JNK modifies neuronal metabolism to promote proteostasis and longevity

Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single-gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. Here, we address this question by comprehensively characterizing age-related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long-lived animals with elevated Jun-N-terminal Kinase signaling. We find that these animals exhibit a delayed age-related decline in protein turnover rates, as well as decreased steady-state neuronal glucose-6-phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose-6-phosphate dehydrogenase (G6PD). Over-expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. Our study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long-lived mutants

The WT1-like transcription factor Klumpfuss maintains lineage commitment of enterocyte progenitors in the Drosophila intestine

In adult epithelial stem cell lineages, the precise differentiation of daughter cells is critical to maintain tissue homeostasis. Notch signaling controls the choice between absorptive and entero-endocrine cell differentiation in both the mammalian small intestine and the Drosophila midgut, yet how Notch promotes lineage restriction remains unclear. Here, we describe a role for the transcription factor Klumpfuss (Klu) in restricting the fate of enteroblasts (EBs) in the Drosophila intestine. Klu is induced in Notch-positive EBs and its activity restricts cell fate towards the enterocyte (EC) lineage. Transcriptomics and DamID profiling show that Klu suppresses enteroendocrine (EE) fate by repressing the action of the proneural gene Scute, which is essential for EE differentiation. Loss of Klu results in differentiation of EBs into EE cells. Our findings provide mechanistic insight into how lineage commitment in progenitor cell differentiation can be ensured downstream of initial specification cues

JNK modifies neuronal metabolism to promote proteostasis and longevity.

Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single-gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. Here, we address this question by comprehensively characterizing age-related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long-lived animals with elevated Jun-N-terminal Kinase signaling. We find that these animals exhibit a delayed age-related decline in protein turnover rates, as well as decreased steady-state neuronal glucose-6-phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose-6-phosphate dehydrogenase (G6PD). Over-expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. Our study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long-lived mutants

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication

Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation

Loss of a proteostatic checkpoint in intestinal stem cells contributes to age-related epithelial dysfunction

Protein homeostasis maintenance (proteostasis) is critical for cell function, but declines during aging. Here the authors detail a proteostatic checkpoint in Drosophila intestinal stem cells coordinating cell cycle arrest with protein aggregate clearance, along with its role in aging related intestinal dysfunction

Homozygous ArfGAP1-null mutants display pleiotropic effects depending on genetic background.

<p>(A-C) Genetic crosses were set up to obtain flies carrying wild-type (+) or null mutant (<i>G3-85</i>) alleles of the <i>ArfGAP1</i> gene in the genetic backgrounds of the wild-type line Canton-S (A), the BLOC-1-subunit mutant <i>blos1</i>^<i>ex2</i> (B) and the Lightoid GTPase mutant <i>ltd</i>^<i>1</i> (C). Notice in (B) that males that were double homozygous for <i>ArfGAP1</i>^<i>G3-85</i> and <i>blos1</i>^<i>ex2</i> did not survive to adulthood (indicated with X). In those cases in which viable adult males were obtained, red pigments were extracted and quantified as described under Materials and Methods. Values were expressed as percentages of the pigment content of wild-type flies. Bars represent means + SD of 3–9 biological replicates. Statistical analyses were performed by means of Student’s t-test (B) or one-way ANOVA followed by Dunnett’s test of each group versus that of flies homozygous for the wild-type <i>ArfGAP1</i> allele in the corresponding genetic background (A and C): ns, not significant; *p<0.05, ***p<0.001. (D and E) eye morphology of adult flies homozygous for <i>ltd</i>^<i>1</i> (D) and for both <i>ltd</i>^<i>1</i> and <i>ArfGAP1</i>^<i>G3-85</i> (E), with the latter displaying a mild interommatidial facets phenotype.</p

<i>ArfGAP1</i> as a modifier of the AP-3 eye pigmentation phenotype.

