With the increasing degree of global aging, AD has become one of the most intractable medical and social problems in mankind. An epidemiological survey showed that there were about 15.07 million dementia patients over 60 years old in China, including 9.83 million AD patients[11]. Data show that the number of AD patients in China is expected to reach 25 million by 2050, which will become an important constraint factor affecting the development of families and society[12]. Despite hundreds of years of research, the exact pathogenesis of AD is still unclear. At present, the "Beta-amyloid cascade theory" is widely recognized as the initial factor leading to the complex pathological events of AD, which is the core factor of the occurrence and development of AD[13, 14]. In recent years, NIA-AA proposed biomarkers Aβ deposition (A), pathological Tau (T), and neurodegeneration (N) as the new diagnostic criteria for AD, emphasizing that Aβ positivity is the first and necessary condition, and it is included in the disease spectrum of AD[15]. Some scholars believe that Aβ can induce apoptosis, stimulate inflammatory cascade, produce oxidative stress, lead to mitochondrial dysfunction, and aggravate the pathological process of AD[16]. Therefore, it is necessary to study the deposition of Aβ amyloid protein.
4.1 Selection of APP/PS1 double transgenic model mice
With the continuous development of transgenic technology, the AD transgenic animal model has gradually matured. APP transgenic animal model is based on APP gene mutation, overexpression or mutation of APP gene leads to the deposition of amyloid beta, which is the central part of AD pathological changes, now has become an ideal model to study the pathogenesis of AD. APP/PS1 transgenic mice were modified with APP/PS gene and belonged to C57BL/6J strain. At about 10 months of age, they developed plaques; gliosis; synaptic and neuronal loss; cerebral vascular amyloidosis; spatial and learning disabilities[17]. As A precursor protein of Aβ, APP affects the production of Aβ. The overexpression of Aβ protein can form senile plaques (SP) and affect the pathological process of AD. APP gene is located on chromosome 21q11.2-22.2, which consists of 18 exons and 17 introns. Its gene product is translated from the APP gene by selective splicing. APP has at least six splicing forms, of which 695, 751 and 770 amino acid residues are the main forms. In the hippocampal gyrus and cortex of AD patients, the ratio of APP695/APP751 was abnormal and positively correlated with the number of plaques. APP mutation can lead to the production of neurotoxic Aβ42 and trigger a variety of pathological mechanisms, which promote apoptosis or death of nerve cells and eventually lead to AD[18]. The PS1 gene is located on chromosome 14 and consists of 10 exons. PS-1 gene is on chromosome 14 and the PS-2 gene is on chromosome 1 jointly control the formation of the γ-secretase complex (APP splice enzyme), which plays an important role in the process of Aβ production. PS1 mutation causes loss of the hydrophilic ring domain of the protein which was encoded by PS1, affects the structural stability of γ-secretase complex, the downstream APP cleavage process, and increases the production of Aβ42, which is one of the main causes of familial AD.
Hippocampus is the most specific part in the course of AD[19], and the first part to change in the early stage of AD[20]. This was consistent with our quantitative proteomics observation that the expression of APP and PSEN was significantly upregulated and the deposition of Aβ amyloid protein in brain region. Alzheimer's disease (AD) is a common neurodegenerative disease with insidious progression. At present, there is no effective treatment. Therefore, early diagnosis and identification are particularly important for patients with AD. Previous studies have indicated that in AD patients, neuropathic lesions of the olfactory cortical structures in the hippocampus are present, Such as reduced neurite connections and loss of neurons. Therefore, some researchers at home and abroad have concluded that the olfactory cortex and hippocampal atrophy may be important sensitive indicators for the early diagnosis of AD[21]. Studies have shown that the hippocampal volume of AD patients is smaller than that of mild cognitive impairment patients and normal subjects, Hippocampal volume size and MMSE score were a significantly positive correlation. In addition, the more obvious hippocampal atrophy is in AD patients, the more severe the degree of cognitive impairment is. Therefore, the hippocampus has a certain reference value in the early diagnosis of AD[21–23]. Therefore, the study of the hippocampus of the APP/PS1 double transgenic rat model is the first choice to study the potential pathogenic mechanism of Aβ and SP in the pathological characteristics of AD[24].
4.2 4D-Label Free quantitative proteomics and PRM results analysis
Quantitative proteomics is a commonly used technology in the study of screening early diagnostic markers of related diseases. Compared with traditional 3D proteomics, 4D label-free quantitative proteomics has the characteristics of rapid, high-throughput and high sensitivity, which is conducive to the early diagnosis of diseases and the screening of candidate markers, has great clinical value and research significance[25, 26]. PRM is a targeted verification proteomics technology based on mass spectrometry, which is a perfect alternative to Western blot and Elisa. The advantage is it can overcome the difficulty of antibody preparation. At the same time, the verification rate has been greatly improved[27].
