Organization Of Neurofilaments Research Articles

Reticulons 1 and 3 are essential for axonal growth and synaptic maintenance associated with intellectual development.

Reticulon (RTN) proteins are a family of proteins biochemically identified for shaping tubular endoplasmic reticulum, a subcellular structure important for vesicular transport and cell-to-cell communication. In our recent study of mice with knockout of both reticulon 1 (Rtn1) and Rtn3, we discovered that Rtn1-/-;Rtn3-/- (brief as R1R3dKO) mice exhibited neonatal lethality, despite the fact that mice deficient in either RTN1 or RTN3 alone exhibit no discernible phenotypes. This has been the first case to find early lethality in animals with deletion of partial members of RTN proteins. The complete penetrance for neonatal lethality can be attributed to multiple defects including the impaired neuromuscular junction found in the diaphragm. We also observed significantly impaired axonal growth in a regional-specific manner, detected by immunohistochemical staining with antibodies to neurofilament light chain and neurofilament medium chain. Ultrastructural examination by electron microscopy revealed a significant reduction in synaptic active zone length in the hippocampus. Mechanistic exploration by unbiased proteomic assays revealed reduction of proteins such as FMR1, Staufen2, Cyfip1, Cullin-4B and PDE2a, which are known components in the fragile X mental retardation pathway. Together, our results reveal that RTN1 and RTN3 are required to orchestrate neurofilament organization and intact synaptic structure of the central nervous system.

GENETICS OF STUTTERING

Stuttering is characterized by frequent repetition or lengthening of sounds, syllables or words, or by frequent hesitations or pauses that disrupt the rhythmic flow of speech. It may be accompanied by movements of the face and/or other parts of the body that coincide in time with the repetitions, prolongations, or pauses in speech flow. The prevalence of stuttering ranges from 0.3% to 5.6%. Usually for 80%-90% of patients stuttering starts gradually when children are 2-7 years old. Longitudinal research shows that 65% -85% of stuttering children recover. The rate of heritability of stuttering is about 70%, indicating that almost one third of the disorder is caused by environmental factors. Currently four stuttering genes have been identified that are involved in intracellular metabolism: GNPTAB, GNPTG, NAGPA and AP4E1. The disorder is caused by single nucleotide synonymous or missense mutations in the exons of these genes. Different people affected by stuttering can carry different sets of mutant variants ‒ from one to several. The effect of the identified genes on neurobiological processes in the human brain and on the development of stuttering remains unknown. So far it has only been discovered that stuttering occurs due to lysosomal dysfunction, which adversely affects the organization of neurofilaments of speech neural networks. The contribution of identified genes to stuttering cumulatively is only 20%. There were identified also 9 candidate stuttering genes (FADS2, PLXNA4, CTNNA3, ARNT2, EYA2, PCSK5, SLC24A3, FMN1 and ADARB2) and 7 regions of 6 chromosomes (2p, 3p, 3q, 12q, 14q, 15q and 16q) reliably linked to this disorder.
 Further successful genetic studies of stuttering are needed to improve our understanding and treatment of this long-enigmatic disorder.

Neurofilament-lysosomal genetic intersections in the cortical network of stuttering

