Temperature and light entrainment of the Drosophila circadian clock

Drosophila melanogaster locomotor activity responds to seasonal conditions by modulating the “evening” activity component. During simulated winters of cold temperature and short days an advanced evening locomotor peak occurs with more daytime locomotor activity; on the other hand long photoperiods and warm temperatures give a delay in the evening peak, thereby avoiding a possible desiccation during the hottest times of the day. This pattern of activity is related to a thermosensitive splicing event that occurs in a 3’ intron in the period gene, with a higher level of splicing and earlier accumulation of PERIOD in short days and low temperatures. A mutation in norpA which encodes a phospholipase C, generates a high level of spliced per at warmer temperature, so mutants behave as if it is colder than it actually is. The relation between norpA, per splicing and the circadian neurons has been analysed. Initially, norpA expression has been investigated via in situ hybridisation and immunocytochemistry. norpA transcript has been localised among the clock pacemakers but not NORPA. Subsequently, norpA expression has been knocked-down by RNAi in specific subset of neurons. The resulting locomotor behaviour shows seasonally related effects implicating the photoreceptors, lateral and dorsal clock neurons as structures involved in timing the locomotor behaviour. In parallel, the thermal role of a second PLCβ, plc21C, has been investigated via RNAi among circadian pacemakers. It has been possible to show that plc21C expression in the photoreceptors, lateral and dorsal neurons is required to set different locomotor behaviours at different temperatures, but not via per and tim splicing. Finally, in contrast to reports that the double photoreceptor mutants involving glass and cryptochrome are “circadian blind”, these flies have been observed to entrain to light-dark cycles at moderate temperatures. Candidate orphan G protein coupled receptors have been screened in order to identify a further set of putative circadian-relevant photoreceptors contributing to this residual entrainment in glass60jcryb mutants. In constant light conditions, the RNAi of CG7497 and CG16958 generates rhythmic or arrhythmic flies depending on the genetic background tested

Rab11 modulates α-synuclein-mediated defects in synaptic transmission and behaviour

A central pathological hallmark of Parkinson's disease (PD) is the presence of proteinaceous depositions known as Lewy bodies, which consist largely of the protein α-synuclein (aSyn). Mutations, multiplications and polymorphisms in the gene encoding aSyn are associated with familial forms of PD and susceptibility to idiopathic PD. Alterations in aSyn impair neuronal vesicle formation/transport, and likely contribute to PD pathogenesis by neuronal dysfunction and degeneration. aSyn is functionally associated with several Rab family GTPases, which perform various roles in vesicle trafficking. Here, we explore the role of the endosomal recycling factor Rab11 in the pathogenesis of PD using Drosophila models of aSyn toxicity. We find that aSyn induces synaptic potentiation at the larval neuromuscular junction by increasing synaptic vesicle (SV) size, and that these alterations are reversed by Rab11 overexpression. Furthermore, Rab11 decreases aSyn aggregation and ameliorates several aSyn-dependent phenotypes in both larvae and adult fruit flies, including locomotor activity, degeneration of dopaminergic neurons and shortened lifespan. This work emphasizes the importance of Rab11 in the modulation of SV size and consequent enhancement of synaptic function. Our results suggest that targeting Rab11 activity could have a therapeutic value in PD

A novel role for kynurenine 3-monooxygenase in mitochondrial dynamics.

The enzyme kynurenine 3-monooxygenase (KMO) operates at a critical branch-point in the kynurenine pathway (KP), the major route of tryptophan metabolism. As the KP has been implicated in the pathogenesis of several human diseases, KMO and other enzymes that control metabolic flux through the pathway are potential therapeutic targets for these disorders. While KMO is localized to the outer mitochondrial membrane in eukaryotic organisms, no mitochondrial role for KMO has been described. In this study, KMO deficient Drosophila melanogaster were investigated for mitochondrial phenotypes in vitro and in vivo. We find that a loss of function allele or RNAi knockdown of the Drosophila KMO ortholog (cinnabar) causes a range of morphological and functional alterations to mitochondria, which are independent of changes to levels of KP metabolites. Notably, cinnabar genetically interacts with the Parkinson's disease associated genes Pink1 and parkin, as well as the mitochondrial fission gene Drp1, implicating KMO in mitochondrial dynamics and mitophagy, mechanisms which govern the maintenance of a healthy mitochondrial network. Overexpression of human KMO in mammalian cells finds that KMO plays a role in the post-translational regulation of DRP1. These findings reveal a novel mitochondrial role for KMO, independent from its enzymatic role in the kynurenine pathway

