Dual Effects of Thyroid Hormone on Neurons and Neurogenesis

Lin et al. Cell Death and Disease (2020) 11:671 https://doi.org/10.1038/s41419-020-02836-9 Cell Death & Disease

ARTICLE Open Access Dual effects of thyroid hormone on neurons and neurogenesis in traumatic brain injury Chao Lin1,2, Nan Li3, Hanxiao Chang1,2,Yuqishen1,2,ZhengLi1,2,Wuwei1,2,HuaChen1,2,HuaLu1,2,JingJi 1,2 and Ning Liu1,2

Abstract Thyroid hormone (TH) plays a crucial role in neurodevelopment, but its function and specific mechanisms remain unclear after traumatic brain injury (TBI). Here we found that treatment with triiodothyronine (T3) ameliorated the progression of neurological deficits in mice subjected to TBI. The data showed that T3 reduced neural death and promoted the elimination of damaged mitochondria via mitophagy. However, T3 did not prevent TBI-induced cell death in phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (Pink1) knockout mice suggesting the involvement of mitophagy. Moreover, we also found that T3 promoted neurogenesis via crosstalk between mature neurons and neural stem cells (NSCs) after TBI. In neuron cultures undergoing oxygen and glucose deprivation (OGD), conditioned neuron culture medium collected after T3 treatment enhanced the in vitro differentiation of NSCs into mature neurons, a process in which mitophagy was required. Taken together, these data suggested that T3 treatment could provide a therapeutic approach for TBI by preventing neuronal death via mitophagy and promoting neurogenesis via neuron–NSC crosstalk. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction treatments focus only on preventing complications or Traumatic brain injury (TBI) is considered to be a providing support in nature. leading cause of substantial mortality and long-term dis- Thyroid hormone (TH) is crucial for neural stem cell ability among young adults worldwide1. In the United (NSC) differentiation and brain development; its defi- States, TBI contributes to >30% of the deaths resulting ciency leads to irreversible neurological alterations6,7. TBI from traumatic injury each year, with ~1.4 million cases is well known to cause changes in systemic TH levels8. requiring emergency treatment and 235,000 being hos- Accumulating evidence from clinical and experimental pitalized2,3. An estimated 85,000 TBI survivors per year studies has indicated that TH treatment showed neuro- suffer from long-term complications, including cognitive protective properties after acute brain injury, including – disorders, chronic disability, and persistent vegetative stroke and TBI9 11. Understanding TH’s actions and the states2,4. The economic burden is serious; about $37.8 mechanisms regulating TH functions in the adult brain is billion is spent annually on treatments, long-term care, essential to design potential therapeutic strategies for TBI, work absences, and premature deaths2,5. However, no due to dysregulated TH signaling during this procession. specific therapies have been developed, while clinical Mitochondria are indispensable organelles for energy production, cell signaling, and cell survival in eukaryotic cells. However, damaged and dysfunctional mitochondria Correspondence: Chao Lin ([email protected]) or Jing Ji ([email protected]) are also responsible for generating the majority of reactive or Ning Liu ([email protected]) 1Department of Neurosurgery, the First Affiliated Hospital of Nanjing Medical oxygen species (ROS) and the release of cytochrome c (cyt University, Nanjing 210029, China c), which results in cell death12. Thus selective elimination 2 Department of Neurosurgery, Jiangsu Province Hospital, Nanjing 210029, China of damaged mitochondria by mitophagy could be a Full list of author information is available at the end of the article These authors contributed equally: Chao Lin, Nan Li potential therapeutic target for TBI. Although several Edited by A. Verkhratsky

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studies have shown that TH coordinately regulates trephine over the right parieto-temporal cortex. The bone mitophagy and mitochondrial function in the lungs and flap was removed and the dura mater was left intact. muscle in a coordinated fashion, none have done so in a Injury was delivered with a 3-mm flat-tip impounder at a TBI model13,14. velocity of 6 m/s, a depth of 0.6 mm, and a duration of In this study, triiodothyronine (T3)-mediated restora- 100 ms. The bone flap was discarded and the scalp was tion of TBI-induced mitochondrial dysfunction was sutured and closed. Then the mice were returned to their associated with the inhibition of mitochondria-regulated cages to recover from anesthesia. Similarly, mice without neuronal apoptosis that depended on phosphatase and impact were considered as sham. tensin homolog (PTEN)-induced putative kinase 1 (Pink1)-mediated mitophagy. Furthermore, we found that Cortical lesion volume T3 indirectly regulated neurogenesis by modifying One section was obtained every 0.5 mm for lesion neuron–NSC crosstalk. Taken together, these findings volume measurement. Lesion volume was calculated from suggested that TH treatment could reduce neuronal the summation of areas of defect on each slice with Image death, promote NSCs to mature neurons, and subse- J (National Institutes of Health, Bethesda, MD, USA) and quently ameliorate behavioral deficits after TBI. multiplied by slice thickness.

