Knocking out Fkbp51 decreases CCl4-induced liver injury through enhancement of mitochondrial function and Parkin activity.

BACKGROUND AND AIMS: Previously, we found that FK506 binding protein 51 (Fkbp51) knockout (KO) mice resist high fat diet-induced fatty liver and alcohol-induced liver injury. The aim of this research is to identify the mechanism of Fkbp51 in liver injury. METHODS: Carbon tetrachloride (CCl4)-induced liver injury was compared between Fkbp51 KO and wild type (WT) mice. Step-wise and in-depth analyses were applied, including liver histology, biochemistry, RNA-Seq, mitochondrial respiration, electron microscopy, and molecular assessments. The selective FKBP51 inhibitor (SAFit2) was tested as a potential treatment to ameliorate liver injury. RESULTS: Fkbp51 knockout mice exhibited protection against liver injury, as evidenced by liver histology, reduced fibrosis-associated markers and lower serum liver enzyme levels. RNA-seq identified differentially expressed genes and involved pathways, such as fibrogenesis, inflammation, mitochondria, and oxidative metabolism pathways and predicted the interaction of FKBP51, Parkin, and HSP90. Cellular studies supported co-localization of Parkin and FKBP51 in the mitochondrial network, and Parkin was shown to be expressed higher in the liver of KO mice at baseline and after liver injury relative to WT. Further functional analysis identified that KO mice exhibited increased ATP production and enhanced mitochondrial respiration. KO mice have increased mitochondrial size, increased autophagy/mitophagy and mitochondrial-derived vesicles (MDV), and reduced reactive oxygen species (ROS) production, which supports enhancement of mitochondrial quality control (MQC). Application of SAFit2, an FKBP51 inhibitor, reduced the effects of CCl4-induced liver injury and was associated with increased Parkin, pAKT, and ATP production. CONCLUSIONS: Downregulation of FKBP51 represents a promising therapeutic target for liver disease treatment.

Sex-Specific Impact of Fkbp5 on Hippocampal Response to Acute Alcohol Injection: Involvement in Alterations of Metabolism-Related Pathways.

High levels of alcohol intake alter brain gene expression and can produce long-lasting effects. FK506-binding protein 51 (FKBP51) encoded by Fkbp5 is a physical and cellular stress response gene and has been associated with alcohol consumption and withdrawal severity. Fkbp5 has been previously linked to neurite outgrowth and hippocampal morphology, sex differences in stress response, and epigenetic modification. Presently, primary cultured Fkbp5 KO and WT mouse neurons were examined for neurite outgrowth and mitochondrial signal with and without alcohol. We found neurite specification differences between KO and WT; particularly, mesh-like morphology was observed after alcohol treatment and confirmed higher MitoTracker signal in cultured neurons of Fkbp5 KO compared to WT at both naive and alcohol-treated conditions. Brain regions that express FKBP51 protein were identified, and hippocampus was confirmed to possess a high level of expression. RNA-seq profiling was performed using the hippocampus of naïve or alcohol-injected (2 mg EtOH/Kg) male and female Fkbp5 KO and WT mice. Differentially expressed genes (DEGs) were identified between Fkbp5 KO and WT at baseline and following alcohol treatment, with female comparisons possessing a higher number of DEGs than male comparisons. Pathway analysis suggested that genes affecting calcium signaling, lipid metabolism, and axon guidance were differentially expressed at naïve condition between KO and WT. Alcohol treatment significantly affected pathways and enzymes involved in biosynthesis (Keto, serine, and glycine) and signaling (dopamine and insulin receptor), and neuroprotective role. Functions related to cell morphology, cell-to-cell signaling, lipid metabolism, injury response, and post-translational modification were significantly altered due to alcohol. In summary, Fkbp5 plays a critical role in the response to acute alcohol treatment by altering metabolism and signaling-related genes.

FKBP51 modulates hippocampal size and function in post-translational regulation of Parkin.

