Journal: bioRxiv

Article Title: FAM84B facilitates tau propagation via RYR3-mediated exocytosis in response to neuroinflammation

doi: 10.64898/2025.12.28.696549

Figure Lengend Snippet: (A) Differentially expressed genes in HEK293 cells in response to FAM84B KO (n=3 each). Genes with p < 0.05 and >2-fold expression change compared to control are marked with red dots. (B) Differentially expressed genes in HEK293 cells in response to FAM84B OE (n=3 each). Genes with p < 0.05 and >2-fold expression change compared to control are marked with red dots. (C) Differentially expressed genes in tau-expressing SH-SY5Y cells in response to FAM84B OE (n=3 each). Genes with p < 0.05 and >1.7-fold expression change compared to cells expressing tau alone are marked with red dots. (D) Venn diagram showing the number of shared genes between RNA-seq datasets in (A–C), with overlapping genes represented in distinct colors (red, green, black, blue). Only the names of membrane-related genes among the shared genes are noted. (E) GO analysis showing cellular compartments of genes identified in (D). The GO terms based on the number of genes are shown. (F) Levels of RYR3 and actin in iPSC-derived CN from HC and patients with AD (n=3 each). (G) Levels of RYR3 and actin in SH-SY5Y cells expressing tau alone or tau with FAM84B-Myc (n=3 each). (H) Colocalization (arrow) of FAM84B and calcium in SH-SY5Y cells expressing tau with mCherry control (C1), and in cells expressing tau with mCherry-FAM84B treated with either DMSO or dantrolene (n=4 each). (I) PHF-1 spread from SH-SY5Y cells expressing Tau-p2a-eGFP with FAM84B to neighboring cells in response to treatment with either DMSO or dantrolene (n=4 each). Arrows indicate recipient cells (red) to which tau has been transmitted. Solid boxes in the images indicate magnified areas. Results are presented as mean ± SEM. Co-localization analysis. (H) Pearson correlation coefficient. Statistical analyses: (A-C, F-I) unpaired two-tailed t -test with Welch’s correction; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: KO, knockout; OE, overexpression; GO, gene ontology; CN, cortical neuron; HC, healthy control; AD, Alzheimer’s disease; PHF-1, phosphorylated tau at residues S396/S404; FOV, field of view.

Article Snippet: Following blocking, membranes were incubated overnight at 4°C with primary antibodies (1:1,000 dilution), including: PHF-1 (Peter Davies Lab), Tau5 (Gail VW Johnson Lab), Tau (Dako, Agilent Technologies, Inc., 0024), Myc-tag (Cell Signaling Technology, 2276), FAM84B (OriGene, TA501992; Proteintech Group, Inc., Rosemont, IL, USA, 18421-1-AP), Alix (Cell Signaling Technology, 2171), Caveolin-1 (Cell Signaling Technology, 3267), RYR3 (Alomone Labs, Jerusalem, Israel, ARR003), Clathrin-heavy chain (CHC, Cell Signaling Technology, 4796), EEA1 (Cell Signaling Technology, 3288), Rab5 (Cell Signaling Technology, 3547), Rab7 (Cell Signaling Technology, 9367), Rab9 (Cell Signaling Technology, 5118), Rab11 (Cell Signaling Technology, 5589), pSTAT3 (Y705) (Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), p62 (Cell Signaling Technology, 8025), LC3 (Cell Signaling Technology, 3868), HSP70 (Cell Signaling Technology, 4876), LAMP-1 (BD Biosciences, Franklin Lakes, NJ, USA, 611042), LAMP-2 (Sigma-Aldrich, L0668), and beta-actin (Merck Millipore, MAB1501).

