Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction

Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction.     

Sagittal Diameter Minus Subcutaneous Thickness. An Easy-to-Obtain Parameter That Improves Visceral Fat Prediction

ARMELLINI FABIO, MAURO ZAMBONI, TAMARA HARRIS, ROCCO MICCIOLO, OTTAVIO BOSELLO. Sagittal diameter minus subcutaneous thickness. An easy-to-obtain parameter that improves visceral fat prediction. Two groups of 99 and 98 women were studied to test if correcting sagittal diameter by subtracting the thickness of subcutaneous abdominal adipose tissue improves its degree of association with visceral adipose tissue. The first group (age, 40 ± 14 years; body mass index [BMI], 36 ± 6 kg/m²) was used to calculate the predictive equations for visceral adipose tissue. The second group (age, 43 ± 14 years; BMI, 37 ± 6 kg/m²) was used for cross-validation. Various anthropometric parameters were measured by ultrasound and computed tomography. Correlation coefficients with single-slice visceral adipose tissue area, after sagittal diameter was corrected by subtracting subcutaneous thickness, rose from 0.63 to 0.72 in the first group and from 0.64 to 0.71 in the second group. The standard error of residuals of the regression formula for visceral adipose tissue area was 10% lower with modified sagittal diameter than with sagittal diameter alone. During cross-validation, the standard error of differences was 5% lower with modified sagittal diameter. The visceral adipose tissue estimate was also less biased by the size of the area when sagittal diameter minus subcutaneous thickness was used. Results show that subtracting the thickness of abdominal subcutaneous adipose tissue from sagittal diameter significantly improves the predictive power of sagittal diameter for visceral adipose tissue and could be a useful tool for epidemiological studies.     

Structure of the human gene for the proliferating cell nuclear antigen

The proliferating cell nuclear antigen (PCNA, cyclin) was originally defined as a nuclear protein whose appearance correlated with the proliferative state of the cell. It is now known to be a co-factor of DNA polymerase delta and to be necessary for DNA synthesis and cell cycle progression. cDNA clones of human PCNA have been isolated and, using one of these cDNA, we have now obtained from a lambda phage library a clone containing the entire human PCNA gene and flanking sequences. The human PCNA gene is a unique copy gene and has 6 exons. It spans, from the cap site to the poly(A) signal 4961 base pairs. We have identified, in the 5'-flanking sequence, a region with promoter activity, a well as other structural elements common to other promoters. An interesting feature of the PCNA gene is the presence of extensive sequence similarities among introns and between introns and exons.     

The promoter of the proliferating cell nuclear antigen (PCNA) gene is active in serum-derived cells.

mRNA levels for the Proliferating Cell Nuclear Antigen (PCNA) gene are growth regulated. PCNA promoters of different lengths were used to drive a linked reporter, the cDNA for human thymidine kinase (TK). After transfection into TK ts13 cells, stable cell lines were obtained. Regardless of promoter length, in all cell lines the levels of TK mRNA were roughly similar in serum-deprived and serum stimulated cells, confirming, by an independent method, that the growth regulation of PCNA mRNA levels doe not depend on the 5' flanking sequence of the PCNA gene.     

The Timing of IFNβ Production Affects Early Innate Responses to Listeria monocytogenes and Determines the Overall Outcome of Lethal Infection

Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a crucial role in the first phase of host defense against infections and tumors. Listeria monocytogenes (Lm) is an intracellular pathogen that colonizes the cytosol of eukaryotic cells. Recent findings have shown Lm specifically in splenic CD8a(+) DCs shortly after intravenous infection. We examined gene expression profiles of mouse DCs exposed to Lm to elucidate the molecular mechanisms underlying DCs interaction with Lm. Using a functional genomics approach, we found that Lm infection induced a cluster of late response genes including type I IFNs and interferon responsive genes (IRGs) in DCs. Type I INFs were produced at the maximal level only at 24 h post infection indicating that the regulation of IFNs in the context of Lm infection is delayed compared to the rapid response observed with viral pathogens. We showed that during Lm infection, IFNγ production and cytotoxic activity were severely impaired in NK cells compared to E. coli infection. These defects were restored by providing an exogenous source of IFNβ during the initial phase of bacterial challenge. Moreover, when treated with IFNβ during early infection, NK cells were able to reduce bacterial titer in the spleen and significantly improve survival of infected mice. These findings show that the timing of IFNβ production is fundamental to the efficient control of the bacterium during the early innate phase of Lm infection.     

