DOI: 10.5061/dryad.4xgxd25r0
title: Data from: Selective loss of primary cilia and neurotrophic signaling in G51D α-synuclein mice highlights a common pathway to Parkinson’s disease

description: Parkinson’s disease is characterized by dopaminergic neuron loss and
 accumulation of α‑synuclein aggregates in the brain. G51D α‑synuclein
 knock‑in mice provide a genetically and clinically relevant model of
 disease, exhibiting early olfactory deficits, age‑dependent motor
 impairment, and progressive phospho‑α‑synuclein accumulation. In multiple
 Parkinson’s disease models, striatal cholinergic and parvalbumin
 interneurons, as well as astrocytes, lose primary cilia and the
 neurotrophic signaling needed to sustain dopaminergic neurons. We show
 here that G51D-α‑synuclein mice share these phenotypes. Phospho‑Ser129
 α‑synuclein accumulation correlates with cilia loss in cholinergic
 interneurons but not in medium spiny neurons that accumulate higher
 phospho‑α‑synuclein levels. In the piriform cortex, parvalbumin neurons
 lose primary cilia and downregulate Neurturin, potentially contributing to
 olfactory dysfunction. Within the peripheral olfactory epithelium,
 horizontal basal cells lose cilia, whereas multi-ciliated olfactory
 sensory neuron cilia remain intact. These findings reveal convergent
 cellular vulnerabilities across Parkinson’s disease models and highlight a
 pathogenic role for impaired ciliary signaling.
description: # Data from: Selective loss of primary cilia and neurotrophic signaling in
 G51D α-synuclein mice highlights a common pathway to Parkinson’s disease
 Dataset DOI:
 [10.5061/dryad.4xgxd25r0](https://doi.org/10.5061/dryad.4xgxd25r0) ##
 Description of the data and file structure This dataset contains the
 primary data supporting the manuscript *“Selective loss of primary cilia
 and neurotrophic signaling in G51D α-synuclein mice highlights a common
 pathway in Parkinson’s disease.”* The dataset includes raw microscopy
 images (.czi), immunoblot images (.tif), quantitative analyses in GraphPad
 Prism format (.pzf), tabulated data files (.csv), and analysis scripts,
 including FIJI/ImageJ macros, R scripts, and CellProfiler pipelines. ###
 Files and variables ### **File list:** **Fig.1.zip file:** *Folder
 Fig_1A_3M_ChAT_cilia* Microscopy images (.czi) of striatal cholinergic
 (ChAT) neurons from 8 animals of 3 months of age. Four animals are wild
 type (WT) mice (Br01, Br02, Br03, Br04), and four are G51D-alpha-synuclein
 (G51D) homozygous knock-in mice (Br05, Br06, Br07, Br08). Note that all
 images of Brains (01 to 08) are split into two files. *Folder
 Fig_1C_12M_ChAT_cilia* Microscopy images (.czi) of striatal cholinergic
 (ChAT) neurons from 14 animals of 12 months of age. Seven animals are wild
 type (WT) mice (Br01_set1, Br02_set1, Br03_set1, Br01_set2, Br02_set2,
 Br03_set2, Br04_set2), and seven are G51D-alpha-synuclein (G51D)
 homozygous knock-in mice (Br04_set1, Br05_set1, Br06_set1, Br05_set2,
 Br06_set2, Br07_set2, Br08_set2). Note that images of Br01_set1 are split
 into 3 files, images of Br04_set1 are split into 2 files, and images of
 Br05_set1 are split into 2 files. *Folder Fig_1E_3M_PV_cilia* Microscopy
 images (.czi) of striatal parvalbumin (PV) neurons from 8 animals of 3
 months of age. Four animals are wild type (WT) mice (Br01, Br02, Br03,
 Br04), and four are G51D-alpha-synuclein (G51D) homozygous knock-in mice
