Excitotoxicity increases the release of 24S-hydroxycholesterol via CYP46A1 activation
Molecular Neurodegeneration volume 8, Article number: O32 (2013)
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 .
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 . 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 ,
- 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.
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.
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.
Ong WY, Tanaka K, Dawe GS, Ittner LM, Farooqui AA: Slow excitotoxicity in Alzheimer’s disease. J Alzheimers Dis. 2013, 35 (4): 643-668.
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.
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.
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.
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.
About this article
Cite this article
Sodero, A., Vriens, J., Ghosh, D. et al. Excitotoxicity increases the release of 24S-hydroxycholesterol via CYP46A1 activation. Mol Neurodegeneration 8 (Suppl 1), O32 (2013). https://doi.org/10.1186/1750-1326-8-S1-O32
- Hippocampal Neuron
- Cerebral Spinal Fluid
- Hippocampal Membrane
- Cell Surface Biotinylation