Sathyanarayanan V. Puthanveettil

Characterization of Small RNAs in Aplysia Reveals a Role for miR-124 in Constraining Synaptic Plasticity through CREB

Memory storage and memory-related synaptic plasticity rely on precise spatiotemporal regulation of gene expression. To explore the role of small regulatory RNAs in learning-related synaptic plasticity, we carried out massive parallel sequencing to profile the small RNAs of Aplysia californica. We identified 170 distinct miRNAs, 13 of which were novel and specific to Aplysia. Nine miRNAs were brain enriched, and several of these were rapidly downregulated by transient exposure to serotonin, a modulatory neurotransmitter released during learning. Further characterization of the brain-enriched miRNAs revealed that miR-124, the most abundant and well-conserved brain-specific miRNA, was exclusively present presynaptically in a sensory-motor synapse where it constrains serotonin-induced synaptic facilitation through regulation of the transcriptional factor CREB. We therefore present direct evidence that a modulatory neurotransmitter important for learning can regulate the levels of small RNAs and present a role for miR-124 in long-term plasticity of synapses in the mature nervous system.

Neuronal Transcriptome of Aplysia: Neuronal Compartments and Circuitry

Molecular analyses of Aplysia, a well-established model organism for cellular and systems neural science, have been seriously handicapped by a lack of adequate genomic information. By sequencing cDNA libraries from the central nervous system (CNS), we have identified over 175,000 expressed sequence tags (ESTs), of which 19,814 are unique neuronal gene products and represent 50%-70% of the total Aplysia neuronal transcriptome. We have characterized the transcriptome at three levels: (1) the central nervous system, (2) the elementary components of a simple behavior: the gill-withdrawal reflex-by analyzing sensory, motor, and serotonergic modulatory neurons, and (3) processes of individual neurons. In addition to increasing the amount of available gene sequences of Aplysia by two orders of magnitude, this collection represents the largest database available for any member of the Lophotrochozoa and therefore provides additional insights into evolutionary strategies used by this highly successful diversified lineage, one of the three proposed superclades of bilateral animals.

Sustained CPEB-Dependent Local Protein Synthesis Is Required to Stabilize Synaptic Growth for Persistence of Long-Term Facilitation in Aplysia

The time course of the requirement for local protein synthesis in the stabilization of learning-related synaptic growth and the persistence of long-term memory was examined using Aplysia bifurcated sensory neuron-motor neuron cultures. We find that, following repeated pulses of serotonin (5-HT), the local perfusion of emetine, an inhibitor of protein synthesis, or a TAT-AS oligonucleotide directed against ApCPEB blocks long-term facilitation (LTF) at either 24 or 48 hr and leads to a selective retraction of newly formed sensory neuron varicosities induced by 5-HT. By contrast, later inhibition of local protein synthesis, at 72 hr after 5-HT, has no effect on either synaptic growth or LTF. These results define a specific stabilization phase for the storage of long-term memory during which newly formed varicosities are labile and require sustained CPEB-dependent local protein synthesis to acquire the more stable properties of mature varicosities required for the persistence of LTF.

Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy

We report here the design, synthesis and application of pyrene binary oligonucleotide probes for selective detection of cellular mRNA. The detection strategy is based on the formation of a fluorescent excimer when two pyrene groups are brought into close proximity upon hybridization of the probes with the target mRNA. The pyrene excimer has a long fluorescence lifetime (>40 ns) compared with that of cellular extracts (∼7 ns), allowing selective detection of the excimer using time-resolved emission spectra (TRES). Optimized probes were used to target a specific region of sensorin mRNA yielding a strong excimer emission peak at 485 nm in the presence of the target and no excimer emission in the absence of the target in buffer solution. While direct fluorescence measurement of neuronal extracts showed a strong fluorescent background, obscuring the detection of the excimer signal, time-resolved emission measurements indicated that the emission decay of the cellular extracts is ∼8 times faster than that of the pyrene excimer probes. Thus, using TRES of the pyrene probes, we are able to selectively detect mRNA in the presence of cellular extracts, demonstrating the potential for application of pyrene excimer probes for imaging mRNAs in cellular environments that have background fluorescence.

Neurexin-neuroligin transsynaptic interaction mediates learning-related synaptic remodeling and long-term facilitation in aplysia.

Neurexin and neuroligin, which undergo heterophilic interactions with each other at the synapse, are mutated in some patients with autism spectrum disorder, a set of disorders characterized by deficits in social and emotional learning. We have explored the role of neurexin and neuroligin at sensory-to-motor neuron synapses of the gill-withdrawal reflex in Aplysia, which undergoes sensitization, a simple form of learned fear. We find that depleting neurexin in the presynaptic sensory neuron or neuroligin in the postsynaptic motor neuron abolishes both long-term facilitation and the associated presynaptic growth induced by repeated pulses of serotonin. Moreover, introduction into the motor neuron of the R451C mutation of neuroligin-3 linked to autism spectrum disorder blocks both intermediate-term and long-term facilitation. Our results suggest that activity-dependent regulation of the neurexin-neuroligin interaction may govern transsynaptic signaling required for the storage of long-term memory, including emotional memory that may be impaired in autism spectrum disorder.

A new component in synaptic plasticity: upregulation of kinesin in the neurons of the gill-withdrawal reflex.

