Nerve Guidance by a Decellularized Fibroblast Extracellular Matrix.

Spinal cord and peripheral nerve injuries require the regeneration of nerve fibers across the lesion site for successful recovery. Providing guidance cues and soluble factors to promote neurite outgrowth and cell survival can enhance repair. The extracellular matrix (ECM) plays a key role in tissue repair by controlling cell adhesion, motility, and growth. In this study, we explored the ability of a mesenchymal ECM to support neurite outgrowth from neurons in the superior cervical ganglia (SCG). Length and morphology of neurites extended on a decellularized fibroblast ECM were compared to those on substrates coated with laminin, a major ECM protein in neural tissue, or fibronectin, the main component of a mesenchymal ECM. Average radial neurite extension was equivalent on laminin and on the decellularized ECM, but contrasted with the shorter, curved neurites observed on the fibronectin substrate. Differences between neurites on fibronectin and on other substrates were confirmed by fast Fourier transform analyses. To control the direction of neurite outgrowth, we developed an ECM with linearly aligned fibril organization by orienting the fibroblasts that deposit the matrix on a polymeric surface micropatterned with a striped chemical interface. Neurites projected from SCGs appeared to reorient in the direction of the pattern. These results highlight the ability of a mesenchymal ECM to enhance neurite extension and to control the directional outgrowth of neurites. This micropatterned decellularized ECM architecture has potential as a regenerative microenvironment for nerve repair.

Stargazin-related protein γ7 is associated with signalling endosomes in superior cervical ganglion neurons and modulates neurite outgrowth.

The role(s) of the newly discovered stargazin-like γ-subunit proteins remains unclear; although they are now widely accepted to be transmembrane AMPA receptor regulatory proteins (TARPs), rather than Ca²âº channel subunits, it is possible that they have more general roles in trafficking within neurons. We previously found that γ7 subunit is associated with vesicles when it is expressed in neurons and other cells. Here, we show that γ7 is present mainly in retrogradely transported organelles in sympathetic neurons, where it colocalises with TrkA-YFP, and with the early endosome marker EEA1, suggesting that γ7 localises to signalling endosomes. It was not found to colocalise with markers of the endoplasmic reticulum, mitochondria, lysosomes or late endosomes. Furthermore, knockdown of endogenous γ7 by short hairpin RNA transfection into sympathetic neurons reduced neurite outgrowth. The same was true in the PC12 neuronal cell line, where neurite outgrowth was restored by overexpression of human γ7. These findings open the possibility that γ7 has an essential trafficking role in relation to neurite outgrowth as a component of endosomes involved in neurite extension and growth cone remodelling.

The death receptor antagonist FLIP-L interacts with Trk and is necessary for neurite outgrowth induced by neurotrophins.

FLICE-inhibitory protein (FLIP) is an endogenous inhibitor of the signaling pathway triggered by the activation of death receptors. Here, we reveal a novel biological function for the long form of FLIP (FLIP-L) in neuronal differentiation, which can be dissociated from its antiapoptotic role. We show that FLIP-L is expressed in different regions of the mouse embryonic nervous system. Immunohistochemistry of mouse brain sections at different stages reveals that, in neurons, FLIP is expressed early during the embryonic neuronal development (embryonic day 16) and decreases at later stages (postnatal days 5-15), when its expression is essentially detected in glial cells. FLIP-L overexpression significantly enhances neurotrophin-induced neurite outgrowth in motoneurons, superior cervical ganglion neurons, and PC12 cells. Conversely, the downregulation of FLIP-L protein levels by specific RNA interference significantly reduces neurite outgrowth, even in the presence of the appropriate neurotrophin stimulus. Moreover, NGF-dependent activation of two main intracellular pathways involved in the regulation of neurite outgrowth, extracellular signal-regulated kinases (ERKs) and nuclear factor kappaB (NF-kappaB), is impaired when endogenous FLIP-L is downregulated, although TrkA remains activated. Finally, we demonstrate that FLIP-L interacts with TrkA, and not with p75(NTR), in an NGF-dependent manner, and endogenous FLIP-L interacts with TrkB in whole-brain lysates from embryonic day 15 mice embryos. Altogether, we uncover a new role for FLIP-L as an unexpected critical player in neurotrophin-induced mitogen-activated protein kinase/ERK- and NF-kappaB-mediated control of neurite growth in developing neurons.

