Doublecortin (Dcx) family proteins regulate filamentous actin structure in developing neurons.

Doublecortin (Dcx) is the causative gene for X-linked lissencephaly, which encodes a microtubule-binding protein. Axon tracts are abnormal in both affected individuals and in animal models. To determine the reason for the axon tract defect, we performed a semiquantitative proteomic analysis of the corpus callosum in mice mutant for Dcx. In axons from mice mutant for Dcx, widespread differences are found in actin-associated proteins as compared with wild-type axons. Decreases in actin-binding proteins α-actinin-1 and α-actinin-4 and actin-related protein 2/3 complex subunit 3 (Arp3), are correlated with dysregulation in the distribution of filamentous actin (F-actin) in the mutant neurons with increased F-actin around the cell body and decreased F-actin in the neurites and growth cones. The actin distribution defect can be rescued by full-length Dcx and further enhanced by Dcx S297A, the unphosphorylatable mutant, but not with the truncation mutant of Dcx missing the C-terminal S/P-rich domain. Thus, the C-terminal region of Dcx dynamically regulates formation of F-actin features in developing neurons, likely through interaction with spinophilin, but not through α-actinin-4 or Arp3. We show with that the phenotype of Dcx/Doublecortin-like kinase 1 deficiency is consistent with actin defect, as these axons are selectively deficient in axon guidance, but not elongation.

Deciphering the axonal transport kinetics of neurofilaments using the fluorescence photoactivation pulse-escape method.

Neurofilaments are transported along axons stochastically in a stop-and-go manner, cycling between brief bouts of rapid movement and pauses that can vary from seconds to hours in length. Presently the only way to analyze neurofilament pausing experimentally on both long and short time scales is the pulse-escape method. In this method, fluorescence photoactivation is used to mark a population of axonal neurofilaments and then the loss of fluorescence from the activated region due to neurofilament movement is monitored by time-lapse imaging. Here we develop a mathematical description of the pulse-escape kinetics in terms of the rate constants of a tested mathematical model and we show how this model can be used to characterize neurofilament transport kinetics from fluorescence photoactivation pulse-escape experiments. This combined experimental and computational approach is a powerful tool for the analysis of the moving and pausing behavior of neurofilaments in axons.

The role of NEFL in cell growth and invasion in head and neck squamous cell carcinoma cell lines.

The neurofilament light polypeptide (NEFL) gene located on chromosome 8q21 is associated with the cancer of several organs and is regarded as a potential tumor suppressor gene. However, the role of the NEFL protein has not yet been studied in cancer cells. Although evidence suggests that there is a correlation between NEFL expression and cancer, studies regarding the role of the NEFL protein have been mostly limited to neurological diseases, such as Charcot-Marie-Tooth's disease (CMT). Most of these studies have not explored the role of NEFL in cancer cell apoptosis and/or invasion. In this study, NEFL expression was manipulated, and apoptosis and invasion were compared in head and neck squamous cell carcinoma cell lines. The results show that the expression of NEFL induces cancer cell apoptosis and inhibits invasion in these cell lines, suggesting that NEFL may play a role in cancer cell apoptosis and invasion.

Normal role of the low-molecular-weight neurofilament protein in mitochondrial dynamics and disruption in Charcot-Marie-Tooth disease.

Intermediate filaments serve important structural roles, but other cellular functions are increasingly recognized. This study demonstrated normal function of the low-molecular-weight neurofilament protein (NFL) in mitochondrial dynamics and disruption in Charcot-Marie-Tooth disease (CMT) due to mutations in the Nefl gene. In motor neurons of spinal cord cultured from Nefl-knockout mice, mitochondrial length and the rate of fusion were decreased concomitant with increased motility. These parameters were normalized after expression of NFL(wt) on the Nefl(-/-) background, but not by overexpression of the profusion protein, mitofusin 2 (MFN2). The effects of CMT-causing NFL mutants bore similarities to and differences from Nefl knockout. In the early phase of toxicity before disruption of the neurofilament network, NFL(Q333P) and NFL(P8R) integrated into neurofilaments and had effects on mitochondria similar to those with Nefl knockout. The reduction of fusion rate by NFL(Q333P) was partly due to interference with the function of the profusion protein MFN2, which is mutated in CMT2A, functionally linking these forms of CMT. In the later phase of toxicity, mitochondria essentially stopped moving in neurons expressing NFL mutants, probably a consequence of cytoskeletal disruption. Overall, the data point to important functions of neurofilaments in mitochondrial dynamics as well as primary involvement in CMT2E/1F.

