Mitochondrial regulation of adult hippocampal neurogenesis: Insights into neurological function and neurodevelopmental disorders.

Mitochondria are essential regulators of cellular energy metabolism and play a crucial role in the maintenance and function of neuronal cells. Studies in the last decade have highlighted the importance of mitochondrial dynamics and bioenergetics in adult neurogenesis, a process that significantly influences cognitive function and brain plasticity. In this review, we examine the mechanisms by which mitochondria regulate adult neurogenesis, focusing on the impact of mitochondrial function on the behavior of neural stem/progenitor cells and the maturation and plasticity of newborn neurons in the adult mouse hippocampus. In addition, we explore the link between mitochondrial dysfunction, adult hippocampal neurogenesis and genes associated with cognitive deficits in neurodevelopmental disorders. In particular, we provide insights into how alterations in the transcriptional regulator NR2F1 affect mitochondrial dynamics and may contribute to the pathophysiology of the emerging neurodevelopmental disorder Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS). Understanding how genes involved in embryonic and adult neurogenesis affect mitochondrial function in neurological diseases might open new directions for therapeutic interventions aimed at boosting mitochondrial function during postnatal life.

Tamoxifen exerts direct and microglia-mediated effects preventing neuroinflammatory changes in the adult mouse hippocampal neurogenic niche.

Tamoxifen-inducible systems are widely used in research to control Cre-mediated gene deletion in genetically modified animals. Beyond Cre activation, tamoxifen also exerts off-target effects, whose consequences are still poorly addressed. Here, we investigated the impact of tamoxifen on lipopolysaccharide (LPS)-induced neuroinflammatory responses, focusing on the neurogenic activity in the adult mouse dentate gyrus. We demonstrated that a four-day LPS treatment led to an increase in microglia, astrocytes and radial glial cells with concomitant reduction of newborn neurons. These effects were counteracted by a two-day tamoxifen pre-treatment. Through selective microglia depletion, we elucidated that both LPS and tamoxifen influenced astrogliogenesis via microglia mediated mechanisms, while the effects on neurogenesis persisted even in a microglia-depleted environment. Notably, changes in radial glial cells resulted from a combination of microglia-dependent and -independent mechanisms. Overall, our data reveal that tamoxifen treatment per se does not alter the balance between adult neurogenesis and astrogliogenesis but does modulate cellular responses to inflammatory stimuli exerting a protective role within the adult hippocampal neurogenic niche.

NR2F1 shapes mitochondria in the mouse brain, providing new insights into Bosch-Boonstra-Schaaf optic atrophy syndrome.

The nuclear receptor NR2F1 acts as a strong transcriptional regulator in embryonic and postnatal neural cells. In humans, mutations in the NR2F1 gene cause Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), a rare neurodevelopmental disorder characterized by multiple clinical features including vision impairment, intellectual disability and autistic traits. In this study, we identified, by genome-wide and in silico analyses, a set of nuclear-encoded mitochondrial genes as potential genomic targets under direct NR2F1 transcriptional control in neurons. By combining mouse genetic, neuroanatomical and imaging approaches, we demonstrated that conditional NR2F1 loss of function within the adult mouse hippocampal neurogenic niche results in a reduced mitochondrial mass associated with mitochondrial fragmentation and downregulation of key mitochondrial proteins in newborn neurons, the genesis, survival and functional integration of which are impaired. Importantly, we also found dysregulation of several nuclear-encoded mitochondrial genes and downregulation of key mitochondrial proteins in the brain of Nr2f1-heterozygous mice, a validated BBSOAS model. Our data point to an active role for NR2F1 in the mitochondrial gene expression regulatory network in neurons and support the involvement of mitochondrial dysfunction in BBSOAS pathogenesis.