 (Br05, Br06, Br07, Br08). Note that all images of Brains (01 to 08) are
 split into two files. *Folder Fig_1G_12M_PV_cilia* Microscopy images
 (.czi) of striatal parvalbumin (PV) neurons from 14 animals of 12 months
 of age. Seven animals are wild type (WT) mice (Br01_set1, Br02_set1,
 Br03_set1, Br01_set2, Br02_set2, Br03_set2, Br04_set2), and seven
 are G51D-alpha-synuclein (G51D) homozygous knock-in mice (Br04_set1,
 Br05_set1, Br06_set1, Br05_set2, Br06_set2, Br07_set2, Br08_set2). Note
 that images of Br01_set1 are split into 2 files, images of Br04_set1 are
 split into 2 files, images of Br05_set1 are split into 2 files, and
 Br06_set1 are split into 2 files. *Folder Fig_1I_3M_Astrocyte_cilia*
 Microscopy images (.czi) of striatal astrocytes from 8 animals of 3 months
 of age. Four animals are wild type (WT) mice (Br01, Br02, Br03, Br04), and
 four are G51D-alpha-synuclein (G51D) homozygous knock-in mice (Br05, Br06,
 Br07, Br08). *Folder Fig_1K_12M_Astrocyte_cilia* Microscopy images (.czi)
 of striatal astrocytes from 14 animals of 12 months of age. Seven animals
 are wild type (WT) mice (Br01_set1, Br02_set1, Br03_set1, Br01_set2,
 Br02_set2, Br03_set2, Br04_set2), and seven are G51D-alpha-synuclein
 (G51D) homozygous knock-in mice (Br04_set1, Br05_set1, Br06_set1,
 Br05_set2, Br06_set2, Br07_set2, Br08_set2). *GraphPad Prism files*
 GraphPad Prism files (.pzf) containing the graphs and analyses used to
 generate Fig. 1B, 1D, 1F, 1H, 1J, and 1L. *Tabular data* * Raw tabular
 data (.csv): Individual (raw) values used for analysis of Fig. 1B, 1D, 1F,
 1H, 1J, and 1L. The number of cells possessing a cilium was manually
 quantified, and values excluded as outliers (e.g., partially imaged cells)
 were labeled as NA. * Processed tabular data for Prism (.csv): Averaged
 values (per mouse brain) were imported into GraphPad Prism to generate
 Fig. 1B, 1D, 1F, 1H, 1J, and 1L. **Fig.2.zip file:** *Folder
 Fig_2A_12M_ChAT_Gdnf_cilia* Microscopy images (.czi) of striatal
 cholinergic (ChAT) neurons from 8 animals of 12 months of age. Four
 animals are wild type (WT) mice (Br01_set2, Br02_set2, Br03_set2,
 Br04_set2), and four are G51D-alpha-synuclein (G51D) homozygous knock-in
 mice (Br05_set2, Br06_set2, Br07_set2, Br08_set2). One additional image
 set (no-probe control) was acquired from a WT animal. Gdnf denotes
 glial-derived neurotrophic factor. *GraphPad Prism files* GraphPad Prism
 files (.pzf) containing the graphs and analyses used to generate Fig. 2B
 and 2C. *Tabular data* * Raw tabular data (.csv): Individual (raw) values
 used for analysis of Fig. 2B and 2C. The number of cells possessing a
 cilium and the number of glial-derived neurotrophic factor (Gdnf) RNA dot
 were manually quantified, and values excluded as outliers (e.g., partially
 imaged cells) were labeled as NA. * Processed tabular data for Prism
 (.csv): Averaged values (per mouse brain) were imported into GraphPad
 Prism to generate Fig. 2B and 2C. **Fig.3.zip file:** *Folder
 Fig_3A_12M_PV_Nrtn_cilia* Microscopy images (.czi) of striatal parvalbumin
 (PV) neurons from 8 animals of 12 months of age. Four animals are wild
 type (WT) mice (Br01_set2, Br02_set2, Br03_set2, Br04_set2), and four
 are G51D-alpha-synuclein (G51D) homozygous knock-in mice (Br05_set2,
 Br06_set2, Br07_set2, Br08_set2). One additional image set (no-probe
 control) was acquired from a WT animal. Note that images of Br02_set2 are
