Progranulin Deficiency Research Articles

Portuguese family with the co-occurrence of frontotemporal lobar degeneration and neuronal ceroid lipofuscinosis phenotypes due to progranulin gene mutation

We and others have reported heterozygous progranulin mutations as an important cause of frontotemporal lobar degeneration (FTLD). It has been identified a complete progranulin deficiency because of a homozygous mutation in a sibling pair with neuronal ceroid lipofuscinosis (NCL). Here, we describe the first case of NCL caused by a homozygous progranulin mutation segregating in a family with neuropathological confirmed FTLD. In this FTLD-NCL family, we detail the clinical phenotype, neuropsychological evaluation and imaging data of our proband harboring a homozygous mutation, c.900_901dupGT, with serum progranulin level (<6 ng/mL). Symptoms included rapidly progressive visual deficit, slightly dysarthria, and cerebellar ataxia. The electroretinogram confirmed a severe attenuation of rod and cone responses compatible with retinal dystrophy diagnosis and magnetic resonance imaging showed severe global cerebellar atrophy. In contrast, heterozygous relatives presented behavioral variant of frontotemporal dementia (FTD) and some also developed extrapyramidal features compatible with corticobasal syndrome. Our findings suggest the importance of assessing serum progranulin levels in suspected recessive adult-onset NCL cases. Overall, a more holistic neurologic intervention is needed to guarantee a proper genetic counseling in cases like the present family where two distinct phenotypes are generated according to the individuals' mutation state.

Commentary: Possible involvement of lysosomal dysfunction in pathological changes of the brain in aged progranulin-deficient mice.

Commentary: Possible involvement of lysosomal dysfunction in pathological changes of the brain in aged progranulin-deficient mice.

Progranulin deficiency leads to severe inflammation, lung injury and cell death in a mouse model of endotoxic shock.

Progranulin (PGRN) is a crucial secreted growth factor involved in various kinds of physiologic and disease processes and often has a protective role in inflammatory diseases. This study was designed to investigate the protective effects of PGRN on endotoxic shock in a mouse model of PGRN deficiency. After lipopolysaccharide (LPS) injection to induce endotoxic shock in mice, PGRN levels were induced in wild-type (WT) mice at 6 and 24 hrs. Survival rate analysis, haematoxylin and eosin staining, immunohistochemical staining, enzyme-linked immunosorbent assay and in situ terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick-end labelling assay were used to reveal the susceptibility, lung injury, inflammatory cell infiltration, production of inflammatory mediators and lung cell death in mice after LPS injection. PGRN-deficient (Grn(-/-) ) mice were highly susceptible to LPS-induced endotoxic shock, with decreased survival, severe lung injury, increased production of pro-inflammatory mediators, and inflammatory cell infiltration and apoptotic death in the lung. Additionally, recombinant PGRN (rPGRN) administration before LPS stimulation ameliorated the survival of and abnormalities in both WT and Grn(-/-) mice. Altogether, these findings indicate that PGRN may be a novel biologic agent with therapeutic potential for endotoxic shock probably by inhibiting LPS-induced systemic and local inflammation in mice for treating endotoxic shock.

Increased Wnt Signaling and Reduced Viability in a Neuronal Model of Progranulin-Deficient Frontotemporal Lobar Degeneration.

