Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most prevalent neurodegenerative disorders, imposing a staggering burden on global health. While clinically distinct—AD primarily affects memory and cognition through degeneration in the hippocampus and cortex, whereas PD impairs motor function via loss of dopaminergic neurons in the substantia nigra—they share a fundamental pathological hallmark: the intracellular or extracellular accumulation of misfolded proteins (1). In AD, this involves amyloid-beta (Aβ) plaques and neurofibrillary tangles of hyperphosphorylated tau; in PD, it is the aggregation of α-synuclein into Lewy bodies (2).

The endoplasmic reticulum (ER) is a crucial organelle responsible for the synthesis, folding, and modification of over a third of the cellular proteome. The accumulation of misfolded proteins disrupts ER homeostasis, triggering a state known as ER stress. In response, the cell activates a sophisticated signaling network called the Unfolded Protein Response (UPR) (3). The UPR is initially a pro-survival mechanism, mediated by three transmembrane sensors: PERK, IRE1α, and ATF6. It aims to restore homeostasis by attenuating protein translation, upregulating ER chaperones like GRP78/BiP, and enhancing ER-associated degradation (ERAD) (4). However, under chronic or overwhelming stress, the UPR can switch to a pro-apoptotic program, primarily through the PERK-eIF2α-ATF4-CHOP signaling axis and the IRE1α-JNK pathway, to eliminate terminally damaged cells (5).

Recent literature has increasingly implicated ER stress in the pathogenesis of both AD and PD. Studies have shown that Aβ oligomers can directly induce UPR activation in neuronal cultures and that UPR markers are elevated in the brains of AD patients and mouse models (6,7). Similarly, α-synuclein aggregates have been demonstrated to impair ER-to-Golgi trafficking and trigger ER stress, with UPR components found co-localized within Lewy bodies in PD brains (8,9).

Despite these links, a significant research gap remains. Most studies have focused on a single disease, and a direct, systematic comparison of the temporal dynamics and pathological contribution of ER stress across both AD and PD models is absent. It is unclear whether ER stress is a common, convergent driver of neurodegeneration that follows a similar activation pattern, or if its role and timing differ fundamentally between Aβ- and α-synuclein-driven pathologies. Understanding this is critical for determining if targeting ER stress could be a unified therapeutic strategy.

Therefore, this study addresses the following research question: Is chronic UPR activation a convergent pathogenic mechanism that precedes and directly correlates with the extent of neuronal degeneration in mechanistically distinct mouse models of AD and PD? Our central hypothesis is that ER stress is an early and progressively intensifying event in both pathologies, and that the magnitude of pro-apoptotic UPR signaling is a direct predictor of neuronal death. Justifying its significance, this research could establish the UPR as a core pathological hub, providing a strong rationale for developing therapies targeting ER homeostasis for a broad spectrum of neurodegenerative diseases.

Study Design

This study employed a controlled, comparative experimental design using two well-established mouse models of neurodegeneration and their respective wild-type (WT) controls. For the AD model, a longitudinal design was used to assess pathology at 3, 6, and 9 months of age. For the PD model, a post-lesion time-course design was implemented to evaluate changes at 1, 3, and 7 days after neurotoxin administration. All experiments were conducted in a blinded manner regarding genotype and treatment group.

Animals and Samples

AD Model: Male 5xFAD transgenic mice (n=24) and age-matched male WT C57BL/6J littermates (n=24) were used. The 5xFAD mice co-express five human familial AD mutations in amyloid precursor protein (APP) and presenilin-1 (PSEN1), leading to aggressive Aβ plaque deposition. Animals were divided into three age groups (3, 6, and 9 months), with n=8 per genotype per time point.

PD Model: Male C57BL/6J mice (8-10 weeks old, n=32) were used. Animals were randomly assigned to receive either MPTP-HCl (Sigma-Aldrich; 20 mg/kg, intraperitoneal injection, four doses at 2-hour intervals; n=16) or an equivalent volume of sterile saline (n=16). Animals were euthanized at 1, 3, or 7 days post-injection (n=8 per treatment group for day 7; n=4 per group for days 1 and 3).

Inclusion/Exclusion Criteria: Only male mice were used to avoid confounding effects of the estrous cycle. Animals showing signs of distress or illness unrelated to the experimental paradigm were excluded.

