Decreased FAK activity and focal adhesion dynamics impair proper neurite formation of medium spiny neurons in Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion in the protein huntingtin (HTT) [55]. While the final pathological consequence of HD is the neuronal cell death in the striatum region of the brain, it is still unclear how mutant HTT (mHTT) causes synaptic dysfunctions at the early stage and during the progression of HD. Here, we discovered that the basal activity of focal adhesion kinase (FAK) is severely reduced in a striatal HD cell line, a mouse model of HD, and the human post-mortem brains of HD patients. In addition, we observed with a FRET-based FAK biosensor [59] that neurotransmitter-induced FAK activation is decreased in HD striatal neurons. Total internal reflection fluorescence (TIRF) imaging revealed that the reduced FAK activity causes the impairment of focal adhesion (FA) dynamics, which further leads to the defect in filopodial dynamics causing the abnormally increased number of immature neurites in HD striatal neurons. Therefore, our results suggest that the decreased FAK and FA dynamics in HD impair the proper formation of neurites, which is crucial for normal synaptic functions [52]. We further investigated the molecular mechanism of FAK inhibition in HD and surprisingly discovered that mHTT strongly associates with phosphatidylinositol 4,5-biphosphate, altering its normal distribution at the plasma membrane, which is crucial for FAK activation [14, 60]. Therefore, our results provide a novel molecular mechanism of FAK inhibition in HD along with its pathological mechanism for synaptic dysfunctions during the progression of HD.

Genetically Encoded Biosensors Based on Fluorescent Proteins.

Genetically encoded biosensors based on fluorescent proteins (FPs) allow for the real-time monitoring of molecular dynamics in space and time, which are crucial for the proper functioning and regulation of complex cellular processes. Depending on the types of molecular events to be monitored, different sensing strategies need to be applied for the best design of FP-based biosensors. Here, we review genetically encoded biosensors based on FPs with various sensing strategies, for example, translocation, fluorescence resonance energy transfer (FRET), reconstitution of split FP, pH sensitivity, maturation speed, and so on. We introduce general principles of each sensing strategy and discuss critical factors to be considered if available, then provide representative examples of these FP-based biosensors. These will help in designing the best sensing strategy for the successful development of new genetically encoded biosensors based on FPs.

Pharmacophore-based virtual screening, biological evaluation and binding mode analysis of a novel protease-activated receptor 2 antagonist.

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor, mediating inflammation and pain signaling in neurons, thus it is considered to be a potential therapeutic target for inflammatory diseases. In this study, we performed a ligand-based virtual screening of 1.6 million compounds by employing a common-feature pharmacophore model and two-dimensional similarity search to identify a new PAR2 antagonist. The common-feature pharmacophore model was established based on the biological screening results of our in-house library. The initial virtual screening yielded a total number of 47 hits, and additional biological activity tests including PAR2 antagonism and anti-inflammatory effects resulted in a promising candidate, compound 43, which demonstrated an IC50 value of 8.22 µM against PAR2. In next step, a PAR2 homology model was constructed using the crystal structure of the PAR1 as a template to explore the binding mode of the identified ligands. A molecular docking method was optimized by comparing the binding modes of a known PAR2 agonist GB110 and antagonist GB83, and applied to predict the binding mode of our hit compound 43. In-depth docking analyses revealed that the hydrophobic interaction with Phe243(5.39) is crucial for PAR2 ligands to exert antagonistic activity. MD simulation results supported the predicted docking poses that PAR2 antagonist blocked a conformational rearrangement of Na(+) allosteric site in contrast to PAR2 agonist that showed Na(+) relocation upon GPCR activation. In conclusion, we identified new a PAR2 antagonist together with its binding mode, which provides useful insights for the design and development of PAR2 ligands.

Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude.

How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, RhoA can promote focal adhesion (FA) growth or disassembly, but how RhoA activity mediates these opposite outcomes is not clear. Here, we develop a photoswitchable RhoA guanine nucleotide exchange factor (GEF), psRhoGEF, to precisely control endogenous RhoA activity. Using this optical tool, we discover that peak FA disassembly selectively occurs upon activation of RhoA to submaximal levels. We also find that Src activation at FAs selectively occurs upon submaximal RhoA activation, identifying Src as an amplitude-dependent RhoA effector. Finally, a pharmacological Src inhibitor reverses the direction of the FA response to RhoA activation from disassembly to growth, demonstrating that Src functions to suppress FA growth upon RhoA activation. Thus, rheostatic control of RhoA activation by psRhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.

Aß modulates actin cytoskeleton via SHIP2-mediated phosphoinositide metabolism.

Emerging evidences suggest that phospholipid metabolism is altered in Alzheimer's disease (AD), but molecular mechanisms on how this affects neurodegeneration in AD is poorly understood. SHIP2 is a phosphoinositide-metabolizing enzyme, which dephosphorylates PI(3,4,5)P3 resulting to PI(3,4)P2, and it has been recently shown that Aß directly increases the activity of SHIP2. Here we monitored, utilizing fluorescent SHIP2 biosensor, real-time increase of PI(3,4)P2-containing vesicles in HT22 cells treated with Aß. Interestingly, PI(3,4)P2 is accumulated at late endosomes and lysosomal vesicles. We further discovered that ARAP3 can be attracted to PI(3,4)P2-positive mature endosomes via its PH domain and this facilitates the degradation of ARAP3. The reduced level of ARAP3 then causes RhoA hyperactivation and filamentous actin, which are critical for neurodegeneration in AD. These results provide a novel molecular link between Aß and actin disruption through dysregulated phosphoinositide metabolism, and the SHIP2-PI(3,4)P2-ARAP3-RhoA signaling pathway can be considered as new therapeutic targets for synaptic dysfunctions in Alzheimer's disease.

Ketanserin, a 5-HT2 antagonist, directly inhibits the ATP-sensitive potassium channel in mouse ventricular myocytes.

The effects of ketanserin, a 5-HT2 antagonist, on the ATP-sensitive K+ (K(ATP)) channels were studied in mouse ventricular myocytes using patch clamp technique. Under the whole-cell voltage clamp conditions, ketanserin (1-100 microM) reversibly inhibited pinacidil-induced K(ATP) current in a concentration-dependent fashion with a Ki value of 9.36 microM and the Hill coefficient was 0.67. This inhibition was developed even with the presence of 5-hydroxytryptamine (100 microM) in the bath. Prazosin, a selective alpha1-antagonist, also failed to mimic the effect of ketanserin. Ketanserin did not affect the channel activity in inside-out configuration under the ATP-free internal solution. Furthermore, ketanserin applied to the external solution did not affect the pinacidil-induced channel activity in the cell-attached patches, but did inhibit it when applied into the pipette. These results suggest that the inhibitory action of ketanserin observed in this study was probably due to a direct action on the K(ATP) channel rather than to an action through the 5-HT2 receptor or alpha1-adrenoceptor blockade, and that the antiarrhythmic activity of ketanserin against cardiac arrhythmias induced in the ischemic/reperfused heart is at least in part attributable to its inhibition of the K(ATP) channel.