FK506-binding protein 51 (encoded by Fkpb51, also known as Fkbp5) has been associated with stress-related mental illness. To investigate its function, we studied the morphological consequences of Fkbp51 deletion. Artificial Intelligence-assisted morphological analysis revealed that male Fkbp51 knock-out (KO) mice possess more elongated dentate gyrus (DG) but shorter hippocampal height in coronal sections when compared to WT. Primary cultured Fkbp51 KO hippocampal neurons were shown to exhibit larger dendritic outgrowth than wild-type (WT) controls and pharmacological manipulation experiments suggest that this may occur through the regulation of microtubule-associated protein. Both in vitro primary culture and in vivo labeling support a role for FKBP51 in the regulation of microtubule-associated protein expression. Furthermore, Fkbp51 KO hippocampi exhibited decreases in ßIII-tubulin, MAP2, and Tau protein levels, but a greater than 2.5-fold increase in Parkin protein. Overexpression and knock-down FKBP51 demonstrated that FKBP51 negatively regulates Parkin in a dose-dependent and ubiquitin-mediated manner. These results indicate a potential novel post-translational regulatory mechanism of Parkin by FKBP51 and the significance of their interaction on disease onset. KO has more flattened hippocampus using AI-assisted measurement Both pyramidal cell layer (PCL) of CA and granular cell layer (GCL) of DG distinguishable as two layers: deep cell layer and superficial layer. Distinct MAP2 expression between deep and superficial layer between KO and WT, Higher Parkin expression in KO brain Mechanism of FKBP51 inhibition resulting in Parkin, MAP2, Tau, and Tubulin expression differences between KO and WT mice, and resulting neurite outgrowth differences.

Over-expression of human PP5 gene in mice induces corneal hyperplasia and leads to ocular surface squamous neoplasia.

Protein phosphatase 5 (PP5) plays an important role in cell proliferation, differentiation, and development. Transgenic PP5 mice (Tg-hPP5 mice) overexpressing human PP5 gene were successfully generated by embryo injection. Tg-hPP5 mice spontaneously developed corneal hyperplasia and ocular surface squamous neoplasia (OSSN). To investigate the mechanism behind PP5-induced corneal hyperplasia, we performed immunohistochemistry, quantitative real-time PCR, and Western Blotting analyses on the corneas of Tg-hPP5 mice at 2 months and 9 months of age. We provide the first demonstration that Tg-hPP5 mice develop corneal hyperplasia at 9-months of age demonstrated via histological analysis and in vitro co-transfection investigation. We also present data that the expression of p53 is significantly reduced while the expression of FGF-7 is significantly increased in Tg-hPP5 mice with corneal hyperplasia. Co-transfection of PP5, p53, and FGF-7-promoter-driven luciferase revealed that PP5 promotes while p53 inhibits FGF-7 expression, which indicates PP5 overexpression inhibits p53 phosphorylation, thereby reducing its tumor suppressor function and increasing FGF-7 expression. In conclusion, PP5 plays a pivotal role in corneal hyperplasia development and its downregulation is a potential target for corneal hyperplasia and OSSN treatment.

Correction to: The mouse and ferret models for studying the novel avian-origin human influenza A (H7N9) virus.

An amendment to this paper has been published and can be accessed via the original article.

Hypotensive Activity of Transgenic Rice Seed Accumulating Multiple Antihypertensive Peptides.

Peptides derived from food protein have the potential to become antihypertensive agents with relatively few negative side effects. Herein, multiple antihypertensive peptides, extracted from the transgenic rice seed, were administered intragastrically into spontaneously hypertensive rats (SHRs) with different dosages, resulting in a significant decrease in the systolic blood pressure (SBP). Furthermore, for a period of 5 weeks, daily intragastric administration of the transgenic rice flour also significantly reduced the SBP of SHRs but not the Wistar Kyoto normotensive rats (WNRs), most importantly, which did not affect the growth, development, or serum chemistry of SHRs or WNRs and did not cause any pathological changes. Our work provides an alternative source of natural antihypertensive agents.

