An anti-sortilin affibody-peptide fusion inhibits sortilin-mediated progranulin degradation
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PROVIDER: S-EPMC11342335 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:The most common inherited form of Frontotemporal Lobar Degeneration (FTLD) known stems from Progranulin (GRN) mutation and exhibits TDP-43 plus ubiquitin aggregates. Despite the causative role of GRN haploinsufficiency in FTLD-TDP, the neurobiology of this secreted glycoprotein is unclear. Here, we examined PGRN binding to the cell surface. PGRN binds to cortical neurons via its C terminus, and unbiased expression cloning identifies Sortilin (Sort1) as a binding site. Sort1?/? neurons exhibit reduced PGRN binding. In the CNS, Sortilin is expressed by neurons and PGRN is most strongly expressed by activated microglial cells after injury. Sortilin rapidly endocytoses and delivers PGRN to lysosomes. Mice lacking Sortilin have elevations in brain and serum PGRN levels of 2.5- to 5-fold. The 50% PGRN decrease causative in FTLD-TDP cases is mimicked in GRN+/? mice, and is fully normalized by Sort1 ablation. Sortilin-mediated PGRN endocytosis is likely to play a central role in FTLD-TDP pathophysiology.
| S-EPMC2990962 | biostudies-literature
Project description:Progranulin (GRN) mutations causing haploinsufficiency are a major cause of frontotemporal lobar degeneration (FTLD-TDP). Recent discoveries demonstrating sortilin (SORT1) is a neuronal receptor for PGRN endocytosis and a determinant of plasma PGRN levels portend the development of enhancers targeting the SORT1-PGRN axis. We demonstrate the preclinical efficacy of several approaches through which impairing PGRN's interaction with SORT1 restores extracellular PGRN levels. Our report is the first to demonstrate the efficacy of enhancing PGRN levels in iPSC neurons derived from frontotemporal dementia (FTD) patients with PGRN deficiency. We validate a small molecule preferentially increases extracellular PGRN by reducing SORT1 levels in various mammalian cell lines and patient-derived iPSC neurons and lymphocytes. We further demonstrate that SORT1 antagonists and a small-molecule binder of PGRN₅₈₈₋₅₉₃, residues critical for PGRN-SORT1 binding, inhibit SORT1-mediated PGRN endocytosis. Collectively, our data demonstrate that the SORT1-PGRN axis is a viable target for PGRN-based therapy, particularly in FTD-GRN patients.
| S-EPMC3929086 | biostudies-literature
Project description:Progranulin (PGRN), a widely secreted growth factor, is involved in multiple biological functions, and mutations located within the PGRN gene (GRN) are a major cause of frontotemporal lobar degeneration with TDP-43-positive inclusions (FLTD-TDP). In light of recent reports suggesting PGRN functions as a protective neurotrophic factor and that sortilin (SORT1) is a neuronal receptor for PGRN, we used a Sort1-deficient (Sort1-/-) murine primary hippocampal neuron model to investigate whether PGRN's neurotrophic effects are dependent on SORT1. We sought to elucidate this relationship to determine what role SORT1, as a regulator of PGRN levels, plays in modulating PGRN's neurotrophic effects.As the first group to evaluate the effect of PGRN loss in Grn knockout primary neuronal cultures, we show neurite outgrowth and branching are significantly decreased in Grn-/- neurons compared to wild-type (WT) neurons. More importantly, we also demonstrate that PGRN overexpression can rescue this phenotype. However, the recovery in outgrowth is not observed following treatment with recombinant PGRN harboring missense mutations p.C139R, p.P248L or p.R432C, indicating that these mutations adversely affect the neurotrophic properties of PGRN. In addition, we also present evidence that cleavage of full-length PGRN into granulin peptides is required for increased neuronal outgrowth, suggesting that the neurotrophic functions of PGRN are contained within certain granulins. To further characterize the mechanism by which PGRN impacts neuronal morphology, we assessed the involvement of SORT1. We demonstrate that PGRN induced-outgrowth occurs in the absence of SORT1 in Sort1-/- cultures.We demonstrate that loss of PGRN impairs proper neurite outgrowth and branching, and that exogenous PGRN alleviates this impairment. Furthermore, we determined that exogenous PGRN induces outgrowth independent of SORT1, suggesting another receptor(s) is involved in PGRN induced neuronal outgrowth.
