Growth arrest specific protein 7 inhibits tau fibrillogenesis.

Here we show that Gas7 inhibits phosphorylated tau fibrillogenesis by binding to phosphorylated tau at its non-WW domain, presumably F-BAR domain. We revealed that Gas7 binds to the third repeat domain of tau, the core element of tau oligomerization and the C-terminal domain of tau and alters the conformation not to form fibrils. These results suggest that Gas7 may serve to protect against Alzheimer's disease and other tauopathies by preventing tau fibrillogenesis.

Fluorescent resonance energy transfer -based biosensor for detecting conformational changes of Pin1.

Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), regulates the activity and stability of various phosphorylated proteins. Pin1 consists of a PPIase domain and WW domain, both of which are required for the function of Pin1. However, how the behavior of these domains changes upon binding to phosphorylated proteins has not been analyzed. We created a Fluorescent Resonance Energy Transfer (FRET)-based biosensor "CPinY", which is composed of Pin1 flanked by CFP and YFP, and analyzed the interaction between Pin1 and c-Myc. Our results indicated that the dual phosphorylation of c-Myc at Thr58 and Ser62 is essential for tight interaction with Pin1. Additionally, this interaction caused a significant conformational change in Pin1. Our CPinY biosensor also detected a novel type of inhibitor of Pin1 function. We believe that his biosensor will be a novel drug screening technology targeting Pin1.

Prolyl isomerase Pin1 is required sperm production by promoting mitosis progression of spermatogonial stem cells.

A prolyl isomerase Pin1 deficient (Pin1-/-) male mice had severe testicular atrophy. We investigated the function of Pin1 in spermatogenesis by analyzing the Pin1-/- mice at reproductive age. Pin1-/- mice had lessαPLZF positive spermatogonia (undifferentiated spermatogonia) than wild type (WT). Nevertheless, the Pin1-/- testis contained approximately the same number of GFRα1 positive spermatogonia (SSCs in steady state) as the WT testis. Furthermore, degeneration of the spermatogenia appeared in seminiferous tubules of 10 months old Pin1-/- mouse testis, and abnormal shape GFRα1 positive spermatogonia were observed. In Pin1-/- spermatogonia, the ratio of the phospho-histone H3 positive cells (mitotic cells) in GFRα1-positive spermatogonia was higher than that of WT. These results suggest that Pin1 promotes the progression of the mitotic cell cycle of SSC in steady-state, which is required for the sperm production from SSCs.

Food polyphenols targeting peptidyl prolyl cis/trans isomerase Pin1.

We searched for inhibitors against prolyl isomerase Pin1 in order to develop functional foods to prevent and cure various Pin1 related diseases such as cancer, diabetes, cardiovascular disease, Alzheimers's disease, and so on. We created a polyphenol library consisting of ingredients in healthy foods and beverages, since polyphenols like epigallocatechin gallate (EGCG) in green tea and 974B in brown algae had been identified as its Pin1 inhibitors. Several polyphenols such as EGCG derivatives, caffeic acid derivatives and tannic acid inhibited Pin1 activity. These results provide a first step in development of the functional foods and beverage targeting Pin1 and its related diseases.

Prolyl isomerase Pin1 promotes proplatelet formation of megakaryocytes via tau.

Here we show that Pin1, a peptidyl-prolyl cis/trans isomerase which catalyzes the isomerization of phosphorylated Ser/Thr-Pro, is a regulatory molecule of thrombopoiesis. We found that mice lacking the Pin1 gene (Pin1-/- mice) formed more megakaryocytes (MKs) than wild type mice (WT mice), and that the proplatelet formation of MKs was poorer in Pin1-/- mice than WT mice. Treatment of Meg-01 cells, a megakaryoblastic floating cell line, with shRNA against Pin1 suppressed the proplatelet formation. Expression of tau, a microtubule associated protein was induced in MKs during proplatelet formation. Pin1 bound tau and promoted microtubule polymerization. Our results show that Pin1 serves as a positive regulatory molecule of proplatelet formation of MKs by enhancing the function of phosphorylated tau.

Prolyl isomerase Pin1 regulates doxorubicin-inducible P-glycoprotein level by reducing Foxo3 stability.

