Active forgetting and neuropsychiatric diseases.

Recent and pioneering animal research has revealed the brain utilizes a variety of molecular, cellular, and network-level mechanisms used to forget memories in a process referred to as "active forgetting". Active forgetting increases behavioral flexibility and removes irrelevant information. Individuals with impaired active forgetting mechanisms can experience intrusive memories, distressing thoughts, and unwanted impulses that occur in neuropsychiatric diseases. The current evidence indicates that active forgetting mechanisms degrade, or mask, molecular and cellular memory traces created in synaptic connections of "engram cells" that are specific for a given memory. Combined molecular genetic/behavioral studies using Drosophila have uncovered a complex system of cellular active-forgetting pathways within engram cells that is regulated by dopamine neurons and involves dopamine-nitric oxide co-transmission and reception, endoplasmic reticulum Ca2+ signaling, and cytoskeletal remodeling machinery regulated by small GTPases. Some of these molecular cellular mechanisms have already been found to be conserved in mammals. Interestingly, some pathways independently regulate forgetting of distinct memory types and temporal phases, suggesting a multi-layering organization of forgetting systems. In mammals, active forgetting also involves modulation of memory trace synaptic strength by altering AMPA receptor trafficking. Furthermore, active-forgetting employs network level mechanisms wherein non-engram neurons, newly born-engram neurons, and glial cells regulate engram synapses in a state and experience dependent manner. Remarkably, there is evidence for potential coordination between the network and cellular level forgetting mechanisms. Finally, subjects with several neuropsychiatric diseases have been tested and shown to be impaired in active forgetting. Insights obtained from research on active forgetting in animal models will continue to enrich our understanding of the brain dysfunctions that occur in neuropsychiatric diseases.

Learning and memory using Drosophila melanogaster: a focus on advances made in the fifth decade of research.

In the last decade, researchers using Drosophila melanogaster have made extraordinary progress in uncovering the mysteries underlying learning and memory. This progress has been propelled by the amazing toolkit available that affords combined behavioral, molecular, electrophysiological, and systems neuroscience approaches. The arduous reconstruction of electron microscopic images resulted in a first-generation connectome of the adult and larval brain, revealing complex structural interconnections between memory-related neurons. This serves as substrate for future investigations on these connections and for building complete circuits from sensory cue detection to changes in motor behavior. Mushroom body output neurons (MBOn) were discovered, which individually forward information from discrete and non-overlapping compartments of the axons of mushroom body neurons (MBn). These neurons mirror the previously discovered tiling of mushroom body axons by inputs from dopamine neurons and have led to a model that ascribes the valence of the learning event, either appetitive or aversive, to the activity of different populations of dopamine neurons and the balance of MBOn activity in promoting avoidance or approach behavior. Studies of the calyx, which houses the MBn dendrites, have revealed a beautiful microglomeruluar organization and structural changes of synapses that occur with long-term memory (LTM) formation. Larval learning has advanced, positioning it to possibly lead in producing new conceptual insights due to its markedly simpler structure over the adult brain. Advances were made in how cAMP response element-binding protein interacts with protein kinases and other transcription factors to promote the formation of LTM. New insights were made on Orb2, a prion-like protein that forms oligomers to enhance synaptic protein synthesis required for LTM formation. Finally, Drosophila research has pioneered our understanding of the mechanisms that mediate permanent and transient active forgetting, an important function of the brain along with acquisition, consolidation, and retrieval. This was catalyzed partly by the identification of memory suppressor genes-genes whose normal function is to limit memory formation.

Early Mitochondrial Defects in the 5xFAD Mouse Model of Alzheimer's Disease.

