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Transcription factor 4 (TCF4) is a crucial regulator of neurodevelopment and has been linked to the pathogenesis of autism, intellectual disability and schizophrenia. As a class I bHLH transcription factor (TF), it is assumed that TCF4 exerts its neurodevelopmental functions through dimerization with proneural class II bHLH TFs. Here, we aim to identify TF partners of TCF4 in the control of interhemispheric connectivity formation. Using a new bioinformatic strategy integrating TF expression levels and regulon activities from single cell RNA-sequencing data, we find evidence that TCF4 interacts with non-bHLH TFs and modulates their transcriptional activity in Satb2+ intercortical projection neurons. Notably, this network comprises regulators linked to the pathogenesis of neurodevelopmental disorders, e.g. FOXG1, SOX11 and BRG1. In support of the functional interaction of TCF4 with non-bHLH TFs, we find that TCF4 and SOX11 biochemically interact and cooperatively control commissure formation in vivo, and regulate the transcription of genes implicated in this process. In addition to identifying new candidate interactors of TCF4 in neurodevelopment, this study illustrates how scRNA-Seq data can be leveraged to predict TF networks in neurodevelopmental processes., Summary: Single-cell RNA sequencing identifies interactions of TCF4 with non-bHLH transcription factors linked to neurodevelopmental and neuropsychiatric disease in the regulation of interhemispheric projection neuron development.

Background: Transcription factor 4 (TCF4) has been linked to human neurodevelopmental disorders such as intellectual disability, Pitt-Hopkins Syndrome (PTHS), autism, and schizophrenia. Recent work demonstrated that TCF4 participates in the control of a wide range of neurodevelopmental processes in mammalian nervous system development including neural precursor proliferation, timing of differentiation, migration, dendritogenesis and synapse formation. TCF4 is highly expressed in the adult hippocampal dentate gyrus – one of the few brain regions where neural stem / progenitor cells generate new functional neurons throughout life.Results: We here investigated whether TCF4 haploinsufficiency, which in humans causes non-syndromic forms of intellectual disability and PTHS, affects adult hippocampal neurogenesis, a process that is essential for hippocampal plasticity in rodents and potentially in humans. Young adult Tcf4 heterozygote knockout mice showed a major reduction in the level of adult hippocampal neurogenesis, which was at least in part caused by lower stem/progenitor cell numbers and impaired maturation and survival of adult-generated neurons. Interestingly, housing in an enriched environment was sufficient to enhance maturation and survival of new neurons and to substantially augment neurogenesis levels in Tcf4 heterozygote knockout mice.Conclusion: Haploinsufficiency for the transcription factor TCF4 has been linked to non-syndromic intellectual disability and PTHS. The present findings raise the possibility that TCF4 haploinsufficiency may have a continuous negative impact on hippocampal function by impeding hippocampal neurogenesis and suggest that behavioural stimulation may be harnessed to ameliorate a subset of TCF4 haploinsufficiency associated neural deficits during adulthood.

ARID1B haploinsufficiency induced by missense or nonsense mutations of ARID1B is a cause of Coffin-Siris syndrome (CSS), a neurodevelopmental disorder associated with intellectual disability. At present, no appropriate human stem cell model for ARID1B-associated CSS has been reported. Here, we describe the generation and validation of ARID1B+/- hESCs by introducing out of frame deletions into exon 5 or 6 of ARID1B with CRISPR/Cas9 genome editing. These ARID1B+/- hESC lines allow to study the pathophysiology of ARID1B-associated CSS in 2D and 3D models of human neurodevelopment.

Intercortical connectivity is important for higher cognitive brain functions by providing the basis for integrating information from both hemispheres. We show that ablation of the neurodevelopmental disorder associated bHLH factor Tcf4 results in complete loss of forebrain commissural systems in mice. Applying a new bioinformatic strategy integrating transcription factor expression levels and regulon activities from single cell RNA-sequencing data predicted a TCF-4 interacting transcription factor network in intercortical projection neurons regulating commissure formation. This network comprises a number of regulators previously linked to the pathogenesis of intellectual disability, autism-spectrum disorders and schizophrenia, e.g. Foxg1, Sox11 and Brg1. Furthermore, we demonstrate that TCF-4 and SOX11 biochemically interact and cooperatively control commissure formation in vivo, and regulate the transcription of genes implied in this process. Our study provides a regulatory transcriptional network for the development of interhemispheric connectivity with potential pathophysiological relevance in neurodevelopmental disorders.

