Immunohistochemical Localization of Huntingtin-associated Protein 1 in Endocrine System of the Rat

Abstract

Huntingtin-associated protein 1 (HAP1) was originally found to be localized in neurons and is thought to play an important role in neuronal vesicular trafficking and/or organelle transport. Based on functional similarity between neuron and endocrine cell in vesicular trafficking, we examined the expression and localization of HAP1 in the rat endocrine system using immunohistochemistry. HAP1-immunoreactive cells are widely distributed in the anterior lobe of the pituitary, scattered in the wall of the thyroid follicles, or clustered in the interfollicular space of the thyroid gland, exclusively but diffusely distributed in the medullae of adrenal glands, and selectively located in the pancreas islets. HAP1-containing cells were also found in the mucosa of stomach and small intestine with a distributive pattern similar to that of gastrointestinal endocrine cells. However, no HAP1-immunoreactive cell was found in the cortex of the adrenal gland, the testis, and the ovary. In the posterior lobe of the pituitary, HAP1-immunoreactive products were not detected in the cell bodies but in many stigmoid bodies, one kind of non-membrane-bound cytoplasmic organelle with a central or eccentric electron-lucent core. HAP1-immunoreactive stigmoid bodies were also found in the cytoplasm of endocrine cells in the thyroid gland, the medullae of adrenal gland, the pancreas islets, the stomach, and small intestine. The present study demonstrates that HAP1 is selectively expressed in part of the small peptide-, protein-, and amino-acid analog and derivative-secreting endocrine cells but not in steroid hormone-secreting cells, suggesting that HAP1 is also involved in intracellular trafficking in certain types of endocrine cells.

Keywords

huntingtin-associated protein intracellular trafficking rat endocrine cell immunohistochemistry

Huntingtin-associated protein-1 (HAP1) was first identified by yeast two-hybrid screening for its association with huntingtin, the protein mutated in Huntington's disease (Li et al. 1995). Huntington's disease (HD) is a neurodegenerative disorder caused by the polyglutamine expansion in the N-terminal region of huntingtin (The Huntington Disease Collaborative Research Group 1993). HAP1 interacts with the N-terminal domain of both wild-type and mutant huntingtin. The binding affinity of HAP1 to huntingtin is enhanced by an expanded polyglutamine repeat, the length of which is known to correlate with the age of HD onset, implying a possible role for HAP1 in the pathogenesis of HD (Li et al. 1995).

HAP1 has been found in several species including the rat, mouse, and human (Li et al. 1995, 1998a; Nasir et al. 1998). Earlier studies showed that HAP1 is a brain-specific protein that is widely expressed in the rat brain (Li et al. 1995, 1996; Gutekunst et al. 1998; Page et al. 1998). However, subsequent in situ hybridization histochemical observation in developing and adult mouse demonstrated that HAP1 is expressed not only in the brain and spinal cord but also in the peripheral nervous system, reproductive glands, and pituitary gland (Dragatsis et al. 2000). Our recent work also detected the localization of HAP1 in the spinal ganglia and retina of the rat (Huang et al. 2004; Xu et al. 2004). In the brain, HAP1 is highly expressed in the limbic-related forebrain regions and midline/periventricular brainstem regions with dramatic enrichment in the pedunculopontine nuclei, the accessory olfactory bulb, and the hypothalamus. In contrast, little expression is detected in the striatum and thalamus (Li et al. 1996; Page et al. 1998; Fujinaga et al. 2004).

HAP1 has been electron microscopically found to be associated with various kinds of neurocytoplasmic organelles and inclusions such as microtubules, synaptic vesicles, and stigmoid bodies (Gutekunst et al. 1998; Li et al. 2000). Stigmoid bodies are distinct, ovoid to circular in shape, non-membrane-bound cytoplasmic inclusions (1–3 μm in diameter) with moderate- to low-electron density and a central or eccentric electron-lucent core (Shinoda et al. 1992), distributed widely and specifically in the limbic forebrain regions (Shinoda et al. 1992, 1993; Li et al. 1998c). By immunohistochemistry, human placental antigen X-P2 has been proven to be an antigen marker of stigmoid body (Shinoda et al. 1993). HAP1 is demonstrated to be localized to the stigmoid body in the rat brain; the brain regions in which HAP1-immunoreactive puncta are most abundant are the same regions where stigmoid bodies are concentrated (Gutekunst et al. 1998). Thus, it implies that HAP1 might be an essential component and another antigen marker of the stigmoid body (Gutekunst et al. 1998; Fujinaga et al. 2004).

