Slow Axonal Transport of Neurofilament Protein in Cultured Neurons

We have investigated the axonal transport of neurofilament protein in cultured neurons by constricting single axons with fine glass fibers. We observed a rapid accumulation of anterogradely and retrogradely transported membranous organelles on both sides of the constrictions and a more gradual accumulation of neurofilament protein proximal to the constrictions. Neurofilament protein accumulation was dependent on the presence of metabolic substrates and was blocked by iodoacetate, which is an inhibitor of glycolysis. These data indicate that neurofilament protein moves anterogradely in these axons by a mechanism that is directly or indirectly dependent on nucleoside triphosphates. The average transport rate was estimated to be at least 130 μm/h (3.1 mm/d), and ∼90% of the accumulated neurofilament protein remained in the axon after detergent extraction, suggesting that it was present in a polymerized form. Electron microscopy demonstrated that there were an abnormally large number of neurofilament polymers proximal to the constrictions. These data suggest that the neurofilament proteins were transported either as assembled polymers or in a nonpolymeric form that assembled locally at the site of accumulation. This study represents the first demonstration of the axonal transport of neurofilament protein in cultured neurons

A proteomic analysis of the statocyst endolymph in common cuttlefish (Sepia officinalis): an assessment of acoustic trauma after exposure to sound

Here, the proteomic analysis of the endolymph was performed before and after sound exposure to assess the efects of exposure to low intensity, low frequency sounds on the statocyst endolymph of the Mediterranean common cuttlefsh (Sepia ofcinalis), determining changes in the protein composition of the statocyst endolymph immediately and 24h after sound exposure. Signifcant diferences in protein expression were observed, especially 24h after exposure. A total of 37 spots were signifcantly diferent in exposed specimens, 17 of which were mostly related to stress and cytoskeletal structure. Among the stress proteins eight spots corresponding to eight hemocyanin isoforms were under-expressed possible due to lower oxygen consumption. In addition, cytoskeletal proteins such as tubulin alpha chain and intermediate flament protein were also down-regulated after exposure. Thus, endolymph analysis in the context of acoustic stress allowed us to establish the efects at the proteome level and identify the proteins that are particularly sensitive to this type of trauma.Postprint (published version

CNS high-grade neuroepithelial tumor with BCOR internal tandem duplication: a comparison with its counterparts in the kidney and soft tissue

学位記番号:医博甲173

Migrating neural crest cells in the trunk of the avian embryo are multipotent

Trunk neural crest cells migrate extensively and give rise to diverse cell types, including cells of the sensory and autonomic nervous systems. Previously, we demonstrated that many premigratory trunk neural crest cells give rise to descendants with distinct phenotypes in multiple neural crest derivatives. The results are consistent with the idea that neural crest cells are multipotent prior to their emigration from the neural tube and become restricted in phenotype after leaving the neural tube either during their migration or at their sites of localization. Here, we test the developmental potential of migrating trunk neural crest cells by microinjecting a vital dye, lysinated rhodamine dextran (LRD), into individual cells as they migrate through the somite. By two days after injection, the LRD-labelled clones contained from 2 to 67 cells, which were distributed unilaterally in all embryos. Most clones were confined to a single segment, though a few contributed to sympathetic ganglia over two segments. A majority of the clones gave rise to cells in multiple neural crest derivatives. Individual migrating neural crest cells gave rise to both sensory and sympathetic neurons (neurofilament-positive), as well as cells with the morphological characteristics of Schwann cells, and other non-neuronal cells (both neurofilament-negative). Even those clones contributing to only one neural crest derivative often contained both neurofilament-positive and neurofilament-negative cells. Our data demonstrate that migrating trunk neural crest cells can be multipotent, giving rise to cells in multiple neural crest derivatives, and contributing to both neuronal and non-neuronal elements within a given derivative. Thus, restriction of neural crest cell fate must occur relatively late in migration or at the final sites of neural crest cell localization

Differences in distribution of the 210 kDa neurofilament subunit and the S-100 protein in the small intestine of a human fetus with trisomy 21 and in that of a normal fetus of the same age

The enteric nervous system of the small intestine of a 22-week-old male fetus with trisomy 21 was examined and compared with gut specimens from a fetus with normal karyotype at the same developmental stage. After the therapeutic termination of the pregnancies, paraffin sections and whole-mounts were prepared, which were processed for histology and immunohistochemistry, with the use of antibodies against the 210 kDA neurofilament subunit and the glial marker S-100 protein. The reduced length of the small intestine, the histologically observed fewer and shorter villi and the frequently appearing pseudostratified epithelium indicate an overall delay in the intestinal development in the trisomic fetus. Both the S-100 protein-immunopositive glial cells and the neurofilament protein-immunopositive nerve cells were distributed differentially in the gut specimens of the trisomic fetus and in the fetus with normal karyotype. While the immunohistochemical expression of the S-100 protein differed only in the circular axis of the gut wall, the distribution of the neurofilament protein-immunoreactive nerve cells also differed along the longitudinal axis of the gastrointestinal tract. Not only the distribution, but also the morphology of the neurofilament protein-immunoreactive myenteric ganglion cells differed in the trisomic fetus as compared with the normal one. The neurofilament protein-immunopositive ganglion cells of the normal fetus possessed lamellar dendrites and one long axon, while the ganglion cells of the trisomic fetus did not exhibit special morphological characteristics. These observations suggest that the enteric nervous system of the fetus with trisomy 21 is involved in the overall delay of the gut development

