Neurofilament Phosphorylation Research Articles

Accumulation of cortical hyperphosphorylated neurofilaments as a marker of neurodegeneration in multiple sclerosis

Background: Axonal loss and grey matter neuronal injury are pathological processes that contribute to disease progression in multiple sclerosis (MS). Axon damage has been associated with changes in the phosphorylation state of neurofilaments and the presence of axonal spheroids. Perikaryal accumulation of abnormally phosphorylated neurofilament proteins has been reported in some neurodegenerative diseases. Objectives: The objective of this article is to determine whether abnormally phosphorylated neurofilament accumulates in neuronal perikarya in demyelinated MS cortex. Methods: We used an antibody to hyperphosphorylated neurofilament-H (SMI-34) to assess the level and distribution of this antigen in paraffin sections of cerebral cortex from cases of neuropathologically confirmed MS and controls. We also examined the relationship of neurofilament phosphorylation to cortical demyelination. Results: The number of SMI-34-positive neuronal somata was significantly higher in the MS cortex than the control cortex. As a proportion of the total number of neurons present (i.e. taking account of neuronal loss), the proportion of SMI-34-positive neurons was also significantly higher in the demyelinated and non-demyelinated MS cortex than the control cortex. Conclusions: MS is associated with the widespread accumulation of hyperphosphorylated neurofilament protein in neuronal somata, with the most marked accumulation in regions of cortical demyelination. This aberrant localisation of hyperphosphorylated neurofilament protein may contribute to neuronal dysfunction and degeneration in MS patients.

Neurofilament protein levels: Quantitative analysis in essential tremor cerebellar cortex

Essential tremor (ET) is among the most prevalent neurological diseases. A substantial increase in the number of Purkinje cell axonal swellings (torpedoes) has been identified in ET brains. We recently demonstrated that torpedoes in ET contain an over-accumulation of disorganized neurofilament (NF) proteins. This now raises the question whether NF protein composition and/or phosphorylation state in cerebellar tissue might differ between ET cases and controls. We used a Western blot analysis to compare the levels and phosphorylation state of NF proteins and α-internexin in cerebellar tissue from 47 ET cases versus 26 controls (2:1 ratio). Cases and controls did not differ with respect to the cerebellar levels of NF-light (NF-L), NF-medium (NF-M), NF-heavy (NF-H), or α-internexin. However, SMI-31 levels (i.e., phosphorylated NF-H) and SMI-32 levels (i.e., non-phosphorylated NF-H) were significantly higher in ET cases than controls (1.28±0.47 vs. 1.06±0.32, p=0.02; and 1.38±0.75 vs. 1.00±0.42, p=0.006). Whether the abnormal phosphorylation state that we observed is a cause of defective axonal transport and/or function of NFs in ET is not known. NF abnormalities have been demonstrated in several neurodegenerative diseases. Regardless of whether these protein aggregates are the cause or consequence of these diseases, NF abnormalities have been shown to be an important factor in the cellular disruption observed in several neurodegenerative diseases. Therefore, further analyses of these NF abnormalities and their mechanisms are important to enhance our understanding of disease pathogenesis in ET.

Neurofilament Phosphorylation during Development and Disease: Which Came First, the Phosphorylation or the Accumulation?

Posttranslational modification of proteins is a ubiquitous cellular mechanism for regulating protein function. Some of the most heavily modified neuronal proteins are cytoskeletal proteins of long myelinated axons referred to as neurofilaments (NFs). NFs are type IV intermediate filaments (IFs) that can be composed of four subunits, neurofilament heavy (NF-H), neurofilament medium (NF-M), neurofilament light (NF-L), and α-internexin. Within wild type axons, NFs are responsible for mediating radial growth, a process that determines axonal diameter. NFs are phosphorylated on highly conserved lysine-serine-proline (KSP) repeats located along the C-termini of both NF-M and NF-H within myelinated axonal regions. Phosphorylation is thought to regulate aspects of NF transport and function. However, a key pathological hallmark of several neurodegenerative diseases is ectopic accumulation and phosphorylation of NFs. The goal of this review is to provide an overview of the posttranslational modifications that occur in both normal and diseased axons. We review evidence that challenges the role of KSP phosphorylation as essential for radial growth and suggests an alternative role for NF phosphorylation in myelinated axons. Furthermore, we demonstrate that regulation of NF phosphorylation dynamics may be essential to avoiding NF accumulations.

