The lipidomes of C. elegans with mutations in asm-3/acid sphingomyelinase and hyl-2/ceramide synthase show distinct lipid profiles during aging.

Lipid metabolism affects cell and physiological functions that mediate animal healthspan and lifespan. Lipidomics approaches in model organisms have allowed us to better understand changes in lipid composition related to age and lifespan. Here, using the model C. elegans, we examine the lipidomes of mutants lacking enzymes critical for sphingolipid metabolism; specifically, we examine acid sphingomyelinase (asm-3), which breaks down sphingomyelin to ceramide, and ceramide synthase (hyl-2), which synthesizes ceramide from sphingosine. Worm asm-3 and hyl-2 mutants have been previously found to be long- and short-lived, respectively. We analyzed longitudinal lipid changes in wild type animals compared to mutants at 1-, 5-, and 10-days of age. We detected over 700 different lipids in several lipid classes. Results indicate that wildtype animals exhibit increased triacylglycerols (TAG) at 10-days compared to 1-day, and decreased lysophoshatidylcholines (LPC). We find that 10-day hyl-2 mutants have elevated total polyunsaturated fatty acids (PUFA) and increased LPCs compared to 10-day wildtype animals. These changes mirror another short-lived model, the daf-16/FOXO transcription factor that is downstream of the insulin-like signaling pathway. In addition, we find that hyl-2 mutants have poor oxidative stress response, supporting a model where mutants with elevated PUFAs may accumulate more oxidative damage. On the other hand, 10-day asm-3 mutants have fewer TAGs. Intriguingly, asm-3 mutants have a similar lipid composition as the long-lived, caloric restriction model eat-2/mAChR mutant. Together, these analyses highlight the utility of lipidomic analyses to characterize metabolic changes during aging in C. elegans.

Temporal Transcriptomics of Gut Escherichia coli in Caenorhabditis elegans Models of Aging.

Host-bacterial interactions over the course of aging are understudied due to complexities of the human microbiome and challenges of collecting samples that span a lifetime. To investigate the role of host-microbial interactions in aging, we performed transcriptomics using wild-type Caenorhabditis elegans (N2) and three long-lived mutants (daf-2, eat-2, and asm-3) fed Escherichia coli OP50 and sampled at days 5, 7.5, and 10 of adulthood. We found host age is a better predictor of the E. coli expression profiles than host genotype. Specifically, host age was associated with clustering (permutational multivariate analysis of variance [PERMANOVA], P = 0.001) and variation (Adonis, P = 0.001, R2 = 11.5%) among E. coli expression profiles, whereas host genotype was not (PERMANOVA, P > 0.05; Adonis, P > 0.05, R2 = 5.9%). Differential analysis of the E. coli transcriptome yielded 22 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 100 KEGG genes enriched when samples were grouped by time point [LDA, linear discriminant analysis; log(LDA), ≥2; P ≤ 0.05], including several involved in biofilm formation. Coexpression analysis of host and bacterial genes yielded six modules of C. elegans genes that were coexpressed with one bacterial regulator gene over time. The three most significant bacterial regulators included genes relating to biofilm formation, lipopolysaccharide production, and thiamine biosynthesis. Age was significantly associated with clustering and variation among transcriptomic samples, supporting the idea that microbes are active and plastic within C. elegans throughout life. Coexpression analysis further revealed interactions between E. coli and C. elegans that occurred over time, building on a growing literature of host-microbial interactions. IMPORTANCE Previous research has reported effects of the microbiome on health span and life span of Caenorhabditis elegans, including interactions with evolutionarily conserved pathways in humans. We build on this literature by reporting the gene expression of Escherichia coli OP50 in wild-type (N2) and three long-lived mutants of C. elegans. The manuscript represents the first study, to our knowledge, to perform temporal host-microbial transcriptomics in the model organism C. elegans. Understanding changes to the microbial transcriptome over time is an important step toward elucidating host-microbial interactions and their potential relationship to aging. We found that age was significantly associated with clustering and variation among transcriptomic samples, supporting the idea that microbes are active and plastic within C. elegans throughout life. Coexpression analysis further revealed interactions between E. coli and C. elegans that occurred over time, which contributes to our growing knowledge about host-microbial interactions.

Using Bacterial Transcriptomics to Investigate Targets of Host-Bacterial Interactions in Caenorhabditis elegans.

The interactions between a host and its resident microbes form complicated networks that can affect host physiology. Disentangling these host-microbe interactions can help us better understand mechanisms by which bacteria affect hosts, while also defining the integral commensal protection that host-associated microbiota offer to promote health. Here we utilize a tractable genetic model organism, Caenorhabditis elegans, to study the effects of host environments on bacterial gene expression and metabolic pathways. First, we compared the transcriptomic profiles of E. coli OP50 in vitro (on agar plates) versus in vivo (fed to C. elegans host). Our data revealed that 110 biosynthetic genes were enriched in host-associated E. coli. Several of these expressed genes code for the precursors and products needed for the synthesis of lipopolysaccharides (LPS), which are important for innate immune and stress responses, as well as pathogenicity. Secondly, we compared the transcriptomic profiles of E. coli fed to hosts with different genetic backgrounds, including the long-lived daf-2/insulin like growth factor (IGF) receptor and short lived daf-16/FOXO transcription factor mutants. We find that hosts genetics also alters bacterial metabolic pathways. Given that bacteria influence host health, this transcriptomics approach can elucidate genes mediating host aging.