 split into 2 files. Nrtn denotes neurturin. *GraphPad Prism files*
 GraphPad Prism files (.pzf) containing the graphs and analyses used to
 generate Fig. 3B and 3C. *Tabular data* * Raw tabular data (.csv):
 Individual (raw) values used for analysis of Fig. 3B and 3C. The number of
 cells possessing a cilium and the number of neurturin (Nrtn) RNA dot were
 manually quantified, and values excluded as outliers (e.g., partially
 imaged cells) were labeled as NA. * Processed tabular data for Prism
 (.csv): Averaged values (per mouse brain) were imported into GraphPad
 Prism to generate Fig. 3B and 3C. **Fig.4.zip file:** *Folder
 Fig_4A_12M_Aldh1l1_Bdnf_cilia* Microscopy images (.czi) of striatal
 Aldh1l1 astrocyte from 8 animals of 12 months of age. Four animals are
 wild type (WT) mice (Br01_set2, Br02_set2, Br03_set2, Br04_set2), and four
 are G51D-alpha-synuclein (G51D) homozygous knock-in mice (Br05_set2,
 Br06_set2, Br07_set2, Br08_set2). One additional image set (no-probe
 control) was acquired from a WT animal. Aldh1l1 denotes Aldehyde
 Dehydrogenase 1 Family Member L1, a marker for the astrocyte. Bdnf
 denotes Brain-derived neurotrophic factor. *GraphPad Prism files* GraphPad
 Prism files (.pzf) containing the graphs and analyses used to generate
 Fig. 4B and 4C. *Tabular data* * Raw tabular data (.csv): Individual (raw)
 values used for analysis of Fig. 4B and 4C. The number of cells possessing
 a cilium and the number of Brain-derived neurotrophic factor (Bdnf) RNA
 dot were manually quantified, and values excluded as outliers (e.g.,
 partially imaged cells) were labeled as NA. * Processed tabular data for
 Prism (.csv): Averaged values (per mouse brain) were imported into
 GraphPad Prism to generate Fig. 4B and 4C. **Fig.5.zip file:** *Folder
 Fig_5A_3M_PCx_PV_cilia* Microscopy images (.czi) of piriform cortex (PCx)
 parvalbumin (PV) neurons from 8 animals of 3 months of age. Four animals
 are wild type (WT) mice (Br01, Br02, Br03, Br04), and four
 are G51D-alpha-synuclein (G51D) homozygous knock-in mice (Br05, Br06,
 Br07, Br08).  *Folder Fig_5C_12M\_ PCx_PV_cilia* Microscopy images (.czi)
 of piriform cortex (PCx) parvalbumin (PV) neurons from 14 animals of 12
 months of age. Seven animals are wild type (WT) mice (Br01_set1,
 Br02_set1, Br03_set1, Br01_set2, Br02_set2, Br03_set2, Br04_set2), and
 seven are G51D-alpha-synuclein (G51D) homozygous knock-in mice (Br04_set1,
 Br05_set1, Br06_set1, Br05_set2, Br06_set2, Br07_set2, Br08_set2). *Folder
 Fig_5E_12M_PCx_PV_Nrtn_cilia* Microscopy images (.czi) of piriform cortex
 (PCx) parvalbumin (PV) neurons from 8 animals of 12 months of age. Four
 animals are wild type (Br01_set2, Br02_set2, Br03_set2, Br04_set2), and
 four are G51D-alpha-synuclein (G51D) homozygous knock-in mice (Br05_set2,
 Br06_set2, Br07_set2, Br08_set2). Nrtn denotes neurturin.  *GraphPad Prism
 files* GraphPad Prism files (.pzf) containing the graphs and analyses used
 to generate Fig. 5B, 5D, 5F and 5G. *Tabular data* * Raw tabular data
 (.csv): Individual (raw) values used for analysis of Fig. 5B, 5D, 5F, and
 5G. The number of cells possessing a cilium and the number of neurturin
 (Nrtn) RNA dot were manually quantified, and values excluded as outliers
 (e.g., partially imaged cells) were labeled as NA. * Processed tabular
 data for Prism (.csv): Averaged values (per mouse brain) imported into