The clinical implications and biologic relevance of neurofilament expression in gastroenteropancreatic neuroendocrine neoplasms.

BACKGROUND: Although gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) exhibit widely divergent behavior, limited biologic information (apart from Ki-67) is available to characterize malignancy. Therefore, the identification of alternative biomarkers is a key unmet need. Given the role of internexin alpha (INA) in neuronal development, the authors assessed its function in neuroendocrine cell systems and the clinical implications of its expression as a GEP-NEN biomarker. METHODS: Functional assays were undertaken to investigate the mechanistic role of INA in the pancreatic BON cell line. Expression levels of INA were investigated in 50 pancreatic NENs (43 primaries, 7 metastases), 43 small intestinal NENs (25 primaries, 18 metastases), normal pancreas (n = 10), small intestinal mucosa (n = 16), normal enterochromaffin (EC) cells (n = 9), mouse xenografts (n = 4) and NEN cell lines (n = 6) using quantitative polymerase chain reaction, Western blot, and immunostaining analyses. RESULTS: In BON cells, decreased levels of INA messenger RNA and protein were associated with the inhibition of both proliferation and mitogen-activated protein kinase (MAPK) signaling. INA was not expressed in normal neuroendocrine cells but was overexpressed (from 2-fold to 42-fold) in NEN cell lines and murine xenografts. In pancreatic NENs, INA was overexpressed compared with pancreatic adenocarcinomas and normal pancreas (27-fold [P = .0001], and 9-fold [P = .02], respectively). INA transcripts were correlated positively with Ki-67 (correlation coefficient [r] = 0.5; P < .0001) and chromogranin A (r = 0.59; P < .0001). INA distinguished between primary tumors and metastases (P = .02), and its expression was correlated with tumor size, infiltration, and grade (P < .05). CONCLUSIONS: INA is a novel NEN biomarker, and its expression was associated with MAPK signaling and proliferation. In clinical samples, elevated INA was correlated with Ki-67 and identified malignancy. INA may provide additional biologic information relevant to delineation of both pancreatic NEN tumor phenotypes and clinical behavior.

Cargo distributions differentiate pathological axonal transport impairments.

Axonal transport is an essential process in neurons, analogous to shipping goods, by which energetic and cellular building supplies are carried downstream (anterogradely) and wastes are carried upstream (retrogradely) by molecular motors, which act as cargo porters. Impairments in axonal transport have been linked to devastating and often lethal neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis, Huntington's, and Alzheimer's. Axonal transport impairment types include a decrease in available motors for cargo transport (motor depletion), the presence of defective or non-functional motors (motor dilution), and the presence of increased or larger cargos (protein aggregation). An impediment to potential treatment identification has been the inability to determine what type(s) of axonal transport impairment candidates that could be present in a given disease. In this study, we utilize a computational model and common axonal transport experimental metrics to reveal the axonal transport impairment general characteristics or "signatures" that result from three general defect types of motor depletion, motor dilution, and protein aggregation. Our results not only provide a means to discern these general impairments types, they also reveal key dynamic and emergent features of axonal transport, which potentially underlie multiple impairment types. The identified characteristics, as well as the analytical method, can be used to help elucidate the axonal transport impairments observed in experimental and clinical data. For example, using the model-predicted defect signatures, we identify the defect candidates, which are most likely to be responsible for the axonal transport impairments in the G93A SOD1 mouse model of ALS.

MSC p43 required for axonal development in motor neurons.

Neuron connectivity and correct neural function largely depend on axonal integrity. Neurofilaments (NFs) constitute the main cytoskeletal network maintaining the structural integrity of neurons and exhibit dynamic changes during axonal and dendritic growth. However, the mechanisms underlying axonal development and maintenance remain poorly understood. Here, we identify that multisynthetase complex p43 (MSC p43) is essential for NF assembly and axon maintenance. The MSC p43 protein was predominantly expressed in central neurons and interacted with NF light subunit in vivo. Mice lacking MSC p43 exhibited axon degeneration in motor neurons, defective neuromuscular junctions, muscular atrophy, and motor dysfunction. Furthermore, MSC p43 depletion in mice caused disorganization of the axonal NF network. Mechanistically, MSC p43 is required for maintaining normal phosphorylation levels of NFs. Thus, MSC p43 is indispensable in maintaining axonal integrity. Its dysfunction may underlie the NF disorganization and axon degeneration associated with motor neuron degenerative diseases.

