Evaluation of a new portable corneal topography system for self-measurement using smartphones: a pilot study.

PURPOSE: Herein, we propose the use of the "KeraVio Ring", which is a portable, selfie-based, smartphone-attached corneal topography system that is based on the Placido ring videokeratoscope. The goal of this study was to evaluate and compare corneal parameters between KeraVio Ring and conventional corneal tomography images. METHODS: We designed the KeraVio Ring as a device comprising 3D-printed LED rings for generating Placido rings that can be attached to a smartphone. Two LED rings are attached to a cone-shaped device, and both corneas are illuminated. Selfies were taken using the KeraVio Ring attached to the smartphone without assistance from any of the examiners. Captured Placido rings on the cornea were analysed by intelligent software to calculate corneal parameters. Patients with normal, keratoconus, or LASIK-treated eyes were included. Anterior segment optical coherence tomography (AS-OCT) was also performed for each subject. RESULTS: We found highly significant correlations between the steepest and flattest keratometry, corneal astigmatism, and vector components obtained with the KeraVio Ring and AS-OCT. In subjects with normal, keratoconus, and LASIK-treated eyes, the mean difference in corneal astigmatism between the two devices was -0.8 ± 1.4 diopters (D) (95% limits of agreement (LoA), -3.6 to 2.0), -1.8 ± 3.7 D (95% LoA, -9.1 to 5.5), and -1.5 ± 1.3 D (95% LoA, -4.0 to 1.1), respectively. CONCLUSIONS: The experimental results showed that the corneal parameters obtained by the KeraVio Ring were correlated with those obtained with AS-OCT. The KeraVio Ring has the potential to address an unmet need by providing a tool for portable selfie-based corneal topography.

Identification of Novel Antimicrobial Compounds Targeting Mycobacterium tuberculosis S-Adenosyl-L-Homocysteine Hydrolase Using Dual Hierarchical In Silico Structure-Based Drug Screening.

The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis (M. tuberculosis) has become a major medical problem. S-adenosyl-L-homocysteine hydrolase (MtSAHH) was selected as the target protein for the identification of novel anti-TB drugs. Dual hierarchical in silico Structure-Based Drug Screening was performed using a 3D compound structure library (with over 150 thousand synthetic chemicals) to identify compounds that bind to MtSAHH's active site. In vitro experiments were conducted to verify whether the nine compounds selected as new drug candidates exhibited growth-inhibitory effects against mycobacteria. Eight of the nine compounds that were predicted by dual hierarchical screening showed growth-inhibitory effects against Mycobacterium smegmatis (M. smegmatis), a model organism for M. tuberculosis. Compound 7 showed the strongest antibacterial activity, with an IC50 value of 30.2 µM. Compound 7 did not inhibit the growth of Gram-negative bacteria or exert toxic effects on human cells. Molecular dynamics simulations of 40 ns using the MtSAHH-Compound 7 complex structure suggested that Compound 7 interacts stably with the MtSAHH active site. These in silico and in vitro results suggested that Compound 7 is a promising lead compound for the development of new anti-TB drugs.

An Inorganic-Organic Hybrid Framework Composed of Polyoxotungstate and Long-Chained Bolaamphiphile.

Surfactants are functional molecules utilized in various situations. The self-assembling property of surfactants enables several molecular arrangements that can be employed to build up nanometer-sized architectures. This is beneficial in the construction of functional inorganic-organic hybrids holding the merits of both inorganic and organic components. Among several surfactants, bolaamphiphile surfactants with two hydrophilic heads are effective, as they have multiple connecting or coordinating sites in one molecule. Here, a functional polyoxotungstate inorganic anion was successfully hybridized with a bolaamphiphile to form single crystals with anisotropic one-dimensional alignment of polyoxotungstate. Keggin-type metatungstate ([H2W12O40]6-, H2W12) was employed as an inorganic anion, and 1,12-dodecamethylenediammonium (C12N2) derived from 1,12-dodecanediamine was combined as an organic counterpart. A simple and general ion-exchange reaction provided a hybrid crystal consisting of H2W12 and C12N2 (C12N2-H2W12). Single crystal X-ray structure analyses revealed a characteristic honeycomb structure in the C12N2-H2W12 hybrid crystal, which is possibly effective for the emergence of conductivity due to the dissociative protons of C12N2.

Computational Screening and Experimental Validation of Inhibitor Targeting the Complex Formation of Grb14 and Insulin Receptor.

