Retrograde signaling from the brain to the retina modulates the termination of the light response in Drosophila

A critical factor in visual function is the speed with which photoreceptors (PRs) return to the resting state when light intensity dims. Several elements subserve this process, many of which promote the termination of the phototransduction cascade. Although the known elements are intrinsic to PRs, we have found that prompt restoration to the resting state of the Drosophila electroretinogram can require effective communication between the retina and the underlying brain. The requirement is seen more dramatically with long than with short light pulses, distinguishing the phenomenon from gross disruption of the termination machinery. The speed of recovery is affected by mutations (in the Hdc and ort genes) that prevent PRs from transmitting visual information to the brain. It is also affected by manipulation (using either drugs like neostigmine or genetic tools to inactivate neurotransmitter release) of cholinergic signals that arise in the brain. Intracellular recordings support the hypothesis that PRs are the target of this communication. We infer that signaling from the retina to the optic lobe prompts a feedback signal to retinal PRs. Although the mechanism of this retrograde signaling remains to be discerned, the phenomenon establishes a previously unappreciated mode of control of the temporal responsiveness of a primary sensory neuron.     

ATM kinase activity modulates Fas sensitivity through the regulation of FLIP in lymphoid cells

Ataxia telangiectasia (A-T) is a rare cancer-predisposing genetic disease, caused by the lack of functional ATM kinase, a major actor of the double strand brakes (DSB) DNA-damage response. A-T patients show a broad and diverse phenotype, which includes an increased rate of lymphoma and leukemia development. Fas-induced apoptosis plays a fundamental role in the homeostasis of the immune system and its defects have been associated with autoimmunity and lymphoma development. We therefore investigated the role of ATM kinase in Fas-induced apoptosis. Using A-T lymphoid cells, we could show that ATM deficiency causes resistance to Fas-induced apoptosis. A-T cells up-regulate FLIP protein levels, a well-known inhibitor of Fas-induced apoptosis. Reconstitution of ATM kinase activity was sufficient to decrease FLIP levels and to restore Fas sensitivity. Conversely, genetic and pharmacologic ATM kinase inactivation resulted in FLIP protein up-regulation and Fas resistance. Both ATM and FLIP are aberrantly regulated in Hodgkin lymphoma. Importantly, we found that reconstitution of ATM kinase activity decreases FLIP protein levels and restores Fas sensitivity in Hodgkin lymphoma-derived cells. Overall, these data identify a novel molecular mechanism through which ATM kinase may regulate the immune system homeostasis and impair lymphoma development.     

Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics

Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p < 0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p = 0.02) and after thrombin (p = 0.01) or S1P (p = 0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p < 0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p < 0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function.     

Rhodopsin kinase: expression in mammalian cells and a two-step purification

A suitable system for expression of the rhodopsin kinase (RK) gene and its mutants is needed for structure-function studies of RK. Previously, investigation of the baculovirus system showed satisfactory production of RK, but posttranslational isoprenylation was deficient. We now report on a comparative study of expression of the RK gene in yeast (Pichia pastoris), COS-1 cells and in an HEK293 stable cell line. Expression in COS-1 cells, by using pCMV5 vector, is the most satisfactory. A two-step procedure for purification of the expressed enzyme with an N-terminal histidine tag has been developed. The purified enzyme has correct posttranslational modifications and shows a somewhat broader pH vs. catalytic activity profile than the wild-type enzyme.     

ATM kinase inhibition in glial cells activates the innate immune response and causes neurodegeneration in Drosophila

To investigate the mechanistic basis for central nervous system (CNS) neurodegeneration in the disease ataxia-telangiectasia (A-T), we analyzed flies mutant for the causative gene A-T mutated (ATM). ATM encodes a protein kinase that functions to monitor the genomic integrity of cells and control cell cycle, DNA repair, and apoptosis programs. Mutation of the C-terminal amino acid in Drosophila ATM inhibited the kinase activity and caused neuron and glial cell death in the adult brain and a reduction in mobility and longevity. These data indicate that reduced ATM kinase activity is sufficient to cause neurodegeneration in A-T. ATM kinase mutant flies also had elevated expression of innate immune response genes in glial cells. ATM knockdown in glial cells, but not neurons, was sufficient to cause neuron and glial cell death, a reduction in mobility and longevity, and elevated expression of innate immune response genes in glial cells, indicating that a non-cell-autonomous mechanism contributes to neurodegeneration in A-T. Taken together, these data suggest that early-onset CNS neurodegeneration in A-T is similar to late-onset CNS neurodegeneration in diseases such as Alzheimer's in which uncontrolled inflammatory response mediated by glial cells drives neurodegeneration.     

