Ken Ichiro Fukuchi

TLR4 mutation reduces microglial activation, increases Aβ deposits and exacerbates cognitive deficits in a mouse model of Alzheimer's disease.

BackgroundAmyloid plaques, a pathological hallmark of Alzheimers disease (AD), are accompanied by activated microglia. The role of activated microglia in the pathogenesis of AD remains controversial: either clearing Aβ deposits by phagocytosis or releasing proinflammatory cytokines and cytotoxic substances. Microglia can be activated via toll-like receptors (TLRs), a class of pattern-recognition receptors in the innate immune system. We previously demonstrated that an AD mouse model homozygous for a loss-of-function mutation of TLR4 had increases in Aβ deposits and buffer-soluble Aβ in the brain as compared with a TLR4 wild-type AD mouse model at 14-16 months of age. However, it is unknown if TLR4 signaling is involved in initiation of Aβ deposition as well as activation and recruitment of microglia at the early stage of AD. Here, we investigated the role of TLR4 signaling and microglial activation in early stages using 5-month-old AD mouse models when Aβ deposits start.MethodsMicroglial activation and amyloid deposition in the brain were determined by immunohistochemistry in the AD models. Levels of cerebral soluble Aβ were determined by ELISA. mRNA levels of cytokines and chemokines in the brain and Aβ-stimulated monocytes were quantified by real-time PCR. Cognitive functions were assessed by the Morris water maze.ResultsWhile no difference was found in cerebral Aβ load between AD mouse models at 5 months with and without TLR4 mutation, microglial activation in a TLR4 mutant AD model (TLR4M Tg) was less than that in a TLR4 wild-type AD model (TLR4W Tg). At 9 months, TLR4M Tg mice had increased Aβ deposition and soluble Aβ42 in the brain, which were associated with decrements in cognitive functions and expression levels of IL-1β, CCL3, and CCL4 in the hippocampus compared to TLR4W Tg mice. TLR4 mutation diminished Aβ-induced IL-1β, CCL3, and CCL4 expression in monocytes.ConclusionThis is the first demonstration of TLR4-dependent activation of microglia at the early stage of β-amyloidosis. Our results indicate that TLR4 is not involved in the initiation of Aβ deposition and that, as Aβ deposits start, microglia are activated via TLR4 signaling to reduce Aβ deposits and preserve cognitive functions from Aβ-mediated neurotoxicity.

Simvastatin enhances learning and memory independent of amyloid load in mice

Normal aging is often associated with a decline in learning and memory functions. This decline is manifested to a much greater extent in Alzheimers disease. Recent studies have indicated statins, a class of cholesterol‐lowering drugs, as a potential therapy for Alzheimers disease. Our objective was to determine whether administering a statin drug (simvastatin) would protect against the development of behavioral deficits in an established mouse model of Alzheimers disease.

Association of Aortic Atherosclerosis with Cerebral β-Amyloidosis and Learning Deficits in a Mouse Model of Alzheimer's Disease

High fat/high cholesterol diets exacerbate beta-amyloidosis in mouse models of Alzheimers disease (AD). It has been impossible, however, to study the relationship between atherosclerosis and beta-amyloidosis in those models because such mice were on atherosclerosis-resistant genetic backgrounds. Here we report the establishment of AD model mice, B6Tg2576, that are prone to atherosclerosis. B6Tg2576 mice were produced by back-crossing Tg2576 mice, an AD mouse model overexpressing human amyloid beta-protein precursor with the Swedish double mutation, to C57BL/6 mice, a strain susceptible to diet-induced atherosclerosis. An atherogenic diet induced aortic atherosclerosis and exacerbated cerebral beta-amyloidosis in B6Tg2576 mice. Compared with age-matched non-transgenic littermates, B6Tg2576 mice developed significantly more diet-induced aortic atherosclerosis. Unexpectedly, normal diet-fed B6Tg2576 mice also developed fatty streak lesions (early atherosclerosis) in the aorta. The aortic atherosclerotic lesion area positively correlated with cerebral beta-amyloid deposits in B6Tg2576 mice on both atherogenic and normal diets. Furthermore, behavioral assessments demonstrated that B6Tg2576 mice fed an atherogenic diet had more spatial learning impairment than those fed a normal diet. Our results suggest that synergistic mechanisms may be involved in the pathogenesis of atherosclerosis and AD. These findings may have important implications in the prevention and treatment of cardiovascular diseases as well as AD.

