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Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2-driven and idiopathic Parkinson's disease - PubMed

  • ️Mon Jan 01 2024

Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2-driven and idiopathic Parkinson's disease

Shahzad S Khan et al. bioRxiv. 2024.

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Abstract

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.

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Conflict of interest statement

The authors declare that they have no competing interests. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials; all primary data is available at: https://doi.org/10.5061/dryad.pk0p2ngvp

Figures

Figure 1.
Figure 1.

Single nucleus RNA sequencing analysis of wild type mouse dorsal striatum. A. tSNE plot showing cell types detected using markers summarized in Table 1. C, D. Comparison of relative LRRK2 (C) or PPM1H (D) RNA levels in cell types color coded as in A. B. RNA scope in situ hybridization to detect LRRK2 transcripts in the dorsal striatum. Green, anti-choline acetyltransferase staining of a cholinergic interneuron surrounded by the much more abundant medium spiny neurons; pink, primary cilia; yellow dots, LRRK2 transcripts, blue, DAPI stained nuclei. Bar, 10μm.

Figure 2.
Figure 2.

Two classes of cells explain ciliation differences in cholinergic interneurons in the dorsal striatum. A. Single nucleus RNA sequencing analysis of cholinergic interneurons (left column) or astrocytes (right column) from wild type mouse dorsal striatum. Cholinergic interneurons and astrocytes sub-clustered according to the markers indicated. Relative LRRK2 and PPM1H expression is shown below for all sub-clusters with purple intensity reflecting abundance in that nucleus. B, C. RNAscope analysis of LRRK2 RNA (white dots in B) in cholinergic interneurons according to ciliation status in 2.5-month-old wild type or PPM1H KO dorsal striatum. Error bars in C represent SEM from 4 WT and 4 PPM1H KO brains, with >25 ChAT+ neurons scored per brain. Statistical significance was determined using an unpaired t-test: ****p <0.0001 for ciliated WT versus unciliated WT; ****p < 0.0001 for ciliated PPM1H KO versus unciliated PPM1H KO. D. Comparison of GDNF expression from RNAseq data between WT and G2019S KI cholinergic interneurons. Statistical significance was determined using a student’s t-test. ****p <0.0001.

Figure 3.
Figure 3.

A. Volcano plot analysis comparing transcripts that increase (right side) or decrease (left side) in LRRK2 G2019S dorsal striatal neurons compared with age matched wild type mouse controls. The cell type analyzed is indicated at the top of each graph. Number of variables for direct spiny projection neurons (7699), indirect spiny projection neurons (7213), eccentric spiny projection neurons (8212), and parvalbumin interneurons (9270). B. Immunofluorescence microscopy of cells stained with antibodies to detect NeuN (green), DRD2 (blue) and CNTN5 (red) expression. Total fluorescence intensity of CNTN5 within 5 pixels of the NeuN or DRD2 was quantified and normalized to either total NeuN staining or total DRD2 staining. Error bars represent SEM from 4 WT and 4 G2019S brains, with >365 NeuN+ neurons and >200 DRD2+ neurons scored per brain. Statistical significance was determined using an unpaired t-test. CNTN5 intensity in NeuN+ neurons: *p = 0.0184 for WT versus G2019S and CNTN5 intensity in DRD2+ neurons: *p = 0.0177 for WT versus G2019S. Bar, 10μm.

Figure 4.
Figure 4.

Loss of tyrosine hydroxylase and GDNF receptor staining in mouse G2019S LRRK2 dorsal striatum. Top panels: Confocal images of tissue labeled with anti-Tyrosine hydroxylase antibodies (green) and anti-GDNF receptor alpha-1 antibodies (magenta) in sections of the dorsal striatum from 5-month-old wild-type (WT) or G2019S LRRK2 KI mice. Bottom graphs: Integrated intensity of Tyrosine hydroxylase and GDNF receptor α−1 was quantified using CellProfiler. Error bars represent SEM from 4 WT and 4 G2019S brains, with >25 fields scored per brain. Statistical significance was determined using an unpaired t-test. Intensity of Tyrosine hydroxylase: *p = 0.0276 for WT versus G2019S and Intensity of GDNF receptor α−1: *p = 0.0327 for WT versus G2019S. Bar, 10μm.

