Search

Summary Adult neurogenesis requires the precise control of neuronal versus astrocyte lineage determination in neural stem cells. While microRNAs (miRNAs) are critically involved in this step during development, their actions in adult hippocampal neural stem cells (aNSCs) has been unclear. As entry point to address that question we chose DICER, an endoribonuclease essential for miRNA biogenesis and other RNAi-related processes. By specific ablation of Dicer in aNSCs in vivo and in vitro, we demonstrate that miRNAs are required for the generation of new neurons, but not astrocytes, in the adult murine hippocampus. Moreover, we identify 11 miRNAs, of which 9 have not been previously characterized in neurogenesis, that determine neurogenic lineage fate choice of aNSCs at the expense of astrogliogenesis. Finally, we propose that the 11 miRNAs sustain adult hippocampal neurogenesis through synergistic modulation of 26 putative targets from different pathways., Graphical Abstract, Highlights • Dicer depletion in aNSCs impairs neurogenesis and stimulates astrogliogenesis • Synergy of 11 miRNAs sustains neuronal fate of aNSCs • miRNA converge on multiple targets in different pathways to induce neurogenesis, In this article, the authors demonstrate that Dicer-dependent miRNAs are required for the generation of new neurons, but not astrocytes, in the adult hippocampus in vivo and in vitro. The authors identify a new set of 11 miRNAs that synergistically converge on multiple targets in different pathways to sustain neurogenic lineage fate commitment in aNSCs.

We studied how the noncompetitive NMDA receptor antagonist MK801 affected different stages of adult hippocampal neurogenesis in mice, and investigated how the activation of benzodiazepine receptors with diazepam interacted with the effects of MK801 on the precursor cells in the adult dentate gyrus. Our findings were: (i) one single MK801 application increased precursor cell proliferation and adult neurogenesis but not gliogenesis 4 weeks later; (ii) the number of label-retaining precursor cells decreased after MK801 (with P = 0.06); (iii) the pro-neurogenic effect included increased cell cycle entry of precursor cells as well as completed cell divisions, except in type-2a cells; (iv) NMDA receptor blockade also increased the number of nestin-GFP-expressing cells expressing calretinin; (v) diazepam alone had a very similar effect on overall precursor cell proliferation to that of MK801 alone; and (vi) diazepam, when co-applied with MK801, abolished the suppression of divisions of type-2a cells induced by MK801 alone, suppressed the MK801-induced effect on proliferation of type-2b cells and had no influence on the effects of MK801 on type-3 cells, but did suppress the increased number of nestin-GFP-positive cells expressing calretinin. From these results we hypothesize that, depending on the precursor cell stage, NMDA-dependent neurotransmission has distinct effects that are partly antagonized and partly enhanced by GABAergic input. We propose that NMDA receptor-dependent signalling maintains the precursor cells in the dentate gyrus while blocking initial stages of development and promoting more advanced stages.

When does adult hippocampal neurogenesis begin? We describe the development of the neurogenic niche in the subgranular zone (SGZ) of the hippocampal dentate gyrus. We did so from the perspective of the situation in the adult. Ontogeny of the dentate gyrus is complex and results in an ectopic neurogenic niche that lifelong generates new granule cells. Neurogenesis during the fetal and early postnatal periods builds the dentate gyrus and gives way to activity-dependent "adult" neurogenesis. We used markers most relevant to adult neurogenesis research to describe this transition: Nestin, Sox2, BLBP, GFAP, Tbr2, Doublecortin (DCX), NeuroD1 and Prox1. We found that massive changes and a local condensation of proliferating precursor cells occurs between postnatal day 7 (P7), near the peak in proliferation, and P14. Before and around P7, the spatial distribution of cells and the co-localization of markers were distinct from the situation in the adult. Unlike the adult SGZ, the marker pair Nestin/Sox2 and the radial glial marker BLBP were not overlapping during embryonic development, presumably indicating different types of radial glia-like cells. Before P7 GFAP-positive cells in the hilus lacked the radial orientation that is characteristic of the adult type-1 cells. DCX, which is concentrated in type-2b and type-3 progenitor cells and early postmitotic neurons in the adult, showed diffuse expression before P7. Intermediate progenitor cell marker Tbr2 became restricted to the SGZ but was found in the granule cell layer (GCL) and hilus before. Lineage markers NeuroD1 and Prox1 confirmed this pattern. We conclude that the neurogenic niche of adult neurogenesis is in place well before true adulthood. This might indicate that consistent with the hypothesized function of adult neurogenesis in activity-dependent plasticity, the early transition from postnatal neurogenesis to adult neurogenesis coincides with the time, when the young mice start to become active themselves.

