DOI: 10.5061/dryad.pk0p2ngvp
title: Data from: Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2driven and idiopathic Parkinson’s disease

description: Activating LRRK2 mutations cause Parkinson’s disease. Previously, we
 showed that cholinergic interneurons and astrocytes but not medium spiny
 neurons of the dorsal striatum lose primary cilia in LRRK2 mutant mice.
 Single nucleus RNA sequencing shows that cilia loss in cholinergic
 interneurons correlates with higher LRRK2 expression and decreased glial
 derived neurotrophic factor transcription. Nevertheless, much higher LRRK2
 expression is seen in medium spiny neurons that have normal cilia in mice
 and humans. In parallel with decreased striatal dopaminergic neurite
 density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion
 protein expression; glial cells show significant loss of ferritin heavy
 chain. Human striatal tissue from LRRK2 pathway mutation carriers and
 idiopathic Parkinson’s disease show similar cilia loss in cholinergic
 interneurons and astrocytes and overall loss of such neurons. These data
 strongly suggest that loss of cilia in specific striatal cell types
 decreases neuroprotection for dopamine neurons in mice and human
 Parkinson’s disease. 
description: <strong>Collection/generation of RNA sequencing
 data</strong>: Mice were PBS perfused and brains were removed by
 surgical dissection. Dorsal striatal tissue was isolated in ice cold PBS
 using a light dissection micrscope. Striatal tissues were placed in 1.5 mL
 eppendorf tubes and quickly frozen in a 50:50 slurry containing 200 proof
 ethanol and dry ice.  1 mL of cold homogenization buffer was added and the
 samples were transferred to dounce homogenizers. The samples were dounced
 10 times with the loose pestle and 10 times with the tight pestle on ice.
  Homogenates were transferred to a conical tube through 30um strainers to
 remove large debris. Tubes were spun for 10 minutes at 4 C, 900xg.
 Supernatant was removed with only ~50 µL of solution remaining.  Samples
 were resuspended in 450 µL of blocking buffer. 10 µL of each sample was
 combined with 10 µL 0.4% Trypan Blue to assess nuclei quality and sample
 yield using a hemocytometer. Concentrations were adjusted to 1000
 nuclei/µL. The Stanford Genomics center prepared libraries, performed
 sequencing using 10x Chromium Single Cell 3', 5', T and B cell
 V(D)J (V2), 10x scATACSeq, 10x 3' WTA (V3), and performed feature
 barcoding.  <strong>Description of
 quality-assurance procedures performed on the RNA sequencing
 data</strong>: Raw fastq files were analyzed using the Seurat 4.0
 package in RStudio. Clusters containing a high percentage of mitochondrial
 content (high percentage of "mt-" reads) were removed before
 gene expression analyses. Additionally, the Doublet Finder package was
 used to remove predicted doublets before the individual sets (e.g. WTA,
 GSA) were merged for gene expression analysis.
 <strong>People involved with sample collection, processing,
 analysis and/or submission</strong>: Sample collection was performed
 by Shahzad Khan. Anthony Fernández-Casteñeda and Ebsy Jaimon assisted with
 mouse perfusions and brain removal. Michael Blanco prepared libraries and
 performed sequencing. Data analysis was performed by Shahzad Khan, Ebsy
 Jaimon, Yu-En Lin, Jonas Nikoloff and Suzanne Pfeffer.
description: # Loss of primary cilia and dopaminergic neuroprotection in pathogenic
 LRRK2driven and idiopathic Parkinson’s disease 
 [https://doi.org/10.5061/dryad.pk0p2ngvp](https://doi.org/10.5061/dryad.pk0p2ngvp) **General Information** Title of Dataset: Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2driven and idiopathic Parkinson’s disease Author/Principal Investigator Information Name: Suzanne Pfeffer ORCID: 0000-0002-6462-984X Institution: Stanford University Address: Beckman Center Room B413 279 Campus Drive Stanford, California 94305-5307 Email: [preffer@stanford.edu](mailto:preffer@stanford.edu) Author/Associate or Co-investigator Information Name: Shahzad Khan ORCID:0000-0003-3962-0226 Present Institution: University of North Carolina at Chapel Hill Address: Beckman Center Room B453 279 Campus Drive Stanford, California 94305-5307 Email: [shahzad_khan@med.unc.edu](mailto:shahzad_khan@med.un) Date of data collection: 2022-01-12 Geographic location of data collection: Stanford, CA **Sharing/Access Information** Licenses/restrictions placed on the data: None Currently Applicable Links to publications that cite or use the data: None Currently Applicable Links to other publicly accessible locations of the data: None Currently Applicable Links/relationships to ancillary data sets: None Currently Applicable Was data derived from another source? No ## Description of the data and file structure DATA & FILE OVERVIEW **File List:** **"10X_files_for_Seurat.zip"** **file**: Fastq files used for Seurat analysis obtained from 10 animals approximately six months of age. Five animals are wild type mice (WTA, WTB, WTC, WTD, WTE) and five are G2019S LRRK2 mice (GSA, GSB, GSC, GSD, GSE). This file has 10 subfolders folders (WTA, WTB, WTC, WTD, WTE, GSA, GSB, GSC, GSD, GSE), each subfolder contains "barcodes.tsv.gz", "features.tsv.gz", and "matrix.mtx.gz" files. WTA - Female - Wild type LRRK2\ WTB - Female - Wild type LRRK2\ WTC - Female - Wild type LRRK2\ WTD - Male - Wild type LRRK2\ WTE - Male - Wild type LRRK2 GSA - Female - G2019S LRRK2\ GSB - Female - G2019S LRRK2\ GSC - Female - G2019S LRRK2\ GSD - Male - G2019S LRRK2\ GSE - Male - G0219S LRRK2 **"10X_snRNAseq_pipeline.Rmd" file**: The R markdown script used to compare gene expression differences between WTA-C and GSA-C files. **"10X_astrocyte_subcluster_pipeline.Rmd" and "10X_cholinergic_neuron_pipeline.Rmd" files**: Pipelines used for subcluster analysis of astrocytes and cholinergic interneurons, respectfully. **Instructions for using R markdown scripts**: Download and install RStudio (RRID:SCR_000432). Launch the R markdown scripts using RStudio (version 4.4.0).  