Interaction of the cytosolic domains of sorLA/LR11 with the amyloid precursor protein (APP) and beta-secretase beta-site APP-cleaving enzyme

sorLA is a recently identified neuronal receptor for amyloid precursor protein (APP) that is known to interact with APP and affect its intracellular transport and processing. Decreased levels of sorLA in the brain of Alzheimer's disease (AD) patients and elevated levels of amyloid-beta peptide (Abeta) in sorLA-deficient mice point to the importance of the receptor in this neurodegenerative disorder. We analyzed APP cleavage in an APP-shedding assay and found that both sorLA and, surprisingly, a sorLA tail construct inhibited APP cleavage in a beta-site APP-cleaving enzyme (BACE)-dependent manner. In line with this finding, sorLA and the sorLA tail significantly reduced secreted Abeta levels when BACE was overexpressed, suggesting that sorLA influences beta-cleavage. To understand the effect of sorLA on APP cleavage by BACE, we analyzed whether sorLA interacts with APP and/or BACE. Because both full-length sorLA and sorLA C-terminal tail constructs were functionally relevant for APP processing, we analyzed sorLA-APP for a potential cytoplasmatic interaction domain. sorLA and C99 coimmunoprecipitated, pointing toward the existence of a new cytoplasmatic interaction site between sorLA and APP. Moreover, sorLA and BACE also coimmunoprecipitate. Thus, sorLA interacts both with BACE and APP and might therefore directly affect BACE-APP complex formation. To test whether sorLA impacts BACE-APP interactions, we used a fluorescence resonance energy transfer assay to evaluate BACE-APP interactions in cells. We discovered that sorLA significantly reduced BACE-APP interactions in Golgi. We postulate that sorLA acts as a trafficking receptor that prevents BACE-APP interactions and hence BACE cleavage of APP

Polarizing a stored proton beam by spin flip?

We discuss polarizing a proton beam in a storage ring, either by selective removal or by spin flip of the stored ions. Prompted by recent, conflicting calculations, we have carried out a measurement of the spin flip cross section in low-energy electron-proton scattering. The experiment uses the cooling electron beam at COSY as an electron target. The measured cross sections are too small for making spin flip a viable tool in polarizing a stored beam. This invalidates a recent proposal to use co-moving polarized positrons to polarize a stored antiproton beam.Comment: 18 pages, 6 figure

Measurement of the Spin-Dependence of the pbar-p Interaction at the AD-Ring

We propose to use an internal polarized hydrogen storage cell gas target in the AD ring to determine for the first time the two total spin-dependent pbar-p cross sections sigma_1 and sigma_2 at antiproton beam energies in the range from 50 to 450 MeV. The data obtained are of interest by themselves for the general theory of pbar-p interactions since they will provide a first experimental constraint of the spin-spin dependence of the nucleon-antinucleon potential in the energy range of interest. In addition, measurements of the polarization buildup of stored antiprotons are required to define the optimum parameters of a future, dedicated Antiproton Polarizer Ring (APR), intended to feed a double-polarized asymmetric pbar-p collider with polarized antiprotons. Such a machine has recently been proposed by the PAX collaboration for the new Facility for Antiproton and Ion Research (FAIR) at GSI in Darmstadt, Germany. The availability of an intense stored beam of polarized antiprotons will provide access to a wealth of single- and double-spin observables, thereby opening a new window on QCD spin physics.Comment: 51 pages, 23 figures, proposal submitted to the SPS committee of CER

Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP's and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway

Formation of Toxic Oligomeric α-Synuclein Species in Living Cells

Background: Misfolding, oligomerization, and fibrillization of α-synuclein are thought to be central events in the onset and progression of Parkinson's disease (PD) and related disorders. Although fibrillar α-synuclein is a major component of Lewy bodies (LBs), recent data implicate prefibrillar, oligomeric intermediates as the toxic species. However, to date, oligomeric species have not been identified in living cells. Methodology/Principal Findings: Here we used bimolecular fluorescence complementation (BiFC) to directly visualize α-synuclein oligomerization in living cells, allowing us to study the initial events leading to α-synuclein oligomerization, the precursor to aggregate formation. This novel assay provides us with a tool with which to investigate how manipulations affecting α-synuclein aggregation affect the process over time. Stabilization of α-synuclein oligomers via BiFC results in increased cytotoxicity, which can be rescued by Hsp70 in a process that reduces the formation of α-synuclein oligomers. Introduction of PD-associated mutations in α-synuclein did not affect oligomer formation but the biochemical properties of the mutant α-synuclein oligomers differ from those of wild type α-synuclein. Conclusions/Significance: This novel application of the BiFC assay to the study of the molecular basis of neurodegenerative disorders enabled the direct visualization of α-synuclein oligomeric species in living cells and its modulation by Hsp70, constituting a novel important tool in the search for therapeutics for synucleinopathies

A Forward Genetic Screen in Mice Identifies Mutants with Abnormal Cortical Patterning

