Humberto Gutierrez

NF-κB signalling regulates the growth of neural processes in the developing PNS and CNS

The proper growth and elaboration of neural processes is essential for the establishment of a functional nervous system during development and is an integral feature of neural plasticity throughout life. Nuclear factor-kappa B (NF-κB) is classically known for its ubiquitous roles in inflammation, immune and stress-related responses and regulation of cell survival in all tissues, including the nervous system. NF-κB participation in other cellular processes remains poorly understood. Here we report a mechanism for controlling the growth of neural processes in developing peripheral and central neurons involving the transcription factor NF-κB. Inhibiting NF-κB activation with super-repressor IκB-α, BAY 11 7082 (IκB-α phosphorylation inhibitor) or N-acetyl-Leu-Leu-norleucinal (proteosomal degradation inhibitor), or inhibiting NF-κB transcriptional activity with κB decoy DNA substantially reduced the size and complexity of the neurite arbors of sensory neurons cultured with brain-derived neurotrophic factor while having no effect on their survival. NF-κB exerted this effect during a restricted period of development following the phase of naturally occurring neuronal death when the processes and connections of the remaining neurons are extensively modified and refined. Inhibiting NF-κB activation or NF-κB transcriptional activity in layer 2 pyramidal neurons in postnatal somatosensory cortical slices reduced dendritic arbor size and complexity. This function of NF-κB has important implications for neural development and may provide an explanation for reported involvement of NF-κB in learning and memory.

Regulation of neural process growth, elaboration and structural plasticity by NF-κB

The nuclear factor-kappa B (NF-κB) family of transcription factors has recently emerged as a major regulator of the growth and elaboration of neural processes. NF-κB signaling has been implicated in controlling axon initiation, elongation, guidance and branching and in regulating dendrite arbor size and complexity during development and dendritic spine density in the adult. NF-κB is activated by a variety of extracellular signals, and either promotes or inhibits growth depending on the phosphorylation status of the p65 NF-κB subunit. These novel roles for NF-κB, together with recent evidence implicating NF-κB in the regulation of neurogenesis in the embryo and adult, have important implications for neural development and for learning and memory in the mature nervous system.

Regulation of axonal and dendritic growth by the extracellular calcium-sensing receptor

The extracellular calcium-sensing receptor (CaSR) monitors the systemic, extracellular, free ionized-calcium level ([Ca2+]o) in organs involved in systemic [Ca2+]o homeostasis. However, CaSR is also expressed in the nervous system, where its role is unknown. We found large amounts of CaSR in perinatal mouse sympathetic neurons when their axons were innervating and branching extensively in their targets. Manipulating CaSR function in these neurons by varying [Ca2+]o, using CaSR agonists and antagonists, or expressing a dominant-negative CaSR markedly affected neurite growth in vitro. Sympathetic neurons lacking CaSR had smaller neurite arbors in vitro, and sympathetic innervation density was reduced in CaSR-deficient mice in vivo. Hippocampal pyramidal neurons, which also express CaSR, had smaller dendrites when transfected with dominant-negative CaSR in postnatal organotypic cultures. Our findings reveal a crucial role for CaSR in regulating the growth of neural processes in the peripheral and central nervous systems.

HGF regulates the development of cortical pyramidal dendrites

Although hepatocyte growth factor (HGF) and its receptor tyrosine kinase MET are widely expressed in the developing and mature central nervous system, little is known about the role of MET signaling in the brain. We have used particle-mediated gene transfer in cortical organotypic slice cultures established from early postnatal mice to study the effects of HGF on the development of dendritic arbors of pyramidal neurons. Compared with untreated control cultures, exogenous HGF promoted a highly significant increase in dendritic growth and branching of layer 2 pyramidal neurons, whereas inactivation of endogenous HGF with function-blocking, anti-HGF antibody caused a marked reduction in size and complexity of the dendritic arbors of these neurons. Furthermore, pyramidal neurons transfected with an MET dominant-negative mutant receptor likewise had much smaller and less complex dendritic arbors than did control transfected neurons. Our results indicate that HGF plays a role in regulating dendritic morphology in the developing cerebral cortex.

