Interactome of the Amyloid Precursor Protein APP in Brain Reveals a Protein Network Involved in Synaptic Vesicle Turnover and a Close Association with Synaptotagmin-1

Knowledge of the protein networks interacting with the amyloid precursor protein (APP) in vivo can shed light on the physiological function of APP. To date, most proteins interacting with the APP intracellular domain (AICD) have been identified by Yeast Two Hybrid screens which only detect direct interaction partners. We used a proteomics-based approach by biochemically isolating tagged APP from the brains of transgenic mice and subjecting the affinity-purified complex to mass spectrometric (MS) analysis. Using two different quantitative MS approaches, we compared the protein composition of affinity-purified samples isolated from wild-type mice versus transgenic mice expressing tagged APP. This enabled us to assess truly enriched proteins in the transgenic sample and yielded an overlapping set of proteins containing the major proteins involved in synaptic vesicle endo- and exocytosis. Confocal microscopy analyses of cotransfected primary neurons showed colocalization of APP with synaptic vesicle proteins in vesicular structures throughout the neurites. We analyzed the interaction of APP with these proteins using pulldown experiments from transgenic mice or cotransfected cells followed by Western blotting. Synaptotagmin-1 (Stg1), a resident synaptic vesicle protein, was found to directly bind to APP. We fused Citrine and Cerulean to APP and the candidate proteins and measured fluorescence resonance energy transfer (FRET) in differentiated SH-SY5Y cells. Differentially tagged APPs showed clear sensitized FRET emission, in line with the described dimerization of APP. Among the candidate APP-interacting proteins, again only Stg1 was in close proximity to APP. Our results strongly argue for a function of APP in synaptic vesicle turnover in vivo. Thus, in addition to the APP cleavage product Aβ, which influences synaptic transmission at the postsynapse, APP interacts with the calcium sensor of synaptic vesicles and might thus play a role in the regulation of synaptic vesicle exocytosis.     

Validating Serum S100B and Neuron-Specific Enolase as Biomarkers for the Human Brain - A Combined Serum, Gene Expression and MRI Study

Introduction

Former studies have investigated the potential of serum biomarkers for diseases affecting the human brain. In particular the glial protein S100B, a neuro- and gliotrophin inducing plasticity, seems to be involved in the pathogenesis and treatment of psychiatric diseases such as major depression and schizophrenia. Neuron-specific enolase (NSE) is a specific serum marker for neuronal damage. However, the specificity of these biomarkers for cell type and brain region has not been investigated in vivo until now.

Methods

We acquired two magnetic resonance imaging parameters sensitive to changes in gray and white matter (T₁-weighted/diffusion tensor imaging) and obtained serum S100B and NSE levels of 41 healthy subjects. Additionally, we analyzed whole brain gene expressions of S100B in another male cohort of three subjects using the Allen Brain Atlas. Furthermore, a female post mortal brain was investigated using double immunofluorescence labelling with oligodendrocyte markers.

Results

We show that S100B is specifically related to white matter structures, namely the corpus callosum, anterior forceps and superior longitudinal fasciculus in female subjects. This effect was observed in fractional anisotropy and radial diffusivity – the latest an indicator of myelin changes. Histological data confirmed a co-localization of S100B with oligodendrocyte markers in the human corpus callosum. S100B was most abundantly expressed in the corpus callosum according to the whole genome Allen Human Brain Atlas. In addition, NSE was related to gray matter structures, namely the amygdala. This effect was detected across sexes.

Conclusion

Our data demonstrates a very high S100B expression in white matter tracts, in particular in human corpus callosum. Our study is the first in vivo study validating the specificity of the glial marker S100B for the human brain, and supporting the assumption that radial diffusivity represents a myelin marker. Our results open a new perspective for future studies investigating major neuropsychiatric disorders.     

CXC Chemokine Receptor 7 (CXCR7) Regulates CXCR4 Protein Expression and Capillary Tuft Development in Mouse Kidney

Background

The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development.

Methodology/Principal Findings

We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 null mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 null mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 null mice was associated with mesangial cell clumping.

Conclusions/Significance

We established that there is a similar glomerular pathology in CXCR7 and CXCR4 null embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.     