Intermediate filaments: new proteins, some answers, more questions.

The past year has seen significant progress in the characterization of intermediate filament proteins. New proteins have been identified and physiologically significant differences between known proteins have been revealed. Changes in intermediate filament organization have been linked to changes in cell behavior, and mutational analyses are beginning to reveal the connection between intermediate filament expression, network formation, cellular behavior and disease.

Slow axonal transport.

New studies provide further evidence that the neuronal cytoskeleton is the product of a dynamic interplay between axonal transport processes and locally regulated assembly mechanisms. These data confirm that the axonal cytoskeleton in mammalian systems is largely stationary and is maintained by a smaller pool of moving subunits or polymers. Slow axonal transport in certain lower species, however, may exhibit quite different features.

Phosphorylation of highly conserved neurofilament medium KSP repeats is not required for myelin-dependent radial axonal growth.

Neurofilament medium (NF-M) is essential for the acquisition of normal axonal caliber in response to a myelin-dependent "outside-in" trigger for radial axonal growth. Removal of the tail domain and lysine-serine-proline (KSP) repeats of NF-M, but not neurofilament heavy, produced axons with impaired radial growth and reduced conduction velocities. These earlier findings supported myelin-dependent phosphorylation of NF-M KSP repeats as an essential component of axonal growth. As a direct test of whether phosphorylation of NF-M KSP repeats is the target for the myelin-derived signal, gene replacement has now been used to produce mice in which all serines of NF-M's KSP repeats have been replaced with phosphorylation-incompetent alanines. This substitution did not alter accumulation of the neurofilaments or their subunits. Axonal caliber and motor neuron conduction velocity of mice expressing KSP phospho-incompetent NF-M were also indistinguishable from wild-type mice. Thus, phosphorylation of NF-M KSP repeats is not an essential component for the acquisition of normal axonal caliber mediated by myelin-dependent outside-in signaling.

Mammalian Navigators are microtubule plus-end tracking proteins that can reorganize the cytoskeleton to induce neurite-like extensions.

Mammalian microtubule plus-end tracking proteins (+TIPs) specifically associate with the ends of growing microtubules. +TIPs are involved in many cellular processes, including mitosis, cell migration and neurite extension. Navigators are mammalian homologues of the C. elegans unc-53 protein, an ATPase that has been linked to the migration and outgrowth of muscles, axons and excretory canals. Here we show that all three mammalian Navigators are +TIPs, consistent with a previous study on Navigator 1 (NAV1) (Martinez-Lopez et al., Mol Cell Neurosci 2005;28:599-612). Overexpression of GFP-tagged Navigators causes displacement of CAP_GLY-motif containing +TIPs, such as CLIP-170, from microtubule ends, suggesting that the Navigator-binding sites on microtubule ends overlap with those of the CAP_GLY-motif proteins. In interphase cells, mammalian Navigators also prominently localize to centrosomes, a localization that does not depend on an intact microtubule network. Fluorescence recovery after photobleaching (FRAP) experiments indicate that NAV1 associates with intracellular structures other than microtubules or centrosomes. Expression of GFP-tagged Navigators induces the formation of neurite-like extensions in non-neuronal cells, showing that Navigators can dominantly alter cytoskeletal behavior. For NAV1 this function depends on its ATPase activity; it is not achieved by a classical type of MT bundling and stabilization. Combined our data suggest that Navigators are +TIPs that can reorganize the cytoskeleton to guide cell shape changes. Our data are consistent with a role for Navigators in neurite outgrowth.

Two distinct functions of the carboxyl-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments.

