The Effect of Fat Intake with Increased Omega-6-to-Omega-3 Polyunsaturated Fatty Acid Ratio in Animal Models of Early and Late Alzheimer's Disease-like Pathogenesis.

This work aims to clarify the effect of dietary polyunsaturated fatty acid (PUFA) intake on the adult brain affected by amyloid pathology. McGill-R-Thy1-APP transgenic (Tg) rat and 5xFAD Tg mouse models that represent earlier or later disease stages were employed. The animals were exposed to a control diet (CD) or an HFD based on corn oil, from young (rats) or adult (mice) ages for 24 or 10 weeks, respectively. In rats and mice, the HFD impaired reference memory in wild-type (WT) animals but did not worsen it in Tg, did not cause obesity, and did not increase triglycerides or glucose levels. Conversely, the HFD promoted stronger microglial activation in Tg vs. WT rats but had no effect on cerebral amyloid deposition. IFN-γ, IL-1ß, and IL-6 plasma levels were increased in Tg rats, regardless of diet, while CXCL1 chemokine levels were increased in HFD-fed mice, regardless of genotype. Hippocampal 3-nitrotyrosine levels tended to increase in HFD-fed Tg rats but not in mice. Overall, an HFD with an elevated omega-6-to-omega-3 ratio as compared to the CD (25:1 vs. 8.4:1) did not aggravate the outcome of AD regardless of the stage of amyloid pathology, suggesting that many neurobiological processes relevant to AD are not directly dependent on PUFA intake.

Early treatment with an M1 and sigma-1 receptor agonist prevents cognitive decline in a transgenic rat model displaying Alzheimer-like amyloid pathology.

The application of the selective allosteric M1 muscarinic and sigma-1 receptor agonist, AF710B (aka ANAVEX3-71), has shown to attenuate Alzheimer's disease-like hallmarks in McGill-R-Thy1-APP transgenic rats when administered at advanced pathological stages. It remains unknown whether preventive treatment strategies applying this compound may be equally effective. We tested whether daily oral administration of AF710B (10 µg/kg) in 7-month-old, preplaque, McGill-R-Thy1-APP rats for 7 months, followed by a 4-week washout period, could prevent Alzheimer's disease-like pathological hallmarks. Long-term AF710B treatment prevented the cognitive impairment of McGill-R-Thy1-APP rats. The effect was accompanied by a reduction in the number of amyloid plaques in the hippocampus and the levels of Aß42 and Aß40 peptides in the cerebral cortex. AF710B treatment also reduced microglia and astrocyte recruitment toward CA1 hippocampal Aß-burdened neurons compared to vehicle-treated McGill-R-Thy1-APP rats, also altering the inflammatory cytokines profile. Lastly, AF710B treatment rescued the conversion of brain-derived neurotrophic factor precursor to its mature and biologically active form. Overall, these results suggest preventive and disease-modifying properties of the compound.

Neuronal loss and inflammation preceding fibrillary tau pathology in a rat model with early human-like tauopathy.

In tauopathies such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), the microtubule associated protein tau undergoes conformational and posttranslational modifications in a gradual, staged pathological process. While brain atrophy and cognitive decline are well-established in the advanced stages of tauopathy, it is unclear how the early pathological processes manifest prior to extensive neurodegeneration. For these studies we have applied a transgenic rat model of human-like tauopathy in its heterozygous form, named McGill-R955-hTau. The goal of the present study was to investigate whether lifelong accumulation of mutated human tau could reveal the earliest tau pathological processes in a context of advanced aging, and, at stages before the overt aggregated or fibrillary tau deposition. We characterized the phenotype of heterozygous R955-hTau rats at three endpoints, 10, 18 and 24-26 months of age, focusing on markers of cognitive capabilities, progressive tau pathology, neuronal health, neuroinflammation and brain ultrastructural integrity, using immunohistochemistry and electron microscopy. Heterozygous R955-hTau transgenic rats feature a modest, life-long accumulation of mutated human tau that led to tau hyperphosphorylation and produced deficits in learning and memory tasks after 24 months of age. Such impairments coincided with more extensive tau hyperphosphorylation in the brain at residues pThr231 and with evidence of oligomerization. Importantly, aged R955-hTau rats presented evidence of neuroinflammation, detriments to myelin morphology and detectable hippocampal neuronal loss in the absence of overt neurofibrillary lesions and brain atrophy. The slow-progressing tauopathy of R955-hTau rats should allow to better delineate the temporal progression of tau pathological events and therefore to distinguish early indicators of tauopathy as having the capability to induce degenerative events in the aged CNS.

