Intergenerational Neuroprotection by an Intestinal Sphingolipid 
in Caenorhabditis elegans

doi:10.21203/rs.3.rs-2058282/v1

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Intergenerational Neuroprotection by an Intestinal Sphingolipid in Caenorhabditis elegans

https://doi.org/10.21203/rs.3.rs-2058282/v1

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In animals, maternal diet and environment can influence the health of offspring. Whether and how maternal dietary choice impacts the nervous system across multiple generations is not well understood. Here, we show that feeding Caenorhabditis elegans with ursolic acid (UA), a natural plant product, reduces adult-onset neurodegeneration intergenerationally. UA provides neuroprotection by enhancing maternal provisioning of sphingosine-1-phosphate (S1P) - a bioactive sphingolipid. Intestine-to-oocyte S1P transfer is required for intergenerational neuroprotection and is dependent on the RME-2 lipoprotein yolk receptor. S1P acts intergenerationally by upregulating transcription of the acid ceramidase-1 (asah-1) gene in the intestine. Spatially regulating sphingolipid metabolism is critical as inappropriate asah-1 expression in neurons causes developmental axon outgrowth defects. Our results show that sphingolipid homeostasis impacts the development and intergenerational health of the nervous system. The ability of specific lipid metabolites to act as messengers between generations may have broad implications for dietary choice during reproduction.

Intergenerational inheritance

maternal diet

sphingolipid homeostasis

axon degeneration

transcription

Animal exposure to environmental and dietary changes can modify the physiology and development of offspring ^{1, 2}. Certain parentally acquired traits may also be inherited over multiple generations ^{3, 4}. In recent years, epigenetic mechanisms of inheritance exploiting small RNAs, DNA methylation and chromatin modifications have been defined ^2–7. However, the potential role of maternal provisioning to offspring as a means of multigenerational inheritance has not been well explored. Maternal provisioning potentially enables inheritance of information beyond nucleic acids – affording transmission of specific lipid, protein and metabolite profiles to offspring ⁸. Such information flow could provide direct reporting of ancestral experience to alter the physiology and development of offspring across generations, and potentially shape evolutionary trajectory.

Whether altered maternal metabolism and provisioning can impact the neuronal health of offspring across generations is an open question and challenging to resolve. The Caenorhabditis elegans model has been effectively used to identify and dissect epigenetic mechanisms due to its short generation time and straightforward genetics ^{3, 4, 7}. C. elegans is also well suited to analyzing the temporal effects of dietary and environmental changes as such conditions can be precisely controlled. In addition, defined C. elegans neurodevelopmental and disease models allow potential multigenerational effects to be rigorously examined ^{9, 10}. We therefore used C. elegans to identify molecules that may regulate neuronal health across generations.

Axons are long cytoplasmic projections that transmit information between neurons. Maintenance of neuronal health requires the transport of cargo (organelles, RNAs, proteins and lipids) along the axonal cytoskeleton ^{11, 12}. Microtubules are major cytoskeletal components comprised of cylindrical structures assembled from α- and β-tubulin heterodimers that are crucial for intracellular transport ^{11, 13}. Defective microtubule structure disrupts the supply of essential materials and is associated with multiple neurodegenerative disorders ¹⁴. Therefore, it is crucial to identify molecular mechanisms that support axonal health under conditions of sub-optimal microtubule-associated intracellular transport. The C. elegans posterior lateral mechanosensory (PLM) neurons extend axons along the length of the worm to coordinate touch responses ¹⁵. A previous study showed that loss of the MEC-17/αTAT1 α-tubulin acetyltransferase causes progressive adult-onset PLM axon degeneration ⁹. MEC-17 loss causes microtubule instability and aberrant axonal transport, leading to reduced number and disrupted spatial distribution of mitochondria, as well as defective synaptic protein localization ⁹. PLM axon degeneration is caused by axon fragility, as the phenotype is robustly suppressed by paralyzing mec-17 mutant animals ⁹. Further, PLM axon degeneration in mec-17 mutants is exacerbated in animals with increased body length (e.g. lon-2 mutant), likely due to the added demand on axonal transport required to maintain a longer axon ⁹. We used the well-defined MEC-17/LON-2-deficient axon degeneration model to identify mechanisms that maintain axon integrity across generations.

