Journal: bioRxiv

Article Title: Atxn2 -CAG100-knock-in affects mouse lifespan and vestibulo-cerebellar function via neural disconnection

doi: 10.1101/333443

Figure Lengend Snippet: Dysregulations of protein and mRNA levels were observed for several factors involved in axon myelination, namely the axon-localized Neurofilament heavy / medium / light chain subunits (NEFH / NEFM / NEFL), the synapse-localized Neuroplastin (NPTN), the synapse-localized alpha-Synuclein (SNCA), versus the oligodendrocyte-localized Myelin Basic Protein (MBP), 2’,3’-Cyclic Nucleotide 3’ Phosphodiesterase (CNP), Proteolipid Protein (PLP1), Myelin oligodendrocyte glycoprotein (MOG) and Myelin associated glycoprotein (MAG). The abundance of the protein of interest was normalized versus beta-Actin (ACTB) as total tissue loading control, and separately versus Calbindin-1 (CALB1) as marker of Purkinje neuron somatodendritic compartment preservation. The mRNA abundance of each factor is shown with normalization versus TATA-box-binding protein ( Tbp ) mRNA, to elucidate if the dysregulation might be triggered at the transcriptional level by the RNA-binding ATXN2 expansion via direct interaction, or represents a post-transcriptional event. T-test was used in general.

Article Snippet: The membranes were blocked in 5% BSA/TBS-T, and incubated overnight at 4 °C with primary antibodies against 1C2 (Chemicon #MAB1574), ATXN2 (monoclonal from BD Biosciences #611378, 1:500; polyclonal from Proteintech #21776-1-AP, 1:500), ACTB (Sigma #A5441, 1:10000), CALB1 (Cell Signaling #13176, 1:2500), CNP (Cell Signaling #5664S, 1:1000), MAG (Cell Signaling #9043S, 1:500), MBP (Merck #05-675, 1:250), MOG (Abcam #ab32760, 1:500), NEFH/M (Proteintech #18934-1-AP, 1:500), NEFL (Cell Signaling #2837S, 1:1000), NPTN (Alomone Labs #ANR-090, 1:500), PLP1 (Abcam #ab28486, 1:1000), SNCA (Cell Signaling #2642S, 1:1000), TUBA4A (Aviva Systems #ARP40179-P050, 1:500).

Techniques: Marker, Preserving, Binding Assay, RNA Binding Assay

Journal: bioRxiv

Article Title: ATXN2 polyglutamine expansion impairs QKI-dependent alternative splicing and oligodendrocyte maintenance

doi: 10.1101/2025.08.08.669189

Figure Lengend Snippet: a, Graph depicting the dysregulation rates of commonly identified proteins in cerebellum and spinal cord samples from WT and KIN animals at terminal stage of 14 months old (r = 0.355, Pearson correlation). Pie charts depict the total number of commonly identified proteins (2999), all dysregulated proteins with >0.5 log 2 (fold change) difference in all directions (196), among which there were 91 proteins commonly upregulated (red), 84 proteins commonly downregulated (green), 12 proteins down in cerebellum, up in spinal cord (blue), and 9 proteins up in cerebellum, down in spinal cord (purple). b, Protein-protein interaction network and pathway enrichment analysis of the commonly downregulated proteins (with <70% abundance in KIN) showed a cluster of multiple myelin (in shades of purple) and axonal/synaptic proteins (in shades of blue and orange), with the term “Myelin sheath” appearing as the strongest dysregulation after the term “Cytoplasm”. c, Protein and transcript levels of neuronal compartment factors in WT and KIN cerebellum (Cb) at pre-onset (3 mo) and terminal (14 mo) stages, showing late onset downregulations for NEFH and NPTN, with an earlier downregulation of NEFL protein. ACTB was used as loading control, and normalizations against CALB1 were performed in order to test the neuropil affection versus Purkinje cell soma. All transcripts showed a significant downregulation at pre-onset stage with progressive reduction during disease course in KIN tissue. Tbp was used as housekeeping gene in qRT-PCR experiments. Each data point represents a single animal. d, Protein and transcript levels of myelin compartment factors in WT and KIN cerebellum at pre-onset (3 mo) and terminal (14 mo) stages, showing prominent downregulations for CNP, MBP and PLP1, with a massive reduction of MOG and MAG proteins. Slight yet significant reductions were observed in MBP and PLP1 levels at the pre-onset stage, accompanying a stronger downregulation of MAG. ACTB was used as loading control, and normalizations against CALB1 were performed in order to test the myelin affection versus Purkinje cell soma. Mbp and Plp1 transcripts showed a progressive downregulation throughout the disease course in KIN tissue, however Cnp , Mog and Mag transcripts interestingly showed a significant upregulation at the pre-onset stage which disappeared at the terminal stage. Tbp was used as housekeeping gene in qRT-PCR experiments. Each data point represents a single animal. Immunohistochemical assessment and quantification of MAG (e) and CNP (e) proteins in WT and KIN cerebellum at pre-onset stage confirms the immunoblot findings with a prominent reduction of MAG abundance and no change in CNP levels in KIN tissue. At least two sections were quantified from two WT and KIN animals. DAPI (blue) marks the nuclei in granular layer.

