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Prokineticin-2 prevents neuronal cell deaths in a model of traumatic brain injury
Results . Prok2 is upregulated after brain injury . A total of three non-contusive brain tissues and five TBI tissues were obtained from the Tissue Bank of the First Affiliated Hospital of Nanjing Medical University, Nanjing, China, to perform transcriptome sequencing. Patient specimen information is shown in Table? 1 . The top 10 genes with the most robust differences based on the log2 (fold change) values were selected (Table? 2 ). The difference in mRNA levels between the human control and TBI groups was confirmed by quantitative reverse transcriptase PCR (qRT-PCR) (Supplementary Fig.? 1a ). Six differentially expressed genes, including IL1RL1, S100A8, S100A9, S100A12, PROK2 , and CXCR1 , were screened out. To determine the neuronal association of the TBI-induced changes, we examined the expression levels of five of these genes— Il1rl1, S100a8, S100a9, Prok2 , and Cxcr1 —in different types of brain cells using the Brain RNA Data Base 33 ( https://web.stanford.edu/group/barres_lab/brain_rnaseq.html ) (Supplementary Fig.? 1b–f ). Prok2 was selected for further analysis because other genes were expressed at low levels in neurons. Table 1 Demographic and clinical characteristics of TBI patients. Full size table Table 2 The top 10 genes with the most significant differences were listed. Full size table Western blot assessments of Prok2 levels demonstrated that its expression was markedly increased after TBI (Fig.? 1a , b). This was confirmed by the immunostaining (Fig.? 1c ). Using a mouse model of controlled cortical impact (CCI) (Fig.? 1d ), we assessed Prok2 expression in the peri-contusional area and found a time-dependent increase in the protein expression ( P ?Prok2, we further explored its role in the brain injury. Fig. 1: Prok2 expression is increased after exposure to TBI, stretch, and Erastin. a , b Western blot analysis and densitometric quantification of Prok2 expression by ImageJ in brain tissue of control ( n ?=?4 samples) and TBI ( n ?=?9 samples) patients. Data presented as mean?±?SD. c Immunofluorescence assessment of Prok2 expression (green) in brain tissue from control and TBI patients. Scale bar is 50?μm. DAPI is used to label nucleus. d Schematic representation of the contusional region (red) and the peri-contusional area (blue) after CCI. Tissues from the peri-contusional area (blue) are collected for western blot and qRT-PCR analysis. e , f Western blot analysis and densitometric quantification of Prok2 expression by ImageJ in control, sham and CCI mouse brain tissue. GAPDH is used as a control. Data presented as mean±SD ( n ?=?3 mice). g Dual immunofluorescence staining shows that Prok2 expression is most prominent in neurons (NeuN-labeled), whereas low levels of Prok2-staining are found in astrocytes (GFAP-labeled) and microglia (iba-1-labeled). Scale bar is 15?μm. Quantification of Prok2 fluorescence intensity by ImageJ is shown in the right panel. Data presented as mean±SD ( n ?=?3). h Representative photomicrographs of NeuN (red) and Map2 (green) expressing primary cortical neurons utilized in the studies. Scale bar is 50?μm for light microscopy image and 20?μm for fluorescence image. i Stretch-induced neuronal injury manifests as the appearance of thin and disrupted neurites and loss of the cytoplasm. Immunofluorescence labeling of tau protein (red) is used to monitor the effects of mechanical stretch on neurites; Hoechst is used to stain cell nuclei. Scale bar is 15 and 8?μm, respectively. j , k Western blot analysis and quantification of Prok2 and cleaved caspase-3 expression by ImageJ in control and stretch groups. GAPDH is used as loading control. Data presented as mean?±?SD ( n ?=?3 experiments). l , m Detection and quantification of Prok2 mRNA in primary cortical neurons exposed to Erastin for 24?h at different concentrations. β-actin is used as control. Data presented as mean?±?SD ( n ?=?3 experiments). n Erastin exposure increases Prok2 protein expression. GAPDH is used as control in western blot assays. For all panels, n indicates biologically independent repeats. P value was determined by a two-tailed unpaired Student’s t test for comparations between two groups. Source data are provided as a Source Data file. Full size image Prok2 affects biological functions by interacting with its receptor, Prokr2. Therefore, we further examined the time course of Prokr2 expression in CCI tissues (Supplementary Fig.? 2a ). Decreased Prokr2 and NeuN expression was found after CCI. As Prokr2 was mainly expressed in neurons (Supplementary Fig.? 2b ), we performed dual immunofluorescence staining for Prokr2 (Green) and NeuN (Red) to specifically confirm that presence of Prokr2 was established almost exclusively in NeuN-positive cells. The intracellular fluorescence intensity of Prokr2 was not affected by CCI (Supplementary Fig.? 2d ). Furthermore, co-immunoprecipitation (Co-IP) assays showed that CCI did not impact the intracellular relationships between Prok2 and Prokr2, in spite of the changes in the average expression of the proteins