Postnatal Rodent Brain Research Articles

Isolation of glia from mice

Glia constitutes a heterogeneous cell population that makes up half of the cells in the central nervous system (CNS). Glial cells include macroglia, astrocytes and oligodendrocytes, and microglia. Their roles are very diverse but overall they orchestrate CNS formation and function by providing neurons with essential support. Although glia-derived immortalized cell lines are now available, primary cultures of glial cells still constitute the most reliable method to study glial functions as the primary cultures retain important characteristics and markers of glia from their normal brain environment. Isolation and culturing of glia from postnatal rodent brain is well-characterized and give higher yield than from adult brain. Therefore, isolation of glial cells from postnatal mouse brains, with an emphasis on microglia, will be described. It will include a protocol describing the steps of isolation and necessary equipments and reagents, as well as the subsequent cell culture monitoring and potential applications.

Acute dose of melatonin via Nrf2 dependently prevents acute ethanol-induced neurotoxicity in the developing rodent brain

BackgroundMelatonin is a well-known potent endogenous antioxidant pharmacological agent with significant neuroprotective actions. Here in the current study, we explored the nuclear factor erythroid 2-related factor 2 (Nrf2) gene-dependent antioxidant mechanism underlying the neuroprotective effects of the acute melatonin against acute ethanol-induced elevated reactive oxygen species (ROS)-mediated neuroinflammation and neurodegeneration in the developing rodent brain.MethodsIn vivo rat pups were co-treated with a single dose of acute ethanol (5 g/kg, subcutaneous (S.C.)) and a single dose of acute melatonin (20 mg/kg, intraperitoneal (I.P.)). Four hours after a single S.C. and I.P. injections, all of the rat pups were sacrificed for further biochemical (Western blotting, ROS- assay, LPO-assay, and immunohistochemical) analyses. In order to corroborate the in vivo results, we used the in vitro murine-hippocampal HT22 and microglial BV2 cells, which were subjected to knockdown with small interfering RNA (siRNA) of Nrf2 genes and exposed with melatonin (100 μM) and ethanol (100 mM) and proceed for further biochemical analyses.ResultsOur biochemical, immunohistochemical, and immunofluorescence results demonstrate that acute melatonin significantly upregulated the master endogenous antioxidant Nrf2 and heme oxygenase-1, consequently reversing the acute ethanol-induced elevated ROS and oxidative stress in the developing rodent brain, and in the murine-hippocampal HT22 and microglial BV2 cells. In addition, acute melatonin subsequently reduced the activated MAPK-p-P38-JNK pathways and attenuated neuroinflammation by decreasing the expression of activated gliosis and downregulated the p-NF-K-B/p-IKKβ pathway and decreased the expression levels of other inflammatory markers in the developing rodent brain and BV2 cells. Of note, melatonin acted through the Nrf2-dependent mechanism to attenuate neuronal apoptosis in the postnatal rodent brain and HT22 cells. Immunohistofluorescence results also showed that melatonin prevented ethanol-induced neurodegeneration in the developing rodent brain. The in vitro results indicated that melatonin induced neuroprotection via Nrf2-dependent manner and reduced ethanol-induced neurotoxicity.ConclusionsThe pleiotropic and potent neuroprotective antioxidant characteristics of melatonin, together with our in vivo and in vitro findings, suppose that acute melatonin could be beneficial to prevent and combat the acute ethanol-induced neurotoxic effects, such as elevated ROS, neuroinflammation, and neurodegeneration in the developing rodent brain.

Downregulation of Sphingosine 1-Phosphate Receptor 1 Promotes the Switch from Tangential to Radial Migration in the OB

