Gene expression fluctuates across the estrous cycle with anxiety-like behavior.




Devin Rocks

Introduction

This project investigated whether the expression of specific Kcnma1 transcript variants fluctuated across the estrous cycle in the ventral hippocampus of female mice. Kcnma1 encodes the alpha subunit of a large voltage-gated calcium-activated potassium channel. Of the 22 reported RefSeq Kcnma1 transcripts, 6 contain a 174bp STRess-axis regulated EXon (STREX). STREX codes for a 58 amino acid insert between domains S8 and S9 of the Kcnma1 protein (Fig. 1) resulting in a more excitable potassium channel [1,3]. Due to the role of potassium channels in the regulation of action potentials, upregulation of the expression of STREX-containing variants is associated with an overall increase in the excitability of the hippocampus [1,2]. Further, previous studies have shown that expression of STREX-containing variants in the uterine myometrium is regulated by sex hormone levels [4]. To determine whether sex hormone fluctuations are regulating the expression of STREX-containing variants in the ventral hippocampus, I designed RT-PCR primers that would amplify all Kcnma1 transcript variants, yielding different sized products depending on the transcripts amplified (Fig 2).

Materials and Methods

Female mice were sacrificed at various stages of the estrous cycle and their ventral hippocampus was dissected. Estrous cycle stage was determined by vaginal-smear cytology.

RT-PCR was performed on RNA isolated from the ventral hippocampus of female mice (2 animals per group, in duplicates) using primer sets specific to targeted genes (Kcnma1, CypA). RT-PCR results were visualized by gel electrophoresis. To confirm that the RT-PCR products were the expected products, bands from RT-PCR gel were purified and sent for sequencing analysis.

RT-PCR results using Kcnma1 primer set 2 were quantified by calculating the ratio of Kcnma1/CypA band intensity for each band (Amersham Imaging software) and normalized by setting the proestrus average relative intensity to 1. Statistical analysis was conducted in SPSS and involved One-way ANOVA (Tukey post-hoc test).

Discussion

Kcnma1 RT-PCR Results: Primer Set 1

RT-PCR performed with the first set of Kcnma1 primers revealed the presence of 4 distinct bands. Sequencing analysis confirmed that these bands corresponded to the expected RT-PCR products (Fig. 2). While the band corresponding to 313/322bp products appeared to have a similar intensity across groups, the top band corresponding to amplification of STREX-containing transcripts appeared to be lighter in metestrus compared to proestrus and diestrus.

Kcnma1 RT-PCR Results: Primer Set 2

In order to specifically target STREX-containing Kcnma1 transcript variants, a second set of primers was designed utilizing a reverse primer in the STREX exon. RT-PCR using this primer set was performed alongside a loading control(CypA) to determine whether the expression of STREX-containing variants in the ventral hippocampus was fluctuating across the estrous cycle. Results from this RT-PCR (Fig. 4, top left) suggest that relative expression of STREX-containing Kcnma1 transcripts is lower in metestrus compared to proestrus and diestrus, while the estrus results displayed some inconsistency between the two samples tested. RT-PCR results were quantified (Fig. 4, right) by the process outlined in Methods. Quantification showed a statistically significant increase in the relative expression of STREX-containing Kcnma1 transcripts in the diestrus phase when compared to estrus and metestrus, and a similar increase in the proestrus phase when compared to metestrus.

Conclusion

Together these results suggest that the fluctuation of sex hormones across the estrous cycle influences gene expression in the ventral hippocampus of female mice. Further, these changes in gene expression can be specific to a certain transcript or set of transcripts, suggesting a possible interplay between sex hormone levels and mRNA splicing machinery. Future work will investigate whether expression levels of STREX-containing Kcnma1 mRNA in the ventral hippocampus have an impact on behavioral phenotypes.

References

1. Ermolinsky BS, Skinner F, Garcia I, et al. “Upregulation of STREX splice variant of the large conductance Ca2+-activated potassium (BK) channel in a rat model of mesial temporal lobe epilepsy.” Neuroscience research. 2011;69(1):73-80. doi:10.1016/j.neures.2010.09.011.
2. Gu N, Vervaeke K, Storm JF. BK potassium channels facilitate high-frequency firing and cause early spike frequency adaptation in rat CA1 hippocampal pyramidal cells. J Physiol. 2007;580:859–882.
3. Chen L, Tian L, MacDonald SH, McClafferty H, Hammond MS, Huibant JM, Ruth P, Knaus HG, Shipston MJ. Functionally diverse complement of large conductance calcium- and voltage-activated potassium channel (BK) alpha-subunits generated from a single site of splicing. J Biol Chem. 2005;280:33599–33609.
4. Zhu N, Eghbali M, Helguera G, Song M, Stefani E, Toro L. Alternative splicing of Slo channel gene programmed by estrogen, progesterone and pregnancy. FEBS Lett. 2005;579:4856–4860.
5. Calderone V. Large-conductance, ca(2+)-activated k(+) channels: function, pharmacology and drugs. Curr Med Chem. 2002;9:1385–1395.

Figures


Figure 1-Kcnma1 protein with the location of the STREX insert.


Figure 2-Table depicting the 4 expected RT-PCR products using the first set of Kcnma1 primers. The STREX-containing variants and the corresponding RT-PCR product are highlighted in red.


Figure 3-RT-PCR results using primers amplifying all Kcnma1 transcripts. Arrows indicate bands with sizes corresponding to the expected products outlined in Figure 2.


Figure 4-RT-PCR results using primers targeting STREX-containing Kcnma1 transcripts (top left), loading control CypA (bottom left), and quantification of results (right). (* = p < 0.05)


Previous work in the Kundakovic lab has demonstrated that the hormonal fluctuations characterizing the estrous cycle are associated with differential gene expression and chromatin accessibility in the ventral hippocampus of female mice. This project aimed to evaluate whether the relative expression of specific mRNA transcript variants fluctuate across the estrous cycle in a hormone-state dependent manner. The target gene, Kcnma1, encodes the alpha subunit of a large voltage-gated calcium-activated potassium channel. Potassium channels function in regulating neuronal excitability through their role in action potentials [5]. Kcnma1 has several transcript variants coding for isoforms with different functional properties [1]. Although it has been shown that certain Kcnma1 transcripts are upregulated in response to sex hormones [4], the influence of sex hormone fluctuations on Kcnma1 transcription in the brain has yet to be explored. Here I show differential expression of specific Kcnma1 transcripts at various estrous cycle stages in the ventral hippocampus of female mice.

Full Paper

Acknowledgments

Thanks to Dr. Kundakovic and her lab for the mice used in this experiment and for guidance in estrous cycle stage determination, dissection of the ventral hippocampus, and RNA isolation. Thanks to Tony and Faaria for their enormous help in the molecular procedures performed in this project. Last, thanks to Dr. Rubin whose guidance and patience made this project possible.


This document was last modified 05/15/2018.
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