Low density lipoprotein receptor-related protein 1 (LRP1) is indispensable for embryonic development. Comparing different genetically engineered mouse models, we found that expression of Lrp1 is essential in the embryo proper. Loss of LRP1 leads to lethal vascular defects with lack of proper investment with mural cells of both large and small vessels. We further demonstrate that LRP1 modulates Gi-dependent sphingosine-1-phosphate (S1P) signaling and integrates S1P and PDGF-BB signaling pathways, which are both crucial for mural cell recruitment, via its intracellular domain. Loss of LRP1 leads to a lack of S1P-dependent inhibition of RAC1 and loss of constraint of PDGF-BB-induced cell migration. Our studies thus identify LRP1 as a novel player in angiogenesis and in the recruitment and maintenance of mural cells. Moreover, they reveal an unexpected link between lipoprotein receptor and sphingolipid signaling that, in addition to angiogenesis during embryonic development, is of potential importance for other targets of these pathways, such as tumor angiogenesis and inflammatory processes.
The RNA-binding protein fused-in-sarcoma (FUS) has been associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two neurodegenerative disorders that share similar clinical and pathological features. Both missense mutations and overexpression of wild-type FUS protein can be pathogenic in human patients. To study the molecular and cellular basis by which FUS mutations and overexpression cause disease, we generated novel transgenic mice globally expressing low levels of human wild-type protein (FUS(WT)) and a pathological mutation (FUS(R521G)). FUS(WT) and FUS(R521G) mice that develop severe motor deficits also show neuroinflammation, denervated neuromuscular junctions, and premature death, phenocopying the human diseases. A portion of FUS(R521G) mice escape early lethality; these escapers have modest motor impairments and altered sociability, which correspond with a reduction of dendritic arbors and mature spines. Remarkably, only FUS(R521G) mice show dendritic defects; FUS(WT) mice do not. Activation of metabotropic glutamate receptors 1/5 in neocortical slices and isolated synaptoneurosomes increases endogenous mouse FUS and FUS(WT) protein levels but decreases the FUS(R521G) protein, providing a potential biochemical basis for the dendritic spine differences between FUS(WT) and FUS(R521G) mice.
Antiphospholipid syndrome patients have antiphospholipid antibodies (aPLs) that promote thrombosis, and they have increased cardiovascular disease risk. Although the basis for the thrombosis has been well delineated, it is not known why antiphospholipid syndrome patients also have an increased prevalence of nonthrombotic vascular occlusion. The aims of this work were to determine if aPLs directly promote medial hypertrophy or neointima formation in mice and to identify the underlying mechanisms.
It is poorly understood why there is greater cardiovascular disease risk associated with the apolipoprotein E4 (apoE) allele vs. apoE3, and also greater risk with the LRP8/apolipoprotein E receptor 2 (ApoER2) variant ApoER2-R952Q. Little is known about the function of the apoE-ApoER2 tandem outside of the central nervous system. We now report that in endothelial cells apoE3 binding to ApoER2 stimulates endothelial NO synthase (eNOS) and endothelial cell migration, and it also attenuates monocyte-endothelial cell adhesion. However, apoE4 does not stimulate eNOS or endothelial cell migration or dampen cell adhesion, and alternatively it selectively antagonizes apoE3/ApoER2 actions. The contrasting endothelial actions of apoE4 vs. apoE3 require the N-terminal to C-terminal interaction in apoE4 that distinguishes it structurally from apoE3. Reconstitution experiments further reveal that ApoER2-R952Q is a loss-of-function variant of the receptor in endothelium. Carotid artery reendothelialization is decreased in ApoER2(-/-) mice, and whereas adenoviral-driven apoE3 expression in wild-type mice has no effect, apoE4 impairs reendothelialization. Moreover, in a model of neointima formation invoked by carotid artery endothelial denudation, ApoER2(-/-) mice display exaggerated neointima development. Thus, the apoE3/ApoER2 tandem promotes endothelial NO production, endothelial repair, and endothelial anti-inflammatory properties, and it prevents neointima formation. In contrast, apoE4 and ApoER2-R952Q display dominant-negative action and loss of function, respectively. Thus, genetic variants of apoE and ApoER2 impact cardiovascular health by differentially modulating endothelial function.
Frontotemporal dementia (FTD) is the most common cause of dementia in people under 60 yr of age and is pathologically associated with mislocalization of TAR DNA/RNA binding protein 43 (TDP-43) in approximately half of cases (FLTD-TDP). Mutations in the gene encoding progranulin (GRN), which lead to reduced progranulin levels, are a significant cause of familial FTLD-TDP. Grn-KO mice were developed as an FTLD model, but lack cortical TDP-43 mislocalization and neurodegeneration. Here, we report retinal thinning as an early disease phenotype in humans with GRN mutations that precedes dementia onset and an age-dependent retinal neurodegenerative phenotype in Grn-KO mice. Retinal neuron loss in Grn-KO mice is preceded by nuclear depletion of TDP-43 and accompanied by reduced expression of the small GTPase Ran, which is a master regulator of nuclear import required for nuclear localization of TDP-43. In addition, TDP-43 regulates Ran expression, likely via binding to its 3'-UTR. Augmented expression of Ran in progranulin-deficient neurons restores nuclear TDP-43 levels and improves their survival. Our findings establish retinal neurodegeneration as a new phenotype in progranulin-deficient FTLD, and suggest a pathological loop involving reciprocal loss of Ran and nuclear TDP-43 as an underlying mechanism.
Members of the low-density lipoprotein (LDL) receptor gene family have a diverse set of biological functions that transcend lipid metabolism. Lipoprotein receptors have broad effects in both the developing and adult brain and participate in synapse development, cargo trafficking, and signal transduction. In addition, several family members play key roles in Alzheimer's disease (AD) pathogenesis and neurodegeneration. This Review summarizes our current understanding of the role lipoprotein receptors play in CNS function and AD pathology, with a special emphasis on amyloid-independent roles in endocytosis and synaptic dysfunction.
Large glycosylating toxins are major virulence factors of various species of pathogenic Clostridia. Prototypes are Clostridium difficile toxins A and B, which cause antibiotics-associated diarrhea and pseudomembranous colitis. The current model of the toxins' action suggests that receptor binding is mediated by a C-terminal domain of combined repetitive oligopeptides (CROP). This model is challenged by the glycosylating Clostridium perfringens large cytotoxin (TpeL toxin) that is devoid of the CROP domain but still intoxicates cells. Using a haploid genetic screen, we identified LDL receptor-related protein 1 (LRP1) as a host cell receptor for the TpeL toxin. LRP1-deficient cells are not able to take up TpeL and are not intoxicated. Expression of cluster IV of LRP1 is sufficient to rescue toxin uptake in these cells. By plasmon resonance spectroscopy, a KD value of 23 nM was determined for binding of TpeL to LRP1 cluster IV. The C terminus of TpeL (residues 1335-1779) represents the receptor-binding domain (RBD) of the toxin. RBD-like regions are conserved in all other clostridial glycosylating toxins preceding their CROP domain. CROP-deficient C. difficile toxin B is toxic to cells, depending on the RBD-like region (residues 1349-1811) but does not interact with LRP1. Our data indicate the presence of a second, CROP-independent receptor-binding domain in clostridial glycosylating toxins and suggest a two-receptor model for the cellular uptake of clostridial glycosylating toxins.
Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a variety of ligands, including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the U.S. National Institute of Allergy and Infectious Diseases, National Institutes of Health to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of non-self or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. The discussion and nomenclature recommendations described in this report only refer to mammalian scavenger receptors. The purpose of this article is to describe the proposed mammalian nomenclature and classification developed at the workshop and to solicit additional feedback from the broader research community.
