Regulation Of Neurotrophins Research Articles

Truncated and full-length TrkB receptors regulate distinct modes of dendritic growth.

Neurotrophin regulation of neuronal morphology is complex and may involve differential action of alternative Trk receptor isoforms. We transfected ferret visual cortical slices with full-length and truncated TrkB receptors to examine their roles in regulating cortical dendrite development. These TrkB isoforms had differential effects on dendritic arborization: whereas full-length TrkB increased proximal dendritic branching, truncated TrkB promoted net elongation of distal dendrites. The morphological effects of each receptor isoform were distinct, yet their actions inhibited one another. Actions of the truncated TrkB receptor did not involve unmasking of endogenous TrkC signaling. These results suggest that TrkB receptors do not regulate dendritic growth per se but, rather, the mode of such growth.

Neurotrophin regulation of sodium and calcium channels in human neuroblastoma cells

Neurotrophins, acting through tyrosine kinase family genes, are essential for neuronal differentiation. The expression of tyrosine kinase family genes is prognostic in neuroblastoma, and neurotrophins reduce proliferation and induce differentiation, indicating that neuroblastomas are regulated by neurotrophins. We tested the effects of nerve growth factor and brain-derived neurotrophic factor on Na + and Ca 2+ currents, using the whole-cell patch-clamp technique, in human neuroblastoma NB69 cells. Control cells exhibited a slow tetrodotoxin-resistant ( ic 50=98 nM) Na + current and a high-voltage-activated Ca 2+ current. Exposure to nerve growth factor (50 ng/ml) and/or brain-derived neurotrophic factor (5 ng/ml) produced the expression of a fast tetrodotoxin-sensitive ( ic 50=10 nM) Na + current after day 3, and suppressed the slow tetrodotoxin-resistant variety. The same type of high-voltage-activated Ca 2+ current was expressed in control and treated cells. The treatment increased the surface density of both Na + and Ca 2+ currents with time after plating, from 17 pA/pF at days 3–5 and 1–5 to 34 and 30 pA/pF after days 6–10, respectively. Therefore, both nerve growth factor and brain-derived neurotrophic factor, acting through different receptors of the tyrosine kinase family and also possibly the tumor necrosis factor receptor-II, were able to regulate differentiation and the expression of Na + and Ca 2+ channels, partially reproducing the modifications induced by diffusible astroglial factors. We show that neurotrophins induced differentiation to a neuronal phenotype and modified the expression of Na + and Ca 2+ currents, partially reproducing the effects of diffusible astroglial factors.

Neurotrophin regulation of beta-actin mRNA and protein localization within growth cones.

Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of beta-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that beta-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of beta-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA. Localization of beta-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.

Reciprocal changes in the expression of neurotrophin mRNAs in target tissues and peripheral nerves of aged rats

trk receptors are downregulated in both dorsal root ganglion (DRG) and spinal motoneurons of aged rats with behavioral sensorimotor deficits. Here we provide evidence, using reverse transcription-polymerase chain reaction (RT-PCR), of decreased levels of neurotrophin (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF; neurotrophin-3, NT-3; and neurotrophin-4, NT-4) mRNAs in target muscles. Moreover, the degree of neurotrophin mRNA decrease in target muscles seems to co-vary with the extent of sensorimotor disturbances. In contrast, the peripheral nerve of aged rats showed a reciprocal regulation of neurotrophins, with increased levels of NGF, BDNF, and NT-4 mRNAs. Taken together, evidence suggest an aging-related attenuation of neurotrophin signaling between target tissues, on one hand, and DRG and motoneurons, on the other, and, furthermore, that target-derived neurotrophins regulate the expression levels of trk mRNAs in both DRG and motoneurons.