The Involvement of Neurofilament Heavy Chain Phosphorylation in the Maturation and Degeneration of Rat Oocytes

Neurofilaments (NF) are intermediate filament proteins that were originally found to be expressed in neurons and are involved in the maintenance of axonal structure. However, there has not been much research on the expression and physiological significance of NF in other organs. In the present study, we examined the expression of NF in rat ovaries and found that NF heavy chain (NF-H) was expressed in oocytes of follicles from the primary to mature stages, ovulated oocytes, fertilized zygotes, and degenerative oocytes of atretic follicles. Cytoplasmic NF-H disappeared at the two-cell stage of embryonic development, whereas degenerative oocytes of atretic follicles continued to express NF-H even after fragmentation. An antibody that specifically recognizes phosphorylated NF-H (pNF-H) was used to examine the pattern of NF-H phosphorylation in oocytes. pNF-H was detected in the cytoplasm and nuclei of oocytes of mature and atretic follicles, ovulated oocytes, and one-cell zygotes. Treatment with 3,3'-iminodipropionitrile, which induces aberrant phosphorylation of NF in the perikarya of neurons and causes neuropathy, induced oocyte degeneration with follicular atresia, phosphorylation of NF-H in oocytes, and ovarian gene expression of cyclin-dependent kinase 5, a candidate kinase of NF-H. However, an indicator of neuron degeneration, Fluoro-Jade C, failed to stain the pNF-H-immunopositive oocytes. Our results indicate that NF-H expressed in oocytes may be involved in the maintenance of oocyte structure during follicular growth and that the phosphorylation of NF-H in ephemeral oocytes may contribute to the degeneration process of oocytes.

Psychosine induces the dephosphorylation of neurofilaments by deregulation of PP1 and PP2A phosphatases

Patients with Krabbe disease, a genetic demyelinating syndrome caused by deficiency of galactosyl-ceramidase and the resulting accumulation of galactosyl-sphingolipids, develop signs of a dying-back axonopathy compounded by a deficiency of large-caliber axons. Here, we show that axonal caliber in Twitcher mice, an animal model for Krabbe disease, is impaired in peripheral axons and is accompanied by a progressive reduction in the abundance and phosphorylation of the three neurofilament (NF) subunits. These changes correlate with an increase in the density of NFs per cross-sectional area in numerous mutant peripheral axons and abnormal increases in the activity of two serine/threonine phosphatases (PP1 and PP2A) in mutant tissue. Similarly, acutely isolated mutant cortical neurons show abnormal phosphorylation of NFs. Psychosine, the neurotoxin accumulated in Krabbe disease, was sufficient to induce abnormal dephosphorylation of NF subunits in a normal motor neuron cell line as well as in acutely isolated normal cortical neurons. This in vitro effect was mediated by PP1 and PP2A, which specifically dephosphorylated NFs. These results demonstrate that the reduced caliber observed in some axons in Krabbe disease involves abnormal dephosphorylation of NFs. We propose that a psychosine-driven pathogenic mechanism through deregulated phosphotransferase activities may be involved in this process.

Arrest of Myelination and Reduced Axon Growth When Schwann Cells Lack mTOR

In developing peripheral nerves, differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years, there has been an increased understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination, together with a growing appreciation of some of the signaling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete. The mammalian target of rapamycin (mTOR) is a core kinase in two major complexes, mTORC1 and mTORC2, that regulate cell growth and differentiation in a variety of mammalian cells. Here we show that elimination of mTOR from murine Schwann cells prevented neither radial sorting nor the initiation of myelination. However, normal postnatal growth of myelinating Schwann cells, both radially and longitudinally, was highly retarded. The myelin sheath in the mutant was much thinner than normal; nevertheless, sheath thickness relative to axon diameter (g-ratio) remained constant in both wild-type and mutant nerves from P14 to P90. Although axon diameters were normal in the mutant at the initiation of myelination, further growth as myelination proceeded was retarded, and this was associated with reduced phosphorylation of neurofilaments. Consistent with thinner axonal diameters and internodal lengths, conduction velocities in mutant quadriceps nerves were also reduced. These data establish a critical role for mTOR signaling in both the longitudinal and radial growth of the myelinating Schwann cell.