The development of drugs targeting gene products associated with insulin resistance holds the potential to enhance our understanding of type 2 diabetes mellitus (T2DM). The virtual screening, based on a three-dimensional (3D) protein structure, is a potential technique to accelerate the development of molecular target drugs. Among the targets implicated in insulin resistance, the genetic characterization and protein function of Grb14 have been clarified without contradiction. The Grb14 gene displays significant variations in T2DM, and its gene product is known to inhibit the function of the insulin receptor (IR) by directly binding to the tyrosine kinase domain. In the present study, a virtual screening, based on a 3D structure of the IR tyrosine kinase domain (IRß) in complex with part of Grb14, was conducted to find compounds that can disrupt the complex formation between Grb14 and IRß. First, ten compounds were selected from 154,118 compounds via hierarchical in silico structure-based drug screening, composed of grid docking-based and genetic algorithm-based programs. The experimental validations suggested that the one compound can affect the blood glucose level. The molecular dynamics simulations and co-immunoprecipitation analysis showed that the compound did not completely suppress the protein-protein interaction between Grb14 and IR, though competitively bound to IR with the tyrosine kinase pseudosubstrate region in Grb14.

2,3-Butandione 2-monoxime inhibits skeletal myosin II by accelerating ATP cleavage.

2,3-Butandione 2-monoxime (BDM) is a widely used myosin inhibitor with an unclear mode of action. In this report, we investigated the mechanism of BDM oxime group nucleophilic reactivity on the phosphoester bond of ATP. BDM increased the ATPase activity of skeletal myosin subfragment 1 (S1) under conditions in which ATP cleavage is the rate-limiting step (K+, EDTA-ATPase activity of native S1 and Mg2+-ATPase activity of trinitrophenylated S1 and partially unfolded S1). Furthermore, the effect of BDM on the S1-bound adenosine 5'-(ß,γ-imido) triphosphate (AMPPNP) 31P NMR spectrum suggests that BDM changes the microenvironment around the phosphorus atoms of myosin-bound nucleotide. A computational search for the BDM-binding site in the adenosine 5'-[γ-thio] triphosphate (myosin-ATPγS) complex predicted that BDM is located adjacent to the nucleotide on myosin. Therefore, we propose that the BDM oxime group catalytically assists in ATP cleavage, thereby enhancing the ATPase activity of myosin in a manner analogous to pralidoxime-mediated reactivation of organophosphate-inactivated acetylcholinesterase. This is the first study suggesting that oxime provides catalytic assistance for ATP cleavage by an ATP-hydrolyzing enzyme.

Time dilation in a perceptually jittering dot pattern.

Although it is known that a moving stimulus appears to dilate in duration compared to a stationary stimulus, whether subjective motion devoid of stimulus motion is sufficient remains unknown. To elucidate this, we used a motion illusion in which an actually static stimulus clearly appears to move, a useful dissociation between actual and subjective motions. We used the jitter aftereffect resulting from adaptation to dynamic noise as such a tool and measured subjective durations of a static random-dot pattern in which illusory jitter was seen, an actually oscillating pattern mimicking the illusory jitter, and a static pattern without illusory jitter. Pattern oscillation as tiny as fixational eye movements robustly evoked time dilation, and time dilation to a similar extent was also induced by an actually static but subjectively jittering pattern. Taken together with the previous knowledge that this subjective jitter is related to a visually based compensation of spurious retinal image motions due to fixational eye movements, these findings demonstrate that visual duration computation is influenced by a representation at a high-level motion processing stage at which a stable visual world despite jittery retinal inputs has been established.

Identification of novel potential antibiotics against Staphylococcus using structure-based drug screening targeting dihydrofolate reductase.

