anti nav1 2α  (Alomone Labs)

Journal: bioRxiv

Article Title: Autism-associated SCN2A deficiency disrupts cortico-striatal circuitry in human brain assembloids

doi: 10.1101/2025.06.02.657036

Figure Lengend Snippet: (A) CRISPR/Cas9 editing introduced the SCN2A p.C959X mutation in human iPSCs. Sanger sequencing confirms successful editing: WT (top) retains cytosine (C), HET (middle) shows overlapping peaks and HOM (bottom) displays a complete C-to-A substitution, introducing a premature stop codon (X). (B) Western blot showing Na V 1.2 ( SCN2A ) expression in WT, HET, and HOM hCOs, with β-actin as a reference. (C) SCN2A mRNA levels in hCO-hStrO assembloids showing reduced expression in HET and HOM SCN2A-C959X mutants vs. WT. n = 8 assembloids/group from 3 hiPSC lines (WT: KOLF2.1J, B07, C03; HET: A02, E04, F01; HOM: A03, D06, F03). One-way ANOVA with Tukey’s test, ****p < 0.0001. (D) Immunostaining of Ankyrin-G (green), Na V 1.2 (red) in hCO-hStrO assembloids on day 154, demonstrating Na V 1.2 localization at the axon initial segment (AIS). n = 3 assembloids. (E) Electrophysiological alterations in neurons carrying SCN2A-C959X mutation. (E(i)) The representative traces of action potentials (APs) in WT and HET at −70 mV. Inset: AP response to +140 pA current injection. (E(ii)) AP generated in response to depolarizing current pulses at −70 mV. (**p < 0.01, two-way ANOVA). (E(iii)) Individual and average maximum AP counts at −70 mV. Mann-Whitney test, ***p < 0.0001. (E(iv)) Input resistance and (E(v)) rheobase measurements at −70 mV. Unpaired t-test. **p < 0.001. (F) Axon projection deficits in SCN2A-C959X assembloids. (F(i)) Experimental timeline and (F(ii)) quantification of axon projections from hCO to hStrO in assembloids on days 10, 20, 30, and 42 post-fusion. One-way ANOVA, Tukey’s test, ****p < 0.0001. (F(iii)) Representative images of axon projections (mScarlet⁺) on day 42 in WT and HET assembloids. (F(iv)) mScarlet coverage quantification in hStrO on days 10, 20, 30, and 42, comparing WT (n = 15 assembloids from B07, KOLF2.1J) and HET (n = 16 assembloids from F01, A02, E04). Mixed-effects model with Geisser-Greenhouse correction, Sidak’s test. *p < 0.05, **p < 0.01. (G) Dendritic morphology deficits in SCN2A-C959X assembloids. (G(i)) Schematic of receiver neuron identification in hStrO. (G(ii)) Representative images of neuronal morphology (Sholl analysis) in WT and HET. (G(iii)) Total dendrite number (unpaired t-test), (G(iv)) dendrite length (unpaired t-test), and (Gv) interaction number (two-way ANOVA) in WT (n = 26 neurons, B07, C03, A11, KOLF2.1J cell lines) and HET (n = 25 neurons, F01, A02, E04 cell lines). ***p < 0.001, ****p < 0.0001. (H) Na V 1.2 localization in dendrites. Immunostaining for MAP2 (green), Na V 1.2 (red), and DAPI (blue) in hCO-hStrO assembloids, confirming Na V 1.2 expression in neuronal dendrites. n = 3 assembloids from two hiPSC lines with consistent results. (I) Dendritic spine deficits in SCN2A-C959X assembloids. (I(i)) Confocal images of striatal receiver (mScarlet⁺) dendritic spines in WT and HET. (I(ii)) Quantification of total receiver spine density in WT (gray, n = 74 dendrites, 9 assembloids, C03, KOLF2.1J, B07 lines) and HET (blue, n = 86 dendrites, 9 assembloids, F01, A02, E04 lines). Mann-Whitney test, ****p < 0.0001. (I(iii)) Spine subtype analysis. ****p < 0.0001 (Multiple Mann-Whitney test). (J) Synaptic transmission deficits in SCN2A-C959X assembloids. (J(i)) Confocal images of receiver neurons (mScarlet⁺, red) in hStrO from hCO-hStrO assembloids. Patch-clamped neurons labeled with neurobiotin (green) confirm that recorded neurons are receivers. (J(ii)) Example sEPSC traces in hStrO receiver neurons from WT (black, top) and HET (blue, bottom). (J(iii)) sEPSC frequency in WT (n = 12 cells, 7 assembloids, A11, KOLF2.1J lines) and HET (n = 16 cells, 7 assembloids, A02, E04 lines). Unpaired t-test, **p < 0.01. (J(iv)) Cumulative probability of sEPSC inter-event intervals. Data are represented as mean ± SEM.