Neuroblast migration is a highly orchestrated process that ensures the proper integration of newborn neurons into complex neuronal circuits. In the postnatal rodent brain, neuroblasts migrate long distances from the subependymal zone of the lateral ventricles to the olfactory bulb (OB) within the rostral migratory stream (RMS). They first migrate tangentially in close contact to each other and later radially as single cells until they reach their final destination in the OB. Sphingosine 1-phosphate (S1P) is a bioactive lipid that interacts with cell-surface receptors to exert different cellular responses. Although well studied in other systems and a target for the treatment of multiple sclerosis, little is known about S1P in the postnatal brain. Here, we report that the S1P receptor 1 (S1P1) is expressed in neuroblasts migrating in the RMS. Using in vivo and in vitro gain- and loss-of-function approaches in both wild-type and transgenic mice, we found that the activation of S1P1 by its natural ligand S1P, acting as a paracrine signal, contributes to maintain neuroblasts attached to each other while they migrate in chains within the RMS. Once in the OB, neuroblasts cease to express S1P1, which results in cell detachment and initiation of radial migration, likely via downregulation of NCAM1 and β1 integrin. Our results reveal a novel physiological function for S1P1 in the postnatal brain, directing the path followed by newborn neurons in the neurogenic niche. The function of each neuron is highly determined by the position it occupies within a neuronal circuit. Frequently, newborn neurons must travel long distances from their birthplace to their predetermined final location and, to do so, they use different modes of migration. In this study, we identify the sphingosine 1-phosphate (S1P) receptor 1 (S1P1) as one of the key players that govern the switch from tangential to radial migration of postnatally generated neuroblasts in the olfactory bulb. Of interest is the evidence that the ligand, S1P, is provided by nearby astrocytes. Finally, we also propose adhesion molecules that act downstream of S1P1 and initiate the transition from tangential chain migration to individual radial migration outside of the stream.

Simplified CLARITY for visualizing immunofluorescence labeling in the developing rat brain.

CLARITY is an innovative technological advance in which intact biological tissue is transformed into a "nanoporous hydrogel-hybridized form" (Chung et al. 2013; Chung and Deisseroth 2013) with markedly improved chemical and optical accessibility, permitting fluorescent visualization and extraction of high-resolution structural data from mm-thick blocks of tissue. CLARITY affords an excellent but as yet unexploited opportunity to visualize the growth and maturation of phenotypically identified neurons and axonal processes in the developing brain. This brief report describes a moderately revised, simplified, and less expensive CLARITY protocol that effectively reveals the structure of chemically identified neurons in whole neonatal/juvenile rat brains and tissue slabs. Rats [postnatal day (P)0-24] were transcardially perfused with one of two fixative/hydrogel solutions, followed by hydrogel polymerization to generate brain hybrids. Whole brain hybrids or 2.0-mm-thick coronal slabs were passively cleared of lipid and then processed for dual immunofluorescence labeling, including labeling using tyramide signal amplification. After refractive index matching using 2,20-Thiodiethanol (60% solution), a Leica confocal microscope equipped with a CLARITY objective was used to view the hypothalamus in whole brain hybrids or slabs. Collected image stacks revealed the distribution and three-dimensional structure of hypothalamic pro-oxyphysin (oxytocin)-, neuropeptide Y-, glucagon-like peptide-1-, and tyrosine hydroxylase-immunopositive neurons and processes within large tissue volumes. Outstanding structural preservation and immunolabeling quality demonstrates the efficacy of this approach for interrogating chemically defined neural circuits as they develop in postnatal rodent brain.

Thrombospondin 4 deficiency in mouse impairs neuronal migration in the early postnatal and adult brain.

In the post-natal rodent brain, neuronal precursors originating from the sub-ventricular zone (SVZ) migrate over a long distance along the rostral migratory stream (RMS) to eventually integrate the olfactory bulb neuronal circuitry. In order to identify new genes specifically expressed in the RMS, we have screened the Allen Brain Atlas Database. We focused our attention on Thrombospondin 4 (Thbs4), one of the 5 members of the Thrombospondin family of large, multidomain, extracellular matrix proteins. In post-natal and adult brain Thbs4 mRNA and protein are specifically expressed in the neurogenic regions, including the SVZ and along the entire RMS. RMS cells expressing Thbs4 are GFAP (Glial Fibrillary Acidic Protein) positive astrocytes. Histological analysis in both wild-type and Thbs4 knock-out mice revealed no major abnormality in the general morphology of these neurogenic regions. Nevertheless, immunostaining for doublecortin demonstrates that in Thbs4-KO, migration of newly formed neurons along the RMS is somehow impaired, with several neurons migrating out of the RMS. This is further supported by a Bromodeoxyuridine-based in vivo approach showing a decrease in the number of newly born neuronal precursors reaching the olfactory bulb, while proliferation in the SVZ is not affected compared to wild-type, both in young animals (P15) and in adults (8 to 12 weeks of age). Corroborating this observation, the number of Parvalbumin- and Calbindin-immunoreactive interneurons in the olfactory bulb is also reduced in Thbs4-KO. Together, these observations support a role for the astrocyte-secreted protein Thbs4 in the migration of newly form neurons within the RMS to the olfactory bulb.

