High Molecular Weight Neurofilament Subunit Research Articles

Low-level blast exposure disrupts gliovascular and neurovascular connections and induces a chronic vascular pathology in rat brain

Much concern exists over the role of blast-induced traumatic brain injury (TBI) in the chronic cognitive and mental health problems that develop in veterans and active duty military personnel. The brain vasculature is particularly sensitive to blast injury. The aim of this study was to characterize the evolving molecular and histologic alterations in the neurovascular unit induced by three repetitive low-energy blast exposures (3 × 74.5 kPa) in a rat model mimicking human mild TBI or subclinical blast exposure. High-resolution two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of purified brain vascular fractions from blast-exposed animals 6 weeks post-exposure showed decreased levels of vascular-associated glial fibrillary acidic protein (GFAP) and several neuronal intermediate filament proteins (α-internexin and the low, middle, and high molecular weight neurofilament subunits). Loss of these proteins suggested that blast exposure disrupts gliovascular and neurovascular interactions. Electron microscopy confirmed blast-induced effects on perivascular astrocytes including swelling and degeneration of astrocytic endfeet in the brain cortical vasculature. Because the astrocyte is a major sensor of neuronal activity and regulator of cerebral blood flow, structural disruption of gliovascular integrity within the neurovascular unit should impair cerebral autoregulation. Disrupted neurovascular connections to pial and parenchymal blood vessels might also affect brain circulation. Blast exposures also induced structural and functional alterations in the arterial smooth muscle layer. Interestingly, by 8 months after blast exposure, GFAP and neuronal intermediate filament expression had recovered to control levels in isolated brain vascular fractions. However, despite this recovery, a widespread vascular pathology was still apparent at 10 months after blast exposure histologically and on micro-computed tomography scanning. Thus, low-level blast exposure disrupts gliovascular and neurovascular connections while inducing a chronic vascular pathology.

Methylphenidate disrupts cytoskeletal homeostasis and reduces membrane-associated lipid content in juvenile rat hippocampus.

Although methylphenidate (MPH) is ubiquitously prescribed to children and adolescents, the consequences of chronic utilization of this psychostimulant are poorly understood. In this study, we investigated the effects of MPH on cytoskeletal homeostasis and lipid content in rat hippocampus. Wistar rats received intraperitoneal injections of MPH (2.0mg/kg) or saline solution (controls), once a day, from the 15th to the 44th day of age. Results showed that MPH provoked hypophosphorylation of glial fibrillary acidic protein (GFAP) and reduced its immunocontent. Middle and high molecular weight neurofilament subunits (NF-M, NF-H) were hypophosphorylated by MPH on KSP repeat tail domains, while NFL, NFM and NFH immunocontents were not altered. MPH increased protein phosphatase 1 (PP1) and 2A (PP2A) immunocontents. MPH also decreased the total content of ganglioside and phospholipid, as well as the main brain gangliosides (GM1, GD1a, and GD1b) and the major brain phospholipids (sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine). Total cholesterol content was also reduced in the hippocampi of juvenile rats treated with MPH. These results provide evidence that disruptions of cytoskeletal and lipid homeostasis in hippocampus of juvenile rats are triggers by chronic MPH treatment and present a new basis for understanding the effects and consequences associated with chronic use of this psychostimulant during the development of the central nervous system.

Vitamin D3 Reverses the Hippocampal Cytoskeleton Imbalance But Not Memory Deficits Caused by Ovariectomy in Adult Wistar Rats.

