GalR2/3 mediates proliferative and trophic effects of galanin on postnatal hippocampal precursors

Understanding how neural activity is functionally linked to the stem cell niche, is assuming ever increasing importance as hippocampal neurogenesis is shown to be important for modulating the behavioural responses to stress and for certain forms of learning and memory. Neuropeptides such as neuropeptide Y and vasoactive intestinal peptide have emerged as important mediators for signalling local interneuron activity to subgranular zone precursors, however, little is known regarding the effects of neuropeptides that are extrinsic modulators of hippocampal information processing. Here, we show that the galanin GalR2/3 agonist Gal2-11 is both trophic and proliferative for postnatal subgranular precursors and proliferating neuroblasts at 10 nM and is purely trophic at doses as low as 100 pM. We found no effect mediated via GalR1. As galanin is co-released from noradrenergic and serotonergic projection neurons to the dentate gyrus, these findings support a direct effect of galanin on hippocampal neurogenesis, which may partly mediate its antidepressant effect via GalR2/3 receptors.     

Origin of exocrine pancreatic cells from nestin-positive precursors in developing mouse pancreas

During pancreatic development, endocrine and exocrine cell types arise from common precursors in foregut endoderm. However, little information is available regarding regulation of pancreatic epithelial differentiation in specific precursor populations. We show that undifferentiated epithelial precursors in E10.5 mouse pancreas express nestin, an intermediate filament also expressed in neural stem cells. Within developing pancreatic epithelium, nestin is co-expressed with pdx1 and p48, but not ngn3. Epithelial nestin expression is extinguished upon differentiation of endocrine and exocrine cell types, and no nestin-positive epithelial cells are observed by E15.5. In E10.5 dorsal bud explants, activation of EGF signaling results in maintenance of undifferentiated nestin-positive precursors at the expense of differentiated acinar cells, suggesting a precursor/progeny relationship between these cell types. This relationship was confirmed by rigorous lineage tracing studies using nestin regulatory elements to drive Cre-mediated labeling of nestin-positive precursor cells and their progeny. These experiments demonstrate that a nestin promoter/enhancer element containing the second intron of the mouse nestin locus is active in undifferentiated E10.5 pancreatic epithelial cells, and that these nestin-positive precursors contribute to the generation of differentiated acinar cells. As in neural tissue, nestin-positive cells act as epithelial progenitors during pancreatic development, and may be regulated by EGF receptor activity.     

强制运动促进成年大鼠海马齿状回神经发生并提高学习能力

为了探讨强制运动对成年大鼠海马齿状回（dentate gyrus,DG）神经发生的影响,强制大鼠在马达驱动的转轮中跑步,用5-溴-2-脱氧尿苷（5-bromo-2-deoxyuridine,BrdU）标记增殖细胞,巢蛋白（neuroepthelial stem cell protein,nestin）标记神经干细胞／前体细胞,然后用免疫细胞化学技术检测大鼠DG中BrdU及nestin阳性细胞。为了解强制运动后DG增殖细胞的功能意义,采用Y-迷宫检测大鼠的学习能力。结果表明,强制运动组DG中BrdU及nestin阳性细胞数均日月显多于对照组（P〈0．05）：强制运动对DG神经发生的效应有强度依赖性。Y-迷宫检测结果显示,强制运动能明显改善大鼠的学习能力。结果提示,在转轮中进行强制跑步能促进成年火鼠DG的神经发生,并改善学习能力。     

GALR2 and Y1R agonists intranasal infusion enhanced adult ventral hippocampal neurogenesis and antidepressant-like effects involving BDNF actions

Dysregulation of adult hippocampal neurogenesis is linked to major depressive disorder (MDD), with more than 300 million people diagnosed and worsened by the COVID-19 pandemic. Accumulating evidence for neuropeptide Y (NPY) and galanin (GAL) interaction was shown in various limbic system regions at molecular-, cellular-, and behavioral-specific levels. The purpose of the current work was to evaluate the proliferating role of GAL2 receptor (GALR2) and Y1R agonists interaction upon intranasal infusion in the ventral hippocampus. We studied their hippocampal proliferating actions using the proliferating cell nuclear antigen (PCNA) on neuroblasts or stem cells and the expression of the brain-derived neurothrophic factor (BDNF). Moreover, we studied the formation of Y1R–GALR2 heteroreceptor complexes and analyzed morphological changes in hippocampal neuronal cells. Finally, the functional outcome of the NPY and GAL interaction on the ventral hippocampus was evaluated in the forced swimming test. We demonstrated that the intranasal infusion of GALR2 and the Y1R agonists promotes neuroblasts proliferation in the dentate gyrus of the ventral hippocampus and the induction of the neurotrophic factor BDNF. These effects were mediated by the increased formation of Y1R–GALR2 heteroreceptor complexes, which may mediate the neurites outgrowth observed on neuronal hippocampal cells. Importantly, BDNF action was found necessary for the antidepressant-like effects after GALR2 and the Y1R agonists intranasal administration. Our data may suggest the translational development of new heterobivalent agonist pharmacophores acting on Y1R–GALR2 heterocomplexes in the ventral hippocampus for the novel therapy of MDD or depressive-affecting diseases.     

