Boards, Advisory Committees, Professional Organizations
Board member, The Nueva School (2017 - Present)
Klein, Robert D., Rosenthal, Arnon, Hynes, Mary A.. "United States Patent 6777196 Klein, Robert D., Rosenthal, Arnon, Hynes, Mary A.", Genentech Inc, Aug 17, 2004
Klein, Robert D., Rosenthal, Arnon, Hynes, Mary A.. "United States Patent 6342348 Neurturin receptor", Genentech Inc, Jan 29, 2002
Hynes, Mary A., Ye, Weilan. "United States Patent 6277820 Method of dopaminergic and serotonergic neuron formation from neuroprogenitor cells", Genentech Inc, Aug 21, 2001
Klein, Robert D., Rosenthal, Arnon, Hynes, Mary A.. "United States Patent 6025157 NTNRα, NTNRα extracellular domain (ECD), NTNRα variants, chimeric NTNRα (e.g., NTNRα immunoadhesion), and antibodies which bind thereto (including agonist and neutralizing antibodies) are disclosed. Various uses for these molecules are described.", Genentech Inc, Feb 15, 2000
Distinct expression of select and transcriptome-wide isolated 3'UTRs suggests critical roles in development and transition states.
2021; 16 (5): e0250669
Mature mRNA molecules are expected to be comprised of a 5'UTR, a 3'UTR and a coding region (CDS). Unexpectedly, however, there have been multiple recent reports of widespread differential expression of mRNA 3'UTRs and their cognate coding regions (CDS), reflecting the expression of isolated 3'UTRs (i3'UTRs); these i3'UTRs can be highly expressed, often in reciprocal patterns to their cognate CDS. As with other long non-coding (lncRNAs), isolated 3'UTRs are likely to play an important role in gene regulation, but little is known about the contexts in which they are deployed. To illuminate the functions of i3'UTRs, here we carry out in vitro, in vivo and in silico analyses of differential 3'UTR/CDS mRNA ratio usage across tissues, development and cell state changes both for a select list of developmentally important genes as well as by unbiased transcriptome-wide analyses. Across two developmental paradigms we find a distinct switch from high i3'UTR expression for stem cell related genes in proliferating cells to high CDS for these genes in newly differentiated cells. Unbiased transcriptome analysis across multiple gene sets shows that regardless of tissue, genes with high 3'UTR to CDS ratios belong predominantly to gene ontology categories related to cell-type specific functions. In contrast, the gene ontology categories of genes with low 3'UTR to CDS ratios are similar across tissues and relate to common cellular functions. We further show that, at least for some genes, traditional transcriptional start site genomic elements correspond to identified RNAseq 3'UTR peak regions, suggesting that some i3'UTRs may be generated by de novo transcription. Our results provide critical information from which detailed hypotheses for individual i3'UTRs can be tested, with a common theme that i3'UTRs appear poised to regulate cell-specific gene expression and state.
View details for DOI 10.1371/journal.pone.0250669
View details for PubMedID 33951080
Widespread Differential Expression of Coding Region and 3 ' UTR Sequences in Neurons and Other Tissues
2015; 88 (6): 1149-1156
Mature messenger RNAs (mRNAs) consist of coding sequence (CDS) and 5' and 3' UTRs, typically expected to show similar abundance within a given neuron. Examining mRNA from defined neurons, we unexpectedly show extremely common unbalanced expression of cognate 3' UTR and CDS sequences; many genes show high 3' UTR relative to CDS, others show high CDS to 3' UTR. In situ hybridization (19 of 19 genes) shows a broad range of 3' UTR-to-CDS expression ratios across neurons and tissues. Ratios may be spatially graded or change with developmental age but are consistent across animals. Further, for two genes examined, a 3' UTR-to-CDS ratio above a particular threshold in any given neuron correlated with reduced or undetectable protein expression. Our findings raise questions about the role of isolated 3' UTR sequences in regulation of protein expression and highlight the importance of separately examining 3' UTR and CDS sequences in gene expression analyses.
View details for DOI 10.1016/j.neuron.2015.10.048
View details for Web of Science ID 000368443900011
View details for PubMedID 26687222
Evidence for topographic guidance of dopaminergic axons by differential Netrin-1 expression in the striatum
MOLECULAR AND CELLULAR NEUROSCIENCE
2014; 61: 85-96
There are two main subgroups of midbrain dopaminergic (DA) neurons: the more medially located ventral tegmental area (VTA) DA neurons, which have axons that innervate the ventral-lateral (VL) striatum, and the more laterally located substantia nigra (SN) DA neurons, which preferentially degenerate in Parkinson's disease (PD) and have axons that project to the dorsal-medial (DM) striatum. DA axonal projections in the striatum are not discretely localized and they arborize widely, however they do not stray from one zone to the other so that VTA axons remain in the VL zone and SN axons in the DM zone. Here we provide evidence that Netrin-1 acts in a novel fashion to topographically pattern midbrain DA axons into these two striatal zones by means of a gradient of Netrin-1 in the striatum and by differential attraction of the axons to Netrin-1. Midbrain DA neurons are attracted to the striatum in culture and this attraction is blocked by an anti-DCC (Netrin receptor) antibody. Mechanistically, outgrowth of both VTA and SN DA axons is stimulated by Netrin-1, but the two populations of DA axons respond optimally to overlapping but distinct concentrations of Netrin-1, with SN axons preferring lower concentrations and VTA axons preferring higher concentrations. In vivo this differential preference is closely mirrored by differences in Netrin-1 expression in their respective striatal target fields. In vivo in mice lacking Netrin-1, DA axons that reach the striatum fail to segregate into two terminal zones and to fully innervate the striatum. Our results reveal novel actions for Netrin-1 and provide evidence for a mechanism through which DA axons can selectively innervate one of two terminal zones in the striatum but have free reign to arborize widely within a terminal zone.