The neurobiological underpinnings of stuttering, a speech disorder characterized by disrupted speech fluency, remain unclear. While recent developments in the field have afforded researchers the ability to pinpoint several genetic profiles associated with stuttering, how these specific genetic backgrounds impact neuronal circuits and how they generate or facilitate the emergence of stuttered speech remains unknown. In this study, we identified the large-scale cortical network that characterizes stuttering using functional connectivity MRI and graph theory. We performed a spatial similarity analysis that examines whether the topology of the stuttering cortical network intersects with genetic expression levels of previously reported genes for stuttering from the protein-coding transcriptome data of the Allen Human Brain Atlas. We found that GNPTG – a gene involved in the mannose-6-phosphate lysosomal targeting pathways – was significantly co-localized with the stuttering cortical network. An enrichment analysis demonstrated that the genes identified with the stuttering cortical network shared a significantly overrepresented biological functionality of Neurofilament Cytoskeleton Organization (NEFH, NEFL and INA). The relationship between lysosomal pathways, cytoskeleton organization, and stuttering, was investigated by comparing the genetic interactome between GNPTG and the neurofilament genes implicated in the current study. We found that genes of the interactome network, including CDK5, SNCA, and ACTB, act as functional links between lysosomal and neurofilament genes. These findings support the notion that stuttering is due to a lysosomal dysfunction, which has deleterious effects on the neurofilament organization of the speech neuronal circuits. They help to elucidate the intriguing, unsolved link between lysosomal mutations and the presence of stuttering.

Ultrastructural immunolocalization of nestin in the regenerating tail of lizards shows its presence during cytoskeletal modifications in the epidermis, muscles and nerves.

Nestin has been considered a neural stem cell marker, and represents an intermediate filament protein likely involved in restructuring the cytoskeleton in different cell types. The present ultrastructural study has immunodetected nestin especially in the wound epidermis, regenerating myotubes and in the growing nerves of the regenerating tail of lizards. In keratinocytes of the stratified wound epidermis nestin is present in the irregular electron-paler meshwork located along the cell perimeter and among keratin bundles converging into desmosomes. In the regenerating muscles nestin-immunoreactivity remains confined to some external regions along the myotubes and in the cytoplasmic ends of the myotubes not occupied by myofibrils. A diffuse nestin immunolabeling is also present among the neurofilaments of growing axons, in Schwann cells and in ependymal cells of the regenerating spinal cord of the tail. The localization of nestin in sites of cytoskeletal remodeling in keratinocytes, myotubes, ependymal cells and axons, suggests that this protein is associated to the reassembling of keratin tonofilaments in moving keratinocytes, assembling of contractile proteins in myotubes, and in the organization of neurofilaments during the growth and myelination of axons within the regenerating lizard tail.

Neurofilament dynamics and involvement in neurological disorders.

Neurons are extremely polarised cells in which the cytoskeleton, composed of microtubules, microfilaments and neurofilaments, plays a crucial role in maintaining structure and function. Neurofilaments, the 10-nm intermediate filaments of neurons, provide structure and mechanoresistance but also provide a scaffolding for the organization of the nucleus and organelles such as mitochondria and ER. Disruption of neurofilament organization and expression or metabolism of neurofilament proteins is characteristic of certain neurological syndromes including Amyotrophic Lateral Sclerosis, Charcot-Marie-Tooth sensorimotor neuropathies and Giant Axonal Neuropathy. Microfluorometric live imaging techniques have been instrumental in revealing the dynamics of neurofilament assembly and transport and their functions in organizing intracellular organelle networks. The insolubility of neurofilament proteins has limited identifying interactors by conventional biochemical techniques but yeast two-hybrid experiments have revealed new roles for oligomeric, nonfilamentous structures including vesicular trafficking. Although having long half-lives, new evidence points to degradation of subunits by the ubiquitin-proteasome system as a mechanism of normal turnover. Although certain E3-ligases ubiquitinating neurofilament proteins have been identified, the overall process of neurofilament degradation is not well understood. We review these mechanisms of neurofilament homeostasis and abnormalities in motor neuron and peripheral nerve disorders. Much remains to discover about the disruption of processes that leads to their pathological aggregation and accumulation and the relevance to pathogenesis. Understanding these mechanisms is crucial for identifying novel therapeutic strategies.

