Local Acceleration of Neurofilament Transport at Nodes of Ranvier.

Myelinated axons are constricted at nodes of Ranvier. These constrictions are important physiologically because they increase the speed of saltatory nerve conduction, but they also represent potential bottlenecks for the movement of axonally transported cargoes. One type of cargo are neurofilaments, which are abundant space-filling cytoskeletal polymers that function to increase axon caliber. Neurofilaments move bidirectionally along axons, alternating between rapid movements and prolonged pauses. Strikingly, axon constriction at nodes is accompanied by a reduction in neurofilament number that can be as much as 10-fold in the largest axons. To investigate how neurofilaments navigate these constrictions, we developed a transgenic mouse strain that expresses a photoactivatable fluorescent neurofilament protein in neurons. We used the pulse-escape fluorescence photoactivation technique to analyze neurofilament transport in mature myelinated axons of tibial nerves from male and female mice of this strain ex vivo Fluorescent neurofilaments departed the activated region more rapidly in nodes than in flanking internodes, indicating that neurofilament transport is faster in nodes. By computational modeling, we showed that this nodal acceleration can be explained largely by a local increase in the duty cycle of neurofilament transport (i.e., the proportion of the time that the neurofilaments spend moving). We propose that this transient acceleration functions to maintain a constant neurofilament flux across nodal constrictions, much as the current increases where a river narrows its banks. In this way, neurofilaments are prevented from piling up in the flanking internodes, ensuring a stable neurofilament distribution and uniform axonal morphology across these physiologically important axonal domains.SIGNIFICANCE STATEMENT Myelinated axons are constricted at nodes of Ranvier, resulting in a marked local decrease in neurofilament number. These constrictions are important physiologically because they increase the efficiency of saltatory nerve conduction, but they also represent potential bottlenecks for the axonal transport of neurofilaments, which move along axons in a rapid intermittent manner. Imaging of neurofilament transport in mature myelinated axons ex vivo reveals that neurofilament polymers navigate these nodal axonal constrictions by accelerating transiently, much as the current increases where a river narrows its banks. This local acceleration is necessary to ensure a stable axonal morphology across nodal constrictions, which may explain the vulnerability of nodes of Ranvier to neurofilament accumulations in animal models of neurotoxic neuropathies and neurodegenerative diseases.

Reversal of neuropathy phenotypes in conditional mouse model of Charcot-Marie-Tooth disease type 2E.

Mutations in the gene encoding for the neurofilament light subunit (NF-L) are responsible for Charcot-Marie-Tooth (CMT) neuropathy type 2E. To address whether CMT2E disease is potentially reversible, we generated a mouse model with conditional doxycycline-responsive gene system that allows repression of mutant hNF-LP22S transgene expression in adult neurons. The hNF-LP22S;tTa transgenic (tg) mice recapitulated key features of CMT2E disease, including aberrant hindlimb posture, motor deficits, hypertrophy of muscle fibres and loss of muscle innervation without neuronal loss. Remarkably, a 3-month treatment of hNF-LP22S;tTa mice with doxycycline after onset of disease efficiently down-regulated expression of hNF-LP22S and it caused reversal of CMT neurological phenotypes with restoration of muscle innervation and of neurofilament protein distribution along the sciatic nerve. These data suggest that therapeutic approaches aimed at abolishing expression or neutralizing hNF-L mutants might not only halt the progress of CMT2E disease, but also revert the disabilities.

A NUDEL-dependent mechanism of neurofilament assembly regulates the integrity of CNS neurons.

The cytoskeleton controls the architecture and survival of central nervous system (CNS) neurons by maintaining the stability of axons and dendrites. Although neurofilaments (NFs) constitute the main cytoskeletal network in these structures, the mechanism that underlies subunit incorporation into filaments remains a mystery. Here we report that NUDEL, a mammalian homologue of the Aspergillus nidulans nuclear distribution molecule NudE, is important for NF assembly, transport and neuronal integrity. NUDEL facilitates the polymerization of NFs through a direct interaction with the NF light subunit (NF-L). Knockdown of NUDEL by RNA interference (RNAi) in a neuroblastoma cell line, primary cortical neurons or post-natal mouse brain destabilizes NF-L and alters the homeostasis of NFs. This results in NF abnormalities and morphological changes reminiscent of neurodegeneration. Furthermore, variations in levels of NUDEL correlate with disease progression and NF defects in a mouse model of neurodegeneration. Thus, NUDEL contributes to the integrity of CNS neurons by regulating NF assembly.

