GRN Expression Research Articles

Progranulin Transcripts with Short and Long 5′ Untranslated Regions (UTRs) Are Differentially Expressed via Posttranscriptional and Translational Repression

Frontotemporal lobar degeneration is associated with cytoplasmic or nuclear deposition of the TAR DNA-binding protein 43 (TDP-43). Haploinsufficiency of progranulin (GRN) is a major genetic risk factor for frontotemporal lobar degeneration associated with TDP-43 deposition. Therefore, understanding the mechanisms that control cellular expression of GRN is required not only to understand disease etiology but also for the development of potential therapeutic strategies. We identified different GRN transcripts with short (38-93 nucleotides) or long (219 nucleotides) 5' UTRs and demonstrate a cellular mechanism that represses translation of GRN mRNAs with long 5' UTRs. The long 5' UTR of GRN mRNA contains an upstream open reading frame (uORF) that is absent in all shorter transcripts. Because such UTRs can be involved in translational control as well as in mRNA stability, we compared the expression of GRN in cells expressing cDNAs with and without 5' UTRs. This revealed a selective repression of GRN translation and a reduction of mRNA levels by the 219-nucleotide-long 5' UTR. The specific ability of this GRN 5' UTR to repress protein expression was further confirmed by its transfer to an independent reporter. Deletion analysis identified a short stretch between nucleotides 76 and 125 containing two start codons within one uORF that is required and sufficient for repression of protein expression. Mutagenesis of the two AUG codons within the uORF is sufficient to reduce translational repression. Therefore initiating ribosomes at the AUGs of the uORF fail to efficiently initiate translation at the start codon of GRN. In parallel the 5' UTR also affects mRNA stability; thus two independent mechanisms determine GRN expression via mRNA stability and translational efficiency.

Progranulin gene (GRN) promoter methylation is increased in patients with sporadic frontotemporal lobar degeneration

Mutations in progranulin gene (GRN) are the most common cause of autosomal dominant familial frontotemporal lobar degeneration (FTLD). In addition, GRN variability influences the risk to develop the disease in non-carriers (sporadic FTLD). We evaluated progranulin gene (GRN) promoter methylation levels in peripheral blood mononuclear cells isolated from 38 patients with sporadic FTLD compared with 38 controls, and correlate them with GRN mRNA expression rate. The percentage of methylation of the GRN promoter was increased in patients with FTLD compared with controls (61.5 vs. 46.3 %, P < 0.001). A trend towards decreased GRN relative expression was observed in patients compared with controls (threefold decrease over controls, P > 0.05), together with a negative correlation between the degree of GRN promoter methylation and mRNA GRN levels (ρ = -0.1, P > 0.05). GRN promoter methylation was not correlated with age. In conclusion, the degree of methylation of the GRN promoter is increased in patients with FTLD as compared with controls, likely leading to a decreased expression of GRN.

Suberoylanilide Hydroxamic Acid (Vorinostat) Up-regulates Progranulin Transcription: RATIONAL THERAPEUTIC APPROACH TO FRONTOTEMPORAL DEMENTIA

Progranulin (GRN) haploinsufficiency is a frequent cause of familial frontotemporal dementia, a currently untreatable progressive neurodegenerative disease. By chemical library screening, we identified suberoylanilide hydroxamic acid (SAHA), a Food and Drug Administration-approved histone deacetylase inhibitor, as an enhancer of GRN expression. SAHA dose-dependently increased GRN mRNA and protein levels in cultured cells and restored near-normal GRN expression in haploinsufficient cells from human subjects. Although elevation of secreted progranulin levels through a post-transcriptional mechanism has recently been reported, this is, to the best of our knowledge, the first report of a small molecule enhancer of progranulin transcription. SAHA has demonstrated therapeutic potential in other neurodegenerative diseases and thus holds promise as a first generation drug for the prevention and treatment of frontotemporal dementia.

TMEM106B regulates progranulin levels and the penetrance of FTLD in GRN mutation carriers

