Introduction
Adamantinomatous craniopharyngioma (ACP) is a rare epithelial tumor that originates from the residual cells of the Rathke sac during embryonic development [
1,
2]. Its occurrence is associated with activation of the Wnt/β-catenin signal transduction pathway, which promotes the occurrence and development of tumors [
2]. ACP is mainly seen in children, and the cystic formation is a typical pathological characteristic which occurs on over 90% of ACP patients [
2‐
4]. ACP is a benign tumor of WHO grade I. At present, total resection is still the main treatment method. However, follow-up data from a large number of ACP cases show that there is a widespread severe quality of life impairment among the survivors , including growth retardation and obesity, which are mainly related to hypothalamic damage [
3,
4].
The hypothalamus, which regulates energy metabolism [
5,
6] and body growth [
7,
8], is vulnerable to injury due to its close proximity to the ACP [
9]. The arcuate nucleus (ARC), an important hypothalamic nucleus that mainly includes neuroendocrine cells and concentrated projection neurons [
6,
10,
11], could regulate GHRH neurons (growth hormone-releasing hormone, GHRH) and SST neurons (somatostatin) to control the secretion mode of growth hormone [
12]. At the same time, ARC activates or inhibits neuropeptide Y (NPY) and agouti-related protein (AgRP) to control feeding behavior and energy metabolism [
13], which is closely related to the onset of obesity and type 2 diabetes [
14].
ACP is mostly composed of cyst fluid and its surrounding cyst wall, with a few substantial components [
15]. The composition of cyst fluid is complex and usually rich in cholesterol and lipids, as well as various proinflammatory factors [
16‐
18]. Our team discovered in previous studies that the production of ACP cystic fluid may be related to the disorder of lipid metabolism in tumor cells [
15]. Lipid metabolism disorder was closely related to inflammation of the hypothalamus [
19,
20]. Compared with those in other brain tumors, the levels of inflammatory factors in ACP cystic fluid and solid components are increased [
21]. It has been reported that the leakage of cyst fluid can cause severe chemical meningitis [
22], and it was previously reported that the injection of cyst fluid into the cortex of rats caused increased expression of markers of inflammation and cell damage and increased body weight [
23]. In vitro experiments have shown that cyst fluid can cause degenerative changes and apoptosis of neurons [
24]. More and more data proved that ACP cystic fluid may be a key factor of hypothalamic inflammatory injury. However, there is currently no reliable ACP cystic fluid-hypothalamus injury animal model for in-depth study of the specific mechanism by which cyst fluid induces hypothalamic injury. The effect of cystic fluid, which is the product of ACP lipid metabolism disorder, on the hypothalamus is still unclear.
The lack of stable cell lines and benign characteristics make it difficult for ACP cells to form the transplanted tumor in hypothalamus; therefore , a study on the cystic fluid, product of ACP cells lipid metabolism disorder, is very necessary and feasible. To explore the effect of ACP cystic fluid on the hypothalamus, this study used stereotactic technology to inject human ACP cystic fluid into the bilateral hypothalamus of 6-week-old male C57 mice as the cystic fluid group, injected the same amount of PBS as the sham operation group, a blank control group received no treatment. After 8 weeks, ACP cystic fluid caused growth retardation, increased the obesity index, and decreased plasma GH and GHRH levels. To determine whether changes in the expression of genes regulating body growth and energy metabolism occurred in hypothalamic neurons, we performed single-cell RNA sequencing on the hypothalamus of mice from the cystic fluid group and the sham operation group and found that ACP cyst fluid significantly affected the expression of the Sst, Fgfr2, and Rnpc3 genes, which regulate body growth and development [
12,
25‐
28], and the expression of the Npy and Pcsk1n genes, which regulate energy metabolism in hypothalamic neurons [
13,
14,
29‐
33], and caused a significant upregulation of the Agrp–Mc3r cellular interaction, which regulate food intake [
34‐
36], between Agrp/Npy neurons and Ghrh neurons. We unexpectedly found that ACP cystic fluid caused inflammatory activation of hypothalamic microglia. We found for the first time that the cellular interaction of CD74–APP, previously found in neurodegenerative diseases [
37], is significantly strengthened between inflammation activated microglia and hypothalamic neurons. Using single-cell RNA sequencing, we also found the same phenomenon of inflammatory activation of microglia in ACP tumor tissue as in mice. Aβ, a marker of neurodegenerative diseases [
37,
38], is deposited in the hypothalamus of mice injected with ACP cyst fluid and in ACP tumor tissues (Additional files
1,
2,
3,
4).
We report here that ACP cystic fluid induces inflammatory activation of mouse hypothalamic microglia. Activated microglia may mediate a mechanism similar to that observed in neurodegeneration of hypothalamic neurons and participate in this process, which may damage the hypothalamus, leading to growth retardation and obesity.