The regulation of the retrograde axonal transport of [formula omitted]-β nerve growth factor is independent of calcium

Calcium has been shown to play a major role in the regulation of endocytosis and exocytosis of synaptic vesicles and retrograde axonal transport of proteins. The role of calcium in the regulation of neurotrophin retrograde axonal transport is unknown. This study aimed to determine if calcium plays a role in the uptake and retrograde axonal transport of 125 I -β nerve growth factor ( 125 I -βNGF) within sympathetic neurons innervating the iris by comparing it with 125 I -anti-dopamine beta hydroxylase (anti-DBH). The nonspecific voltage-sensitive calcium channel (VSCC) antagonists, cadmium (200 nmol/eye) and nickel (100 nmol/eye) reduced the amount of 125 I -anti-DBH retrograde axonal transport by 90 and 70%, respectively. In contrast, cadmium (200 nmol/eye) had no effect on 125 I -βNGF retrograde axonal transport, while nickel (100 nmol/eye) caused a significant increase in the amount transported to the ganglia. The L-type VSCC antagonist nifedipine (10 nmol/eye) and N-type VSCC antagonist omega-conotoxin (1.5 nmol/eye) both had no effect on 125 I -anti-DBH retrograde axonal transport which suggests that these types of calcium channels are not involved in the exocytosis/endocytosis of anti-DBH containing vesicles. Thapsigargin (0.2 nmol/eye), an inhibitor of sarcoplasmic reticulum Ca 2+-ATPases also significantly inhibited 125 I -anti-DBH transport but had no effect on 125 I -βNGF retrograde transport. This suggests that 125 I -anti-DBH and 125 I -βNGF are internalized into different vesicle types and that the endocytosis and retrograde axonal transport of 125 I -βNGF are not dependent upon calcium.

An improved method for dissociation and aggregate culture of human fetal brain cells in serum-free medium

Moscona, in the early sixties [A.A. Moscona, Recombination of dissociated cells and the development of cell aggregates, in: B.M. Willmer (Ed.), Cells and Tissues in Culture, Academic Press, New York, 1965, pp. 489–529.] [16], discovered that aggregation of dissociated cells is a property of embryonal cells. Several features of the aggregate culture system are particularly attractive for the conduct of biochemical and molecular studies on the human fetal brain. (i) All the pertinent procedural parameters can be readily controlled and standardized, resulting in a consistently reproducible system suitable for quantitative analyses. (ii) Neuronal enriched aggregates can be readily obtained, with minimal neurotoxicity. (iii) Aggregates can be easily harvested for biochemical and molecular studies. Aggregate cultures, generated from rodent fetal brains, have been extensively utilized as a tool to study regulation of aminergic neurons [P. Honegger, E. Richelson, Biochemical differentiation of mechanically dissociated brain in aggregating cell culture, Brain Res. 109 (1976) 335–354; P. Honegger, E. Richelson, Biochemical differentiation of aggregating cell cultures of different fetal rat brain regions, Brain Res. 133 (1977) 329–339.] [11, 12]and peptidergic neurons (neuropeptide Y (NPY) and somatostatin (SRIF) [A. Barnea, E. Anthony, G. Lu, G. Cho, Morphological differentiation of neuropeptide Y neurons in aggregate cultures of dissociated fetal cortical cells: a model system for glia–neuron paracrine interactions, Brain Res. 625 (1993) 313–322; A. Barnea, G. Cho, G. Lu, M. Mathis, Brain-derived neurotrophic factor induces functional expression and phenotypic differentiation of cultured fetal neuropeptide Y producing neurons, J. Neurosci. Res. 42 (1995) 638–647; A. Barnea, A. Hajibeigi, G. Cho, P. Magni, Regulated production and secretion of immunoreactive neuropeptide Y by aggregating fetal brain cells in culture, Neuroendocrinology 54 (1991) 7–13; P. Magni, A. Barnea, Forskolin and phorbol ester stimulation of neuropeptide Y (NPY) production and secretion by aggregating fetal brain cells in culture: evidence for regulation of NPY biosynthesis at transcriptional and posttranscriptional levels, Endocrinology 130 (1992) 976–984.]) [4–6, 14]. However, very few studies have utilized this system to study regulatory processes of human fetal neurons/glia [M. McCarthy, L. Resnik, F. Taub, R.V. Stewart, R.D. Dix, Infection of human neural cell aggregate cultures with a clinical isolate of cytomegalovirus, J. Neuropathol. Exp. Neurol. 50 (1991) 441–450; L. Pulliam, M.E. Berens, M.L. Rosenblum, A normal human brain cell aggregate model for neurobiological studies, J. Neurosci. Res. 21 (1988) 521–530.] [15, 17]. In a series of studies in our laboratory [N. Aguila-Mansilla, A. Barnea, Human fetal brain cells in aggregate culture: a model system to study regulatory processes of the developing human neuropeptide Y (NPY) producing neuron, Int. J. Dev. Neurosci. 14 (1996) 531–539; A. Barnea, N. Aguila-Mansilla, H.T. Chute, A.A. Welcher, Comparison of neurotrophin regulation of human and rat neuropeptide Y (NPY) neurons: induction of NPY production in aggregate cultures derived from rat but not from human fetal brains, Brain Res. 732 (1996) 52–60; A. Barnea, N. Aguila-Mansilla, G. Lu, R.H. Ho, Opposite effects of astrocyte-derived soluble factor(s) on the functional expression of fetal peptidergic neurons in aggregate cultures: enhancement of neuropeptide Y and suppression of somatostatin, J. Neurosci. Res. 50 (1997) 605–617; A. Barnea, J. Roberts, R.H. Ho, Evidence for a synergistic effect of the HIV-1 envelope protein gp120 and brain-derived neurotrophic factor (BDNF) leading to enhanced expression of somatostatin neurons in aggregate cultures derived from the human fetal cortex, Brain Res. 815 (1999) 349–357.] [1–3, 7], we have established a human-derived aggregate culture system, maintained in serum-free medium for up to 28 days, in which expression of NPY and SRIF genes is a regulated process. Herein, we outline our protocols for (i) generating dissociated cells from the human fetal cortex with a high yield (85–95%) of viable cells and (ii) generating and maintaining aggregates comprised of differentiating neurons and astrocytes or predominantly neurons (astrocyte-deficient). We provide examples demonstrating differential regulation of the expression of NPY and SRIF. We propose that this culture system can serve as a model to study regulatory processes of the human fetal cortex. Themes: Cell biology Topic: Gene structure and function, general

