Sortilin Overexpression Research Articles

Sortilin-Mediated Inhibition of TREK1/2 Channels in Primary Sensory Neurons Promotes Prediabetic Neuropathic Pain.

Neuropathic pain can occur during the prediabetic stage, even in the absence of hyperglycemia. The presence of prediabetic neuropathic pain (PDNP) poses challenges to the management of individuals with prediabetes. However, the mechanisms underlying this pain remain unclear. This study aims to investigate the underlying mechanism and identify potential therapeutic targets of PDNP. A prediabetic animal model induced by a high-energy diet exhibits both mechanical allodynia and thermal hyperalgesia. Furthermore, hyperexcitability and decreased potassium currents are observed in the dorsal root ganglion (DRG) neurons of these rats. TREK1 and TREK2 channels, which belong to the two-pore-domain K+ channel (K2P) family and play an important role in controlling cellular excitability, are downregulated in DRG neurons. Moreover, this alteration is modulated by Sortilin, a molecular partner that modulates the expression of TREK1. The overexpression of Sortilin negatively affects the expression of TREK1 and TREK2, leading to increased neuronal excitability in the DRG and enhanced peripheral pain sensitivity in rats. Moreover, the downregulation of Sortilin or activation of TREK1 and TREK2 channels by genetic or pharmacological approaches can alleviate PDNP. Therefore, targeting the Sortilin-mediated TREK1/2 pathway may provide a therapeutic approach for ameliorating PDNP.

Sortilin enhances secretion of apolipoprotein(a) through effects on apolipoprotein B secretion and promotes uptake of lipoprotein(a)

Elevated plasma lipoprotein(a) (Lp(a)) is an independent, causal risk factor for atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Lp(a) is formed in or on hepatocytes from successive noncovalent and covalent interactions between apo(a) and apoB, although the subcellular location of these interactions and the nature of the apoB-containing particle involved remain unclear. Sortilin, encoded by the SORT1 gene, modulates apoB secretion and LDL clearance. We used a HepG2 cell model to study the secretion kinetics of apo(a) and apoB. Overexpression of sortilin increased apo(a) secretion, while siRNA-mediated knockdown of sortilin expression correspondingly decreased apo(a) secretion. Sortilin binds LDL but not apo(a) or Lp(a), indicating that its effect on apo(a) secretion is likely indirect. Indeed, the effect was dependent on the ability of apo(a) to interact noncovalently with apoB. Overexpression of sortilin enhanced internalization of Lp(a), but not apo(a), by HepG2 cells, although neither sortilin knockdown in these cells or Sort1 deficiency in mice impacted Lp(a) uptake. We found several missense mutations in SORT1 in patients with extremely high Lp(a) levels; sortilin containing some of these mutations was more effective at promoting apo(a) secretion than WT sortilin, though no differences were found with respect to Lp(a) internalization. Our observations suggest that sortilin could play a role in determining plasma Lp(a) levels and corroborate in vivo human kinetic studies which imply that secretion of apo(a) and apoB are coupled, likely within the hepatocyte.

Diagnostic and Therapeutic Implications of Sortilin Expressed on the Surface of Bladder Carcinoma Cells.

Background & Objective:Cell surface expression of sortilin in different types of cancer signifies it as a therapeutic target for cancer therapy. The aim of this study was to detect sortilin expression in bladder cancer cells using an anti-sortilin monoclonal antibody (mAb) to evaluate sortilin as a target for developing diagnostic and therapeutic agents against bladder carcinoma. Methods:The protein expression of sortilin in bladder cancer tissues and cell lines (5637 and EJ138) was investigated by immunohistochemistry (IHC), immune-cytochemistry (ICC), and flow cytometry. Furthermore, the capability of anti-sortilin mAb in apoptosis induction in bladder cancer cells was evaluated.Results:A high expression level was observed in bladder carcinoma tissues (P≤0.001) and cell lines, using IHC and ICC, respectively. Flow cytometry results showed cell surface expression of 27.5±3% (P≤0.01), 74.4±7.8% (P≤0.001), and 4.2±0.4% of sortilin in EJ138, 5637, and HFFF cells, respectively. In EJ138 anti-sortilin mAb induced apoptosis in 25.2±11.5% (P≤0.05) (early) and 4.5±1.1% (P>0.05) (late) after 6 h incubation, while for 12 h, the values of 11.6±3.8% (P>0.05) and 20.7±4.4% (P≤0.05) were achieved. In 5637 cells, 6 h incubation resulted in 10.2±0.3% (P>0.05) and 6.6±1.4% (P>0.05) apoptosis induction, while these values were 12.1±0.8% (P>0.05) and 27.4±4.5% (P≤0.01) after 12 h. The HFFF cells did not show significant apoptosis. Conclusion:The overexpression of sortilin in bladder tumor cells and its potential in inducing apoptosis via directed targeting with the specific monoclonal antibody may represent this protein as a potential candidate of targeted therapy in bladder carcinoma.

