Gamma frequency entrainment attenuates amyloid load and modifies microglia

Changes in gamma oscillations (20-50 Hz) have been observed in several neurological disorders. However, the relationship between gamma oscillations and cellular pathologies is unclear. Here we show reduced, behaviourally driven gamma oscillations before the onset of plaque formation or cognitive decline in a mouse model of Alzheimer's disease. Optogenetically driving fast-spiking parvalbumin-positive (FS-PV)-interneurons at gamma (40 Hz), but not other frequencies, reduces levels of amyloid-β (Aβ)[subscript 1-40] and Aβ [subscript 1-42] isoforms. Gene expression profiling revealed induction of genes associated with morphological transformation of microglia, and histological analysis confirmed increased microglia co-localization with Aβ. Subsequently, we designed a non-invasive 40 Hz light-flickering regime that reduced Aβ[subscript 1-40] and Aβ[subscript 1-42] levels in the visual cortex of pre-depositing mice and mitigated plaque load in aged, depositing mice. Our findings uncover a previously unappreciated function of gamma rhythms in recruiting both neuronal and glial responses to attenuate Alzheimer's-disease-associated pathology.National Institutes of Health (U.S.) (Grant 1R01EY023173)National Institutes of Health (U.S.) (Grant 1DP1NS087724)National Institutes of Health (U.S.) (Grant RF1AG047661)National Institutes of Health (U.S.) (Grant ROIGM104948

Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer’s Disease Phenotypes

The dismal success rate of clinical trials for Alzheimer’s disease (AD) motivates us to develop model systems of AD pathology that have higher predictive validity. The advent of induced pluripotent stem cells (iPSCs) allows us to model pathology and study disease mechanisms directly in human neural cells from healthy individual as well as AD patients. However, two-dimensional culture systems do not recapitulate the complexity of neural tissue, and phenotypes such as extracellular protein aggregation are difficult to observe. We report brain organoids that use pluripotent stem cells derived from AD patients and recapitulate AD-like pathologies such as amyloid aggregation, hyperphosphorylated tau protein, and endosome abnormalities. These pathologies are observed in an age-dependent manner in organoids derived from multiple familial AD (fAD) patients harboring amyloid precursor protein (APP) duplication or presenilin1 (PSEN1) mutation, compared to controls. The incidence of AD pathology was consistent amongst several fAD lines, which carried different mutations. Although these are complex assemblies of neural tissue, they are also highly amenable to experimental manipulation. We find that treatment of patient-derived organoids with β- and γ-secretase inhibitors significantly reduces amyloid and tau pathology. Moreover, these results show the potential of this model system to greatly increase the translatability of pre-clinical drug discovery in AD

Organoids created from different lines of AD patient iPSCs exhibit AD phenotypes.

<p>(A) Tissue sections from fAD (<i>APP</i>^Dp2-3, ND34732, AG068840) and control (Ctrl; CS-0020-01, AG09173) organoids were processed for immunoreactivity against Aβ (D45D2, white), MAP2 (red), and pTau (S396, green) and labeled with the nuclear dye Hoechst. (B) Quantification of Aβ immunoreactivity in fAD and Ctrl organoids following 90 days of culture. Values between the two control lines did not significantly differ. Number of Aβ-positive aggregates in two size classes (Particle Counts): one-way ANOVA with post-hoc Tukey’s multiple comparisons test; <i>F</i> (4,21) = 6.15, **p = 0.0019, R² = 0.5396 (1–3μm); <i>F</i> (4,21) = 7.95, ***p = 0.0005, R² = 0.6024 (3–6 μm). (C) Quantification of the average intensity of pTau Ser396 immunoreactivity as a fold change of Ctrl in fAD and Ctrl organoids following 90 days of culture. Values between the two control lines did not significantly differ. (Each data point represent one organoid). One-way ANOVA with post-hoc Tukey’s multiple comparisons test; <i>F</i> (4,20) = 9.629, ***p = 0.0002, R² = 0.6582. On charts: *p < 0.05, **p < 0.01, ***p < 0.001.</p

Organoids created from patient-derived iPSCs exhibit robust Alzheimer’s disease (AD)-like pathology.