in the brain tissue (Supplementary Fig.? 2e ). This is in line with the results of dual immunofluorescence staining for Prokr2 (Green) and Prok2 (Red), demonstrating that CCI caused increased Prok2 levels but no changes in Prokr2 expression in NeuN-positive neurons (Supplementary Fig.? 2 f). These results indicate that the decreased total expression of Prokr2 was due to the reduced number of NeuN-positive cells without changes in its intracellular expression in NeuN-positive cells. In vitro studies of ferroptosis-associated enhanced Prok2 expression in neurons . Analysis of Prok2 in the brain tissue may be complicated by different levels of its expression in various types of cells. Immunofluorescence showed a more robust elevation of Prok2 in neurons (labeled by NeuN) 24?h after CCI than in astrocytes (labeled by GFAP) and microglia cells (labeled by iba-1). In fact, astrocytes and microglia showed low levels of Prok2 (Fig.? 1g ). As increased Prok2 levels may be important contributors to the CCI-induced neuronal injury, we performed in vitro mechanical stretch experiments with primary cortical neurons (Fig.? 1h , i). Primary neurons were identified by immunostaining assays using dual-labeling with NeuN and Map2. The cells were extracted from newborn mouse brain cortices and cultured for 5 days before use. An early (3?h) increase in Prok2 levels was detected after the stretch. At 9?h after stretch, increased levels of cleaved caspase-3 were also found (Fig.? 1j , k). Further, several other insults were used to test their capacity to change Prok2 expression. LPS (2?μg/ml) (Supplementary Fig.? 3a , b) or corticosterone (1?μM) (Supplementary Fig.? 3c , d) did not alter Prok2 expression after treatments for 24?h. However, exposure to H 2 O 2 (12?h) (Supplementary Fig.? 3e–g ) or excitotoxic glutamate (Supplementary Fig.? 3h–j ) increased Prok2 expression. Notably, both H 2 O 2 -induced oxidative stress and high levels of glutamate are known to trigger lipid peroxidation and cause cell ferroptosis 14 , 34 . Therefore, we speculated that Prok2 was involved in the regulation of ferroptosis. Notably, treatment of cells with a specific ferroptosis inducer, Erastin (24?h), increased Prok2 expression (Fig.? 1l–n , Supplementary Fig.? 4 c, d). In contrast, the levels of cleaved caspase-3 were not altered during Erastin-induced ferroptosis, as demonstrated by western blots (Supplementary Fig.? 4a , b) and immunostaining (Supplementary Fig.? 4c–e ). Interestingly, H 2 O 2 , excitotoxic glutamate and Erastin did not affect the expression of Prokr2 (Supplementary Fig.? 4f–k ), suggesting that ferroptosis induced increase in Prok2 levels was not driven by Prokr2-mediated regulation. Fer-1, a specific ferroptosis inhibitor, alleviated the Erastin-driven increase in Prok2 mRNA levels and lactate dehydrogenase (LDH) release (Supplementary Fig.? 5a–c ). This was confirmed by immunostaining which also demonstrated that cleaved caspase-3 levels were not changed after Erastin treatment (Supplementary Fig.? 5d–f ). Fer-1 also prevented elevation of Prok2 mRNA levels and cell death triggered by glutamate (Supplementary Fig.? 5g–i ). In contrast, Fer-1 did not affect the H 2 O 2 -induced enhancement of Prok2 mRNA levels, but reduced LDH release (Supplementary Fig.? 5j–l ). Combined, these data are compatible with the involvement of Prok2 in regulation of ferroptosis in primary neurons. Upregulation of Prok2 decreases Erastin- or stretch-induced neuronal cytotoxicity and lipid peroxidation . We further explored the role of Prok2 as a regulator of ferroptosis in primary neuronal cells. We used lentivirus containing Prok2 (Flagged) to increase Prok2 mRNA levels and shProk2 to knock down Prok2 . One out of three Prok2 -interfering sequences tested was found to decrease Prok2 mRNA expression most efficiently (Fig.? 2a , b). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay indicated that Lv-Prok2 inhibited Erastin-induced cell damage, whereas shProk2 aggravated this effect. Fer-1 inhibited ferroptosis and prevented injury induced by Prok2 downregulation (Fig.? 2c , d). Stretch-induced cell death was also alleviated by Prok2 overexpression. Lowering Prok2 expression enhanced cell death after stretch, and this effect was inhibited by Fer-1 administration (Supplementary Fig.? 6a , b). In primary neurons incubated for 24?h with Erastin, disruption and thinning of the neurites with large vacuoles and bright spots as well as decreased cytoplasm were observed. Cellular and neurite fragments were also detectable in the extracellular compartment. Lv-Prok2 administration resulted in morphologically distinct protection against Erastin-induced injury of primary cortical neurons (Fig.? 2e ). Both Erastin administration or stretch injury caused aggregation of Fe3 + in the neurons, and this effect was suppressed by Prok2 overexpression. Low levels of Prok2 were associated with the increased contents of iron, preventable by Fer-1 treatment (Fig.? 2f , g and Supplementary Fig.? 6c , d). Exogenous Prok2 improved viability of cells treated with Erastin or exposed to stretch, as assayed by CCK-8. In contrast, shProk2-treated neuronal