Resting GABA concentration predicts peak gamma frequency and fMRI amplitude in response to visual stimulation in humans

Functional imaging of the human brain is an increasingly important technique for clinical and cognitive neuroscience research, with functional MRI (fMRI) of the blood oxygen level-dependent (BOLD) response and electroencephalography or magnetoencephalography (MEG) recordings of neural oscillations being 2 of the most popular approaches. However, the neural and physiological mechanisms that generate these responses are only partially understood and sources of interparticipant variability in these measures are rarely investigated. Here, we test the hypothesis that the properties of these neuroimaging metrics are related to individual levels of cortical inhibition by combining magnetic resonance spectroscopy to quantify resting GABA concentration in the visual cortex, MEG to measure stimulus-induced visual gamma oscillations and fMRI to measure the BOLD response to a simple visual grating stimulus. Our results demonstrate that across individuals gamma oscillation frequency is positively correlated with resting GABA concentration in visual cortex (R = 0.68; P < 0.02), BOLD magnitude is inversely correlated with resting GABA (R = −0.64; P < 0.05) and that gamma oscillation frequency is strongly inversely correlated with the magnitude of the BOLD response (R = −0.88; P < 0.001). Our results are therefore supportive of recent theories suggesting that these functional neuroimaging metrics are dependent on the excitation/inhibition balance in an individual''s cortex and have important implications for the interpretation of functional imaging results, particularly when making between-group comparisons in clinical research.     

Rhodopsin kinase: two mAbs binding near the carboxyl terminus cause time-dependent inactivation

Two mAbs generated against rhodopsin kinase (RK) were characterized for their epitopes. Both antibodies recognize short peptide sequences, overlapping but distinct, close to the carboxyl terminus. Binding of RK to the antibodies is slow. Attempts were made to use the antibodies immobilized on protein A-Sepharose beads to bind and purify the enzyme. Time-dependent inactivation of the enzyme occurred after its binding to the antibodies. Studies using different conditions to maintain the enzyme in the active form during binding or to reactivate the purified inactivated enzyme were unsuccessful.     

Rhodopsin expressed in Chinese hamster ovary cells regulates adenylyl cyclase activity

The cDNA encoding bovine opsin was transfected into Chinese hamster ovary (CHO) cells to generate stable clones expressing the rod cell photoreceptor protein. Cells expressing opsin, when incubated in 11-cis retinal and exposed to light, inhibited forskolin-stimulated adenylyl cyclase activity. Rhodopsin-mediated inhibition of adenylyl cyclase was prevented by treatment of cells with pertussis toxin. In the same cells, thrombin stimulated phosphatidylinositol hydrolysis through G protein-mediated pathways, but rhodopsin neither significantly influenced the action of thrombin nor stimulated phosphatidylinositol hydrolysis. Our findings indicate that rhodopsin selectively regulates a Gi protein in intact CHO cells that is coupled to adenylyl cyclase but not to phospholipase C.     

