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Diazepam Binding Inhibitor Promotes Progenitor Proliferation in the Postnatal SVZ by Reducing GABA Signaling
Abstract
The subventricular zone (SVZ) of the lateral ventricles is the largest neurogenic niche of the postnatal brain. New SVZ-generated neurons migrate via the rostral migratory stream to the olfactory bulb (OB) where they functionally integrate into preexisting neuronal circuits. Nonsynaptic GABA signaling was previously shown to inhibit SVZ-derived neurogenesis. Here we identify the endogenous protein diazepam binding inhibitor (DBI) as a positive modulator of SVZ postnatal neurogenesis by regulating GABA activity in transit-amplifying cells. We performed DBI loss- and gain-of-function experiments in vivo at the peak of postnatal OB neuron generation in mice and demonstrate that DBI enhances proliferation by preventing SVZ progenitors to exit the cell cycle. Furthermore, we provide evidence that DBI exerts its effect on SVZ progenitors via its octadecaneuropeptide proteolytic product (ODN) by inhibiting GABA-induced currents. Together our data reveal a regulatory mechanism by which DBI counteracts the inhibitory effect of nonsynaptic GABA signaling on subventricular neuronal proliferation.
... Specifically, we demonstrated that DBI modulates GABA A -receptor-mediated currents, both in the SVZ of the lateral ventricle and in the dentate gyrus, thereby regulating the proliferation of neural progenitors in juvenile and adult mice (Alfonso et al., 2012;Dumitru et al., 2017). ...
... DBI is a small protein (10 kDa) that, when secreted to the extracellular space (Abrahamsen and Stenmark, 2010;Loomis et al., 2010), binds to the benzodiazepine binding site of the GABA A receptor and modulates its activity (Bormann, 1991;Bormann et al., 1985;Christian et al., 2013;Gray et al., 1986). The DBI cleavage product octadecaneuropeptide (ODN) also binds with high affinity to the GABA A receptor (Slobodyansky et al., 1989), mimicking the effect of DBI in vitro and in vivo (Alfonso et al., 2012;Dumitru et al., 2017). Given the crucial function of DBI in adult neurogenesis and considering the aforementioned differences between embryonic and adult neurogenesis, we wondered whether DBI modulates neurogenesis during embryonic development. ...
... In contrast to the extensive knowledge regarding the modulation of GABA A receptor activity via the benzodiazepine binding site in the adult brain (Brickley and Mody, 2012;Sigel and Ernst, 2018), very little is known about whether and, if so, how such fine-tuning occurs during brain development. We reasoned that DBI is a promising candidate, as we previously demonstrated that the small versatile molecule modulates GABAergic currents of neural stem cells in the neurogenic niches of the postnatal brain (Alfonso et al., 2012;Dumitru et al., 2017). Indeed, in this study we identified DBI as a We provide in vivo evidence that DBI and its cleavage product ODN modulate GABA A -receptor-mediated currents in embryonic progenitor cells and thereby decrease progenitor cell proliferation. ...
Of the neurotransmitters that influence neurogenesis, gamma-aminobutyric acid (GABA) plays an outstanding role, and GABA receptors support non-synaptic signaling in progenitors and migrating neurons. Here, we report that expression levels of diazepam binding inhibitor (DBI), an endozepine that modulates GABA signaling, regulate embryonic neurogenesis, affecting the long-term outcome regarding the number of neurons in the postnatal mouse brain. We demonstrate that DBI is highly expressed in radial glia and intermediate progenitor cells in the germinal zones of the embryonic mouse brain that give rise to excitatory and inhibitory cells. The mechanism by which DBI controls neurogenesis involves its action as a negative allosteric modulator of GABA-induced currents on progenitor cells that express GABAA receptors containing γ2 subunits. DBI’s modulatory effect parallels that of GABAA-receptor-mediating signaling in these cells in the proliferative areas, reflecting the tight control that DBI exerts on embryonic neurogenesis.
... This is consistent with ECs constituting the major part of the important spinal CSF-CNS barrier (Cousins et al., 2021), as well as being a potential source of growth factors (Del Bigio, 2010). In brain neurogenic niches, endogenous signaling regulates the production of cells so that the demand for new cells of different lineages can be tuned to meet requirements (Alfonso et al., 2012;Daynac et al., 2013;Fabbiani et al., 2020;Miyajima et al., 2021;Morizur et al., 2018;Nguyen et al., 2003;Wang et al., 2003). One signaling mechanism is through GABA, which negatively regulates proliferative activity in the subventricular zone (SVZ) and subgranular zone (SGZ) (Bolteus and Bordey, 2004;Catavero et al., 2018;Ge et al., 2007;Nguyen et al., 2003). ...
... The exact mechanism of how GABA controls NSC cycle entry in the brain remained elusive until recently. Negative allosteric modulation of GABAergic neurotransmission by the endogenous ligand diazepam binding inhibitor (DBI) is an important mechanism regulating NSC cell cycle exit and the levels of this protein, and its breakdown products, therefore enable a precise equilibrium between quiescence and maintenance of proliferation (Alfonso et al., 2012;Dumitru et al., 2017). ...
... Sox2 + nuclei of ECs are located in the cytoplasm of DBI-IR cells (Fig. 4D-Dii). The full length DBI pro-peptide is a precursor to octadecaneuropeptide (ODN), a smaller fragment of DBI which also has high affinity for GABAaR/CBR (Slobodyansky et al., 1989) and replicates the effects of DBI at CBR (Alfonso et al., 2012;Dumitru et al., 2017). There was also colocalization of ODN with Sox2 + ECs ( Fig. 4E-Eii). ...
Manipulation of neural stem cell proliferation and differentiation in the postnatal CNS is receiving significant attention due to therapeutic potential. In the spinal cord, such manipulations may promote repair in conditions such as multiple sclerosis or spinal cord injury, but may also limit excessive cell proliferation contributing to tumours such as ependymomas. Here we show that when ambient GABA is increased in vigabatrin-treated or decreased in glutamic acid decarboxylase67-green fluorescent protein (GAD67-GFP) mice, the numbers of proliferating cells respectively decreased or increased. Thus, intrinsic spinal cord GABA levels are correlated with the extent of cell proliferation, providing important evidence for the possibility of manipulating these levels. Diazepam binding inhibitor, an endogenous protein that interacts with GABA receptors and its breakdown product, octadecaneuropeptide, which preferentially activates central benzodiazepine (CBR) sites, were highly expressed in the spinal cord, especially in ependymal cells surrounding the central canal. Furthermore, animals with reduced CBR activation via treatment with flumazenil or Ro15-4513, or with a G2F77I mutation in the CBR binding site had greater numbers of Ethynyl-2’-deoxyuridine positive cells compared to control, which maintained their stem cell status since the proportion of newly proliferated cells becoming oligodendrocytes or astrocytes was significantly lower. Altering endogenous GABA levels or modulating GABAergic signaling through specific sites on the GABA receptors therefore influences NSC proliferation in the adult spinal cord. These findings provide a basis for further study into how GABAergic signaling could be manipulated to enable spinal cord self-regeneration and recovery or limit pathological proliferative activity.
... Point mutation targeting the benzodiazepine site of GABA A R renders neuronal cells insensitive to ODN (Dumitru et al., 2017). At micromolar concentrations, electrophysiological studies show that ODN acts as a NAM on GABA A Rs, that is, reduces GABA A R-mediated inhibition (Guidotti et al., 1983;Ferrero et al., 1986;Barmack et al., 2004;Alfonso et al., 2012;Dumitru et al., 2017) with no epileptogenic effect (Vezzani et al., 1991). ODN appears to be a relatively new astroglial modulator of GABA A R signaling and should therefore be considered for its potential to correct the imbalance between excitation and inhibition that arises as a consequence of a stroke. ...
... To address the general hypothesis that extrinsic ODN can be used to safely manipulate neuronal excitability and influence postischemic repair, we first checked whether, as reported previously in vitro, ODN induced a measurable enhancement of cortical activity in vivo. In neuronal cell culture, the GABA A R NAM effect of ODN was observed in the micromolar to millimolar range (Guidotti et al., 1983;Ferrero et al., 1986;Alfonso et al., 2012). We therefore targeted this range to test ODN in vivo. ...
... The endozepine, ODN, has been repeatedly categorized as a GABA A R NAM (Bormann et al., 1985;Costa and Guidotti, 1991;Alfonso et al., 2012;Dumitru et al., 2017). Yet, all these experiments have been conducted in vitro. ...
Following stroke, the survival of neurons and their ability to re-establish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABAA receptors (GABAAR). Conversely, in the late phase, negative allosteric modulation of GABAAR can correct the sub-optimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABAAR synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which make it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that during the sub-acute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.SIGNIFICANCE STATEMENTStroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide (ODN), can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.
... This effect is flumazenil-dependent, consistent with a NAM effect on GABA A R. Concurrently, using the same preparation, another laboratory reported similar results with DBI and did not observe any activity induced by ODN or OP (Macdonald, Weddle, & Gross, 1986). Twenty-six years later, in two consecutive papers demonstrating the potency of endozepines to stimulate neurogenesis in the subventricular zone (SVZ), the research group of Monyer reported in acute brain slices that ODN induces a NAM effect on GABAmediated inhibitory current in neuronal stem cells (NSC; Alfonso, Le Magueresse, Zuccotti, Khodosevich, & Monyer, 2012). The NAM effect induced by ODN (about -50%) is achieved with a fairly high concentration of ODN (20 M). ...
... It is generally accepted that neurogenesis persists in several areas of the adult brain, notably in the SVZ of the lateral ventricles, the subgranular zone (SGZ) of the hippocampus, and in the hypothalamus (Gould, 2007;Kempermann et al., 2018;Spalding et al., 2013, for review). In the postnatal mouse brain, the DBI/ACBP gene is expressed in stem cells in the neurogenic niches of the SVZ and SGZ (Alfonso et al., 2012;Dumitru et al., 2017;Nochi et al., 2013). DBI/ACBP-expressing cells are labeled with antibodies against nestin and SOX2, two NSC markers, and Mash1, a transit-amplifying cell marker. ...
... Transfection of short hairpin RNA sequences (shRNA) against DBI/ACBP induces a decrease of neuronal proliferation in the SVZ (Alfonso et al., 2012). Quantification of BrdU + neurons in the OB, the final destination of the NSC-derived cells of the SVZ, 12 days post-injection, reveals a reduction of newborn neurons in this area (Alfonso et al., 2012). ...
The existence of specific binding sites for benzodiazepines (BZs) in the brain has prompted the search for endogenous BZ receptor ligands designated by the generic term « endozepines ». This has led to the identification of an 86-amino acid polypeptide capable of displacing [3H]diazepam binding to brain membranes, thus called diazepam-binding inhibitor (DBI). It was subsequently found that the sequence of DBI is identical to that of a lipid carrier protein termed acyl-CoA-binding protein (ACBP). The primary structure of DBI/ACBP has been well preserved, suggesting that endozepines exert vital functions. The DBI/ACBP gene is expressed by astroglial cells in the central nervous system, and by various cell types in peripheral organs. Endoproteolytic cleavage of DBI/ACBP generates several bioactive peptides including a triakontatetraneuropeptide that acts as a selective ligand of peripheral BZ receptors/translocator protein (PBR/TSPO), and an octadecaneuropeptide that activates a G protein-coupled receptor and behaves as an allosteric modulator of the GABAAR. Although DBI/ACBP is devoid of a signal peptide, endozepines are released by astrocytes in a regulated manner. Consistent with the diversity and wide distribution of BZ-binding sites, endozepines appear to exert a large array of biological functions and pharmacological effects. Thus, intracerebroventricular administration of DBI or derived peptides induces proconflict and anxiety-like behaviors, and reduces food intake. Reciprocally, the expression of DBI/ACBP mRNA is regulated by stress and metabolic signals. In vitro, endozepines stimulate astrocyte proliferation and protect neurons and astrocytes from apoptotic cell death. Endozepines also regulate neurosteroid biosynthesis and neuropeptide expression, and promote neurogenesis. In peripheral organs, endozepines activate steroid hormone production, stimulate acyl chain ceramide synthesis and trigger pro-inflammatory cytokine secretion. The expression of the DBI/ACBP gene is enhanced in addiction/withdrawal animal models, in patients with neurodegenerative disorders and in various types of tumors. We review herein the current knowledge concerning the various actions of endozepines and discusses the physiopathological implications of these regulatory gliopeptides.
... The bleak prognosis following initial diagnosis ($14 months) (Wen and Kesari, 2008) lends urgency to the need for a better understanding of the specific players and molecular pathways that orchestrate the growth and malignancy of brain tumors. We previously described that acyl-CoA-binding protein (ACBP) (originally named Diazepam Binding Inhibitor, DBI) is a potent regulator of neural stem and progenitor cell proliferation via central benzodiazepine receptor (i.e., GABA A receptor) signaling in the adult rodent neurogenic niches (Alfonso et al., 2012;Dumitru et al., 2017). Interestingly, ACBP shows very high expression in human gliomas, which prompted us to investigate whether this protein could also regulate cell proliferation in malignant brain tumors. ...
... We have previously reported that ACBP in neural progenitor cells from the adult neurogenic niches acts as a positive regulator of neurogenesis by negatively modulating GABA A receptor signaling (Alfonso et al., 2012;Dumitru et al., 2017). Therefore, we first investigated whether modulation of GABAergic signaling could be the mechanism by which high levels of ACBP expression promotes GBM proliferation. ...
... In the adult brain, ACBP is expressed only in astrocytes, neural stem cells, and progenitor cells (Dumitru et al., 2017). We have previously demonstrated that ACBP acts as a pro-proliferative agent in the adult neurogenic niches through GABAergic signaling modulation (Alfonso et al., 2012;Dumitru et al., 2017). Thus, the overall cellular functions of ACBP in neural progenitors and GBM cells converge on the similar outcome of proliferation support under both physiological and GBM conditions. ...
Glioblastoma multiforme (GBM) undergoes metabolic reprogramming to meet the high ATP and anabolic demands of the tumor cells. However, the role of fatty acid oxidation (FAO) and its regulators in the GBM context has been largely unknown. Here, we show that the neural stem cell pro-proliferative factor acyl-CoA-binding protein (ACBP, also known as DBI) is highly expressed in GBM, and by binding to acyl-CoAs, it cell-autonomously maintains high proliferation rates, promoting tumor growth and poor survival in several preclinical models. Mechanistic experiments using ACBP-acyl-CoA binding affinity variants and pharmacological FAO modulators suggest that ACBP supports tumor growth by controlling the availability of long-chain fatty acyl-CoAs to mitochondria, promoting FAO in GBM. Thus, our findings uncover a critical link between lipid metabolism and GBM progression established by ACBP and offer a potential therapeutic strategy for an effective anti-proliferative metabolic management of GBM.
... Ces cellules sont caractérisées par un profil électrophysiologique similaire à celui des neurones immatures, ainsi que par l'expression, même à l'âge adulte, du marqueur double cortine (DCX) [46]. Dans la moelle spinale adulte, la plupart de ces cellules neuronales sont des neurones GABAergiques suggérant qu'ils sont des neurones fonctionnels [47]. ...
... SCI causes massive cell death and inflammation, leading to free radical and cytokine production which exacerbates neurons and oligodendrocyte apoptosis at the lesion site and axonal degeneration above the injury [46,47]. Following SCI, a scar is formed which is composed of a fibrotic and a glial component. ...
