PF-06447475 Molecule Attenuates the Neuropathology of Familial Alzheimer's and Coexistent Parkinson's Disease Markers in PSEN1 I416T Dopaminergic-like Neurons

Abstract

Familial Alzheimer's disease (FAD) is a complex multifactorial disorder clinically characterized by cognitive impairment and memory loss. Pathologically, FAD is characterized by intracellular accumulation of the protein fragment Aβ42 (iAβ), hyperphosphorylated microtubule-associated protein TAU (p-TAU), and extensive degeneration of basal forebrain cholinergic neurons of the nucleus basalis of Meynert (NbM) and the medial septal nucleus (MSN), mainly caused by mutations in the amyloid precursor protein (APP), presenilin 1 (PSEN1), and PSEN2 gene. Since the dopaminergic system may contribute to FAD symptoms, alterations in the nigro-hippocampal pathway may be associated with cognitive impairment in FAD. Interestingly, p-α-synuclein (p-α-Syn), Aβ, and p-TAU have been found to coexist in vulnerable regions of postmortem AD brains. However, the mechanism by which Aβ, p-TAU, and α-Syn coexist in DAergic neurons in AD brains has not been determined. We generated PSEN1 I416T dopaminergic-like neurons (DALNs) from I416T menstrual stromal cells (MenSCs) in NeuroForsk 2.0 medium for 7 days and then cultured them in minimal culture medium (MCm) for another 4 days. On day 11, DALNs were analyzed for molecular and pathological markers by flow cytometry and fluorescence microscopy. We found that mutant DALNs showed increased accumulation of iAβ as well as increased phosphorylation of TAU at S202/T205 compared to WT DALNs. Thus, mutant DALNs exhibited typical pathological hallmarks of Alzheimer's disease. Furthermore, PSEN1 I416T DALNs showed concomitant signs of OS as evidenced by the appearance of oxidized sensor protein DJ-1 (i.e., DJ-1C106-SO₃) and apoptotic markers TP53, pS63-c-JUN, PUMA, and cleavage caspase 3 (CC3). Notably, these DALNs exhibited PD-associated proteins such as intracellular accumulation of α-Syn (detected as aggregates of pS129-α-Syn) and phosphorylation of LRRK2 kinase at residue S935. In addition, mutant DALNs showed a 17.16- and 6.17-fold decrease in DA-induced Ca²⁺ flux, compared to WT DALNs. These observations suggest that iAβ and p-TAU, together with p-α-Syn, and p-LRRK2 kinase, may damage DAergic neurons and thereby contribute to the exacerbation of neuropathologic processes in FAD. Remarkably, the LRRK2 inhibitor PF-06447475 (PF-475) significantly reversed PSEN1 I416T-induced neuropathological markers in DAergic neurons. PF-465 inhibitor reduced iAβ, oxDJ-1C106-SO₃, and p-TAU. In addition, this inhibitor reduced pS935-LRRK2, pS129-αSYN, pS63-c-JUN, and CC3. We conclude that the observed neuroprotective effects of PF-475 are due to direct inhibition of LRRK2 activity and that the LRRK2 protein is upstream of the molecular cascade of apoptosis and proteinopathy. Our results suggest that PF-475 is an effective neuroprotective agent against endogenous PSEN1 I416T-induced neurotoxicity in DALNs coexisting with Parkinson's disease markers. Therefore, PF-475 may be of great therapeutic value in FAD.

Keywords: Alzheimer’s disease; I416T; PF-06447475; apoptosis; dopaminergic neurons; menstrual mesenchymal stromal cells; mutation; presenilin.

