Journal: Alzheimer's & Dementia

Article Title: Single‐cell analysis reveals neuroprotective histone deacetylase inhibitor pathways

doi: 10.1002/alz.71108

Figure Lengend Snippet: Integrative multi‐cell type analysis workflow identifies DISC1 as a convergent target of TSA in AD. Schematic representation of the multi‐pronged analytical framework used to identify and validate TSA as a therapeutic candidate for AD. The workflow began with cell‐type‐specific drug repurposing analysis of scRNA‐seq data from Grubman et al., ¹³ Mathys et al., ¹⁴ and Green et al. ¹⁵ datasets, resulting in the identification of TSA as a promising compound. We strategically investigated TSA's effects through two parallel cell type‐specific pathways: neurons and microglia. These cell types were specifically selected based on their known vulnerability in AD pathology and significant drug scores in our initial ASGARD analysis. For neurons, we applied DEGAS analysis to identify AD‐associated neuronal subpopulations, followed by differential expression analysis of TSA‐treated neurons in mice. Concurrently, we examined microglia, which showed high TSA drug scores in doublet interactions, and conducted differential expression analysis across microglial subtypes. Both analytical branches converged on DISC1 as a key upregulated gene, which was subsequently validated in human iPSC‐derived neuronal models through both phenotypic assays and transcriptomic analysis. This cell type‐specific approach enabled the identification of DISC1 as a potential mechanistic target underlying TSA's neuroprotective effect in AD. Created in BioRender. Peyton, M. (2025) https://biorender.com/73irecf . AD, Alzheimer's disease; ASGARD, A Single Cell Guided Pipeline to Aid Repurposing of Drugs; DEGAS, Diagnostic Evidence Gauge of Single cells; DISC1, Disrupted‐In‐Schizophrenia 1; iPSC, induced pluripotent stem cell; scRNA‐seq, single‐cell RNA sequencing; TSA, trichostatin‐A.

Article Snippet: For experimental treatments, neurons were cultured until in vitro day (DIV) 4, at which point they were treated with various concentrations of TSA (TSA, Sigma–Aldrich, cat. #: T1952) and Aβ oligomers (StressMarq, cat. #: SPR‐488).

Techniques: Quantitative Proteomics, Derivative Assay, Diagnostic Assay, RNA Sequencing

Journal: Alzheimer's & Dementia

Article Title: Single‐cell analysis reveals neuroprotective histone deacetylase inhibitor pathways

doi: 10.1002/alz.71108

Figure Lengend Snippet: Single‐cell analysis identifies TSA as top drug repurposing candidate across cortical brain regions. (A) UMAP projections of all cells from six control and six AD samples in the Grubman et al. ¹³ entorhinal cortex dataset, with clusters representing cell‐type‐specific groupings. (B) Pathway enrichment analysis highlighting key signaling pathways significantly enriched within each cell type cluster in the Grubman dataset, with significance represented as ‐log10(FDR). (C) Drug Score analysis for AD samples in the Grubman dataset, displaying compounds with FDR < 0.1 and Drug Score ranking within the 90th percentile. TSA (trichostatin‐A) emerges among the top‐ranked candidates. (D) UMAP visualizations of all cells from 24 control and 24 AD samples in the Mathys et al. ¹⁴ prefrontal cortex dataset, with distinct clusters representing cell types. (E) Pathway enrichment analysis for the Mathys dataset showing key signaling pathways significantly enriched within each cell type cluster, with significance represented as ‐log10(FDR). (F) Drug Score analysis for AD samples in the Mathys dataset, displaying compounds with FDR < 0.1 and Drug Score values within the 90th percentile. (G) UMAP projections from the Green et al. ¹⁵ aged prefrontal cortex dataset showing cell‐type‐specific clustering across AD and control samples. (H) Pathway enrichment analysis for the Green dataset, highlighting significantly enriched signaling pathways across cell types, with significance represented as ‐log10(FDR). (I) Drug Score analysis for the Green dataset showing top‐ranked therapeutic candidates with FDR < 0.1 and Drug Scores within the 90th percentile. Cell types: Ast, astrocytes; CUX2+, CUX2‐positive excitatory neurons; CUX2‐, CUX2‐negative excitatory neurons; Dou, doublets; End, endothelial cells; Ex, excitatory neurons; In, inhibitory neurons; Inh, inhibitory neurons; Mic, microglia; Neu, neurons; Oli, oligodendrocytes; Opc, oligodendrocyte progenitor cells; Per, pericytes; unID, unidentified cells. AD, Alzheimer's disease; FDR, false discovery rate; TSA, trichostatin‐A; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: For experimental treatments, neurons were cultured until in vitro day (DIV) 4, at which point they were treated with various concentrations of TSA (TSA, Sigma–Aldrich, cat. #: T1952) and Aβ oligomers (StressMarq, cat. #: SPR‐488).