The objective of study was to investigate changes caused by ovariectomy (OVX) on aversive and non-aversive memories, as well as on cytoskeleton phosphorylating system and on vitamin D receptor (VDR) immunocontent in hippocampus. The neuroprotective role of vitamin D was also investigated. Ninety-day-old female Wistar rats were divided into four groups: SHAM, OVX, VITAMIN D and OVX+VITAMIN D; 30days after the OVX, vitamin D supplementation (500IU/kg), by gavage, for 30days was started. Results showed that OVX impaired short-term and long-term recognition, and long-term aversive memories. OVX altered hippocampal cytoskeleton phosphorylating system, evidenced by the hyperphosphorylation of glial fibrillary acidic protein (GFAP), low molecular weight neurofilament subunit (NFL), medium molecular weight neurofilament subunit (NFM) and high molecular weight neurofilament subunit (NFH), and increased the immunocontent of c-Jun N-terminal protein kinases (JNK), Ca2+/calmodulin-dependent protein kinase II (PKCaMII) and of the sites phosphorylated lysine-serine-proline (KSP) repeats, Ser55 and Ser57. Vitamin D reversed the effects caused by OVX on cytoskeleton in hippocampus, but it was not able to reverse the effects on memory.

Early expression of the high molecular weight neurofilament subunit attenuates axonal neurite outgrowth

Phospho-dependent interactions of the C-terminal region of the high molecular weight NF subunit (NF-H) with each other and with other cytoskeletal elements stabilize the axonal cytoskeleton and contribute to an increase in axonal caliber. The same kinase cascades that mediate axonal pathfinding via growth cone dynamics are those that foster NF-mediated axonal stabilization, yet there is a developmental delay in the accumulation of NF C-terminal phosphorylation. Moreover, the phospho-mediated C-terminal NF-H interactions that stabilize the axonal cytoskeleton also inhibit axonal elongation. We hypothesized that a delay in expression and/or accumulation of NF-H within developing axons is essential to allow axonal elongation and pathfinding.We tested this hypothesis in differentiating NB2a/d1 cells. The first 3 days of differentiation of NB2a/d1 cells is normally accompanied by rapid elongation of axonal neurites. This period is followed by the accumulation of C-terminally phosphorylated NF-H, cessation of axonal elongation and an increase in axonal caliber.Herein, overexpression of GFP-tagged NF-H simultaneously with induction of differentiation fostered accumulation of C-terminally phosphorylated NF-H within developing axonal neurites within 48hr, which was accompanied by retardation of axonal elongation and a hastened increase in caliber. These effects were prevented by treatment with inhibitors of kinases that mediate the association of NFs with other cytoskeletal elements. Overexpression of GFP-NF-H lacking the C-terminal 187 amino acids (which mediate NF–NF interactions) did not retard elongation nor increase caliber.These findings support the hypothesis that a developmental delay in NF-H C-terminal phosphorylation is essential to allow appropriate axonal elongation prior to stabilization.

Phosphorylated neurofilament subunit levels in the serum of cervical compressive myelopathy patients

We investigated the serum levels of the phosphorylated form of the high molecular weight neurofilament subunit (pNF-H) in patients with cervical compressive myelopathy. pNF-H is becoming increasingly recognized as a biomarker for axonal injury, however, it remains unclear whether serum pNF-H is elevated in chronic spinal cord compression. We examined 26 patients who underwent surgery for cervical compressive myelopathy. Peripheral blood samples were obtained both preoperatively and 1week after surgery to evaluate the serum pNF-H levels using an enzyme-linked immunosorbent assay. A history of recent aggravation of myelopathy was also investigated. Of the 26 myelopathy patients, the preoperative serum pNF-H level was negative in 20 patients and moderately elevated in six. Patients who were positive for pNF-H were more likely to have had a recent aggravation of myelopathy compared with the pNF-H negative patients (83 versus 25%; p=0.02). All patients who were positive for pNF-H before surgery remained positive after surgery. Two patients who became positive after surgery demonstrated a neurologic deterioration associated with the surgery. In conclusion, the serum pNF-H level was negative in the majority of patients with cervical compressive myelopathy. Our results suggest that an elevated serum level of pNF-H is associated with an acute worsening of myelopathy and that a positive conversion of pNF-H after surgery is a marker of perioperative neural damage.