The death receptor antagonist FAIM promotes neurite outgrowth by a mechanism that depends on ERK and NF-κB signaling

Fas apoptosis inhibitory molecule (FAIM) is a protein identified as an antagonist of Fas-induced cell death. We show that FAIM overexpression fails to rescue neurons from trophic factor deprivation, but exerts a marked neurite growth–promoting action in different neuronal systems. Whereas FAIM overexpression greatly enhanced neurite outgrowth from PC12 cells and sympathetic neurons grown with nerve growth factor (NGF), reduction of endogenous FAIM levels by RNAi decreased neurite outgrowth in these cells. FAIM overexpression promoted NF-κB activation, and blocking this activation by using a super-repressor IκBα or by carrying out experiments using cortical neurons from mice that lack the p65 NF-κB subunit prevented FAIM-induced neurite outgrowth. The effect of FAIM on neurite outgrowth was also blocked by inhibition of the Ras–ERK pathway. Finally, we show that FAIM interacts with both Trk and p75 neurotrophin receptor NGF receptors in a ligand-dependent manner. These results reveal a new function of FAIM in promoting neurite outgrowth by a mechanism involving activation of the Ras–ERK pathway and NF-κB.

Blockade of N-methyl-D-aspartate receptors in the insular cortex disrupts taste aversion and spatial memory formation.

The present experiments examined the effects of direct intracortical microinjections of the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid directly into the insular cortex of rats, before or immediately after training of conditioned taste aversion and the water maze spatial learning task. In the first series of experiments animals received bilateral injections of 2-amino-5-phosphonovaleric acid prior to taste aversion conditioning or spatial training. A strong disruptive effect was found in the acquisition of training tasks. To determine the possible involvement of N-methyl-D-aspartate receptors in the early post-training processes taking place in the cortex during both learning paradigms, in a second series of experiments, animals received bilateral 2-amino-5-phosphonovaleric acid microinjections 30, 60 or 120 min after the acquisition trial, and 15 min before the retention test. For spatial learning successive treatments were independently done either starting at the onset of the asymptotic phase of the learning curve, 0, 30 or 120 min after finishing the training session, as well as 15 min before the retention test trial. The conditioned taste aversion task remained sensitive to N-methyl-D-aspartate blockade during a period of at least 2 h after the first presentation of the gustatory stimulus, while in the case of the spatial learning task, a gradually decreasing effect was observed from the onset of the asymptotic phase onwards. Taken together, these results provide direct evidence for N-methyl-D-aspartate receptor involvement in cortical regulation of memory formation. Furthermore, our results suggest that in the same cortical region, a different time-course for the activation of N-methyl-D-aspartate-dependent mechanisms occurs during the early formation of cortically mediated memories, depending on the particular behavioural task.

Nuclear factor kappa B signaling either stimulates or inhibits neurite growth depending on the phosphorylation status of p65/RelA.

Nuclear factor κB (NF-κB) signaling is known to promote neurite growth from developing sensory neurons and to enhance the size and complexity of pyramidal neuron dendritic arbors in the developing cerebral cortex. In marked contrast, here we show that NF-κB signaling can also exert a potent inhibitory influence on neurite growth in certain neurons, and can either promote or inhibit neurite growth in the same neurons depending on the mechanism of NF-κB activation. In neonatal superior cervical ganglion sympathetic neurons, enhancing NF-κB transcriptional activity by overexpressing either the p65 NF-κB subunit or the IκB kinase-β (IKKβ) subunit of the IκB kinase complex, or by tumor necrosis factor α (TNFα) treatment, strongly inhibits neurite growth. Paradoxically in neonatal nodose ganglion sensory neurons, enhancing NF-κB transcriptional activity by p65/p50 overexpression increases neurite growth, whereas enhancing NF-κB transcriptional activity by IKKβ overexpression inhibits neurite growth. In addition to activating NF-κB, IKKβ overexpression leads to phosphorylation of p65 on serine 536. Blockade of serine 536 phosphorylation by a S536A-p65 mutant protein prevents the growth-inhibitory effects of IKKβ overexpression in both sensory and sympathetic neurons and the growth-inhibitory effects of TNFα on sympathetic neurons. Furthermore, expression of a p65 S536D phosphomimetic mutant inhibits neurite growth from sensory neurons. These results demonstrate that NF-κB can either stimulate or inhibit neurite growth in developing neurons depending on the phosphorylation status of p65.

