Neuronal and glial beta-secretase (BACE) protein expression in transgenic Tg2576 mice with amyloid plaque pathology

We measured tissue distribution and expression pattern of the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE) in the brains of transgenic Tg2576 mice that show amyloid pathology. BACE protein was expressed at high levels in brain; at lower levels in heart and liver; and at very low levels in pancreas, kidney, and thymus and was almost absent in spleen and lung when assayed by Western blot analysis. We observed strictly neuronal expression of BACE protein in the brains of nontransgenic control mice, with the most robust immunocytochemical labeling present in the cerebral cortex, hippocampal formation, thalamus, and cholinergic basal forebrain nuclei. BACE protein levels did not differ significantly between control and transgenic mice or as a result of aging. However, in the aged, 17-month-old Tg2576 mice there was robust amyloid plaque formation, and BACE protein was also present in reactive astrocytes present near amyloid plaques, as shown by double immunofluorescent labeling and confocal laser scanning microscopy. The lack of astrocytic BACE immunoreactivity in young transgenic Tg2576 mice suggests that it is not the APP overexpression but rather the amyloid plaque formation that stimulates astrocytic BACE expression in Tg2576 mice. Our data also suggest that the neuronal overexpression of APP does not induce the overexpression of its metabolizing enzyme in neurons. Alternatively, the age-dependent accumulation of amyloid plaques in the Tg2576 mice does not require increased neuronal expression of BACE. Our data support the hypothesis that neurons are the primary source of beta-amyloid peptides in brain and that astrocytic beta-amyloid generation may contribute to amyloid plaque formation at later stages or under conditions when astrocytes are activated.     

β-Amyloid-induced glial expression of both pro- and anti-inflammatory cytokines in cerebral cortex of aged transgenic Tg2576 mice with Alzheimer plaque pathology

To elucidate the mechanisms involved in β-amyloid-mediated inflammation in Alzheimer’s disease, transgenic Tg2576 mice containing as transgene the Swedish double mutation of human amyloid precursor protein 695, were examined for the expression pattern of various cytokines using double immunocytochemistry and laser scanning microscopy. Tg2576 mice studied at postnatal ages of 13, 16 and 19 months demonstrated an age-related accumulation of both senile and diffuse β-amyloid plaques in neocortex and hippocampus. Reactive interleukin (IL)-1β-immunoreactive astrocytes were found in close proximity to both fibrillary and diffuse β-amyloid deposits detectable at very early stages of plaque development, while activated microglia appeared in and around fibrillary β-amyloid plaques only. Subpopulations of reactive astrocytes also demonstrated immunolabeling for transforming growth factor (TGF)-β1, TGF-β3, and IL-10, already detectable in 13-month-old transgenic mouse brain, while a few IL-6-immunoreactive astrocytes were observed only at later stages of plaque development. The early β-amyloid-mediated upregulation of IL-1β, TGF-β, and IL-10 in surrounding reactive astrocytes indicates the induction of both pro- and anti-inflammatory mechanisms. The transgenic approach used in this study may thus provide a useful tool to further disclose the in vivo mechanisms by which pro- and anti-inflammatory cytokines interact and/or contribute to the progression of Alzheimer’s disease.     

Muscarinic acetylcholine receptor inhibition in transgenic Alzheimer-like Tg2576 mice by scopolamine favours the amyloidogenic route of processing of amyloid precursor protein

The molecular mechanisms of the interrelationship between cholinergic neurotransmission, processing of amyloid precursor protein (APP) and beta-amyloid (Abeta) production in vivo are still less understood. To reveal any effect of cholinergic dysfunction on APP processing in vivo, 11-month-old transgenic Tg2576 mice with Abeta plaque pathology received intraperitoneal injections of scopolamine at a daily dosage of 2mg/kg body weight for 14 days in order to suppress cortical cholinergic transmission by chronic inhibition of muscarinic acetylcholine receptors. Scopolamine treatment of transgenic Tg2576 mice resulted in increased levels of fibrillar Abeta(1-40) and Abeta(1-42), while the soluble, SDS-extractable Abeta level remained unchanged as compared to vehicle-injected Tg2576 mice. alpha-Secretase activity determined in cortical tissue from scopolamine-treated Tg2576 mice was lower by about 30% as compared to that assayed in control mice, while beta-secretase activity and BACE1 protein expression appeared unaffected by scopolamine treatment. The amount of sAPPalpha, the product secreted by alpha-secretase-mediated APP cleavage, and the unprocessed APP were assayed in the soluble and membrane fraction, respectively, of cortical tissue preparations from treated and control mice by Western blotting. Using the anti antibody 6E10 which specifically labels human sAPPalpha and full length APP in transgenic Tg2576, an enhanced APP level was detected in the membrane fraction from treated mice as compared to controls, while in the soluble fraction scopolamine treatment did not affect the protein level of sAPPalpha. These data indicate an accumulation of APP in cortical membrane fraction in scopolamine-treated Tg2576 mice presumably due to the decreased level of alpha-secretase-mediated APP cleavage, and further suggest that chronic suppression of cortical muscarinic cholinergic transmission may alter the balance between alpha- and beta-secretory APP processing by favouring the amyloidogenic route.     

