Calcium-activated proteolysis of neurofilament proteins in goldfish Mauthner axons

We have examined the proteolytic breakdown of neurofilament proteins (NFPs) in isolated Mauthner axoplasm (M-axoplasm). Documentation of proteolytic breakdown of NFPs in M-axoplasm is important because NFPs are not degraded in distal segments of severed Mauthner axons (M-axons) maintained in vivo for up to 62 days at 20°C. By incubating M-axoplasm with 2 mM calcium in vitro, we have demonstrated that M-axoplasm contains an endogenous calcium-activated neutral protease that degrades NFPs. This calcium-activated proteolysis of M-axoplasm NFPs produced novel bands on silver-stained gels. These novel bands were presumed to be NFP breakdown products because they reacted with antibodies to the α-intermediate filament antigen (anti-IFA) on immunoblots from these gels. Incubations of M-axoplasm with 2 mM calcium plus exogenous calpain produced novel bands similar to those observed for M-axoplasm incubated with 2 mM calcium. Incubations of M-axoplasm with 2m M calcium plus calpain inhibitors did not produce these novel bands. These in vitro data indicate that M-axoplasm contains calpain that degrades NFPs and produces novel bands similar to those observed from distal segments of severed M-axons maintained in vivo longer than 62 days postseverance. Factors that affect the activity of calpain or affect the ability of calpain to degrade NFPs could account for the delayed degradation of NFPs in distal segments of severed M-axons maintained in vivo. © 1995 John Wiley & Sons, Inc.     

Delaminating myelin membranes help seal the cut ends of severed earthworm giant axons

Transected axons are often assumed to seal by collapse and fusion of the axolemmal leaflets at their cut ends. Using photomicroscopy and electronmicroscopy of fixed tissues and differential interference contrast and confocal fluorescence imaging of living tissues, we examined the proximal and distal cut ends of the pseudomyelinated medial giant axon of the earthworm, Lumbricus terrestris, at 5–60 min post-transection in physiological salines and Ca²⁺-free salines. In physiological salines, the axolemmal leaflets at the cut ends do not completely collapse, much less fuse, for at least 60 min post-transection. In fact, the axolemma is disrupted for 20–100 μm from the cut end at 5–60 min post-transection. However, a barrier to dye diffusion is observed when hydrophilic or styryl dyes are placed in the bath at 15–30 min post-transection. At 30–60 min post-transection, this barrier to dye diffusion near the cut end is formed amid an accumulation of some single-layered and many multilayered vesicles and other membranous material, much of which resembles delaminated pseudomyelin of the glial sheath. In Ca²⁺-free salines, this single and multilayered membranous material does not accumulate, and a dye diffusion barrier is not observed. These and other data are consistent with the hypothesis that plasmalemmal damage in eukaryotic cells is repaired by Ca²⁺-induced vesicles arising from invaginations or evaginations of membranes of various origin which form junctional contacts or fuse with each other and/or the plasmalemma. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 945–960, 1997     

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.     

Age-related atrophy of motor axons in mice deficient in the mid-sized neurofilament subunit.

Neurofilaments are central determinants of the diameter of myelinated axons. It is less clear whether neurofilaments serve other functional roles such as maintaining the structural integrity of axons over time. Here we show that an age-dependent axonal atrophy develops in the lumbar ventral roots of mice with a null mutation in the mid-sized neurofilament subunit (NF-M) but not in animals with a null mutation in the heavy neurofilament subunit (NF-H). Mice with null mutations in both genes develop atrophy in ventral and dorsal roots as well as a hind limb paralysis with aging. The atrophic process is not accompanied by significant axonal loss or anterior horn cell pathology. In the NF-M-null mutant atrophic ventral root, axons show an age-related depletion of neurofilaments and an increased ratio of microtubules/neurofilaments. By contrast, the preserved dorsal root axons of NF-M-null mutant animals do not show a similar depletion of neurofilaments. Thus, the lack of an NF-M subunit renders some axons selectively vulnerable to an age-dependent atrophic process. These studies argue that neurofilaments are necessary for the structural maintenance of some populations of axons during aging and that the NF-M subunit is especially critical.     