A critical reevaluation of the stationary axonal cytoskeleton hypothesis

Neurofilaments are transported along axons in a rapid intermittent and bidirectional manner but there is a long-standing controversy about whether this applies to all axonal neurofilaments. Some have proposed that only a small proportion of axonal neurofilaments are mobile and that most are deposited into a persistently stationary and extensively cross-linked cytoskeleton that remains fixed in place for many months without movement, turning over very slowly. In contrast, others have proposed that this hypothesis is based on a misinterpretation of the experimental data and that, in fact, all axonal neurofilaments move. These contrary perspectives have distinct implications for our understanding of how neurofilaments are organized and reorganized in axons both in health and disease. Here, we discuss the history and substance of this controversy. We show that the published data on the kinetics and distribution of neurofilaments along axons favor a simple "stop and go" transport model in which axons contain a single population of neurofilaments that all move in a stochastic, bidirectional and intermittent manner. Based on these considerations, we propose a dynamic view of the neuronal cytoskeleton in which all neurofilaments cycle repeatedly between moving and pausing states throughout their journey along the axon. The filaments move infrequently, but the average pause duration is on the order of hours rather than weeks or months. Against this fluid backdrop, the action of molecular motors on neurofilaments can have dramatic effects on neurofilament organization that would not be possible if the neurofilaments were extensively cross-linked into a truly stationary network.

Increased EID1 nuclear translocation impairs synaptic plasticity and memory function associated with pathogenesis of Alzheimer's disease

Though loss of function in CBP/p300, a family of CREB-binding proteins, has been causally associated with a variety of human neurological disorders, such as Rubinstein–Taybi syndrome, Huntington's disease and drug addiction, the role of EP300 interacting inhibitor of differentiation 1 (EID1), a CBP/p300 inhibitory protein, in modulating neurological functions remains completely unknown. Through the examination of EID1 expression and cellular distribution, we discovered that there is a significant increase of EID1 nuclear translocation in the cortical neurons of Alzheimer's disease (AD) patient brains compared to that of control brains. To study the potential effects of EID1 on neurological functions associated with learning and memory, we generated a transgenic mouse model with a neuron-specific expression of human EID1 gene in the brain. Overexpression of EID1 led to an increase in its nuclear localization in neurons mimicking that seen in human AD brains. The transgenic mice had a disrupted neurofilament organization and increase of astrogliosis in the cortex and hippocampus. Furthermore, we demonstrated that overexpression of EID1 reduced hippocampal long-term potentiation and impaired spatial learning and memory function in the transgenic mice. Our results indicated that the negative effects of extra nuclear EID1 in transgenic mouse brains are likely due to its inhibitory function on CBP/p300 mediated histone and p53 acetylation, thus affecting the expression of downstream genes involved in the maintenance of neuronal structure and function. Together, our data raise the possibility that alteration of EID1 expression, particularly the increase of EID1 nuclear localization that inhibits CBP/p300 activity in neuronal cells, may play an important role in AD pathogenesis.

Smn deficiency causes neuritogenesis and neurogenesis defects in the retinal neurons of a mouse model of spinal muscular atrophy

The eye is an excellent model for the study of neuronal development and pathogenesis of central nervous system disorders because of its relative ease of accessibility and the well-characterized cellular makeup. We have used this model to study spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease caused by deletions or mutations in the survival of motor neuron 1 gene (SMN1). We have investigated the expression pattern of mouse Smn mRNA and protein in the neural retina and the optic nerve of wild type mice. Smn protein is present in retinal ganglion cells and amacrine cells within the neural retina as well as in glial cells in the optic nerve. Histopathological analysis in phenotype stage SMA mice revealed that Smn deficiency is associated with a reduction in ganglion cell axon and glial cell number in the optic nerve, as well as compromised cellular processes and altered organization of neurofilaments in the neural retina. Whole mount preparation and retinal neuron primary culture provided further evidence of abnormal synaptogenesis and neurofilament accumulation in the neurites of Smn-deficient retinal neurons. A subset of amacrine cells is absent, in a cell-autonomous fashion, in the retina of SMA mice. Finally, the retinas of SMA mice have altered electroretinograms. Altogether, our study has demonstrated defects in axodendritic outgrowth and cellular composition in Smn-depleted retinal neurons, indicating a role for Smn in neuritogenesis and neurogenesis, and providing us with an insight into pathogenesis of SMA.