Data Collection Procedures

At the designated endpoints, mice were deeply anesthetized with isoflurane and transcardially perfused with ice-cold phosphate-buffered saline (PBS). Brains were harvested; one hemisphere was snap-frozen in liquid nitrogen and stored at -80°C for biochemical analysis, while the other was fixed in 4% paraformaldehyde for 24 hours for immunohistochemistry. The hippocampus was dissected from the AD model brains, and the ventral midbrain containing the substantia nigra pars compacta (SNpc) was dissected from the PD model brains for Western blotting.

Instruments and Techniques

Western Blotting: Frozen tissue was homogenized in RIPA buffer with protease and phosphatase inhibitors. Protein concentration was determined using a BCA assay (Thermo Fisher). Equal amounts of protein (20 μg) were resolved by SDS-PAGE, transferred to PVDF membranes, and incubated overnight at 4°C with the following primary antibodies: anti-GRP78/BiP (1:1000, Cell Signaling), anti-phospho-PERK (1:1000, Cell Signaling), anti-XBP1s (1:500, BioLegend), anti-CHOP (1:1000, Cell Signaling), anti-NeuN (1:1000, Millipore), anti-Tyrosine Hydroxylase (TH, 1:2000, Millipore), and anti-β-actin (1:5000, Sigma-Aldrich). After incubation with HRP-conjugated secondary antibodies, bands were visualized using an enhanced chemiluminescence (ECL) kit and quantified using ImageJ software. Protein levels were normalized to β-actin.

Immunohistochemistry (IHC) and TUNEL Staining: Fixed hemispheres were cryoprotected in 30% sucrose, sectioned at 40 μm on a cryostat, and stored as free-floating sections. For IHC, sections were incubated with primary antibodies against CHOP (1:500), NeuN (1:500), or TH (1:1000), followed by appropriate fluorescent secondary antibodies (Alexa Fluor 488/594). To detect apoptosis, a TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions. Slices were counterstained with DAPI and imaged on a Zeiss LSM 880 confocal microscope. Cell counts were performed on three sections per animal in the CA1 region of the hippocampus (AD) or the SNpc (PD).

Statistical Analysis

All data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9. For comparisons between genotypes/treatments across multiple time points, a two-way ANOVA followed by Tukey’s post-hoc test was used. For single time-point comparisons, an unpaired Student’s t-test was used. Pearson correlation analysis was performed to determine the relationship between CHOP protein levels and neuronal marker levels (NeuN or TH). A p-value of < 0.05 was considered statistically significant.

Progressive Activation of the UPR in the 5xFAD Mouse Hippocampus

To assess the temporal profile of ER stress in the AD model, we measured key UPR markers in hippocampal lysates from 3, 6, and 9-month-old 5xFAD and WT mice. Two-way ANOVA revealed a significant effect of both genotype and age. As shown in Table 1, the ER chaperone GRP78 was significantly upregulated in 5xFAD mice as early as 3 months (p < 0.05) and increased further at 6 and 9 months compared to WT controls (p < 0.001). The UPR initiator p-PERK and the downstream pro-apoptotic factor CHOP followed a similar pattern, showing significant elevation by 6 months and peaking at 9 months (p < 0.001). The IRE1α pathway marker, spliced XBP1 (XBP1s), also showed a significant increase at 6 and 9 months (p < 0.01). These data indicate a sustained and escalating activation of all three UPR branches during AD pathology progression.

UPR Activation Precedes and Correlates with Neuronal Loss in the 5xFAD Model

We next quantified neuronal loss by measuring levels of the neuronal marker NeuN. A significant reduction in NeuN protein was first observed at 6 months (p < 0.05) and was more pronounced at 9 months in 5xFAD mice (p < 0.001) (Table 1). This demonstrates that initial UPR activation (e.g., GRP78 at 3 months) precedes substantial neuronal loss. Pearson correlation analysis revealed a highly significant negative correlation between hippocampal CHOP levels and NeuN levels (r = -0.89, p < 0.001), suggesting a strong association between pro-apoptotic ER stress and neuronal demise (Table 2). IHC analysis confirmed a reduction in NeuN-positive cells in the CA1 region and showed co-localization of CHOP within these remaining, stressed neurons.

Acute and Robust UPR Activation in the MPTP Mouse Substantia Nigra

In the MPTP model of PD, a rapid induction of the UPR was observed in the substantia nigra. GRP78 and p-PERK levels were significantly elevated as early as 1 day post-injection (p < 0.05) and remained high at 3 and 7 days compared to saline controls (Table 1). CHOP expression increased significantly by day 3 (p < 0.01) and peaked at day 7 (p < 0.001). Similar to the AD model, these findings point to an early and sustained ER stress response following neurotoxic insult.

