ApoE Receptor 2 Regulates Synapse and Dendritic Spine Formation

Apolipoprotein E receptor 2 (ApoEr2) is a postsynaptic protein involved in long-term potentiation (LTP), learning, and memory through unknown mechanisms. We examined the biological effects of ApoEr2 on synapse and dendritic spine formation-processes critical for learning and memory.In a heterologous co-culture synapse assay, overexpression of ApoEr2 in COS7 cells significantly increased colocalization with synaptophysin in primary hippocampal neurons, suggesting that ApoEr2 promotes interaction with presynaptic structures. In primary neuronal cultures, overexpression of ApoEr2 increased dendritic spine density. Consistent with our in vitro findings, ApoEr2 knockout mice had decreased dendritic spine density in cortical layers II/III at 1 month of age. We also tested whether the interaction between ApoEr2 and its cytoplasmic adaptor proteins, specifically X11α and PSD-95, affected synapse and dendritic spine formation. X11α decreased cell surface levels of ApoEr2 along with synapse and dendritic spine density. In contrast, PSD-95 increased cell surface levels of ApoEr2 as well as synapse and dendritic spine density.These results suggest that ApoEr2 plays important roles in structure and function of CNS synapses and dendritic spines, and that these roles are modulated by cytoplasmic adaptor proteins X11α and PSD-95

Cardiac Transcription Factor Nkx2.5 Is Downregulated under Excessive O-GlcNAcylation Condition

Post-translational modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is linked the development of diabetic cardiomyopathy. We investigated whether Nkx2.5 protein, a cardiac transcription factor, is regulated by O-GlcNAc. Recombinant Nkx2.5 (myc-Nkx2.5) proteins were reduced by treatment with the O-GlcNAcase inhibitors STZ and O-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-N-phenylcarbamate; PUGNAC) as well as the overexpression of recombinant O-GlcNAc transferase (OGT-flag). Co-immunoprecipitation analysis revealed that myc-Nkx2.5 and OGT-flag proteins interacted and myc-Nkx2.5 proteins were modified by O-GlcNAc. In addition, Nkx2.5 proteins were reduced in the heart tissue of streptozotocin (STZ)-induced diabetic mice and O-GlcNAc modification of Nkx2.5 protein increased in diabetic heart tissue compared with non-diabetic heart. Thus, excessive O-GlcNAcylation causes downregulation of Nkx2.5, which may be an underlying contributing factor for the development of diabetic cardiomyopathy

Structural aspects and physiological consequences of APP/APLP trans-dimerization

The amyloid precursor protein (APP) is one of the key proteins in Alzheimer’s disease (AD), as it is the precursor of amyloid β (Aβ) peptides accumulating in amyloid plaques. The processing of APP and the pathogenic features of especially Aβ oligomers have been analyzed in detail. Remarkably, there is accumulating evidence from cell biological and structural studies suggesting that APP and its mammalian homologs, the amyloid precursor-like proteins (APLP1 and APLP2), participate under physiological conditions via trans-cellular dimerization in synaptogenesis. This offers the possibility that loss of synapses in AD might be partially explained by dysfunction of APP/APLPs cell adhesion properties. In this review, structural characteristics of APP trans-cellular interaction will be placed critically in context with its putative physiological functions focusing on cell adhesion and synaptogenesis

Secreted Human Amyloid Precursor Protein Binds Semaphorin 3a and Prevents Semaphorin-Induced Growth Cone Collapse

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-β peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα695 (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα695 binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα695 inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (Kd≤8·10−9 M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins

Stroke awareness decreases prehospital delay after acute ischemic stroke in korea