Clonal Analysis in Recurrent Astrocytic, Oligoastrocytic and Oligodendroglial Tumors Implicates IDH1- Mutation as Common Tumor Initiating Event

Background

To investigate the dynamics of inter- and intratumoral molecular alterations during tumor progression in recurrent gliomas.

Methodology/Principal Findings

To address intertumoral heterogeneity we investigated non- microdissected tumor tissue of 106 gliomas representing 51 recurrent tumors. To address intratumoral heterogeneity a set of 16 gliomas representing 7 tumor pairs with at least one recurrence, and 4 single mixed gliomas were investigated by microdissection of distinct oligodendroglial and astrocytic tumor components. All tumors and tumor components were analyzed for allelic loss of 1p/19q (LOH 1p/19q), for TP53- mutations and for R132 mutations in the IDH1 gene. The investigation of non- microdissected tumor tissue revealed clonality in 75% (38/51). Aberrant molecular alterations upon recurrence were detected in 25% (13/51). 64% (9/14) of these were novel and associated with tumor progression. Loss of previously detected alterations was observed in 36% (5/14). One tumor pair (1/14; 7%) was significant for both. Intratumoral clonality was detected in 57% (4/7) of the microdissected tumor pairs and in 75% (3/4) of single microdissected tumors. 43% (3/7) of tumor pairs and one single tumor (25%) revealed intratumoral heterogeneity. While intratumoral heterogeneity affected both the TP53- mutational status and the LOH1p/19q status, all tumors with intratumoral heterogeneity shared the R132 IDH1- mutation as a common feature in both their microdissected components.

Conclusions/Significance

The majority of recurrent gliomas are of monoclonal origin. However, the detection of divertive tumor cell clones in morphological distinct tumor components sharing IDH1- mutations as early event may provide insight into the tumorigenesis of true mixed gliomas.     

Defining the Antigenic Diversity of Plasmodium falciparum Apical Membrane Antigen 1 and the Requirements for a Multi-Allele Vaccine against Malaria

Apical Membrane Antigen 1 (AMA1) is a leading malaria vaccine candidate and a target of naturally-acquired human immunity. Plasmodium falciparum AMA1 is polymorphic and in vaccine trials it induces strain-specific protection. This antigenic diversity is a major roadblock to development of AMA1 as a malaria vaccine and understanding how to overcome it is essential. To assess how AMA1 antigenic diversity limits cross-strain growth inhibition, we assembled a panel of 18 different P. falciparum isolates which are broadly representative of global AMA1 sequence diversity. Antibodies raised against four well studied AMA1 alleles (W2Mef, 3D7, HB3 and FVO) were tested for growth inhibition of the 18 different P. falciparum isolates in growth inhibition assays (GIA). All antibodies demonstrated substantial cross-inhibitory activity against different isolates and a mixture of the four different AMA1 antibodies inhibited all 18 isolates tested, suggesting significant antigenic overlap between AMA1 alleles and limited antigenic diversity of AMA1. Cross-strain inhibition by antibodies was only moderately and inconsistently correlated with the level of sequence diversity between AMA1 alleles, suggesting that sequence differences are not a strong predictor of antigenic differences or the cross-inhibitory activity of anti-allele antibodies. The importance of the highly polymorphic C1-L region for inhibitory antibodies and potential vaccine escape was assessed by generating novel transgenic P. falciparum lines for testing in GIA. While the polymorphic C1-L epitope was identified as a significant target of some growth-inhibitory antibodies, these antibodies only constituted a minor proportion of the total inhibitory antibody repertoire, suggesting that the antigenic diversity of inhibitory epitopes is limited. Our findings support the concept that a multi-allele AMA1 vaccine would give broad coverage against the diversity of AMA1 alleles and establish new tools to define polymorphisms important for vaccine escape.     

