Organotypic Slice Cultures from Transgenic Mice As Disease Model Systems

Journal of Molecular Neuroscience : MN. Dec, 2002  |  Pubmed ID: 12540058

Organotypic slice cultures have been prepared from the brains of transgenic mice with Alzheimer's disease-type pathology. Cell types within the slice undergo differentiation and slices can be maintained in culture for up to 6 mo when prepared from young neonates. Slices have been prepared from mice overexpressing genes of relevance to Alzheimer's disease, including mutant or wild-type tau. Neurons in these slices develop neurons that are immunoreactive for a number of markers of abnormal tau. Organotypic slice models are currently being used to test the impact of tangle enhancers or inhibitors as a prescreen for efficacy before testing drugs in vivo.

Imaging Brain Amyloid of Alzheimer Disease in Vivo in Transgenic Mice with an Abeta Peptide Radiopharmaceutical

Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism. Feb, 2002  |  Pubmed ID: 11823720

Abeta 1-40 is a potential peptide radiopharmaceutical that could be used to image the brain Abeta amyloid of Alzheimer disease in vivo, should this peptide be made transportable through the blood-brain barrier in vivo. The blood-brain barrier transport of [ 125 I]-Abeta 1-40 in a transgenic mouse model was enabled by conjugation to the rat 8D3 monoclonal antibody to the mouse transferrin receptor. The Abeta 1-40 -8D3 conjugate is a bifunctional molecule that binds the blood-brain barrier TfR and undergoes transport into brain and binds the Abeta amyloid plaques of Alzheimer disease. App SW /Psen1 double-transgenic and littermate control mice were administered either unconjugated Abeta 1-40 or the Abeta 1-40 -8D3 conjugate intravenously, and brain scans were obtained 6 hours later. Immunocytochemical analysis showed abundant Abeta immunoreactive plaques in the brains of the App SW /Psen1 transgenic mice and there was a selective retention of radioactivity in the brains of these mice at 6 hours after intravenous administration of the conjugate. In contrast, there was no selective sequestration either of the conjugate in control littermate mouse brain or of unconjugated Abeta 1-40 in transgenic mouse brain. In conclusion, the results show that it is possible to image the Abeta amyloid burden in the brain in vivo with an amyloid imaging agent, provided the molecule is conjugated to a blood-brain barrier drug-targeting system.

Statin Therapy for Alzheimer's Disease: Will It Work?

Journal of Molecular Neuroscience : MN. Aug-Oct, 2002  |  Pubmed ID: 12212773

Disease-modifying therapies are being developed for Alzheimer's disease (AD). These are expected to slow the clinical progression of the disease or delay its onset. Cerebral accumulation of amyloid beta (A beta) peptides is an early and perhaps necessary event for establishing AD pathology. Consequently therapies aimed at attenuating brain amyloidosis are expected to be disease modifying. Based on the epidemiological evidence pointing to a link between cholesterol metabolism and AD and the numerous laboratory studies implicating cholesterol in the process of A beta production and accumulation, it is now believed that cholesterol-lowering therapies will be of value as disease modifying agents. Several epidemiological studies revealed that statin use for the treatment of coronary arterial disease is associated with a decreased prevalence or a decreased risk of developing AD. These observations require both preclinical and clinical validation. The former involves testing statins in one or more animal models of AD in order to establish which disease features are affected by statin treatment, the relative efficacy with which different statins modify these features and the mechanism(s) by which statins affect AD phenotypes. The latter requires prospective, randomized, placebo controlled trials to evaluate the effect of statin treatment on cognitive and AD biomarker outcomes. We have initiated a study aimed at determining the effects of atorvastatin (Lipitor), a statin with the largest US market share, on brain A beta deposition in the PSAPP transgenic mouse model of Alzheimer's amyloidosis. Our results indicate that Lipitor treatment markedly attenuates A beta deposition in this animal model.

Cholesterol in Alzheimer's Disease and Tauopathy

Annals of the New York Academy of Sciences. Nov, 2002  |  Pubmed ID: 12480774

Cholesterol has been implicated in the pathogenesis of amyloid plaques in Alzheimer's disease (AD) and in the formation of neurofibrillary pathology in Niemann-Pick disease. Several epidemiology studies have implicated high cholesterol as a risk factor for AD and have shown that the use of cholesterol-reducing agents (statins) can be protective against the disease. We and others have shown that cholesterol levels modulate the processing of the amyloid precursor protein (APP) both in vivo and in vitro, affecting the accumulation of A-beta (Abeta) peptides that may directly impact the risk of AD. Mutations in the Niemann-Pick C gene (NPC) result in deficient cholesterol transport/storage. Clinically, Niemann-Pick disease causes a severe childhood lipidosis, with neurodegeneration characterized by the presence of AD-type neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau. Studies of mouse models of NPC show that defects in cellular cholesterol trafficking are associated with enhanced generation of Abeta and the hyperphosphorylation of tau, further implicating the cholesterol homeostasis pathway as a risk factor for amyloidosis.

Novel Therapeutic Approach for the Treatment of Alzheimer's Disease by Peripheral Administration of Agents with an Affinity to Beta-amyloid

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2003  |  Pubmed ID: 12514198

Plaques containing beta-amyloid (Abeta) peptides are one of the pathological features of Alzheimer's disease, and the reduction of Abeta is considered a primary therapeutic target. Amyloid clearance by anti-Abeta antibodies has been reported after immunization, and recent data have shown that the antibodies may act as a peripheral sink for Abeta, thus altering the periphery/brain dynamics. Here we show that peripheral treatment with an agent that has high affinity for Abeta (gelsolin or GM1) but that is unrelated to an antibody or immune modulator reduced the level of Abeta in the brain, most likely because of a peripherally acting effect. We propose that in general, compounds that sequester plasma Abeta could reduce or prevent brain amyloidosis, which would enable the development of new therapeutic agents that are not limited by the need to penetrate the brain or evoke an immune response.

Co-localization of Cholesterol, Apolipoprotein E and Fibrillar Abeta in Amyloid Plaques

Brain Research. Molecular Brain Research. Jan, 2003  |  Pubmed ID: 12573540

Recent evidence strongly suggests a role for cholesterol and apolipoprotein E in the etiology of Alzheimer's disease. We have demonstrated the co-localization of cholesterol and apolipoprotein E with beta-amyloid immunoreactivity and thioflavin S immunofluorescence in AD type plaques of a transgenic mouse model. Cholesterol and apolipoprotein E co-localized to the core of thioflavin S-positive (fibrillar) plaques, but not thioflavin S-negative (diffuse) plaques from an early age. By 18 months of age, there was extensive coverage of fibrillar plaques immunopositive for apolipoprotein E and cholesterol oxidase. These findings support evidence that cholesterol and apolipoprotein E are involved in fibrillar plaque formation or maintenance, and suggest that cholesterol may impact amyloid formation extracellularly, as well as through an intracellular effect.

Brain Damage Results in Down-regulation of N-acetylaspartate As a Neuronal Osmolyte

Neuromolecular Medicine. 2003  |  Pubmed ID: 12728192

N-acetyl-L-aspartate (NAA) is present in the vertebrate brain, where its concentration is one of the highest of all free amino acids. Although NAA is synthesized and stored primarily in neurons, it is not hydrolyzed in these cells. However, after its regulated release into extracellular fluid, neuronal NAA is hydrolyzed by amidohydrolase II that is present in oligodendrocytes. About 30% of neurons do not contain appreciable amounts of NAA, but its prominence in 1H nuclear magnetic resonance spectroscopic (MRS) studies has led to its wide use as a neuronal marker in diagnostic human medicine as both an indicator of brain pathology, and of disease progression in a variety of central nervous system (CNS) diseases. Loss of NAA has been interpreted as indicating either loss of neurons, or loss of neuron viability. In this investigation, the upregulation of NAA in early stages of construction of the CNS, and its downregulation in experimentally induced damage models of the CNS is reported. The results of this study indicate that the buildup of NAA is not required for viability of neurons in monocellular cultures, and that NAA is lost from multicellular cultured brain slice explants that contain viable neurons. Thus, loss of NAA does not necessarily indicate either loss of neurons or their function. The NAA system, when present in the brain, appears to reflect a high degree of cellular integration, and therefore may be a unique metabolic construct of the intact vertebrate brain.

Use of in Vivo Models to Study the Role of Cholesterol in the Etiology of Alzheimer's Disease

Neurochemical Research. Jul, 2003  |  Pubmed ID: 12737522

Cholesterol has been implicated in the pathogenesis of Alzheimer's disease, both through intracellular effects, and through an extracellular effect due to its physical interaction with plaque associated amyloid. Epidemiology studies have implicated high cholesterol as a risk factor for AD, and have shown that the use of cholesterol reducing agents (statins) can be protective against the disease. We, and others have shown that cholesterol levels modulate the processing of the amyloid precursor protein (APP) both in vivo and in vitro, affecting the accumulation of Abeta (Abeta) peptides which may directly impact the risk of AD. This review describes the biology of sterols, and identifies how cholesterol may exacerbate the pathogenesis of AD. Data from in vivo and in vitro studies will then be presented to describe how treatments aimed at modulating lipid levels may be efficacious in treating AD.

Cdk5 is a Key Factor in Tau Aggregation and Tangle Formation in Vivo

Neuron. May, 2003  |  Pubmed ID: 12765608

Tau aggregation is a common feature of neurodegenerative diseases such as Alzheimer's disease, and hyperphosphorylation of tau has been implicated as a fundamental pathogenic mechanism in this process. To examine the impact of cdk5 in tau aggregation and tangle formation, we crossed transgenic mice overexpressing the cdk5 activator p25, with transgenic mice overexpressing mutant (P301L) human tau. Tau was hyperphosphorylated at several sites in the double transgenics, and there was a highly significant accumulation of aggregated tau in brainstem and cortex. This was accompanied by increased numbers of silver-stained neurofibrillary tangles (NFTs). Insoluble tau was also associated with active GSK. Thus, cdk5 can initiate a major impact on tau pathology progression that probably involves several kinases. Kinase inhibitors may thus be beneficial therapeutically.

Presenilin Redistribution Associated with Aberrant Cholesterol Transport Enhances Beta-amyloid Production in Vivo

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jul, 2003  |  Pubmed ID: 12843267

Epidemiology, in vitro, and in vivo studies strongly implicate a role for cholesterol in the pathogenesis of Alzheimer's disease (AD). We have examined the impact of aberrant intracellular cholesterol transport on the processing of the amyloid precursor protein (APP) in a mouse model of Niemann-Pick type C (NPC) disease. In the NPC mouse brain, cholesterol accumulates in late endosomes/lysosomes. This was associated with the accumulation of beta-C-terminal fragments (CTFs) of APP, but the level of beta-secretase and its activity were not affected. Alpha-secretase activity and secreted APPalpha generation were also not affected, suggesting CTFs increased because of decreased clearance. The level of presenilin-1 (PS-1) was unchanged, but gamma-secretase activity was greatly enhanced, which correlated with an increase in Abeta40 and Abeta42 levels. These events were associated with abnormal distribution of PS-1 in the endosomal system. Our results show that aberrant cholesterol trafficking is associated with the potentiation of APP processing components in vivo, leading to an overall increase in Abeta levels.