Sortilin deletion in the prefrontal cortex and hippocampus ameliorates depressive-like behaviors in mice via regulating ASM/ceramide signaling.

Major depressive disorder (MDD) is a common psychiatric disorder characterized by persistent mood despondency and loss of motivation. Although numerous hypotheses have been proposed, the possible pathogenesis of MDD remains unclear. Several recent studies show that a classic transporter protein, sortilin, is closely associated with depression. In the present study, we investigated the role of sortilin in MDD using a well-established rodent model of depression. Mice were subjected to chronic unpredictable mild stress (CUMS) for 6 weeks. We showed that the expression levels of sortilin were significantly increased in the prefrontal cortex and hippocampus of CUMS mice. The depressive-like behaviors induced by CUMS were alleviated by specific knockdown of sortilin in the prefrontal cortex and hippocampus. We revealed that sortilin facilitated acid sphingomyelinase (ASM)/ceramide signaling, which activated RhoA/ROCK2 signaling, ultimately causing the transformation of dendritic spine dynamics. Specific overexpression of sortilin in the prefrontal cortex and hippocampus induced depressive-like behaviors, which was mitigated by injection of ASM inhibitor SR33557 (4 µg/μL) into the prefrontal cortex and hippocampus. In conclusion, sortilin knockdown in the prefrontal cortex and hippocampus plays an important role in ameliorating depressive-like behavior induced by CUMS, which is mainly evidenced by decreasing the trafficking of ASM from the trans-Golgi network to the lysosome and reducing the ceramide levels. Our results provide a new insight into the pathology of depression, and demonstrate that sortilin may be a potential therapeutic target for MDD.

Overexpression of sortilin is associated with 5-FU resistance and poor prognosis in colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide. Even if 5‐fluorouracil (5‐FU) is used as the first‐line chemotherapeutic drug, responsiveness is only 20‐30%. Acquired resistance to 5‐FU contributes to both poor patient prognosis and relapse, emphasizing the need to identify biomarkers. Sortilin, a vacuolar protein sorting 10 protein (Vps10p), implicated in protein trafficking, is over expressed in CRC cell lines cultured 72 hours in presence of 5‐FU. This overexpression was also observed in 5‐FU‐resistant cells derived from these cell lines as well as in CRC primary cultures (or patients derived cell lines). A significantly higher expression of sortilin was observed in vivo, in 5‐FU‐treated tumours engrafted in Nude mice, as compared with non‐treated tumour. A study of transcriptional regulation allowed identifying a decrease in ATF3 expression, as an explanation of sortilin overexpression following 5‐FU treatment. In silico analysis revealed SORT1 expression correlation with poor prognosis. Moreover, sortilin expression was found to be positively correlated with CRC tumour grades. Collectively, our findings identify sortilin as a potential biomarker of 5‐FU resistance associated with poor clinical outcomes and aggressiveness in CRC. As a new prognostic factor, sortilin expression could be used to fight against CRC.

Sortilin promotes macrophage cholesterol accumulation and aortic atherosclerosis through lysosomal degradation of ATP-binding cassette transporter A1 protein.

