IFITM3 is involved in mNRPC proliferation
Initially, to obtain the variation and distribution of IFITM3 gene during the development of mouse retina in vivo, we integrated a published dataset of the developing murine retina in single cell RNA-sequencing and visualized the cell-type identification in Figure 1A[24]. During the embryonic stage, the IFITM3 gene was mainly expressed in the early RPCs at embryological day 11(E11) and E12 (Figure 1B). For the postnatal stage (P0, P2, P8), fewer positive expressed in the detected cells, while for the postnatal day 14, the RPCs and muller cells also are positive expression of IFITM3. These results suggest the IFITM3 are important for the retinal progenitor cells in the retina development.
To investigate the function of IFITM3 in mNRPCs, we transfected siRNA IFITM3 into mNRPCs and examined cell viability. Cell growth (Figure 1C-1E and Supplementary Figure S1A-1C) was clearly inhibited in the IFITM3 siRNA transfected group (siRNA IFITM3) compared with c group and scramble control (SC) group, with significantly decreased expression of IFITM3 at the RNA (Figure 1F) and protein levels (Figure 1G) after IFITM3 knockdown, but there was no significant decrease in ARPE19 cell growth (Supplementary Figure S1D-E). The cell viability was greatly decreased after IFITM3 knockdown, shown by the CCK-8 assay, significantly differed among the groups (Figure 1H), and the protein expression of Cyclin D1 (CCND1) decreased in the corresponding cell groups (Figure 1I), These results indicated that IFITM3 is involved in RPC proliferation through the regulation of cell division and cell viability in vivo and in vitro.
Cell membrane structure and function were deeply changed after IFITM3 knockdown
To further explore the regulatory effect of IFITM3 knockdown on the cells, RNA-seq analysis was performed. Among the GO terms, cell membrane and extracellular matrix (ECM)-related, transmembrane transport-related and synaptic transmission enrichment-related terms were the most enriched after knockdown of the IFITM3 gene. These results revealed that genes related to ion transmembrane transport, vacuoles, lysosomes and cellular calcium ion homeostasis were upregulated in the knockdown group, while genes involved in regulation of the ECM, WNT pathway and cell proliferation were significantly downregulated (Figure 2A), suggesting that the stability of the cell membrane system was seriously damaged and that its material transport function was blocked, ultimately leading to cell death due to a lack of nutrients, and these findings further reveal how IFITM3 is involved in regulating mNRPC proliferation. The KEGG pathway enrichment assay further revealed that the signaling pathways enriched in the DEGs mainly included the synaptic vesicle cycle, the lysosome pathway, the calcium signaling pathway, the MAPK signaling pathway and other upregulated pathways (Figure 2B and Supplementary Figure S2A), as well as downregulated pathways, such as the WNT, amino acid biosynthesis and fatty acid metabolism pathways, among others (Figure 2C and Supplementary Figure S2B).
Furthermore, GO analysis of the ECM pathway showed that the expression of ion channel-related genes, including the genes encoding calcium (such as CACNB2), potassium (such as KCNB1, KCNF1 and KCNC3) and chloride (such as CLCN1) ion channels, was clearly changed after IFITM3 knockdown (Supplementary Figure S2C); additionally, genes that regulate cell proliferation involved in metabolism (such as TNC and MVD) and proliferation-related pathway genes (such as CCND3, STAT1 and IL6) (Supplementary Figure S2D) also showed significant changes in expression, indicating that inhibition of IFITM3 expression led to damage to the cell membrane, abnormal material transport, abnormal metabolism of intracellular substances, decreased cell proliferation and ultimately cell death. Taken together, these results indicate that the integrity of the membrane system (including the cell membrane, vacuole, lysosome and ECM) was impaired after IFITM3 knockdown and that cells could no longer survive without sufficient nutrition, eventually leading to cell death.
