Formation of memory assemblies through the DNA-sensing TLR9 pathway.

As hippocampal neurons respond to diverse types of information1, a subset assembles into microcircuits representing a memory2. Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage3-5. Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes6. Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.

Regulation of Zbp1 by miR-99b-5p in microglia controls the development of schizophrenia-like symptoms in mice.

Current approaches to the treatment of schizophrenia have mainly focused on the protein-coding part of the genome; in this context, the roles of microRNAs have received less attention. In the present study, we analyze the microRNAome in the blood and postmortem brains of schizophrenia patients, showing that the expression of miR-99b-5p is downregulated in both the prefrontal cortex and blood of patients. Lowering the amount of miR-99b-5p in mice leads to both schizophrenia-like phenotypes and inflammatory processes that are linked to synaptic pruning in microglia. The microglial miR-99b-5p-supressed inflammatory response requires Z-DNA binding protein 1 (Zbp1), which we identify as a novel miR-99b-5p target. Antisense oligonucleotides against Zbp1 ameliorate the pathological effects of miR-99b-5p inhibition. Our findings indicate that a novel miR-99b-5p-Zbp1 pathway in microglia might contribute to the pathogenesis of schizophrenia.

Formin 2 links neuropsychiatric phenotypes at young age to an increased risk for dementia.

Age-associated memory decline is due to variable combinations of genetic and environmental risk factors. How these risk factors interact to drive disease onset is currently unknown. Here we begin to elucidate the mechanisms by which post-traumatic stress disorder (PTSD) at a young age contributes to an increased risk to develop dementia at old age. We show that the actin nucleator Formin 2 (Fmn2) is deregulated in PTSD and in Alzheimer's disease (AD) patients. Young mice lacking the Fmn2 gene exhibit PTSD-like phenotypes and corresponding impairments of synaptic plasticity, while the consolidation of new memories is unaffected. However, Fmn2 mutant mice develop accelerated age-associated memory decline that is further increased in the presence of additional risk factors and is mechanistically linked to a loss of transcriptional homeostasis. In conclusion, our data present a new approach to explore the connection between AD risk factors across life span and provide mechanistic insight to the processes by which neuropsychiatric diseases at a young age affect the risk for developing dementia.

HDAC1 links early life stress to schizophrenia-like phenotypes.

Schizophrenia is a devastating disease that arises on the background of genetic predisposition and environmental risk factors, such as early life stress (ELS). In this study, we show that ELS-induced schizophrenia-like phenotypes in mice correlate with a widespread increase of histone-deacetylase 1 (Hdac1) expression that is linked to altered DNA methylation. Hdac1 overexpression in neurons of the medial prefrontal cortex, but not in the dorsal or ventral hippocampus, mimics schizophrenia-like phenotypes induced by ELS. Systemic administration of an HDAC inhibitor rescues the detrimental effects of ELS when applied after the manifestation of disease phenotypes. In addition to the hippocampus and prefrontal cortex, mice subjected to ELS exhibit increased Hdac1 expression in blood. Moreover, Hdac1 levels are increased in blood samples from patients with schizophrenia who had encountered ELS, compared with patients without ELS experience. Our data suggest that HDAC1 inhibition should be considered as a therapeutic approach to treat schizophrenia.

K-Lysine acetyltransferase 2a regulates a hippocampal gene expression network linked to memory formation.

Neuronal histone acetylation has been linked to memory consolidation, and targeting histone acetylation has emerged as a promising therapeutic strategy for neuropsychiatric diseases. However, the role of histone-modifying enzymes in the adult brain is still far from being understood. Here we use RNA sequencing to screen the levels of all known histone acetyltransferases (HATs) in the hippocampal CA1 region and find that K-acetyltransferase 2a (Kat2a)--a HAT that has not been studied for its role in memory function so far--shows highest expression. Mice that lack Kat2a show impaired hippocampal synaptic plasticity and long-term memory consolidation. We furthermore show that Kat2a regulates a highly interconnected hippocampal gene expression network linked to neuroactive receptor signaling via a mechanism that involves nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In conclusion, our data establish Kat2a as a novel and essential regulator of hippocampal memory consolidation.

A hippocampal insulin-growth factor 2 pathway regulates the extinction of fear memories.

