Final Report Summary - DENDRITIC PROCESSING (Local Processing of Dendritically Synthesized Membrane Proteins)
N-glycosylation – the sequential addition of complex sugars to membrane and secreted proteins as they progress through the endoplasmic reticulum and the Golgi apparatus – is one of the most frequent protein posttranslational modifications and regulates virtually every aspect of membrane protein biology. In mammals, most organ-specific N-glycosylation events occur in the brain. Yet, little is known about the nature, function and regulation of N-glycosylation in neurons.
Recent studies indicate that the dendrites of hippocampal neurons may contain up to hundreds of messenger RNAs encoding membrane proteins such as neurotransmitter receptors and voltage-gated ion channels, suggesting that most – if not all – types of dendritic proteins can be synthesized locally where they are needed. Intriguingly, only a subset of the compartments of the secretory pathway – the array of intracellular membranes where proteins of the cell-surface are synthesized and N-glycosylated – is found in dendrites. In particular, most dendrites lack generic Golgi membranes. As N-glycosylation normally occurs in sequential steps as protein progress through secretory compartments, we sought to determine whether the membrane proteins that are locally translated in dendrites may follow a distinct secretory pathway than those made in the soma, and, thereby, acquire a specific glycosylation status.
Our work shows that, as a result of unconventional secretory processing up to hundreds of surface-expressed neurotransmitter receptors, voltage-gated ion channels and synaptic adhesion proteins display atypical and insofar unrecognized glycosylation profiles that are typically associated with immature proteins before their export to the cell surface. This core-glycosylation is associated with a faster protein turnover and is regulated by synaptic activity, unraveling a novel mechanism controlling the electrical and chemical sensing properties of the neuronal membrane.
Another objective of the project was to develop a new transgenic mouse line to enable genetically targeted protein metabolic in the mammalian brain. Building up on tools that were developed in the lab, I designed and outsourced the generation of a knock-in mouse where the conditional expression of a mutant enzyme (methionyl tRNA synthetase L274G or MetRS*) enables the incorporation of non-canonical “tag” amino-acids in the proteins made by specific cells, that can hence be subsequently purified or visualized.
Our work shows that MetRS* expression enables targeted protein metabolic labeling for direct visualization in specific cells in complex tissues in vivo, and hopefully in the near future, to purify and characterize cell-specific proteomes for basic and clinical research.
Novelty and significance
Perhaps not surprisingly given the central importance of N-glycosylation in the biology of membrane and secreted proteins, congenital N-glycosylation defects, especially in brain, result in severe and usually lethal developmental disorders. Interestingly, some glutamate receptors and GABAA receptors display abnormally increased or decreased N-glycosylation profiles in schizophrenic patients. Our work challenges the notion that this merely reflects altered intracellular levels of immature receptors – as commonly assumed insofar - and rather suggests that these glycosylation defects impact functional synaptic receptors present on the plasma membrane.
Despite a general awareness that a better characterization of the N-glycosylation status of key pharmaceutical targets would be instrumental to design new drugs to alleviate hyperactivity and insomnia (GABA A receptors), depression (serotonine transporters), schizophrenia (NMDA receptors), autism (mGluR5), inflammation and neuropathic pain (cannabinoid receptors) etc, we know surprisingly little on the glycosylation of these proteins. While the huge variability of N-glycan structure hinders their identification and attribution to specific proteins by mass spectrometry in complex samples, we believe that our knowledge of what to look for (core-glycosylated profiles of specific proteins) and where (plasma membrane) will allow us to successfully determine the glycosylation profiles of candidate proteins.
In a broader context, it is worth noting that alterations in a number of glycosylation profiles are commonly used for clinical diagnosis, notably to determine cancer prognosis, and are thought to determine the drug resistance of some tumors. Yet, the cell biological bases for these changes are still completely unknown. Studying the unconventional glycosylation of neuronal surface membrane proteins thus has a conceptual and technical relevance that extends far beyond neurobiology. Most notably, an increased branching of complex N-glycans is typically associated with a poor prognosis for breast and colon cancers in humans. Consistently, studies in mice show that Golgi-associated glycosyltransferases such as N-GlcNac, sialyl- and fucosyl-transferases are instrumental to tumor invasiveness. The prevalence of core-glycosylated surface glycoproteins that we unrivalled in neurons may thus provide important insights on how N-glycans terminal branching is regulated and can be opposed.
Protein synthesis consumes a large amount of cell’s energy, requiring a tight control of which and how many proteins are being synthesized at a given time. In the brain, protein expression is directly regulated by neuronal activity, enabling dynamic changes of neuron proteome during learning and memory formation - a process that is disrupted in pathologies such as autism. In the recent years, non-radioactive strategies have been developed by the Schuman lab and other groups to tag nascent proteins for imaging and/or biochemical detection. Yet, there is still a need for techniques to selectively label, visualize and purify proteins made in specific cell types in vivo. The new transgenic mouse that we developed thus represents a technical advance that will be of interest for many researchers inside and outside the field.
