Enhancement of Neprilysin Activity by Natural Polyphenolic Compounds and Their Derivatives in Cultured Neuroglioma Cells.

The onset of Alzheimer's disease (AD) is characterized by accumulation of amyloid ß peptide (Aß) in the brain. Neprilysin (NEP) is one of the major Aß-degrading enzymes. Given findings that NEP expression in the brain declines from the early stage of AD before apparent neuronal losses are observed, enhancement of NEP activity and expression may be a preventive and therapeutic strategy relevant to disease onset. We screened for compounds that could enhance the activity and expression of NEP using a polyphenol library previously constructed by our research group and investigated the structure-activity relationships of the identified polyphenols. We found that amentoflavone, apigenin, kaempferol, and chrysin enhanced the activity and expression of NEP, suggesting that chemical structures involving a double bond between positions 2 and 3 in the C ring of flavones are important for NEP enhancement, while catechol or pyrogallol structures, except for the galloyl group of catechins, abolished these effects. Moreover, natural compounds, such as quercetin, were not effective per se, but were changed to effective compounds by adding a lipophilic moiety. Using our study findings, we propose improvements for dietary habits with experimental evidence, and provide a basis for the development of novel small molecules as disease-modifying drugs for AD.

Activity-dependent cleavage of dyskinesia-related proline-rich transmembrane protein 2 (PRRT2) by calpain in mouse primary cortical neurons.

Mutations of PRRT2 (proline-rich transmembrane protein 2) cause several neurological disorders, represented by paroxysmal kinesigenic dyskinesia (PKD), which is characterized by attacks of involuntary movements triggered by sudden voluntary movements. PRRT2 is reported to suppress neuronal excitation, but it is unclear how the function of PRRT2 is modulated during neuronal excitation. We found that PRRT2 is processed to a 12 kDa carboxy-terminal fragment (12K-CTF) by calpain, a calcium-activated cysteine protease, in a neuronal activity-dependent manner, predominantly via NMDA receptors or voltage-gated calcium channels. Furthermore, we clarified that 12K-CTF is generated by sequential cleavages at Q220 and S244. The amino-terminal fragment (NTF) of PRRT2, which corresponds to PKD-related truncated mutants, is not detected, probably due to rapid cleavage at multiple positions. Given that 12K-CTF lacks most of the proline-rich domain, this cleavage might be involved in the activity-dependent enhancement of neuronal excitation perhaps through transient retraction of PRRT2's function. Therefore, PRRT2 might serve as a buffer for neuronal excitation, and lack of this function in PKD patients might cause neuronal hyperexcitability in their motor circuits.

In vitro enzymatic electrochemical monitoring of glucose metabolism and production in rat primary hepatocytes on highly O2 permeable plates.

In situ continuous glucose monitoring under physiological culture conditions is imperative in understanding the dynamics of cell and tissue behaviors and their physiological responses since glucose plays an important role in principal source of biological energy. We therefore examined physiologically relevant dynamic changes in glucose levels based on glucose metabolism and production during aerobic culture (10% O2) of rat primary hepatocytes stimulated with insulin or glucagon on a highly O2 permeable plate, which can maintain the oxygen concentration close to the periportal zone of the liver. As glucose monitoring devices, we used oxygen-independent glucose dehydrogenase-modified single-walled carbon nanotube electrodes placed close to the surface of the hepatocytes. The current response of glucose oxidation slightly decreased after the addition of insulin in the presence of glucose due to the acceleration of glucose uptake by the hepatocytes, whereas that significantly increased after the addition of glucagon and fructose even in the absence of glucose due to the conversion of fructose to glucose based on gluconeogenesis. These phenomena might be consistent relatively with the physiological behaviors of hepatocytes in the periportal region. The present monitoring system would be useful for the studies of glucose homeostasis and diabetes in vitro.

Aminophospholipids are signal-transducing TREM2 ligands on apoptotic cells.

Variants of triggering receptor expressed on myeloid cells 2 (TREM2) are associated with an increased incidence of Alzheimer's disease, as well as other neurodegenerative disorders. Using a newly developed, highly sensitive reporter cell model, consisting of Jurkat T cells stably overexpressing a reporter gene and a gene encoding TREM2DAP12 fusion protein, we show here that TREM2-dependent signal transduction in response to apoptotic Neuro2a cells is mediated by aminophospholipid ligands, phosphatidylserine and phosphatidylethanolamine, which are not exposed on the intact cell surface, but become exposed upon apoptosis. We also show that signal-transducing TREM2 ligands different from aminophospholipids, which appear to be derived from neurons, might be present in membrane fractions of mouse cerebral cortex. These results may suggest that TREM2 regulates microglial function by transducing intracellular signals from aminophospholipids on apoptotic cells, as well as unidentified ligands in the membranes of the cerebral cortex.