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David Vilchez,
Leah Boyer,
Margaret Lutz,
Carsten Merkwirth,
Ianessa Morantte,
Chris Tse,
Brian Spencer,
Lesley Page,
Eliezer Masliah,
William Travis Berggren, Fred H Gage,
Andrew Dillin
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ABSTRACT: Proteostasis is critical for maintaining cell function and proteome stability may play an important role in human embryonic stem cell (hESC) immortality. Notably, hESC populations exhibit a high assembly of active proteasomes, a key node of the proteostasis network. FOXO4, an insulin/IGF-1 responsive transcription factor, regulates proteasome activity in hESCs. We find that loss of FOXO4 reduces the potential of hESCs to differentiate into neural lineages. Therefore, FOXO4 crosses evolutionary boundaries and links hESC function to invertebrate longevity modulation.
Aging Cell 06/2013; 12(3):518-522. · 6.26 Impact Factor
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ABSTRACT: New neurons, which have been implicated in pattern separation, are continually generated in the dentate gyrus in the adult hippocampus. Using a genetically modified rabies virus, we demonstrated that molecular layer perforant pathway (MOPP) cells innervated newborn granule neurons in adult mouse brain. Stimulating the perforant pathway resulted in the activation of MOPP cells before the activation of dentate granule neurons. Moreover, activation of MOPP cells by focal uncaging of glutamate induced strong inhibition of granule cells. Together, these results indicate that MOPP cells located in the molecular layer of the dentate gyrus contribute to feed-forward inhibition of granule cells via perforant pathway activation.
Proceedings of the National Academy of Sciences 05/2013; · 9.68 Impact Factor
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David Vilchez,
Leah Boyer,
Margaret Lutz,
Carsten Merkwirth,
Ianessa Morantte,
Chris Tse,
Brian Spencer,
Lesley Page,
Eliezer Masliah,
W Travis Berggren, Fred H Gage,
Andrew Dillin
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David Vilchez,
Leah Boyer,
Margaret Lutz,
Carsten Merkwirth,
Ianessa Morantte,
Chris Tse,
Brian Spencer,
Lesley Page,
Eliezer Masliah,
W Travis Berggren, Fred H Gage,
Andrew Dillin
[show abstract]
[hide abstract]
ABSTRACT: Proteostasis is critical for maintaining cell function and proteome stability may play an important role in human embryonic stem cell (hESC) immortality. Notably, hESC populations exhibit a high assembly of active proteasomes, a key node of the proteostasis network. FOXO4, an insulin/IGF-1 responsive transcription factor, regulates proteasome activity in hESCs. We find that loss of FOXO4 reduces the potential of hESCs to differentiate into neural lineages. Therefore, FOXO4 crosses evolutionary boundaries and links hESC function to invertebrate longevity modulation. © 2013 The Authors Aging Cell © 2013 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.
Aging cell 03/2013; · 7.55 Impact Factor
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David Vilchez,
Leah Boyer,
Margaret Lutz,
Carsten Merkwirth,
Ianessa Morantte,
Chris Tse,
Brian Spencer,
Lesley Page,
Eliezer Masliah,
W Travis Berggren, Fred H Gage,
Andrew Dillin
[show abstract]
[hide abstract]
ABSTRACT: Proteostasis is critical for maintaining cell function and proteome stability may play an important role in human embryonic stem cell (hESC) immortality. Notably, hESC populations exhibit a high assembly of active proteasomes, a key node of the proteostasis
Aging Cell 03/2013; · 6.26 Impact Factor
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ABSTRACT: Newborn neurons are generated from neural stem cells (NSCs) in two major niches of the adult brain. Maintenance of self-renewal and multipotency of adult NSCs is controlled by multiple transcription factor networks. We show here that paired related homeobox protein Prx1 (MHox1/Prrx1) plays an important role in the maintenance of adult NSCs. Prx1 works with the transcription factor Sox2 as a coactivator, and depletion of Prx1 in cultured adult mouse NSCs reduces their self-renewal. In addition, we find that Prx1 protein is expressed in Sox2/GFAP/Nestin astrocytes in the germinal regions of the adult mouse forebrain. The continuous expression of Prx1 in proliferating adult mouse hippocampal stem/progenitor cells in vivo leads to the generation of radial/horizontal-shaped astrocyte progenitor- and oligodendrocyte progenitor-like cells with no newborn neurons in the neurogenic niche. These data suggest that Prx1 plays an important role as a key switch for neural cell lineage determination and the maintenance of the self-renewal of adult NSCs at several stages in the adult brain.
