Oxidative stress affects processing of amyloid precursor protein in vascular endothelial cells.

BACKGROUND: Oxidative stress is thought to be a key player in the pathogenesis of neurodegenerative dementia, including Alzheimer's disease (AD). It has been assumed that oxidative stress contributes to the ß-amyloid deposition in cerebral blood vessels. METHODS: In order to prove this hypothesis, we examined the effect of oxidative stress on the processing of amyloid precursor protein (APP) in primary endothelial cells (EC) derived from cerebral cortical tissue of transgenic Tg2576 mice. Following exposure of EC by 1 µM hydrogen peroxide for up to 48 hours, formation and secretion of APP cleavage products sAPPα and sAPPß into the culture medium as well as the expression of endothelial APP were assessed. RESULTS: Oxidative stress resulted in enhanced secretion of sAPPß into the culture medium as compared to controls (absence of hydrogen peroxide), which was accompanied by an increased APP expression, induction of VEGF synthesis, nitric oxide and oxygen free radicals productions, and differential changes of endothelial phospo-p42/44 MAPK expression. CONCLUSION: The data suggest that oxidative stress may represent a major risk factor in causing Aß deposition in the brain vascular system by initiating the amyloidogenic route of endothelial APP processing. The enhanced ß-secretase activity following oxidative stress exposure, possibly promoted by phosphorylation of p42/44 MAPK.

AßPP-Transgenic 2576 Mice Mimic Cell Type-Specific Aspects of Acetyl-CoA-Linked Metabolic Deficits in Alzheimer's Disease.

The pyruvate-derived acetyl-CoA is a principal direct precursor substrate for bulk energy synthesis in the brain. Deficits of pyruvate dehydrogenase in the neocortex are common features of Alzheimer's disease and other age-related encephalopathies in humans. Therefore, amyloid-ß overload in brains of diverse transgenic Alzheimer's disease model animals was investigated as one of neurotoxic compounds responsible for pyruvate dehydrogenase inhibition yielding deficits of cholinergic neurotransmission and cognitive functions. Brains of aged, 14-16-month-old Tg2576 mice contained 0.6 µmol/kg levels of amyloid-ß1 - 42. Activities of pyruvate dehydrogenase complex, choline acetyltransferase, and several enzymes of acetyl-CoA and energy metabolism were found to be unchanged in both forebrain mitochondria and synaptosomes of Tg2576 mice, indicating preservation of structural integrity at least in cholinergic neuronal cells. However, in transgenic brain synaptosomes, pyruvate utilization, mitochondrial levels, and cytoplasmic acetyl-CoA levels, as well as acetylcholine content and its quantal release, were all found to be decreased by 25-40% . On the contrary, activation of pyruvate utilization was detected and no alterations in acetyl-CoA content and citrate or α-ketoglutarate accumulation were observed in transgenic whole brain mitochondria. These data indicate that amyloid-ß evoked deficits in acetyl-CoA are confined to mitochondrial and cytoplasmic compartments of Tg2576 nerve terminals, becoming early primary signals paving the path for further stages of neurodegeneration. On the other hand, acetyl-CoA synthesis in mitochondrial compartments of glial cells seems to be activated despite amyloid-ß accumulated in transgenic brains.

Expression of vascular endothelial growth factor (VEGF) mRNA, VEGF receptor 2 (Flk-1) mRNA, and of VEGF co-receptor neuropilin (Nrp)-1 mRNA in brain tissue of aging Tg2576 mice by in situ hybridization.