View details for DOI 10.1016/j.mcn.2014.05.003
View details for Web of Science ID 000340690400009
View details for PubMedID 24867253
Characterization of axon guidance cue sensitivity of human embryonic stem cell-derived dopaminergic neurons
MOLECULAR AND CELLULAR NEUROSCIENCE
2010; 45 (4): 324-334
Dopaminergic neurons derived from human embryonic stem cells will be useful in future transplantation studies of Parkinson's disease patients. As newly generated neurons must integrate and reconnect with host cells, the ability of hESC-derived neurons to respond to axon guidance cues will be critical. Both Netrin-1 and Slit-2 guide rodent embryonic dopaminergic (DA) neurons in vitro and in vivo, but very little is known about the response of hESC-derived DA neurons to any axonal guidance cues. Here we examined the ability of Netrin-1 and Slit-2 to affect human ESC DA axons in vitro. hESC DA neurons mature over time in culture with the developmental profile of DA neurons in vivo, including expression of the DA neuron markers FoxA2, En-1 and Nurr-1, and receptors for both Netrin and Slit. hESC DA neurons respond to exogenous Netrin-1 and Slit-2, showing an increased responsiveness to Netrin-1 as the neurons mature in culture. These responses were maintained in the presence of pro-inflammatory cytokines that might be encountered in the diseased brain. These studies are the first to evaluate and confirm that suitably matured human ES-derived DA neurons can respond appropriately to axon guidance cues.
View details for DOI 10.1016/j.mcn.2010.07.004
View details for Web of Science ID 000283970100002
View details for PubMedID 20637284
Gene targeting using a promoterless gene trap vector ("targeted trapping") is an efficient method to mutate a large fraction of genes
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2005; 102 (37): 13188-13193
A powerful tool for postgenomic analysis of mammalian gene function is gene targeting in mouse ES cells. We report that homologous recombination using a promoterless gene trap vector ("targeting trapping") yields targeting frequencies averaging above 50%, a significant increase compared with current approaches. These high frequencies appear to be due to the stringency of selection with promoterless constructs, because most random insertions are silent and eliminated by drug selection. The promoterless design requires that the targeted gene be expressed in ES cells at levels exceeding a certain threshold (which we estimate to be approximately 1% of the transferrin receptor gene expression level, for the secretory trap vector used here). Analysis of 127 genes that had been trapped by random (nontargeted) gene trapping with the same vector shows that virtually all are expressed in ES cells above this threshold, suggesting that targeted and random trapping share similar requirements for expression levels. In a random sampling of 130 genes encoding secretory proteins, about half were expressed above threshold, suggesting that about half of all secretory genes are accessible by either targeted or random gene trapping. The simplicity and high efficiency of the method facilitate systematic targeting of a large fraction of the genome by individual investigators and large-scale consortia alike.
View details for DOI 10.1073/pnas.0505474102
View details for Web of Science ID 000231916300034
View details for PubMedID 16129827
View details for PubMedCentralID PMC1193537
- Embryonic stern cells go dopaminergic NEURON 2000; 28 (1): 11–14
Broad specificity of GDNF family receptors GFR alpha 1 and GFR alpha 2 for GDNF and NTN in neurons and transfected cells
JOURNAL OF NEUROSCIENCE RESEARCH
2000; 61 (1): 1–9
The glial cell line-derived neurotrophic factor (GDNF) family of ligands binds to lipid anchored proteins termed GDNF family receptor (GFR)alphas, and then activates the RET receptor tyrosine kinase, by ligand GFRalpha. The binding of soluble GFRalphas to transfected cells suggested that different GFRalphas were dedicated to particular ligands, with GDNF acting primarily or entirely through GFRalpha1, and neurturin (NTN), through GFRalpha2. More recent evidence has suggested the possibility of cross-talk between these ligands and the two receptors. We examined here whether crosstalk between the GDNF ligands and the GFRalphas is biologically relevant, using midbrain dopaminergic, and parasympathetic, submandibular gland neurons. By biochemical and genetic addition and/or deletion of GFRalpha1 and 2, we show that in both neuronal cell types, robust biological activities of GDNF or NTN can be mediated by either GFRalpha1 or GFRalpha2, although GDNF is slightly more potent in dopaminergic (DA) neurons which normally express GFRalpha1, and NTN in submandibular neurons which normally express GFRalpha2. Throughout the body, GDNF and NTN are likely to have important biological actions on both GFRalpha1- and GFRalpha2-expressing cells.
View details for Web of Science ID 000087787400001
View details for PubMedID 10861794
Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system
2000; 25 (1): 239-252
alpha-Synuclein (alpha-Syn) is a 14 kDa protein of unknown function that has been implicated in the pathophysiology of Parkinson's disease (PD). Here, we show that alpha-Syn-/- mice are viable and fertile, exhibit intact brain architecture, and possess a normal complement of dopaminergic cell bodies, fibers, and synapses. Nigrostriatal terminals of alpha-Syn-/- mice display a standard pattern of dopamine (DA) discharge and reuptake in response to simple electrical stimulation. However, they exhibit an increased release with paired stimuli that can be mimicked by elevated Ca2+. Concurrent with the altered DA release, alpha-Syn-/- mice display a reduction in striatal DA and an attenuation of DA-dependent locomotor response to amphetamine. These findings support the hypothesis that alpha-Syn is an essential presynaptic, activity-dependent negative regulator of DA neurotransmission.