Cerebrospinal Fluid Phosphorylated Neurofilament Subunit NF-H (pNF-H) is a Specific Biomarker and Its Pattern is Predictive for Spinal Cord Injury

Introduction In this study, we measured the phosphorylated neurofilament subunit NF-H (pNF-H) in the cerebrospinal fluid (CSF) of patients with spinal cord injury (SCI) and we set the correlation between the pNF-H levels and the severity of the injury. Patients and Methods The study included 20 patients with acute traumatic SCI: 12 patients with complete SCI and 8 patients with incomplete SCI. All patients were classified according to the American Spinal Injury Association Impairment Scale and all patients underwent surgery during the first 24 hours (decompression, stabilization). We measured daily the heavy phosphorylated neurofilament subunit (pNF-H) concentration by sandwich ELISA test in CSF of all patients and we correlated the values of pNF-H with the clinical evolution. Results For all patients with SCI, pNF-H was detectable in CSF samples and the values were different in the cases of complete SCI compared with the cases of incomplete SCI and the cerebrospinal pNF-H level was more elevated in cases of complete SCI. The level of CSF pNF-H was 10 to 100 times higher in complete SCI than in incomplete SCI, where the level was close to normal. The patients with a favorable neurological evolution after treatment had a specific pattern of daily values of NFP-H: a sudden increase up to a maximum value then a progressive decrease until normal. The maximum values were different in each case, from 10 times up to 200 times higher than the normal. Conclusions The phosphorylated form of the high molecular weight neurofilament subunit NF-H (pNF-H) in CSF is a specific biomarker for SCI and it can distinguish the severity of SCI. pNF-H is a predictive biomarker because its value pattern can show the reducing or stopping of the secondary lesion and the favorable result.

The high molecular weight neurofilament subunit plays an essential role in axonal outgrowth and stabilization.

ABSTRACTNeurofilaments (NFs) are thought to provide structural support to mature axons via crosslinking of cytoskeletal elements mediated by the C-terminal region of the high molecular weight NF subunit (NF-H). Herein, we inhibited NF-H expression in differentiating mouse NB2a/d1 cells with shRNA directed against murine NF-H without affecting other NF subunits, microtubules or actin. shRNA-mediated NF-H knockdown not only in compromised of late-stage axonal neurite stabilization but also compromised early stages of axonal neurite elongation. Expression of exogenous rat NF-H was able to compensate for knockdown of endogenous NF-H and restored the development and stabilization of axonal neurites. This rescue was prevented by simultaneous treatment with shRNA that inhibited both rat and murine NF-H, or by expression of exogenous rat NF-H lacking the C-terminal sidearm during knockdown of endogenous NF-H. Demonstration of a role for NF-H in the early stages of axonal elaboration suggests that axonal stabilization is not delayed until synaptogenesis, but rather that the developing axon undergoes sequential NF-H-mediated stabilization along its length in a proximal–distal manner, which supports continued pathfinding in distal, unstabilized regions.

Disrupted cytoskeletal homeostasis, astrogliosis and apoptotic cell death in the cerebellum of preweaning rats injected with diphenyl ditelluride