The emergence of multidrug-resistant Staphylococcus aureus (S. aureus) makes the treatment of infectious diseases in hospitals more difficult and increases the mortality of the patients. In this study, we attempted to identify novel potent antibiotic candidate compounds against S. aureus dihydrofolate reductase (saDHFR). We performed three-step in silico structure-based drug screening (SBDS) based on the crystal structure of saDHFR using a 154,118 chemical compound library. We subsequently evaluated whether candidate chemical compounds exhibited inhibitory effects on the growth of the model bacterium: Staphylococcus epidermidis (S. epidermidis). The compound KB1 showed a strong inhibitory effect on the growth of S. epidermidis. Moreover, we rescreened chemical structures similar to KB1 from a 461,397 chemical compound library. Three of the four KB1 analogs (KBS1, KBS3, and KBS4) showed inhibitory effects on the growth of S. epidermidis and enzyme inhibitory effects on saDHFR. We performed structure-activity relationship (SAR) analysis of active chemical compounds and observed a correlative relationship among the IC50 values, interaction residues, and structure scaffolds. In addition, the active chemical compounds (KB1, KBS3, and KBS4) had no inhibitory effects on the growth of model enterobacteria (E. coli BL21 and JM109 strains) and no toxic effects on cultured mammalian cells (MDCK cells). Results obtained from Protein Ligand Interaction Fingerprint (PLIF) and Ligand Interaction (LI) analyses suggested that all of the active compounds exhibited potential inhibitory effects on mutated saDHFR of the drug-resistant strains. The structural and experimental information concerning these novel chemical compounds will likely contribute to the development of new antibiotics for both wild-type and drug-resistant S. aureus.

Data on molecular docking and molecular dynamics targeting Mycobacterium tuberculosis shikimic acid kinase.

We have previously performed a hierarchical in silico screening of a Mycobacterium tuberculosis shikimic acid kinase [1]. Specifically, 11 compounds were screened from a library of 154,118 compounds provided by ChemBridge [2] using UCSF DOCK [3] and the GOLD [4] program in the first and second steps, respectively. Molecular dynamic simulations were further performed on compound 2 (2-[(5Z)-5-(1-benzyl-5bromo-2-oxoindol-3-(5Z)-5-(1-benzyl-5-bromo-2-oxoindol-3-(5Z)-4-oxo-2 ylidene)-4oxo-2-sulfanylidene-1,3-thiazolidin-3-yl] acetic acid), which showed antimicrobial efficacy. These processes yielded ligand docking scores and trajectories. In this data article, we have added solvent-accessible surface area and PCA analyses, which were calculated from the raw docking scores and trajectories. Data obtained from molecular docking and molecular dynamic simulations are useful in two ways: (1) Further support for previous work (2) Provides a stepping stone for experimental scientists to conduct in silico studies and research ideas for other drug discovery researchers and computational biologists. We believe that this article will provide an opportunity to develop new Mycobacterium tuberculosis therapeutics through searching for analogs and inhibitors against new targets.

Identification of compounds with potential antibacterial activity against Mycobacterium through structure-based drug screening.

To identify novel antibiotics against Mycobacterium tuberculosis, we performed a hierarchical structure-based drug screening (SBDS) targeting the enoyl-acyl carrier protein reductase (InhA) with a compound library of 154,118 chemicals. We then evaluated whether the candidate hit compounds exhibited inhibitory effects on the growth of two model mycobacterial strains: Mycobacterium smegmatis and Mycobacterium vanbaalenii. Two compounds (KE3 and KE4) showed potent inhibitory effects against both model mycobacterial strains. In addition, we rescreened KE4 analogs, which were identified from a compound library of 461,383 chemicals through fingerprint analysis and genetic algorithm-based docking simulations. All of the KE4 analogs (KES1-KES5) exhibited inhibitory effects on the growth of M. smegmatis and/or M. vanbaalenii. Based on the predicted binding modes, we probed the structure-activity relationships of KE4 and its analogs and found a correlative relationship between the IC50 values and the interaction residues/LogP values. The most potent inhibitor, compound KES4, strongly and stably inhibited the long-term growth of the model bacteria and showed higher inhibitory effects (IC50 = 4.8 µM) than isoniazid (IC50 = 5.4 µM), which is a first-line drug for tuberculosis therapy. Moreover, compound KES4 did not exhibit any toxic effects that impede cell growth in several mammalian cell lines and enterobacteria. The structural and experimental information of these novel chemical compounds will likely be useful for the development of new anti-TB drugs. Furthermore, the methodology that was used for the identification of the effective chemical compound is also likely to be effective in the SBDS of other candidate medicinal drugs.

In silico Identification of Potential Inhibitors Against Staphylococcus Aureus Tyrosyl-tRNA Synthetase.