Neuropeptide Y stimulates neuronal precursor proliferation in the post-natal and adult dentate gyrus

Adult dentate neurogenesis is important for certain types of hippocampal-dependent learning and also appears to be important for the maintenance of normal mood and the behavioural effects of antidepressants. Neuropeptide Y (NPY), a peptide neurotransmitter released by interneurons in the dentate gyrus, has important effects on mood, anxiety-related behaviour and learning and memory. We report that adult NPY receptor knock-out mice have significantly reduced cell proliferation and significantly fewer immature doublecortin-positive neurons in the dentate gyrus. We also show that the neuroproliferative effect of NPY is dentate specific, is Y1-receptor mediated and involves extracellular signal-regulated kinase (ERK)1/2 activation. NPY did not exhibit any effect on cell survival in vitro but constitutive loss of the Y1 receptor in vivo resulted in greater survival of newly generated neurons and an unchanged total number of dentate granule cells. These results show that NPY stimulates neuronal precursor proliferation in the dentate gyrus and suggest that NPY-releasing interneurons may modulate dentate neurogenesis.     

Regulation of peptidergic vesicle mobility by secretagogues

Neuropeptides are released into the extracellular space from large secretory granules. In order to reach their release sites, these granules are translocated on microtubules and thought to interact with filamentous actin as they approach the cell membrane. We have used a green fluorescent protein-tagged neuropeptide prohormone (prepro-orphanin FQ) to visualize vesicle trafficking dynamics in NS20Y cells and cultures of primary hippocampal neurons. We found that the majority of secretory granules were mobile and accumulated at both the tips of neurites as well as other apparently specialized cellular sites. We also used live-cell imaging to test the notion that peptidergic vesicle mobility was regulated by secretagogues. We show that treatment with forskolin appeared to increase vesicle rates of speed, while depolarization with high K⁺ had no effect, even though both treatments stimulated neuropeptide secretion. In cultured hippocampal neurons the green fluorescent protein-tagged secretory vesicles were routed to both dendrites and axons, indicating that peptidergic vesicle transport was not polarized. Basal peptidergic vesicle mobility rates in hippocampal neurons were the same as those in NS20Y cells. Taken together, these studies suggest that secretory vesicle mobility is regulated by specific classes of secretagogues and that neuropeptide containing secretory vesicles may be released from dendritic structures.     

Intracellular nitric oxide mediates neuroproliferative effect of neuropeptide y on postnatal hippocampal precursor cells

Neuropeptide Y (NPY) is widely expressed in the central and peripheral nervous systems and is proliferative for a range of cells types in vitro. NPY plays a key role in regulating adult hippocampal neurogenesis in vivo under both basal and pathological conditions, although the underlying mechanisms are largely unknown. We have investigated the role of nitric oxide (NO) on the neurogenic effects of NPY. Using postnatal rat hippocampal cultures, we show that the proliferative effect of NPY on nestin(+) precursor cells is NO-dependent. As well as the involvement of neuronal nitric-oxide synthase, the proliferative effect is mediated via an NO/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) and extracellular signal-regulated kinase (ERK) 1/2 signaling pathway. We show that NPY-mediated intracellular NO signaling results in an increase in neuroproliferation. By contrast, extracellular NO had an opposite, inhibitory effect on proliferation. The importance of the NO-cGMP-PKG signaling pathway in ERK1/2 activation was confirmed using Western blotting. This work unites two significant modulators of hippocampal neurogenesis within a common signaling framework and provides a mechanism for the independent extra- and intracellular regulation of postnatal neural precursors by NO.     

Characterization and regulation of peptidylglycine α-amidating monooxygenase (PAM) expression in H9c2 cardiac myoblasts

Nitric oxide (NO) is a gaseous, radical molecule that plays a role in various physiological processes in the nervous system such as learning and hippocampal plasticity. It is generated from l-arginine by nitric oxide synthases (NOS), which come in three isoforms depending on the tissue of origin, namely inducible-NOS (iNOS in macrophages), endothelial-NOS (eNOS in endothelial cells) and neural-NOS (nNOS in neural cells). We used epidermal growth factor (EGF)-responsive nestin-positive neural precursor cells originating from the mouse E16 embryonic striatum, and studied the relative expression of NOS isoforms probed with isoform-specific antibody using the avidin-biotin immunohistochemical method. Our data revealed both nNOS and eNOS to be expressed in both neurospheres and desegregated neural precursor cells. However, iNOS signals were virtually undetectable in both cell categories. When the neural precursor cells were carried in the presence of poly-l-ornithine (PLO), there was a strong induction of the expression of iNOS proteins, indicating the possibility that this isoform is amenable to modulation by extracellular cues. These preliminary results suggest both nNOS and eNOS to be important in the physiology of neural precursor cells, and that iNOS might also play a role at certain stages in the life of these cells.     

