|
Staining for huntingtin-associated protein 1 (Hap1) consistently reveals the presence of stigmoid bodies (SBs), inclusions of unknown function in the cytoplasm,
axons, and dendrites in a particular set of neurons in the peripheral and central nervous system. SBs are highly enriched in the hypothalamic region where they
are present in the lateral hypothalamus, ventromedial hypothalamus, and paraventricular nucleus. They are also numerous in the amygdala and central gray.
Studies have suggested that SBs and Hap1 are important contributors in feeding control. More recently Hap1 levels have been shown to be elevated in
hypothalamic nuclei of food deprived rodents and decreased following intraventricular insulin injections. The first aim of our study was to examine the effect of
food restriction and water deprivation on Hap1 levels and SB numbers in the hypothalamus, amygdala, and central gray areas. Mice underwent periods of food
deprivation for 24 or 48 hours, food restriction for 7 days, or water deprivation for 24 hours. Brain sections were immunostained for Hap1. Initial data confirm the
finding that food deprivation results in increased Hap1 expression in the hypothalamus. In addition, Hap1 levels increased in the medial amygdala and the central
gray. Food restriction and water deprivation also altered Hap1 levels, but conclusive findings cannot be articulated at this point. The second aim of our study
was to evaluate the co-localization of Hap1 and orexin A, a neurotransmitter which controls feeding behavior and is found in many of the same brain regions as
Hap1. Due to technical issues, further work is needed to attempt to co-localize orexin A and neurons with SBs.
Huntingtin-associated protein 1 (Hap1), a protein implicated with the motion disorder Huntington’s Disease, can also be used to identify stigmoid bodies.
Stigmoid bodies (SBs) are inclusions of unknown function found in the neurons of many structures of the brain, including the hypothalamus, thalamus and
amygdala. SBs are typically 1-4µm spherical structures found in the cytoplasm, dendrites or axon terminals. However, the location, size, and presence of SBs
can be influenced by external factors (Chan et al, 2002; Sheng et al, 2006). In addition, Hap1 has also been localized to innervations to specific organs in the
periphery of the body such as the duodenum and pancreas (Liao et al, 2006). Hap1 has also been found to play a vital role in maintenance of GABA receptors
(Kittler et al, 2004). Additional research (Sheng et al, 2006) seems to relate the levels of Hap1 to food intake. Fasted mice showed higher levels of Hap1 in the
arcuate nucleus, lateral hypothalamus, and dorsomedial hypothalamus, while those fed high carbohydrate diets showed less Hap1 in comparison to controls.
However, the effects of fasting on the Hap1 levels in the rest of the brain have not been clearly addressed. The fact that such trends are seen in the hypothalamus
has heavy implications, as it plays a key role in the regulation of feeding behavior in the mammalian brain. Indeed, it has been found that Hap1 knockout mice
show an inability to feed (Chan et al, 2002). These knockouts develop normally from conception till birth, but die shortly after, presumably due to a lack of
feeding behavior. Findings such as these have led many to posit that Hap1 may play an important role in feeding behavior. On a related note, orexin A is an
important neurotransmitter already known to invoke feeding behavior. Studies have found that concentrations of orexin undergo alterations during fasting,
though the literature shows some contradictions as to when and how (Park et al, 2004; Mondal et al, 1999). Given the correlations outlined above, there is a
distinct possibility that orexin A and Hap1 may be somehow linked in the control and evocation of feeding behavior. Furthermore, the hypothalamus is also
known to play regulatory effects on drinking behavior, though no literature exists regarding SBs and drinking behavior.
Animals:
C57/Bl6 mice were set into groups of three, and each group underwent a period of controlled ingestion:
- Food deprivation for 24 hours
- Food deprivation for 48 hours
- Water deprivation for 24 hours
- Food restriction to 85% of original body weight for 7 days
- Food and water ad libitum
Each mouse was perfused with physiological saline and 4% paraformaldehyde, and the brain extracted.
Immunocytochemistry:
Brains were cut into 50 micrometer slices by freezing microtome. Brain sections from all test conditions were washed with 3% hydrogen peroxide in 0.1% triton,
then with a blocking buffer of 4% normal goat serum (NGS). Sections then underwent a staining procedure, with a primary antibody against Hap1 at a
concentration of 1:100, then a secondary biotinylated antibody against the primary at 1:750, and finally a tertiary (Vector Laboratories). Diaminobenzadine
(DAB) was used as the final step to stain for Hap1 and SBs. Sections were then mounted and cover slipped. Peripheral organs were sectioned and mounted
on slides and then underwent staining for Hap1 as described above. Sections from the control as well as 48 hours of food deprivation were then selected for
double staining. These sections underwent immunostaining and nickel DAB for Hap1, and were then restained with orexin A antibody and regular DAB.
Hap1 staining significantly increased in the PVN and VMH of fasted animals. Fasting did not cause any significant Hap1 levels change in other parts of the brain.
