HMB360 Padlet: Exploring Beta-Catenin and its link to Depression and Anxiety by Kylie Kalpakis

Targeting Beta-Catenin in GLAST-Expressing Cells: Impact on Anxiety and Depression-Related Behaviour and Hippocampal Proliferation

Kyliekal — 2020-11-28 16:53:37 UTC

METHODS AND TECHNIQUES

Kyliekal — 2020-11-28 17:06:18 UTC

Subjects: Transgenic Mice

The animal models in this experiment underwent a series of behavioural and histochemical analysis to examine the effects of beta-catenin expression on anxiety and depression-like behaviour

Generating Transgenic Mice

Two transgenic mouse models were generated in which the activity of beta-catenin was either inactivated or stabilized in the hippocampus
The GLAST-Cre mouse line was crossed with mice who carry a gene that either inactivates or stabilizes beta-catenin

 cKO mice (knockout mice, inactivate beta-catenin)

GLAST-Cre x Ctnnb1^flox/flox

cST mice (stabilizing mice, stabilize beta-catenin)

GLAST-Cre x Ctnnb1^(ex3)Fl/Fl

Wild-type mice were also included in this study as a control (do not carry genes that inactivate or stabilize beta-catenin)

INTRODUCTION

Kyliekal — 2020-11-28 17:06:39 UTC

EXPERIMENTAL DESIGN

Kyliekal — 2020-11-28 18:07:07 UTC

This experiment was split into two groups: Group A and Group B

Group A: cKO/cST Phenotype Induction

This group consists of both knockout and stabilizing mice, as well as their respective wild-types for experimental controls
These mice underwent treatment with either tamoxifen or corn oil
Tamoxifen is a drug that induces either the inactive or stabilized phenotype
Following induction of phenotype, the mice were subject to either immunohistochemical analyses (Figure 1, set 1), or a series of behavioural tests (Figure 1, set 2)

Group B: Chronic Corticosterone Model in cST Mice

This group only contained the stabilizing and wild-type mice, not the knockout
These mice were subject to the corticosterone depression model in which doses of this hormone were administered to the animal over time
Corticosterone is a stress hormone, in which when administered to the mice creates an animal model of depression
From this, the researchers could determine the influence of beta-catenin in this mouse depression model
After treatment, these mice were analyzed in a different subset of behavioural tests (Figure 1, set 3)

Image from Vidal et. al (2018) - Figure 1

BEHAVIOURAL ASSAYS

Kyliekal — 2020-11-28 19:23:34 UTC

A number of behavioural assays were used to compare any changes in behaviour associated with beta-catenin inactivation or stabilization in these mice.

These behavioural tests induce stress upon the mice in the study in order to gain a better understanding of any patterns associated with depression-like behaviour correlated to beta-catenin expression in these models.

The types of behavioural tests used in this study are as follows:

Open-Field Test (OF)
Light/Dark Box Test (LBD)
Novelty Suppressed Feeding (NSF)
Sucrose Preference Test (SP)
Forced Swimming Test (FST)

All of these tests inflict the mice with different stressors in order to assess whether they exhibit normal behaviour or anxiety and depression-like behaviours in response to encountering the stressor.

The mice in Group A were subject to all five of the behavioural assays listed above, whereas Group B was only subject to a subset of these assays, including the Novelty Suppressed Feeding, Sucrose Preference and the Forced Swimming Test.

IMMUNOHISTOCHEMICAL ANALYSES

Kyliekal — 2020-11-28 19:39:50 UTC

Studying immunohistochemistry in these models is important for understanding the relationship between beta-catenin expression and hippocampal proliferation.

They used a number of markers in attempt to visualize the pattern of hippocampal proliferation in these mouse models.

