Concentration-dependent regulation of neuronal gene expression by nerve growth factor

NGF is a neurotrophic protein that promotes the survival, growth, and differentiation of developing sympathetic neurons. To directly determine the effects of different concentrations of NGF on neuronal gene expression, we examined mRNAs encoding the p75 low-affinity NGF (LNGF) receptor, T alpha 1 alpha-tubulin (T alpha 1), and tyrosine hydroxylase (TH) in pure cultures of rat sympathetic neurons from postnatal day 1 superior cervical ganglia. Studies of the timecourse of gene expression during 2 wk in culture indicated that a 5-d incubation period would be optimal for the concentration-effect studies. Analysis of RNA isolated from neurons cultured in 2-200 ng/ml 2.5S NGF for 5 d revealed that, as the NGF concentration increased, neurons expressed correspondingly increased levels of all three mRNAs. Both LNGF receptor and TH mRNAs increased seven-fold, and T alpha 1 mRNA increased four-fold in neurons cultured in 200 versus 10 ng/ml NGF. In contrast, T26 alpha-tubulin mRNA, which is constitutively expressed, did not alter as a function of NGF concentration. When neurons were initially cultured in 10 ng/ml NGF for 5 d, and then 200 ng/ml NGF was added, LNGF receptor, T alpha 1, and TH mRNAs all increased within 48 h. The timecourse of induction differed: T alpha 1 mRNA was maximal by 5 h, whereas LNGF receptor and TH mRNAs first began to increase at 12 h after the NGF increase. These experiments show that NGF regulates expression of a subset of mRNAs important to neuronal growth and differentiation over a broad concentration range, suggesting that the effects of NGF may be mediated by more than just a single receptor operating at one fixed affinity. These results also suggest a mechanism for coupling neuronal synthesis of axonal proteins to increases in size of the innervated target territory during growth of the organism.     

Isotypes of alpha-tubulin are differentially regulated during neuronal maturation

The mRNAs for two isotypes of alpha-tubulin, termed T alpha 1 and T26, are known to be expressed in the rat nervous system. We have compared the expression of these two alpha-tubulin mRNAs during neural development, using RNA blotting and in situ hybridization techniques with probes directed against unique sequences of each mRNA. T alpha 1 mRNA is highly enriched in the embryonic nervous system but is markedly less abundant in the adult brain; T26 mRNA is expressed in many embryonic tissues with little change in abundance during development. Within the nervous system, T alpha 1 mRNA is enriched in regions with neurons actively undergoing neurite extension, such as the cortical plate, whereas T26 mRNA is relatively homogeneous in distribution, with some enrichment in proliferative zones. Expression of T alpha 1 mRNA is also increased in PC12 cells induced to differentiate and extend neurite processes by nerve growth factor. Taken together, the data indicate that T alpha 1-tubulin mRNA is expressed at high levels during the extension of neuronal processes. The abundant expression of T alpha 1-tubulin mRNA may therefore reflect either a means to increase the available pool of alpha-tubulin or a specific requirement for the T alpha 1 isotype for neurite extension.     

Blocking Cytochrome c Activity within Intact Neurons Inhibits Apoptosis

Cytochrome c has been shown to play a role in cell-free models of apoptosis. During NGF withdrawal–induced apoptosis of intact rat superior cervical ganglion (SCG) neurons, we observe the redistribution of cytochrome c from the mitochondria to the cytoplasm. This redistribution is not inhibited by the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVADfmk) but is blocked by either of the neuronal survival agents 8-(4-chlorophenylthio)adenosine 3′:5′-cyclic monophosphate (CPT-cAMP) or cycloheximide. Moreover, microinjection of SCG neurons with antibody to cytochrome c blocks NGF withdrawal–induced apoptosis. However, microinjection of SCG neurons with cytochrome c does not alter the rate of apoptosis in either the presence or absence of NGF. These data suggest that cytochrome c is an intrinsic but not limiting component of the neuronal apoptotic pathway.     

