Molecular and Cellular Neuroscience 63 (2014) 114–123

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Role of synucleins in traumatic brain injury — An experimental in vitro and in vivo study in mice☆ Irina Surgucheva a,b, Shuangteng He c, Megan C. Rich d,e, Ram Sharma f, Natalia N. Ninkina g, Philip F. Stahel d,e, Andrei Surguchov a,b,⁎ a

Retinal Biology Research Laboratory, VA Medical Center, 4801 East Linwood, Boulevard, Kansas City, MO 64128, USA Department of Neurology, Kansas University Medical Center, Kansas City, 3901, Rainbow Boulevard, Kansas City, KS 66160, USA Stroke Research Laboratory, Kansas City Department of Veterans Affairs Medical Center, 4801 East Linwood Boulevard, Kansas City, MO 64128, USA d Department of Orthopaedics, University of Colorado, School of Medicine, Denver Health Medical Center, Denver, CO 80204, USA e Department of Neurosurgery, University of Colorado, School of Medicine, Denver Health Medical Center, Denver, CO 80204, USA f Research Service, VA Medical Center, 4801 East Linwood Boulevard, Kansas City, MO 64128, USA g School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK b c

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Article history: Received 22 July 2014 Revised 16 October 2014 Accepted 23 October 2014 Available online 24 October 2014 Keywords: Traumatic brain injury In vitro scratch injury model Protein aggregation Synucleins Cofilin-actin rods

a b s t r a c t Synucleins are small prone to aggregate proteins associated with several neurodegenerative diseases (NDDs), however their role in traumatic brain injury (TBI) is an emerging area of investigation. Using in vitro scratch injury model and in vivo mouse weight-drop model we have found that the injury causes alterations in the expression and localization of synucleins near the damaged area. Before injury, α-synuclein is diffused in the cytoplasm of neurons and γ-synuclein is both in the cytoplasm and nucleus of oligodendrocytes. After the scratch injury of the mixed neuronal and glial culture, α-synuclein forms punctate structures in the cytoplasm of neurons and γ-synuclein is almost completely localized to the nucleus of the oligodendrocytes. Furthermore, the amount of post-translationally modified Met38-oxidized γ-synuclein is increased 3.8 fold 24 h after the scratch. α- and γ-synuclein containing cells increased in the initially cell free scratch zone up to 24 h after the scratch. Intracellular expression and localization of synucleins are also changed in a mouse model of focal closed head injury, using a standardized weight drop device. γ-Synuclein goes from diffuse to punctate staining in a piriform cortex near the amygdala, which may reflect the first steps in the formation of deposits/inclusions. Surprisingly, oxidized γ-synuclein co-localizes with cofilin-actin rods in the thalamus, which are absent in all other regions of the brain. These structures reach their peak amounts 7 days after injury. The changes in γ-synuclein localization are accompanied by injury-induced alterations in the morphology of both astrocytes and neurons. Published by Elsevier Inc.

1. Introduction Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. In the US alone, over 1.7 million individuals suffer from TBI (Faul et al., 2010; Murray and Lopez, 1997; Hyder et al., 2007) at an estimated cost of over $60 billion and 200,000 hospitalizations each year (Finkelstein et al., 2006; Langlois et al., 2006). Primary injury occurs at the moment of trauma causing immediate contusion, hemorrhage, diffuse axonal injury, and ischemia (Vink and Nimmo, 2009; Kumar and Loane, 2012). TBI patients display many of the same pathologies that are associated with several neurodegenerative diseases (NDDs) (Guo et al., 2000; Lye and Shores, 2000; Ikonomovic et al., 2004; Goldman et al., 2006). A link ☆ The authors declare no competing financial interests. ⁎ Corresponding author at: Retinal Biology Research Laboratory, VA Medical Center, 4801 East Linwood, Boulevard, Kansas City, MO 64128, USA. E-mail address: [email protected] (A. Surguchov).

http://dx.doi.org/10.1016/j.mcn.2014.10.005 1044-7431/Published by Elsevier Inc.

between TBI and the subsequent development of AD has been documented (Guo et al., 2000; Lye and Shores, 2000; Ikonomovic et al., 2004). Likewise, TBI is an epidemiological risk factor for the development of sporadic PD (Goldman et al., 2006) and other NDDs (Blennow et al., 2012). Various biochemical changes following TBI include altered protein trafficking, protein aggregation, complement activation and altered cytoskeletal organization (Blennow et al., 2012; Neher et al., 2014; Smith et al., 2003; Uryu et al., 2007). Axonal transport disruption in TBI contributes to the subsequent prolonged period of axon loss (Higuchi et al., 2002; Morfini et al., 2002) and causes the rapid and long-term accumulation of several proteins that compose key pathologic aggregates similar to those found in AD and PD (Lye and Shores, 2000; Ikonomovic et al., 2004; Goldman et al., 2006). Of these proteins, the most widely studied in TBI are β-amyloid precursor protein (APP), amyloid-β (Aβ) peptides, neurofilament proteins, Tau and synucleins (Smith et al., 2003; Uryu et al., 2007; Chen et al., 2009; Blennow et al., 2012). Deposits of aggregated α-synuclein have been demonstrated in

