Biomedical subjects

Ernest Arenas

Publications and source records attributed to Ernest Arenas.

At least 19 recordsLinked to original sources

PubMed2006

Microarray analyses support a role for Nurr1 in resistance to oxidative stress and neuronal differentiation in neural stem cells.

Nurr1 is an orphan nuclear receptor required for the development of midbrain dopaminergic neurons. To better understand the molecular consequences of Nurr1 expression, we compared the transcriptomes of two independent control and Nurr1-expressing NSC lines using Affymetrix cDNA microarrays. These data reveal the regulation of genes involved in promoting cell survival (trophic/growth factors and stress response genes) and in preventing cell death (decreased caspase-3 and caspase-11 expression). We found that conditioned medium from Nurr1-expressing NSC lines enhanced the survival of midbrain dopaminergic neurons in primary cultures and that Nurr1-expressing NSC lines themselves were more resistant to oxidative stress. These findings are accompanied by a dynamic pattern of gene regulation that is consistent with a role for Nurr1 in promoting both the acquisition of brain-region-specific identity (Engrailed-1) and neuronal differentiation (tubulin beta III). Interestingly, our gene expression profiles suggested that tenascin-C was regulated by Nurr1 in developing dopaminergic neurons. This was further confirmed in vitro and in Nurr1 knockout mice where low levels of tenascin-C mRNA were observed. Analysis of tenascin-C-null mice revealed an increase in the number of Nurr1(+) cells that become tyrosine hydroxylase-positive (TH(+)) dopaminergic neurons at embryonic day 11.5, suggesting that tenascin-C normally delays the acquisition of TH by Nurr1(+) precursors. Thus, our results confirm the presence of both secreted and cell-intrinsic survival signals modulated by Nurr1 and suggest that Nurr1 is a key regulator of both survival and dopaminergic differentiation.

Animals↗

PubMed2006

Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate beta-catenin.

The current view of canonical Wnt signalling is that following Wnt binding to its receptors (Frizzled-Lrp5/6), dishevelled (Dvl) becomes hyperphosphorylated, and the signal is transduced to the APC-GSK3beta-axin-beta-catenin multiprotein complex, which subsequently dissociates. As a result beta-catenin is not phosphorylated, escapes proteosomal degradation and activates its target genes after translocation to the nucleus. Here, we analyzed the importance of the Wnt-3a-induced phosphorylation and shift in electrophoretic migration of Dvl (PS-Dvl) for the activation of beta-catenin. Analysis of Wnt-3a time- and dose-responses in a dopaminergic cell line showed that beta-catenin is activated rapidly (within minutes) and at a low dose of Wnt-3a (1 ng/ml). Surprisingly, PS-Dvl appeared only after 30 min and at greater doses (> or =20 ng/ml) of Wnt-3a. Moreover, we found that a casein kinase 1 inhibitor (D4476) or siRNA for casein kinase 1 delta/epsilon (CK1delta/epsilon) blocked the Wnt-3a-induced PS-Dvl. Interestingly, CK1 inhibition or siRNA for CK1delta/epsilon did not ablate the activation of beta-catenin by Wnt-3a, indicating that there is a PS-Dvl-independent path to activate beta-catenin. The increase in beta-catenin activation by Wnt-3a (PS-Dvl-dependent or -independent) were blocked by Dickkopf1 (Dkk1), suggesting that the effect of Wnt-3a is in both cases mediated by Lrp5/6 receptors. Thus, our results show that Wnt-3a rapidly induce a partial activation of beta-catenin in the absence of PS-Dvl at low doses, while at high doses induce a full activation of beta-catenin in a PS-Dvl-dependent manner.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Dynamic temporal and cell type-specific expression of Wnt signaling components in the developing midbrain.

