The majority of transcripts of a second gene (SMN2) generate an alternative spliced isoform that lacks exon 7 and produces a truncated nonfunctional form of SMN. A major function of SMN is the biogenesis of spliceosomal snRNPs, which are essential components of the pre-mRNA splicing machinery, the spliceosome. In recent years, new potential therapies have been developed to increase SMN levels, including treatment with antisense oligonucleotides (ASOs). The ASO-nusinersen (Spinraza) promotes the inclusion of exon 7 in SMN2 transcripts and notably enhances the production of full-length SMN in mouse models of SMA. In this work, we used the intracerebroventricular injection of nusinersen in the SMN∆7 mouse model of SMA to evaluate the effects of this ASO on the behavior of Cajal bodies (CBs), nuclear structures involved in spliceosomal snRNP biogenesis, and the cellular distribution of polyadenylated mRNAs in αMNs. The administration of nusinersen at postnatal day (P) 1 normalized SMN expression in the spinal cord but not in skeletal muscle, rescued the growth curve and improved motor behavior at P12 (late symptomatic stage). Importantly, this ASO recovered the number of canonical CBs in MNs, significantly reduced the abnormal accumulation of polyadenylated RNAs in nuclear granules, and normalized the expression of the pre-mRNAs encoding chondrolectin and choline acetyltransferase, two key factors for αMN homeostasis. We propose that the splicing modulatory function of nusinersen in SMA αMN is mediated by the rescue of CB biogenesis, resulting in enhanced polyadenylated pre-mRNA transcription and splicing and nuclear export of mature mRNAs for translation. Our results support that the selective restoration of SMN expression in the spinal cord has a beneficial impact not only on αMNs but also on skeletal myofibers. However, the rescue of SMN expression in muscle appears to be necessary for the complete recovery of motor function.Spinal muscular atrophy (SMA) is a devastating autosomal recessive neuromuscular disease characterized by degeneration of spinal cord alpha motor neurons (αMNs). SMA is caused by the homozygous deletion or mutation of the  SMN depletion triggers the selective degeneration of lower alpha motor neurons (αMNs), resulting in preferential atrophy of the proximal skeletal muscles with weakness and muscle paralysis3.Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease that is considered the main genetic cause of infant mortality. SMA is caused by the homozygous loss or mutation of the SMN1 gene, humans ubiquitously express one or several copies of a closely related paralog gene called SMN2. This gene encodes a full-length SMN protein but presents one consistent difference, that is, the transition C to T (C6T) in exon 7. This change deactivates a splicing enhancer and introduces a splicing silencer, resulting in the exclusion of exon 7 in most SMN2 transcripts5. These transcripts are translated into a truncated SMN (SMNΔ7) protein that is rapidly ubiquitylated and degraded by the proteasome system7. Although the expression of the SMN2 gene is residual under physiological conditions, the deletion or mutation of SMN1 confers the number of copies of SMN2 considerable importance in modifying the severity of SMA phenotypes. Thus, the most severe and frequent SMA is that of type I, which usually has two copies of the SMN2 gene. Patients with type II or III SMA have a higher copy number of SMN211.In addition to the 12. The SMN complex acts as a molecular chaperone to assist the cytoplasmic stage of the assembly of spliceosomal U snRNPs, which are essential splicing factors of the spliceosome14. Linked to this function of the SMN complex, the Cajal body (CB) is particularly relevant, given its implication in the biogenesis of both spliceosomal U snRNPs and nucleolar snoRNPs18. In particular, the enrichment of the CB in spliceosomal U snRNPs is consistent with the growing evidence of a spatial association of this organelle with certain transcriptionally active genomic loci of snRNAs21. Moreover, the physical association of CBs with the dense fibrillar component of the nucleolus, the site of synthesis and early processing of pre-rRNAs provides a molecular link for a nucleolus-CB interaction24.The functional full-length SMN protein homo-oligomerizes and forms a stable complex (SMN-complex) with Gemin2-8 and Unrip proteins26. The dysfunction of mRNA processing in this SMA mouse is also characterized by (i) the early and progressive accumulation of polyadenylated mRNAs in nuclear granules called PARGs (“poly(A) RNA granules”), (ii) the increase in intron-containing pre-mRNAs, and (iii) the disruption of the protein synthesis machinery27. In fact, the dysfunction of RNA metabolism has enabled us to redefine the SMA as a RNA pathology, although it could also be considered a spliceopathy28.In this context, recent studies from our laboratory have shown that reduced SMN levels in αMNs of a type I SMA patient and the SMN∆7 mouse model induce nuclear features of αMN degeneration, including depletion of canonical CBs, nucleolar stress and altered mRNA processingO-methoxyethyl (MOE)-modified ASO was designed to increase full-length SMN protein levels by promoting the inclusion of exon 7 in SMN2 mRNAs. Nusinersen binds to a target site within intron 7, ≅ 10 nucleotides downstream of the 5′-splice site, known as ISS-N1 (intronic splicing silencer N1)29. This interaction displaces the splicing suppressor proteins hnRNP A1/A2 and enables spliceosomal U1 snRNPs to bind to the 5′-splice site, thereby promoting the inclusion of exon 7 in SMN2 transcripts35. Previous studies in mouse SMA models have demonstrated that nusinersen, when administered directly into the cerebrospinal fluid (CSF), prolongs animal survival and prevents αMN and skeletal muscle pathology37.To explore whether normalizing SMN expression in SMN∆7 αMNs during the neonatal period, prior to αMN degeneration, prevents CB depletion and splicing alterations with nuclear retention of unspliced pre-mRNAs in PARGs, we treated SMN∆7 mice with the antisense oligonucleotide (ASO) nusinersen (Spinraza). This 2′-Chodl) and choline acetyltransferase (Chat), two key factors for αMN homeostasis38, and (iv) reduces the nuclear