Figure 1

PSEN1 I416T MenSCs transdifferentiated into dopaminergic-like neurons (DALNs) express dopaminergic markers Tyrosine hydroxylase (TH) and dopamine transporter (DAT). Representative density plot figures showing TH/DAT double-positive population in WT PSEN1 and PSEN1 I416T DALNs after (A) 7 and (B) 11 days of transdifferentiation. (C) Percentage of TH/DAT double-positive population. (D–S) Representative fluorescence microscopy photographs showing dopaminergic markers DAT (red fluorescence) and TH (green fluorescence). The nuclei were stained with Hoechst 33,342 (blue). (T,U) Mean fluorescence intensity (MFI) quantification of images of DAT (red fluorescence) and TH (green fluorescence). The figures represent 1 out of 3 independent experiments. One-way ANOVA followed by Tukey’s test (** p < 0.002 (C), *** p < 0.001 (C)) or post hoc test Bonferroni (* p < 0.033 (T), ** p < 0.002 (T)). The data are presented as the mean ± SD of three independent experiments (n = 3). Bars in blue represent WT cells. Bars in red represent mutant cells. Image magnification, 200×.

Figure 1

PSEN1 I416T MenSCs transdifferentiated into dopaminergic-like neurons (DALNs) express dopaminergic markers Tyrosine hydroxylase (TH) and dopamine transporter (DAT). Representative density plot figures showing TH/DAT double-positive population in WT PSEN1 and PSEN1 I416T DALNs after (A) 7 and (B) 11 days of transdifferentiation. (C) Percentage of TH/DAT double-positive population. (D–S) Representative fluorescence microscopy photographs showing dopaminergic markers DAT (red fluorescence) and TH (green fluorescence). The nuclei were stained with Hoechst 33,342 (blue). (T,U) Mean fluorescence intensity (MFI) quantification of images of DAT (red fluorescence) and TH (green fluorescence). The figures represent 1 out of 3 independent experiments. One-way ANOVA followed by Tukey’s test (** p < 0.002 (C), *** p < 0.001 (C)) or post hoc test Bonferroni (* p < 0.033 (T), ** p < 0.002 (T)). The data are presented as the mean ± SD of three independent experiments (n = 3). Bars in blue represent WT cells. Bars in red represent mutant cells. Image magnification, 200×.

Figure 2

PSEN1 I416T DALNs express abundant iAβ and shows oxidation of DJ-1 after 11 days of transdifferentiation. Representative flow cytometry contour plots show intracellular amyloid-beta (iAβ) in (A) WT PSEN1 and (B) PSEN1 I416T DALNs. (C) Percentage of intracellular amyloid-beta (iAβ). Representative flow cytometry contour plots show oxidized protein DJ-1 (DJ-1 C106-SO₃) in (D) WT PSEN1 and (E) PSEN1 I416T DALNs. (F) Percentage of oxDJ-1 (DJ-1 C106-SO₃). WT PSEN1 and PSEN1 I416T DALNs were stained with primary antibodies against iAβ (G,H), oxDJ-1 (I,J), Hoechst (K,L), and merged (M,N). Positive red fluorescence reflects the cytoplasmic presence of iAβ protein, positive green fluorescence reflects the cytoplasmic presence of oxDJ-1, and positive blue fluorescence reflects nuclei. Quantification of the (O) iAβ and (P) oxDJ-1 mean fluorescence intensity (MFI). The figures represent one out of three independent experiments. One-way ANOVA followed by Tukey’s test. The data are expressed as the mean ± SD of three independent experiments (n = 3); * p < 0.033 (P), ** p < 0.01. (C,F), ** p < 0.002 (O). Bars in blue represent WT cells. Bars in red represent mutant cells. Image magnification, 200×.