White matter loss in a mouse model of periventricular leukomalacia is rescued by trophic factors.

Periventricular leukomalacia (PVL) is the most frequent cause of cerebral palsy and other intellectual disabilities, and currently there is no treatment. In PVL, glutamate excitotoxicity (GME) leads to abnormal oligodendrocytes (OLs), myelin deficiency, and ventriculomegaly. We have previously identified that the combination of transferrin and insulin growth factors (TSC1) promotes endogenous OL regeneration and remyelination in the postnatal and adult rodent brain. Here, we produced a periventricular white matter lesion with a single intracerebral injection of N-methyl-d-aspartate (NMDA). Comparing lesions produced by NMDA alone and those produced by NMDA + TSC1 we found that: NMDA affected survival and reduced migration of OL progenitors (OLPs). In contrast, mice injected with NMDA + TSC1 proliferated twice as much indicating that TSC1 supported regeneration of the OLP population after the insult. Olig2-mRNA expression showed 52% OLP survival in mice receiving a NMDA injection and increased to 78% when TSC1 + NMDA were injected simultaneously and ventricular size was reduced by TSC1. Furthermore, in striatal slices TSC1 reduced the inward currents induced by NMDA in medium-sized spiny neurons, demonstrating neuroprotection. Thus, white matter loss after excitotoxicity can be partially rescued as TSC1 conferred neuroprotection to preexisting OLP and regeneration via OLP proliferation. Furthermore, we showed that early TSC1 administration maximizes neuroprotection.

Primary Culture of Cortical Neurons

Primary culture of neurons from cerebral cortex is a popular model to study neuronal function in vitro and to explore the molecular mechanism of neurite outgrowth in the developing and adult central nervous system. This protocol is for preparing a culture of cerebral cortical neurons from postnatal rodent brain (Muramatsu et al., 2012). One day after cell plating, we can observe neurite outgrowth by microscope.

The serotonin 5-HT3 receptor: a novel neurodevelopmental target.

Serotonin (5-hydroxytryptamine, 5-HT), next to being an important neurotransmitter, recently gained attention as a key-regulator of pre- and postnatal development in the mammalian central nervous system (CNS). Several receptors for 5-HT are expressed in the developing brain including a ligand-gated ion channel, the 5-HT3 receptor. Over the past years, evidence has been accumulating that 5-HT3 receptors are involved in the regulation of neurodevelopment by serotonin. Here, we review the spatial and temporal expression patterns of 5-HT3 receptors in the pre- and early postnatal rodent brain and its functional implications. First, 5-HT3 receptors are expressed on GABAergic interneurons in neocortex and limbic structures derived from the caudal ganglionic eminence. Mature inhibitory GABAergic interneurons fine-tune neuronal excitability and thus are crucial for the physiological function of the brain. Second, 5-HT3 receptors are expressed on specific glutamatergic neurons, Cajal–Retzius cells in the cortex and granule cells in the cerebellum, where they regulate morphology, positioning, and connectivity of the local microcircuitry. Taken together, the 5-HT3 receptor emerges as a potential key-regulator of network formation and function in the CNS, which could have a major impact on our understanding of neurodevelopmental disorders in which 5-HT plays a role.

Developmental distribution pattern of metabotropic glutamate receptor 5 in prenatal human hippocampus

Metabotropic glutamate receptor 5 (mGluR5) is concentrated in zones of active neurogenesis in the prenatal and postnatal rodent brain and plays an important role in the regulation of neurogenesis. However, little is known about mGluR5 in the prenatal human brain. Here, we aimed to explore the expression pattern and cellular distribution of mGluR5 in human fetal hippocampus. Thirty-four human fetuses were divided into four groups according to gestational age: 9-11, 14-16, 22-24 and 32-36 weeks. The hippocampus was dissected out and prepared. The protein and mRNA expression of mGluR5 were evaluated by Western blot and immunohistochemistry or real-time PCR. The cellular distribution of mGluR5 was observed with double-labeling immunofluorescence. Both mGluR5 mRNA and protein were detected in the prenatal human hippocampus by real-time PCR and immunoblotting, and the expression levels increased gradually over time. The immunohistochemistry results were consistent with immunoblotting and showed that mGluR5 immunoreactivity was mainly present in the inner marginal zone (IMZ), hippocampal plate (HP) and ventricular zone (VZ). The double-labeling immunofluorescence showed that mGluR5 was present in neural stem cells (nestin-positive), neuroblasts (DCX-positive) and mature neurons (NeuN-positive), but not in typical astrocytes (GFAP-positive). The cells co-expressing mGluR5 and nestin were mainly located in the IMZ, HP and subplate at 11 weeks, all layers at 16 weeks, and CA1 at 24 weeks. As development proceeded, the number of mGluR5/nestin double-positive cells decreased gradually so that there were only a handful of double-labeled cells at 32 weeks. However, mGluR5/DCX double-positive cells were only found in the HP, IZ and IMZ at 11 weeks. The pattern of mGluR5 expression by neural stem/progenitor cells, neuroblasts and neurons provides important anatomical evidence for the role of mGluR5 in the regulation of human hippocampal development.

