Paulette Bernd

GABAergic and cholinergic neurons exhibit high-affinity nerve growth factor binding in rat basal forebrain

We have used dissociated, rat basal forebrain cultures to identify specific cell types that are potentially responsive to nerve growth factor (NGF). Expression of high-affinity NGF binding sites was examined. A subpopulation of cells containing choline acetyltransferase (CAT), the acetylcholine-synthesizing enzyme, exhibited high-affinity binding, employing combined immunocytochemistry and 125I-NGF radioautography. Unexpectedly, a gamma-aminobutyric acid (GABA)-containing cell group also expressed high-affinity binding. These cells that exhibit high-affinity binding appear to be neurons since they stain positively with the neuron marker, neuron-specific enolase, and negatively with the nonneuron marker, glial fibrillary acidic protein. Our observations suggest that NGF may regulate multiple brain systems and functions that have yet to be explored. Conversely, only subsets of cholinergic or GABA neurons expressed high-affinity binding, suggesting that these transmitter populations are composed of differentially responsive subpopulations.

Nerve growth factor and serum differentially regulate development of the embryonic otic vesicle and cochleovestibular ganglion in vitro.

The preceding paper (P. Bernd and J. Represa, 1989, Dev. Biol. 134) describes the characterization and localization of nerve growth factor (NGF) receptors in inner ear primordia, the otic vesicle (OV) and cochleovestibular ganglion (CVG), obtained from 72-hr (stage 19-20) quail embryos. The studies described in this paper investigated whether NGF serves as a mitogen, a survival factor, and/or a differentiation factor in this system. Explants of isolated OV and CVG were maintained for 24 hr in serum-free medium alone (M-199), M-199 containing serum, M-199 containing NGF, or M-199 containing both serum and NGF. [3H]Thymidine was also present for the entire culture period. Both OV and CVG incorporated greater amounts of [3H]thymidine in the presence of serum or NGF, and their combined effect was additive. NGFs effects were dose dependent, saturable, and specific (blocked by anti-NGF). NGF caused little or no morphological differentiation of OV and no increase in protein levels, in contrast to OV grown in the presence of serum. CVG had both cochlear and vestibular portions present in all cases, but the apparent size and protein content of CVG was increased in the presence of either serum or NGF. Effects of serum and NGF were completely, but reversibly, blocked by amiloride, suggesting that the Na+-H+ exchange system had been activated. In order to determine whether increases in [3H]thymidine incorporation were due to increased cell survival or perhaps to an increase in proliferation, explants were initially grown for a 24-hr period in serum-free medium, followed by reactivation for an additional 24 hr in medium containing serum and/or NGF. It is likely that cells requiring either serum or NGF for survival would die during a 24-hr period in their absence. Our results revealed that the level of [3H]thymidine incorporation in OV was the same after reactivation. In the case of CVG, only NGF treatment yielded similar results; [3H]thymidine incorporation was lower in CVG reactivated with serum. It appears, therefore, that serum has probable proliferative effects upon OV and CVG, as well as survival effects for CVG. NGF, however, does not appear to affect survival in either OV or CVG, so that increases in [3H]thymidine incorporation in response to NGF are most likely due to proliferative effects upon OV or CVG, at least at this embryonic stage.

Characterization and localization of nerve growth factor receptors in the embryonic otic vesicle and cochleovestibular ganglion

We have investigated the possibility that nerve growth factor (NGF) may play a role in the development of the inner ear. Primordia of the inner ear, the otic vesicle (OV) and cochleovestibular ganglion (CVG), were isolated from 72-hr (stage 19-20) quail embryos and examined for the presence of NGF receptors. Quantitative binding studies revealed that both OV and CVG exhibited specific 125I-NGF binding; levels of nonspecific binding were 6 to 26% of total binding. Scatchard analysis yielded a linear plot, indicating the presence of a single class of NGF receptor. The average binding constant (Kd) was 8.0 nM for OV and 8.6 nM for CVG, corresponding to the low affinity (site II) NGF receptor. Examination of light microscopic radioautographs indicated that most of the specific 125I-NGF binding was located in the ventromedial wall of the OV, with little or no binding in the lateral wall and endolymphatic primordia. These studies were corroborated by microdissection of OV, in which 70% of the radioactivity was found to be localized in the medial half of the OV. In CVG, specific 125I-NGF binding was more concentrated in the cochlear portion of the ganglion, with silver grains primarily over areas containing support cells and immature neurons. Quantitative binding studies with isolated cochlear and vestibular ganglia obtained from 144-hr (stage 29-30) quail embryos revealed that the cochlear ganglion exhibited three times more specific 125I-NGF binding than the vestibular ganglion. The presence of NGF receptors on OV and CVG suggests that these structures are responsive to and/or dependent upon NGF. The following paper (J. Represa and P. Bernd, 1989, Dev. Biol. 134) examines the question of whether NGF serves either as a mitogen, a survival factor, or a differentiation factor in this system.