Progranulin (PGRN) deficiency is considered the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Recent work unveiled a relationship between Wnt signaling and PGRN in cellular models of FTLD and cells of patients carrying loss-of-function GRN mutations. This study was undertaken to explore the relationship between PGRN deficit and Wnt signaling in the regulation of survival of GRN knockdown neuroblastoma SH-SY5Y cells (GRN KD). We report here that both canonical and noncanonical Wnt signaling cascades are overactivated in GRN KD cells. We detected increased expression levels of Wnt1 and Wnt5a ligands of the Frizzled receptors, as well as evidence for increased signaling of the Wnt/β-catenin and Wnt/Ca2+ cascades in PGRN deficient cells, such as increased nuclear content of β-catenin and higher levels of cyclin D1, or increased levels of the active form of the NFAT1 transcription factor, respectively. Upregulation of either Wnt/β-catenin or Wnt/Ca2+ signaling in GRN KD cells leads to the stimulation of BrdU incorporation into DNA, hyperphosphorylation of the pRb family of proteins and reduced cell viability over time. Blocking the Wnt cascades by specific canonical or noncanonical inhibitors of Wnt-dependent signaling, normalized the rate of DNA synthesis, and what it is more important restored the viability of GRN KD cells. Our results suggest an important role of Wnt activation inducing cell cycle disturbance-mediated neuronal loss in the pathogenesis of PGRN deficiency-linked FTLD-TDP. Therefore, it is plausible that modulation of Wnt signaling could be a promising strategy for developing of new disease-modifying treatments for FTLD-TDP.

Core neuropathological abnormalities in progranulin-deficient mice are penetrant on multiple genetic backgrounds

Loss-of-function mutations in the progranulin gene (GRN) are a common cause of familial frontotemporal lobar degeneration (FTLD). A high degree of heterogeneity in the age-of-onset, duration of disease, and clinical presentation of FTLD, even among families carrying the same GRN mutation, suggests that additional modifying genes may be important to pathogenesis. Progranulin-knockout mice display subtle behavioral abnormalities and progressive neuropathological changes, as well as altered dendritic morphology and synaptic deficits in the hippocampus. In this study we evaluated multiple neuropathological endpoints in aged progranulin knockout mice and their wild-type littermates on two different genetic backgrounds: C57Bl/6 and 129/SvImJ. We find that in most brain regions, both strains are susceptible to progranulin-mediated neuropathological phenotypes, including astrogliosis, microgliosis, and highly accelerated deposition of the aging pigment lipofuscin. Neuroinflammation due to progranulin deficiency is exaggerated in the B6 strain and present, but less pronounced, in the 129 strain. Differences between the strains in hippocampal neuron counts and neuronal morphology suggest a complex role for progranulin in the hippocampus. We conclude that core progranulin-mediated neurodegenerative phenotypes are penetrant on multiple inbred mouse strains, but that genetic background modulates progranulin’s role in neuroinflammation and hippocampal biology.

Progranulin Reduced Neuronal Cell Death by Activation of Sortilin 1 Signaling Pathways After Subarachnoid Hemorrhage in Rats.

Progranulin has been reported to have neuroprotective actions in cultured neurons. This study investigated the effect of recombinant rat progranulin on early brain injury after subarachnoid hemorrhage. Controlled in vivo laboratory study. Animal research laboratory. Two hundred thirty adult male Sprague-Dawley rats weighing 280-320 g. Subarachnoid hemorrhage was induced in rats by endovascular perforation. Rat recombinant progranulin (1 and 3 ng) was administrated intracerebroventricularly at 1.5 hours after subarachnoid hemorrhage. Progranulin small interfering RNA was administrated by intracerebroventricularly at 1 day before subarachnoid hemorrhage induction. Subarachnoid hemorrhage grade, neurologic score, and brain water content were measured at 24 and 72 hours after subarachnoid hemorrhage. Neural apoptosis was evaluated by double immunofluorescence staining using terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick-end labeling and neuronal nuclei. For mechanistic study, the expression of progranulin, phosphorylated Akt, Akt, p-Erk, Erk, Bcl-2, and cleaved caspase-3 were analyzed by Western blot at 24 hours after subarachnoid hemorrhage. siRNA for sortilin 1 (a progranulin receptor) was used to intervene the downstream pathway. The expression of progranulin decreased and reached the lowest point at 24 hours after subarachnoid hemorrhage. Administration of rat recombinant progranulin decreased brain water content and improved neurologic functions at both 24 and 72 hours after subarachnoid hemorrhage, while knockdown of endogenous progranulin aggravated neurologic deficits after subarachnoid hemorrhage. Rat recombinant progranulin treatment reduced neuronal apoptosis, while progranulin deficiency promoted neuronal apoptosis at 24 hours after subarachnoid hemorrhage. Rat recombinant progranulin promoted Akt activation, increased Bcl-2 level, but reduced caspase-3 level. Knockdown of progranulin binding factor sortilin 1 abolished the beneficial effects of rat recombinant progranulin at 24 hours after subarachnoid hemorrhage. Rat recombinant progranulin alleviated neuronal death via sortilin 1-mediated and Akt-related antiapoptosis pathway. Rat recombinant progranulin may have potentials to ameliorate early brain injury for subarachnoid hemorrhage patients.