The phagocytosis of apoptotic cells (ACs), or efferocytosis, by DCs is critical for self-tolerance and host defense. Although many efferocytosis-associated receptors have been described in vitro, the functionality of these receptors in vivo has not been explored in depth. Using a spleen efferocytosis assay and targeted genetic deletion in mice, we identified a multiprotein complex--composed of the receptor tyrosine kinase AXL, LDL receptor-related protein-1 (LRP-1), and RAN-binding protein 9 (RANBP9)--that mediates DC efferocytosis and antigen cross-presentation. We found that AXL bound ACs, but required LRP-1 to trigger internalization, in murine CD8?+ DCs and human-derived DCs. AXL and LRP-1 did not interact directly, but relied on RANBP9, which bound both AXL and LRP-1, to form the complex. In a coculture model of antigen presentation, the AXL/LRP-1/RANBP9 complex was used by DCs to cross-present AC-associated antigens to T cells. Furthermore, in a murine model of herpes simplex virus-1 infection, mice lacking DC-specific LRP-1, AXL, or RANBP9 had increased AC accumulation, defective viral antigen-specific CD8+ T cell activation, enhanced viral load, and decreased survival. The discovery of this multiprotein complex that mediates functionally important DC efferocytosis in vivo may have implications for future studies related to host defense and DC-based vaccines.
We created an Nse-CreERT2 mouse line expressing the tamoxifen-inducible CreERT2 recombinase under the control of the neuron-specific enolase (Nse) promoter. By using Cre reporter lines we could show that this Nse-CreERT2 line has recombination activity in the granule cells of all cerebellar lobules as well as in postmitotic granule cell precursors in the external granular layer of the developing cerebellum. A few hippocampal dentate gyrus granule cells showed Cre-mediated recombination as well. Cre activity could be induced in both the developing and adult mouse brain. The established mouse line constitutes a valuable tool to study the function of genes expressed by cerebellar granule cells in the developing and adult brain. In combination with reporter lines it is a useful model to analyze the development and maintenance of the cerebellar architecture including granule cell distribution, migration, and the extension of granule cell fibers in vivo.
Reelin is a glycoprotein that is critical for proper layering of neocortex during development as well as dynamic regulation of glutamatergic postsynaptic signaling in mature synapses. Here, we show that Reelin also acts presynaptically, resulting in robust rapid enhancement of spontaneous neurotransmitter release without affecting properties of evoked neurotransmission. This effect of Reelin requires a modest but significant increase in presynaptic Ca(2+) initiated via ApoER2 signaling. The specificity of Reelin action on spontaneous neurotransmitter release is encoded at the level of vesicular SNARE machinery as it requires VAMP7 and SNAP-25 but not synaptobrevin2, VAMP4, or vti1a. These results uncover a presynaptic regulatory pathway that utilizes the heterogeneity of synaptic vesicle-associated SNAREs and selectively augments action potential-independent neurotransmission.
The primary role of apolipoprotein E (apoE) is to mediate the cellular uptake of lipoproteins. However, a new role for apoE as a regulator of bone metabolism in mice has recently been established. In contrast to mice, the human APOE gene is characterized by three common isoforms APOE ?2, ?3, and ?4 that result in different metabolic properties of the apoE isoforms, but it remains controversial whether the APOE polymorphism influences bone traits in humans. To clarify this, we investigated bone phenotypes of apoE knock-in (k.i.) mice, which express one human isoform each (apoE2?k.i., apoE3?k.i., apoE4?k.i.) in place of the mouse apoE. Analysis of 12-week-old female k.i. mice revealed increased levels of biochemical bone formation and resorption markers in apoE2?k.i. animals as compared to apoE3?k.i. and apoE4?k.i., with a reduced osteoprotegerin (OPG)/receptor activator of NF-?B ligand (RANKL) ratio in apoE2?k.i., indicating increased turnover with prevailing resorption in apoE2?k.i. Accordingly, histomorphometric and micro-computed tomography (µCT) analyses demonstrated significantly lower trabecular bone mass in apoE2 than in apoE3 and apoE4?k.i. animals, which was reflected by a significant reduction of lumbar vertebrae maximum force resistance. Unlike trabecular bone, femoral cortical thickness, and stability was not differentially affected by the apoE isoforms. To extend these observations to the human situation, plasma from middle-aged healthy men homozygous for ?2/?2, ?3/?3, and ?4/?4 (n?=?21, n?=?80, n?=?55, respectively) was analyzed with regard to bone turnover markers. In analogy to apoE2?k.i. mice, a lower OPG/RANKL ratio was observed in the serum of ?2/?2 carriers as compared to ?3/?3 and ?4/?4 individuals (p?=?0.02 for ?2/?2 versus ?4/?4). In conclusion, the current data strongly underline the general importance of apoE as a regulator of bone metabolism and identifies the APOE ?2 allele as a potential genetic risk factor for low trabecular bone mass and vertebral fractures in humans.
GABAergic inhibitory interneurons (IN) represent a heterogeneous population with different electrophysiological, morphological, and molecular properties. The correct balance between interneuronal subtypes is important for brain function and is impaired in several neurological and psychiatric disorders. Here we show the data of 123 molecularly and electrophysiologically characterized neurons of juvenile rat barrel cortex acute slices, 48 of which expressed Reelin (Reln). Reln mRNA was exclusively detected in Gad65/67-positive cells but was found in interneuronal subtypes in different proportions: all cells of the adapting-Somatostatin (SST) cluster expressed Reln, whereas 63% of the adapting-neuropeptide Y (NPY, 50% of the fast-spiking Parvalbumin (PVALB), and 27% of the adapting/bursting-Vasoactive Intestinal Peptide (VIP) cluster were Reln-positive. Silhouette analysis revealed a high impact of the parameter Reln on cluster quality. By analyzing the co-localization of RELN immunoreactivity with those of different IN-markers, we found that RELN is produced layer-independently in SST-, NPY-, and NOS1-expressing INs, whereas co-localization of RELN and VIP was mostly absent. Of note, RELN co-localized with PVALB, predominantly in INs of layers IV/V (>30%). Our findings emphasize RELNs role as an important IN-marker protein and provide a basis for the functional characterization of Reln-expressing INs and its role in the regulation of inhibitory IN networks.
The lipoprotein receptor LRP1 is essential in neurons of the central nervous system, as was revealed by the analysis of conditional Lrp1-deficient mouse models. The molecular basis of its neuronal functions, however, is still incompletely understood. Here we show by immunocytochemistry, electron microscopy, and postsynaptic density preparation that LRP1 is located postsynaptically. Basal and NMDA-induced phosphorylation of the transcription factor cAMP-response element-binding protein (CREB) as well as NMDA target gene transcription are reduced in LRP1-deficient neurons. In control neurons, NMDA promotes ?-secretase-dependent release of the LRP1 intracellular domain (LRP1-ICD). However, pull-down and chromatin immunoprecipitation (ChIP) assays showed no direct interaction between the LRP1-ICD and either CREB or target gene promoters. On the other hand, NMDA-induced degradation of the postsynaptic scaffold protein PSD-95 was impaired in the absence of LRP1, whereas its ubiquitination was increased, indicating that LRP1 influences the composition of postsynaptic protein complexes. Accordingly, NMDA-induced internalization of the AMPA receptor subunit GluA1 was impaired in LRP1-deficient neurons. These results show a role of LRP1 in the regulation and turnover of synaptic proteins, which may contribute to the reduced dendritic branching and to the neurological phenotype observed in the absence of LRP1.
