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. 2007 May 25;146(3):890-900.
doi: 10.1016/j.neuroscience.2007.02.027. Epub 2007 Mar 27.

Astrogliosis and behavioral changes in mice lacking the neutral cysteine protease bleomycin hydrolase

S E Montoya  1 , E Thiels, J P Card, J S Lazo
Affiliations

Astrogliosis and behavioral changes in mice lacking the neutral cysteine protease bleomycin hydrolase

S E Montoya et al. Neuroscience. .

Abstract

Bleomycin hydrolase (BLMH) is a multifaceted neutral cysteine protease with a suggested role in antigen presentation, homocysteine-thiolactone metabolism, and Alzheimer's disease pathogenesis. Deletion of the protease in mice results in increased neonatal mortality and dermatopathology. Immunohistochemical and behavioral studies of BLMH knockout mice were undertaken to further evaluate the role of the protease in the brain. No gross abnormalities in the CNS were observed upon preliminary histological examination of B6.129Blmhtm1Geh/J null animals. However, glial fibrillary acid protein immunohistochemistry revealed a global reactive astrogliosis in the aged null animals, indicative of undefined brain pathology. The role of BLMH in the brain was further explored by characterizing the behavioral phenotype of hybrid [129S6-Blmhtm1Geh/JxB6.129 Blmhtm1Geh/J]F1 null and littermate controls using multiple behavioral paradigms. In the water maze, deletion of BLMH resulted in poorer performance during water maze probe trials without detectable effect of the mutation on sensorimotor function. In addition, no age-dependent decline in discriminative performance on probe trials was observed in null animals. These data suggest a physiological non-redundant function for BLMH in the CNS.

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Figures

Figure 1
Figure 1. Nissl staining of B6.129 Blmhtm1Geh/J null and wild-type controls
Serially sectioned 4 % paraformaldehyde fixed brain tissue from male aged littermate wild-type and BLMH null B6.129 Blmhtm1Geh/J mice were stained with Nissl. A. Nissl staining of the hippocampus. B. Nissl staining of cerebral cortex. (N = 3 +/+, −/−).
Figure 2
Figure 2. GFAP staining in cortex and hippocampus of B6.129 Blmhtm1Geh/J null and wild-type controls
Serially sectioned 4 % paraformaldehyde-fixed brain tissue from littermate male aged wild-type and BLMH null B6.129 Blmhtm1Geh/J mice were immunostained with GFAP to assess astrogliosis. Representative GFAP staining of the cortex (A), hippocampus (B), CA 1 region of hippocampus (C) and the CA3 region of hippocampus (D) (N = 3 +/+, −/−). Bar graph representation of the number of GFAP stained cells in the CA1 and CA3 regions of the hippocampus from wild-type and null hybrid mice (E). *P < 0.05, t-test for independent groups; **P < 0.005, t-test for independent groups).
Figure 3
Figure 3. Rotarod testing of BLMH null F1 hybrids and littermate controls
Young and aged F1 hybrid BLMH null and wild-type mice were tested over seven days on an accelerating rotarod apparatus (3–19 rpm). Latency to fall from the apparatus was measured daily during a 180-s trial and represented as mean ± SEM for each genotype. A. Seven-day accelerating rotarod test for 4-to 5-month old young males (+/+ N = 8, −/−N = 7). B. Seven-day accelerating rotarod retesting for males at 14 to 15 months of age (+/+ N = 8, −/− N = 7). C. Seven-day accelerating rotarod test for naïve 20- to 23- month old female mice (+/+ N = 6, −/− N = 6). * Post-hoc test P< 0.05.
Figure 4
Figure 4. Contextual fear conditioning of BLMH null F1 hybrids and littermate controls
Contextual fear conditioning was evaluated in young and aged F1 hybrid BLMH null and wild-type mice. On the day of training, animals were exposed for 2 min to the training context (train-context) and a 30-s tone (train–tone). Immediately following the tone, the animals received a 0.75-mA scrambled foot shock. Tone and shock pairing was repeated once after a 2-min interval. Twenty-four hours later, the animals were evaluated for freezing when exposed to the original training context (context) or when exposed to the tone in a novel context (tone). Data are represented for pooled male and female mice by genotype as mean ± SEM. Aged animals showed significantly more freezing than young animals to the context (P < 0.0005) and the tone (P < 0.005) during testing.
Figure 5
Figure 5. Acquisition phase of water maze testing for BLMH null and littermate controls
Young and aged F1 hybrid BLMH null and wild-type animals were evaluated for learning and memory of spatial information using the water maze paradigm. On days 1–5, each animal received four training trials in which the animal was allowed to swim freely about a pool in which a submerged, hidden platform was located. On days 6 and 7, the task was modified in that the platform was made visible. Average escape latency from the water was measured for each animal in s and is represented for pooled male and female mice as mean ± SEM. A. Latency results for young animals across the 5 days of training and the visible-platform testing (days 6 and 7) (+/+ N = 12, −/− N = 13). B. Latency results for aged animals across the 5 days of training and the visible-platform testing (days 6 and 7). (+/+ N = 10, −/− N = 12).
Figure 6
Figure 6. Probe trial testing of BLMH null and littermate controls
Probe trials were conducted to evaluate retention of the submerged platform location following 5 days of training. Young and aged F1 hybrid BLMH null and wild-type animals were allowed to swim freely for 60 s in the water tank with the platform removed. Time spent in each quadrant was recorded in s and expressed as mean (± SEM) percent time in the respective quadrants. Target represents the quadrant that formerly contained the submerged platform. A. Percent time spent in each of the 4 test quadrants by pooled male and female young mice. B. Percent time spent in each of the 4 test quadrants by pooled male and female aged animals. Animals in all groups tested show preference for the target quadrant over control quadrants. C. Percent time in the target quadrant for wild-type and BLMH null mice across age. Young null animals spent significantly less time in the target quadrant than did young wild-type controls (P < 0.005). In addition, aged wild-type animals spent significantly less time in the target quadrant than young wild-type mice (P < 0.005). * P<0.05, **P < 0.005, *** P < 0.001
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