Sortilin is closely associated with hyperlipidemia and the risk of atherosclerosis (AS). The role of sortilin and the underlying mechanism in peripheral macrophage are not fully understood. In this study, we investigated the effect of macrophage sortilin on ATP-binding cassette transporter A1 (ABCA1) expression, ABCA1-mediated cholesterol efflux, and aortic AS. Macrophage sortilin expression was upregulated by oxidized low-density lipoproteins (ox-LDLs) in both concentration- and time-dependent manners. Its expression reached the peak level when cells were incubated with 50μg/ml ox-LDL for 24h. Overexpression of sortilin in macrophage reduced cholesterol efflux, leading to an increase in intracellular total cholesterol, free cholesterol, and cholesterol ester. Sortilin was found to bind with ABCA1 protein and suppress macrophage ABCA1 expression, resulting in a decrease in cholesterol efflux from macrophages. The inhibitory effect of sortilin in cholesterol efflux was partially reversed by treatment with chloroquine, a lysosomal inhibitor. On the contrary, the ABCA1 protein level and ABCA1-mediated cholesterol efflux is increased by sortilin short hairpin RNA transfection. The fecal and biliary cholesterol 3H-sterol from cholesterol-laden mouse peritoneal macrophage was reduced by sortilin overexpression through lentivirus vector (LV)-sortilin in low-density lipoprotein receptor knockout mice, which was prevented by co-treatment with chloroquine. Treatment with LV-sortilin reduced plasma high-density lipoprotein and increased plasma ox-LDL levels. Accordingly, aortic lipid deposition and plaque area were exacerbated, and ABCA1 expression was reduced in mice in response to infection with LV-sortilin alone. These effects of LV-sortilin were partially reversed by chloroquine. Sortilin enhances lysosomal degradation of ABCA1 protein and suppresses ABCA1-mediated cholesterol efflux from macrophages, leading to foam cell formation and AS development.

Abstract 087: Hepatic Sortilin Regulates Apolipoprotein B Secretion only Under Conditions of Secretory StressDependent of the Presence of Secretory Stress

SORT1 is strongly associated with plasma lipid traits and coronary artery disease. The protein it encodes, sortilin is a multi-ligand receptor involved in trafficking of proteins from the Golgi to the lysosome. Hepatic sortilin overexpression reduces VLDL secretion, but the effects of loss of sortilin function on apolipoprotein B100 (apoB) and VLDL secretion have been contradictory and perplexing. Conflicting studies have variously reported increased and decreased apoB/VLDL secretion in response to loss of sortilin, but used a variety of different models and methods of ablating sortilin function. To resolve this, we first measured VLDL secretion in Sort1 knockout (KO) mice on a chow diet and observed no differences in either apoB or TG secretion. ApoB secretion was similarly unaffected in Sort1 KO primary hepatocytes or in McA-RH7777 cells treated with siRNA to knockdown sortilin. We concluded that in a basal secretory state, loss of sortilin alone does not impact apoB secretion, leading us to test if differences in secretory state might explain the discrepancies in published results. We found that when hAPOB was overexpressed, Sort1 knockdown further increased its secretion. The reports of increased VLDL secretion with Sort1 knockout occurred with apoB overexpression, or on high fat diet (HFD); consistent with our hypothesis that secretory overload or metabolic stress are required to uncover the deficiency phenotype. We placed Sort1 KO mice on a 45% HFD for 12 weeks and observed a significant increase in VLDL secretion compared to controls. In addition, when we lipid-loaded hepatocytes with oleic acid or palmitic acid, apoB secretion was increased, and Sort1 knockdown further increased this compared to control. We treated cells with Tunicamycin to induce ER stress, and found that apoB secretion was decreased in the control group was observed, but not in the absence of Sort1, suggesting that sortilin regulates apoB secretion the cell is stressed. Based on these data, we propose that hepatic sortilin regulates the post-ER fate of apoB for either degradation or export; thereby coordinating intracellular apoB metabolism in response to the number and quality of apoB particles that reach the Golgi, and the level of post-ER pre-secretory proteolysis activity.

Abstract 022: Hepatic Sortilin Regulates Apolipoprotein B Secretion only Under Conditions of Secretory StressDependent of the Presence of Secretory Stress

