Brain Proteome Changes Induced by Olfactory Learning in <i>Drosophila</i>

For more than 30 years, the study of learning and memory in Drosophila melanogaster (fruit fly) has used an olfactory learning paradigm and has resulted in the discovery of many genes involved in memory formation. By varying learning programs, the creation of different memory types can be achieved, from short-term memory formation to long-term. Previous studies in the fruit fly used gene mutation methods to identify genes involved in memory formation. Presumably, memory creation involves a combination of genes, pathways, and neural circuits. To examine memory formation at the protein level, a quantitative proteomic analysis was performed using olfactory learning and ¹⁵N-labeled fruit flies. Differences were observed in protein expression and relevant pathways between different learning programs. Our data showed major protein expression changes occurred between short-term memory (STM) and long-lasting memory, and only minor changes were found between long-term memory (LTM) and anesthesia-resistant memory (ARM)

Heterochromatin Formation Promotes Longevity and Represses Ribosomal RNA Synthesis

<div><p>Organismal aging is influenced by a multitude of intrinsic and extrinsic factors, and heterochromatin loss has been proposed to be one of the causes of aging. However, the role of heterochromatin in animal aging has been controversial. Here we show that heterochromatin formation prolongs lifespan and controls ribosomal RNA synthesis in <em>Drosophila</em>. Animals with decreased heterochromatin levels exhibit a dramatic shortening of lifespan, whereas increasing heterochromatin prolongs lifespan. The changes in lifespan are associated with changes in muscle integrity. Furthermore, we show that heterochromatin levels decrease with normal aging and that heterochromatin formation is essential for silencing rRNA transcription. Loss of epigenetic silencing and loss of stability of the rDNA locus have previously been implicated in aging of yeast. Taken together, these results suggest that epigenetic preservation of genome stability, especially at the rDNA locus, and repression of unnecessary rRNA synthesis, might be an evolutionarily conserved mechanism for prolonging lifespan.</p> </div

Heterochromatin levels are important for longevity and muscle integrity.

<p>Percent survival of adult female flies of indicated genotypes at 25°C. Flies had been made coisogenic by extensive outcrossing (see Methods). n donates the number of flies counted. p values are from Log rank analysis. (A) Flies carrying one copy of <i>hsp70-HP1</i> (expressing more HP1) were longer lived (p = 6.31×10⁻²⁴), and flies heterozygous for <i>Su(var)205⁵</i> (loss-of-function allele) were shorter lived (p = 2.03×10⁻⁸⁶) when compared with wild-type “+/+” controls. (B) Flies heterozygous for <i>hop^Tum-l</i> (gain-of-function mutation) and <i>stat92E⁰⁶³⁴⁶</i> (loss-of-function allele) were both shorter lived (p = 8.87×10⁻²³; 2.92×10⁻⁵³, respectively) than control, and flies heterozygous for <i>hop³</i> (loss-of-function allele) had longer lifespan compared to control flies (p = 7.34×10⁻²⁵). (C) Flies of indicated genotype and age were confined in food vials and their movements were recorded by video and then analyzed as average velocity. Each data point is the average of >3 recordings of different groups of flies. Error bars are S.E.M. Note that <i>Su(var)205^+/−</i> flies lose mobility precipitously as they age, whereas <i>hsp70-HP1</i> flies maintain high mobility for a longer period of time. (D) Top: adult large instestines of indicated age and genotype were stained with phalloidin-fluorescein to reveal the longitudinal and circular intestinal body wall muscle fibers. Images are partions of representative midgut showing 3 longitudinal fibers (wide bands) and circular fibers (thin bands). Note that <i>Su(var)205^+/−</i> flies exhibit premature muscle degeneration: the longitudinal fibers are discontinuous with “loose ends”. Bottom: each fluorescein-stained gut was assigned a morphology score of 0 to 10 based on the integrity of the longitudinal muscle fibers (see Methods). The muscle integrity index was calculated by averaging the scores of 10 guts for each indicated genotype and age. <i>Su(var)205^5/+</i> and <i>Su(var)205^2/+</i> showed similar phenotypes; the results were combined. Error bars are standard deviations.</p

Heterochromatin controls rRNA transcription.

