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.     

Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers II: Sigma-2/PGRMC1 Receptors Mediate Abeta 42 Oligomer Binding and Synaptotoxicity

Amyloid beta (Abeta) 1–42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer''s disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer''s disease patients'' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.     

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.     

Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation and ameliorates learning and memory in Alzheimer's disease

Cyclophilin D (CypD, encoded by Ppif) is an integral part of the mitochondrial permeability transition pore, whose opening leads to cell death. Here we show that interaction of CypD with mitochondrial amyloid-beta protein (Abeta) potentiates mitochondrial, neuronal and synaptic stress. The CypD-deficient cortical mitochondria are resistant to Abeta- and Ca(2+)-induced mitochondrial swelling and permeability transition. Additionally, they have an increased calcium buffering capacity and generate fewer mitochondrial reactive oxygen species. Furthermore, the absence of CypD protects neurons from Abeta- and oxidative stress-induced cell death. Notably, CypD deficiency substantially improves learning and memory and synaptic function in an Alzheimer's disease mouse model and alleviates Abeta-mediated reduction of long-term potentiation. Thus, the CypD-mediated mitochondrial permeability transition pore is directly linked to the cellular and synaptic perturbations observed in the pathogenesis of Alzheimer's disease. Blockade of CypD may be a therapeutic strategy in Alzheimer's disease.     

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 dual phosphatase activity of synaptojanin1 is required for both efficient synaptic vesicle endocytosis and reavailability at nerve terminals

Phosphoinositides have been implicated in synaptic vesicle recycling largely based on studies of enzymes that regulate phosphoinositide synthesis and hydrolysis. One such enzyme is synaptojanin1, a multifunctional protein conserved from yeast to humans, which contains two phosphoinositol phosphatase domains and a proline-rich domain. Genetic ablation of synaptojanin1 leads to pleiotropic defects in presynaptic function, including accumulation of free clathrin-coated vesicles and delayed vesicle reavailability, implicating this enzyme in postendocytic uncoating of vesicles. To further elucidate the role of synaptojanin1 at nerve terminals, we performed quantitative synaptic vesicle recycling assays in synj1(-/-) neurons. Our studies show that synaptojanin1 is also required for normal vesicle endocytosis. Defects in both endocytosis and postendocytic vesicle reavailability can be fully restored upon reintroduction of synaptojanin1. However, expression of synaptojanin1 with mutations abolishing catalytic activity of each phosphatase domain reveals that the dual action of both domains is required for normal synaptic vesicle internalization and reavailability.     

A Time Course Analysis of the Electrophysiological Properties of Neurons Differentiated from Human Induced Pluripotent Stem Cells (iPSCs)

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca²⁺ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.     

Differential Dynamic Microscopy: A High-Throughput Method for Characterizing the Motility of Microorganisms

We present a fast, high-throughput method for characterizing the motility of microorganisms in three dimensions based on standard imaging microscopy. Instead of tracking individual cells, we analyze the spatiotemporal fluctuations of the intensity in the sample from time-lapse images and obtain the intermediate scattering function of the system. We demonstrate our method on two different types of microorganisms: the bacterium Escherichia coli (both smooth swimming and wild type) and the biflagellate alga Chlamydomonas reinhardtii. We validate the methodology using computer simulations and particle tracking. From the intermediate scattering function, we are able to extract the swimming speed distribution, fraction of motile cells, and diffusivity for E. coli, and the swimming speed distribution, and amplitude and frequency of the oscillatory dynamics for C. reinhardtii. In both cases, the motility parameters were averaged over ∼ 10⁴ cells and obtained in a few minutes.     

Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission

Endophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.