Disease modifying therapy for AD?1

Alzheimer's disease (AD) is the most common form of dementia in industrialized nations. If more effective therapies are not developed that either prevent AD or block progression of the disease in its very early stages, the economic and societal cost of caring for AD patients will be devastating. Only two types of drugs are currently approved for the treatment of AD: inhibitors of acetyl cholinesterase, which symptomatically enhance cognitive state to some degree but are not disease modifying; and the adamantane derivative, memantine. Memantine preferentially blocks excessive NMDA receptor activity without disrupting normal receptor activity and is thought to be a neuroprotective agent that blocks excitotoxicty. Memantine therefore may have a potentially disease modifying effect in multiple neurodegenerative conditions. An improved understanding of the pathogeneses of AD has now led to the identification of numerous therapeutic targets designed to alter amyloid beta protein (Abeta) or tau accumulation. Therapies that alter Abeta and tau through these various targets are likely to have significant disease modifying effects. Many of these targets have been validated in proof of concept studies in preclinical animal models, and some potentially disease modifying therapies targeting Abeta or tau are being tested in the clinic. This review will highlight both the promise of and the obstacles to developing such disease modifying AD therapies.     

C-terminal PAL motif of presenilin and presenilin homologues required for normal active site conformation

The Alzheimer's disease-associated beta-amyloid peptide is produced through cleavage of amyloid precursor protein by beta-secretase and gamma-secretase. gamma-Secretase is a complex containing presenilin (PS) as the catalytic component and three essential cofactors: Nicastrin, anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2). PS and signal peptide peptidase (SPP) define a novel family of aspartyl proteases that cleave substrates within the transmembrane domain presumptively using two membrane-embedded aspartic acid residues for catalysis. Apart from the two aspartate-containing active site motifs, the only other region that is conserved between PS and SPP is a PAL sequence at the C-terminus. Although it has been well documented that this motif is essential for gamma-secretase activity, the mechanism underlying such a critical role is not understood. Here we show that mutations in this motif affect the conformation of the active site of gamma-secretase resulting in a complete loss of PS binding to a gamma-secretase transition state analog inhibitor, Merck C. Analogous mutations in SPP significantly inhibit its enzymatic activity. Furthermore, these mutations also abolish SPP binding to Merck C, indicating that SPP and gamma-secretase share a similar active site conformation, which is dependent on the PAL motif. Exploring the amino acid requirements within this motif reveals a very small side chain requirement, which is conserved during evolution. Together, these observations strongly support the hypothesis that the PAL motif contributes to the active site conformation of gamma-secretase and of SPP.     

Distinct Patterns of DNA Damage Response and Apoptosis Correlate with Jak/Stat and PI3Kinase Response Profiles in Human Acute Myelogenous Leukemia

Background

Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in intracellular signaling responses. Here SCNP was used to characterize Acute Myeloid Leukemia (AML) disease subtypes based on survival, DNA damage response and apoptosis pathways.

Methodology and Principal Findings

Thirty four diagnostic non-M3 AML samples from patients with known clinical outcome were treated with a panel of myeloid growth factors and cytokines, as well as with apoptosis-inducing agents. Analysis of induced Jak/Stat and PI3K pathway responses in blasts from individual patient samples identified subgroups with distinct signaling profiles that were not seen in the absence of a modulator. In vitro exposure of patient samples to etoposide, a DNA damaging agent, revealed three distinct “DNA damage response (DDR)/apoptosis” profiles: 1) AML blasts with a defective DDR and failure to undergo apoptosis; 2) AML blasts with proficient DDR and failure to undergo apoptosis; 3) AML blasts with proficiency in both DDR and apoptosis pathways. Notably, AML samples from clinical responders fell within the “DDR/apoptosis” proficient profile and, as well, had low PI3K and Jak/Stat signaling responses. In contrast, samples from clinical non responders had variable signaling profiles often with in vitro apoptotic failure and elevated PI3K pathway activity. Individual patient samples often harbored multiple, distinct, leukemia-associated cell populations identifiable by their surface marker expression, functional performance of signaling pathway in the face of cytokine or growth factor stimulation, as well as their response to apoptosis-inducing agents.

Conclusions and Significance

Characterizing and tracking changes in intracellular pathway profiles in cell subpopulations both at baseline and under therapeutic pressure will likely have important clinical applications, potentially informing the selection of beneficial targeted agents, used either alone or in combination with chemotherapy.     

Longitudinal axons are guided by Slit/Robo signals from the floor plate

Longitudinal axons grow long distances along precise pathways to connect major CNS regions. However, during embryonic development, it remains largely undefined how the first longitudinal axons choose specific positions and grow along them. Here, we review recent evidence identifying a critical role for Slit/Robo signals to guide pioneer longitudinal axons in the embryonic brain stem. These studies indicate that Slit/Robo signals from the floor plate have dual functions: to repel longitudinal axons away from the ventral midline, and also to maintain straight longitudinal growth. These dual functions likely cooperate with other guidance cues to establish the major longitudinal tracts in the brain.Key words: Slit, Robo, longitudinal axon, hindbrain, axon guidance     

Behaviour and physiology: the thermal strategy of leatherback turtles

Background

Adult leatherback turtles (Dermochelys coriacea) exhibit thermal gradients between their bodies and the environment of ≥8°C in sub-polar waters and ≤4°C in the tropics. There has been no direct evidence for thermoregulation in leatherbacks although modelling and morphological studies have given an indication of how thermoregulation may be achieved.

