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. 2010 Jun 30;31(1):351–361. doi: 10.1038/jcbfm.2010.99

Age-dependent effect of apolipoprotein E4 on functional outcome after controlled cortical impact in mice

Rebekah C Mannix 1,*, Jimmy Zhang 2,3, Juyeon Park 2,3, Xuan Zhang 2,3, Kiran Bilal 2,3, Kendall Walker 4, Rudolph E Tanzi 5, Giuseppina Tesco 4, Michael J Whalen 2,3
PMCID: PMC3049498  PMID: 20588316

Abstract

The apolipoprotein E4 (APOE4) gene leads to increased brain amyloid beta (Aβ) and poor outcome in adults with traumatic brain injury (TBI); however, its role in childhood TBI is controversial. We hypothesized that the transgenic expression of human APOE4 worsens the outcome after controlled cortical impact (CCI) in adult but not immature mice. Adult and immature APOE4 mice had worse motor outcome after CCI (P<0.001 versus wild type (WT)), but the Morris water maze performance was worse only in adult APOE4 mice (P=0.028 at 2 weeks, P=0.019 at 6 months versus WT), because immature APOE4 mice had performance similar to WT for up to 1 year after injury. Brain lesion size was similar in adult APOE4 mice but was decreased (P=0.029 versus WT) in injured immature APOE4 mice. Microgliosis was similar in all groups. Soluble brain Aβ40 was increased at 48 hours after CCI in adult and immature APOE4 mice and in adult WT (P<0.05), and was dynamically regulated during the chronic period by APOE4 in adults but not immature mice. The data suggest age-dependent effects of APOE4 on cognitive outcome after TBI, and that therapies targeting APOE4 may be more effective in adults versus children with TBI.

Keywords: traumatic brain injury, genetics, developmental biology, Alzheimer's disease

Introduction

Traumatic brain injury (TBI) is a leading cause of death and disability in children and young adults worldwide that lacks specific therapy and is associated with a rehabilitative cost of 60 billion dollars annually in the United States (Schneier et al, 2006). The observation that recovery from TBI of a similar mechanism and injury level may differ markedly from one patient to another suggests a contribution of genetic risk factors to neurologic outcome (Nicoll, 1996). The best known genetic risk factor for poor outcome after TBI in adults is the E4 allele of the apolipoprotein E (APOE) gene (Jordan et al, 1997; Teasdale et al, 1997).

Apolipoprotein E is the predominant cholesterol and lipid transport protein in the brain. Three allelic variants of APOE yield three distinct protein isoforms (namely APOE2, APOE3, and APOE4, respectively). Apolipoprotein E allele frequencies in Caucasians are 7, 78, and 15%, respectively. Apolipoprotein E3, the most common APOE isoform, has a role in maintaining synaptic integrity and function, promotes neural recovery and repair, and may limit inflammation after acute central nervous system injury (Chen et al, 1997; Lynch et al, 2003; Vitek et al, 2009). Apolipoprotein E3 facilitates amyloid beta (Aβ) transport into the microglia for degradation or across the blood–brain barrier for removal from the brain, whereas APOE4 promotes Aβ accumulation and is associated with an increased risk of Alzheimer's disease (AD) (Kim et al, 2009).

In adults with TBI, the presence of at least one APOE4 allele is associated with early mortality (Teasdale et al, 1997), prolonged coma (Sorbi et al, 1995), worse functional outcome (Jordan et al, 1997), and an increased risk of developing AD (Mayeux et al, 1995). In contrast to these well-established detrimental effects in adults, few studies have examined the impact of APOE4 on outcome in pediatric TBI. In the largest prospective study published to date, the presence of at least one APOE4 allele was associated with unfavorable outcome (such as death, vegetative survival, or severe disability at 6 months) in children aged 0 to 15 years (Teasdale et al, 2005). However, in a study of 71 children (mean age 13 years) with TBI severe enough to require in-patient rehabilitation, Blackman et al (2005) found improved functional outcomes in children with at least one E4 allele (Blackman et al, 2005). More recently, Moran et al (2009) showed that the APOE4 allele is not consistently associated with outcome in children aged 8 to 15 years with mild TBI defined as a brief loss of consciousness, Glasgow Coma Score 13 or 14, or two or more acute signs or symptoms of concussion (Moran et al, 2009). Other studies have suggested a benefit of APOE4 on cognition in children in developing countries (Wright et al, 2003). Thus, APOE4 might not be detrimental to outcome after TBI in children as it is in adults.

