Conceived and designed the experiments: TH. Performed the experiments: TH NM. Analyzed the data: TH. Contributed reagents/materials/analysis tools: TH MK. Wrote the paper: TH. Discussion and development of 3xTg-AD model mice: FML.
N.M. is employed by Kyudo Ltd; this company had no role in this study design, data collection and analysis, decision to publish, or preparation of this manuscript. All other authors declare no competing interests. Parts of the experiment, such as feeding model mice, were supported by Arfresa Pharmcology (
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 (Aβ) 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 Aβ 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.
Amyloid plaques and neurofibrillary tangles are the two pathophysiological hallmarks of Alzheimer's disease (AD). Intracellular amyloid-beta 42 (Aβ42) is increasingly being recognized as an early pathological trigger that can lead to amyloid plaques and may even induce neurofibrillary tangles. We previously reported that homocysteic acid (HA) induces intracellular accumulation of Aβ42 and that the production of α-synuclein in the presence of methionine results in cell death
HA affects the two pathophysiological hallmarks of AD and may be involved in its etiology. HA is also known as an NMDA (N-methyl-D-glutamate) receptor agonist
On the basis of these HA toxicities, few researchers have studied the possibility of HA pathogenicity in the onset of AD
The 3xTg-AD mouse model showed memory impairment at an early age because of Aβ accumulation in neuronal cells
The mouse germline used in this study was a kind gift from Professor F. M. Laferla (University of California, Irvine). The housing environment (12 h/12 h light/dark cycle) was a germ-free clean room. Seven 3xTg-AD hemizygous male mice (3 and 7 months old) were studied. Also, four nontransgenic (non-Tg) mice were studied. The 3xTg-AD mice developed both plaque and tangle pathology in AD-relevant brain regions. Despite an equivalent overexpression of the human sAPP and human tau transgenes, the 3xTg-AD mice developed extracellular As deposits before tangle formation, consistent with the amyloid cascade hypothesis. In addition, these mice exhibited deficits in synaptic plasticity, including long-term potentiation, which occurs before extracellular As deposition and tau pathology but is associated with intracellular As immunoreactivity. These results support the view that synaptic dysfunction is a proximal defect in the pathobiology of AD and precede extracellular plaque formation and neurofibrillary pathology. As these 3xTg-AD mice phenocopy critical aspects of AD neuropathology, this model will be useful in preclinical intervention trials, particularly because the efficacy of anti-AD compounds in mitigating the neurodegenerative effects mediated by both signature lesions can be evaluated.
Vitamin B6-deficient food was purchased from Kyudo Ltd.
Nutrient composition will be described further.
Anti-HA antibodies were purchased from MoBiTec Co. (Germany). Polyclonal antisera were raised in rabbits after immunization with a glutaraldehyde-containing HA conjugate, following which antibody specificity was determined by performing ELISA with competition experiments involving HA-G-BSA, cysteine-G-BSA, and homocysteine-G-BSA (compound cross-reactivity ratios 1∶1, 1∶85, and 1∶231, respectively).
We synthesized a glutaraldehyde-containing HA conjugate KLH (Keyhole limpet hemocyanin) compound, and mice were immunized twice by IP route with this compound and BCG(Bacillus Calmette-Guérin). Two weeks later KLH and BCG immunized mice again. Immunization volume was 20 µL.
The apparatus used for Morris water maze task comprised a circular aluminum tank (1.5 m in diameter) painted white and filled with water maintained at 26–29°C. The maze was located in a room containing several simple, visual extramaze cues. To reduce stress, mice were placed on a platform in both the hidden and cued versions of the task for 10 s before the first training trial.
Mice were trained to swim to a 14-cm circular clear Plexiglas platform placed 1.5 cm below the water surface that was invisible to the mice while swimming. Platform location was randomly selected for each mouse but was kept constant for that mouse throughout the training period. In each trial, mouse was placed in the tank at one of the four designated starting points in a pseudorandom order. Mice were allowed to search for and escape to the submerged platform. If a mouse failed to find the platform within 60 s, it was manually guided towards it and allowed to remain there for 10 s. Then, each mouse was placed in a holding cage under a warming lamp for 25 s until the start of the next trial. To ensure that memory differences were not due to the lack of task learning, the mice underwent four trials a day for as many days as required to meet the criterion, which was defined as a <20-s mean escape latency before the first probe trial was run. To prevent overtraining, probe trials were run for each group as soon as they met the group criterion and stopped after all the groups met the criterion. Retention of spatial training was assessed 1.5 and 24 h after the last training trial. Both probe trials consisted of a 60-s free swim in the pool without the platform. Mice were monitored by a camera mounted on the ceiling directly above the pool and all trials were stored on videotape (burnt onto a DVD) for subsequent analysis. Parameters measured during the probe trial comprised initial latency time to reach the platform (1).
