Yoshitsugu Shitaka

Differential Effects between γ-Secretase Inhibitors and Modulators on Cognitive Function in Amyloid Precursor Protein-Transgenic and Nontransgenic Mice

γ-Secretase inhibitors (GSIs) reduce amyloid-β (Aβ) peptides but inevitably increase the β-C-terminal fragment (β-CTF) of amyloid precursor protein (APP), potentially having undesirable effects on synapses. In contrast, γ-secretase modulators (GSMs) reduce Aβ42 without increasing β-CTF. Although the Aβ-lowering effects of these compounds have been extensively studied, little effort has been made to investigate their effects on cognition. Here, we compared the effects of two GSIs—(2S)-2-hydroxy-3-methyl-N-[(2S)-1-{[(1S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-3-benzazepin-1-yl]amino}-1-oxopropan-2-yl]butanamide (LY450139, semagacestat) and (2R)-2-[[(4-chlorophenyl)sulfonyl][[2-fluoro-4-(1,2,4-oxazol-3-yl)phenyl]methyl]amino-5,5,5-trifluoropentanamide (BMS-708163)—and a second-generation GSM [{(2S,4R)-1-[(4R)-1,1,1-trifluoro-7-methyloctan-4-yl]-2-[4-(trifluoromethyl)phenyl]piperidin-4-yl}acetic acid (GSM-2)] on spatial working memory in APP-transgenic (Tg2576) and nontransgenic mice using the Y-maze task. While acute dosing with either GSI ameliorated memory deficits in 5.5-month-old Tg2576 mice, these effects disappeared after 8 d subchronic dosing. Subchronic dosing with either GSI rather impaired normal cognition in 3-month-old Tg2576 mice, with no inhibition on the processing of other γ-secretase substrates, such as Notch, N-cadherin, or EphA4, in the brain. LY450139 also impaired normal cognition in wild-type mice; however, the potency was 10-fold lower than that in Tg2576 mice, indicating an APP-dependent mechanism likely with β-CTF accumulation. Immunofluorescence studies revealed that the β-CTF accumulation was localized in the presynaptic terminals of the hippocampal stratum lucidum and dentate hilus, implying an effect on presynaptic function in the mossy fibers. In contrast, both acute and subchronic dosing with GSM-2 significantly ameliorated memory deficits in Tg2576 mice and did not affect normal cognition in wild-type mice. We demonstrated a clear difference between GSI and GSM in effects on functional consequences, providing new insights into strategies for developing these drugs against Alzheimers disease.

Neuroprotective effects of the selective type 1 metabotropic glutamate receptor antagonist YM-202074 in rat stroke models

We describe in vitro properties and in vivo neuroprotective effects of a newly synthesized, high-affinity, selective allosteric metabotropic glutamate receptor type 1 (mGluR(1)) antagonist, N-cyclohexyl-6-{[(2-methoxyethyl)(methyl)amino]methyl}-N-methylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-202074). YM-202074 bound an allosteric site of rat mGluR(1) with a K(i) value of 4.8+/-0.37 nM. YM-202074 also inhibited the mGluR(1)-mediated inositol phosphates production in rat cerebellar granule cells with an IC(50) value of 8.6+/-0.9 nM, while showing selectivity over mGluR(2-7). When YM-202074 was infused intravenously at an initial dose of 20 mg/kg/h for 0.5 h followed by a dose of 5 mg/kg/h for 7.5 h, the free concentration of YM-202074 in the brain rapidly (<12 min) reached approximately 0.3 microM, reaching a steady-state phase within 1.5 h. We first treated rats such that they developed transient middle cerebral artery (MCA) occlusion. Results clearly demonstrate a dose-dependent improvement of neurological deficit and reduction of the infarct volume in both the hemisphere and cortex when YM-202074 was infused intravenously immediately after occlusion at a dose of 10 or 20 mg/kg/h for 0.5 h followed by a dose of 2.5 or 5 mg/kg/h for 23.5 h, respectively. Significant neuroprotection was maintained even when the administration of drugs was delayed by up to 2 h following the onset of ischemia. Furthermore, the improvement of neurological deficit and the reduction of infarct volume were sustained for 1 week following the onset of ischemia. These results suggest that YM-202074 exhibits great potential as a novel neuroprotective agent for the treatment of stroke.