Assessment of general anaesthetic cytotoxicity in murine cortical neurones in dissociated culture

General anaesthetics are proposed to cause unconsciousness by modulating neuronal excitability in the mammalian brain through mechanisms that include enhancement of inhibitory GABA A receptor currents and suppression of excitatory glutamate receptor responses. Both intravenous and volatile agents may produce neurotoxic effects during early postnatal rodent brain development through similar mechanisms. In the following study, we investigated anaesthetic cytotoxicity in primary cortical neurones and glia from postnatal day 2–8 mice. Cultures at 4–20 days in vitro were exposed to combinations of ketamine (100 μM to 3 mM), nitrous oxide (75%, v/v) and/or isoflurane (1.5–5%, v/v) for 6–12 h. Neuronal survival and cell death were measured via microtubule associated protein 2 immunoassay and lactate dehydrogenase release assays, respectively. Clinically relevant anaesthetic concentrations of ketamine, nitrous oxide and isoflurane had no significant neurotoxic effects individually or when given as anaesthetic cocktails, even with up to 12 h exposure. This lack of neurotoxicity was observed regardless of whether cultures were prepared from postnatal day 0–2 or day 8 mice, and was also unaffected by number of days in vitro (DIV 4–20). Significant neurotoxic effects were only observed at supraclinical concentrations ( e.g. 1–3 mM ketamine). Our study suggests that neurotoxicity previously reported in vivo is not due to direct cytotoxicity of anaesthetic agents, but results from other impacts of the anaesthetised state during early brain development.

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved (1). The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

How DISC1 Regulates Postnatal Brain Development: Girdin Gets In on the AKT

In this issue of Neuron, Kim et al. and Enomoto et al. show that DISC1 plays a key role in regulating postnatal brain development though interaction with Girdin. Girdin in turn regulates AKT signaling. Thus, another facet of the role of DISC1 is established, shedding more light on fundamental brain processes and the developmental basis of major psychiatric disorders.

Glial cells are born with synapses

In postnatal rodent brain, certain NG2-expressing oligodendroglial precursor cells (OPCs) are contacted by synaptic terminals from local neurons. However, it has remained elusive whether and when NG2(+) cells are integrated into neuronal circuits. Here we use patch-clamp recordings from mitotic cells in murine brain slices to show that, unlike any other cell in the central nervous system (CNS), cortical NG2(+) cells divide and relocate while being linked to synaptic junctions. Together with bromodeoxyuridine (BrdU) labeling, our recordings imply that cellular processes that bear synaptic junctions are surprisingly kept during cytokinesis and are inherited by the daughter cells. Cell cycle time (78 h) and relocation speed (5 microm/day) are slowed, and NG2(+) cells largely divide symmetrically. Inheritance of synapses enables newborn glial cells to establish synaptic connections much faster than newborn neurons and ensures that the entire population of NG2(+) cells is exposed to synaptic signals from local axons. The results suggest that synapses do not only transmit neuronal activity but also act as environmental cues for the development of glial cells. Inheritance of synapses allows for the direct transfer of environmental interactions to clonal descendants of OPCs, which might be important for effective colonization and myelination of the developing brain.

