MicroRNAs 124 and 137 Cooperate
after Seizures and Affect Proliferation,
Migration and Morphology of Nestin+
Neural Stem/Progenitor Cells in the
Mouse Dentate Gyrus
~ An exploratory study ~
Lynnet Frijling
10020012
Assessors:
Pascal Bielefeld
Carlos Fitzsimons
Swammerdam Institute for Life Sciences
University of Amsterdam
Index
Abstract ... 2
Introduction ... 3
Adult Hippocampal Neurogenesis and Seizures ... 3
MicroRNAs ... 3
MiR Cooperativity ... 4
Research Questions and Hypotheses ... 4
Material and Methods ... 5
MicroRNA Injection and SE Induction ... 5
Brain Sectioning ... 5 Immunohistochemistry ... 5 Confocal Microscopy ... 6 Quantification ... 6 Analysis ... 6 Results ... 7 Discussion... 11
Balance between miR-124 and miR-137 ... 11
Cell types with Nestin Expression ... 12
Morphological Effects ... 12
Effects on Excitability of the Network ... 14
Ectopic Cell Placement ... 14
Methodological Concerns ... 15
Suggestions for future Studies ... 15
Literature ... 17
Abstract
Aberrant adult hippocampal neurogenesis (AHN) has often been associated with epileptic seizures. The exact mechanisms by which AHN is affected remain unknown, but a role is reserved for microRNAs (miRs) which regulate messengerRNAs (mRNAs). MiR-124 and miR-137 have previously been shown to cooperate after status epilepticus (SE). In this study we demonstrate in vivo that miR-124 increases the number of Nestin+ cells and miR-137 decreases the number of Nestin+ cells in the dentate gyrus (DG) after Kainic Acid (KA) induced seizures. Moreover, an equimolar mix of both miRs restores the original equilibrium. Besides these results concerning the quantity of cells, the Nestin+ cells showed morphological changes when miR-124 was increased in the DG. Neural stem
cells/proliferating cells show aberrant sprouting and ectopic cell placement. These results indicate a significant role for miR-124, miR-137 and specifically for the cooperation of the two miRs in AHN.
Introduction
Adult Hippocampal Neurogenesis and Seizures
Adult hippocampal neurogenesis (AHN) is the process in which new neurons are formed out of neural stem/progenitor cells (NSPC) in the subgranular zone (SGZ) of the adult dentate gyrus (DG). During this process cells will proliferate, migrate, differentiate and integrate into the hippocampal network. AHN can be influenced by many processes, among which epileptic seizures. Bengzon et al. (1997) found that seizures stimulate the proliferation of neural stem cells in the DG and that they induce apoptosis in the DG. The function of these often-seen effects remains unclear, as AHN may be as well protective and facilitating towards the epileptogenic process, as described by Danzer (2012).
It is suggested that the integrated new neurons have a net inhibitory effect on the hippocampal network (Ikrar et al., 2013). Due to this property, AHN might have a repressing function in epileptic seizures, which are known to affect the hippocampus. Iyengar et al. show that a reduction of AHN makes the hippocampus more susceptible to the effects of Kainic Acid (KA), an often-used
pharmacological model to simulate temporal lobe epilepsy (Ben-Ari, 2012). Accordingly, it is possible that AHN helps to stabilize the hippocampal network after seizures and makes it less susceptible to recurrent seizures. Herein lies the opportunity to make the hippocampal network more resilient to seizures and create possibilities for epileptic patients. To use AHN to the advantage of people with temporal lobe epilepsy, the process and all its molecular features must be well understood.
