Activity-dependent p25 generation regulates synaptic plasticity and Aß-induced cognitive impairment.

Cyclin-dependent kinase 5 regulates numerous neuronal functions with its activator, p35. Under neurotoxic conditions, p35 undergoes proteolytic cleavage to liberate p25, which has been implicated in various neurodegenerative diseases. Here, we show that p25 is generated following neuronal activity under physiological conditions in a GluN2B- and CaMKIIα-dependent manner. Moreover, we developed a knockin mouse model in which endogenous p35 is replaced with a calpain-resistant mutant p35 (Δp35KI) to prevent p25 generation. The Δp35KI mice exhibit impaired long-term depression and defective memory extinction, likely mediated through persistent GluA1 phosphorylation at Ser845. Finally, crossing the Δp35KI mice with the 5XFAD mouse model of Alzheimer's disease (AD) resulted in an amelioration of ß-amyloid (Aß)-induced synaptic depression and cognitive impairment. Together, these results reveal a physiological role of p25 production in synaptic plasticity and memory and provide new insights into the function of p25 in Aß-associated neurotoxicity and AD-like pathology.

State-dependent memory retrieval: insights from neural dynamics and behavioral perspectives.

Memory retrieval is strikingly susceptible to external states (environment) and internal states (mood states and alcohol), yet we know little about the underlying mechanisms. We examined how internally generated states influence successful memory retrieval using the functional magnetic resonance imaging (fMRI) of laboratory mice during memory retrieval. Mice exhibited a strong tendency to perform memory retrieval correctly only in the reinstated mammillary body-inhibited state, in which mice were trained to discriminate auditory stimuli in go/no-go tasks. fMRI revealed that distinct auditory cues engaged differential brain regions, which were primed by internal state. Specifically, a cue associated with a reward activated the lateral amygdala, while a cue signaling no reward predominantly activated the postsubiculum. Modifying these internal states significantly altered the neural activity balance between these regions. Optogenetic inhibition of those regions in the precue period blocked the retrieval of type-specific memories. Our findings suggest that memory retrieval is under the control of two interrelated neural circuits underlying the neural basis of state-dependent memory retrieval.

Suv39h1 regulates memory stability by inhibiting the expression of Shank1 in hippocampal newborn neurons.

Adult newborn neurons are involved in memory encoding and extinction, but the neural mechanism is unclear. We found the adult newborn neurons at 4 weeks are recruited by learning and subjected to epigenetic regulations, consequently reducing their ability to be re-recruited later. After removal of the epigenetic blockage, Suv39h1 KO mice showed an increased recruiting number of aged newborn neurons and enhanced flexibility in learning tasks. Besides NRXN1, we found SHANK1, the synaptic scaffold protein, is one of the major targets of Suv39h1, regulating memory stability. Expression of Shank1 is transiently engaged to enhance synaptogenesis during learning and is strongly suppressed by Suv39h1 from 5 h after learning. Exogenously overexpression of Shank1 in dentate gyrus increased the density of mushroom spines and decreased the persistency of old memories. Our study indicated the activity-regulated epigenetic modification in newly matured newborn neurons in hippocampus insulates temporally distinct experiences and stabilizes old memories.

Mutations in ASH1L confer susceptibility to Tourette syndrome.

Tourette syndrome (TS) is a childhood-onset neuropsychiatric disorder characterized by repetitive motor movements and vocal tics. The clinical manifestations of TS are complex and often overlap with other neuropsychiatric disorders. TS is highly heritable; however, the underlying genetic basis and molecular and neuronal mechanisms of TS remain largely unknown. We performed whole-exome sequencing of a hundred trios (probands and their parents) with detailed records of their clinical presentations and identified a risk gene, ASH1L, that was both de novo mutated and associated with TS based on a transmission disequilibrium test. As a replication, we performed follow-up targeted sequencing of ASH1L in additional 524 unrelated TS samples and replicated the association (P value = 0.001). The point mutations in ASH1L cause defects in its enzymatic activity. Therefore, we established a transgenic mouse line and performed an array of anatomical, behavioral, and functional assays to investigate ASH1L function. The Ash1l+/- mice manifested tic-like behaviors and compulsive behaviors that could be rescued by the tic-relieving drug haloperidol. We also found that Ash1l disruption leads to hyper-activation and elevated dopamine-releasing events in the dorsal striatum, all of which could explain the neural mechanisms for the behavioral abnormalities in mice. Taken together, our results provide compelling evidence that ASH1L is a TS risk gene.