Glycation potentiates neurodegeneration in models of Huntington's disease

Protein glycation is an age-dependent posttranslational modification associated with several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. By modifying amino-groups, glycation interferes with folding of proteins, increasing their aggregation potential. Here, we studied the effect of pharmacological and genetic manipulation of glycation on huntingtin (HTT), the causative protein in Huntington’s disease (HD). We observed that glycation increased the aggregation of mutant HTT exon 1 fragments associated with HD (HTT72Q and HTT103Q) in yeast and mammalian cell models. We found that glycation impairs HTT clearance thereby promoting its intracellular accumulation and aggregation. Interestingly, under these conditions autophagy increased and the levels of mutant HTT released to the culture medium decreased. Furthermore, increased glycation enhanced HTT toxicity in human cells and neurodegeneration in fruit flies, impairing eclosion and decreasing life span. Overall, our study provides evidence that glycation modulates HTT exon-1 aggregation and toxicity, and suggests it may constitute a novel target for therapeutic intervention in HD

Tryptophan-2,3-dioxygenase (TDO) inhibition ameliorates neurodegeneration by modulation of kynurenine pathway metabolites

Metabolites of the kynurenine pathway (KP) of tryptophan (TRP) degradation have been closely linked to the pathogenesis of several neurodegenerative disorders. Recent work has highlighted the therapeutic potential of inhibiting two critical regulatory enzymes in this pathway-kynurenine-3-monooxygenase (KMO) and tryptophan-2,3-dioxygenase (TDO). Much evidence indicates that the efficacy of KMO inhibition arises from normalizing an imbalance between neurotoxic [3-hydroxykynurenine (3-HK); quinolinic acid (QUIN)] and neuroprotective [kynurenic acid (KYNA)] KP metabolites. However, it is not clear if TDO inhibition is protective via a similar mechanism or if this is instead due to increased levels of TRP-the substrate of TDO. Here, we find that increased levels of KYNA relative to 3-HK are likely central to the protection conferred by TDO inhibition in a fruit fly model of Huntington's disease and that TRP treatment strongly reduces neurodegeneration by shifting KP flux toward KYNA synthesis. In fly models of Alzheimer's and Parkinson's disease, we provide genetic evidence that inhibition of TDO or KMO improves locomotor performance and ameliorates shortened life span, as well as reducing neurodegeneration in Alzheimer's model flies. Critically, we find that treatment with a chemical TDO inhibitor is robustly protective in these models. Consequently, our work strongly supports targeting of the KP as a potential treatment strategy for several major neurodegenerative disorders and suggests that alterations in the levels of neuroactive KP metabolites could underlie several therapeutic benefits

Genetic and pharmacological induction of denitrosylation reverses and prevents nitrergic effects on transmitter release and vesicle pools.

<p>(A) NO-induced suppression of evoked release (<i>w</i>^<i>1118</i> Ctrl eEJCs data in grey from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003611#pbio.2003611.g001" target="_blank">Fig 1A</a>) can be reversed after 15 min of GSH application (150 μM, blue). Wash out of NO alone shows mild nonsignificant recovery (green). (B) Mean QC for conditions indicated (OE, <i>elav</i> > UAS-<i>fdh</i>31 [GSNOR], <i>elav</i> > UAS-<i>GCLm</i>, <i>elav</i> > UAS-<i>GCLc</i>, all ± NO). NO has no effects on QC in genotypes indicated (<i>w</i>^<i>1118</i> + NO data in grey from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003611#pbio.2003611.g001" target="_blank">Fig 1</a> for comparisons, ***<i>p</i> < 0.0001 <i>w</i>^<i>1118</i> + NO versus <i>w</i>^<i>1118</i>), #<i>p</i> < 0.05, ##<i>p</i> < 0.01, ###<i>p</i> < 0.001, ####<i>p</i> < 0.0001 versus <i>w</i>^<i>1118</i> + NO. (C) Cumulative QC graphs for genotypes indicated (±NO) with linear regression to the last 200 ms, Ctrl in grey taken from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003611#pbio.2003611.g002" target="_blank">Fig 2</a> for comparison. (D) Mean RRP sizes, NO has no effects on vesicle pool size in tested genotypes (<i>w</i>^<i>1118</i> data in grey from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003611#pbio.2003611.g001" target="_blank">Fig 1</a>, <i>w</i>^<i>1118</i> + NO versus <i>w</i>^<i>1118</i>: ***<i>p</i> < 0.0001), ##<i>p</i> < 0.01, ###<i>p</i> < 0.01, ####<i>p</i> < 0.01 versus <i>w</i>^<i>1118</i> + NO. (E) PPR at 20 ms ISI for indicated conditions, ****<i>p</i> < 0.0001 versus Ctrl. (F) mEJCs analysis: left, mean mEJC amplitudes (±NO), middle, mean mEJC frequencies (±NO), right, mean mEJC decay kinetics for genotypes indicated (±NO). OE of GSNOR, GCLm, or GCLc prevents NO effects on the frequency of mEJCs. The raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003611#pbio.2003611.s014" target="_blank">S4 Data</a>. Data denote mean ± SEM for all data comparisons. ANOVA with post hoc Tukey-Kramer. Ctrl, control; eEJC, evoked EJC; EJC, excitatory junction current; <i>fdh</i>, formaldehyde dehydrogenase; <i>GCLc</i>, glutamate-cysteine ligase catalytic subunit C; <i>GCLm</i>, glutamate-cysteine ligase catalytic subunit M; GSH, glutathione; GSNOR, S-nitrosoglutathione reductase; ISI, interspike interval; mEJC, miniature EJC; NO, nitric oxide; n.s., nonsignificant; OE, overexpression; PPR, paired pulse ratio; QC, quantal content; RRP, readily releasable pool.</p

