Long-term normalization of calcineurin activity in model mice rescues Pin1 and attenuates Alzheimer's phenotypes without blocking peripheral T cell IL-2 response.

BACKGROUND: Current treatments for Alzheimer's disease (AD) have largely failed to yield significant therapeutic benefits. Novel approaches are desperately needed to help address this immense public health issue. Data suggests that early intervention at the first stages of mild cognitive impairment may have a greater chance for success. The calcineurin (CN)-Pin1 signaling cascade can be selectively targeted with tacrolimus (FK506), a highly specific, FDA-approved CN inhibitor used safely for > 20 years in solid organ transplant recipients. AD prevalence was significantly reduced in solid organ recipients treated with FK506. METHODS: Time release pellets were used to deliver constant FK506 dosage to APP/PS1 mice without deleterious manipulation or handling. Immunofluorescence, histology, molecular biology, and behavior were used to evaluate changes in AD pathology. RESULTS: FK506 can be safely and consistently delivered into juvenile APP/PS1 mice via time-release pellets to levels roughly seen in transplant patients, leading to the normalization of CN activity and reduction or elimination of AD pathologies including synapse loss, neuroinflammation, and cognitive impairment. Pin1 activity and function were rescued despite the continuing presence of high levels of transgenic Aß42. Indicators of neuroinflammation including Iba1 positivity and IL-6 production were also reduced to normal levels. Peripheral blood mononuclear cells (PBMC) obtained during treatment or splenocytes isolated at euthanasia activated normally after mitogens. CONCLUSIONS: Low-dose, constant FK506 can normalize CNS CN and Pin1 activity, suppress neuroinflammation, and attenuate AD-associated pathology without blocking peripheral IL-2 responses making repurposed FK506 a viable option for early, therapeutic intervention in AD.

Pin1 and Alzheimer's disease.

Alzheimer's disease (AD) is an immense and growing public health crisis. Despite over 100 years of investigation, the etiology remains elusive and therapy ineffective. Despite current gaps in knowledge, recent studies have identified dysfunction or loss-of-function of Pin1, a unique cis-trans peptidyl prolyl isomerase, as an important step in AD pathogenesis. Here I review the functionality of Pin1 and its role in neurodegeneration.

Alterations in the RB Pathway With Inactivation of RB1 Characterize Glioblastomas With a Primitive Neuronal Component.

A primitive neuronal component is a feature of some glioblastomas but defining molecular alterations of this histologic variant remains uncertain. We performed next-generation sequencing of 1500 tumor related genes on tissue from 9 patients with glioblastoma with a primitive component (G/PN) and analyzed 27 similar cases from the Cancer Genome Atlas (TCGA) dataset. Alterations in the RB pathway were identified in all of our patients' tumors and 81% of TCGA tumors with the retinoblastoma tumor suppressor gene (RB1) commonly affected. Although RB1 mutations were observed in some conventional glioblastomas, the allelic fractions of these mutations were significantly higher in tumors with a primitive neuronal component in both our and TCGA cohorts (median, 72% vs 25%, p < 0.001 and 80% vs 40%, p < 0.02, respectively). Further, in 78% of patients in our cohort, RB expression was lost by immunohistochemistry. Our findings indicate that alterations in the RB pathway are common in G/PNs and suggest that inactivation of RB1 may be a driving mechanism for the phenotype.

Digital Droplet PCR for SARS-CoV-2 Resolves Borderline Cases.

OBJECTIVES: The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR-in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results. METHODS: We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer's specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription-polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated. RESULTS: The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate. CONCLUSIONS: The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.

Pin1 Regulates IL-5 Induced Eosinophil Polarization and Migration.

Eosinophils become polarized in response to cytokines such IL-5 or eotaxin prior to directional migration. Polarization is preceded by F-actin assembly, but the mechanisms that regulate these events and how the shape change influences cell migration from the peripheral blood into the lung remain unclear. In this study, we show that the prolyl isomerase, Pin1, is required for IL-5-induced Eos polarization and migration. Co-immunoprecipitation and immunofluorescence analysis revealed that Pin1 directly interacts with members of Rho GTPase family. Mouse eosinophils lacking Pin1 or human cells treated with Pin1 inhibitors showed significantly reduced IL-5-induced GTPase activity and cofilin phosphorylation, resulting in reduced F-actin polymerization, cell polarization, and directional migration to chemokines. Our result suggests that Pin1 regulates cytoskeletal re-organization, eosinophil morphology, and cell migration through the modulation of Rho GTPase activity. Targeting Pin1 along with GTPases could provide a new approach to reduce pulmonary Eos accumulation during asthmatic exacerbations.