Article Snippet: Primary antibodies utilized in this study were: ATXN2 (BD Biosciences #611378, 1:100; Proteintech #21776-1-AP, 1:200), CALB1 (Cell Signaling #13176, 1:1000), CC1 (Millipore #OP80, 1:50), CNP (Cell Signaling #5664S, 1:100), MAG (Cell Signaling #9043S, 1:100), OLIG2 (Millipore #MABN50, 1:300), PABP (Abcam #ab21060, 1:200), PDGFRα (Novus, #AF1062, 1:100), QKI6 (Neuromab #75-190, 1:200).

Techniques: Control, Quantitative RT-PCR, Immunohistochemical staining, Western Blot

Journal: bioRxiv

Article Title: ATXN2 polyglutamine expansion impairs QKI-dependent alternative splicing and oligodendrocyte maintenance

doi: 10.1101/2025.08.08.669189

Figure Lengend Snippet: A, Transcript and protein levels of neuronal and myelin compartment factors in WT and KIN spinal cord at the terminal stage (14 mo), showing a dysregulation pattern similar to that of cerebellum. Reduced abundance of many neuronal and myelin proteins is mirrored by their transcript level, except the prominent downregulations of CNP, MOG and MAG proteins without a reduction of their transcripts. ACTB was used as loading control in quantitative immunoblots. Tbp was used as housekeeping gene in qRT- PCR experiments. Each data point represents a single animal. b, Immunohistochemical assessment of MAG protein in spinal cord sections from WT and KIN animals at terminal stage showed a marked reduction in KIN in agreement with immunoblot results. Each data point represents a technical replicate. c, Transcript levels of additional neuronal and myelin compartment factors in WT and KIN cerebellum (Cb, 3 mo and 14 mo) and spinal cord (SC, 14 mo), display early and progressive dysregulations of neuronal Ina , oligodendroglial Mobp and neuron-secreted Hapln4 . Later onset dysregulations were observed in oligodendroglial Mal , Rtn4 , and neuron-secreted Hapln1-3 . Tbp was used as housekeeping gene in qRT- PCR experiments. Each data point represents a single animal.

Article Snippet: Primary antibodies utilized in this study were: ATXN2 (BD Biosciences #611378, 1:100; Proteintech #21776-1-AP, 1:200), CALB1 (Cell Signaling #13176, 1:1000), CC1 (Millipore #OP80, 1:50), CNP (Cell Signaling #5664S, 1:100), MAG (Cell Signaling #9043S, 1:100), OLIG2 (Millipore #MABN50, 1:300), PABP (Abcam #ab21060, 1:200), PDGFRα (Novus, #AF1062, 1:100), QKI6 (Neuromab #75-190, 1:200).