High levels of HAP1 expression in the hypothalamus and the pituitary gland imply that HAP1 may be important for endocrine function. Endocrine cells are similar to neurons to some extent; however, an immunohistochemical observation of HAP1 in endocrine system remains to be attempted. In the present study, we carry out immunohistochemistry to examine the distribution of HAP1 in endocrine organs and tissues including the pituitary, thyroid, adrenal medulla, pancreas islet, the mucosa of the stomach, and small intestine. The results demonstrate that HAP1 is selectively expressed in some endocrine cells.

Materials and Methods

Animals and Tissue Preparation

A total of ten 10- to 12-week-old Sprague-Dawley rats, six males and four females, weighing 220–250 g, were used in this study. All rats were treated in accordance with the Guidelines for Animal Experimentation of the Experimental Animal Science Center of Tongji Medical College. They were deeply anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg/kg bodyweight) and then perfused via ascending aorta with 100 ml of 0.01 M sodium phosphate-buffered 0.85% saline (PBS; pH 7.4), followed by 300 ml of 0.1 M sodium phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde. After perfusion, pituitary glands, thyroid glands, adrenal glands, pancreas, testes or ovaries, stomachs, duodenum, jejunum, and ileum were removed and postfixed in the same fixative at 4C for 4–6 hr. The removed organs were soaked in cold 0.1 M PB containing 30% sucrose at 4C overnight for cryoprotection, quickly frozen, and cut into 10- to 30-μm sections on a cryostat. Frozen sections were collected in ice-cold PBS or directly mounted onto gelatin-coated slides and kept at 4C until use.

Immunohistochemistry

Immunostaining of pituitary glands, thyroid glands, and adrenal glands was performed on free-floating sections, whereas immunostaining of the testes, ovaries, pancreas, stomachs, and small intestines was done on the sections mounted on slides. After being rinsed in PBS, the sections were processed for immunohistochemical localization of HAP1 by the avidin-biotin-peroxidase method (Hsu et al. 1981). To reduce endogenous peroxidase activity and to prevent nonspecific antibody binding, sections were treated in 1% hydrogen peroxide for 2 hr and 5% normal goat serum (NGS) for 30 min in PBS after incubation in PBS containing 1% Triton X-100. Thereafter, sections were incubated with a guinea pig polyclonal antibody to a glutathione S-transferase (GST) fusion protein containing amino acids 278–445 of rat HAP1 (1:5000), which was generated as described previously (Li et al. 2003), at 4C for 35–40 hr, followed by incubation of biotinylated goat anti-guinea pig IgG (1:200; Vector Labs, Burlingame, CA) at room temperature for 2 hr and avidin-biotin complex (1:100, Vector ABC Elite; Vector Labs) at room temperature for 2 hr. Primary and secondary antibodies and avidin-biotin complex were diluted with PBS containing 3% Triton X-100 and 5% NGS. Between incubations the tissues were rinsed in PBS. Finally, the HAP1-immunoreactive products were visualized by incubation with 0.02% diaminobenzidine (DAB; Sigma) and 0.005% hydrogen peroxide in 0.05 M Tris-HCl buffer for 10–15 min. Sections were then dehydrated, coverslipped, and examined using light microscopy. The stomach and small intestine sections were lightly counterstained with hematoxylin before dehydration. For the controls, the primary antibody was omitted or absorbed with excess GST-HAP1 fusion protein, or normal serum was used.

Results

Light microscopic immunohistochemistry revealed that HAP1 immunoreactivites were present in a series of endocrine organs and mucosa of gastrointestinal tracts but not in the testes and ovaries.

