Postmortem AD Brain Research Articles

The role of the blood‐brain barrier in brain insulin signaling

AbstractBackgroundDefects in central response to insulin is suspected to play a role in AD. However, to act on the brain, pancreas‐secreted insulin must interact first with the blood‐brain barrier (BBB). The insulin receptor (INSR) consists in 2 insulin‐binding extracellular α chains, either short (INSRα‐A) or long (INSRα‐B) isoforms, as well as 2 intracellular β‐chains (INSRβ) containing auto‐phosphorylation sites. The aim of the present study was to better understand the role of INSR located on the BBB in AD.MethodWe used microvessel‐enriched brain samples from individuals classified as Controls, MCI or AD and from 3xTg‐AD mice from different ages. In situ cerebral perfusion was used to directly assess the interaction between circulating insulin and the BBB.ResultsWe first show that INSR in human and mouse brains are concentrated in blood microvessels, with INSRα‐B being almost absent from the parenchymal fractions. Lower levels of INSRα‐B were observed in microvessel extracts from the parietal cortex of AD persons, associated with cognitive decline. The resulting shift toward a higher INSRα‐A:INSRα‐B ratio is consistent with insulin resistance situated on the BBB. Intracarotid infusion of insulin induced a rapid phosphorylation of INSRβ retrieved from cerebrovascular extracts, but not from parenchymal extracts of the mouse brain. In the 3xTg‐AD mouse model of AD neuropathology, lower levels of cerebrovascular INSRα‐B were observed whereas activation of INSRβ was blunted upon intracarotid administration of insulin. Finally, we provide some insights on a possible association between BACE1 activation and the reduction of cerebrovascular INSR.ConclusionOverall, data gathered from postmortem AD brains and an animal model of AD neuropathology suggest that brain insulin resistance in AD may occur at the level of INSR located on microvessels. These results also indicate that INSR and its downstream signaling within endothelial cells of the BBB are systemically accessible drug targets in AD.

Investigating the functional significance of presynaptic tau in ageing and disease

AbstractBackgroundSynaptic dysfunction is an early pathological feature of neurodegenerative disease and is a close correlate of cognitive decline. Modified tau mislocalises to the synaptic compartment in disease, where some forms of tau interact with synaptic proteins and disrupt synapse function. This study seeks to further explore synaptic tau accumulation, and the impact on synaptic health and tau release, during ageing and tauopathy progression.MethodPost‐mortem Brodmann area 9 (BA9) pre‐frontal cortex from control or Braak stage I‐VI tissues were obtained. Formalin‐fixed paraffin‐embedded sections were subject to immunohistochemistry. Total lysates, cytosolic and synaptoneurosome fractions were isolated for western blotting, imaging and analysis of synaptic protein interactions.Organotypic brain slice cultures were prepared from P9‐10 wild‐type mice and Tau35 mice in which a disease‐associated tau fragment is expressed. In addition, some wild‐type slices were transduced with AAV2/TM8‐WT‐htau0N4R‐EGFP (WT) or AAV2/TM8‐P301L/S320F‐htau0N4R‐EGFP. Slices were cultured for 21‐28 DIV. Total lysate, cytosolic, and synaptoneurosome fractions were isolated for western blot, immunocytochemistry, or immunoprecipitation. Culture medium was used for tau sandwich ELISA.ResultWe observed progressive neurodegenerative changes, including the accumulation of sarkosyl‐insoluble tau, in organotypic brain slices expressing Tau35 and mutant (AAV‐expressed) human tau. Mislocalisation of phosphorylated and aggregated tau to the synaptic fraction in the tauopathy slice models mirrors, at least in part, changes observed in postmortem AD brain. Tau mislocalisation was associated with significant loss of specific pre‐synaptic proteins in Tau35 mice and AD brain. Mutant AAV‐expressed tau, but not WT AAV‐expressed tau, interacts with specific presynaptic vesicle proteins. Furthermore, alterations in basal tau release from slices was observed when disease‐like tau was expressed.ConclusionSlice cultures from mice in which disease‐associated tau is expressed at physiological levels (Tau35) or in which mutant tau is virally induced recapitulate key elements of human tauopathy. These long‐term brain slice cultures allow sensitive characterisation of presynaptic alterations that occur early in tauopathy. These models are now being used to determine if interactions between tau and pre‐synaptic proteins are essential for tau‐mediated synaptic dysfunction and/or tau release.