Les lésions de la moelle spinale constituent un problème de santé public d’une ampleur grandissante. Bien que l’espérance de vie ait été améliorée, les patients médullo-lésés souffrent de certains handicaps entraînant une perte partielle ou complète des fonctions sensorielles et/ou motrices. La moelle spinale lésée entreprend aussitôt une réponse à cette lésion. Chronologiquement, la lésion se divise en deux grandes phases : la phase primaire qui se caractérise par la destruction tissulaire induite par le traumatisme mécanique, suivie d’une destruction cellulaire. Alors que la phase secondaire est la conséquence moléculaire et cellulaire de la phase primaire. Durant plusieurs années, différentes stratégies thérapeutiques ont été proposé principalement la thérapie cellulaire qui a prouvé ses effets bénéfiques dans différents modèles expérimentaux de la lésion , mais de nombreux obstacles sont à prendre en considération tel que, principalement, son caractère invasif. afin de pouvoir l’appliquer chez l’homme d’une manière efficace et reproductible . A la vue de ces contraintes cliniques, nous avons décidé d’explorer un traitement non invasif connu pour ses effets neuroprotecteurs et neurotrophiques dans le SNC ; la stimulation magnétique répétitive trans-spinale (rTSMS). Etonnement, peu d’études ont exploré cette thérapie dans le cadre des LMTs, et rare sont celles qui l’ont utilisé d’une manière focale, c’est à dire directement au niveau du site de la lésion. A ce jour, les mécanismes et les voies sous-jacentes de ces effets dans ce cadre restent toujours inconnus. C’est pourquoi nous avons entrepris de caractériser ces effets dans le cadre de mes travaux de Thèse. En effet, en premier lieu, nous avons évalué les effets de la rTSMS sur la réparation tissulaire, via la modulation de la cicatrice médullaire et de ces différentes composantes in vivo, ainsi que sur la récupération fonctionnelle dans différents paradigmes (aigue et chronique) et à différents âges (juvénile, adulte et vieux) chez des souris WT ayant subi une transsection complète de la moelle spinale. En second lieu, l’objectif était de décrire les mécanismes à l’origine des effets de la rTSMS. Pour ce faire, des analyses protéomiques ont été réalisées, puis nous avons évalué l’effet de la rTSMS sur la réactivité des cellules souches endogènes de la moelle, ainsi que, la contribution de ces dernières dans la mise en place de la cicatrice gliale in vitro et in vivo via un modèle de souris transgénique hFoxJ1-CreER T2 ::tdTomato. L’objectif global était d’étudier, pour la première fois, l’effet de la rTSMS sur la réponse des différentes composantes cellulaires résidentes de la moelle spinale, les mécanismes à l’origine de ces effets, ainsi que la capacité à restaurer les fonctions motrices perdues suite à la lésion médullaire.
... DBI and its derived peptides, including ODN, are collectively termed endozepines (Tonon et al. 2006). In the brain of vertebrates, DBI mRNA is predominantly expressed by glial cells (Tonon et al. 2006) and the occurrence of DBI-like immunoreactivity has been visualized in astroglial cells in various regions of the CNS, notably in ependymal cells bordering the cerebral ventricles, in tanycytes of the median eminence, in pituicytes of the pituitary, and in Bergmann cells of the cerebellum (Vidnyanszky et al. 1994;Tonon et al. 2006;Matsuda et al. 2007;Alfonso et al. 2012). It was initially reported that the endozepines ODN and DBI act as inverse agonists of centraltype benzodiazepine receptors (CBR) (Ferrero et al. 1986). ...
... It is now established that reactive astrocytes contribute to the defense of surrounding neurons under moderate oxidative stress by releasing neuroprotective factors, suggesting that overproduction of endozepines by astroglial cells observed in neurodegenerative disorders may be involved in the protection of neurons. Besides its neuroprotective effect, a recent study reported that ODN stimulates neurogenesis in adult mouse brain, and that DBI inhibition with siRNA leads to growth arrest and death of neural progenitors (Alfonso et al. 2012). Altogether, these data suggest that ODN might have a therapeutic potential for treatment of cerebral injuries involving oxidative neurodegeneration. ...
... ODN, via activation of the central-type BZ receptors, stimulates the incorporation of [ 3 H]thymidine into cultured rat astrocytes (74). Consistent with this proliferative effect of ODN, in vivo studies have demonstrated that ODN promotes the proliferation of neuronal progenitor stem cells from the germinative sub-ventricular zone in adult rat (75). Conversely, inhibition of DBI gene transcription by shRNA transfection in vivo reduces the number of proliferating cells and the number of neurons newly formed at the level of olfactory bulbs but does not affect stem cell survival (75). ...
... Consistent with this proliferative effect of ODN, in vivo studies have demonstrated that ODN promotes the proliferation of neuronal progenitor stem cells from the germinative sub-ventricular zone in adult rat (75). Conversely, inhibition of DBI gene transcription by shRNA transfection in vivo reduces the number of proliferating cells and the number of neurons newly formed at the level of olfactory bulbs but does not affect stem cell survival (75). Furthermore, the fact that ODN can inhibit neuronal cell death and stimulate neurogenesis suggests that it could also play a key role during brain development. ...
Octadecaneuropeptide (ODN) and its precursor diazepam-binding inhibitor (DBI) are peptides belonging to the family of endozepines. Endozepines are exclusively produced by astroglial cells in the central nervous system of mammals, and their release is regulated by stress signals and neuroactive compounds. There is now compelling evidence that the gliopeptide ODN protects cultured neurons and astrocytes from apoptotic cell death induced by various neurotoxic agents. In vivo, ODN causes a very strong neuroprotective action against neuronal degeneration in a mouse model of Parkinson's disease. The neuroprotective activity of ODN is based on its capacity to reduce inflammation, apoptosis, and oxidative stress. The protective effects of ODN are mediated through its metabotropic receptor. This receptor activates a transduction cascade of second messengers to stimulate protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) signaling pathways, which in turn inhibits the expression of proapoptotic factor Bax and the mitochondrial apoptotic pathway. In N2a cells, ODN also promotes survival and stimulates neurite outgrowth. During the ODN-induced neuronal differentiation process, numerous mitochondria and peroxisomes are identified in the neurites and an increase in the amount of cholesterol and fatty acids is observed. The antiapoptotic and neurotrophic properties of ODN, including its antioxidant, antiapoptotic, and pro-differentiating effects, suggest that this gliopeptide and some of its selective and stable derivatives may have therapeutic value for the treatment of some neurodegenerative diseases.
... In fact, ODN is involved in the control of food intake, sleep, aggressiveness, and anxiety . At the cellular level, numerous data indicate that ODN acts both as an autocrine and paracrine factor modulating proliferation and differentiation of astrocytes and neurons (Alfonso et al. 2012;Dumitru et al. 2017). In addition, ODN exerts a potent protective effect against the deleterious action of oxidative stress in cultured astrocytes Hamdi et al. 2011;Hamdi et al. 2012) and neurons (Kaddour et al. 2013). ...
... Several studies have shown that astroglialderived peptides, i.e. activity-dependent neurotrophic factor, activity-dependent neuroprotective protein and endozepine ODN, provide neuroprotection against various neurotoxins including 6-OHDA (Dejda et al. 2005;Kaddour et al. 2013;Masmoudi-Kouki et al. 2007), MPTP and alcohol (Sari and Gozes 2006). Besides its neuroprotective action, ODN is able to stimulate neurogenesis in the adult mouse brain, and silencing the ODN precursor, DBI, leads to growth arrest and neuro-progenitor cell death (Alfonso et al. 2012;Dumitru et al. 2017), suggesting that ODN plays a major role during brain development and on neuron-glia interaction. In support of this notion, it has been shown that, in the cerebellar cortex, the ODN precursor is exclusively expressed by Bergmann glia (Tonon et al. 1990), and alteration of these cells by 6-OHDA treatment reduces DBI expression and disturbs maturation and migration of granule neuron precursors (Podkletnova et al. 2001). ...
Oxidative stress, associated with various neurodegenerative diseases, induces imbalance in ROS generation, impairs cellular antioxidant defences and finally triggers both neurons and astroglial cell death by apoptosis. Astrocytes specifically synthesize and release endozepines, a family of regulatory peptides, including the octadecaneuropeptide (ODN). We have previously reported that ODN is a potent neuroprotective agent that prevents 6-OHDA-induced apoptotic neuronal death. The purpose of the present study was to investigate the potential glioprotective effect of ODN on 6-OHDA-induced oxidative stress and cell death in cultured rat astrocytes. Incubation of astrocytes with graded concentrations of ODN (10 ⁻¹⁴ to 10 ⁻⁸ M) inhibited 6-OHDA-evoked cell death in a concentration- and time-dependent manner. In addition, ODN prevented the decrease of mitochondrial activity and caspase-3 activation induced by 6-OHDA. Toxin-treated cells exhibited high level of ROS associated with a generation of H 2 O 2 and O 2 °- and a reduction of both SOD and catalase activities. Co-treatment of astrocytes with low concentrations of ODN dose dependently blocked 6-OHDA-evoked ROS production and inhibition of antioxidant enzymes activities. Taken together, these data demonstrate that ODN is a potent glioprotective agent that prevents 6-OHDA-induced oxidative stress and apoptotic cell death. ODN is thus a potential candidate to delay neuronal damages in various pathological conditions involving oxidative neurodegeneration.
... Point mutation targeting the benzodiazepine site of GABA A R renders neuronal cells insensitive to ODN (Dumitru et al., 2017). At micromolar concentrations, electrophysiological studies show that ODN acts as a NAM on GABA A Rs, i.e. reducing GABA A R mediated inhibition (Guidotti et al., 1983;Ferrero et al., 1986;Barmack et al., 2004;Alfonso et al., 2012;Dumitru et al., 2017) with no epileptogenic effect (Vezzani et al., 1991). ODN appears to be a relatively new astroglial modulator of GABA A R signaling and should therefore be considered for its potential to correct the imbalance between excitation and inhibition that arises as a consequence of a stroke. ...
... The endozepine, ODN, has been repeatedly categorized as a GABA A R NAM (Bormann et al., 1985;Costa and Guidotti, 1991;Alfonso et al., 2012;Dumitru et al., 2017). Yet, all these experiments have been conducted in vitro. ...
Following stroke, the survival of neurons and their ability to re-establish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABA A receptors (GABA A R). Conversely, in the late phase, negative allosteric modulation of GABA A R can correct the sub-optimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABA A R synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which make it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that during the sub-acute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.
SIGNIFICANCE STATEMENT
Stroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide (ODN), can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.
... Growing evidence suggests a role for stem cells as niche actors that regulate their own homeostasis and influence differentiation of their progeny ( Figure 2B). In the subventricular zone, NSCs and progenitors produce an endozepine called diazepam-binding inhibitor that inhibits GABA-A receptors and whose proteolytic product blocks the differentiation-promoting effects of neuroblast-derived GABA (Alfonso et al., 2012). As such, NSCs and progenitors maintain a proliferative state by antagonizing signals from their descendants (Alfonso et al., 2012). ...
... In the subventricular zone, NSCs and progenitors produce an endozepine called diazepam-binding inhibitor that inhibits GABA-A receptors and whose proteolytic product blocks the differentiation-promoting effects of neuroblast-derived GABA (Alfonso et al., 2012). As such, NSCs and progenitors maintain a proliferative state by antagonizing signals from their descendants (Alfonso et al., 2012). In the adult hippocampus, NSPCs regulate their own activation and quiescence through distinct autocrine and paracrine mechanisms. ...
Radial glia-like neural stem cells (RGLs) in the dentate gyrus subregion of the hippocampus give rise to dentate granule cells (DGCs) and astrocytes throughout life, a process referred to as adult hippocampal neurogenesis. Adult hippocampal neurogenesis is sensitive to experiences, suggesting that it may represent an adaptive mechanism by which hippocampal circuitry is modified in response to environmental demands. Experiential information is conveyed to RGLs, progenitors, and adult-born DGCs via the neurogenic niche that is composed of diverse cell types, extracellular matrix, and afferents. Understanding how the niche performs its functions may guide strategies to maintain its health span and provide a permissive milieu for neurogenesis. Here, we first discuss representative contributions of niche cell types to regulation of neural stem cell (NSC) homeostasis and maturation of adult-born DGCs. We then consider mechanisms by which the activity of multiple niche cell types may be coordinated to communicate signals to NSCs. Finally, we speculate how NSCs integrate niche-derived signals to govern their regulation.
... Whether GABA causes depolarization or hyperpolarization depends, at least in part, on the expression of Na + -K + -2Cl − co-transporter NKCC1 and a consequent accumulation of intracellular chloride ions 34 . Thus, activation of GABA A receptors in neural precursors results in chloride efflux and calcium influx 34,35 and attenuates the proliferation of NSCs 33,36,37 . It is thought that neuroblast-derived local GABA provides negative feedback for NSC proliferation 31,33 . ...
... It is thought that neuroblast-derived local GABA provides negative feedback for NSC proliferation 31,33 . This feedback loop is further adjusted by modulators such as diazepam-binding inhibitor (DBI), which is highly expressed in the SVZ and other neurogenic areas and inhibits GABA A -receptor (GABA A -R) mediated currents 37,38 (Fig. 2a). ...
The establishment of neuronal and glial networks in the brain depends on the activities of neural progenitors, which are influenced by cell-intrinsic mechanisms, interactions with the local microenvironment and long-range signaling. Progress in neuroscience has helped identify key factors in CNS development. In parallel, studies in recent years have increased our understanding of molecular and cellular factors in the development and growth of primary brain tumors. To thrive, glioma cells exploit pathways that are active in normal CNS progenitor cells, as well as in normal neurotransmitter signaling. Furthermore, tumor cells of incurable gliomas integrate into communicating multicellular networks, where they are interconnected through neurite-like cellular protrusions. In this Review, we discuss evidence that CNS development, organization and function share a number of common features with glioma progression and malignancy. These include mechanisms used by cells to proliferate and migrate, interact with their microenvironment and integrate into multicellular networks. The emerging intersections between the fields of neuroscience and neuro-oncology considered in this review point to new research directions and novel therapeutic opportunities.
... Together, these findings strongly suggest that the anorectic and metabolic effects of ACBP and its derived peptide ODN are mediated via the ARC melanocortin system. ODN activates POMC neurons through a GABAA independent but ODN GPCR dependent 8 mechanism ODN has been shown to act as a negative allosteric modulator of the GABAA receptor (39). Importantly, POMC neurons of the ARC receive strong inhibitory GABAergic inputs from neighboring neurons (40,41) suggesting that ODN-induced POMC neurons activation could be due to inhibition of GABAergic inputs. ...
... Interestingly, pan-brain ACBP overexpression leads to hydrocephalus (enlargement of lateral ventricles) in mice, suggesting that ependymal ACBP may regulate cerebrospinal fluid production and/or circulation (51). In addition, ACBP is expressed in the subventricular zone, comprising ependymocytes, where it promotes neuroprogenitor proliferation via GABAA inhibition (39,52). Additional work and genetic models will be needed to assess specifically the role of ACBP in tanycytes. ...
Glial cells have emerged as key players in the central control of energy balance and etiology of obesity. Astrocytes play a central role in neural communication via the release of gliotransmitters. Acyl-CoA binding protein (ACBP)-derived endozepines are secreted peptides that modulate the GABAA receptor. In the hypothalamus, ACBP is enriched in arcuate nucleus (ARC) astrocytes, ependymocytes and tanycytes. Central administration of the endozepine octadecaneuropeptide (ODN) reduces feeding and improves glucose tolerance, yet the contribution of endogenous ACBP in energy homeostasis is unknown. We demonstrated that ACBP deletion in GFAP+ astrocytes, but not in Nkx2.1-lineage neural cells, promoted diet-induced hyperphagia and obesity in both male and female mice, an effect prevented by viral rescue of ACBP in ARC astrocytes. ACBP-astrocytes were observed in apposition with proopiomelanocortin (POMC) neurons and ODN selectively activated POMC neurons through the ODN-GPCR but not GABAA, and supressed feeding while increasing carbohydrate utilization via the melanocortin system. Similarly, ACBP overexpression in ARC astrocytes reduced feeding and weight gain. Finally, the ODN-GPCR agonist decreased feeding and promoted weight loss in ob/ob mice. These findings uncover ACBP as an ARC gliopeptide playing a key role in energy balance control and exerting strong anorectic effects via the central melanocortin system.
... It is The copyright holder for this preprint this version posted May 5, 2024. ; https://doi.org/10.1101/2024.05.03.592384 doi: bioRxiv preprint ventricular zone 36 and in CA1 pyramidal cells 37 . However, endozepines are also reported to have positive allosteric effects on the GABAA receptor in dorsal root ganglia 38 and the dentate gyrus 37 indicating that endozepine action on GABAA receptors is not yet fully understood. ...
The central nervous system (CNS) plays a key role in regulating metabolic functions, but conditions like obesity and diabetes can disrupt this balance. Within the CNS, the nucleus of the solitary tract (NTS) in the dorsal vagal complex (DVC) controls glucose metabolism and feeding behaviour. In rodents, the NTS senses insulin and communicates with the liver to regulate glucose production. Even short term exposure to a high fat diet (HFD) can lead to insulin resistance and impair NTS function. However, we still know little about which cells in the NTS are sensitive to insulin. Our study aimed to identify these insulin sensitive cells and understand how they affect glucose metabolism.