A fast and accurate procedure for deriving the Sholl profile in quantitative studies of neuronal morphology

The growth and elaboration of neural processes underpins the establishment of neural architecture during development and is a key facet of neural plasticity throughout life. Sholl analysis is a valuable and widely used method for quantifying the extent and complexity of neural processes in the vicinity of the neuronal soma, such as the dendritic arbors of individual neurons in vivo and neuritic arbors of individual neurons in vitro. It consists of tracing a series of concentric rings with regular radial increments centred in the neuronal soma and counting the number X(i) of processes intersecting each ring. This is a laborious and time-consuming procedure that consequently can only be applied to relatively small numbers of neurons. We propose a simpler and accurate method for deriving exactly the same information based only on the relative position, with respect to the cell soma, of the bifurcation (B(i)) and terminal points (T(i)) of processes. By means of the iterative equation X(i)=X(i-1)+B(i)-T(i,) it is possible to automatically reconstruct the complete pattern of intersections between neurites and the concentric rings. We compared our method with the conventional Sholl analysis and found that our simplified procedure is approximately five times faster permitting numerically larger samples to be analyzed. We further tested the sensitivity of our method of analysis by looking at the effect of preventing NF-kappaB signaling on BDNF-dependent neuritic growth in sensory neurons.

Nuclear factor-kappaB activation via tyrosine phosphorylation of inhibitor kappaB-alpha is crucial for ciliary neurotrophic factor-promoted neurite growth from developing neurons

The cytokine ciliary neurotrophic factor (CNTF) promotes the growth of neural processes from many kinds of neurons in the developing and regenerating adult nervous system, but the intracellular signaling mechanisms mediating this important function of CNTF are poorly understood. Here, we show that CNTF activates the nuclear factor-κB (NF-κB) transcriptional system in neonatal sensory neurons and that blocking NF-κB-dependent transcription inhibits CNTF-promoted neurite growth. Selectively blocking NF-κB activation by the noncanonical pathway that requires tyrosine phosphorylation of inhibitor κB-α (IκB-α), but not by the canonical pathway that requires serine phosphorylation of IκB-α, also effectively inhibits CNTF-promoted neurite growth. CNTF treatment activates spleen tyrosine kinase (SYK) whose substrates include IκB-α. CNTF-induced SYK phosphorylation is rapidly followed by increased tyrosine phosphorylation of IκB-α, and blocking SYK activation or tyrosine phosphorylation of IκB-α prevents CNTF-induced NF-κB activation and CNTF-promoted neurite growth. These findings demonstrate that NF-κB signaling by an unusual activation mechanism is essential for the ability of CNTF to promote the growth of neural processes in the developing nervous system.

NGF-promoted axon growth and target innervation requires GITRL-GITR signaling.

Nerve growth factor (NGF) has an important role in regulating sympathetic neuron survival and target field innervation during development. Here we show that glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR), a member of the TNF superfamily, and its ligand (GITRL) are co-expressed in mouse sympathetic neurons when their axons are innervating their targets under the influence of target-derived NGF. In culture, GITRL enhanced NGF-promoted neurite growth from neonatal sympathetic neurons, and preventing GITR-GITRL interaction in these neurons or knocking down GITR inhibited NGF-promoted neurite growth without affecting neuronal survival. Tnfrsf18−/− (Gitr) neonates have reduced sympathetic innervation density in vivo compared with Gitr+/+ littermates. GITR activation is required for the phosphorylation of extracellular signal–regulated kinases 1 and 2 by NGF that is necessary for neurite growth. Our results reveal a previously unknown signaling loop in developing sympathetic neurons that is crucial for NGF-dependent axon growth and target innervation.