BACKGROUND: Drug-resistant Staphylococcus aureus (S. aureus) has spread from nosocomial to community-acquired infections. Novel antimicrobial drugs that are effective against resistant strains should be developed.　S. aureus tyrosyl-tRNA synthetase (saTyrRS) is considered essential for bacterial survival and is an attractive target for drug screening. OBJECTIVE: The purpose of this study was to identify potential new inhibitors of saTyrRS by screening compounds in silico and evaluating them using molecular dynamics (MD) simulations. METHODS: A 3D structural library of 154,118 compounds was screened using the DOCK and GOLD docking simulations and short-time MD simulations. The selected compounds were subjected to MD simulations of a 75-ns time frame using GROMACS. RESULTS: Thirty compounds were selected by hierarchical docking simulations. The binding of these compounds to saTyrRS was assessed by short-time MD simulations. Two compounds with an average value of less than 0.15 nm for the ligand RMSD were ultimately selected. The long-time (75 ns) MD simulation results demonstrated that two novel compounds bound stably to saTyrRS in silico. CONCLUSION: Two novel potential saTyrRS inhibitors with different skeletons were identified by in silico drug screening using MD simulations. The in vitro validation of the inhibitory effect of these compounds on enzyme activity and their antibacterial effect on drug-resistant S. aureus would be useful for developing novel antibiotics.

Identification of a Chemical Inhibitor with a Novel Scaffold Targeting Decaprenylphosphoryl-ß-D-Ribose Oxidase (DprE1).

BACKGROUND: Tuberculosis is the second leading cause of death from infectious diseases worldwide. Multidrug-resistant Mycobacterium tuberculosis is spreading throughout the world, creating a crisis. Hence, there is a need to develop anti-tuberculosis drugs with novel structures and versatile mechanisms of action. OBJECTIVE: In this study, we identified antimicrobial compounds with a novel skeleton that inhibits mycobacterium decaprenylphosphoryl-ß-D-ribose oxidase (DprE1). METHODS: A multi-step, in silico, structure-based drug screening identified potential DprE1 inhibitors from a library of 154,118 compounds. We experimentally verified the growth inhibitory effects of the eight selected candidate compounds against Mycobacterium smegmatis. Molecular dynamics simulations were performed to understand the mechanism of molecular interactions between DprE1 and ompound 4. RESULTS: Eight compounds were selected through in silico screening. Compound 4 showed strong growth inhibition against M. smegmatis. Molecular dynamics simulation (50 ns) predicted direct and stable binding of Compound 4 to the active site of DprE1. CONCLUSION: The structural analysis of the novel scaffold in Compound 4 can pave way for antituberculosis drug development and discovery.

Identification of novel antimicrobial compounds targeting Mycobacterium tuberculosis shikimate kinase using in silico hierarchical structure-based drug screening.

The development of new anti-TB drugs to prevent the spread of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains is imperative. Mtb shikimate kinase (MtSK) was selected as the target protein to screen for new anti-TB drugs. We performed hierarchical in silico screening using a library of 154,118 compounds to search for novel compounds that could bind to the active site of MtSK. The growth-inhibitory effects of the candidate compounds on Mycobacterium smegmatis were evaluated in vitro. Nine of the 11 candidate compounds exhibited inhibitory effects against mycobacteria in vitro. The inhibitory activity of Compound 2 (IC50 = 1.39 µM) was higher than that of isoniazid, the first-line drug for TB treatment. Moreover, Compound 2 did not exhibit toxicity against mammalian cells and Escherichia coli. Molecular dynamics simulations using the MtSK-Compound 2 complex structure in a timeframe of 100 ns suggested that Compound 2 could stably bind to MtSK. The binding free energy of Compound 2 was estimated to be -37.96 kcal/mol using the MM/PBSA method, demonstrating that Compound 2 can stably bind to MtSK. These in silico and in vitro results indicated that Compound 2 is a promising hit compound for the development of novel anti-TB drugs.

Identification of α-Tocopherol succinate as an RFFL-substrate interaction inhibitor inducing peripheral CFTR stabilization and apoptosis.

The E3 ubiquitin ligase RFFL is an apoptotic inhibitor highly expressed in cancers and its knockdown suppresses cancer cell growth and sensitizes to chemotherapy. RFFL also participates in peripheral protein quality control which removes the functional cell surface ΔF508-CFTR channel and reduces the efficacy of pharmaceutical therapy for cystic fibrosis (CF). Although RFFL inhibitors have therapeutic potential for both cancer and CF, they remain undiscovered. Here, a chemical array screening has identified α-tocopherol succinate (αTOS) as an RFFL ligand. NMR analysis revealed that αTOS directly binds to RFFL's substrate-binding region without affecting the E3 enzymatic activity. Consequently, αTOS inhibits the RFFL-substrate interaction, ΔF508-CFTR ubiquitination and elimination from the plasma membrane of epithelial cells, resulting in the increased functional CFTR channel. Among the α-tocopherol (αTOL) analogs we tested, only αTOS inhibited the RFFL-substrate interaction and increased the cell surface ΔF508-CFTR, depending on RFFL expression. Similarly, the unique proapoptotic effect of αTOS was dependent on RFFL expression. Thus, unlike other αTOL analogs, αTOS acts as an RFFL protein-protein interaction inhibitor which may explain its unique biological properties among αTOL analogs. Moreover, αTOS may act as a CFTR stabilizer, a novel class of drugs that extend cell surface ΔF508-CFTR lifetime.

