Parkinson's disease and multiple system atrophy patient iPSC-derived oligodendrocytes exhibit alpha-synuclein-induced changes in maturation and immune reactive properties.

SignificanceOur results demonstrate the existence of early cellular pathways and network alterations in oligodendrocytes in the alpha-synucleinopathies Parkinson's disease and multiple system atrophy. They further reveal the involvement of an immune component triggered by alpha-synuclein protein, as well as a connection between (epi)genetic changes and immune reactivity in multiple system atrophy. The knowledge generated in this study could be used to devise novel therapeutic approaches to treat synucleinopathies.

Upregulation of APP endocytosis by neuronal aging drives amyloid-dependent synapse loss.

Neuronal aging increases the risk of late-onset Alzheimer's disease. During normal aging, synapses decline, and ß-amyloid (Aß) accumulates intraneuronally. However, little is known about the underlying cell biological mechanisms. We studied neuronal aging using normal-aged brain and aged mouse primary neurons that accumulate lysosomal lipofuscin and show synapse loss. We identified the upregulation of amyloid precursor protein (APP) endocytosis as a neuronal aging mechanism that potentiates APP processing and Aß production in vitro and in vivo. The increased APP endocytosis may contribute to the early endosome enlargement observed in the aged brain. Mechanistically, we showed that clathrin-dependent APP endocytosis requires F-actin and that clathrin and endocytic F-actin increase with neuronal aging. Finally, Aß production inhibition reverts synaptic decline in aged neurons, whereas Aß accumulation, promoted by endocytosis upregulation in younger neurons, recapitulates aging-related synapse decline. Overall, we identify APP endocytosis upregulation as a potential mechanism of neuronal aging and, thus, a novel target to prevent late-onset Alzheimer's disease. This article has an associated First Person interview with the first author of the paper.

APOE4 Affects Basal and NMDAR-Mediated Protein Synthesis in Neurons by Perturbing Calcium Homeostasis.

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans, APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the predisposition for Alzheimer's disease (AD). The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4-induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons or synaptoneurosomes to APOE4 leads to a significant decrease in global protein synthesis. Primary cortical neurons were derived from male and female embryos of Sprague Dawley (SD) rats or C57BL/6J mice. Synaptoneurosomes were prepared from P30 male SD rats. APOE4 treatment also abrogates the NMDA-mediated translation response indicating an alteration of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure of neurons to APOE3 causes a short burst of calcium through NMDA receptors (NMDARs) leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-type voltage-gated calcium channels (L-VGCCs), thereby causing sustained translation inhibition through eukaryotic translation elongation factor 2 (eEF2) phosphorylation, which in turn disrupts the NMDAR response. Thus, we show that APOE4 affects basal and activity-mediated protein synthesis responses in neurons by affecting calcium homeostasis.SIGNIFICANCE STATEMENT Defective protein synthesis has been shown as an early defect in familial Alzheimer's disease (AD). However, this has not been studied in the context of sporadic AD, which constitutes the majority of cases. In our study, we show that Apolipoprotein E4 (APOE4), the predominant risk factor for AD, inhibits global protein synthesis in neurons. APOE4 also affects NMDA activity-mediated protein synthesis response, thus inhibiting synaptic translation. We also show that the defective protein synthesis mediated by APOE4 is closely linked to the perturbation of calcium homeostasis caused by APOE4 in neurons. Thus, we propose the dysregulation of protein synthesis as one of the possible molecular mechanisms to explain APOE4-mediated synaptic and cognitive defects. Hence, the study not only suggests an explanation for the APOE4-mediated predisposition to AD, it also bridges the gap in understanding APOE4-mediated pathology.

Galectin-3 is elevated in CSF and is associated with Aß deposits and tau aggregates in brain tissue in Alzheimer's disease.