Techniques: Single-cell Analysis, Control, Protein-Protein interactions

Journal: Alzheimer's & Dementia

Article Title: Single‐cell analysis reveals neuroprotective histone deacetylase inhibitor pathways

doi: 10.1002/alz.71108

Figure Lengend Snippet: Doublet cell analysis reveals cellular interaction patterns and drug targeting opportunities in AD. (A) UMAP projections of all cells from six control and six AD samples in the Grubman et al. ¹³ dataset, showing cell‐type‐specific clusters with doublets re‐annotated into their most likely two contributing cell types (e.g., ast‐mic for astrocyte–microglia doublets). (B) Bar chart displaying the counts of identified cell types, including doublets without splitting into their component cell types. (C) Pie chart illustrating the distribution of identified doublet cell types, providing an overview of the most common cellular interactions observed in the dataset. (D) Pathway enrichment analysis for re‐annotated cell type clusters, highlighting key signaling pathways with significant enrichment, represented as ‐log10(FDR). (E) Drug Score analysis for AD samples, highlighting compounds with FDR < 0.1 and Drug Scores within the 90th percentile. TSA shows particularly strong enrichment in doublet populations involving microglia. Cell types: Ast, astrocytes; Dou, doublets; End, endothelial cells; Mic, microglia; Neu, neurons; Oli, oligodendrocytes; Opc, oligodendrocyte progenitor cells; unID, unidentified cells. AD, Alzheimer's disease; FDR, false discovery rate; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: For experimental treatments, neurons were cultured until in vitro day (DIV) 4, at which point they were treated with various concentrations of TSA (TSA, Sigma–Aldrich, cat. #: T1952) and Aβ oligomers (StressMarq, cat. #: SPR‐488).

Techniques: Cell Analysis, Control, Protein-Protein interactions

Journal: Alzheimer's & Dementia

Article Title: Single‐cell analysis reveals neuroprotective histone deacetylase inhibitor pathways