Maturation and long-term hypoxia alters Ca2+-induced Ca2+ release in sheep cerebrovascular sympathetic neurons.

The contribution of sympathetic nerves arising from the superior cervical ganglia (SCG) toward the growth and function of cerebral blood vessels is pertinent throughout maturation as well as in response to cardiovascular stress imposed by high-altitude long-term hypoxia (LTH). The function of SCG sympathetic neurons is dependent on intracellular Ca2+ concentration ([Ca2+]i) signaling, which is strongly influenced by a process known as Ca(2+)-induced Ca2+ release (CICR) from the smooth endoplasmic reticulum (SER). In this study, we used the sheep SCG neuronal model to test the hypotheses that maturation decreases CICR and high-altitude LTH depresses CICR in fetal SCG neurons but not in those of the adult. We found that the contribution of CICR to electric field stimulation (EFS)-evoked [Ca2+]i transients was greatest in SCG cells from normoxic fetuses and was abolished by LTH. The decline in CICR was associated with a reduction in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in fetal SCG cells during LTH, reducing SER Ca2+ levels below the threshold needed for the coupling of Ca2+ influx and CICR. With respect to the maturation from the fetus to adult, the decrease in CICR may reflect both a reduction in the levels of ryanodine receptor isoforms 2 and 3 and SERCA function. In response to LTH and in contrast to the fetus, CICR function in adult SCG cells is maintained and may reflect alterations in other mechanisms that modulate the CICR process. As CICR is instrumental in the function of sympathetic neurons within the cerebrovasculature, the loss of this signaling mechanism in the fetus may have consequences for the adaptation to LTH in terms of fetal susceptibility to vascular insults.

Synergistic effects of osteonectin and NGF in promoting survival and neurite outgrowth of superior cervical ganglion neurons.

Schwann cells (SCs) play a major role in the successful regeneration of peripheral nerves regeneration. Here we examined the effects of osteonectin (ON), a major factor secreted by SCs, on survival and neuritogenesis of mouse superior cervical ganglion (SCG) neurons. SC conditioned medium (SCCM) not only promoted the survival and neuritogenesis of SCG neurons at a level comparable to nerve growth factor (NGF) but also doubled the neurite length of NGF-treated SCG neurons. SCCM neuritogenic effects were not blocked by the tyrosine kinase receptor (Trk) inhibitor K252a demonstrating that these are not due solely to classical neurotrophic factors. Anti-ON neutralizing antibody diminished the SCCM-induced survival and neuritogenesis significantly. In the presence of K252a, the SCCM neuritogenic effects were blocked completely by anti-ON which suggests synergistic effects of ON with Trk-mediated growth factors. ON alone increased the survival and neurite outgrowth of SCG neurons significantly at high density cultures. ON at low concentration acts synergistically with NGF which induced maximum survival and neurite outgrowth (>50% increase). However, ON at high concentration was detrimental to survival (64% decrease) and neurite outgrowth (87% decrease) even in the presence of NGF. The well documented counter-adhesive effect of ON may account for this observation. Nevertheless, the growth promoting effects of ON became more pronounced as the cell density increased which suggests a possible interaction of ON with growth factors secreted by SCG neurons (autocrine or paracrine effects). Taken together, our study indicates that ON plays important roles in nervous system repair through its synergistic effects with growth factors.

Developmental profile of cholinergic and purinergic traits and receptors in peripheral chemoreflex pathway in cats.