Modeling of Neuropeptide Receptors Y1, Y4, Y5, and Docking Studies with Neuropeptide Antagonist Analogues: Implications for Selectivity

Abstract Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y(1)-Y(5) and y(6). Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Modeling of neuropeptide receptors Y1, Y4, Y5, and docking studies with neuropeptide antagonist

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y1-Y5 and y6. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase

Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused a rapid and transient increase in the concentration of free calcium in the cytoplasm as measured by the fluorescent probe, Fura-2. The effect of both peptides was independent of extracellular calcium as addition of EGTA or manganese neither changed the size nor the shape of the calcium response. The calcium response to NPY was abolished by pretreatment with thapsigargin, which can selectively deplete a calcium store in the endoplasmic reticulum. Y1 receptor stimulation, by both NPY and [Leu31,Pro34]NPY, also inhibited the forskolin-stimulated cAMP production with an EC50 of 3.5 nM. There was a close relation between the receptor binding and the cellular effects as half-maximal displacement of [125I-Tyr36]monoiodoNPY from the receptor was obtained with 2.1 nM NPY. The Y2-specific ligand NPY(16-36)peptide had no effect on either intracellular calcium or cAMP levels in the SK-N-MC cells. It is concluded that Y1 receptor stimulation is associated with both mobilization of intracellular calcium and inhibition of adenylate cyclase activity.     

Modeling of Neuropeptide Receptors Y1, Y4, Y5, and Docking Studies with Neuropeptide Antagonist Analogues: Implications for Selectivity

Abstract

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y₁-Y₅ and y₆. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

A subpopulation of mushroom body intrinsic neurons is generated by protocerebral neuroblasts in the tobacco hornworm moth, Manduca sexta (Sphingidae, Lepidoptera)

Subpopulations of Kenyon cells, the intrinsic neurons of the insect mushroom bodies, are typically sequentially generated by dedicated neuroblasts that begin proliferating during embryogenesis. When present, Class III Kenyon cells are thought to be the first born population of neurons by virtue of the location of their cell somata, farthest from the position of the mushroom body neuroblasts. In the adult tobacco hornworm moth Manduca sexta, the axons of Class III Kenyon cells form a separate Y tract and dorsal and ventral lobelet; surprisingly, these distinctive structures are absent from the larval Manduca mushroom bodies. BrdU labeling and immunohistochemical staining reveal that Class III Kenyon cells are in fact born in the mid-larval through adult stages. The peripheral position of their cell bodies is due to their genesis from two previously undescribed protocerebral neuroblasts distinct from the mushroom body neuroblasts that generate the other Kenyon cell types. These findings challenge the notion that all Kenyon cells are produced solely by the mushroom body neuroblasts, and may explain why Class III Kenyon cells are found sporadically across the insects, suggesting that when present, they may arise through de novo recruitment of neuroblasts outside of the mushroom bodies. In addition, lifelong neurogenesis by both the Class III neuroblasts and the mushroom body neuroblasts was observed, raising the possibility that adult neurogenesis may play a role in mushroom body function in Manduca.     

Timelines in the insect brain: fates of identified neural stem cells generating the central complex in the grasshopper Schistocerca gregaria

This study employs labels for cell proliferation and cell death, as well as classical histology to examine the fates of all eight neural stem cells (neuroblasts) whose progeny generate the central complex of the grasshopper brain during embryogenesis. These neuroblasts delaminate from the neuroectoderm between 25 and 30 % of embryogenesis and form a linear array running from ventral (neuroblasts Z, Y, X, and W) to dorsal (neuroblasts 1-2, 1-3, 1-4, and 1-5) along the medial border of each protocerebral hemisphere. Their stereotypic location within the array, characteristic size, and nuclear morphologies, identify these neuroblasts up to about 70 % of embryogenesis after which cell shrinkage and shape changes render progressively more cells histologically unrecognizable. Molecular labels show all neuroblasts in the array are proliferative up to 70 % of embryogenesis, but subsequently first the more ventral cells (72–75 %), and then the dorsal ones (77–80 %), cease proliferation. By contrast, neuroblasts elsewhere in the brain and optic lobe remain proliferative. Apoptosis markers label the more ventral neuroblasts first (70–72 %), then the dorsal cells (77 %), and the absence of any labeling thereafter confirms that central complex neuroblasts have exited the cell cycle via programmed cell death. Our data reveal appearance, proliferation, and cell death proceeding as successive waves from ventral to dorsal along the array of neuroblasts. The resulting timelines offer a temporal blueprint for building the neuroarchitecture of the various modules of the central complex.     

