Prevention of age-related spatial memory deficits in a transgenic mouse model of Alzheimer's disease by chronic Ginkgo biloba treatment

Alzheimer's disease (AD) is characterized by cognitive decline and deposition of beta-amyloid (Abeta) plaques in cortex and hippocampus. A transgenic mouse AD model (Tg2576) that overexpresses a mutant form of human Abeta precursor protein exhibits age-related cognitive deficits, Abeta plaque deposition, and oxidative damage in the brain. We tested the ability of Ginkgo biloba, a flavonoid-rich antioxidant, to antagonize the age-related behavioral impairment and neuropathology exhibited by Tg2576 mice. At 8 months of age, 16 female Tg2576 and 15 female wild-type (wt) littermate mice were given ad lib access to tap water or Ginkgo biloba (70 mg/kg/day in water). After 6 months of treatment, all mice received Morris water maze training (4 trials/day for 10 days) to assess hippocampal dependent spatial learning. All mice received a 60-s probe test of spatial memory retention 24 h after the 40th trial. Untreated Tg2576 mice exhibited a spatial learning impairment, relative to wt mice, while Ginkgo biloba-treated Tg2576 mice exhibited spatial memory retention comparable to wt during the probe test. Spatial learning was not different between Ginkgo biloba-treated and untreated wt mice. There were no group differences in learning to swim to a visible platform. Soluble Abeta and hippocampal Abeta plaque burden did not differ between the Tg2576 groups. Brain levels of protein carbonyls were paradoxically elevated in Ginkgo biloba-treated mice. These data indicate that chronic Ginkgo biloba treatment can block an age-dependent decline in spatial cognition without altering Abeta levels and without suppressing protein oxidation in a transgenic mouse model of AD.     

Induction of cytokines in glial cells surrounding cortical beta-amyloid plaques in transgenic Tg2576 mice with Alzheimer pathology.

beta-Amyloid plaque deposition observed in brains from Alzheimer patients, might function as immune stimulus for glial/macrophages activation, which is supported by observations of activated microglia expressing interleukin (IL)-1beta and elevated IL-6 immunoreactivity in close proximity to amyloid plaques. To elucidate the mechanisms involved in beta-amyloid-mediated inflammation, transgenic mice (Tg2576) expressing high levels of the Swedish double mutation of human amyloid precursor protein and progressively developing typical beta-amyloid plaques in cortical brain regions including gliosis and astrocytosis, were examined for the expression pattern of a number of cytokines.Using ribonuclease protection assay, interleukin (IL)-1alpha,-beta, IL-1 receptor antagonist, IL-6, IL-10, IL-12, IL-18, interferon-gamma, and macrophage migration inhibitory factor (MIF) mRNA were not induced in a number of cortical areas of Tg2576 mice regardless of the postnatal ages studied ranging between 2 and 13 months. Using immunocytochemistry for IL-1alpha,beta, IL-6, tumor necrosis factor (TNF)-alpha, and macrophage chemotactic protein (MCP)-1, only IL-1beta was found to be induced in reactive astrocytes surrounding beta-amyloid deposits detected in 14-month-old Tg2576 mice. Using non-radioactive in situ hybridization glial fibrillary acidic protein (GFAP) mRNA was detected to be expressed by reactive astrocytes in close proximity to beta-amyloid plaques. The local immune response detected around cortical beta-amyloid deposits in transgenic Tg2576 mouse brain is seemingly different to that observed in brains from Alzheimer patients but may represent an initial event of chronic neuroinflammation at later stages of the disease.     