Analysis of Student Attitudes of a Neurobiology Themed Inquiry Based Research Experience in First Year Biology Labs.

Inquiry based research experiences are thought to increase learning gains in biology, STEM retention, and confidence in students of diverse backgrounds. Furthermore, such research experiences within the first year of college may foster increased student retention and interest in biology. However, providing first year students in biology labs with inquiry-based experiences is challenging given demands of large student enrollments, restricted lab space, and instructor time. Thus, we aimed to integrate a small neurobiology themed research experience within a three-week modular, first-year biology laboratory setting. For this, students first performed a whole class lab examining the effects of ethanol on movement and associative learning. Using skills they acquired, the students devised, executed, and presented their self-designed experiments and results. Using pre-and post-course surveys, we analyzed student attitudes on their experiences, including technical skills, inquiry-based learning styles in which experimental outcomes are often unknown, and research in their first year of biology. Analyzing data collected for three years, we found that students self-reported gains in technical skills and positive attitudes toward inquiry-based learning. In contrast, we found that students did not self-report increased interest in research experiences in general.

Loss of Sphingosine Kinase Alters Life History Traits and Locomotor Function in Caenorhabditis elegans.

Sphingolipid metabolism is important to balance the abundance of bioactive lipid molecules involved in cell signaling, neuronal function, and survival. Specifically, the sphingolipid sphingosine mediates cell death signaling, whereas its phosphorylated form, sphingosine-1-phosphate (S1P), mediates cell survival signaling. The enzyme sphingosine kinase produces S1P, and the activity of sphingosine kinase impacts the ability of cells to survive under stress and challenges. To examine the influence of sphingolipid metabolism, particularly enzymes regulating sphingosine and S1P, in mediating aging, neuronal function and stress response, we examined life history traits, locomotor capacities and heat stress responses of young and old animals using the model organism Caenorhabditis elegans. We found that C. elegans sphk-1 mutants, which lack sphingosine kinase, had shorter lifespans, reduced brood sizes, and smaller body sizes compared to wild type animals. By analyzing a panel of young and old animals with genetic mutations in the sphingolipid signaling pathway, we showed that aged sphk-1 mutants exhibited a greater decline in neuromuscular function and locomotor behavior. In addition, aged animals lacking sphk-1 were more susceptible to death induced by acute and prolonged heat exposure. On the other hand, older animals with loss of function mutations in ceramide synthase (hyl-1), which converts sphingosine to ceramide, showed improved neuromuscular function and stress response with age. This phenotype was dependent on sphk-1. Together, our data show that loss of sphingosine kinase contributes to poor animal health span, suggesting that sphingolipid signaling may be important for healthy neuronal function and animal stress response during aging.

Extrasynaptic muscarinic acetylcholine receptors on neuronal cell bodies regulate presynaptic function in Caenorhabditis elegans.

Acetylcholine (ACh) is a potent neuromodulator in the brain, and its effects on cognition and memory formation are largely performed through muscarinic acetylcholine receptors (mAChRs). mAChRs are often preferentially distributed on specialized membrane regions in neurons, but the significance of mAChR localization in modulating neuronal function is not known. Here we show that the Caenorhabditis elegans homolog of the M1/M3/M5 family of mAChRs, gar-3, is expressed in cholinergic motor neurons, and GAR-3-GFP fusion proteins localize to cell bodies where they are enriched at extrasynaptic regions that are in contact with the basal lamina. The GAR-3 N-terminal extracellular domain is necessary and sufficient for this asymmetric distribution, and mutation of a predicted N-linked glycosylation site within the N-terminus disrupts GAR-3-GFP localization. In transgenic animals expressing GAR-3 variants that are no longer asymmetrically localized, synaptic transmission at neuromuscular junctions is impaired and there is a reduction in the abundance of the presynaptic protein sphingosine kinase at release sites. Finally, GAR-3 can be activated by endogenously produced ACh released from neurons that do not directly contact cholinergic motor neurons. Together, our results suggest that humoral activation of asymmetrically localized mAChRs by ACh is an evolutionarily conserved mechanism by which ACh modulates neuronal function.

Neuropeptide secreted from a pacemaker activates neurons to control a rhythmic behavior.