Beta-Caryophyllene Modifies Intracellular Lipid Composition in a Cell Model of Hepatic Steatosis by Acting through CB2 and PPAR Receptors.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease; however, no specific pharmacological therapy has yet been approved for this condition. Plant-derived extracts can be an important source for the development of new drugs. The aim of this study was to investigate the effects of (E)-ß-caryophyllene (BCP), a phytocannabinoid recently found to be beneficial against metabolic diseases, on HepG2 steatotic hepatocytes. Using a fluorescence-based lipid quantification assay and GC-MS analysis, we show that BCP is able to decrease lipid accumulation in steatotic conditions and to change the typical steatotic lipid profile by primarily reducing saturated fatty acids. By employing specific antagonists, we demonstrate that BCP action is mediated by multiple receptors: CB2 cannabinoid receptor, peroxisome proliferator-activated receptor α (PPARα) and γ (PPARγ). Interestingly, BCP was able to counteract the increase in CB2 and the reduction in PPARα receptor expression observed in steatotic conditions. Moreover, through immunofluorescence and confocal microscopy, we demonstrate that CB2 receptors are mainly intracellularly localized and that BCP is internalized in HepG2 cells with a maximum peak at 2 h, suggesting a direct interaction with intracellular receptors. The results obtained with BCP in normal and steatotic hepatocytes encourage future applications in the treatment of NAFLD.

Chronic thinner inhalation alters olfactory behaviors in adult mice.

Volatile solvents exposure can result in various behavioral impairments that have been partly associated to altered adult hippocampal neurogenesis. Despite recent evidence supporting this association, few studies have been devoted to examine the impact on olfactory functioning and olfactory bulb (OB) neurogenesis, although olfactory system is directly in contact with volatile molecules. Thus, this study was designed to evaluate in adult mice the potential modifications of the olfactory functioning after acute (1 day), subchronic (6 weeks) and chronic (12 weeks) exposure to thinner vapor at both behavioral and cellular levels. Firstly, behavioral evaluations showed that chronic thinner exposure impacts on odor detection ability of treated mice but does not affect mice ability to efficiently discriminate between two different odors. Moreover, chronic thinner exposure produces impairment in the olfactory-mediated associative memory. Secondly, analysis of the effects of thinner exposure in the subventricular zone (SVZ) of the lateral ventricle and in the OB revealed that thinner treatments do not induce apoptosis nor glial activation. Thirdly, immunohistochemical quantification of different markers of adult olfactory neurogenesis showed that inhalant treatments do not change the number of proliferating progenitors in the SVZ and the rostral migratory stream (RMS), as well as the number of newborn cells reaching and integrating in the OB circuitry. Altogether, our data highlight that the impaired olfactory performances in chronically-exposed mice are not associated to an alteration of adult neurogenesis in the SVZ-OB system.

Sex Steroids and the Shaping of the Peripubertal Brain: The Sexual-Dimorphic Set-Up of Adult Neurogenesis.

Steroid hormones represent an amazing class of molecules that play pleiotropic roles in vertebrates. In mammals, during postnatal development, sex steroids significantly influence the organization of sexually dimorphic neural circuits underlying behaviors critical for survival, such as the reproductive one. During the last decades, multiple studies have shown that many cortical and subcortical brain regions undergo sex steroid-dependent structural organization around puberty, a critical stage of life characterized by high sensitivity to external stimuli and a profound structural and functional remodeling of the organism. Here, we first give an overview of current data on how sex steroids shape the peripubertal brain by regulating neuroplasticity mechanisms. Then, we focus on adult neurogenesis, a striking form of persistent structural plasticity involved in the control of social behaviors and regulated by a fine-tuned integration of external and internal cues. We discuss recent data supporting that the sex steroid-dependent peripubertal organization of neural circuits involves a sexually dimorphic set-up of adult neurogenesis that in turn could be relevant for sex-specific reproductive behaviors.

Neurogranin Regulates Adult-Born Olfactory Granule Cell Spine Density and Odor-Reward Associative Memory in Mice.

Neurogranin (Ng) is a brain-specific postsynaptic protein, whose role in modulating Ca2+/calmodulin signaling in glutamatergic neurons has been linked to enhancement in synaptic plasticity and cognitive functions. Accordingly, Ng knock-out (Ng-ko) mice display hippocampal-dependent learning and memory impairments associated with a deficit in long-term potentiation induction. In the adult olfactory bulb (OB), Ng is expressed by a large population of GABAergic granule cells (GCs) that are continuously generated during adult life, undergo high synaptic remodeling in response to the sensory context, and play a key role in odor processing. However, the possible implication of Ng in OB plasticity and function is yet to be investigated. Here, we show that Ng expression in the OB is associated with the mature state of adult-born GCs, where its active-phosphorylated form is concentrated at post-synaptic sites. Constitutive loss of Ng in Ng-ko mice resulted in defective spine density in adult-born GCs, while their survival remained unaltered. Moreover, Ng-ko mice show an impaired odor-reward associative memory coupled with reduced expression of the activity-dependent transcription factor Zif268 in olfactory GCs. Overall, our data support a role for Ng in the molecular mechanisms underlying GC plasticity and the formation of olfactory associative memory.

Quantal Release of Dopamine and Action Potential Firing Detected in Midbrain Neurons by Multifunctional Diamond-Based Microarrays.

Micro-Graphitic Single Crystal Diamond Multi Electrode Arrays (µG-SCD-MEAs) have so far been used as amperometric sensors to detect catecholamines from chromaffin cells and adrenal gland slices. Besides having time resolution and sensitivity that are comparable with carbon fiber electrodes, that represent the gold standard for amperometry, µG-SCD-MEAs also have the advantages of simultaneous multisite detection, high biocompatibility and implementation of amperometric/potentiometric protocols, aimed at monitoring exocytotic events and neuronal excitability. In order to adapt diamond technology to record neuronal activity, the µG-SCD-MEAs in this work have been interfaced with cultured midbrain neurons to detect electrical activity as well as quantal release of dopamine (DA). µG-SCD-MEAs are based on graphitic sensing electrodes that are embedded into the diamond matrix and are fabricated using MeV ion beam lithography. Two geometries have been adopted, with 4 × 4 and 8 × 8 microelectrodes (20 µm × 3.5 µm exposed area, 200 µm spacing). In the amperometric configuration, the 4 × 4 µG-SCD-MEAs resolved quantal exocytosis from midbrain dopaminergic neurons. KCl-stimulated DA release occurred as amperometric spikes of 15 pA amplitude and 0.5 ms half-width, at a mean frequency of 0.4 Hz. When used as potentiometric multiarrays, the 8 × 8 µG-SCD-MEAs detected the spontaneous firing activity of midbrain neurons. Extracellularly recorded action potentials (APs) had mean amplitude of ∼-50 µV and occurred at a mean firing frequency of 0.7 Hz in 67% of neurons, while the remaining fired at 6.8 Hz. Comparable findings were observed using conventional MEAs (0.9 and 6.4 Hz, respectively). To test the reliability of potentiometric recordings with µG-SCD-MEAs, the D2-autoreceptor modulation of firing was investigated by applying levodopa (L-DOPA, 20 µM), and comparing µG-SCD-MEAs, conventional MEAs and current-clamp recordings. In all cases, L-DOPA reduced the spontaneous spiking activity in most neurons by 70%, while the D2-antagonist sulpiride reversed this effect. Cell firing inhibition was generally associated with increased APs amplitude. A minority of neurons was either insensitive to, or potentiated by L-DOPA, suggesting that AP recordings originate from different midbrain neuronal subpopulations and reveal different modulatory pathways. Our data demonstrate, for the first time, that µG-SCD-MEAs are multi-functional biosensors suitable to resolve real-time DA release and AP firing in in vitro neuronal networks.

Neuron-Astroglia Cell Fate Decision in the Adult Mouse Hippocampal Neurogenic Niche Is Cell-Intrinsically Controlled by COUP-TFI In Vivo.

In the dentate gyrus (DG) of the mouse hippocampus, neurogenesis and astrogliogenesis persist throughout life. Adult-born neurons and astrocytes originate from multipotent neural stem cells (NSCs) whose activity is tightly regulated within the neurogenic niche. However, the cell-intrinsic mechanisms controlling neuron-glia NSC fate choice are largely unknown. Here, we show COUP-TFI/NR2F1 expression in DG NSCs and its downregulation upon neuroinflammation. By using in vivo inducible knockout lines, a retroviral-based loss-of-function approach and genetic fate mapping, we demonstrate that COUP-TFI inactivation in adult NSCs and/or mitotic progenitors reduces neurogenesis and increases astrocyte production without depleting the NSC pool. Moreover, forced COUP-TFI expression in adult NSCs/progenitors decreases DG astrogliogenesis and rescues the neuro-astrogliogenic imbalance under neuroinflammation. Thus, COUP-TFI is necessary and sufficient to promote neurogenesis by suppressing astrogliogenesis. Our data propose COUP-TFI as a central regulator of the neuron-astroglia cell fate decision and a key modulator during neuroinflammation in the adult hippocampus.

Decreased Hippocampal Neuroplasticity and Behavioral Impairment in an Animal Model of Inhalant Abuse.

Thinners are highly toxic chemicals widely employed as organic solvents in industrial and domestic use. They have psychoactive properties when inhaled, and their chronic abuse as inhalants is associated with severe long-term health effects, including brain damage and cognitive-behavioral alterations. Yet, the sites and mechanisms of action of these compounds on the brain are far from being fully understood. Here, we investigated the consequences of paint thinner inhalation in adult male mice. Depression-like behaviors and an anxiolytic effect were found following repeated exposure in chronic treatments lasting 12 weeks. Both subchronic (6 weeks) and chronic treatments impaired learning and memory functions, while no changes were observed after acute treatment. To investigate possible molecular/structural alterations underlying such behavioral changes, we focused on the hippocampus. Notably, prolonged, but not acute thinner inhalation strongly affected adult neurogenesis in the dentate gyrus (DG), reducing progenitor cell proliferation after chronic treatments and impairing the survival of newborn neurons following both chronic and subchronic treatments. Furthermore, a down-regulation in the expression of BDNF and NMDA receptor subunits as well as a reduction in CREB expression/phosphorylation were found in the hippocampi of chronically treated mice. Our findings demonstrate for the first time significant structural and molecular changes in the adult hippocampus after prolonged paint thinner inhalation, indicating reduced hippocampal neuroplasticity and strongly supporting its implication in the behavioral dysfunctions associated to inhalant abuse.

Stage-specific functions of Semaphorin7A during adult hippocampal neurogenesis rely on distinct receptors.

The guidance protein Semaphorin7A (Sema7A) is required for the proper development of the immune and nervous systems. Despite strong expression in the mature brain, the role of Sema7A in the adult remains poorly defined. Here we show that Sema7A utilizes different cell surface receptors to control the proliferation and differentiation of neural progenitors in the adult hippocampal dentate gyrus (DG), one of the select regions of the mature brain where neurogenesis occurs. PlexinC1 is selectively expressed in early neural progenitors in the adult mouse DG and mediates the inhibitory effects of Sema7A on progenitor proliferation. Subsequently, during differentiation of adult-born DG granule cells, Sema7A promotes dendrite growth, complexity and spine development through ß1-subunit-containing integrin receptors. Our data identify Sema7A as a key regulator of adult hippocampal neurogenesis, providing an example of how differential receptor usage spatiotemporally controls and diversifies the effects of guidance cues in the adult brain.

Detecting Neuronal Differentiation Markers in Newborn Cells of the Adult Brain.

Adult neurogenesis is the lifelong generation of new neurons that occurs into restricted regions of the adult mammalian brain, namely the dentate gyrus of the hippocampus and the olfactory bulb. In this chapter, we describe the procedures aimed to investigate adult neurogenesis in the murine brain. Specifically, we explain how to inject into animals exogenous markers of proliferation (i.e., BrdU) and prepare brain tissues to perform immunofluorescence reactions for neuronal markers in combination with BrdU staining. As BrdU is incorporated in the DNA during the S-phase of the cell cycle of proliferating cells and is then inherited by daughter cells, by coupling BrdU-immunoreactivity together with the immunolabeling for neuronal markers, we provide the general procedures that can be applied to identify adult-born neurons and to characterize their specific phenotypes in different brain regions, under physiological condition or in pathological states.

Neuregulin1 alpha activates migration of neuronal progenitors expressing ErbB4.

Deficits in neuronal migration during development in the central nervous system may contribute to psychiatric diseases. The ligand neuregulin1 (NRG1) and its receptor ErbB4 are genes conferring susceptibility to schizophrenia, playing a key role in the control of neuronal migration both during development and adulthood. Several NRG1 and ErbB4 isoforms were identified, which deeply differ in their characteristics. Here we focused on the four ErbB4 isoforms and the two NRG1 isoforms differing in their EGF-like domain, namely α and ß. We hypothesized that these isoforms, which are differently regulated in schizophrenic patients, could play different roles in neuronal migration. Our hypothesis was strengthened by the observation that both NRG1α and NRG1ß and the four ErbB4 isoforms are expressed in the medial and lateral ganglionic eminences and in the cortex during development in rat. We analysed in vitro the signal transduction pathways activated by the different ErbB4 isoforms following the treatment with soluble recombinant NRG1α or NRG1ß and the ability to stimulate migration. Our data show that two ErbB4 isoforms, namely JMa-cyt2 and JMb-cyt1, following NRG1α and NRG1ß treatment, strongly activate AKT phosphorylation, conferring high migratory activity to neuronal progenitors, thus demonstrating that both NRG1α and NRG1ß can play a role in neuronal migration.

Opposite-sex attraction in male mice requires testosterone-dependent regulation of adult olfactory bulb neurogenesis.

Opposite-sex attraction in most mammals depends on the fine-tuned integration of pheromonal stimuli with gonadal hormones in the brain circuits underlying sexual behaviour. Neural activity in these circuits is regulated by sensory processing in the accessory olfactory bulb (AOB), the first central station of the vomeronasal system. Recent evidence indicates adult neurogenesis in the AOB is involved in sex behaviour; however, the mechanisms underlying this function are unknown. By using Semaphorin 7A knockout (Sema7A ko) mice, which show a reduced number of gonadotropin-releasing-hormone neurons, small testicles and subfertility, and wild-type males castrated during adulthood, we demonstrate that the level of circulating testosterone regulates the sex-specific control of AOB neurogenesis and the vomeronasal system activation, which influences opposite-sex cue preference/attraction in mice. Overall, these data highlight adult neurogenesis as a hub for the integration of pheromonal and hormonal cues that control sex-specific responses in brain circuits.

Adult Born Olfactory Bulb Dopaminergic Interneurons: Molecular Determinants and Experience-Dependent Plasticity.

The olfactory bulb (OB) is a highly plastic brain region involved in the early processing of olfactory information. A remarkably feature of the OB circuits in rodents is the constitutive integration of new neurons that takes place during adulthood. Newborn cells in the adult OB are mostly inhibitory interneurons belonging to chemically, morphologically and functionally heterogeneous types. Although there is general agreement that adult neurogenesis in the OB plays a key role in sensory information processing and olfaction-related plasticity, the contribution of each interneuron subtype to such functions is far to be elucidated. Here, we focus on the dopaminergic (DA) interneurons: we highlight recent findings about their morphological features and then describe the molecular factors required for the specification/differentiation and maintenance of the DA phenotype in adult born neurons. We also discuss dynamic changes of the DA interneuron population related to age, environmental stimuli and lesions, and their possible functional implications.

Semaphorin7A regulates neuroglial plasticity in the adult hypothalamic median eminence.

Reproductive competence in mammals depends on the projection of gonadotropin-releasing hormone (GnRH) neurons to the hypothalamic median eminence (ME) and the timely release of GnRH into the hypothalamic-pituitary-gonadal axis. In adult rodents, GnRH neurons and the specialized glial cells named tanycytes periodically undergo cytoskeletal plasticity. However, the mechanisms that regulate this plasticity are still largely unknown. We demonstrate that Semaphorin7A, expressed by tanycytes, plays a dual role, inducing the retraction of GnRH terminals and promoting their ensheathment by tanycytic end feet via the receptors PlexinC1 and Itgb1, respectively. Moreover, Semaphorin7A expression is regulated during the oestrous cycle by the fluctuating levels of gonadal steroids. Genetic invalidation of Semaphorin7A receptors in mice induces neuronal and glial rearrangements in the ME and abolishes normal oestrous cyclicity and fertility. These results show a role for Semaphorin7A signalling in mediating periodic neuroglial remodelling in the adult ME during the ovarian cycle.

Odour enrichment increases adult-born dopaminergic neurons in the mouse olfactory bulb.

