Shal/K(v)4 channels are required for maintaining excitability during repetitive firing and normal locomotion in Drosophila.

Rhythmic behaviors, such as walking and breathing, involve the coordinated activity of central pattern generators in the CNS, sensory feedback from the PNS, to motoneuron output to muscles. Unraveling the intrinsic electrical properties of these cellular components is essential to understanding this coordinated activity. Here, we examine the significance of the transient A-type K(+) current (I(A)), encoded by the highly conserved Shal/K(v)4 gene, in neuronal firing patterns and repetitive behaviors. While I(A) is present in nearly all neurons across species, elimination of I(A) has been complicated in mammals because of multiple genes underlying I(A), and/or electrical remodeling that occurs in response to affecting one gene.In Drosophila, the single Shal/K(v)4 gene encodes the predominant I(A) current in many neuronal cell bodies. Using a transgenically expressed dominant-negative subunit (DNK(v)4), we show that I(A) is completely eliminated from cell bodies, with no effect on other currents. Most notably, DNK(v)4 neurons display multiple defects during prolonged stimuli. DNK(v)4 neurons display shortened latency to firing, a lower threshold for repetitive firing, and a progressive decrement in AP amplitude to an adapted state. We record from identified motoneurons and show that Shal/K(v)4 channels are similarly required for maintaining excitability during repetitive firing. We then examine larval crawling, and adult climbing and grooming, all behaviors that rely on repetitive firing. We show that all are defective in the absence of Shal/K(v)4 function. Further, knock-out of Shal/K(v)4 function specifically in motoneurons significantly affects the locomotion behaviors tested.Based on our results, Shal/K(v)4 channels regulate the initiation of firing, enable neurons to continuously fire throughout a prolonged stimulus, and also influence firing frequency. This study shows that Shal/K(v)4 channels play a key role in repetitively firing neurons during prolonged input/output, and suggests that their function and regulation are important for rhythmic behaviors

Light-induced recruitment of INAD-signaling complexes to detergent-resistant lipid rafts in Drosophila photoreceptors

Here, we reveal a novel feature of the dynamic organization of signaling components in Drosophila photoreceptors. We show that the multi-PDZ protein INAD and its target proteins undergo light-induced recruitment to detergent-resistant membrane (DRM) rafts. Reduction of ergosterol, considered to be a key component of lipid rafts in Drosophila, resulted in a loss of INAD-signaling complexes associated with DRM fractions. Genetic analysis demonstrated that translocation of INAD-signaling complexes to DRM rafts requires activation of the entire phototransduction cascade, while constitutive activation of the light-activated channels resulted in recruitment of complexes to DRM rafts in the dark. Mutations affecting INAD and TRP showed that PDZ4 and PDZ5 domains of INAD, as well as the INAD-TRP interaction, are required for translocation of components to DRM rafts. Finally, selective recruitment of phosphorylated, and therefore activatable, eye-PKC to DRM rafts suggests that DRM domains are likely to function in signaling, rather than trafficking

Linking Aβ42-Induced Hyperexcitability to Neurodegeneration, Learning and Motor Deficits, and a Shorter Lifespan in an Alzheimer’s Model

<div><p>Alzheimer’s disease (AD) is the most prevalent form of dementia in the elderly. β-amyloid (Aβ) accumulation in the brain is thought to be a primary event leading to eventual cognitive and motor dysfunction in AD. Aβ has been shown to promote neuronal hyperactivity, which is consistent with enhanced seizure activity in mouse models and AD patients. Little, however, is known about whether, and how, increased excitability contributes to downstream pathologies of AD. Here, we show that overexpression of human Aβ42 in a <i>Drosophila</i> model indeed induces increased neuronal activity. We found that the underlying mechanism involves the selective degradation of the A-type K+ channel, Kv4. An age-dependent loss of Kv4 leads to an increased probability of AP firing. Interestingly, we find that loss of Kv4 alone results in learning and locomotion defects, as well as a shortened lifespan. To test whether the Aβ42-induced increase in neuronal excitability contributes to, or exacerbates, downstream pathologies, we transgenically over-expressed Kv4 to near wild-type levels in Aβ42-expressing animals. We show that restoration of Kv4 attenuated age-dependent learning and locomotor deficits, slowed the onset of neurodegeneration, and partially rescued premature death seen in Aβ42-expressing animals. We conclude that Aβ42-induced hyperactivity plays a critical role in the age-dependent cognitive and motor decline of this Aβ42-<i>Drosophila</i> model, and possibly in AD.</p></div

Premature death is partially rescued by expression of K_v4 in Aβ42-expressing flies.

