Immunohistochemical analysis of huntingtin-associated protein 1 in adult rat spinal cord and its regional relationship with androgen receptor.

Huntingtin-associated protein 1 (HAP1) is a neuronal interactor with causatively polyglutamine (polyQ)-expanded huntingtin in Huntington's disease and also associated with pathologically polyQ-expanded androgen receptor (AR) in spinobulbar muscular atrophy (SBMA), being considered as a protective factor against neurodegenerative apoptosis. In normal brains, it is abundantly expressed particularly in the limbic-hypothalamic regions that tend to be spared from neurodegeneration, whereas the areas with little HAP1 expression, including the striatum, thalamus, cerebral neocortex and cerebellum, are targets in several neurodegenerative diseases. While the spinal cord is another major neurodegenerative target, HAP1-immunoreactive (ir) structures have yet to be determined there. In the current study, HAP1 expression was immunohistochemically evaluated in light and electron microscopy through the cervical, thoracic, lumbar, and sacral spinal cords of the adult male rat. Our results showed that HAP1 is specifically expressed in neurons through the spinal segments and that more than 90% of neurons expressed HAP1 in lamina I-II, lamina X, and autonomic preganglionic regions. Double-immunostaining for HAP1 and AR demonstrated that more than 80% of neurons expressed both in laminae I-II and X. In contrast, HAP1 was specifically lacking in the lamina IX motoneurons with or without AR expression. The present study first demonstrated that HAP1 is abundantly expressed in spinal neurons of the somatosensory, viscerosensory, and autonomic regions but absent in somatomotor neurons, suggesting that the spinal motoneurons are, due to lack of putative HAP1 protectivity, more vulnerable to stresses in neurodegenerative diseases than other HAP1-expressing neurons probably involved in spinal sensory and autonomic functions.

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.

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 Huntington's 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.

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.

Wolfram syndrome 1 (Wfs1) mRNA expression in the normal mouse brain during postnatal development.

Wolfram syndrome is a rare genetic disorder accompanying diabetes insipidus, sensorineural hearing loss, neurological complications, and psychiatric illness. This syndrome has been attributed to mutations in the WFS1 gene. In this study, we made a detailed histochemical analysis of the distribution of Wfs1 mRNA in the brain of developing mice. There were three patterns of change in the strength of Wfs1 mRNA signals from birth to early adulthood. In type 1, the signals were weak or absent in neonates but strong or moderate in young adults. This pattern was observed in the CA1 field, parasubiculum, and entorhinal cortex. In type 2, the signals were of a relatively constant strength during development. This pattern was seen in limbic structures (e.g. subiculum and central amygdaloid nucleus) and brainstem nuclei (e.g. facial and chochlear nuclei). In type 3, the signals peaked in the second week of age. This pattern was observed in the thalamic reticular nucleus. Thus, Wfs1 mRNA was widely distributed in the normal mouse brain during postnatal development. This evidence may provide clues as to the physiological role of the Wfs1 gene in the central nervous system, and help to explain endocrinological, otological, neurological, and psychiatric symptoms in Wolfram syndrome patients.

Sex-steroidal regulation of aromatase mRNA expression in adult male rat brain: a quantitative non-radioactive in situ hybridization study.

Neuronal aromatase, the enzyme that catalyzes the conversion of androgens to estrogens, is involved in brain sexual differentiation, the regulation of reproductive behavior, and gonadotropin secretion. We have previously reported that aromatase P450 (AromP450) protein expression is enhanced by both androgens and estrogens in the principal nucleus of the bed nucleus of the stria terminalis (prBST) and posterodorsal part of the medial amygdaloid nucleus (pdMAm) of the adult rat but is not altered in the central amygdaloid nucleus (CeAm) even after sex-steroid withdrawal or supplementation. Here, we have evaluated, via in situ hybridization with digoxigenin-labeled cRNA probes, the sex-steroidal regulation of brain AromP450 mRNA in the prBST, pdMAm, and CeAm of orchidectomized and adrenalectomized adult male rats treated with sesame oil, testosterone (1 mg/rat/day), dihydrotestosterone (1 mg/rat/day), or 17beta-estradiol (2 microg/rat/day) for 6 days. AromP450-mRNA expression in the prBST and pdMAm was markedly reduced in orchidectomized/adrenalectomized rats treated with sesame oil but strongly enhanced by testosterone or dihydrotestosterone and significantly reinstated by 17beta-estradiol. These results are essentially consistent with those of AromP450 protein expression and thus indicate that enhanced AromP450-protein expression in the prBST and pdMAm reflects transcriptional upregulation and/or post-transcriptional stabilization of its mRNA by sex steroids. In the CeAm, despite moderate AromP450-protein expression, the mRNA has never been detected with or without sex-steroidal manipulations, indicating that the putative sex-steroid-insensitive AromP450 mRNA in the CeAm may be distinct from that in the prBST and pdMAm or, if it occurs at all, expressed at much lower levels.