Depression of fast axonal transport in axons demyelinated by intraneural injection of a neurotoxin fromK. humboldtiana

Tullidinol, a neurotoxin extracted from the Karwinskia humboldtiana fruit, dissolved in peanut oil was injected into the right sciatic nerve of adult cats. The contralateral sciatic nerve received an equivalent volume of peanut oil alone. The fast axonal transport of labeled ([³H]Leucine) protein was studied in sensory and motor axons of both sciatic nerves. The radioactive label was pressure injected either into the L7 dorsal root ganglion or the ventral region of the same spinal cord segment. Several days after the toxin injection, the cat limped and the Achilles tendon reflex was nearly absent in the right hind limb. The amount of transported label was decreased distal to the site of toxin injection. Proximal to this site, the transported material was dammed. Sensory and motor axons showed similar changes. In addition, the toxin produced demyelination and axonal degeneration. Axonal transport and the structure of the axons were normal in the contralateral nerve. Both, Schwann cells and axons of the right sciatic nerve showed globular inclusions, presumably oil droplets containing the toxin. We conclude that Schwann cells and axons as well are tullidinol targets.Departamento de Química. Centro de Investigación y de Estudios Avanzados del IPN.Special issue dedicated to Dr. Sidney Ochs.     

Processing of neurofilament proteins from perikaryal to axonal type

In previous studies, neuronal cell bodies, excised by hand from bovine spinal ganglia, were analyzed and heterogeneous intermediate and high molecular weight neurofilament proteins that differed in electrophoretic mobility from their axonal counterparts were demonstrated (1, 2). In the present experiment, intermediate and high molecular weight neurofilament proteins of the axonal type were treated with alkaline phosphatase, and neurofilament proteins enriched in perikaryal type proteins were labeled with³²P. Results showed that neurofilament proteins were phosphorylated after their translation, in the perikarya and the proximal portion of the axon, and suggested that phosphorylation was responsible for the differences between axonal and perikaryal neurofilament proteins.Special Issue dedicated to Prof. Holger Hydén.     

Heterophilic binding of L1 on unmyelinated sensory axons mediates Schwann cell adhesion and is required for axonal survival.

This study investigated the function of the adhesion molecule L1 in unmyelinated fibers of the peripheral nervous system (PNS) by analysis of L1- deficient mice. We demonstrate that L1 is present on axons and Schwann cells of sensory unmyelinated fibers, but only on Schwann cells of sympathetic unmyelinated fibers. In L1-deficient sensory nerves, Schwann cells formed but failed to retain normal axonal ensheathment. L1-deficient mice had reduced sensory function and loss of unmyelinated axons, while sympathetic unmyelinated axons appeared normal. In nerve transplant studies, loss of axonal-L1, but not Schwann cell-L1, reproduced the L1-deficient phenotype. These data establish that heterophilic axonal-L1 interactions mediate adhesion between unmyelinated sensory axons and Schwann cells, stabilize the polarization of Schwann cell surface membranes, and mediate a trophic effect that assures axonal survival.     

Regeneration of motor axons in crayfish limbs: Distal stump activation followed by synaptic reformation

Summary Servered distal stumps of limb motor axons in the crayfish Procambarus clarkii remain ultrastructurally intact for at least 2–3 ms after being severed from their cell body. Initial regeneration of a motor axon is associated with the appearance of up to 200 small profiles (satellite axons) having no glial sheath adjacent to the large surviving stump for about 1 cm distal to the lesion at 4–5 wks postoperatively. These satellite axons are seen 2–4 cm distally at the target muscles 3–4 ms postoperatively. By 14–15 ms postoperative, the motor sheaths from the lesion site to the target muscles contain small axonal processes having thick glial sheaths. Behavioral tests show that some axons that are reconnected to the CNS at 4–5 wks may not be connected at 14–15 ms, whereas other axons not connected by 3–4 ms may be connected at 14–15 ms when the original distal stumps have degenerated.We suggest that all these data can best be explained by the view that motor axons in crayfish limbs initially regenerate via activation of the surviving distal stump by satellite axons which grow out from proximal stump. In most cases, these satellite axons continue to activate the surviving distal stump as they slowly grow to the target muscle. Eventually the satellite axons reform synapses on the target muscle and the original distal stump degenerates.This work was supported by NSF grants BNS 77-27678 and 80-22248 and an NIH RCDA 00070 to GDB. The authors would like to thank Mr. Martis Ballinger, Mr. Robert Reiss, and Mrs. Mary Raymond for their excellent technical assistance. We would also like to thank Dr. Wesley Thompson and Mr. Douglas Baxter for helpful discussions.     

The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate

The phosphorylated carboxyl-terminal "tail" domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002. J. Cell Biol. 158:681-693), we investigated possible functions of the NF-M tail domain by constructing NF-M tail-deleted (NF-MtailDelta) mutant mice using an embryonic stem cell-mediated "gene knockin" approach that preserves normal ratios of the three neurofilament subunits. Mutant NF-MtailDelta mice exhibited severely inhibited radial growth of both motor and sensory axons. Caliber reduction was accompanied by reduced spacing between neurofilaments and loss of long cross-bridges with no change in neurofilament protein content. These observations define distinctive functions of the NF-M tail in regulating axon caliber by modulating the organization of the neurofilament network within axons. Surprisingly, the average rate of axonal transport of neurofilaments was unaltered despite these substantial effects on axon morphology. These results demonstrate that NF-M tail-mediated interactions of neurofilaments, independent of NF transport rate, are critical determinants of the size and cytoskeletal architecture of axons, and are mediated, in part, by the highly phosphorylated tail domain of NF-M.     

Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain.

The degradation of phosphorylated and dephosphorylated neurofilament proteins by the Ca2+-activated neutral proteinase calpain was studied. Neurofilaments were isolated from bovine spinal cord, dephosphorylated by alkaline phosphatase (from Escherichia coli) and radioiodinated with [125I]-Bolton-Hunter reagent. The radioiodinated neurofilament proteins (untreated and dephosphorylated) were incubated in the presence and absence of calpain from rabbit skeletal muscle, and the degradation rates of large (NF-H), mid-sized (NF-M) and small (NF-L) neurofilament polypeptides were analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. The degradation of dephosphorylated neurofilament proteins occurred at a higher rate, and to a greater extent, than did that of the phosphorylated (untreated) neurofilament proteins. The dephosphorylated high-molecular-mass neurofilament (NF-HD) was proteolyzed 6 times more quickly than the untreated NF-H. The degradation rate of the NF-M and NF-L neurofilament proteins was also enhanced after dephosphorylation, but less than that of NF-H. This indicates that the dephosphorylation of neurofilament proteins can increase their sensitivity to calpain degradation.     

Purification and degradation of purified neurofilament proteins by the brain calcium-activated neutral proteases

The effect of the three forms (CANP1, CANP2 & CANP3) of calf brain calcium activated neutral protease (CANP) on the hydrolysis of purified neurofilament triplet proteins was investigated. It was observed that: each of the purified neurofilament proteins, was hydrolyzed slowly by CANP2 whereas the hydrolysis of 150 KDa and 68 kDa proteins by CANP1 & CANP3 was rapid; when assembled neurofilaments were used as a substrate, again differences in the rate and extent of degradation of the triplet proteins by the three proteases were observed. For example, little cleavage of the 68 kDa protein by CANP2 and CANP3 was noted whereas 210 kDa and 150 kDa proteins remained largely intact. CANP1 degraded the 150 kDa and 68 kDa proteins more rapidly than 210 kDa protein, where only a slight effect was noted. These data provide further proof of the existence of three different forms of CANP in the brain, and indications of the resistance of 210 kDa protein to proteolysis which may be compatible with its proposed special role in crossbridge formation.     