Symbiont recruitment versus ant-symbiont co-evolution in the attine ant-microbe symbiosis

The symbiosis between fungus-farming ants (Attini, Formicidae), their cultivated fungi, garden-infecting Escovopsis pathogens, and Pseudonocardia bacteria on the ant integument has been popularized as an example of ant-Escovopsis-Pseudonocardia co-evolution. Recent research could not verify earlier conclusions regarding antibiotic-secreting, integumental Pseudonocardia that co-evolve to specifically suppress Escovopsis disease in an ancient co-evolutionary arms-race. Rather than long-term association with a single, co-evolving Pseudonocardia strain, attine ants accumulate complex, dynamic biofilms on their integument and in their gardens. Emerging views are that the integumental biofilms protect the ants primarily against ant diseases, whereas garden biofilms protect primarily against garden diseases; attine ants selectively recruit ('screen in') microbes into their biofilms; and the biofilms of ants and gardens serve diverse functions beyond disease-suppression.     

DAF-fluorescence without NO: elicitor treated tobacco cells produce fluorescing DAF-derivatives not related to DAF-2 triazol

Diaminofluorescein-dyes (DAFs) are widely used for visualizing NO· production in biological systems. Here it was examined whether DAF-fluorescence could be evoked by other means than nitrosation. Tobacco (Nicotiana tabacum) suspension cells treated with the fungal elicitor cryptogein released compound(s) which gave a fluorescence increase in the cell-free filtrate after addition of DAF-2 or DAF-FM or DAR-4M. DAF-reactive compounds were relatively stable and identified as reaction products of H(2)O(2) plus apoplastic peroxidase (PO). CPTIO prevented formation of these products. Horseradish-peroxidase (HR-PO) plus H(2)O(2) also generated DAF-fluorescence in vitro. Using RP-HPLC with fluorescence detection, DAF derivatives were further analyzed. In filtrates from cryptogein-treated cells, fluorescence originated from two novel DAF-derivatives also obtained in vitro with DAF-2+HR-PO+H(2)O(2). DAF-2T was only detected when an NO donor (DEA-NO) was present. Using high resolution mass spectrometry, the two above-described novel DAF-reaction products were tentatively identified as dimers. In cells preloaded with DAF-2 DA and incubated with or without cryptogein, DAF-fluorescence originated from a complex pattern of multiple products different from those obtained in vitro. One specific peak was responsive to exogenous H(2)O(2), and another, minor peak eluted at or close to DAF-2T. Thus, in contrast to the prevailing opinion, DAF-2 can be enzymatically converted into a variety of highly fluorescing derivatives, both inside and outside cells, of which none (outside) or only a minor part (inside) appeared NO· dependent. Accordingly, DAF-fluorescence and its prevention by cPTIO do not necessarily indicate NO· production.     

Metal-induced DNA translocation leads to DNA polymerase conformational activation

Binding of the catalytic divalent ion to the ternary DNA polymerase β/gapped DNA/dNTP complex is thought to represent the final step in the assembly of the catalytic complex and is consequently a critical determinant of replicative fidelity. We have analyzed the effects of Mg(2+) and Zn(2+) on the conformational activation process based on NMR measurements of [methyl-(13)C]methionine DNA polymerase β. Unexpectedly, both divalent metals were able to produce a template base-dependent conformational activation of the polymerase/1-nt gapped DNA complex in the absence of a complementary incoming nucleotide, albeit with different temperature thresholds. This conformational activation is abolished by substituting Glu295 with lysine, thereby interrupting key hydrogen bonds necessary to stabilize the closed conformation. These and other results indicate that metal-binding can promote: translocation of the primer terminus base pair into the active site; expulsion of an unpaired pyrimidine, but not purine, base from the template-binding pocket; and motions of polymerase subdomains that close the active site. We also have performed pyrophosphorolysis studies that are consistent with predictions based on these results. These findings provide new insight into the relationships between conformational activation, enzyme activity and polymerase fidelity.     

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37°C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.     

CCL3 induced migration occurs independently of intracellular calcium release

The molecular signalling pathway of cell migration and whether it can occur independently of the release of intracellular calcium is still not completely understood. Therefore we investigated here the molecular mechanisms of CCL3 induced cell migration and the importance of intracellular calcium for chemotaxis in more detail. We show that CCL3 induced cell migration is dependent on activation of PLC. Several PKC inhibitors block the release of intracellular calcium independently of CCL3 activation and do not affect cell migration. This confirms that the release of intracellular calcium is not necessary for chemotaxis towards CCL3 and that PKC inhibitors should be used with caution in calcium release assays.