SORT1 is strongly associated with plasma lipid traits and coronary artery disease. The protein it encodes, sortilin is a multi-ligand receptor involved in trafficking of proteins from the Golgi to the lysosome. Hepatic sortilin overexpression reduces VLDL secretion, but the effects of loss of sortilin function on apolipoprotein B100 (apoB) and VLDL secretion have been contradictory and perplexing. Conflicting studies have variously reported increased and decreased apoB/VLDL secretion in response to loss of sortilin, but used a variety of different models and methods of ablating sortilin function. To resolve this, we first measured VLDL secretion in Sort1 knockout (KO) mice on a chow diet and observed no differences in either apoB or TG secretion. ApoB secretion was similarly unaffected in Sort1 KO primary hepatocytes or in McA-RH7777 cells treated with siRNA to knockdown sortilin. We concluded that in a basal secretory state, loss of sortilin alone does not impact apoB secretion, leading us to test if differences in secretory state might explain the discrepancies in published results. We found that when hAPOB was overexpressed, Sort1 knockdown further increased its secretion. The reports of increased VLDL secretion with Sort1 knockout occurred with apoB overexpression, or on high fat diet (HFD); consistent with our hypothesis that secretory overload or metabolic stress are required to uncover the deficiency phenotype. We placed Sort1 KO mice on a 45% HFD for 12 weeks and observed a significant increase in VLDL secretion compared to controls. In addition, when we lipid-loaded hepatocytes with oleic acid or palmitic acid, apoB secretion was increased, and Sort1 knockdown further increased this compared to control. We treated cells with Tunicamycin to induce ER stress, and found that apoB secretion was decreased in the control group was observed, but not in the absence of Sort1, suggesting that sortilin regulates apoB secretion the cell is stressed. Based on these data, we propose that hepatic sortilin regulates the post-ER fate of apoB for either degradation or export; thereby coordinating intracellular apoB metabolism in response to the number and quality of apoB particles that reach the Golgi, and the level of post-ER pre-secretory proteolysis activity.

Prions amplify through degradation of the VPS10P sorting receptor sortilin.

Prion diseases are a group of fatal neurodegenerative disorders caused by prions, which consist mainly of the abnormally folded isoform of prion protein, PrPSc. A pivotal pathogenic event in prion disease is progressive accumulation of prions, or PrPSc, in brains through constitutive conformational conversion of the cellular prion protein, PrPC, into PrPSc. However, the cellular mechanism by which PrPSc is progressively accumulated in prion-infected neurons remains unknown. Here, we show that PrPSc is progressively accumulated in prion-infected cells through degradation of the VPS10P sorting receptor sortilin. We first show that sortilin interacts with PrPC and PrPSc and sorts them to lysosomes for degradation. Consistently, sortilin-knockdown increased PrPSc accumulation in prion-infected cells. In contrast, overexpression of sortilin reduced PrPSc accumulation in prion-infected cells. These results indicate that sortilin negatively regulates PrPSc accumulation in prion-infected cells. The negative role of sortilin in PrPSc accumulation was further confirmed in sortilin-knockout mice infected with prions. The infected mice had accelerated prion disease with early accumulation of PrPSc in their brains. Interestingly, sortilin was reduced in prion-infected cells and mouse brains. Treatment of prion-infected cells with lysosomal inhibitors, but not proteasomal inhibitors, increased the levels of sortilin. Moreover, sortilin was reduced following PrPSc becoming detectable in cells after infection with prions. These results indicate that PrPSc accumulation stimulates sortilin degradation in lysosomes. Taken together, these results show that PrPSc accumulation of itself could impair the sortilin-mediated sorting of PrPC and PrPSc to lysosomes for degradation by stimulating lysosomal degradation of sortilin, eventually leading to progressive accumulation of PrPSc in prion-infected cells.

Abstract 18667: Lipoprotein(a) Catabolism and Apolipoprotein(a) Secretion is Regulated by Sortilin

Elevated levels of lipoprotein(a) (Lp(a)) in plasma have been identified as an independent, causal risk factor for coronary heart disease. Lp(a) consists of a low-density lipoprotein (LDL)-like particle whose apolipoproteinB-100 (apoB-100) moiety is covalently linked to the unique glycoprotein apolipoprotein(a) (apo(a)). Recently, Lp(a) internalization by the LDL-receptor (LDLr) in hepatic cells and primary human fibroblasts has been shown to be regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9). However, Lp(a)/apo(a) internalization still occurs even with a defective LDLr or in the presence of PCSK9 (in fibroblasts and hepatic cells, respectively), indicating a role for receptors other than the LDLr in Lp(a) catabolism. Hepatic sortilin has been identified as a potential receptor mediating LDL catabolism as well as the regulation of apoB-100 secretion. Sortilin localizes to the Golgi apparatus where it mediates trafficking of specific bound ligands to the lysosome. Sortilin also localizes to clathrin-coated pits in the plasma membrane where it can act as an internalization receptor. We demonstrate in the current study that Lp(a), but not apo(a), internalization in hepatic cells is influenced by sortilin overexpression. In the presence of PCSK9, Lp(a) internalization greatly increases with sortilin overexpression compared to control. These results suggest that hepatic sortilin has the ability to act as a receptor for Lp(a) catabolism, through the LDL-like moiety, in a manner that is not dependent on the LDLr. Furthermore, Lp(a) internalization increases with sortilin overexpression in primary human fibroblasts with a defective LDLr, again emphasizing an LDLr-independent role for sortilin as a receptor for Lp(a). Interestingly, an increase in apo(a) secretion is observed with sortilin overexpression in hepatic cells. Removal of the carboxyl-terminal tail of sortilin results in an inability to promote the secretion of apo(a), indicating a direct role for the sorting motifs present in this region of sortilin for the regulation of apo(a) secretion. Taken together, these results indicate novel roles for sortilin in both Lp(a) catabolism and apo(a) secretion.