The glucocorticoid receptor-FKBP51 complex contributes to fear conditioning and posttraumatic stress disorder.

Posttraumatic stress disorder (PTSD) can develop after exposure to severe psychological trauma, leaving patients with disabling anxiety, nightmares, and flashbacks. Current treatments are only partially effective, and development of better treatments is hampered by limited knowledge of molecular mechanisms underlying PTSD. We have discovered that the glucocorticoid receptor (GR) and FK506 binding protein 51 (FKBP51) form a protein complex that is elevated in PTSD patients compared with unaffected control subjects, subjects exposed to trauma without PTSD, and patients with major depressive disorder (MDD). The GR-FKBP51 complex is also elevated in fear-conditioned mice, an aversive learning paradigm that models some aspects of PTSD. Both PTSD patients and fear-conditioned mice had decreased GR phosphorylation, decreased nuclear GR, and lower expression of 14-3-3ε, a gene regulated by GR. We created a peptide that disrupts GR-FKBP51 binding and reverses behavioral and molecular changes induced by fear conditioning. This peptide reduces freezing time and increases GR phosphorylation, GR-FKBP52 binding, GR nuclear translocation, and 14-3-3ε expression in fear-conditioned mice. These experiments demonstrate a molecular mechanism contributing to PTSD and suggest that the GR-FKBP51 complex may be a diagnostic biomarker and a potential therapeutic target for preventing or treating PTSD.

Loss of FKBP5 Affects Neuron Synaptic Plasticity: An Electrophysiology Insight.

FKBP5 (FKBP51) is a glucocorticoid receptor (GR) binding protein, which acts as a co-chaperone of heat shock protein 90 (HSP90) and negatively regulates GR. Its association with mental disorders has been identified, but its function in disease development is largely unknown. Long-term potentiation (LTP) is a functional measurement of neuronal connection and communication, and is considered one of the major cellular mechanisms that underlies learning and memory, and is disrupted in many mental diseases. In this study, a reduction in LTP in Fkbp5 knockout (KO) mice was observed when compared to WT mice, which correlated with changes to the glutamatergic and GABAergic signaling pathways. The frequency of mEPSCs was decreased in KO hippocampus, indicating a decrease in excitatory synaptic activity. While no differences were found in levels of glutamate between KO and WT, a reduction was observed in the expression of excitatory glutamate receptors (NMDAR1, NMDAR2B and AMPAR), which initiate and maintain LTP. The expression of the inhibitory neurotransmitter GABA was found to be enhanced in Fkbp5 KO hippocampus. Further investigation suggested that increased expression of GAD65, but not GAD67, accounted for this increase. Additionally, a functional GABAergic alteration was observed in the form of increased mIPSC frequency in the KO hippocampus, indicating an increase in presynaptic GABA release. Our findings uncover a novel role for Fkbp5 in neuronal synaptic plasticity and highlight the value of Fkbp5 KO as a model for studying its role in neurological function and disease development.

Estrogen-Dependent Upregulation of Adcyap1r1 Expression in Nucleus Accumbens Is Associated With Genetic Predisposition of Sex-Specific QTL for Alcohol Consumption on Rat Chromosome 4.