Transgenic mice expressing the βAPP695SWE mutation: effects on exploratory activity, anxiety, and motor coordination

The functional consequences of the betaAPP transgene with the Swedish mutation in mice were assessed in tests of exploratory activity and motor coordination. The betaAPP(695)SWE (Tg2576) transgenic mice are characterized by Abeta plaque formation in the neocortex and hippocampus. By comparison to non-transgenic mice controlled for age and gender, 17-month-old betaAPP(695)SWE transgenic mice displayed impaired spontaneous alternation, increased activity levels in the peripheral part of the open-field, and reduced anxiety in the elevated plus-maze. These results are similar to the loss of inhibitory control observed in some patients with Alzheimers disease. These measures may be added to cognitive dysfunctions as testing ground for Abeta vaccination and other attempts at experimental therapies.

Overexpression of amyloid precursor protein alters its normal processing and is associated with neurotoxicity

The recent discovery that point mutations in the beta/A4 amyloid precursor protein may be the cause of certain forms of familial Alzheimers disease provides strong support for the view that a thorough understanding of the metabolism of this protein may elucidate the pathogenesis of most forms of the disease and thus serve as a basis for rational prevention and therapy. Here we show that overexpression of a portion of the amyloid precursor protein molecule produces at least four distinct fragments of the COOH-terminus of amyloid precursor protein, suggesting altered proteolysis of amyloid precursor protein, and that such overexpression is associated with cytotoxicity. The degree of toxicity in the P19 cell culture model (differentiating mouse embryonal carcinoma cells) is shown to be related to the two larger novel COOH-terminal protein fragments (16 and 14 kilodalton), as well as to levels of expression of these two fragments. The toxicity is manifested in several differentiated cell lineages, including neuronal cells.

Accumulation of protein O-GlcNAc modification inhibits proteasomes in the brain and coincides with neuronal apoptosis in brain areas with high O-GlcNAc metabolism

All tissues contain the enzymes that modify and remove O‐GlcNAc dynamically from nucleocytoplasmic proteins. These enzymes have been shown to play a role in the control of transcription, vesicular trafficking and, more recently, proteasome function. Modification by O‐GlcNAc of the 19S cap of the proteasome inhibits proteasomal function. Transcripts of both O‐GlcNAc transferase and O‐GlcNAcase are very abundant in the brain, with the highest concentrations in hippocampal neurons and Purkinje cells. When the on‐rate of modification is favored over the off‐rate by intraventricular administration of a drug, streptozocin, these areas of the brain display the most rapid accumulation of O‐GlcNAc. Cerebral proteasome function is reduced and ubiquitin and p53 accumulate in these brain regions, with the subsequent activation of a p53‐dependent transgene and the endogenous Mdm2 gene. Later, some hippocampal cells, but not Purkinje cells, undergo apoptosis. These observations suggest that the O‐GlcNAc system may participate in neurodegeneration, particularly in the hippocampus.

Exploratory activity, anxiety, and motor coordination in bigenic APPswe + PS1/ΔE9 mice

Bigenic APPswe + PS1/DeltaE9 mice with Abeta plaque formation in neocortex and hippocampus were evaluated in three tests measuring exploratory activity. By comparison to a non-transgenic group controlled for age and gender, 7-month-old APPswe + PS1/DeltaE9 mice spent more time in the open arms and had higher open/total entries and duration in the elevated plus-maze, indicative of disinhibitory tendencies. On the contrary, the groups did not differ in T-maze and open-field tests. Moreover, the motor coordination of the bigenic group was equivalent to that of controls in stationary beam, coat-hanger, rotorod, and grip strength tests. No mouse displayed neurological signs, such as pathological reflexes, myoclonus, or convulsions. The results in the elevated plus-maze test are akin to the loss of inhibitory control observed in some patients with Alzheimers disease.

Increased frequency of 6-thioguanine-resistant peripheral blood lymphocytes in Werner syndrome patients.

