FKBP51 inhibition ameliorates neurodegeneration and motor dysfunction in the neuromelanin-SNCA mouse model of Parkinson's disease

Abstract

Parkinson's disease (PD) is characterized by the loss of neuromelanin (NM)-containing dopaminergic (DA) neurons in the substantia nigra (SN) pars compacta (SNpc) and the buildup of α-synuclein (α-syn) inclusions, called Lewy bodies. To investigate the roles of NM and α-syn in DA neuron degeneration, we modeled PD by inducing NM accumulation in a humanized α-syn mouse model (Snca^-; PAC-Tg(SNCA^WT)) via the expression of human tyrosinase in the SN. We found that this mouse strain develops naturally progressive motor dysfunction and dopaminergic neuronal loss in the SN with aging. Upon tyrosinase injection, NM-containing neurons developed p62 and ubiquitin inclusions. Furthermore, the upregulation of genes associated with microglial activation in the midbrain indicated a role of pro-inflammatory factors in neurodegeneration. Midbrain RNA sequencing confirmed the microglial response and identified Fkbp5 as one of the more dysregulated genes. Next, we showed that FKBP51(51 kDa) was significantly upregulated with aging and in PD human brains. Pharmacological treatment with SAFit2, a potent FKBP51 inhibitor, led to a reduction in ubiquitin-positive inclusions, prevention of neurodegeneration in the SNpc, and improved motor function in NM-SNCAWT mice. These results highlight the critical role of FKBP51 in PD and propose SAFit2 as a promising therapeutic candidate for reducing neurodegeneration in PD.

Keywords: FKBP5; PAC-Tg-SNCAWT; Parkinson's disease; SAFit2; Snca-; aging; neuroinflammation; neuromelanin; neuroprotection; synuclein.

Graphical abstract

Figure 1

SNCAWT mice showed motor impairments and dopaminergic neuronal loss with aging (A) Motor function in SNCAWT mice was assessed between 3 and 22 months of age (n = 10–12 per group). Motor coordination was analyzed measuring the latency to fall in accelerating rotarod test (rotarod test), the time spent to turn down and descend the pole (pole test), and the time that the mice took to recover the position of the upper paws (catalepsy test). (B) Representative photomicrographs showing TH⁺ dopaminergic neurons in the midbrain and quantification of TH⁺ cells in the midbrain of 3-, 13-, and 22-month-old SNCAWT mice (n = 6 per group). (C) Representative photomicrographs showing TH immunoreactivity in the striatum and quantification of TH⁺ fibers measured by optical densitometry in 3-, 13-, and 22-month-old SNCAWT mice (n = 4-5 per group). Magnification bar: (B) 0,7 mm, (C) 1 mm. Data are presented as mean ± standard error of the mean (SEM). One-way ANOVA ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 2

AAV9-mediated hTyr overexpression in the SN induces motor deficits, nigrostriatal neurodegeneration, and LB-like formation in SNCAWT mice (A) Schedule of motor tests, AAV9 administration, and procedures performed in 3- and 13-month-old SNCAWT mice. (B) Motor behavior was evaluated using the rotarod, pole, and catalepsy tests in 3- and 13-month-old SNCAWT mice during the fourth week after either AAV-hTyr or AAV-null injection (n = 6–10 per group). (C) Representative photomicrographs showing TH⁺ dopaminergic neurons in the midbrain. Quantification of TH⁺ cells in the midbrain of 3- and 13-month-old mice sacrificed 4 weeks after either AAV-hTyr or AAV-null injection (n = 5–6 per group). (D) Representative photomicrographs showing TH immunoreactivity in the striatum. Quantification of TH⁺ fibers in 3- and 13-month-old SNCAWT mice sacrificed 5 weeks after AAV-hTyr or AAV-null injection measured by optical densitometry. (E) Representative images of Lewy body-like formation in NM-laden dopaminergic neurons in 3- and 13-month-old AAV9-hTyr-injected SNCAWT mice detected by multiple IHC with anti-TH/p62 antibodies, anti-TH/ubiquitin antibodies, anti-p62/ubiquitin, and anti-TH/p62/ubiquitin antibodies. (F) Representative images of ubiquitin (red) and P62 (green) immunostaining in the midbrain of 3- and 13-month-old SNCAWT mice injected with AAV9-hTyr. The square indicates magnified images (a and b). Data are presented as mean ± SEM. One-way ANOVA followed by Tukey post hoc test was used. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. Magnification bar: (C) 0,7 mm, (D) 1 mm, (E) 0.01 mm, (F) 0.2 mm, and 0.02 mm in sets with high magnification.