Quantitative analysis of amyloid plaques in a mouse model of Alzheimer’s disease by phase-contrast X-ray computed tomography

Densely aggregated beta-amyloid peptides are believed to play a key role in the pathogenesis of Alzheimers disease. Amyloid plaques are a potential target for molecular imaging to determine the clinical status of Alzheimers disease. Phase-contrast X-ray imaging combined with computed tomography is a promising technique that can be used to visualize the physical density of structures in biological tissues non-invasively, and without the use of imaging agents. Using brain tissue isolated from a mouse model of Alzheimers disease, we show that beta-amyloid 40-positive/beta-amyloid 42-positive amyloid plaques, but not beta-amyloid 40-negative/beta-amyloid 42-positive amyloid plaques, exist as high-density aggregates that can be specifically detected by phase-contrast X-ray computed tomography. The phase-contrast X-ray computed tomography detected beta-amyloid 40-positive/beta-amyloid 42-positive amyloid plaques in three-dimensions with an extremely high sensitivity comparable to that of histological analysis, and also enabled the load of amyloid plaques to be quantified. Furthermore, the use of phase-contrast X-ray computed tomography reveals that the physical density of beta-amyloid 40-positive/beta-amyloid 42-positive amyloid plaques increases with age, and that the large volume, high-density, amyloid plaques that are specifically observed in aged Alzheimers disease mice are closely associated with neuritic dystrophy. These results demonstrate that phase-contrast X-ray computed tomography is a highly sensitive imaging technique for analyzing dense-cored amyloid plaques in postmortem samples, and is beneficial in elucidating amyloid pathophysiology in Alzheimers disease.

Characterization of cognitive deficits in a transgenic mouse model of Alzheimer's disease and effects of donepezil and memantine.

Alzheimers disease is characterized by a progressive decline in cognitive function and involves β-amyloid (Aβ) in its pathogenesis. To characterize cognitive deficits associated with Aβ accumulation, we analyzed PS1/APP mice overexpressing mutant presenilin-1 (PS1, M146L; line 6.2) and amyloid precursor protein (APP, K670N/M671L; line Tg2576), a mouse model of Alzheimers disease with accelerated Aβ production. Age-dependent changes in working and spatial memory behaviors were investigated using Y-maze and Morris water maze tasks, respectively, in female PS1/APP mice at ages of 2, 4, 6, and 12 months. Significant deficits in working and spatial memory were observed from 4 and 6 months of age, respectively. Acute single-dose administrations of memantine, a low-to-moderate-affinity N-methyl-d-aspartate (NMDA) antagonist, showed improvements in working memory deficits at 4 months of age, whereas donepezil, an acetylcholinesterase (AChE) inhibitor, did not. However, both drugs improved spatial memory dysfunction at 6 months of age at therapeutically relevant doses. No age-related dramatic changes were observed in expression levels of several proteins relating to memory dysfunction and also the mechanisms of donepezil and memantine in the cerebral cortex of PS1/APP mice until 6 months of age. Taken together, these results suggest dysfunctions in cholinergic and/or glutamatergic transmissions may be involved in the cognitive deficits associated with Aβ toxicity. Since donepezil and memantine have been widely used for treating patients of Alzheimers disease, these results also suggest that cognitive deficits in PS1/APP mice assessed in the Y-maze and Morris water maze tasks are a useful animal model for evaluating novel Alzheimers disease therapeutics.

Oxidative stress accelerates amyloid deposition and memory impairment in a double-transgenic mouse model of Alzheimer's disease

Oxidative stress is known to play a prominent role in the onset and early stage progression of Alzheimers disease (AD). For example, protein oxidation and lipid peroxidation levels are increased in patients with mild cognitive impairment. Here, we created a double-transgenic mouse model of AD to explore the pathological and behavioral effects of oxidative stress. Double transgenic (APP/DAL) mice were constructed by crossing Tg2576 (APP) mice, which express a mutant form of human amyloid precursor protein (APP), with DAL mice expressing a dominant-negative mutant of mitochondrial aldehyde dehydrogenase 2 (ALDH2), in which oxidative stress is enhanced. Y-maze and object recognition tests were performed at 3 and 6 months of age to evaluate learning and memory. The accumulation of amyloid plaques, deposition of phosphorylated-tau protein, and number of astrocytes in the brain were assessed histopathologically at 3, 6, 9, and 12-15 months of age. The life span of APP/DAL mice was significantly shorter than that of APP or DAL mice. In addition, they showed accelerated amyloid deposition, tau phosphorylation, and gliosis. Furthermore, these mice showed impaired performance on Y-maze and object recognition tests at 3 months of age. These data suggest that oxidative stress accelerates cognitive dysfunction and pathological insults in the brain. APP/DAL mice could be a useful model for exploring new approaches to AD treatment.