retention of polyadenylated mRNAs into PARGs at the symptomatic P12 stage. These effects are accompanied by the restoration of the protein synthesis machinery, supporting an effective recovery of mRNA processing and translation.Our results in SMN∆7 αMNs show that the intracerebroventricular (ICV) injection of nusinersen at the neonatal period  (i) improves motor function, (ii) rescues the CB number, (iii) increases the expression of pre-mRNAs encoding chondrolectin  the preclinical phase (from P0 to P6) and (ii) the clinical phase (from P7 to P12), when the clinical symptoms appear and progress, commonly leading to the death of mice between P12 and P14. = 84.23, p < 0.0001; “body weight follow-up × nusinersen treatment”: F = 57.64, p < 0.0001).The ICV administration of nusinersen Fig. A at P1 w39. Whereas the SMNΔ7 mice did not acquire the righting reflex during the neonatal period studied (P1-P12)  = 14.85, p < 0.0001; “righting reflex follow-up × nusinersen treatment”: F = 72.77, p < 0.0001). Moreover, whereas SMNΔ7 mice exhibited very severe muscular atrophy, which affects the ability of moving, nusinersen-treated SMNΔ7 mice exhibited moderate hindlimb atrophy and preserved ambulation capacity; however, moderate hindlimb paresis was noticeable in these animals compared to WT mice  muscle lysates revealed that the neonatal ICV delivery of nusinersen in SMNΔ7 mice did not increase SMN expression in muscle at P12  immunostained for ChAT, a marker of αMNsorn Fig. I,K. Thusorn Fig. P. In conorn Fig. J–K,P.40. Interestingly, despite the reduced SMN expression in the muscle of nusinersen-treated SMNΔ7 mice, nonsignificant differences in myofiber diameter were found between WT mice and nusinersen-treated SMNΔ7 mice at P12 , a marker of mitochondrial oxidative activity18. Consistent with this notion, our previous studies in αMNs from an SMA patient and the SMNΔ7 mouse have shown that the αMN degeneration caused by SMN deficiency includes an early depletion of canonical CBs and relocalization of coilin as perinucleolar caps and/or within the nucleolus25. Based on this observation, we considered that the disruption of CBs is an early nuclear sign of αMN degeneration in SMA.The CB is a transcription-dependent nuclear organelle whose number and size accommodate the cellular demands of pre-mRNA splicing18, in both WT and nusinersen-treated SMNΔ7 αMNs    and ChAT (choline acetyltransferase) are highly expressed in αMNs, playing crucial functions in motor axon growth and neurotransmission, respectively52. Moreover, Chodl and ChAT are major gene products with dysregulated expression in SMA mouse αMNs38.Previous studies have demonstrated widespread defects in transcription and pre-mRNA splicing of essential genes for αMN homeostasis in cellular and animal models of SMAChodl and ChAT, we analyzed the expression levels of both pre-mRNAs by qRT-PCR in spinal cord RNA extracts at P12. We found a significant reduction in Chodl and ChAT pre-mRNAs in SMNΔ7 samples compared with WT samples  distributed throughout the cytoplasm in both WT and nusinersen-treated SMNΔ7 αMNs  RNA hybridization signal, suggesting a decreased availability of mRNA for translation  RNA into PARGs can be a neuroprotective mechanism to prevent the nuclear export and translation of unprocessed or aberrant polyadenylated mRNAs.Next, we analyzed whether nusinersen treatment preserves the normal distribution of poly(A) RNAs, which include all mRNAs with the exception of histone mRNAs, as an indicator of proper nuclear mRNA processing and export to the cytoplasm. In situ hybridization with the poly-dT probe revealed that poly(A) RNA appeared diffusely distributed throughout the nucleus (excluding the nucleolus), highly concentrated in nuclear speckles and accumulated in extensive cytoplasmic areas of WT αMNs Fig. F,I. In cion Fig. G,J. Inteion Fig. H,K. Furtion Fig. B. Since 58. Degeneration of αMNs leads to severe muscular atrophy by denervation, muscle paralysis and premature death by respiratory failure in type I SMA, the most severe form of the disease.It is well established that αMNs are the main physiopathological cellular targets of SMA, although increasing evidence has revealed the primary importance of other peripheral tissues26. Moreover, polyadenylated mRNAs are aggregated in PARGs, reflecting an alteration of mRNA splicing with increased intron retention, abnormal accumulation of incorrectly processed mRNAs and inhibition of the nuclear export of mature mRNAs27. This neuronal dysfunction leads to a neurodegenerative process, which is characterized by reduced availability of mature mRNAs for translation, progressive disruption of the protein synthesis machinery and, ultimately, neuronal death53.In our previous studies in the SMNΔ7 mouse model of SMA, we observed that SMN depletion in αMNs during the symptomatic postnatal stage severely impacts nuclear compartments involved in pre-rRNA and pre-mRNA processing37. However, a recent study using the systemic administration of ASO10-27 in SMA mice supports that peripheral tissues play an essential role in SMA pathology41. The authors indicate that the increase in SMN exclusively in peripheral tissues compensates for its deficiency in the spinal cord and preserves αMNs. In our study, the improvements in body weight, motor function and survival in SMN∆7 mice treated with nusinersen were associated with a notable increase in SMN levels in the spinal cord but not in the TA muscle, which exhibited reduced SMN levels, similar to those detected in untreated SMN∆7 mice. Although a fraction of nusinersen injected into cerebral ventricles may be cleared with CSF into the blood and influence peripheral tissues, our results and those from other authors59 support that the increase in SMN in the spinal cord is required for the rescue of αMN and skeletal myofiber homeostasis. In fact, we observed that both the skeletal myofiber size distribution and the SDH oxidative staining pattern of myofibers are rescued, despite having reduced SMN levels in muscle. However, the relative importance of SMN restoration in spinal cord versus peripheral tissues in the therapeutic outcome reported in separate studies may be notably influenced by differences in transgenic mouse models of SMA and treatment