Approximately 5% of patients with malignant glioma achieve complete response (CR) after first-line combined modality treatment. Although these patients will invariably suffer from tumor recurrence, they usually do not receive any further treatment to maintain remission. According to in vitro and in vivo clinical studies, 13-cis retinoic acid (cRA) may be a promising agent for maintenance therapy in these patients.We initiated a clinical study to evaluate the feasibility and toxicity of high-dose cRA as maintenance therapy in patients with high-grade glioma in complete remission after first-line multimodal treatment.A prospective single-arm phase-II study in patients with CR after combined first-line therapy (neurosurgery, radio- and chemotherapy) was performed. Patients were treated with cRA at 60 mg/m2 BS from day 1 to 21 in four-weekly cycles with a dose escalation of up to 100 mg/m2 BS until tumor recurrence. Clinical controls were performed every 4 weeks, magnetic resonance imaging every 8 weeks.Twenty-three patients (10, grade IV; 13, grade III) were evaluable using an intention-to-treat analysis. Treatment was well tolerated for up to 149 weeks with moderate dermatological symptoms in all patients. No grade 4 toxicities were observed. Median time to progression was 41 weeks, median overall survival 74 weeks after inclusion in the protocol.There is an urgent need for strategies maintaining remission in patients with malignant glioma. Maintenance therapy with high-dose cRA is feasible and well tolerated over long periods of time. A controlled clinical trial to test the efficacy of cRA as a maintenance treatment in malignant glioma is warranted.

BACKGROUND Doxorubicin exhibits high efficacy in malignant glioma cell cultures. Nonetheless, as a standard formulation, doxorubicin has not been used clinically, due to poor penetration of the blood-brain barrier. Furthermore, doxorubicin is known to induce tumor resistance genes. To address both of these issues, the authors investigated the use of pegylated liposomal doxorubicin (Caelyx™; Essex Pharma, Munich, Germany) alone (Trial 1) and in combination with tamoxifen (Trial 2) in two sequentially performed nonrandomized prospective Phase II trials involving patients with recurrent high-grade glioma. METHODS Twenty patients were included in each trial. Progression-free survival at 6 months (PFS-6) and toxicity were the primary endpoints. Expression of the tumor resistance proteins multidrug resistance protein 1 (MDR-1) and multiple resistance protein (MRP) was evaluated by immunohistochemical methods and by sestamibi–single-photon emission computed tomography (SPECT). RESULTS The overall response rate (including cases of disease stabilization) was 40% in both Trial 1 and Trial 2. PFS-6 was 15%, and the median time to disease progression was 17 weeks. It is noteworthy that 40% of patients with Grade III tumors had long-term responses, which lasted for up to 3 years. There was no significant difference between Trial 1 and Trial 2 in terms of efficacy. Both regimens were well tolerated, with the main side effect being palmoplantar erythrodysesthesia. The authors found no correlation between clinical response and expression of tumor resistance genes or between clinical response and SPECT data. CONCLUSIONS Pegylated liposomal doxorubicin administered alone or in combination with tamoxifen is safe and moderately effective in patients with recurrent high-grade glioma. None of the putative predictors for response that were evaluated proved to be significant in this setting. Cancer 2004. © 2004 American Cancer Society.