Switching From Fear to No Fear by Different Neural Ensembles in Mouse Retrosplenial Cortex.

Fear extinction is generally considered a form of new learning that inhibits previously acquired fear memories. Here, by tracking immediate early gene expression in vivo, we found that contextual fear extinction training evoked distinct neural ensembles in mouse retrosplenial cortex (RSC). The optogenetic reactivation of these extinction-activated neurons in the RSC was sufficient to suppress a fear response, while the reactivation of conditioning-activated neurons in the same area promoted a fear response. The generation of such an extinction-memory-related neural ensemble was associated with adult neurogenesis, as abolishing newborn neurons in the adult hippocampus via X-ray irradiation eliminated both the extinction-activated neurons in the RSC and the optogenetic-reactivation-induced suppression of contextual fear memory. Therefore, switching from fear to no fear in response to the same context is modulated by the RSC through an extinction-activated neural ensemble, the generation of which might require adult neurogenesis in the hippocampus.

An epigenetic blockade of cognitive functions in the neurodegenerating brain.

Cognitive decline is a debilitating feature of most neurodegenerative diseases of the central nervous system, including Alzheimer's disease. The causes leading to such impairment are only poorly understood and effective treatments are slow to emerge. Here we show that cognitive capacities in the neurodegenerating brain are constrained by an epigenetic blockade of gene transcription that is potentially reversible. This blockade is mediated by histone deacetylase 2, which is increased by Alzheimer's-disease-related neurotoxic insults in vitro, in two mouse models of neurodegeneration and in patients with Alzheimer's disease. Histone deacetylase 2 associates with and reduces the histone acetylation of genes important for learning and memory, which show a concomitant decrease in expression. Importantly, reversing the build-up of histone deacetylase 2 by short-hairpin-RNA-mediated knockdown unlocks the repression of these genes, reinstates structural and synaptic plasticity, and abolishes neurodegeneration-associated memory impairments. These findings advocate for the development of selective inhibitors of histone deacetylase 2 and suggest that cognitive capacities following neurodegeneration are not entirely lost, but merely impaired by this epigenetic blockade.

In vivo imaging of immediate early gene expression reveals layer-specific memory traces in the mammalian brain.

The dynamic processes of formatting long-term memory traces in the cortex are poorly understood. The investigation of these processes requires measurements of task-evoked neuronal activities from large numbers of neurons over many days. Here, we present a two-photon imaging-based system to track event-related neuronal activity in thousands of neurons through the quantitative measurement of EGFP proteins expressed under the control of the EGR1 gene promoter. A recognition algorithm was developed to detect GFP-positive neurons in multiple cortical volumes and thereby to allow the reproducible tracking of 4,000 neurons in each volume for 2 mo. The analysis revealed a context-specific response in sparse layer II neurons. The context-evoked response gradually increased during several days of training and was maintained 1 mo later. The formed traces were specifically activated by the training context and were linearly correlated with the behavioral response. Neuronal assemblies that responded to specific contexts were largely separated, indicating the sparse coding of memory-related traces in the layer II cortical circuit.

A novel pathway regulates memory and plasticity via SIRT1 and miR-134.

