Aiko Ishii

Selective loss of nigral dopamine neurons induced by overexpression of truncated human α-synuclein in mice

Parkinsons disease is characterized by loss of nigral dopaminergic neurons and presence of Lewy bodies, whose major component is alpha-synuclein. In the present study, we generated transgenic mice termed Syn130m that express truncated human alpha-synuclein (amino acid residue number: 1-130) in dopaminergic neurons. Notably, dopaminergic neurons were selectively diminished in the substantia nigra pars compacta of Syn130m, while transgenic mice that expressed comparable amount of full-length human alpha-synuclein did not develop such pathology. Therefore, the truncation of human alpha-synuclein seems to be primarily responsible for the loss of nigral dopaminergic neurons. The nigral pathology resulted in impairment of axon terminals in the striatum and concomitant decrease in striatal dopamine content. Behaviorally, spontaneous locomotor activities of Syn130m were reduced, but the abnormality was ameliorated by treatment with L-DOPA. The loss of nigral dopaminergic neurons was not progressive and seemed to occur during embryogenesis along with the onset of expression of the transgene. Our results indicate that truncated human alpha-synuclein is deleterious to the development and/or survival of nigral dopaminergic neurons.

Accumulation of phosphorylated α‐synuclein in dopaminergic neurons of transgenic mice that express human α‐synuclein

Parkinsons disease is neuropathologically characterized by the presence of Lewy bodies, whose major component is α‐synuclein. We had previously generated transgenic mice that expressed human α‐synuclein carrying an Ala53Thr point mutation (hα‐syn140m) under the control of the rat tyrosine hydroxylase promoter and found that hα‐syn140m was localized not only in the cytoplasm but also in the nuclei of mesencephalic dopaminergic neurons. In the present study, we carried out immunohistochemical analysis of the brain of Tg mice using anti‐PSer129, an antibody that specifically recognizes α‐synuclein phosphorylated at Ser129. The antibody detected only phosphorylated hα‐syn140m, whereas phosphorylation of endogenous α‐synuclein, if any, was below the detection limit of the method employed. The analysis showed that approximately one‐third of the hα‐syn140m‐positive neurons in the midbrain of heterozygous Tg mice were concomitantly reactive to anti‐PSer129. The ratio almost doubled in homozygotes, indicating that the phosphorylation level depends directly on the amount of substrate. In addition, the ratio did not change at least up to 48 weeks of age. These data strongly suggest that hα‐syn140m underwent constitutive phosphorylation and that the phosphorylation level was maintained to a certain level until the aged stages. Remarkably, hα‐syn140m localized in the nuclei seemed to be preferentially phosphorylated compared with that in the cytoplasm. Among kinases that have been reported to be involved in the phosphorylation of α‐synuclein, the β subunit of casein kinase‐2 was detected in the nuclei by immunohistochemistry. These data imply that at least casein kinase‐2 is involved in the phosphorylation of hα‐syn140m in the Tg mice.

Molecular Pathological Evaluation of Clusterin in a Rat Model of Unilateral Ureteral Obstruction as a Possible Biomarker of Nephrotoxicity

In the present study, to determine the validity of considering clusterin as a possible biomarker of nephrotoxicity, the expression and distribution of clusterin in the rat UUO kidney were investigated. Real-time RT-PCR revealed an immediate increase in the clusterin mRNA level in the kidney, within 6 hours after UUO, and also maintenance of the mRNA expression level from day-1 to day-3 was 60-fold higher in the UUO kidney than in the sham kidney. ISH analysis revealed clusterin mRNA signals in the UUO renal tubular epithelium, whereas no signal was observed in the sham kidney. Detection of clusterin-α and -β was conducted using the subtype-specific antibodies, by both of western blotting and immunohistochemistry. Although clusterin-α was predominant in the UUO urine, only faint signals were noted at the brush border of the tubular epithelium or intraductal. On the other hand, strong signals of clusterin-β were detected in the UUO kidney homogenate, and the molecule was localized in the renal tubular epithelium. These results suggest that clusterin was translated in the renal tubular epithelium after de novo expression induced by renal injury. Thus, detection of clusterin mRNA and clusterin-β in the kidney or clusterin-α in the urine may be useful for predicting nephrotoxicity.