Monoclonal Antibody Identification of Subpopulations of Cerebral Cortical Neurons Affected in Alzheimer disease

Neuronal degeneration is one of the hallmarks of Alzheimer disease (AD). Given the paucity of molecular markers available for the identification of neuronal subtypes, the specificity of neuronal loss within the cerebral cortex has been difficult to evaluate. With a panel of four monoclonal antibodies (mAbs) applied to central nervous system tissues from AD patients, we have immunocytochemically identified a population of vulnerable cortical neurons; a subpopulation of pyramidal neurons is recognized by mAbs 3F12 and 44.1 in the hippocampus and neocortex, and clusters of multipolar neurons in the entorhinal cortex reactive with mAb 44.1 show selective degeneration. Closely adjacent stellate-like neurons in these regions, identified by mAb 6A2, show striking preservation in AD. The neurons recognized by mAbs 3F12 and 44.1, to the best of our knowledge, do not comprise a single known neurotransmitter system. mAb 3A4 identifies a phosphorylated antigen that is undetectable in normal brain but accumulates early in the course of AD in somas of vulnerable neurons. Antigen 3A4 is distinct from material reactive with thioflavin S or antibody generated against paired helical filaments. Initially, antigen 3A4 is localized to neurons in the entorhinal cortex and subiculum, later in the association neocortex, and, ultimately in cases of long duration, in primary sensory cortical regions. mAb 3F12 recognizes multiple bands on immunoblots of homogenates of normal and Ad cortical tissues, whereas mAb 3A4 does not bind to immunoblots containing neurofilament proteins or brain homogenates from AD patients. Ultrastructurally, antigen 3A4 is localized to paired-helical filaments. Using these mAbs, further molecular characterization of the affected cortical neurons is now possible

Fast Transport of Neurofilament Protein along Microtubules in Squid Axoplasm

Using squid axoplasm as a model system, we have visualized the fast transport of non-filamentous neurofilament protein particles along axonal microtubules. This transport occurs at speeds of 0.5-1.0 microm/second and the majority of neurofilament particles stain with kinesin antibody. These observations demonstrate, for the first time, that fast (0.5-1.0 microm/second) transport of neurofilament proteins occurs along microtubules. In addition, our studies suggest that neurofilament protein can be transported as non-membrane bound, nonfilamentous subunits along axons, and that the transport is kinesin-dependent. Microtubule-based fast transport might therefore provide a mechanism for the distribution and turnover of neurofilament, and perhaps other cytoskeletal proteins, throughout neurons

Palisade endings have an exocytotic machinery but lack acetylcholine receptors and distinct acetylcholinesterase activity

Purpose: The purpose of this work was to test whether palisade endings express structural and molecular features of exocytotic machinery, and are associated with acetylcholine receptors, and enzymes for neurotransmitter breakdown. Methods: Extraocular rectus muscles from six cats were studied. Whole-mount preparations of extraocular muscles (EOMs) were immunolabeled with markers for exocytotic proteins, including synaptosomal-associated protein of 25 kDa (SNAP25), syntaxin, synaptobrevin, synaptotagmin, and complexin. Acetylcholine receptors (AChRs) were visualized with α-bungarotoxin and with an antibody against AChRs, and acetylcholinesterase (AChE) was tagged with anti-AChE. Molecular features of multicolor labeled palisade endings were analyzed in the confocal scanning microscope, and their ultrastructural features were revealed in the transmission electron microscope. Results: All palisade endings expressed the exocytotic proteins SNAP25, syntaxin, synaptobrevin, synaptotagmin, and complexin. At the ultrastructural level, vesicles docked at the plasma membrane of terminal varicosities of palisade endings. No AChRs were associated with palisade endings as demonstrated by the absence of α-bungarotoxin and anti-AChR binding. AChE, the degradative enzyme for acetylcholine exhibited low, if any, activity in palisade endings. Axonal tracking showed that axons with multiple en grappe motor terminals were in continuity with palisade endings. Conclusions: This study demonstrates that palisade endings exhibit structural and molecular characteristics of exocytotic machinery, suggesting neurotransmitter release. However, AChRs were not associated with palisade endings, so there is no binding site for acetylcholine, and, due to low/absent AChE activity, insufficient neurotransmitter removal. Thus, the present findings indicate that palisade endings belong to an effector system that is very different from that found in other skeletal muscles.Austrian Science Fund (FWF) grant P32463-BMinisterio de Ciencia, Innovación y Universidades (PGC2018-094654-B-100

Fast Transport of Neurofilament Protein along Microtubules in Squid Axoplasm

Using squid axoplasm as a model system, we have visualized the fast transport of non-filamentous neurofilament protein particles along axonal microtubules. This transport occurs at speeds of 0.5-1.0 microm/second and the majority of neurofilament particles stain with kinesin antibody. These observations demonstrate, for the first time, that fast (0.5-1.0 microm/second) transport of neurofilament proteins occurs along microtubules. In addition, our studies suggest that neurofilament protein can be transported as non-membrane bound, nonfilamentous subunits along axons, and that the transport is kinesin-dependent. Microtubule-based fast transport might therefore provide a mechanism for the distribution and turnover of neurofilament, and perhaps other cytoskeletal proteins, throughout neurons