Methods Brain water content Mice and protocol The mouse brain was immediately removed and This study used male C57BL/6 mice (12 weeks old) weighed after sacrifice to obtain the wet weight. Then it − − weighing 22–26 g. We obtained Pink1 / mice from the was dried at 70 °C for 72 h to obtain the dry weight. Lastly, Model Animal Resource Center (MARC; Nanjing, the brain water content was measured in accordance to China)15. Pink1 knockout resulted in the impairment of water content (%) = [(wet weight − dry weight)/wet mitophagy. These mice did not present other specific weight] × 100%. abnormalities at baseline. Green fluorescent protein (GFP) microtubule-associated protein light chain 3 (LC3) trans- Cell culture genic mice were obtained from CasGene Biotech. Co., Ltd Primary neurons were isolated from 17-day-old − − (Beijing, China)16. We crossed GFP-LC3 mice and Pink1 / embryonic mouse brain cerebral cortex and plated on −/− 6 mice to generate GFP-LC3 Pink1 mice. All transgenic poly-D-lysine-coated culture dishes (2.0 × 10 cells/mL). In mice were of C57BL/6J background. Standard laboratory accordance with this study protocol, we used primary animal food and water were provided by the Animal neurons for experiments at 7 days. NSCs were isolated Center of Nanjing Medical University (NMU; Nanjing from 14-day-old embryonic mouse brains (the lateral China). Mice were maintained in a pathogen-free envir- ganglionic eminence and the ventral midbrain of onment and housed on 12-h day/night cycles at an embryonic) and plated on laminin- and poly-L-ornithine- ambient temperature of 22 ± 2 °C. All animal experiments coated dishes. In accordance with the study protocol, were approved by the Animal Care and Use Committee NSCs were grown to 70% confluence before designed of NMU. experiments. Cortical lesion volume and brain water content were measured on day 3 after brain injury. Behavioral tests Oxygen–glucose deprivation (OGD) were done on post-TBI days 7–13. On day 14 after brain In brief, the medium was changed to Dulbecco’s mod- injury, mice were sacrificed and samples were obtained for ified Eagle’s medium (DMEM) without glucose and other designed experiments. serum. Hypoxia was induced by placing the cells in a hypoxic chamber (Billups–Rothenberg, Inc., San Diego, Drug treatment CA, US), which we perfused by 90% N2,5%H2, and 5% We subcutaneously administered T3 (catalog no. CO2 for 30 min to obtain <0.5% O2. Then the chamber T2877; Sigma-Aldrich, St. Louis, MO, US) to mice at was sealed and kept at 37 °C. Primary neurons were 20 μg/100 g/day for 14 consecutive days, starting 24 h incubated at 37 °C for 2 h before reperfusion, while for after brain injury. Saline (vehicle) was used in the sham NSCs, OGD duration was 8 h. group. In vitro, cells were treated with 10 nM T3 for 72 h. Conditioned medium (CM) preparation Primary neurons were incubated in DMEM without Traumatic brain injury serum for 2 h, with or without OGD, and then treated Controlled cortical impact was applied to induce cere- with 10 nM T3 for 24 h. Next, the conditioned medium bral contusion as previously described16. Briefly, a cra- (referred as OGD-T3-N-CM or control-T3-N-CM) was niotomy was done with a portable drill and 4-mm collected. In accordance with the experimental protocols,

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NSCs with or without OGD were treated with CM and Signaling Technology, catalog no. 3700), and NeuN normal medium at a ratio of 1:1 at 37 °C for 24 h. (1:1000; Abcam, catalog no. ab104224).

