cGMP-Dependent Protein Kinase Inhibition Extends the Upper Temperature Limit of Stimulus-Evoked Calcium Responses in Motoneuronal Boutons of <i>Drosophila melanogaster</i> Larvae

<div><p>While the mammalian brain functions within a very narrow range of oxygen concentrations and temperatures, the fruit fly, <i>Drosophila melanogaster</i>, has employed strategies to deal with a much wider range of acute environmental stressors. The <i>foraging</i> (<i>for</i>) gene encodes the cGMP-dependent protein kinase (PKG), has been shown to regulate thermotolerance in many stress-adapted species, including <i>Drosophila</i>, and could be a potential therapeutic target in the treatment of hyperthermia in mammals. Whereas previous thermotolerance studies have looked at the effects of PKG variation on <i>Drosophila</i> behavior or excitatory postsynaptic potentials at the neuromuscular junction (NMJ), little is known about PKG effects on presynaptic mechanisms. In this study, we characterize presynaptic calcium ([Ca²⁺]_i) dynamics at the <i>Drosophila</i> larval NMJ to determine the effects of high temperature stress on synaptic transmission. We investigated the neuroprotective role of PKG modulation both genetically using RNA interference (RNAi), and pharmacologically, to determine if and how PKG affects presynaptic [Ca²⁺]_i dynamics during hyperthermia. We found that PKG activity modulates presynaptic neuronal Ca²⁺ responses during acute hyperthermia, where PKG activation makes neurons more sensitive to temperature-induced failure of Ca²⁺ flux and PKG inhibition confers thermotolerance and maintains normal Ca²⁺ dynamics under the same conditions. Targeted motoneuronal knockdown of PKG using RNAi demonstrated that decreased PKG expression was sufficient to confer thermoprotection. These results demonstrate that the PKG pathway regulates presynaptic motoneuronal Ca²⁺ signaling to influence thermotolerance of presynaptic function during acute hyperthermia.</p></div

Pharmacological modulation of PKG influences thermotolerance.

<p>Average temperature of stimulus-induced Ca²⁺ response failure due to acute hyperthermia was compared for HL3 controls (n = 13), 40μM 8-Br-cGMP PKG activation (n = 10) and 50μM Rp-8-Br-PET-cGMPS PKG inhibition (n = 6). The temperature of Ca²⁺ response failure was significantly different between control HL3, PKG activation and PKG inhibition (Holm-Sidak, df = 2, <i>P</i><0.001). Letters represent significance between groups where A is assigned to the group with the highest mean and bars are represented as mean+/-SEM.</p

PKG modulation influences temperature-dependent decline in peak Ca²⁺ responses.

<p>(<b>A</b>) Peak Ca²⁺ fluorescence declined as a result of temperature. Images from a representative HL3 control trial demonstrate the decline in response to stimulation as temperature increased (labeled below image). (<b>B</b>) Pharmacological modulation of PKG shows no significant effect on F₀ or ΔF during hyperthermia (bottom panel and top panel respectively). Average F₀ Ca²⁺ fluorescence prior to stimulation (bottom panel) is not affected by the addition of PKG activator or inhibitor. For each trial, baseline Ca²⁺ was averaged from ROIs prior to stimulation at each temperature. Baseline Ca²⁺ was then averaged between trials of the same treatment for each temperature in the hyperthermia ramp. A significant difference was seen between +PKG and HL3 at 22°C, however this was the only temperature where a drug treatment had any effect on F₀ at a given temperature (two-way ANOVA, Holm-Sidak, F_(10,105) = 1.770, P = 0.05; * = P<0.05). Average maximum ΔF values were of ROIs showed little difference between treatments at a given temperature (top panel). Average ΔF showed no difference between HL3 and +PKG trials over the entire temperature ramp, however, average ΔF of –PKG was significantly different compared to both HL3 and +PKG ΔF at 30°C and 40°C (two-way ANOVA, Holm-Sidak, F_(10,105) = 1.113, P = 0.05; * = P<0.05). (<b>C</b>) Temperature-dependent change in average peak Ca²⁺ decline was altered by PKG modulation. Average peak Ca²⁺changes were averaged for ROIs per temperature per trial. Trial averages were then averaged for each condition and temperature to determine the average Ca²⁺ response for drug and control conditions at each temperature. Activation of the PKG pathway with 40μM 8-Br-cGMP (green, n = 5) showed an increased decline in Ca²⁺ dynamics and failed to elicit stimulus-induced Ca²⁺ responses at a much lower temperature than HL3 controls (black, n = 6) or PKG inhibition trials (red, n = 5). Significantly different changes in Ca²⁺ flux between drug treatments for each temperature are shown using brackets with an * (two-way ANOVA, Holm-Sidak, F_(2,105) = 36.95, P = 0.05; * = P<0.05).</p

Measuring temperature-dependent changes in stimulus-induced Ca²⁺ dynamics at the <i>Drosophila</i> NMJ.

<p>(<b>A</b>) Ca²⁺ levels at the NMJ before, during and after 5s stimulation. Dotted lines indicate expanded region shown in the inset to the right. Type 1b boutons were randomly selected as regions of interest (ROIs) to measure changes in Ca²⁺ levels during stimulation. (<b>B</b>) Changes in Ca²⁺ dynamics measured by changes in pixel intensity for individual ROI over time, before, during and after stimulation. The bar indicates the onset and duration of stimulation. (<b>C</b>) Representative traces demonstrate Ca²⁺ response to stimulation decays as temperature increases. Representative ROI traces of a single HL3 preparation show the change in pixel intensity as Ca²⁺ rises and falls in response to 5s stimulation over the time course of the experiment. The bar above the Ca²⁺ curve shows the duration of stimulation. Temperature ramp increases are shown in descending rows and motoneuronal failure can be seen for each treatment as the Ca²⁺ trace flat lines. Data traces were imaged and extracted using NIS Elements (Nikon Instruments, Inc.).</p

Motoneuronal inhibition of PKG using <i>for</i> RNAi is sufficient to confer thermoprotection.

<p>(<b>A</b>) Temperature-dependent change in average peak Ca²⁺ decay was attenuated by <i>foraging</i> knockdown using RNAi. Peak Ca²⁺changes were averaged as outlined in pharmacology experiments. Homozygous expression of for RNAi exhibited significantly different Ca²⁺ dynamics in response to stimulus compared to +/+ controls at 40°C (Holm-Sidak, df = 10, * = P<0.05). (<b>B</b>) Expression of <i>for</i> RNAi to prevent the expression of PKG solely in motoneurons extended the temperature at which Ca²⁺ dynamics fail. Expression of one copy of <i>for</i> RNAi (n = 7) showed no difference in failure temperature from animals with two copies of <i>for</i> RNAi (n = 6) (Holm-Sidak, df = 2, <i>P</i> = 0.929). Both experimental groups showed an extension in the permissible functional temperature compared to the control group that lacked any copies of <i>for</i> RNAi (n = 16) (Holm-Sidak, df = 2, <i>P</i><0.001). Letters represent significance between groups where A is assigned to the highest mean and bars are represented as mean+/-SEM.</p