Galectin-3 (Gal-3) is a beta-galactosidase binding protein involved in microglial activation in the central nervous system (CNS). We previously demonstrated the crucial deleterious role of Gal-3 in microglial activation in Alzheimer's disease (AD). Under AD conditions, Gal-3 is primarily expressed by microglial cells clustered around Aß plaques in both human and mouse brain, and knocking out Gal-3 reduces AD pathology in AD-model mice. To further unravel the importance of Gal-3-associated inflammation in AD, we aimed to investigate the Gal-3 inflammatory response in the AD continuum. First, we measured Gal-3 levels in neocortical and hippocampal tissue from early-onset AD patients, including genetic and sporadic cases. We found that Gal-3 levels were significantly higher in both cortex and hippocampus in AD subjects. Immunohistochemistry revealed that Gal-3+ microglial cells were associated with amyloid plaques of a larger size and more irregular shape and with neurons containing tau-inclusions. We then analyzed the levels of Gal-3 in cerebrospinal fluid (CSF) from AD patients (n = 119) compared to control individuals (n = 36). CSF Gal-3 levels were elevated in AD patients compared to controls and more strongly correlated with tau (p-Tau181 and t-tau) and synaptic markers (GAP-43 and neurogranin) than with amyloid-ß. Lastly, principal component analysis (PCA) of AD biomarkers revealed that CSF Gal-3 clustered and associated with other CSF neuroinflammatory markers, including sTREM-2, GFAP, and YKL-40. This neuroinflammatory component was more highly expressed in the CSF from amyloid-ß positive (A+), CSF p-Tau181 positive (T+), and biomarker neurodegeneration positive/negative (N+/-) (A + T + N+/-) groups compared to the A + T-N- group. Overall, Gal-3 stands out as a key pathological biomarker of AD pathology that is measurable in CSF and, therefore, a potential target for disease-modifying therapies involving the neuroinflammatory response.

Sphingosine 1-Phoshpate Receptors are Located in Synapses and Control Spontaneous Activity of Mouse Neurons in Culture.

Sphingosine-1-phosphate (S1P) is best known for its roles as vascular and immune regulator. Besides, it is also present in the central nervous system (CNS) where it can act as neuromodulator via five S1P receptors (S1PRs), and thus control neurotransmitter release. The distribution of S1PRs in the active zone and postsynaptic density of CNS synapses remains unknown. In the current study, we investigated the localization of S1PR1-5 in synapses of the mouse cortex. Cortical nerve terminals purified in a sucrose gradient were endowed with all five S1PRs. Further subcellular fractionation of cortical nerve terminals revealed S1PR2 and S1PR4 immunoreactivity in the active zone of presynaptic nerve terminals. Interestingly, only S1PR2 and S1PR3 immunoreactivity was found in the postsynaptic density. All receptors were present outside the active zone of nerve terminals. Neurons in the mouse cortex and primary neurons in culture showed immunoreactivity against all five S1PRs, and Ca2+ imaging revealed that S1P inhibits spontaneous neuronal activity in a dose-dependent fashion. When testing selective agonists for each of the receptors, we found that only S1PR1, S1PR2 and S1PR4 control spontaneous neuronal activity. We conclude that S1PR2 and S1PR4 are located in the active zone of nerve terminals and inhibit neuronal activity. Future studies need to test whether these receptors modulate stimulation-induced neurotransmitter release.

Correlative imaging to resolve molecular structures in individual cells: Substrate validation study for super-resolution infrared microspectroscopy.

Light microscopy has been a favorite tool of biological studies for almost a century, recently producing detailed images with exquisite molecular specificity achieving spatial resolution at nanoscale. However, light microscopy is insufficient to provide chemical information as a standalone technique. An increasing amount of evidence demonstrates that optical photothermal infrared microspectroscopy (O-PTIR) is a valuable imaging tool that can extract chemical information to locate molecular structures at submicron resolution. To further investigate the applicability of sub-micron infrared microspectroscopy for biomedical applications, we analyzed the contribution of substrate chemistry to the infrared spectra acquired from individual neurons grown on various imaging substrates. To provide an example of correlative immunofluorescence/O-PTIR imaging, we used immunofluorescence to locate specific organelles for O-PTIR measurement, thus capturing molecular structures at the sub-cellular level directly in cells, which is not possible using traditional infrared microspectroscopy or immunofluorescence microscopy alone.