Farnesyltransferase inhibitor LNK-754 attenuates axonal dystrophy and reduces amyloid pathology in mice

BackgroundAmyloid plaque deposition and axonal degeneration are early events in AD pathogenesis. Aβ disrupts microtubules in presynaptic dystrophic neurites, resulting in the accumulation of impaired endolysosomal and autophagic organelles transporting β-site amyloid precursor protein cleaving enzyme (BACE1). Consequently, dystrophic neurites generate Aβ42 and significantly contribute to plaque deposition. Farnesyltransferase inhibitors (FTIs) have recently been investigated for repositioning toward the treatment of neurodegenerative disorders and block the action of farnesyltransferase (FTase) to catalyze farnesylation, a post-translational modification that regulates proteins involved in lysosome function and microtubule stability. In postmortem AD brains, FTase and its downstream signaling are upregulated. However, the impact of FTIs on amyloid pathology and dystrophic neurites is unknown.MethodsWe tested the effects of the FTIs LNK-754 and lonafarnib in the 5XFAD mouse model of amyloid pathology.ResultsIn 2-month-old 5XFAD mice treated chronically for 3 months, LNK-754 reduced amyloid plaque burden, tau hyperphosphorylation, and attenuated the accumulation of BACE1 and LAMP1 in dystrophic neurites. In 5-month-old 5XFAD mice treated acutely for 3 weeks, LNK-754 reduced dystrophic neurite size and LysoTracker-Green accumulation in the absence of effects on Aβ deposits. Acute treatment with LNK-754 improved memory and learning deficits in hAPP/PS1 amyloid mice. In contrast to LNK-754, lonafarnib treatment was less effective at reducing plaques, tau hyperphosphorylation and dystrophic neurites, which could have resulted from reduced potency against FTase compared to LNK-754. We investigated the effects of FTIs on axonal trafficking of endolysosomal organelles and found that lonafarnib and LNK-754 enhanced retrograde axonal transport in primary neurons, indicating FTIs could support the maturation of axonal late endosomes into lysosomes. Furthermore, FTI treatment increased levels of LAMP1 in mouse primary neurons and in the brains of 5XFAD mice, demonstrating that FTIs stimulated the biogenesis of endolysosomal organelles.ConclusionsWe show new data to suggest that LNK-754 promoted the axonal trafficking and function of endolysosomal compartments, which we hypothesize decreased axonal dystrophy, reduced BACE1 accumulation and inhibited amyloid deposition in 5XFAD mice. Our results agree with previous work identifying FTase as a therapeutic target for treating proteinopathies and could have important therapeutic implications in treating AD.

Single-Cell Transcriptional Profiling and Gene Regulatory Network Modeling in Tg2576 Mice Reveal Gender-Dependent Molecular Features Preceding Alzheimer-Like Pathologies

Alzheimer’s disease (AD) onset and progression is influenced by a complex interplay of several environmental and genetic factors, one of them gender. Pronounced gender differences have been observed both in the relative risk of developing AD and in clinical disease manifestations. A molecular level understanding of these gender disparities is still missing, but could provide important clues on cellular mechanisms modulating the disease and reveal new targets for gender-oriented disease-modifying precision therapies. We therefore present here a comprehensive single-cell analysis of disease-associated molecular gender differences in transcriptomics data from the neocortex, one of the brain regions most susceptible to AD, in one of the most widely used AD mouse models, the Tg2576 model. Cortical areas are also most commonly used in studies of post-mortem AD brains. To identify disease-linked molecular processes that occur before the onset of detectable neuropathology, we focused our analyses on an age with no detectable plaques and microgliosis. Cell-type specific alterations were investigated at the level of individual genes, pathways, and gene regulatory networks. The number of differentially expressed genes (DEGs) was not large enough to build context-specific gene regulatory networks for each individual cell type, and thus, we focused on the study of cell types with dominant changes and included analyses of changes across the combination of cell types. We observed significant disease-associated gender differences in cellular processes related to synapse organization and reactive oxygen species metabolism, and identified a limited set of transcription factors, including Egr1 and Klf6, as key regulators of many of the disease-associated and gender-dependent gene expression changes in the model. Overall, our analyses revealed significant cell-type specific gene expression changes in individual genes, pathways and sub-networks, including gender-specific and gender-dimorphic changes in both upstream transcription factors and their downstream targets, in the Tg2576 AD model before the onset of overt disease. This opens a window into molecular events that could determine gender-susceptibility to AD, and uncovers tractable target candidates for potential gender-specific precision medicine for AD.

Selective Detection of Misfolded Tau From Postmortem Alzheimer's Disease Brains.