In the present report 15 day-old rats were injected with 0.3μmol of diphenyl ditelluride (PhTe)2/kg body weight and parameters of neurodegeneration were analyzed in slices from cerebellum 3 and 6 days afterwards. The earlier responses, at day 3 after injection, included hyperphosphorylation of intermediate filament (IF) proteins from astrocyte (glial fibrillary acidic protein – GFAP – and vimentin) and neuron (low-, medium- and high molecular weight neurofilament subunits: NF-L, NF-M and NF-H); increased mitogen-activated protein kinase (MAPK) (Erk and p38MAPK) and cAMP-dependent protein kinase (PKA) activities. Also, reactive astrogliosis takes part of the early responses to the insult with (PhTe)2, evidenced by upregulated GFAP in Western blot, PCR and immunofluorescence analysis. Six days after (PhTe)2 injection we found persistent astrogliosis, increased propidium iodide (PI) positive cells in NeuN positive population evidenced by flow cytometry and reduced immunofluorescence for NeuN, suggesting that the in vivo exposure to (PhTe)2 progressed to neuronal death. Moreover, activated caspase 3 suggested apoptotic neuronal death. Neurodegeneration was related with decreased [3H]glutamate uptake and decreased Akt immunoreactivity, however phospho-GSK-3-β (Ser9) was not altered in (PhTe)2 injected rat. Therefore, the present results show that the earlier cerebellar responses to (PhTe)2 include disruption of cytoskeletal homeostasis that could be related with MAPK and PKA activation and reactive astrogliosis. Akt inhibition observed at this time could also play a role in the neuronal death evidenced afterwards. The later events of the neurodegenerative process are characterized by persistent astrogliosis and activation of apoptotic neuronal death through caspase 3 mediated mechanisms, which could be related with glutamate excitotoxicity. The progression of these responses are therefore likely to be critical for the outcome of the neurodegeneration provoked by (PhTe)2 in rat cerebellum.

In vivo treatment with diphenyl ditelluride induces neurodegeneration in striatum of young rats: Implications of MAPK and Akt pathways

In the present report 15day-old Wistar rats were injected with 0.3μmol of diphenyl ditelluride (PhTe)2/kg body weight and parameters of neurodegeneration were analyzed in slices from striatum 6days afterwards. We found hyperphosphorylation of intermediate filament (IF) proteins from astrocyte (glial fibrillary acidic protein—GFAP and vimentin) and from neuron (low-, medium- and high molecular weight neurofilament subunits: NF-L, NF-M and NF-H, respectively) and increased MAPK (Erk, JNK and p38MAPK) as well as PKA activities. The treatment induced reactive astrogliosis in the striatum, evidenced by increased GFAP and vimentin immunocontent as well as their mRNA overexpression. Also, (PhTe)2 significantly increased the propidium iodide (PI) positive cells in NeuN positive population without altering PI incorporation into GFAP positive cells, indicating that in vivo exposure to (PhTe)2 provoked neuronal damage. Immunohistochemistry showed a dramatic increase of GFAP staining characteristic of reactive astrogliosis. Moreover, increased caspase 3 in (PhTe)2 treated striatal slices suggested apoptotic cell death. (PhTe)2 exposure decreased Akt immunoreactivity, however phospho-GSK-3-β (Ser9) was unaltered, suggesting that this kinase is not directly implicated in the neurotoxicity of this compound. Therefore, the present results shed light into the mechanisms of (PhTe)2-induced neurodegeneration in rat striatum, evidencing a critical role for the MAPK and Akt signaling pathways and disruption of cytoskeletal homeostasis, which could be related with apoptotic neuronal death and astrogliosis.

Signaling mechanisms downstream of quinolinic acid targeting the cytoskeleton of rat striatal neurons and astrocytes

The studies of signaling mechanisms involved in the disruption of the cytoskeleton homeostasis were performed in a model of quinolinic acid (QUIN) neurotoxicity in vitro. This investigation focused on the phosphorylation level of intermediate filament (IF) subunits of astrocytes (glial fibrillary acidic protein — GFAP) and neurons (low, medium and high molecular weight neurofilament subunits — NFL, NFM and NFH, respectively). The activity of the phosphorylating system associated with the IFs was investigated in striatal slices of rat exposed to QUIN or treated simultaneously with QUIN plus glutamate receptor antagonists, calcium channel blockers or kinase inhibitors. Results showed that in astrocytes, the action of 100μM QUIN was mainly due to increased Ca2+ influx through NMDA and L-type voltage-dependent Ca2+ channels (L-VDCC). In neuronal cells QUIN acted through metabotropic glutamate receptor (mGluR) activation and influx of Ca2+ through NMDA receptors and L-VDCC, as well as Ca2+ release from intracellular stores. These mechanisms then set off a cascade of events including activation of PKA, PKCaMII and PKC, which phosphorylate head domain sites on GFAP and NFL. Also, Cdk5 was activated downstream of mGluR5, phosphorylating the KSP repeats on NFM and NFH. mGluR1 was upstream of phospholipase C (PLC) which, in turn, produced diacylglycerol (DAG) and inositol 3,4,5 triphosphate (IP3). DAG is important to activate PKC and phosphorylate NFL, while IP3 contributed to Ca2+ release from internal stores promoting hyperphosphorylation of KSP repeats on the tail domain of NFM and NFH. The present study supports the concept of glutamate and Ca2+ contribution in excitotoxic neuronal damage provoked by QUIN associated to dysfunction of the cytoskeleton homeostasis and highlights the differential signaling mechanisms elicited in striatal astrocytes and neurons.

