Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons.

The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.

Psychosine induces the dephosphorylation of neurofilaments by deregulation of PP1 and PP2A phosphatases.

Patients with Krabbe disease, a genetic demyelinating syndrome caused by deficiency of galactosyl-ceramidase and the resulting accumulation of galactosyl-sphingolipids, develop signs of a dying-back axonopathy compounded by a deficiency of large-caliber axons. Here, we show that axonal caliber in Twitcher mice, an animal model for Krabbe disease, is impaired in peripheral axons and is accompanied by a progressive reduction in the abundance and phosphorylation of the three neurofilament (NF) subunits. These changes correlate with an increase in the density of NFs per cross-sectional area in numerous mutant peripheral axons and abnormal increases in the activity of two serine/threonine phosphatases (PP1 and PP2A) in mutant tissue. Similarly, acutely isolated mutant cortical neurons show abnormal phosphorylation of NFs. Psychosine, the neurotoxin accumulated in Krabbe disease, was sufficient to induce abnormal dephosphorylation of NF subunits in a normal motor neuron cell line as well as in acutely isolated normal cortical neurons. This in vitro effect was mediated by PP1 and PP2A, which specifically dephosphorylated NFs. These results demonstrate that the reduced caliber observed in some axons in Krabbe disease involves abnormal dephosphorylation of NFs. We propose that a psychosine-driven pathogenic mechanism through deregulated phosphotransferase activities may be involved in this process.

Efficacy of immunohistological methods in detecting functionally viable mechanoreceptors in the remnant stumps of injured anterior cruciate ligaments and its clinical importance.

PURPOSE: Various histological and immunological methods have been used to detect the mechanoreceptors and nerve fibers on the intact ACLs as well as on the remnant stumps. However, some of these methods lack standardization, and the variable thickness of slices used often leads to misinterpretation. The study was based on the hypothesis that immunohistological methods are easier and more reliable means to demonstrate mechanoreceptors in the remnant ACL stumps as compared with the conventional methods. We also attempted to validate the methodology of immunohistology as a means of characterizing functional mechanoreceptors in the residual stump of an injured ACL. METHODS: The remnants of the ruptured ACL in 95 patients were harvested during arthroscopic ACL reconstruction and evaluated immunohistologically using hematoxylin and eosin (H&E), and monoclonal antibodies to S-100 and NFP. Multiple sections from each specimen were serially examined by two histologists. RESULTS: The positivity of monoclonal antibody against NFP showed a statistically significant relationship with the presence of morphologically normal mechanoreceptors, whereas the positivity of monoclonal antibody against S-100 showed a statistically significant relationship with the presence of free nerve ending in the residual stump of an injured ACL. CONCLUSIONS: Immunological methods are more reliable and easier to use as compared with the conventional methods of histological staining for identifying remnant stumps likely to be of some proprioceptive benefit after an ACL injury. Such an identification might help us preserve certain remnant stumps during ACL reconstruction which might in turn improve the postoperative functional outcomes.

Pin1-dependent prolyl isomerization modulates the stress-induced phosphorylation of high molecular weight neurofilament protein.

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H(2)O(2)) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H(2)O(2) and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases.

Effects of unoprostone on phosphorylated extracellular signal-regulated kinase expression in endothelin-1-induced retinal and optic nerve damage.

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been implicated in the development of normal- and high-tension glaucoma. We investigated the effects of unoprostone on extracellular signal-regulated kinase (ERK) in ET-1-induced retinal ganglion cell (RGC) death and optic nerve injury. Our morphometric study showed that intravitreal injection of ET-1 led to cell loss in the RGC layer (RGCL) in 28 days. Western blot analysis showed decreased neurofilament (NF) protein in the optic nerve 28 days after ET-1 injection. In this in vivo model, increased phosphorylated ERK (p-ERK) was observed in the retina on 1 day and subsequently in the optic nerve from 7 days after ET-1 injection. Simultaneous injection of M1, as a metabolite of unoprostone, showed further increased p-ERK levels compared with ET-1 injection alone. Our morphometric study of flat-mount preparations stained with cresyl violet or retrograde labeling with a neuro-tracer and Western blot analysis of NF showed that inhibition of ERK phosphorylation led to acceleration of ET-1-induced RGC death and optic nerve damage. In addition, M1 significantly attenuated both RGC loss and the decrease in NF protein induced by ET-1. The protective effects of M1 were significantly inhibited by U0126, an ERK inhibitor. These results suggest that unoprostone has neuroprotective effects against ET-1-induced neuronal injury through ERK phosphorylation.

Defective axonal transport of neurofilament proteins in neurons overexpressing peripherin.

Peripherin is a type III neuronal intermediate filament detected in motor neuron inclusions of amyotrophic lateral sclerosis (ALS) patients. We previously reported that overexpression of peripherin provokes late-onset motor neuron dysfunction in transgenic mice. Here, we show that peripherin overexpression slows down axonal transport of neurofilament (NF) proteins, and that the transport defect precedes by several months the appearance of axonal spheroids in adult mice. Defective NF transport by peripherin up-regulation was further confirmed with dorsal root ganglia (DRG) neurons cultured from peripherin transgenic embryos. Immunofluorescence microscopy and western blotting revealed that excess peripherin provokes reduction in levels of hyperphosphorylated NF-H species in DRG neurites. Similarly the transport of a green fluorescent protein (GFP)-tagged NF-M, delivered by means of a lentiviral construct, was impaired in DRG neurites overexpressing peripherin. These results demonstrate that peripherin overexpression can cause defective transport of type IV NF proteins, a phenomenon that may account for the progressive formation of ALS-like spheroids in axons.