Identification of novel inhibitors for mycobacterial polyketide synthase 13 via in silico drug screening assisted by the parallel compound screening with genetic algorithm-based programs.

Identifying small compounds capable of inhibiting Mycobacterium tuberculosis polyketide synthase 13 (Pks13), in charge of final step of mycolic acid biosynthesis, could lead to the development of a novel antituberculosis drug. This study screened for lead compounds capable of targeting M. tuberculosis Pks13 from a chemical library comprising 154,118 compounds through multiple in silico docking simulations. The parallel compound screening (PCS), conducted via two genetic algorithm-based programs was applied in the screening strategy. Out of seven experimentally validated compounds, four compounds showed inhibitory effects on the growth of the model mycobacteria (Mycobacterium smegmatis). Subsequent docking simulation of analogs of the promising leads with the assistance of PCS resulted in the identification of three additional compounds with potent antimycobacterial effects (compounds A1, A2, and A5). Further, molecular dynamics simulation predicted stable interaction between M. tuberculosis Pks13 active site and compound A2, which showed potent antimycobacterial activity comparable to that of isoniazid. The present study demonstrated the efficacy of in silico structure-based drug screening through PCS in antituberculosis drug discovery.

Computer-assisted screening of mycobacterial growth inhibitors: Exclusion of frequent hitters with the assistance of the multiple target screening method.

Background: The emergence of frequent hitters (FHs) remains a challenge in drug discovery. We have previously used in silico structure-based drug screening (SBDS) to identify antimycobacterial candidates. However, excluding FHs has not been integrated into the SBDS system. Methods: A dataset comprising 15,000 docking score (protein-compound affinity matrix) was constructed by multiple target screening (MTS): DOCK-GOLD two-step docking simulations with 154,118 compounds versus the 30 target proteins essential for mycobacterial survival. After extraction of 141 compounds from the protein-compound affinity matrix, compounds determined to be FHs or false positives were excluded. Antimycobacterial properties of the top nine compounds selected through SBDS were experimentally evaluated. Results: Nine compounds designated KS1-KS9 were selected for experimental evaluation. Among the selected compounds, KS3, identified as adenosylhomocysteinase inhibitor, showed a potent inhibitory effect on antimycobacterial growth (inhibitory concentration [IC]50 = 1.2 M). However, the compound also showed potent cytotoxicity. Conclusion: The MTS method is applicable in SBDS for the identification of enzyme-specific inhibitors.

Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. The I93M mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is associated with familial PD, and we have previously shown that the I93M UCH-L1-transgenic mice exhibit dopaminergic cell loss. Over 90% of neurodegenerative diseases, including PD, occur sporadically. However, the molecular mechanisms underlying sporadic PD as well as PD associated with I93M UCH-L1 are largely unknown. UCH-L1 is abundant (1-5% of total soluble protein) in the brain and is a major target of oxidative/carbonyl damage associated with sporadic PD. As well, abnormal microtubule dynamics and tubulin polymerization are associated with several neurodegenerative diseases including frontotemporal dementia and parkinsonism linked to chromosome 17. Here we show that familial PD-associated mutant UCH-L1 and carbonyl-modified UCH-L1 display shared aberrant properties: compared with wild-type UCH-L1, they exhibit increased insolubility and elevated interactions with multiple proteins, which are characteristics of several neurodegenerative diseases-linked mutants. Circular dichroism analyses suggest similar structural changes in both UCH-L1 variants. We further report that one of the proteins interacting with UCH-L1 is tubulin, and that aberrant interaction of mutant or carbonyl-modified UCH-L1 with tubulin modulates tubulin polymerization. These findings may underlie the toxic gain of function by mutant UCH-L1 in familial PD. Our results also suggest that the carbonyl modification of UCH-L1 and subsequent abnormal interactions of carbonyl-modified UCH-L1 with multiple proteins, including tubulin, constitute one of the causes of sporadic PD.