It has been known that the phosphoSer/Thr-Pro-specific peptidyl prolyl cis/trans isomerase Pin1 regulates a variety of intracellular signaling pathways, including the response to the genotoxic drug doxorubicin. Pin1 binds phosphorylated p53 and stabilizes p53 to cause cell cycle arrest and apoptosis quickly in response to doxorubicin. Here we show another mechanism of Pin1 to maintain cell sensitivity to genotoxic stress, irrespective of whether p53 is present or not. In response to the genotoxic drug, Pin1 binds and decreases levels of the phosphorylated Foxo3, the positive transcription factor of P-glycoprotein (P-gp) gene. Through this mechanism of action, Pin1 decreases the level of P-gp and signals the cell to pump the genotoxic drugs out. This shows that Pin1 is implemented in maintaining the susceptibility to the genotoxic drugs by controlling P-gp level as well as p53-dependent apoptosis and cell cycle signaling pathways.

A high-throughput screen for inhibitors of the prolyl isomerase, Pin1, identifies a seaweed polyphenol that reduces adipose cell differentiation.

The peptidyl prolyl cis/trans isomerase Pin1 enhances the uptake of triglycerides and the differentiation of fibroblasts into adipose cells in response to insulin stimulation. Pin1 downregulation could be a potential approach to prevent and treat obesity-related disorders. In order to identify an inhibitor of Pin1 that exhibited minimal cytotoxicity, we established a high-throughput screen for Pin1 inhibitors and used this method to identify an inhibitor from 1,056 crude fractions of two natural product libraries. The candidate, a phlorotannin called 974-B, was isolated from the seaweed, Ecklonia kurome. 974-B inhibited the differentiation of mouse embryonic fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity. We discovered the Pin1 inhibitor, 974-B, from the seaweed, E. kurome, and showed that it blocks the differentiation of fibroblasts into adipose cells, suggesting that 974-B could be a lead drug candidate for obesity-related disorders.

Establishment of adipose-derived mesenchymal stem cell lines from a p53-knockout mouse.

Mesenchymal stem cells (MSCs) can differentiate into a variety of cell types. MSCs exist in several tissues such as the bone marrow, adipose, muscle, cartilage, and tendon. This differentiation potential makes MSCs candidates for cell-based therapeutic strategies for mesenchymal tissue injuries. MSCs can be prepared from bone marrow (BM-MSCs) and adipose (AD-MSCs); however, these MSCs exhibit senescence-associated growth arrest and display inevitable heterogeneity. We established several AD-MSC cell lines from a p53-knockout (KO) mouse. These cell lines were immortalized, but no cell lines grew anchorage-independently, suggesting that they are not cancerous. They differentiated into adipocytes, osteoblasts, and chondrocytes by treatment with certain stimuli. Moreover, following injection into the tail vein, the cells migrated into the wounded region of the liver and differentiated into hepatocytes. We succeeded in establishing several AD-MSC clonal cell lines that maintain the tissue-specific markers and characteristics of the developmental phase. These clonal cell lines will serve as important tools to study the mechanism of differentiation of MSCs.

Effect of Pin1 or microtubule binding on dephosphorylation of FTDP-17 mutant Tau.

Neurodegenerative tauopathies, including Alzheimer disease, are characterized by abnormal hyperphosphorylation of the microtubule-associated protein Tau. One group of tauopathies, known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), is directly associated with mutations of the gene tau. However, it is unknown why mutant Tau is highly phosphorylated in the patient brain. In contrast to in vivo high phosphorylation, FTDP-17 Tau is phosphorylated less than wild-type Tau in vitro. Because phosphorylation is a balance between kinase and phosphatase activities, we investigated dephosphorylation of mutant Tau proteins, P301L and R406W. Tau phosphorylated by Cdk5-p25 was dephosphorylated by protein phosphatases in rat brain extracts. Compared with wild-type Tau, R406W was dephosphorylated faster and P301L slower. The two-dimensional phosphopeptide map analysis suggested that faster dephosphorylation of R406W was due to a lack of phosphorylation at Ser-404, which is relatively resistant to dephosphorylation. We studied the effect of the peptidyl-prolyl isomerase Pin1 or microtubule binding on dephosphorylation of wild-type Tau, P301L, and R406W in vitro. Pin1 catalyzes the cis/trans isomerization of phospho-Ser/Thr-Pro sequences in a subset of proteins. Dephosphorylation of wild-type Tau was reduced in brain extracts of Pin1-knockout mice, and this reduction was not observed with P301L and R406W. On the other hand, binding to microtubules almost abolished dephosphorylation of wild-type and mutant Tau proteins. These results demonstrate that mutation of Tau and its association with microtubules may change the conformation of Tau, thereby suppressing dephosphorylation and potentially contributing to the etiology of tauopathies.

A novel role for hGas7b in microtubular maintenance: possible implication in tau-associated pathology in Alzheimer disease.

