MicroRNA-29b regulates the expression level of human progranulin, a secreted glycoprotein implicated in frontotemporal dementia

Progranulin deficiency is thought to cause some forms of frontotemporal dementia (FTD), a major early-onset age-dependent neurodegenerative disease. How progranulin (PGRN) expression is regulated is largely unknown. We identified an evolutionarily conserved binding site for microRNA-29b (miR-29b) in the 3\u27 untranslated region (3\u27UTR) of the human PGRN (hPGRN) mRNA. miR-29b downregulates the expression of luciferase through hPGRN or mouse PGRN (mPGRN) 3\u27UTRs, and the regulation was abolished by mutations in the miR-29b binding site. To examine the direct effect of manipulating endogenous miR-29b on hPGRN expression, we established a stable NIH3T3 cell line that expresses hPGRN under the control of the cytomegalovirus promoter. Ectopic expression of miR-29b decreased hPGRN expression at the both mRNA and protein levels. Conversely, knockdown of endogenous miR-29b with locked nucleic acid increased the production and secretion of hPGRN in NIH3T3 cells. Endogenous hPGRN in HEK 293 cells was also regulated by miR-29b. These findings identify miR-29b as a novel posttranscriptional regulator of PGRN expression, raising the possibility that miR-29b or other miRNAs might be targeted therapeutically to increase hPGRN levels in some FTD patients

Signal peptide peptidase (SPP) dimer formation as assessed by fluorescence lifetime imaging microscopy (FLIM) in intact cells

BACKGROUND: Signal peptide peptidase (SPP) is an intramembrane cleaving protease identified by its cleavage of several type II membrane signal peptides. Conservation of intramembrane active site residues demonstrates that SPP, SPP family members, and presenilins (PSs) make up a family of intramembrane cleaving proteases. Because SPP appears to function without additional protein cofactors, the study of SPP may provide structural insights into the mechanism of intramembrane proteolysis by this biomedically important family of proteins. Previous studies have shown that SPP isolated from cells appears to be a homodimer, but some evidence exists that in vitro SPP may be active as a monomer. We have conducted additional experiments to determine if SPP exists as a monomer or dimer in vivo. RESULTS: Fluorescence lifetime imaging microscopy (FLIM) can be is used to determine intra- or intermolecular interactions by fluorescently labeling epitopes on one or two different molecules. If the donor and acceptor fluorophores are less than 10 nm apart, the donor fluorophore lifetime shortens proportionally to the distance between the fluorophores. In this study, we used two types of fluorescence energy transfer (FRET) pairs; cyan fluorescent protein (CFP) with yellow fluorescent protein (YFP) or Alexa 488 with Cy3 to differentially label the NH2- or COOH-termini of SPP molecules. A cell based SPP activity assay was used to show that all tagged SPP proteins are proteolytically active. Using FLIM we were able to show that the donor fluorophore lifetime of the CFP tagged SPP construct in living cells significantly decreases when either a NH2- or COOH-terminally YFP tagged SPP construct is co-transfected, indicating close proximity between two different SPP molecules. These data were then confirmed in cell lines stably co-expressing V5- and FLAG-tagged SPP constructs. CONCLUSION: Our FLIM data strongly suggest dimer formation between two separate SPP proteins. Although the tagged SPP constructs are expressed throughout the cell, SPP dimer detection by FLIM is seen predominantly at or near the plasma membrane

The Vein Patterning 1 (VEP1) Gene Family Laterally Spread through an Ecological Network

Lateral gene transfer (LGT) is a major evolutionary mechanism in prokaryotes. Knowledge about LGT— particularly, multicellular— eukaryotes has only recently started to accumulate. A widespread assumption sees the gene as the unit of LGT, largely because little is yet known about how LGT chances are affected by structural/functional features at the subgenic level. Here we trace the evolutionary trajectory of VEin Patterning 1, a novel gene family known to be essential for plant development and defense. At the subgenic level VEP1 encodes a dinucleotide-binding Rossmann-fold domain, in common with members of the short-chain dehydrogenase/reductase (SDR) protein family. We found: i) VEP1 likely originated in an aerobic, mesophilic and chemoorganotrophic α-proteobacterium, and was laterally propagated through nets of ecological interactions, including multiple LGTs between phylogenetically distant green plant/fungi-associated bacteria, and five independent LGTs to eukaryotes. Of these latest five transfers, three are ancient LGTs, implicating an ancestral fungus, the last common ancestor of land plants and an ancestral trebouxiophyte green alga, and two are recent LGTs to modern embryophytes. ii) VEP1's rampant LGT behavior was enabled by the robustness and broad utility of the dinucleotide-binding Rossmann-fold, which provided a platform for the evolution of two unprecedented departures from the canonical SDR catalytic triad. iii) The fate of VEP1 in eukaryotes has been different in different lineages, being ubiquitous and highly conserved in land plants, whereas fungi underwent multiple losses. And iv) VEP1-harboring bacteria include non-phytopathogenic and phytopathogenic symbionts which are non-randomly distributed with respect to the type of harbored VEP1 gene. Our findings suggest that VEP1 may have been instrumental for the evolutionary transition of green plants to land, and point to a LGT-mediated ‘Trojan Horse’ mechanism for the evolution of bacterial pathogenesis against plants. VEP1 may serve as tool for revealing microbial interactions in plant/fungi-associated environments

Early retinal neurodegeneration and impaired Ran-mediated nuclear import of TDP-43 in progranulin-deficient FTLD

Frontotemporal dementia (FTD) is the most common cause of dementia in people under 60 yr of age and is pathologically associated with mislocalization of TAR DNA/RNA binding protein 43 (TDP-43) in approximately half of cases (FLTD-TDP). Mutations in the gene encoding progranulin (GRN), which lead to reduced progranulin levels, are a significant cause of familial FTLD-TDP. Grn-KO mice were developed as an FTLD model, but lack cortical TDP-43 mislocalization and neurodegeneration. Here, we report retinal thinning as an early disease phenotype in humans with GRN mutations that precedes dementia onset and an age-dependent retinal neurodegenerative phenotype in Grn-KO mice. Retinal neuron loss in Grn-KO mice is preceded by nuclear depletion of TDP-43 and accompanied by reduced expression of the small GTPase Ran, which is a master regulator of nuclear import required for nuclear localization of TDP-43. In addition, TDP-43 regulates Ran expression, likely via binding to its 3′-UTR. Augmented expression of Ran in progranulin-deficient neurons restores nuclear TDP-43 levels and improves their survival. Our findings establish retinal neurodegeneration as a new phenotype in progranulin-deficient FTLD, and suggest a pathological loop involving reciprocal loss of Ran and nuclear TDP-43 as an underlying mechanism

Identification of a miR-29b target site in the 3′UTR of hPGRN mRNA.

<p>(<b>A</b>) Schematic representation of hPGRN mRNA (NM_002087.2 showing with the predicted miR-29b binding site in the 3′UTR. (<b>B</b>) The actual nucleotide sequences in hPGRN mRNA and miR-29b show partial match. (<b>C</b>) Sequence alignment indicates that miR-29b is 100% conserved in vertebrates. (<b>D</b>) The predicted miR-29b binding site in hPGRN 3′UTR is highly conserved in mammals. The miR-29b seed sequences and their predicted binding sites in the hPGRN 3′UTR are shown underlined. The conserved nucleotides are in grey.</p