Methyl-CpG binding domain 2 (Mbd2) is an epigenetic regulator of autism-risk genes and cognition.

The Methyl-CpG-Binding Domain Protein family has been implicated in neurodevelopmental disorders. The Methyl-CpG-binding domain 2 (Mbd2) binds methylated DNA and was shown to play an important role in cancer and immunity. Some evidence linked this protein to neurodevelopment. However, its exact role in neurodevelopment and brain function is mostly unknown. Here we show that Mbd2-deficiency in mice (Mbd2-/-) results in deficits in cognitive, social and emotional functions. Mbd2 binds regulatory DNA regions of neuronal genes in the hippocampus and loss of Mbd2 alters the expression of hundreds of genes with a robust down-regulation of neuronal gene pathways. Further, a genome-wide DNA methylation analysis found an altered DNA methylation pattern in regulatory DNA regions of neuronal genes in Mbd2-/- mice. Differentially expressed genes significantly overlap with gene-expression changes observed in brains of Autism Spectrum Disorder (ASD) individuals. Notably, downregulated genes are significantly enriched for human ortholog ASD risk genes. Observed hippocampal morphological abnormalities were similar to those found in individuals with ASD and ASD rodent models. Hippocampal Mbd2 knockdown partially recapitulates the behavioral phenotypes observed in Mbd2-/- mice. These findings suggest that Mbd2 is a novel epigenetic regulator of genes that are associated with ASD in humans. Mbd2 loss causes behavioral alterations that resemble those found in ASD individuals.

Progressive human-like tauopathy with downstream neurodegeneration and neurovascular compromise in a transgenic rat model.

Tauopathies, including frontotemporal dementia (FTD) and Alzheimer's disease (AD), clinically present with progressive cognitive decline and the deposition of neurofibrillary tangles (NFTs) in the brain. Neurovascular compromise is also prevalent in AD and FTD however the relationship between tau and the neurovascular unit is less understood relative to other degenerative phenotypes. Current animal models confer the ability to recapitulate aspects of the CNS tauopathies, however, existing models either display overaggressive phenotypes, or do not develop neuronal loss or genuine neurofibrillary lesions. In this report, we communicate the longitudinal characterization of brain tauopathy in a novel transgenic rat model, coded McGill-R955-hTau. The model expresses the longest isoform of human P301S tau. Homozygous R955-hTau rats displayed a robust, progressive accumulation of mutated human tau leading to the detection of tau hyperphosphorylation and cognitive deficits accelerating from 14 months of age. This model features extensive tau hyperphosphorylation with endogenous tau recruitment, authentic neurofibrillary lesions, and tau-associated neuronal loss, ventricular dilation, decreased brain volume, and gliosis in aged rats. Further, we demonstrate how neurovascular integrity becomes compromised at aged life stages using a combination of electron microscopy, injection of the tracer horseradish peroxidase and immunohistochemical approaches.

Long-term nucleus basalis cholinergic depletion induces attentional deficits and impacts cortical neurons and BDNF levels without affecting the NGF synthesis.

Basal forebrain cholinergic neurons (BFCNs) represent the main source of cholinergic innervation to the cortex and hippocampus and degenerate early in Alzheimer's disease (AD) progression. Phenotypic maintenance of BFCNs depends on levels of mature nerve growth factor (mNGF) and mature brain-derived neurotrophic factor (mBDNF), produced by target neurons and retrogradely transported to the cell body. Whether a reciprocal interaction where BFCN inputs impact neurotrophin availability and affect cortical neuronal markers remains unknown. To address our hypothesis, we immunolesioned the nucleus basalis (nb), a basal forebrain cholinergic nuclei projecting mainly to the cortex, by bilateral stereotaxic injection of 192-IgG-Saporin (the cytotoxin Saporin binds p75ntr receptors expressed exclusively by BFCNs) in 2.5-month-old Wistar rats. At 6 months post-lesion, Saporin-injected rats (SAP) showed an impairment in a modified version of the 5-Choice Serial Reaction Time Task (5-choice task). Postmortem analyses of the brain revealed a reduction of Choline Acetyltransferase-immunoreactive neurons compared to wild-type controls. A diminished number of cortical vesicular acetylcholine transporter-immunoreactive boutons was accompanied by a reduction in BDNF mRNA, mBDNF protein levels, markers of glutamatergic (vGluT1), and GABAergic (GAD65) neurons in the SAP-group compared to the controls. NGF mRNA, NGF precursor, and mNGF protein levels were not affected. Additionally, cholinergic markers correlated with the attentional deficit and BDNF levels. Our findings demonstrate that while cholinergic nb loss impairs cognition and reduces cortical neuron markers, it produces differential effects on neurotrophin availability, affecting BDNF but not NGF levels.

