Journal: iScience

Article Title: Genetic dissection of the role of Piga and Pgap2 in the embryonic mouse brain

doi: 10.1016/j.isci.2026.116299

Figure Lengend Snippet: Late onset hypomyelination in Olig2 ; Piga mutant brains (A) Percent genotype ratios of wildtype, Olig2 Cre/wt ; Piga flox/X female and Olig2 Cre/wt ; Piga flox/Y male survival to P21 ( n = 96, Chi-square p = 0.609). Immunostaining of MBP (B–D), CONTACTIN1 (E–G), SOX10 (H–J) and PDGFRα (K–M) in wild type (B, E, H, and K) Olig2 Cre/wt ; Piga flox/X female (C, F, I, and L) and Olig2 Cre/wt ; Piga flox/Y male (D, G, J, and M) animals. MBP and CONTACTIN1 data are from P96 (scale bar, 500 μm) while SOX10 and PDGFRα are from P21 (scale bar, 25 μm) ( n = 4 animals per genotype). Quantification of SOX10 (N, n = 3 animals per genotype, ANOVA p = 0.170), PGDFRα (O, n = 4, ANOVA p = 0.564) positive cells and the PDGFRα/SOX10 ratio (P, n = 3, ANOVA p = 0.430). Data represent individual values and the mean ± s.e.m. White arrows indicate SOX10 and PDGFRα positive cells.

Article Snippet: Mouse: Olig2 -Cre; B6.129- Olig2 tm1.1(cre)Wdr /J , The Jackson Laboratory , RRID:IMSR_JAX:025567.

Techniques: Mutagenesis, Immunostaining

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) SLC35A2 encodes a transporter for UDP-galactose, which is required for the synthesis of O-GalNAc glycans. B) The lectin VVA binds terminal alpha-GalNAc (Tn-antigen), while PNA binds terminal Gal of Core 1 O-GalNAc glycans (T-antigen), which can be removed by O-glycosidase (OGA). C) Breeding strategy of Emx1-Cre mice with floxed- Slc35a2 mice, with blue regions highlighting the forebrain expression Emx1 . D) VVA binding is minimal in protein blots of wild-type mouse cortex; female (+/+) and male (+/y) with floxed- Slc35a2 . In contrast, mice lacking Slc35a2 in cells expressing the forebrain-specific promoter Emx1 ( Emx1-Cre ) display robust VVA binding in both females (+/-) and males (-/y), consistent with inhibition of O-GalNAc extension. Binding of PNA after Neuraminidase A (NeuA) treatment was significantly reduced in both +/- and -/y lines, and specificity of PNA binding was confirmed via OGA treatment of a +/+ sample. ConA, which binds all N-glycans, was unaffected in all mouse lines, and specificity was confirmed using peptide N-glycosidase F (PNGase F) treatment of a +/+ sample. Each lane contains 15 µg cortical protein lysate from an individual mouse of the corresponding genotype. Experiment was replicated three times with similar results. E) Quantification of lectin binding from D normalized to total protein (TP) staining within the same lane of each blot. Data presented as the mean binding intensity +/- SEM, with data points for each individual mouse included, N = 3/genotype. One-way ANOVA confirmed significant group differences ( p < 0.05) for VVA and PNA but not ConA, followed by post hoc Student’s t-tests to confirm genotype effects in each sex: ** p -value = 0.01, **** p -value = 0.0001. F) Schematic of coronal mouse brain section highlighting the cortex (CTX), corpus callosum (CC), and basal ganglia (BG). G) VVA binding is minimal across all regions in wildtype mouse cortex (+/y), while PNA binding localizes to neuronal tracts of the CC and axon bundles in the BG. In contrast, mice lacking forebrain expression of Slc35a2 (-/y) display a build-up of truncated O-GalNAc glycans in cortex that fail to properly localize. Mature O-GalNAc glycans are entirely absent from the CTX and CC, but preserved in the BG, which does not express Emx1 -Cre. H) Relative quantification of lectin binding across brain regions (CTX, CC, BG) from G. Data presented as the mean binding intensity +/- SEM, with individual points representing the average of 6 measures across independent mice. Colored bars correspond with the specific brain region indicated in (F): CTX, purple; CC, orange; BG, blue. N = 2 for +/y VVA; N = 4 for +/- PNA, -/y VVA, and -/y PNA. Fluorescence was quantified as arbitrary intensity (a.i.) after subtracting background signal from each sample.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Expressing, Binding Assay, Inhibition, Staining, Quantitative Proteomics, Fluorescence