Insulin Resistance Causes Posttranslational Downregulation of Hepatic Sortilin 1 in Diabetic Mice

Dyslipidemia increases the risk of cardiovascular diseases in diabetes. Hepatic Sortilin 1 decreases hepatic apoB production, and its liver expression is associated with plasma LDL cholesterol and cardiovascular disease risk in humans. The goal of this study is to investigate the insulin regulation of hepatic Sortilin 1 in diabetes. Insulin treatment rapidly induced Sortilin 1 protein, but not mRNA, in AML12 cells. A PI3K inhibitor or an AKT inhibitor treatment rapidly reduced, while overexpression of a constitutively active AKT induced Sortilin 1 protein in AML12 cells. LC-MS/MS analysis showed that Sortilin 1 was phosphorylated at serine 825. Previous studies have shown that phosphorylation of S825 by casein kinase II promotes the cellular trafficking function of Sortilin 1 via enhancing its interaction with other trafficking adaptor proteins. Interestingly, abolishing S825 phosphorylation by S825A mutation or by a casein kinase 2 inhibitor prevented insulin induction of Sortilin1 in AML12 cells. High fat diet-fed obese mice and streptozocin-treated mice showed decreased hepatic Sortilin 1 protein. Treating mice with a PI3K inhibitor rapidly decreased hepatic Sortilin 1 protein and increased plasma LDL-cholesterol and triglyceride levels. Furthermore, over-expression of Sortilin 1 in mouse livers with adenovirus-mediated gene delivery prevented PI3K inhibitor inhibition of hepatic Sortilin 1. This prevented the PI3K inhibitor-induced elevation of plasma triglyceride, but not cholesterol in mice, suggesting that Sortilin 1-independent mechanisms also mediate the cholesterol-raising effect of PI3K inhibitor. In summary, insulin regulation of Sortilin 1 may be a novel link between insulin resistance and increased hepatic apoB production in insulin resistance.

Macrophage sortilin promotes LDL uptake, foam cell formation, and atherosclerosis.

Noncoding gene variants at the SORT1 locus are strongly associated with low-density lipoprotein cholesterol (LDL-C) levels, as well as with coronary artery disease. SORT1 encodes a protein called sortilin, and hepatic sortilin modulates LDL metabolism by targeting apolipoprotein B-containing lipoproteins to the lysosome. Sortilin is also expressed in macrophages, but its role in macrophage uptake of LDL and in atherosclerosis independent of plasma LDL-C levels is unknown. To determine the effect of macrophage sortilin expression on LDL uptake, foam cell formation, and atherosclerosis. We crossed Sort1(-/-) mice onto a humanized Apobec1(-/-); hAPOB transgenic background and determined that Sort1 deficiency on this background had no effect on plasma LDL-C levels but dramatically reduced atherosclerosis in the aorta and aortic root. To test whether this effect was a result of macrophage sortilin deficiency, we transplanted Sort1(-/-);LDLR(-/-) or Sort1(+/+);LDLR(-/-) bone marrow into Ldlr(-/-) mice and observed a similar reduction in atherosclerosis in mice lacking hematopoetic sortilin without an effect on plasma LDL-C levels. In an effort to determine the mechanism by which hematopoetic sortilin deficiency reduced atherosclerosis, we found no effect of sortilin deficiency on macrophage recruitment or lipopolysaccharide-induced cytokine release in vivo. In contrast, sortilin-deficient macrophages had significantly reduced uptake of native LDL ex vivo and reduced foam cell formation in vivo, whereas sortilin overexpression in macrophages resulted in increased LDL uptake and foam cell formation. Macrophage sortilin deficiency protects against atherosclerosis by reducing macrophage uptake of LDL. Sortilin-mediated uptake of native LDL into macrophages may be an important mechanism of foam cell formation and contributor to atherosclerosis development.