The intellectual disability gene, Sox11, encodes for a critical neurodevelopmental transcription factor with functions in precursor survival, neuronal fate determination, migration and morphogenesis. The mechanisms regulating SOX11’s activity remain largely unknown. Mass spectrometric analysis uncovered that SOX11 can be post-translationally modified by phosphorylation. Here, we report that phosphorylatable serines surrounding the high-mobility group box modulate SOX11’s transcriptional activity. Through Mass Spectrometry (MS), co-immunoprecipitation assays and in vitro phosphorylation assays followed by MS we verified that protein kinase A (PKA) interacts with SOX11 and phosphorylates it on S133. In vivo replacement of SoxC factors in developing adult-generated hippocampal neurons with SOX11 S133 phospho-mutants indicated that phosphorylation on S133 modulates dendrite development of adult-born dentate granule neurons, while reporter assays suggested that S133 phosphorylation fine-tunes the activation of select target genes. These data provide novel insight into the control of the critical neurodevelopmental regulator SOX11 and imply SOX11 as a mediator of PKA-regulated neuronal development.

The autophagy-lysosome pathway (ALP) regulates intracellular homeostasis of the cytosolic protein SNCA/alpha-synuclein and is impaired in synucleinopathies, including Parkinson disease and dementia with Lewy bodies (DLB). Emerging evidence suggests that ALP influences SNCA release, but the underlying cellular mechanisms are not well understood. Several studies identified SNCA in exosome/extracellular vesicle (EV) fractions. EVs are generated in the multivesicular body compartment and either released upon its fusion with the plasma membrane, or cleared via the ALP. We therefore hypothesized that inhibiting ALP clearance 1) enhances SNCA release via EVs by increasing extracellular shuttling of multivesicular body contents, 2) alters EV biochemical profile, and 3) promotes SNCA cell-to-cell transfer. Indeed, ALP inhibition increased the ratio of extra- to intracellular SNCA and upregulated SNCA association with EVs in neuronal cells. Ultrastructural analysis revealed a widespread, fused multivesicular body-autophagosome compartment. Biochemical characterization revealed the presence of autophagosome-related proteins, such as LC3-II and SQSTM1. This distinct “autophagosome-exosome-like” profile was also identified in human cerebrospinal fluid (CSF) EVs. After a single intracortical injection of SNCA-containing EVs derived from CSF into mice, human SNCA colocalized with endosome and neuronal markers. Prominent SNCA immunoreactivity and a higher number of neuronal SNCA inclusions were observed after DLB patient CSF EV injections. In summary, this study provides compelling evidence that a) ALP inhibition increases SNCA in neuronal EVs, b) distinct ALP components are present in EVs, and c) CSF EVs transfer SNCA from cell to cell in vivo. Thus, macroautophagy/autophagy may regulate EV protein composition and consequently progression in synucleinopathies.

In adult hippocampal neurogenesis, stem/progenitor cells generate dentate granule neurons that contribute to hippocampal plasticity. The establishment of a morphologically defined dendritic arbor is central to the functional integration of adult‐born neurons. We investigated the role of canonical Wnt/β‐catenin signaling in dendritogenesis of adult‐born neurons. We show that canonical Wnt signaling follows a biphasic pattern, with high activity in stem/progenitor cells, attenuation in immature neurons, and reactivation during maturation, and demonstrate that this activity pattern is required for proper dendrite development. Increasing β‐catenin signaling in maturing neurons of young adult mice transiently accelerated dendritic growth, but eventually produced dendritic defects and excessive spine numbers. In middle‐aged mice, in which protracted dendrite and spine development were paralleled by lower canonical Wnt signaling activity, enhancement of β‐catenin signaling restored dendritic growth and spine formation to levels observed in young adult animals. Our data indicate that precise timing and strength of β‐catenin signaling are essential for the correct functional integration of adult‐born neurons and suggest Wnt/β‐catenin signaling as a pathway to ameliorate deficits in adult neurogenesis during aging., Precise timing and strength of β‐catenin signaling determines correct functional integration of adult‐born neurons, implicating Wnt/β‐catenin activity as protective pathway against aging‐related deficits in adult neurogenesis.

