sAPP rescues deficits in amyloid precursor ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2012 | 122 | 208–220

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doi: 10.1111/j.1471-4159.2012.07761.x

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*Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide South Australia, Australia Centre for Neurological Diseases, Hanson Institute, South Australia, Australia àMental Health Research Institute, University of Melbourne, Victoria, Australia §Department of Pathology and Bio21 Molecular Science and BioTechnology Institute, The University of Melbourne, Victoria, Australia

Abstract The amyloid precursor protein (APP) is thought to be neuroprotective following traumatic brain injury (TBI), although definitive evidence at moderate to severe levels of injury is lacking. In the current study, we investigated histological and functional outcomes in APP)/) mice compared with APP+/+ mice following a moderate focal injury, and whether administration of sAPPa restored the outcomes in knockout animals back to the wildtype state. Following moderate controlled cortical impact injury, APP)/) mice demonstrated greater impairment in motor and cognitive outcome as determined by the ledged beam and Barnes Maze tests respectively (p < 0.05). This corresponded with the degree of neuronal damage, with APP)/) mice having significantly greater lesion volume (25.0 ± 1.6 vs. 20.3 ± 1.6%, p < 0.01) and hippocampal damage, with less remaining CA neurons (839 ± 245

vs. 1353 ± 142 and 1401 ± 263). This was also associated with an impaired neuroreparative response, with decreased GAP-43 immunoreactivity within the cortex around the lesion edge compared with APP+/+ mice. The deficits observed in the APP)/) mice related to a lack of sAPPa, as treatment with exogenously added sAPPa post-injury improved APP)/) mice histological and functional outcome to the point that they were no longer significantly different to APP+/+ mice (p < 0.05). This study shows that endogenous APP is potentially protective at moderate levels of TBI, and that this neuroprotective activity is related to the presence of sAPPa. Importantly, it indicates that the mechanism of action of exogenously added sAPPa is independent of the presence of endogenous APP. Keywords: amyloid precursor protein, sAPPa, traumatic brain injury. J. Neurochem. (2012) 122, 208–220.

Traumatic brain injury (TBI) remains a significant health problem, with an estimated 10 million people affected annually by a head injury serious enough to result in death or hospitalization (Hyder et al. 2007). Following the initial impact cell death is ongoing because of the initiation of a number of secondary injury factors such as inflammation and excitotoxicity (Gaetz 2004). In response, a number of endogenous neuroprotective pathways are activated in an attempt to limit this damage (Keyvani and Schallert 2002). Our recent studies indicate that the amyloid precursor protein (APP) is involved in these pathways, as APP)/) mice had an exacerbation of cognitive and motor deficits following a mild diffuse TBI when compared with wildtype mice (Corrigan

et al. 2012a). This is likely because of the lack of the APP metabolite, sAPPa, which has neuroprotective and neurotrophic activity (Mattson 1997). Although, APP)/) mice

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Received March 01, 2012; revised manuscript received March 27, 2012; accepted April 12, 2012. Address correspondence and reprint requests to A/Prof Corinna van den Heuvel, Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005, Australia. E-mail: [email protected] 1 co-senior authors. Abbreviations used:(APP), amyloid precursor protein; (CCI), controlled cortical impact; (DCX), doublecortin; (MAP2), microtubule associated protein-2; (TBI), traumatic brain injury.

2012 The Authors Journal of Neurochemistry 2012 International Society for Neurochemistry, J. Neurochem. (2012) 122, 208–220

sAPPa rescues deficits in APP)/) mice post TBI | 209

were found to be more vulnerable following a mild diffuse TBI, it is unknown whether the presence of endogenous APP would remain protective after a more severe focal injury. Indeed, other endogenous neuroprotective agents, including oestrogen and progesterone in females, have been shown experimentally to be more effective against diffuse (Kupina et al. 2003), rather than focal injuries (Hall et al. 2005). Our previous studies have indicated that sAPPa improves functional and histological outcome following diffuse TBI in Sprague Dawley rats (Thornton et al. 2006) and controlled cortical impact injury (CCI) in wildtype mice (Corrigan et al. 2012b). However, it is unclear whether endogenously expressed APP is required for sAPPa to exert its neuroprotective effects. A previous in vitro study found that the presence of endogenous APP was required for sAPPa to improve survival of cells exposed to metabolic stress (Gralle et al. 2009), whereas knock-in of sAPPa was shown to be sufficient to rescue physiological deficits in APP)/) mice. Therefore the current study aims to assess whether the expression of endogenous APP is protective following a moderate to severe focal TBI and whether treatment with exogenous sAPPa is effective in APP)/) mice.

