Impaired spatial reference memory and increased exploratory behavior in P301L tau transgenic mice

The neuropathological hallmark shared between Alzheimer's disease (AD) and familial frontotemporal dementia (FTDP-17) are neurofibrillary tangles (NFT) which are composed of filamentous aggregates of the microtubule-associated protein tau. Their formation has been reproduced in transgenic mice, which express the FTDP-17-associated mutation P301L of tau. In these mice, tau aggregates are found in many brain areas including the hippocampus and the amygdala, both of which are characterized by NFT formation in AD. Previous studies using an amygdala-specific test battery revealed an increase in exploratory behavior and an accelerated extinction of conditioned taste aversion in these mice. Here, we assessed P301L mice in behavioral tests known to depend on an intact hippocampus. Morris water maze and Y-maze revealed intact spatial working memory but impairment in spatial reference memory at 6 and 11 months of age. In addition, a modest disinhibition of exploratory behavior at 6 months of age was confirmed in the open field and the elevated O-maze and was more pronounced during aging.     

Impaired sexual behavior in male mice deficient for the beta1-3 N-acetylglucosaminyltransferase-I gene

The beta1-3 N-acetylglucosaminyltransferase-1 (B3gnt1) gene encodes a poly-N-acetyllactosamine synthase which can initiate and extend poly-N-acetyllactosamine chains [Gal(beta1-4)GlcNAc (beta1-3)(n)]. Previous investigations with heterozygous and homozygous null mice for this gene have revealed the importance of poly-N-acetyllactosamine chains for the formation of olfactory axon connections with the olfactory bulb and the migration of gonadotropin releasing hormone neurons to the hypothalamus. The possible long-term effects of these developmental defects, however, has not yet been studied. Here we have examined a reproductive phenotype observed in B3gnt1-null mice. Whereas the B3gnt1 null females were fertile, the B3gnt1 null males were not able to sire litters at the expected rate when mated to either wildtype or B3gnt1-null females. We assessed male sexual behavior as well as male reproduction parameters such as testes size, spermatogenesis, sperm number, morphology, and the development of early embryos in order to identify the source of a reduced rate of reproduction. Our findings show that the B3gnt1 null male reproductive organs were functional and could not account for the lower rate at which they produced offspring with wildtype conspecifics. Hence, we propose that the phenotype observed resulted from an impaired sexual response to female mating partners.     

Diminished pheromone-induced sexual behavior in neurokinin-1 receptor deficient (TACR1(-/-)) mice

Studies in mice with targeted deletions of tachykinin genes suggest that tachykinins and their receptors influence emotional behaviors such as aggression, depression and anxiety. Here, we investigated whether TAC1- and TAC4-encoded peptides (substance P and hemokinin-1, respectively) and the neurokinin-1 receptor (NK-1R) are involved in the modulation of sexual behaviors. Male mice deficient for the NK-1R (TACR1 (-/-)) exhibited decreased exploration of female urine in contrast to C57BL/6 control mice and mice deficient for NK-1R ligands such as TAC1 (-/-), TAC4 (-/-) and the newly generated TAC1 (-/-) /TAC4 (-/-) mice. In comparison to C57BL/6 mice, mounting frequency and duration were decreased in male TACR1 (-/-) mice, while mounting latency was increased. Decreased preference for sexual pheromones was also seen in female TACR1 (-/-) mice. Furthermore, administration of the NK-1R-antagonist L-703,606 decreased investigation of female urine by male C57BL/6 mice, suggesting an involvement of NK-1R in urine sniffing behavior. Our results provide evidence for the NK-1R in facilitating sexual approach behavior, as male TACR1 (-/-) mice exhibited blunted approach behavior toward females following the initial interaction compared with C57BL/6 mice. NK-1R signaling may therefore play an important role in pheromone-induced sexual behavior.     

Impaired working memory,cognitive flexibility and reward processing in mice genetically lacking Gpr88: Evidence for a key role for Gpr88 in multiple cortico-striatal-thalamic circuits

The GPR88 orphan G protein-coupled receptor is expressed throughout the striatum, being preferentially localised in medium spiny neurons. It is also present in lower densities in frontal cortex and thalamus. Rare mutations in humans suggest a role in cognition and motor function, while common variants are associated with psychosis. Here we evaluate the influence of genetic deletion of GPR88 upon performance in translational tasks interrogating motivation, reward evaluation and cognitive function. In an automated radial arm maze ‘N-back’ working memory task, Gpr88 KO mice showed impaired correct responding, suggesting a role for GPR88 receptors in working memory circuitry. Associative learning performance was similar to wild-type controls in a touchscreen task but performance was impaired at the reversal learning stage, suggesting cognitive inflexibility. Gpr88 KO mice showed higher breakpoints, reduced latencies and lengthened session time in a progressive ratio task consistent with enhanced motivation. Simultaneously, locomotor hyperactivity was apparent in this task, supporting previous findings of actions of GPR88 in a cortico-striatal-thalamic motor loop. Evidence for a role of GPR88 in reward processing was demonstrated in a touchscreen-based equivalent of the Iowa gambling task. Although both Gpr88 KO and wild-type mice showed a preference for an optimum contingency choice, Gpr88 KO mice selected more risky choices at the expense of more advantageous lower risk options. Together these novel data suggest that striatal GPR88 receptors influence activity in a range of procedures integrated by prefrontal, orbitofrontal and anterior cingulate cortico-striatal-thalamic loops leading to altered cognitive, motivational and reward evaluation processes.     