protocols with ASO.In this study, we demonstrate that the therapeutic outcome of the ICV administration of nusinersen (Spinraza) in the SMNΔ7 mouse model of type I SMA is mediated by the selective restoration of SMN levels in the spinal cord but not in muscle. Nusinersen treatment in neonatal SMNΔ7 mice normalizes the growth curve and improves motor function at the late symptomatic stage (P12). These effects are mediated by preserving the normal population of αMNs, as well as the nuclear organization of mRNA processing compartments and the protein synthesis machinery. Consistent with these findings, previous studies in mouse SMA models have demonstrated that nusinersen, when administered directly into the CSF, prolongs animal survival and prevents αMN and skeletal muscle pathologySMN2 gene, promoting the inclusion of exon 7 in the SMN2 mRNAs and their translation in full-length SMN protein60, we detected a recovery in the number and molecular composition of CBs. This neuroprotective mechanism, mediated by the increased expression of SMN in αMNs, can promote the molecular assembly of canonical CBs, which are enriched in coilin, SMN and spliceosomal snRNPs43. CBs are transcription-dependent nuclear organelles involved in spliceosomal snRNP biogenesis required for pre-mRNA splicing61. Depletion of CBs and coilin relocation in the form of perinucleolar caps or within the nucleolus are reliable early signs of neuronal dysfunction in many neurodegenerative disorders, including SMA62. It is noteworthy that snRNPs are essential components of the spliceosome, the macromolecular machine for pre-mRNA splicing14, and their biogenesis is considerably more efficient in CBs than in the nucleoplasm63. In fact, depletion of CBs in cells derived from SMA patients correlates with reduced U4/U6-U5 tri-snRNP assembly, a key maturation step of spliceosomal snRNPs, and with splicing alterations64. A recent study reported an increase in the number of Gems (SMN-positive and coilin- and snRNP-negative nuclear bodies of unknown function46) in αMNs following the subcutaneous administration of ASO10-27 in a mouse model of SMA41. However, in our study, we have never observed Gems but canonical CBs induced by nusinersen treatment in SMA mice.Consistent with the essential role of nusinersen in the splicing regulation of the Chodl and ChAT, two genes dysregulated in SMA that are essential for αMN survival52, is consistent with the increase in CBs, whose numbers must be accommodated to the neuronal demand for pre-mRNA processing44. In this context, it was also noteworthy that nusinersen treatment in SMNΔ7 mice also rescued the normal organization of the protein synthesis machinery (Nissl bodies) and substantially recovered the nuclear and cytoplasmic compartmentalization of polyadenylated mRNAs. It is well established that all mRNAs, except for histone mRNAs, are polyadenylated in the nucleus, and the poly(A) tail is necessary for efficient mRNA export and translation65. The regulation of the nuclear and cytoplasmic compartmented transcriptomes is essential for cell homeostasis. Indeed, in this study and previously27, we have demonstrated the nuclear accumulation of polyadenylated RNAs into PARGs, which is associated with intron retention. Nuclear retention of polyadenylated mRNAs may prevent the nuclear export of unspliced or inappropriately expressed transcripts and attenuate translation and energy consumption to maintain energy homeostasis under stress conditions66. Moreover, nuclear retention of polyadenylated mRNAs may be linked to defective snRNP biogenesis in CBs. In this sense, a recent study67 reported that the inhibition of U4 snRNP biogenesis, a key component for U4/U6-U5 tri-snRNP assembly in CBs68, results in the abnormal nuclear accumulation of polyadenylated mRNAs. Importantly, the ICV administration of nusinersen in SMN∆7 mice substantially reduced the formation of PARGs and restored the nuclear and cytoplasmic compartmentalization of polyadenylated mRNAs in αMNs. This neuronal response is consistent with a substantial recovery of CB number and pre-mRNA processing and export of mature polyadenylated mRNAs for their translation.In nusinersen-treated SMNΔ7 mice, the transcription rate activation of Chodl and ChAT primary transcripts, (iii) nuclear and cytoplasmic distribution of polyadenylated mRNAs, and (iv) organization of protein synthesis machinery in αMNs of the SMNΔ7 mouse appears to be essential for maintaining neuronal homeostasis and survival. The increase in SMN expression in the spinal cord partially prevents the severe SMA myopathy induced by αMN degeneration. We suggest that the incomplete recovery of neuromuscular performance in nusinersen-treated SMNΔ7 mice with moderate hindlimb paresis may result from reduced SMN expression in muscle. Further studies will be addressed to investigate the potential role of nusinersen treatment on skeletal myofibers, particularly on the focal disruption of the sarcomere architecture and actin dynamics observed in the SMNΔ7 mice (our unpublished results). In this vein, the combined use of subcutaneous and ICV administration of nusinersen may improve the therapeutic effects of this drug in SMA myopathy41.In conclusion, the nusinersen (Spinraza)-dependent rescue of the (i) molecular assembly of canonical CBs, (ii) expression of Smn/−+;SMN2/++;SMN∆7/++, heterozygous knockouts for mouse Smn , which were purchased from the Jackson Laboratory , were crossed to generate Smn−/−;SMN2/++;SMN∆7/++ (SMN∆7) mice and Smn/++;SMN2/++;SMN∆7/++ mice that were wild-type for Smn (WT). SMN∆7 mice exhibit a severe postnatal SMA phenotype with a mean lifespan of approximately 2 weeks70. Animal care and handling were performed in accordance with the Spanish legislation  and the guidelines of the European Commission for the Accommodation and Care of Laboratory Animals (revised in Appendix A of the Council Directive 2010/63/UE). The experimental plan was approved by the Ethics Committee of the University of Cantabria and the Committee for Animal Care and Use of the University of Lleida. On P0, the identification of WT and SMN∆7 mice performed by genotyping with PCR. DNA was extracted from the tail, as previously described69. Age-matched WT littermates of mutant