Neurotrophin regulation of synaptic transmission

Examples of signaling molecules that are devoted to neuronal development at the exclusion of other functions are scarce. It may then come as no surprise to learn that a family of molecules that promote neuronal survival, differentiation and outgrowth also regulate synaptic transmission at both developing and mature synapses. Indeed, many studies over the past five years have shown that neurotrophins, including nerve growth factor (NGF), neurotrophin-3 (NT-3), NT-4/5 and brain-derived neurotrophic factor (BDNF), have both rapid and long-latency influences on synaptic strength. New research has highlighted the enormous range of neurotrophin actions at both developing and mature synapses, demonstrating that transmission can be enhanced or reduced at excitatory and inhibitory synapses by either pre- or postsynaptic mechanisms.

Differential Regulation of Neurotrophin andtrkReceptor mRNAs in Catecholaminergic Nuclei during Chronic Opiate Treatment and Withdrawal

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their receptors trkB and trkC, respectively, are expressed in the locus coeruleus (LC) and ventral tegmental area (VTA), brain regions known to be involved in opiate addiction. Previously, administration of exogenous neurotrophins has been shown to oppose effects of chronic morphine treatment on LC and VTA neurons. However, the response of endogenous neurotrophins in LC and VTA to opiate treatment is unknown. In this study, BDNF, NT-3, trkB, and trkC mRNAs were analyzed in these regions after chronic morphine treatment and during antagonist precipitated withdrawal. Although chronic morphine exposure resulted in only modest increases in BDNF and NT-3 mRNA expression in LC, precipitated withdrawal led to a marked, rapid, and prolonged increase in BDNF mRNA and a delayed decrease in NT-3 mRNA. Levels of trkB and trkC mRNAs, which were unchanged by chronic morphine treatment, were elevated in LC at 2 and 6 hr of withdrawal. By 20 hr, trkB mRNA levels in LC had returned to control, whereas trkC mRNA levels fell below control values. In contrast to the substantial alterations observed in LC, there was no regulation of the neurotrophins or trk mRNAs within the VTA during chronic opiate treatment or withdrawal, with the exception of an increase in trkB mRNA at 6 hr of withdrawal. These results suggest that neurotrophins and their receptors per se may be involved in opiate-induced plasticity of the LC, whereas other mechanisms would appear to be involved in the VTA.