<p>(A) Concentration of Aβ_1–40 and Aβ_1–42 from supernatant of control (Ctrl; CS-0020-01) and familial AD (fAD; <i>APP</i>^Dp1-1) organoid cultures, measured by ELISA, as well as the ratio of Aβ_1–42 to Aβ_1–40 concentrations. Unpaired two-tailed t-test with equal variance: *p = 0.047 (Aβ_1–40), unpaired two-tailed t-test with Welch’s correction for unequal variance: **p = 0.004 (Aβ_1–42), p = 0.48 (Aβ_1-42/Aβ_1–40). (B) Tissue sections from fAD (<i>APP</i>^Dp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against amyloid β (Aβ) using two antibodies (D54D2: white, 4G8: green), as well as antibodies against the neuronal marker MAP2 (red) and stained with the nuclear dye Hoechst (blue). Insets demonstrate Aβ immunoreactivity that appears both extracellular (i, arrow) and intracellular (ii, arrowhead) based upon MAP2 co-localization. (C) Z-projection of immunolabeled tissue sections from 90 day old Ctrl and fAD organoids showing immunoreactivity for Aβ (D45D2: white) and MAP2 (red). The edge of the tissue section is visible at the left bottom corner of each example. (D) Quantification of Aβ immunoreactivity in fAD and Ctrl organoids following 60d and 90d culture. Particle Counts: one-way ANOVA with post-hoc Fishers Least Significant Difference (LSD) test for multiple comparisons; <i>F</i> (3,28) = 4.385, ***p = 0 0.0008, R² = 0.32 (i-60 days); <i>F</i> (5,43) = 3.346, *p = 0 0.012, R² = 0.28 (90 days). Particle Size: Two-tailed Mann Whitney test for non-normal distributions (normality α < 0.05), **p = 0.006 (60 days), ***p = 0.001 (90 days). (E) Tissue sections from fAD (<i>APP</i>^Dp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against phosphorylated Tau (pTau, green) at Serine 396 (S396) and MAP2 (red) following 90d culture. Hoechst (blue) labels cell nuclei. (F) Quantification of pTau immunoreactivity for the Ser396 at 60d and 90d, and for the Threonine 181 (Thr181) pTau at 90d. Values are plotted as mean intensity of immunoreactivity as fold change of Ctrl. Unpaired two-tailed t-test with equal variance: p = 0.67 (60 day Ser396), **p = 0.001 (90 day Ser396), *p = 0.03 (90 day Thr181). (G) Sections from fAD (<i>APP</i>^Dp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against the early endosome antigen 1 (EEA1, green) and MAP2 (red). The dotted white line outlines the region of higher magnification to show EEA1 detail. (H) Quantification of EEA1 immunoreactivity in fAD and Ctrl organoids following 90d culture. EEA1 Particle Counts: one-way ANOVA with post-hoc Fisher’s LSD test for multiple comparisons; <i>F</i> (3,16) = 4.0, *p = 0.026, R² = 0.43. EEA1 Particle size: unpaired two-tailed t-test with Welch’s correction: *p = 0.041. (I) Organoids from Ctrl and fAD lines were subjected to the transferrin endocytosis assay to label pools of clathrin-coated early endosomes. (Each data point represent one organoid) Quantification of the average size of transferrin-positive particles: unpaired two-tailed t-test with equal variance, **p = 0.005. Average number (count) of transferrin-positive particles, unpaired two-tailed t-test with equal variance, p = 0.64. On charts: *p < 0.05, **p < 0.01, ***p < 0.001.</p

Organoids created from AD patient iPSCs respond to compound treatment.

<p>(A) Schematic of beta (BACE-1) and gamma (Comp-E) secretase inhibitor treatment (top). At 30 days of culture, fAD (<i>APP</i>^Dp1-1) organoids were treated with low dose (BACE-1, 1μM and Comp-E, 3nM) or high dose (BACE-1, 5 μM and Comp-E 6nM) combined compounds, or equivalent DMSO vehicle. Following 30 or 60 days of culture and drug treatment, organoids at 60 and 90 days of culture, respectively, were processed for immunohistochemistry (IHC). Tissue sections from fAD (<i>APP</i>^Dp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against Aβ (D45D2, white), pTau (Ser396, green), and MAP2 (red). Examine images are from 90 day organoids. (B) Quantification of Aβ particle number and size in compound treated and fAD organoids following 30 days of administration. Number of Aβ-positive aggregates in two size classes (Particle Counts): one-way ANOVA with Fishers LSD test for multiple comparisons; <i>F</i> (5,24) = 3.58, *p = 0.014, R² = 0.4296. Particle size: one-way ANOVA with Kruskal-Wallis test for non-normal distribution (α < 0.05), p = 0.475. (C) Quantification of Aβ particle number and size in treated (high dose) and untreated fAD organoids following 60 days of compound administration. Number of Aβ-positive aggregates in three size classes (Particle Counts): one-way ANOVA with Fishers LSD test for multiple comparisons; <i>F</i> (5,19) = 5.02, **p = 0.004, R² = 0.5691. Particle size: Mann-Whitney two-tailed test for non-normal distribution (α < 0.05), p = 0.09. (D) Quantification of the average intensity of pTau Ser396 immunoreactivity as a fold change of Ctrl in fAD organoids following 30 and 60 days of compound treatment. 30 day treatment. (Each data point represent one organoid). Unpaired two-tailed t-test with equal variance, p = 0.69. 60 day treatment: one-way ANOVA with Tukey’s multiple comparisons test, F (2,13) = 19.82, ***p = 0.0001, R² = 0.7530. On charts: *p < 0.05, **p < 0.01, ***p < 0.001.</p

