Alexey G. Ryazanov

Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes

Double-stranded RNA interference (RNAi) is an effective method for disrupting expression of specific genes in Caenorhabditis elegans and other organisms. Applications of this reverse-genetics tool, however, are somewhat restricted in nematodes because introduced dsRNA is not stably inherited. Another difficulty is that RNAi disruption of late-acting genes has been generally less consistent than that of embryonically expressed genes, perhaps because the concentration of dsRNA becomes lower as cellular division proceeds or as developmental time advances. In particular, some neuronally expressed genes appear refractory to dsRNA-mediated interference. We sought to extend the applicability of RNAi by in vivo expression of heritable inverted-repeat (IR) genes. We assayed the efficacy of in vivo-driven RNAi in three situations for which heritable, inducible RNAi would be advantageous: (i) production of large numbers of animals deficient for gene activities required for viability or reproduction; (ii) generation of large populations of phenocopy mutants for biochemical analysis; and (iii) effective gene inactivation in the nervous system. We report that heritable IR genes confer potent and specific gene inactivation for each of these applications. We suggest that a similar strategy might be used to test for dsRNA interference effects in higher organisms in which it is feasible to construct transgenic animals, but impossible to directly or transiently introduce high concentrations of dsRNA.

Elongation Factor 2 and Fragile X Mental Retardation Protein Control the Dynamic Translation of Arc/Arg3.1 Essential for mGluR-LTD

Group I metabotropic glutamate receptors (mGluR) induce long-term depression (LTD) that requires protein synthesis. Here, we demonstrate that Arc/Arg3.1 is translationally induced within 5 min of mGluR activation, and this response is essential for mGluR-dependent LTD. The increase in Arc/Arg3.1 translation requires eEF2K, a Ca(2+)/calmodulin-dependent kinase that binds mGluR and dissociates upon mGluR activation, whereupon it phosphorylates eEF2. Phospho-eEF2 acts to slow the elongation step of translation and inhibits general protein synthesis but simultaneously increases Arc/Arg3.1 translation. Genetic deletion of eEF2K results in a selective deficit of rapid mGluR-dependent Arc/Arg3.1 translation and mGluR-LTD. This rapid translational mechanism is disrupted in the fragile X disease mouse (Fmr1 KO) in which mGluR-LTD does not require de novo protein synthesis but does require Arc/Arg3.1. We propose a model in which eEF2K-eEF2 and FMRP coordinately control the dynamic translation of Arc/Arg3.1 mRNA in dendrites that is critical for synapse-specific LTD.

TRPM7, a novel regulator of actomyosin contractility and cell adhesion

Actomyosin contractility regulates various cell biological processes including cytokinesis, adhesion and migration. While in lower eukaryotes, α‐kinases control actomyosin relaxation, a similar role for mammalian α‐kinases has yet to be established. Here, we examined whether TRPM7, a cation channel fused to an α‐kinase, can affect actomyosin function. We demonstrate that activation of TRPM7 by bradykinin leads to a Ca2+‐ and kinase‐dependent interaction with the actomyosin cytoskeleton. Moreover, TRPM7 phosphorylates the myosin IIA heavy chain. Accordingly, low overexpression of TRPM7 increases intracellular Ca2+ levels accompanied by cell spreading, adhesion and the formation of focal adhesions. Activation of TRPM7 induces the transformation of these focal adhesions into podosomes by a kinase‐dependent mechanism, an effect that can be mimicked by pharmacological inhibition of myosin II. Collectively, our results demonstrate that regulation of cell adhesion by TRPM7 is the combined effect of kinase‐dependent and ‐independent pathways on actomyosin contractility.

Autophagy regulates ageing in C. elegans

The role of autophagy in ageing regulation has been suggested based on studies in C. elegans, in which knockdown of the expression of bec-1 (ortholog of the yeast and mammalian autophagy genes ATG6/VPS30 and beclin 1, respectively) shortens the lifespan of the daf-2(e1370) mutant C. elegans. However, Beclin1/ATG6 is also known to be involved in other cellular functions in addition to autophagy. In the current study, we knocked down two other autophagy genes, atg-7 and atg-12, in C. elegans using RNAi. We showed that RNAi shortened the lifespan of both wild type and daf-2 mutant C. elegans, providing strong support for a role of autophagy in ageing regulation.

Phosphorylation of Annexin I by TRPM7 Channel-Kinase

TRPM7 is an unusual bifunctional molecule consisting of a TRP ion channel fused to a protein kinase domain. It has been shown that TRPM7 plays a key role in the regulation of intracellular magnesium homeostasis as well as in anoxic neuronal death. TRPM7 channel has been characterized using electrophysiological techniques; however, the function of the kinase domain is not known and endogenous substrates for the kinase have not been reported previously. Here we have identified annexin 1 as a substrate for TRPM7 kinase. Phosphorylation of annexin 1 by TRPM7 kinase is stimulated by Ca2+ and is dramatically increased in extracts from cells overexpressing TRPM7. Phosphorylation of annexin 1 by TRPM7 kinase occurs at a conserved serine residue (Ser5) located within the N-terminal amphipathic α-helix of annexin 1. The N-terminal region plays a crucial role in interaction of annexin 1 with other proteins and membranes, and therefore, phosphorylation of annexin 1 at Ser5 by TRPM7 kinase may modulate function of annexin 1.

