Potentiation of neurotoxicity in double-mutant mice with Pink1 ablation and A53T-SNCA overexpression

Abstract

The common age-related neurodegeneration of Parkinson's disease can result from dominant causes like increased dosage of vesicle-associated alpha-synuclein (SNCA) or recessive causes like deficiency of mitophagy factor PINK1. Interactions between these triggers and their convergence onto shared pathways are crucial, but currently conflicting evidence exists. Here, we crossed previously characterized mice with A53T-SNCA overexpression and with Pink1 deletion to generate double mutants (DMs). We studied their lifespan and behavior, histological and molecular anomalies at late and early ages. DM animals showed potentiated phenotypes in comparison with both single mutants (SMs), with reduced survival and strongly reduced spontaneous movements from the age of 3 months onwards. In contrast to SMs, a quarter of DM animals manifested progressive paralysis at ages >1 year and exhibited protein aggregates immunopositive for pSer129-SNCA, p62 and ubiquitin in spinal cord and basal brain. Brain proteome quantifications of ubiquitination sites documented altered degradation of SNCA and the DNA-damage marker H2AX at the age of 18 months. Global brain transcriptome profiles and qPCR validation experiments identified many consistent transcriptional dysregulations already at the age of 6 weeks, which were absent from SMs. The observed downregulations for Dapk1, Dcaf17, Rab42 and the novel SNCA-marker Lect1 as well as the upregulations for Dctn5, Mrpl9, Tmem181a, Xaf1 and H2afx reflect changes in ubiquitination, mitochondrial/synaptic/microtubular/cell adhesion dynamics and DNA damage. Thus, our study confirmed that SNCA-triggered neurotoxicity is exacerbated by the absence of PINK1 and identified a novel molecular signature that is detectable early in the course of this double pathology.

Figure 1.

Survival curves of both SM versus DM. (A) While male FVB/N mice (starting n = 61) showed aggressive behavior at young age and inbreeding multimorbidity at advanced age resulting in constant high mortality across all ages independent from WT or mutant genotype, the homozygous PrPmtA line with overexpression of human A53T-SNCA (n = 77) resulted in some early mortality (significant P-value = 0.0002 in Mantel–Cox test, median survival reduced from 372 to 226 days). (B) WT 129/SvEv mice (n = 75) showed a moderate number of animal deaths at young age and slowly progressive mortality, as expected from an inbred line. The SM animals with Pink1^−/− (n = 104) exhibited an extended lifespan (significant P-value = 0.0159, median survival lengthened from 545 to 592 days). (C) WT-F1-hybrids with a 50 : 50 ratio of FVB/N and 129/SvEv background (n = 34) showed few deaths during young age owing to aggressive behavior, and a much increased lifespan as often seen after outbreeding. In comparison, DM animals (n = 51) showed some mortality early on, which increased in a steeply progressive manner after age 450 days (significant P-value < 0.0001, median survival reduced from 723 to 546 days).

Figure 2.

Spontaneous motor activity of DM and SM mice at the age of 3 months. Documentation of spontaneous open-field behavior as the most sensitive and reliable measure of abnormal movements in PD mouse models revealed highly significant marked reductions of horizontal activity, vertical activity and stereotype movements for the DM mice (n = 45) relative to their appropriate WT controls (n = 43), already at the age of 3 months (columns at the right side). These deficits were not observed in both SM lines versus their appropriate WT controls (n = 10 PrPmtA, 22 FVB/N, 16 Pink1^−/− and 16 129Sv/Ev).

Figure 3.