Tau aggregates are present in multiple neurodegenerative diseases known as “tauopathies,” including Alzheimer’s disease, Pick’s disease, progressive supranuclear palsy, and corticobasal degeneration. Such misfolded tau aggregates are therefore potential sources for selective detection and biomarker discovery. Six human tau isoforms present in brain tissues and both 3R and 4R isoforms have been observed in the neuronal inclusions. To develop selective markers for AD and related rare tauopathies, we first used an engineered tau protein fragment 4RCF as the substrate for ultrasensitive real-time quaking-induced conversion analyses (RT-QuIC). We showed that misfolded tau from diseased AD and other tauopathy brains were able to seed recombinant 4RCF substrate. We further expanded to use six individual recombinant tau isoforms as substrates to amplify misfolded tau seeds from AD brains. We demonstrated, for the first time to our knowledge, that misfolded tau from the postmortem AD brain tissues was able to specifically seed all six full-length human tau isoforms. Our results demonstrated that RT-QuIC analysis can discriminate AD and other tauopathies from non-AD normal controls. We further uncovered that 3R-tau isoforms displayed significantly faster aggregation kinetics than their 4R-tau counterparts under conditions of both no seeding and seeding with AD brain homogenates. In summary, our work offers potential new avenues of misfolded tau detection as potential biomarkers for diagnosis of AD and related tauopathies and provides new insights into isoform-specific human tau aggregation.

A\u03b2 oligomers trigger necroptosis-mediated neurodegeneration via microglia activation in Alzheimer\u2019s disease

Alzheimer’s disease (AD) is a major adult-onset neurodegenerative condition with no available treatment. Compelling reports point amyloid-β (Aβ) as the main etiologic agent that triggers AD. Although there is extensive evidence of detrimental crosstalk between Aβ and microglia that contributes to neuroinflammation in AD, the exact mechanism leading to neuron death remains unknown. Using postmortem human AD brain tissue, we show that Aβ pathology is associated with the necroptosis effector pMLKL. Moreover, we found that the burden of Aβ oligomers (Aβo) correlates with the expression of key markers of necroptosis activation. Additionally, inhibition of necroptosis by pharmacological or genetic means, reduce neurodegeneration and memory impairment triggered by Aβo in mice. Since microglial activation is emerging as a central driver for AD pathogenesis, we then tested the contribution of microglia to the mechanism of Aβo-mediated necroptosis activation in neurons. Using an in vitro model, we show that conditioned medium from Aβo-stimulated microglia elicited necroptosis in neurons through activation of TNF-α signaling, triggering extensive neurodegeneration. Notably, necroptosis inhibition provided significant neuronal protection. Together, these findings suggest that Aβo-mediated microglia stimulation in AD contributes to necroptosis activation in neurons and neurodegeneration. As necroptosis is a druggable degenerative mechanism, our findings might have important therapeutic implications to prevent the progression of AD.

ATAD3A oligomerization promotes neuropathology and cognitive deficits in Alzheimer\u2019s disease models

Predisposition to Alzheimer’s disease (AD) may arise from lipid metabolism perturbation, however, the underlying mechanism remains elusive. Here, we identify ATPase family AAA-domain containing protein 3A (ATAD3A), a mitochondrial AAA-ATPase, as a molecular switch that links cholesterol metabolism impairment to AD phenotypes. In neuronal models of AD, the 5XFAD mouse model and post-mortem AD brains, ATAD3A is oligomerized and accumulated at the mitochondria-associated ER membranes (MAMs), where it induces cholesterol accumulation by inhibiting gene expression of CYP46A1, an enzyme governing brain cholesterol clearance. ATAD3A and CYP46A1 cooperate to promote APP processing and synaptic loss. Suppressing ATAD3A oligomerization by heterozygous ATAD3A knockout or pharmacological inhibition with DA1 restores neuronal CYP46A1 levels, normalizes brain cholesterol turnover and MAM integrity, suppresses APP processing and synaptic loss, and consequently reduces AD neuropathology and cognitive deficits in AD transgenic mice. These findings reveal a role for ATAD3A oligomerization in AD pathogenesis and suggest ATAD3A as a potential therapeutic target for AD.

