Altered Synaptic Vesicle Release and Ca2+ Influx at Single Presynaptic Terminals of Cortical Neurons in a Knock-in Mouse Model of Huntington’s Disease

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by the abnormal expansion of CAG repeats in the huntingtin (HTT) gene, which leads to progressive loss of neurons starting in the striatum and cortex. One possible mechanism for this selective loss of neurons in the early stage of HD is altered neurotransmission at synapses. Despite the recent finding that presynaptic terminals play an important role in HD, neurotransmitter release at synapses in HD remains poorly understood. Here, we measured synaptic vesicle release in real time at single presynaptic terminals during electrical field stimulation. We found the increase in synaptic vesicle release at presynaptic terminals in primary cortical neurons in a knock-in mouse model of HD (zQ175). We also found the increase in Ca2+ influx at presynaptic terminals in HD neurons during the electrical stimulation. Consistent with increased Ca2+-dependent neurotransmission in HD neurons, the increase in vesicle release and Ca2+ influx was rescued with Ca2+ chelators or by blocking N-type voltage-gated Ca2+ channels, suggesting N-type voltage-gated Ca2+ channels play an important role in HD. Taken together, our results suggest that the increased synaptic vesicles release due to increased Ca2+ influx at presynaptic terminals in cortical neurons contributes to the selective neurodegeneration of these neurons in early HD and provide a possible therapeutic target

Altered exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of cultured striatal neurons in a knock-in mouse model of Huntington’s disease

Huntington’s disease (HD) is a progressive dominantly inherited neurodegenerative disease caused by the expansion of a cytosine-adenine-guanine (CAG) trinucleotide repeat in the huntingtin gene, which encodes the mutant huntingtin protein containing an expanded polyglutamine tract. One of neuropathologic hallmarks of HD is selective degeneration in the striatum. Mechanisms underlying selective neurodegeneration in the striatum of HD remain elusive. Neurodegeneration is suggested to be preceded by abnormal synaptic transmission at the early stage of HD. However, how mutant huntingtin protein affects synaptic vesicle exocytosis at single presynaptic terminals of HD striatal neurons is poorly understood. Here, we measured synaptic vesicle exocytosis at single presynaptic terminals of cultured striatal neurons (mainly inhibitory neurons) in a knock-in mouse model of HD (zQ175) during electrical field stimulation using real-time imaging of FM 1-43 (a lipophilic dye). We found a significant decrease in bouton density and exocytosis of synaptic vesicles at single presynaptic terminals in cultured striatal neurons. Real-time imaging of VGAT-CypHer5E (a pH sensitive dye conjugated to an antibody against vesicular GABA transporter (VGAT)) for inhibitory synaptic vesicles revealed a reduction in bouton density and exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of HD striatal neurons. Thus, our results suggest that the mutant huntingtin protein decreases bouton density and exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of striatal neurons, causing impaired inhibitory synaptic transmission, eventually leading to the neurodegeneration in the striatum of HD

Facile and versatile ligand analysis method of colloidal quantum dot

Colloidal quantum-dots (QDs) are highly attractive materials for various optoelectronic applications owing to their easy maneuverability, high functionality, wide applicability, and low cost of mass-production. QDs usually consist of two components: the inorganic nano-crystalline particle and organic ligands that passivate the surface of the inorganic particle. The organic component is also critical for tuning electronic properties of QDs as well as solubilizing QDs in various solvents. However, despite extensive effort to understand the chemistry of ligands, it has been challenging to develop an efficient and reliable method for identifying and quantifying ligands on the QD surface. Herein, we developed a novel method of analyzing ligands in a mild yet accurate fashion. We found that oxidizing agents, as a heterogeneous catalyst in a different phase from QDs, can efficiently disrupt the interaction between the inorganic particle and organic ligands, and the subsequent simple phase fractionation step can isolate the ligand-containing phase from the oxidizer-containing phase and the insoluble precipitates. Our novel analysis procedure ensures to minimize the exposure of ligand molecules to oxidizing agents as well as to prepare homogeneous samples that can be readily analyzed by diverse analytical techniques, such as nuclear magnetic resonance spectroscopy and gas-chromatography mass-spectrometry. © 2021, The Author(s).1

