Estrogen and BDNF Interactions

Estrogen and BDNF Interaction Pathways
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A: Induction; B: Convergence [1]

Brain-derived neurotrophic factor (BDNF) and estrogen are both important neuromodulators of synaptic plasticity which have been thoroughly studied individually. They have been shown to induce structural changes to and within neurons, as well as promote neuronal proliferation and differentiation within the hippocampus. Interestingly, they were found to be complimentarily expressed and to trigger similar cascading pathways, second messenger systems and have genomic effects in the hippocampus leading to dendritic growth. [1] These commonalities have led scientists to look further into estrogen-BDNF interactions. Possible direct and convergent pathways have been identified to address this link. An estrogen response element (ERE) identified on BDNF may serve to directly trigger its expression when estrogen bound; conversely, these two neuromodulators may work in concert to trigger the same intracellular transcription factors which eventually increase dendritic growth.[2] These dendritic changes equate to improvements in memory and learning tasks which represents the ultimate effect of estrogen and BDNF on individuals.

1. Relationship Origins

1.1 Common signalling pathways

A major factor which led to studies of estrogen and BDNF in concert was common signalling pathways within the hippocampus; an area especially important for memory formation and learning. They both interact with a number of common receptors, enzymes and proteins such as MAP kinase, ERKs, PI3 kinase, CaMKII, CREB and Src/Fyn. [2] Numerous studies have documented these interactions with BDNF and estrogen hinting at possible converging pathways between the two neuromodulators. Of particular interest is their interaction with CREB; a protein which is intricately linked with memory formation and learning and is activated downstream of the MAP kinases, ERK’s, PI3 kinases, CaMKII and Src/Fyn.

Estrogen and BDNF Interaction Pathways
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A: Convergence; B:Induction; Common signalling pathways and proteins triggered by estrogen and BDNF and downstream effects.[2]

The link between estrogen and CREB is established via a signaling cascade; estrogen does not directly enhance it. Generally, it involves estrogen binding a receptor which leads to the release of calcium which subsequently leads to the release of CREB .[3] Therefore, estrogen treatment has the ability to increase levels of CREB-positive neurons and induce its expression in cells. This specifically applies to areas within the hippocampus. Studies have shown that estrogen treated ovariectomized (OVX) rats that previously had lower levels of CREB production due to hypoglycemia, were able to increase and maintain their levels with estrogen treatment. [4] Estrogen is also able to significantly increase the levels of CREB in otherwise normal OVX rats. [4] Moreover, not only does estrogen have a rescuing effect on the levels of CREB in the hippocampus when estrogen is deplete but it also has an enhancing effect on CREB stores when there is existing estrogen.

Various intermediates regulate the pathway between estrogen and CREB. CaMKII is one such intermediate. Estrogen increases levels of CaMKII in a time and dose dependent manner. Optimal estrogen dosage to increase CaMKII was 0.1 mg/kg and maximum effects are seen after one hour of treatment, with a gradual decline after that time point. [3] Estrogen also activates MAP kinase and ERK2 via Src tyrosine kinase. When levels of estrogen are elevated there is greater activation of this pathway. In these models estrogen replacement of OVX rats is also able to restore normal functioning of this pathway. [5]

Similar to estrogen, BDNF triggers a calcium based signalling cascade with leads to the activation of CREB. Studies show that the inhibition of BDNF in the hippocampus leads to significantly lower levels of CREB whereas infusion of BDNF significantly increased CREB levels. [6] BDNF also triggers the CaMKII signalling pathway leading to the phosphorylation of a regulatory sequence on the CREB gene. [7] MAPKAPK-2, which is regulated by a MAP kinase, is another intermediary between CaMKII and CREB. In addition, ERK1 AND 2 levels are
significantly elevated with BDNF treatment in the hippocampus and ERK2 is decreased when BDNF is blocked. [6]

Clearly BDNF and estrogen share numerous downstream signalling pathways and affect the expression of intermediate proteins in these pathways. Their influence on these particular signalling pathways is especially important because they both lead to the activation of CREB which is needed for neuronal and synaptic enhancement and subsequently learning and memory.

1.2 Effects on Dendritic Spines

The formation, growth and pruning of dendritic spines are all necessary to facilitate learning and memory. Studies have shown that both estrogen and BDNF play a role in the maintenance of these dendritic spines. An increase in spine density occurs when either BDNF or estrogen levels are modified. Studies show that the ovariectomy or elimination of endogenous estrogen in rats decreases spine density in brain areas such as the hippocampus, prefrontal cortex and somatosensory cortex. These studies also found that treatment with estrogen subsequently increases the number and density of spines in comparison to controls which did not receive estrogen. [8] Similarly, studies using CaMK-BNDF knockouts show the ongoing necessity of BDNF to maintain spine density in adult mice. Mice that contained the CaMK-BNDF knockout showed a decrease in spine density in comparison to wild type mice. There was also a progressive decrease in BDNF levels as the mice matured. They had a 29% reduction in spine density at the 12 week mark in comparison to levels at the 5 week mark. [9]

Effects on Dendritic Spines
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A/C: typical dendritic structure in wild type mice;
B/D: CaMK-BNDF KO mice have reduced dendritic structure compared to wild type;
E-F: significant decrease in number of spines between 5 and 12 weeks [9]

Morphological traits of spines are also affected by BDNF and estrogen. When particular versions of BDNF are mutated or knocked out the dendritic spines adopt thinner and denser configurations which are both characteristics of immature spines. Without BDNF to regulate spine morphology the spine heads do not grow and the synapses are not properly pruned, both of which are important to a mature spine. Estrogen treatment on OVX mice is also accompanied by morphological changes such as an increased proportion of mushroom shaped, thin, double headed and stubby dendritc spines. Interestingly, the estrogen treated spines of OVX subjects had a greater proportion of the mushroom-shaped spines than thin spines whereas the OVX control subjects had a greater proportion of thin spines. [8] The growth of a mushroom spine is an increase in the size of the spine head, the same change which was seen with BDNF treatment. In addition, the OVX control mice having a greater proportion of thinner spines is the sign of an immature spine. exactly what was seen when BDNF was inhibited.

2. Possible Links:

2.1 Genomic Interaction: ERE (estrogen response element) on BDNF

Using a computerized gene homology program, a gene sequence which closely resembles the typical estrogen response element was identified on BDNF. The standard estrogen response element and the one found on BDNF differ by one base pair mismatch and the BDNF ERE is divided into two pentamers separated by a 9 nucleotide spacer in comparison to the 3 nucleotide spacer usually seen. [10] Gel shift techniques were used to determine if there was an interaction between the proposed estrogen response element sequence and an estrogen ligand-receptor complex. The complex successfully bound to the ERE and had a protective effect, inhibiting the cleaving function of DNase. [9] Therefore, this ERE provides estrogen with a way to regulate the genomic production of BDNF by initiating and protecting it.

A genomic effect means that there will be an alteration to the genome in terms of the rate of gene transcription, in this case the transcription of BDNF RNA. Studies have shown the up-regulation of BDNF mRNA levels in the hippocampus following estrogen treatment exemplifying the genomic effects of estrogen on BDNF. Researchers had a control group of non-gonadectomised rats and two treatment groups; one group of gonadectomised rats was treated with vehicle and the other was given estrogen at postnatal day one. The gonadectomised rats that were given the vehicle had a significant decrease in BDNF mRNA levels. Those given the estrogen replacement had levels similar to control rats. [11] In addition, there was a linear increase in BDNF mRNA levels with increased does of estrogen. Other studies have found similar results that mRNA levels of BDNF increase with estrogen treatment most dramatically in areas within the hippocampus. [12]

Though there are clear genomic effects of estrogen on BDNF, there are certain scientific results pertaining to estrogen and BDNF interaction that cannot be explained using this mechanism. For this reason scientists began to look for alternate interaction mechanisms including non-genomic factors which can be attributed to the differing study results.

2.2 Non-genomic Interaction: Induction and Convergence

GABAergic interneurons and Granule Cell (Pyramidal Cell) Signalling
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The estrogen will bind estrogen receptors on GABAergic cells and uninhibit the granule cell leading to BDNF release [18]

An alternate interaction pathway between BDNF and estrogen involves GABAergic interneurons. Numerous estrogen receptors are found on the surface of these interneurons. The interneurons in turn are found in close proximity with granule cells which are the BDNF producing cells in the hippocampus. Therefore, when estrogen binds the GABAergic interneurons it suppresses their inhibitory effect on the granule cells allowing them to increase their production of BDNF. [2] This mechanism is plausible because multiple studies have shown that BDNF levels can be increased with neuronal activity alone. [13] Therefore, when the inhibitory effect of the GABAergic interneurons is removed the granule cells can be stimulated by other neurons to produce BDNF. In normal functioning cells estrogen takes the place of the artificial neuronal stimulation, inhibiting the GABAergic interneurons so BDNF is produced.

Rather than induction of BDNF by estrogen, there may be a convergent pathway which links these two neuromodulators. As stated above in section 1.1 Common Signalling Pathways, they both effect a number of common enzymes, receptors and proteins leading to increased CREB transcription and ultimately an enhancement in memory and learning. The pathway begins with BDNF and estrogen binding to their receptors, TrkB and ER respectively, which then trigger a signalling cascade. Both of these receptors trigger kinase intracellular cascades such as MAPK/ERK, PI3 kinase, CaMKII. These cascades eventually lead to the activation of CREB which triggers transcription factors to initiate transcription. [1] This then leads to a positive feedback loop in which more BDNF is transcribed. Therefore, BDNF and estrogen trigger parallel pathways which lead to the eventual increase of BDNF.

3. Ultimate Application of the Estrogen-BDNF Interaction:

3.1 Effect on memory and learning

Numerous studies have documented the memory and learning benefits of estrogen and BDNF. Usually researchers will use spatial memory tests to assess both learning and memory in animal models. The animals are forced to make associations between cues in the environment and some desired outcome and remember these associations in subsequent trials. An example would be the Morris Water Maze (MWM); animals must use visual cues to locate a platform which is underwater after an initial trial run. Ovariectomized rats were either treated with estrogen or left as controls and then trained on the MWM. The estrogen treated OVX rats spent significantly more time in the quadrant where the platform had been hidden than the controls. [14] This suggests that they were better able to use the visual cues to identify where the platform would be (learning) and then remember that that is where it would be found in successive trials (memory). In addition, the estrogen treated rats all had increased levels of BDNF. [14]

Estrogen Increased BDNF levels
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Estrogen significantly increases the levels of BDNF in all age
categories in comparison to the control and treatment groups [14]

In a object recognition test with similar OVX treatment and control groups as stated above, OVX plus estrogen rats had higher levels of BDNF compared to control. These rats also spent significantly more time exploring new areas and objects than control mice. Rats are innately curious creatures and those treated with estrogen remembered encountering the old places and objects from previous trials so they spent more time with the new objects and places. [15] Even after several days, the estrogen replaced rats recognized the old from the new judging by which they spent more time with. In addition, increasing the estrogen dose or number of estrogen treatments further enhanced the levels of BDNF and results in the object recognition test.

Effects on Learning and Memory
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Rats spend significantly more time exploring new objects and places
in the object placement and object recognition tests [1]

Their effect on learning and memory can also be assessed by looking at rats performance on these tests at different stages of the estrous cycle. During late diestrus and proestrus when estrogen levels are at their highest, more hippocampal BDNF is stimulated and rats are able to more easily distinguish between old and new objects in object recognition tests. [16] Therefore they can identify the ones which they have already encountered much more easily. Similar results are seen during other spatial memory tests when estrogen levels are at their peak. Moreover, the phases in the estrus cycle when estrogen and BDNF are highest are also the points at which dendritic spine density is at its peak in the hippocampal regions. This would explain the enhancements in memory. Doses of estrogen which are able to increase dendritic spine density will show improvements in memory and learning. [17]

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