Sensory Transduction Channel Subunits, tax-4 and tax-2, Modify Presynaptic Molecular Architecture in C. elegans

During development, neural activity is important for forming proper connections in neural networks. The effect of activity on the gross morphology and synaptic strength of neurons has been well documented, but little is known about how activity affects different molecular components during development. Here, we examine the localization of four fluorescently-tagged presynaptic proteins, RAB-3, SNG-1/synaptogyrin, SYD-2/Liprin-α, and SAD-1/SAD kinase, in the C. elegans thermosensory neuron AFD. We show that tax-4 and tax-2, two genes that encode the cyclic nucleotide-gated channel necessary for sensory transduction in AFD, disrupt the localization of all four proteins. In wild-type animals, the synaptic vesicle (SV) markers RAB-3 and SNG-1 and the active zone markers SYD-2 and SAD-1 localize in a stereotyped, punctate pattern in the AFD axon. In tax-4 and tax-2 mutants, SV and SYD-2 puncta are more numerous and less intense. Interestingly, SAD-1 puncta are also less intense but do not increase in number. The change in puncta number can be rescued cell-autonomously in AFD. These results suggest that sensory transduction genes tax-4 and tax-2 are necessary for the proper assembly of presynapses

Development and validation of the Australian Aboriginal racial identity and self-esteem survey for 8-12 year old children (IRISE-C)

Introduction: In Australia, there is little empirical research of the racial identity of Indigenous children and youth as the majority of the current literature focuses on adults. Furthermore, there are no instruments developed with cultural appropriateness when exploring the identity and self-esteem of the Australian Aboriginal population, especially children. The IRISE-C (Racial Identity and Self-Esteem of children) inventory was developed to explore the elements of racial identity and self-esteem of urban, rural and regional Aboriginal children. This paper describes the development and validation of the IRISE-C instrument with over 250 Aboriginal children aged 8 to 12 years. Methods: A pilot of the IRISE C instrument was combined with individual interviews and was undertaken with 35 urban Aboriginal children aged 8-12 years. An exploratory factor analysis was performed to refine the survey and reduce redundant items in readiness for the main study. In the main study, the IRISE C was employed to 229 Aboriginal children aged 6-13 years across three sites (rural, regional and urban) in Western Australia. An exploratory factor analysis using Principal axis factoring was used to assess the fit of items and survey structure. A confirmatory factor analysis was then employed using LISREL (diagonally weighted least squares) to assess factor structures across domains. Internal consistency and reliability of subscales were assessed using Cronbach's co-efficient alpha. Results: The pilot testing identified two key concepts - children's knowledge of issues related to their racial identity, and the importance, or salience, that they attach to these issues. In the main study, factor analyses showed two clear factors relating to: Aboriginal culture and traditions; and a sense of belonging to an Aboriginal community. Principal Axis Factoring of the Knowledge items supported a 2-factor solution, which explained 38.7 % of variance. Factor One (Aboriginal culture) had a Cronbach's alpha of 0.835; Factor 2 (racial identity) had a Cronbach's alpha of 0.800, thus demonstrating high internal reliability of the scales. Conclusion: The IRISE-C has been shown to be a valid instrument useful of exploring the development of racial identity of Australian Aboriginal children across the 8-12 year old age range and across urban, rural and regional geographical locations

The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

Opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated by direct binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus. Here, we show for the first time that in the HCN2 channel cGMP can also exert an inhibitory effect on gating via cGMP-dependent protein kinase II (cGKII)-mediated phosphorylation. Using coimmunoprecipitation and immunohistochemistry we demonstrate that cGKII and HCN2 interact and colocalize with each other upon heterologous expression as well as in native mouse brain. We identify the proximal C-terminus of HCN2 as binding region of cGKII and show that cGKII phosphorylates HCN2 at a specific serine residue (S641) in the C-terminal end of the CNBD. The cGKII shifts the voltage-dependence of HCN2 activation to 2–5 mV more negative voltages and, hence, counteracts the stimulatory effect of cGMP on gating. The inhibitory cGMP effect can be either abolished by mutation of the phosphorylation site in HCN2 or by impairing the catalytic domain of cGKII. By contrast, the inhibitory effect is preserved in a HCN2 mutant carrying a CNBD deficient for cGMP binding. Our data suggest that bidirectional regulation of HCN2 gating by cGMP contributes to cellular fine-tuning of HCN channel activity

Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo.

Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNAAla mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin

Mediator Subunit 12 Is Required for Neutrophil Development in Zebrafish

Hematopoiesis requires the spatiotemporal organization of regulatory factors to successfully orchestrate diverse lineage specificity from stem and progenitor cells. Med12 is a regulatory component of the large Mediator complex that enables contact between the general RNA polymerase II transcriptional machinery and enhancer bound regulatory factors. We have identified a new zebrafish med12 allele, syr, with a single missense mutation causing a valine to aspartic acid change at position 1046. Syr shows defects in hematopoiesis, which predominantly affect the myeloid lineage. Syr has identified a hematopoietic cell-specific requirement for Med12, suggesting a new role for this transcriptional regulator

High-Pass Filtering of Input Signals by the Ih Current in a Non-Spiking Neuron, the Retinal Rod Bipolar Cell

Hyperpolarization–activated cyclic nucleotide–sensitive (HCN) channels mediate the If current in heart and Ih throughout the nervous system. In spiking neurons Ih participates primarily in different forms of rhythmic activity. Little is known, however, about its role in neurons operating with graded potentials as in the retina, where all four channel isoforms are expressed. Intriguing evidence for an involvement of Ih in early visual processing are the side effects reported, in dim light or darkness, by cardiac patients treated with HCN inhibitors. Moreover, electroretinographic recordings indicate that these drugs affect temporal processing in the outer retina. Here we analyzed the functional role of HCN channels in rod bipolar cells (RBCs) of the mouse. Perforated–patch recordings in the dark–adapted slice found that RBCs exhibit Ih, and that this is sensitive to the specific blocker ZD7288. RBC input impedance, explored by sinusoidal frequency–modulated current stimuli (0.1–30 Hz), displays band–pass behavior in the range of Ih activation. Theoretical modeling and pharmacological blockade demonstrate that high–pass filtering of input signals by Ih, in combination with low–pass filtering by passive properties, fully accounts for this frequency–tuning. Correcting for the depolarization introduced by shunting through the pipette–membrane seal, leads to predict that in darkness Ih is tonically active in RBCs and quickens their responses to dim light stimuli. Immunohistochemistry targeting candidate subunit isoforms HCN1–2, in combination with markers of RBCs (PKC) and rod–RBC synaptic contacts (bassoon, mGluR6, Kv1.3), suggests that RBCs express HCN2 on the tip of their dendrites. The functional properties conferred by Ih onto RBCs may contribute to shape the retina's light response and explain the visual side effects of HCN inhibitors

Francisella tularensis Uses Cholesterol and Clathrin-Based Endocytic Mechanisms to Invade Hepatocytes

Francisella tularensis are highly infectious microbes that cause the disease tularemia. Although much of the bacterial burden is carried in non-phagocytic cells, the strategies these pathogens use to invade these cells remains elusive. To examine these mechanisms we developed two in vitro Francisella-based infection models that recapitulate the non-phagocytic cell infections seen in livers of infected mice. Using these models we found that Francisella novicida exploit clathrin and cholesterol dependent mechanisms to gain entry into hepatocytes. We also found that the clathrin accessory proteins AP-2 and Eps15 co-localized with invading Francisella novicida as well as the Francisella Live Vaccine Strain (LVS) during hepatocyte infections. Interestingly, caveolin, a protein involved in the invasion of Francisella in phagocytic cells, was not required for non-phagocytic cell infections. These results demonstrate a novel endocytic mechanism adopted by Francisella and highlight the divergence in strategies these pathogens utilize between non-phagocytic and phagocytic cell invasion