Neurotrophin regulation of gene expression.

The neurotrophins comprise a family of secreted proteins that elicit profound responses in cells of the developing and mature vertebrate nervous system including the regulation of neuronal survival and differentiation. The molecular mechanisms by which the neurotrophins exert their effects have been the subject of intense investigation. The neurotrophins elicit responses in neurons via members of the Trk family of receptors and the p75 neurotrophin receptor. Once activated, neurotrophin receptors trigger a large number of biochemical events that propagate the neurotrophin signal from the plasma membrane to the interior of the cell. An important target of the neurotrophin-induced signaling pathways is the nucleus, where neurotrophin-induced signals are coupled to alterations in gene expression. These neurotrophin-induced changes in gene expression are critical for many of the phenotypic effects of neurotrophins including the regulation of neuronal survival and differentiation.

Neurotrophin regulation of ionic currents and cell size depends on cell context.

Trk receptor activation by neurotrophins is often considered to have a defined set of actions on target neurons, including supporting neuronal survival, inducing morphological differentiation, and regulating a host of target genes that specify neuronal phenotype. It is not known if all such regulatory effects are obligatory, or if some may vary depending on the cell context in which the receptors are expressed. We have examined this issue by comparing neurotrophin effects on the regulation of electrical excitability and morphological differentiation in two strains of PC12 cells. We found that while neurotrophins induced neurite extension and increased calcium currents in both PC12 cell types, sodium current levels were regulated in only one of these strains. Moreover, we found little correlation between calcium current levels and the extent of morphological differentiation when compared in individual cells of a single strain. Thus, the regulatory effects of neurotrophins on cell phenotype are not fully determined by the Trk receptors that they activate; rather, they can vary with differences in cell context that arise not only between different cell lineages, but also between individual cells of clonal relation.

Neurotrophins Differentially Regulate Voltage-Gated Ion Channels

Neurotrophic factors profoundly affect neuronal differentiation, but whether they influence neuronal phenotype in instructive ways remains unclear: do different neurotrophic factors always trigger identical programs of differentiation or can each impose distinct functional properties even when acting upon the same population of target neurons? We addressed this issue by examining the regulatory effects of the four neurotrophins on the molecular components of electrical excitability, voltage-gated ion channels, within a single cellular context. Using patch clamp methods, we studied neurotrophin regulation of voltage-gated sodium, calcium, and potassium currents in SK-N-SH neuroblastoma cells. We found that each neurotrophin induced a unique pattern of expression of ionic currents despite similar activation of initial signal transduction events. Thus, each neurotrophin imposed a different excitable phenotype even when acting upon the same target cells

Neurotrophin regulation of the developing nervous system: analyses of knockout mice.

The neurotrophins, NGF, BDNF, NT3 and NT4, are one family in a growing repertoire of neurotrophic factors. The neurotrophins have long been implicated in neuronal survival and recent studies from mice with targeted disruptions of the neurotrophin genes confirm this role, but also reveal that the action of the neurotrophins is more complex, and in some instances more interactive, than originally envisaged. Lack of functional NGF, BDNF and NT3 genes results in severe neuronal deficits and an early postnatal death. However, NT4 is unique among the neurotrophins and while the absence of NT4 does result in limited sensory neuron loss these mice do not die early, suggesting that NT4-dependent neurons are not critical for survival. Phenotypic analyses of mice lacking neurotrophin receptors, TrkA, B and C, confirm that TrkA is the functional receptor for NGF, TrkB acts as the primary receptor for BDNF and NT4, and NT3 signals primarily through TrkC. However, the finding that TrkC mutant mice have a less dramatic phenotype than their NT3 counterparts implicates NT3 in signaling via receptors other than TrkC. Further studies, using combinatorial Trk and neurotrophin deletions, reveal that while BDNF and NT4 subserve distinct neuron populations in most cases, other neuron sub-populations can be supported by either BDNF or NT4, providing evidence for compensatory actions between neurotrophins. As a mechanism to explain programmed cell death that occurs in the developing nervous system, recent studies examining neurotrophin gene-dosage effects suggest that the availability of neurotrophins, NGF, BDNF and NT3, may be limiting for some neuron populations. In addition, the proposed switch in neurotrophin dependency for some neuron populations is now being determined using neurotrophin mutant mice. We discuss these and other recent findings on neurotrophin requirements for the developing nervous system.