| S-EPMC3508877 | biostudies-other
Project description:Mutations in the progranulin (PGRN) gene have been linked to two distinct neurodegenerative diseases, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests a critical role of PGRN in lysosomes. However, how PGRN is trafficked to lysosomes is still not clear. Here we report a novel pathway for lysosomal delivery of PGRN. We found that prosaposin (PSAP) interacts with PGRN and facilitates its lysosomal targeting in both biosynthetic and endocytic pathways via the cation-independent mannose 6-phosphate receptor and low density lipoprotein receptor-related protein 1. PSAP deficiency in mice leads to severe PGRN trafficking defects and a drastic increase in serum PGRN levels. We further showed that this PSAP pathway is independent of, but complementary to, the previously identified PGRN lysosomal trafficking mediated by sortilin. Collectively, our results provide new understanding on PGRN trafficking and shed light on the molecular mechanisms behind FTLD and NCL caused by PGRN mutations.
| S-EPMC4576858 | biostudies-literature
Project description:RATIONALE:Genome-wide association studies identified single-nucleotide polymorphisms near the SORT1 locus strongly associated with decreased plasma LDL-C (low-density lipoprotein cholesterol) levels and protection from atherosclerotic cardiovascular disease and myocardial infarction. The minor allele of the causal SORT1 single-nucleotide polymorphism locus creates a putative C/EBPα (CCAAT/enhancer-binding protein α)-binding site in the SORT1 promoter, thereby increasing in homozygotes sortilin expression by 12-fold in liver, which is rich in this transcription factor. Our previous studies in mice have showed reductions in plasma LDL-C and its principal protein component, apoB (apolipoprotein B) with increased SORT1 expression, and in vitro studies suggested that sortilin promoted the presecretory lysosomal degradation of apoB associated with the LDL precursor, VLDL (very-low-density lipoprotein). OBJECTIVE:To determine directly that SORT1 overexpression results in apoB degradation and to identify the mechanisms by which this reduces apoB and VLDL secretion by the liver, thereby contributing to understanding the clinical phenotype of lower LDL-C levels. METHODS AND RESULTS:Pulse-chase studies directly established that SORT1 overexpression results in apoB degradation. As noted above, previous work implicated a role for lysosomes in this degradation. Through in vitro and in vivo studies, we now demonstrate that the sortilin-mediated route of apoB to lysosomes is unconventional and intersects with autophagy. Increased expression of sortilin diverts more apoB away from secretion, with both proteins trafficking to the endosomal compartment in vesicles that fuse with autophagosomes to form amphisomes. The amphisomes then merge with lysosomes. Furthermore, we show that sortilin itself is a regulator of autophagy and that its activity is scaled to the level of apoB synthesis. CONCLUSIONS:These results strongly suggest that an unconventional lysosomal targeting process dependent on autophagy degrades apoB that was diverted from the secretory pathway by sortilin and provides a mechanism contributing to the reduced LDL-C found in individuals with SORT1 overexpression.
| S-EPMC5815905 | biostudies-literature
Project description:Progranulin (PGRN) is a growth factor implicated in several neurodegenerative diseases, such as frontotemporal lobar degeneration. Despite its important role in the central nervous system (CNS), the mechanisms controlling PGRN expression in the CNS are largely unknown. Recent evidence, however, suggested that several stressors, such as hypoxia, acidosis, or oxidative stress, induce PGRN expression. The present study was mainly aimed at determining whether and, if so, how glucose deprivation affects PGRN expression in PC12 cells. Initially, it was found that glucose deprivation gradually induced PGRN gene expression in PC12 cells. To elucidate the underlying molecular mechanisms, several intracellular signalings that were modified in response to glucose deprivation were examined. Both adenosine monophosphate kinase (AMPK) activation and changes in osmotic pressure, which are modified by extracellular glucose concentration, had no effect on PGRN gene expression; on the other hand, p38 activation in response to glucose deprivation played an important role in inducing PGRN gene expression. It was also found that expression of sortilin, a PGRN receptor implicated in PGRN endocytosis, was dramatically reduced by glucose deprivation. In contrast to glucose-dependent regulation of PGRN gene expression, AMPK activation played a central role in reducing sortilin expression. Overall, the present study suggests that the PGRN-sortilin axis is modulated by glucose deprivation via two distinct mechanisms. As PGRN is neuroprotective, this system may represent a new neuroprotective mechanism activated by glucose deprivation in the CNS.