Nerve Growth Factor Compromise in Down Syndrome.

The basal forebrain cholinergic system relies on trophic support by nerve growth factor (NGF) to maintain its phenotype and function. In Alzheimer's disease (AD), basal forebrain cholinergic neurons (BFCNs) undergo progressive atrophy, suggesting a deficit in NGF trophic support. Within the central nervous system, NGF maturation and degradation are tightly regulated by an activity-dependent metabolic cascade. Here, we present a brief overview of the characteristics of Alzheimer's pathology in Down syndrome (DS) with an emphasis on this NGF metabolic pathway's disruption during the evolving Alzheimer's pathology. Such NGF dysmetabolism is well-established in Alzheimer's brains with advanced pathology and has been observed in mild cognitive impairment (MCI) and non-demented individuals with elevated brain amyloid levels. As individuals with DS inexorably develop AD, we then review findings that support the existence of a similar NGF dysmetabolism in DS coinciding with atrophy of the basal forebrain cholinergic system. Lastly, we discuss the potential of NGF-related biomarkers as indicators of an evolving Alzheimer's pathology in DS.

The Nerve Growth Factor Metabolic Pathway Dysregulation as Cause of Alzheimer's Cholinergic Atrophy.

The cause of the loss of basal forebrain cholinergic neurons (BFCNs) and their terminal synapses in the cerebral cortex and hippocampus in Alzheimer's disease (AD) has provoked a decades-long controversy. The cholinergic phenotype of this neuronal system, involved in numerous cognitive mechanisms, is tightly dependent on the target-derived nerve growth factor (NGF). Consequently, the loss of BFCNs cholinergic phenotype in AD was initially suspected to be due to an NGF trophic failure. However, in AD there is a normal NGF synthesis and abundance of the NGF precursor (proNGF), therefore the NGF trophic failure hypothesis for the atrophy of BCNs was abandoned. In this review, we discuss the history of NGF-dependency of BFCNs and the atrophy of these neurons in Alzheimer's disease (AD). Further to it, we propose that trophic factor failure explains the BFCNs atrophy in AD. We discuss evidence of the occurrence of a brain NGF metabolic pathway, the dysregulation of which, in AD explains the severe deficiency of NGF trophic support for the maintenance of BFCNs cholinergic phenotype. Finally, we revise recent evidence that the NGF metabolic dysregulation in AD pathology starts at preclinical stages. We also propose that the alteration of NGF metabolism-related markers in body fluids might assist in the AD preclinical diagnosis.

Connecting the "Dots": From Free Radical Lipid Autoxidation to Cell Pathology and Disease.

Our understanding of lipid peroxidation in biology and medicine is rapidly evolving, as it is increasingly implicated in various diseases but also recognized as a key part of normal cell function, signaling, and death (ferroptosis). Not surprisingly, the root and consequences of lipid peroxidation have garnered increasing attention from multiple disciplines in recent years. Here we "connect the dots" between the fundamental chemistry underpinning the cascade reactions of lipid peroxidation (enzymatic or free radical), the reactive nature of the products formed (lipid-derived electrophiles), and the biological targets and mechanisms associated with these products that culminate in cellular responses. We additionally bring light to the use of highly sensitive, fluorescence-based methodologies. Stemming from the foundational concepts in chemistry and biology, these methodologies enable visualizing and quantifying each reaction in the cascade in a cellular and ultimately tissue context, toward deciphering the connections between the chemistry and physiology of lipid peroxidation. The review offers a platform in which the chemistry and biomedical research communities can access a comprehensive summary of fundamental concepts regarding lipid peroxidation, experimental tools for the study of such processes, as well as the recent discoveries by leading investigators with an emphasis on significant open questions.

Early intraneuronal amyloid triggers neuron-derived inflammatory signaling in APP transgenic rats and human brain.