Techniques: Control, Western Blot, Quantitative RT-PCR, Immunohistochemical staining

Journal: bioRxiv

Article Title: ATXN2 polyglutamine expansion impairs QKI-dependent alternative splicing and oligodendrocyte maintenance

doi: 10.1101/2025.08.08.669189

Figure Lengend Snippet: Expression levels of Cnp transcript isoforms, produced by an unknown splice factor, were measured by qRT-PCR in KIN cerebellum (Cb) at 14 months. No alteration of its splicing pattern was observed even at the terminal disease stage, suggesting that the alterations of Mag and Plp1 levels observed earlier were specific outcomes of ATXN2 pathology. Actb was used as housekeeping gene. Each data point represents a single animal.

Article Snippet: Primary antibodies utilized in this study were: ATXN2 (BD Biosciences #611378, 1:100; Proteintech #21776-1-AP, 1:200), CALB1 (Cell Signaling #13176, 1:1000), CC1 (Millipore #OP80, 1:50), CNP (Cell Signaling #5664S, 1:100), MAG (Cell Signaling #9043S, 1:100), OLIG2 (Millipore #MABN50, 1:300), PABP (Abcam #ab21060, 1:200), PDGFRα (Novus, #AF1062, 1:100), QKI6 (Neuromab #75-190, 1:200).

Techniques: Expressing, Produced, Quantitative RT-PCR

Journal: bioRxiv

Article Title: ATXN2 polyglutamine expansion impairs QKI-dependent alternative splicing and oligodendrocyte maintenance

doi: 10.1101/2025.08.08.669189

Figure Lengend Snippet: Progressive aggregation of ATXN2 in neurons is a hallmark of its toxicity across multiple neurodegeneration syndromes. These aggregates also sequester other RNA-binding proteins, including PABP, TDP-43, and FUS, disrupting a multitude of subcellular processes ranging from altered autophagy to axonal cargo transport, which ultimately lead to the degeneration of axon terminals and cell somata. Our findings from the Atxn2 -CAG100-KIN mouse that expresses the mutant protein throughout the body at an endogenous level show that Purkinje cell somata are largely preserved even at the terminal stages of the disease course, whereas demyelination, and consequently, axon-myelin disconnection are among the first events of pathogenesis. Damaged myelin stimulates OPC proliferation and differentiation, however with unsuccessful maturation. We show that ATXN2 aggregates also form in cerebellar oligodendrocytes, sequestering the splice factor QKI, which leads to a dramatic loss of key mature myelin proteins, such as L-Mag and Plp1. Additionally, pronounced astrogliosis and microgliosis are also observed in KIN tissue, likely as a consequence of myelin and axonal damage.

Article Snippet: Primary antibodies utilized in this study were: ATXN2 (BD Biosciences #611378, 1:100; Proteintech #21776-1-AP, 1:200), CALB1 (Cell Signaling #13176, 1:1000), CC1 (Millipore #OP80, 1:50), CNP (Cell Signaling #5664S, 1:100), MAG (Cell Signaling #9043S, 1:100), OLIG2 (Millipore #MABN50, 1:300), PABP (Abcam #ab21060, 1:200), PDGFRα (Novus, #AF1062, 1:100), QKI6 (Neuromab #75-190, 1:200).

Techniques: RNA Binding Assay, Mutagenesis

Journal: PLoS ONE

Article Title: Talpid3-Binding Centrosomal Protein Cep120 Is Required for Centriole Duplication and Proliferation of Cerebellar Granule Neuron Progenitors

doi: 10.1371/journal.pone.0107943

Figure Lengend Snippet: Wild type and Ta3 mutant primary mouse embryonic fibroblasts (pMEFs) were coimmunostained for Cep120 and γ ˜tubulin (A) or Odf2 and Cep120 (B), together with DAPI (for nuclei). Arrowheads indicate the specific staining at centrioles. The Cep120 signal at the daughter and mother centrioles (DC and MC, respectively) was quantified for 21 randomly chosen cells, using NIH image J. Cep120 signal ratios were then calculated, specifically the DC to MC ratio and the wild type to Ta3 mutant ratio (average + standard deviation) (C). (D) No significant changes in Cep120 levels in Ta3 mutant MEFs. Immunoblots show the expression of endogenous Ta3 and Cep120 in wild type and Ta3 mutant MEFs, with tubulin as a loading control.

