Aberrant DJ-1 expression underlies L-type calcium channel hypoactivity in dendrites in tuberous sclerosis complex and Alzheimer's disease

Abstract

L-type voltage-gated calcium (Ca²⁺) channels (L-VGCC) dysfunction is implicated in several neurological and psychiatric diseases. While a popular therapeutic target, it is unknown whether molecular mechanisms leading to disrupted L-VGCC across neurodegenerative disorders are conserved. Importantly, L-VGCC integrate synaptic signals to facilitate a plethora of cellular mechanisms; however, mechanisms that regulate L-VGCC channel density and subcellular compartmentalization are understudied. Herein, we report that in disease models with overactive mammalian target of rapamycin complex 1 (mTORC1) signaling (or mTORopathies), deficits in dendritic L-VGCC activity are associated with increased expression of the RNA-binding protein (RBP) Parkinsonism-associated deglycase (DJ-1). DJ-1 binds the mRNA coding for the alpha and auxiliary Ca²⁺ channel subunits Ca_V1.2 and α2δ2, and represses their mRNA translation, only in the disease states, specifically preclinical models of tuberous sclerosis complex (TSC) and Alzheimer's disease (AD). In agreement, DJ-1-mediated repression of Ca_V1.2/α2δ2 protein synthesis in dendrites is exaggerated in mouse models of AD and TSC, resulting in deficits in dendritic L-VGCC calcium activity. Finding of DJ-1-regulated L-VGCC activity in dendrites in TSC and AD provides a unique signaling pathway that can be targeted in clinical mTORopathies.

Keywords: Alzheimer’s disease; RNA-binding protein; mammalian target of rapamycin; tuberous sclerosis complex; voltage-gated calcium channels.

Fig. 1.

DJ-1 associates with Cacna2d2 and Cacna1c in Tsc1 cKO. (A) List of predicted DJ-1 binding mRNA targets coding for Ca²⁺ channel subunits (coding region, CR) (B) (Top) qRT-PCR analyses of Tsc1^Cre/Cre: KO and Tsc1 ^fl/fl: WT from DJ-1-RNA-immunoprecipitation. Cacna1c: WT = 0.05 ± 0.03, Tsc1 cKO = 0.95 ± 0.3, P = 0.03. Akt1: WT = 0.04 ± 0.02, Tsc1 cKO = 0.44 ± 0.05, P = 0.001. Gapdh: WT = 0.04 ± 0.03, Tsc1 cKO = 0.09 ± 0.06, P = 0.47. (Bottom) qRT-PCR of 5% of the input. Cacna1c: WT = 1 ± 0.09, Tsc1 cKO = 0.83 ± 0.1251, P = 0.29. Akt1: WT = 1 ± 0.05, Tsc1 cKO = 1.22 ± 0.17, P = 0.24. Gapdh: WT = 1 ± 0.01, Tsc1 cKO = 1.15 ± 0.05, P = 0.006. n = 3 technical replicates, 3 independent mice. Student’s t test, two-tailed. (C) (Top) Representative images of Cacna2d2 mRNA. (Bottom) Magnified dendrites (dashed-lines) Cacna2d2 mRNA, indicated by green puncta/yellow arrowheads. (D) (Top) qRT-PCR of mRNA isolated from DJ-1 RNA-IP. Cacna2d2: WT = 0.07 ± 0.04, Tsc1 cKO = 0.4 ± 0.05, P = 0.01. Cacna2d1: WT = 0.002 ± 0.002, Tsc1 cKO = 0.04 ± 0.04, P = 0.4. (Bottom) qRT-PCR of 5% input RNA from DJ-1 RNA-IP. Cacna2d2: WT = 1 ± 0.03, Tsc1 cKO = 0.8 ± 0.09, P = 0.08. Cacna2d1: WT = 1 ± 0.03, Tsc1 cKO = 1.23 ± 0.21, P = 0.26. N = 3 technical replicates, 3 independent mice, t test, two-tailed.

Fig. 2.