Chronic inflammation during Alzheimer's disease (AD) is most often attributed to sustained microglial activation in response to amyloid-ß (Aß) plaque deposits and cell death. However, cytokine release and microgliosis are consistently observed in AD transgenic animal models devoid of such pathologies, bringing into question the underlying processes that may be at play during the earliest AD-related immune response. We propose that this plaque-independent inflammatory reaction originates from neurons burdened with increasing levels of soluble and oligomeric Aß, which are known to be the most toxic amyloid species within the brain. Laser microdissected neurons extracted from preplaque amyloid precursor protein (APP) transgenic rats were found to produce a variety of potent immune factors, both at the transcript and protein levels. Neuron-derived cytokines correlated with the extent of microglial activation and mobilization, even in the absence of extracellular plaques and cell death. Importantly, we identified an inflammatory profile unique to Aß-burdened neurons, since neighboring glial cells did not express similar molecules. Moreover, we demonstrate within disease-vulnerable regions of the human brain that a neuron-specific inflammatory response may precede insoluble Aß plaque and tau tangle formation. Thus, we reveal the Aß-burdened neuron as a primary proinflammatory agent, implicating the intraneuronal accumulation of Aß as a significant immunological component in the AD pathogenesis.

NP03, a Microdose Lithium Formulation, Blunts Early Amyloid Post-Plaque Neuropathology in McGill-R-Thy1-APP Alzheimer-Like Transgenic Rats.

Epidemiological, preclinical, and clinical studies have suggested a role for microdose lithium in reducing Alzheimer's disease (AD) risk by modulating key mechanisms associated with AD pathology. The novel microdose lithium formulation, NP03, has disease-modifying effects in the McGill-R-Thy1-APP transgenic rat model of AD-like amyloidosis at pre-plaque stages, before frank amyloid-ß (Aß) plaque deposition, during which Aß is primarily intraneuronal. Here, we are interested in determining whether the positive effects of microdose lithium extend into early Aß post-plaque stages. We administered NP03 (40µg Li/kg; 1 ml/kg body weight) to McGill-R-Thy1-APP transgenic rats for 12 weeks spanning the transition phase from plaque-free to plaque-bearing. The effect of NP03 on remote working memory was assessed using the novel object recognition task. Levels of human Aß38, Aß40, and Aß42 as well as levels of pro-inflammatory mediators were measured in brain-extracts and plasma using electrochemiluminescent assays. Mature Aß plaques were visualized with a thioflavin-S staining. Vesicular acetylcholine transporter (VAChT) bouton density and levels of chemokine (C-X-C motif) ligand 1 (CXCL1), interleukin-6 (IL-6), and 4-hydroxynonenal (4-HNE) were probed using quantitative immunohistochemistry. During the early Aß post-plaque stage, we find that NP03 rescues functional deficits in object recognition, reduces loss of cholinergic boutons in the hippocampus, reduces levels of soluble and insoluble cortical Aß42 and reduces hippocampal Aß plaque number. In addition, NP03 reduces markers of neuroinflammation and cellular oxidative stress. Together these results indicate that microdose lithium NP03 is effective at later stages of amyloid pathology, after appearance of Aß plaques.

Early Long-Term Memory Impairment and Changes in the Expression of Synaptic Plasticity-Associated Genes, in the McGill-R-Thy1-APP Rat Model of Alzheimer's-Like Brain Amyloidosis.

Accruing evidence supports the hypothesis that memory deficits in early Alzheimer Disease (AD) might be due to synaptic failure caused by accumulation of intracellular amyloid beta (Aß) oligomers, then secreted to the extracellular media. Transgenic mouse AD models provide valuable information on AD pathology. However, the failure to translate these findings to humans calls for models that better recapitulate the human pathology. McGill-R-Thy1-APP transgenic (Tg) rat expresses the human amyloid precursor protein (APP751) with the Swedish and Indiana mutations (of familial AD), leading to an AD-like slow-progressing brain amyloid pathology. Therefore, it offers a unique opportunity to investigate learning and memory abilities at early stages of AD, when Aß accumulation is restricted to the intracellular compartment, prior to plaque deposition. Our goal was to further investigate early deficits in memory, particularly long-term memory in McGill-R-Thy1-APP heterozygous (Tg+/-) rats. Short-term- and long-term habituation to an open field were preserved in 3-, 4-, and 6-month-old (Tg+/-). However, long-term memory of inhibitory avoidance to a foot-shock, novel object-recognition and social approaching behavior were seriously impaired in 4-month-old (Tg+/-) male rats, suggesting that they are unable to either consolidate and/or evoke such associative and discriminative memories with aversive, emotional and spatial components. The long-term memory deficits were accompanied by increased transcript levels of genes relevant to synaptic plasticity, learning and memory processing in the hippocampus, such as Grin2b, Dlg4, Camk2b, and Syn1. Our findings indicate that in addition to the previously well-documented deficits in learning and memory, McGill-R-Thy1-APP rats display particular long-term-memory deficits and deep social behavior alterations at pre-plaque early stages of the pathology. This highlights the importance of Aß oligomers and emphasizes the validity of the model to study AD-like early processes, with potentially predictive value.