Journal of Neuroscience 02/2013; 33(9):4066-75. · 7.11 Impact Factor
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ABSTRACT: Faithful execution of developmental gene expression programs occurs at multiple levels and involves many different components such as transcription factors, histone-modification enzymes, and mRNA processing proteins. Recent evidence suggests that nucleoporins, well known components that control nucleo-cytoplasmic trafficking, have wide-ranging functions in developmental gene regulation that potentially extend beyond their role in nuclear transport. Whether the unexpected role of nuclear pore proteins in transcription regulation, which initially has been described in fungi and flies, also applies to human cells is unknown. Here we show at a genome-wide level that the nuclear pore protein NUP98 associates with developmentally regulated genes active during human embryonic stem cell differentiation. Overexpression of a dominant negative fragment of NUP98 levels decreases expression levels of NUP98-bound genes. In addition, we identify two modes of developmental gene regulation by NUP98 that are differentiated by the spatial localization of NUP98 target genes. Genes in the initial stage of developmental induction can associate with NUP98 that is embedded in the nuclear pores at the nuclear periphery. Alternatively, genes that are highly induced can interact with NUP98 in the nuclear interior, away from the nuclear pores. This work demonstrates for the first time that NUP98 dynamically associates with the human genome during differentiation, revealing a role of a nuclear pore protein in regulating developmental gene expression programs.
PLoS Genetics 02/2013; 9(2):e1003308. · 8.69 Impact Factor
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ABSTRACT: The hippocampus is critical for episodic memory and computational studies have predicted specific functions for each hippocampal subregion. Particularly, the dentate gyrus (DG) is hypothesized to perform pattern separation by forming distinct representations of similar inputs. How pattern separation is achieved by the DG remains largely unclear. By examining neuronal activities at a population level, we revealed that, unlike CA1 neuron populations, dentate granule cell (DGC) ensembles activated by learning were not preferentially reactivated by memory recall. Moreover, when mice encountered an environment to which they had not been previously exposed, a novel DGC population-rather than the previously activated DGC ensembles that responded to past events-was selected to represent the new environmental inputs. This selection of a novel responsive DGC population could be triggered by small changes in environmental inputs. Therefore, selecting distinct DGC populations to represent similar but not identical inputs is a mechanism for pattern separation. DOI:http://dx.doi.org/10.7554/eLife.00312.001.
eLife. 01/2013; 2:e00312.
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Leo Kurian,
Ignacio Sancho-Martinez,
Emmanuel Nivet,
Aitor Aguirre,
Krystal Moon,
Caroline Pendaries,
Cecile Volle-Challier,
Francoise Bono,
Jean-Marc Herbert,
Julian Pulecio, [......],
Sergio Ruiz,
Ilir Dubova,
Concepcion Rodriguez,
Ahmet M Denli,
Francesca S Boscolo,
Rathi D Thiagarajan, Fred H Gage,
Jeanne F Loring,
Louise C Laurent,
Juan Carlos Izpisua Belmonte
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ABSTRACT: Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct, in the absence of proliferation and multipotent progenitor generation, or indirect, by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state, induced by brief exposure to reprogramming factors, followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells, including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies.
Nature Methods 12/2012; · 19.28 Impact Factor
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Beate Winner,
Martin Regensburger,
Sebastian Schreglmann,
Leah Boyer,
Iryna Prots,
Edward Rockenstein,
Michael Mante,
Chunmei Zhao,
Jürgen Winkler,
Eliezer Masliah, Fred H Gage
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ABSTRACT: α-Synuclein has been reported to be important in modulating brain plasticity and to be a key protein in neurodegenerative diseases, including Lewy body dementia (LBD). We investigated how α-synuclein levels modulate adult neurogenesis and the development of dendritic arborization and spines in the dentate gyrus, in which new neurons are constantly added. In the human hippocampus, levels of endogenous α-synuclein were increased in LBD, and the numbers of SOX2-positive cells were decreased. We investigated whether newly generated neurons were modulated by endogenous α-synuclein, and we found increased adult neurogenesis in α/β-synuclein knock-out mice. In contrast, overexpression of human wild-type α-synuclein (WTS) decreased the survival and dendritic development of newborn neurons. Endogenous α-synuclein expression levels increased the negative impact of WTS on dendrite development, suggesting a toxic effect of increasing amounts of α-synuclein. To attempt a rescue of the dendritic phenotype, we administered rolipram to activate the cAMP response element-binding protein pathway, which led to a partial rescue of neurite development. The current work provides novel insights into the role of α-synuclein in adult hippocampal neurogenesis.