To determine whether TMEM106B single nucleotide polymorphisms (SNPs) are associated with frontotemporal lobar degeneration (FTLD) in patients with and without mutations in progranulin (GRN) and to determine whether TMEM106B modulates GRN expression. We performed a case-control study of 3 SNPs in TMEM106B in 482 patients with clinical and 80 patients with pathologic FTLD-TAR DNA-binding protein 43 without GRN mutations, 78 patients with FTLD with GRN mutations, and 822 controls. Association analysis of TMEM106B with GRN plasma levels was performed in 1,013 controls and TMEM106B and GRN mRNA expression levels were correlated in peripheral blood samples from 33 patients with FTLD and 150 controls. In our complete FTLD patient cohort, nominal significance was identified for 2 TMEM106B SNPs (top SNP rs1990622, p(allelic) = 0.036). However, the most significant association with risk of FTLD was observed in the subgroup of GRN mutation carriers compared to controls (corrected p(allelic) = 0.0009), where there was a highly significant decrease in the frequency of homozygote carriers of the minor alleles of all TMEM106B SNPs (top SNP rs1990622, CC genotype frequency 2.6% vs 19.1%, corrected p(recessive) = 0.009). We further identified a significant association of TMEM106B SNPs with plasma GRN levels in controls (top SNP rs1990622, corrected p = 0.002) and in peripheral blood samples a highly significant correlation was observed between TMEM106B and GRN mRNA expression in patients with FTLD (r = -0.63, p = 7.7 × 10(-5)) and controls (r = -0.49, p = 2.2 × 10(-10)). In our study, TMEM106B SNPs significantly reduced the disease penetrance in patients with GRN mutations, potentially by modulating GRN levels. These findings hold promise for the development of future protective therapies for FTLD.

Dysregulation of the Mitogen Granulin in Human Cancer through the miR-15/107 microRNA Gene Group

Granulin (GRN) is a potent mitogen and growth factor implicated in many human cancers, but its regulation is poorly understood. Recent findings indicate that GRN is regulated strongly by the microRNA miR-107, which functionally overlaps with miR-15, miR-16, and miR-195 due to a common 5' sequence critical for target specificity. In this study, we queried whether miR-107 and paralogs regulated GRN in human cancers. In cultured cells, anti-argonaute RNA coimmunoprecipitation with downstream microarray analyses indicates that GRN mRNA is directly targeted by numerous miR-15/107 miRNAs. We further tested this association in human tumors. MiR-15 and miR-16 are known to be downregulated in chronic lymphocytic leukemia (CLL). Using pre-existing microarray datasets, we found that GRN expression is higher in CLL relative to nonneoplastic lymphocytes (P < 0.00001). By contrast, other prospective miR-15/miR-16 targets in the dataset (BCL-2 and cyclin D1) were not upregulated in CLL. Unlike in CLL, GRN was not upregulated in chronic myelogenous leukemia (CML) where miR-107 paralogs are not known to be dysregulated. Prior studies have shown that GRN is also upregulated, and miR-107 downregulated, in prostate carcinoma. Our results indicate that multiple members of the miR-107 gene group indeed repress GRN protein levels when transfected into prostate cancer cells. At least a dozen distinct types of cancer have the pattern of increased GRN and decreased miR-107 expression. These findings indicate for the first time that the mitogen and growth factor GRN is dysregulated via the miR-15/107 gene group in multiple human cancers, which may provide a potential common therapeutic target.

Progranulin expression in the developing and adult murine brain

Frontotemporal lobar degeneration (FTLD) is a neurodegenerative condition characterized by focal degeneration of the frontal and temporal lobes of the brain. Autosomal dominantly inherited mutations of the progranulin gene (GRN) have been identified as the cause of a subset of cases of familial FTLD. In order to better understand the function of progranulin in the central nervous system (CNS), we have assessed the spatiotemporal expression pattern of both the murine progranulin gene (Grn) and the protein (Grn) by using transgenic knock-in mice expressing a reporter gene from the Grn locus and by immunohistochemistry, respectively. We compared Grn expression with a panel of established markers for distinct neuronal developmental stages and specific cell lineages at time points ranging from embryonic day 13.5 through to the mature adult. We find that Grn is expressed in both neurons and microglia within the CNS, but that it shows a different developmental expression pattern in each cell type. Grn expression in neurons increases as the cells mature, whereas expression in microglia varies with the cells' state of activation, being specifically upregulated in microglia in response to excitotoxic injury. Our results suggest that progranulin plays distinct roles in neurons and microglia, both of which likely contribute to overall neuronal health and function.