In this study, the hippocampus of APP/PS1 double transgenic mice was used as the research object, and 4D label-free quantitative proteomics and targeted proteomics technology were used as the research means. Hippocampus was excised from the mice and subjected to 4D label-free and high-resolution LC-MS/MS analyses for quantitative proteomics studies. A total of 4928 were quantifiable. After the database was searched, the quality control evaluation was carried out from the distribution of peptide length, peptide per protein, protein coverage distribution, and protein mass distribution to ensure that the quality of the results met the standard. Then PCA and RSD were performed to detect and visualize variations in the hippocampus between the two comparison groups. The PCA results showed that both groups shared good similarities within the group, and the difference between the groups is obvious. The RSD results also indicated the intra-group differences were small. If a fold change ≥ 1.5 and ≤ 1/1.5 were set as the thresholds of up-regulated and down-regulated proteins, respectively. In the model group vs. the control group, twenty-nine proteins were up-regulated and twenty-five proteins were down-regulated. The fifty-four DE proteins were further enriched and analyzed, we performed a functional classification based on Subcellular localization, GO terms, and a KOG analysis, we found that ribosomal structure and biogenesis possibly play critical roles in AD.
Next, we conducted GO classification and KEGG pathway enrichment analyses for DE proteins in each comparison group, aiming to find out whether differentially expressed proteins have significant enrichment trends in certain functional types. Then, we visualized the protein-protein interaction (PPI) based on a confidence score > 0.7 (high confidence) for DE proteins. At last, we used PRM to validate the target Proteins. Combined with the results of BP\MF\CC\KEGG enrichment analysis, PPI interaction visualization, and PRM validation, it is suggested that ribosomal-related proteins Rpl18, Rpl17, Rpl19, Rpl24, Rpl35, and Rpl6 may be related to the potential pathogenesis of AD.
4.3 Ribosomal proteins and Alzheimer's disease
Ribosomes are mainly synthesized in the nucleolus (the largest subnuclear organelle), which is a membraneless and highly dynamic structure. Nucleolus components include rRNA, rDNA, and ribonucleoprotein. A nucleolus is a place for rRNA gene storage, rRNA synthesis and processing, and assembly of ribosomal subunits. In biological cells, ribosomes act as factories that move along mRNA templates to perform protein synthesis functions. The number of ribosomes in eukaryotic cells can reach 106.Ribosomes are complex enzyme systems composed of Ribosomal RNA (rRNA) and Ribosomal protein (Rp). Only within this overall structure can individual enzymes or proteins have catalytic activities and jointly undertake the task of protein biosynthesis. In eukaryotes, the 60S large subunit and the 40S small subunit assemble together to form the mature ribosome, the large subunit was composed of 5S, 5.8S, 28S rRNA, and 49 RPs, and the small subunit was composed of 18S rRNA and 33 RPs[28, 29]. The large ribosomal subunit is responsible for carrying Aminoacyl-tRNA (AA-tRNA), peptide bond formation, and binding of AA-tRNA to peptide chains. The A site, P site, and transpeptidase center are also located in the large subunit. The small ribosomal subunit is responsible for the specific recognition of mRNAs, such as the recognition of the initiation part and the interaction between codons and anticodons. The mRNA binding site is also on the small subunit.
Ribosomal RNA (rRNA) is the most abundant type of RNA in cells, accounting for 82% of the total RNA. It is also the type of RNA with the largest molecular weight. Combined with proteins, it can form ribosomes, whose function is to synthesize amino acids to peptide chains under the guidance of mRNA. Just only rRNA itself cannot perform its function, but also needs to combine with a variety of proteins to form ribosomes, which serve as the "assemble machine" for protein biosynthesis. In humans, rRNA genes are located in the short arm's proximal centromere of chromosomes 13, 14, 15, 21, 22. The exception is the 5S RNA gene, which is located in the nuclear plasma. The rRNA gene was transcribed to produce precursor 47S rRNA, which was modified and processed to form 28S, 18S and 5.8S rRNA. These rRNAs are assembled with ribosomal proteins to form large and small subunits, which are then exported from the nucleus. Finally, mature ribosomes are generated in the cytoplasm. Rp is an important component of the ribosome, which participates in ribosome assembly and stabilizes rRNA structure to improve translation efficiency and accuracy[30]. Studies have shown that Rp not only participates in protein synthesis but also plays an important role in the cell cycle, cell division, cell apoptosis and DNA damage repair[31].Ribosome synthesis is rigorous and orderly, and damage to any step may affect protein synthesis[32–34], therefore, strictly controlling the expression of the Rp and rRNA for normal cell physiology function is very important.