Techniques: Expressing, Control, RNA Sequencing, Membrane, Derivative Assay, Two Tailed Test, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: FAM84B facilitates tau propagation via RYR3-mediated exocytosis in response to neuroinflammation

doi: 10.64898/2025.12.28.696549

Figure Lengend Snippet: (A) Venn diagram showing the seven shared transcriptional cofactors between the ENCODE ChIP-seq and CHEA ChIP-seq datasets. The genes are categorized into functional terms and noted. (B) Genomic information of the FAM84B gene obtained from the UCSC Genome Browser. Transcription cofactor clusters identified from the ENCODE ChIP-seq data, with those likely to bind to the regulatory elements of FAM84B, as identified by FANTOM5, highlighted. (C) Levels of phosphorylated STAT3 (Y705) in the cerebral cortex of HC (n=3) and patients with VD (n=3), CBD (n=3), PSP (n=4), PART (n=3), and AD (n=6). (D) Levels of phosphorylated STAT3 (Y705) and total STAT3 in iPSC-derived CN from HC and patients with AD (n=3 each). (E) Levels of phosphorylated STAT3 (Y705), total STAT3, FAM84B, and actin in SH-SY5Y cells treated with either control vehicle or IL-6 (n=3 each). (F) Changes in the levels of phosphorylated STAT3 (Y705), total STAT3, FAM84B, and actin in SH-SY5Y cells expressing STAT3-WT or STAT3-DN in response to treatment with either control vehicle or IL-6 (n=3 each). (G) PHF-1 spread from Tau-p2a-eGFP expressing SH-SY5Y cells to neighboring cells in response to treatment with either control vector or IL-6 (n=4 each). Arrows indicate recipient cells (red) to which tau has been transmitted. Solid boxes in the images indicate magnified areas. (H) PHF-1 spread from IL-6 treated SH-SY5Y cells expressing Tau-p2a-eGFP to neighboring cells in response to treatment with either DMSO or dantrolene (n=4 each). Arrows indicate recipient cells (red) to which tau has been transmitted. Solid boxes in the images indicate magnified areas. (I) Changes in the levels of PHF-1, total tau, phosphorylated STAT3 (Y705), total STAT3, FAM84B (FAM84B-Myc), RYR3, and actin in iPSC-derived CN from HC after infection with AAV and subsequent treatments (n=3 each). (J) Changes in the levels of phosphorylated tau (T181 and S396) in the CM of iPSC-derived CN from HC after infection with AAV and subsequent treatments (n=8 each). In (I–J), the experimental groups are: (1) CN infected with AAV containing tau, (2) CN infected with AAV containing tau and FAM84B-Myc, (3) CN infected with AAV containing tau and treated with IL-6 and TNF-α, (4) CN infected with AAV containing tau and FAM84B-Myc and treated with dantrolene, and (5) CN infected with AAV containing tau, treated with IL-6 and TNF-α, followed by treatment with dantrolene. Results are presented as mean ± SEM. Statistical analyses: (C) ordinary two-way ANOVA; (D-J) unpaired two-tailed t -test with Welch’s correction; ns: not significant, *p < 0.05, **p < 0.01, and ***p < 0.001. Abbreviations: HC, healthy control; VD, vascular dementia; CBD, corticobasal degeneration; PSP, progressive supranuclear palsy; PART, primary age-related tauopathy; AD, Alzheimer’s disease; CN, cortical neuron; WT, wild type; DN, dominant negative; PHF-1, phosphorylated tau at S396/S404 residue; FOV, field of view; AAV, adeno-associated virus; CM, conditioned media.

Article Snippet: Following blocking, membranes were incubated overnight at 4°C with primary antibodies (1:1,000 dilution), including: PHF-1 (Peter Davies Lab), Tau5 (Gail VW Johnson Lab), Tau (Dako, Agilent Technologies, Inc., 0024), Myc-tag (Cell Signaling Technology, 2276), FAM84B (OriGene, TA501992; Proteintech Group, Inc., Rosemont, IL, USA, 18421-1-AP), Alix (Cell Signaling Technology, 2171), Caveolin-1 (Cell Signaling Technology, 3267), RYR3 (Alomone Labs, Jerusalem, Israel, ARR003), Clathrin-heavy chain (CHC, Cell Signaling Technology, 4796), EEA1 (Cell Signaling Technology, 3288), Rab5 (Cell Signaling Technology, 3547), Rab7 (Cell Signaling Technology, 9367), Rab9 (Cell Signaling Technology, 5118), Rab11 (Cell Signaling Technology, 5589), pSTAT3 (Y705) (Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), p62 (Cell Signaling Technology, 8025), LC3 (Cell Signaling Technology, 3868), HSP70 (Cell Signaling Technology, 4876), LAMP-1 (BD Biosciences, Franklin Lakes, NJ, USA, 611042), LAMP-2 (Sigma-Aldrich, L0668), and beta-actin (Merck Millipore, MAB1501).

