Genetics of cystogenesis in base-edited human organoids reveal therapeutic strategies for polycystic kidney disease.

In polycystic kidney disease (PKD), microscopic tubules expand into macroscopic cysts. Among the world's most common genetic disorders, PKD is inherited via heterozygous loss-of-function mutations but is theorized to require additional loss of function. To test this, we establish human pluripotent stem cells in allelic series representing four common nonsense mutations, using CRISPR base editing.

Targeting G542X CFTR nonsense alleles with ELX-02 restores CFTR function in human-derived intestinal organoids.

Background: Promoting full-length protein production is a requisite step to address some of the remaining unmet medical need for those with Cystic Fibrosis (CF) nonsense alleles. ELX-02 promotes read-through of mRNA transcripts bearing nonsense mutations, including the most common CF nonsense allele G542X, in several different preclinical models including human bronchial epithelial cells. Here we evaluate ELX-02 mediated read-through using the CFTR-dependent Forskolin-induced swelling (FIS) assay across a selection of G542X genotype patient derived organoids (PDOs).

Intravitreal administration of small molecule read-through agents demonstrate functional activity in a nonsense mutation mouse model.

The prevalence of nonsense mutations as a class within genetic diseases such as inherited retinal disorders (IRDs) presents an opportunity to develop a singular, common therapeutic agent for patients whose treatment options are otherwise limited. We propose a novel approach to addressing IRDs utilizing Eukaryotic Ribosome Selective Glycosides, ELX-01 and ELX-06, delivered to the eye by intravitreal (IVT) injection. We assessed read-through activity in vitro using a plasmid-based dual luciferase assay and in vivo in a mouse model of oculocutaneous albinism type 2.

ELX-02 Generates Protein via Premature Stop Codon Read-Through without Inducing Native Stop Codon Read-Through Proteins.

ELX-02 is a clinical stage, small-molecule eukaryotic ribosomal selective glycoside acting to induce read-through of premature stop codons (PSCs) that results in translation of full-length protein. However, improved read-through at PSCs has raised the question of whether native stop codon (NSC) fidelity would be impacted. Here, we compare read-through by ELX-02 in PSC and NSC contexts.

Estrogen receptor β ligand therapy activates PI3K/Akt/mTOR signaling in oligodendrocytes and promotes remyelination in a mouse model of multiple sclerosis.

The identification of a drug that stimulates endogenous myelination and spares axon degeneration during multiple sclerosis (MS) could potentially reduce the rate of disease progression. Using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we have previously shown that prophylactic administration of the estrogen receptor (ER) β ligand 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) decreases clinical disease, is neuroprotective, stimulates endogenous myelination, and improves axon conduction without altering peripheral cytokine production or reducing central nervous system (CNS) inflammation. Here, we assessed the effects of therapeutic DPN treatment during peak EAE disease, which represents a more clinically relevant treatment paradigm.

Attenuation of corpus callosum axon myelination and remyelination in the absence of circulating sex hormones.

Sex differences in the structure and organization of the corpus callosum (CC) can be attributed to genetic, hormonal or environmental effects, or a combination of these factors. To address the role of gonadal hormones on axon myelination, functional axon conduction and immunohistochemistry analysis of the CC in intact, gonadectomized and hormone-replaced gonadectomized animals were used. These groups were subjected to cuprizone diet-induced demyelination followed by remyelination.

An animal model of cortical and callosal pathology in multiple sclerosis.

The pathological and radiological hallmarks of multiple sclerosis (MS) include multiple demyelinated lesions disseminated throughout the white matter of the central nervous system (CNS). More recently, the cerebral cortex has been shown to be affected in MS, but the elucidation of events causing cortical demyelination has been hampered by the lack of animal models reflecting such human cortical pathology. In this report, we have described the presence of cortical gray matter and callosal white matter demyelinating lesions in the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of MS.

Molecular targets and mechanisms for ethanol action in glycine receptors.

Glycine receptors (GlyRs) are recognized as the primary mediators of neuronal inhibition in the spinal cord, brain stem and higher brain regions known to be sensitive to ethanol. Building evidence supports the notion that ethanol acting on GlyRs causes at least a subset of its behavioral effects and may be involved in modulating ethanol intake. For over two decades, GlyRs have been studied at the molecular level as targets for ethanol action.

Loop 2 structure in glycine and GABA(A) receptors plays a key role in determining ethanol sensitivity.