The type III neurofilament peripherin is expressed in the tuberomammillary neurons of the mouse.

BACKGROUND: Peripherin, a type III neuronal intermediate filament, is widely expressed in neurons of the peripheral nervous system and in selected central nervous system hindbrain areas with projections towards peripheral structures, such as cranial nerves and spinal cord neurons. Peripherin appears to play a role in neurite elongation during development and axonal regeneration, but its exact function is not known. We noticed high peripherin expression in the posterior hypothalamus of mice, and decided to investigate further the exact location of expression and function of peripherin in the mouse posterior hypothalamus. RESULTS: In situ hybridization indicated expression of peripherin in neurons with a distribution reminiscent of the histaminergic neurons, with little signal in any other part of the forebrain. Immunocytochemical staining for histidine decarboxylase and peripherin revealed extensive colocalization, showing that peripherin is produced by histaminergic neurons in all parts of the tuberomammillary nucleus. We next used histamine immunostaining in peripherin knockout, overexpressing and wild type mice to study if altered peripherin expression affects these neurons, but could not detect any visible difference in the appearance of these neurons or their axons. Peripherin knockout mice and heterozygotic littermates were used for measurement of locomotor activity, feeding, drinking, and energy expenditure. Both genotypes displayed diurnal rhythms with all the parameters higher during the dark period. The respiratory quotient, an indicator of the type of substrate being utilized, also exhibited a significant diurnal rhythm in both genotypes. The diurnal patterns and the average values of all the recorded parameters for 24 h, daytime and night time were not significantly different between the genotypes, however. CONCLUSION: In conclusion, we have shown that peripherin is expressed in the tuberomammillary neurons of the mouse hypothalamus. Monitoring of locomotor activity, feeding, drinking, and energy expenditure in mice either lacking or overexpressing peripherin did not reveal any difference, so the significance of peripherin in these neurons remains to be determined. The complete overlap between histidine decarboxylase and peripherin, both the protein and its mRNA, renders peripherin a useful new marker for histaminergic neurons in the hypothalamus.

Functions of intermediate filaments in neuronal development and disease.

Five major types of intermediate filament (IF) proteins are expressed in mature neurons: the three neurofilament proteins (NF-L, NF-M, and NF-H), alpha-internexin, and peripherin. While the differential expression of IF genes during embryonic development suggests potential functions of these proteins in axogenesis, none of the IF gene knockout experiments in mice caused gross developmental defects of the nervous system. Yet, deficiencies in neuronal IF proteins are not completely innocuous. Substantial developmental loss of motor axons was detected in mice lacking NF-L and in double knockout NF-M;NF-H mice, supporting the view of a role for IFs in axon stabilization. Moreover, the absence of peripherin resulted in approximately 30% loss of small sensory axons. Mice lacking NF-L had a scarcity of IF structures and exhibited a severe axonal hypotrophy, causing up to 50% reduction in conduction velocity, a feature that would be very detrimental for large animal species. Unexpectedly, the NF-M rather than NF-H protein turned out to be required for proper radial growth of large myelinated axons. Studies with transgenic mice suggest that some types of IF accumulations, reminiscent of those found in amyotrophic lateral sclerosis (ALS), can have deleterious effects and even cause neurodegeneration. Additional evidence for the involvement of IFs in pathogenesis came from the recent discovery of neurofilament gene mutations linked to ALS and Charcot-Marie-Tooth disease (CMT2E). Conversely, we discuss how certain types of perikaryal neurofilament aggregates might confer protection in motor neuron disease.

Peripherin is not a contributing factor to motor neuron disease in a mouse model of amyotrophic lateral sclerosis caused by mutant superoxide dismutase.

Peripherin is a type III intermediate filament protein detected in axonal spheroids associated with amyotrophic lateral sclerosis (ALS). The overexpression of peripherin induces degeneration of spinal motor neurons during aging in transgenic mice and in cultured neuronal cells derived from peripherin transgenic embryos. Here, we investigated whether peripherin is a contributor of pathogenesis in mice overexpressing a mutant superoxide dismutase 1 (SOD1(G37R)) gene linked to familial ALS. This was done by the generation and analysis of SOD1(G37R) mice that either overexpress a peripherin transgene (G37R;TgPer mice) or lack the endogenous peripherin gene (G37R;Per-/- mice). Surprisingly, upregulation or suppression of peripherin expression had no effects on disease onset, mortality, and loss of motor neurons in SOD1(G37R) mice. These results provide compelling evidence that peripherin is not a key contributor of motor neuron degeneration associated with toxicity of mutant SOD1.