Biomedical subjects

R D McKay

Publications and source records attributed to R D McKay.

At least 37 records · Page 2Linked to original sources

PubMed1998

Platelet-derived growth factor induces chemotaxis of neuroepithelial stem cells.

The ability of differentiating cells to migrate within the developing central nervous system (CNS) depends on extrinsic guidance signals, some of which are growth factors. In this study we have investigated the chemotactic response of cultured stem cells from the embryonic rat cortex to platelet-derived growth factor (PDGF). Nestin-positive stem cells from the developing CNS can be maintained and expanded in vitro under serum-free conditions in the presence of basic fibroblast growth factor (bFGF). Northern blot analysis of PDGF receptor expression revealed both alpha- and beta-receptors on bFGF-treated neural stem cells. Both PDGF-AA and PDGF-BB readily induced directed migration of cultured neuroepithelial cells as measured in a microchemotaxis assay. Blocking of the migratory response was achieved by incubation with PDGF isoform-specific antibodies. More than 90% of the migrating cells were nestin-positive and incorporation of BrdU was also seen suggesting the cells to be immature and not yet committed to a specific cell lineage. These findings suggest a role for PDGF in cell migration in the developing cortex.

Animals↗

PubMed1998

A rod end deletion in the intermediate filament protein nestin alters its subcellular localization in neuroepithelial cells of transgenic mice.

Neuroepithelial and radial glial cells span between the ventricular and the pial surfaces of the neural tube and express two intermediate filaments (IFs), nestin and vimentin, which form a filamentous network throughout the length of the cells. In this report we study the polymerization characteristics of nestin and examine how mutations affect the assembly and localization of the nestin protein in cultured cells and in the developing CNS of transgenic mice. A wild-type rat nestin gene transfected into the IF-free SW13 cell line failed to assemble into a filamentous network but was incorporated into the existing IF network of a subclone expressing vimentin, demonstrating that nestin requires vimentin for proper assembly. In transgenic mice, rat nestin formed a network indistinguishable from that formed by endogenous nestin and vimentin, but a mutant form lacking five amino acids at the carboxy terminus of the rod domain was largely restricted to the pial endfeet. Since nestin mRNA is localized to the pial endfoot region we propose that both transgenes are translated there, but that the wild-type protein is preferentially incorporated into the IF network. These observations provide evidence for hierarchical assembly and a complex organization of the IF network along the ventricular-pial axis in the early CNS.

Amino Acid Sequence↗

PubMed1998

Hippocampal synaptic plasticity in mice overexpressing an embryonic subunit of the NMDA receptor.

The effects of changing NMDA receptor subunit composition on synaptic plasticity in the hippocampus were analyzed by creating transgenic mice overexpressing NR2D, a predominantly embryonic NMDA receptor subunit. NMDA-evoked currents in the transgenic mice had smaller amplitudes and slower kinetics. The transgenics also displayed age-dependent deficits in synaptic plasticity in area CA1 of the hippocampus. Long-term depression was selectively impaired in juvenile mice when NR2D overexpression was moderate. In mature mice, overexpression of NR2D was associated with a reduction of both NR2B and Ca2+-independent activity of Ca2+- and calmodulin-dependent protein kinase II. These biochemical changes were correlated with a marked impairment of NMDA-dependent long-term potentiation, but spatial behavior was normal in these mice. These results show that the developmental regulation of NMDA receptor subunit composition alters the frequency at which modification of synaptic responses occur after afferent stimulation.

2-Amino-5-phosphonovalerate↗

PubMed1998

Multiple routes to astrocytic differentiation in the CNS.

Ciliary neurotrophic factor (CNTF) acts instructively to switch multipotent stem cells of the CNS to an astrocytic fate. Here we show that CNTF causes activation of janus kinase-signal transducers and activators of transcription and mitogen-activated protein kinase (MAPK) pathways with differential kinetics in these cells. Inhibition studies indicate that activation of the MAPK pathway is required early in the differentiation process, whereas activation of signal transducer and activator of transcription (STAT) proteins is required for commitment to an astrocytic fate. Bone morphogenetic proteins have also been shown to cause astrocytic differentiation but do not cause STAT activation or astrocytic differentiation in fibroblast growth factor 2-expanded fetal stem cells used here. These results show that there are two distinct routes to initiate astrocytic commitment in multipotent CNS precursors.