BACKGROUND: Mitochondrial (MT) dysfunction is a hallmark of Alzheimer's disease (AD). Amyloid-ß protein precursor and amyloid-ß peptides localize to MT and lead to MT dysfunction in familial forms of AD. This dysfunction may trigger subsequent types of pathology. OBJECTIVE: To identify the MT phenotypes that occur early in order to help understand the cascade of AD pathophysiology. METHODS: The 5xFAD mouse model was used to explore the time course of MT pathologies in both sexes. Protein biomarkers for MT dynamics were measured biochemically and MT function was measured using oxygen consumption and ATP assays. RESULTS: We discovered progressive alterations in mitochondrial dynamics (biogenesis, fission, fusion, and mitophagy) and function (O2 consumption, ATP generation, and Ca2+ import) in the hippocampus of 5xFAD mice in both sexes as early as 2 months of age. Thus, mitochondrial dynamics and function become altered at young ages, consistent with an early role for mitochondria in the AD pathological cascade. CONCLUSION: Our study offers the baseline information required to understand the hierarchical relationship between the multiple pathologies that develop in this mouse model and provides early biomarkers for MT dysfunction. This will aid in dissecting the temporal cascade of pathologies, understanding sex-specific differences, and in testing the efficacy of putative mitochondrial therapeutics.

Innate and learned odor-guided behaviors utilize distinct molecular signaling pathways in a shared dopaminergic circuit.

Odor-based learning and innate odor-driven behavior have been hypothesized to require separate neuronal circuitry. Contrary to this notion, innate behavior and olfactory learning were recently shown to share circuitry that includes the Drosophila mushroom body (MB). But how a single circuit drives two discrete behaviors remains unknown. Here, we define an MB circuit responsible for both olfactory learning and innate odor avoidance and the distinct dDA1 dopamine receptor-dependent signaling pathways that mediate these behaviors. Associative learning and learning-induced MB plasticity require rutabaga-encoded adenylyl cyclase activity in the MB. In contrast, innate odor preferences driven by naive MB neurotransmission are rutabaga independent, requiring the adenylyl cyclase ACXD. Both learning and innate odor preferences converge on PKA and the downstream MBON-γ2α'1. Importantly, the utilization of this shared circuitry for innate behavior only becomes apparent with hunger, indicating that hardwired innate behavior becomes more flexible during states of stress.

Genetics and Molecular Biology of Memory Suppression.

The brain is designed not only with molecules and cellular processes that help to form memories but also with molecules and cellular processes that suppress the formation and retention of memory. The latter processes are critical for an efficient memory management system, given the vast amount of information that each person experiences in their daily activities and that most of this information becomes irrelevant with time. Thus, efficiency dictates that the brain should have processes for selecting the most critical information for storage and suppressing the irrelevant or forgetting it later should it escape the initial filters. Such memory suppressor molecules and processes are revealed by genetic or pharmacologic insults that lead to enhanced memory expression. We review here the predominant memory suppressor molecules and processes that have recently been discovered. They are diverse, as expected, because the brain is complex and employs many different strategies and mechanisms to form memories. They include the gene-repressive actions of small noncoding RNAs, repressors of protein synthesis, cAMP-mediated gene expression pathways, inter- and intracellular signaling pathways for normal forgetting, and others. A deep understanding of memory suppressor molecules and processes is necessary to fully comprehend how the brain forms, stabilizes, and retrieves memories and to reveal how brain disorders disrupt memory.

Active forgetting requires Sickie function in a dedicated dopamine circuit in Drosophila.

Forgetting is an essential component of the brain's memory management system, providing a balance to memory formation processes by removing unused or unwanted memories, or by suppressing their expression. However, the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. Here we show that the memory suppressor gene, sickie, functions in a single dopamine neuron (DAn) by supporting the process of active forgetting in Drosophila. RNAi knockdown (KD) of sickie impairs forgetting by reducing the Ca2+ influx and DA release from the DAn that promotes forgetting. Coimmunoprecipitation/mass spectrometry analyses identified cytoskeletal and presynaptic active zone (AZ) proteins as candidates that physically interact with Sickie, and a focused RNAi screen of the candidates showed that Bruchpilot (Brp)-a presynaptic AZ protein that regulates calcium channel clustering and neurotransmitter release-impairs active forgetting like sickie KD. In addition, overexpression of brp rescued the impaired forgetting of sickie KD, providing evidence that they function in the same process. Moreover, we show that sickie KD in the DAn reduces the abundance and size of AZ markers but increases their number, suggesting that Sickie controls DAn activity for forgetting by modulating the presynaptic AZ structure. Our results identify a molecular and circuit mechanism for normal levels of active forgetting and reveal a surprising role of Sickie in maintaining presynaptic AZ structure for neurotransmitter release.

Associative learning drives longitudinally graded presynaptic plasticity of neurotransmitter release along axonal compartments.

Anatomical and physiological compartmentalization of neurons is a mechanism to increase the computational capacity of a circuit, and a major question is what role axonal compartmentalization plays. Axonal compartmentalization may enable localized, presynaptic plasticity to alter neuronal output in a flexible, experience-dependent manner. Here, we show that olfactory learning generates compartmentalized, bidirectional plasticity of acetylcholine release that varies across the longitudinal compartments of Drosophila mushroom body (MB) axons. The directionality of the learning-induced plasticity depends on the valence of the learning event (aversive vs. appetitive), varies linearly across proximal to distal compartments following appetitive conditioning, and correlates with learning-induced changes in downstream mushroom body output neurons (MBONs) that modulate behavioral action selection. Potentiation of acetylcholine release was dependent on the CaV2.1 calcium channel subunit cacophony. In addition, contrast between the positive conditioned stimulus and other odors required the inositol triphosphate receptor, which maintained responsivity to odors upon repeated presentations, preventing adaptation. Downstream from the MB, a set of MBONs that receive their input from the γ3 MB compartment were required for normal appetitive learning, suggesting that they represent a key node through which reward learning influences decision-making. These data demonstrate that learning drives valence-correlated, compartmentalized, bidirectional potentiation, and depression of synaptic neurotransmitter release, which rely on distinct mechanisms and are distributed across axonal compartments in a learning circuit.

Memory suppressor genes: Modulating acquisition, consolidation, and forgetting.

The brain has a remarkable but underappreciated capacity to limit memory formation and expression. The term "memory suppressor gene" was coined in 1998 as an attempt to explain emerging reports that some genes appeared to limit memory. At that time, only a handful of memory suppressor genes were known, and they were understood to work by limiting cAMP-dependent consolidation. In the intervening decades, almost 100 memory suppressor genes with diverse functions have been discovered that affect not only consolidation but also acquisition and forgetting. Here we highlight the surprising extent to which biological limits are placed on memory formation through reviewing the literature on memory suppressor genes. In this review, we present memory suppressors within the framework of their actions on different memory operations: acquisition, consolidation, and forgetting. This is followed by a discussion of the reasons why there may be a biological need to limit memory formation.

High-Throughput Phenotypic Assay for Compounds That Influence Mitochondrial Health Using iPSC-Derived Human Neurons.

There is a critical need to develop high-throughput assays to identify compounds that offer therapy for individuals suffering from neurodegenerative diseases. Most brain disorders, including neurodegenerative diseases, share the common neuropathology of mitochondria dysfunction, which can lead to apoptosis of neurons, overproduction of reactive oxygen species (ROS), and other cellular neuropathologies characteristic of these diseases. Human induced pluripotent stem cells (iPSCs) with a stable genomic insertion of the neurogenin-2 transcription factor under the control of the TetOn promoter can be differentiated into excitatory human neurons (i3Neurons) within 3 days of exposure to doxycycline. These neurons have been used to develop and validate a live-cell assay for parameters of mitochondrial dynamics and function using two compounds known to promote mitochondrial elongation in mouse neurons, 4-hydroxychalcone and 2,4-dihyrdroxychalcone. The assay involves plating the neurons in 384-well microtiter plates, treating them with known or unknown substances, and then capturing morphological information for the neuronal mitochondria using a lentivirus vector to express a mitochondrial-targeted fluorescence reporter. The i3Neuron cultures exposed to these two compounds for 24 h exhibit significantly decreased circularity and significantly increased length compared to controls, two morphological parameters correlated with increased mitochondrial health. The assay is rapid, with results obtained after a one-week-long i3Neuron culture or one month if neurons are co-cultured with astrocytes. This live-cell, mitochondrial phenotypic assay can be used for high-throughput screening or as an orthogonal assay for compounds obtained via other high-throughput screening campaigns.

