Takeshi Nishio

Alpha-Klotho as a regulator of calcium homeostasis

α-klotho was identified as a gene associated with premature aging–like phenotypes characterized by short lifespan. In mice, we found the molecular association of α-Klotho (α-Kl) and Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) and provide evidence for an increase of abundance of Na+,K+-ATPase at the plasma membrane. Low concentrations of extracellular free calcium ([Ca2+]e) rapidly induce regulated parathyroid hormone (PTH) secretion in an α-Kl- and Na+,K+-ATPase–dependent manner. The increased Na+ gradient created by Na+,K+-ATPase activity might drive the transepithelial transport of Ca2+ in cooperation with ion channels and transporters in the choroid plexus and the kidney. Our findings reveal fundamental roles of α-Kl in the regulation of calcium metabolism.

Tenascin-C regulates proliferation and migration of cultured astrocytes in a scratch wound assay

Tenascin-C (TNC), an extracellular matrix glycoprotein, is involved in tissue morphogenesis like embryogenesis, wound healing or tumorigenesis. Astrocytes are known to play major roles in wound healing in the CNS. To elucidate the roles of TNC in wound closure by astrocytes, we have examined the morphological changes of cultured astrocytes in a scratch wound assay and measured the content of soluble TNC released into the medium. We have also localized the expression of TNC mRNA, TNC, glial fibrillary acidic protein (GFAP), vimentin and integrin beta1. After wounding, glial cells rapidly released the largest TNC isoform and proliferated in the border zones. Subsequently, they became polarized with unidirectional processes and finally migrated toward the denuded area. The proliferating border zone cells and pre-migratory cells intensely expressed TNC mRNA, TNC-, vimentin-, GFAP- and integrin beta1-like immunoreactivity, while the migratory cells showed generally reduced expression except the front. Exogenous TNC enhanced cell proliferation and migration, while functional blocking with anti-TNC or anti-integrin beta1 antibody reduced both of them. These results suggest that mechanical injury induces boundary astrocytes to produce and release TNC that promotes cell proliferation and migration via integrin beta1 in an autocrine/paracrine fashion.

Cellular localization of nerve growth factor-like immunoreactivity in adult rat brain: quantitative and immunohistochemical study.

To elucidate the role and the mechanism of action of nerve growth factor in the adult central nervous system, we investigated the localization of nerve growth factor-like immunoreactivity in adult rat brain, both quantitatively and immunohistochemically, using polyclonal anti-nerve growth factor immunoglobulin G. We raised rabbit polyclonal anti-mouse nerve growth factor antibody with an extremely high titer as 10(-9) determined by an enzyme immunoassay. The affinity-purified anti-nerve growth factor immunoglobulin G specifically recognized nerve growth factor with no cross-reaction to recombinant brain-derived neurotrophic factor and neurotrophin-3 evaluated by an enzyme immunoassay. We quantified nerve growth factor content in each layer of the adult rat cerebral cortex and in each small piece (0.225 mg wet weight tissue) of the diencephalon, brainstem and cerebellum with a highly sensitive two-site enzyme immunoassay. Nerve growth factor content was unevenly distributed in the cerebral cortex (dense in layers II/III and V/VI and sparse in layers I and IV). Moderate to high levels of nerve growth factor were registered in the habenular nuclei, zona incerta, ventral tegmental area, substantia nigra, locus coeruleus, ventral cochlear nucleus, trapezoid body, lateral vestibular nucleus, cerebellar nuclei and paraflocculus. Immunohistochemically, the nerve growth factor-like immunoreactivity was found in the cell bodies, dendrites and axons of adult rat central neurons, not only in the cerebral cortex, hippocampus and basal forebrain, but also in the diencephalon, brainstem and cerebellum. The population of neurons with nerve growth factor-like immunoreactivity was limited, but unexpectedly widespread, and the density of these cells correlated well with the content determined by an enzyme immunoassay in the present and a previous study [Nishio T. et al. (1992) Expl Neurol. 116, 76-84]. The monoamine neurons, including dopaminergic, noradrenergic and serotonergic neurons, showed intense nerve growth factor-like immunoreactivity, indicating that the central monoaminergic neuronal system may also be involved in the nerve growth factor trophic system. To visualize nerve growth factor transported in the axons and to enhance the immunostaining in the nerve growth factor-producing cells, we injected colchicine, a potent inhibitor of microtubule polymerization and a blocker of axoplasmic transport, into the lateral ventricle of adult Wistar rat brain. Colchicine treatment enhanced the intensities of nerve growth factor-like immunoreactivity in the axons and cell bodies, especially in the axon hillocks and the proximal axons of the nerve growth factor-producing neurons. This observation may suggest the existence of an orthograde axonal transport system for nerve growth factor in the central neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Spontaneous regeneration of the corticospinal tract after transection in young rats: a key role of reactive astrocytes in making favorable and unfavorable conditions for regeneration.