Marker #1: beta-catenin +

immunolabelling of the beta-catenin positive cells illustrates their abundance in the hippocampus, providing a clear comparison between the two models

Marker #2 and #3: Ki-67 and Brd-U

the Ki-67 and Brd-U assays are both used as a marker for proliferation, and here they are particularly looking at the hippocampus

Marker #4: doublecortin (dcx)

doublecortin is used as a marker for neurogenesis in these mouse models

Image from Vidal et. al (2018) - Figure 5

This image is illustrating the immunohistochemical analyses from the various markers used in this study. Each row is representative of the results from a different marker. The left column shows the marker in wild-type mice, whereas the images on the right show that same marker for cKO mice. Parts A and B show the marker Ki-67, parts C and D show the BrdU staining, and parts E and F show the marker for doublecortin .

WHAT IS BETA-CATENIN?

hollyjiogo — 2020-11-28 21:34:58 UTC

Beta-catenin is a highly conserved dual function protein

Function 1= involved in inter-cellular adhesion
- Acts as anchor for cadherin, which is subsequently linked to the actin cytoskeleton of cell via Alpha-catenin, binding 2 cells together (3)
- Also affects synaptic stability between hippocampal neurons (linked to emotional memory) and neurons within the amygdala (also linked to emotional memories) (2)
Function 2= acts as a transriptional activator in the canonical Wnt signalling pathways
Beta-catenin is crucial for the formation of memory via neural plasticity (3)

Want to know more?

In the mammalian cell, it can be found in the prefrontal cortex, the dorsal hippocampus, the nucleus accumbens, the basal amygdala and the hypothalamus (2)
Overexpression can cause neural out-growth and decrease of differentiation (2)
Premature inhibition can cause progenitor neurons to exit cell cycle and increase differentiation (2)

Introducing the star of the article: BETA-CATENIN!

hollyjiogo — 2020-11-28 21:41:38 UTC

Beta-catenin's function is dependent on which of its partners bind to its armadillo (ARM) domain.
Wikiwand/B-catenin. Accessed from https://www.wikiwand.com/en/Beta-catenin

WNT/BETA-CATENIN SIGNALLIG PATHWAY (2)

hollyjiogo — 2020-11-28 21:47:26 UTC

B-catenin is involved in 1 of 3 Wnt pathways, the canonical Wnt signalling pathway

So what does Beta-catenin activity look like ?
1. Wnt proteins bind to Frizzled transmembrane (co-) receptors low density lipoprotein related protein 5/6 (LRP5/6)-> Wnt pathway is now active
2. Destruction complex (degrades B-catenin in absence of Wnt activation) is bound to activated Frizzled receptors
3. Phosphorylated Beta-catenin is no longer ubiquitinated/phosphorylated by B-transducin repeats containing proteins (B-TrCP)/ glycogen serine kinase 3-beta (GSK3b)
4. B-catenin allowed to increase in levels to saturate the cytoplasm
5. B-catenin translocates into the nucleus, acts as a coactivator and is associated with TCF/LEF1 (multifunctional transcription factors)
6. You get transcription of Wnt pathway target genes, depending on what isoforms of TCF/LEF1 bind to B-catenin

hollyjiogo — 2020-11-28 21:53:53 UTC

If the explanation of the Wnt signalling pathway left you with questions, watch this video. If not, why don't you find out the main aim of the article?
HusseinBiology (2018). Wnt/B-Catenin Signaling Pathway [Video]

WNT/BETA-CATENIN AND DEPRESSION

hollyjiogo — 2020-11-29 00:59:50 UTC

Brain samples of patients with MDD are found to exhibit dysregulation of Wnt signalling pathway and alterations of beta-catenin levels (1)
The inhibition of glycogen synthase kinase 3-beta (GSK3b) via phosphorylation rescues depressive like behaviour (1)
- In resting state, it phosphorylates B-catenin, causing its degradation
Overexpression of beta-catenin has similar effects, rescuing depressive like behaviour (1)
Lowered beta-catenin protein levels in nucleus even when mRNA levels remain the same (2)
- Depression may be linked to alterations in the functionality of the beta-catenin peptide in the nucleus causing a reduction in a viable detectable beta-catenin product
Key role of beta-catenin in relation to depression may be linked to Dicer1 (1)
- B-catenin target gene
- Codes for Dicer protein, which is involved in cleaving and formation of miRNA
- Different miRNA play a role in stress adaptation
- Lower overall mRNA linked to lower activation of prefrontal cortex (linked to cognitive behaviour, personality expression, decision making, and social behaviour)
- Upregulation of miR016 miRNA in raphe nuclei and hippocampus has been found to induce depressive behaviour
  - miRNA may act on untranslated regions of genes and the serotonin transporter

