Bianxiao Cui, Ph.D. - US grants

Our broad objective is to understand the mechanisms by which the nerve growth factor (NGF) signal is propagated from the axon terminal to the cell body. NGF retrograde signaling is critical for the survival, differentiation, and maintenance of certain types neurons. Disrupted NGF retrograde transport was reported to contribute to the loss of the basal forebarin cholinergic (BFC) neurons in the brains of patients with Alzheimer's Disease or Down's Syndrome. This project will use advanced imaging techniques to directly visualize NGF transport in live neruons in real time. We focus on exploring dynamic features of NGF transport in normal and Down's Syndrome mice. The aims are: 1. Characterize the movement of NGF-containing endosomes in axons and define their pausing mechanism(s), by using quantum dot conjugated NGF to track endosomal movements with nanometer resolution. 2. Determine whether NGF-lacking endosomes are present, whether they are relevant for NGF signaling, and whether there are alternative signaling pathways independent of endosomal transport, by marking the NGF-lacking endosomes with photo-activatable green fluorescence proteins that are fused to the C-terminal of TrkA receptor. 3. Identify the abnormal features of disrupted NGF transport in Down Syndrome mouse neurons, by characterizing individual features of transport dynamics, which inlcude the average speed, the moving speed, the pausing duration, and the pausing frequency. 4. Determine how amyloid precursor protein overexpression leads to the abnormal NGF retrograde transport in Down Syndrome mouse by examining how overexpression of amyloid precursor protein in DS mice might cause defective structural or axonal features that lead to disrupted NGF transport. Achieving those aims will increase our understanding of how NGF signal is propagated in normal and degenerative neurons. More broadly, those studies will contribute to elucidate the pathogenesis of Alzheimer's disease and Down syndrome.

Our broad objective is to understand the mechanisms by which the nerve growth factor (NGF) signal is propagated from the axon terminal to the cell body. NGF retrograde signaling is critical for the survival, differentiation, and maintenance of certain types neurons. Disrupted NGF retrograde transport was reported to contribute to the loss of the basal forebarin cholinergic (BFC) neurons in the brains of patients with Alzheimer's Disease or Down's Syndrome. This project will use advanced imaging techniques to directly visualize NGF transport in live neruons in real time. We focus on exploring dynamic features of NGF transport in normal and Down's Syndrome mice. The aims are: 1. Characterize the movement of NGF-containing endosomes in axons and define their pausing mechanism(s), by using quantum dot conjugated NGF to track endosomal movements with nanometer resolution. 2. Determine whether NGF-lacking endosomes are present, whether they are relevant for NGF signaling, and whether there are alternative signaling pathways independent of endosomal transport, by marking the NGF-lacking endosomes with photo-activatable green fluorescence proteins that are fused to the C-terminal of TrkA receptor. 3. Identify the abnormal features of disrupted NGF transport in Down Syndrome mouse neurons, by characterizing individual features of transport dynamics, which inlcude the average speed, the moving speed, the pausing duration, and the pausing frequency. 4. Determine how amyloid precursor protein overexpression leads to the abnormal NGF retrograde transport in Down Syndrome mouse by examining how overexpression of amyloid precursor protein in DS mice might cause defective structural or axonal features that lead to disrupted NGF transport. Achieving those aims will increase our understanding of how NGF signal is propagated in normal and degenerative neurons. More broadly, those studies will contribute to elucidate the pathogenesis of Alzheimer's disease and Down syndrome.

CAREER: Nanopillar Electrode Arrays for Highly Sensitive Detection of Neuroelectric Activities

Abstract: Neurons encode information by electrical signals. To understand information processing and storage in neuronal networks, one of the most important tactics is to accurately record the small electrical signals generated by individual neurons over time. However, challenges associated with sensitive and noninvasive detection of small neuroelectric activities have hampered the effort for a better understanding of this complex process. Intracellular recording is very sensitive in detecting minuscule neuroelectric signals, but it is highly invasive and causes severe damage to the cell membrane. On the other hand, extracellular recording is non-invasive and very powerful in measuring large numbers of neurons, but it suffers poor signal-to-noise ratio and is unable to detect small synaptic neuroelectric potentials. This project will develop a novel electrical sensor -- nanopillar electrode arrays -- for highly-sensitive and non-invasive recording of neuroelectric activities. Nanopillars protruding from the planar surface will enhance the electric coupling between the neuron and the recording electrode. The new sensor will improve the detection sensitivity by two-orders of magnitude over the current extracellular recording technique. With the combined advantages of high sensitivity, non-invasiveness, and simultaneous multi-cell recording, the integrated device will enable new studies aimed to understand the mechanisms that govern how neurons in a network coordinate their activities in space and time and how those activities might encode external information.