Cytokine Receptor-Like Factor 1 is Highly Expressed in Damaged Human Knee Osteoarthritic Cartilage and Involved in Osteoarthritis Downstream of TGF-β

Osteoarthritis (OA) is the most prevalent joint disease and is characterized by pain and functional loss of the joint. However, the pathogenic mechanism of OA remains unclear, and no drug therapy for preventing its progress has been established. To identify genes related to the progress of OA, the gene expression profiles of paired intact and damaged cartilage obtained from OA patients undergoing joint substitution were compared using oligo microarrays. Using functional categorization combined with gene ontology and a statistical analysis, five genes were found to be highly expressed in damaged cartilage (HBEGF, ASUS, CRLF1, LOX, CDA), whereas three genes were highly expressed in intact tissues (CHST2, PTPRD, CPAN6). Among these genes, the upregulated expression of CRLF1 was reconfirmed using real-time PCR, and the in vivo expression of CRLF1 was detected in clusters of chondrocytes and fibrocartilage-like cells in damaged OA cartilages using in situ hybridization. In vitro, the transcriptional level of CRLF1 was positively regulated by TGF-β1 in the mouse chondrogenic cell line ATDC5. Additionally, the CRLF1/CLC complex promoted the proliferation of ATDC5 cells and suppressed the expression level of aggrecan and type II collagen. Our data suggest that the CRLF1/CLC complex disrupts cartilage homeostasis and promotes the progress of OA by enhancing the proliferation of chondrocytes and suppressing the production of cartilage matrix. A component of the complex, CRLF1, may be useful as a biomarker of OA; and the corresponding receptor is a potential new drug target for OA.

Suppression of AGE Precursor Formation Following Unilateral Ureteral Obstruction in Mouse Kidneys by Transgenic Expression of α-Dicarbonyl/ L -Xylulose Reductase

Unilateral ureteral obstruction (UUO) of kidneys causes acute generation of carbonyl stress. By electrospray ionization/liquid chromatography/mass spectrometry (ESI/LC/MS) we measured the content of methyl glyoxal, glyoxal, and 3-deoxyglucosone in mouse kidney extracts following UUO. UUO resulted in elevation of these dicarbonyls in the obstructed kidneys. Furthermore, the accumulation of 3-deoxyglucosone was significantly reduced in the kidneys of mice transgenic for α-dicarbonyl/L-xylulose reductase (DCXR) as compared to their wild-type littermates, demonstrating 4.91±2.04 vs. 6.45±1.85 ng/mg protein (P=0.044) for the obstructed kidneys, and 3.68±1.95 vs. 5.20±1.39 ng/mg protein (P=0.026) for the contralateral kidneys. On the other hand, collagen III content in kidneys showed no difference as monitored by in situ hybridization. Collectively, DCXR may function in the removal of renal α-dicarbonyl compounds under oxidative circumstances, but it was not sufficient to suppress acute renal fibrosis during 7 d of UUO by itself.

Suppression of Renal α-Dicarbonyl Compounds Generated following Ureteral Obstruction by Kidney-Specific α-Dicarbonyl/l-Xylulose Reductase

Renal unilateral ureteral obstruction (UUO) causes acute generation of α‐dicarbonyl stress substances, such as glyoxal, 3‐deoxyglucosone, and methylglyoxal, in the kidneys. These α‐dicarbonyl compounds are prone to form advanced glycation end products (AGEs) via the nonenzymatic Maillard reaction. Using transgenic (Tg) mice overexpressing a kidney‐specific short‐chain oxidoreductase, α‐dicarbonyl/L‐xylulose reductase (DCXR), we measured generation of α‐dicarbonyls following UUO by means of electrospray ionization/liquid chromatography/mass spectrometry in their kidney extracts. The accumulation of 3‐deoxyglucosone was significantly reduced in the kidneys of the mice Tg for DCXR compared to their wild‐type littermates, demonstrating 4.91 ± 2.04 vs. 6.45 ± 1.85 ng/mg protein (P = 0.044) for the obstructed kidneys, and 3.68 ± 1.95 vs. 5.20 ± 1.39 ng/mg protein (P = 0.026) for the contralateral kidneys. Despite the reduction in accumulated α‐dicarbonyls, collagen III content in kidneys of the Tg mice and their wild‐type littermates showed no difference as monitored by in situ hybridization. Collectively, DCXR may function in the removal of renal α‐dicarbonyl compounds under oxidative circumstances, but it is not sufficient to suppress acute renal fibrosis during 7 days UUO.

Downregulated expression in high IgA (HIGA) mice and the renal protective role of meprinβ

This study discusses the critical role of the metalloproteinase meprinbeta in the progression of glomerulonephritis. Using a microarray technique, the gene expression profiles in glomeruli isolated from high serum IgA (HIGA) mice with a purity of 97% or greater were examined. HIGA mice are a valid model of human IgA nephropathy (IgAN), with the typical pathological features of this condition, including a consistently high serum IgA level as well as dominant mesangial IgA deposition and mesangial enlargement. Among the many upregulated/downregulated genes after the development of IgAN, the downregulation of meprinbeta was intriguing. The expression level of the meprinbeta gene at 40 weeks of age was 52% of that observed at 8 weeks of age (prior to the development of IgAN), although in the control BALB/c mice, a 2.19-fold elevation was seen. These results were also confirmed by semi-quantitative RT-PCR and immunostaining analyses. As meprinbeta is a subunit of metalloproteinase meprins (meprin A, meprin B) and meprins are capable of proteolytically degrading extracellular matrix (ECM) components and proteolytically processing bioactive peptides, the downregulation of meprinbeta may contribute to the progression of glomerulonephritis and the eventual glomerular scarring. This working hypothesis was examined using an in vivo meprinbeta inhibition study. The inhibition of meprins by actinonin exacerbated some parameters of renal injury in mice afflicted with anti-glomerular basement membrane (anti-GBM) antibody-associated nephritis. These in vitro and in vivo results suggest that meprinbeta may play a protective role against the progression of renal injury through the degradation of ECM and bioactive peptides.