cells displayed the low viability, which could be recovered by Fer-1 (Fig.? 2h and Supplementary Fig.? 6e ). Lv-Prok2 alleviated the Erastin- or stretch-induced LDH release. Downregulation of Prok2 was associated with additional cytotoxicity, which was reduced by Fer-1 (Fig.? 2i and Supplementary Fig.? 6f ). Western blot results indicated that Erastin increased Acsl4 contents but reduced Gpx4 levels. After Erastin treatment, Lv-Prok2 decreased Acsl4 expression but increased Gpx4 expression. On the contrary, shProk2 significantly inhibited Gpx4 but promoted Acsl4 expression. Fer-1 inhibited shProk2-induced Gpx4 downregulation and Acsl4 upregulation (Fig.? 2j ). In contrast to Erastin, stretch did not change Gpx4 expression, but shProk2 decreased Gpx4 after stretch in a Fer-1 preventable way (Supplementary Fig.? 6g ). Furthermore, exogenous Prok2 reduced Erastin-induced or stretch-induced lipid peroxidation assessed by ROS generation, BODIPY 581/591 C11 oxidation, MDA content and GPX activity (Supplementary Fig.? 7a–d and Supplementary Fig.? 8a–e ). Levels of oxygenated AA metabolites, 15-hydroxyeicosatetraenoic acid (15-HETE), and 12-hydroxyeicosatetraenoic acid (12-HETE), were significantly increased after Erastin or stretch treatment. Notably, Lv-Prok2 reduced the levels of these metabolites (Supplementary Fig.? 7e , f and Supplementary Fig.? 8f , g), whereas shProk2 enhanced lipid peroxidation. Inhibition of ferroptosis by Fer-1 prevented activation of lipid peroxidation. Fig. 2: Overexpression of Prok2 attenuates Erastin-induced cytotoxicity. Primary cortical neurons are treated with 20??M Erastin for 24?h, lentivirus containing Prok2 (Lv-Prok2) or control (vector), control RNAi (shCtrl) or RNAi against Prok2 (shProk2), and ferrostatin-1 (Fer-1). a shProk2 decreases Prok2 mRNA levels vs. shCtrl. One of the three Prok2-interfereing shRNAs, the sh-Prok2-3#, is found to decrease Prok2 mRNA expression most efficiently. b Quantification of Prok2 mRNA levels by ImageJ. Data presented as mean?±?SD ( n ?=?3 experiments). c , d Cell death measured by TUNEL staining of primary neurons. Scale bar is 50?μm. Data presented as mean±SD ( n ?=?4 experiments). e Representative photomicrographs of primary cortical neurons in different groups. Characteristic of neuronal injury are disruption and thinning of neurites with large vacuoles and bright spots as well as decreased cytoplasm. Cellular and neurite fragments are also observed in extracellular compartment. Scale bar is 20?μm. f , g Prussian blue staining shows that Erastin stimulates the formation of Fe3 + (blue) in neurons. Lv-Prok2 suppresses this effect. Scale bar is 10?μm. The iron levels are determined based on the color intensities measured by ImageJ. Data presented as mean±SD ( n ?=?3 independent experiments). h , i Cell viability analyzed by CCK-8. Cytotoxicity induced by Erastin is measured by LDH assay. Data presented as mean?±?SD ( n ?=?5 experiments). j Expression of Prok2, Gpx4 and Acsl4 in primary neurons under various treatment conditions. GAPDH is used as control in western blot assays. For all panels, n indicates biologically independent repeats. P value was determined by two-tailed unpaired Student’s t test for comparations between two groups. Source data are provided as a Source Data file. Full size image We further examined whether Prok2 upregulation could change neuronal electrophysiological activity after exposure to Erastin. In the control group, action potentials, appearing as continued waves, were induced under appropriate stimulus (pA) (Supplementary Fig.? 9a upper graph). Erastin-induced damage to primary neurons resulted in a drastic decrease of action potentials in the patch clamp recordings. Prok2 overexpression caused alleviation of Erastin-induced damage and continued action potentials were detectable in the Erastin?+?Lv-Prok2 group (Supplementary Fig.? 9a , b). Prok2-induced protection of mitochondrial functions is mediated by Acsl4 . Ferroptosis is commonly accompanied by mitochondrial injury 35 , 36 . We examined the morphology and distribution of mitochondria 24?h after treatment with Erastin using Mito-Tracker (Fig.? 3a ). In general, Mito-Tracker staining exhibited a fusiform structure, a small rod-like form in neurites, and an interconnected network in the cytoplasm, which were the dominant morphologies in the normal primary neurons. Upon Erastin treatment, the mitochondrial network looked disrupted and mitochondria appeared as small fragmented punctiform structures and small circles. Injury and disruption of neurites were accompanied by a decline in mitochondrial length as well. Prok2 overexpression attenuated Erastin-induced changes of mitochondrial morphology (Fig.? 3b , c). Dual immunofluorescence labeling using Prok2 and Tomm20 indicated that Prok2 protected mitochondria and promoted their migration to neurites (Fig.? 3d ), which was also observed in the stretch model (Supplementary Fig.? 10a ). Transmission electron microscopy (TEM) studies revealed shrunken mitochondria, outer mitochondrial membrane (OMM) rupture, and the formation of light vacuoles likely related to the mitochondrial collapse in Erastin-treated cells, which are the characteristic changes in ferroptosis 17 , 37 ; Prok2 overexpression prevented the appearance of these changes (Fig.? 3e ). Fig. 3: Prok2 protects mitochondrial function in an Acsl4-dependent manner. 