Piwil2 (Mili) sustains neurogenesis and prevents cellular senescence in the postnatal hippocampus.

Adult neural progenitor cells (aNPCs) ensure lifelong neurogenesis in the mammalian hippocampus. Proper regulation of aNPC fate has thus important implications for brain plasticity and healthy aging. Piwi proteins and the small noncoding RNAs interacting with them (piRNAs) have been proposed to control memory and anxiety, but the mechanism remains elusive. Here, we show that Piwil2 (Mili) is essential for proper neurogenesis in the postnatal mouse hippocampus. RNA sequencing of aNPCs and their differentiated progeny reveal that Mili and piRNAs are dynamically expressed in neurogenesis. Depletion of Mili and piRNAs in the adult hippocampus impairs aNPC differentiation toward a neural fate, induces senescence, and generates reactive glia. Transcripts modulated upon Mili depletion bear sequences complementary or homologous to piRNAs and include repetitive elements and mRNAs encoding essential proteins for proper neurogenesis. Our results provide evidence of a critical role for Mili in maintaining fitness and proper fate of aNPCs, underpinning a possible involvement of the piRNA pathway in brain plasticity and successful aging.

Context-dependent effects of IL-2 rewire immunity into distinct cellular circuits.

Interleukin 2 (IL-2) is a key homeostatic cytokine, with therapeutic applications in both immunogenic and tolerogenic immune modulation. Clinical use has been hampered by pleiotropic functionality and widespread receptor expression, with unexpected adverse events. Here, we developed a novel mouse strain to divert IL-2 production, allowing identification of contextual outcomes. Network analysis identified priority access for Tregs and a competitive fitness cost of IL-2 production among both Tregs and conventional CD4 T cells. CD8 T and NK cells, by contrast, exhibited a preference for autocrine IL-2 production. IL-2 sourced from dendritic cells amplified Tregs, whereas IL-2 produced by B cells induced two context-dependent circuits: dramatic expansion of CD8+ Tregs and ILC2 cells, the latter driving a downstream, IL-5-mediated, eosinophilic circuit. The source-specific effects demonstrate the contextual influence of IL-2 function and potentially explain adverse effects observed during clinical trials. Targeted IL-2 production therefore has the potential to amplify or quench particular circuits in the IL-2 network, based on clinical desirability.

Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation.

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

Glucocorticoids Promote Fear Generalization by Increasing the Size of a Dentate Gyrus Engram Cell Population.

BACKGROUND: Traumatic experiences, such as conditioned threat, are coded as enduring memories that are frequently subject to generalization, which is characterized by (re-) expression of fear in safe environments. However, the neurobiological mechanisms underlying threat generalization after a traumatic experience and the role of stress hormones in this process remain poorly understood. METHODS: We examined the influence of glucocorticoid hormones on the strength and specificity of conditioned fear memory at the level of sparsely distributed dentate gyrus (DG) engram cells in male mice. RESULTS: We found that elevating glucocorticoid hormones after fear conditioning induces a generalized contextual fear response. This was accompanied by a selective and persistent increase in the excitability and number of activated DG granule cells. Selective chemogenetic suppression of these sparse cells in the DG prevented glucocorticoid-induced fear generalization and restored contextual memory specificity, while leaving expression of auditory fear memory unaffected. CONCLUSIONS: These results implicate the sparse ensemble of DG engram cells as a critical cellular substrate underlying fear generalization induced by glucocorticoid stress hormones.

Early life stress decreases cell proliferation and the number of putative adult neural stem cells in the adult hypothalamus.