Beta-amyloid-induced glial expression of both pro- and anti-inflammatory cytokines in cerebral cortex of aged transgenic Tg2576 mice with Alzheimer plaque pathology

To elucidate the mechanisms involved in beta-amyloid-mediated inflammation in Alzheimer's disease, transgenic Tg2576 mice containing as transgene the Swedish double mutation of human amyloid precursor protein 695, were examined for the expression pattern of various cytokines using double immunocytochemistry and laser scanning microscopy. Tg2576 mice studied at postnatal ages of 13, 16 and 19 months demonstrated an age-related accumulation of both senile and diffuse beta-amyloid plaques in neocortex and hippocampus. Reactive interleukin (IL)-1beta-immunoreactive astrocytes were found in close proximity to both fibrillary and diffuse beta-amyloid deposits detectable at very early stages of plaque development, while activated microglia appeared in and around fibrillary beta-amyloid plaques only. Subpopulations of reactive astrocytes also demonstrated immunolabeling for transforming growth factor (TGF)-beta1, TGF-beta3, and IL-10, already detectable in 13-month-old transgenic mouse brain, while a few IL-6-immunoreactive astrocytes were observed only at later stages of plaque development. The early beta-amyloid-mediated upregulation of IL-1beta, TGF-beta, and IL-10 in surrounding reactive astrocytes indicates the induction of both pro- and anti-inflammatory mechanisms. The transgenic approach used in this study may thus provide a useful tool to further disclose the in vivo mechanisms by which pro- and anti-inflammatory cytokines interact and/or contribute to the progression of Alzheimer's disease.     

Increased neuronal and glial expression of protein kinase C isoforms in neocortex of transgenic Tg2576 mice with amyloid pathology

We investigated the influence of five- to sevenfold neuronal overexpression of the Swedish mutation of human APP695 (APPsw) in the transgenic mouse strain Tg2576 on neocortical protein kinase C (PKC) expression and subcellular distribution. Using specific antibodies to PKC alpha, PKC beta, PKC gamma, PKC epsilon and PKC zeta isoforms for Western blot analysis, we observed increased immunoreactivity for PKC alpha and PKC gamma isoforms in crude tissue homogenates from the neocortex of 16-month-old APPsw mice as compared with nontransgenic littermates, which was not present in 6 month-old Tg2576 mice. We also observed elevated levels of PKC alpha, PKC beta, PKC gamma and PKC zeta in membrane fractions and reduced concentrations of PKC alpha and PKC gamma in cytosolic fractions of aged Tg2576 mice, indicating that these PKC isoforms are in their activated state. In young, 6-month-old Tg2576 mice, however, the increase in membrane-bound PKC isoforms and concomitant decrease in cytosolic PKC isoforms was much less pronounced, demonstrating the age-dependent nature of alterations in PKC isoforms. Immunocytochemistry of brain sections supported these findings and revealed increased neuronal labelling for PKC alpha, PKC gamma and PKC lambda isoforms in neocortex of 16-month-old APPsw mice compared with nontransgenic littermates, with the increase being strongest for PKC gamma and PKC lambda isoforms. Additionally, PKC gamma and to a lesser extent PKC lambda isoforms were induced in reactive astrocytes in proximity to amyloid plaques. Our data indicate that neuronal overexpression of APPsw causes a dynamic change in neuronal expression and activation of multiple PKC isoforms known to be regulators of proteolytic amyloid precursor protein (APP) processing (PKC alpha) and of neuronal survival (PKC lambda and PKC zeta). The induction of the PKC gamma and PKC lambda isoforms in reactive astrocytes surrounding amyloid plaques might be required for astrocyte activation and astrocytic cytokine expression in response to amyloid plaque formation.     