Hyperphosphorylation and Aggregation of Tau in Mice Expressing Normal Human Tau Isoforms

Journal of Neurochemistry. Aug, 2003  |  Pubmed ID: 12859672

Neurofibrillary tangles are composed of insoluble aggregates of the microtubule-associated protein tau. In Alzheimer's disease the accumulation of neurofibrillary tangles occurs in the absence of tau mutations. Here we present mice that develop pathology from non-mutant human tau, in the absence of other exogenous factors, including beta-amyloid. The pathology in these mice is Alzheimer-like, with hyperphosphorylated tau accumulating as aggregated paired helical filaments. This pathologic tau accumulates in the cell bodies and dendrites of neurons in a spatiotemporally relevant distribution.

Detection of Alzheimer's Amyloid in Transgenic Mice Using Magnetic Resonance Microimaging

Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. Aug, 2003  |  Pubmed ID: 12876705

The presence of amyloid-beta (Abeta) plaques in the brain is a hallmark pathological feature of Alzheimer's disease (AD). Transgenic mice overexpressing mutant amyloid precursor protein (APP), or both mutant APP and presenilin-1 (APP/PS1), develop Abeta plaques similar to those in AD patients, and have been proposed as animal models in which to test experimental therapeutic approaches for the clearance of Abeta. However, at present there is no in vivo whole-brain imaging method to detect Abeta plaques in mice or men. A novel method is presented to detect Abeta plaques in the brains of transgenic mice by magnetic resonance microimaging (muMRI). This method uses Abeta1-40 peptide, known for its high binding affinity to Abeta, magnetically labeled with either gadolinium (Gd) or monocrystalline iron oxide nanoparticles (MION). Intraarterial injection of magnetically labeled Abeta1-40, with mannitol to transiently open the blood-brain barrier (BBB), enabled the detection of many Abeta plaques. Furthermore, the numerical density of Abeta plaques detected by muMRI and by immunohistochemistry showed excellent correlation. This approach provides an in vivo method to detect Abeta in AD transgenic mice, and suggests that diagnostic MRI methods to detect Abeta in AD patients may ultimately be feasible.

Evidence for Peripheral Clearance of Cerebral Abeta Protein Following Chronic, Active Abeta Immunization in PSAPP Mice

Neurobiology of Disease. Oct, 2003  |  Pubmed ID: 13678662

Immunization with amyloid-beta (Abeta) peptide in mouse models of Alzheimer's disease has been reported to decrease cerebral Abeta levels and improve behavioral deficits. Several mechanisms have been proposed, including antibody-induced phagocytosis of Abeta by cerebral microglia and increased efflux of Abeta from the brain to the periphery. The latter mechanism was suggested in mice undergoing acute, passive transfer of an Abeta monoclonal antibody. Here, PSAPP transgenic mice were actively immunized by a single intraperitoneal injection of synthetic Abeta followed by chronic intranasal administration of Abeta with the mucosal adjuvant, Escherichia coli heat-labile enterotoxin, LT, twice weekly for 8 weeks. Serum from Abeta-immunized mice had an average of 240 microg/ml of anti-Abeta-specific antibodies; these antibodies had epitope(s) within Abeta1-15 and were of immunoglobulin (Ig) isotypes IgG2b, IgG2a, and IgG1. Immunization led to a 75% decrease in plaque number (P < 0.0001) and a 58% decrease in Abetax-42 levels (P < 0.026) in brain, and gliosis and neuritic dystrophy were diminished. No pathological effects of the immunization were observed in kidney, spleen, or snout. Serum Abeta levels increased 28-fold in immunized mice (53.06 ng/ml) compared to controls (1.87 ng/ml). Most of the Abeta in the serum of the immunized mice was bound to antibodies. We conclude that following active immunization, anti-Abeta antibodies sequester serum Abeta and may increase central nervous system to serum Abeta clearance.

Changes in Apolipoprotein E Expression in Response to Dietary and Pharmacological Modulation of Cholesterol

Journal of Molecular Neuroscience : MN. 2003  |  Pubmed ID: 14501024

Apolipoprotein E (ApoE) influences the risk of late onset Alzheimer's disease (AD) in an isoform-dependent manner, such that the presence of the apoE epsilon4 allele increases the risk of AD while the presence of the apoE epsilon2 allele appears to be protective. Although a number of ApoE functions are isoform dependent and may underlie the "risk factor" activity of AD, its ability to bind amyloid beta peptides and influence their clearance and/or deposition has gained strong experimental support. Evidence suggests that in addition to genotype, increased ApoE transcription can contribute to AD risk. There is growing evidence in support of the hypothesis that disrupted cholesterol metabolism is an early risk factor for AD. Studies in animal models have shown that chronic changes in cholesterol metabolism associate with changes in brain Abeta accumulation, a process instrumental for establishing AD pathology. ApoE mediates cholesterol homeostasis in the body and is a major lipid carrier in brain. As such, its expression in the periphery and in brain changes in response to changes in cholesterol metabolism. Here, we used a transgenic mouse model of Alzheimer's amyloidosis to examine whether the diet-induced or pharmacologically induced changes in plasma cholesterol that result in altered brain amyloidosis also affect ApoE content in liver and in brain. We found that chronic changes in total cholesterol in plasma lead to changes in ApoE mRNA levels in brain. We also found that cholesterol loading of primary glial cells increases cellular and secreted ApoE levels and that long-term treatment of astrocytes and microglia with statins leads to a decrease in the cellular and/or secreted ApoE. These observations suggest that disrupted cholesterol metabolism may increase the risk of developing AD in part due to the effect of cholesterol on brain ApoE expression.

Rapid Neurofibrillary Tangle Formation After Localized Gene Transfer of Mutated Tau

The American Journal of Pathology. Jan, 2004  |  Pubmed ID: 14695347

Neurofibrillary pathology was produced in the brains of adult rats after localized gene transfer of human tau carrying the P301L mutation, which is associated with frontotemporal dementia with parkinsonism. Within 1 month of in situ transfection of the basal forebrain region of normal rats, tau-immunoreactive and argyrophilic neuronal lesions formed. The fibrillar lesions had features of neurofibrillary tangles and tau immunoreactivity at light and electron microscopic levels. In addition to neurofibrillary tangles, other tau pathology, including pretangles and neuropil threads, was abundant and widespread. Tau gene transfer to the hippocampal region of amyloid-depositing transgenic mice produced pretangles and threads, as well as intensely tau-immunoreactive neurites in amyloid plaques. The ability to produce neurofibrillary pathology in adult rodents makes this a useful method to study tau-related neurodegeneration.

MRI Assessment of Neuropathology in a Transgenic Mouse Model of Alzheimer's Disease

Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. Apr, 2004  |  Pubmed ID: 15065253

The cerebral deposition of amyloid beta-peptide, a central event in Alzheimer's disease (AD) pathogenesis, begins several years before the onset of clinical symptoms. Noninvasive detection of AD pathology at this initial stage would facilitate intervention and enhance treatment success. In this study, high-field MRI was used to detect changes in regional brain MR relaxation times in three types of mice: 1). transgenic mice (PS/APP) carrying both mutant genes for amyloid precursor protein (APP) and presenilin (PS), which have high levels and clear accumulation of beta-amyloid in several brain regions, starting from 10 weeks of age; 2). transgenic mice (PS) carrying only a mutant gene for presenilin (PS), which show subtly elevated levels of Abeta-peptide without beta-amyloid deposition; and 3). nontransgenic (NTg) littermates as controls. The transverse relaxation time T(2), an intrinsic MR parameter thought to reflect impaired cell physiology, was significantly reduced in the hippocampus, cingulate, and retrosplenial cortex, but not the corpus callosum, of PS-APP mice compared to NTg. No differences in T(1) values or proton density were detected between any groups of mice. These results indicate that T(2) may be a sensitive marker of abnormalities in this transgenic mouse model of AD.

Loss of Presenilin Function Causes Impairments of Memory and Synaptic Plasticity Followed by Age-dependent Neurodegeneration

Neuron. Apr, 2004  |  Pubmed ID: 15066262

Mutations in presenilins are the major cause of familial Alzheimer's disease, but the pathogenic mechanism by which presenilin mutations cause memory loss and neurodegeneration remains unclear. Here we demonstrate that conditional double knockout mice lacking both presenilins in the postnatal forebrain exhibit impairments in hippocampal memory and synaptic plasticity. These deficits are associated with specific reductions in NMDA receptor-mediated responses and synaptic levels of NMDA receptors and alphaCaMKII. Furthermore, loss of presenilins causes reduced expression of CBP and CREB/CBP target genes, such as c-fos and BDNF. With increasing age, mutant mice develop striking neurodegeneration of the cerebral cortex and worsening impairments of memory and synaptic function. Neurodegeneration is accompanied by increased levels of the Cdk5 activator p25 and hyperphosphorylated tau. These results define essential roles and molecular targets of presenilins in synaptic plasticity, learning and memory, and neuronal survival in the adult cerebral cortex.

Transgenic Mouse Models of Alzheimer's Disease: How Useful Have They Been for Therapeutic Development?

Briefings in Functional Genomics & Proteomics. Apr, 2004  |  Pubmed ID: 15163359

Transgenic mice have been created in an attempt to generate models of human Alzheimer's disease, but success has been partial and unpredictable. The overall aim of this paper is to illustrate how genomics can be used in translational research, turning genetic information in the form of pathogenic mutations into clinically useful drugs against a major human disease. This paper will illustrate how genetic information allows researchers to dissect the aetiology of a disease and then replicate the disease in vivo through the process of transgenesis. The limitations of recreating a condition like Alzheimer's disease in a transgenic mouse, how far the mice have advanced understanding of the disease and how useful they have been for the development of therapeutics will then be discussed.

Links Between the Pathology of Alzheimer's Disease and Vascular Dementia

Neurochemical Research. Jun, 2004  |  Pubmed ID: 15176482

The major neuropathological lesions defining Alzheimer's disease (AD) include neurofibrillary tangles and amyloid plaques, which are mainly composed of abnormally phosphorylated tau and amyloid-beta (A beta), respectively. Numerous neuropathological and neuroimaging studies indicate that at least one-third of AD cases are complicated by some degree of vascular pathology, whereas in a similar proportion of patients clinically diagnosed with vascular dementia, AD pathology is also present. Many classical vascular risk factors such as hypertension, diabetes mellitus, and hypercholesterolemia have recently been shown also to increase the risk of AD. Growing evidence suggests that vascular pathology lowers the threshold for the clinical presentation of dementia at a given level of AD-related pathology and potentially directly promotes AD lesions such as A beta plaques. Cerebral ischemia, chronically up-regulates expression of the amyloid precursor protein (APP), which is the precursor to the amyloid beta peptide and damages the blood-brain barrier (BBB), affecting A beta peptide clearance from the brain. Recognition of the importance of these vascular risk factors for AD-related dementia and their treatment will be beneficial not only for preventing cardiac, cerebral, and peripheral complications of vascular disease, but also will likely have a direct impact on the occurrence of sporadic AD in older subjects. In this paper, we review some of the links between vascular risk factors and AD pathology and present data on the direct effect of ischemia on cognitive function and A beta deposition in a mouse model of AD.