3D mapping reveals network-specific amyloid progression and subcortical susceptibility in mice

© 2019, The Author(s). Alzheimer’s disease (AD) is a progressive, neurodegenerative dementia with no cure. Prominent hypotheses suggest accumulation of beta-amyloid (Aβ) contributes to neurodegeneration and memory loss, however identifying brain regions with early susceptibility to Aβ remains elusive. Using SWITCH to immunolabel intact brain, we created a spatiotemporal map of Aβ deposition in the 5XFAD mouse. We report that subcortical memory structures show primary susceptibility to Aβ and that aggregates develop in increasingly complex networks with age. The densest early Aβ occurs in the mammillary body, septum, and subiculum- core regions of the Papez memory circuit. Previously, early mammillary body dysfunction in AD had not been established. We also show that Aβ in the mammillary body correlates with neuronal hyper-excitability and that modulation using a pharmacogenetic approach reduces Aβ deposition. Our data demonstrate large-tissue volume processing techniques can enhance biological discovery and suggest that subcortical susceptibility may underlie early brain alterations in AD

Multi-sensory Gamma Stimulation Ameliorates Alzheimer’s-Associated Pathology and Improves Cognition

© 2019 Elsevier Inc. We previously reported that inducing gamma oscillations with a non-invasive light flicker (gamma entrainment using sensory stimulus or GENUS) impacted pathology in the visual cortex of Alzheimer's disease mouse models. Here, we designed auditory tone stimulation that drove gamma frequency neural activity in auditory cortex (AC) and hippocampal CA1. Seven days of auditory GENUS improved spatial and recognition memory and reduced amyloid in AC and hippocampus of 5XFAD mice. Changes in activation responses were evident in microglia, astrocytes, and vasculature. Auditory GENUS also reduced phosphorylated tau in the P301S tauopathy model. Furthermore, combined auditory and visual GENUS, but not either alone, produced microglial-clustering responses, and decreased amyloid in medial prefrontal cortex. Whole brain analysis using SHIELD revealed widespread reduction of amyloid plaques throughout neocortex after multi-sensory GENUS. Thus, GENUS can be achieved through multiple sensory modalities with wide-ranging effects across multiple brain areas to improve cognitive function. Auditory stimulation combined with light-induced gamma oscillations in the hippocampus CA1 and auditory cortex regions of the brain reduces amyloid levels and improves memory in animal models of Alzheimer's disease

APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer’s Disease Phenotypes in Human iPSC-Derived Brain Cell Types

The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimer's disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function (neurons), lipid metabolism (astrocytes), and immune response (microglia-like cells). APOE4 neurons exhibited increased synapse number and elevated Aβ42 secretion relative to isogenic APOE3 cells while APOE4 astrocytes displayed impaired Aβ uptake and cholesterol accumulation. Notably, APOE4 microglia-like cells exhibited altered morphologies, which correlated with reduced Aβ phagocytosis. Consistently, converting APOE4 to APOE3 in brain cell types from sAD iPSCs was sufficient to attenuate multiple AD-related pathologies. Our study establishes a reference for human cell-type-specific changes associated with the APOE4 variant. Video Abstract: [Figure presented] By generating and characterizing isogenic APOE3- or APOE4-carrying human brain cell types, Lin et al. show that the APOE4 variant can lead to extensive gene expression alterations, and multiple cellular phenotypes potentially related to AD pathogenesis, in neurons, astrocytes, and microglia.National Institutes of Health (NIH) (Grants RF1-AG048056, RC1-AG036106, and RF1-AG048029