IPSC-derived cortical neurons to study sporadic Alzheimer disease: A transcriptome comparison with post-mortem brain samples

Alzheimer’s disease (AD) is the most common cause of dementia, characterized by the progressive impairment of cognition and memory loss. Sporadic AD (sAD) represents approximately 95 % of the AD cases and is induced by a complex interplay between genetic and environmental factors called “Alzheimerogens”. Heavy metals (e.g. copper) and pesticides (e.g. fipronil) can affect many AD-related processes, including neuroinflammation (considered as AD-inducing factor). Research would benefit from in vitro models to investigate effects of Alzheimerogens. We compared transcriptomics changes in sAD induced pluripotent stem cell (iPSC) derived cortical neurons to differentially expressed genes (DEGs) identified in post-mortem AD brain tissue. These analyses showed that many AD-related processes could be identified in the sAD iPSC-derived neurons, and furthermore, could even identify more DEGs functioning in these processes than post-mortem AD-brain tissue. Thereafter, we exposed the iPSCs to AD-inducing factors (copper(II)chloride, fipronil sulfone and an inflammatory cytokine cocktail). Cytokine exposure induced expression of immune related genes while copper-exposure affected genes involved in lipid and cholesterol metabolism, which are known AD-related processes. Fipronil-exposure did not result in significant transcriptomic changes, although prolonged exposures or higher doses may be necessary. Overall, we show that iPSC-derived cortical neurons can be beneficial in vitro models to identify Alzheimerogens and AD-related molecular mechanisms.

Supporting anti‐tau antibody trials using computer simulation of spatial tau propagation

AbstractBackgroundAlthough tau pathology is a major driver of AD pathology, the mere complexity of tau biology significantly hampers the identification and validation of the most impactful target. Recent clinical trials using antibodies against various forms of tau have come up short in improving cognitive readout.MethodA Quantitative Systems Pharmacology computer platform of tau biology has been developed including tau species diversity, neuronal activity‐dependent tau secretion, tau binding to membrane surface receptors, subsequent internalization at the synaptic cleft and along the axonal membrane following a diffusion gradient, slow motor‐mediated axonal transport, intracellular degradation and templating of monomeric tau by seed‐competent tau to form higher‐order aggregates. Polysynaptic progression is implemented by modeling secretion from the first synapse and uptake in the second axon.ResultThe platform was calibrated to reproduce preclinical experiments from in‐vitro cellular assays and after injection of brain extract in Tg models. The model is humanized using published data on seed‐competent tau from postmortem AD brain at different Braak stages and spatio‐temporal progression over neuronal projections. By exploring the effect of target mediated drug disposal of antibodies, we show that depending upon the disease state only between 15 and 30% of misfolded tau protein can be captured by antibodies currently in clinical trials. Introducing PSP specific uptake parameters from preclinical experiments, the simulations suggest an even lower efficacy due to the higher affinity of PSP‐tau for membrane surface receptors. We further identify conditions under which specificity (i.e. monomeric vs misfolded tau) of antibodies outweigths sensitivity (affinity). Finally, the simulations suggest that reduced oligomerization and increased degradation have a greater impact on the accumulation of seed‐competent tau in distal regions, likely because they act during the long intracellular residence of misfolded tau protein.ConclusionAdvanced Quantitative Systems Pharmacology computer modeling combining drug exposure with mechanistic modeling allows to ‘humanize’ preclinical experiments and aims to support tauopathy R&D projects.

Transcriptome analyses of 7-day-old zebrafish larvae possessing a familial Alzheimer\u2019s disease-like mutation in psen1 indicate effects on oxidative phosphorylation, ECM and MCM functions, and iron homeostasis

BackgroundEarly-onset familial Alzheimer’s disease (EOfAD) is promoted by dominant mutations, enabling the study of Alzheimer’s disease (AD) pathogenic mechanisms through generation of EOfAD-like mutations in animal models. In a previous study, we generated an EOfAD-like mutation, psen1Q96_K97del, in zebrafish and performed transcriptome analysis comparing entire brains from 6-month-old wild type and heterozygous mutant fish. We identified predicted effects on mitochondrial function and endolysosomal acidification. Here we aimed to determine whether similar effects occur in 7 day post fertilization (dpf) zebrafish larvae that might be exploited in screening of chemical libraries to find ameliorative drugs.ResultsWe generated clutches of wild type and heterozygous psen1Q96_K97del 7 dpf larvae using a paired-mating strategy to reduce extraneous genetic variation before performing a comparative transcriptome analysis. We identified 228 differentially expressed genes and performed various bioinformatics analyses to predict cellular functions.ConclusionsOur analyses predicted a significant effect on oxidative phosphorylation, consistent with our earlier observations of predicted effects on ATP synthesis in adult heterozygous psen1Q96_K97del brains. The dysregulation of minichromosome maintenance protein complex (MCM) genes strongly contributed to predicted effects on DNA replication and the cell cycle and may explain earlier observations of genome instability due to PSEN1 mutation. The upregulation of crystallin gene expression may be a response to defective activity of mutant Psen1 protein in endolysosomal acidification. Genes related to extracellular matrix (ECM) were downregulated, consistent with previous studies of EOfAD mutant iPSC neurons and postmortem late onset AD brains. Also, changes in expression of genes controlling iron ion transport were observed without identifiable changes in the prevalence of transcripts containing iron responsive elements (IREs) in their 3′ untranslated regions (UTRs). These changes may, therefore, predispose to the apparent iron dyshomeostasis previously observed in 6-month-old heterozygous psen1Q96_K97del EOfAD-like mutant brains.