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neurons and axons following a single episode of TBI in humans (Newell et al., 1999; Smith et al., 2003; Ikonomovic et al., 2004; Uryu et al., 2007) and in animal models of TBI (Uryu et al., 2003). α-Synuclein aggregates disrupt cell function and are accompanied by a neuroinflammatory response (Holmin et al., 1995; Holmin and Hojeberg, 2004) similar to what is observed in PD and other NDDs (Braak et al., 2002, 2006). The neuroinflammatory response leads to increased reactive oxygen species (ROS), oxidation and aggregation of proteins thus causing synaptic dysfunction (Nakamura et al., 2012). Furthermore, α-synuclein is elevated in the cerebrospinal fluid of TBI patients and can be used as a biomarker for the diagnosis and prognosis of TBI patients (Mondello et al., 2013). While the role of α-synuclein in NDDs and in TBI appears to be somewhat established (reviewed in Surguchov, 2013; Trojanowski and Lee, 1998) the function of another member of the family, γsynuclein, in pathology remains to be elucidated. Similar to α-synuclein, γ-synuclein is an aggregation prone protein which forms intracellular inclusions in NDDs (Galvin et al., 2000; Surgucheva et al., 2002, 2012; Ninkina et al., 2009; Bachurin et al., 2012; Busch and Morgan, 2012). Here we show that TBI causes significant increase of aggregated oxidized γ-synuclein near the injury site. We also describe for the first time a colocalization of oxidized γ-synuclein with another product of oxidative stress, cofilin-actin rods. 2. Results We sought to determine the effect of TBI on the expression and localization of native α- and γ-synucleins and their post-translationally modified isoforms (phosphorylated α-synuclein and oxidized γsynuclein) using two models of this pathology, i.e. a scratch cellular model and a weight-drop mouse model. An in vitro confluent primary brain cell culture was subjected to a scratch injury as described elsewhere (Lööv et al., 2012, 2013). It produced a localized and distinct injury with clear borders (Fig. 1, B, arrowheads) to surrounding uninjured cells. Significant changes in the morphology of both astrocytes (red, A and B, GFAP positive staining) and neurons (green, C and D, α-synuclein positive staining) were observed in this scratch injury

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model. In a control culture of primary brain cells, astrocyte processes extend in all directions (A, the mean angle from vertical is 47 ± 34°), whereas after the scratch the astrocyte processes predominantly extend vertically (B, the mean angle from vertical is 28 ± 20°, p b 0.001). These results suggest that after the scratch the astrocyte processes are oriented towards the injured area. Neurons in control culture have long processes (C, arrows) with a total length of neurites per α-synuclein immunopositive cell equal to 26.9 μm/cell. Here, αsynuclein is diffusely distributed in both the cell body and in the processes. After the scratch the neuronal processes became shorter or disappeared (D, green) with a total length of neurites per cell reduced to 5.5 μm/cell. α-Synuclein arranged in a punctate pattern in the cytoplasm of the cell body in scratch injured tissue (D, arrowheads; D2, arrows). Similarly, in astrocytes the injury induced a punctate pattern of α-synuclein accumulation as well, predominantly in the perinuclear area (D1, arrows). No significant changes in the level of α-synuclein immunoreactivity were observed as a result of the injury. Alterations in the localization of γ-synuclein in response to the injury occurred mainly in oligodendrocytes (Fig. 2). In a control primary culture, γ-synuclein is present predominantly in the cell body (A, arrowheads) and to a lesser extent in processes (arrows), whereas after the scratch injury it is translocated to the nuclei (E, H, green, arrowheads). In control samples CNPase (B) is distributed throughout the cell body (arrowheads) and processes (arrows) of oligodendrocytes, while after the injury it is present predominantly in the cytoplasm (F, H, red, arrows). The injury does not affect the quantity of non-modified γ-synuclein (not shown), but increases 3.79 fold the level of oxidized γ-synuclein which is present as an oligomeric form with molecular weight ~48 kDa (Fig. 2I and J, lanes 5–8). Using a scratch assay with confluent primary brain cell culture, we then determined whether cells could be found within the injury site. We detected α-synuclein positive cells in the scratched area beginning 6 h after the injury (Fig. 3B, arrows), increasing at 24 h (C, green, arrows) and 48 h (D) after the injury. α-Synuclein positive cells in the scratch area were identified as neurons by double staining with

Fig. 1. IF staining of primary cell culture from mouse brain in control samples (A, C) and 24 h after the injury (B, D). A–D: red is GFAP, blue is DAPI. A and B: green is γsynuclein. C and D: green is α-synuclein. The scalpel cut creates a clear injury (B, between arrowheads) with surrounding cells that remain unharmed. Significant changes in the morphology of astrocytes (red, A, B) and neurons (green, C and D) occur after the injury. Cells immunopositive for α-synuclein in C and D were identified as neurons since they were stained by antibody to neuronal marker βIII-tubulin. Astrocytes become more elongated and their processes are located perpendicular to the cut after the injury (arrows, B). After the scratch neurons have lost their axons and become spread on the surface, punctate α-synuclein staining appears in the cytoplasm (D, arrowheads and D2, arrows). Accumulation of punctate α-synuclein in astrocytes is shown on D1. Bars on panels A and B correspond to 50 μM, on panels C and D — 20 μM, on panels D1 and D2 — 10 μM.

