SorLA Expression Research Articles

The Alzheimer's disease gene SORL1 regulates lysosome function in human microglia.

The SORL1 gene encodes the sortilin related receptor protein SORLA, a sorting receptor that regulates endo-lysosomal trafficking of various substrates. Loss of function variants in SORL1 are causative for Alzheimer's disease (AD) and decreased expression of SORLA has been repeatedly observed in human AD brains. SORL1 is highly expressed by microglia, the tissue resident immune cells of the brain. Loss of SORLA leads to enlarged lysosomes in hiPSC-derived microglia like cells (hMGLs). However, whether SORLA deficiency contributes to microglia dysfunction and how this is relevant to AD is not known. In this study, we show that loss of SORLA results in decreased lysosomal degradation and lysosomal enzyme activity due to altered trafficking of lysosomal enzymes in hMGLs. Furthermore, lysosomal exocytosis, an important process involved in immune responses and cellular signaling, is also impaired in SORL1 deficient microglia. Phagocytic uptake of fibrillar amyloid beta 1-42 and synaptosomes is increased in SORLA deficient hMGLs, but due to reduced lysosomal degradation, these substrates aberrantly accumulate in lysosomes. Overall, these data highlight the microglial endo-lysosomal network as a potential novel pathway through which SORL1 may increase AD risk and contribute to development of AD. Additionally, our findings may inform development of novel lysosome and microglia associated drug targets for AD.

Morphological profiling in human neural progenitor cells classifies hits in a pilot drug screen for Alzheimer's disease.

Alzheimer's disease accounts for 60-70% of dementia cases. Current treatments are inadequate and there is a need to develop new approaches to drug discovery. Recently, in cancer, morphological profiling has been used in combination with high-throughput screening of small-molecule libraries in human cells in vitro. To test feasibility of this approach for Alzheimer's disease, we developed a cell morphology-based drug screen centred on the risk gene, SORL1 (which encodes the protein SORLA). Increased Alzheimer's disease risk has been repeatedly linked to variants in SORL1, particularly those conferring loss or decreased expression of SORLA, and lower SORL1 levels are observed in post-mortem brain samples from individuals with Alzheimer's disease. Consistent with its role in the endolysosomal pathway, SORL1 deletion is associated with enlarged endosomes in neural progenitor cells and neurons. We, therefore, hypothesized that multi-parametric, image-based cell phenotyping would identify features characteristic of SORL1 deletion. An automated morphological profiling method (Cell Painting) was adapted to neural progenitor cells and used to determine the phenotypic response of SORL1-/- neural progenitor cells to treatment with compounds from a small internationally approved drug library (TargetMol, 330 compounds). We detected distinct phenotypic signatures for SORL1-/- neural progenitor cells compared to isogenic wild-type controls. Furthermore, we identified 16 compounds (representing 14 drugs) that reversed the mutant morphological signatures in neural progenitor cells derived from three SORL1-/- induced pluripotent stem cell sub-clones. Network pharmacology analysis revealed the 16 compounds belonged to five mechanistic groups: 20S proteasome, aldehyde dehydrogenase, topoisomerase I and II, and DNA synthesis inhibitors. Enrichment analysis identified DNA synthesis/damage/repair, proteases/proteasome and metabolism as key pathways/biological processes. Prediction of novel targets revealed enrichment in pathways associated with neural cell function and Alzheimer's disease. Overall, this work suggests that (i) a quantitative phenotypic metric can distinguish induced pluripotent stem cell-derived SORL1-/- neural progenitor cells from isogenic wild-type controls and (ii) phenotypic screening combined with multi-parametric high-content image analysis is a viable option for drug repurposing and discovery in this human neural cell model of Alzheimer's disease.

Functional characterization of SORL1 variants in cell-based assays to investigate variant pathogenicity.