Supporting Literature: Beta-catenin in Post-mortem brains (1)

hollyjiogo — 2020-11-29 01:03:50 UTC

GSK3B is associated with a protein complex to phosphorylate and degrade Beta-catenin

Accumulation of Beta-catenin is considered a marker for in-vivo inhibition of GSK3 -> ratio between active and inhibited form of GSK3B is an important factor in the accumulation of Beta-catenin

This study looked at postmortem brains of 20 depressed and non depressed subjects, and quantified Beta-catenin, total GSK3b, ser9-pGSK3b (inhibited), and activated GSK3b levels in the prefrontal cortex

Beta-catenin: decreased in depressed patients
Inhibited GSK3b: decreased in depressed patients
Ratio of total to activated GSK3b was increased (inverse correlation between active GSK3b and Beta-catenin levels)

Beta-catenin can serve as a marker for antidepressant behaviour provoked by the GSK3b inhibition

Supporting Literature: Beta-catenin links to anxiety-like behaviour (4)

hollyjiogo — 2020-11-29 15:18:07 UTC

There is potential involvement of hippocampal neurogenesis in modulating chronic pain (which can lead to anxiety)

Mice suffering persistent neuropathic pain show a reduction of active neural stem cells in the ventral senate gyrus, then the decrease of neurogenesis and the appearance of anxiety like behaviour
Wnt/Beta-catenin signaling, which is necessary for sustaining neural stem cells was suppressed
Depleting Beta-catenin by Nestin-Cre reduces the number of active neural stem cells and makes anxiety development easier

hollyjiogo — 2020-11-29 15:22:30 UTC

Depression not only affects individuals and their loved ones, it has the potential to impact the economy! Check out this short video to learn more:
CBC News (2016). Depression, anxiety cost Canadian economy billions {Video].

hollyjiogo — 2020-11-29 15:24:30 UTC

Here are some Canadian, depression related facts. It's more common than you think, making it a topic of research with a lot of potential benefits!
Walk Along (2010) Major Depressive Disorder (Depressio) [Infographic]. Accessed from https://www.walkalong.ca/explore/encyclopedia/major-depressive-disorder-depression

What is Depression?

hollyjiogo — 2020-11-29 15:28:16 UTC

Depression is a mood disorder that results in persistent feelings of sadness or helplessness, and a loss of interest in life and its daily activities (6).

What's the difference between sadness and depression (6)?

Sadness is a normal reaction associated with disappointment, frustration, a difficult problem etc
Depression is a more intense feeling, affecting the way one views themselves and those around them, and affecting the way they go about their lives
Sadness comes in waves, while depression is a persistent feeling that often times cannot be sourced to one event

Depression cannot be completely cured, but symptoms can be alleviated through different treatments (2).

One of these is the use of antidepressants, which usually act by balancing neurotransmitters in the brain, but can have a wide array of mechanisms of action.
In this study we are using GLAST cells, which are glutamate transporters. Glutamate is a neurotransmitter often found to be less expressed in depressed individuals -> does Beta-catenin have a relation to antidepressants?

Depression is also often co-morbid with anxiety, which is a disorder in which people feel intense and persistent fear or worry over everyday activities (2).