Sortilin regulates progranulin action in castration-resistant prostate cancer cells.

The growth factor progranulin is as an important regulator of transformation in several cellular systems. We have previously demonstrated that progranulin acts as an autocrine growth factor and stimulates motility, proliferation, and anchorage-independent growth of castration-resistant prostate cancer cells, supporting the hypothesis that progranulin may play a critical role in prostate cancer progression. However, the mechanisms regulating progranulin action in castration-resistant prostate cancer cells have not been characterized. Sortilin, a single-pass type I transmembrane protein of the vacuolar protein sorting 10 family, binds progranulin in neurons and negatively regulates progranulin signaling by mediating progranulin targeting for lysosomal degradation. However, whether sortilin is expressed in prostate cancer cells and plays any role in regulating progranulin action has not been established. Here, we show that sortilin is expressed at very low levels in castration-resistant PC3 and DU145 cells. Significantly, enhancing sortilin expression in PC3 and DU145 cells severely diminishes progranulin levels and inhibits motility, invasion, proliferation, and anchorage-independent growth. In addition, sortilin overexpression negatively modulates Akt (protein kinase B, PKB) stability. These results are recapitulated by depleting endogenous progranulin in PC3 and DU145 cells. On the contrary, targeting sortilin by short hairpin RNA approaches enhances progranulin levels and promotes motility, invasion, and anchorage-independent growth. We dissected the mechanisms of sortilin action and demonstrated that sortilin promotes progranulin endocytosis through a clathrin-dependent pathway, sorting into early endosomes and subsequent lysosomal degradation. Collectively, these results point out a critical role for sortilin in regulating progranulin action in castration-resistant prostate cancer cells, suggesting that sortilin loss may contribute to prostate cancer progression.

σ1B adaptin regulates adipogenesis by mediating the sorting of sortilin in adipose tissue

ABSTRACT Here, we describe altered sorting of sortilin in adipocytes deficient for the σ1B-containing AP-1 complex, leading to the inhibition of adipogenesis. The AP-1 complex mediates protein sorting between the trans-Golgi network and endosomes. Vertebrates express three AP1 σ1 subunit isoforms – σ1A, σ1B and σ1C (also known as AP1S1, AP1S2 and AP1S3, respectively). σ1B-deficient mice display impaired recycling of synaptic vesicles and lipodystrophy. Here, we show that sortilin is overexpressed in adipose tissue from σ1B−/− mice, and that its overexpression in wild-type cells is sufficient to suppress adipogenesis. σ1B-specific binding of sortilin requires the sortilin DxxD-x12-DSxxxL motif. σ1B deficiency does not lead to a block of sortilin transport out of a specific organelle, but the fraction that reaches lysosomes is reduced. Sortilin binds to the receptor DLK1, an inhibitor of adipocyte differentiation, and the overexpression of sortilin prevents DLK1 downregulation, leading to enhanced inhibition of adipogenesis. DLK1 and sortilin expression are not increased in the brain tissue of σ1B−/− mice, although this is the tissue with the highest expression of σ1B and sortilin. Thus, adipose-tissue-specific and σ1B-dependent routes for the transport of sortilin exist and are involved in the regulation of adipogenesis and adipose-tissue mass.

The Hypercholesterolemia-Risk Gene SORT1 Facilitates PCSK9 Secretion

Circulating PCSK9 destines low-density lipoprotein receptor for degradation in lysosomes, resulting in increased LDL cholesterol. Accordingly, it is an attractive drug target for hypercholesterolemia, and results from clinical trials are promising. While the physiological role of PCSK9 in cholesterol metabolism is well described, its complex mechanism of action remains poorly understood, although it is known to depend on intracellular trafficking. We here identify sortilin, encoded by the hypercholesterolemia-risk gene SORT1, as a high-affinity sorting receptor for PCSK9. Sortilin colocalizes with PCSK9 in the trans-Golgi network and facilitates its secretion from primary hepatocytes. Accordingly, sortilin-deficient mice display decreased levels of circulating PCSK9, while sortilin overexpression in the liver confers increased plasma PCSK9. Furthermore, circulating PCSK9 and sortilin were positively correlated in a human cohort of healthy individuals, suggesting that sortilin is involved in PCSK9 secretion in humans. Taken together, our findings establish sortilin as a critical regulator of PCSK9 activity.