Animals↗

PubMed1998

Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats.

In vitro expansion of central nervous system (CNS) precursors might overcome the limited availability of dopaminergic neurons in transplantation for Parkinson's disease, but generating dopaminergic neurons from in vitro dividing precursors has proven difficult. Here a three-dimensional cell differentiation system was used to convert precursor cells derived from E12 rat ventral mesencephalon into dopaminergic neurons. We demonstrate that CNS precursor cell populations expanded in vitro can efficiently differentiate into dopaminergic neurons, survive intrastriatal transplantation and induce functional recovery in hemiparkinsonian rats. The numerical expansion of primary CNS precursor cells is a new approach that could improve both the ethical and the technical outlook for the use of human fetal tissue in clinical transplantation.

Animals↗

PubMed1998

Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats.

Limited experimental access to the central nervous system (CNS) is a key problem in the study of human neural development, disease, and regeneration. We have addressed this problem by generating neural chimeras composed of human and rodent cells. Fetal human brain cells implanted into the cerebral ventricles of embryonic rats incorporate individually into all major compartments of the brain, generating widespread CNS chimerism. The human cells differentiate into neurons, astrocytes, and oligodendrocytes, which populate the host fore-, mid-, and hindbrain. These chimeras provide a unique model to study human neural cell migration and differentiation in a functional nervous system.

Animals↗

PubMed1998

Survival and synaptogenesis of hippocampal neurons without NMDA receptor function in culture.

Physiological and morphological properties of cultured hippocampal neurons were measured to investigate whether NMDA receptors play a role in survival and differentiation. Neurons dissociated from mouse embryos with different NMDAR1 genotypes were grown in culture. Electrophysiological analysis verified the absence of NMDA receptor-mediated currents in neurons taken from homozygous mutant (NR1-/-) embryos. The number of surviving hippocampal neurons was 2.5-fold higher in cultures from the NR1-/- embryos compared with wild type (NR1 +/+) and heterozygous (NR1+/-) controls. Despite the lack of NMDA receptor function, NR1-/- neurons formed synapsin I-positive presynaptic boutons associated with MAP2ab-positive dendrites in culture. Confocal microscopic analysis of Dil labelled neurons confirmed the presence of dendritic spines on NR1-/- neurons with 80% of the density found in NR1 +/+ neurons. These results suggest that the NMDA receptor has little effect on general features of neuronal differentiation. In contrast, there is clear effect on neuronal survival. This finding establishes neuron number in standard culture conditions as a measure of NMDA receptor activity.

Animals↗

PubMed1998

POU transcription factors control expression of CNS stem cell-specific genes.

Multipotential stem cells throughout the developing central nervous system have common properties. Among these is expression of the intermediate filament protein nestin and the brain fatty acid binding protein (B-FABP). To determine if common mechanisms control transcription in CNS stem cells, the regulatory elements of these two genes were mapped in transgenic mice. A 257 basepair enhancer of the rat nestin gene is sufficient for expression throughout the embryonic neuroepithelium. This enhancer contains two sites bound by the class III POU proteins Brn-1, Brn-2, Brn-4, and Tst-1. Only one of the two POU sites is required for CNS expression. An adjacent hormone response element is necessary for expression in the dorsal midbrain and forebrain. The regulatory sites of the B-FABP gene are strikingly similar to those of the nestin gene. A hybrid POU/Pbx binding site is recognized in vitro by Pbx-1, Brn-1 and Brn-2. This site is essential for expression in most of the CNS. In addition, a hormone response element is necessary for forebrain expression. Both the nestin and B-FABP genes therefore depend on POU binding sites for general CNS expression, with hormone response elements additionally required for activity in the anterior CNS. These data indicate that regulation by POU proteins and hormone receptors is a general mechanism for CNS stem cell-specific transcription.