Eosinophils, Pin1 and the Response to Respiratory Viral Infection and Allergic Stimuli.

Eosinophils (Eos) are prominent inflammatory cells found in the sputum, airways, and airway walls during and after exacerbations of allergic asthma. These cells are potent secretors of a wide array of cytotoxic granule proteins, cytokines, and lipid mediators involved in the initiation and maintenance of the Th2-type inflammatory reaction. Even though respiratory viral and bacterial infections are known risk factors contributing to severity and prognosis, the induction of anti-microbial Th1 type responses can offset allergic Th2 responses. Emerging evidence suggests that the prolyl isomerase Pin1 plays important roles in both Th1 and Th2 immunity in the lung and especially during allergic disease. In this review, we summarize recent findings related to the role of Pin1 in the regulation of Eos differentiation, survival, migration, cytokine expression, and antiviral immunity in asthma.

TLR-7 Stress Signaling in Differentiating and Mature Eosinophils Is Mediated by the Prolyl Isomerase Pin1.

The response of eosinophils (Eos) to respiratory virus has emerged as an important link between pulmonary infection and allergic asthmatic exacerbations. Eos activate innate immune responses through TLR signaling. In this study, using mouse and human Eos and mice lacking the prolyl isomerase Pin1 selectively in Eos, we show that Pin1 is indispensable for eosinophilopoiesis in the bone marrow (BM) and mature cell function in the presence of TLR7 activation. Unbiased in vivo analysis of mouse models of allergic airway inflammation revealed that TLR7 activation in knockout mice resulted in systemic loss of Eos, reduced IFN production, and an inability to clear respiratory viruses. Consistent with this finding, BM mouse Eos progenitors lacking Pin1 showed markedly reduced cell proliferation and survival after TLR7 activation. Mechanistically, unlike wild-type cells, Pin1 null mouse Eos were defective in the activation of the endoplasmic reticulum stress-induced unfolded protein response. We observed significant reductions in the expression of unfolded protein response components and target genes, aberrant TLR7 cleavage and trafficking, and reduced granule protein production in knockout Eos. Our data strongly suggest that Pin1 is required for BM Eos generation and function during concurrent allergen challenge and viral infection.

Alzheimer's Disease, Dendritic Spines, and Calcineurin Inhibitors: A New Approach?

Therapeutics to effectively treat Alzheimer's disease (AD) are lacking. In vitro, animal and human studies have implicated the excessive activation of the protein phosphatase calcineurin (CN) as an early step in the pathogenesis of AD. We discuss recent data showing that the prolyl isomerase Pin1 is suppressed by CN-mediated dephosphorylation induced by Aß42 signaling. Pin1 loss directly leads to the reductions in dendritic spines and synapses characteristic of early AD pathology. Pin1 activity, and synapse and dendritic spine numbers are rescued by FK506, a highly specific and United States Food and Drug Administration approved CN inhibitor. Solid organ transplant recipients chronically treated with FK506 showed much lower AD incidence than expected. As such, we suggest prospective clinical trials to determine if systemic FK506 can normalize CN activity in the brain, preserve Pin1 function and support synaptic health in early AD.

Pin1 mediates Aß42-induced dendritic spine loss.

Early-stage Alzheimer's disease is characterized by the loss of dendritic spines in the neocortex of the brain. This phenomenon precedes tau pathology, plaque formation, and neurodegeneration and likely contributes to synaptic loss, memory impairment, and behavioral changes in patients. Studies suggest that dendritic spine loss is induced by soluble, multimeric amyloid-ß (Aß42), which, through postsynaptic signaling, activates the protein phosphatase calcineurin. We investigated how calcineurin caused spine pathology and found that the cis-trans prolyl isomerase Pin1 was a critical downstream target of Aß42-calcineurin signaling. In dendritic spines, Pin1 interacted with and was dephosphorylated by calcineurin, which rapidly suppressed its isomerase activity. Knockout of Pin1 or exposure to Aß42 induced the loss of mature dendritic spines, which was prevented by exogenous Pin1. The calcineurin inhibitor FK506 blocked dendritic spine loss in Aß42-treated wild-type cells but had no effect on Pin1-null neurons. These data implicate Pin1 in dendritic spine maintenance and synaptic loss in early Alzheimer's disease.

Protein Translation and Signaling in Human Eosinophils.