Pro-Apoptotic ER Stress Correlates with Dopaminergic Neuron Degeneration

The neurotoxin MPTP induces specific loss of dopaminergic neurons. We quantified this by measuring levels of Tyrosine Hydroxylase (TH). A significant reduction in TH protein was observed by day 3 (p < 0.05) and became severe by day 7 (p < 0.001) (Table 1). Again, the initial UPR activation preceded major neurodegeneration. Correlation analysis showed a very strong negative correlation between CHOP levels and TH levels in the substantia nigra (r = -0.92, p < 0.001) (Table 2). This was supported by TUNEL staining, which revealed a significant increase in apoptotic, TH-positive cells at day 7, many of which also expressed CHOP.

Table 1: Quantitative Analysis of UPR and Neuronal Markers in AD and PD Models

Table 2: Correlation Between Pro-Apoptotic ER Stress and Neuronal Loss

This study provides direct comparative evidence that chronic ER stress is a convergent pathogenic mechanism in mechanistically distinct mouse models of AD and PD. Our primary finding is that the UPR is activated early in the disease course in both models, and the magnitude of its pro-apoptotic signaling, specifically via CHOP, is strongly correlated with the severity of neuronal degeneration. This supports our hypothesis and positions ER stress as a critical link between protein misfolding and neuronal death.

The temporal analysis revealed a crucial insight: UPR activation is not merely a consequence of end-stage disease but an early pathological event. In 5xFAD mice, the upregulation of the chaperone GRP78 at 3 months, an age with minimal Aβ plaque load and no significant neuronal loss, suggests that ER stress is an initial response to soluble oligomeric Aβ species, consistent with previous reports (7,10). Similarly, in the MPTP model, ER stress markers were elevated within 24 hours, preceding the peak of dopaminergic cell death. This early activation implies that the UPR is an integral part of the pathogenic cascade rather than a bystander effect.

Our study's key contribution is the direct comparison between AD and PD models, which reveals a striking similarity in the UPR's progression from an adaptive to a pro-apoptotic response. Despite the different causative insults (chronic genetic-driven Aβ accumulation vs. acute neurotoxicant-induced mitochondrial dysfunction), both pathologies converge on the activation of the PERK-CHOP axis. The extremely high correlation coefficients between CHOP and neuronal markers (NeuN/TH) in both models underscore the pathological relevance of this pathway. CHOP is a transcription factor known to promote apoptosis by downregulating the anti-apoptotic protein Bcl-2 and upregulating pro-apoptotic factors like BIM and death receptors (11,12). Our findings align with studies that have shown that genetic deletion of CHOP is neuroprotective in various models of neurodegeneration (13).

The results must be interpreted within the context of the study's limitations. First, mouse models, while invaluable, do not fully recapitulate the complexity of human sporadic AD and PD. The 5xFAD model represents familial AD, and the MPTP model is an acute toxicant model, not a model of chronic α-synucleinopathy. Future studies should validate these findings in models based on tau or α-synuclein transgenes. Second, our study demonstrates a strong correlation, but it does not definitively prove causation. Future interventional studies using pharmacological inhibitors of UPR pathways (e.g., PERK inhibitors) in these models would be necessary to establish a causal link. Finally, we focused on the hippocampus and substantia nigra; exploring other affected brain regions would provide a more complete picture.

The theoretical and practical implications of our findings are significant. Theoretically, they reinforce the concept of "proteotoxicity" as a shared disease mechanism, with the ER serving as a central sensor and effector of this stress. This suggests that distinct proteinopathies may utilize a common final pathway to execute cell death. Practically, our results highlight the UPR as a promising therapeutic target. Rather than developing disease-specific drugs against Aβ or α-synuclein, which has proven challenging, modulating ER stress could offer a broader therapeutic window. Strategies could involve either inhibiting the pro-death arms (e.g., PERK/CHOP) or enhancing the adaptive arms of the UPR to restore protein homeostasis (14,15,16).

In conclusion, this study demonstrates that ER stress is an early, progressive, and pathologically significant event in both AD and PD mouse models. The transition from an adaptive to a pro-apoptotic UPR, marked by the upregulation of CHOP, is a key determinant of neuronal fate in both disorders. These findings identify ER stress as a critical convergent point in the pathogenesis of distinct neurodegenerative diseases. This work contributes to our understanding of shared disease mechanisms and provides a strong rationale for exploring ER stress modulators as a unified therapeutic strategy to combat neurodegeneration. Future research should focus on validating these mechanisms in other disease models and human tissues, and on testing the efficacy of UPR-targeting compounds in preventing neuronal loss.

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