Evaluation of potential activity of luseogliflozin on vascular proliferation in the mesenteric lymph node with or without vascular tumors in Sprague-Dawley rats in a carcinogenicity study

The incidence of mesenteric lymph node vascular tumors can vary in rats, and appropriate assessment of potential risk of tumorigenicity is needed when the incidence is higher in treated groups than in a control group. In a 2-year rat carcinogenicity study of luseogliflozin, a selective sodium-dependent glucose co-transporter 2 inhibitor for the treatment of type 2 diabetes mellitus, there was a slight but statistically significant increase in the total number of hemangiomas and hemangiosarcomas in the mesenteric lymph nodes in males at a high-dose. As part of the risk assessment for luseogliflozin, its effect on the vascular proliferation potential in the mesenteric lymph nodes was examined in a rat carcinogenicity study by performing an image analysis using specimens with double immunohistochemical staining for PCNA and CD34 in control and high-dose males. In addition, immunohistochemical staining for VEGF was performed to detect enhanced angiogenesis. In the high-dose males that did not have a hemangioma/hemangiosarcoma, neither an increased number of PCNA/CD34-positive cells nor changes in the expression pattern of VEGF was observed. On the other hand, in the high-dose males that had a hemangioma/hemangiosarcoma, the number of PCNA-positive cells was increased in the tumor areas, and the number in the hemangioma/hemangiosarcoma was approximately one-half of that in the hemangiosarcoma in the control male. In conclusion, no potential change leading to vascular proliferation/tumors was detected in the mesenteric lymph nodes of high-dose males receiving luseogliflozin.

Influence of the estrus cycle on the evaluation of a vaginal irritation study in intact and ovariectomized rats

When conducting vaginal irritation studies, ovariectomized rats or rabbits are typically used according to practical reports. In the present study, we evaluated the influence of the estrus cycle in a vaginal irritation study using intact rats and ovariectomized rats, which exhibit a late diestrus-like condition, to determine whether intact rats can be useful for evaluating vaginal irritancy. Rats were divided into 4 groups: proestrus, estrus, and metestrus or diestrus in intact rats and ovariectomized rats. All the rats in each group were treated with a vehicle or sodium dodecyl sulfate, as the irritant, in single-dose and 4-day repeat-dose vaginal irritation studies. Each rat’s vagina was examined histopathologically, and the irritation score was calculated using a semiquantitative scoring system. In the single-dose study, the irritation scores for the proestrus or ovariectomized groups treated with sodium dodecyl sulfate were higher than those of the estrus group or metestrus or diestrus group. In the 4-day repeat-dose study, a significant histopathological difference was not found among the intact rats (proestrus, estrus, and metestrus or diestrus groups), and the irritation score range of the intact rats was similar to that of the ovariectomized rats, though the mean score of the intact rats was slightly lower than that of the ovariectomized rats. These results suggest that intact rats might be well suited for 4-day vaginal irritation studies and useful for evaluating vaginal irritancy using not only the mean score, but also individual irritation score ranges, whereas the estrus cycle would need to be identified in single-dose vaginal irritation studies.

Morphological analysis of patchy thickening and reddish discoloration of active hair growth areas in the skin of New Zealand White rabbits

Patchy thickening and reddish discoloration of active hair growth areas of skin in rabbits are occasionally found, and this gross feature could affect precise evaluation when conducting a dermal irritation test. Since little is known about the mechanism of this phenomenon, we examined the dorsal skin of New Zealand White rabbits morphologically and immunohistochemically in order to identify the possible mechanism responsible for developing these skin changes in relation to the hair cycle. Skin samples from 4 rabbits were divided into three groups (5 samples/group) based on their macroscopic characteristics: a thickened skin, erythematous skin, and smooth skin group. Histomorphological examination revealed that the percentage of hair follicles in the anagen phase, hair follicle length, hair follicle area, and proliferating cell nuclear antigen-positive cells in the hair follicles were greater in the thickened skin and erythematous skin groups than in the smooth skin group. Unlike mice and rats, the dermis was nearly adjacent to the muscular layer with a thin hypodermis, and the whole lengths of hair follicles in the anagen phase were located in the dermis in the rabbit skin. These results suggest that large hair follicles in the anagen phase compressed the surrounding dermis; therefore, the skin was grossly raised and showed thickening. A higher number of CD31-positive blood vessels, suggesting the occurrence of angiogenesis, was observed around the hair follicles in the erythematous skin group, and they seemed to affect the reddish discoloration of skin noted grossly.