animals were used as controls.41. All mice were sacrificed at the late symptomatic neurodegenerative stage (P12)27.To investigate the effects of nusinersen (Spinraza) treatment in the αMNs of SMN∆7 mice, we used 32 animals (16 WT mice and 16 SMN∆7 mice) that were divided into four groups. WT and SMN∆7 mice were treated with 10 µl of Spinraza (Biogen) at a dose of 6.5 mg/kg (nusinersen group), or with 10 µl of a 0.9% saline solution (vehicle group). Spinraza or the vehicle was injected at the day of birth (P0) via ICV at the precise point indicated in Fig. 39. Briefly, this test assesses the motor capacity such that a mouse pup can stand up from a supine position. The average age for the rodent straightening reflex to appear is P5. As this test is a reflex, there is no learning component, and it can be repeated throughout the period of experimentation. This test consists of placing the pups face up on a cotton sheet or bench pad and keeping them in this position for 5 s. The pups are then released, and the time it takes to return to the prone position is recorded, as well as the direction of the correction (left or right). A total of 1 min is given for each test, if necessary. The test is repeated for a total of three trials.To analyze the effect of nusinersen treatment on motor functions, we used the surface righting test (righting reflex), according to the protocol of Feather-Schussler and Ferguson71. Briefly, transverse cryosections were incubated in an SDH reaction mixture . During the electron transfer from succinic acid to nitroblue tetrazolium by SDH, a color change occurs, the rate of which represents SDH activity. As controls, we used cryosections incubated in a medium lacking succinic acid. Then, the samples were air-dried and mounted, and images were captured with a microscope  using 40 × or 63 × oil objectives. The morphometric analysis of myofiber diameter was performed on transversal cryosections of the TA muscle stained with the histochemical SDH assay. The measurement of the diameter of each myofiber in the field was performed on randomly collected microscope images using a 40 × objective and ImageJ software . At least 100 myofibers per animal  were measured.To investigate the effect of the ICV administration of nusinersen on the phenotype type of TA muscle myofibers in the SMNΔ7 mouse, we used the histochemical SDH assay, a specific mitochondrial marker of myofiber oxidative activity. Thus, most SDH-positive myofibers correspond to small type 1 myofibers, which have slow contraction and high aerobic metabolism. Less SDH-positive myofibers of type 2X of greater caliber are associated with faster contraction and more anaerobic muscle. Finally, type 2A myofibers have an intermediate signal and activity between type 1 and 2X myofibers43.For immunofluorescence analysis of αMNs at P12, 4 mice per group  were fixed by perfusion with 3.7% paraformaldehyde (freshly prepared) in phosphate-buffered saline (PBS) under deep anesthesia with pentobarbital (50 mg/kg). The spinal cords were rapidly dissected, removed, postfixed for 6 h and washed in PBS. Transverse sections (160-µm thick) of the spinal cord were obtained with a vibratome, and small tissue fragments from the anterior horn were dissected out. The samples were transferred to a drop of PBS on a positively charged slide , and squash preparations of dissociated MNs were generated following the previously reported procedure2, 6 mM K3PO4, 1 mM EGTA, and 0.1% glucose; pH 7.0) and relaxed at 4 °C until fixed in 3.7% paraformaldehyde72. Muscle fragments were cryoprotected in 15% and 30% sucrose in PBS  until they sank, embedded in Tissue-Tek OCT Compound (Sakura FineTek USA), and frozen on a chilled metal block in dry ice. Cryosections measuring 8 μm in thickness were mounted on SuperFrost slides and stored at − 80 °C until use.For muscle, tissue fragments of the TA were pinned to cork, immersed in ice-cold relaxing buffer  were used. Some samples were counterstained with propidium iodide (PI) or Phalloidin-FITC. The slides were mounted with ProLong Anti-Fading Medium (Invitrogen).For immunofluorescence, all samples were processed as described in Pena et al.We used an LSM510  laser scanning microscope using a 63 × oil (1.4 NA) objective. To avoid overlapping signals, images were obtained by sequential excitation at 488 nm and 543 nm. The fluorescence profiles of confocal intensity signals across a line shown in Fig. S2 were generated using confocal images of double immunolabeled samples. Images were processed using Adobe Photoshop CS6 software.The following primary antibodies were used for immunofluorescence: mouse monoclonal anti-SMN ; mouse monoclonal anti-TMG-cap ; mouse monoclonal anti-fibrillarin ; rabbit polyclonal anti-coilin ; and goat polyclonal anti-choline acetyltransferase (ChAT) .27. Preparations of MNs were permeabilized with TBS-E-SDS for 15 min at 37 °C, washed three times in 6 × SSPE-0.1% Tween 20 for 15 min, and incubated with the probe containing tRNA for 3 h at 42 °C in a humidified chamber. An oligo(dT)(50)-mer, 5′-end-labeled with biotin , was used as a probe for fluorescence in situ hybridization (FISH) to poly(A) RNA. The hybridization mixture contained 80 ng of oligo dT(50), 2 × SSC, 1 mg/ml tRNA, 10% dextran sulfate and 25% formamide. After hybridization, preparations of MNs were washed in 6 × SSC for 15 min and then washed in 4 × SSC-0.1% Tween 20 for 15 min at room temperature. The hybridization signal was detected with FITC-avidin for 30 min. All samples were mounted with Vectashield .Fluorescence in situ hybridization (FISH) was performed according to the protocol described by Narcís et al.The quantitative analysis of the proportion of SMN∆7 αMNs containing PARGs was performed in squash preparations processed for FISH with the poly(dT) probe. The proportion of neurons containing these granules was estimated by direct examination of the different focal planes throughout neuronal nuclei using a 40 × objective. Quantification was performed on at least 100 αMNs from WT (n = 4), SMN∆7 (n = 4) and nusinersen-treated SMN∆7 (n = 4) mice.For qRT-PCR, we used WT (n = 4), SMN∆7 (n = 4) and nusinersen-treated SMN∆7 (n = 4) mice. Mice were decapitated after being anesthetized, and the lumbar spinal