Summary Swiprosin-1/Efhd2 (Efhd2) is highly expressed in the CNS during development and in the adult. EFHD2 is regulated by Ca2+ binding, stabilizes F-actin, and promotes neurite extension. Previous studies indicated a dysregulation of EFHD2 in human Alzheimer's disease brains. We hypothesized a detrimental effect of genetic ablation of Efhd2 on hippocampal integrity and specifically investigated adult hippocampal neurogenesis. Efhd2 was expressed throughout adult neuronal development and in mature neurons. We observed a severe reduction of the survival of adult newborn neurons in Efhd2 knockouts, starting at the early neuroblast stage. Spine formation and dendrite growth of newborn neurons were compromised in full Efhd2 knockouts, but not upon cell-autonomous Efhd2 deletion. Together with our finding of severe hippocampal tauopathy in Efhd2 knockout mice, these data connect Efhd2 to impaired synaptic plasticity as present in Alzheimer's disease and identify a role of Efhd2 in neuronal survival and synaptic integration in the adult hippocampus., Highlights • Efhd2 is expressed in the dentate gyrus and its loss reduces adult neurogenesis • Reduced neurite complexity and spine density in new neurons of Efhd2 knockout mice • Role of cell-extrinsic EFHD2 for dendrite morphology of adult newborn neurons • Increased levels of pathological TAU in the hippocampus of Efhd2 knockout mice, In this report, Mielenz, Winner, and colleagues show a novel impact of Efhd2 on survival and integration of adult-born hippocampal neurons. This is of particular significance since EFHD2 regulates cytoskeletal transport and synaptic plasticity and since levels of pathological TAU are increased in Efhd2 knockout mice.

Adult neurogenesis is tightly regulated by the neurogenic niche. Cellular contacts between niche cells and neural stem cells are hypothesized to regulate stem cell proliferation or lineage choice. However, the structure of adult neural stem cells and the contact they form with niche cells are poorly described. Here, we characterized the morphology of radial glia-like (RGL) cells, their molecular identity, proliferative activity, and fate determination in the adult mouse hippocampus. We found the coexistence of two morphotypes of cells with prototypical morphological characteristics of RGL stem cells: Type α cells, which represented 76% of all RGL cells, displayed a long primary process modestly branching into the molecular layer and type β cells, which represented 24% of all RGL cells, with a shorter radial process highly branching into the outer granule cell layer-inner molecular layer border. Stem cell markers were expressed in type α cells and coexpressed with astrocytic markers in type β cells. Consistently, in vivo lineage tracing indicated that type α cells can give rise to neurons, astrocytes, and type β cells, whereas type β cells do not proliferate. Our results reveal that the adult subgranular zone of the dentate gyrus harbors two functionally different RGL cells, which can be distinguished by simple morphological criteria, supporting a morphofunctional role of their thin cellular processes. Type β cells may represent an intermediate state in the transformation of type α, RGL stem cells, into astrocytes.