Progranulin (PGRN) is a secreted glycoprotein expressed in neurons and glia that is implicated in neuronal survival on the basis that mutations in the GRN gene causing haploinsufficiency result in a familial form of frontotemporal dementia (FTD). Recently, a direct interaction between PGRN and tumor necrosis factor receptors (TNFR I/II) was reported and proposed to be a mechanism by which PGRN exerts anti-inflammatory activity, raising the possibility that aberrant PGRN-TNFR interactions underlie the molecular basis for neuroinflammation in frontotemporal lobar degeneration pathogenesis. Here, we report that we find no evidence for a direct physical or functional interaction between PGRN and TNFRs. Using coimmunoprecipitation and surface plasmon resonance (SPR) we replicated the interaction between PGRN and sortilin and that between TNF and TNFRI/II, but not the interaction between PGRN and TNFRs. Recombinant PGRN or transfection of a cDNA encoding PGRN did not antagonize TNF-dependent NF?B, Akt, and Erk1/2 pathway activation; inflammatory gene expression; or secretion of inflammatory factors in BV2 microglia and bone marrow-derived macrophages (BMDMs). Moreover, PGRN did not antagonize TNF-induced cytotoxicity on dopaminergic neuroblastoma cells. Last, co-addition or pre-incubation with various N- or C-terminal-tagged recombinant PGRNs did not alter lipopolysaccharide-induced inflammatory gene expression or cytokine secretion in any cell type examined, including BMDMs from Grn+/- or Grn-/- mice. Therefore, the neuroinflammatory phenotype associated with PGRN deficiency in the CNS is not a direct consequence of the loss of TNF antagonism by PGRN, but may be a secondary response by glia to disrupted interactions between PGRN and Sortilin and/or other binding partners yet to be identified.
During dentate gyrus development, the early embryonic radial glial scaffold is replaced by a secondary glial scaffold around birth. In contrast to neocortical and early dentate gyrus radial glial cells, these postnatal glial cells are severely altered with regard to position and morphology in reeler mice lacking the secreted protein Reelin. In this study, we focus on the functional impact of these defects. Most radial glial cells throughout the nervous system serve as scaffolds for migrating neurons and precursor cells for both neurogenesis and gliogenesis. Precursor cell function has been demonstrated for secondary radial glial cells but the exact function of these late glial cells in granule cell migration and positioning is not clear. No data exist concerning the interplay between granule neurons and late radial glial cells during dentate gyrus development. Herein, we show that despite the severe morphological defects in the reeler dentate gyrus, the precursor function of secondary radial glial cells is not impaired during development in reeler mice. In addition, selective ablation of Disabled-1, an intracellular adaptor protein essential for Reelin signaling, in neurons but not in glial cells allowed us to distinguish effects of Reelin signaling on radial glial cells from possible secondary effects based on defective granule cells positioning.
Progranulin is a secreted glycoprotein, and the GRN gene is mutated in some cases of frontotemporal dementia. Progranulin has also been implicated in cell growth, wound healing, inflammation, and cancer. We investigated the molecular nature of secreted progranulin and provide evidence that progranulin exists as a homodimer. Although recombinant progranulin has a molecular mass of ?85 kDa by SDS-PAGE, it elutes in fractions corresponding to ?170-180 kDa by gel-filtration chromatography. Additionally, recombinant progranulin can be intermolecularly cross-linked, yielding a complex corresponding to a dimer (?180 kDa), and progranulins containing different epitope tags physically interact. In plasma, progranulin similarly forms complexes of ?180-190 kDa. Although progranulin partially co-fractionated with high density lipoproteins (HDL) by gel-filtration chromatography, we found no evidence that progranulin in mouse or human plasma is a component of HDL either by ultracentrifugation or by lipid binding assays. We conclude that circulating progranulin exists as a dimer and is not likely a component of HDL.
The integration of newborn neurons into functional neuronal networks requires migration of cells to their final position in the developing brain, the growth and arborization of neuronal processes and the formation of synaptic contacts with other neurons. A central player among the signals that coordinate this complex sequence of differentiation events is the secreted glycoprotein Reelin, which also modulates synaptic plasticity, learning and memory formation in the adult brain. Binding of Reelin to ApoER2 and VLDL receptor, two members of the LDL receptor family, initiates a signaling cascade involving tyrosine phosphorylation of the intracellular cytoplasmic adaptor protein Disabled-1, which targets the neuronal cytoskeleton and ultimately controls the positioning of neurons throughout the developing brain. However, it is possible that Reelin signals interact with other receptor-mediated signaling cascades to regulate different aspects of brain development and plasticity. EphB tyrosine kinases regulate cell adhesion and repulsion-dependent processes via bidirectional signaling through ephrin B transmembrane proteins. Here, we demonstrate that Reelin binds to the extracellular domains of EphB transmembrane proteins, inducing receptor clustering and activation of EphB forward signaling in neurons, independently of the classical Reelin receptors, ApoER2 and VLDLR. Accordingly, mice lacking EphB1 and EphB2 display a positioning defect of CA3 hippocampal pyramidal neurons, similar to that in Reelin-deficient mice, and this cell migration defect depends on the kinase activity of EphB proteins. Together, our data provide biochemical and functional evidence for signal integration between Reelin and EphB forward signaling.
Apolipoprotein E (APOE) is the major risk factor for sporadic Alzheimers disease. Among other functions, APOE is proposed to sequester neurotoxic amyloid-? (A?) peptides in the brain, delivering them to cellular catabolism via neuronal APOE receptors. Still, the receptors involved in this process remain controversial. Here, we identified the pro-neurotrophin receptor sortilin as major endocytic pathway for clearance of APOE/A? complexes in neurons. Sortilin binds APOE with high affinity. Lack of receptor expression in mice results in accumulation of APOE and of A? in the brain and in aggravated plaque burden. Also, primary neurons lacking sortilin exhibit significantly impaired uptake of APOE/A? complexes despite proper expression of other APOE receptors. Despite higher than normal brain APOE levels, sortilin-deficient animals display anomalies in brain lipid metabolism (e.g., accumulation of sulfatides) seen in APOE-deficient mice, indicating functional deficiency in cellular APOE uptake pathways. Together, our findings identified sortilin as an essential neuronal pathway for APOE-containing lipoproteins in vivo and suggest an intriguing link between A? catabolism and pro-neurotrophin signaling converging on this receptor.
ApoE, ApoE receptors and APP cooperate in the pathogenesis of Alzheimers disease. Intriguingly, the ApoE receptor LRP4 and APP are also required for normal formation and function of the neuromuscular junction (NMJ). In this study, we show that APP interacts with LRP4, an obligate co-receptor for muscle-specific tyrosine kinase (MuSK). Agrin, a ligand for LRP4, also binds to APP and co-operatively enhances the interaction of APP with LRP4. In cultured myotubes, APP synergistically increases agrin-induced acetylcholine receptor (AChR) clustering. Deletion of the transmembrane domain of LRP4 (LRP4 ECD) results in growth retardation of the NMJ, and these defects are markedly enhanced in APP(-/-);LRP4(ECD/ECD) mice. Double mutant NMJs are significantly reduced in size and number, resulting in perinatal lethality. Our findings reveal novel roles for APP in regulating neuromuscular synapse formation through hetero-oligomeric interaction with LRP4 and agrin and thereby provide new insights into the molecular mechanisms that govern NMJ formation and maintenance. DOI:http://dx.doi.org/10.7554/eLife.00220.001.