Matrix metalloproteinase-9 promotes nerve growth factor-induced neurite elongation but not new sprout formation in vitro.

Matrix metalloproteinase-9 (MMP-9) is a basal-lamina-degrading protease that we have recently shown to be localized in regenerating sciatic nerve. We now demonstrate that MMP-9 colocalizes with growth-associated protein GAP-43 in regenerating nerves in vivo and is involved in vitro in axonal sprouting. By using a PC12 cell model for neuronal sprouting, we analyzed the effects of recombinant MMP-9, MMP-9-neutralizing antibody, and a broad-spectrum MMP inhibitor (Ro 31-9790) on sprout formation, elongation, and branching. Quantitative phase-contrast microscopy showed that MMP-9 elongated neuronal sprouts by 67% and increased their branching by 14% but did not change the number of sprouts relative to nerve growth factor (NGF) treatment. Double immunofluorescence for GAP-43, a marker for growth cones, and alpha-tubulin, a marker for axonal microtubules, showed that MMP-9-treated cells had increased distribution of alpha-tubulin but no effect on GAP-43. Western blot analyses of cell lysates demonstrated that the NGF-induced increase in GAP-43 was unchanged with MMP-9 treatment or inhibition, confirming that MMP-9 had no effect on new sprout formation. However, Ro 31-9790 reduced GAP-43 levels to those seen in untreated cells, suggesting that an MMP other than MMP-9 is important for sprout formation. Finally, phosphorylated neurofilament M (NFM-p), a marker for regenerative elongation, was induced with MMP-9 treatment and was inhibited by the anti-MMP-9 antibody treatment, confirming the role of MMP-9 in axonal elongation. NFM-p colocalized with MMP-9 in regenerating sciatic nerve fibers. These findings suggest that MMP-9 regulates neurite extension in regenerating peripheral nerve fibers and, therefore, might be of therapeutic value in promoting regeneration in vivo.

Ligand-dependent inhibition and reversal of tau filament formation.

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule associated protein tau. Because animal model studies suggest that a toxic gain of function accompanies tau aggregation in neurons, selective pharmacological inhibitors of the process may have utility in slowing neurodegeneration. Here, the properties of a candidate small molecule inhibitor of tau fibrillization, 3-(2-hydroxyethyl)-2-[2-[[3-(2-hydroxyethyl)-5-methoxy-2-benzothiazolylidene]methyl]-1-butenyl]-5-methoxybenzothiazolium (N744), were characterized in vitro using transmission electron microscopy. N744 inhibited arachidonic acid-induced aggregation of full-length, four-repeat tau protein at substoichiometric concentrations relative to total tau and with an IC(50) of approximately 300 nM. Inhibition was accompanied by a dose-dependent decrease in the number concentration of filaments, suggesting that N744 interfered with tau filament nucleation. Stoichiometric concentrations of N744 also promoted tau disaggregation when added to mature synthetic filaments. Disaggregation followed first-order kinetics and was accompanied by a steady decrease in filament number, suggesting that N744 promoted endwise loss of tau molecules with limited filament breakage. N744 at substoichiometric concentrations did not inhibit Abeta and alpha-synuclein aggregation, indicating it was tau selective under these conditions. Because of its activity in vitro, N744 may offer a pharmacological approach to the role of tau fibrillization in neurodegeneration.

Loss of neurofilaments alters axonal growth dynamics.

The highly regulated expression of neurofilament (NF) proteins during axon outgrowth suggests that NFs are important for axon development, but their contribution to axon growth is unclear. Previous experiments in Xenopus laevis embryos demonstrated that antibody-induced disruption of NFs stunts axonal growth but left unresolved how the loss of NFs affects the dynamics of axon growth. In the current study, dissociated cultures were made from the spinal cords of embryos injected at the two-cell stage with an antibody to the middle molecular mass NF protein (NF-M), and time-lapse videomicroscopy was used to study early neurite outgrowth in descendants of both the injected and uninjected blastomeres. The injected antibody altered the growth dynamics primarily in long neurites (>85 microm). These neurites were initiated just as early and terminated growth no sooner than did normal ones. Rather, they spent relatively smaller fractions of time actively extending than normal. When growth occurred, it did so at the same velocity. In very young neurites, which have NFs made exclusively of peripherin, NFs were unaffected, but in the shaft of older neurites, which have NFs that contain NF-M, NFs were disrupted. Thus growth was affected only after NFs were disrupted. In contrast, the distributions of alpha-tubulin and mitochondria were unaffected; thus organelles were still transported into neurites. However, mitochondrial staining was brighter in descendants of injected blastomeres, suggesting a greater demand for energy. Together, these results suggest a model in which intra-axonal NFs facilitate elongation of long axons by making it more efficient.

Axotomy-induced neurofilament phosphorylation is inhibited in situ by microinjection of PKA and PKC inhibitors into identified lamprey neurons.

Close axotomy of identified lamprey neurons induces phosphorylation of somatodendritic neurofilaments (NFs), followed by ectopic regeneration of neurofilamentous sprouts from the dendrites. We used in situ intracellular microinjection to study the mechanism of axotomy-induced NF phosphorylation. We found that inhibitors of protein kinase C (PKC) and protein kinase A (PKA) block somatodendritic NF phosphorylation for up to 15 days when injected at the time of axotomy. Injection of PKA catalytic subunit, diacylglycerol, or okadaic acid induces somatodendritic NF phosphorylation in intact neurons with the same time course as close axotomy. These results suggest that transient activation of PKC, PKA, and/or serine phosphatase inhibition by axotomy triggers persistent intracellular changes that may be related to polarity loss in these neurons.