Comparison of size distribution and (Pro249-Ser258) epitope exposure in in vitro and in vivo derived Tau fibrils

BackgroundAlthough several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood. The aim of this study is to characterize Tau seeds by their size and seeding capacity.MethodsTau aggregates were isolated from postmortem AD brain tissue and separated from low molecular weight species by sucrose gradient ultracentrifugation. Biochemical characterization of the different fractions was done by non-reducing Western blotting and aggregate-specific immuno-assays using in house developed anti-Tau monoclonal antibodies, including PT76 which binds to an epitope close to the microtubule-binding domain and, hence, also to K18. Seeding efficiency was then assessed in HEK293 cells expressing K18 FRET sensors.ResultsWe observed that upon sonication of Tau aggregates different size-distributed tau aggregates are obtained. In biochemical assays, these forms show higher signals than the non-sonicated material in some aggregation-specific Tau assays. This could be explained by an increased epitope exposure of the smaller aggregates created by the sonication. By analyzing human brain derived and recombinant (K18) Tau aggregates in a cellular FRET assay, it was observed that, in the absence of transfection reagent, sonicated aggregates showed higher aggregation induction. Preparations also showed altered profiles on native PAGE upon sonication and we could further separate different aggregate species based on their molecular weight via sucrose gradients.ConclusionsThis study further elucidates the molecular properties regarding relative aggregate size and seeding efficiency of sonicated vs. non-sonicated high molecular weight Tau species. This information will provide a better knowledge on how sonication, a commonly used technique in the field of study of Tau aggregation, impacts the aggregates. In addition, the description of PT76-based aggregation specific assay is a valuable tool to quantify K18 and human AD Tau fibrils.

The lipid phosphatase Synaptojanin 1 undergoes a significant alteration in expression and solubility and is associated with brain lesions in Alzheimer\u2019s disease

Synaptojanin 1 (SYNJ1) is a brain-enriched lipid phosphatase critically involved in autophagosomal/endosomal trafficking, synaptic vesicle recycling and metabolism of phosphoinositides. Previous studies suggest that SYNJ1 polymorphisms have significant impact on the age of onset of Alzheimer’s disease (AD) and that SYNJ1 is involved in amyloid-induced toxicity. Yet SYNJ1 protein level and cellular localization in post-mortem human AD brain tissues have remained elusive. This study aimed to examine whether SYNJ1 localization and expression are altered in post-mortem AD brains. We found that SYNJ1 is accumulated in Hirano bodies, plaque-associated dystrophic neurites and some neurofibrillary tangles (NFTs). SYNJ1 immunoreactivity was higher in neurons and in the senile plaques in AD patients carrying one or two ApolipoproteinE (APOE) ε4 allele(s). In two large cohorts of APOE-genotyped controls and AD patients, SYNJ1 transcripts were significantly increased in AD temporal isocortex compared to control. There was a significant increase in SYNJ1 transcript in APOEε4 carriers compared to non-carriers in AD cohort. SYNJ1 was systematically co-enriched with PHF-tau in the sarkosyl-insoluble fraction of AD brain. In the RIPA-insoluble fraction containing protein aggregates, SYNJ1 proteins were significantly increased and observed as a smear containing full-length and cleaved fragments in AD brains. In vitro cleavage assay showed that SYNJ1 is a substrate of calpain, which is highly activated in AD brains. Our study provides evidence of alterations in SYNJ1 mRNA level and SYNJ1 protein degradation, solubility and localization in AD brains.