View details for Web of Science ID 000085043200025
View details for PubMedID 10707987
The seven-transmembrane receptor Smoothened cell-autonomously induces multiple ventral cell types
2000; 3 (1): 41–46
Sonic Hedgehog (Shh) is a secreted protein that controls cell fate and mitogenesis in the developing nervous system. Here we show that a constitutively active form of Smoothened (Smo-M2) mimics concentration-dependent actions of Shh in the developing neural tube, including activation of ventral marker genes (HNF3beta, patched, Nkx2.2, netrin-1), suppression of dorsal markers (Pax-3, Gli-3, Ephrin A5) and induction of ventral neurons (dopaminergic, serotonergic) and ventrolateral motor neurons (Islet-1+, Islet-2+, HB9+) and interneurons (Engrailed-1+, CHX10+). Furthermore, Smo-M2's patterning activities were cell autonomous, occurring exclusively in cells expressing Smo-M2. These findings suggest that Smo is a key signaling component in the Hh receptor and that Shh patterns the vertebrate nervous system as a morphogen, rather than through secondary relay signals.
View details for DOI 10.1038/71114
View details for Web of Science ID 000085810200010
View details for PubMedID 10607393
Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system
1999; 126 (12): 2785–97
RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5-15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.
View details for Web of Science ID 000081366200019
View details for PubMedID 10331988
Neurturin exerts potent actions on survival and function of midbrain dopaminergic neurons
JOURNAL OF NEUROSCIENCE
1998; 18 (13): 4929–37
Glial cell line-derived neurotrophic factor (GDNF) exhibits potent effects on survival and function of midbrain dopaminergic (DA) neurons in a variety of models. Although other growth factors expressed in the vicinity of developing DA neurons have been reported to support survival of DA neurons in vitro, to date none of these factors duplicate the potent and selective actions of GDNF in vivo. We report here that neurturin (NTN), a homolog of GDNF, is expressed in the nigrostriatal system, and that NTN exerts potent effects on survival and function of midbrain DA neurons. Our findings indicate that NTN mRNA is sequentially expressed in the ventral midbrain and striatum during development and that NTN exhibits survival-promoting actions on both developing and mature DA neurons. In vitro, NTN supports survival of embryonic DA neurons, and in vivo, direct injection of NTN into the substantia nigra protects mature DA neurons from cell death induced by 6-OHDA. Furthermore, administration of NTN into the striatum of intact adult animals induces behavioral and biochemical changes associated with functional upregulation of nigral DA neurons. The similarity in potency and efficacy of NTN and GDNF on DA neurons in several paradigms stands in contrast to the differential distribution of the receptor components GDNF Family Receptor alpha1 (GFRalpha1) and GFRalpha2 within the ventral mesencephalon. These results suggest that NTN is an endogenous trophic factor for midbrain DA neurons and point to the possibility that GDNF and NTN may exert redundant trophic influences on nigral DA neurons acting via a receptor complex that includes GFRalpha1.
View details for Web of Science ID 000074290000012
View details for PubMedID 9634558
View details for PubMedCentralID PMC6792569
GFR alpha 1 is an essential receptor component for GDNF in the developing nervous system and kidney
1998; 21 (1): 53–62
Glial cell line-derived neurotrophic factor (GDNF) is a distant member of the TGFbeta protein family that is essential for neuronal survival and renal morphogenesis. We show that mice who are deficient in the glycosyl-phosphatidyl inositol (GPI) -linked protein GFRalpha1 (GDNFRalpha) display deficits in the kidneys, the enteric nervous system, and spinal motor and sensory neurons that are strikingly similar to those of the GDNF- and Ret-deficient mice. GFRalpha1-deficient dopaminergic and nodose sensory ganglia neurons no longer respond to GDNF or to the structurally related protein neurturin (NTN) but can be rescued when exposed to GDNF or neurturin in the presence of soluble GFRalpha1. In contrast, GFRalpha1-deficient submandibular parasympathetic neurons retain normal response to these two factors. Taken together with the available genetic and biochemical data, these findings support the idea that GFRalpha1 and the transmembrane tyrosine kinase Ret are both necessary receptor components for GDNF in the developing kidney and nervous system, and that GDNF and neurturin can mediate some of their activities through a second receptor.
View details for DOI 10.1016/S0896-6273(00)80514-0
View details for Web of Science ID 000075061900006
View details for PubMedID 9697851
View details for PubMedCentralID PMC2710137
FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate
1998; 93 (5): 755-766
During development, distinct classes of neurons are specified in precise locations along the dorso-ventral and anterior-posterior axes of the neural tube. We provide evidence that intersections of Shh, which is expressed along the ventral neural tube, and FGF8, which is locally produced at the mid/hindbrain boundary and in the rostral forebrain, create induction sites for dopaminergic neurons in the midbrain and forebrain. The same intersection, when preceded by a third signal, FGF4, which is expressed in the primitive streak, defines an inductive center for hindbrain 5-HT neurons. These findings illustrate that cell patterning in the neural plate is a multistep process in which early inducers, which initially divide the neural plate into crude compartments, are replaced by multiple local organizing centers, which specify distinct neuronal cell types within these compartments.