We have recently reported that, unlike IL-5 and GM-CSF, IL-3 induces increased translation of a subset of mRNAs. In addition, we have demonstrated that Pin1 controls the activity of mRNA binding proteins, leading to enhanced mRNA stability, GM-CSF protein production and prolonged eosinophil (EOS) survival. In this review, discussion will include an overview of cap-dependent protein translation and its regulation by intracellular signaling pathways. We will address the more general process of mRNA post-transcriptional regulation, especially regarding mRNA binding proteins, which are critical effectors of protein translation. Furthermore, we will focus on (1) the roles of IL-3-driven sustained signaling on enhanced protein translation in EOS, (2) the mechanisms regulating mRNA binding proteins activity in EOS, and (3) the potential targeting of IL-3 signaling and the signaling leading to mRNA binding activity changes to identify therapeutic targets to treat EOS-associated diseases.

Peptidyl-Prolyl Isomerase 1 Regulates Ca2+ Handling by Modulating Sarco(Endo)Plasmic Reticulum Calcium ATPase and Na2+/Ca2+ Exchanger 1 Protein Levels and Function.

BACKGROUND: Aberrant Ca2+ handling is a prominent feature of heart failure. Elucidation of the molecular mechanisms responsible for aberrant Ca2+ handling is essential for the development of strategies to blunt pathological changes in calcium dynamics. The peptidyl-prolyl cis-trans isomerase peptidyl-prolyl isomerase 1 (Pin1) is a critical mediator of myocardial hypertrophy development and cardiac progenitor cell cycle. However, the influence of Pin1 on calcium cycling regulation has not been explored. On the basis of these findings, the aim of this study is to define Pin1 as a novel modulator of Ca2+ handling, with implications for improving myocardial contractility and potential for ameliorating development of heart failure. METHODS AND RESULTS: Pin1 gene deletion or pharmacological inhibition delays cytosolic Ca2+ decay in isolated cardiomyocytes. Paradoxically, reduced Pin1 activity correlates with increased sarco(endo)plasmic reticulum calcium ATPase (SERCA2a) and Na2+/Ca2+ exchanger 1 protein levels. However, SERCA2a ATPase activity and calcium reuptake were reduced in sarcoplasmic reticulum membranes isolated from Pin1-deficient hearts, suggesting that Pin1 influences SERCA2a function. SERCA2a and Na2+/Ca2+ exchanger 1 associated with Pin1, as revealed by proximity ligation assay in myocardial tissue sections, indicating that regulation of Ca2+ handling within cardiomyocytes is likely influenced through Pin1 interaction with SERCA2a and Na2+/Ca2+ exchanger 1 proteins. CONCLUSIONS: Pin1 serves as a modulator of SERCA2a and Na2+/Ca2+ exchanger 1 Ca2+ handling proteins, with loss of function resulting in impaired cardiomyocyte relaxation, setting the stage for subsequent investigations to assess Pin1 dysregulation and modulation in the progression of heart failure.

Sampling strategies to capture single-cell heterogeneity.

Advances in single-cell technologies have highlighted the prevalence and biological significance of cellular heterogeneity. A critical question researchers face is how to design experiments that faithfully capture the true range of heterogeneity from samples of cellular populations. Here we develop a data-driven approach, illustrated in the context of image data, that estimates the sampling depth required for prospective investigations of single-cell heterogeneity from an existing collection of samples.

Aggressive Behavior in Silent Subtype III Pituitary Adenomas May Depend on Suppression of Local Immune Response: A Whole Transcriptome Analysis.

Silent subtype III pituitary adenomas (SS-3) are clinically nonfunctional adenomas that are more aggressive in terms of invasion and risk of recurrence than their conventional null cell counterparts. We previously showed that these tumors can be distinguished by immunohistochemistry based on the identification of a markedly enlarged and fragmented Golgi apparatus. To understand the molecular correlates of differential aggressiveness, we performed whole transcriptome sequencing (RNAseq) on 4 SS-3 and 4 conventional null cell adenomas. The genes that were highly upregulated in all the SS-3 adenomas included 2 secreted proteins involved in the suppression of T-lymphocyte activity, i.e., ARG2 (multiple testing adjusted padj = 1.5 × 10-3) and SEMA3A (padj = 3.3 × 10-3). Highly downregulated genes in all the SS-3 adenomas included HLA-B (padj = 3.3 × 10-6), suggesting reduced antigen presentation by the adenoma to cytotoxic T-cells. Quantitative RT-PCR of these genes performed on the adenoma samples supported the RNAseq results. We also found a relative decrease in the overall concentration of T-lymphocytes in the SS-3 tumors. These results suggest that SS-3 adenomas actively suppress the immune system and raise the possibility that they may be treatable with immune checkpoint inhibitors or nonspecific cancer immunotherapies.

