Marcus J. Allen

Inhibition of GSK-3 ameliorates Abeta pathology in an adult-onset Drosophila model of Alzheimer's disease.

Aβ peptide accumulation is thought to be the primary event in the pathogenesis of Alzheimers disease (AD), with downstream neurotoxic effects including the hyperphosphorylation of tau protein. Glycogen synthase kinase-3 (GSK-3) is increasingly implicated as playing a pivotal role in this amyloid cascade. We have developed an adult-onset Drosophila model of AD, using an inducible gene expression system to express Arctic mutant Aβ42 specifically in adult neurons, to avoid developmental effects. Aβ42 accumulated with age in these flies and they displayed increased mortality together with progressive neuronal dysfunction, but in the apparent absence of neuronal loss. This fly model can thus be used to examine the role of events during adulthood and early AD aetiology. Expression of Aβ42 in adult neurons increased GSK-3 activity, and inhibition of GSK-3 (either genetically or pharmacologically by lithium treatment) rescued Aβ42 toxicity. Aβ42 pathogenesis was also reduced by removal of endogenous fly tau; but, within the limits of detection of available methods, tau phosphorylation did not appear to be altered in flies expressing Aβ42. The GSK-3–mediated effects on Aβ42 toxicity appear to be at least in part mediated by tau-independent mechanisms, because the protective effect of lithium alone was greater than that of the removal of tau alone. Finally, Aβ42 levels were reduced upon GSK-3 inhibition, pointing to a direct role of GSK-3 in the regulation of Aβ42 peptide level, in the absence of APP processing. Our study points to the need both to identify the mechanisms by which GSK-3 modulates Aβ42 levels in the fly and to determine if similar mechanisms are present in mammals, and it supports the potential therapeutic use of GSK-3 inhibitors in AD.

Molecular Mechanism of Rectification at Identified Electrical Synapses in the Drosophila Giant Fiber System

Summary Electrical synapses are neuronal gap junctions that mediate fast transmission in many neural circuits [1–5]. The structural proteins of gap junctions are the products of two multigene families. Connexins are unique to chordates [3–5]; innexins/pannexins encode gap-junction proteins in prechordates and chordates [6–10]. A concentric array of six protein subunits constitutes a hemichannel; electrical synapses result from the docking of hemichannels in pre- and postsynaptic neurons. Some electrical synapses are bidirectional; others are rectifying junctions that preferentially transmit depolarizing current anterogradely [11, 12]. The phenomenon of rectification was first described five decades ago [1], but the molecular mechanism has not been elucidated. Here, we demonstrate that putative rectifying electrical synapses in the Drosophila Giant Fiber System [13] are assembled from two products of the innexin gene shaking-B. Shaking-B(Neural+16) [14] is required presynaptically in the Giant Fiber to couple this cell to its postsynaptic targets that express Shaking-B(Lethal) [15]. When expressed in vitro in neighboring cells, Shaking-B(Neural+16) and Shaking-B(Lethal) form heterotypic channels that are asymmetrically gated by voltage and exhibit classical rectification. These data provide the most definitive evidence to date that rectification is achieved by differential regulation of the pre- and postsynaptic elements of structurally asymmetric junctions.

The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter

The largest central synapse in adult Drosophila is a mixed electro‐chemical synapse whose gap junctions require the product of the shaking‐B (shak‐B) gene. Shak‐B2 mutant flies lack gap junctions at this synapse, which is between the giant fibre (GF) and the tergotrochanteral motor neuron (TTMn), but it still exhibits a long latency response upon GF stimulation. We have targeted the expression of the light chain of tetanus toxin to the GF, to block chemical transmission, in shak‐B2 flies. The long latency response in the tergotrochanteral muscle (TTM) was abolished indicating that the chemical component of the synapse mediates this response. Attenuation of GAL4‐mediated labelling by a cha‐GAL80 transgene, reveals the GF to be cholinergic. We have used a temperature‐sensitive allele of the choline acetyltransferase gene (chats2) to block cholinergic synapses in adult flies and this also abolished the long latency response in shak‐B2 flies. Taken together the data provide evidence that both components of this mixed synapse are functional and that the chemical neurotransmitter between the GF and the TTMn is acetylcholine. Our findings show that the two components of this synapse can be separated to allow further studies into the mechanisms by which mixed synapses are built and function.

Dietary restriction delays aging, but not neuronal dysfunction, in Drosophila models of Alzheimer's disease.

Dietary restriction (DR) extends lifespan in diverse organisms and, in animal and cellular models, can delay a range of aging-related diseases including Alzheimers disease (AD). A better understanding of the mechanisms mediating these interactions, however, may reveal novel pathways involved in AD pathogenesis, and potential targets for disease-modifying treatments and biomarkers for disease progression. Drosophila models of AD have recently been developed and, due to their short lifespan and susceptibility to genetic manipulation, we have used the fly to investigate the molecular connections among diet, aging and AD pathology. DR extended lifespan in both Arctic mutant Aβ42 and WT 4R tau over-expressing flies, but the underlying molecular pathology was not altered and neuronal dysfunction was not prevented by dietary manipulation. Our data suggest that DR may alter aging through generalised mechanisms independent of the specific pathways underlying AD pathogenesis in the fly, and hence that lifespan-extending manipulations may have varying effects on aging and functional declines in aging-related diseases. Alternatively, our analysis of the specific effects of DR on neuronal toxicity downstream of Aβ and tau pathologies with negative results may simply confirm that the neuro-protective effects of DR are upstream of the initiating events involved in the pathogenesis of AD.

