Subcellular localization of Cofilin 2 in neurons

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A summary over all performed colocalization studies and the localization of Cofilin 2 in defined neuronal subtypes is listed in the following table 2:

Brain area Marker protein Cell type Cofilin 2

expression

cortex Calbindin

Parvalbumin

smooth nonpyramidal interneurons in layer II/III

of the motor cortex

+

cerebellum Calbindin

Parvalbumin

Purkinje cells +

Parvalbumin Stellate and basket cells +

striatum Calbindin Medium spiny neurons +

Parvalbumin Fast-spiking interneurons - thalamus Parvalbumin Principal sensory and

motor relay nuclei

+ Substantia nigra Tyrosine hydroxylase Dopaminergic neurons of

the nigrostriatal pathway

+ Ventral tegmental

area

Tyrosine hydroxylase Dopaminergic neurons of the mesolimbic pathway

+ Midbrain (dorsal and

median raphe nuclei)

Tryptophan hydroxylase

Serotonergic neurons +- (subset)

hypothalamus Choline

acetyltransferase

Cholinergic neurons involved in memory

function

+

Brain stem Choline

acetyltransferase

Cholinergic neurons in the tegmental cholinergic

system

+- (subset)

cortex vGLUT1 Glutamatergic neurons

with low-release probability

+

thalamus vGLUT2 Glutamatergic neurons

with high-release probability

-

Table 2: Overview over the colocalization studies with a Cofilin 2-specific antibody FHU-I and different markers for neuronal subtypes in defined brain regions.

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accompanied by a marked shrinkage of spines. Thereby spine shrinkage is mediated by the activity of Cofilin, which implicates actin depolymerization to activity-induced reduction in the number of synaptic connections (Zhou et al., 2004). Additionally, actin is localized to the presynaptic site and there seems to be important for the formation of the active zone and the clustering of synaptic vesicles into defined pools (Nelson et al., 2013). Although the loss of Cofilin 1 in the forebrain only affected postsynaptic mechanisms (Rust et al., 2010), a double knockout of ADF and Cofilin 1 also displayed a presynaptic phenotype (Gorlich et al., 2011).

Thereby an enrichment of ADF in presynaptic terminals was observed, but the analysis of ADF^-/- mutant mice revealed no changes in presynaptic recruitment or exocytosis of synaptic vesicles. Additionally, postsynaptic mechanisms were unchanged in mice lacking ADF.

Görlich et al found an elevated level of Cofilin 1 in mice deficient for ADF, suggesting a compensatory effect of Cofilin 1 and a cooperated function of ADF and Cofilin 1 in regulation actin rearrangement at synapses. Additionally in mice deficient for ADF and Cofilin 1 a disturbed morphology of striatal excitatory neurons, accompanied by an increased glutamate release was observed, resulting in hyper-locomotion of ADF^-/- Cofilin 1^fl/fl CaMKII-Cre knockout animals. This phenotype was not detected in single mutants for Cofilin 1^fl/fl CaMKII-Cre or ADF^-/-, showing a functional redundancy for both proteins in synapses (Zimmermann et al., 2015). In this study the pre- and postsynaptic localization of Cofilin 2 was examined, which could play a role in specific synaptic functions.

4.2.1. Cofilin 2 is localized in pre and postsynaptic compartments

To analyze the expression of Cofilin 2 in synapses synaptosomes were prepared from the cortex of adult wt animals. Thereby synaptosomes are isolated synaptic terminals that have detached from the axon. After homogenization of cortical tissue and subsequent fractionation by centrifugation steps, a reseal of the plasma membrane encloses parts of the pre- and postsynaptic membrane, leaving behind functional synapses, which could be examined to study synaptic transmission. The synaptosomes were further fractionated to separate the pre- and postsynaptic site, as well as extrasynaptic proteins. The obtained lysates were incubated with PSD-95, as a marker for the postsynaptic site, Syntaxin-I as a marker for the presynaptic site and Synapthophysin as a general marker for synaptosomes. The expression of ADF/Cofilin family members was analyzed with specific antibodies for all three isoforms.

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Fig. 29: Cofilin 2 is expressed in pre- and post-synaptic terminals. A To examine the synaptic localization of Cofilin 2 synaptosomes from adult wt cortices were prepared. 10µg of every probe were loaded on a 10% and 15% gel and analyzed with antibodies against the ADF/Cofilin family and marker for the presynaptic site (Syntaxin), postsynaptic site (PSD-95) and a general marker for synaptosomes (synaptophysin). Controls for every protein of the ADF/Cofilin family were also loaded on the gel: brain control for Cofilin 1, muscle control for Cofilin 2 and a uterus control for ADF. B Immunofluorescence analysis on adult wt vibratome sections with an excitatory postsynaptic marker PSD-95 (Alexa594 red) and a Cofilin 2 specific antibody FHU-I (Alexa488 green) in the cortex. Nuclei were stained with Draq5 (blue). Yellow spots indicate and overlap between both signals. Images were taken at a magnification of 63x. Scale bar: 20µm The right panel shows a 3x zoom. Scale bar: 10µm C Colocalization studies between Cofilin 2 (Alexa488 green) and Gephyrin (Alexa594 red), which is an inhibitory postsynaptic marker. Yellow spots indicate an overlap between both signals. Adult wt vibratome sections were stained and the striatum was analyzed. Nuclei were stained with Draq5 (blue). Images were taken at a magnification of 63x. Scale bar: 20µm The right panel shows a 3x zoom. Scale bar: 10µm