NGF inhibits M/KCNQ currents and selectively alters neuronal excitability in subsets of sympathetic neurons depending on their M/KCNQ current background

M/KCNQ currents play a critical role in the determination of neuronal excitability. Many neurotransmitters and peptides modulate M/KCNQ current and neuronal excitability through their G protein-coupled receptors. Nerve growth factor (NGF) activates its receptor, a member of receptor tyrosine kinase (RTK) superfamily, and crucially modulates neuronal cell survival, proliferation, and differentiation. In this study, we studied the effect of NGF on the neuronal (rat superior cervical ganglion, SCG) M/KCNQ currents and excitability. As reported before, subpopulation SCG neurons with distinct firing properties could be classified into tonic, phasic-1, and phasic-2 neurons. NGF inhibited M/KCNQ currents by similar proportion in all three classes of SCG neurons but increased the excitability only significantly in tonic SCG neurons. The effect of NGF on excitability correlated with a smaller M-current density in tonic neurons. The present study indicates that NGF is an M/KCNQ channel modulator and the characteristic modulation of the neuronal excitability by NGF may have important physiological implications.     

NGF mRNA expression in developing cutaneous epithelium related to innervation density

To determine if the initial level of NGF mRNA in developing cutaneous epithelium is correlated with its final innervation density, we measured the concentration of NGF mRNA in the epithelia of the maxillary, mandibular and ophthalmic territories of trigeminal ganglion in the embryonic mouse. At the onset of neuronal death in the ganglion there were marked differences in the concentration of NGF mRNA in these epithelia: the level was highest in the epithelium of the densely innervated maxillary territory, it was lower in the epithelium of the moderately innervated mandibular territory and was lowest in the epithelium of the sparsely innervated ophthalmic territory. These regional differences in the level of NGF mRNA during the early stages of target field innervation suggest that the level of NGF production in target field cells, rather than regional differences in the access of innervating neurons to NGF, governs the number of neurons that survive. Because the same percentage cell death occurs in each of the subsets of trigeminal neurons that innervate the maxillary, mandibular and ophthalmic territories, regional differences in NGF synthesis are not responsible for establishing differences in innervation density, rather they maintain differences that arise earlier in development.     

Expression of NGF receptor and GAP-43 mRNA in DRG neurons during collateral sprouting and regeneration of dorsal cutaneous nerves

The collateral sprouting of intact sensory axons and the regenertion of damaged ones differ in a number of respects. Regeneration is triggered by axotomy-induced damage, probably involves the loss of a peripheral signal, and appears to occur independently of NGF, while collateral sprouting is evoked and sustained by an increase in a target-driven signal, namely NGF. New findings strengthen the distinction between these two phenomena. Nerve growth factor receptor (NGFR) mRNA is increased in undamaged DRG neurons whose axons are sprouting into denervated skin. This response is related to an increased availablity of target-derived NGF, a proposal supported by a number of findings including increased NGF mRNA in the denervated target. In contrast, we observed little or no change in the NGFR mRNA levels in regenerating neurons, consistent with the observations that NGF does not play a role in this process. However, increases in neuronal GAP-43 mRNA are found during both regeneration and collateral sprouting, a result in keeping with the proposal that GAP-43 is primarily associated with nerve growth, and the observation that GAP-43 expression is not especially influenced by NGF. 1994 John Wiley & Sons, Inc.     

Reduction in nerve growth factor availability leads to a conditioning lesion‐like effect in sympathetic neurons