Axonopathy is associated with complex axonal transport defects in a model of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease characterized by myelin and axonal pathology. In a viral model of MS, we tested whether axonopathy initiation and development are based on an impaired transport of neurofilaments. Spinal cords of Theiler's murine encephalomyelitis virus (TMEV)-infected and mock-infected mice and TMEV infected neuroblastoma N1E-115 cells were analyzed by microarray analysis, light microscopy and electron and laser confocal microscopy. In vivo axonal accumulation of non-phosphorylated neurofilaments after TMEV infection revealed a temporal development caused by the impairments of the axonal traffic consisting of the downregulation of kinesin family member 5A, dynein cytoplasmic heavy chain 1, tau-1 and β-tubulin III expression. In addition, alterations of the protein metabolism were also noticed. In vitro, the TMEV-infected N1E-115 cells developed tandem-repeated swellings similar to in vivo alterations. Furthermore, the hypothesis of an underlying axonal self-destruction program involving nicotinamide adenine dinucleotide depletion was supported by molecular findings. The obtained data indicate that neurofilament accumulation in TME is mainly the result of dysregulation of their axonal transport machinery and impairment of neurofilament phosphorylation and protein metabolism. The present findings allow a more precise understanding of the complex interactions responsible for initiation and development of axonopathies in inflammatory degenerative diseases.

Neurofilament phosphorylation regulates axonal transport by an indirect mechanism: A merging of opposing hypotheses

Neurofilaments (NFs) are among the most abundant constituents of the axonal cytoskeleton. NFs consist of four subunits, termed NF-H, NF-M and NF-L, corresponding to heavy, medium and light in reference to their molecular mass and α-internexin. Phosphorylation of the C-terminal "sidearms" of NF-H and NF-M regulates the ability of NFs to form a cytoskeletal lattice that supports the mature axon. C-terminal phosphorylation events have classically been considered to regulate NF axonal transport. By contrast, studies demonstrating that NF axonal transport was not accelerated following sidearm deletion provided evidence that phosphorylation does not regulate NF transport. Herein, we demonstrate how comparison of transport and distribution of differentially phosphorylated NFs along axons identify common ground between these hypotheses and may resolve this controversy.

Axonal damage in the making: Neurofilament phosphorylation, proton mobility and magnetisation transfer in multiple sclerosis normal appearing white matter

AimsMultiple sclerosis (MS) leaves a signature on the phosphorylation and thus proton binding capacity of axonal neurofilament (Nf) proteins. The proton binding capacity in a tissue is the major determinant for exchange between bound and free protons and thus the magnetisation transfer ratio (MTR). This study investigated whether the MTR of non-lesional white matter (NLWM) was related to the brain tissue concentration of neurofilament phosphoforms. MethodsUnfixed post-mortem brain slices of 12 MS patients were analysed using MTR, T1 at 1.5T. Blocks containing NLWM were processed for embedding in paraffin and inspected microscopically. Adjacent tissue was microdissected, homogenised and specific protein levels were quantified by ELISA for the Nf heavy chain (NfH) phosphoforms, glial fibrillary acidic protein (GFAP), S100B and ferritin. ResultsAveraged hyperphosphorylated NfH (SMI34) but not phosphorylated NfH (SMI35) levels were different between individual patients NLWM. The concentration of hyperphosphorylated NfH-SMI34 correlated with T1 (R=0.70, p=0.0114) and — inversely — with MTR (R=−0.73, p=0.0065). NfH-SMI35 was not correlated to any of the MR indices. ConclusionsPost-translational modifications of axonal proteins such as phosphorylation of neurofilaments occur in NLWM and may precede demyelination. The resulting change of proton mobility influences MTR and T1. This permits the in vivo detection of these subtle tissue changes on a proteomic level in patients with MS.