The immediate-early 63-kDa (IE63) protein of varicella-zoster virus (VZV) is a phosphoprotein encoded by open reading frame (ORF) ORF63/ORF70. To identify functional domains, 22 ORF63 mutations were evaluated for effects on IE63 binding to the major VZV transactivator, IE62, and on IE63 phosphorylation and nuclear localization in transient transfections, and after insertion into the viral genome with VZV cosmids. The IE62 binding site was mapped to IE63 amino acids 55 to 67, with R59/L60 being critical residues. Alanine substitutions within the IE63 center region showed that S165, S173, and S185 were phosphorylated by cellular kinases. Four mutations that changed two putative nuclear localization signal (NLS) sequences altered IE63 distribution to a cytoplasmic/nuclear pattern. Only three of 22 mutations in ORF63 were compatible with recovery of infectious VZV from our cosmids, but infectivity was restored by inserting intact ORF63 into each mutated cosmid. The viable IE63 mutants had a single alanine substitution, altering T171, S181, or S185. These mutants, rOKA/ORF63rev[T171], rOKA/ORF63rev[S181], and rOKA/ORF63rev[S185], produced less infectious virus and had a decreased plaque phenotype in vitro. ORF47 kinase protein and glycoprotein E (gE) synthesis was reduced, indicating that IE63 contributed to optimal expression of early and late gene products. The three IE63 mutants replicated in skin xenografts in the SCIDhu mouse model, but virulence was markedly attenuated. In contrast, infectivity in T-cell xenografts was not altered. Comparative analysis suggested that IE63 resembled the herpes simplex virus type 1 U S 1.5 protein, which is expressed colinearly with ICP22 (U S 1). In summary, most mutations of ORF63 made with our VZV cosmid system were lethal for infectivity. The few IE63 changes that were tolerated resulted in VZV mutants with an impaired capacity to replicate in vitro. However, the IE63 mutants were attenuated in skin but not T cells in vivo, indicating that the contribution of the IE63 tegument/regulatory protein to VZV pathogenesis depends upon the differentiated human cell type which is targeted for infection within the intact tissue microenvironment.

Declining learning and memory function is associated with the attenuation of adult hippocampal neurogenesis. As in humans, chronic stress or depression in animals is accompanied by hippocampal dysfunction, and neurogenesis is correspondingly down regulated, in part, by the activity of the hypothalamic-pituitary-adrenal axis as well as glutamatergic and serotonergic networks. Antidepressants can reverse this effect over time but one of the most clinically effective moderators of stress or depression and robust stimulators of neurogenesis is simple voluntary physical exercise such as running. Curiously, running also elevates circulating stress hormone levels yet neurogenesis is doubled in running animals. In evaluating the signalling that running provides to the central nervous system in mice, we have found that peripheral vascular endothelial growth factor (VEGF) is necessary for the effects of running on adult hippocampal neurogenesis. Peripheral blockade of VEGF abolished running-induced neurogenesis but had no detectable effect on baseline neurogenesis in non-running animals. These data suggest that VEGF is an important element of a 'somatic regulator' of adult neurogenesis and that these somatic signalling networks can function independently of the central regulatory networks that are typically considered in the context of hippocampal neurogenesis.

Angiopoietins play a pivotal role in tumor angiogenesis by modulating vascular endothelial proliferation and survival. The expression of angiopoietins 1 and 2 (Ang-1 and Ang-2) and vascular endothelial growth factor (VEGF) has been documented in human malignant glioma. The expression of Ang-1, Ang-2, VEGF, and Tie-2, a member of the receptor tyrosine kinases and the natural receptor for both Ang-1 and Ang-2, follows a distinct transcriptional profile in vivo. Ang-2 and VEGF were expressed early in tumor formation and their levels increased throughout tumor growth. Their expression coincided with the expansion of the tumor mass and the formation of the vascular tree. There was no significant change in the expression of Tie-2 and Ang-1. The expression of Ang-1 and Tie-2 was more noticeable at the periphery of the tumor. The expression of Ang-2 was more robust at the periphery and within the tumor mass, and VEGF was more concentrated within the center of the tumor. This distinct expression profile may explain the morphology of the newly formed vessels at various times and regions of the tumor. The lack of concomitant expression of Ang-1 may underscore the unopposed endovascular induction by Ang-2 and VEGF resulting in the chaotic appearance and fragility of tumor vessels.