SORLA, the protein encoded by the SORL1 gene, has an important role in recycling cargo proteins to the cell surface. While SORLA loss-of-function variants occur almost exclusively in Alzheimer's disease cases, the majority of SORL1 variants are missense variants that are individually rare and can have individual mechanisms how they impair SORLA function as well as have individual effect size on disease risk. However, since carriers mostly come from small pedigrees, it is challenging to determine variant penetrance, leaving clinical significance associated with most missense variants unclear. In this article, we present functional approaches to evaluate the pathogenicity of a SORL1 variant, p.D1105H. First, we generated our mutant receptor by inserting the D1105H variant into the full-length SORLA-WT receptor. Then using western blot analysis we quantified the effect of the mutation on maturation and shedding of the receptor for transfected cells, and finally applied a flow cytometry approach to quantify SORLA expression at the cell surface. The results showed decreased maturation, decreased shedding, and decreased cell surface expression of D1105H compared with wild-type SORLA. We propose how these approaches can be used to functionally assess the pathogenicity of SORL1 variants in the future. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

SorLA in astrocytes regulates blood-brain barrier integrity

The brain`s homeostasis depends heavily on the integrity of the blood-brain barrier (BBB). Astrocytes are an essential part of the BBB in modulating and maintaining the barrier properties of the brain endothelial cells (BECs). Despite decades of research, the elements of glial regulation are not fully elucidated. SorLA/SorL1/LR11, a multifunctional receptor, is the most composite member of the Vps10p domain receptor family. In this study, we characterize the expression and function of SorLA in the cells of the BBB. The applied in vitro approaches describe BBB functions in primary cells isolated from wild-type and Sorl1−/− knock-out rats. Here, we present that Sorl1 gene is highly expressed in wild-type astrocytes but not in BECs and pericytes. Furthermore, we show that SorLA in astrocytes is an important regulator of the BBB’s tightness. The primary rat BBB models where astrocytes lack SorLA protein proved leaky, which correlated well with the decrease in claudin-5 tight junction protein in BECs. Meanwhile, other junctional proteins, i.e., occludin and zonula occludens-1 are unaffected. Collectively, these data suggest that the absence of SorLA in astrocytes affects the tight junctions of BECs, thereby disturbing the BBB. Our results add another layer to understanding astrocyte-endothelial interactions in the healthy and diseased BBB.

LncRNA NONMMUT055714 acts as the sponge of microRNA-7684-5p to protect against postoperative cognitive dysfunction.

Postoperative cognitive dysfunction (POCD) is a neurological complication of surgery especially common in elderly patients. In this study, we investigated the role of NONMMUT055714 in POCD via regulation of miR-7684-5p. In a POCD mouse model, we induced overexpression of NONMUTT055714 via transfection of lentivrus into the hippocampus, and used the Morris water maze for assessment of cognitive function. Silencing of NONMUTT055714 and miR-7684-5p was induced in primary hippocampal neurons to observe the effects of these regulatory RNAs on cellular processes. Bioinformatics analysis and a double luciferase reporter experiment were performed to further explore the relationship between NONMMUT055714, miR-7684-5p, and SORLA. Cell and animal rescue experiments were performed to verify the ability of miR-7684-5p to reverse the protective effects of NONMMUT055714 overexpression in POCD. We observed that NONMMUT055714 has decreased expression in the POCD mouse model. Overexpression of NONMMUT055714 protected against cognitive impairment of the POCD mouse model in vivo. We identified miR-7684-5p as a NONMMUT055714-related miRNA and in turn as an upstream regulator of SORLA. We found that NONMMUT055714 downregulation is associated with decreased SORLA, increased Aβ and p-tau expression, increased inflammatory biomarkers, increased markers of oxidative stress, and increased neuronal apoptosis in vitro. The effects of NONMMUT055714 downregulation were reversed by silencing miR-7684-5p in vitro and in vivo. Taken together, our findings suggest that NONMMUT055714 is protective against the development of POCD via its function as a ceRNA (or miRNA sponge) in the regulation of miR-7684-5p and SORLA. We therefore propose NONMMUT055714 as a novel target for the investigation and prevention of POCD.

SORLA Expression in Synaptic Plexiform Layers of Mouse Retina.

