Screening bioactive food compounds in honey bees suggests curcumin blocks alcohol-induced damage to longevity and DNA methylation

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Identification of 121 variants of honey bee Vitellogenin protein sequences with structural differences at functional sites

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In the Laboratory and during Free-Flight: Old Honey Bees Reveal Learning and Extinction Deficits that Mirror Mammalian Functional Decline

Loss of brain function is one of the most negative and feared aspects of aging. Studies of invertebrates have taught us much about the physiology of aging and how this progression may be slowed. Yet, how aging affects complex brain functions, e.g., the ability to acquire new memory when previous experience is no longer valid, is an almost exclusive question of studies in humans and mammalian models. In these systems, age related cognitive disorders are assessed through composite paradigms that test different performance tasks in the same individual. Such studies could demonstrate that afflicted individuals show the loss of several and often-diverse memory faculties, and that performance usually varies more between aged individuals, as compared to conspecifics from younger groups. No comparable composite surveying approaches are established yet for invertebrate models in aging research. Here we test whether an insect can share patterns of decline similar to those that are commonly observed during mammalian brain aging. Using honey bees, we combine restrained learning with free-flight assays. We demonstrate that reduced olfactory learning performance correlates with a reduced ability to extinguish the spatial memory of an abandoned nest location (spatial memory extinction). Adding to this, we show that learning performance is more variable in old honey bees. Taken together, our findings point to generic features of brain aging and provide the prerequisites to model individual aspects of learning dysfunction with insect models

Metabolic enzymes in glial cells of the honeybee brain and their associations with aging, starvation and food response

<div><p>The honey bee has been extensively studied as a model for neuronal circuit and memory function and more recently has emerged as an unconventional model in biogerontology. Yet, the detailed knowledge of neuronal processing in the honey bee brain contrasts with the very sparse information available on glial cells. In other systems glial cells are involved in nutritional homeostasis, detoxification, and aging. These glial functions have been linked to metabolic enzymes, such as glutamine synthetase and glycogen phosphorylase. As a step in identifying functional roles and potential differences among honey bee glial types, we examined the spatial distribution of these enzymes and asked if enzyme abundance is associated with aging and other processes essential for survival. Using immunohistochemistry and confocal laser microscopy we demonstrate that glutamine synthetase and glycogen phosphorylase are abundant in glia but appear to co-localize with different glial sub-types. The overall spatial distribution of both enzymes was not homogenous and differed markedly between different neuropiles and also within each neuropil. Using semi-quantitative Western blotting we found that rapid aging, typically observed in shortest-lived worker bees (foragers), was associated with declining enzyme levels. Further, we found enzyme abundance changes after severe starvation stress, and that glutamine synthetase is associated with food response. Together, our data indicate that aging and nutritional physiology in bees are linked to glial specific metabolic enzymes. Enzyme specific localization patterns suggest a functional differentiation among identified glial types.</p></div

Gustatory responsiveness (GRS) to sucrose was associated with relative abundance of glutamine synthetase (GS) but not with glycogen phosphorylase (GP).

<p>(A) Relative GS abundance was higher in brains of bees with high sucrose responsiveness (High GRS), as compared to the group with low responsiveness (low GRS). (B) No such association was evident for GP. Box plots indicate medians and 25/75 percentiles for ranked protein abundance values. Asterisks depict significance (* P<0.05; for detailed statistics see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198322#sec010" target="_blank">Results</a> section).</p

Colocalization of glutamine synthetase (GS) with two types of neuropil glia.

<p>(A) Non-glomerular neuropiles (NP), such as proto- and tritocerebral lobes, are surrounded by somacortices (SC). DAPI staining shows the localization of almost all neuronal and glial somata within the soma cortex and the interface to the neuropiles (SC, arrows). Only few cell bodies lie deep in the neuropil (asterisks). (B and C) The glial-specific marker α-repo (magenta) reveals that glial cells are mostly found at the boundary between soma cortex and synaptic neuropil (arrowheads in B). Many of these peripheral glial cells show intense α-GS staining (green). Location and co-localization with α-repo and arborizations that extend into the neuropil (arrows) support that these α-GS positive cells are astrocyte-like glia, a type of neuropil glia. Note that not all α-repo positive glial cells show α-GS staining (arrowheads in C). Yet, α-repo positive glia within the neuropil did show adjacent α-GS, arborization-like staining (asterisks in B). (D) In glomerular neuropiles, such as the antennal lobes (shown), DAPI reveals somata that surround the entire neuropil and also single glomeruli (Glo, arrows). (E and F) α-repo staining (magenta) marks glial cells that outline single glomeruli (arrows), lack arborizations and hence resemble ensheathing, rather than astrocyte glia. Scale bar 20μm in A, B, D, E, 5μm in C, F.</p

The distribution of glycogen phosphorylase (GP) in the brain.