MicroRNAs
The process of neurogenesis is strictly controlled by regulatory proteins, which in turn can be regulated by molecular factors such as microRNAs (miRs). These miRs recognize specific
messengerRNAs (mRNAs) and can target them to be cleaved or repressed, thereby influencing the gene expression posttranscriptionally (Wilczynska & Buschell, 2014) (figure 1). Henshall (2014) stated that miRs are a potentially important mechanism that controls protein levels in epilepsy. Moreover, miR expression levels are affected after kainate seizures (Liu et al., 2010). Still, it has not been found easy to discriminate the individual effects of a certain miRs, as miRs frequently interact and
sometimes even might work solely in combinations (Wilczynska & Bushell, 2014). In addition, a consequence of miR cooperativity is that their targets could be more sensitive to smaller changes in miRs. Hence, it is crucial to study miRs not only individually but also in combinations, to unravel their complete functioning regarding the regulation of neurogenesis. It would be insightful to study miR control in this process and thereby pay special attention to miR cooperativity.
MiR Cooperativity
The miR most frequently linked to neurogenesis is miR-124 (Vreugdenhil & Berezikov, 2011). It has been suggested that this miR is involved in directing cells to a neuronal phenotype (Lim et al., 2005). Furthermore, Lim et al. showed that miR-124 downregulates a great number of targets involved in neurogenesis. It is likely that miR-124 does not control all its targets by itself, but rather cooperates with other miRs. Santos et al. (2015) demonstrated that 124 cooperates with 137 and miR-128 to control neurogenesis by targeting overlapping sets of genes.
Schouten et al. (2015) show that miR-124 targets the BCL2L13 protein, a protein involved with apoptosis. Moreover, they demonstrate that miR-137 is most likely to pair up with miR-124. These miRs were shown to share 336 common targets, 61 of which are targets in the process of apoptosis. In vitro, Schouten et al. show that after KA treatment, only an equimolar combination of miR-124 and miR-137 affects the levels of apoptotic protein, while miR-124 or miR-137 separately have no effect. It is hypothesized that KA causes an increase of these miRs, leading to less BCL2L13 pro-apoptotic protein, and thereby promoting proliferation in neurogenesis. In vivo, Schouten et al. demonstrate that BCL2L13 in the DG of mice is expressed in Nestin-GFP positive cells (Nestin-GFP+) and more so, in Nestin-GFP+ cells positive for polysialic acid form of neural cell adhesion molecules (Nestin-GFP+/PSA-NCAM+), classified as intermediate neuronal progenitors (Fukuda et al., 2003).
To effectively study miR cooperativity in the process of neurogenesis after status epilepticus (SE), miR-124 and miR-137 are a starting point since they have been proven to work in cooperation in vitro and are responsive to epileptic seizures. It would be interesting to see how these miRs work together in vivo, and how they affect the process of neurogenesis after SE. As neurogenesis starts with NSPCs proliferating in the SGZ of the DG, the first step is to see how SE and these miRs affect the Nestin-GFP+ cells. To study subsequent steps such as apoptosis and proliferation, it is also
valuable to know how the first step of neurogenesis is affected. In this exploratory study, mice will be exposed to KA and to miR-124, miR-137 or an equimolar mix. Using Nestin as a marker, the number, location and morphology of NSPCs in the DG can be assessed.
Research Questions and Hypotheses
The questions addressed in this exploratory study shall be:
- What is the effect of KA treatment on the amount of NSPCs in the subgranular zone (SGZ), the
granular cell layer (GCL) and the molecular layer (mol L)?
- What is the effect of infusing miR-124 and miR-137 both separately and combined into the
DG, prior to SE onset, on the amount of Nestin-GFP positive cells in the SGZ, the GCL and the mol L after seizures?
KA treatment is expected to increase the number of NSPCs in the SGZ, GCL and mol L, and possibly affect their migration. Since this is an exploratory study into the effects of miRs in vivo, no real predictions can be made about the effects of miRs on neurogenesis. As miR-124 is related to proliferating cells, a raise of miR could be expected to cause more NSPCs. In addition, a mix of both miRs is expected to show deviating results from one of the miRs when infused separately.