Sorting protein-related receptor containing LDLR class A repeats (SORLA; also known as LR11) exerts intraneuronal trafficking functions in the central nervous system. Recently, involvement of SORLA in retinogenesis was proposed, but no studies have examined yet in detail the expression pattern of this sorting receptor in the retina. Here, we provide a spatio-temporal characterization of SORL1 mRNA and its translational product SORLA in the postnatal mouse retina. Using stereological analysis, we confirmed previous studies showing that receptor depletion in knockout mice significantly reduces the number of cells in the inner nuclear layer (INL), suggesting that functional SORLA expression is essential for the development of this retinal strata. qPCR and Western blot analyses showed that SORL1/SORLA expression peaks at postnatal day 15, just after eye opening. Interestingly, we found that transcripts are somatically located in several neuronal populations residing in the INL and the ganglion cell layer, whereas SORLA protein is also present in the synaptic plexiform layers. In line with receptor expression in dendritic terminals, we found delayed stratification of the inner plexiform layer in knockout mice, indicating an involvement of SORLA in neuronal connectivity. Altogether, these data suggest a novel role of SORLA in synaptogenesis. Receptor dysfunctions may be implicated in morphological and functional impairments of retinal inner layer formation associated with eye disorders.

INVESTIGATING THE ROLE OF SORTILIN-RELATED RECEPTOR (SORL1/SORLA) IN HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED NEURAL CELLS AS A THERAPEUTIC TARGET FOR ALZHEIMER’S DISEASE

Recent compelling genetic evidence has identified genes involved in endocytosis and vesicular trafficking including SORL1 as AD risk genes. Decreased expression of SORLA has been repeatedly observed in postmortem human AD brains especially in late onset sporadic AD (SAD) which is the most common form of AD. Eventually, several studies linked SORLA expression with production and deposition of the pathogenic protein β amyloid peptide which is one of the hallmarks of AD pathogenesis. However, most of these studies were done using mouse models and non-human cell lines and role of SORLA in amyloid β production in human neural cells remains unclear. SORL1 was overexpressed in clonal populations of human pluripotent stem cells (hiPSCs) using the piggybac transposon system. Control and SORL1overexpressed hiPSCs were then differentiated into neural cells. Amount of Aβ-40 and Aβ-42 released was measured using ELISA in hiPSC derived neural cells. Additionally, intra-cellular trafficking was examined using immunocytochemistry and cell biology assays. Specifically, to assess transport into lysosomes, pH sensitive fluorescent dye Phrodo conjugated with Aβ was used. Overexpression of SORL1 in hiPSC derived human neural cells resulted in decreased Aβ-40 and Aβ-42 released into media as measured by ELISA. This decrease can be caused by a possible increase in Aβ transport into lysosomes for degradation. In support of this idea, increased Aβ transport into lysosomes was observed in SORL1 overexpressed neural cells as compared to control cells using the Phrodo assay. Decreased Aβ release could also result from increased degradation of Amyloid precursor protein (APP). In support of this hypothesis, we showed increased colocalization of APP with a lysosomal marker, Lysosome associated membrane protein (LAMP1) using immunocytochemistry. Overall, these results suggest that overexpression of SORL1 could lead to decreased amyloid β production in part due to increased transport of Aβ and APP into lysosomes for degradation in human neural cells. Our findings reveal a previously unidentified role of SORL1 in hiPSC derived neural cells, information that may be useful in exploring not just SORLA, but other sorting receptors expressed in human neurons as therapeutic targets for AD.

Amyloidosis causes downregulation of SorLA, SorCS1 and SorCS3 expression in mice.

Accumulation of β-amyloid peptide (Aβ) is regarded as a primary cause of Alzheimer's disease (AD). Aβ is derived by sequential cleavage of the amyloid precursor protein (APP). Alterations in the subcellular targeting of APP are thought to affect the degree of Aβ production. Sorting receptors, such as SorLA, convey subcellular targeting of APP. Dysfunction of SorLA, and likely of the related receptors SorCS1 and SorCS3, cause AD. Nevertheless, disease progression could also provoke altered expression of the receptors. Here, we assessed if Aβ plaque formation promotes altered expression of SorLA, SorCS1 and SorCS3. We analyzed transcript levels during aging and after amyloidosis in brain areas characterized by early amyloid plaque formation in an AD mouse model (APPPS1) and wild types. We observed stable expression levels during aging (1-12 months). After plaque formation, SorCS1 and SorLA expression were markedly reduced in the frontal cerebral cortex and to a minor extent in the hippocampus, whereas SorCS3 expression was solely reduced in the frontal cerebral cortex. Our results indicate that disease progression, associated with Aβ accumulation, can negatively regulate expression of the receptors.

