Barbara Hausott

The neurotoxic effects of amitriptyline are mediated by apoptosis and are effectively blocked by inhibition of caspase activity

Oral tricyclic antidepressants, widely used as adjuncts in the treatment of chronic pain, block sodium channels in vitro and nerve conduction in vivo. However, toxicity of amitriptyline has been observed after neural application. We therefore investigated the mechanism and possible prevention of amitriptyline neurotoxicity. To assess dose-dependent neurotoxicity of amitriptyline, we incubated neuron cultures from adult rat dorsal root ganglia with amitriptyline and quantified neuronal survival. Additionally, we investigated accepted markers of apoptosis (mitochondrial membrane potential, cytosolic cytochrome c, and activated caspase-3) and co-incubated amitriptyline with an inhibitor of caspase activity, z-vad-fmk, to assess the effect on cell survival. We found a dose-dependent neurotoxic effect of amitriptyline. Neurons incubated with amitriptyline exhibited loss of mitochondrial membrane potential, release of cytochrome c into the cytoplasm, and activation of caspase-3. Co-incubation with z-vad-fmk substantially improved neuronal survival in culture. In conclusion, amitriptyline-induced neurotoxicity is mediated by apoptosis and is attenuated by inhibition of caspase activity, suggesting that inhibition of apoptotic pathways may be efficient at alleviating local anesthetic–induced neurotoxicity. In vivo studies will have to corroborate whether the co-injection of anti-apoptotic drugs with local anesthetics decreases neurotoxic side effects.

Rho GTPases as regulators of morphological neuroplasticity.

Summary GTPases function as intracellular, bimolecular switches by adopting different conformational states in response to binding GDP or GTP. Their activation is mediated through cell-surface receptors. Rho GTPases act on several downstream effectors involved in cellular morphogenesis, cell polarity, migration and cell division. In neurons, Rho GTPases regulate various features of dendritic and axonal outgrowth during development and regeneration mainly through their effects on the cytoskeleton. This review summarizes the main functions of Rho, Rac and Cdc42 GTPases as key regulators of morphological neuroplasticity under normal and pathological conditions.

Neurotoxicity of Lidocaine Involves Specific Activation of the p38 Mitogen-activated Protein Kinase, but Not Extracellular Signal–regulated or c-jun N-terminal Kinases, and Is Mediated by Arachidonic Acid Metabolites

Background:Pharmacologic inhibition of the p38 mitogen-activated protein kinase (MAPK) leads to a reduction in lidocaine neurotoxicity in vitro and in vivo. The current study investigated in vitro the hypotheses that lidocaine neurotoxicity is specific for dorsal root ganglion cells of different size or phenotype, involves time-dependent and specific activation of the p38 MAPK, that p38 MAPK inhibitors are only effective if applied with local anesthetic, and that p38 MAPK activation triggers activation of lipoxygenase pathways. Methods:The authors used primary sensory neuron cultures and pheochromocytoma cell line cultures to detect time-dependent activation of the p38 MAPK or related pathways such as extracellular signal–regulated kinases and c-jun N-terminal kinases. Cells were divided by size or by immunoreactivity for calcitonin gene–related peptide or isolectin B4, indicative of nociceptive phenotype. The authors also investigated whether arachidonic acid pathways represent a downstream effector of the p38 MAPK in local anesthetic–induced neurotoxicity. Results:All types of dorsal root ganglion cells were subject to neurotoxic effects of lidocaine, which were mediated by specific activation of the p38 MAPK but not extracellular signal–regulated kinases or c-jun N-terminal kinases. Neuroprotective efficacy of p38 MAPK inhibitors declined significantly when administered more than 1 h after lidocaine exposure. Activation of p38 MAPK preceded activation of arachidonic acid pathways. Neurotoxicity of lidocaine, specific activation of p38 MAPK, and neuroprotective effects of a p38 MAPK inhibitor were further confirmed in pheochromocytoma cell line cultures. Conclusions:Specific and time-dependent activation of the p38 MAPK is involved in lidocaine-induced neurotoxicity, most likely followed by activation of lipoxygenase pathways.

The pros and cons of growth factors and cytokines in peripheral axon regeneration.

Injury to a peripheral nerve induces a complex cellular and molecular response required for successful axon regeneration. Proliferating Schwann cells organize into chains of cells bridging the lesion site, which is invaded by macrophages. Approximately half of the injured neuron population sends out axons that enter the glial guidance channels in response to secreted neurotrophic factors and neuropoietic cytokines. These lesion-associated polypeptides create an environment that is highly supportive for axon regrowth, particularly after acute injury, and ensure that the vast majority of regenerating axons are directed toward the distal nerve stump. Unfortunately, most neurotrophic factors and neuropoietic cytokines are also strong stimulators of axonal sprouting. Although some of the axonal branches will withdraw at later stages, the sprouting effect contributes to the misdirection of reinnervation that results in the lack of functional recovery observed in many patients with peripheral nerve injuries. Here, we critically review the role of neuronal growth factors and cytokines during axon regeneration in the peripheral nervous system. Their differential effects on axon elongation and sprouting were elucidated in various studies on intraneuronal signaling mechanisms following nerve lesion. The present data define a goal for future therapeutic strategies, namely, to selectively stimulate a Ras/Raf/ERK-mediated axon elongation program over an intrinsic PI3K-dependent axonal sprouting program in lesioned motor and sensory neurons. Instead of modulating growth factor or cytokine levels at the lesion site, targeting specific intraneuronal molecules, such as the negative feedback inhibitors of ERK signaling, has been shown to promote long-distance regeneration while avoiding sprouting of regenerating axons until they have reached their target areas.