Material and Methods
MicroRNA Injection and SE Induction
Twenty-four male, transgenic Nestin-GFP mice were housed in groups in a twelve-hour day/night cycle (lights on at 6.30). Animals had free access to food and water and were kept in a room with constant temperature and humidity. The mice were randomly divided in three groups: miR 124, miR 137 and miR 124 & 137, with n=8 per group. Within these groups, animals were split in two
conditions: Kainic Acid or Saline control. Animals were six weeks old when they underwent
stereotaxic surgery. 1.0 µL of 50µM miR-124, miR-137 or both (mirVana®) was injected into the right DG at the coordinates of -2.0 (anterior-posterior), 2.0 (medial-lateral) and -2.0 (dorsal-ventral). In the contralateral DG, 1.0 µL of NT-miR (non-targeting) was infused.
After forty-eight hours, a second stereotaxic surgery was performed. Bilaterally, either 50 nL of 2.22mM Kainic Acid or Saline control was injected at the same coordinates as the miR injections. 72 hours after this second surgery, the animals were sacrificed by perfusion. Brains were extracted and kept in PFA for post fixation.
Brain Sectioning
Brains were sliced coronally at 40 µM and slices were divided into eight series. Each contained representative slices of the hippocampus over the anterior-posterior axis. The slices were kept in PB with 0.01% azide in order to protect them from bacteria. The jars were kept at four degrees in the fridge.
Immunohistochemistry
On the tissue, two stainings were done, meaning two out of the eight series were used. The first staining was for BCL RAMBO, PSA-NCAM and GFP, using antibodies in the following concentrations:
- Chicken anti GFP (1:500) - Mouse anti PSA-NCAM (1:500) - Rabbit anti BCL RAMBO (1:200)
A second staining was done for Cleaved Caspase 3, PSA-NCAM and GFP. The results of this experiment are not included in this article. The methods of this experiment are annexed as an addendum.
For both stainings the following secondary antibodies were used: - Goat anti chicken (wavelength of 488)
- Goat anti mouse (wavelength of 647) - Goat anti rabbit (wavelength of 568)
After a two-day protocol slices were mounted onto superfrost glasses using vectashield with dapi, to counterstain for DNA.
Confocal Microscopy
Images were acquired using a confocal microscope. A 40x water corrected objective was used. Slices that were visibly disrupted by the injection of microRNAs or Kainic Acid were not used. Only slices from the Kainic Acid condition were observed. Slices from the Saline condition were disregarded due to lack of time.
Pictures were taken of the slices at least one slice away from the injection site. In each slice, eight pictures were taken. Two of the suprapyramidal blade of the right DG, two of the infrapyramidal blade of the right DG, and the same amount of the left DG. Less images were acquired if the DG was incomplete due to rupture, folding, etc. The images obtained were z-stacks, allowing cells to be quantified throughout of the slice. In order to prevent searching for desired results, the location of the image was established based on the blue dapi signal.
Quantification
The z-stacks obtained with confocal microscopy were loaded into image-j. The complete scan was examined over the z-axis while counting the positive cell bodies in the SGZ, GCL and mol L.
Analysis
The number of Nestin-GFP+ cells of all pictures of the left or right DG were added togetherm and then averaged over the number of pictures taken per DG. These values were analysed per group: NT, miR-124, miT-137 and mix, and a one-way anova was performed over the groups per zone: SGZ, GCL and mol L.
Results
Table 1. Average number of cells and standard deviation observed in the pictures of the DG per experimental group.
SGZ GCL Mol L All layers
NT 235,3 ± 61,5 84,8 ± 26,5 132,4 ± 24,7 452,5 ± 84,8
miR-124 248,5 ± 45,1 123,1 ± 63,3 145,4 ± 45,8 517,0 ± 113,4
miR-137 185,5 ± 32,1 46,3 ± 11,7 105,5 ± 33,6 337,3 ± 51,6
Mix 261,2 ± 22,6 81,8 ± 45,1 143,5 ± 25,6 486,5 ± 54,0
On average, the dentate gyrus targeted with NT miRs counted 235,3 cells positive for Nestin-GFP in the SGZ, 84,8 in the GCL and 132,4 in the mol L. When targeted with miR 124, 248,5 cells were found positive for Nestin-GFP in the SGZ, 123,1 in the GCL, and 145,4 in the mol L. When targeted with miR 137, 185,5 cells were positive for Nestin-GFP in the SGZ, 46,25 in the GCL and 105,5 in the mol L. When targeted with both miR 124 and 137, 261,2 cells were found positive in the SGZ, 81,8 in the GCL and 143,5 in the mol L.
