Makoto Michikawa

Tooth loss induces memory impairment and neuronal cell loss in APP transgenic mice.

Tooth loss is a known risk factor of Alzheimers disease (AD). However, the association of tooth loss with the molecular pathogenesis of AD is still unknown. The hypothesis that the molecular pathogenesis of AD is enhanced by molar tooth loss was tested. Seventeen female transgenic mice (J20) were divided into the experimental (EX, n=10) and control (C, n=7) groups. In the EX group, maxillary bilateral molar teeth were extracted at the age of 6 months. In the C group, however, these teeth remained intact. Passive avoidance test was performed to evaluate learning and memory abilities right after tooth extraction (6 months old) and 4 months later (10 months old). After the test at 10 months, amyloid beta (Aβ) deposition and changes of neuronal cell number and area in the hippocampus were investigated using half of the brains. The other half was homogenized and used to determine Aβ40 and Aβ42 levels by ELISA. At the 10 months of age, learning and memory abilities were significantly impaired in the EX group compared to the C group (P<0.05). The neuronal cell number in the CA1 and CA3 regions was significantly lower in the EX group than in the C group (P<0.05). Total Aβ, Aβ40, and Aβ42 levels showed no significant intergroup difference. Molar tooth loss may cause neuronal cell loss in the hippocampus, leading to memory impairment; this process may be independent of the amyloid cascade.

Aβ43 Is the Earliest-Depositing Aβ Species in APP Transgenic Mouse Brain and Is Converted to Aβ41 by Two Active Domains of ACE

Amyloid-β protein (Aβ) varies in length at its carboxyl terminus. The longer Aβ species, Aβ43 and Aβ42, are highly amyloidogenic and deposit more frequently than Aβ40 in the brain of Alzheimer disease (AD) patients. However, the characterization of Aβ43 deposition in the brain and the relationship between Aβ43 and Aβ42 or Aβ40 remain unclear. We provide evidence that Aβ43 deposition appears earlier than Aβ42 and Aβ40 deposition in the brain of mutant amyloid precursor protein transgenic (APPtg) mice, suggesting that Aβ43 is the earliest-depositing species. In addition, we found increased Aβ43 levels and Aβ43/Aβ42 ratios in the serum of AD patients, suggesting their use as diagnostic blood biomarkers for AD. We further show that angiotensin-converting enzyme (ACE) converts Aβ43 to Aβ41. Notably, this Aβ43-to-Aβ41 converting activity requires two active domains of ACE. Inhibition of ACE activity significantly enhanced Aβ43 deposition in APPtg mouse brain. Our results suggest that Aβ43 is the earliest-depositing species in brain parenchyma and that Aβ43 may trigger later Aβ42 and Aβ40 deposition or may be converted to Aβ42 and Aβ40 plaques. Activities of both ACE domains may be important for reducing Aβ43 levels in serum and reducing brain Aβ43 deposition.

KSGal6ST is essential for the 6-sulfation of galactose within keratan sulfate in early postnatal brain

Keratan sulfate (KS) comprises repeating disaccharides of galactose (Gal) and N-acetylglucosamine (GlcNAc). Residues of Gal and GlcNAc in KS are potentially modified with sulfate at their C-6 positions. The 5D4 monoclonal antibody recognizes KS structures containing Gal and GlcNAc, both 6-sulfated, and has been used most extensively to evaluate KS expression in mammalian brains. We previously showed that GlcNAc6ST1 is an enzyme responsible for the synthesis of the 5D4 epitope in developing brain and in the adult brain, where it is induced after injury. It has been unclear which sulfotransferase is responsible for Gal-6-sulfation within the 5D4 KS epitope in developing brains. We produced mice deficient in KSGal6ST, a Gal-6-sulfotransferase. Western blotting and immunoprecipitation revealed that all 5D4-immunoreactivity to proteins, including phosphacan, were abolished in KSGal6ST-deficient postnatal brains. Likewise, the 5D4 epitope, expressed primarily in the cortical marginal zone and subplate and dorsal thalamus, was eliminated in KSGal6ST-deficient mice. Disaccharide analysis showed the loss of Gal-6-sulfate in KS of the KSGal6ST-deficient brains. Transfection studies revealed that GlcNAc6ST1 and KSGal6ST cooperated in the expression of the 5D4 KS epitope in HeLa cells. These results indicate that KSGal6ST is essential for C-6 sulfation of Gal within KS in early postnatal brains.