Figure 3

PSEN1 I416T DALNs shows high levels of phosphorylated protein TAU (pS202/T205-TAU) after 11 days of transdifferentiation. Representative flow cytometry contour plots show intracellular pS202/T205-TAU in (A) WT PSEN1 and (B) PSEN1 I416T DALNs. Representative flow cytometry contour plots show total TAU protein (t-TAU) in (A’) WT PSEN1 and (B’) PSEN1 I416T DALNs. (C) Percentage of p-TAU/t-TAU ratio. WT PSEN1 and PSEN1 I416T DALNs were stained with primary antibodies against pS202/T205-TAU (D,E), t-TAU (F,G), Hoechst (H,I), and merged (J,K). Positive red fluorescence reflects the cytoplasmic presence of pS202/T205-TAU protein, positive green fluorescence reflects the cytoplasmic presence of t-TAU, and positive blue fluorescence reflects nuclei. (L) Quantification of the pS202/T205-TAU/t-TAU ratio mean fluorescence intensity (MFI). The figures represent one out of three independent experiments. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SD of three independent experiments (n = 3); * p < 0.05, ** p < 0.002. Bars in blue represent WT cells. Bars in red represent mutant cells. Image magnification, 200×.

Figure 4

PSEN1 I416T DALNs increase levels of phosphorylated protein LRRK2 (pS935-LRRK2) after 11 days of transdifferentiation. Representative flow cytometry contour plots show intracellular pS935-LRRK2 in (A) WT PSEN1 and (B) PSEN1 I416T DALNs. Representative flow cytometry contour plots show total LRRK2 protein (t-LRRK2) in (A’) WT PSEN1 and (B’) PSEN1 I416T DALNs. (C) Percentage of pS935-LRRK2/t-LRRK2 ratio. WT PSEN1 and PSEN1 I416T DALNs were stained with primary antibodies against pS935-LRRK2 (D,E), t-LRRK2 (F,G), Hoechst (H,I), and merged (J,K). Positive green fluorescence reflects the cytoplasmic presence of pS935-LRRK2 protein, positive red fluorescence reflects the cytoplasmic presence of t-LRRK2, and positive blue fluorescence reflects nuclei. (L) Quantification of the pS935-LRRK2/t-LRRK2 ratio mean fluorescence intensity (MFI). The figures represent one out of three independent experiments. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SD of three independent experiments (n = 3); * p < 0.033. Bars in blue represent WT cells. Bars in red represent mutant cells. Image magnification, 200×.

Figure 5

PSEN1 I416T DALNs increase levels of phosphorylated protein alpha synuclein (pS129-αSYN) after 11 days of transdifferentiation. Representative flow cytometry contour plots show intracellular pS129-αSYN in (A) WT PSEN1 and (B) PSEN1 I416T DALNs. Representative flow cytometry contour plots show total alpha synuclein protein (t-αSYN) in (A’) WT PSEN1 and (B’) PSEN1 I416T DALNs. (C) Percentage of pS129-αSYN/t-αSYN ratio. WT PSEN1 and PSEN1 I416T DALNs were stained with primary antibodies against pS129-αSYN (D,E), t-αSYN (F,G), Hoechst (H,I), and merged (J,K). Positive green fluorescence reflects the cytoplasmic presence of pS129-αSYN, positive red fluorescence reflects the cytoplasmic presence of t-αSYN protein, and positive blue fluorescence reflects nuclei. (L) Quantification of the pS129-αSYN/t-αSYN ratio mean fluorescence intensity (MFI). The figures represent one out of three independent experiments. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SD of three independent experiments (n = 3); ** p < 0.002, *** p < 0.001. Bars in blue represent WT cells. Bars in red represent mutant cells. Image magnification, 200×.

