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Alzheimer's Disease: Exploring the Hippocampal Proteome with DIA Technology Under Chronic Stress

Proteomics research based on Data-independent acquisition (DIA) technology plays a crucial role in Alzheimer's disease (AD) studies. AD is a common neurodegenerative disorder, often referred to as primary age-related dementia. It is clinically characterized by memory impairment, cognitive decline, and diminishing language skills, significantly impacting individuals' lives and well-being. The pathophysiological mechanisms of AD are highly complex, and its etiology remains elusive.

The utilization of DIA technology in proteomics research for AD offers several significant advantages:

1. Comprehensive Protein Analysis: DIA technology allows for the simultaneous analysis of multiple proteins in a sample, rather than being limited to specific precursor ions. This provides a more comprehensive view of protein information, potentially revealing protein changes associated with AD, including potential biomarkers.

2. Quantitative Analysis: DIA technology not only provides qualitative protein information but also enables relative and absolute protein quantification. This is instrumental in understanding changes in protein expression in the context of AD pathogenesis and disease progression.

3. Discovery of New Biomarkers: The comprehensiveness and high sensitivity of DIA technology make it an ideal tool for the discovery of novel AD biomarkers. By comparing protein expression in AD patients and control groups, potential disease-related protein differences can be identified, aiding in early diagnosis and disease monitoring.

4. Data Re-analysis: Data generated by DIA can be re-analyzed at later stages to uncover new insights or address new research questions. This flexibility contributes to an ongoing deepening of our understanding of AD.

Case. Multi-Omics Analysis of Glioblastoma Reveals Key Oncogenic Pathways and Therapeutic Opportunities ^[1]

Background

The pathophysiological mechanisms of Alzheimer's disease (AD) are intricate, and its root causes remain elusive. Over the past few decades, the use of mass spectrometry-based proteomics methods has proven to be an effective approach for fundamental research, the discovery of potential biomarkers, and the identification of drug targets.

In this particular study, chronic stress was induced in two groups of mice, one comprising APPPS1 transgenic mice and the other containing wild-type (WT) mice, over a span of four weeks. Subsequently, a three-week period of diurnal disruption was implemented. To delve deeper into the molecular changes associated with this stress, LC-MS/MS-based proteomic analysis was carried out on the hippocampal tissue. This analysis employed label-free quantitative Data-Independent Acquisition (DIA) methodology. By comparing the results with those of the control group, the study successfully identified proteins that exhibited significant upregulation and downregulation in both the APPPS1 and WT mice exposed to chronic stress.

Research Objectives

The aim of this study is to investigate the impact of chronic stress on the hippocampal proteome of male APPPS1 transgenic mice and wild-type (WT) littermate mice.

Research Findings

Plasma Corticosterone

In this research, the investigation revealed that three weeks after the conclusion of the chronic stress period, no statistically significant impact on plasma corticosterone levels was observed (as depicted in Figure 1B). These findings align with previous observations, indicating the consistency of the results.

Transgenic-Induced Nesting Deficits

Before the onset of chronic stress, the overall well-being of both APPPS1 and WT mice was assessed through a nesting activity test. This assessment unveiled significant main effects attributed to genotype and time, alongside a noteworthy interaction effect (illustrated in Figure 1C). These findings point to a notable impairment in nesting behavior among the APPPS1 mice. Notably, all the APPPS1 mice displayed substantial deficits when compared to their WT counterparts. Previous research has also suggested a potential link between nesting deficits and hippocampal damage.

Figure 1 Plasma Corticosterone Level Measurement and Nesting Activity Assessment in Stress-Induced Plaque Pathology

Stress-Induced Plaque Pathology:

Utilizing thioflavin S staining, an examination of the prefrontal cortex and hippocampus in the APPPS1 cohort mice was conducted to assess the impact of chronic stress. The results indicated that chronic stress did not exert a substantial effect on the prefrontal cortex plaque pathology, as depicted in Figure 2B and 2D. Furthermore, it was observed that the quantity of plaques in the hippocampal tissue remained unchanged following exposure to chronic stress, as illustrated in Figure 2A and 2C.

Figure 2 Effects of chronic stress on prefrontal cortex and hippocampal plaque pathology

Identification of Differential Proteins

Bottom-up quantitative proteomic analysis of hippocampal tissue identified a total of 1153 quantifiable unique proteins. Of these, 1106 were identified in all groups, and the remaining 47 were unidentified in at least one group (Fig. 3D). 3 proteins were unique to the WT control group (MRS2, UGGT1, and FXYD6), 7 proteins were unique to the stress group (1 protein was unique to the WT stress group, 3 proteins were unique to the APPPS1 stress group, and the WT stress and APPPS1 stress groups a total of 3 proteins) and 2 proteins specific to the APPPS1 control group.