Temporal pattern of nerve growth factor (NGF) binding in vivo and the in vitro effects of NGF on cultures of developing auditory and vestibular neurons.

NGF binding patterns reflect the presence of receptors for this growth factor. High specific binding of 125I 2.5 S-NGF was observed for the 11 gestation day (gd) statoacoustic ganglion (SAG) with lower levels recorded for both 14 gd acoustic ganglion (AG) and vestibular ganglion (VG) samples. Fourteen day AG cells were more than twice as active for binding NGF when compared to VG samples of the same gestational age. Both whole ganglion explants and dissociated cell cultures were grown in chemically defined medium for short term culture to assay changes in neurite outgrowth and survival of neurons in response to the addition of exogenous 2.5 S-NGF. The most vigorous neurite outgrowth and neuronal survival responses were produced by 11 gd SAG samples treated with NGF. Acoustic ganglion specimens of both 11 gd and 14 gd embryos were much more responsive to the neurotrophic effects of NGF when compared to the responses of their VG counterparts. There was a correlation between NGF binding ability and in vitro responsiveness to exogenous NGF. We hypothesize based on the results of this study that NGF (and/or a member of the NGF family of growth factors) is involved in the control of developmentally regulated neuronal cell death of SAG neurons and may play a role in the innervation of developing inner ear sensory structures.

Role for basic fibroblast growth factor (FGF-2) in tyrosine kinase (TrkB) expression in the early development and innervation of the auditory receptor: in vitro and in situ studies.

A previous study showed that basic fibroblast growth factor (FGF-2) promotes the effects of brain-derived neurotrophic factor (BDNF) on migration and neurite outgrowth from the cochleovestibular ganglion (CVG). This suggests that FGF-2 may up-regulate the receptor for BDNF. Thus we have examined TrkB expression during CVG formation and otic innervation in vitro and in the chicken embryo using immunohistochemistry. Following anatomical staging according to Hamburger-Hamilton, results were compared with mRNA expression in vitro using in situ hybridization. In the embryo at stage 16 (E2+) clusters of either lightly stained or immunonegative cells occurred within the otocyst and among those migrating to the CVG. By stage 22 (E3.5), immunostaining was concentrated in the CVG perikarya and invaded the processes growing into the otic epithelium but not into the rhombencephalon. Subsequently TrkB expression decreased in the perikarya and became localized in the leading processes of the fibers invading the epithelium and in the structures participating in synapse formation with the hair cells. In vitro there was moderate immunostaining and modest in situ hybridization for trkB in the neuroblasts migrating from the otocyst under control conditions. In contrast, neuroblasts previously exposed to FGF-2 exhibited accelerated migration and differentiation, with increased trkB mRNA expression. Morphological differentiation was associated with more intense immunostaining of processes than cell bodies. Evidently TrkB shifts its expression sequentially from sites engaged in migration, ganglion cell differentiation, axonal outgrowth, epithelial innervation, and synapse formation. FGF-2 may promote the role of BDNF in these developmental events by upregulating the TrkB receptor.