Arachidonic or Docosahexaenoic Acid Diet Prevents Memory Impairment in Tg2576 Mice

It is believed that the amyloid β-protein (Aβ) plays a causative role in the development of Alzheimers disease (AD). The amyloid-β protein precursor (AβPP), a substrate of Aβ, and β-secretase and γ-secretase complex proteins, which process AβPP to generate Aβ, are all membrane proteins. Thus, it is reasonable to assume that alterations in brain lipid metabolism modulate AβPP and/or Aβ metabolism. However, the role of cellular polyunsaturated fatty acids in AβPP processing has not been completely understood yet. We report here that 4 months of treatment of Tg2576 mice with an arachidonic acid (ARA)- or a docosahexaenoic acid (DHA)-containing (ARA+ or DHA+) diet prevented memory impairment at 13 months of age. Although, AβPP processing to generate soluble AβPP and induce Aβ synthesis was enhanced, Aβ(1- 42)/Aβ(1- 40) ratio decreased in 14-month-old Tg2576 mice fed with the ARA+ or DHA+ diet. The ARA+ or DHA+ diet did not alter the AβPP levels and the expression levels of Aβ-degrading enzymes. In cortical primary neuron cultures, ARA or DHA treatment also increased soluble AβPP and Aβ(1- 40) levels, and decreased Aβ(1- 42)/Aβ(1- 40) ratio, which are similar to what were observed in Tg2576 mice fed with ARA+ or DHA+ diet. These findings suggest that not only the DHA+ diet, but also the ARA+ diet could prevent cognitive dysfunction in Tg2576 mice through the alteration of AβPP processing.

Arachidonic acid diet attenuates brain Aβ deposition in Tg2576 mice.

The amyloid β-protein (Aβ) is believed to play a causative role in the development of Alzheimers disease (AD). Because the amyloid precursor protein (APP), a substrate of Aβ, and β-secretase and γ-secretase complex proteins, which process APP to generate Aβ, are all membrane proteins, it is possible to assume that alterations in brain lipid metabolism modulate APP and/or Aβ metabolism. However, the role of polyunsaturated fatty acids in Aβ metabolism remains unknown. We report here that 9 months-treatment of Tg2576 mice with arachidonic acid (ARA)-containing (ARA+) diet prevented brain Aβ deposition in 17-month-old Tg2576 mice. APP processing to generate soluble APPα, CTF-β, and Aβ synthesis was attenuated in Tg2576 mice fed with the ARA+ diet. These findings suggest that ARA+ diet could prevent Aβ deposition through the alteration of APP processing in Tg2576 mice.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological changes in intracellular organellae in cultured rat astrocytes

3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) is widely used for cell viability and cytotoxicity assays, but cell biological effects of MTT itself have not been investigated. In this paper we show that MTT induces a morphological change in an intracellular membranous compartment labeled with anti‐Rab5 antibody, dissociation of early endosomal auto‐antigen (EEA1) from the membrane fraction, and phosphorylation of Akt probably through a phosphatidylinositol‐3‐OH kinase [PI(3)K] pathway in cultured rat astrocytes. These findings suggest that MTT affects cellular functions and conditions to some extent, and such effects of MTT may cause some discrepancies of measurement of cell viability using MTT assay and other assays. That is, the effects of MTT on cells could influence the results of cell viability assay. Moreover, MTT or other tetrazolium salts could be used as interesting activators of Akt to investigate the mechanism by which Akt or PI(3)K is activated.

Angiotensin type 1a receptor deficiency decreases amyloid β-protein generation and ameliorates brain amyloid pathology

Alzheimer’s disease is characterized by neuronal loss and cerebral accumulation of amyloid-β protein (Aβ) and lowering the generation of Aβ is a pivotal approach in the strategy of Alzheimer’s disease treatment. Midlife hypertension is a major risk factor for the future onset of sporadic Alzheimer’s disease and the use of some antihypertensive drugs may decrease the incidence of Alzheimer’s disease. However, it is largely unknown how the blood pressure regulation system is associated with the pathogenesis of Alzheimer’s disease. Here we found that the deficiency of angiotensin type 1a receptor (AT1a), a key receptor for regulating blood pressure, significantly decreased Aβ generation and amyloid plaque formation in a mouse model of Alzheimer’s disease. The lack of AT1a inhibited the endocleavage of presenilin-1 (PS1), which is essential for γ-secretase complex formation and Aβ generation. Notably, the ligand of AT1a, angiotensin II, enhanced Aβ generation, PS1 endocleavage and γ-secretase complex formation. Our results suggest that AT1a activation is closely associated with Aβ generation and brain amyloid accumulation by regulating γ-secretase complex formation. Thus, removal of life style factors or stresses that stimulate AT1a to elevate blood pressure may decrease Aβ generation and brain amyloid accumulation, thereby preventing the pathogenesis of Alzheimer’s disease.