Development of Abeta Terminal End-specific Antibodies and Sensitive ELISA for Abeta Variant

Biochemical and Biophysical Research Communications. Jul, 2004  |  Pubmed ID: 15184044

Alzheimer's disease (AD) is a neurodegenerative affliction associated with memory dysfunction. Senile plaques are a pathological hallmark of AD, and amyloid beta (Abeta) peptides are a major component of these plaques. Abeta peptides are derived from proteolytic cleavage of the Abeta protein precursor (APP) by beta- and gamma-secretases to generate two principal species, Abeta1-40 and Abeta1-42. We have developed antibodies against the N- and C-termini of these peptides, and an ELISA for accurate and sensitive quantitative assessment. Sandwich ELISA composed of N-terminus (Abeta1) end-specific antibody, clone 82E1, and C-termini end-specific antibodies, and clones 1A10 and 1C3 for Abeta40 and Abeta42, respectively, detects full-length Abeta1-40 and 1-42 with a sensitivity in the sub single digit fmol/ml (equivalent to single digit pg/ml) range with no cross-reactivity to APP. A combination of C-termini antibodies and an antibody against the middle region of Abeta detects mouse Abeta in non-transgenic mouse brains.

Amyloid-beta Deposition is Associated with Decreased Hippocampal Glucose Metabolism and Spatial Memory Impairment in APP/PS1 Mice

Journal of Neuropathology and Experimental Neurology. May, 2004  |  Pubmed ID: 15198121

In Alzheimer disease (AD) patients, early memory dysfunction is associated with glucose hypometabolism and neuronal loss in the hippocampus. Double transgenic (Tg) mice co-expressing the M146L presenilin 1 (PS1) and K670N/M671L, the double "Swedish" amyloid precursor protein (APP) mutations, are a model of AD amyloid-beta deposition (Abeta) that exhibits earlier and more profound impairments of working memory and learning than single APP mutant mice. In this study we compared performance on spatial memory tests, regional glucose metabolism, Abeta deposition, and neuronal loss in APP/PS1, PS1, and non-Tg (nTg) mice. At the age of 2 months no significant morphological and metabolic differences were detected between 3 studied genotypes. By 8 months, however, APP/PS1 mice developed selective impairment of spatial memory, which was significantly worse at 22 months and was accompanied by reduced glucose utilization in the hippocampus and a 35.8% dropout of neurons in the CA1 region. PS1 mice exhibited a similar degree of neuronal loss in CA1 but minimal memory deficit and no impairment of glucose utilization compared to nTg mice. Deficits in 22 month APP/PS1 mice were accompanied by a substantially elevated Abeta load, which rose from 2.5% +/- 0.4% at 8 months to 17.4% +/- 4.6%. These findings implicate Abeta or APP in the behavioral and metabolic impairments in APP/PS1 mice and the failure to compensate functionally for PS1-related hippocampal cell loss.

Rat Transgenic Models with a Phenotype of Intracellular Abeta Accumulation in Hippocampus and Cortex

Journal of Alzheimer's Disease : JAD. Jun, 2004  |  Pubmed ID: 15201476

In this communication we report the characterization of several transgenic rat lines expressing human AbetaPP carrying the Swedish and Indiana mutations (coded UKUR28), the human presenilin 1 transgene with the 'Finn' mutation (coded UKUR19) and double transgenic rats expressing both transgenes (coded UKUR25). In these Tg rats, the AbetaPP and PS1 transgene expression was largely restricted to the hippocampus and neocortex. The PS1 transgenic rats did not produce visible changes in Abeta immunoreactivity. The AbetaPP transgenic rats (both the single Tg UKUR28, and double Tg UKUR25) generated a phenotype of intra-neuronalbeta accumulation without plaque formation and with no increased immunoreactivity for AbetaPP amino and carboxyl-terminal epitopes. This phenotype was apparent as early as 6 months of age in the transgenic rat lines carrying the human AbetaPP transgene. No senile plaques of aggregated Abeta were observed in any of the transgenic lines generated, up to 24 months of age. The hAbetaPP single homozygous Tg line (UKUR28) showed an increase in ERK2, without changes in glycogen synthase kinase 3 (GSK3) activity. A preliminary protein analysis of the hippocampus of the double transgenic rat (UKUR25) by mass spectrometry showed differences in the protein profile between this transgenic line and controls.

Mostly Separate Distributions of CLAC- Versus Abeta40- or Thioflavin S-reactivities in Senile Plaques Reveal Two Distinct Subpopulations of Beta-amyloid Deposits

The American Journal of Pathology. Jul, 2004  |  Pubmed ID: 15215182

Collagenous Alzheimer amyloid plaque component (CLAC) is a unique non-Abeta amyloid component of senile plaques (SP) derived from a transmembrane collagen termed CLAC-precursor. Here we characterize the chronological and spatial relationship of CLAC with other features of SP amyloid in the brains of patients with Alzheimer's disease (AD), Down syndrome (DS), and of PSAPP transgenic mice. In AD and DS cerebral cortex, CLAC invariably colocalized with Abeta42 but often lacked Abeta40- or thioflavin S (thioS)-reactivities. Immunoelectron microscopy of CLAC-positive SP showed labeling of fibrils that are more loosely dispersed compared to typical amyloid fibrils in CLAC-negative SP. In DS cerebral cortex, diffuse plaques in young patients were negative for CLAC, whereas a subset of SP became CLAC-positive in patients aged 35 to 50 years, before the appearance of Abeta40. In DS cases over 50 years of age, Abeta40-positive SP dramatically increased, whereas CLAC burden remained at a constant level. In PSAPP transgenic mice, CLAC was positive in the diffuse Abeta deposits surrounding huge-cored plaques. Thus, CLAC and Abeta40 or thioS exhibit mostly separate distribution patterns in SP, suggesting that CLAC is a relatively early component of SP in human brains that may have inhibitory effects against the maturation of SP into beta-sheet-rich amyloid deposits.

Brain on Steroids Resists Neurodegeneration

Nature Medicine. Jul, 2004  |  Pubmed ID: 15229510

Presenilin Mutations in Familial Alzheimer Disease and Transgenic Mouse Models Accelerate Neuronal Lysosomal Pathology

Journal of Neuropathology and Experimental Neurology. Aug, 2004  |  Pubmed ID: 15330337

The neuronal lysosomal system is a major degradative pathway, induced by cell stress and closely linked to Alzheimer disease (AD) and other neurodegenerative diseases. Here, we show that mutations of presenilin (PS) 1 and 2, which cause familial early-onset AD (FAD), induce more severe lysosomal system neuropathology in humans than does sporadic AD (SAD). Cathepsin D and B levels were higher in PS-FAD neocortex than in SAD and, unlike neurons in SAD, expressed higher levels of the cation-independent mannose-6-phosphate receptor. Lysosomal pathology was also evident in more populations of neurons in PS-FAD brains, including the less vulnerable neurons in laminae II and IV and affected neurons contained high numbers of hydrolase-positive vesicular compartments with a broader range of abnormal morphology. In transgenic mice expressing mutant amyloid precursor protein (APPswe), introducing mutant PSI significantly upregulated the lysosomal system in neocortical and hippocampal neurons. This upregulation, though milder in severity, resembled that seen in human PS-FAD. Accumulation of hydrolases in dystrophic neurites in senile plaques was particularly strong, suggesting that amyloid deposition may be a stimulus for local mobilization of the lysosomal system. PS1 mice lacking the APPswe transgene also had a mild lysosomal response in some neuronal populations, which was not seen in the APPswe mice. Our findings suggest that presenilin mutations have amyloid-independent effects on the lysosomal system, which are synergistic with the lysosomal system pathology that is associated with beta-amyloid.

Visualization of Beta-amyloid Plaques in a Transgenic Mouse Model of Alzheimer's Disease Using MR Microscopy Without Contrast Reagents

Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. Sep, 2004  |  Pubmed ID: 15334572

The visualization of beta-amyloid plaque deposition in brain, a key feature of Alzheimer's disease (AD), is important for the evaluation of disease progression and the efficacy of therapeutic interventions. In this study, beta-amyloid plaques in the PS/APP transgenic mouse brain, a model of human AD pathology, were detected using MR microscopy without contrast reagents. beta-Amyloid plaques were clearly visible in the cortex, thalamus, and hippocampus of fixed brains of PS/APP mice. The distribution of plaques identified by MRI was in excellent agreement with those found in the immunohistological analysis of the same brain sections. It was also demonstrated that image contrast for beta-amyloid plaques was present in freshly excised nonfixed brains. Furthermore, the detection of beta-amyloid plaques was achieved with a scan time as short as 2 hr, approaching the scan time considered reasonable for in vivo imaging.

Fibrillar Amyloid Deposition Leads to Local Synaptic Abnormalities and Breakage of Neuronal Branches

Nature Neuroscience. Nov, 2004  |  Pubmed ID: 15475950

Amyloid plaques are a hallmark of Alzheimer disease, but their importance in its pathogenesis is controversial. By neuronal labeling and transcranial two-photon imaging, we show in a transgenic mouse model of Alzheimer disease that dendrites passing through or near fibrillar amyloid deposits undergo spine loss and shaft atrophy, and nearby axons develop large varicosities, together leading to neurite breakage and large-scale, permanent disruption of neuronal connections. Thus, fibrillar amyloid deposition is more detrimental to neuronal circuitry than previously thought, underscoring the importance of prevention and early clearance of plaques.

Inhibition of Glycogen Synthase Kinase-3 by Lithium Correlates with Reduced Tauopathy and Degeneration in Vivo

Proceedings of the National Academy of Sciences of the United States of America. May, 2005  |  Pubmed ID: 15867159

Neurofibrillary tangles composed of hyperphosphorylated, aggregated tau are a common pathological feature of tauopathies, including Alzheimer's disease. Abnormal phosphorylation of tau by kinases or phosphatases has been proposed as a pathogenic mechanism in tangle formation. To investigate whether kinase inhibition can reduce tauopathy and the degeneration associated with it in vivo, transgenic mice overexpressing mutant human tau were treated with the glycogen synthase kinase-3 (GSK-3) inhibitor lithium chloride. Treatment resulted in significant inhibition of GSK-3 activity. Lithium administration also resulted in significantly lower levels of phosphorylation at several epitopes of tau known to be hyperphosphorylated in Alzheimer's disease and significantly reduced levels of aggregated, insoluble tau. Administration of a second GSK-3 inhibitor also correlated with reduced insoluble tau levels, supporting the idea that lithium exerts its effect through GSK-3 inhibition. Levels of aggregated tau correlated strongly with degree of axonal degeneration, and lithium-chloride-treated mice showed less degeneration if administration was started during early stages of tangle development. These results support the idea that kinases are involved in tauopathy progression and that kinase inhibitors may be effective therapeutically.