 GraphPad Prism to generate Fig. 5B, 5D, 5F, and 5G. **Fig.6.zip file:**
 *Folder Fig_6A_12M_WT_G51D_HBC_data* Microscopy images (.czi) of
 horizontal basal cells (HBSs) in the olfactory epithelium from 8 animals
 of 12 months of age. Four animals are wild type (WT) mice (wt1, wt2, wt3,
 wt4), and four are G51D-alpha-synuclein (G51D) homozygous knock-in mice
 (g51d1, g51d2, g51d3, g51d4). Note that images of wt3 are split into 2
 files. *Folder Fig_6D_12M_OSN_ac-tubulin_regular_staining* Microscopy
 images (.czi) of olfactory sensory neurons (OSNs) in the olfactory
 epithelium from 8 animals of 12 months of age. Four animals are wild type
 (WT) mice (wt1, wt2, wt3, wt4), and four are G51D-alpha-synuclein (G51D)
 homozygous knock-in mice (g51d1, g51d2, g51d3, g51d4). Note that images of
 wt3 are split into 2 files. *Folder
 Fig_6G_6H_12M_expansion_microscopy_WT_G51D* Expansion microscopy images
 (.czi) of olfactory sensory neurons (OSNs) in the olfactory epithelium
 from 4 animals of 12 months of age. Two animals are wild type (WT) mice
 (wt1, wt2), and two are G51D-alpha-synuclein (G51D) homozygous knock-in
 mice (g51d1, g51d2). Note that each mouse contributes 25 image files.
 *GraphPad Prism files* GraphPad Prism files (.pzf) containing the graphs
 and analyses used to generate Fig. 6B, 6C, 6E, 6F and 6I. *Tabular data* *
 Raw tabular data (.csv): Individual (raw) values used for analysis of Fig.
 6B, 6C, 6E, 6F, and 6I. The number of cells possessing a cilium was
 manually quantified. The length used to quantify the number of HBCs was
 measured in millimeters (mm). The intensity of acetylated tubulin was
 manually quantified. The area used to normalize the number of HBCs was
 measured in square micrometers (µm²). * Processed tabular data for Prism
 (.csv): Averaged values (per mouse epithelium) were imported into GraphPad
 Prism to generate Fig. 6B, 6C, 6E, 6F, and 6I. For visualization in Fig.
 6F, values originally measured in square micrometers (µm^2^) were
 converted to square millimeters (mm^2^). **Fig.7.zip file:** *Folder
 Fig_7A_7C_pSNCA_ChAT_WT_and_G51D* Microscopy images (.czi) of striatal
 cholinergic (ChAT) neurons from 8 animals of 12 months of age. Four
 animals are wild type (WT) mice (Br01_set2, Br02_set2, Br03_set2,
 Br04_set2), and four are G51D-alpha-synuclein (G51D) homozygous knock-in
 mice (Br05_set2, Br06_set2, Br07_set2, Br08_set2). Note that images of
 Br02_set2 are split into 2 files. pSNCA denotes phospho–α-synuclein.
 *Folder Fig_7E_pSNCA_ChAT_MSN_in_G51D_only* Microscopy images (.czi) of
 striatal cholinergic (ChAT) and medium spiny neurons (MSNs) from 4 animals
 of 12 months of age. Four animals are G51D-alpha-synuclein (G51D)
 homozygous knock-in mice (Br05_set2, Br06_set2, Br07_set2, Br08_set2).
 *GraphPad Prism files* GraphPad Prism files (.pzf) containing the graphs
 and analyses used to generate Fig. 7B and 7F. *Tabular data* * Raw tabular
 data (.csv): Individual (raw) values used for analysis of Fig. 7B, 7D, and
 7F. The number of cells possessing a cilium was manually quantified.
 Quantification of phospho–α-synuclein (pSNCA) in cholinergic (ChAT) and
 medium spiny neurons (MSNs) was performed using FIJI/ImageJ macros, R
 scripts, and CellProfiler pipelines, which are included in the Fig. 7
 pipeline files. * Processed tabular data for Prism (.csv): Averaged values
 (per mouse brain) imported into GraphPad Prism to generate Fig. 7B and 7F.