Alzheimer's disease is a neurodegenerative disorder of the elderly and advancing age is the major risk factor for Alzheimer's disease development. Telomere shortening represents one of the molecular causes of ageing that limits the proliferative capacity of cells, including neural stem cells. Studies on telomere lengths in patients with Alzheimer's disease have revealed contrary results and the functional role of telomere shortening on brain ageing and Alzheimer's disease is not known. Here, we have investigated the effects of telomere shortening on adult neurogenesis and Alzheimer's disease progression in mice. The study shows that aged telomerase knockout mice with short telomeres (G3Terc(-/-)) exhibit reduced dentate gyrus neurogenesis and loss of neurons in hippocampus and frontal cortex, associated with short-term memory deficit in comparison to mice with long telomere reserves (Terc(+/+)). In contrast, telomere shortening improved the spatial learning ability of ageing APP23 transgenic mice, a mouse model for Alzheimer's disease. Telomere shortening was also associated with an activation of microglia in ageing amyloid-free brain. However, in APP23 transgenic mice, telomere shortening reduced both amyloid plaque pathology and reactive microgliosis. Together, these results provide the first experimental evidence that telomere shortening, despite impairing adult neurogenesis and maintenance of post-mitotic neurons, can slow down the progression of amyloid plaque pathology in Alzheimer's disease, possibly involving telomere-dependent effects on microglia activation.

We asked whether cell-cycle associated protein p27kip1 might be involved in the transition of precursor cells to postmitotic maturation in adult hippocampal neurogenesis. p27kip1 was expressed throughout the dentate gyrus with a strong nuclear expression in early postmitotic, calretinin-positive neurons and neuronally determined progenitor cells (type-3 and some type-2b), lower or absent expression in radial glia-like precursor cells (type-1) and type-2a cells and essentially no expression in granule cells. This suggested a transitory role in late proliferative and early postmitotic phases of neurogenesis. Inconsistent with a role limited to cell cycle arrest the acute stimuli, voluntary wheel running (RUN), environmental enrichment (ENR) and kainate-induced seizures increased p27kip1 expressing cells. Sequential short-term combination of RUN and ENR yielded more p27kip1 cells than either stimulus alone, indicating an additive effect. In vitro, p27kip1 was lowly expressed by proliferating precursor cells but increased upon differentiation. In p27kip1−/− mice neurogenesis was reduced in vivo, whereas the number of proliferating cells was increased. Accordingly, the microdissected dentate gyrus of p27kip1−/− mice generated more colonies in the neurosphere assay and an increased number of larger spheres with the differentiation potential unchanged. In p27kip1−/− monolayer cultures, proliferation was increased and cell cycle genes were upregulated. In the Morris water maze p27kip1−/− mice learned the task but were specifically impaired in the reversal phase explainable by the decrease in adult neurogenesis. We conclude that p27kip1 is involved in the decisive step around cell-cycle exit and plays an important role in activity-regulated and functionally relevant adult hippocampal neurogenesis.

Neural stem/progenitor cells (NSPCs) generate new neurons throughout life in the mammalian dentate gyrus (DG), part of the hippocampal formation. Here we review recent advances in our understanding of intrinsic and extrinsic mechanisms regulating the distinct developmental steps from the quiescent NSPCs toward cell proliferation, neuronal fate determination, and functional integration into the DG circuitry. Further, we discuss how the hippocampal microenvironment regulates the activity of NSPCs and subsequent steps of neuronal maturation and integration. Finally, we will summarize some of the current key open questions and suggest potential future directions for the field with the aim to (1) improve our understanding of the neurogenic process in the adult brain, and (2) to test for the translational relevance of adult neurogenesis for human physiology and disease.

Summary We describe the labeling of adult neural stem cells (aNSCs) in the mouse and human dentate gyrus (DG) by the combinatorial expression of glial fibrillary acidic protein (GFAP) and Prominin1, as revealed by immunohistochemistry. Split-Cre-based genetic fate mapping of these double-positive cells in the adult murine DG reveals their NSC identity, as they are self-renewing and contribute to neurogenesis over several months. Their progeny reacts to stimuli such as voluntary exercise with increased neurogenesis. Prominin1+/GFAP+ cells also exist in the adult human DG, the only region in the human brain for which adult neurogenesis has been consistently reported. Our data, together with previous evidence of such double-positive NSCs in the developing murine brain and in neurogenic regions of vertebrates with widespread neurogenesis, suggest that Prominin1- and GFAP-expressing cells are NSCs in a wide range of species in development and adulthood., Highlights • Prominin1 is expressed in radial and nonradial NSCs in the adult hippocampus • Fate mapping reveals the long-term neurogenic lineage of Prominin1+/hGFAP+ NSCs • Prominin1 labels GFAP+ radial glia processes in the adult human hippocampus, The combinatorial expression of GFAP and Prominin1 labels adult neural stem cells (NSCs) in the mouse dentate gyrus as revealed by immunohistochemistry and split-Cre-based fate mapping. Additionally, Prominin1+/GFAP+ cells exist in the adult human dentate gyrus. Such double-positive cells also represent NSCs in the embryonic forebrain and the adult subependymal zone, suggesting Prominin1+/GFAP+ expression as a general property of NSCs.