The number of SBs in the hypothalamus did not significantly change following 24 and 48 hrs fasting. In the central gray and amygdala, the number of SBs
decreased significantly after 48 hours of fasting in comparison to controls. Food restriction and water deprivation results cannot yet be elaborated on. In
addition to confirming the work of Sheng et al (2006) in regards to Hap1 levels in the hypothalamus, these findings also show that levels of Hap1 in the other
structures examined are not altered by fasting. These data strengthen the argument that Hap1 plays a vital role in feeding behavior. However, counterintuitively,
the number of SBs is only affected after 48 hours of fasting outside of the hypothalamus (in the CG and amygdala). Although a larger study with more animals
is needed to further confirm our findings, it seems that SBs may serve a different function outside of the hypothalamus than Hap1 serves in it.
Future Plans
Due to time constraints and immunostaining difficulties, the co-localization of Hap1 and orexin A was not thoroughly investigated; only single staining was
accomplished and suggests a potential interaction between Hap1 and Orexin A. Weight loss is a common effect of Huntington’s disease, and it has been
found that in HD mouse models, as well as HD human cases, Orexin positive neurons of the hypothalamus degenerate (Petersen et al, 2005). Western
blotting and immunocytochemistry will be further used to determine if the our new Hap1 antibody detects a phosphorylated or dephosphorylated Hap1 protein.
Furthermore, due to background staining problems, the localization of Hap1 in the peripheral organs will require more exploration as well. Finally, the size of
SBs will be determined under the various conditions studied.
This material is based upon work supported by the Howard Hughes Medical Institute under Grant No.52003727. Additional funding came from NSI grand number 525990.
1 Chan, E.Y.W., Nasir, J., Gutekunst, C.A., Coleman, S., Maclean, A., Maas, A., Metzler, M., Gertsenstein, M., Ross, C.A., Nagy, A. and Hayden, M.R. (2002) Targeted disruption of Huntingtin-associated protein-1 (Hap1) results in postnatal death due to depressed feeding behavior. Human Molecular Genetics, 11, 945-959.
2 Kittler, J.F., Thomas, P., Tretter, V., Bogdanov, Y.D., Haucke, V., Smart, T.G. and Moss, S.J. (2004) Huntingtin-associated protine 1 regulates inhibitory synaptic transmission by modulating γ-aminobutyric acid type A recepter membrane trafficking. Proceedings of the National Academy of Science, 34, 12736-12741.
3 Liao, M., Shen, J., Zhang, Y., Li, S.H., Li, X.J. and Li, H. ( 2005) Immunohistochemical localization of Huntingtin-associated protein 1 in endocrine system of the rat. Journal of Histochemistry & Cytochemistry, 53, 1517-1524.
4 Mai, J.K., Assheur, J., Paxinos, G. (2004) Atlas of the Human Brain. Elsevier Academic Press: Boston.
5 Mondal, M.S., Nakazato, M., Date, Y., Murakami, N., Yanagisawa, M. and Matsukura, S. (1999) Widespread distribution of orexin in rat brain and its regulation upon fasting. Biochemical and Biophysical Research Communications, 256, 495-499.
6 Park, E.S., Yi, S.J., Kim, J.S., Lee, H.S., Lee, I.S., Seong, J.K., Jin, H.K. and Yoon, Y.S. (2004) Changes in orexin-A and neuropeptide Y expression in the hypothalamus of the fasted and high-fat diet fed rats. J. Vet. Sci., 5, 295-302.
7 Paxinos, G., Watson, C. (1998) The Rat Brain in Stereotaxic Coordinates. Academic Press: New York.
8 Petersen, A., Gil, J., Maat-Schieman, M.L.C., Bjorkqvist, M., Tanila, H., Araujo, I.M., Smith, R., Popovic, N., Wierup, N., Norlen, P., Li, J.Y., Roos, R.A.C., Sundler, F., Mulder, H. and Brundin, P. (2005) Orexin loss in Huntington’s disease. Human Molecular Genetics, 14, 39-47.
9 Sheng, G., Chang, G., Lin, J.Y., Yu, Z.X., Fang, Z.H., Rong, J., Lipton, S.A., Li, S.H, Tong, G., Leibowitz, S.F. and Li, X.J. (2006) Hypothalamic huntingtin-associated protein 1 as a mediator of feeding behavior. Nature Medicine, 12, 526-533.
Hap1 is a protein that may serve several functions in the brain, though conclusive functions have not yet been elucidated. Hap1 can be used to stain for
stigmoid bodies, small structures of unknown function found in neurons of several structures in the brain. Recent research has implied that Hap1 may have to do
with the control of feeding behavior. My main goal was to determine if Hap1 interacted with an important messenger protein in the brain, Orexin A, to control feeding
behavior. I expected that the numbers of stigmoid bodies and the amount of Hap1 would vary if I changed how much food and water animals consumed. I
found that the number of stigmoid bodies decreased after periods of fasting, while Hap1 increased.
Immunocytochemistry, perfusion, and histology
Hap1, immunocytochemistry, stigmoid bodies, Orexin
|