Histological Co-localization of Iron in Abeta Plaques of PS/APP Transgenic Mice

Neurochemical Research. Feb, 2005  |  Pubmed ID: 15895823

This study confirms the presence of iron, co-localized with Abeta plaques, in PS/APP mouse brain, using Perls' stain for Fe3+ supplemented by 3,3'-diaminobenzidine (DAB) and Abeta immunohistochemistry in histological brains sections fixed with formalin or methacarn. In this study, the fixation process and the slice thickness did not interfere with the Perls' technique. The presence of iron in beta-amyloid plaques in PS/APP transgenic mice, a model of Alzheimer's disease (AD) pathology, may explain previous reports of reductions of transverse relaxation time (T2) in MRI studies and represent the source of the intrinsic Abeta plaque MR contrast in this model.

Cell-cycle Reentry and Cell Death in Transgenic Mice Expressing Nonmutant Human Tau Isoforms

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2005  |  Pubmed ID: 15930395

Mutations in the microtubule-associated protein tau gene have been linked to neurofibrillary tangle (NFT) formation in several neurodegenerative diseases known as tauopathies; however, no tau mutations occur in Alzheimer's disease, although this disease is also characterized by NFT formation and cell death. Importantly, the mechanism of tau-mediated neuronal death remains elusive. Aged mice expressing nonmutant human tau in the absence of mouse tau (htau mice) developed NFTs and extensive cell death. The mechanism of neuron death was investigated in htau mice, and surprisingly, the presence of tau filaments did not correlate directly with death within individual cells, suggesting that cell death can occur independently of NFT formation. Our observations show that the mechanism of neurodegeneration involved reexpression of cell-cycle proteins and DNA synthesis, indicating that nonmutant tau pathology and neurodegeneration may be linked via abnormal, incomplete cell-cycle reentry.

An Abeta Sequestration Approach Using Non-antibody Abeta Binding Agents

Current Alzheimer Research. Apr, 2005  |  Pubmed ID: 15974928

Amyloid beta (Abeta) has been considered as a primary cause of Alzheimer's disease (AD), and Abeta lowering approaches have been tested. Active immunization against Abeta is one of several promising Abeta-lowering approaches. Two mechanisms have been proposed: enhancement of microglial phagocytosis and Abeta sequestration (also called "peripheral sink"). We hypothesized that Abeta sequestration without immune modulation is sufficient to reduce the brain Abeta load and have demonstrated effective sequestration with Abeta binding agents that do not stimulate an immune reaction. Recent reports from other groups showed two other non-immune related Abeta binding agents, which have no structural relation to compounds we previously tested, reduced brain Abeta after peripheral administration. Congo red is a chemically synthesized small molecule that has binding affinity to Abeta. In the present study, we tested three Congo red derivatives in Abeta plaque-forming mice at an early pathological stage. Unfortunately, peripheral administration for three weeks did not substantially alter brain Abeta load. Optimized Abeta binding agents with high affinity to soluble Abeta are necessary for the sequestration approach.

Dense-core Plaques in Tg2576 and PSAPP Mouse Models of Alzheimer's Disease Are Centered on Vessel Walls

The American Journal of Pathology. Aug, 2005  |  Pubmed ID: 16049337

Occurrence of amyloid beta (Abeta) dense-core plaques in the brain is one of the chief hallmarks of Alzheimer's disease (AD). It is not yet clear what factors are responsible for the aggregation of Abeta in the formation of these plaques. Using Tg2576 and PSAPP mouse models that exhibit age-related development of amyloid plaques similar to that observed in AD, we showed that approximately 95% of dense plaques in Tg2576 and approximately 85% in PSAPP mice are centered on vessel walls or in the immediate perivascular regions. Stereoscopy and simulation studies focusing on smaller plaques suggested that vascular associations for both Tg2576 and PSAPP mice were dramatically higher than those encountered by chance alone. We further identified ultrastructural microvascular abnormalities occurring in association with dense plaques. Although occurrence of gross cerebral hemorrhage was infrequent, we identified considerable infiltration of the serum proteins immunoglobulin and albumin in association with dense plaques. Together with earlier evidence of vascular clearance of Abeta, our data suggest that perturbed vascular transport and/or perivascular enrichment of Abeta leads to the formation of vasocentric dense plaques in Tg2576 and PSAPP mouse models of AD.

Untangling Memory Deficits

Nature Medicine. Aug, 2005  |  Pubmed ID: 16079873

Macroautophagy--a Novel Beta-amyloid Peptide-generating Pathway Activated in Alzheimer's Disease

The Journal of Cell Biology. Oct, 2005  |  Pubmed ID: 16203860

Macroautophagy, which is a lysosomal pathway for the turnover of organelles and long-lived proteins, is a key determinant of cell survival and longevity. In this study, we show that neuronal macroautophagy is induced early in Alzheimer's disease (AD) and before beta-amyloid (Abeta) deposits extracellularly in the presenilin (PS) 1/Abeta precursor protein (APP) mouse model of beta-amyloidosis. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of intracellular Abeta. Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity. Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production. Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.

Using Proteomics and Network Analysis to Elucidate the Consequences of Synaptic Protein Oxidation in a PS1 + AbetaPP Mouse Model of Alzheimer's Disease

Journal of Alzheimer's Disease : JAD. Dec, 2005  |  Pubmed ID: 16340081

Increasing evidence suggests that oxidative injury is involved in the pathogenesis of many age-related neurodegenerative disorders, including Alzheimer's disease (AD). Identifying the protein targets of oxidative stress is critical to determine which proteins may be responsible for the neuronal impairments and subsequent cell death that occurs in AD. In this study, we have applied a high-throughput shotgun proteomic approach to identify the targets of protein carbonylation in both aged and PS1 + AbetaPP transgenic mice. However, because of the inherent difficulties associated with proteomic database searching algorithms, several newly developed bioinformatic tools were implemented to ascertain a probability-based discernment between correct protein assignments and false identifications to improve the accuracy of protein identification. Assigning a probability to each identified peptide/protein allows one to objectively monitor the expression and relative abundance of particular proteins from diverse samples, including tissue from transgenic mice of mixed genetic backgrounds. This robust bioinformatic approach also permits the comparison of proteomic data generated by different laboratories since it is instrument- and database-independent. Applying these statistical models to our initial studies, we detected a total of 117 oxidatively modified (carbonylated) proteins, 59 of which were specifically associated with PS1 + AbetaPP mice. Pathways and network component analyses suggest that there are three major protein networks that could be potentially altered in PS1 + AbetaPP mice as a result of oxidative modifications. These pathways are 1) iNOS-integrin signaling pathway, 2) CRE/CBP transcription regulation and 3) rab-lyst vesicular trafficking. We believe the results of these studies will help establish an initial AD database of oxidatively modified proteins and provide a foundation for the design of future hypothesis driven research in the areas of aging and neurodegeneration.

The Effects of ABCA1 on Cholesterol Efflux and Abeta Levels in Vitro and in Vivo

Journal of Neurochemistry. Aug, 2006  |  Pubmed ID: 16771834

ABCA1 promotes cholesterol efflux from cells and is required for maintaining plasma cholesterol levels. Cholesterol homeostasis is important in the production of beta-amyloid (Abeta), a peptide that is overproduced in Alzheimer's disease (AD). Overexpression of ABCA1 can be achieved by stimulating Liver X Receptors (LXR), and changes in Abeta have been reported after LXR stimulation in vitro. To determine whether ABCA1 could alter endogenous Abeta levels, we used two different in vivo systems. We first examined the effects of an LXR agonist (TO-901317) on wild-type mice and found an increase in brain ABCA1 and apoE levels, which caused an increase in plasma cholesterol. This was accompanied by a decrease in brain Abeta levels. We then examined endogenous Abeta levels in ABCA1 knockout mice and found that, despite having no ABCA1, lowered brain apoE levels, and lowered plasma cholesterol, there was no change in Abeta levels. To assess these in vivo models in an in vitro system, we designed a model in which cholesterol transport via ABCA1 (or related transporters) was prevented. Switching off cholesterol efflux, even in the presence of TO-901317, caused no change in Abeta levels. However, when efflux capability was restored, TO-901317 reduced Abeta levels. These data show that promoting cholesterol efflux is a viable target for Abeta reducing strategies; however, knockout of cholesterol transporters is not sufficient to alter Abeta in vitro or in vivo.

Cholesterol Distribution, Not Total Levels, Correlate with Altered Amyloid Precursor Protein Processing in Statin-treated Mice

Neuromolecular Medicine. 2006  |  Pubmed ID: 16775383

There are now a number of studies that suggest that cholesterol might regulate the processing of the amyloid precursor protein to form the neurotoxic peptide Abeta. This research has opened the possibility that cholesterol-lowering drugs might be efficacious as anti-Abeta drugs for use in Alzheimer's disease. The use of HMG-CoA reductase inhibitors (commonly called statins) in vitro and in vivo has proven them to be Abeta-lowering agents, however, the mechanism of action of these drugs is not yet known. One possible mechanism is that they reduce Abeta levels indirectly by reducing cholesterol in the central nervous system (CNS). In this study, we administered three different statins (simvastatin, lovastatin, and atorvastatin) to nontransgenic mice. We found that all three compounds had similar effects on Abeta, reducing both Abeta40 and Abeta42. The statins decreased beta-cleaved C-terminal fragment (CTF) although having no effect on alpha-CTF levels. However, the drugs did not have a similar effect on cholesterol in the CNS. Only lovastatin significantly reduced total cholesterol in isolated plasma membranes. As cholesterol is not distributed evenly in the plasma membrane, we examined bilayer distribution of cholesterol and found that all three statins caused CNS cholesterol to translocate from the cytofacial leaflet to the exofacial leaflet. This data suggests that cholesterol distribution and not total cholesterol levels may be important to Abeta production in the CNS.