Extinction learning refers to the phenomenon that a previously learned response to an environmental stimulus, for example, the expression of an aversive behaviour upon exposure to a specific context, is reduced when the stimulus is repeatedly presented in the absence of a previously paired aversive event. Extinction of fear memories has been implicated with the treatment of anxiety disease but the molecular processes that underlie fear extinction are only beginning to emerge. Here, we show that fear extinction initiates upregulation of hippocampal insulin-growth factor 2 (Igf2) and downregulation of insulin-growth factor binding protein 7 (Igfbp7). In line with this observation, we demonstrate that IGF2 facilitates fear extinction, while IGFBP7 impairs fear extinction in an IGF2-dependent manner. Furthermore, we identify one cellular substrate of altered IGF2 signalling during fear extinction. To this end, we show that fear extinction-induced IGF2/IGFBP7 signalling promotes the survival of 17-19-day-old newborn hippocampal neurons. In conclusion, our data suggest that therapeutic strategies that enhance IGF2 signalling and adult neurogenesis might be suitable to treat disease linked to excessive fear memory.

microRNA-34c is a novel target to treat dementias.

MicroRNAs are key regulators of transcriptome plasticity and have been implicated with the pathogenesis of brain diseases. Here, we employed massive parallel sequencing and provide, at an unprecedented depth, the complete and quantitative miRNAome of the mouse hippocampus, the prime target of neurodegenerative diseases such as Alzheimer's disease (AD). Using integrative genetics, we identify miR-34c as a negative constraint of memory consolidation and show that miR-34c levels are elevated in the hippocampus of AD patients and corresponding mouse models. In line with this, targeting miR-34 seed rescues learning ability in these mouse models. Our data suggest that miR-34c could be a marker for the onset of cognitive disturbances linked to AD and indicate that targeting miR-34c could be a suitable therapy.

HDAC1 regulates fear extinction in mice.

Histone acetylation has been implicated with the pathogenesis of neuropsychiatric disorders and targeting histone deacetylases (HDACs) using HDAC inhibitors was shown to be neuroprotective and to initiate neuroregenerative processes. However, little is known about the role of individual HDAC proteins during the pathogenesis of brain diseases. HDAC1 was found to be upregulated in patients suffering from neuropsychiatric diseases. Here, we show that virus-mediated overexpression of neuronal HDAC1 in the adult mouse hippocampus specifically affects the extinction of contextual fear memories, while other cognitive abilities were unaffected. In subsequent experiments we show that under physiological conditions, hippocampal HDAC1 is required for extinction learning via a mechanism that involves H3K9 deacetylation and subsequent trimethylation of target genes. In conclusion, our data show that hippocampal HDAC1 has a specific role in memory function.

Recovery of learning and memory is associated with chromatin remodelling.

Neurodegenerative diseases of the central nervous system are often associated with impaired learning and memory, eventually leading to dementia. An important aspect in pre-clinical research is the exploration of strategies to re-establish learning ability and access to long-term memories. By using a mouse model that allows temporally and spatially restricted induction of neuronal loss, we show here that environmental enrichment reinstated learning behaviour and re-established access to long-term memories after significant brain atrophy and neuronal loss had already occurred. Environmental enrichment correlated with chromatin modifications (increased histone-tail acetylation). Moreover, increased histone acetylation by inhibitors of histone deacetylases induced sprouting of dendrites, an increased number of synapses, and reinstated learning behaviour and access to long-term memories. These data suggest that inhibition of histone deacetylases might be a suitable therapeutic avenue for neurodegenerative diseases associated with learning and memory impairment, and raises the possibility of recovery of long-term memories in patients with dementia.

The anaphase promoting complex is required for memory function in mice.

Learning and memory processes critically involve the orchestrated regulation of de novo protein synthesis. On the other hand it has become clear that regulated protein degradation also plays a major role in neuronal plasticity and learning behavior. One of the key pathways mediating protein degradation is proteosomal protein destruction. The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that targets proteins for proteosomal degradation by the 26S proteasome. While the APC/C is essential for cell cycle progression it is also expressed in postmitotic neurons where it has been implicated with axonal outgrowth and neuronal survival. In this study we addressed the role of APC/C in learning and memory function by generating mice that lack the essential subunit APC2 from excitatory neurons of the adult forebrain. Those animals are viable but exhibit a severe impairment in the ability to extinct fear memories, a process critical for the treatment of anxiety diseases such as phobia or post-traumatic stress disorder. Since deregulated protein degradation and APC/C activity has been implicated with neurodegeneration we also analyzed the effect of Apc2 deletion in a mouse model for Alzheimer's disease. In our experimental setting loss of APC2 form principle forebrain neurons did not affect the course of pathology in an Alzheimer's disease mouse model. In conclusion, our data provides genetic evidence that APC/C activity in the adult forebrain is required for cognitive function.

A hippocampal Cdk5 pathway regulates extinction of contextual fear.