Methods Mice Generation of APP)/) mice has been described previously (Zheng et al. 1995), with both the APP+/+ and APP)/) mice on the same background strain, C57BLbj x 129sv. Male mice were used aged between 10–14 weeks and randomly assigned into sham, injured and treatment groups. Experimental protocols were approved by the Experimental Ethics committee of the Institute of Medical and Veterinary Science and conducted according to guidelines established for the use of animals in experimental research as outlined by the Australian National Health and Medical Research Council. Controlled cortical impact injury model An intraperitoneal injection of 2, 2, 2 tribromoethanol ethanol (250 mg/kg) was used to anaesthetize the mice, with surgery commencing once pedal foot reflexes were absent. Following a midline scalp incision, a 3 mm craniotomy was performed in the centre of the right parietal bone, with impact delivered with a 2 mm flat impactor tip at a depth of 1.5 mm in APP+/+ mice or 1.3 mm in APP)/) mice at a velocity of 5 m/s with a dwell time of 100 ms. Sham mice underwent craniotomy, but not CCI injury. The difference in deformation depths was to compensate for the smaller brain size of the APP)/) mice, with previous studies showing a reduction in brain weight of about 10% in APP)/) mice (Ring et al. 2007). Impact depth was varied as computer modelling has suggested that this parameter is more important in determining injury severity than the diameter of the impactor (Mao et al. 2010). Using these parameters at 5 h post-injury histological outcome in APP)/) and APP+/+ mice was the same, indicating a similar level of primary injury (Supplementary Data). At 15 min post-injury, a randomized subset of the APP)/) mice received an intracerebroventricular (ICV) injection of 2 lL of

sAPPa (APP18-611, 25 lM), with all other mice receiving an equal volume artificial CSF vehicle. The sAPPa was produced as previously described (Henry et al. 1997), expressed as a secreted protein in the methylotrophic yeast, Pichia pastoris. To facilitate treatment a 0.3 mm craniotomy was performed on the left side at the stereotaxic coordinates relative to bregma: posterior 0.5 mm, lateral 1 mm (Paxinos and Franklin 2007), with the needle lowered 2.5 mm and retracted 0.3 mm to allow injection at a rate of 0.5 lL/min. Motor outcome For assessment of motor deficits following focal injury, we used the ledged beam which has been previously shown to be a sensitive test of unilateral deficits (Bye et al. 2007; Semple et al. 2010). The beam was 1 m in length tapering from 3.5 to 0.5 cm, with underhanging ledges 1.0 cm in width on either side. Mice were pretrained for 3 days prior to injury to habituate them to the task, and then tested each day for 7 days following injury, with each mouse given two trials which were videotaped for later analysis by a blinded observer. The number of times the underhanging ledge was used (foot faults) by limbs on the left side, which was contralateral to the CCI injury, were counted and averaged across the two trials. Cognitive outcome Cognitive deficits were assessed using the Barnes Maze, which has been described in detail elsewhere (Koopmans et al. 2003). Briefly the Barnes Maze consists of a white circular platform with 40 holes evenly spaced around the perimeter, to which one is connected to an escape box. Animals were pre-trained for 5 days prior to injury, with their best time taken to find and enter the escape box with front paws and trunk recorded as their pre-injury baseline level. Assessment was conducted on days 2, 4 and 6 post-injury, with escape latency (time in seconds) recorded by a blinded observer. Tissue processing For histological analysis animals were perfusion fixed with 10% formalin at 24 h or 7 days after CCI (n = 5 per group), with 7 day animals randomly chosen from those undergoing functional assessment. Assessment of cortical tissue damage To determine the extent of damaged tissue after CCI, 5 sections per brain, 400 lm apart, were stained with Haematoxylin and Eosin (H&E), representing the region Bregma 0 to )4 because of the shrinkage associated with processing. The unaffected area of the cortex of each hemisphere was outlined within the NDP View software, with damaged tissue defined by a decrease in H&E staining intensity. The volume of undamaged tissue in each hemisphere was then calculated by summing the area of each section and multiplying it by 0.4. The percentage of cortical tissue damage was then calculated as (uninjured cortical volume – injured cortical volume)/ uninjured cortical volume · 100 (Semple et al. 2010). Assessment of hippocampal damage Three sections located 200 lm apart representing the region Bregma )1.2 to )2.1 from each animal at 24 h post-injury were stained with Flouro Jade-C (FJC) to determine the extent of hippocampal cell damage, with corresponding sections from 7 day post-injury mice stained with H&E to assess the number of remaining neurons within


210 | F. Corrigan et al.

