Keiji Kokubu

Interaction of ataxin-3 with huntingtin-associated protein 1 through Josephin domain.

Huntingtin-associated protein 1 (HAP1) is an essential component of the stigmoid body (STB) and known as a possible neuroprotective interactor with causative proteins for Huntingtons disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 17 (SCA17), and Joubert syndrome. To clarify what other causative molecules HAP1/STB could interact with, we cloned normal causative genes for several neural disorders from human brain RNA library and evaluated their subcellular interaction with HAP1/STB by immunocytochemistry and immunoprecipitation after cotransfection into Neuro2a cells. The results clearly showed that HAP1/STB interacts with the normal ataxin-3 through Josephin domain and polyglutamine-expanded mutants derived from SCA3 as well. The findings suggest that HAP1/STB could modify the physiological function of normal ataxin-3 and pathogenesis of SCA3 attributable to the mutant ataxin-3.

Anti-human placental antigen complex X-P2 (hPAX-P2) anti-serum recognizes C-terminus of huntingtin-associated protein 1A common to 1B as a determinant marker for the stigmoid body

The anti-serum against an unknown human placental antigen complex X-P2 (hPAX-P2) immunohistochemically recognizes three putative molecules (hPAX-P2S, hPAX-P2N, and hPAX-P2R), each of which is associated with the stigmoid bodies (STBs), necklace olfactory glomeruli (NOGs), or reticulo-filamentous structures (RFs) in the rat brain. The STBs also contain huntingtin-associated protein 1 (HAP1), and the HAP1-cDNA transfection induces STB-like inclusions in cultured cells. In order to clarify the relationship between hPAX-P2S and HAP1 isoforms (A/B), we performed Western blotting, immuno-histo/cytochemistry for light- and electron-microscopy and pre-adsorption tests with HAP1 deletion fragments. The results showed that the anti-hPAX-P2 anti-serum recognizes HAP1474–577 of HAP1A/B in Western blotting and strongly immunostains HAP1A-induced STB-like inclusions but far weakly detects HAP1B-induced diffuse structures in HAP1-transfected HEK 293 cells. In the rat brain, immunoreactivity of the anti-hPAX-P2 anti-serum for the STBs was eliminated by pre-adsorption with HAP1474–577, whereas no pre-adsorption with any different HAP1 fragments can suppress immunoreactivity for the NOGs and RFs, which were not immunoreactive to anti-HAP1 anti-serum. These findings indicate that hPAX-P2S, which is distinct from hPAX-P2N and hPAX-P2R, is identical with STB-constituted HAP1 and that the HAP1-induced/immunoreactive inclusions correspond to the hPAX-P2-immunoreactive STBs previously identified in the brain.

Microtubule-dependent formation of the stigmoid body as a cytoplasmic inclusion distinct from pathological aggresomes.

The stigmoid body (STB) is a neurocytoplasmic inclusion containing huntingtin-associated protein 1 (HAP1), an interactor of huntingtin, and its formation is induced by transfection of HAP1-cDNA into cultured cells. Although STB is believed to play a protective role in polyglutamine diseases, including Huntington’s disease and spinal and bulbar muscular atrophy, by sequestering the causative proteins, huntingtin and androgen receptor, respectively, its physiological function and formation remain poorly understood. Therefore, STB is occasionally confused with another cytoplasmic inclusion observed in polyglutamine diseases, the aggresome. Here we examined the subcellular dynamics of STB and compared it immunohistochemically and cytochemically with the aggresome in the rat brain and COS-7 or HeLa cells transfected with HAP1 and/or polyglutamine disease-associated genes. In time-lapse image analysis of HAP1-transfected cells, the HAP1-induced STB is formed from multiple fusions of small HAP1 inclusions characterized by vigorous cytoplasmic movement. In HAP1-transfected cells treated with a microtubule-depolymerizing drug, although the formation of small HAP1 inclusions was not affected, their fusion was critically inhibited. Immunohistochemistry and cytochemistry revealed the absence of association between STB and aggresomal markers, such as ubiquitin/proteasome, intermediate filaments, and the centrosome. Taken together, we concluded that STB is formed by a two-step process comprising microtubule-independent formation of small HAP1 inclusions and microtubule-dependent fusion of these inclusions, and that STB is distinct from pathological aggresomes.

