In Vivo Longitudinal Monitoring of Changes in the Corpus Callosum Integrity During Disease Progression in a Mouse Model of Alzheimer's Disease.

The corpus callosum is the largest commissural fiber connecting left and right hemisphere of the brain. Emerging evidence suggests that a variety of abnormalities detected in the microstructure of this white matter fiber can be an early event in Alzheimer's disease (AD) pathology. However, little is known about tissue characteristics of these abnormalities and how these abnormalities evolve during AD progression. In this study, we measured in vivo magnetic resonance transverse relaxation times (T2) to longitudinally monitor changes in tissue integrity and abnormalities related to myelination and demyelination processes in corpus callosum of AD mouse models. The most striking finding of our study was a significant elongation of T2 values in the corpus callosum at 10, 14, 16 and 18 months of age compared to age-matched wild-type mice. In contrast, the gray matter regions surrounding the corpus callosum, such as the cortex and hippocampus, showed a significant T2 decrease compared to wild-type mice. Histological analyses clearly revealed demyelination, gliosis and amyloid-plaque deposition in the corpus callosum. Our results suggest that demyelinating and inflammatory pathology may result in prolonged relaxation time during AD progression. To our knowledge, this is the first in vivo T2 study assessing the microstructural changes with age in the corpus callosum of the Tg2576 mouse model and it demonstrates the application of T2 measurement to noninvasively detect tissue integrity of the corpus callosum, which can be an early event in disease progression.

Monitoring blood flow alterations in the Tg2576 mouse model of Alzheimer's disease by in vivo magnetic resonance angiography at 17.6 T.

Many neurodegenerative diseases including Alzheimer's disease are linked to abnormalities in the vascular system. In AD, the deposition of amyloid ß (Aß) peptide in the cerebral vessel walls, known as cerebral amyloid angiopathy (CAA) is frequently observed, leading to blood flow abnormalities. Visualization of the changes in vascular structure is important for early diagnosis and treatment. Blood vessels can be imaged non-invasively by magnetic resonance angiography (MRA). In this study we optimized high resolution MRA at 17.6 T to longitudinally monitor morphological changes in cerebral arteries in a Tg2576 mouse model, a widely used model of AD. Our results at 17.6 T show that MRA significantly benefits from the ultra-high magnetic field strength especially to visualize smaller vessels. Visual and quantitative analysis of MRA results revealed severe blood flow defects in large and medium sized arteries in Tg2576 mice. In particular blood flow defects were observed in the middle cerebral artery (MCA) and in the anterior communicating artery (AComA) in Tg2576 mice. Histological data show that Aß levels in the vessel wall may be responsible for impaired cerebral blood flow, thereby contributing to the early progression of AD. To our knowledge this is the first ultra-high field MRA study monitoring blood flow alterations longitudinally in living Tg2576 mice, consequently providing a powerful tool to test new therapeutic intervention related to CAA in a mouse model of AD.

Changes in hippocampal NMDA-R subunit composition induced by exposure of neonatal rats to L-glutamate.

Overactivation of NMDA-Rs may mediate excitotoxic cell death associated with epileptic seizures, and hypoxic-ischemic conditions. We assessed whether repeated subcutaneous administration of l-glutamate to neonatal rats affects the subunit composition of NMDA-Rs. Accordingly, cortical and hippocampal tissue from 14-day-old rats was analyzed by Western blotting and RT-PCR to quantify the protein and mRNA expression of different NMDA-R subunits. In addition, tissue sections were Nissl stained to assess the cell damage in this tissue. Early exposure of neonatal rats to L-glutamate differentially affects the expression of mRNA transcripts for NMDA-R subunits in the cerebral cortex and hippocampus. In the cerebral cortex, a decrease in NR2B subunit mRNA expression was observed, as well as a loss of NR1 and NR2A protein. By contrast, neonatal L-glutamate administration augmented the transcripts encoding the NR1, NR2B, and NR2C subunits in the hippocampal formation. The expression of mRNA encoding the NR2A subunit was not affected by neonatal L-glutamate administration in either of the brain regions examined. This differential expression of NMDA-R subunits following neonatal exposure to L-glutamate may represent an adaptive response of the glutamate receptors to overactivation in order to reduce the effect of high L-glutamate during the early period of life when the animal is more vulnerable to excitotoxicity.