An anova test showed no significance in the SGZ (p=0.1765) (figure 2), in the GCL (p=0.1134) (figure 3) and in the mol L (p=0.3640) (figure 4). As can be seen in all three layers, a trend seems visible wherein more Nestin+ cells are visible in the miR-124 group compared to the NT group. Also, the amount of Nestin+ cells seems to be decreased in the miR-137 group compared to the NT group, especially in the GCL. Moreover, the amount of Nestin+ cells in the mix group seems to be comparable with the NT group in all three layers.
Figure 2. Number of Nestin-GFP+ cells in the SGZ in all experimental groups, with standard deviations.
Figure 3. Number of Nestin-GFP+ cells in the GCL of all experimental groups, with standard deviations.
Figure 4. Number of Nestin-GFP+ cells in the mol L of all experimental groups, with standard deviations.
Figure 5 shows the data of all layers of the DG combined, per experimental group. An one-way anova denonstrates p=0.047 for all layers combined. Post Hoc test LSD shows significant difference
between MiR-124 and MiR-137 (p=0.009), and between MiR-137 and mix (p=0.035). Levenes test showed homogeneity of variances could be assumed (p=0.213).
Figure 6 displays examples of the Nestin-GFP+ cells in all experimental groups. It can be observed that the shape of the sprouting of the cells in the miR-124 group is more exorbitant than in the other groups. Figure 6 shows the number of Nestin-GFP+ cells of all zones in the non-targeting control group, and thereby the effect of the KA treatment.
Figure 7 shows the effect of KA on Nestin+ cells in the three layers. As Saline data are missing in this study, the net effect of KA is not visible. However, in the discussion these data can be compared to results from previous studies.
Figure 6. Examples of the pictures taken of the DG in all experimental groups. Green are the Nestin-GFP+ cells. Blue are the cell bodies counterstained with Dapi.
Discussion
Balance between miR-124 and miR-137
In the total DG, the number of Nestin+ cells is found to be significantly lower in the miR-137 + KA group than in the miR-124 + KA group and the mix + KA group. As the number of Nestin+ cells of the miR-137 group is lower than the miR-124, but not lower than the NT group, it can be concluded that miR-124 and miR-137 have opposite effects on the number of stem/progenitor cells after KA-induced SE. Moreover, as the miR-137 group has significantly less Nestin+ cells than the mix group, it can be stated that miR-137 has a discriminating effect on the number of stem/progenitor cells compared to a combination of both miRs. This does demonstrate that miR-137 and miR-124 do indeed cooperate.
Sun et al. (2011) elucidated that miR-137 reduces proliferation in neural stem cells (figure 8). The number of Nestin+ cells found in the miR-137 group does confirm that increased levels of miR-137 reduce Nestin+ cell numbers compared to both miRs increased in an equimolar concentration. No significant difference was found between increased levels of miR-137 and the NT group, but with p=0.059, a trend is visible. It is remarkable that these significances and trends are more evident with miR-137 than miR-124, suggesting that miR-137 has a greater
momentum in the cooperation and probably needs lower concentrations than miR-124 in proportion to create an equilibrium.