Immunohistochemical analysis For analysis the slides were digitally scanned using a Nanazoomer slide scanner (Hamamatsu, Hamamatusu City, Shizuoka, Japan) and the image exported as a jpeg file using the software associated with the slide scanner (NDP-view). To assess the reparative response, post-injury levels of GAP-43 were assessed with sequential stepwise 40· images of the cortex taken at 400, 800 and 1200 lm from the edge of the lesion and exported as jpeg files. The MAP-2 immunostaining was used to assess dendritic integrity post-injury, with sequential 40· images of the CA2 stratum radiatum layer, the CA3 region and the dentate hilus taken. For both GAP-43 and MAP-2 analysis, images then underwent colour deconvolution using Ruifork and Johnston’s method with DAB and haematoxylin separated to allow objective analysis of staining intensity (Ruifrok and Johnston 2001). The algorithm was coded as an NIH Image J macro, with background subtraction and colour correction applied to the images before processing. A histogram analysis of the deconvolved DAB channel allowed the calculation of a percentage of DAB weight through the summation of pixel frequency as a product of pixel intensity, allowing the comparison of this value to that of other images [20]. For analysis of neurogenesis, the number of DCX-positive cells embedded within the granule cell layer of the dentate gyrus was counted in three sections spanning the dorsal hippocampus, as increased rate of neurogenesis correlates with an increased number of cells expressing DCX (Couillard-Despres et al. 2005). The length of the dentate gyrus was measured, and the number of cells/mm counted. Statistical analysis All data were analysed using either a one or two-way ANOVA as appropriate, (repeated measures for functional outcome analysis),

Results Motor and cognitive outcome Motor deficits following CCI injury were assessed using the ledged beam (Fig. 1a). Both APP+/+ and APP)/) mice had an increase in the number of contralateral foot faults following injury when compared with their respective uninjured sham controls, which was significant from days 1–4 for the APP+/+ mice (p < 0.05) and from days 1–7 in APP)/) mice (p < 0.01). Notably, there was a clear exacerbation of motor deficits in APP)/) mice, with an obviously slower rate of improvement. Indeed, they were significantly different from APP+/+ mice on days 2–4 postinjury (p < 0.05). Similarly APP)/) mice demonstrated a greater cognitive impairment following TBI, as determined on the Barnes Maze, than APP+/+ mice (Fig. 1b). They had an increased escape latency when compared with APP+/+ mice on all days tested post-injury (68.3 ± 24.3, 65.3 ± 34.2

(a) 10 No. foot faults (L)

Immunohistochemistry Immunohistochemistry was performed on three sections per mouse to analyse levels of GAP-43, MAP-2 and DCX. Briefly, for GAP43, immunohistochemistry slides were used that were 400 lm apart representing Bregma )1.2 to )3.5, whilst for MAP-2 and DCX staining, slides were used that were 200 lm apart representing Bregma )1.2 to )2.1 to allow specific analysis of the dorsal hippocampus. Sections were incubated with either biotintylated anti-mouse GAP-43 (1 : 1000; Novocastra, Newcastle, England, UK), MAP-2 (1 : 1000; Abcam, Cambridge, MA, USA) or antiguinea pig doublecortin (DCX) (1 : 8000; Millipore Corporation, Bedford, MA, USA), followed by the appropriate secondary antibody, then streptavidin peroxidase conjugate, with bound antibody finally detected with 3,3-diaminobenzidene tetrahydrochloride and sections counter-stained with haematoxylin.

followed by Bonferonni t tests using Graphpad Prism software. A p value of < 0.05 was considered significant in all experiments.

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this area. Images were then taken of the CA region and granular dentate gyrus layer which were sequentially photographed at 40· magnification. The images were imported into Image J, and either the number of FJC +ve neurons or remaining neurons was manually counted using the Image J cell count software by a blinded observer. To determine the effects of injury on the dentate gyrus at 7 days, the area of the granular layer of the dentate gyrus was determined in five sections located 200 lm apart (Bregma )1.5 to )3), with the volume calculated by summing the area of each section and multiplying it by 0.2.

