(-)- Gossypol Inhibition of Musashi-Mediated Forgetting Improves Memory and Age-Dependent Memory Decline in Caenorhabditis elegans.

Musashi RNA-binding proteins (MSIs) retain a pivotal role in stem cell maintenance, tumorigenesis, and nervous system development. Recently, we showed in C. elegans that Musashi (MSI-1) actively promotes forgetting upon associative learning via a 3'UTR-dependent translational expression of the Arp2/3 actin branching complex. Here, we investigated the evolutionary conserved role of MSI proteins and the effect of their pharmacological inhibition on memory. Expression of human Musashi 1 (MSI1) and Musashi 2 (MSI2) under the endogenous Musashi promoter fully rescued the phenotype of msi-1(lf) worms. Furthermore, pharmacological inhibition of human MSI1 and MSI2 activity using (-)- gossypol resulted in improved memory retention, without causing locomotor, chemotactic, or learning deficits. No drug effect was observed in msi-1(lf) treated worms. Using Western blotting and confocal microscopy, we found no changes in MSI-1 protein abundance following (-)- gossypol treatment, suggesting that Musashi gene expression remains unaltered and that the compound exerts its inhibitory effect post-translationally. Additionally, (-)- gossypol suppressed the previously seen rescue of the msi-1(lf) phenotype in worms expressing human MSI1 specifically in the AVA neuron, indicating that (-)- gossypol can regulate the Musashi pathway in a memory-related neuronal circuit in worms. Finally, treating aged worms with (-)- gossypol reversed physiological age-dependent memory decline. Taken together, our findings indicate that pharmacological inhibition of Musashi might represent a promising approach for memory modulation.

Phosphorylation of MSI-1 is implicated in the regulation of associative memory in Caenorhabditis elegans.

The Musashi family of RNA-binding proteins controls several biological processes including stem cell maintenance, cell division and neural function. Previously, we demonstrated that the C. elegans Musashi ortholog, msi-1, regulates forgetting via translational repression of the Arp2/3 actin-branching complex. However, the mechanisms controlling MSI-1 activity during the regulation of forgetting are currently unknown. Here we investigated the effects of protein phosphorylation on MSI-1 activity. We showed that MSI-1 function is likely controlled by alterations of its activity rather than its expression levels. Furthermore, we found that MSI-1 is phosphorylated and using mass spectrometry we identified MSI-1 phosphorylation at three residues (T18, S19 and S34). CRISPR-based manipulations of MSI-1 phosphorylation sites revealed that phosphorylation is necessary for MSI-1 function in both short- and long-term aversive olfactory associative memory. Thus, our study provides insight into the mechanisms regulating memory-related MSI-1 activity and may facilitate the development of novel therapeutic approaches.

Genome-Wide Temporal Expression Profiling in Caenorhabditis elegans Identifies a Core Gene Set Related to Long-Term Memory.

The identification of genes related to encoding, storage, and retrieval of memories is a major interest in neuroscience. In the current study, we analyzed the temporal gene expression changes in a neuronal mRNA pool during an olfactory long-term associative memory (LTAM) in Caenorhabditis elegans hermaphrodites. Here, we identified a core set of 712 (538 upregulated and 174 downregulated) genes that follows three distinct temporal peaks demonstrating multiple gene regulation waves in LTAM. Compared with the previously published positive LTAM gene set (Lakhina et al., 2015), 50% of the identified upregulated genes here overlap with the previous dataset, possibly representing stimulus-independent memory-related genes. On the other hand, the remaining genes were not previously identified in positive associative memory and may specifically regulate aversive LTAM. Our results suggest a multistep gene activation process during the formation and retrieval of long-term memory and define general memory-implicated genes as well as conditioning-type-dependent gene sets.SIGNIFICANCE STATEMENT The identification of genes regulating different steps of memory is of major interest in neuroscience. Identification of common memory genes across different learning paradigms and the temporal activation of the genes are poorly studied. Here, we investigated the temporal aspects of Caenorhabditis elegans gene expression changes using aversive olfactory associative long-term memory (LTAM) and identified three major gene activation waves. Like in previous studies, aversive LTAM is also CREB dependent, and CREB activity is necessary immediately after training. Finally, we define a list of memory paradigm-independent core gene sets as well as conditioning-dependent genes.