We found that insulin receptors in astrocytes are crucial for the NTS ability to regulate glucose production in the liver. Insulin evokes the release of endozepines from astrocytes, and injecting endozepines into the NTS reduces glucose production. The effect of endozepines within the NTS is mimicked by GABAA antagonists and prevented by an agonist, suggesting that insulin prompts astrocytes to release endozepines, which then attenuate GABAA receptor activity, ultimately reducing glucose production in the liver.
Our study is the first to show that insulin dependent release of endozepines from NTS astrocytes is fundamental to control blood glucose levels, providing valuable insights into the mechanisms underlying insulin function within this specific region of the CNS.
... The expression of the Na/K/2Cl co-transporter NKCC1, which causes intracellular chloride accumulation, is mandatory for the GABA-A receptor to induce cell depolarization instead of hyperpolarization, thus reducing cell proliferation [16]. This regulatory mechanism is further influenced by modifiers like diazepam-binding inhibitor (DBI), which is abundantly present in neurogenic areas and which inhibits the GABA-A-receptor induced cell depolarization [53]. The depolarizing activity of GABA-A receptors has been reported to decrease cell growth in low-grade gliomas (LGGs) [27]. ...
In recent years, research on brain cancers has turned towards the study of the interplay between the tumor and its host, the normal brain. Starting from the establishment of a parallelism between neurogenesis and gliomagenesis, the influence of neuronal activity on the development of brain tumors, particularly gliomas, has been partially unveiled. Notably, direct electrochemical synapses between neurons and glioma cells have been identified, paving the way for new approaches for the cure of brain cancers. Since this novel field of study has been defined “cancer neuroscience”, anticancer therapeutic approaches exploiting these discoveries can be referred to as “cancer neuromodulation”. In the present review, we provide an up-to-date description of the novel findings and of the therapeutic neuromodulation perspectives in cancer neuroscience. We focus both on more traditional oncologic approaches, aimed at modulating the major pathways involved in cancer neuroscience through drugs or genetic engineering techniques, and on electric stimulation proposals; the latter is at the cutting-edge of neuro-oncology.
... Of the DEGs identified by bulk RNA-seq on ErbB4 +/+ and ErbB4 −/− embryos, CRYM, PRSS12 and DBi were found to be regulated by ErbB4-JMa. Interestingly, DBi has been shown to influence NPC proliferation through regulation of γ-aminobutyric acid type A receptors (GABAaRs) 44,45 , whose expression is also regulated by ErbB4 46,47 , raising the possibility that these three molecules interact during brain development. The decrease in DBi expression due to loss of ErbB4-JMa may play a role in the early transition from progenitor maintenance to astrogenesis. ...
Like all receptor tyrosine kinases (RTKs), ErbB4 signals through a canonical signaling involving phosphorylation cascades. However, ErbB4 can also signal through a non-canonical mechanism whereby the intracellular domain is released into the cytoplasm by regulated intramembrane proteolysis (RIP) and translocates to the nucleus where it regulates transcription. These different signaling mechanisms depend on the generation of alternative spliced isoforms, a RIP cleavable ErbB4-JMa and an uncleavable ErbB4-JMb. Non-canonical signaling by ErbB4-JMa has been implicated in the regulation of brain, heart, mammary gland, lung, and immune cell development. However, most studies on non-canonical ErbB4 signaling have been performed in vitro due to the lack of an adequate mouse model. We created an ErbB4-JMa specific knock out mouse and demonstrate that RIP-dependent, non-canonical signaling by ErbB4-JMa is required for the regulation of GFAP expression during cortical development. We also show that ErbB4-JMa signaling is not required for the development of the heart, mammary glands, sensory ganglia. Furthermore, we identify genes whose expression during cortical development is regulated by ErbB4, and show that the expression of three of them, CRYM and DBi, depend on ErbB4-JMa whereas WDFY1 relies on ErbB4-JMb. Thus, we provide the first animal model to directly study the roles of ErbB4-JMa and non-canonical ErbB4 signaling in vivo.
... Evidence from both the DG and SVZ suggests that NSCs may selfregulate their own response to ambient neurotransmitters via their secretome. In the adult SVZ, in vivo antibody staining revealed that type B NSCs and type C IPCs express diazepam binding inhibitor (DBI), a secreted, soluble molecule that can competitively bind to GABA receptors (Alfonso et al., 2012). SVZ type B NSCs and type C IPCs both express GABA receptors and are well established to decrease their proliferation in response to GABA (Fernando et al., 2011;Liu et al., 2005). ...
Neural stem cell (NSC) based therapies are at the forefront of regenerative medicine strategies to combat illness and injury of the central nervous system (CNS). In addition to their ability to produce new cells, NSCs secrete a variety of products, known collectively as the NSC secretome, that have been shown to ameliorate CNS disease pathology and promote recovery. As pre-clinical and clinical research to harness the NSC secretome for therapeutic purposes advances, a more thorough understanding of the endogenous NSC secretome can provide useful insight into the functional capabilities of NSCs. In this review, we focus on research investigating the autocrine and paracrine functions of the endogenous NSC secretome across life. Throughout development and adulthood, we find evidence that the NSC secretome is a critical component of how endogenous NSCs regulate themselves and their niche. We also find gaps in current literature, most notably in the clinically-relevant domain of endogenous NSC paracrine function in the injured CNS. Future investigations to further define the endogenous NSC secretome and its role in CNS tissue regulation are necessary to bolster our understanding of NSC-niche interactions and to aid in the generation of safe and effective NSC-based therapies.
... A previous study showed that this metabolic reprogramming mediated by DBI can promote tumor development (Duman et al., 2019). Interestingly, several studies have shown that DBI can maintain the proliferation of stem and progenitor cells (Alfonso et al., 2012;Dumitru et al., 2017). Interestingly, DBI expression is not restricted to gliomas but is also present in most tumor types pointing to the possibility of a general mechanism of DBI in other cancers. ...
Background: Drug resistance and recurrence often develop during the treatment of muscle-invasive bladder cancer (MIBC). The existence of cancer stem cells (CSCs) in MIBC makes the formulation of effective treatment strategies extremely challenging. We aimed to use single-cell RNA sequencing approaches to identify CSCs and evaluate their molecular characteristics and to discover possible therapeutic measures.
Methods: GEO data sets GSE130001 and GSE146137 were used to construct an expression matrix. After cells were identified by type, malignant epithelial cells inferred by InferCNV were extracted for stemness evaluation. The subset of cells with the highest stemness was subjected to weighted gene coexpression network analysis (WGCNA) and pseudotime analysis to identify key genes. In addition, we predicted drug sensitivity relationships for key genes in CTD and predicted the correlation between drugs and survival through siGDC.
Results: We found that there were some CSCs in MIBC samples. The CSC population was heterogeneous during tumor development and was divided into quiescent and proliferating CSCs. We identified DBI as the key gene in quiescent CSCs. Analysis of a TCGA data set showed that higher DBI expression indicated higher histological grade. In addition, we predicted that acetaminophen can reduce DBI expression, thereby reducing the stemness of CSCs. Thus, we identified a potential new use of acetaminophen.
Conclusion: We systematically explored CSCs in tumors and determined that DBI may be a key gene and potential therapeutic target in quiescent CSCs. In addition, we confirmed that acetaminophen may be a candidate drug targeting CSCs, improving our understanding of CSC-targeting therapeutic strategies.
... For instance, NOTCH and BMP are pro-quiescence signals while WNT and SHH are considered proactivation ones (Ahn and Joyner, 2005;Lie et al., 2005;Imayoshi et al., 2010;Mira et al., 2010). Metabolic cues and neurotransmitters such as GABA are also heavily involved (Alfonso et al., 2012;Berg et al., 2013;Knobloch and Jessberger, 2017;Paul et al., 2017;Catavero et al., 2018;Adusumilli et al., 2020). But in reality, cells rarely respond to a single signal, instead receiving a complex mix of signalling and metabolic cues simultaneously. ...
The majority of adult neural stem cells (aNSCs) are in a distinct metabolic state of reversible cell cycle exit also known as quiescence. The rate of aNSC activation determines the number of new neurons generated and directly influences the long-term maintenance of neurogenesis. Despite its relevance, it is still unclear how aNSC quiescence is regulated. Many factors contribute to this, like aNSC heterogeneity, the lack of reliable quiescence markers, the complexity of the neurogenic niches or the intricacy of the transcriptional and post-transcriptional mechanisms involved. In this perspective article I discuss possible solutions to these problems. But, first and foremost, I believe we require a model that goes beyond a simple transition toward activation. Instead, we must acknowledge the full complexity of aNSC states, which include not only activation but also differentiation and survival as behavioural outcomes. I propose a model where aNSCs dynamically transition through a cloud of highly interlinked cellular states driven by intrinsic and extrinsic cues. I also show how a new perspective enables us to integrate current results into a coherent framework leading to the formulation of new testable hypothesis. This model, like all others, is still far from perfect and will be reshaped by future findings. I believe that having a more complete view of aNSC transitions and embracing their complexity will bring us closer to understand how aNSC activity and neurogenesis are controlled throughout life.
... In addition, we also identified a small population of local CR + GABAergic interneurons that inhibit subep-ChAT + neurons. Previously, GABA was shown to be involved in various functions in the SVZ niche such as enhancing neuroblast maturation 37 , and preserving the postnatal/adult NSCs by inhibiting their proliferation and differentiation , but the source of GABA was largely unclear 22,38 . We demonstrated GABAergic CR + interneurons are a local source of GABA (Fig. 2G). ...
The maintenance and differentiation of the adult neural stem cells (NSCs) in the subventricular zone (SVZ) are controlled by cell-intrinsic molecular pathways that interact with extrinsic signaling cues. How neurogenesis in the SVZ is regulated by neural circuit activity remains poorly understood. Here we identified a novel neural circuit that regulates the state of lateral ventricular wall (LV) NSCs. Our results demonstrate that direct glutamatergic inputs from the frontal cortex, as well as local inhibitory interneurons, control the activity of subependymal cholinergic neurons. In vivo optogenetic and chemogenetic stimulation of defined neuronal populations within this circuit were sufficient to control LV NSC proliferation and SVZ neurogenesis. Moreover, acetylcholine (ACh), which activates M1 muscarinic ACh receptors, triggers the activation of quiescent NSCs. These findings shed light on neural activity-dependent regulation of postnatal and adult LV NSCs activation and SVZ neurogenesis.
... N eurotransmitter signaling is traditionally associated with communication between neurons. However, several reports suggest that neurotransmitters also influence critical aspects of neurogenesis, including proliferation, migration, and differentiation, under both physiological and pathological conditions [1][2][3][4][5][6][7][8][9][10][11][12] . The association between neurotransmitter signaling and neurogenesis appears to be primarily dependent on transmitter receptors that are not confined to neurons. ...
Physical exercise stimulates adult neurogenesis, yet the underlying mechanisms remain poorly understood. A fundamental component of the innate neuroregenerative capacity of zebrafish is the proliferative and neurogenic ability of the neural stem/progenitor cells. Here, we show that in the intact spinal cord, this plasticity response can be activated by physical exercise by demonstrating that the cholinergic neurotransmission from spinal locomotor neurons activates spinal neural stem/progenitor cells, leading to neurogenesis in the adult zebrafish. We also show that GABA acts in a non-synaptic fashion to maintain neural stem/progenitor cell quiescence in the spinal cord and that training-induced activation of neurogenesis requires a reduction of GABAA receptors. Furthermore, both pharmacological stimulation of cholinergic receptors, as well as interference with GABAergic signaling, promote functional recovery after spinal cord injury. Our findings provide a model for locomotor networks’ activity-dependent neurogenesis during homeostasis and regeneration in the adult zebrafish spinal cord. The mechanisms stimulating adult neurogenesis are unclear. Here, the authors show the contribution of cholinergic and GABAergic signalling within the locomotor network to spinal cord neurogenesis during homeostasis and regeneration, showing neurogenesis depends on circuit activity in the adult zebrafish.
... The B1 cells can give rise either to proliferating C cells, which finally differentiate to neuroblasts (A cells) migrating to the olfactory bulb, or to B2 cells which are similar to B1 cells but do not enter the ependymal wall. The B1 cells receive signals from young neurons [151,152], from C cells [153], from epithelial cells [154] as well as from the vasculature [155,156]. By the contact to the CSF, the B1 cells have also the opportunity to sense blood-born molecules entering the CSF by the CP or to signals secreted by the latter [150,157]. ...
The blood-brain barrier (BBB) protects the vertebrate central nervous system from harmful blood-borne, endogenous and exogenous substances to ensure proper neuronal function. The BBB describes a function that is established by endothelial cells of CNS vessels in conjunction with pericytes, astrocytes, neurons and microglia, together forming the neurovascular unit (NVU). Endothelial barrier function is crucially induced and maintained by the Wnt/β-catenin pathway and requires intact NVU for proper functionality. The BBB and the NVU are characterized by a specialized assortment of molecular specializations, providing the basis for tightening, transport and immune response functionality.
... GABA can inhibit both cell proliferation and neuronal differentiation in this neurogenic region. However, type-B1 and type-C cells express the diazepambinding inhibitor protein, which competitively inhibits the binding of GABA to its receptors, thus preventing GABAergic neurotransmission and promoting cell proliferation (Liu et al., 2005;Fernando et al., 2011;Alfonso et al., 2012). Lastly, the population of cholinergic neurons (which expresses choline acetyl transferase) present in the SVZ is also thought to regulate neuroblast proliferation through activation of fibroblast growth factor receptor (FGFR)-mediated signaling (Paez-Gonzalez et al., 2014). ...
Convincing evidence has repeatedly shown that new neurons are produced in the mammalian brain into adulthood. Adult neurogenesis has been best described in the hippocampus and the subventricular zone (SVZ), in which a series of distinct stages of neuronal development has been well characterized. However, more recently, new neurons have also been found in other brain regions of the adult mammalian brain, including the hypothalamus, striatum, substantia nigra, cortex, and amygdala. While some studies have suggested that these new neurons originate from endogenous stem cell pools located within these brain regions, others have shown the migration of neurons from the SVZ to these regions. Notably, it has been shown that the generation of new neurons in these brain regions is impacted by neurologic processes such as stroke/ischemia and neurodegenerative disorders. Furthermore, numerous factors such as neurotrophic support, pharmacologic interventions, environmental exposures, and stem cell therapy can modulate this endogenous process. While the presence and significance of adult neurogenesis in the human brain (and particularly outside of the classical neurogenic regions) is still an area of debate, this intrinsic neurogenic potential and its possible regulation through therapeutic measures present an exciting alternative for the treatment of several neurologic conditions. This review summarizes evidence in support of the classic and novel neurogenic zones present within the mammalian brain and discusses the functional significance of these new neurons as well as the factors that regulate their production. Finally, it also discusses the potential clinical applications of promoting neurogenesis outside of the classical neurogenic niches, particularly in the hypothalamus, cortex, striatum, substantia nigra, and amygdala.
... These reports suggest that the expression dynamics and protein stability of bHLH factors might contribute to differential outputs of Notch signaling. The quiescent state of adult NSCs is also regulated by other signaling receptors, for example, activation of gamma-aminobutyric acid (GABA) receptors by GABA secreted from NSC niche cells in the SVZ and DG [38,39] and activation of integrin receptors by binding of Mfge8, a phagocytosis factor, which is secreted from quiescent NSCs of the DG [40]. Adult NSCs express GABA A receptors and tonically respond to GABA from niche cells, which are parvalbumin-expressing (PV + ) interneurons in the DG or neuroblasts in the SVZ that can dictate the NSC choice between quiescence and activation through nonsynaptic GABA signaling [39,41]. ...
Quiescence is a cellular strategy for maintaining somatic stem cells in a specific niche in a low metabolic state without senescence for a long period of time. During development, neural stem cells (NSCs) actively proliferate and self‐renew, and their progeny differentiate into both neurons and glial cells to form mature brain tissues. On the other hand, most NSCs in the adult brain are quiescent and arrested in G0/G1 phase of the cell cycle. Quiescence is essential in order to avoid the precocious exhaustion of NSCs, ensuring a sustainable source of available stem cells in the brain throughout the lifespan. After receiving activation signals, quiescent NSCs reenter the cell cycle and generate new neurons. This switching between quiescence and proliferation is tightly regulated by diverse signaling pathways. Recent studies suggest significant involvement of cellular proteostasis (homeostasis of the proteome) in the quiescent state of NSCs. Proteostasis is the result of integrated regulation of protein synthesis, folding, and degradation. In this review, we discuss regulation of quiescence by multiple signaling pathways, especially bone morphogenetic protein and Notch signaling, and focus on the functional involvement of the lysosome, an organelle governing cellular degradation, in quiescence of adult NSCs.