Axotomized peripheral neurons are capable of regeneration, and the rate of regeneration can be enhanced by a conditioning lesion (i.e., a lesion prior to the lesion after which neurite outgrowth is measured). A possible signal that could trigger the conditioning lesion effect is the reduction in availability of a target‐derived factor resulting from the disconnection of a neuron from its target tissue. We tested this hypothesis with respect to nerve growth factor (NGF) and sympathetic neurons by administering an antiserum to NGF to adult mice for 7 days prior to explantation or dissociation of the superior cervical ganglion (SCG) and subsequently measuring neurite outgrowth. The antiserum treatment dramatically lowered the concentration of NGF in the SCG and increased the rate of neurite outgrowth in both explants and cell cultures. The increase in neurite outgrowth was similar in magnitude to that seen after a conditioning lesion. To determine if exogenous NGF could block the effect of a conditioning lesion, mice were injected with NGF or cytochrome C immediately prior to unilateral axotomy of the SCG, and for 7 days thereafter. A conditioning lesion effect of similar magnitude was seen in NGF‐treated and control animals. While NGF treatment increased NGF levels in the contralateral control ganglion, it did not significantly elevate levels in the axotomized ganglion. The results suggest that the decreased availability of NGF after axotomy is a sufficient stimulus to induce the conditioning lesion effect in sympathetic neurons. While NGF administration did not prevent the conditioning lesion effect, this may be due to the markedly decreased ability of sympathetic neurons to accumulate the growth factor after axotomy. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Reduction in nerve growth factor availability leads to a conditioning lesion-like effect in sympathetic neurons

Axotomized peripheral neurons are capable of regeneration, and the rate of regeneration can be enhanced by a conditioning lesion (i.e., a lesion prior to the lesion after which neurite outgrowth is measured). A possible signal that could trigger the conditioning lesion effect is the reduction in availability of a target-derived factor resulting from the disconnection of a neuron from its target tissue. We tested this hypothesis with respect to nerve growth factor (NGF) and sympathetic neurons by administering an antiserum to NGF to adult mice for 7 days prior to explantation or dissociation of the superior cervical ganglion (SCG) and subsequently measuring neurite outgrowth. The antiserum treatment dramatically lowered the concentration of NGF in the SCG and increased the rate of neurite outgrowth in both explants and cell cultures. The increase in neurite outgrowth was similar in magnitude to that seen after a conditioning lesion. To determine if exogenous NGF could block the effect of a conditioning lesion, mice were injected with NGF or cytochrome C immediately prior to unilateral axotomy of the SCG, and for 7 days thereafter. A conditioning lesion effect of similar magnitude was seen in NGF-treated and control animals. While NGF treatment increased NGF levels in the contralateral control ganglion, it did not significantly elevate levels in the axotomized ganglion. The results suggest that the decreased availability of NGF after axotomy is a sufficient stimulus to induce the conditioning lesion effect in sympathetic neurons. While NGF administration did not prevent the conditioning lesion effect, this may be due to the markedly decreased ability of sympathetic neurons to accumulate the growth factor after axotomy.     

Developmental changes of nerve growth factor levels in sympathetic ganglia and their target organs

The predominant source of nerve growth factor (NGF) used by mature sympathetic neurons originates in their target organs (Heumann, R., Korsching, S., Scott, J., and Thoenen, H. (1984), EMBO J. 3, 3183-3189; Korsching, S., and Thoenen, H. (1985), J. Neurosci. 5, 1058-1061). We have determined the NGF content of two sympathetically innervated mouse organs, submandibular gland and heart ventricle, and of sympathetic ganglia from mouse and rat between embryonic Day 12 (E12) and adulthood. NGF levels were measured by a two-site enzyme immunassay (Korsching, S., and Thoenen, H. (1983), Proc. Natl. Acad. Sci. USA 80, 3513-3516). In heart ventricle and submandibular gland, NGF first became detectable around the time of initial innervation by sympathetic neurons (E12 and E13, respectively) and increased respectively 14- and 7-fold in the following 2 days, to reach adult levels already at E14 for heart ventricle (1.4 +/- 0.2 ng NGF/g wet wt). NGF in the superior cervical ganglion (SCG) was first detected at the same time as in its target organ, the submandibular gland. NGF content in the SCG then increased 6-fold during the next 2 days and continued to increase until the end of the third postnatal week, when adult levels were reached. Although the levels of NGF in the adult mouse submandibular gland are sexually dimorphic and six orders of magnitude higher than those in other sympathetic target organs, no sex difference in the NGF content was found in either developing submandibular gland or SCG until the end of the third postnatal week. Moreover, the steep NGF increase observed in the male submandibular gland after postnatal Day 18 (250-fold within the following 3 days and up to the 55,000-fold in the next 7 days) was not reflected in a corresponding increase in the NGF content of the male SCG. These data indicate that, in accordance with earlier findings (see Levi-Montalcini, R., and Angeletti, P. U. (1968), Physiol. Rev. 48, 534-569), SCG neurons do not have access to the large amounts of NGF synthesized during and after adolescence in the mouse submandibular gland. Our results support the concept that initial fiber outgrowth of sympathetic neurons is neither dependent on NGF nor mediated by it. The time course of NGF levels in the SCG is consistent with the concept that sympathetic neurons are provided with NGF by means of retrograde axonal transport from the innervated organs already early in development.     