Oxygen consumption rate (OCR) Transmission electron microscopy We assessed the OCR of primary neurons using a For microscopic examination, mice were euthanized microplate (type XF24) extracellular analyzer (Seahorse with 10% choral hydrate administration and perfused Bioscience, Billerica, MA, US). Briefly, we added 1 μM using 4% paraformaldehyde. The ultrastructure of the oligomycin to inhibit adenosine triphosphate synthesis. samples (100-nm ultrathin sections) was observed with a Then 1 μM carbonyl cyanide-4-(trifluoromethoxy) phe- transmission electron microscope (Quanta 10, FEI Co.). nylhydrazone (FCCP) was added to uncouple the mitochondrial membrane. Finally, we added 1 μM of each Determination of ROS rotenone and antimycin A (R+A) to inhibit complexes I We determined ROS in brain tissues using dihy- and III. Basal OCR was computed as [OCRinitial − OCRR+A]; droethidium (DHE; catalog no. D11347; Thermo Fisher) maximum respiration rate was measured as [OCRFCCP − staining. On day 14 after brain injury, mice were sacrificed OCRR+A]. and samples were obtained for DHE experiment. Briefly, we obtained 20-μm-thick cryosections of frozen brain Confocal microscopy tissues. The samples were incubated with 10 μM DHE at Briefly, coronal frozen sections were embedded onto the 37 °C for 30 min in a humidified, dark chamber and then slides and then incubated by primary or secondary anti- counterstained with DAPI. Fluorescent images were bodies. Multiple fields from one coverslip/well were obtained using the Zeiss confocal microscope and deter- selected to get an average at random and imaged for fur- mined with Image J. ther analysis using the Image-Pro Plus 7.0 software. Each experiment was repeated independently in triplicate or Morris water maze (MWM) more. GFP-LC3 mice were used in this experiment. The MWM was performed by the same experimenter to following primary antibody was used: cyclooxygenase-4 evaluate spatial learning and memory at the same time (COXIV; 1:1000; Abcam, catalog no. ab16056). each day between 08:30 and 12:00 hours. For each trial, mice underwent this experiment using a random start Terminal deoxynucleotidyl transferase dUTP nick-end locations. If mice failed to reach the platform within set- labeling (TUNEL) assay ting time, they were picked up and placed on the platform TUNEL assay was used to evaluate apoptotic cell death for 15 s. For probe trials, mice were placed in the tank in accordance with the manufacturer’s instruction opposite the target quadrant and looked for the platform (#12156792910, Roche, Germany). Briefly, coronal frozen for 60 s. The hidden platform trial was done on test days sections (7-μm thick) were embedded onto the slides and 1–5. On test day 5, a probe trial was performed 2 h after incubated with 50 μL TUNEL reaction mixture. The slides the hidden platform trial. Both visible platform trials were were observed and quantified using a Nikon fluorescent done on test day 6 or 7. The latencies to find the platform microscope. The apoptosis was calculated as TUNEL- and the number of entries were analyzed with a tracking positive cells (red)/4,6-diamidino-2-phenylindole (DAPI) device and software (Chromotrack 3.0, San Diego (blue). Instruments).

Immunoblot analysis Elevated plus maze Extracts for western blot were homogenized in RIPA The elevated plus maze was done to evaluate anxiety/ buffer. In brief, proteins were separated by electrophoresis risk-taking behavior (Noldus Ethovision). The elevated and then transferred to polyvinylidene difluoride mem- plus maze is raised 85 cm above the floor and comprised branes, which were processed with primary antibodies. of the open center (decision zone), the two open arms Next, we incubated these membranes in the appropriate (aversive arms), and the two closed arms (safe arms). diluted secondary antibody at room temperature for 1 h. These four arms were extended out opposite from each The follow primary antibodies were used: p62 (1:1000; other to create a plus shape. Mice were placed on the Abcam, catalog no. ab56416), LC3 (1:1000; Cell Signaling center platform, facing a closed arm, and allowed to Technology, catalog no. 2775s), translocase of outer explore the maze for 5 min. Time in the open arms was mitochondrial membrane 40 homolog (TOM40; 1:1000; used to quantify anxiety-like behavior. Abcam, catalog no. ab51884), COXIV (1:1000; Cell Sig- naling Technology, catalog no. 11967), manganese Novel object recognition (NOR) superoxide dismutase (MnSOD; 1:1000; Cell Signaling Briefly, mice were placed on an empty surface for 5 min Technology, catalog no. 13141), β-actin (1:1000; Cell on test day 1. On test day 2, mice were placed on the same

