[Show abstract][Hide abstract] ABSTRACT: Almost since the discovery of long-term potentiation (LTP) in the hippocampus, its locus of expression has been debated. Throughout the years, convincing evidence has accumulated to suggest that LTP can be supported either presynaptically, by an increase in transmitter release, or postsynaptically, by an increase in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor number. However, whereas postsynaptic enhancement appears to be consistently obtained across studies following LTP induction, presynaptic enhancement is not as reliably observed. Such discrepancies, along with the failure to convincingly identify a retrograde messenger required for presynaptic change, have led to the general view that LTP is mainly supported postsynaptically, and certainly, research within the field for the past decade has been heavily focused on the postsynaptic locus. Here, we argue that LTP can be expressed at either synaptic locus, but that pre- and postsynaptic forms of LTP are dissociable phenomena mediated by distinct mechanistic processes, which are sensitive to different patterns of neuronal activity. This view of LTP helps to reconcile discrepancies across the literature and may put to rest a decades-long debate.
Philosophical Transactions of The Royal Society B Biological Sciences 01/2014; 369(1633):20130154.
[Show abstract][Hide abstract] ABSTRACT: Much excitement surrounded the proposal that a family of endo-lysosomal channels, the two-pore channels (TPCs) were the long sought after targets of the Ca(2+) -mobilising messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, the role of TPCs in NAADP signalling may be more complex than originally envisaged. First, NAADP may not bind directly to TPCs but via an accessory protein. Second, two papers recently challenged the notion that TPCs are NAADP-regulated Ca(2+) channels by suggesting that they are highly selective Na(+) channels regulated by the lipid phosphatidylinositol 3,5-bisphosphate and by ATP. This paper aims critically to evaluate the evidence for TPCs as NAADP targets and to discuss how the new findings fit in with what we know about endo-lysosomal Ca(2+) stores.
[Show abstract][Hide abstract] ABSTRACT: Niemann-Pick type C (NPC) is a neurodegenerative lysosomal storage disorder caused by defects in the lysosomal proteins NPC1 or NPC2. NPC cells are characterised by reduced lysosomal calcium levels and impaired sphingosine transport from lysosomes. Natural Killer (NK) cells kill virally infected/transformed cells via degranulation of lysosome-related organelles. Their trafficking from lymphoid tissues into the circulation is dependent on sphingosine-1-phosphate (S1P) gradients, sensed by S1P receptor 5 (S1P5). We hypothesised that NK cell function and trafficking could be affected in NPC disease due to the combined effects of the lysosomal calcium defect and sphingosine storage. In an NPC1 mouse model we found the frequency of NK cells was altered and phenocopied S1P5 deficient mice, consistent with defects in S1P levels. NK cells from NPC1 mice also had a defect in cytotoxicty due to a failure in degranulation of cytotoxic granules, which was associated with reduced lysosomal calcium levels. Affected NPC1 patients and NPC1 heterozygote carriers had reduced NK cell numbers in their blood and showed similar phenotypic and developmental changes to those observed in the NPC1 mouse. These findings highlight the effects of lysosomal storage on the peripheral immune system.