Distal to proximal development of peripheral nerves requires the expression of neurofilament heavy

At the initiation of radial growth, neurofilaments are likely to consist primarily of neurofilament light and medium as neurofilament heavy expression is developmentally delayed. To better understand the role of neurofilament heavy in structuring axons, axonal diameter and neurofilament organization were measured in proximal and distal segments of the sciatic nerve and along the entire length of the phrenic nerve. Deletion of neurofilament heavy reduced axonal diameters and neurofilament number in proximal nerve segments. However, neurofilament spacing was greater in proximal versus distal phrenic nerve segments. Taken together, these results suggest that loss of neurofilament heavy reduces radial growth in proximal axonal segments by reducing the accumulation of neurofilaments. As neurofilament heavy expression is developmentally delayed, these results suggest that without neurofilament heavy, the neurofilament network is established in a distal to proximal gradient perhaps to allow distal axonal segments to develop prior to proximal segments.

Embryonic radial glia bridge spinal cord lesions and promote functional recovery following spinal cord injury

Radial glial cells are neural stem cells (NSC) that are transiently found in the developing CNS. To study radial glia, we isolated clones following immortalization of E13.5 GFP rat neurospheres with v-myc. Clone RG3.6 exhibits polarized morphology and expresses the radial glial markers nestin and brain lipid binding protein. Both NSC and RG3.6 cells migrated extensively in the adult spinal cord. However, RG3.6 cells differentiated into astroglia slower than NSC, suggesting that immortalization can delay differentiation of radial glia. Following spinal cord contusion, implanted RG3.6 cells migrated widely in the contusion site and into spared white matter where they exhibited a highly polarized morphology. When injected immediately after injury, RG3.6 cells formed cellular bridges surrounding spinal cord lesion sites and extending into spared white matter regions in contrast to GFP fibroblasts that remained in the lesion site. Behavioral analysis indicated higher BBB scores in rats injected with RG3.6 cells than rats injected with fibroblasts or medium as early as 1 week after injury. Spinal cords transplanted with RG3.6 cells or dermal fibroblasts exhibited little accumulation of chondroitin sulfate proteoglycans (CSPG) including NG2 proteoglycans that are known to inhibit axonal growth. Reduced levels of CSPG were accompanied by little accumulation in the injury site of activated macrophages, which are a major source of CSPG. However, increased staining and organization of neurofilaments were found in injured rats transplanted with RG3.6 cells suggesting neuroprotection or regrowth. The combined results indicate that acutely transplanted radial glia can migrate to form bridges across spinal cord lesions in vivo and promote functional recovery following spinal cord injury by protecting against macrophages and secondary damage.

Modulation of repulsive forces between neurofilaments by sidearm phosphorylation

Recent studies have advanced the notion that the axonal organization of neurofilaments (NFs) is based on mutual steric repulsion between the unstructured “sidearm” domains of adjacent NFs. Here, we present experimental evidence that these repulsive forces are modulated by the degree of sidearm phosphorylation. When NFs are sedimented into a gelatinous pellet, pellet volume falls with increasing ionic strength and enzymatic dephosphorylation; sedimentation of phosphorylated NFs in the presence of divalent cations also dramatically reduces pellet volume. Further, atomic force microscopy imaging of isolated mammalian NFs reveals robust exclusion of colloidal particles from the NF backbone that is reduced at high ionic strength and attenuated when the filaments are enzymatically dephosphorylated. Phosphate–phosphate repulsion on the NF sidearm appears to modulate NF excluded volume in a graded fashion, thereby controlling axonal NF organization through interfilament forces.

