Neurofilament Dynamics Research Articles

Neurofilament L dynamics description in the model of tauopathy

AbstractBackgroundNeurofilament proteins are important constituents of neuronal cytoskeleton along with microtubules. Increased concentration of neurofilament light protein in cerebrospinal fluid(CSF) and plasma is considered as a potential biomarker of axonal degeneration [1]. Together with tau, neurofilaments are contained in the neurofibrillary tangles (NFT), the biomarker of Alzheimer’s disease and other tauopathies [2]. The goal of this study was to develop a quantitative systems pharmacology (QSP) model to describe neurofilament dynamics during progression of tauopathies both in human patients and in transgenic P301L Tau mice.MethodThe quantitative model correctly describes the dynamics of neurofilament proteins during neurodegeneration processes, which include cytoskeletal degradation by calpain, proteasome, autophagy and caspase, formation of pathological aggregates, release of neurofilament proteins from degenerated axons to cerebrospinal fluid and plasma. These processes are dependent on pathological processes in tauopathy models, such as changes in protein degradation, axonal transport deficits, pathological hyperphosphorylated tau accumulation. To calibrate the model, the literature data on neurofilament concentration in brain, cerebrospinal fluid and plasma, phosphorylation and degradation level were used. Description of the processes is partially based on neuron homeostasis model developed earlier [3]. The model was validated on taupathy P301L transgenic mice, amyloid APP/PS1 mice and on Alzheimer’s disease patients’ data.ResultThe described model correctly predicts the increase of neurofilament light protein concentration in CSF and plasma both in P301L transgenic mice model of tauopathy and in human patients versus controls. Pathological changes of microtubule dynamics and cytoskeleton breakdown are also described. Model predicts the reduction of NfL concentration in CSF during potential tau targeting therapy in P301L transgenic mice and humans.ConclusionThe developed model can be helpful in simulating potential effects of therapeutics used in neurodegeneration diseases treatment.

Annual Plasma Neurofilament Dynamics Is a Sensitive Biomarker of Disease Activity in Patients with Multiple Sclerosis.

Background and Objectives: Neurofilament light chain (NfL) is a sensitive biomarker of neuroaxonal damage. This study aimed to assess the relationship between the annual change in plasma NfL (pNfL) and disease activity in the past year, as defined by the concept no evidence of disease activity (NEDA) in a cohort of multiple sclerosis (MS) patients. Materials and Methods: Levels of pNfL (SIMOA) were examined in 141 MS patients and analyzed in relationship to the NEDA-3 status (absence of relapse, disability worsening, and MRI activity) and NEDA-4 (NEDA-3 extended by brain volume loss ≤ 0.4%) during the last 12 months. Patients were divided into two groups: annual pNfL change with an increase of less than 10% (group 1), and pNfL increases of more than 10% (group 2). Results: The mean age of the study participants (n = 141, 61% females) was 42.33 years (SD, 10.17), and the median disability score was 4.0 (3.5-5.0). The ROC analysis showed that a pNfL annual change ≥ 10% correlates with the absence of the NEDA-3 status (p < 0.001; AUC: 0.92), and the absence of the NEDA-4 status (p < 0.001; AUC: 0.839). Conclusions: Annual plasma NfL increases of more than 10% appear to be a useful tool for assessing disease activity in treated MS patients.

Intraindividual Neurofilament Dynamics in Serum Mark the Conversion to Sporadic Parkinson's Disease.

With disease-modifying treatment strategies on the horizon, stratification of individual patients at the earliest stages of Parkinson's disease (PD) is key-ideally already at clinical disease onset. Blood levels of neurofilament light chain (NfL) provide an easily accessible fluid biomarker that might allow capturing the conversion from prodromal to manifest PD. We assessed longitudinal serum NfL levels in subjects converting from prodromal to manifest sporadic PD (converters), at-risk subjects, and matched controls (72 participants with ≈4 visits), using single-molecule array (Simoa) technique. While NfL levels were not increased at the prodromal stage, subjects converting to the manifest motor stage showed a significant intraindividual acceleration of the age-dependent increase of NfL levels. The temporal dynamics of intraindividual NfL blood levels might mark the conversion to clinically manifest PD, providing a potential stratification biomarker for individual disease onset in the advent of precision medicine for PD. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Influence of a GSK3β phosphorylation site within the proximal C-terminus of Neurofilament-H on neurofilament dynamics.

ABSTRACTPhosphorylation of the C-terminal tail of the heavy neurofilament subunit (NF-H) impacts neurofilament (NF) axonal transport and residence within axons by fostering NF-NF associations that compete with transport. We tested the role of phosphorylation of a GSK-3β consensus site (S493) located in the proximal portion of the NF-H tail in NF dynamics by transfection of NB2a/d1 cells with NF-H, where S493 was mutated to aspartic acid (S493D) or to alanine (S493A) to mimic constitutive phosphorylation and non-phosphorylation. S493D underwent increased transport into axonal neurites, while S493A displayed increased perikaryal NF aggregates that were decorated by anti-kinesin. Increased levels of S493A co-precipitated with anti-kinesin indicating that reduced transport of S493A was not due to reduced kinesin association but due to premature NF-NF interactions within perikarya. S493D displayed increased phospho-immunoreactivity within axonal neurites at downstream C-terminal sites attributable to mitogen-activated protein kinase and cyclin-dependent kinase 5. However, S493D was more prone to proteolysis following kinase inhibition, suggesting that S493 phosphorylation is an early event that alters sidearm configuration in a manner that promotes appropriate NF distribution. We propose a novel model for sidearm configuration.

Morphine causes persistent induction of nitrated neurofilaments in cortex and subcortex even during abstinence

Morphine has a profound role in neurofilament (NF) expression. However, there are very few studies on the fate of NFs during morphine abstinence coinciding with periods of relapse. Mice were treated chronically with morphine to render them tolerant to and dependent on morphine and sacrificed thereafter while another group, treated similarly, was left for 2months without morphine. A long-lasting alteration in the stoichiometric ratio of the three NFs was observed under both conditions in both the cortex and subcortex. Morphine abstinence caused significant alterations in the phosphorylated and nitrated forms of the three NF subunits. Nitrated neurofilament light polypeptide chain (NFL) was significantly increased during chronic morphine treatment which persisted even after 2months of morphine withdrawal. Mass spectrometric analysis following two-dimensional gel electrophoresis (2DE)-gel electrophoresis of cytoskeleton fractions of both cortex and subcortex regions identified enzymes associated with energy metabolism, cytoskeleton-associated proteins as well as NFs which showed sustained regulation even after abstinence of morphine for 2months. It is suggestive that alteration in the levels of some of these proteins may be instrumental in the increased nitration of NFL during morphine exposure. Such gross alteration in NF dynamics is indicative of a concerted biological process of neuroadaptation during morphine abstinence.

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.

Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics.

C-terminal neurofilament phosphorylation mediates cation-dependent self-association leading to neurofilament incorporation into the stationary axonal cytoskeleton. Multiple kinases phosphorylate the C-terminal domains of the heavy neurofilament subunit (NF-H), including cyclin-dependent protein kinase 5 (CDK5), mitogen-activated protein kinases (MAPKs), casein kinase 1 and 2 (CK1 and CK2) and glycogen synthase kinase 3β (GSK3β). The respective contributions of these kinases have been confounded because they phosphorylate multiple substrates in addition to neurofilaments and display extensive interaction. Herein, differentiated NB2a/d1 cells were transfected with constructs expressing GFP-tagged NF-H, isolated NF-H sidearms and NF-H lacking the distal-most 187 amino acids. Cultures were treated with roscovitine, PD98059, Li(+), D4476, tetrabromobenzotriazole and calyculin, which are active against CDK5, MKK1 (also known as MAP2K1), GSK3β, CK1, CK2 and protein phosphatase 1 (PP1), respectively. Sequential phosphorylation by CDK5 and GSK3β mediated the neurofilament-neurofilament associations. The MAPK pathway (i.e. MKK1 to ERK1/2) was found to downregulate GSK3β, and CK1 activated PP1, both of which promoted axonal transport and restricted neurofilament-neurofilament associations to axonal neurites. The MAPK pathway and CDK5, but not CK1 and GSK3β, inhibited neurofilament proteolysis. These findings indicate that phosphorylation of neurofilaments by the proline-directed MAPK pathway and CDK5 counterbalance the impact of phosphorylation of neurofilaments by the non-proline-directed CK1 and GSK3β.

Fast rearrangement of the neuronal growth cone's actincytoskeleton following VEGF stimulation.

The neuronal growth cone plays a crucial role in the development of the nervous system. This highly motile structure leads the axon to its final destination by translating guidance cues into cytoskeletal rearrangements. Recently, vascular endothelial growth factor (VEGF), which is essential for angiogenesis and vascular sprouting, has been found to exert a trophic activity also on neurons, leading to an increased axonal outgrowth, similar to the well-known nerve growth factor (NGF). The neurotrophic properties of VEGF are likely to be promoted via the VEGF receptor 2 (VEGFR-2) and neuropilin-1 (NRP-1). In the long term, VEGF attracts and influences the growth cone velocity and leads to growth cone enlargement. The present study focuses on immediate VEGF effects using RFP-actin and GFP-NF-M microinjected chicken dorsal root ganglia for live cell imaging of the neuronal growth cone. We analyzed actin and neurofilament dynamics following VEGF and NGF treatment and compared the effects. Furthermore, key signaling pathways of VEGF were investigated by specific blocking of VEGFR-2 or NRP-1. With the aid of confocal laser scanning microscopy and stimulated emission depletion microscopy, we show for the first time that VEGF has a quick effect on the actin-cytoskeleton, since actin rearrangements were identifiable within a few minutes, leading to a dramatically increased motion. Moreover, these effects were strongly enhanced by adding both VEGF and NGF. Most notably, the effects were inhibited by blocking VEGFR-2, therefore we propose that the immediate effects of VEGF on the actin-cytoskeleton are mediated through VEGFR-2.

Axonal regeneration is compromised in NFH-LacZ transgenic mice but not in NFH-GFP mice

To investigate neurofilament (NF) dynamics during the cytoskeleton reorganization in regenerating axons, and their electrophysiological and histological consequences, we used two transgenic lines of mice: neurofilament high (NFH)-LacZ and NFH-green fluorescent protein (GFP). In NFH-LacZ mice, NFs are retained in cell bodies and deficient in axons (Eyer and Peterson, 1994), while in NFH-GFP mice the fluorescent fusion protein is normally transported along axons (Letournel et al., 2006). Following a crush of the sciatic nerve, conduction recovery in NFH-GFP mice is similar to wild-type (wt) mice, but it is reduced in NFH-LacZ mice. Moreover, changes of axonal calibres following regeneration are similar between NFH-GFP and wt mice, but they are systematically reduced in NFH-LacZ mice. Finally, the axonal transport of NFH-GFP fusion protein and NFs is re-initiated after the crush as evidenced by the fluorescent and immunolabelling of axons distal from the crushed point, but NFs and the fusion protein are not transported along axons during regeneration in NFH-LacZ mice. Together, these results argue that the absence of axonal NFs in NFH-LacZ mice compromises the axonal regeneration, and that the NFH-GFP reporter fusion protein represents an efficient model to evaluate the NF dynamics during axonal regeneration.

Fast rearrangement of the neuronal growth cone’s actin cytoskeleton following VEGF stimulation

The neuronal growth cone plays a crucial role in the development of the nervous system. This highly motile structure leads the axon to its final destination by translating guidance cues into cytoskeletal rearrangements. Recently, vascular endothelial growth factor (VEGF), which is essential for angiogenesis and vascular sprouting, has been found to exert a trophic activity also on neurons, leading to an increased axonal outgrowth, similar to the well-known nerve growth factor (NGF). The neurotrophic properties of VEGF are likely to be promoted via the VEGF receptor 2 (VEGFR-2) and neuropilin-1 (NRP-1). In the long term, VEGF attracts and influences the growth cone velocity and leads to growth cone enlargement. The present study focuses on immediate VEGF effects using RFP-actin and GFP-NF-M microinjected chicken dorsal root ganglia for live cell imaging of the neuronal growth cone. We analyzed actin and neurofilament dynamics following VEGF and NGF treatment and compared the effects. Furthermore, key signaling pathways of VEGF were investigated by specific blocking of VEGFR-2 or NRP-1. With the aid of confocal laser scanning microscopy and stimulated emission depletion microscopy, we show for the first time that VEGF has a quick effect on the actin-cytoskeleton, since actin rearrangements were identifiable within a few minutes, leading to a dramatically increased motion. Moreover, these effects were strongly enhanced by adding both VEGF and NGF. Most notably, the effects were inhibited by blocking VEGFR-2, therefore we propose that the immediate effects of VEGF on the actin-cytoskeleton are mediated through VEGFR-2.

