Targeting progranulin alleviated silica particles-induced pulmonary inflammation and fibrosis via decreasing Il-6 and Tgf-ß1/Smad.

Long term exposure to silica particles leads to various diseases, among which silicosis is of great concern. Silicosis is an interstitial lung disease caused by inhalation of silica particles in production environments. However, the mechanisms underlying silicosis remains unclear. Our previous studies revealed that progranulin (Pgrn) promoted the expression of pro-inflammatory factors in alveolar macrophages treated with silica particles and the secretion of extracellular matrix of pulmonary fibroblasts. Nevertheless, the role of Pgrn in silica particles-induced silicosis in vivo was unknown. This study found that silica particles increased Pgrn expression in silicosis patients. Pgrn deficiency reduced lung inflammation and fibrosis in silica particles-induced silicosis mouse models. Subsequently, based on transcriptional sequencing and interleukin (Il) -6 knockout mouse models, results demonstrated that Pgrn deficiency might decrease silicosis inflammation by reducing the production of Il-6, thereby modulating pulmonary fibrosis in the early stage of silicosis mouse models. Furthermore, another mechanism through which Pgrn deficiency reduced fibrosis in silicosis mouse models was the regulation of the transforming growth factor (Tgf) -ß1/Smad signaling pathway. Conclusively, Pgrn contributed to silicosis inflammation and fibrosis induced by silica particles, indicating that Pgrn could be a promising therapeutic target.

Interaction with ERp57 is required for progranulin protection against Type 2 Gaucher disease.

Gaucher disease (GD), one of the most common lysosomal storage diseases, is caused by GBA1 mutations resulting in defective glucocerebrosidase (GCase) and consequent accumulation of its substrates ß-glucosylceramide (ß-GlcCer). We reported progranulin (PGRN), a secretary growth factor-like molecule and an intracellular lysosomal protein was a crucial co-factor of GCase. PGRN binds to GCase and recruits Heat Shock Protein 70 (Hsp70) to GCase through its C-terminal Granulin (Grn) E domain, termed as ND7. In addition, both PGRN and ND7 are therapeutic against GD. Herein we found that both PGRN and its derived ND7 still displayed significant protective effects against GD in Hsp70 deficient cells. To delineate the molecular mechanisms underlying PGRN's Hsp70-independent regulation of GD, we performed a biochemical co-purification and mass spectrometry with His-tagged PGRN and His-tagged ND7 in Hsp70 deficient cells, which led to the identification of ERp57, also referred to as protein disulfide isomerase A3 (PDIA3), as a protein that binds to both PGRN and ND7. Within type 2 neuropathic GD patient fibroblasts L444P, bearing GBA1 L444P mutation, deletion of ERp57 largely abolished the therapeutic effects of PGRN and ND7, as manifested by loss of effects on lysosomal storage, GCase activity, and ß-GlcCer accumulation. Additionally, recombinant ERp57 effectively restored the therapeutic effects of PGRN and ND7 in ERp57 knockout L444P fibroblasts. Collectively, this study reports ERp57 as a previously unrecognized binding partner of PGRN that contributes to PGRN regulation of GD.

Progranulin improves neural development via the PI3K/Akt/GSK-3ß pathway in the cerebellum of a VPA-induced rat model of ASD.

Autism spectrum disorder (ASD) is a neurodevelopmental disease featuring social interaction deficits and repetitive/stereotyped behaviours; the prevalence of this disorder has continuously increased. Progranulin (PGRN) is a neurotrophic factor that promotes neuronal survival and differentiation. However, there have not been sufficient studies investigating its effect in animal models of autism. This study investigated the effects of PGRN on autistic phenotypes in rats treated with valproic acid (VPA) and assessed the underlying molecular mechanisms. PGRN was significantly downregulated in the cerebellum at postnatal day 14 (PND14) and PND35 in VPA-exposed rats, which simultaneously showed defective social preference, increased repetitive behaviours, and uncoordinated movements. When human recombinant PGRN (r-PGRN) was injected into the cerebellum of newborn ASD model rats (PND10 and PND17), some of the behavioural defects were alleviated. r-PGRN supplementation also reduced cerebellar neuronal apoptosis and rescued synapse formation in ASD rats. Mechanistically, we confirmed that PGRN protects neurodevelopment via the PI3K/Akt/GSK-3ß pathway in the cerebellum of a rat ASD model. Moreover, we found that prosaposin (PSAP) promoted the internalisation and neurotrophic activity of PGRN. These results experimentally demonstrate the therapeutic effects of PGRN on a rat model of ASD for the first time and provide a novel therapeutic strategy for autism.

