Specific Neurochemical Derangements of Brain Projecting Neurons in Apolipoprotein E-Deficient Mice

Abstract: Apolipoprotein E (apoE)-deficient mice provide a useful system for studying the role of apoE in neuronal maintenance and repair. Previous studies revealed specific memory impairments in these mice that are associated with presynaptic derangements in projecting forebrain cholinergic neurons. In the present study we examined whether dopaminergic, noradrenergic, and serotonergic projecting pathways of apoE-deficient mice are also affected and investigated the mechanisms that render them susceptible. The densities of nerve terminals of forebrain cholinergic projections were monitored histochemically by measurements of acetylcholinesterase activity, whereas those of the dopaminergic nigrostriatal pathway, the noradrenergic locus coeruleus cortical projection, and the raphe-cortical serotonergic tract were measured autoradiographically using radioligands that bind specifically to the respective presynaptic transporters of these neuronal tracts. The results obtained revealed that synaptic densities of cholinergic, noradrenergic, and serotonergic projections in specific brain regions of apoE-deficient mice are markedly lower than those of controls. Furthermore, the extent of presynaptic derangement within each of these tracts was found to be more pronounced the further away the nerve terminal is from its cell body. In contrast, the nerve terminal density of the dopaminergic neurons that project from the substantia nigra to the striatum was unaffected and was similar to that of the controls. The rank order of these presynaptic derangements at comparable distances from the respective cell bodies was found to be septohippocampal cholinergic > nucleus basalis cholinergic > locus coeruleus adrenergic > raphe serotonergic ? nigrostriatal dopaminergic, which interestingly is similar to that observed in Alzheimer's disease. These results suggest that two complementary factors determine the susceptibility of brain projecting neurons to apoE deficiency: pathway-specific differences and the distance of the nerve terminals from their cell body.     

Cortactin releases the brakes in actin- based motility by enhancing WASP-VCA detachment from Arp2/3 branches

Cortactin is involved in invadopodia and podosome formation [1], pathogens and endosome motility [2], and persistent lamellipodia protrusion [ [3] and [4] ]; its overexpression enhances cellular motility and metastatic activity [ [5] , [6] , [7] and [8] ]. Several mechanisms have been proposed to explain cortactin's role in Arp2/3-driven actin polymerization [ [9] and [10] ], yet its direct role in cell movement remains unclear. We use a biomimetic system to study the mechanism of cortactin-mediated regulation of actin-driven motility [11]. We tested the role of different cortactin variants that interact with Arp2/3 complex and actin filaments distinctively. We show that wild-type cortactin significantly enhances the bead velocity at low concentrations. Single filament experiments show that cortactin has no significant effect on actin polymerization and branch stability, whereas it strongly affects the branching rate driven by Wiskott-Aldrich syndrome protein (WASP)-VCA fragment and Arp2/3 complex. These results lead us to propose that cortactin plays a critical role in translating actin polymerization at a bead surface into motion, by releasing WASP-VCA from the new branching site. This enhanced release has two major effects: it increases the turnover rate of branching per WASP molecule, and it decreases the friction-like force caused by the binding of the moving surface with respect to the growing actin network.     

A de novo protein binding pair by computational design and directed evolution

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.     

Pathogen detection using short-RNA deep sequencing subtraction and assembly

MOTIVATION: Early and accurate detection of human pathogen infection is critical for treatment and therapeutics. Here we describe pathogen identification using short RNA subtraction and assembly (SRSA), a detection method that overcomes the requirement of prior knowledge and culturing of pathogens, by using degraded small RNA and deep sequencing technology. We prove our approach's efficiency through identification of a combined viral and bacterial infection in human cells.     

Identification of Anaplasma marginale type IV secretion system effector proteins

Background

Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.

Results

By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141) of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system.

Conclusions

The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work.     

Core promoter binding by histone-like TAF complexes

A major function of TFIID is core promoter recognition. TFIID consists of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Most of them contain a histone fold domain (HFD) that lacks the DNA-contacting residues of histones. Whether and how TAF HFDs contribute to core promoter DNA binding are yet unresolved. Here we examined the DNA binding activity of TAF9, TAF6, TAF4b, and TAF12, which are related to histones H3, H4, H2A, and H2B, respectively. Each of these TAFs has intrinsic DNA binding activity adjacent to or within the HFD. The DNA binding domains were mapped to evolutionarily conserved and essential regions. Remarkably, HFD-mediated interaction enhanced the DNA binding activity of each of the TAF6-TAF9 and TAF4b-TAF12 pairs and of a histone-like octamer complex composed of the four TAFs. Furthermore, HFD-mediated interaction stimulated sequence-specific binding by TAF6 and TAF9. These results suggest that TAF HFDs merge with other conserved domains for efficient and specific core promoter binding.     

The adipocyte as an important target cell for Trypanosoma cruzi infection

Adipose tissue plays an active role in normal metabolic homeostasis as well as in the development of human disease. Beyond its obvious role as a depot for triglycerides, adipose tissue controls energy expenditure through secretion of several factors. Little attention has been given to the role of adipocytes in the pathogenesis of Chagas disease and the associated metabolic alterations. Our previous studies have indicated that hyperglycemia significantly increases parasitemia and mortality in mice infected with Trypanosoma cruzi. We determined the consequences of adipocyte infection in vitro and in vivo. Cultured 3T3-L1 adipocytes can be infected with high efficiency. Electron micrographs of infected cells revealed a large number of intracellular parasites that cluster around lipid droplets. Furthermore, infected adipocytes exhibited changes in expression levels of a number of different adipocyte-specific or adipocyte-enriched proteins. The adipocyte is therefore an important target cell during acute Chagas disease. Infection of adipocytes by T. cruzi profoundly influences the pattern of adipokines. During chronic infection, adipocytes may represent an important long-term reservoir for parasites from which relapse of infection can occur. We have demonstrated that acute infection has a unique metabolic profile with a high degree of local inflammation in adipose tissue, hypoadiponectinemia, hypoglycemia, and hypoinsulinemia but with relatively normal glucose disposal during an oral glucose tolerance test.     

Confocal-based cell-specific finite element modeling extended to study variable cell shapes and intracellular structures: The example of the adipocyte

This communication extends the recently reported cell-specific finite element (FE) method in Slomka and Gefen (2010) in which geometrically realistic FE cell models are created from confocal microscopy scans for large deformation analyses. The cell-specific FE method is extended here in the following aspects: (i) we demonstrate that cell-specific FE is versatile enough to deal with cells of substantially different geometrical shapes. The examples of an “elongated” pre-adipocyte and a “round” mature adipocyte are used to demonstrate this feature. (ii) We demonstrate that cell-specific FE can be used to analyze the mechanical behavior of cells that incorporate complex intracellular structures and are subjected to large deformations—again through the example of an adipocyte which contains a multitude of lipid droplets, each having a different size and shape. By demonstrating feasibility of inclusion of such inhomogeneities in the cytoplasm, the present work paves the way for modeling cellular organelles such as Golgi bodies, lysosomes and mitochondria in mechanically loaded cells using cell-specific FE.