The Channel Kinases TRPM6 and TRPM7 Are Functionally Nonredundant

TRPM7 and its closest homologue, TRPM6, are the only known fusions of an ion channel pore with a kinase domain. Deletion of TRPM7 in DT40 B-lymphocytes causes growth arrest, Mg2+ deficiency, and cell death within 24–48 h. Amazingly, in analogy to TRPM6-deficient patients who can live a normal life if provided with a Mg2+-rich diet, TRPM7-deficient DT40 B-lymphocytes show wild type cell growth if supplied with 5–10 mm Mg2+ concentrations in their extracellular medium. Here we have investigated the functional relationship between TRPM6 and TRPM7. We show that TRPM7 deficiency in DT40 cells cannot be complemented by heterologously expressed TRPM6. Nevertheless, both channels can influence each others biological activity. Our data demonstrate that TRPM6 requires TRPM7 for surface expression in HEK-293 cells and also that TRPM6 is capable of cross-phosphorylating TRPM7 as assessed using a phosphothreonine-specific antibody but not vice versa. TRPM6 and TRPM7 coexpression studies in DT40 B-cells indicate that TRPM6 can modulate TRPM7 function. In conclusion, although TRPM6 and TRPM7 are closely related and deficiency in either one of these molecules severely affects Mg2+ homeostasis regulation, TRPM6 and TRPM7 do not appear to be functionally redundant but rather two unique and essential components of vertebrate ion homeostasis regulation.

Alpha-kinases: a new class of protein kinases with a novel catalytic domain

Colin Blakemore What is your advice to a neuroscience PhD student? Think long and hard about whether you want to commit yourself to a career in scientific neuroscience. There are limited resources, grants, jobs and ideas. The sort of people who can’t live without research are the sort of people who will have the enthusiasm to continue. Also, it is very easy to get bogged down in the intricacies of the brain. Never forget that the brain is a cognitive machine, not just a piece of tissue. That is especially relevant to neurogeneticists, who will look at a gene in isolation from everything else. What, in your opinion, is the greatest unanswered question? What exactly are qualia? Qualia are the qualities of conscious experience — the redness of red, for example. But what are they in physical terms? For example, think in terms of explaining the conscious experience of the bat. We may be able to give a description of the conscious thought of a bat, but we will never be able to experience what it is to be that bat.

Elongation factor-2 kinase and its newly discovered relatives

Phosphorylation of elongation factor‐2 (eEF‐2) by the highly specific eEF‐2 kinase results in eEF‐2 inactivation and, therefore, may regulate the global rate of protein synthesis in animal cells. Cloning and sequencing of eEF‐2 kinase led to the discovery of a new family of protein kinases, named α‐kinases, whose catalytic domains display no sequence homology to conventional eukaryotic protein kinases. Several mammalian α‐kinases have recently been cloned. Two of these α‐kinases, named channel‐kinases 1 and 2 (ChaK1 and ChaK2) represent a new type of signaling molecules that are protein kinases fused to ion channels.

Ca2+/calmodulin-dependent phosphorylation of elongation factor 2

Incubation of a ribosome‐free extract of rabbit reticulocytes or rat liver with [γ‐32P]ATP and Ca2+ results in incorporation of 32P predominantly into a single polypeptide of Mr ∼ 100 000. This polypeptide is identified as elongation factor 2 (EF‐2). Phosphorylation of EF‐2 is strictly Ca2+‐dependent and can be inhibited by the calmodulin antagonist trifluoperazine. It is suggested that the Ca2+/calmodulin‐dependent phosphorylation of EF‐2 is involved in regulation of protein biosynthesis.

Acute Suppression of Spontaneous Neurotransmission Drives Synaptic Potentiation

The impact of spontaneous neurotransmission on neuronal plasticity remains poorly understood. Here, we show that acute suppression of spontaneous NMDA receptor-mediated (NMDAR-mediated) neurotransmission potentiates synaptic responses in the CA1 regions of rat and mouse hippocampus. This potentiation requires protein synthesis, brain-derived neurotrophic factor expression, eukaryotic elongation factor-2 kinase function, and increased surface expression of AMPA receptors. Our behavioral studies link this same synaptic signaling pathway to the fast-acting antidepressant responses elicited by ketamine. We also show that selective neurotransmitter depletion from spontaneously recycling vesicles triggers synaptic potentiation via the same pathway as NMDAR blockade, demonstrating that presynaptic impairment of spontaneous release, without manipulation of evoked neurotransmission, is sufficient to elicit postsynaptic plasticity. These findings uncover an unexpectedly dynamic impact of spontaneous glutamate release on synaptic efficacy and provide new insight into a key synaptic substrate for rapid antidepressant action.