View details for Web of Science ID 000073956700012
View details for PubMedID 9630220
Persephin, a novel neurotrophic factor related to GDNF and neurturin
1998; 20 (2): 245–53
A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.
View details for DOI 10.1016/S0896-6273(00)80453-5
View details for Web of Science ID 000072154700010
View details for PubMedID 9491986
Activating Smoothened mutations in sporadic basal-cell carcinoma
1998; 391 (6662): 90–92
Basal-cell carcinomas (BCCs) are the commonest human cancer. Insight into their genesis came from identification of mutations in the PATCHED gene (PTCH) in patients with the basal-cell nevus syndrome, a hereditary disease characterized by multiple BCCs and by developmental abnormalities. The binding of Sonic hedgehog (SHH) to its receptor, PTCH, is thought to prevent normal inhibition by PTCH of Smoothened (SMO), a seven-span transmembrane protein. According to this model, the inhibition of SMO signalling is relieved following mutational inactivation of PTCH in basal-cell nevus syndrome. We report here the identification of activating somatic missense mutations in the SMO gene itself in sporadic BCCs from three patients. Mutant SMO, unlike wild type, can cooperate with adenovirus E1A to transform rat embryonic fibroblast cells in culture. Furthermore, skin abnormalities similar to BCCs developed in transgenic murine skin overexpressing mutant SMO. These findings support the role of SMO as a signalling component of the SHH-receptor complex and provide direct evidence that mutated SMO can function as an oncogene in BCCs.
View details for Web of Science ID 000071326100055
View details for PubMedID 9422511
Control of cell pattern in the neural tube by the zinc finger transcription factor and oncogene Gli-1
1997; 19 (1): 15–26
Sonic hedgehog (Shh) is a putative morphogen secreted by the floor plate and notochord, which specifies the fate of multiple cell types in the ventral aspect of the vertebrate nervous system. Since in Drosophila the actions of Hh have been shown to be transduced by Cubitus interruptus (Ci), a zinc finger transcription factor, we examined whether a vertebrate homolog of this protein can mediate the functions of Shh in the vertebrate nervous system. Here, we demonstrate that expression of Gli-1, one of three vertebrate homologs of Ci, can be induced by Shh in the neural tube. Further, ectopic expression of Gli-1 in the dorsal midbrain and hindbrain of transgenic mice mimics the effects of ectopically expressed Shh-N, leading to the activation of ventral neural tube markers such as Ptc, HNF-3beta, and Shh; to the suppression of dorsal markers such as Pax-3 and AL-1; and to the formation of ectopic dorsal clusters of dopaminergic and serotonergic neurons. These findings demonstrate that GLI-1 can reproduce the cell patterning actions of Shh in the developing nervous system and provide support for the hypothesis that it is a mediator of the Shh signal in vertebrates.
View details for DOI 10.1016/S0896-6273(00)80344-X
View details for Web of Science ID A1997XN39400003
View details for PubMedID 9247260
The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog
1996; 384 (6605): 129-134
The protein Sonic hedgehog (Shh) controls patterning and growth during vertebrate development. Here we demonstrate that it binds Patched (vPtc), which has been identified as a tumour-suppressor protein in basal cell carcinoma, with high affinity. We show that Ptc can form a physical complex with a newly cloned vertebrate homologue of the Drosophila protein Smoothened (vSmo), and that vSmo is coexpressed with vPtc in many tissues but does not bind Shh directly. These findings, combined with available genetic evidence from Drosophila, support the hypothesis that Ptc is a receptor for Shh, and that vSmo could be a signalling component that is linked to Ptc.
View details for Web of Science ID A1996VT33600055
View details for PubMedID 8906787
INDUCTION OF MIDBRAIN DOPAMINERGIC-NEURONS BY SONIC HEDGEHOG
1995; 15 (1): 35-44
Midbrain dopaminergic neurons, whose loss in adults results in Parkinson's disease, can be specified during embryonic development by a contact-dependent signal from floor plate cells. Here we show that the amino-terminal product of Sonic hedgehog autoproteolysis (SHH-N), an inductive signal expressed by floor plate cells, can induce dopaminergic neurons in vitro. We show further that manipulations to increase the activity of cyclic AMP-dependent protein kinase A, which is known to antagonize hedgehog signaling, can block dopaminergic neuron induction by floor plate cells. Our results and those of other studies indicate that SHH-N can function in a dose-dependent manner to induce different cell types within the neural tube. Our results also provide the basis for a potential cell transplantation therapy for Parkinson's disease.
View details for Web of Science ID A1995RL75600007
View details for PubMedID 7619528
CONTROL OF NEURONAL DIVERSITY BY THE FLOOR PLATE - CONTACT-MEDIATED INDUCTION OF MIDBRAIN DOPAMINERGIC-NEURONS
1995; 80 (1): 95-101
The notochord and floor plate contribute to patterning the ventral neural tube in part by expressing a diffusible factor that induces motoneurons. To determine the mechanisms that direct the development of other classes of ventral neurons, we studied the development of dopaminergic neurons that reside near motoneurons in the ventral midbrain. We provide evidence that dopaminergic neurons develop in the vicinity of the floor plate and that they can be specified by the floor plate in vitro and in vivo. Unlike motoneurons, efficient induction of dopaminergic neurons requires contact with floor plate cells. These results suggest that neuronal diversification along the dorsal-ventral axis may be achieved partly through the concerted action of diffusible and contact-dependent signals from a single organizing center, the floor plate.