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The Western blots of synaptosomes indicated an expression of Cofilin 2 in pre- and postsynaptic terminals (figure 29A). Also a pre- and postsynaptic expression of Cofilin 1 could be verified as already shown by Rust et al (2010). Only for ADF no signal could be observed in pre- and postsynaptic lysates. This is unexpected since the expression of ADF was already examined by Görlich et al via immunofluorescence analysis between ADF and synaptophysin or PSD-95, indicating a pre- and postsynaptic localization of ADF (Gorlich et al., 2011). Additionally, Rust et al also obtained a band for ADF in the presynaptic fraction of synaptosomal lysates (Rust et al., 2010). The functionality of the used antibody against ADF could be proven by the loaded uterus control, which exhibited a band at the height of ADF.

The successful preparation of synaptosomes was controlled with the antibody synapthophysin, which is a general marker for nerve terminals. This membrane glycoprotein of synaptic vesicles is ubiquitously expressed in all neurons and should give a signal in the total lysate of synaptosomes and the soluble extrasynaptic fraction. A band for synapthophysin could be examined in the total lysate of synaptosomes and the soluble extrasynaptic fraction, as expected. As a presynaptic marker served the membrane-protein syntaxin, which is associated with the docking of synaptic vesicles to the active zone. A signal for syntaxin could be observed in the total lysate, the extrasynaptic lysate and the presynaptic fractionation, which is expected since syntaxin is a soluble protein. PSD-95 was used as postsynaptic marker, due to its function in the anchoring of NMDA receptors in the postsynaptic membrane. Enrichment in the PSD-95 signal could be observed from the total synaptosomal lysate to the postsynaptic fractionation. Also a weak band for PSD-95 could be seen in the presynaptic lysate, showing that the first fractionation between the pre- and postsynaptic proteins was not sufficient enough and a small amount of postsynaptic proteins could be found in the presynaptic lysate. A fractionation of presynaptic proteins still occurred since the staining of syntaxin-1 displayed a signal in the presynaptic fraction. Additionally, Rust et al already confirmed the presynaptic localization of Cofilin 1 in synaptosomal preparations, which could be also seen in this blot (Rust et al., 2010). To confirm the obtained results from the Western blot, immunofluorescence analyses on adult wt vibratome sections were performed. In a colocalization staining between vGLUT1 and FHU-I a presynaptic localization of Cofilin 2 could be further affirmed (figure 28). The localization of Cofilin 2 to postsynaptic sites was further examined with a staining of PSD-95 for excitatory synapses and FHU-I. Also in this staining a colocalization between Cofilin 2 and PSD-95 was observed (figure 29B). To also analyze inhibitory synapses stainings with FHU-I and Gephyrin were performed, which anchors GABA and Glycine receptors in the postsynaptic membrane. A few yellow spots were examined in this staining, but more frequently the signals were in close proximity to one another and only displayed a small overlap (figure 29C).

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In summary all three ADF/Cofilin family members are found in synapses and display a pre- and postsynaptic localization. Thereby Cofilin 2 seemed to be localized only in a subset of excitatory and inhibitory postsynaptic compartments.

4.2.2. Cofilin 2 displays a broad expression in primary cortical cultures

To gain insight into the distribution of Cofilin 2 to specific neuronal structures, which exhibit different functions, the subcellular localization of Cofilin 2 in neurons was further analyzed in vitro in primary cultures. Therefore primary cortical neurons were prepared and the cells were fixed after 4 days in culture. Immunostainings with the neuronal marker betaIII-tubulin and a Cofilin 2-specific marker FHU-I were prepared. Thereby tubulin is the major component of microtubules and is a dimer composed of one alpha and one beta tubulin molecule. There are several isoforms of beta tubulin, which are expressed in a tissue-specific manner. BetaIII-tubulin is regarded to be a neuron-specific marker and has been suggested to be one of the earliest markers to signal neuronal commitment in the primitive neuroepithelium (Fanarrage et al., 1999). Thereby BetaIII-tubulin stained the cell soma, neurites and fine neurite branches. As seen in figure 30 Cofilin 2 displayed a broad expression in cortical neurons and was not restricted to a specific sub-compartment. Cofilin 2 expression was observed in every neurite, including fine branches and growth cones at the tips of neurites. Additionally a strong signal for Cofilin 2 was detected in the cell soma.

Fig.30: Subcellular localization of Cofilin 2 in primary cortical neurons. To analyze the localization of Cofilin 2 in neurons primary cortical cultures were prepared and the cells were fixed at div4 (4 days in vitro). The cells were stained with the neuronal marker betaIII-tubulin (A594 red) and the Cofilin 2-specific marker FHU-I (A488 green). Cofilin 2 was localized all over the cell, with a staining in the cell soma and neurites, as well as fine branches at the end of neurites. Images were taken at a magnification of 40x. The image was overexposed to analyze the localization of Cofilin 2 also in fine neurite branches. Scale bar: 20µm

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