Complex roles of myoglianin in regulating adult performance and lifespan

Myoglianin, the Drosophila homolog of the secreted vertebrate proteins Myostatin and GDF-11, is an important regulator of neuronal modelling, and synapse function and morphology. While Myoglianin suppression during development elicits positive effects on the neuromuscular system, genetic manipulations of myoglianin expression levels have a varied effect on the outcome of performance tests in aging flies. Specifically, Myoglianin preserves jumping ability, has no effect on negative geotaxis, and negatively regulates flight performance in aging flies. In addition, Myoglianin exhibits a tissue-specific effect on longevity, with myoglianin upregulation in glial cells increasing the median lifespan. These findings indicate complex role for this TGF-β-like protein in governing neuromuscular signalling and consequent behavioural outputs and lifespan in adult flies

Cell type-specific modulation of healthspan by Forkhead family transcription factors in the nervous system

Reduced activity of insulin/insulin-like growth factor signaling (IIS) increases healthy lifespan among diverse animal species. Downstream of IIS, multiple evolutionarily conserved transcription factors (TFs) are required; however, distinct TFs are likely responsible for these effects in different tissues. Here we have asked which TFs can extend healthy lifespan within distinct cell types of the adult nervous system in Drosophila. Starting from published single-cell transcriptomic data, we report that forkhead (FKH) is endogenously expressed in neurons, whereas forkhead-box-O (FOXO) is expressed in glial cells. Accordingly, we find that neuronal FKH and glial FOXO exert independent prolongevity effects. We have further explored the role of neuronal FKH in a model of Alzheimer’s disease-associated neuronal dysfunction, where we find that increased neuronal FKH preserves behavioral function and reduces ubiquitinated protein aggregation. Finally, using transcriptomic profiling, we identify Atg17, a member of the Atg1 autophagy initiation family, as one FKH-dependent target whose neuronal overexpression is sufficient to extend healthy lifespan. Taken together, our results underscore the importance of cell type-specific mapping of TF activity to preserve healthy function with age

Enhancing autophagy by redox regulation extends lifespan in <i>Drosophila</i>

Redox signalling is an important modulator of diverse biological pathways and processes, and operates through specific post-translational modification of redox-sensitive thiols on cysteine residues 1–4. Critically, redox signalling is distinct from irreversible oxidative damage and functions as a reversible ‘redox switch’ to regulate target proteins. H2O2 acts as the major effector of redox signalling, both directly and through intracellular thiol redox relays 5,6. Dysregulation of redox homeostasis has long been implicated in the pathophysiology of many age-related diseases, as well as in the ageing process itself, however the underlying mechanisms remain largely unclear 7,8. To study redox signalling by H2O2in vivo and explore its involvement in metabolic health and longevity, we used the fruit fly Drosophila as a model organism, with its tractable lifespan and strong evolutionary conservation with mammals 9. Here we report that inducing an endogenous redox-shift, by manipulating levels of the H2O2-degrading enzyme catalase, improves health and robustly extends lifespan in flies, independently of oxidative stress resistance and dietary restriction. We find that the catalase redox-shifted flies are acutely sensitive to starvation stress, which relies on autophagy as a vital survival mechanism. Importantly, we show that autophagy is essential for the lifespan extension of the catalase flies. Furthermore, using redox-inactive knock-in mutants of Atg4a, a major effector of autophagy, we show that the lifespan extension in response to catalase requires a key redox-regulatory cysteine residue, Cys102 in Atg4a. These findings demonstrate that redox regulation of autophagy can extend lifespan, confirming the importance of redox signalling in ageing and as a potential pro-longevity target.</jats:p

Activating transcription factor 4-dependent lactate dehydrogenase activation as a protective response to amyloid beta toxicity

