Time：2023-02-03 10:14:24 View:
Chemotherapy-related cognitive impairment (CRCI) is a kind of cognitive impairment induced after chemotherapy administration. Its common clinical manifestations include deficits in attention, memory, multitasking ability, decision-making ability, and learning and language abilities [1,2]. Previous studies have reported that over 60% of patients treated with systemic chemotherapy have developed chemotherapy-related cognitive dysfunction, and this condition may persist for several years even after chemotherapy is discontinued [3]. The existing mechanisms mostly revolve around the direct toxic effects of drugs on nerve cells, including the destruction of the blood-brain barrier (BBB), the loss of neurons in the hippocampal region, white matter damage, neuronal inflammatory damage, oxidative stress, etc. However, the exact underlying mechanisms have not been fully elucidated [4]. Therefore, analyzing the new mechanisms of the occurrence and development of CRCI will be conducive to drug research and development and the optimization of clinical treatment plans, thereby alleviating the great pain caused by chemotherapy to patients.
The neurovascular unit (NVU) refers to the study of cerebral blood vessels (endothelial cells, pericytes) and nerve cells (glial cells, neurons) as a whole. Since its proposal, it has provided new opportunities for the research on the pathogenesis of various neurological diseases and the screening of drug targets [5]. It is reported that the chemotherapy drug bortezomib cannot pass through the BBB, but there are clinical cases and imaging results indicating that the use of bortezomib can induce central adverse reactions including cognitive impairment [6-8]. Then, does bortezomib indirectly cause chemotherapy-related cognitive dysfunction by inducing vasogenic injury, and what are the molecular and cellular mechanisms behind it? It awaits further in-depth exploration.
January 1, 2023 The research group of Professor Yingmei Lu from the School of Basic Medical Sciences of Nanjing Medical University and the research group of Professor Feng Han from the School of Pharmacy jointly published an article titled "Endothelial TFEB signaling-mediated autophagic disturbance initiates" in Autophagy Research on "microglial activation and cognitive dysfunction" Professors Lu Yingmei and Han Feng are the co-corresponding authors of the paper, and doctoral student Lu Yaping is the first author. This study reports the relationship between cognitive dysfunction induced by the chemotherapy drug bortezomib and autophagy disorders in cerebrovascular endothelial cells. It also revealed that abnormal TFEB-STAT3-IL23A signaling in cerebrovascular endothelium plays a crucial role in the activation of microglia and the disease process of cognitive dysfunction. (Extended Reading: Research progress of Lu Yingmei/Han Feng's team, for details, see the "Logic Neuroscience" report (click to read) : STTT/Lu Yingmei/Han Feng's team collaboration Reveals the Molecular Mechanism of ataxia caused by abnormal circuits from the IV/V lobes to the parietal nucleus of the cerebellum)
In this article, researchers found that after mice were injected with the chemotherapy drug bortezomib (1 mg/kg), cognitive-related behavioral abnormalities occurred (Figure 1A-C). At the same time, the number of excitatory and inhibitory synapses in the hippocampal brain region decreased (Figure 1D-F), while quantitative and morphological analyses of microglia in the hippocampal and prefrontal cortex indicated that microglia were overactivated (Figure 1G-M) and had enhanced synaptic phagocytic activity (Figure 2F-I). The above results suggest that bortezomib treatment leads to the activation of microglia, as well as the loss of synaptic and cognitive functions.
Figure 1 Bortezomib treatment mediates microglial activation, synaptic and cognitive function loss（Pictrue：Lu YP, et al., Autophagy, 2023）
Here, researchers used the CSF1R inhibitor PLX5622 to clear microglia in the brain and found that eliminating overactivated microglia reversed bortezomi-mediated synaptic and cognitive deficits (Figure 2). It is concluded that the activation of microglia induced by bortezomib is a key factor in synaptic and cognitive dysfunction.
Figure 2 shows that the clearance of microglia restores the synaptic and cognitive deficits induced by bortezomib（Pictrue：Lu YP, et al., Autophagy, 2023）
Cerebrovascular endothelial cell injury is associated with a variety of neurological diseases [9,10]. The RNA-seq results indicated that bortezomib treatment led to abnormal autophagy-lysosomal pathways in cerebrovascular endothelial cells (Figures 3A, B). The researchers used double-labeled LC3 adenovirus to label the changes in autophagic flux, and the results indicated that bortezomib treatment inhibited autophagic flux in cerebrovascular endothelial cells (Figure 3C-E); Furthermore, bortezomib treatment caused excessive accumulation of the autophagy receptor molecule SQSTM1 in cerebrovascular endothelial cells (Figure 3F-I). It was demonstrated that bortezomib mediates endodermal autophagy disorders, including the inhibition of autophagic flux and the accumulation of autophagic substrates, suggesting that the above phenomena may be caused by the functional damage of lysosomes.