20?μM Erastin for 24?h is used to induce ferroptosis in these studies. a Mito-Tracker staining exhibits a fusiform structure, a small rod-like form in neurites, and an interconnected network in the cytoplasm in the normal primary neurons. Erastin treatment disrupts the mitochondrial network and mitochondria appear as small fragmented punctiform structures and small circles. Prok2 overexpression reduces mitochondrial circularity ( b ) and increases mitochondrial length ( c ). Data presented as mean?±?SD ( n ?=?5 experiments). d Immunofluorescence assessment of the expression and intracellular distribution of Tomm20 and Prok2. Prok2 overexpression increases Tomm20 positivity and promotes migration of mitochondria to neurites. Scale bar is 10?μm. e Representative electron microscopy images show shrunken mitochondria and outer membrane rupture upon exposure to Erastin (red arrow), which is inhibited by Prok2 overexpression. Scale bar is 500?nm. f – l Expression levels of Acsl4, Gpx4, Tfam, Tomm20, and MT-ND1 (mitochondrial DNA copy number) in Acsl4 deficient (shAcsl4) and control (shCtrl) primary neurons. GAPDH is used as control. Data presented as mean?±?SD ( n ?=?3 experiments). m ATP levels are decreased upon exposure to Erastin. Overexpression of Prok2 prevented the decrease in mtDNA copy number and ATP levels in Erastin-treated cells. Data presented as mean?±?SD ( n ?=?5 experiments). n Representative TEM images illustrating that Erastin administration does not cause marked changes of mitochondrial morphology in shAcsl4 primary neurons. Scale bar is 500?nm. For all panels, n indicates biologically independent repeats. P value was determined by two-tailed unpaired Student’s t test for comparations between two groups. Source data are provided as a Source Data file. Full size image We observed that Acsl4 expression was decreased and Gpx4 expression was increased in ferroptotic cells overexpressing Prok2 however the underlying mechanism(s) of these effects remained unclear. In the CCI mouse model, injury increased Acsl4 levels but Gpx4 levels remained stable (Supplementary Fig.? 11a–c ), suggesting differences in regulation of Acsl4 and Gpx4 in this model vs. Erastin. And earlier studies established that Gpx4 expression was higher in Acsl4 KO ( Acsl4 ?/?) cells than in WT ( Acsl4 +/+) cells 17 . Therefore, we focused our efforts on exploring the role of Acsl4 as a possible mechanism of Prok2-mediated control of mitochondrial metabolism and ferroptosis. Previous studies have shown that Acsl4 KO cells are resistant to RSL3-induced OMM rupture and lipid peroxidation compared to WT cells 17 . We chose to extend this work by using shCtrl and shAcsl4 in primary cortical neurons treated with Erastin or exposed to stretch. In Erastin treated or stretch exposed cells, we found a compensatory increase of Prok2. But overexpression of Prok2 did not affect Gpx4, Tomm20, Tfam, ATP and MT-ND1 levels in shAcsl4 cells (Fig.? 3f–m (the left part) and Supplementary Fig.? 10b–h ). In shCtrl cells, Erastin-induced ferroptosis was inhibited by Lv-Prok2 treatment, associated with decreased Acsl4 and increased Gpx4 expression (Fig.? 3f–i (the right part)). Gpx4 levels were not changed by a stretch but were elevated by Prok2 (Supplementary Fig.? 10d ). In addition, elevated levels of Tomm20, Tfam, MT-ND1, and ATP were found in shCtrl cells (Fig.? 3j–m (the right part) and Supplementary Fig.? 10e–h ). No characteristic for ferroptosis morphological changes of mitochondria was observed in shAcsl4 primary neurons after Erastin treatment (Fig. 3n ). While stretch-induced mild mitochondrial swelling was detected by TEM, ferroptosis-related characteristic morphological changes were not observed (Supplementary Fig.? 10i ). These results suggest that Prok2-induced protection of mitochondrial functions is mediated by Acsl4. Prok2 promotes Acsl4 ubiquitination and degradation . Assuming that Prok2-mediated decreased Acsl4 content was an important contributor to suppress neuronal ferroptosis, we explored possible mechanisms by which Prok2 downregulated Acsl4 in vitro. We assessed Acsl4 mRNA stability in vector- and Lv-Prok2 cells treated with Erastin by blocking mRNA synthesis with a transcription inhibitor, actinomycin D. Prok2 overexpression had no effect on the steady-state levels of Acsl4 mRNA (Fig.? 4a ). We next explored whether Prok2-mediated decrease of Acsl4 levels occurred via the protein degradation pathway. When CHX, a protein synthesis inhibitor, was added to vector- and Lv-Prok2 cells at different time points, western blot analysis revealed a higher Acsl4 degradation rate in Lv-Prok2 cells (Fig.? 4b , c). Considering two major mechanisms of protein degradation—the autophagy-lysosomal pathway and the ubiquitin-proteasomal pathway—we pretreated primary cortical neurons with bafilomycin A1 (a specific lysosomal inhibitor) and bortezomib (a proteasomal inhibitor), either separately or in