Comparison of neurotrophin regulation of human and rat neuropeptide Y (NPY) neurons: induction of NPY production in aggregate cultures derived from rat but not from human fetal brains

Previous studies established that brain-derived neurotrophic factor (BDNF) induces neuropeptide Y (NPY) production and accumulation of NPY-mRNA in cultures of rat fetal brain tissues. In this study, we addressed the question: Are cultured human NPY neurons regulated by BDNF and/or by another member of the neurotrophin (NT) family of growth factors? Using aggregate cultures derived from human fetal cortical hemispheres, we assessed the effect of BDNF on NPY production varying the following experimental conditions: fetal and culture age; medium composition (with and without serum), dose and duration of exposure to BDNF, and neurotrophin species tested (BDNF, NT-4/5, NT-3 or NGF). Under none of these conditions did BDNF, NT-4/5, NT-3 or NGF induce an increase in NPY production. This was in contrast to forskolin + phorbol 12 myristate 13-acetate (PMA) which were highly effective in inducing NPY production, verifying that expression of NPY is a regulated process in these cultures. None of these neurotrophins enhanced the response to forskolin + PMA. By comparison, using aggregate cultures derived from rat fetal cortices, BDNF and NT-4/5 were equipotent in inducing NPY production but NT-3 and NGF were essentially ineffective. Moreover, the effects of BDNF or NT-4/5 and forskolin + PMA on NPY production were additive, indicating the involvement of distinct intracellular signalling pathways. Western blot analyses of human- and rat-derived aggregates indicated the presence of full-length Trk receptors which are tyrosine-phosphorylated in response to either BDNF, NT-4/5 or NT-3. Primary cultures of astrocytes (rat as well as human) were devoid of a functional TrkB receptor, strongly suggesting a neuronal expression of TrkB in the aggregates. Thus, a functional TrkB receptor is expressed by both the human and rat aggregates, but only the rat aggregates responded to BDNF or NT-4/5. These results are consistent with a difference in a post TrkB-receptor event(s) mediating BDNF action in the cultured human and rat fetal NPY neurons.

Unilateral LTP triggers bilateral increases in hippocampal neurotrophin andtrk receptor mRNA expression in behaving rats: Evidence for interhemispheric communication

Induction of long-term potentiation (LTP) in the dentate gyrus of awake rats triggered a rapid (2 hour) elevation in tyrosine kinase receptor (trkB and trkC) gene expression and a delayed (6-24 hour) increase in brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) gene expression. Depending on the mRNA species, LTP induction led to highly selective unilateral or bilateral increases in gene expression. Specifically, trkB and NT-3 mRNA elevations were restricted to granule cells in the ipsilateral dentate gyrus, whereas bilateral increases in trkC, BDNF, and nerve growth factor (NGF) mRNA levels occurred in granule cells and hippocampal pyramidal cells. Both unilateral and bilateral changes in gene expression were N-methyl-D-aspartate (NMDA) receptor-dependent and LTP-specific. Bilateral electrophysiological recordings demonstrated that LTP was unilaterally induced; this was corroborated by a dramatic unilateral increase in the expression of the immediate early gene zif/268, a marker for LTP, restricted to the ipsilateral granule cells. The results indicate that LTP triggers an interhemispheric communication manifested as selective, bilateral increases in gene expression at multiple sites in the hippocampal network. Furthermore, our findings suggest that physiological plastic changes in the adult brain may involve coordinated, time-dependent regulation of multiple neurotrophin and trk receptor genes.

