Dendritic spines are postsynaptic protrusions at excitatory synapses that are critical for proper neuronal synaptic transmission. While lipid and protein membrane components are necessary for spine formation, it is largely unknown how they are recruited to developing spines. Endosomal trafficking is one mechanism that may influence this development. We recently reported that Lemur kinase 1A (LMTK1A), a membrane-bound Ser/Thr kinase, regulates trafficking of endosomes in neurons. LMTK1 has been shown to be a p35 Cdk5 activator-binding protein and a substrate for Cdk5-p35; however, its neuronal function has not been sufficiently studied. Here, we investigate the role of LMTK1 in spine formation. Depletion of LMTK1 increases spine formation, maturation, and density in primary cultured neurons and in mouse brain of either sex. Additionally, expression of kinase-negative LMTK1 stimulates spine formation in primary neurons andin vivo. LMTK1 controls spine formation through Rab11, a regulator of recycling endosome trafficking. We identify TBC1D9B, a Rab11A GTPase-activating protein (Rab11A GAP), as a LMTK1 binding protein, and find that TBC1D9B mediates LMTK1 activity on Rab11A. TBC1D9B inactivates Rab11A under the control of LMTK1A. Further, by analyzing the effect of decreased TBC1D9B expression in primary neurons, we demonstrate that TBC1D9B indeed regulates spine formation. This is the first demonstration of the biological function of TBC1D9B. Together, with the regulation of LMTK1 by Cdk5-p35, we propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel signaling mechanism regulating endosomal transport for synapse formation and function.SIGNIFICANCE STATEMENTDendritic spines are postsynaptic specializations essential for synaptic transmission. However, it is not known how critical membrane components are recruited to spines for their formation. Endosomal trafficking is one such mechanism that may mediate this process. Here we investigate regulators of endosomal trafficking and their contribution to spine formation. We identify two novel factors, LMTK1 and TBC1D9B, which regulate spine formation upstream of Rab11A, a small GTPase. LMTK1 is a membrane bound Ser/Thr kinase regulated by Cdk5-p35, and TBC1D9B is a recently identified Rab11 GAP. LMTK1 controls the GAP activity of TBC1D9B on Rab11A, and TBC1D9B mediates the LMTK1 activity on Rab11A. We propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel mechanism controlling spine formation and function.

Trafficking of recycling endosomes (REs) is regulated by the small GTPase, Rab11A; however, the regulatory mechanism remains elusive. Apoptosis-associated tyrosine kinase 1A (AATYK1A) is a Ser/Thr kinase expressed highly in brain. We have recently shown that AATYK1A localizes to Rab11A-positive RE and is phosphorylated at Ser34 by cyclin-dependent kinase 5 (Cdk5). Here, we have investigated a role of AATYK1A and its phosphorylation in recycling endosomal trafficking using Chinese hamster ovary-K1 (CHO-K1) cells. AATYK1A localizes predominantly to Rab11A-positive pericentrosomal endocytic recycling compartment (ERC). Phosphorylation at Ser34 of AATYK1A disrupts its accumulation in the pericentrosomal ERC. Consistently, phosphorylation-mimic mutant (AATYK1A-S34D) did not accumulate in the ERC and additionally attenuated ERC formation. ERC formation suppression can be reversed by constitutively active Rab11A-Q70L, suggesting a functional link between AATYK1A phosphorylation and Rab11A activity. Although no direct interaction between AATYK1A and Rab11A could be detected, the exchange of guanine nucleotides bound to Rab11A was significantly reduced in the presence of the phosphorylation-mimic AATYK1A-S34D. Together, our results reveal a regulatory role for AATYK1A in the formation of pericentrosomal ERC. They furthermore indicate that Cdk5 can disrupt ERC formation via Ser34 phosphorylation of AATYK1A. Finally, our data suggest a mechanism by which AATYK1A signaling couples Cdk5 to Rab11A activity.