Inflammation and oxidative stress as biomarkers of premature aging in persons with intellectual disability

The decline in cognitive ability and physical performance in older adults with intellectual disabilities (ID) is accompanied by less participation in social activities and a sedentary lifestyle; however the pathogenesis is not clear yet. It was recently suggested that chronic disease, adverse drug reactions, and aging create a cascade of events that can be best characterized as an asymptomatic inflammatory process. This cascade of events is mediated by cytokine interleukins 1 and 6 (IL-1α, and IL-6), nitric oxide (NO) and total oxidative stress (OS). Our hypothesis was that chronic inflammation in the bloodstream of persons with ID contributes to their “premature aging”. To test this hypothesis, we measured and compared the levels of inflammatory molecules in persons with and without ID. Fifteen adults with, and 15 adults without ID (control group) participated in this study. The levels of NO metabolites (NOx), IL-1α, and IL-6 were obtained from participants’ serum. OS markers were drawn from participants’ capillary. Western blot, RT-PCR and specific chemical analysis were used as measurement tools. The levels of inflammatory molecules and OS were significantly higher in persons with ID compared to the control group. Asymptomatic inflammation in the bloodstream of the older adults with ID might explain the “premature aging” of these individuals. Monitoring the levels of inflammatory molecules could serve as biomarkers of “premature aging” which may allow early diagnosis and intervention, and improve the quality of care for persons with ID.     

Modulation of cortical nitric oxide synthase,glutamate, and redox state by nifedipine and taurine in PTZ-kindled mice

PURPOSE: Correlation between pentylenetetrazol (PTZ)-induced kindling and the cortical nitric oxide synthase (NOS), intracellular calcium [Ca2+]i, glutamate, and free radicals was studied in mice, as well as the modulatory action of nifedipine and taurine on these parameters. METHODS: Male Swiss albino mice were used. Mice in one group received a single convulsive dose of PTZ (50 mg/kg, i.p), and were killed 24 h later. To induce kindling, PTZ was injected in a subconvulsive dose (40 mg/kg, i.p.) every other day for 3 weeks. One kindled group was used as control, whereas two other groups were injected 30 min before PTZ with either nifedipine (30 mg/kg, i.p) or taurine (100 mg/kg, i.p). All three kindled groups were killed 24 h after the last injection. RESULTS: Compared with normal control group, PTZ-kindled mice had significantly higher levels of [Ca2+]i, malonaldehyde (MDA), NOS, and lactate dehydrogenase (LDH) but had lower levels of superoxide dismutase (SOD) and reduced glutathione (GSH). Acute seizures of the same intensity did not induce these alterations, indicating their link to the kindling phenomenon and not to seizure activity. The effect of taurine, known as an antioxidant, was more pronounced than that of the Ca2+-channel blocker, nifedipine. The first drug reversed the PTZ-kindled action on [Ca2+]i, NOS, LDH, GSH, and SOD, whereas nifedipine restored only LDH and GSH levels. However, both drugs did not restore the elevated MDA level. CONCLUSIONS: This study suggests that free radicals, as well as NOS, are implicated in PTZ-induced kindling, and that antioxidants could play a role in controlling the accompanying changes.     

Neuropeptide alterations in the hippocampal formation and cortex of transgenic mice overexpressing beta-amyloid precursor protein (APP) with the Swedish double mutation (APP23)

The role of neuropeptides and the significance of peptidergic mechanisms in neurodegenerative diseases are still unclear. In the periphery, nerve injury results in dramatic changes in the expression of neuropeptides. An important question regards to what extent similar changes occur, and similar mechanisms operate, after lesions and/or degeneration in the brain. The purpose of this work is, therefore, to study neuropeptides with regard to their presence and distribution in the APP23 mouse (HuAPP(751) K670M/N671L under the murine Thy-1 promoter), a model for Alzheimer's disease, or cerebral amyloidosis, using the immunohistochemical technique. In addition, tyrosine hydroxylase and acetylcholinesterase were analyzed. This study shows marked neuropeptide changes in the hippocampal formation and the ventral cortex, whereas the dorsolateral neocortex was less affected. There was a considerable variation with regard to peptide expression among animals of the same age which was related to the variation in Abeta deposition. Dystrophic and varicose fibers containing galanin, neuropeptide Y, enkephalin, and especially cholecystokinin were commonly seen in close proximity to amyloid plaques. In addition, generalized changes were observed, such as increases of enkephalin and neuropeptide Y in stratum lacunosum moleculare and of neuropeptide Y, enkephalin, and dynorphin in mossy fibers. In contrast, cholecystokinin was decreased in mossy fibers. Comparatively small differences were observed between wild-type and transgenic mice with regard to tyrosine hydroxylase (noradrenergic but also dopaminergic fibers) and acetylcholine esterase (mainly cholinergic fibers). The increase of neuropeptides in dystrophic fibers in this model may represent a response to nerve injury caused by the amyloid accumulation and may reflect attempts to counteract degeneration by initiating protective and/or regenerative processes.