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) Fluorescent images of +/y and -/y primary neuron cultures at 11 days in vitro (DIV11) show robust VVA binding in Slc35a2 -deleted cultures only. Scale bar = 75 μm. B) Quantification of raw integrated density values of VVA fluorescence (VVA Int.). ROIs were selected via automated module. N = 5 independent cultures/genotype. C) High magnification of the ROI from A (white box), highlighting that while binding is high in many MAP2+ neurons, some appear VVA negative. C) Representative raster plots from 2 minutes of high-density multi-electrode array (HD-MEA) recordings of DIV11 +/y and -/y primary cultures, with active units representing cells firing ≥0.1 spikes/second. Each point represents an individual detected action potential, and colored vertical lines indicate a time when > 20% of active units were recruited during a network bursting event. E) Quantification of spontaneous network activity measured by HD-MEA shows that while +/y cultures show clear periods of uniform network activity that recruit many active units, -/y cultures are hyperactive and demonstrate aberrant network activity. N = 4 and 5 independent cultures for +/y and -/y cultures, respectively. F) Colocalization of the axon initial segment (AIS) marker Ankyrin G (AnkG) with PNA shows a reduction of properly localized O-GalNAc glycans to the AIS (white arrow, AnkG+/MAP2-). Scale bar = 10μm. G) Quantification of the mean PNA value along the AIS from F. N = 3 independent cultures for +/y and -/y cultures, respectively, where 31-32 AISs were measured per condition and average PNA profiles at the AIS were log transformed to fit the normality assumption of the statistical test. Data are shown as mean ± SEM. p value = *<0.05, **<0.01, ***<0.001, ****<0.0001 via unpaired two-tailed t-test.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: In Vitro, Binding Assay, Fluorescence, Activity Assay, Marker, Transformation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) Schematic depicting affinity purification and elution of VVA-bound glycoproteins in mouse cortex, including controls for elution (+ GlcNAc) and binding (no lectin), analyzed by LC-MS/MS. B) The total number of peptides with spectral count > 0 detected in each sample. Biological triplicates for +/y and -/y were included, as well as a single +/- sample, all eluted with 200 mM GalNAc. Controls for lectin specificity and non-specific binding of a -/y sample were performed in parallel, specifically elution with 200 mM GlcNAc and sample with no VVA lectin. C) Among the detected 443 distinct glycopeptides detected, HexNAc(1) modified peptides (+ 203.079 Da) were almost exclusively detected in Slc35a2- deleted samples. D) HexNAc modified glycopeptides mapped to 60 genes, of which lecticans including Vcan, Bcan, and Ncan were most abundant. E) Volcano plot of significantly upregulated (blue) and downregulated (red) proteins in +/y vs -/y cortex, mutants compared to WT. Dotted line indicates significance threshold of p = 0.05. F) Ingenuity Pathway Analysis (IPA) highlighting the top 10 canonical pathways increased by Slc35a2 deletion.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Affinity Purification, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Modification

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) Sagittal adult mouse brain sections labeled with WFA for CSPGs and PNA for O-GalNAc glycans display almost no colocalization despite both being bound to lecticans in the brain. B) High magnification images of a deep cerebellar nuclei (ROI from A; white box) where both WFA and PNA are present show non-overlapping signals, with WFA densely surrounding cells where PNA is excluded. Scale bar = 20 μm. Nuclei are stained with Hoechst-33324. C) ConA, GNL, AAL, and PNA + Neuraminidases A (NeuA) binding (top) from brain cortex samples of wild-type male mice (+/y) and human brain cortex that does not harbor SLC35A2 mutations. Treatment with PNGase F (#) and O-Glycosidase (#*) of a human sample in the last lane confirms the lectin specificity. D) Quantification of lectin binding from C normalized to TP staining within the same lane of each blot. Compared to mouse cortex, human cortex had significantly reduced ConA binding of 27%. GNL staining was decreased even further in humans relative to mice by 55%, while AAL showed no significant difference. In contrast, PNA binding after NeuA treatment was significantly increased in human cortex relative to mice by 44%. Data presented as the mean binding intensity +/- SEM, with data points for each individual mouse and human samples included. N = 4 independent samples per group, with 15 µg cortical protein lysate loaded per lane. Experiment replicated twice with similar results. Student’s t-tests: *p-value = 0.05, **p-value = 0.01, ***p-value = 0.001.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Labeling, Staining, Binding Assay