| S-EPMC5292667 | biostudies-literature
Project description:Haploinsufficiency of the progranulin protein is a leading cause of frontotemporal lobar degeneration. Accumulating evidence support a crucial role of progranulin in the lysosome. Progranulin comprises 7.5 granulin repeats and is known to traffic to lysosomes via direct interactions with prosaposin or sortilin. Within the lysosome, progranulin gets processed into granulin peptides. Here, we report that sortilin and prosaposin independently regulate lysosomal trafficking of progranulin in vivo. The deletion of either prosaposin or sortilin alone results in a significant decrease in the ratio of granulin peptides versus full-length progranulin in mouse brain lysates. This decrease is further augmented by the deficiency of both prosaposin and sortilin. A concomitant increase in the levels of secreted progranulin in the serum was observed. Interestingly, while the deletion of both prosaposin and sortilin totally abolishes lysosomal localization of progranulin in neurons, it has a limited effect on lysosomal trafficking of progranulin in microglia, suggesting the existence of a novel sortilin and prosaposin independent pathway mediating progranulin lysosomal trafficking. In summary, our studies shed light on the regulation of lysosomal trafficking and processing of progranulin in vivo.
| S-EPMC8833632 | biostudies-literature
Project description:Progranulin haplo-insufficiency is a main cause of frontotemporal lobar degeneration (FTLD) with TDP-43 aggregates. Previous studies have shown that sortilin regulates progranulin trafficking and is a main determinant of progranulin level in the brain. In this study, we mapped the binding site between progranulin and sortilin. Progranulin binds to the beta-propeller region of sortilin through its C-terminal tail. The C-terminal progranulin fragment is fully sufficient for sortilin binding and progranulin C-terminal peptide displaces progranulin binding to sortilin. Deletion of the last 3 residues of progranulin (QLL) abolishes its binding to sortilin and also sortilin dependent regulation of progranulin trafficking. Since progranulin haplo-insufficiency results in FTLD, these results may provide important insights into future studies of progranulin trafficking and signaling and progranulin based therapy for FTLD.
| S-EPMC3115958 | biostudies-literature
Project description:Brain positron emission tomography (PET) imaging with radiolabelled proteins is an emerging concept that potentially enables visualization of unique molecular targets in the brain. However, the pharmacokinetics and protein radiolabelling methods remain challenging. Here, we report the performance of an engineered, blood-brain barrier (BBB)-permeable affibody molecule that exhibits rapid clearance from the brain, which was radiolabelled using a unique fluorine-18 labelling method, a cell-free protein radiosynthesis (CFPRS) system. AS69, a small (14 kDa) dimeric affibody molecule that binds to the monomeric and oligomeric states of α-synuclein, was newly designed for brain delivery with an apolipoprotein E (ApoE)-derived brain shuttle peptide as AS69-ApoE (22 kDa). The radiolabelled products 18F-AS69 and 18F-AS69-ApoE were successfully synthesised using the CFPRS system. Notably, 18F-AS69-ApoE showed higher BBB permeability than 18F-AS69 in an ex vivo study at 10 and 30 min post injection and was partially cleared from the brain at 120 min post injection. These results suggest that small, a brain shuttle peptide-fused fluorine-18 labelled protein binders can potentially be utilised for brain molecular imaging.
| S-EPMC7844286 | biostudies-literature
Project description:Background: Pathological angiogenesis is the hallmark of many vision-threatening diseases. Anti-VEGF is a primary treatment with substantial beneficial effects. However, such agents require frequent intravitreal injections. Our previous work established a method for effectively modifying exosomes (EXOs) for loading therapeutic peptides. Here, we used this system to load the anti-angiogenic peptide KV11, aiming to establish an EXO-based therapy strategy to suppress neovascularization in the retina. Methods: Using an anchoring peptide, CP05, we linked KV11 to endothelial cell (EC) derived EXOs, yielding EXOKV11. We tested the delivery efficiency of EXOKV11 via two commonly used ocular injection methods: retro-orbital injection and intravitreal injection. Deploying an oxygen-induced retinopathy (OIR) model and a VEGF injection model, we tested the effects of EXOKV11 on neovascular formation, EC proliferation, and vascular permeability. In vitro experiments were used to test the mechanism and to analyze the effects of EXOKV11 on EC proliferation, migration, and sprouting. Results: By using the EXO loading system, KV11 was more efficiently delivered to the blood vessels of the mouse retina via retro-orbital injection. In both OIR model and VEGF injection model, EXOKV11 was more effective than KV11 alone in inhibiting neovascularization and vessel leakage. The therapeutic effect of retro-orbital injection of EXOKV11 was comparable to the intravitreal injection of VEGF-trap. Mechanistically, KV11 alone inhibited VEGF-downstream signaling, while EXOKV11 showed a stronger effect. Conclusions: We used EXOs as a carrier for intraocular delivery of KV11. We showed that KV11 itself has an anti-angiogenic effect through retro-orbital injection, but that this effect was greatly enhanced when delivered with EXOs. Thus, this system has the potential to treat proliferative retinopathy via retro-orbital injection which is a less invasive manner compared with intravitreal injection.
| S-EPMC8039955 | biostudies-literature