The NAD-dependent deacetylase Sir2 was initially identified as a mediator of replicative lifespan in budding yeast and was subsequently shown to modulate longevity in worms and flies. Its mammalian homologue, SIRT1, seems to have evolved complex systemic roles in cardiac function, DNA repair and genomic stability. Recent studies suggest a functional relevance of SIRT1 in normal brain physiology and neurological disorders. However, it is unknown if SIRT1 has a role in higher-order brain functions. We report that SIRT1 modulates synaptic plasticity and memory formation via a microRNA-mediated mechanism. Activation of SIRT1 enhances, whereas its loss-of-function impairs, synaptic plasticity. Surprisingly, these effects were mediated via post-transcriptional regulation of cAMP response binding protein (CREB) expression by a brain-specific microRNA, miR-134. SIRT1 normally functions to limit expression of miR-134 via a repressor complex containing the transcription factor YY1, and unchecked miR-134 expression following SIRT1 deficiency results in the downregulated expression of CREB and brain-derived neurotrophic factor (BDNF), thereby impairing synaptic plasticity. These findings demonstrate a new role for SIRT1 in cognition and a previously unknown microRNA-based mechanism by which SIRT1 regulates these processes. Furthermore, these results describe a separate branch of SIRT1 signalling, in which SIRT1 has a direct role in regulating normal brain function in a manner that is disparate from its cell survival functions, demonstrating its value as a potential therapeutic target for the treatment of central nervous system disorders.

HDAC2 negatively regulates memory formation and synaptic plasticity.

Chromatin modifications, especially histone-tail acetylation, have been implicated in memory formation. Increased histone-tail acetylation induced by inhibitors of histone deacetylases (HDACis) facilitates learning and memory in wild-type mice as well as in mouse models of neurodegeneration. Harnessing the therapeutic potential of HDACis requires knowledge of the specific HDAC family member(s) linked to cognitive enhancement. Here we show that neuron-specific overexpression of HDAC2, but not that of HDAC1, decreased dendritic spine density, synapse number, synaptic plasticity and memory formation. Conversely, Hdac2 deficiency resulted in increased synapse number and memory facilitation, similar to chronic treatment with HDACis in mice. Notably, reduced synapse number and learning impairment of HDAC2-overexpressing mice were ameliorated by chronic treatment with HDACis. Correspondingly, treatment with HDACis failed to further facilitate memory formation in Hdac2-deficient mice. Furthermore, analysis of promoter occupancy revealed an association of HDAC2 with the promoters of genes implicated in synaptic plasticity and memory formation. Taken together, our results suggest that HDAC2 functions in modulating synaptic plasticity and long-lasting changes of neural circuits, which in turn negatively regulates learning and memory. These observations encourage the development and testing of HDAC2-selective inhibitors for human diseases associated with memory impairment.

Ash1L ameliorates psoriasis via limiting neuronal activity-dependent release of miR-let-7b.

BACKGROUND AND PURPOSE: Psoriasis is a common autoimmune skin disease that significantly diminishes patients' quality of life. Interactions between primary afferents of the somatosensory system and the cutaneous immune system mediate the pathogenesis of psoriasis. This study aims to elucidate the molecular mechanisms of how primary sensory neurons regulate psoriasis formation. EXPERIMENTAL APPROACH: Skin and total RNA were extracted from wild-type (WT) and ASH1-like histone lysine methyltransferase (Ash1l+/- ) mice in both naive and imiquimod (IMQ)-induced psoriasis models. Immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescence-activated cell sorting (FACS) were then performed. Microfluidic chamber coculture was used to investigate the interaction between somatosensory neurons and bone marrow dendritic cells (BMDCs) ex vivo. Whole-cell patch clamp recordings were used to evaluate neuronal excitability after Ash1L haploinsufficiency in primary sensory neurons. KEY RESULTS: The haploinsufficiency of ASH1L, a histone methyltransferase, in primary sensory neurons causes both neurite hyperinnervation and increased neuronal excitability, which promote miR-let-7b release from primary afferents in the skin in a neuronal activity-dependent manner. With a 'GUUGUGU' core sequence, miR-let-7b functions as an endogenous ligand of toll-like receptor 7 (TLR7) and stimulates the activation of dermal dendritic cells (DCs) and interleukin (IL)-23/IL-17 axis, ultimately exacerbating the symptoms of psoriasis. Thus, by limiting miR-let-7b release from primary afferents, ASH1L prevents dermal DC activation and ameliorates psoriasis. CONCLUSION AND IMPLICATIONS: Somatosensory neuron ASH1L modulates the cutaneous immune system by limiting neuronal activity-dependent release of miR-let-7b, which can directly activate dermal DCs via TLR7 and ultimately lead to aggravated psoriatic lesion.