Figure 5.
Figure 5.

Volcano plot analysis comparing transcripts that increase (right side) or decrease (left side) in LRRK2 G2019S dorsal striatal astrocytes, oligodendrocytes or microglia compared with age matched wild type mouse controls. The cell type analyzed is indicated at the top of each graph. The tSNE plot at the upper left is the same as that presented in Fig. 2 and is included here for reference purposes. Number of genes for total astrocytes (4791), GFAP+ astrocytes (4463), ALDH1A1+ astrocytes (4901), CRYM+ astrocytes (4027), NRG1+ astrocytes (4011), Oligodendrocytes (3522 genes), Microglia (3606).

Figure 6.
Figure 6.

Loss of primary cilia in human striatal cholinergic neurons but not medium spiny neurons from postmortem brain sections from Parkinson’s disease patients and age matched controls. A. Confocal microscopy of tissue labeled with anti-ChAT antibody (green) and anti-AC3 antibody (magenta) to identify primary cilia. B. Quantitation of the percentage of ChAT+ neurons containing a cilium. Error bars represent SEM from 4 Control, 3 G2019S, 3 sporadic PD, and 1 PPM1H mutation brains, with >50 ChAT+ neurons scored. Statistical significance was determined using one-way ANOVA. ***p = 0.0009 for Control versus G2019S PD and **p = 0.0014 for Control versus Sporadic PD. Bar, 10μm. C. Confocal microscopy of human striatal tissue labeled with anti-DARPP-32 antibody (green) to label medium spiny neurons and anti-AC3 antibody (magenta) to identify primary cilia. D. Quantitation of the percentage of DARPP32+ neurons containing a cilium. Error bars represent SEM from 4 Control, 3 G2019S, 3 sporadic PD, and 1 PPM1H mutation brains, with >60 DARPP32+ neurons scored. All samples were scored blind and evaluated by two independent scientists. Bar, 10μm.

Figure 7.
Figure 7.

Loss of primary cilia in striatal astrocytes from postmortem brain sections from Parkinson’s disease patients. Left panels. Confocal images of astrocytes labeled using anti-GFAP (green) and primary cilia labeled using anti-Arl13b (magenta). Nuclei were labeled using DAPI (blue). Right panels. Quantitation of the percentage of GFAP+ astrocytes containing a cilium. Error bars represent SEM from 4 Control, 3 G2019S, 3 sporadic PD, and 1 PPM1H mutation brains, with >40 GFAP+ astrocytes scored. All samples were scored blind and evaluated by two independent scientists. Statistical significance was determined using one-way ANOVA. **p = 0.0015 for Control versus G2019S and ***p = 0.0009 for Control versus Sporadic PD. Bar, 10μm.

Figure 8.
Figure 8.

Loss of cholinergic neurons in the striatum of mice and of Parkinson’s patients. A. Representative tile scan images of the mouse dorsal striatum from 5-month-old wild-type or G2019S LRRK2 KI mice. Cholinergic interneurons were labeled using anti-ChAT (green). B. Quantitation of ChAT+ neurons detected per sq mm. Error bars represent SEM from 4 WT and 4 G2019S brains, with 4 sections scored per mouse. C. Representative tile scan images of the striatum from postmortem brain sections from Parkinson’s disease patients, identified geographically and molecularly using anti-DARPP-32 antibodies (cholinergic interneurons were labeled using anti-ChAT (green). D. Quantitation of ChAT+ neurons detected per sq mm. Error bars represent SEM from 4 Control, 3 genetic PD, 3 sporadic PD, and 1 PPM1H mutated brains, with >100 tiles scored per brain. Areas boxed with dashed lines are enlarged and shown. Statistical significance was determined using one-way ANOVA. ***p = 0.0009 for Control versus G2019S and ***p = 0.0007 for Control versus Sporadic PD. Bar, 100μm.

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