... Mice were injected i.p. with 1 mg of tamoxifen twice a day for 5 consecutive days. BrdU injections were performed i.p., 50 mg/kg for adult mice and 30 mg/kg for pups, as described before [102,103]. ...
In mammals, most adult neural stem cells (NSCs) are located in the ventricular–subventricular zone (V-SVZ) along the wall of the lateral ventricles and they are the source of olfactory bulb interneurons. Adult NSCs exhibit an apico-basal polarity; they harbor a short apical process and a long basal process, reminiscent of radial glia morphology. In the adult mouse brain, we detected extremely long radial glia-like fibers that originate from the anterior–ventral V-SVZ and that are directed to the ventral striatum. Interestingly, a fraction of adult V-SVZ-derived neuroblasts dispersed in close association with the radial glia-like fibers in the nucleus accumbens (NAc). Using several in vivo mouse models, we show that newborn neurons integrate into preexisting circuits in the NAc where they mature as medium spiny neurons (MSNs), i.e., a type of projection neurons formerly believed to be generated only during embryonic development. Moreover, we found that the number of newborn neurons in the NAc is dynamically regulated by persistent pain, suggesting that adult neurogenesis of MSNs is an experience-modulated process.
... They are reported to regulate the development and differentiation of specific organs (Mandrup et al., 1998). For example, ACBP modulates signaling pathways in neurogenesis (Alfonso et al., 2012) and hepatic cells differentiation (Petrescu et al., 2003) processes. ...
Malaria is an infectious disease that is caused by different species of Plasmodium. Several antimalarial drugs are used to counter the spread and infectivity of Plasmodium species. However, humans are also vulnerable to many of the antimalarial drugs, including the quinine- and quinoline-based drugs. In particular, the antimalarial quinoline mefloquine has been reported to show adverse neuropsychiatric effects in humans. Though mefloquine is known to be neurotoxic, the molecular mechanisms associated with this phenomenon are still obscure. In this study, we show that mefloquine binds to and inactivates the human acyl-CoA binding protein (hACBP), potentially inducing redox stress in human neuroblastoma cells (IMR-32). Mefloquine occupies the acyl-CoA binding pocket of hACBP by interacting with several of the critical acyl-CoA binding amino acids. This leads to the competitive inhibition of acyl-CoA(s) binding to hACBP and to the accumulation of lipid droplets inside the IMR-32 cells. The accumulation of cytosolic lipid globules and oxidative stress finally correlates with the apoptotic death of cells. Taken together, our study deciphers a mechanistic detail of how mefloquine leads to the death of human cells by perturbing the activity of hACBP and lipid homeostasis.
... Finally it was reported that in other CNS niches GABAergic signaling regulates quiescence and proliferation, so it has been suggested that, those CSF-N could have a similar role in the ependymal niche [148], [149]. ...
Over the last 10 years, JP Hugnot’s lab has been focusing on the different pools of progenitors and stem cells found in the adult spinal cord both in human and mouse. This is important to conduct this kind of research as the spinal cord is affected by several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and traumatic lesions for which there is no cure. In anamniotes such as Zebrafish, the spinal cord can regenerate after lesion due to endogenous progenitors/stem cells activation. So by investigating the presence and properties of such cells in mammals especially human, one could possibly harness those cells toward regeneration including neurons. We conducted RNA profiling to compare human vs mouse stem cell niche and lesioned vs non lesioned spinal cord mouse stem cell niche. This niche is particularly interesting as in anamniotes, radial ependymoglia cells located in this region are multipotent and can generate new motoneurons after lesion. And similar, albeit non identical, cells are present in mouse. In mammals, after lesion, these niche cells actively proliferate and migrate to generate mainly astrocytic cells and few oligodendrocytes which participate to the glial scar and regeneration by providing neurotrophic factor such as CNTF, HGF, and IGF-1. This niche contains at least 5 cell types and here a new dorsal cell type expressing Msx1 and Id4 transcription factors was identified. These results indicated that the adult spinal cord niche in mouse and human is a mosaic of cells with different developmental origin and maintaining high levels of neural developmental genes. Glial-neuronal interactions supporting and keeping neurons intact can be influence neurodegenerative diseases. One of these glial cells is the satellite oligodendrocyte or so called perineuronal satellite cells (PNCs). PNCs are tightly associated to the soma of large neurons and widely spread in the grey matter of the CNS both cortex and spinal cord. However the cellular properties and functional roles of these unmyelinating oligodendrocytes are not yet discovered. In this study, nestin-GFP positive cells are associated to neurons immunostained for neuronal nuclear antigen in both cortex and spinal cord. We identified PNCs as CNPase positive cells that are neither oligodendrocyte progenitor cells (PDGFRa) nor myelinating oligodendrocytes (MBP). These data suggest that PNCs might affect neuronal survival as well as the myelination process in demyelinating conditions. Also it could be implicated in neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis due to their interaction with motor neurons.
... In this context, ODN, which is exclusively produced by astroglial cells in the central nervous system of mammals [14], and which stimulates neurogenesis in adult mouse brains [15], along with nerve regeneration [16], could be a promising factor. ODN is a gliopeptide generated through proteolytic cleavage of an 86-amino acid precursor called diazepam-binding inhibitor (DBI), and is able to prevent both neuron and astroglia cell death induced by several neurotoxic substances including hydrogen peroxide (H2O2) [17,18] and 6-hydroxydopamine (6-OHDA) [19,20]. ...
Neurodegenerative diseases are characterized by oxidative stress, mitochondrial damage, and death of neuronal cells. To counteract such damage and to favor neurogenesis, neurotrophic factors could be used as therapeutic agents. Octadecaneuropeptide (ODN), produced by astrocytes, is a potent neuroprotective agent. In N2a cells, we studied the ability of ODN to promote neuronal differentiation. This parameter was evaluated by phase contrast microscopy, staining with crystal violet, cresyl blue, and Sulforhodamine 101. The effect of ODN on cell viability and mitochondrial activity was determined with fluorescein diacetate and DiOC6(3), respectively. The impact of ODN on the topography of mitochondria and peroxisomes, two tightly connected organelles involved in nerve cell functions and lipid metabolism, was evaluated by transmission electron microscopy and fluorescence microscopy: detection of mitochondria with MitoTracker Red, and peroxisome with an antibody directed against the ABCD3 peroxisomal transporter. The profiles in fatty acids, cholesterol, and cholesterol precursors were determined by gas chromatography, in some cases coupled with mass spectrometry. Treatment of N2a cells with ODN (10−14 M, 48 h) induces neurite outgrowth. ODN-induced neuronal differentiation was associated with modification of topographical distribution of mitochondria and peroxisomes throughout the neurites and did not affect cell viability and mitochondrial activity. The inhibition of ODN-induced N2a differentiation with H89, U73122, chelerythrine and U0126 supports the activation of a PKA/PLC/PKC/MEK/ERK-dependent signaling pathway. Although there is no difference in fatty acid profile between control and ODN-treated cells, the level of cholesterol and some of its precursors (lanosterol, desmosterol, lathosterol) was increased in ODN-treated cells. The ability of ODN to induce neuronal differentiation without cytotoxicity reinforces the interest for this neuropeptide with neurotrophic properties to overcome nerve cell damage in major neurodegenerative diseases.
... Contact between cells enables further nuanced regulation of NSCs within the V-SVZ. Through negative feedback mechanisms, more differentiated cells are hypothesized to limit the differentiation of progenitors within the adult V-SVZ [81,192,193]. For example, neuroblasts release gamma-aminobutyric acid (GABA), which helps preserve the quiescent state of NSCs; upon neuroblast removal (and thus removal of GABA), NSCs enter a more proliferative state [76][77][78][79][80]. Cell-cell contact likewise remains important for proper migration of more differentiated neuroblasts from the V-SVZ to the OB [194]. ...
In this chapter, heterogeneity is explored in the context of the ventricular–subventricular zone, the largest stem cell niche in the mammalian brain. This niche generates up to 10,000 new neurons daily in adult mice and extends over a large spatial area with dorso-ventral and medio-lateral subdivisions. The stem cells of the ventricular–subventricular zone can be subdivided by their anatomical position and transcriptional profile, and the stem cell lineage can also be further subdivided into stages of pre- and post-natal quiescence and activation. Beyond the stem cells proper, additional differences exist in their interactions with other cellular constituents of the niche, including neurons, vasculature, and cerebrospinal fluid. These variations in stem cell potential and local interactions are discussed, as well as unanswered questions within this system.
... Adult NSCs also receive feedback signals from cells at later stages in the lineage. For instance, neuroblasts secrete non-synaptic γ-aminobutyric acid (GABA) that binds to GABA type A receptor (GABA A R) expressed by qNSCs and inhibits their proliferation [67,68]. Interestingly, it was also shown that adult neurogenesis could be modulated depending on hunger or satiety, via hypothalamic control. ...
Brain and nervous system cancers in children represent the second most common neoplasia after leukemia. Radiotherapy plays a significant role in cancer treatment; however, the use of such therapy is not without devastating side effects. The impact of radiation-induced damage to the brain is multifactorial, but the damage to neural stem cell populations seems to play a key role. The brain contains pools of regenerative neural stem cells that reside in specialized neurogenic niches and can generate new neurons. In this review, we describe the advances in radiotherapy techniques that protect neural stem cell compartments, and subsequently limit and prevent the occurrence and development of side effects. We also summarize the current knowledge about neural stem cells and the molecular mechanisms underlying changes in neural stem cell niches after brain radiotherapy. Strategies used to minimize radiation-related damages, as well as new challenges in the treatment of brain tumors are also discussed.
... B1 cells, thereby reducing proliferation (Liu et al., 2005;Fernando et al., 2011). In contrast, the release of the diazepam-binding inhibitor protein (DBI) from B1 cells and C cells increases V-SVZ proliferation and A cell production (Alfonso et al., 2012). Through their lateral processes, B1 cells contact each other and likely exchange signals that are associated with their state of activation and possibly their mode of division. ...
In the adult rodent brain, neural stem cells (NSCs) persist in the ventricular-subventricular zone (V-SVZ) and the subgranular zone (SGZ), which are specialized niches in which young neurons for the olfactory bulb (OB) and hippocampus, respectively, are generated. Recent studies have significantly modified earlier views on the mechanisms of NSC self-renewal and neurogenesis in the adult brain. Here, we discuss the molecular control, heterogeneity, regional specification and cell division modes of V-SVZ NSCs, and draw comparisons with NSCs in the SGZ. We highlight how V-SVZ NSCs are regulated by local signals from their immediate neighbors, as well as by neurotransmitters and factors that are secreted by distant neurons, the choroid plexus and vasculature. We also review recent advances in single cell RNA analyses that reveal the complexity of adult neurogenesis. These findings set the stage for a better understanding of adult neurogenesis, a process that one day may inspire new approaches to brain repair.
... It remains to be determined whether NSCs are capable of releasing potent chemicals such as gliotransmitters and other secretory molecules into the neurogenic niche, which could in turn influence both mature and newborn progeny. Supporting this view, NSCs and their immediate progeny are found to secrete diazepam-binding inhibitor in the adult rodent brain; this antagonizes GABA signaling and promotes proliferation of neuroblasts through paracrine signaling [61]. In addition, NSCs might directly influence each other through autocrine signaling; NSCs in the adult SGZ express both the vascular endothelial growth factor (VEGF) receptor 3 and its ligand VEGF-C, and VEGF receptor stimulation promotes NSC activation [62]. ...
Adult neurogenesis, a developmental process of generating functionally integrated neurons from neural stem cells, occurs throughout life in the hippocampus of the mammalian brain and highlights the plastic nature of the mature central nervous system. Substantial evidence suggests that new neurons participate in cognitive and affective brain functions and aberrant adult neurogenesis contributes to various brain disorders. Focusing on adult hippocampal neurogenesis, we review recent findings that advance our understanding of the key properties and potential functions of adult neural stem cells. We further discuss the key evidence demonstrating the causal role of aberrant hippocampal neurogenesis and various brain disorders. Finally, we propose strategies aimed at simultaneously correcting stem cells and their niche for treating brain disorders.
Nutrient availability is a major selective force in the evolution of metazoa, and thus plasticity in tissue function and morphology is shaped by adaptive responses to nutrient changes. Utilizing Drosophila, we reveal that distinct calibration of acyl-CoA metabolism, mediated by Acbp6 (Acyl-CoA binding-protein 6), is critical for nutrient-dependent tissue plasticity. Drosophila Acbp6, which arose by evolutionary duplication and binds acyl-CoA to tune acetyl-CoA metabolism, is required for intestinal resizing after nutrient deprivation through activating intestinal stem cell proliferation from quiescence. Disruption of acyl-CoA metabolism by Acbp6 attenuation drives aberrant ‘switching’ of metabolic networks in intestinal enterocytes during nutrient adaptation, impairing acetyl-CoA metabolism and acetylation amid intestinal resizing. We also identified STAT92e, whose function is influenced by acetyl-CoA levels, as a key regulator of acyl-CoA and nutrient-dependent changes in stem cell activation. These findings define a regulatory mechanism, shaped by acyl-CoA metabolism, that adjusts proliferative homeostasis to coordinately regulate tissue plasticity during nutrient adaptation.
Neurogenesis and differentiation of neural stem cells (NSCs) are controlled by cell-intrinsic molecular pathways that interact with extrinsic signaling cues. In this study, we identify a circuit that regulates neurogenesis and cell proliferation in the lateral ventricle-subventricular zone (LV-SVZ). Our results demonstrate that direct glutamatergic projections from the anterior cingulate cortex (ACC), as well as inhibitory projections from calretinin+ local interneurons, modulate the activity of cholinergic neurons in the subependymal zone (subep-ChAT+). Furthermore, in vivo optogenetic stimulation and inhibition of the ACC-subep-ChAT+ circuit are sufficient to control neurogenesis in the ventral SVZ. Both subep-ChAT+ and local calretinin+ neurons play critical roles in regulating ventral SVZ neurogenesis and LV-SVZ cell proliferation.
Background:
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by amyloid-β peptide (Aβ) deposition. Aβ accumulation induces oxidative stress, leading to mitochondrial dysfunction, apoptosis, and so forth. Octadecaneuropeptide (ODN), a diazepam-binding inhibitor (DBI)-derived peptide, has been reported to have antioxidant properties. However, it is unclear whether ODN has neuroprotective effects in AD.
Objective:
To profile the potential effects of ODN on AD.
Methods:
We established a mouse model of AD via microinjection of Aβ in the lateral ventricle. Utilizing a combination of western blotting assays, electrophysiological recordings, and behavioral tests, we investigated the neuroprotective effects of ODN on AD.
Results:
DBI expression was decreased in AD model mice and cells. Meanwhile, ODN decreased Aβ generation by downregulating amyloidogenic AβPP processing in HEK-293 cells stably expressing human Swedish mutant APP695 and BACE1 (2EB2). Moreover, ODN could inhibit Aβ-induced oxidative stress in primary cultured cells and mice, as reflected by a dramatic increase in antioxidants and a decrease in pro-oxidants. We also found that ODN could reduce oxidative stress-induced apoptosis by restoring mitochondrial membrane potential, intracellular Ca2 + and cleaved caspase-3 levels in Aβ-treated primary cultured cells and mice. More importantly, intracerebroventricular injection of ODN attenuated cognitive impairments as well as long-term potentiation in Aβ-treated mice.
Conclusion:
These results suggest that ODN may exert a potent neuroprotective effect against Aβ-induced neurotoxicity and memory decline via its antioxidant effects, indicating that ODN may be a potential therapeutic agent for AD.
Adult somatic stem cells, also called progenitors, are essential for tissue regeneration and homeostasis. Stem cells have been identified in almost all tissues including those with little regenerative capacity. It is a central interest in the field of regenerative medicine to understand the signals that govern stem cell function, particularly in tissues that typically do not regenerate. Progenitors are present in small numbers in most tissues and reside in a specific cellular context that is referred to as the stem cell niche. In this article, we survey the stem cell niche in several tissues and examine its role in stem cell function. While the anatomical appearance of the stem cell niche differs among tissues, common features exist that serve similar roles. We review here the stem cell niche in two tissues capable of robust regeneration (skeletal muscle and skin) and two tissues that display poor regenerative capacity (CNS and heart). An increased understanding of the stem cell niche, particularly in tissues that are “poor regenerators”, will have a significant impact upon regenerative medicine. Dissecting the molecular signals transmitted between resident progenitors and neighboring niche cells can lead to the identification of new therapeutic targets to augment regenerative capacity as well as increase our fundamental understanding of these important cellular players.
Manipulation of neural stem cell proliferation and differentiation in the postnatal CNS is receiving significant attention due to therapeutic potential. In the spinal cord, such manipulations may promote repair in conditions such as multiple sclerosis or spinal cord injury, but may also limit excessive cell proliferation contributing to tumours such as ependymomas. We show that when ambient γ-aminobutyric acid (GABA) is increased in vigabatrin-treated or decreased by GAD67 allele haplodeficiency in glutamic acid decarboxylase67-green fluorescent protein (GAD67-GFP) mice of either sex, the numbers of proliferating cells respectively decreased or increased. Thus, intrinsic spinal cord GABA levels are correlated with the extent of cell proliferation, providing important evidence for manipulating these levels. Diazepam binding inhibitor, an endogenous protein that interacts with GABA receptors and its breakdown product, octadecaneuropeptide, which preferentially activates central benzodiazepine (CBR) sites, were highly expressed in spinal cord, especially in ependymal cells surrounding the central canal. Furthermore, animals with reduced CBR activation via treatment with flumazenil or Ro15-4513, or with a G2F77I mutation in the CBR binding site had greater numbers of Ethynyl-2′-deoxyuridine positive cells compared to control, which maintained their stem cell status since the proportion of newly proliferated cells becoming oligodendrocytes or astrocytes was significantly lower. Altering endogenous GABA levels or modulating GABAergic signaling through specific sites on GABA receptors therefore influences NSC proliferation in the adult spinal cord. These findings provide a basis for further study into how GABAergic signaling could be manipulated to enable spinal cord self-regeneration and recovery or limit pathological proliferative activity.
Earlier work has implicated the neurotransmitter GABA in controlling forebrain progenitor proliferation. In this issue of Neuron, Everlien et al. (2022) demonstrate that diazepam binding inhibitor acts to keep the neurogenesis-promoting effect of GABA at bay.
The discovery of postnatal neurogenesis, the isolation and cultivation of neural stem cells (NSCs) in the adult brain, and the subsequent production of autologous neurons from them in vitro gave scientists the hope that new regenerative technologies for the restoration of the central nervous system (CNS) functions lost in case of diseases and injuries would be created soon. However, over the next 30 years, the excitement and charm of the new discovery gave us a way to understand the functions of NSCs and critical assessment of these cells. The latest data on the functioning of stem cell niches in the adult brain and clonal studies of adult NSC derivatives allow us to conclude that their functions in the adult brain are most likely not related to the reparative regeneration of CNS structures. Young neurons formed in the subventricular and subgranular zones of the adult brain, embedded in neural networks, perform quite specific functions: the modulation of odor recognition and the functioning of learning and memory, respectively. In higher primates and humans, neurogenesis in the subventricular zone is not determined, and the level of neurogenesis in the dentate gyrus is comparable to the level in mice only in the early postnatal period, when the young neurons formed participate in cognitive plasticity, memory modulation and other functions of the developing hippocampus. The presence of neurogenesis in adult humans is not confirmed in recent studies. Under conditions of pathology, global changes in homeostasis occur in the stem cell niches of the adult brain, accompanied by activation of dormant NSCs and increased proliferation of surviving NSCs and all subsequent progenitor clones; however, in the case of pronounced cell death even of nearby brain structures, epiform regeneration involving newly formed neuroblasts does not occur even in lower mammals. In humans, taking into account the pronounced age-related involution of neurogenesis in the dentate gyrus, the restoration of cerebral functions is carried out only through neuronal plasticity. Understanding the biological role of adult NSCs allows us to conclude that the creation of technologies for cellular regeneration for diseases and injuries of the human central nervous system, even if possible, is attainable only by reprogramming adult somatic cells.
Spinal cord injury (SCI), a serious disease of the central nervous system, often with irreversible loss of motor or sensory functions. Failure of axon connection and inhibition of microenvironment after SCI severely hinder the regeneration of damaged tissue and neuron function. Therefore, the new perspective of treatment of spinal cord injury is the reconstruction of neural circuit. Stem cells are a kind of cells with differentiation potential. They reconstruct local circulation by differentiating into neurons to replace damaged cells. It can also secrete various factors to regulate the host microenvironment and play a therapeutic role. Biomaterials can fill the cavity at the site of spinal cord injury, load therapeutic drugs, provide adsorption sites for transplanted cells and play a bridging role. In this review, the therapeutic role of stem cells and biomaterials is discussed, together with their properties, advantages, limitations, and future perspectives, providing a reference for basic and clinical research on SCI treatment.
Recent studies have revealed an altered expression of NKCC1 and KCC2 in prefrontal cortex (PFC) and hippocampus of schizophrenic patients. Despite extensive considerations, the alteration of NKCC1 and KCC2 co-transporters at different stages of development has not been fully studied. Therefore, we evaluated the expression of these transporters in PFC and hippocampus at time points of four, eight, and twelve weeks in post-weaning social isolation rearing rat model. For this purpose, 23–25 days-old rats were classified into social- or isolation-reared groups. The levels of NKCC1 and KCC2 mRNA expression were evaluated at hippocampus or PFC regions at the time-points of four, eight, and twelve weeks following housing. Post-weaning isolation rearing decreased the hippocampal KCC2 mRNA expression level, but does not affect the NKCC1 mRNA expression. However, no significant difference was observed in the PFC mRNA levels of NKCC1 and KCC2 in the isolation-reared group compared to the socially-reared group during the course of modeling. Further, we assessed the therapeutic effect of selective NKCC1 inhibitor bumetanide (10 mg/kg), on improvement of prepulse inhibition (PPI) test on twelve weeks isolation-reared rats. Intraperitoneal administration of bumetanide (10 mg/kg) did not exert beneficial effects on PPI deficit. Our findings show that isolation rearing reduces hippocampal KCC2 expression level and may underlie hippocampal GABA excitatory. In addition, 10 mg/kg bumetanide is not effective in improving the reduced PPI of twelve weeks isolation-reared rats.
Collectively, our findings show that hippocampal chloride transporter KCC2 contributes to excitatory GABA dysregulation in the developmental rat model of schizophrenia.
Four decades ago Costa and colleagues identified a small, secreted polypeptide in the brain that can displace the benzodiazepine diazepam from the GABAA receptor, and was thus termed diazepam binding inhibitor (DBI). Shortly after, an identical polypeptide was identified in liver by its ability to induce termination of fatty acid synthesis, and was named acyl-CoA binding protein (ACBP). Since then, ACBP/DBI has been studied in parallel without a clear and integrated understanding of its dual roles. The first genetic loss-of-function models have revived the field, allowing targeted approaches to better understand the physiological roles of ACBP/DBI in vivo. We discuss the roles of ACBP/DBI in central and tissue-specific functions in mammals, with an emphasis on metabolism and mechanisms of action.
While unrestrained cell growth characterizes all cancers, the attributes of the tumor microenvironment or host organ provide unique trophic factors and mechanism towards this growth. Especially for the brain, synapse-related biology are among these. Particularly in the last decade, a surge of ground-breaking research has explored the interactions and contributions of synapses towards gliomagenesis. This new wave of investigations has greatly added to a multidisciplinary, hybrid field dubbed ‘cancer neuroscience’. Here we will explore how mechanisms in healthy brain have correlates in the glioma microenvironment. In turn, we explore a pathological consequence in glioma brains, seizures. Through this process, we explore how work in electrophysiology, mouse modeling, glial biology, and genomics come together to lend novel perspective and insight towards this most deadly of cancers, high grade glioma.
The contribution of neuroglial interactions to the regulation of energy balance has gained
increasing acceptance in recent years. In this context, endozepines, endogenous analogs of benzodiazepine derived from diazepam-binding inhibitor, are now emerging as major players. Produced by glial cells (astrocytes and tanycytes), endozepines have been known for two decades to exert potent anorexigenic effects by acting at the hypothalamic level. However, it is only recently that their modes of action, including the mechanisms by which they modulate energy metabolism, have begun to be elucidated. The data available today are abundant, significant and sometimes contradictory, revealing a much more complex regulation than initially expected. Several mechanisms of action of endozepines seem to coexist at the central level, particularly in the hypothalamus. The brainstem has also recently emerged as a potential site of action for endozepines. In addition to their central anorexigenic effects, endozepines may also display peripheral effects promoting orexigenic actions, adding to their complexity and raising yet more questions. In this review, we attempt to provide an overview of our current knowledge in this rapidly evolving field and to pinpoint questions that remain unanswered.
In the adult ventricular-subventricular zone (V-SVZ), neural stem cells (NSCs) generate new olfactory bulb (OB) neurons and glia throughout life. To map adult neuronal lineage progression, we profiled >56,000 V-SVZ and OB cells by single-cell RNA sequencing (scRNA-seq). Our analyses reveal the molecular diversity of OB neurons, including fate-mapped neurons, lineage progression dynamics, and an NSC intermediate enriched for Notum, which encodes a secreted WNT antagonist. SCOPE-seq technology, which links live-cell imaging with scRNA-seq, uncovers cell-size transitions during NSC differentiation and preferential NOTUM binding to proliferating neuronal precursors. Consistently, application of NOTUM protein in slice cultures and pharmacological inhibition of NOTUM in slice cultures and in vivo demonstrated that NOTUM negatively regulates V-SVZ proliferation. Timely, context-dependent neurogenesis demands adaptive signaling among neighboring progenitors. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, which attenuates WNT-stimulated proliferation in NSC progeny.
Les maladies neurodégénératives sont caractérisées par un stress oxydatif associé à des dommages mitochondriaux aboutissant à la mort des cellules neuronales. Pour atténuer ces dommages et favoriser la cytoprotection neuronale ainsi que la neurogenèse, des facteurs neurotrophiques naturels de type endogènes (Neuropeptide : octadécaneuropeptide (ODN)) ou exogènes (Polyphénols : resvératrol (RSV) et apigénine (API)) pourraient être utilisés comme agents thérapeutiques permettant de favoriser la différenciation neuronale des cellules souches immatures et pluripotentes. L’ODN est un peptide produit par les astrocytes et connu comme agent neuroprotecteur puissant d’où l’intérêt d’étudier ses effets sur la mobilisation du calcium, sa capacité à protéger les cellules neuronales contre la mort par apoptose générée par le peroxyde d’hydrogène (H2O2) et d’évaluer son pouvoir à stimuler la neurogenèse en favorisant la différenciation neuronale. Les effets des polyphénols (RSV, API), composés majeurs du régime méditerranéen, sur la neurogenèse ont été également évalués.Les propriétés cytoprotectrices et/ou différenciatrices de l’ODN (10-16 -10-8 M) et des polyphénols (RSV : 6.25 -50 µM ; API : 6.25 -50 µM) ont été essentiellement étudiées sur des cellules de neuroblastomes murins N2a mais aussi sur d’autres lignées nerveuses murines (BV-2, C6) et humaines (SK-N-BE ; CCF-STTG1). La cytoprotection a été mesurée par différents tests de viabilité (FDA, MTT, DiOC6(3), iodure de propidium). La différenciation a été évaluée morphologiquement par la présence de neurites (axones et dendrites) visualisés grâce à différentes techniques de microscopie. L’acide rétinoïque (AR : 6.25 -50 µM) a été utilisé comme contrôle positif d’induction de différenciation. Les voies de signalisation impliquées dans la différenciation neuronale ont été caractérisées. Nous avons aussi étudié l’effet de l’ODN (10-14 M, 48 h) sur la morphologie, la topographie et l’activité des mitochondries et des peroxysomes au cours de la différenciation. Ces deux organites sont impliqués dans le métabolisme des lipides (acides gras, cholestérol).Les résultats obtenus montrent que l’ODN est capable de promouvoir la survie des cellules N2a cultivées en condition de stress oxydatif aigue induit par le H2O2. De plus, l’ODN ainsi que les polyphénols RSV et l’API, qui sont dépourvus d’effet cytotoxique intrinsèque stimulent la croissance des neurites indiquant qu’ils exercent des effets neurotrophiques pro-différenciateurs. Cet effet de l’ODN met en jeu l’activation de son récepteur métabotropique associé aux voies de transduction intracellulaire PKA, PKC ainsi que MAPK / ERKs. De plus, l’ODN stimule la biogenèse des mitochondries et des peroxysomes, organites essentiels dans l’activité axonale (transport axonal et renouvellement). L’étude des voies de signalisation démontre que les effets trophiques du RSV et API mettent en jeu l’activation des voies de transduction de la PKC, PKA ainsi que celle des MAPK / ERKs.Sur la base de ces résultats, la libération d’ODN pourrait être un mécanisme endogène de protection en réponse aux attaques oxydatives et au processus de neurodégénérescence, empêchant la mort cellulaire et favorisant la différenciation des cellules neuronales. Nos travaux mettent aussi en évidence pour la première fois que les polyphénols en plus de leur action antioxydante stimulent la formation, la maturation et l’élongation des neurites des cellules N2a non différenciées. L’ensemble de ces travaux indique que le neuropeptide ODN et les polyphénols RSV et API sont de puissants agents neurotrophiques. Ces molécules présentent donc un intérêt pharmacologique en vue de leur utilisation et/ou de leurs analogues synthétiques pour traiter des maladies neurodégénératives en favorisant la neuroprotection, la neuro-réparation et la neurogenèse.
Neurogenesis persists in the adult brain of mammals in the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone (SGZ) of the dentate gyrus (DG). The complex interactions between intrinsic and extrinsic signals provided by cells in the niche but also from distant sources regulate the fate of neural stem/progenitor cells (NPCs) in these sites. This fine regulation is perturbed in ageing and in pathological conditions leading to a different NPCs behavior tailored to the specific physio-pathological feature. Indeed, NPCs exert in physiological and pathological conditions important neurogenic and non-neurogenic regulatory functions and participate in maintaining and protecting brain tissue homeostasis. In this review, we discuss intrinsic and extrinsic signals that regulate NPC activation and NPC functional role in various homeostatic and non-homeostatic conditions.
Altered lipid metabolism is common in glioblastoma, but its role in tumorigenesis is not well understood. In this issue of Cell Metabolism, Duman et al. (2019) provide new insight into this process, demonstrating that acyl-CoA-binding protein (ACBP) drives glioblastoma growth by promoting mitochondrial long fatty acyl-CoA accumulation and β-oxidation.
Learning and memory are fundamental processes that are disrupted in many neurological disorders including Alzheimer's disease and epilepsy. The hippocampus plays an integral role in these functions, and modulation of synaptic transmission mediated by γ‐aminobutyric acid (GABA) type‐A receptors (GABAARs) impacts hippocampus‐dependent learning and memory. The protein diazepam binding inhibitor (DBI) differentially modulates GABAARs in various brain regions, including hippocampus, and changes in DBI levels may be linked to altered learning and memory. The effects of genetic loss of DBI signaling on these processes, however, have not been determined. In these studies, we examined male and female constitutive DBI knockout mice and wild‐type littermates to investigate the role of DBI signaling in modulating multiple forms of hippocampus‐dependent spatial learning and memory. DBI knockout mice did not show impaired discrimination of objects in familiar and novel locations in an object location memory test, but did exhibit reduced time spent exploring the objects. Multiple parameters of Barnes maze performance, testing the capability to utilize spatial reference cues, were disrupted in DBI knockout mice. Furthermore, whereas most wild‐type mice adopted a direct search strategy upon learning the location of the target hole, knockout mice showed higher rates of using an inefficient random strategy. In addition, DBI knockout mice displayed typical levels of contextual fear conditioning, but lacked a sex difference observed in wild‐type mice. Together, these data suggest that DBI selectively influences certain forms of spatial learning and memory, indicating novel roles for DBI signaling in modulating hippocampus‐dependent behavior in a task‐specific manner.
Neurons of neocortical layers II–VI in the dorsomedial cortex of the mouse arise in the pseudostratified ventricular epithelium (PVE) through 11 cell cycles over the six embryonic days 11–17 (E11–E17). The present experiments measure the proportion of daughter cells that leave the cycle (quiescent or Q fraction or Q) during a single cell cycle and the complementary proportion that continues to proliferate (proliferative or P fraction or P; P = 1 − Q). Q and P for the PVE become 0.5 in the course of the eighth cycle, occurring on E14, and Q rises to ∼0.8 (and P falls to ∼0.2) in the course of the 10th cycle occurring on E16. This indicates that early in neuronogenesis, neurons are produced relatively slowly and the PVE expands rapidly but that the reverse happens in the final phase of neuronogenesis. The present analysis completes a cycle of analyses that have determined the four fundamental parameters of cell proliferation: growth fraction, lengths of cell cycle, and phases Q and P. These parameters are the basis of a coherent neuronogenetic model that characterizes patterns of growth of the PVE and mathematically relates the size of the initial proliferative population to the neuronal population of the adult neocortex.
The expression of peripheral-type benzodiazepine receptor (PBR) and diazepam binding inhibitor (DBI) were studied in human astrocytic tu mors using immunocytochemistry and in situ hybridization. Both PBR and DBI were prominently expressed in neoplastic cells, whereas in nor mal brain their amount was low or undetectable. Immunocytochemical double staining demonstrated that PBR and DBI were present in the same cells, suggesting that DBI may act in an autocrine manner in these cells. Analysis of 86 cases showed that PBR expression was statistically signif icantly associated with tumor malignancy grade (P = 0.004) and the proliferative index as determined by immunocytochemistry with the MUM antibody (P = 0.004). Patients having tumors with high levels of PBR-immunoreactive cells had a shorter life expectancy than patients whose tumors showed lower PBR contents (/' = 0.024). In conclusion, these results show that PBR expression is higher in neoplastic cells than in normal brain tissue. They also suggest that PBR immunocytochemistry might be useful in evaluating malignancy in brain tumors.
The relative contribution of intrinsic and extrinsic cues in the regulation of cortical neurogenesis remains a crucial challenge
in developmental neurobiology. We previously reported that a transient population of glutamatergic neurons, the cortical plate
(CP) transient neurons, migrates from the ventral pallium (VP) over long distances and participate in neocortical development.
Here, we show that the genetic ablation of this population leads to a reduction in the number of cortical neurons especially
fated to superficial layers. These defects result from precocious neurogenesis followed by a depletion of the progenitor pools.
Notably, these changes progress from caudolateral to rostrodorsal pallial territories between E12.5 and E14.5 along the expected
trajectory of the ablated cells. Conversely, we describe enhanced proliferation resulting in an increase in the number of
cortical neurons in the Gsx2 mutants which present an expansion of the VP and a higher number of CP transient neurons migrating into the pallium. Our
findings indicate that these neurons act to maintain the proliferative state of neocortical progenitors and delay differentiation
during their migration from extraneocortical regions and, thus, participate in the extrinsic control of cortical neuronal
numbers.
Adult neural stem cell proliferation is dynamic and has the potential for massive self-renewal yet undergoes limited cell division in vivo. Here, we report an epigenetic mechanism regulating proliferation and self-renewal. The recruitment of the PI3K-related kinase signaling pathway and histone H2AX phosphorylation following GABA(A) receptor activation limits subventricular zone proliferation. As a result, NSC self-renewal and niche size is dynamic and can be directly modulated in both directions pharmacologically or by genetically targeting H2AX activation. Surprisingly, changes in proliferation have long-lasting consequences on stem cell numbers, niche size, and neuronal output. These results establish a mechanism that continuously limits proliferation and demonstrates its impact on adult neurogenesis. Such homeostatic suppression of NSC proliferation may contribute to the limited self-repair capacity of the damaged brain.
The acyl-CoA-binding protein (ACBP)/diazepam binding inhibitor is an intracellular protein that binds C(14)-C(22) acyl-CoA esters and is thought to act as an acyl-CoA transporter. In vitro analyses have indicated that ACBP can transport acyl-CoA esters between different enzymatic systems; however, little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP(-/-)). These mice are viable and fertile and develop normally. However, around weaning, the ACBP(-/-) mice go through a crisis with overall weakness and a slightly decreased growth rate. Using microarray analysis, we show that the liver of ACBP(-/-) mice displays a significantly delayed adaptation to weaning with late induction of target genes of the sterol regulatory element-binding protein (SREBP) family. As a result, hepatic de novo cholesterogenesis is decreased at weaning. The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors, leading to reduced binding of SREBP to target sites in chromatin. In conclusion, lack of ACBP interferes with the normal metabolic adaptation to weaning and leads to delayed induction of the lipogenic gene program in the liver.
The subventricular zone (SVZ) is one of two regions where neurogenesis persists in the postnatal brain. The SVZ, located along the lateral ventricle, is the largest neurogenic zone in the brain that contains multiple cell populations including astrocyte-like cells and neuroblasts. Neuroblasts migrate in chains to the olfactory bulb where they differentiate into interneurons. Here, we discuss the experimental approaches to record the electrophysiology of these cells and image their migration and calcium activity in acute slices. Although these techniques were in place for studying glial cells and neurons in mature networks, the SVZ raises new challenges due to the unique properties of SVZ cells, the cellular diversity, and the architecture of the region. We emphasize different methods, such as the use of transgenic mice and in vivo electroporation that permit identification of the different SVZ cell populations for patch clamp recording or imaging. Electroporation also permits genetic labeling of cells using fluorescent reporter mice and modification of the system using either RNA interference technology or floxed mice. In this review, we aim to provide conceptual and technical details of the approaches to perform electrophysiological and imaging studies of SVZ cells.
The integration of adult-born neurons into the circuitry of the adult hippocampus suggests an important role for adult hippocampal neurogenesis in learning and memory, but its specific function in these processes has remained elusive. In this article, we summarize recent progress in this area, including advances based on behavioural studies and insights provided by computational modelling. Increasingly, evidence suggests that newborn neurons might be involved in hippocampal functions that are particularly dependent on the dentate gyrus, such as pattern separation. Furthermore, newborn neurons at different maturation stages may make distinct contributions to learning and memory. In particular, computational studies suggest that, before newborn neurons are fully mature, they might function as a pattern integrator by introducing a degree of similarity to the encoding of events that occur closely in time.
The adult mouse subependymal zone (SEZ) harbors neural stem cells that are thought to exclusively generate GABAergic interneurons of the olfactory bulb. We examined the adult generation of glutamatergic juxtaglomerular neurons, which had dendritic arborizations that projected into adjacent glomeruli, identifying them as short-axon cells. Fate mapping revealed that these originate from Neurog2- and Tbr2-expressing progenitors located in the dorsal region of the SEZ. Examination of the progenitors of these glutamatergic interneurons allowed us to determine the sequential expression of transcription factors in these cells that are thought to be hallmarks of glutamatergic neurogenesis in the developing cerebral cortex and adult hippocampus. Indeed, the molecular specification of these SEZ progenitors allowed for their recruitment into the cerebral cortex after a lesion was induced. Taken together, our data indicate that SEZ progenitors not only produce a population of adult-born glutamatergic juxtaglomerular neurons, but may also provide a previously unknown source of progenitors for endogenous repair.
The rat brain increases >6x in mass from birth to adulthood, presumably through the addition of glial cells and increasing neuronal size, without the addition of neurons. To test this hypothesis, here we investigate quantitatively the postnatal changes in the total number of neuronal and non-neuronal cells in the developing rat brain, and examine how these changes correlate with brain growth. Total numbers of cells were determined with the isotropic fractionator in the brains of 53 Wistar rats, from birth to young adulthood. We find that at birth, >90% of the cells in the rat brain are neurons. Following a dormant period of approximately 3 days after birth, the net number of neurons in the cerebral cortex, hippocampus, and remaining tissue (excluding cerebellum and olfactory bulb) doubles during the first week, then is reduced by 70% during the second postnatal week, concurrently with net gliogenesis. A second round of net addition of 6 million neurons is observed in the cerebral cortex over the following 2 weeks. During the first postnatal week, brain growth relates mainly to increased numbers of neurons of larger average size. In the second and third weeks, it correlates with increased numbers of non-neuronal cells that are smaller in size than the preexisting neurons. Postnatal rat brain development is thus characterized by dramatic changes in the cellular composition of the brain, whose growth is governed by different combinations of cell addition and loss, and changes in average cell size during the first months after birth.
Each of the five cellular layers of the cerebral neocortex is composed of a specific number of a single predominant 'class' of projection neuron. The projection neuron class is defined by its unique morphology and axonal projections to other areas of the brain. Precursor cell populations lining the embryonic lateral ventricles produce the projection neurons. The mechanisms regulating precursor cell proliferation also regulate total numbers of neurons produced at specific developmental periods and destined to a specific neocortical layer. Because the newborn neurons migrate relatively long distances to reach their final layer destinations, it is often assumed that the mechanisms governing acquisition of neuronal-class-specific characteristics, many of which become evident after neuron production, are independent of the mechanisms governing neuron production. We review evidence that suggests that the two mechanisms might be linked via operations of Notch1 and p27(Kip1), molecules known to regulate precursor cell proliferation and neuron production.
In the developing brain, cell migration is a crucial process for structural organization, and is therefore highly regulated to allow the correct formation of complex networks, wiring neurons, and glia. In the early postnatal brain, late developmental processes such as the production and migration of astrocyte and oligodendrocyte progenitors still occur. Although the brain is completely formed and structured few weeks after birth, it maintains a degree of plasticity throughout life, including axonal remodeling, synaptogenesis, but also neural cell birth, migration and integration. The subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus are the two main neurogenic niches in the adult brain. Neural stem cells reside in these structures and produce progenitors that migrate toward their ultimate location: the olfactory bulb and granular cell layer of the DG respectively. The aim of this review is to synthesize the increasing information concerning the organization, regulation and function of cell migration in a mature brain. In a normal brain, proteins involved in cell-cell or cell-matrix interactions together with secreted proteins acting as chemoattractant or chemorepellant play key roles in the regulation of neural progenitor cell migration. In addition, recent data suggest that gliomas arise from the transformation of neural stem cells or progenitor cells and that glioma cell infiltration recapitulates key aspects of glial progenitor migration. Thus, we will consider glioma migration in the context of progenitor migration. Finally, many observations show that brain lesions and neurological diseases trigger neural stem/progenitor cell activation and migration toward altered structures. The factors involved in such cell migration/recruitment are just beginning to be understood. Inflammation which has long been considered as thoroughly disastrous for brain repair is now known to produce some positive effects on stem/progenitor cell recruitment via the regulation of growth factor signaling and the secretion of a number of chemoattractant cytokines. This knowledge is crucial for the development of new therapeutic strategies. One of these strategies could consist in increasing the mobilization of endogenous progenitor cells that could replace lost cells and improve functional recovery.
Most forebrain GABAergic interneurons in rodents are born during embryonic development in the ganglionic eminences (GE) and migrate tangentially into the cortical plate. A subset, however, continues to be generated postnatally in the subventricular zone (SVZ). These interneurons populate the olfactory bulb (OB) reached via migration in the rostral migratory stream (RMS). Employing transgenic mice expressing EGFP in 5-HT3-positive neurons, we identified additional migratory pathways in the early postnatal brain. Time-lapse imaging experiments revealed massive migration of EGFP-positive cells from the SVZ into numerous forebrain regions, including cortex, striatum, and nucleus accumbens. The neuronal fate of the migratory EGFP-labeled cells was indicated by their doublecortin (DCX) expression. Birthdating experiments, by using 5-bromo-2′-deoxyuridine (BrdU) and retrovirus-based experiments, provided evidence that migrating neuroblasts were born in the SVZ postnatally and developed a distinct GABAergic phenotype. Our results demonstrate that the SVZ is a reservoir of GABAergic interneurons not only for the OB, but also for other cortical and subcortical areas.
• calretinin
• EGFP
• 5-HT3A receptor
• inhibitory interneurons
• neuroblast migration
The early development of the mammalian forebrain involves the massive proliferation of the ventricular zone cells lining the lateral ventricles. A remnant of this highly proliferative region persists into adult life, where it is known as the subependymal layer. We examined the proliferation kinetics and fates of the mitotically active cells in the subependyma of the adult mouse. The medial edge, the lateral edge, and the dorsolateral corner of the subependymal layer of the rostral portion of the lateral ventricle each contained mitotically active cells, but the dorsolateral region had the highest percentage of bromodeoxyuridine (BrdU)-labeled cells per unit area. Repeated injections of BrdU over 14 hr revealed a proliferation curve for the dorsolateral population with a growth fraction of 33%, indicating that 33% of the cells in this subependymal region make up the proliferating population. The total cell cycle time in this population was approximately 12.7 hr, with an S-phase of 4.2 hr. To examine the fate of these proliferating cells, we injected low concentrations of a replication-deficient, recombinant retrovirus directly into the lateral ventricles of adult mice for uptake by mitotically active subependymal cells. Regardless of the survival time postinjection (10 hr, 1 d, 2 d, or 8 d), the number of retrovirally labeled cells per clone remained the same (1 or 2 cells/clone). This suggests that one of the progeny from each cell division dies. Moreover, the clones remained confined to the subependyma and labeled cells were not seen in the surrounding brain tissue. Thus, while 33% of the dorsolateral subependymal cells continue to proliferate in adult life, the fate of the postmitotic progeny is death.
A cumulative labelling protocol using 5-bromo-2'-deoxyuridine (BUdR) was followed to determine: (1) the growth fraction (i.e., the proportion of cells that comprise the proliferating population), (2) the length of the cell cycle, and (3) the length of the DNA-synthetic phase (S-phase) for proliferative cells in the dentate gyrus of the mouse. On postnatal day 20 (P20), C57BL/6J mice were injected with BUdR at two hour intervals for a total period of 12 hours. Animals were sacrificed at selected intervals, and the brains were processed for immunohistochemistry using a monoclonal antibody directed against single-stranded DNA containing BUdR. The numbers of BUdR-labelled and unlabelled cells in sections through the hilus of the dentate gyrus were counted. The number of BUdR-labelled cells increased linearly from an initial value of about 12% of the total number of cells to a maximum value of just over 24% of the total. These findings indicate that, at P20, a maximum of 24.2 +/- 1.2% of the cells in the dentate hilus are part of the proliferating population. The calculated length of the cell cycle of the cells comprising the intrahilar proliferative zone was estimated to be 16.1 +/- 0.8 h. The length of the S-phase was estimated at 8.0 +/- 0.4 h. In addition, mathematical analysis, using one and two population models, indicates that over 90% of the proliferating cells in the dentate hilus at this age comprise a single population at least in terms of the lengths of the cell cycle and the S-phase.(ABSTRACT TRUNCATED AT 250 WORDS)
Diazepam binding inhibitor (DBI), an endogenous 10-kDa polypeptide was isolated from rat and human brain by monitoring displacement of radioactive diazepam bound to specific recognition sites in brain synaptic and mitochondrial membranes. The cellular location of DBI mRNA was studied in rat brain and selected peripheral tissues by in situ hybridization histochemistry with a 35S-labeled single-stranded complementary RNA probe. DBI mRNA was heterogeneously distributed in rat brain, with particularly high levels in the area postrema, the cerebellar cortex, and ependyma of the third ventricle. Intermediate levels were found in the olfactory bulb, pontine nuclei, inferior colliculi, arcuate nucleus, and pineal gland. Relatively low but significant levels of silver grains were observed overlying many mesencephalic and telencephalic areas that have previously been shown to contain numerous DBI-immunoreactive neurons and a high density of central benzodiazepine receptors. In situ hybridizations also revealed high levels of DBI mRNA in the posterior lobe of the pituitary gland, liver, and germinal center of the white pulp of spleen, all tissues that are rich in peripheral benzodiazepine binding sites. The tissue-specific pattern of DBI gene expression described here could be exploited to further understand the physiological function of DBI in the brain and periphery.
During the development of the mammalian brain, neuronal precursors migrate to their final destination from their site of birth
in the ventricular and subventricular zones (VZ and SVZ, respectively). SVZ cells in the walls of the lateral ventricle continue
to proliferate in the brain of adult mice and can generate neurons in vitro, but their fate in vivo is unknown. Here SVZ cells
from adult mice that carry a neuronal-specific transgene were grafted into the brain of adult recipients. In addition, the
fate of endogenous SVZ cells was examined by microinjection of tritiated thymidine or a vital dye that labeled a discrete
population of SVZ cells. Grafted and endogenous SVZ cells in the lateral ventricle of adult mice migrate long distances and
differentiate into neurons in the olfactory bulb.