Approaches to improve the prognosis of patients with high-grade glioma, which is still grim despite combined therapy and major advances in the development of new drugs, are highly important. Chemotherapeutic agents have not consistently produced favorable results in the relapse setting so far, with tumor responses reported in a minority of cases. Among them, doxorubicin has not shown significant efficacy, despite being one of the most effective substances in vitro and in animal models. Nevertheless, encapsulation of doxorubicin using polyethylene-glycol significantly improves penetration across the blood-brain barrier. Moreover, recent clinical trials in other entities have demonstrated that similar clinical responses can be achieved using approximately half of the dose of polyethylene-glycol-liposomes compared with conventional liposomes. Considering these facts, polyethylene-glycol-liposomal doxorubicin with and without tamoxifen was evaluated within two sequential Phase II trials performed at our institution. Polyethylene-glycol-liposomal doxorubicin (Caelyx) was efficient in a reasonable number of patients with equivalent results in comparison with other successful Phase II studies. This article will discuss the literature and our own results on polyethylene-glycol-liposomal doxorubicin, with a special focus on toxicity and efficacy data raised in clinical trials on patients with high-grade glioma.

Adult hippocampal neurogenesis is to a large degree controlled at the level of cell survival, and a number of potential mediators of this effect have been postulated. Here, we investigated the small heat shock protein Hspb8, which, because of its pleiotropic prosurvival effects in other systems, was considered a particularly promising candidate factor. Hspb8 is, for example, found in plaques of Alzheimer disease but exerts neuroprotective effects. We found that expression of Hspb8 increased during differentiationin vitroand was particularly associated with later stages (48–96 h) of differentiation. Gain-of-function and loss-of-function experiments supported the hypothesis that Hspb8 regulates cell survival of new neuronsin vitro. In the dentate gyrus of adult micein vivo, lentiviral overexpression of Hspb8 doubled the surviving cells and concomitantly promoted differentiation and net neurogenesis without affecting precursor cell proliferation. We also discovered that the truncated form of the crystallin domain of Hspb8 was sufficient to affect cell survival and neuronal differentiationin vitroandin vivo. Precursor cell experimentsin vitrorevealed that Hspb8 increases the phosphorylation of Akt and suggested that the prosurvival effect can be produced by a cell-autonomous mechanism. Analysis of hippocampal Hspb8 expression in mice of 69 strains of the recombinant inbred set BXD revealed thatHspb8is acis-acting gene whose expression was associated with clusters of transcript enriched in genes linked to growth factor signaling and apoptosis. Our results strongly suggest that Hspb8 and its α-crystallin domain might act as pleiotropic prosurvival factor in the adult hippocampus.

Transforming growth factors-beta 1 and -beta 2 (TGF-beta 1 and -beta 2) are important growth-regulatory proteins for astroglial neoplasms. We analyzed their role in tumor-cell proliferation in 12 glioma cell lines, employing phosphorothioate antisense oligodeoxynucleotides (S-ODNs, 14 mer), specifically targeted against the coding sequences of TGF-beta 1-mRNA and TGF-beta 2-mRNA. TGF-beta 1-S-ODNs inhibited cell proliferation in 5 of 12 gliomas, whereas TGF-beta 2-S-ODNs reduced the cell proliferation in all glioma cell lines, compared to nonsense-S-ODN-treated and S-ODN-untreated cells as controls. The efficacy and specificity of antisense effects was validated by Northern-blot analysis and determination of protein concentrations in culture supernatants (ELISA). Exogenous hrTGF-beta 1 either stimulated or inhibited the cell lines, whereas pnTGF-beta 2 stimulated the proliferation of most glioma cells. Blocking the extracellular pathway of TGF-beta by neutralizing antibodies only slightly inhibited those cell lines, which were markedly stimulated by TGF-betas. As the effects of TGF-beta 2-S-ODNs were much stronger than those of TGF-beta neutralizing antibodies, we postulate that the endogenously produced TGF-beta 2 control glioma-cell proliferation, in part by an intracellular loop.