View details for Web of Science ID A1995QB91000013
View details for PubMedID 7813022
TGF-BETA-2 AND TCF-BETA-3 ARE POTENT SURVIVAL FACTORS FOR MIDBRAIN DOPAMINERGIC-NEURONS
1994; 13 (5): 1245–52
The vertebrate ventral midbrain contains 3-4 x 10(4) dopaminergic neurons that influence motor activity, emotional behavior, and cognition. Recently, glial cell line-derived neurotrophic factor (GDNF) was shown to be a potent survival factor for these dopaminergic neurons in culture. However, many midbrain dopaminergic neurons project to targets that do not express GDNF. We report here that transforming growth factors (TGFs) TGF beta 2 and TGF beta 3, which are distantly related to GDNF, also prevent the death of cultured rat embryonic midbrain dopaminergic neurons at picomolar concentrations. Furthermore, we find that TGF beta 2, TGF beta 3, and GDNF are expressed sequentially as local and target-derived trophic factors and that subpopulations of dopaminergic neurons projecting to distinct targets have access to only one of these factors. These findings are consistent with the idea that GDNF, TGF beta 2, and TGF beta 3 are physiological survival factors for developing midbrain dopaminergic neurons and may have applications as therapeutics for Parkinson's disease, a neurodegenerative disorder of dopaminergic neurons.
View details for DOI 10.1016/0896-6273(94)90062-0
View details for Web of Science ID A1994PU76300020
View details for PubMedID 7946360
TAG-1 CAN MEDIATE HOMOPHILIC BINDING, BUT NEURITE OUTGROWTH ON TAG-1 REQUIRES AN L1-LIKE MOLECULE AND BETA-1 INTEGRINS
1994; 12 (3): 675–90
Subsets of axons in the embryonic nervous system transiently express the glycoprotein TAG-1, a member of the subfamily of immunoglobulin (Ig)-like proteins that contain both C2 class Ig and fibronectin type III domains. TAG-1 is attached to the cell surface by a glycosylphosphatidylinositol linkage and is secreted by neurons. In vitro studies have shown that substrate-bound TAG-1 promotes neurite outgrowth. We have examined the nature of axonal receptors that mediate the neurite-outgrowth promoting properties of TAG-1. Although TAG-1 can mediate homophilic binding, neurite outgrowth on a substrate of TAG-1 does not depend on the presence of TAG-1 on the axonal surface. Instead, neurite outgrowth on TAG-1 is inhibited by polyclonal antibodies directed against L1 and, independently, by polyclonal and monoclonal antibodies against beta 1-containing integrins. These results provide evidence that TAG-1 can interact with cell surfaces in both a homophilic and heterophilic manner and suggest that neurite extension on TAG-1 requires the function of both integrins and an L1-like molecule.
View details for DOI 10.1016/0896-6273(94)90222-4
View details for Web of Science ID A1994NC76900018
View details for PubMedID 7512353
NEUROTROPHIN-4/5 IS A SURVIVAL FACTOR FOR EMBRYONIC MIDBRAIN DOPAMINERGIC-NEURONS IN ENRICHED CULTURES
JOURNAL OF NEUROSCIENCE RESEARCH
1994; 37 (1): 144–54
Parkinson's disease is a prevalent neurological disease characterized by profound and incapacitating movement disorders. A common pathology in Parkinson's patients is degeneration of substantia nigra dopaminergic neurons that innervate the striatum and a corresponding decrease in striatal dopamine content. We now report that NT-4/5 can prevent the death of rat embryonic substantia nigra dopaminergic neurons in low density, enriched, primary cultures. Furthermore, these neurons express messenger RNA encoding the trkB receptor for NT-4/5 and transcripts for NT-4/5 are present in their environment. In addition, we demonstrate that NT-4/5 protects embryonic dopaminergic neurons from the toxic effects of the neurotoxin MPP+. Thus, NT-4/5 could be a physiological survival factor for midbrain dopaminergic neurons and may be useful as a therapeutic agent for Parkinson's disease.
View details for DOI 10.1002/jnr.490370118
View details for Web of Science ID A1994MR52400016
View details for PubMedID 7908342
ONTOGENY OF GLUCAGON MESSENGER-RNA AND ENCODED PRECURSOR IN THE RAT INTESTINE
1990; 29 (2-3): 117–31
The ontogeny of proglucagon mRNA and encoded precursor was studied in rat intestine from day 11 of fetal gestation (E11) to maturity. The earliest time point for detection of proglucagon antigenic determinants by immunocytochemistry, and of proglucagon mRNA by in situ hybridization histochemistry, was day 14 of fetal gestation (E14), suggesting this time as the point of onset of intestinal proglucagon gene expression and mRNA translation. Between day 17 and 18 of gestation (E17 and E18) there was a significant 10 fold increase in intestinal L cell density, indicating that this time in gestation is one of increased L cell differentiation and/or proliferation. Proglucagon mRNA abundance in developing rat intestine showed a major 8 fold increase between E17 and E18. Similar magnitude of increases in L cell density and proglucagon mRNA abundance suggests that the increase in proglucagon mRNA abundance reflects an increase in L cell numbers rather than increases in proglucagon gene transcription or mRNA stability per cell.