Trib3 Is Elevated in Parkinson's Disease and Mediates Death in Parkinson's Disease Models

Parkinsons disease (PD) is characterized by the progressive loss of select neuronal populations, but the prodeath genes mediating the neurodegenerative processes remain to be fully elucidated. Trib3 (tribbles pseudokinase 3) is a stress-induced gene with proapoptotic activity that was previously described as highly activated at the transcriptional level in a 6-hydroxydopamine (6-OHDA) cellular model of PD. Here, we report that Trib3 immunostaining is elevated in dopaminergic neurons of the substantia nigra pars compacta (SNpc) of human PD patients. Trib3 protein is also upregulated in cellular models of PD, including neuronal PC12 cells and rat dopaminergic ventral midbrain neurons treated with 6-OHDA, 1-methyl-4-phenylpyridinium (MPP+), or α-synuclein fibrils (αSYN). In the toxin models, Trib3 induction is substantially mediated by the transcription factors CHOP and ATF4. Trib3 overexpression is sufficient to promote neuronal death; conversely, Trib3 knockdown protects neuronal PC12 cells as well as ventral midbrain dopaminergic neurons from 6-OHDA, MPP+, or αSYN. Mechanism studies revealed that Trib3 physically interacts with Parkin, a prosurvival protein whose loss of function is associated with PD. Elevated Trib3 reduces Parkin expression in cultured cells; and in the SNpc of PD patients, Parkin levels are reduced in a subset of dopaminergic neurons expressing high levels of Trib3. Loss of Parkin at least partially mediates the prodeath actions of Trib3 in that Parkin knockdown in cellular PD models abolishes the protective effect of Trib3 downregulation. Together, these findings identify Trib3 and its regulatory pathways as potential targets to suppress the progression of neuron death and degeneration in PD. SIGNIFICANCE STATEMENT Parkinsons disease (PD) is the most common neurodegenerative movement disorder. Current treatments ameliorate symptoms, but not the underlying neuronal death. Understanding the core neurodegenerative processes in PD is a prerequisite for identifying new therapeutic targets and, ultimately, curing this disease. Here, we describe a novel pathway involving the proapoptotic protein Trib3 in neuronal death associated with PD. These findings are supported by data from multiple cellular models of PD and by immunostaining of postmortem PD brains. Upstream, Trib3 is induced by the transcription factors ATF4 and CHOP; and downstream, Trib3 interferes with the PD-associated prosurvival protein Parkin to mediate death. These findings establish this new pathway as a potential and promising therapeutic target for treatment of PD.

Distribution of cholinergic neuronal differentiation factor/leukemia inhibitory factor binding sites in the developing and adult rat nervous system in vivo

Cholinergic neuronal differentiation factor/leukemia inhibitory factor (CDF/LIF) is a multifunctional cytokine that affects neurons as well as many other cell types. Toward elucidating its neural functions in vivo, we previously investigated the distribution of CDF/LIF binding sites with iodinated native CDF/LIF in embryonic to postnatal day 0 (P0) rats. In the present study, we have extended our examination to postnatal ages and find that specific CDF/LIF binding sites are present at defined developmental stages in additional brain regions not previously exhibiting binding by P0. High levels of binding are detected in all P7 sensory and autonomic ganglia examined, but only in restricted postnatal central nervous system structures. Cranial motor and mesencephalic trigeminal neurons maintain high levels throughout, while binding to spinal motor neurons, which decreases to low levels at P0, reappears by P14 and increases with age. Most other structures, which show detectable binding by P0, exhibit higher levels at postnatal ages, including the red, deep, ventral cochlear, trapezoid, superior olivary, vestibular, ventral tegmental, and ventral posterior thalamic nuclei as well as the glomerular layer of the olfactory bulb. High levels are also detected in several structures for the first time after P0, including the cerebellar cortex (molecular and Purkinje cell layers), lateral reticular nucleus of the medulla and reticular formation, as well as the reticulotegmental, medial geniculate, solitary (rostral, dorsomedial, and commissural regions), medial septal, lateral mammillary, and lateral habenular nuclei. These results not only identify regions of potential CDF/LIF-responsive neurons and glia throughout development but suggest new CDF/LIF roles in the nervous system.