Most studies investigating possible mechanisms of APOE4 and outcome after experimental and human TBI have focused on Aβ, which is a 40 to 42 amino-acid peptide implicated in synaptic dysfunction and cell death in AD (Lue et al, 1999; Venkitaramani et al, 2007). As the APOE4 phenotype is characterized in part by accumulation of Aβ in the brain, the APOE4 mutation could lead to increased brain Aβ after TBI and thus increased risk for AD (Johnson et al, 2010). Strategies targeting Aβ improved histopathological and functional outcome after TBI in aged mice (Loane et al, 2009); however, the possibility that Aβ may influence outcome after pediatric TBI has not been reported. This is a particularly relevant issue in children because some evidence suggests that Aβ is less toxic to the immature brain (Brewer, 1998).

The goal of this study was to test the hypothesis that, after controlled cortical impact (CCI), APOE4 would be associated with worse functional outcome in adult but not immature mice, and to examine whether differences in soluble brain Aβ correspond with functional outcome.

Materials and methods

Mice

All experiments were approved by the Massachusetts General Hospital Institutional Review Board and complied with the NIH Guide for the Care and Use of Laboratory Animals. Mice were given free access to food and water and were housed in laminar flow racks in a temperature-controlled room with 12-hour day/night cycles. Transgenic mice that express targeted replacement of the mouse APOE allele with human APOE4 under the direction of the human glial fibrillary acidic protein promoter (Holtzman et al, 2000) were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Homozygous APOE4 transgenic mice do not express endogenous mouse APOE, develop normally, are fertile, are grossly phenotypically normal, and are congenic with C57Bl/6 (at least six backcrosses, Jackson Laboratories, 21 January 2010). The murine APOE primary sequence is the same as that of human APOE4 in the polymorphic region (Arg 112), but is believed to behave like human APOE3, because it lacks the Arg-61 domain interactions that confer the functional properties of APOE4 (Raffai et al, 2001). Heterozygous APOE4 mice have one copy of the wild-type (WT) murine APOE allele. Male and female adult (aged 2 to 4 months) and immature (aged 20 to 21 days) APOE4 mice were used in the two experimental protocols described below. Wild-type age- and gender-matched C57Bl/6 mice were used as controls. In all experiments, male and female APOE4 and WT mice were distributed equally between groups.

Experimental Protocols

A total of 57 adult APOE4 homozygous, 9 APOE4 heterozygous, and 46 WT mice were used in protocol 1. For protocol 2 (immature mouse studies), 50 APOE4 homozygotes and 54 WT mice were used (Figure 1).

Figure 1.

Figure 1

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Flow chart for experimental protocols in adult and immature mice. (A) Protocol 1 describing the use of adult homozygous and heterozygous APOE4 and WT mice. (B) Protocol 2 describing the use of immature homozygous APOE4 and WT mice. APOE4, apolipoprotein E4; WT, wild type.

Mouse Controlled Cortical Impact Model

The mouse CCI model was used as described previously (Bermpohl et al, 2006) because this model reproduces cell death and cognitive deficits experienced by children and adults with severe TBI (Blackman et al, 2005). Mice were anesthetized with 3% isoflurane, N2O, and O2 (2:1) and placed in a stereotactic frame. A 5-mm craniotomy was performed over the left parietotemporal cortex and the bone flap was removed. Controlled cortical impact was then produced using a pneumatic cylinder with a 3-mm flat-tip impounder, velocity 6 m/s, and impact depth of 0.6 mm. The scalp was sutured closed and mice were allowed to recover from anesthesia in their cages.

Assessment of Motor Function

Gross vestibulomotor function was assessed using a wire grip test (Bermpohl et al, 2006). The test consisted of placing the mouse on a wire suspended between two poles and grading the degree of attachment and movement of the mouse. Scores were as follows: 0 was given to a mouse that fell from the wire within 30 seconds; 1 point for unilateral grasp of either upper or lower extremities, 2 points for midline grasp of both upper and lower extremities but not the tail; 3 points for midline grasp of all extremities plus the tail; 4 points for movement along the wire after achieving a score of 3; and 5 points for climbing down the pole within 60 seconds.

