Biology subjects

Green, W.

Publications and source records attributed to Green, W..

4 recordsLinked to original sources

bioRxiv2025

Engineering a human-based translational activator for targeted protein expression restoration

Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate mRNA would be an advantageous approach to correct gene expression, but has not been evaluated in an in vivo disease model. Here, we investigated if a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identifiy and engineere human proteins capable of increasing mRNA translation using the CRISPR-Cas Inspired RNA-targeting System (CIRTS) platform to enable programmable, guide RNA (gRNA)-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3), that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold - key phenotypic indicators of Dravet syndrome. This work validates a new strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential translational activation has to address neurological haploinsufficiency. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=36 SRC="FIGDIR/small/663984v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@9fd35aorg.highwire.dtl.DTLVardef@ecf89borg.highwire.dtl.DTLVardef@1b591d2org.highwire.dtl.DTLVardef@17873e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Evaluation of an image-derived input function for kinetic modeling of nicotinic acetylcholine receptor-binding PET ligands

Development of positron emission tomography (PET) radiotracers that bind with high-affinity to 4{beta}2-type nicotinic receptors (4{beta}2Rs) allows for in vivo investigations of the mechanisms underlying nicotine addiction and smoking cessation. One challenge associated with preclinical PET imaging involves the lack of true tissue reference regions free of specific tracer binding in the rodent brain, impeding accurate quantification of the tracer binding potential. Here, we investigate the use of an image-derived arterial input function for kinetic analysis of radiotracer binding in male and female mice. Two radiotracers were explored in this study: 2-[18F]FA85380 (2-FA), which displays similar pKa and binding affinity to the smoking cessation drug varenicline (Chantix), and [18F]Nifene, which displays similar pKa and binding affinity to nicotine. For both radiotracers, time-activity curves of the left ventricle of the heart displayed similar standardized uptake values (SUVs) across wild type mice, mice lacking the {beta}2 subunit for tracer binding, and acute nicotine-treated mice, whereas typical reference tissue SUVs displayed high variation between groups. Binding potential values estimated from a two-tissue compartment model (2TCM) fit of the data with the image-derived input function were significantly higher than estimates from reference tissue-based estimations. Rate constants of radiotracer dissociation were very slow for 2-FA and very fast for Nifene, similar to the in vitro dissociation rates reported for varenicline and nicotine, respectively. We conclude that use of an image-derived input function for kinetic modeling of nicotinic PET ligands improves quantification compared to reference tissue-based methods, and that the chemical properties of 2-FA and Nifene are suitable to study receptor response to nicotine addiction and smoking cessation therapies.

neuroscience↗

bioRxiv2021

Trapping of Nicotinic Acetylcholine Receptor Ligands Assayed by in vitro Cellular Studies and in vivo PET Imaging

A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membranepermeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pKa and high-affinity for 4{beta}2-type nicotinic receptors (4{beta}2Rs) are trapped in intracellular acidic vesicles containing 4{beta}2Rs in vitro. Nicotine, with lower pKa and 4{beta}2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligands in vivo in mouse brain and identifying the trapping brain organelle in vitro as Golgi satellites (GSats). Two PET 18F-labelled imaging ligands were chosen: [18F]2-FA85380 (2-FA) with varenicline-like pKa and affinity and [18F]Nifene with nicotine-like pKa and affinity. [18F]2-FA PET-imaging kinetics were very slow consistent with 2-FA trapping in 4{beta}2R-containing GSats. In contrast, [18F]Nifene kinetics were rapid, consistent with its binding to 4{beta}2Rs but no trapping. Specific [18F]2-FA and [18F]Nifene signals were eliminated in {beta}2 subunit knockout mice or by acute nicotine injections demonstrating binding to sites on {beta}2-containing receptors. Chloroquine, which dissipates GSat pH gradients, reduced [18F]2-FA distributions while having little effect on [18F]Nifene distributions in vivo consistent with only [18F]2-FA trapping in GSats. These results are further supported by in vitro findings where dissipation of GSat pH gradients blocks 2-FA trapping in GSats without affecting Nifene. By combining in vitro and in vivo imaging, we mapped both the brain-wide and subcellular distributions of weak-base nicotinic receptor ligands. We conclude that ligands, such as varenicline, are trapped in neurons in 4{beta}2R-containing GSats, which results in very slow release long after nicotine is gone after smoking. SignificanceMechanisms of nicotine addiction remain poorly understood. An earlier study using in vitro methods found that the anti-smoking nicotinic ligand, varenicline (Chantix) was trapped in 4{beta}2R-containing acidic vesicles. Using a fluorescent labeled high-affinity nicotinic ligand, this study provided evidence that these intracellular acidic vesicles were 4{beta}2R-containing Golgi satellites. In vivo PET imaging with F-18 labeled nicotinic ligands provided additional evidence that differences in PET ligand trapping in acidic vesicles were the cause of differences in PET ligand kinetics and subcellular distributions. These findings combining in vitro and in vivo imaging revealed new mechanistic insights into the kinetics of weak base PET imaging ligands and the subcellular mechanisms underlying nicotine addiction.

neuroscience↗

bioRxiv2018

INPP5E controls ciliary localization of phospholipids and odor response kinetics in a mouse model of Joubert syndrome

Ciliopathies manifested in part by a dysfunction of several phosphoinositide 5phosphatases constitute Lowes, Dent disease 2 and Joubert syndromes through critical involvement of properly functioning primary cilia (PC). We showed that deletion of INPP5E under the control of OMP-Cre in mature mouse olfactory sensory neurons (OSNs) led to a dramatic redistribution of PI(4,5)P2 (PIP2) in cilia, significant reduction of PI(3,4)P2 and enrichment of PI(3,4,5)P3 in knobs. Redistribution of the phospholipids accompanied marked elongation of cilia in INPP5E-OMP knockout (KO) OSNs. Such a dramatic remodeling of phospholipid composition however did not affect other integral membrane lipids (cholesterol, sphingomyelin, glycosylated phosphaditylinositol, phosphatidylserine). Proteins known to bind with high affinity PIP2 entered the cilia of the KO OSNs. Loss of INPP5E did not affect ciliary localization of endogenous olfactory receptor M71/M72 or distribution and movement of IFT122 particles implicating independent of phospholipids mechanism of retrograde protein transport in cilia of mature OSNs. Net odor sensitivity and response magnitude as measured by EOG was not affected by the mutation. However, odor adaptation in the KO mouse was significantly impaired resulting in less efficient recovery and altered inactivation kinetics of the odor response at the EOG and single-cell level. These findings implicate phosphoinositide-dependent regulation of active Ca2+ extrusion in OSNs whereby controlling the rate of sensory adaptation.\n\nSignificance statementCurrently there are little if any available treatment to cure congenital ciliopathies. This is in part due to lack of basic knowledge of cilia biology. Olfactory cilia as well as primary cilia appear to be a phospholipid privileged organelle distinct from the rest of plasma membrane albeit sharing its continuity. We characterized distribution of several critically important for cell biology phospholipids and showed that their balance, especially of PIP2, is disrupted in Joubert syndrome animal model and has functional implications. Virally assisted delivery of wild type INPP5E to the mutant OSNs was able to restore localization of PIP2 and rescued impaired response to odor.

neuroscience↗