Neurons of neocortical layers II-VI in the dorsomedial cortex of the mouse arise in the pseudostratified ventricular epithelium (PVE) through 11 cell cycles over the six embryonic days 11-17 (E11-E17). The present experiments measure the proportion of daughter cells that leave the cycle (quiescent or Q fraction or Q) during a single cell cycle and the complementary proportion that continues to proliferate (proliferative or P fraction or P; P = 1 - Q). Q and P for the PVE become 0.5 in the course of the eighth cycle, occurring on E14, and Q rises to approximately 0.8 (and P falls to approximately 0.2) in the course of the 10th cycle occurring on E16. This indicates that early in neuronogenesis, neurons are produced relatively slowly and the PVE expands rapidly but that the reverse happens in the final phase of neuronogenesis. The present analysis completes a cycle of analyses that have determined the four fundamental parameters of cell proliferation: growth fraction, lengths of cell cycle, and phases Q and P. These parameters are the basis of a coherent neuronogenetic model that characterizes patterns of growth of the PVE and mathematically relates the size of the initial proliferative population to the neuronal population of the adult neocortex.
We have found that, during the early stages of cortical neurogenesis, both GABA and glutamate depolarize cells in the ventricular zone of rat embryonic neocortex. In the ventricular zone, glutamate acts on AMPA/kainate receptors, while GABA acts on GABAA receptors. GABA induces an inward current at resting membrane potentials, presumably owing to a high intracellular Cl- concentration maintained by furosemide-sensitive Cl- transport. GABA and glutamate also produce increases in intracellular Ca2+ in ventricular zone cells, in part through activation of voltage-gated Ca2+ channels. Furthermore, GABA and glutamate decrease the number of embryonic cortical cells synthesizing DNA. Depolarization with K+ similarly decreases DNA synthesis, suggesting that the neurotransmitters act via membrane depolarization. Applied alone, GABAA and AMPA/kainate receptor antagonists increase DNA synthesis, indicating that endogenously released amino acids influence neocortical progenitors in the cell cycle. These results demonstrate a novel role for amino acid neurotransmitters in regulating neocortical neurogenesis.
The adult mammalian forebrain contains a population of multipotential neural stem cells in the subependyma of the lateral ventricles whose progeny are the constitutively proliferating cells, which divide actively throughout life. The adult mammalian brain is ideal for examining the kinetics of the stem cells due to their strict spatial localization and the limited and discrete type of progeny generated (constitutively proliferating cells). Clonal lineage analyses 6 days after retrovirus infection revealed that under baseline conditions 60% of the constitutively proliferating cells undergo cell death, 25% migrate to the olfactory bulb and 15% remain confined to the lateral ventricle subependyma (where they reside for approximately 15 days). Analysis of single cell clones 31 days after retroviral infection revealed that the stem cell divides asymmetrically to self-renew and give rise to constitutively proliferating cells. Following repopulation of the depleted subependyma the average clone size is 2.8 times larger than control, yet the absolute number of cells migrating to the olfactory bulb is maintained and the stem cell retains its asymmetric mode of division. The number of neural stem cells in the adult forebrain 33 days after repopulation of the subependyma was estimated using bromodeoxyuridine labeling of subepenydmal cells. There were calculated to be 1200-1300 cells between the rostral corpus callosum and rostral anterior commissure; these data support a lineage model similar to those based on stem cell behavior in other tissue types.
Widespread cell migrations are the hallmark of vertebrate brain development. In the early embryo, morphogenetic movements of precursor cells establish the rhombomeres of the hindbrain, the external germinal layer of the cerebellum, and the regional boundaries of the forebrain. In midgestation, after primary neurogenesis in compact ventricular zones has commenced, individual postmitotic cells undergo directed migrations along the glial fiber system. Radial migrations establish the neuronal layers. Three molecules have been shown to function in glial guided migration--astrotactin, glial growth factor, and erbB. In the postnatal period, a wave of secondary neurogenesis produces huge numbers of interneurons destined for the cerebellar cortex, the hippocampal formation, and the olfactory bulb. Molecular analysis of the genes that mark stages of secondary neurogenesis show similar expression patterns of a number of genes. Thus these three regions may have genetic pathways in common. Finally, we consider emerging studies on neurological mutant mice, such as reeler, and human brain malformations. Positional cloning and identification of mutated genes has led to new insights on laminar patterning in brain.
Recent studies have implicated the classical neurotransmitters GABA and glutamate in the regulation of neural progenitor proliferation. We now show that GABA and glutamate have opposite effects on the two neural progenitor populations in the ventricular zones (VZs) and subventricular zones (SVZs) of the embryonic cerebrum. Application of either molecule to organotypic slice cultures dramatically increases proliferation in the VZ by shortening the cell cycle, whereas proliferation in the SVZ is decreased. These disparate effects, measured both by bromodeoxyuridine uptake and the expansion of retrovirally labeled progenitor clones, are mimicked by the application of specific GABA and glutamate agonists and are blocked by antagonists. Thus, the relative contributions of the VZ and SVZ to neocortical growth may be regulated by differential responsiveness to GABA and glutamate.
The subventricular zone (SVZ) is the largest germinal layer in the adult mammalian brain and comprises stem cells, transit-amplifying progenitors, and committed neuroblasts. Although the SVZ contains the highest concentration of dividing cells in the adult brain, the intracellular mechanisms controlling their proliferation have not been elucidated. We show here that loss of the cyclin-dependent kinase inhibitor p27Kip1 has very specific effects on a population of CNS progenitors responsible for adult neurogenesis. Using bromodeoxyuridine and [(3)H]thymidine incorporation to label cells in S phase and cell-specific markers and electron microscopy to identify distinct cell types, we compared the SVZ structure and proliferation characteristics of wild-type and p27Kip1-null mice. Loss of p27Kip1 had no effect on the number of stem cells but selectively increased the number of the transit-amplifying progenitors concomitantly with a reduction in the number of neuroblasts. We conclude that cell-cycle regulation of SVZ adult progenitors is remarkably cell-type specific, with p27Kip1 being a key regulator of the cell division of the transit-amplifying progenitors.
Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.
The migration of neuronal precursor cells is essential for the formation of the embryonic nervous system and for the maintenance of the adult nervous system. Modern approaches have greatly facilitated molecular and cellular studies of mechanisms underlying neuronal migration. Here we use the cells migrating from the anterior subventricular zone to the olfactory bulb as a model to discuss in some detail how neuronal migration can be studied. These methods can be adapted to other models of neuronal (or somatic cell) migration.
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Glioma cells in acute slices and in primary culture, and glioma-derived human cell lines were screened for the presence of functional GABAA receptors. Currents were measured in whole-cell voltage clamp in response to γ-aminobutyric acid (GABA). While cells from the most malignant glioma, the glioblastoma multiforme, did not respond to GABA, an inward current (under our experimental conditions with high Cl− concentration in the pipette) was induced in gliomas of lower grades, namely in 71% of oligodendroglioma cells and in 62% of the astrocytoma cells. Glioma cell lines did not express functional GABAA receptors, irrespective of the malignancy of the tumour they originate from. The currents elicited by application of GABA were due to activation of GABAA receptors; the specific agonist muscimol mimicked the response, the antagonists bicuculline and picrotoxin blocked the GABA-activated current and the benzodiazepine receptor agonist flunitrazepam augmented the GABA-induced current and the benzodiazepine inverse agonist DMCM decreased the GABA current. Cells were heterogeneous with respect to the direction of the current flow as tested in gramicidin perforated patches: in some cells GABA hyperpolarized the membrane, while in the majority it triggered a depolarization. Moreover, GABA triggered an increase in [Ca2+]i in the majority of the tumour cells due to the activation of Ca2+ channels. Our results suggest a link between the expression of GABA receptors and the growth of glioma cells as the disappearance of functional GABAA receptors parallels unlimited growth typical for malignant tumours and immortal cell lines.
The whole-cell patch clamp technique was used to characterize voltage- and neurotransmitter-activated currents in the medulloblastoma cell line MHH-MED-3 and cells from tissue slices and primary cultures of two medulloblastoma biopsies. These preparations revealed similar electrophysiological properties. All tested cells displayed 4-aminopyridine-sensitive delayed rectifying K+ currents, γ-aminobutyric acidA receptor-mediated Cl− currents and most of them inward rectifier K+ currents. Transient inward currents were mainly carried by low-voltage activated T-type Ca2+ channels in MHH-MED-3 cells, and tetrodotoxin-sensitive Na+ channels in cells from the primary culture. From these characteristics we conclude that medulloblastoma cells share physiological features with developing cerebellar granule cells at an immature stage.
Certain secreted proteins bypass the canonical exit pathway from cells. Two studies now shed light on the unconventional secretion route taken by the yeast acyl-coenzyme A-binding protein: this protein is sequestered into autophagic vesicles that are re-routed to the plasma membrane where their content is released to the extracellular space.
Like the liver or other peripheral organs, two regions of the adult brain possess the ability of self-renewal through a process called neurogenesis. This raises tremendous hope for repairing the damaged brain, and it has stimulated research on identifying signals controlling neurogenesis. Neurogenesis involves several stages from fate determination to synaptic integration via proliferation, migration, and maturation. While fate determination primarily depends on a genetic signature, other stages are controlled by the interplay between genes and microenvironmental signals. Here, we propose that neurotransmitters are master regulators of the different stages of neurogenesis. In favor of this idea, a description of selective neurotransmitter signaling and their functions in the largest neurogenic zone, the subventricular zone (SVZ), is provided. In particular, we emphasize the interactions between neuroblasts and astrocyte-like cells that release gamma-aminobutyric acid (GABA) and glutamate, respectively. However, we also raise several limitations to our knowledge on neurotransmitters in neurogenesis. The function of neurotransmitters in vivo remains largely unexplored. Neurotransmitter signaling has been viewed as uniform, which dramatically contrasts with the cellular and molecular mosaic nature of the SVZ. How neurotransmitters are integrated with other well-conserved molecules, such as sonic hedgehog, is poorly understood. In an effort to reconcile these differences, we discuss how specificity of neurotransmitter functions can be provided through their multitude of receptors and intracellular pathways in different cell types and their possible interactions with sonic hedgehog.
Gamma-amino butyric acid (GABA) is the most abundant inhibitory neurotransmitter in the central nervous system (CNS) and many physiological actions are modulated by GABA(A) receptors. These chloride channels can be opened by GABA and are a target for a variety of important drugs such as benzodiazepines, barbiturates, neuroactive steroids, convulsants and anaesthetics. GABA(A) receptors are involved in anxiety, feeding and drinking behaviour, circadian rhythm, cognition, vigilance, and learning and memory. Moreover, deficits in the functional expression of GABA(A) receptors have been implicated in multiple neurological and psychiatric diseases. This review aims to discuss the unique physiological and pharmacological properties of the multitude of GABA(A) receptor subtypes present in the CNS, making this receptor an important target for novel rational drug therapy.
Brain size variation among different mammals is tightly associated with different levels of cerebral function. Mechanisms that regulate the number of neurons and hence the size of the brain must be at least partially embedded within the very early phase of neocortical development, that is, embedded in proliferation/differentiation characteristics of the neural progenitor cells (NPCs) of the neocortex. Here we review a sequence of critical events through which the neocortex is formed in the embryonic forebrain, with particular emphasis on cell cycle kinetics of the NPCs that produce non-GABAergic projection neurons, the majority of neurons in the neocortex. In general, the critical parameters that determine the total number of cells produced by a given progenitor population through a sequence of cell cycles are (1) the number of cell cycles that constitute the production period and (2) the probability of cell cycle exit (Q fraction or Q) of progenitor cells for each of the cell cycles. We will also review molecular mechanisms that modulate the critical parameters above, with a special reference to the cell cycle regulatory protein p27(Kip1), inhibitor of G1 phase progression of the cell cycle. Finally the neocortical dysgenesis caused by genetic modification in mice where p27(Kip1) is either deleted or overexpressed is presented as examples of neuron number changes and resultant neocortical dysgenesis by Q fraction alteration.
Sedative and anticonvulsant drugs, which inhibit N-methyl-D-aspartate receptor-mediated excitation or enhance GABA-mediated action, may cause apoptotic neurodegeneration in the developing mammalian brain. Here we explored whether such agents influence early postnatal neurogenesis.
The N-methyl-D-aspartate antagonist MK801 and the GABA subtype A agonists phenobarbital and diazepam were administered to infant rats, and cell proliferation and neurogenesis were studied in the brain using 5-bromo-2'-deoxyuridine and doublecortin immunohistochemistry and stereology. Using confocal microscopy, we quantified neurogenesis in the dentate gyrus on postnatal day 15 (P15) after treatment with MK801 or phenobarbital on P6 to P10. Learning and memory were assessed at the age of 6 months after early postnatal treatment with phenobarbital.
MK801, phenobarbital, and diazepam reduced numbers of newly born cells in the brain. We found no evidence that these agents caused apoptosis of 5-bromo-2'-deoxyuridine-positive cells. In the dentate gyrus, many of the newly formed cells differentiated toward a neuronal phenotype. Phenobarbital and MK801 reduced numbers of newly formed neurons in the dentate gyrus. At the age of 6 months, phenobarbital-treated rats had fewer neurons in the dentate gyrus and performed worse than saline-treated littermates in water maze learning and memory task.
These findings show that blockade of N-methyl-D-aspartate receptor-mediated excitation and enhancement of GABA subtype A receptor activation impair cell proliferation and inhibit neurogenesis in the immature rat brain. Because many sedative and antiepileptic drugs used in pediatric medicine act via these mechanisms, our findings raise concerns about their potential impact on human brain development.
Diazepam binding inhibitor (DBI) is a 9-kD polypeptide that was first isolated in 1983 from rat brain by monitoring its ability to displace diazepam from the benzodiazepine (BZD) recognition site located on the extracellular domain of the type A receptor for gamma-aminobutyric acid (GABAA receptor) and from the mitochondrial BZD receptor (MBR) located on the outer mitochondrial membrane. In brain, DBI and its two major processing products [DBI 33-50, or octadecaneuropeptide (ODN) and DBI 17-50, or triakontatetraneuropeptide (TTN)] are unevenly distributed in neurons, with the highest concentrations of DBI (10 to 50 microMs) being present in the hypothalamus, amygdala, cerebellum, and discrete areas of the thalamus, hippocampus, and cortex. DBI is also present in specialized glial cells (astroglia and Bergmann glia) and in peripheral tissues. In the periphery, the highest concentration of DBI occurs in cells of the zona glomerulosa and fasciculata of the adrenal cortex and in Leydig cells of the testis; interestingly, these are the same cell types in which MBRs are highly concentrated. Stimulation of MBRs by appropriate ligands (including DBI and TTN) facilitates cholesterol influx into mitochondria and the subsequent formation of pregnenolone, the parent molecule for endogenous steroid production; this facilitation occurs not only in peripheral steroidogenic tissues, but also in glial cells, the steroidogenic cells of the brain. Some of the steroids (pregnenolone sulfate, dehydroepiandrosterone sulfate, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one) produced in brain (neurosteroids) function as potent (with effects in the nanomolar concentration range) positive or negative allosteric modulators of GABAA receptor function. Thus, accumulating evidence suggests that the various neurobiological actions of DBI and its processing products may be attributable to the ability of these peptides either to bind to BZD recognition sites associated with GABAA receptors or to bind to glial cell MBRs and modulate the rate and quality of neurosteroidogenesis. The neurobiological effects of DBI and its processing products in physiological and pathological conditions (hepatic encephlopaty, depression, panic) concentrations may therefore be explained by interactions with different types of BZD recognition site. In addition, recent reports that DBI and some of its fragments inhibit (in nanomolar concentrations) glucose-induced insulin release from pancreatic islets and bind acyl-coenzyme A with high affinity support the hypothesis that DBI isa precursor of biologically active peptides with multiple actions in the brain and in peripheral tissues.