Normal and Abnormal Tau Neurobiology

Alzheimer Disease and Associated Disorders. Oct-Dec, 2006  |  Pubmed ID: 17132962

The Amyloid Pathology Progresses in a Neurotransmitter-specific Manner

Neurobiology of Aging. Nov, 2006  |  Pubmed ID: 16271419

Past studies using transgenic models of early-staged amyloid pathology, have suggested that the amyloid pathology progresses in a neurotransmitter-specific manner where cholinergic terminals appear most vulnerable, followed by glutamatergic terminals and finally by somewhat more resistant GABAergic terminals. To determine whether this neurotransmitter-specific progression persists at later pathological stages, presynaptic bouton densities, and the areas of occupation and localization of plaque adjacent dystrophic neurites were quantified in 18-month-old APP(K670N, M671L)+PS1(M146L) doubly transgenic mice. Quantification revealed that transgenic animals had significantly lower cholinergic, glutamatergic and GABAergic presynaptic bouton densities. Cholinergic and glutamatergic dystrophic neurites appear to be heavily influenced by fibrillar Abeta as both types displayed a decreasing area of occupation with respect to increasing plaque size. Furthermore, cholinergic dystrophic neurites reside in closer proximity to plaques than glutamatergic dystrophic neurites, while GABAergic dystrophic neurites were minimal regardless of plaque size. To investigate whether similarities exist in the human AD pathology, a monoclonal antibody (McKA1) against the human vesicular glutamate transporter 1 (VGluT1) was developed. Subsequent staining in AD brain tissue revealed the novel presence of glutamatergic dystrophic neurites, to our knowledge the first evidence of a structural glutamatergic deficit in the AD pathology.

Insulin Dysfunction Induces in Vivo Tau Hyperphosphorylation Through Distinct Mechanisms

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Dec, 2007  |  Pubmed ID: 18077675

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease (AD) brains, and tau hyperphosphorylation is thought to be a critical event in the pathogenesis of the disease. The large majority of AD cases is late onset and sporadic in origin, with aging as the most important risk factor. Insulin resistance, impaired glucose tolerance, and diabetes mellitus (DM) are other common syndromes in the elderly also strongly age dependent, and there is evidence supporting a link between insulin dysfunction and AD. To investigate the possibility that insulin dysfunction might promote tau pathology, we induced insulin deficiency and caused DM in mice with streptozotocin (STZ). A mild hyperphosphorylation of tau could be detected 10, 20, and 30 d after STZ injection, and a massive hyperphosphorylation of tau was observed after 40 d. The robust hyperphosphorylation of tau was localized in the axons and neuropil, and prevented tau binding to microtubules. Neither mild nor massive tau phosphorylation induced tau aggregation. Body temperature of the STZ-treated mice did not differ from control animals during 30 d, but dropped significantly thereafter. No change in beta-amyloid (Abeta) precursor protein (APP), APP C-terminal fragments, or Abeta levels were observed in STZ-treated mice; however, cellular protein phosphatase 2A activity was significantly decreased. Together, these data indicate that insulin dysfunction induced abnormal tau hyperphosphorylation through two distinct mechanisms: one was consequent to hypothermia; the other was temperature-independent, inherent to insulin depletion, and probably caused by inhibition of phosphatase activity.

Antibody Against C-terminal Abeta Selectively Elevates Plasma Abeta

Neuroreport. Feb, 2007  |  Pubmed ID: 17314674

Accumulation of amyloid beta in the brain is a pathological hallmark of Alzheimer's disease, and the reduction of amyloid beta has been proposed as a primary therapeutic target. Mice immunized against amyloid beta and mice infused with anti-amyloid beta antibody (active and passive immunization, respectively) have reduced brain amyloid beta levels, and two mechanisms have been proposed: microglial phagocytosis in the brain and enhancement of amyloid beta efflux by antibodies present in the periphery (sequestration). The optimal antibody for microglial phagocytosis has been shown to be N-terminal-specific antibody; however, the potency of C-terminal-specific antibody in sequestration remains unclear. In this study, we found that anti-amyloid beta 40-specific antibody induces amyloid beta sequestration. These results indicate that C-terminal antibodies may be useful in amyloid beta sequestration therapy.

Anesthesia Leads to Tau Hyperphosphorylation Through Inhibition of Phosphatase Activity by Hypothermia

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Mar, 2007  |  Pubmed ID: 17376970

Postoperative cognitive dysfunction, confusion, and delirium are common after general anesthesia in the elderly, with symptoms persisting for months or years in some patients. Even middle-aged patients are likely to have postoperative cognitive dysfunction for months after surgery, and Alzheimer's disease (AD) patients appear to be particularly at risk of deterioration after anesthesia. Several investigators have thus examined whether general anesthesia is associated with AD, with some studies suggesting that exposure to anesthetics may increase the risk of AD. However, little is known on the biochemical consequences of anesthesia on pathogenic pathways in vivo. Here, we investigated the effect of anesthesia on tau phosphorylation and amyloid precursor protein (APP) metabolism in mouse brain. We found that, regardless of the anesthetic used, anesthesia induced rapid and massive hyperphosphorylation of tau, rapid and prolonged hypothermia, inhibition of Ser/Thr PP2A (protein phosphatase 2A), but no changes in APP metabolism or Abeta (beta-amyloid peptide) accumulation. Reestablishing normothermia during anesthesia completely rescued tau phosphorylation to normal levels. Our results indicate that changes in tau phosphorylation were not a result of anesthesia per se, but a consequence of anesthesia-induced hypothermia, which led to inhibition of phosphatase activity and subsequent hyperphosphorylation of tau. These findings call for careful monitoring of core temperature during anesthesia in laboratory animals to avoid artifactual elevation of protein phosphorylation. Furthermore, a thorough examination of the effect of anesthesia-induced hypothermia on the risk and progression of AD is warranted.

Collapsin Response Mediator Protein-2 Hyperphosphorylation is an Early Event in Alzheimer's Disease Progression

Journal of Neurochemistry. Nov, 2007  |  Pubmed ID: 17683481

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that can regulate microtubule assembly in neurons. This function of CRMP2 is regulated by phosphorylation by glycogen synthase kinase 3 (GSK3) and cyclin-dependent kinase 5 (Cdk5). Here, using novel phosphospecific antibodies, we demonstrate that phosphorylation of CRMP2 at Ser522 (Cdk5-mediated) is increased in Alzheimer's disease (AD) brain, while CRMP2 expression and phosphorylation of the closely related isoform CRMP4 are not altered. In addition, CRMP2 phosphorylation at the Cdk5 and GSK3 sites is increased in cortex and hippocampus of the triple transgenic mouse [presenilin-1 (PS1)(M146V)KI; Thy1.2-amyloid precursor protein (APP)(swe); Thy1.2tau(P301L)] that develops AD-like plaques and tangles, as well as the double (PS1(M146V)KI; Thy1.2-APP(swe)) transgenic mouse. The hyperphosphorylation is similar in magnitude to that in human AD and is evident by 2 months of age, ahead of plaque or tangle formation. Meanwhile, there is no change in CRMP2 phosphorylation in two other transgenic mouse lines that display elevated amyloid beta peptide levels (Tg2576 and APP/amyloid beta-binding alcohol dehydrogenase). Similarly, CRMP2 phosphorylation is normal in hippocampus and cortex of Tau(P301L) mice that develop tangles but not plaques. These observations implicate hyperphosphorylation of CRMP2 as an early event in the development of AD and suggest that it can be induced by a severe APP over-expression and/or processing defect.

Increased Dopaminergic Neuron Sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in Transgenic Mice Expressing Mutant A53T Alpha-synuclein

Neurochemical Research. May, 2008  |  Pubmed ID: 17999181

Familial Parkinson's disease (PD) has been linked to point mutations and duplication of the alpha-synuclein gene and mutant alpha-synuclein expression increases the vulnerability of neurons to exogenous insults. In this study, we analyzed the levels of dopamine and its metabolites in the olfactory bulb (OB), and nigrostriatal regions of transgenic mice expressing human, mutant A53T alpha-synuclein (alpha-syn tg) and their non-transgenic (ntg) littermates using a sub-toxic, moderate dose of MPTP to determine if mutant human alpha-synuclein sensitizes the central dopaminergic systems to oxidative stress. We observed that after a single, sub-lethal MPTP injection, dopamine levels were reduced in striatum and SN in both the alpha-syn tg and ntg mice. In the olfactory bulb, a region usually resistant to MPTP toxicity, levels were reduced only in the alpha-syn tg mice. In addition, we identified a significant increase in dopamine metabolism in the alpha-syn transgenic, but not ntg mice. Finally, MPTP treatment of alpha-syn tg mice was associated with a marked elevation in the oxidative product, 3-nitrotyrosine that co-migrated with alpha-synuclein. Cumulatively, the data support the hypothesis that mutant alpha-synuclein sensitizes dopaminergic neurons to neurotoxic insults and is associated with greater oxidative stress. The alpha-syn tg line is therefore useful to study the genetic and environmental inter-relationship in PD.

Is Tau Aggregation Toxic or Protective?

Journal of Alzheimer's Disease : JAD. Aug, 2008  |  Pubmed ID: 18688098

Abnormal protein deposits are a common feature of many human diseases including Alzheimer's disease. In Alzheimer's disease, the appearance of tangles, composed of the microtubule associated protein tau, correlates with both cell death and symptom severity. However, are tau filaments simply markers of disease progression, or are they directly responsible for cell death? Due to conflicting findings from cell and animal models, it remains controversial whether tau polymers or smaller pre-fibrillar aggregates or tau monomers are the toxic species. Indeed, if monomeric or oligomeric species are mediators of disease, formation of larger tau filaments may prove beneficial to affected cells. This review will examine the findings regarding the toxicity of various tau species.

Anesthesia-induced Hyperphosphorylation Detaches 3-repeat Tau from Microtubules Without Affecting Their Stability in Vivo

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2008  |  Pubmed ID: 19036972

In Alzheimer's disease, tau is hyperphosphorylated, which is thought to detach it from microtubules (MTs), induce MT destabilization, and promote aggregation. Using a previously described in vivo model, we investigated whether hyperphosphorylation impacts tau function in wild-type and transgenic mice. We found that after anesthesia-induced hypothermia, MT-free tau was hyperphosphorylated, which impaired its ability to bind MTs and promote MT assembly. MT-bound tau was more resistant to hyperphosphorylation compared with free tau and tau did not dissociate from MTs in wild-type mice. However, 3-repeat tau detached from MT in the transgenic mice. Surprisingly, dissociation of tau from MTs did not lead to overt depolymerization of tubulin, and there was no collapse, or disturbance of axonal MT networks. These results indicate that, in vivo, a subpopulation of tau bound to MTs does not easily dissociate under conditions that extensively phosphorylate tau. Tau remaining on the MTs under these conditions is sufficient to maintain MT network integrity.

