The Journal of Neuroscience, February 6, 2008, 28(6):1452-1459; doi:10.1523/JNEUROSCI.3253-07.2008
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Neurobiology of Disease
Intron-3 Retention/Splicing Controls Neuronal Expression of Apolipoprotein E in the CNS
Qin Xu,1,2
David Walker,1
Aubrey Bernardo,1
Jens Brodbeck,1,2
Maureen E. Balestra,1 and
Yadong Huang1,2,3,4
1Gladstone Institute of Neurological Disease and 2Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158, and Departments of 3Pathology and 4Neurology, University of California, San Francisco, California 94143
Correspondence should be addressed to Dr. Yadong Huang, Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158. Email: yhuang{at}gladstone.ucsf.edu
Neuronal expression of apolipoprotein (apo) E4 may contribute to the pathogenesis of Alzheimer's disease (AD). In studying how apoE expression is regulated in neurons, we identified a splicing variant of apoE mRNA with intron-3 retention (apoE-I3). ApoE-I3 mRNA was detected in neuronal cell lines and primary neurons, but not in astrocytic cell lines or primary astrocytes, from humans and mice by reverse transcription (RT)-PCR. In both wild-type and human apoE knock-in mice, apoE-I3 was found predominantly in cortical and hippocampal neurons by in situ hybridization. Cell fractionation and quantitative RT-PCR revealed that over 98% of the apoE-I3 mRNA was retained in the nucleus without protein translation. In transfected primary neurons, apoE expression increased dramatically when intron-3 was deleted from a genomic DNA construct and decreased markedly when intron-3 was inserted into a cDNA construct, suggesting that intron-3 retention/splicing controls apoE expression in neurons. In response to excitotoxic challenge, the apoE-I3 mRNA was markedly increased in morphologically normal hippocampal neurons but reduced in degenerating hippocampal neurons in mice; apoE mRNA showed the opposite pattern. This apparent precursor–product relationship between apoE-I3 and apoE mRNA was supported by a transcriptional inhibition study. Thus, neuronal expression of apoE is controlled by transcription of apoE-I3 under normal conditions and by processing of apoE-I3 into mature apoE mRNA in response to injury.
Key words: apolipoprotein E; Alzheimer's disease; excitotoxicity; gene regulation; intron retention; knock-in mice
Received April 17, 2007;
revised Nov. 7, 2007;
accepted Dec. 15, 2007.
Correspondence should be addressed to Dr. Yadong Huang, Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158. Email: yhuang{at}gladstone.ucsf.edu