Many researchers believe that more in-depth studies on novel molecular mechanisms and pathological changes in AD are crucial for identifying new drug targets. However, the mechanism by which CIRP affects the development of AD is still unclear.
To gain novel insights into the functions of CIRP in the pathogenesis of astrocytes, we comprehensively analyzed the mRNA profiles of 3 pairs of control and ov-CIRP human astrocytoma cell lines. We identified the significantly differentially expressed mRNAs in all 3 pairs of cells and annotated their functions. Previous studies have reported that CIRP is widely involved in various biological processes, including circadian modulation, cell proliferation and survival, telomere maintenance, cellular stress response, inflammation and cancer [14]. Here, the microarray data also revealed that the changes in mRNAs induced by CIRP overexpression were implicated in the response to stimuli, inflammatory bowel disease, cell growth and so on, which was in accordance with previous studies (Fig. 2, 3). Moreover, our work revealed a comprehensive transcriptional network implying that the abnormal expression of CIRP in astrocytes was associated with a series of nervous system diseases, such as autism spectrum disorder, Parkinson's disease (motor and cognitive disorders), bipolar disorder, schizophrenia, and AD (Table 2).
Table 2
The relative disease of CNS after CIRP over-expression
Term | Database | Input gene symbols | P-Value |
Autism spectrum disorder | NHGRI GWAS Catalog | IFI44,MAP4K4 | 0.0171048995095 |
Parkinson's disease (motor and cognition) | NHGRI GWAS Catalog | C8orf | 0.0253314272708 |
Normalized brain volume | NHGRI GWAS Catalog | BICD1 | 0.0253314272708 |
Response to antidepressant treatment | NHGRI GWAS Catalog | DTWD1 | 0.0581276711223 |
Bipolar disorder and schizophrenia | NHGRI GWAS Catalog | LRRIQ3 | 0.1204990915480 |
Alzheimer's disease | GAD | TCN2,PLAU,PSEN2, | 0.0278533148062 |
Hippocampal atrophy | GAD | PRUNE2 | 0.0800292664413 |
Depression | GAD | ARGLU1 | 0.1578254639020 |
Attention deficit disorder with hyperactivity | GAD | URB2 | 0.1673380884350 |
Neuro-degenerative diseases | KEGG DISEASE | ANO10, PSEN2, | 0.3888821624070 |
It is well known that the pathological damage of AD is related to the accumulation of extracellular senile plaques composed of aggregates of Aβ peptides and the hyperphosphorylation of Tau [15]. Wang et al reported that eCIRP activated STAT3 via IL-6Rα and speculated that eCIRP-derived microglia may be important mediators of neuronal tau phosphorylation after exposure to alcohol [10]. Here, to explore the function of CIRP in astrocyte-induced neuronal injury, the phosphorylation of p-Tau and the expression of BACE and Aβ1–42 in neurons were measured after they were cocultured with ov-CIRP or control astrocytes. Our data revealed for the first time that the overexpression of CIRP in astrocytes greatly promoted the expression of p-Tau (Ser396), Aβ1–42, and BACE in neurons in a coculture system. These results proved that CIRP in astrocytes could act as a vital mediator of AD.
Finally, we explored the molecular mechanisms by which CIRP in astrocytes regulates neuronal tau phosphorylation. Microarray analysis revealed three mRNAs in astrocytes that are candidates for AD after CIRP overexpression. Among them, uPAs gained our attention. uPA encodes a secreted serine protease that converts plasminogen to plasmin, and mutation of uPA is closely associated with the onset of Alzheimer's disease. Thus, we measured the expression levels of uPA in control and ov-CIRP astrocytoma cells by q-PCR and WB assays. The results confirmed a significant decrease in uPA in ov-CIRP astrocytes compared with control astrocytes at both the mRNA and protein levels.
Merino et al. reported that uPA and its receptor were mainly expressed in neuronal extensions, growth cones and a subpopulation of astrocytes in the mature brain, and the release of uPA in the mature central nervous system could improve axonal and synaptic function after injury[16, 17]. Moreover, increasing evidence has shown that the plasminogen activator urokinase can protect neurons from amyloid β-triggered synaptic injury[18, 19]. On the basis of this evidence, we speculated that the neuronal damage induced by CIRP overexpression in astrocytes may involve decreased expression of uPA. Thus, we added uPA to an astrocyte neuron coculture system and found that uPA treatment significantly alleviated ov-CIRP astrocyte-induced dysfunction of neurons, as evidenced by decreased Aβ1‒42 accumulation and tau hyperphosphorylation in the ov-CIRP + uPA group. These results implied that the overexpression of CIRP in astrocytes could cause AD-like alterations in neurons partly through the downregulation of uPA. Notably, previous studies have focused mainly on the function of neuronal uPAs in AD. Here, we revealed for the first time that abnormal expression of uPAs in astrocytes is another cause of neuron dysfunction. Our data confirmed the significant role of CIRP in the development of AD, provided insight into the molecular signaling involved in CIRP-induced neuronal damage, and provided a possible new therapeutic target for alleviating cognitive decline during AD. However, the current work explored the roles of CIRP only in astrocytes at the cellular level, and further studies should be carried out in animals. Moreover, the mechanism by which CIRP affects the levels of uPA is still unclear.
In summary, this study revealed that the overexpression of CIRP in astrocytes exerts a harmful impact on neurons by elevating the expression levels of BACE, Aβ1–42 and p-Tau (Ser396) in neurons via the downregulation of uPA (Figure. 7). Thus, the downregulation of CIRP expression may be a useful method for alleviating synaptic dysfunction during AD.