Dopamine-based mechanism for transient forgetting.

Active forgetting is an essential component of the memory management system of the brain1. Forgetting can be permanent, in which prior memory is lost completely, or transient, in which memory exists in a temporary state of impaired retrieval. Temporary blocks on memory seem to be universal, and can disrupt an individual's plans, social interactions and ability to make rapid, flexible and appropriate choices. However, the neurobiological mechanisms that cause transient forgetting are unknown. Here we identify a single dopamine neuron in Drosophila that mediates the memory suppression that results in transient forgetting. Artificially activating this neuron did not abolish the expression of long-term memory. Instead, it briefly suppressed memory retrieval, with the memory becoming accessible again over time. The dopamine neuron modulates memory retrieval by stimulating a unique dopamine receptor that is expressed in a restricted physical compartment of the axons of mushroom body neurons. This mechanism for transient forgetting is triggered by the presentation of interfering stimuli immediately before retrieval.

Early Mitochondrial Fragmentation and Dysfunction in a Drosophila Model for Alzheimer's Disease.

Many different cellular systems and molecular processes become compromised in Alzheimer's disease (AD) including proteostasis, autophagy, inflammatory responses, synapse and neuronal circuitry, and mitochondrial function. We focused in this study on mitochondrial dysfunction owing to the toxic neuronal environment produced by expression of Aß42, and its relationship to other pathologies found in AD including increased neuronal apoptosis, plaque deposition, and memory impairment. Using super-resolution microscopy, we have assayed mitochondrial status in the three distinct neuronal compartments (somatic, dendritic, axonal) of mushroom body neurons of Drosophila expressing Aß42. The mushroom body neurons comprise a major center for olfactory memory formation in insects. We employed calcium imaging to measure mitochondrial function, immunohistochemical and staining techniques to measure apoptosis and plaque formation, and olfactory classical conditioning to measure learning. We found that mitochondria become fragmented at a very early age along with decreased function measured by mitochondrial calcium entry. Increased apoptosis and plaque deposition also occur early, yet interestingly, a learning impairment was found only after a much longer period of time-10 days, which is a large fraction of the fly's lifespan. This is similar to the pronounced delay between cellular pathologies and the emergence of a memory dysfunction in humans. Our studies are consistent with the model that mitochondrial dysfunction and/or other cellular pathologies emerge at an early age and lead to much later learning impairments. The results obtained further develop this Drosophila model as a useful in vivo system for probing the mechanisms by which Aß42 produces mitochondrial and other cellular toxicities that produce memory dysfunction.

Pathologic Complete Response to Neoadjuvant Nivolumab/Ipilimumab in a Patient with Metastatic Renal Cell Carcinoma.

Nivolumab plus ipilimumab represents an effective combination of checkpoint inhibitors that can lead to a durable response with minimal toxicity in patients with metastatic renal cell carcinoma (mRCC). We present a case of a pathologic complete response to neoadjuvant nivolumab plus ipilimumab in a patient with a 13.9 cm left renal mass and significant retroperitoneal and iliac lymphadenopathy, classified as intermediate-risk mRCC. We discuss and review the literature on complete responses after systemic therapy and the ability to predict who has undergone a complete response in the face of residual radiographic evidence of disease.

Rac1 Impairs Forgetting-Induced Cellular Plasticity in Mushroom Body Output Neurons.