σ1B-adaptin sorts sortilin in adipose tissue regulating adipogenesis

Here, we describe altered sorting of sortilin in adipocytes deficient for the σ1B-containing AP-1 complex, leading to the inhibition of adipogenesis. The AP-1 complex mediates protein sorting between the trans-Golgi network and endosomes. Vertebrates express three AP1 σ1 subunit isoforms - σ1A, σ1B and σ1C (also known as AP1S1, AP1S2 and AP1S3, respectively). σ1B-deficient mice display impaired recycling of synaptic vesicles and lipodystrophy. Here, we show that sortilin is overexpressed in adipose tissue from σ1B(-/-) mice, and that its overexpression in wild-type cells is sufficient to suppress adipogenesis. σ1B-specific binding of sortilin requires the sortilin DxxD-x12-DSxxxL motif. σ1B deficiency does not lead to a block of sortilin transport out of a specific organelle, but the fraction that reaches lysosomes is reduced. Sortilin binds to the receptor DLK1, an inhibitor of adipocyte differentiation, and the overexpression of sortilin prevents DLK1 downregulation, leading to enhanced inhibition of adipogenesis. DLK1 and sortilin expression are not increased in the brain tissue of σ1B(-/-) mice, although this is the tissue with the highest expression of σ1B and sortilin. Thus, adipose-tissue-specific and σ1B-dependent routes for the transport of sortilin exist and are involved in the regulation of adipogenesis and adipose-tissue mass.

Abstract 444: The Liver-Specific Role of Sortilin in VLDL Secretion

Introduction Sortilin, the protein product of the SORT1 gene, is a multi-ligand sorting receptor involved in Golgi to lysosome trafficking that is expressed in multiple tissues. Genome-wide association studies have shown an association between elevated SORT1 expression in human liver and a reduction in low-density lipoprotein cholesterol (LDL-C) and in myocardial infarction (MI) risk. Our lab has previously shown liver specific sortilin overexpression in mice reduces LDL by facilitating LDL plasma clearance and by promoting the lysosomal degradation of very-low density lipoprotein (VLDL). Interestingly, we and others have also shown that total body Sort1 deficiency in mice is associated with a paradoxical reduction in VLDL secretion (48%, P = 0.004). One major difference is that the overexpression studies were hepatic specific whereas the loss-of-function studies were in total body knockout (KO) mice. Methods/Results We used liver-specific Sort1 conditional KO mice and reconstitution of hepatic Sort1 in total body KO mice to further probe the specific role of hepatic sortilin in VLDL secretion. Sort1 fl/fl mice were injected with a control adeno-associated virus (AAV) or an AAV expressing Cre recombinase driven by a liver specific promoter and in vivo VLDL secretion studies were performed. Liver-specific Sort1 deletion reduced VLDL secretion by 51% (P = 0.005). We also used AAV to test the hypothesis that sortilin serves as a chaperone to facilitate VLDL secretion. Sort1 reconstitution in total body Sort1 KO mice reduced VLDL secretion by 29% (P = 0.002). Interestingly, expression of a sortilin mutant that cannot traffic to the lysosome but does traffic to the plasma membrane increased VLDL secretion by 88% (P = 0.001). Conclusion These findings suggest that liver-specificity is not responsible for the paradoxical reduction in secretion seen in the total body Sort1 KO mouse, which is reproduced in the hepatic-specific sortilin KO mouse. The differential effects of wild-type and lysosomal defective sortilin on VLDL secretion and are consistent with a model in which sortilin serves a dual function in VLDL trafficking, with low levels of sortilin facilitating VLDL export and higher levels of sortilin promoting pre-secretory lysosomal VLDL degradation.