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Fig. 2. A–H — Intracellular localization of γ-synuclein in the primary culture of brain cells before (A–D) and after (E–H) the scratch. γ-Synuclein (green) in control samples (A) is localized in oligodendrocyte cell bodies (arrowheads) and processes (arrows). It is relocated to the nuclei 24 h after the scratch (E, H, arrowheads). CNPase, a marker for oligodendrocytes (red) is located in the cell body of control cells (B) and predominantly in the cytoplasm after the scratch (F, arrows). Scale bars correspond to 10 μm. I and J — Immunoblotting of extracts of primary cell culture from mouse brain after the scratch injury and control uninjured samples. I — Western blots immunostained with monoclonal C4 antibody to actin (green) and oxidized γsynuclein (red). MW — Molecular weight standards. BE — Extracts of mouse brain were used as controls. 1–4 — extracts from primary cell cultures from control samples; 5–8 — cultures harvested 24 h after the scratch. J — Quantification of oxidized γ-synuclein bands compared to actin in 4 independent experiments. Standard errors show that the difference between two means (0.125 ± 0.06 for combined controls and 0.357 ± 0.015 for combined scratch samples) is statistically significant (p b 0.05). Error bars for each mean bar were calculated after combining results of three independent Western blots.

βIII-tubulin antibody (E and F). In addition to neurons, we also observed astrocytes in the wound area at 24 h and these increased in number at 48 h (C and D, red, arrowheads). γ-Synuclein-positive cells were also found in the scratched area 48 h after the scratch (Fig. 4B, green, arrows) together with astrocytes (red, arrowhead). Some of γ-synuclein-positive cells are β-tubulin-positive neurons, others are CNPase positive oligodendrocytes. We then examined synuclein localization in brains of mice subjected to a weight-drop model of closed head injury described in detail

elsewhere (Leinhase et al., 2006; Flierl et al., 2009). We identified oligodendrocytes (CNPase positive) and astrocytes (GFAP positive) in brain sections harvested 24 h after TBI or control sham surgery (Fig. 5). After the TBI a CNPase immunoreactivity calculated as percent of labeled area is increased from 16.1 ± 0.9% to 28.2 ± 1.2% (Fig. 5, red, C compared with A). The immunoreactivity of GFAP is also increased from 7.2 ± 0.3% to 14.3 ± 0.6% pointing to a glial cell reactivity, neuroinflammatory and astroglial response; percent of labeled area is increased from 16.1 ± 0.9% to 28.2 ± 1.2% (Fig. 5, red, C compared

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Fig. 3. Relocation of α-synuclein positive cells into the injured area in the primary culture of mouse E14 brain cells after wounding. The cells were immunolabeled for-α-synuclein (green) and GFAP (red). Images were captured immediately (0 h, A), 6 h (B), 24 h (C) and 48 h (D) after wounding. The presence of α-synuclein in the wound area was first noticed 6 h after the injury (B, green, arrows). The number of cells in the wound area increased after 24 h and remained the same at 48 h after the injury (D). By 48 h after the injury astrocytes were also found in the area of the wound (D, red, arrowheads). E and F. α-Synuclein is localized in the same cells (E) as the neuronal marker βIII-tubulin (F). Similar data were obtained in 3 other independent experiments. Wound areas on panels A–D are marked by thick vertical arrows at the left parts of panels A–D. Scale bars on E and F correspond to 10 μM.

with A). The immunoreactivity of GFAP is also increased from 7.2 ± 0.3% to 14.3 ± 0.6% pointing to a glial cell reactivity, neuroinflammatory and astroglial response after injury (red, D compared to B). We then sought to determine if changes in α- or γ-synuclein localization occurred after injury by immunolabeling. We did not find significant alterations in α-synuclein immunoreactivity (data not shown); however, injury-induced alterations in γ-synuclein immunoreactivity occurred (Fig. 5, green, B, D). γ-Synuclein is diffusely stained in the nucleus and cytoplasm before injury but after injury γsynuclein forms in a punctate pattern intracellularly predominantly in the nuclei (D, D1), which may reflect a step toward the formation of inclusions described elsewhere (Surgucheva et al., 2002, 2012). After the injury 67.8% of cells have granular γ-synuclein deposits (D); such cells were not found in the control samples (B). The deposits are localized to the piriform cortex near the amygdala. Therefore, the transition of γ-synuclein from diffuse staining (B and B1) to punctate

pattern of staining (D1) occurs in the area with pronounced glial reaction revealed by GFAP and CNPase. These results suggest that γ-synuclein may play a role in TBI similar to its role in NDDs where it is upregulated and forms inclusions and deposits (Galvin et al., 2000; Surgucheva et al., 2002; Ninkina et al., 2009). Because TBI is accompanied by increased ROS production and ROS has been shown to be important for oxidation of the actin binding protein, cofilin and formation of cofilin-actin rods in neurons (Bamburg et al., 2010; Bernstein and Bamburg, 2010), we examined the localization of oxidized γ-synuclein along with cofilin or actin immunostaining. Surprisingly, we found oxidized γ-synuclein associated with cofilin and actin stained structures within the mouse brain (Fig. 6). The stained structures are found only in the thalamus and appear to be localized to the edge of the nuclear compartment, probably at the nuclear surface (b1–b3). It is worth noting that the appearance of these structures is not a consequence of post-mortem changes, since they are present in

Fig. 4. Presence of γ-synuclein positive cells in the wound area. The cells were immunolabeled for γ-synuclein (green) and GFAP (red). A — Control cultured cells before scratch. B — Presence of γ-synuclein positive cells (arrows) and astrocytes (arrowheads) in the scratch area 48 h after the injury. Similar data were obtained in 3 other independent experiments.