Diphenyl ditelluride induces hypophosphorylation of intermediate filaments through modulation of DARPP-32-dependent pathways in cerebral cortex of young rats

We studied the effect of different concentrations of diphenyl ditelluride (PhTe)(2) on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and neurofilament (NF) subunits from cerebral cortex and hippocampus of rats during development. (PhTe)(2)-induced hypophosphorylation of GFAP and NF subunits only in cerebral cortex of 9- and 15-day-old animals but not in hippocampus. Hypophosphorylation was dependent on ionotropic glutamate receptors, as demonstrated by the specific inhibitors 10μM DL-AP5 and 50μM MK801, 100μM CNQX and 100μM DNQX. Also, 10μM verapamil and 10μM nifedipine, two L-voltage-dependent Ca(2+) channels (L-VDCC) blockers; 50μM dantrolene, a ryanodine channel blocker, and the intracellular Ca(2+) chelator Bapta-AM (50μM) totally prevented this effect. Results obtained with 0.2μM calyculin A (PP1 and PP2A inhibitor), 1μM Fostriecin a potent protein phosphatase 2A (PP2A) inhibitor, 100μM FK-506 or 100μM cyclosporine A, specific protein phosphatase 2B inhibitors, pointed to PP1 as the protein phosphatase directly involved in the hypophosphorylating effect of (PhTe)(2). Finally, we examined the activity of DARPP-32, an important endogenous Ca(2+)-mediated inhibitor of PP1 activity. Western blot assay using anti-DARPP-32, anti-pThr34DARPP-32, and anti-pThr75DARPP-32 antibodies showed a decreased phosphorylation level of the inhibitor at Thr34, compatible with inactivation of protein kinase A (PKA) by pThr75 DARPP-32. Decreased cAMP and catalytic subunit of PKA support that (PhTe)(2) acted on neuron and astrocyte cytoskeletal proteins through PKA-mediated inactivation of DARPP-32, promoting PP1 release and hypophosphorylation of IF proteins of those neural cells. Moreover, in the presence of Bapta, the level of the PKA catalytic subunit was not decreased by (PhTe)(2), suggesting that intracellular Ca(2+) levels could be upstream the signaling pathway elicited by this neurotoxicant and targeting the cytoskeleton.

Structural and Functional Components of the Feline Enteric Nervous System

Neurohistological and immunohistochemical examinations of the feline enteric nervous system (ENS) were performed by using antibodies against neuron-specific enolase (NSE), phosphorylated neurofilaments (PN), non-phosphorylated neurofilaments (NPN) and vasoactive intestinal peptide (VIP), whereas glial cells were investigated by using antibodies against glial fibrillary acidic protein (GFAP). The study included full-thickness biopsies of the stomach, duodenum, jejunum, ileum and colon of 11 healthy cats. In this study, immunohistochemical staining of feline ENS with antibodies to NSE, PN and NPN revealed the presence of different ganglionated and aganglionated plexus. The two ganglionated plexus were arranged in a plexus submucosus internus & externus and a plexus myentericus. Furthermore, plexus mucosus and subserosal plexus represented two aganglionated plexus. GFAP-stained cellular elements were smaller than and in close contact to enteric neurons possibly resembling astrocytes of the central nervous system. VIP is one of the major neurotransmitters of enteric inhibitory neurons, and immunoreactivity was present in all layers of the gut, especially in ganglionated plexus. This is the first report, describing feline ENS by using immunohistochemical methods.