Spatial Memory Acquisition Assessment

Experimenters blinded to the mouse genotype evaluated the spatial memory performance of mice using the Morris water maze (MWM) task, as described previously (Bermpohl et al, 2006). A white pool (83 cm diameter, 60 cm deep) was filled with water to 29 cm depth. Several highly visible intramaze and extramaze cues that remained constant throughout the trials were located in and around the pool. Water temperature was maintained at ∼24°C. The goal platform (a round, clear, plastic platform 10 cm in diameter) was positioned 1 cm below the surface of water. Each mouse was subjected to a maximum of two series of four trials per day. For each trial, mice were randomized to one of the four starting locations (namely north, south, east, or west) and placed in the pool facing the wall. Mice were given a maximum of 60 or 90 seconds to find and rest upon the submerged platform. If the mouse failed to reach the platform by the allotted time, it was placed on the platform by the experimenter and allowed to remain there for 10 seconds. Mice were warmed and dried with a lamp between trials. For probe trials, mice were placed in the pool with the platform removed and the time that the animal swam in the target quadrant was recorded (maximum 60 seconds). For visible platform trials, the goal platform was marked by red tape and placed 0.5 cm above the water level. Performance in the MWM was quantitated by latency to the platform or latency in the target quadrant (probe trials).

Lesion Volume

Morphometric image analysis was used to determine the lesion size after CCI. Mice were anesthetized with isoflurane and killed by decapitation and the brains removed. Coronal sections (12 μm) were cut at 0.5 mm distances from the anterior to the posterior brain and mounted on poly--lysine-coated slides. The area of both hemispheres was determined using image analysis (Nikon Eclipse Ti 2000, MS Elements, MVI, Avon, MA, USA). Lesion volume was obtained by subtracting the volume of brain tissue remaining in the left (injured) hemisphere from that of the right (uninjured) hemisphere and expressed in mm3.

Assessment of Brain Water Content

The brains were divided into left and right hemispheres. A small amount of the brain tissue anterior and posterior to the contusion was cut in the coronal plane and discarded. After recording the wet weight of the remaining brain tissue in each hemisphere, the brains were dried in an oven at 90°C for 48 hours and dry brain weight was obtained. Percentage brain water content of each hemisphere was calculated as (wet−dry/wet) weight × 100%. Brain edema was estimated as the difference in the percentage brain water content (injured−uninjured hemisphere).

Brain Aβ40 Levels

We assessed detergent soluble Aβ40 levels in the brain tissue because soluble but not insoluble (fibrillar) Aβ levels correlate strongly with the extent of synaptic loss and severity of cognitive impairment in AD (Lue et al, 1999; McLean et al, 1999), and Aβ40 levels are normally higher in the brain compared with Aβ42. Cortical and hippocampal brain tissues in naive animals, pericontusional tissue including the cortex and the underlying hippocampus in acutely injured animals (48 hours), or cortical and hippocampal brain tissues surrounding the cavitary lesion from animals in chronic periods after CCI were used for determination of Aβ40. At various times after CCI, the brains were removed and bisected into injured and uninjured hemispheres. The brains were frozen in liquid nitrogen and stored at −80°C until processing. Tissues samples were rapidly homogenized in 250 μL of RIPA buffer with protease inhibitor tablet (Sigma-Aldrich, St Louis, MO, USA). After centrifugation (14,000 r.p.m., 4°C for 15 minutes), the supernatant was collected (fraction 1), and the pellet was dissolved in 250 μL RIPA buffer and centrifuged again to obtain the supernatant (fraction 2). The two fractions were combined and protein content determined using the Bio-Rad (Hercules, CA, USA) assay. Soluble Aβ40 was measured by sandwich enzyme-linked immunosorbent assay (Wako, Richmond, VA, USA) according to the manufacturer's instructions using sample protein concentrations of 1 to 2.5 mg/mL, as described previously (Bales et al, 1997). Data were expressed as pmol/g protein.