Wnt1 and -5a have been shown to modulate the proliferation and differentiation of midbrain dopaminergic (DA) neurons. However, it is not known whether other Wnts or which Frizzled (Fz) receptors are expressed in the developing midbrain. We found that 13 out of 19 Wnts, all 10 Fzs, and several intracellular Wnt signaling modulators, including Axin, FRAT, Naked, Par-1, and Ltap are developmentally regulated between embryonic days (E) 10.5 and 15.5. Next, we studied whether Fzs are differentially expressed in different cell types and examined neuronal-progenitor- or glial-enriched cultures and DA neurons isolated from TH-GFP reporter mice. We found that Fz8 is expressed at high levels in DA neurons at E11.5 and E13.5. Fz6 and -7 are the predominant transcripts in glial precursors, and Fz9, which is absent in DA neurons at E11.5, is the main receptor expressed in neuronal precursors. We therefore examined the function of Fz9 in DA cells and found that overexpression of Fz9 reduced Wnt5a- but not Wnt3a-induced hyperphosphorylation of Dishevelled. Thus, our results show that Fzs are developmentally regulated and differentially expressed in VM precursors, DA neurons, and glia. These findings suggest that Fz expression contributes to provide specificity to Wnt-mediated effects.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Neural progenitor cells engineered to secrete GDNF show enhanced survival, neuronal differentiation and improve cognitive function following traumatic brain injury.

We sought to evaluate the potential of C17.2 neural progenitor cells (NPCs) engineered to secrete glial cell line-derived neurotrophic factor (GDNF) to survive, differentiate and promote functional recovery following engraftment into the brains of adult male Sprague-Dawley rats subjected to lateral fluid percussion brain injury. First, we demonstrated continued cortical expression of GDNF receptor components (GFRalpha-1, c-Ret), suggesting that GDNF could have a physiological effect in the immediate post-traumatic period. Second, we demonstrated that GDNF over-expression reduced apoptotic NPC death in vitro. Finally, we demonstrated that GDNF over-expression improved survival, promoted neuronal differentiation of GDNF-NPCs at 6 weeks, as compared with untransduced (MT) C17.2 cells, following transplantation into the perilesional cortex of rats at 24 h post-injury, and that brain-injured animals receiving GDNF-C17.2 transplants showed improved learning compared with those receiving vehicle or MT-C17.2 cells. Our results suggest that transplantation of GDNF-expressing NPCs in the acute post-traumatic period promotes graft survival, migration, neuronal differentiation and improves cognitive outcome following traumatic brain injury.

Animals↗

PubMed2006

BMPs, FGF8 and Wnts regulate the differentiation of locus coeruleus noradrenergic neuronal precursors.

In the present study, we investigated the involvement of rhombomere 1 patterning proteins in the regulation of the major noradrenergic centre of the brain, the locus coeruleus. Primary cultures of rat embryonic day 13.5 locus coeruleus were treated with fibroblast growth factor-8, noggin and members of the bone morphogenetic and Wnt protein families. We show that bone morphogenetic proteins 2, 5 and 7 increase and noggin decreases the number of tyrosine hydroxylase-positive locus coeruleus neurons. Interestingly, from all Wnts expressed in the first rhombomere by embryonic day 12.5 in the mice, we only found expression of wnt5a mRNA in the vicinity of the locus coeruleus. In agreement with this finding, from all Wnts studied in vitro, only Wnt5a increased the number of tyrosine hydroxylase-positive neurons in locus coeruleus cultures. Finally, we also found that fibroblast growth factor-8 increased the number of tyrosine hydroxylase-positive cells in locus coeruleus cultures. Neither of the identified factors affected the survival of tyrosine hydroxylase-positive locus coeruleus noradrenergic neurons or the proliferation of their progenitors or neurogenesis. Instead, our results suggest that these patterning signals of rhombomere 1 may work to promote the differentiation of noradrenergic progenitors at later stages of development.

Animals↗

PubMed2006

Function of Wnts in dopaminergic neuron development.

Dopaminergic (DA) neurons in the ventral midbrain (VM) are one of the major cell types lost in Parkinson's disease (PD). Proof of principle exists for cell replacement therapies for PD, but wider application is halted by the unavailability of abundant sources of DA neurons. Stem cells might constitute one of these sources. However, efficient protocols promoting their specific differentiation into a DA neuronal phenotype are required. In this review, we summarize the latest findings concerning the contribution of the Wnt family of glycolipoproteins in the development of VM DA neurons. Regulators of Wnt signaling are involved in several neurodevelopmental processes. Recent results indicate that Wnts are key regulators of proliferation and differentiation of DA precursors during VM neurogenesis and different Wnts have specific and unique activity profiles. Interestingly, chemical inhibitors of glycogen synthase kinase-3beta stabilize beta-catenin and increase DA differentiation in VM precursor cultures. We hereby propose that Wnts are likely to contribute in the future to improve stem/precursor cell replacement therapy approaches to PD.