Progranulin promotes hippocampal neurogenesis and alleviates anxiety-like behavior and cognitive impairment in adult mice subjected to cerebral ischemia.

AIMS: Cerebral ischemia can lead to anxiety and cognitive impairment due to the loss of hippocampal neurons. Facilitation of endogenous neurogenesis in the hippocampus is a potential therapeutic strategy for alleviating ischemia-induced anxiety and cognitive impairment. Progranulin (PGRN), a secretory glycoprotein, has been reported to have a mitogentic effect on many cell types. However, it is not clear whether PGRN enhances hippocampal neurogenesis and promotes functional recovery. METHODS: Adult male C57BL/6 mice were subjected to permanent middle cerebral artery occlusion (pMCAO) and injected intracerebroventricularly with recombinant mouse PGRN 30 min after pMCAO. Anxiety-like behavior was detected by the open field and the elevated plus maze tests, and spatial learning and memory abilities were evaluated by Morris water maze. Neurogenesis was examined by double labeling of BrdU and neural stem cells or neurons markers. For mechanism studies, the level of ERK1/2 and AKT phosphorylation were assessed by western blotting. RESULTS: Progranulin significantly alleviated anxiety-like behavior and spatial learning and memory impairment induced by cerebral ischemia in mice. Consistent with the functional recovery, PGRN promoted neural stem cells (NSCs) proliferation and neuronal differentiation in the dentate gyrus (DG) after cerebral ischemia. PGRN upregulated the expression of phosphorylated ERK1/2 and Akt in the DG after cerebral ischemia. CONCLUSIONS: Progranulin alleviates ischemia-induced anxiety-like behavior and spatial learning and memory impairment in mice, probably via stimulation of hippocampal neurogenesis mediated by activation of MAPK/ERK and PI3K/Akt pathways. PGRN might be a promising candidate for coping with ischemic stroke-induced mood and cognitive impairment in clinic.

Injectable recombinant block polymer gel for sustained delivery of therapeutic protein in post traumatic osteoarthritis.

Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. Here, we demonstrate that E5C, a recombinant protein block polymer, consisting of five repeats of elastin like polypeptide (E) and a coiled-coil domain of cartilage oligomeric matrix protein (C), is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E5C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin, provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E5C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E5C gels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the progranulin-derivative Atsttrin, for therapeutic application in OA.

Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

Progranulin promotes bone fracture healing via TNFR pathways in mice with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) significantly increases bone fragility and fracture risk. Progranulin (PGRN) promotes bone fracture healing in both physiological and type 1 diabetic conditions. The present study aimed to investigate the role of PGRN in T2DM bone fracture healing. MKR mice (with an FVB/N genetic background) were used as the T2DM model. Drill-hole and Bonnarens and Einhorn models were used to investigate the role of PGRN in T2DM fracture healing in vivo. Primary bone marrow cells were isolated for molecular and signaling studies, and reverse transcription-polymerase chain reaction, immunohistochemical staining, and western blotting were performed to assess PGRN effects in vitro. PGRN mRNA and protein expression were upregulated in the T2DM model. Local administration of recombinant PGRN effectively promoted T2DM bone fracture healing in vivo. Additionally, PGRN could induce anabolic metabolism during endochondral ossification through the TNFR2-Akt and Erk1/2 pathways. Furthermore, PGRN showed anti-inflammatory activity in the T2DM bone regeneration process. These findings suggest that local administration of exogenous PGRN may be an alternative strategy to support bone regeneration in patients with T2DM. Additionally, PGRN might hold therapeutic potential for other TNFR-related metabolic disorders.

