Proteomic analysis across patient iPSC-based models and human post-mortem hippocampal tissue reveals early cellular dysfunction and progression of Alzheimer's disease pathogenesis.

The hippocampus is a primary region affected in Alzheimer's disease (AD). Because AD postmortem brain tissue is not available prior to symptomatic stage, we lack understanding of early cellular pathogenic mechanisms. To address this issue, we examined the cellular origin and progression of AD pathogenesis by comparing patient-based model systems including iPSC-derived brain cells transplanted into the mouse brain hippocampus. Proteomic analysis of the graft enabled the identification of pathways and network dysfunction in AD patient brain cells, associated with increased levels of Aß-42 and ß-sheet structures. Interestingly, the host cells surrounding the AD graft also presented alterations in cellular biological pathways. Furthermore, proteomic analysis across human iPSC-based models and human post-mortem hippocampal tissue projected coherent longitudinal cellular changes indicative of early to end stage AD cellular pathogenesis. Our data showcase patient-based models to study the cell autonomous origin and progression of AD pathogenesis.

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.

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.

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.

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.

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.

APOE in the bullseye of neurodegenerative diseases: impact of the APOE genotype in Alzheimer's disease pathology and brain diseases.

ApoE is the major lipid and cholesterol carrier in the CNS. There are three major human polymorphisms, apoE2, apoE3, and apoE4, and the genetic expression of APOE4 is one of the most influential risk factors for the development of late-onset Alzheimer's disease (AD). Neuroinflammation has become the third hallmark of AD, together with Amyloid-ß plaques and neurofibrillary tangles of hyperphosphorylated aggregated tau protein. This review aims to broadly and extensively describe the differential aspects concerning apoE. Starting from the evolution of apoE to how APOE's single-nucleotide polymorphisms affect its structure, function, and involvement during health and disease. This review reflects on how APOE's polymorphisms impact critical aspects of AD pathology, such as the neuroinflammatory response, particularly the effect of APOE on astrocytic and microglial function and microglial dynamics, synaptic function, amyloid-ß load, tau pathology, autophagy, and cell-cell communication. We discuss influential factors affecting AD pathology combined with the APOE genotype, such as sex, age, diet, physical exercise, current therapies and clinical trials in the AD field. The impact of the APOE genotype in other neurodegenerative diseases characterized by overt inflammation, e.g., alpha- synucleinopathies and Parkinson's disease, traumatic brain injury, stroke, amyotrophic lateral sclerosis, and multiple sclerosis, is also addressed. Therefore, this review gathers the most relevant findings related to the APOE genotype up to date and its implications on AD and CNS pathologies to provide a deeper understanding of the knowledge in the APOE field.

Aß/Amyloid Precursor Protein-Induced Hyperexcitability and Dysregulation of Homeostatic Synaptic Plasticity in Neuron Models of Alzheimer's Disease.

Alzheimer's disease (AD) is increasingly seen as a disease of synapses and diverse evidence has implicated the amyloid-ß peptide (Aß) in synapse damage. The molecular and cellular mechanism(s) by which Aß and/or its precursor protein, the amyloid precursor protein (APP) can affect synapses remains unclear. Interestingly, early hyperexcitability has been described in human AD and mouse models of AD, which precedes later hypoactivity. Here we show that neurons in culture with either elevated levels of Aß or with human APP mutated to prevent Aß generation can both induce hyperactivity as detected by elevated calcium transient frequency and amplitude. Since homeostatic synaptic plasticity (HSP) mechanisms normally maintain a setpoint of activity, we examined whether HSP was altered in AD transgenic neurons. Using methods known to induce HSP, we demonstrate that APP protein levels are regulated by chronic modulation of activity and that AD transgenic neurons have an impaired adaptation of calcium transients to global changes in activity. Further, AD transgenic compared to WT neurons failed to adjust the length of their axon initial segments (AIS), an adaptation known to alter excitability. Thus, we show that both APP and Aß influence neuronal activity and that mechanisms of HSP are disrupted in primary neuron models of AD.

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.

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.

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.

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.

Monitoring the interactions between alpha-synuclein and Tau in vitro and in vivo using bimolecular fluorescence complementation.

