Ataxin2 functions via CrebA to mediate Huntingtin toxicity in circadian clock neurons

Disrupted circadian rhythms is a prominent and early feature of neurodegenerative diseases including Huntington???s disease (HD). In HD patients and animal models, striatal and hypothalamic neurons expressing molecular circadian clocks are targets of mutant Huntingtin (mHtt) pathogenicity. Yet how mHtt disrupts circadian rhythms remains unclear. In a genetic screen for modifiers of mHtt effects on circadian behavior in Drosophila, we discovered a role for the neurodegenerative disease gene Ataxin2 (Atx2). Genetic manipulations of Atx2 modify the impact of mHtt on circadian behavior as well as mHtt aggregation and demonstrate a role for Atx2 in promoting mHtt aggregation as well as mHtt-mediated neuronal dysfunction. RNAi knockdown of the Fragile X mental retardation gene, dfmr1, an Atx2 partner, also partially suppresses mHtt effects and Atx2 effects depend on dfmr1. Atx2 knockdown reduces the cAMP response binding protein A (CrebA) transcript at dawn. CrebA transcript level shows a prominent diurnal regulation in clock neurons. Loss of CrebA also partially suppresses mHtt effects on behavior and cell loss and restoration of CrebA can suppress Atx2 effects. Our results indicate a prominent role of Atx2 in mediating mHtt pathology, specifically via its regulation of CrebA, defining a novel molecular pathway in HD pathogenesis

A Conserved Bicycle Model for Circadian Clock Control of Membrane Excitability

SummaryCircadian clocks regulate membrane excitability in master pacemaker neurons to control daily rhythms of sleep and wake. Here, we find that two distinctly timed electrical drives collaborate to impose rhythmicity on Drosophila clock neurons. In the morning, a voltage-independent sodium conductance via the NA/NALCN ion channel depolarizes these neurons. This current is driven by the rhythmic expression of NCA localization factor-1, linking the molecular clock to ion channel function. In the evening, basal potassium currents peak to silence clock neurons. Remarkably, daily antiphase cycles of sodium and potassium currents also drive mouse clock neuron rhythms. Thus, we reveal an evolutionarily ancient strategy for the neural mechanisms that govern daily sleep and wake

Dual PDF signaling pathways reset clocks via TIMELESS and acutely excite target neurons to control circadian behavior.

Molecular circadian clocks are interconnected via neural networks. In Drosophila, PIGMENT-DISPERSING FACTOR (PDF) acts as a master network regulator with dual functions in synchronizing molecular oscillations between disparate PDF(+) and PDF(-) circadian pacemaker neurons and controlling pacemaker neuron output. Yet the mechanisms by which PDF functions are not clear. We demonstrate that genetic inhibition of protein kinase A (PKA) in PDF(-) clock neurons can phenocopy PDF mutants while activated PKA can partially rescue PDF receptor mutants. PKA subunit transcripts are also under clock control in non-PDF DN1p neurons. To address the core clock target of PDF, we rescued per in PDF neurons of arrhythmic per⁰¹ mutants. PDF neuron rescue induced high amplitude rhythms in the clock component TIMELESS (TIM) in per-less DN1p neurons. Complete loss of PDF or PKA inhibition also results in reduced TIM levels in non-PDF neurons of per⁰¹ flies. To address how PDF impacts pacemaker neuron output, we focally applied PDF to DN1p neurons and found that it acutely depolarizes and increases firing rates of DN1p neurons. Surprisingly, these effects are reduced in the presence of an adenylate cyclase inhibitor, yet persist in the presence of PKA inhibition. We have provided evidence for a signaling mechanism (PKA) and a molecular target (TIM) by which PDF resets and synchronizes clocks and demonstrates an acute direct excitatory effect of PDF on target neurons to control neuronal output. The identification of TIM as a target of PDF signaling suggests it is a multimodal integrator of cell autonomous clock, environmental light, and neural network signaling. Moreover, these data reveal a bifurcation of PKA-dependent clock effects and PKA-independent output effects. Taken together, our results provide a molecular and cellular basis for the dual functions of PDF in clock resetting and pacemaker output

Circadian Clocks Function in Concert with Heat Shock Organizing Protein to Modulate Mutant Huntingtin Aggregation and Toxicity

Summary: Neurodegenerative diseases commonly involve the disruption of circadian rhythms. Studies indicate that mutant Huntingtin (mHtt), the cause of Huntington’s disease (HD), disrupts circadian rhythms often before motor symptoms are evident. Yet little is known about the molecular mechanisms by which mHtt impairs circadian rhythmicity and whether circadian clocks can modulate HD pathogenesis. To address this question, we used a Drosophila HD model. We found that both environmental and genetic perturbations of the circadian clock alter mHtt-mediated neurodegeneration. To identify potential genetic pathways that mediate these effects, we applied a behavioral platform to screen for clock-regulated HD suppressors, identifying a role for Heat Shock Protein 70/90 Organizing Protein (Hop). Hop knockdown paradoxically reduces mHtt aggregation and toxicity. These studies demonstrate a role for the circadian clock in a neurodegenerative disease model and reveal a clock-regulated molecular and cellular pathway that links clock function to neurodegenerative disease. : Disruption of circadian rhythms is frequently observed across a range of neurodegenerative diseases. Here, Xu et al. demonstrate that perturbation of circadian clocks alters the toxicity of the mutant Huntingtin protein, the cause of Huntington’s disease (HD). Moreover, they reveal a key mechanistic link between the clock and HD. Keywords: circadian, Huntington’s disease, heat shock, transcriptome, genetic scree

Non-PDF (E-cell) pacemaker neuron specific modulation of PKA activity can phenocopy or rescue <i>pdfr</i>- behaviors.

