Behavioral Defects in Chaperone-Deficient Alzheimer's Disease Model Mice

Molecular chaperones protect cells from the deleterious effects of protein misfolding and aggregation. Neurotoxicity of amyloid-beta (Aβ) aggregates and their deposition in senile plaques are hallmarks of Alzheimer's disease (AD). We observed that the overall content of αB-crystallin, a small heat shock protein molecular chaperone, decreased in AD model mice in an age-dependent manner. We hypothesized that αB-crystallin protects cells against Aβ toxicity. To test this, we crossed αB-crystallin/HspB2 deficient (CRYAB-/-HSPB2-/-) mice with AD model transgenic mice expressing mutant human amyloid precursor protein. Transgenic and non-transgenic mice in chaperone-sufficient or deficient backgrounds were examined for representative behavioral paradigms for locomotion and memory network functions: (i) spatial orientation and locomotion was monitored by open field test; (ii) sequential organization and associative learning was monitored by fear conditioning; and (iii) evoked behavioral response was tested by hot plate method. Interestingly, αB-crystallin/HspB2 deficient transgenic mice were severely impaired in locomotion compared to each genetic model separately. Our results highlight a synergistic effect of combining chaperone deficiency in a transgenic mouse model for AD underscoring an important role for chaperones in protein misfolding diseases

Fear conditioning test.

<p>Percent freezing of mice over time during Training (Top), Cued test (middle) and Contextual test (bottom). Mean values ± SEM are plotted. Data were analyzed by two-way repeated measure ANOVA. Differences between groups were not significant for training and cued tests. In contextual tests, WT performed significantly better than WTTg (p = 0.003); difference between WTTg and KOTg was significant (p = 0.014); other groups were not significantly different. Blue hatched boxes represent two separate auditory signals (80dB) of 30 seconds each during training and a single auditory signal for 2 minutes during cued test followed by 2 sec foot shock. Symbol representations are - KOTg (red circle), KO (orange circle), WTTg (yellow triangle) and WT (green triangle). (n = 7 for KOTg; n = 15 for KO; n = 7 for WTTg and n = 9 for WT).</p

Hot plate test.

<p>The duration of time to elicit a thermo-sensitive reflex response in mice is shown on the left axis (black bars). The temperature at which the response was recorded is shown on the right axis (grey bars). Mean values ± SEM are plotted. Data for different groups were compared by t-tests. Both the time and temperature required elicit a response in KOTg were significantly higher than those for KO (p = 0.022 and 0.031, respectively), WT (p = 0.001 and 0.001, respectively) and WTTg (p = 0.05 and 0.05, respectively). All other groups were statistically similar. WT is <i>CRYAB^+/+, Tg^0/0</i>; WTTg is <i>CRYAB^+/+, Tg^+/0</i>; KO is <i>CRYAB^-/-, Tg^0/0</i> and KOTg is <i>CRYAB^-/-, Tg^+/0</i>. (n = 7 for KOTg; n = 15 for KO; n = 7 for WTTg and n = 9 for WT).</p

Generation of the required genotypes of mice.

<p>(A) Schematic diagram showing the mouse crosses that lead to the required genotypes. (B) Immunoblots showing αB-crystallin and Hsp27 expression in two sets of mice at 7 months of age. WT is <i>CRYAB^+/+HspB2^+/+, Tg^0/0</i>; WTTg is <i>CRYAB^+/+HspB2^+/+, Tg^+/0</i>; KO is <i>CRYAB^-/-HspB2^-/-, Tg^0/0</i> and KOTg is <i>CRYAB^-/-HspB2^-/-, Tg^+/0</i>.</p

Chaperone levels in AD model mice.

<p>(A) Immunoblots showing age-dependence of αB-crystallin expression in mice expressing mutant human APP transgene. Brain lysates were prepared from two individual non-transgenic (NTg1 & NTg2) or transgenic (Tg1 & Tg2) mice each at 3 months or 7 months of age. Samples were immunoblotted for αB-crystallin, Hsp70, Hsp27, Aβ and actin. 40 µg of total protein was analyzed. Similar levels of Hsp70, Hsp27 and actin in all samples show equal protein loading. (B) Densitometric analysis of band intensities in A. Intensities were compared by two-sample Student's t-test for statistical signifcance. APP expression in Tg mice were significantly greater than NTg mice as expected (p = 0.006 at 3 months, p = 0.017 at 7 months). αB-crystallin expression was significantly lower at 7-months compared to 3-months old transgenic mice brain (p = 0.044). αB-crystallin levels in 7-month old transgenic mice was also significantly lower than non-transgenic mice at similar age (p = 0.018). All other comparisons showed no significant differences.</p