Article Snippet: For Western blotting, primary antibodies included Rabbit anti-SCN2A (Na V 1.2) (Alomone Labs, ASC-002, 1:200), Rabbit anti- SCN2A (Sigma-Aldrich, ZRB1300, 1:200), and Mouse anti-β-Actin (Thermo Fisher, MA5-15739, 1:1000).

Techniques: CRISPR, Mutagenesis, Sequencing, Western Blot, Expressing, Immunostaining, Injection, Generated, MANN-WHITNEY, Transmission Assay, Labeling

Journal: bioRxiv

Article Title: Autism-associated SCN2A deficiency disrupts cortico-striatal circuitry in human brain assembloids

doi: 10.1101/2025.06.02.657036

Figure Lengend Snippet: (A) Schematic representation of SCN2A highlighting the p.C959X nonsense mutation site. (B) AIS morphology. (B(i)) Schematic and representative images of AISs labeled by Ankyrin-G (green) in WT and HET SCN2A-C959X neurons. (B(ii)) AIS length quantification (WT: n = 42 neurons from B07, C03, KOLF2.1J hiPSC lines; HET: n = 56 neurons from F01, A02, E04 hiPSC lines). Unpaired Welch’s t test, **p < 0.01. (C) Impaired excitatory synapse formation in SCN2A -mutant hCOs. (C(i)) Schematic of excitatory synapses labeled by Syn1 (green, presynaptic) and PSD95 (red, postsynaptic). (C(ii)) Representative images of excitatory synapses (PSD95 + /Syn1 + colocalization) in WT and HET SCN2A-C959X hCOs. Scale bar: 10 µm. (C(iii)) Quantification of excitatory synapses. WT: 29 images from 9 organoids (KOLF2.1J, B07, A11); HET: 37 images from 10 organoids (A02, E04). Unpaired t-test, ***p < 0.001. (D) Immunostaining of NeuN (green), Na V 1.2 (red), and DAPI (blue) in hCO-hStrO assembloids, showing colocalization of Na V 1.2 with NeuN in both hCO and hStrO, indicating Na V 1.2 expression in neuronal cell bodies. (E) Reduced dendritic spine density in SCN2A -mutant striatal neurons in hCO-hStrO assembloid slices. (E(i)) Schematic of patch-clamp recording on hStrO receiver neurons and dendritic spine density quantification. (E(ii)) Representative images of patched neurons labeled with neurobiotin (magenta). (E(iii)) Representative images of dendritic spines from patched neurons. (E(iv)) Quantification of total receiver spine density. WT (gray, n = 24 dendrites, from 4 assembloids, A11, KOLF2.1J); HET SCN2A-C959X (blue, n = 27 dendrites, from 5 assembloids, A02, E04). Mann-Whitney test: ****pc

Article Snippet: For Western blotting, primary antibodies included Rabbit anti-SCN2A (Na V 1.2) (Alomone Labs, ASC-002, 1:200), Rabbit anti- SCN2A (Sigma-Aldrich, ZRB1300, 1:200), and Mouse anti-β-Actin (Thermo Fisher, MA5-15739, 1:1000).

Techniques: Mutagenesis, Labeling, Immunostaining, Expressing, Patch Clamp, MANN-WHITNEY

Journal: bioRxiv

Article Title: Autism-associated SCN2A deficiency disrupts cortico-striatal circuitry in human brain assembloids