We demonstrated the occurrence of marked regeneration of the corticospinal tract (CST) after a single transection and failure of regeneration after a repeated transection in young rats. To provide convincing evidence for the complete transection and regeneration we used retrograde neuronal double labeling. Double-labeled neurons that took up the first tracer from the transection site and the second tracer from the injection site caudal to the transection site were observed in the sensorimotor cortex. The anterograde tracing method revealed various patterns of regeneration. In the most successful cases the vast majority of regenerated fibers descended in the normal tract and terminated normally whereas a trace amount of fibers coursed aberrantly. In the less successful cases fibers descended partly normally and partly aberrantly or totally aberrantly. To clarify the role of astrocytes in determining the success or failure of regeneration we compared expression of glial fibrillary acidic protein (GFAP), vimentin and neurofilament (NF) immunoreactivity (IR) in the lesion between single and repeated transections. In either transection, astrocytes disappeared from the CST near the lesion site as early as 3 h after lesioning. However, by 24 h after a single transection, immature astrocytes coexpressing GFAP- and vimentin-IR appeared in the former astrocyte-free area and NF-positive axons crossed the lesion. By contrast, after a repeated transection the astrocyte-free area spread and NF-positive axons never crossed the lesion. It appears likely that the major sign, and possibly cause of failure of regeneration is the prolonged disappearance of astrocytes in the lesioned tract area.

Medial nigral dopamine neurons have rich neurotrophin support in humans.

: To assess the action of neurotrophin in human dopaminergic neurons, we studied the immunolocalization of neurotrophins or trks in human substantia nigra pars compacta (SNc). The neuromelanin-containing neurons in the SNc showed immunoreactivities for neurotrophins or trks, suggesting an autocrine/paracrine regulation. Quantitative analysis revealed that the percentage of those expressing NGF-like immunoreactivity (NGF-LI), BDNF-LI, NT3-LI, trkA-LI, trkB-LI, or trkC-LI was 66%, 74%, 85%, 66%, 71% or 86%, respectively. The percentage of cells expressing neurotrophins or trks was higher in the medial part than in the lateral part of the SNc. The preferential expression of neurotrophin-trk systems in the medial neurons may, at least partially, explain the differential susceptibility in Parkinsons disease.

Small increase in triplet repeat length of cerebellum from patients with myotonic dystrophy

Myotonic dystrophy (DM) is genetically characterized by abnormal expansion of an unstable CTG trinucleotide repeat, located in the 3′-untranslated region of mRNA encoding the family of serine-threonine protein kinases. DNA extracted from various organs of patients with DM was analyzed by the Southern blotting method. We identified differently expanded bands in DNAs from various tissues from patients with DM. In studying the length of the CTG repeat in different regions of the brain, we found a noticeably small increase in repeat length in the cerebellum compared with other tissues. While this phenomenon has been reported in other triplet repeat diseases such as Huntington disease, spinocerebellar ataxia type 1, and dentatorubral-pallidoluysian atrophy, we are the first to describe it in DM. Although the mechanism of expansion of the triplet repeat remains to be defined, the tissue-dependent somatic mosaicism suggests that its occurrence may depend on the differentiated state of each tissue.

Neutrophin switching in spinal motoneurons of amyotrophic lateral sclerosis.

TO clarify the roles of neurotrophins in the human spinal motoneurons, with special reference to amyotrophic lateral sclerosis (ALS), we studied the immunohistochemical localizations of neurotrophins and their receptors in spinal cords of patients with ALS and compared them with controls. In the controls, the majority of motoneurons showed BDNF-, NT3-, trkB-and trkC-like immunoreactivity (-LI) suggesting that the motoneurons receive an autocrine regulation by both BDNF and NT3. In ALS patients, about three-quarters of the motoneurons had degenerated and the remaining motoneurons showed significantly decreased BDNF-LI, increased NGF-and trkA-LI. These findings indicated neurotrophin-switching in the remaining spinal motoneurons of ALS patients from BDNF and NT3 responsive to NGF responsive.

