Fig 1.
Tamoxifen-injected iMfn2DA mice show impaired locomotion and DA neurodegeneration.
Control and iMfn2DA mice were analyzed at 3, 6 and 9 weeks after tamoxifen or vehicle injection. (A) Spontaneous motor activity (horizontal activity, vertical activity and total distance) was measured in open field. ***p< 0.001, n>14. (B-C) Representative images of TH-like immunoreactivity in section from midbrain (Scale bars: 100 μm) and striatum (Scale bars: 200 μm), right panels. Quantification of TH-positive DA neurons in the midbrain and TH-immunoreactive nerve terminals in the striatum, left panels. ***p< 0.001 n = 3. (D) Analysis of DA and HVA levels in the striatum. Data are shown as mean ± SD. ***p<0.001 n≥5.
Fig 2.
Loss of Mfn2 in adult DA neurons affects mitochondrial morphology and cristae structure.
Analyses were performed 3, 6, and 9 weeks after tamoxifen injection in control and iMfn2DA mice. (A) Representative confocal microscopy images of YFP-labelled mitochondria (green) in TH immunoreactive neurons (red) (Scale bars: 10 μm). (B) Quantification of aspect ratio and mitochondrial circularity from confocal images in TH+ DA neurons. AR data are shown as mean ± SD and circularity data as median of individual mitochondria. ***p< 0.00, n = 3 for each genotype. (C) Representative electron microscopy images of mitochondrial cristae structure (Scale bars: 1 μm).
Fig 3.
Tamoxifen-injected iMfn2DA mice exhibit impaired axonal mitochondrial transport and OXPHOS deficiency.
(A) MitoYFP-labelled mitochondria in TH+ nerve terminals of the striatum at 3, 6 and 9 weeks after tamoxifen injection (Scale bars: 10 μm). (B) Quantificantion of mitoYFP-labelled mitochondria total intensity in the striatum of control and tamoxifen-injected iMfn2DA mice at 3, 6 and 9 weeks after tamoxifen injection. Data are shown as mean ± SD. *p< 0.05, ***p< 0.001, n = 3 for genotype. (C) Cytochrome c oxidase and succinate dehydrogenase (COX/SDH) double-labelling enzyme histochemistry of the midbrain (Scale bars: 100 μm).
Fig 4.
Immune response and mtDNA depletion in tamoxifen-injected iMfn2DA mouse brains.
(A) Experimental workflow: vibratome sections were enzymatically digested and mechanically triturated resulting in a single cell suspension; the bulk of mitoYFP positive (mitoYFP+) cells and mitoYFP negative (mitoYFP-) cells were collected by FACS; samples were used for either mtDNA quantification or for RNA library preparation followed by RNAseq. (B) Hierarchical clustering analysis of RNAseq data from DA neurons isolated from n = 5 iMfn2DA and n = 6 control mice. (C) Heatmap showing that RNA levels (Reads Per Kilobase Million, RPKM) of genes encoding DA neuron markers (Th, Ddc, Scl6a3, and Aldh1a1) and transcription factors (Nr4a2, En1, Pitx3, Foxa1 and Foxa2) are highly enriched in mitoYFP+ samples. Data are shown as mean of the RPKM in control (n = 6), iMfn2DA (n = 5), and mitoYFP- (n = 3) mice. (D) Relative mtDNA levels (ND1/18S rRNA and ND6/18S rRNA) measured in FACS-sorted DA neurons. *p< 0.05, **p<0.01, n = 4. Data are shown as mean ± SD. (E) Gene ontology (GO) analysis showing the most dysregulated biological processes in FACS-sorted DA neurons isolated from iMfn2DA mice at 3 weeks after tamoxifen injection.
Fig 5.
Activation of glial cells surrounding DA neurons in in tamoxifen-injected iMfn2DA mice.
(A) Volcano plot displaying differential gene expression in iMfn2DA FACS-sorted DA neurons: the 439 differentially expressed genes are represented in black and blue. The genes involved in the immune response are represented in blue. (B) Heatmap showing the gene expression of NF-kB signaling pathway in DA neurons isolated from iMfn2DA and control mice. (C) RNA expression levels (RPKM) of the inflammation markers Tnf-α and IL1β in mitoYFP+ cells from iMfn2DA and control mice. Data are shown as mean ± SD. (D-E). Representative confocal microscopy image of control and iMfn2DA mouse brains at 3, 6, and 9 weeks after injection. DA neurons were labelled with an antibody against TH (red) and the brain sections were additionally labelled with antibody against IBA1 (green) in panel (D) or GFAP (green) in panel (E) (Scale bars: 100 μm). (F) IBA1 and GFAP immunoreactivities quantified as total intensity in the stained areas of the midbrain from control and tamoxifen-injected iMfn2DA mice at 3, 6 and 9 weeks after injection. Data are shown as mean ± SD. n≥3.*p< 0.05, **p<0.01. (G) Log2(FC) of RNA expression of genes involved in signaling pathways that potentially could drive neuroinflammation in iMfn2DA mice.