The cellular and molecular mechanisms responsible for the development of inner retinal circuitry are poorly understood. Reelin and apolipoprotein E (apoE), ligands of apoE receptor 2 (ApoER2), are involved in retinal development and degeneration, respectively. Here we describe the function of ApoER2 in the developing and adult retina. ApoER2 expression was highest during postnatal inner retinal synaptic development and was considerably lower in the mature retina. Both during development and in the adult, ApoER2 was expressed by A-II amacrine cells. ApoER2 knock-out (KO) mice had rod bipolar morphogenic defects, altered A-II amacrine dendritic development, and impaired rod-driven retinal responses. The presence of an intact ApoER2 NPxY motif, necessary for binding Disabled-1 and transducing the Reelin signal, was also necessary for development of the rod bipolar pathway, while the alternatively spliced exon 19 was not. Mice deficient in another Reelin receptor, very low-density lipoprotein receptor (VLDLR), had normal rod bipolar morphology but altered A-II amacrine dendritic development. VLDLR KO mice also had reductions in oscillatory potentials and delayed synaptic response intervals. Interestingly, age-related reductions in rod and cone function were observed in both ApoER2 and VLDLR KOs. These results support a pivotal role for ApoER2 in the establishment and maintenance of normal retinal synaptic connectivity.
The correct number and shape of teeth are critical factors for an aesthetic and functional dentition. Understanding the molecular mechanisms regulating tooth number and shape are therefore important in orthodontics. Mice have only one incisor and three molars in each jaw quadrant that are divided by a tooth-less region, the diastema. Although mice lost teeth in the diastema during evolution, the remnants of the evolutionary lost teeth are observed as transient epithelial buds in the wild-type diastema during early stages of development. Shh and Fgf signaling pathways that are essential for tooth development have been shown to be repressed in the diastema. It remains unclear however how Wnt signaling, that is also required for tooth development, is regulated in the diastema. In this study we found that in the embryonic diastema, Wnt5a expression was observed in mesenchyme, whereas Wnt4 and Wnt10b were expressed in epithelium. The expression of Wnt6 and Wnt11 was found in both tissues. The Wnt co-receptor, Lrp6, was weakly expressed in the diastema overlapping with weak Lrp4 expression, a co-receptor that inhibits Wnt signaling. Secreted Wnt inihibitors Dkk1, Dkk2, and Dkk3 were also expressed in the diastema. Lrp4 mutant mice develop supernumerary teeth in the diastema that is accompanied by upregulation of Wnt signaling and Lrp6 expression. Wnt signaling is thus usually attenuated in the diastema by these secreted and membrane bound Wnt inhibitors.
Progranulin (GRN) haploinsufficiency is a frequent cause of familial frontotemporal dementia, a currently untreatable progressive neurodegenerative disease. By chemical library screening, we identified suberoylanilide hydroxamic acid (SAHA), a Food and Drug Administration-approved histone deacetylase inhibitor, as an enhancer of GRN expression. SAHA dose-dependently increased GRN mRNA and protein levels in cultured cells and restored near-normal GRN expression in haploinsufficient cells from human subjects. Although elevation of secreted progranulin levels through a post-transcriptional mechanism has recently been reported, this is, to the best of our knowledge, the first report of a small molecule enhancer of progranulin transcription. SAHA has demonstrated therapeutic potential in other neurodegenerative diseases and thus holds promise as a first generation drug for the prevention and treatment of frontotemporal dementia.
The low density lipoprotein receptor-related protein (LRP1) is a transmembrane receptor that integrates multiple signaling pathways. Its cytoplasmic domain serves as docking sites for several adaptor proteins such as the Src homology 2/?-collagen (ShcA), which also binds to several tyrosine kinase receptors such as the insulin-like growth factor 1 (IGF-1) receptor. However, the physiological significance of the physical interaction between LRP1 and ShcA, and whether this interaction modifies tyrosine kinase receptor signaling, are still unknown. Here we report that LRP1 forms a complex with the IGF-1 receptor, and that LRP1 is required for ShcA to become sensitive to IGF-1 stimulation. Upon IGF-1 treatment, ShcA is tyrosine phosphorylated and translocates to the plasma membrane only in the presence of LRP1. This leads to the recruitment of the growth factor receptor-bound protein 2 (Grb2) to ShcA, and activation of the Ras/MAP kinase pathway. Conversely, in the absence of ShcA, IGF-1 signaling bifurcates toward the Akt/mammalian target of rapamycin pathway and accelerates adipocyte differentiation when cells are stimulated for adipogenesis. These results establish the LRP1-ShcA complex as an essential component in the IGF-1-regulated pathway for MAP kinase and Akt/mammalian target of rapamycin activation, and may help to understand the IGF-1 signaling shift from clonal expansion to growth-arrested cells and differentiation during adipogenesis.
Disabled-1 (Dab1) is an adaptor protein that is essential for the intracellular transduction of Reelin signaling, which regulates the migration and differentiation of postmitotic neurons during brain development in vertebrates. Dab1 function depends on its tyrosine phosphorylation by Src family kinases, especially Fyn.
Obesity is a growing epidemic characterized by excess fat storage in adipocytes. Although lipoprotein receptors play important roles in lipid uptake, their role in controlling food intake and obesity is not known. Here we show that the lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis. Conditional deletion of the Lrp1 gene in the brain resulted in an obese phenotype characterized by increased food intake, decreased energy consumption, and decreased leptin signaling. LRP1 directly binds to leptin and the leptin receptor complex and is required for leptin receptor phosphorylation and Stat3 activation. We further showed that deletion of the Lrp1 gene specifically in the hypothalamus by Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results demonstrate that the lipoprotein receptor LRP1, which is critical in lipid metabolism, also regulates food intake and energy homeostasis in the adult central nervous system.
Trypanosoma cruzi, an intracellular protozoan parasite that infects humans and other mammalian hosts, is the etiologic agent in Chagas disease. This parasite can invade a wide variety of mammalian cells. The mechanism(s) by which T. cruzi invades its host cell is not completely understood. The activation of many signaling receptors during invasion has been reported; however, the exact mechanism by which parasites cross the host cell membrane barrier and trigger fusion of the parasitophorous vacuole with lysosomes is not understood.
TDP-43, or TAR DNA-binding protein 43, is a pathological marker of a spectrum of neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. TDP-43 is an RNA/DNA-binding protein implicated in transcriptional and posttranscriptional regulation. Recent work also suggests that TDP-43 associates with cytoplasmic stress granules, which are transient structures that form in response to stress. In this study, we establish sorbitol as a novel physiological stressor that directs TDP-43 to stress granules in Hek293T cells and primary cultured glia. We quantify the association of TDP-43 with stress granules over time and show that stress granule association and size are dependent on the glycine-rich region of TDP-43, which harbors the majority of pathogenic mutations. Moreover, we establish that cells harboring wild-type and mutant TDP-43 have distinct stress responses: mutant TDP-43 forms significantly larger stress granules, and is incorporated into stress granules earlier, than wild-type TDP-43; in striking contrast, wild-type TDP-43 forms more stress granules over time, but the granule size remains relatively unchanged. We propose that mutant TDP-43 alters stress granule dynamics, which may contribute to the progression of TDP-43 proteinopathies.
The low-density lipoprotein (LDL) receptor family is a large evolutionarily conserved group of transmembrane proteins. It has been shown that LDL receptor family members can also function as direct signal transducers or modulators for a broad range of cellular signaling pathways. We have identified a novel mode of signaling pathway integration/coordination that occurs outside cells during development that involves an LDL receptor family member. Physical interaction between an extracellular protein (Wise) that binds BMP ligands and an Lrp receptor (Lrp4) that modulates Wnt signaling, acts to link these two pathways. Mutations in either Wise or Lrp4 in mice produce multiple, but identical abnormalities in tooth development that are linked to alterations in BMP and Wnt signaling. Teeth, in common with many other organs, develop by a series of epithelial-mesenchymal interactions, orchestrated by multiple cell signaling pathways. In tooth development, Lrp4 is expressed exclusively in epithelial cells and Wise mainly in mesenchymal cells. Our hypothesis, based on the mutant phenotypes, cell signaling activity changes and biochemical interactions between Wise and Lrp4 proteins, is that Wise and Lrp4 together act as an extracellular mechanism of coordinating BMP and Wnt signaling activities in epithelial-mesenchymal cell communication during development.