Acquiring new memories in neocortex of hippocampal-lesioned mice.

The hippocampus interacts with the neocortical network for memory retrieval and consolidation. Here, we found the lateral entorhinal cortex (LEC) modulates learning-induced cortical long-range gamma synchrony (20-40 Hz) in a hippocampal-dependent manner. The long-range gamma synchrony, which was coupled to the theta (7-10 Hz) rhythm and enhanced upon learning and recall, was mediated by inter-cortical projections from layer 5 neurons of the LEC to layer 2 neurons of the sensory and association cortices. Artificially induced cortical gamma synchrony across cortical areas improved memory encoding in hippocampal lesioned mice for originally hippocampal-dependent tasks. Mechanistically, we found that activities of cortical c-Fos labeled neurons, which showed egocentric map properties, were modulated by LEC-mediated gamma synchrony during memory recall, implicating a role of cortical synchrony to generate an integrative memory representation from disperse features. Our findings reveal the hippocampal mediated organization of cortical memories and suggest brain-machine interface approaches to improve cognitive function.

ASH1L haploinsufficiency results in autistic-like phenotypes in mice and links Eph receptor gene to autism spectrum disorder.

ASD-associated genes are enriched for synaptic proteins and epigenetic regulators. How those chromatin modulators establish ASD traits have remained unknown. We find haploinsufficiency of Ash1l causally induces anxiety and autistic-like behavior, including repetitive behavior, and alters social behavior. Specific depletion of Ash1l in forebrain induces similar ASD-associated behavioral defects. While the learning ability remains intact, the discrimination ability of Ash1l mutant mice is reduced. Mechanistically, deletion of Ash1l in neurons induces excessive synapses due to the synapse pruning deficits, especially during the post-learning period. Dysregulation of synaptic genes is detected in Ash1l mutant brain. Specifically, Eph receptor A7 is downregulated in Ash1l+/- mice through accumulating EZH2-mediated H3K27me3 in its gene body. Importantly, increasing activation of EphA7 in Ash1l+/- mice by supplying its ligand, ephrin-A5, strongly promotes synapse pruning and rescues discrimination deficits. Our results suggest that Ash1l haploinsufficiency is a highly penetrant risk factor for ASD, resulting from synapse pruning deficits.

Single Image-Based Vignetting Correction for Improving the Consistency of Neural Activity Analysis in 2-Photon Functional Microscopy.

High-resolution functional 2-photon microscopy of neural activity is a cornerstone technique in current neuroscience, enabling, for instance, the image-based analysis of relations of the organization of local neuron populations and their temporal neural activity patterns. Interpreting local image intensity as a direct quantitative measure of neural activity presumes, however, a consistent within- and across-image relationship between the image intensity and neural activity, which may be subject to interference by illumination artifacts. In particular, the so-called vignetting artifact-the decrease of image intensity toward the edges of an image-is, at the moment, widely neglected in the context of functional microscopy analyses of neural activity, but potentially introduces a substantial center-periphery bias of derived functional measures. In the present report, we propose a straightforward protocol for single image-based vignetting correction. Using immediate-early gene-based 2-photon microscopic neural image data of the mouse brain, we show the necessity of correcting both image brightness and contrast to improve within- and across-image intensity consistency and demonstrate the plausibility of the resulting functional data.

Detecting Abnormal Neuronal Activity in a Chronic Migraine Model by Egr1-EGFP Transgenic Mice.