Single-Molecule Fluorescence Studies of ReAsH and Myosin VI

119 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006.Single-molecule fluorescence spectroscopy has become one of the most powerful techniques in chemistry and biology. Recently, a new fluorescence technique called fluorescence imaging with one nanometer accuracy (FIONA) was shown to localize the position of a fluorescent molecule within a nanometer. Using FIONA, we examined photophysical properties of ReAsH - a novel fluorescent molecule. We showed that ReAsH is more photostable and brighter than eGFP, indicating that ReAsH can be used for in vivo tracking and localization of specific proteins. We also have used FIONA to study myosin VI, the only myosin to move in a direction opposite that of other myosins. This reverse directionality enables myosin VI to be involved in endocytosis. We investigated its structural and functional mechanisms. First, we discovered the stepping mechanism of myosin VI. By placing fluorescent molecules in two different positions and using FIONA, we showed that myosin VI walks hand-over-hand. Second, we revealed the dimerization mechanism of myosin VI. We demonstrated that myosin VI can be dimerized by clustering monomers through binding them either to actin or to antibodies, which implies that cargo-binding induces the dimerization of myosin VI inside cells. Third, we showed that the 53 amino acids unique to myosin VI (the unique insert) determine its directionality. We constructed several chimeric myosins with or without the unique insert, and tested them with gliding assay, which showed that the unique insert causes reverse directionality. Using FIONA, we found that it has a broad distribution similar to that of myosin VI, suggesting that the converter domain is the source of its highly variable step size. Fourth, we measured the processivity of myosin VI at different temperatures, and found that the average step size was smaller at higher temperatures, suggesting that the lever arm becomes less rigid at higher temperatures. Circular dichroism spectrum confirmed a structural change at ∼30°C. We found that additional ADP increases the run-length of myosin VI, implying that the preference of myosin VI for ADP and competition between ADP and ATP for catalytic sites prevents myosin VI from detaching from actin and allows it to walk long distances.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Single-Molecule Fluorescence Studies of ReAsH and Myosin VI

119 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006.Single-molecule fluorescence spectroscopy has become one of the most powerful techniques in chemistry and biology. Recently, a new fluorescence technique called fluorescence imaging with one nanometer accuracy (FIONA) was shown to localize the position of a fluorescent molecule within a nanometer. Using FIONA, we examined photophysical properties of ReAsH - a novel fluorescent molecule. We showed that ReAsH is more photostable and brighter than eGFP, indicating that ReAsH can be used for in vivo tracking and localization of specific proteins. We also have used FIONA to study myosin VI, the only myosin to move in a direction opposite that of other myosins. This reverse directionality enables myosin VI to be involved in endocytosis. We investigated its structural and functional mechanisms. First, we discovered the stepping mechanism of myosin VI. By placing fluorescent molecules in two different positions and using FIONA, we showed that myosin VI walks hand-over-hand. Second, we revealed the dimerization mechanism of myosin VI. We demonstrated that myosin VI can be dimerized by clustering monomers through binding them either to actin or to antibodies, which implies that cargo-binding induces the dimerization of myosin VI inside cells. Third, we showed that the 53 amino acids unique to myosin VI (the unique insert) determine its directionality. We constructed several chimeric myosins with or without the unique insert, and tested them with gliding assay, which showed that the unique insert causes reverse directionality. Using FIONA, we found that it has a broad distribution similar to that of myosin VI, suggesting that the converter domain is the source of its highly variable step size. Fourth, we measured the processivity of myosin VI at different temperatures, and found that the average step size was smaller at higher temperatures, suggesting that the lever arm becomes less rigid at higher temperatures. Circular dichroism spectrum confirmed a structural change at ∼30°C. We found that additional ADP increases the run-length of myosin VI, implying that the preference of myosin VI for ADP and competition between ADP and ATP for catalytic sites prevents myosin VI from detaching from actin and allows it to walk long distances.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Single-molecule fluorescence to study molecular motors

