Dendritic spine maturation could be affected by the loss of Cofilin 2

Within the first week of murine development dendritic protrusions begin to increase in density. These protrusions are also capable of extreme movements, especially early during development with an accelerated synaptogenesis starting around postnatal day 7.

Interestingly Cofilin 2 expression analysis during different developmental time points indicated the highest expression of Cofilin 2 at P7, when the complete knockout of Cofilin 2 is starting to became lethal. At that time point the Cofilin 2 expression reaches a peak in most brain areas, except for the cortex and cerebellum, which showed the highest expression at P0 and adulthood (figure 22). The protrusion of dendritic spines for the establishment of synapses needs a constant rearrangement of the actin cytoskeleton, since actin is the most prominent cytoskeletal protein in spines. Spines are highly dynamic structures and their formation and maturation depends on actin remodeling (Cingolani and Goda 2008).

ADF/Cofilin family members are localized at spines and enable the rapid rearrangement of actin, due to the depolymerization of filaments (Hotulainen et al., 2005; Racz and Weinberg 2006). During the second and third week of life, the density of dendritic spines further increases, in coincidence with the rate of synaptogenesis. During this time period (P15 – P21) a prominent expression of Cofilin 2 could be observed in all brain regions, although weaker in comparison to P7. The motility of spines is developmentally regulated and decreases during the first postnatal weeks as synaptic circuits mature. During the next month a pruning process with a loss of spines is observed in animals, which generates the mature spine density after several months. An expression of Cofilin 2 is also found at P90 showing a broad expression in all brain regions (figure 22). Additionally also the spine morphology changes during the development. Early in development most spines are stubby or thin with quite long filopodia. Over the next weeks the number of thin spines decreases and the spine maturation can be seen in the occurrence of mushroom shaped spines, which display the site of strong synapses. Thereby the spine volume is proportional to the area of the PSD and PSD area correlates with the number of incorporated receptors. In Cofilin 2 deficient animals a reduction in the number of mushroom-shaped spines could be observed (figure 41), which could implicate Cofilin 2 in the maturation process of dendritic spines. The overall number of spines was not altered and an concurrent increase in the number of thin spines was also detected. This suggests that Cofilin 2 was not necessary for the formation of spines, but rather was important for the transition from immature to mature spines. The incorporation of

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new receptors as well as the enlargement of the PSD needs a dynamic actin cytoskeleton, which allows the expansion of the spine volume. A loss of Cofilin 2 could lead to an impaired rearrangement of actin filaments blocking the transport of proteins for the increment in spine volume and also the transport of receptors to the postsynaptic membrane.

Glutamate is the main excitatory neurotransmitter in the brain and can fulfill its function via the activation of three receptors: AMPA, NMDA and kainate receptors. In this study the frequency and amplitude of mEPSCs in the Ca1 region of the hippocampus were examined.

These mEPSCs are generated by spontaneous glutamate vesicle release and the binding of glutamate to the AMPA receptor, which is an ionotropic transmembrane receptor for the fast excitatory synaptic transmission that enables the opening of further glutamate-gated receptors. The AMPA receptor number seemed to be not affected in Cofilin 2^fl/fl Nestin-Cre animals, since no changes in the amplitude of mEPSCs in the Ca1 region of the hippocampus was detected (figure 43). To directly analyze the lateral diffusion and clustering of AMPA receptors, hippocampal cultures should be incubated with quantum dots conjugated with a primary antibody directed against the AMPA receptor subunit GluR2. Live cell imaging of stained hippocampal neurons should be performed to track the transport and clustering of AMPA receptors to the postsynaptic membrane in Cofilin 2^fl/fl Nestin-Cre animals. An altered lateral diffusion of AMPA receptors was observed in Cofilin 1^fl/fl CaMKII-Cre mice, while no alterations in the surface expression of NMDA receptors could be observed (Rust et al., 2010). NMDA-receptors are a second class of ionotropic glutamate receptors that mediates the fast and slow components of excitatory synaptic transmission. Since an increased number of immature (thin) spines, accompanied with a reduced level of mushroom shaped spines, were observed in Cofilin 2^fl/fl Nestin-Cre animals and thin spines mainly contain NMDA-receptors, this class of glutamate receptors should also be analyzed. The NMDA receptor surface expression should be analyzed by radio-ligand binding of [³H]-labelled MK-801, an uncompetitive antagonist of the NMDA receptor. Changes in the number of NMDA receptors cannot be detected in mEPSCs, since these receptors are voltage-gated and open when the cell is depolymerized to -40mV. During the electrophysiological recordings cells were patched at -70mV, restricting the opening of NMDA receptors and their contribution to the recorded mEPSCs. Another possibility would be to examine the ratio between the NMDA- and AMPA response in electrophysiological studies. These studies could show that different types of glutamate receptors depend differently on Cofilin 2 for their transport and clustering and are therefore distinctly affected by the loss of Cofilin 2. Further it was also shown that NMDA and AMPA receptors are differently sensitive to drugs altering actin dynamics (Allison et al., 1998; Kirsch and Betz 1995). This indicates that actin contributes to the differential organization of distinct pools of postsynaptic receptors to obtain a postsynaptic specialization. Thin spines represent immature spines and mainly contain