Techniques: ChIP-sequencing, Functional Assay, Derivative Assay, Control, Expressing, Plasmid Preparation, Infection, Two Tailed Test, Dominant Negative Mutation, Residue, Virus

Journal: Nature Communications

Article Title: A spatial single-cell atlas of the claustro-insular region uncovers key regulators of neuronal identity and excitability

doi: 10.1038/s41467-025-63138-2

Figure Lengend Snippet: Images of the CLA in coronal sections from Nr4a2 wt/wt ( a ) and Nr4a2 del/wt ( b ) mice, with Nr4a2 mRNA transcripts labeled by smFISH (white). Insets show magnified regions (white squares). Nuclei are stained with DAPI (blue). Scale bar: 200 μm. c Mean number of Nr4a2 mRNA puncta per cell in the claustro-insular area. Each dot represents one section of an Nr4a2 del/wt mouse relative to the mean of all sections of Nr4a2 wt/wt control mice processed in the same batch. Data are mean ± SD. n = 4 mice per genotype; ***p < 0.001; χ 2 -based LRT applied to a negative binomial GLMM with quadratic parametrization. d Total number of significantly modulated genes in Nr4a2 del/wt mice in each cell type from the scRNAseq dataset (n = 5 Nr4a2 wt/wt and 8 Nr4a2 del/wt mice). Two-sided Wilcoxon rank sum test; p -values were adjusted for multiple comparisons using the Bonferroni method. Violin plots illustrating the distribution of expression for downregulated ( e ) and upregulated ( f ) genes across claustro-insular neuronal populations from Nr4a2 wt/wt and Nr4a2 del/wt mice in the scRNAseq dataset (normalized data are shown on a log 10 scale). Mean number of mRNA puncta per cell of downregulated ( g ) and upregulated ( h ) genes in Nr4a2 + and/or Oprk1 + cells from the claustro-insular region. Each dot represents one section of an Nr4a2 del/wt mouse relative to the mean of all sections of Nr4a2 wt/wt control mice processed in the same batch. Data are mean ± SD. n = 2-3 mice per genotype. *p < 0.05, ***p < 0.001; χ 2 -based LRT applied to a negative binomial GLMM with quadratic parametrization; p -values were adjusted for multiple comparisons using the Holm method. Cell type-specific modulation of Ntm ( i ) and Ryr2 ( j ) expression. Top: Violin plots show the distribution of expression of marker genes used for cell type identification in the smFISH data (raw data are shown on a linear scale). Bottom: Box plots show the mean Ntm and Ryr2 expression levels within specific cell populations from Nr4a2 wt/wt and Nr4a2 del/wt mice (n = 2 mice per genotype, 6–8 sections). Each dot represents one section. Box limits: Q1–Q3; line: median; whiskers: ±1.5*IQR. *p < 0.05, ***p < 0.001; χ 2 -based LRT applied to a negative binomial GLMM; p -values adjusted using the Holm method. Source data are provided as a Source Data file. Exact sample details are provided in Supplementary Data .