<p>(A-D) Longevity assays, using populations of ∼200 male flies, for indicated genotypes (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005025#sec012" target="_blank">Materials and Methods</a>). (A) Survival plots for wild-type, two different transgenic insertions of <i>UAS-DNK</i>_<i>v</i><i>4</i> (#14 and #20), and <i>elav-GAL4;;UAS-DNK</i>_<i>v</i><i>4</i> (elav-GAL4::UAS-DNK_v4) lines are shown. Neuronal expression of <i>UAS-DNK</i>_<i>v</i><i>4</i> leads to a significantly reduced (<i>P</i> < 0.0001 by log-rank analyses) lifespan at 25°C compared to <i>UAS</i> background controls. (B) Longevity assays performed for flies raised at 18°C during development, then shifted to 30°C for the entirety of the adult lifespan. Induced expression of DNK_v4 resulted in a significantly reduced lifespan (<i>elav-GAL4;tub-GAL80ts;UAS-DNK</i>_<i>v</i><i>4</i>; median age of 32 days), compared to wild-type (median age of 39 days) and <i>UAS-DNK</i>_<i>v</i><i>4</i> (median age of 37 days); significance of <i>P</i> < 0.0001 by log-rank analyses. (C-D) Survival plots for <i>elav-GAL4;+</i> (elav-GAL4), <i>elav-GAL4;UAS-Aβ42/+</i> (elav-GAL4::UASAβ42), <i>elav-GAL4;UAS-Aβ42/UAS-K</i>_<i>v</i><i>4</i> (elav-GAL4::UASAβ42,UAS-K_v4), <i>elav-GAL4;UAS-Aβ42/UAS-K</i>_<i>v</i><i>1</i> (elav-GAL4::UASAβ42,UAS-K_v1), <i>elav-GAL4;UAS-Aβ42/+;UAS-CD8-GFP/+</i> (elav-GAL4::UASAβ42,UAS-CD8GFP), and <i>elav-GAL4;UAS-Aβ42/+;UAS-EKO/+</i> (elav-GAL4::UASAβ42,UAS-EKO); all of these assays were performed at 25°C. <i>elav-GAL4;UAS-Aβ42/+</i> flies show a severely reduced lifespan (median age of 41 days) at 25°C, compared with the <i>UAS-Aβ42/+</i> background control (median age of 70 days; significance of <i>P</i> < 0.0001 by log-rank analyses). <i>elav-GAL4;UAS-Aβ42/UAS-K</i>_<i>v</i><i>4</i> flies showed a partial rescue of lifespan (median age of 46 days; P < 0.0001 by log-rank and Gehan-Breslow-Wilcoxon analyses), while <i>elav-GAL4;UAS-Aβ42/+;UAS-EKO/+</i>, <i>elav-GAL4;UAS-Aβ42/K</i>_<i>v</i><i>1</i>, <i>and elav-GAL4;UAS-Aβ42/+;UAS-CD8-GFP/+</i> flies did not show a significant improvement in lifespan from <i>elav-GAL4;UAS-Aβ42/+</i> flies.</p

Aβ42-induced changes in voltage-dependent K⁺ currents in primary neurons.

<p>Representative K⁺ current traces (A) and quantification of current amplitudes (B) from MB neuron recordings from <i>UAS-Aβ42/+</i> (Ctr) and <i>elav-GAL4;UAS-Aβ42/+</i> (Aβ42) embryos cultured for 1, 5 and 9 days. The K_v2-K_v3 DR component was recorded using a prepulse of -45 mV to completely inactivate K_v4 channels, before stepping to a test potential of +50 mV; the K_v4 current was then isolated by subtracting this DR component from the total whole-cell current elicited from a prepulse of -125 mV, stepping to a test potential of +50 mV. Current density was obtained by dividing peak current amplitude by cell capacitance. K_v4 current density was significantly reduced in Aβ42 MB neurons in 9-day old cultures, while no difference in K_v2-K_v3 currents was observed. (n = 8–10 for each group, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, Student’s t-test). Scale bars, 100 pA, 50 ms. (C-D) Steady-state inactivation and activation properties of Kv4 compared between neurons from Ctr and Aβ42 cultures. For steady-state inactivation analyses, we used a prepulse from -125 to -5 mV, in 5 mV intervals, then stepped to a test potential of +50 mV; representative current traces are shown (C, <i>Left</i>). For G –V analyses, we used voltage jumps from -40 to 50 mV, in 10 mV intervals, and either a prepulse of -125 mV or -45 mV for the total current or DR component, respectively; K_v4 current amplitudes were obtained by subtracting the DR component from the total current. Steady-state inactivation and activation curves were fitted with Boltzmann functions, I/I_max = 1/(1 + exp[(V – V_1/2)/k]) and G/G_max = 1/(1 + exp[(V_1/2 – V)/k]), respectively. V_1/2 is the half-maximal voltage, and k is the slope factor. Note that steady-state inactivation was fitted by two Boltzmann equations, and K_v4 is represented by the first Boltzmann (more negative operating range)[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005025#pgen.1005025.ref020" target="_blank">20</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005025#pgen.1005025.ref021" target="_blank">21</a>]. There was no significant difference in half-maximal inactivation potential values (Ctr, V_1/2 = -69.3 ± 2.1 mV, k = 9.1 ± 0.5; Aβ42, V_1/2 = -71.2 ±3.4 mV, k = 9.7 ± 0.5). There was also no difference in the half-maximal activation potential values (Ctr, V_1/2 = -5.7 ± 0.4 mV, k = 12.5 ± 0.8; Aβ42, V_1/2 = -6.8 ± 0.4 mV, k = 13.6 ± 0.6). n = 10 or each group. *** P < 0.001. All neurons were from cultures 9 days old.</p