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Fig. 5. Immunohistochemical staining of mouse brain cortex 24 h after experimental weight-drop TBI. A, B sham controls. C, D — After brain injury. γ-synuclein immunoreactivity is green. In A and C red is CNPase. In B and D, red is GFAP. Immunolabeling of both CNPase and GFAP are significantly increased after experimental TBI. γ-Synuclein which shows mainly diffuse staining in controls (B1) forms intra-cellular deposits in some cells after experimental TBI (D1). γ-Synuclein-positive intracellular inclusions are shown by arrows in D1. Scale bar is 40 μM for panels A and C, 20 μM for panels B and D, 5 μM for panels B1 and D1.

samples after animal perfusion with 4% formaldehyde containing solution. The presence of oxidized γ-synuclein in actin–cofilin rods was further confirmed in experiments with double staining of these structures using a combination of different antibodies (C, D and E). The rods have 11.2 ± 1.4 μm lengths. Then we compared the number of rods relatively to nuclei in thalamus samples at different time intervals after the injury with the number of rods in sham-operated mice (Fig. 7). The amount of rods after the injury compared to their number in sham operated mice is increased 24 h and 7 days post-injury, however at 48 h post-injury the quantity of rods is reduced both in experimental group and in sham-operated animals (C). 3. Discussion Despite the failures of TBI clinical trials to date, progress has been made in identifying the numerous alterations in molecular and cellular processes associated with TBI pathophysiology, including oxidative stress (Hall et al., 2010) and protein aggregation. TBI results in an array of pathophysiological responses and clinical consequences similar to a subset of those observed in PD and other NDDs including accumulation of aggregated synucleins and disturbances in the synuclein metabolism (Smith et al., 2003; Uryu et al., 2007; Beauchamp et al., 2008; Blennow et al., 2012). α-Synuclein is by far the best known and most studied among the three members of the synuclein family, due to its strong link with PD, the second most common human NDDs. In the case of α-synucleinopathies including PD, many lines of evidence demonstrate a strong association between α-synuclein aggregation and neurodegeneration (Trojanowski and Lee, 1998; Galvin et al., 2000). An important role of post-translationally modified synucleins in neurodegenerative processes has been also described (Oueslati et al., 2010; Surgucheva et al., 2012; Beyer and Ariza, 2013). The alterations in αsynuclein expression and localization in brain cells after TBI recapitulate some of the key features of α-synuclein pathologies that are seen in the brains of patients with synucleinopathies, such as aggregation of α-synuclein and the generation of post-translationally and conformationally modified forms of this protein. Interestingly, increased

immunoreactivity of not only α-, but also γ-synuclein has been reported after TBI in mammals, including humans (Uryu et al., 2003, 2007) and in spinal cord injury (Sakurai et al., 2009). Using two models of TBI we investigated a role of two members of the synuclein family involved in NDDs, α- and γ-synuclein in postinjury pathology. We describe here alterations in synuclein localization and increase in the level of post-translationally modified γ-synuclein. Previously, accumulation of γ-synuclein in the form of aggregated intracellular inclusions after injury was shown in association with cellspecific neuronal death in lamprey brain. Cells that accumulated synuclein also exhibited more ubiquitin-containing inclusions similar to what occurs during disease states. Importantly, γ-synuclein accumulation preceded and strongly correlated with subsequent neuronal death (Busch and Morgan, 2012). In another study using 24-month old mice that underwent cortical impact injury, α-synuclein immunoreactivity increased in the neutrophil of the cortex, stratum and hippocampus, while γ-synuclein increased in the striatal corticofugal fibers (Uryu et al., 2003). These results are in agreement with our data presented here pointing to the involvement of synucleins in secondary events in TBI. γ-Synuclein may play a role of a trigger linking protein aggregation and oxidative stress in TBI, since it easily oxidizes, forms aggregates and induces α-synuclein aggregation (Surgucheva et al., 2012). We found significant alterations in the localization of γ-synuclein in response to the injury in oligodendrocytes. Similar observations about predominant accumulation of synuclein in glial cytoplasmic inclusions (GCI) in oligodendrocytes were described previously (Tu et al., 1998; Papp et al., 1989; Wakabayashi et al., 1998). Such GCIs are considered as a center for the recruitment of misfolded proteins to form aggresomes. Some authors assume that oligodendrocytes adopt a modified process of aggresome formation and may upgrade the expression of synucleins selectively in response to some diseasespecific signal. Another possibility is that oligodendrocytes may actively take up synucleins released from adjacent neurons (Chiba et al., 2012). Previously it has been shown that both α-synuclein (Guo et al., 2000; Kanthasamy et al., 2011; Ding et al., 2013) and γ-synuclein

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Fig. 6. Immunolabeling of mouse thalamus 24 h after experimental TBI. Met38-oxidized-γ-synuclein (B, green) is in structures that co-immunolabel for cofilin (A, green) and actin (A, red). Nuclei in both images are stained with DAPI (blue). Boxed areas a1, a2, and a3 are taken from A; b1, b2, and b3 are taken from B. C, D and E: Structures that stain for the oxidized γ-synuclein (C) also immunostain for cofilin (D) as shown by the virtually complete overlay of the merged image (E).