NODding off in acute kidney injury with progranulin?

Inflammation is a common feature of murine models of AKI. The endogenous mechanisms which serve to limit inflammation and reduce the severity of AKI are poorly understood. Zhou et al identify progranulin as one such protective mediator. Deficiency of progranulin was associated with increased inflammation and increased injury in both ischemic and nephrotoxic models of AKI. Moreover, administration of exogenous progranulin reduced AKI even when delivered after AKI was established. Interference in NOD2 pathways is suggested as a possible mechanism for protection. PRGN-based therapeutics are under development and might have application in the treatment or prevention of AKI.

Progranulin protects against renal ischemia/reperfusion injury in mice

Progranulin (PGRN), an autocrine growth factor, has multiple physiological functions and is widely involved in the pathogenesis of many types of diseases. The pivotal anti-inflammatory function of PGRN in rheumatoid arthritis encouraged us to examine the role of PGRN in acute kidney injury (AKI). We found that levels of PGRN were significantly reduced in the kidney in a mouse model of renal ischemia/reperfusion injury. We also observed that PGRN deficiency (Grn(-/-) mice) significantly aggravated renal injury as evidenced by higher serum creatinine, more severe morphological injury, increased tubular epithelial cell death, and tubulointerstitial neutrophil and macrophage infiltration versus wild-type mice. In vitro, we found that recombinant human PGRN attenuated hypoxia-induced inflammatory actions and apoptosis in proximal tubule epithelial cells, at least in part associated with a nucleotide-binding oligomerization domain containing 2 (NOD2)-mediated immune response. Importantly, pretreatment with or delayed administration of recombinant human PGRN protected against or promoted recovery from renal ischemia/reperfusion injury in wild-type and Grn(-/-) mice. Similar protective effects were also found in cisplatin-induced AKI. Thus, our findings provide a better understanding of the biological activities of PGRN in the kidney and suggest that PGRN may be an innovative therapeutic strategy for treating patients with AKI.

Are the inflammatory mechanism differentialy regulated across different osteoarthritic joints

Are the inflammatory mechanism differentialy regulated across different osteoarthritic joints

Gremlin1 induced by excessive mechanical stress loading enhances cartilage degradation

Gremlin1 induced by excessive mechanical stress loading enhances cartilage degradation

Progranulin Facilitates Conversion and Function of Regulatory T Cells under Inflammatory Conditions

The progranulin (PGRN) is known to protect regulatory T cells (Tregs) from a negative regulation by TNF-α, and its levels are elevated in various kinds of autoimmune diseases. Whether PGRN directly regulates the conversion of CD4+CD25-T cells into Foxp3-expressing regulatory T cells (iTreg), and whether PGRN affects the immunosuppressive function of Tregs, however, remain unknown. In this study we provide evidences demonstrating that PGRN is able to stimulate the conversion of CD4+CD25-T cells into iTreg in a dose-dependent manner in vitro. In addition, PGRN showed synergistic effects with TGF-β1 on the induction of iTreg. PGRN was required for the immunosuppressive function of Tregs, since PGRN-deficient Tregs have a significant decreased ability to suppress the proliferation of effector T cells (Teff). In addition, PGRN deficiency caused a marked reduction in Tregs number in the course of inflammatory arthritis, although no significant difference was observed in the numbers of Tregs between wild type and PGRN deficient mice during development. Furthermore, PGRN deficiency led to significant upregulation of the Wnt receptor gene Fzd2. Collectively, this study reveals that PGRN directly regulates the numbers and function of Tregs under inflammatory conditions, and provides new insight into the immune regulatory mechanism of PGRN in the pathogenesis of inflammatory and immune-related diseases.

