High content screening in neurodegenerative diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency(1-4). Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation(5). For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known(6, 7). As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity(8). This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention. High-throughput screening (HTS) is an ideal methodology to address these issues(9-12). but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties(13, 14). This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)(4, 15-17). HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. HCS has many advantages over HTS(18, 19), but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing(20) and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism(21). Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets

Caltubin, a Novel Molluscan Tubulin-Interacting Protein, Promotes Axonal Growth and Attenuates Axonal Degeneration of Rodent Neurons

Axotomized central neurons of most invertebrate species demonstrate a strong regenerative capacity, and as such may provide valuable molecular insights and new tools to promote axonal regeneration in injured mammalian neurons. In this study, we identified a novel molluscan protein, caltubin, ubiquitously expressed in central neurons of Lymnaea stagnalis and locally synthesized in regenerating neurites. Reduction of caltubin levels by gene silencing inhibits the outgrowth and regenerative ability of adult Lymnaea neurons and decreases local α- and β-tubulin levels in neurites. Caltubin binds to α- and/or β-tubulin in both Lymnaea and rodent neurons. Expression of caltubin in PC12 cells and mouse cortical neurons promotes NGF-induced axonal outgrowth and attenuates axonal retraction after injury. This is the first study illustrating that a xenoprotein can enhance outgrowth and prevent degeneration of injured mammalian neurons. These results may open up new avenues in molecular repair strategies through the insertion of molecular components of invertebrate regenerative pathways into mammalian neurons. ©2011 the authors

Synaptic proteome changes in a DNA repair deficient Ercc1 mouse model of accelerated aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used Ercc1 mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged Ercc1 mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and Ercc1 mutant hippocampal neurons displayed normal outgrowth and synapse formation in vitro. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline

Exciton Spin Dynamics in Semiconductor Quantum Wells

In this paper we will review Exciton Spin Dynamics in Semiconductor Quantum Wells. The spin properties of excitons in nanostructures are determined by their fine structure. We will mainly focus in this review on GaAs and InGaAs quantum wells which are model systems.Comment: 55 pages, 27 figure

Somatic TARDBP variants as a cause of semantic dementia

The aetiology of late-onset neurodegenerative diseases is largely unknown. Here we investigated whether de novo somatic variants for semantic dementia can be detected, thereby arguing for a more general role of somatic variants i