Accumulation of amyloid beta peptides is thought to initiate the pathogenesis of Alzheimer’s disease. However, the precise mechanisms mediating their neurotoxicity are unclear. Our microarray analyses show that, in Drosophila models of amyloid beta 42 toxicity, genes involved in the unfolded protein response and metabolic processes are upregulated in brain. Comparison with the brain transcriptome of early-stage Alzheimer’s patients revealed a common transcriptional signature, but with generally opposing directions of gene expression changes between flies and humans. Among these differentially regulated genes, lactate dehydrogenase (Ldh) was up-regulated by the greatest degree in amyloid beta 42 flies and the human orthologs (LDHA and LDHB) were down-regulated in patients. Functional analyses revealed that either over-expression or inhibition of Ldh by RNA interference (RNAi) slightly exacerbated climbing defects in both healthy and amyloid beta 42-induced Drosophila. This suggests that metabolic responses to lactate dehydrogenase must be finely-tuned, and that its observed upregulation following amyloid beta 42 production could potentially represent a compensatory protection to maintain pathway homeostasis in this model, with further manipulation leading to detrimental effects. The increased Ldh expression in amyloid beta 42 flies was regulated partially by unfolded protein response signalling, as ATF4 RNAi diminished the transcriptional response and enhanced amyloid beta 42-induced climbing phenotypes. Further functional studies are required to determine whether Ldh upregulation provides compensatory neuroprotection against amyloid beta 42-induced loss of activating transcription factor 4 activity and endoplasmatic reticulum stress.Our study thus reveals dysregulation of lactate dehydrogenase signalling in Drosophila models and patients with Alzheimer’s disease, which may lead to a detrimental loss of metabolic homeostasis. Importantly, we observed that down-regulation of ATF4-dependent endoplasmic reticulum-stress signalling in this context appears to prevent Ldh compensation and to exacerbate amyloid beta 42-dependent neuronal toxicity. Our findings therefore suggest caution in the use of therapeutic strategies focused on down-regulation of this pathway for treatment of Alzheimer’s disease, since its natural response to the toxic peptide may induce beneficial neuroprotective effects

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

Over-expression of recycling Rabs rescues age-related loss of gap junctions (GJs) and giant fiber system (GFS) function.

<p>(A and B) Over-expression of Rab4 (wild type [WT]) or Rab11(WT) in the GFS led to increased levels of shaking-B protein (SHAK-B) in the thorax of old flies. Representative confocal images for Rab4(WT) are shown in (A), the quantification in (B) (<i>n</i> = 5–10). (C) Tergotrochanteral muscle pathway (TTM) response latencies from young ([y] days 5–7) and old ([o] days 45–50) flies of various genotypes. WT and constitutively active (CA) construct over-expressed WT or CA forms of Rab4 and Rab11, respectively. Bars with different first letters indicate significant difference (irrespective of the subscript). The letters in the parentheses indicate a lack of significance with the specified bar (<i>n</i> = 4–8). (D) Quantification of the SHAK-B signal intensity in the bilateral tracts of young (7-day-old) flies with silenced Rab4 or Rab11 expression (<i>n</i> = 8–10). (E) Rab11 is indispensable for the effect of reduced signaling on the transmission through the TTM branch of the GF circuit (<i>n</i> = 5–13). Error bars denote SEM.</p

Lowered insulin/insulin-like growth factor signaling (IIS) increases connexin 43 (Cx43) gap junction (GJ) formation in human cells.

<p>(A and B) Confocal pictures of human retinal pigment epithelial (RPE1) cell monolayers upon elevated or reduced IIS, stimulated or not with insulin (1 hour) or insulin receptor (IR)/insulin-like growth factor-1 receptor (IGF1R) dual inhibitor (“IR inhib”), as indicated, and immunostained for Cx43 (green), integrin α3 (ITGA3, red), and DNA (DAPI, blue), images are representative of at least 30 captures from 3 independent experiments). Bar, 10 μm. (C, D, G) Quantification from high-throughput microscopy images of the total levels of Cx43 in RPE1 cells upon elevated or reduced IIS, stimulated or not with insulin, IR/IGF1R dual inhibitor (“IR inhib”), protein kinase C (PKC) activator or inhibitor or lysosomal inhibitors (NH₄Cl or bafilomycin A [BafA]) or transfected with the indicated wild-type (WT), constitutively active (CA) or dominant-negative (DN) Rab constructs, and normalized as indicated. (Data are shown as means ± SEM from 3 independent experiments (over 12,000 Cx43 punctae per condition); <i>n</i>.<i>s</i>., not significant; *<i>P</i> < 0.05, **<i>P</i> < 0.1, ***<i>P</i> < 0.001; 1-way ANOVA and Dunnett test versus “−insulin,” “+insulin,” or enhanced green fluorescent protein [EGFP], as appropriate). (E and H) Percentage of Cx43 colocalizing with ITGA3 (“Surface”), early endosome antigen 1 (EEA1; “Early Endosomes”) or lysosomal-associated membrane protein 1 (Lamp1; “Lysosomes”) in cells treated as indicated (Data are shown as means ± SEM from 3 independent experiments (over 12,000 Cx43 punctae per condition); <i>n</i>.<i>s</i>. = not significant; *<i>P</i> < 0.05, **<i>P</i> < 0.1, ***<i>P</i> < 0.001; 1-way ANOVA and Dunnett test versus “−insulin,” “+insulin,” or EGFP, as appropriate). (F) Transferrin efflux (endosomal recycling) measured by flow cytometry from RPE1 cells upon elevated or reduced IIS, stimulated or not with insulin (1 hour), as indicated. (Data are shown as means ± SEM from 3 independent experiments and normalized to “elevated IIS” at 7.5 minutes. Over 10,000 cells were analyzed per condition and per experiment; <i>n</i>.<i>s</i>., not significant; ***<i>P</i> < 0.001; 1-way ANOVA and Dunnett test.</p