In this paper, we show that supplying the natural product ursolic acid (UA), specifically during oocyte production, prevents axon degeneration intergenerationally in C. elegans. UA protects axons by upregulating expression of the sphingolipid biosynthetic enzyme acid ceramidase-1 (asah-1) gene in the intestine. Intestine-to-oocyte transport of sphingosine-1-phosphate (S1P), a downstream bioactive sphingolipid metabolite, in maternal yolk is required for intergenerational axon protection by upregulating asah-1 expression in subsequent generations. We further show that transcriptional regulation of asah-1 is dependent on the intestinal transcription factors PQM-1 (GATA zinc finger) and CEH-60 (TALE class). Together, our study shows that a short-term dietary supplement during the maternal reproductive period can have neuroprotective effects over multiple generations – providing a new paradigm for intergenerational inheritance of health-promoting traits. We propose that intergenerational regulation of metabolism and metabolic gene expression may be a universal principle underlying intergenerational inheritance across evolution.

Ursolic Acid Prevents Axon Degeneration Intergenerationally

To identify molecules that suppress axon degeneration, we expressed green fluorescent protein (GFP) specifically in the C. elegans mechanosensory neurons and examined PLM morphology in wild-type and mec-17(ok2109); lon-2(e678) animals (Figure 1A-B). As shown previously, mec-17(ok2109); lon-2(e678) mutant animals exhibit ~50% penetrant PLM axon degeneration in day 3 adults (Figure 1B) ⁹. In a screen of natural products, we identified ursolic acid (UA) as a suppressor of axon degeneration in mec-17(ok2109); lon-2(e678) mutant animals (Figure 1). UA is a lipophilic pentacyclic triterpenoid acid found in plants that has broad biological functions, acting as an anti-inflammatory, antioxidant, and neuroprotective molecule ^{16, 17} (Figure 1C). To assess the potency of UA-induced neuroprotection, we fed mec-17(ok2109); lon-2(e678) animals with different UA concentrations and examined PLM axon degeneration in day 3 adults (Figure 1A-D and S1A). We found that incubating P0 larval stage 4 (L4) animals with 50 mM UA for one generation robustly reduced axon degeneration in mec-17(ok2109); lon-2(e678) F1 progeny (Figure 1D and S1A). To determine if the UA-induced suppression of axon degeneration was caused by reduced body length or motility we used Wormlab tracking (MBF Bioscience LLC, Williston, VT USA). We found no change in motility or body length of mec-17(ok2109); lon-2(e678) animals exposed to UA compared to controls, suggesting a molecular rather than physical effect (Figure S1B-E).

We wondered whether there was a critical functional period during development for UA to reduce PLM axon degeneration. To investigate this, we exposed mec-17(ok2109); lon-2(e678) animals to UA at the following stages of C. elegans development: P0 L3 larvae-L4 larvae (during sperm generation and before oocyte production), P0 L4 larvae-adult (oocytes and sperm present), embryo-L1 larvae (embryogenesis) or L1 larvae-adult (all larval stages and adult) (Figure 1E). We found that axon degeneration in F1 adult progeny was only reduced when P0 hermaphrodites containing oocytes and sperm (P0 L4-adult) were exposed to UA (Figure 1E-F). This result suggested that the axonal health-promoting effect of UA is deposited in the gametes to maintain axonal health through to adulthood. This prompted us to examine whether UA could also protect the nervous system in subsequent generations. We incubated P0 L4 hermaphrodites with DMSO (control) or UA for 16 hours, then transferred them to untreated plates to lay eggs for 3 hours (Figure 1G). These F1 eggs therefore underwent oocyte maturation during UA exposure. When F1 animals reached the L4 stage, a cohort were transferred to lay eggs for analysis of the next generation and the remainder were matured until day 3 of adulthood to examine PLM axon health (Figure 1G-H). We repeated this process for subsequent generations. We found that UA incubation during P0 oocyte maturation reduced PLM axon degeneration in the F1 and F2 generations but not the F3 generation (Figure 1G-H). This reveals an intergenerational neuroprotective effect of UA.