Animals↗

PubMed2006

A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo.

Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinson's disease.

Animals↗

PubMed2006

Neurogenin 2 is required for the development of ventral midbrain dopaminergic neurons.

Proneural genes are crucial regulators of neurogenesis and subtype specification in many areas of the nervous system; however, their function in dopaminergic neuron development is unknown. We report that proneural genes have an intricate pattern of expression in the ventricular zone of the ventral midbrain, where mesencephalic dopaminergic neurons are generated. Neurogenin 2 (Ngn2) and Mash1 are expressed in the ventral midline, while Ngn1, Ngn2 and Mash1 are co-localized more laterally in the ventricular zone. Ngn2 is also expressed in an intermediate zone immediately adjacent to the ventricular zone at the ventral midline. To examine the function of these genes, we analyzed mutant mice in which one or two of these genes were deleted (Ngn1, Ngn2 and Mash1) or substituted (Mash1 in the Ngn2 locus). Our results demonstrate that Ngn2 is required for the differentiation of Sox2(+) ventricular zone progenitors into Nurr1(+) postmitotic dopaminergic neuron precursors in the intermediate zone, and that it is also likely to be required for their subsequent differentiation into tyrosine hydroxylase-positive dopaminergic neurons in the marginal zone. Although Mash1 normally has no detectable function in dopaminergic neuron development, it could partially rescue the generation of dopaminergic neuron precursors in the absence of Ngn2. These results demonstrate that Ngn2 is uniquely required for the development of midbrain dopaminergic neurons.

Animals↗

PubMed2005

An efficient method for the derivation of mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) represent a unique tool for many researchers; however, the process of ESC derivation is often very inefficient and requires high specialization, training, and expertise. To circumvent these limitations, we aimed to develop a simple and efficient protocol based on the use of commercially available products. Here, we present an optimized protocol that we successfully applied to derive ESCs from several knockout mouse strains (Wnt-1, Wnt-5a, Lrp6, and parkin) with 50%-75% efficiency. The methodology is based on the use of mouse embryonic fibroblast feeders, knockout serum replacement (SR), and minimal handling of the blastocyst. In this protocol, all centrifugation steps (as well as the use of trypsin inhibitor) were avoided and replaced by an ESC medium containing fetal calf serum (FCS) after the trypsinizations. We define the potential advantages and disadvantages of using SR and FCS in individual steps of the protocol. We also characterize the ESCs for the expression of ESC markers by immunohistochemistry, Western blot, and a stem cell focused microarray. In summary, we provide a simplified and improved protocol to derive mESCs that can be useful for laboratories aiming to isolate transgenic mESCs for the first time.

Animals↗

PubMed2005

Ventral midbrain glia express region-specific transcription factors and regulate dopaminergic neurogenesis through Wnt-5a secretion.

Glial cells have been classically described as supporting cells for neurons. Recently, additional roles during neural development have begun to emerge. Here, we report that ventral midbrain glia, including astrocytes and radial glia, are the source of signals required by neural precursors to acquire a dopaminergic phenotype. We found that ventral midbrain glia, but not cortical glia, secrete high levels of the glycolipoprotein Wnt-5a, express region-specific transcription factors such as Pax-2, En-1 and Otx-2 and increase the differentiation of cortical or ventral midbrain Nurr1 precursors into tyrosine hydroxylase-positive neurons. Moreover, blocking experiments using a Wnt-5a blocking antibody indicated that the effects of ventral midbrain glia on Nurr1-positive neural precursors are partially mediated by Wnt-5a. Thus, our results identify Wnt-5a as an important component of the dopaminergic inductive activity of the ventral midbrain glia.

Animals↗

PubMed2005

Increased Wnt levels in the neural tube impair the function of adherens junctions during neurulation.