Figure 3

AAV9-mediated hTyr overexpression in the SN induces a robust inflammatory response (A) Representative images of Iba1 immunostaining in the midbrain of 3- and 13-month-old SNCAWT mice injected with AAV9-null or AAV9-hTyr. Magnification bar: 0.2 mm and 0.02 mm in sets. (B) Quantification of Iba1⁺ cells in the midbrain of 3- and 13-month-old mice sacrificed 4 weeks after AAV9-hTyr or AAV9-null injection (n = 5–6 per group). (C) Representative images of Iba immunofluorescence (green) showing the microglial phagocytosis of NM in the midbrain of AAV9-hTyr-injected mice. Magnification bar: 0.01 mm. (D) Representative magnified images of NM-containing dopaminergic neurons from 3- and 13-month-old AAV9-hTyr-injected SNCAWT mice immunostained with Iba1 (yellow), TH (red), and p62 (green). Magnification bar: 0.01 mm (E) qPCR assay showed the changes in expression of neuroinflammatory-related genes in the mesencephalon of the 3- and 13-month-old AAV-hTyr mice compared to AAV-null mice (∗p < 0.05, ∗∗p < 0.01). Data are presented as mean ± SEM. Unpaired two-tailed Student’s t test was used to compare two groups. One-way ANOVA followed by Tukey post hoc test to compare more than two groups. ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 4

RNA-seq analysis indicates that an exacerbated microglial activation may contribute to chronic neurodegeneration in the PD model of NM accumulation (A) Volcano plot of RNA-seq data showing up-and downregulated genes (DEGs) as red and green dots, respectively (p < 0.01 and log2 fold change >0.5). Non-DEGs are represented as gray dots. Arrows show genes of interest with the lowest p value and/or highest FC. (B) GO Biological Process (BP) enrichment analysis of the DEGs in the mesencephalon of AAV-hTyr-injected mice compared to AAV-null-injected mice. (C) Analysis of RNA-seq data using CIBERSOTx program to quantify the proportion of cells in each sample. A significant increase of the percentage of microglial (mMgl) cells and decrease of dopaminergic neurons (mDA) are detected in AAV-hTyr samples. mRgl1-3, radial glia type 1–3; mEndo, endothelial; mPeri, pericyte; mEpend, ependymal; mGaba1a, 1b, and 2, GABAergic neurons 1a, 1b, and 2; mSert, serotonergic neurons; mNbL1-2, lateral neuroblasts; mNbML1-5 mediolateral neuroblasts; mRN, red nucleus, mOMTN OMTN; mNProg, neuronal progenitor; mNbM, medial neuroblast; mNbDA, DA neuroblast; mDA0-2, DA neurons 1 and 2. (D) Volcano plot of RNA-seq data analyzed using Seurat that allows the deconvolution of bulk data. The color-coding of the genes corresponds to the cell type in (C).

Figure 5

FKBP51 levels increase with aging in the midbrain of SNCAWT mice and in dopaminergic and microglial cells in AAV9-hTyr-injected mice (A) Representative images of FKBP51/TH/DAPI immunofluorescence in the SN of 3- and 13-month-old mice injected with AAV-hTyr or AAV-null accompanied with FKBP51 fluorescence intensity (FI) quantification (n = 7–9 per group). (B) Representative images of FKBP51/IBA1/DAPI immunofluorescence in the SN of 3- and 13-month-old mice injected with AAV-hTyr or AAV-null accompanied with FKBP51 FI quantification (n = 7–9 per group). (C) Representative images of FKBP51/HSP90/DAPI immunofluorescence in the SN of SNCAWT mice injected with AAV-hTyr or AAV-null accompanied with Hsp90 FI quantification (n = 7–9 per group). Data are presented as mean ± SEM. One-way ANOVA followed by Tukey post hoc test was used. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. Magnification bar: 0.01 mm.