<p>(A) Schematic representation of a pre-rRNA transcript, with an R2 element inserted into the 28S gene. Arrows above the 5′ETS region represent PCR primers used for qPCR analysis. (B) RNA was isolated from 3^rd instar larvae of indicated genotypes and was subjected to Northern blotting with an R2 5′ antisense probe. Transcripts from the <i>Adh</i> gene were used as a loading control. Levels of the transcripts were quantified with a phospho-imager. The full-length (FL) R2 transcript is 3.6 kb. The lower bands are all degradation products, which appear soon after transcription <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002473#pgen.1002473-Eickbush1" target="_blank">[42]</a>. A density plot is shown to the right with arbitrary units (a.u.). (C) The levels of pre-rRNA in 2-day-old male flies of indicated genotypes were measured by qRT-PCR. Relative pre-rRNA levels are shown with standard deviations. (D) Representative larvae of indicated genotypes. <i>Su(var)205^−/−</i> were transheterozygous for <i>Su(var)205²</i> and <i>Su(var)205⁵</i>. (E) Flies of indicated genotypes were outcrossed to minimize genetic background effects and were raised in parallel at 25°C with similar larval density. Top: Representative male flies of indicated genotypes. Bottom: The fly body weight was measured as the average of 10 2-day old male flies. <i>Su(var)205^2/+</i> and <i>Su(var)205^5/+</i> male flies had similar body weights, the results were combined and shown as <i>Su(var)205^+/−</i>. Standard deviations and p values (compared with wild-type control; Student's <i>t</i>-Test) are shown.</p

Heterochromatin levels decline with aging.

<p>(A) Gut tissues from young (3-day old) and old (35-day old) female flies were dissected and stained with anti-HP1 (magenta). Images were scanned at identical settings with confocal microscopy. Note that HP1 forms prominent foci in the young gut (one pointed by an arrow), whereas in the old gut HP1 staining seems more diffuse and lacks the prominent foci. (B) Male flies of indicated age (in weeks) were homogenized and the protein extracts were subjected to SDS-PAGE and blotted sequentially with antibodies for H3K9m2, HP1, H3, and α-Tubulin. Representative images for one of the three experiments are shown. Lower panels show intensity ratios as indicated. Note that total H3 levels decrease with age, and that H3K9m2 signals decrease with age even when normalized to total H3. (C) Chromatin immunoprecipitation (ChIP) was carried out with anti-HP1 antibodies using extracts from young (3-day old) and old (35-day old) male wild-type or <i>hsp70-HP1/+</i> flies. Note that HP1 is enriched in <i>1360</i> (representative heterochromatin sequence) of young but not old wild-type flies (lane 2, top two panels), and is detectable in both young and old <i>hsp70-HP1/+</i> flies (middle panels). (D) Ovaries from young (3-day old) and old (35-day old) female <i>DX1</i> flies were stained with anti-ßgal. Note the much increased ßgal levels in old ovaries. (E) Total protein from single young (3-day old) and old (35-day old) female flies of indicated genotypes were blotted by anti-ß-gal antibodies or anti-aTublin (control). Note the appearance of ß-gal in old <i>DX1</i> flies (arrow). The band below ß-gal is a nonspecific band.</p

Heterochromatin affects stability of the nucleolus and rDNA locus.

<p>(A) 3^rd instar larval salivary glands of indicated genotypes were stained with anti-Fibrillarin. <i>Stat92E^F</i> is a hypomorphic allele. Images were scanned with a confocal microscope at identical settings, and representative nuclei and quantifications are shown. N represents the number of nuclei scored. (B) 3^rd instar larvae of indicated genotypes were processed for ECC (left) or genomic (right; control) DNA. The presence of indicated sequences were detected by PCR. <i>Rp49</i> (lane 1) and <i>5S rDNA</i> (lane 2) are non-repeated sequences and are used as negative controls for ECC. 1: rp49. 2: 5S rDNA. 3: Satellite 1.688. 4: 5′ 18S rDNA. 5: 3′ 18S rDNA. 6: 18 to 5.8S spacer. 7: 5.8S rDNA. 8: 28S rDNA. 9: Mid 28S rDNA. 10: 3′ 28S rDNA. Each genotype was analyzed three times; representative PCR results are shown. ECC levels were quantified by calculating the ECC index for each genotype (see Methods) and the results are shown (bottom). Error bars indicate S.E.M., and p values (Student's <i>t</i>-Test) indicate statistical significance of the differences compared with wild type. Note that ECC DNA (lane 3–10) was detected at high levels in <i>Stat92E</i>, <i>Su(var)205</i>, and <i>hop^Tum-l</i> heterozygotes, only minimally in wild-type controls, but not in <i>hop</i> loss-of-function heterozygotes.</p