Active memory forgetting is a well-regulated biological process thought to be adaptive and to allow proper cognitive functions. Recent efforts have elucidated several molecular players involved in the regulation of olfactory forgetting in Drosophila, including the small G protein Rac1, the dopamine receptor DAMB, and the scaffold protein Scribble. Similarly, we recently reported that dopaminergic neurons mediate both learning- and forgetting-induced plasticity in the mushroom body output neuron MBON-γ2α'1. Two open questions remain: how does forgetting affect plasticity in other, highly plastic, mushroom body compartments and how do genes that regulate forgetting affect this cellular plasticity? Here, we show that forgetting reverses short-term synaptic depression induced by aversive conditioning in the highly plastic mushroom body output neuron MBON-γ1pedc>α/ß. In addition, our results indicate that genetic tampering with normal forgetting by inhibition of small G protein Rac1 impairs restoration of depressed odor responses to learned odor by intrinsic forgetting through time passing and forgetting induced acutely by shock stimulation after conditioning or reversal learning. These data further indicate that some forms of forgetting truly erase physiological changes generated by memory encoding.

Mechanism of Action and Target Identification: A Matter of Timing in Drug Discovery.

Two opposing viewpoints are held regarding the need for understanding a drug's molecular target and mechanism of action. One extreme viewpoint is that it is unnecessary, because, after all, there are many beneficial drugs in use for which the target and mechanism of action remain unknown. A second extreme viewpoint is that target identification and mechanism of action should be elucidated very early in the drug discovery process due to the tangible benefits provided by this knowledge. I offer an intermediate perspective that considers the complexity of the disease of interest, the existence of a standard-of-care treatment, and the resources available to the investigator.

miR-92a Suppresses Mushroom Body-Dependent Memory Consolidation in Drosophila.

MicroRNAs (miRNAs) fine tune gene expression to regulate many aspects of nervous system physiology. Here, we show that miR-92a suppresses memory consolidation that occurs in the αß and γ mushroom body neurons (MBns) of Drosophila, making miR-92a a memory suppressor miRNA. Bioinformatics analyses suggested that mRNAs encoding kinesin heavy chain 73 (KHC73), a protein that belongs to Kinesin-3 family of anterograde motor proteins, may be a functional target of miR-92a. Behavioral studies that employed expression of khc73 with and without its 3' untranslated region (UTR) containing miR-92a target sites, luciferase assays in HEK cells with reporters containing wild-type and mutant target sequences in the khc73 3'UTR, and immunohistochemistry experiments involving KHC73 expression with and without the wild-type khc73 3'UTR, all point to the conclusion that khc73 is a major target of miR-92a in its functional role as a miRNA memory suppressor gene.

Erratum: High-Throughput Small Molecule Screen Identifies Modulators of Mitochondrial Function in Neurons.

[This corrects the article DOI: 10.1016/j.isci.2020.100931.].

High-Throughput Small Molecule Screen Identifies Modulators of Mitochondrial Function in Neurons.

We developed a high-throughput assay for modulators of mitochondrial function in neurons measuring inner mitochondrial membrane potential (ΔΨm) and ATP production. The assay was used to screen a library of small molecules, which led to the identification of structural/functional classes of mitochondrial modulators such as local anesthetics, isoflavones, COXII inhibitors, adrenergic receptor blockers, and neurotransmitter system effectors. Our results show that some of the isolated compounds promote mitochondrial health, enhance oxygen consumption rate, and protect neurons against toxic insults found in the cellular environment of Alzheimer disease. These studies offer a set of compounds that may provide efficacy in protecting the mitochondrial system in neurodegenerative disorders.

Neuron-based high-content assay and screen for CNS active mitotherapeutics.

Impaired mitochondrial dynamics and function are hallmarks of many neurological and psychiatric disorders, but direct screens for mitotherapeutics using neurons have not been reported. We developed a multiplexed and high-content screening assay using primary neurons and identified 67 small-molecule modulators of neuronal mitostasis (MnMs). Most MnMs that increased mitochondrial content, length, and/or health also increased mitochondrial function without altering neurite outgrowth. A subset of MnMs protected mitochondria in primary neurons from Aß(1-42) toxicity, glutamate toxicity, and increased oxidative stress. Some MnMs were shown to directly target mitochondria. The top MnM also increased the synaptic activity of hippocampal neurons and proved to be potent in vivo, increasing the respiration rate of brain mitochondria after administering the compound to mice. Our results offer a platform that directly queries mitostasis processes in neurons, a collection of small-molecule modulators of mitochondrial dynamics and function, and candidate molecules for mitotherapeutics.

