Protein mishandling and impaired lysosomal proteolysis generated through calcium dysregulation in Alzheimer's disease

Abstract

Impairments in neural lysosomal- and autophagic-mediated degradation of cellular debris contribute to neuritic dystrophy and synaptic loss. While these are well-characterized features of neurodegenerative disorders such as Alzheimer's disease (AD), the upstream cellular processes driving deficits in pathogenic protein mishandling are less understood. Using a series of fluorescent biosensors and optical imaging in model cells, AD mouse models and human neurons derived from AD patients, we reveal a previously undescribed cellular signaling cascade underlying protein mishandling mediated by intracellular calcium dysregulation, an early component of AD pathogenesis. Increased Ca²⁺ release via the endoplasmic reticulum (ER)-resident ryanodine receptor (RyR) is associated with reduced expression of the lysosome proton pump vacuolar-ATPase (vATPase) subunits (V1B2 and V0a1), resulting in lysosome deacidification and disrupted proteolytic activity in AD mouse models and human-induced neurons (HiN). As a result of impaired lysosome digestive capacity, mature autophagosomes with hyperphosphorylated tau accumulated in AD murine neurons and AD HiN, exacerbating proteinopathy. Normalizing AD-associated aberrant RyR-Ca²⁺ signaling with the negative allosteric modulator, dantrolene (Ryanodex), restored vATPase levels, lysosomal acidification and proteolytic activity, and autophagic clearance of intracellular protein aggregates in AD neurons. These results highlight that prior to overt AD histopathology or cognitive deficits, aberrant upstream Ca²⁺ signaling disrupts lysosomal acidification and contributes to pathological accumulation of intracellular protein aggregates. Importantly, this is demonstrated in animal models of AD, and in human iPSC-derived neurons from AD patients. Furthermore, pharmacological suppression of RyR-Ca²⁺ release rescued proteolytic function, revealing a target for therapeutic intervention that has demonstrated effects in clinically-relevant assays.

Keywords: Alzheimer's disease; calcium; lysosome; ryanodine receptor; vATPase.

Fig. 1.

Decreased v-ATPase in AD mice are restored after normalizing intracellular Ca²⁺ levels. Hippocampus from 3–4-mo-old 3xTg-AD mice and age-matched NTg controls were immunolabeled for vATPase membrane-embedded V0a1 and cytosolic V1B2 subunits along with lysosomal surface marker (Lamp1). Representative high-resolution images of V0a1 (A) and V1B2 (B) were taken on Leica SP8 63X with 441X zoom of the primary cellular layer (PCL) and stratum radiatum (SR) of the CA1 field. Quantification showed decreased fluorescence density for (C) V0a1, (D) V1B2 in multiple regions of the hippocampus (CA1, CA3, DG) and cortex of 3xTg-AD mice, that was rescued with Ryanodex treatment (10mg/kg; 30-d i.p.). (E) There were no significant differences in Lamp1 density throughout the hippocampus and cortex. Colocalization of (F) V0a1 and (G) V1B2 to lysosomes specifies that deficits seen in vATPase are localized to lysosomes (NTg Vehicle, black bar, n = 8 animals; NTg Ryanodex, black pattern bar, n = 8 animals; 3xTg Vehicle, grey bar, n = 8 animals; 3xTg Ryanodex, grey pattern bar, n = 8 animals) *P <0.05

Fig. 2.

Ryanodine receptor 2 (RyR2)-evoked Ca²⁺ release alkalizes lysosomal pH through v-ATPase proton pump interference in model cells. (A) Representative traces of RyR2-evoked Ca²⁺ release (10mM caffeine) on HEK293T cells overexpressing hippocampal-relevant RyR2 treated with control, Ryanodex or bafilomycin. (B) Cells were incubated with Fura-2AM and ER-Ca²⁺ release was measured as peak change in 340/380 ratio over baseline (n= 6 coverslips/treatment). Normalizing RyR2-Ca²⁺ release with Ryanodex treatment (10μM; 1h) attenuated RyR2-evoked Ca²⁺ release. Inhibiting vATPase with bafilomycin (25nM; 4h) did not affect RyR2-evoked Ca²⁺ release. Lysosomal pH was measured using LysoSensor Yellow/Blue dextran (n = 27 wells/treatment). (D–F) Representative traces overlaying RyR2-Ca²⁺ release and lysosomal pH indicate immediate lysosomal pH fluctuations with a rise in intracellular Ca²⁺ levels. (C and  D) RyR2-evoked Ca²⁺ release alkalized lysosomal pH. (C and  E) Normalizing RyR2-Ca²⁺ release with Ryanodex treatment (10μM; 1h) attenuated the RyR2-mediated lysosomal alkalization. (C and  F) Inhibiting vATPase with bafilomycin (25nM; 4h) alkalized lysosomal pH, and no additional pH changes occurred in response to RyR2-evoked Ca²⁺ release. *P < 0.05