Sympathetic Neurons Express and Secrete MMP-2 and MT1-MMP to Control Nerve Sprouting via Pro-NGF Conversion

Recently, we have shown that high frequency electrical field stimulation (HFES) of sympathetic neurons (SN) induces nerve sprouting by up-regulation of nerve growth factor (NGF) which targets the tyrosine kinase A receptor (TrkA) in an autocrine/paracrine manner. There is increasing evidence that matrix metalloproteinase-2 (MMP-2) is not only involved in extracellular matrix (ECM) turnover but may also exert beneficial effects during neuronal growth. Therefore, this study aimed to investigate the regulation and function of MMP-2 and its major activator membrane type 1-matrix metalloproteinase (MT1-MMP) as well its inhibitor TIMP-1 in SN under conditions of HFES. Moreover, we analyzed molecular mechanisms of the beneficial effect of losartan, an angiotensin II type I receptor (AT-1)blocker on HFES-induced nerve sprouting. Cell cultures of SN from the superior cervical ganglia (SCG) of neonatal rats were electrically stimulated for 48 h with a frequency of 5 or 50 Hz. HFES increased MMP-2 and MT1-MMP mRNA and protein expression, whereas TIMP-1 expression remained unchanged. Under conditions of HFES, we observed a shift from pro- to active-MMP-2 indicating an increase in MMP-2 enzyme activity. Specific pharmacological MMP-2 inhibition contributed to an increase in pro-NGF amount in the cell culture supernatant and significantly reduced HFES-induced neurite outgrowth. Losartan abolished HFES-induced nerve sprouting in a significant manner by preventing HFES-induced NGF, MMP-2, and MT1-MMP up-regulation. In summary, specific MMP-2 blockade prevents sympathetic nerve sprouting (SNS) by inhibition of pro-NGF conversion while losartan abolishes HFES-induced SNS by reducing total NGF, MMP-2 and MT1-MMP expression.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulation of Sympathetic Neuron Neuropeptide Y and Catecholamine Expression

Abstract: Two forms of pituitary adenylate cyclase-activating polypeptide (PACAP), the 38- and 27-amino-acid forms (PACAP38 and PACAP27, respectively), which share amino acid sequence homology with vasoactive intestinal peptide (VIP), were evaluated for their abilities to regulate sympathetic neuron catecholamine and neuropeptide Y (NPY) expression. PACAP38 and PACAP27 potently and efficaciously stimulated NPY and catecholamine secretion in primary cultured superior cervical ganglion (SCG) neurons; 100- to 1,000-fold higher concentrations of VIP were required to modulate secretion, suggesting that SCG neurons express the PACAP-selective type I receptor. PACAP38 elicited a sustained seven- to ninefold increase in the rate of NPY secretion and three-fold stimulation in the rate of catecholamine release. PACAP38 and PACAP27 produced parallel neuronal NPY and catecholamine release, but cellular levels of NPY and catecholamines were differentially regulated. Sympathetic neuron NPY content was decreased, whereas cellular total catecholamine levels were elevated by the PACAP peptides; total NPY and catecholamine levels (secreted plus cellular content) were increased. In concert with the increased total peptide and transmitter production, pro-NPY and tyrosine hydroxylase mRNA levels were elevated. Furthermore, PACAP38 was more efficacious than PACAP27 in regulating pro-NPY and tyrosine hydroxylase mRNA. SCG neuronal expression of mRNA encoding the type I PACAP receptor further supported the studies demonstrating that sympathetic neuronal levels of NPY and catecholamine content and secretion and mRNA are differentially regulated by the PACAP peptides.     

