Tomoko Tashiro

Two Populations of Axonally Transported Tubulin Differentiated by Their Interactions with Neurofilaments

Abstract: In the sensory fibers of the rat sciatic nerve (fibers of the dorsal root ganglion cells), two components of tubulin transport were observed that differed in the rate of transport, solubility in Triton, and subunit composition. The faster component, migrating ahead of the neurofilament proteins, was soluble in 1% Triton. The slower component, migrating with the neurofilament proteins, was insoluble in 1% Triton and contained a unique polypeptide, “NAP,” in the tubulin region that was not present in the faster component. “NAP” was not a subspecies of tubulin as evidenced by peptide mapping. It seems to be a neurofilament‐associated protein. When a complete separation of the main tubulin wave from the neurofilament wave was achieved in the motor axons of the same nerve (axons of the ventral motoneurons) under the effect of ββ‐iminodipropionitrile, a portion of tubulin was still found associated with the retarded neurofilament wave. The subunit composition of this portion was similar to the slower, neurofilament‐associated component in the sensory fibers under normal conditions, i.e., enriched in “NAP” and the most acidic subtype of (β‐tubulin. It is suggested that two populations of transported tubulin exist that are differentiated by the extent of their interaction with neurofilaments.

A calcium-dependent protease selectively degrading the 160 000 Mr component of neurofilaments is associated with the cytoskeletal preparation of the spinal cord and has an endogenous inhibitory factor

A class of calcium-activated proteases has been found in a wide variety of tissues (reviews [ 1,2]). In vertebrate [3-71 and invertebrate [8,9] nerves, similar type of proteolytic activity has been detected in the soluble compartment, acting preferentially on the neurofilament proteins. As neurofilaments are the major structural elements of the axoplasm, significant roles for this enzyme in pathological [3-51 and developmental [6] aspects have been suggested. in addition. The homogenate was stirred at room temperature for 1 h, which was effective in increasing the yield of neurofilaments. The homogenate was then layered on TKM buffer containing 1 M sucrose, and centrifuged at 78 000 X g for 2 h. The neurofilamentenriched pellet from three animals was usually suspended in 10 ml TKM buffer. It was either assayed immediately for the proteolytic activity or stored at -80°C.

Increased Solubility of High‐Molecular‐Mass Neurofilament Subunit by Suppression of Dephosphorylation: Its Relation to Axonal Transport

Abstract: To investigate the role of phosphorylation in the turnover and transport of neurofilament (NF) proteins in vivo, we studied their solubility properties and axonal transport in the rat sciatic nerve using phosphatase inhibitors to minimize dephosphorylation during preparation. About 20% of the 200‐kDa subunit (NF‐H) in the axon was soluble in the 1% Triton‐containing buffer under the present conditions, whereas this amount was less and more variable in the absence of phosphatase inhibitors. The 68‐kDa subunit (NF‐L) was exclusively insoluble and not affected by the inhibitors. Such selective solubilization of NF‐H by phosphorylation differed significantly from the in vitro phosphorylation with cyclic AMP‐dependent protein kinase, which resulted in NF disassembly. The carboxy‐terminal phosphorylation state of NF‐H probed with the phosphorylation‐sensitive antibodies was also not directly related to solubility. The solubility of NF‐H did not differ along the nerve. In contrast, the solubility of l‐[35S]methionine‐labeled, transported NF‐H was lowest at the peak of radioactivity. Higher solubility at the leading edge, regardless of its location along the nerve, indicates that NF‐H solubility is positively correlated with the rate of NF transport.

Neurofilaments of Klotho, the mutant mouse prematurely displaying symptoms resembling human aging

We reported previously that neurofilaments (NFs) of aged rats were highly packed in the axon and contained a smaller amount of NF‐M as compared with those of young rats (Uchida et al. [ 1999 ] J. Neurosci. Res. 58:337–348). We studied NFs of the mutant mouse, named Klotho, which displays prematurely symptoms resembling human aging. The transport of axonal cytoskeletal proteins, including NFs, tubulin and actin, was decreased at the leading portion of the peak of transported proteins in Klotho during the process of premature aging. The nearest neighbor inter‐NF distance in Klotho axons (35–39 nm) was shorter than that of the wild‐type mouse (48–49 nm), indicating the packing of NFs in Klotho. The ratio of NF‐M to NF‐L was slightly decreased in cytoskeletons from the spinal cords of Klotho. These changes are similar, though not identical, to those observed in aged rats, and are the first evidence of age‐related changes in the neurons of Klotho. J. Neurosci. Res. 64:364–370, 2001.