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surface including two identical objects for 5 min to 1-cm wide beam. One point is given for failing to recognize them. Then mice were placed on this surface complete each of the tasks. including two objects, one is “old” object and the other is “new” object, for 5 min on test day 3. The amount of time Statistical analysis spent in exploring “new” object was recorded to evaluate All results are reported as the mean ± standard deviation the extent of learning and memory. (SD). Gray levels were detected with Image J. The unpaired Student’s t test was applied to compare the differences Neurological severity score (NSS) between two groups with the Prism Software 6.04 (Graph- In brief, a ten-point NSS was used to assess motor Pad Software, Inc.). Motor and MWM data were analyzed function and reflexes of the mice at different time by two-way analysis of variance for an overall statistic and points: (i) exit circle: ability to exit circle of 30 cm dia- followed by Tukey’s test for between-group comparisons. meter (time limit: 3 min); (ii) hemiparesis: paresis of Significant differences were defined as P value < 0.05. upper and/or lower limb of the contralateral side; (iii) straight walk: ability to walk straight on the floor; (iv) Results startle reflex: innate reflex; the mouse will bounce in T3 treatment rescued behavioral deficits after TBI response to a loud hand clap; (v) seeking behavior: To evaluate the effect of T3 on outcome after TBI, we physiological behavior as a sign of “interest” in the examined lesion volume and brain edema. First, we found environment; (vi) beam balancing: ability to balance on a that cortical lesion volume was smaller in the T3-treated beamof7mmwidthforatleast10s;(vii)gripona mice than the mice that received vehicle (Fig. 1a, b). round stick: ability to grip on a beam of 5 mm width for Furthermore, the brain water content was significantly at least 10 s; (viii) beam walk: ability to cross a 30-cm decreased in the TBI group treated with T3 versus those long beam of 3 cm width; (ix) beam walk: same task, on a treated by vehicle (Fig. 1c), suggesting that T3 had the 2-cm wide beam; and (x) beam walk: same task, on a effect of ameliorating brain edema induced by TBI. Taken

Fig. 1 T3 treatment rescued histological and functional deficits after TBI. a, b Representative images (a, arrow) and quantification (b) of cortical lesion volume (n = 5, **P < 0.01). c Analysis of water content (n = 5, *P < 0.05). d–g Morris water maze (MWM). Graph showing hidden platform trial (d) and visible platform trials (e). Representative images (f) and quantification of probe tests (g, #P > 0.05, *P < 0.05). h–k Elevated plus maze. In the open arm, T3-treated TBI mice had similar performance to sham in the present time (h), but vehicle-treated TBI mice spent more time (h,*P < 0.05). i Velocity in the open arm (#P > 0.05). j, k All groups had similar velocity in the closed arm (j, #P > 0.05) and traveled similar total distance (k, #P > 0.05). l Novel object recognition (NOR, **P < 0.01). m Neurological severity score (NSS, **P < 0.01, ***P < 0.001 between T3- and vehicle-treated TBI mice). Sham mice, n = 20; T3-treated sham mice, n = 20; vehicle-treated TBI mice, n = 20; T3-treated TBI mice, n = 22. All data are mean ± SD. V vehicle.

