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

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

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

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

Larval olfactory associative learning and locomotion is defective in Aβ42-expressing larvae, and rescued by K_v4.

<p>(A) The following genotypes were trained and tested for learning function: wild-type (wt), <i>UAS-DNK</i>_<i>v</i><i>4</i> (UAS-DNK_v4), <i>elav-GAL4;;UAS-DNK</i>_<i>v</i><i>4</i> (elav-GAL4::DNK_v4), <i>elav-GAL4</i> (elav), <i>UAS-Aβ42/+</i> (UAS-Aβ42), <i>UAS-K</i>_<i>v</i><i>4</i> (UAS-K_v4), <i>elav-GAL4;UAS-Aβ42/+</i> (elav::Aβ42), <i>elav-GAL4;UAS-Aβ42/UAS-K</i>_<i>v</i><i>4</i> (elav::Aβ42+K_v4), <i>elav-GAL4;UAS-Aβ42/UAS-K</i>_<i>v</i><i>1</i> (elav::Aβ42+K_v1), <i>elav-GAL4;UAS-Aβ42/+;UAS-CD8-GFP</i> (elav::Aβ42+GFP), <i>elav-GAL4;UAS-Aβ42/+;UAS-EKO/+</i> (elav::Aβ42+EKO), <i>dnc</i>^<i>1</i> (dnc), <i>201y-GAL4</i> (201y), and <i>201y-GAL4;UAS-DNK</i>_<i>v</i><i>4</i> (201y::DNK_v4). Learning was tested by training larvae to associate AM or OCT with fructose in the AM+/OCT- or AM-/OCT+ paradigms described in the text and Materials and Methods. After training, larvae were placed on a plain agarose test plate, with AM and OCT sources at opposite sides; after 20 minutes, larvae on each half of the plate were counted. Preference scores and a learning index (LI) were calculated, as described in Materials and Methods (n = 4–15 pairs of groups for each genotype, with one pair consisting of one group for AM+/OCT- training and one group for AM-/OCT+ training; 5 larvae per individual group). <i>elav-GAL4;UAS-DNK</i>_<i>v</i><i>4</i>, <i>201y-GAL4;UAS-DNK</i>_<i>v</i><i>4</i>, and <i>dnc</i> all showed significantly reduced learning indices, compared to wild-type and background controls. <i>elav-GAL4;UAS-Aβ42/+</i> also had a significantly reduced LI compared to background controls, which was rescued in <i>elav-GAL4;UAS-Aβ42/UAS-K</i>_<i>v</i><i>4</i>, but not in <i>elav-GAL4;UAS-Aβ42/+;UAS-CD8-GFP/+</i>, <i>elav-GAL4;UAS-Aβ42/UAS-K</i>_<i>v</i><i>1</i>, or <i>elav-GAL4;UAS-Aβ42/+;UAS-EKO/+</i>. (B) Locomotor activity was tested in a standard climbing assay on wild-type (wt), <i>elav-GAL4</i> (elav), <i>UAS-Aβ42/+</i> (UAS), <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-K</i>_<i>v</i><i>1</i> (Aβ42+K_v1), <i>elav-GAL4;UAS-Aβ42/+;UAS-CD8-GFP/+</i> (Aβ42+GFP), and <i>elav-GAL4;UAS-Aβ42/+;UAS-EKO/+ (</i>Aβ42+EKO) at 1–2 days and 14–15 days after eclosion, as described in text; each fly was given one point for every two tubes they climbed out of. The mean score of flies from each group (30–35 flies) was calculated; this was then repeated for 5–15 groups for each genotype with averages shown. <i>elav-GAL4;UAS-Aβ42/+</i> flies showed a significant impairment at 14–15 days, compared with <i>UAS-Aβ42/+</i> background controls. Co-expression of <i>UAS-K</i>_<i>v</i><i>4</i> or <i>UAS-K</i>_<i>v</i><i>1</i> rescues this impairment, while expression of <i>UAS-EKO</i> did not. * denotes <i>P</i> < 0.05, Student’s t-test.</p

Restoration of excitability in Aβ42-expressing flies ameliorates age-dependent neuronal loss in MBs.

