Tet2 loss and enhanced ciliogenesis suppress α-synuclein pathology

Abstract

There are no approved treatments that slow Parkinson's disease (PD) progression and therefore it is important to identify novel pathogenic mechanisms that can be targeted. Loss of the epigenetic marker, Tet2 appears to have some beneficial effects in PD models, but the underlying mechanism of action is not well understood. We performed an unbiased transcriptomic analysis of cortical neurons isolated from patients with PD to identify dysregulated pathways and determine their potential contributions to the disease process. We discovered that genes associated with primary cilia, non-synaptic sensory and signaling organelles, are upregulated in both early and late stage PD patients. Enhancing ciliogenesis in primary cortical neurons via sonic hedgehog signaling suppressed the accumulation of α-synuclein pathology in vitro. Interestingly, deletion of Tet2 in mice also enhanced the expression of primary cilia and sonic hedgehog signaling genes and reduced the accumulation of α-synuclein pathology and dopamine neuron degeneration in vivo. Our findings demonstrate the crucial role of TET2 loss in regulating ciliogenesis and potentially affecting the progression of PD pathology.

Keywords: Parkinson’s disease; Primary cilia; Tet2; α-synuclein.

Fig. 1

Dysregulation of primary cilia gene networks in cortical neurons from PD patients. Differentially expressed genes (DEGs) in the prefrontal cortex of PD neurons were identified in our RNAseq dataset using a robust linear regression model a Significantly altered genes in PD cortical neurons are highlighted in orange (q < 0.05, log(FC) ≥ 0.5, robust linear regression, multiple testing corrected). b Number of significantly dysregulated (up- and down-regulated) genes in all PD neurons relative to controls (q < 0.05). c Heatmap of dysregulated primary cilia-associated genes under the ‘ciliopathies’ hub. d Network and clustering of the significantly enriched pathways obtained via GSEA of the transcriptomic changes in PD neurons, with nodes representing altered pathways clustered into networks by EnrichmentMap and node colors constituting normalized enrichment score e, f Enrichment plots for ciliopathies (e) and oxidative phosphorylation (f), g Heatmap of dysregulated pathways in PD neurons as revealed by Cell component and Wiki pathways

Fig. 2

Enhancing ciliogenesis via SHH signaling suppresses αSyn pathology in vitro. Altered primary cilia and αSyn pathology in cortical neurons exposed to αSyn PFF for 14 days and incubated with different doses of purmophamine (PM). a, b Representative images showing ACIII⁺ (a) and pS129 αSyn (b) puncta (scale bar 50 µM). Analysis of c NeuN⁺ cortical neuron count normalized to vehicle (DMSO) control, d Area covered by MAP2⁺ neurons, e Area covered by ACIII⁺ primary cilia normalized to NeuN⁺ cell count, f pS129 αSyn pathology area normalized to total MAP2 area. Data are mean ± s.e.m., for 9 independent wells from 3 separate experiments, *P < 0.05, **P < 0.01, ***P < 0.001, one way ANOVA with Tukey’s multiple comparisons test relative to respective controls

Fig. 3

Tet2 inactivation reduces αSyn pathology. Representative images (scale bar 250 µM) and quantification of pS129 αSyn pathology in the frontal cortex, striatum, and substantia nigra at a, b 3 months (n = 5–7 mice per PFF group, n = 15 mice per PBS group) and c, d 6 months (n = 11–12 mice per PFF group, n = 13–14 mice per PBS group) after αSyn PFF injection. The percentage area with pS129 αSyn immunoreactive staining was quantified using the Aiforia platform. Data are mean ± s.e.m., analyzed using two-way ANOVA with Tukey’s multiple comparisons test relative to the respective controls, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

Tet2 deletion enhances the expression of genes linked with ciliogenesis and SHH signaling. Six months after striatal αSyn PFF inoculation, we examined the cortical expression of multiple genes using qPCR, a mRNA expression of Tet1, bTet2, cTet3, dTalpid3, e, Gli1, fGli2, gGli3, and hPtch1 (n = 3–5 mice per group). Data are mean ± s.e.m., analyzed using two-sided t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

Tet2 inactivation protects against αSyn-induced dopamine neuron loss. a Representative image (scale bar 500 µM) and b quantification of TH-positive neurons in the substantia nigra 6 months after αSyn PFF injection (n = 11–13 mice per PFF group, n = 9 mice per PBS group). TH immunoreactive staining was quantified using the Aiforia platform. c, d Behavioral assessment i.e., wire hang test (c) and rotarod (d) were performed 6 months after αSyn PFF injection (n = 15–16 mice per PFF group, n = 17–20 mice per PBS group). Data are mean ± s.e.m., analyzed using two-way ANOVA with Tukey’s multiple comparisons test relative to the respective controls, **P < 0.01