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together, these data demonstrated that T3 treatment via mitophagy13,17. Pink1 is a positive regulator of mito- diminished histological defects after TBI. phagy; therefore, to confirm the role of mitophagy, we +/+ − − To further determine the impact of T3 on behavioral or subjected Pink1 or Pink1 / mice to TBI18. Compared functional outcomes after TBI, we assessed the mice using with controls, we observed an increased amount of MWM, elevated plus maze, NOR, and NSS after injury. autophagic vacuoles containing damaged mitochondria in +/+ − − Mice from different groups showed no differences in the the Pink1 mice but not in the Pink1 / mice (Fig. 3a). hidden- or visible-platform trial performance (Fig. 1d–g). Next, we found that T3-induced decreases in mitochon- However, we observed a significant improvement during drial marker proteins from inner mitochondrial mem- the probe test in the TBI + T3 group compared with the brane (COXIV), outer mitochondrial membrane TBI + vehicle group (Fig. 1f, g). Latency in visible- (TOM40), and matrix (MnSOD) subcompartments were platform trials did not differ among groups, indicating partially reversed by genetic deletion of Pink1 (Fig. 3b–d). that MWM performance was not influenced by differ- Using GFP-LC3 transgenic mice, we found that T3 ences in swimming ability or vision (Fig. 1e). During the treatment enhanced co-localization of GFP-LC3-positive elevated plus maze test, T3-treated mice exhibited mini- puncta with mitochondria and decreased the expression mal risk-taking performance, similar to sham mice of mitochondrial markers, which suggested an increase in (Fig. 1h–k). We found that TBI mice treated with mitophagy (Fig. 3e–g). Furthermore, we found that T3- T3 spent less time in the open arm than vehicle-treated induced mitophagy was blocked by genetic deletion of TBI mice (Fig. 1h). All groups had similar velocity in the Pink1 (Fig. 3e, g). Taken together, these data showed that open (Fig. 1i) and closed arms (Fig. 1j). Nest, we con- T3 treatment significantly promoted mitophagy after TBI ducted an NOR test to further evaluate memory retention. dependent on the Pink1 pathway. T3-treated mice spent significantly more time in exploring the novel object than the vehicle-treated mice (Fig. 1l). T3 treatment reversed TBI-induced mitochondrial There was a tendency for T3 treatment to normalize the dysfunction via mitophagy performance of mice on these tests. Finally, mice treated Given the role of T3 in promoting mitophagy, we with T3 showed improved motor skills (Fig. 1m). Taken examined its effect on mitochondrial dysfunction caused together, these data suggested that T3 administration by TBI. One pathway that neurons employ to prevent resulted in a possible reduction of behavioral deficits and apoptosis is via effective elimination of damaged mito- restored some functional outcomes after TBI. chondria, thus preventing excessive ROS production through mitophagy. First, we found that T3 treatment T3 treatment promoted autophagy after TBI increased basal OCR and maximum respiratory capacity We have recently shown that autophagy can attenuate in primary neurons with OGD (Fig. 4a, b). Next, we neuronal death and behavioral deficits after TBI12,16.As assessed ROS levels in the cortex or hippocampus using other studies have reported, T3 induces autophagy in DHE staining, which is commonly applied to detect ROS brown adipose tissue and lungs13,17. We wondered whe- production. As expected, we observed DHE signal in the ther T3 plays an important role in autophagy after TBI. cortex (Fig. 4c up, Fig. 4d) and hippocampus (Fig. 4c Western blot analysis showed an increase in p62 down, Fig. 4e) after TBI, and it was significantly atte- − − (SQSMT1) and a decrease in lipidated microtubule- nuated by T3 treatment. However, ROS level in Pink1 / associated protein LC3–phosphatidylethanolamine con- mice was not reduced by such treatment (Fig. 4c–e). jugate (LC3-II) in TBI versus control brain tissue, sug- Taken together, these data showed that T3 treatment gesting inhibition of autophagy at least from 3 to 14 days could ameliorate mitochondrial dysfunction induced by after head injury (Fig. 2a–c). Strikingly, we found that T3 TBI in a manner that depended on the mitophagy treatment restored the autophagic activity as evidenced by pathway. decreased p62 and increased LC3-II (Fig. 2d–f). Further- more, representative immunofluorescence data showed T3 treatment suppressed mitochondria-regulated that T3 treatment promoted the expression of GFP-LC3 in apoptosis in a mitophagy-dependent manner the cortex (Fig. 2g, h) and hippocampus (Fig. 2i, j), sug- Given the ability of T3 to restore mitochondrial func- gesting an increase in autophagy. Taken together, these tion, we asked whether it could prevent TBI-induced data showed that T3 treatment could restore the autop- neuronal death. OGD of primary cortical neuron cultures hagic activity and promote the brain autophagy after TBI. in vitro upregulated the level of cleaved-caspase-3 and caused cytotoxicity (Fig. 5a, b). The addition of T3 at the T3-promoted mitophagy was dependent on the Pink1 onset of OGD significantly blocked induction of this pathway pathway (Fig. 5a, b) and rescued neuron viability (Fig. 5c). As previously reported, T3 improved mitochondrial However, we did not find this change in primary cortical − − bioenergetics and ameliorated mitochondrial dysfunction neuron from Pink1 / mice (Fig. 5a–c).