Wnt7a has been reported to signal via the canonical pathway, but also in non-canonical pathways acting on the cytoskeleton. Since Wnt7a is expressed after neurulation, we set to investigate the effects of Wnt7a on brain regionalization. We engineered transgenic mouse embryos that, under control of the nestin second intron, overexpressed Wnt7a in neural stem/progenitor cells. Surprisingly, transgenic embryos failed to complete cranial neurulation due to reduced levels and an impaired distribution of actin microfilaments, beta-catenin, and N-cadherin at the neural tube adherens junctions. These transgenic embryos expressed high levels of Vangl2, an essential component of non-canonical Wnt signaling. In agreement with a disregulation of this pathway, aberrant spinal neurulation was detected in the transgenic embryos, revealing a novel function regulated by Wnts. Thus, our findings suggest that Wnt7a overexpression disrupts normal Wnt signaling in the neural tube, resulting in defective adherens junctions and neurulation.

Actin Cytoskeleton↗

PubMed2005

NTera2: a model system to study dopaminergic differentiation of human embryonic stem cells.

NTera2, a human embryonal carcinoma (EC) stem cell line, shares many characteristics with human embryonic stem cells (hESCs). To determine whether NTera2 can serve as a useful surrogate for hESCs, we compared global gene expression between undifferentiated NTera2, multiple undifferentiated hESC cell lines, and their differentiated derivatives, and we showed that NTera2 cells share multiple markers with hESCs. Similar to hESCs, NTera2 cells differentiated into TH-positive cells that express dopaminergic markers including AADC, DAT, Nurr1, TrkB, TrkC, and GFRA1 when co-cultured with PA6 cells. Flow cytometry analysis showed that tyrosine hydroxylase (TH) and neural cell adhesion molecule (NCAM) expression increased, whereas SSEA4 expression decreased as cells differentiated. Medium conditioned by PA6 cells stimulated differentiation of NTera2 cells to generate TH-positive cells that expressed dopaminergic markers. Flow cytometry selected polysialylated (PSA-NCAM) cells responded to medium conditioned by PA6 cells by differentiating into TH-positive cells and expressed dopaminergic markers. Sorted cells differentiated for 4 weeks in PA6 cell conditioned media included functional neurons that responded to neurotransmitters and exhibited electronic excitability. Therefore, NTera2 cell dopaminergic neuronal differentiation and PSA-NCAM enrichment provides a useful system for the future study of hESCs.

Animals↗

PubMed2005

Purified Wnt-5a increases differentiation of midbrain dopaminergic cells and dishevelled phosphorylation.

The Wnt family of lipoproteins regulates several aspects of the development of the nervous system. Recently, we reported that Wnt-3a enhances the proliferation of midbrain dopaminergic precursors and that Wnt-5a promotes their differentiation into dopaminergic neurones. Here we report the purification of hemagglutinin-tagged Wnt-5a using a three-step purification method similar to that previously described for Wnt-3a. Haemagglutinin-tagged Wnt-5a was biologically active and induced the differentiation of immature primary midbrain precursors into tyrosine hydroxylase-positive dopaminergic neurones. Using a substantia nigra-derived dopaminergic cell line (SN4741), we found that Wnt-5a, unlike Wnt-3a, did not promote beta-catenin phosphorylation or stabilization. However, both Wnt-5a and Wnt-3a activated dishevelled, as assessed by a phosphorylation-dependent mobility shift. Moreover, the activity of Wnt-5a on dishevelled was blocked by pre-treatment with acyl protein thioesterase-1, indicating that palmitoylation of Wnt-5a is necessary for its function. Thus, our results suggest that Wnt-3a and Wnt-5a, respectively, activate canonical and non-canonical Wnt signalling pathways in ventral midbrain dopaminergic cells. Furthermore, we identify dishevelled as a key player in transducing both Wnt canonical and non-canonical signals in dopaminergic cells.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The antimicrobial peptide rCRAMP is present in the central nervous system of the rat.

