Biology subjects

Tree, M. O.

Publications and source records attributed to Tree, M. O..

5 recordsLinked to original sources

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

Spheroid architecture strongly induces miR-221/222 expression and promotes oxidative phosphorylation and survival of mobile tumor cells through a mechanism that includes restriction of miR-9 expression

Tumor cell spheroids are three dimensional multicellular structures that form during the expansive growth of carcinoma cells. Spheroids support tumor metastasis as vehicles of dissemination, promoting growth and survival of bulk tumor and cancer stem cells within the mobile tumor cell population. Deciphering how spheroid architecture affects tumor cell phenotype will be essential for the development of therapeutics to inhibit transperitoneal metastasis and the development of peritoneal carcinomatosis by ovarian cancers. We investigated how spheroid formation directly affects OXPHOS activity and microRNA expression in a cultured ovarian carcinoma cell system. The rate of oxidative phosphorylation/respiration per cell in spheroids was nearly double that of the same cell type growing in suspension as single cells. Cells growing as spheroids showed greatly enhanced expression of miR-221/222, an oncomiR that targets multiple tumor suppressor genes, promotes invasion, as well as reduced expression of miR-9, which targets mitochondrial tRNA-modification enzymes and inhibits OXPHOS. Consistent with the greater efficiency of ATP generation afforded by OXPHOS phosphorylation, tumor cells growing as spheroids injected into the nutrient-poor environment of the murine peritoneum survived longer than the cells growing in suspension as loosely associated aggregates. The data suggest that in addition to the reported effects of spheroid formation on cancer cell growth and phenotype, including promotion of stem cell generation, spheroid architecture increases the OXPHOS activity of constituent tumor cells. During the mobile phase of metastasis, when ovarian tumor cells disperse through nutrient-poor environments such as the peritoneum, enhanced OXPHOS activity afforded by spheroid architecture would enhance survival and thereby contribute to metastatic potential.

cancer 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↗