Exosome-associated AAV vector as a robust and convenient neuroscience tool.

Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to their widespread use in the neuroscience laboratory is the cost, labor, skill and time-intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when the vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared with standard AAV. Here, we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood-brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV-transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing three-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex vivo serial two-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable the widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.

Genetic overlap between Alzheimer's disease and Parkinson's disease at the MAPT locus.

We investigated the genetic overlap between Alzheimer's disease (AD) and Parkinson's disease (PD). Using summary statistics (P-values) from large recent genome-wide association studies (GWAS) (total n=89 904 individuals), we sought to identify single nucleotide polymorphisms (SNPs) associating with both AD and PD. We found and replicated association of both AD and PD with the A allele of rs393152 within the extended MAPT region on chromosome 17 (meta analysis P-value across five independent AD cohorts=1.65 × 10(-7)). In independent datasets, we found a dose-dependent effect of the A allele of rs393152 on intra-cerebral MAPT transcript levels and volume loss within the entorhinal cortex and hippocampus. Our findings identify the tau-associated MAPT locus as a site of genetic overlap between AD and PD, and extending prior work, we show that the MAPT region increases risk of Alzheimer's neurodegeneration.

Isolation and characterization of APLP2 encoding a homologue of the Alzheimer's associated amyloid beta protein precursor.

Familial Alzheimer's disease (FAD) is a genetically heterogeneous disorder that includes a rare early-onset form linked to mutations in the amyloid b protein precursor (APP) gene. Clues to the function of APP derive from the recent finding that it is a member of a highly conserved protein family that includes the mammalian amyloid precursor-like protein (APLP1) gene which maps to the same general region of human chromosome 19 linked to late-onset FAD. Here we report the isolation of the human APLP2 gene. We show that APLP2 is a close relative of APP and exhibits a very similar pattern of expression in the brain and throughout the body. Like APP, APLP2 contains a cytoplasmic domain predicted to couple with the GTP-binding protein G(o) indicating that it may be an additional cell surface activator of this G protein.

Alpha-2 macroglobulin is genetically associated with Alzheimer disease.

Alpha-2-macroglobulin (alpha-2M; encoded by the gene A2M) is a serum pan-protease inhibitor that has been implicated in Alzheimer disease (AD) based on its ability to mediate the clearance and degradation of A beta, the major component of beta-amyloid deposits. Analysis of a deletion in the A2M gene at the 5' splice site of 'exon II' of the bait region (exon 18) revealed that inheritance of the deletion (A2M-2) confers increased risk for AD (Mantel-Haenzel odds ratio=3.56, P=0.001). The sibship disequilibrium test (SDT) also revealed a significant association between A2M and AD (P=0.00009). These values were comparable to those obtained for the APOE-epsilon4 allele in the same sample, but in contrast to APOE-epsilon4, A2M-2 did not affect age of onset. The observed association of A2M with AD did not appear to account for the previously published linkage of AD to chromosome 12, which we were unable to confirm in this sample. A2M, LRP1 (encoding the alpha-2M receptor) and the genes for two other LRP ligands, APOE and APP (encoding the amyloid beta-protein precursor), have now all been genetically linked to AD, suggesting that these proteins may participate in a common neuropathogenic pathway leading to AD.

Activation of glycogen synthase kinase-3 beta mediates ß-amyloid induced neuritic damage in Alzheimer's disease.

ß-Amyloid (Aß) plaques in Alzheimer (AD) brains are surrounded by severe dendritic and axonal changes, including local spine loss, axonal swellings and distorted neurite trajectories. Whether and how plaques induce these neuropil abnormalities remains unknown. We tested the hypothesis that oligomeric assemblies of Aß, seen in the periphery of plaques, mediate the neurodegenerative phenotype of AD by triggering activation of the enzyme GSK-3ß, which in turn appears to inhibit a transcriptional program mediated by CREB. We detect increased activity of GSK-3ß after exposure to oligomeric Aß in neurons in culture, in the brain of double transgenic APP/tau mice and in AD brains. Activation of GSK-3ß, even in the absence of Aß, is sufficient to produce a phenocopy of Aß-induced dendritic spine loss in neurons in culture, while pharmacological inhibition of GSK-3ß prevents spine loss and increases expression of CREB-target genes like BDNF. Of note, in transgenic mice GSK-3ß inhibition ameliorated plaque-related neuritic changes and increased CREB-mediated gene expression. Moreover, GSK-3ß inhibition robustly decreased the oligomeric Aß load in the mouse brain. All these findings support the idea that GSK3ß is aberrantly activated by the presence of Aß, and contributes, at least in part, to the neuronal anatomical derangement associated with Aß plaques in AD brains and to Aß pathology itself.

Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid beta-peptide.

The pathogenic event common to all forms of Alzheimer's disease is the abnormal accumulation of the amyloid beta-peptide (Abeta). Here we provide strong evidence that intracellular cholesterol compartmentation modulates the generation of Abeta. Using genetic, biochemical and metabolic approaches, we found that cholesteryl-ester levels are directly correlated with Abeta production. Acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyses the formation of cholesteryl esters, modulates the generation of Abeta through the tight control of the equilibrium between free cholesterol and cholesteryl esters. We also show that pharmacological inhibitors of ACAT, developed for the treatment of atherosclerosis, are potent modulators of Abeta generation, indicating their potential for use in the treatment of Alzheimer's disease.

Alzheimer-associated presenilins 1 and 2: neuronal expression in brain and localization to intracellular membranes in mammalian cells.

Mutations in two recently identified genes appear to cause the majority of early-onset familial Alzheimer's disease (FAD). These two novel genes, presenilin 1 (PS1) and presenilin 2 (PS2) are members of an evolutionarily conserved gene family. The normal biological role(s) of the presenilins and the mechanism(s) by which the FAD-associated mutations exert their effect remain unknown. Employing in situ hybridization, we demonstrate that the expression patterns of PS1 and PS2 in the brain are extremely similar to each other and that messages for both are primarily detectable in neuronal populations. Immunochemical analyses indicate that PS1 and PS2 are similar in size and localized to similar intracellular compartments (endoplasmic reticulum and Golgi complex). FAD-associated mutations in PS1 and PS2 do not significantly modify either their migration patterns on SDS-polyacrylamide gel electrophoresis or their overall subcellular localization, although subtle differences in perinuclear staining were noted for mutant PS1.

Triflusal reduces dense-core plaque load, associated axonal alterations and inflammatory changes, and rescues cognition in a transgenic mouse model of Alzheimer's disease.

Inflammation has been associated with the two classic lesions in the Alzheimer's (AD) brain, amyloid deposits and neurofibrillary tangles. Recent data suggest that Triflusal, a compound with potent anti-inflammatory effects in the central nervous system in vivo, might delay the conversion from amnestic mild cognitive impairment to a fully established clinical picture of dementia. In the present study, we investigated the effect of Triflusal on brain Abeta accumulation, neuroinflammation, axonal curvature and cognition in an AD transgenic mouse model (Tg2576). Triflusal treatment did not alter the total brain Abeta accumulation but significantly reduced dense-cored plaque load and associated glial cell proliferation, proinflammatory cytokine levels and abnormal axonal curvature, and rescued cognitive deficits in Tg2576 mice. Behavioral benefit was found to involve increased expression of c-fos and BDNF, two of the genes regulated by CREB, as part of the signal transduction cascade underlying the molecular basis of long-term potentiation. These results add preclinical evidence of a potentially beneficial effect of Triflusal in AD.

Interaction of the apolipoprotein E receptors low density lipoprotein receptor-related protein and sorLA/LR11.

In this study, we examined protein-protein interactions between two neuronal receptors, low density lipoprotein receptor-related protein (LRP) and sorLA/LR11, and found that these receptors interact, as indicated by three independent lines of evidence: co-immunoprecipitation experiments on mouse brain extracts and mouse neuronal cells, surface plasmon resonance analysis with purified human LRP and sorLA, and fluorescence lifetime imaging microscopy (FLIM) on rat primary cortical neurons. Immunocytochemistry experiments revealed widespread co-localization of LRP and sorLA within perinuclear compartments of rat primary neurons, while FLIM analysis showed that LRP-sorLA interactions take place within a subset of these compartments.

Alzheimer's disease: cell-specific pathology isolates the hippocampal formation.