Figure 6

FKBP51 levels increase in neurons and microglial cells of PD patients (A) Annotated uniform manifold approximation and projection (UMAP) of the single-cell RNA-seq GEO: GSE243639 dataset. (B) Feature plot of FKBP5 expression levels in the GEO: GSE243639 dataset. (C) Violin plot of FKBP5 expression in the brains of healthy individuals vs. PD patients across all cell types (FC = 1.71; p < 0.0001). (D) Violin plot of FKBP5 expression in the brains of healthy individuals vs. PD patients in microglia cells (FC = 1.87; p < 0.0001). (E) FKBP51 expression analyzed in postmortem human midbrain samples from PD and healthy age-matched controls (n = 5 per condition). Representative western blot images are shown. (F) Representative images of FKBP51/IBA1/DAPI immunofluorescence in postmortem human midbrain samples obtained from PD patients and healthy age-matched individuals. (G) Representative images of FKBP51 (green) immunostaining in NM neurons of postmortem human midbrain samples from PD patients and healthy age-matched individuals. Magnification bar: (F) 0.01 mm; (G) 0.05 mm and 0.02 mm in sets with high magnification.

Figure 7

FKBP51 inhibition protects against nigrostriatal neurodegeneration linked to a reduce ubiquitin and α-synuclein-positive inclusions at the NM-containing neurons within the SNpc (A) Schedule of motor tests, AAV-hTyr administration, and SAFit2 treatment (20 mg/kg) or vehicle performed in 3-month-old SNCAWT mice. (B) Motor behavior was evaluated using the rotarod, pole, and catalepsy tests in SNCAWT mice during the fourth week after AAV-hTyr injection (n = 6–8 per group) in mice treated with SAFit2 or vehicle for 2 weeks. (C) Representative photomicrographs showing TH⁺ dopaminergic neurons in the midbrain. Quantification of TH⁺ cells in the midbrain of AAV-hTyr-injected mice treated with SAFit2 or vehicle and sacrificed 4 weeks after AAV-hTyr injection (n = 6 per group). (D) Representative photomicrographs showing TH-immunoreactivity in the striatum. Quantification of TH⁺ fibers in the striatum of AAV-hTyr-injected mice treated with SAFit2 or vehicle and sacrificed 4 weeks after AAV-hTyr injection and measured by optical densitometry. (E) Representative images of Iba1 immunostaining and quantification of Iba1⁺ cells in the midbrain of 3-month-old SNCAWT mice injected with AAV9-hTyr and treated with SAFit2 or vehicle for 12 days (n = 4–5/group). (F) Representative images of FKBP51/TH/DAPI immunofluorescence in the SN of AAV-hTyr-injected mice treated with SAFit2 or vehicle and sacrificed 4 weeks after AAV-hTyr injection accompanied with FKBP51 FI quantification. (G) Representative images of FKBP51/IBA1/DAPI immunofluorescence in the SN of AAV-hTyr-injected mice treated with SAFit2 or vehicle and sacrificed 4 weeks after AAV-hTyr injection accompanied with FKBP51 fluorescence intensity quantification. Data are presented as mean ± SEM. One-way ANOVA followed by Tukey post hoc test was used. ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. Magnification bar: (C and D) 1 mm, (E) 0.02 mm, (F and G) 0.01 mm.

Figure 8

SAFit2 reduced FKBP51 expression in the midbrain of SNCAWT mice injected with AAV9-hTyr (A) Representative images of FKBP51/HSP90/DAPI immunofluorescence in the SN of AAV-hTyr-injected mice treated with SAFit2 or vehicle and sacrificed 4 weeks after AAV-hTyr injection accompanied with Hs90 and FKBP51 FI correlation. (B) Representative images of the midbrain of AAV-hTyr-injected mice treated with SAFit2 or vehicle and immunostained with ubiquitin (red) and quantification of the corresponding intracellular optical density (n = 100 neurons/condition). (C) Representative images of the midbrain of AAV-hTyr-injected mice treated with SAFit2 or vehicle and immunostained with α-synuclein (red) and quantification of the corresponding intracellular optical density (n = 200 neurons/condition). (D) Correlation between intracellular optical density of ubiquitin and α-synuclein immunostaining in SN dopaminergic neurons of AAV-hTyr-injected mice treated with SAFit2 or vehicle and sacrificed 4 weeks after AAV-hTyr injection (n = 100 neurons/condition). (E) Representative images of Iba1 (yellow) and ubiquitin (red) immunostaining in the midbrain of 3-month-old SNCAWT mice injected with AAV9-hTyr and treated with SAFit2 or vehicle for 2 weeks. Data are presented as mean ± SEM. One-way ANOVA followed by Tukey post hoc test was used. ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. Magnification bar: (A) 0.01 mm, (B and C) 0.2 mm and 0.01 in sets with high magnification, (E) 0.02 mm.