Chronic migraine (CM) is a highly disabling neurological disorder characterized by recurrent headache accompanied by a variety of sensory and/or emotional symptoms. However, the mechanisms of migraine onset and its chronicity have not been elucidated. The present study was designed to search for brain regions and neurons that were abnormally activated by CM and might be related to its pathogenesis and different concomitant symptoms. CM models were established here by repeated intraperitoneal injection of nitroglycerin (NTG) every other day for 9 days to early growth response gene 1 (Egr1)-enhanced green fluorescent protein (EGFP) transgenic mice, which allowed monitoring of neuronal activities in the whole brain. CM-related behaviors were recorded through head grooming test and light aversion assay. Elevation of Egr1 expression signals was detected in trigeminal nucleus caudalis (TNC), primary somatosensory cortex (SSp), lateral amygdala nucleus (LA), primary visual area (VISp), and temporal association areas (TEa) 2 h after the last injection of NTG by immunofluorescence and digital slice scanning technology. Meanwhile, no change of Egr1 expression was found in auditory areas (AUD), CA1, ectorhinal area (ECT), piriform (PIR), and anterior cingulate area (ACC). Furthermore, with the strongest support by evidence-based medicine among the current limited oral treatments of CM, topiramate was administrated every day for 11 days from 2 days before the first NTG injection. The results showed that topiramate partially improved the photophobia behavior of CM models in the short-term with gradually weakened efficacy as the course of the disease prolonged. Meanwhile, NTG-induced increase in Egr1 expression was completely reversed in TNC, SSp, and VISp and partially reduced in LA and TEa by topiramate at the same time point mentioned above. In conclusion, the current results suggested that the abnormal hyperactivities in TNC, SSp and VISp were associated with the pathogenesis of CM.

Egr1-EGFP transgenic mouse allows in vivo recording of Egr1 expression and neural activity.

BACKGROUND: Immediate-early genes (IEGs) have been serving as markers of active neurons for their rapid responses to stimulation. With the development of IEG-EGFP reporters by the GENSAT project, application of the IEGs have been greatly expanded. However, detailed validations for these systems are still lacking, causing trouble in the interpretation of the fluorescence signals. NEW METHOD: In this work, taken Egr1-EGFP transgenic mice as an example, we proposed an improvement for the usage of the Egr1-EGFP reporter system based on detailed validation of its fluorescence signals. RESULTS: Firstly, the exogenous EGFP mRNA levels were linearly correlated with the endogenous Egr1 mRNA levels in neurons. Secondly, the 3-hr-changes of the Egr1-EGFP signals before and after the stimulus were positively correlated with the stimulus-induced neuronal activities. Interestingly, persistent neuronal activity patterns in the post-stimulus phase also showed correlation with the stimulus-induced Egr1-EGFP signal changes. Furthermore, enriched environments engaged dramatic neuronal activations, allowing detailed characterization of Egr1-EGFP expression dynamics. COMPARISON WITH EXISTING METHOD(S): People used to infer the neuronal activities based on the raw fluorescence signals of IEG-EGFP reporter system, which was strongly obstructed by distinct protein regulation or dynamic properties between the EGFP and the IEGs. We demonstrated a better way for data analysis and experimental design. CONCLUSIONS: Taken together, this work proves that Egr1-EGFP signal is weakly but significantly correlated to task-induced neural activity and gives detailed characterization of the signal dynamics. It not only provides basis for the understanding of the IEG-EGFP fluorescence signals but also offers instructions for proper experimental design with IEG-EGFP reporter systems.

Rett syndrome linked to defects in forming the MeCP2/Rbfox/LASR complex in mouse models.

Rett syndrome (RTT) is a severe neurological disorder and a leading cause of intellectual disability in young females. RTT is mainly caused by mutations found in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). Despite extensive studies, the molecular mechanism underlying RTT pathogenesis is still poorly understood. Here, we report MeCP2 as a key subunit of a higher-order multiunit protein complex Rbfox/LASR. Defective MeCP2 in RTT mouse models disrupts the assembly of the MeCP2/Rbfox/LASR complex, leading to reduced binding of Rbfox proteins to target pre-mRNAs and aberrant splicing of Nrxns and Nlgn1 critical for synaptic plasticity. We further show that MeCP2 disease mutants display defective condensate properties and fail to promote phase-separated condensates with Rbfox proteins in vitro and in cultured cells. These data link an impaired function of MeCP2 with disease mutation in splicing control to its defective properties in mediating the higher-order assembly of the MeCP2/Rbfox/LASR complex.