Mitogen-activated protein kinase regulates neurofilament axonal transport

Mitogen-activated protein kinase (MAP) kinase plays a pivotal role in the development of the nervous system by mediating both neurogenesis and neuronal differentiation. Here we examined whether p42/44 MAP kinase plays a role in axonal transport and the organization of neurofilaments (NFs) in axonal neurites. Dominant-negative p42/44 MAP kinase, anti-MAP kinase antisense oligonucleotides and the MAP kinase inhibitor PD98059 all reduced NF phospho-epitopes and inhibited anterograde NF axonal transport of GFP-tagged NF subunits in differentiated NB2a/d1 neuroblastoma cells. Expression of constitutively active MAP kinase and intracellular delivery of active enzyme increased NF phospho-epitopes and increased NF axonal transport. Longer treatment with PD98059 shifted NF transport from anterograde to retrograde. PD98059 did not inhibit overall axonal transport nor compromise overall axonal architecture or composition. The p38 MAP kinase inhibitor SB202190 did not inhibit NF transport whereas the kinase inhibitor olomoucine inhibited both NF and mitochondrial transport. Axonal transport of NFs containing NF-H whose C-terminal region was mutated to mimic extensive phosphorylation was substantially less affected by PD98059 compared to a wild-type construct. These data suggest that p42/44 MAP kinase regulates NF anterograde transport by NF C-terminal phosphorylation. MAP kinase may therefore stabilize developing axons by promoting the accumulation of NFs within growing axonal neurites.

Phosphorylated MAP1B is induced in central sprouting of primary afferents in response to peripheral injury but not in response to rhizotomy.

A peripheral nerve lesion induces sprouting of primary afferents from dorsal root ganglion (DRG) neurons into lamina II of the dorsal horn. Modifications of the environment in consequence to the axotomy provide an extrinsic stimulus. A potential neuron-intrinsic factor that may permit axonal sprouting is microtubule-associated protein 1B (MAP1B) in a specific phosphorylated form (MAP1B-P), restricted to growing or regenerating axons. We show here that both in rat and mouse, a sciatic nerve cut is rapidly followed by the appearance of MAP1B-P expression in lamina II, increasing to a maximum between 8 and 15 days, and diminishing after three months. Evidence is provided that sprouting and induction of MAP1B-P expression after peripheral injury are phenomena concerning essentially myelinated axons. This is in accordance with in situ hybridization data showing especially high MAP1B-mRNA levels in large size DRG neurons that give rise to myelinated fibers. We then employed a second lesion model, multiple rhizotomy with one spared root. In this case, unmyelinated CGRP expressing fibers do indeed sprout, but coexpression of MAP1B-P and CGRP is never observed in lamina II. Finally, because a characteristic of myelinated fibers is their high content in neurofilament protein heavy subunit (NF-H), we used NF-H-LacZ transgenic mice to verify that MAP1B-P induction and central sprouting were not affected by perturbing the axonal organization of neurofilaments. We conclude that MAP1B-P is well suited as a rapidly expressed, axon-intrinsic marker associated with plasticity of myelinated fibers.

Role of long-range repulsive forces in organizing axonal neurofilament distributions: evidence from mice deficient in myelin-associated glycoprotein.

When the axon of a motor neuron is sectioned and visualized by electron microscopy, a two-dimensional distribution of neurofilaments (NFs) with nonrandom spacing is revealed; this ordered arrangement implies the presence of physical interactions between the NFs. To gain insight into the molecular basis of this organization, we characterized NF distributions from mouse sciatic nerve cross sections using two statistical mechanical measures: radial distribution functions and occupancy probability distributions. Our analysis shows that NF organization may be described in terms of effective pairwise interactions. In addition, we show that these statistical mechanical measures can detect differences in NF architecture between wild-type and myelin-associated glycoprotein null mutant mice. These differences are age dependent, with marked contrast between the NF distributions by 9 months of age. Finally, using Monte Carlo simulations, we compare the experimental results with predictions for models in which adjacent NFs interact through rigid cross bridges, deformable cross bridges, and long-range repulsive forces. Among the models tested, a model in which the filaments interact through a long-range repulsive force is most consistent with the results of our analysis.