Figure 6

PSEN1 I416T DALNs increase levels of TP53 and P53 upregulated modulator of apoptosis (PUMA) after 11 days of transdifferentiation. Representative flow cytometry contour plots show TP53 protein level in (A) WT PSEN1 and (B) PSEN1 I416T DALNs. (C) Percentage of TP53 expression. Representative flow cytometry contour plots show P53 upregulated modulator of apoptosis (PUMA) protein in (D) WT PSEN1 and (E) PSEN1 I416T DALNs. (F) Percentage of PUMA. In addition, WT PSEN1 and PSEN1 I416T DALNs were stained with primary antibodies against TP53 (G,H), PUMA (I,J), Hoechst (K,L), and merged (M,N). Positive green fluorescence reflects the cytoplasmic presence of TP53 protein, positive red fluorescence reflects the cytoplasmic presence of PUMA, and positive blue fluorescence reflects nuclei. Quantification of the of (O) TP53 and (P) PUMA mean fluorescence intensity (MFI) in WT PSEN1 and PSEN1 I416T DALNs. The figures represent one out of three independent experiments. The data are presented as the mean ± SD of three independent experiments (n = 3). One-way ANOVA followed by Tukey’s test. The data are expressed as the mean ± SD; * p < 0.033, **** p < 0.0001. Bars in blue represent WT cells. Bars in red represent mutant cells. Image magnification, 200×.

Figure 7

PSEN1 I416T DALNs increase levels of c-JUN phosphorylated at serine 63 (pS63-c-JUN) and cleaved caspase-3 (CC3) after 11 days of transdifferentiation. Representative flow cytometry contour plots show pS63-c-JUN protein level in (A) WT PSEN1 and (B) PSEN1 I416T DALNs. (C) Percentage of pS63-c-JUN expression. Representative flow cytometry contour plots show cleaved caspase-3 (CC3) protein in (D) WT PSEN1 and (E) PSEN1 I416T DALNs. (F) Percentage of CC3. WT PSEN1 and PSEN1 I416T DALNs were stained with primary antibodies against pS63-c-JUN (G,H), CC3 (I,J), Hoechst (K,L), and merged (M,N). Positive red fluorescence reflects the cytoplasmic presence of pS63-c-JUN, positive green fluorescence reflects the cytoplasmic presence of CC3, and positive blue fluorescence reflects nuclei. Quantification of the of (O) pS63-c-JUN and (P) CC3 mean fluorescence intensity (MFI). The figures represent one out of three independent experiments. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SD of three independent experiments (n = 3); * p < 0.033. ** p < 0.02, *** p < 0.0002. Bars in blue represent WT cells. Bars in red represent mutant cells. Image magnification, 200×.

Figure 8

PSEN1 I416T DALNs exhibit dysfunctional Ca²⁺ influx response to dopamine (DA) at day 11 of transdifferentiation. Representative time-lapse images (0, 10, 20, 30, 40, 60, 70, 80, 90, 100, 110, 120 s) of Ca²⁺ fluorescence in response to DA (arrow) in (A) WT PSEN1 and (B) PSEN1 I416T DALNs. Color contrast indicates fluorescence intensity: dark blue < light blue < green < red. (C) Graph showing ΔF/F and (D) area under the curve (AUC). The figures represent 1 out of 3 independent experiments. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SD of three independent experiments (n = 3); * p < 0.033,. Bars in blue represent WT cells. Bars in red represent mutant cells. Image magnification, 200×.

Figure 9

The LRRK2 inhibitor PF-06447475 (PF-475) blunts the expression of iAβ and oxidation of DJ-1 in PSEN1 I416T DALNs. Representative flow cytometry contour plots show intracellular amyloid-beta (iAβ) in (A,C) untreated or (B,D) treated cells with PF-06447475 LRRK2 inhibitor (PF-475). (E) Percentage of intracellular amyloid-beta (iAβ). (F–I) Representative flow cytometry contour plots show oxidized protein DJ-1 (DJ-1 C106-SO₃). (J) Percentage of oxDJ-1 (DJ-1 C106-SO₃). One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SD of three independent experiments (n = 3); * p < 0.033, ** p < 0.002.