Intergenerational Axon Protection Requires Intestine-Oocyte Transport

How does UA intergenerationally protect the nervous system? As the UA functional period is during oocyte production and maturation, we hypothesized that UA may affect maternal yolk provisioning to oocytes. In C. elegans, yolk is synthesized in the hermaphrodite intestine and contains lipids and lipoproteins that provide oocytes, and thus embryos, with nutrients for development ⁸. Oocyte yolk import occurs through endocytosis and requires RME-2 (Receptor-Mediated Endocytosis-2), a low-density lipoprotein receptor ¹⁸. We found that RME-2 is required for UA to reduce PLM neuron degeneration, supporting a role for intestine-oocyte transport (Figure 2A). Animals lacking RME-2 are also defective in transport of RNAs that are major transmitters of epigenetic inheritance ¹⁹. The nuclear HRDE-1 (Heritable RNAi Deficient 1) Argonaute is required for inheritance of small RNAs, however, we found that hrde-1 is dispensable for UA to reduce PLM degeneration (Figure 2B) ²⁰. These data suggest that UA stimulates alternative epigenetic factors in the maternal yolk and that this information optimizes the oocyte/embryonic environment to promote axon health.

Ursolic Acid Induces Acid Ceramidase (ASAH-1) Expression in the Intestine

To identify factor(s) regulated by UA to prevent PLM axon degeneration, we examined the transcriptomes of synchronized L4 larvae that were exposed to UA (or DMSO as a control) for 12 hours (Figure 2C, S2 and Table S1). We identified 49 dysregulated genes (FDR 0.02) (8 upregulated and 41 downregulated) and surveyed this dataset for genes expressed in the intestine (the source of yolk) that potentially control lipid metabolism. We detected increased asah-1 transcript abundance in animals exposed to UA, which we confirmed by quantitative polymerase chain reaction (qPCR) analysis of independent RNA samples (Figure 2D). asah-1 encodes an acid ceramidase that hydrolyzes ceramide into fatty acid and sphingosine ²¹. To assess whether asah-1 is required for UA to reduce PLM axon degeneration, we performed RNA-mediated interference (RNAi) to knock down asah-1 in mec-17(ok2109); lon-2(e678) animals incubated with UA (Figure 2E). We found that UA does not reduce PLM axon degeneration in asah-1 RNAi knockdown animals, suggesting that asah-1 expression is required for the UA neuroprotective effect (Figure 2E). To identify the tissue in which asah-1 is expressed and potentially regulated by UA, we monitored the spatial and temporal expression pattern of asah-1 using 2000 bp of upstream sequence to drive nuclear-localized GFP expression (Figure 2F and S3). We detected nuclear GFP expression exclusively in intestinal cells from late embryos through to adult in this Pasah-1::gfp transcriptional reporter (Figure 2F and S3). At all stages, we observed a bias in nuclear GFP expression in the anterior intestine (Figure 2F and S3). We imaged nuclear GFP expression in Pasah-1::gfp L4 animals after 12 h of UA exposure and detected increased fluorescence in intestinal nuclei (Figure 2G). Thus, UA induces asah-1 transcription in the intestine.

Intestinal ASAH-1 Expression Protects Axons

As asah-1 is upregulated in animals exposed to UA, we wondered whether transgenic overexpression of asah-1 in mec-17(ok2109); lon-2(e678) animals could mimic the neuroprotective effect of UA. Single cell sequencing studies corroborate that asah-1 is predominantly expressed in the intestine, however low-level expression is detected other cells/tissues, including the PLM neurons ^{22, 23}. We therefore overexpressed asah-1 in mec-17(ok2109); lon-2(e678) animals using the following heterologous promoters: intestine (ges-1 promoter), hypodermis (dpy-7 promoter), muscle (myo-3 promoter) and the mechanosensory neurons (mec-4 promoter) (Figure 3A and S4) ^24-27. Overexpressing asah-1 in the intestine, but not in hypodermis or muscle, reduced PLM axon degeneration in mec-17(ok2109); lon-2(e678) animals (Figure 3A). Overexpressing asah-1 in the mechanosensory neurons caused PLM axon outgrowth defects in mec-17(ok2109); lon-2(e678) animals (Figure S4A and C), precluding analysis of PLM degeneration. Overexpression of asah-1 in neurons of wild-type animals, either in the mechanosensory neurons (mec-4 promoter) or pan-neuronally (rab-3 promoter), also caused extensive axon outgrowth defects in the ALM and PLM neurons (Figure 3B-C and S4A-C). However, no defects were detected in PVM guidance (Figure S4E). These data reveal that neuronal asah-1 overexpression disrupts axon outgrowth of the ALM and PLM axons, which extend axons embryonically, but not the PVM axons which extend post-embryonically. This potential embryonic effect is supported by the observation of axon guidance defects in the PVQ ventral nerve cord neurons when asah-1 is overexpressed in the nervous system (Figure S4F-G). However, animals overexpressing asah-1 in the nervous system did not exhibit overt motility defects, suggesting that global nervous system architecture was intact. Together, these data reveal that intestinal asah-1 induction reduces PLM axon degeneration in animals with defective microtubule stability, and that inappropriate neuronal expression of asah-1, with likely associated disruption of sphingolipid homeostasis, cell-autonomously causes neurodevelopmental defects.

