Spatially and temporally distinct patterns of expression for VPS10P domain receptors in human cerebral organoids

Vacuolar protein sorting 10 protein (VPS10P) domain receptors are a unique class of intracellular sorting receptors that emerge as major risk factors associated with psychiatric and neurodegenerative diseases, including bipolar disorders, autism, schizophrenia, as well as Alzheimer’s disease and frontotemporal dementia. Yet, the lack of suitable experimental models to study receptor functions in the human brain has hampered elucidation of receptor actions in brain disease. Here, we have adapted protocols using human cerebral organoids to the detailed characterization of VPS10P domain receptor expression during neural development and differentiation, including single-cell RNA sequencing. Our studies uncovered spatial and temporal patterns of expression unique to individual receptor species in the human brain. While SORL1 expression is abundant in stem cells and SORCS1 peaks in neural progenitors at onset of neurogenesis, SORT1 and SORCS2 show increasing expression with maturation of neuronal and non-neuronal cell types, arguing for distinct functions in development versus the adult brain. In neurons, subcellular localization also distinguishes between types of receptor species, either mainly localized to the cell soma (SORL1 and SORT1) or also to neuronal projections (SORCS1 and SORCS2), suggesting divergent functions in protein sorting between Golgi and the endo-lysosomal system or along axonal and dendritic tracks. Taken together, our findings provide an important resource on temporal, spatial, and subcellular patterns of VPS10P domain receptor expression in cerebral organoids for further elucidation of receptor (dys) functions causative of behavioral and cognitive defects of the human brain

Multi-compartmental modeling of SORLA’s influence on amyloidogenic processing in Alzheimer’s disease

BACKGROUND: Proteolytic breakdown of the amyloid precursor protein (APP) by secretases is a complex cellular process that results in formation of neurotoxic Aβ peptides, causative of neurodegeneration in Alzheimer’s disease (AD). Processing involves monomeric and dimeric forms of APP that traffic through distinct cellular compartments where the various secretases reside. Amyloidogenic processing is also influenced by modifiers such as sorting receptor-related protein (SORLA), an inhibitor of APP breakdown and major AD risk factor. RESULTS: In this study, we developed a multi-compartment model to simulate the complexity of APP processing in neurons and to accurately describe the effects of SORLA on these processes. Based on dose–response data, our study concludes that SORLA specifically impairs processing of APP dimers, the preferred secretase substrate. In addition, SORLA alters the dynamic behavior of β-secretase, the enzyme responsible for the initial step in the amyloidogenic processing cascade. CONCLUSIONS: Our multi-compartment model represents a major conceptual advance over single-compartment models previously used to simulate APP processing; and it identified APP dimers and β-secretase as the two distinct targets of the inhibitory action of SORLA in Alzheimer’s disease

Soluble Alpha-APP (sAPPalpha) Regulates CDK5 Expression and Activity in Neurons

A growing body of evidence suggests a role for soluble alpha-amyloid precursor protein (sAPPalpha) in pathomechanisms of Alzheimer disease (AD). This cleavage product of APP was identified to have neurotrophic properties. However, it remained enigmatic what proteins, targeted by sAPPalpha, might be involved in such neuroprotective actions. Here, we used high-resolution two- dimensional polyacrylamide gel electrophoresis to analyze proteome changes downstream of sAPPalpha in neurons. We present evidence that sAPPalpha regulates expression and activity of CDK5, a kinase that plays an important role in AD pathology. We also identified the cytoprotective chaperone ORP150 to be induced by sAPPalpha as part of this protective response. Finally, we present functional evidence that the sAPPalpha receptor SORLA is essential to mediate such molecular functions of sAPPalpha in neurons

An Endocytic Pathway Essential for Renal Uptake and Activation of the Steroid 25-(OH) Vitamin D3

AbstractSteroid hormones may enter cells by diffusion through the plasma membrane. However, we demonstrate here that some steroid hormones are taken up by receptor-mediated endocytosis of steroid-carrier complexes. We show that 25-(OH) vitamin D3 in complex with its plasma carrier, the vitamin D–binding protein, is filtered through the glomerulus and reabsorbed in the proximal tubules by the endocytic receptor megalin. Endocytosis is required to preserve 25-(OH) vitamin D3 and to deliver to the cells the precursor for generation of 1,25-(OH)2 vitamin D3, a regulator of the calcium metabolism. Megalin−/− mice are unable to retrieve the steroid from the glomerular filtrate and develop vitamin D deficiency and bone disease

GAS1 is required for NOTCH-dependent facilitation of SHH signaling in the ventral forebrain neuroepithelium

Growth arrest-specific 1 (GAS1) acts as a co-receptor to patched 1, promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in induced pluripotent stem cell-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating NOTCH signaling, which is essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives NOTCH pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating NOTCH and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations

Elevated sortilin expression discriminates functional from non-functional neuroendocrine tumors and enables therapeutic targeting

A subset of neuroendocrine tumors (NETs) can cause an excessive secretion of hormones, neuropeptides, and biogenic amines into the bloodstream. These so-called functional NETs evoke a hormone-related disease and lead to several different syndromes, depending on the factors released. One of the most common functional syndromes, carcinoid syndrome, is characterized mainly by over-secretion of serotonin. However, what distinguishes functional from non-functional tumors on a molecular level remains unknown. Here, we demonstrate that the expression of sortilin, a widely expressed transmembrane receptor involved in intracellular protein sorting, is significantly increased in functional compared to non-functional NETs and thus can be used as a biomarker for functional NETs. Furthermore, using a cell line model of functional NETs, as well as organoids, we demonstrate that inhibition of sortilin reduces cellular serotonin concentrations and may therefore serve as a novel therapeutic target to treat patients with carcinoid syndrome

Identification of novel regulators of developmental hematopoiesis using Endoglin regulatory elements as molecular probes.

Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the -8/+7/+9-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/-8-kb enhancer targeted TIE2+/c-KIT+/CD41- endothelial cells that were enriched for hematopoietic potential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters, LRP2, a multiligand receptor, was the only gene that had not previously been associated with hematopoiesis. We show that LRP2 is indeed involved in definitive hematopoiesis and by doing so validate the use of reporter gene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.National Health and Medical Research Council of Australia, Australian Research Council, Dr Tom Bee Stem Cell Research Fund, Cancer Research UK, Biotechnology and Biological Sciences Research Council, Leukaemia and Lymphoma Research, The Leukaemia and Lymphoma Society, core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust - MRC Cambridge Stem Cell Institute (Grant IDs: R01 HL04880, P015PO1HL32262-32, 5P30 DK49216, 5R01 DK53298, 5U01 HL10001-05, R24 DK092760)This is the author accepted manuscript. The final version is available from the American Society of Hematology via http://dx.doi.org/10.1182/blood-2016-02-69787

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