Although no significant differences were found within the three layers, a trend seems visible in which miR-124 injection raises the amount of Nestin+ cells, whereas miR-137 injection lowers the number of Nestin+ cells. Notably, the equimolar mix of both miRs seems to create an equilibrium and restore the balance. The amount of Nestin+ cells in the mix group is comparable with the NT control group. Judging by this outcome, it seems that miR-124 and miR-137 cancel out each other’s effect. It could be possible that miR-124 and miR-137 work together in a counterpoise. A correct balance between the two miRs could bring about a standard amount of Nestin+ cells in the SGZ, the GCL and the mol L after seizures. This would suggest a possible role for miR-124 and miR-137 in neurogenesis and proliferation, as the numbers of Nestin+ cells are directly influenced by proliferation. Moreover, cooperativity is needed to ensure healthy proliferation and neurogenesis, because infusion of only one of the miRs leads to a divergent number of new born cells in the DG.
To know to what extend the miRs affect the apoptotic process, it is necessary to analyse the scans of the same animals, that were stained for PSA-NCAM. Since this marker would show the number of cells during and after the apoptotic process, a decrease with respect to the Nestin+ cells is expected.
Figure 8. BrdU-labeled (proliferating) cells in a control group and in miR-137 transfected neural stem cells. Bar graphs shows significantly less BrdU-labeled cells in the miR-137 group compared to the control group. Figure adapted from Sun et al. (2011).
It would be interesting to see how these miRs influence this decrease, both separately and in the mix.
Cell types with Nestin Expression
It is important to note that the positive cells counted in this study are not necessarily neural stem/progenitor cells. Clarke et al. (1994) already observed that Nestin+ cells can also be reactive astrocytes. Moreover, Filippov et al. (2003) show that Nestin+ cells can be colabeled for GFAP, an astrocytic marker. Consequently, in counting Nestin+ cells, not only stem/progenitor cells but also astrocytes (or their precursors) could be counted. This could mean an overestimation of the number of progenitor cells. As according to Filippov et al. (2003) these colabeled cells are mostly present in the SGZ, this overestimation could be most prominent in the SGZ. Also, Ogata & Kosaka (2002) make clear that the distribution of astrocytes over the DG is not even across layers. The number of
astrocytes is lowest in the GCL compared to the rest of the DG. This could mean that this overestimation of stem/progenitor cells is least existing in the GCL.
There is no evidence on how miR-124 or miR-137 affect the development of progenitor cells or astrocytes, so it is not clear whether this altered the ratios between the experimental groups. To test if and what part of the Nestin+ cells are in fact astrocyte, a counterstain should be performed with GFAP.
Moreover, the Nestin marker does not distinguish between neural stem and progenitor cells. It is unclear how the miRs in combination with the KA affect these two groups. To control for these differences, counterstains can be performed with for instance SOX2 for neural stem cells, and KI67 for progenitor cells.
Morphological Effects
Even though the numbers of Nestin+ cells showed no significance between groups, great morphological differences in the Nestin+ cells stood out. Especially miR-124 infusion resulted in abnormal and aberrant sprouting of dendrites (and axons). Infusions with miR-137 and the mix do not lead to the same effect, but rather resemble the NT group. This would again suggest that cooperativity is needed between both miRs to ensure the proper dendritic/axonal sprouting.
Vreugdenhil & Berezikov (2010) did previously summarize that miR-124 could act as a master-switch, repressing genes that keep cells in a non-neuronal state, and thus promoting differentiation (figure 9). This concept could apply, as these are indeed new-born cells in or around the process of
proliferation. An excess of miR-124 could indeed lead to an excess in neuronal differentiation and therefore to an aberrantly sprouting phenotype. However, the Nestin+ cells in the current study are not yet in the process of differentiating. Yet, an excessive amount of miR-124 during the proliferation phase could perhaps still work as a switch by repressing gene transcription and stimulating
axonal/dendritic growth. It would be interesting to see if the genes summarized by Vreugdenhil & Berezikov (2010) are indeed suppressed in Nestin+ progenitor cells.
Moreover, this study also suggests that miR-124 does not work as a master-switch on its own. An equilibrium between miR-124 and miR-137 seems to restore axonal/dendritic sprouting. It could be that miR-137 promotes the same targets that miR-124 represses, thereby balancing out the effect. Or, miR-137 could target and repress miR-124 directly.