Axonal Transport Rates in Vivo Are Unaffected by Tau Deletion or Overexpression in Mice

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Feb, 2008  |  Pubmed ID: 18272688

Elevated tau expression has been proposed as a possible basis for impaired axonal transport in Alzheimer's disease. To address this hypothesis, we analyzed the movement of pulse radiolabeled proteins in vivo along retinal ganglion cell (RGC) axons of mice that lack tau or overexpress human tau isoforms. Here, we show that the global axonal transport rates of slow and fast transport cargoes in axons are not significantly impaired when tau expression is eliminated or increased. In addition, markers of slow transport (neurofilament light subunit) and fast transport (snap25) do not accumulate in retinas and are distributed normally along optic axons in mice that lack or overexpress tau. Finally, ultrastructural analyses revealed no abnormal accumulations of vesicular organelles or neurofilaments in RGC perikarya or axons in mice overexpressing or lacking tau. These results suggest that tau is not essential for axonal transport and that transport rates in vivo are not significantly affected by substantial fluctuations in tau expression.

Interplay Between Cyclin-dependent Kinase 5 and Glycogen Synthase Kinase 3 Beta Mediated by Neuregulin Signaling Leads to Differential Effects on Tau Phosphorylation and Amyloid Precursor Protein Processing

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Mar, 2008  |  Pubmed ID: 18322105

Cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase 3beta (GSK3beta) have been implicated in pathogenic processes associated with Alzheimer's disease because both kinases regulate tau hyperphosphorylation and enhance amyloid precursor protein (APP) processing leading to an increase in amyloid beta (Abeta) production. Here we show that young p25 overexpressing mice have enhanced cdk5 activity but reduced GSK3beta activity attributable to phosphorylation at the inhibitory GSK3beta-serine 9 (GSK3beta-S9) site. Phosphorylation at this site was mediated by enhanced activity of the neuregulin receptor complex, ErbB, and activation of the downstream phosphatidylinositol 3 kinase/Akt pathway. Young p25 mice had elevated Abeta peptide levels, but phospho-tau levels were decreased overall. Thus, cdk5 appears to play a dominant role in the regulation of amyloidogenic APP processing, whereas GSK3beta plays a dominant role in overall tau phosphorylation. In older mice, GSK3beta inhibitory phosphorylation at S9 was reduced relative to young mice. Abeta peptide levels were still elevated but phospho-tau levels were either unchanged or showed a trend to increase, suggesting that GSK3beta activity increases with aging. Inhibition of cdk5 by a specific inhibitor reduced cdk5 activity in p25 mice, leading to reduced Abeta production in both young and old mice. However, in young mice, cdk5 inhibition reversed GSK3beta inhibition, leading to an increase in overall tau phosphorylation. When cdk5 inhibitor was administered to very old, nontransgenic mice, inhibition of cdk5 reduced Abeta levels, and phospho-tau levels showed a trend to increase. Thus, cdk5 inhibitors may not be effective in targeting tau phosphorylation in the elderly.

Transcriptional Regulation of Beta-secretase by P25/cdk5 Leads to Enhanced Amyloidogenic Processing

Neuron. Mar, 2008  |  Pubmed ID: 18341989

Cyclin-dependent kinase 5 (cdk5) has been implicated in Alzheimer's disease (AD) pathogenesis. Here, we demonstrate that overexpression of p25, an activator of cdk5, led to increased levels of BACE1 mRNA and protein in vitro and in vivo. A p25/cdk5 responsive region containing multiple sites for signal transducer and activator of transcription (STAT1/3) was identified in the BACE1 promoter. STAT3 interacts with the BACE1 promoter, and p25-overexpressing mice had elevated levels of pSTAT3 and BACE1, whereas cdk5-deficient mice had reduced levels. Furthermore, mice with a targeted mutation in the STAT3 cdk5 responsive site had lower levels of BACE1. Increased BACE levels in p25 overexpressing mice correlated with enhanced amyloidogenic processing that could be reversed by a cdk5 inhibitor. These data demonstrate a pathway by which p25/cdk5 increases the amyloidogenic processing of APP through STAT3-mediated transcriptional control of BACE1 that could have implications for AD pathogenesis.

Retromer Deficiency Observed in Alzheimer's Disease Causes Hippocampal Dysfunction, Neurodegeneration, and Abeta Accumulation

Proceedings of the National Academy of Sciences of the United States of America. May, 2008  |  Pubmed ID: 18480253

Although deficiencies in the retromer sorting pathway have been linked to late-onset Alzheimer's disease, whether these deficiencies underlie the disease remains unknown. Here we characterized two genetically modified animal models to test separate but related questions about the effects that retromer deficiency has on the brain. First, testing for cognitive defects, we investigated retromer-deficient mice and found that they develop hippocampal-dependent memory and synaptic dysfunction, which was associated with elevations in endogenous Abeta peptide. Second, testing for neurodegeneration and amyloid deposits, we investigated retromer-deficient flies expressing human wild-type amyloid precursor protein (APP) and human beta-site APP-cleaving enzyme (BACE) and found that they develop neuronal loss and human Abeta aggregates. By recapitulating features of the disease, these animal models suggest that retromer deficiency observed in late-onset Alzheimer's disease can contribute to disease pathogenesis.

Females Exhibit More Extensive Amyloid, but Not Tau, Pathology in an Alzheimer Transgenic Model

Brain Research. Jun, 2008  |  Pubmed ID: 18486110

Epidemiological studies indicate that women have a higher risk of Alzheimer's disease (AD) even after adjustment for age. Though transgenic mouse models of AD develop AD-related amyloid beta (Abeta) and/or tau pathology, gender differences have not been well documented in these models. In this study, we found that female 3xTg-AD transgenic mice expressing mutant APP, presenilin-1 and tau have significantly more aggressive Abeta pathology. We also found an increase in beta-secretase activity and a reduction of neprilysin in female mice compared to males; this suggests that a combination of increased Abeta production and decreased Abeta degradation may contribute to higher risk of AD in females. In contrast to significantly more aggressive Abeta pathology in females, gender did not affect the levels of phosphorylated tau in 3xTg-AD mice. These results point to the involvement of Abeta pathways in the higher risk of AD in women. In addition to comparison of pathology between genders at 9, 16 and 23 months of age, we examined the progression of Abeta pathology at additional age points; i.e., brain Abeta load, intraneuronal oligomeric Abeta distribution and plaque load, in male 3xTg-AD mice at 3, 6, 9, 12, 16, 20 and 23 months of age. These findings confirm progressive Abeta pathology in 3xTg-AD transgenic mice, and provide guidance for their use in therapeutic research.

A Transgenic Rat That Develops Alzheimer's Disease-like Amyloid Pathology, Deficits in Synaptic Plasticity and Cognitive Impairment

Neurobiology of Disease. Jul, 2008  |  Pubmed ID: 18504134

In the last decade, multiple lines of transgenic APP overexpressing mice have been created that recapitulate certain aspects of Alzheimer's disease (AD). However, none of the previously reported transgenic APP overexpressing rat models developed AD-like beta-amyloid (Abeta) deposits, or age-related learning and memory deficits. In the present study, we have characterized a transgenic rat model overexpressing transgenes with three, familial AD mutations (two in APP and one in PS1) that were developed by Flood et al. [Flood, D.G., et al., Abeta deposition in a transgenic rat model of Alzheimer's disease. Society for Neuroscience 2003, Washington, DC, 2003]. From the age of 9 months, these rats develop Abeta deposits in both diffuse and compact forms, with the latter being closely associated with activated microglia and reactive astrocytes. Impaired long-term potentiation (LTP) was revealed by electrophysiological recordings performed on hippocampal slices from rats at 7 months of age, which is 2 months before the appearance of amyloid plaques. The deficit in LTP was accompanied by impaired spatial learning and memory in the Morris water maze, which became more pronounced in transgenic rats of 13 months of age. For Tg rats of both ages, there was a trend for cognitive impairment to correlate with total Abeta42 levels in the hippocampus. The rat model therefore recapitulates AD-like amyloid pathology and cognitive impairment. The advantage of the rat model over the available mouse models is that rats provide better opportunities for advanced studies, such as serial CSF sampling, electrophysiology, neuroimaging, cell-based transplant manipulations, and complex behavioral testing.

New Directions for Frontotemporal Dementia Drug Discovery

Alzheimer's & Dementia : the Journal of the Alzheimer's Association. Mar, 2008  |  Pubmed ID: 18631953

This report summarizes the recommendations of the Frontotemporal Dementia (FTD) Working Group on FTD Drug Discovery that was part of an international FTD Workshop held on January 18 and 19, 2007, in Miami, Florida. The workshop was sponsored by the National Institute of Neurological Disorders and Stroke (NINDS) and the National Institute on Aging (NIA) of the National Institutes of Health (NIH) and the Association for Frontotemporal Dementia (AFTD) with the express purpose of defining opportunities to improve the diagnosis and treatment of patients affected by a neurodegenerative disorder classified as one of the many variants of FTD. The recognition that almost all forms of FTD are due to TDP-43 proteinopathies and tauopathies creates new opportunities for FTD drug discovery targeting pathways of TDP-43 and tau-mediated neurodegeneration.

Linking Abeta and Tau in Late-onset Alzheimer's Disease: a Dual Pathway Hypothesis

Neuron. Nov, 2008  |  Pubmed ID: 19038212

Alzheimer's disease is characterized by abnormal elevation of Abeta peptide and abnormal hyperphosphorylation of the tau protein. The "amyloid hypothesis," which is based on molecular defects observed in autosomal-dominant early-onset Alzheimer's disease (EOAD), suggests a serial model of causality, whereby elevation of Abeta drives other disease features including tau hyperphosphorylation. Here, we review recent evidence from drug trials, genetic studies, and experimental work in animal models that suggests that an alternative model might exist in late-onset AD (LOAD), the complex and more common form of the disease. Specifically, we hypothesize a "dual pathway" model of causality, whereby Abeta and tau can be linked by separate mechanisms driven by a common upstream driver. This model may account for the results of recent drug trials and, if confirmed, may guide future drug development.

Noninvasive and Transient Blood-brain Barrier Opening in the Hippocampus of Alzheimer's Double Transgenic Mice Using Focused Ultrasound

Ultrasonic Imaging. Jul, 2008  |  Pubmed ID: 19149463

The spatio-temporal nature of focused ultrasound-induced blood-brain barrier (BBB) opening as a brain drug delivery method was investigated in Alzheimer's disease model mice. The left hippocampus of transgenic (APP/PS1, n = 3) and nontransgenic (n = 3) mice was sonicated (frequency: 1.525 MHz, peak-negative pressure: 600 kPa, pulse length: 20 ms, duty cycle: 20%, duration: 1 min) in vivo, through their intact skin and skull, after intravenous injection of microbubbles (SonoVue; 25 microl). Sequential, high-field MR images (9.4 Tesla) were acquired before and after injection of gadolinium (Omniscan, 0.75 ml, molecular weight: 573.7 Da) on two separate days for each mouse. Gadolinium deposits through the ultrasound-induced BBB opening in the left hippocampus revealed significant contrast-enhancement in the MRI. On the following day, MRI revealed significant BBB closure within the same region. However, the BBB opening extent and BBB closing timeline varied in different regions within the same sonicated location. This indicates that opening and closing were dependent on the brain region targeted. No significant difference in BBB opening or closing behaviors was observed between the APP/PS1 and the nontransgenic mice. In conclusion, a BBB-impermeable molecule was noninvasively, transiently and reproducibly delivered to the hippocampus of Alzheimer's APP/PS1 mice.