This article will look at both depression and anxiety related behaviours

AIM OF THE STUDY: What do researchers want to know?

hollyjiogo — 2020-11-29 15:41:06 UTC

1. Researchers accept the idea that adult born neurons contribute to the efficacy of antidepressants (1)
2. Many studies have shown that changes in Beta-catenin expression levels are linked to depression and anxiety related behaviours (1)
3. Researchers also accept the fact that Beta-catenin plays a key role in the balance between neural progenitor cell expansion and differentiation during brain development (2)

What is still unclear is the role of Beta-catenin in the development of new neurons in the adult hippocampus, and of course its behavioural applications.

Therefore, the main hypothesis or big question of this paper is:
What is the implication of Beta-catenin in adult hippocampal proliferation, and consequently its implications in the manifestations of depression/anxiety related behaviours?

Understanding this relationship has major implications on being able to develop more efficacious treatments.

Conclusions

aryanjalali — 2020-12-05 17:23:59 UTC

Significance
The results of this paper speak to the importance of B-catenin in modulating depression. Better understanding of the role that B-catenin plays in depression may provide insights into possible therapies for depressed humans that involve controlling B-catenin. More knowledge of B-catenin and its role in depression may also be relevant when trying to understand how current anti-depressants function and what these drugs change to alleviate depressive symptoms.

Limitations
The conclusions made in this paper are made a lot weaker due to the low sample size. Each separate experimental group contained between 3 and 10 mice. Such a low sample size leads to issues of chance skewing the results. There is also the obvious limitation of using mice models which is that the results may not necessarily extend to humans.

Another potential issue is with regards to how well the transgenic mice lines were created. The knockout mice still had traces of B-catenin positive cells in their hippocampus while the stabilized mice had both wild type beta catenin and the stabilized version of beta catenin. Questions about how well the transgenic mice lines were created may weaken the results from this paper.

Future directions
Future studies may choose to further investigate the molecular mechanisms that facilitate link between lack of B-catenin and depression. Although this study found neuronal growth to be a factor, this one mechanism is not enough to fully explain the behavioral differences between the cST and cKO mice (as admitted by the researchers in this study). Further investigation of the mechanism behind B-catenin may provide further insight into how depression affects the brain and how to treat it.

Another possible study may involve the clinical significance of B-catenin. Small clinical trials involving treating depressed humans with B-catenin may prove to be useful for alleviating depressive symptoms. If such a study proves successful, development of a new drug for depressed individuals would provide doctors with a new tool for treating depressed people.

Discussion

aryanjalali — 2020-12-05 17:27:46 UTC

Results summary
The results of the paper by Vidal et al. all point towards the importance of B-catenin in modulating depressive behavior. Knocking out B-catenin lead to increased depressive symptoms compared to the WT mice as measured by the LDB, NFS, SP, FST, and immunostaining test. On the other hand, stabilizing B-catenin lead to increased resistance to depression as measured by the NFS, FST, and SP test. DCX immunostaining also showed decreased DCX in cST mice compared to WT mice after depression was induced, presumably since DCX is a marker for immature neurons and neuronal precursor cells and there was more neurogenesis and differentiation into adult neurons in the cST mice.

One of the major mechanisms modulating this difference was thought to be related to the role of B-catenin in controlling neuron growth. cKO mice had more immature neurons with fewer proliferation markers, less hippocampal neural proliferation, and abnormal dendrite morphology compared to WT mice. On the other hand, cST mice were found to have normal neuron morphology with neurogenesis actually increasing compared to WT.

Strengths of this paper
The authors of this paper used a variety of behavioral tests to measure the effects of modulating B-catenin in mice. Almost all these tests showed consistent results in that they all suggested B-catenin stabilization lead to resilience to depression and a reduction in depressive symptoms while B-catenin knockout lead to increased depressive symptoms. We think that the results of this paper is strengthened by the consistent conclusions drawn from a variety of tests.

Comparison to previous literature
In general, the results of this paper align with previous literature. Previous work on mice has also found a negative relationship between B-catenin and depressive symptoms⁶. One study found that intentionally inducing stress in mice reduced the B-catenin levels found in their brain by up to 50%⁶. In addition, post-mortem analysis of human brains has found significantly less B-catenin in the brains of depressed individuals compared to healthy ones⁷.

