In this study, we used WGCNA method to construct a co-expression network of 2713 differential expression genes in 56 AD patients and 44 healthy controls, and finally identified 7 gene modules ranging in size from 32 to 1619. Correlation analysis of gene modules and disease phenotype showed that the turquoise module and the brown module were significantly associated with age. The turquoise module has obvious biological significance, which is related to synaptic signaling and MAPK signaling pathway. The hub gene in this module is ATL1. The Brown module is mainly related to molecular metabolism and cell growth. The hub gene of this module is CIRBP.
The algorithm of WGCNA software is to construct a gene co-expression network based on the similarity of expression profiles between samples, so as to systematically describe the gene expression information. It has advantages over traditional differential expression analysis and has been widely used in complex diseases. In this study, we identified seven gene modules using WGCNA, and the two modules with the strongest association with AD are the turquoise module and the brown module. The turquoise module has significant biological significance which is associated with synaptic signaling and MAPK signaling pathways. Synapses are the basic unit of memory storage and information transmission in the brain. Previous analysis of brain samples from AD patients showed that a large number of synaptic losses are closely related to cognitive decline. Various synaptic proteins, such as SNAP-25 (a presynaptic protein), PSD-95 (a post-synaptic protein), synapsin 1 and chromogranin B (synaptic vesicle protein), are reduced in AD patients brain[9, 10]. AD animal models also show defects in synaptic transmission as well as impairment of Long-term potentiation and Long-term depression. These results indicate an imbalance in synaptic function in AD.
MAPK is a serine/threonine protein kinase. The MAPKs pathway is an important signal transduction pathway in mammalian cells. It is a key bridge connecting internal and external cells. It transduces extracellular stimulation signals into cells and their nucleus and causes cellular biological response. It is involved in physiological processes such as cell proliferation, differentiation, apoptosis and stress response. In mammals, several different MAPKs have been identified, including p38 MAPK, c-junN-terminal kinase (JNK), extracellular signal-regulated kinase (EERK1/2) and ERK5/BMK-1. Amongst MAPK, p38 MAPK is widely involved in signaling pathways of different biological functions. In the central nervous system, p38 MAPK is highly expressed in areas critical for learning and memory and may be a key component of advanced brain function. By studying the brain tissue of the control group and AD patients after death, it was found that p38 MAPK was activated in the early stage of AD[13, 14]. Besides, the upstream activation molecule MAPK6 of p38 MAPK was also found to be up-regulated in the brain tissue of autopsy in AD patients. Several studies have found that pericytes are the main component of cerebral vascular composition, and they are degraded and reduced in the hippocampus and cortex of AD patients[16-18]. Xu et al. found that Aβ inhibits the differentiation of mesenchymal stem cells into pericytes by activating ERK1/2MAPK signaling pathway. Recent studies have confirmed that autophagy plays a role in AD. Interestingly, MAPK has a special function as a positive regulator and a negative regulator of autophagy[21, 22]. MAPK can promote autophagy through phosphorylation of its BCL2. These findings suggest that MAPK may be involved in the pathogenesis of AD. This is consistent with our results and reflects the reliability of WGCNA for identifying gene modules.
We use the TopHub in R to calculate the hub gene in the candidate module. The hub gene of the turquoise module is ATL1. Atlastin (ATL) belongs to the dynamin GTPase superfamily and is present in all vertebrates, as well as homologous proteins in many other organisms. Novel studies have found that membrane protein atlastin is very important for homologous membrane fusion of tubular endoplasmic reticulum[25, 26]. In neurons, the endoplasmic reticulum is tubular in the axons, and in the dendrites, it contains a network and a tubular form[28, 29]. Impaired endoplasmic reticulum function is associated with many diseases, such as hereditary spastic paraplegia(HSP). HSP is a degenerative disease of the nervous system. It is classified into the simple type and variant type according to clinical features. The simple type is characterized by bilateral lower extremity spastic paralysis. Variants may include other nervous system manifestations such as optic atrophy, retinal pigmentation, epilepsy, Deafness and neurodevelopmental delay. More than 50 genetic loci are genetics of HSP. Interestingly, many mutations in ATL-1 were found in HSP patients, suggesting that neuronal ER morphology may be associated with HSP pathogenesis. However, neuronal ER injury, the relationship between ATL-1 and HSP has not been determined, and how ATL-1 causes a neurodevelopmental delay in patients remains to be further studied. Interestingly, some studies have found that genetic mutations encoding spastin-SPAST can cause not only HSP but also AD. In HSP with SPAST mutation, spastin cuts down microtubule activity, whereas, in AD patient cell model, spastin is abnormally activated and induces microtubule breakdown. Therefore, whether there is similar pathogenesis between HSP and AD, whether ATL1 is also associated with the pathogenesis of AD remains to be further studied, which may provide a new direction for the diagnosis and treatment of AD.
CIRBP (cold-inducible RNA-binding protein) is an RNA-binding protein, also known as nuclear heterogeneous ribonucleoprotein, which is an 18kD protein of the glycine-rich RNA-binding protein family. CIRBP is the first cold shock protein found in mammalian cells, and it can regulate cell growth and apoptosis under cold induction. Similar to CIRBP, RBM3 (RNA-binding morif protein 3), which is also a cold-induced protein, is up-regulated under cold stimulation and can regulate transcription and translation . The reduction in the number of synapses is an early feature of neurodegenerative diseases. Diego Peretti found that in prion-infected mice and 5XFAD (AD) mice, the ability to regenerate synapses after hypothermia is reduced, which is related to the failure of RBM3 induction. RBM3 over-expression can be achieved by reducing the temperature to increase endogenous levels or by lentiviral delivery before RBM3 loses response, which can provide continuous synaptic protection for 5XFAD mice and prion-infected mice, thereby preventing behavioral defects and neurons lost and significantly prolonged survival. Therefore, the cold shock pathway can be used as a potential protective therapy in neurodegenerative diseases. Although much is known about the processes leading to synaptic dysfunction and loss, how it affects synaptic regeneration is still unknown. CIRBP, which is also a cold-inducible protein, can protect neuronal cells when its expression is increased. Whether it can also provide protection to synapses like RBM3 is yet to be further studied. It may bring new therapeutic targets for the neuroprotection of neurodegenerative diseases.
Our results indicate that the identification of specific modules and hub genes in AD by WGCNA provides new clues for further research in the future. But our study also has some limitations. Firstly, AD can be divided into early-onset and late-onset according to the age of onset. The pathogenesis of these two types is not consistent. As we lacked more clinical data, we did not classify AD in our study. Secondly, we identified the key genes related to AD from microarray data analysis, which should be further verified in vitro or vivo experiments. Although we found some positive results, the sample size is still small. A large number of clinical samples will be needed in the future to validate our clinical results and to clarify the underlying mechanisms by which key genes affect AD.