Immunohistochemical Detection of Activated Microglia

At 48 hours after CCI, mice were anesthetized and transcardially perfused with 4% paraformaldehyde 6 to 72 hours after injury. The brain was postfixed for 24 hours in 4% paraformaldehyde and cryoprotected in 30% sucrose for 24 hours. Coronal sections were cut (20 mm) and mounted on poly--lysine-coated slides. Sections were washed in phosphate-buffered saline, blocked in 3% normal goat serum in phosphate-buffered saline for 1 hour, and incubated overnight at 4°C with rabbit anti-Iba-1 antibody (1:200; Wako Pure Chemical Industries, Osaka, Japan). Slides were washed in phosphate-buffered saline and incubated with the appropriate Cy3-conjugated secondary antibody (1:300; Jackson ImmunoResearch, West Grove, PA, USA) for 60 minutes, washed in phosphate-buffered saline, and coverslipped. Brain sections were photographed on a Nikon Eclipse T300 fluorescence microscope (Nikon, Tokyo, Japan), using excitation/emission filters of 568/585 nm. For comparisons between groups, × 400 fields from the inferolateral aspect of the cortex underlying the contusion were randomly selected from brain regions at the level of the anterior hippocampus and photographed with identical camera settings by an observer blinded to the genotype, compared, and representative fields were shown for qualitative analysis.

Statistical Analyses

Data are mean±s.e.m. Motor and MWM data were analyzed by repeated measures analysis of variance (group × time). Aβ40 levels, brain edema, and volumetric data were analyzed using nonparametric analyses as appropriate (signed rank for comparisons between the ipsilateral and contralateral hemispheres within groups, rank sum or Kruskal–Wallis for comparisons between groups). The gene–dose effect of the probe trials was assessed using linear regression. Mortality data for immature mice were measured by χ2 test. For each test, P<0.05 was considered significant.

Results

All animals survived CCI and the experimental periods, except for six of the immature APOE4 homozygous mice that died at 9 to 10 months after CCI (compared with none of the injured immature WT mice; P=0.009).

Motor Outcome

Before CCI, the wire grip test performance was similar in naive adult APOE4 and WT mice. After CCI, the performance in all groups (WT, homozygous APOE4, and heterozygous APOE4) was impaired compared with baseline and improved over the test period (P<0.001 for time effect). However, WT performance was significantly better than APOE4 homozygotes and heterozygotes (P<0.001 group effect), because neither APOE4 group returned to WT performance levels by day 21 (Figure 2A). Similar to adult animals, motor performance after CCI was significantly worse in immature homozygous APOE4 compared with WT mice (P<0.001 group effect, Figure 2B).

Figure 2.

Figure 2

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Motor performance after CCI. Motor performance in (A) adult and (B) immature mice after CCI. Adult and immature APOE4 mice performed worse than did WT (P<0.001 group effect, both experiments). APOE4, apolipoprotein E4; CCI, controlled cortical impact; WT, wild type.

Cognitive Outcome

Naive (uninjured) adult APOE4 homozygous and WT mice (aged 2 to 4 months) showed a time-dependent improvement in MWM performance (P<0.001), with no differences observed between groups (Figure 3A). After CCI in a separate group of animals, MWM performance was significantly better in WT mice compared with that in APOE4 homozygotes and heterozygotes (P=0.028 group effect) (Figure 3B). Probe trial latencies also differed significantly among groups with regard to the number of animals performing above chance (defined as 20/60 seconds): APOE4 homozygous, 1/9; APOE4 heterozygous, 4/9; WT 8/12 (P=0.002 for gene–dose effect).

Figure 3.

Figure 3

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Morris water maze (MWM) performance in adult mice. (A) Naive (2 to 4 months) APOE4 and WT mice did not differ with regard to MWM performance. (B) APOE4 heterozygous and homozygous mice performed significantly worse than did WT after CCI (P=0.028 group effect). APOE4, apolipoprotein E4; CCI, controlled cortical impact; WT, wild type.