Ras acts as a molecular switch between two forms of consolidated memory in Drosophila.

Long-lasting, consolidated memories require not only positive biological processes that facilitate long-term memories (LTM) but also the suppression of inhibitory processes that prevent them. The mushroom body neurons (MBn) in Drosophila melanogaster store protein synthesis-dependent LTM (PSD-LTM) as well as protein synthesis-independent, anesthesia-resistant memory (ARM). The formation of ARM inhibits PSD-LTM but the underlying molecular processes that mediate this interaction remain unknown. Here, we demonstrate that the Ras→Raf→rho kinase (ROCK) pathway in MBn suppresses ARM consolidation, allowing the formation of PSD-LTM. Our initial results revealed that the effects of Ras on memory are due to postacquisition processes. Ras knockdown enhanced memory expression but had no effect on acquisition. Additionally, increasing Ras activity optogenetically after, but not before, acquisition impaired memory performance. The elevated memory produced by Ras knockdown is a result of increased ARM. While Ras knockdown enhanced the consolidation of ARM, it eliminated PSD-LTM. We found that these effects are mediated by the downstream kinase Raf. Similar to Ras, knockdown of Raf enhanced ARM consolidation and impaired PSD-LTM. Surprisingly, knockdown of the canonical downstream extracellular signal-regulated kinase did not reproduce the phenotypes observed with Ras and Raf knockdown. Rather, Ras/Raf inhibition of ROCK was found to be responsible for suppressing ARM. Constitutively active ROCK enhanced ARM and impaired PSD-LTM, while decreasing ROCK activity rescued the enhanced ARM produced by Ras knockdown. We conclude that MBn Ras/Raf inhibition of ROCK suppresses the consolidation of ARM, which permits the formation of PSD-LTM.

Aversive Training Induces Both Presynaptic and Postsynaptic Suppression in Drosophila.

The α'ß' subtype of Drosophila mushroom body neurons (MBn) is required for memory acquisition, consolidation and early memory retrieval after aversive olfactory conditioning. However, in vivo functional imaging studies have failed to detect an early forming memory trace in these neurons as reflected by an enhanced G-CaMP signal in response to presentation of the learned odor. Moreover, whether cellular memory traces form early after conditioning in the mushroom body output neurons (MBOn) downstream of the α'ß' MBn remains unknown. Here, we show that aversive olfactory conditioning suppresses the calcium responses to the learned odor in both α'3 and α'2 axon segments of α'ß' MBn and in the dendrites of α'3 MBOn immediately after conditioning using female flies. Notably, the cellular memory traces in both α'3 MBn and α'3 MBOn are short-lived and persist for <30 min. The suppressed response in α'3 MBn is accompanied by a reduction of acetylcholine (ACh) release, suggesting that the memory trace in postsynaptic α'3 MBOn may simply reflect the suppression in presynaptic α'3 MBn. Furthermore, we show that the α'3 MBn memory trace does not occur from the inhibition of GABAergic neurons via GABAA receptor activation. Because activation of the α'3 MBOn drives approach behavior of adult flies, our results demonstrate that aversive conditioning promotes avoidance behavior through suppression of the α'3 MBn-MBOn circuit.SIGNIFICANCE STATEMENTDrosophila learn to avoid an odor if that odor is repeatedly paired with electric shock. Mushroom body neurons (MBns) are known to be major cell types that mediate this form of aversive conditioning. Here we show that aversive conditioning causes a reduced response to the conditioned odor in an axon branch of one subtype of the MBn for no more than 30 min after conditioning, and in the dendrites of postsynaptic, MB output neurons (MBOns). Because experimenter-induced activation of the MBOn induces approach behavior by the fly, our data support a model that aversive learning promotes avoidance by suppressing the MBn-MBOn synapses that normally promote attraction.