Pan-neuronal expression of K_v4 slows the onset and severity of Aβ42-induced neurodegeneration.

<p>(A) Representative confocal images, from MB position described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005025#pgen.1005025.g007" target="_blank">Fig. 7</a>, show nuclear (DAPI) staining (upper panels, blue) from five genotypes, including <i>UAS- Aβ42/+</i> (Ctr), <i>elav-GAL4;UAS-Aβ42/+</i> (Aβ42), <i>elav-GAL4;UAS-Aβ42/UAS-K</i>_<i>v</i><i>4</i> (Aβ42+K_v4), <i>elav-GAL4;UAS-Aβ42/UAS-GFP</i> (Aβ42+GFP) and <i>elav-GAL4;UAS-Aβ42/UAS-K</i>_<i>v</i><i>1</i> (Aβ42+K_v1). Co-immunolabeling for Aβ42 is shown (lower panels, red) from these genotypes. Brains were dissected and labeled from flies aged 15 days (15d), 25 days (25d) and 40 days (40d) AE. Note that Aβ42 immunostaining is clearly seen in all the genotypes except Ctr. (B) Quantification of cell density (DAPI-positive) from confocal images described in (A). Note that there is significant cell loss in Aβ42, Aβ42+GFP, and Aβ42+K_v1 flies at 25d, but not in Aβ42+K_v4. n = 6–8 for each genotype, * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t-test. (C) Relative anti-Aβ42 signal was quantified in the same aged genotypes described in (A); background fluorescence is reflected in Ctr samples. Note that no significant difference in Aβ42 signal was seen in Aβ42+K_v4 brains, compared to Aβ42 at 15 and 25 days AE; at 40d AE, Aβ42+K_v4 brains show an increase in Aβ42 signal; quantification, however, was difficult due to the number of degenerative “holes” seen in confocal sections at this age. n = 6–8 for each genotype. All data are expressed as means ± SEM.</p

Aβ42 induces increased neuronal excitability in cultures 9 days old.

