Anton Roebroek - Academia.edu (original) (raw)

Papers by Anton Roebroek

Molecular Neurobiology

The low-density lipoprotein receptor-related protein-1 (LRP1) has a dual role in the metabolism o... more The low-density lipoprotein receptor-related protein-1 (LRP1) has a dual role in the metabolism of the amyloid precursor protein (APP). In cellular models, LRP1 enhances amyloid-β (Aβ) generation via APP internalization and thus its amyloidogenic processing. However, conditional knockout studies in mice define LRP1 as an important mediator for the clearance of extracellular Aβ from brain via cellular degradation or transcytosis across the blood-brain barrier (BBB). In order to analyze the net effect of LRP1 on production and clearance of Aβ in vivo, we crossed mice with impaired LRP1 function with a mouse model of Alzheimer's disease (AD). Analysis of Aβ metabolism showed that, despite reduced Aβ clearance due to LRP1 inactivation in vivo, less Aβ was found in cerebrospinal fluid (CSF) and brain interstitial fluid (ISF). Further analysis of APP metabolism revealed that impairment of LRP1 in vivo shifted APP processing from the Aβ-generating amyloidogenic cleavage by beta-secretase to the non-amyloidogenic processing by alpha-secretase as shown by a decrease in extracellular Aβ and an increase of soluble APP-α (sAPP-α). This shift in APP processing resulted in overall lower Aβ levels and a reduction in plaque burden. Here, we present for the first time clear in vivo evidence that global impairment of LRP1's endocytosis function favors non-amyloidogenic processing of APP due to its reduced internalization and subsequently, reduced amyloidogenic processing. By inactivation of LRP1, the inhibitory effect on Aβ generation overrules the simultaneous impaired Aβ clearance, resulting in less extracellular Aβ and reduced plaque deposition in a mouse model of AD.

Behavioural brain research, Jan 27, 2018

Mild traumatic brain injury (mTBI) can lead to diffuse neurophysical damage as well as cognitive ... more Mild traumatic brain injury (mTBI) can lead to diffuse neurophysical damage as well as cognitive and affective alterations. The nature and extent of behavioral changes after mTBI are still poorly understood and how strong an impact force has to be to cause long-term behavioral changes is not yet known. Here, we examined spatial learning acquisition, retention and reversal in a Morris water maze, and assessed search strategies during task performance after a single, mild, closed-skull traumatic impact referred to as "minimal" TBI. Additionally, we investigated changes in conditioned learning in a contextual fear-conditioning paradigm. Results show transient deficits in spatial memory retention, which, although limited, are indicative of deficits in long-term memory reconsolidation. Interestingly, minimal TBI causes animals to relapse to less effective search strategies, affecting performance after a retention pause. Apart from cognitive deficits, results yielded a sub-acute...

Molecular Biology Reports, 1996

The proprotein processing enzyme furin is the mammalian prototype of a novel family of subtilisin... more The proprotein processing enzyme furin is the mammalian prototype of a novel family of subtilisin-like serine endoproteases which possess cleavage specificity for sites involving multiple basic amino acid residues and are involved in the processing of precursor proteins of a variety of regulatory peptides and proteins. One of the limiting steps in the engineering of mammalian cells designed for the overproduction of secreted proteins is the endoproteolytic cleavage of the precursor molecule to its mature biologically active form. The extremely low level of endogenous furin is likely the reason why cells are not able to fully mature overexpressed precursor proteins to their mature form. Here, we report a CHO-derived cell line genetically engineered for the production of high levels of recombinant proteins that need such endoproteolytic maturation. First, the human furin cDNA under the control of the cytomegalovirus early promoter and enhancer was introduced and overexpressed in a DHFR-deficient CHO cell line. A permanent cell line CHO-D3-FUR was established that expressed biologically active furin. Subsequently, to demonstrate the capacity of CHO-D3-FUR cells to produce recombinant proteins in a fully matured form, two derivative cell lines were established that overexpressed the von Willebrand factor (vWF) and transforming growth factor beta 1 (TGF beta 1); CHO-D3-vWF and CHO-D3-TGF beta 1, respectively. Both derivative cell lines were able to produce relatively high levels of recombinant protein in a fully matured and biologically active form. Our results illustrate the potential of the CHO-D3-FUR cell line in the production of recombinant secretory proteins that need endoproteolytic activation at the consensus furin cleavage sequence Arg-X-Lys/Arg-Arg.

Cytogenet Genome Res, 1994

Genetic sequences of the novel NSP gene, which encodes neuroendocrine-specific proteins, were iso... more Genetic sequences of the novel NSP gene, which encodes neuroendocrine-specific proteins, were isolated from cDNA libraries constructed with mRNA isolated from human lung carcinoma cells. Hybridization analysis of a panel of human x mouse cell hybrids with an 0.8-kb NSP cDNA probe indicated that the human NSP gene is probably located on chromosome 14. Fluorescence in situ hybridization analysis of metaphase chromosomes using overlapping genomic clones of NSP as a probe localized the NSP gene to chromosome region 14q21-->q22.

