Riassunto analitico
In this thesis, it has been identified the transcripts of putative enzymes of the Endocannabinoid pathway in
the central ring ganglia of the pond snail Lymnaea stagnalis, a valid model organism for Translational
Medicine.
The Endocannabinoid pathway shows a high level of conservation between vertebrates and invertebrates
and plays a key role in neuroplasticity, neuroprotection, stress regulation, energy homeostasis, and
immune responses, acting as pivotal player between the brain and peripheral body regions.
To date, the primary organisms utilized for studying the Endocannabinoid pathway have predominantly
been primates and rodents. However, research employing invertebrate models has shown notable
experimental efficiency because of shorter experiment durations and lower animal care costs, facilitated by
their rapid generation times, high offspring numbers, and greater experimental manipulability. While
invertebrates cannot fully replace mammalian models in preclinical studies, the characterization of the
Endocannabinoid pathway in these models could be extremely useful in unraveling its complexity while
translating results to mammals.
Although the genome and transcriptome of Lymnaea have been sequenced, their characterization has not
been completed thus far. Instead of generating a new transcriptome, we compared a recent L. stagnalis
genome assembly with annotated proteins from Homo sapiens, Mus musculus, and Biomphalaria glabrata,
a molluscan model whose genome and transcriptome have been characterized. This approach allowed the
identification of L. stagnalis contigs corresponding to the enzymes of the Endocannabinoid pathway in the
annotated genome of B. glabrata.
To identify transcripts within these contigs, we retrieved putative exonic sequences and visualized RNA-seq
reads on the L. stagnalis genome with Integrative Genomics Viewer. Subsequently we aligned these
putative exonic sequences with the available transcriptome shotgun assembly (TSA) of L. stagnalis central
nervous system transcriptome. This method allowed the identification of one specific transcript per enzyme
of the Endocannabinoid pathway: FX181219.1 (Lym-DAGL-like), FX189644.1 (Lym-MAGL-like), FX191515.1
(Lym-NAPE-PLD-like), and FX195089.1 (Lym-FAAH-like).
All the identified potential transcripts of all endocannabinoid system enzymes contained open reading
frames whose identity was confirmed by comparing each sequence with amino acid sequences of
corresponding enzymes from H. sapiens, M. musculus, A. californica, and B. glabrata. In H. sapiens, we
identified four orthologs exhibiting an average homology of approximately 40% with the corresponding L.
stagnalis sequences. Similar degrees of homology were observed with M. musculus sequences, while higher
homology was found with mollusc sequences (55% for A. californica and 65% for B. glabrata). Phylogenetic
analyses revealed a high level of conservation between L. stagnalis sequences of Lym-DAGL-like, Lym-FAAH-
like, and Lym-NapePLD-like to those of Mollusca. Interestingly, Lym-MAGL-like amino acid sequences were
closely related to those of humans.
The transcripts predicted in silico were then validated using Sanger sequencing and the expression patterns
of the enzymes of the Endocannabinoid pathway were quantified.
This study represents the first identification of putative enzymes of the Endocannabinoid pathway in a
molluscan model organism.
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