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NMDA receptors, while the maturation of spines is accompanied with an occurrence of AMPA receptors in the postsynaptic density. Thereby the maturation of spines results from the lateral transport of AMPA-receptors from the extrasynaptic space into the postsynaptic density (Fortin et al., 2012; Takahashi et al., 2009). The finding of an increased number of immature spines suggests a role for Cofilin 2 in AMPA receptor transport during the maturation process, although our preliminary electrophysiology findings did not reveal changes in the number of AMPA receptors in Cofilin2^fl/fl Nestin-Cre animals. One possibility would be the fact that the recordings were performed on P21 old mice and therefore at a developmental time point were synaptogenesis was still not completed. At that early time point the pruning of synaptic connections takes place removing synaptic contacts that are not used rather than the maturation of spines to strengthen synaptic contacts. Therefore the transport of AMPA receptors could be more important and frequently at a later time point, masking a defect in AMPA receptor number in electrophysiological studies performed on young Cofilin 2 deficient animals. Zhou et al showed that F-actin stabilization by jasplakinolide results in a reduced endocytosis of AMPA receptors from the PSD, while actin depolymerization via latrunculin accelerates AMPA receptor internalization into the PSD (Zhou et al., 2001). In Cofilin 1^fl/fl CamKII-Cre mutants an altered number of AMPA receptors were found, implicating Cofilin 1 in the lateral diffusion of AMPA-receptors (Rust et al., 2010).

Additionally an increase in mushroom-shaped spines was observed, highlighting the possibility that the loss of Cofilin 1 blocks AMPA receptor endocytosis, which leads to an increase in the number of AMPA receptors and therefore to an increased PSD area. This increase in the PSD area then elevates the number of mushroom-shaped spines.

In the Cofilin 2^fl/fl Nestin-Cre knockout mice no changes in the number of AMPA-receptors (figure 43) and further the opposite effect on mushroom shaped spines was detected, with a decreased number on cortical pyramidal neurons (figure 41). Studies with GFP-tagged actin revealed two distinct actin pools in dendritic spines (Honkura et al., 2008). On the one hand stable longitudinal filaments present along the core of spines that are involved in the exo - endocytic trafficking of receptors, as well as the overall stability of the spine. On the other hand the dynamic sub-plasmalemmal actin network at the postsynaptic scaffold responsible for the anchoring and organization of receptor pools and thereby for the morphological remodeling of the spine head. Differences in the turnover rates and structures of these filaments are important for different postsynaptic mechanisms. One possibility is that the transport of AMPA-receptors was not affected due to the loss of Cofilin 2, but the increase in spine size upon maturation was blocked due to the increased level of F-actin by the loss of an actin depolymerizing factor. The more static actin filaments in dendritic spines could interfere with the anchoring and organization of AMPA receptors, necessary for the maturation of spines, while the basic synaptic transmission could be fulfilled. Thereby it was

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also shown that the trafficking of AMPA receptors and the internalization can occur independently of spine shape changes (Cingolani and Goda 2008). The loss of Cofilin 2 could have a stronger impact on the more dynamic sub-plasmalemmal actin network, responsible for the anchoring and clustering of receptors, than the more static longitudinal filaments along the core of spines for the trafficking of receptors. Thereby Cofilin 2 could fulfill a role in receptor anchoring and clustering rather than in the transport, while Cofilin 1 seems to be involved in both processes.