Molecular motors, which use energy from ATP hydrolysis to take nanometer-scale steps with run-lengths on the order of micrometers, have important roles in areas such as transport and mitosis in living organisms. New techniques have recently been developed to measure these small movements at the single-molecule level. In particular, fluorescence imaging has contributed to the accurate measurement of this tiny movement. We introduce three single-molecule fluorescence imaging techniques which can find the position of a fluorophore with accuracy in the range of a few nanometers. These techniques are named after Hollywood animation characters: Fluorescence Imaging with One Nanometer Accuracy (FIONA), Single-molecule High-REsolution Colocalization (SHREC), and Defocused Orientation and Position Imaging (DOPI). We explain new understanding of molecular motors obtained from measurements using these techniques

Influence of synaptic vesicle position on release probability and exocytotic fusion mode

Neurotransmission depends on movements of transmitter-laden synaptic vesicles, but accurate, nanometer-scale monitoring of vesicle dynamics in presynaptic terminals has remained elusive. Here, we report three-dimensional, real-time tracking of quantum dot-loaded single synaptic vesicles with an accuracy of 20 to 30 nanometers, less than a vesicle diameter. Determination of the time, position, and mode of fusion, aided by trypan blue quenching of Qdot fluorescence, revealed that vesicles starting close to their ultimate fusion sites tended to fuse earlier than those positioned farther away. The mode of fusion depended on the prior motion of vesicles, with long-dwelling vesicles preferring kiss-and-run rather than full-collapse fusion. Kiss-and-run fusion events were concentrated near the center of the synapse, whereas full-collapse fusion events were broadly spread

Application of a Terrestrial LIDAR System for Elevation Mapping in Terra Nova Bay, Antarctica

A terrestrial Light Detection and Ranging (LIDAR) system has high productivity and accuracy for topographic mapping, but the harsh conditions of Antarctica make LIDAR operation difficult. Low temperatures cause malfunctioning of the LIDAR system, and unpredictable strong winds can deteriorate data quality by irregularly shaking co-registration targets. For stable and efficient LIDAR operation in Antarctica, this study proposes and demonstrates the following practical solutions: (1) a lagging cover with a heating pack to maintain the temperature of the terrestrial LIDAR system; (2) co-registration using square planar targets and two-step point-merging methods based on extracted feature points and the Iterative Closest Point (ICP) algorithm; and (3) a georeferencing module consisting of an artificial target and a Global Navigation Satellite System (GNSS) receiver. The solutions were used to produce a topographic map for construction of the Jang Bogo Research Station in Terra Nova Bay, Antarctica. Co-registration and georeferencing precision reached 5 and 45 mm, respectively, and the accuracy of the Digital Elevation Model (DEM) generated from the LIDAR scanning data was ±27.7 cm

Precise and Long-Term Tracking of Mitochondria in Neurons using a Bioconjugatable and Photostable AIE Luminogen

Tracking mitochondrial movement in neurons is an attractive research field as dysregulation of mitochondrial motion is associated with multiple neurological diseases. To attain the precise trajectory of a single mitochondrion and achieve long-term imaging of mitochondria in neurons, specific and photostable fluorescent probes with a long emission lifetime are required. Existing mitochondrial targeting fluorescent dyes suffer from poor photostability, high toxicity, “always-on” behavior, and aggregation-caused quenching effect, which limit their use in studying mitochondria in neurons. To overcome these challenges, we designed and synthesized an aggregation-induced emission (AIE)-active luminogen, TPAP-C5-yne, which consists of an activated alkyne terminus for bioconjugation with amines, and a cationic pyridinium moiety to selectively target mitochondria. For the first time using TPAP-C5-yne, we successfully tracked and analyzed the motion of a single mitochondrion in live primary hippocampal neurons accurately using real-time fluorescence images acquired by a sensitive EMCCD camera. In addition, long-term imaging of mitochondria in live neurons for a week is achieved by TPAP-C5-yne, which was not feasible with a commercially available mitochondrial targeting probe before

Real-time three-dimensional tracking of single vesicles reveals abnormal motion and pools of synaptic vesicles in neurons of Huntington's disease mice

10.1016/j.isci.2021.103181iScience241010318