Maintained synaptophysin immunoreactivity in Tg2576 transgenic mice during aging: correlations with cognitive impairment

Regional loss of synapses, particularly within the neocortex and hippocampus, is characteristic of Alzheimer's Disease (AD) and strongly correlated with extent of cognitive impairment. The Tg2576 transgenic mouse model of AD develops Abeta-containing neuritic plaques by 10-16 months of age and shows cognitive impairment in several tasks. In the present study, synaptophysin immunoreactivity (SYN-IR; a marker for synaptic terminals) was evaluated in the neocortex and hippocampus of behaviorally-tested Tg2576 transgenic (Tg+) mice aged 3, 9, 14, and 19 months of age. In control non-transgenic (Tg-) mice, SYN-IR in both neocortex and hippocampus tended to decrease with age, while SYN-IR in Tg+ mice was maintained with age. Thus, 19M Tg+ mice exhibited significantly greater synaptophysin immunostaining compared to 19M Tg- mice in both inner and outer neocortical regions, as well as in the dentate gyrus' outer molecular layer and polymorphic layer. Over all four age groups collectively, outer cortical SYN-IR was also greater in Tg+ compared to Tg- mice. Multiple factors could be responsible for maintained SYN-IR in aged Tg+ mice, including compensatory changes in synaptic morphology and staining of dystrophic neuritics associated with Abeta deposition. For all animals combined (Tg+ and Tg-), as well as for aged 19M animals alone, hippocampal SYN-IR was correlated with impaired acquisition and spatial reference memory in the Morris water maze task, suggestive that elevated hippocampal SYN-IR is a manifestation of pathophysiologic synaptic processing within the hippocampus. Also for 19M animals alone, hippocampal SYN-IR was highly correlated with impaired visible platform recognition, indicative that elevated SYN-IR is linked to visual agnosia. The results of this study are consistent with the premise that maintained SYN-IR in Tg2576 mice during aging is associated with impaired synaptic function, resulting in cognitive deficits.     

The beta-amyloid-related proteins presenilin 1 and BACE1 are axonally transported to nerve terminals in the brain

In this study, we show that removal of entorhinal cortex (ERC) afferents to hippocampus reduces levels of presenilin 1 (PS1) in the dentate gyrus of APPswe/PS1DeltaE9 transgenic (Tg) mice. PS1 immunoreactivity on the deafferented dentate gyrus decreases by approximately 25% and 50%, 2 and 4 weeks post-lesion compared to the contralateral side; by Western blotting, there is an approximately 40% decrease of the 43 kDa (full length) PS1 and an approximately 80% decrease of the 28 kDa (N-terminal fragment) PS1 on the lesioned dentate gyrus. Levels of beta-site APP Cleavage Enzyme 1 (BACE1) immunoreactivity also decrease by approximately 50% and 65% 2 and 4 weeks post-lesion. Together, these data demonstrate that PS1 and BACE1 are transported from the entorhinal cortex to the hippocampus via axons of the perforant pathway.     

Distribution of chromogranin B-like immunoreactivity in the human hippocampus and its changes in Alzheimer’s disease

Synapse loss is crucially involved in cognitive decline in Alzheimer’s disease (AD). This study was performed to investigate the distribution and density of chromogranin B-like immunoreactivity in the hippocampus of control compared to AD brain. Chromogranin B is a large precursor molecule found in large dense-core vesicles. For immunocytochemistry we used an antiserum raised against a synthetic peptide (PE-11) present in the chromogranin B molecule. Chromogranin B-like immunoreactivity was concentrated in the terminal field of mossy fibers, the inner molecular layer of the dentate gyrus and in layer II of the entorhinal cortex. In AD, chromogranin B was detected in neuritic plaques. The density of chromogranin B-like immunoreactivity was significantly reduced in the inner molecular layer of the dentate gyrus and in layers II, III and V of the entorhinal cortex in AD brains. The present study demonstrates that chromogranin B is a marker for human hippocampal pathways. It is particularly suitable for studying nerve fibers terminating at the inner molecular layer of the dentate gyrus. It is present in neuritic plaques, and its density is reduced in a layer-specific manner. Received: 14 February 2000 / Accepted: 6 March 2000     

An increase in Abeta42 in the prefrontal cortex is associated with a reversal-learning impairment in Alzheimer's disease model Tg2576 APPsw mice