SummaryThe frequency of spontaneous 6-thioguanine (TG)-resistant peripheral blood lymphocytes in five unrelated Werner syndrome (WS) patients was determined using an autoradiographic labeling assay. The average frequency of TG-resistant lymphocytes was eightfold higher in WS patients than in sex- and age-matched normal control donors. This finding and previous identification of increased spontaneous chromosomal rearrangements and deletions in WS cells or cell lines suggest that WS is a human genomic instability or mutator syndrome.

The Alternatively Spliced Kunitz Protease Inhibitor Domain Alters Amyloid β Protein Precursor Processing and Amyloid β Protein Production in Cultured Cells

The insoluble amyloid deposited extracellularly in the brains of patients with Alzheimers disease (AD) is composed of amyloid β protein, a ∼4-kDa secreted protein that is derived from a set of large proteins collectively referred to as the amyloid β protein precursor (βAPP). During normal processing the βAPP is cleaved by β secretase, producing a large NH2-terminal secreted derivative (sAPPβ) and a COOH-terminal fragment beginning at Aβ1, which is subsequently cleaved by γ secretase releasing secreted Aβ. Most secreted Aβ is Aβ1-40, but ∼10% of secreted Aβ is Aβ1-42. Alternative βAPP cleavage by α secretase produces a slightly longer NH2-terminal secreted derivative (sAPPα) and a COOH-terminal fragment beginning at Aβ17, which is subsequently cleaved by γ secretase releasing a ∼3-kDa secreted form of Aβ (P3). Several of the βAPP isoforms that are produced by alternative splicing contain a 56-amino acid Kunitz protease inhibitor (KPI) domain known to inhibit proteases such as trypsin and chymotrypsin. To determine whether the KPI domain influences the proteolytic cleavages that generate Aβ, we compared Aβ production in transfected cells expressing human KPI-containing βAPP751 or KPI-free βAPP695. We focused on Aβs ending at Aβ42 because these forms appear to be most relevant to AD. Using specific sandwich enzyme-linked immunosorbent assays, we analyzed full-length Aβ1-42 and total Aβ ending at Aβ42 (Aβ1-42 + P3(42)). In addition, we analyzed the large secreted derivatives produced by α secretase (sAPPα) and β secretase (sAPPβ). In mouse teratocarcinoma (P19) cells expressing βAPP695 or βAPP751, expression of the KPI-containing βAPP751 resulted in the secretion of a lower percentage of P3(42) and sAPPα and a correspondingly higher percentage of Aβ1-42 and sAPPβ. Similar results were obtained in human embryonic kidney (293) cells. These results indicate that expression of the KPI domain reduces α secretase cleavage so that less P3 and relatively more full-length Aβ are produced. Thus, in human brain and in animal models of AD, the amount of KPI-containing βAPP produced may be an important factor influencing Aβ deposition.

LMNA mutation in a 45 year old Japanese subject with Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder phenotypically characterised by many features of premature aging.1–3 The characteristic features include short stature, prominent eyes, micrognathia, craniofacial disproportion, loss of subcutaneous fat, alopecia, beaked nose, “plucked-bird” appearance, coax valga, pathologic bone fractures, atherosclerosis, and cardiovascular disorders. At birth, the appearance of patients with HGPS is generally normal, but by 1 year of age patients show severe growth retardation, balding, and sclerodermatous skin changes. They average ~1 m in height and usually weigh less than 15 kg even as teenagers. The age at death ranges from 7 to 28 years, with a median age of 13.4 years. Over 80% of deaths are due to heart attacks or congestive heart failure. We previously reported an extraordinarily long-lived patient with HGPS who survived to age 45.4 At birth he appeared normal and his large head was noted at 1 year. Growth retardation was first noticed at the age of 12. He began to lose his hair during childhood and had total alopecia at age 20. When we examined him at the age of 44, he was 132 cm in height and 24.5 kg in weight and had all of the characteristic features listed above. He died of myocardial infarction at age 45. Thus, compared to classical cases of HGPS, the onset of the disease in our patient was late and it took a longer time to develop full-blown HGPS. Recently, mutations in the lamin A/C gene ( LMNA ) which cause classical cases of HGPS have been identified.5–7 The molecular mechanism by which these mutations produce the characteristic manifestations of HGPS remains to be elucidated. Therefore, it is of great interest to investigate the molecular basis of our patient, which allowed him to live about three fold longer than classical …