WP6: Alternative splicing modulations under imbalanced cholinergic signaling: from spliceosome components to target transcripts

Scientific team: Sophie Diamant, Shani Ben-Arie, Debra Toiber, Amit Berson, Inbal Mor, Tama Evron, Ari Meerson, Boris Byrk, Hermona Soreq

Objectives: To determine alternative splicing contributions to gene-environment interactions, delineate dynamic changes in spliceosome components and develop approaches for retrieving balanced splicing in mouse models with conditionally manipulated cholinergic balance.

Description of the work: Rapid, yet long-lasting alternative splicing changes occur in stress and anxiety . Acetylcholine modulates several different neurotransmission circuits, and the pre-mRNA of the acetylcholine hydrolyzing enzyme acetylcholinesterase (AChE) is subject to splice variations at both its 5’ and 3’ ends. To explore the implications of such variations, we designed and tested an in-house cDNA spotted microarray for over a 100 components of the mammalian spliceosome. Preliminary analyses demonstrate that this microarray is sensitive enough to detect significant stress-induced changes in spliceosome components in the mouse striatum. The impact of cholinergic signaling on alternative splicing will be tested in a novel mouse strain with tetracycline-controlled antisense suppression of the stress-induced AChE-R variant, along the following specific aims:

1. To explore and characterize expression changes in spliceosome components under environmental, psychological and disease stress;

2. To investigate how such stress-induced changes influence alternative splicing of target transcripts associated with cholinergic neurotransmission;

3. To search for causal association(s) between such splicing variations and resultant phenotype by manipulating the splicing events and following the resultant physiology;

4. To test the impact of cholinergic imbalance on alternative splicing and vice versa.
   
   Aim 1: We will test the effects of environmental (e.g. exposure to anticholinesterases), psychological (e.g. confined swim stress) or disease stress (e.g. exposure to bacterial lipopolysaccharide) on the expression levels of spliceosome components in various cells, tissues and brain regions. Bioinformatics analysis of the spliceosome components and their validated target sequences will be performed in WP1. We will also test the effects of the splicing manipulations outlined under WP2-5 on the feedback reaction of spliceosome components in cell culture systems. MALDI-TOF analysis (WP4) of the corresponding proteins will complement this analysis.
   Aim 2: RNA will be extracted from experimental animals with constitutively or conditionally manipulated cholinergic neurotransmission (e.g. by over- or under-expression of AChE, or under exposure to anticholinesterases or stress). Validation will involve real-time PCR, in situ hybridization and immunohistochemistry.
   Aim 3: To identify functionally relevant secondary and tertiary RNA structures, we shall develop a fluorescence polarization assay measuring synthetic RNA interactions with recombinant splicing factors and combine the use of spliceosome microarrays with the emerging animal models.
   Aim 4: Association of spliceosome composition with specific physiological effects (WP3) will be challenged by manipulating stress-modified spliceosome transcripts using antisense or siRNA tools and measuring long-term potentiation . Finally, spliceosome composition will be tested in Alzheimer’s disease brains from the Amsterdam Brain Bank.

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Nijholt, I., et al., Stress-induced alternative splicing of acetylcholinesterase results in enhanced fear memory and long-term potentiation. Mol Psychiatry, 2004. 9(2): p. 174-83.

Brenner, T., et al., The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis. Faseb J, 2003. 17(2): p. 214-22.

Meshorer, E., et al., Alternative splicing and neuritic mRNA translocation under long-term neuronal hypersensitivity. Science, 2002. 295(5554): p. 508-12.

Kaufer, D., et al., Acute stress facilitates long-lasting changes in cholinergic gene expression. Nature, 1998. 393(6683): p. 373-7. Accompanied by: News & Views: Sapolsky, R.M., The stress of Gulf War syndrome. Nature, 1998. 393(6683): p. 308-9