On the role of NR3A in human NMDA receptors

In the present paper we describe our on-going project investigating the functional roles of the N-methyl- d-aspartate (NMDA) receptor subunit NR3A. We find that NR3A mRNA is abundant both in embryonic and adult human brain, in contrast to the almost non-existing expression in adult rodent brain. Human NR3A (hNR3A) protein expression is particularly abundant in the cerebral cortex, as shown by western blot using NR3A-specific antibodies. Distribution of hNR3A in adult human brain shows a similar pattern as NR3A in post-natal rodent brain. We have previously reported that NR3A contains a glycine binding site, with similar affinity as the glycine binding site of NR1 subunits. This suggests that NR3A may replace one of the two NR1 subunits in native NMDA receptors. Cloning of hNR3A showed a human-specific polyproline-sequence in the intracellular C-terminus, that may bind to SH3-domains. We hypothesized that the significant differences in expression in the adult human and rodent brain could be due to an atypical interaction of hNR3A with the SH3 domain of the synaptic scaffolding protein PSD-95, that binds to NR2 subunits through its PDZ domains. However, using a number of different protein interaction assays, binding of PSD-95 to hNR3A could no be demonstrated either in vitro or in vivo. To identify intracellular signaling pathways for NR3A-containing NMDA receptors, we screened for proteins interacting with hNR3A and identified three proteins: plectin, CARP-1 and GPS2. The possible physiological roles of these interactions are discussed.

Neurogenesis as a potential therapeutic strategy for neurodegenerative disorders

The discovery that new neurons could be observed in post-natal rodent brain was reported in the early 1960s [1–3], but only gained wider acceptance when a potential source of this “neurogenesis” was identified. Since the 1990s, progenitors for the main neural cell types had been isolated from rodent [45,49,58,59] and human adult or fetal brain tissue [11,63]. These isolated progenitors, or stem cells, are unique in their ability to self-renew and to differentiate, in a multipotential manner, into astrocytes, oligodendrocytes and neurons. In mouse and human stem cells, single growth factor treatment allows for continued proliferation or for differentiation toward a particular neural fate [28, 33,60,64]. At the same time that investigators were beginning to understand the multipotential nature of the isolated progenitors, the localization of in vivo neurogenesis to discrete areas of the brain were described. In rodents [4,32] and in non-human [23] and human primates [16], immunohistological examination determined that the areas of greatest concentration of new neurons in the adult brain are located in the subventricular zone, where they migrate to the olfactory bulb and in the subgranular zone of dentate gyrus. Results from the isolation of post-mortem human brain tissue, suggest that new neurons can be formed in the dentate gyrus well into adulthood [46]. It is important to note, when discussing neurogenesis, that three apparent stages exist, first the point of neural stem cell proliferation in the subgranular zone or subventricular zone, then the migration, on differentiation, to the granule cell layers, and finally the maturation with generation of functional neural networks for surviving neurons. Mitogen, growth factor and cytokine control of each neurogenesis step in isolated fetal neural progenitors [9,19,28] imply that each stage is finely regulated and deviations in for instance growth factor levels, could have physiological consequences. Accordingly, increases in proliferation of stem cells or neuronal progenitors promoted by the mitogen, basic fibroblast growth factor (bFGF), followed by differentiation to neurons, subsequently requires addition of brain-derived neurotrophic factor (BDNF) to maintain survival and arborization of the new neurons. It appears that only 50% of new neurons in the granule layer of dentate gyrus or new neurons in the olfactory bulb survive to maturity, normally a 2–4 week process. This observation could be important if neurogenesis is going to be exploited for therapeutic purposes.

Nestin expression persists in astrocytes of organotypic slice cultures from rat cortex

Nestin is an intermediate filament protein typical for neural precursor cells that is down-regulated in the post-natal rodent brain. Re-expression of nestin has been observed in reactive astrocytes after injury. In this study, organotypic slice cultures from rat cortex were examined for expression of nestin and glial fibrillary acidic protein between 2 and 8 weeks in culture. Immunoreactivity for nestin and glial fibrillary acidic protein was seen in astrocytes which persisted throughout the observation period. Immunofluorescence double labeling showed widespread co-localization of nestin and glial fibrillary acidic protein. Image analysis revealed that levels of nestin-immunoreactivity plateaued after 5 weeks in culture. By comparison nestin immunoreactivity was absent from glial cells of the cortex in mature rats. These immunohistochemical findings of a persistent expression of nestin in glial cells of organotypic slice culture of the rat cortex indicate a different time course of glial maturation in vitro. This difference could be related to the altered trophic stimulation in vitro; differences in neuronal maturation, activity or survival; slow degeneration of the vasculature; or intrinsic properties of astrocytes.