<p>(A) Converged confocal z-stack images of a typical mushroom body from the transgenic line, <i>elav-GAL4;;GFP</i>.<i>S65T</i>.<i>T10/+</i> (<i>Left</i>, <i>Top</i>), and a corresponding diagram with regions containing cell bodies, dendrites (in calyx), and axons shown (<i>Left</i>, <i>bottom</i>); MB neurons were GFP labeled by the <i>GFP</i>.<i>S65T</i>.<i>T10</i> transgene (regardless of GAL4 driver present, see text) and counted from single images taken 25 μm from the posterior-most edge of MB, as depicted by the dotted line shown. Representative confocal images used for counting neurons are shown from <i>elav-GAL4;;GFP</i>.<i>S65T</i>.<i>T10/+</i> (Ctr), <i>elav-GAL4;UAS-K</i>_<i>v</i><i>4/+;GFP</i>.<i>S65T</i>.<i>T10/+</i> (K_v4), <i>elav-GAL4;UAS-Aβ42/+;GFP</i>.<i>S65T</i>.<i>T10/+</i> (Aβ42), and <i>elav-GAL4;UAS-Aβ42/UAS-K</i>_<i>v</i><i>4;GFP</i>.<i>S65T</i>.<i>T10/+</i> (Aβ42 + K_v4) flies at indicated ages: 15, 25, 40 and 50 days AE (<i>Middle</i>); in confocal images, the calyx (C) is seen above the region containing MB cell somas (s). Quantitative analyses show that restoration of normal excitability by transgenic <i>UAS-K</i>_<i>v</i><i>4</i> expression significantly increases neuronal survival at 40 and 50 days, delaying the onset of neurodegeneration (no significant (n.s,) difference between Ctr and Aβ42 + K_v4 at 25 d) (<i>Right</i>). (n = 6–7 for each group, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, Student’s t-test). (B) Local expression of <i>UAS-Aβ42</i> was induced using the MB driver <i>201y-GAL4</i>. Representative confocal images and quantification, as described in (A), are shown from <i>201y-GAL4;GFP</i>.<i>S65T</i>.<i>T10/+</i> (Ctr), <i>201y-GAL4/UAS-Aβ42/+;GFP</i>.<i>S65T</i>.<i>T10/+</i> (201y-Gal4+Aβ42), <i>201y-GAL4/UAS-Aβ42; GFP</i>.<i>S65T</i>.<i>T10/UAS-K</i>_<i>v</i><i>4</i> (201y-Gal4+Aβ42+K_v4), and <i>201y-GAL4/UAS-Aβ42;UAS-EKO/GFP</i>.<i>S65T</i>.<i>T10</i> (201y-Gal4+Aβ42+EKO) heads at 15 and 40 days after eclosion. Local expression of Aβ42 results in a significant reduction in MBNs at 40 days (110.30 +/- 4.03), compared to Ctr (132.10 +/- 3.17), which is rescued by K_v4 expression (129.40 +/- 4.18), but not expression of EKO (111.30 +/- 4.76); n = 8–9 brains for each condition, *<i>P</i> < 0.05, Student’s t-test.</p

Aβ42-induced down-regulation of K_v4 protein is mediated by a proteasome and lysosome-dependent pathway.