To determine the effect of APOE4 and aging on cognitive outcome, a separate group of adult (aged 2 to 4 months) APOE4 homozygous and WT mice were first tested as naive (uninjured) mice and performed similarly in the MWM test (Figure 4A). Naive probe trials for APOE4 (25±2 seconds) also did not differ from WT (27±5 seconds). These mice were then subjected to CCI and tested in the MWM test with the goal platform in different locations at 2 weeks and 6 months after injury. At 2 weeks after CCI, APOE4 homozygotes performed similarly to WT (P=0.098 group effect, Figure 4B). At 6 months, APOE4 mice performed strikingly worse than did WT mice (Figure 4B, P=0.019 group effect). In contrast to APOE4 homozygotes, MWM performance in WT mice was similar at 2 weeks and 6 months after CCI (Figure 4B). In probe trials conducted at 2 weeks, there was no statistical difference between APOE4 (16±10 seconds) and WT (21±6 seconds) mice, but at 6 months, APOE4 (8.8±2.9 seconds) performed significantly worse than did WT (18±2.9 seconds, P=0.001). Figure 4C shows that MWM performance in a separate group of aged naive APOE4 and WT mice (aged 9 to 12 months) did not differ between APOE4 homozygotes and WT. Probe trials for naive aged APOE4 (25±2 seconds) also did not differ from WT (24±2 seconds). Thus, after TBI, adult homozygous APOE4 mice have progressive worsening of cognitive function that is not observed in injured WT or aged naive APOE4 mice.

Figure 4.

Figure 4

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Effect of aging on Morris water maze (MWM) performance after controlled cortical impact (CCI) in adult WT and homozygous APOE4 mice. (A) Naive WT and homozygous APOE4 mice (2 to 4 months) performed similarly in the MWM before CCI. (B) After CCI, injured homozygous APOE4 adults show no significant impairment in performance compared with WT at 2 weeks after injury (P=0.098) but performed significantly worse than did WT at 6 months after injury (P=0.019 group effect; P=0.004 for time effect). (C) Morris water maze performance did not differ between groups of naive (uninjured) aged (9 to 12 months) WT and homozygous APOE4 mice. APOE4, apolipoprotein E4; WT, wild type.

In contrast to adult mice, MWM performance in injured immature APOE4 and WT mice did not differ at 5 weeks, 6 months, or 1 year after injury (the latter time point including mice from the 5 week and 6 months cohort retested with the goal platform in a new location) (Figure 5).

Figure 5.

Figure 5

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Morris water maze performance after controlled cortical impact in immature mice. Immature homozygous APOE4 mice had similar performance as WT at (A) 5 weeks, (B) 6 months, and (C) 1 year after injury. APOE4, apolipoprotein E4; WT, wild type.

Histopathology

At 21 days after CCI, adult and immature APOE4 homozygotes and WT mice showed well-demarcated cavitary lesions involving the cortex and hippocampus in the injured hemispheres (data not shown). Total brain tissue loss did not differ between adult homozygous APOE4 and WT mice (Figure 6, P=0.2). In contrast, brain tissue loss was significantly decreased after CCI in immature APOE4 versus WT mice (Figure 6, P=0.029). Brain edema did not differ at 24 hours after injury between adult homozygous APOE4 (2.7%±0.3%) and WT (2.8%±0.2%, P=0.58) mice, and between immature homozygous APOE4 (1.5%±0.6%) and WT (1.2%±0.4%, P=0.37) mice.

Figure 6.

Figure 6

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Lesion volume in adult and immature mice after controlled cortical impact. Postinjury brain lesion size did not differ between adult APOE4 and WT mice. In contrast, immature mice had significantly less total brain tissue loss compared with WT (*P=0.029). APOE4, apolipoprotein E4; WT, wild type.

Immunohistochemical analysis of Iba-1-positive microglia showed a robust increase in activation of the microglia in the cortical and subcortical (hippocampal) regions of the injured hemisphere at 48 hours after CCI compared with noninjured hemispheres (Figure 7) and naıve control mice (not shown). No gross differences in Iba-1 staining were observed between adult or immature APOE4 homozygotes and WT mice.

Figure 7.

Figure 7

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Microglial activation after controlled cortical impact in APOE4 versus wild-type (WT) mice. Immature and adult homozygous APOE4 or WT mice (n=2 to 4 per group) were subjected to controlled cortical impact and microgliosis was assessed qualitatively by Iba-1 staining at 48 hours. Immature and adult APOE4 mice had no gross differences in microgliosis compared with WT. Representative images are shown here. APOE4, apolipoprotein E4.