Regulation of neurofilament axonal transport by phosphorylation in optic axons in situ

Axonal transport of neurofilament (NFs) is considered to be regulated by phosphorylation. While existing evidence for this hypothesis is compelling, supportive studies have been largely restricted to correlative evidence and/or experimental systems involving mutants. We tested this hypothesis in retinal ganglion cells of normal mice in situ by comparing subunit transport with regional phosphorylation state coupled with inhibition of phosphatases. NF subunits were radiolabeled by intravitreal injection of 35S-methionine. NF axonal transport was monitored by following the location of the peak of radiolabeled subunits immunoprecipitated from 9x1.1 mm segments of optic axons. An abrupt decline transport rate was observed between days 1 and 6, which corresponded to translocation of the peak of radiolabeled subunits from axonal segment 2 into segment 3. Notably, this is far downstream from the only caliber increase of optic axons at 150 mu from the retina. Immunoblot analysis demonstrated a unique threefold increase between segments 2 and 3 in levels of a "late-appearing" C-terminal NF-H phospho-epitope (RT97). Intravitreal injection of the phosphatase inhibitor okadaic acid increased RT97 immunoreactivity within retinas and proximal axons, and markedly decreased NF transport rate out of retinas and proximal axons. These findings provide in situ experimental evidence for regulation of NF transport by site-specific phosphorylation.     

A dynamical system model of neurofilament transport in axons

We develop a dynamical system model for the transport of neurofilaments in axons, inspired by Brown's "stop-and-go" model for slow axonal transport. We use fast/slow time-scale arguments to lower the number of relevant parameters in our model. Then, we use experimental data of Wang and Brown to estimate all but one parameter. We show that we can choose this last remaining parameter such that the results of our model agree with pulse-labeling experiments from three different nerve cell types, and also agree with stochastic simulation results.     

Depression-like behavior in the forced swimming test in PACAP-deficient mice: amelioration by the atypical antipsychotic risperidone

Axonal degeneration is a key component of many neurodegenerative diseases. Injured axons undergo a program of self-destruction termed Wallerian degeneration that is an active, well-regulated process. The pathways leading to axon fragmentation are uncharacterized, but experiments with wld ^s mutant mice led to the discovery that over-expression of NMN adenylyltransferase 1 or treatment with NAD⁺ can inhibit axonal degeneration. In this study, we show that the purine nucleosides adenosine and guanosine, but not inosine, inhibit injury-induced axonal degeneration in cultured dorsal root ganglia neurons. Axons can be preserved by adding adenosine within 6 h of the axonal injury. The presence of adenosine was required continuously after the injury to maintain axonal protection. Together these results suggest that adenosine does not alter the neuronal response to injury, but instead inhibits a local axonal pathway necessary for the commitment and/or execution of the axon destructive program.     

Electron microscopy of severed motor fibers in the crayfish

Summary Cut and crushed crayfish claw nerves were examined with the electron microscope at intervals up to 6 months after lesion. In sections 1 centimeter distal to the lesion there were no signs of degeneration among the giant motor axons even after many months. Swelling of glial wrappings was observed within 48 hours of nerve severance and was particularly notable in the innermost glial layer, the adaxonal layer. Golgi elements, rough endoplasmic reticulum, and mitochondria accumulated in the glia. These changes were perhaps indicative of a greater supportive role required by the severed axons. Regeneration from the proximal stumps of the giant axons began within one week and had proceeded across the lesion gap by 4 weeks. Axon sprouts appeared to travel toward the terminals within the glial sheaths of the distal giant axon segments. Before regeneration was complete, as determined by a simple behaviour test, the regenerating axons occupied increasing proportions of the sheath space. After regeneration was complete occasional degenerations were seen among the sprouts. These degenerations may have occurred in regenerating axons which had grown to the incorrect muscles. The original distal giant axons probably degenerated, as well, after regeneration was complete. There was no evidence of rehealing of proximal and distal segments of the axons.This work was supported by NIH postdoctoral fellowship number 1F2 NB 32, 723 N RB awarded to RHN and grants in aid from the Multiple Sclerosis Society, The American Cancer Society and The National Institutes of Health.     