Recent studies indicate that the dendrites of hippocampal neurons may contain up to hundreds of messenger RNAs encoding membrane proteins such as neurotransmitter receptors and voltage-gated ion channels, suggesting that most – if not all – types of dendritic proteins can be synthesized locally where they are needed. Intriguingly, only a subset of the compartments of the secretory pathway – the array of intracellular membranes where proteins of the cell-surface are synthesized and N-glycosylated – is found in dendrites. In particular, most dendrites lack generic Golgi membranes. As N-glycosylation normally occurs in sequential steps as protein progress through secretory compartments, we sought to determine whether the membrane proteins that are locally translated in dendrites may follow a distinct secretory pathway than those made in the soma, and, thereby, acquire a specific glycosylation status.
Our work shows that, as a result of unconventional secretory processing up to hundreds of surface-expressed neurotransmitter receptors, voltage-gated ion channels and synaptic adhesion proteins display atypical and insofar unrecognized glycosylation profiles that are typically associated with immature proteins before their export to the cell surface. This core-glycosylation is associated with a faster protein turnover and is regulated by synaptic activity, unraveling a novel mechanism controlling the electrical and chemical sensing properties of the neuronal membrane.
Another objective of the project was to develop a new transgenic mouse line to enable genetically targeted protein metabolic in the mammalian brain. Building up on tools that were developed in the lab, I designed and outsourced the generation of a knock-in mouse where the conditional expression of a mutant enzyme (methionyl tRNA synthetase L274G or MetRS*) enables the incorporation of non-canonical “tag” amino-acids in the proteins made by specific cells, that can hence be subsequently purified or visualized.
Our work shows that MetRS* expression enables targeted protein metabolic labeling for direct visualization in specific cells in complex tissues in vivo, and hopefully in the near future, to purify and characterize cell-specific proteomes for basic and clinical research.
Novelty and significance
Perhaps not surprisingly given the central importance of N-glycosylation in the biology of membrane and secreted proteins, congenital N-glycosylation defects, especially in brain, result in severe and usually lethal developmental disorders. Interestingly, some glutamate receptors and GABAA receptors display abnormally increased or decreased N-glycosylation profiles in schizophrenic patients. Our work challenges the notion that this merely reflects altered intracellular levels of immature receptors – as commonly assumed insofar - and rather suggests that these glycosylation defects impact functional synaptic receptors present on the plasma membrane.
Despite a general awareness that a better characterization of the N-glycosylation status of key pharmaceutical targets would be instrumental to design new drugs to alleviate hyperactivity and insomnia (GABA A receptors), depression (serotonine transporters), schizophrenia (NMDA receptors), autism (mGluR5), inflammation and neuropathic pain (cannabinoid receptors) etc, we know surprisingly little on the glycosylation of these proteins. While the huge variability of N-glycan structure hinders their identification and attribution to specific proteins by mass spectrometry in complex samples, we believe that our knowledge of what to look for (core-glycosylated profiles of specific proteins) and where (plasma membrane) will allow us to successfully determine the glycosylation profiles of candidate proteins.
In a broader context, it is worth noting that alterations in a number of glycosylation profiles are commonly used for clinical diagnosis, notably to determine cancer prognosis, and are thought to determine the drug resistance of some tumors. Yet, the cell biological bases for these changes are still completely unknown. Studying the unconventional glycosylation of neuronal surface membrane proteins thus has a conceptual and technical relevance that extends far beyond neurobiology. Most notably, an increased branching of complex N-glycans is typically associated with a poor prognosis for breast and colon cancers in humans. Consistently, studies in mice show that Golgi-associated glycosyltransferases such as N-GlcNac, sialyl- and fucosyl-transferases are instrumental to tumor invasiveness. The prevalence of core-glycosylated surface glycoproteins that we unrivalled in neurons may thus provide important insights on how N-glycans terminal branching is regulated and can be opposed.
Protein synthesis consumes a large amount of cell’s energy, requiring a tight control of which and how many proteins are being synthesized at a given time. In the brain, protein expression is directly regulated by neuronal activity, enabling dynamic changes of neuron proteome during learning and memory formation - a process that is disrupted in pathologies such as autism. In the recent years, non-radioactive strategies have been developed by the Schuman lab and other groups to tag nascent proteins for imaging and/or biochemical detection. Yet, there is still a need for techniques to selectively label, visualize and purify proteins made in specific cell types in vivo. The new transgenic mouse that we developed thus represents a technical advance that will be of interest for many researchers inside and outside the field.