The proposed research focuses on developing sensitive instrument to facilitate the quantitative measurements of signal propagation in neuronal networks. The education component of this proposal complements the research goal in (1) incorporating instrumentation fundamentals in an undergraduate laboratory course, (2) introducing research writing and presentation skills to undergraduate lab reports, and (3) mentoring under-privileged high school students in the research laboratory.

DESCRIPTION (Provided by the applicant) Abstract: The axon acts as a conduit for organized transport of materials between the cell body and the synapse, a process that is essential for the function and survival of neurons. Defective axonal transport, such as accumulation of axonal cargoes, has been linked with a range of neurodegenerative diseases by extensive genetic and biochemical studies. However, it is still unclear whether and how defective axonal transport might play a role in the progression of neuronal degeneration. Genetic and biochemical approaches lack precise control over when and where the cargo accumulations will happen along the axon, which makes it difficult to pinpoint the role of transport defect in the process of neuronal degeneration. In this proposal, we propose to engineer magnetic and optical forces that specifically stall a population of axonal cargoes that contain magnetic or optical nanoparticle probes at the trapping area. Physically stalling the cargoes would be one of the most direct means to perturb a cargo transport process, which, however, are technically challenging in live cells. We will overcome those challenges using advanced nanofabrication, imaging techniques and novel nanoparticle probes. Inside the narrow axon, stalled cargoes will act as roadblocks to slow down the trafficking of other probe-free cargoes that are not affected by external forces. Such force-induced traffic jams afford new approaches to investigate whether blocking the axonal transport is sufficient to induce neuronal degeneration and how cellular processes response to axonal traffic blockage. Public Health Relevance: Age-related neurodegenerative diseases, such as Alzheimer's disease, impact the lives of millions and pose a growing public health challenge. This study aims to investigate how defective axonal transport might cause or contribute to the progression of those neurodegenerative diseases. The findings of this research will advance our understanding of age-related neuronal death and assist therapeutic interventions for the treatment of these disorders.

This INSPIRE award is partially funded by the Electronic and Photonic Materials Program and the Biomaterials Program in the Division of Materials Research in the Directorate for Mathematical and Physical Sciences; the Neural Systems Program in the Division of Integrative Organismal Systems in the Directorate for Biological Sciences; the Instrument Development for Biological Research Program in the Division of Biological Infrastructure in the Directorate for Biological Sciences; and the Nano-Biosensing Program in the Division of Chemical, Bioengineering, Environmental, and Transport Systems in the Directorate for Engineering.

Technical Description: This research project aims to develop a novel technique to detect electric activities of a neuronal network using voltage-gated optical transitions in graphene. A neuron receives, analyzes and conveys information in the form of electrical signals such as action potentials. The decoding of neural data requires accurate recording of those electrical signals. This project exploits unique electrical and optical properties of graphene to detect electric activities of a neuronal network in a highly parallel and non-invasive manner. It advances scientific understanding in several important fronts, including (1) fundamental understanding of graphene as a unique electronic and optical material for high-sensitivity bio-imaging, (2) interfacing graphene with neuron cells, (3) non-invasive optical detection of action potential, and (4) highly parallel imaging of neuron activity in a neuronal network. This project builds on the complimentary expertise of Professor Wang and Professor Cui on graphene optical spectroscopy and neural electrophysiology, respectively.

Non-technical Description: This project captures the tremendous opportunities provided by graphene for optical imaging of neural network activities, which has a potential to become an important tool in neuroscience. The research requires extensive collaboration and exchange of students between Cui and Wang labs, which provides a unique opportunity for graduate and undergraduate students to be exposed to sciences in different disciplines including biology, engineering, materials science, optics, and physics.