Biochemical and cognitive effects of docosahexaenoic acid differ in a developmental and SorLA dependent manner

Beneficial effects of omega-3 fatty acid intake on cognition are under debate as some studies show beneficial effects while others show no effects of omega-3 supplementation. These inconsistencies may be a result of inter-individual response variations, potentially caused by gene and diet interactions. SorLA is a multifunctional receptor involved in ligand trafficking including lipoprotein lipase and amyloid precursor protein. Decreased SorLA levels have been correlated to Alzheimer’s disease, and omega-3 fatty acid supplementation is known to increase SorLA expression in neuronal cell lines and mouse models. We therefore addressed potential correlations between Sorl1 and dietary omega-3 in SorLA deficient mice (Sorl1−/−) and controls exposed to diets supplemented with or deprived of omega-3 during their entire development and lifespan (lifelong) or solely from the time of weaning (post weaning).Observed diet-induced effects were only evident when exposed to lifelong omega-3 supplementation or deprivation as opposed to post weaning exposure only. Lifelong exposure to omega-3 supplementation resulted in impaired spatial learning in Sorl1−/− mice. The vitamin C antioxidant capacity in the brains of Sorl1−/− mice was reduced, but reduced glutathione and vitamin E levels were increased, leaving the overall antioxidant capacity of the brain inconclusive. No gross morphological differences of hippocampal neurons were found to account for the altered behavior.We found a significant adverse effect in cognitive performance by combining SorLA deficiency with lifelong exposure to omega-3. Our results stress the need for investigations of the underlying molecular mechanisms to clarify the precise circumstances under which omega-3 supplementation may be beneficial.

SORLA facilitates insulin receptor signaling in adipocytes and exacerbates obesity.

In humans, genetic variation of sortilin-related receptor, L(DLR class) A repeats containing (SORL1), which encodes the intracellular sorting receptor SORLA, is a major genetic risk factor for familial and sporadic forms of Alzheimer's disease. Recent GWAS analysis has also associated SORL1 with obesity in humans and in mouse models, suggesting that this receptor may play a role in regulating metabolism. Here, using mouse models with genetic loss or tissue-specific overexpression of SORLA as well as data from obese human subjects, we observed a gene-dosage effect that links SORLA expression to obesity and glucose tolerance. Overexpression of human SORLA in murine adipose tissue blocked hydrolysis of triacylglycerides and caused excessive adiposity. In contrast, Sorl1 gene inactivation in mice accelerated breakdown of triacylglycerides in adipocytes and protected animals from diet-induced obesity. We then identified the underlying molecular mechanism whereby SORLA promotes insulin-induced suppression of lipolysis in adipocytes. Specifically, we determined that SORLA acts as a sorting factor for the insulin receptor (IR) that redirects internalized receptor molecules from endosomes to the plasma membrane, thereby enhancing IR surface expression and strengthening insulin signal reception in target cells. Our findings provide a molecular mechanism for the association of SORL1 with human obesity and confirm a genetic link between neurodegeneration and metabolism that converges on the receptor SORLA.

Sex moderates the effects of the Sorl1 gene rs2070045 polymorphism on cognitive impairment and disruption of the cingulum integrity in healthy elderly.

The SORL1 rs2070045 polymorphism was reported to be associated with SorLA expression in the brain and the risk of late-onset Alzheimer's disease (AD). However, the influence of this polymorphism on cognitive functioning is likely to be moderated by sex. This study aimed to examine the sex moderation on the effects of rs2070045 on neuropsychological performance and the cingulum integrity in Chinese Han population. In this study, 780 non-demented older adults completed a battery of neuropsychological scales. Diffusion tensor images (DTI) of 126 subjects were acquired. We adopted the atlas-based segmentation strategy for calculating the DTI indices of the bilateral cingulum and cingulum hippocampal part for each subject. We used a multivariate analysis of variance (MANOVA) to compare the cognitive performance and DTI differences between the rs2070045 genotype. Controlling for age, education, and the APOE ɛ4 status, the influence of sex on the effects of the rs2070045 polymorphism on executive function was observed. We also found an interaction between sex and the rs2070045 polymorphism on the white matter (WM) microstructure of the left cingulum hippocampal part. Furthermore, the mean diffusivity and axial diffusivity of the tract were associated with Trail Making Test performance in T/T men. These results hint that sex moderates the association between the rs2070045 polymorphism and executive function, as well as the WM integrity of the left cingulum hippocampal part. Our findings underscore the importance of considering the influence of sex when examining the candidate genes for cognitive abilities and AD.