The medial temporal lobe-dependent memory loss associated with Alzheimer's disease (AD) is often accompanied by a loss of prefrontal cortex-dependent cognitive domains that fall under the broad category of executive function. In this study, we examined the relationship between one type of prefrontal-dependent executive function, discrimination reversal-learning, and levels of the amyloid beta protein (Abeta) of 40 and 42 residues in a transgenic mouse model (Tg2576) of the over-expression of the familial AD mutant form of the amyloid precursor protein (APPsw). Tg2576 and their non-transgenic (NTg) littermates were assessed at 3 and 6 months of age when there is little to no amyloid plaque deposition. After reversal-learning assessment, Abeta40 and Abeta42 were quantified in the prefrontal cortex and hippocampus. Tg2576 mice were impaired in reversal-learning at 6 but not 3 months of age when compared to the NTg group. Coincidently, there was a corresponding approximately 3-fold increase of Abeta42 levels in the prefrontal cortex of 6- compared to 3-month-old Tg2576 mice. In addition, the prefrontal cortex contained higher levels of Abeta42 compared to the hippocampus at both 3 and 6 months of age, regardless of genotype, indicating a high vulnerability of this brain region to Abeta42 accumulation. These data suggest that the early emergence of reversal-learning deficits in the Tg2576 mouse may be due to the localized increase of Abeta42 in the prefrontal cortex.     

Apolipoprotein E deposition and astrogliosis are associated with maturation of beta-amyloid plaques in betaAPPswe transgenic mouse: Implications for the pathogenesis of Alzheimer's disease

A transgenic mouse expressing the human beta-amyloid precursor protein with the 'Swedish' mutation, Tg2576, was used to investigate the mechanism of beta-amyloid (Abeta) deposition. Previously, we have reported that the major species of Abeta in the amyloid plaques of Tg2576 mice are Abeta1-40 and Abeta1-42. Moreover, Abeta1-42 deposition precedes Abeta1-40 deposition, while Abeta1-40 accumulates in the central part of the plaques later in the pathogenic process. Those data indicate that Abeta deposits in Tg2576 mice have similar characteristics to those in Alzheimer's disease. In the present study, to understand more fully the amyloid deposition mechanism implicating Alzheimer's disease pathogenesis, we examined immunohistochemically the distributions of apolipoprotein E (apoE) and Abeta in amyloid plaques of aged Tg2576 mouse brains. Our findings suggest that Abeta1-42 deposition precedes apoE deposition, and that Abeta1-40 deposition follows apoE deposition during plaque maturation. We next examined the relationship between apoE and astrogliosis associated with amyloid plaques using a double-immunofluorescence method. Extracellular apoE deposits were always associated with reactive astrocytes whose processes showed enhancement of apoE-immunoreactivity. Taken together, the characteristics of amyloid plaques in Tg2576 mice are similar to those in Alzheimer's disease with respect to apoE and astrogliosis. Furthermore, apoE deposition and astrogliosis may be necessary for amyloid plaque maturation.     

Cortical glucose metabolism is altered in aged transgenic Tg2576 mice that demonstrate Alzheimer plaque pathology

Alzheimer's disease is associated with markedly impaired cerebral glucose metabolism as detected by reduced cortical desoxyglucose utilization, by altered activities of key glycolytic enzymes or by reduced densities of cortical glucose transporter subtypes. To determine whether formation and/or deposition of beta-amyloid plays a role in the pathology of glucose metabolism, transgenic Tg2576 mice that overexpress the Swedish mutation of the human amyloid precursor protein and demonstrate a progressive, age-related cortical and hippocampal deposition of beta-amyloid plaques, were used to study expression and activity of key enzymes of brain glycolysis (phosphofructokinase, PFK) and glyconeogenesis (fructose1,6-bisphosphatase; FbPase). Quantitative RT-PCR revealed high expression levels of both C- and M-type PFK mRNA in non-transgenic mouse cerebral cortex, whilst there was little expression of the L-type. In 24-month-old transgenic Tg2576 mouse cortex, but not in 7-, 13-, and 17-month-old mice, the copy number of PFK-C mRNA was significantly reduced in comparison to non-transgenic littermates, while the mRNA level of the other PFK isoforms and FbPase did not differ between transgenic and non-transgenic tissue samples. In situ hybridization in brain sections from aged Tg2576 mice revealed reduced PFK-C mRNA expression in beta-amyloid plaque-associated neurons and upregulation in reactive astrocytes surrounding beta-amyloid deposits. The decreased PFK-C protein level detected by Western analysis in cerebral cortical tissue from 24-month-old transgenic Tg2576 mice was accompanied by reduced enzyme activity of PFK in comparison to non-transgenic littermates. Our data demonstrate that impairment of cerebral cortical glucose metabolism occurs only due to the long-lasting high beta-amyloid burden. This results from a reduction in glycolytic activity in beta-amyloid plaque-associated neurons and a concomitant upregulation in reactive, plaque-surrounding astrocytes.     