The brain is protected against invading pathogens by the blood-brain barrier, and also by its own innate defence system consisting of microglia and neurons in a coordinated network. Antimicrobial peptides are a part of the innate immune system at epithelial surfaces, and may also have important functions in the brain. Recently, we characterized the rat homologue of the human cathelicidin LL-37, designated rCRAMP. Here we present several lines of evidence for this peptide being expressed in rat CNS. (1) A peptide/protein extract of rat brain is active against bacteria in a salt-dependent manner. (2) Western blot analysis demonstrates the presence of rCRAMP in rat brain extract. (3) rCRAMP peptide and mRNA are present mainly in specific CNS regions (olfactory bulb, cerebellum, medulla oblongata and spinal cord). (4) rCRAMP-like immunoreactivity is detected in olfactory bulb, cerebellum and spinal cord by immunohistochemistry. (5) Moreover, the transcript of rCRAMP is detected in primary cultures from hippocampus, striatum, cerebellum and spinal cord, as shown with RT-PCR and Southern blot analyses. In addition, the rCRAMP peptide exhibits in vitro activity against the neuropathogenic bacterium Neisseria meningitidis. Taken together, these data suggest that the cathelicidin rCRAMP may play a role in the innate immunity of the CNS.

Animals↗

PubMed2005

Engineering a dopaminergic phenotype in stem/precursor cells: role of Nurr1, glia-derived signals, and Wnts.

Recent results from clinical trials using fetal tissue grafts in patients with Parkinson's disease (PD) have indicated that current surgical strategies for dopamine cell replacement therapy need to be improved in order to achieve better functional integration of the grafts and to avoid dyskinesias. Previous studies using rich dopaminergic (DA) cell suspensions have provided proof-of-concept that PD patients can benefit from cell replacement therapy. Stem cells have been proposed as better candidates for cell replacement therapy in PD since they can be standardized, expanded, and engineered in vitro. Recent developments indicate that cell preparations enriched in DA neurons can be generated in vitro, but their functional integration in animal models of disease is still far from optimal. This is not entirely surprising considering our limited knowledge of the development of DA neurons and the reduced number of factors that have been implemented in stem cell differentiation protocols. This review will focus on three aspects of DA neuron development: (1) the function of Nurr1 and retinoid X receptors (RXR) in the differentiation of DA precursors and in the survival of DA neurons; (2) the role of glia in DA neurogenesis and the differentiation of DA precursors; and (3) the function of the Wnt family of lipoproteins in the proliferation and differentiation of DA precursors. A greater understanding of the cellular and molecular mechanisms that control DA neuron development, as well as their functional integration in vivo, are likely to ultimately contribute to the development of novel stem cell replacement therapies for PD.

Animals↗

PubMed2005

Cripto as a target for improving embryonic stem cell-based therapy in Parkinson's disease.

Embryonic stem (ES) cells have been suggested as candidate therapeutic tools for cell replacement therapy in neurodegenerative disorders. However, limitations for the use of these cells lie in our restricted knowledge of the molecular mechanisms involved in their specialized differentiation and in the risk of tumor formation. Recent findings suggest that the EGF-CFC protein Cripto is a key player in the signaling pathways controlling neural induction in ES cells. Here we show that in vitro differentiation of Cripto(-/-) ES cells results in increased dopaminergic differentiation and that, upon transplantation into Parkinsonian rats, they result in behavioral and anatomical recovery with no tumor formation. The use of knockout ES cells that can generate dopamine cells while eliminating tumor risk holds enormous potential for cell replacement therapy in Parkinson's disease.

Animals↗

PubMed2004

Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity.

The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF(II)135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo.

Alternative Splicing↗

PubMed2004

GSK-3beta inhibition/beta-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons.

Wnts are important regulators of dopamine (DA) neuron differentiation in the developing ventral mesencephalon and could thus serve as potential tools in the treatment of Parkinson's disease. In this study, we investigate whether established intracellular Wnt signalling components could modulate the development of DA neurons. Two chemical inhibitors of glycogen synthase kinase (GSK)-3beta, indirubin-3-monoxime and kenpaullone, were found to increase neuronal differentiation in ventral mesencephalon precursor cultures. In addition, the GSK-3beta-specific inhibitor kenpaullone increased the size of the DA neuron population through conversion of precursors expressing the orphan nuclear receptor-related factor 1 into tyrosine hydroxylase positive neurons, thereby mimicking an effect of Wnts. We show that GSK-3beta inhibitors stabilized beta-catenin and that overexpression of beta-catenin in ventral mesencephalic precursors resulted in increased DA differentiation. The three- to fivefold increase in DA differentiation of precursor cells by GSK-3beta inhibitors suggests that such compounds could be used to improve stem/precursor cell therapy approaches in Parkinson's disease.

Animals↗