Journal of Neuroscience 11/2012; 32(47):16906-16916. · 7.11 Impact Factor
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ABSTRACT: Worldwide increases in life expectancy have been paralleled by a greater prevalence of chronic and age-associated disorders, particularly of the cardiovascular, neural and metabolic systems. This has not been met by commensurate development of new drugs and therapies, which is in part owing to the difficulty in modelling human diseases in laboratory assays or experimental animals. Patient-specific induced pluripotent stem (iPS) cells are an emerging paradigm that may address this. Reprogrammed somatic cells from patients are already applied in disease modelling, drug testing and drug discovery, thus enabling researchers to undertake studies for treating diseases 'in a dish', which was previously inconceivable.
Nature Reviews Molecular Cell Biology 10/2012; 13(11):713-26. · 39.12 Impact Factor
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ABSTRACT: Adult hippocampal neurogenesis is considered important for cognition. The integration of newborn dentate gyrus granule cells into the existing network is regulated by afferent neuronal activity of unspecified origin. Here we combine rabies virus-mediated retrograde tracing with retroviral labelling of new granule cells (21, 30, 60, 90 days after injection) to selectively identify and quantify their monosynaptic inputs in vivo. Our results show that newborn granule cells receive afferents from intra-hippocampal cells (interneurons, mossy cells, area CA3 and transiently, mature granule cells) and septal cholinergic cells. Input from distal cortex (perirhinal (PRH) and lateral entorhinal cortex (LEC)) is sparse 21 days after injection and increases over time. Patch-clamp recordings support innervation by the LEC rather than from the medial entorhinal cortex. Mice with excitotoxic PRH/LEC lesions exhibit deficits in pattern separation but not in water maze learning. Thus, PRH/LEC input is an important functional component of new dentate gyrus neuron circuitry.
Nature Communications 10/2012; 3:1107. · 7.40 Impact Factor
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David Vilchez,
Leah Boyer,
Ianessa Morantte,
Margaret Lutz,
Carsten Merkwirth,
Derek Joyce,
Brian Spencer,
Lesley Page,
Eliezer Masliah,
W Travis Berggren, Fred H Gage,
Andrew Dillin
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[hide abstract]
ABSTRACT: Embryonic stem cells can replicate continuously in the absence of senescence and, therefore, are immortal in culture 1,2 . Although genome stability is essential for the survival of stem cells, proteome stability may have an equally important role in stem-cell identity and function. Furthermore, with the asymmetric divisions invoked by stem cells, the passage of damaged proteins to daughter cells could potentially destroy the resulting lineage of cells. Therefore, a firm understanding of how stem cells maintain their proteome is of central importance. Here we show that human embryonic stem cells (hESCs) exhibit high proteasome activity that is correlated with increased levels of the 19S proteasome subunit PSMD11 (known as RPN-6 in Caenorhabditis elegans) 3–5 and a corresponding increased assembly of the 26S/30S proteasome. Ectopic expression of PSMD11 is sufficient to increase proteasome assembly and activity. FOXO4, an insulin/insulin-like growth factor-I (IGF-I) responsive transcription factor associated with long lifespan in invertebrates 6,7 , regulates proteasome activity by modulating the expression of PSMD11 in hESCs. Proteasome inhibition in hESCs affects the expression of pluripotency markers and the levels of specific markers of the distinct germ layers. Our results suggest a new regulation of proteostasis in hESCs that links longevity and stress resistance in invertebrates to hESC function and identity. Embryonic stem cells are unique among all stem-cell populations examined in that they do not seem to undergo replicative senescence 1,2 . Because proteostasis is crucial for maintaining proper cell function 8,9 , hESCs could provide a new model to define proteostasis regulation and its demise in ageing. Central to proteostasis is the ubiquitin proteasome system and we examined whether proteasome activity changes as hESCs differentiate into several cell lineages. To evaluate differences in the 26S/30S proteasome activity, we monitored the degradation of specific fluorogenic peptide substrates 10 . We differentiated H9 hESCs into neural progenitors cells (NPCs) and observed a marked decrease in the chymotrypsin-like proteasome activity (Fig. 1a). Moreover, when NPCs were differentiated into neurons, we detected a further decrease in proteasome activity during the differentiation process that was observable after 2 weeks (Fig. 1a and Supplementary Fig. 1). Consistent with enhanced proteasome activity in hESCs, we found increased levels of polyubiquitinated