<p>(A) Abundant α-GP immunostaining was detected in different brain areas. The image stack shows α-GP staining in similar brain regions as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198322#pone.0198322.g001" target="_blank">Fig 1A</a> for α-GS (antennal, AL, central complex, CC, mushroom bodies, Mb, optic lobes, OL). (B and C) Higher resolution images confirming α-GP signals (arrows) for the protocerebral lobes (B, inferior medial protocerebrum, imPC, lateral horn, LH) and the central complex (C; central complex, CC, central body, CB, ellipsoid body, EB). (D-F) Co-localization with the nuclear marker (DAPI, cyan) demonstrates that most intense α-GP signals (magenta, arrows) outline the neuropil of non-glomerular protocerebral lobes (D, lateral horn, LH, inferior medial protocerebrum, imPC) and mark borders of the antennal lobe’s (AL) glomerular neuropiles (E, arrowheads). These localization features suggest that GP is present in ensheathing glia (arrows in D, E, compare A). In addition, α-GP staining at the brain’s periphery suggests GP is present in surface glial subtypes that form the blood brain barrier (asterisks in F). The lower resolution inset in F depicts the location of neuropiles shown in F (lateral protocerebrum, lPC, medulla, Me). (G and H) Co-staining for α-GP and α-GS in cell bodies located between the AL and protocerebral neuropil regions. In contrast to α-GS immunostaining (green), arborizations (arrows) into the neuropil were not evident with α-GP, suggesting different cellular localization for both enzymes. Inset in H with a high-resolution image showing that α-GP and α-GS do not co-localize, suggesting that the enzymes are specific for different glial types. I: Western blots of brain tissue with the α-GP antibody reveal a single band with the expected size of about 97kDa (arrow, for the complete blot compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198322#pone.0198322.s003" target="_blank">S3B Fig</a>). (J-O) Using the same microscopy settings, we only detected immunosignals for samples incubated with the α-GP primary antibody (J, L, N) but not in controls for unspecific secondary antibody staining and autofluorescence (K, M, O). Scale bar = 200μm in A, 50μm in B-F, 20μm in G, H, 200μm in O for J-O.</p

The relative abundance of glutamine synthetase (GS) and glycogen phosphorylase (GP) was reduced in the old group of foragers.

<p>(A and B) Relative protein abundance of GS (A) and GP (B) in brains of the old group (foraging duration ≥ 15 days) as compared to the young group of foragers (foraging duration ≥ 5 days). Box plots indicate medians and 25/75 percentiles for rank values assessed by semi-quantitative Western blotting. Ranks were calculated from GS and GP densitometric values, normalized to the total protein staining (Sypro Ruby). Asterisks depict significance (* P<0.05; for detailed statistics see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198322#sec010" target="_blank">Results</a> section).</p

Severe starvation stress reduced the relative abundance of glutamine synthetase (GS) and glycogen phosphorylase (GP).

<p>(A) Mortality after 12hrs was significantly higher in the starved group as compared to controls that were allowed to feed ad libitum (detailed statistics in the Results section). (B and C) Relative protein abundance of GS (B) and GP (C) in brains of the starved (ST) and the satiated control group (SA). Box plots indicate medians and 25/75 percentiles for ranked protein abundance values. Asterisks depict significance (* P<0.05; for detailed statistics see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198322#sec010" target="_blank">Results</a> section).</p

The distribution of glutamine synthetase (GS) in the honey bee brain.

<p>(A) Confocal microscopic image stack showing abundant α-GS immunolabeling for different brain areas, including central regions with the mushroom bodies (Mb), central complex (CC), as well as antennal (AL), and optic lobes (OL). The faint green line indicates brain and tissue borders of the brain section. (B-G) Higher resolution images reveal marked α-GS staining (arrows) for the lateral and medial protocerebrum (B; lateral protocerebrum, lPC, inferior medial PC, imPC, lateral horn, LH), for the central complex (C; central complex, CC with central body, CB and ellipsoid body, EB), and for output regions of the mushroom bodies (D, E; vertical lobe, vL and γ lobe, γL, medial lobe, mL). Note that staining was most intense at the periphery of protocerebral neuropiles (B; imPC, lPC, LH; D; superior medial protocerebrum, smPC). In contrast, weaker α-GS immunolabeling was detected in the antennal lobes (G), and only sparse staining in the modules of the mushroom bodies’ calyx regions (F; calyx, Cal, collar, Co, lip, Li, basal ring, BR, neck, Ne). H: Western blots of brain tissue with the α-GS antibody reveal a single band (arrow) close to the expected size of 41kDa (for the complete blot compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198322#pone.0198322.s003" target="_blank">S3A Fig</a>). (I-N) With immunohistochemistry we detected a signal only if the α-GS primary antibody was present (I, K, M), whereas controls for unspecific secondary antibody staining and autofluorescence did not reveal marked signals (J, L, N). Scale bar 200μm in A, 50μm in B-G, 200μm in N for I-N.</p