Human iPSC-Derived Neuronal Model of Tau-A152T Frontotemporal Dementia Reveals Tau-Mediated Mechanisms of Neuronal Vulnerability

Frontotemporal dementia (FTD) and other tauopathies characterized by focal brain neurodegeneration and pathological accumulation of proteins are commonly associated with tau mutations. However, the mechanism of neuronal loss is not fully understood. To identify molecular events associated with tauopathy, we studied induced pluripotent stem cell (iPSC)-derived neurons from individuals carrying the tau-A152T variant. We highlight the potential of in-depth phenotyping of human neuronal cell models for pre-clinical studies and identification of modulators of endogenous tau toxicity. Through a panel of biochemical and cellular assays, A152T neurons showed accumulation, redistribution, and decreased solubility of tau. Upregulation of tau was coupled to enhanced stress-inducible markers and cell vulnerability to proteotoxic, excitotoxic, and mitochondrial stressors, which was rescued upon CRISPR/Cas9-mediated targeting of tau or by pharmacological activation of autophagy. Our findings unmask tau-mediated perturbations of specific pathways associated with neuronal vulnerability, revealing potential early disease biomarkers and therapeutic targets for FTD and other tauopathies

FXR1P but not FMRP regulates the levels of mammalian brain-specific microRNA-9 and microRNA-124

Mammalian brain-specific miR-9 and miR-124 have been implicated in several aspects of neuronal development and function. However, it is not known how their expression levels are regulated in vivo. We found that the levels of miR-9 and miR-124 are regulated by FXR1P but not by the loss of FXR2P or FMRP in vivo, a mouse model of fragile X syndrome. Surprisingly, the levels of miR-9 and miR-124 are elevated in fmr1/fxr2 double-knock-out mice, in part reflecting posttranscriptional upregulation of FXR1P. Indeed, FXR1P is required for efficient processing of pre-miR-9 and pre-miR-124 in vitro and forms a complex with Dicer and pre-miRNAs. These findings reveal differential roles of FMRP family proteins in controlling the expression levels of brain-specific miRNAs

Beta-Catenin Phosphorylated at Threonine 120 Antagonizes Generation of Active Beta-Catenin by Spatial Localization in trans-Golgi Network

The stability and subcellular localization of beta-catenin, a protein that plays a major role in cell adhesion and proliferation, is tightly regulated by multiple signaling pathways. While aberrant activation of beta-catenin signaling has been implicated in cancers, the biochemical identity of transcriptionally active beta-catenin (ABC), commonly known as unphosphorylated serine 37 (S37) and threonine 41 (T41) β-catenin, remains elusive. Our current study demonstrates that ABC transcriptional activity is influenced by phosphorylation of T120 by Protein Kinase D1 (PKD1). Whereas the nuclear β-catenin from PKD1-low prostate cancer cell line C4-2 is unphosphorylated S37/T41/T120 with high transcription activity, the nuclear β-catenin from PKD1-overexpressing C4-2 cells is highly phosphorylated at T120, S37 and T41 with low transcription activity, implying that accumulation of nuclear β-catenin alone cannot be simply used as a read-out for Wnt activation. In human normal prostate tissue, the phosphorylated T120 β-catenin is mainly localized to the trans-Golgi network (TGN, 22/30, 73%), and this pattern is significantly altered in prostate cancer (14/197, 7.1%), which is consistent with known down regulation of PKD1 in prostate cancer. These in vitro and in vivo data unveil a previously unrecognized post-translational modification of ABC through T120 phosphorylation by PKD1, which alters subcellular localization and transcriptional activity of β-catenin. Our results support the view that β-catenin signaling activity is regulated by spatial compartmentation and post-translational modifications and protein level of β-catenin alone is insufficient to count signaling activity

Socializing One Health: an innovative strategy to investigate social and behavioral risks of emerging viral threats

In an effort to strengthen global capacity to prevent, detect, and control infectious diseases in animals and people, the United States Agency for International Development’s (USAID) Emerging Pandemic Threats (EPT) PREDICT project funded development of regional, national, and local One Health capacities for early disease detection, rapid response, disease control, and risk reduction. From the outset, the EPT approach was inclusive of social science research methods designed to understand the contexts and behaviors of communities living and working at human-animal-environment interfaces considered high-risk for virus emergence. Using qualitative and quantitative approaches, PREDICT behavioral research aimed to identify and assess a range of socio-cultural behaviors that could be influential in zoonotic disease emergence, amplification, and transmission. This broad approach to behavioral risk characterization enabled us to identify and characterize human activities that could be linked to the transmission dynamics of new and emerging viruses. This paper provides a discussion of implementation of a social science approach within a zoonotic surveillance framework. We conducted in-depth ethnographic interviews and focus groups to better understand the individual- and community-level knowledge, attitudes, and practices that potentially put participants at risk for zoonotic disease transmission from the animals they live and work with, across 6 interface domains. When we asked highly-exposed individuals (ie. bushmeat hunters, wildlife or guano farmers) about the risk they perceived in their occupational activities, most did not perceive it to be risky, whether because it was normalized by years (or generations) of doing such an activity, or due to lack of information about potential risks. Integrating the social sciences allows investigations of the specific human activities that are hypothesized to drive disease emergence, amplification, and transmission, in order to better substantiate behavioral disease drivers, along with the social dimensions of infection and transmission dynamics. Understanding these dynamics is critical to achieving health security--the protection from threats to health-- which requires investments in both collective and individual health security. Involving behavioral sciences into zoonotic disease surveillance allowed us to push toward fuller community integration and engagement and toward dialogue and implementation of recommendations for disease prevention and improved health security