The significance of the cholinergic system in the brain during aging and in Alzheimer's disease.

Acetylcholine is widely distributed in the nervous system and has been implicated to play a critical role in cerebral cortical development, cortical activity, controlling cerebral blood flow and sleep-wake cycle as well as in modulating cognitive performances and learning and memory processes. Cholinergic neurons of the basal forebrain complex have been described to undergo moderate degenerative changes during aging, resulting in cholinergic hypofunction that has been related to the progressing memory deficits with aging. Basal forebrain cholinergic cell loss is also a consistent feature of Alzheimer's disease, which has been suggested to cause, at least partly, the cognitive deficits observed, and has led to the formulation of the cholinergic hypotheses of geriatric memory dysfunction. Impaired cortical cholinergic neurotransmission may also contribute to beta-amyloid plaque pathology and increase phosphorylation of tau protein the main component of neurofibrillar tangles in Alzheimer's disease. Understanding the molecular mechanisms underlying the interrelationship between cortical cholinergic dysfunction, beta-amyloid formation and deposition, and tau pathology in Alzheimer's disease, would allow to derive potential treatment strategies to pharmacologically intervene in the disease-causing signaling cascade.

Neuronal damage and changes in the expression of muscarinic acetylcholine receptor subtypes in the neonatal rat cerebral cortical upon exposure to sparteine, a quinolizidine alkaloid.

Sparteine is a quinolizidine alkaloid (QA) produced by Lupine species that has generated much interest due to its anti-hypertensive, anti-pyretic, and anti-inflammatory properties. In the nervous system, sparteine has been shown to display anti-cholinergic and depressive activity, although how sparteine exerts its toxic effects in the brain remains unclear. We have addressed this issue by administering subcutaneous injections of sparteine (25 mg/kg of body weight) to rats on postnatal days 1 and 3, and then examining the expression of the muscarinic acetylcholine receptor (mAChR) subunits m1-m4 in the brains of the neonatal rats 14-60 days later. Administration of sparteine to neonatal rats caused neuronal damage in the cerebral motor cortex accompanied by transient changes in the expression of m1-m4 mAChR subunits as revealed by both RT-PCR and Western blotting. This effect could be prevented by pre-treatment with atropine (10 mg/kg) 1 h prior to the injection of sparteine, suggesting that the cytotoxic activity of sparteine is mediated through mAChRs.

Zinc uptake is mediated by M1 muscarinic acetylcholine receptors in differentiated SK-SH-SY5Y cells.

Alzheimer's disease (AD), the most prevalent form of dementia, is characterized by several major morphological hallmarks such as senile plaques, neurofibrillary tangles and a loss of cholinergic basal forebrain neurons. Apart from cholinergic markers like choline acetyltransferase and acetylcholinesterase, there have been reports on changes in muscarinic acetylcholine receptors (mAChR) as well as on influences of zinc metabolism in the disease. As recent studies gave hints about a possible link between mAChRs and zinc uptake, the human neuroblastoma cell line SK-SH-SY5Y was used to evaluate the role of M1-mAChR on zinc uptake. Zinc levels were semi-quantitatively detected by using the zinc-specific fluorophor Zn-AF2-DA. In the presence of 1 microM extracellular zinc, M1-mAChR stimulation with talsaclidine increased intracellular zinc levels as did stimulation of PKC by phorbol esters. Furthermore, the effect of extracellular zinc on the expression of the zinc finger protein PNUTS (protein phosphatase 1 nuclear targeting subunit 10) was investigated and revealed an upregulation of PNUTS expression in the presence of 1 microM extracellular zinc by 294% when compared to incubation in zinc free medium. In summary, this report demonstrates that intracellular zinc uptake in SK-SH-SY5Y cells is controlled by M1-mAChR mediated signalling pathways and that zinc may act as a cofactor for transcriptional regulation of zinc finger genes such as PNUTS.