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Fig. 2 T3 treatment promoted mitophagy after TBI. a–c Western blot analysis of autophagy after TBI. β-Actin was used as a loading control (*P < 0.05, **P < 0.01, #P > 0.05 versus sham). d–f T3 restored the activity of autophagy after TBI (*P < 0.05, **P < 0.01). In a–f, β-actin was used as loading control and protein was obtained from cortical injured lesion. g Representative fluorescence micrographs of mouse cortex. Scale bar: up 50 µm, down 20 µm. h Quantifications of LC3 puncta in the mouse cortex, as an indicator of autophagosome (*P < 0.05). i Representative fluorescence micrographs of the mouse hippocampus. Scale bar: up 50 µm, down 20 µm. j Quantifications of LC3 puncta in the mouse hippocampus, as an indicator of autophagosome (**P < 0.01).

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Fig. 3 T3 treatment promoted mitophagy via Pink1 pathway. a Representative electron micrographs of the mouse cortex. Mitochondrion was engulfed by vacuolar structures (arrow). Mt mitochondrion. Scale bar, 1 µm. b–d T3 decreased of the mitochondrial markers of TOM40, MnSOD, and COXIV in Pink1+/+ mice but not in Pink1−/− mice (*P < 0.05, #P > 0.05). e–g T3 increased co-localization of GFP-LC3 and mitochondria (arrows) versus control in Pink1+/+ mice after TBI but not in Pink1−/− mice (Scale bar, 20 µm. *P < 0.05, #P > 0.05).

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Fig. 4 T3 treatment attenuated mitochondrial dysfunction after TBI. a Seahorse analysis of OCR for neurons subjected to hypoxia and then treated with or without 10 nM T3 for 24 h. b A graph showing basal and maximal OCR (*P < 0.05, **P < 0.01). c Representative images of DHE staining on the cortex (up) and hippocampus (down), Scale bar, 100 µm. d Quantifications of DHE fluorescence intensity on the cortex (*P < 0.05, #P > 0.05). e Quantifications of DHE fluorescence intensity on the hippocampus (*P < 0.05, #P > 0.05).

+/+ To confirm this effect of T3, we subjected Pink1 or T3 treatment enhanced neurogenesis after TBI − − Pink1 / mice to TBI. Cyt c released into the cytosol is TBI is well known to induce delayed processes of an apoptogenic factor that triggers neuronal death12. endogenous neurogenesis19. We asked whether T3 could Our previous study showed that the elimination of exert these additional beneficial effects by stimulating damaged mitochondria reduced the release of cyt c and neurogenesis. We found that T3 treatment seemed to subsequently prevented mitochondria-dependent apop- increase the number of cells that were positive for both tosis after TBI12. As expected, T3 could significantly NueN and 5-bromo-2′-deoxyuridine (BrdU) in the sub- +/+ + inhibit cyt c release in Pink1 mice subjected to TBI ventricular zone (Fig. 6a). Quantification of the NeuN / + (Fig. 5d–f). Consistent with the data in vitro, we did not BrdU cells showed that T3 treatment significantly observe this inhibitory activity of T3 on TBI-induced cell increased these surrogate markers of neurogenesis, com- − − death in Pink1 / mice, suggesting a central role for pared with that in vehicle-treated mice that were sub- mitophagy in this process (Fig. 5d–f). Compared with jected to TBI (Fig. 6b). Similarly, we observed more + + mice treated by vehicle, T3 treatment reduced the NeuN /BrdU cells in hippocampus of mice treated by +/+ number of TUNEL-positive cells in Pink1 mice after T3 than those treated by vehicle (Fig. 6c, d). In addition, − − TBI but not in Pink1 / mice (Fig. 5g, h). In addition, we we also found that T3 treatment could promote neuro- found that Pink1 deficiency significantly increased the genesis on day 3 after brain injury (Supplementary Fig. S2). death of dopaminergic neurons in the ventral mesen- Taken together, these data showed the effect of T3 on cephalon after TBI but did not cause more neuronal adult neurogenesis in our model. death in the injured cortex (Fig. 5 and Supplementary Fig. S1). Taken together, these data showed that T3 T3 treatment promoted neurogenesis via the crosstalk treatment could significantly prevent TBI-induced neu- between mature neurons and NSCs in our model ronal death via clearance of damaged mitochondria by To explore the specific molecular mechanism of mitophagy. neurogenesis, we first subjected cultured primary