Sprouty2 down-regulation promotes axon growth by adult sensory neurons

Fibroblast growth factors (FGFs) play a prominent role in axonal growth during development and repair. Treatment with FGF-2 or overexpression of FGF receptors promotes peripheral axon regeneration mainly by activation of extracellular signal-regulated kinase (ERK). The Ras/Raf/ERK pathway is under the control of Sprouty proteins acting as negative feedback inhibitors. We investigated the expression of Sprouty isoforms in adult sensory neurons of dorsal root ganglia (DRG) as well as the effects of Sprouty inhibition on axon growth by small interfering RNAs (siRNAs). Sprouty2 revealed the highest expression level in DRG neurons. Down-regulation of Sprouty2 promoted elongative axon growth by adult sensory neurons accompanied by enhanced FGF-2-induced activation of ERK and Ras, whereas Sprouty2 overexpression inhibited axon growth. Sprouty2 was not regulated in vivo in response to a sciatic nerve lesion. Together, our results imply that Sprouty2 is highly expressed in adult peripheral neurons and its down-regulation strongly promotes elongative axon growth by activation of the Ras/Raf/ERK pathway.

Promotion of neurite outgrowth by fibroblast growth factor receptor 1 overexpression and lysosomal inhibition of receptor degradation in pheochromocytoma cells and adult sensory neurons

Basic fibroblast growth factor (FGF-2) is up-regulated in response to a nerve lesion and promotes axonal regeneration by activation of the tyrosine kinase receptor fibroblast growth factor receptor 1 (FGFR1). To determine the effects of elevated FGFR1 levels on neurite outgrowth, overexpression was combined with lysosomal inhibition of receptor degradation. In pheochromocytoma (PC12) cells, FGFR1 overexpression resulted in flattened morphology, increased neurite outgrowth and activation of extracellular signal-regulated kinase (ERK) and AKT. Degradation of FGFR1 was inhibited by the lysosomal inhibitor leupeptin and by the proteasomal inhibitor lactacystin. In rat primary adult neurons, FGFR1 overexpression enhanced FGF-2-induced axon growth which was further increased by co-treatment with leupeptin. Lysosomal inhibition of receptor degradation concomitant with ligand stimulation of neurons overexpressing FGFR1 provides new insight in tyrosine kinase receptor-mediated promotion of axon regeneration and demonstrates that adult sensory neurons express sub-optimal levels of tyrosine kinase receptors for neurotrophic factors.

Constitutively expressed catalytic proteasomal subunits are up‐regulated during neuronal differentiation and required for axon initiation, elongation and maintenance

Inhibition of the proteasome by lactacystin, a specific blocker of the catalytic β‐subunits, results in transient neurite outgrowth by neuronal cell lines. Vice versa, as demonstrated in this study, treatment of pheochromocytoma (PC12) cells with nerve growth factor (NGF) or other differentiating agents reduces proteasomal activity. This is accompanied by an increase in mRNA and protein levels of the catalytically active subunits β1, β2 and β5, but not of their inducible counterparts, indicating changes in subunit composition of the proteasome during neuronal differentiation. In contrast to neuronal cell lines, however, pre‐treatment of primary neurons with proteasome inhibitors completely prevents axon formation, and lower concentrations of lactacystin (0.5–5 µm) significantly reduce axonal elongation and branching in vitro. Furthermore, established axonal networks degenerate rapidly and long‐term survival of peripheral neurons is impaired in the presence of proteasome inhibitors. Axonal pathology is reminiscent of the morphological changes observed in neurodegenerative disorders and supports a crucial role of the constitutive catalytic subunits in axon initiation, maintenance and regeneration.

ERK-Mediated Phosphorylation of Fibroblast Growth Factor Receptor 1 on Ser777 Inhibits Signaling