Hyperhomocysteinemia selectively alters expression and stoichiometry of intermediate filament and induces glutamate- and calcium-mediated mechanisms in rat brain during development

The aim of the present work was to investigate the actions of a chemically induced chronic hyperhomocysteinemia model on intermediate filaments (IFs) of cortical and hippocampal neural cells and explore signaling mechanisms underlying such effects. Results showed that in hyperhomocysteinemic rats the expression of neural IF subunits was affected. In cerebral cortex, glial fibrillary acidic protein (GFAP) expression was donwregulated while in hippocampus high and middle molecular weight neurofilament subunits (NF-H and NF-M, respectively) were up-regulated. Otherwise, the immunocontent of IF proteins was unaltered in cerebral cortex while in hippocampus the immunocontent of cytoskeletal-associated low molecular weight neurofilament (NF-L) and NF-H subunits suggested a stoichiometric ratio consistent with a decreased amount of core filaments enriched in lateral projections. These effects were not accompanied by an alteration in IF phosphorylation. In vitro results showed that 500μM Hcy-induced protein phosphatases 1-, 2A- and 2B-mediated hypophosphorylation of NF subunits and GFAP in hippocampal slices of 17-day-old rats without affecting the cerebral cortex, showing a window of vulnerability of cytoskeleton in developing hippocampus. Ionotropic and metabotropic glutamate receptors were involved in this action, as well as Ca2+ release from intracellular stores through ryanodine receptors. We propose that the mechanisms observed in the hippocampus of 17-day-old rats could support the neural damage observed in these animals.

The Phosphorylated Axonal Form of the Neurofilament Subunit NF-H (pNF-H) as a Blood Biomarker of Traumatic Brain Injury

The detection of neuron-specific proteins in blood might allow quantification of the degree of neuropathology in experimental and clinical contexts. We have been studying a novel blood biomarker of axonal injury, the heavily phosphorylated axonal form of the high molecular weight neurofilament subunit NF-H (pNF-H). We hypothesized that this protein would be released from damaged and degenerating neurons following experimental traumatic brain injury (TBI) in amounts large enough to allow its detection in blood and that the levels detected would reflect the degree of injury severity. An enzyme-linked immunosorbent assay (ELISA) capture assay capable of detecting nanogram amounts of pNF-H was used to test blood of rats subjected to experimental TBI using a controlled cortical impact (CCI) device. Animals were subjected to a mild (1.0 mm), moderate (1.5 mm), or severe (2.0 mm) cortical contusion, and blood samples were taken at defined times post-injury. The assay detected the presence of pNF-H as early as 6 h post-injury; levels peaked at 24-48 h, and then slowly decreased to baseline over several days post-injury. No signal above baseline was detectable in control animals. Analysis of variance (ANOVA) showed a significant effect of lesion severity, and post hoc analysis revealed that animals given a moderate and severe contusion showed higher levels of blood pNF-H than controls. In addition, the peak levels of pNF-H detected at both 24 and 48 h post-injury correlated with the degree of injury as determined by volumetric analysis of spared cortical tissue. Relative amounts of pNF-H were also determined in different areas of the central nervous system (CNS) and were found to be highest in regions containing large-diameter axons, including spinal cord and brainstem, and lowest in the cerebral cortex and hippocampus. These findings suggest that the measurement of blood levels of pNF-H is a convenient method for assessing neuropathology following TBI.

