Amyloid formation of α-synuclein (α-Syn) and its familial mutations are directly linked with Parkinson's disease (PD) pathogenesis. Recently, a new familial α-Syn mutation (A53E) was discovered, associated with an early onset aggressive form of PD, which delays α-Syn aggregation. When we overexpressed wild-type (WT) and A53E proteins in cells, showed neither toxicity nor aggregate formation, suggesting merely overexpression may not recapitulate the PD phenotype in cell models. We hypothesized that cells expressing the A53E mutant might possess enhanced susceptibility to PD-associated toxicants compared to that of the WT. When cells were treated with PD toxicants (dopamine and rotenone), cells expressing A53E showed more susceptibility to cell death along with compromised mitochondrial potential and an increased production of reactive oxygen species. The higher toxicity of A53E could be due to more oligomers being formed in cells as confirmed by a dot blot assay using amyloid specific OC and A11 antibody and using an in vitro aggregation study. The cellular model presented here suggests that along with familial mutation, environmental and other cellular factors might play a crucial role in dictating PD pathogenesis.
A crucial property of circadian clocks is the ability to regulate the shape of an oscillation over its cycle length (waveform) appropriately, thus enhancing Darwinian fitness. Many studies over the past decade have revealed interesting ways in which the waveform of rodent behavior could be manipulated, one of which is that the activity bout bifurcates under environments that have 2 light/dark cycles within one 24-h day (LDLD). It has been observed that such unique, although unnatural, environments reveal acute changes in the circadian clock network. However, although adaptation of waveforms to different photoperiods is well studied, modulation of waveforms under LDLD has received relatively less attention in research on insect rhythms. Therefore, we undertook this study to ask the following questions: what is the extent of waveform plasticity that Drosophila melanogaster exhibits, and what are the neuronal underpinnings of such plasticity under LDLD? We found that the activity/rest rhythms of wild-type flies do not bifurcate under LDLD. Instead, they show similar but significantly different behavior from that under a long-day LD cycle. This behavior is accompanied by differences in the organization of the circadian neuronal network, which include changes in waveforms of a core clock component and an output molecule. In addition, to understand the functional significance of such variations in the waveform, we examined laboratory selected populations that exhibit divergent eclosion chronotypes (and therefore, waveforms). We found that populations selected for predominant eclosion in an evening window ( late chronotypes) showed reduced amplitude plasticity and increased phase plasticity of activity/rest rhythms. This, we argue, is reflective of divergent evolution of circadian neuronal network organization in our laboratory selected flies.
Summary A neuronal circuit of ∼150 neurons modulates rhythmic activity-rest behavior of Drosophila melanogaster . While it is known that coherent ∼24-hr rhythms in locomotion are brought about when 7 distinct neuronal clusters function as a network due to chemical communication amongst them, there are no reports of communication via electrical synapses made up of gap junctions. Here, we report that gap junction proteins, Innexins play crucial roles in determining the intrinsic period of activity-rest rhythms in flies. We show the presence of Innexin2 in the ventral lateral neurons, wherein RNAi-based knockdown of its expression slows down the speed of activity-rest rhythm along with alterations in the oscillation of a core-clock protein PERIOD and the output molecule pigment dispersing factor. Specifically disrupting the channel-forming ability of Innexin2 causes period lengthening, suggesting that Innexin2 may function as hemichannels or gap junctions in the clock circuit.
30The circadian neuronal circuit of Drosophila melanogaster is made up of about 150 neurons, 31 distributed bilaterally and distinguished into 7 clusters. Multiple lines of evidence suggest 32 that coherent rhythms in behaviour are brought about when these clusters function as a 33 network. Although chemical modes of communication amongst circadian neurons have 34 been well-studied, there has been no report of communication via electrical synapses made 35 up of gap junctions. Here, we report for the first time that gap junction proteins -Innexins 36 play crucial roles in determining the period of free-running activity rhythms in flies. Our 37 experiments reveal the presence of gap junction protein INNEXIN2 in the ventral lateral 38 neurons. RNA-interference based knockdown of its expression in circadian pacemakers 39 slows down the speed of locomotor activity rhythm. Concomitantly, we find alterations in 40 the oscillation of a core-clock protein PERIOD and in the output molecule Pigment 41 Dispersing Factor in the circadian pacemaker neuron network. 42 43 44 45 membrane potential, activity-rest. 46 47 48 150 neurons distributed bilaterally in the brain. Based on their location, they can be divided 53 into lateral neurons (LN) and dorsal neurons (DN). The lateral neurons are further divided 54 into the small ventral lateral neurons (s-LNv), large ventral lateral neurons (l-LNv), the 55 lateral dorsal neurons (LNd) and the lateral posterior neurons (LPN). The dorsal cluster of 56 neurons are further divided into 3 groups as dorsal neurons 1-3 (DN1-3) (reviewed in 57 Sheeba, 2008). 58 Each of these neurons have a ticking molecular clock composed of a self-sustained 59 transcriptional translational feedback loop (TTFL) made up of four core clock genes Clock, 60 Cycle, Period and Timeless. The period of these molecular oscillations in mRNA and protein 61 within the pacemaker circuit in the fly brain mirror the period of rhythmic activity-rest 62 behaviour reviewed in (Hardin, 2005). Although molecular circadian clocks in individual 63 neurons can be thought of as ticking cell autonomously due to the precisely timed cycling of 64 their mRNA and proteins, one interesting question that remains to be fully understood is 65 how these distinct neuronal clusters, with distinct intrinsic periodicities (Yoshii et al., 2009) 66 together bring about one coherent period of the behavioural activity rhythm. Early studies 67 of Drosophila clock neuronal network have shown that under constant darkness and 68 constant temperature (DD 25 °C), s-LNv neurons and clocks in these cells are necessary and 69 sufficient for the persistence of activity-rest rhythms (Helfrich-Förster, 1998, Renn et al., 70 1999). s-LNv release neuropeptide Pigment Dispersing factor (PDF) in the dorsal part of the 71 brain via their projections in a time-of-day dependent manner (Park et al., 2000). Lack of 72 PDF results in arrhythmicity of activity-rest rhythms under constant conditions (Renn et al., 73 1999) suggesting that PDF is necessary for persistence...
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