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) Surgically resected tissue from human SLC35A2 -associated focal epilepsy was analyzed for VVA positivity using fluorescence microscopy, lectin blots, and confirmatory DNA sequencing. B) VVA binding across the full tissue block of a high mutation case (VAF = 62.6%) shows variable intensity. Nuclei are stained with Hoechst-33324, and the green signal represents auto-fluorescent red blood cells in the 488ƛ channel. Scale bar = 1 mm. C) ROI from B shows scattered binding, with areas of both isolated and diffuse signal. Autofluorescent red blood cells can be seen in the lumen of vessels at the 488ƛ channel. Scale bar = 100 µm. D) High magnification images of VVA staining from the same sample, which shows areas of diffuse mesh-like binding around multiple cells and some individual cells. Scale bar = 10 µm. Complementary images with individual channels are available in the Supplementary Material. E) Random 500 x 500 µm fluorescent intensity measures of VVA and H-33324 signal from B, plotted as arbitrary intensity (a.i.) normalized to average background signal adjacent to tissue block. Bar indicates median, N = 82 and 80.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Fluorescence, Microscopy, DNA Sequencing, Binding Assay, Blocking Assay, Mutagenesis, Staining, Isolation

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) Surgically resected tissue from a SLC35A2 -associated focal epilepsy patient (VAF = 3.4-5.2%) were analyzed for VVA positivity via lectin-based histochemistry. Using laser capture microdissection (LCM), VVA+ areas were isolated from VVA-areas and both were re-sequenced for the patient specific variant. B) Variant allele frequency (VAF) from areas positive and negative for VVA binding was measured using amplicon sequencing of DNA isolated via laser capture microdissection (LCM). Areas of diffuse VVA+ show enrichment for the patient-specific variant in SLC35A2 , which were absent in both VVA-areas and reference. C) Image of the SLC35A2- associated epilepsy tissue sample stained with VVA, visualized using DAB histochemistry and counterstained with hematoxylin/eosin. Scale bar = 2000 μm. D) High magnification images from (C) of areas of both low density VVA+ with individual cells (i), diffuse high density VVA+ (ii), and hypercellularity in white matter (iii). Scale bar = 100 μm.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Laser Capture Microdissection, Isolation, Variant Assay, Binding Assay, Amplification, Sequencing, Staining

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) VVA lectin blot (both high and low exposure) from human cortex lysate from surgical biopsies of eight cases of SLC35A2 -associated epilepsy, as well as multiple unrelated controls. The percentage of SLC35A2 variant allele frequency (VAF) measured on the same same is indicated, as well as controls (-). 15 µg of brain protein lysate was loaded per lane, with total protein (TP) stain indicated in the right panel. B) Simple linear regression plot of normalized VVA intensity vs VAF % shows a strong positive linear correlation in SLC35A2 -associated focal epilepsy. C) Linear regression after removal of the highest VAF sample (62.6%) as a potential outlier in B maintains a strong correlation.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Variant Assay, Staining

Journal: bioRxiv

Article Title: PIEZOs regulate oligodendrocyte sheath formation, expansion, and myelination potential

doi: 10.64898/2026.04.23.720488

Figure Lengend Snippet: A) Representative trace from whole-cell patch clamp electrophysiology of a primary rat OPC in culture. The top of the graph is the stimulation trace showing the 300 ms stimulation trace with 0.4 µm step-wise increases in membrane displacement using a glass stimulation rod. The bottom portion of the graph shows the current (pA) trace at each stimulation. 13 of the 14 tested cells showed detectable MA currents. Scale bar: 10 μm B) Maximal current (Imax) for each cell in response to indentation stimulation N = 2 animals, 14 cells, 2 independent cell isolations. The red data point is associated with the representative trace in . C) Inactivation time constant for each cell responsive to indentation stimulation N = 2 animals, 13 cells, 2 independent cell isolations. The red data point is associated with the representative trace in . D) qRT-PCR of Piezo1 , Tmem63a , Mag , and Cspg4 in cultured rat OLs during differentiation (0-192 h). Values are normalized to the transcript’s highest expression value during differentiation N = 3 animals, 3 independent cell isolations. E) Western blot analysis of PIEZO1-tdTOMATO, PDGFRA, MBP, and ACTB expression in Piezo1 tdT/tdT and Piezo1 Wt/Wt control murine OPCs and OLs at 24-72h differentiation. F) Quantification of tdTOMATO intensity relative to ACTB in western blots. N = 3 animals per genotype, 3 independent cell isolations. Red dots indicate the data points corresponding to the example image in E.