Progranulin Deficiency Reduces CDK4/6/pRb Activation and Survival of Human Neuroblastoma SH-SY5Y Cells.

Null mutations in GRN are associated with frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). However, the influence of progranulin (PGRN) deficiency in neurodegeneration is largely unknown. In neuroblastoma cells, silencing of GRN gene causes significantly reduced cell survival after serum withdrawal. The following observations suggest that alterations of the CDK4/6/retinoblastoma protein (pRb) pathway, secondary to changes in PI3K/Akt and ERK1/2 activation induced by PGRN deficiency, are involved in the control of serum deprivation-induced apoptosis: (i) inhibiting CDK4/6 levels or their associated kinase activity by sodium butyrate or PD332991 sensitized control SH-SY5Y cells to serum deprivation-induced apoptosis without affecting survival of PGRN-deficient cells; (ii) CDK4/6/pRb seems to be downstream of the PI3K/Akt and ERK1/2 signaling pathways since their specific inhibitors, LY294002 and PD98059, were able to decrease CDK6-associated kinase activity and induce death of control SH-SY5Y cells; (iii) PGRN-deficient cells show reduced stimulation of PI3K/Akt, ERK1/2, and CDK4/6 activities compared with control cells in the absence of serum; and (iv) supplementation of recombinant human PGRN was able to rescue survival of PGRN-deficient cells. These observations highlight the important role of PGRN-mediated stimulation of the PI3K/Akt-ERK1/2/CDK4/6/pRb pathway in determining the cell fate survival/death under serum deprivation.

Possible involvement of lysosomal dysfunction in pathological changes of the brain in aged progranulin-deficient mice.

IntroductionIt has been shown that progranulin (PGRN) deficiency causes age-related neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Previous studies also suggested that PGRN is involved in modulating lysosomal function. To elucidate the pathophysiological role of PGRN in the aged brain, in the present study, lysosomal function and pathological changes of the brain were investigated using 10- and 90-week-old wild-type and PGRN-deficient mice.ResultsWe showed that PGRN deficiency caused enhanced CD68 expression in activated microglia and astrogliosis in the cortex and thalamus, especially in the ventral posteromedial nucleus/ventral posterolateral nucleus (VPM/VPL), in the aged brain. Immunoreactivity for Lamp1 (lysosome marker) in the VPM/VPL and expression of lysosome-related genes, i.e. cathepsin D, V-type proton ATPase subunit d2, and transcription factor EB genes, were also increased by PGRN deficiency. Aggregates of p62, which is selectively degraded by the autophagy-lysosomal system, were observed in neuronal and glial cells in the VPM/VPL of aged PGRN-deficient mice. TAR DNA binding protein 43 (TDP-43) aggregates in the cytoplasm of neurons were also observed in aged PGRN-deficient mice. PGRN deficiency caused enhanced expression of glial cell-derived cytotoxic factors such as macrophage expressed gene 1, cytochrome b-245 light chain, cytochrome b-245 heavy chain, complement C4, tumor necrosis factor-α and lipocalin 2. In addition, neuronal loss and lipofuscinosis in the VPM/VPL and disrupted myelination in the cerebral cortex were observed in aged PGRN-deficient mice.ConclusionsThe present study shows that aged PGRN-deficient mice present with NCL-like pathology as well as TDP-43 aggregates in the VPM/VPL, where a particular vulnerability has been reported in NCL model mice. The present results also suggest that these pathological changes in the VPM/VPL are likely a result of lysosomal dysfunction. How PGRN prevents lysosomal dysfunction with aging remains to be elucidated.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-014-0078-x) contains supplementary material, which is available to authorized users.

