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. 

Collection/generation of RNA sequencing data: 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. 

Description of quality-assurance procedures performed on the RNA sequencing data: 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.

People involved with sample collection, processing, analysis and/or submission: 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.