Elsevier

Brain Research

Volume 1276, 18 June 2009, Pages 11-21
Brain Research

Research Report
A neuronal VLDLR variant lacking the third complement-type repeat exhibits high capacity binding of apoE containing lipoproteins

https://doi.org/10.1016/j.brainres.2009.04.030Get rights and content

Abstract

Very-low-density lipoprotein receptor (VLDLR) is a multi ligand apolipoprotein E (apoE) receptor and is involved in brain development through Reelin signaling. Different forms of VLDLR can be generated by alternative splicing. VLDLR-I contains all exons. VLDLR-II lacks an O-linked sugar domain encoded by exon 16, while VLDLR-III lacks the third complement-type repeat in the ligand binding domain encoded by exon 4. We quantitatively compared lipoprotein binding to human VLDLR variants and analyzed their mRNA expression in both human cerebellum and mouse brain. VLDLR-III exhibited the highest capacity in binding to apoE enriched β-VLDL in vitro and was more effective in removing apoE containing lipoproteins from the circulation than other variants in vivo. In human cerebellum, the major species was VLDLR-II, while the second most abundant species was a newly identified VLDLR-IV which lacks both exon 4 and 16. VLDLR-I was present at low levels. In adult mice, exon 4 skipping varied between 30 and 47% in different brain regions, while exon 16 skipping ranged by 51–76%. Significantly higher levels of VLDLR proteins were found in mouse cerebellum and cerebral cortex than other regions. The deletions of exon 4 and exon 16 frequently occurred in primary neurons, indicating that newly identified variant VLDLR-IV is abundant in neurons. In contrast, VLDLR mRNA lacking exon 4 was not detectable in primary astrocytes. Such cell type-specific splicing patterns were found in both mouse cerebellum and cerebral cortex. These results suggest that a VLDLR variant lacking the third complement-type repeat is generated by neuron-specific alternative splicing. Such differential splicing may result in different lipid uptake in neurons and astrocytes.

Introduction

The very-low-density-lipoprotein receptor (VLDLR) was first isolated as a member of the LDLR gene family that binds apolipoprotein E (apoE) containing lipoproteins. This receptor family has diverse roles beyond lipoprotein metabolism and development (Takahashi et al., 2004, Willnow et al., 2007). The phenotype of VLDLR deficiency in humans is heterogeneous. In the Hutterite population, VLDLR deficiency causes autosomal recessive cerebellar hypoplasia, a nonprogressive neurological disorder known as disequilibrium syndrome, which is characterized by moderate to profound mental retardation, delayed ambulation and ataxia (Boycott et al., 2005, Moheb et al., 2008). In humans with quadrupedal gait known as Unertan syndrome, VLDLR deficiency is associated with dysarthric speech, mental retardation, cerebrocerebellar hypoplasia as well as quadrupedal locomotion (Ozcelik et al., 2008b). However, it is debated whether VLDLR plays a key role in the transition from quadrupedal to bipedal locomotion in humans (Herz et al., 2008, Humphrey et al., 2008, Ozcelik et al., 2008a). The brain abnormality found in VLDLR deficiency is likely caused by impaired Reelin signaling. The development of the neocortex requires a coordinated migration of neurons in both radial and tangential directions to their final laminar positions. Reelin plays a critical role in the coordination of this migration via its binding to apoE receptor 2 (apoER2) and VLDLR (D'Arcangelo et al., 1999, Hiesberger et al., 1999) by providing a positional cue that determines the development of the normal cortical layering pattern (D'Arcangelo et al., 1995). The binding of Reelin to these receptors recruits the adaptor protein Dab1 through intracellular NPxY motif, which subsequently induces tyrosine phosphorylation of Dab1 and activates downstream events (Beffert et al., 2004). It has been reported that VLDLR mediates a stop signal for migrating neurons, while apoER2 plays an essential role for the migration of late generated neocortical neurons (Hack et al., 2007).

