Visual function restoration in a mouse model of Leber congenital amaurosis via therapeutic base editing.

Leber congenital amaurosis (LCA), an inherited retinal degeneration, causes severe visual dysfunction in children and adolescents. In patients with LCA, pathogenic variants, such as RPE65, are evident in specific genes, related to the functions of retinal pigment epithelium and photoreceptors. In contrast to the original Cas9, base editing tools can correct pathogenic substitutions without generation of DNA double-stranded breaks (DSBs). In this study, dual adeno-associated virus (AAV) vectors containing split adenine base editors (ABEs) with trans-splicing intein were prepared for in vivo base editing in retinal degeneration of 12 (rd12) mice, an animal model of LCA, possessing a nonsense mutation of C to T transition in the Rpe65 gene (p.R44X). Subretinal injection of AAV-ABE in retinal pigment epithelial cells of rd12 mice resulted in an A to G transition. The on-target editing was sufficient for recovery of wild-type mRNA, RPE65 protein, and light-induced electrical responses from the retina. Compared with our previous therapeutic editing strategies using Cas9 and prime editing, or with the gene transfer strategy shown in the current study, our results suggest that, considering the editing efficacy and functional recovery, ABEs could be a strong, reliable method for correction of pathogenic variants in the treatment of LCA.

Neuroprotective Activity of Methanolic Extract of Lysimachia christinae against Glutamate Toxicity in HT22 Cell and Its Protective Mechanisms.

PURPOSE: Excessive glutamate amount can give oxidative stress to neuronal cells, and the accumulation of cell death can trigger the neurodegenerative disorders. In this study, we discovered the neuroprotective effect of Lysimachia christinae Hance in the mouse hippocampal HT22 cell line. METHOD: Overnight incubated HT22 cells were pretreated with L. christinae extract dose dependently (1, 10, and 100 µg/ml). Followed by then, glutamate was treated. These treated cells were incubated several times again, and cell viability, accumulation of reactive oxygen species (ROS) and Ca2+, mitochondrial membrane potential (MMP), and glutathione-related enzyme amount were measured. RESULTS: As a result, L. christinae increases the cell viability by inhibiting the ROS and Ca2+ formation, recovering the level of MMP and enhancing the activity of glutathione production compared with only vehicle-treated groups. CONCLUSION: These draw that L. christinae may remarkably decelerate the neurodegeneration by minimizing neuronal cell damage via oxidative stress.

Neuroprotective effects of Magnoliae Flos extract in mouse hippocampal neuronal cells.

Magnoliae Flos (MF) is a traditional medicinal herb used for managing rhinitis, sinusitis and headache. The purpose of the present study was to determine the neuroprotective effect of MF against glutamate-induced oxidative stress and to assess the underlying mechanism. Glutamate is a major endogenous excitatory neurotransmitter in the brain and contributes to the development of neurodegenerative diseases by excessive activation. MF extract was subjected to a neuroprotective effect assay in HT22 mouse hippocampal cells. The mechanism underlying the neuroprotective effect of MF extract was evaluated by assaying reactive oxygen species (ROS) levels, intracellular Ca2+ levels, mitochondrial membrane potential, glutathione level and antioxidant enzyme activity in HT22 cells. MF extract significantly decreased glutamate-induced death of HT22 cells (80.83 ± 7.34% relative neuroprotection). MF extract reduced the intracellular ROS and Ca2+ levels and increased the glutathione level and glutathione reductase and glutathione peroxide activities. Moreover, MF extract attenuated the mitochondrial membrane potential in HT22 cells. These results suggested that MF extract exerts a neuroprotective effect against oxidative stress HT22 cells, which was mediated by its antioxidant activity.

Neuroprotective effect of Aronia melanocarpa extract against glutamate-induced oxidative stress in HT22 cells.

BACKGROUND: Glutamate (an endogenous excitatory neurotransmitter) at high concentrations contributes to the development of neurodegenerative diseases. Aronia melanocarpa (A. melanocarpa) berries contain anthocyanins and have high antioxidant activities. In this study, we evaluated whether A. melanocarpa berries could protect neuronal cells against glutamate-induced oxidative stress. METHOD: A. melanocarpa berries exerted a protective effect against cytotoxicity in HT22 mouse hippocampal cells by MTT assay. We evaluated oxidative stress parameters including ROS level, intracellular Ca2+ level, glutathione level and antioxidant enzyme activity in HT22 cells to elucidate the mechanism of its neuroprotective effect. RESULTS: A. melanocarpa berries decreased glutamate-induced death of HT22 cells. In addition, A. melanocarpa berries reduced ROS and intracellular Ca2+ levels. Glutathione level, antioxidant enzymes, glutathione reductase and glutathione peroxide activities and mitochondrial membrane potential were also increased in HT22 cells. CONCLUSION: These results suggested that A. melanocarpa berries protected HT22 cells by exerting an antioxidant effect.