Results from a pilot study on amiodarone administration in monogenic frontotemporal dementia with granulin mutation

Frontotemporal dementia (FTD) is one of the most important neurodegenerative conditions and Granulin (GRN) is one of the major genes associated to the disease. FTD-GRN patients are still orphan for any evidence-based target-therapy approach. Interestingly, it has been recently found that alkalizing agents rescued haploinsufficiency in cellular models expressing FTD-GRN mutations. We set up a pilot phase II clinical trial in five FTD patients with GRN Thr272s(g.1977_1980delCACT) mutation, to determine if amiodarone (200 mg/day) may (1) reverse progranulin deficiency and (2) delay disease progression. Each patient was scheduled for 7 study visits over 12 months period. We assessed GRN levels at baseline and after amiodarone administration during the treatment course. Somatic and neurologic examinations, along with cognitive and behavioral assessment were recorded as well. No significant effect on peripheral GRN levels was observed. In treated FTD, disease course did not differ when compared with a group of untreated FTD-GRN patients. This is the first trial targeting progranulin rescue in FTD-GRN patients using amiodarone. Despite the negative findings, it may be interesting to extend this attempt to a larger sample of subjects and to other alkalizing agents to restore granulin haploinsufficiency.

Possible involvement of lysosomal dysfunction in pathological changes of the brain in aged progranulin-deficient mice

It has been shown that progranulin (PGRN) deficiency causes age-related neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Previous studies also suggested that PGRN is involved in modulating lysosomal function. To elucidate the pathophysiological role of PGRN in the aged brain, in the present study, lysosomal function and pathological changes of the brain were investigated using 10- and 90-week-old wild-type and PGRN-deficient mice. We showed that PGRN deficiency caused enhanced CD68 expression in activated microglia and astrogliosis in the cortex and thalamus, especially in the ventral posteromedial nucleus/ventral posterolateral nucleus (VPM/VPL), in the aged brain. Immunoreactivity for Lamp1 (lysosome marker) in the VPM/VPL and expression of lysosome-related genes, i.e. cathepsin D, V-type proton ATPase subunit d2, and transcription factor EB genes, were also increased by PGRN deficiency. Aggregates of p62, which is selectively degraded by the autophagy-lysosomal system, were observed in neuronal and glial cells in the VPM/VPL of aged PGRN-deficient mice. TAR DNA binding protein 43 (TDP-43) aggregates in the cytoplasm of neurons were also observed in aged PGRN-deficient mice. PGRN deficiency caused enhanced expression of glial cell-derived cytotoxic factors such as macrophage expressed gene 1, cytochrome b-245 light chain, cytochrome b-245 heavy chain, complement C4, tumor necrosis factor-α and lipocalin 2. In addition, neuronal loss and lipofuscinosis in the VPM/VPL and disrupted myelination in the cerebral cortex were observed in aged PGRN-deficient mice. The present study shows that aged PGRN-deficient mice present with NCL-like pathology as well as TDP-43 aggregates in the VPM/VPL, where a particular vulnerability has been reported in NCL model mice. The present results also suggest that these pathological changes in the VPM/VPL are likely a result of lysosomal dysfunction. How PGRN prevents lysosomal dysfunction with aging remains to be elucidated.

