G protein‐gated inwardly‐rectifying K+ (GIRK/family 3 of inwardly‐rectifying K+) channels are cou... more G protein‐gated inwardly‐rectifying K+ (GIRK/family 3 of inwardly‐rectifying K+) channels are coupled to neurotransmitter action and can play important roles in modulating neuronal excitability. We investigated the temporal and spatial expression of GIRK1, GIRK2 and GIRK3 subunits in the developing and adult brain of mice and rats using biochemical, immunohistochemical and immunoelectron microscopic techniques. At all ages analysed, the overall distribution patterns of GIRK1‐3 were very similar, with high expression levels in the neocortex, cerebellum, hippocampus and thalamus. Focusing on the hippocampus, histoblotting and immunohistochemistry showed that GIRK1‐3 protein levels increased with age, and this was accompanied by a shift in the subcellular localization of the subunits. Early in development (postnatal day 5), GIRK subunits were predominantly localized to the endoplasmic reticulum in the pyramidal cells, but by postnatal day 60 they were mostly found along the plasma memb...
The histoblot (in situ immunoblotting) technique is a simple, reproducible, and sensitive method ... more The histoblot (in situ immunoblotting) technique is a simple, reproducible, and sensitive method for protein detection that allows both protein quantitation and analysis of tissue distribution. This easy and fast method allows the direct transfer of native proteins from unfixed frozen tissue sections by mechanical pressure to an immobilizing matrix. Proteins are directly blotted onto nitrocellulose membranes that are then immunolabelled similar to a Western blot, but the result is an immunohistochemical imprint of the section retaining all proteins. The histoblot combines advantages of western blot and immunohistochemical methods and yields optimal accessibility of proteins blotted on membranes whilst also preserving anatomical resolution. In addition, it avoids chemical modifications, crosslinking, or semi-denaturation of proteins, which can alter the access of antibody to epitopes, as introduced by conventional immunohistochemistry. Therefore, the histoblot often enables the use of antibodies that do not recognise the target protein in fixed tissue samples. This method has become a trusted alternative to reveal and compare the regional distribution and expression profile of different proteins in the brain in physiological and pathological conditions. In addition, the technique exhibits a high subregional resolution, although is not suitable to unravel protein distribution at the cellular and subcellular levels. In this review, we introduce the histoblot procedure used in our laboratory on brain sections for the identification of quantitative changes of neurotransmitter receptors, ion channels and other signalling molecules in the brain. We also discuss the potentialities, limitations, and fundamental principles of this technique.
G protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels are critical to brain function. The... more G protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels are critical to brain function. They hyperpolarize neurons in response to activation of different G protein-coupled receptors, reducing cell excitability. Molecular cloning has revealed four distinct mammalian genes (GIRK1-4), which, with the exception of GIRK4, are broadly expressed in the central nervous system (CNS) and have been implicated in a variety of neurological disorders. Although the molecular structure and composition of GIRK channels are key determinants of their biophysical properties, their cellular and subcellular localization patterns and densities on the neuronal surface are just as important to nerve function. Current data obtained with high-resolution quantitative localization techniques reveal complex, subcellular compartment-specific distribution patterns of GIRK channel subunits. Recent efforts have focused on determining the associated proteins that form macromolecular complexes with GIRK channels. Demonstration of the precise subcellular compartmentalization of GIRK channels and their associated proteins represents a crucial step in understanding the contribution of these channels to specific aspects of neuronal function under both physiological and pathological conditions. Here, we present an overview of studies aimed at determining the cellular and subcellular localization of GIRK channel subunits in mammalian brain neurons and discuss implications for neuronal physiology.
Tau pathology is a hallmark of Alzheimer’s disease (AD) and other tauopathies, but how pathologic... more Tau pathology is a hallmark of Alzheimer’s disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the...
ABSTRACTKufs disease/CLN4 is an autosomal dominant neurodegenerative disorder that affects young ... more ABSTRACTKufs disease/CLN4 is an autosomal dominant neurodegenerative disorder that affects young adults, caused by mutations in the DNAJC5 gene that encodes the synaptic vesicle co-chaperone Cysteine String Protein α (CSPα/DNAJC5). The Leu115Arg and Leu116Δ mutations in humans are known to independently cause the disease, although the underlying mechanisms are unknown. To investigate the disease mechanisms in vivo, we generated three independent mouse lines overexpressing different versions of CSPα/DNAJC5 under the neuron-specific Thy1 promoter: wild-type (WT), Leu115Arg, and Leu116Δ. Mice expressing mutant CSPα/DNAJC5 are viable and do not show any significant increase in morbidity or mortality. However, we observed the presence of pathological lipofuscinosis in the mutants, indicated by autofluorescent punctate structures labeled with antibodies against ATP synthase subunit C, which were absent in the WT transgenic line. Additionally, transmission electron microscopy revealed intr...