 **Fig.7_pipeline.zip file:** *Folder scripts_for_macro* FIJI/ImageJ
 scripts used to generate non-overlapping 3-slice maximum-intensity
 projections from microscopy z-stacks. *Folder cellprofiler* A CellProfiler
 pipeline used to quantify phospho–α-synuclein (pSNCA) in cholinergic
 (ChAT) and medium spiny neurons (MSNs). *Folder Rscripts* R scripts used
 to analyze the output tables generated by the CellProfiler pipeline.
 **Fig.S1.zip file:** *Folder Fig_S1A_3M_MSN_cilia* Microscopy images
 (.czi) of striatal medium spiny neurons (MSNs) from 8 animals of 3 months
 of age. Four animals are wild type (WT) mice (Br01, Br02, Br03, Br04), and
 four are G51D-alpha-synuclein (G51D) homozygous knock-in mice (Br05, Br06,
 Br07, Br08). *Folder Fig_S1C_12M_MSN_cilia* Microscopy images (.czi) of
 striatal medium spiny neurons (MSNs) from 14 animals of 12 months of age.
 Seven animals are wild type (WT) mice (Br01_set1, Br02_set1, Br03_set1,
 Br01_set2, Br02_set2, Br03_set2, Br04_set2), and seven
 are G51D-alpha-synuclein (G51D) homozygous knock-in mice (Br04_set1,
 Br05_set1, Br06_set1, Br05_set2, Br06_set2, Br07_set2, Br08_set2).
 *GraphPad Prism files* GraphPad Prism files (.pzf) containing the graphs
 and analyses used to generate Fig. S1B and S1D. Dopamine- and
 cAMP-Regulated Phosphoprotein (DARPP32) is a cell marker for medium spiny
 neuron (MSN). *Tabular data* * Raw tabular data (.csv): Individual (raw)
 values used for analysis of Fig. S1B and S1D. The number of cells
 possessing a cilium was manually quantified, and values excluded as
 outliers (e.g., partially imaged cells) were labeled as NA. * Processed
 tabular data for Prism (.csv): Averaged values (per mouse brain) were
 imported into GraphPad Prism to generate Fig. S1B and S1D. **Fig.S2.zip
 file:** *Folder Fig_S2A_2B_2C_2D_12M_expansion_microscopy_WT_G51D_images*
 Expansion microscopy images (.czi) of olfactory sensory neurons (OSNs) in
 the olfactory epithelium from 4 animals of 12 months of age. Two animals
 are wild type (WT) mice (wt1, wt2), and two are G51D-alpha-synuclein
 (G51D) homozygous knock-in mice (g51d1, g51d2). Note that each mouse
 contributes 25 image files. **Fig.S3.zip file:** *Folder
 Fig_S3A_WB_pRab10_pRab12_images* Western blotting (WB) images (.tif) of
 the whole brain lysates from 8 animals of 9 months of age. Four animals
 are wild type (WT) mice (WT1, WT2, WT3, WT4), and four
 are G51D-alpha-synuclein (G51D) homozygous knock-in mice (G51D1, G51D2,
 G51D3, G51D4). *Tabular data* * Raw tabular data (.csv): Individual (raw)
 values used for analysis of Fig. S2B. Quantitative immunoblotting analysis
 with the indicated antibodies (pRab10, total Rab10, pRab12, total Rab12)
 and data analyzed using the LI-COR Odyssey IRdye imaging and the gel
 scanning plugin in ImageJ. ## Code/software * Microsoft Excel (or
 equivalent spreadsheet software) to open .csv files * GraphPad Prism 10 or
 newer to open Prism files (.pzf) * ZEISS ZEN 3.4 or newer to open .czi
 files (alternatively, FIJI/ImageJ can also be used) * FIJI/ImageJ to run
 the macros and view .tif files. * RStudio to open and run the R scripts *
 CellProfiler 4.2.6 or newer to open the CellProfiler pipeline