This study describes the developmental profile of specific aspects of cholinergic and purinergic neurotransmission in key organs of the peripheral chemoreflex: the carotid body (CB), petrosal ganglion (PG) and superior cervical ganglion (SCG). Using real time RT-PCR and Western blot analyses, we assessed both mRNA and protein expression levels for choline-acetyl-transferase (ChAT), nicotinic receptor (subunits alpha3, alpha4, alpha7, and beta2), ATP and purinergic receptors (P2X2 and P2X3). These analyses were performed on tissue from 1- and 15-day-old, 2-month-old, and adult cats. During development, ChAT protein expression level increased slightly in CB; however, this increase was more important in PG and SCG. In CB, mRNA level for alpha4 nicotinic receptor subunit decreased during development (90% higher in 1-day-old cats than in adults). In the PG, mRNA level for beta2 nicotinic receptor subunit increased during development (80% higher in adults than in 1-day-old cats). In SCG, mRNA for alpha7 nicotinic receptor levels increased (400% higher in adults vs. 1-day-old cats). Conversely, P2X2 receptor protein level was not altered during development in CB and decreased slightly in PG; a similar pattern was observed for the P2X3 receptor. Our findings suggest that in cats, age-related changes in cholinergic and purinergic systems (such as physiological expression of receptor function) are significant within the afferent chemoreceptor pathway and likely contribute to the temporal changes of O2-chemosensitivity during development.

Bone morphogenetic protein down-regulation of neuronal pituitary adenylate cyclase-activating polypeptide and reciprocal effects on vasoactive intestinal peptide expression.

Among bone morphogenetic proteins (BMPs), the decapentaplegic (Dpp; BMP2, BMP4) and glass bottom boat (Gbb/60A; BMP5, BMP6, BMP7) subgroups have well-described functions guiding autonomic and sensory neuronal development, fiber formation and neurophenotypic identities. Evaluation of rat superior cervical ganglia (SCG) post-ganglionic sympathetic neuron developmental regulators identified that selected BMPs of the transforming growth factor beta superfamily have reciprocal effects on neuronal pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) expression. Dpp and Gbb/60A BMPs rapidly down-regulated PACAP expression, while up-regulating other sympathetic neuropeptides, including PACAP-related VIP. The suppressive effects of BMP on PACAP mRNA and peptide expression were potent, efficacious and phosphorylated mothers against decapentaplegic homolog (Smad) signaling-dependent. Axotomy of SCG dramatically increases PACAP expression, and the possibility that abrogation of inhibitory retrograde target tissue BMP signaling may contribute to this up-regulation of sympathetic neuron PACAP was investigated. Replacement of BMP6 to SCG explant preparations significantly blunted the injury-induced elevated PACAP expression, with a concomitant decrease in sympathetic PACAP-immunoreactive neuron numbers. These studies suggested that BMPs modulate neuropeptide identity and diversity by stimulating or restricting the expression of specific peptidergic systems. Furthermore, the liberation of SCG neurons from target-derived BMP inhibition following axotomy may be one participating mechanism associated with injury-induced neuropeptidergic plasticity.

Differential expression and regulation of the high-affinity choline transporter CHT1 and choline acetyltransferase in neurons of superior cervical ganglia.

Previous studies revealed that leukemia inhibitory factor (LIF) and retinoic acid (RA) induce a noradrenergic to cholinergic switch in cultured sympathetic neurons of superior cervical ganglia (SCG) by up-regulating the coordinate expression of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. Here, we examined the effect of both factors on high-affinity choline uptake (HACU) and on expression of the high-affinity choline transporter CHT1. We found that HACU and CHT1-mRNA levels are up-regulated by LIF and down-regulated by RA in these neurons. Thus, in contrast to LIF, RA differentially regulates the expression of the presynaptic cholinergic proteins. Moreover, we showed that untreated SCG neurons express HACU and CHT1-mRNAs at much higher levels than ChAT activity and transcripts. In intact SCG, CHT1-mRNAs are abundant and synthesized by the noradrenergic neurons themselves. This study provides the first example of CHT1 expression in neurons which do not use acetylcholine as neurotransmitter.

HGF promotes survival and growth of maturing sympathetic neurons by PI-3 kinase- and MAP kinase-dependent mechanisms.