proNGF Inhibits Neurogenesis and Induces Glial Activation in Adult Mouse Dentate Gyrus

Recent studies have shown that the precursor of nerve growth factor (proNGF) is highly elevated in aging brains and in the brains of patients with Alzheimer’s Disease. proNGF accumulates in hippocampus which is an important neurogenic region related to learning and memory. However, it remains unclear whether proNGF has an influence on hippocampal neurogenesis. In this study, we demonstrated that the high-affinity receptor of proNGF, p75 neurotrophic factor (p75NTR), was expressed both on cells undergoing mitosis and postmitotic mature cells in mouse hippocampus. proNGF infusion into adult mouse hippocampus significantly reduced the density of BrdU-incorporating cells and the density of BrdU/Doublecortin double positive cells in the subgranular zone of hippocampus, indicating an inhibitory effect of proNGF on hippocampal neurogenesis. proNGF infusion also induced prominent cell apoptosis and activated residential astrocyte and microglia, which might further impair the hippocampal neurogenesis. These results implied that proNGF played a pivotal role in regulating the hippocampal neurogenesis and might account for the memory deficit and cognitive impairment.     

Suppressor of Cytokine Signalling 2 (SOCS2) Regulates Numbers of Mature Newborn Adult Hippocampal Neurons and Their Dendritic Spine Maturation

Overexpression of suppressor of cytokine signalling 2 (SOCS2) has been shown to promote hippocampal neurogenesis in vivo and promote neurite outgrowth of neurons in vitro. In the adult mouse brain, SOCS2 is most highly expressed in the hippocampal CA3 region and at lower levels in the dentate gyrus, an expression pattern that suggests a role in adult neurogenesis. Herein we examine generation of neuroblasts and their maturation into more mature neurons in SOCS2 null (SOCS2KO) mice. EdU was administered for 7 days to label proliferative neural precursor cells. The number of EdU-labelled doublecortin⁺ neuroblasts and NeuN⁺ mature neurons they generated was examined at day 8 and day 35, respectively. While no effect of SOCS2 deletion was observed in neuroblast generation, it reduced the numbers of EdU-labelled mature newborn neurons at 35 days. As SOCS2 regulates neurite outgrowth and dentate granule neurons project to the CA3 region, alterations in dendritic arborisation or spine formation may have correlated with the decreased numbers of EdU-labelled newborn neurons. SOCS2KO mice were crossed with Nes-CreER^T2/mTmG mice, in which membrane eGFP is inducibly expressed in neural precursor cells and their progeny, and the dendrite and dendritic spine morphology of newborn neurons were examined at 35 days. SOCS2 deletion had no effect on total dendrite length, number of dendritic segments, number of branch points or total dendritic spine density but increased the number of mature “mushroom” spines. Our results suggest that endogenous SOCS2 regulates numbers of EdU-labelled mature newborn adult hippocampal neurons, possibly by mediating their survival and that this may be via a mechanism regulating dendritic spine maturation.     

Distinct Changes in Peptide YY Binding to, and mRNA Levels of, Y1 and Y2 Receptors in the Rat Hippocampus Associated with Kindling Epileptogenesis

Abstract: Electrical kindling of the rat dorsal hippocampus induced significant changes in the binding of ¹²⁵I-peptide YY to Y1 and Y2 subtypes of neuropeptide Y receptors and in their mRNA levels in the area dentata as assessed by quantitative receptor autoradiography and in situ hybridization histochemistry. Binding to Y1 receptor sites decreased by 50% ( p < 0.05) in the molecular layer of the stimulated dentate gyrus, 2 days after preconvulsive stage 2 and 1 week or 1 month after generalized stage 5 seizures compared with sham-stimulated rats. Binding to Y2 receptor sites increased bilaterally by 36–87% ( p < 0.05) in the hilus at stage 2 and 1 week or 1 month after stage 5. No significant changes were observed after one afterdischarge or in the other hippocampal subfields or in the cortex. Y1 receptor mRNA signal decreased bilaterally by 50–64% ( p < 0.01) in the granule cell layer, 6 h but not 24 h after stages 2 and 5. The Y2 receptor mRNA signal was enhanced by 283% ( p < 0.01) in the stimulated granule cell layer 24 h after stage 2. At 6 and 24 h after stage 5, mRNA levels were increased both ipsilaterally (283 and 360%, respectively; p < 0.01) and contralaterally (190 and 260%, respectively; p < 0.05). No significant changes in level of either mRNA was found following one afterdischarge. These modifications, and the enhanced neuropeptide Y release previously shown in the hippocampus, suggest that kindling is associated with lasting changes in neuropeptide Y-mediated neurotransmission.