Lipoprotein receptor signaling regulates the positioning and differentiation of postmitotic neurons during development and modulates neuronal plasticity in the mature brain. Depending on the contextual situation, the lipoprotein receptor ligand Reelin can have opposing effects on cortical neurons. We show that Reelin increases growth cone motility and filopodia formation, and identify the underlying signaling cascade. Reelin activates the Rho GTPase Cdc42, known for its role in neuronal morphogenesis and directed migration, in an apolipoprotein E receptor 2-, Disabled-1-, and phosphatidylinositol 3-kinase-dependent manner. We demonstrate that neuronal vesicle trafficking, a Cdc42-controlled process, is increased after Reelin treatment and further provide evidence that the peptidergic VIP/PACAP38 system and Reelin can functionally interact to promote axonal branching. In conclusion, Reelin-induced activation of Cdc42 contributes to the regulation of the cytoskeleton of individual responsive neurons and converges with other signaling cascades to orchestrate Rho GTPase activity and promote neuronal development. Our data link the observation that defects in Rho GTPases and Reelin signaling are responsible for developmental defects leading to neurological and psychiatric disorders.
TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)(n) is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)(n)TA(TG)(m), is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.
The evolutionarily ancient low-density lipoprotein (LDL) receptor gene family represents a class of widely expressed cell surface receptors. Since the dawn of the first primitive multicellular organisms, several structurally and functionally distinct families of lipoprotein receptors have evolved. In accordance with the now obsolete one-gene-one-function hypothesis, these cell surface receptors were originally perceived as mere transporters of lipoproteins, lipids, and nutrients or as scavenger receptors, which remove other kinds of macromolecules, such as proteases and protease inhibitors from the extracellular environment and the cell surface. This picture has since undergone a fundamental change. Experimental evidence has replaced the perception that these receptors serve merely as cargo transporters. Instead it is now clear that the transport of macromolecules is inseparably intertwined with the molecular machinery by which cells communicate with each other. Lipoprotein receptors are essentially sensors of the extracellular environment that participate in a wide range of physiological processes by physically interacting and coevolving with primary signal transducers as co-regulators. Furthermore, lipoprotein receptors modulate cellular trafficking and localization of the amyloid precursor protein (APP) and the ?-amyloid peptide (A?), suggesting a role in the pathogenesis of Alzheimers disease. Moreover, compelling evidence shows that LDL receptor family members are involved in tumor development and progression.
In antiphospholipid syndrome (APS), antiphospholipid antibodies (aPL) binding to ?2 glycoprotein I (?2GPI) induce endothelial cell-leukocyte adhesion and thrombus formation via unknown mechanisms. Here we show that in mice both of these processes are caused by the inhibition of eNOS. In studies of cultured human, bovine, and mouse endothelial cells, the promotion of monocyte adhesion by aPL entailed decreased bioavailable NO, and aPL fully antagonized eNOS activation by diverse agonists. Similarly, NO-dependent, acetylcholine-induced increases in carotid vascular conductance were impaired in aPL-treated mice. The inhibition of eNOS was caused by antibody recognition of domain I of ?2GPI and ?2GPI dimerization, and it was due to attenuated eNOS S1179 phosphorylation mediated by protein phosphatase 2A (PP2A). Furthermore, LDL receptor family member antagonism with receptor-associated protein (RAP) prevented aPL inhibition of eNOS in cell culture, and ApoER2-/- mice were protected from aPL inhibition of eNOS in vivo. Moreover, both aPL-induced increases in leukocyte-endothelial cell adhesion and thrombus formation were absent in eNOS-/- and in ApoER2-/- mice. Thus, aPL-induced leukocyte-endothelial cell adhesion and thrombosis are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by ?2GPI, apoER2, and PP2A. Our results suggest that novel therapies for APS can now be developed targeting these mechanisms.
The low-density lipoprotein receptor-related protein (LRP1) is a multifunctional cell surface receptor that belongs to the LDL receptor (LDLR) gene family and that is widely expressed in several tissues. LRP1 consists of an 85-kDa membrane-bound carboxyl fragment (? chain) and a non-covalently attached 515-kDa (? chain) amino-terminal fragment. Through its extracellular domain, LRP1 binds at least 40 different ligands ranging from lipoprotein and protease inhibitor complex to growth factors and extracellular matrix proteins. LRP-1 has also been shown to interact with scaffolding and signaling proteins via its intracellular domain in a phosphorylation-dependent manner and to function as a co-receptor partnering with other cell surface or integral membrane proteins. LRP-1 is thus implicated in two major physiological processes: endocytosis and regulation of signaling pathways, which are both involved in diverse biological roles including lipid metabolism, cell growth/differentiation processes, degradation of proteases, and tissue invasion. The embryonic lethal phenotype obtained after target disruption of the LRP-1 gene in the mouse highlights the biological importance of this receptor and revealed a critical, but yet undefined role in development. Tissue-specific gene deletion studies also reveal an important contribution of LRP1 in vascular remodeling, foam cell biology, the central nervous system, and in the molecular mechanisms of atherosclerosis.
The dentate gyrus is a brain region where neurons are continuously born throughout life. In the adult, the role of its radial glia in neurogenesis has attracted much attention over the past years; however, little is known about the generation and differentiation of glial cells and their relationship to radial glia during the ontogenetic development of this brain structure. Here, we combine immunohistochemical phenotyping using antibodies against glial marker proteins with BrdU birthdating to characterize the development of the secondary radial glial scaffold in the dentate gyrus and its potential to differentiate into astrocytes. We demonstrate that the expression of brain lipid-binding protein, GLAST, and glial fibrillary acidic protein (GFAP) characterizes immature differentiating cells confined to an astrocytic fate in the early postnatal dentate gyrus. On the basis of our studies, we propose a model where immature astrocytes migrate radially through the granule cell layer to adopt their final positions in the molecular layer of the dentate gyrus. Time-lapse imaging of acute hippocampal slices from hGFAP-eGFP transgenic mice provides direct evidence for such a migration mode of differentiating astroglial cells in the developing dentate gyrus.
Apolipoprotein E (ApoE) genotype is a powerful genetic modifier of Alzheimers disease (AD). The ApoE4 isoform significantly reduces the mean age-of-onset of dementia through unknown mechanisms. Here, we show that ApoE4 selectively impairs synaptic plasticity and NMDA receptor phosphorylation by Reelin, a regulator of brain development and modulator of synaptic strength. ApoE4 reduces neuronal surface expression of Apoer2, a dual function receptor for ApoE and for Reelin, as well as NMDA and AMPA receptors by sequestration in intracellular compartments, thereby critically reducing the ability of Reelin to enhance synaptic glutamate receptor activity. As a result, the ability of Reelin to prevent LTP suppression by extracts from AD-afflicted human brains in hippocampal slices from knockin mice expressing the human ApoE4 isoform is severely impaired. These findings show an isoform-specific role of ApoE in the localization and intracellular trafficking of lipoprotein and glutamate receptors and thereby reveal an alternative mechanism by which ApoE4 may accelerate onset of dementia and neuronal degeneration by differentially impairing the maintenance of synaptic stability.
Reelin signalling in the early developing cortex regulates radial migration of cortical neurons. Later in development, Reelin promotes maturation of dendrites and dendritic spines. Finally, in the mature brain, it is involved in modulating synaptic function. In recent years, efforts to identify downstream signalling events induced by binding of Reelin to lipoprotein receptors led to the characterization of novel components of the Reelin signalling cascade. In the present review, we first address distinct functions of the Reelin receptors Apoer2 and Vldlr in cortical layer formation, followed by a discussion on the recently identified downstream effector molecule n-cofilin, involved in regulating actin cytoskeletal dynamics required for coordinated neuronal migration. Next, we discuss possible functions of the recently identified Reelin-Notch signalling crosstalk, and new aspects of the role of Reelin in the formation of the dentate radial glial scaffold. Finally, progress in characterizing the function of Reelin in modulating synaptic function in the adult brain is summarized. The present review has been inspired by a session entitled Functions of Reelin in the developing and adult hippocampus, held at the Spring Hippocampal Research Conference in Verona/Italy, June 2009.