Pituitary Glands

In the rat pituitary glands, HAP1-immunoreactive cells were widely distributed in the anterior lobe, but not all anterior pituitary cells were labeled. Immunoreactivity, which was often moderate, was involved the entire cytoplasm (Figure 1A). In some HAP1-postive cells, one or more cytoplasmic puncta with varied sizes were intensely labeled (Figures 1A and 1B), which were previously identified as stigmoid bodies (Gutekunst et al. 1998). In the posterior lobe, no HAP1-positive cell body was seen, but strong HAP1-immunoreactive stigmoid bodies were found. These punctuate or dot-like bodies were distributed throughout the posterior lobe, some being arranged roughly in a linear way (Figure 1C). In the pars intermedia, cells showed very weak or negative HAP1 immunoreactivity (Figure 1A).

Figure 1.

Immunohistochemical staining for HAP1 in pituitary gland of the rat. (A) The anterior lobe (Ant) contains numerous immunopositive cells, the posterior lobe (Post) has a large number of immunopositive stigmoid bodies, and the cells in pars intermedia (Int) are negative or weakly positive. (B) High-power photomicrograph showing the diffuse immunoreactive products of HAP1 in the cytoplasm of HAP1-positive anterior lobe cells and the cells containing stigmoid bodies (arrows). (C) HAP1-immunopositive stigmoid bodies (arrows) in the posterior lobe. Bars = 20 μm.

Thyroid Glands

Immunohistochemical staining showed that the thyroid gland contained HAP1-positive cells, which were similar to parafollicular cells in their pattern of distribution, i.e., they occurred as solitary cells scattered in the wall of the thyroid follicles or small clusters in the interfollicular space (Figure 2A). Strongly HAP1-immunoreactive stigmoid bodies were also detected in the HAP1-positive cells (Figure 2B). In contrast, no HAP1 was detected in follicular cells.

Adrenal Glands

Moderate to strong HAP1 immunoreactivity was exclusively but diffusely distributed in the medullae of adrenal glands. A large number of HAP1-positive cells were arranged in nests or short cords (Figure 3). On the other hand, cortical cells including cells in zona glomerulosa, zona fasculata, and zona reticularis did not show any HAP1 immunoreactivity.

Figure 2.

Immunohistochemical staining for HAP1 in the thyroid gland of the rat. (A) Immunopositive cells are scattered between the follicular cells or distributed in the interfollicular space in small clusters. (B) High-power photomicrograph showing HAP1-positive stigmoid bodies (arrows). Bars = 20 μm.

Figure 3.

Immunohistochemical staining for HAP1 in adrenal gland of the rat. Immunoreactivity of HAP1 is exclusively distributed in the medulla (Med). Cort, cortex of adrenal gland. Bar = 50 μm.

Pancreas Islets

Moderate HAP1 immunoreactivity was also found exclusively in the endocrine portion of the pancreas, the pancreas islets (Figure 4). They were distributed throughout the islets and localized in the cytoplasm. Occasionally, stigmoid bodies with intense HAP1 immunoreactivity were observed. Like the cortex of the adrenal gland, the exocrine portion of the pancreas did not contain any HAP1-immunoreactive product.

Stomachs and Small Intestines

HAP1 immunoreactivity was found in the gastrointestinal tract. In the gastric mucosa, many HAP1-positive cells were densely distributed in the glands but were rare in the surface epithelium (Figure 5). HAP1-immunoreactive stigmoid bodies were detected in some glandular HAP1-positive cells (Figure 5B). There were regional differences in positive cell numbers: they were few in the cardiac region, moderate in the fundic region, and numerous in the pyloric region. In the pyloric glands they tended to be located at the bottom of the glands, whereas the body region remained unlabeled. In the duodenum, HAP1-immunoreactive cells were scattered in the villi and intestine glands (Figure 5C), the number being lower than in the stomach. HAP1-positive cells also occurred in the jejunum and ileum in the same way as in the duodenum but were fewer in number than in the duodenum.

Figure 4.