MiR-107 Regulates Granulin/Progranulin with Implications for Traumatic Brain Injury and Neurodegenerative Disease

Granulin (GRN, or progranulin) is a protein involved in wound repair, inflammation, and neoplasia. GRN has also been directly implicated in frontotemporal dementia and may contribute to Alzheimer's disease pathogenesis. However, GRN regulation expression is poorly understood. A high-throughput experimental microRNA assay showed that GRN is the strongest target for miR-107 in human H4 neuroglioma cells. miR-107 has been implicated in Alzheimer's disease pathogenesis, and sequence elements in the open reading frame-rather than the 3' untranslated region-of GRN mRNA are recognized by miR-107 and are highly conserved among vertebrate species. To better understand the mechanism of this interaction, FLAG-tagged Argonaute constructs were used following miR-107 transfection. GRN mRNA interacts preferentially with Argonaute 2. In vitro and in vivo studies indicate that regulation of GRN by miR-107 may be functionally important. Glucose supplementation in cultured cells that leads to increased miR-107 levels also results in decreased GRN expression, including changes in cell compartmentation and decreased secretion of GRN protein. This effect was eliminated following miR-107 transfection. We also tested a mouse model where miR-107 has been shown to be down-regulated. In brain tissue subjacent to 1.0 mm depth controlled cortical impact, surviving hippocampal neurons show decreased miR-107 with augmentation of neuronal GRN expression. These findings indicate that miR-107 contributes to GRN expression regulation with implications for brain disorders.

P354 A NOVEL HDL-ASSOCIATED PROTEIN, PROGRANULIN, IS INVOLVED IN LIPID METABOLISM AND MACROPHAGE RECRUITMENT INTO ADIPOSE TISSUES

Frontotemporal dementia (FTD) arises from neurodegeneration in the frontal, insular, and anterior temporal lobes. Autosomal dominant causes of FTD include heterozygous mutations in the GRN gene causing haploinsufficiency of progranulin (PGRN) protein. Recently, histone deacetylase (HDAC) inhibitors have been identified as enhancers of PGRN expression, although the mechanisms through which GRN is epigenetically regulated remain poorly understood. Using a chemogenomic toolkit, including optoepigenetic probes, we show that inhibition of class I HDACs is sufficient to upregulate PGRN in human neurons, and only inhibitors with apparent fast binding to their target HDAC complexes are capable of enhancing PGRN expression. Moreover, we identify regions in the GRN promoter in which elevated H3K27 acetylation and transcription factor EB (TFEB) occupancy correlate with HDAC-inhibitor-mediated upregulation of PGRN. These findings have implications for epigenetic and cis-regulatory mechanisms controlling human GRN expression and may advance translational efforts to develop targeted therapeutics for treating PGRN-deficient FTD.

The enigmatic roles of microglial versus neuronal progranulin in neurological disease

Progranulin is an ancient, highly conserved gene that regu-lates cell division, survival, and migration across a widevariety of species. Until the publication of two papersshowing that mutations in a single copy of the human GRNgene was the cause of frontotemporal lobar dementia withubiquitinated TDP-43 inclusions(FTLD-TDP)[2, 5] in 2006,very little was known about the importance of progranulinwithin the central nervous system. These reports created astrong impetus to identify new mutations and to explore thecontributionofthisproteininbrainhealthanddisease.Within3 years of the first publications, the Alzheimer Disease andFrontotemporal Dementia Mutation Database (http://www.molgen.ua.ac.be/FTDMutations/) has grown to encompass68 distinct GRN mutations in 218 families.In contrast to many neurodegenerative disorders wheremutations result in a toxic gain-of function, most of theGRN-dependent FTLD-TDPs are thought to be caused by aglobal decrease in the levels of expressed or secretedprogranulin. The vast majority of reported mutations leadto nonsense-mediated mRNA decay, a mechanism forelimination of mutant transcripts, resulting in complete lossof mRNA expression from one allele [7]. Although avariety of different mutations have been described, themost common genetic mutations result from prematureinsertion of stop codons or frame shifts, causing destructionof mRNA through nonsense-mediated decay. The prevail-ing wealth of mutations that have been recently discovered,all ascribed with a similar mode of action, have stronglysupported the hypothesis that GRN haploinsufficiencyresults in insufficient GRN protein, leading to disease.Althoughalargenumberofmutationshavebeenidentifiedsince 2006, there have been a paucity of studies character-izing GRN expression in FTLD-TDP patients, and themechanisms describing the pathogenic loss of progranulinhave been developed primarily from studies of patient-derived lymphoblastoid cell lines. While these mechanismsclearlyshowtheeffectofthegeneticmutationsonproductionof the protein, there is a widespread variation in the ageof disease onset; some carriers with pathological GRNmutations remain cognitively unimpaired into their 70s. Twopublications in this issue of Acta Neuropathologica, one byChen-Plotkin et al. [4] and the other by Naphade et al. [6],on the dynamic regulation of progranulin in the CNS suggestthat the prevailing hypothesis of global GRN haploinsuffi-ciency leading to a loss of protein may be too simplistic anexplanation to completely account for development ofFTLD-TDP.The paper by Chen-Plotkin et al. [4] has filled anoticeable gap in the FTLD-TDP literature by providing adetailed comparison of regional progranulin expression inthe brains of affected patients that span 11 different GRNvariants, comparing expression in frontal and temporalcortex, 2 histopathologically involved regions, and theoccipital cortex and cerebellum, 2 spared regions, againstthe brains of both control patients and FTLD-TDP patientswithout GRN mutations. Similar to previous studies, theauthors reported a notable reduction in GRN transcripts inlymphoblastoid cell lines derived from the GRN mutationcarriers. However, when comparing levels of progranulintranscript between the normal controls and FTLD-TDPpatients with GRN mutations, they made the surprisingdiscovery that progranulin transcripts were not significantlydecreased in any of the brain regions surveyed, andreported that the frontal cortex showed strongly elevatedlevels of GRN transcript compared with control. By