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and gamma-aminobutyric acid type A receptors (GABA(A)Rs). To test this, we mutated Loop 2 in the alpha1 subunit of GlyRs and in the gamma subunit of alpha1beta2gamma2GABA(A)Rs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of alpha1GlyR subunits with Loop 2 from the deltaGABA(A)R (deltaL2), but not the gammaGABA(A)R subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function.

Assaying the functional effects of demyelination and remyelination: revisiting field potential recordings.

The occurrence and histopathological characteristics of demyelination and neurodegeneration have been well described in different demyelinating mouse models. However, histopathological analysis is limiting in that it is unable to describe the functional consequences of demyelination and recovery after remyelination. Establishing the functional correlates of axon demyelination and remyelination is an important goal and can be used to measure axon function and develop neuroprotective therapies.

Roles for loop 2 residues of alpha1 glycine receptors in agonist activation.

The present study tested the hypothesis that several residues in Loop 2 of alpha1 glycine receptors (GlyRs) play important roles in mediating the transduction of agonist activation to channel gating. This was accomplished by investigating the effect of cysteine point mutations at positions 50-60 on glycine responses in alpha1GlyRs using two-electrode voltage clamp of Xenopus oocytes. Cysteine substitutions produced position-specific changes in glycine sensitivity that were consistent with a beta-turn structure of Loop 2, with odd-numbered residues in the beta-turn interacting with other agonist-activation elements at the interface between extracellular and transmembrane domains.

Targets for ethanol action and antagonism in loop 2 of the extracellular domain of glycine receptors.

The present studies used increased atmospheric pressure in place of a traditional pharmacological antagonist to probe the molecular sites and mechanisms of ethanol action in glycine receptors (GlyRs). Based on previous studies, we tested the hypothesis that physical-chemical properties at position 52 in extracellular domain Loop 2 of alpha1GlyRs, or the homologous alpha2GlyR position 59, determine sensitivity to ethanol and pressure antagonism of ethanol. Pressure antagonized ethanol in alpha1GlyRs that contain a non-polar residue at position 52, but did not antagonize ethanol in receptors with a polar residue at this position.

Evidence that ethanol acts on a target in Loop 2 of the extracellular domain of alpha1 glycine receptors.

Considerable evidence indicates that ethanol acts on specific residues in the transmembrane domains of glycine receptors (GlyRs). In this study, we tested the hypothesis that the extracellular domain is also a target for ethanol action by investigating the effect of cysteine substitutions at positions 52 (extracellular domain) and 267 (transmembrane domain) on responses to n-alcohols and propyl methanethiosulfonate (PMTS) in alpha1GlyRs expressed in Xenopus oocytes. In support of the hypothesis: (i) The A52C mutation changed ethanol sensitivity compared to WT GlyRs; (ii) PMTS produced irreversible alcohol-like potentiation in A52C GlyRs; and (iii) PMTS binding reduced the n-chain alcohol cutoff in A52C GlyRs.

Multiple sites of ethanol action in alpha1 and alpha2 glycine receptors suggested by sensitivity to pressure antagonism.

The current study used an ethanol antagonist, increased atmospheric pressure, to test the hypothesis that ethanol acts on multiple sites in glycine receptors (GlyRs). The effects of 12 times normal atmospheric pressure of helium-oxygen gas (pressure) on ethanol-induced potentiation of GlyR function in Xenopus oocytes expressing human alpha1, alpha2 or the mutant alpha1(A52S) GlyRs were measured using two-electrode voltage clamp. Pressure reversibly antagonized potentiation of glycine in alpha1 GlyR by 40-200 mm ethanol, but did not antagonize 10 and 25 mm ethanol in the same oocytes.

Ethanol potentiation of glycine receptors expressed in Xenopus oocytes antagonized by increased atmospheric pressure.

Background: Behavioral and biochemical studies indicate that exposure to 12 times normal atmospheric pressure (12 ATA) of helium-oxygen gas (heliox) is a direct, selective ethanol antagonist. The current study begins to test the hypothesis that ethanol acts by a common mechanism on ligand-gated ion channels by expanding previous hyperbaric investigations on gamma-aminobutyric acid type A (GABA(A)) receptors (GABA(A)Rs) at the biochemical level to alpha(1)glycine (GlyRs) expressed in Xenopus oocytes.

Methods: Oocytes expressing wild-type alpha(1) homomeric GlyRs were voltage-clamped (-70 mV) and tested in the presence of glycine (EC(2)) +/- ethanol (50-200 mM) under 1 ATA control and 3 to 12 ATA heliox conditions.