In order to study the morphological localization of the endogenous benzodiazepine ligand octadecaneuropeptide (ODN) in rat brain, we have developed antibodies against this peptide. Using a radioimmunoassay for ODN, we have observed that synthetic ODN and serial dilutions of several brain areas gave parallel displacement curves. By light microscope immunocytochemistry, ODN-immunoreactive material was only detected in glial and ependymal cells. Immunolabelled cells were found in high concentrations in the olfactory bulb, hypothalamus, hippocampus, periaqueductal gray, cerebral cortex and the circumventricular organs. In the cerebellar cortex, immunostaining was associated with Bergmann cells. The studies performed at the electron microscopic level confirmed the association of immunoreactive material with glial and ependymal cells. The present results suggest that ODN might play a role in the function of glial cells which have been shown to contain benzodiazepine receptors of the "peripheral type".
The gamma-aminobutyric acid (GABAA) receptor complex is a hetero-oligomeric protein which contains an integral chloride channel and several modulatory domains. The ligands of benzodiazepine recognition sites can up- or down-regulate the activity of the GABAA receptor. The effects of DBI (diazepam binding inhibitor) on GABAA receptors have been studied in cultured mammalian central neurons. Experiments performed with patch-clamp techniques, as well as with conventional intracellular microelectrodes, have revealed a reversible reduction of GABA-induced responses by micromolar concentrations of DBI. This effect was prevented by Ro 15-1788 (flumazenil), a selective benzodiazepine receptor antagonist. From these data, DBI is capable of reducing the activity of the GABAA receptor complex by specifically interacting with the benzodiazepine recognition site. The idea of DBI being a negative allosteric modulator of GABAA receptor channels is in agreement with biochemical, as well as behavioral, pharmacology data.
An endogenous peptide, named diazepam-binding inhibitor (DBI) capable of displacing benzodiazepines from binding sites has been recently fully characterized. In order to clearly identify the cell types responsible for the biosynthesis of DBI in the rat central nervous system, we have performed high resolution in situ hybridization in the area postrema, hypothalamus and cerebellum, using a [35S]-labeled single stranded RNA probe. Hybridization signal was detected in both semithin and ultrathin sections. In all the brain areas examined, specific labeling was exclusively observed in non-neuronal cells including ependymal and subependymal cells bordering the third ventricle. The results obtained clearly establish that DBI is synthesized by non-neuronal cells in the rat brain.
The participation of GABA receptors in the inhibitory transmission at mammalian central synapses was demonstrated experimentally two decades ago. Whilst the ‘classical’ action of GABA involves the opening of Cl− channels, pharmacologically distinct effects of GABA on cation channels were detected later. This led to the notion of GABAA and GABAB receptor subtypes. The GABAA receptor complex contains an integral Cl− ionophore, whereas GABAB receptors couple to Ca2+ and K+ channels via GTP-binding proteins. The physiological and pharmacological properties of GABAA and GABAB receptors will be discussed below in terms of ion channels that are activated by the two receptor subtypes.
This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17-50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octadecaneuropeptide, DBI 33-50) antibodies coupled to CNBr-Sepharose 4B followed by two reverse-phase HPLC steps. The amino acid sequence of the purified peptide is: Thr-Gln-Pro-Thr-Asp-Glu-Glu-Met-Leu-Phe-Ile-Tyr-Ser-His-Phe-Lys-Gln-Ala-Thr-Val - Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. Synthetic TTN injected intracerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific "peripheral" benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the "central" BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5-4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 microM. TTN also enhances picrotoxinin inhibition of gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at "Ro 5-4864 preferring" BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor-mediated effects.
Time of origin (final cell division) of neurons and neuroglia of the mouse olfactory and accessory olfactory formations was determined by autoradiography. Animals were injected with thymidine-H3 at various developmental stages and killed at or near maturity. In the olfactory formation mitral cells (the largest neurons) arise first, mainly over the three day period from the eleventh day of gestation (E11) to E13, tufted cells chiefly from E13 to E18, and granule cells (the smallest neurons) mainly from E18 to postnatal day 20. Most of the smaller and more superficial peripheral tufted cells arise later than the deeper and larger middle and internal tufted cells. All three types of granule cells have a time of origin extending well into postnatal life, with internal granule cells arising over a longer and later period than periglomerular cells or granule cells of the mitral cell layer. Neuroglial precursors undergo final cell division chiefly between E17 and P10. In the phylogenetically less evolved accessory olfactory formation, mitral cells originate earlier than their homologues in the olfactory formation; mitral cells principally from E10 to E12 and granule cells chiefly from E12 to E18. The results support the concept that some germinal layers of the central nervous system are programmed to produce a succession of cell types, larger cells before smaller ones.
Neurogenesis in the rat olfactory bulb was examined with 3H-thymidine-radiography. For the animals in the prenatal groups, the initial 3H-thymidine exposures were separated by 24 h; they were the offspring of pregnant females given two injections on consecutive embryonic (E) days (E12-E13, E13-E14, . . . E21-E22). For the animals in the postnatal (P) groups, the initial 3H-thymidine injections were separated by 48 h, each group receiving either four (PO-P3, P2-P4, . . . P6-P9) or two (P8-P9, P10-P11, . . . P20-P21) consecutive daily injections. On P60, the percentage of labeled cells and the proportion of cells added during either 24 h or 48 h periods were quantified at several anatomical levels for each neuronal population in the main olfactory bulb (mitral cells, tufted cells, granule cells, interneurons in the external plexiform layer, periglomerular granule cells) and accessory olfactory bulb (output neurons, granule cells, periglomerular granule cells). The total time span of neurogenesis extends from E12 to beyond P20. Output neurons are prenatally generated over 5-9 day periods (with most neurogenesis occurring over 2-4 days) in a strict sequential order beginning with the accessory bulb output neurons (E13-E14) and ending with the interstitial tufted cells lying between the glomeruli in the main bulb (E20-E22). These data are correlated with the main and accessory bulb projection fields in the amygdala and with the chronology of amygdala neurogenesis. With the exception of the granule cells in the accessory bulb (88% generated between E15-E22), the rest of the interneuronal populations are generated postnatally and nearly simultaneously. While most neurons (75-80%) originate during the first three weeks of life, all interneuronal populations, including accessory bulb granule cells, show some neurogenesis beyond P20. Injections of 3H-thymidine in juvenile and adult rats indicates neurogenesis up to P60 in the accessory bulb and up to P180 in the main bulb, especially in the main bulb granule cell population. There is circumstantial evidence for turnover of main bulb granule cells during adult life.
Benzodiazepines, which are in extensive clinical use, can regulate neoplastic growth via benzodiazepine receptors. We have studied the expression of the diazepam binding inhibitor (DBI) polypeptide, a putative endogenous ligand for benzodiazepine receptors in normal and pathological human brain. In normal brain, DBI immunoreactivity (IR) and mRNA were detected in all brain areas, with the highest levels in the cerebellum, amygdala, and hippocampus. In light and electron microscope immunohistochemistry, DBI-IR was only detected in glial and ependymal cells. In brain tumors, such as astrocytomas, glioblastomas and medulloblastomas, a much higher content of DBI-IR and -mRNA was found in normal tissues. The highest level of DBI expression was found in the most anaplastic tumors. DBI-IR was virtually undetectable in meningiomas and pituitary adenomas. The high expression of DBI in brain tumors might play a role in the neoplastic growth of glial cells via the mitochondrial benzodiazepine receptor, or it may be involved in the regulation of the high energy consumption of these tumors via acyl-CoA metabolism.
The subventricular zone of the postnatal forebrain produces mainly glia, although it supports limited neurogenesis. To determine whether the subventricular zone is positionally specified, the phenotype and destination of the progeny of subventricular zone cells along the anterior-posterior axis of the lateral ventricles were analyzed. A retroviral lineage tracer containing the E. coli reporter gene lacZ was injected into different parts of the subventricular zone of neonatal rat pups, and at various times thereafter, the expression of beta-galactosidase was detected histochemically or immunohistochemically in the descendants of infected cells. A discrete region of the anterior part of the subventricular zone (SVZa) generated an immense number of neurons that differentiated into granule cells and periglomerular cells of the olfactory bulb-the two major types of interneurons. Thus, the SVZa appears to constitute a specialized source of neuronal progenitor cells. To reach the olfactory bulb, neurons arising in the SVZa migrate several millimeters along a highly restricted route. Guidance cues must be involved to prohibit widespread dispersion of these migrating neurons.
High concentrations of diazepam-binding inhibitor (DBI) mRNA have been detected in astrocytoma, suggesting that DBI-derived peptides may play a role in glial cell proliferation. In the present study, we have investigated the effect of a processing product of DBI, the octadecaneuropeptide ODN, on DNA synthesis in cultured rat astrocytes. At very low concentrations (10(-14) to 10(-11) M), ODN caused a dose-dependent increase of [3H]thymidine incorporation. At higher doses (10(-10) to 10(-5) M), the effect of ODN gradually declined. The central-type benzodiazepine receptor antagonist flumazenil (10(-6) M) completely suppressed the stimulatory action of ODN whereas the peripheral-type benzodiazepine receptor ligand, PK11195 (10(-6) M) had no effect. The ODN-induced stimulation of [3H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The GABAA receptor antagonist bicuculline (10(-4) M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABAA agonist 3APS (10(-10) to 10(-4) M) also induced a dose-related increase of [3H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABAA receptor complex, stimulates DNA synthesis in rat glial cells. These data provide evidence for an autocrine role of endozepines in the control of glial cell proliferation.
Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. This germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. Here we show that SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, we show that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. We conclude that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain.
Astrocytes and astrocytoma cells actively express the diazepam-binding inhibitor (DBI) gene, suggesting that DBI-processing products may regulate glial cell activity. In the present study, we have investigated the possible effect of one of the DBI-derived peptides, the triakontatetraneuropeptide (TTN), on [(3)H]thymidine incorporation in cultured rat astrocytes. Reversed-phase HPLC analysis of incubation media indicated that TTN is the major form of DBI-derived peptides released by cultured astrocytes. At very low concentrations (10(-14)-10(-11) M), TTN induced a dose-dependent increase in [(3)H]thymidine incorporation, whereas at higher concentrations (10(-10)-10(-5) M) the effect of TTN gradually declined. In the same range of concentrations, the specific peripheral-type benzodiazepine receptor (PBR) agonist Ro 5-4864 mimicked the bell-shaped stimulatory effect of TTN on [(3)H]thymidine incorporation. The PBR antagonist PK11195 (10(-6) M) suppressed the stimulatory action of both TTN and Ro 5-4864 on [(3)H]thymidine incorporation, whereas the central-type benzodiazepine receptor antagonist flumazenil (10(-6) M) had no effect. The present study demonstrates that the endozepine TTN stimulates DNA synthesis in rat glial cells through activation of PBRs. These data strongly suggest that TTN exerts an autocrine/paracrine stimulatory effect on glial cell proliferation.
The diazepam binding inhibitor (DBI) was originally isolated from the brain as an intrinsic ligand of the benzodiazepine binding site on the type-A gamma-aminobutyric acid receptor (GABA(A) receptor). Its wide-spread distribution in non-neural tissues outside the brain suggests that DBI has various functions other than GABA-mediated neurotransmission. Since DBI is identical with the acyl-CoA binding protein, which has the ability to bind long chain acyl-CoA esters, the major function of DBI may possibly be related to lipid metabolism. This idea was supported by our previous study showing the consistent coexpression of DBI and fatty acid binding proteins (FABPs) in epithelia throughout the gastrointestinal tract. The present histochemical study focused on the distribution of DBI in neural tissues, and revealed a definite existence of DBI in non-neuronal supporting cells in both the central and peripheral nervous systems. In the brain, intense immunoreactivity for DBI was detected in the cerebellar Bergmann glia, olfactory ensheathing glia, subgranular layer of the dentate gyrus, and retinal Muller cells. In the peripheral nervous system, satellite cells in sensory/autonomic ganglia, Schwann cells, and sustentacular cells in the adrenal medulla were immunoreactive to a DBI antibody. Moreover, the colocalization of DPI and brain-type FABP (B-FABP) was observed in most of the non-neuronal supporting cells mentioned above, indicating that DBI and B-FABP are cooperatively involved in the energy metabolism of astrocytes and related cells, which are thought to support neuronal development and functions.
Previous studies have reported the presence of migrating and dividing neuronal progenitors in the subventricular zone (SVZ) and rostral migratory stream (RMS) of the postnatal mammalian brain. Although the behaviour of these progenitors is thought to be influenced by local signals, the nature and mode of action of the local signals are largely unknown. One of the signalling molecules known to affect the behaviour of embryonic neurons is the neurotransmitter GABA. In order to determine whether GABA affects neuronal progenitors via the activation of specific receptors, we performed cell-attached, whole-cell and gramicidin perforated patch-clamp recordings of progenitors in postnatal mouse brain slices containing either the SVZ or the RMS. Recorded cells displayed a morphology typical of migrating neuronal progenitors had depolarized zero-current resting potentials, and lacked action potentials. A subset of progenitors contained GABA and stained positive for glutamic acid decarboxylase 67 (GAD-67) as shown by immunohistochemistry. In addition, every neuronal progenitor responded to GABA via picrotoxin-sensitive GABAA receptor (GABAAR) activation. GABAARs displayed an ATP-dependent rundown and a low sensitivity to Zn2+. GABA responses were sensitive to benzodiazepine agonists, an inverse agonist, as well as a barbiturate agonist. While GABA was hyperpolarizing at the zero-current resting potentials, it was depolarizing at the cell resting potentials estimated from the reversal potential of K+ currents through a cell-attached patch. Thus, our study demonstrates that neuronal progenitors of the SVZ/RMS contain GABA and are depolarized by GABA, which may constitute the basis for a paracrine signal among neuronal progenitors to dynamically regulate their proliferation and/or migration.
Certain metabolites of progesterone and deoxycorticosterone are established as potent and selective positive allosteric modulators of the gamma-aminobutyric acid type A (GABA(A)) receptor. Upon administration these steroids exhibit clear behavioural effects that include anxiolysis, sedation and analgesia, they are anticonvulsant and at high doses induce a state of general anaesthesia, a profile consistent with an action to enhance neuronal inhibition. Physiologically, peripherally synthesised pregnane steroids derived from endocrine glands such as the adrenals and ovaries function as hormones by crossing the blood brain barrier to influence neuronal signalling. However, the demonstration that certain neurons and glial cells within the central nervous system (CNS) can synthesize these steroids either de novo, or from peripherally derived progesterone, has led to the proposal that these steroids (neurosteroids) can additionally function in a paracrine manner, to locally influence GABAergic transmission. Steroid levels are known to change dynamically, for example in stress and during pregnancy. Given that GABA(A) receptors are ubiquitously expressed throughout the central nervous system, such changes in steroid levels would be predicted to cause a global enhancement of inhibitory neurotransmission throughout the brain, a scenario that would seem incompatible with a physiological role as a selective neuromodulator. Here, we will review emerging evidence that the GABA-modulatory actions of the pregnane steroids are highly selective, with their actions being brain region and indeed neuron dependent. Furthermore, the sensitivity of GABA(A) receptors is not static but can dynamically change. The molecular mechanisms underpinning this neuronal specificity will be discussed with particular emphasis being given to the role of GABA(A) receptor isoforms, protein phosphorylation and local steroid metabolism and synthesis.
The role of Zn(2+) in the CNS has remained enigmatic for several decades. This divalent cation is accumulated by specific neurons into synaptic vesicles and can be released by stimulation in a Ca(2+)-dependent manner. Using Zn(2+) fluorophores, radiolabeled Zn(2+), and selective chelators, the location of this ion and its release pattern have been established across the brain. Given the distribution and possible release under physiological conditions, Zn(2+) has the potential to act as a modulator of both excitatory and inhibitory neurotransmission. Excitatory N-methyl-D-aspartate (NMDA) receptors are directly inhibited by Zn(2+), whereas non-NMDA receptors appear relatively unaffected. In contrast, inhibitory transmission mediated via GABA(A)receptors can be potentiated via a presynaptic mechanism, influencing transmitter release; however, although some tonic GABAergic inhibition may be suppressed by Zn(2+), most synaptic GABA receptors are unlikely to be modulated directly by this cation. In the spinal cord, glycinergic transmission may also be affected by Zn(2+) causing potentiation. Recently, the penetration of synaptically released Zn(2+) into neurons suggests that this ion has the potential to act as a direct transmitter, by affecting postsynaptic signaling pathways. Taken overall, present studies are broadly supportive of a neuromodulatory role for Zn(2+) at specific excitatory and inhibitory synapses.