G protein‐gated inwardly‐rectifying K+ (GIRK/family 3 of inwardly‐rectifying K+) channels are cou... more G protein‐gated inwardly‐rectifying K+ (GIRK/family 3 of inwardly‐rectifying K+) channels are coupled to neurotransmitter action and can play important roles in modulating neuronal excitability. We investigated the temporal and spatial expression of GIRK1, GIRK2 and GIRK3 subunits in the developing and adult brain of mice and rats using biochemical, immunohistochemical and immunoelectron microscopic techniques. At all ages analysed, the overall distribution patterns of GIRK1‐3 were very similar, with high expression levels in the neocortex, cerebellum, hippocampus and thalamus. Focusing on the hippocampus, histoblotting and immunohistochemistry showed that GIRK1‐3 protein levels increased with age, and this was accompanied by a shift in the subcellular localization of the subunits. Early in development (postnatal day 5), GIRK subunits were predominantly localized to the endoplasmic reticulum in the pyramidal cells, but by postnatal day 60 they were mostly found along the plasma memb...
The histoblot (in situ immunoblotting) technique is a simple, reproducible, and sensitive method ... more The histoblot (in situ immunoblotting) technique is a simple, reproducible, and sensitive method for protein detection that allows both protein quantitation and analysis of tissue distribution. This easy and fast method allows the direct transfer of native proteins from unfixed frozen tissue sections by mechanical pressure to an immobilizing matrix. Proteins are directly blotted onto nitrocellulose membranes that are then immunolabelled similar to a Western blot, but the result is an immunohistochemical imprint of the section retaining all proteins. The histoblot combines advantages of western blot and immunohistochemical methods and yields optimal accessibility of proteins blotted on membranes whilst also preserving anatomical resolution. In addition, it avoids chemical modifications, crosslinking, or semi-denaturation of proteins, which can alter the access of antibody to epitopes, as introduced by conventional immunohistochemistry. Therefore, the histoblot often enables the use of antibodies that do not recognise the target protein in fixed tissue samples. This method has become a trusted alternative to reveal and compare the regional distribution and expression profile of different proteins in the brain in physiological and pathological conditions. In addition, the technique exhibits a high subregional resolution, although is not suitable to unravel protein distribution at the cellular and subcellular levels. In this review, we introduce the histoblot procedure used in our laboratory on brain sections for the identification of quantitative changes of neurotransmitter receptors, ion channels and other signalling molecules in the brain. We also discuss the potentialities, limitations, and fundamental principles of this technique.
G protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels are critical to brain function. The... more G protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels are critical to brain function. They hyperpolarize neurons in response to activation of different G protein-coupled receptors, reducing cell excitability. Molecular cloning has revealed four distinct mammalian genes (GIRK1-4), which, with the exception of GIRK4, are broadly expressed in the central nervous system (CNS) and have been implicated in a variety of neurological disorders. Although the molecular structure and composition of GIRK channels are key determinants of their biophysical properties, their cellular and subcellular localization patterns and densities on the neuronal surface are just as important to nerve function. Current data obtained with high-resolution quantitative localization techniques reveal complex, subcellular compartment-specific distribution patterns of GIRK channel subunits. Recent efforts have focused on determining the associated proteins that form macromolecular complexes with GIRK channels. Demonstration of the precise subcellular compartmentalization of GIRK channels and their associated proteins represents a crucial step in understanding the contribution of these channels to specific aspects of neuronal function under both physiological and pathological conditions. Here, we present an overview of studies aimed at determining the cellular and subcellular localization of GIRK channel subunits in mammalian brain neurons and discuss implications for neuronal physiology.
Tau pathology is a hallmark of Alzheimer’s disease (AD) and other tauopathies, but how pathologic... more Tau pathology is a hallmark of Alzheimer’s disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the...
ABSTRACTKufs disease/CLN4 is an autosomal dominant neurodegenerative disorder that affects young ... more ABSTRACTKufs disease/CLN4 is an autosomal dominant neurodegenerative disorder that affects young adults, caused by mutations in the DNAJC5 gene that encodes the synaptic vesicle co-chaperone Cysteine String Protein α (CSPα/DNAJC5). The Leu115Arg and Leu116Δ mutations in humans are known to independently cause the disease, although the underlying mechanisms are unknown. To investigate the disease mechanisms in vivo, we generated three independent mouse lines overexpressing different versions of CSPα/DNAJC5 under the neuron-specific Thy1 promoter: wild-type (WT), Leu115Arg, and Leu116Δ. Mice expressing mutant CSPα/DNAJC5 are viable and do not show any significant increase in morbidity or mortality. However, we observed the presence of pathological lipofuscinosis in the mutants, indicated by autofluorescent punctate structures labeled with antibodies against ATP synthase subunit C, which were absent in the WT transgenic line. Additionally, transmission electron microscopy revealed intr...
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Papers by Rafael Lujan