Members of the cyclin-dependent kinase (CDK)-inhibitory protein (CIP)/kinase-inhibitory protein (KIP) family of cyclin-dependent kinase inhibitors regulate proliferation and cell cycle exit of mammalian cells. In the adult brain, the CIP/KIP protein p27kip1 has been related to the regulation of intermediate progenitor cells located in neurogenic niches. Here, we uncover a novel function of p27kip1 in the adult hippocampus as a dual regulator of stem cell quiescence and of cell-cycle exit of immature neurons. In vivo, p27kip1 is detected in radial stem cells expressing SOX2 and in newborn neurons of the dentate gyrus. In vitro, the Cdkn1b gene encoding p27kip1 is transcriptionally upregulated by quiescence signals such as BMP4. The nuclear accumulation of p27kip1 protein in adult hippocampal stem cells encompasses the BMP4-induced quiescent state and its overexpression is able to block proliferation. p27kip1 is also expressed in immature neurons upon differentiation of adult hippocampal stem cell cultures. Loss of p27kip1 leads to an increase in proliferation and neurogenesis in the adult dentate gyrus, which results from both a decrease in the percentage of radial stem cells that are quiescent and a delay in cell cycle exit of immature neurons. Analysis of animals carrying a disruption in the cyclin-CDK interaction domain of p27kip1 indicates that the CDK inhibitory function of the protein is necessary to control the activity of radial stem cells. Thus, we report that p27kip1 acts as a central player of the molecular program that keeps adult hippocampal stem cells out of the cell cycle. Stem Cells 2015;33:219–229

SummaryNeurons of the cerebral cortex are organized in layers and columns. Unlike laminar patterning, the mechanisms underlying columnar organization remain largely unexplored. Here, we show that ephrin-B1 plays a key role in this process through the control of nonradial steps of migration of pyramidal neurons. In vivo gain of function of ephrin-B1 resulted in a reduction of tangential motility of pyramidal neurons, leading to abnormal neuronal clustering. Conversely, following genetic disruption of ephrin-B1, cortical neurons displayed a wider lateral dispersion, resulting in enlarged ontogenic columns. Dynamic analyses revealed that ephrin-B1 controls the lateral spread of pyramidal neurons by limiting neurite extension and tangential migration during the multipolar phase. Furthermore, we identified P-Rex1, a guanine-exchange factor for Rac3, as a downstream ephrin-B1 effector required to control migration during the multipolar phase. Our results demonstrate that ephrin-B1 inhibits nonradial migration of pyramidal neurons, thereby controlling the pattern of cortical columns.

Background and Purpose— Adult neurogenesis in the dentate gyrus is a unique form of brain plasticity that is strongly stimulated after stroke. We investigate the morphological properties of new granule cells, which are born and develop after the ischemic insult, and query whether these adult-born neurons properly integrate into the pre-existing hippocampal circuitries. Methods— Two well-established models were used to induce either small cortical infarcts (photothrombosis model) or large territorial infarcts (transient middle cerebral artery occlusion model). New granule cells were labeled 4 days after the initial insult by intrahippocampal injection of a retroviral vector encoding green fluorescent protein and newborn neurons were morphologically analyzed using a semiautomatic Neurolucida system and confocal laser scanning microscopy at 6 weeks. Results— Approximately 5% to 10% of newborn granule cells displayed significant morphological abnormalities comprising additional basal dendrites and, after middle cerebral artery occlusion, also ectopic cell position. The extent of morphological abnormalities was higher after large territorial infarcts and seems to depend on the severity of ischemic damage. An increased portion of mushroom spines in aberrant neurons suggests stable synaptic integration. However, poststroke generated granule cells with regular appearance also demonstrate alterations in dendritic complexity and spine morphology. Conclusions— The remarkable stimulation of dentate neurogenesis after stroke coincides with an increased rate of aberrantly integrated neurons, which may contribute to functional impairments and, hypothetically, favor pathogenesis of adjustment disorders, cognitive deficits, or epilepsy often seen in stroke patients.