GRN 3′UTR+78 C&gt;T is not associated with risk for Parkinson’s disease

A single nucleotide polymorphism in the 3'-untranslated region of the progranulin gene (GRN; 3'UTR+78C>T; rs5848) was reported to alter the risk for frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). rs5848 is located within a micro-RNA binding site and affects the expression of GRN. As FTLD-U patients often present with parkinsonism, we investigated the association of GRN rs5848 and risk of Parkinson's disease in two Caucasian patient-control series (n = 1413) from the US and Poland. No association was observed between rs5848 and susceptibility to Parkinson's disease (individual series and combined analysis). This finding shows that GRN rs5848 does not affect the risk of Parkinson's disease in the US and Polish populations.

Plasma progranulin levels predict progranulin mutation status in frontotemporal dementia patients and asymptomatic family members

Mutations in the progranulin gene (GRN) are an important cause of frontotemporal lobar degeneration (FTLD) with ubiquitin and TAR DNA-binding protein 43 (TDP43)-positive pathology. The clinical presentation associated with GRN mutations is heterogeneous and may include clinical probable Alzheimer's disease. All GRN mutations identified thus far cause disease through a uniform disease mechanism, i.e. the loss of functional GRN or haploinsufficiency. To determine if expression of GRN in plasma could predict GRN mutation status and could be used as a biological marker, we optimized a GRN ELISA and studied plasma samples of a consecutive clinical FTLD series of 219 patients, 70 control individuals, 72 early-onset probable Alzheimer's disease patients and nine symptomatic and 18 asymptomatic relatives of GRN mutation families. All FTLD patients with GRN loss-of-function mutations showed significantly reduced levels of GRN in plasma to about one third of the levels observed in non-GRN carriers and control individuals (P < 0.001). No overlap in distributions of GRN levels was observed between the eight GRN loss-of-function mutation carriers (range: 53–94 ng/ml) and 191 non-GRN mutation carriers (range: 115–386 ng/ml). Similar low levels of GRN were identified in asymptomatic GRN mutation carriers. Importantly, ELISA analyses also identified one probable Alzheimer's disease patient (1.4%) carrying a loss-of-function mutation in GRN. Biochemical analyses further showed that the GRN ELISA only detects full-length GRN, no intermediate granulin fragments. This study demonstrates that using a GRN ELISA in plasma, pathogenic GRN mutations can be accurately detected in symptomatic and asymptomatic carriers. The ∼75% reduction in full-length GRN, suggests an unbalanced GRN metabolism in loss-of-function mutation carriers whereby more GRN is processed into granulins. We propose that plasma GRN levels could be used as a reliable and inexpensive tool to identify all GRN mutation carriers in early-onset dementia populations and asymptomatic at-risk individuals.

Common variation in the miR-659 binding-site of GRN is a major risk factor for TDP43-positive frontotemporal dementia

Loss-of-function mutations in progranulin (GRN) cause ubiquitin- and TAR DNA-binding protein 43 (TDP-43)-positive frontotemporal dementia (FTLD-U), a progressive neurodegenerative disease affecting ∼10% of early-onset dementia patients. Here we expand the role of GRN in FTLD-U and demonstrate that a common genetic variant (rs5848), located in the 3′-untranslated region (UTR) of GRN in a binding-site for miR-659, is a major susceptibility factor for FTLD-U. In a series of pathologically confirmed FTLD-U patients without GRN mutations, we show that carriers homozygous for the T-allele of rs5848 have a 3.2-fold increased risk to develop FTLD-U compared with homozygous C-allele carriers (95% CI: 1.50–6.73). We further demonstrate that miR-659 can regulate GRN expression in vitro, with miR-659 binding more efficiently to the high risk T-allele of rs5848 resulting in augmented translational inhibition of GRN. A significant reduction in GRN protein was observed in homozygous T-allele carriers in vivo, through biochemical and immunohistochemical methods, mimicking the effect of heterozygous loss-of-function GRN mutations. In support of these findings, the neuropathology of homozygous rs5848 T-allele carriers frequently resembled the pathological FTLD-U subtype of GRN mutation carriers. We suggest that the expression of GRN is regulated by miRNAs and that common genetic variability in a miRNA binding-site can significantly increase the risk for FTLD-U. Translational regulation by miRNAs may represent a common mechanism underlying complex neurodegenerative disorders.