Sphingolipids are amphipathic bioactive molecules with multiple cellular functions, including cell adhesion and migration, cell death and cell proliferation ²⁸. Sphingolipid homeostasis is maintained through the de novo or salvage pathways (Figure 4A) ²⁹. Serine palmitoyltransferase (SPT) is the rate-limiting enzyme in the de novo pathway that generates ceramide - the ASAH-1 substrate (Figure 4A). In C. elegans, sptl-1 knockout causes embryonic lethality and larval arrest, likely due to loss of sphingolipid complexity. Therefore, to assess the importance of SPTL-1 in regulating PLM axon degeneration, we overexpressed sptl-1 cDNA in the intestine. We found that sptl-1 overexpression reduces PLM axon degeneration of mec-17(ok2109); lon-2(e678) animals, confirming that ceramide or its derivatives are important for axon health (Figure 4B). In the salvage pathway, lysosomal membrane ceramide is hydrolyzed to sphingosine by acid ceramidases (CDase), and sphingosine phosphorylation by sphingosine kinases (SphK) generates sphingosine-1-phosphate (S1P) (Figure 4A) ²⁹. We found that driving intestinal sphk-1 expression reduces PLM axon degeneration of mec-17(ok2109); lon-2(e678) animals, as we previously showed by overexpressing sptl-1 or asah-1 (Figure 4B-D). Further, sphk-1 loss increases PLM axon degeneration and prevents UA-induced reduction of PLM axon degeneration of mec-17(ok2109); lon-2(e678) animals (Figure S5A-B). We have shown that intestinal overexpression of asah-1 reduces PLM axon degeneration (Figure 4C). We next determined whether sphk-1, and therefore S1P generation, is required for ASAH-1 to perform this function. We therefore knocked down sphk-1 expression in animals overexpressing asah-1 in the intestine and evaluated PLM axon degeneration in mec-17(ok2109); lon-2(e678) animals (Figure 4E). We found that sphk-1 RNAi suppressed the beneficial effect of asah-1 overexpression on PLM axon degeneration (Figure 4E). Together, these data suggest that the protective role of UA in reducing PLM axon degeneration is dependent on S1P generation.

Sphingosine-1-Phosphate (S1P) Prevents Axon Degeneration Intergenerationally

We directly examined the role of S1P in PLM axonal health by incubating mec-17(ok2109); lon-2(e678) animals for one generation (P0 L4 - F1 3-day old adult) with different S1P concentrations. We found that 20 mM S1P reduced PLM axon degeneration (Figure 5A-B). To determine the S1P functional period, we exposed mec-17(ok2109); lon-2(e678) animals to S1P from P0 L4 larvae-adult (oocytes present) or L1 larvae-adult (all larval stages and adult). S1P only reduced axon degeneration in F1 progeny when provided to hermaphrodites containing oocytes (P0 L4-adult) - the same functional period as UA (Figure 5C compared to 1F). Additionally, incubating P0 animals from L4 larvae-adult (16 hours) with S1P reduced axonal degeneration for two generations - revealing an epigenetic intergenerational effect of this lipid (Figure 5D-E). These data suggest that intestinal S1P is transported within the yolk to oocytes to promote PLM axonal health in subsequent generations. To examine whether S1P can undergo intestine-oocyte transport, we fed wild-type L4 larvae with heat-killed OP50 Escherichia coli containing 20 mM S1P-Fluorescein, a fluorescently labelled S1P analog (Figure 5F). Fluorescence was observed in the intestinal tract within 1h of feeding, suggesting that S1P-Fluorescein is not immediately metabolized (Figure S5C). After 16h of S1P-Fluorescein feeding, we detected fluorescence in proximal oocytes, suggesting yolk-dependent intestine-oocyte transport (Figure 5F). To determine the importance of intestine-oocyte S1P transport for preventing neurodegeneration, we knocked down rme-2 by RNAi in mec-17(ok2109); lon-2(e678) animals incubated with S1P. We found that S1P neuroprotection requires rme-2 (Figure 5G). These data reveal that S1P-dependent neuroprotection requires maternal information transfer from the intestine and oocyte through the low-density receptor RME-2 receptor.