Neuronal spreading and plaque induction of intracellular Aß and its disruption of Aß homeostasis.

The amyloid-beta peptide (Aß) is thought to have prion-like properties promoting its spread throughout the brain in Alzheimer's disease (AD). However, the cellular mechanism(s) of this spread remains unclear. Here, we show an important role of intracellular Aß in its prion-like spread. We demonstrate that an intracellular source of Aß can induce amyloid plaques in vivo via hippocampal injection. We show that hippocampal injection of mouse AD brain homogenate not only induces plaques, but also damages interneurons and affects intracellular Aß levels in synaptically connected brain areas, paralleling cellular changes seen in AD. Furthermore, in a primary neuron AD model, exposure of picomolar amounts of brain-derived Aß leads to an apparent redistribution of Aß from soma to processes and dystrophic neurites. We also observe that such neuritic dystrophies associate with plaque formation in AD-transgenic mice. Finally, using cellular models, we propose a mechanism for how intracellular accumulation of Aß disturbs homeostatic control of Aß levels and can contribute to the up to 10,000-fold increase of Aß in the AD brain. Our data indicate an essential role for intracellular prion-like Aß and its synaptic spread in the pathogenesis of AD.

Amyloid Structural Changes Studied by Infrared Microspectroscopy in Bigenic Cellular Models of Alzheimer's Disease.

Alzheimer's disease affects millions of lives worldwide. This terminal disease is characterized by the formation of amyloid aggregates, so-called amyloid oligomers. These oligomers are composed of ß-sheet structures, which are believed to be neurotoxic. However, the actual secondary structure that contributes most to neurotoxicity remains unknown. This lack of knowledge is due to the challenging nature of characterizing the secondary structure of amyloids in cells. To overcome this and investigate the molecular changes in proteins directly in cells, we used synchrotron-based infrared microspectroscopy, a label-free and non-destructive technique available for in situ molecular imaging, to detect structural changes in proteins and lipids. Specifically, we evaluated the formation of ß-sheet structures in different monogenic and bigenic cellular models of Alzheimer's disease that we generated for this study. We report on the possibility to discern different amyloid signatures directly in cells using infrared microspectroscopy and demonstrate that bigenic (amyloid-ß, α-synuclein) and (amyloid-ß, Tau) neuron-like cells display changes in ß-sheet load. Altogether, our findings support the notion that different molecular mechanisms of amyloid aggregation, as opposed to a common mechanism, are triggered by the specific cellular environment and, therefore, that various mechanisms lead to the development of Alzheimer's disease.

APP depletion alters selective pre- and post-synaptic proteins.

The normal role of Alzheimer's disease (AD)-linked amyloid precursor protein (APP) in the brain remains incompletely understood. Previous studies have reported that lack of APP has detrimental effects on spines and electrophysiological parameters. APP has been described to be important in synaptic pruning during development. The effect of APP knockout on mature synapses is complicated by this role in development. We previously reported on differential changes in synaptic proteins and receptors in APP mutant AD transgenic compared to wild-type neurons, which revealed selective decreases in levels of pre- and post-synaptic proteins, including of surface glutamate receptors. In the present study, we undertook a similar analysis of synaptic composition but now in APP knockout compared to wild-type mouse neurons. Here we demonstrate alterations in levels of selective pre- and post-synaptic proteins and receptors in APP knockout compared to wild-type mouse primary neurons in culture and brains of mice in youth and adulthood. Remarkably, we demonstrate selective increases in levels of synaptic proteins, such as GluA1, in neurons with APP knockout and with RNAi knockdown, which tended to be opposite to the reductions seen in AD transgenic APP mutant compared to wild-type neurons. These data reinforce that APP is important for the normal composition of synapses.

Poly(propylene imine) dendrimers with histidine-maltose shell as novel type of nanoparticles for synapse and memory protection.

