Immunhistochemical and functional characterisation of the mitogen-activated protein kinase p38 in the endogenous clock of Drosophila melanogaster

Circadianes und Stress-System sind zwei physiologische Systeme, die dem Organismus helfen sich an Veränderungen ihrer Umwelt anzupassen. Während letzteres spontane und schnelle Antworten auf akute, unvorhersehbare Umweltreize liefert, sagt das circadiane System täglich wiederkehrende Ereignisse vorher and bereitet den Organismus so vorzeitig auf diese nahende Umweltveränderung vor. Dennoch, trotz dieser unterschiedlichen Reaktionsmechanismen agieren beide Systeme nicht komplett autonom. Studien der vergangen Jahre belegen vielmehr eine Interaktion beider Systeme. So postulieren sie zum einem Unterschiede in der Stressantwort in Abhängigkeit von der Tageszeit zu der der Reiz auftritt und weisen zugleich auf eine Zunahme von gestörten biologischen Tagesrhythmen, wie zum Beispiel Schlafstörungen, in Folge von unkontrollierten oder exzessiven Stress hin. Ebenso liefern kürzlich durchgeführte Studien an Vertebraten und Pilzen Hinweise, dass mit p38, eine Stress-aktivierte Kinase, an der Signalweiterleitung zur inneren Uhr beteiligt ist (Hayashi et al., 2003), sogar durch dieses endogene Zeitmesssystem reguliert wird (Vitalini et al., 2007; Lamb et al., 2011) und deuten damit erstmals eine mögliche Verbindung zwischen Stress-induzierten und regulären rhythmischen Anpassungen des Organismus an Umweltveränderungen an. Molekulare und zelluläre Mechanismen dieser Verknüpfung sind bisher noch nicht bekannt. Während die Rolle von p38 MAPK bei der Stress- und Immunantwort in Drosophila melanogaster gut charakterisiert ist, wurden Expression und Funktion von p38 in der inneren Uhr hingegen bislang nicht untersucht. Die hier vorliegende Arbeit hatte daher zum Ziel mittels immunhistochemischer, verhaltensphysiologischer und molekularer Methoden eine mögliche Rolle der Stress-aktivierten Kinase im circadianen System der Fliege aufzudecken. Antikörperfärbungen sowie Studien mit Reporterlinien zeigen deutliche Färbesignale in den s-LNv, l-LNv und DN1a und erbringen erstmals einen Nachweis für p38 Expression in den Uhrneuronen der Fliege. Ebenso scheint die Aktivität von p38 MAPK in den DN1a uhrgesteuert zu sein. So liegt p38 vermehrt in seiner aktiven Form in der Dunkelphase vor und zeigt, neben seiner circadian regulierten Aktivierung, zusätzlich auch eine Inaktivierung durch Licht. 15-Minuten-Lichtpulse in der subjektiven Nacht führen zu einer signifikanten Reduktion von aktivierter, phosphorylierter p38 MAPK in den DN1a von Canton S Wildtypfliegen im Vergleich zu Fliegen ohne Lichtpuls-Behandlung. Aufzeichnungen der Lokomotoraktivität offenbaren zusätzlich die Notwendigkeit von p38 MAPK für wildtypisches Timing der Abendaktivität sowie zum Erhalt von 24-Stunden-Verhaltensrhythmen unter konstanten Dauerdunkel-Bedindungen. So zeigen Fliegen mit reduzierten p38 Level in Uhrneuronen einen verzögerten Beginn der Abendaktivität und stark verlängerte Freilaufperioden. In Übereinstimmung mit Effekten auf das Laufverhalten scheint darüber hinaus die Expression einer dominant-negativen Form von p38b in Drosophila’s wichtigsten Uhrneuronen eine verspätete nukleäre Translokation von Period zur Folge zu haben. Westernblots legen zusätzlich einen Einfluss von p38 auf den Phosphorylierungsgrad von Period nahe und liefern damit einen mögliche Erklärung für den verspäteten Kerneintritt des Uhrproteins. Abschließende Stützung der Westernblotergebnisse bringen in vitro Kinasenassays und deuten auf p38 als eine potentielle „Uhrkinase“ hin, welche auch in vivo Period an Serin 661 sowie weiteren potentiellen Phosphorylierungsstellen phosphorylieren könnte. Zusammengenommen deuten die Ergebnisse der hier vorliegenden Arbeit eindeutig auf eine bedeutende Rolle von p38, neben dessen Funkion im Stress-System, auch im circadianen System der Fliege hin und offenbaren damit die Möglichkeit, dass p38 als Schnittstelle zwischen beider Systeme fungiert.The circadian and the stress system are two distinct physiological systems that help the organism to adapt to environmental challenges. While the latter elicits reactive responses to acute environmental changes, the circadian system predicts daily occurring alterations and prepares the organism in advance. However, despite of these differences both responses are not mutually exclusive. Studies in the last years obviously prove a strong interaction between both systems showing a strong time-related stress response depending on the time of day of stressor presentation on the one hand and increased disturbances of daily rhythms, like sleep disorders, in consequence of uncontrolled or excessive stress on the other. In line with this fact, recent studies in vertebrates and fungi indicate that p38, a stress-activated Kinase, is involved in signaling to the circadian clock (Hayashi et al., 2003) and in turn is additionally regulated by this timekeeping system (Vitalini et al., 2007; Lamb et al., 2011) providing an interesting link between stress-induced and regularly rhythmic adaptations of the organism to environmental changes. However, little is known about molecular and cellular mechanisms of this interconnection. In Drosophila melanogaster the role of p38 MAPK is well characterized in terms of immune and stress response, p38 expression and function in the circadian clock has not been reported so far. Therefore, the present thesis aimed to elucidate a putative role of the stress-activated Kinase in the fly’s circadian system using an immunohistochemical, behavioral as well as molecular approach. Surprisingly, for the first time antibody as well as reporterline studies cleary prove p38 expression in Drosophila clock neurons showing visible staining in s-LNvs, l-LNvs and DN1as. Moreover p38 MAPK in DN1as seems to be activated in a clock-dependent manner. p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. 15 minutes light pulse applied during the dark phase lead to a significant reduction in phosphorylated and activated p38 MAPK in Canton S wildtype flies compared to flies without light pulse treatment. In addition, locomotor activity recordings reveal that p38 is essential for a wild-type timing of evening activity and for maintaining ~24h behavioral rhythms under constant darkness. Flies with reduced p38 activity in clock neurons show delayed evening activity onsets and drastically lengthened the period of their free-running rhythms. In line with these effects on locomotor behavior, the nuclear translocation of the clock protein Period is significantly delayed on the expression of a dominant-negative form of p38b in Drosophila’s most important clock neurons. Western Blots reveal that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays additionally confirm the Western Blot results and point to p38 as a potential “clock kinase” phosphorylating Period at Serin 661 and putative phosphorylation sites. Taken together, the results of the present thesis clearly indicate a prominent role of p38 in the circadian system of the fly besides its function in stress-input pathways und open up the possibility of p38 MAPK being a nodal point of both physiological systems