Serine phosphorylation limits signaling by a fibroblast growth factor receptor. Limiting Growth Factor Signaling Binding of fibroblast growth factors (FGFs) to receptor tyrosine kinases in the FGFR family triggers activation of these receptors through phosphorylation of tyrosine residues, thereby initiating signaling that stimulates cell proliferation and differentiation in various developmental processes, such as axonal growth. Zakrzewska et al. investigated the effect of phosphorylation of serine residues in FGFR1 on receptor activity. FGF-dependent activation of the mitogen-activated protein kinases ERK1 and ERK2 (ERK1/2) resulted in phosphorylation of Ser777 in the C-terminal region of the receptor, and this phosphorylation event was associated with decreased activation of FGFR1 and attenuated signaling. Dorsal root ganglion neurons expressing a S777A mutant FGFR1 exhibited enhanced and sustained receptor activation and extended longer axons than those expressing the wild-type receptor. In addition, previous stimulation of cells with epidermal growth factor, a ligand for a distinct receptor tyrosine kinase, resulted in the ERK1/2-mediated serine phosphorylation of FGFR1 and a reduction in subsequent FGF-dependent signaling. Together, these data suggest that an ERK-dependent mechanism initiated by activation of FGFR1 or other growth factor receptors prevents excessive FGFR1 signaling. Fibroblast growth factor 1 (FGF1) controls cellular activities through the activation of specific cell-surface FGF receptors (FGFRs). Transphosphorylation of tyrosine residues in the kinase domain of FGFRs leads to activation of intracellular signaling cascades, including those mediated by mitogen-activated protein kinases (MAPKs). FGFRs also contain a serine-rich C-terminal tail. We identified a regulatory mechanism of FGFR signaling involving phosphorylation of Ser777 in the C-terminal region of FGFR1 by the MAPKs extracellular signal–regulated kinase 1 (ERK1) and ERK2. Prevention of the phosphorylation of Ser777 in FGFR1 or mutation of Ser777 to alanine enhanced FGF-stimulated receptor tyrosine phosphorylation and increased cell proliferation, cell migration, and axonal growth. A form of FGFR1 with a phosphomimetic mutation at Ser777 exhibited reduced signaling. Activation of MAPKs by other receptor tyrosine kinases also resulted in phosphorylation of Ser777 in FGFR1, thereby enabling crosstalk regulation of FGFR activity by other signaling pathways. Our data reveal a negative feedback mechanism that controls FGF signaling and thereby protects the cell from excessive activation of FGFR.

In Zucker Diabetic Fatty rats, subclinical diabetic neuropathy increases in vivo lidocaine block duration but not in vitro neurotoxicity

Background and Objectives Application of local anesthetics may lead to nerve damage. Increasing evidence suggests that risk of neurotoxicity is higher in patients with diabetic peripheral neuropathy. In addition, block duration may be prolonged in neuropathy. We sought to investigate neurotoxicity in vitro and block duration in vivo in a genetic animal model of diabetes mellitus type 2. Methods In the first experiments, neurons harvested from control Zucker diabetic fatty (ZDF) rats were exposed to acute (24 hours) or chronic (72 hours) hyperglycemia, followed by incubation with lidocaine 40 mM (approximately 1%). In a second experiment, neurons harvested from control ZDF rats, or diabetic ZDF rats, were incubated with lidocaine, with or without SB203580, an inhibitor of the p38 mitogen-activated protein kinase. Finally, we performed sciatic nerve block (lidocaine 2%, 0.2 mL) in control or diabetic ZDF rats and measured motor and nociceptive block duration. Results In vitro, neither acute nor chronic hyperglycemia altered neurotoxic properties of lidocaine. In vitro, incubation of neurons with lidocaine resulted in a slightly decreased survival ratio when neurons were harvested from diabetic (57% ± 19%) as compared with control (64% ± 9%) rats. The addition of SB203580 partly reversed this enhanced neurotoxic effect and raised survival to 71% ± 12% in diabetic neurons and 66% ± 9% in control rats, respectively. In vivo, even though no difference was detected at baseline testing, motor block was significantly prolonged in diabetic as compared with control rats (137 ± 16 vs 86 ± 17 min). Conclusions In vitro, local anesthetic neurotoxicity was more pronounced on neurons from diabetic animals, but the survival difference was small. In vivo, subclinical neuropathy leads to substantial prolongation of block duration. We conclude that early diabetic neuropathy increases block duration, whereas the observed increase in toxicity was small.

Activation of TrkB receptors by NGFβ mimetic peptide conjugated polymersome nanoparticles

Activation of tyrosine kinase receptor B (TrkB), a neurotrophin receptor, has been shown to increase neuronal cell survival and promote regeneration. Stimulation of the TrkB receptor by neurotrophic growth factors has been identified as a possible therapeutic target for the treatment of neurodegenerative disorders. However, growth factor delivery is problematic because of a short half-life in vivo. We have conjugated hNgf-EE, a short peptide mimetic of NGFβ to the surface of polymersome nanoparticles and shown that they are capable of activating the TrkB receptor in vitro in the SHSY-G7 cell line. We propose that polymersomes could act as a scaffold for the delivery of TrkB activating moieties and that the polymersome size and polyethylene glycol surface have been shown to increase in vivo retention time. These multifunctional nanoparticles have potential for the treatment of neurodegenerative disorders by TrkB activation. From the ClinicaL Editor: Tyrosine kinase receptor B activation has been shown to promote regeneration and survival of neurons. However, growth factor delivery to stimulate these receptors remains problematic. The authors demonstrate that a peptide mimetic of NGFβ conjugated to the surface of polymersome nanoparticles is capable of activating the TrkB receptors. These nanoparticles may offer a novel treatment strategy for a variety of neurodegenerative disorders.