Yoo et al. (2009) have previously elucidated that miR-124 plays a role in repressing the BAF53a gene, which is essential for the proliferation of neural progenitor cells. They showed that disturbed repression by miR-124 leads to persistent expression of BAF53a, which defected dendritic outgrowth. The results of the current study could be in line with this, as an overexpression of miR-124 leads to aberrant sprouting, possibly mediated by over-repression of BAF53a. Also, Yoo et al. (2009) state that miR-124 is suppressed by transcription factor REST, while Vreugdenhil & Berezikov (2010) mentioned REST to instead be repressed by miR-124. In this theory, the infusion of miR-124 in this study would have led to repression of REST, which would have led to even higher concentrations of miR-124, which then leads to dendritic outgrowth instead of proliferation and
Figure 9. miR-124 works as switch on multiple genes. Figure adapted from Vreugdenhil & Berezikov (2010).
Figure 10. REST represses miR-9 and miR-124, which repress BAF53a, which represses BAF53b and stimulates proliferation, while BAF53b stimulates dendritic outgrowth. Graphs depict the situation in the presence (green) and absence (red) of REST. Figure adapted from Yoo et al. (2009).
thereby steering the cells towards differentiation (figure 10).
Even though we see slightly higher numbers of Nestin+ cells in the DG in the miR-124 group compared to the NT group, these numbers were not significant. This does concur with the theory proposed by Yoo et al. (2009) that the presence of miR-124 leads towards outgrowth and not proliferation (figure 8).
As it is suggested that miR-124 and REST repress each other, the problem arises in which both factors undo each other’s effects. Or even: if one of the factors is dominant, it could continuously repress its own repressor, without restoring a balance. This could be what we are witnessing in the current study: dominant levels of miR-124 lead aberrant outgrowth. In this study we see miR-137 acting as a factor that balances the scale. It could be hypothesised that miR-137 represses miR-124 or
upregulates REST. It would be interesting to further study the relationship between miR-124, REST and miR-137.
Effects on Excitability of the Network
This aberrant sprouting in the DG could possibly influence the network in such a way that it affects its susceptibility to seizures. The Nestin+ cells observed in this study are merely stem/progenitor cells, but their location and morphology could possibly affect the location and morphology of the subsequently formed neurons. Morgan & Soltesz (2008) showed that the epileptic activity of a network is greatly determined by the connectivity of a microcircuit. Highly interconnected granule cell hubs can contribute to the hyperexcitability of the network. However, Morgan & Soltesz (2008) subscribe this effect specifically to the larger basal dendrites that extend into the hilus, and not to aberrant apical dendrites in the mol L. It remains unclear how an extended dendritic network in the mol L affects the network and affects seizures. To speculate: more complex dendritic arborisations in the mol L would allow for a higher susceptibility to input from the entorhinal cortex onto the granule cells. This again would lead to more output via mossy fibres onto the CA3.
Moreover, it is not yet clear how the location and morphology of neural stem/progenitor cells later influences the development of neurons in the DG. To study this, the effects of KA and miRs should also be studied in later stages of neurogenesis.
Ectopic Cell Placement
In all groups, Nestin+ cells were found ectopically placed in the mol L. Previously, Heine et al. (2004) showed that the ratio of new born cells in the DG of 6 weeks old animals is approximately 5.5 : 2,5 : 1, respectively SGZ :
GCL : mol L. This would mean that in a healthy 6-week-old
animal, the GCL should have 2.5 times as much new-born cells than the mol L (figure 11). However, this current study shows that the ratio of SGZ : GCL : mol L is
approximately 3 : 1 : 1.6 (figure 7). It shows that the ratio of Nestin+ cells in the mol L compared to the SGZ and the GCL is much higher after these animals were treated with KA. This study confirms that epileptic seizures lead to ectopic new born cell migration into the mol L, as previously seen by Parent et al. (1997). Between miR
Figure 11. Numbers and ratio of new born cells in the hilus, SGX, GCL and mol L of the DG of 2 weeks, 6 weeks, 12 months and 24 months old animals. Figure adapted from Heine et al. (2004).
groups, no significant difference could be found in the amount of Nestin+ cells. This suggests that both miR-124 and miR-137 have no significant role in the ectopic placement of the cells, although they do again seem to balance each other and cooperate to effectuate this rise in ectopic placement.