doi: 10.1002/alz.71108

Figure Lengend Snippet: TSA modulates synaptic and developmental gene programs and prevents Aβ‐induced neurotoxicity in human iPSC‐derived cortical neurons. (A) Volcano plot of DEGs in TSA‐treated mouse hippocampal neurons versus control. Blue points represent significantly downregulated genes, while red points represent significantly upregulated genes (adjusted p ‐value < 0.05). (B) GO Biological Process enrichment analysis for genes with positive log2 fold change (upregulated by TSA treatment). (C) GO Biological Process enrichment analysis for genes with negative log2 fold change (downregulated by TSA treatment). (D) Venn diagram showing overlapping upregulated genes across three independent analyses: TSA‐treated mouse hippocampal neurons (red, 5015 unique genes), microglial subtypes from the Lee et al. human dataset (blue, 419 unique genes), and AD‐associated neurons identified via DEGAS cell prioritization analysis (green, 142 unique genes). The diagram reveals 104 genes shared between the TSA and Lee datasets, 54 genes shared between the TSA and DEGAS, 4 genes shared between Lee and DEGAS, and critically, 1 gene (DISC1) upregulated across all three experimental contexts, identifying it as a convergent therapeutic target. (E) MTS cell viability assay results in human iPSC‐derived cortical neurons. Left panel: Dose‐response curve showing Aβ oligomer‐induced toxicity at concentrations of 0.2, 1, and 5 µM compared to control. Right panel: TSA neuroprotection against 5 µM Aβ oligomers, with neurons pre‐treated with varying TSA concentrations (0.066, 0.2, 0.33) showing dose‐dependent rescue of cell viability. Statistical significance: ns (not significant), ** p < 0.01, *** p < 0.001, **** p < 0.0001. (F) Quantification of synaptic cluster density (number of clusters per 20 µm dendrite) across treatment conditions. NT neurons show baseline synaptic density (gray), 5 µM) cause significant synaptic loss (red), and co‐treatment with 0.2 µM TSA (Aβo + TSA, green) significantly rescues synaptic density, demonstrating TSA's protective effect on synaptic integrity. Each dot represents an individual measurement. Statistical significance: ns (not significant), **** p < 0.0001. (G) Representative confocal immunofluorescence images of synapses in human iPSC‐derived cortical neurons. Neurons were immunostained for the postsynaptic marker PSD95 (red, left column) and presynaptic marker Syn1 (green, middle column), with colocalization (yellow, right column) indicating functional synapses. Rows show: NT controls (top), 5 µM Aβo treatment (middle), and combined treatment with Aβo plus 0.2 µM TSA (Aβo+TSA, bottom). TSA treatment preserves synaptic density and colocalization despite Aβ exposure. Scale bar = 2 µm. Aβ, β‐amyloid; Aβo, Aβ oligomers; AD, Alzheimer's disease; DEG, differentially expressed genes; DEGAS, Diagnostic Evidence Gauge of Single cells; GO, gene ontology; iPSC, induced pluripotent stem cell; NT, non‐treated; TSA, trichostatin‐A.

Article Snippet: For experimental treatments, neurons were cultured until in vitro day (DIV) 4, at which point they were treated with various concentrations of TSA (TSA, Sigma–Aldrich, cat. #: T1952) and Aβ oligomers (StressMarq, cat. #: SPR‐488).

Techniques: Derivative Assay, Control, Viability Assay, Immunofluorescence, Marker, Functional Assay, Diagnostic Assay

Journal: Alzheimer's & Dementia

Article Title: Single‐cell analysis reveals neuroprotective histone deacetylase inhibitor pathways

doi: 10.1002/alz.71108

Figure Lengend Snippet: Transcriptomic analysis of TSA effects on iPSC‐derived cortical neurons reveals distinct gene expression patterns . (A) Volcano plot displaying DEGs between control and TSA‐treated human iPSC‐derived cortical neurons. Significantly upregulated genes are shown in red and downregulated genes in blue (adjusted p ‐value < 0.05, |log 2 FC| > 0.58). (B) Box plots showing DISC1 expression levels (normalized log2CPM) across four treatment conditions: Control, Amyloid_beta (Aβ alone), TSA (TSA alone), and Combined (TSA + Aβ). Statistical comparisons are indicated with brackets and significance levels. (C) GO Biological Process enrichment analysis for genes upregulated by TSA treatment. Dot size represents gene count, and color indicates ‐log 10 (FDR). (D) GO Biological Process enrichment analysis for genes downregulated by TSA treatment. (E) GO Molecular Function enrichment for genes upregulated by TSA treatment. (F) GO Molecular Function enrichment for genes downregulated by TSA treatment. (G) Heatmap showing expression patterns of top differentially expressed genes across all samples. Samples are grouped by treatment condition (Control, Amyloid_beta, TSA, Combined) with color‐coded bars at the top. Gene expression is displayed as normalized z ‐scores, with red indicating high expression and blue indicating low expression. Aβ, β‐amyloid; DEG, differentially expressed genes; DISC1, Disrupted‐In‐Schizophrenia 1; FDR, false discovery rate; GO, gene ontology; iPSC, induced pluripotent stem cell; TSA, trichostatin‐A.