Animals↗

PubMed1998

An immortalized, type-1 astrocyte of mesencephalic origin source of a dopaminergic neurotrophic factor.

Rat embryonic d 14 (E14) mesencephalic cells, 2.5% of which are glioblasts, were incubated in medium containing 10% of fetal bovine serum for 12 h and subsequently expanded in a serum-free medium using basic fibroblast growth factor (bFGF) as the mitogen. On a single occasion, after more than 15 d in culture, several islets of proliferating, glial-like cells were observed in one dish. The cells, when isolated and passaged, proliferated rapidly in either a serum-free or serum-containing growth medium. Subsequent immunocytochemical analysis showed that they stained positive for GFAP and vimentin, and negative for A2B5, O4, GalC, and MAP2. Serum-free conditioned medium (CM) prepared from these cells caused a fivefold increase in survival and promoted neuritic expansion of E14 mesencephalic dopaminergic neurons in culture. These actions are similar to those exerted by CM derived from primary, mesencephalic type-1 astrocytes. The pattern of expression of the region-selective genes; wnt-1, en-1, sis showed that 70% of the cells were heteroploid, and of these, 50% were tetraploid. No apparent decline in proliferative capacity has been observed after 25 passages. The properties of this cell line, named ventral mesencephalic cell line one (VMCL1), are consistent with those of an immortalized, type-1 astrocyte. The mesencephalic origin of the cell line, and the pattern and potency of the neurotrophic activity exerted by the CM, strongly suggest that the neurotrophic factor(s) identified are novel, and will likely be strong candidates with clinical utility for the treatment of Parkinson's disease.

Animals↗

PubMed1998

Regulation of neurogenesis by growth factors and neurotransmitters.

The generation of neurons and glia in the developing nervous system is likely to be regulated by extrinsic factors, including growth factors and neurotransmitters. Evidence from in vivo and/or in vitro systems indicates that basic fibroblast growth factor, transforming growth factor (TGF)-alpha, insulin-like growth factor-1, and the monoamine neurotransmitters act to increase proliferation of neural precursors. Conversely, glutamate, gamma-aminobutyric acid, and opioid peptides are likely to play a role in down-regulating proliferation in the developing nervous system. Several other factors, including the neuropeptides vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide, as well as the growth factors platelet-derived growth factor, ciliary neurotrophic factor, and members of the TGF-beta family, have different effects on proliferation and differentiation depending on the system examined. Expression of many of these factors and their receptors in germinal regions of the central nervous system suggests that they can act directly on precursor populations to control their proliferation. Together, the findings discussed here indicate that proliferation and cell fate determination in the developing brain are regulated extrinsically by complex interactions between a relatively large number of growth factors and neurotransmitters.

Aging↗

PubMed1997

In vitro-generated neural precursors participate in mammalian brain development.

During embryogenesis, pluripotent stem cells segregate into daughter lineages of progressively restricted developmental potential. In vitro, this process has been mimicked by the controlled differentiation of embryonic stem cells into neural precursors. To explore the developmental potential of these cell-culture-derived precursors in vivo, we have implanted them into the ventricles of embryonic rats. The transplanted cells formed intraventricular neuroepithelial structures and migrated in large numbers into the brain tissue. Embryonic-stem-cell-derived neurons, astrocytes, and oligodendrocytes incorporated into telencephalic, diencephalic, and mesencephalic regions and assumed phenotypes indistinguishable from neighboring host cells. These observations indicate that entirely in vitro-generated neural precursors are able to respond to environmental signals guiding cell migration and differentiation and have the potential to reconstitute neuronal and glial lineages in the central nervous system.

Animals↗

PubMed1997

Id genes encoding inhibitors of transcription are expressed during in vitro astrocyte differentiation and in cell lines derived from astrocytic tumors.

