LDLR-related protein 10 (LRP10) regulates amyloid precursor protein (APP) trafficking and processing: evidence for a role in Alzheimer's disease.

BACKGROUND: The Aß peptide that accumulates in Alzheimer's disease (AD) is derived from amyloid precursor protein (APP) following proteolysis by ß- and γ-secretases. Substantial evidence indicates that alterations in APP trafficking within the secretory and endocytic pathways directly impact the interaction of APP with these secretases and subsequent Aß production. Various members of the low-density lipoprotein receptor (LDLR) family have been reported to play a role in APP trafficking and processing and are important risk factors in AD. We recently characterized a distinct member of the LDLR family called LDLR-related protein 10 (LRP10) that shuttles between the trans-Golgi Network (TGN), plasma membrane (PM), and endosomes. Here we investigated whether LRP10 participates in APP intracellular trafficking and Aß production. RESULTS: In this report, we provide evidence that LRP10 is a functional APP receptor involved in APP trafficking and processing. LRP10 interacts directly with the ectodomain of APP and colocalizes with APP at the TGN. Increased expression of LRP10 in human neuroblastoma SH-SY5Y cells induces the accumulation of mature APP in the Golgi and reduces its presence at the cell surface and its processing into Aß, while knockdown of LRP10 expression increases Aß production. Mutations of key motifs responsible for the recycling of LRP10 to the TGN results in the aberrant redistribution of APP with LRP10 to early endosomes and a concomitant increase in APP ß-cleavage into Aß. Furthermore, expression of LRP10 is significantly lower in the post-mortem brain tissues of AD patients, supporting a possible role for LRP10 in AD. CONCLUSIONS: The present study identified LRP10 as a novel APP sorting receptor that protects APP from amyloidogenic processing, suggesting that a decrease in LRP10 function may contribute to the pathogenesis of Alzheimer's disease.

Amyloid-ß(1-15/16) as a marker for γ-secretase inhibition in Alzheimer's disease.

Amyloid-ß (Aß) producing enzymes are key targets for disease-modifying Alzheimer's disease (AD) therapies since Aß trafficking is at the core of AD pathogenesis. Development of such drugs might benefit from the identification of markers indicating in vivo drug effects in the central nervous system. We have previously shown that Aß(1-15) is produced by concerted ß-and α-secretase cleavage of amyloid-ß protein precursor (AßPP). Here, we test the hypothesis that this pathway is more engaged upon γ-secretase inhibition in humans, and cerebrospinal fluid (CSF) levels of Aß(1-15/16) represent a biomarker for this effect. Twenty healthy men were treated with placebo (n = 5) or the γ-secretase inhibitor semagacestat (100 mg [n = 5], 140 mg [n = 5], or 280 mg [n = 5]). CSF samples were collected hourly over 36 hours and 10 time points were analyzed by immunoassay for Aß(1-15/16), Aß(x-38), Aß(x-40), Aß(x-42), sAßPPα, and sAßPPß. The CSF concentration of Aß(1-15/16) showed a dose-dependent response over 36 hours. In the 280 mg treatment group, a transient increase was seen with a maximum of 180% relative to baseline at 9 hours post administration of semagacestat. The concentrations of Aß(x-38), Aß(x-40), and Aß(x-42) decreased the first 9 hours followed by increased concentrations after 36 hours relative to baseline. No significant changes were detected for CSF sAßPPα and sAßPPß. Our data shows that CSF levels of Aß(1-15/16) increase during treatment with semagacestat supporting its feasibility as a pharmacodynamic biomarker for drug candidates aimed at inhibiting γ-secretase-mediated AßPP-processing.

Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.

We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.

Sequential expression and redundancy of Pitx2 and Pitx3 genes during muscle development.

The myogenic program is controlled by different groups of transcription factors acting during muscle development, including bHLH muscle regulatory factors (MRFs), the paired factors Pax3 and Pax7 and the homeobox factors Six1 and Six4. This program is critically dependent on MRFs that target downstream muscle-specific genes. We now report the expression of Pitx2 and Pitx3 transcription factors throughout muscle development. Pitx2 is first expressed in muscle progenitor cells of the dermomyotome and myotome. The onset of myoblast differentiation is concomitant with expression of Pitx3; its expression is maintained in all skeletal muscles while Pitx2 expression decreases thereafter. We have generated Pitx3 mutant mice and this deficiency does not significantly perturb muscle development but it is completely compensated by the maintenance of Pitx2 expression in all skeletal muscles. These experiments suggest that Pitx genes are important for myogenesis and that Pitx2 and Pitx3 may have partly redundant roles.

A methodology for global validation of microarray experiments.

BACKGROUND: DNA microarrays are popular tools for measuring gene expression of biological samples. This ever increasing popularity is ensuring that a large number of microarray studies are conducted, many of which with data publicly available for mining by other investigators. Under most circumstances, validation of differential expression of genes is performed on a gene to gene basis. Thus, it is not possible to generalize validation results to the remaining majority of non-validated genes or to evaluate the overall quality of these studies. RESULTS: We present an approach for the global validation of DNA microarray experiments that will allow researchers to evaluate the general quality of their experiment and to extrapolate validation results of a subset of genes to the remaining non-validated genes. We illustrate why the popular strategy of selecting only the most differentially expressed genes for validation generally fails as a global validation strategy and propose random-stratified sampling as a better gene selection method. We also illustrate shortcomings of often-used validation indices such as overlap of significant effects and the correlation coefficient and recommend the concordance correlation coefficient (CCC) as an alternative. CONCLUSION: We provide recommendations that will enhance validity checks of microarray experiments while minimizing the need to run a large number of labour-intensive individual validation assays.

Pitx1 in vivo promoter activity and mechanisms of positive autoregulation.

During early mouse embryogenesis, Pitx1 (pituitary homeobox 1), a member of the bicoid subgroup of PAIRED homeobox-containing transcription factors, marks the stomodeum, oral ectoderm, pituitary and first branchial arch in the anterior part of the embryo and lateral plate mesoderm only in the posterior half of the embryo. We have now defined PITX1 promoter fragments that mimic the anterior but not posterior expression of PITX1 in transgenic mice. In addition, we show positive regulation of this promoter in transfection studies by three members of the Pitx1 family (Pitx1, Pitx1b, Pitx2), as well as by a related factor, Otx1. PITX1 autoregulation depends on DNA-binding and trans-activation domains of Pitx1 and it may be responsible for establishment and/or maintenance of the Pitx1 expression domain.

Pitx1 and Pitx2 are required for development of hindlimb buds.

Two closely related homeobox transcription factors, Pitx1 and Pitx2, have been implicated in patterning of lateral plate mesoderm derivatives: Pitx1 for specification of hindlimb identity and Pitx2 for determination of laterality. We show that, together, Pitx1 and Pitx2 are required for formation of hindlimb buds and, when present in limited doses, for development of proximal (femur) and anterior (tibia and digit 1) hindlimb structures. Although Pitx1 is expressed throughout developing hindlimb buds, Pitx2 is not expressed in limb bud mesenchyme itself, but is co-expressed with Pitx1 in the presumptive hindlimb field before bud growth. Thus, Pitx1 and Pitx2 genes are required for sustained hindlimb bud growth and formation of hindlimbs.