Hepatic steatosis, characterized by lipid accumulation in hepatocytes, is a key diagnostic feature in patients with chronic hepatitis C virus (HCV) infection. This study aimed to clarify the involvement of phospholipid metabolic pathways in the pathogenesis of HCV-induced steatosis. The expression and distribution of lipid species in the livers of human liver chimeric mice were analyzed using imaging mass spectrometry. Triglyceride accumulation and lipid droplet formation were studied in phospholipase A2 group 4C (PLA2G4C) knockout or overexpressing cells. Imaging mass spectrometry of the infected mouse model revealed increased lysophosphatidylcholine levels and decreased phosphatidylcholine levels in HCV-positive regions of the liver. Among the transcripts associated with phosphatidylcholine biosynthesis, upregulation of PLA2G4C mRNA was most pronounced following HCV infection. Activation of the transcription factor NF-κB and upregulation of c-Myc were important for activation of PLA2G4C transcription by HCV infection and expression of the viral proteins Core-NS2. The amount and size of lipid droplets were reduced in PLA2G4C-knockout cells. Inhibition of NF-κB or c-Myc activity suppressed lipid droplet formation in HCV-infected cells. HCV infection promoted the stabilization of lipid droplets, but this stability was reduced in PLA2G4C-knockout cells. Overexpression of PLA2G4C decreased the levels of phosphatidylcholine species in the lipid droplet fraction and led to lower levels of key factors involved in lipolysis (breakdown of triglycerides into glycerol and free fatty acids), such as ATGL, PLIN1 and ABHD5 on the lipid droplets. HCV infection markedly increases PLA2G4C expression. This may alter the phospholipid composition of the lipid droplet membrane, leading to stabilization and enlargement of the droplets. The involvement of phospholipid metabolism pathways in the pathogenesis of hepatitis C virus (HCV)-related liver diseases remains unclear. We found that PLA2G4C expression is upregulated through NF-κB and c-Myc activation upon HCV infection, and this upregulation is associated with a decrease in phosphatidylcholine species. The increased expression of PLA2G4C resulted in changes in the phospholipid composition of lipid droplets, led to the dissociation of lipolysis-related factors from the lipid droplet surface and the accumulation of lipid content within the droplets. These findings suggest that the disruption of the phospholipid metabolism pathway caused by HCV infection may contribute to the development of HCV-associated fatty liver. It would be interesting to determine whether alcohol- and/or metabolic dysfunction-associated steatohepatitis are also associated with increased PLA2 activity, altered phospholipid composition and decreased levels of ATGL and its cofactors in lipid droplet membranes.

The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that forms the two different protein complexes, known as mTORC1 and mTORC2. mTOR signaling is activated in a variety of tumors, including glioma that is one of the malignant brain tumors. FilGAP (ARHGAP24) is a negative regulator of Rac, a member of Rho family small GTPases. In this study, we found that FilGAP interacts with mTORC1/2 and is involved in tumor formation in glioma. FilGAP interacted with mTORC1 via Raptor and with mTORC2 via Rictor and Sin1. Depletion of FilGAP in KINGS-1 glioma cells decreased phosphorylation of S6K and AKT. Furthermore, overexpression of FilGAP increased phosphorylation of S6K and AKT, suggesting that FilGAP activates mTORC1/2. U-87MG, glioblastoma cells, showed higher mTOR activity than KINGS-1, and phosphorylation of S6K and AKT was not affected by suppression of FilGAP expression. However, in the presence of PI3K inhibitors, phosphorylation of S6K and AKT was also decreased in U-87MG by depletion of FilGAP, suggesting that FilGAP may also regulate mTORC2 in U-87MG. Finally, we showed that depletion of FilGAP in KINGS-1 and U-87MG cells significantly reduced spheroid growth. These results suggest that FilGAP may contribute to tumor growth in glioma by regulating mTORC1/2 activities.

A high integrated SiGe-MMIC transceiver having voltage controlled oscillator (VCO), phase locked loop (PLL), dual-transmit block for amplitude shift keying (ASK) mode and pi/4-quadrature phase shift keying (QPSK) mode, and common receiver block for the dual modes is developed for 5.8GHz dedicated short range communications (DSRC) terminals. To obtain stability of transceiver using differential configuration, which has on-chip closed loops generated by common ground/V CC pad arrangement and differential pairs, the stabilized MMIC design method based on the loop analysis is introduced to it. In the loop analysis, the both on-chip loops of differential mode and common mode are considered, the maximum loop gain of less than -3dB all over the frequency range is defined as the absolute stabilized condition. The simulated results for each loops show that isolation resistors inserted in bias feed line are effective for improving the stability. The effectiveness of the design method is evaluated by the measured results. The SiGe-MMIC transceiver having the stabilized amplifier chain is fabricated in 0.35μm SiGe-BiCMOS, and it achieves the adjacent channel power ratio (ACPR) of −38.2dBc under the ASK modulation and that of −47.2dBc under the pi/4-QPSK modulation.