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Fig. 1 Motor and cognitive outcome following CCI injury, as assessed on the ledged beam (a) and Barnes Maze (b). APP)/) mice were significantly impaired in both tasks when compared with APP+/+ mice, with this rescued following treatment with sAPPa (n = 10 per group) (^p < 0.05; ^^p < 0.01, ^^^p < 0.001 compared with sAPPa treated APP)/) mice; **p < 0.01, *p < 0.05 compared with APP+/+ injured mice).



and 45.1 ± 21 s vs. 51.5 ± 23.23, 41.0 ± 16.6 and 26.4 ± 9.6 s), which reached significance on day 4 (p < 0.01). Following sAPPa treatment, the motor and cognitive performance of APP)/) mice was no longer significantly different compared with the APP+/+ mice. Indeed, they demonstrated a significant reduction in the number of foot faults when compared with untreated injured APP)/) mice on days 1–5 days post-injury and a significant improvement in escape latency on days 2 and 4 following TBI on the Barnes Maze. Cortical injury To determine the role of APP in secondary cortical degeneration following focal TBI, the extent of tissue damage after CCI injury was assessed by H&E staining (Fig. 2). At 24 h post-injury there was a small increase in lesion volume in the APP)/) mice, at 21.0 ± 2.7% compared with the APP+/+ mice, at 18.2 ± 0.9%. From 24 h to 7 days post-injury the progression of cortical damage in APP)/) mice was greater than in APP+/+ mice, with a significant increase in tissue damage between the groups (25.0 ± 1.8% vs. 20.3 ± 1.8%, p < 0.01). The sAPPa treatment of APP)/) mice had a negligible effect on the extent of tissue damage at 24 h post-injury, although by day 7 sAPPa treatment had completely inhibited the lesion expansion as compared with the untreated APP)/) mice, with cortical damage at 19.6 ± 2.3% (p < 0.01). There was no significant difference in lesion volume between the APP+/+ and APP)/) sAPPa treated mice at either time point. Hippocampal injury The level of hippocampal cell damage was determined by assessing both the number of FJC +ve neurons at 24 h (Fig. 3) and the number of remaining neurons within H&E stained sections at 7 days following CCI injury (Fig. 4). Although, considerable neuronal degeneration within the hippocampus was evident in all injured mice following CCI, qualitatively it appeared to be far greater in the vehicle treated APP)/) mice (Fig. 3a–e). This increase was quan-

Fig. 2 Cortical damage, as expressed as a percentage of damage when compared with the uninjured left cortex. (n = 5 per group) (**p < 0.01 compared to APP+/+ mice; ^^p < 0.01 compared to APP)/) sAPPa treated mice).

titatively confirmed, with an increase in FJC +ve neurons in APP)/) mice within both the CA region (Fig. 3f) and the dentate gyrus (Fig. 3g) when compared to APP+/+ mice (p < 0.05) and sAPPa treated APP)/) mice (p < 0.05). By 7 days post-injury there was an obvious reduction in the number of remaining neurons within the CA region of the hippocampus in APP)/) mice compared with APP+/+ mice and sAPPa treated APP)/) mice, with the CA2/3 region appearing particularly vulnerable (Fig. 4a–e). Quantitation of hippocampal CA neurons at 7 days found a significant increase in both APP+/+ mice and sAPPa treated APP)/) mice following injury with 1353 ± 142 and 1401 ± 263, respectively, when compared with APP)/) mice, with 839 ± 245 (p < 0.01). Nonetheless, both these groups showed significant neuronal loss when compared to their respective shams (APP+/+ 2129 ± 83, APP)/) 2034 ± 137 p < 0.01) (Fig. 4f). In addition, APP)/) mice had a significantly greater reduction in the volume of the granular layer of the dentate gyrus than APP+/+ or sAPPa treated APP)/) mice (p < 0.05). MAP-2 Immunohistochemistry To assess hippocampal dendritic integrity following CCI injury, immunohistochemistry was performed using the dendritic marker MAP-2 (Fig. 5). Decreases in MAP-2 staining within the dentate hilus were evident in all mice following injury, although this was greater in APP)/) mice, with an almost complete loss of dendrites within this region by 7 days post-Injury. The APP)/) mice also had a greater decrease in MAP-2 immunoreactivity within the CA2 and CA3 regions of the hippocampus in comparison with APP+/+ and sAPPa treated APP)/) mice. However, although there was an overall decrease in the amount of dendrites in APP)/) mice, those that remained appeared thickened. These observations were confirmed by colour deconvolution (5p–r). In APP)/) mice the loss of MAP-2 staining was exacerbated compared with the APP+/+ and sAPPa treated APP)/) mice within the dentate hilus, CA3 region and the stratum radiatum layer of the CA2 region at day 7 post-injury (p < 0.05), with an almost 70% reduction in staining in comparison to APP+/+ and sAPPa treated APP)/) mice within the dentate hilus. DCX Immunohistochemistry To determine the effects of endogenous APP expression on neurogenesis following CCI injury, the numbers of DCXpositive cells within the subgranular layer of the dentate gyrus were quantified (Fig. 6). By 24 h following injury, there was a clear reduction in the number of DCX-positive cells in all injured mice when compared with shams, reflecting their vulnerability following CCI injury. By 7 days post-injury, the number of DCX-positive cells had significantly increased in both the APP+/+ mice and the sAPPa treated APP)/) mice. However in APP)/) mice, the