Vascular endothelial growth factor (VEGF) has been characterized as a heparin binding angiogenic growth factor displaying high specificity for endothelial cells. It is profoundly accumulated and co-localized with amyloid beta (Aß) plaques in the brain of Alzheimer's disease patients. In order to examine the effect of Aß plaques on the expression level of VEGF mRNA and its receptors, brain tissue of both transgenic Tg2576 and wild type mice at ages ranging from 13 to 22 months was subjected to in situ hybridization followed by densitometric assessment using computer-assisted image analysis. Strong expression of VEGF mRNA, fetal liver kinase (Flk)-1 mRNA, and neuropilin (Nrp)-1 mRNA in the piriform, entorhinal, somatosensory, frontal cortex and hippocampal formation of both transgenic and non-transgenic mice brain was detected. Developmentally, only expression of VEGF mRNA was increased with age in the entorhinal, and somatosensory cortex of wild type mice. In 20-month-old transgenic Tg2576 mice, up-regulation of VEGF mRNA, Flk-1 mRNA, and Nrp-1 mRNA transcripts was observed in the entorhinal cortex compared to age-matched wild type mice. Our data suggest up-regulation of VEGF mRNA, Flk-1 mRNA and Nrp-1 mRNA, at least in the entorhinal cortex at ages when Aß deposition in Tg2576 is typically increasing.

Hypoxic stress, brain vascular system, and ß-amyloid: a primary cell culture study.

This study stresses the hypothesis whether hypoxic events contribute to formation and deposition of ß-amyloid (Aß) in cerebral blood vessels by affecting the processing of endothelial amyloid precursor protein (APP). Therefore, cerebral endothelial cells (ECs) derived from transgenic Tg2576 mouse brain, were subjected to short periods of hypoxic stress, followed by assessment of formation and secretion of APP cleavage products sAPPα, sAPPß, and Aß as well as the expression of endothelial APP. Hypoxic stress of EC leads to enhanced secretion of sAPPß into the culture medium as compared to normoxic controls, which is accompanied by increased APP expression, induction of vascular endothelial growth factor (VEGF) synthesis, nitric oxide production, and differential changes in endothelial p42/44 (ERK1/2) expression. The hypoxia-mediated up-regulation of p42/44 at a particular time of incubation was accompanied by a corresponding down-regulation of the phosphorylated form of p42/44. To reveal any role of hypoxia-induced VEGF in endothelial APP processing, ECs were exposed by VEGF. VEGF hardly affected the amount of sAPPß and Aß(1-40) secreted into the culture medium, whereas the suppression of the VEGF receptor action by SU-5416 resulted in decreased release of sAPPß and Aß(1-40) in comparison to control incubations, suggesting a role of VEGF in controlling the activity of γ-secretase, presumably via the VEGF receptor-associated tyrosine kinase. The data suggest that hypoxic stress represents a mayor risk factor in causing Aß deposition in the brain vascular system by favoring the amyloidogenic route of endothelial APP processing. The hypoxic-stress-induced changes in ß-secretase activity are presumably mediated by altering the phosphorylation status of p42/44, whereas the stress-induced up-regulation of VEGF appears to play a counteracting role by maintaining the balance of physiological APP processing.

Neuroprotective role of γ-enolase in microglia in a mouse model of Alzheimer's disease is regulated by cathepsin X.

γ-Enolase is a neurotrophic-like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C-terminal end of the molecule. We have investigated the expression and colocalization of γ-enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ-enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C-terminally cleaved form of γ-enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ-enolase in microglial cells in response to amyloid-ß peptide (Aß) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ-enolase proved to be neuroprotective against Aß toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ-enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid-ß-related neurodegeneration.

ß-Amyloid, cholinergic transmission, and cerebrovascular system -- a developmental study in a mouse model of Alzheimer's disease.