Diphenyl ditelluride- and methylmercury-induced hyperphosphorilation of the high molecular weight neurofilament subunit is prevented by organoselenium compounds in cerebral cortex of young rats

Organotellurides are important intermediates in organic synthesis and, consequently, the occupational exposure to them is a constant risk for laboratory workers. These compounds can elicit many neurotoxic events in the central nervous system (CNS) that are associated with several neurological symptoms. In contrast, organoselenium compounds are considered to exert neuroprotective actions on such effects. Neurofilaments (NF) are important cytoskeletal proteins and phosphorylation/dephosphorylation of NF is important to stabilize the cytoskeleton. In this work we investigated the potential protective ability of the selenium compounds ebselen and diphenyl diselenide (PhSe) 2 against the effect of diphenyl ditelluride (PhTe) 2 and methylmercury (MeHg) on the total (phosphorylated plus nonphosphorylated) and phosphorylated immunocontent of the high molecular weight neurofilament subunit (NF-H) from slices of cerebral cortex of 17-day-old rats. We observed that 1 μM MeHg induced hyperphosphorylation, increasing the total immunocontent of this subunit of the high-salt Triton insoluble NF-H. Otherwise, 15 μM (PhTe) 2 induced hyperphosphorylation of the high-salt Triton insoluble NF-H without altering the total immunocontent of this protein into the cytoskeletal fraction. Concerning the selenium compounds, 15 μM (PhSe) 2 and 5 μM ebselen did not induce alteration per se on the in vitro phosphorylation of NF-H. In addition, (PhSe) 2 and ebselen at these concentrations, presented a protective effect against the action of (PhTe) 2 and MeHg, on the immunoreactivity of NF-H. Considering that hyperphosphorylation of NF-H is associated with neuronal dysfunction it is probable that the effects of (PhTe) 2 and MeHg could be related to the remarkable neurotoxicity of these organocalcogenides. Furthermore the neuroprotective action of selenium compounds against (PhTe) 2 and MeHg effects could be a promising route to be exploited for a possible treatment of calcogenides poisoning.

The high and middle molecular weight neurofilament subunits regulate the association of neurofilaments with kinesin: Inhibition by phosphorylation of the high molecular weight subunit

Kinesin participates in axonal transport of neurofilaments (NFs), but the mode by which they attach to kinesin is unclear. We compared the association of NFs with kinesin in mice expressing or lacking NF-H or NF-M. In normal and M−/− mice, the leading edge of metabolically labeled NF subunits was selectively co-precipitated with kinesin. By contrast, the entire wave of radiolabeled subunits co-precipitated with kinesin in H−/− mice. Similar bulk levels of NFs co-precipitated with kinesin from normal and H−/− mice, but reduced levels co-precipitated from M−/− mice. These data suggest that both NF-H and NF-M regulate the association of NFs with kinesin. They further indicate that phosphorylation of NF-H dissociates NFs from kinesin and provides a mechanism by which NF-H phosphorylation can contribute to the slowing of NF axonal transport.

Myelin-associated glycoprotein modulates expression and phosphorylation of neuronal cytoskeletal elements and their associated kinases.