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Fig. 3 Hippocampal neuronal degeneration as assessed by FJC staining at 24 h following injury. The APP+/+ sham (a), APP+/+ injured (b), APP)/) sham (c), APP)/) injured (d) and sAPPa treated APP)/) mice (e). Evidence of increased FJC staining in APP)/) mice was

confirmed by a count of the number of positive neurons within the CA region (f) and dentate gyrus (g). (n = 5 per group) (Scale bar = 200lm) (***p < 0.001, *p < 0.01 compared to APP+/+ injured mice, ^^^p < 0.001, ^p < 0.05 compared to sAPPa treated APP)/) mice).

numbers of DCX-positive cells remained low, at 12.1 ± 4.6 cells/mm, such that they were now significantly decreased when compared with the APP+/+ (26.6 ± 5.6 cells/mm, p < 0.01) and sAPPa treated APP)/) mice (22.66 ± 5.4 cells/mm, p < 0.05). Representative images seen in Fig. 7 show that within the APP+/+ and sAPPa treated APP)/) mice some of these cells appear to be migrating into the granular layer of the dentate gyrus and extending their dendrites towards the molecular layer, whilst there was little evidence of the incorporation of any immature neurons into the dentate gyrus of the APP)/) mice.

sAPPa treated APP)/) mice had a significant increase in the amount of GAP-43 staining up to 1.2 mm from the lesion site when compared to their respective shams. This was confirmed by colour deconvolution (p < 0.001 for APP+/+; p < 0.01 for sAPPa treated APP)/) mice) with APP+/+ mice having significantly higher %DAB weight of GAP-43 than APP)/) mice up to 1.2 mm from the injury site, including the region closest to the contusion (p < 0.01). Treatment with sAPPa meant that the APP)/) mice were no longer significantly different to APP+/+ mice.

GAP-43 Immunohistochemistry To assess the reparative response following CCI injury, GAP43 was examined via immunohistochemistry at 7 days postinjury. The GAP-43 is known to promote neurite sprouting and neurite extension which will facilitate synaptic plasticity (Bendotti et al. 1997). Following injury, APP)/) mice only had a significant increase in GAP-43 immunoreactivity when compared with their uninjured shams in the region closest to the lesion. However, GAP-43 immunoreactivity was similar between injured APP)/) mice and shams as the distance from the lesion increased (Fig. 8). In contrast, both APP+/+ and

Discussion This study demonstrates that following a moderate focal injury the absence of APP expression makes APP)/) mice more vulnerable to injury resulting in greater cognitive and motor deficits. These deficits correspond with increased levels of cortical and hippocampal cell damage, as well as an impaired reparative response. These differences between wildtype and APP)/) mice in the moderate focal injury were similar to those observed using a mild diffuse TBI model (Corrigan et al. 2012a). Demonstrating that endogenous APP expression is neuroprotective in two different TBI models



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Fig. 4 Hippocampal neurodegeneration as assessed with H&E staining at 7 days post-injury. Normal hippocampal architecture is observed in an APP+/+ (a) and APP)/) sham (c). There is a clear reduction in the number of remaining hippocampal neurons in APP)/) mice (d) when compared with APP+/+ (b) and APP)/) sAPPa treated animals (e) following injury. These observations were confirmed with a

count of the number of remaining neurons within, the CA region (f), with a significant reduction in the volume of the granular layer of the dentate gyrus also noted in APP)/) mice (g). (n = 5 per group) (Scale bar = 100 lm) (*p < 0.05, ***p < 0.001 compared to APP+/+ mice; ^^p < 0.01, ^^^p < 0.001 compared to sAPPa treated APP)/) mice).