Estimation of the mean from sums with unknown numbers of summands

Using dye intensity measurements at synaptic terminals to examine neurotransmitter release leads to the problem of estimating an expectation when, instead of observing independent random variables with the same expectation, one observes, with error, independent random sums of independent variables with the same expectation--but with the number of summands in the random sums unobserved. Here, a relatively convenient nonparametric approach to estimation is presented. Data from an experiment in which cationic styrylpyridinium dye FM4-64 was used in cultured mouse hippocampal neurons are used to illustrate the approach.     

Predicting Pathological Features at Radical Prostatectomy in Patients with Prostate Cancer Eligible for Active Surveillance by Multiparametric Magnetic Resonance Imaging

Purpose

The aim of this study was to investigate the prognostic performance of multiparametric magnetic resonance imaging (mpMRI) and Prostate Imaging Reporting and Data System (PIRADS) score in predicting pathologic features in a cohort of patients eligible for active surveillance who underwent radical prostatectomy.

Methods

A total of 223 patients who fulfilled the criteria for “Prostate Cancer Research International: Active Surveillance”, were included. Mp–1.5 Tesla MRI examination staging with endorectal coil was performed at least 6–8 weeks after TRUS-guided biopsy. In all patients, the likelihood of the presence of cancer was assigned using PIRADS score between 1 and 5. Outcomes of interest were: Gleason score upgrading, extra capsular extension (ECE), unfavorable prognosis (occurrence of both upgrading and ECE), large tumor volume (≥0.5ml), and seminal vesicle invasion (SVI). Receiver Operating Characteristic (ROC) curves and Decision Curve Analyses (DCA) were performed for models with and without inclusion of PIRADS score.

Results

Multivariate analysis demonstrated the association of PIRADS score with upgrading (P<0.0001), ECE (P<0.0001), unfavorable prognosis (P<0.0001), and large tumor volume (P = 0.002). ROC curves and DCA showed that models including PIRADS score resulted in greater net benefit for almost all the outcomes of interest, with the only exception of SVI.

Conclusions

mpMRI and PIRADS scoring are feasible tools in clinical setting and could be used as decision-support systems for a more accurate selection of patients eligible for AS.     

The study of ontogenetic trajectory reveals the timing of reproductive events in Ancylus fluviatilis (Gastropoda: Planorbidae)

We analysed ontogenetic shape change in the planorbid limpet Ancylus fluviatilis (Müller, 1774) in two rivers in Southern Italy. We developed a new method to discriminate among different cohorts in Ancylus, based on principal component analysis. The method is useful when shape change during growth is allometric, as in our study model. We discovered that bivoltinism occurs in Ancylus in Southern Italy, contrary to previous accounts, which invariably describe A. fluviatilis as a semelparous and univoltine species, although acknowledging difficulty in discriminating among cohorts. The methods presented here may potentially help research in reproductive traits in many other mollusc populations where shape change during ontogeny is demonstrated to be allometric.     

APP heterozygosity averts memory deficit in knockin mice expressing the Danish dementia BRI2 mutant

An autosomal dominant mutation in the BRI2/ITM2B gene causes familial Danish dementia (FDD). Analysis of FDD(KI) mice, a mouse model of FDD genetically congruous to the human disease since they carry one mutant and one wild-type Bri2/Itm2b allele, has shown that the Danish mutation causes loss of Bri2 protein, synaptic plasticity and memory impairments. BRI2 is a physiological interactor of Aβ-precursor protein (APP), a gene associated with Alzheimer disease, which inhibits processing of APP. Here, we show that APP/Bri2 complexes are reduced in synaptic membranes of FDD(KI) mice. Consequently, APP metabolites derived from processing of APP by β-, α- and γ-secretases are increased in Danish dementia mice. APP haplodeficiency prevents memory and synaptic dysfunctions, consistent with a role for APP metabolites in the pathogenesis of memory and synaptic deficits. This genetic suppression provides compelling evidence that APP and BRI2 functionally interact, and that the neurological effects of the Danish form of BRI2 only occur when sufficient levels of APP are supplied by two alleles. This evidence establishes a pathogenic sameness between familial Danish and Alzheimer's dementias.