Development of the axon cap neuropil of the Mauthner cell in the goldfish

Summary Development of the axon cap neuropil of the Mauthner neuron in post-hatching larval goldfish brains was observed electron-microscopically. The axonal initial segment of newly hatched (day-4) larvae is completely covered with synaptic terminals containing clear spherical synaptic vesicles. Profiles of thin terminal axons, the spiral fibers, containing similar synaptic vesicles, rapidly increase in number around the initial segment and form glomerular neuropil similar to the central core of the adult axon cap by day 7. Three types of synapses are formed in the core neuropil. Bouton-type synapses contacting the initial segment are most abundant in day-4 to-14 larvae; they decrease thereafter and are rare on the distal half of the initial segment of day-40 larvae. Asymmetric axo-axonic synapses are commonly observed between spiral fibers in the core neuropil of day-7 to -19 larvae, but become fewer by day 40. Unique symmetrical axo-axonic synapses showing accumulation of synaptic vesicles on either side of apposed membrane thickenings first appear in day-14 core neuropil, gradually increase in number, and become the predominant type in day-40 core neuropil. Thick myelinated axons, which lose their myelin sheaths in the glial cap cell layer, start to penetrate into the axon cap on day 10. They gradually increase in number and form the peripheral part of the axon cap together with the cap dendrites, which finally grow into the axon cap from the axon hillock region of the Mauthner cell by day 40.     

Topography of retinal axons in the diencephalon of goldfish

Summary The projections of horseradish peroxidase-filled axons from each quadrant of the retina were studied to determine whether retinal projections of goldfish are topographically organized in diencephalic target nuclei. A distinct topography of the dorsal, nasal, ventral and temporal retina exists in the lateral geniculate nucleus and in the dorsolateral optic nucleus of the thalamus. The projections of retinal quadrants show minimal spatial overlap in each of these nuclei. The suprachiasmatic nucleus of the hypothalamus is extensively innervated by ventral retinal fibers, whereas the nucleus is sparsely innervated by fibers from the other three retinal quadrants. A rudimentary topography also exists in the pretectum where the dorsal pretectal area receives projections primarily from the ventral retina and the ventral pretectal area receives projections mostly from the dorsal retina. These data show that retinal projections to some diencephalic nuclei are topographically organized.This work was supported by Research Grant EY-01426 to S.C.S.     

Ultrastructural studies of severed medial giant and other CNS axons in crayfish

Summary The distal stumps of severed medial giant axons (MGAs) and of non-giant axons (NGAs) in the CNS of the crayfish Procambarus clarkii show long-term (5–9 months) survival associated with disorientation of mitochondria and thickening of the glial sheath. However, the morphological responses of the two axonal types differ in that neither the proximal nor the distal stump of severed MGAs ever fills with mitochondria as is observed in some severed NGAs. Furthermore, the adaxonal glial layer never completely encircles portions of MGA axoplasm as occurs in many severed NGAs; in fact, ultrastructural changes in the adaxonal layer around severed MGAs are often difficult to detect. No multiple axonal profiles are ever seen within the glial sheath of the proximal or distal stumps of severed MGAs whereas these structures are easily located within severed NGAs.This work was supported by NIH research grant #NS-14412 and an RCDA#00070 to GDB