Changes in Organization and Axonal Transport of Cytoskeletal Proteins During Regeneration

Abstract: Changes in solubility and transport rate of cyto‐skeletal proteins during regeneration were studied in the motor fibers of the rat sciatic nerve. Nerves were injured by freezing at the midthigh level either 1–2 weeks before (experiment I) or 1 week after radioactive labeling of the spinal cord with l‐[35S]methionine (experiment II). Labeled proteins in 6‐mm consecutive segments of the nerve 2 weeks after labeling were analyzed following fractionation into soluble and insoluble populations with 1° Triton at 4°C. When ax‐onal transport of newly synthesized cytoskeleton was examined in the regenerating nerve in experiment I, a new faster component enriched in soluble tubulin and actin was observed that was not present in the control nerve. The rate of the slower main component containing most of the insoluble tubulin and actin together with neurofilament proteins was not affected. A smaller but significant peak of radioactivity enriched in soluble tubulin and actin was also detected ahead of the main peak when the response of the preexisting cytoskeleton was examined in experiment II. It is thus concluded that during regeneration changes in the organization take place in both the newly synthesized and the preexisting axonal cytoskeleton, resulting in a selective acceleration in rate of transport of soluble tubulin and actin.

Characteristics of cerebral non-histone chromatin proteins as revealed by polyacrylamide gel electrophoresis

A rapid method has been developed for the polyacrylamide gel electrophoretic angdysis of the whole chromatin proteins in small amounts, and comparison of non-histone proteins has been made among chromatins prepared from neurone-rich, oligodendroglial as well as liver nuclei. Neuronal chromatin is characterized by its unusual wealth of high molecular weight non-histone proteins. Neuronal chromatin is also provided with a group of non-histone proteins of higher molecular weights, which is apparently absent in oligodendroglial and liver chromatins.

Selective Solubilization of High-Molecular-Mass Neurofilament Subunit During Nerve Regeneration

Abstract: A reduction in neurofilament (NF) protein synthesis and changes in their phosphorylation state are observed during nerve regeneration. To investigate how such metabolic changes are involved in the reorganization of the axonal cytoskeleton, we studied the injury‐induced changes in the solubility and axonal transport of NF proteins as well as their phosphorylation states in the rat sciatic nerve. In the control nerve, 15‐25% of high‐molecular‐mass NF subunit (NF‐H) was recovered in the 1% Triton‐soluble fraction when fractionated in the presence of phosphatase inhibitors. After a complete loss of NF proteins distal to the injury site (70‐75 mm from the spinal cord) 1 week after injury, NF‐H detected in the regenerating sprouts at 2 weeks or later exhibited higher solubility (>50%) and lower C‐terminal phosphorylation level than NF‐H in the control nerve. Solubility increase was also apparent with L‐[35S]methionine‐labeled NF‐H that was in transit in the proximal axon at the time of injury. The low‐molecular‐mass subunit remained in the insoluble fraction in both the normal and the regenerating nerves, indicating that selective solubilization of NF‐H rather than total filament disassembly occurs during regeneration.

Two 68-kDa proteins in slow axonal transport belong to the 70-kDa heat shock protein family and the annexin family

Abstract: The major 68‐kDa protein found selectively in the faster of the two subcomponents of slow axonal transport [group IV or slow component b (SCb)] in the rat sciatic nerve has been characterized. It was found to contain two distinct classes of proteins, S1 and S2, both of which have isoelectric points of 5.7, but differ in their solubility in the presence of calcium. The S1 protein, which contributes up to 70% of the 68‐kDa component, was soluble in the presence or absence of calcium, whereas the S2 protein was bound to the cytoskeleton in a calcium‐dependent manner. Further characterization of the two proteins by peptide mapping and immunological methods revealed that the S1 protein belonged to a family of proteins related to the 70‐kDa heat shock protein, whereas the S2 protein was identical to 68‐kDa calelec‐trin (annexin VI). Selective occurrence in SCb of these proteins with potential abilities to regulate protein‐protein or protein‐membrane interactions suggests that they may play important roles in the control of cytoskeletal organization in the axon, because SCb contains mainly cytoskeletal proteins in a more dynamic form compared with the slowest rate component, slow component a, which is enriched in the stably polymerized form of these proteins.

Early Effects of β,β′‐Iminodipropionitrile on Tubulin Solubility and Neurofilament Phosphorylation in the Axon

Abstract: To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of β,β′‐iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with l‐[35S]methionine revealed that transport inhibition occurred all along the nerve within 1–2 days. Transport of cold‐insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold‐soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200‐kDa neurofilament protein was detected in the ventral root using phosphorylation‐dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.

Impairment of Cytoskeletal Protein Transport due to Aging or ββ’-lminodipropionitrile Intoxication in the Rat Sciatic Nerve

Three major age-related changes in cytoskeletal organization and metabolism in the axon were observed by comparing slow axonal transport in the sciatic nerves of rats aged 7-80 weeks: (a) a progressiv