Neurofilaments are the major cytoskeletal elements in the axon that take highly ordered structures composed of parallel arrays of 10-nm filaments linked to each other with frequent cross-bridges, and they are believed to maintain a highly polarized neuronal cell shape. Here we report the function of rat NF-M in this characteristic neurofilament assembly. Transfection experiments were done in an insect Sf9 cell line lacking endogenous intermediate filaments. NF-L and NF-M coassemble to form bundles of 10-nm filaments packed in a parallel manner with frequent cross-bridges resembling the neurofilament domains in the axon when expressed together in Sf9 cells. Considering the fact that the expression of either NF-L or NF-M alone in these cells results in neither formation of any ordered network of 10-nm filaments nor cross-bridge structures, NF-M plays a crucial role in this parallel filament assembly. In the case of NF-H the carboxyl-tail domain has been shown to constitute the cross-bridge structures. The similarity in molecular architecture between NF-M and NF-H suggests that the carboxyl-terminal tail domain of NF-M also constitutes cross-bridges. To examine this and to further investigate the function of the carboxyl-terminal tail domain of NF-M, we made various deletion mutants that lacked part of their tail domains, and we expressed these with NF-L. From this deletion mutant analysis, we conclude that the carboxyl-terminal tail domain of NF-M has two distinct functions. First, it is the structural component of cross-bridges, and these cross-bridges serve to control the spacing between core filaments. Second, the portion of the carboxyl-terminal tail domain of NF-M that is directly involved in cross-bridge formation affects the core filament assembly by helping them to elongate longitudinally so that they become straight.

Neurofilament-dependent radial growth of motor axons and axonal organization of neurofilaments does not require the neurofilament heavy subunit (NF-H) or its phosphorylation.

Neurofilaments are essential for establishment and maintenance of axonal diameter of large myelinated axons, a property that determines the velocity of electrical signal conduction. One prominent model for how neurofilaments specify axonal growth is that the 660-amino acid, heavily phosphorylated tail domain of neurofilament heavy subunit (NF-H) is responsible for neurofilament-dependent structuring of axoplasm through intra-axonal crossbridging between adjacent neurofilaments or to other axonal structures. To test such a role, homologous recombination was used to generate NF-H-null mice. In peripheral motor and sensory axons, absence of NF-H does not significantly affect the number of neurofilaments or axonal elongation or targeting, but it does affect the efficiency of survival of motor and sensory axons. Loss of NF-H caused only a slight reduction in nearest neighbor spacing of neurofilaments and did not affect neurofilament distribution in either large- or small-diameter motor axons. Since postnatal growth of motor axon caliber continues largely unabated in the absence of NF-H, neither interactions mediated by NF-H nor the extensive phosphorylation of it within myelinated axonal segments are essential features of this growth.

Requirement of heavy neurofilament subunit in the development of axons with large calibers.

Neurofilaments (NFs) are prominent components of large myelinated axons. Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber. We created NF-H knockout mice to assess the contribution of NF-H to the development of axon size as well as its effect on the amounts of low and mid-sized NF subunits (NF-L and NF-M respectively). Surprisingly, we found that NF-L levels were reduced only slightly whereas NF-M and tubulin proteins were unchanged in NF-H-null mice. However, the calibers of both large and small diameter myelinated axons were diminished in NF-H-null mice despite the fact that these mice showed only a slight decrease in NF density and that filaments in the mutant were most frequently spaced at the same interfilament distance found in control. Significantly, large diameter axons failed to develop in both the central and peripheral nervous systems. These results demonstrate directly that unlike losing the NF-L or NF-M subunits, loss of NF-H has only a slight effect on NF number in axons. Yet NF-H plays a major role in the development of large diameter axons.

Neurofilament subunit NF-H modulates axonal diameter by selectively slowing neurofilament transport.

To examine the mechanism through which neurofilaments regulate the caliber of myelinated axons and to test how aberrant accumulations of neurofilaments cause motor neuron disease, mice have been constructed that express wild-type mouse NF-H up to 4.5 times the normal level. Small increases in NF-H expression lead to increased total neurofilament content and larger myelinated axons, whereas larger increases in NF-H decrease total neurofilament content and strongly inhibit radial growth. Increasing NF-H expression selectively slow neurofilament transport into and along axons, resulting in severe perikaryal accumulation of neurofilaments and proximal axonal swellings in motor neurons. Unlike the situation in transgenic mice expressing modest levels of human NF-H (Cote, F., J.F. Collard, and J.P. Julien. 1993. Cell. 73:35-46), even 4.5 times the normal level of wild-type mouse NF-H does not result in any overt phenotype or enhanced motor neuron degeneration or loss. Rather, motor neurons are extraordinarily tolerant of wild-type murine NF-H, whereas wild-type human NF-H, which differs from the mouse homolog at > 160 residue positions, mediates motor neuron disease in mice by acting as an aberrant, mutant subunit.

