Cofilin 2 PCR
4. Results
7.1. Cofilin 2 expression and upregulation
7.1.1. The loss of Cofilin 2 leads to the upregulation of Cofilin 1 and ADF in distinct brain
The expression of Cofilin 2 was analyzed in different brain regions of P7 animals via Western blot. Lysates from Cofilin 2+/+ animals at P7 were prepared, due to the fact that it is the latest time point before the complete deletion of Cofilin 2 is lethal. In every analyzed brain region a signal for the Cofilin 2 antibody could be observed, showing a broad expression of Cofilin 2 in the brain of 7 day old newborn (figure 21). The highest expression of Cofilin 2 at P7 was found in the cortex and cerebellum, which are the brain regions with the highest migrational rates during development, followed by the midbrain and the hippocampus. The brain-specific knockout of Cofilin 1 affects migrational processes of postmitotic neurons from the subventricular zone to the cortical layers (Bellenchi et al., 2007). The areas with lowest Cofilin 2 expression at P7 were the striatum and olfactory bulb. Interestingly, the areas with an increased Cofilin 2 expression also showed an upregulation of ADF and Cofilin 1 upon the loss of Cofilin 2. An upregulation of another ADF/Cofilin family member upon the deletion of one isoform is a common mechanism in knockout animals of this family. All three members display 82% sequence homology and share therefore the same basic function, which could compensate a loss and attenuate a specific phenotype. Nevertheless all three members also show distinct biochemical properties. For example Cofilin 2 has a higher binding affinity to F-actin than Cofilin 1 or ADF and therefore an increased severing activity, while the depolymerization rate is lower, compared to ADF and Cofilin 1. Therefore the loss of Cofilin 2 could still lead to a specific phenotype, since some actin-dependent processes could rely more on a high severing activity, which cannot be achieved by ADF or Cofilin 1.
Since a broad expression of Cofilin 2 was examined in the brain, the next step was the analysis of different developmental time points for their Cofilin 2 expression. The expression profile of Cofilin 2 during different developmental time points ranking from P0, P7, P15, P21 to adult animals were examined in distinct brain regions of wt animals (figure 22).
Interestingly in almost all brain regions the expression of Cofilin 2 was highest at P7, the time point when the deletion of Cofilin 2 was starting to become lethal. The only exception was the cortex where the expression peaks at P0 and the cerebellum where the highest expression was found in adult animals. A conditional deletion of Cofilin 1 in the brain using Nestin-Cre expression leads to a cortical migrational defect, resulting in the absence of the layers II – IV
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(Bellenchi et al., 2007). These result link actin depolymerization factors with cortical layer formation defects and resemble the pathology of type 1 lissencephaly patients (Bellenchi et al., 2007). The cortical proliferation and migration wave starts at E13.5 and the layering is completed until E19.5. Cofilin 2 could have a role during these processes and the expression could then decline after birth when the development of the cortex is terminated. Therefore it would be interesting to analyze also the expression of Cofilin 2 during embryonic stages that are important for cortical development. Since the Cofilin 2fl/fl Nestin-Cre brains did not showed a translucent appearance of the cerebral cortex (figure 34), the loss of Cofilin 2 seemed to be compensated by the expression of ADF and Cofilin 1. An elevated level of ADF and Cofilin 1 was detected in cortical lysates of Cofilin 2-/- animals around P7 (figure 21), which could lead to a milder phenotype.
Also the process of synaptogenesis starts at P7 with an elevated number of synaptic contacts that are built, followed by a fine-tuning around P15 leading to the pruning of synapses that are not frequently used. The establishment of synapses requires a breakup of the actin cytoskeleton for the protrusion of filopodia-like structures, which are then reassembled to dendritic spines or presynaptic terminals. Actin is also the main cytoskeletal protein in dendritic spines and the assembly of the postsynaptic compartment needs a highly dynamic actin cytoskeleton. Thereby Cofilin 2 is important to depolymerize existing actin filaments for the protrusion of new filaments and also the recycling of older filaments to maintain the G/F-actin pool. Further the severing activity of Cofilin 2 is important to obtain a high number of shorter actin filaments, which can be used to assemble the branched actin cytoskeleton in dendritic spines and presynaptic terminals. Only this branched actin network can fulfill the important function of actin filaments in this specialized compartments. Rust et al analyzed the phosphorylation level of Cofilin 1 during synaptogenesis and also found higher phosphorylation levels at early postnatal stages, which dramatically decreased during synaptogenesis (P21 – P50) and therefore confirm the observed results here (Rust et al., 2010).
