Elsevier

Neuroscience

Volume 190, 5 September 2011, Pages 289-300
Neuroscience

Neurodegeneration, Neuroprotection, and Disease-Oriented Neuroscience
Research Paper
Orexin neurons in hypothalamic slice cultures are vulnerable to endoplasmic reticulum stress

https://doi.org/10.1016/j.neuroscience.2011.06.026Get rights and content

Abstract

Narcolepsy results from disruption of orexin neurons in the hypothalamus that play a key role in maintenance of the arousal state. Underlying mechanisms leading to selective loss of orexin neurons remain unknown. On the other hand, endoplasmic reticulum stress, namely, conditions associated with impairment of endoplasmic reticulum functions such as proper folding and sorting of newly synthesized proteins, is implicated in pathogenesis of several types of neurodegenerative disorders. Here we found that application of endoplasmic reticulum stress inducers such as tunicamycin (that prevents protein N-glycosylation) and thapsigargin (that inhibits Ca2+-ATPase) to organotypic slice cultures of the hypothalamus caused preferential loss of orexin-immunoreactive neurons, as compared to melanin-concentrating hormone- or calcitonin gene-related peptide-immunoreactive neurons. The decrease in orexin-immunoreactive neurons at early time points (6–24 h) was not accompanied by induction of cell death as indicated by the absence of caspase-3 activation and no significant change in the number of NeuN-positive cells, whereas sustained treatment with tunicamycin for 72 h induced cell death. At 24- h treatment, tunicamycin and thapsigargin did not decrease expression of prepro-orexin mRNA, suggesting that post-transcriptional mechanisms were responsible for depletion of orexin peptides. In addition, inhibition of axonal transport by colchicine and inhibition of proteasomal activity by MG132 significantly prevented the decrease in orexin immunoreactivity by tunicamycin. Comparative examinations of expression of unfolded protein response-related proteins revealed that C/EBP-homologous protein (a transcription factor that promotes induction of apoptosis) as well as phosphorylated form of RNA-dependent protein kinase-like endoplasmic reticulum kinase (a protein kinase that mediates inhibition of protein translation) was expressed more prominently in orexin neurons than in melanin-concentrating hormone neurons, in response to tunicamycin. These results indicate that orexin neurons are particularly sensitive to endoplasmic reticulum stress, which may be relevant to pathogenic events in narcolepsy.

Highlights

▶Effect of ER stress inducers was examined in hypothalamic slice cultures. ▶ER stress inducers caused selective loss of orexin. ▶Loss of orexin at early stage resulted from depletion of stored orexin. ▶Loss of orexin at later stage resulted from degeneration of orexin neurons. ▶ER stress might be relevant to pathogenic events in narcolepsy.

Section snippets

Preparation of hypothalamic slice cultures

Organotypic slice cultures of rat hypothalamus were prepared as described in our previous studies (Katsuki and Akaike, 2004, Michinaga et al., 2010b. Experimental procedures were approved by our institutional ethical committee concerning animal experiments, and were conformed to the Guide and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health. Briefly, brains were obtained from 7–8-day-old Wistar/ST rats (Nihon SLC, Shizuoka, Japan) under deep

Orexin neurons decrease more prominently than other neuropeptide-containing neurons during ER stress

To examine whether ER stress could affect orexin-immunoreactive neurons, we used conventional ER stress inducers such as tunicamycin and thapsigargin. Tunicamycin, by preventing protein N-glycosylation (Kaneko and Tsukamoto, 1994), promotes accumulation of misfolded proteins that leads to ER stress and initiation of UPR. Thapsigargin inhibits Ca2+-ATPase of ER (Perez-Sala and Mollinedo, 1995) and depletes ER luminal Ca2+, which leads to protein unfolding and misfolding due to Ca2+-dependent

Discussion

Here we explored potential relationship between ER stress and the integrity of orexin neurons. For this purpose, we prepared hypothalamic slice cultures from rats at 7 or 8 days old, and after 18 days of cultivation, treated them with various drugs. This corresponds to the usage of brains of 25 or 26-day-old rats, provided that developmental events proceed in vitro in the same manner as those in vivo. In this context, a study on developmental changes of orexin expression has shown that

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology, Japan.

References (54)

  • Y.Y. Lee et al.

    Tauroursodeoxycholate (TUDCA), chemical chaperone, enhances function of islets by reducing ER stress

    Biochem Biophys Res Commun

    (2010)
  • L. Lin et al.

    Narcolepsy and the HLA region

    J Neuroimmunol

    (2001)
  • Y. Ma et al.

    ER chaperone functions during normal and stress conditions

    J Chem Neuroanat

    (2004)
  • S. Michinaga et al.

    An anti-Parkinson drug ropinirole depletes orexin from rat hypothalamic slice culture

    Neurosci Res

    (2010)
  • N. Naidoo

    Cellular stress/the unfolded protein response: relevance to sleep and sleep disorders

    Sleep Med Rev

    (2009)
  • R.V. Rao et al.

    Misfolded proteins, endoplasmic reticulum stress and neurodegeneration

    Curr Opin Cell Biol

    (2004)
  • T.C. Thannickal et al.

    Reduced number of hypocretin neurons in human narcolepsy

    Neuron

    (2000)
  • Y. Yamamoto et al.

    Postnatal development of orexin/hypocretin in rats

    Brain Res Mol Brain Res

    (2000)
  • A. Yamanaka et al.

    Hypothalamic orexin neurons regulate arousal according to energy balance in mice

    Neuron

    (2003)
  • Y. Yang et al.

    p38 and JNK MAPK, but not ERK1/2 MAPK, play important role in colchicine-induced cortical neurons apoptosis

    Eur J Pharmacol

    (2007)
  • J.M. Zeitzer et al.

    The neurobiology of hypocretins (orexins), narcolepsy and related therapeutic interventions

    Trends Pharmacol Sci

    (2006)
  • L. Zhao et al.

    Endoplasmic reticulum stress in health and disease

    Curr Opin Cell Biol

    (2006)
  • A. Fontana et al.

    Narcolepsy: autoimmunity, effector T cell activation due to infection, or T cell independent, major histocompatibility complex class II induced neuronal loss?

    Brain

    (2010)
  • J. Hallmayer et al.

    Narcolepsy is strongly associated with the T-cell receptor alpha locus

    Nat Genet

    (2009)
  • K. Haze et al.

    Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress

    Mol Biol Cell

    (1999)
  • M. Høyer-Hansen et al.

    Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium

    Cell Death Differ

    (2007)
  • Y. Ishida et al.

    Autophagic elimination of misfolded procollagen aggregates in the endoplasmic reticulum as a means of cell protection

    Mol Biol Cell

    (2009)
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