combination, and subsequently exposed them to Lv-Prok2 or vector. The CHX was used as a positive control in these experiments. Bortezomib alone or in combination with bafilomycin A1 largely blocked the Prok2-mediated degradation of Acsl4. On the contrary, treatment of cells with bafilomycin A1 alone had no effect (Fig.? 4d , e). These data suggested that Prok2 primarily used the ubiquitin–proteasomal pathway for Acsl4 degradation. We co-transfected primary cortical neurons with a plasmid expressing Flagged-Acsl4 and a plasmid expressing an HA-Ubiquitin (Ub). In parallel, cells were transfected with empty vectors as negative controls. Whole-cell lysates were subjected to IP with anti-Flag or anti-HA conjugated to agarose beads. The anti-Flag and anti-HA immunoprecipitates were subjected to SDS–PAGE followed by visualization of blots with anti-HA, anti-Acsl4, and anti-Flag antibodies. Ubiquitinated species of Acsl4 were detected (Fig.? 4f–h ). Next, we used MG132 to examine the ubiquitination status of endogenous Acsl4 in vector-treated and Lv-Prok2-treated primary cortical neurons triggered to ferroptosis by Erastin. Based on the detection of Acsl4 and Ub in total lysates of cells treated with Erastin or exposed to stretch, we showed that Prok2 promoted Acsl4 ubiquitination/degradation, and this effect was abolished by MG132 (Fig.? 4i and Supplementary Fig.? 12a ). Equal amounts of whole-cell lysate were subjected to IP with an anti-Acsl4 or -IgG, followed by western blotting using anti-Acsl4 or anti-Ub. Importantly, after Acsl4 IP, the amount of pulled-down non-ubiquitinated Acsl4 was lower in the precipitates of the Lv-Prok2 plus MG132 samples than in those of the vector-plus MG132 sample (Fig.? 4j , compare lanes 3 vs. 2). However, the Lv-Prok2 plus MG132 cells clearly had a higher amount of polyubiquitinated Acsl4 than the vector-plus MG132 samples. In contrast, western blotting with anti-Acsl4 or anti-Ub did not reveal specific bands in the IgG immunoprecipitates. Prok2 upregulation also led to higher amounts of Acsl4 ubiquitination in the stretch model (Supplementary Fig.? 12b ). Altogether, these results provide direct evidence that Prok2 overexpression enhanced Acsl4 ubiquitination and possibly channeled Acsl4 toward its proteasomal degradation. Fig. 4: Prok2 promotes Acsl4 ubiquitination degradation. a Vector and Prok2 overexpressing (Lv-Prok2) primary cortical neurons are treated with 20?μM Erastin for 24?h. Then, actinomycin D (Act D), a transcription inhibitor blocking mRNA synthesis, is added at a concentration of 6?μg/ml, and total RNA is isolated at the indicated time points for semi-qRT-PCR analysis of Acsl4 and β-actin . Prok2 overexpression has no effect on the steady-state levels of Acsl4 mRNA. b Vector and Prok2 (Lv-Prok2) overexpressing primary cortical neurons are treated with 20?μM Erastin for 24?h after which CHX, a protein synthesis inhibitor, is added to cells at a concentration of 5?μg/ml. Total cell lysates are isolated at the indicated times and subjected to western blotting. Bands are visualized using antibodies against Acsl4 and GAPDH. c The line graph (left panel) shows the expressions of Acsl4 analyzed by ImageJ and the bar graph (right panel) indicates a higher Acsl4 degradation rate observed in Lv-Prok2 cells vs. vector after Erastin treatment. Data are presented as mean values?±?SD ( n ?=?3 experiments). d , e Primary cortical neurons are treated with 5?μg/ml CHX, 200?nM bortezomib (Bort), 50?nM bafilomycin A1 (Baf A1), or a combination of Bort and Baf A1 for 1?h prior to the addition of 20?μM Erastin. Cell lysates are obtained after 24?h of Erastin administration and immunoblotted for Acsl4 and GAPDH. Bar graph shows Acsl4 expression normalized to GAPDH under different conditions. Bort alone or in combination with Baf A1 blocks Prok2-mediated degradation of Acsl4. Data are presented as mean values?±?SD ( n ?=?3 experiments). f – h Plasmids encoding Flag-tagged Acsl4 and HA-Ubiquitin (Ub) as well as empty vectors as controls are co-transfected into primary cortical neurons. Then cell lysates are immunoprecipitated with anti-Flag or anti-HA and western blot analysis is performed for Acsl4, Flag, and HA showing ubiquitinated species of Acsl4. i Primary cortical neurons expressing control empty vector or Lv-Prok2 are treated with 20?μM MG132 for 6?h to block proteasomal degradation and then are exposed to 20?μM Erastin for 24?h. Total lysates are analyzed for Acsl4 and ubiquitinated proteins by immunoblotting using anti-Acsl4 and anti-Ub antibodies. The decrease in Acsl4 protein observed upon Prok2 overexpression is abolished by MG132 and ubiquitination of Acsl4 is increased. j , k IP with Acsl4 antibody and western blot with anti-Ub show that ubiquitination of Acsl4 is higher in Prok2 overexpressing plus MG132-treated cells vs. control untreated cells as well as empty vector transfected plus MG132-treated cells. IgG is used as a negative control. Bar graph shows quantification of ubiquitinated Acsl4. Data are presented as mean values?±?SD ( n ?