Over‐expressed MST1 impaired spatial memory via disturbing neural oscillation patterns in mice

The activated mammalian Ste20‐like serine/threonine kinases 1 (MST1) was found in the central nervous system diseases, such as cerebral ischemia, stroke and ALS, which were related with cognitions. The aim of this study was to examine the effect of elevated MST1 on memory functions in C57BL/6J mice. We also explored the underlying mechanism about the pattern alteration of neural oscillations, closely associated with cognitive dysfunctions, at different physiological rhythms, which were related to a wide range of basic and higher‐level cognitive activities. A mouse model of the adeno‐associated virus (AAV)‐mediated overexpression of MST1 was established. The behavioral experiments showed that spatial memory was significantly damaged in MST1 mice. The distribution of either theta or gamma power was clearly disturbed in MST1 animals. Moreover, the synchronization in both theta and gamma rhythms, and theta‐gamma cross‐frequency coupling were significantly weakened in MST1 mice. In addition, the expressions of GABAA receptor, GAD67 and parvalbumin (PV) were obviously increased in MST1 mice. Meanwhile, blocking MST1 activity could inhibit the activation of FOXO3a and YAP. The above data suggest that MST1‐overexpression may induce memory impairments via disturbing the patterns of neural activities, which is possibly associated with the abnormal GABAergic expression level.     

GISP binding to TSG101 increases GABA receptor stability by down-regulating ESCRT-mediated lysosomal degradation

The neuron-specific G protein-coupled receptor interacting scaffold protein (GISP) is a multidomain, brain-specific protein derived from the A-kinase anchoring protein-9 gene. We originally isolated GISP as an interacting partner for the GABA(B) receptor subunit GABA(B1). Here, we show that the protein tumour susceptibility gene 101 (TSG101), an integral component of the endosomal sorting machinery that targets membrane proteins for lysosomal degradation, also interacts with GISP. TSG101 co-immunoprecipitates with GISP from adult rat brain, and using GST pull-downs, we identified that the eighth coiled-coiled region of GISP is critical for TSG101 association. Intriguingly, although there is no direct interaction between GISP and the GABA(B2) subunit, their co-expression in HEK293 cells increases levels of GABA(B2). GISP also inhibits TSG101-dependent GABA(B2) down-regulation in human embryonic kidney 293 cells whereas over-expression of a mutant GISP lacking the TSG101 binding domain has no effect on GABA(B2) degradation. These data suggest that GISP can function as a negative regulator of TSG101-dependent lysosomal degradation of transmembrane proteins in neurons to promote receptor stability.     

Akt1 deficiency modulates reward learning and reward prediction error in mice

In contemporary reinforcement learning models, reward prediction error (RPE), the difference between the expected and actual reward, is thought to guide action value learning through the firing activity of dopaminergic neurons. Given the importance of dopamine in reward learning and the involvement of Akt1 in dopamine-dependent behaviors, the aim of this study was to investigate whether Akt1 deficiency modulates reward learning and the magnitude of RPE using Akt1 mutant mice as a model. In comparison to wild-type littermate controls, the expression of Akt1 proteins in mouse brains occurred in a gene-dosage-dependent manner and Akt1 heterozygous (HET) mice exhibited impaired striatal Akt1 activity under methamphetamine challenge. No genotypic difference was found in the basal levels of dopamine and its metabolites. In a series of reward-related learning tasks, HET mice displayed a relatively efficient method of updating reward information from the environment during the acquisition phase of the two natural reward tasks and in the reverse section of the dynamic foraging T-maze but not in methamphetamine-induced or aversive-related reward learning. The implementation of a standard reinforcement learning model and the Bayesian hierarchical parameter estimation show that HET mice have higher RPE magnitudes and that their action values are updated more rapidly among all three test sections in T-maze. These results indicate that Akt1 deficiency modulates natural reward learning and RPE. This study showed a promising avenue for investigating RPE in mutant mice and provided evidence for the potential link from genetic deficiency, to neurobiological abnormalities, to impairment in higher-order cognitive functioning.     