The MAP Kinase p38 Is Part of Drosophila melanogaster's Circadian Clock

All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways

p38b promotes PER phosphorylation during the dark phase.

<p>To analyze daily phosphorylation of PER in flies that express the dominant-negative form of p38b in clock neurons and photoreceptor cells, we performed Western blots on head extracts after 4 days entrainment to LD 12∶12 cycles. According to our behavioral data, timing of PER accumulation was not affected in experimental flies (<i>UAS-p38b^DN-S;cry-Gal4/+;+</i>) in comparison with their respective control (A). However, regarding the degree of PER phosphorylation we observed differences at all time points when we compared both genotypes. For better comparison Western blots were repeated and samples of control and <i>UAS-p38b^DN-S;cry-Gal4/+;+</i> flies were plotted side by side for each ZT (B). Interestingly, flies with impaired p38 signaling indeed had less phosphorylated PER, showing the largest differences to the controls at the end of the night. Western blots were repeated 4 times and always gave similar results. Bars above the blots depict the light regime of the LD 12∶12. The “<i>C</i>” refers to respective control, <i>DNS</i> to <i>UAS-p38b^DN-S;cry-Gal4/+;+</i>.</p

Locomotor activity rhythms of <i>p38b</i> and <i>p38a</i> null mutants and hypomorphic double mutant flies.