Poly(propylene imine) dendrimers have been shown to be promising 3-dimensional polymers for the use in the pharmaceutical and biomedical applications. Our aims of this study were first, to synthesize a novel type of dendrimer with poly(propylene imine) core and maltose-histidine shell (G4HisMal) assessing if maltose-histidine shell can improve the biocompatibility and the ability to cross the blood-brain barrier, and second, to investigate the potential of G4HisMal to protect Alzheimer disease transgenic mice from memory impairment. Our data demonstrate that G4HisMal has significantly improved biocompatibility and ability to cross the blood-brain barrier in vivo. Therefore, we suggest that a maltose-histidine shell can be used to improve biocompatibility and ability to cross the blood-brain barrier of dendrimers. Moreover, G4HisMal demonstrated properties for synapse and memory protection when administered to Alzheimer disease transgenic mice. Therefore, G4HisMal can be considered as a promising drug candidate to prevent Alzheimer disease via synapse protection.

Prion-like seeding and nucleation of intracellular amyloid-ß.

Alzheimer's disease (AD) brain tissue can act as a seed to accelerate aggregation of amyloid-ß (Aß) into plaques in AD transgenic mice. Aß seeds have been hypothesized to accelerate plaque formation in a prion-like manner of templated seeding and intercellular propagation. However, the structure(s) and location(s) of the Aß seeds remain unknown. Moreover, in contrast to tau and α-synuclein, an in vitro system with prion-like Aß has not been reported. Here we treat human APP expressing N2a cells with AD transgenic mouse brain extracts to induce inclusions of Aß in a subset of cells. We isolate cells with induced Aß inclusions and using immunocytochemistry, western blot and infrared spectroscopy show that these cells produce oligomeric Aß over multiple replicative generations. Further, we demonstrate that cell lysates of clones with induced oligomeric Aß can induce aggregation in previously untreated N2a APP cells. These data strengthen the case that Aß acts as a prion-like protein, demonstrate that Aß seeds can be intracellular oligomers and for the first time provide a cellular model of nucleated seeding of Aß.

ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-ß accumulation.

Intracellular amyloid-ß (Aß) accumulation is a key feature of early Alzheimer's disease and precedes the appearance of Aß in extracellular plaques. Aß is generated through proteolytic processing of amyloid precursor protein (APP), but the intracellular site of Aß production is unclear. APP has been localized to multivesicular bodies (MVBs) where sorting of APP onto intraluminal vesicles (ILVs) could promote amyloidogenic processing, or reduce Aß production or accumulation by sorting APP and processing products to lysosomes for degradation. Here, we show that APP localizes to the ILVs of a subset of MVBs that also traffic EGF receptor (EGFR), and that it is delivered to lysosomes for degradation. Depletion of the endosomal sorting complexes required for transport (ESCRT) components, Hrs (also known as Hgs) or Tsg101, inhibited targeting of APP to ILVs and the subsequent delivery to lysosomes, and led to increased intracellular Aß accumulation. This was accompanied by dramatically decreased Aß secretion. Thus, the early ESCRT machinery has a dual role in limiting intracellular Aß accumulation through targeting of APP and processing products to the lysosome for degradation, and promoting Aß secretion.

Plaque formation and the intraneuronal accumulation of ß-amyloid in Alzheimer's disease.

Amyloid plaques and neurofibrillary tangles (NFTs) in the brain are the neuropathological hallmarks of Alzheimer's disease (AD). Amyloid plaques are composed of ß-amyloid peptides (Aß), while NFTs contain hyperphosphorylated tau proteins. Patients with familial AD who have mutations in the amyloid precursor protein (APP) gene have either increased production of Aß or generate more aggregation-prone forms of Aß. The findings of familial AD mutations in the APP gene suggest that Aß plays a central role in the pathophysiology of AD. Aß42, composed of 42 amino acid residues, aggregates readily and is considered to form amyloid plaque. However, the processes of plaque formation are still not well known. It is generally thought that Aß is secreted into the extracellular space and aggregates to form amyloid plaques. Aß as extracellular aggregates and amyloid plaques are thought to be toxic to the surrounding neurons. The intraneuronal accumulation of Aß has more recently been demonstrated and is reported to be involved in synaptic dysfunction, cognitive impairment, and the formation of amyloid plaques in AD. We herein provide an overview of the process of the intraneuronal accumulation of Aß and plaque formation, and discuss its implications for the pathology, early diagnosis, and therapy of AD.