The majority of patients suffering from Alzheimer's disease (AD) demonstrate cerebral vascular changes and impaired regulation of cerebral blood flow, which has been assumed to play an important role in AD pathogenesis (vascular hypothesis of AD). There is strong evidence that both ß-amyloid (Aß) oligomers and plaques contribute to vascular injuries and functional impairments of the neurovascular unit. Vice versa, Aß lesions can be triggered by hypertension and ischemic brain injury, while Aß aggregates appear to have anti-angiogenic properties. Cholinergic dysfunction may result in impaired cerebral blood flow with consequences on normal function of the neurovascular unit including processing of the amyloid precursor protein (APP). To characterize in vivo the developmental relationship between Aß formation and deposition, cortical cholinergic innervation and cerebrovascular abnormalities, transgenic Tg2576 mice that overexpress the Swedish double mutation of human APP, and demonstrate significant cerebral cortical deposition of Aß plaques at ages from 9 months onwards, were considered as an appropriate animal model. Using the somatosensory cortex as a representative region, serial cryocut sections, were obtained from mice at ages ranging from 4 up to 18 months. These were subjected to immunohistochemistry to label vascular endothelial cells (anti-glucose transporter 1 (GluT1) immunostaining), cholinergic nerve terminals (anti-vesicular acetylcholine transporter (VAChT) immunostaining) and ß-amyloid plaques (thioflavin S, and/or Solanum tuberosum lectin staining). This was followed by a thorough quantitative evaluation of the age-related spatial relationship between cerebral cortical capillaries, Aß plaques and cholinergic terminals, using computer-assisted image analysis. The density of cholinergic terminals estimated by evaluation of VAChT immunohistochemistry in somatosensory cortical sections of wild type mice did not change with aging regardless of the cortical layer examined, while in cortical layers II/III and IV of somatosensory cortex of transgenic Tg2576 mice age-related decreases in cholinergic fiber densities were assessed. However, quantitative morphometric analysis demonstrated an age-related reduction in the number of varicosities on cholinergic fibers, particularly in layer IV, in both transgenic Tg2576 mice and non-transgenic littermates. Cholinergic innervation of microvessels in the somatosensory cortex decreased with aging in both Tg2576 mice and non-transgenic littermates, as revealed by estimating the ratio of the number of cholinergic vascular contacts and total length of blood vessel. There was no significant difference in the perivascular cholinergic innervation in areas that demonstrated significant plaque load and those with no plaque deposits regardless of the cortical layer examined. The density of blood vessels estimated in the somatosensory cortex of transgenic mice by anti-GluT-1 immunohistochemistry did not differ to that obtained in wild type mice before the onset of plaque deposition (younger than 10 months). However, in aged, 18-month-old Tg2576 mice, demonstrating high plaque loads, decreased blood vessel densities, particularly in layer IV of the somatosensory cortex, were observed. The data obtained in this study strongly support the idea of an age-related interplay between Aß accumulation, cholinergic dysfunction, and vascular impairments. However, it remains to be elucidated as to which processes play a causative role and which events are secondary. A potential mechanism is provided by the vascular hypothesis of AD. Aging-, and life-style-associated damage of the brain microvasculature may affect Aß clearance and perivascular drainage, promoting cerebrovascular Aß deposition, inducing partial loss of cholinergic vascular innervation and changes in vascular function, angiogenesis and upregulation of the vesicular endothelial growth factor (VEGF) with consequences on APP processing and Aß accumulation.

Cholinergic denervation exacerbates amyloid pathology and induces hippocampal atrophy in Tg2576 mice.

The main pathological hallmarks of Alzheimer's disease (AD) consist of amyloid plaques and neurofibrillary tangles. Hippocampal cell loss, atrophy and cholinergic dysfunction are also features of AD. The present work is aimed at studying the interactions between cholinergic denervation, APP processing and hippocampal integrity. The cholinergic immunotoxin mu p-75-saporin was injected into the 3rd ventricle of 6- to 8-month-old Tg2576 mice to induce a cholinergic denervation. Four weeks after cholinergic immunolesion, a significant 14-fold increase of soluble Aß1-42 was observed. Cholinergically lesioned Tg2576 mice showed hippocampal atrophy together with degenerating FluoroJade-B-stained neurons and reduction of synaptophysin expression in CA1-3 pyramidal layers. We also found that cholinergic denervation led to reduced levels of ADAM17 in hippocampus of Tg2576 mice. Inhibition of ADAM17 with TAPI-2 (5 µM) decreased viability of hippocampal primary neurons from Tg2576 brains and decreased phosphorylation of downstream effectors of trophic signalling (ERK and Akt). The cholinergic agonist carbachol (100 µM) rescued these effects, suggesting that cholinergic deficits might render hippocampus more vulnerable to neurotoxicity upon certain toxic environments. The present work proposes a novel model of AD that worsens the patent amyloid pathology of Tg2576 mice together with hippocampal synaptic pathology and neurodegeneration. Drugs aimed at favoring cholinergic transmission should still be considered as potential treatments of AD.