The low-density lipoprotein (LDL) receptor gene family is a highly conserved group of membrane receptors with diverse functions in developmental processes, lipoprotein trafficking, and cell signaling. The low-density lipoprotein (LDL) receptor-related protein 1b (LRP1B) was reported to be deleted in several types of human malignancies, including non-small cell lung cancer. Our group has previously reported that a distal extracellular truncation of murine Lrp1b that is predicted to secrete the entire intact extracellular domain (ECD) is fully viable with no apparent phenotype.
Development of the kidney is initiated when the ureteric bud (UB) branches from the Wolffian duct and invades the overlying metanephric mesenchyme (MM) triggering the mesenchymal/epithelial interactions that are the basis of organ formation. Multiple signaling pathways must be integrated to ensure proper timing and location of the ureteric bud formation.
Cenani-Lenz syndrome (CLS) is an autosomal-recessive congenital disorder affecting distal limb development. It is characterized mainly by syndactyly and/or oligodactyly and is now shown to be commonly associated with kidney anomalies. We used a homozygosity-mapping approach to map the CLS1 locus to chromosome 11p11.2-q13.1. By sequencing candidate genes, we identified recessive LRP4 mutations in 12 families with CLS. LRP4 belongs to the low-density lipoprotein (LDL) receptor-related proteins (LRPs), which are essential for various developmental processes. LRP4 is known to antagonize LRP6-mediated activation of canonical Wnt signaling, a function that is lost by the identified mutations. Our findings increase the spectrum of congenital anomalies associated with abnormal lipoprotein receptor-dependent signaling.
TDP-43 is a DNA/RNA-binding protein implicated in multiple steps of transcriptional and post-transcriptional regulation of gene expression. Alteration of this multifunctional protein is associated with a number of neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin positive inclusions. Whereas a pathological link to neurodegenerative disorders has been established, the cellular and physiological functions of TDP-43 remain unknown. In this study, we show that TDP-43 is a nuclear protein with persistent high-level expression during embryonic development and with progressively decreased protein levels during postnatal development. In mice where the TDP-43 gene (Tardbp) was disrupted using a gene trap that carries a beta-galactosidase marker gene, heterozygous (Tardbp(+/-)) mice are fertile and healthy, but intercrosses of Tardbp(+/-) mice yielded no viable homozygotic null (Tardbp(-/-)) mice. Indeed, Tardbp(-/-) embryos die between 3.5 and 8.5 days of development. Tardbp(-/-) blastocysts grown in cell culture display abnormal expansion of their inner cell mass. The pattern of beta-galactosidase staining at E9.5 Tardbp(+/-) embryos is predominantly restricted to the neuroepithelium and remains prominent in neural progenitors at E10.5-12.5. TDP-43 is detected in spinal cord progenitors and in differentiated motor neurons as well as in the dorsal root ganglia at E12.5. Beta-galactosidase staining of tissues from adult Tardbp(+/-) mice shows widespread expression of TDP-43, including prominent levels in various regions of the central nervous system afflicted in neurodegenerative disorders. These results indicate that TDP-43 is developmentally regulated and indispensible for early embryonic development.
Changes in tooth shape have played a major role in vertebrate evolution with modification of dentition allowing an organism to adapt to new feeding strategies. The current view is that molar teeth evolved from simple conical teeth, similar to canines, by progressive addition of extra "cones" to form progressively complex multicuspid crowns. Mammalian incisors, however, are neither conical nor multicuspid, and their evolution is unclear. We show that hypomorphic mutation of a cell surface receptor, Lrp4, which modulates multiple signaling pathways, produces incisors with grooved enamel surfaces that exhibit the same molecular characteristics as the tips of molar cusps. Mice with a null mutation of Lrp4 develop extra cusps on molars and have incisors that exhibit clear molar-like cusp and root morphologies. Molecular analysis identifies misregulation of Shh and Bmp signaling in the mutant incisors and suggests an uncoupling of the processes of tooth shape determination and morphogenesis. Incisors thus possess a developmentally suppressed, cuspid crown-like morphogenesis program similar to that in molars that is revealed by loss of Lrp4 activity. Several mammalian species naturally possess multicuspid incisors, suggesting that mammals have the capacity to form multicuspid teeth regardless of location in the oral jaw. Localized loss of enamel may thus have been an intermediary step in the evolution of cusps, both of which use Lrp4-mediated signaling.
Insulin stimulates the translocation of intracellular GLUT4 to the plasma membrane where it functions in adipose and muscle tissue to clear glucose from circulation. The pathway and regulation of GLUT4 trafficking are complicated and incompletely understood and are likely to be contingent upon the various proteins other than GLUT4 that comprise and interact with GLUT4-containing vesicles. Moreover, not all GLUT4 intracellular pools are insulin-responsive as some represent precursor compartments, thus posing a biochemical challenge to the purification and characterization of their content. To address these issues, we immunodepleted precursor GLUT4-rich vesicles and then immunopurified GLUT4 storage vesicle (GSVs) from primary rat adipocytes and subjected them to semi-quantitative and quantitative proteomic analysis. The purified vesicles translocate to the cell surface almost completely in response to insulin, the expected behavior for bona fide GSVs. In total, over 100 proteins were identified, about 50 of which are novel in this experimental context. LRP1 (low density lipoprotein receptor-related protein 1) was identified as a major constituent of GSVs, and we show it interacts with the lumenal domains of GLUT4 and other GSV constituents. Its cytoplasmic tail interacts with the insulin-signaling pathway target, AS160 (Akt substrate of 160 kDa). Depletion of LRP1 from 3T3-L1 adipocytes reduces GLUT4 expression and correspondingly results in decreased insulin-stimulated 2-[(3)H]deoxyglucose uptake. Furthermore, adipose-specific LRP1 knock-out mice also exhibit decreased GLUT4 expression. These findings suggest LRP1 is an important component of GSVs, and its expression is needed for the formation of fully functional GSVs.
Lrp4 is a multifunctional member of the low density lipoprotein-receptor gene family and a modulator of extracellular cell signaling pathways in development. For example, Lrp4 binds Wise, a secreted Wnt modulator and BMP antagonist. Lrp4 shares structural elements within the extracellular ligand binding domain with Lrp5 and Lrp6, two established Wnt co-receptors with important roles in osteogenesis. Sclerostin is a potent osteocyte secreted inhibitor of bone formation that directly binds Lrp5 and Lrp6 and modulates both BMP and Wnt signaling. The anti-osteogenic effect of sclerostin is thought to be mediated mainly by inhibition of Wnt signaling through Lrp5/6 within osteoblasts. Dickkopf1 (Dkk1) is another potent soluble Wnt inhibitor that binds to Lrp5 and Lrp6, can displace Lrp5-bound sclerostin and is itself regulated by BMPs. In a recent genome-wide association study of bone mineral density a significant modifier locus was detected near the SOST gene at 17q21, which encodes sclerostin. In addition, nonsynonymous SNPs in the LRP4 gene were suggestively associated with bone mineral density. Here we show that Lrp4 is expressed in bone and cultured osteoblasts and binds Dkk1 and sclerostin in vitro. MicroCT analysis of Lrp4 deficient mutant mice revealed shortened total femur length, reduced cortical femoral perimeter, and reduced total femur bone mineral content (BMC) and bone mineral density (BMD). Lumbar spine trabecular bone volume per total volume (BV/TV) was significantly reduced in the mutants and the serum and urinary bone turnover markers alkaline phosphatase, osteocalcin and desoxypyridinoline were increased. We conclude that Lrp4 is a novel osteoblast expressed Dkk1 and sclerostin receptor with a physiological role in the regulation of bone growth and turnover, which is likely mediated through its function as an integrator of Wnt and BMP signaling pathways.