Spinal cord repair in neonatal rats: a correlation between axonal regeneration and functional recovery

The present study aimed to analyse how anatomical regeneration contributes to functional recovery after experimental spinal cord repair. Thoracic spinal cord of neonatal rats was completely transected to make a gap and repaired by grafting a section of embryonic spinal cord. Six weeks after surgery, outcome of locomotor performance was assessed using an open field locomotor scale (BBB scale). Axonal regeneration across the repaired site was quantitatively assessed in the raphe, vestibular, and red nuclei and the sensorimotor cortex by a retrograde tracing method. The rats that had no labelled neurons in any of the supraspinal nuclei showed no hind–forelimb coordination. The rats that had labelled neurons in the brainstem nuclei but not in the sensorimotor cortex showed hind–forelimb coordination of varying grades depending on the amount of regeneration. The rats that had labelled neurons in all of the examined nuclei showed almost normal locomotion. In addition to a relationship between distribution of the labelled neurons and functional recovery, a positive correlation was observed between number of the labelled neurons in each of the supraspinal nuclei and locomotor performance of the rat. Thus the grade of restored function appeared to be regulated by distribution and number of fibres regenerated across the repaired site and into the target region. These results suggest that accurate reconstruction of neural connections is essential for significant functional recovery after spinal cord repair.

Detailed distribution of nerve growth factor in rat brain determined by a highly sensitive enzyme immunoassay

We modified a previously reported enzyme immunoassay method to make it more sensitive for quantification of nerve growth factor (NGF), and succeeded in measuring the NGF content in as small as 2 mg (wet weight) of rat brain tissue. Rat brain was cut into about 600 pieces of the same size, and the NGF content in each piece was determined by this method. The findings were as follows: (i) In the cerebral cortex, NGF contents were unevenly distributed, ranging from less than 0.1 to 1.8 ng/g wet wt. The level was highest in the caudal parietal and rostral occipital cortices and lowest in the lateral parietal cortex. (ii) Areas comprising the limbic system such as the cingulate gyrus, pyriform cortex, amygdala, anterior and medial thalamus, hippocampus, septum, and diagonal band of Broca contained high levels of NGF. (iii) In the brain stem and cerebellum, the levels were low; however, a relatively high level was registered in the cerebellar nuclei, lateral vestibular nucleus, ventral cochlear nucleus, superior olive, and pontine reticular nuclei. These findings, taken together with previously published information, suggest that the neurons in the anterior and medial thalamus, pontine reticular nuclei, superior olive, ventral cochlear nucleus, and cerebellar Purkinje cells may be additional populations of NGF-responsive neurons in the rat brain.

Immunohistochemical and ultrastructural characterization of ubiquitinated eosinophilic fibrillary neuronal inclusions in sporadic amyotrophic lateral sclerosis

Abstract We found eosinophilic fibrillary neuronal inclusions (EFNI) that were argyrophilic and immunoreactive for anti-ubiquitin in the cerebral cortex of a patient with sporadic amyotrophic lateral sclerosis (ALS) and mild personality changes. Both hematoxylin and eosin and Bodian’s preparations revealed the EFNI to be rod-, flame-shaped, or spherical structures existing within the swollen neuronal perinuclear region in the third, fifth, and sixth layers of the fronto-parieto-temporal cortices including the primary motor cortex. On electron microscopy, filamentous profiles aggregated and formed a single bundle or globule in the neuronal perikaryon without any limiting membrane. Most EFNI had a characteristic multiple layer arrangement. The inner core consisted of randomly oriented granule-free tubules with a fuzzy outer contour, measuring 15–20 nm in diameter. The surrounding layer was made up of granule-associated filaments, electron-dense free granules, and small vesicular profiles. Large autolysosome-like membrane-bound vesicular profiles were found scattered at the periphery. Neurofilaments were usually mingled with in the surrounding cytoplasm. Many EFNI were also found in dendrites, but only a few in axons. Both granule-free tubules and granule-associated filaments expressed ubiquitin protein epitopes. Aberrant phosphorylation of neurofilament protein and induction of αB-crystallin were shown to exist in EFNI-bearing swollen neurons. Despite having a variety of histological appearances, our observations revealed that EFNI all have common immunocytochemical and ultrastructural characteristics, and thus we assume that EFNI represent a series of cytological alterations in the motor and extra-motor cortices of ALS patients.