The drug development pipeline requires that potential drugs pass through a gamut of tests which demonstrate the drugs' efficacy and safety. The drug safety test starts from cellular studies before moving into animals and finally humans. As a drug moves from cellular measurements to animal measurements, the time and money involved increase dramatically. Therefore, it is essential to detect potential drug toxicity early on, preferably at the cellular level. The drug toxicity manifests mostly as non-specific binding to ion channels and therefore affecting cardiac action potentials. The current golden standard technique to detect action potential changes is patch clamps. However, the patch clamp has very low throughput and is challenging to implement, which limit the number of drug trials or drug candidates that can be screened. The proposed nanoelectrode device allows these companies to expedite testing and reduce the cost of cellular tests. In particular, nanoelectrodes can measure electrophysiology in high throughput and low toxicity and therefore allowing testers to measure more drugs with greater ease.

Current drug toxicology screens inevitably involve patch clamp electrophysiology to test off-target cardiac activity. Patch clamp is technically challenging to use and has limited multiplexing capability. Over the last six years, this group has worked to develop a new nanotechnology tool for electrophysiology. The proposed tool measures intracellular recordings of mammalian cells with high throughput and low requirements on technical capability in the experimenter. Signals recorded by these nanoelectrodes are carried out from the culture under test on metal leads and amplified at an external multi-electrode array (MEA) amplifier. This team expects to have customers in both pharmaceutical toxicology and academic laboratories who are interested in pursuing electrophysiological questions. This team also believes that anyone interested in the initial screening of new drugs for their toxicological side effects would be interested in the proposed technology that could be integrated into automated cell culture systems and offers simple handling and experimentation.

Project Summary / Abstract: Action potentials of electrogenic cells, such as neurons and cardiomyocytes, are crucial for their physiological functions. Neurons use action potential to transmit signals over long distances, and cardiomyocytes use action potentials to synchronize the contraction of millions of cells during each heartbeat. To understand these important physiological functions, one of the most important tactics is to accurately record the electrical potentials from cells. However, the current two major classes of electrophysiological methods, intracellular and extracellular recording, suffer severe limitations in their applications. Intracellular recording such as patch clamp suffers from extremely low throughput and toxic intracellular dialysis. Extracellular recording such as planar electrode array suffers from poor signal and lack of one-to-one cell-to-electrode coupling. In the last decade, much effort has been focused on developing new generation of electrophysiology tools to achieve high throughput intracellular recording. In particular, nanotechnology-based electrode sensors developed independently in several groups has shown great promise in achieving highly sensitive and high throughput intracellular recording. However, developing these nascent technologies into robust electrophysiological tools would require extensive studies for characterization, validation, and optimization. This proposal aims to develop the nanoelectrode technology into robust electrophysiological tools for biomedical research. When accomplished, this new technology will enable users to (a) perform sensitive, intracellular recording of action potentials from tens to hundreds of individual cells simultaneously; (b) achieve long-term, minimally-invasive recording of the cells for days to weeks; and (c) afford stable culture and recording of the hSC-CMs under optimal environmental conditions.

Project Summary / Abstract: Many biomedical applications require direct contact between the cells and non-biological materials. For example, medical implants inserted into the patient?s body have intimate contact with adjacent cells and tissues. Synthetic materials ?show? only their surfaces to the biological environment. These surfaces are recognized by cells through their chemical composition and their physical properties. Surface chemistry has been studied extensively and many surface functionalization strategies have been developed to promote cell adhesion and integration. The importance of physical properties in modulating cellular behavior, such as surface topography and material rigidity are increasingly recognized. In particular, studies show that surface topography in the scale of tens of nanometers to a few micrometers significantly affect cell adhesion and tissue integration. As topographic features are stable over long-term and easier to control, they offer unique advantages for modulating cell responses for tissue engineering. However, the grant challenge is how to optimize surface topology to achieve a desired function among a high-dimensional space of topological features. To address this challenge, it is imperative to ask the fundamental question ?how do cells detect its environmental topology??. Despite a large body of observations, little is known about the origin or underlying mechanisms of the effect of topographical cues on cell behavior. This proposal aims to answer the fundamental question by proposing and testing a new hypothesis, the curvature hypothesis, based on very recent studies in the PI?s lab. A comprehensive research program will be built to validate the curvature hypothesis by understanding how positive and negative membrane curvatures differentially regulate the intracellular signaling, confirming that membrane curvature is a critical player in topography-induced cell adhesion and stem cell differentiation, and visualizing membrane deformations on a variety of complex surface topography. These studies will not only contribute to mechanistic understandings of the interaction between living cells and synthetic materials, but also accelerate the effort to design material surfaces for desired applications.