Val97Leu mutant presenilin-1 induces tau hyperphosphorylation and spatial memory deficit in mice and the underlying mechanisms

Although the pathological role of presenilin-1 mutation in early onset familial Alzheimer's disease has been widely studied, few focused on how the presenilin-1 mutations result in memory impairment and tau hyperphosphorylation. In the present study, we expressed human Val97Leu mutant presenilin-1, which is reported in Chinese pedigrees by our group, in transgenic mice and found that the mutant presenilin-1 induced spatial memory deficit and tau hyperphosphorylation at PHF-1, pS199/202, pT231 and pS396 epitopes, but not at pS214 and pS422 epitopes. Pearson analysis showed that the memory deficit was only significantly correlated with tau phosphorylation level at PHF-1, pS199/202, pT231 and pS396 epitopes. Additionally, the hyperphosphorylated tau and tangle-like argentophilic structures were detected at CA3 and CA4, but not CA1, region of hippocampus, and we also found tangle-like structure and wizened degenerative neurons in frontal cortex. We demonstrated the tau hyperphosphorylation at the same epitopes in N2a cells expressing the mutant presenilin-1, which is caused by inhibition of phosphoinositol-3 kinase/Akt and activation of glycogen synthase kinase-3 specifically. Our data demonstrated that human Val97Leu mutant presenilin-1 causes spatial memory deficit in mice and increases tau phosphorylation level in glycogen synthase kinase-3-dependent manner.     

Genetic analysis of G protein-coupled receptor expression in Escherichia coli: inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor

The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production.     

Computational analysis of the CB1 carboxyl‐terminus in the receptor‐G protein complex

Despite the important role of the carboxyl‐terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1‐Gi complex (Shim, Ahn, and Kendall, The Journal of Biological Chemistry 2013;288:32449–32465) to incorporate a complete CB1 Ct (Glu416^Ct–Leu472^Ct). Based on the structural constraints from NMR studies, we employed ROSETTA to predict tertiary folds, ZDOCK to predict docking orientation, and molecular dynamics (MD) simulations to obtain two distinct plausible models of CB1 Ct in the CB1‐Gi complex. The resulting models were consistent with the NMR‐determined helical structure (H9) in the middle region of the CB1 Ct. The CB1 Ct directly interacted with both Gα and Gβ and stabilized the receptor at the Gi interface. The results of site‐directed mutagenesis studies of Glu416^Ct, Asp423^Ct, Asp428^Ct, and Arg444^Ct of CB1 Ct suggested that the CB1 Ct can influence receptor‐G protein coupling by stabilizing the receptor at the Gi interface. This research provided, for the first time, models of the CB1 Ct in contact with the G protein. Proteins 2016; 84:532–543. © 2016 Wiley Periodicals, Inc.     

Age-related decrease in stimulated glutamate release and vesicular glutamate transporters in APP/PS1 transgenic and wild-type mice

We assessed baseline and KCl-stimulated glutamate release by using microdialysis in freely moving young adult (7 months) and middle-aged (17 months) transgenic mice carrying mutated human amyloid precursor protein and presenilin genes (APdE9 mice) and their wild-type littermates. In addition, we assessed the age-related development of amyloid pathology and spatial memory impaired in the water maze and changes in glutamate transporters. APdE9 mice showed gradual spatial memory impairment between 6 and 15 months of age. The stimulated glutamate release declined very robustly in 17-month-old APdE9 mice as compared to 7-month-old APdE9 mice. This age-dependent decrease in stimulated glutamate release was also evident in wild-type mice, although it was not as robust as in APdE9 mice. When compared to individual baselines, all aged wild-type mice showed 25% or greater increase in glutamate release upon KCl stimulation, but none of the aged APdE9 mice. There was an age-dependent decline in VGLUT1 levels, but not in the levels of VGLUT2, GLT-1 or synaptophysin. Astrocyte activation as measured by glial acidic fibrillary protein was increased in middle-aged APdE9 mice. Blunted pre-synaptic glutamate response may contribute to memory deficit in middle-aged APdE9 mice.     

Exposure to AC and DC magnetic fields induces changes in 5-HT1B receptor binding parameters in rat brain membranes

The binding properties of the G-protein coupled receptor (GPCR) serotonin 5-HT1B receptor were studied under exposure to AC (50 and 400 Hz) and DC magnetic fields (MF) in rat brain membranes. This was an attempt at replicating the positive findings of Massot et al. In saturation experiments using [3H]5-HT, 1-h exposures at 1.1 mT(rms) 50 Hz caused statistically significant increases in both the K(D) and B(max) binding parameters, from 1.74 +/- 0.3 to 4.51 +/- 0.86 nM and from 1428 +/- 205 to 2137 +/- 399 CPM, respectively, in good agreement with previous results. Exposure of the membranes at 400 Hz 0.675 mT(rms) did not elicit a larger increase in K(D) in spite of a much larger induced current density. DC fields (1.1 and 11 mT) had a lesser effect compared to AC fields at low values of K(Dsham), but decreased the affinity at higher values of K(Dsham). Modeling of the receptor-ligand-G protein interactions using the extended ternary complex model yielded good fits for all our data and that of Massot et al., showing that the AC field may act by decreasing the ability of the G-protein to alter the ligand-receptor affinity. The hypothesis is that the bipolar nature of the AC field explains the different nature of the effects observed with AC and DC exposures. These findings constitute one of the few documented pieces of evidence for cell-free effects of DC and extremely low frequency (ELF) AC MFs in the mT range.     

