Mitochondrial biology in Drosophila, mouse and hiPSC-derived neurons
Mitochondria participate in many different physiological processes in neurons; most notable is the synthesis of ATP to provide energy for the demanding cellular processes underlying neuron function that include synaptic transmission, action potential generation, and maintenance of membrane potentials. In addition, they function as calcium buffers through the action of the calcium uniporter that localizes to mitochondria. Current literature indicates that mitochondria become defective in the vast majority of neurological and psychiatric disorders.
We probed the function of the calcium uniporter in Drosophila memory formation by knocking down two of its key components, MCU and MiCU1. Not surprisingly, our results show that this insult impairs adult memory formation. But in a surprising twist, the results demonstrated that normal uniporter function is required during development, and not during adulthood, for normal adult memory formation! Thus, it appears that uniporter function is critical at a particular stage during development in order for adult neurons to develop the competence for supporting normal memory formation.
We have also actively probed mitochondrial function and dynamics in mouse primary neurons through small molecule screening and high-content imaging. We have developed small molecule screens to test the effects of small molecules on several parameters of mitochondrial dynamics including biogenesis, fission, fusion, branching, protein import, and health. We are now pursuing follow up collaborative experiments on our lead compounds, that include analog development and pharmacokinetics. In addition, we have ongoing experiments to test the in vivo efficacy of these molecules in animal models for neurodegenerative diseases. We have also developed the required technology and experimental expertise to screen and test small molecules in neurons derived from human IPSCs.
Biology of active forgetting
The vast majority of learning and memory research has focused on mechanisms for how the brain acquires (learns) information and then stabilizes and consolidates it. Yet, sound arguments can be made for the hypothesis that the positive processes for encoding memories must be balanced by processes for forgetting memories. We have recently made excellent progress in unraveling this overlooked aspect of brain function. We discovered a few years ago that a small number of dopamine neurons innervate the Drosophila mushroom bodies and provide a chronic signal for forgetting. Thus, these dopamine neurons can be considered as “forgetting cells;” functioning to erode the memory traces formed from prior learning in a subset of mushroom body neurons – the “engram cells.” We also found that this dopamine-based forgetting signal is communicated to the mushroom body neurons through a specialized dopamine receptor, which mobilizes a signaling pathway in the mushroom bodies involving the scaffolding protein Scribble to activate the small G-protein, Rac, for memory deterioration. In addition, we found that the potency of this forgetting signal is modulated by both internal and external factors. Sleep turns down the signal whereas sensory stimulation turns it up. These observations explain, in part, how sleep enhances memory formation and sensory stimulation after learning attenuates it. Much remains to be discovered about the biology of active forgetting and we are pursuing experiments to understand it further.
Memory and Aging
We have a keen interest in the changes that occur with aging that impair memory formation. Using Drosophila, we demonstrated that intermediate-term memory becomes compromised with age and that activating a specific neuron known as the dorsal posterior medial neuron (DPMn) reverses this impairment. This suggests an age-dependent defect in DPMn function or connectivity. In addition, protein-synthesis-dependent long-term memory is also impaired in aged flies, and this impairment might also be due to lowered function or connectivity of DPMn with mushroom body or other neurons. We are continuing our aging studies with a focus on the DPMn and how age alters its function and connectivity.
Imaging neural activity and molecular and cellular memory traces in living Drosophila
We have made terrific progress in visualizing neural activity and molecular and cellular memory traces in living Drosophila. This line of experimentation began 20 years ago with our then fantasy that it would be wonderful to see memory traces form in a living brain after learning. Our functional imaging studies have progressed through several stages, starting with a rather crude preparation made by cutting an optical window in the head of living flies fixed in plastic pipette tips, and using transgenically-supplied reporters to visualize and quantitate neural activity before and after learning. We have used protein-based reporters that allow us to visualize calcium influx into neurons, synaptic release, cAMP levels, and others. Some of our more recent improvements for functional imaging experiments include utilizing head-fixed flies touching a floating ball. This allows us to simultaneously monitor neural activity and fly locomotor activity.
With these preparations, we have measured neural activity in response to odorants, electric shock, and other types of stimulation. Most importantly, these experimental strategies have allowed us to visualize the changes in synaptic transmission, or calcium influx, or other properties that occur with conditioning. This has defined about half a dozen molecular memory traces that form after conditioning with odors and either aversive or appetitive stimuli. The memory traces identified in this way occur in different types of neurons in the olfactory nervous system and occur at different times after conditioning. For instance, a very early memory trace forms in the projection neurons of the antennal lobe that persists only for a few minutes. The dorsal paired medial neurons form a delayed memory trace that forms at 30 min after training and persists for about an hour, correlating with intermediate-term memory. The alpha/beta neurons of the mushroom bodies form a memory trace by 9 hours after training that persists for at least 24 hours. This memory trace depends upon the activity of CREB and normal protein synthesis at the time of training. These strategies have provided a literal window into the dynamics of memory trace formation in a living organism.
Memory suppressor genes
Drosophila mushroom bodies.
(Image courtesy of Ron Davis Laboratory)
The last 20 years of learning and memory research have identified numerous genes and their protein products in worms, flies, and mice that are required for the processes of acquisition and memory stabilization. However, much less is known about the biological constraints on memory formation. Are there genes and gene products that limit acquisition, or consolidation? Indeed there are and these are known as memory suppressor genes, defined as those genes and their products that place biological limits on the formation of memories. They are identified from gene inactivation experiments that produce organisms with enhanced memory formation.
Through a large, RNAi-expression based behavioral screen using Drosophila, we have identified several dozen new memory suppressor genes. Each one of these is important to understand at a deep level because they tell us about the features in brain design and function that limit memory formation. Furthermore, genes are generally well conserved between Drosophila and human and so screens of this type offer a filter for human genes that may limit human memory. Obviously, these products could prove very important in our projects designed to identify new drugs for cognitive improvement in human brain disorders. Several of these memory suppressor genes are currently under study. However, the scribble gene described above was identified from our memory suppressor screen along with a gene encoding a plasma membrane transporter of the SLC22A family. Our studies of this transporter indicate that it participates in the termination of neurotransmitter signal by importing acetylcholine from the synaptic cleft.
MicroRNA function in memory formation
MicroRNAs are small RNAs that regulate mRNA translation and/or stability, thereby altering gene expression at a post-transcriptional level. We used a novel technology to inactivate ~130 different Drosophila miRNA genes one at a time. This involves expressing a complementary sequence to the miRNA (“sponge”) in order to titrate and limit the concentration of the miRNA in neurons and then testing these flies for memory. From this “sponge” screen, we identified several memory suppressor miRNA genes that have been and are currently under study.
One such important memory suppressor miRNA gene is miR-980. When miR-980 abundance is reduced, memory performance in flies goes up. Surprisingly, this enhanced memory was observed when the miR-980 sponge was expressed in a variety of subsets of neurons in the Drosophila brain, which indicated that its function is to increase the excitability of neurons in general. Conversely, overexpression of miR-980 impairs memory formation. One of the genes regulated by miR-980 is A2bp1, a gene previously implicated in human epilepsy and autism. Overexpression of A2bp1 by itself enhances memory, indicating that it is a memory-enhancing gene.