ASAH-1 Expression is Regulated by the PQM-1/CEH-60 Transcription Factors

How does UA induce asah-1 expression in the intestine to enable intergenerational protection of the nervous system? As UA induces expression of the asah-1 transcriptional reporter (Figure 2F-G), we reasoned that transcription factors (TFs) related to intestinal stress or metabolic control may regulate asah-1 expression. We examined the asah-1 promoter for conserved TF binding motifs and surveyed publicly available ChIP-seq data for TFs exhibiting strong peaks upstream of the asah-1 coding sequence (Figure 6A and S6A). We identified ChIP-seq peaks for two TFs in the asah-1 promoter: PQM-1, a GATA zinc-finger TF, and CEH-60/PBX, a TALE class (Three Amino Acid Loop Extension) TF (Figure 6A and S6A). These ChIP-seq peaks coincide with putative binding sites we identified in silico (Figure 6A and S6A). PQM-1 and CEH-60 function in the intestine to balance transcriptional networks governing stress responses and nutrient supply to progeny ^{30, 31}. We therefore investigated the potential role of these TFs in controlling PLM axon degeneration and asah-1 expression.

To directly assess whether PQM-1/CEH-60 transcriptionally regulate endogenous asah-1, we generated a fluorescent reporter by inserting a f2A-gfp-h2B cassette immediately downstream of the asah-1 coding sequence using CRISPR-Cas9 (Figure 6A-B). Ribosomal skipping occurs at the viral f2A sequence, and thus independently translated GFP-H2B protein is visualized in nuclei (Figure 6B and S6B) ³². We detected GFP-H2B expression in anterior intestinal nuclei (except the most anterior nuclei pair), with weak/undetectable expression in the posterior intestine (Figure 6B and S6B). This anteriorly biased expression was also observed in the asah-1 transcriptional reporter (Figure 6B compared to S3), suggesting that spatial expression of asah-1 is transcriptionally regulated. Exposing the asah-1 endogenous reporter to UA induced GFP-H2B expression (Figure 6C and S7A), supporting our previous finding with the asah-1 transcriptional reporter (Figure 2F-G). We next crossed pqm-1(ok485) and ceh-60(ok1485) loss-of-function mutants into asah-1::f2A-gfp-h2B animals and measured GFP-H2B fluorescence in intestinal nuclei. Loss of PQM-1 elevated GFP-H2B expression and loss of either PQM-1 or CEH-60 prevented GFP-H2B induction by UA (Figure 6D). To examine the potential role of PQM-1 and CEH-60 in UA-induced neuroprotection, we knocked down their expression using RNAi (Figure 6E-F). ceh-60 and pqm-1 RNAi inhibited UA neuroprotection in mec-17(ok2109); lon-2(e678) animals (Figure 6E-F). Hence, PQM-1 and CEH-60 enable asah-1 induction and neuroprotection in response to UA exposure.