Here, we report a novel role for hGas7b (human growth arrest specific protein 7b) in the regulation of microtubules. Using a bioinformatic approach, we studied the actin-binding protein hGas7b with a structural similarity to the WW domain of a peptidyl prolyl cis/trans isomerase, Pin1, that facilitates microtubule assembly. Thus, we have demonstrated that hGas7b binds Tau at the WW motif and that the hGas7b/Tau protein complex interacts with the microtubules, promoting tubulin polymerization. Tau, in turn, contributes to protein stability of hGas7b. Furthermore, we observed decreased levels of hGas7b in the brains from patients with Alzheimer disease. These results suggest an important role for hGas7b in microtubular maintenance and possible implication in Alzheimer disease.

Apathetic and withdrawing students in Japanese universities--with regard to Hikikomori and student apathy.

UNLABELLED: In recent years, the increasing number of young people withdrawing from society, so called Hikikomori, has been a cause for concern in Japan. These are people who stay at home and do not work or attend school for more than 6 months. Most of them are not regarded as having any psychotic illness such as schizophrenia. With respect to college students, "student apathy" syndrome has been discussed since the 1960's. OBJECTIVES: To evaluate the proportion of apathetic and withdrawing students among those who leave school, take off, or repeat academic years; to see how these situations have changed in the last 20 years; and to identify the characteristics of such high risk groups so as to provide them with effective psychiatric support services. METHODS: First I examined the mean rates of the academic events mentioned above among students of Japanese national universities. I then compared those rates statistically between males and females, and among 6 groups according to gender and academic majors. Then the reasons for those academic events were examined. RESULTS: The rates of each event have continually increased over the last 21 years, and a considerable number of the students were shown to have been in a state of "student apathy." Male 4-year course students had a high risk, especially male science course students had serious problems.

Potential benefits and harms of a peer support social network service on the internet for people with depressive tendencies: qualitative content analysis and social network analysis.

BACKGROUND: Internet peer support groups for depression are becoming popular and could be affected by an increasing number of social network services (SNSs). However, little is known about participant characteristics, social relationships in SNSs, and the reasons for usage. In addition, the effects of SNS participation on people with depression are rather unknown. OBJECTIVE: The aim was to explore the potential benefits and harms of an SNS for depression based on a concurrent triangulation design of mixed methods strategy, including qualitative content analysis and social network analysis. METHODS: A cross-sectional Internet survey of participants, which involved the collection of SNS log files and a questionnaire, was conducted in an SNS for people with self-reported depressive tendencies in Japan in 2007. Quantitative data, which included user demographics, depressive state, and assessment of the SNS (positive vs not positive), were statistically analyzed. Descriptive contents of responses to open-ended questions concerning advantages and disadvantages of SNS participation were analyzed using the inductive approach of qualitative content analysis. Contents were organized into codes, concepts, categories, and a storyline based on the grounded theory approach. Social relationships, derived from data of "friends," were analyzed using social network analysis, in which network measures and the extent of interpersonal association were calculated based on the social network theory. Each analysis and integration of results were performed through a concurrent triangulation design of mixed methods strategy. RESULTS: There were 105 participants. Median age was 36 years, and 51% (36/71) were male. There were 37 valid respondents; their number of friends and frequency of accessing the SNS were significantly higher than for invalid/nonrespondents (P = .008 and P = .003). Among respondents, 90% (28/31) were mildly, moderately, or severely depressed. Assessment of the SNS was performed by determining the access frequency of the SNS and the number of friends. Qualitative content analysis indicated that user-selectable peer support could be passive, active, and/or interactive based on anonymity or ease of use, and there was the potential harm of a downward depressive spiral triggered by aggravated psychological burden. Social network analysis revealed that users communicated one-on-one with each other or in small groups (five people or less). A downward depressive spiral was related to friends who were moderately or severely depressed and friends with negative assessment of the SNS. CONCLUSIONS: An SNS for people with depressive tendencies provides various opportunities to obtain support that meets users' needs. To avoid a downward depressive spiral, we recommend that participants do not use SNSs when they feel that the SNS is not user-selectable, when they get egocentric comments, when friends have a negative assessment of the SNS, or when they have additional psychological burden.

Prolyl Isomerase Pin1 Directly Regulates Calcium/Calmodulin-Dependent Protein Kinase II Activity in Mouse Brains.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is abundant in the brain and functions as a mediator of calcium signaling. We found that the relative activity of CaMKII was significantly lower in the WT mouse brains than in the Pin1-/- mouse brains. Pin1 binds to phosphorylated CaMKII and weakens its activity. For this reason, the phosphorylation level of tau in the presence of Pin1 is lower than that in the absence of Pin1, and microtubule polymerization is not downregulated by CaMKII when Pin1 is present. These results suggest a novel mechanism of action of Pin1 to prevent neurodegeneration.