Hepatocyte growth factor (HGF) is a pleiotrophic factor whose many functions include promoting neuronal survival and growth. Hitherto, these effects have been observed in the presence of other neurotrophic factors like NGF and CNTF, and this requirement for an accessory factor has made it difficult to elucidate the signaling pathways that mediate its survival and growth-enhancing effects. Here, we show that HGF promotes the survival of mature sympathetic neurons of the superior cervical ganglion (SCG) grown at low density in defined medium lacking other neurotrophic factors. This effect was first clearly observed in cultures established from postnatal day 20 (P20) mice and became maximal by P40. HGF also enhanced the growth of neurite arbors from neurons throughout postnatal development and in the adult. HGF treatment resulted in phosphorylation of Akt and ERK1/ERK2. Preventing Akt activation with the phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 blocked the HGF survival response, and inhibition of ERK activation with the MEK inhibitors PD98059 or U0126 reduced the HGF survival response and the neurite growth-promoting effects of HGF. These results indicate that HGF promotes the survival and growth of maturing sympathetic neurons by both PI-3 kinase- and MAP kinase-dependent mechanisms.

The effects of deafferentation and exogenous NGF on neurotrophins and neurotrophin receptor mRNA expression in the adult superior cervical ganglion.

Levels of nerve growth factor (NGF) and neurotrophin-3 (NT-3) protein and neurotrophin receptor mRNA in adult sympathetic neurons were investigated following surgical removal of preganglionic input and/or in vivo administration of NGF. Expression of trkC and p75, but not trkA, was significantly decreased following a 3-week deafferentation of the superior cervical ganglion (SCG). Protein levels of NGF and NT-3 in the SCG were unchanged by deafferentation. A 2-week intracerebroventricular infusion of NGF without deafferentation resulted in enhanced mRNA levels of trkA, trkC, and p75 as well as significantly increased NGF and NT-3 protein in the SCG. When NGF infusion followed deafferentation, both trkA and p75 showed significant increases while trkC levels were similar to control values. NGF protein was not increased in the SCG when deafferentation preceded exogenous NGF, yet NT-3 was elevated and levels were similar to cases receiving NGF infusion only. These results support a role for preganglionic input in trkC and p75 expression in adult sympathetic neurons. The increased levels of NT-3 protein and trkC gene expression observed following NGF infusion suggest that NGF influences NT-3 regulation in adult sympathetic neurons. In addition, the present findings provide evidence that, when preganglionic input is removed prior to the NGF infusion, NT-3 effectively competes with NGF for trkA binding. Taken together, we propose that NT-3 may play a role in the robust sprouting of sympathetic cerebrovascular axons previously observed following NGF administration, particularly when deafferentation precedes the NGF infusion period.

c-jun is essential for sympathetic neuronal death induced by NGF withdrawal but not by p75 activation.

Sympathetic neurons depend on NGF binding to TrkA for their survival during vertebrate development. NGF deprivation initiates a transcription-dependent apoptotic response, which is suggested to require activation of the transcription factor c-Jun. Similarly, apoptosis can also be induced by selective activation of the p75 neurotrophin receptor. The transcriptional dependency of p75-mediated cell death has not been determined; however, c-Jun NH2-terminal kinase has been implicated as an essential component. Because the c-jun-null mutation is early embryonic lethal, thereby hindering a genetic analysis, we used the Cre-lox system to conditionally delete this gene. Sympathetic neurons isolated from postnatal day 1 c-jun-floxed mice were infected with an adenovirus expressing Cre recombinase or GFP and analyzed for their dependence on NGF for survival. Cre immunopositive neurons survived NGF withdrawal, whereas those expressing GFP or those uninfected underwent apoptosis within 48 h, as determined by DAPI staining. In contrast, brain-derived neurotrophic factor (BDNF) binding to p75 resulted in an equivalent level of apoptosis in neurons expressing Cre, GFP, and uninfected cells. Nevertheless, cycloheximide treatment prevented BDNF-mediated apoptosis. These results indicate that whereas c-jun is required for apoptosis in sympathetic neurons on NGF withdrawal, an alternate signaling pathway must be induced on p75 activation.

Postnatal expression of VEGF and its receptor flk-1 in peripheral ganglia.

The postnatal (P0-P12) and adult expression of vascular endothelial growth factor and its receptor flk-1 was investigated in superior cervical (SCG) and dorsal root ganglia (DRG) in mice by immunocytochemistry. At P0 all neurons in SCG and DRG contained VEGF. The number of VEGF-immunoreactive neurons in DRG but not in SCG, decreased postnatally and reached adult levels (34%) at P12. At P0 flk-1 was found in virtually all neurons in the SCG and in roughly half of the neurons in DRG. The number of flk-1 positive neurons then decreased and reached adult levels at P12. The findings demonstrate temporal changes in VEGF and flk-1 expression, suggesting developmental regulation of VEGF activity in peripheral ganglia.