Treatment of emotional disorders involves the promotion of extinction processes, which are defined as the learned reduction of fear. The molecular mechanisms underlying extinction have only begun to be elucidated. By employing genetic and pharmacological approaches in mice, we show here that extinction requires downregulation of Rac-1 and cyclin-dependent kinase 5 (Cdk5), and upregulation of p21 activated kinase-1 (PAK-1) activity. This is physiologically achieved by a Rac-1-dependent relocation of the Cdk5 activator p35 from the membrane to the cytosol and dissociation of p35 from PAK-1. Moreover, our data suggest that Cdk5/p35 activity prevents extinction in part by inhibition of PAK-1 activity in a Rac-1-dependent manner. We propose that extinction of contextual fear is regulated by counteracting components of a molecular pathway involving Rac-1, Cdk5 and PAK-1. Our data suggest that this pathway could provide a suitable target for therapeutic treatment of emotional disorders.

Primary cilia are required for the persistence of memory and stabilization of perineuronal nets.

It is well established that the formation of episodic memories requires multiple hippocampal mechanisms operating on different time scales. Early mechanisms of memory formation (synaptic consolidation) have been extensively characterized. However, delayed mechanisms, which maintain hippocampal activity as memories stabilize in cortical circuits, are not well understood. Here we demonstrate that contrary to the transient expression of early- and delayed-response genes, the expression of cytoskeleton- and extracellular matrix-associated genes remains dynamic even at remote time points. The most profound expression changes clustered around primary cilium-associated and collagen genes. These genes most likely contribute to memory by stabilizing perineuronal nets in the dorsohippocampal CA1 subfield, as revealed by targeted disruptions of the primary cilium or perineuronal nets. The findings show that nonsynaptic, primary cilium-mediated mechanisms are required for the persistence of context memory.

Exercise as a model to identify microRNAs linked to human cognition: a role for microRNA-409 and microRNA-501.

MicroRNAs have been linked to synaptic plasticity and memory function and are emerging as potential biomarkers and therapeutic targets for cognitive diseases. Most of these data stem from the analysis of model systems or postmortem tissue from patients which mainly represents an advanced stage of pathology. Due to the in-accessibility of human brain tissue upon experimental manipulation, it is still challenging to identify microRNAs relevant to human cognition, which is however a key step for future translational studies. Here, we employ exercise as an experimental model for memory enhancement in healthy humans with the aim to identify microRNAs linked to memory function. By analyzing the circulating smallRNAome we find a cluster of 18 microRNAs that are highly correlated to cognition. MicroRNA-409-5p and microRNA-501-3p were the most significantly regulated candidates. Functional analysis revealed that the two microRNAs are important for neuronal integrity, synaptic plasticity, and morphology. In conclusion, we provide a novel approach to identify microRNAs linked to human memory function.

Epigenetic gene expression links heart failure to memory impairment.

In current clinical practice, care of diseased patients is often restricted to separated disciplines. However, such an organ-centered approach is not always suitable. For example, cognitive dysfunction is a severe burden in heart failure patients. Moreover, these patients have an increased risk for age-associated dementias. The underlying molecular mechanisms are presently unknown, and thus, corresponding therapeutic strategies to improve cognition in heart failure patients are missing. Using mice as model organisms, we show that heart failure leads to specific changes in hippocampal gene expression, a brain region intimately linked to cognition. These changes reflect increased cellular stress pathways which eventually lead to loss of neuronal euchromatin and reduced expression of a hippocampal gene cluster essential for cognition. Consequently, mice suffering from heart failure exhibit impaired memory function. These pathological changes are ameliorated via the administration of a drug that promotes neuronal euchromatin formation. Our study provides first insight to the molecular processes by which heart failure contributes to neuronal dysfunction and point to novel therapeutic avenues to treat cognitive defects in heart failure patients.

A microRNA signature that correlates with cognition and is a target against cognitive decline.

While some individuals age without pathological memory impairments, others develop age-associated cognitive diseases. Since changes in cognitive function develop slowly over time in these patients, they are often diagnosed at an advanced stage of molecular pathology, a time point when causative treatments fail. Thus, there is great need for the identification of inexpensive and minimal invasive approaches that could be used for screening with the aim to identify individuals at risk for cognitive decline that can then undergo further diagnostics and eventually stratified therapies. In this study, we use an integrative approach combining the analysis of human data and mechanistic studies in model systems to identify a circulating 3-microRNA signature that reflects key processes linked to neural homeostasis and inform about cognitive status. We furthermore provide evidence that expression changes in this signature represent multiple mechanisms deregulated in the aging and diseased brain and are a suitable target for RNA therapeutics.

Opposing roles of transient and prolonged expression of p25 in synaptic plasticity and hippocampus-dependent memory.