establishes APP as an important modulator of neuronal injury following TBI. These changes are not because of a greater amount of primary injury in APP)/) mice as the level of injury was adjusted, with depth of impact decreased from 1.5 to 1.3 mm to compensate for the reported reduction in brain size in APP)/) mice (Ring et al. 2007). It should be noted that this is not associated with major neuronal loss within the cortex or hippocampus of adult APP)/) mice (Phinney et al. 1999; Herms et al. 2004). This is supported by our findings that there was no significant difference in cell numbers within the CA region of the hippocampus nor in the volume of the dentate gyrus between APP+/+ and APP)/) mice. With adjustment of the impact parameters, cortical and hippocampal damage were not significantly different between the APP+/+ and APP)/) mice at 5 h post-injury. As such the exacerbation of deficits in APP)/) mice is presumably because of an increase in the level of secondary injury received, rather than because of the primary insult. Moreover, the APP)/) phenotype, which includes reduced grip strength (Zheng et al. 1995) and age related cognitive deficits (Dawson et al. 1999; Phinney et al. 1999; Senechal et al. 2008) is not the cause of the impairments observed in

these mice following injury, as there was no difference in performance on either the ledged beam or the Barnes Maze between APP+/+ and APP)/) shams. Following treatment with sAPPa APP)/) mice were no longer significantly different to APP+/+ mice, indicating that a lack of sAPPa is the most likely cause of the enhanced deficits in APP)/) mice. The sAPPa treatment led to a significant improvement in functional outcome, a reduction in cortical and hippocampal cell damage and a restoration of the reparative response when compared with untreated APP)/) mice, supporting the role of sAPPa as a neuroprotective agent (Goodman and Mattson 1994; Smith-Swintosky et al. 1994; Thornton et al. 2006; Copanaki et al. 2010). Its mechanism of action, at this stage, remains unknown. However it has been shown to reduce excitotoxicity by activating high conductance potassium channels which hyperpolarize the cell and prevent calcium influx (Furukawa and Mattson 1998) and also activate a number of antiapoptotic pathways (Cheng et al. 2002; Stein et al. 2004). The neuroprotective properties of APP, are indicated by the greater loss of hippocampal MAP-2 immunohistochemistry seen in APP)/) mice compared with their APP+/+ and sAPPa treated counterparts following injury. The cytoskel-



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Fig. 5 Representative images of MAP-2 immunohistochemistry demonstrating normal staining in APP+/+ (a–c) and APP)/) shams (d–f) within the CA2, CA3 and dentate hilus regions. A clear reduction in MAP-2 staining in these areas is noted in APP)/) (j–l) mice following injury when compared with APP+/+ (g–i) and sAPPa treated APP)/) mice (m–o) at 7 days post-injury. These observations were confirmed

with colour deconvolution within the stratum radiatum layer of the CA2 region (p), the CA3 region (q) and the dentate hilus (r).(n = 5 per group) (*p < 0.05, p < 0.01 compared to APP+/+ mice, ^p < 0.05, ^^p < 0.01 compared to sAPPa treated APP)/) mice) (a, d, j, m scale bar = 300 lm, otherwise scale bar = 100 lm).

etal protein MAP-2 is expressed in neuronal cells, predominantly within dendrites and the perikarya. It is particularly vulnerable to degradation by calcium-activated neural proteases, such as calpain, with a reduction in MAP-2 staining

previously reported immediately following injury (Lewen et al. 1996; Folkerts et al. 1998; Saatman et al. 2001). Although this loss of MAP-2 immunoreactivity is not always indicative of neuronal cell loss (Lewen et al. 1996; Folkerts



phosphoprotein which increases following injury (Christman et al. 1997; Hulsebosch et al. 1998; Emery et al. 2000; Thompson et al. 2006) and is an important regulator of synaptic plasticity by promoting neuronal sprouting and neurite outgrowth (Bendotti et al. 1997). During regeneration GAP-43 is transported along neuronal fibres and distributed in growth cones (Thompson et al. 2006), with its up-regulation following injury reflecting an attempt by the injured CNS to promote axonal regeneration and compensate for neuronal loss by creating new synaptic connections (Emery et al. 2003). Although, GAP-43 expression is normally low in neuronal cell bodies, injury is associated with intense labelling of the perikarya, suggestive of changes in the growth status of the neuron (Doster et al. 1991; Harris et al. 2010). Indeed, following focal TBI, GAP-43 positive perikarya, often with visible processes, have been noted around the contusional edge (Harris et al. 2010), with this consistent with the staining seen in APP+/+ mice within this study. The reduced immunostaining of GAP-43 in APP)/) mice therefore, reflects an inhibition of intrinsic recovery mechanisms that can facilitate improvement in motor performance following TBI. Interestingly, the up-regulation of GAP-43 is known to be impaired with increasing injury severity. This may relate to the post-traumatic neural repair response being overwhelmed, resulting in a failure to elicit a plasticity response, as well as increased degradation of plasticity related proteins (Thompson et al. 2006). It is