Article Snippet: For experimental treatments, neurons were cultured until in vitro day (DIV) 4, at which point they were treated with various concentrations of TSA (TSA, Sigma–Aldrich, cat. #: T1952) and Aβ oligomers (StressMarq, cat. #: SPR‐488).

Techniques: Derivative Assay, Gene Expression, Control, Expressing

Journal: Alzheimer's & Dementia

Article Title: Single‐cell analysis reveals neuroprotective histone deacetylase inhibitor pathways

doi: 10.1002/alz.71108

Figure Lengend Snippet: Differential gene expression analysis of TSA and amyloid‐beta treatment in neural cells. (A) Volcano plot showing the main effect of Aβ treatment on gene expression (Amyloid‐beta Main Effect). Significantly downregulated genes are shown in blue and upregulated genes in red (adjusted p ‐value < 0.05, |log 2 FC| > 0.58). (B) Volcano plot displaying the main effect of TSA treatment on gene expression (TSA Main Effect). Significantly downregulated genes are shown in blue and upregulated genes in red. (C) Volcano plot illustrating the interaction effect between TSA and Aβ treatments (TSA:Amyloid‐beta Interaction Effect). Significantly downregulated genes are shown in blue and upregulated genes in red. (D) Heatmap displaying expression patterns of top differentially expressed genes across experimental conditions, clustered by effect type. Samples are organized by treatment condition: Control, AB (Aβ), TSA, and TSA+AB (Combined). Left sidebar color bars indicate Condition (Control, Amyloid_beta, TSA, Combined). Right sidebar color bars indicate Direction (Up, Down) and Effect (TSA, Amyloid‐beta, Interaction). Expression values are shown as normalized z ‐scores with yellow indicating high expression and blue indicating low expression. Aβ, β‐amyloid; TSA, trichostatin‐A.

Article Snippet: For experimental treatments, neurons were cultured until in vitro day (DIV) 4, at which point they were treated with various concentrations of TSA (TSA, Sigma–Aldrich, cat. #: T1952) and Aβ oligomers (StressMarq, cat. #: SPR‐488).

Techniques: Gene Expression, Expressing, Control

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of siRNA transfection of iMG. Cells were transfected 24 hours after iMG plating. After an additional 24 hours, cells were incubated with 2 μg/mL of pHrodo-labeled oligomerized amyloid (1-42) and imaged over a 20-hour window. (B) Confocal fluorescence images of iMG assessed for internalization of pHrodo-oligomerized amyloid beta treated as in (A). Digital phase contrast was used to visualize iMG. Images are stitched from four 20X images. Scale bar represents 400 μm. (C) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG per hour. Data are normalized to mock transfected control. (D) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG at endpoint. Data are normalized to mock transfected control. (E) RNA isolated from iMG 24 hours after transfection was used to quantify the percent CD33 mRNA expression by qPCR. (F) Change in TREM2 secretion in conditioned media from iMG was quantified 24 hours after transfection by MSD. (G) Protein lysates isolated from iMG 24 hours after transfection were used to quantify percent SYK phosphorylation by AlphaLISA. Data represent mean ± SEM using one-way ANOVA with uncorrected Fisher’s LSD (D) and Dunnett’s multiple comparison test (E-G). Data points (n-numbers) are plotted on each bar graph and scatter plot each representing an independent experimental replicate. Line plots are representative of three independent experimental replicates (C).

Article Snippet: Oligomerized amyloid-beta (1-42) particles (StressMarg) were obtained and pHrodo-labeled using pHrodo iFL Green STP ester labeling kit (Invitrogen), following the manufacturer’s instructions.