Lysosome Activation in IFITM3-Knockdown Cells
Because membrane-related systems were severely damaged after IFITM3 knockdown, we studied membranous organelles, including the endoplasmic reticulum (ER), mitochondria and lysosomes, in the cell. The lysosome is an important site of regulation for mTORC1 signaling, which mainly controls eukaryotic cell growth. We treated cells with 100 nM RAMP, a specific mTOR inhibitor and an autophagy activator, and then treated the cells with 50 nM LysoTracker, 50 nM MitoTracker and 1 µM ER-Tracker for 30 min. Lysosomes showed agglomeration in the IFITM3-knockdown cells without RAMP treatment, while lysosomes showed agglomeration in all groups when with RAMP treatment, but there was no significant difference in IFITM3-knockdown cells with or without RAMP treatment (Figure 3A). Mitochondria and the ER were increased in IFITM3-knockdown cells treated with RAMP, but there was no significant difference among the groups (Figure 3B-C), suggesting that mitochondria and the ER were not involved in regulating mNRPC proliferation after IFITM3 knockdown and microautophagy (mA) was not activated.
The lysosome-specific markers lysosome-associated membrane protein 1 (LAMP1) and LAMP2A were detected in RAMP-treated and untreated cells, which showed that the expression of LAMP1 and LAMP2A was significantly increased and accompanied by increased lysosome agglomeration in RAMP-treated cells, especially IFITM3-knockdown cells (Figure 4A-C), indicating activation of the lysosome system followed by destruction of the cell membrane system to initiate cell cleanup, eventually causing cell death. These results suggest that IFITM3 in the cell membrane provides the first protective barrier for mNRPCs, and its decreased expression leads to the breakdown of cell homeostasis and initiation of lysosome formation to eliminate damaged cells. Furthermore, there was no significant difference among the IFITM3-knockdown cells with or without RAMP treatment (Figure 4C), suggesting that MA was not activated in the cells when the IFITM3 gene was knocked down. However, IFITM3 knockdown led to a cascade of effects causing increased membrane permeability that eventually led to cell death through activation of lysosomes and the CMA pathway.
Activation of the CMA Pathway when Knockdown of IFITM3
Cell viability was significantly inhibited and eventually caused cell death after IFITM3 knockdown. However, the MA pathway was not the main pathway responsible for this regulatory effect, and lysosomes were significantly activated, while the expression of key CMA pathway-related proteins was obviously increased. Based on these data, we examined apoptotic cells by staining the cells with calcein-AM. The results showed no obvious apoptosis in IFITM3-knockdown cells (Supplementary Figure 3A-C), and no apoptotic bodies were detected by Hoechst 33342 staining (Supplementary Figure 3D-F).
Additionally, the results of western blot analysis showed significantly increased expression of the antiapoptotic protein BCL2 and decreased expression of P53 (Figure 5A) in IFITM3-knockdown cells, which suggested that the apoptotic pathway was not activated when IFITM3 was knocked down. Cell activity was greatly decreased, and the expression of CCND1, the main function of which is to promote cell proliferation, was significantly decreased in the knockdown group. Further study showed obviously decreased expression of ERK1/2 among the groups (Figure 5B), suggesting that the downregulation of IFITM3 resulted in decreased cell survival and that the ability of the cells to proliferate was severely reduced. Our further investigation focused on the MA and CMA pathways. Expression of the related proteins P62 and LAMP1 was significantly upregulated (Figure 5A), but there was no significant change in the MA pathway-related proteins ATG13, BECLIN1, ATG5, ATG7 and LC3A/B (Figure 5A). Meanwhile, the expression of LAMP2A and HSC70 (Figure 5C) was significantly upregulated, which indicated and confirmed the activation of CMA in the cells after IFITM3 knockdown. There was also no significant difference in the expression of LC3A/B in IFITM3-knockdown cells (Figure 5D).
To study the generation of autophagic flux upon IFITM3 knockdown, stable LC3-GFP-mCherry-expressing mNRPCs were constructed. The results showed that no autophagic flux was generated in the cells (Figure 5D and Supplementary Figure 4). When IFITM3 was knocked down (Figure 6A-C), the results of immunohistochemical (IHC) analysis showed lightly increased expression of BCL2 (Figure 6D-F) and no significant changes in ATG7 expression (Figure 6G-I), while the expression of LAMP1 (Figure 6J-L), P62 (Figure 6M-O) and LAMP2A (Figure 6P-R) was significantly changed. The data were consistent with the WB results, indicating no activation of MA or the apoptotic pathway, while the CMA pathway was activated in IFITM3-knockdown cells. According to the above data, we believe that IFITM3 regulates cell viability mainly by regulating the CMA pathway, suggesting that the IFITM3 gene has a protective function that increases progenitor cell survival and self-renewal.