doi: 10.1101/2025.06.02.657036

Figure Lengend Snippet: (A) Experimental workflow. n = 7 assembloids/group from WT (iPSC lines: KOLF2.1J, B07, C03), HET (iPSC lines: A02, E04, F01), and HOM (iPSC lines: A03, D06, F03). (B) Bar graph showing counts per million (CPM) of SCN2A expression in WT, HET, and HOM in bulk RNA sequencing. One-way ANOVA with Tukey’s comparisons, ****p < 0.0001. (C) Differential gene expression analysis. Volcano plots displaying differentially expressed genes (DEGs) in HET vs. WT (C(i)) and HOM vs. WT (C(ii)) comparisons. Upregulated genes (magenta), downregulated (blue), and non-significant (gray) (FDR < 0.05). Key downregulated genes associated with axon and synapse pathways are labeled. (D) Downregulated gene pathways. (D(i)) Venn diagram illustrating overlapping downregulated genes in HET vs. WT and HOM vs. WT. (D(ii)) KEGG pathway enrichment analysis of downregulated genes in the nervous system and development and regeneration (marked with ’#’) classification. (D(iii)) Axonal deficits. Heatmap of top downregulated axon-related genes, categorized into axon initial segments (AIS) (top 10) and axon terminal genes (top 10), indicating axonal deficits in HET and HOM. (D(iv)) Synaptic deficits. Heatmap of top downregulated synapse-related genes, classified into excitatory (top 10) and inhibitory (GABAergic, top 10) synapses. (E) Upregulated gene pathways. (E(i)) Venn diagram showing overlapping upregulated genes in HET vs. WT and HOM vs. WT. (E(ii)) Reactome pathway enrichment analysis of the top 10 overlapping upregulated pathways, with key pathways associated with nonsense-mediated decay (NMD) highlighted in bold. (E(iii)) Heatmap of top 20 upregulated NMD-related genes in HET and HOM vs. WT (CPM: Counts per million). (F) Autism-associated gene alterations. Heatmap of the top 10 ASD-associated DEGs in HET vs. WT and HOM vs. WT. Scale: -log 10 (P).

Article Snippet: For Western blotting, primary antibodies included Rabbit anti-SCN2A (Na V 1.2) (Alomone Labs, ASC-002, 1:200), Rabbit anti- SCN2A (Sigma-Aldrich, ZRB1300, 1:200), and Mouse anti-β-Actin (Thermo Fisher, MA5-15739, 1:1000).

Techniques: Expressing, RNA Sequencing, Gene Expression, Labeling

Journal: bioRxiv

Article Title: Autism-associated SCN2A deficiency disrupts cortico-striatal circuitry in human brain assembloids

doi: 10.1101/2025.06.02.657036

Figure Lengend Snippet: (A) Global transcriptomic alterations. Principal component analysis (PCA) of transcriptomic data showing clustering of WT (KOLF2.1J, B07, C03 lines), HET (A02, E04, F01 lines), and HOM (A03, D06, F03 lines) assembloids based on global gene expression. Each dot represents a sample (n = 7 assembloids/group). (B) Gene ontology (GO) cellular component (CC) analysis of overlapping downregulated genes, revealing reductions in axon terminal and axon initial segments; excitatory synapses, and GABAergic synapses. (C) Disrupted synapse-related pathways in SCN2A-C959X assembloids. Top 10 overlapping downregulated pathways in GO biological processes (BP) in HET vs. WT and HOM vs. WT comparisons. (D) Disrupted ion channel-related pathways in SCN2A-C959X assembloids. (D(i)) The top 10 overlapping downregulated pathways in GO molecular functions (MF) reveal ion channel gene dysregulation in HET and HOM assembloids. Heatmaps of overlapping downregulated ion channel genes (blue), categorized into ligand-gated channel-related (D(ii)), voltage-gated sodium channel-related (D(iii)), and voltage-gated potassium channel-related (D(iv). Highlighted SCN3A showed no significant changes in the HET group but was significantly reduced in the HOM group. (E) Synaptic dysfunction in HET and HOM assembloids. Network plot of the top 5 Reactome pathways enriched in downregulated DEGs, primarily affecting NMDA receptor activity, neurotransmitter signaling, synaptic transmission, and neuronal interactions, suggesting disrupted synaptic function in SCN2A -deficient assembloids. (F) Increased RNA surveillance in SCN2A-C959X assembloids. Network plot of the top 5 Reactome pathways enriched in upregulated DEGs, revealing associations with mRNA splicing, nonsense-mediated decay (NMD), and exon junction complex (EJC) function. (G) Network analysis of ASD-associated genes, depicting interactions between differentially expressed ASD-related genes in SCN2A -mutant assembloids. Top ten hub genes are highlighted in red.

Article Snippet: For Western blotting, primary antibodies included Rabbit anti-SCN2A (Na V 1.2) (Alomone Labs, ASC-002, 1:200), Rabbit anti- SCN2A (Sigma-Aldrich, ZRB1300, 1:200), and Mouse anti-β-Actin (Thermo Fisher, MA5-15739, 1:1000).

Techniques: Gene Expression, Activity Assay, Transmission Assay, Mutagenesis