pT120 β-catenin blocks generation of nuclear ABC and accumulates in trans Golgi network.

<p>(A) C4-2 and C4-2/PKD1 cells were fractionated into fractions enriched for cytoplasmic, nuclear or membrane proteins and analyzed by Western blot with antibodies as noted. E-cadherin, lamin A/C and b-actin were used as plasma membrane, nuclear and cytosol markers, respectively. The * indicates a leftover band from previous blotting. The densitometric data of each β-catenin isoform in a cell was the average of three assays and was expressed as percentage. (B) Overexpression of PKD1 prevents Wnt-induced β-catenin accumulation. C4-2 and C4-2/PKD1 cells were cultured with recombinant Wnt3a for indicated times. The relative amount of β-catenin at each time point was expressed as a ratio to the amount of β-catenin at starting time 0 (data was from a single assay). (C) Immunofluorescence staining of C4-2/PKD1 cells with pT120 and TGN p230 antibodies The TGN and pT120 colocalization are indicated by white arrows.</p

Analysis of pT120 antibody staining in prostate cancer.

<p>Analysis of pT120 antibody staining in prostate cancer.</p

Characterization of pT120 antibody.

<p>(A) NIH 3T3 lysates that were transfected with either HA-tagged wild type, or T102I/T112R/T120I triple mutant, or T120I mutant β-catenin and blotted by pT120 antibody. (B) Cell lysates from C4-2 (lanes 1 and 3) and C4-2/PKD1 (lanes 2 and 4 to 7) cells were blotted with either β-catenin conventional antibody H102 (lanes 1 and 2) or pT120 antibody (lanes 3–7). Competition assay was performed in C4-2/PKD1 cell lysate in the presence of 20 nM of antigenic phospho-peptide (lane 6) or the nonphospho-peptide (lane 7). (C) Inhibition of PKD1 activity decreases T120 phosphorylation. LNCaP cells were transiently transfected with two shRNA targeting PKD1 or a control shRNA vector. Cell lysates were blotted with antibodies indicated. The relative protein levels quantified by densitometry are expressed as fraction of the control group on the average of two assays.</p

pT120 β-catenin is significantly decreased in TGN of prostate cancer samples.

<p>Immunohistological staining was performed with H102 (panel A) and pT120 (panel B) antibodies on serial section of tissues. 3 and 4 are higher magnification of 1 and 2, respectively. 1 and 3 are normal prostate tissue. 5 and 6 show that pT120 β-catenin predominantly distributes to plasma membrane and nucleus in tumor samples. Bars in panels 1 and 2 equal to 50 micrometer. (C) Scatter plot for pT120 staining pattern in normal and prostate cancer samples. Normal and tumor samples were counted for the “particle” staining pattern as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033830#pone-0033830-g004" target="_blank">Fig. 4B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033830#pone-0033830-g005" target="_blank">5B3</a>. The number on Y-axis represents the average of two independent counts. A cutoff at 30 (dashline) is used to as arbitrary definition of positive and negative staining.</p

Comparison of H102 and pT120 staining patterns.

<p>Comparison of H102 and pT120 staining patterns.</p

PKD1 represses Wnt/β-catenin transcription activity.

<p>(A) PKD1 inhibits β-catenin/TCF transcription activity. Topflash assay was performed in LNCaP cells. Data were normalized by <i>Renilla</i> luciferase activity based on average of triple samples. Error bars represent standard deviation. Significant difference between the control and tested groups were determined by Student's t-test. (B) Overexpression of PKD1 in C4-2 cells results in inhibition of Wnt target genes. Representative results of semi-quantitative RT-PCR for Wnt/β-catenin target genes from two independent assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033830#s4" target="_blank">Materials and Methods</a>). (C) β-catenin in C4-2/PKD1 cells is co-localized with TGN marker p230 (white arrows). When the cells were treated with PKD1 small molecule inhibitor CID 755763 (50 nM) for 3days, the pattern is decreased (D). Anti β-catenin antibody H102 was used to stain endogenous β-catenin (red). TGN marker p230 and nuclei were labeled green. Images represent typical results from two tests.</p