The 03/31/2020 updated version of DoubletFinder was used for this study. The fastq files required to run the R markdown scripts are located in the "**10X_files_for_Seurat.zip**" file. "**10X_dorsalstriatum_snRNAseq_gene_expression_analyses.xlsx**" **file**: Gene expression analysis results for each cluster and subcluster compared in this study. The columns lists the gene names (Names), p values (p_val), log2 fold changes (log2FoldChange), percentage of nuclei from G2019S KI mice expressing the listed gene (pct.1), percentage of nuclei from WT mice expressing the listed gene (pct.2), the adjustive p values (padj), the number of reads obtained from G2019S KI mice for the listed gene (Counts_GS), the number of reads obtained from WT mice for the listed gene (Counts_WT). **Figure 1B zip file:** Image captured from 5-month-old WT mouse brain. **Figure 2BC zip file:** Images from 2.5-month-old WT and PPM1H KO animals. Brains 8,9,11,12 are PPM1H KO and Brains 13,14,15,17 are WT. **Figure 3 zip file**: Images from 2.5-month-old WT animals. Brain 13,14,15, and 17 are WT. Raw image files used to generate figure. **Figure 4B zip file:** Images from 5-month-old WT and G2019S KI animals. Brains 1,4,7,8 are WT and Brains 2,3,5,6 are G2019S KI. **Figure 5 zip file:** \ Images from 5-month-old WT and G2019S KI animals. Brains 1,4,7,8 are WT and Brains 2,3,5,6 are G2019S KI. **Figure 7 zip file:** Images from postmortem brain sections from Parkinson’s disease patients and age-matched controls. Brain 01-31, Brain 01-46, Brain 02-27, Brain 03-63, Brain 249, Brain 418, Brain 100 are controls. Brain 01-39, Brain 13-60, Brain 10-37 are G2019S. Brain 10-28, Brain 16-23, Brain 18-72, Brain 10-27, Brain 19-73, Brain 21-81, Brain 12-56, Brain 12-03 are Sporadic PD. Brain 14-03 is PPM1H truncation. Note that the data for Brain 12-56 is split into two files (Brain12_56_1_chat_488_darpp32_568_AC3_647, Brain12_56_2_chat_488_darpp32_568_AC3_647). **Figure 8 zip file:** Images from postmortem brain sections from Parkinson’s disease patients and age-matched controls. Brain 01-31, Brain 01-46, Brain 02-27, Brain 03-63, Brain 249, Brain 100 are controls. Brain 01-39, Brain 13-60, Brain 10-37 are G2019S. Brain 10-28, Brain 16-23, Brain 18-72, Brain 1047, Brain 643 are Sporadic PD. Brain 14-03 is PPM1H truncation. Note that the data for Brain 01-46 is split into two files (Brain01_46_gfap_488_darpp32_568_Arl13b_647-01.czi, Brain01_46_gfap_488_darpp32_568_Arl13b_647-02.czi). **Figure 9 zip file:** Figure 9AB- Images from 5-month-old WT and G2019S KI animals. Brains 1,4,7,8 are WT and Brains 2,3,5,6 are G2019S KI. Figure 9CD-Images from postmortem brain sections from Parkinson’s disease patients and age-matched controls. Brain 01-31, Brain 01-46, Brain 02-27, Brain 03-63 are controls. Brain 01-39, Brain 13-60, Brain 10-37 are G2019S. Brain 10-28, Brain 16-23, Brain 18-72 are Sporadic PD. Brain 14-03 is PPM1H truncation. **Raw Figure files zip**: Raw Image files used to generate figures. **Tables and graphs.prism**: Tabular data and graphs used to generate graphs shown in Figure 2C,3B,4,6B,6D,7,8B,8D. \**Pipelines zip:  **CellProfiler pipelines used for quantification in Figure 4B and 5. **Relationship between files, if important:** "WT" files represent sequencing performed on tissue obtained from wild type animals. "GS" files represent sequencing performed on tissue obtained from G2019S KI animals. WTA, WTB, WTC, GSA, GSB, and GSC files were used for Seurat analyses. WTD, WTE, GSD, and GSE were not used for gene expression analysis. **Are there multiple versions of the dataset?** No ### Change log #### May 9, 2024 A new version of the repository was uploaded as a result of changes made during the manuscript review process. The changes made are detailed below: * We added additional human subjects to our cholinergic neuron and astrocyte cilia analyses shown in **Figures 7 and 8** (former Figures 6 and 7), respectively * Added data used to generate a new figure (**Figure 3_zip)** * Renamed previous figure files (**Figures 3, 6, 7 and 8**) to reflect their respective new order following revisions (**Figures 4, 7, 8 and 9**) * Updated the following data files to include additional human subjects: **Figure_7.zip**; **Figure_8.zip** * Updated the following analysis file to incorporate new data: **Tables_and_Graphs.prism** * Updated **Raw Figure files zip** to incorporate images from new Figure 3 #### June 26, 2024 A new version of the repository was uploaded as a result of changes made during the manuscript review process. The changes made to the **Figure 5 zip file** are detailed below: * This folder has the updated Cell Profiler pipeline, raw values in Excel and Prism, raw images, and images used to generate the revised figure.