cord was quickly removed and frozen in liquid nitrogen. RNA was isolated with TRIzol following the manufacturer’s instructions  and purified with the RNeasy kit .27, 1 μg of RNA was reverse-transcribed to first-strand cDNA utilizing a High Capacity cDNA Reverse Transcription Kit (Life Technologies) using random hexamers as primers. The expression of the mRNA candidates ChAT and Chodl was determined by qRT-PCR using gene-specific SYBR Green-based primers (Invitrogen). The threshold cycle (Ct) for each well was determined and the results were normalized to Gapdh mRNA. Relative gene expression was calculated according to the 2-(ΔΔCt) equation73. Each value in this work represents the mean ± SD of independent samples obtained under the same conditions and compared to two replicated qRT-PCR analyses. The SYBR Green-based specific primers for murine pre-RNAs analyzed were as follows: for the pre-mRNA of ChAT 5′-CTTGGGGCCAGTCTGATAGC-3′ and 5′-GGACACATGGCTAGAAGGGG-3′ and for the pre-mRNA of Chodl 5′-GCTGTTGTCTCCCGCATCTT-3′ and 5′-AAGTGGAAGCGTTTGGGATT-3′.According to our laboratory protocols27. Briefly, spinal cords and skeletal TA muscle samples from WT (n = 3), SMN∆7 (n = 3) and nusinersen-treated SMN∆7 (n = 4) mice were lysed at 4 °C in a buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 2% Nonidet NP-40, 1 mM MgCl2, 1 mM dithiothreitol, and 10% glycerol and supplemented with EDTA-free complete protease inhibitor cocktail and PhosphoSTOP (Roche). Samples were sonicated and cleared by centrifugation at 14,000 rpm for 10 min at 4 °C. The proteins were separated on 4–20% NuPage TG SDS–PAGE gels (Invitrogen) and transferred to nitrocellulose membranes using standard procedures. Mouse monoclonal anti-SMN (diluted 1:500) and rabbit polyclonal anti-Lamin A/C  were used. Protein bands were detected with an Odyssey Infrared-Imaging System (Li-Cor Biosciences) according to the Odyssey Western-Blotting Protocol. For the quantitative analysis of the blots, ImageJ software was used .Analysis of the level of SMN protein were performed according to the protocol described by Narcís et al. t test (GraphPad). In all cases, differences were considered to be statistically significant if p < 0.05.For comparisons among the experimental groups, data were analyzed using GraphPad Prism 7 software and PASW Statistics 22  packages. Data are expressed as mean ± SD. The statistical analysis was assessed by one-way ANOVA or mixed-design two-way ANOVA (Split-Plot ANOVA), followed by the Bonferroni post hoc test (SPSS) or Student’s Supplementary information 1Supplementary information 2Supplementary video


Accordingly, TNBC low EHD2 expressers were found to benefit the most from chemotherapy when compared to all subtypes of breast cancers. Our study validates EHD2 expression level as an independent prognostic factor of metastasis-free survival and as a new predictive marker of chemotherapy efficacy in TNBC patients.EHD2 is a mechanotransducing ATPase localized in caveolae invaginations at the plasma membrane. EHD2 has recently been associated with several human cancers, however the significance of EHD2 transcript levels in cancer prognosis remains debated. Breast cancer is the most commonly occurring cancer in women and prognosis is variable depending on the subtypes. Triple negative breast cancer (TNBC) often has a poor therapeutic response. The aim of this study was to assess the prognostic significance of EHD2 transcripts and protein expression levels in breast carcinomas. We found that low EHD2 levels were associated with enhanced proliferation, migration and invasion of TNBC cells. EHD2 expression was significantly reduced in TNBC tissues and the loss of EHD2 led to higher expression of the pro-tumoral cytokine IL-8. In apparent contradiction with  We recently established EHD2 as a key element in mechanotransduction by connecting caveolae mechanosensing with gene transcription under mechanical stress3. EHD2 has recently been associated with several types of cancer. It has been shown that EHD2 protein levels were reduced in esophageal squamous cell carcinoma compared to normal tissues4. Low expression of EHD2 increased the migration of human esophageal squamous cell carcinoma TE1 cells4 and similar results have also been reported in ovarian cancer, malignant melanoma and hepatocellular carcinoma7. In line with these findings, it was also shown that EHD2 can inhibit metastasis of breast cancer9.Eps15 homology domain containing protein 2 (EHD2) is a cytosolic ATPase localized at the neck of small plasma membrane invaginations called caveolae, where it controls their stability and dynamics at the cell surface10. Breast cancer prognosis is variable, depending mostly on the molecular features of the tumor and on tumor stage at diagnosis. Breast tumors can be separated into different molecular subtypes: (1) the luminal A and B subtypes, overexpressing progesterone (PR) and/or estrogen receptors (ER), (2) the HER2 + subtype, expressing high levels of the human epidermal growth factor receptor 2 (HER2) protein and (3) the triple negative breast cancers (TNBC) expressing no PR and/or ER and without HER2 overexpression and/or amplification13. Although TNBC account for only 15% of primary breast cancers14, they remain an important therapeutic challenge because of their poor prognosis, with variable responses to chemotherapy16. An important challenge is therefore to identify new targets and associated biomarkers for TNBC therapy. In this study, we found that the expression of EHD2 was significantly reduced in TNBC as compared to the other subtypes of breast cancers, and that the loss of EHD2 significantly enhanced the migration and the invasion of TNBC cells. Moreover IL-8, a cytokine endowed with a pro-tumorigenic function, was negatively regulated by EHD2. Low EHD2 expression was associated with good prognosis in TNBC in two independent cohorts. Finally, TNBC low EHD2 expressers showed a positive response to chemotherapy compared with all subtypes of breast cancers. Altogether, our data establish EHD2 as a tumor suppressor which is an independent prognostic factor of metastasis-free survival and a new predictive biomarker of chemotherapy efficacy in TNBC.Breast cancer is the most commonly occurring cancer