The next step was the confirmation of the complete Cofilin 2 knockout and the respected altered levels of ADF or Cofilin 1. Therefore lysates for every region of Cofilin 2+/+, Cofilin 2 +/-and Cofilin 2-/- animals were loaded on one gel and the blot was incubated with antibodies against all three ADF/Cofilin family members. The Cofilin 2+/- lysates should contain a 50%
reduction in the Cofilin 2 expression, due to the loss of Cofilin 2 on only one allele.
Densitometric analysis revealed an almost 50% reduction in the cortex, hippocampus, olfactory bulb and midbrain lysates. Only in the striatum (90%) and cerebellum (71%) a higher Cofilin 2 expression rate was left. This could result from an unsteady Cofilin 2 expression level around P7 in these brain regions compared with the other brain regions.
Additionally the Cofilin 2 protein has a long half-life and could result in an elevated protein
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level, although the reduction in the Cofilin 2 expression is there, due to the loss of one Cofilin 2 allele. Another explanation would be that every animal is unique and has its own metabolism and hormone balance and reacts to environmental stimuli differently. Therefore the number of analyzed animals should be increased to obtain a representative mean.
In figure 20 the complete knockout of Cofilin 2 was verified, since in Cofilin 2-/- lysates no band at the height of Cofilin 2 was detectable. Additionally a slight upregulation of ADF and Cofilin 1 in the cortex, midbrain and olfactory bulb could be observed, but the strongest upregulation of ADF and Cofilin 1 at P7 was reflected in the hippocampus (figure 21). In the hippocampus actin plays an important role for learning processes like LTP and the establishment of synapses (Fukazawa et al., 2003). An increased F-actin content was found in cortical and hippocampal synaptosomes of Cofilin 1fl/fl CaMKII-Cre animals, which leads to an enlargement of dendritic spines and altered postsynaptic parameters like L-LTP and LTD (Rust et al., 2010). Görlich et al detected an upregulation of Cofilin 1 in synaptic structures of ADF-/- mice and suggested that the lack of synaptic defects in ADF-/- mice could be explained by the elevated Cofilin 1 levels that compensate for the loss of ADF (Gorlich et al., 2011).
This hypothesis was confirmed in ADF-/- Cofilin 1fl/fl CaMKII-Cre animals, which showed an additive effect of ADF, since the F-actin level in synaptosomes was further increased in double knockout animals in comparison to Cofilin 1fl/fl CaMKII-Cre animals (Zimmermann et al., 2015). Additionally a presynaptic phenotype was observed in double knockout animals for ADF and Cofilin 1, while the single brain-specific knockout of Cofilin 1 only leads to postsynaptic defects. The observed upregulation of ADF and Cofilin 1 in certain brain regions could attenuate a possible Cofilin 2 based brain phenotype and indicates that Cofilin 2 expression is important in these brain regions, since the loss needs to be compensated by an upregulation of other ADF/Cofilin family members.
Since the complete deletion of Cofilin 2 is lethal around P7, a brain specific deletion of Cofilin 2 using Cre-expression under the Nestin promotor was analyzed. In this mouse line Cofilin 2 was deleted in neuronal and glial cell precursors of the forebrain, starting around E10.5 (Tronche et al., 1999). Cofilin 2fl/fl Nestin-Cre animals were viable, but smaller than their littermates, and allowed the study of adult animals. With the help of this conditional mouse line it was examined whether the conditional deletion of Cofilin 2 was restricted to specific brain regions and if the deletion rate of the Nestin-Cre recombinase was sufficient enough in every brain region. Additionally a possible upregulation of ADF or Cofilin 1 was analyzed in adult animals. Therefore lysates from different brain regions from adult Cofilin 2fl/fl and Cofilin
2fl/fl Nestin-Cre animals were prepared. In adult animals the highest expression level of Cofilin
2 was found again in the cortex, midbrain and cerebellum, followed by the striatum, hippocampus and olfactory bulb (figure 33). The deletion of Cofilin 2 was efficient in almost every brain region, since no band for Cofilin 2 was detected in the Cofilin 2fl/fl Nestin-Cre
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lysates. Only for the midbrain a faint band at the height of Cofilin 2 was detected, indicating that in the midbrain the deletion of Cofilin 2 was not complete. Only the midbrain and hippocampus showed an upregulation of ADF and Cofilin 1 in both P7 and adult animals, whereas the hippocampus showed the highest upregulation of ADF and Cofilin 1 at both time points, highlighting an important function of ADF/Cofilin family members in this brain region.