NOD2 Suppresses Colorectal Tumorigenesis via Downregulation of the TLR Pathways.

Although NOD2 is the major inflammatory bowel disease susceptibility gene, its role in colorectal tumorigenesis is poorly defined. Here, we show that Nod2-deficient mice are highly susceptible to experimental colorectal tumorigenesis independent of gut microbial dysbiosis. Interestingly, the expression of inflammatory genes and the activation of inflammatory pathways, including NF-κB, ERK, and STAT3 are significantly higher in Nod2-/- mouse colons during colitis and colorectal tumorigenesis, but not at homeostasis. Consistent with higher inflammation, there is greater proliferation of epithelial cells in hyperplastic regions of Nod2-/- colons. In vitro studies demonstrate that, while NOD2 activates the NF-κB and MAPK pathways in response to MDP, it inhibits TLR-mediated activation of NF-κB and MAPK. Notably, NOD2-mediated downregulation of NF-κB and MAPK is associated with the induction of IRF4. Taken together, NOD2 plays a critical role in the suppression of inflammation and tumorigenesis in the colon via downregulation of the TLR signaling pathways.

Epstein-Barr Virus-induced Gene 2 Mediates Allergen-induced Leukocyte Migration into Airways.

RATIONALE: Leukocyte recruitment to sites of allergic inflammation depends on the local production of priming cytokines, chemokines, and potentially other mediators. Previously, we showed that eosinophils (Eos) express numerous orphan G-protein-coupled receptors, including Epstein-Barr virus-induced gene 2 (EBI2). Despite its contribution to inflammatory diseases, the role of EBI2 in pulmonary eosinophilia is unknown. OBJECTIVES: To determine whether oxysterol ligands for EBI2 are increased in asthma exacerbation, and if or how they promote Eos pulmonary migration. METHODS: EBI2 ligands and pulmonary eosinophilia were measured in the bronchoalveolar lavage fluid from patients with mild asthma 48 hours after acute allergen challenge. In vitro, the ability of EBI2 ligands alone or in combination with IL-5 priming to induce the migration of human blood Eos was assessed. MEASUREMENTS AND MAIN RESULTS: EBI2 was constitutively and stably expressed in peripheral blood Eos. Eos treated with the EBI2 ligands showed significantly increased transwell migration that was enhanced by priming with physiologic doses of IL-5. Migration was suppressed by inhibitors of the prolyl isomerase Pin1 or extracellular signal-regulated kinases (ERK) 1/2 or by pertussis toxin. EBI2 signaling activated Pin1 isomerase activity through a cascade that was sensitive to ERK inhibitors and pertussis toxin. The concentration of EBI2 ligands was significantly increased in the bronchoalveolar lavage fluid 48 hours after segmental allergen challenge and strongly correlated with the increased numbers of Eos, lymphocytes, and neutrophils in the airways. CONCLUSIONS: Oxysterols are increased in inflamed airways after allergen challenge and, through G-protein subunit α, ERK, and Pin1 signaling, likely participate in the regulation of Eos migration into the lung in people with asthma.

Erratum: Publisher Correction: Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

[This corrects the article DOI: 10.1038/ncomms5802.].

Human eosinophil activin A synthesis and mRNA stabilization are induced by the combination of IL-3 plus TNF.

Eosinophils contribute to immune regulation and wound healing/fibrosis in various diseases, including asthma. Growing appreciation for the role of activin A in such processes led us to hypothesize that eosinophils are a source of this transforming growth factor-ß superfamily member. Tumor necrosis factor-α (TNF) induces activin A by other cell types and is often present at the site of allergic inflammation along with the eosinophil-activating common ß (ßc) chain-signaling cytokines (interleukin (IL)-5, IL-3, granulocyte-macrophages colony-stimulating factor (GM-CSF)). Previously, we established that the combination of TNF plus a ßc chain-signaling cytokine synergistically induces eosinophil synthesis of the remodeling enzyme matrix metalloproteinase-9. Therefore, eosinophils were stimulated ex vivo by these cytokines and in vivo through an allergen-induced airway inflammatory response. In contrast to IL-5+TNF or GM-CSF+TNF, the combination of IL-3+TNF synergistically induced activin A synthesis and release by human blood eosinophils. IL-3+TNF enhanced activin A mRNA stability, which required sustained signaling of pathways downstream of p38 and extracellular signal-regulated kinase mitogen-activated protein kinases. In vivo, following segmental airway allergen challenge of subjects with mild allergic asthma, activin A mRNA was upregulated in airway eosinophils compared with circulating eosinophils, and ex vivo, circulating eosinophils tended to release more activin A in response to IL-3+TNF. These data provide evidence that eosinophils release activin A and that this function is enhanced when eosinophils are present in an allergen-induced inflammatory environment. Moreover, these data provide the first evidence for posttranscriptional control of activin A mRNA. We propose that an environment rich in IL-3+TNF will lead to eosinophil-derived activin A, which has an important role in regulating inflammation and/or fibrosis.