Article Snippet: Piezo1 F/F mice were purchased from Jackson Laboratories (B6.Cg-Piezo1 tm2.1Apat/J; Jax strain: 029213) , and crossed to a Olig2-Cre (Olig2-TVA-IRES-Cre KIKO; Jax strain: 011103) mice which were a gift from Dr. David Rowitch.

Techniques: Patch Clamp, Membrane, Quantitative RT-PCR, Cell Culture, Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: PIEZOs regulate oligodendrocyte sheath formation, expansion, and myelination potential

doi: 10.64898/2026.04.23.720488

Figure Lengend Snippet: A. Raw images from Fura2 calcium imaging after the application of DMSO, 30 µM Yoda1, and 200 mM KCl. Scale bar: 25 μm B. Representative calcium imaging trace from control ( Piezo1 Wt/Wt ;Olig2 Cre/Wt ) OPCs loaded with Fura2 and sequentially exposed to DMSO, 30 µM Yoda1, and 200 mM KCl. Trace averaged from 72 cells imaged from 1 coverslip. C. Representative calcium imaging trace from Piezo1 CKO cells ( Piezo1 Fl/Fl ;Olig2 Cre/Wt ) loaded with Fura2 and exposed to DMSO, 30 µM Yoda1, and 200 mM KCl. Trace averaged from 121 CKO cells from a single coverslip. D. Quantification of area under the 340/380 curve for each animal during application of Yoda1. The imaging data from 3 coverslips per animal were pooled for analysis for 3 animals per genotype. Data shown as mean ± SEM. p = 0.024 by student’s t-test, N = 3 animals per genotype, 971 control cells and 1043 cells from Piezo1 CKO cells, 3 independent isolations. E. Representative images from EdU proliferation assay of control or Piezo1 CKO cells. OLIG2 is a marker of OLCs, and EdU labels cells that divided over the 6.5-hour time course. F. Experimental design for the detection of proliferating cells in vitro . G. Quantification of the proportion of OLIG2+ cells labelled for EdU. N = 3 independent OPC isolations per genotype, with 818 cells assessed for controls and 947 cells for Piezo1 CKOs . p = 0.643 by paired t-test. H. Representative images from control or Piezo1 CKO cells fixed 48 hours into differentiation and stained for OLIG2, PDGFRA, and MBP. I. Quantification of the percentage of OLIG2+ cells that are PDGFRA+ OPCs. Control experimental totals: 859 cells, 3 animals, 3 independent OPC isolations CKO experimental totals: 824 cells, 3 animals, 3 independent OPC isolations. p = 0.207 by paired, 2-tailed t-test. J. Quantification of the percentage of OLIG2+ cells that are actively differentiating (PDGFRA-, MBP-). Control experimental totals: 859 cells, 3 animals, 3 independent OPC isolations CKO experimental totals: 824 cells, 3 animals, 3 independent OPC isolations. p = 0.101 by paired, 2-tailed t-test. K. Quantification of the percentage of OLIG2+ cells that are MBP+ OLs. Control experimental totals: 859 cells, 3 animals, 3 independent OPC isolations CKO experimental totals: 824 cells, 3 animals, 3 independent OPC isolations. p = 0.097 by paired, 2-tailed t-test. L. Representative images of a control and Piezo1 CKO cell plated onto nanofibers and allowed to differentiate for 7 days before being fixed and stained with DAPI and MBP. M. The number of sheaths per OL for Control and Piezo1 CKO cells. p = 0.767 by paired, 2-tailed, t-test. N. Average sheath length per OL for Control and Piezo1 CKO cells. p = 0.019 by paired, 2-tailed t-test. O. Average myelin output (summed sheath lengths per OL) for control ( Piezo1 wt/wt ;Olig2 Cre/wt ) and Piezo1 CKO cells. p = 0.032 by paired, 2-tailed t-test. For M-O, Control experimental totals: 50 cells, 3 animals, 3 independent cell isolations. CKO experimental totals: 48 cells, 3 animals, 3 independent cell isolations. For E, H, and L – Scale bar: 50 μm

Article Snippet: Piezo1 F/F mice were purchased from Jackson Laboratories (B6.Cg-Piezo1 tm2.1Apat/J; Jax strain: 029213) , and crossed to a Olig2-Cre (Olig2-TVA-IRES-Cre KIKO; Jax strain: 011103) mice which were a gift from Dr. David Rowitch.