Mice were sacrificed by CO2 asphyxiation, and the brains were rapidly removed and fixed for 48 h in 4% paraformaldehyde. Sections (50-µm thick) were processed for free-floating immunohistochemistry as previously described
HA was extracted from mouse brain (hippocampus and cortex) with trichloroacetic acid. Brains (1.50−2.00 g) were isolated from 4-month-old 3xTg-AD homozygous male mice. Mice were killed by rapid decapitation and their brains were quickly excised and placed on an ice-cold petri dish. For the gradient high-performance liquid chromatography (HPLC) method, tissue samples were weighed and homogenized using a sonicator for 10 s in ice in 4 mL of ice-cold 10% (w/v) trichloroacetic acid per 100 mg tissue (wet weight). HA (4 µg) was added as an internal standard. For isocratic HPLC, tissue samples were divided into six aliquots. The samples were homogenized as described above. The homogenates for isocratic or gradient HPLC were left on ice for 1 h and centrifuged at 20,000×
Urine of the 3xTg-AD male mice (15-month-old) was collected for 24 h. The urinary HA level was measured according to the method of the HPLC system.
Mice were anesthetized as follows: 2-mm-wide incisions were made in the left hemisphere and a guide cannula was inserted into the left ventricular space using a Teflon tube (1 mm in diameter). This operation did not impair learning and memory performance, and the abilities of the operated mice were similar to those of mice that did not undergo surgery.
Statistical significance was estimated with Student's
All animals experiments have been done according to the accepted international guideline methods and Saga Woman Junior College approved this work according to animal experimental guideline.
We measured HA levels in the brains of 4-month-old 3xTg-AD-homozygous mice. At this age, mice display an intracellular accumulation of Aβ in the brain regions affected by AD. This accumulation also appears to be associated with the early memory deficit exhibited by these mice
Category | Results | P value |
Hippocampus | ||
HA level pmoles/mg wet weight | ||
Control | 23.63±9.2 (n = 5) | |
3xTg-AD | 41.25±5.4 (n = 4) | <0.001 |
Cortex | ||
Control | 20.41±6.6 (n = 5) | |
3xTg-AD | 32.16±4.8 (n = 4) | <0.01 |
HA level in the brain of 3xTg-AD male mice at 4 months was measured by the method described in
We then fed 3-month-old 3xTg-AD mice vitamin B6-deficient food for 3 weeks to increase HA levels in the brain. The brain HA levels were significantly increased following the 3-week vitamin B6-deficient diet (
Category | Results | P value |
Control (n = 10) | 42.0±8.1 pmoles/mg brain | |
B6 deficient (n = 10) | 65.2±15.0 pmoles/mg brain | <0.001 |
Anti-HA antibody Treatment (n = 10) | 20.1±7.1 pmoles/mg brain | <0.001 |
Control mice: 3xTg-AD mice at 3 months and 3 weeks with normal feeding. Control mice showed good memory performance, but B6-deficient feeding group showed memory impairment. Whole brain was homogenized with physiological saline and HA level was measured, after the memory task, according to the method described in
To further examine the role of HA in the pathogenesis of AD, we administered anti-HA antibody intracranially through guide cannula during the course of the vitamin B6-deficient diet. Anti-HA antibody significantly decreased the HA level in 3xTg-AD mice (
A. HA Level in Urine of 3xTg-AD Mice at 15 Months (n = 5) | ||
Category | Results | P value |
Control | 22.5±8.5 µM | |
Vaccine treatment | 7.8±9.1 µM (35% of control) | <0.001 |
B. HA Level in 3xTg-AD Mice Brain at 12 Months | ||
Category | Results | P value |
Control (n = 10) | 71.5±20.3 pmoles/mg brain | |
Vaccine treatment (n = 10) | 30.6±18.4 pmoles/mg brain (43% of control) | <0.001 |
Note: A: Vaccine treatment was started at 12 months. Three months later, urine was collected an entire day and HA level was measured. 3xTg-AD male mice were all homozygous. HA level with vaccine treatment decreased with time, indicating that vaccine efficacy became stronger with time.
