Pitx3 deficiency promotes age-dependent alterations in striatal medium spiny neurons

Abstract

Background: The classical motor symptoms of Parkinson's disease (PD) are tightly linked to the gradual loss of dopamine within the striatum. Concomitantly, medium spiny neurons (MSNs) also experience morphological changes, such as reduced dendritic complexity and spine density, which may be potentially associated with motor dysfunction as well. Thus, MSNs may serve as the emerging targets for PD therapy besides the midbrain dopaminergic neurons.

Results: To comprehensively examine pathological alterations of MSNs longitudinally, we established a TH ^Cre/ Pitx3 ^fl/fl (Pitx3^cKO ) mouse model that developed canonical PD features, including a significant loss of SNc DAergic neurons and motor deficits. During aging, the targeted neurotransmitter, MSNs morphology and DNA methylation profile were significantly altered upon Pitx3 deficiency. Specifically, dopamine, GABA and glutamate decreased in the model at the early stage. While nuclear, soma and dendritic atrophy, as well as nuclear invaginations increased in the aged MSNs of Pitx3^cko mice. Furthermore, more nuclear DNA damages were characterized in MSNs during aging, and Pitx3 deficiency aggravated this phenomenon, together with alterations of DNA methylation profiling associated with lipoprotein and nucleus pathway at the late stage.

Conclusion: The early perturbations of the neurotransmitters within MSNs may potentially contribute to the alterations of metabolism, morphology and epigenetics within the striatum at the late stage, which may provide new perspectives on the diagnosis and pathogenesis of PD.

Keywords: DNA methylation; Parkinson’s disease; aging; medium spiny neurons; neuronal morphology.

FIGURE 1

Neurodegeneration in 12- and 18-month-old Pitx3^cKO mice. (A) IFC co-staining of TH and NeuN in the ventral midbrain sections from 6-, 12-, and 18-month-old Pitx3^cWT and Pitx3^cKO mice. SNc and VTA were outlined, respectively (scale bar: 200 μm; high-magnification, 20 μm). (B) Quantification of TH⁺/NeuN⁺ and NeuN⁺ neurons in the SNc and VTA from 6-, 12-, and 18-month-old Pitx3^cWT and Pitx3^cKO mice (N = 3–4 mice per genotype; all males except for two females in 6-month-old Pitx3^cKO and 12-month-old Pitx3^cWT). 2way ANOVA analysis with Sidak’s multiple comparisons test, ****p < 0.0001 (12 months for TH⁺/NeuN⁺ co-staining), ****p < 0.0001 (12 months for NeuN⁺ staining), **p = 0.0041 (18 months for TH⁺/NeuN⁺ co-staining), **p = 0.0064 (18 months for NeuN⁺ staining).

FIGURE 2

Striatal pathology triggered movement abnormalities in Pitx3^cKO mice. (A) IFC staining of TH in the striatal sections from 12- to 18-month-old Pitx3^cWT and Pitx3^cKO mice (scale bar: 200 μm; high-magnification, 10 μm). (B) Quantification of relative TH intensity in the striatum from 12- to 18-month-old Pitx3^cWT and Pitx3^cKO mice (N = 4 mice per genotype; all males except for two females in 6-month-old Pitx3^cKO and 12-month-old Pitx3^cWT). Unpaired t-test, ***p = 0.0006 (18 months). (C) Levels of neurotransmitter in 6- (N = 6 mice per genotype; all males) and 12-month-old Pitx3^cKO and Pitx3^cWT mice (N = 6 mice per genotype; all males). The scaled intensity of three metabolites is relatively depicted according to the color key shown on the above. Red indicates high intensity levels; blue, low intensity levels. Unpaired t-test, *p = 0.026 (Dopamine, 6M), **p = 0.0083 (GABA, 6M), *p = 0.012 (Glutamate, 6M), *p = 0.0202 (Dopamine, 12M). (D) The latency to fall from rotarod was recorded from Pitx3^cWT and Pitx3^cKO mice at 6 (N = 11–13 mice per genotype; all males), 12 (N = 11–15 per genotype; all males) and 18 months of age (N = 9–11 mice per genotype; all males). 2way ANOVA analysis with Sidak’s multiple comparisons test at 12 and 18 months, *p = 0.0171 (day 4, 12 months), *p = 0.0202 (day 5, 12 months), *p = 0.0376 (day 6, 12 months), *p = 0.0326 (day 6, 18 months). (E) Center-corner preference analyses for Pitx3^cWT and Pitx3^cKO mice at 6 (N = 11–13 mice per genotype; all males), 12 (N = 12–14 per genotype; all males), and 18 (N = 9–10 mice per genotype; all males) months of age. 2way ANOVA analysis with Sidak’s multiple comparisons test, *p = 0.05 (time in corner at 18 months).