(Surguchov et al., 2001; Surgucheva et al., 2003, 2008) affect gene expression on a transcriptional level. Therefore, TBI-induced shuffling of synucleins between cytoplasmic and nuclear compartments described here may have a significant role in the reprograming of gene expression and signal transduction in response to injury. After γ-synuclein is translocated to the nucleus in response to the injury, it may affect gene expression as described elsewhere (Surgucheva et al., 2003). Cell migration is a key event during the repair of damaged tissue after an injury. We can speculate how the movement of synuclein containing cells to an injured area can be involved in wound recovery. Although both α- and γ-synuclein are associated with several NDDs and participate in pathological alterations in brain cells, their deleterious functions are usually linked to overexpression, mutations or post-translational modifications. At the same time, non-modified synucleins expressed at a moderate level exhibit chaperone activity and may be neuroprotective (Manning-Bog et al., 2003; Albani et al., 2004; reviewed in Surguchov, 2008). We assume that the migration of non-modified synucleins to the injured area has a protective effect, whereas the role of post-translationally modified proteins depends on the nature of their post-translational modification. Several authors have hypothesized that synucleins possess prion-like properties and

participate in cell-to-cell transmission (Dunning et al., 2013; Lee et al., 2010) the mechanism and role of which is not completely understood. Another common feature of α- and γ-synuclein is that both proteins possess numerous sites for post-translational modifications that regulate their substrate affinity and can stabilize intermediate conformations (Esposito et al., 2007). We found here that oxidized γ-synuclein is present in actin–cofilin rods. These structures disrupt synaptic function, cause synaptic loss and are associated with several neurodegenerative diseases (Bamburg et al., 2010; Bernstein and Bamburg, 2010; Bernstein et al., 2012). Oxidized γ-synuclein is present in actin–cofilin rods located near the nuclei but not inside the nuclei (Fig. 6). To the best of our knowledge there is no previous report on γ-synuclein association with the actin–cofilin rods. Cofilin is a critical actin-depolimerizing factor, and the function of which is regulated by phosphorylation. TBI causes cofilin dephosphorylation and the resulting increase in cofilin activity could degrade the actin-rich cytoskeleton (Campbell et al., 2012). Injury-induced modulation of the actin cytoskeleton (Garland et al., 2012) and a role of another member of the synuclein family, α-synuclein in these processes have been described previously (Esposito et al., 2007; Sousa et al., 2009). Furthermore, quantitative proteomics assays suggest that αsynuclein interacts with many components of the actin cytoskeleton

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Therefore, new mechanisms underlying TBI induced pathogenesis may be due to the modulation of actin–cofilin rod–γ-synuclein interaction, the shifting of γ-synuclein state from diffuse to punctate form and its translocation to the nucleus. It is a matter of further investigation to reveal whether the delivery of cells expressing synucleins to the damaged sites described here plays a role in the repair of the injured area. The characterization of the association of synucleins with cytoskeletal components could reveal new insight into their physiological role. 4. Experimental methods 4.1. Animals Male mice of the C57BL/6 strain (Jackson Laboratory) were used. All experiments with animals were approved by the Institutional Animal Care and Use Committee (Animal Welfare Assurance Number A3752-01). 4.2. Scratch model Fig. 7. Percent of nuclei with adjacent rods. The experiments were repeated four times; asterisks show statistically significant differences (p b 0.05) between sham operated mice and animals after injury.

(e.g. actin, cofilin, destrin, F-actin capping proteins) (Zhou et al., 2004) however, axonal transport of α-synuclein does not depend on actin cytoskeleton (Utton et al., 2005; Roy et al., 2008). The role of γ-synuclein in the modulation of actin cytoskeleton is unknown. It is intriguing to determine what role γ-synuclein plays in actin–cofilin rod formation and whether it is implicated in the reorganization of the actin cytoskeleton. Presumably γ-synuclein may play a role in the modulation of actin–cofilin rod formation similar to α-synuclein regulating actin cytoskeletal structure and dynamics (Sousa et al., 2009). The kinetic of changes in the amount of rods after the injury (elevation after 24 h and 7 days post-injury, but reduction at 48 h post-injury) is similar to responses to trauma described for other proteins. For example, TBI causes some upregulation of phosphorylated (pSer3-cofilin) shortly after the trauma, then the amount of the protein decreases significantly after 24 h and finally considerably increases after 7 days (Campbell et al., 2012). The kinetic of such changes reflects acute cell/tissue reaction on injury, then a period of some adaptation, after which the reaction returns again. This role of γ-synuclein can be identified in future experiments using overexpression and knockdown approaches that we have used previously (Surgucheva et al., 2008). Interestingly, while nonphosphorylated cofilin is a regulator of actin dynamics, phosphorylated cofilin binds to membrane and is implicated in membrane and lipid metabolism, as well as in phospholipase D1 regulation (Han et al., 2007). Similar properties of synucleins in membrane binding and interaction with phospholipase are described for members of the synuclein family, including γ-synuclein (Shaw et al., 2004; Ducas and Rhoades, 2012; Guo et al., 2012). Thus, we can assume that cofilin–γ-synuclein interaction may be mediated by a membrane. The existence of oxidized γ-synuclein in cofilin-actin rods exclusively in thalamus, but not in other brain regions may be explained by a combination of several reasons: the highest level of γ-synuclein expression in this part of the brain (Su et al., 2004 and http://commons. wikimedia.org/wiki/File:PBB_GE_SNCG_209877_at_fs.png), a relatively high level of cofilin in thalamus (Li et al., 2006) and elevated oxidative stress and high protein oxidation level in the thalamus (Benedict et al., 2007). Interestingly, we did not find colocalization of non-modified γ-synuclein in actin–cofilin rods, suggesting that oxidative stress may modulate γ-synuclein binding to these rods. Regulation of actin cytoskeleton by post-translational modifications of the cytoskeletal proteins and its connection with human diseases is described previously (Sun et al., 2013).