<p>(A-C) Red pigments were extracted from the heads of adult male flies of the indicated genetic backgrounds carrying wild-type (+) or mutant (<i>G3-85</i>) alleles of the <i>ArfGAP1</i> gene on chromosome 3. The extracted pigments were quantified as described under Materials and Methods, and the resulting values expressed as percentages of the pigment content of wild-type (Canton-S) flies. Bars represent means + SD of 3–17 biological replicates. Statistical analyses were performed by means of Student’s t-test (A and B) or one-way ANOVA followed by Dunnett’s test of each group versus <i>g</i>^<i>2</i> flies carrying no deletion (C): *p<0.05, **p<0.01, ***p<0.001. Notice that a single copy of <i>ArfGAP1</i>^<i>G3-85</i> mutant allele over wild-type <i>ArfGAP1</i> was sufficient to ameliorate the pigmentation defects of both <i>g</i>^<i>2</i> (A) and <i>rb</i>^<i>1</i> (B) AP-3-subunit mutants. Notice in (C) that such partial suppressor effect was not exacerbated in flies homozygous for the <i>ArfGAP1</i>^<i>G3-85</i> allele or heterozygous for this allele over any of two deficiencies in which the deleted genomic regions include the entire <i>ArfGAP1</i> gene, namely <i>Df(3L)eyg</i>^<i>C1</i> (Df1) and <i>Df(3L)BSC380</i> (Df2). The deficiency <i>Df(3L)BSC413</i>, in which the deleting genomic region excludes the <i>ArfGAP1</i> gene, was used as a control (Df3).</p

Attempts to validate selected deficiencies carrying the <i>w</i>^<i>+mC</i> marker as genetic modifiers of the <i>g</i>^<i>2</i> mutation.

<p>(A) Red pigments were extracted from the heads of AP-3-deficient (<i>g</i>^<i>2</i>) or White-negative (<i>w</i>^<i>1118</i>) adult males carrying single copies of the indicated deficiencies with their associated <i>w</i>^<i>+mC</i> marker. The extracted pigments were quantified as described under Materials and Methods, and the resulting values expressed as percentages of the pigment content of wild-type (Canton-S) flies. Bars represent means + SD of 2–10 biological replicates. Notice that the activity of the <i>w</i>^<i>+mC</i> marker associated with deficiency <i>Df(3L)ED4978</i> resulted in a red pigment content (arrow) higher than that of <i>g</i>^<i>2</i> flies (black bar). (B-D) Analyses of red pigment content in the eyes of adult <i>g</i>^<i>2</i> mutant males carrying no deletions (—), single copies of the deficiencies <i>Df(3L)ED4978</i> (B), <i>Df(3R)Exel6195</i> (C) and <i>Df(2L)XE-3801</i> (D) that had been identified through screening, or single copies of deficiencies with partially overlapping deletions and devoid of the <i>w</i>^<i>+mC</i> marker. Schematic representations of the extent of overlap between the chromosomal regions deleted in the deficiencies identified through screening (blue) and the others (grey) are included in each figure panel. Notice in (C) that a small portion of the chromosomal region deleted in <i>Df(3R)Exel6195</i> (dashed box) did not overlap with any of those deleted in other available deficiencies. One-way ANOVA followed by Dunnett’s test of each group versus control <i>g</i>^<i>2</i> flies carrying no deletion (black bars): **p<0.01; ***p<0.001.</p

Effects of the <i>Atg2</i>^{<i>EP3697</i>} allele on red pigment content in various genetic backgrounds.

<p>Red pigments were extracted from the heads of adult male flies of the indicated genetic backgrounds lacking (open bars) or carrying (filled bars) one copy of the <i>Atg2</i>^{<i>EP3697</i>} allele over a normal chromosome 3. The extracted pigments were quantified as described under Materials and Methods, and the resulting values expressed as percentages of the pigment content of wild-type (Canton-S) flies. Bars represent means + SD of 7–15 biological replicates. One-way ANOVA followed by Bonferroni comparison of selected group pairs: *p<0.05, **p<0.01, ***p<0.001. Notice that a single copy of <i>Atg2</i>^{<i>EP3697</i>} over a normal <i>Atg2</i> allele increased pigmentation of two AP-3-subunit mutants (<i>g</i>^<i>2</i> and <i>rb</i>^<i>1</i>), a BLOC-1-subunit mutant (<i>blos1</i>^<i>ex2</i>), and a mutant in the Rab-GTPase Lightoid (<i>ltd</i>^<i>1</i>), though it also increased the red pigment content of wild-type flies. Although the transposon inserted in <i>Atg2</i>^{<i>EP3697</i>} carries the <i>w</i>^<i>+mC</i> marker, its weak activity led to barely detectable red pigments in a White-null background (<i>w</i>^<i>1118</i>).</p