Periodic Reporting for period 4 - NanoBioNext (Nanoscale Biomeasurements of Nerve Cells and Vesicles: Molecular Substructure and the Nature of Exocytosis)
Período documentado: 2023-02-01 hasta 2024-07-31
Problem/issue addressed. The emphasis of the project was on partial release during exocytosis. This was recently discovered and at the time this project began, it was not clear if it was a general phenomenon.
It was proposed to develop novel analytical tools at the micro to nanoscale level that are needed to understand critically important parts of neurotransmitter release in the brain. These include, 1) the vesicle closing during partial release, 2) the effect of substructure of transmitter vesicles on communication and plasticity, and 3) cell membrane lipid changes that might strengthen or weaken cell-to-cell communication via changing the rate of exocytosis or the fraction released in partial exocytosis (plasticity). Several completely new electrochemical methods, a completely new combination of electrochemistry with STED super-resolution microscopy, and a model for the initial plasticity in short-term memory has been examined.
Importance to society. The combined methods open new doorways to study protein-lipid interactions, drug sequestration in endosomes, and the analysis of organelles in living systems. The nanoscale biomeasurement paradigms developed here make it possible to quantify and localize lipids and metals in cell models, neurons, and vesicles with a spatial resolution of 40 nm. The combination of electrochemistry with STED and NanoSIMS open new directions in nanobio analytical chemistry. This provides unique possibilities to understand chemical communication and the plasticity of neurotransmitter release that is the likely starting point or initiator of short-term memory. Short-term memory is a key component lost in aging and neurodegenerative disease so the initiation of this process is highly important. The concept that drugs might affect lipid distribution in the brain as part of their mechanism of action could be very important, leading to new strategies for medicine. Showing that partial release during exocytosis is ubiquitous or common is important as it it provides a mechanistic view to better understand misfunction of cells in the brain during degenerative or early-age disorders.
It was proposed to develop novel analytical tools at the micro to nanoscale level that are needed to understand critically important parts of neurotransmitter release in the brain. These include, 1) the vesicle closing during partial release, 2) the effect of substructure of transmitter vesicles on communication and plasticity, and 3) cell membrane lipid changes that might strengthen or weaken cell-to-cell communication via changing the rate of exocytosis or the fraction released in partial exocytosis (plasticity). Several completely new electrochemical methods, a completely new combination of electrochemistry with STED super-resolution microscopy, and a model for the initial plasticity in short-term memory has been examined.
Importance to society. The combined methods open new doorways to study protein-lipid interactions, drug sequestration in endosomes, and the analysis of organelles in living systems. The nanoscale biomeasurement paradigms developed here make it possible to quantify and localize lipids and metals in cell models, neurons, and vesicles with a spatial resolution of 40 nm. The combination of electrochemistry with STED and NanoSIMS open new directions in nanobio analytical chemistry. This provides unique possibilities to understand chemical communication and the plasticity of neurotransmitter release that is the likely starting point or initiator of short-term memory. Short-term memory is a key component lost in aging and neurodegenerative disease so the initiation of this process is highly important. The concept that drugs might affect lipid distribution in the brain as part of their mechanism of action could be very important, leading to new strategies for medicine. Showing that partial release during exocytosis is ubiquitous or common is important as it it provides a mechanistic view to better understand misfunction of cells in the brain during degenerative or early-age disorders.
Final report
i. Determine if partial exocytotic release is ubiquitous in nature. We have carried out experiments with both pancreatic beta cells and at an octopamine-containing neuron in the fly larva to show that partial release is the predominant form of exocytosis for insulin/serotonin and octopamine, respectively. We published this in two papers in Angewandte Chemie. See figure 1. We published another paper in Angewandte Chemie examining the release of serotonin from gut BON cells, which is partial. We recently examined the serotonergic fly neurons in Drosophila and found some vesicles release about 30% of their cargo and some essentially 100%. The general theme has been that the majority of exocytosis is partial.