The neuron/glia co-culture scratch model was used as described previously (Lööv et al., 2012, 2013) with slight modifications. In this cell culture model the neurons and glia were cultured together in the same medium throughout the experiment. A microglia-free mixed culture of primary neurons, astrocytes and oligodendrocytes was used as described previously (Lööv et al., 2012, 2013). Briefly, E14 mouse cortices were first grown into neurospheres, seeded as single cells on glass coverslips and differentiated for 8 days into neurons, astrocytes and oligodendrocytes. The cell layer was injured by scratching with a scalpel 20 times in two directions. After injury, the plates were incubated for 6, 24 or 48 h, fixed and stained by immunocytochemistry against specific markers for neurons (βIII tubulin), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (2′,3′-cyclic-nucleotide 3′-phosphodiesterase, CNPase). To generate a neural cell culture, dissected cortices from E14 mice were dissociated in GIBCO Hank's Balanced Salt Solution supplemented with 8 mM HEPES buffer, 50 units of penicillin and 50 μg/ml of streptomycin (Invitrogen/Life Technologies, Carlsbad, CA), hereafter referred to as HBSS. The cell suspension was centrifuged and cells were resuspended in a medium (GIBCO Dulbecco's Modified Eagle Medium, DMEM/F12) with GlutaMAX supplemented with 50 units/ml penicillin and 50 μg/ml streptomycin, 8 mM HEPES buffer, B-27 serum free supplement fortified with 10 ng/ml Fibroblast Growth Factor 2 (FGF2) (Invitrogen/Life Technologies) and 20 ng/ml natural mouse Epidermal Growth Factor (EGF, Becton Dickinson, Franklin Lakes, NJ). The cells were grown non-adherently into neurospheres and passaged every 3rd to 5th days by dissociation in HBSS and resuspension in new medium supplemented with mitogens at concentrations described above. Prior to the experiment, cells were dissociated in HBSS and plated as a monolayer on coverslips coated with poly-Lornithine (Sigma-Aldrich, St. Louis, MO) and laminin (Invitrogen/Life Technologies, Carlsbad, CA). The first 24–48 h cells were maintained in EGF and FGF2 supplemented medium and thereafter replaced with mitogen-free medium to initiate cell differentiation. The medium was replaced every second to third days during the differentiation period of 8–11 days. The cell layer was injured by a scratch with a scalpel tip and 24 h after the scratch the culture was washed 3 × 5 min with Dulbecco's Phosphate-Buffered Saline (DPBS) and fixed in freshly prepared 2% paraformaldehyde (PA) for 60 min at RT. After additional washing, cells were permeabilized and non-specific sites blocked with DPBS, containing 0.1% Triton X-100 and 5% goat serum (NGS) for 45 min at RT. Incubation in primary antibody was carried out overnight at +4 °C followed by washing with DPBS (3 × 10 min) with shaking. Samples were then incubated for 2 h at RT with alexa-488-donkey-anti-rabbit

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antibody (1:200) and/or alexa-594-goat anti-mouse antibody (1:250). Washing in DPBS was repeated and the samples mounted in Vectashield media (Vector, Olean, NY) supplemented with DAPI. 4.3. Microscopy Images were taken using a Nikon Eclipse 80i microscope (Melville, NY) with an Olympus DP72 camera; cellSense Standard software was used for image analysis (Center Valley, PA). 4.4. Mouse weight drop model of TBI The experiments were performed in compliance with the standards of the NIH Guide for Care and Use of Laboratory Animals (NIH publication no. 86-23, 1996) and were approved by the Institutional Animal Care and Use Committee of the University of Colorado (IACUC, protocol no. B-79612(01)1E). All mice used in this study were pathogen free males of the C57BL/6 strain (Jackson Laboratory, cat. no. 000664), 10–12 weeks of age, weighing 25–35 g and were housed in single cages for at least 7 days before experimental procedures. A standardized weight-drop device was used to produce a focal closed head injury to the left hemisphere as previously described (Leinhase et al., 2006; Flierl et al., 2009). The animals were random sorted into experimental groups (trauma, sham operated and control groups). After adequate isoflurane anesthesia, a midline longitudinal scalp incision was performed, the skin was retracted, and the skull exposed. The head was immobilized and a 333 g weight was dropped on the skull from a height of 2.5 cm, resulting in a focal blunt injury to the left hemisphere. After trauma, the mice received supporting oxygenation with 100% O2 until fully awake. Analgesia was provided by intraperitoneal injection of 0.05 mg/kg fentanyl immediately prior to the experimental procedure, followed by the injection of 0.01 mg/kg fentanyl every 12 h until euthanization. Sham operated animals received all procedures with regard to analgesia, anesthesia and scalp incision but were not subjected to TBI. The extent of posttraumatic neurological impairment was assessed at defined time intervals after trauma (t = 24 h, 48 h and 7 days). At corresponding time-points, mice were euthanized, and brain tissue was surgically removed, snap-frozen in liquid nitrogen and stored at − 80 °C for future analysis by immunohistochemistry. Assessment of injury severity and modality was carried out as described previously (Leinhase et al., 2006; Beauchamp et al., 2008; Flierl et al., 2009). Frozen slides with coronal brain cryosections of 10 μm thickness were fixed in 3.7% PA at PBS, pH = 7.2 for 20 min at RT and washed 3 times in PBS for 5 min on an orbital shaker. For antigen retrieval, the slides were treated in 10 mM citrate buffer with 0.05% Tween (pH = 6) for 1.5 min in a microwave oven and cooled for 20 min at RT. The samples were then permeabilized in a 0.2% Triton X-100 solution for 45 min at RT washed in PBS 3 × 5 min and incubated in blocking solution (15% NHS with 0.2% Triton) for 2 h at RT. Then the samples were incubated overnight in primary antibody at + 4 °C in 1% NHS and 0.05% Triton. 4.5. Antibodies (Abs) for WB and immunofluorescence (IF) The following primary Abs were used; α-synuclein — polyclonal anti-rabbit (C-20, Cat. # sc-7011-R, Santa Cruz, Dallas, TX, dilution 1:300), polyclonal γ-synuclein anti-rabbit antibody (Abcam Cambridge, MA, ab55424, dilution 1:250), γ-synuclein — monoclonal antimouse, clone 1H10D2 (Santa Cruz: sc-65979, dilution 1:350), anti-β-tubulin III — rabbit monoclonal, clone EP 1569Y, Cat# 04-1049 (Millipore, Billerica, MA, dilution 1:700), mouse monoclonal antibodies to mouse CNPase-clone 11-5B (Sigma, St. Louis, MO, Cat# C5922, dilution 1:400), rabbit muscle actin clone C4 (Millipore, Billerica, MA, Cat # Mab 1501R, dilution 1:7000 for WB) and chicken cofilin (MAb22; Shaw et al., 2004, dilution 1:600 for IF and 1:6000 for WB). Generation