Decreased phosphorylation of neurofilaments in mice lacking myelin-associated glycoprotein (MAG) was shown to be associated with decreased activities of extracellular-signal regulated kinases (ERK1/2) and cyclin-dependent kinase-5 (cdk5). These in vivo changes could be caused directly by the absence of a MAG-mediated signaling pathway or secondary to a general disruption of the Schwann cell-axon junction that prevents signaling by other molecules. Therefore, in vitro experimental paradigms of MAG interaction with neurons were used to determine if MAG directly influences expression and phosphorylation of cytoskeletal proteins and their associated kinases. COS-7 cells stably transfected with MAG or with empty vector were co-cultured with primary dorsal root ganglion (DRG) neurons. Total amounts of the middle molecular weight neurofilament subunit (NF-M), microtubule-associated protein 1B (MAP1B), MAP2, and tau were up-regulated significantly in DRG neurons in the presence of MAG. There was also increased expression of phosphorylated high molecular weight neurofilament subunit (NF-H), NF-M, and MAP1B. Additionally, in similar in vitro paradigms, total and phosphorylated NF-M were increased significantly in PC12 neurons co-cultured with MAG-expressing COS cells or treated with a soluble MAG Fc-chimera. The increased expression of phosphorylated cytoskeletal proteins in the presence of MAG in vitro was associated with increased activities of ERK 1/2 and cdk5. We propose that interaction of MAG with an axonal receptor(s) induces a signal transduction cascade that regulates expression of cytoskeletal proteins and their phosphorylation by these proline-directed protein kinases.

Effect of propionic and methylmalonic acids on the high molecular weight neurofilament subunit (NF-H) in rat cerebral cortex.

Propionic and methylmalonic acidemias are inherited neurometabolic disorders biochemically characterized by tissue accumulation of propionic (PA) and methylmalonic (MMA) acids, respectively. Neurofilaments (NF) are important cytoskeletal proteins and phosphorylation/dephosphorylation of NF is important to stabilize the cytoskeleton. We investigated the effects of PA and MMA on the high molecular weight neurofilament subunit associated with the cytoskeletal fraction of rat cerebral cortex along development. Cortical slices from 9- to 60-day-old rats were incubated with 2.5 mM PA or MMA. The cytoskeletal fraction was extracted and the immunoreactivity for phosphorylated or total NF-H was analyzed by immunoblotting using specific antibodies. Results showed that treatment of tissue slices with the acids induced an increased Triton-insoluble phosphorylated NF-H immunoreactivity in up to 17-day-old rats. Furthermore, treatments significantly increased the total amount of NF-H in 12-day-old rats. These findings indicate that PA and MMA alter the dynamic regulation of NF-H assembly in the cytoskeletal fraction.

Effect of the branched-chain alpha-ketoacids accumulating in maple syrup urine disease on the high molecular weight neurofilament subunit (NF-H) in rat cerebral cortex.

In this study we investigated the effects of the branched chain alpha-ketoacids accumulating in maple syrup urine disease (MSUD) on the concentrations of the high molecular weight neurofilament subunit (NF-H) associated with the cytoskeletal fraction of the cerebral cortex of 12-day-old rats. Cortical slices were incubated with alpha-ketoisocaproic acid (KIC), alpha-keto beta-methylvaleric acid (KMV) and alpha-ketoisovaleric acid (KIV) at concentrations ranging from 0.5 to 1.0 mM. The cytoskeletal fraction was extracted and the immunoreactivity for phosphorylated and total NF-H was analyzed by immunoblotting. The in vitro 32P incorporation into NF-H was also determined. Results showed that treatment of tissue slices induced with KMV increased Triton-insoluble phosphorylated NF-H immunoreactivity, with no alteration in total NF-H immunoreactivity. Furthermore, KIC treatment drastically increased the total amount of NF-H, whereas KIV did not change either phosphorylated or total NF-H immunoreactivity. KMV also increased the in vitro 32P incorporation into NF-H, confirming the highly phosphorylated NF-H levels detected in the immunoblot. These findings demonstrate that KIC and KMV alter the dynamic regulation of NF-H assembly in the cytoskeletal fraction. Therefore we may suggest that cytoskeletal disorganization may be one of the factors associated with the neurodegeneration characteristic of MSUD disease.

Selective accumulation of the high molecular weight neurofilament subunit within the distal region of growing axonal neurites.