Ca²⁺ dependency of evoked release is reduced by NO.

<p>(A) Representative raw eEJC recordings at different [Ca²⁺]_e (from 0.5 to 3 mM). (B) Time course of single eEJCs for 2 NMJs (<i>w</i>^<i>1118</i> Ctrl and NO exposure) at indicated [Ca²⁺]_e. (C) Parabolic fits to the variance-mean relationships for the conditions indicated. (D) <i>N</i> estimated from fluctuation analysis in both conditions. (E) Ca²⁺ cooperativity of evoked release is shown on a double logarithmic plot for <i>w</i>^<i>1118</i> Ctrl and NO. (F) Raw traces for PPRs of a Ctrl and NO-treated NMJ at 1.0 and 1.5 mM [Ca²⁺]_e, illustrating a reduced release probability following NO exposure. (G) Summary of PPR at 1 and 1.5 mM [Ca²⁺]_e for various ISI for Ctrl and NO (<i>n</i> = 5 larvae for Ctrl, <i>n</i> = 3 larvae for NO, with 2–3 NMJs per larva). (H) Images of myrGCaMP-expressing NMJs from a Ctrl (top) and a NO-treated larva (bottom) before, during (at 3 s, 60 Hz), and after the 8-s train. Changes in GCaMP5 fluorescence were analyzed for each bouton to calculate ΔF/F₀. The raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003611#pbio.2003611.s013" target="_blank">S3 Data</a>. Values per NMJ were averaged and data denoting mean ± SEM for each condition of 6–8 NMJs (3–4 larvae) are shown in I. *<i>p</i> < 0.05, ***<i>p</i> < 0.001, Student <i>t</i> test. [Ca²⁺]_e, extracellular calcium concentration; Ctrl, control; eEJC, evoked EJC; EJC, excitatory junction current; ISI, interspike interval; myrGCaMP, <i>N</i>-myristoylated GCaMP; <i>N</i>, number of release-ready vesicles; NMJ, neuromuscular junction; NO, nitric oxide; PPR, paired pulse ratio; stim, stimulation.</p

Nitrergic effects on evoked and spontaneous release require cpx.

<p>(A) Raw eEJC recordings of a 50-Hz train, 500 ms in cpx^-/- larvae (±NO). (B) Mean QC for genotypes indicated, showing the lack of NO effects in cpx^-/- larvae (<i>w</i>^<i>1118</i> Ctrl in grey from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003611#pbio.2003611.g002" target="_blank">Fig 2</a>). (C) Cumulative QC for the same conditions as in (B). (D) Mean QC and vesicle pool sizes for the genotypes indicated (<i>w</i>^<i>1118</i> Ctrl data from Figs <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003611#pbio.2003611.g001" target="_blank">1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003611#pbio.2003611.g002" target="_blank">2</a>, ****<i>p</i> < 0.0001 versus <i>w</i>^<i>1118</i> Ctrl). (E) mEJCs recordings from m6 and m5 in cpx^-/- and cpx^+/- ± NO. (F) Mean mEJC frequencies for conditions and genotypes indicated (****<i>p</i> < 0.0001 versus m5 <i>w</i>^<i>1118</i> Ctrl, #<i>p</i> < 0.05 versus m6 cpx^+/- Ctrl). (G) Raw traces of mEJC recordings before (left) and after (right) high frequency stimulation in <i>w</i>^<i>1118</i> (±NO) and NOS^C larvae. (H) Average fold change of mEJC frequency during 50 s after stimulation compared to baseline frequency before stimulation for conditions and genotypes indicated (NOS “null” comprised of data from NOS^C and NOS^Δ15). NO treatment (40 min) suppresses increases in frequency, NOS “null” potentiates relative increases and enhanced denitrosylation (GCLm and GSNOR OE) prevents NO-induced suppression (*<i>p</i> < 0.05, **<i>p</i> < 0.01 versus Ctrl, ##<i>p</i> < 0.01, ####<i>p</i> < 0.001 versus NO). The raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003611#pbio.2003611.s015" target="_blank">S5 Data</a>. Data denote mean ± SEM in all graphs, ANOVA with post hoc Tukey-Kramer. Ctrl, control; cpx, complexin; eEJC, evoked EJC; EJC, excitatory junction current; GCLm, glutamate-cysteine ligase catalytic subunit M; GSNOR, S-nitrosoglutathione reductase; mEJC, miniature EJC; m5, muscle 5; m6, muscle 6; NO, nitric oxide; OE, overexpression; QC, quantal content.</p