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 HAP1(474-577) 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 HAP1(474-577), 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.

Region-specific expression and sex-steroidal regulation on aromatase and its mRNA in the male rat brain: immunohistochemical and in situ hybridization analyses.

The brain has an estrogen-biosynthetic potential resulting from the presence of neuronal aromatase, which controls the intraneural sex-steroidal milieu and is involved in brain sexual differentiation, psychobehavioral regulation, and neuroprotection. In the rat brain, three distinct aromatase-P450-immunoreactive (AromP450-I) neural groups have been categorized in terms of their peak expression time (fetal, fetoneonatal, and young-to-adult groups), suggesting the presence of region-specific regulation on brain AromP450. In the present study, we compared the expressions between AromP450 protein and mRNA by using immunohistochemistry and in situ hybridization with an ovary-derived cRNA probe in serial sections of fetal, fetoneonatal, and adult male rat brains and then performed steroidal manipulations to evaluate the sex-steroidal effects on AromP450 in adult orchiectomized and adrenalectomized (OCX + ADX) male rats. As a result, prominent mRNA signals were detected in the fetal (i.e., the anterior medial preoptic nucleus) and fetoneonatal (i.e., the medial preopticoamygdaloid neuronal arc) groups, although no detectable signal was found in the "young-to-adult" group (i.e., the central amygdaloid nucleus). In addition, the "fetoneonatal" AromP450-I neurons were prominently reduced in number and intensity after OCX + ADX and then were reinstated by the administration of dihydrotestosterone, testosterone, or 17beta-estradiol. In contrast, none of the sex steroids had any significant effects on the young-to-adult group. Several possible explanations were explored for why the young-to-adult group may differ in aromatase expression and regulation, including the possibility that distinct splicing variants or isozymes for aromatase exist in the rat brain.

Huntingtin-associated protein 1 (HAP1) interacts with androgen receptor (AR) and suppresses SBMA-mutant-AR-induced apoptosis.

Huntingtin-associated protein 1 (HAP1), an interactor of huntingtin, has been known as an essential component of the stigmoid body (STB) and recently reported to play a protective role against neurodegeneration in Huntington's disease (HD). In the present study, subcellular association between HAP1 and androgen receptor (AR) with a long polyglutamine tract (polyQ) derived from spinal-and-bulbar-muscular-atrophy (SBMA) was examined using HEp-2 cells cotransfected with HAP1 and/or normal ARQ25, SBMA-mutant ARQ65 or deletion-mutant AR cDNAs. The results provided the first clear evidence that HAP1 interacts with AR through its ligand-binding domain in a polyQ-length-dependent manner and forms prominent inclusions sequestering polyQ-AR, and that addition of dihydrotestosterone reduces the association strength of HAP1 with ARQ25 more dramatically than that with ARQ65. Furthermore, SBMA-mutant-ARQ65-induced apoptosis was suppressed by cotransfection with HAP1. Our findings strongly suggest that HAP1/STB is relevant to polyQ-length-dependent modification on subcellular AR functions and critically involved in pathogenesis of not only HD but also SBMA as an important intrinsic neuroprotectant determining the threshold for cellular vulnerability to apoptosis. Taking together with previous reports that HAP1/STB is selectively expressed in the brain regions spared from degenerative targets in HD and SBMA, the current study might explain the region-specific occurrence of neurodegeneration in both diseases, shedding light on common aspects of their molecular pathological mechanism and yet-to-be-uncovered diagnostic or therapeutic applications for HD and SBMA patients.

Neuroanatomical distribution of Huntingtin-associated protein 1-mRNA in the male mouse brain.