Amelioration of cognitive deficits in plaque-bearing Alzheimer's disease model mice through selective reduction of nascent soluble Aβ42 without affecting other Aβ pools.

Given that amyloid‐β 42 (Aβ42) is believed to be a culprit in Alzheimers disease (AD), reducing Aβ42 production should be a potential therapeutic approach. γ‐Secretase modulators (GSMs) cause selective reduction of Aβ42 or both reduction of Aβ42 and Aβ40 without affecting total Aβ through shifting the γ‐cleavage position in amyloid precursor protein. We recently reported on GSM‐2, one of the second‐generation GSMs, that selectively reduced brain Aβ42 level and significantly ameliorated cognitive deficits in plaque‐free 5.5‐month‐old Tg2576 AD model mice. Here, we investigated the effects of GSM‐2 on 10‐, 14‐, and 18‐month‐old mice which had age‐dependent increase in amyloid plaques. Eight‐day treatment with GSM‐2 significantly ameliorated cognitive deficits measured by Y‐maze task in the mice of any age. However, GSM‐2 reduced brain soluble Aβ42 only in 10‐month‐old mice. In contrast, GSM‐2 markedly reduced newly synthesized soluble Aβ42 in both 10‐ and 18‐month‐old mice with similar efficacy when measured using the stable isotope‐labeling technique, suggesting that nascent Aβ42 plays a more significant role than plaque‐associated soluble Aβ42 in the cognitive deterioration of Tg2576 mice. These findings further indicate the potential utility of approach to reducing Aβ42 synthesis in AD therapeutic regimens.

Intravenous transplantation of bone marrow-derived mononuclear cells prevents memory impairment in transgenic mouse models of Alzheimer's disease.

Stem cell transplantation therapy is currently in clinical trials for the treatment of ischemic stroke, and several beneficial aspects have been reported. Similarly, in Alzheimers disease (AD), stem cell therapy is expected to provide an efficient therapeutic approach. Indeed, the intracerebral transplantation of stem cells reduced amyloid-β (Aβ) deposition and rescued memory deficits in AD model mice. Here, we show that intravenous transplantation of bone marrow-derived mononuclear cells (BMMCs) improves cognitive function in two different AD mouse models, DAL and APP mice, and prevents neurodegeneration. GFP-positive BMMCs were isolated from tibiae and femurs of 4-week-old mice and then transplanted intravenously into DAL and APP mice. Transplantation of BMMCs suppressed neuronal loss and restored memory impairment of DAL mice to almost the same level as in wild-type mice. Transplantation of BMMCs to APP mice reduced Aβ deposition in the brain. APP mice treated with BMMCs performed significantly better on behavioral tests than vehicle-injected mice. Moreover, the effects were observed even with transplantation after the onset of cognitive impairment in DAL mice. Together, our results indicate that intravenous transplantation of BMMCs has preventive effects against the cognitive decline in AD model mice and suggest a potential therapeutic effect of BMMC transplantation therapy.

IC-P-049

non-AD dementia were excluded, HPV and ERCV predicted the progression to AD, but the CDR-SB was not a significant predictor. In the group of carriers of ApoE 4 allele, progressive MCI subjects had significantly reduced HPV and ERCV compared to stable MCI subjects. There were no differences in volumes between the stable and progressive MCI groups without 4 allele. Conclusions: This study supports the use of MRI scan for volumetric assessment of HP and ERC in elderly subjects with memory problems to assess the risk of progression to dementia. Although not a significant predictor of dementia, the effect of ApoE 4 allele was clearly seen on the medial temporal lobe morphology in MCI subjects. Acknowledgements: The study was supported by the Research Council for Health of the Academy of Finland, and an EVO grant 5772720 from the Kuopio University Hospital.