Arsenic affects expression and processing of amyloid precursor protein (APP) in primary neuronal cells overexpressing the Swedish mutation of human APP.

Arsenic poisoning due to contaminated water and soil, mining waste, glass manufacture, select agrochemicals, as well as sea food, affects millions of people world wide. Recently, an involvement of arsenic in Alzheimer's disease (AD) has been hypothesized (Gong and O'Bryant, 2010). The present study stresses the hypothesis whether sodium arsenite, and its main metabolite, dimethylarsinic acid (DMA), may affect expression and processing of the amyloid precursor protein (APP), using the cholinergic cell line SN56.B5.G4 and primary neuronal cells overexpressing the Swedish mutation of APP, as experimental approaches. Exposure of cholinergic SN56.B5.G4 cells with either sodium arsenite or DMA decreased cell viability in a concentration- and exposure-time dependent manner, and affected the activities of the cholinergic enzymes acetylcholinesterase and choline acetyltransferase. Both sodium arsenite and DMA exposure of SN56.B5.G4 cells resulted in enhanced level of APP, and sAPP in the membrane and cytosolic fractions, respectively. To reveal any effect of arsenic on APP processing, the amounts of APP cleavage products, sAPPß, and ß-amyloid (Aß) peptides, released into the culture medium of primary neuronal cells derived from transgenic Tg2576 mice, were assessed by ELISA. Following exposure of neuronal cells by sodium arsenite for 12h, the membrane-bound APP level was enhanced, the amount of sAPPß released into the culture medium was slightly higher, while the levels of Aß peptides in the culture medium were considerably lower as compared to that assayed in the absence of any drug. The sodium arsenite-induced reduction of Aß formation suggests an inhibition of the APP γ-cleavage step by arsenite. In contrast, DMA exposure of neuronal cells considerably increased formation of Aß and sAPPß, accompanied by enhanced membrane APP level. The DMA-induced changes in APP processing may be the result of the enhanced APP expression. Alternatively, increased Aß production may also be due to stimulation of caspase activity by arsenic compounds, or failure in Aß degradation. In summary, the present report clearly demonstrates that sodium arsenite and DMA affect processing of APP in vitro.

Glutaminyl cyclase contributes to the formation of focal and diffuse pyroglutamate (pGlu)-Aß deposits in hippocampus via distinct cellular mechanisms.

In the hippocampal formation of Alzheimer's disease (AD) patients, both focal and diffuse deposits of Aß peptides appear in a subregion- and layer-specific manner. Recently, pyroglutamate (pGlu or pE)-modified Aß peptides were identified as a highly pathogenic and seeding Aß peptide species. Since the pE modification is catalyzed by glutaminyl cyclase (QC) this enzyme emerged as a novel pharmacological target for AD therapy. Here, we reveal the role of QC in the formation of different types of hippocampal pE-Aß aggregates. First, we demonstrate that both, focal and diffuse pE-Aß deposits are present in defined layers of the AD hippocampus. While the focal type of pE-Aß aggregates was found to be associated with the somata of QC-expressing interneurons, the diffuse type was not. To address this discrepancy, the hippocampus of amyloid precursor protein transgenic mice was analysed. Similar to observations made in AD, focal (i.e. core-containing) pE-Aß deposits originating from QC-positive neurons and diffuse pE-Aß deposits not associated with QC were detected in Tg2576 mouse hippocampus. The hippocampal layers harbouring diffuse pE-Aß deposits receive multiple afferents from QC-rich neuronal populations of the entorhinal cortex and locus coeruleus. This might point towards a mechanism in which pE-Aß and/or QC are being released from projection neurons at hippocampal synapses. Indeed, there are a number of reports demonstrating the reduction of diffuse, but not of focal, Aß deposits in hippocampus after deafferentation experiments. Moreover, we demonstrate in neurons by live cell imaging and by enzymatic activity assays that QC is secreted in a constitutive and regulated manner. Thus, it is concluded that hippocampal pE-Aß plaques may develop through at least two different mechanisms: intracellularly at sites of somatic QC activity as well as extracellularly through seeding at terminal fields of QC expressing projection neurons.