Fig. 6 Quantitative counts of the number of DCX-positive cells within the dentate gyrus expressed as cells/mm (n = 5 per group) (**p < 0.01 compared to APP+/+ mice; ^p < 0.05 compared to APP)/) sAPPa treated mice; #p < 0.05 compared to 24 h time point).

et al. 1998), within this study it corresponded with the degree of hippocampal damage noted histologically, suggested that the decrease in MAP-2 immunoreactivity was primarily the result of neuronal cell death. As well as providing neuroprotection, a neurotrophic role for sAPPa has been extensively characterized (Saitoh et al. 1989; Araki et al. 1991; Bhasin et al. 1991; Qiu et al. 1995; Ohsawa et al. 1997). Evidence for the loss of the neurotrophic role of sAPPa in APP)/) mice is seen through the reduction in GAP-43 immunoreactivity within the cortex following injury, which is restored following sAPPa treatment. The GAP-43 is a neural specific membrane associated 24 h

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that are extending dendrites into the molecular layer of the dentate gyrus. (Images are representative of n = 5 per group, scale bar = 100lm).



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Fig. 8 Representative images of GAP-43 immunohistochemistry in an APP+/+ (a) and APP)/) (b) sham, with images c–k representing sections taken at increasing distance from the lesion from APP+/+ injured (c–e), APP)/) injured (f – h) and sAPPa treated APP)/) mice (i–k), with the images on the left the closest and those on the right furthest away from the

lesion. Levels of GAP-43 are also expressed as %DAB weight (i), confirming the decrease in levels observed in APP)/) mice compared with APP+/+ mice and sAPPa treated APP)/) mice post-injury. (n = 5 per group) (**p < 0.01, ***p < 0.001 compared to APP+/+ mice, ^^p < 0.01 compared to sAPPa treated APP)/) mice) (Scale bar = 100 lm).

unclear whether ablating APP expression in this instance creates an environment which is less conducive for repair, or whether APP itself, through sAPPa, is required to facilitate GAP-43 production and trafficking following TBI.

To examine the effects of APP ablation on neurogenesis, DCX immunohistochemistry was performed, as it is a reliable marker of immature neurons (Couillard-Despres et al. 2005; von Bohlen Und Halbach 2007). Within the



subgranular zone of the dentate gyrus new neurons are born throughout life (Eriksson et al. 1998), and these neurons are capable of migrating into the granular cell layer where they can connect to the CA3 region, their target area (Markakis and Gage 1999). Predictably, at 24 h post-injury all mice exhibited a decrease in the number of DCX cells within the dentate gyrus, with these cells particularly vulnerable following TBI (Rola et al. 2006). However, by 7 days post-injury the APP+/+ and sAPPa treated APP)/) mice had shown a significant increase in the number of DCX cells, consistent with reports elsewhere (Ramaswamy et al. 2005; Rola et al. 2006), which was not evident in untreated APP)/) mice. The DCX expressing late neural progenitors are normally more susceptible to injury than early neural progenitors that do not express DCX (Yu et al. 2008). However, given the significant amount of cell damage evident within the dentate gyrus of the APP)/) mice when compared with APP+/+ mice, these results may simply reflect a loss of early progenitors rather than an inhibition of maturation of these precursors. Regardless of the mechanism, given the time frame, it is highly unlikely that the decrease in neurogenesis observed in the APP)/) mice contributed to the increase in functional deficits, with further studies needed to assess the long-term effects of APP knockout on neurogenesis following TBI. As sAPPa was able to rescue the exacerbation of deficits seen in APP)/) mice, this reveals that the presence of endogenous full length APP or its metabolites are not required for sAPPa to exert its protective effects. This is contrary to previous in vitro reports which found that sAPPa was unable to provide protection from metabolic stress (Gralle et al. 2009) or stimulate neurite outgrowth in the absence of APP (Young-Pearse et al. 2008). However in vivo, knock-in of sAPPa was sufficient to mediate the postnatal physiological actions of APP in APP)/) mice (Ring et al. 2007). Thus sAPPa may act via alternative mechanisms of action in vivo compared with those reported in vitro, to account for the difference in results. Although this study demonstrates that endogenous APP, via sAPPa, is protective immediately after a moderate focal injury this does not preclude a role for APP, through its metabolite Ab, from worsening long-term neurodegeneration following TBI. The APP is processed via two mutually exclusive pathways, cleavage via a secretase releases sAPPa, whilst cleavage via b and c secretases produces Ab (Suh and Checler 2002). Damaged axons are thought to be a key source of Ab generation following TBI, as they allow longterm pathological co-localisation of APP with the b-secretase, BACE-1, and the c-secretase, PS1, assisting in a shift towards amyloidogenic processing (Smith et al. 1999; Kamal et al. 2001; Uryu et al. 2007). Indeed it has been proposed that ongoing axonal injury allows the continued generation of Ab peptides (Johnson et al. 2010), with long-term progressive axonal degeneration and intra-axonal Ab accu-