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of Ab to γ-synuclein with an oxidized methionine-38 (Met38) (21st Century Biochemicals, Marlboro, MA, dilution 1:200 for IF and 1:600 for WB) is described previously (Surgucheva et al., 2012). Briefly, Ab was raised in rabbits using as an antigen peptide Ac-TKEGV [M-O] YVGAKT-Ahx-C-amide, where [M − O] is methionine sulfoxide. The Ab to γ-synuclein with oxidized methionine was affinity purified on a column containing antigenic peptide. Purified Ab was immunodepleted using an immunodepletion column with unmodified peptide without oxidized methionine. The absence of cross reaction between Abs to different synuclein isoforms was confirmed by Western blotting (Surgucheva et al., 2014). Rabbit polyclonal antibody 1439 that recognizes both cofilin and phospho-cofilin (dilution 1:2000) was used as described (Shaw et al., 2004). The following secondary antibodies were used; Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 594 donkey anti-mouse (Jackson ImmunoResearch Lab, West Grove, PA,) and donkey anti-sheep-IgGTR (sc-3913; Santa Cruz, Dallas, TX). Controls without secondary antibodies were used for each IF staining. 4.6. Preparation of mouse brain homogenates Brains were extracted following euthanasia, separated into left and right hemispheres and immediately homogenized in lysis buffer (Sigma, St. Louis, MO) containing 100 mM Tris–HCl pH = 7.5, 150 mM NaCl, 0.5% SDSNa, 0.5% Nonidet P-40, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 5 μg/ml pepstatin, and 1 mM phenyl–methyl– sulphonyl fluoride in deionized water. Homogenization was carried out in an Ultra Turrax Homogenizer. The brain homogenate was centrifuged at 13,000 g for 15 min and the protein content of the supernatant was determined by a BCA protein assay kit (Pierce Biotechnology, Rockford, IL). 4.7. Western blotting Extracts from a whole brain and thalamus for Western blotting were prepared as described elsewhere (Flierl et al., 2009). The brain extracts (30 μg total protein) were analyzed by WB in a 12% polyacrylamide gel (PAAG) in the presence of SDS as described previously (Surgucheva et al., 2002, 2012). After electrophoresis, proteins were transferred onto an Immobilon-FL transfer membrane (0.45 μm; Millipore, Chelmsford, MA). Nonspecific binding sites were blocked by immersing the membrane in PBS with 5% nonfat dry milk for 1 h at room temperature (RT) on an orbital shaker. The membrane was incubated in primary antibody diluted in 5% dry milk with 0.1% Tween 20 overnight at + 4 °C. As a secondary antibody we used IR Dye 680RD, goat antirabbit and IR Dye800 CW goat anti-rabbit. For quantitative imaging of bands an infrared imaging system (Odyssey, LiCor, Lincoln, NE) was used. Other details have been described previously (Surgucheva et al., 2008, 2012). 4.8. Calculation of labeled area on images Labeled area was analyzed using seven sections from each mouse; a minimum of three mice were included in sham and experimental groups. The percentage of labeled area on images was calculated using MetaMorph Software (Molecular Devices, LLC, Sunnyvale, CA). The CNPase immunoreactive area was measured in an 8 bit RGB color range of 0–255 at threshold above 90 in the red channel. The GFAP immunoreactive area was measured at threshold above 70 in the red channel. The ratio of labeled area above threshold to total tissue area was calculated. 4.9. Measurements of neurite and rod length The length of neurites was determined as follows. The total length of neurites was measured on at least four independent images taking into