Characterization and regulation of reductase kinase, a protein kinase that modulates the enzymic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase

The activity of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase [HMG-CoA reductase; mevalonate:NADP(+) oxidoreductase (CoA-acylating), EC 1.1.1.34] can be modulated in vitro by a phosphorylation-dephosphorylation reaction sequence. A microsomal reductase kinase catalyzes the phosphorylation of HMG-CoA reductase and histones. Histone phosphorylation was enhanced 2- to 3-fold by cyclic AMP. Reductase kinase exists in interconvertible active and inactive forms. Incubation of reductase kinase with phosphoprotein phosphatase resulted in a time-dependent decrease in the ability of reductase kinase to catalyze the phosphorylation of histones and to inactivate HMG-CoA reductase. Incubation of phosphoprotein phosphatase-inactivated reductase kinase with [gamma-(32)P]ATP plus Mg(2+) and a partially purified protein kinase designated reductase kinase kinase resulted in parallel increases in protein-bound (32)P radioactivity and ability to inactivate HMG-CoA reductase. Incubation of (32)P-labeled reductase kinase with phosphoprotein phosphatase resulted in a time-dependent loss of protein-bound (32)P radioactivity and a decrease in the ability to inactivate HMG-CoA reductase. Polyacrylamide gel electrophoresis of purified reductase kinase incubated with reductase kinase kinase and [gamma-(32)P]ATP plus Mg(2+) revealed that the (32)P radioactivity and reductase kinase enzymic activity were located in a single electrophoretic position. Dephosphorylation of (32)P-labeled purified reductase kinase with phosphoprotein phosphatase was associated with significant loss of radioactivity and enzymic activity in the protein band ascribed to reductase kinase. These results provide evidence that the activity of reductase kinase, like HMG-CoA reductase, is modulated by a reversible phosphorylation-dephosphorylation reaction sequence.     

big bang gene modulates gut immune tolerance in Drosophila

Chronic inflammation of the intestine is detrimental to mammals. Similarly, constant activation of the immune response in the gut by the endogenous flora is suspected to be harmful to Drosophila. Therefore, the innate immune response in the gut of Drosophila melanogaster is tightly balanced to simultaneously prevent infections by pathogenic microorganisms and tolerate the endogenous flora. Here we describe the role of the big bang (bbg) gene, encoding multiple membrane-associated PDZ (PSD-95, Discs-large, ZO-1) domain-containing protein isoforms, in the modulation of the gut immune response. We show that in the adult Drosophila midgut, BBG is present at the level of the septate junctions, on the apical side of the enterocytes. In the absence of BBG, these junctions become loose, enabling the intestinal flora to trigger a constitutive activation of the anterior midgut immune response. This chronic epithelial inflammation leads to a reduced lifespan of bbg mutant flies. Clearing the commensal flora by antibiotics prevents the abnormal activation of the gut immune response and restores a normal lifespan. We now provide genetic evidence that Drosophila septate junctions are part of the gut immune barrier, a function that is evolutionarily conserved in mammals. Collectively, our data suggest that septate junctions are required to maintain the subtle balance between immune tolerance and immune response in the Drosophila gut, which represents a powerful model to study inflammatory bowel diseases.     

Ghrelin modulates sympathetic nervous system activity and stress response in lean and overweight men

Ghrelin is a growth hormone-releasing peptide secreted by the stomach with potent effects on appetite. Experimental and clinical studies indicate that ghrelin also influences cardiovascular regulation and metabolic function and mediates behavioral responses to stress. We investigated the effects of ghrelin on blood pressure (BP), sympathetic nervous system activity, and mental stress responses in lean (n=13) and overweight or obese (n=13) individuals. Subjects received an intravenous infusion of human ghrelin (5 pmol/kg per minute for 1 hour) and saline in a randomized fashion. Ghrelin decreased systolic (-6 and -11 mm Hg) and diastolic BP (-8 mm Hg for both), increased muscle sympathetic nervous system activity (18±2 to 28±3 bursts per min, P<0.05 and from 21±2 to 32±3 bursts per min, P<0.001) in lean and overweight or obese subjects, respectively, without a significant change in heart rate, calf blood flow, or vascular resistance. Ghrelin induced a rise in plasma glucose concentration in lean individuals (P<0.05) and increased cortisol levels in both groups (P<0.05). Stress induced a significant change in mean BP (+22 and +27 mm Hg), heart rate (+36 and +29 bpm), and muscle sympathetic nervous system activity (+6.1±1.6 and +6.8±2.7 bursts per min) during saline infusion in lean and overweight or obese subjects, respectively. During ghrelin infusion, the changes in BP and muscle sympathetic nerve activity in response to stress were significantly reduced in both groups (P<0.05). In conclusion, ghrelin exerts unique effects in that it reduces BP and increases muscle sympathetic nervous system activity and blunts cardiovascular responses to mental stress. These responses may represent a combination of peripheral (baroreflex-mediated) and central effects of ghrelin.     

