Biology subjects

Hochgeschwender, U.

Publications and source records attributed to Hochgeschwender, U..

13 recordsLinked to original sources

bioRxiv2025

Magneto-Photonic Gene Circuit for Minimally Invasive Control of Gene Expression in Mammalian Cells

Precise control of gene expression is one of the fundamental goals of synthetic biology. Whether the objective is to modify endogenous cellular function or induce the expression of molecules for diagnostic and therapeutic purposes, gene regulation remains a key aspect of biological systems. Over time, advances in protein engineering and molecular biology have led to the creation of gene circuits capable of inducing the expression of specific proteins in response to external stimulus such as light. These optogenetic, or light-activated circuits hold significant potential for gene therapy as a tool for regulating the expression of therapeutic genes within cells. However, the applications of optogenetic systems can be limited by the lack of efficient ways for light delivery inside cells or tissue. Our approach to address this challenge is to harness the power of bioluminescence to produce light directly inside cells using a luminescent enzyme. Combined with a photosensitive transcription factor, we report the development of a fully genetically encoded optogenetic circuit for control of gene expression. Furthermore, we utilized a magneto sensitive protein to engineer a split protein version of this luminescent enzyme, where its reconstitution is driven by a 50mT magnetic stimulus. Thus, resulting in a first-of-its-kind gene circuit activated by a combination of light and magnetic stimulus. We expect this work to advance the implementation of light-controlled systems without the need of external light sources, as well as serve as a basis for the development of future magneto-sensitive tools.

synthetic biology↗

bioRxiv2025

Control of Synaptic Communication through Molecularly Engineered Bioluminescence Light Emission and Sensing

Synapses are sites of intercellular communication between neurons and from neurons to target organs, and of signal integration that underly physiological and behavioral responses. We have developed a modular platform, Interluminescence (Int), for experimental control of synaptic transmission: bioluminescent light, generated by a luciferase oxidizing a luciferin, from a presynaptic neuron is used to activate transsynaptic optogenetic ion channels in the postsynaptic neuron. Two strategies can modulate postsynaptic neurons in the presence of luciferin. In the Act-Int approach, a luciferase is genetically expressed in synaptic vesicles and released during depolarization-induced presynaptic vesicle fusion and exocytosis. In the Persist-Int approach, a luciferase is tethered to the presynaptic membrane where it can support sustained transsynaptic signaling. Both strategies can activate postsynaptic neurons with comparable efficacy under the conditions tested. By design, the modularity of the platform permits the use of luciferases and opsins ranging in brightness and light sensitivity, with the luciferase targeted to different subcellular regions of the presynaptic neuron, and the postsynaptic opsin being excitatory or inhibitory. Our results demonstrate the utility and versatility of Interluminescence to mediate synapse-specific transmission that is either activity-dependent or activity-independent.

neuroscience↗

bioRxiv2024

Presymptomatic Targeted Circuit Manipulation for Ameliorating Huntington's Disease Pathogenesis

Early stages of Huntingtons disease (HD) before the onset of motor and cognitive symptoms are characterized by imbalanced excitatory and inhibitory output from the cortex to striatal and subcortical structures. The window before the onset of symptoms presents an opportunity to adjust the firing rate within microcircuits with the goal of restoring the impaired E/I balance, thereby preventing or slowing down disease progression. Here, we investigated the effect of presymptomatic cell-type specific manipulation of activity of pyramidal neurons and parvalbumin interneurons in the M1 motor cortex on disease progression in the R6/2 HD mouse model. Our results show that dampening excitation of Emx1 pyramidal neurons or increasing activity of parvalbumin interneurons once daily for 3 weeks during the pre-symptomatic phase alleviated HD-related motor coordination dysfunction. Cell-type-specific modulation to normalize the net output of the cortex is a potential therapeutic avenue for HD and other neurodegenerative disorders. O_FIG O_LINKSMALLFIG WIDTH=156 HEIGHT=200 SRC="FIGDIR/small/604946v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@11e546borg.highwire.dtl.DTLVardef@38676eorg.highwire.dtl.DTLVardef@10e9e43org.highwire.dtl.DTLVardef@b8779e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsCortical excitatory pyramidal neurons and inhibitory parvalbumin interneurons are affected in Huntingtons disease Repeated bioluminescence-mediated opto-chemogenetic inhibition/excitation of affected cell types in the motor cortex improved motor coordination and inter-limb gait parameters in HD mice Early manipulation of select microcircuits before the onset of symptoms presents an avenue to slow HD disease progression