ADAM10 and BACE1 are localized to synaptic vesicles.

Synaptic degeneration and accumulation of the neurotoxic amyloid ß-peptide (Aß) in the brain are hallmarks of Alzheimer disease. Aß is produced by sequential cleavage of the amyloid precursor protein (APP), by the ß-secretase ß-site APP cleaving enzyme 1 (BACE1) and γ-secretase. However, Aß generation is precluded if APP is cleaved by the α-secretase ADAM10 instead of BACE1. We have previously shown that Aß can be produced locally at the synapse. To study the synaptic localization of the APP processing enzymes we used western blotting to demonstrate that, compared to total brain homogenate, ADAM10 and BACE1 were greatly enriched in synaptic vesicles isolated from rat brain using controlled-pore glass chromatography, whereas Presenilin1 was the only enriched component of the γ-secretase complex. Moreover, we detected ADAM10 activity in synaptic vesicles and enrichment of the intermediate APP-C-terminal fragments (APP-CTFs). We confirmed the western blotting findings using in situ proximity ligation assay to demonstrate close proximity of ADAM10 and BACE1 with the synaptic vesicle marker synaptophysin in intact mouse primary hippocampal neurons. In contrast, only sparse co-localization of active γ-secretase and synaptophysin was detected. These results indicate that the first step of APP processing occurs in synaptic vesicles whereas the final step is more likely to take place elsewhere.

The inside-out amyloid hypothesis and synapse pathology in Alzheimer's disease.

Cumulative evidence in brains and cultured neurons of Alzheimer's disease (AD) transgenic mouse models, as well as in human postmortem AD brains, highlights that age-related increases in ß-amyloid peptide (Aß), particularly in endosomes near synapses, are involved in early synapse dysfunction. Our immunoelectron microscopy and high-resolution immunofluorescence microscopy studies show that this early subcellular Aß accumulation leads to progressive Aß aggregation and pathology, particularly within dystrophic neurites and synapses. These studies confirm that neuritic/synaptic Aß accumulation is the nidus of plaque formation. Aß-dependent synapse pathology in AD models is modulated by synaptic activity and is plaque independent. The amyloid precursor protein (APP) is normally transported down neurites and appears to be preferentially processed to Aß at synapses. Synapses are sites of early Aß accumulation and aberrant tau phosphorylation in AD, which alter the synaptic composition at early stages of the disease. Elucidating the normal role of APP, and potentially of Aß, at synapses should provide important insights into the mechanism(s) of Aß-induced synapse dysfunction in AD and how to therapeutically mitigate these dysfunctions.

Intracellular Amyloid-ß in the Normal Rat Brain and Human Subjects and Its relevance for Alzheimer's Disease.

BACKGROUND: Amyloid-ß (Aß) is a normal product of neuronal activity, including that of the aggregation-prone Aß42 variant that is thought to cause Alzheimer's disease (AD). Much knowledge about AD comes from studies of transgenic rodents expressing mutated human amyloid-ß protein precursor (AßPP) to increase Aß production or the Aß42/40 ratio. Yet, little is known about the normal expression of Aß42 in rodent brains. OBJECTIVE: To characterize the brain-wide expression of Aß42 throughout the life span of outbred Wistar rats, and to relate these findings to brains of human subjects without neurological disease. METHODS: Aß42 immunolabeling of 12 Wistar rat brains (3-18 months of age) and brain sections from six human subjects aged 20-88 years. RESULTS: In healthy Wistar rats, we find intracellular Aß42 (iAß42) in neurons throughout the brain at all ages, but levels vary greatly between brain regions. The highest levels are in neurons of entorhinal cortex layer II, alongside hippocampal neurons at the CA1/subiculum border. Concerning entorhinal cortex layer II, we find similarly high levels of iAß42 in the human subjects. CONCLUSION: Expression of iAß42 in healthy Wistar rats predominates in the same structures where iAß accumulates and Aß plaques initially form in the much used, Wistar based McGill-R-Thy1-APP rat model for AD. The difference between wild-type Wistar rats and these AD model rats, with respect to Aß42, is therefore quantitative rather that qualitative. This, taken together with our human results, indicate that the McGill rat model in fact models the underlying wild-type neuronal population-specific vulnerability to Aß42 accumulation.