Biological responses in Balb/c mice after long‐term parenteral administration of the light subunit of mushroom tyrosinase

Light subunit of mushroom tyrosinase (LSMT) is a protein of unknown function from mushroom Agaricus bisporus that has been demonstrated to permeate through rat intestine ex vivo. Thus, it can be absorbed in the intestine, thereby holding a promise as a drug carrier for oral administration, similar to HA‐33 protein from botulinum, one of the closest structural homologs of LSMT. However, the safety of LSMT should be ensured prior to its use. Here, we described biological response of LSMT upon weekly intraperitoneal administration of 50 μg/day to the Balb/c mice for 12 weeks. Motoric and behavior profiles, as well as the index of main organs (liver, spleen, lung, heart, and kidney), and body weight, were not significantly changed as compared with the control group. Also, no IgG was detected in the serum. The results suggest that LSMT is safe for further development.     

Cryo-EM structure of dodecameric Vps4p and its 2:1 complex with Vta1p

The type I AAA (ATPase associated with a variety of cellular activities) ATPase Vps4 and its co-factor Vta1p/LIP5 function in membrane remodeling events that accompany cytokinesis, multivesicular body biogenesis, and retrovirus budding, apparently by driving disassembly and recycling of membrane-associated ESCRT (endosomal sorting complex required for transport)-III complexes. Here, we present electron cryomicroscopy reconstructions of dodecameric yeast Vps4p complexes with and without their microtubule interacting and transport (MIT) N-terminal domains and Vta1p co-factors. The ATPase domains of Vps4p form a bowl-like structure composed of stacked hexameric rings. The two rings adopt dramatically different conformations, with the “upper” ring forming an open assembly that defines the sides of the bowl and the lower ring forming a closed assembly that forms the bottom of the bowl. The N-terminal MIT domains of the upper ring localize on the symmetry axis above the cavity of the bowl, and the binding of six extended Vta1p monomers causes additional density to appear both above and below the bowl. The structures suggest models in which Vps4p MIT and Vta1p domains engage ESCRT-III substrates above the bowl and help transfer them into the bowl to be pumped through the center of the dodecameric assembly.     

Signaling mechanisms and physiological functions of G‐protein Gα13 in blood vessel formation,bone homeostasis,and cancer

Heterotrimeric G‐proteins are cellular signal transducers. They mainly relay signals from G‐protein‐coupled receptors (GPCRs). GPCRs function as guanine nucleotide‐exchange factors to active these G‐proteins. Based on the sequence and functional similarities, these G‐proteins are grouped into four subfamilies: G_s, G_i, G_q, and G_12/13. The G_12/13 subfamily consists of two members: G₁₂ and G₁₃. G_12/13‐mediated signaling pathways play pivotal roles in a variety of physiological processes, while aberrant regulation of this pathway has been identified in various human diseases. Here we summarize the signaling mechanisms and physiological functions of Gα₁₃ in blood vessel formation and bone homeostasis. We further discuss the expanding roles of Gα₁₃ in cancers, serving as oncogenes as well as tumor suppressors.     

N‐linked glycosylation of AtVSR1 is important for vacuolar protein sorting in Arabidopsis

Vacuolar sorting receptors (VSRs) in Arabidopsis mediate the sorting of soluble proteins to vacuoles in the secretory pathway. The VSRs are post‐translationally modified by the attachment of N‐glycans, but the functional significance of such a modification remains unknown. Here we have studied the role(s) of glycosylation in the stability, trafficking and vacuolar protein transport of AtVSR1 in Arabidopsis protoplasts. AtVSR1 harbors three complex‐type N‐glycans, which are located in the N‐terminal ‘PA domain’, the central region and the C‐terminal epidermal growth factor repeat domain, respectively. We have demonstrated that: (i) the N‐glycans do not affect the targeting of AtVSR1 to pre‐vacuolar compartments (PVCs) and its vacuolar degradation; and (ii) N‐glycosylation alters the binding affinity of AtVSR1 to cargo proteins and affects the transport of cargo into the vacuole. Hence, N‐glycosylation of AtVSR1 plays a critical role in its function as a VSR in plants.