Axonal maturation in situ is accompanied by the transition of neurofilaments (NFs) comprised of only NF-M and NF-L to those also containing NF-H. Since NF-H participates in interactions of NFs with each other and with other cytoskeletal constituents, its appearance represents a critical event in the stabilization of axons that accompanies their maturation. Whether this transition is effected by replacement of "doublet" NFs with "triplet" NFs, or by incorporation of NF-H into existing doublet NFs is unclear. To address this issue, we examined the distribution of NF subunit immunoreactivity within axonal cytoskeletons of differentiated NB2a/d1 cell and DRG neurons between days 3-7 of outgrowth. Endogenous immunoreactivity either declined in a proximal-distal gradient or was relatively uniform along axons. This distribution was paralleled by microinjected biotinylated NF-L. By contrast, biotinylated NF-H displayed a bipolar distribution, with immunoreactivity concentrated within the proximal- and distal-most axonal regions. Proximal biotinylated NF-H accumulation paralleled that of endogenous NF immunoreactivity; however, distal-most biotinylated NF-H accumulation dramatically exceeded that of endogenous NFs and microinjected NF-L. This phenomenon was not due to co-polymerization of biotin-H with vimentin or alpha-internexin. This phenomenon declined with continued time in culture. These data suggest that NF-H can incorporate into existing cytoskeletal structures, and therefore suggest that this mechanism accounts for at least a portion of the accumulation of triplet NFs during axonal maturation. Selective NF-H accumulation into existing cytoskeletal structures within the distal-most region may provide de novo cytoskeletal stability for continued axon extension and/or stabilization.

C-terminal phosphorylation of the high molecular weight neurofilament subunit correlates with decreased neurofilament axonal transport velocity

We probed the relationship of NF axonal transport of neurofilaments (NFs) to their phosphorylation state by comparing these parameters in two closely-aged groups of young adult mice — 2 and 5 months of age. This particular time interval was selected since prior studies demonstrate that optic axons have already completed axonal caliber expansion and attained adult NF levels by 2 months but, as shown herein, continue to increase NF-H C-terminal phosphorylation. NF axonal transport was monitored by autoradiographic analysis of the distribution of radiolabeled subunits immunoprecipitated from optic axon segments at intervals following intravitreal injection of 35 S -methionine. Both the peak and front of radiolabeled NFs translocated faster in 2- vs. 5-month-old mice. This developmental decline in NF transport rate was not due to reduced incorporation of NFs into the cytoskeleton, nor to an overall decline in slow axonal transport. By excluding or minimizing other factors, these findings support previous conclusions that C-terminal NF phosphorylation regulates NF axonal transport.

Triton-soluble phosphovariants of the heavy neurofilament subunit in developing and mature mouse central nervous system

The low abundance of soluble neurofilament (NF) subunits in mature axons has suggested that newly synthesized NF proteins rapidly assemble into highly stable polymers and associate with the Triton X-100-insoluble cytoskeleton. The dynamic nature of these subunit associations in vivo remains unresolved, and the applicability of this assembly model to NFs in other neuronal compartments or to developing neurons is unknown. Here, we report that a unique pool of Triton X-100-soluble, extensively phosphorylated, high molecular weight NF subunits (NF-H, or H-200) are abundantly expressed in the mouse CNS during early postnatal development and persist in the perikaryal compartment of some mature neurons. Triton-soluble H-200 subunits appeared at postnatal day 14 (P14) and remained high through P60, beyond which the percentage declined to marginal levels by P120. Medium and low molecular weight NF (NF-M and NF-L, respectively) were at all times only detectable within the cytoskeleton. Comparison of soluble and cytoskeleton-associated H-200 immunoreactivity indicated that certain phosphorylation-dependent epitopes were confined to the cytoskeleton. Pulse-chase radiolabeling analyses in optic pathway demonstrated that some Triton-soluble NF-H subunits are extensively phosphorylated within retinal perikarya before they are incorporated into Triton-insoluble structures. These findings indicate that the assembly behaviors of NF-H differ substantially from those of NF-M and NF-L, and that the interaction of NF-H with NFs may be more dynamic than is generally recognized, especially during brain development and within specific compartments of mature neurons.