Motor Unit Abnormalities in Dystonia musculorum Mice

Dystonia musculorum (dt) is a mouse inherited sensory neuropathy caused by mutations in the dystonin gene. While the primary pathology lies in the sensory neurons of dt mice, the overt movement disorder suggests motor neurons may also be affected. Here, we report on the contribution of motor neurons to the pathology in dt27J mice. Phenotypic dt27J mice display reduced alpha motor neuron cell number and eccentric alpha motor nuclei in the ventral horn of the lumbar L1 spinal cord region. A dramatic reduction in the total number of motor axons in the ventral root of postnatal day 15 dt27J mice was also evident. Moreover, analysis of the trigeminal nerve of the brainstem showed a 2.4 fold increase in number of degenerating neurons coupled with a decrease in motor neuron number relative to wild type. Aberrant phosphorylation of neurofilaments in the perikaryon region and axonal swellings within the pre-synaptic terminal region of motor neurons were observed. Furthermore, neuromuscular junction staining of dt27J mouse extensor digitorum longus and tibialis anterior muscle fibers showed immature endplates and a significant decrease in axon branching compared to wild type littermates. Muscle atrophy was also observed in dt27J muscle. Ultrastructure analysis revealed amyelinated motor axons in the ventral root of the spinal nerve, suggesting a possible defect in Schwann cells. Finally, behavioral analysis identified defective motor function in dt27J mice. This study reveals neuromuscular defects that likely contribute to the dt27J pathology and identifies a critical role for dystonin outside of sensory neurons.

Neurofilament dot blot assays: Novel means of assessing axon viability in culture

Axonal structure and integrity are vital to overall neuronal maintenance and action potential propagation. Neurofilaments (NFs) are one of the main cytoskeletal components of axons and phosphorylation of NF subunits regulates speed of NF transport through axons and determines optimal axonal calibre required for signal propagation. Many previous studies of neuroprotective agents have focussed on neuronal viability in models of neurodegenerative disease, without specifically considering axon function as an indicator of neuronal damage. In this study, we have focused on developing novel assays for determining axon viability by measuring levels of neurofilament phosphorylation in cultured cortical neurons. The nitric oxide donor DETANONOate (NO) was used as an inflammatory insult and glial cell line-derived neurotrophic factor (GDNF) and superoxide dismutase (SOD) were tested as potential axonal protective agents. Using ‘dot blot’ methodologies, we show a decrease in NF phosphorylation in cortical neurons exposed to NO-mediated cell toxicity and an attenuation of NO-mediated changes in NF phosphorylation associated with GDNF and SOD treatment. These results correlated well with immunocytochemical counts. We propose therefore that the dot blot assay is a novel method for assessing axonal integrity in vitro and may play a useful role in the future for testing the effects of agents on axonal viability, providing a reliable and reproducible screening method for potential therapeutics for neurodegenerative diseases.

Changes of protein oxidation, calpain and cytoskeletal proteins (alpha tubulin and pNF-H) levels in rat brain after nerve agent poisoning

Highly toxic organophosphorus (OP) nerve agents, sarin and soman act by inhibiting acetylcholinesterase (AChE) function at neuronal synapses and cause many toxic effects including death within minutes. The effect of nerve agents on protein oxidation, calpain, and cytoskeletal protein levels was not well known. In the present study we investigated these parameters after subcutaneous injection of sarin (120 μg/kg) and soman (80 μg/kg) in the rat brain. Results indicate that several rat brain proteins were intensely oxidized after nerve agent poisoning. Immunoreactivity levels of μ-calpain were significantly elevated in cerebral cortex and cerebellum regions of rat brain from 2.5 h to 30 days. Alpha tubulin levels reduced from 1 to 7 days in the supernatant and 1 to 3 days in the pellet fractions of cerebellum and cerebral cortex, where as phosphorylation of high molecular weight neurofilament (pNF-H) was increased significantly in nerve agent intoxicated rat brains as compared to control rats. AChE activity was inhibited up to 3 days after nerve agent exposure in plasma and brain. Results suggest that altered protein oxidation, calpain and cytoskeletal protein levels are due to multiple mechanisms of nerve agents actions and these changes might be involved in nerve agent induced complex neurotoxicity.