Suppressing aberrant phospholipase D1 signaling in 3xTg Alzheimer\u2019s disease mouse model promotes synaptic resilience

Current approaches in treatment of Alzheimer’s disease (AD) is focused on early stages of cognitive decline. Identifying therapeutic targets that promote synaptic resilience during early stages may prevent progressive memory deficits by preserving memory mechanisms. We recently reported that the inducible isoform of phospholipase D (PLD1) was significantly increased in synaptosomes from post-mortem AD brains compared to age-matched controls. Using mouse models, we reported that the aberrantly elevated neuronal PLD1 is key for oligomeric amyloid driven synaptic dysfunction and underlying memory deficits. Here, we demonstrate that chronic inhibition using a well-tolerated PLD1 specific small molecule inhibitor is sufficient to prevent the progression of synaptic dysfunction during early stages in the 3xTg-AD mouse model. Firstly, we report prevention of cognitive decline in the inhibitor-treated group using novel object recognition (NOR) and fear conditioning (FC). Secondly, we provide electrophysiological assessment of better synaptic function in the inhibitor-treated group. Lastly, using Golgi staining, we report that preservation of dendritic spine integrity as one of the mechanisms underlying the action of the small molecule inhibitor. Collectively, these studies provide evidence for inhibition of PLD1 as a potential therapeutic strategy in preventing progression of cognitive decline associated with AD and related dementia.

INVESTIGATING THE ROLE OF SORTILIN-RELATED RECEPTOR (SORL1/SORLA) IN HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED NEURAL CELLS AS A THERAPEUTIC TARGET FOR ALZHEIMER’S DISEASE

Recent compelling genetic evidence has identified genes involved in endocytosis and vesicular trafficking including SORL1 as AD risk genes. Decreased expression of SORLA has been repeatedly observed in postmortem human AD brains especially in late onset sporadic AD (SAD) which is the most common form of AD. Eventually, several studies linked SORLA expression with production and deposition of the pathogenic protein β amyloid peptide which is one of the hallmarks of AD pathogenesis. However, most of these studies were done using mouse models and non-human cell lines and role of SORLA in amyloid β production in human neural cells remains unclear. SORL1 was overexpressed in clonal populations of human pluripotent stem cells (hiPSCs) using the piggybac transposon system. Control and SORL1overexpressed hiPSCs were then differentiated into neural cells. Amount of Aβ-40 and Aβ-42 released was measured using ELISA in hiPSC derived neural cells. Additionally, intra-cellular trafficking was examined using immunocytochemistry and cell biology assays. Specifically, to assess transport into lysosomes, pH sensitive fluorescent dye Phrodo conjugated with Aβ was used. Overexpression of SORL1 in hiPSC derived human neural cells resulted in decreased Aβ-40 and Aβ-42 released into media as measured by ELISA. This decrease can be caused by a possible increase in Aβ transport into lysosomes for degradation. In support of this idea, increased Aβ transport into lysosomes was observed in SORL1 overexpressed neural cells as compared to control cells using the Phrodo assay. Decreased Aβ release could also result from increased degradation of Amyloid precursor protein (APP). In support of this hypothesis, we showed increased colocalization of APP with a lysosomal marker, Lysosome associated membrane protein (LAMP1) using immunocytochemistry. Overall, these results suggest that overexpression of SORL1 could lead to decreased amyloid β production in part due to increased transport of Aβ and APP into lysosomes for degradation in human neural cells. Our findings reveal a previously unidentified role of SORL1 in hiPSC derived neural cells, information that may be useful in exploring not just SORLA, but other sorting receptors expressed in human neurons as therapeutic targets for AD.

Optimization of solid-phase extraction (SPE) in the preparation of [18F]D3FSP: A new PET imaging agent for mapping Aβ plaques