In adult hippocampus, new neurons are continuously generated from neural stem cells (NSCs), but the molecular mechanisms regulating adult neurogenesis remain elusive. We found that Wnt signaling, together with the removal of Sox2, triggered the expression of NeuroD1 in mice. This transcriptional regulatory mechanism was dependent on a DNA element containing overlapping Sox2 and T-cell factor/lymphoid enhancer factor (TCF/LEF)-binding sites (Sox/LEF) in the promoter. Notably, Sox/LEF sites were also found in long interspersed nuclear element 1 (LINE-1) elements, consistent with their critical roles in the transition of NSCs to proliferating neuronal progenitors. Our results describe a previously unknown Wnt-mediated regulatory mechanism that simultaneously coordinates activation of NeuroD1 and LINE-1, which is important for adult neurogenesis and survival of neuronal progenitors. Moreover, the discovery that LINE-1 retro-elements embedded in the mammalian genome can function as bi-directional promoters suggests that Sox/LEF regulatory sites may represent a general mechanism, at least in part, for relaying environmental signals to other nearby loci to promote adult hippocampal neurogenesis.

Adult-generated dentate granule neurons have emerged as major contributors to hippocampal plasticity. New neurons are generated from neural stem cells through a complex sequence of proliferation, differentiation, and maturation steps. Development of the new neuron is dependent on the precise temporal activity of transcription factors, which coordinate the expression of stage-specific genetic programs. Here, we review current knowledge in transcription factor-mediated regulation of mammalian neural stem cells and neurogenesis and will discuss potential mechanisms of how transcription factor networks, on one hand, allow for precise execution of the developmental sequence and, on the other hand, allow for adaptation of the rate and timing of adult neurogenesis in response to complex stimuli. Understanding transcription factor-mediated control of neuronal development will provide new insights into the mechanisms underlying neurogenesis-dependent plasticity in health and disease.

Neural stem/progenitor cells in the adult brain are able to generate both the brain's major cell types: glial cells, which are non-neuronal, and the active nerve cells or neurons. Neurons are produced in just two regions of the brain. Lie et al. have now identified a protein family that instructs the adult neural stem cells to produce neurons, rather than glial cells. The signal molecule Wnt3 is shown to be crucial for the production of neurons in the adult hippocampus, a region believed to be involved in learning and memory formation. Ultimately these studies may help develop therapies to repair brain damage caused by disease or trauma. The generation of new neurons from neural stem cells is restricted to two regions of the adult mammalian central nervous system: the subventricular zone of the lateral ventricle, and the subgranular zone of the hippocampal dentate gyrus1. In both regions, signals provided by the microenvironment regulate the maintenance, proliferation and neuronal fate commitment of the local stem cell population1. The identity of these signals is largely unknown. Here we show that adult hippocampal stem/progenitor cells (AHPs) express receptors and signalling components for Wnt proteins, which are key regulators of neural stem cell behaviour in embryonic development2. We also show that the Wnt/β-catenin pathway is active and that Wnt3 is expressed in the hippocampal neurogenic niche. Overexpression of Wnt3 is sufficient to increase neurogenesis from AHPs in vitro and in vivo. By contrast, blockade of Wnt signalling reduces neurogenesis from AHPs in vitro and abolishes neurogenesis almost completely in vivo. Our data show that Wnt signalling is a principal regulator of adult hippocampal neurogenesis and provide evidence that Wnt proteins have a role in adult hippocampal function.