=?3 experiments). For all panels, n indicates biologically independent repeats. P value was determined by two-tailed unpaired Student’s t test for comparations between two groups. Source data are provided as a Source Data file. Full size image Fbxo10 is crucial for Prok2-induced Acsl4 ubiquitination/degradation . To directly examine the role of E3 ubiquitin ligase in the control of Acsl4 ubiquitination and degradation, we examined the proteins pulled down after IP of neuronal lysates with Acsl4 antibody using silver staining followed by LC–MS (Fig.? 5a , b). Fbxo10 was the only ubiquitin ligase identified (Fig.? 5b ). Immunofluorescence staining confirmed that Acsl4 and Fbxo10 are co-localized in the mitochondria (Fig.? 5c ). Erastin caused upregulation of Acsl4 and a decrease of Fbxo10 staining. The intensity of mitochondrial staining decreased upon Erastin exposure, particularly in the neurites. Transfection with Myc-amplified Fbxo10 suppressed Acsl4 expression with or without Erastin stimulation and enhanced Ub binding to Acsl4 (Fig.? 5d ). Similar results were observed in the stretch model (Supplementary Fig.? 12c ). Fbxo10 has several functional domains which can interact with different signaling proteins. To determine the binding domain of Fbxo10 for Acsl4, three Myc-tagged Fbxo10 domains (aa 6–49, aa 460–867 and the whole aa 1–951; Fig.? 5e , the upper part) were synthesized and integrated into plasmid, respectively. A plasmid carrying only Myc was used as a control. Specific protein–protein interaction was detected between the Fbxo10 domain (aa 460–867) and Acsl4 (Fig.? 5e the lower part). Fig. 5: Fbxo10 is crucial in Prok2-induced Acsl4 ubiquitination. a Cell lysates obtained from primary cortical neurons are immunoprecipitated with Acsl4 antibody. Silver staining is used to reveal all proteins bound to Acsl4 antibody. Mass spectrometry analysis identified Fbxo10 as the only ubiquitin ligase in the extracted protein from primary cortical neurons. b Mass spectrogram of Fbxo10. c Immunofluorescence staining shows co-localization of Fbxo10 (green), Acsl4 (red) and Mito-tracker (blue) in primary cortical neurons. Scale bar is 10?μm. d IP with Acsl4 followed by western blot with anti-Ub shows that overexpression of Fbxo10 increases ubiquitination of Acsl4 in the presence or absence of Erastin. e Schematic representation of Fbxo10 fusion proteins (upper part). Interaction is detected between Fbxo10 domains (aa 460–867) and Acsl4 (lower part). f Overexpression of Prok2 in primary cortical neurons increases Fbxo10 protein levels in the presence or absence of Erastin. g , h Primary neurons are co-transfected with Lv-Prok2-Flag and Fbxo10 shRNA. Cell lysates are obtained and immunoprecipitated with Acsl4 antibody followed by western blot with anti-Ub. Prok2-induced ubiquitination of Acsl4 is decreased in Fbxo10-knockdown cells both in the presence and absence of Erastin. Acsl4 is used as a control in IP lysates; GAPDH is used as a control in input lysates. Source data are provided as a Source Data file. Full size image To further investigate the relationship between Prok2 and Fbxo10, we examined the expression of Fbxo10 in cells overexpressing Prok2. Elevated Fbxo10 levels were observed after Prok2 overexpression under the normal, Erastin-stimulated or stretch conditions (Fig.? 5f and Supplementary Fig.? 12d ). Based on these results, we hypothesized that Fbxo10 may be involved in Prok2-induced Acsl4 ubiquitination and degradation. To further test this hypothesis, we knocked down Fbxo10 expression in Prok2-overexpressing cells and examined Acsl4 expression after vehicle (DMSO) control, Erastin, or stretch exposure (Fig.? 5g , h and Supplementary Fig.? 12e ). Fbxo10 deficiency attenuated Acsl4 ubiquitination/degradation induced by Prok2 overexpression. Thus, Prok2 overexpression alleviates ferroptosis by promoting expression of Fbxo10 and accelerating Acsl4 ubiquitination/degradation. Of note, in contrast to the effects of Erastin treatment or stretch exposure, upregulation of Fbxo10 by Prok2 was not equally efficient in the control (DMSO) group. Neuroprotective effects of intracerebroventricular (ICV) injection of adeno-associated virus (AAV)-Prok2 depend on Acsl4 regulation . To examine the validity of the proposed mechanism in vivo, we performed AAV-Prok2 and AAV-shFbxo10 transfections before conducting CCI at the time points displayed in Supplementary Fig.? 13a . Flag-conjugated AAV-Prok2 was injected into the lateral ventricle of the mouse brain. Increased Prok2 expression was observed after 7 days (Fig.? 6a , b). In order to exclude the effects of injection on neuronal mitochondria, we examined Tomm20 expression and mitochondrial morphology by immunostaining and TEM, respectively, and found no significant changes (Supplementary Fig.? 13b–d ). In the CCI model, Prok2 upregulation by AAV-Prok2 was associated with an increase in the levels of Prokr2 (Supplementary Fig.? 13e, f ). Fig. 6: AAV-Prok2 intracerebroventricular injection (i.c.v) decreases CCI-induced lesion volume in a Fbxo10-dependent way. a GFP-tagged Prok2-AAV is injected into mouse brain at 1 week before CCI. Dual-labeled immunofluorescence staining with Prok2-eGFP (green) and NeuN (red) is used to test the efficiency of AAV transfection in neurons. Scale bar is 100?