neuroscience↗

bioRxiv2023

Engineering luminopsins with improved coupling efficiencies

SignificanceLuminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. AimWe investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. ApproachWe quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array (MEAs) recordings in primary neurons. ResultsLuciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. ConclusionsWhole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Forster resonance energy transfer (FRET), the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

molecular biology↗

bioRxiv2023

A Bioluminescent Activity Dependent (BLADe) Platform for Converting Neuronal Activity to Photoreceptor Activation

Genetically encoded sensors and actuators have advanced the ability to observe and manipulate cellular activity, yet few non-invasive strategies enable cells to directly couple their intracellular states to user-defined outputs. We promote a bioluminescent activity-dependent (BLADe) platform that facilitates programmable feedback through genetically encoded light generation. Using calcium (Ca{superscript 2}) flux as a model, we engineered a Ca{superscript 2}-dependent luciferase that functions as an activity-gated light source capable of photoactivating light-sensing actuators. As an initial demonstration of the versatility of this platform we present two separate use cases in neurons. In the first application, the presence of luciferin triggers Ca{superscript 2} dependent local illumination that provides activity dependent gene expression by activating a light-sensitive transcription factor. In the second application, neuronal activity-driven Ca{superscript 2} fluctuations via locally generated bioluminescence control neural dynamics through opsin activation in single cells, populations and intact tissue. BLADe can be expanded to couple any signal that bioluminescent enzymes can be engineered to detect with the wide variety of photosensing actuators. This modular strategy of coupling an activity dependent light emitter to a light sensing actuator offers, in principle, a generalizable framework for state dependent cell-autonomous control across biological systems.

neuroscience↗

bioRxiv2023

CaBLAM! A high-contrast bioluminescent Ca2+ indicator derived from an engineered Oplophorus gracilirostris luciferase

Measuring ongoing cellular activity is essential to understanding the dynamic functions of biological organisms. The most popular current approach is imaging fluorescence-based genetically encoded Ca2+ indicators (GECIs). While fluorescent probes are useful in many contexts, bioluminescence-based GECIs--probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein--have several distinct advantages. Because bioluminescent (BL) GECIs do not use the bright extrinsic excitation light required for fluorescence, BL GECIs do not photobleach, do not suffer from nonspecific autofluorescent background, and do not cause phototoxicity. Further, BL GECIs can be applied in contexts where directly shining photons on an imaging target is not possible. Despite these advantages, the use of BL GECIs has to date been limited by their small changes in bioluminescence intensity, high baseline signal at resting Ca2+ concentrations, and suboptimal Ca2+ affinities. Here, we describe a new BL GECI, CaBLAM (Ca2+ BioLuminescence Activity Monitor), that displays much higher dynamic range than previous BL GECIs and has a Ca2+ affinity suitable for capturing physiological changes in cytosolic Ca2+ concentration. With these improvements, CaBLAM captures single-cell and subcellular resolution activity at high frame rates in cultured neurons and in vivo, and allows multi-hour recordings in mice and behaving zebrafish. This new advance provides a robust alternative to traditional fluorescent GECIs that can enable or enhance imaging across many experimental conditions.

neuroscience↗

bioRxiv2023

A FRET-based excitatory luminopsin with improved efficacy of bioluminescence-driven activation

SignificanceBioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). AimTo further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence- driven) LMO activation. ApproachWe developed novel luciferases optimized for Forster resonance energy transfer (FRET) when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). ResultsA new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard), and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. ConclusionsWe report a robust new option for achieving multiple modes of control in a single actuator, and a promising engineering strategy for continued improvement of BL-OG.

neuroscience↗

bioRxiv2021

Selective Control of Synaptically-Connected Circuit Elements by All-Optical Synapses