Relating Interactions between Neurofilaments to the Structure of Axonal Neurofilament Distributions through Polymer Brush Models

Neurofilaments (NFs) have been proposed to interact with one another through mutual steric exclusion of their unstructured C-terminal “sidearm” domains, producing order in axonal NF distributions and conferring mechanical strength to the axon. Here we apply theory developed for polymer brushes to examine the relationship between the brush properties of the sidearms and NF organization in axons. We first measure NF–NF radial distribution functions and occupancy probability distributions for adult mice. Interpreting the probability distributions using information theory, we show that the NF distributions may be represented by a single pair potential of mean force. Then, to explore the relationship between model parameters and NF architecture, we conduct two-dimensional Monte Carlo simulations of NF cross-sectional distributions. We impose purely repulsive interaction potentials in which the sidearms are represented as neutral and polyelectrolyte chains. By treating the NFs as telechelic polymer brushes, we also incorporate cross-bridging interactions. Both repulsive potentials are capable of reproducing NF cross-sectional densities and their pair correlations. We find that NF structure is sensitive to changes in brush thickness mediated by chain charge, consistent with the experimental observation that sidearm phosphorylation regulates interfilament spacing. The presence of attractive cross-bridging interactions contributes only modestly to structure for moderate degrees of cross-bridging and leads to NF aggregation for extensive cross-bridging.

Neurofilaments of aged rats: The strengthened interneurofilament interaction and the reduced amount of NF‐M

Amyotrophic lateral sclerosis is an age-related neurological disease, characterized by neurofilament (NF) accumulation in primary axons followed by degeneration of motor neurons. To elucidate age-related factors that might lead to pathological NF accumulation, NFs were compared between young and aged rats. Electron microscopic examination of sciatic nerve axons revealed that NFs were more than twice as densely packed in aged rat axons (542 ± 180 NFs/mm2) as in young adult rat axons (211 ± 73 NFs/mm2). The NFs isolated from aged rats also appeared to be more aggregated than those from young rats. Phosphorylation at the head or tail domains was studied as a possible candidate affecting NF organization. Western blotting with phosphorylation-dependent antibodies showed higher phosphorylation of NF-H in the tail domains of aged rat spinal cord NFs, but dephosphorylation did not diminish the differences in aggregation between aged and young rat NFs. On the other hand, when NFs were phosphorylated by A-kinase on their head domains, the extent of phosphorylation in NF-M of aged rat NFs was only one-third of young rat NFs. We found that aged rat NFs contained only 60% of the NF-M of young rat NFs in molar ratio compared to NF-L. These results raise a possibility that the decreased amount of NF-M induces the aggregates of isolated NFs and the higher packing density of NF in aged rat axons. J. Neurosci. Res. 58:337–348, 1999. © 1999 Wiley-Liss, Inc.

Neurofilaments of aged rats: The strengthened interneurofilament interaction and the reduced amount of NF-M

Amyotrophic lateral sclerosis is an age-related neurological disease, characterized by neurofilament (NF) accumulation in primary axons followed by degeneration of motor neurons. To elucidate age-related factors that might lead to pathological NF accumulation, NFs were compared between young and aged rats. Electron microscopic examination of sciatic nerve axons revealed that NFs were more than twice as densely packed in aged rat axons (542 +/- 180 NFs/mm2) as in young adult rat axons (211 +/- 73 NFs/mm2). The NFs isolated from aged rats also appeared to be more aggregated than those from young rats. Phosphorylation at the head or tail domains was studied as a possible candidate affecting NF organization. Western blotting with phosphorylation-dependent antibodies showed higher phosphorylation of NF-H in the tail domains of aged rat spinal cord NFs, but dephosphorylation did not diminish the differences in aggregation between aged and young rat NFs. On the other hand, when NFs were phosphorylated by A-kinase on their head domains, the extent of phosphorylation in NF-M of aged rat NFs was only one-third of young rat NFs. We found that aged rat NFs contained only 60% of the NF-M of young rat NFs in molar ratio compared to NF-L. These results raise a possibility that the decreased amount of NF-M induces the aggregates of isolated NFs and the higher packing density of NF in aged rat axons.