proteins in differentiated cells compared with hESCs (Fig. 1b). Because hESCs are known to vary in their characteristics despite unlimited capacity of self-renewal 11 , we differentiated a distinct hESC line, HUES-6 cells, and obtained similar results (Supplementary Figs 1 and 2). Proteasome inhibitors blocked activity from extracts of hESCs, NPCs and neurons (Supplementary Fig. 3), indicating that the increased peptidase activity was indeed due to the proteasome. In addition, the other two activities of the proteasome, the caspase-like and trypsin-like activities, were also increased in hESCs (Supplementary Fig. 4). Proteasome activity did not differ depending on the passage number (Supplementary Fig. 5). The decrease in proteasome activity was not a specific phenomenon associated with the neural lineage as differentiation into either trophoblasts or fibroblasts induced a similar decrease (Fig. 1c, d). Notably, hESCs lost their high proteasome activity in a continuous progressive manner during the differentiation process (Fig. 1c). Moreover, we examined other cell lines extracted from human tissues, such as astrocytes, BJ fibroblasts or immortalized HEK293T cells, and found that these cells also had lower proteasome activity compared with hESCs (Supplementary Fig. 6). We tested whether high proteasome activity in hESCs was associated with increased proliferation and found
Nature 09/2012; 489(7415-7415):304-308. · 36.28 Impact Factor
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David Vilchez,
Leah Boyer,
Ianessa Morantte,
Margaret Lutz,
Carsten Merkwirth,
Derek Joyce,
Brian Spencer,
Lesley Page,
Eliezer Masliah,
W Travis Berggren, Fred H Gage,
Andrew Dillin
[show abstract]
[hide abstract]
ABSTRACT: Embryonic stem cells can replicate continuously in the absence of senescence and, therefore, are immortal in culture. Although genome stability is essential for the survival of stem cells, proteome stability may have an equally important role in stem-cell identity and function. Furthermore, with the asymmetric divisions invoked by stem cells, the passage of damaged proteins to daughter cells could potentially destroy the resulting lineage of cells. Therefore, a firm understanding of how stem cells maintain their proteome is of central importance. Here we show that human embryonic stem cells (hESCs) exhibit high proteasome activity that is correlated with increased levels of the 19S proteasome subunit PSMD11 (known as RPN-6 in Caenorhabditis elegans) and a corresponding increased assembly of the 26S/30S proteasome. Ectopic expression of PSMD11 is sufficient to increase proteasome assembly and activity. FOXO4, an insulin/insulin-like growth factor-I (IGF-I) responsive transcription factor associated with long lifespan in invertebrates, regulates proteasome activity by modulating the expression of PSMD11 in hESCs. Proteasome inhibition in hESCs affects the expression of pluripotency markers and the levels of specific markers of the distinct germ layers. Our results suggest a new regulation of proteostasis in hESCs that links longevity and stress resistance in invertebrates to hESC function and identity.
Nature 09/2012; 489(7415):304-8. · 36.28 Impact Factor
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Naohiro Egawa,
Shiho Kitaoka,
Kayoko Tsukita,
Motoko Naitoh,
Kazutoshi Takahashi,
Takuya Yamamoto,
Fumihiko Adachi,
Takayuki Kondo,
Keisuke Okita,
Isao Asaka, [......],
Takashi Nonaka,
Masato Hasegawa,
Akihiro Kawata,
Minoru Yoshida,
Tatsutoshi Nakahata,
Ryosuke Takahashi,
Maria C N Marchetto, Fred H Gage,
Shinya Yamanaka,
Haruhisa Inoue
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ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a late-onset, fatal disorder in which the motor neurons degenerate. The discovery of new drugs for treating ALS has been hampered by a lack of access to motor neurons from ALS patients and appropriate disease models. We generate motor neurons from induced pluripotent stem cells (iPSCs) from familial ALS patients, who carry mutations in Tar DNA binding protein-43 (TDP-43). ALS patient-specific iPSC-derived motor neurons formed cytosolic aggregates similar to those seen in postmortem tissue from ALS patients and exhibited shorter neurites as seen in a zebrafish model of ALS. The ALS motor neurons were characterized by increased mutant TDP-43 protein in a detergent-insoluble form bound to a spliceosomal factor SNRPB2. Expression array analyses detected small increases in the expression of genes involved in RNA metabolism and decreases in the expression of genes encoding cytoskeletal proteins. We examined four chemical compounds and found that a histone acetyltransferase inhibitor called anacardic acid rescued the abnormal ALS motor neuron phenotype. These findings suggest that motor neurons generated from ALS patient-derived iPSCs may provide a useful tool for elucidating ALS disease pathogenesis and for screening drug candidates.