Id proteins belong to a class of nuclear transcription factors known as helix-loop-helix proteins. It has been reported that Id genes function as negative regulators of differentiation, and Id gene expression is down-regulated during cell differentiation. We examined the regulation of Id genes during astrocyte differentiation in a murine nervous system precursor cell line, NSEHip2-28, which is able to differentiate along the astroglial lineage, as well as in human astroglial tumor cell lines. Upon induction of NSEHip2-28 differentiation, at a time when glial fibrillary acidic protein expression became detectable, the expression of all four Id family members initially increased dramatically, and subsequently decreased. Furthermore, varying levels of Id gene expression were found in astroglial tumor cell lines displaying variable degrees of lineage-specific differentiation. These results suggest that the expression of Id family members may play an important role in the control of astrocyte differentiation.

Animals↗

PubMed1997

Survival, integration, and differentiation of neural stem cell lines after transplantation to the adult rat striatum.

The in vivo properties of four different neural stem cell lines, generated from embryonic striatum or hippocampus by immortalization with the temperature-sensitive (s) A58/U19 allele of the SV40 Large T-antigen, have been studied with respect to their ability to survive, differentiate, and integrate after transplantation to the adult rat striatum. The cells were labeled with [3H]thymidine prior to grafting, and combined autoradiography and immunohistochemistry was used to characterize their phenotypic differentiation within the adult brain environment. The results show that all four types of cells survived well, up to at least 1.5-6 months postgrafting, without any signs of tissue perturbation or tumor formation. The cells underwent, on average, 2-3 cell divisions during the first 5 days after implantation and exhibited extensive migration over a distance of 1-1.5 mm from the injection site to become morphologically integrated with the surrounding host striatum. The cell number and tissue distribution attained by 2 weeks remained stable for up to 6 months postgrafting with the exception of one cell line, which showed a 40% loss of cells between 2 and 6 weeks. Twice the number of [3H]thymidine-labeled cells were recovered when the cells were grafted into a 1-week-old excitotoxic striatal lesion, probably due to an increased proliferation of the cells in response to the neuron-depleting depleting lesion. The immortalized cells behaved as multipotent neural progenitors. The vast majority of the cells developed a glial-like morphology, 6-14% being clearly GFAP-positive; however, a small but consistent proportion of them (1-3%) expressed MAP-2 and exhibited neuron-like morphology. In mature transplants about 75-80% of the grafted cells were located in the striatal grey matter, and 10-15% in white matter, some of which are proposed to have differentiated into oligodendrocytes. Remaining 5-10% occurred around small blood vessels (resembling pericytes) and in the subventricular zone underneath the ependyma of the lateral ventricle. It is concluded that the ts cell lines are highly suitable for intracerebral transplantation and that they allow the creation of a regionally confined cellular chimeras where the graft-derived glial cells become stably integrated with the resident glial cell matrix.

Animals↗

PubMed1997

Novel differentially expressed genes induced by kainic acid in hippocampus: putative molecular effectors of plasticity and injury.

Systemic kainic acid administration in rats induces acute limbic status epilepticus and subsequent neuronal degeneration and development of chronic hyperexcitability with similarities to human temporal lobe epilepsy. The mechanisms mediating the responses to kainic acid likely involve transcriptional changes in genes of importance for cellular injury, protection, and plasticity. We have used an arbitrarily primed PCR technique to identify such changes in the rat dentate gyrus. Three previously uncharacterized transcripts were found to be upregulated in the dentate gyrus 4 h following systemic kainic acid. In situ hybridization using riboprobes transcribed from the cloned PCR fragments were used to confirm differential expression specifically in dentate granule neurons following seizure. Basal expression for all three transcripts is widespread throughout the rat brain, with the highest levels seen in the hippocampal pyramidal and granule cell layers. The novel sequences do not match any known full-length cDNAs and may belong to novel gene families. However, they all showed high homology to human partial cDNA sequences (ESTs) that are expressed in brain as well as several other tissues. Two additional transcripts identified in this study corroborate earlier findings on differential expression of heat-shock proteins after seizure. The novel transcripts found in this study may be involved in epileptogenesis and neuronal responses to damage following seizure.

Animals↗

PubMed1997

Neurotrophins stimulate chemotaxis of embryonic cortical neurons.