<p>Both <i>p38</i> null mutants, <i>p38b^Δ45</i> (upper panels in A) and <i>p38a^Δ1</i> (upper panels in B), displayed wildtype-like behavior with activity bouts around lights-on and lights-off when recorded in LD 12∶12. Even if evening activity onset of <i>p38a^Δ1</i>seems to be delayed compared to <i>w¹¹¹⁸</i>, this delay did not result in a longer free-running period under constant darkness (lower panels in B). Similarly, flies, lacking the <i>p38b</i> gene, also showed comparable free-running rhythms as their respective controls (lower panels in A). Activity data in C show two representative single actograms of a double mutant strain with a hypomorphic <i>p38b</i> allele (<i>p38b^Δ25;p38a^Δ1</i>). Since these flies are hardly viable and die within 3–6 days after emergence of the pupa, flies were already entrained to LD12∶12 during pupal stage and subsequently monitored in DD conditions after eclosion. Even if periodogram analysis was not possible due to the short recording period, <i>p38b^Δ25;p38a^Δ1</i> flies clearly showed a long free-running period when kept in constant darkness (C). For recording and processing of activity data as well as for figure labeling see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004565#pgen-1004565-g003" target="_blank">Figure 3</a>.</p

Daily oscillations of nuclear PER in s-LN_vs and l-LN_vs of flies expressing a dominant negative form of p38b in these cells.

<p>Flies were entrained in LD 12∶12, dissected every one to two hours and staining intensity of nucleus and cell body was measured as described in Material and Methods. Nuclear PER staining intensities were normalized to total staining and tested for statistically significance. Expression of the dominant negative form of p38b phase delayed nuclear accumulation of PER in the s-LN_vs (A) and l-LN_vs (B). Arrows indicate the maxima of nuclear PER staining that occurred significantly later in <i>UAS-p38b^DN-S;Pdf-Gal4/+;+</i> flies than in control flies. This delay in nuclear PER accumulation in PDF-positive clock neurons is well consistent with the shifted evening activity in these flies. Bars above the graphs depict the light regime of the LD 12∶12.</p

Daily p38 mRNA (A) and protein expression (B–D) in <i>Canton S</i> wildtype.

<p>A: Quantitative real-time PCR on head extracts revealed constant mRNA expression throughout the day with allover higher levels of <i>p38b</i> compared to <i>p38a</i> (p<0.001). B: Antibody staining with anti-p-p38 on adult brains displayed rhythmic phosphorylation of p38 in DN_1as in LD with significant higher p-p38 levels occurring during the night than in the day (p<0.05). C: A highly significant reduction of active p38 in DN_1as at CT6 compared to CT18 in DD indicates a clock-controlled activation of p38 (p<0.001) D: Only a 15 minute light pulse (LP) during subjective night (CT18) and not during the subjective day (CT6) leads to a reduction in active p38 in DN_1as, suggesting a clock-dependent photic reduction of active p38. The “C” in D indicates control brains without 15 minute light pulse (LP). Error bars show SEM. Significant differences (p<0.05) are indicated by *, highly significant differences (p<0.001) by **.</p

p38b phosphorylates PER <i>in vitro</i>.

