The discovery that piRNAs exist outside the germline, in several major organs of Aplysia, but significantly in the nervous system, suggests much broader roles for piRNAs than has been previously appreciated. In addition to their presence and in certain cases enrichment in neurons, Aplysia piRNAs are unique from those previously described in that they derive from hotspots in the genome where they are abundantly expressed and, notably, several piRNAs are regulated by neuromodulators important for learning-related synaptic plasticity, suggesting functions in memory storage.
An understanding of the role of piRNAs in the epigenetic regulation of long-term memory is significant for several reasons. First, the role of epigenetic modifications in differentiated cells, especially in adult neurons, has been controversial. It is commonly thought that changes in gene expression during development are permanent, but that they are not permanent in adult neurons where the plastic nature of synaptic connections, by definition, requires bi-direction and reversible changes in gene expression. In recent years, the identification of DNA demethylase activity in adult neurons (Baretto et al. 2007
, Rai et al. 2008
, Ma et al. 2009
) brought forth the possibility that epigenetic changes in the adult brain may not necessarily be permanent, but may simply be more long-lasting, and more permanent than the other known modifications so far described. Subsequent studies have identified individual gene loci that are methylated in response to neurotransmitter activity, though the time course of onset and persistence of methylation are unclear and require further study. Our study provides a piRNA-mediated mechanism for epigenetic regulation in neurons, and further, explores the electrophysiological properties of DNA Methyltransferase and of Piwi in synaptic plasticity.
The findings that the piwi/piRNA complex regulates the CREB2 promoter by DNA methylation in an activity-dependent manner provides an attractive explanation for how neurons translate transient stimuli into stable internal representations, and is consistent with several earlier studies that show a role for epigenetic regulation in memory (Miller and Sweatt 2007
, Miller et al. 2010
, Feng et al. 2010
). Our data on the variability in baseline CREB2 methylation levels suggests further that each neuron may have a different basal level of CREB2 expression, which reflects its experience and immediate history. This would be consistent with earlier observations showing that variations in baseline levels of CREB1 across populations of neurons in the amygdala determine the sequence in which these neurons are recruited for memory and for recall (Han et al. 2007
). Since CREB2 is antagonistic to CREB1, long-lasting changes in CREB2 levels could set up this CREB1 distribution in neuronal cells, based on experience, which in turn can dictate which neurons are already holding a memory trace and which neurons are readily drawn into new memory traces (Han et al. 2007
, Won et al. 2008
). The likelihood that CREB2 set-points within a neuron can dictate its functional capacity for both memory and flexibility is further supported by a ubiquitin ligase over-expressing mouse model that is phenotypically much smarter than wild-type mice (Pavlopoulos et al. 2011
), and these mice show significantly reduced baseline CREB2 expression when compared with wildtype mice (Unpublished Observations, Pavlopoulos et a. 2011
The discussion above, however, does not address the question as to how piwi mediated transcriptional, and therefore cell-wide changes in neurons (intrinsic plasticity), effectively mediate synapse-specific events (synaptic plasticity)? As studies previously have emphasized, it is likely that both forms of plasticity co-exist such that one can fine-tune the other, but it is also possible, that in certain contexts the two exist entirely independently. While synaptic plasticity affords orders of magnitude more computational power and is therefore ideal for storage of explicit memories requiring attention to detail, intrinsic plasticity such as those driven by piRNA mediated epigenetics has the advantage of priming memories and allowing for robust generalized learning where the same association rules are applicable to experiential learning in various contexts. Since human life is characterized by a great deal of habit-formation and repetition based associative learning, the use of intrinsic plasticity alone in some parts of the human brain may turn out to be an efficient method for this type of memory storage.
Future work on the role of small RNAs in learning and memory should provide further insight into the varying roles of miRNAs versus piRNAs. While notable exceptions exist (Wayman et al. 2008
, Fiore et al. 2009
), we and others have previously found a rapid turn-over of several neuronal miRNAs in response to neuromodulators and neuronal activity (Rajasethupathy et al. 2009
, Krol et al. 2010
), which contrasts to the slow but more enduring up-regulation of the few neuronal piRNAs observed in this study. In addition, while aca-miR-124 (Rajasethupathy et al. 2009
) and aca-miR-22 (In Preparation) constrain serotonin dependent long-term facilitation, piwi-dependent piR-F enhances it. We currently have very few cases from which to draw generalizable conclusions, but future large-scale studies of small RNA function in neurons may highlight the possible existence of two distinct classes of small RNAs that are bi-directionally regulated by neuromodulators, and that act on a functionally segregated population of targets, to effect either facilitation or constraint on memory related synaptic plasticity. Further studies would also benefit from genome wide analysis of piRNA/piwi occupied promoter regions during serotonin-mediated synaptic plasticity to obtain a more complete picture of the epigenetic landscape during memory. One attractive possibility is that piRNAs are directed only toward inhibitors of plasticity, and that with each repeated training trial (either behavioral training or pulses of serotonin) the promoters of more inhibitory genes are silenced, such that eventually the cell is maximally primed and excitable, allowing for the strongest associative memories. Finally, future experiments with chromatin IP of RNA Polymerase and/or of piwi at the CREB2 locus would greatly increase our understanding of the mechanisms governing piwi dependent methylation. It would substantiate the idea that piwi is recruited to CREB2 in an activity dependent manner, and further that methylation of the promoter is directly responsible for the observed reduction in transcription of the gene. It is also possible that other small RNAs play a role in epigenetic regulation during plasticity. Irrespective of their biogenesis properties, small RNAs confer versatile sequence specificity to mechanisms of gene regulation, and therefore, any small RNA that evolves functionality for its guide protein to recruit methylation elements to the target promoter could prove equally effective. It is possible therefore, that one of the many rapidly multiplying classes of nuclear small RNAs take over the same task in other species.
In summary, we find that piwi/piRNAs control the activity dependent epigenetic regulation of the transcription factor CREB2, which may prove to be an important and general mechanism of small RNA mediated long-lasting regulation of gene-expression in neurons that contributes to long-term memory storage. This initial study compels the further exploration of a genome-wide approach toward understanding the extent of small-RNA-mediated epigenetic regulation in neurons during learning and memory.