ii. Vesicle measurements and nanoscale subvesicular measurements. We have developed two types of nanopore electrodes that can be used to measure vesicle content and size, one for extracellular (see figure 2) and the other for intracellular experiments. These works have both been published in the Journal of the American Chemical Society. We used nanoscale secondary ion mass spectrometry (NanoSIMS) to quantify the content of transmitter vesicles and to view the difference between the dense core and the halo of the vesicle. We then used it for the analysis of partial release from vesicles by showing that labeled drug was taken up during exocytotic release and transmitter was decreased by 60% after stimulation of PC12 cells. These works were both published in ACS Nano. We then used NanoSIMS to quantitatively probe the halo and dense core in a paper in Int J Molec Sci. We used correlative transmission electron microscopy (TEM) and NanoSIMS imaging to examine the relation between fraction of partial exocytotic release and vesicle diameter to test the hypothesis that they are dependent. We found they are not (Angewandte Chemie). We also made great progress with electrochemistry showing that the Hofmeister effect can be used to regulate exocytosis and to develop compartment-selective sensors for vesicle content. We published these works in the J Am Chem Soc and two in Angewandte Chemie. We have investigated the pore opening of single vesicles at electrified interfaces, showing that vesicles open via electroporation towards the electrode surface (ACS Nano) and used combined STED and NanoSIMS to examine stress granule turnover in neuronal progenitor cells (Int J Mol Sci). Furthermore, we developed a method to measure the contents of exosomes and found that those from adrenal chromaffin cells contain catecholamine neurotransmitters (J Am Chem Soc). We also used our methods to discover that reactive oxygen species are formed by stress granules and went on to show that this release apparently controls homotypic fusion of vesicles in vitro, both works published in Angewandte Chemie.
iii. Lipids in signaling plasticity, nanoimaging. We have developed a paradigm for plasticity in exocytotic release from a cell model by use of repeated stimulations and shown a key parameter is partial release (published in Proceedings of the National Academy of Sciences, USA) and followed this with mass spectrometry imaging of model cells with similar paradigms. This comparison has been published in the International Journal of Chemical Sciences. We have also developed NanoSIMS strategies to quantitatively examine the contents of single nanometer vesicles across their structure, published in ACS Nano. We have also developed combined electrochemistry and NanoSIMS strategies to examine the effects of iron homeostasis on partial release (Angewandte Chemie). Finally, we found that omega-3 and-6 fatty acids alter the lipid composition of membranes and vesicle size thereby regulating exocytotic release and catecholamine storage (ACS Chem Neurosci). We used combined confocal fluorescence microscopy and electrochemistry to develop a method to examine the dynamics of vesicle opening on the electrode surface (ACS Measurement Science Gold). This is shown in Figure 3. This led to a new hypothesis of the surface reaction chemistry required for the vesicle to undergo electroporation.
iv. Drugs, zinc, lipids, and learning/memory. We have been highly successful here showing that drugs that affect cognitive ability (cocaine, methylphenidate, zinc, barbiturates, lidocaine, chemo treatment drugs, etc) all affect exocytosis and several have now been shown to affect the lipid membrane structure providing a potential mechanism for understanding the initiation of plasticity. These studies have been published in numerous recent papers. We showed that the drug modafinil changes the lipid composition of the brain (Angewandte Chemie) and then proposed a mechanism for the action of modafinil on cognition (QRB Discovery, see Figure 4). We have found that on our model systems, all drugs that are known or suspected to diminish cognitive ability in humans cause a decrease in the partial release fraction in our model cell systems. In contrast, to date, all drugs that are known or suspected to increase cognitive ability in humans result in an increase in the fraction of exocytotic release. This forms the basis for a preliminary model of the initiation of plasticity and we feel could be important in the mechanism of short-term memory.
i. Determine if partial exocytotic release is ubiquitous in nature. We have carried out experiments with both pancreatic beta cells and at an octopamine-containing neuron in the fly larva to show that partial release is the predominant form of exocytosis for insulin/serotonin and octopamine, respectively. We published this in two papers in Angewandte Chemie. See figure 1. We published another paper in Angewandte Chemie examining the release of serotonin from gut BON cells, which is partial. We recently examined the serotonergic fly neurons in Drosophila and found some vesicles release about 30% of their cargo and some essentially 100%. The general theme has been that the majority of exocytosis is partial.