Perturbed mitochondria-ER contacts in live neurons that model the amyloid pathology of Alzheimer's disease.

The use of fixed fibroblasts from familial and sporadic Alzheimer's disease patients has previously indicated an upregulation of mitochondria-ER contacts (MERCs) as a hallmark of Alzheimer's disease. Despite its potential significance, the relevance of these results is limited because they were not extended to live neurons. Here we performed a dynamic in vivo analysis of MERCs in hippocampal neurons from McGill-R-Thy1-APP transgenic rats, a model of Alzheimer's disease-like amyloid pathology. Live FRET imaging of neurons from transgenic rats revealed perturbed 'lipid-MERCs' (gap width <10 nm), while 'Ca2+-MERCs' (10-20 nm gap width) were unchanged. In situ TEM showed no significant differences in the lipid-MERCs:total MERCs or lipid-MERCs:mitochondria ratios; however, the average length of lipid-MERCs was significantly decreased in neurons from transgenic rats as compared to controls. In accordance with FRET results, untargeted lipidomics showed significant decreases in levels of 12 lipids and bioenergetic analysis revealed respiratory dysfunction of mitochondria from transgenic rats. Thus, our results reveal changes in MERC structures coupled with impaired mitochondrial functions in Alzheimer's disease-related neurons.This article has an associated First Person interview with the first author of the paper.

Experimental Pharmacology in Transgenic Rodent Models of Alzheimer's Disease.

This Mini Review discusses the merits and shortfalls of transgenic (tg) rodents modeling aspects of the human Alzheimer's disease (AD) pathology and their application to evaluate experimental therapeutics. It addresses some of the differences between mouse and rat tg models for these investigations. It relates, in a condensed fashion, the experience of our research laboratory with the application of anti-inflammatory compounds and S-adenosylmethionine (SAM) at the earliest stages of AD-like amyloid pathology in tg mice. The application of SAM was intended to revert the global brain DNA hypomethylation unleashed by the intraneuronal accumulation of amyloid-ß-immunoreactive material, an intervention that restored levels of DNA methylation including of the bace1 gene. This review also summarizes experimental pharmacology observations made in the McGill tg rat model of AD-like pathology by applying "nano-lithium" or a drug with allosteric M1 muscarinic and sigma 1 receptor agonistic properties (AF710B). Extremely low doses of lithium (up to 400 times lower than used in the clinic) had remarkable beneficial effects on lowering pathology and improving cognitive functions in tg rats. Likewise, AF710B treatment, even at advanced stages of the pathology, displayed remarkable beneficial effects. This drug, in experimental conditions, demonstrated possible "disease-modifying" properties as pathology was frankly diminished and cognition improved after a month of "wash-out" period. The Mini-Review ends with a discussion on the predictive value of similar experimental pharmacological interventions in current rodent tg models. It comments on the validity of some of these approaches for early interventions at preclinical stages of AD, interventions which may be envisioned once definitive diagnosis of AD before clinical presentation is made possible.

Neuropathological changes and cognitive deficits in rats transgenic for human mutant tau recapitulate human tauopathy.

The assembly of tau protein into abnormal filaments and brain cell degeneration are characteristic of a number of human neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Several murine models have been generated to better understand the mechanisms contributing to tau assembly and neurodegeneration. Taking advantage of the more elaborate central nervous system and higher cognitive abilities of the rat, we generated a model expressing the longest human tau isoform (2N4R) with the P301S mutation. This transgenic rat line, R962-hTau, exhibits the main features of human tauopathies, such as: age-dependent increase in inclusions comprised of aggregated-tau, neuronal loss, global neurodegeneration as reflected by brain atrophy and ventricular dilation, alterations in astrocytic and microglial morphology, and myelin loss. In addition, substantial deficits across multiple memory and learning paradigms, including novel object recognition, fear conditioning and Morris water maze tasks, were observed at the time of advanced tauopathy. These results support the concept that progressive tauopathy correlates with brain atrophy and cognitive impairment.