Protein kinase C modulates ventilatory patterning in the developing rat

Protein kinase C (PKC) mediates important components of signal transduction pathways underlying neuronal excitability and modulates respiratory timing mechanisms in adult rats. To determine ventilatory effects of systemic PKC inhibition during development, whole-body plethysmographic recordings were conducted in 2-3-d (n = 11), 5-6-d (n = 19), 10-12-d (n = 14), and 20-21-d-old (n = 14) rat pups after treatment with vehicle and Ro 32-0432 (100 mg/kg, intraperitoneally). Ro 32-0432 decreased minute ventilation (V E) by 51.0 +/- 5.5% (mean +/- SEM) in youngest pups (p < 0.01) but only 19.1 +/- 6.8% in 20-21-d-old pups (p < 0.01). V E decreases were always due to frequency reductions with tidal volume (VT) remaining unaffected. Respiratory rate decreases primarily resulted from marked expiratory time (TE) prolongations being more pronounced in 2-3-d-old (115.5 +/- 28.9%) compared with 20-21-d old (36.6 +/- 10.9%; p < 0.002 analysis of variance [ANOVA] ). Expression of the PKC isoforms alpha, beta, gamma, delta, iota, and mu was further examined in brainstem and cortex by immunoblotting and revealed different patterns with postnatal age and location. We conclude that endogenous PKC inhibition elicits age-dependent ventilatory reductions which primarily affect timing mechanisms rather than changes in volume drive. This effect on ventilation abates with increasing postnatal age suggesting that the neural substrate mediating overall respiratory output may be more critically dependent on PKC activity in the immature animal.     

A protein kinase activity tightly associated with Drosophila type II DNA topoisomerase.

A protein kinase activity has been identified that is tightly associated with the purified Drosophila type II DNA topoisomerase. The kinase and topoisomerase activities are not separated when the enzyme is subjected to analytical chromatography (phosphocellulose, single-strand DNA agarose, and Sephacryl S-300) and analytical glycerol gradient sedimentation. These two activities are also inactivated to the same extent by either heat or N-ethylmaleimide treatment. The evidence, however, does not rule out the possibility that the kinase activity resides in a polypeptide other than the topoisomerase polypeptide. The topoisomerase-associated protein kinase activity is not stimulated by Ca2+ or cyclic nucleotides. It shows a broad substrate range, including the DNA topoisomerase itself, casein, phosvitin, and histones. Phosphoamino acid analysis identified phosphoserine and phosphothreonine in polypeptides modified by the topoisomerase-associated protein kinase. No similar activity has been identified previously in Drosophila melanogaster.     

Longevity and the stress response in Drosophila

The concept that lifespan is a function of the capacity to withstand extrinsic stress is very old. In concordance with this, long-lived individuals often have increased resistance against a variety of stresses throughout life. Genes underlying the stress response may therefore have the ability to affect lifespan. The progress in modern genetic techniques has allowed researchers to test this idea. The general stress response involves the expression of stress proteins, such as chaperones and antioxidative proteins, downregulation of genes involved in energy metabolism and the release of protective substances. Do these same changes in patterns of expression have the ability to mitigate ageing and prolong lifespan? It appears that parts of this response indeed are also associated with extended longevity, whereas some elements are not, due to their high cost or long-term deleterious consequences. Here we briefly review the state of the art of research on ageing and longevity in the model organism Drosophila, with focus on the role of the general stress response. We will conclude by contemplating some of the implications of the findings in this research and will suggest several directions for future research.     