Tsc1 cKO mouse model exhibits decreased de novo protein synthesis of Ca_V1.2 and α2δ2, but not α2δ1. De novo protein synthesis, visualized by SUnSET-PLA (green) in hippocampal dendrites (MAP2, red). WT (W) and TSC (T) dendrites are outlined by broken lines. (A) Basal Ca_V1.2 protein synthesis is detected in dendrites of WT is markedly reduced in TSC, quantified below (WT = 1.00 ± 0.07, 117 total ROIs, 12 to 18 ROIs/slice, 2 to 3 slices/animal, 3 animals; Tsc1 cKO = 0.01 ± 0.01, 106 total ROIs, 12 to 18 ROIs/slice, 2 to 3 slices/animal, 3 animals). (B) α2δ2 basal new protein synthesis is detected in dendrites of WT but is attenuated in TSC as quantified below (WT = 1.00 ± 0.15, n = 77 total ROIs, 12 to 18 ROIs/slice, 2 to 3 slices/animal, 3 animals; Tsc1 cKO = 0.12 ± 0.02, n = 73 total ROIs, 12 to 18 ROIs/slice, 2 to 3 slices/animal, 3 animals). (C) Basal α2δ1 protein synthesis in dendrites of WT is lower than TSC as quantified below (WT = 1.00 ± 0.17, n = 28 total ROIs, 12 to 18 ROIs/slice, 2 to 3 slices/animal, 3 animals; Tsc1 cKO = 3.97 ± 0.90, n = 17 total ROIs, 12 to 18 ROIs/slice, 2 to 3 slices/animal, 3 animals). For representative images in A through C, Ca_V1.2, α2δ2, and α2δ1 puncta were dilated once using ImageJ. Bar values represent mean ± SEM. ***P < 0.001, ****P < 0.0001.

Fig. 3.

Overexpression of DJ-1 represses Ca_V1.2 and α2δ2 expression in neuronal dendrites and reduces L-VGCC activity in response to BayK. Protein of interest density is denoted by pseudo coloring (fire) and Map2 (grayscale). Representative images of control (CTL, Left) and overexpression (DJ-1 OE, Middle). (A) DJ-1 expression (Left) and quantification (Right); Scale bar, 20 µm, (Right) DJ-1/Map2 intensity ratio (control = 1.00 ± 0.08; OE = 2.99 ± 0.40; n per condition = 5 ROI/slice, 2 slices/animal, 4 animals; P < 0.0001). (B) (Top) Ca_V1.2. (Top, Right) quantification of Ca_V1.2/Map2 ratio (control = 1.00 ± 0.09; OE = 0.66 ± 0.06, n per condition = 5 ROI/slice, 2 slices/animal, 4 animals; P < 0.002). (Middle) α2δ2. (Middle, Right) quantification of α2δ2/Map2 ratio (control = 1.00 ± 0.13; OE = 0.67 ± 0.06, n per condition = 5 ROI/slice, 2 slices/animal, 4 animals; P = 0.02). (Bottom) α2δ1. (Bottom, Right) quantification of α2δ1/Map2 ratio (control = 1.00 ± 0.12; OE = 1.06 ± 0.11, n per condition = 5 ROI/slice, 2 slices/animal, 4 animals; P = 0.72). Bar values are shown as mean ± SEM; statistical tests: Student’s t test. (C) L-VGCC activity detected by L-type agonist, BayK-8644 (BayK, 5 μM). (Left) Average traces of Ca²⁺ fluorescence signal before (baseline; 0 to 60 s) and after (520 to 580 s) the addition of vehicle or BayK at ~90 s. In pcDNA-transfected neurons (control), Ca2+ fluorescence increases with BayK (black) compared to vehicle treatment (gray). In DJ-1 overexpressing neurons, addition of BayK (red) has similar Ca2+ fluorescence as vehicle treatment (pink). (Right) Quantification of change in fluorescence (ΔF) normalized to baseline (F). Significance determined by two-way ANOVA (F_1,34 = 12.04, P = 0.0014) and genotype (F_1,34 = 7.22, P = 0.0111). pcDNA-Vehicle (gray, 5.80 ± 3.16, n = 4 dendrites, 4 neurons) vs. pcDNA-BayK (black, 57.07 ± 14.81, n = 6 dendrites, 4 neurons), DJ-1-OE-Vehicle (pink, −3.10 ± 6.04, n = 17 dendrites, 11 neurons) vs. DJ-1-OE-BayK (red, 13.44 ± 7.57, n = 11 dendrites, 9 neurons). Statistical tests: A and B by Student’s t test; C by two-way ANOVA. Bar values represent mean ± SEM. *P < 0.05, **P < 0.01.

Fig. 4.