The olfactory bulb (OB) is the first brain region involved in the processing of olfactory information. In adult mice, the OB is highly plastic, undergoing cellular/molecular dynamic changes that are modulated by sensory experience. Odour deprivation induces down-regulation of tyrosine hydroxylase (TH) expression in OB dopaminergic interneurons located in the glomerular layer (GL), resulting in decreased dopamine in the OB. Although the effect of sensory deprivation is well established, little is known about the influence of odour enrichment on dopaminergic cells. Here we report that prolonged odour enrichment on C57BL/6J strain mice selectively increases TH-immunopositive cells in the GL by nearly 20%. Following odour enrichment on TH-green fluorescent protein (GFP) transgenic mice, in which GFP identified both mature TH-positive cells and putative immature dopaminergic cells expressing TH mRNA but not TH protein, we found a similar 20% increase in GFP-expressing cells, with no changes in the ratio between TH-positive and TH-negative cells. These data suggest that enriched conditions induce an expansion in the whole dopaminergic lineage. Accordingly, by using 5-bromo-2-deoxyuridine injections to label adult-generated cells in the GL of TH-GFP mice, we found an increase in the percentage of 5-bromo-2-deoxyuridine-positive dopaminergic cells in enriched compared with control conditions, whereas no differences were found for calretinin- and calbindin-positive subtypes. Strikingly, the fraction of newborn cells among the dopaminergic population doubled in enriched conditions. On the whole, our results demonstrate that odour enrichment drives increased integration of adult-generated dopaminergic cells that could be critical to adapt the OB circuits to the environmental incoming information.

The interplay between reproductive social stimuli and adult olfactory bulb neurogenesis.

Adult neurogenesis is a striking form of structural plasticity that adapts the brain to the changing world. Accordingly, new neuron production is involved in cognitive functions, such as memory, learning, and pattern separation. Recent data in rodents indicate a close link between adult neurogenesis and reproductive social behavior. This provides a key to unravel the functional meaning of adult neurogenesis in biological relevant contexts and, in parallel, opens new perspectives to explore the way the brain is processing social stimuli. In this paper we will summarize some of the major achievements on cues and mechanisms modulating adult neurogenesis during social behaviors related to reproduction and possible role/s played by olfactory newborn neurons in this context. We will point out that newborn interneurons in the accessory olfactory bulb (AOB) represent a privileged cellular target for social stimuli that elicit reproductive behaviors and that such cues modulate adult neurogenesis at two different levels increasing both proliferation of neuronal progenitors in the germinative regions and integration of newborn neurons into functional circuits. This dual mechanism provides fresh neurons that can be involved in critical activities for the individual fitness, that is, the processing of social stimuli driving the parental behavior and partner recognition.

COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons.

COUP-TFI is an orphan nuclear receptor acting as a strong transcriptional regulator in different aspects of forebrain embryonic development. In this study, we investigated COUP-TFI expression and function in the mouse olfactory bulb (OB), a highly plastic telencephalic region in which continuous integration of newly generated inhibitory interneurons occurs throughout life. OB interneurons belong to different populations that originate from distinct progenitor lineages. Here, we show that COUP-TFI is highly expressed in tyrosine hydroxylase (TH)-positive dopaminergic interneurons in the adult OB glomerular layer (GL). We found that odour deprivation, which is known to downregulate TH expression in the OB, also downregulates COUP-TFI in dopaminergic cells, indicating a possible correlation between TH- and COUP-TFI-activity-dependent action. Moreover, we demonstrate that conditional inactivation of COUP-TFI in the EMX1 lineage results in a significant reduction of both TH and ZIF268 expression in the GL. Finally, lentiviral vector-mediated COUP-TFI deletion in adult-generated interneurons confirmed that COUP-TFI acts cell-autonomously in the control of TH and ZIF268 expression. These data indicate that COUP-TFI regulates TH expression in OB cells through an activity-dependent mechanism involving ZIF268 induction and strongly argue for a maintenance rather than establishment function of COUP-TFI in dopaminergic commitment. Our study reveals a previously unknown role for COUP-TFI in the adult brain as a key regulator in the control of sensory-dependent plasticity in olfactory dopaminergic neurons.