Humans show sex differences related to alcohol use disorders (AUD). Animal model research has the potential to provide important insight into how sex differences affect alcohol consumption, particularly because female animals frequently drink more than males. In previous work, inbred strains of the selectively bred alcohol-preferring (P) and non-preferring (NP) rat lines revealed a highly significant quantitative trait locus (QTL) on rat chromosome 4, with a logarithm of the odds score of 9.2 for alcohol consumption. Recently, interval-specific congenic strains (ISCS) were developed by backcrossing the congenic P.NP line to inbred P (iP) rats to further refine the chromosome 4 QTL region. Two ISCS sub-strains, ISCS-A and ISCS-B, were obtained with a narrowed QTL, where the smallest region of overlap consisted of 8.9 Mb in ISCS-B. Interestingly, we found that females from both ISCS lines consumed significantly less alcohol than female iP controls (p < 0.05), while no differences in alcohol consumption were observed between male ISCS and iP controls. RNA-sequencing was performed on the nucleus accumbens of alcohol-naïve female ISCS-B and iP rats, which revealed differentially expressed genes (DEG) with greater than 2-fold change and that were functionally relevant to behavior. These DEGs included down-regulation of Oxt, Asb4, Gabre, Gabrq, Chat, Slc5a7, Slc18a8, Slc10a4, and Ngfr, and up-regulation of Ttr, Msln, Mpzl2, Wnt6, Slc17a7, Aldh1a2, and Gstm2. Pathway analysis identified significant alterations in gene networks controlling nervous system development and function, as well as cell signaling, GABA and serotonin receptor signaling and G-protein coupled receptor signaling. In addition, ß-estradiol was identified as the most significant upstream regulator. The expression levels of estrogen-responsive genes that mapped to the QTL interval and have been previously associated with alcohol consumption were measured using RT-qPCR. We found that expression of the Adcyap1r1 gene, encoding the pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor, was upregulated in female ISCS-B compared to female iP controls, while no differences were exhibited in males. In addition, sequence variants in the Adcyap1r1 promoter region showed a differential response to estrogen stimulation in vitro. These findings demonstrate that rat chromosome 4 QTL contains genetic variants that respond to estrogen and are associated with female alcohol consumption.

Protein phosphatase 5 and the tumor suppressor p53 down-regulate each other's activities in mice.

Protein phosphatase 5 (PP5), a serine/threonine phosphatase, has a wide range of biological functions and exhibits elevated expression in tumor cells. We previously reported that pp5-deficient mice have altered ataxia-telangiectasia mutated (ATM)-mediated signaling and function. However, this regulation was likely indirect, as ATM is not a known PP5 substrate. In the current study, we found that pp5-deficient mice are hypersensitive to genotoxic stress. This hypersensitivity was associated with the marked up-regulation of the tumor suppressor tumor protein p53 and its downstream targets cyclin-dependent kinase inhibitor 1A (p21), MDM2 proto-oncogene (MDM2), and phosphatase and tensin homolog (PTEN) in pp5-deficient tissues and cells. These observations suggested that PP5 plays a role in regulating p53 stability and function. Experiments conducted with p53+/-pp5+/- or p53+/-pp5-/- mice revealed that complete loss of PP5 reduces tumorigenesis in the p53+/- mice. Biochemical analyses further revealed that PP5 directly interacts with and dephosphorylates p53 at multiple serine/threonine residues, resulting in inhibition of p53-mediated transcriptional activity. Interestingly, PP5 expression was significantly up-regulated in p53-deficient cells, and further analysis of pp5 promoter activity revealed that p53 strongly represses PP5 transcription. Our results suggest a reciprocal regulatory interplay between PP5 and p53, providing an important feedback mechanism for the cellular response to genotoxic stress.

Ablation of protein phosphatase 5 (PP5) leads to enhanced both bone and cartilage development in mice.