As PQM-1 and CEH-60 have key roles of in controlling metabolic homeostasis and yolk production ^{30, 31}, asah-1 regulation in these TF mutants may be secondary consequence of disrupted lipid homeostasis. Therefore, to directly examine the importance of PQM-1 and CEH-60 binding on asah-1 expression, we used CRISPR-Cas9 to mutate putative binding motifs that coincide with the PQM-1/CEH-60 ChIP peaks in the asah-1 promoter (Figure 6A and S6A). We found that mutating the PQM-1 binding motif strongly induced GFP-H2B intestinal expression, with mutation of the CEH-60 site having a weaker effect (Figure 6G and S7B). These data support a direct role for these TFs in asah-1 transcriptional repression and suggests that PQM-1/CEH-60 occupancy at the asah-1 promoter can control sphingolipid homeostasis. We also noticed that mutating the PQM-1 motif caused a posterior shift in GFP-H2B expression, revealing spatial regulation of intestinal expression (Figure 6G). As mutating the PQM-1 motif strongly increased expression from the asah-1 locus, we wondered if this was neuroprotective. When the asah-1::f2A-gfp-h2B (PQM-1 motif mutant) strain was crossed into mec-17(ok2109); lon-2(e678) animals, we observed robust reduction in PLM axon degeneration (Figure 6H). This reveals that de-repression of asah-1 transcription has a functionally relevant outcome for axonal health.

ASAH-1 Expression is Regulated Intergenerationally by S1P Levels

Ceramide hydrolysis is the only catabolic source of sphingosine, and therefore ceramidase (e.g. ASAH-1) activity is deemed a rate-limiting step in governing intracellular sphingosine and S1P levels ³³. We hypothesized that S1P intergenerational inheritance may be triggered by an imbalance in sphingolipid homeostasis, such as increased S1P levels. Thus, detection and recycling of S1P to sphingosine and then ceramide may feedback to induce asah-1 expression in progeny. To explore this possibility, we exposed asah-1::f2A-gfp-h2B P0 animals to S1P for 16 h (L4 - young adult) and measured GFP-H2B intestinal expression in subsequent generations. Remarkably, GFP-H2B levels were increased in F1 and F2 progeny following P0 S1P exposure (Figure 6I), establishing a regulatory mechanism for S1P-induced intergenerational inheritance.

Here, we show that acute supplementation to the maternal diet (as short at 16 hr) can prevent axon degeneration intergenerationally in C. elegans (Figure 7). Dietary supplementation with UA induces intestinal expression of the asah-1 gene, which encodes a rate-limiting enzyme in the sphingolipid salvage pathway. Our genetic evidence reveals that induction of either the de novo or salvage sphingolipid pathway prevents axon degeneration, and that expression of sphingosine kinase expression is required for promoting axon health. The metabolite generated by sphingosine kinase is S1P. We found that supplementing the diet of reproductive hermaphrodites with S1P also prevented axon degeneration intergenerationally. The neuroprotection provided by UA and S1P was dependent on RME-2, a low-density lipoprotein receptor that transports intestinally derived yolk into oocytes ¹⁸. Supporting a role for intestinal yolk in neuroprotection, we observed that a fluorescently labelled S1P analog (S1P-Fluoroscein) is transported from the intestine to oocytes. Our analysis of the asah-1 promoter revealed putative binding motifs for the PQM-1 (GATA zinc finger) and CEH-60 (TALE class) TFs that coincide with published ChIP-seq peaks. PQM-1 and CEH-60 are required for intestinal lipid homeostasis ^{30, 31}, and we found that both TFs are required for UA neuroprotection. Mutating the PQM-1 binding motif in the asah-1 promoter caused increased endogenous asah-1 expression in the intestine – suggesting disruption of a repressive element. Remarkably, mutation of this PQM-1 binding motif was sufficient to reduce axon degeneration, further confirming the critical role for asah-1 regulation of neuronal health.

Spatial Regulation of Sphingolipid Pathway Genes Impact Neuronal Health and Development

Our work shows that neuronal development and health require correct spatial regulation of sphingolipid biosynthetic enzymes. In the mec-17; lon-2 neurodegeneration model, elevated intestinal asah-1 expression reduced PLM axon degeneration in a sphingosine kinase dependant manner. In contrast, overexpressing asah-1 in the nervous system caused developmental axon outgrowth defects in the ALM and PLM neurons. However, we did not observe overt motility defects in animals overexpressing asah-1 in neurons, suggesting that axon outgrowth/guidance is largely intact, and that disrupted sphingolipid homeostasis has context-specific effects. Analysis of embryonic single cell sequencing reveals that asah-1 is expressed at low levels in ALM/PLM neurons, as are other members of the sphingolipid salvage pathway such as sphk-1 ²². In contrast, expression of sptl-1 that encodes the rate-limiting enzyme of the sphingolipid de novo pathway was undetected in these neurons. Perhaps asah-1 overexpression in the embryonic ALM/PLM neurons causes depletion of ceramide and/or other ceramide derivates that cannot be replenished by the de novo pathway. Such disruption of ceramide homeostasis in neurons could affect a plethora of signalling pathways in addition to membrane health and therefore disrupt axon outgrowth.