BACKGROUND: Delayed arrival at hospital is one of the major obstacles in enhancing the rate of thrombolysis therapy in patients with acute ischemic stroke. Our study aimed to investigate factors associated with prehospital delay after acute ischemic stroke in Korea. METHODS: A prospective, multicenter study was conducted at 14 tertiary hospitals in Korea from March 2009 to July 2009. We interviewed 500 consecutive patients with acute ischemic stroke who arrived within 48 hours. Univariate and multivariate analyses were performed to evaluate factors influencing prehospital delay. RESULTS: Among the 500 patients (median 67 years, 62% men), the median time interval from symptom onset to arrival was 474 minutes (interquartile range, 170-1313). Early arrival within 3 hours of symptom onset was significantly associated with the following factors: high National Institutes of Health Stroke Scale (NIHSS) score, previous stroke, atrial fibrillation, use of ambulance, knowledge about thrombolysis and awareness of the patient/bystander that the initial symptom was a stroke. Multivariable logistic regression analysis indicated that awareness of the patient/bystander that the initial symptom was a stroke (OR 4.438, 95% CI 2.669-7.381), knowledge about thrombolysis (OR 2.002, 95% CI 1.104-3.633) and use of ambulance (OR 1.961, 95% CI 1.176-3.270) were significantly associated with early arrival. CONCLUSIONS: In Korea, stroke awareness not only on the part of patients, but also of bystanders, had a great impact on early arrival at hospital. To increase the rate of thrombolysis therapy and the incidence of favorable outcomes, extensive general public education including how to recognize stroke symptoms would be important.ope

Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor

The tissue inhibitor of metalloproteinases-3 (TIMP-3) is a major regulator of extracellular matrix turnover and protein shedding by inhibiting different classes of metalloproteinases, including disintegrin metalloproteinases (ADAMs). Tissue bioavailability of TIMP-3 is regulated by the endocytic receptor low-density-lipoprotein receptor-related protein-1 (LRP-1). TIMP-3 plays protective roles in disease. Thus, different approaches have been developed aiming to increase TIMP-3 bioavailability, yet overall effects of increased TIMP-3 in vivo have not been investigated. Herein, by using unbiased mass-spectrometry we demonstrate that TIMP-3-overexpression in HEK293 cells has a dual effect on shedding of transmembrane proteins and turnover of soluble proteins. Several membrane proteins showing reduced shedding are known as ADAM10 substrates, suggesting that exogenous TIMP-3 preferentially inhibits ADAM10 in HEK293 cells. Additionally identified shed membrane proteins may be novel ADAM10 substrate candidates. TIMP-3-overexpression also increased extracellular levels of several soluble proteins, including TIMP-1, MIF and SPARC. Levels of these proteins similarly increased upon LRP-1 inactivation, suggesting that TIMP-3 increases soluble protein levels by competing for their binding to LRP-1 and their subsequent internalization. In conclusion, our study reveals that increased levels of TIMP-3 induce substantial modifications in the cellular secretome and that TIMP-3-based therapies may potentially provoke undesired, dysregulated functions of ADAM10 and LRP-1

Amyloid Precursor Protein Is Required for Normal Function of the Rod and Cone Pathways in the Mouse Retina

Amyloid precursor protein (APP) is a transmembrane glycoprotein frequently studied for its role in Alzheimer's disease. Our recent study in APP knockout (KO) mice identified an important role for APP in modulating normal neuronal development in the retina. However the role APP plays in the adult retina and whether it is required for vision is unknown. In this study we evaluated the role of APP in retinal function and morphology comparing adult wildtype (WT) and APP-KO mice. APP was expressed on neuronal cells of the inner retina, including horizontal, cone bipolar, amacrine and ganglion cells in WT mice. The function of the retina was assessed using the electroretinogram and although the rod photoreceptor responses were similar in APP-KO and WT mice, the post-photoreceptor, inner retinal responses of both the rod and cone pathways were reduced in APP-KO mice. These changes in inner retinal function did not translate to a substantial change in visual acuity as assessed using the optokinetic response or to changes in the gross cellular structure of the retina. These findings indicate that APP is not required for basic visual function, but that it is involved in modulating inner retinal circuitry

Inactivation of DltA Modulates Virulence Factor Expression in Streptococcus pyogenes