and the number one cause of cancer mortality in women in the worldWe examined EHD2 mRNA expression in a retrospective cohort of 526 breast cancer patients that was collected over the last 30 years  of 423 invasive breast tumors  of cell lines derived from triple negative basal-like breast cancers had low EHD2 mrna expression compared to non-cancer epithelial cell lines Fig. . We nextTo better understand the significance of variations of EHD2 expression in breast cancers, we selected the Hs578T, MDA-MB-231 and MDA-MB-436 TNBC cell lines that respectively express three representative levels of EHD2 transcripts i.e. high, medium, and low Fig. .in vitro cell migration/wounding assay to investigate the role of EHD2 in the aggressiveness of Hs578T, MDA-MB-231 and MDA-MB-436 TNBC cells that are respectively defined as moderately (Hs578T) and highly invasive (MDA-MB-231 and MDA-MB-436). As expected from the transcript levels, immunoblot analyses confirmed that Hs578T, MDA-MB-231 and MDA-MB-436 TNBC cell lines express high, medium and low levels of EHD2 protein, respectively  ; Mesenchymal (M); Mesenchymal stem–like (MSL); Immunomodulatory (IM), and Luminal androgen receptor (LAR). It has been shown that TNBC subtypes can respond differentially to treatments29. Accordingly, we found that low EHD2 was a new independent prognostic factor in the subgroup of TNBC patients treated with chemotherapy compared to all other breast cancer subgroups. These results not only establish EHD2 as a metastatic suppressor, they further define EHD2 as a new independent prognostic factor of metastasis-free survival that can be used as a biomarker to predict chemotherapy efficacy in TNBC.The fact that low EHD2 expression in the TNBC subtype is associated with good prognosis, but also with an increase in cell proliferation, migration and invasion, may be seen as an apparent contradiction. It is nevertheless known that highly proliferative breast cancer cells respond better to chemotherapyin vitro. However, analysis of TNBC patient samples revealed that low EHD2 was predictive of chemotherapy efficacy. We propose that the increased proliferation observed in TNBC cells with low levels of EHD2 allows a better therapeutic response since chemotherapy is more efficient on highly proliferative cells. Altogether, our study not only reveals that EHD2 acts as a tumor suppressor, it is also a new independent prognostic factor of metastasis-free survival and a new biomarker of chemotherapy response in TNBC patients.In this study, we have found that low EHD2 expression in TNBC cells correlated with enhanced proliferation, migration and invasion Analyses of human samples were performed in accordance with the French Bioethics Law 2004–800, the French National Institute of Cancer (INCa) Ethics Charter and after approval by the Institut Curie review board and ethics committee (Comité de Pilotage du Groupe Sein). Women were informed of the research use of their tissues. Informed written consent was obtained from all participants after detailed explanation of the study. Data were analyzed anonymously.30; ER − PR − HER2 + (HER2), ER < 10%, PR < 10%, HER2 2+ amplified or 3+; ER − PR − HER2 − (TNBC), ER < 10%, PR < 10%, HER2 0/1+ or 2+ nonamplified according to the American Society of Clinical Oncology (ASCO) guidelines31. TMA consisted of 1 mm diameter tumor core replicates selected from whole-tumor tissue sections in the most representative tumor areas (high tumor cell density) of each tumor sample and a matched tissue core from adjacent non-tumoral breast epithelium. For EHD2 immunohistochemistry, EnVision FLEX, High pH kit  was used according to the manufacturer’s instructions32. Analyses were performed in a blinded manner with 3 independent observers. H-score (IHC intensity multiplied by the percentage of positively stained cells) in the different molecular subtypes of breast cancers were quantified.423 samples of primary breast tumors surgically removed before any radiation, hormonal or chemotherapy treatment at Institut Curie from 2005 to 2006 have been analyzed. 235 samples of luminal A and B, 83 samples of HER2 + and 105 samples of TNBC were analyzed in this study  is a double monoclonal antibody-based assay. The two monoclonal antibodies, designated TAB 257 and TAB 259, are specific for different epitopes of the external domain of the c-erbB-2 molecule35.We analyzed 526 samples of primary unilateral invasive breast tumors excised from women at the Institut Curie/René Huguenin Hospital  from 1978 to 2008 (Supplementary Table The population was divided into four groups according to HR (ER and PR) and HER2 status  or from the German Resource Centre for Biological Material . They were cultured as per conditions recommended by the suppliers. mRNAs were provided by the Transfer Department of the Curie Institute (Paris).For qPCR, quantitative values were obtained from the cycle number  at which the increase in the fluorescence signal associated with exponential growth of PCR products began to be detected by the laser detector of the ABI Prism 7900 sequence detection system , using PE biosystems analysis software according to the manufacturer’s instructions.36.The precise amount of total mRNA added to each reaction mix  and its quality (i.e. lack of extensive degradation) are both difficult to assess. Therefore, we also quantified transcripts of the TBP gene (Genbank accession NM_003194) encoding the TATA box-binding protein (a component of the DNA-binding protein complex TFIID) as an endogenous RNA control and normalized each sample on the basis of its TBP content. We selected TBP as an endogenous control because the prevalence of its transcripts is moderate, and because there are no known TBP retropseudogenes ΔCtsample, where the ΔCt value of the sample was determined by subtracting the Ct value of the specific target gene from the Ct value of the TBP gene. The NTARGET values of the samples were subsequently normalized such that the median of the NTARGET values for the 16 normal breast tissues was 1 in breast tumors, and such that the value for the “basal mRNA level”  was 1 in cell lines. Values of 0.33 or less was considered to represent underexpression, of the EHD2 gene in our tumor sample series. We have previously