Science translational medicine 08/2012; 4(145):145ra104. · 7.80 Impact Factor
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ABSTRACT: Here we describe protocols for the dopaminergic differentiation of pluripotent stem cells. We have optimized and compared two distinct protocols, both of which are chemically defined and applicable to both embryonic and induced pluripotent stem cells. First, we present a five-step method based on rosette formation (Basic Protocol 1); then we describe a monolayer paradigm based on inhibition of alternate developmental pathways (Basic Protocol 2). Directed differentiation of pluripotent cells into specific cell types is a crucial step towards understanding human development and realizing the biomedical relevance of these cells, whether for replacement therapy or disease modeling.
Current protocols in stem cell biology 08/2012; Chapter 1:Unit1H.6.
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Alessandra Giorgetti,
Maria C. N. Marchetto,
Mo Li,
Diana Yu,
Raffaella Fazzina,
Yangling Mu,
Antonio Adamo,
Ida Paramonov,
Julio Castaño Cardoso,
Montserrat Barragan Monasterio,
Cedric Bardy,
Riccardo Cassiani-Ingoni,
Guang-Hui Liu, Fred H. Gage,
Juan Carlos Izpisua Belmonte
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ABSTRACT: The finding that certain somatic cells can be directly converted into cells of other lineages by the delivery of specific
sets of transcription factors paves the way to novel therapeutic applications. Here we show that human cord blood (CB) CD133+ cells lose their hematopoietic signature and are converted into CB-induced neuronal-like cells (CB-iNCs) by the ectopic expression
of the transcription factor Sox2, a process that is further augmented by the combination of Sox2 and c-Myc. Gene-expression
analysis, immunophenotyping, and electrophysiological analysis show that CB-iNCs acquire a distinct neuronal phenotype characterized
by the expression of multiple neuronal markers. CB-iNCs show the ability to fire action potentials after in vitro maturation
as well as after in vivo transplantation into the mouse hippocampus. This system highlights the potential of CB cells and
offers an alternative means to the study of cellular plasticity, possibly in the context of drug screening research and of
future cell-replacement therapies.
Proceedings of the National Academy of Sciences 07/2012; 109(31):12556-12561. · 9.68 Impact Factor
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ABSTRACT: Cellular programming and reprogramming technology (CPART) presents a novel approach for understanding disease progression and mechanism. In addition, CPART provides an innovative opportunity for developing diagnostic tools and novel drug candidates for therapy. In this Forum, we will discuss obstacles and solutions for modeling brain disease using CPART.
Cell stem cell 06/2012; 10(6):642-5. · 23.56 Impact Factor
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ABSTRACT: In the adult brain, neural stem cells have been found in two major niches: the dentate gyrus and the subventricular zone [corrected]. Neurons derived from these stem cells contribute to learning, memory, and the autonomous repair of the brain under pathological conditions. Hence, the physiology of adult neural stem cells has become a significant component of research on synaptic plasticity and neuronal disorders. In addition, the recently developed induced pluripotent stem cell technique provides a powerful tool for researchers engaged in the pathological and pharmacological study of neuronal disorders. In this review, we briefly summarize the research progress in neural stem cells in the adult brain and in the neuropathological application of the induced pluripotent stem cell technique.
Protein & Cell 04/2012; 3(4):251-61.
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Oliver Bracko,
Tatjana Singer,
Stefan Aigner,
Marlen Knobloch,
Beate Winner,
Jasodhara Ray,
Gregory D Clemenson,
Hoonkyo Suh,
Sebastien Couillard-Despres,
Ludwig Aigner, Fred H Gage,
Sebastian Jessberger
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ABSTRACT: Neural stem cells (NSCs) generate neurons throughout life in the hippocampal dentate gyrus (DG). How gene expression signatures differ among NSCs and immature neurons remains largely unknown. We isolated NSCs and their progeny in the adult DG using transgenic mice expressing a GFP reporter under the control of the Sox2 promoter (labeling NSCs) and transgenic mice expressing a DsRed reporter under the control of the doublecortin (DCX) promoter (labeling immature neurons). Transcriptome analyses revealed distinct gene expression profiles between NSCs and immature neurons. Among the genes that were expressed at significantly higher levels in DG NSCs than in immature neurons was the growth factor insulin-like growth factor 2 (IGF2). We show that IGF2 selectively controls proliferation of DG NSCs in vitro and in vivo through AKT-dependent signaling. Thus, by gene expression profiling of NSCs and their progeny, we have identified IGF2 as a novel regulator of adult neurogenesis.
Journal of Neuroscience 03/2012; 32(10):3376-87. · 7.11 Impact Factor