FIGURE 3

Analyses of neuronal morphology in Pitx3^cKO mice during aging. (A) The GFP-labeled individual MSN in 12-month-old Pitx3^cWT and Pitx3^cKO mice (scale bar: 10 μm). (B,C) Sholl analysis of dendritic complexity of GFP-labeled MSNs in 12-month-old Pitx3^cWT and Pitx3^cKO mice (N = 3 mice per genotype; 5–8 neurons per mouse were counted). Benjamin- Hochberg multiple comparison test of dendritic complexity at 18, 24, 30, 36, 42, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, and 114 μm from soma, q ≤ 0.05. (D) Analyses of primary dendrites (N = 3 mice per genotype; 5–8 neurons per mouse were counted; all males). (E) Dendritic length of GFP-labeled MSNs in 12-month-old Pitx3^cWT and Pitx3^cKO mice (N = 3 mice per genotype; 5–8 neurons per mouse were counted) unpaired t-test, ^****p < 0.0001. (F) Co-staining of Darpp32 and DAPI in MSNs of 12-month-old Pitx3^cWT and Pitx3^cKO mice (scale bar: 500 μm; high-magnification, 5 μm). (G) The soma and nucleus size of MSNs in 12-month-old Pitx3^cWT and Pitx3^cKO mice (N = 4 mice per genotype; all males). Conditional logistic regression test, ^****p < 0.0001. (H) Cumulative frequency of the soma and nuclear size distribution in MSNs of 12-month-old Pitx3^cWT and Pitx3^cKO mice. (I) The soma and nucleus size of MSNs in Pitx3^cWT and Pitx3^cKO mice at 6 (N = 4 mice per genotype; all males), 12 (N = 4 mice per genotype; all males) and 18 months (N = 3 mice per genotype; all males) of age. 2way ANOVA analysis with Sidak’s multiple comparisons test, ^**p = 0.0075 (soma, 12 months), ^****p < 0.0001 (soma, 18 months), ^**p = 0.0022 (nuclei, 12 months), ^****p < 0.0001 (nuclei, 18 months). (J) The nuclear size and soma size ratio (N/C ratio) of MSNs in Pitx3^cWT and Pitx3^cKO mice. DS, dorsal striatum.

FIGURE 4

Nuclear invaginations increase accompanied with genomic instability in Pitx3^cKO mice. (A) Co-staining of LaminB and Ctip2 in MSNs of 18-month-old Pitx3^cWT and Pitx3^cKO mice (scale bar: 10 μm) white arrow points to nuclear invagination. (B) Ratio of MSN nuclei containing ≥ 1 invagination in Pitx3^cWT and Pitx3^cKO mice at 12 (N = 3 mice per genotype; all males) and 18 (N = 3 mice per genotype; all males) month of age. Unpaired t-test, **p = 0.0041. (C) Co-staining of Nuclear Pore and LaminB (scale bar: 5 μm). (D) Costaining of Nuclear Pore and TOM20 (scale bar: 5 μm). White arrow points to a cluster of mitochondria. (E) Co- staining of γH2A.X and Ctip2 in the striatal sections of 12- and 18-month-old Pitx3^cWT and Pitx3^cKO mice (scale bar: 10 μm). (F) The ratios of MSNs with 10 or more γH2A.X-positive foci in the nuclei (N = 3 mice per genotype). Unpaired t-test, ***p = 0.0005.

FIGURE 5

Comparative analysis of the DMGs in Pitx3^cWT and Pitx3^cKO during aging. (A) The volcano plots of DNA methylation data collected from the striatum of Pitx3^cWT and Pitx3^cKO mice at 12 (N = 3 mice per genotype; all males) and 18 months of age (N = 3 mice per genotype; all males). (B) Supervised clustering for the DMGs data collected from the 12- and 18-month-old Pitx3^cWT mice. (C) Integrated map of GO terms enriched among the DMGs of Pitx3^cWT and Pitx3^cKO mice at 12 (background with white color) and 18 months of age (background with pink color). Red circles represented DMGs identified from our data. (D,E) 448 DMGs were identified during aging independent of genotype. They were analyzed with GO terms (D) and metabolic pathway (E), respectively.