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Fig. 5 T3 treatment reduced cell death after TBI. a Representative western blot analysis for the expression of caspase-3 and cleaved-caspase-3 in cortical neuronal cultures that were treated as indicated. β-Actin was used as a loading control. b Densitometry of cleaved-caspase-3 (*P < 0.05, #P > 0.05). c Pericyte viability, as determined by the Cell Counting Kit (CCK)-8, in cortical neuronal cultures that were treated as indicated (*P < 0.05, #P > 0.05). d Representative western blot analysis of cytochrome c (cyt c) in the injured cortex. e Densitometry of cyt c in cytosolic fraction (*P < 0.05, ****P < 0.0001, #P > 0.05). f Densitometry of cyt c in mitochondrial fraction (**P < 0.01, #P > 0.05). g, h Representative ﬂuorescence micrographs of the mouse cortex after TBI. Apoptotic cell death was assessed by TUNEL staining (scale bar, 50 µm. *P < 0.05, **P < 0.01, #P > 0.05). In d–h, samples were obtained from the cortical injured lesion on day 14 after brain injury.

mouse NSCs to hypoxia–reoxygenation. T3 treatment The Pink1 pathway was required for T3-promoted could not promote differentiation of these NSCs to neurogenesis in our model mature neurons (Fig. 7a–c). Next, we wanted to know Mitochondrial metabolism is involved in NSC differ- whether mature neurons could promote NSC differ- entiation20. We wanted to understand the implication of entiation. The addition of neuron-conditioned medium mitophagy in T3 control of NSCs’ fates. The results (with or without OGD) could not shift NSCs toward a showed that conditioned medium favored NSCs shifting neuronal phenotype (Fig. 7d–f). However, conditioned toward a neuronal phenotype, whereas we did not − − medium from cultured neurons that were subjected to observed this change in NSCs from Pink1 / mice OGD and treated with T3 enhanced the differentiation (Fig. 8a–c). Interestingly, we found that conditioned − − of NSCs into mature neurons (Fig. 7d–f). Taken toge- medium from Pink1 / neurons also could not enhance ther, these data showed that T3 treatment promoted the differentiation of NSCs into mature neurons neurogenesis via crosstalk between mature neurons and (Fig. 8d–f). Furthermore, there were no differences in NSCs. NOR (Fig. 8g) or NSS (Fig. 8h) between the T3 and

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Fig. 6 T3 treatment promoted neurogenesis after TBI. a Representative images of BrdU+NeuN+ double-positive cells in subventricular zone (scale bar, 50 µm). b Quantiﬁcation of double-positive cells in subventricular zone (**P < 0.01, #P > 0.05). c Representative images of BrdU+NeuN+ double-positive cells in the hippocampus (scale bar, 50 µm). d Quantiﬁcation of double-positive cells in the hippocampus (*P < 0.05, #P > 0.05).

− − vehicle treatment groups in Pink1 / mice after TBI. damaged mitochondria via Pink1-dependent mitophagy. Taken together, these data showed the involvement of In addition, T3 regulated NSC differentiation toward a mitophagy in T3-promoted neurogenesis and recovery in neuronal phenotype via crosstalk between mature neu- our model. rons and NSCs. Taken together, these findings suggested that T3 treatment might provide a therapeutic approach Discussion for TBI by preventing cell death and promoting Mitochondria are dynamic organelles that play key roles neurogenesis. in many cellular functions, including energy production, Mitophagy plays a key role in mitochondrial quality oxidative stress, metabolism, and cell death21. One path- control with fusion and fission dynamics that eliminate way that neurons employ to prevent excessive ROS pro- dysfunctional and damaged organelles16,22. The mito- duction and avoid cell death is effective elimination of phagic machinery could eliminate damaged mitochondria dysfunctional or damaged mitochondria via a specialized that would have otherwise possibly induced apoptosis. Cyt autophagic process, mitophagy16. Impaired mitophagy c released from damaged mitochondria into the cytosol is causes an accumulation of dysfunctional mitochondria, an apoptogenic factor that generates death signals in resulting in the neuronal death12,16. In this study, our data TBI3,16. The removal of damaged mitochondria by mito- showed that T3 markedly reduced ROS production and phagy significantly attenuates mitochondria-dependent prevented neuronal death by promoting the clearance of apoptosis and subsequently reduces neuronal death16.