Fig. 3.

Modulating RyR-Ca²⁺ release regulates lysosomal pH in primary hippocampal murine cultures. (A) Representative traces of RyR-evoked Ca²⁺ release (10mM caffeine) on 3xTg-AD and NTg hippocampal primary neuronal cultures (12–14d) treated with control, Ryanodex, or bafilomycin. (B) Neurons were incubated with Fura-2AM and ER-Ca²⁺ release was measured as peak change in 340/380 ratio over baseline (n= 19 coverslips/treatment). Normalizing RyR-Ca²⁺ release with Ryanodex treatment (10μM; 1h) attenuated RyR-evoked Ca²⁺ release. Inhibiting vATPase with bafilomycin (25nM; 4h) did not affect RyR-evoked Ca²⁺ release. Lysosomal pH was measured using LysoSensor Yellow/Blue dextran on hippocampal primary neuronal culture (n = 8 coverslips/treatment). (C) Baseline lysosomal pH is increased in 3xTg-AD neurons as compared to NTg neurons. RyR-evoked Ca²⁺ release alkalized lysosomal pH in NTg neurons, while no further lysosomal pH changes occurred in 3xTg-AD neurons. Ryanodex treatment (10μM; 1h) attenuated the RyR-mediated lysosomal alkalization and restored baseline lysosomal pH in 3xTg-AD neurons. (D) Bafilomycin (25nM; 4h) alkalized lysosomal pH, and no additional pH changes occurred in response to RyR-evoked Ca²⁺ release in NTg nor 3xTg-AD neurons. *P < 0.05

Fig. 4.

Modulating RyR-Ca²⁺ regulates vATPase expression and lysosomal pH in HiN derived from AD and non-AD patients. HiN were immunolabeled for vATPase membrane-embedded V0a1 and cytosolic V1B2 subunits along with a lysosomal marker (LysoTracker). Representative images and quantification showed decreased fluorescence density for V0a1 (A, C; n = 9 coverslips/treatment), V1B2 (B, D; n = 9 coverslips/treatment), and LysoTracker (E; n = 14 coverslips/treatment), that was rescued with Ryanodex (10µM; 6h). (F) Representative traces of RyR-evoked Ca²⁺ release (10mM caffeine) on AD and non-AD HiN treated with control, Ryanodex (10µM; 1h), or bafilomycin (25nM; 4h). (G) Neurons were incubated with ratiometric indicator Fura-2AM, and ER-Ca²⁺ release was measured as peak change in 340/380 ratio over baseline (n = 8 coverslips/treatment). Normalizing RyR-Ca²⁺ release with Ryanodex treatment (10μM; 1h) decreased RyR-evoked Ca²⁺ release. Inhibiting vATPase with bafilomycin (25nM; 4h) did not affect RyR-evoked Ca²⁺ release. Lysosomal pH was measured using LysoSensor Yellow/Blue dextran on HiN (n = 11 coverslips/treatment). (H) Baseline lysosomal pH is increased in AD HiN as compared to non-AD HiN. RyR-evoked Ca²⁺ release alkalized lysosomal pH in non-AD HiN, while no further pH changes occurred in AD HiN. Ryanodex treatment (10μM; 1h) attenuated the RyR-mediated lysosomal alkalization and restored baseline lysosomal pH in AD HiN. (I) Bafilomycin (25nM; 4h) alkalized lysosomal pH, and no additional pH changes occurred in response to RyR-evoked Ca²⁺ release in non-AD nor AD HiN. *P < 0.05

Fig. 5.