Apoptotic protein expression and activation of caspases is changed following cholinergic denervation and hippocampal sympathetic ingrowth in rat hippocampus.

Following cholinergic denervation of the hippocampus by medial septal lesions, an unusual neuronal reorganization occurs in which peripheral adrenergic fibers arising from superior cervical ganglia grow into the hippocampus (hippocampal sympathetic ingrowth). Recent studies suggest that a similar process, in which sympathetic noradrenergic axons invade the hippocampus, can occur in Alzheimer's disease patients. In the last few years, the occurrence of apoptotic cell death has been studied in Alzheimer's disease patients and in animal models of this disorder. Several studies suggest that the hippocampus is an important area to be considered for apoptotic cell death. In our studies in the rat hippocampus, we have measured the expression of inducers and blockers of apoptosis in membrane, cytosolic and mitochondrial fractions, and the activity of caspases. The level of cytosolic Fas was increased in cholinergic denervation compared to control and hippocampal sympathetic ingrowth groups. The membrane Fas ligand expression was significantly increased in hippocampal sympathetic ingrowth and in cholinergic denervation compared to the control group. The level of caspase-3 (CPP32) was increased in the cholinergic denervation group compared to control and hippocampal sympathetic ingrowth groups. The cytosolic expression of bcl-x was increased in hippocampal sympathetic ingrowth compared to control and cholinergic denervation. The cytosolic activity of caspase-3 appeared to be significantly decreased in hippocampal sympathetic ingrowth and increased in cholinergic denervation groups compared to control and cholinergic denervation/hippocampal sympathetic ingrowth, respectively. From the present results, we suggest that cholinergic denervation may be responsible for pro-apoptotic responses, while hippocampal sympathetic ingrowth may protect neurons from apoptosis in rat dorsal hippocampus.

Characterization of an NGF-P-TrkA retrograde-signaling complex and age-dependent regulation of TrkA phosphorylation in sympathetic neurons.

Nerve growth factor (NGF) is a target-derived trophic factor for developing sympathetic and cutaneous sensory neurons. NGF promotes growth and survival of neurons via activation of the receptor tyrosine kinase TrkA. We used compartmentalized cultures of sympathetic neurons to address the mechanism of NGF signaling from distal axons and terminals to proximal axons and cell bodies. Our results demonstrate that an NGF-phospho-TrkA (NGF-P-TrkA)-signaling complex forms in distal axons and is retrogradely transported as a complex to cell bodies of sympathetic neurons. Although a minor fraction of both NGF and TrkA is retrogradely transported, a large fraction of the NGF that is retrogradely transported is found complexed with retrogradely transported TrkA. Interestingly, the metabolism of the P-TrkA complex is dramatically different in young, NGF-dependent sympathetic neurons as compared to older, NGF-independent sympathetic neurons. After withdrawal of NGF from distal axons of young neurons, P-TrkA within distal axons, as well as within proximal axons and cell bodies, dephosphorylates rapidly. In contrast, after withdrawal of NGF from distal axons of older neurons, P-TrkA within distal axons dephosphorylates completely, although more slowly than that in young neurons, whereas dephosphorylation of P-TrkA within proximal axons and cell bodies occurs markedly more slowly, with at least one-half of the level of P-TrkA remaining 2 d after NGF withdrawal. Thus, P-TrkA within the cell bodies of young, NGF-dependent sympathetic neurons is derived from distal axons. A more stable P-TrkA complex within cell bodies of mature sympathetic neurons may contribute to the acquisition of NGF independence for survival of mature sympathetic neurons.

Differences in the ways sympathetic neurons and endocrine cells process, store, and secrete exogenous neuropeptides and peptide-processing enzymes.