Note: B: After Morris water maze test, these mice were sacrificed and their brains were immediately freezed in liquid nitrogen. Next, HA level was measured with the method described in Materials and Method.
We next evaluated the hippocampus-dependent memory with the Morris water maze task. Three-month old 3xTg-AD mice given a vitamin B6-deficient diet exhibited significant memory impairment compared with 3-month-old control mice (non-Tg and Tg with a normal diet exhibited good memory performance;
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 (
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
After 3 weeks, we measured memory performance in the Morris water maze tasks (
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
Mice were treated the same as those shown in
Category | Results | Average | P value |
Control | 9, 8.8, 7.8, 9 8.2 (cm) | 8.6±0.5 | |
Experiment | 9.6, 10.1, 9.6, 10.3 (cm) | 9.9±0.4 | <0.01 |
Control: 7 month-old hemizygous + B6 deficient.
Experimental: 7 month-old hemizygous male +B6 deficient + anti-HA antibody.
5 different sections were observed and its major axis was measured.
Average (n = 2) is shown.
Non-Tg control showed Hippocampal major axis of 10±0.5 cm.
Category | Results (sec) | P value |
Control | 28.5±2 s (n = 5) | |
Vaccine treatment | 56±8 s (n = 5) | <0.001 |
Mice tails were clipped by a tiny metal clipper and the time taken by the mice to turn back and take off these clippers was measured. 3xTg-AD male mice were 18-month-old. Vaccine treatment was started at 12 months. 3xTg-AD male mice were all homozygous.
Our results were obtained by vitamin B6-deficient feeding to mice, which induced the vitamin B6-deficient burden on 3xTg-AD mice. We then investigated whether the cure effect of anti-HA antibody can be observed on 3xTg-AD mice fed with normal food. The result is shown in
Mice were 12 months old. Memory performance was measured via the Morris water experiment described in
We developed the HA vaccine according to the anti-HA antibody procedure described in
We demonstrated the specificity of anti-HA antibody or HA vaccine to destroy HA. From
The results of our transgenic experiments confirmed that anti-HA antibody suppressed memory impairment induced by B6-deficient food. This hypothesis is supported by our data suggesting that higher levels of HA are present in 3xTg-AD homozygous mice compared with non-Tg mice (
Now, we would like to clarify whether the HA vaccine (anti-HA antibody) induced neurogenesis. We are interested in the finding that the anti-pain effect induced the neurogenesis of hippocampus
In conclusion, (i) HA accelerates pathological changes in AD; and (ii) HA toxicity decreases with anti-HA antibody, which induces strong neurogenesis in the hippocampus, resulting in marked recovery of memory performance. Our hypothesis is also supported by the observation that anti-HA antibodies induced marked recovery in 7-month-old hemizygous mice However, recovery was observed on the second trial day and not the third trial day. This observation could be probably induced by the fact that the control hemizygous mice did not impair partially their memory ability at 7 months old and that control mice exhibited partial good memory performance on the third trial day. We should note the cure effect of anti-HA antibody or HA vaccine on memory impairment of 3xTg-AD mice. Homozygous 3xTg-AD mice exhibited complete memory impairment at 12 months old with normal food intake (
This is the first study, to our knowledge, to demonstrate marked recovery from AD induced by treatment with anti-HA antibody or HA vaccine. Our findings prove the strong curative effect of anti-HA antibody treatment and HA vaccine and support the idea that HA is a true etiological agent and an accelerator in the pathogenesis of AD. However, one may think that vitamin B6-deficient burden 3xTg-AD mice to have HA pathogen artificially. We thus needed to investigate whether the effect of anti-HA antibody or HA vaccine could be observed in the normal feeding of 3xTg-AD mice. The result shows clearly the strong cure effect of anti-HA antibody and HA vaccine in the normal feeding of 3xTg-AD mice (12-month-old;
But how does HA induce neurodegeneration? HA affects the two pathophysiological hallmarks of AD and may be involved in its etiology. Moreover, HA itself can induce neurodegeneration at a higher level with no amyloid
Dr. Masayasu Ohyagi (Kyushu University, Japan) is acknowledged for his technical support in the histochemical and immunochemical staining observations and for his scientific advice.