Figure 10

The LRRK2 inhibitor PF-06447475 reduces the levels of phosphorylated protein TAU (pS202/T205-TAU) in PSEN1 I416T DALNs. Representative flow cytometry contour plots show intracellular pS202/T205-TAU in (A,C) untreated or (B,D) treated cells with PF-475. Representative flow cytometry contour plots show total-TAU protein in (A’,C’) untreated or (B’–D’) treated cells with PF-475. (E) Percentage of p-TAU/t-TAU ratio in untreated or treated cells with PF-475. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SD of three independent experiments (n = 3); * p < 0.033, and ** p < 0.002.

Figure 11

The LRRK2 inhibitor PF-06447475 reduces the levels of phosphorylated protein LRRK2 (pS935-LRRK2) in PSEN1 I416T DALNs. Representative flow cytometry contour plots show intracellular pS935-LRRK2 in (A,C) untreated or (B,D) treated cells with PF-475. Representative flow cytometry contour plots show total LRRK2 protein (A’,C’) untreated or (B’,D’) treated with PF-475. (E) Percentage of pS935-LRRK2/t-LRRK2 ratio untreated or treated cells with PF-475. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SD of three independent experiments (n = 3); * p< 0.033.

Figure 12

The LRRK2 inhibitor PF-06447475 reduces the levels of phosphorylated protein pS129-α-Syn in PSEN1 I416T DALNs. Representative flow cytometry contour plots show intracellular pS129-αSYN in (A,C) untreated or (B,D) treated with PF-475. Representative flow cytometry contour plots show total alpha synuclein protein (t-αSYN) in (A’,C’) untreated or (B’,D’) treated cells with PF-475. (E) Percentage of pS129-αSYN/t-αSYN ratio untreated or treated cells with PF-475. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SD of three independent experiments (n = 3), * p < 0.033.

Figure 13

The LRRK2 inhibitor PF-06447475 reduces the levels of c-JUN phosphorylated at serine 63 (pS63-c-JUN) and cleaved caspase-3 (CC3) in PSEN1 I416T DALNs. Representative flow cytometry contour plots show pS63-c-JUN protein level in (A,C) untreated or (B,D) treated with PF-475. (E) Percentage of pS63-c-JUN expression in untreated or treated cells with PF-475. Representative flow cytometry contour plots show cleaved caspase-3 (CC3) protein in (F,H) untreated or (G,I) treated cells with PF-475. (J) Percentage of CC3 in untreated or treated cells with PF-475. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SD of three independent experiments (n = 3); * p < 0.034, and ** p < 0.008.

Figure 14

Schematic model of cell signaling induced by PSEN1/γ-secretase and effect of LRRK2 inhibitor PF475 in PSEN1 I416T DALNs. (A) PSEN1/γ-secretase signaling mechanism. The PSEN1+/- gene encodes a 467 aa catalytic and non-catalytic PSEN1 I146T protein that alters the metabolism of the type I transmembrane protein APP by a mechanism that is not fully understood, resulting in the overproduction of iAβ (1). The iAβ peptide acts on mitochondrial complex I (NADH: ubiquinone oxidoreductase), complex III (Q-cytochrome c oxidoreductase), or complex IV (cytochrome c oxidase) (2), interfering with the electron transport chain and simultaneously generating the anion superoxide (O2^.-) and hydrogen peroxide (H₂O₂, 3). The latter is not only capable of oxidizing the stress sensor protein DJ-1Cys106-SH to DJ-1Cys106-SO₃ (4), but also directly activates leucine-rich repeat kinase 2 (LRRK2) kinase directly through autophosphorylation or indirectly through kinase (e.g., via IKK) signaling (5). Once LRRK2 is phosphorylated at S935, the active pS935-LRRK2 kinase activates at least six major targets: (i) alpha-synuclein (αSyn) at the pathological residue S129 (6); (ii) inactivates protein peroxiredoxin 3 (PRDX3, 7), preventing H₂O₂ catalysis; (iii) activates the mitochondrial fission protein DLP-1 (dynamin-like protein 1, 8), which together with the fission protein-1 (Fis-1) receptor induces loss of mitochondrial potential (ΔΨm, 9); (iv) pS935-LRRK2 indirectly phosphorylates JNK (10), which, in turn, represses nuclear factor erythroid 2-related factor 2 (Nrf2)-associated expression of antioxidant proteins (11), phosphorylates the protein TAU (12), and activates c-JUN (13); (v) activates the transcription factors TP53 (14). Both pS63-c-Jun and TP53 factors transcribe PUMA (15), a Bcl-2-only protein involved in further mitochondrial depolarization (9). Impairment of mitochondrial potential leads to the release of apoptogenic proteins (e.g., cytochrome C), resulting in the production of cleaved caspase 3 (16), which is responsible for chromatin condensation and DNA fragmentation (17), a typical apoptotic feature in PSEN1 I416T DALNs. (B) Upon exposure to PF475, p-LRRK2-associated apoptosis signaling is drastically reduced (faint black line). As a result, PRDX3 activity is restored (7), Nfr2-induced antioxidant protein expression is increased (11), Aβ-induced ROS/H₂O₂ is decreased (2,3), and ΔΨm is increased (9). These actions result in global neuronal recovery and survival of PSEN1 I416T DALNs (18).