Temporal pattern of nerve growth factor receptor expression in developing cochlear and vestibular ganglia in quail and mouse

SummaryWe have previously demonstrated the presence of specific receptors for nerve growth factor (NGF) in cochleovestibular ganglia of 72 h (stage 19–20) quail embryos, with a greater density of NGF receptors in the cochlear portion of the ganglion. The present study was conducted to determine the temporal pattern of NGF receptor expression in cochlear and vestibular ganglia throughout development, and was conducted in two species, quail and mouse. As in the quail, specific binding of125I-NGF was detected in cochleovestibular ganglia of mouse embryos from an embryonic age equivalent to 72 h quail embryos (embryonic day 11, E11), with a similar concentration of125I-NGF binding in the cochlear portion. Quantitative studies revealed that125I-NGF binding continued to increase, in both cochlear and vestibular ganglia, for several days of development, and then began to decrease to minimal levels. Maximal levels were achieved at E7 in the quail, and E14 to E16 in the mouse, while minimal levels were reached by E13 in the quail, and E18 in the mouse. The level of125I-NGF binding in cochlear ganglia was two to three times higher than in vestibular ganglia; a finding corroborated by radioautographic studies. In both quail and mouse, NGF receptors were more heavily concentrated in the ventromedial portion of the cochlear ganglion, adjacent to the cochlear duct; an area containing both support cells and peripheral neuronal processes. In the vestibular ganglion,125I-NGF binding was more homogeneous, although small areas containing high densities of silver grains were observed. The presence of NGF receptors in cochlear and vestibular ganglia suggests that these ganglia may be responsive to and/or dependent upon NGF during their development.

The potential role of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in avian cochlear and vestibular ganglia development

The role of the nerve growth factor family of neurotrophins in the development of cochlear and vestibular ganglia is unclear. In order to predict the potential importance of nerve growth factor, brain‐derived neurotrophic factor or neurotrophin‐3, we examined the expression of neurotrophin mRNA and full‐length neurotrophin receptor mRNA by in‐situ hybridization and reverse transcription‐polymerase chain reaction, as well as whether high affinity 125I‐nerve growth factor binding was present, in cochlear and vestibular ganglia of the quail at several stages of development (stages 26, 31 and 36). Nerve growth factor, brain‐derived neurotrophic factor and neurotrophin‐3 mRNA was detected at all ages examined, suggesting that these neurotrophins may serve an autocrine or paracrine function, especially prior to target contact. In addition, we found full‐length trkA and trkC mRNA was expressed, the products of which are the functional neuronal receptors for nerve growth factor and neurotrophin‐3, respectively. Although full‐length trkA mRNA was found, physiologically important high affinity 125I‐nerve growth factor binding was not detected. Since nerve growth factors effects on survival and neurite outgrowth are mediated through high affinity binding, nerve growth factor may serve an as yet unidentified role in this system. Full‐length trkB mRNA, the product of which is the functional neuronal receptor for brain‐derived neurotrophic factor, was not detected using reverse transcription‐polymerase chain reaction, however, truncated (non‐catalytic) trkB was present, at least in cochlear ganglia at stage 31. It is not known what function may be subserved by these truncated receptors.

EXPRESSION OF NEUROTROPHIN TRK AND P75 RECEPTORS IN QUAIL EMBRYOS UNDERGOING GASTRULATION AND NEURULATION

We have previously demonstrated the presence of mRNA for the full‐length neurotrophin receptors trkA, trkB and trkC in quail embryos from stages 1 through 6 using reverse transcription followed by the polymerase chain reaction (RT‐PCR; Yao et al. [1994] Dev. Biol. 165: 727–730). Furthermore, we showed that mRNA for the neurotrophins brain‐derived neurotrophic factor and neurotrophin‐3 was present from stage 1 onward, while nerve growth factor mRNA began to be expressed at stage 5. In the present study, wholemount in situ hybridization was used to localize full‐length trk mRNA in embryos from stages 3 through 10. Structures expressing trkC mRNA included the primitive streak and Hensens node, the neural plate or notochord, somites and the rostral neural tube. trkA and trkB mRNA were expressed at much lower levels than trkC mRNA; however, staining was detected on the primitive streak and Hensens node. In addition to trk mRNA, we have also demonstrated the presence of full‐length Trk protein in embryos from stages 3 through 11, suggesting that the trk mRNA detected at these early stages is translated into functional cell surface receptors. To support this hypothesis, we have shown that neurotrophins can induce phosphorylation of Trk on tyrosine residues, at least at stage 11. We also detected mRNA and protein for the nontyrosine kinase neurotrophin receptor, p75, at similar stages. The presence of neurotrophin receptors, as well as neurotrophin mRNA, in embryos undergoing gastrulation and neurulation leads to speculation that neurotrophins may be playing a role in these processes.