D-alanylated lipoteichoic acid is a virtually ubiquitous component of gram-positive cell walls. Mutations in the dltABCD operon of numerous species exhibit pleiotropic effects, including reduced virulence, which has been attributed to increased binding of cationic antimicrobial peptides to the more negatively charged cell surface. In this study, we have further investigated the effects that mutating dltA has on virulence factor expression in Streptococcus pyogenes.Isogenic Delta dltA mutants had previously been created in two distinct M1T1 isolates of S. pyogenes. Immunoblots, flow cytometry, and immunofluorescence were used to quantitate M protein levels in these strains, as well as to assess their ability to bind complement. Bacteria were tested for their ability to interact with human PMN and to grow in whole human blood. Message levels for emm, sic, and various regulatory elements were assessed by quantitative RT-PCR. Cell walls of Delta dltA mutants contained much less M protein than cell walls of parent strains and this correlated with reduced levels of emm transcripts, increased deposition of complement, increased association of bacteria with polymorphonuclear leukocytes, and reduced bacterial growth in whole human blood. Transcription of at least one other gene of the mga regulon, sic, which encodes a protein that inactivates antimicrobial peptides, was also dramatically reduced in Delta dltA mutants. Concomitantly, ccpA and rofA were unaffected, while rgg and arcA were up-regulated.This study has identified a novel mechanism for the reduced virulence of dltA mutants of Streptococcus pyogenes in which gene regulatory networks somehow sense and respond to the loss of DltA and lack of D-alanine esterification of lipoteichoic acid. The mechanism remains to be determined, but the data indicate that the status of D-alanine-lipoteichoic acid can significantly influence the expression of at least some streptococcal virulence factors and provide further impetus to targeting the dlt operon of gram-positive pathogens in the search for novel antimicrobial compounds

The ε3 and ε4 Alleles of Human APOE Differentially Affect Tau Phosphorylation in Hyperinsulinemic and Pioglitazone Treated Mice

Impaired insulin signalling is increasingly thought to contribute to Alzheimer's disease (AD). The ε4 isoform of the APOE gene is the greatest genetic risk factor for sporadic, late onset AD, and is also associated with risk for type 2 diabetes mellitus (T2DM). Neuropathological studies reported the highest number of AD lesions in brain tissue of ε4 diabetic patients. However other studies assessing AD pathology amongst the diabetic population have produced conflicting reports and have failed to show an increase in AD-related pathology in diabetic brain. The thiazolidinediones (TZDs), peroxisome proliferator-activated receptor gamma agonists, are peripheral insulin sensitisers used to treat T2DM. The TZD, pioglitazone, improved memory and cognitive functions in mild to moderate AD patients. Since it is not yet clear how apoE isoforms influence the development of T2DM and its progression to AD, we investigated amyloid beta and tau pathology in APOE knockout mice, carrying human APOEε3 or ε4 transgenes after diet-induced insulin resistance with and without pioglitazone treatment.Male APOE knockout, APOEε3-transgenic and APOEε4-transgenic mice, together with background strain C57BL6 mice were kept on a high fat diet (HFD) or low fat diet (LFD) for 32 weeks, or were all fed HFD for 32 weeks and during the final 3 weeks animals were treated with pioglitazone or vehicle.All HFD animals developed hyperglycaemia with elevated plasma insulin. Tau phosphorylation was reduced at 3 epitopes (Ser396, Ser202/Thr205 and Thr231) in all HFD, compared to LFD, animals independent of APOE genotype. The introduction of pioglitazone to HFD animals led to a significant reduction in tau phosphorylation at the Ser202/Thr205 epitope in APOEε3 animals only. We found no changes in APP processing however the levels of soluble amyloid beta 40 was reduced in APOE knockout animals treated with pioglitazone

Identification of Stk25 as a genetic modifier of Tau phosphorylation in Dab1-mutant mice.

Hyperphosphorylation of the microtubule binding protein Tau is a feature of a number of neurodegenerative diseases, including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. The dab1 gene encodes a docking protein that is required for normal brain lamination and dendritogenesis as part of the Reelin signaling pathway. We show that dab1 gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. Genomic regions that regulate the phospho Tau phenotype in dab1 mutants have previously been identified. Using a microarray gene expression comparison between dab1-mutants from the high-phospho Tau expressing and low-phospho Tau expressing strains, we identified Stk25 as a differentially expressed modifier of dab1-mutant phenotypes. Stk25 knockdown reduces Tau phosphorylation in embryonic neurons. Furthermore, Stk25 regulates neuronal polarization and Golgi morphology in an antagonistic manner to Dab1. This work provides insights into the complex regulation of neuronal behavior during brain development and provides insights into the molecular cascades that regulate Tau phosphorylation