Recombinant Progranulin Prevents the Loss of Proteoglycan in Surgically Induced Osteoarthritis Model

Osteoarthritis (OA) is a degenerative joint disease that affects more than 46 million people in the United States alone. Since mechanisms by which OA ensues are largely unknown, there are no therapeutic targets that effectively prevent and treat the disease. However, growth factors, cytokines and matrix-degrading enzymes are strongly implicated in initiating and aggravating OA lesions. Thus, a molecular understanding of these molecules will provide invaluable information toward the search for novel therapeutic targets for OA. Our genome-wide screen for novel, differentially expressed genes in OA led to the isolation of progranulin (PGRN) as a novel OA-associated growth factor [1]. In subsequent global screen for the binding proteins of PGRN, we found that PGRN bound to TNF Receptors (TNFR). In addition, PGRN blocks the binding of TNFα to TNFR and inhibits TNFα-induced ADAMTS cleavage of cartilage oligomeric matrix protein (COMP) [2]. These previous findings led us to determine whether recombinant PGRN prevents cartilage degradation in the progression of OA in vivo. For this purpose, we took advantage of PGRN knockout mice to generate anterior cruciate ligament (ACL) transection induced OA models which develop severe OA due to the deficiency of PGRN. Importantly, intra-articular injection of rPGRN growth factor significantly prevented the degeneration of cartilage in PGRN-deficient OA model. PGRN treated mice retained cartilage integrity and showed little or no degradation of cartilage matrix in comparison to highly degraded cartilage of non-treated mice (Figure).

Progranulin Deficiency Promotes Post-Ischemic Blood–Brain Barrier Disruption

Loss-of-function mutations of progranulin (PGRN) have been linked to frontotemporal dementia, but little is known about the effects of PGRN deficiency on the brain in health and disease. PGRN has been implicated in neurovascular development, inflammation, and Wnt signaling, a pathway involved in the formation of the blood-brain barrier (BBB). Because BBB alterations and inflammation contribute to ischemic brain injury, we examined the role of PGRN in the brain damage produced by ischemia-reperfusion. PGRN(+/-) and PGRN(-/-) mice underwent middle cerebral artery occlusion (MCAO) with monitoring of cerebral blood flow. Infarct volume and motor deficits were assessed 72 h later. Post-ischemic inflammation was examined by expression of inflammatory genes and flow cytometry. BBB structure and permeability were examined by electron microscopy (EM) and Evans blue (EB) extravasation, respectively. MCAO resulted in ~60% larger infarcts in PGRN(+/-) and PGRN(-/-) mice, an effect independent of hemodynamic factors or post-ischemic inflammation. Rather, massive hemorrhages and post-ischemic BBB disruption were observed, unrelated to degradation of tight junction (TJ) proteins or matrix metalloproteinases (MMPs). By EM, TJ were 30-52% shorter, fewer, and less interlocking, suggesting a weaker seal between endothelial cells. Intracerebral injection of platelet-derived growth factor-CC (PDGF-CC), which increases BBB permeability, resulted in a more severe BBB breakdown in PGRN(+/-) and PGRN(-/-) than wild-type mice. We describe a previously unrecognized involvement of PGRN in the expression of key ultrastructural features of the BBB. Such a novel vasoprotective role of PGRN may contribute to brain dysfunction and damage in conditions associated with reduced PGRN function.

PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from frontotemporal lobar degeneration-progranulin mutation carriers

Loss-of-function progranulin (PGRN) mutations have been identified as the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Previously, we reported cell cycle–related alterations in lymphoblasts from FTLD-TDP patients, carrying the c.709-1G>A null PGRN mutation, suggesting aberrant cell cycle activation in affected neurons. Here we report that PGRN haploinsufficiency activates the extracellular signal-regulated protein kinases 1 and 2 pathway in a Ca2+, protein kinase C–dependent, and pertussis toxin–sensitive manner. Addition of exogenous PGRN or conditioned medium from control cells normalized the response of PGRN-deficient lymphoblasts to serum activation. Our data indicated that noncanonical Wnt5a signaling might be overactivated by PGRN deficiency. We detected increased cellular and secreted levels of Wnt5a in PGRN-deficient lymphoblasts associated with enhanced phosphorylated calmodulin kinase II. Moreover, treatment of control cells with exogenous Wingless-type 5a (Wnt5a)-activated Ca2+/calmodulin kinase II (CaMKII), increased extracellular signal-regulated protein kinases 1 and 2 activity and cell proliferation up to the levels found in c.709-1G>A carrier cells. PGRN knockdown SH-SY5Y neuroblastoma cells also show enhanced Wnt5a content and signaling. Taken together, our results revealed an important role of Wnt signaling in FTLD-TDP pathology and suggest a novel target for therapeutic intervention.