AbetaPP induces cdk5-dependent tau hyperphosphorylation in transgenic mice Tg2576

Previous studies of Abeta-induced neuronal damage of hippocampal cells in culture have provided strong evidence that deregulation of the Cdk5/p35 kinase system is involved in the neurodegeneration pathway. Cdk5 inhibitors and antisense probes neuroprotected hippocampal cells against the neurotoxic action of Abeta. To further investigate the mechanisms underlying the participation of Cdk5 in neuronal degeneration, the transgenic mouse containing the Swedish mutations, Tg2576, was used as an animal model. Immunocytochemical studies using anti-Abeta(1-17) antibody evidenced the presence of labeled small-clustered core plaques in the hippocampus and cortex of 18-month-old transgenic mice brains. The loss of granular cells without a compressed appearance was detected in the vicinity of the cores in the dentate gyrus of the hippocampus. Immunostaining of Tg2576 brain sections with antibodies AT8, PHF1 and GFAP labeled punctuate dystrophic neurites in and around the amyloid core. Reactive astrogliosis around the plaques in the hippocampus was also observed. Studies at the molecular level showed differences in the expression of the truncated Cdk5 activator p25 in the transgenic animal, as compared with wild type controls. However no differences in Cdk5 levels were detected, thus corroborating previous cellular findings. Interestingly, hyperphosphorylated tau epitopes were substantially increased as assessed with the AT8 and PHF1 antibodies, in agreement with the observation of a p25 increase in the transgenic animal. These observations strongly suggest that the increased exposure of Alzheimer's type tau phosphoepitopes in the transgenic mice correlated with deregulation of Cdk5 leading to an increase in p25 levels. These studies also provide further evidence on the links between extraneuronal amyloid deposition and tau pathology.     

Stereological analysis of the reorganization of the dentate gyrus following entorhinal cortex lesion in mice

Denervation of the dentate gyrus by entorhinal cortex lesion has been widely used to study the reorganization of neuronal circuits following central nervous system lesion. Expansion of the non-denervated inner molecular layer (commissural/associational zone) of the dentate gyrus and increased acetylcholinesterase-positive fibre density in the denervated outer molecular layer have commonly been regarded as markers for sprouting following entorhinal cortex lesion. However, because this lesion extensively denervates the outer molecular layer and causes tissue shrinkage, stereological analysis is required for an accurate evaluation of sprouting. To this end we have performed unilateral entorhinal cortex lesions in adult C57BL/6J mice and have assessed atrophy and sprouting in the dentate gyrus using modern unbiased stereological techniques. Results revealed the expected increases in commissural/associational zone width and density of acetylcholinesterase-positive fibres on single brain sections. Yet, stereological analysis failed to demonstrate concomitant increases in layer volume or total acetylcholinesterase-positive fibre length. Interestingly, calretinin-positive fibres did grow beyond the border of the commissural/associational zone into the denervated layer and were regarded as sprouting axons. Thus, our data suggest that in C57BL/6J mice shrinkage of the hippocampus rather than growth of fibres underlies the two morphological phenomena most often cited as evidence of regenerative sprouting following entorhinal cortex lesion. Moreover, our data suggest that regenerative axonal sprouting in the mouse dentate gyrus following entorhinal cortex lesion may be best assessed at the single-fibre level.