Keiya Nakajima

Mesencephalic Neural Stem (Progenitor) Cells Develop to Dopaminergic Neurons More Strongly in Dopamine-Depleted Striatum than in Intact Striatum

Epidermal growth factor (EGF)/fibroblast growth factor (FGF)-responsive stem (progenitor) cells from embryonic brain have self-renewing and multipotent properties and thus are good candidates for donor cells in neural transplantation. However, the survival and differentiation to mature neurons after grafting of stem cells into adult brain are rather poor. We hypothesize that the differentiation of stem cells to mature neurons, such as dopaminergic (DAergic) neurons, is dependent on environmental cues that control the ontogenic development. We compared the survival and differentiation between mesencephalic (MS) and cortical (CTx) stem (progenitor) cells, following grafting into bilateral striata of hemiparkinsonian model rats. MS and CTx stem cells were prepared from E12 rats and proliferated in serum-free medium with EGF or basic FGF for 2 weeks. One day after being primed to differentiate, the cell suspensions of both origins were grafted into the bilateral striata of adult rats that had unilateral 6-OHDA lesions in the substantia nigra. MS cells differentiated to tyrosine hydroxylase (TH)-positive neurons more strongly in DA-depleted striatum than in intact striatum, and methamphetamine-induced rotation was ameliorated in half of the grafted animals. Rosette-like cell aggregation and dysfunction of the blood-brain barrier (BBB) were less in and around the grafts in DA-depleted striatum, suggesting less proliferation and more differentiation of MS stem cells in DA-depleted striatum. Neither TH-positive neurons nor behavioral amelioration were detected following CTx stem (progenitor) cell transplantation in the striata. Data suggest that the DA-depleted striatum offers a suitable environment for MS stem (progenitor) cells to differentiate into mature DAergic neurons.

GDNF is a major component of trophic activity in DA-depleted striatum for survival and neurite extension of DAergic neurons

Extracts from dopamine (DA)-depleted striatal tissue (lesion extract) and from intact striatal tissue (intact extract) were prepared, and trophic activities in these extracts were evaluated using survival and neurite extension of DAergic neurons as indices. Levels of brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), glial cell-line derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) in extracts were measured using enzyme-linked immunosorbent assay (ELISA). The lesion extract exhibited a stronger trophic activity on survival and neurite extension of DAergic neurons than intact extract. In lesion extract, bFGF was slightly and GDNF was significantly increased, while BDNF and NT-3 were the same level in each extract. The peak increase of bFGF and GDNF was during 2 to 3 weeks after DA depletion. Trophic activity of extract was strongly attenuated after immunoprecipitation of GDNF and partly attenuated after immunoprecipitation of bFGF. In parallel immunohistological study, no significant variations were found for striatal microtubule-associated protein-2 (MAP-2)- nor OX-41-immunoreactive cells, while the number of strongly labeled glial fibrillary acidic protein (GFAP)-immunoreactive cells were increased in DA-depleted striatum, suggesting reactive gliosis. Data suggest that bFGF is a minor, while GDNF is a major component of trophic activity for DAergic neurons in DA-depleted striatum, and increased bFGF and GDNF levels may be mediated partly by reactive gliosis.

Estrogen protects against while testosterone exacerbates vulnerability of the lateral striatal artery to chemical hypoxia by 3-nitropropionic acid

Gender differences in the vulnerability of the lateral striatal artery (1STR artery) to systemic intoxication with 3-nitropropionic acid (3-NPA, succinate dehydrogenase inhibitor) were studied. Subcutaneous injection of 3-NPA (20 mg/kg once a day for 2 days) induced striatal selective lesions in half of male rats associated with motor symptoms (rolling, paddling, recumbency, etc) while female rats were resistant. Lesions were located in the lateral striata and characterized by astroglial necrotic cell death, enhanced immunoreaction to factor VIII-related antigen, edema, extravasation of IgG and sometimes bleeding. The motor and histological disturbances were highly sex-dependent and modulated by changes in hormonal levels. Males were more susceptible than females. Castration had little effect but ovariectomy enhanced the vulnerability. Replacement therapy with testosterone increased while estradiol or tamoxifen suppressed the vulnerability in ovariectomized females. Investigation of the arterial architecture of the brain often revealed rectangular and acute angled branchings in the centrolateral striatum where the ISTR artery feeds. A parallel in vitro toxicity study demonstrated that an extreme Ca++ overload and a strong cellular swelling resulted in astrocytic cell death. Data suggest that 1STR artery and astrocytes are highly vulnerable to 3-NPA intoxication in males. The greater vulnerability of the ISTR artery may contribute to the pathogenesis of neurodegenerative diseases, striatal bleeding, etc. Protective effects of estrogen and tamoxifen may mediate gender differences often observed in these disorders and suggest their potential use as therapeutic agents for these disorders.