View details for Web of Science ID A1990DT60200006
View details for PubMedID 2217900
SELECTIVE EXPRESSION OF AN ENDOGENOUS LACTOSE-BINDING LECTIN GENE IN SUBSETS OF CENTRAL AND PERIPHERAL NEURONS
JOURNAL OF NEUROSCIENCE
1990; 10 (3): 1004–13
Cellular interactions in a variety of vertebrate non-neural tissues are thought to be mediated by cell surface carbohydrate structures. The detection of cell-specific surface carbohydrates and carbohydrate-binding proteins within the embryonic nervous system has raised the possibility that carbohydrate recognition may also contribute to the interactions of developing neurons. Soluble lactose-binding lectins constitute one class of carbohydrate-binding proteins expressed in the vertebrate nervous system. We describe here the isolation of cDNAs from rat brain libraries encoding one of these lectins, RL-14.5, and demonstrate that this protein is not only homologous to other soluble lectins, but also identical in primary sequence to a lectin present in at least one non-neural tissue. RNA blot analysis and in situ hybridization reveal a restricted pattern of expression of RL-14.5 mRNA within the rat nervous system. High levels of RL-14.5 mRNA are present in primary sensory neurons and motoneurons in the spinal cord and brain stem. Moreover, expression of RL-14.5 mRNA in sensory and motoneurons is detectable soon after neuronal differentiation. These findings, together with previous studies demonstrating the selective expression of oligosaccharide ligands for RL-14.5 on the same neurons, are consistent with the idea that carbohydrate-mediated interactions contribute to the development of this subset of mammalian neurons.
View details for Web of Science ID A1990CV82500026
View details for PubMedID 2319298
View details for PubMedCentralID PMC6570114
- In situ hybridization using 32P labelled oligodeoxyribonucleotides for the cellular localization of mRNA in neuronal and endocrine tissue. An analysis of procedural variables. Histochemistry 1988
INSULIN-LIKE GROWTH FACTOR-II MESSENGER RIBONUCLEIC-ACIDS ARE SYNTHESIZED IN THE CHOROID-PLEXUS OF THE RAT-BRAIN
1988; 2 (1): 47–54
Previous studies demonstrating the presence of immunoreactive insulin-like growth factors (IGFs) and their receptors in the brain suggest a role of the IGFs in the central nervous system. IGF-II has been implicated as the predominant IGF in brain of mature animals based on studies of immunoreactive peptide and of IGF-II mRNAs. To obtain information about the sites of synthesis of IGF-II in adult rat brain, a 32P-labeled 31 base long synthetic oligodeoxyribonucleotide complementary in sequence to trailer peptide coding sequences in rat IGF-II mRNA (IGF-II 31 mer) was hybridized with coronal sections of fixed rat brain. The IGF-II 31 mer showed specific hybridization with the choroid plexus throughout rat brain, whereas in other brain regions, structures or cells, hybridization was not discernibly above background. These findings suggest that the choroid plexus is a primary site of synthesis of IGF-II, a probable source of IGF-II in cerebrospinal fluid, and a potential source of IGF-II for actions on target cells within the adult rat brain.
View details for DOI 10.1210/mend-2-1-47
View details for Web of Science ID A1988M124600006
View details for PubMedID 3398842
- Characterization, localization and regulation of extrapancreatic proglucagon mRNAs. Biomedical Research 1988
ISOLATION OF RAT TESTIS CDNAS ENCODING AN INSULIN-LIKE GROWTH FACTOR-I PRECURSOR
DNA-A JOURNAL OF MOLECULAR & CELLULAR BIOLOGY
1987; 6 (4): 325–30
We have characterized rat testis cDNAs encoding insulin-like growth factor I (IGF-I) precursor to facilitate studies of IGF-I expression in the male reproductive system. Two clones, P2 and P3, with inserts of 786 and 1200 bp, respectively, were isolated from a lambda gt11 library of rat testis cDNAs. The longest open reading frame of cDNA P2 predicts a 153-amino-acid residue IGF-I precursor that has only 11 amino acid substitutions compared with a human IGF-IA precursor encoded by a human liver mRNA. Three substitutions are within the predicted rat IGF-I sequence: a Pro for Asp in the B domain, an Ile for Ser in the C domain, and Thr for Ala in the D domain. Only two substitutions distinguish the predicted rat sequence from a mouse liver IGF-IA precursor: Thr for Ala in the signal peptide and Ala for Ser in the D domain. P2 hybridizes with poly(A)+ mRNAs of 7.5, 4.7, 1.7, and 1.2-0.9 kb in rat liver and testis. The other testis cDNA, P3, appears to represent a partially processed rat IGF-I mRNA precursor. By comparing the sequence of cDNA P2 with that of cDNA P3 and a 2.3-kb rat IGF-I genomic fragment, we predict exon splice sites within the codon for residue 26 and between residues 86-87 of the rat IGF-I precursor. Both of the predicted splice sites align with exon-intron junctions in the human IGF-I gene. We conclude, therefore, that IGF-I is synthesized as a precursor in the rat testis and that the structure of IGF-I genes, mRNAs, and precursors are highly conserved across species.