Serine-23 is a major protein kinase A phosphorylation site on the amino-terminal head domain of the middle molecular mass subunit of neurofilament proteins.

We have shown previously that phosphate groups on the amino-terminal head domain region of the middle molecular mass subunit of neurofilament proteins (NF-M) are added by second messenger-dependent protein kinases. Here, we have identified Ser23 as a specific protein kinase A phosphorylation site on the native NF-M subunit and on two synthetic peptides, S1 (14RRVPTETRSSF24) and S2 (21RSSFSRVSGSPSSGFRSQSWS41), localized within the amino-terminal head domain region. Ser23 was identified as a phosphorylation site on the 32P-labeled alpha-chymotryptic peptide that carried >80% of the 32P-phosphates incorporated into the NF-M subunit by protein kinase A. The synthetic peptides S1 and S2 were phosphorylated 18 and two times more efficiently by protein kinase A than protein kinase C, respectively. Neither of the peptides was phosphorylated by casein kinase II. The sequence analyses of the chemically modified phosphorylated serine residues showed that Ser23 was the major site of phosphorylation for protein kinase A on both S1 and S2 peptides. Low levels of incorporation of 32P-phosphates into Ser22, Ser28, and Ser32 by protein kinase A were also observed. Protein kinase C incorporated 32P-phosphates into Ser22, Ser23, Ser25, Ser28, Ser32, and a threonine residue, but none of these sites could be assigned as a major site of phosphorylation. Analyses of the phosphorylated synthetic peptides by liquid chromatography-tandem mass spectrometry also showed that protein kinase A phosphorylated only one site on peptide S1 and that ions with up to four phosphates were detected on peptide S2. Analysis of the data from the tandem ion trap mass spectrometry by using the computer program PEPSEARCH did not unequivocally identify the specific sites of phosphorylation on these serine-rich peptides. Our data suggest that Ser23 is a major protein kinase A-specific phosphorylation site on the amino-terminal head region of the NF-M subunit. Phosphorylation of Ser23 on the NF-M subunit by protein kinase A may play a regulatory role in neurofilament assembly and/or the organization of neurofilaments in the axon.

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.

Expression of neurofilament proteins by horizontal cells in the rabbit retina varies with retinal location

Classical neurofibrillar staining methods and immunocytochemistry with antibodies to the light, medium and heavy chain subunits of the neurofilament triplet have been used for in situ and in vitro investigation of the organization of neurofilaments in A- and B-type horizontal cells of the adult rabbit retina. Surprisingly, their expression and organization within a cell is dependent on its location along the dorso-ventral axis of the retina. A-type horizontal cells in superior retina consistently stained with a wide variety of neurofibrillar methods to reveal neurofibrillar bundles, which immunocytochemistry showed to contain all three neurofilament subunits. A-type horizontal cells in inferior retina were uniformly refractory to neurofibrillar staining, although they expressed all three subunits. However, there was less of the light and medium subunits; the organization of the filaments into bundles (neurofibrils) is minimal. B-type horizontal cells could not be stained with any neurofibrillar method and were not recognizable by in situ immunocytochemistry. However, B-type cells could be seen to express all three subunits in vitro, but the expression of the light and medium subunits was weak. There was only a slight difference between B-type cells taken from superior and inferior retina. Combined with the results of recent transfection studies, these findings suggest that the amount of the light neurofilament subunit present in a horizontal cell determines its content of neurofibrillar bundles, and that rabbit horizontal cells may contain more neurofilament protein, particularly of the heavy subunit, than is used for neurofilament formation.(ABSTRACT TRUNCATED AT 250 WORDS)