To explore how gene products, required for the initiation of synaptic growth, move from the cell body of the sensory neuron to its presynaptic terminals, and from the cell body of the motor neuron to its postsynaptic dendritic spines, we have investigated the anterograde transport machinery in both the sensory and motor neurons of the gill-withdrawal reflex of Aplysia. We found that the induction of long-term facilitation (LTF) by repeated applications of serotonin, a modulatory transmitter released during learning in Aplysia, requires upregulation of kinesin heavy chain (KHC) in both pre- and postsynaptic neurons. Indeed, upregulation of KHC in the presynaptic neurons alone is sufficient for the induction of LTF. However, KHC is not required for the persistence of LTF. Thus, in addition to transcriptional activation in the nucleus and local protein synthesis at the synapse, our studies have identified a third component critical for long-term learning-related plasticity: the coordinated upregulation of kinesin-mediated transport.

Consolidation of learning strategies during spatial working memory task requires protein synthesis in the prefrontal cortex

Working memory is a temporary memory store where information is held briefly until the appropriate behavior is produced. However, the improvement in the performance of working memory tasks with practice over days points to the existence of a long-lasting component associated with learning strategies that lead to optimal performance. Here we show that the improvement in the performance of mice in a radial maze working memory task required the integrity of the medial prefrontal cortex (mPFC). We further demonstrate that this improvement of working memory performance requires the synthesis of de novo proteins in the mPFC. We suggest that in addition to storing memory briefly the mPFC is also involved in the consolidation and storage of the long-term learning strategies used in working memory.

Characterization of prion-like conformational changes of the neuronal isoform of Aplysia CPEB

The neuronal isoform of cytoplasmic polyadenylation element–binding protein (CPEB) is a regulator of local protein synthesis at synapses and is critical in maintaining learning-related synaptic plasticity in Aplysia. Previous studies indicate that the function of Aplysia CPEB can be modulated by conversion to a stable prion-like state, thus contributing to the stabilization of long-term memory on a molecular level. Here, we used biophysical methods to demonstrate that Aplysia CPEB, like other prions, undergoes a conformational switch from soluble α-helix–rich oligomer to β-sheet–rich fiber in vitro. Solid-state NMR analyses of the fibers indicated a relatively rigid N-terminal prion domain. The fiber form of Aplysia CPEB showed enhanced binding to target mRNAs as compared to the soluble form. Consequently, we propose a model for the Aplysia CPEB fibers that may have relevance for functional prions in general.Although significant knowledge of cellular and molecular mechanisms underlying the acquisition and early storage of implicit and explicit long-term memory has been gained, the mechanisms by which memories are maintained for long periods of time are still not fully understood. Because proteins normally have relatively short half-lives, of hours or days, the question remains: How can the change in molecular composition of a synapse be maintained for long periods of time, as is required for long-term memory? We previously found one answer to this conundrum in a work describing a prion-like regulator of local protein synthesis at the synapse in the marine snail Aplysia californica: the cytoplasmic polyadenylation element–binding protein Aplysia CPEB. This provided physiological evidence that the prion-like properties of Aplysia CPEB might explain the self-sustained, continuous molecular turnover at the synapse.

Pharmacological Selectivity Within Class I Histone Deacetylases Predicts Effects on Synaptic Function and Memory Rescue

Histone deacetylases (HDACs) are promising therapeutic targets for neurological and psychiatric disorders that impact cognitive ability, but the relationship between various HDAC isoforms and cognitive improvement is poorly understood, particularly in mouse models of memory impairment. A goal shared by many is to develop HDAC inhibitors with increased isoform selectivity in order to reduce unwanted side effects, while retaining procognitive effects. However, studies addressing this tack at the molecular, cellular and behavioral level are limited. Therefore, we interrogated the biological effects of class I HDAC inhibitors with varying selectivity and assessed a subset of these compounds for their ability to regulate transcriptional activity, synaptic function and memory. The HDAC-1, -2, and -3 inhibitors, RGFP963 and RGFP968, were most effective at stimulating synaptogenesis, while the selective HDAC3 inhibitor, RGFP966, with known memory enhancing abilities, had minimal impact. Furthermore, RGFP963 increased hippocampal spine density, while HDAC3 inhibition was ineffective. Genome-wide gene expression analysis by RNA sequencing indicated that RGFP963 and RGFP966 induce largely distinct transcriptional profiles in the dorsal hippocampus of mature mice. The results of bioinformatic analyses were consistent with RGFP963 inducing a transcriptional program that enhances synaptic efficacy. Finally, RGFP963, but not RGFP966, rescued memory in a mouse model of Alzheimer’s Disease. Together, these studies suggest that the specific memory promoting properties of class I HDAC inhibitors may depend on isoform selectivity and that certain pathological brain states may be more receptive to HDAC inhibitors that improve network function by enhancing synapse efficacy.

A strategy to capture and characterize the synaptic transcriptome

Here we describe a strategy designed to identify RNAs that are actively transported to synapses during learning. Our approach is based on the characterization of RNA transport complexes carried by molecular motor kinesin. Using this strategy in Aplysia, we have identified 5,657 unique sequences consisting of both coding and noncoding RNAs from the CNS. Several of these RNAs have key roles in the maintenance of synaptic function and growth. One of these RNAs, myosin heavy chain, is critical in presynaptic sensory neurons for the establishment of long-term facilitation, but not for its persistence.