Figure 14

Schematic model of cell signaling induced by PSEN1/γ-secretase and effect of LRRK2 inhibitor PF475 in PSEN1 I416T DALNs. (A) PSEN1/γ-secretase signaling mechanism. The PSEN1+/- gene encodes a 467 aa catalytic and non-catalytic PSEN1 I146T protein that alters the metabolism of the type I transmembrane protein APP by a mechanism that is not fully understood, resulting in the overproduction of iAβ (1). The iAβ peptide acts on mitochondrial complex I (NADH: ubiquinone oxidoreductase), complex III (Q-cytochrome c oxidoreductase), or complex IV (cytochrome c oxidase) (2), interfering with the electron transport chain and simultaneously generating the anion superoxide (O2^.-) and hydrogen peroxide (H₂O₂, 3). The latter is not only capable of oxidizing the stress sensor protein DJ-1Cys106-SH to DJ-1Cys106-SO₃ (4), but also directly activates leucine-rich repeat kinase 2 (LRRK2) kinase directly through autophosphorylation or indirectly through kinase (e.g., via IKK) signaling (5). Once LRRK2 is phosphorylated at S935, the active pS935-LRRK2 kinase activates at least six major targets: (i) alpha-synuclein (αSyn) at the pathological residue S129 (6); (ii) inactivates protein peroxiredoxin 3 (PRDX3, 7), preventing H₂O₂ catalysis; (iii) activates the mitochondrial fission protein DLP-1 (dynamin-like protein 1, 8), which together with the fission protein-1 (Fis-1) receptor induces loss of mitochondrial potential (ΔΨm, 9); (iv) pS935-LRRK2 indirectly phosphorylates JNK (10), which, in turn, represses nuclear factor erythroid 2-related factor 2 (Nrf2)-associated expression of antioxidant proteins (11), phosphorylates the protein TAU (12), and activates c-JUN (13); (v) activates the transcription factors TP53 (14). Both pS63-c-Jun and TP53 factors transcribe PUMA (15), a Bcl-2-only protein involved in further mitochondrial depolarization (9). Impairment of mitochondrial potential leads to the release of apoptogenic proteins (e.g., cytochrome C), resulting in the production of cleaved caspase 3 (16), which is responsible for chromatin condensation and DNA fragmentation (17), a typical apoptotic feature in PSEN1 I416T DALNs. (B) Upon exposure to PF475, p-LRRK2-associated apoptosis signaling is drastically reduced (faint black line). As a result, PRDX3 activity is restored (7), Nfr2-induced antioxidant protein expression is increased (11), Aβ-induced ROS/H₂O₂ is decreased (2,3), and ΔΨm is increased (9). These actions result in global neuronal recovery and survival of PSEN1 I416T DALNs (18).