<p>E-cells were targeted using <i>cry13-G4</i> and <i>pdf-G80</i>. <i>U-PKA-mC</i>* is a constitutively active PKA catalytic subunit that lacks the ability to bind to regulatory subunits. Graphs are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g001" target="_blank">Figure 1</a>. Genotype (N). (A) pdf-G80/+;cry13-G4/+ (15), (B) pdf-G80/+;cry13-G4/U-PKA-R1dn (10), (C) pdfr-;pdf-G80/+;cry13-G4/U-PKA-R1dn (26), (F) pdf-G80/U-PKA-mC*;cry13-G4/+ (15). (G) pdfr-;U-PKA-mC*/+ (22), (H) pdfr-;pdf-G80/U-PKA-mC*;cry13-G4/+ (48). (D,E,I,J) magnified overlays of morning (D,I) and evening (E,J) for the indicated color-coded genotypes as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g001" target="_blank">Figure 1</a>. *<i>p</i><0.05 versus both G4 and UAS parental controls. a, <i>p</i><0.05 versus UAS parental control, <i>p</i> = 0.056 versus G4 parental control. b, <i>p</i><0.05 versus <i>pdfr</i>-;<i>U-PKA-mC</i>*/+, not significant versus <i>pdf-G80</i>/U<i>-PKA-mC</i>*;<i>cry13-G4</i>/+. Complete quantification appears in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-t001" target="_blank">Table 1</a>.</p

<i>cwo-G4</i>><i>U-PKA-R1dn</i> expression reduces TIM levels in the absence of PER in LD.

<p>PKA-R1dn was expressed broadly in the circadian system using <i>cwo-G4</i> and restricted from PDF cells using <i>pdf-G80</i>. Data are displayed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g004" target="_blank">Figure 4</a>. Cell group (N): (A) sLNv (36–90), (B) LNd (49–130), (C) DN1 (42–195). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

PDF cell clocks specifically target TIM in LNds and DN1s in constant darkness.

<p>Single confocal slices showing PDF, PER, and TIM staining. <i>per⁰¹</i>;;<i>U-per</i>/+ (control, black lines) and <i>per⁰¹</i>;<i>pdf-G4</i>/+;<i>U-per</i>/+ (pdfPER, blue lines). sLNvs are marked with anti-PDF. TIM and PER images are displayed in NIH ImageJ lookup table 5 Ramps (inverted) for visibility. PDF images used to identify sLNv are in gray scale. Cells (N): (A) sLNv (53–87), (B) LNd (74–122), (C) DN1 (57–208). Average cell intensities were normalized to PPP CT6 = 1 before combining measurements from three (TIM) experiments. In some cases error bars (SEM) are very small and obscured by the data point. In no case were error bars omitted. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

PKA function in DN1p neurons.

<p>(A) Reduction of PKA activity in DN1p has no effect on the phase of evening activity, but reduces morning anticipation. Graphs are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g001" target="_blank">Figure 1</a>. Genotype (N). (A) Clk4.1-G4/+ (19), (B) Clk4.1-G4/U-PKA-R1dn (14). (C and D) are overlays of morning and evening activity as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g001" target="_blank">Figure 1</a>. PKA subunits are transcriptionally regulated by the circadian clock in DN1ps. DN1ps were marked with <i>Clk4.1-G4</i>><i>U-GFP</i> in wt (black) or <i>per⁰¹</i> (blue) background, dissociated, sorted, and analyzed by quantitative RT-PCR for PKA subunit transcripts (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#s4" target="_blank">Materials and Methods</a>). (E) PKA-C1, (F) PKA-R1, (G) PKA-R2. Error bars are SEM.</p

PDF induces an increase in [Ca²⁺]_i in the DN1p neurons.

<p>Representative current clamp recordings obtained in cell-attached mode showing on the same cell (A) depolarization, (B) increase in instant firing frequency, and (C) increase in [Ca²⁺]_i in <i>U-GcaMP6f</i>/+;<i>Clk4.1-G4</i>/+ male flies (<i>n</i> = 3).</p

DD circadian behavior parameters.

<p>See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#s4" target="_blank">Materials and Methods</a> for details of behavior quantification.</p>a<p>Only one rhythmic fly, therefore there is no SEM.</p><p>*<i>p</i><0.014 versus both parental controls.</p