Behavioral and other phenotypes in a cytoplasmic Dynein light intermediate chain 1 mutant mouse

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system (1-5). To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex (6-8). Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions. Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method( 9). However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice (10-11) (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA (12). These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C). Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines

Signatures of natural selection between life cycle stages separated by metamorphosis in European eel

Received: 16 December 2014, Accepted: 6 July 2015, Published: 13 August 2015[Background] Species showing complex life cycles provide excellent opportunities to study the genetic associations between life cycle stages, as selective pressures may differ before and after metamorphosis. The European eel presents a complex life cycle with two metamorphoses, a first metamorphosis from larvae into glass eels (juvenile stage) and a second metamorphosis into silver eels (adult stage). We tested the hypothesis that different genes and gene pathways will be under selection at different life stages when comparing the genetic associations between glass eels and silver eels.[Results] We used two sets of markers to test for selection: first, we genotyped individuals using a panel of 80 coding-gene single nucleotide polymorphisms (SNPs) developed in American eel; second, we investigated selection at the genome level using a total of 153,423 RAD-sequencing generated SNPs widely distributed across the genome. Using the RAD approach, outlier tests identified a total of 2413 (1.57 %) potentially selected SNPs. Functional annotation analysis identified signal transduction pathways as the most over-represented group of genes, including MAPK/Erk signalling, calcium signalling and GnRH (gonadotropin-releasing hormone) signalling. Many of the over-represented pathways were related to growth, while others could result from the different conditions that eels inhabit during their life cycle.[Conclusions] The observation of different genes and gene pathways under selection when comparing glass eels vs. silver eels supports the adaptive decoupling hypothesis for the benefits of metamorphosis. Partitioning the life cycle into discrete morphological phases may be overall beneficial since it allows the different life stages to respond independently to their unique selection pressures. This might translate into a more effective use of food and niche resources and/or performance of phase-specific tasks (e.g. feeding in the case of glass eels, migrating and reproducing in the case of silver eels).We acknowledge funding from the Danish Council for Independent Reasearch, Natural Sciences (grant 09-072120 to MMH).Peer reviewe

Behavioral and Other Phenotypes in a Cytoplasmic Dynein Light Intermediate Chain 1 Mutant Mouse

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system

Two-way engagement and communication strategies for health and medical research participants: a rapid evidence review

This project is seeking to determine what two-way engagement and communication strategies have been trialled in health and medical research context. It focuses on implementations that have been evaluated, and what the outcomes were for participants and research projects

MicroRNAs: Not "Fine-Tuners" but Key Regulators of Neuronal Development and Function

No grant i

Defining terms associated with genomic findings beyond scope of the original test: A scoping review

Using scoping review methodology, we aim to identify and map current terminology use related to genomic findings that are unrelated to original intent of the test (e.g. secondary/incidental/additional findings)

The ethics approval process for multisite research studies in Australia: changes sought by the Australian Genomics initiative

Australian Genomics is calling for a change in research ethics and governance frameworks Australian Genomics is a national initiative building evidence to ensure the effective implementation of genomic medicine into Australian health care (www.australiangenomics.org.au). The research program is embedded in clinical practice, with 5000 patients with rare diseases and cancers being prospectively recruited for genomic testing into clinical flagship projects through 31 hospitals across Australia (Box 1). Achieving national recruitment will ensure that the clinical, diagnostic and research pathways are developed through the infrastructure and workforce in each jurisdiction. We initiated the research ethics and governance approval process for our multisite human research project, which was eligible for single ethical review by one Human Research Ethics Committee under the Australian National Mutual Acceptance (NMA) framework (Box 2), and recorded details relating to our experience in navigating the research ethics and governance system. This included any site‐specific assessment (SSA) requirements, review time, personnel costs, and causes of delay. When NMA was introduced, it was envisaged that the reform would consolidate a nationalised ethics review system.1 Internationally, Australia's NMA ethics review process has been lauded as a streamlined system, leading the way for other countries.2, 3 In the United States and Canada, the institutional review board system requires researchers to apply to each institution in a multicentre study. Researchers report little harmonisation in application requirements, considerable expense and time to prepare applications, and a lack of consistency in institutional review board response to projects in multicentre studies.2 However, Canada and the US have initiated single multisite review systems. Implementation in Canada will be relevant to Australia's situation, as they share a similar federated model of government. Until recently, in the United Kingdom, multicentre studies were served by Research Ethics Committees, with local Research Ethics Committees charged with subsequently reviewing projects for local issues. Three years after the introduction of this system in 1997, one study, in which a multicentre Research Ethics Committee‐approved project was then propagated to 125 local Research Ethics Committees, found that while approval times had improved, 67% of changes requested by local Research Ethics Committees were considered non‐local and thus outside their prerogative. Issues also remained around costs to prepare applications and transparency of information for researchers.4 These issues resonate with our experience of NMA in Australia. In 2015, a new Health Research Authority run by the National Health Service and backed by new legislation was introduced in the UK to manage ethics approvals nationally, in a model reform Australia could consider. The Human Heredity and Health in Africa initiative is undertaking the ethics review process for genomic studies across Africa. A recent report on the challenges faced by this initiative suggested that the main barrier to ethics approval is a lack of genomic expertise within ethics committees.5 With increasing international data sharing efforts, internationally compatible solutions to research ethics issues need to be developed. The Global Alliance for Genomics and Health has developed an ethics review recognition policy,6 and Australia could continue to demonstrate leadership internationally if remaining challenges to multijurisdictional research were addressed.The Australian Genomics Health Alliance is funded by a National Health and Medical Research Council grant (Grant Reference No. 1113531) and the Australian Government’s Medical Research Future Fun