Techniques: Imaging, Control, Proliferation Assay, Marker, In Vitro, Staining

Journal: bioRxiv

Article Title: PIEZOs regulate oligodendrocyte sheath formation, expansion, and myelination potential

doi: 10.64898/2026.04.23.720488

Figure Lengend Snippet: A. Schematic displaying the features of the knockdown Cas13d targeting system. Tol2 allows integration into the genome, eGFP-CAAX enables morphological characterization of cells, and 4 sites for crRNA guides mediate gene-specific knockdown. B. Experimental timeline starting with embryo harvest on the first day of the experiment, followed by longitudinal imaging of zebrafish embryos at 3, 5, and 7 dpf. C. Representative images of non-targeted control and piezo1-KD cell tracked through time from OL age 1-5, with day 1 being the first day of sheath formation. D. Number of sheaths per OL for non-targeted control and piezo1-KD cells across the experimental timeline. Control: OL1/3: N = 13 cells; 9 fish. OL5: 7 cells; 5 fish. piezo1-KD : OL1/3: N = 12 cells; 7 fish OL5: 9 cells; 6 fish. LMEM (Linear Mixed Effects Model) p cond : 0.002 p cell age:cond :0.009 t-test p OL1 : 0.024, p OL3 : 0.059, p OL5 : 0.039 E. Average sheath length per OL for non-targeted control and piezo1-KD cells. Control: OL1/3: N = 13 cells; 9 fish. OL5: 7 cells; 5 fish. piezo1-KD : OL1/3: N = 12 cells; 7 fish OL5: 9 cells; 6 fish. LMEM p cond : 0.121 p cell age:cond : 0.248 t-test p OL1 : 0.448, p OL3 : 0.194, p OL5 : 0.665 F. Total myelin output per OL for non-targeted control and piezo1-KD cells. Control: OL1/3: N = 13 cells; 9 fish. OL5: 7 cells; 5 fish. piezo1-KD : OL1/3: N = 12 cells; 7 fish OL5: 9 cells; 6 fish. LMEM p cond : 0.123 p cell age:cond :0.234 t-test p OL1 : 0.545, p OL3 : 0.135, p OL5 : 0.052 G. Representative images of non-targeted control and piezo2-KD cells tracked from OL age 1-5, with day 1 being the first day of sheath formation. H. Number of sheaths per OL for non-targeted control and piezo2-KD cells. Control : OL1/3: N = 13 cells; 9 fish. OL5: 12 cells; 8 fish. piezo2-KD : OL1/3: N = 14 cells; 10 fish OL5: 9 cells; 8 fish. LMEM p cond : 0.046 p cell age:cond : 0.371 t-test p OL1 : 0.981, p OL3 : 0.456, p OL5 : <0.001 I. Average sheath length per OL for non-targeted control and piezo2-KD cells. Control : OL1/3: N = 13 cells; 9 fish. OL5: 12 cells; 8 fish. piezo2-KD : OL1/3: N = 14 cells; 10 fish OL5: 9 cells; 8 fish. LMEM p cond : 0.207 p cell age:cond : 0.377 t-test p OL1 : 0.364, p OL3 : 0.850, p OL5 : 0.669 J. Total myelin output per OL for non-targeted controls and piezo2-KD cells. Control : OL1/3: N = 13 cells; 9 fish. OL5: 12 cells; 8 fish. piezo2-KD : OL1/3: N = 14 cells; 10 fish OL5: 9 cells; 8 fish. LMEM p cond : 0.634 p cell age:cond : 0.033 t-test p OL1 : 0.864, p OL3 : 0.115, p OL5 : 0.003 For C/G – Scale bar: 25 μm. For E-F and H-J graphs are mean ± SEM and * represent statistically significant timepoints.

Article Snippet: Piezo1 F/F mice were purchased from Jackson Laboratories (B6.Cg-Piezo1 tm2.1Apat/J; Jax strain: 029213) , and crossed to a Olig2-Cre (Olig2-TVA-IRES-Cre KIKO; Jax strain: 011103) mice which were a gift from Dr. David Rowitch.

Techniques: Knockdown, Imaging, Control

Journal: bioRxiv

Article Title: PIEZOs regulate oligodendrocyte sheath formation, expansion, and myelination potential

doi: 10.64898/2026.04.23.720488

Figure Lengend Snippet: A. Example tracing of OLs used to quantify the number of sheaths per OL for non-targeted control and double knockdown (dKD) cells tracked from OL1-5 (3 to 7 dpf). Arrowhead indicates the formation of a new sheath generated outside the normal developmental window. B. Change in the number of sheaths per OL between 5 to 7 dpf for each condition. The targeting of piezo1 , piezo2 , and dKD were performed as independent experiments; therefore, corresponding control groups are shown for each experiment. Control P1 : N = 7 cells, 7 animals; piezo1-KD : N = 11 cells; 8 fish; p = 0.536 Control P2 : N = 13 cells, 10 animals; piezo2-KD : N = 9 cells, 8 fish; p = 0.781 Control dKD :11 cells, 11 fish; dKD: 10 cells, 8 fish; p = 0.014

Article Snippet: Piezo1 F/F mice were purchased from Jackson Laboratories (B6.Cg-Piezo1 tm2.1Apat/J; Jax strain: 029213) , and crossed to a Olig2-Cre (Olig2-TVA-IRES-Cre KIKO; Jax strain: 011103) mice which were a gift from Dr. David Rowitch.