Protein interacting with NIMA (never in mitosis A)-1 regulates axonal growth cone adhesion and spreading through myristoylated alanine-rich C kinase substrate isomerization.

Axonal growth cone motility requires precise regulation of adhesion to navigate the complex environment of the nervous system and reach its target. Myristoylated alanine-rich C kinase substrate (MARCKS) protein is enriched in the developing brain and plays an important, phosphorylation-dependent role in the modulation of axonal growth cone adhesion. The ratio of phospho-MARCKS (MARCKS-P) to total MARCKS controls adhesion modulation and spreading of the axonal growth cone. Pin1, a peptidyl-prolyl cis/trans isomerase (PPIase) that recognizes and binds to phosphorylated serine/threonine residues preceded by a proline (pSer/Thr-Pro) is also expressed in the developing brain. Here, we show that Pin1 is present in the growth cone, interacts with MARCKS-P, and regulates its dephosphorylation. We also described morphological alterations in the corpus callosum and cerebral cortex fibers of the Pin1 knockout mouse brain that may be caused by the misregulation of MARCKS-P and alterations of neuronal adhesion. We have shown that MARCKS, a critical protein in the movement of neuronal growth cones, is in turn regulated by both phosphorylation and cis-trans peptidyl isomerization mediated by Pin1. In the absence of Pin1, MARCKS is hyperphosphorylated, leading to loss of adhesions, and collapse of the growth cone. The Pin1 KO mice exhibited disturbed neuronal projections from the cerebral cortex and reduced white matter tracks such as the corpus callosum. This study highlights a novel function of Pin1 in neurodevelopment.

Phosphate-Induced Renal Fibrosis Requires the Prolyl Isomerase Pin1.

Tubulo-interstitial fibrosis is a common, destructive endpoint for a variety of kidney diseases. Fibrosis is well correlated with the loss of kidney function in both humans and rodents. The identification of modulators of fibrosis could provide novel therapeutic approaches to reducing disease progression or severity. Here, we show that the peptidyl-prolyl isomerase Pin1 is an important molecular contributor that facilitates renal fibrosis in a well-characterized animal model. While wild-type mice fed a high phosphate diet (HPD) for 8-12 weeks developed calcium deposition, macrophage infiltration and extracellular matrix (ECM) accumulation in the kidney interstitium, Pin1 null mice showed significantly less pathology. The serum Pi in both WT and KO mice were significantly increased by the HPD, but the serum Ca was slightly decreased in KO compared to WT. In addition, both WT and KO HPD mice had less weight gain but exhibited normal organ mass (kidney, lung, spleen, liver and heart). Unexpectedly, renal function was not initially impaired in either genotype irrespective of the HPD. Our results suggest that diet containing high Pi induces rapid renal fibrosis before a significant impact on renal function and that Pin1 plays an important role in the fibrotic process.

The DNA Sensor AIM2 Maintains Intestinal Homeostasis via Regulation of Epithelial Antimicrobial Host Defense.

Microbial pattern molecules in the intestine play immunoregulatory roles via diverse pattern recognition receptors. However, the role of the cytosolic DNA sensor AIM2 in the maintenance of intestinal homeostasis is unknown. Here, we show that Aim2(-/-) mice are highly susceptible to dextran sodium sulfate-induced colitis that is associated with microbial dysbiosis as represented by higher colonic burden of commensal Escherichia coli. Colonization of germ-free mice with Aim2(-/-) mouse microbiota leads to higher colitis susceptibility. In-depth investigation of AIM2-mediated host defense responses reveals that caspase-1 activation and IL-1ß and IL-18 production are compromised in Aim2(-/-) mouse colons, consistent with defective inflammasome function. Moreover, IL-18 infusion reduces E. coli burden as well as colitis susceptibility in Aim2(-/-) mice. Altered microbiota in inflammasome-defective mice correlate with reduced expression of several antimicrobial peptides in intestinal epithelial cells. Together, these findings implicate DNA sensing by AIM2 as a regulatory mechanism for maintaining intestinal homeostasis.