A study of the lipoprotein lipase inhibitory mechanism ofPoncirus trifoliatawater extracts

Purpose: Poncirus trifoliata has been reported to have anti-inflammatory, antioxidant, and immune activities. However, its anti-obesity activity and the mechanism by which the water extract of dried, immature fruit of Poncirus trifoliata (PF-W) acts are not clear. This study suggests a potential mechanism associated with the anti-obesity activity of PF-W. Methods: We measured the effect of PF-W on lipoprotein lipase (LPL) regulation using enzyme-linked immunosorbent assay (ELISA) and an activity assay. The LPL regulation mechanism was examined by reverse transcription polymerase chain reaction (RTPCR) to measure the mRNA expression of biomarkers related to protein transport and by western blot for analysis of the protein expression of the transcription factor CCAAT-enhancer-binding protein (C/EBPβ). Results: The total polyphenol and flavonoid content of PF-W was 52.15 ± 4.02 and 6.56 ± 0.47 mg/g, respectively. PF-W treatment decreased LPL content in media to 58 ± 5% of that in control adipocyte media, and increased LPL content to 117 ± 3.5% of that in control adipocytes, but did not affect the mRNA expression of LPL. PF-W also increased the mRNA expression of sortilin-related receptor (SorLA), a receptor that induces endocytosis and intracellular trafficking of LPL, in a concentration- and timedependent manner. Finally, cell fractionation revealed that PF-W treatment induced the expression of C/EBPβ, a SorLA transcription factor, in the nuclei of 3T3-L1 adipocytes. Conclusion: The LPL secretion and activity assay showed PF-W to be an LPL secretion inhibitor, and these results suggest the potential mechanism of PF-W involving inhibition of LPL secretion through C/EBPβ-mediated induction of SorLA expression.

REGULATION OF SORLA IN GENERAL AND IN ALZHEIMER'S DISEASE

The Sortillin-related receptor (sorLA) is involved in cellular trafficking and processing of the Amyloid precursor protein (APP). A decrease in sorLA expression has been identified in brain tissue from patients suffering from Alzheimer's disease (AD), suggesting that sorLA may be a key regulator of Aβ production and that sorLA activity protects against AD. Furthermore, association between Single Nucleotide Polymorphisms (SNPs) from the gene encoding sorLA (gene name: SORL1) and AD was recently identified; suggesting that transcription of SORL1 could be linked to the development of AD. The mechanism behind the regulation of SORL1 expression in general and how AD-associated SNPs influence sorLA levels during AD, has not yet been characterized. To identify cis - and trans -regulatory factors important for the expression of sorLA, we studied the proximal promoter of SORL1 using in silico analysis and generated promoter constructs for Dual Luciferase Assay. We precipitated proteins from nuclear cell extracts binding to the SORL1 promoter using Streptavidin magnetic beads and biotinylated oligomers. By introduction of constructs for over expression and siRNA molecules for under expression of transcription factors, we determined their effects on sorLA expression by qPCR and Western blotting. Using site-directed mutagenesis we mapped regulatory cis-element binding sites for verified transcription factors. We have identified regions in the SORL1 promoter, important for induction as well as repression of sorLA expression. We identified several trans cription factors that is directly involved in the regulation of sorLA expression, and mapped their binding sites within the proximal SORL1 promoter. Finally, we determined the level of these transcription factors in brain tissue from patients with Alzheimer's disease. We have investigated the regulation of sorLA expression and identified several cis - and trans-regulatory elements important for the proper expression of sorLA. These studies may help to elucidate the mechanisms underlying the observ ed down regulation of sorLA and the linkage to disease onset of SORL1 SNPs in AD patients.

The role of SorLA in neurotrophic activity and complex behavior

SorLA binds amyloid precursor protein (APP) in the trans-Golgi network and protects it against proteolysis by both the alpha- and beta-secretases, preventing the generation of toxic amyloid-beta peptide. We have recently found that SorLA also affects neuronal function in a different manner by acting as sorting receptor for the neurotrophic factor GDNF and its receptors GFRα1 and RET, directing them from the cell surface to endosomes. Through this mechanism, GDNF is targeted to lysosomes and degraded while GFRα1 and RET recycles, creating an efficient GDNF clearance pathway. SorLA thereby influences a number of neuronal systems known to depend on GDNF activity including the dopaminergic system and the hippocampus. Accordingly, mice with altered SorLA expression exhibit altered reward and memory function, and a behavioral phenotype characterized by altered anxiety, hyperactivity, and response to psychostimulants.