Figure 3 Bortezomib treatment mediates autophagy disorder in cerebrovascular endothelial cells（Pictrue：Lu YP, et al., Autophagy, 2023）
The transcription factor EB (TFEB) regulates the expression of lysosomal biosynthesis and autophagy-related genes in organisms [11,12]. Researchers found that bortezomib treatment reduced nuclear transfer of TFEB and down-regulated the expression of its downstream lysosomal membrane-associated proteins LAMP1 and LAMP2A. The number of LGALS3-labeled damaged lysosomes increased, while the number of LAMP1-positive healthy lysosomes decreased (Figure 4). The above results indicate that bortezomib treatment causes lysosomal renewal disorders, and the damaged lysosomes accumulate excessively within the cells, suggesting that the lysosomal dysfunction mediated by abnormal expression of endothellum-derived TFEB is the fundamental cause of the disorder of autophagy in the cerebral vascular endothelium.
Figure 4 Bortezomib injury in cerebrovascular endothelial cells promotes TFEB nuclear transfer and lysosomal dysfunction（Pictrue：Lu YP, et al., Autophagy, 2023）
Previous studies have shown that an increase in Ca2+ influx can activate calcineurin PPP3, thereby mediating the nuclear transfer of TFEB and promoting the enhancement of lysosomal function [13]. Researchers conducted whole-cell patch clamp recording of cerebrovascular endothelium and found that the cardiac glycoside drug digoxin could restore the reduction of Ca2+ influx mediated by bortezomib (Figure 5A-E). Reverse the inactivation of calcium-regulated phosphate PPP3 caused by bortezomib (Figure 5F-H). The above results suggest that digoxin enhances PPP3 activity by increasing Ca2+ current in cerebrovascular endothelial cells and participates in regulating the TFEB-mediated autophagolysosomal pathway.
Figure 5 shows that digoxin induces an increase in intracellular Ca2+ current to restore bortezomi-mediated PPP3 inactivation（Picture：Lu YP, et al., Autophagy, 2023）
The researchers analyzed the RNA-seq results and found that bortezomib treatment activated the inflammatory signaling pathway in cerebrovascular endothelial cells, significantly upregulated the expression of IL23A, and increased the content of IL23A in serum and the supernatant of cerebrovascular endothelial cell culture (Figure 6A-D). In vitro and in vivo experiments demonstrated that combined digoxin treatment significantly inhibited bortezomib-mediated increase in cerebrovascular endothelial IL23A expression, and restored bortezomib-mediated microglial activation and cognitive dysfunction (Figure 6E-M). The results of Golgi staining also showed that digoxin treatment effectively restored bortezomi-mediated reduction in spine number (Figure 6 N-R). Therefore, the above results indicate that digoxin inhibits the expression and secretion of IL23A, reverses the activation of microglia caused by bortezomib in vivo, and alleviates the loss of synaptic and cognitive functions.
Figure 6 Digoxin inhibits the expression of IL23A in cerebrovascular endothelial cells and restores bortezomi-mediated cognitive loss（Picture：Lu YP, et al., Autophagy, 2023）
To further clarify the relationship between the upregulation of IL23A expression in cerebrovascular endothelium and abnormal autophagy mediated by TFEB, researchers confirmed through JASPAR database screening and dual-luciferase reporter gene analysis experiments that there is a STAT3 binding site in the IL23A promoter region and it can promote IL23A transcription. Western blotting experiments indicated that bortezomib increased the expression of the transcriptional activation form of STAT3, p-STAT3 (Y705, S727), while combined digoxin treatment inhibited its upregulation. To further clarify the relationship between TFEB and STAT3, researchers knocked out TFEB in HEK293 cells using CRISPR-Cas9 technology and found that TFEB knockout increased the expression of p-STAT3 (Y705, S727), while restoring TFEB expression inhibited the upregulation of p-STAT3. The above results demonstrate that TFEB negatively regulates the transcriptional activity of STAT3 and promotes the expression of IL23A.
Figure 7 shows that TFEB negatively regulates the transcriptional activity of STAT3 to promote the expression of IL23A（Pic：Lu YP, et al., Autophagy, 2023）