Article Snippet: Other antibodies used for immunofluorescence and Western blotting included Flag M2 mAb (1∶1000), acetylated tubulin (1∶2000), γ-tubulin (1∶2000) (Sigma), Arl13b (1∶1500) , Calb1 (1∶200, cat #13176, Cell signaling), Pax6 (1∶50), and BrdU (1∶200) (DSHB, University of Iowa).

Techniques: Mutagenesis, Staining, Standard Deviation, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Talpid3-Binding Centrosomal Protein Cep120 Is Required for Centriole Duplication and Proliferation of Cerebellar Granule Neuron Progenitors

doi: 10.1371/journal.pone.0107943

Figure Lengend Snippet: Wild type and Cep120 -/- pMEFs were coimmunostained for the indicated proteins. Most Cep120 mutant cells lacked either one or both centrioles, as indicated by γ ˜tubulin staining. Staining for Cep164, Ta3, and Odf2 showed that the missing centriole was the mother centriole. Staining for Arl13b and acetylated α ˜tubulin indicated no cilia being formed in Cep120 mutant cells. Arrowheads point to the staining of γ ˜tubulin (a centriole marker), the indicated proteins, or cilia.

Article Snippet: Other antibodies used for immunofluorescence and Western blotting included Flag M2 mAb (1∶1000), acetylated tubulin (1∶2000), γ-tubulin (1∶2000) (Sigma), Arl13b (1∶1500) , Calb1 (1∶200, cat #13176, Cell signaling), Pax6 (1∶50), and BrdU (1∶200) (DSHB, University of Iowa).

Techniques: Mutagenesis, Staining, Marker

Journal: PLoS ONE

Article Title: Talpid3-Binding Centrosomal Protein Cep120 Is Required for Centriole Duplication and Proliferation of Cerebellar Granule Neuron Progenitors

doi: 10.1371/journal.pone.0107943

Figure Lengend Snippet: (A–D) Sagittal brain sections of P14 mice with the indicated genotypes were coimmunostained for acetylated α ˜tubulin and Arl13b, cilia markers. The fourth ventricular choroid plexus is circled. Framed areas in panels A and B are enlarged in A′/A″ and B′/B″. Panels A″ and B″ show representative areas of ependyma near the fourth ventricle. Panels C and D show representative areas of CGNPs. Arrowheads indicate representative cilia. Note that cilia develop in the Cep120 f/- ; nes-Cre choroid plexus, but not in ependymal cells and CGNPs. (G–L) Sagittal cerebellar sections of P14 mice with the indicated genotypes were coimmunostained for γ ˜tubulin and Cep120, Odf2, or Ta3, as shown. Note that very few centrioles are present in Cep120 f/- ; nes-Cre CGNPs, relative to wild type CGNPs. Arrowheads indicate one or two representative centrioles in each panel.

Article Snippet: Other antibodies used for immunofluorescence and Western blotting included Flag M2 mAb (1∶1000), acetylated tubulin (1∶2000), γ-tubulin (1∶2000) (Sigma), Arl13b (1∶1500) , Calb1 (1∶200, cat #13176, Cell signaling), Pax6 (1∶50), and BrdU (1∶200) (DSHB, University of Iowa).

Techniques:

Journal: bioRxiv

Article Title: Atxn2 -CAG100-knock-in affects mouse lifespan and vestibulo-cerebellar function via neural disconnection