Structural and functional changes of neuronal and glial components of the feline enteric nervous system in cats with chronic inflammatory and non-inflammatory diseases of the gastrointestinal tract

Immunohistochemical examinations of the enteric nervous system (ENS) were performed on biopsies of healthy cats and compared to findings in cats suffering from inflammatory bowel disease or intestinal lymphoma. In lymphocytic–plasmacytic enterocolitis all affected samples had significant reductions in glial fibrillary acidic protein and vasoactive intestinal peptide (VIP) and mostly of neuron-specific enolase (NSE) possibly reflecting alterations in enteric glial cells and neurons. In cases with eosinophilic gastroenterocolitis significantly reduced phosphorylated neurofilament (PN) expression was present suggesting a disturbance in neuronal cytoskeleton, whereas cats with fibrosing enteropathy had reduced expression of NSE, non-phosphorylated neurofilaments (NPN), PN and VIP, possibly reflecting neuronal disturbances. In cases with intestinal lymphoma only the reduction in PN and the increase in NPN were obvious suggesting direct damage or interference of neoplastic cells with enteric neurons. In conclusion, structural and functional alterations of the ENS may contribute to clinically evident signs of vomiting and/or diarrhea.

Axonal Loss and Neurofilament Phosphorylation Changes Accompany Lesion Development and Clinical Progression in Multiple Sclerosis

Neuroaxonal damage and loss are increasingly recognized as disability determining features in multiple sclerosis (MS) pathology. However, little is known about the long-term sequelae of inflammatory demyelination on neurons and axons. Spinal cord tissue of 31 MS patients was compared to three amyotrophic lateral sclerosis (ALS) and 10 control subjects. MS lesions were staged according to the density of KiM-1P positive macrophages and microglia and the presence of myelin basic protein (MBP) positive phagocytes. T cells were quantified in the parenchyma and meninges. Neuroaxonal changes were studied by immunoreactivity (IR) for amyloid precursor protein (APP) and variably phosphorylated neurofilaments (SMI312, SMI31, SMI32). Little T cell infiltration was still evident in chronic inactive lesions. The loss of SMI32 IR in ventral horn neurons correlated with MS lesion development and disease progression. Similarly, axonal loss in white matter (WM) lesions correlated with disease duration. A selective reduction of axonal phosphorylated neurofilaments (SMI31) was observed in WM lesions. In ALS, the loss of neuronal SMI32 IR was even more pronounced, whereas the relative axonal reduction resembled that found in MS. Progressive neuroaxonal neurofilament alterations in the context of chronic inflammatory demyelination may reflect changes in neuroaxonal metabolism and result in chronic neuroaxonal dysfunction as a putative substrate of clinical progression.

Evidence of endoplasmic reticular stress in the spinal motor neurons exposed to CSF from sporadic amyotrophic lateral sclerosis patients

We have earlier reported that intrathecal injection of cerebrospinal fluid (CSF) from sporadic Amyotrophic Lateral Sclerosis patients (ALS-CSF) into neonatal rats and supplementation of rat spinal cord cultures with ALS-CSF induces motor neuron degeneration via aberrant neurofilament phosphorylation and Golgi apparatus fragmentation. Intracellular aggregates immunoreactive to ubiquitin, phosphorylated neurofilaments and choline acetyl transferase (ChAT) were prominently seen in NSC-34 cells exposed to ALS-CSF. Protein aggregation could cause stress on endoplasmic reticulum (ER) and may precede Golgi fragmentation. Here we assessed the effect of ALS-CSF on the expression of GRP-78 and caspase-12 proteins, the markers of ER stress responses, in NSC-34 cells and rat spinal cords by immunochemistry and immunoblotting. Both in vitro and in vivo, increased expression of these proteins accompanied elevated active caspase-12 levels. Apoptotic nuclei and nuclear translocation of caspase-12 were noted in some cells. In vitro, the occurrence of ER stress was supported by electron microscopic observations of numerous free polyribosomes and fragmented ER cisternae. Aggregated mSOD1 protein causes ER stress in familial ALS. ER stress is also reported in the autopsy samples of sporadic ALS. Thus our observation of ER stress may be linked to the protein aggregation, viz. phosphorylated neurofilaments and ChAT, reported earlier.