Tsc1 cKO neurons have reduced levels of calcium channel-associated proteins and L-VGCC function. (A–C) Map2 staining (red) marks dendrites. (A) Ca_V1.2 (blue). Compared to wild type (WT, Top), TSC1cKO dendrites (Bottom) have less Ca_V1.2 as quantified (Right). CaV1.2/Map2: (WT = 1.00 ± 0.10, n = 27 neurons; Tsc1 cKO = 0.66 ± 0.09, n = 23 neurons; P = 0.0129). (B) α2δ2 (blue) expression is greater in WT (Top) than Tsc1 cKO as quantified (Right). α2δ2/Map2 (control = 1.00 ± 0.10, n = 15 neurons; Tsc1 cKO = 0.52 ± 0.08, n = 12 neurons; P = 0.0017). (C) Representative WT (Top) and Tsc1 cKO (Bottom) neurons express α2δ1 (blue) in dendrites. α2δ1/Map2 (WT = 1.00 ± 0.12, n = 30 neurons; Tsc1 cKO = 1.69 ± 0.21, n = 34 neurons; P < 0.0085). (D) (Left) Average traces of calcium fluorescence before (baseline; 0 to 60 s) and after (520 to 580 s) the addition of vehicle or BayK at ~90 s. With BayK, dendritic calcium fluorescence increases in WT (black) but not in Tsc1 cKO (red). (Right) Quantification of change in fluorescence (ΔF) normalized to baseline (F). Two-way ANOVA revealed a significant main effect of treatment (F_1,76 = 8.15, P = 0.0055), genotype (F_1,76 = 9.64, P = 0.0027), and a significant genotype × treatment interaction (F_1,76 = 6.591, P = 0.0122). WT-Vehicle (gray, −0.96 ± 1.12, n = 20 dendrites, 15 neurons) vs. WT-BayK (black, 27.92 ± 6.39, n = 14 dendrites, 11 neurons) Tsc1cKO-Vehicle (pink, −3.82 ± 2.98, n = 28 dendrites, 20 neurons) vs. Tsc1cKO-BayK (red, −2.29 ± 2.44, n = 18 dendrites, 14 neurons). Statistical tests: A through C by t test; D by two-way ANOVA, Tukey post hoc test. Bar values represent mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 5.

The APP/PS1 preclinical model of AD exhibits aberrant DJ-1 expression and CaV1.2 and a2d2 de novo translation. (A) In hippocampal neuronal cultures, expression of DJ-1 (blue) in dendrites (Map2, red), yellow arrows indicate puncta, is low in WT (Top) compared to APP/PS1 (Bottom) as quantified (Right). Quantification: WT = 1.00 ± 0.088, n = 133 dendrites, 48 neurons; APP/PS1 = 1.89 ± 0.10, n = 143 dendrites, 50 neurons, ****P < 0.0001. (B) (Top). qRT-PCR from DJ-1-RIP from APP/PS1 and WT animals, showing Cacna1c and Cacna2d2 bind to DJ-1. Cacna1c: WT = 0.06 ± 0.01, APP/PS1 = 0.87 ± 0.2, P = 0.01. Cacna2d2: WT = 0.02 ± 0.01, APP/PS1 = 0.89 ± 0.3, P = 0.03. Cacna2d1: WT = 0.26 ± 0.1, APP/PS1 = 0.27 ± 0.07, P = 0.99. Akt1: WT = 0.01 ± 0.01, APP/PS1 = 0.61 ± 0.05, P = 0.0004. Gapdh: WT = 0.11 ± 0.06, APP/PS1 = 0.15 ± 0.08, P = 0.71. (Bottom) qRT-PCR of 5% of the input from APP/PS1 or WT. Cacna1c: WT = 1 ± 0.06, APP/PS1 = 0.91 ± 0.14, P = 0.61. Cacna2d2: WT = 1 ± 0.08, APP/PS1 = 0.57 ± 0.10, P = 0.0042. Cacna2d1: WT = 1 ± 0.04, APP/PS1 = 1.3 ± 0.22, P = 0.23. Akt1: WT = 1 ± 0.04, APP/PS1 = 1.2 ± 0.11, P = 0.14. Gapdh: WT = 1 ± 0.05, APP/PS1 = 1.28 ± 0.03, P = 0.0001. n = 3 technical replicates, 3 independent mice. t test, two-tailed. (C) De novo protein synthesis, SUnSET-PLA (green) in dendrites (MAP2, red). (Left) Ca_V1.2 is basally translated in WT but is repressed in APP/PS1 as quantified in rightmost panel. Quantification (Bottom): WT = 1.00 ± 0.09, n = 110 total ROIs, 15 to 21 ROIs/slice, 3 slices/animal, 3 animals; APP/PS1 = 0.01 ± 0.003, n = 114 total ROIs, 15 to 21 ROIs/slice, 3 slices/animal, 3 animal. (Middle) Basal translation of α2δ2 is robust in WT but is attenuated in APP/PS1 as quantified in rightmost panel. Quantification (Bottom): WT = 1.00 ± 0.13, n = 100 total ROIs, 15 to 21 ROIs/slice, 3 slices/animal, 3 animals; APP/PS1 = 0.03 ± 0.005, n = 100 total ROIs, 15 to 21 ROIs/slice, 3 slices/animal, 3 animals. (Right) New synthesis of α2δ1 protein in WT is lower than APP/PS1 as quantified in rightmost panel. Quantification (Bottom): WT = 1.00 ± 0.14, n = 104 total ROIs, 15 to 21 ROIs/slice, 3 slices/animal, 3 animals; APP/PS1 = 2.00 ± 0.14, n = 112 total ROIs, 15 to 21 ROIs/slice, 3 slices/animal, 3 animals. For representative images, CaV1.2, α2δ2, and α2δ1 puncta were dilated once using ImageJ. Statistical tests: A and C by Student’s t test. Bar values represent mean ± SEM. ****P < 0.0001.