doi: 10.1101/333443

Figure Lengend Snippet: Dysregulations of protein and mRNA levels were observed for several factors involved in axon myelination, namely the axon-localized Neurofilament heavy / medium / light chain subunits (NEFH / NEFM / NEFL), the synapse-localized Neuroplastin (NPTN), the synapse-localized alpha-Synuclein (SNCA), versus the oligodendrocyte-localized Myelin Basic Protein (MBP), 2’,3’-Cyclic Nucleotide 3’ Phosphodiesterase (CNP), Proteolipid Protein (PLP1), Myelin oligodendrocyte glycoprotein (MOG) and Myelin associated glycoprotein (MAG). The abundance of the protein of interest was normalized versus beta-Actin (ACTB) as total tissue loading control, and separately versus Calbindin-1 (CALB1) as marker of Purkinje neuron somatodendritic compartment preservation. The mRNA abundance of each factor is shown with normalization versus TATA-box-binding protein ( Tbp ) mRNA, to elucidate if the dysregulation might be triggered at the transcriptional level by the RNA-binding ATXN2 expansion via direct interaction, or represents a post-transcriptional event. T-test was used in general.

Article Snippet: The membranes were blocked in 5% BSA/TBS-T, and incubated overnight at 4 °C with primary antibodies against 1C2 (Chemicon #MAB1574), ATXN2 (monoclonal from BD Biosciences #611378, 1:500; polyclonal from Proteintech #21776-1-AP, 1:500), ACTB (Sigma #A5441, 1:10000), CALB1 (Cell Signaling #13176, 1:2500), CNP (Cell Signaling #5664S, 1:1000), MAG (Cell Signaling #9043S, 1:500), MBP (Merck #05-675, 1:250), MOG (Abcam #ab32760, 1:500), NEFH/M (Proteintech #18934-1-AP, 1:500), NEFL (Cell Signaling #2837S, 1:1000), NPTN (Alomone Labs #ANR-090, 1:500), PLP1 (Abcam #ab28486, 1:1000), SNCA (Cell Signaling #2642S, 1:1000), TUBA4A (Aviva Systems #ARP40179-P050, 1:500).

Techniques: Marker, Preserving, Binding Assay, RNA Binding Assay

Journal: bioRxiv

Article Title: Atxn2 -CAG100-knock-in affects mouse lifespan and vestibulo-cerebellar function via neural disconnection

doi: 10.1101/333443

Figure Lengend Snippet: Dysregulations of protein and mRNA levels were observed for several factors involved in axon myelination, namely the axon-localized Neurofilament heavy / medium / light chain subunits (NEFH / NEFM / NEFL), the synapse-localized Neuroplastin (NPTN), the synapse-localized alpha-Synuclein (SNCA), versus the oligodendrocyte-localized Myelin Basic Protein (MBP), 2’,3’-Cyclic Nucleotide 3’ Phosphodiesterase (CNP), Proteolipid Protein (PLP1), Myelin oligodendrocyte glycoprotein (MOG) and Myelin associated glycoprotein (MAG). The abundance of the protein of interest was normalized versus beta-Actin (ACTB) as total tissue loading control, and separately versus Calbindin-1 (CALB1) as marker of Purkinje neuron somatodendritic compartment preservation. The mRNA abundance of each factor is shown with normalization versus TATA-box-binding protein ( Tbp ) mRNA, to elucidate if the dysregulation might be triggered at the transcriptional level by the RNA-binding ATXN2 expansion via direct interaction, or represents a post-transcriptional event. T-test was used in general.

Article Snippet: The membranes were blocked in 5% BSA/TBS-T, and incubated overnight at 4 °C with primary antibodies against 1C2 (Chemicon #MAB1574), ATXN2 (monoclonal from BD Biosciences #611378, 1:500; polyclonal from Proteintech #21776-1-AP, 1:500), ACTB (Sigma #A5441, 1:10000), CALB1 (Cell Signaling #13176, 1:2500), CNP (Cell Signaling #5664S, 1:1000), MAG (Cell Signaling #9043S, 1:500), MBP (Merck #05-675, 1:250), MOG (Abcam #ab32760, 1:500), NEFH/M (Proteintech #18934-1-AP, 1:500), NEFL (Cell Signaling #2837S, 1:1000), NPTN (Alomone Labs #ANR-090, 1:500), PLP1 (Abcam #ab28486, 1:1000), SNCA (Cell Signaling #2642S, 1:1000), TUBA4A (Aviva Systems #ARP40179-P050, 1:500).

Techniques: Marker, Preserving, Binding Assay, RNA Binding Assay