Immunohistochemical staining for HAP1 in the rat pancreas. Immunoreactivity of HAP1 is selectively localized in the islets of the pancreas. Arrows indicate immunopositive stigmoid bodies. Bar = 50 μm.

Figure 5.

Immunohistochemical staining for HAP1 in the pylorus and duodenum. (A) HAP1-immunopositive cells are densely distributed in the pyloric glands and dispersively in the surface epithelium. Arrowheads indicate HAP1-immunopositive cells in the surface epithelium. (B) High-power photomicrograph showing the localization of HAP1-immunoreactive products in the pyloric glands and surface epithelium with arrows indicating immunopositive stigmoid bodies. (C) Scattered distribution of HAP1-immunoreactive cells in the villi (V, arrow) and the intestine glands (IG, arrowhead) of the duodenum. Bars = 20 μm.

Discussion

To understand the normal function of huntingtin and the mechanisms by which mutant huntingtin causes the specific neurodegeneration in HD, efforts in the search for binding partners of huntingtin have led to the identification of several huntingtin-interacting or -associated proteins (Li et al. 1995; Kalchman et al. 1997; Wanker et al. 1997). HAP1 is the first identified partner for huntingtin. Previous studies on HAP1 were mainly concentrated on its relations to HD pathology and its role in neurons. The early biochemical, in situ hybridization histochemical, and immunohistochemical studies on HAP1 expression and distribution have indicated that HAP1 is a brain-specific protein, though its expression in reproductive glands and pituitary gland is also detected by in situ hybridization histochemistry (Li et al. 1995, 1996; Gutekunst et al. 1998; Page et al. 1998; Dragatsis et al. 2000; Fujinaga et al. 2004). The present immunohistochemical study shows for the first time that HAP1 is present in the following endocrine organs or tissues: pituitary, thyroid, adrenal medulla, pancreas islet, and mucosa of gastrointestinal tract.

The present study reveals that stigmoid bodies are present not only in the brain but also in the endocrine organs or tissues; some amino acid-derived hormone-secreting endocrine cells that express HAP1 contain one to two stigmoid bodies with strong HAP1-immunoreactivity. Abundant HAP1-positive stigmoid bodies but no HAP1-positive cell bodies are found in the posterior lobe of the pituitary. The HAP1-positive stigmoid bodies in the region seem to be located within the unmyelinated nerve fibers that come from the neuroendocrine cells or the secretory neurons in the supraoptic and paraventricular nuclei of the hypothalamus. By double-labeling immunofluorescence, we have found that the neurons in both supraoptic and paraventricular nuclei express HAP1, which colocalized with vasopressin (Shen et al., unpublished data). HAP1 has been regarded as an essential component and molecular marker of the stigmoid body (Gutekunst et al. 1998; Fujinaga et al. 2004). The presence of HAP1-immunoreactive stigmoid bodies in the endocrine cells also supports the idea that HAP1 is expressed in these cells.

Chemically, hormones secreted by endocrine cells are divided into two classes: amino acid-derived hormones and steroid hormones. The amino acid-derived hormones include small peptides, proteins, amino acid analogs, and derivatives, e.g., catecholamines; the steroid hormones are cholesterol-derived compounds. Accordingly, endocrine cells are classified as either amino acid-derived hormone-secreting cells or steroid hormone-secreting cells. The former includes the endocrine cells in the anterior and intermediate lobes of the pituitary gland, neuroendocrine cells in the hypothalamus, parafollicular cells in the thyroid gland, medullary cells of the adrenal gland, endocrine cells in pancreas islet, and the diffuse endocrine cells distributed in the gastrointestinal and respiratory tracts. The latter or steroid hormone-secreting cells include the adrenal cortical cells, interstitial cells in the testis, and theca and lutein cells in the ovary. Intriguingly, our study demonstrates that HAP1 is expressed in amino acid-derived hormone-secreting endocrine cells but not in the steroid hormone-secreting endocrine cells, suggesting that HAP1 might be highly relevant to the functions of the amino acid-derived hormone-secreting endocrine cells but not important for steroid-secreting endocrine cells. However, whereas the present immunohistochemical examination fails to detect HAP1 in rat testis and ovary, previous in situ hybridization histochemical study has shown the expression of HAP1 mRNA in mouse testis and ovary (Dragatsis et al. 2000). The negative immunhistochemical staining of HAP1 in rat testis and ovary is likely due to the low level of HAP1 expression and/or the species difference.