Fluorescently Guided Optical Photothermal Infrared Microspectroscopy for Protein-Specific Bioimaging at Subcellular Level.

Infrared spectroscopic imaging is widely used for the visualization of biomolecule structures, and techniques such as optical photothermal infrared (OPTIR) microspectroscopy can achieve <500 nm spatial resolution. However, these approaches lack specificity for particular cell types and cell components and thus cannot be used as a stand-alone technique to assess their properties. Here, we have developed a novel tool, fluorescently guided optical photothermal infrared microspectroscopy, that simultaneously exploits epifluorescence imaging and OPTIR to perform fluorescently guided IR spectroscopic analysis. This novel approach exceeds the diffraction limit of infrared microscopy and allows structural analysis of specific proteins directly in tissue and single cells. Experiments described herein used epifluorescence to rapidly locate amyloid proteins in tissues or neuronal cultures, thus guiding OPTIR measurements to assess amyloid structures at the subcellular level. We believe that this new approach will be a valuable addition to infrared spectroscopy providing cellular specificity of measurements in complex systems for studies of structurally altered protein aggregates.

Lowering levels of reelin in entorhinal cortex layer II-neurons results in lowered levels of intracellular amyloid-ß.

Projection neurons in the anteriolateral part of entorhinal cortex layer II are the predominant cortical site for hyper-phosphorylation of tau and formation of neurofibrillary tangles in prodromal Alzheimer's disease. A majority of layer II projection neurons in anteriolateral entorhinal cortex are unique among cortical excitatory neurons by expressing the protein reelin. In prodromal Alzheimer's disease, these reelin-expressing neurons are prone to accumulate intracellular amyloid-ß, which is mimicked in a rat model that replicates the spatio-temporal cascade of the disease. Two important findings in relation to this are that reelin-signalling downregulates tau phosphorylation, and that oligomeric amyloid-ß interferes with reelin-signalling. Taking advantage of this rat model, we used proximity ligation assay to assess whether reelin and intracellular amyloid-ß directly interact during early, pre-plaque stages in anteriolateral entorhinal cortex layer II reelin-expressing neurons. We next made a viral vector delivering micro-RNA against reelin, along with a control vector, and infected reelin-expressing anteriolateral entorhinal cortex layer II-neurons to test whether reelin levels affect levels of intracellular amyloid-ß and/or amyloid precursor protein. We analysed 25.548 neurons from 24 animals, which results in three important findings. First, in reelin-expressing anteriolateral entorhinal cortex layer II-neurons, reelin and intracellular amyloid-ß engage in a direct protein-protein interaction. Second, injecting micro-RNA against reelin lowers reelin levels in these neurons, amounting to an effect size of 1.3-4.5 (Bayesian estimation of Cohen's d effect size, 95% credible interval). This causes a concomitant reduction of intracellular amyloid-ß ranging across three levels of aggregation, including a reduction of Aß42 monomers/dimers amounting to an effect size of 0.5-3.1, a reduction of Aß prefibrils amounting to an effect size of 1.1-3.5 and a reduction of protofibrils amounting to an effect size of 0.05-2.1. Analysing these data using Bayesian estimation of mutual information furthermore reveals that levels of amyloid-ß are dependent on levels of reelin. Third, the reduction of intracellular amyloid-ß occurs without any substantial associated changes in levels of amyloid precursor protein. We conclude that reelin and amyloid-ß directly interact at the intracellular level in the uniquely reelin-expressing projection neurons in anteriolateral entorhinal cortex layer II, where levels of amyloid-ß are dependent on levels of reelin. Since amyloid-ß is known to impair reelin-signalling causing upregulated phosphorylation of tau, our findings are likely relevant to the vulnerability for neurofibrillary tangle-formation of this entorhinal neuronal population.