Perivascular drainage of solutes is impaired in the ageing mouse brain and in the presence of cerebral amyloid angiopathy.

The deposition of amyloid-ß (Aß) peptides in the walls of leptomeningeal and cortical blood vessels as cerebral amyloid angiopathy (CAA) is present in normal ageing and the majority of Alzheimer's disease (AD) brains. The failure of clearance mechanisms to eliminate Aß from the brain contributes to the development of sporadic CAA and AD. Here, we investigated the effects of CAA and ageing on the pattern of perivascular drainage of solutes in the brains of naïve mice and in the Tg2576 mouse model of AD. We report that drainage of small molecular weight dextran along cerebrovascular basement membranes is impaired in the hippocampal capillaries and arteries of 22-month-old wild-type mice compared to 3- and 7-month-old animals, which was associated with age-dependent changes in capillary density. Age-related alterations in the levels of laminin, fibronectin and perlecan in vascular basement membranes were also noted in wild-type mice. Furthermore, dextran was observed in the walls of veins of Tg2576 mice in the presence of CAA, suggesting that deposition of Aß in vessel walls disrupts the normal route of elimination of solutes from the brain parenchyma. These data support the hypothesis that perivascular solute drainage from the brain is altered both in the ageing brain and as a consequence of CAA. These findings have implications for the success of therapeutic strategies for the treatment of AD that rely upon the health of the ageing cerebral vasculature.

The cholinergic system in aging and neuronal degeneration.

The basal forebrain cholinergic complex comprising medial septum, horizontal and vertical diagonal band of Broca, and nucleus basalis of Meynert provides the mayor cholinergic projections to the cerebral cortex and hippocampus. The cholinergic neurons of this complex have been assumed to undergo moderate degenerative changes during aging, resulting in cholinergic hypofunction that has been related to the progressing memory deficits with aging. However, the previous view of significant cholinergic cell loss during aging has been challenged. Neuronal cell loss was found predominantly in pathological aging, such as Alzheimer's disease, while normal aging is accompanied by a gradual loss of cholinergic function caused by dendritic, synaptic, and axonal degeneration as well as a decrease in trophic support. As a consequence, decrements in gene expression, impairments in intracellular signaling, and cytoskeletal transport may mediate cholinergic cell atrophy finally leading to the known age-related functional decline in the brain including aging-associated cognitive impairments. However, in pathological situations associated with cognitive deficits, such as Parkinsons's disease, Down-syndrome, progressive supranuclear palsy, Jakob-Creutzfeld disease, Korsakoff's syndrome, traumatic brain injury, significant degenerations of basal forebrain cholinergic cells have been observed. In presenile (early onset), and in the advanced stages of late-onset Alzheimer's disease (AD), a severe loss of cortical cholinergic innervation has extensively been documented. In contrast, in patients with mild cognitive impairment (MCI, a prodromal stage of AD), and early forms of AD, apparently no cholinergic neurodegeneration but a loss of cholinergic function occurs. In particular imbalances in the expression of NGF, its precursor proNGF, the high and low NGF receptors, trkA and p75NTR, respectively, changes in acetylcholine release, high-affinity choline uptake, as well as alterations in muscarinic and nicotinic acetylcholine receptor expression may contribute to the cholinergic dysfunction. These observations support the suggestion of a key role of the cholinergic system in the functional processes that lead to AD. Malfunction of the cholinergic system may be tackled pharmacologically by intervening in cholinergic as well as neurotrophic signaling cascades that have been shown to ameliorate the cholinergic deficit at early stages of the disease, and slow-down the progression. However, in contrast to many other, dementing disorders, in AD the cholinergic dysfunctions are accompanied by the occurrence of two major histopathological hallmarks such as ß-amyloid plaques and neurofibrillary tangles, provoking the question whether they play a particular role in inducing or mediating cholinergic dysfunction in AD. Indeed, there is abundant evidence that ß-amyloid may trigger cholinergic dysfunction through action on α7 nicotinic acetylcholine receptors, affecting NGF signaling, mediating tau phosphorylation, interacting with acetylcholinesterase, and specifically affecting the proteome in cholinergic neurons. Therefore, an early onset of an anti ß-amyloid strategy may additionally be potential in preventing aging-associated cholinergic deficits and cognitive impairments.