Ribosomes are the main sites for the synthesis of nascent peptides. Compared with mature proteins, partially folded nascent peptides are metastable and more prone to misfolding [35, 36]. When the protein is misfolded, it loses its normal function, clogging cellular processes and creating toxic aggregates. Protein aggregation has been linked to a variety of aging-related diseases, like Alzheimer's disease[37]. Since the speed of ribosomes varies depending on their location during translational elongation, elongation deceleration leads to ribosome collisions and degradation of nascent peptides and transcripts[38–41], destruction of translation dynamics or co-translation processing, and reduction of cell fitness, leading to aggregation of nascent proteins and folding of co-translated proteins[42–44], eventually leading to neurological degeneration[45–47].
Yingchao Li et al[48] through hippocampal, cortical proteomics and bioinformatic analysis indicated that aging is relevant to abnormal expression of proteins related to the ribosome (RPL4, RPS3). The researches show that dozens of transcripts encoding ribosome biogenesis and protein synthesis machinery components were specifically down-regulated with age at the translational level, consistent with the decline in protein synthesis with age[49]. Similarly, researchers also discovered that a large amount of ribosomal proteins had different expression with older age. Especially, all 60S and 40S ribosomal proteins were entirely reduced in aging muscle; except 60S ribosomal proteins RPL12 and RPL3, which were overrepresented in aging muscle[50]. These findings show that ribosomal protein expression decreases with age. However, in this research, the ribosomal protein expression found in the AD samples was elevated, we speculate that the increase was not due to age, but due to the pathological factors of AD. Masayoshi et.al[51] by employing comprehensive and accurate quantitative proteomics in Alzheimer’s disease (AD)to explore the AD molecular mechanism. The results indicate that of the 29 ribosomal proteins that were quantified, 28 (RPLP0, RPL4, RPL6, RPL7A, RPL8, RPL10A, RPL11, RPL12, RPL14, RPL15, RPL18, RPL23, RPL27, RPL27A, RPL31, RPL35A, RPS2, RPS3, RPS3A, RPS4X, RPS7, RPS8, RPS14, RPS16, RPS20, RPS24, RPS25 and RPSA) were significantly upregulated in AD patients. This is in preliminary agreement with our experimental results. Therefore, they put forward that the increase of ribosome function is a common phenomenon in Alzheimer's disease and drug treatment of the disease is positively correlated with the inhibition of ribosome biosynthesis[51]. Especially, Donepezil, which is clinically used AD drugs, has also been reported to inhibit ribosome biosynthesis[52]. Ribosomal protein L6 (Rpl6) is a large subunit of ribosomal constituent proteins, and abnormal expression is related to cell damage and cell proliferation[53]. RPL6 encodes 60S ribosomal subunit that plays a vital role in oxidative phosphorylation, synaptic transmission, and neuronal signaling in Parkinson Disease (PD)[54]. When a DNA double-strand break occurs, Rpl6 is recruited to the DNA damage site, enhanced by interaction with histone H2a, and participated in the DNA damage response (DDR)[55], H2a was also differentially expressed in this study, suggesting that Alzheimer's disease may be pathogenic by affecting the DNA damage response.
The present experimental data indicated that ribosomal proteins Rpl18, Rpl17, Rpl19, Rpl24, Rpl35, and Rpl6 were significantly up-regulated in the hippocampus of APP/PS1 mice, which may be a potential pathogenic mechanism. However, the underlying regulatory association mechanism of ribosomal proteins remains unclear. And how to cause or aggravate AD and the sequence of occurrence with AD still need to be further explored. In addition, abundant cerebral blood supply is a decisive factor for maintaining normal function of the brain region. Previous studies have shown that hippocampal blood flow is positively correlated with cognitive function, and the hippocampus is particularly sensitive to hypoperfusion. Studies also shown that the ribosome protein in the hippocampus of AD patients is significantly upregulated, but it is only found in the brain capillary region, not in the brain parenchyma region, Therefore, the relationship between ribosomal proteins and brain capillaries needs further investigation in the future. In addition, each subregion of hippocampus encodes different memory information, which is related to various factors such as Aβ deposition and Tau protein hyperphosphorylation. However, the research on the hippocampal subregion is still in the stage of basic research, which can be further studied in the future.