used the same cutoff point for tumor gene underexpression37.Results, expressed as N-fold differences in target gene expressions relative to the TBP gene (and termed “NTARGET”) were determined as NTARGET = 233.The primers for TBP and EHD2 were chosen with the assistance of the Oligo 6.0 program . We scanned the dbEST database to confirm the total gene specificity of the nucleotide sequences chosen for the primers and the absence of single nucleotide polymorphisms. The nucleotide sequences of the primers used were as follows: EHD2-U (5′-TTT GCG AAG ATT CAG CTG GAA CAT-3′) and EHD2-L (5′-GGC TTC AGC GAG TGA AAC TTG GT-3′) PCR product of 113 bp and TBP-U (5′-TGC ACA GGA GCC AAG AGT GAA-3′) and TBP-L (5′-CAC ATC ACA GCT CCC CAC CA-3′) PCR product of 132 bp. To avoid amplification of contaminating genomic DNA, one of the two primers was placed at the junction between two exons. Agarose gel electrophoresis was used to verify the specificity of PCR amplicons. The conditions of total RNA extraction, complementary DNA synthesis and PCR were as previously describedwww.atcc.org) and cultured according to recommended conditions. All cells used in this study were within 10 passages after thawing. All cell lines were Mycoplasma free.All cell lines were obtained from the American Type Culture Collection  according to manufacturer’s instructions and cultured for the next 48 hours. Experiments were performed upon validation of depletion efficiency by immunoblot analysis using specific antibodies and normalizing to the total level of clathrin heavy chain (CHC) used as control. Control siRNA (SI03650325 5′-AATTCTCCGAACGTGTCACGT) was purchased from Qiagen and served as reference point. The EHD2 siRNA sequences were used at the final concentration of 20 nM .The following antibodies were purchased from the indicated suppliers: mouse monoclonal anti-EHD2 ; mouse monoclonal anti-clathrin heavy chain ; mouse monoclonal anti-GFP ; secondary antibody conjugated to horseradish peroxidase (HRP) (Beckman Coulter and Invitrogen).Cells were transfected with EGFP or EHD2-EGFP using Lipofectamine 2000  according to manufacturer’s instructions. Experiments were performed 6–24 hours after transfection. pEGFP was purchased from Clontech. EHD2-EGFP was generously provided by Dr. A. Helenius . pmEmerald was purchased from Addgene. EHD2-mEmerald was prepared by amplifying EHD2 from EHD2-EGFP using primers: F:AAAAAAAAGCTTCGATGTTCAGCTGGCTGAAGCGG; R: AAAAAAGGATCCCGCTCGGCGGAGCCCTTGT.The product was inserted into pmEmerald using HindIII and BamHI enzymes. pmEmerald and EHD2-mEmerald plasmids were electroporated using Ingenio electroporation kit (Mirus) following manufacturer’s instructions. Electroporation of cells was performed with a pulse at 220 V and 975 μF with a Gene Pulser® II module (Bio-Rad). Experiments were performed 24-48 hours after transfection.5 cells/ml) was seeded into each well of the insert. After allowing cell attachment for 24 hours, the culture inserts were removed, and the cells were incubated with fresh culture medium. Images of the cell migration into the defined cell free gap (500 μm width) were captured after 16 hours using Nikon phase contrast 2. Cells tracks were analyzed using the manual tracking software component of the Icy program.Wound healing assays were performed using specific wound assay chambers (Ibidi). Cells were trypsinised and resuspended, 70 μl of cell suspension (5 × 10Transwell chambers were assembled using a 8 µm pore Thincert insert (Greiner Bio-One) as upper chambers and 24-well plates as lower chambers. For the invasion assay, cell culture inserts were coated with 100 µl Matrigel (1:1 diluted with PBS). Experiments were performed according to the manufacturer’s protocol. Briefly, cells were harvested, washed in PBS, and resuspended in serum free DMEM medium with 0.2% BSA. 600 µl of serum-free or complete DMEM medium with 0.2% BSA was added to the lower chamber. 200 µl of the cell suspension was added into the insert and incubated for 20 hours at 37 °C and 5% CO2. Subsequently, the medium was removed and replaced with 450 µl DMEM containing 0.2% BSA and 8 µM Calcein-AM. The plate with inserts was incubated for 45 min at 37 °C and 5% CO2. The cells were then detached using 600 µl of Trypsine-EDTA. Finally, migratory cells were quantified with a fluorescence plate reader  at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.4 cells/well in a 96-well plate and cultured for 24 hours prior to the experiment. All conditions were performed in triplicate. Cells transfected with pmEmerald or control siRNA served as control. Proliferation was measured using the Cell Proliferation ELISA BrdU kit (Roche Diagnostics GmbH), according to the manufacturer’s instructions. Briefly, cells were labeled with BrdU at a final concentration of 10 μM/well, for 12 h at 37 °C. The cells were then denatured with FixDenat solution and incubated for 120 min with 1:100 diluted mouse anti-BrdU conjugated to peroxidase. After two washes (PBS 1×), the substrate solution was added for 25 min and, after this period, the reaction was stopped with 1 M H2SO4 solution. Absorbance was measured within 5 min at 450 nm with a reference wavelength at 690 nm using an ELISA plate reader.24 hours after electroporation or 48 hours after siRNA transfection, cells were seeded at 5 × 10All analyses were performed using Prism 6.0 software (GraphPad Inc.). A two-tailed t-test was used when two conditions were compared. For more than two conditions, one-way ANOVA was used with Bonferroni’s multiple comparison test or Dunnett’s multiple comparison test . Significance of mean comparison is marked on the graphs by asterisks. Error bars denote SEM.EHD2 and the clinical parameters was assessed by nonparametric tests, chi-squared test  and the Kruskal-Wallis H test . Differences were considered significant at confidence levels greater than 95% (p < 0.05). To visualize the efficacy of a molecular marker to discriminate between two populations (patients that developed/did not develop metastases) in the absence of an arbitrary cut-off value, data were summarized in a ROC (receiver operating characteristic) curve38. The AUC (area under curve) was calculated as a single measure to discriminate efficacy. Survival distributions were estimated by the Kaplan-Meier method, and the significance of differences between survival rates were ascertained with the log-rank test: Cox’s proportional hazard regression model was used to assess prognostic significance and the results are presented as hazard ratio and 95% confidence intervals. Metastasis-free survival (MFS) was determined as the interval between initial diagnosis and detection of the first metastasis.The correlation between mRNA expression of Supplementary Information.