Defective smooth muscle expression of LDL receptor-related protein-1 (Lrp1) increases atherosclerosis in hypercholesterolemic mice. This study explored the importance of smooth muscle Lrp1 expression under normolipidemic conditions.
Abnormal processing of the amyloid precursor protein (APP) and beta-amyloid (Abeta) plaque accumulation are defining features of Alzheimer disease (AD), a genetically complex neurodegenerative disease that is characterized by progressive synapse loss and neuronal cell death. Abeta induces synaptic dysfunction in part by altering the endocytosis and trafficking of AMPA and NMDA receptors. Reelin is a neuromodulator that increases glutamatergic neurotransmission by signaling through the postsynaptic ApoE receptors Apoer2 and Vldlr and thereby potently enhances synaptic plasticity. Here we show that Reelin can prevent the suppression of long-term potentiation and NMDA receptors, which is induced by levels of Abeta comparable to those present in an AD-afflicted brain. This reversal is dependent upon the activation of Src family tyrosine kinases. At high concentrations of Abeta peptides, Reelin can no longer overcome the Abeta induced functional suppression and this coincides with a complete blockade of the Reelin-dependent phosphorylation of NR2 subunits. We propose a model in which Abeta, Reelin, and ApoE receptors modulate neurotransmission and thus synaptic stability as opposing regulators of synaptic gain control.
Lipoprotein receptors are evolutionarily ancient proteins that are expressed on the surface of many cell types. Beginning with the appearance of the first primitive multicellular organisms, several structurally and functionally distinct families of lipoprotein receptors evolved. Originally, these cell surface proteins were thought to merely mediate the traffic of lipids and nutrients between cells and, in some cases, by functioning as scavenger receptors, remove other kinds of macromolecules, such as proteases and protease inhibitors from the extracellular space and the cell surface. Over the last decade, this picture has fundamentally changed. We now appreciate that many of these receptors are not mere cargo transporters; they are deeply embedded in the machinery by which cells communicate with each other. By physically interacting and coevolving with fundamental signaling pathways, lipoprotein receptors have occupied essential and surprisingly diverse functions that are indispensable for integrating the complex web of cellular signal input during development and in differentiated tissues.
ATP-binding cassette transporter A1 mediates apolipoprotein AI-dependent efflux of cholesterol and thereby removes cholesterol from peripheral tissues. ABCA1 expression is tightly regulated and deficiency of this cholesterol transporter results in cholesterol accumulation within cells. Low-density lipoprotein receptor-related protein 1 (LRP1) participates in lipid metabolism and energy homeostasis by endocytosis of apolipoprotein E-containing lipoproteins and modulation of cellular proliferation signals.
Low density lipoprotein receptor-related protein 1 (LRP1) protects against atherosclerosis by regulating the activation of platelet-derived growth factor receptor beta (PDGFRbeta) in vascular smooth muscle cells (SMCs). Activated PDGFRbeta undergoes tyrosine phosphorylation and subsequently interacts with various signaling molecules, including phosphatidylinositol 3-kinase (PI3K), which binds to the phosphorylated tyrosine 739/750 residues in mice, and thus regulates actin polymerization and cell movement.
Over the past few years, apolipoprotein E (ApoE) receptors, also known as LDL receptor-related proteins, have distinguished themselves as functionally diverse signaling receptors with pivotal roles not only in the vascular system but also in the nervous system and during development.
Dysequilibrium syndrome is a genetically heterogeneous condition that combines autosomal recessive, nonprogressive cerebellar ataxia with mental retardation. Here, we report the first patient heterozygous for 2 novel mutations in VLDLR. An 18-month-old girl presented with significant hypotonia, global developmental delay, and truncal and peripheral ataxia. Magnetic resonance imaging of the brain demonstrated hypoplasia of the inferior cerebellar vermis and hemispheres, small pons, and a simplified cortical sulcation pattern. Sequence analysis of the VLDLR gene identified a nonsense and missense mutation. Six mutations in VLDLR have now been identified in 5 families with a phenotype characterized by moderate-to-profound mental retardation, delayed ambulation, truncal and peripheral ataxia, and occasional seizures. Neuroanatomically, the loss-of-function effect of the different mutations is indistinguishable. VLDLR-associated cerebellar hypoplasia is emerging as a panethnic, clinically, and molecularly well-defined genetic syndrome.
Apolipoprotein E receptor 2 (Apoer2) is a multifunctional transport and signaling receptor that regulates the uptake of selenium into the mouse brain and testis through endocytosis of selenoprotein P (Sepp1). Mice deficient in Apoer2 or Sepp1 are infertile, with kinked and hypomotile spermatozoa. They also develop severe neurological defects on a low selenium diet, due to a profound impairment of selenium uptake. Little is known about the function of Apoer2 in the testis beyond its role as a Sepp1 receptor. By contrast, in the brain, Apoer2 is an essential component of the Reelin signaling pathway, which is required for proper neuronal organization and synapse function. Using knock-in mice, we have functionally dissociated the signaling motifs in the Apoer2 cytoplasmic domain from Sepp1 uptake. Selenium concentration of brain and testis was normal in the knock-in mutants, in contrast to Apoer2 knock-outs. Thus, the neurological defects in the signaling impaired knock-in mice are not caused by a selenium uptake defect, but instead are a direct consequence of a disruption of the Reelin signal. Reduced sperm motility was observed in some of the knock-in mice, indicating a novel signaling role for Apoer2 in sperm development and function that is independent of selenium uptake.
The low-density lipoprotein receptor-related protein LRP1 is a cell surface receptor with functions in diverse physiological pathways, including lipid metabolism. Here we show that LRP1-deficient fibroblasts accumulate high levels of intracellular cholesterol and cholesteryl-ester when stimulated for adipocyte differentiation. We demonstrate that LRP1 stimulates a canonical Wnt5a signaling pathway that prevents cholesterol accumulation. Moreover, we show that LRP1 is required for lipolysis and stimulates fatty acid synthesis independently of the noradrenergic pathway, through inhibition of GSK3beta and its previously unknown target acetyl-CoA carboxylase (ACC). As a result of ACC inhibition, mature LRP1-deficient adipocytes of adult mice are hypotrophic, and lower uptake of fatty acids into adipose tissue leads to their redistribution to the liver. These results establish LRP1 as a novel integrator of adipogenic differentiation and fat storage signals.
Birthdate-dependent neuronal layering is fundamental to neocortical functions. The extracellular protein Reelin is essential for the establishment of the eventual neuronal alignments. Although this Reelin-dependent neuronal layering is mainly established by the final neuronal migration step called "terminal translocation" beneath the marginal zone (MZ), the molecular mechanism underlying the control by Reelin of terminal translocation and layer formation is largely unknown. Here, we show that after Reelin binds to its receptors, it activates integrin ?5?1 through the intracellular Dab1-Crk/CrkL-C3G-Rap1 pathway. This intracellular pathway is required for terminal translocation and the activation of Reelin signaling promotes neuronal adhesion to fibronectin through integrin ?5?1. Since fibronectin is localized in the MZ, the activated integrin ?5?1 then controls terminal translocation, which mediates proper neuronal alignments in the mature cortex. These data indicate that Reelin-dependent activation of neuronal adhesion to the extracellular matrix is crucial for the eventual birth-date-dependent layering of the neocortex.