This study aimed to investigate the role of protein phosphatase 5 (PP5) on bone and cartilage development using both in vivo and in vitro approaches. Six- to 8-week- old male PP5 knockout mice (KO) and their wild-type (WT) littermate controls were randomly selected for this study, and their body weights and bone (femur) lengths were measured. Micro-computed tomography scanning (Micro-CT) was performed to determine femoral bone density and micro-architecture. Mesenchymal stem cells (MSCs) isolated from bone marrow were used to examine the effects of PP5 on osteogenesis in vitro. Whole-mount Alcian blue and Alizarin red staining were used to detect cartilage formation in newborn vertebrae, limbs, and feet. Hematoxylin and eosin (H&E) staining was performed to determine growth plate thickness. Real-time PCR analysis, western blotting, and immunohistochemistry were used to detect the expression of genes and proteins in bone marrow-derived MSCs as well as in bone and cartilage tissues. The results showed PP5 KO mice exhibited significantly reduced body weight and shorter femur length compared to WT controls. The KO mice also had significantly higher volumetric bone mineral density (BMD), trabecular bone volume, and cortical thickness in the femur. The deficiency of PP5 significantly enhanced the formation of cartilage in vertebrae, limbs, and feet. In addition, KO mice possessed a wider distal femur growth plates containing significantly more chondrocytes than WT mice. Furthermore, higher expressions of several cartilage-specific genes were observed in the articular cartilage of PP5 KO mice. Immunohistochemical labeling of growth plates demonstrated that phospho-PPARγ, Runx1, and Runx2 levels were considerably higher in the KO mice. In conclusion, PP5 is a significant negative regulator on the regulation of bone and cartilage development.

Loss of FKBP5 impedes adipocyte differentiation under both normoxia and hypoxic stress.

FK506-binding protein 51 (FKBP51) is one of the most important regulators in the GR-mediated stress response, and we previously demonstrated that loss of FKBP5 arrests adipogenesis and renders mice resistant to diet-induced obesity (DIO). However, the exact role of FKBP5 in the process of adipocyte differentiation under hypoxic conditions (the common microenvironment where adipocytes reside in obese individuals) is still unclear. Here, by isolating and culturing WT- and Fkbp5-knockout mouse embryonic fibroblasts (MEFs), and treat them at normal oxygen environment (21% O2, nomorxia) or low oxygen environment (5% O2, hypoxia). Enhanced adipogenesis were observed at hypoxia when compared to normal oxygen environment. The loss of FKBP5 significantly prevents the adipogenesis from KO MEFs under nomorxia condition, with subtle enhancement of adipogenesis at hypoxia condition, which is similar as observed in WT-MEFs at hypoxia condition but with obvious enhancement of adipogenesis. Importantly, the protein level of FKBP5 reduced in undifferentiated MEFs under acute hypoxic stress (24 h), but drastically increased during the mid-late stage of adipocyte (Day 6) differentiation from WT-MEFs under chronic hypoxia. Furthermore, we find under normal and hypoxic conditions that FKBP5 deletion alters the expression profile of adipogenesis-related genes, including those involved in lipogenesis, lipolysis, and energy metabolism, which partially explains the compromised adipocyte differentiation in FKBP51-KO MEFs. Taken together, our findings identify a novel role of FKBP5 in hypoxia-regulated adipogenesis, and provide a candidate for anti-obesity strategies targeting FKBP51.

The FKBP5 Gene Affects Alcohol Drinking in Knockout Mice and Is Implicated in Alcohol Drinking in Humans.

FKBP5 encodes FK506-binding protein 5, a glucocorticoid receptor (GR)-binding protein implicated in various psychiatric disorders and alcohol withdrawal severity. The purpose of this study is to characterize alcohol preference and related phenotypes in Fkbp5 knockout (KO) mice and to examine the role of FKBP5 in human alcohol consumption. The following experiments were performed to characterize Fkpb5 KO mice. (1) Fkbp5 KO and wild-type (WT) EtOH consumption was tested using a two-bottle choice paradigm; (2) The EtOH elimination rate was measured after intraperitoneal (IP) injection of 2.0 g/kg EtOH; (3) Blood alcohol concentration (BAC) was measured after 3 h limited access of alcohol; (4) Brain region expression of Fkbp5 was identified using LacZ staining; (5) Baseline corticosterone (CORT) was assessed. Additionally, two SNPs, rs1360780 (C/T) and rs3800373 (T/G), were selected to study the association of FKBP5 with alcohol consumption in humans. Participants were college students (n = 1162) from 21-26 years of age with Chinese, Korean or Caucasian ethnicity. The results, compared to WT mice, for KO mice exhibited an increase in alcohol consumption that was not due to differences in taste sensitivity or alcohol metabolism. Higher BAC was found in KO mice after 3 h of EtOH access. Fkbp5 was highly expressed in brain regions involved in the regulation of the stress response, such as the hippocampus, amygdala, dorsal raphe and locus coeruleus. Both genotypes exhibited similar basal levels of plasma corticosterone (CORT). Finally, single nucleotide polymorphisms (SNPs) in FKBP5 were found to be associated with alcohol drinking in humans. These results suggest that the association between FKBP5 and alcohol consumption is conserved in both mice and humans.