Optical Visualization of Alzheimer's Pathology Via Multiphoton-excited Intrinsic Fluorescence and Second Harmonic Generation

Optics Express. Mar, 2009  |  Pubmed ID: 19259208

Intrinsic optical emissions, such as autofluorescence and second harmonic generation (SHG), are potentially useful for functional fluorescence imaging and biomedical disease diagnosis for neurodegenerative diseases such as Alzheimer's disease (AD). Here, using multiphoton and SHG microscopy, we identified sources of intrinsic emissions in ex vivo, acute brain slices from AD transgenic mouse models. We observed autofluorescence and SHG at senile plaques as well as characterized their emission spectra. The utility of intrinsic emissions was demonstrated by imaging senile plaque autofluorescence in conjunction with SHG from microtubule arrays to assess the polarity of microtubules near pathological lesions. Our results suggest that tissues from AD transgenic models contain distinct intrinsic emissions, which can provide valuable information about the disease mechanisms.

Acceleration and Persistence of Neurofibrillary Pathology in a Mouse Model of Tauopathy Following Anesthesia

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Aug, 2009  |  Pubmed ID: 19279139

Alzheimer's disease and other tauopathies are characterized by the presence of intracellular neurofibrillary tangles composed of hyperphosphorylated, insoluble tau. General anesthesia has been shown to be associated with increased risk of Alzheimer's disease, and we have previously demonstrated that anesthesia induces hypothermia, which leads to overt tau hyperphosphorylation in the brain of mice regardless of the anesthetic used. To investigate whether anesthesia enhances the long-term risk of developing pathological forms of tau, we exposed a mouse model with tauopathy to anesthesia and monitored the outcome at two time points-during anesthesia, or 1 wk after exposure. We found that exposure to isoflurane at clinically relevant doses led to increased levels of phospho-tau, increased insoluble, aggregated forms of tau, and detachment of tau from microtubules. Furthermore, levels of phospho-tau distributed in the neuropil, as well as in cell bodies increased. Interestingly, the level of insoluble tau was increased 1 wk following anesthesia, suggesting that anesthesia precipitates changes in the brain that provoke the later development of tauopathy. Overall, our results suggest that anesthesia-induced hypothermia could lead to an acceleration of tau pathology in vivo that could have significant clinical implications for patients with early stage, or overt neurofibrillary tangle pathology.

Structure-activity Relationship of Cyanine Tau Aggregation Inhibitors

Journal of Medicinal Chemistry. Jun, 2009  |  Pubmed ID: 19432420

A structure-activity relationship for symmetrical cyanine inhibitors of human tau aggregation was elaborated using a filter trap assay. Antagonist activity depended on cyanine heterocycle, polymethine bridge length, and the nature of meso- and N-substituents. One potent member of the series, 3,3'-diethyl-9-methylthiacarbocyanine iodide (compound 11), retained submicromolar potency and had calculated physical properties consistent with blood-brain barrier and cell membrane penetration. Exposure of organotypic slices prepared from JNPL3 transgenic mice (which express human tau harboring the aggregation prone P301L tauopathy mutation) to compound 11 for one week revealed a biphasic dose response relationship. Low nanomolar concentrations decreased insoluble tau aggregates to half those observed in slices treated with vehicle alone. In contrast, high concentrations (> or =300 nM) augmented tau aggregation and produced abnormalities in tissue tubulin levels. These data suggest that certain symmetrical carbocyanine dyes can modulate tau aggregation in the slice biological model at concentrations well below those associated with toxicity.

Inhibition of Tau Polymerization with a Cyanine Dye in Two Distinct Model Systems

The Journal of Biological Chemistry. Jul, 2009  |  Pubmed ID: 19478088

In a host of neurodegenerative diseases Tau, a microtubule-associated protein, aggregates into insoluble lesions within neurons. Previous studies have utilized cyanine dyes as Tau aggregation inhibitors in vitro. Herein we utilize cyanine dye 3,3'-diethyl-9-methyl-thiacarbocyanine iodide (C11) to modulate Tau polymerization in two model systems, an organotypic slice culture model derived from Tau transgenic mice and a split green fluorescent protein complementation assay in Tau-expressing cells. In slice cultures, submicromolar concentrations (0.001 microm) of C11 produced a significant reduction of aggregated Tau and a corresponding increase in unpolymerized Tau. In contrast, treatment with a 1 microm dose promoted aggregation of Tau. These results were recapitulated in the complementation assay where administration of 1 microm C11 produced a significant increase in polymerized Tau relative to control, whereas treatment of cells with 0.01 microm C11 resulted in a marked reduction of aggregated Tau. In the organotypic slice cultures, modulation of Tau aggregation was independent of changes in phosphorylation at disease and microtubule binding relevant epitopes for both dosing regimes. Furthermore, treatment with 0.001 microm C11 resulted in a decrease in both total filament mass and number. There was no evidence of apoptosis or loss of synaptic integrity at either dose, however, whereas submicromolar concentrations of C11 did not interfere with microtubule binding, higher doses resulted in a decrease in the levels of microtubule-bound Tau. Overall, a cyanine dye can dissociate aggregated Tau in an ex vivo model of tauopathy with little toxicity and exploration of the use of these type of dyes as therapeutic agents is warranted.

Metabolic Activity Determines Efficacy of Macroautophagic Clearance of Pathological Oligomeric Alpha-synuclein

The American Journal of Pathology. Aug, 2009  |  Pubmed ID: 19628769

Macroautophagy is an essential degradative pathway that can be induced to clear aggregated proteins, such as those found in Parkinson's disease and dementia with Lewy bodies, a form of Parkinsonism. This study found that both LC3-II and beclin were significantly increased in brains from humans with Dementia with Lewy bodies and transgenic mice overexpressing mutant alpha-synuclein, as compared with respective controls, suggesting that macroautophagy is induced to remove alpha-syn, particularly oligomeric or mutant forms. Aged mutant animals had higher autophagy biomarker levels relative to younger animals, suggesting that with aging, autophagy is less efficient and requires more stimulation to achieve the same outcome. Disruption of autophagy by RNA interference significantly increased alpha-syn oligomer accumulation in vitro, confirming the significance of autophagy in alpha-syn clearance. Finally, rotenone-induced alpha-syn aggregates were cleared following rapamycin stimulation of autophagy. Chronic rotenone exposure and commensurate reduction of metabolic activity limited the efficacy of rapamycin to promote autophagy, suggesting that cellular metabolism is critical for determining autophagic activity. Cumulatively, these findings support the concept that neuronal autophagy is essential for protein homeostasis and, in our system, reduction of autophagy increased the accumulation of potentially pathogenic alpha-synuclein oligomers. Aging and metabolic state were identified as important determinants of autophagic activity. This study provides therapeutic and pathological implications for both synucleinopathy and Parkinson's disease, identifying conditions in which autophagy may be insufficient to degrade alpha-syn aggregates.

Age-dependent Impairment of Cognitive and Synaptic Function in the Htau Mouse Model of Tau Pathology

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Aug, 2009  |  Pubmed ID: 19710325

A hallmark feature of Alzheimer's disease pathology is the presence of neurofibrillary tangles (NFTs), which are intracellular aggregates of conformationally abnormal and hyperphosphorylated tau. The presence of NFTs in the forebrain is associated with impairments of cognitive function, supporting a central role for tau in dementia. The significance of the accumulation of NFTs for neuronal and cognitive function is still obscure. It is possible that NFTs disrupt synaptic transmission and plasticity, leading to memory deficits and cognitive malfunction. To elucidate the relationship between the development of tau pathology and synaptic and cognitive functions, we performed behavioral tests and electrophysiological experiments in the htau mouse. Here we report age-dependent cognitive and physiological impairments in htau mice that preceded neurodegeneration. Twelve-month-old htau mice with moderate tau pathology, but not 4-month-old mice with early-stage tau pathology, presented cognitive deficits in an object recognition memory task in which the visual recognition memory of a novel object was disrupted. Moreover, only 12-month-old htau mice exhibit spatial memory deficits, as indicated by the impaired performance in the Morris water maze. In addition, we report that basal synaptic transmission and induction of long-term potentiation with high-frequency stimulation, but not theta burst stimulation, is perturbed in hippocampal CA1 region of old but not young htau mice. Our results suggest that tau pathology may underlie an age-dependent learning impairment through disruption of synaptic function.

Presenilins Are Enriched in Endoplasmic Reticulum Membranes Associated with Mitochondria

The American Journal of Pathology. Nov, 2009  |  Pubmed ID: 19834068

Presenilin-1 (PS1) and -2 (PS2), which when mutated cause familial Alzheimer disease, have been localized to numerous compartments of the cell, including the endoplasmic reticulum, Golgi, nuclear envelope, endosomes, lysosomes, the plasma membrane, and mitochondria. Using three complementary approaches, subcellular fractionation, gamma-secretase activity assays, and immunocytochemistry, we show that presenilins are highly enriched in a subcompartment of the endoplasmic reticulum that is associated with mitochondria and that forms a physical bridge between the two organelles, called endoplasmic reticulum-mitochondria-associated membranes. A localization of PS1 and PS2 in mitochondria-associated membranes may help reconcile the disparate hypotheses regarding the pathogenesis of Alzheimer disease and may explain many seemingly unrelated features of this devastating neurodegenerative disorder.

Lowering Beta-amyloid Levels Rescues Learning and Memory in a Down Syndrome Mouse Model

PloS One. 2010  |  Pubmed ID: 20532168

beta-amyloid levels are elevated in Down syndrome (DS) patients throughout life and are believed to cause Alzheimer's disease (AD) in adult members of this population. However, it is not known if beta-amyloid contributes to intellectual disability in younger individuals. We used a gamma-secretase inhibitor to lower beta-amyloid levels in young mice that model DS. This treatment corrected learning deficits characteristic of these mice, suggesting that beta-amyloid-lowering therapies might improve cognitive function in young DS patients.

Phenothiazine-mediated Rescue of Cognition in Tau Transgenic Mice Requires Neuroprotection and Reduced Soluble Tau Burden

Molecular Neurodegeneration. 2010  |  Pubmed ID: 21040568

It has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection.