Techniques: Transfection, Incubation, Labeling, Fluorescence, Control, Isolation, Expressing, Phospho-proteomics, Comparison

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of AAV6-mediated transduction of iMG. Cells were transduced with an MOI of 125000 at the time of plating. On day 3, cells were incubated with 2 μg/mL of pHrodo-labeled oligomerized amyloid (1-42) and imaged over a 20-hour window. (B) Protein lysates isolated from iMG 72 hours after transduction were used to quantify percent CD33 protein expression by MSD. Data are normalized to no AAV6 transduction control. (C) Confocal fluorescence images of iMG assessed for internalization of pHrodo-oligomerized amyloid beta treated as in (A). Digital phase contrast was used to visualize iMG. Images are stitched from four 20X images. Scale bar represents 400 μm. (D) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry per hour. Data are normalized to no AAV6 transduction control. (E) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry at endpoint. Data are normalized to no AAV6 transduction control. (F) TREM2 secretion in conditioned media was quantified from iMG 72 hours after transduction by MSD. (G)(left) Percent change in TREM2 secretion in conditioned media was quantified from HMC3 cells 48 hours after transduction with increasing AAV6-CD33 concentrations by MSD. Data are represented as a percent change relative to dose-matched AAV6-mCherry. (right) TREM2 secretion in conditioned media was quantified from HMC3 cells 48 hours after transduction of AAV6-CD33 by MSD. (MOI = 250000). (H) Protein lysates isolated from HMC3 cells 48 hours after transduction were used to quantify percent full length TREM2 protein expression by MSD. (MOI = 250000). Data represent mean ± SEM using one-way ANOVA with post-hoc Tukey’s multiple comparisons test (B), with uncorrected Fisher’s LSD (E), with post-hoc Dunnett multiple comparisons test (F, G) and one-tailed unpaired t-test (H). Data points (n-numbers) are plotted on each bar graph and scatter plot each representing an independent experimental replicate. Line plots represent three independent experimental replicates (D, G).

Article Snippet: Oligomerized amyloid-beta (1-42) particles (StressMarg) were obtained and pHrodo-labeled using pHrodo iFL Green STP ester labeling kit (Invitrogen), following the manufacturer’s instructions.

Techniques: Transduction, Incubation, Labeling, Isolation, Expressing, Control, Fluorescence, One-tailed Test

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of AAV6-mediated transduction of iMG. Cells were transduced with an MOI of 125000 at the time of plating. On day 1, cells were transfected with a di-siRNA targeting CD33 for 48 hours. On day 3, cells were incubated with 2 μg/mL of pHrodo-labeled oligomerized amyloid (1-42) and imaged over a 20-hour window. (B) Confocal fluorescence images of iMG assessed for internalization of pHrodo-oligomerized amyloid beta treated as in (A). Digital phase contrast was used to visualize iMG. Images are stitched from four 20X images. Scale bar represents 400 μm. (C) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry per hour after transfection of increasing concentrations of CD33-targeting di-siRNA per hour. Data are normalized to mock transfected no AAV6 transduction control. (D) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry per hour after transfection of increasing concentrations of CD33-targeting di-siRNA at endpoint. Data are normalized to mock transfected no AAV6 transduction control. (E) Protein lysates isolated from iMG 72 hours after transduction and 48 hours after transfection were used to quantify percent CD33 protein expression by MSD. Data are normalized to mock transfected AAV6-CD33-treated iMG. (F) Linear regression of data from (D) and (E) was plotted to demonstrate the correlation between percent CD33 expression levels and oligomerized amyloid-beta uptake. Data represent mean ± SEM using one-way ANOVA with uncorrected Fisher’s LSD (D) and Dunnett’s multiple comparison test (E). Data points (numbers) are plotted on each bar graph and scatter plot, each representing an independent experimental replicate. Line plots are representative of three independent experimental replicates (C, F).

Article Snippet: Oligomerized amyloid-beta (1-42) particles (StressMarg) were obtained and pHrodo-labeled using pHrodo iFL Green STP ester labeling kit (Invitrogen), following the manufacturer’s instructions.

Techniques: Transduction, Transfection, Incubation, Labeling, Fluorescence, Control, Isolation, Expressing, Comparison