Insulin signaling regulates giant fiber system (GFS) function during aging, and gap junctional density.

<p>(A) GFS-specific over-expression of <i>InR</i>^<i>dn</i> abolished the age-related response latency decline (tergotrochanteral muscle [TTM] pathway age x genotype interaction: <i>P</i> = 0.0004; <i>n</i> = 6–9). (B) Representative images of thoracic shaking-B protein (SHAK-B) staining in 45-day-old control (<i>A307-GAL4/+</i>) flies (left), and 45-day-old <i>A307-GAL4/UAS-InR</i>^<i>dn</i> flies (right). Scale bar: 15 μm. (C) Quantification of SHAK-B signal intensities in the bilateral tracts of the GFS (<i>n</i> = 4–7). (D) Top: Over-expression of <i>SHAK-B(N+16)</i> prevented functional decline in the GFS with age (interaction <i>P</i> = 0.015; <i>n</i> = 6–14 per genotype/age). Bottom: Representative TTM traces from 45-day-old control (left) and <i>SHAK-B(n+16)</i>-over-expressing (right) flies. Red arrows indicate response latency periods. All panels: error bars denote SEM.</p

Ubiquitously reduced insulin/insulin-like growth factor signaling (IIS) prevents age-associated decline in transmission through the giant fiber system (GFS).

<p>(A) The GFS, showing insertion sites for recording and stimulating electrodes. The monosynaptic tergotrochanteral muscle (TTM) pathway involves the large electrochemical giant fiber (GF)- tergotrochanteral muscle motor neuron (TTMn) synapse. The electrochemical GF-peripherally synapsing interneurons (PSI), PSI, and chemical (cholinergic) PSI-dorsal longitudinal muscle motor neuron (DLMn) synapses comprise the bisynaptic dorsal longitudinal muscle (DLM) pathway. All neuromuscular synapses are chemical (glutamatergic). (B) Response latencies significantly increased with age when recorded from the TTMs (left) and DLMs (right) (<i>n</i> = 8–9). Representative TTM and DLM traces are shown below. Red bars indicate the time between brain stimulus and muscle response. (C) Reduced IIS (<i>da-GAL4/UAS-InR</i>^<i>dn</i>) prevented age-associated decline in GFS transmission (age x genotype interaction between the control genotypes and <i>da-GAL4/UAS-InR</i>^<i>dn</i> is significant, <i>P</i> < 0.03; <i>n</i> = 6–16; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001655#pbio.2001655.s008" target="_blank">S1 Table</a>). Error bars denote SEM.</p

Insulin signaling manipulation in the adult nervous system.

<p>(A) Nervous system-specific insulin/insulin-like growth factor signaling (IIS) down-regulation (RU+) prevented age-associated loss of transmission in the tergotrochanteral muscle (TTM) pathway (<i>n</i> = 7–10). (B) TTM response latency deteriorated faster with age in flies over-expressing IIS (RU+) (age x treatment interaction: <i>P</i> = 0.0105; <i>n</i> = 6–9). Both panels: error bars denote SEM.</p