Immunohistochemistry in spinal cord at ages 15–17 months. Bright-field immunohistochemical stainings were performed in age-matched WT-F1-hybrids, PrPmtA and DM mouse (for Pink1^−/−, see Supplementary Material, Fig. S3 upper panels) spinal cord (focused on one anterior horn with ventral surface below and midline central canal to the right) in comparison. Protein aggregation was detected by antibodies against phospho-Serine129-alpha-synuclein (pSer129-SNCA), p62 or ubiquitin. In WT spinal cord at old age, no protein aggregates were detected. In PrPmtA, the pSer129-SNCA antibody detected few neuronal nuclei owing to the overexpression of SNCA (3D, open arrow). In DM, strong immunoreactivity throughout the gray substance in neuronal cytoplasm with extension along neurites can be observed. In high-magnification panels (panel row below), the granular and thread-like composition of the signals and the corkscrew-appearance of some immunopositive neurites become apparent (3J, black arrows), as well as the higher sensitivity of the pSer129-SNCA antibody for this pattern.

Figure 4.

Immunohistochemistry in midbrain at ages 15–17 months. Bright-field immunohistochemical stainings were performed in age-matched WT-F1-hybrids, PrPmtA and DM mouse (for Pink1^−/−, see Supplementary Material, Fig. S3 lower panels) midbrain (focused on a region between VTA and SNc, with ventral surface below and midline to the left) in comparison. Protein aggregation was detected by antibodies against phospho-Serine129-alpha-synuclein (pSer129-SNCA), p62 and ubiquitin. In WT spinal cord at old age, no protein aggregates were detected. In PrPmtA, the pSer129-SNCA antibody again detected few neuronal nuclei owing to the overexpression of SNCA in the basal brain (4D, open arrow). In DM, strong immunoreactivity throughout these areas in neuronal cytoplasm with extension along neurites can be observed. In high-magnification panels (panel row below), the granular and thread-like composition of the signals and the corkscrew-appearance of some immunopositive neurites become apparent (4J, black arrows), as well as the higher sensitivity of the pSer129-SNCA antibody for this pattern.

Figure 5.

Immunoblots in protein extracts from brain hemisphere at the age of 1 year. Quantitative comparison of proteins with soluble (RIPA fraction) or more insoluble (SDS fraction) distribution, in brain hemispheres from WT (F1-hybrids between FVB/N and 129/SvEv), both SM lines and DM mice. Altered abundance of p62 could not be detected in any condition; beta-actin was used as a loading control. (A) RIPA fraction shows the increased dosage of alpha-synuclein owing to the overexpression of human A53T-SNCA in the DM and the PrPmtA mice, with the appearance of phospho-Serine129-alpha-synuclein immunoreactivity (pSer129) in these two lines. (B) SDS fraction confirms the increased abundance of alpha-synuclein owing to the presence of human A53T-SNCA in these two lines and documents the appearance of additional pSer129 immunoreactive bands of higher molecular for both lines. The antibody against the alpha-synuclein C-term epitope detected a putative light increase of immunoreactivity in DM tissue.

Figure 6.

Transcriptome validation by qPCR in DM midbrain at the age of 6 months. (A) The global transcriptome survey was assessed by the independent technique qPCR in midbrain of additional animals (six DM versus six WT-F1-hybrids). Tissues from an earlier age were employed to define expression dysregulations that play a role at disease stages before the formation of aggregates. This demonstrated significant downregulations for Adora2b, Dapk1, Dcaf17, Homer1, Rab42 and significant upregulations for Dctn5, Mrpl9, Tmem181a, Xaf1 mRNA levels. The bar graphs show average 2^−ΔΔCt values ± SEM, relative to Tbp loading control, normalized to WT. These data identify factors with a role in early pathogenesis, which reflect anomalies in mitochondrial translation/apoptosis, ubiquitination, microtubular membrane transport, synaptic vesicles/receptors and DNA-damage repair. (B) On the basis of transcriptome bioinformatics identifying significant Lect1 transcript downregulation in the DM midbrain, qPCR was employed to demonstrate Lect1 levels to correlate inversely with the gain-of-function (left panel, five DM versus five F1-hybrid WT mice) (middle panel, four PrPmtA versus four FVB/N WT mice) and the loss-of-function (right panel, five Snca-KO versus five 129S6/SvEvTac WT mice) of alpha-synuclein in brain hemispheres of 6-month-old animals.