Techniques: Control, Knockdown, Generated

Journal: bioRxiv

Article Title: WWOX deficiency uncovers a cell-autonomous mechanism impairing myelin repair

doi: 10.1101/2025.11.22.689900

Figure Lengend Snippet: a Myelination and remyelination are essential processes for neuronal survival, function, and homeostasis of the brain. We assessed the cellular and molecular basis of impaired remyelination by relating cell-type-preferential expression and genetic predisposition to demyelinating diseases. b 2D representation of 17,799 nuclei from Multiple Sclerosis (MS) and control (CTR) postmortem white matter samples. Single nuclei are represented as points and labeled by cell type (left) or lesion type (right) (CTR: Control; NA: Normal Appearing white matter; RM: Remyelinating; CI: Chronically Inactive; A: Active; CA: Chronically Active). c Cell-type MS genetic variability association analysis. Bar lengths define the strength of the association as quantified via MAGMA (-log10(adjusted p-value)) using cell-type-preferential expression in CTR samples (NEBULA, Differential Expression Analysis (DEA) between cell types in CTR samples, adjusted p-value < 0.01). Positive (negative) bars display positive (negative) associations between cell-type-preferential expression and MS genetic variability. Vertical dashed lines highlight significance thresholds for the association (p-value < 0.05, MAGMA gene-covar, linear regression). d Candidate genes driving cell-type specificity and GWAS associations in microglia or oligodendroglia. Candidate genes were identified by prioritization in the original MS GWAS study and for their cell-type preferential expression. Candidate genes are ranked based on MS GWAS genetic variability (MAGMA gene-level score), and MS transcriptional variability was considered for further prioritization (NEBULA, differential expression MS vs CTR, adjusted p-value < 0.01). e Cell-type composition across MS lesions and CTR samples. f Relative expression of oligodendroglial candidate genes differentially expressed across lesions (Wilcoxon signed-rank test, log2(FoldChange) > 0.05, detected in at least 25% of the nuclei, and adjusted p-value < 0.01). h Expression of WWOX and canonical marker genes across human brain development.

Article Snippet: Wwox conditional ablation from OPCs was achieved through crossing Wwox flox/flox mice with Olig2-Cre mice purchased from Jackson Laboratory (Stock: 025567, B6.129-Olig2tm.11(cre)Wdr/J).

Techniques: Expressing, Control, Labeling, Quantitative Proteomics, Marker

Journal: bioRxiv

Article Title: WWOX deficiency uncovers a cell-autonomous mechanism impairing myelin repair

doi: 10.1101/2025.11.22.689900

Figure Lengend Snippet: a Schematics of control (CTR) and Multiple Sclerosis (MS) white matter lesion types. b 2D representation of nuclei from both CTR and MS postmortem samples. Each dot is a single nucleus and is labeled by the original annotation. Side annotations compares the original and our refined annotation. c Transcriptional signature expression of the top 5 signature genes for each cell type in the control condition. d Cell-type marker gene expression on the whole dataset (logcounts). e Number of cell-type-preferential genes prioritized in the original MS GWAS publication in each cell type. f Expression of Wwox in cell-sorted oligodendroglial bulk transcriptomics.

Article Snippet: Wwox conditional ablation from OPCs was achieved through crossing Wwox flox/flox mice with Olig2-Cre mice purchased from Jackson Laboratory (Stock: 025567, B6.129-Olig2tm.11(cre)Wdr/J).