Examination of temporal lobe structures from Alzheimer patients reveals a specific cellular pattern of pathology of the subiculum of the hippocampal formation and layers II and IV of the entorhinal cortex. The affected cells are precisely those that interconnect the hippocampal formation with the association cortices, basal forebrain, thalamus, and hypothalamus, structures crucial to memory. This focal pattern of pathology isolates the hippocampal formation from much of its input and output and probably contributes to the memory disorder in Alzheimer patients.

Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactions.

An increased prevalence of apolipoprotein E (ApoE) epsilon 4 allele exists in late onset familial Alzheimer's disease. We found, in sporadic Alzheimer's disease, that 62% of patients possessed an ApoE-epsilon 4 allele, compared with 20% of controls. ApoE-epsilon 4/4 patients had more senile plaques (SPs) than epsilon 3/3 patients. ApoE immunoreactivity of SPs was equivalent in both groups. Two receptors bind ApoE complexes, the low density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP). In normal brain, anti-LRP antibodies strongly stained neurons and lightly stained astrocytes; anti-LDL receptor antibodies stained only the neuropil and astrocytes. In Alzheimer's disease, SPs and reactive astrocytes were also strongly LRP immunoreactive. Colocalization of ApoE and LRP to SPs implies that these molecules may be involved in metabolism of components of SPs.

Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice.

We investigated synaptic communication and plasticity in hippocampal slices from mice overexpressing mutated 695-amino-acid human amyloid precursor protein (APP695SWE), which show behavioral and histopathological abnormalities simulating Alzheimer's disease. Although aged APP transgenic mice exhibit normal fast synaptic transmission and short term plasticity, they are severely impaired in in-vitro and in-vivo long-term potentiation (LTP) in both the CA1 and dentate gyrus regions of the hippocampus. The LTP deficit was correlated with impaired performance in a spatial working memory task in aged transgenics. These deficits are accompanied by minimal or no loss of presynaptic or postsynaptic elementary structural elements in the hippocampus, suggesting that impairments in functional synaptic plasticity may underlie some of the cognitive deficits in these mice and, possibly, in Alzheimer's patients.

Entorhinal Cortex: Antemortem Cortical Thickness and Postmortem Neurofibrillary Tangles and Amyloid Pathology.

BACKGROUND AND PURPOSE: The entorhinal cortex, a critical gateway between the neocortex and hippocampus, is one of the earliest regions affected by Alzheimer disease-associated neurofibrillary tangle pathology. Although our prior work has automatically delineated an MR imaging-based measure of the entorhinal cortex, whether antemortem entorhinal cortex thickness is associated with postmortem tangle burden within the entorhinal cortex is still unknown. Our objective was to evaluate the relationship between antemortem MRI measures of entorhinal cortex thickness and postmortem neuropathological measures. MATERIALS AND METHODS: We evaluated 50 participants from the Rush Memory and Aging Project with antemortem structural T1-weighted MR imaging and postmortem neuropathologic assessments. Here, we focused on thickness within the entorhinal cortex as anatomically defined by our previously developed MR imaging parcellation system (Desikan-Killiany Atlas in FreeSurfer). Using linear regression, we evaluated the association between entorhinal cortex thickness and tangles and amyloid-ß load within the entorhinal cortex and medial temporal and neocortical regions. RESULTS: We found a significant relationship between antemortem entorhinal cortex thickness and entorhinal cortex (P = .006) and medial temporal lobe tangles (P = .002); we found no relationship between entorhinal cortex thickness and entorhinal cortex (P = .09) and medial temporal lobe amyloid-ß (P = .09). We also found a significant association between entorhinal cortex thickness and cortical tangles (P = .003) and amyloid-ß (P = .01). We found no relationship between parahippocampal gyrus thickness and entorhinal cortex (P = .31) and medial temporal lobe tangles (P = .051). CONCLUSIONS: Our findings indicate that entorhinal cortex-associated in vivo cortical thinning may represent a marker of postmortem medial temporal and neocortical Alzheimer disease pathology.

Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases?

The pathogenesis of nerve cell death in neurodegenerative diseases is unknown. An attractive hypothesis is that an impairment of energy metabolism may underlie slow excitotoxic neuronal death. Several studies have demonstrated mitochondrial or oxidative defects in neurodegenerative diseases. Impaired energy metabolism results in decreases in high-energy phosphate stores and a deteriorating membrane potential. Under these conditions, the voltage-sensitive Mg2+ block of NMDA receptors is relieved, allowing the receptors to be persistently activated by endogenous concentrations of glutamate. In this way, metabolic defects may lead to neuronal death by a slow 'excitotoxic' mechanism. Recent studies indicate that such a mechanism occurs in vivo, and it may play a role in animal models of Huntington's disease and Parkinson's disease. If a similar mechanism occurs in neurodegenerative diseases in humans it may be possible to use either excitatory amino acid antagonists or agents to improve neuronal bioenergetics as therapeutic treatments for these disorders.

Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain.

Adeno-associated virus (AAV) vectors have gained a preeminent position in the field of gene delivery to the normal brain through their ability to achieve extensive transduction of neurons and to mediate long-term gene expression with no apparent toxicity. In adult animals direct infusion of AAV vectors into the brain parenchyma results in highly efficient transduction of target structures. However AAV-mediated global delivery to the adult brain has been an elusive goal. In contrast, widespread global gene delivery has been obtained by i.c.v. injection of AAV1 or AAV2 in neonates. Among the novel AAV serotypes cloned and engineered for production of recombinant vectors, AAV8 has shown a tremendous potential for in vivo gene delivery with nearly complete transduction of many tissues in rodents after intravascular infusion. Here we compare the efficiency of an AAV8 serotyped vector with that of AAV1 and AAV2 serotyped vectors for the extent of gene delivery to the brain after neonatal injection into the lateral ventricles. The vectors all encoded green fluorescent protein (GFP) under control of a hybrid CMV enhancer/chicken beta-actin promoter with AAV2 inverted terminal repeats, but differed from each other with respect to the capsid type. A total of 6.8 x 10(10) genome copies were injected into the lateral ventricles of postnatal day 0 mice. Mice were killed at postnatal day 30 and brains analyzed for distribution of GFP-positive cells. AAV8 proved to be more efficient than AAV1 or AAV2 vectors for gene delivery to all of the structures analyzed, including the cerebral cortex, hippocampus, olfactory bulb, and cerebellum. Moreover the intensity of gene expression, assessed using a microarray reader, was considerably higher for AAV8 in all structures analyzed. In conclusion, the enhanced transduction achieved by AAV8 compared with AAV1 and AAV2 indicates that AAV8 is the superior serotype for gene delivery to the CNS.

Early detection of cryptic memory and glucose uptake deficits in pre-pathological APP mice.

Earlier diagnosis and treatment of Alzheimer's disease would greatly benefit from the identification of biomarkers at the prodromal stage. Using a prominent animal model of aspects of the disease, we here show using clinically relevant methodologies that very young, pre-pathological PDAPP mice, which overexpress mutant human amyloid precursor protein in the brain, exhibit two cryptic deficits that are normally undetected using standard methods of assessment. Despite learning a spatial memory task normally and displaying normal brain glucose uptake, they display faster forgetting after a long delay following performance to a criterion, together with a strong impairment of brain glucose uptake at the time of attempted memory retrieval. Preliminary observations suggest that these deficits, likely caused by an impairment in systems consolidation, could be rescued by immunotherapy with an anti-ß-amyloid antibody. Our data suggest a biomarker strategy for the early detection of ß-amyloid-related abnormalities.

Growth arrest of individual senile plaques in a model of Alzheimer's disease observed by in vivo multiphoton microscopy.

In Alzheimer's disease, amyloid-beta peptide aggregates in the extracellular space to form senile plaques. The process of plaque deposition and growth has been modeled on the basis of in vitro experiments in ways that lead to divergent predictions: either a diffusion-limited growth model in which plaques grow by first-order kinetics, or a dynamic model of continual deposition and asymmetrical clearance in which plaques reach a stable size and stop growing but evolve morphologically over time. The models have not been tested in vivo because plaques are too small (by several orders of magnitude) for conventional imaging modalities. We now report in vivo multiphoton laser scanning imaging of thioflavine S-stained senile plaques in the Tg2576 transgenic mouse model of Alzheimer's disease to test these biophysical models and show that there is no detectable change in plaque size over extended periods of time. Qualitatively, geometric features remain unchanged over time in the vast majority of the 349 plaques imaged and re-imaged. Intervals as long as 5 months were obtained. Nonetheless, rare examples of growth or shrinkage of individual plaques do occur, and new plaques appear between imaging sessions. These results indicate that thioflavine S-positive plaques appear and then are stable, supporting a dynamic feedback model of plaque growth.