Effect of VEGF and its receptor antagonist SU-5416, an inhibitor of angiogenesis, on processing of the ß-amyloid precursor protein in primary neuronal cells derived from brain tissue of Tg2576 mice.

A large number of Alzheimer patients demonstrate cerebrovascular pathology, which has been assumed to be related to ß-amyloid (Aß) deposition. Aß peptides have been described to inhibit angiogenesis both in vitro and in vivo, and deregulation of angiogenic factors may contribute to various neurological disorders including neurodegeneration. One of the key angiogenic factor is the vascular endothelial growth factor (VEGF). Increased levels of VEGF have been observed in brains of Alzheimer patients, while the functional significance of VEGF up-regulation in the pathogenesis and progression of AD is still a matter of debate. To test whether VEGF may affect neuronal APP processing, primary neuronal cells derived from brain tissue of E16 embryos of Tg2576 mice were exposed with 1 ng/ml VEGF for 6, 12, and 24h, followed by monitoring formation and secretion of soluble Aß peptides, release of the human APP cleavage products, sAPPßswe and sAPPα, into the culture medium as well as the activities of α- and ß-secretases in neuronal cell extracts. Exposure of primary neuronal cells by VEGF for 24h led to slightly reduced sAPPß release, accompanied by decreased ß-secretase activity 12h after VEGF exposure. Incubation of neurons by the VEGF receptor antagonist and angiogenesis inhibitor SU-5416 for 24h resulted in increased release of sAPPßswe, and strikingly enhanced secretion of Aß peptides into the culture medium, which was accompanied by a significant increase in ß-secretase activity, as compared to control incubations. The SU-5416-induced effects on APP processing could not be suppressed by the additional presence of VEGF, suggesting that SU-5416 affects pathways that are apparently independent of VEGF receptor signaling. The data obtained indicate that VEGF-driven mechanisms may affect APP processing, suggesting a link of angiogenesis and pathogenesis of Alzheimer's disease.

Insulin-resistant brain state after intracerebroventricular streptozotocin injection exacerbates Alzheimer-like changes in Tg2576 AbetaPP-overexpressing mice.

For studying rare hereditary Alzheimer's disease (AD), transgenic (Tg) animal models overexpressing amyloid-beta protein precursor (AbetaPP) followed by increased amyloid-beta (Abeta) formation are used. In contrast, sporadic AD has been proposed to start with an insulin-resistant brain state (IRBS).We investigated the effect of IRBS induced by intracerebroventricularly (icv) administered streptozotocin (STZ) on behavior, glycogen synthase kinase-3 (GSK) alpha/beta content, and the formation of AD-like morphological hallmarks Abeta and tau protein in AbetaPP Tg2576 mice. Nine-month-old Tg mice were investigated 6 months after a single icv injection of STZ or placebo. Spatial cognition was analyzed using the Morris water maze test. Soluble and aggregated Abeta40/42 fragments, total and phosphorylated tau protein, and GSK-3alpha/beta were determined by ELISA. Cerebral (immuno)histological analyses were performed. In Tg mice, STZ treatment increased mortality, reduced spatial cognition, and increased cerebral aggregated Abeta fragments, total tau protein, and congophilic amyloid deposits. These changes were associated with decreased GSK-3alpha/beta ratio (phosphorylated/total). A linear negative correlation was detected between Abeta42 and cognition, and between GSK-3alpha/beta ratio and aggregated Abeta40+42. No marked necrotic and apoptotic changes were observed. In conclusion, IRBS may aggravate AD-like changes such as behavioral and increase the formation of pathomorphological AD hallmarks via GSK-3alpha/beta pathway in AbetaPP-overexpressing mice.