L-glutamate and phorbol ester stimulate the release of secretory amyloid precursor protein from rat cortical synaptosomes.

Treatment of rat cortical synaptosomes with micromolar concentrations of L-glutamate stimulated the release of the secreted form of amyloid precursor protein in a concentration-dependent, however biphasic manner as assayed by semiquantitative Western blot analysis. The secreted amyloid precursor protein released from synaptosomes into the incubation medium was highest in the presence of 500 microM L-glutamate (about 64% over the level assayed in the incubation medium in the absence of any drug). In contrast, direct stimulation of protein kinase C by phorbol-12-myristate-13-acetate resulted in a concentration-independent increase in secretory amyloid precursor protein release by about 100% already detectable at a concentration of 0.1 microM but with no significant change at higher concentrations up to 10 microM. The presented data show that there is a constitutive release of secretory amyloid precursor protein from synaptosomes and suggest that (i) processing of amyloid precursor protein at the synaptic level is controlled by L-glutamate presumably via activation of protein kinase C, and (ii) isolated cortical synaptosomes represent a useful experimental approach to selectively study amyloid precursor protein metabolism at the synaptic level.

Experimental traumatic brain injury in rats stimulates the expression, production and activity of Alzheimer's disease beta-secretase (BACE-1).

Traumatic brain injury (TBI) is a risk factor for the development of Alzheimer's disease (AD). After a traumatic brain injury depositions of amyloid beta (Abeta) in the brain parenchyma were found. In this study we investigated the expression pattern of beta-secretase (BACE-1) in ipsi- or contralateral hippocampus and cortex following controlled cortical TBI in rats. BACE-1 mRNA levels, estimated by real time RT-PCR, were elevated 24 h post injury, and persisting up to 72 h, in the ipsi- and contralateral hippocampus and cerebral cortex as compared to the sham-treated animals (p<0.01). The TBI-induced changes in BACE-1 mRNA are due to enhanced hippocampal and cortical expression of BACE-1 mRNA in neurons and reactive astrocytes as revealed by in situ hybridization. The alterations in hippocampal BACE-1 mRNA levels are accompanied by corresponding increases in BACE-1 protein levels in ipsi- and contralateral hippocampus and ipsilateral cortex as demonstrated by Western blot analysis. In contrast, in the contralateral cortex only a weak increase of traumatically induced BACE-1 protein production was found. The activity of BACE-1 as measured by the formation of the cleavage product of amyloid beta precursor protein, transiently increased up to 48 h after injury, but returned to basal level 7 days post injury. This study demonstrates that the beta-secretase is stimulated following TBI and may suggest a mechanism for the temporal increase of Abeta levels observed in patients with brain trauma.

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.

Expression of beta-Secretase mRNA in the brain of rats with immunohistochemical destruction of basal forebrain cholinergic system.

We studied properties of cloned BACE mRNA (beta-site of the enzyme cleaving amyloid precursor protein) and evaluated the possibility of using this clone for identification and/or prediction of neurodegenerative disorders associated with cholinergic deficiency. Wistar rats subjected to immunohistochemical destruction of the basal forebrain cholinergic system were used as the experimental model. Nonradioactive in situ hybridization and immunohistochemical visualization of the astroglia revealed strong hybridization signal of BACE mRNA in neurons of the cortex, hippocampus, and thalamus. Astrocytes remained unstained. Immunohistochemical destruction of the basal forebrain produced no significant changes in the distribution of BACE mRNA.

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.

Expression of fructose-1,6-bisphosphatase mRNA isoforms in normal and basal forebrain cholinergic lesioned rat brain.