μm (left) and 30?μm (right). b Increased brain tissue Prok2 expression is detected by western blot at 7 days after i.c.v. injection of GFP-tagged Prok2-AAV. GAPDH is used as control. c , d AAV-shFbxo10 carrying luciferase is injected into mouse brain tissue. Cri Maestro In-vivo Imaging Systems is used to screen for successful transfection. Fbxo10 knockdown in brain tissue is confirmed by western blot assays. GAPDH is used as control. e – g 2 days after CCI, protein expression of Gpx4 and Acsl4 proteins is examined by western blot analysis. Fer-1(1?mg/kg per day) is given i.p. once daily for 7 days before CCI and continued until euthanasia. While Acsl4 levels increases. Gpx4 levels do not change after CCI. AAV-Prok2 administration increases Gpx4 but decreases Acsl4 expression, which is blocked by Fbxo10 knockdown after CCI. Fer-1 administration suppresses CCI-induced increases in Acsl4 levels and alleviates AAV-shFbxo10-induced decrease in Gpx4 expression. Data are presented as mean values?±?SD ( n ?=?3 mice per group). h and i Representative T2 weighted MR images showing lesion volume in mouse brain after CCI in different experimental groups. While AAV-Prok2 transfection reduces lesion volume, co-transfection of AAV-Prok2 and AAV-shFbxo10 abolishes this effect. Administration of Fer-1 on the other hand decreases lesion volume in CCI mice expressing AAV-Prok2 and AAV-shFbxo10. Data are presented as mean values?±?SD ( n ?=?4 mice per group). j Mitochondrial morphology under different conditions is evaluated by electron microscopy. CCI-induced shrunken mitochondria and rupture of OMM, ferroptosis-related morphological changes of mitochondria (red arrow), are prevented by AAV-Prok2. k Immunostaining is used to examine the spatial distribution Gpx4 and Acsl4 expression in pericontusional area and shows similar treatment effect that is seen in western blot analysis observed in panels f , g . l , m Cell death response in the pericontusional area is quantified using TUNEL. Data are presented as mean values?±?SD ( n ?=?4 mice per group). For all panels, n indicates biologically independent repeats. P value was determined by two-tailed unpaired Student’s t test for comparations between two groups. Source data are provided as a Source Data file. Full size image Assuming that the effects of Prok2 were realized via regulation of Acsl4, we explored whether this mechanism occurred in vivo. We downregulated Acsl4 expression by intracranial injection of AAV-shAcsl4 tagged with HA. To monitor the transfection efficiency, we used immunofluorescence microscopy (Supplementary Fig.? 14a ), as well as immunoblotting (Supplementary Fig.? 14b , c). Acsl4 content was reduced by AAV-shAcsl4 and remained low after CCI. In the AAV-shAcsl4 group, Gpx4 levels did not change after CCI but increased in the Prok2 overexpression group (Supplementary Fig.? 14b–d ). We found that overexpression of Prok2 prevented CCI-induced: (i) decreases in GPX activity, as well as total GSH levels and GSH:GSSG ratio, (ii) increases in MDA levels and the number of TUNEL- and NeuN-positive cells in an Acsl4-depended manner (Supplementary Fig.? 14e–j ). Effects of AAV-Prok2 and AAV-shFbxo10 on CCI-induced ferroptosis . We further assessed in vivo effects of AAV-Prok2 and AAV-shFbxo10 on CCI-induced ferroptosis. To this end, mice were injected with luciferase-conjugated AAV-shFbxo10 and knockdown of Fbxo10 was confirmed (Fig.? 6c , d). The contribution of ferroptosis was evaluated by the treatment with Fer-1 (1?mg/kg) which was administered i.p. before CCI injury once daily until euthanasia or the Morris water maze (MWM) test. Additionally, the expression of Gpx4 and Acsl4 was examined by western blot assays within 2 days post CCI (Fig.? 6e–g ). We observed that Acsl4 levels were increased in the CCI group. However, Gpx4 levels did not change significantly ( P ?=?0.7852). AAV-Prok2 administration increased Gpx4 expression but decreased Acsl4 levels, and this effect was blocked by Fbxo10 knockdown. Fer-1 alleviated ferroptosis, suppressing Acsl4 and elevating Gpx4 levels, regardless of Fbxo10 deficiency. Immunohistochemical staining for Acsl4 and Gpx4 showed similar results (Fig.? 6k ). AAV-Prok2 reduced the lesion volume assessed by MRI. Furthermore, AAV-shFbxo10 administration increased the lesion volume even in the presence of enhanced Prok2 expression, and this effect was suppressed by Fer-1 (Fig.? 6h , i). Prok2 overexpression or Fer-1 treatment protected against the mitochondrial shrinkage and outer membrane rupture after CCI (Fig.? 6j ). TUNEL staining showed that Prok2 overexpression attenuated, while Fbxo10 deficiency enhanced CCI-induced cell death. The increase in AAV-shFbxo10-induced cell death after CCI was decreased by Fer-1 administration (Fig.? 6l , m). CCI resulted in ferroptotic changes as evidenced by decreased GSH, GSH levels, GSH:GSSG ratio, and GPX activity, and increased lipid peroxidation assessed by MDA, 12-HETE, and 15-HETE levels (Supplementary Fig.? 15a–f ). Pretreatment with AAV-Prok2 attenuated these changes in a Fbxo10 dependent manner. Detrimental effects of AAV-shFbxo10 co-treatment on GSH levels, GPx4 activity and lipid peroxidation after CCI were attenuated by Fer-1. Increased levels of Prok2 improve and AAV-shFbxo10 suppresses motor ability and learning performance after CCI . We further studied the effects of Prok2 in vivo using several neurocognitive tests. A schematic timeline of experiments is displayed in Fig.? 7a . The rotarod test was employed to assess motor abilities 2 days after CCI. Motor activity of AAV-Prok2-injected mice exposed to CCI was markedly improved compared to the CCI-alone. This improvement was not observed in the CCI?