Brain Aβ40 Levels

In naive animals (Figure 8A), brain Aβ40 concentration increased with aging in APOE4 but not in WT mice, consistent with a known role for APOE4 in brain Aβ accumulation. In adult mice, brain Aβ40 increased by 25% to 30% at 48 hours after CCI in the injured hemispheres (Figure 8B) in WT and APOE4 mice (P=0.046 and P=0.028, respectively) versus uninjured hemispheres (Figure 8C). At 1 month after CCI, adult homozygous APOE4 mice maintained increased soluble Aβ40 levels in the injured and uninjured hemispheres, whereas soluble Aβ40 was decreased in injured (Figure 8B) compared with uninjured hemispheres (Figure 8C) of adult WT (injured, 3.8±0.45 pmol/g; uninjured 7.2±1.1pmol/g protein) and APOE4 heterozygous (injured, 4.4±1.3 pmol/g; uninjured 7.6±1.7 pmol/g protein) mice (P=0.04 injured versus contralateral hemisphere for both groups).

Figure 8.

Figure 8

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Soluble Aβ40 levels in the brain assessed by ELISA. (A) Data from naive (uninjured) animals. Naive APOE4 but not WT animals had increased brain Aβ40 with aging. (B) Data from injured hemispheres. At 48 hours after controlled cortical impact (CCI), immature APOE4 but not WT mice show increased Aβ40 in injured compared with noninjured hemispheres (P=0.047). One month after injury, Aβ40 levels in immature WT and APOE4 animals remained increased compared with naive controls (P=0.01 and P=0.008, respectively). By 1 year after injury, however, injured immature APOE4 and WT mice had Aβ40 levels similar to those of naive immature animals but significantly less than aged naive APOE4 mice (P<0.001). (C) Data from uninjured hemispheres. At 48 hours after CCI, neither immature nor adult APOE4 or WT mice had significant increases in soluble Aβ40 compared with naive age- and genotype-matched animals. At 1 month after injury, both immature WT and APOE4 animals had increased Aβ40 in uninjured hemispheres compared with naive controls (P=0.0069 and P=0.028, respectively). Only WT adults had increased Aβ40 in uninjured cortices 1 month after injury compared with naive WT (P=0.0012). 1 year after injury in the immature period, levels of Aβ40 in uninjured cortices had decreased to that of naive uninjured immature animals. APOE4, apolipoprotein E4; ELISA, enzyme-linked immunosorbent assay; WT, wild type.

After CCI in immature mice, brain Aβ40 was significantly increased at 48 hours in the injured versus noninjured hemispheres in APOE4 homozygotes (P=0.04), but the increase in the injured hemispheres of WT mice did not reach statistical significance (P=0.14 versus uninjured hemispheres). Immature APOE4 homozygotes (48 hours) also had increased Aβ40 in the contralateral hemisphere compared with WT (P=0.047). At 1 month after CCI, immature homozygous APOE4 and WT mice maintained increased soluble Aβ40 levels in uninjured hemispheres compared with naive (Figures 8A and 8C). Soluble Aβ40 was decreased in injured compared with uninjured hemispheres of immature WT (P=0.005) but not APOE4 mice (Figures 8B and 8C), but soluble Aβ40 was increased in the injured hemispheres of both APOE4 and WT mice compared with their naive controls (P=0.008 and P=0.01, respectively) (Figures 8A and 8B). At 1 year after CCI, brain Aβ40 levels were similar in injured and uninjured hemispheres of immature homozygous APOE4 and WT mice, and were similar to 1-year-old naive WT mice but significantly less than naive age-matched APOE4 mice (P=0.002 for injured APOE4 versus naive aged APOE4).

Discussion

To our knowledge, this is the first report of the effect of APOE4 in an immature TBI model. We found that after CCI, transgenic human APOE4 mice had progressive deterioration in cognitive function when injured during adulthood but not during the immature period. Interestingly, TBI was associated with increased brain Aβ40 at 1 month in the contralateral hemispheres of adult WT and APOE4 heterozygous mice, and both hemispheres of adult homozygous APOE4 mice, whereas TBI in immature mice inhibited the accumulation of soluble brain Aβ40 observed in the aging brains of naive homozygous APOE4 mice. Unexpectedly, immature APOE4 mice had decreased brain tissue damage versus WT, suggesting a protective effect of APOE4 in this regard.