Synaptic defects in spinal muscular atrophy animal models

Proximal spinal muscular atrophy, the most frequent genetic cause of childhood lethality, is caused by homozygous loss or mutation of the SMN1 gene on human chromosome 5, which codes for the survival motor neuron (SMN) protein. SMN plays a role in the assembly of small nuclear ribonucleoproteins and, additionally, in synaptic function. SMN deficiency produces defects in motor neuron β-actin mRNA axonal transport, neurofilament dynamics, neurotransmitter release, and synapse maturation. The underlying molecular mechanisms and, in particular, the role of the cytoskeleton on the pathogenesis of this disease are starting to be revealed.

Neurofilament Dynamics: A Tug of War by Microtubule Motors

Structural support for axons, which can consist of volumes thousands of times larger than the neuronal perikaryon, is provided in part by neurofilaments (NFs), the major fibrous constituent of the axonal cytoskeleton. Most NFs undergo anterograde transport (towards the synapse or growth cone), while a few undergo retrograde transport (back towards the perikaryon). Some NFs exhibit an extended residence time along axons, which allows NFs to provide structural support to the axon yet minimizes NF turnover, which would otherwise impart a prohibitive metabolic burden upon the neuron. Herein, we explore known and hypothesized roles for microtubule motors in transport and distribution of NFs along axons. We present evidence that those NFs that display extended residence along axons are critically dependent upon surrounding microtubules, and that simultaneous interaction with multiple microtubule motors provides the architectural force regulating their distribution.

Regulation of neurofilament dynamics by phosphorylation

Neurofilament (NF) phosphorylation has long been considered to regulate axonal transport rate and in doing so to provide stability to mature axons. Studies utilizing mice in which the C-terminal region of NF subunits (which contains the vast majority of phosphorylation sites) has been deleted has prompted an ongoing challenge to this hypothesis. We evaluate the collective evidence to date for and against a role for NF C-terminal phosphorylation in regulation of axonal transport and in providing structural support for axons, including some novel studies from our laboratory. We present a few suggestions for further experimentation in this area, and expand upon previous models for axonal NF dynamics. Finally, we address how C-terminal phosphorylation is regionally and temporally regulated by a balance of kinase and phosphatase activities, and how misregulation of this balance can contribute to motor neuron disease.

Potentiation of arsenic neurotoxicity by folate deprivation: Protective role of S-adenosyl methionine

Folate deficiency contributes to a variety of age-related neurological and psychological disorders including amyotrophic lateral sclerosis (ALS). The environmental neurotoxin arsenic has recently been linked with decreased neurofilament (NF) content in peripheral nerve. We examined herein, whether or not folate deprivation potentiated the impact of arsenic on NF dynamics. Arsenic inhibited translocation of NFs into axonal neurites in culture and increased perikaryal NF phosphoepitopes. Folate deprivation potentiated the impact of arsenic on these phenomena. Supplementation with S-adenosyl methionine (SAM) attenuated the impact of folate deprivation on arsenic neurotoxicity, consistent with the decrease in SAM following folate deprivation and the requirement for SAM-mediated methylation for arsenic bioelimination. These findings demonstrate how key nutritional deficiencies can potentiate the impact of enrivonmental neurotoxins.