Somatic stem cells have been claimed to possess an unexpectedly broad differentiation potential (referred to here as plasticity) that could be induced by exposing stem cells to the extracellular developmental signals of other lineages in mixed-cell cultures1,2,3,4,5,6. Recently, this and other experimental evidence supporting the existence of stem-cell plasticity have been refuted because stem cells have been shown to adopt the functional features of other lineages by means of cell-fusion-mediated acquisition of lineage-specific determinants (chromosomal DNA) rather than by signal-mediated differentiation1,2,5,7,8. In this study we co-cultured mouse neural stem cells (NSCs), which are committed to become neurons and glial cells9,10, with human endothelial cells, which form the lining of blood vessels11. We show that in the presence of endothelial cells six per cent of the NSC population converted to cells that did not express neuronal or glial markers, but instead showed the stable expression of multiple endothelial markers and the capacity to form capillary networks. This was surprising because NSCs and endothelial cells are believed to develop from the ectoderm and mesoderm, respectively. Experiments in which endothelial cells were killed by fixation before co-culture with live NSCs (to prevent cell fusion) and karyotyping analyses, revealed that NSCs had differentiated into endothelial-like cells independently of cell fusion. We conclude that stem-cell plasticity is a true characteristic of NSCs and that the conversion of NSCs to unanticipated cell types can be accomplished without cell fusion.

We investigated the survival, distribution and differentiation capabilities of adult rat hippocampus-derived progenitor cells (AHPs) by grafting them into either the intact or dopamine (DA)-denervated adult rat striatum (ST). Furthermore, we tested the effects of the in vivo administration of retinoic acid (RA) on the differentiation of the grafted cells. AHPs, prelabeled in vitro with bromodeoxyuridine (BrdU) and primed with RA, were transplanted bilaterally into the ST of hemiparkinsonian rats. Twenty animals were divided in four groups: three groups received i.p. injections of RA (1.5 mg/kg/day) for 1, 2 or 4 weeks and one group received vehicle injections for 4 weeks. Approximately 60% of the implanted BrdU-immunoreactive (BrdU+) cells were present in either intact or lesioned ST after 5 weeks of transplantation, with a striking widespread radial distribution from the implantation site. The cells became morphologically integrated with the surrounding host tissue, with no evidence of tumor formation. Approximately 18% of the BrdU+ cells were immunoreactive for the glial precursor marker NG2 and occasionally BrdU+ cells co-expressed the neuronal marker TuJ1. This differentiation pattern was similar in the intact and DA-denervated ST. Although further research is needed to find more adequate methods to drive the differentiation of these cells toward the desired phenotypes, the survival, differentiation potential and widespread distribution throughout the ST observed in this study suggest that AHPs may be useful in treatment of degenerative disorders affecting the nervous system.