Stimulus repetition modulates gamma-band synchronization in primate visual cortex

When a sensory stimulus repeats, neuronal firing rate and functional MRI blood oxygen level-dependent responses typically decline, yet perception and behavioral performance either stay constant or improve. An additional aspect of neuronal activity is neuronal synchronization, which can enhance the impact of neurons onto their postsynaptic targets independent of neuronal firing rates. We show that stimulus repetition leads to profound changes of neuronal gamma-band (∼40–90 Hz) synchronization. Electrocorticographic recordings in two awake macaque monkeys demonstrated that repeated presentations of a visual grating stimulus resulted in a steady increase of visually induced gamma-band activity in area V1, gamma-band synchronization between areas V1 and V4, and gamma-band activity in area V4. Microelectrode recordings in area V4 of two additional monkeys under the same stimulation conditions allowed a direct comparison of firing rates and gamma-band synchronization strengths for multiunit activity (MUA), as well as for isolated single units, sorted into putative pyramidal cells and putative interneurons. MUA and putative interneurons showed repetition-related decreases in firing rate, yet increases in gamma-band synchronization. Putative pyramidal cells showed no repetition-related firing rate change, but a decrease in gamma-band synchronization for weakly stimulus-driven units and constant gamma-band synchronization for strongly driven units. We propose that the repetition-related changes in gamma-band synchronization maintain the interareal stimulus signaling and sharpen the stimulus representation by gamma-synchronized pyramidal cell spikes.Stimulus repetition typically leads to reduced neuronal firing rates and reduced functional MRI blood oxygen level-dependent signals, whereas behavior that is based on stimulus processing is not affected or is enhanced (1). Different models have been proposed to reconcile these behavioral and neurophysiological findings (1). In a “fatigue model,” neuronal responses are reduced in proportion to their amplitude, leaving relative response patterns unchanged; in a “sharpening model,” neurons that code features irrelevant to identification of a stimulus exhibit repetition suppression, leading to a sparser and sharpened representation of the repeated stimulus; and in a “facilitation model,” stimulus repetition leads to faster stimulus processing, and thereby smaller overall neuronal activity. Gotts and coworkers (2–4) suggested a “synchronization model” in which stimulus repetition leads to reduced firing rates accompanied by increased synchronization. The increased synchronization might explain how less-activated neuronal groups can maintain their impact onto postsynaptic neurons and, ultimately, behavior, while reducing metabolic costs at the same time. The synchronization model has received support from a number of studies in human subjects, using source-localized magnetoencephalography. Ghuman et al. (5) report enhanced frontotemporal 14-Hz synchronization for repeated vs. novel stimuli. Gilbert et al. (3) found that stimulus repetition leads to enhanced 5- to 15-Hz power in the right fusiform gyrus and enhanced 15- to 35-Hz power in striate and extrastriate cortex. Corresponding data were also reported for multisite microelectrodes recordings in striate and parietal cortex of awake cats, where von Stein et al. (6) found that interareal alpha-band synchronization was stronger for repeated compared with novel stimuli. The common finding across these studies is enhanced alpha/beta activity or coupling for repeated stimuli. The alpha coupling reported by von Stein et al. (6) occurs in a behavioral context and has a phase relationship and layer specificity that suggests a top-down–directed interaction. Thus, enhanced alpha/beta activity or coupling for repeated stimuli might reflect enhanced top-down signaling, perhaps related to enhanced predictability of repeated stimuli. However, increased synchronization with stimulus repetition according to the model of Gotts and coworkers (2–4) should also serve the maintenance of feedforward signaling of repeated stimuli in the face of reduced firing rates. Feedforward signaling has been strongly linked to local and interareal gamma-band synchronization (7–9). Local gamma-band synchronization likely enhances the postsynaptic impact of the precisely synchronized output spikes (10). Interareal gamma-band synchronization likely aligns excitability cycles such that inputs arrive when postsynaptic target neurons are receptive (11, 12). However, whether multiple presentations of a stimulus result in enhanced gamma-band synchronization remains unknown (details are provided in SI Discussion). We investigated gamma-band synchronization within and between macaque monkey areas V1 and V4 and report that stimulus repetition leads to profound changes in gamma-band synchronization within and between these areas.