Effects of SNO formation on farnesylation and cpx function.

<p>(A) The farnesyl transferase facilitates the addition of a farnesyl group to cpx (green) containing the CAAX motif. Upon S-nitrosylation, this motif is not recognized and the protein will not be farnesylated, resulting in lack of endomembrane targeting. (B) The schematic shows the effects of S-nitrosylation of cpx Cys within CAAX, resulting in fewer proteins being farnesylated and tethered to endomembranes. This allows a greater proportion of cytosolic cpx to be able to bind to the SNARE complex (including syntaxin [yellow]/Munc-18 [red], SNAP-25 [dark purple], synaptobrevin [purple]) to compete with synaptotagmin [blue] binding and prevent fusion because of its clamping function, which results in reduced transmitter release. This process is reversible and depends on the availability of GSH-dependent de-nitrosylation of cpx. GSH is generated by GCL and GSNO is broken down into G-SS-G and NH₂OH by GSNOR activity. Cys, cysteine; cpx, complexin; G-SS-G, glutathione disulphide; GCL, glutamate-cysteine ligase; GluR, glutamate receptor; GSH, glutathione; GSNO, S-nitrosoglutathione; GSNOR, S-nitrosoglutathione reductase; NH₂OH, hydroxylamine; SNARE, soluble <i>N</i>-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor; SNO, S-nitrosothiol.</p

NO reduces evoked release and frequency of spontaneous release in a cGMP-independent manner.

<p>(A) NO suppresses evoked release (eEJC) over a time course of 55 min. Insets show representative single eEJCs at 40 min for both conditions. (B) Mean eEJC amplitudes (left axis) and QC (right axis) of <i>w</i>^<i>1118</i> NMJs are reduced following NO exposure (at 40 min). The sGC inhibitor ODQ (50 μM) did not affect the response to NO. (C) Mean eEJC amplitudes (left axis, black) and QC (right axis, grey) of NOS^C and NOS^Δ15 NMJs. (D) Raw mEJC recordings of <i>w</i>^<i>1118</i> NMJs and mEJC parameters (top to bottom: amplitude, frequency, decay). Top insets show representative mEJC recordings. Bottom insets show single mEJCs (grey) and averaged mEJC (red) with single exponential fit to the decay. (E) Raw mEJC recordings for both NOS^C and NOS^Δ15 genotypes. Below, mEJC quantal parameters: amplitude and frequency, Student <i>t</i> test each relative to <i>w</i>^<i>1118</i> Ctrl, *<i>p</i> = 0.04, ***<i>p</i> = 0.001. (F) cGMP content of larval brains under the conditions indicated (NO: 40 min NO exposure, NO + ODQ: 40 min NO exposure in the presence of 50 μM ODQ, NO + Zap: 40 min NO exposure + PDE inhibitor Zap, 20 μM). (G) FlincG3 fluorescence images of a Ctrl and stimulated NMJ (20 Hz for 10 s, duty cycle: 1 min for total of 10 min). (H) Summary of FlincG3 fluorescence (in a.u.’s). The raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003611#pbio.2003611.s011" target="_blank">S1 Data</a>. Data denote mean ± SEM in all graphs, ANOVA with post hoc Tukey-Kramer, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ****<i>p</i> < 0.0001. a.u., arbitrary unit; cGMP, cyclic guanosine monophosphate; Ctrl, control; eEJC, evoked EJC; EJC, excitatory junction current; mEJC, miniature EJC; NMJ, neuromuscular junction; NO, nitric oxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PDE, phosphodiesterase; QC, quantal content; sGC, soluble guanylyl cyclase; Zap, zaprinast.</p