Abstract 5: Sortilin Regulates Hepatic VLDL Secretion and LDL Uptake in a Lysosome-Dependent Manner

Sortilin, the protein product of the SORT1 gene, is a multi-ligand sorting receptor involved in Golgi to lysosome and plasma membrane to lysosome protein trafficking. Genome wide association studies for lipid traits have identified the 1p13 locus harboring the SORT1 gene as strongly associated both with plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) risk in humans. Adeno-associated virus (AAV)-mediated hepatic sortilin overexpression in LDL receptor deficient mice reduced plasma cholesterol by 30% at two weeks ( n = 6 mice per group, P = 0.02), with a concomitant reduction in LDL-C. In vivo VLDL production studies demonstrated a 50% reduction in the VLDL triglyceride secretion rate ( P = 0.007) and a 50% reduction in apoB secretion ( P = 0.02) with sortilin overexpression. In vivo LDL turnover studies demonstrated a 3-fold increase in the LDL fractional catabolic rate (FCR) with sortilin overexpression ( n = 6 mice per group, P = 0.00002). Sortilin deficiency both alone and on an LDL receptor deficient background led to a 40% and 50% reduction in FCR ( n = 6 mice per group, P = 0.002 and P = 0.01). The effect of sortilin on both VLDL secretion and LDL turnover is dependent on the ability of sortilin to traffic to the lysosome, as sortilin mutants that cannot traffic to the lysosome do not affect VLDL secretion or LDL uptake in vivo or in vitro . Surface plasmon reasonance demonstrated a high affinity interaction between sortilin and the apoB in LDL particles at physiological pH with a K d of ∼2 nM, and this affinity virtually disappears at the acidic lysosomal pH. In sum, these data are consistent with a model in which sortilin binds apoB-containing lipoprotein particles in the Golgi apparatus and at the plasma membrane and traffics them to the endolysosomal compartment for degradation, thereby reducing VLDL secretion and facilitating LDL uptake, explaining the strong association of hepatic sortilin overexpression in humans with reduced plasma cholesterol.

Sortilin Associates with Transforming Growth Factor-β Family Proteins to Enhance Lysosome-mediated Degradation

Transforming growth factor (TGF)-β family proteins are synthesized as precursors that are cleaved to generate an active ligand. Previous studies suggest that TGF-β activity can be controlled by lysosomal degradation of both precursor proteins and ligands, but how these soluble proteins are trafficked to the lysosome is incompletely understood. The current studies show that sortilin selectively co-immunoprecipitates with the cleaved prodomain and/or precursor form of TGF-β family members. Furthermore, sortilin co-localizes with, and enhances accumulation of a nodal family member in the Golgi. Co-expression of sortilin with TGF-β family members leads to decreased accumulation of precursor proteins and cleavage products and this is attenuated by lysosomal, but not proteosomal inhibitors. In Xenopus embryos, overexpression of sortilin leads to a decrease in phospho-Smad2 levels and phenocopies loss of nodal signaling. Conversely, down-regulation of sortilin expression in HeLa cells leads to an up-regulation of endogenous bone morphogenic protein pathway activation, as indicated by an increase in phospho-Smad1/5/8 levels. Our results suggest that sortilin negatively regulates TGF-β signaling by diverting trafficking of precursor proteins to the lysosome during transit through the biosynthetic pathway.

BACE1 Retrograde Trafficking Is Uniquely Regulated by the Cytoplasmic Domain of Sortilin

BACE1 (β-site β-amyloid precursor protein (APP)-cleaving enzyme 1) mediates the first proteolytic cleavage of APP, leading to amyloid β-peptide (Aβ) production. It has been reported that BACE1 intracellular trafficking, in particular endosome-to-TGN sorting, is mediated by adaptor complexes, such as retromer and Golgi-localized γ-ear-containing ARF-binding proteins (GGAs). Here we investigated whether sortilin, a Vps10p domain-sorting receptor believed to participate in retromer-mediated transport of select membrane cargoes, contributes to the subcellular trafficking and activity of BACE1. Our initial studies revealed increased levels of sortilin in post-mortem brain tissue of AD patients and that overexpression of sortilin leads to increased BACE1-mediated cleavage of APP in cultured cells. In contrast, RNAi suppression of sortilin results in decreased BACE1-mediated cleavage of APP. We also found that sortilin interacts with BACE1 and that a sortilin construct lacking its cytoplasmic domain, which contains putative retromer sorting motifs, remains bound to BACE1. However, expression of this truncated sortilin redistributes BACE1 from the trans-Golgi network to the endosomes and substantially reduces the retrograde trafficking of BACE1. Site-directed mutagenesis and chimera experiments reveal that the cytoplasmic tail of sortilin, but not those from other VPS10p domain receptors (e.g. SorCs1b and SorLA), plays a unique role in BACE1 trafficking. Our studies suggest a new function for sortilin as a modulator of BACE1 retrograde trafficking and subsequent generation of Aβ.