Arsenic inhibits neurofilament transport and induces perikaryal accumulation of phosphorylated neurofilaments: Roles of JNK and GSK-3β

The environmental neurotoxin arsenic has recently been associated with altered neurofilament (NF) content in sciatic nerve. We examined herein the impact of sodium arsenite (the inorganic form of arsenic) on NF dynamics. Treatment of differentiated NB2/d1 cells and cultured dorsal root ganglion neurons decreased NF transport into axonal neurites and increased perikaryal phospho-NF immunoreactivity. Both of these effects were prevented by a pharmacological inhibitor (SP600125) of c-jun terminal kinase and by expression of a dominant-negative form of this kinase. Arsenic-induced inhibition of NF transport was prevented by treatment with lithium, a selective inhibitor of glycogen synthase kinase-3beta. Pharmacological inhibitors of cyclin-dependent kinase 5 and p38 mitogen-activated protein kinase did not attenuate the effects of arsenic on NF dynamics. These latter findings suggest that this environmental neurotoxin could contribute to peripheral neuropathy by perturbing NF dynamics.

Cyclin-dependent kinase 5 increases perikaryal neurofilament phosphorylation and inhibits neurofilament axonal transport in response to oxidative stress.

Cyclin-dependent kinase 5 (cdk5) phosphorylates the high molecular weight neurofilament (NF) protein. Overexpression of cdk5 inhibits NF axonal transport and induces perikaryal accumulation of disordered phospho-NF cables. Experimental and clinical motor neuron disease is characterized by oxidative stress, increased cdk5 activity, and accumulation of phospho-NFs within perikarya or proximal axons. Because oxidative stress increases cdk5 activity in experimental motor neuron disease, we examined whether oxidative stress induced cdk5-mediated NF phosphorylation. Treatment of cultured neuronal cells with hydrogen peroxide inhibited axonal transport of green fluorescent protein-tagged NF subunits and induced perikaryal accumulation of NF phosphoepitopes normally confined to axons. These effects were prevented by treatment with the cdk5 inhibitor roscovitine or transfection with a construct expressing the endogenous cdk5 inhibitor peptide. These findings indicate that oxidative stress can compromise NF dynamics via hyperactivation of cdk5 and suggest that antioxidants may alleviate multiple aspects of neuropathology in motor neuron disease.

Cdk5 regulates axonal transport and phosphorylation of neurofilaments in cultured neurons.

Phosphorylation has long been considered to regulate neurofilament (NF) interaction and axonal transport, and, in turn, to influence axonal stability and their maturation to large-caliber axons. Cdk5, a serine/threonine kinase homologous to the mitotic cyclin-dependent kinases, phosphorylates NF subunits in intact cells. In this study, we used two different haptenized NF subunits and manipulated cdk5 activity by microinjection, transfection and pharmacological inhibition to monitor the effect of Cdk5-p35 on NF dynamics and transport. We demonstrate that overexpression of cdk5 increases NF phosphorylation and inhibits NF axonal transport, whereas inhibition both reduces NF phosphorylation and enhances NF axonal transport in cultured chicken dorsal-root-ganglion neurons. Large phosphorylated-NF 'bundles' were prominent in perikarya following cdk5 overexpression. These findings suggest that Cdk5-p35 activity regulates normal NF distribution and that overexpression of Cdk5-p35 induces perikaryal accumulation of phosphorylated-NFs similar to those observed under pathological conditions.

The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation.

The forms in which neurofilament (NF) subunits undergo axonal transport is controversial. Recent studies from have provided real-time visualization of the slow axonal transport of NF subunits by transfecting neuronal cultures with constructs encoding green fluorescent protein (GFP)-conjugated NF-M subunits. In our studies in differentiated NB2a/d1 cells, the majority NF subunits underwent transport in the form of punctate NF precursors, while studies in cultured neurons have demonstrated transport of NF subunits in predominantly filamentous form. Although different constructs were used in these studies, transfection of the same cultured neurons with our construct yielded the filamentous pattern observed by others, while transfection of our cultures with their construct generated punctate structures, confirming that the observed differences did not reflect variances in assembly-competence among the constructs. Manipulation of intracellular kinase, phosphatase, and protease activities shifted the predominant form of GFP-conjugated subunits between punctate and filamentous, confirming, as shown previously for vimentin, that punctate structures represent precursors for intermediate filament formation. Since these prior studies were conducted at markedly differing neuronal differentiation states, we tested the alternate hypothesis that these differing results reflected developmental alterations in NF dynamics that accompany various stages of neuritogenesis. We conducted time-course analyses of transfected NB2a/d1 cells, including monitoring of transfected cells over several days, as well as transfecting cells at varying intervals prior to and following induction of differentiation and axonal neurite outgrowth. GFP-conjugated subunits were predominantly filamentous during the period of most robust axonal outgrowth and NF accumulation, and presented a mixed profile of punctate and filamentous forms prior to neuritogenesis and following the developmental slowing of neurite outgrowth. These analyses demonstrate that NF subunits are capable of undergoing axonal transport in multiple forms, and that the predominant form in which NF subunits undergo axonal transport varies in accord with the rate of axonal elongation and accumulation of NFs within developing axons.