Methodological Concerns
In this study, the number of pictures taken of the DG per animal and condition were averaged, as from some brains, more pictures could be made than from others. Therefore, the number of cells counted in some DGs is extrapolated. Moreover, not all pictures were taken with the DG in the same orientation. When the GCL crossed the picture diagonally, it displayed a larger surface of the GCL, and thereby possibly led to a higher cell count. Likewise, if the picture displayed a larger proportion of the mol L, it evidently allowed more cells to be counted. It would be a better idea to average the number of cells over the surface of the DG, and when possible, use a similar number of pictures per brain.
Suggestions for future Studies
Firstly, it would be interesting to scan the slices not only for GFP, but also for the other stained markers: PSA-NCAM, BCL RAMBO and Cleaved Caspase 3 (figure 12). Due to a limited amount of time, only the Nestin-GFP+ cells were counted in this study. This led to some insights on new-born cells after KA, and the involvement of miRs on neurogenesis and proliferation. However, to further study the effects of seizures and different miRs on apoptosis, differentiation and migration, the number of PSA-NCAM+ cells in the DG should also be counted. This marker shows cells in the process of fate-specification and differentiation; cells that could go into apoptosis.
To compare the numbers of Nestin+ and PSA-NCAM+ cells would be especially interesting, as it would allow for predictions on how apoptosis affected the number of cells, and how specific miRs affected this. The amount of Cleaved Caspase 3 in combination with BCL-RAMBO would also be interesting to study, as it could offer insight into how different miRs could (cooperatively) influence BCL-RAMBO and thereby Cleaved Caspase 3. Together with the cell count on the PSA-NCAM marker, a conclusion can be drawn on how these miRs affect the apoptotic process.
Secondly, the aberrant sprouting of dendrites of new-born cells with higher levels of miR-124 is worth looking into. Especially since this sprouting is not observed in the mix group, which suggests that miR-124 and miR-137 should be balanced for a healthy proliferation of cells. It would be interesting to know via what mechanism miR-124 and miR-137 cooperate, and thereby control proliferation, differentiation and possibly even migration.
Lastly, the ratios by which miR-124 and miR-137 cooperate should be explored further. It has become clear that miR-124 and miR-137 have different effects, but also that they somehow cooperate. This cooperation could be vital to neurogenesis in the hippocampus. This study also indicated that miR-124 and miR-137 cooperate in certain proportions. To know how the amount of miRs relate to each other in the stages of neurogenesis could be vital to understanding the regulation of the process and how the process could be adjusted. This could provide possible ways to alter neurogenesis after seizures and make the hippocampus more resilient.
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Addendum
A second staining was performed for Cleaved Caspase 3, PSA-NCAM and GFP. For this staining, the concentration of Cleaved Caspase 3 antibody, and also as if the results are best with or without antigen retrieval, still had to be determined. So first, a test staining was done on the tissue of animal six. Since this animal received both miR-124 and 137, as well as Kainic Acid, the most Cleaved Caspase 3 was expected here. Animal six was chosen over the other animals in this group and condition by parameters as how well the surgeries and perfusion went. After analysing the results of the test staining, it was determined that the following antibodies should be used in the following concentrations:
- Chicken anti GFP (1:500) - Mouse anti PSA-NCAM (1:500) - Rabbit anti Cleaved Caspase 3 (1:600)
No antigen retrieval was performed on the tissue for the second staining, as this did not improve the quality of the staining.