Genomics, 1998

This paper describes the cDNA cloning, genomic orroendocrine-specific gene mapped to human chromo... more This paper describes the cDNA cloning, genomic orroendocrine-specific gene mapped to human chromoganization, and expression of the human RTN2 gene some 14q21-q22 (Kools et al., 1994), has multiple proon chromosome 19q13.3, which was recognized by virmoters, and is transcribed in three mRNA variants tue of its high similarity with the human RTN1 (forencoding three NSP protein isoforms with unique merly called NSP) gene on chromosome 14q21-q22. In amino-terminal parts, but common carboxy-terminal a region of about 12 kb in total, 11 RTN2 exons could parts. The proteins are associated with the endoplasbe identified. Like the RTN1 gene, the RTN2 gene is mic reticulum and therefore are named NSP reticulons transcribed into different mRNA variants. Two have a (Senden et al., 1994a,b, 1996; Van de Velde et al., size of about 2.3 kb, and a third has a size of about 1.3 1994a,b,c). The predicted transmembrane domains kb. The two 2.3-kb transcripts differ because of alterpresent in the common parts of the NSP proteins were native splicing of exon 5. Transcription of the 1.3-kb shown to play an essential role in this association (Van transcript starts presumably from an internal prode Velde et al., 1994a). Furthermore, the NSP reticumoter within exon 5. The three mRNAs encode three lons have been classified into a novel category of neurodifferent proteins, RTN2-A (545 aa), RTN2-B (472 aa), endocrine markers with respect to human lung cancer and RTN2-C (205 aa), which share a common carboxyterminal segment of 201 aa. In this common segment, (Senden et al., 1994b; Van de Velde et al., 1994c). Howthe homology with the RTN1 proteins, with yet un-ever, the function of the NSP reticulons remains obknown function, is found. Two hydrophobic subrescure. The association of the proteins with the endogions are present, which are thought to be responsible plasmic reticulum, a particular component of the neufor the association of the RTN1 and RTN2 proteins roendocrine secretory pathway, suggests that they with the endoplasmic reticulum. The amino-terminal constitute novel components of it. It was speculated regions of the RTN2-A and RTN2-B proteins are rich in that the reticulons are associated with the endoplasmic negatively charged residues and in proline and serine reticulum as channel-like complexes (Van de Velde et residues and contain multiple potential phosphorylaal., 1994a). tion sites. Analysis of the expression of the RTN2 gene Comparison of NSP genomic and cDNA sequences shows differential expression in human tissues with a with databank nucleotide sequences resulted in the disstrikingly high expression of the 1.3-kb transcript in covery of three other members of this novel family of skeletal muscle. ᭧ 1998 Academic Press reticulon-encoding genes in human (van de Velde et al., 1994a; Roebroek et al., 1996). Homology was found with INTRODUCTION genomic sequences present in a DNA region of about 106 kb around the ERCC1 locus on human chromosome Recently, the cDNA cloning, expression, and genomic 19q13.3 (Martin-Gallardo et al., 1992) and with partial organization of the human NSP (RTN1) gene was de-cDNA sequences of so-called expressed sequence tags (ESTs). These genes were tentatively called NSP-like Sequence data from this article have been deposited with the genes I, II, and III to emphasize their relationship with EMBL/GenBank Data Libraries under Accession Nos. AF004222 to AF004224. the NSP gene, the prototype of this novel gene family 1 To whom correspondence should be addressed at the Laboratory encoding reticulons. Of the NSP-like gene I, six exons for Molecular Oncology, Center for Human Genetics, University of corresponding to the six 3-end NSP exons (exon 4 to Louvain and Flanders Interuniversity Institute for Biotechnology, exon 9) could be identified in the genomic sequence on

Cellular and Molecular Life Sciences, 2010

The proximal NPXY and distal NPXYXXL motifs in the intracellular domain of LRP1 play an important... more The proximal NPXY and distal NPXYXXL motifs in the intracellular domain of LRP1 play an important role in regulation of the function of the receptor. The impact of single and double inactivating knock-in mutations of these motifs on receptor maturation, cell surface expression, and ligand internalization was analyzed in mutant and control wild-type mice and MEFs. Single inactivation of the proximal NPXY or in combination with inactivation of the distal NPXYXXL motif are both shown to be associated with an impaired maturation and premature proteasomal degradation of full-length LRP1. Therefore, only a small mature LRP1 pool is able to reach the cell surface resulting indirectly in severe impairment of ligand internalization. Single inactivation of the NPXYXXL motif revealed normal maturation, but direct impairment of ligand internalization. In conclusion, the proximal NPXY motif proves to be essential for early steps in the LRP1 biosynthesis, whereas NPXYXXL appears rather relevant for internalization.