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consideration the magnification. The value obtained was divided by the number of α-synuclein-immunoreactive cells. Rod length was calculated by a similar method. 4.10. Statistical analysis All experiments were performed at least in triplicate. The data were expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA). One way ANOVA followed by Dunnett's multiple comparison tests (versus control) was used to analyze the statistical significance among multiple groups. p values b0.05 were considered statistically significant. A Student's t test was used to assess significant differences between groups. Acknowledgments This work was supported by VA Merit review grant 1I01BX000361 and the Glaucoma Foundation grant QB42308. We thank Dr. James R Bamburg from the Department of Biochemistry and Molecular Biology, Colorado State University for a generous gift of cofilin antibodies and many helpful suggestions. References Albani, D., Peverelli, E., Rametta, R., Batelli, S., Veschini, L., Negro, A., Forloni, G., 2004. Protective effect of TAT-delivered alpha-synuclein: relevance of the C-terminal domain and involvement of HSP70. FASEB J. 18 (14), 1713–1715. Bachurin, S.O., Shelkovnikova, T.A., Ustyugov, A.A., Peters, O., Khritankova, I., Afanasieva, M.A., Tarasova, T.V., Alentov, I.I., Buchman, V.L., Ninkina, N.N., 2012. Dimebon slows progression of proteinopathy in γ-synuclein transgenic mice. Neurotox. Res. 22 (1), 33–42. Bamburg, J.R., Bernstein, B.W., Davis, R.C., Flynn, K.C., Goldsbury, C., Jensen, J.R., Maloney, M.T., Marsden, I.T., Minamide, L.S., Pak, C.W., Shaw, A.E., Whiteman, I., Wiggan, O., 2010. ADF/cofilin-actin rods in neurodegenerative diseases. Curr. Alzheimers Res. 7 (3), 241–250. Beauchamp, K., Mutlak, H., Smith, W.R., Shohami, E., Stahel, P.F., 2008. Pharmacology of traumatic brain injury: where is the “golden bullet”? Mol. Med. (Camb. Mass) 14 (11–12), 731–740. Benedict, J.W., Sommers, C.A., Pearce, D.A., 2007. Progressive oxidative damage in the central nervous system of a murine model for juvenile Batten disease. J. Neurosci. Res. 85 (13), 2882–2891. Bernstein, B.W., Bamburg, J.R., 2010. ADF/cofilin: a functional node in cell biology. Trends Cell Biol. 20 (4), 187–195. Bernstein, B.W., Shaw, A.E., Minamide, L.S., Pak, C.W., Bamburg, J.R., 2012. Incorporation of cofilin into rods depends on disulfide intermolecular bonds: implications for actin regulation and neurodegenerative disease. J. Neurosci. 32 (19), 6670–6681. Beyer, K., Ariza, A., 2013. α-Synuclein posttranslational modification and alternative splicing as a trigger for neurodegeneration. Mol. Neurobiol. 47 (2), 509–524. Blennow, K., Hardy, J., Zetterberg, H., 2012. The neuropathology and neurobiology of traumatic brain injury. Neuron 76, 886–899. Braak, H., Del Tredici, K., Bratzke, H., Hamm-Clement, J., Sandmann-Keil, D., Rub, U., 2002. Staging of the intracerebral inclusion body pathology associated with idiopathic Parkinsons disease (preclinical and clinical stages). J. Neurol. 249 (Suppl. 3), III/1–III/5. Braak, H., Bohl, J.R., Muller, C.M., Rub, U., de Vos, R.A., Del Tredici, K., 2006. Stanley Fahn Lecture: the staging procedure for the inclusion body pathology associated with sporadic Parkinson's disease reconsidered. Mov. Disord. 21, 2042–2051. Busch, D.J., Morgan, J.R., 2012. Synuclein accumulation is associated with cell-specific neuronal death after spinal cord injury. J. Comp. Neurol. 520 (8), 1751–1771. Campbell, J.N., Low, B., Kurz, J.E., Patel, S.S., Young, M.T., Churn, S.B., 2012. Mechanisms of dendritic spine remodeling in a rat model of traumatic brain injury. J. Neurotrauma 29 (2), 218–234 (20). Chen, X.H., Johnson, V.E., Uryu, K., Trojanowski, J.Q., Smith, D.H., 2009. A lack of amyloid beta plaques despite persistent accumulation of amyloid beta in axons of long-term survivors of traumatic brain injury. Brain Pathol. 19 (2), 214–223. Chiba, Y., Takei, S., Kawamura, N., Kawaguchi, Y., Sasaki, K., Hasegawa-Ishii, S., Furukawa, A., Hosokawa, M., Shimada, A., 2012. Immunohistochemical localization of aggresomal proteins in glial cytoplasmic inclusions in multiple system atrophy. Neuropathol. Appl. Neurobiol. 38, 559–571. Ding, H., Fineberg, N.S., Gray, M., Yacoubian, T.A., 2013. α-Synuclein overexpression represses 14-3-3θ transcription. J. Mol. Neurosci. 51 (3), 1000–1009. Ducas, V.C., Rhoades, E., 2012. Quantifying interactions of β-synuclein and γ-synuclein with model membranes. J. Mol. Biol. 423 (4), 528–539. Dunning, C.J., George, S., Brundin, P., 2013. What's to like about the prion-like hypothesis for the spreading of aggregated α-synuclein in Parkinson disease? Prion 7 (1), 92–97.

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