Parkinson's disease (PD) and Alzheimer's disease (AD) are characterized by pathological accumulation and aggregation of different amyloidogenic proteins, α-synuclein (aSyn) in PD, and amyloid-ß (Aß) and Tau in AD. Strikingly, few PD and AD patients' brains exhibit pure pathology with most cases presenting mixed types of protein deposits in the brain. Bimolecular fluorescence complementation (BiFC) is a technique based on the complementation of two halves of a fluorescent protein, which allows direct visualization of protein-protein interactions. In the present study, we assessed the ability of aSyn and Tau to interact with each other. For in vitro evaluation, HEK293 and human neuroblastoma cells were used, while in vivo studies were performed by AAV6 injection in the substantia nigra pars compacta (SNpc) of mice and rats. We observed that the co-expression of aSyn and Tau led to the emergence of fluorescence, reflecting the interaction of the proteins in cell lines, as well as in mouse and rat SNpc. Thus, our data indicates that aSyn and Tau are able to interact with each other in a biologically relevant context, and that the BiFC assay is an effective tool for studying aSyn-Tau interactions in vitro and in different rodent models in vivo.

Nano-Infrared Imaging of Primary Neurons.

Alzheimer's disease (AD) accounts for about 70% of neurodegenerative diseases and is a cause of cognitive decline and death for one-third of seniors. AD is currently underdiagnosed, and it cannot be effectively prevented. Aggregation of amyloid-ß (Aß) proteins has been linked to the development of AD, and it has been established that, under pathological conditions, Aß proteins undergo structural changes to form ß-sheet structures that are considered neurotoxic. Numerous intensive in vitro studies have provided detailed information about amyloid polymorphs; however, little is known on how amyloid ß-sheet-enriched aggregates can cause neurotoxicity in relevant settings. We used scattering-type scanning near-field optical microscopy (s-SNOM) to study amyloid structures at the nanoscale, in individual neurons. Specifically, we show that in well-validated systems, s-SNOM can detect amyloid ß-sheet structures with nanometer spatial resolution in individual neurons. This is a proof-of-concept study to demonstrate that s-SNOM can be used to detect Aß-sheet structures on cell surfaces at the nanoscale. Furthermore, this study is intended to raise neurobiologists' awareness of the potential of s-SNOM as a tool for analyzing amyloid ß-sheet structures at the nanoscale in neurons without the need for immunolabeling.

Astrocytic and Neuronal Apolipoprotein E Isoforms Differentially Affect Neuronal Excitability.

Synaptic changes and neuronal network dysfunction are among the earliest changes in Alzheimer's disease (AD). Apolipoprotein E4 (ApoE4), the major genetic risk factor in AD, has been shown to be present at synapses and to induce hyperexcitability in mouse knock-in brain regions vulnerable to AD. ApoE in the brain is mainly generated by astrocytes, however, neurons can also produce ApoE under stress conditions such as aging. The potential synaptic function(s) of ApoE and whether the cellular source of ApoE might affect neuronal excitability remain poorly understood. Therefore, the aim of this study was to elucidate the synaptic localization and effects on neuronal activity of the two main human ApoE isoforms from different cellular sources in control and AD-like in vitro cultured neuron models. In this study ApoE is seen to localize at or near to synaptic terminals. Additionally, we detected a cellular source-specific effect of ApoE isoforms on neuronal activity measured by live cell Ca2+ imaging. Neuronal activity increases after acute but not long-term administration of ApoE4 astrocyte medium. In contrast, ApoE expressed by neurons appears to induce the highest neuronal firing rate in the presence of ApoE3, rather than ApoE4. Moreover, increased neuronal activity in APP/PS1 AD transgenic compared to wild-type neurons is seen in the absence of astrocytic ApoE and the presence of astrocytic ApoE4, but not ApoE3. In summary, ApoE can target synapses and differentially induce changes in neuronal activity depending on whether ApoE is produced by astrocytes or neurons. Astrocytic ApoE induces the strongest neuronal firing with ApoE4, while the most active and efficient neuronal activity induced by neuronal ApoE is caused by ApoE3. ApoE isoforms also differentially affect neuronal activity in AD transgenic compared to wild-type neurons.

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.