Npy deletion in an alcohol non-preferring rat model elicits differential effects on alcohol consumption and body weight.

Neuropeptide Y (NPY) is widely expressed in the central nervous system and influences many physiological processes. It is located within the rat quantitative trait locus (QTL) for alcohol preference on chromosome 4. Alcohol-nonpreferring (NP) rats consume very little alcohol, but have significantly higher NPY expression in the brain than alcohol-preferring (P) rats. We capitalized on this phenotypic difference by creating an Npy knockout (KO) rat using the inbred NP background to evaluate NPY effects on alcohol consumption. Zinc finger nuclease (ZNF) technology was applied, resulting in a 26-bp deletion in the Npy gene. RT-PCR, Western blotting and immunohistochemistry confirmed the absence of Npy mRNA and protein in KO rats. Alcohol consumption was increased in Npy(+/-) but not Npy(-/-) rats, while Npy(-/-) rats displayed significantly lower body weight when compared to Npy(+/+) rats. In whole brain tissue, expression levels of Npy-related and other alcohol-associated genes, Npy1r, Npy2r, Npy5r, Agrp, Mc3r, Mc4r, Crh and Crh1r, were significantly greater in Npy(-/-) rats, whereas Pomc and Crhr2 expressions were highest in Npy(+/-) rats. These findings suggest that the NPY-system works in close coordination with the melanocortin (MC) and corticotropin-releasing hormone (CRH) systems to modulate alcohol intake and body weight.

FKBP51 Null Mice Are Resistant to Diet-Induced Obesity and the PPARγ Agonist Rosiglitazone.

FK506-binding protein-51 (FKBP51) is a molecular cochaperone recently shown to be a positive regulator of peroxisome proliferator-activated receptor (PPAR)γ, the master regulator of adipocyte differentiation and function. In cellular models of adipogenesis, loss of FKBP51 not only reduced PPARγ activity but also reduced lipid accumulation, suggesting that FKBP51 knock-out (KO) mice might have insufficient development of adipose tissue and lipid storage ability. This model was tested by examining wild-type (WT) and FKBP51-KO mice under regular and high-fat diet conditions. Under both diets, FKBP51-KO mice were resistant to weight gain, hepatic steatosis, and had greatly reduced white adipose tissue (WAT) but higher amounts of brown adipose tissue. Under high-fat diet, KO mice were highly resistant to adiposity and exhibited reduced plasma lipids and elevated glucose and insulin tolerance. Profiling of perigonadal and sc WAT revealed elevated expression of brown adipose tissue lineage genes in KO mice that correlated increased energy expenditure and a shift of substrate oxidation to carbohydrates, as measured by indirect calorimetry. To directly test PPARγ involvement, WT and KO mice were fed rosiglitazone agonist. In WT mice, rosiglitazone induced whole-body weight gain, increased WAT mass, a shift of substrate oxidation to lipids, and elevated expression of PPARγ-regulated lipogenic genes in WAT. In contrast, KO mice had reduced rosiglitazone responses for these parameters. Our results identify FKBP51 as an important regulator of PPARγ in WAT and as a potential new target in the treatment of obesity and diabetes.

Establishment and phenotypic analysis of an Mstn knockout rat.