Normalizing RyR-Ca²⁺ release rescues disrupted proteolytic activity in HiN. (A) Representative images of lysosomal proteolytic activity, measured by DQ^TM-Red BSA, in HiN treated with control, Ryanodex and bafilomycin are shown. (B) Quantification of fluorescence intensity showed decreased protease activity in AD HiN, which was rescued with Ryanodex (10µM; 6h) treatment. vATPase inhibition with bafilomycin (25nM; 4h) demolished protease activity in both non-AD and AD HiN (n = 5 coverslips/treatment) *P < 0.05.

Fig. 6.

Autophagic clearance of hyperphosphorylated tau is dependent on regulated RyR-Ca²⁺ signaling in mice. Hippocampus from 3–4-mo-old 3xTg-AD mice and age-matched NTg controls were immunolabled for autophagosomes (LC3B) and early-stage phosphorylated tau (S262). (A) Representative high-resolution images were taken on Leica SP8 63X with 441X zoom of the primary cellular layer (PCL) and stratum radiatum (SR) of the CA1 field. Quantification showed decreased fluorescence density of (B) LC3B and increased fluroesence density of(C) p-tau S262 in multiple regions of the hippocampus (CA1, CA3, DG) and cortex of 3xTg-AD mice, which was rescued with Ryanodex treatment (10mg/kg; 30-d i.p.). (NTg Vehicle, black bar, n = 8 animals; NTg Ryanodex, black pattern bar, n = 8 animals; 3xTg Vehicle, grey bar, n = 8 animals; 3xTg Ryanodex, grey pattern bar, n = 8 animals) *P <0.05.

Fig. 7.

Regulated RyR-Ca²⁺ release is necessary for autophagy-mediated clearance of intracellular protein aggregates, like hyperphosphorylated tau. (A) Representative images of autophagic vesicles, measured by DAP-green autophagy detection dye in non-AD and AD HiN. (B) Quantification of fluorescence density of autophagic vesicles shows increased density of autophagic vesicles in AD HiN, which are rescued with Ryanodex treatment (10µM; 6h; n = 5 coverslips/treatment). (C) Representative images of fixed HiN cultures immunolabeled for hyperphosphorylated tau (AT8). (D) Quantification of fluorescence density of hyperphosphorylated tau shows increased AT8 in AD HiN. Inhibition of vATPase with bafilomycin (25nM; 4h) exacerbated p-tau in non-AD HiN. Ryanodex (10µM; 6h) treatment reduced the elevated AD-associated hyperphosphorylated tau in AD HiN. (n = 10 coverslips/treatment). (E–G) Aβ₄₂- and Aβ₄₀-specific ELISA of the supernatant from the non-AD and AD HiN showed increased secreted Aβ₄₂/Aβ₄₀ in AD neurons. (F) No difference was seen in Aβ₄₀ levels across treatments. Bafilomycin treatment (25nM; 4h) did not exacerbate secreted Aβ₄₂/Aβ₄₀ levels in non-AD HiN, showing that blocking autophagy has a more profound effect on intracellular deposits than extracellular deposits. Additionally, Ryanodex (10µM; 6h) treatment significantly reduced extracellular Aβ₄₂/Aβ₄₀ levels in AD HiN comparable to non-AD HiN (n = 8 wells/treatment). *P <0.05.

Fig. 8.

Protein handling via lysosomal-autophagosome pathway is disrupted in early stages of Alzheimer’s disease. Dysregulated RyR-Ca²⁺ signaling leads to elevated cytosolic Ca²⁺ concentration. This rise of Ca²⁺ disrupts lysosomal vATPase proton pump, resulting in alkalization of the lysosomal lumen. The alkaline environment is not conducive for lysosomal protease, cathepsin, and hydrolase activity, thereby disrupting proteolysis of cellular debris. Autophagic clearance is halted, and turnover of bulky, misfolded proteins, and damaged organelles is disrupted. Intracellular aggregates such as hyperphosphorylated tau accumulate within autophagic vacuoles furthering AD proteinopathy.