Alterations in cyclin-dependent protein kinase 5 (CDK5) protein levels, activity and immunocytochemistry in canine motor neuron disease.

Hereditary canine spinal muscular atrophy (HCSMA) is a dominantly inherited motor neuron disease in Brittany spaniels that is clinically characterized by progressive muscle weakness leading to paralysis. Histopathologically, degeneration is confined to motor neurons with accumulation of phosphorylated neurofilaments in axonal internodes. Cyclin-dependent kinase 5 (CDK5), a kinase related to the cell cycle kinase cdc2, phosphorylates neurofilaments and regulates neurofilament dynamics. We examined CDK5 activity, protein levels, and cellular immunoreactivity in nervous tissue from dogs with HCSMA, from closely age-matched controls and from dogs with other neurological diseases. On immunoblot analysis, CDK5 protein levels were increased in the HCSMA dogs (by approximately 1.5-fold in both the cytosolic and the particulate fractions). CDK5 activity was significantly increased (by approximately 3-fold) in the particulate fractions in the HCSMA dogs compared to all controls. The finding that CDK5 activity was increased in the young HCSMA homozygotes with the accelerated form of the disease, who do not show axonal swellings histologically, suggests that alterations in CDK5 occurs early in the pathogenesis, prior to the development of significant neurofilament pathology. Immunocytochemically, there was strong CDK5 staining of the nuclei, cytoplasm and axonal processes of the motor neurons in both control dogs and dogs with HCSMA. Further immunocytochemical studies demonstrated CDK5 staining where neurofilaments accumulated, in axonal swellings in the dogs with HCSMA. Our observations suggest phosphorylation-dependent events mediated by CDK5 occur in canine motor neuron disease.

Aluminum inhibits neurofilament assembly, cytoskeletal incorporation, and axonal transport. Dynamic nature of aluminum-induced perikaryal neurofilament accumulations as revealed by subunit turnover.

The mechanism by which aluminum induces formation of perikaryal neurofilament (NF) inclusions remains unclear. Aluminum treatment inhibits: 1. The incorporation of newly synthesized NF subunits into Triton-insoluble cytoskeleton of axonal neurites; 2. Their degradation and dephosphorylation; 3. Their translocation into axonal neurites. It also fosters the accumulation of phosphorylated NFs within perikarya. In the present study, we addressed the relationship among these effects. Aluminum reduced the assembly of newly synthesized NF subunits into NFs. During examination of those subunits that did assemble in the presence of aluminum, it was revealed that aluminum also interfered with transport of newly assembled NFs into axonal neurites. Similarly, a delay in axonal transport of microinjected biotinylated NF-H was observed in aluminum-treated cells. Aluminum also inhibited the incorporation of newly synthesized and microinjected subunits into the Triton-insoluble cytoskeleton within both perikarya and neurites. Once incorporated into Triton-insoluble cytoskeletons, however, biotinylated subunits were retained within perikarya of aluminum-treated cells to a greater extent than within untreated cells. Notably, these subunits were depleted in the presence and absence of aluminum within 48 h, despite the persistence of the aluminum-induced perikaryal accumulation itself, suggesting that individual NF subunits undergo turnover even within aluminum-induced perikaryal accumulations. These findings demonstrate that aluminum interferes with multiple aspects of neurofilament dynamics and furthermore leaves open the possibility that aluminum-induced perikaryal NF whorls may not represent permanent structures, but rather may require continued recruitment of cytoskeletal constituents.