Characteristics and Risk Factors for Suicide and Deaths Among College Students: A 23-Year Serial Prevalence Study of Data From 8.2 Million Japanese College Students.

OBJECTIVE: Suicide is a leading cause of death for college students. The aim of this study was to identify risk factors of suicide among college students that could improve university services to help prevent college suicide. METHODS: We conducted a 23-year serial prevalence study of the prevalence and characteristics of death and suicide among 8,262,314 Japanese college students. We analyzed rates of suicide from the 1989 to 1990 academic year through the 2011-2012 academic year and characterized suicide among this population, focusing on students' sex and psychiatric and academic backgrounds to identify risk factors for suicide. RESULTS: Suicide rates increased throughout the 23 years, and suicide was the leading cause of death every year from 1996 onward. Suicide accounted for 42.4% of all deaths that happened in the 23 years. Male students, medicine majors, students in the final year of their program, and students who completed extra years of schooling or took academic leaves of absence were at higher risk for suicide. Only 16.4% had received an official psychiatric diagnosis and 16.0% had received services through the university health center prior to the suicides. CONCLUSIONS: Results suggest the need for a stronger support system for college students. Areas for improvement could include better advertising of mental health services, student and staff education about suicide risk factors, and mentorship and outreach programs for students in their final year of classes, those majoring in medicine, and those who have taken leaves of absence or failed classes. Accommodations at the administrative level would also be helpful for students who need to retake classes or transfer credit.

Characteristics and Risk Factors for Negative Academic Events: A 27-Year Serial Prevalence Study of 9.7 Million Japanese College Students.

OBJECTIVE: To examine the prevalence of and the factors contributing to leaves of absence and school discontinuation in Japanese college students over a 27-year period. Trends in these academic events over time were assessed, and students at elevated risk and psychosocial difficulties in need of supportive intervention were identified. METHODS: Surveys were collected from the majority of Japanese national universities between 1985 and 2012, yielding data on a total of 9.7 million Japanese university students. Each year, data collected included the number of students enrolled at a university and the number of students who discontinued school and took leaves of absence. The reasons for these academic events were also collected in the surveys. RESULTS: We found that instances of these academic events have become prevalent over the past decades among Japanese university students. The rates of leaves of absence and school discontinuation for men were consistently higher than that for women throughout the study. Negative reasons such as apathetic state were the dominant reason for these academic events. Males, especially in 4-year programs (liberal arts and sciences), were more likely to have negative events due to negative reasons such as apathetic state. These students were not diagnosed psychiatrically. CONCLUSION: The population of students at elevated risk should receive psychosocial interventions and be provided mental health support.

Loss of Pin1 Suppresses Hedgehog-Driven Medulloblastoma Tumorigenesis.

Medulloblastoma is the most common malignant brain tumor in children. Therapeutic approaches to medulloblastoma (combination of surgery, radiotherapy, and chemotherapy) have led to significant improvements, but these are achieved at a high cost to quality of life. Alternative therapeutic approaches are needed. Genetic mutations leading to the activation of the Hedgehog pathway drive tumorigenesis in ~30% of medulloblastoma. In a yeast two-hybrid proteomic screen, we discovered a novel interaction between GLI1, a key transcription factor for the mediation of Hedgehog signals, and PIN1, a peptidylprolyl cis/trans isomerase that regulates the postphosphorylation fate of its targets. The GLI1/PIN1 interaction was validated by reciprocal pulldowns using epitope-tagged proteins in HEK293T cells as well as by co-immunoprecipiations of the endogenous proteins in a medulloblastoma cell line. Our results support a molecular model in which PIN1 promotes GLI1 protein abundance, thus contributing to the positive regulation of Hedgehog signals. Most importantly, in vivo functional analyses of Pin1 in the GFAP-tTA;TRE-SmoA1 mouse model of Hedgehog-driven medulloblastoma demonstrate that the loss of Pin1 impairs tumor development and dramatically increases survival. In summary, the discovery of the GLI1/PIN1 interaction uncovers PIN1 as a novel therapeutic target in Hedgehog-driven medulloblastoma tumorigenesis.

Comparative analysis of enzyme activities and mRNA levels of peptidyl prolyl cis/trans isomerases in various organs of wild type and Pin1-/- mice.