A positive correlation between the dimension of the spine head and the PSD area, and between the PSD area and the number of synaptic glutamate receptors exists. Therefore spine head size and synaptic efficiency are highly correlated and a reduced number of mushroom-shaped spines, which represent ´strong synapses´, could lead to a reduced synaptic efficiency. Additionally alternations in actin dynamics in dendritic spines are associated with mental retardation (Meng et al., 2002; Pontrello and Ethell 2009). Therefore reduced actin rearrangements at spines can impact on learning and memory processes.

Impairment in working memory processes were detected in ADF^-/- Cofilin 2^fl/fl Nestin-Cre animals (figure 57). The single knockout of Cofilin 2 did not lead to significant changes in working memory, although a tendency for an impaired working memory was detected (figure 50). In the hippocampus of newborn and adult animals an upregulation of ADF and Cofilin 1 was detected in Cofilin 2^fl/fl Nestin-Cre animals, which could compensate the loss of Cofilin 2 and milder the effect on the working memory (figure 21 and 33). Additionally a link exists between morphological changes and increased synaptic efficiency due to the process of LTP. After induction of LTP an increase in the PSD area could be observed (Hering and Sheng 2001), which also reflects an increase in the number of mushroom-shaped spines.

Therefore the reduced number of mushroom-shaped spines in Cofilin 2 deficient animals could also indicate an impaired induction of LTP. Actin is the major cytoskeletal protein found in pre- and postsynaptic terminals and a modulation of actin is likely to drive cytoarchitectural changes that are associated within synaptic plasticity. Rust et al showed with electrophysiological studies that the deletion of Cofilin 1 impairs the late phase of LTP, although the induction of LTP is not affected (Rust et al., 2010). The analysis of LTP in Cofilin 2^fl/fl Nestin-Cre animals should be conducted in further electrophysiological experiments to clarify the role of Cofilin 2 for LTP.

Further the presence of F-actin at synapses is most critical during early development, while the disruption of actin in mature synapses does not disrupt clustering of synaptic proteins (Yao et al., 2006). This observation implicates actin in the establishment and maturation of synapses during development. In young synapses the depolymerization of F-actin results in dispersion of synaptic vesicles and N-cadherin clusters. This shows that during early developmental stages adhesion molecules are both recruited and stabilized by actin and

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actin clustering is required downstream of neuronal adhesion for the clustering of vesicles.

The specification of synapses is mediated via cell-cell contact between transmembrane molecules and therefore specifies the sites for presynaptic assembly. Cadherins are adhesion molecules that form trans-synaptic homophilic dimers and engage F-actin rearrangements through catenins both on pre- and postsynaptic sides. Thereby cadherin-mediated adhesion directly triggers presynaptic actin polymerization and vesicle trapping through Rac GEFßPix. Reduced actin dynamics through the loss of Cofilin 2 could inhibit the active actin reorganization upon signaling of cell-cell contact and thereby prevent the assembly of presynaptic compartments. This could result in a decreased number of functional synapses and could explain the increased number of thin immature spines. The maturation of spines also depends on synaptic activity. An increased neuronal excitability by application of a GABA_A receptor antagonist, results in an increased spine density and area, while an inhibition of vesicle release by botulinum toxin causes a significant decrease in spine density (McKinney et al., 1999). This means that thin spines represent immature spines without a presynaptic partner. Therefore the increased number of thin spines could indicate a decreased number of functional synapses. This hypothesis could be tested with a staining of vGLUT1 and PSD-95, whose colocalization indicates a functional synapse or by EM micrographs.

7.3.3. Cofilin 2 fulfills distinct functions in the presynaptic