<p>(A) Representative traces of separated K_v4 and K_v2-K_v3 currents recorded from MB neurons from <i>UAS-Aβ42/+</i> (Ctr) and <i>elav-GAL4;UAS-Aβ42/+</i> (Aβ42) cultures at 9 days, after incubation with MG132 (10 μM). 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. Quantitative analyses of K_v4 current density shows that MG132 blocked the Aβ42-induced down-regulation of K_v4 (<i>Right;</i> n = 8–9 for each group, ** <i>P</i> < 0.01, Student’s t-test). Scale bars represent 100 pA and 50 ms. (B) Representative immunoblots and quantitative analyses of K_v4 protein levels in cultured brains. The Aβ42-induced decrease in K_v4 is blocked when brains were incubated with MG132 for 4 days. K_v4 levels were normalized to the loading control signal from anti-syntaxin (syn) (n = 3 for each group, * <i>P</i> < 0.05, Student’s t-test). (C) Down-regulation of K_v4 protein by Aβ42 is absent when proteasome activity was inhibited by expression of the dominant temperature-sensitive <i>UAS-Pros26</i>^<i>1</i><i>and UASProsβ</i>^<i>2</i> transgenes. <i>elav-GAL4;UAS-pros26</i>^<i>1</i>/+;<i>UAS-prosβ</i>^<i>2</i>/+ flies (pros), <i>elav-GAL4;UAS-Aβ42/UAS-pros26</i>^<i>1</i>;<i>UAS-prosβ</i>^<i>2</i>/+ flies (pros + Aβ42), <i>UAS-Aβ42/+</i> (Ctr), and <i>elav-GAL4;UAS-Aβ42/+</i> (Aβ42) were all raised at 18°C during development, then shifted to 29°C for 7–8 days after adult eclosion. Immunoblot analyses were performed for K_v4, and normalized to syn (<i>Left</i>). For quantitative analyses (<i>Right</i>), n = 4 for each condition, * <i>P</i> < 0.05, Student’s t-test. (D) Aβ42-induced decrease in K_v4 current is also absent by the genetic inhibition of the proteasome described in (C) (<i>right</i>). <i>elav-GAL4;UAS-Pros26</i>^<i>1</i>/<i>UAS-Aβ42;UAS-Prosβ</i>^<i>2</i>/+ and <i>elav-GAL4;UAS-Pros26</i>^<i>1</i>/+;<i>UAS-Prosβ</i>^<i>2</i>/+ flies were raised at 18°C during development, then shifted as newly-eclosed adult flies to 29°C for 7–8 days (n = 4 for immunoblots, and n = 8 for each current recordings). Scale bars represent 25 pA and 50 ms. (E) Representative K_v4 and K_v2–3 currents in Ctrl and <i>elav-GAL4;UAS-Aβ42</i> (Aβ42) neurons from cultures treated with 20 μM leupeptin for 8 days. 20 μM leupeptin blocked the Aβ42-induced decrease in K_v4 current density seen in Ctrl neurons.</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

Transgenic over-expression of K_v4 in Aβ42-expressing flies restores K_v4 protein levels and normal excitability to neurons.

<p>(A) Immunoblot analyses of <i>UAS-Aβ42/+</i> (Ctrl), <i>elav-GAL4;UAS-Aβ42/+</i> (Aβ42), and <i>elav-GAL4;UAS-Aβ42/UAS-K</i>_<i>v</i><i>4</i> (Aβ42 + K_v4) fly heads at 7–8 days are shown. Transgenic expression of <i>UAS-K</i>_<i>v</i><i>4</i> restores K_v4 protein levels to wild type levels. (n = 3 for each group, * <i>P</i> < 0.05, Student’s t test). (B) Representative K_v4 currents isolated in voltage-clamp mode from primary neurons, from cultures 9 days old, from Ctrl, Aβ42 and <i>elav-GAL4;UASAβ42/UAS-K</i>_<i>v</i><i>4</i> (Aβ42+K_v4) flies are shown (<i>Right</i>). 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. Quantitative analyses (<i>Left</i>) show that K_v4 current density from Aβ42 neurons (194.3 +/- 23.68 pA, <i>n</i> = 9) were significantly smaller than from Ctrl neurons (320.2 +/- 33.52 pA, <i>n</i> = 25). K_v4 current density in Aβ42+K_v4 neurons showed a near complete restoration (334.3 ± 48.97 pA, n = 18). * <i>P</i> < 0.05, Student’s <i>t</i>-test. Scale bars represent 100 pA and 20 ms. (C) Representative current-clamp recordings from Aβ42 and the Aβ42+K_v4 neurons in response to ramped stimulation protocol described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005025#pgen.1005025.g001" target="_blank">Fig. 1A</a>, show that the time to first peak was significantly prolonged by the restoration of K_v4 to Aβ42-expressing neurons. Scale bar represents 10 mV and 50 ms. (D) Quantification of the average current injected and charge transfer required to elicit the first AP are shown. Both were significantly increased with transgenic restoration of K_v4: 58.9 +/- 4.54 pA, <i>n</i> = 9 (<i>Left</i>), and 11,108.0 +/- 920.49 pAms, <i>n</i> = 9 (<i>Right</i>); note that quantification for Ctrl and Aβ42 values represent the same data set shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005025#pgen.1005025.g001" target="_blank">Fig. 1B</a>. * <i>P</i> < 0.05, Student’s <i>t</i>-test.</p