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Fig. 7 T3 treatment promoted neurogenesis via crosstalk between mature neuron and NSCs. a–c Representative western blot and quantification of NeuN (b, #P > 0.05) and growth-associated protein 43 (GAP43; c, #P > 0.05) expression in NSC cultures that were treated with T3 or vehicle for 3 days in a reoxygenation state after 8 h of hypoxia (H/R). d–f Representative western blot and quantification of the indicated proteins in NSC cultures that were subjected to H/R and treated with mature neuron conditioned medium from the experimental treatment groups. e Quantification of NEUN (*P < 0.05, #P > 0.05). f Quantification of GAP43 (*P < 0.05, #P > 0.05).

Dynamin-related protein 1 (Drp1) is involved in mito- TH has been speculated to be a putative cure that chondrial fission and clearance of damaged mitochondria could determine the cell fate, eventual survival, and via mitophagy; it is activated and then recruited to differentiation of NSCs6,7.Thisnotionisinspiredby mitochondria from the cytosol in response to stress. T3 several studies wherein a role for TH has been identified promotes Drp1 activation and localization to mitochon- as playing a key role in the modification of mitochon- dria23. In this study, our data demonstrated that T3 drial metabolism and promotion of differentiation20. treatment reduced cell death and improved outcomes Recent evidence now suggests that mitochondrial dys- after TBI. Mitophagic agonists might show potential as function is characterized by impaired mitochondrial new neuroprotective drugs for TBI, but the specific respiration and increased ROS production, both of mechanisms involved in promoting the degradation of which have been shown to impair adult neurogen- damaged mitochondria remain to be delineated. esis6,7,24. In this study, T3 promoted adult neurogenesis The microenvironment is critical for fate determi- via neuron–NSC crosstalk, in which mitophagy was nation and cell differentiation of NSCs24.Emerging required. evidence reveals that TBI markedly augments the In summary, the present study showed that T3 treatment proliferation of these cells, but a microenvironment of promoted mitophagy to reduce cell death but also regulated accumulated toxins, ROS, and reduced oxygen leads to NSC differentiation toward a neuronal phenotype after TBI. low survival rates for mature neurons24,25.Inthis Furthermore, we found that T3-induced neurogenesis was study, T3 treatment enhanced the clearance of via crosstalk between mature neurons and NSCs, in which damaged mitochondria via mitophagy and reduced mitophagy was required. Given that mitophagy is affected ROS production. Surprisingly, Pink1 knockout mice in the development of several diseases, further investigation demonstrated little recovery after T3 treatment, sug- is required to determine how to mediate mitophagy in gesting the involvement of mitophagy in T3-induced order to attenuate cell death and improve outcomes in TBI neurogenesis and neuroprotection. and other mitophagy-related conditions.

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Fig. 8 Mitophagy was essential for T3-mediated neurogenesis. a–c Representative western blot and quantification of NeuN and GAP43 in NSC cultures isolated from Pink1+/+ or Pink1−/− mice. b Quantification of NeuN (**P < 0.01, #P > 0.05). c Quantification of GAP43 (*P < 0.05, #P > 0.05). d–f Representative western blot analysis of NeuN and GAP43 in NSC cultures treated by different conditioned mediums. e Quantification of NeuN (*P < 0.05, #P > 0.05). f Quantification of GAP43 (**P < 0.01, #P > 0.05). g Novel object recognition (NOR, #P > 0.05). h Neurological severity score (NSS, #P > 0.05 versus vehicle-treated TBI mice). Sham mice, n = 15; T3-treated sham mice, n = 15; vehicle-treated TBI mice, n = 15; T3-treated TBI mice, n = 15.

Acknowledgements Publisher’s note This work was supported by a grant from the National Natural Science Springer Nature remains neutral with regard to jurisdictional claims in Foundation of China (81901258 to C.L. and 81972153 J.J.). published maps and institutional afﬁliations.

Author details Supplementary Information accompanies this paper at (https://doi.org/ 1 Department of Neurosurgery, the First Afﬁliated Hospital of Nanjing Medical 10.1038/s41419-020-02836-9). University, Nanjing 210029, China. 2Department of Neurosurgery, Jiangsu Province Hospital, Nanjing 210029, China. 3Department of Nephrology, Drum Received: 9 April 2020 Revised: 28 July 2020 Accepted: 28 July 2020 Tower Hospital, Nanjing 210029, China

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