Alterations of ATG4A and LC3B in neurons derived from Alzheimer's disease patients.

We investigated the alterations in autophagy-related molecules in neurons differentiated from induced pluripotent stem cells obtained from patients with Alzheimer's disease (AD). Consistent with our previous microarray data, ATG4A protein was upregulated in the neurons derived from a familial AD patient with an APP-E693Δ mutation who showed accumulation of intracellular amyloid ß peptide (Aß). This upregulation was reversed by inhibiting Aß production, suggesting that the intracellular Aß may be responsible for the upregulation of ATG4A. The LC3B-II/LC3B-I ratio, an index of autophagosome formation, was lower in the neurons derived from the AD patient with APP-E693Δ as well as the neurons derived from other familial and sporadic AD patients. These findings indicate that dysregulation of autophagy-related molecules may accelerate the pathogenesis of AD.

Identification and Characterization of Synaptic Vesicle Membrane Protein VAT-1 Homolog as a New Catechin-Binding Protein.

(-)-Epigallocatechin-3-gallate (EGCg), a major constituent of green tea extract, is well-known to exhibit many beneficial actions for human health by interacting with numerous proteins. In this study we identified synaptic vesicle membrane protein VAT-1 homolog (VAT1) as a novel EGCg-binding protein in human neuroglioma cell extracts using a magnetic pull-down assay and LC-tandem mass spectrometry. We prepared recombinant human VAT1 and analyzed its direct binding to EGCg and its alkylated derivatives using surface plasmon resonance. For EGCg and the derivative NUP-15, we measured an association constant of 0.02-0.85 ×103 M-1s-1 and a dissociation constant of nearly 8 × 10-4 s-1. The affinity Km(affinity) of their binding to VAT1 was in the 10-20 µM range and comparable with that of other EGCg-binding proteins reported previously. Based on the common structure of the compounds, VAT1 appeared to recognize a catechol or pyrogallol moiety around the B-, C- and G-rings of EGCg. Next, we examined whether VAT1 mediates the effects of EGCg and NUP-15 on expression of neprilysin (NEP). Treatments of mock cells with these compounds upregulated NEP, as observed previously, whereas no effect was observed in the VAT1-overexpressing cells, indicating that VAT1 prevented the effects of EGCg or NUP-15 by binding to and inactivating them in the cells overexpressing VAT1. Further investigation is required to determine the biological significance of the VAT1-EGCg interaction.

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.

Performance Evaluation of IMU and DVL Integration in Marine Navigation.

Global navigation satellite system (GNSS) spoofing poses a significant threat to maritime logistics. Many maritime electronic devices rely on GNSS time, positioning, and speed for safe vessel operation. In this study, inertial measurement unit (IMU) and Doppler velocity log (DVL) devices, which are important in the event of GNSS spoofing or outage, are considered in conventional navigation. A velocity integration method using IMU and DVL in terms of dead-reckoning is investigated in this study. GNSS has been widely used for ship navigation, but IMU, DVL, or combined IMU and DVL navigation have received little attention. Military-grade sensors are very expensive and generally cannot be utilized in smaller vessels. Therefore, this study focuses on the use of consumer-grade sensors. First, the performance of a micro electromechanical system (MEMS)-based yaw rate angle with DVL was evaluated using 60 min of raw data for a 50 m-long ship located in Tokyo Bay. Second, the performance of an IMU-MEMS using three gyroscopes and three accelerometers with DVL was evaluated using the same dataset. A gyrocompass, which is equipped on the ship, is used as a heading reference. The results proved that both methods could achieve less than 1 km horizontal error in 60 min.

Proline-rich transmembrane protein 2 knock-in mice present dopamine-dependent motor deficits.

Mutations of proline-rich transmembrane protein 2 (PRRT2) lead to dyskinetic disorders such as paroxysmal kinesigenic dyskinesia (PKD), which is characterized by attacks of involuntary movements precipitated by suddenly initiated motion, and some convulsive disorders. Although previous studies have shown that PKD might be caused by cerebellar dysfunction, PRRT2 has not been sufficiently analyzed in some motor-related regions, including the basal ganglia, where dopaminergic neurons are most abundant in the brain. Here, we generated several types of Prrt2 knock-in (KI) mice harboring mutations, such as c.672dupG, that mimics the human pathological mutation c.649dupC and investigated the contribution of Prrt2 to dopaminergic regulation. Regardless of differences in the frameshift sites, all truncating mutations abolished Prrt2 expression within the striatum and cerebral cortex, consistent with previous reports of similar Prrt2 mutant rodents, confirming the loss-of-function nature of these mutations. Importantly, administration of l-dopa, a precursor of dopamine, exacerbated rotarod performance, especially in Prrt2-KI mice. These findings suggest that dopaminergic dysfunction in the brain by the PRRT2 mutation might be implicated in a part of motor symptoms of PKD and related disorders.

The role of TREM2 N-glycans in trafficking to the cell surface and signal transduction of TREM2.

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. TREM2 is glycosylated in vitro and in vivo, but the significance of the modification is unknown. We previously established a sensitive and specific reporter cell model involving cultured Jurkat cells stably expressing a luciferase reporter gene and a gene encoding a TREM2DAP12 fusion protein to monitor TREM2-dependent signalling. In the present study, we prepared modified reporter cells to investigate the role of the N-glycans at N20 and N79. We show that the N-glycans at N79 have a requisite role in translocation of TREM2 to the cell surface, while the N-glycans at both N20 and N79 have a critical role in intracellular signal transduction. Our results indicate that structural changes to the TREM2 N-glycans may cause microglial dysfunction that contributes to the pathogenesis of neurodegenerative disorders and that maintaining the integrity of TREM2 N-glycosylation and the responsible glycosyltransferases may be a novel therapeutic strategy to treat these disorders.