Fructose-1,6-bisphosphatase is one of the key enzymes in the gluconeogenic pathway predominantly occurring in liver, kidney and muscle. In the brain, fructose-1,6-bisphosphatase has been suggested to be an astrocyte-specific enzyme but the functional importance of glyconeogenesis in the brain is still unclear. To further elucidate the cellular source of fructose-1,6-bisphosphatase in the brain, non-radioactive in situ hybridizations were performed using digoxigenin-labeled RNA probes based on the sequence of recently cloned rat liver and muscle fructose-1,6-bisphosphatase cDNAs. In situ hybridization using a riboprobe for the liver isoform revealed a location of the hybridization signal mainly in neurons, while rat muscle fructose-1,6-bisphosphatase mRNA was detected in both neurons and astrocytes in the hippocampal formation and in layer I of the cerebral cortex.RT-PCR using RNA preparations of rat astrocytes, neurons, and adult whole brain demonstrated a localization of liver fructose-1,6-bisphosphatase mRNA isoform in neurons but not in astrocytes. The muscle fructose-1,6-bisphosphatase mRNA isoform could be detected by RT-PCR in total rat brain, astrocytic, and neuronal mRNA preparations. The isoforms of fructose-1,6-bisphosphatase mRNA seemingly demonstrate a distinct cellular expression pattern in rat brain suggesting a role of glyconeogenesis in both neurons and glial cells.

Ontogenetic changes in protein level of amyloid precursor protein (APP) in growth cones and synaptosomes from rat brain and prenatal expression pattern of APP mRNA isoforms in developing rat embryo.

To elucidate the functional role of the amyloid precursor protein (APP) during brain ontogeny, developmental changes of APP levels in growth cones and synaptosomes were studied from embryonic day 14 up to postnatal day (PD) 400 using Western analysis. APP level in growth cones was low during prenatal stages of development, but demonstrating a continuous increase from PD 3 up to PD 10. Highest concentration of APP in synaptosomes was found between PD 7 and 10, followed by a considerable decrease up to PD 30 and persisting at this level up to PD 400. In situ hybridization to differentiate between APP695 mRNA, APP751 mRNA and APP770 mRNA revealed distinct age-related expression pattern of various APP isoforms. During prenatal brain development APP695 mRNA is maximally expressed in brain structures, containing differentiating nerve cells. APP751 and APP770 mRNA isoforms are diffusely distributed in the embryo throughout the prenatal period examined and their expression is higher in peripheral organs such as skin, lung, liver and bones as compared to the brain. The increase of APP level during synaptogenesis suggests a functional role of APP in the processes of neurite outgrowth and cell targeting as well as in the maintenance of the functional integrity of synapses in the mature brain. The APP695 isoform seems to be the major form involved in embryonic brain maturation.

Protein kinase Calpha and beta1 isoforms are regulators of alpha-secretory proteolytic processing of amyloid precursor protein in vivo.

We have recently shown that in utero treatment of guinea pigs with the DNA methylating substance methylazoxymethanol acetate (MAM) results in neocortical microencephalopathy, increased protein kinase C (PKC) activity and altered processing of the amyloid precursor protein (APP) in neocortex of offspring. Here we show that PKCalpha and PKCbeta1 are the key regulators of alpha-secretory APP processing in guinea pig neocortex under these experimental conditions in vivo. This conclusion is based on the selective translocation of PKCalpha and PKCbeta1 isoforms to the cell membrane in MAM-treated guinea pigs, as revealed by Western blot analysis and by immunocytochemistry. Additionally, we observed that [3H]phorbol ester binding to protein kinase C increased by 38% and enhanced basal PKC activity by 58% in the neocortex of microencephalic guinea pigs. Inhibition of PKCalpha/PKCbeta1 by Gö6976 abolished this difference, suggesting that constitutive overactivation of these PKC isoforms accounts for the increase in total PKC activity. We also observed a strong positive correlation between levels of alpha-secretase-processed APP and PKC activity in the neocortex of individual animals, providing further evidence for a significant role of classical PKC isoforms in nonamyloidogenic APP processing.

Fibrillary beta-amyloid deposits are closely associated with atrophic nitric oxide synthase (NOS)-expressing neurons but do not upregulate the inducible NOS in transgenic Tg2576 mouse brain with Alzheimer pathology.