Phenotypic Diversity in Neuronal Cell Lines Derived from Raphe Nucleus by Retroviral Transduction

Two neuronal cell lines, RN33B and RN46A, were derived from E12 rat medullary raphe neuroepithelial cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen (T-ag). At permissive temperature (33°C), both cell lines express nuclear T-ag immunoreactivity, low levels of neuron-specific enolase (NSE), and low (NF-L) and medium (NFM), but not high (NF-H), MW neurofilament proteins. Once differentiated, both cell lines cease dividing, take on a neuronal morphology, and express enhanced levels of NSE and all three NF proteins. The complex interaction of variables which mediates the differentiation of these neuronal cell lines is discussed. RN33B and RN46A cells show both similar and different physiological properties, and those features are highlighted to demonstrate the utility of these cell lines in studying neuronal differentiation, both in vitro and in vivo. For example, the expression of NF-L and NF-M in differentiating RN33B and RN46A cells can be potentiated by activation of protein kinase A. RN33B cells are glutamatergic while RN46A cells are serotonergic. 5-HT uptake and synthesis in RN46A cells are differentially regulated, as synthesis is maximal only in differentiated RN46A cells which have been treated with BDNF followed by partial depolarization. 5-HT uptake is seen at similar levels in both undifferentiated and differentiated RN46A cells and is independent of neurotrophin regulation. Following transplantation into the CNS, RN33B cells survive ≥6 months and differentiate according to local environmental signals; RN46A cells survive ≤2 months and do not show similar plasticity in vivo. These data are modeled to provide a conceptual framework as to the generation and differentiation capacity of neuronal cell lines derived by retroviral transduction.

An improved method detects differential NGF and BDNF gene expression in response to depolarization in cultured hippocampal neurons

Differential regulation of individual neurotrophins by impulse activity potentially allows transformation of instantaneous signalling into diverse, long-lasting neural alterations. To define the temporal profiles of trophin gene expression we examined nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNAs in dissociated cell cultures of rat hippocampus using an improved solution hybridization technique. Traditional methods lack the precision and sensitivity to detect small changes during brief intervals and the facility to process large sample numbers simultaneously. This improved method has now allowed us to better define the dynamics of depolarization-induced changes in expression of individual trophin genes. Using elevated K + as a depolarizing stimulus, NGF mRNA increased 40% after 48 h. In contrast, BDNF message rose almost 4-fold within 3 h and attained a maximal 6-fold increase within 6 h. Similar increases in BDNF mRNA levels were exhibited following treatment of cultures with glutamate, an excitatory neurotransmitter. To document the sensitivity of BDNF mRNA to depolarizing conditions, we examined expression after K + withdrawal. BDNF message began decreasing within one hour post-depolarization, and returned to basal levels after 6 h. Observations indicate that BDNF and NGF mRNAs are induced differentially in response to impulse activity; BDNF message is acutely responsive to ongoing changes, whereas NGF mRNA responds more slowly and sluggishly. The physiological implications of this differential regulation are discussed.

Nicotine effects on the regulation of amyloid precursor protein splicing, neurotrophin and glucose transporter RNA levels in aged rats

It has been reported that an inverse relationship exists between nicotine intake and the incidence of Alzheimer's Disease (AD). Although nicotine has been reported to induce c-fos, in the present study it was shown that this induction does not alter the accumulation of a number of transcripts associated with AD. Altered splicing patterns of Amyloid Precursor Protein (APP) and changes in neurotrophin and glucose transporter expression have been implicated in AD and behavioral deficits in rats. The effects of subacute administration of nicotine (12 mg/ml at 2.3 μl/hr for 14 days) on the abundance levels of APP, glucose transporter(GLUT) and neurotrophin transcripts were determined by rtPCR in the hippocampus, cortex, and striatum of aged (22–24 months) male Wistar rats. No significant differences between saline and nicotine infused rats were detected for APP abundance levels or ratio of the various isoforms. However, both groups had a higher level of APP transcripts containing the Kunitz Protease Inhibitor (KPI) domain in the hippocampus than in either the cortex or striatum. The mean percentages of APP 695 for the two groups were 75% in the hippocampus and 82 and 81% in the cortex and striatum, respectively (P<0.01). No changes in the abundance of GLUT1, GLUT3, nerve growth factor (NGF) or brain derived neurotrophic factor (BDNF) transcripts were detected. However, since both APP and GLUT1 are thought to be regulated post-transcriptionally, the present results do not rule out a change at the protein level. Further work will be required to determine whether nicotine can influence the expression of these proteins which affect neuronal function.