Understanding percepts, engrams and actions requires methods for selectively modulating synaptic communication between specific subsets of interconnected cells. Here, we develop an approach to control synaptically connected elements using bioluminescent light: Luciferase-generated light, originating from a presynaptic axon terminal, modulates an opsin in its postsynaptic target. Vesicular-localized luciferase is released into the synaptic cleft in response to presynaptic activity, creating a real-time Optical Synapse. Light production is under experimenter-control by introduction of the small molecule luciferin. Signal transmission across this optical synapse is temporally defined by the presence of both the luciferin and presynaptic activity. We validate synaptic Interluminescence by multi-electrode recording in cultured neurons and in mice in vivo. Interluminescence represents a powerful approach to achieve synapse-specific and activity-dependent circuit control during behavior in vivo.

neuroscience↗

bioRxiv2021

Bioluminescent Genetically Encoded Glutamate Indicator for Molecular Imaging of Neuronal Activity

Genetically encoded optical sensors and advancements in microscopy instrumentation and techniques have revolutionized the scientific toolbox available for probing complex biological processes such as release of specific neurotransmitters. Most genetically encoded optical sensors currently used are based on fluorescence and have been highly successful tools for single-cell imaging in superficial brain regions. However, there remains a need to develop new tools for reporting neuronal activity in vivo within deeper structures without the need for hardware such as lenses or fibers to be implanted within the brain. Our approach to this problem is to replace the fluorescent elements of the existing biosensors with bioluminescent elements. This eliminates the need of external light sources to illuminate the sensor and overcomes several drawbacks of fluorescence imaging such as limited light penetration depth, excitation scattering, and tissue heating that are all associated with the external light needed for fluorescence imaging. Here we report the development of the first genetically encoded neurotransmitter indicators based on bioluminescent light emission. These probes exhibit robust changes in light output in response to extracellular presentation of the excitatory neurotransmitter glutamate. We expect this new approach to neurotransmitter indicator design to enable the engineering of specific bioluminescent probes for multiple additional neurotransmitters in the future, ultimately allowing neuroscientists to monitor activity associated with a specific neurotransmitter as it relates to behavior in a variety of neuronal and psychiatric disorders, among many other applications.

neuroscience↗

bioRxiv2020

Miniaturized Devices for Bioluminescence Imaging in Freely Behaving Animals

Fluorescence miniature microscopy in vivo has recently proven a major advance, enabling cellular imaging in freely behaving animals. However, fluorescence imaging suffers from autofluorescence, phototoxicity, photobleaching and non-homogeneous illumination artifacts. These factors limit the quality and time course of data collection. Bioluminescence provides an alternative kind of activity-dependent light indicator. Bioluminescent calcium indicators do not require light input, instead generating photons through chemiluminescence. As such, limitations inherent to the requirement for light presentation are eliminated. Further, bioluminescent indicators also do not require excitation light optics: the removal of this component should make lighter and lower cost microscope with fewer assembly parts. While there has been significant recent progress in making brighter and faster bioluminescence indicators, parallel advances in imaging hardware have not yet been realized. A hardware challenge is that despite potentially higher signal-to-noise of bioluminescence, the signal strength is lower than that of fluorescence. An open question we address in this report is whether fluorescent miniature microscopes can be rendered sensitive enough to detect bioluminescence. We demonstrate this possibility in vitro and in vivo by implementing optimizations of the UCLA fluorescent miniscope. These optimizations yielded a miniscope (BLmini) which is 22% lighter in weight, has 45% fewer components, is up to 58% less expensive, offers up to 15 times stronger signal (as dichroic filtering is not required) and is sensitive enough to capture spatiotemporal dynamics of bioluminescence in the brain with a signal-to-noise ratio of 34 dB.

bioengineering↗

bioRxiv2020

Selective Postnatal Excitation of Neocortical Pyramidal Neurons Results in Distinctive Behavioral and Circuit Deficits in Adulthood