Stress is a potent environmental factor that can confer potent and enduring effects on brain structure and function. Exposure to stress during early life (ELS) has been linked to a wide range of consequences later in life. In particular, ELS exerts lasting effects on neurogenesis in the adult hippocampus, suggesting that ELS is a significant regulator of adult neural stem cell numbers and function. Here, we investigated the effect of ELS on cell proliferation and the numbers of neural stem/precursor cells in another neurogenic region: the hypothalamus of adult mice. We show that ELS has long-term suppressive effects on cell proliferation in the hypothalamic parenchyma and reduces the numbers of putative hypothalamic neural stem/precursor cells at 4 months of age. Specifically, ELS reduced the number of PCNA + cells present in hypothalamic areas surrounding the 3rd ventricle with a specific reduction in the proliferation of Sox2+/Nestin-GFP + putative stem cells present in the median eminence at the base of the 3rd ventricle. Furthermore, ELS reduced the total numbers of ß-tanycytes lining the ventral 3rd ventricle, without affecting α-tanycyte numbers in more dorsal areas. These results are the first to indicate that ELS significantly reduces proliferation and ß-tanycyte numbers in the adult hypothalamus, and may have (patho)physiological consequences for metabolic regulation or other hypothalamic functions in which ß-tanycytes are involved.

LAY SUMMARYWe show for the first time, long-lasting effects of exposure to early life stress on cellular plasticity in the hypothalamus of adult mice.Stress in the first week of life resulted in reduced numbers of (proliferating) stem cells in specific subregions of the hypothalamus at an adult age.This loss of stem cells and decreased proliferation highlights how early life stress can affect hypothalamic functions in later life.

Adult Neural Stem Cell Regulation by Small Non-coding RNAs: Physiological Significance and Pathological Implications.

The adult neurogenic niches are complex multicellular systems, receiving regulatory input from a multitude of intracellular, juxtacrine, and paracrine signals and biological pathways. Within the niches, adult neural stem cells (aNSCs) generate astrocytic and neuronal progeny, with the latter predominating in physiological conditions. The new neurons generated from this neurogenic process are functionally linked to memory, cognition, and mood regulation, while much less is known about the functional contribution of aNSC-derived newborn astrocytes and adult-born oligodendrocytes. Accumulating evidence suggests that the deregulation of aNSCs and their progeny can impact, or can be impacted by, aging and several brain pathologies, including neurodevelopmental and mood disorders, neurodegenerative diseases, and also by insults, such as epileptic seizures, stroke, or traumatic brain injury. Hence, understanding the regulatory underpinnings of aNSC activation, differentiation, and fate commitment could help identify novel therapeutic avenues for a series of pathological conditions. Over the last two decades, small non-coding RNAs (sncRNAs) have emerged as key regulators of NSC fate determination in the adult neurogenic niches. In this review, we synthesize prior knowledge on how sncRNAs, such as microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs), may impact NSC fate determination in the adult brain and we critically assess the functional significance of these events. We discuss the concepts that emerge from these examples and how they could be used to provide a framework for considering aNSC (de)regulation in the pathogenesis and treatment of neurological diseases.

Co-administration of Anti microRNA-124 and -137 Oligonucleotides Prevents Hippocampal Neural Stem Cell Loss Upon Non-convulsive Seizures.

Convulsive seizures promote adult hippocampal neurogenesis (AHN) through a transient activation of neural stem/progenitor cells (NSPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG). However, in a significant population of epilepsy patients, non-convulsive seizures (ncSZ) are observed. The response of NSPCs to non-convulsive seizure induction has not been characterized before. We here studied first the short-term effects of controlled seizure induction on NSPCs fate and identity. We induced seizures of controlled intensity by intrahippocampally injecting increasing doses of the chemoconvulsant kainic acid (KA) and analyzed their effect on subdural EEG recordings, hippocampal structure, NSPC proliferation and the number and location of immature neurons shortly after seizure onset. After establishing a KA dose that elicits ncSZ, we then analyzed the effects of ncSZ on NSPC proliferation and NSC identity in the hippocampus. ncSZ specifically triggered neuroblast proliferation, but did not induce proliferation of NSPCs in the SGZ, 3 days post seizure onset. However, ncSZ induced significant changes in NSPC composition in the hippocampus, including the generation of reactive NSCs. Interestingly, intrahippocampal injection of a combination of two anti microRNA oligonucleotides targeting microRNA-124 and -137 normalized neuroblast proliferation and prevented NSC loss in the DG upon ncSZ. Our results show for the first time that ncSZ induce significant changes in neuroblast proliferation and NSC composition. Simultaneous antagonism of both microRNA-124 and -137 rescued seizure-induced alterations in NSPC, supporting their coordinated action in the regulation of NSC fate and proliferation and their potential for future seizure therapies.

The orphan nuclear receptor TLX: an emerging master regulator of cross-talk between microglia and neural precursor cells.

Neuroinflammation and neurogenesis have both been the subject of intensive investigation over the past 20 years. The sheer complexity of their regulation and their ubiquity in various states of health and disease have sometimes obscured the progress that has been made in unraveling their mechanisms and regulation. A recent study by Kozareva et al. (Neuronal Signaling (2019) 3), provides evidence that the orphan nuclear receptor TLX is central to communication between microglia and neural precursor cells and could help us understand how inflammation, mediated by microglia, influences the development of new neurons in the adult hippocampus. Here, we put recent studies on TLX into the context of what is known about adult neurogenesis and microglial activation in the brain, along with the many hints that these processes must be inter-related.

Glucocorticoid-mediated modulation of morphological changes associated with aging in microglia.

Microglia dynamically adapt their morphology and function during increasing age. However, the mechanisms behind these changes are to date poorly understood. Glucocorticoids (GCs) are long known and utilized for their immunomodulatory actions and endogenous GC levels are described to alter with advancing age. We here tested the hypothesis that age-associated elevations in GC levels implicate microglia function and morphology. Our data indicate a decrease in microglial complexity and a concomitant increase in GC levels during aging. Interestingly, enhancing GC levels in young mice enhanced microglial ramifications, while the knockdown of the glucocorticoid receptor expression in old mice aggravated age-associated microglial amoebification. These data suggest that GCs increase ramification of hippocampal microglia and may modulate age-associated changes in microglial morphology.

A Standardized Protocol for Stereotaxic Intrahippocampal Administration of Kainic Acid Combined with Electroencephalographic Seizure Monitoring in Mice.

Lack of scientific reproducibility is a growing concern and weak experimental practices may contribute to irreproducibility. Here, we describe an optimized and versatile protocol for stereotaxic intrahippocampal administration of Kainic Acid (KA) in mice with a C57Bl6 background. In this protocol, KA administration is combined with in vivo recording of neuronal activity with wired and wireless setups. Following our protocol, KA administration results in a robust dose-dependent induction of low-level epileptiform activity or Status Epilepticus (SE) and induces previously characterized hallmarks of seizure-associated pathology. The procedure consists of three main steps: Craniotomy, stereotaxic administration of KA, and placement of recording electrodes in intrahippocampal, and subdural locations. This protocol offers extended possibilities compared to the systemic administration of KA, as it allows the researcher to accurately regulate the local dose of KA and resulting seizure activity, and permits the use and study of convulsive and non-convulsive KA doses, resulting in higher reproducibility and lower inter-individual variability and mortality rates. Caution should be taken when translating this procedure to different strains of mice as inter-strain sensitivity to KA has been described before. The procedure can be performed in ~1 h by a trained researcher, while intrahippocampal administration of KA without placing recording electrodes can be done in 25 min, and can be easily adapted to the titrated intrahippocampal administration of other drugs.

Transcription factor oscillations in neural stem cells: Implications for accurate control of gene expression.

Naturally occurring oscillations in glucocorticoids induce a cyclic activation of the glucocorticoid receptor (GR), a well-characterized ligand-activated transcription factor. These cycles of GR activation/deactivation result in rapid GR exchange at genomic response elements and GR recycling through the chaperone machinery, ultimately generating pulses of GR-mediated transcriptional activity of target genes. In a recent article we have discussed the implications of circadian and high-frequency (ultradian) glucocorticoid oscillations for the dynamic control of gene expression in hippocampal neural stem/progenitor cells (NSPCs) (Fitzsimons et al., Front. Neuroendocrinol., 2016). Interestingly, this oscillatory transcriptional activity is common to other transcription factors, many of which regulate key biological functions in NSPCs, such as NF-kB, p53, Wnt and Notch. Here, we discuss the oscillatory behavior of these transcription factors, their role in a biologically accurate target regulation and the potential importance for a dynamic control of transcription activity and gene expression in NSPCs.

miRNA-Mediated Regulation of Adult Hippocampal Neurogenesis; Implications for Epilepsy.

Hippocampal neural stem/progenitor cells (NSPCs) proliferate and differentiate to generate new neurons across the life span of most mammals, including humans. This process takes place within a characteristic local microenvironment where NSPCs interact with a variety of other cell types and encounter systemic regulatory factors. Within this microenvironment, cell intrinsic gene expression programs are modulated by cell extrinsic signals through complex interactions, in many cases involving short non-coding RNA molecules, such as miRNAs. Here we review the regulation of gene expression in NSPCs by miRNAs and its possible implications for epilepsy, which has been linked to alterations in adult hippocampal neurogenesis.

Multi-omics profile of the mouse dentate gyrus after kainic acid-induced status epilepticus.

Temporal lobe epilepsy (TLE) can develop from alterations in hippocampal structure and circuit characteristics, and can be modeled in mice by administration of kainic acid (KA). Adult neurogenesis in the dentate gyrus (DG) contributes to hippocampal functions and has been reported to contribute to the development of TLE. Some of the phenotypical changes include neural stem and precursor cells (NPSC) apoptosis, shortly after their birth, before they produce hippocampal neurons. Here we explored these early phenotypical changes in the DG 3 days after a systemic injection of KA inducing status epilepticus (KA-SE), in mice. We performed a multi-omics experimental setup and analyzed DG tissue samples using proteomics, transcriptomics and microRNA profiling techniques, detecting the expression of 2327 proteins, 13401 mRNAs and 311 microRNAs. We here present a description of how these data were obtained and make them available for further analysis and validation. Our data may help to further identify and characterize molecular mechanisms involved in the alterations induced shortly after KA-SE in the mouse DG.

MicroRNA-124 and -137 cooperativity controls caspase-3 activity through BCL2L13 in hippocampal neural stem cells.

Adult neurogenesis continuously contributes new neurons to hippocampal circuits and the programmed death of a subset of immature cells provides a primary mechanism controlling this contribution. Epileptic seizures induce strong structural changes in the hippocampus, including the induction of adult neurogenesis, changes in gene expression and mitochondrial dysfunction, which may all contribute to epileptogenesis. However, a possible interplay between this factors remains largely unexplored. Here, we investigated gene expression changes in the hippocampal dentate gyrus shortly after prolonged seizures induced by kainic acid, focusing on mitochondrial functions. Using comparative proteomics, we identified networks of proteins differentially expressed shortly after seizure induction, including members of the BCL2 family and other mitochondrial proteins. Within these networks, we report for the first time that the atypical BCL2 protein BCL2L13 controls caspase-3 activity and cytochrome C release in neural stem/progenitor cells. Furthermore, we identify BCL2L13 as a novel target of the cooperative action of microRNA-124 and microRNA-137, both upregulated shortly after seizure induction. This cooperative microRNA-mediated fine-tuning of BCL2L13 expression controls casp3 activity, favoring non-apoptotic caspase-3 functions in NSPC exposed to KA and thereby may contribute to the early neurogenic response to epileptic seizures in the dentate gyrus.

Optogenetic inhibition of chemically induced hypersynchronized bursting in mice.

Synchronized activity is common during various physiological operations but can culminate in seizures and consequently in epilepsy in pathological hyperexcitable conditions in the brain. Many types of seizures are not possible to control and impose significant disability for patients with epilepsy. Such intractable epilepsy cases are often associated with degeneration of inhibitory interneurons in the cortical areas resulting in impaired inhibitory drive onto the principal neurons. Recently emerging optogenetic technique has been proposed as an alternative approach to control such seizures but whether it may be effective in situations where inhibitory processes in the brain are compromised has not been addressed. Here we used pharmacological and optogenetic techniques to block inhibitory neurotransmission and induce epileptiform activity in vitro and in vivo. We demonstrate that NpHR-based optogenetic hyperpolarization and thereby inactivation of a principal neuronal population in the hippocampus is effectively attenuating seizure activity caused by disconnected network inhibition both in vitro and in vivo. Our data suggest that epileptiform activity in the hippocampus caused by impaired inhibition may be controlled by optogenetic silencing of principal neurons and potentially can be developed as an alternative treatment for epilepsy.