Background Adult neurogenesis is a particular example of brain plasticity that is partially modulated by the endocannabinoid system. Whereas the impact of synthetic cannabinoids on the neuronal progenitor cells has been described, there has been lack of information about the action of plant-derived extracts on neurogenesis. Therefore we here focused on the effects of Δ9-tetrahydrocannabinol (THC) and Cannabidiol (CBD) fed to female C57Bl/6 and Nestin-GFP-reporter mice on proliferation and maturation of neuronal progenitor cells and spatial learning performance. In addition we used cannabinoid receptor 1 (CB1) deficient mice and treatment with CB1 antagonist AM251 in Nestin-GFP-reporter mice to investigate the role of the CB1 receptor in adult neurogenesis in detail. Results THC and CBD differed in their effects on spatial learning and adult neurogenesis. CBD did not impair learning but increased adult neurogenesis, whereas THC reduced learning without affecting adult neurogenesis. We found the neurogenic effect of CBD to be dependent on the CB1 receptor, which is expressed over the whole dentate gyrus. Similarly, the neurogenic effect of environmental enrichment and voluntary wheel running depends on the presence of the CB1 receptor. We found that in the absence of CB1 receptors, cell proliferation was increased and neuronal differentiation reduced, which could be related to CB1 receptor mediated signaling in Doublecortin (DCX)-expressing intermediate progenitor cells. Conclusion CB1 affected the stages of adult neurogenesis that involve intermediate highly proliferative progenitor cells and the survival and maturation of new neurons. The pro-neurogenic effects of CBD might explain some of the positive therapeutic features of CBD-based compounds.

Neurogenesis occurs constitutively within the periventricular region (PVR) of the lateral ventricles (LV) of the adult mammalian brain. The occurrence of adult neurogenesis within the PVR outside the neurogenic niche of the LV remains controversial, but neural stem cells can be isolated from PVR of the whole ventricular system. The histological basis of this phenomenon including the regional differences of cellular phenotypes within the PVRs is still enigmatic. The occurrence of neurogenesis or manipulable progenitor cells in caudal parts of the adult brain is however one prerequisite for orthotopic regenerative approaches in Parkinson's disease (PD) and other disorders of the midbrain/brainstem. Using quantitative immunohistochemical techniques and electron microscopy, we found a rostro-caudal gradual loss of cellular diversity within the PVR throughout the whole ventricular axis with loss of transit amplifying epidermal growth factor-receptor+ type C cells in all parts caudal to the LV, a gradual reduction from rostral to caudal of both stem cells (type B cells or astrocytes) without signs of proliferation outside the PVR of the LV as well as neuroblasts-like cells (polysialylated neural cell adhesion molecule [PSA-NCAM]+, but doublecortin negative cells) with a different morphology compared with neuroblasts of the PVR of the LV. Electron microscopy confirmed these immunohistochemical data. The proportion of Nestin+/CD24+ cells and Nestin+/S100β+ ependymal cells were consecutively increased in the PVR from rostral to caudal, and ultrastructural analysis showed a region-specific morphology with darker cytoplasm with occasional large lipid droplets as well as indented nuclei within the caudal PVRs. The strong correlation of neuroblast-like cells with the number of neurosphere-forming cells suggests that a quiescent subtype of PSA-NCAM+ cells might be a source of neurosphere-forming cells. We did not find any evidence for neurogenesis or the occurrence of neuroprogenitors within the substantia nigra or other parts of the midbrain/brainstem outside the PVR. Our data provide the histological framework for future studies on orthotopic regenerative approaches in PD by recruiting endogenous predopaminergic progenitors from the midbrain PVR. Disclosure of potential conflicts of interest is found at the end of this article.