Our findings confirm and extend previous studies that show the importance of APOE to functional recovery after experimental TBI. In a closed head cerebral contusion model, adult APOE null mice had worse motor function and increased spatial memory deficits (up to 40 days after injury) compared with WT (Chen et al, 1997), suggesting that the loss of the normal APOE allele alone may be detrimental. Compared with APOE null mice and transgenic human APOE3 mice (human APOE3 on a null murine APOE background), transgenic human APOE4 mice had increased mortality, motor deficits, brain tissue damage, and decreased expression of soluble alpha amyloid precursor protein (α-APP) after closed head contusion TBI (Ezra et al, 2003; Sabo et al, 2000). Our data suggest a modest detrimental effect of APOE4 on cognitive outcome 2 weeks after CCI (Figures 3B and 4B) but a more profound effect during the aging process (6 months, Figure 4B), which has not been reported previously.

We found a dominant negative effect of APOE4 on postinjury motor performance (Figure 2), and perhaps cognitive function (Figure 3) but not chronic brain Aβ40 accumulation after CCI (Figure 8). A dominant negative function of APOE4 has been shown in cultured microglia from APOE4/APOE3 mice (Vitek et al, 2009), and in neurodegenerative disease mouse models as well (Buttini et al, 2000). Why APOE4 exerts a dominant negative effect on postinjury neurologic function but not Aβ clearance is unknown; however, it is possible that APOE4 exerts toxicity independent of Aβ40. It is also possible that a toxic effect of Aβ42, which we did not measure in the current study, may contribute to the detrimental effects of APOE4 in adult carriers after TBI. Further experiments examining the temporal course of Aβ42 are required to resolve this issue.

The progressive cognitive deterioration observed in injured adult APOE4 mice was associated with high levels of Aβ40 in both cerebral hemispheres at 1 month after injury (Figure 8). These data suggest a contribution of Aβ40 to cognitive outcome after TBI in adult homozygous APOE4 mice. The lack of cognitive decline in injured immature APOE4 homozygotes is especially striking, given the strong detrimental effects of APOE4 in injured adult mice (Figure 4B, 6 months) and the potential for synergism between childhood TBI (Anderson et al, 2005) and APOE4. Studies in human AD and in human APP transgenic mice show that functional neurologic deficits correlate with increasing levels of soluble and deposited levels of Aβ, which were highest in adult APOE4 mice in the current study (Holtzman et al, 2000; Lue et al, 1999; McLean et al, 1999). However, soluble Aβ40 levels in the brain after CCI are relatively low compared with Aβ levels in transgenic AD mice, suggesting that an interaction between TBI and Aβ40/APOE4 is required to induce cognitive deficits. This tenet is supported by our finding that naive 9-to-12-month-old APOE4 mice performed similarly to WT in MWM testing, despite a nearly three-fold increase in Aβ40 levels (Figure 8, 9-to-12-month-old naive groups). Alternatively, mechanisms other than soluble Aβ40 may mediate the detrimental effects of APOE4 in TBI.

Apolipoprotein E4 may be detrimental to TBI by increasing brain inflammation (Lynch et al, 2003), altering APP processing (Ezra et al, 2003), and by accelerating brain Aβ deposition (Hartman et al, 2002). Genetic and pharmacological strategies targeting Aβ have been reported to reduce functional deficits and histopathological damage after CCI in older adult (aged 9 to 11 months) mice (Loane et al, 2009), lending support for a causative role for Aβ in postinjury cognitive deficits. Nonetheless, strategies to eliminate Aβ production in APOE4 mice are required to convincingly elucidate the relationship between Aβ and APOE4 in the pathogenesis of cognitive (and motor) deficits after experimental TBI (Loane et al, 2009).

We found dynamic regulation of brain Aβ40 by TBI and APOE4 in the chronic period after injury, depending on the age of mice at the time of CCI. In adult WT and APOE4 mice, Aβ40 levels were increased by TBI in contralateral hemispheres by ∼50%, whereas only APOE4 homozygotes had a significant increase in Aβ40 in injured hemispheres at 1 month (Figure 8). In contrast to adult mice, chronic brain Aβ40 levels in immature mice were regulated solely by the presence or absence of TBI, because injured immature WT and homozygous APOE4 mice failed to accumulate Aβ40 in either hemisphere even after 1 year (e.g., compare injured immature APOE4 at 1 year with 1-year-old naive APOE4 mice in Figure 8). This striking finding in immature mice suggests age-dependent (and APOE4-independent) regulatory mechanisms for accumulation of Aβ40 in the brain after TBI.

