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

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

Rapamycin acts in the brain to decrease body weight gain.

<p>A and B: RAP i.c.v. injection (Day 0, broken line) induces a transient decrease in body weight gain (A), which results in prolonged shift in body weight (B). C: Food intake is inhibited by RAP i.c.v. D: Food efficiency is inhibited by RAP i.c.v. *p<0.05, **p<0.01, ***p<0.001 (two-way Mixed-ANOVA).</p

Rapamycin does not induce malaise or illness.

<p>A: During the two-bottle test, RAP group ingested significantly less fluid (sum of water and 0.1% Saccharin solution). *p<0.05 (unpaired t-test). B: There was no difference in saccharin preference (unpaired t-test). C: During the two-bottle test, there was no difference in total fluid intake in LiCl- and VEH-treated rats (sum of water and 0.1% Saccharin solution). D: LiCl-treated rats showed a significantly lower saccharin preference compared to VEH-treated rats. ****p<0.0001 (unpaired t-test)</p

Single systemic injection of rapamycin induces prolonged decrease in body weight gain.

<p>A: Rapamycin (RAP) i.p. injection on Day 0 (vertical broken line) induces a transient decrease in daily food intake. B: Three-day cumulative food intake (Day 1–3 post-injection) shows a dose-dependent inhibition. C: RAP induces a transient decrease in food efficiency. D: Three-day cumulative food efficiency (Day 1–3) shows a dose-dependent suppression. E: RAP induces a transient decrease in daily weight gain. For panel A, C and E, *p<0.001 for 10 mg/kg vs.VEH; **p<0.05 for 1 and 10 mg/kg vs. VEH; ***p<0.05 for all RAP doses vs.VEH (two-way Mixed-ANOVA). For panels B and D, ##p<0.01, ###p<0.001 (one-way ANOVA with Tukey's test). F: Cumulative body weight gain curve depicting that RAP injection results in a downward shift in body weight. The first 2 weeks (box) is expanded and shown in the inset. The effect is dose-dependent. VEH vs.10 mg/kg, p<0.01 on Day 3–74; VEH vs. 1 mg/kg, p<0.01 on Day 2–11, p<0.05 on Day 14 and 18; VEH vs. 0.1 mg/kg, not significant (two-way Mixed-ANOVA). G, H: Averaged daily water intake (H) and water intake normalized to/body weight (G) shows no difference between RAP (10 mg/kg)-treated animals compared to VEH (two-way Mixed-ANOVA).</p

Rapamycin does not affect glucose tolerance.

<p>A: There were no differences in blood glucose levels measured at 15, 30, 60, 90 and 120 minutes post-glucose injection in RAP and VEH animals (two-way Mixed-ANOVA). B: There is no difference in non-fasted blood glucose in rats administered with VEH or RAP (10 mg/kg i.p.) at 2 weeks post-injection (unpaired t-test).</p

Single Rapamycin Administration Induces Prolonged Downward Shift in Defended Body Weight in Rats

Manipulation of body weight set point may be an effective weight loss and maintenance strategy as the homeostatic mechanism governing energy balance remains intact even in obese conditions and counters the effort to lose weight. However, how the set point is determined is not well understood. We show that a single injection of rapamycin (RAP), an mTOR inhibitor, is sufficient to shift the set point in rats. Intraperitoneal RAP decreased food intake and daily weight gain for several days, but surprisingly, there was also a long-term reduction in body weight which lasted at least 10 weeks without additional RAP injection. These effects were not due to malaise or glucose intolerance. Two RAP administrations with a two week interval had additive effects on body weight without desensitization and significantly reduced the white adipose tissue weight. When challenged with food deprivation, vehicle and RAP-treated rats responded with rebound hyperphagia, suggesting that RAP was not inhibiting compensatory responses to weight loss. Instead, RAP animals defended a lower body weight achieved after RAP treatment. Decreased food intake and body weight were also seen with intracerebroventricular injection of RAP, indicating that the RAP effect is at least partially mediated by the brain. In summary, we found a novel effect of RAP that maintains lower body weight by shifting the set point long-term. Thus, RAP and related compounds may be unique tools to investigate the mechanisms by which the defended level of body weight is determined; such compounds may also be used to complement weight loss strategy

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