Purpose of review Failing adult neurogenesis is increasingly considered a factor in the pathogenesis and course of psychiatric disorders. The level of evidence in favor of such hypotheses varies, but disturbed cellular plasticity in the hippocampus may be a common aspect of several neuropsychiatric diseases. Recent findings This review covers the literature from mid-2006 to the end of 2007. We discuss studies and theoretical papers dealing with the contribution of adult neurogenesis to dementias and neurodegeneration, major depression, schizophrenia, and alcohol and drug abuse. Of these disorders, most progress has recently been made with schizophrenia for which, in contrast to the other conditions, suggestive genetic evidence exists (e.g. Disc1, Npas3). Summary Failing adult hippocampal neurogenesis may not explain major depression, addiction or schizophrenia, but contributes to the hippocampal aspects of the disease. We propose that the key to a more thorough understanding of this contribution will come from increased knowledge on the functional relevance of new neurons in the hippocampus and better clinical data relating to symptoms possibly related to such function. Research on the molecular basis of adult hippocampal neurogenesis may help to explain how hippocampal aspects of these disorders develop.

Melanoma inhibitory activity (MIA) is an 11 kD protein secreted by malignant melanomas. Recent studies revealed an interaction of MIA with epitopes of extracellular matrix proteins including fibronectin. Structural homology of MIA with the binding sites of alpha4beta1 integrin results in complex interactions of MIA with molecules binding to alpha4beta1 integrin. As cells of the immune system express alpha4beta1 integrins (VLA-4), we investigated whether MIA may modulate the function of human leukocytes. Here we describe the effects of MIA on the activation of human PBMCs and auto-/allogeneic lymphokine-activated killer cell (LAK) cytotoxicity in human MIA-negative glioma cell lines and MIA-positive melanoma cell lines in vitro. MIA inhibits PHA- or IL-2-induced human PBMC proliferation in a dose-dependent manner up to 63% ((3)H-Tdr incorporation) and 59% (cell count), respectively, when added to the cell culture prior to mitogen stimulation. In addition, both autologous (GL and HW) and allogeneic (HTZ-17, HTZ-243 and HTZ-374) antitumor LAK cytotoxicity was reduced by the addition of exogenous rhMIA (500 ng/ml, f.c.). Consequently, endogenous inhibition of MIA expression in human melanoma cells by MIA-specific phosphorothioate antisense oligonucleotides enhanced the autologous LAK-cell activity to the same level as observed in MIA-negative human HMB melanoma cells expressing an MIA-antisense construct. Our results indicate that MIA may contribute to immunosuppression frequently seen in malignant melanomas by inhibiting cellular antitumor immune reactions. Antagonization of MIA activity using antisense techniques may represent a novel therapeutic strategy for treatment of malignant melanomas.

Varicella-zoster virus (VZV) establishes latency in sensory ganglia and causes herpes zoster upon reactivation. These investigations in a nonobese diabetic severe combined immunodeficient mouse-human neural cell model showed that VZV infected both neurons and glial cells and spread efficiently from cell to cell in vivo . Neural cell morphology and protein synthesis were preserved, in contrast to destruction of epithelial cells by VZV. Expression of VZV genes in neural cells was characterized by nuclear retention of the major viral transactivating protein and a block in synthesis of the predominant envelope glycoprotein. The attenuated VZV vaccine strain retained infectivity for neurons and glial cells in vivo . VZV gene expression in differentiated human neural cells in vivo differs from neural infection by herpes simplex virus, which is characterized by latency-associated transcripts, and from lytic VZV replication in skin. The chimeric nonobese diabetic severe combined immunodeficient mouse model may be useful for investigating other neurotropic human viruses.

In the adult, neurogenesis occurs where constellations of signaling molecules are correctly orchestrated and where competent cells are present to interpret these signals. As the instruments used to observe adult neurogenesis become more sophisticated, the concept of a discrete competent “stem cell” has become less concrete. Neural progenitor cells once thought committed to a single lineage can be influenced to become multipotent and somatic tissues appear to yield cells capable of tremendous peripheral and central lineage potential. The variety of cell types that appear competent to generate neurons suggests that the “Hilios” of adult neurogenesis may not necessarily be a single cellular entity but rather the sum of signals that dictate, “Make a new neuron here.” These signals may not be limited to the recruitment of preexisting neural stem cells but may also, in some subtle way, reprogram local precursors to create “stem-like cells,” where needed. © 2002 Wiley-Liss, Inc.