Techniques: Control, Labeling, Expressing, Marker, Gene Expression

Journal: bioRxiv

Article Title: WWOX deficiency uncovers a cell-autonomous mechanism impairing myelin repair

doi: 10.1101/2025.11.22.689900

Figure Lengend Snippet: a. Experimental design used to assess primary cultures of Oligodendrocyte Precursor Cells (OPCs) and their differentiation (DIV: Days In Vitro). b Relative expression (z-score) of cell-type marker genes for Radial Glia (RG), OPCs, and Oligodendrocytes (Oli) across samples. c Volcano plot showing differential expression of leading edges from gene programs in d . For each gene program, the first five leading edges were visualized on the volcano plot if differentially expressed (adjusted p-value < 0.05 and log2(FC) > 1). Upregulated (downregulated) leading edges with respect to WWOX-deficient cultures were annotated in red (blue). All other genes, except Wwox , have been colored in grey even if differentially expressed. Dashed lines define differential expression thresholds for adjusted p-value and log2(Fold Change). d Inferred activity of selected gene programs. Gene programs were selected from the top 5 most over- and underrepresented gene programs (based on adjusted p-value) for each tested gene set (KEGG, WikiPathways, and Tabula Muris). e Immunofluorescence staining (IF) of OPCs (PDGFRa+) and Olis (MBP+) in WT and KO cultures (data are shown as means +/− SEM; two-tailed t-test). f Morphological analysis of Oli. From left to right: IF of CTR and KO Oli (MBP+); quantification of Oli surface area (Kolmogorov-Smirnov test); Sholl analysis of branching complexity based on the number of intersections with concentric circles drawn from the cell soma (data are shown as means +/− SEM, ANOVA).

Article Snippet: Wwox conditional ablation from OPCs was achieved through crossing Wwox flox/flox mice with Olig2-Cre mice purchased from Jackson Laboratory (Stock: 025567, B6.129-Olig2tm.11(cre)Wdr/J).

Techniques: In Vitro, Expressing, Marker, Quantitative Proteomics, Activity Assay, Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: WWOX deficiency uncovers a cell-autonomous mechanism impairing myelin repair

doi: 10.1101/2025.11.22.689900

Figure Lengend Snippet: a Schematic of the Cre-LoxP-based genetic mouse model used to conditionally delete Wwox in oligodendrocyte lineage under Olig2-Cre expression. b IF staining for WWOX and CC1 (mature Oli) in the corpus callosum of O-CTR and O-KO mice at Postnatal day 17 (P17). WWOX expression is markedly reduced in CC1 + Olis of O-KO brains, confirming efficient Cre-mediated recombination. c IF staining and quantification of OPCs (PDGFRα+) and Olis (CC1+) in the corpus callosum (data are shown as means ± SEM; two-tailed t-test). d IF for MBP in O-CTR and O-KO brain regions at 2 months of age. Quantification was based on N=2 mice per genotype, with n=3 sections analyzed per animal (data are shown as means ± SEM; two-tailed t-test). e Representative TEM images of the midsagittal corpus callosum from adult O-CTR and O-KO mice. White squares indicate magnified regions. Middle: Quantification of myelin thickness by g-ratio analysis (linear regression). Right: Quantification of the number of myelinated axons per FOV shows no significant difference between groups (data are shown as means ± SEM; two-tailed t-test). f Repeated analysis shown in e with 18-month-old O-CTR and O-KO mice.

Article Snippet: Wwox conditional ablation from OPCs was achieved through crossing Wwox flox/flox mice with Olig2-Cre mice purchased from Jackson Laboratory (Stock: 025567, B6.129-Olig2tm.11(cre)Wdr/J).

Techniques: Expressing, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: WWOX deficiency uncovers a cell-autonomous mechanism impairing myelin repair

doi: 10.1101/2025.11.22.689900

Figure Lengend Snippet: a Experimental design of cuprizone-induced remyelination in mouse models. b Representative immunofluorescence (IF) for MBP in brain sections following demyelination and remyelination periods. c Quantification of MBP intensity in conditions shown in b for three anatomically matched sagittal sections per animal (data are shown as means +/− SEM; two-tailed t-test). d Transmission Electron Microscopy (TEM) analysis of myelin. Left: representative images of the midsagittal corpus callosum following remyelination; white squares indicate magnified regions. Middle: g-ratio analysis quantifying differences due to oligodendroglial WWOX deficiency (O-KO) in the average amount of myelin and the effect of axonal diameter (linear regression). Right: quantification of the number of myelinated axons per Field Of View (FOV) (data are shown as means +/-SEM; two-tailed t-test; n.s., not significant). e Representative IF for OPCs (PDGFRa+) and mature oligodendrocytes (CC1+) in the corpus callosum. f Quantification of PDGFRa+ and CC1+ cells in anatomically matched sagittal sections (data are shown as means +/− SEM; two-tailed t-test).

Article Snippet: Wwox conditional ablation from OPCs was achieved through crossing Wwox flox/flox mice with Olig2-Cre mice purchased from Jackson Laboratory (Stock: 025567, B6.129-Olig2tm.11(cre)Wdr/J).