Phospholipase D2 Ablation Ameliorates Alzheimer's Disease-linked Synaptic Dysfunction and Cognitive Deficits

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Dec, 2010  |  Pubmed ID: 21147981

Growing evidence implicates aberrant lipid signaling in Alzheimer's disease (AD). While phospholipases A2 and C have been recently shown to mediate key actions of amyloid β-peptide (Aβ) through a dysregulation of arachidonic acid and phosphatidylinositol-4,5-bisphosphate metabolism, respectively, the role of phospholipase D (PLD) has so far remained elusive. PLD produces phosphatidic acid (PA), a bioactive lipid involved in multiple aspects of cell physiology, including signaling and membrane trafficking processes. Here we show that oligomeric Aβ enhances PLD activity in cultured neurons and that this stimulatory effect does not occur upon ablation of PLD2 via gene targeting. Aβ fails to suppress long-term potentiation in PLD2-deficient hippocampal slices, suggesting that PLD2 is required for the synaptotoxic action of this peptide. In vivo PLD activity, as assessed by detection of phosphatidylethanol levels using mass spectrometry (MS) following ethanol injection, is also increased in the brain of a transgenic mouse model of AD (SwAPP). Furthermore, Pld2 ablation rescues memory deficits and confers synaptic protection in SwAPP mice despite a significant Aβ load. MS-based lipid analysis of Pld2 mutant brains in the presence or absence of the SwAPP transgene unmasks striking crosstalks between different PA species. This lipid analysis shows an exquisite acyl chain specificity and plasticity in the perturbation of PA metabolism. Collectively, our results point to specific molecular species of PA as key modulators of AD pathogenesis and identify PLD2 as a novel potential target for therapeutics.

The Phospholipase D1 Pathway Modulates Macroautophagy

Nature Communications. 2010  |  Pubmed ID: 21266992

Although macroautophagy is known to be an essential degradative process whereby autophagosomes mediate the engulfment and delivery of cytoplasmic components into lysosomes, the lipid changes underlying autophagosomal membrane dynamics are undetermined. Here, we show that phospholipase D1 (PLD1), which is primarily associated with the endosomal system, partially relocalizes to the outer membrane of autophagosome-like structures upon nutrient starvation. The localization of PLD1, as well as the starvation-induced increase in PLD activity, are altered by wortmannin, a phosphatidylinositol 3-kinase inhibitor, suggesting PLD1 may act downstream of Vps34. Pharmacological inhibition of PLD and genetic ablation of PLD1 in mouse cells decreased the starvation-induced expansion of LC3-positive compartments, consistent with a role of PLD1 in the regulation of autophagy. Furthermore, inhibition of PLD results in higher levels of Tau and p62 aggregates in organotypic brain slices. Our in vitro and in vivo findings establish a role for PLD1 in autophagy.

Age and α-synuclein Expression Interact to Reveal a Dependence of Dopaminergic Axons on Endogenous Akt/PKB Signaling

Neurobiology of Disease. Nov, 2011  |  Pubmed ID: 21782946

The mechanisms underlying the chronic neurodegeneration that occurs in Parkinson's disease (PD) are unknown. One emerging hypothesis is that neural systems deteriorate and eventually degenerate due to a primary failure of either extrinsic neurotrophic support or the intrinsic cellular pathways that mediate such support. One of the cellular pathways that have been often identified in mediating neurotrophic effects is that of PI3K/Akt signaling. In addition, recent observations have suggested a primary failure of PI3K/Akt signaling in animal models and in PD patients. Therefore, to explore the possible role of endogenous Akt signaling in maintaining the viability and functionality of substantia nigra (SN) dopamine neurons, one of the principal systems affected in PD, we have used an adeno-associated viral vector to transduce them with a dominant negative (DN) form of Akt, the pleckstrin homology (PH) domain alone (DN(PH)-Akt). In addition, we have examined the effect of DN(PH)-Akt in murine models of two risk factors for human PD: advanced age and increased expression of α-synuclein. We find that transduction of these neurons in normal adult mice has no effect on any aspect of their morphology at 4 or 7weeks. However, in both aged mice and in transgenic mice with increased expression of human α-synuclein we observe decreased phenotypic expression of the catecholamine synthetic enzyme tyrosine hydroxylase (TH) in dopaminergic axons and terminals in the striatum. In aged transgenic α-synuclein over-expressing mice this reduction was 2-fold as great. We conclude that the two principal risk factors for human PD, advanced age and increased expression of α-synuclein, reveal a dependence of dopaminergic neurons on endogenous Akt signaling for maintenance of axonal phenotype.

Modulation of ABCA1 by an LXR Agonist Reduces β-amyloid Levels and Improves Outcome After Traumatic Brain Injury

Journal of Neurotrauma. Feb, 2011  |  Pubmed ID: 21175399

Traumatic brain injury (TBI) increases brain beta-amyloid (Aβ) in humans and animals. Although the role of Aβ in the injury cascade is unknown, multiple preclinical studies have demonstrated a correlation between reduced Aβ and improved outcome. Therefore, therapeutic strategies that enhance Aβ clearance may be beneficial after TBI. Increased levels of ATP-binding cassette A1 (ABCA1) transporters can enhance Aβ clearance through an apolipoprotein E (apoE)-mediated pathway. By measuring Aβ and ABCA1 after experimental TBI in C57BL/6J mice, we found that Aβ peaked early after injury (1-3 days), whereas ABCA1 had a delayed response (beginning at 3 days). As ABCA1 levels increased, Aβ levels returned to baseline levels-consistent with the known role of ABCA1 in Aβ clearance. To test if enhancing ABCA1 levels could block TBI-induced Aβ, we treated TBI mice with the liver X-receptor (LXR) agonist T0901317. Pre- and post-injury treatment increased ABCA1 levels at 24 h post-injury, and reduced the TBI-induced increase in Aβ. This reduction in Aβ was not due to decreased amyloid precursor protein processing, or a shift in the solubility of Aβ, indicating enhanced clearance. T0901317 also limited motor coordination deficits in injured mice and reduced brain lesion volume. These data indicate that activation of LXR can reduce Aβ accumulation after TBI, and is accompanied by improved functional recovery.

Microarray Analysis of CA1 Pyramidal Neurons in a Mouse Model of Tauopathy Reveals Progressive Synaptic Dysfunction

Neurobiology of Disease. Feb, 2012  |  Pubmed ID: 22079237

The hTau mouse model of tauopathy was utilized to assess gene expression changes in vulnerable hippocampal CA1 neurons. CA1 pyramidal neurons were microaspirated via laser capture microdissection followed by RNA amplification in combination with custom-designed microarray analysis and qPCR validation in hTau mice and nontransgenic (ntg) littermates aged 11-14months. Statistical analysis revealed ~8% of all the genes on the array platform were dysregulated, with notable downregulation of several synaptic-related markers including synaptophysin (Syp), synaptojanin, and synaptobrevin, among others. Downregulation was also observed for select glutamate receptors (GluRs), Psd-95, TrkB, and several protein phosphatase subunits. In contrast, upregulation of tau isoforms and a calpain subunit were found. Microarray assessment of synaptic-related markers in a separate cohort of hTau mice at 7-8months of age indicated only a few alterations compared to the 11-14month cohort, suggesting progressive synaptic dysfunction occurs as tau accumulates in CA1 pyramidal neurons. An assessment of SYP and PSD-95 expression was performed in the hippocampal CA1 sector of hTau and ntg mice via confocal laser scanning microscopy along with hippocampal immunoblot analysis for protein-based validation of selected microarray observations. Results indicate significant decreases in SYP-immunoreactive and PSD-95-immunoreactive puncta as well as downregulation of SYP-immunoreactive and PSD-95-immunoreactive band intensity in hTau mice compared to age-matched ntg littermates. In summary, the high prevalence of downregulation of synaptic-related genes indicates that the moderately aged hTau mouse may be a model of tau-induced synaptodegeneration, and has profound effects on how we perceive progressive tau pathology affecting synaptic transmission in AD.

Comparative Lipidomic Analysis of Mouse and Human Brain with Alzheimer Disease

The Journal of Biological Chemistry. Jan, 2012  |  Pubmed ID: 22134919

Lipids are key regulators of brain function and have been increasingly implicated in neurodegenerative disorders including Alzheimer disease (AD). Here, a systems-based approach was employed to determine the lipidome of brain tissues affected by AD. Specifically, we used liquid chromatography-mass spectrometry to profile extracts from the prefrontal cortex, entorhinal cortex, and cerebellum of late-onset AD (LOAD) patients, as well as the forebrain of three transgenic familial AD (FAD) mouse models. Although the cerebellum lacked major alterations in lipid composition, we found an elevation of a signaling pool of diacylglycerol as well as sphingolipids in the prefrontal cortex of AD patients. Furthermore, the diseased entorhinal cortex showed specific enrichment of lysobisphosphatidic acid, sphingomyelin, the ganglioside GM3, and cholesterol esters, all of which suggest common pathogenic mechanisms associated with endolysosomal storage disorders. Importantly, a significant increase in cholesterol esters and GM3 was recapitulated in the transgenic FAD models, suggesting that these mice are relevant tools to study aberrant lipid metabolism of endolysosomal dysfunction associated with AD. Finally, genetic ablation of phospholipase D(2), which rescues the synaptic and behavioral deficits of an FAD mouse model, fully normalizes GM3 levels. These data thus unmask a cross-talk between the metabolism of phosphatidic acid, the product of phospholipase D(2), and gangliosides, and point to a central role of ganglioside anomalies in AD pathogenesis. Overall, our study highlights the hypothesis generating potential of lipidomics and identifies novel region-specific lipid anomalies potentially linked to AD pathogenesis.

Trans-synaptic Spread of Tau Pathology in Vivo

PloS One. 2012  |  Pubmed ID: 22312444

Tauopathy in the brain of patients with Alzheimer's disease starts in the entorhinal cortex (EC) and spreads anatomically in a defined pattern. To test whether pathology initiating in the EC spreads through the brain along synaptically connected circuits, we have generated a transgenic mouse model that differentially expresses pathological human tau in the EC and we have examined the distribution of tau pathology at different timepoints. In relatively young mice (10-11 months old), human tau was present in some cell bodies, but it was mostly observed in axons within the superficial layers of the medial and lateral EC, and at the terminal zones of the perforant pathway. In old mice (>22 months old), intense human tau immunoreactivity was readily detected not only in neurons in the superficial layers of the EC, but also in the subiculum, a substantial number of hippocampal pyramidal neurons especially in CA1, and in dentate gyrus granule cells. Scattered immunoreactive neurons were also seen in the deeper layers of the EC and in perirhinal and secondary somatosensory cortex. Immunoreactivity with the conformation-specific tau antibody MC1 correlated with the accumulation of argyrophilic material seen in old, but not young mice. In old mice, axonal human tau immunoreactivity, especially at the endzones of the perforant pathway, was greatly reduced. Relocalization of tau from axons to somatodendritic compartments and propagation of tauopathy to regions outside of the EC correlated with mature tangle formation in neurons in the EC as revealed by thioflavin-S staining. Our data demonstrate propagation of pathology from the EC and support a trans-synaptic mechanism of spread along anatomically connected networks, between connected and vulnerable neurons. In general, the mouse recapitulates the tauopathy that defines the early stages of AD and provides a model for testing mechanisms and functional outcomes associated with disease progression.