Discussion
Although ACP is a benign tumor, the poor quality of life caused by hypothalamic injury has been difficult to solve [
3]. The hypothalamus, a center that maintains hormone balance, energy metabolism, and homeostasis, is closely adjacent to the ACP, making it the most susceptible structure to be violated by the ACP, causing hormonal, metabolic, and electrolyte disorders and leading to lifelong medical care for patients [
58]. Although the mainstream view is that the tumor compression and surgical trauma cause damage to the hypothalamus [
59,
60], the effect of cyst fluid, a unique component of ACP, on the hypothalamus is unknown. To eliminate the interference of surgical trauma and tumor compression, we pushed the tip of the microinjector into the upper part of the bilateral hypothalamus of the mouse and then slowly injected the cyst fluid or PBS to avoid mechanical damage as much as possible to allow the fluid to penetrate into the hypothalamus. After 8 weeks, the mice in cystic fluid group showed growth retardation and obesity tendency, and the decrease in serum GH and GHRH and the short-term increase in food intake were corresponded to the appropriate phenotype. Growth retardation and obesity are the most intuitive manifestations of hypothalamic injury [
58]. In the rat hypothalamic electrical damage model [
61], the phenotypes of shorter body length, abnormal obesity, and continuous increased food intake implying that although the damage caused by ACP cystic fluid in the hypothalamus is not as strong as electrical damage, it still has a deleterious effect on the hypothalamus. ARC is a nucleus that regulates energy metabolism and body growth [
12,
13]. We found that the expression of the Fgfr2 and Rnpc3 genes was significantly decreased in the ARC neurons of mice in the cystic fluid group. Their knockout and mutation caused dwarfism in mice and humans [
25‐
28]. At the same time, the Npy and Pcsk1n genes were significantly highly expressed in ARC neurons in the cyst fluid group. In addition, ACP cystic fluid caused high expression of the Sst gene in the mouse hypothalamus, and the growth inhibitory hormone encoded by Sst antagonizes growth hormone [
12] and the activation of Sst neurons promote food intake and obesity [
49]. Pcsk1n is an endogenous inhibitor of Pcsk1. The high expression of proSAAS encoded by Pcsk1n causes obesity and diabetes in mice [
29]. Pcsk1 gene mutations are common in obese human populations [
30,
32]. The significant changes in the expression of the above genes in ARC neurons of mice in the cystic fluid group may help explain the growth retardation and increased obesity index observed in mice and provide new target genes for the further study of ACP. The hypothalamic orexin system includes Npy/Agrp, Pomc, Mc4r, and Mc3r [
36]. Pomc inhibits food intake by exciting Mc4r and Mc3r; in contrast, Npy/Agrp promotes food intake by inhibiting Mc4r and Mc3r [
35]. Npy/Agrp and Pomc maintain the balance of energy metabolism through Mc4r and Mc3r [
34,
36]. The significant enhancement of the Agrp–Mc3r interaction between Npy/Agrp neurons and Ghrh neurons seems to imply that ACP cystic fluid disrupts the orexin system in the hypothalamus.
As previously shown, ACP cystic fluid has a damaging effect on neurons in vitro [
24] ; the abovementioned gene changes may be caused by the direct damage of cystic fluid to ARC neurons. However, through pseudochronological analysis and GO pathway enrichment analysis, we found that ACP cystic fluid caused inflammatory activation of microglia and increased expression of IL-6, which was also verified in PCR experiments. Pseudochronological analysis suggested that activated microglia mainly exhibit inflammation activation, increased synthesis of proinflammatory factors, activation of inflammation-related pathways, increased response to lipoprotein particle stimulation, and beta-amyloid (Aβ) synthesis and response. The signal that positively regulates neuronal apoptosis is significantly strengthened in ARC neurons, which is similar to the results of in vitro experiments previously reported by Ghosh et al. [
24]. It is worth noting that beta-amyloid (Aβ) produced by APP, a pathological sign of Alzheimer's disease, causes inflammatory damage to neurons and is responsible for the degeneration of synapses and the apoptosis of neurons [
38]. In a study of Alzheimer’s disease (AD), authors discovered the cell interaction between CD74 and APP for the first time and proposed that the high expression of CD74 in the brains of AD patients may be caused by the overexpression of APP in AD patients, which may contribute to a biological negative feedback mechanism to resist the upregulation of harmful APP processing [
37]. This hypothesis suggests that the CD74–APP interaction is a way for the body to fight neurodegenerative diseases. In this study, the significantly enhanced CD74–APP interaction between inflammatory activated microglia and ARC neurons in the mouse hypothalamus suggested that ACP cystic fluid may cause neurodegenerative lesions in the hypothalamus. The COPI coat plays a role in the processing and transport of amyloid precursor protein (APP), and α-COP (COPA) is a member of the heptameric COPI outer shell complex. Studies have shown that knocking down COPA inhibits the maturation and cleavage of APP, resulting in a decrease in the release of Aβ, and the high expression of COPA promotes the release of Aβ, increasing the toxic effect on neurons [
54]. In our study, the high expression of Aβ and the enhanced interaction of CD74–COPA in the mouse hypothalamus further suggested that ACP cystic fluid may cause pathological changes similar to those observed in neurodegenerative diseases.