Bacurd2 is a novel interacting partner to Rnd2 which controls radial migration within the developing mammalian cerebral cortex.

BACKGROUND: During fetal brain development in mammals, newborn neurons undergo cell migration to reach their appropriate positions and form functional circuits. We previously reported that the atypical RhoA GTPase Rnd2 promotes the radial migration of mouse cerebral cortical neurons (Nature 455(7209):114-8, 2008; Neuron 69(6):1069-84, 2011), but its downstream signalling pathway is not well understood. RESULTS: We have identified BTB-domain containing adaptor for Cul3-mediated RhoA degradation 2 (Bacurd2) as a novel interacting partner to Rnd2, which promotes radial migration within the developing cerebral cortex. We find that Bacurd2 binds Rnd2 at its C-terminus, and this interaction is critical to its cell migration function. We show that forced expression or knockdown of Bacurd2 impairs neuronal migration within the embryonic cortex and alters the morphology of immature neurons. Our in vivo cellular analysis reveals that Bacurd2 influences the multipolar-to-bipolar transition of radially migrating neurons in a cell autonomous fashion. When we addressed the potential signalling relationship between Bacurd2 and Rnd2 using a Bacurd2-Rnd2 chimeric construct, our results suggest that Bacurd2 and Rnd2 could interact to promote radial migration within the embryonic cortex. CONCLUSIONS: Our studies demonstrate that Bacurd2 is a novel player in neuronal development and influences radial migration within the embryonic cerebral cortex

Identification and characterization of a population of motile neurons in long-term cortical culture

The specific phenotypes and progression to maturity of primary cortical neurons in long-term culture correlate well with neurons in vivo. Utilizing a model of neuronal injury in long-term cultures at 21 days in vitro (DIV), we have identified a distinct population of neurons that translocate into the injury site. 5-Bromo-2'-deoxyUridine (BrdU) incorporation studies demonstrated that neurons with the capacity to translocate were 21 days old. However, this motile ability is not consistent with the traditional view of the maturation and structural stability of neurons in long-term culture. Therefore, we examined the neurons' cytoskeletal profile using immunocytochemistry, to establish relative stage of maturation and phenotype. Expression of marker proteins including β-III-tubulin, α-internexin, NF-L and NF-M, tau and L1 indicated the neurons were differentiated, and in some cases polarized. The neurons did not immunolabel with NF-H or MAP2, which might suggest they had not reached the level of maturity of other neurons in culture. They did not express the microtubule-associated migration marker doublecortin (DCX). Cytoskeletal disrupting agents were used to further investigate the role of the microtubule cytoskeleton in translocation, and microtubule destabilization significantly enhanced aspects of their motility. Finally, molecular guidance cues affected their motility in a similar manner to that reported for both axon guidance and early neuron migration. Therefore, this study has identified and characterized a population of motile neurons in vitro that have the capacity to migrate into a site of injury. These studies provide new information on the structurally dynamic features of subsets of neurons.14 page(s