[Show abstract][Hide abstract] ABSTRACT: S-adenosylmethionine (SAM) is synthesized from methionine, which is abundant in animal-derived protein, in an energy-consuming reaction. SAM and S-adenosylhomocysteine (SAH) correlate with body mass index (BMI). Plasma total concentration of the SAM-associated product cysteine (tCys) correlates with fat mass in humans and cysteine promotes adiposity in animals. In a cross-sectional study of 610 participants, we investigated whether SAM and SAH are associated with BMI via lean mass or fat mass and dietary protein sources as determinants of SAM and tCys concentrations. Plasma SAM was not associated with lean mass, but mean adjusted fat mass increased from 24 kg (95% CI: 22.6, 25.1) to 30 kg (95% CI: 28.7, 31.3) across SAM quartiles (P < 0.001) and trunk fat:total fat ratio increased from 0.48 to 0.52 (P < 0.001). Erythrocyte SAM was also positively associated with fat mass and trunk fat:total fat ratio. The association of SAM with fat mass was not weakened by adjustment for serum tCys, lipids, creatinine, or dietary or lifestyle confounders. Concentrations of the SAM precursor, methionine, and the SAM product, SAH, were not independently associated with adiposity. Intake of animal-derived protein was not related to serum methionine but was positively associated with plasma SAM (partial r = 0.11) and serum tCys (partial r = 0.13; P < 0.05 for both after adjustment for age, gender, and total energy intake). In conclusion, plasma SAM, but not methionine, is independently associated with fat mass and trunkal adiposity, suggesting increased conversion of methionine to SAM in obese individuals. Prospective studies are needed to investigate the interactions among dietary energy and animal protein content, SAM concentrations, and change in body weight and cardiometabolic risk.
[Show abstract][Hide abstract] ABSTRACT: Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) plays an important role in the intracellular survival of the microorganism inside macrophages. Medicinal chemistry efforts to optimize inhibitors of the TBNAT enzyme have been hampered by the lack of a three-dimensional structure of the enzyme. In this paper, the first structure of TBNAT, determined using a lone crystal produced using cross-seeding with the homologous protein from M. marinum, is reported. Despite the similarity between the two enzymes (74% sequence identity), they show distinct physical and biochemical characteristics. The structure elegantly reveals the characteristic features of the protein surface as well as details of the active site of TBNAT relevant to drug-discovery efforts. The crystallographic analysis of the diffraction data presented many challenges, since the crystal was twinned and the habit possessed pseudo-translational symmetry.
Acta Crystallographica Section D Biological Crystallography 08/2013; 69(Pt 8):1433-46.
[Show abstract][Hide abstract] ABSTRACT: Mounting scientific and clinical evidence supports the key role played by phospholipase C zeta (PLCζ), a sperm-specific protein, in the activation of oocytes following fertilisation. Lacking a pleckstrin homology domain, PLCζ remains the smallest known mammalian PLC and was first identified in 2002. Since then, PLCζ has been the target for a multitude of studies in both mammalian and non-mammalian species focused upon its fundamental biochemical activity and crucial role as the mediator of oocyte activation. The earliest event subsequent to gamete fusion is the onset of a series of intracellular calcium oscillations within the oocyte, which are known to modulate cortical granule exocytosis, release meiotic arrest, regulate gene expression, recruit maternal mRNA, and initiate embryogenesis. Collectively these processes are known as 'oocyte activation' and together, represent a fundamental mechanism for early embryonic development. Evidence suggests that these processes are initiated and controlled by calcium release from ooplasmic sources in response to PLCζ activity via the inositol-1,4,5-triphosphate (IP3) pathway. While the biochemical action of PLCζ has been extensively studied, especially in relation to the EF-hands, X-Y linker, and C2 domain, all of which play critical roles for in vivo activity, there are still key gaps in our knowledge, particularly in terms of regulation and interaction with other proteins within the oocyte. Moreover, increasing clinical evidence has revealed a strong correlation between certain types of male infertility and the aberrant expression, localisation, structure and function of PLCζ in human sperm, particularly in cases of recurrent intracytoplasmic sperm injection (ICSI) failure, globozoospermia, and oocyte activation deficiency (OAD). In addition, two heterozygous substitution mutations have been identified in the coding sequence of PLCζ in one particular patient causing disruption to the catalytic X and Y domains and resulting in infertility. Although, such cases can be treated via the use of artificial oocyte activators (AOAs) such as calcium ionophores, significant concern remains over the use of such chemical agents, largely due to the fact that calcium release manifests as a single transient, rather than a series of oscillations as observed during normal fertilisation. Current interest in PLCζ is thus to develop a series of prognostic, diagnostic and therapeutic approaches which could first identify male patients that are deficient in PLCζ and then rescue oocyte activation ability via assisted reproductive technology (ART) and a pure, functionally-active, recombinant human PLCζ protein. While significant progress has been made in such areas over recent years, there is a clear need to translate scientific findings to clinical settings in order to maximise successful outcome for patients.