Article Snippet: Finally, the mechanism underlying calcium-induced calcium release modulation of BK channels was dissected using the following pharmacological channel blockers: Cav1: Nifedipine (10 μM), Cav2.1: ω-Agatoxin IVA (100 nM, Alomone Labs), Cav2.2: ω-Conotoxin GVIA (3 μM, Alomone Labs), Cav2.3: SNX-482 (400 nM, Alomone Labs), Cav3: ML218 (2 μM, Alomone Labs), BKCa channels: Iberiotoxin (100 nM, Alomone Labs), RyR2: Ryanodine (10–20 μM, Alomone Labs).

Techniques: Labeling, Staining, Control, Expressing, Marker

Journal: Nature Communications

Article Title: A spatial single-cell atlas of the claustro-insular region uncovers key regulators of neuronal identity and excitability

doi: 10.1038/s41467-025-63138-2

Figure Lengend Snippet: a Significantly modulated genes encoding channels, neurotransmitter receptors, and related signaling proteins in CLA cells of Nr4a2 del/wt mice when compared to wild-type littermates. b Retrogradely labeled CLA neurons projecting to the mPFC were recorded. Bottom : example of a patched cell filled with biocytin. The same procedure was performed on n = 585 CLA neurons. c Example of the spiking activity evoked by a 1s 250 pA depolarizing step. d Input/Output functions of CLA neurons recorded in artificial cerebrospinal fluid (ACSF) or in blockers of the fast glutamatergic (NBQX and APV) and GABAergic (Gabazine) synaptic transmission (* indicates significant using Fisher’s LSD test corrected with a 5% FDR Benjamini–Hochberg). e Area under the curve (AUC) of the input/output function for each Nr4a2 wt/wt and Nr4a2 del/wt cell in ACSF and synaptic blockers. f Resting membrane potential is recorded at the opening of cells. g Examples of recorded action potentials (AP), (bottom traces: truncated AP). HW half-width, RT rise time, DT decay time, AHP afterhyperpolarization potential. h Quantifications of the different AP parameters (each circle represents the mean parameter value computed on all spikes of all steps for a given cell). i Example of the firing frequency evoked by a 250 pA current step under various pharmacological conditions. j Input/Output functions of CLA neurons recorded in the presence of VGCC blockers (10 µM nifedipine, 100 nM ω-agatoxin IVA, 3 µM ω-conotoxin GVIA, 400 nM SNX-482, 2 µM ML218), BK channels blocker (100 nM iberiotoxin), and RyR2 channel blocker (10–20 µM ryanodine). k Comparison of the firing functions between Nr4a2 wt/wt and Nr4a2 del/wt cells, expressed as a percentage of the mean wild-type AUC calculated for each pharmacological condition (see “Methods” section). Cav2&3: ω-Agatoxin IVA, ω-Conotoxin GVIA, SNX-482, and ML218. Cav2.1&2.2: ω-Agatoxin IVA and ω-Conotoxin GVIA. Cav2.3: SNX-482. Cav3: ML218. *p < 0.05, ***p < 0.001; Mann–Whitney U test. l Comparison of the relative AHP amplitude between Nr4a2 wt/wt and Nr4a2 del/wt cells under each pharmacological condition. **p < 0.01; Mann–Whitney test. m Summary diagram of the mechanism controlling changes in firing frequency through the activation of BK channels. Data are shown as mean ± SEM. In e , f , h , k , and l , each circle represents one recorded neuron. Source data are provided as a Source Data file. See Supplementary Data for more details.

Article Snippet: Finally, the mechanism underlying calcium-induced calcium release modulation of BK channels was dissected using the following pharmacological channel blockers: Cav1: Nifedipine (10 μM), Cav2.1: ω-Agatoxin IVA (100 nM, Alomone Labs), Cav2.2: ω-Conotoxin GVIA (3 μM, Alomone Labs), Cav2.3: SNX-482 (400 nM, Alomone Labs), Cav3: ML218 (2 μM, Alomone Labs), BKCa channels: Iberiotoxin (100 nM, Alomone Labs), RyR2: Ryanodine (10–20 μM, Alomone Labs).

Techniques: Labeling, Activity Assay, Transmission Assay, Membrane, Comparison, MANN-WHITNEY, Activation Assay