In ACP tumor tissues, we also verified the same phenomenon of inflammatory activation of microglia observed in mice. Activated microglia also showed inflammatory activation, increased synthesis of proinflammatory factors, activation of inflammation-related pathways, increased response to lipoprotein granule stimulation, and increased synthesis and response of beta-amyloid (Aβ). Interestingly, in ACP tumor tissues, we found that CD74–APP and CD74–COPA interactions were also significantly strengthened between inflammatory activated microglia and neurons. CD74–APP, an intercellular interaction that is unique to Alzheimer's disease [
37], was discovered for the first time in ACP tumor tissue. Although there are some gliosis bands in ACP tumor tissues, there is very little nerve tissue, so single-cell RNA sequencing cannot identify the specific types of neurons identified. The expression of MHC-II, APOE, and CD74 was also found on microglia (IBA1+) in the ACP nerve junction area. The expression of Aβ, the pathological marker of Alzheimer's disease [
38], in the whorl-like cells and gliosis zone of ACP tumor tissues suggests that a pathological change similar to neurodegenerative disease may also occur in the ACP tumor microenvironment. This is the first time that Aβ has been discovered in ACP and provides a new research direction for further study of ACP.
In addition, the single-cell RNA sequencing data indicated that the expression of CD68, an index of phagocytosis of microglia [
62], was increased in cluster 7 microglia in the cystic fluid group, and this was verified in tissue samples of ACP and hypothalamic tissues of mice. ACP cyst fluid is rich in cholesterol and other lipid particles. In the hypothalamus, these foreign bodies inevitably recruit a large number of immune cells for elimination. The pseudochronological analysis and GO enrichment analysis indicated that the expression of APOE in inflammation activated microglia was enhanced, and lipoprotein granule responses and Myd88-TLR-related pathway responses were also enhanced. We speculate that microglia can clear lipids in cyst fluid while activating inflammation-related pathways. Therefore, if we can control inflammation while enhancing the "noninflammatory phagocytosis" of microglia, it may be possible to inhibit the occurrence of hypothalamic-related inflammation caused by ACP cyst fluid. Notably, the senescent cells, which secrete SASPs (senescence-associated secretory phenotypes), including IL-6, are present in the ACP tumors [
63]. Martinez et al. [
64] found that SASPs play a critical role in the initiation of the ACP and promote inflammation in the nerve tissues surrounding tumors via the paracrine function. At the same time, the SASPs are associated with the pathogenesis of the neurodegenerative diseases [
65] ; therefore, the influences of the senescent cells in the ACP tumors on the microglia activation and on the Aβ deposition could not be excluded.
The ACP cystic fluid we injected into the hypothalamus of mice is present only temporarily. Although we simulated the leakage process of cyst fluid during ACP resection, Aβ, observed in the mouse hypothalamus injected with ACP cystic fluid, is also expressed in whorl-like cells and gliotic tissues of ACP tumors. In current studies, no Aβ was found in the analysis of the composition of ACP cystic fluid [
21,
66], which implies that the leakage of cystic fluid not only occurs during surgical resection but also may occur slowly during prolonged disease progression and lead to the production of Aβ. Interestingly, our team previously found that the key protein PPAR-γ [
15], induces adipogenic differentiation and promotes the formation of ACP cyst fluid, is also expressed in whorl-like cells. It was found that APOE uses cholesterol to transport neuronal amyloid precursor protein (APP) in and out of lipid clusters to promote the production of Aβ [
67]. In our experiment, the expression of APOE was upregulated in microglia both in ACP gliosis tissue and the hypothalamus of mice injected with ACP cystic fluid. This seems to imply that the lipid component (cholesterol) in cystic fluid is the key factor leading to the deposition of Aβ.
In short, we have an unprecedented understanding of ACP cystic fluid. Avoiding leakage of cystic fluid during surgical resection is a good measure to protect the hypothalamus from hormonal and energy metabolism disorders. The animal model established in this study is helpful for the research and treatment of ACP-related hypothalamic injury. At the same time, the Npy, Fgfr2, Rnpc3, Sst, and Pcsk1n genes, and the cell-to-cell interactions CD74–APP, CD74–COPA, and Agrp–Mc3r, and Aβ may provide potential targets for further study of ACP. In this study, a pathological phenomenon very similar to neurodegenerative diseases was found in the ACP tumor tissue, implying that medicines used to treat neurodegenerative diseases may help prevent ACP damage to the hypothalamus.
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