Neurotrophin regulation of energy homeostasis in the central nervous system.

Our hypothesis is that one cause of neuronal cell death and shrinkage in the aged central nervous system is an inability of neurons to maintain oxidant homeostasis in the face of increased levels of reactive oxygen species, decreased endogenous antioxidants, and impaired energy metabolism associated with physiological senescence, Alzheimer's, and Parkinson's diseases. Since treatment with nerve growth factor (NGF) reverses behavioral impairments in aged rats and stimulates cholinergic activity in the basal forebrain, while brain-derived neurotrophic factor appears to play a similar role in the striatum, we propose that neurotrophin-mediated cell-sparing reflects effects on oxidant homeostasis. Neurotrophins may play a similar cell-sparing role in hypoxic/ischemic injury to the nervous system, which also is mediated in part by reactive oxygen species. The degradation of one such species, H2O2, is catalyzed by catalase and glutathione peroxidase (GSH Px). The activity of the latter enzyme is dependent on glutathione reductase and the availability of NADPH for regeneration of reduced GSH. The GSH redox cycle is also regulated by enzymes of the hexose monophosphate shunt. NGF protects PC12 cells from H2O2 injury by stimulating the synthesis of antioxidant enzymes including catalase, GSH Px, glucose-6-phosphate dehydrogenase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis. NGF also enhances recovery from the NAD+ losses occurring as a consequence of H2O2 treatment.

Expression and regulation of neurotrophins and their receptors in hippocampal systems

Expression and regulation of neurotrophins and their receptors in hippocampal systems

Regulation of neurotrophin and traka, trkb and trkc tyrosine kinase receptor messenger RNA expression in kindling

Levels of messenger RNA for nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and the tyrosine kinase receptors trakA, trkB and trkC have been studied using in situ hybridization in the rat brain 2 h and four weeks after kindling-induced seizures. Epileptiform activity evoked by hippocampal stimulation and exceeding 70 s lead to a concomitant and transient increase of brain- derived neurotrophic factor, nerve growth factor, trkB and trkC messenger RNA expression in dentate granule cells after both focal and generalized seizures. Brain-derived neurotrophic factor messenger RNA levels were also increased bilaterally in the CA1-CA3 regions, amygdala and the piriform, entorhinal, perirhinal, retrosplenial and temporal cortices after generalized seizures. The magnitude of the increases was similar throughout the development of kindling and in the fully kindled brain. No changes of trkA messenger RNA were observed. In amygdalar kindling, elevated brain-derived neurotrophic factor messenger RNA levels developed more rapidly in the amygdala—piriform cortex than after stimulation in the hippocampus but changes in the hippocampal formation were only seen in few animals. Intraventricular 6-hydroxydopamine or a bilateral fimbria—fornix lesion did not alter basal expression or seizure-evoked changes in messenger RNA levels for neurotrophins or trk receptors but increased the number of animals exhibiting elevated levels after the first stimulation, probably due to a prolongation of seizure activity. Both in sham-operated and fimbria—fornix-lesioned rats seizure activity caused a marked reduction of neurotrophin-3 messenger RNA levels in dentate granule cells. The results indicate that activation of the brain-derived neurotrophic factor gene, at least in dentate granule cells, is an “all-or-none” type of response and dependent on the duration but not the severity of seizures or the stage of kindling epileptogenesis. Changes in brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and trkB and trkC were observed concomitantly in the dentate gyrus, which suggests that seizure activity sets in motion a cascade of genomic events possibly mediated via a common mechanism. Since altered messenger RNA levels outside hippocampus were detected only for brain-derived neurotrophic factor, neurotrophin and trk gene expression in these regions seems to be regulated differently.