Formation of a 6-layered cortical plate and axon tract patterning are key features of cerebral cortex development. Abnormalities of these processes may be the underlying cause for a range of functional disabilities seen in human neurodevelopmental disorders. To identify mouse mutants with defects in cortical lamination or corticofugal axon guidance, N-ethyl-N-nitrosourea (ENU) mutagenesis was performed using mice expressing LacZ reporter genes in layers II/III and V of the cortex (Rgs4-lacZ) or in corticofugal axons (TAG1-tau-lacZ). Four lines with abnormal cortical lamination have been identified. One of these was a splice site mutation in reelin (Reln) that results in a premature stop codon and the truncation of the C-terminal region (CTR) domain of reelin. Interestingly, this novel allele of Reln did not display cerebellar malformation or ataxia, and this is the first report of a Reln mutant without a cerebellar defect. Four lines with abnormal cortical axon development were also identified, one of which was found by whole-genome resequencing to carry a mutation in Lrp2. These findings demonstrated that the application of ENU mutagenesis to mice carrying transgenic reporters marking cortical anatomy is a sensitive and specific method to identify mutations that disrupt patterning of the developing brain

LRP10 interacts with SORL1 in the intracellular vesicle trafficking pathway in non-neuronal brain cells and localises to Lewy bodies in Parkinson's disease and dementia with Lewy bodies

Loss-of-function variants in the low-density lipoprotein receptor-related protein 10 (LRP10) gene have been associated with autosomal-dominant Parkinson's disease (PD), PD dementia, and dementia with Lewy bodies (DLB). Moreover, LRP10 variants have been found in individuals diagnosed with progressive supranuclear palsy and amyotrophic lateral sclerosis. Despite this genetic evidence, little is known about the expression and function of LRP10 protein in the human brain under physiological or pathological conditions. To better understand how LRP10 variants lead to neurodegeneration, we first performed an in-depth characterisation of LRP10 expression in post-mortem brains and human-induced pluripotent stem cell (iPSC)-derived astrocytes and neurons from control subjects. In adult human brain, LRP10 is mainly expressed in astrocytes and neurovasculature but undetectable in neurons. Similarly, LRP10 is highly expressed in iPSC-derived astrocytes but cannot be observed in iPSC-derived neurons. In astrocytes, LRP10 is present at trans-Golgi network, plasma membrane, retromer, and early endosomes. Interestingly, LRP10 also partially co-localises and interacts with sortilin-related receptor 1 (SORL1). Furthermore, although LRP10 expression and localisation in the substantia nigra of most idiopathic PD and DLB patients and LRP10 variant carriers diagnosed with PD or DLB appeared unchanged compared to control subjects, significantly enlarged LRP10-positive vesicles were detected in a patient carrying the LRP10 p.Arg235Cys variant. Last, LRP10 was detected in Lewy bodies (LB) at late maturation stages in brains from idiopathic PD and DLB patients and in LRP10 variant carriers. In conclusion, high LRP10 expression in non-neuronal cells and undetectable levels in neurons of control subjects indicate that LRP10-mediated pathogenicity is initiated via cell non-autonomous mechanisms, potentially involving the interaction of LRP10 with SORL1 in vesicle trafficking pathways. Together with the specific pattern of LRP10 incorporation into mature LBs, these data support an important mechanistic role for disturbed vesicle trafficking and loss of LRP10 function in neurodegenerative diseases

Megalin/LRP2 Expression Is Induced by Peroxisome Proliferator-Activated Receptor -Alpha and -Gamma: Implications for PPARs' Roles in Renal Function

BACKGROUND: Megalin is a large endocytic receptor with relevant functions during development and adult life. It is expressed at the apical surface of several epithelial cell types, including proximal tubule cells (PTCs) in the kidney, where it internalizes apolipoproteins, vitamins and hormones with their corresponding carrier proteins and signaling molecules. Despite the important physiological roles of megalin little is known about the regulation of its expression. By analyzing the human megalin promoter, we found three response elements for the peroxisomal proliferator-activated receptor (PPAR). The objective of this study was to test whether megalin expression is regulated by the PPARs. METHODOLOGY/PRINCIPAL FINDINGS: Treatment of epithelial cell lines with PPARα or PPARγ ligands increased megalin mRNA and protein expression. The stimulation of megalin mRNA expression was blocked by the addition of specific PPARα or PPARγ antagonists. Furthermore, PPAR bound to three PPAR response elements located in the megalin promoter, as shown by EMSA, and PPARα and its agonist activated a luciferase construct containing a portion of the megalin promoter and the first response element. Accordingly, the activation of PPARα and PPARγ enhanced megalin expression in mouse kidney. As previously observed, high concentrations of bovine serum albumin (BSA) decreased megalin in PTCs in vitro; however, PTCs pretreated with PPARα and PPARγ agonists avoided this BSA-mediated reduction of megalin expression. Finally, we found that megalin expression was significantly inhibited in the PTCs of rats that were injected with BSA to induce tubulointerstitial damage and proteinuria. Treatment of these rats with PPARγ agonists counteracted the reduction in megalin expression and the proteinuria induced by BSA. CONCLUSIONS: PPARα/γ and their agonists positively control megalin expression. This regulation could have an important impact on several megalin-mediated physiological processes and on pathophysiologies such as chronic kidney disease associated with diabetes and hypertension, in which megalin expression is impaired

Mutations in Zebrafish lrp2 Result in Adult-Onset Ocular Pathogenesis That Models Myopia and Other Risk Factors for Glaucoma

The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, Bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals—but not all—develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease