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Abstract Dysregulation of the kynurenine pathway (KP) is implicated in many human diseases and disorders, from immunological, metabolic, neurodegenerative, and neuropsychiatric conditions to cancer, and represents an appealing target for new therapeutic approaches. In this intricate scenario, invertebrates, like Lymnaea stagnalis (LS), provide a flexible tool to unravel the complexity of the KP. Starting from the available LS genome and transcriptome, we identified putative transcripts of all KP enzymes containing an ORF; each predicted protein possessed a high degree of sequence conservation to known orthologues of other invertebrate and vertebrate model organisms. Sequences were confirmed by qualitative PCR and sequencing. At the same time, the qRT-PCR analysis revealed that Lym IDO-like, Lym TDO-like, Lym AFMID-like, Lym KMO-like, Lym AADAT-like, Lym KYAT I/III-like, Lym KYNU-like, Lym HAAO-like, and Lym ACMSD-like showed widespread tissue expression. Then, tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxy-kynurenine, xanthurenic acid, picolinic acid, and quinolinic acid were identified in the hemolymph of LS by UHPLC-Q exactive mass spectrometer. Our study provides the most thorough characterization to date of the KP in an invertebrate model, supporting the value of LS for future functional studies of this pathway at the cellular, synaptic, and behavioral levels.

The endocannabinoid system (ECS) plays an important role in neuroprotection, neuroplasticity, energy balance, modulation of stress, and inflammatory responses, acting as a critical link between the brain and the body's peripheral regions, while also offering promising potential for novel therapeutic strategies. Unfortunately, in humans, pharmacological inhibitors of different ECS enzymes have led to mixed results in both preclinical and clinical studies. As the ECS has been highly conserved throughout the eukaryotic lineage, the use of invertebrate model organisms like the pond snail Lymnaea stagnalis may provide a flexible tool to unravel unexplored functions of the ECS at the cellular, synaptic, and behavioral levels. In this study, starting from the available genome and transcriptome of L. stagnalis, we first identified putative transcripts of all ECS enzymes containing an open reading frame. Each predicted protein possessed a high degree of sequence conservation to known orthologues of other invertebrate and vertebrate organisms. Sequences were confirmed by qualitative PCR and sequencing. Then, we investigated the transcriptional effects induced by different stress conditions (i.e., bacterial LPS injection, predator scent, food deprivation, and acute heat shock) on the expression levels of the enzymes of the ECS in Lymnaea's central ring ganglia. Our results suggest that in Lymnaea as in rodents, the ECS is involved in mediating inflammatory and anxiety‐like responses, promoting energy balance, and responding to acute stressors. To our knowledge, this study offers the most comprehensive analysis so far of the ECS in an invertebrate model organism. [ABSTRACT FROM AUTHOR]

Almost all individuals with Down's syndrome (DS) show the characteristic neuropathological features of Alzheimer's disease (AD) by the age of 40, yet not every individual with DS experiences symptoms of AD later in life. Similar to neurotypical developing subjects, AD in people with DS lasts for a long preclinical phase in which biomarkers follow a predictable order of changes. Hence, a prolonged asymptomatic period precedes the onset of dementia, underscoring the importance of identifying new biomarkers for the early detection and monitoring of cognitive decline in individuals with DS. Blood-based biomarkers may offer an alternative non-invasive strategy for the detection of peripheral biological alterations paralleling nervous system pathology in an early phase of the AD continuum. In the last few years, a strong neurobiological link has been demonstrated between the deficit of transforming growth factor-β1 (TGF-β1) levels, an anti-inflammatory cytokine endowed with neuroprotective activity, and early pro-inflammatory processes in the AD brain. In this clinical prospective observational study, we found significant lower plasma TGF-β1 concentrations at the first neuropsychological evaluation (baseline = T0) both in young adult DS individuals (19-35 years) and older DS subjects without AD (35-60 years) compared to age- and sex-matched healthy controls. Interestingly, we found that the lower TGF-β1 plasma concentrations at T0 were strongly correlated with the following cognitive decline at 12 months. In addition, in young individuals with DS, we found, for the first time, a negative correlation between low TGF-β1 concentrations and high TNF-α plasma concentrations, a pro-inflammatory cytokine that is known to be associated with cognitive impairment in DS individuals with AD. Finally, adopting an ex vivo approach, we found that TGF-β1 concentrations were reduced in parallel both in the plasma and in the peripheral blood mononuclear cells (PBMCs) of DS subjects, and interestingly, therapeutic concentrations of fluoxetine (FLX) applied to cultured PBMCs (1 µM for 24 h) were able to rescue TGF-β1 concentrations in the culture media from DS PBMCs, suggesting that FLX, a selective serotonin reuptake inhibitor (SSRI) endowed with neuroprotective activity, might rescue TGF-β1 concentrations in DS subjects at higher risk to develop cognitive decline. [ABSTRACT FROM AUTHOR]

Different populations of organisms occurring across varying thermal regimes show diversity in responses to heat stress. We use a "common garden experimental" approach designed to deal with phenotypic plasticity to study in Lymnaea stagnalis (Linnaeus, 1758) the behavioural and molecular responses to a heat shock in laboratory-inbred snails (W-strain) and freshly collected snails (Stony strain) from ponds. In the W-strain, which has been reared under standardized temperatures for generations, the exposure to 30 °C for 1 h (heat shock, HS) when experienced after a novel "taste" results in a taste-specific aversion known as the "Garcia effect". This learned avoidance requires the upregulation of heat shock proteins (HSPs). In contrast, freshly collected Stony strain, which experiences temperature fluctuations regularly, does not exhibit a Garcia effect. Here, we found that (1) Stony-strain snails have higher basal mRNA levels of HSPs than W-strain ones; (2) in the W-strain, the training procedure to cause the Garcia effect upregulates the mRNA levels of HSPs and key neuroplasticity-related genes such as CREB1 and GRIN1; (3) in Stony-strain snails, the same training procedure fails to alter the mRNA levels of those targets. These data suggest that Stony-strain snails do not perceive the HS as a stressor because of the higher HSP basal mRNA levels, which may confer a higher thermal tolerance. [ABSTRACT FROM AUTHOR]

Introduction: The Garcia effect, a solid learning paradigm, was used to investigate the molecular and behavioral effects induced by different lengths of fasting on the cognitive functions in the pond snail Lymnaea stagnalis, a valid model system. Methods: Three experimental groups were used: moderately hungry snails, food-deprived for 1 day (D1 snails), severely hungry snails (D5 snails), fasting for 5 days, and satiated snails with ad libitum access to food (AL snails). In the Garcia effect, a single pairing of an appetitive stimulus with a heat stressor results in a learned taste-specific negative hedonic shift. D5 snails were injected with bovine insulin and D1 snails with the insulin receptor antibody (Ab). As a control group, AL snails were injected with saline. Gene expression analyses were performed by real-time PCR in snails' central nervous system (CNS). Results: AL snails are "average learners," D1 snails are the best performers, whereas the D5 ones do not show the Garcia effect. Severely fasting snails injected with insulin 3 h before the training procedure show the Garcia effect, whereas injecting 1-day fasting snails with insulin receptor Ab blocks their ability to express memory. The differences in memory performances are associated with changes in the expression levels of selected targets involved in neuronal plasticity, energy homeostasis, and stress response. Discussion: Our results suggest that short-term fasting creates an optimal internal state in L. stagnalis' CNS, allowing a spike in insulin release and an upregulation of genes involved in neuroplasticity. Long-term fasting, instead, upregulates genes involved in energy homeostasis and animal survival. [ABSTRACT FROM AUTHOR]