Between the APPPS1 stress group and the APPPS1 control group, 198 significantly dysregulated proteins were identified (79 upregulated and 119 downregulated). Compared to the WT control group, 82 proteins were significantly upregulated and 211 proteins were significantly downregulated in the WT stress group. And compared to WT control, there were 61 significantly up-regulated proteins and 117 significantly down-regulated proteins in APPPS1 control group. Compared to the WT control group, 84 proteins were significantly up-regulated and 177 proteins were significantly down-regulated in the APPPS1 stress group.

Pathway Enrichment Analysis

Based on the KEGG database, we analyzed the functional enrichment of different proteins, and found that the up-regulated proteins were enriched into 16 KEGG pathways and down-regulated proteins were enriched into 40 pathways in APPPS1 stress group vs APPPS1 control group, and the up-regulated proteins were enriched into 94 KEGG pathways and down-regulated proteins were enriched into 79 pathways in WT stress group vs WT control group. Meanwhile, APPPS1 control group vs WT control group had up-regulated proteins enriched to 71 pathways and down-regulated proteins enriched to 29 pathways. Finally, upregulated proteins were enriched to 26 pathways and downregulated proteins were enriched to 48 pathways in APPPS1 stress group vs WT control group.

In addition to the pathway enrichment results for down-regulated proteins in the APPPS1 control group vs WT control group, oxidative phosphorylation (OXPHOS) was ranked in the top 10 of the differential protein-enriched pathways in each of the other differential subgroups. Careful observation of the differential proteins enriched by this pathway revealed that four proteins belonged to the respiratory chain and vacuolar-type ATPase (v-ATPase; Fig. 3A), V- ATPase enzymes pump protons into the lumen of organelles such as vesicles, lysosomes, and nuclear endosomes through ATP hydrolysis, thereby generating an acidic luminal environment. The Alzheimer's disease pathway was similarly identified in all comparative subgroups (Fig. 3 B). Of the proteins enriched for this pathway, 23 of them were also enriched for the OXPHOS pathway, and 9 proteins were annotated to intercellular synapses/dendrites (Gene ontology). Finally, metabolic pathways were also among the top 10 KEGG pathways in all comparative subgroups. Thirty-one of these proteins were enriched for the OXPHOS pathway, and 19 were enriched for the TCA cycle and pyruvate metabolism pathways.

Interestingly, most of these proteins enriched in the OXPHOS pathway in the WT stress group belonged to the complex I of the respiratory chain and the V1 structural domain of the V-ATPase complex, whereas the differential proteins in the APPPS1 control group and the APPPS1 stress group were more broadly distributed in the OXPHOS pathway. Thus, in nonamyloid lesion brains, chronic stress may more directly affect the V1 structural domains of complex I and the V-ATPase complex, whereas amyloid lesions more broadly affect the OXPHOS pathway. In conclusion, analysis of the data suggests that both amyloid pathology and chronic stress may lead to dysregulation of the OXPHO pathway S, and that these two factors combine to have a superimposed effect on this dysregulation.

Figure 3 KEGG pathway analysis of differential proteins

Conclusion

The data herein show that chronic stress modulates mitochondrial function in the hippocampus at the protein level and has a strong effect on complex I in WT mice. The data also suggest that AD-like pathology has a widespread effect on the mitochondrial OXPHOS pathway, and that chronic stress enhances this detrimental effect, possibly leading to reduced hippocampal synaptic plasticity. Based on the data in this paper and previous literature, it can be hypothesized that chronic stress impairs mitochondrial function through higher neuronal energy demands and adaptive mechanisms. In the AD brain, amyloid lesions and reduced mitochondrial function worsen these energy-demanding adaptive mechanisms, and loss of mitochondrial function enhances ROS and neuroinflammation, which ultimately will worsen behavioral outcomes and accelerate the development of neuropsychiatric disorders (NPDs). This hypothesis remains to be fully substantiated, and the effects of chronic stress on functional brain regions in AD, as well as systemic effects, require further study.

Reference

Clement, Amalie, et al. "Chronic stress induces hippocampal mitochondrial damage in APPPS1 model mice and wildtype littermates." Journal of Alzheimer's Disease 87.1 (2022): 259-272.

* For Research Use Only. Not for use in diagnostic procedures.

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