Treatment of Alzheimer's disease with anti-homocysteic acid antibody in 3xTg-AD male mice

Abstract

Alzheimer's disease (AD) is an age-associated progressive neurodegenerative disorder with dementia, the exact pathogenic mechanisms of which remain unknown. We previously reported that homocysteic acid (HA) may be one of the pathological biomarkers in the brain with AD and that the increased levels of HA may induce the accumulation of intraneuronal amyloid-beta (Abeta) peptides. In this study, we further investigated the pathological role of HA in a mouse model of AD. Four-month-old prepathological 3xTg-AD mice exhibited higher levels of HA in the hippocampus than did age-matched nontransgenic mice, suggesting that HA accumulation may precede both Abeta and tau pathologies. We then fed 3-month-old 3xTg-AD mice with vitamin B6-deficient food for 3 weeks to increase the HA levels in the brain. Concomitantly, mice received either saline or anti-HA antibody intraventricularly via a guide cannula every 3 days during the course of the B6-deficient diet. We found that mice that received anti-HA antibody significantly resisted cognitive impairment induced by vitamin B6 deficiency and that AD-related pathological changes in their brains was attenuated compared with the saline-injected control group. A similar neuroprotective effect was observed in 12-month-old 3xTg-AD mice that received anti-HA antibody injections while receiving the regular diet. We conclude that increased brain HA triggers memory impairment and that this condition deteriorates with amyloid and leads to subsequent neurodegeneration in mouse models of AD.

Figure 1. Long-Term Memory Test in the Morris Water Maze task.

Non-transgenic mice had an average score of 3 mice each day. Hemizygous transgenic control (hemizygous + B6 deficient) mice had an average score of 3 transgenic control mice each day. Transgenic experimental (hemizygous + B6 deficient + anti-HA antibody) mice had an average score of 3 transgenic experimental mice each day. 3xTg-AD mice were aged 3 months and 3 weeks. Statistically significant difference was observed after 2 trial days between hemizygous + B6 deficient and hemizygous + B6 deficient + antibody (P<0.001) and after 3 trial days between hemizygous + B6 deficient and hemizygous + B6 deficient + antibody (P<0.05).

Figure 2. Immunohistochemical observations of Amygdalar, Cortical, and Hippocampal Neurons.

Anti-HA antibody was diluted 100-fold. Immunohistochemical observations were repeated thrice and each observation gave the same result. Ten homozygous transgenic 3-month-old mice were fed B6-deficient food for 3 weeks. Transgenic experimental mice were injected with anti-HA antibody every 3 days. For details, see Materials and Methods. CTL, transgenic control mice; Ex, transgenic experimental mice.

Figure 3. Curative Effect of Anti-HA Antibody as shown by Long-Term Memory Performance.

Seven-month-old male 3xTg-AD homozygous mice mice fed B6-deficient food for 3 weeks served as the control. Experimental mice were treated with anti-HA antibody every 3 days; antibody (100-fold dilution) was injected into the brain as described in Materials and Methods. The figure shows average data for five male mice. To demonstrate the strong curative effect of the antibody, for comparison we show the results for 2-month-old hemizygous male mice, whose memory performance was normal. Statistically significant difference was observed after 2 trial days (P<0.001). However after 3 trial days, a statistically significant difference was not observed (^* P<0.001). The difference between control and experiment mice (P<0.01). The difference between experimental mice and 7-month-old mice with normal food (n = 5).

Figure 4. Hematoxylin-Eosin Staining of Cortical and Hippocampal Neurons in Control and Experimental Groups.

Mice were treated the same as those shown in Fig. 3.

Figure 5. The Effect on Memory Performance of HA Vaccine on Normal Feeding of 3xTg-AD Male Mice.

Mice were 12 months old. Memory performance was measured via the Morris water experiment described in Materials and Methods. Statistically significant difference was observed on the third, fourth, and fifth trial days (P<0.001). 3xTg-AD male mice were all homozygous (n = 15).