Volume 8 Supplement 1

Molecular Neurodegeneration: Basic biology and disease pathways

Open Access

Excitotoxicity increases the release of 24S-hydroxycholesterol via CYP46A1 activation

  • Alejandro Sodero1, 7,
  • Joris Vriens2,
  • Debapriya Ghosh2,
  • David Stegner3,
  • Anna Brachet4,
  • Marta Pallotto5,
  • Marco Sassoe-Pognetto5,
  • Jos Brouwers6,
  • Bernd Helms6,
  • Bernard Nieswandt3,
  • Thomas Voets2 and
  • Carlos Dotti1, 4
Molecular Neurodegeneration20138(Suppl 1):O32

https://doi.org/10.1186/1750-1326-8-S1-O32

Published: 13 September 2013

Background

Excitotoxicity, a common hallmark of different neurological disorders including Alzheimer’s disease (AD), is the consequence of exacerbated neuronal stimulation and leads to a high influx of calcium trough membrane glutamate receptors [1].

On the other hand, high levels of the cholesterol metabolite 24S-hydroxycholesterol (24-HC) have been found in cerebral spinal fluid (CSF) from AD patients [2, 3]. Brain cholesterol homeostasis is an essential, tightly-regulated process that ensures neuronal integrity, viability, and function. One of the mechanisms that neurons put to work to regulate the amount of cellular cholesterol is its conversion into the metabolite 24-HC by the 24-cholesterol hydroxylase CYP46A1 [4]. In this work, we explored the possibility that excitotoxicity, a common process that precedes neurodegeneration, could be a direct modulator of the 24-HC production via a CYP46A1-mediated hydroxylation of cholesterol.

Materials and methods

Cholesterol levels were measured by fluorimetric detection (Amplex Red Kit, Invitrogen) in the 100,000g membrane fraction of cultured hippocampal neurons and mouse hippocampal tissue, and in purified synaptosomes.

- 24-HC release was measured in the medium of cultured hippocampal neurons by LC/MS analysis.

- Cell surface biotinylation was performed using a membrane-impermeable biotin (EZ-link Sulfo-NHS-Biotin, Pierce), and cell surface CYP46A1 was detected by western immunoblotting using two different antibodies.

- STIM2 knockout mice were generated in the lab of Prof. Bernard. Nieswandt [5],

- Electron microscopy was performed in mouse hippocampal slices.

- TIRF microscopy was performed in HEK293T cells and hippocampal neurons expressing CYP46A1 fused to GFP.

- Intracellular calcium concentration was monitored in cultured hippocampal neurons using ratiometric Fura-2-based fluorimetry.

Results

We observed that excessive stimulation of glutamate receptors induces a significant loss of membrane cholesterol, which is paralleled by the release to the extracellular milieu of the metabolite 24-HC. Cholesterol loss was induced by depolarization of cultured hippocampal neurons with high potassium or stimulation of postsynaptic NMDA receptors. Importantly, purified synapses showed a similar reduction of this sterol after in vitro stimulation. Moreover, we observed a significant reduction in the content of cholesterol of hippocampal membranes of C57BL/6J mice treated with kainic acid for only 30 minutes (supra-epileptic condition). Consistent with a cause-effect relationship, knockdown of CYP46A1 prevented the glutamate-mediated cholesterol loss in cultured hippocampal neurons. Mechanistically, we found that the cholesterol reduction requires high levels of intracellular calcium, a functional Stromal Interaction Molecule 2 (STIM2) and mobilization of CYP46A1 from the endoplasmic reticulum, its natural sub-cellular compartment, towards the plasma membrane. Imaging studies with Fura-2 showed that the cholesterol loss is able to modulate the intracellular calcium response induced by depolarization.

Conclusions

This study underscores the key role of excitatory neurotransmission in the control of neuronal cholesterol content and suggests that excitotoxicity is one of the causes for the increased levels of 24-HC observed in the CSF of AD patients. Whether or not the observed cholesterol catabolism and the reduction in the magnitude of the calcium peaks that parallel excitotoxicity are part of a protective response to fight against injury remains elusive and merits further investigation.

Authors’ Affiliations

(1)
VIB Center for Biology of Disease, Katholieke Universiteit Leuven
(2)
Department of Molecular Cell Biology, Katholieke Universiteit Leuven
(3)
Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg
(4)
Centro de Biología Molecular Severo Ochoa, CSIC-UAM
(5)
Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin and National Institute of Neuroscience
(6)
Department of Biochemisty and Cell Biology, Faculty of Veterinary Medicine, University of Utrecht
(7)
Currently at Institute of Neuroscience, Université Catholique de Louvain

References

  1. Ong WY, Tanaka K, Dawe GS, Ittner LM, Farooqui AA: Slow excitotoxicity in Alzheimer’s disease. J Alzheimers Dis. 2013, 35 (4): 643-668.PubMedGoogle Scholar
  2. Papassotiropoulos A, Lütjohann D, Bagli M, Locatelli S, Jessen F, Buschfort R, Ptok U, Björkhem I, von Bergmann K, Heun R: 24S-hydroxycholesterol in cerebrospinal fluid is elevated in early stages of dementia. J Psychiatr Res. 2002, 36 (1): 27-32. 10.1016/S0022-3956(01)00050-4.View ArticlePubMedGoogle Scholar
  3. Schönknecht P, Lütjohann D, Pantel J, Bardenheuer H, Hartmann T, von Bergmann K, Beyreuther K, Schröder J: Cerebrospinal fluid 24S-hydroxycholesterol is increased in patients with Alzheimer’s disease compared to healthy controls. Neurosci Lett. 2002, 324 (1): 83-85. 10.1016/S0304-3940(02)00164-7.View ArticlePubMedGoogle Scholar
  4. Russell DW, Halford RW, Ramirez DM, Shah R, Kotti T: Cholesterol 24-hydroxylase: an enzyme of cholesterol turnover in the brain. Annu Rev Biochem. 2009, 78: 1017-1040. 10.1146/annurev.biochem.78.072407.103859.PubMed CentralView ArticlePubMedGoogle Scholar
  5. Berna-Erro A, Braun A, Kraft R, Kleinschnitz C, Schuhmann MK, Stegner D, Wultsch T, Eilers J, Meuth SG, Stoll G, Nieswandt B: STIM2 regulates capacitive Ca2+ entry in neurons and plays a key role in hypoxic neuronal cell death. Sci Signal. 2009, 2 (93): ra67-10.1126/scisignal.2000522.View ArticlePubMedGoogle Scholar

Copyright

© Sodero et al; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.