Myostatin (Mstn) is an inhibitor of myogenesis, regulating the number and size of skeletal myocytes. In addition to its myogenic regulatory function, Mstn plays important roles in the development of adipose tissues and in metabolism. In the present study, an Mstn knockout rat model was generated using the zinc finger nuclease (ZFN) technique in order to further investigate the function and mechanism of Mstn in metabolism. The knockout possesses a frame shift mutation resulting in an early termination codon and a truncated peptide of 109 amino acids rather than the full 376 amino acids. The absence of detectable mRNA confirmed successful knockout of Mstn. Relative to wild-type (WT) littermates, Knockout (KO) rats exhibited significantly greater body weight, body circumference, and muscle mass. However, no significant differences in grip force was observed, indicating that Mstn deletion results in greater muscle mass but not greater muscle fiber strength. Additionally, KO rats were found to possess less body fat relative to WT littermates, which is consistent with previous studies in mice and cattle. The aforementioned results indicate that Mstn knockout increases muscle mass while decreasing fat content, leading to observed increases in body weight and body circumference. The Mstn knockout rat model provides a novel means to study the role of Mstn in metabolism and Mstn-related muscle hypertrophy.

Combination of ESI and MALDI mass spectrometry for qualitative, semi-quantitative and in situ analysis of gangliosides in brain.

Gangliosides are a family of complex lipids that are abundant in the brain. There is no doubt the investigations about the distribution of gangliosides in brian and the relationship between gangliosides and Alzheimer's disease is profound. However, these investigations are full of challenges due to the structural complexity of gangliosides. In this work, the method for efficient extraction and enrichment of gangliosides from brain was established. Moreover, the distribution of gangliosides in brain was obtained by matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). It was found that 3-aminoquinoline (3-AQ) as matrix was well-suited for MALDI MS analysis of gangliosides in negative ion mode. In addition, the pretreatment by ethanol (EtOH) cleaning brain section and the addition of ammonium formate greatly improved the MS signal of gangliosides in the brain section when MALDI MSI analysis was employed. The distribution of ganliosides in cerebral cortex, hippocampus and cerebellum was respectively acquired by electrospray ionization (ESI) MS and MALDI MSI, and the data were compared for reliability evaluation of MALDI MSI. Further, applying MALDI MSI technology, the distribution of gangliosides in amyloid precursor protein transgenic mouse brain was obtained, which may provide a new insight for bioresearch of Alzheimer's disease (AD).

Mitochondria-nucleus shuttling FK506-binding protein 51 interacts with TRAF proteins and facilitates the RIG-I-like receptor-mediated expression of type I IFN.

Virus-derived double-stranded RNAs (dsRNAs) are sensed in the cytosol by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs). These induce the expression of type I IFN and proinflammatory cytokines through signaling pathways mediated by the mitochondrial antiviral signaling (MAVS) protein. TNF receptor-associated factor (TRAF) family proteins are reported to facilitate the RLR-dependent expression of type I IFN by interacting with MAVS. However, the precise regulatory mechanisms remain unclear. Here, we show the role of FK506-binding protein 51 (FKBP51) in regulating the dsRNA-dependent expression of type I IFN. The binding of FKBP51 to TRAF6 was first identified by "in vitro virus" selection and was subsequently confirmed with a coimmunoprecipitation assay in HEK293T cells. The TRAF-C domain of TRAF6 is required for its interaction, although FKBP51 does not contain the consensus motif for interaction with the TRAF-C domain. Besides TRAF6, we found that FKBP51 also interacts with TRAF3. The depletion of FKBP51 reduced the expression of type I IFN induced by dsRNA transfection or Newcastle disease virus infection in murine fibroblasts. Consistent with this, the FKBP51 depletion attenuated dsRNA-mediated phosphorylations of IRF3 and JNK and nuclear translocation of RelA. Interestingly, dsRNA stimulation promoted the accumulation of FKBP51 in the mitochondria. Moreover, the overexpression of FKBP51 inhibited RLR-dependent transcriptional activation, suggesting a scaffolding function for FKBP51 in the MAVS-mediated signaling pathway. Overall, we have demonstrated that FKBP51 interacts with TRAF proteins and facilitates the expression of type I IFN induced by cytosolic dsRNA. These findings suggest a novel role for FKBP51 in the innate immune response to viral infection.