Project Summary Understanding how a network of interconnected neurons receives, stores and processes information requires parallel and high quality recording of neuroelectric signals. Intracellular recording techniques such as patch clamp are invasive and limited to recording 1-2 cells. While extracellular multielectrode arrays can record multiple cells, they are pre-fabricated and thus can only probe fixed locations. Optical detection of electric activities provides the needed spatial flexibility. Calcium sensors such as GcaMP have a slow time response and not suitable to record fast-spiking pacemaker neurons such as dopaminergic neurons. Voltage-sensitive fluorescence proteins and dyes have much faster time response, but their recording time is usually limited by photobleaching. In this project, we will demonstrate an orthogonal approach of optical recording. This method, Electrochromic Optical Recording of Electric potentials (ECORE) makes use of a unique material property ? optical absorption of an electrochromic film depends on applied voltages. We detect the optical reflection of an electrochromic film to read out cellular electrical activities. The method is truly label-free, i.e. free of any molecular probes that need to be incorporated into cells and perturb cellular physiology, and not limited by photobleaching or photo-toxicity. In preliminary work, we have built a sensitive optical setup that is able to detect the reflectivity change of the electrochromic film in response to electrical potentials as small as 10 microvolts. Indeed, we have used ECORE to successfully record single-cell action potentials in neurons, cardiomyocytes, and brain tissues. With this project, we plan to dramatically expand ECORE capabilities by developing a scanning ECORE platform for parallel detection and an ECORE microscope for subcellular measurement of neuroelectric activities. We will use ECORE to probe the functional connectivity of dopaminergic neurons in midbrain area. Accomplishment of this work will result in a new class of electrophysiological tools that can be used by other research groups.

Project Summary / Abstract: This MIRA proposal merges two distinct projects supported by R01GM128142, ?The role of membrane curvature in surface nanotopography-induced cell functions?, and R01GM125737, ?Developing nanoscale electrophysiology sensors for robust intracellular recording?. While the two projects focus on different biological questions, the unifying theme is to develop nanoscale probes to elucidate the cellular machinery in the intricate environment of living cells. In this proposal, we discuss topics along the lines of the parent grants, focusing on the significance of the biological problems, our recent and evolving results, and directions for the future. For the first project, the long-term goal is to understand how membrane curvature regulates biochemical signals that are transmitted through the cell-matrix interface. At the cell-matrix interface, where the cells make physical contact with extracellular matrices, the membrane may be locally deformed by matrix topography or mechanical forces. As it remains a challenge to manipulate nanoscale membrane curvature in live cells, our current understanding of how local membrane curvature affects signal transmission is limited. We propose to use nanotechnology-based precision engineering to control interface membrane curvature in live cells. We seek to understand how cellular processes are affected by membrane curvature and the underlying molecular mechanisms. The knowledge gained will help us understanding how cells interact with extracellular matrix and also help us designing biomaterials for better integration with cells. For the second project, we are developing vertical nanoelectrodes into a robust and easy-to-use electrophysiology tool that can reliably achieve parallel intracellular recording of cardiomyocytes with minimal perturbation. Simultaneous nanoelectrode and patch clamp recordings on same cells confirmed that nanoelectrodes accurately record action potential waveforms for classification and characterization of stem-cell-derived cardiomyocytes. These nanoelectrodes will enable us to understand how in vitro interventions accelerate the maturation of stem-cell-derived cardiomyocyte. Furthermore, nanoelectrodes provide an ideal tool for monitoring the generation and resealing of membrane pores on cardiomyocytes that are prone to membrane rupture due to their large size and strong mechanical contraction. We will use nanoelectrode to investigate how proteins participate in the membrane resealing process. We hope to achieve a broad impact by combining the development of new tools with applications to specific biological systems.