Liquid diet induces memory impairment accompanied by a decreased number of hippocampal neurons in mice

It is suggested that masticatory dysfunction affects the central nervous system; however, the underlying mechanism remains unknown. Brain‐derived neurotrophic factor (BDNF) and its receptor, TrkB, are known to play important roles in memory and learning. In this study, we examined the effects of mastication on memory, the expression levels of BDNF and TrkB, and the number of neurons in the hippocampus of mice. Male C57 BL/6J mice (3 weeks old) were randomly divided into the control group (Nu2009=u20097) fed chow pellets and the experimental group (Nu2009=u20097) fed a liquid diet, which reduces mastication during eating. At 14 weeks of age, we performed a passive avoidance test and found that memory and learning ability were impaired in the experimental group compared with the control group. After the behavioral experiment, brains were harvested and analyzed morphologically and biochemically. In the hippocampus of the experimental group, the expression levels of BDNF were significantly higher, whereas those of TrkB were lower than those of the control group. In the cerebral cortex, these levels remained unchanged between the two groups. The ratio of phospho‐p44/42 ERK/pan ERK, a downstream molecule of BDNF/TrkB signaling, in the experimental group was significantly lower than that of the control group in the cortex and hippocampus. The number of pyramidal neurons in the hippocampus was lower in the experimental group than in the control group. These findings suggest that reduced mastication induced by a liquid diet in early childhood may impair memory and learning ability, accompanied by neuronal loss in the hippocampus.

Astrocyte׳s endogenous apoE generates HDL-like lipoproteins using previously synthesized cholesterol through interaction with ABCA1

Apolipoprotein E (apoE) in the brain is predominantly synthesized in and secreted from astrocytes to generate apoE-containing high-density lipoprotein-like particles (apoE/HDL). However, the mechanism underlying generation of apoE/HDL has not been completely understood. The newly synthesized cholesterol, which is synthesized in rat astrocytes within 24 h using [(3)H]-acetate as a cholesterol precursor, was assembled as lipoproteins with densities of 1.12-1.17 g/mL (higher density HDL), although apoE was secreted as lipoproteins with lower densities of 1.08-1.12 g/mL from the cells. This finding suggests that the newly synthesized cholesterol is released without the association with apoE, which is like that from apoE-deficient mouse (apoE-KO) astrocytes. The cholesterol released from rat astrocytes at 3 days after the onset of its synthesis (previously synthesized cholesterol) was assembled as apoE/HDL with the densities of 1.08-1.12 g/mL (lower density HDL). These findings indicate that the endogenous apoE participates in the release of previously synthesized cholesterol but not newly synthesized one. Whereas, exogenously added human apoE induced release of both newly synthesized and previously synthesized cholesterols to generate apoE/HDL with lower density, suggesting that the cellular pool of cholesterol released by endogenous and exogenous apoE is different. The endogenous apoE was distributed in the caveolin-1-rich domain along with ATP-binding cassette transporter A1 (ABCA1) in the membrane fraction and immuno-precipitated using an anti-ABCA1 antibody. However, this is not the case for ABCA1-KO astrocytes. These findings suggest that endogenous apoE generates lower density HDL to produce more lipidated HDL using previously synthesized cholesterol through the interaction with ABCA1 in caveolin-1-rich domain of astrocytes.

Enhancement of FGF-1 release along with cytosolic proteins from rat astrocytes by hydrogen peroxide

We previously observed that the production and release of fibroblast growth factor (FGF-1) are increased in rat astrocytes during in vitro long-term culture, that FGF-1 enhances the generation of apoE-containing high density lipoproteins (apoE/HDL), and that the wound healing of brain cryoinjury delays in apoE-deficient mouse. The detail mechanism underlying these phenomena remains unknown. In this study, we examined effects of oxidative stress on release of FGF-1 from cultured rat astrocytes. The treatment of rat astrocytes with 100µM hydrogen peroxide (H2O2) for 10min enhanced FGF-1 release without inducing apoptosis. The conditioned medium prepared from the cells cultured in a fresh medium after the treatment with H2O2 had the FGF-1-like activities, which enhanced cholesterol synthesis, signalings to phosphorylate Akt and ERK, and apoE secretion. The oxidative stress induced by H2O2 enhanced the release of cytosolic proteins such as HSP70 and HSP90 in addition to FGF-1. Antioxidants such as ascorbic acid and ebselen suppressed the release of cytosolic proteins induced by H2O2 treatment. The addition of lipoproteins such as low density lipoproteins (LDL), furthermore, canceled H2O2-induced release of FGF-1 and cytosolic proteins. Proteolysis of cytosolic proteins in the H2O2-treated rat astrocytes was enhanced in the presence of exogenous trypsin, which was attenuated by the pretreatment with LDL, suggesting that H2O2 increases the permeability of the membrane of cells, which was prevented by the addition of lipoproteins. These findings suggest that oxidative stress is one of the candidates which triggers FGF-1 release from astrocytes in the brain, and that the lipid homeostasis in the cell membrane may regulate H2O2-induced release of FGF-1.