Fig. 6.

APP/PS1 neurons express reduced levels of calcium channel-associated proteins and L-type VGCC function. (A–C) Map2 staining (red) marks dendrites. (A) Ca_V1.2 proteins (blue) in dendrites, wild type (WT, Top), APP/PS1 dendrites (Bottom) have less Ca_V1.2. (Right) Quantification of Ca_V1.2/Map2: WT = 1.00 ± 0.06, n = 114 dendrites, 39 neurons; APP/PS1 = 0.45 ± 0.05, n = 108 dendrites, 38 neurons. (B) α2δ2 (blue) expression is greater in WT (Top) than APP/PS1 (Bottom). (Right) Quantification of α2δ2/Map2: WT = 1.00 ± 0.10, n = 104 dendrites, 38 neurons; APP/PS1 = 0.79 ± 0.08, n = 98 dendrites, 39 neurons. (C) Representative WT (Top) and APP/PS1 (Bottom) neurons express α2δ1 (blue) in dendrites. APP/PS1 dendrites express more α2δ1. (Right) Quantification of α2δ1/Map2 WT = 1.00 ± 0.11, n = 91 dendrites, 35 neurons; APP/PS1 = 1.30 ± 0.10, n = 85 dendrites, 32 neurons. (D) L-VGCC function as measured by BayK-induced calcium fluorescence is absent in APP/PS1. (Left) Average traces of calcium fluorescence before (baseline; 0 to 60 s) and after (520 to 580 s) the addition of vehicle or BayK at ~90 s. In BayK, dendritic calcium fluorescence increases in WT (black) but not in APP/PS1 (red). (Right) Quantification of change in fluorescence (ΔF) normalized to baseline (F). ΔF/F was determined by normalizing ΔF of each condition/genotype to its respective baseline fluorescence (F). Two-way ANOVA revealed a significant main effect of treatment (F_1,60 = 9.962, P = 0.0025) but not genotype (F_1,60 = 3.512, P = 0.0658). There was a significant genotype × treatment interaction (F_1,60 = 16.72, P = 0.0001). Tukey’s multiple comparisons post hoc test revealed a significant difference between WT-Vehicle (gray, −9.86 ± 3.57, n = 14 dendrites, 11 neurons) vs. WT-BayK (black, 26.90 ± 7.27, n = 16 dendrites, 12 neurons), but no significant difference between APP/PS1-Vehicle (pink, 1.3810 ± 3.87, n = 19 dendrites, 15 neurons) vs. APP/PS1-BayK (red, −4.51 ± 4.51, n = 15 dendrites, 12 neurons). There is a significant difference between WT-BayK vs. APP/PS1-BayK. Statistical tests: A through C by Student’s t test; D by two-way ANOVA. Bar values represent mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 7.

Synapses isolated from prefrontal cortex of AD individuals express upregulation of mTORC1 signaling and DJ-1 levels, and downregulation of Ca_V1.2 and α2δ2 levels. (A) Western blot of phosphorylated S6 ribosomal protein (Ser240/244) (control = 1.00 ± 0.07, n = 3; AD = 1.94 ± 0.34, n = 3) (B) DJ-1 protein (control = 1.00 ± 0.15, n = 5; AD = 1.56 ± 0.18, n = 5;). (C) AD patient synapses have decreased Ca_V1.2 (control = 1.00 ± 0.20, n = 5; AD = 0.42 ± 0.21, n = 5) and (D) decreased α2δ2 levels (control = 1.00 ± 0.15, n = 4; AD = 0.45 ± 0.20, n = 3). Statistical tests: Student’s t test. Bar values represent mean ± SEM. *P < 0.05. For pathological details, see SI Appendix, Fig. S6.