SORLA-Mediated Trafficking of TrkB Enhances the Response of Neurons to BDNF

Stimulation of neurons with brain-derived neurotrophic factor (BDNF) results in robust induction of SORLA, an intracellular sorting receptor of the VPS10P domain receptor gene family. However, the relevance of SORLA for BDNF-induced neuronal responses has not previously been investigated. We now demonstrate that SORLA is a sorting factor for the tropomyosin-related kinase receptor B (TrkB) that facilitates trafficking of this BDNF receptor between synaptic plasma membranes, post-synaptic densities, and cell soma, a step critical for neuronal signal transduction. Loss of SORLA expression results in impaired neuritic transport of TrkB and in blunted response to BDNF in primary neurons; and it aggravates neuromotoric deficits caused by low BDNF activity in a mouse model of Huntington’s disease. Thus, our studies revealed a key role for SORLA in mediating BDNF trophic signaling by regulating the intracellular location of TrkB.

SorLA Deficiency Dissects Amyloid Pathology from Tau and Cholinergic Neurodegeneration in a Mouse Model of Alzheimer's Disease

Sortilin-related receptor with A-type repeats (SorLA, also known as LR11) has been implicated in Alzheimer's disease (AD). Thus, genetic studies associated SorLA gene variants with the risk of sporadic AD. Also, in vitro and in vivo studies showed that SorLA impairs processing of the amyloid-β protein precursor (AβPP) to amyloid-β. In particular, it has been found that loss of SorLA accelerates senile plaque deposition in mouse models overexpressing mutant forms of human AβPP and presenilin 1. Here we tested the possibility that SorLA deficiency also interferes with behavioral and neuropathological endpoints in an alternative murine AD model, the AD10 anti-nerve growth factor (NGF) mouse, in which amyloid-β accumulation derives from the altered processing of endogenous AβPP. In addition to alterations in AβPP processing, AD10 mice also show cholinergic deficit and tau hyperphosphorylation resulting in behavioral deficits in learning and memory paradigms. We found that the loss of SorLA not only exacerbates early amyloid pathology but, at the same time, protects from cholinergic deficit and from early phospho-tau mislocalization. The results show that in the AD10 anti-NGF mouse model the AβPP processing-related aspects of neurodegeneration can be dissociated from those related to tau posttranslational processing and to cholinergic phenotypic maintenance by modulation of SorLA expression. We suggest that SorLA regulates different aspects of neurodegeneration in a complex way, supporting the hypothesis that SorLA expression might be critical not only for amyloid-related pathology but also for other cellular processes altered in AD.

SorLA in Glia: Shared Subcellular Distribution Patterns with Caveolin-1

SorLA is an established sorting and trafficking protein in neurons with demonstrated relevance to Alzheimer's disease (AD). It shares these roles with the caveolins, markers of membrane rafts microdomains. To further our knowledge on sorLA's expression and traffic, we studied sorLA expression in various cultured glia and its relation to caveolin-1 (cav-1), a caveolar microdomain marker. RT-PCR and immunoblots demonstrated sorLA expression in rat C6 glioma, primary cultures of rat astrocytes (PCRA), and human astrocytoma 1321N1 cells. PCRA were determined to express the highest levels of sorLA's message. Induction of differentiation of C6 cells into an astrocyte-like phenotype led to a significant decrease in sorLA's mRNA and protein expression. A set of complementary experimental approaches establish that sorLA and cav-1 directly or indirectly interact in glia: (1) co-fractionation in light-density membrane raft fractions of rat C6 glioma, PCRA, and human 1321N1 astrocytoma cells; (2) a subcellular co-localization distribution pattern in vesicular perinuclear compartments seen via confocal imaging in C6 and PCRA; (3) additional confocal analysis in C6 cells suggesting that the perinuclear compartments correspond to their co-localization in early endosomes and the trans-Golgi; and; (4) co-immunoprecipitation data strongly supporting their direct or indirect physical interaction. These findings further establish that sorLA is expressed in glia and that it shares its subcellular distribution pattern with cav-1. A direct or indirect cav-1/sorLA interaction could modify the trafficking and sorting functions of sorLA in glia and its proposed neuroprotective role in AD.