VLDLR gene is subjected to alternative splicing. The full-length VLDLR cDNA encoded by all exons is a type I VLDLR (Fig. 1) (Oka et al., 1994, Sakai et al., 1994, Takahashi et al., 1992). The first variant identified in a human monocytic leukemia cell line THP-1 is a type II lacking the O-linked sugar domain that is encoded by exon 16 (VLDLR-II) (Sakai et al., 1994). The second variant lacks the third complement-type cysteine-rich repeat in the ligand binding domain (VLDLR-III) (Jokinen et al., 1994). This domain is encoded by exon 4 and has been reported to be involved in rhinovirus binding (Verdaguer et al., 2004). The VLDLR-III appears to be brain-specific (Jokinen et al., 1994). In addition, a VLDLR cDNA clone lacking exon 9 that encodes 42 amino acids in the epidermal growth factor precursor homology domain has been reported in mice (Gafvels et al., 1994). β-migrating triglyceride-rich lipoproteins (β-VLDL) are enriched by apoE and are ligands for VLDLR (Takahashi et al., 1992). The uptake of β-VLDL by VLDLR-II has been reported to be relatively low compared with VLDLR-I (Iijima et al., 1998). Rapid degradation and secretion into the culture medium has been reported for VLDLR-II (Iijima et al., 1998, Magrane et al., 1999). The VLDLR is a multi ligand receptor and binds to a variety of ligands including proteinases, proteinase–inhibitor complexes (Argraves et al., 1995, Heegaard et al., 1995), lipoprotein lipase (Argraves et al., 1995) and viruses (Marlovits et al., 1998). Although VLDLR-I and VLDLR-II bind receptor-associated protein (RAP) and serine proteinase–inhibitor complexes with similar affinity (Heegaard et al., 1995, Martensen et al., 1997), VLDLR-III displayed lower binding of RAP but similar binding of urokinase-type plasminogen activator (uPA)/plasminogen activator inhibitor-1 or uPA/protease nexin-1 (Rettenberger et al., 1999). Lipoprotein binding to VLDLR-III, however, has not been characterized.

In this study, we analyzed the binding capacity of VLDLR variants to β-VLDL in vitro and tested their efficacy in lipoprotein uptake in mice. We found that VLDLR-III was more effective in both capacities than the other variants. These results led us to examine which variants are expressed in the human brain. We determined structures of VLDLR mRNA species in human cerebellum by RT-PCR cloning. We found that most VLDLR mRNA species in human cerebellum lacked exon 16 (VLDLR-III), but also found VLDLR mRNA species lacking exon 4 as well as exon 16 (VLDLR-IV). To determine whether alternative splicing of the VLDLR gene is brain region- or cell type-specific, we analyzed exon skipping by RNase protection assay (RPA) and protein expression by semi-quantitative immunoblot analysis in mice. We prepared primary neurons and astrocytes from mouse cerebral cortex and cerebellum and analyzed exon skipping by RT-PCR. VLDLR mRNA showed both exon 4 and 16 skipping in neurons suggesting that VLDLR-II and -IV are the major species in neurons, while the majority of VLDLR mRNA species in astrocytes contained exon 4 and were therefore VLDLR-I and -II. Finally, we analyzed developmental regulation of alternative splicing of mouse VLDLR gene. Unlike in human brain, exon skipping was not highly regulated in mouse brain during development and maturity.

Section snippets

VLDLR-III exhibited high capacity binding of β-VLDL

It has been reported that VLDLR-I and -II have a different binding capacity of β-VLDL (Iijima et al., 1998), but lipoprotein binding to VLDLR-III has not been examined. We constructed first generation adenoviral vectors expressing human VLDLR-I to -III (Ad-VLDLR) and examined their binding of apoE containing lipoproteins in vitro as well as in vivo. We used CHO-ldlA7 cells, which are CHO cells lacking LDLR. As LDLR is the major receptor for lipoprotein uptake in CHO cells, CHO-ldlA7 cells have

Discussion

VLDLR was initially thought to have a primary function via delivery of triglyceride-rich apoE containing lipoproteins into tissues that are active in fatty acid metabolism (Takahashi et al., 1992). However, VLDLR−/− mice displayed only a modest decrease in body mass index and adipose tissue mass in epididymal fat pads (Frykman et al., 1995), indicating a minor role of VLDLR in triglyceride metabolism. Instead, VLDLR was found to play a major role in the development of the brain via Reelin

Recombinant adenoviral vector

The cDNAs for the full-length human VLDLR (type I), VLDLR lacking exon 16 (type II) or exon 4 (type III) as described previously (Rettenberger et al., 1999). VLDLR cDNAs were subcloned into the BglII/ClaI sites of pAvCvSv and first generation Ads were generated as described (Kobayashi et al., 1996).

125I Labeling of β-VLDL

β-VLDL was isolated by ultracentrifugation from the plasma of adult female New Zealand White rabbits that were fed a diet of normal chow supplemented with 1% cholesterol (Roth et al., 1983). Blood

Acknowledgments

This work was supported by HL73144 (KO), P50-HL59314, P30-DK79638 (LC) and NS04884 (HCL). We thank Monty Krieger (MIT) for providing CHO-ldlA7 cells.

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    Current address: Department of Pharmacology, Dong-A University College of Medicine, Busan 602-714, South Korea.

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