Dopa-producing astrocytes generated by adenoviral transduction of human tyrosine hydroxylase gene: in vitro study and transplantation to hemiparkinsonian model rats.

Astrocytes secreting a large amount of 3,4-dihydroxyphenylalanine (dopa) were generated by adenoviral transduction of the human tyrosine hydroxylase (TH) gene. After characterizing in vitro, the effect of transplantation of these astrocytes to the striatum of hemiparkinsonian model rats was investigated. Subconfluent cortical astrocytes were infected by replication-defect adenovirus type 5 carrying the human TH-1 gene or the LacZ reporter gene under the promoter of the glial fibrillary acidic protein (AdexGFAP-HTH-1, AdexGFAP-NL-LacZ). Dopa secretion was not evident at 3 days after the transduction of the HTH-1 gene but it increased from 7 days up to at least 4 months. The secretion was substrate (tyrosine)-dependent, and was enhanced by loading tetrahydrobioputerin (BH4) concentration-dependently. One-third of the hemiparkinsonian model rats, that were transplanted the HTH-1 gene-transduced astrocytes or introduced the direct injection of the viral vector to the striatum, showed a reduction of methamphetamine-induced rotations for at least 6 weeks. Apomorphine-induced rotation was decreased to the 50% level of the controls, but the reduction was obtained equally by the transplantation of HTH-1 gene-transduced or LacZ reporter gene-transduced astrocytes, or by the introduction of HTH-1 or LacZ gene carrying adenovirus. Treatment with FK506 for 3 weeks improved the late-phase apomorphine-induced rotations following the introduction of the HTH-1 gene carrying adenovirus. Histological examination revealed that, in animals that showed a reduction of methamphetamine-rotation, the TH positive astrocytes-like cells were distributed widely in the host striatum for at least 4 weeks. The number of TH positive astrocytes-like cells and their immunoreactivity decreased after 6 weeks when OX-41 positive microglias/macrophages were infiltrated. Data indicate that the adenoviral transduction of the human TH gene to astrocytes and its introduction to the striatum is a promising approach for the treatment of Parkinsons disease. However, the further technical improvements are required to optimize the adenoviral gene delivery, such as the control of viral toxicity and the regulation of the immune response.

ARG-VASOPRESSIN CONTENT IN THE SUPRACHIASMATIC NUCLEUS OF RAT PUPS : CIRCADIAN RHYTHM AND ITS DEVELOPMENT

To investigate the development of Arg-vasopressin (AVP) content and its diurnal rhythm in the suprachiasmatic nucleus (SCN), AVP was measured by enzyme immunoassay in the rat pup SCN punched out from tissue slices obtained at postnatal day (PD) 1, 5, 10, 12 and 20 from animals maintained under a light-dark cycle (LD). The AVP levels, measured at a restricted time of day, increased from PD1 reaching the adult level at PD10-12. Diurnal rhythm similar to that in adults was evident at PD10-12 under the LD conditions used. The peak value of AVP was observed at the earlier light period, and its trough occurred at the end of the light period. The circadian rhythm remained for 7 days under constant dark (DD) or constant light (LL) conditions. These results indicate that the AVP, one of the output signals from the SCN, starts to oscillate after PD10-12, and shows a free-running rhythm during the nursing period.

Dopamine-denervation enhances the trophic activity in striatum: evaluation by morphological and electrophysiological development in PC12D cells.

To evaluate the possibility that dopamine (DA) denervation enhances the trophic activity in striatum, normal or DA-depleted striatal tissue extract (N- or L-extract, respectively) was obtained, and their trophic effects on PC12D cells were investigated from the viewpoints of differentiation using morphological and electrophysiological analyses. Treatment with N- or L-extract induced neurite outgrowth in a concentration-dependent manner, and induced the enlargement of cell size. These effects were stronger in L-extract than in N-extract. Cation currents were investigated in whole cell patch-clamp mode. Development of cation current started with delayed-rectifier type K+ current (IK) and transient type K+ current (IA), followed by Ca2+ current (ICa) and tetrodotoxin-sensitive Na+ current (INa). INa was expressed more frequently in L-extract treated cells than N-extract treated cells at D7-9. The larger IK amplitude in L-extract treatment at D7-9 seemed to be related to the expression of INa. Development of IA was similar at any stage for both treatments. ICa development started at D3-5 after treatments, and the amplitude and current density were similar in both treatments. ICa was strongly blocked by omega-conotoxin GVIA (3 microM), indicating that N-type channels were mainly expressed after treatments. The data suggests that L-extract has stronger effects to hasten the differentiation of PC12D cells than N-extract by promoting the neurite outgrowth, cell enlargement and expression of voltage-dependent cation channels, especially INa and IK.