Vascular calcification is a hallmark of advanced atherosclerosis. Here we show that deletion of the nuclear receptor PPAR? in vascular smooth muscle cells of low density lipoprotein receptor (LDLr)-deficient mice fed an atherogenic diet high in cholesterol, accelerates vascular calcification with chondrogenic metaplasia within the lesions. Vascular calcification in the absence of PPAR? requires expression of the transmembrane receptor LDLr-related protein-1 in vascular smooth muscle cells. LDLr-related protein-1 promotes a previously unknown Wnt5a-dependent prochondrogenic pathway. We show that PPAR? protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2, which functions as a Wnt5a antagonist. Targeting this signalling pathway may have clinical implications in the context of common complications of atherosclerosis, including coronary artery calcification and valvular sclerosis.
From the earliest stages of embryogenesis and throughout life, transcriptional regulation is carefully orchestrated in order to generate, shape, and reshape the central nervous system (CNS). TAR DNA-binding protein 43 (TDP-43) is identified as a regulator of essential transcriptional events in the CNS. Evidence for its importance comes from the identification of TDP-43 protein aggregates and genetic mutations in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Efforts are being made to learn more about the biological function of TDP-43 and gain a better understanding of its role in neurodegeneration. TDP-43 RNA targets and protein interactions have now been identified, and in vivo evidence shows that TDP-43 is essential in CNS development and function. This review will highlight aspects of these findings.
Mothers milk is widely accepted as nutritious and protective to the newborn mammals by providing not only macronutrients but also immune-defensive factors. However, the mechanisms accounting for these benefits are not fully understood. Here we show that maternal very-low-density-lipoprotein (VLDL) receptor deletion in mice causes the production of defective milk containing diminished levels of platelet-activating factor acetylhydrolase (PAFAH). As a consequence, the nursing neonates suffer from alopecia, anaemia and growth retardation owing to elevated levels of pro-inflammatory platelet-activating factors. VLDL receptor deletion significantly impairs the expression of phospholipase A2 group 7 (Pla2g7) in macrophages, which decreases PAFAH secretion. Exogenous oral supplementation of neonates with PAFAH effectively rescues the toxicity. These findings not only reveal a novel role of VLDL receptor in suppressing inflammation by maintaining macrophage PAFAH secretion, but also identify the maternal VLDL receptor as a key genetic program that ensures milk quality and protects the newborns.
GRN mutations cause frontotemporal lobar degeneration with TDP-43-positive inclusions. The mechanism of pathogenesis is haploinsufficiency. Recently, homozygous GRN mutations were detected in two patients with neuronal ceroid lipofuscinosis, a lysosomal storage disease. It is unknown whether the pathogenesis of these two conditions is related. Progranulin is cleaved into smaller peptides called granulins. Progranulin and granulins are attributed with roles in cancer, inflammation, and neuronal physiology. Cell surface receptors for progranulin, but not granulin peptides, have been reported. Revealing the cell surface receptors and the intracellular functions of granulins and progranulin is crucial for understanding their contributions to neurodegeneration.
The RNA-binding protein TDP-43 is strongly linked to neurodegeneration. Not only are mutations in the gene encoding TDP-43 associated with ALS and FTLD, but this protein is also a major constituent of pathological intracellular inclusions in these diseases. Recent studies have significantly expanded our understanding of TDP-43 physiology. TDP-43 is now known to play important roles in neuronal RNA metabolism. It binds to and regulates the splicing and stability of numerous RNAs encoding proteins involved in neuronal development, synaptic function and neurodegeneration. Thus, a loss of these essential functions is an attractive hypothesis regarding the role of TDP-43 in neurodegeneration. Moreover, TDP-43 is an aggregation-prone protein and, given the role of toxic protein aggregates in neurodegeneration, a toxic gain-of-function mechanism is another rational hypothesis. Importantly, ALS related mutations modulate the propensity of TDP-43 to aggregate in cell culture. Several recent studies have documented that cytoplasmic TDP-43 aggregates co-localize with stress granule markers. Stress granules are cytoplasmic inclusions that repress translation of a subset of RNAs in times of cellular stress, and several proteins implicated in neurodegeneration (i.e. Ataxin-2 and SMN) interact with stress granules. Thus, understanding the interplay between TDP-43 aggregation, stress granules and the effect of ALS-associated TDP-43 mutations may be the key to understanding the role of TDP-43 in neurodegeneration. We propose two models of TDP-43 aggregate formation. The "independent model" stipulates that TDP-43 aggregation is independent of stress granule formation, in contrast to the "precursor model" which presents the idea that stress granule formation contributes to a TDP-43 aggregate "seed" and that chronic stress leads to concentration-dependent TDP-43 aggregation. This article is part of a Special Issue entitled: RNA-Binding Proteins.
Apolipoprotein E (APOE) genotype is the major genetic risk factor for Alzheimer disease (AD); the ?4 allele increases risk and the ?2 allele is protective. In the central nervous system (CNS), apoE is produced by glial cells, is present in high-density-like lipoproteins, interacts with several receptors that are members of the low-density lipoprotein receptor (LDLR) family, and is a protein that binds to the amyloid-? (A?) peptide. There are a variety of mechanisms by which apoE isoform may influence risk for AD. There is substantial evidence that differential effects of apoE isoform on AD risk are influenced by the ability of apoE to affect A? aggregation and clearance in the brain. Other mechanisms are also likely to play a role in the ability of apoE to influence CNS function as well as AD, including effects on synaptic plasticity, cell signaling, lipid transport and metabolism, and neuroinflammation. ApoE receptors, including LDLRs, Apoer2, very low-density lipoprotein receptors (VLDLRs), and lipoprotein receptor-related protein 1 (LRP1) appear to influence both the CNS effects of apoE as well as A? metabolism and toxicity. Therapeutic strategies based on apoE and apoE receptors may include influencing apoE/A? interactions, apoE structure, apoE lipidation, LDLR receptor family member function, and signaling. Understanding the normal and disease-related biology connecting apoE, apoE receptors, and AD is likely to provide novel insights into AD pathogenesis and treatment.
This study investigated the possible role of the ApoE receptors Lrp1 and Apoer2 in mediating the pathological effects of ApoE4 in ApoE-targeted-replacement mice expressing either the human ApoE3 or ApoE4 allele. In this study we show that activation of the amyloid cascade by inhibition of the A?-degrading enzyme neprilysin results in up-regulation of the ApoE receptor Lrp1 in the CA1 hippocampal neurons of 4-month-old ApoE4 mice, but not in the corresponding ApoE3 or ApoE-deficient (KO) mice. These results are in accordance with the previous findings that activation of the amyloid cascade induces A? accumulation in the CA1 neurons of ApoE4 mice, but not in ApoE3 or ApoE-KO mice. This suggests that the apoE4-driven elevation of Lrp1 is mediated via a gain of function mechanism and may play a role in mediating the effects of ApoE4 on A?. In contrast, no changes were observed in the levels of the corresponding Apoer2 receptor following the neprilysin inhibition. The ApoE receptors of naive ApoE4 mice were also affected differentially and isoform specifically by ApoE4. However, under these conditions, the effect was an ApoE4-driven reduction in the levels of Apoer2 in CA1 and CA3 pyramidal neurons, whereas the levels of Lrp1 were not affected. RT-PCR measurements revealed that the levels of Apoer2 and Lrp1 mRNA in the hippocampus of naïve and neprilysin-inhibited mice were not affected by ApoE4, suggesting that the observed effects of ApoE4 on the levels of these receptors is post-transcriptional. In conclusion, this study shows that the levels of hippocampal ApoE receptors Lrp1 and Apoer2 in vivo are affected isoform specifically by ApoE4 and that the type of receptor affected is context dependent.
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