Huntingtin-associated protein 1 (HAP1) was identified as an interactor of the gene product (Huntingtin) responsible for Huntington's disease and found to be a core component of the stigmoid body. Even though HAP1 is highly expressed in the brain, detailed information on HAP1 distribution has not been fully described. Focusing on the neuroanatomical analysis of HAP1-mRNA expression using in situ hybridization histochemistry, the present study clarified its detailed regional distribution in the entire mouse brain. Mouse HAP1 (Hap1)-mRNAs were abundantly expressed in the limbic-related forebrain regions and midline/periventricular brainstem regions including the olfactory bulb, limbic-associated cortices, hippocampus, septum, amygdala, bed nucleus of the stria terminalis, preoptico-hypothalamic regions, central gray, raphe nuclei, locus coeruleus, parabrachial nuclei, nucleus of the solitary tract, and area postrema. In contrast, little expression was detected in the striatum and thalamus, implying that Hap1 is associated with neurodegeneration-sparing regions rather than target lesions in Huntington's disease. The distribution pattern, resembling that of the stigmoid body, suggests that HAP1 and the stigmoid body are implicated in protection from neuronal death rather than induction of neurodegeneration in Huntington's disease, and that they play an important role in integrating instinct behaviors and underlying autonomic, visceral, arousal, drive, memory, and neuroendocrinergic functions, particularly during extensive homeostatic or emotional processes. These data will provide an important morphological base for a future understanding of functions of HAP1 and the stigmoid body in the brain.

Expression of estrogen receptors (alpha, beta) and androgen receptor in serotonin neurons of the rat and mouse dorsal raphe nuclei; sex and species differences.

Sex steroids have been inferred to be involved in the regulation of affective status at least partly through the serotonergic (5-HT) system, particularly in the dorsal raphe nucleus (DRN), which innervates enormous projections to the cerebral cortex and limbic system. In the present study, the expression of estrogen receptors-alpha and -beta (ERalpha, ERbeta), androgen receptor (AR) and 5-HT was examined immunohistochemically in the rat and mouse DRN in both sexes. The results showed that large numbers of ERalpha- and/or ERbeta-immunoreactive (ERalpha-I, ERbeta-I) cells were found in the DRN of both male and female mice, whereas only small numbers of ERalpha-I cells and no ERbeta-I cells were seen in the rat DRN of each sex. With respect to AR-immunoreactive (AR-I) cells, moderate numbers of such cells were present only in male rats and mice, and no or very few could be observed in female ones. The ERalpha-I, ERbeta-I, and AR-I cells were mainly distributed in the rostral DRN. In double-immunostaining, many 5-HT-I neurons were found to show ERalpha and/or ERbeta expression specifically in the rostral DRN (particularly dorsal, ventral and interfascicular parts) of mice of both sexes, but not in that of rats. In contrast, only a few 5-HT neurons were observed to show AR expression in the DRN of both rodents. The current results strongly suggest that sex steroids can modulate the affective regulation of the serotonergic system through ERalpha and/or ERbeta in 5-HT neurons of the mouse rostral DRN (but not so much through AR), and that such effects might be different depending on the sex and species, as shown by the prominent sex differences in AR expression and prominent species differences in ERalpha and ERbeta expression.

Gonadal and adrenal effects on the glucocorticoid receptor in the rat hippocampus, with special reference to regulation by estrogen from an immunohistochemical view-point.

Focusing on the hippocampal CA1 region, effects of peripheral gonadal and adrenal steroids on the glucocorticoid receptor (GR) were immunohistochemically evaluated in male and female adult rat brains after adrenalectomy (ADX), gonadectomy (GDX), and administration of estradiol (E2) and/or corticosterone (CS). In ADXed male rats, the hippocampal nuclear GR decreased and turned back to the cytoplasm, whereas in females, nuclear localization persisted even after ADX. In GDX+ADXed female rats, the GR was dispersedly translocated from the nucleus to the cytoplasm as well as in GDX+ADXed males. The dispersed cytoplasmic GR was again translocated into the nucleus by administration of CS. In addition, administration of a small dose of E2 for 4-13 days was found to sufficiently recover the nuclear location of GR in GDX+ADXed rat brains, whereas medium-to-large doses could not do this. Also, a longer administration more strongly enhances the nuclear GR location and expression. The present study provided strong immunohistochemical evidence that the sexually dimorphic effects of ADX on hippocampal GR are attributable to gonadal hormones, and that E2 is implicated in the effects in inversely-dose- and directly-duration-dependent manner. Taken together, intriguing gonadal and adrenal crosstalk is considered to play some important role in regulating hippocampal GR morphology and to have a possibly crucial influence on stress-related disorders such as depression.