Tissue extract from dopamine-depleted striatum enhances differentiation of cultured striatal type-1 astrocytes

The effects of tissue extract from dopamine (DA)-depleted striatum (lesion extract, L-ext) on morphological and electrophysiological natures of cultured striatal astrocytes were investigated. L-ext treatment suppressed the proliferation of type-1 astrocytes. They became fibrous in a concentration-dependent manner. These changes were not observed in type-2 astrocytes. By whole cell patch-clamp recording, two kinetically and pharmacologically distinct voltage-activated potassium currents, A current and delayed rectifier, were identified. L-ext treatment enhanced both currents in type-1 astrocytes, but only A current in type-2. Data suggest that in tissue extract from DA-depleted striatum, there are increased trophic activities that promote the differentiation of type-1 astrocytes.

Gender-Related Difference of the Effect of 3-Nitropropionic Acid on Striatal Artery

Systemic intoxication of rats with 3-nitropropionic acid (3-NPA), a mycotoxin that inhibits succinate dehydrogenase in the mitochondrial respiratory chain, induces the striatum-selective lesions and motor symptoms (hyperkinesia, rolling, paddling, hypotonia, recumbency, etc.) reminiscent of Huntington’s disease (1–4). The lesions are localized in the centrolateral striatum and not in other parts of the brain. Inside the lesions, glial fibrillary acidic protein (GFAP) positive astroglia and microtubule-associated protein (MAP)-2-positive neurons are lost and the extravasation of immunoglobulin G (IgG) (the blood—brain barrier dysfunction) is detected (58). The mechanisms for the striatum selective lesions have been proposed as follows: the enhancement of excitotoxicity (decrease in the threshold for excitotoxicity) due to the deficiency of high-energy ATP (9–11), the dopamine (DA) toxicity by excessive release and turnover (6,12), the high vulnerability of the lateral striatal artery (1STR artery) accompanying the dysfunction of the BBB (13), or the involvement of high 3-NPA uptake activity by an acid transporter (glial glutamate transporter). The motor disturbances and histological (striatal) damages by 3-NPA were often detected in adult male rats but not in adult females or younger rats (6). Thus, the 3-NPA intoxication is a good model to investigate the pathophysiology of neuronal/glial cell death, neurodegenerative disorders, autoimmune diseases, stroke, or that of any disorders in which gender difference is often observed. Here, we review the recent finding of the gender differences of 3-NPA intoxication.

3-Nitropropionic acid (3-NPA)-induced toxicity of endothelial cells in vitro is mediated by nitric oxide dependent and independent mechanisms

’ The Center of Japan Biol. Chem., Kaizu, Gifu 503-0651, * Dept. of Physiol., Nagoya City Univ. Med. Sch., Nagoya 467-8601 and 3 Department of Physiology, Inst. Med.Sci., Banaras Hindu Univ., Varanasi, India Systemic administration of 3-NPA produces striatal specific astrocyte loss associated with destruction of blood-brain barrier. The in viva application of 3-NPA to the mixed cultures demonstrated greater vulnerability of astrocytes than neurons. Since endothelial cells are the first ones to be exposed to the toxin when it enters the circulation, we examined the effect of 3NPA on these cells. The human brain microvascular endothelial cells were exposed to 3-NPA or sodium nitroprusside (SNP) or their combination for 24 h and the nitrite concentration of medium (indicator of NO activity) and cell numbers were determined. The 3-NPA (1.7 mM) increased nitrite levels in presence or absence of cells in the medium and was associated with 50 % decrease in number of cells. SNP (0.004-0.4 uM) also increased the nitrite concentration in dose-dependent manner in presence of cells, but without cells the increase was only seen with 0.4 PM. The increase of nitrite by SNP was positively correlated with the cell loss. The nitrite concentration remained almost same with SNP (0.004-0.4 t&l) in presence of 3-NPA but there was greater cell loss for the same concentration of nitrite. The results indicate that the 3-NPAinduced toxicity of endothelial cells mediated by NO and non-NO mechanisms.

513 Characterization of neurotrophic factors and genes expressed in the striatum of hemiparkinsonian model rats

Kunio Koshimura, Junko Tanaka, Yoshio Murakami, Yuzuru Kato It has been revealed that erythropoietin (EPO) receptors are located in some brain areas such as hippocampus. We investigated the effects of EPO on Ca2+ uptake, dopamine release and cell survival in PC12 cells which possess EPO receptors. EPO (10-‘3-10-10 M) increased 45Ca2+ uptake into PC12 cells (1 min, 37C), which was inhibited by nicardipine(1 microM) or anti-EPO antibody (1:lOO diluted). At the same concentrations, EPO increased dopamine release from PC12 cells (10 min, 37C), which was sensitive to nicardipine or anti-EPO antibody. After 5-day culture without serum and NGF, number of viable cells decreased to one third of that of the control cells cultured with serum and NGF. Administration of EPO to the culture medium (10-‘3-10-10 M) increased viable number of cells cultured without serum and NGF. EPO (10-13-10-‘o M) stimulated MAP kinase activity in PC12 cells. These results suggest that EPO stimulates neuronal function and viability via activation of Ca2+ channels.