ii. Vesicle measurements and nanoscale subvesicular measurements. We have developed two types of nanopore electrodes that can be used to measure vesicle content and size, one for extracellular (see figure 2) and the other for intracellular experiments. These works have both been published in the Journal of the American Chemical Society. We used nanoscale secondary ion mass spectrometry (NanoSIMS) to quantify the content of transmitter vesicles and to view the difference between the dense core and the halo of the vesicle. We then used it for the analysis of partial release from vesicles by showing that labeled drug was taken up during exocytotic release and transmitter was decreased by 60% after stimulation of PC12 cells. These works were both published in ACS Nano. We then used NanoSIMS to quantitatively probe the halo and dense core in a paper in Int J Molec Sci. We used correlative transmission electron microscopy (TEM) and NanoSIMS imaging to examine the relation between fraction of partial exocytotic release and vesicle diameter to test the hypothesis that they are dependent. We found they are not (Angewandte Chemie). We also made great progress with electrochemistry showing that the Hofmeister effect can be used to regulate exocytosis and to develop compartment-selective sensors for vesicle content. We published these works in the J Am Chem Soc and two in Angewandte Chemie. We have investigated the pore opening of single vesicles at electrified interfaces, showing that vesicles open via electroporation towards the electrode surface (ACS Nano) and used combined STED and NanoSIMS to examine stress granule turnover in neuronal progenitor cells (Int J Mol Sci). Furthermore, we developed a method to measure the contents of exosomes and found that those from adrenal chromaffin cells contain catecholamine neurotransmitters (J Am Chem Soc). We also used our methods to discover that reactive oxygen species are formed by stress granules and went on to show that this release apparently controls homotypic fusion of vesicles in vitro, both works published in Angewandte Chemie.
iii. Lipids in signaling plasticity, nanoimaging. We have developed a paradigm for plasticity in exocytotic release from a cell model by use of repeated stimulations and shown a key parameter is partial release (published in Proceedings of the National Academy of Sciences, USA) and followed this with mass spectrometry imaging of model cells with similar paradigms. This comparison has been published in the International Journal of Chemical Sciences. We have also developed NanoSIMS strategies to quantitatively examine the contents of single nanometer vesicles across their structure, published in ACS Nano. We have also developed combined electrochemistry and NanoSIMS strategies to examine the effects of iron homeostasis on partial release (Angewandte Chemie). Finally, we found that omega-3 and-6 fatty acids alter the lipid composition of membranes and vesicle size thereby regulating exocytotic release and catecholamine storage (ACS Chem Neurosci). We used combined confocal fluorescence microscopy and electrochemistry to develop a method to examine the dynamics of vesicle opening on the electrode surface (ACS Measurement Science Gold). This is shown in Figure 3. This led to a new hypothesis of the surface reaction chemistry required for the vesicle to undergo electroporation.
iv. Drugs, zinc, lipids, and learning/memory. We have been highly successful here showing that drugs that affect cognitive ability (cocaine, methylphenidate, zinc, barbiturates, lidocaine, chemo treatment drugs, etc) all affect exocytosis and several have now been shown to affect the lipid membrane structure providing a potential mechanism for understanding the initiation of plasticity. These studies have been published in numerous recent papers. We showed that the drug modafinil changes the lipid composition of the brain (Angewandte Chemie) and then proposed a mechanism for the action of modafinil on cognition (QRB Discovery, see Figure 4). We have found that on our model systems, all drugs that are known or suspected to diminish cognitive ability in humans cause a decrease in the partial release fraction in our model cell systems. In contrast, to date, all drugs that are known or suspected to increase cognitive ability in humans result in an increase in the fraction of exocytotic release. This forms the basis for a preliminary model of the initiation of plasticity and we feel could be important in the mechanism of short-term memory.