor properties and has been coupled to activity dependent neurodegeneration. The GluR2 subunit plays a major role in receptor trafficking mediating the continuous replacement of synaptic receptors and is important for receptor tetramerization in the endoplasmatic reticulum. Given the many important properties of the GluR2 subunit, it was of great interest to investigate and compare synaptic properties in neuronal populations expressing AMPARs with or without the GluR2 subunits. Earlier findings suggested that neurons cultured from spinal cord were devoid of GluR2 and expressed high amounts of GluR4. In contrast, GluR2 was detected in almost all cells from cortical cultures (Dai et al., 2001). To investigate differences in synaptic currents of receptors from these neuronal preparations, miniature excitatory postsynaptic currents (mEPSCs) were recorded followed by single cell RT-PCR of the same neuron. Unfortunately, no population of GluR2 lacking neurons was detected by single cell RT-PCR, but a higher detection frequency of GluR3 in cortical neurons was found, while GluR4 was more abundant in spinal cord neurons. Developmental changes of AMPA receptor subunit expression was investigated in an attempt to explain these contrasting findings. A decrease in the presence of the GluR4 subunit for older embryos as well as for longer days in vitro(DIV) was found. However, the GluR2 subunit was detected in about half of the neurons regardless of culture type, embryonic age, and DIV. Analysis of the kinetics of detected mEPSCs showed no correlation to GluR1-GluR4 mRNA levels estimated by single cell RT-PCR. To create a pool of GluR2 negative neurons, the newly described method of acute knock down of specific mRNAs, RNA interference (RNAi), was established in the laboratory and shown to work in cultured primary neurons. The effectiveness of seven plasmids expressing short hairpin RNAs (shRNAs), targeting the GluR2 mRNA, was estimated in BHK cells expressing GluR2. Quantitative RT-PCR on purified RNA showed that all of the constructs down regulated GluR2 mRNA two and three days after transfection, while a mutated shRNA did not. Knockdown of GluR2 protein expression was estimated by immunohistochemistry, which showed that all but one construct mediated GluR2 protein knockdown four days after transfection. Due to the low transfection efficiency of cultured primary neurons, GluR2 knockdown was estimated at the single cell level. Rectification properties in transfected neurons were analyzed followed by single cell RTPCR. No inward rectification was seen in control neurons, while several neurons transfected with the same construct showed inward rectification, indicating GluR2 knockdown in surface expressed AMPARs. In addition, because no mEPSCs were observed at positive holding potentials in these neurons, synaptic AMPARs seemingly lacked GluR2. 1-naphthyl acetyl spermine (Naspm) selectively block AMPARs lacking GluR2 and was seen to reduced responses to exogenously applied AMPA in some transfected neurons. However, single cell RT-PCR still detected GluR2 mRNA in some of the neurons transfected with the knockdown constructs. Therefore, a larger number of transfected neurons for each construct have to be investigated by single cell RT-PCR and patch clamp recordings before final conclusions can be drawn. The ability to down regulate GluR2 mRNA enables the study of neuronal responses to the expected sudden loss of GluR2 in regard to synaptic currents and receptor trafficking. The RNA interference technique is effective in primary neuronal cultures and can readily be employed in knockdown of other components of the AMPAR trafficking pathway. To increase the number of affected neurons, lentiviral mediated gene delivery have been shown to be very effective and would facilitate the analysis of effective gene knockdown.

KW - Ionotrof glutamat receptor

KW - GluR2

KW - AMPA receptor

KW - Enkel celle RT-PCR

KW - RNA interferens

KW - Ionotropic glutamate receptor

KW - GluR2

KW - AMPA receptor

KW - Single cell RT-PCR

KW - RNA interference

M3 - Ph.D. thesis

BT - Molecular Characterization of Native and Recombinant Ionotrophic Glutamate Receptors Expressed in Neurons and Heterologous Systems

CY - Det Sundhedsvidenskabelige Fakultet, Aarhus universitet

ER -