Effect of antioxidant supplements on lipid peroxidation levels in primary cortical neuron cultures.

Oxidative stress, specifically lipid peroxidation, is a major driving force in neurodegenerative processes. However, the exact role of lipid peroxidation remains elusive as reliable real-time detection and quantification of lipid peroxyl radicals proves to be challenging in vitro and in vivo. Motivated by this methodological limitation, we have optimized conditions for real-time imaging and quantification of lipid peroxyl radical generation in primary neuron cultures using the lipophilic fluorogenic antioxidant H4BPMHC (8-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-methyl)-1,5-di(3-chloropropyl)-pyrromethene fluoroborate), an α-tocopherol analog probe. By subjecting neurons to different antioxidant conditions in the presence and absence of lipid peroxidation inducing stressors (Haber-Weiss reagents), we maximized H4BPMHC sensitivity and confirmed its potential to temporally resolve subtle and marked differences in lipid peroxidation levels in real-time. Herein we report imaging and quantification of homeostatic and induced lipid peroxidation in primary neuron cultures, supporting the use of this probe for investigating healthy and diseased states. Overall these results provide the necessary foundation and impetus towards using H4BPMHC for elucidating and mapping lipid peroxyl radical contributions to ROS-associated pathological processes in neurons.

Platelets Bioenergetics Screening Reflects the Impact of Brain Aß Plaque Accumulation in a Rat Model of Alzheimer.

Alzheimer's disease (AD) is associated to depressed brain energy supply and impaired cortical and hippocampal synaptic function. It was previously reported in McGill-R-Thy1-APP transgenic (Tg(+/+)) rats that Aß deposition per se is sufficient to cause abnormalities in glucose metabolism and neuronal connectivity. These data support the utility of this animal model as a platform for the search of novel AD biomarkers based on bioenergetic status. Recently, it has been proposed that energy dysfunction can be dynamically tested in platelets (PLTs) of nonhuman primates. PLTs are good candidates to find peripheral biomarkers for AD because they may reflect in periphery the bioenergetics deficits and the inflammatory and oxidative stress processes taking place in AD brain. In the present study, we carried out a PLTs bioenergetics screening in advanced-age (12-14 months old) control (WT) and Tg(+/+) rats. Results indicated that thrombin-activated PLTs of Tg(+/+) rats showed a significantly lower respiratory rate, as compared to that measured in WT animals, when challenged with the same dose of FCCP (an uncoupler of oxidative phosphorylation). In summary, our results provide original evidence that PLTs bioenergetic profiling may reflect brain bioenergetics dysfunction mediated by Aß plaque accumulation. Further studies on human PLTs from control and AD patients are required to validate the usefulness of PLTs bioenergetics as a novel blood-based biomarker for AD.

Evidence of intraneuronal Aß accumulation preceding tau pathology in the entorhinal cortex.

Growing evidence gathered from transgenic animal models of Alzheimer's disease (AD) indicates that the intraneuronal accumulation of amyloid-ß (Aß) peptides is an early event in the AD pathogenesis, producing cognitive deficits before the deposition of insoluble plaques. Levels of soluble Aß are also a strong indicator of synaptic deficits and concurrent AD neuropathologies in post-mortem AD brain; however, it remains poorly understood how this soluble amyloid pool builds within the brain in the decades leading up to diagnosis, when a patient is likely most amenable to early therapeutic interventions. Indeed, characterizing early intracellular Aß accumulation in humans has been hampered by the lack of Aß-specific antibodies, variability in the quality of available human brain tissue and the limitations of conventional microscopy. We therefore sought to investigate the development of the intraneuronal Aß pathology using extremely high-quality post-mortem brain material obtained from a cohort of non-demented subjects with short post-mortem intervals and processed by perfusion-fixation. Using well-characterized monoclonal antibodies, we demonstrate that the age-dependent intraneuronal accumulation of soluble Aß is pervasive throughout the entorhinal cortex and hippocampus, and that this phase of the amyloid pathology becomes established within AD-vulnerable regions before the deposition of Aß plaques and the formation of tau neurofibrillary tangles. We also show for the first time in post-mortem human brain that Aß oligomers do in fact accumulate intraneuronally, before the formation of extracellular plaques. Finally, we validated the origin of the Aß-immunopositive pool by resolving Aß- and APP/CTF-immunoreactive sites using super resolution structured illumination microscopy. Together, these findings indicate that the lifelong accrual of intraneuronal Aß may be a potential trigger for downstream AD-related pathogenic events in early disease stages.