Alcohol-preferring rats show decreased corticotropin-releasing hormone-2 receptor expression and differences in HPA activation compared to alcohol-nonpreferring rats.

BACKGROUND: Corticotropin-releasing hormone (CRH) and urocortins (UCNs) bind to corticotropin-releasing hormone type 2 receptor (CRF2 receptor ), a Gs protein-coupled receptor that plays an important role in modulation of anxiety and stress responses. The Crhr2 gene maps to a quantitative trait locus (QTL) for alcohol preference on chromosome 4 previously identified in inbred alcohol-preferring (iP) and-nonpreferring (iNP) F2 rats. METHODS: Real-time polymerase chain reaction was utilized to screen for differences in Crhr2 mRNA expression in the central nervous system (CNS) of male iP and iNP rats. DNA sequence analysis was then performed to screen for polymorphism in Crhr2 in order to identify genetic variation, and luciferase reporter assays were then applied to test their functional significance. Next, binding assays were used to determine whether this polymorphism affected CRF2 receptor binding affinity as well as CRF2 receptor density in the CNS. Finally, social interaction and corticosterone levels were measured in the P and NP rats before and after 30-minute restraint stress. RESULTS: Crhr2 mRNA expression studies found lower levels of Crhr2 mRNA in iP rats compared to iNP rats. In addition, DNA sequencing identified polymorphisms in the promoter region, coding region, and 3'-untranslated region between the iP and iNP rats. A 7 bp insertion in the Crhr2 promoter of iP rats altered expression in vitro as measured by reporter assays, and we found that CRF2 receptor density was lower in the amygdala of iP as compared to iNP rats. Male P rats displayed decreased social interaction and significantly higher corticosterone levels directly following 30-minute restraint when compared to male NP rats. CONCLUSIONS: This study identified Crhr2 as a candidate gene of interest underlying the chromosome 4 QTL for alcohol consumption that was previously identified in the P and NP model. Crhr2 promoter polymorphism is associated with reduced mRNA expression in certain brain regions, particularly the amygdala, and lowered the density of CRF2 receptor in the amygdala of iP compared to iNP rats. Together, these differences between the animals may contribute to the drinking disparity as well as the anxiety differences of the P and NP rats.

Phosphatase of regenerating liver 2 (PRL2) deficiency impairs Kit signaling and spermatogenesis.

The Phosphatase of Regenerating Liver (PRL) proteins promote cell signaling and are oncogenic when overexpressed. However, our understanding of PRL function came primarily from studies with cultured cell lines aberrantly or ectopically expressing PRLs. To define the physiological roles of the PRLs, we generated PRL2 knock-out mice to study the effects of PRL deletion in a genetically controlled, organismal model. PRL2-deficient male mice exhibit testicular hypotrophy and impaired spermatogenesis, leading to decreased reproductive capacity. Mechanistically, PRL2 deficiency results in elevated PTEN level in the testis, which attenuates the Kit-PI3K-Akt pathway, resulting in increased germ cell apoptosis. Conversely, increased PRL2 expression in GC-1 cells reduces PTEN level and promotes Akt activation. Our analyses of PRL2-deficient animals suggest that PRL2 is required for spermatogenesis during testis development. The study also reveals that PRL2 promotes Kit-mediated PI3K/Akt signaling by reducing the level of PTEN that normally antagonizes the pathway. Given the strong cancer susceptibility to subtle variations in PTEN level, the ability of PRL2 to repress PTEN expression qualifies it as an oncogene and a novel target for developing anti-cancer agents.