Cell proliferation and total granule cell number in dentate gyrus of transgenic Tg2576 mouse.

This report provides in vivo evidence of adult neurogenesis and the total granule cell count in the dentate gyrus of the Tg2576 mouse model of Alzheimer's disease. Mice were deeply anaesthetized and perfused with 4 percent buffered paraformaldehyde. Brains were removed and post-fixed in the same fixative overnight. Following equilibration in 30 percent sucrose, 30 micrometer sections were cut in sagittal plane in freezing microtome for immunohistochemistry and 20 micrometer from plastic embedded brains. Thioflavin-S confirmed the presence of amyloid plaques in the Tg2576 mice. Cell proliferation in the subventricular zone and dentate gyrus of hippocampus were observed with Ki-67 and doublecortin markers. Using optical fractionator, total granule number was estimated to be 445,280 per hemisphere in the 18-month-old Tg2576 mouse. Cell proliferation tends to end in the dentate gyrus but continues in the SVZ and the total granule cell number was less compared to normal laboratory and wild rodents.

Effects of lateral fluid percussion injury on cholinergic markers in the newborn piglet brain.

Traumatic brain injury is a leading cause of death and disability in children. Studies using adult animal models showed alterations of the central cholinergic neurotransmission as a result of trauma. However, there is a lack of knowledge about consequences of brain trauma on cholinergic function in the immature brain. It is hypothesized that trauma affects the relative acetylcholine esterase activity and causes a loss of cholinergic neurons in the immature brain. Severe fluid percussion trauma (FP-TBI, 3.8+/-0.3atm) was induced in 15 female newborn piglets, monitored for 6h and compared with 12 control animals. The hemispheres ipsilateral to FP-TBI obtained from seven piglets were used for acetylcholine esterase histochemistry on frozen sagittal slices, while regional cerebral blood flow and oxygen availability was determined in the remaining eight FP-TBI animals. Post-fixed slices were immunohistochemically labelled for choline acetyltransferase as well as for low-affinity neurotrophin receptor in order to characterize cholinergic neurons in the basal forebrain. Regional cerebral blood flow and brain oxygen availability were reduced during the first 2h after FP-TBI (P<0.05). In addition, acetylcholine esterase activity was significantly increased in the neocortex, basal forebrain, hypothalamus and medulla after trauma (P<0.05), whereas the number of choline acetyltransferase and low-affinity neurotrophin receptor positive cells in the basal forebrain were unaffected by the injury. Thus, traumatic brain injury evoked an increased relative activity of the acetylcholine esterase in the immature brain early after injury, without loss of cholinergic neurons in the basal forebrain. These changes may contribute to developmental impairments after immature traumatic brain injury.

Recurrent systemic infections with Streptococcus pneumoniae do not aggravate the course of experimental neurodegenerative diseases.

Neurological symptoms of patients suffering from neurodegenerative diseases such as Alzheimer's dementia (AD), Parkinson's disease (PD), or amyotrophic lateral sclerosis (ALS) often worsen during infections. We assessed the disease-modulating effects of recurrent systemic infections with the most frequent respiratory pathogen, Streptococcus pneumoniae, on the course of AD, PD, and ALS in mouse models of these neurodegenerative diseases [transgenic Tg2576 mice, (Thy1)-[A30P]alpha SYN mice, and Tg(SOD1-G93A) mice]. Mice were repeatedly challenged intraperitoneally with live S. pneumoniae type 3 and treated with ceftriaxone for 3 days. Infection caused an increase of interleukin-6 concentrations in brain homogenates. The clinical status of (Thy1)-[A30P]alpha SYN mice and Tg(SOD1-G93A) mice was monitored by repeated assessment with a clinical score. Motor performance was controlled by the tightrope test and the rotarod test. In Tg2576 mice, spatial memory and learning deficits were assessed in the Morris water maze. In none of the three mouse models onset or course of the disease as evaluated by the clinical tests was affected by the recurrent systemic infections performed. Levels of alpha-synuclein in brains of (Thy1)-[A30P]alpha SYN mice did not differ between infected animals and control animals. Plaque sizes and concentrations of A beta 1-40 and A beta 1-42 were not significantly different in brains of infected and uninfected Tg2576 mice. In conclusion, onset and course of disease in mouse models of three common neurodegenerative disorders were not influenced by repeated systemic infections with S. pneumoniae, indicating that the effect of moderately severe acute infections on the course of neurodegenerative diseases may be less pronounced than suspected.