While deregulation of cyclin-dependent kinase 5 (Cdk5) has been implicated in neurodegenerative diseases, its precise role in synaptic plasticity and memory remains elusive. Proteolytic cleavage of p35, a regulatory subunit of Cdk5, by calpain results in the generation of the truncated p25 protein, which causes hyperactivation of Cdk5. Using region-specific and inducible transgenic mice, we show that transiently increased p25 expression in the hippocampus enhanced long-term potentiation (LTP) and facilitated hippocampus-dependent memory. Moreover, p25 expression increased the number of dendritic spines and synapses. Importantly, enhanced memory achieved by a transient expression of p25 followed by its repression did not cause neurodegeneration. In contrast, prolonged p25 production caused severe cognitive deficits, which were accompanied by synaptic and neuronal loss and impaired LTP. Our data suggest a role for p25 in synaptic plasticity, synaptogenesis, learning, and memory and provide a model whereby deregulation of a plasticity factor can contribute to neurodegeneration.

The epigenetic bottleneck of neurodegenerative and psychiatric diseases.

The orchestrated expression of genes is essential for the development and survival of every organism. In addition to the role of transcription factors, the availability of genes for transcription is controlled by a series of proteins that regulate epigenetic chromatin remodeling. The two most studied epigenetic phenomena are DNA methylation and histone-tail modifications. Although a large body of literature implicates the deregulation of histone acetylation and DNA methylation with the pathogenesis of cancer, recently epigenetic mechanisms have also gained much attention in the neuroscientific community. In fact, a new field of research is rapidly emerging and there is now accumulating evidence that the molecular machinery that regulates histone acetylation and DNA methylation is intimately involved in synaptic plasticity and is essential for learning and memory. Importantly, dysfunction of epigenetic gene expression in the brain might be involved in neurodegenerative and psychiatric diseases. In particular, it was found that inhibition of histone deacetylases attenuates synaptic and neuronal loss in animal models for various neurodegenerative diseases and improves cognitive function. In this article, we will summarize recent data in the novel field of neuroepigenetics and discuss the question why epigenetic strategies are suitable therapeutic approaches for the treatment of brain diseases.

FOXG1 Regulates PRKAR2B Transcriptionally and Posttranscriptionally via miR200 in the Adult Hippocampus.

Rett syndrome is a complex neurodevelopmental disorder that is mainly caused by mutations in MECP2. However, mutations in FOXG1 cause a less frequent form of atypical Rett syndrome, called FOXG1 syndrome. FOXG1 is a key transcription factor crucial for forebrain development, where it maintains the balance between progenitor proliferation and neuronal differentiation. Using genome-wide small RNA sequencing and quantitative proteomics, we identified that FOXG1 affects the biogenesis of miR200b/a/429 and interacts with the ATP-dependent RNA helicase, DDX5/p68. Both FOXG1 and DDX5 associate with the microprocessor complex, whereby DDX5 recruits FOXG1 to DROSHA. RNA-Seq analyses of Foxg1cre/+ hippocampi and N2a cells overexpressing miR200 family members identified cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) as a target of miR200 in neural cells. PRKAR2B inhibits postsynaptic functions by attenuating protein kinase A (PKA) activity; thus, increased PRKAR2B levels may contribute to neuronal dysfunctions in FOXG1 syndrome. Our data suggest that FOXG1 regulates PRKAR2B expression both on transcriptional and posttranscriptional levels.

Regulatory mechanisms of fear extinction and depression-like behavior.

Human anxiety is frequently accompanied by depression, and when they co-occur both conditions exhibit greater severity and resistance to treatment. Little is known, however, about the molecular processes linking these emotional and mood disorders. Based on previously reported phosphorylation patterns of extracellular signal-regulated kinase (ERK) in the brain, we hypothesized that ERK's upstream activators intertwine fear and mood regulation through their hippocampal actions. We tested this hypothesis by studying the upstream regulation of ERK signaling in behavioral models of fear and depression. Wild-type and ERK1-deficient mice were used to study the dorsohippocampal actions of the putative ERK activators: mitogen-activated and extracellular signal-regulated kinase (MEK), protein kinase C (PKC), and cAMP-dependent protein kinase (PKA). Mice lacking ERK1 exhibited enhanced fear extinction and reduced depression caused by overactivation of ERK2. Both behaviors were reversed by inhibition of MEK, however the extinction phenotype depended on hippocampal, whereas the depression phenotype predominantly involved extrahippocampal MEK. Unexpectedly, inhibition of PKC accelerated extinction and decreased depression by ERK-independent mechanisms, whereas inhibition of PKA did not produce detectable molecular or behavioral effects in the employed paradigm. These results indicate that, contrary to fear conditioning but similar to mood stabilization, extinction of fear required upregulation of MEK/ERK and downregulation of ERK-independent PKC signaling. The dissociation of these pathways may thus represent a common mechanism for fear and mood regulation, and a potential therapeutic option for comorbid anxiety and depression.