In the pituitary, only some but not all anterior cells are labeled by HAP1 antibody; thus, it seems HAP1 is expressed in specific amino acid-derived hormone-secreting endocrine cells, which is in line with our preliminary double-labeling immunfluorescent study in the rat. In the pituitary, HAP1 is selectively expressed in thyrotrophs but not in corticotrophs and somatotrophs. In the pancreas islets, B cells are HAP1 positive, but A cells and D cells are HAP1 negative. In the mucosae of stomach and duodenum, HAP1 is expressed in the gastrin cells but not in the somatostatin cells (Liao et al., unpublished data).

The amino acid-derived hormone-secreting endocrine cells are similar to neurons in many aspects. For example, all amino acid-derived hormone-secreting endocrine cells contain secretory granules that are similar to large dense core vesicles contained in axonal terminals of neurons. The medullary cells of the adrenal gland are modified postganglionic sympathetic neurons containing numerous large dense core vesicles. Synthesized hormone is packed in and transported by secretory granules in the same way as neurotransmitter is and by synaptic vesicles of neurons. Both hormone and neurotransmitter are released by exocytosis. Similar to neuroexocytosis, hormone release from granules or large dense core vesicles in endocrine cell is triggered by calcium influx through voltage-dependent channels (Zhang et al. 1998). Finally, amino acid-derived hormone-secreting endocrine cells also contain membrane receptors that hormones and neurotransmitters act on to control or to regulate the cell activities.

Growing evidence has shown that HAP1 is involved in intracellular trafficking in neurons (Li and Li 2005). HAP1 has been electron microscopically found to be localized to various kinds of membranous organelles in neurons, including endosomes, tubulovesicular structures, and synaptic vesicles (Martin et al. 1999; Li et al. 2000; Penzes et al. 2001). A study with a stop-flow, double-crush ligation approach in rat sciatic nerve has shown that HAP1 may have a role in both anterograde and retrograde transport of axons (Block-Galarza et al. 1997). HAP1 has also been shown to interact with p150glued, a dynactin subunit that participates in the intracellular transport of organelles and structures along microtubules (Engelender et al. 1997; Li et al. 1998b), and hepatocyte growth factor-regulated tyrosine kinase substrate, a mammalian homolog of yeast vacuolar protein sorting 27 proteins involved in formation or functions of early endosome via regulation of endocytotic pathway or membrane trafficking (Li et al. 2002). Furthermore, it has been reported that HAP1 binds to type I inositol (1, 4, 5)-triphosphate receptor, an intracellular Ca²⁺ release channel that plays an important role in neuronal function (Tang et al. 2003). Kittler and colleagues (2004) have confirmed that HAP1 binds the gamma-aminobutyric acid type A receptors [GABA(A)R], modulating synaptic GABA(A)R number by inhibiting receptor degradation and facilitating receptor recycling. Stigmoid bodies or similar organelles have been generally thought to be involved in protein synthesis and transport (Shinoda et al. 1993; Kind et al. 1997). In our previous work, we have found that transfection of HAP1-cDNA into HEK293 cells results in formation of HAP1-immunoreactive cytoplasmic inclusions with a very similar ultrastructure to that of the stigmoid body (Li et al. 1998c), which further implicates that HAP1 is likely to contribute to formation of HAP1-immunoreactive inclusions and transporting molecular constituents or products of these inclusions.

Secretion of amino acid-derived hormones from endocrine cells involves active trafficking of secretory granules and transport of hormones and membrane receptors. The expression of HAP1 in some classes of the amino acid-derived hormone-secreting endocrine cells strongly suggests that HAP1 also plays an important role in the trafficking of secretory granules, associated hormones or molecules, and membrane receptors in these specific endocrine cells.

Footnotes

Acknowledgements

This work was supported by the National Natural Science Foundation of China (30225024).

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