In leading models of Autism Spectrum Disorder, and in human data, the efficacy of outgoing cortical connectivity transitions from overly exuberant to languid from early development to adulthood. This transition begs the question of whether the early enhancement in excitation might be a common driver, across etiologies, of these symptoms. We directly tested this concept by chemogenetically driving neuronal activity in neocortical neurons during postnatal days 4-14. Hyperexcitation of Emx1-, but not dopamine transporter-, parvalbumin-, or Dlx5/6-expressing neurons led to decreased social interaction and increased grooming activity in adult animals. In vivo optogenetic interrogation in adults revealed decreased baseline but increased stimulus-evoked firing rates of pyramidal neurons, impaired recruitment of inhibitory neurons and reduced cortico-striatal communication. These results directly support the prediction that changed firing in developing circuits irreversibly alters adult circuit function that leads to maladaptive changes in behaviors. This experimental approach offers a valuable platform to study the impact of disruption of developmental neural activity on the formation and function of adult neural circuits and behavior.

neuroscience↗

bioRxiv2019

The BioLuminescent-OptoGenetic in vivo Response to Coelenterazine is Proportional, Sensitive and Specific in Neocortex

BioLuminescent (BL) light production can modulate neural activity and behavior through coexpressed OptoGenetic (OG) elements, an approach termed BL-OG. Yet, the relationship between BL-OG effects and bioluminescent photon emission has not been characterized in vivo. Further, the degree to which BL-OG effects strictly depend on optogenetic mechanisms driven by bioluminescent photons is unknown. Crucial to every neuromodulation method is whether the activator shows a dynamic concentration range driving robust, selective, and non-toxic effects. We systematically tested the effects of four key components of the BL-OG mechanism (luciferin, oxidized luciferin, luciferin vehicle, and bioluminescence), and compared these against effects induced by the Luminopsin-3 (LMO3) BL-OG molecule, a fusion of slow burn Gaussia luciferase (sbGLuc) and Volvox ChannelRhodopsin-1 (VChR1). We performed combined bioluminescence imaging and electrophysiological recordings while injecting specific doses of Coelenterazine (substrate for sbGluc), Coelenteramide (CTM, the oxidized product of CTZ), or CTZ vehicle. CTZ robustly drove activity in mice expressing LMO3, with photon production proportional to firing rate. In contrast, low and moderate doses of CTZ, CTM, or vehicle did not modulate activity in mice that did not express LMO3. We also failed to find bioluminescence effects on neural activity in mice expressing an optogenetically non-sensitive LMO3 variant. We observed weak responses to the highest dose of CTZ in control mice, but these effects were significantly smaller than those observed in the LMO3 group. These results show that in neocortex in vivo, there is a large CTZ range wherein BL-OG effects are specific to its active chemogenetic mechanism.

neuroscience↗

bioRxiv2019

Restoring Function After Severe Spinal Cord Injury Through Bioluminescence-Driven Optogenetics

The ability to manipulate specific neuronal populations of the spinal cord following spinal cord injury (SCI) could prove highly beneficial for rehabilitation in patients through maintaining and strengthening still existing neuronal connections and/or facilitating the formation of new connections. A non-invasive and highly specific approach to neuronal stimulation is bioluminescent-optogenetics (BL-OG), where genetically expressed light emitting luciferases are tethered to light sensitive channelrhodopsins (luminopsins, LMO); neurons are activated by the addition of the luciferase substrate coelenterazine (CTZ). This approach utilizes ion channels for current conduction while activating the channels through application of a small chemical compound, thus allowing non-invasive stimulation and recruitment of all targeted neurons. Rats were transduced in the lumbar spinal cord with AAV2/9 to express the excitatory LMO3 under control of a pan-neuronal or motor neuron-specific promoter. A day after contusion injury of the thoracic spine, rats received either CTZ or vehicle every other day for 2 weeks. Activation of either interneuron or motor neuron populations below the level of injury significantly improved locomotor recovery lasting beyond the time of stimulation. Utilizing histological and gene expression methods we identified neuronal plasticity as a likely mechanism underlying the functional recovery. These findings provide a foundation for a rational approach to spinal cord injury rehabilitation, thereby advancing approaches for functional recovery after SCI.

neuroscience↗