Multimodal Memory Components and Their Long-Term Dynamics Identified in Cortical Layers II/III but Not Layer V.

Activity patterns of cerebral cortical regions represent the current environment in which animals receive multi-modal inputs. These patterns are also shaped by the history of activity that reflects learned information on past multimodal exposures. We studied the long-term dynamics of cortical activity patterns during the formation of multimodal memories by analyzing in vivo high-resolution 2-photon mouse brain imaging data of Immediate Early Gene (IEG) expression, resolved by cortical layers. Strikingly, in superficial layers II/III, the patterns showed similar dynamics across structurally and functionally distinct cortical areas and the consistency of dynamic patterns lasted for one to several days. By contrast, in deep layer V, the activity dynamics varied across different areas, and the current activities were sensitive to the previous activities at different time points, depending on the cortical locations, indicating that the information stored in the cortex at different time points was distributed across different cortical areas. These results suggest different roles of superficial and deep layer neurons in the long-term multimodal representation of the environment.

Discrimination of the hierarchical structure of cortical layers in 2-photon microscopy data by combined unsupervised and supervised machine learning.

The laminar organization of the cerebral cortex is a fundamental characteristic of the brain, with essential implications for cortical function. Due to the rapidly growing amount of high-resolution brain imaging data, a great demand arises for automated and flexible methods for discriminating the laminar texture of the cortex. Here, we propose a combined approach of unsupervised and supervised machine learning to discriminate the hierarchical cortical laminar organization in high-resolution 2-photon microscopic neural image data of mouse brain without observer bias, that is, without the prerequisite of manually labeled training data. For local cortical foci, we modify an unsupervised clustering approach to identify and represent the laminar cortical structure. Subsequently, supervised machine learning is applied to transfer the resulting layer labels across different locations and image data, to ensure the existence of a consistent layer label system. By using neurobiologically meaningful features, the discrimination results are shown to be consistent with the layer classification of the classical Brodmann scheme, and provide additional insight into the structure of the cerebral cortex and its hierarchical organization. Thus, our work paves a new way for studying the anatomical organization of the cerebral cortex, and potentially its functional organization.

Activation of delta opioid receptors induces receptor insertion and neuropeptide secretion.

Here we describe a novel mechanism for plasma membrane insertion of the delta opioid receptor (DOR). In small dorsal root ganglion neurons, only low levels of DORs are present on the cell surface, in contrast to high levels of intracellular DORs mainly associated with vesicles containing calcitonin gene-related peptide (CGRP). Activation of surface DORs caused Ca(2+) release from IP(3)-sensitive stores and Ca(2+) entry, resulting in a slow and long-lasting exocytosis, DOR insertion, and CGRP release. In contrast, membrane depolarization or activation of vanilloid and P2Y(1) receptors induced a rapid DOR insertion. Thus, DOR activation induces a Ca(2+)-dependent insertion of DORs that is coupled to a release of excitatory neuropeptides, suggesting that treatment of inflammatory pain should include blockade of DORs.

Distinct subcellular distribution of delta-opioid receptor fused with various tags in PC12 cells.

In small dorsal root ganglion neurons, delta-opioid receptors (DORs) have been found to be mainly distributed in the cytoplasm and often associated with the membrane of large dense-core vesicles (LDCVs) that contain neuropeptides. To study the distribution of DORs under various physiological or pharmacological conditions, the receptors fused with different tags are constructed, transfected into cells or animals, and examined with microscopy. In this study, we show that DOR with different tags have distinct patterns of subcellular distribution in neuroendocrine cells, PC12 cells. Both immunostaining and vesicle fraction analysis showed that the native DORs expressed in PC12 cells were mainly associated with LDCVs. In transfected PC12 cells, DOR tagged with Myc or hemagglutinin exhibited LDCV localization. However, DOR fused with GFP at N- or C-terminus was found to be mainly localized on the cell surface, and mediated the function of DOR agonist. Therefore, the distribution of DOR fused with GFP differs from the native DORs. These results suggest that the subcellular distribution of the receptor could be better presented by the fused tag with smaller molecular size.