We investigated the enzyme activity of peptidyl prolyl cis/trans isomerases (PPIases) in brain, testis, lung, liver, and mouse embryonic fibroblasts (MEF) of Pin1+/+ and Pin1-/- mice. The aim of this study is to determine if other PPIases can substitute for the loss of Pin1 activity in Pin1-/- mice and what influence Pin1 depletion has on the activities of other PPIases members. The results show that high PPIase activities of Pin1 are found in organs that have the tendency to develop Pin1 knockout phenotypes and, therefore, provide for the first time an enzymological basis for these observations. Furthermore we determined the specific activity (k(cat)/K(M)) of endogenous Pin1 and found that it is strongly reduced as compared with the recombinant protein in all investigated organs. These results suggest that posttranslational modifications may influence the PPIase activity in vivo. The activities originating from cyclophilin and FKBP are not influenced by the Pin1 knockout, but a basal enzymatic activity towards phosphorylated substrates could be found in Pin1-/- lysates. Real time PCR experiments of all PPIases in different mouse organs and MEF of Pin1+/+ and Pin1-/- mice support the finding and reveal the specific expression profiles of PPIases in mice.

Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes.

BACKGROUND: While screening for an inhibitor of the peptidyl prolyl cis/trans isomerase, Pin1, we came across a brown algae polyphenol that blocks the differentiation of fibroblasts into adipocytes. However, its effectiveness on the accumulation of fat in the body has never been studied. METHODOLOGY/PRINCIPAL FINDINGS: Oral administration of brown algae polyphenol to mice fed with a high fat diet, suppressed the increase in fat volume to a level observed in mice fed with a normal diet. We speculate that Pin1 might be required for the differentiation of stem cell to adipocytes. We established wild type (WT) and Pin1-/- (Pin1-KO) adipose-derived mesenchymal stem cell (ASC) lines and found that WT ASCs differentiate to adipocytes but Pin1-KO ASCs do not. CONCLUSION AND SIGNIFICANCE: Oral administration of brown algae polyphenol, a Pin1 inhibitor, reduced fat buildup in mice. We showed that Pin1 is required for the differentiation of stem cells into adipocytes. We propose that oral intake of brown algae polyphenol is useful for the treatment of obesity.

Role of prolyl isomerase pin1 in pathogenesis of diseases and remedy for the diseases from natural products.

The peptidyl prolyl cis/trans isomerase Pin1, the human ortholog of yeast Ess1 specifically isomerizes peptide bindings of pSer/pThr-Pro residues in various proteins, and regulates the expression levels and functions of phosphorylated proteins. Activation of Pin1 is associated with pathology of a variety of diseases, such as cancer, Alzheimer's disease, infectious diseases and so on. Therefore, regulatory compounds for Pin1 can be applied as a clinical medicine against these diseases. Many chemists have exerted themselves to synthesize the inhibitors based on the 3D structure of Pin1. We have screened for the inhibitors against Pin1 from the natural products including the functional foods. Here we review the Pin1-associated pathology and the known inhibitors identified from natural products. And we introduce the screening methods targeting Pin1 activity.

Prolyl isomerase Pin1 regulates mouse embryonic fibroblast differentiation into adipose cells.

BACKGROUND: A peptidyl prolyl cis/trans isomerase, Pin1, regulates insulin signal transduction. Pin1 reduces responses to insulin stimulation by binding CRTC2 (CREB-regulated transcriptional co-activator 2) and PPARγ (peroxisome prolifereator- activated receptor γ), but conversely enhances insulin signaling by binding IRS-1 (insulin receptor substrate-1), Akt kinase, and Smad3. Therefore, it is still unclear whether Pin1 inhibits or enhances adipose cell differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Pin1(-/-) and wild-type mice were fed with high fat diets and adipose tissue weight was measured. Compared to wild-type mice, Pin1(-/-) mice had lower adipose tissue weight, while the weight of other tissues was similar. Mouse embryo fibroblasts (MEFs), prepared from both groups of mice, were induced to differentiate into adipose cells by stimulation with insulin. However, the rate of differentiation of MEFs from Pin1(-/-) mice was less than that of MEFs from wild-type mice. The rate of insulin-induced MEF cell differentiation in Pin1(-/-) mice was restored by increasing expression of Pin1. We found that Pin1 binds to phosphoThr172- and phosphoSer271-Pro sites in CREB suppress the activity in COS-7 cells. CONCLUSION AND SIGNIFICANCE: Pin1 enhanced the uptake of triglycerides and the differentiation of MEF cells into adipose cells in response to insulin stimulation. Results of this study suggest that Pin1 down-regulation could be a potential approach in obesity-related dysfunctions, such as high blood pressure, diabetes, non-alcoholic steatohepatitis.