Monoamine oxidase-B (MAO-B) has recently emerged as a potential therapeutic target for Alzheimer's disease (AD) because of its association with aberrant gamma-aminobutyric acid (GABA) production in reactive astrocytes. Although short-term treatment with irreversible MAO-B inhibitors, such as selegiline, improves cognitive deficits in AD patients, long-term treatments have shown disappointing results. We show that prolonged treatment with selegiline fails to reduce aberrant astrocytic GABA levels and rescue memory impairment in APP/PS1 mice, an animal model of AD, because of increased activity in compensatory genes for a GABA-synthesizing enzyme, diamine oxidase (DAO). We have developed a potent, highly selective, and reversible MAO-B inhibitor, KDS2010 (IC(50) = 7.6 nM; 12,500-fold selectivity over MAO-A), which overcomes the disadvantages of the irreversible MAO-B inhibitor. Long-term treatment with KDS2010 does not induce compensatory mechanisms, thereby significantly attenuating increased astrocytic GABA levels and astrogliosis, enhancing synaptic transmission, and rescuing learning and memory impairments in APP/PS1 mice.
Title: Rapid identification of unknown carboxyl esterase activity in Corynebacterium glutamicum using RNA-guided CRISPR interference Lee SS, Shin H, Jo S, Lee SM, Um Y, Woo HM Ref: Enzyme Microb Technol, 114:63, 2018 : PubMed
RNA-guided genome engineering technologies have been developed for the advanced metabolic engineering of microbial cells to enhance production of value-added chemicals in Corynebacterium glutamicum as an industrial host. In this study, the RNA-guided CRISPR interference (CRISPRi) was applied to rapidly identify of unknown genes for native esterase activity in C. glutamicum. Combining with the carboxyl esterase (MekB) protein sequence alignment, two target genes (the cg0961 and cg0754) were selected for the CRISPRi application to investigate the possible native esterase in C. glutamicum. The recombinant strain with repressed expression of the cg0961 gene exhibited almost no capability on degradation of methyl acetate as a substrate of carboxyl esterase. This result was also confirmed in the cg0961 gene deletion mutant. Thus, we concluded that Cg0961 plays a major role of the native carboxyl esterase activity in C. glutamicum. In addition, CRISPRi demonstrated an application for gene identification and its function as another genetic tool for metabolic engineering in C. glutamicum.
Title: Reciprocal regulation of acetylcholinesterase and butyrylcholinesterase in mammalian skeletal muscle Berman HA, Decker MM, Jo S Ref: Developmental Biology, 120:154, 1987 : PubMed
Developmental regulation, from the fetal period to 11 months of age, and the influence of denervation on the appearance and disappearance of the molecular forms of acetylcholinesterase (AchE) and butyrylcholinesterase (BuchE) in rat skeletal muscle were examined. The enzyme forms were extracted from anterior tibialis in 0.01 M sodium phosphate buffer, pH 7.0, containing 1 N NaCl, 0.01 M EGTA, 1% Triton X-100, and a cocktail of antiproteases, and analyzed by velocity sedimentation on 5-20% linear sucrose gradients. Three principal forms, denoted by sedimentation coefficients of 4, 10.8, and 16 S, were observed in muscle from all age groups. The amounts of each of the molecular forms of AchE and BuchE in skeletal muscle exhibited distinct and reciprocal patterns of appearance and disappearance during pre- and postnatal development. In tissue derived from animals less than 2 weeks of age, BuchE represented the predominant component of activity in the 4 S form, was present equally with AchE in the 10.8 S form, and was subordinate to AchE in the 16 S form. Between 1 and 2 weeks of age a progressive increase in AchE activities coincident with a reduction in BuchE activities resulted in inversion in the amounts of the two enzymes present in adult muscle. Denervation of muscle caused a dramatic reduction in the presence of AchE molecular forms with no discernable influence on the presence of BuchE molecular forms. These results indicate that biosynthesis of BuchE is strictly regulated in a reciprocal manner with that of AchE, and that BuchE metabolism is independent of the state of muscle innervation. Increased synthesis of AchE and either reduced synthesis or increased degradation of BuchE can account for the reciprocal regulation of these enzymes. These characteristics of mammalian muscle contrast sharply with characteristics deduced for avian tissue (Silman et al. (1979) Nature (London) 280, 160-162). The innervation-independent metabolism of BuchE and the diverse modes of its regulation in different tissue from different species signify that BuchE function may be unrelated to cholinergic neurotransmission.