View details for DOI 10.1089/dna.1987.6.325
View details for Web of Science ID A1987J730900005
View details for PubMedID 3652906
GROWTH-HORMONE DEPENDENCE OF SOMATOMEDIN-C INSULIN-LIKE GROWTH FACTOR-I AND INSULIN-LIKE GROWTH FACTOR-II MESSENGER RIBONUCLEIC-ACIDS
1987; 1 (3): 233–42
The GH dependence of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) and insulin like growth factor II (IGF-II) mRNAs was investigated by Northern blot hybridizations of polyadenylated RNAs from liver, pancreas, and brain of normal rats, untreated hypophysectomized rats, and hypophysectomized rats 4 h or 8 h after an ip injection of human GH (hGH). Using a 32P-labeled human Sm-C/IGF-I cDNA as probe, four Sm-C/IGF-I mRNAs of 7.5, 4.7, 1.7, and 1.2 kilobases (kb) were detected in rat liver and pancreas but were not detectable in brain. In both liver and pancreas, the abundance of these Sm-C/IGF-I mRNAs was 8- to 10-fold lower in hypophysectomized rats than in normal rats. Within 4 h after injection of hGH into hypophysectomized animals, the abundance of liver and pancreatic Sm-C/IGF-I mRNAs was restored to normal. A human IGF-II cDNA was used as a probe for rat IGF-II mRNAs which were found to be very low in abundance in rat liver and showed no evidence of regulation by GH status. In pancreas, IGF-II mRNA abundance was below the detection limit of the hybridization procedures. The brain contained two IGF-II mRNAs of 4.7 and 3.9 kb that were 5-fold lower in abundance in hypophysectomized rats than in normal rats. These brain IGF-II mRNAs were not, however, restored to normal abundance at 4 or 8 h after ip hGH injection into hypophysectomized animals. To investigate further, the effect of GH status on abundance of Sm-C/IGF-I and IGF-II mRNAs in rat brain, a second experiment was performed that differed from the first in that hypophysectomized rats were given an injection of hGH into the lateral ventricle (intracerebroventricular injection) and a rat Sm-C/IGF-I genomic probe was used to analyze Sm-C/IGF-I mRNAs. In this experiment, a 7.5 kb Sm-C/IGF-I mRNA was detected in brain polyadenylated RNAs. The abundance of the 7.5 kb mRNA was 4-fold lower in hypophysectomized rats than in normal rats and was increased to 80% of normal within 4 h after icv administration of hGH to hypophysectomized animals. As in the first experiment, the abundance of the 4.7 and 3.9 kb brain IGF-II mRNAs was lower than normal in hypophysectomized rats. Brain IGF-II mRNAs were increased to 50% of normal in hypophysectomized rats given an icv injection of hGH but within 8 h after the injection rather than at 4 h as with Sm-C/IGF-I mRNAs.
View details for DOI 10.1210/mend-1-3-233
View details for Web of Science ID A1987K944400004
View details for PubMedID 3453890
INVOLVEMENT OF N-METHYL-D-ASPARTATE RECEPTORS IN EPILEPTIFORM BURSTING IN THE RAT HIPPOCAMPAL SLICE
JOURNAL OF PHYSIOLOGY-LONDON
1986; 380: 175–89
The effects of the N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosphonovaleric acid (D-APV), and other excitatory amino acid antagonists, were studied on CA1 pyramidal neurones treated with picrotoxin or bicuculline to reduce synaptic inhibition mediated by gamma-aminobutyric acid (GABA). Under these conditions epileptiform burst firing is readily produced by orthodromic stimulation of the pyramidal cell population. D-APV reduced the plateau amplitude and duration of the depolarization underlying evoked and spontaneous bursts without affecting membrane potential, input resistance or the ability of the cell to fire a Ca2+ spike or a short train of Na+ spikes. A late component of the subthreshold excitatory post-synaptic potential (e.p.s.p.) was voltage dependent, being reduced in amplitude on membrane hyperpolarization. D-APV selectively removed this component of the e.p.s.p. in disinhibited slices. In contrast, in the absence of GABA antagonists, D-APV had no noticeable effect on the e.p.s.p. as studied with field potential recordings. The concentration-response relationship of the inhibitory effect of D-APV and L-APV on population spike bursts was studied. The action of APV was highly stereoselective; the EC50 of D-APV was approximately 700 nM, whereas a similar inhibition required 540 microM-L-APV. A number of other excitatory amino acid antagonists were tested at a fixed concentration (100 microM). Among them, the quisqualate antagonist gamma-D-glutamylaminomethyl sulphonic acid was ineffective against epileptiform bursts. In the low nanomolar concentration range both D- and L-APV potentiated bursting. These results suggest that in the absence of GABAergic inhibition, a significant component of the slow depolarization underlying burst firing is voltage dependent, synaptic in origin and mediated by NMDA receptors. We propose that, under normal (non-epileptic) physiological conditions, the balance between synaptic inhibition mediated by GABA receptors and synaptic excitation mediated by NMDA receptors may modulate the excitability of pyramidal cell dendrites.
View details for DOI 10.1113/jphysiol.1986.sp016279
View details for Web of Science ID A1986E981200012
View details for PubMedID 2886653
View details for PubMedCentralID PMC1182931
- Somatomedin-C/IGF-I and IGF-II mRNAs in rat fetal and adult tissues Journal of Biological Chemistry 1986
- Cellular localization of Proglucagon/Glucagon -like peptide I mRNAs in Rat Brain Journal of Neuroscience Research 1986
DIRECT ACTION OF MAZINDOL ON GUINEA-PIG VENTROMEDIAL HYPOTHALAMIC NEURONS - INTRACELLULAR STUDIES IN SLICE PREPARATION
BRAIN RESEARCH BULLETIN
1985; 15 (1): 29–31
Neuronal responses in the ventromedial hypothalamic nucleus (VMH) to mazindol (MZD) were examined by intracellular recordings in hypothalamic slices in vitro. Bath application of MZD caused depolarization of the membrane by 8.0 +/- 3.4 mV (mean +/- S.D.) associated with an increase in the input membrane resistance (23.0 +/- 5.8%) among 20% of the cells examined. Current-voltage plots, obtained simultaneously, showed the reversal potential between -80 and -90 mV. These results indicate that MZD depolarizes VMH neurons by reducing the K+ conductance, through a mechanism similar to that of glucose, and the anorectic action of MZD can be explained by its direct effects on the VMH.