<p>(A) Representative immunoblots of fly heads (<i>Top</i>) and immunostaining of cultured neurons (<i>Bottom</i>) showing expression of Aβ42 in <i>elav-GAL4;UAS-Aβ42/+</i> (Aβ42) heads and <i>elav-GAL4;UAS-Aβ42/+;UAS-GFP</i> neurons, respectively; absence of Aβ42 expression in wild-type (wt) is shown as a negative control. Note that immunostaining is from a large cluster of neurons identified by GFP expression. Scale bar represents 12.5 μm. (B) Shown are representative wild-type and <i>elav-GAL4;UAS-Aβ42/+</i> (Aβ42) cell cultures, both with neurons expressing GFP as a marker, at 9 days. At lower power (<i>Top</i>), whole cultures from a single-embryo can be seen (scale bar represents 25 μm); at higher power (<i>Bottom</i>), neuronal processes between clusters of neurons can be seen (scale bar represents 8 μm). No obvious differences in growth of cultured neurons between wt and Aβ42 were observed. (C) Representative current-clamp recordings from wild-type (Ctrl) and <i>elav-GAL4;UAS-Aβ42/+</i> (Aβ42) neurons, showing relative times to the first action potential (AP) firing in response to ramped current stimulation. Current injection was ramped from 0 to 150 pA over 1 sec; voltage responses to the first 336 ms (50 pA) of the ramp protocol are shown. Scale bars represent 50 ms and 10 mV. (D) Quantification of the injected current (<i>Left</i>) and the charge transfer (<i>Right</i>) required to elicit the first AP in Ctrl and Aβ42 neurons during ramp stimulation; all ramp protocols were initiated from a membrane potential of -50 to -60 mV. The average charge transfer to elicit AP firing was also significantly reduced in Aβ42 neurons (2197.4 +/- 484.46 pAms, n = 6), compared to Ctrl (5132.8 +/- 1091.32 pAms, n = 13; <i>P</i> < 0.05, Student’s t-test). (E) Representative traces (<i>Left</i>) from a wild-type neuron, showing the latency to AP firing in response to a 500 ms pulse of 20 pA current injection, following pre-injections of current that generated a membrane potential of -115, -65, or -30 mV. Latencies to AP firing was normalized to average latency at -30 to -40 mV are shown for wild-type neurons from the indicated membrane potentials (<i>Right</i>). Note the negative correlation between AP latency and membrane potential (* denotes a significant difference in latency compared to that at V_m = -30 to -40mV; <i>P</i> < 0.05, Student’s t-test). Scale bar represents 100 ms and 20 mV. (F) AP latencies, in response to 500 ms, 40 pA current pulses, from single events are plotted against membrane potential for Ctrl and Aβ42 neurons. Ctrl cells showed a significant correlation between latency and membrane potential (<i>r</i> = -0.5633, <i>P</i> < 0.0005, n = 37), while Aβ42 cells did not (<i>r</i> = -0.4111, <i>P</i> = 0.0574, n = 22); the coefficient of correlation for each genotype was calculated by the Pearson product-moment correlation coefficients (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005025#sec012" target="_blank">Materials and Methods</a>).</p

Expression of Aβ42 decreases K_v4 protein and currents in the intact brain.

<p>(A) Representative neuronal cluster from 9 day old culture from <i>elav-GAL4;UAS-Aβ42/UAS-GFP-K</i>_<i>v</i><i>4</i> embryos show co-localization of Aβ42 (red) and GFP-K_v4 (green) immunostaining. Scale bar represents 8.3 μm. (B) Representative K_v4 and K_v2-K_v3 traces (<i>Left</i>), and K_v4 current density quantification (<i>Right</i>) from GFP-labeled MB neurons in <i>UAS-Aβ42/+</i> (Ctr) and <i>elav-GAL4;UAS-Aβ42/+</i> (Aβ42) intact brains at 3 and 8 days; Ctr1 and Ctr2 on right correspond to <i>UAS-Aβ42/+</i> and <i>elav-GAL4;+</i>, respectively. The K_v2-K_v3 DR component was recorded using a prepulse of -45 mV to completely inactivate K_v4 channels, before stepping to a test potential of +50 mV; the K_v4 current was then isolated by subtracting this DR component from the total whole-cell current elicited from a prepulse of -125 mV, stepping to a test potential of +50 mV. Current density was obtained by dividing peak current amplitude by cell capacitance. K_v4 current density in Aβ42 neurons at 8 days were significantly reduced compared to Ctr1 and Ctr2 neurons (<i>P</i> < 0.05, n = 6–13 for each genotype). Scale bars represent 50 pA and 50 ms. (C) Representative immunoblots (<i>Left</i>) and quantitative analyses (<i>Right</i>) of K_v4 protein levels in <i>UAS-Aβ42/+</i> (Ctr) and <i>elav-GAL4;UAS-Aβ42/+</i> (Aβ42) heads at 2–3, 7–8, and 14–15 days after eclosion. Protein levels were normalized to the loading control signal from anti-syntaxin (syn) (n = 4 for each group, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, Student’s t-test). (D) Representative immunoblots (<i>Left</i>) and quantitative analyses (<i>Right</i>) of K_v4 protein from <i>elav</i> (Ctr), <i>elav;;UAS-Aβ40/+</i> (Aβ40), and <i>elav;UAS-Aβ42/+</i> heads at 14–15 days AE. (n = 3–4 for each group, * <i>P</i> < 0.05, Student’s t-test indicates significant difference between Aβ40 and Aβ42) (E) Representative blots (<i>Left</i>) and quantitative results (<i>Right</i>) from RT-PCR analyses for <i>K</i>_<i>v</i><i>4</i> and <i>actin</i> RNA levels from <i>elav</i> (Ctr), <i>UAS-Aβ42</i> (UAS), and <i>elav;UAS-Aβ42/+</i> (Aβ42) flies aged 2, 7, and 15 days AE. Quantitation was performed from RT-PCR results from three independent RNA extractions; RT minus controls were always performed in parallel with RT plus reactions as indicated. No significant differences were seen across the three genotypes.</p