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

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

The Role of Progranulin in CNS Injury and Disease

Loss-of-function mutations in progranulin (GRN) that result in haploinsufficiency of the progranulin protein (PGRN) have been causally linked to frontotemporal dementia (FTD). PGRN levels are tightly regulated, as high levels cause cancer and low levels cause FTD. PGRN is a secreted glycoprotein implicated in cell survival, proliferation, and inflammation. In the central nervous system (CNS), PGRN is expressed by neurons and microglia, but its cellular functions remain unclear. We generated a murine model of PGRN-deficiency in order to begin to elucidate the CNS function of PGRN, and to model aspects of human FTD. We explored the cell type contributions of PGRN deficiency to CNS injury using the acute toxin model of 1-methyl-4-(2'methylphenyl)-1,2,3,6-tetrahydrophine (MPTP), which is known to cause both neuroinflammation and neuron death. We determined that PGRN deficiency leads to uncontrolled neuroinflammation that is detrimental to neuron survival. Therefore, microglial PGRN is critical for regulating neuroinflammation following acute brain injury. The PGRN deficient mice were aged in order to determine whether they model FTD behaviorally and/or pathologically. The homozygous PGRN deficient mice developed behavioral changes that may be linked to their age-dependent CNS inflammatory phenotype. These data suggest the importance of microglial PGRN in normal aging. Interestingly, the heterozygous and homozygous PGRN deficient mice have similar early behavioral changes, but the heterozygous mice lack any gross neuroinflammatory pathology. These early behavioral phenotypes in PGRN-deficient animals are linked to alterations in neuronal activity, suggesting that PGRN is important for neuronal function. It is likely that PGRN deficiency causes early neuron dysfunction that triggers gliosis. The gliosis then proceeds uncontrolled due to the lack of microglial PGRN that exacerbates the neuronal phenotype, thereby causing a vicious positive feedback loop that is detrimental to neuronal function and survival. Our data suggests that PGRN is important for both neuronal and microglial function in normal aging. Further characterization of these aging phenotypes in cell-type specific models of PGRN deficiency are critical for exploring this hypothesis. Overall, the development of therapeutic strategies to regulate PGRN levels in both neurons and microglia has implications for both neurodegeneration and CNS injury

MiR-29b decreases the levels of intracellular and secreted hPGRN.

<p>(<b>A</b>) The relative levels of mature miR-29b in hPGRN-3T3 cells were increased by transient expression of the miR-29b precursor or miR-29b mimic. (<b>B</b>) Western blot analysis showing that the lower intracellular levels of hPGRN in hPGRN-3T3 cells after ectopic expression of pre-miR-29b-1. Actin was used as the loading control. (<b>C</b>) Quantification of relative intracellular hPGRN levels with or without the overexpression of pre-miR-29b-1. n = 4. WB: western blot. (<b>D</b>) The relative intracellular levels of hPGRN with or without overexpression of pre-miR-29b-1 were also measured by ELISA.</p

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

Figure 5

<p>(<b>A</b>, <b>B</b>) hPGRN-3T3 cells were transfected with miR-29b or negative control mimics, and the relative hPGRN levels in total cell lysates (<b>A</b>) and medium (<b>B</b>) were determined by ELISA. In all cases, the relative level of the control was set as 1.0. Values are mean ± SEM. *<i>P</i><0.05. **<i>P</i><0.01. ***<i>P</i><0.001. (<b>C</b>) Quantitative RT-PCR analysis revealed that the level of hPGRN mRNA was decreased by miR-29b mimics. (<b>D, E</b>) The relative hPGRN levels in total cell lysates (<b>D</b>) and medium (<b>E</b>) of HEK293 cells as determined by ELISA after transfection with miR-29b or control mimics. Value are mean ± SEM. ** <i>P</i><0.01, *** <i>P</i><0.001. n = 12.</p

MiR-29b suppresses the expression of the luciferase reporter with hPGRN 3′UTR.

<p>(<b>A</b>) Schematic representation of the constructs used in the luciferase reporter assay. hPGRN 3′UTR was cloned into the luciferase vector containing the SV40 promoter, which was cotransfected with the vector encoding pre-miR-29b-1 and H1 promoter. (<b>B</b>) Both pre-miR-29b-1 and pre-miR-29b-2, two genes located on different chromosomes that produce the identical mature miR-29b, had similar effects on luciferase expression. (<b>C</b>) Coexpression of miR-29b but not miR-9 suppressed the expression of luciferase with hPGRN 3′UTR. miR-29b also suppressed the expression of luciferase with mPGRN 3′UTR. (<b>D</b>) Luciferase activity was also reduced by a miR-29b mimic but not by a miR-9 mimic. Cel-miR-67 (Dharmacon), which doesn't exist in mammals, was used as the control. Values are mean ± SEM. **<i>P</i><0.01. ***<i>P</i><0.001.</p