Different subsets of newborn granule cells: a possible role in epileptogenesis?

Several factors, including epileptic seizures, can strongly stimulate ongoing neurogenesis in the adult hippocampus. Although adult-born granule cells generated after seizure activity have different physiological properties from their normal counterparts, they integrate into the existing, mature network of the adult hippocampal dentate gyrus. However, the exact role of the neurogenic response during epilepsy and its possible involvement in epileptogenesis have remained elusive. Here, we discuss recent studies shedding new light on the interplay between epilepsy and neurogenesis, and try to explain discrepancies in this literature by proposing seizure severity-dependent induction of two subsets of newborn cells with different properties. We hypothesise that a low seizure intensity would stimulate neurogenesis to a 'physiological plasticity' level and have few pathological consequences. In contrast, a high initial seizure intensity may induce a specific subset of altered and/or ectopically located new granule cells with different electrophysiological properties that could initiate hyperexcitatory recurrent networks that could, in turn, contribute to chronic epilepsy. This hypothesis may clarify previously contradictory data in the literature, and could thereby aid in our understanding of the role of neurogenesis in epileptogenesis, and open up promising avenues for therapeutic intervention.

Focal neocortical epilepsy affects hippocampal volume, shape, and structural integrity: a longitudinal MRI and immunohistochemistry study in a rat model.

PURPOSE: Extratemporal epilepsy often coincides with cognitive decline, which may be associated with hippocampal dysfunction. Severe hippocampal sclerosis can be detected with conventional neuroimaging in some patients with chronic extrahippocampal epilepsy (so-called dual pathology). However, subtle structural hippocampal changes may already develop at a much earlier phase, and in a larger number of patients. Our goal was to longitudinally characterize the development of bilateral hippocampal pathology in an experimental neocortical focal epilepsy model. METHODS: Focal unilateral neocortical epilepsy was induced by microinjection of tetanus toxin in the primary motor cortex in adult male Sprague-Dawley rats. Another group of age-matched rats served as controls. In both groups, structural magnetic resonance imaging (MRI) was performed at 1, 3, 7, and 10 weeks of follow-up. Bilateral hippocampi were outlined and macroscopically analyzed using a state-of-the-art point-based morphometry model. Hippocampal microstructural changes at the end of follow-up, 10 weeks after epilepsy induction, were assessed with postmortem standard cresyl-violet, Fluoro-Jade, proteolipid protein 1, vimentin, glial fibrillary acidic protein, and ionized calcium binding adaptor molecule 1 stainings. KEY FINDINGS: All rats in the injected group developed seizures. The ipsilateral hippocampal volume was on average 8.76 (mean) ± 3.32% (standard deviation) smaller in the epileptic animals as compared to controls (p = 0.01) during the 10 weeks of follow-up. The contralateral hippocampus showed a similar reduction of 8.49 (mean) ± 3.27% (standard deviation) in total volume (p = 0.02). Clear hippocampal shape differences were found between the two groups. The most affected areas after epilepsy induction were the bilateral dorso-mediorostral, dorsolateral, and ventrolateral areas of the hippocampi. Normal developmental shape changes of the hippocampus, as detected in control rats, were largely absent in the ipsilateral hippocampus of epileptic rats. Quantitative histologic analysis revealed significant neuronal loss in the hippocampus, most pronounced in the hilar subregion, globally impaired myelination, reactive astrocytosis, and activated microglia. We found a weak but significant correlation between the number of neurons and hippocampal volume (r = 0.25, p = 0.0025). SIGNIFICANCE: We found evidence of hippocampal pathology in both hemispheres following experimental focal neocortical epilepsy. The observed development of bilateral hippocampal pathology, with onset in the early stages of focal neocortical epilepsy, may be a significant factor in comorbidities, such as cognitive dysfunction, found in patients with extratemporal localization-related epilepsy.