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.

Disruption of the NF-H gene increases axonal microtubule content and velocity of neurofilament transport: relief of axonopathy resulting from the toxin beta,beta'-iminodipropionitrile.

To investigate the role of the neurofilament heavy (NF-H) subunit in neuronal function, we generated mice bearing a targeted disruption of the gene coding for the NF-H subunit. Surprisingly, the lack of NF-H subunits had little effect on axonal calibers and electron microscopy revealed no significant changes in the number and packing density of neurofilaments made up of only the neurofilament light (NF-L) and neurofilament medium (NF-M) subunits. However, our analysis of NF-H knockout mice revealed an approximately 2.4-fold increase of microtubule density in their large ventral root axons. This finding was further corroborated by a corresponding increase in the ratio of assembled tubulin to NF-L protein in insoluble cytoskeletal preparations from the sciatic nerve. Axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed an increased transport velocity of newly synthesized NF-L and NF-M proteins in motor axons of NF-H knockout mice. When treated with beta,beta'-iminodipropionitrile (IDPN), a neurotoxin that segregates microtubules and retards neurofilament transport, mice heterozygous or homozygous for the NF-H null mutation did not develop neurofilamentous swellings in motor neurons, unlike normal mouse littermates. These results indicate that the NF-H subunit is a key mediator of IDPN-induced axonopathy.

Iron is increased in the brains of ageing mice lacking the neurofilament light gene.

There has been strong interest in the role of metals in neurodegeneration, and how ageing may predispose the brain to related diseases such as Alzheimer's disease. Recent work has also highlighted a potential interaction between different metal species and various components of the cytoskeletal network in the brain, which themselves have a reported role in age-related degenerative disease and other neurological disorders. Neurofilaments are one such class of intermediate filament protein that have a demonstrated capacity to bind and utilise cation species. In this study, we investigated the consequences of altering the neurofilamentous network on metal ion homeostasis by examining neurofilament light (NFL) gene knockout mice, relative to wildtype control animals, at adulthood (5 months of age) and advanced age (22 months). Inductively coupled plasma mass spectroscopy demonstrated that the concentrations of iron (Fe), copper (Cu) and zinc (Zn) varied across brain regions and peripheral nerve samples. Zn and Fe showed statistically significant interactions between genotype and age, as well as between genotype and region, and Cu demonstrated a genotype and region interaction. The most substantial difference between genotypes was found in Fe in the older animals, where, across many regions examined, there was elevated Fe in the NFL knockout mice. This data indicates a potential relationship between the neurofilamentous cytoskeleton and the processing and/or storage of Fe through ageing.

Neurofilaments switch between distinct mobile and stationary states during their transport along axons.

We have developed a novel pulse-escape fluorescence photoactivation technique to investigate the long-term pausing behavior of axonal neurofilaments. Cultured sympathetic neurons expressing a photoactivatable green fluorescent neurofilament fusion protein were illuminated with violet light in a short segment of axon to create a pulse of fluorescent neurofilaments. Neurofilaments departed from the photoactivated regions at rapid velocities, but the overall loss of fluorescence was slow because many of the neurofilaments paused for long periods of time before moving. The frequency of neurofilament departure was more rapid initially and slower at later times, resulting in biphasic decay kinetics. By computational simulation of the kinetics, we show that the neurofilaments switched between two distinct states: a mobile state characterized by intermittent movements and short pauses (average = 30 s) and a stationary state characterized by remarkably long pauses (average = 60 min). On average, the neurofilaments spent 92% of their time in the stationary state. Combining short and long pauses, they paused for 97% of the time, resulting in an average transport rate of 0.5 mm/d. We speculate that the relative proportion of the time that neurofilaments spend in the stationary state may be a principal determinant of their transport rate and distribution along axons, and a potential target of mechanisms that lead to abnormal neurofilament accumulations in disease.