Most neurons store peptides in large dense core vesicles (LDCVs) and release the neuropeptides in a regulated manner. Although LDCVs have been studied in endocrine cells, less is known about these storage organelles in neurons. In this study we use the endogenous peptide NPY (neuropeptide Y) and the endogenous peptide-processing enzyme PAM (peptidylglycine alpha-amidating monooxygenase) as tools to study the peptidergic system in cultured neurons from the superior cervical ganglion (SCG). Once mature, SCG neurons devote as much of their biosynthetic capabilities to neurotransmitter production as endocrine cells devote to hormone production. Unlike pituitary and atrium, SCG neurons cleave almost all of the bifunctional PAM protein they produce into soluble monofunctional enzymes. Very little PAM or NPY is secreted under basal conditions, and the addition of secretagogue dramatically stimulates the secretion of PAM and NPY to a similar extent. Although endocrine cells typically package "foreign" secretory products together with endogenous products, pro-opiomelanocortin- and PAM-derived products encoded by adenovirus in large part were excluded from the LDCVs of SCG neurons. When expressed in corticotrope tumor cells and primary anterior pituitary cultures, the same virally encoded products were metabolized normally. The differences that were observed could reflect differences in the properties of neuronal and endocrine peptidergic systems or differences in the ability of neurons and endocrine cells to express viral transcripts.

Leukemia inhibitory factor and ciliary neurotrophic factor regulate dendritic growth in cultures of rat sympathetic neurons.

Cytokines such as leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have previously been shown to regulate neurotransmitter and neuropeptide synthesis in sympathetic neurons [P.H. Patterson, Leukemia inhibitory factor, a cytokine at the interface between neurobiology and immunology, Proc. Natl. Acad. Sci. USA 91 (1994) 7833-7835]. We considered the possibility that these agents may also affect the development of neuronal cell shape. Intracellular dye injection and immunocytochemistry were used to assess dendritic growth in cultures of perinatal rat sympathetic neurons and the effects of LIF and CNTF were compared to those of osteogenic protein-1 (OP-1), a growth factor that induces profuse dendritic growth in these neurons [P. Lein, M. Johnson, X. Guo, D. Rueger, D. Higgins, Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons, Neuron 15 (1995) 597-605]. Under control conditions, sympathetic neurons formed only axons. Exposure to either LIF or OP-1 stimulated dendritic growth, but the magnitude of the response to LIF was much less than that obtained with OP-1 with respect to both dendritic number and length. Simultaneous exposure to LIF and OP-1 resulted in dendritic growth equivalent to that observed in the presence of LIF alone, suggesting that LIF inhibits the response of neurons to OP-1. Both the stimulatory and inhibitory effects of LIF were mimicked by CNTF, but not by other growth factors. These data suggest that LIF and CNTF regulate dendritic development in a complex manner that is dependent on both the morphological state of the neuron and the presence of other growth factors. However, the net effect of exposure to these cytokines appears to be the production of a population of neurons with rudimentary arbors consisting of only one or two short dendrites.

Pituitary adenylate cyclase-activating peptide (PACAP) and PACAP type 1 receptor expression in regenerating adult mouse and rat superior cervical ganglia in vitro.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a regulatory peptide belonging to the vasoactive intestinal peptide (VIP) family, is widely distributed in the central and peripheral nervous system. Recent studies have shown that PACAP expression is upregulated in sensory neurons in response to axonal injury. Here we report that PACAP and PACAP type 1 receptors are located in rat and mouse superior cervical ganglia (SCG). PACAP-immunoreactivity (-IR) was demonstrated in preganglionic fibers, whereas only occasional PACAP-IR cell bodies could be observed. In situ hybridization histochemistry using 35S-labeled deoxyribonucleotide probes confirmed that PACAP mRNA was present only in occasional cell bodies. In contrast, PACAP type 1 receptor mRNA was expressed in virtually all cell bodies within the ganglia. After removal and culturing of the SCG for 24 h, there was a marked increase in PACAP mRNA, whilst PACAP type 1 receptor mRNA expression appeared to be downregulated in most nerve cell bodies except for a few scattered neurons displaying a strong upregulation. The total specific binding of PACAP to isolated SCG membranes as assayed by [125I]PACAP-27 binding showed an increase in SCG cultured for 48 h. PACAP-27 neither affected axonal outgrowth from the cultured SCG nor the survival of cells within the SCG. We conclude that PACAP and PACAP receptors are rapidly upregulated in sympathetic ganglia in response to axonal injury and that PACAP may play a role during nerve regeneration.