Previous reports have described increased Aβ clearance after experimental TBI in the adult mouse brain (Nakagawa et al, 1999). Transgenic mice overexpressing APP had reduced accumulation of Aβ in the injured brain of mice injured at 4 months of age, and regression of established Aβ plaques in mice injured at 2 years of age, through unknown mechanisms (Nakagawa et al, 1999). Our findings suggest that TBIs in the immature period also induce mechanism(s) that prevent later accumulation of brain Aβ, despite the known detrimental effects of APOE4 on brain Aβ clearance (Deane et al, 2008). Soluble Aβ may be cleared by vascular transport across the blood–brain barrier (Shibata et al, 2000), uptake by the microglia (Mandrekar et al, 2009), extracellular degradation by proteases (Wang et al, 2006), and deposition into insoluble Aβ aggregation forms (Nagano et al, 2004). The inversion of Aβ levels of WT mice between the ipsilateral and contralateral hemispheres of adult mice at 48 hours and 1 month after CCI might be explained by TBI-induced clearance of Aβ by the microglia (or by induction of other clearance mechanisms), whereas the persistent increase in injured hemispheres in homozygous APOE4 mice could be explained by the known effects of APOE4 on microglial Aβ metabolism (Vitek et al, 2009). Further studies are required to determine why immature mice do not accumulate Aβ40 (and perhaps other Aβ species) in the brain after CCI. Nonetheless, our data raise the question of whether Aβ-targeted therapy would be effective in younger patients with TBI (Loane et al, 2009).

Apolipoprotein E4 may also influence outcome after TBI by mechanisms altering immune regulation/inflammation, calcium homeostasis, excitotoxicity, and neurotransmission (Kerr et al, 2003; Lynch et al, 2003; Small, 2009). Previous studies have shown progressive deficits in working memory in the absence of age-related neurodegeneration in glial fibrillary acidic protein-APOE4 mice (Hartman et al, 2001), and APOE4 is associated with age-related decreases in synaptophysin-immunoreactive presynaptic terminals, decreased choline acetyltransferase activity, and reduced cholinergic fibers in mutant hAPP mice expressing APOE4 under the direction of neuron-specific enolase (Buttini et al, 2002).

The postinjury behavioral phenotypes associated with APOE4 seem to be independent of brain tissue damage per se, as brain tissue loss and brain edema did not differ between adult APOE4 and WT mice, and immature APOE4 mice had significantly decreased brain tissue loss compared with WT (Figure 6). Moreover, no gross differences were found in microglia activation at 48 hours between APOE4 versus WT immature or adult mice (Figure 7). Adult transgenic APOE4 mice had increased brain edema after intracerebral hemorrhage (James et al, 2009) and worse brain tissue damage in a closed head TBI model (Sabo et al, 2000). Our findings in adult homozygous APOE4 mice are consistent with some studies in human patients, which have failed to show differences in TBI severity in adult APOE4 carriers using magnetic resonance imaging (Hiekkanen et al, 2007). Our study also raises the interesting possibility that APOE4 may exert protective effects on acute cell death or other mechanisms of brain tissue loss after TBI in the immature period, through mechanisms yet to be determined. However, further studies are required to explore the possibility that the presently observed ability of APOE4 to sustain brain insult in immature mice may be lost under conditions in which APOE4 can be produced in neurons versus astrocytes.

In conclusion, we show significant age-related differences in functional outcome after CCI in mice carrying the APOE4 allele with the glial fibrillary acidic protein promoter. The data raise a number of questions regarding the potential differences in mechanism(s) of APOE4 toxicity in adult versus immature mice after TBI, and question the relevance of brain Aβ as a unifying mechanism of APOE4 toxicity across the age spectrum of TBI. The data also suggest that therapies targeting the APOE4 allele may need to be tailored according to age, because their use may not effectively prevent functional deficits after TBI in younger patients.

The authors declare no conflict of interest.

Footnotes

This work was supported by grants from NICHD 5K12HD052896 (RCM), the Cure Alzheimer's Fund (RET), NIA 1RO1AGO33016 (GT), and NINDS 5RO1NS047447 (MJW).

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