<p>To test whether p38b phosphorylates PER <i>in vitro</i>, either non-radioactive kinase assays followed by urea-PAGE (A–D) or radioactive kinase assays with autoradiography (E–F) were performed. A–D: Non-radioactive kinase assays were conducted with poly-histidine tagged p38b (His₆-p38b) and two truncated GST-tagged PER isoforms, GST-PER^1–700 (A,B) and GST-PER^658–1218 (C,D). Samples were subsequently separated with urea-PAGE and visualized by Coomassie staining (A,C). To further confirm PER's position in the gel two samples of the same gel were additionally blotted onto nitrocellulose membrane and immunolabeled using an anti-PER antibody and a secondary fluorescent antibody (B,D). While GST-PER^1–700 without kinase did not shift within 60 minutes, the addition of His₆-p38b induced a downward shift of GST-PER^1–700 indicating phosphorylation of PER (A; dotted line). Immunoblots with anti-PER further confirmed the size as well as the shift of the GST-PER^1–700 band (B). In addition to GST-PER^1–700, GST-PER^658–1218 also displayed band shifts after incubation with His₆-p38b (C). This was most prominent after 60 minutes, when addition of His₆-p38b resulted in two distinct shifted bands (black arrowheads), which could be additionally confirmed by Western blots (D). Time scale below graphs represents minutes after addition of His₆-p38b, the “C” refers to control and represents substrate samples without kinase and ATP. (E) Radioactive <i>in vitro</i> kinase assays were conducted with the indicated GST-PER fusion proteins and GST-p38b. Control reactions were performed in the absence of GST-p38b or with GST in combination with GST-p38b. Coomassie staining proved loading of the indicated protein combinations. Below, phosphorylation of GST-PER proteins was detected by autoradiography. (F) For quantitative analysis five independent <i>in vitro</i> kinase assay experiments were performed and analyzed. For each reaction within a single experiment, autoradiography signal intensities were normalized to the corresponding Coomassie stained protein band. Values in the graph are shown as percentages of GST-PER^658–1218 phosphorylation (100%; * p<0.05, ** p<0.005).</p

Locomotor activity rhythms of flies expressing a dominant-negative form of p38b <i>(p38b^DN-S)</i> in <i>Drosophila</i> clock neurons and respective controls.

<p>Both, expression of a dominant negative form of p38b in either all clock neurons (<i>UAS-p38b^DN-S;tim(UAS)-Gal4/+;+</i>) or just in a subset of clock cells, the PDF-positive LN_vs (<i>UAS</i>-<i>p38b^DN-S;Pdf-Gal4/+;+</i>), resulted in a diurnal activity profile with a significantly delayed evening activity onset in comparison with respective controls (upper panels in A and B). This delay in evening activity is accompanied by a significantly prolonged free-running period in <i>UAS</i>-<i>p38b^DN-S;tim(UAS)-Gal4/+;+</i> (lower panels in A) as well as in <i>UAS</i>-<i>p38b^DN-S;Pdf-Gal4/+;+</i> flies(lower panels in B), when released into constant darkness. For recording and processing of activity data as well as for figure labeling see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004565#pgen-1004565-g003" target="_blank">Figure 3</a>.</p

Rhythmicity and period length of all investigated genotypes in constant darkness (DD) according to χ²-periodogram analysis.

<p><i>n</i> indicates the number of tested flies per genotype that survived locomotor recordings. Power and period values were averaged over all rhythmic flies for each genotype.</p>1<p>Flies with power values <20 were defined as arrhythmic.</p>2<p>Power is a measure of rhythmicity and is given in % of variance.</p

p38 MAPK expression pattern in adult male <i>Canton S</i> brains.

<p>p38 MAPK distribution within the circadian clock was investigated immunohistochemically with an antibody directed against <i>Drosophila</i> p38b (A–C) and against phosphorylated human p38 (D–G). A–C: Staining with anti-p38b (green) in <i>Canton S</i> wildtype brains was visible in many cell bodies close to the lateral clock neurons, but co-labeling with anti-VRI (magenta) and anti-PDF (blue) revealed clear p38b expression in the l-LN_vs (white stars in B3) and the s-LN_vs (white stars in C3). B1–B4 represents a close-up of l-LN_vs, C1–C4 a shows close-up of s-LN_vs. Furthermore, we found staining in the entire cortex including the region of the dorsal neurons (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004565#pgen.1004565.s001" target="_blank">Fig. S1</a>). D–G: Staining with anti-p-p38 (green) was restricted to fewer neurons, but revealed again staining in the entire cortex that was stronger at night (F, ZT21) than during the day (D, ZT9). Double-labeling with anti-VRI (magenta) and anti-p-p38 antibody (green) revealed active p38 only in 2 clock neurons, the DN_1as (white stars in G2). Also in these cells, p-p38 staining intensity depended on the time of day, showing a higher level of active p38 at ZT 21 (G2, white stars) than at ZT9 (E2). E1–E3 and G1–G3 represent a close-up of DN_1as. Scale bar = 10 µm.</p