Febs Letters, May 7, 2004

Frontiers in Pharmacology, 2015

The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1) has been attribute... more The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1) has been attributed a role in cancer. However, its presumably often indirect involvement is far from understood. LRP1 has both endocytic and signaling activities. As a matricellular receptor it is involved in regulation, mostly by clearing, of various extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases, protease inhibitor complexes, and the endoglycosidase heparanase. Furthermore, by binding extracellular ligands including growth factors and subsequent intracellular interaction with scaffolding and adaptor proteins it is involved in regulation of various signaling cascades. LRP1 expression levels are often downregulated in cancer and some studies consider low LRP1 levels a poor prognostic factor. On the contrary, upregulation in brain cancers has been noted and clinical trials explore the use of LRP1 as cargo receptor to deliver cytotoxic agents. This mini-review focuses on LRP1's role in tumor growth and metastasis especially by modulation of the extracellular tumor environment. In relation to this role its diagnostic, prognostic and therapeutic potential will be discussed.

Critical reviews in oncogenesis

ABSTRACT

Proceedings of the National Academy of Sciences

A U2 small nuclear RNA-associated protein, designated B", was recently identified as the target a... more A U2 small nuclear RNA-associated protein, designated B", was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies

Enzyme, 1991

Furin, the translational product of the recently discovered fur gene, appears to be the first kno... more Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues. Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC 3.4.21.61), which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2. We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases. Furthermore, the biosynthesis o...

Methods in molecular biology (Clifton, N.J.), 2014

Molecular genetic strategies applying embryonic stem cell (ES cell) technologies to study the fun... more Molecular genetic strategies applying embryonic stem cell (ES cell) technologies to study the function of a gene in mice or to generate a mouse model for a human disease are continuously under development. Next to (conditional) inactivation of genes the application and importance of approaches to generate knock-in mutations are increasing. In this chapter the principle and application of recombinase-mediated cassette exchange (RMCE) are discussed as being a new emerging knock-in strategy, which enables easy generation of a series of different knock-in mutations within one gene. An RMCE protocol, which was used to generate a series of different knock-in mutations in the Lrp1 gene of ES cells, is described in detail as an example of how RMCE can be used to generate highly efficiently an allelic series of differently modified ES cell clones from a parental modified ES cell clone. Subsequently the differently modified ES cell clones can be used to generate an allelic series of mutant kn...

PloS one, 2013

Members of the Nuclear eXport Factor (NXF) family are involved in the export of mRNA from the nuc... more Members of the Nuclear eXport Factor (NXF) family are involved in the export of mRNA from the nucleus to the cytoplasm, or hypothesized to play a role in transport of cytoplasmic mRNA. We previously reported on the loss of NXF5 in a male patient with a syndromic form of intellectual disability. To study the functional role of NXF5 we identified the mouse counterpart. Based on synteny, mouse Nxf2 is the ortholog of human NXF5. However, we provide several lines of evidence that mouse Nxf7 is the actual functional equivalent of NXF5. Both Nxf7 and NXF5 are predominantly expressed in the brain, show cytoplasmic localization, and present as granules in neuronal dendrites suggesting a role in cytoplasmic mRNA metabolism in neurons. Nxf7 was primarily detected in the pyramidal cells of the hippocampus and in layer V of the cortex. Similar to human NXF2, mouse Nxf2 is highly expressed in testis and shows a nuclear localization. Interestingly, these findings point to a different evolutionary...

Methods in molecular biology (Clifton, N.J.), 2011

Recombinase-mediated cassette exchange (RMCE) is a powerful tool to generate a series of knock-in... more Recombinase-mediated cassette exchange (RMCE) is a powerful tool to generate a series of knock-in mutations into a particular gene of the mouse. It uses standard ES cell technologies to introduce an exchangeable cassette into the gene of interest by homologous recombination. Because the introduced exchangeable cassette is flanked by heterotypic specific recombination target sequences, site-specific recombinases can be used in a subsequent RMCE step to exchange the cassette by other sequences. Here, an experimental procedure for the application of RMCE in E14 ES cells using heterotypic FRT recombination target sequences and the site-specific recombinase Flp is elaborated.

FEBS letters, Jan 7, 2004

The intracellular domain (ICD) of the low-density lipoprotein receptor-related protein (LRP) func... more The intracellular domain (ICD) of the low-density lipoprotein receptor-related protein (LRP) functionally interacts with adaptor proteins both as an integral part of the receptor polypeptide and after proteolytic release. Identification of such adaptors has been difficult because the ICD is self-activating in conventional transcription factor-based yeast two-hybrid screens. We adopted an alternative screen for the ICD that depends on the activation of the Ras-signaling pathway and uncovered the transcription factor MafB as novel ICD interacting protein. MafB is a regulator of hindbrain segmentation and interacts with the ICD through a leucine zipper domain. The ICD co-localizes with MafB to the nucleus and negatively regulates its transcriptional activity, suggesting a possible role for LRP in brain development.