BACKGROUND: Rhythmic behaviors are driven by endogenous biological clocks in pacemakers, which must reliably transmit timing information to target tissues that execute rhythmic outputs. During the defecation motor program in C. elegans, calcium oscillations in the pacemaker (intestine), which occur about every 50 s, trigger rhythmic enteric muscle contractions through downstream GABAergic neurons that innervate enteric muscles. However, the identity of the timing signal released by the pacemaker and the mechanism underlying the delivery of timing information to the GABAergic neurons are unknown. RESULTS: Here, we show that a neuropeptide-like protein (NLP-40) released by the pacemaker triggers a single rapid calcium transient in the GABAergic neurons during each defecation cycle. We find that mutants lacking nlp-40 have normal pacemaker function, but lack enteric muscle contractions. NLP-40 undergoes calcium-dependent release that is mediated by the calcium sensor, SNT-2/synaptotagmin. We identify AEX-2, the G-protein-coupled receptor on the GABAergic neurons, as the receptor for NLP-40. Functional calcium imaging reveals that NLP-40 and AEX-2/GPCR are both necessary for rhythmic activation of these neurons. Furthermore, acute application of synthetic NLP-40-derived peptide depolarizes the GABAergic neurons in vivo. CONCLUSIONS: Our results show that NLP-40 carries the timing information from the pacemaker via calcium-dependent release and delivers it to the GABAergic neurons by instructing their activation. Thus, we propose that rhythmic release of neuropeptides can deliver temporal information from pacemakers to downstream neurons to execute rhythmic behaviors.

Localized sphingolipid signaling at presynaptic terminals is regulated by calcium influx and promotes recruitment of priming factors.

Activity-dependent changes in presynaptic function represent a critical mechanism by which synaptic strength is controlled. However, how changes in synaptic activity couple to presynaptic components to control synaptic vesicle release and recycling are poorly understood. Sphingosine kinase (SphK) is a sphingolipid metabolic enzyme whose activity-dependent recruitment to membrane regions within presynaptic terminals promotes neurotransmitter release. Here, we show that synaptic recruitment of SPHK-1, the SphK ortholog in Caenorhabditis elegans, is mediated by presynaptic calcium influx. Quantitative fluorescence imaging of live presynaptic terminals reveals that blocking presynaptic calcium influx reduces synaptic SPHK-1 abundance whereas increasing calcium influx increases SPHK-1 synaptic abundance. CALM-1, the calcium and integrin binding protein ortholog, colocalizes with SPHK-1 at release sites and regulates muscarinic-mediated synaptic SPHK-1 recruitment. We identify two additional sphingolipid metabolic enzymes that are concentrated at presynaptic terminals, and mutants lacking one of these, HYL-1/ceramide synthase, have defects in synaptic transmission and in synaptic vesicle cycling. Finally, we show that SPHK-1 activity is required for the recruitment of the priming protein UNC-13/Munc13 to presynaptic terminals following activation by muscarinic signaling. These findings suggest that calcium-dependent regulation of local S1P metabolism at synapses may be an important mechanism by which synaptic vesicle priming factors are recruited to release sites to promote synaptic transmission.

Recruitment of sphingosine kinase to presynaptic terminals by a conserved muscarinic signaling pathway promotes neurotransmitter release.

Sphingolipids are potent lipid second messengers that regulate cell differentiation, migration, survival, and secretion, and alterations in sphingolipid signaling have been implicated in a variety of diseases. However, how sphingolipid levels are regulated, particularly in the nervous system, remains poorly understood. Here, we show that the generation of sphingosine-1-phosphate by sphingosine kinase (SphK) promotes neurotransmitter release. Electrophysiological, imaging, and behavioral analyses of Caenorhabditis elegans mutants lacking sphingosine kinase sphk-1 indicate that neuronal development is normal, but there is a significant defect in neurotransmitter release from neuromuscular junctions. SPHK-1 localizes to discrete, nonvesicular regions within presynaptic terminals, and this localization is critical for synaptic function. Muscarinic agonists cause a rapid increase in presynaptic SPHK-1 abundance, whereas reduction of endogenous acetylcholine production results in a rapid decrease in presynaptic SPHK-1 abundance. Muscarinic regulation of presynaptic SPHK-1 abundance is mediated by a conserved presynaptic signaling pathway composed of the muscarinic acetylcholine receptor GAR-3, the heterotrimeric G protein Gαq, and its effector, Trio RhoGEF. SPHK-1 activity is required for the effects of muscarinic signaling on synaptic transmission. This study shows that SPHK-1 promotes neurotransmitter release in vivo and identifies a novel muscarinic pathway that regulates SphK abundance at presynaptic terminals.

Depletion of central BDNF in mice impedes terminal differentiation of new granule neurons in the adult hippocampus.

Granule neurons generated in the adult mammalian hippocampus synaptically integrate to facilitate cognitive function and antidepressant efficacy. Here, we investigated the role of BDNF in facilitating their maturation in vivo. We found that depletion of central BDNF in mice elicited an increase in hippocampal cell proliferation without affecting cell survival or fate specification. However, new mutant neurons failed to fully mature as indicated by their lack of calbindin, reduced dendritic differentiation and an accumulation of calretinin(+) immature neurons in the BDNF mutant dentate gyrus. Furthermore, the facilitating effects of GABA(A) receptor stimulation on neurogenesis were absent in the mutants, suggesting that defects might be due to alterations in GABA signaling. Transcriptional analysis of the mutant hippocampal neurogenic region revealed increases in markers for immature neurons and decreases in neuronal differentiation facilitators. These findings demonstrate that BDNF is required for the terminal differentiation of new neurons in the adult hippocampus.