This leads to the suggestion that especially in the hippocampus the deletion of Cofilin 2 seems to be compensated by other ADF/Cofilin family members. A compensatory effect of other ADF/Cofilin family members upon the loss of one member was also detected in ADF -/-mice and Cofilin 1fl/fl CaMKII-Cre mouse models, suggesting a functional redundancy (Gorlich et al., 2011; Rust et al., 2010). In the cerebellum only an upregulation of Cofilin 1 was detected in adult animals, indicating that both Cofilins could be necessary for synaptic processes in the adult cerebellar cortex. The upregulation of ADF and Cofilin 1 was lost between P7 and adulthood in the cortex and olfactory bulb, leading to the suggestion that ADF/Cofilin family members could contribute to migrational processes that assemble brain structures.
Since an upregulation of other ADF/Cofilin family members occurred upon the loss of Cofilin 2, the analysis of double knockout animals was important and necessary to check whether these upregulations prevent or attenuate a possible Cofilin 2-based phenotype. To analyze if the combined loss of ADF and Cofilin 2 in the brain displayed a stronger phenotype than the single conditional knockout of Cofilin 2, lysates of adult ADF-/- Cofilin 2fl/fl Nestin-Cre knockout animals were analyzed for a possible upregulation of Cofilin 1. The complete knockout of ADF was viable and showed no obvious brain phenotype, but an additive effect of ADF was detected in ADF-/- Cofilin 1fl/fl CaMKII-Cre animals (Gorlich et al., 2011; Wolf et al., 2015;
Zimmermann et al., 2015). Total brain lysates of adult ADF-/- Cofilin 2fl/fl control animals and ADF-/- Cofilin 2fl/fl Nestin-Cre knockout animals were prepared. In adult animals the dual loss of both ADF and Cofilin 2 does not lead to an upregulation of Cofilin 1 in total brain lysates (figure 51). This could suggest overlapping functions or a subcellular localization for ADF and Cofilin 2 that is distinct to the brain specific function and localization of Cofilin 1. The upregulation of ADF was not sufficient to compensate for the loss of Cofilin 1 in Cofilin 1fl/fl CaMKII-Cre animals, which displayed a postsynaptic phenotype (Rust et al., 2010). An explanation for this observation could be the fact that ADF is predominantly enriched in presynaptic terminals and therefore fails to countervail the loss of Cofilin 1 in postsynaptic structures (Gorlich et al., 2011). On the other hand displayed ADF-/- Cofilin 1fl/fl CaMKII-Cre animals an additive effect of ADF, since in knockout animals also a presynaptic phenotype could be observed (Wolf et al., 2015; Zimmermann et al., 2015). This could suggest that the loss of Cofilin 2 might be more compensated by ADF than Cofilin 1. The fact that ADF and Cofilin 2 could have overlapping functions in the brain makes the analysis of double knockout
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animals quite interesting, since it could have a more severe phenotype than the single knockout of Cofilin 2. A second explanation could be that the normal expression of Cofilin 1 is sufficient to maintain the brain morphology and function. In this study only total brain lysates of ADF-/- Cofilin 2fl/fl and ADF-/- Cofilin 2fl/fl Nestin-Cre animals were analyzed for alterations in the Cofilin 1 level. A better insight into the possible upregulation of Cofilin 1, due to the loss of ADF and Cofilin 2, should be analyzed in brain lysates of different brain regions, since the complete lysate could hide a weak upregulation of Cofilin 1 in distinct brain regions. Görlich et al also detected no upregulation of Cofilin 1 in ADF-/- mice in brain area lysates of the cortex and hippocampus, while an upregulation of Cofilin 1 was detected in synaptosomal lysates (Gorlich et al., 2011). As seen in figure 51 a complete knockout of ADF and Cofilin 2 was verified, since no band for ADF or Cofilin 2 was detected in the brain lysate of ADF-/- Cofilin 2fl/fl Nes-Cre animals.
Since Cofilin 2 displayed a broad expression profile in different brain regions, a further examination of distinct neuronal subtypes in these brain regions was performed. A possible role of Cofilin 2 in certain brain regions will be further discussed in the next chapters.