Acknowledgements

hollyjiogo — 2020-12-05 17:28:31 UTC

Introduction and Hypothesis: Holly Jiogo
Methods and Techniques: Kylie Kalpakis
Results: Daphne Gonnet
Discussion and conclusion: Aryan Jalali

References

hollyjiogo — 2020-12-05 17:33:55 UTC

1. Karege F, Perroud N, Burkhardt S, Fernandez R, Ballmann E, La Harpe R, Malafosse A. Protein levels of β-catenin and activation state of glycogen synthase kinase-3β in major depression. A study with postmortem prefrontal cortex. J Affect Disord. 2012 Jan;136(1-2):185-188.
2. Mostany R, Valdizán EM, Pazos A. A role for nuclear beta-catenin in SNRI antidepressant-induced hippocampal cell proliferation. Neuropharmacology. 2008 Jul;55(1):18-26.
3. Vidal R., Garro-Martínez E, Díaz Á, Castro E, Florensa-Zanuy E, Taketo M. M, Pilar-Cuéllar F. (2018). Targeting β-Catenin in GLAST-Expressing Cells: Impact on Anxiety and Depression-Related Behavior and Hippocampal Proliferation. Molecular Neurobiology, 56(1), 553-566.
4. Youyi Z, Li Z, Wang M, Yu J, Yang J, Liu A, Yao H, Liu X, Shen Y, Guo B, Wang Y, Wu S. Anxiety Specific Response and Contribution of Active Hippocampal Neural Stem Cells to Chronic Pain Through Wnt/β-Catenin Signaling in Mice. Frontiers in Molecular Neuroscience 2018; 11:296.
5. National Institute of Mental Health (2013). Major Depression. Accessed from www.nimh.nih.gov/health/statistics/prevalence/major-depression-among-adults.shtml
6. Dai J, Pan JY, Liao N, Shi J, Zeng Q, Huang L, Chen LP. Influence of miR-155 on behaviors of depression mice through regulating Wnt/β-catenin signaling pathway. Eur Rev Med Pharmacol Sci. 2020 Feb;24(3):1398-1407.
7. Karege F, Perroud N, Burkhardt S, Fernandez R, Ballmann E, La Harpe R, Malafosse A. Protein levels of β-catenin and activation state of glycogen synthase kinase-3β in major depression. A study with postmortem prefrontal cortex. J Affect Disord. 2012 Jan;136(1-2):185-188.

Keep scrolling down for more on Beta-Catenin and Depression!

hollyjiogo — 2020-12-05 18:14:18 UTC

RESULT #1: BETA-CATENIN MODULATES ANXIETY-LIKE BEHAVIOUR

daphnegonnet — 2020-12-05 21:20:15 UTC

The researchers found that beta-catenin modulates anxiety-like behaviour from the behaviour tests they performed on wild-type (WT), cKO (knock-out), and cST (stabilizing) mice from group A, set 2.

Figure 3, A-H

daphnegonnet — 2020-12-05 21:32:58 UTC

Figure 3, I-N

daphnegonnet — 2020-12-05 21:34:53 UTC

Results

daphnegonnet — 2020-12-05 21:36:33 UTC

LDB test: They observed that the cKO mice (figure 3A) spent increased time in dark areas and decreased time in light areas compared to the WT group. When performed with the cST mice (figure 3D), no significant differences were found between the cST and WT group. All other measures for this test, namely the distance travelled (figures 3B and 3E) and the number of transitions (figures 3C and 3F) saw no remarkable differences between both the cKO and cST mice when compared to WT.
OF test: The OF test produced no real differences between any of the groups (figures 3G and 3H).
NFS test: They observed that the cKO had increased latency of feeding (figures 3I and 3J), while the cST had decreased latency of feeding compared to WT (figures 3L and 3M). The researchers also measured the amount of food eaten by the mice during the post-test, but found no statistically significant differences between the cKO or cST and the WT (figures 3K and 3N).