Methodology/Principal Findings

We show for the first time that leatherbacks are indeed capable of thermoregulation from studies on juvenile leatherbacks of 16 and 37 kg. In cold water (< 25°C), flipper stroke frequency increased, heat loss through the plastron, carapace and flippers was minimized, and a positive thermal gradient of up to 2.3°C was maintained between body and environment. In warm water (25 – 31°C), turtles were inactive and heat loss through their plastron, carapace and flippers increased. The thermal gradient was minimized (0.5°C). Using a scaling model, we estimate that a 300 kg adult leatherback is able to maintain a maximum thermal gradient of 18.2°C in cold sub-polar waters.

Conclusions/Significance

In juvenile leatherbacks, heat gain is controlled behaviourally by increasing activity while heat flux is regulated physiologically, presumably by regulation of blood flow distribution. Hence, harnessing physiology and behaviour allows leatherbacks to keep warm while foraging in cold sub-polar waters and to prevent overheating in a tropical environment.     

A Forward Chemical Screen in Zebrafish Identifies a Retinoic Acid Derivative with Receptor Specificity

Background

Retinoids regulate key developmental pathways throughout life, and have potential uses for differentiation therapy. It should be possible to identify novel retinoids by coupling new chemical reactions with screens using the zebrafish embryonic model.

Principal Findings

We synthesized novel retinoid analogues and derivatives by amide coupling, obtaining 80–92% yields. A small library of these compounds was screened for bioactivity in living zebrafish embryos. We found that several structurally related compounds significantly affect development. Distinct phenotypes are generated depending on time of exposure, and we characterize one compound (BT10) that produces specific cardiovascular defects when added 1 day post fertilization. When compared to retinoic acid (ATRA), BT10 shows similar but not identical changes in the expression pattern of embryonic genes that are known targets of the retinoid pathway. Reporter assays determined that BT10 interacts with all three RAR receptor sub-types, but has no activity for RXR receptors, at all concentrations tested.

Conclusions

Our screen has identified a novel retinoid with specificity for retinoid receptors. This lead compound may be useful for manipulating components of retinoid signaling networks, and may be further derivatized for enhanced activity.     

Real-Time Decision Fusion for Multimodal Neural Prosthetic Devices

Background

The field of neural prosthetics aims to develop prosthetic limbs with a brain-computer interface (BCI) through which neural activity is decoded into movements. A natural extension of current research is the incorporation of neural activity from multiple modalities to more accurately estimate the user''s intent. The challenge remains how to appropriately combine this information in real-time for a neural prosthetic device.

Methodology/Principal Findings

Here we propose a framework based on decision fusion, i.e., fusing predictions from several single-modality decoders to produce a more accurate device state estimate. We examine two algorithms for continuous variable decision fusion: the Kalman filter and artificial neural networks (ANNs). Using simulated cortical neural spike signals, we implemented several successful individual neural decoding algorithms, and tested the capabilities of each fusion method in the context of decoding 2-dimensional endpoint trajectories of a neural prosthetic arm. Extensively testing these methods on random trajectories, we find that on average both the Kalman filter and ANNs successfully fuse the individual decoder estimates to produce more accurate predictions.

Conclusions

Our results reveal that a fusion-based approach has the potential to improve prediction accuracy over individual decoders of varying quality, and we hope that this work will encourage multimodal neural prosthetics experiments in the future.     

Characterization of Coastal Urban Watershed Bacterial Communities Leads to Alternative Community-Based Indicators

Background

Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring.

Methodology/Principal Findings

Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and α-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC∶A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies.

Conclusions/Significance

This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health.     

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

Background

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species.

Methods and Findings

We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity.

Conclusions

The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.     

Identification of a new uracil-DNA glycosylase family by expression cloning using synthetic inhibitors

BACKGROUND: The cellular environment exposes DNA to a wide variety of endogenous and exogenous reactive species that can damage DNA, thereby leading to genetic mutations. DNA glycosylases protect the integrity of the genome by catalyzing the first step in the base excision-repair of lesions in DNA. RESULTS: Here, we report a strategy to conduct genome-wide screening for expressed DNA glycosylases, based on their ability to bind to a library of four synthetic inhibitors that target the enzyme's active site. These inhibitors, used in conjunction with the in vitro expression cloning procedure, led to the identification of novel Xenopus and human proteins, xSMUG1 and hSMUG1, respectively, that efficiently excise uracil residues from DNA. Despite a lack of statistically significant overall sequence similarity to the two established classes of uracil-DNA glycosylases, the SMUG1 enzymes contain motifs that are hallmarks of a shared active-site structure and overall protein architecture. The unusual preference of SMUG1 for single-stranded rather than double-stranded DNA suggests a unique biological function in ridding the genome of uracil residues, which are potent endogenous mutagens. CONCLUSIONS: The 'proteomics' approach described here has led to the isolation of a new family of uracil-DNA glycosylases. The three classes of uracil-excising enzymes (SMUG1 being the most recently discovered) represent a striking example of structural and functional conservation in the almost complete absence of sequence conservation.