Effects of Perinatal Exposure to Phthalate/Adipate Esters on Hypothalamic Gene Expression and Sexual Behavior in Rats

Our previous research has identified the granulin (grn) and p130 genes as sex steroid-regulated genes in the neonatal rat hypothalamus that might be involved in sexual differentiation of the brain. Since phthalate/adipate esters such as di-n-butyl phthalate (DBP), diisononyl phthalate (DINP), and di-2-ethylhexyl adipate (DEHA) are suspected to interfere with the endocrine system as environmental endocrine disruptors having estrogenic or antiandrogenic properties, these chemicals may affect sexual differentiation of the brain. The present study assessed the effects of perinatal exposure to DBP, DINP, and DEHA on grn and p130 mRNA expressions in the hypothalamus on postnatal day (PND) 7 and sexual behaviors after maturation in rats. Maternal rats were given a phytoestrogen-free diet containing different doses of DBP (20, 200, 2,000, and 10,000 ppm), DINP (40, 400, 4,000, and 20,000 ppm) and DEHA (480, 2,400, and 12,000 ppm) from gestational day 15 to the day of weaning (PND 21). DBP and DINP exposure during the perinatal period resulted in an increase in hypothalamic grn and p130 mRNA levels in females and males, respectively, but DEHA exposure decreased expression levels of grn in males and p130 in females, although the effects were not dose-dependent. After maturation, male rats that were exposed to several doses of DBP, DINP, and DEHA displayed decreased copulatory behavior. The lordosis quotient was decreased in females perinatally exposed to DBP, DINP, and DEHA at all the doses used. On the other hand, serum levels of LH and FSH in both sexes and the estrous cycles in females were not affected by the treatments. These results suggest that inappropriate expression of grn and/or p130 genes in the brains of male and female neonatal rats by perinatal exposure to these chemicals may exert permanent effects on the hypothalamus, thereby decreasing sexual behavior after maturation.

In vivo acrogranin uterine biological activity is mediated by ovarian steroids in mouse uterus

In rodents, ovarian steroid hormones deeply influence the uterus during the estrous cycle, receptivity period, and implantation by triggering the expression of different sets of genes important for these processes. Since acrogranin (Grn), a growth factor present in many tissues, is expressed particularly highly in the uterus and the trophectoderm of mouse preimplantation embryos, our objective was determine whether steroids regulate Grn in a stage-specific manner. Prospective experimental laboratory study, using an in-vivo mouse model with physiologic and pharmacological treatments. The levels of acrogranin mRNA and protein were examined in the uteri of immature and adult mice following hormonal physiologic (estrous cycle) and pharmacological treatments (in oophorectomized animals) using in-situ hybridization, real time RT-PCR, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunohistochemistry. Acrogranin mRNA and protein expression progressively increased in the uteri from immature (young pups) to adult sexually mature animals. A recurrent, variable pattern of Grn gene and protein expression was determined to occur during the estrous cycle with particularly high levels at diestrus. The uteri of ovariectomized adult mice displayed a dose-dependent and additive acrogranin expression pattern in response to estradiol alone, progesterone, and estradiol plus rogesterone together. The blockage of progesterone and estrogen receptors with RU-486 and ICI 182,780, respectively, in ovariectomized animals followed by progesterone and estrogen stimulation resulted in a decreased Grn mRNA and protein expression when compared with the controls treated with a placebo. These findings indicate that Grn is a mediator of steroid action in the mouse uterus in vivo. However, low levels of Grn mRNA and protein were found in the uteri of young pups and gonadectomized animals without any hormonal stimulus whatsoever, suggesting that a small part of acrogranin gene expression is gonadal steroid hormone independent. Two important and interesting implications derived from these results are (1) the robustness of Grn expression during the diestrous stage and implantation window coupled with its crucial role during blastocyst development, indicate that acrogranin may be important for embryo receptivity and implantation and (2) the identification of acrogranin as a mediator of ovarian steroid action in uterus, along with its characteristics as a regulator of tumor growth and invasiveness, place it as a strong candidate for a gene involved in common uterine hormone-dependent pathologies like fibroids and endometrial cancer.