SorLA regulates the activity of lipoprotein lipase by intracellular trafficking

Many different tissues and cell types exhibit regulated secretion of lipoprotein lipase (LPL). However, the sorting of LPL in the trans Golgi network has not, hitherto, been understood in detail. Here, we characterize the role of SorLA (officially known as SorLA-1 or sortilin-related receptor) in the intracellular trafficking of LPL. We found that LPL bound to SorLA under neutral and acidic conditions, and in cells this binding mainly occurred in vesicular structures. SorLA expression changed the subcellular distribution of LPL so it became more concentrated in endosomes. From the endosomes, LPL was further routed to the lysosomes, which resulted in a degradation of newly synthesized LPL. Consequently, an 80% reduction of LPL activity was observed in cells that expressed SorLA. By analogy, SorLA regulated the vesicle-like localization of LPL in primary neuronal cells. Thus, LPL binds to SorLA in the biosynthetic pathway and is subsequently transported to endosomes. As a result of this SorLA mediated-transport, newly synthesized LPL can be routed into specialized vesicles and eventually sent to degradation, and its activity thereby regulated.

SorLA modulates atheroprotective properties of CLA by regulating monocyte migration

Objective We have previously shown that CLA induces regression of pre-established atherosclerotic lesions in apoE −/− mice. CLA is a known ligand of peroxisome proliferator activated receptors (PPARs) and it is postulated that CLA mediates its atheroprotective effects through activation of PPARs. Earlier work in our group identified the monocyte/macrophage cell as the primary cellular target of CLA. In this study we identified novel genes regulated by CLA during the regression of atherosclerosis and characterised a role for one of these, SorLA. SorLA is a member of the vacuolar protein sorting 10 protein (Vps10p) domain receptor family, which has structural homology with the LDLR family. Methods and results Expression of SorLA was identified with its Vps10p family member Sort1 by transcriptomic analysis of murine aorta following CLA-induced regression of atherosclerosis. Decreased expression of both receptors was confirmed by real-time PCR in the aorta of CLA-supplemented mice. SorLA protein expression was predominantly localised to monocyte/macrophage cells in the vasculature by immunohistochemistry. CLA and the PPAR-γ agonists, troglitazone, and 15-deoxy-prostaglandin (PG) J 2, decreased protein and RNA expression of SorLA in THP-1 monocytes; while pre-treatment with a PPAR-γ antagonist established a PPAR-γ dependent role for CLA regulation of SorLA. CLA inhibits monocyte migration. Consistent with a role for SorLA in mediating this response, overexpression of SorLA increased migration of THP-1 monocytes to monocyte chemoattractant protein-1 with a coincident increase in UPAR expression. Conclusion CLA may mediate its atheroprotective effects in part through reduced expression of SorLA and a resulting inhibition of monocyte migration in vitro.

SorLA: a membrane raft protein in glial cells

SorLA is a sorting protein in neurons with relevance to Alzheimer's disease (AD). It shares roles with the caveolins, markers of the lipid membrane rafts microdomains. To further our knowledge on sorLA's expression and traffic, we studied sorLA expression in various cultured glia and its relation to caveolin‐1 (cav1), a caveolar microdomain marker. RT‐PCR and immunoblots demonstrated sorLA expression in rat cells: C6 glioma, primary cultures astrocytes (PCRA) and 1321N1 human astrocytoma cells. Differentiation of C6 cells to an astrocyte phenotype (C6 diff) led to a significant decrease in sorLA's mRNA and protein expression. Sucrose density gradient revealed cofractionation of sorLA with cav1 in light density membrane raft fractions of undifferentiated C6, C6 diff and 1321N1 cells. Immunofluorescences analysis of C6 and PCRA revealed a cellular distribution akin to sorLA's staining pattern in neurons. In C6 and PCRA cells a significant percent of sorLA colocalized with cav1, mostly in perinuclear compartments, cytoplasmic vesicles and plasmalemma. These findings establish that sorLA is expressed in glia and is a membrane raft protein involved with cav1 in glial intracellular membrane microdomains trafficking. This study supports a role for glia and its caveolar membrane raft microdomains to the sorting functions of sorLA in the neurodegenerative cascade of AD, unveiling new venues for disease treatment.