RESULT #2: BETA-CATENIN MODULATES DEPRESSION-RELATED BEHAVIOUR

daphnegonnet — 2020-12-05 21:53:59 UTC

They also found that beta-catenin modulates depression-related behaviour from the behaviour tests they performed on wild-type (WT), cKO (knock-out), and cST (stabilizing) mice from group A, set 2.

Figure 4, A-D

daphnegonnet — 2020-12-05 21:56:29 UTC

Results

daphnegonnet — 2020-12-05 21:58:13 UTC

SP test: They observed that the cKO mice had decreased sucrose preference when compared to the WT mice (figure 4A). The same test for the cST mice, and we see that there were no significant differences between the two groups (figure 4C).
FST: The cKO mice showed increased immobility during the last two minutes of the test and increased immobility over time (figure 4B). In the first 3 to 4 minutes of the test there were no real differences between the WT and cKO mice, but that in the last two minutes there was an increase in the immobility of the cKO mice. The corresponding data for the cST showed no significant differences in immobility times when compared to wild type (figure 4D).

RESULT #3: LOCALIZATION TO SGZ & MODULATION OF HIPPOCAMPAL PROLIFERATION

daphnegonnet — 2020-12-05 22:09:32 UTC

From their initial experiments, they determined that beta-catenin localizes mainly to the subgranular zone (SGZ) of the hippocampus. They found that there was an overall decrease in hippocampal proliferation in cKO mice from the assays they performed on wild-type (WT), cKO (knock-out), and cST (stabilizing) mice from group A, set 1.

Figure 5, A-F

daphnegonnet — 2020-12-05 22:30:40 UTC

Figure 5, G-N

daphnegonnet — 2020-12-05 22:32:10 UTC

Results

daphnegonnet — 2020-12-05 22:33:59 UTC

BrdUassay: They observed a decrease in the number of BrdU⁺ cells in the SGZ of cKO mice (figure 5B) compared to the WT (figure 5A), as shown in figure 5G. They saw no significant differences in the number of these cells in the cST mice when compared to the WT (figure 5K).
Ki-67 assay: They observed a decrease in the number of Ki67⁺ cells in the SGZ of cKO mice (figure 5D) compared to the WT (figure 5C), as shown in figure 5H. They also saw a significant increase in the number of these cells in the cST mice when compared to the WT (figure 5L).
DCX assay: They observed a decrease in the number of DCX⁺ cells in the SGZ of cKO mice (figure 5F) compared to the WT (figure 5E), as shown in figure 5I. They saw no significant differences in the number of these cells in the cST mice when compared to the WT (figure 5M).

RESULT #4: CORTICOSTERONE EFFECTS ON cST MICE

daphnegonnet — 2020-12-05 22:47:18 UTC

The researchers performed multiple behavioural tests on cST mice from group B, set 3 that were treated with either water or chronic corticosterone to build a model of depression. They found that the cST mice seemed to build a greater resistance to stress than the WT mice.

Figure 8, A-D

daphnegonnet — 2020-12-05 22:52:21 UTC

Results

daphnegonnet — 2020-12-05 22:54:13 UTC

NFS test: The cST mice in both conditions (water and corticosterone) had decreased latency of feeding compared to WT both conditions (figure 8A).
FST: The corticosterone-treated cST mice and the water-treated cST mice had decreased immobility compared to WT (figure 8B). Interestingly, we see no significant changes between the water and corticosterone cST mice themselves. The cST mice seem to have some sort of resilience to stress and depression.
SP test: A anhedonic effect was observed in corticosterone WT mice, but not in corticosterone cST mice (figure 8C), pointing to a potential resilience to stress and depression.
DCX assay: They observed a decrease in the number of DCX⁺cells in the SGZ of cST mice in both conditions compared to WT in both conditions (figure 8D).