In the adult mammalian brain, new neurons are continuously generated from neural stem cells in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus [1]. Increasing evidence for the functional importance of adult-generated neurons in these regions for learning, complex behavior, and mood regulation, as well as a potential link of impaired adult neurogenesis to cognitive deficits in ageing and neurodegeneration have sparked great interest in the regulatory mechanisms underlying the coordinated generation of new functional neurons. Neurogenesis includes self-renewal and fate specification of neural stem cells, migration and maturation of young neurons, and functional integration of new neurons into the neural circuitry [1]. A large body of work has demonstrated that neurogenesis is regulated in a complex manner by the dynamic interplay of cell-extrinsic signals derived from the neurogenic “niche” and cell-intrinsic transcriptional and epigenetic regulators. More recently, microRNAs (miRNAs) have emerged as potent modulators of adult neurogenesis. miRNA 132 has been linked to the maturation and functional integration of newly generated hippocampal neurons and – in this context – is potentially regulated by hippocampal network activity and CREB-signalling [2]. Several miRNAs have also been implied in controlling the balance of stem cell maintenance and differentiation - a crucial checkpoint for sustaining adult neurogenesis throughout adulthood. In this context, miRNA-137 [3] and miRNA-184 [4] have been found to promote stem cell maintenance at the expense of differentiation, while miRNA-9 [5], miRNA lethal-7b (let-7b) [6, 7], and miRNA-124 [8] tip the balance from stem cell maintenance towards differentiation through the negative regulation of cyclin D1 and the stem cell maintenance factors TLX, Hmga2, and Sox9. Intriguingly, the latter factors are transcriptional regulators and thus are likely to control the expression of larger sets of genes, which may explain the profound effects of those miRNAs in stem cell maintenance and differentiation. In the February issue of Aging Brett and colleagues link the microRNA cluster miR-106b~25 to neural stem cell expansion and neuronal differentiation [9]. In this work, they demonstrate that miR-106b~25 promotes proliferation in primary neural stem cell cultures. Moreover they report that overexpression of miR-106b~25 enhances neural stem cell differentiation toward the neuronal lineage. Although the in vitro modulatory effects of miRNA-106b~25 on neural stem cell behaviour are relatively modest, the authors made several notable observations that imply this miRNA cluster as a critical regulator of adult neurogenesis and warrant further investigation of its function and regulation: Firstly, in silico predictions suggest that miRNA-25 may be involved in the modulation of transforming growth factor β (TGFβ)/bone morphogenic protein (BMP) and insulin/IGF signalling, i.e., signalling pathways that control neural stem cell quiescence, proliferation, and fate as well as age-related stem cell dysfunction in other organs. Secondly, the authors identify a functional FoxO3 binding site near the promoter for miR-106b~25, which modulates the activity of the miRNA cluster. Recent work identified FoxO transcription factors as crucial regulators of stem cell maintenance in the hematopoietic and the adult central nervous system, whose loss results in premature depletion of the stem cell pool. Notably, FoxO transcription factor-dependent pathways control ageing and longevity in C.elegans and Drosophila melanogaster and certain FoxO3 gene variants are associated with increased lifespan in people [10]. Hence, the miRNA-106b~25 cluster may emerge as an important modulator of ageing in neural stem cells and the neurogenic niches. The in vivo function of miRNA-106b~25 in neural stem cell behaviour and neurogenesis has to be determined and it will be particularly interesting to determine the impact of ageing on the activity of the miRNA-106b~25 cluster in neural stem cells and their progeny. Moreover, it will be important to understand if and how the miRNA-106b~25 cluster is controlled by signals derived from the neurogenic environment, given the evidence that major shifts in pathway activities contribute to stem cell dysfunction during ageing. In the long run, determining functional targets of miRNA-106b~25 in the adult neurogenic lineage may reveal novel pathways in the control of neurogenesis, which may be harnessed for treatment of age-associated cognitive deficits.

The aim of this study was to establish a cellular system to investigate the requirement for cell surface and diffusible molecules in the differentiation of fetal mesencephalic cells toward the dopamine lineage. Toward this end, we immortalized rat embryonic day 14 (E14) mesencephalon with a regulatable retroviral vector encoding v-myc. The stably transduced cells were pooled and designated as VME14 cells. VME14 cells proliferated rapidly, stopped proliferating, extended processes, and expressed GFAP after suppression of the v-mycexpression with tetracycline, suggesting that VME14 cells differentiated into glial cells. Dissociated cells derived from the E11 rat mesencephalon gave rise to only a small number of tyrosine hydroxylase (TH)-positive neurons. However, when grown on a monolayer of the differentiated VME14 cells, a significantly higher number of cells differentiated into TH-positive neurons. VME14 cells were transduced with the secreted N-terminal cleavage product of the Sonic hedgehog gene (SHH-N), an inducer of mesencephalic dopaminergic neurons. This monoclonal, SHH-N-overexpressing cell line further enhanced dopaminergic differentiation of E11 rat mesencephalon cells. Thus, SHH-N and signals derived from fetal mesencephalic glia act cooperatively to facilitate dopaminergic differentiation. These fetal mesencephalon-derived cell lines will provide tools for the study of signals involved in dopaminergic differentiation.