Suzanne R. Pfeffer

Multiple lysosomal stresses activate Parkinson’s-linked LRRK2 kinase, and these involve the so-called “CASM” pathway to help recruit LRRK2 to lysosome surfaces. Clegg et al. (https://doi.org/10.1083/jcb.202511137) show that LRRK2, activated upon lysosome swelling caused by PIKfyve inhibition, instead relies solely on Rab GTPase binding for lysosome localization and activation.

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 spiny projection 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 multiciliated 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.

The endolysosomal phospholipid bis(monoacylglycerol)phosphate (BMP) is aberrantly elevated in urine from Parkinson’s patients carrying mutations in leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GCase). Because BMP resides on, and regulates biogenesis of, endolysosomal intralumenal membranes that become extracellular vesicles (EVs) upon release, we hypothesized that increased urinary BMP reflects enhanced exocytosis of BMP-enriched EVs. We analyzed BMP metabolism and EV-associated BMP release in wild-type (WT) and R1441G LRRK2 mouse embryonic fibroblasts (MEFs). Immunofluorescence and transmission electron microscopy revealed structural alterations in endolysosomes and the antibody-accessible BMP pool, indicating disrupted endolysosomal homeostasis. Biochemical analysis of isolated EV fractions showed increased release of LAMP2-positive EVs by mutant cells, partially restored by LRRK2 kinase inhibition but further, variably, increased by GCase inhibition. Mass spectrometry detected higher total di-22:6-BMP and di-18:1-BMP in mutant LRRK2 MEFs compared to WT. Inhibition of LRRK2 partially restored cellular BMP, whereas GCase inhibition further elevated it. In EVs from mutant cells, LRRK2 inhibition reduced BMP content, while GCase inhibition tended to increase it. Metabolic labeling showed elevated BMP was not due to increased synthesis, despite higher levels of the BMP-synthesizing enzyme CLN5 in mutant MEFs and patient fibroblasts. Finally, pharmacological modulation of EV release and live total internal reflection fluorescence imaging in human G2019S LRRK2 fibroblasts further confirmed that BMP release is likely associated with EV secretion. Together, these results establish LRRK2 as a regulator of BMP in cells and its release through EVs and suggest that GCase activity further modulates this process in LRRK2 mutant cells. Mechanistic insights from these studies have implications for the use of BMP-positive EVs as potential biomarkers for Parkinson’s disease.

Here we describe a method for monitoring CD63+ endo-lysosome exocytosis in hTERT-RPE cells stably expressing CD63-pHluorin. The fluorescence emission of pHluorin, a pH‑sensitive fluorescent probe, is quenched within acidic compartments such as lysosomes and multi-vesicular bodies (Miesenböck et al., 1998; Verweij et al., 2018). When these compartments fuse with the plasma membrane, the sudden shift in pH from acidic to neutral causes the probe to fluoresce. CD63-pHluorin enables monitoring of fusion events at the plasma membrane using Total internal reflection fluorescence (TIRF) microscopy. Powerful ExoJ software (Liu et al., 2024) enables automated quantitation of individual events. This protocol includes siRNA transfection to monitor the effects of specific protein depletion on endo-lysosome exocytosis events.

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. Teaser: Loss of primary cilia may contribute to dopamine neuron loss in both inherited and common Parkinson’s disease.

Here, we present a protocol for 4-5× expansion of mouse olfactory epithelium (OE), optimized for high-resolution imaging of olfactory sensory neuron (OSN) cilia. The method combines rapid dissection of the OE on a cold platform to preserve delicate ciliary structures with hydrogel embedding and expansion microscopy (ExM) to achieve isotropic 4-5 fold expansion (based on references 2-4). This approach enables detailed visualization of OSN ciliary architecture using conventional confocal microscopy, without the need for specialized super-resolution instrumentation. OSNs were labeled using anti-Olfactory Marker Protein (OMP) and their cilia were labeled using anti-acetyl-tubulin antibodies.

This technique expands mouse brain tissue 4.5-fold in all spatial dimensions by embedding brain slices in swellable acrylamide gels. This allows enhanced resolution compared to traditional microscopy to precisely investigate subcellular targets in the brain. Here we adapted previously developed expansion protocols to reliably image enlarged Arl13b-GFP-positive cilia of striatal cholinergic interneurons in Arl13b-GFP knock-in mouse brain slices.

This protocol describes an automated image analysis workflow using CellProfiler (1) to quantify Ser129 phosphorylated α-synuclein (pSNCA) within Cholinergic and Medium Spiny Neurons (MSNs) in the striatum of Synuclein G51D mutant mice (2). Brain sections are immunostained for Ser129 phospho-α-synuclein, DARPP-32 to identify MSNs, choline acetyltransferase (ChAT) to label cholinergic neurons, and DAPI to identify nuclei. Confocal Z-stacks are processed into consecutive three-slice maximum-intensity projections (MIPs) using a custom FIJI/ImageJ macro prior to segmentation and analysis. Quantitative outputs include integrated pSNCA intensity and neuronal mask area, which are normalised using a custom R script to yield cell type specific pSNCA burden per unit area. This workflow enables cell type resolved quantification of α-synuclein pathology from fluorescence imaging datasets. (1) Stirling DR, Swain-Bowden MJ, Lucas AM, Carpenter AE, Cimini BA, Goodman A (2021). CellProfiler 4:improvements in speed, utility and usability. BMC Bioinformatics, 22 (1), 433.. PMID: 34507520 PMCID: PMC8431850. (2) Kim Y, McInnes J, Kim J, Liang YHW, Veeraragavan S, Garza AR, Belfort BDW, Arenkiel B, Samaco R, Zoghbi HY. Olfactory deficit and gastrointestinal dysfunction precede motor abnormalities in alpha-Synuclein G51D knock-in mice. Proc Natl Acad Sci U S A. 2024 Sep 24;121(39):e2406479121. doi: 10.1073/pnas.2406479121.

The overall aim of this workflow is to generate three-slice maximum-intensity projections (MIPs) in an effort to preserve weak, spatially localized signals that can be missed when projecting across the entire Z-stack. For this purpose, a two-step ImageJ/FIJI workflow was developed. In the first step, a multi-series .czi file is treated as a container of independent image datasets, and each embedded dataset is extracted and exported as a standalone OME-TIFF file, preserving all acquired channels as full Z-stacks. In the second step, these per-dataset stacks are processed to generate three-slice maximum-intensity projections for user-defined channels. The resulting two-dimensional images are saved with a consistent naming scheme, enabling straightforward downstream grouping and high-throughput quantitative analysis using CellProfiler.