Transgenic mice (Tg2576) that express the Swedish double mutation of human amyloid precursor protein and develop Alzheimer-like beta-amyloid deposits in the aged brain, were used to study the effect of beta-amyloid deposition on expression of both neuronal (nNOS) and inducible nitric oxide synthase (iNOS) in cells surrounding beta-amyloid plaques. Nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry and double immunofluorescent labeling revealed that most of the fibrillary, thioflavine-S-positive cortical beta-amyloid deposits in 13-, 17-, and 21-month-old transgenic animals were closely associated with dystrophic nNOS-positive neurons, while nNOS-bearing neurons located more distal to plaques appeared to be unaffected. There was no significant expression of iNOS in transgenic mouse brain. The data suggest enhanced vulnerability of nNOS-containing neocortical neurons to beta-amyloid toxicity. Alternatively, expression of nNOS may also be a response to plaque-mediated damage of neurons, consistent with a neuroprotective role of nitric oxide.

Sequential upregulation of cell adhesion molecules in degenerating rat basal forebrain cholinergic neurons and in phagocytotic microglial cells.

To study the functional role of adhesion molecules in neurodegenerative events in vivo, the basal forebrain cholinergic lesion-induced expression of the intercellular adhesion molecule (ICAM)-1 and leukocyte function-associated antigen (LFA)-1 was studied by double immunocytochemistry and Western blot analysis. A single intracerebroventricular application of the cholinergic immunotoxin, 192IgG-saporin, produced a selective cholinergic cell loss in rat basal forebrain nuclei detectable by gradual loss of choline acetyltransferase (ChAT)-immunoreactive cells starting 3 days but being nearly complete 7 days after injection of the toxin. The degeneration of cholinergic neurons was accompanied by a striking appearance of activated microglial cells in the lesioned areas. Four days following injection of 192IgG-saporin, ICAM-1 immunoreactivity was predominantly observed in ChAT-positive neurons and partly in activated microglia in the basal forebrain nuclei, while LFA-1 expression at this time point was restricted to neurons. However, 7 days after cholinergic lesion, only a few, shrunken neuronal somata were found to be immunoreactive for ICAM-1 and LFA-1, while activated microglial cells demonstrated strong immunoreactivity for ICAM-1 and LFA-1 in the lesioned forebrain areas, persisting up to 14 days after lesion while no immunoreactivity was observed in neurons at this time point. Western blot analysis demonstrated increased ICAM-1 level in the basal forebrain already detectable 4 days after surgery but being more pronounced 7 days post lesion. The data suggest that ICAM-1 and LFA-1 may act as intercellular recognition signals by which degenerating cholinergic neurons actively participate in the sequence of events leading to their targeting and elimination by phagocytotic microglia.

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.

Rat basal forebrain cholinergic lesion affects neuronal nitric oxide synthase activity in hippocampal and neocortical target regions.

Nitric oxide (NO)-mediated mechanisms have been assigned a role in cortical perfusion, learning and memory as well as in neuronal plasticity. Dysfunction of cortical cholinergic transmission has also been associated with reduced cortical cerebral blood flow and impaired performance in learning and memory tasks suggesting a link between the basal forebrain cholinergic system and cortical NO-mediated mechanisms. The aim of this study was therefore to study the influence of cholinergic input on neuronal NO-synthase (nNOS) activity in cortical cholinoceptive target neurons. A nearly complete loss of rat basal forebrain cholinergic cells was induced by a single intracerebroventricular application of the cholinergic immunotoxin 192IgG-saporin. Basal forebrain cholinergic hypofunction resulted in reduced catalytic and substrate binding activity of nNOS in a number of hippocampal and neocortical subregions 7 days after lesion as revealed by NADPH-diaphorase enzyme histochemistry and quantitative autoradiography of [3H]L-N(G)-nitro-arginine binding, respectively. The total amount of nNOS protein assayed by Western analysis, was not affected in the cortical and hippocampal regions examined. The data indicate that cortical cholinergic deafferentation results in reduced nNOS activity in select cholinoceptive neocortical and hippocampal neurons. As the total amount of cortical nNOS protein was not affected by basal forebrain cholinergic lesion, the results suggest that the ratio of catalytically active and inactive cortical nNOS is driven by basal forebrain cholinergic input presumably via M1-muscarinic cholinergic receptors.

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.