The promotion of bone healing by progranulin, a downstream molecule of BMP-2, through interacting with TNF/TNFR signaling

Endochondral ossification plays a key role in the bone healing process, which requires normal cartilage callus formation. Progranulin (PGRN) growth factor is known to enhance chondrocyte differentiation and endochondral ossification during development, yet whether PGRN also plays a role in bone regeneration remains unknown. In this study we established surgically-induced bone defect and ectopic bone formation models based on genetically-modified mice. Thereafter, the bone healing process of those mice was analyzed through radiological assays including X-ray and micro CT, and morphological analysis including histology and immunohistochemistry. PGRN deficiency delayed bone healing, while recombinant PGRN enhanced bone regeneration. Moreover, PGRN was required for BMP-2 induction of osteoblastogenesis and ectopic bone formation. Furthermore, the role of PGRN in bone repair was mediated, at least in part, through interacting with TNF-α signaling pathway. PGRN-mediated bone formation depends on TNFR2 but not TNFR1, as PGRN promoted bone regeneration in deficiency of TNFR1 but lost such effect in TNFR2 deficient mice. PGRN blocked TNF-α-induced inflammatory osteoclastogenesis and protected BMP-2-mediated ectopic bone formation in TNF-α transgenic mice. Collectively, PGRN acts as a critical mediator of the bone healing process by constituting an interplay network with BMP-2 and TNF-α signaling, and this represents a potential molecular target for treatment of fractures, especially under inflammatory conditions.

Progranulin Does Not Bind Tumor Necrosis Factor (TNF) Receptors and Is Not a Direct Regulator of TNF-Dependent Signaling or Bioactivity in Immune or Neuronal Cells

Progranulin (PGRN) is a secreted glycoprotein expressed in neurons and glia that is implicated in neuronal survival on the basis that mutations in the GRN gene causing haploinsufficiency result in a familial form of frontotemporal dementia (FTD). Recently, a direct interaction between PGRN and tumor necrosis factor receptors (TNFR I/II) was reported and proposed to be a mechanism by which PGRN exerts anti-inflammatory activity, raising the possibility that aberrant PGRN-TNFR interactions underlie the molecular basis for neuroinflammation in frontotemporal lobar degeneration pathogenesis. Here, we report that we find no evidence for a direct physical or functional interaction between PGRN and TNFRs. Using coimmunoprecipitation and surface plasmon resonance (SPR) we replicated the interaction between PGRN and sortilin and that between TNF and TNFRI/II, but not the interaction between PGRN and TNFRs. Recombinant PGRN or transfection of a cDNA encoding PGRN did not antagonize TNF-dependent NFκB, Akt, and Erk1/2 pathway activation; inflammatory gene expression; or secretion of inflammatory factors in BV2 microglia and bone marrow-derived macrophages (BMDMs). Moreover, PGRN did not antagonize TNF-induced cytotoxicity on dopaminergic neuroblastoma cells. Last, co-addition or pre-incubation with various N- or C-terminal-tagged recombinant PGRNs did not alter lipopolysaccharide-induced inflammatory gene expression or cytokine secretion in any cell type examined, including BMDMs from Grn+/- or Grn-/- mice. Therefore, the neuroinflammatory phenotype associated with PGRN deficiency in the CNS is not a direct consequence of the loss of TNF antagonism by PGRN, but may be a secondary response by glia to disrupted interactions between PGRN and Sortilin and/or other binding partners yet to be identified.