Differential Expression of Collapsin Response Mediator Proteins (CRMP/ULIP) in Subsets of Oligodendrocytes in the Postnatal Rodent Brain

The family of collapsin response mediator protein/Unc-33-like protein (CRMP/Ulip), composed of four homologous members, is specifically and highly expressed in the nervous system during embryonic neuronal development and dramatically down-regulated in the adult. Members of this family have been proposed to be part of the semaphorins signal transduction pathway involved in axonal outgrowth. Here, we show by in situ hybridization and immunohistochemistry that CRMP2/Ulip2, and to a lesser extent CRMP3/Ulip4, are expressed in immature and mature oligodendrocytes, but not in astrocytes. Transcripts encoding the other CRMP/Ulip members are also detectable by RT-PCR in highly purified mature oligodendrocytes. Interestingly, in the adult, the protein CRMP2/Ulip2 is mainly detectable in subsets of oligodendrocytes distributed according to an increasing rostrocaudal gradient, with the largest number of positive cells being present in the brain stem and spinal cord. In cultures of highly purified oligodendrocytes, however, CRMP2/Ulip2 was detectable in all the cells. Addition of Sema3A in the culture medium completely inhibited the emergence of oligodendrocyte processes suggesting that, as in neurons, a Sema3A signaling pathway mediated via CRMP2/Ulip2 may be involved in the regulation of oligodendroglial process outgrowth.

Gtx, an oligodendrocyte-specific homeodomain protein, has repressor activity.

Myelin, a multilamellar membrane structure that facilitates nerve conduction, is synthesized in the central nervous system (CNS) by oligodendrocytes. Gtx, a member of the homeodomain family of transcriptional factors, is a candidate regulator of myelin gene expression, because it is uniquely expressed in myelinating oligodendrocytes in postnatal rodent brain. To analyze the regulatory activity of Gtx, we first identified the optimal Gtx-binding sequence using an in vitro DNA-binding assay. This sequence, (A/T)TTAATGA, contains a TAAT core and is similar, but not identical, to that of other homeodomain protein binding sites. When coexpressed in cultured cells along with a minimal promoter containing five tandem repeats of this optimal Gtx-binding sequence, Gtx demonstrated repressor activity, which was also present when Gtx was tethered to DNA by way of the strong GAL4 DNA-binding domain. Truncations of the GAL4-Gtx fusion identified a portable repressor domain within a relatively proline/alanine-rich region N-terminal to the Gtx homeodomain. Cotransfection of a Gtx expression vector into a variety of cell lines, including oligodendrocytes, along with constructs containing portions of the PLP, MBP, or Gtx promoters fused to a reporter gene, however, did not modulate transcription from any of these promoter constructs. These data support the notion that the oligodendrocyte-specific homeodomain protein Gtx can act as a transcriptional repressor. In addition, they suggest that interaction of Gtx with other, as yet undefined, transcriptional regulators modifies Gtx activity in oligodendrocytes.

Gtx, an oligodendrocyte-specific homeodomain protein, has repressor activity

Myelin, a multilamellar membrane structure that facilitates nerve conduction, is synthesized in the central nervous system (CNS) by oligodendrocytes. Gtx, a member of the homeodomain family of transcriptional factors, is a candidate regulator of myelin gene expression, because it is uniquely expressed in myelinating oligodendrocytes in postnatal rodent brain. To analyze the regulatory activity of Gtx, we first identified the optimal Gtx-binding sequence using an in vitro DNA-binding assay. This sequence, (A/T)TTAATGA, contains a TAAT core and is similar, but not identical, to that of other homeodomain protein binding sites. When coexpressed in cultured cells along with a minimal promoter containing five tandem repeats of this optimal Gtx-binding sequence, Gtx demonstrated repressor activity, which was also present when Gtx was tethered to DNA by way of the strong GAL4 DNA-binding domain. Truncations of the GAL4-Gtx fusion identified a portable repressor domain within a relatively proline/alanine-rich region N-terminal to the Gtx homeodomain. Cotransfection of a Gtx expression vector into a variety of cell lines, including oligodendrocytes, along with constructs containing portions of the PLP, MBP, or Gtx promoters fused to a reporter gene, however, did not modulate transcription from any of these promoter constructs. These data support the notion that the oligodendrocyte-specific homeodomain protein Gtx can act as a transcriptional repressor. In addition, they suggest that interaction of Gtx with other, as yet undefined, transcriptional regulators modifies Gtx activity in oligodendrocytes.