Perineuronal nets are largely unaffected in Alzheimer model Tg2576 mice.

Changes in the molecular organization of the extracellular matrix are key factors in neuropathology. We investigated aggrecan-based perineuronal nets (PNs) in relation to neurodegeneration and activation of glial cells in a transgenic mouse (Tg2576) model of Alzheimer's disease. The formation of amyloid plaques in the cerebral cortex occurred independently of the area-specific distribution of PNs. Matrix components were only affected in the core of plaques in advanced stages of pathology. PNs remained unchanged in the large marginal zone occupied by reactive astrocytic processes. We conclude that the aggrecan-based extracellular matrix of PNs is not enzymatically altered in peripheral plaque territories and is only removed after neuronal death.

Synaptotagmins in neurodegeneration.

Synaptotagmins (Syts) are transmembrane proteins involved in the regulation of membrane trafficking. Here, we summarize literature data that provide growing evidence that several Syts are involved in the pathophysiological mechanisms of temporal lobe epilepsy and Parkinson's disease, as well as few reports related to brain ischemia and Alzheimer's disease (AD). We also report new data from our laboratories, showing changes of the expression of several Syts in Tg2576 mouse model of AD that may be related to neuroinflammation surrounding the beta-amyloid plaques. Furthermore, we demonstrate N-methyl-D-aspartate receptor-mediated upregulation of Syt 4 mRNA in a model of excitotoxic striatal lesion induced by unilateral striatal injection of quinolinic acid, associating the upregulation of Syt 4 with mechanisms of excitotoxicity. We propose that pharmacological manipulation of Syt expression in animal models of neurodegeneration should be further explored, as it may help to clarify the role of individual Syt isoforms in the regulation of membrane trafficking in neurodegeneration.

Vascular endothelial growth factor (VEGF) affects processing of amyloid precursor protein and beta-amyloidogenesis in brain slice cultures derived from transgenic Tg2576 mouse brain.

The up-regulation of the angiogenic vascular endothelial growth factor (VEGF) in brains of Alzheimer patients in close relationship to beta-amyloid (Abeta) plaques, suggests a link of VEGF action and processing of the amyloid precursor protein (APP). To reveal whether VEGF may affect APP processing, brain slices derived from 17-month-old transgenic Tg2576 mice were exposed with 1ng/ml VEGF for 6, 24, and 72h, followed by assessing cytosolic and membrane-bound APP expression, level of both soluble and fibrillar Abeta-peptides, as well as activities of alpha- and beta-secretases in brain slice tissue preparations. Treatment of brain slices with VEGF did not significantly affect the expression level of APP, regardless of the exposure time studied. In contrast, VEGF exposure of brain slices for 6h reduced the formation of soluble, SDS extractable Abeta(1-40) and Abeta(1-42) as compared to brain slice cultures incubated in the absence of any drug, while the fibrillar Abeta peptides did not change significantly. This effect was less pronounced 24h after VEGF exposure, but was no longer detectable when brain slices were exposed by VEGF for 72h, which indicates an adaptive response to chronic VEGF exposure. The VEGF-mediated reduction in Abeta formation was accompanied by a transient decrease in beta-secretase activity peaking 6h after VEGF exposure. To reveal whether the VEGF-induced changes in soluble Abeta-level may be due to actions of VEGF on Abeta fibrillogenesis, the fibrillar status of Abeta was examined using the thioflavin-T binding assay. Incubation of Abeta preparations obtained from Tg2576 mouse brain cortex, in the presence of VEGF slightly decreased the fibrillar content with increasing incubation time up to 72h. The data demonstrate that VEGF may affect APP processing, at least in vitro, suggesting a role of VEGF in the pathogenesis of Alzheimer's disease.