mulation found to persist for years following the initial trauma in humans (Chen et al. 2009). This may underlie the observation that a history of previous TBI may increase the risk for the later development of Alzheimer’s disease (AD) (Mortimer et al. 1991; Salib and Hillier 1997; Fleminger et al. 2003), especially in susceptible individuals with the APOE e4 allele (Mayeux et al. 1995; Guo et al. 2000). This supposition is yet to be conclusively proven with contradictory epidemiological reports on whether a positive association exists between a history of TBI and AD (Guo et al. 2000; Rasmusson et al., 1995; Launer et al. 1999; Fratiglioni et al. 1993). Furthermore, while some histopathological studies of individuals who died after suffering a single severe TBI demonstrate widespread Ab deposition irrespective of age (Roberts et al. 1991; Gentleman et al. 1997; Ikonomovic et al. 2004), others have concluded that Ab deposition in victims below the age of 60 is a rare occurrence (Adle-Biassette et al. 1996; Braak and Braak 1997). As well as an association with AD, TBI induced increases in levels of Ab, may also play a role in the development of chronic traumatic encephalopathy (CTE) (Gavett et al. 2011). CTE is a form of neurodegeneration that is believed to result from repeated head injuries, and is associated with a number of sports including boxing, American football and professional wrestling (McKee et al. 2010). This link is also yet to be conclusively proven, with Ab deposits only found in 40– 45% of individuals with CTE, whilst neurofibrillary tangles, comprised of hyperphosphorylated tau are found in nearly 100% of cases (McKee et al. 2009), suggesting that tauopathy could be the primary cause of neuronal injury. Nonetheless, toxic unaggregated oligomeric forms of Ab could be contributing to toxicity in CTE without formation of Ab deposits (Lambert et al. 1998; Walsh et al. 2002; Cleary et al. 2005). It is clear that further investigations will be necessary to determine whether endogenous APP remains neuroprotective following models of repetitive TBI replicating the situation in CTE, as well as its effects on long-term functional and histological outcome post-TBI. Nonetheless, this study firmly demonstrates that endogenous APP is a neuroprotective agent immediately following TBI, with this activity mediated by sAPPa. Although, smaller domains of sAPPa have been identified with similar levels of neuroprotective activity to sAPPa (Corrigan et al. 2011), delineating the mechanism of action of sAPPa will assist in the development of sAPPa, or fragments and analogues thereof, as therapeutic agents against TBI.

Acknowledgements We thank Prof. Hui Zheng for providing the APP knockout mice and Dr Mark Habgood (University of Melbourne), Dr Jenna Zeibell and Jim Manavis for expert technical assistance. We thank Dr. Chi Pham (The University of Melbourne) for purifying the sAPPa. We also thank the staff within the IMVS animal care facility. This work



was funded, in part, by grants from the Brain Foundation and the Neurosurgical Research Foundation. RC is a National Health and Medical Research Council Senior Research Fellow. No authors have a conflict of interest to declare.

Supporting information Additional supporting information may be found in the online version of this article: Figure S1. Lesion volume (a) and degenerating neurons as detected by FJC staining within the CA region of the hippocampus (b) and dentate gyrus (c) at 5 h post-injury demonstrating that the primary injury level is the same between APP+/+ and APP)/) mice. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

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