Reduced insulin signaling maintains electrical transmission in a neural circuit in aging flies

Lowered insulin/insulin-like growth factor (IGF) signaling (IIS) can extend healthy lifespan in worms, flies, and mice, but it can also have adverse effects (the “insulin paradox”). Chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber system (GFS), a simple escape response neuronal circuit, by increasing targeting of the gap junctional protein innexin shaking-B to gap junctions (GJs). Endosomal recycling of GJs was also stimulated in cultured human cells when IIS was reduced. Furthermore, increasing the activity of the recycling small guanosine triphosphatases (GTPases) Rab4 or Rab11 was sufficient to maintain GJs upon elevated IIS in cultured human cells and in flies, and to rescue age-related loss of GJs and of GFS function. Lowered IIS thus elevates endosomal recycling of GJs in neurons and other cell types, pointing to a cellular mechanism for therapeutic intervention into aging-related neuronal disorders.

A modifier screen in the Drosophila eye reveals that aPKC interacts with Glued during central synapse formation

BackgroundThe Glued gene of Drosophila melanogaster encodes the homologue of the vertebrate p150Glued subunit of dynactin. The Glued1 mutation compromises the dynein-dynactin retrograde motor complex and causes disruptions to the adult eye and the CNS, including sensory neurons and the formation of the giant fiber system neural circuit.ResultsWe performed a 2-stage genetic screen to identify mutations that modified phenotypes caused by over-expression of a dominant-negative Glued protein. We screened over 34,000 flies and isolated 41 mutations that enhanced or suppressed an eye phenotype. Of these, 12 were assayed for interactions in the giant fiber system by which they altered a giant fiber morphological phenotype and/or altered synaptic function between the giant fiber and the tergotrochanteral muscle motorneuron. Six showed interactions including a new allele of atypical protein kinase C (aPKC). We show that this cell polarity regulator interacts with Glued during central synapse formation. We have mapped the five other interacting mutations to discrete chromosomal regions.ConclusionOur results show that an efficient way to screen for genes involved in central synapse formation is to use a two-step strategy in which a screen for altered eye morphology precedes the analysis of central synaptogenesis. This has highlighted a role for aPKC in the formation of an identified central synapse.

Impact of insulin signaling and proteasomal activity on physiological output of a neuronal circuit in aging Drosophila melanogaster

The insulin family of growth factors plays an important role in development and function of the nervous system. Reduced insulin and insulin-growth-factor signaling (IIS), however, can improve symptoms of neurodegenerative diseases in laboratory model organisms and protect against age-associated decline in neuronal function. Recently, we showed that chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber escape response circuit. Here, we expand our initial findings by demonstrating that reduced functional output in the giant fiber system of aging flies can be prevented by increasing proteasomal activity within the circuit. Manipulations of IIS in neurons can also affect longevity, underscoring the relevance of the nervous system for aging.

Erratum to “Dietary restriction delays aging, but not neuronal dysfunction, in Drosophila models of Alzheimer's disease.” [Neurobiol. Aging 32 (2011) 1977–1989]

Fig. 4. Analysis of fully fed vs DR food effects on tau levels and phosphorylation in flies over-expressing WT human tau. (A) Tau expression and phosphorylation levels were measured by western blotting in control flies (welav/+, w;UAS-4Rtau/+), and at the indicated time points in welav/+;UAS-4Rtau/+flies treated on1.0 vs 2.0 Y medium. Primary antibodies were as follows: Anti-tau (total tau; Dako, UK), PHF-1 (phospho-Ser396/404 tau), AT8 (phospho-Ser199/202 tau), pS422(phospho-Ser422 tau) and anti-actin. (B) Phospho-tau levels, in welav/+;UAS-4R tau/+flies, were normalised to total tau protein for each sample, and are expressed as average relative intensities SEM. Dietary manipulation did not alter the level or pattern of tau phosphorylation across age at Ser396/404 (P 1⁄4 0.412), Ser199/202 (P 1⁄4 0.838) or Ser422 (P 1⁄4 0.677) epitopes (two-way ANOVA).

02-P015 Dcamsap (ssp4) is a member of a family of membrane-skeleton proteins essential for the life of animals

The CAMSAP1 gene in vertebrates is a spectrin associated protein that is regulated by Ca2+/calmodulin. It is a member of a family of protein in animals, of unknown function, that contain a calponin homology (CH) domain and a conserved C-terminal DUF1781 domain. Invertebrates have single CAMSAP genes and vertebrates generally have three. The Drosophila homologue of CAMSAP1 is Dcamsap (ssp4). Transposon insertion mutations in Dcamsap are lethal. Fly embryos die before hatching with defects in head involution. We have generated an antibody to the C-terminus of Dcamsap and investigated the expression of the gene using western blotting and by immunocytochemistry to whole-mount embryos. Expression is elevated in discrete cells that may be undergoing apoptosis. Analysis of both the insertion mutations and also dsRNAi knockdown of Dcamsap in the eye indicate that Dcamsap is required for regulation of cell number during development.