Apolipoprotein E intersects with amyloid-ß within neurons.

Apolipoprotein E4 (ApoE4) is the most important genetic risk factor for Alzheimer's disease (AD). Among the earliest changes in AD is endosomal enlargement in neurons, which was reported as enhanced in ApoE4 carriers. ApoE is thought to be internalized into endosomes of neurons, whereas ß-amyloid (Aß) accumulates within neuronal endosomes early in AD. However, it remains unknown whether ApoE and Aß intersect intracellularly. We show that internalized astrocytic ApoE localizes mostly to lysosomes in neuroblastoma cells and astrocytes, whereas in neurons, it preferentially localizes to endosomes-autophagosomes of neurites. In AD transgenic neurons, astrocyte-derived ApoE intersects intracellularly with amyloid precursor protein/Aß. Moreover, ApoE4 increases the levels of endogenous and internalized Aß42 in neurons. Taken together, we demonstrate differential localization of ApoE in neurons, astrocytes, and neuron-like cells, and show that internalized ApoE intersects with amyloid precursor protein/Aß in neurons, which may be of considerable relevance to AD.

Cerebral Aß deposition precedes reduced cerebrospinal fluid and serum Aß42/Aß40 ratios in the AppNL-F/NL-F knock-in mouse model of Alzheimer's disease.

BACKGROUND: Aß42/Aß40 ratios in cerebrospinal fluid (CSF) and blood are reduced in preclinical Alzheimer's disease (AD), but their temporal and correlative relationship with cerebral Aß pathology at this early disease stage is not well understood. In the present study, we aim to investigate such relationships using App knock-in mouse models of preclinical AD. METHODS: CSF, serum, and brain tissue were collected from 3- to 18-month-old AppNL-F/NL-F knock-in mice (n = 48) and 2-18-month-old AppNL/NL knock-in mice (n = 35). The concentrations of Aß42 and Aß40 in CSF and serum were measured using Single molecule array (Simoa) immunoassays. Cerebral Aß plaque burden was assessed in brain tissue sections by immunohistochemistry and thioflavin S staining. Furthermore, the concentrations of Aß42 in soluble and insoluble fractions prepared from cortical tissue homogenates were measured using an electrochemiluminescence immunoassay. RESULTS: In AppNL-F/NL-F knock-in mice, Aß42/Aß40 ratios in CSF and serum were significantly reduced from 12 and 16 months of age, respectively. The initial reduction of these biomarkers coincided with cerebral Aß pathology, in which a more widespread Aß plaque burden and increased levels of Aß42 in the brain were observed from approximately 12 months of age. Accordingly, in the whole study population, Aß42/Aß40 ratios in CSF and serum showed a negative hyperbolic association with cerebral Aß plaque burden as well as the levels of both soluble and insoluble Aß42 in the brain. These associations tended to be stronger for the measures in CSF compared with serum. In contrast, no alterations in the investigated fluid biomarkers or apparent cerebral Aß plaque pathology were found in AppNL/NL knock-in mice during the observation time. CONCLUSIONS: Our findings suggest a temporal sequence of events in AppNL-F/NL-F knock-in mice, in which initial deposition of Aß aggregates in the brain is followed by a decline of the Aß42/Aß40 ratio in CSF and serum once the cerebral Aß pathology becomes significant. Our results also indicate that the investigated biomarkers were somewhat more strongly associated with measures of cerebral Aß pathology when assessed in CSF compared with serum.