During mammalian cortical development, neuronal precursors proliferate within ventricular regions then migrate to their target destinations in the cortical plate, where they organize into layers. In the rat, most cortical neuronal migration occurs during the final week of gestation (Bayer et al, 1991; Jacobson, 1991). At this time (E15-E21), reverse transcriptase-polymerase chain reaction demonstrated that cortical homogenates contain mRNA encoding brain derived neurotrophic factor (BDNF) and the catalytic form of its high-affinity receptor, TrkB. Immunocytochemistry and in situ hybridization of sections revealed that the catalytic TrkB receptors predominantly localize to regions containing migratory cells. Many TrkB+ cells exhibited the classic morphology of migrating neurons, suggesting that TrkB ligands play a role in cortical neuronal migration. We analysed whether TrkB ligands influence the motility of embryonic cortical cells (from E15-E21) using a quantitative in vitro chemotaxis assay. High-affinity TrkB ligands (BDNF and NT4/5) stimulated chemotaxis (directed migration) of embryonic neurons at concentrations ranging from 1 to 100 ng/ml. NT-3, a low-affinity TrkB ligand, only stimulated significant migration at high concentrations (> or =100 ng/ml). Peak migration to BDNF was observed at gestational day 18 (E18). BDNF-induced chemotaxis was blocked by either tyrosine kinase inhibitor, K252a, or the Ca2+-chelator, BAPTA-AM, suggesting that BDNF-induces motility via autophosphorylation of TrkB receptor proteins and involves Ca2+-dependent mechanisms. BDNF-stimulation of increased cytosolic Ca2+ was confirmed with optical recordings of E18 cortical cells loaded with Ca2+ indicator dye. Thus, signal transduction through the TrkB receptor complex directs neuronal migration, suggesting that, in vivo, BDNF exerts chemotropic effects that are critical to morphogenesis of the cortex.

Animals↗

PubMed1996

Single factors direct the differentiation of stem cells from the fetal and adult central nervous system.

Identifying the signals that regulate stem cell differentiation is fundamental to understanding cellular diversity in the brain. In this paper we identify factors that act in an instructive fashion to direct the differentiation of multipotential stem cells derived from the embryonic central nervous system (CNS). CNS stem cell clones differentiate to multiple fates: neurons, astrocytes, and oligodendrocytes. The differentiation of cells in a clone is influenced by extracellular signals: Platelet-derived growth factor (PDGF-AA, -AB, and -BB) supports neuronal differentiation. In contrast, ciliary neurotrophic factor and thyroid hormone T3 act instructively on stem cells to generate clones of astrocytes and oligodendrocytes, respectively. Adult stem cells had remarkably similar responses to these growth factors. These results support a simple model in which transient exposure to extrinsic factors acting through known pathways initiates fate decisions by multipotential CNS stem cells.

Animals↗

PubMed1996

Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro.

To understand the mechanism of the sequential restriction of multipotency of stem cells during development, we have established culture conditions that allow the differentiation of neuroepithelial precursor cells from embryonic stem (ES) cells. A highly enriched population of neuroepithelial precursor cells derived from ES cells proliferates in the presence of basic fibroblast growth factor (bFGF). These cells differentiate into both neurons and glia following withdrawal of bFGF. By further differentiating the cells in serum-containing medium, the neurons express a wide variety of neuron-specific genes and generate both excitatory and inhibitory synaptic connections. The expression pattern of position-specific neural markers suggests the presence of a variety of central nervous system (CNS) neuronal cell types. These findings indicate that neuronal precursor cells can be isolated from ES cells and that these cells can efficiently differentiate into functional post-mitotic neurons of diverse CNS structures.

Animals↗

PubMed1996

Neuronal progenitors as tools for cell replacement in the nervous system.

The clinical prospect of using neural precursor cells for reconstructive approaches in the nervous system has received strong impetus from a recent series of important experimental findings. Transplantation studies in the developing brain have demonstrated that migration and differentiation of neural precursor cells are regulated predominantly by environmental signals. Several observations suggest that the mature CNS retains at least some of these guidance cues. These findings, together with recent evidence for the persistence of neural stem cells in the adult mammalian brain, have made precursor cell recruitment a new focus in CNS reconstruction.

Animals↗