Our data show that maternal S1P transport to oocytes reduces neurodegeneration in axons with impaired cytoskeletal function (mec-17; lon-2 mutant). We propose that elevated S1P in the oocyte/embryo influences the axonal environment to limit axon fragility. The important roles of sphingolipid homeostasis in plasma membrane fluidity, lipid raft integrity and the molecular trafficking may be central to the protective role S1P plays in this context ^{34, 35}. Disrupted sphingolipid metabolism has been implicated in multiple neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) ³⁶. The distinct autonomous and non-autonomous effects of sphingolipid homeostasis we revealed in C. elegans may also be relevant in these disease contexts.

Sphingolipid Regulation of Intergenerational Inheritance

The neuroprotective effect of intestinal asah-1 is dependent on expression of sphingosine kinase (SPHK-1), an enzyme that phosphorylates sphingosine to generate S1P. In support of this maternally supplied S1P is transferred from the intestine to oocytes to protect axons. Maternal S1P is likely transported within low-density yolk lipoproteins, as removal of the RME-2 yolk receptor, expressed on oocytes, inhibits S1P neuroprotection. In mammals, plasma lipoproteins also carry S1P, highlighting a conserved mode of carriage ³⁷.

How does S1P act intergenerationally? We noticed that intestinal asah-1 expression was elevated for two generations following acute exposure of reproductive stage hermaphrodites with S1P. This suggests that mechanisms exist within intestinal cells to detect changes in sphingolipid levels, including S1P, and to alter the expression of sphingolipid pathway genes (e.g. asah-1) in response. The increased level of asah-1 in the intestine of F1 animals could generate high S1P that would be transferred to the F2 generation to maintain the intergenerational effect. We suggest that a threshold of intestinal S1P level must be maintained to initiate asah-1 upregulation and this threshold may be lost over time – hence the reason for the intergenerational rather than transgenerational effect. At the level of asah-1 gene regulation, subtle changes in the subcellular localisation or promoter occupancy of asah-1 regulators, including the PQM-1 and CEH-60 TFs, could be responsible. Indeed, PQM-1 nuclear localization is responsive to certain stressors and perturbed insulin growth factor signalling ³¹.

In mammals, periods of prenatal and postnatal interactions enable mothers to influence offspring ³⁸. Alterations in reproductive behavior of offspring permits transmission of these effects to succeeding generations ³⁸ We have shown that transmission of a lipid metabolite in the yolk can intergenerationally protect specific neurons in C. elegans. We suggest that our findings point to a broader underlying role for metabolism and metabolic gene expression in controlling intergenerational effects across species.

ACKNOWLEDGEMENTS

We thank Brent Neumann and members of the Pocock laboratory for comments on the manuscript. Some strains used in this study were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research and Infrastructure Programs (P40 OD10440) and the National BioResource Project (NBRP) Japan. This work was supported by the following funding: National Health and Medical Research Council grants GNT1105374, GNT1137645 and GNT2000766 (RP); Apex Biotech Research Pty Ltd donation 281589068 (ZX)

AUTHOR CONTRIBUTIONS

Conceptualization: WW, ZX, RP; Methodology: WW, JL, RP; Investigation: WW, TS, XC, QF, RC, RP; Visualization: WW, XC, RC, RP; Funding acquisition: ZX, RP; Project administration: WW, RP; Supervision: JL, ZX, RP; Writing – original draft: RP; Writing – review & editing: WW, TS, XC, QF, RC, JL, ZX, RP.

DECLARATION OF INTERESTS

The authors declare no competing interests.

DATA AND MATERIALS AVAILABLITY

All data are available in the main text or the supplementary materials.

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Intergenerational Neuroprotection by an Intestinal Sphingolipid in Caenorhabditis elegans

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