Structural modification of a novel inhibitor for mycobacterium enoyl-acyl carrier protein reductase assisted by In silico structure-based drug screening.

Background: Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (mtInhA) is involved in the biosynthesis of mycolic acids, a major component of mycobacterial cell walls, and has been targeted in the development of anti-tuberculosis (TB) drugs. In our previous in silico structure-based drug screening study, we identified KES4, a novel class of mtInhA inhibitor. KES4 is composed of four ring structures (A-D-rings) and molecular dynamic simulation predicted that the D-ring is essential for the interaction with mtInhA. Methods: The structure-activity relationship study of the D-ring was attempted and aided by in silico docking simulations to improve the mtInhA inhibitory activity of KES4. A virtual chemical library of the D-ring-modified KES4 was then constructed and subjected to in silico docking simulation against mtInhA using the GOLD program. The candidate compound showing the highest GOLD score, referred to as KEN1, was synthesized, and its biological properties were compared with those of the lead compound KES4. Results: We achieved the synthesis of KEN1 and evaluated its effects on InhA activity, mycobacterial growth, and cytotoxicity. The antimycobacterial activity of KEN1 was comparable to that of the lead compound (KES4), although it exhibited superior activity in mtInhA inhibition. \. Conclusions: We obtained a KES4 derivative with high mtInhA inhibitory activity by in silico docking simulation with a chemical library consisting of a series of D-ring-modified KES4.

Improvement of the novel inhibitor for Mycobacterium enoyl-acyl carrier protein reductase (InhA): a structure-activity relationship study of KES4 assisted by in silico structure-based drug screening.

InhA or enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis (mtInhA), which controls mycobacterial cell wall construction, has been targeted in the development of antituberculosis drugs. Previously, our in silico structure-based drug screening study identified a novel class of compounds (designated KES4), which is capable of inhibiting the enzymatic activity of mtInhA, as well as mycobacterial growth. The compounds are composed of four ring structures (A-D), and the MD simulation predicted specific interactions with mtInhA of the D-ring and methylene group between the B-ring and C-ring; however, there is still room for improvement in the A-ring structure. In this study, a structure-activity relationship study of the A-ring was attempted with the assistance of in silico docking simulations. In brief, the virtual chemical library of A-ring-modified KES4 was constructed and subjected to in silico docking simulation against mtInhA using the GOLD program. Among the selected candidates, we achieved synthesis of seven compounds, and the bioactivities (effects on InhA activity and mycobacterial growth and cytotoxicity) of the synthesized molecules were evaluated. Among the compounds tested, two candidates (compounds 3d and 3f) exhibited superior properties as mtInhA-targeted anti-infectives for mycobacteria than the lead compound KES4.

G-protein-coupled receptor screen reveals a role for chemokine receptor CCR5 in suppressing microglial neurotoxicity.

G-protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins, and several GPCRs have been implicated in signaling between neurons and glia to protect neurons from pathological stresses. Here, we have used a screening strategy to investigate GPCRs that are involved in neuronal protection. The real-time PCR was performed using 274 primers targeting nonsensory GPCR mRNAs, which were listed on the database. The cDNAs from control and nerve-injured hypoglossal nuclei of mouse brain were used, and the alterations of PCR products were compared. This screen and the subsequent in situ hybridization screen exhibited six GPCR mRNAs which were prominently and convincingly induced in nerve-injured hypoglossal nuclei. Among these candidates, the chemokine receptor CCR5 was selected, based on the marked induction in CCR5 mRNA in microglia after nerve injury. The mRNA expression of ligands for CCR5, such as regulated on activation normal T-cell expressed and secreted (RANTES/CCL5), MIP-1alpha, and MIP-1beta, were induced in injured motor neurons, indicating that CCR5 and its ligands were expressed in microglia and neurons, respectively, in response to nerve injury. In vitro, lipopolysaccharide (LPS)-induced expression of mRNAs for inflammatory cytokines (IL-1beta, IL-6, and tumor necrosis factor-alpha) and inducible nitric oxide synthase (iNOS) in microglia were all suppressed by RANTES. Those suppressions were not observed in microglia from CCR5 null mice. In addition, nerve injury-induced motor neuron death seen in wild type C56BL/6J mice was accelerated in CCR5 knock-out C57BL/6J. These results may suggest that CCR5-mediated neuron-glia signaling functions to protect neurons by suppressing microglia toxicity.

Solo/Trio8, a membrane-associated short isoform of Trio, modulates endosome dynamics and neurite elongation.

With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.