IntroductionAlzheimer's disease is a common neurodegenerative disease that is characterized by the presence of Aβ plaques in the brain. The FDA has approved the use of Amyvid (florbetapir f18, AV-45) as a PET imaging agent for detecting Aβ plaques in the living human brain. In an attempt to reduce N-demethylation in vivo by taking advantage of more stable C-D bonds, an analog of AV-45, [18F]D3FSP ([18F]7), was synthesized to improve image contrast for detecting and monitoring the Aβ plaques. A convenient and improved preparation of [18F]D3FSP ([18F]7) is needed for widespread clinical application. We report herein the optimization of the radiosynthesis and solid-phase extraction (SPE) procedure. MethodsRadiosyntheses of [18F]D3FSP ([18F]7) under different fluorination conditions were evaluated, and the intermediate, containing an N-Boc protecting group, was deprotected using different acids. One of the major objectives was to simplify the final purification step via SPE to avoid the commonly employed HPLC purification and maximize the radiochemical yields of [18F]D3FSP ([18F]7) while simultaneously removing several chemical impurities (pseudocarriers). Washing various solid-phase cartridges with different combinations of ethanol/water and acetonitrile/water was explored to optimize the purification step. To evaluate the potential interference in Aβ plaques imaging from the presence of pseudocarriers, each chemical was identified and quantified by LC/MS and HPLC. An in vitro binding assay was employed to evaluate the binding affinities of [18F]D3FSP ([18F]7) and the pseudocarriers to Aβ plaques using postmortem AD brain tissue. ResultsUsing the optimized radiosynthesis method and SPE purification, the final dose of [18F]D3FSP ([18F]7) was obtained in 50 min with a very low content of pseudocarriers (21.7 ± 5.5 μg). The radiochemical yield was 44.4 ± 5.7% (decay corrected), and the radiochemical purity was >95%. SPE-purified doses of [18F]D3FSP ([18F]7) displayed excellent binding affinity and specificity for Aβ plaques as measured in an in vitro binding assay using AD brain homogenates, and the OH-pseudocarrier, 8 (Ki = 19.5 ± 0.5 nM), and the Cl-pseudocarrier, 10 (Ki = 18.6 ± 3.9 nM), showed lower binding affinities for Aβ plaques than those of AV-45 (Ki = 8.6 ± 0.5 nM) and D3FSP, 7 (Ki = 9.8 ± 0.5 nM). ConclusionsAn optimized radiosynthesis and fast SPE purification method suitable for the preparation of clinical doses of [18F]D3FSP ([18F]7) was accomplished. The results of quality control tests and binding studies suggested that the SPE-purified doses of [18F]D3FSP ([18F]7) are appropriate for imaging Aβ plaques in the human brain.

Elevated cellular cholesterol in Familial Alzheimer's presenilin 1 mutation is associated with lipid raft localization of β-amyloid precursor protein.

Familial Alzheimer’s disease (FAD)-associated presenilin 1 (PS1) serves as a catalytic subunit of γ-secretase complex, which mediates the proteolytic liberation of β-amyloid (Aβ) from β-amyloid precursor protein (APP). In addition to its proteolytic role, PS1 is involved in non-proteolytic functions such as protein trafficking and ion channel regulation. Furthermore, postmortem AD brains as well as AD patients showed dysregulation of cholesterol metabolism. Since cholesterol has been implicated in regulating Aβ production, we investigated whether the FAD PS1-associated cholesterol elevation could influence APP processing. We found that in CHO cells stably expressing FAD-associated PS1 ΔE9, total cholesterol levels are elevated compared to cells expressing wild-type PS1. We also found that localization of APP in cholesterol-enriched lipid rafts is substantially increased in the mutant cells. Reducing the cholesterol levels by either methyl-β-cyclodextrin or an inhibitor of CYP51, an enzyme mediating the elevated cholesterol in PS1 ΔE9-expressing cells, significantly reduced lipid raft-associated APP. In contrast, exogenous cholesterol increased lipid raft-associated APP. These data suggest that in the FAD PS1 ΔE9 cells, the elevated cellular cholesterol level contributes to the altered APP processing by increasing APP localized in lipid rafts.

GJA1 (connexin43) is a key regulator of Alzheimer\u2019s disease pathogenesis

GJA1 (connexin43) has been predicted as the top key driver of an astrocyte enriched subnetwork associated with Alzheimer’s disease (AD). In this study, we comprehensively examined GJA1 expression across 29 transcriptomic and proteomic datasets from post-mortem AD and normal control brains. We demonstrated that GJA1 was strongly associated with AD amyloid and tau pathologies and cognitive functions. RNA sequencing analysis of Gja1−/− astrocytes validated that Gja1 regulated the subnetwork identified in AD, and many genes involved in Aβ metabolism. Astrocytes lacking Gja1 showed reduced Apoe protein levels as well as impaired Aβ phagocytosis. Consistent with this, wildtype neurons co-cultured with Gja1−/− astrocytes contained higher levels of Aβ species than those with wildtype astrocytes. Moreover, Gja1−/− astrocytes was more neuroprotective under Aβ stress. Our results underscore the importance of GJA1 in AD pathogenesis and its potential for further investigation as a promising pharmacological target in AD.