Distribution Patterns of Uterine Glands and Embryo Spacing in the Mouse

To clarify the mechanism of implantation, relationship between positioning of the mouse embryo in the uterus and distribution of uterine glands along the long axis of the uterine horn was examined by three‐dimensional remodelling of the uterine endometrium. There were two unique regions in the endometrium. Uterine glands were distributed widely from mesometrial to anti‐mesometrial side in one region. It was localized from lateral to anti‐mesometrial side in another. These different regions were alternately aligned throughout the uterine horn. The number and position of embryos was consistent with that of the latter region. This study suggests that the type of distribution of uterine glands is closely related to the positioning of the embryo in mice.

Intracellular colocalization of HAP1/STBs with steroid hormone receptors and its enhancement by a proteasome inhibitor

The stigmoid body (STB) is a cytoplasmic inclusion containing huntingtin-associated protein 1 (HAP1), and HAP1/STB formation is induced by transfection of the HAP1 gene into cultured cells. In the present study, we examined the intracellular colocalization of HAP1/STBs with steroid hormone receptors (SHRs), including the androgen receptor (AR), estrogen receptor, glucocorticoid receptor (GR), and mineralocorticoid receptor, in COS-7 cells cotransfected with HAP1 and each receptor. We found that C-terminal ligand-binding domains of all SHRs had potential for colocalization with HAP1/STBs, whereas only AR and GR were clearly colocalized with HAP1/STBs when each full-length SHR was coexpressed with HAP1. In addition, it appeared that HAP1/STBs did not disrupt GR and AR functions because the receptors on HAP1/STBs maintained nuclear translocation activity in response to their specific ligands. When the cells were treated with a proteasome inhibitor, GR and AR localized outside HAP1/STBs translocated into the nucleus, whereas the receptors colocalized with HAP1/STBs persisted in their colocalization even after treatment with their ligands. Therefore, HAP1/STBs may be involved in cytoplasmic modifications of the nuclear translocation of GR and AR in a ubiquitin-proteasome system.

Androgen receptor expression in the preoptic and anterior hypothalamic areas of the adult Wistar rat and C57BL/6 mouse

cess of the severe stress response. To study the physiological roles of OT in the hypothalamo–neurohypophysial system, we adopted the experimental paradigm of SPS. In this paradigm, Sprague-Dawley male rats were exposed to SPS (immobilization for 2 hr, forced swimming for 20 min, followed by ether anesthesia). The rats were then maintained in an undisturbed condition for either 3-day or 7-day recovery period. Fluorescent immunohistochemistry for OT, in combination with morphometrical analysis, revealed that the OT immunoreactivity in magnocellular neurons in the SON was significantly decreased by Day 7. Neuronal OT levels in the SON on Day 7 were reduced by about 21% compared with those of control. This study suggests that SPS exposure causes long-term suppression of OT expression in the SON neurons. Further studies on expression and function of central OT in SPS-exposed rats will lead to a better understanding of patho-physiological roles of OT in stress-related mental disorders.

Relaying function of the STB/HAP1 on nuclear translocation of androgen and glucocorticoid receptors and its proteasome-dependent regulation

The stigmoid body (STB) is the neurocytoplasmic inclusion abundantly and specifically distributed in the limbic and hypothalamic regions of the normal brain. The STB has been also determined to contain huntingtin-associated protein 1 (HAP1), an interactor of huntingtin, and to be induced by the transfection of HAP1-cDNA into cultured cells (STB/HAP1). Although STB/HAP1 has been reported to have protective functions in Huntington?s disease and spinal-and-bulbar-muscular-atrophy by sequestering the causative proteins, huntingtin and androgen receptor (AR), its physiological functions remain unclear. In the present study, using immunocytochemistry, immunoprecipitation and time-lapse image analysis, we examined subcellular interaction of STB/HAP1 with the steroid-hormone receptors, including AR, estrogen receptors (ER and ER ), glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in COS-7 cells cotransfected with HAP1 and each receptor. The results clearly showed that STB/HAP1 sequesters GR and AR via a ligand-binding domain and that the receptors maintain nucleartranslocation activity in response to their specific ligands. When the cells were treated with MG132, a proteasome inhibitor, GR and AR localized outside of STB/HAP1 were clearly translocated into the nucleus as seen in the cells without MG132, while the receptors localized in the STB/HAP1 persisted in their association even after the treatment of their specific ligands. These results suggest that STB/HAP1 has a relaying function to modify the nuclear translocation of GR and AR, which is closely related to the proteasome activity.