Progranulin Promotes Regeneration of Inflammatory Periodontal Bone Defect in Rats via Anti-inflammation, Osteoclastogenic Inhibition, and Osteogenic Promotion.

Progranulin (PGRN) has been proved to play a crucial role in anti-inflammation and osteogenesis promotion; thus, it was hypothesized that PGRN could promote bone regeneration in periodontal disease. In this experiment, the periodontal bone defects were established in periodontitis rats; recombinant human progranulin (rhPGRN), tumor necrosis factor alpha inhibitor (anti-TNF-α), or phosphate buffer saline (PBS)-loaded collagen membrane scaffolds were implanted within defects and the rats were sacrificed at scheduled time points. Volume of new bone was assessed by radiological and histomorphometric analyses. Expression of osteogenesis-related markers and tumor necrosis factor-α (TNF-α) was evaluated using immunohistochemistry. Tartrate-resistant acid phosphatase (TRAP) staining was also performed to determine the number of osteoclasts. Immunofluorescence (IF) staining was performed to explore the interaction between rhPGRN and tumor necrosis factor receptors (TNFRs). The results showed that the rhPGRN group had significantly superior quantity and quality of newly formed bone, higher expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and TNFR2 compared with the PBS group and the anti-TNF-α group. Similarly to the anti-TNF-α group, the rhPGRN group also exhibited the significant inhibitory effect on the expression of TNF-α and the number of TRAP-positive cells compared with the PBS group. Hence, our experiment suggests that PGRN promotes regeneration of inflammatory periodontal bone defect in rats via anti-inflammation, osteoclastogenic inhibition, and osteogenic promotion. Local administration of PGRN may provide a new therapeutic strategy for periodontal bone regeneration.

Methods to Investigate the Protection Against Neurodegenerative Disorders Provided by Progranulin Gene Transfer in the Brain.

Progranulin (PGRN) is a multifunctional protein that is widely expressed throughout the brain, where it has been shown to be a critical regulator of CNS inflammation (Ahmed et al., J Neuroinflammation 4:7, 2007; Yin et al., J Exp Med 207:117-128, 2010; Martens et al., J Clin Investig 122:3955-3959; Inestrosa and Arenas). PGRN functions as an autocrine neuronal growth factor, important for long-term neuronal survival (Ahmed et al., J Neuroinflammation 4:7, 2007; Nat Rev Neurosci 11:77-86, 2009). Together, these critical roles in the CNS suggest that enhancing PGRN expression may provide neuronal support and protection for neurodegenerative disorders, such as Parkinson's disease (PD). Here, we describe the application of PGRN gene transfer using in vivo delivery of lentiviral expression vectors in a rodent model of PD.

Progranulin ameliorates coxsackievirus-B3-induced viral myocarditis by downregulating Th1 and Th17 cells.

Viral myocarditis, which is caused by Coxsackievirus B3 (CVB3) infection, is a leading reason of sudden cardiac death in young adults. Progranulin (PGRN), a pleiotropic growth factor, has been shown to exert anti-inflammatory function in a variety of inflammatory diseases. However, the expression and function of PGRN in the pathogenesis of viral myocarditis remain largely unknown. In this study, we found that PGRN levels in plasma and cardiac tissues were significantly upregulated post CVB3 infection, and negative correlated with disease severity. PGRN deficiency significantly exacerbated, whereas recombinant PGRN treatment attenuated CVB3-induced myocarditis in mice. PGRN downregulated Th1 and Th17 cell responses and cytokine production in vitro and in vivo, whereas its effect on viral myocarditis was Treg cell independent. Furthermore, PGRN regulated Th1 and Th17 cells differentiation through inhibition of the JAK/STAT pathway. Therefore, our findings reveal a critical role for PGRN in reducing CVB3-induced myocarditis and suggest that PGRN maybe a novel therapeutic treatment for viral myocarditis.