+?AAV-Prok2+AAV-shFbxo10 group ( P ?=?0.0887, n ?=?10 mice/group). Notably, Fer-1 effectively suppressed the harmful effects of AAV-shFbxo10 and enhanced motor abilities (Fig.? 7b ). The maximally tolerated rotation speed was not significantly different between the groups, with the notable exception of the sham and CCI groups (Fig.? 7c ). The MWM test was performed 14 days after CCI. Mice were subjected to 3 days of visible training sessions, during which the platform was on the surface of the water and indicated by black staining. Latency, distance, and swimming speed were equal in different groups of mice exposed to the treatments, suggesting that motor abilities did not interfere with the hidden training part (Fig.? 7d–f ). After elimination of the possibility of interferences due to motor differences, mice were trained to find a submerged platform during a 5-day hidden training session. The motion curves on the 5th day of hidden training session are shown in Fig.? 7g . CCI-exposed mice spent more time and traveled longer distances to reach the platform than sham-operated mice during the training. AAV-Prok2 mice exhibited a decreased latency and distance as the training progressed. Mice injected with AAV-shFbxo10 demonstrated a significant decrease in their ability to learn the location of the submerged platform. However, Fer-1 administration significantly reduced the latency and distance required for searching for the hidden platform, in spite of the AAV-shFbxo10 administration (Fig.? 7h , i). Different treatments did not cause changes in the swimming speed of mice in any of the tested groups during the hidden training sessions (Fig.? 7j ). Fig. 7: AAV-Prok2 improves neurobehavioral outcome after CCI. a Schematic outlining the timeline for the neurobehavioral testing. Fer-1 (1?mg/kg) is given i.p. once daily for 7 days before CCI and continued until euthanasia or the MWM test. Motor function is evaluated using Rotarod 2 days after CCI. MWM is utilized to examine spatial memory acquisition. MWM consisted of d – f visible platform testing for 3 days (4 trials per day) to assess motor and visual capabilities, followed by h – j hidden platform testing for 5 days (4 trials per day) to assess spatial learning ability. b A two-tailed unpaired Student’s t test and one-way ANOVA plus Tukey’s test revealed motor activity of inured AAV-Prok2-injected mice is improved versus CCI-alone ( P ?=?0.0232). Addition of AAV-shFbxo10 abolishes this effect ( P ?=?0.0222). Fer-1 attenuated the negative effect of AAV-shFbxo10 and enhanced motor function ( P ?=?0.0263). Data are presented as mean values?±?SD ( n ?=?8 mice in sham group and 10 mice per group in other groups). c The rotation speed tolerated is not significantly different between groups except between sham?+?AAV-NC and CCI?+?AAV-NC. P ?=?0.0395 versus CCI?+?AAV-NC group. Data are presented as mean values?±?SD ( n ?=?8 mice in sham?+?AAV-NC group; n ?=?10 mice in CCI?+?AAV-NC group). d – f Latency to platform, distance to platform and swimming speed in the visible platform testing. Data are presented as mean values?±?SD ( n ?=?8 mice in sham?+?AAV-NC group; n ?=?10 mice per group in other groups). g Representative swimming tracks of the mice in all five groups on the 8th day of the MWM task. h – j During the hidden platform testing, time spent to reach the platform ( h ), swimming distance ( i ) and swimming speed ( j ) are recorded. One-way ANOVA followed by Tukey post hoc test for different groups on the same time point are carried out. Among of them, (red) means CCI?+?AAV-NC group versus sham?+?AAV-NC group; (blue) means CCI?+?AAV-Prok2 group versus CCI?+?AAV-NC group; (green) means CCI?+?AAV-Prok2?+?shFbxo10 group versus CCI?+?AAV-Prok2 group; (orange) means CCI?+?AAV-Prok2?+?shFbxo10+Fer-1 group versus CCI?+?AAV-Prok2?+?shFbxo10 group. Mice in CCI group spend more time ( P (19d) ?Fbxo10 group exhibits a significant decline in the ability to learn the spatial location of the submerged platform ( P (19d) ?=?0.0499, P (20d) ?Fer-1 administration significantly reduces the latency and distance spent on searching for the hidden platform despite AAV-shFbxo10 administration ( P (19d) ?=?0.0009, P (20d) ?two-way ANOVA with repeated measures followed by Tukey post hoc test is used for the whole groups, which reveals group by day interaction effect in latency to platform ( F 16,215 ?=?3.524, P ?MWM analysis, data are presented as mean values?±?SD ( n ?=?8 mice in sham?+?AAV-NC group; n ?=?10 mice per group in other groups). Full size image .
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