Retrograde Transport of Endogenous Nerve Growth Factor in Superior Cervical Ganglion of Adult Rats

The concentration of naturally synthesized nerve growth factor (NGF) was measured in various tissues of adult rats, using a highly sensitive two-site enzyme immunoassay. The highest concentration was found in the superior cervical sympathetic ganglion (SCG). Transection of the postganglionic external carotid nerve (ECN) reduced the ganglionic level of NGF more than did section of the internal carotid nerve (ICN). When both the preganglionic nerve and the ECN were cut, the ganglionic NGF level decreased even more. On the other hand, when the preganglionic nerve and the ICN were both sectioned, leaving the ECN intact, endogenous NGF content in the SCG was significantly enhanced 3-9 h after operation. Bilateral extirpation of submaxillary gland produced a rapid decrease in ganglionic NGF 3-6 h after operation, and even unilateral removal of one salivary gland caused a decrease in both ganglia, which was however much greater in the ipsi- than in the contralateral ganglion. Removal of the eyeballs caused a much smaller reduction in ganglionic NGF than did removal of the glands. These results suggest that the endogenous NGF that accumulates in the SCG is mostly synthesized in the submaxillary gland rather than in the iris, and that it is transported to the SCG, mostly via the ipsilateral ECN.     

Cortical Overexpression of Neuronal Calcium Sensor-1 Induces Functional Plasticity in Spinal Cord Following Unilateral Pyramidal Tract Injury in Rat

Following trauma of the adult brain or spinal cord the injured axons of central neurons fail to regenerate or if intact display only limited anatomical plasticity through sprouting. Adult cortical neurons forming the corticospinal tract (CST) normally have low levels of the neuronal calcium sensor-1 (NCS1) protein. In primary cultured adult cortical neurons, the lentivector-induced overexpression of NCS1 induces neurite sprouting associated with increased phospho-Akt levels. When the PI3K/Akt signalling pathway was pharmacologically inhibited the NCS1-induced neurite sprouting was abolished. The overexpression of NCS1 in uninjured corticospinal neurons exhibited axonal sprouting across the midline into the CST-denervated side of the spinal cord following unilateral pyramidotomy. Improved forelimb function was demonstrated behaviourally and electrophysiologically. In injured corticospinal neurons, overexpression of NCS1 induced axonal sprouting and regeneration and also neuroprotection. These findings demonstrate that increasing the levels of intracellular NCS1 in injured and uninjured central neurons enhances their intrinsic anatomical plasticity within the injured adult central nervous system.     

Neuronal nicotinic acetylcholine receptor alpha3 subunit protein in rat brain and sympathetic ganglion measured using a subunit-specific antibody: regional and ontogenic expression

A synthetic peptide corresponding to the C-terminus of the alpha 3 subunit of the rat neuronal nicotinic acetylcholine receptor (nAChR) was used to generate a rabbit polyclonal alpha 3 antibody. The specificity of this antibody was characterized by immunoblotting, immunohistochemical and immunoprecipitation techniques. Using this antibody, the relative densities of the alpha 3 subunit were quantitatively determined in different brain regions and in superior cervical ganglion (SCG). Among these regions, SCG, interpeduncular nucleus (IPN) and pineal gland showed the highest levels of alpha 3 protein expression. Habenula and superior colliculi had intermediate levels of expression. Low levels were found in cerebral cortex, hippocampus and cerebellum. The ontogenic profile of the alpha 3 subunit in the SCG was also determined. The alpha 3 protein level is low at postnatal day (P 1), but increases rapidly during the first seven postnatal days. This level then plateaus and remains stable through postnatal day 35. These findings suggest that neuronal nAChRs containing the alpha 3 subunit participate in important roles in specific regions of the rat brain and the SCG.