[Show abstract][Hide abstract] ABSTRACT: The major afferent innervation of the basal ganglia is derived from the cortex and the thalamus. These excitatory inputs mainly target the striatum where they innervate the principal type of striatal neuron, the medium-sized spiny neurons (MSNs), and are critical in the expression of basal ganglia function. The aim of this work was to test directly whether corticostriatal and thalamostriatal terminals make convergent synaptic contact with individual direct and indirect pathway MSNs. Individual MSNs were recorded in vivo and labelled by the juxtacellular method in the striatum of BAC transgenic mice in which green fluorescent protein reports the expression of dopamine D1 or D2 receptors. After recovery of the neurons, the tissue was immunolabelled for vesicular glutamate transporters type 1 and 2, as markers of cortical and thalamic terminals, respectively. Three of each class of MSNs were reconstructed in 3D and second-order dendrites selected for electron microscopic analysis. Our findings show that direct and indirect pathway MSNs, located in the matrix compartment of the striatum, receive convergent input from cortex and thalamus preferentially on their spines. There were no differences in the pattern of innervation of direct and indirect pathway MSNs, but the cortical input is more prominent in both and synaptic density is greater for direct pathway neurons. The 3D reconstructions revealed no morphological differences between direct and indirect MSNs. Overall, our findings demonstrate that direct and indirect pathway MSNs located in the matrix receive convergent cortical and thalamic input and suggest that both cortical and thalamic inputs are involved in the activation of MSNs.
[Show abstract][Hide abstract] ABSTRACT: While sensorimotor adaptation to prisms that displace the visual field takes minutes, adapting to an inversion of the visual field takes weeks. In spite of a long history of the study, the basis of this profound difference remains poorly understood. Here, we describe the computational issue that underpins this phenomenon and presents experiments designed to explore the mechanisms involved. We show that displacements can be mastered without altering the updated rule used to adjust the motor commands. In contrast, inversions flip the sign of crucial variables called sensitivity derivatives-variables that capture how changes in motor commands affect task error and therefore require an update of the feedback learning rule itself. Models of sensorimotor learning that assume internal estimates of these variables are known and fixed predicted that when the sign of a sensitivity derivative is flipped, adaptations should become increasingly counterproductive. In contrast, models that relearn these derivatives predict that performance should initially worsen, but then improve smoothly and remain stable once the estimate of the new sensitivity derivative has been corrected. Here, we evaluated these predictions by looking at human performance on a set of pointing tasks with vision perturbed by displacing and inverting prisms. Our experimental data corroborate the classic observation that subjects reduce their motor errors under inverted vision. Subjects' accuracy initially worsened and then improved. However, improvement was jagged rather than smooth and performance remained unstable even after 8 days of continually inverted vision, suggesting that subjects improve via an unknown mechanism, perhaps a combination of cognitive and implicit strategies. These results offer a new perspective on classic work with inverted vision.
[Show abstract][Hide abstract] ABSTRACT: When cytotoxic T-lymphocytes (CTLs) kill infected or cancerous cells they secrete cytolytic proteins (perforin and granzymes) into the target cell. These "death factors" are pre-stored in cytolytic granules within the CTL until an increase in the intracellular Ca(2+) drives granule exocytosis. However, not all sources of Ca(2+) stimulate exocytosis: we have recently demonstrated that it is the cytolytic granules themselves that are the source of the Ca(2+) that most efficiently drives their own exocytosis; release of Ca(2+) from the granules is only activated by the Ca(2+)-mobilizing messenger NAADP (nicotinic acid adenine dinucleotide phosphate) that acts upon target two-pore channels (TPCs) present on the granules. That NAADP is a unique stimulus of exocytosis may be of fundamental importance not only to immunology but to cell biology in general.
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