Techniques: Immunofluorescence, Two Tailed Test, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: WWOX deficiency uncovers a cell-autonomous mechanism impairing myelin repair

doi: 10.1101/2025.11.22.689900

Figure Lengend Snippet: a Experimental design of single-nucleus RNA-sequencing (snRNA-seq) data generation in cuprizone-induced remyelination mouse models. The number of samples, and absolute number and fraction of nuclei retrieved for each combination of genotype and condition are reported. b 2D representation of the whole snRNA-seq dataset (40,906 nuclei) labeled by broad cell type populations. c 2D representation labeled by condition. d 2D representation with associated quantification of transcriptional shifts due to oligodendroglial Wwox deficiency in each condition. 2D representations are labeled by genotype. e Number of differentially expressed genes per cell type in each condition. (ExN: Excitatory Neurons; InN: Inhibitory Neurons; OPC: Oligodendrocyte Precursor Cells; Oli: Oligodendrocytes; Ast: Astrocytes; Mic: Microglia; End: Endothelial cells)

Article Snippet: Wwox conditional ablation from OPCs was achieved through crossing Wwox flox/flox mice with Olig2-Cre mice purchased from Jackson Laboratory (Stock: 025567, B6.129-Olig2tm.11(cre)Wdr/J).

Techniques: RNA Sequencing, Labeling

Journal: bioRxiv

Article Title: WWOX deficiency uncovers a cell-autonomous mechanism impairing myelin repair

doi: 10.1101/2025.11.22.689900

Figure Lengend Snippet: a Volcano plot showing differentially expressed oligodendroglial TFs in primary OPC cultures. b Expression trends of Wwox and Sox10 between KO and CTR OPCs across conditions in snRNA-seq data. c Representative IF and quantification of SOX10+ cells in the corpus callosum of KO and CTR mice following remyelination (data are presented as mean ± SEM; two-tailed t-test). d Gene set enrichment analysis of the SOX10 positive regulon in OPCs and oligodendrocytes across conditions in snRNA-seq data. e Western blots of GST pull-down assay for physical interaction of WWOX and SOX10 in the HEK293T cell line. The role of the WW domain in the physical interaction was tested with the WFPA mutant WWOX protein carrying a defect in the WW domain. f Western blots of Cycloheximide (CHX) chase assay of SOX10. Cells were subjected to a protein synthesis inhibitor, CHX (20 μg/ml), to measure SOX10 stability in both Wwox KO and CTR immortalised OPC cell lines (Oli-neu). Proteasome inhibitor MG132 (10 μM) was used to assess proteasome-mediated degradation of SOX10. g Quantification of SOX10 protein levels from CHX chase assay for each genotype.

Article Snippet: Wwox conditional ablation from OPCs was achieved through crossing Wwox flox/flox mice with Olig2-Cre mice purchased from Jackson Laboratory (Stock: 025567, B6.129-Olig2tm.11(cre)Wdr/J).

Techniques: Expressing, Two Tailed Test, Western Blot, Pull Down Assay, Mutagenesis

Journal: bioRxiv

Article Title: WWOX deficiency uncovers a cell-autonomous mechanism impairing myelin repair

doi: 10.1101/2025.11.22.689900

Figure Lengend Snippet: a IF staining and quantification of SOX10+ cells in the corpus callosum of O-CTR and O-KO mice across conditions (normal diet, demyelination, and remyelination).(data are presented as mean ± SEM; two-tailed t-test; n.s., not significant). b AlphaFold structural predictions of WWOX and SOX10 proteins. The WW1 domain in WWOX and the PPxY motif in SOX10 are highlighted, suggesting a potential interaction interface. c Western blot of HA pull-down assay using cortical lysates from HA-tagged WWOX mice. d Western blot analysis of WT and Wwox-KO Oli-neu cells transfected with HA-Ubiquitin (Addgene plasmid #18712). Lysates were subjected to immunoprecipitation using anti-SOX10 antibody followed by immunoblotting with anti-HA to assess ubiquitination of SOX10.

Article Snippet: Wwox conditional ablation from OPCs was achieved through crossing Wwox flox/flox mice with Olig2-Cre mice purchased from Jackson Laboratory (Stock: 025567, B6.129-Olig2tm.11(cre)Wdr/J).

Techniques: Staining, Two Tailed Test, Western Blot, Pull Down Assay, Transfection, Ubiquitin Proteomics, Plasmid Preparation, Immunoprecipitation