Graphical Abstract Highlights d Islet C3 expression is upregulated in human T2D and rodent models of diabetes d C3 is present within the cytosol and binds autophagy-related protein 16-1 (ATG16L1) d b cells lacking C3 have impaired autophagy d Intracellular C3 protects b cells from palmitic acid/IAPPmediated apoptosis In Brief King et al. show that the main complement protein, C3, is expressed intracellularly in pancreatic b cells. C3 binds ATG16L1, thus regulating autophagy and protecting b cells from death from various insults. These findings highlight a novel intracellular protective role of this major immunological protein. SUMMARYWe show here that human pancreatic islets highly express C3, which is both secreted and present in the cytosol. Within isolated human islets, C3 expression correlates with type 2 diabetes (T2D) donor status, HbA1c, and inflammation. Islet C3 expression is also upregulated in several rodent diabetes models. C3 interacts with ATG16L1, which is essential for autophagy. Autophagy relieves cellular stresses faced by b cells during T2D and maintains cellular homeostasis. C3 knockout in clonal b cells impaired autophagy and led to increased apoptosis after exposure of cells to palmitic acid and IAPP. In the absence of C3, autophagosomes do not undergo fusion with lysosomes. Thus, C3 may be upregulated in islets during T2D as a cytoprotective factor against b cell dysfunction caused by impaired autophagy. Therefore, we revealed a previously undescribed intracellular function for C3, connecting the complement system directly to autophagy, with a broad potential importance in other diseases and cell types.

CD59 is a glycosylphosphatidylinositol (GPI)‐anchored cell surface inhibitor of the complement membrane attack complex (MAC). We showed previously that CD59 is highly expressed in pancreatic islets but is down‐regulated in rodent models of diabetes. CD59 knockdown but not enzymatic removal of cell surface CD59 led to a loss of glucose‐stimulated insulin secretion (GSIS), suggesting that an intracellular pool of CD59 is required. In this current paper, we now report that non‐GPI‐anchored CD59 is present in the cytoplasm, colocalizes with exocytotic protein vesicle‐associated membrane protein 2, and completely rescues GSIS in cells lacking endogenous CD59 expression. The involvement of cytosolic non–GPI‐anchored CD59 in GSIS is supported in phosphatidylinositol glycan class A knockout GPI anchor–deficient β‐cells, in which GSIS is still CD59 dependent. Furthermore, site‐directed mutagenesis demonstrated different structural requirements of CD59 for its 2 functions, MAC inhibition and GSIS. Our results suggest that CD59 is retrotranslocated from the endoplasmic reticulum to the cytosol, a process mediated by recognition of trimmed N‐linked oligosaccharides, supported by the partial glycosylation of non‐GPI‐anchored cytosolic CD59 as well as the failure of N‐linked glycosylation site mutant CD59 to reach the cytosol or rescue GSIS. This study thus proposes the previously undescribed existence of non–GPI‐anchored cytosolic CD59, which is required for insulin secretion.—Golec, E., Rosberg, R., Zhang, E., Renström, E., Blom, A. M., King, B. C. A cryptic non–GPI‐anchored cytosolic isoform of CD59 controls insulin exocytosis in pancreatic β‐cells by interaction with SNARE proteins. FASEB J. 33, 12425–12434 (2019). http://www.fasebj.org

Glioblastoma is characterized by extensive necrotic areas with surrounding hypoxia. The cancer cell response to hypoxia in these areas is well-described; it involves a metabolic shift and an increase in stem cell-like characteristics. Less is known about the hypoxic response of tumor-associated astrocytes, a major component of the glioma tumor microenvironment. Here, we used primary human astrocytes and a genetically engineered glioma mouse model to investigate the response of this stromal cell type to hypoxia. We found that astrocytes became reactive in response to intermediate and severe hypoxia, similarly to irradiated and temozolomide-treated astrocytes. Hypoxic astrocytes displayed a potent hypoxia response that appeared to be driven primarily by hypoxia-inducible factor 2-alpha (HIF-2α). This response involved the activation of classical HIF target genes and the increased production of hypoxia-associated cytokines such as TGF-β1, IL-3, angiogenin, VEGF-A, and IL-1 alpha. In vivo, astrocytes were present in proximity to perinecrotic areas surrounding HIF-2α expressing cells, suggesting that hypoxic astrocytes contribute to the glioma microenvironment. Extracellular matrix derived from hypoxic astrocytes increased the proliferation and drug efflux capability of glioma cells. Together, our findings suggest that hypoxic astrocytes are implicated in tumor growth and potentially stemness maintenance by remodeling the tumor microenvironment.

Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite treatment through surgery, irradiation and chemotherapy, all patients suffer recurrence of treatment-resistant tumors. Previous studies from our lab showed that astrocytes become reactive and generate tumor-supportive conditions for glioma cells, when exposed to hypoxia or irradiation. Reactive astrocytes are known to upregulate proteins of the complement system, especially complement component 3 (C3), in several neurological disorders. However, it remains unexplored how these complement proteins are expressed in stromal astrocytes in glioblastoma. Tissue sections from a glioma mouse model showed presence of C3 in the invasive front, and in hypoxic, and perivascular spaces of the tumors, where there is an abundance of astrocytes. C3 expression co-localized with Nestin and CD44, two markers of mesenchymal and/or stem-like glioma cells. Primary human astrocytes grown in hypoxic conditions upregulated C3 as well as other proteins associated with a more extensive infiltrative phenotype of glioblastoma. In general, a strong correlation between hypoxia (195 genes) and complement (200 genes) gene signatures were found in TCGA GBM dataset (R= 0.82, p-value= 0). Single-cell sequencing data from primary GBM tumors showed a subpopulation of highly C3-expressing astrocytes, which were enriched for cellular pathways comprising epithelial mesenchymal transition (EMT), TNF-alpha, complement, hypoxia, and interferon signaling. Human glioma cell lines from proneural, classical and mesenchymal subtypes all showed increased CAIX and GLUT1 expression grown under lower oxygen tensions, while two out of three subtypes showed upregulation of stemness markers (OCT4 and NANOG1) as well as C3 and C3aR. Overall, our data indicate a strong link between hypoxia and complement expression in the brain tumor microenvironment, where a local expression of complement proteins could possibly lead to tumor promoting signaling, which will be further explored.

Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite treatment through surgery, irradiation and chemotherapy all patients suffer recurrence of treatment-resistant tumors and the survival prognosis remains poor. The recurrence of tumors is driven by the invasive nature of the tumor and appears to be related to cells with stem like characteristics that are present in perivascular and hypoxic niches. Previous studies from our lab showed that astrocytes grown in hypoxic or irradiated conditions increase the stemness of glioma cells. The altered behavior of the astrocytes leads to increased cell size and a change in secreted cytokines. Reactive astrocytes are important in other central nervous system (CNS) diseases involved in tissue repair such as traumatic brain injury and Alzheimer’s disease. Interestingly, in several neurological diseases, reactive astrocytes upregulate complement proteins, especially complement component 3 (C3). However, it remains relatively unexplored how these complement proteins in stromal astrocytes are expressed in glioblastoma. Tissue sections from our glioma mouse model shows presence of C3 around hypoxic areas where there is an abundance of astrocytes. We have also shown that astrocytes grown at 21%, 1% and 0.1% oxygen upregulate complement protein C3 as well as other proteins associated with a more extensive infiltrative phenotype of glioblastoma. Datasets with human patients showed that C3 expression was correlated with higher grade tumors and that patients with tumors expressing C3 had more risk to get new tumors after primary treatment (including but not limited to radiotherapy). In this ongoing project, we are investigating whether activation of the complement system in the tumor microenvironment contributes to tumor progression. The upregulation of C3 in astrocytes in hypoxic conditions could therefore through local complement activation possibly led to tumor promoting signaling leading to beneficial survival of therapies of nearby glioma cells.