View details for DOI 10.1016/0361-9230(85)90057-7
View details for Web of Science ID A1985ANX5400005
View details for PubMedID 4027704
CORTICOTROPIN RELEASING-FACTOR (CRF) - ORIGIN AND COURSE OF AFFERENT PATHWAYS TO THE MEDIAN-EMINENCE (ME) OF THE RAT HYPOTHALAMUS
1984; 39 (4): 296–306
8-10 days after making various lesions in the rat hypothalamus, the presence of corticotropin releasing factor (CRF) immunoreactive neural structures was studied in paraffin and vibratome sections with CRF immunocytochemistry. Bilateral anterolateral deafferentation of the medial basal hypothalamus (MBH) caused complete disappearance of CRF immunoreactivity from the median eminence (ME) in brains where the posterior edge of the cut reached the level of the pituitary stalk. A shorter cut resulted in positive immunostaining caudal to the caudal edge of the cut. Unilateral deafferentation of the MBH caused significant decrease in CRF immunostaining in the ipsilateral ME. Unilateral posterolateral deafferentation of the MBH caused no changes in CRF immunostaining in the rostral ME, while fewer CRF-containing processes were observed in the more caudal regions. A horizontal cut ventral to the paraventricular nuclei (PVN) caused a slight decrease in the number of CRF-immunoreactive profiles in the ME. A wider and complete unilateral horizontal cut resulted in a significant decrease in CRF immunoreactivity on the operated side. Following various surgical interventions, hormone accumulation in cell bodies was detected in the paraventricular, periventricular preoptic, dorsomedial, periventricular, lateral and posterior hypothalamic, and premammillary nuclei. Fibers arising from most of these nuclei formed a fan-like projection to the ME. The majority of the CRF-fibers ran through the lateral tract of the fan, and reached the ME by the lateral-basal retrochiasmatic area (LBRCA). Scattered fibers were detected in the lateral-basal hypothalamus as far caudally as the level of the pituitary stalk. Unilateral anterolateral and horizontal cuts did not result in complete disappearance of CRF immunoreactivity from the ipsilateral ME, indicating the existence of CRF-fibers of contralateral origin in the ME.
View details for DOI 10.1159/000123996
View details for Web of Science ID A1984TH93400002
View details for PubMedID 6387524
CHOLINERGIC ROLE IN MONKEY DORSOLATERAL PREFRONTAL CORTEX DURING BAR-PRESS FEEDING-BEHAVIOR
1983; 278 (1-2): 185–94
Cholinergic involvement in the neuronal activity of the dorsolateral (DL) prefrontal cortex in the monkey was investigated during bar-press feeding behavior. Iontophoretic application of ACh increased the firing rate of more than half of the cells through muscarinic receptors. Activity of ACh-sensitive cells did not correlate with any particular event during the feeding task. Continuous application of ACh markedly enhanced increases in response to events during the feeding task, and application of an ACh antagonist diminished response levels. This indicates that ACh release may occur during the feeding task and affect cortical cells to improve the signal-to-noise ratio of the excitatory input. Decreased responses to events during the feeding task diminished during ACh application. Driven discharges in the cells were evoked by stimulation of the basal nucleus of Meynert (BNM) where ACh containing cells are localized. This response was specifically blocked by iontophoretic application of atropine. In conclusion, cholinergic inputs arising from the BNM are distributed profusely among the cortical cells and modulate their excitability during bar-press feeding behavior.
View details for DOI 10.1016/0006-8993(83)90237-8
View details for Web of Science ID A1983RQ57100017
View details for PubMedID 6640306
IMMUNOCYTOCHEMICAL LOCALIZATION OF CORTICOTROPIN RELEASING-FACTOR (CRF) IN THE RAT SPINAL-CORD
1983; 275 (2): 373–77
The presence of corticotropin releasing factor (CRF)-immunoreactive nerve fibers and cell bodies in the spinal cord is demonstrated. Immunopositive fibers were found in the lateral column of the white matter, in laminae I, V-VII, X, and in the intermediolateral column of the spinal cord. Complete transection of the spinal cord showed that the majority of the fibers in the lateral funiculus formed an ascending pathway; however, a few descending fibers were also detected. Hypophysectomy resulted in enhanced immunoreactivity of the fibers and staining of CRF-immunoreactive cell bodies in laminae V-VII, X, and in the intermediolateral sympathetic column. The results suggest that CRF is not merely an ACTH releasing factor, but also a regulatory peptide which may be involved in several stress-related neural responses.
View details for DOI 10.1016/0006-8993(83)91001-6
View details for Web of Science ID A1983RH85300019
View details for PubMedID 6194860
- SYSTEMIC AND INTRAVENTRICULAR NALOXONE ADMINISTRATION - EFFECTS ON FOOD AND WATER-INTAKE BEHAVIORAL AND NEURAL BIOLOGY 1981; 32 (3): 334–42