Phosphorylation-Mediated Conformational Changes in the Mouse Neurofilament Architecture: Insight from a Neurofilament Brush Model

Neurofilaments (NFs) are important cytoskeletal filaments that consist of long flexible C-terminal tails that are abundant with charges. The tails attain additional negative charges through serine phosphorylation of Lys-Ser-Pro (KSP) repeat motifs that are particularly found in neurofilament heavy (NF-H) and neurofilament medium (NF-M) proteins. These side-arm protrusions mediate the interaction between neighboring filaments and maintain axonal diameter. However, the precise role of NF proteins and their phosphorylation in regulating interfilament distances and axonal diameter still remains unclear. In this regard, a recent gene replacement study revealed that the phosphorylation of mouse NF-M KSP repeats does not affect axonal cytoarchitecture, challenging the conventional viewpoint on the role of NF phosphorylation. To better understand the effect of phosphorylation, particularly NF-M phosphorylation, we applied a computational method to reveal phosphorylation-mediated conformational changes in mouse NF architecture. We employed a three-dimensional sequence-based coarse-grained NF brush model to perform Monte Carlo simulations of mouse NF by using the sequence and stoichiometry of mouse NF proteins. Our result shows that the phosphorylation of mouse NF-M does not change the radial extension of NF-M side arms under a salt-free condition and in ionic solution, highlighting a structural factor that supports the notion that NF-M KSP phosphorylation has no effect on the axonal diameter of mouse. On the other hand, significant phosphorylation-mediated conformational changes were found in NF-H side arms under the salt-free condition, while the changes in ionic solution are not significant. However, NF-H side arms are found at the periphery of mouse NF architecture, implying a role in linking neighboring filaments.

Down regulation of trophic factors in neonatal rat spinal cord after administration of cerebrospinal fluid from sporadic amyotrophic lateral sclerosis patients

Accumulating evidence supports neuroprotective role of trophic factors in amyotrophic lateral sclerosis (ALS). Previous studies from our laboratory report that the CSF of patients with sporadic ALS (ALS-CSF) induces degenerative changes in the rat spinal motor neurons and reactive astrogliosis in the surrounding gray matter. The present study was aimed to investigate if the ALS-CSF affected the expression of trophic factors namely, brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) in the newborn rat spinal cords. ALS-CSF was intrathecally injected into the neonatal rats and the mRNA levels of the trophic factors were determined by quantitative real-time polymerase chain reaction. Here, we report significant down regulation in the gene expression of trophic factors for BDNF, FGF2 and IGF1. BDNF mRNA levels were found to be reduced by 6.8-fold in the ALS-CSF injected group compared to control groups. The levels of IGF1 and FGF2 mRNA were also decreased by 3.91- and 2.13-fold, respectively, in the ALS group. We further found that exogenous supplementation of BDNF considerably reduced the aberrant phosphorylation of neurofilaments, complementing our earlier findings of restored expression of voltage gated sodium channel. Reduced expression of trophic factors indicates an altered microenvironment of the motor neurons and could possibly be one of the contributing factors in the degeneration process.

C‐terminal neurofilament phosphorylation fosters neurofilament–neurofilament associations that compete with axonal transport

Neurofilaments (NFs) associate with each other and with other cytoskeletal elements to form a lattice that supports the mature axon. Phosphorylation contributes to formation of this structure by fostering cation-dependent interactions among NF sidearms. By inducing NF bundling, phosphorylation impedes their axonal transport. To examine the impact of the known NF kinase cdk5 on these phenomena, transfected cells with constructs expressing GFP-tagged NF-H sidearms (lacking the rod domain to preclude assembly) with and without site-directed mutagenesis of 7 cdk5 consensus sites, and monitored the impact on NF transport and association with the axonal NF bundle. These mutations did not alter transport but pseudo-phosphorylated mutants displayed a greater association with axonal NF bundles. By contrast, these same mutations in full-length NF-H altered NF transport as well as bundling. Since isolated sidearms cannot assemble, they can only interact with NFs via a single sidearm-sidearm interaction, while assembled NFs can form multiple such interactions. These finding suggest that individual sidearm-sidearm interactions are dynamic and do not persist long enough to slow NF transport, and that bundle formation and maintenance depends upon both the long half-life of NF polymers and the establishment of multiple phosphorylation-dependent sidearm-mediated interactions among NFs.