Novel human neuronal tau model exhibiting neurofibrillary tangles and transcellular propagation

Tauopathies are a class of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia and progressive supranuclear palsy, which are associated with the pathological aggregation of tau protein into neurofibrillary tangles (NFT). Studies have characterized tau as a “prion-like” protein given its ability to form distinct, stable amyloid conformations capable of transcellular and multigenerational propagation in clonal fashion. It has been proposed that progression of tauopathy could be due to the prion-like propagation of tau, suggesting the possibility that end-stage pathologies, like NFT formation, may require an instigating event such as tau seeding. To investigate this, we applied a novel human induced pluripotent stem cell (hiPSC) system we have developed to serve as a human neuronal model. We introduced the tau repeat domain (tau-RD) with P301L and V337M (tau-RD-LM) mutations into hiPSC-derived neurons and observed expression of tau-RD at levels similar to total tau in postmortem AD brains. Tau aggregation occurred without the addition of recombinant tau fibrils. The conditioned media from tau-RD cultures contained tau-RD seeds, which were capable of inducing aggregate formation in homotypic mode in non-transduced recipient neuronal cultures. The resultant NFTs were thioflavin-positive, silver stain-positive, and assumed fibrillary appearance on transmission electron microscopy (TEM) with immunogold, which revealed paired helical filament 1 (PHF1)-positive NFTs, representing possible recruitment of endogenous tau in the aggregates. Functionally, expression of tau-RD caused neurotoxicity that manifested as axon retraction, synaptic density reduction, and enlargement of lysosomes. The results of our hiPSC study were reinforced by the observation that Tau-RD-LM is excreted in exosomes, which mediated the transfer of human tau to wild-type mouse neurons in vivo. Our hiPSC human neuronal system provides a model for further studies of tau aggregation and pathology as well as a means to study transcellular propagation and related neurodegenerative mechanisms.

Clinical Aspects of Glucose Metabolism and Chronic Disease.

The burden of chronic disease is an emerging world health problem. Advances made in the treatment of individual disease states often fail to consider multimorbidity patterns in clinical research models. Adjusting for age as a confounder ignores its contribution as a powerful risk factor for most chronic diseases. Sarcopenia is an age-related loss of skeletal muscle mass, which is accelerated by chronic inflammation and its resulting cascade of cytokines. Skeletal muscle loss results in insulin resistance, hyperglycemia, and altered mitochondrial glucose signaling pathways. Vascular disease in the brain may alter blood-brain barrier function, allowing transport of substances into the brain which adversely affect the "astrocyte-centric" subunit. Neurogenesis that provides neuronal plasticity is impaired in the diabetic brain, while insulin resistance markers such as insulin-like growth factor (IGF-1) and insulin receptor substrate (IRS-1) are associated with poor cognitive performance. Advanced glycation end products generated by chronic hyperglycemia are found in postmortem AD brain. Intranasal insulin administration, a preferential route for CNS delivery, improved cognitive function in healthy adults, without affecting circulating levels of insulin or glucose. Exercise has demonstrated a neuroprotective effect through induction of antioxidative enzymes, neurotrophic, and vascular endothelial growth factors. Sarcopenia appears to be a dynamic process and is potentially reversible with attention to nutrition and cardiovascular fitness. Early detection and intervention may slow the progression of multimortality disease states and should be a focus of worldwide health systems.

RGS2 expression predicts amyloid-β sensitivity, MCI and Alzheimer's disease: genome-wide transcriptomic profiling and bioinformatics data mining.

Alzheimer's disease (AD) is the most frequent cause of dementia. Misfolded protein pathological hallmarks of AD are brain deposits of amyloid-β (Aβ) plaques and phosphorylated tau neurofibrillary tangles. However, doubts about the role of Aβ in AD pathology have been raised as Aβ is a common component of extracellular brain deposits found, also by in vivo imaging, in non-demented aged individuals. It has been suggested that some individuals are more prone to Aβ neurotoxicity and hence more likely to develop AD when aging brains start accumulating Aβ plaques. Here, we applied genome-wide transcriptomic profiling of lymphoblastoid cells lines (LCLs) from healthy individuals and AD patients for identifying genes that predict sensitivity to Aβ. Real-time PCR validation identified 3.78-fold lower expression of RGS2 (regulator of G-protein signaling 2; P=0.0085) in LCLs from healthy individuals exhibiting high vs low Aβ sensitivity. Furthermore, RGS2 showed 3.3-fold lower expression (P=0.0008) in AD LCLs compared with controls. Notably, RGS2 expression in AD LCLs correlated with the patients' cognitive function. Lower RGS2 expression levels were also discovered in published expression data sets from postmortem AD brain tissues as well as in mild cognitive impairment and AD blood samples compared with controls. In conclusion, Aβ sensitivity phenotyping followed by transcriptomic profiling and published patient data mining identified reduced peripheral and brain expression levels of RGS2, a key regulator of G-protein-coupled receptor signaling and neuronal plasticity. RGS2 is suggested as a novel AD biomarker (alongside other genes) toward early AD detection and future disease modifying therapeutics.