To explore the epidemiological characteristics, clinical characteristics, treatment strategies, and clinical results of non-dislocated hyperextension tibial plateau fracture.A total of 25 cases of non-dislocated hyperextension tibial plateau fracture patients were collected , aged 27–79 years. Preoperative tibial plateau posterior slope angle was − 10~0° (average − 5.2°). Preoperative MRI showed 5 cases of MCL injury, 3 cases of PLC complex injury, and 2 cases of PLC + PCL injury. The change of tibial plateau posterior slope angle was more than 10° in patients with ligament injury, and the patients with a tibial plateau posterior slope angle change less than 10° had no ligament injury; 6 patients with simple column fracture had a ligament injury, 2 patients with bilateral column fracture had a ligament injury, and 2 patients with three column fracture had a ligament injury.Patients were followed up for 12–24 months (average 16.4 months). The operative time was 65–180 min (average 124 min), and the blood loss was 20–200 ml (average 106 ml). The plate was placed on the anterior part of tibial plateau. Evaluation of postoperative fracture reduction was as follows: 20 cases reached anatomic reduction, 5 cases reached good reduction (between 2 and 5 mm articular surface collapse), and the excellent rate of fracture reduction was 100%. The fracture healing time was 3–6 months (average 3.3 months). The postoperative knee Rasmussen score was 18–29 (average 24.9), and the postoperative knee joint mobility was 90–130° (average 118°). Two patients suffered superficial infection.The main imaging characteristic of “non-dislocated hyperextension tibial plateau fracture” is the change of tibial plateau posterior slope angle. The injury of single anteromedial column/anterolateral column fracture is easy to combine with “diagonal” injury, and when the tPSA changes more than 10°, it is easy to be combined with ligament injury. By reducing the joint articular surface and lower limb force line, repairing the soft tissue structure, and reconstructing the knee joint stability, we can get satisfactory results.20180145.It was a retrospective study. This study was consistent with the ethical standards of the Second Affiliated Hospital of Zhejiang University Medical College and was approved by the hospital ethics committee and the trial registration number of our hospital was  More and more attention is paid to the tibial plateau fracture injury mechanism at present, it is clear that tibial plateau fracture injury mechanism can carry out targeted reduction and treatment of plateau fracture, and the existing mechanism of injury speculation is generally based on Luo CF’s three-column theoretical classification , 2. AccoHyperextension tibial plateau fracture is rare in clinical practice. In 2001, Chiba et al.  firstly We retrospectively analyzed patients with hyperextension tibial plateau fracture treated in our hospital in recent years. Due to the injury mechanism greatly differenced from dislocated knee joint injury, the fracture-dislocation type tibial plateau fracture was excluded. By collection of clinical data of patients with non-dislocated hyperextension tibial plateau fracture, we hope to clarify (1) the clinical diagnostic criteria of non-dislocated hyperextension tibial plateau fracture; (2) the bone and soft tissue damage evaluation in non-dislocated hyperextension tibial plateau fracture; and (3) clinical treatment plan and prognosis of non-dislocated hyperextension tibial plateau fracture.The patient inclusion criteria were as follows: (1) tibial plateau pTSA change  with anterior compression of the bone mass, (2) injury to surgery time less than 3 weeks, and (3) patient follow-up time greater than 1 year. The exclusion criteria were as follows: (1) the fracture-dislocation type tibial plateau fractures (Moore classification), (2) evaluation of postoperative function severely affected by psychiatric or other cognitive disorders, (3) pathological fractures, and (4) open tibial plateau fractures.This study included a total of 25 patients  in our hospital from January 2018 to December 2018, aged 27–79 years (average 51 years). Causes of injury were as follows: 18 cases of traffic accidents, 2 cases of heavy crushes, and 5 cases of falls. Schatzker classification of tibial plateau fractures were as follows: 4 cases of type II, 5 cases of type IV, 13 cases of type V, and 3 cases of type VI; three-column theory classification were as follows: 4 cases of simple lateral column, 5 cases of simple medial column, 7 cases of medial column + lateral columns, and 9 cases of three columns. The tibial plateau posterior slope angle was − 10 to 0° before surgery (average − 5.2°) ; the medial collateral ligament attachment tear was sutured with a suture anchor ; the body injury of medial collateral ligament was repair with a direct suture. For meniscus damage, 2–0 absorbable sutures were used for suture repair. If sutures could not be sutured, meniscus plasty was performed.For the posterolateral structural injury, the knee joint was still unstable after the reduction and fixation of the medial plateau fracture during the operation. Then, another 10–15 cm long lateral incision was made to show the iliotibial bundle, the biceps femoris tendon, and the fibula head. The damaged tissue was sutured during the operation, and the edge of the joint capsule was sutured with a suture anchor ; the little fibula head fracture with avulsion at the lateral collateral ligament attachment point was fixed with a suture anchor; the large fibula head fracture with a large avulsion at the attachment point of the biceps femoris tendon was fixed with a wire tension band. For meniscus damage, 2–0 absorbable sutures were used for suture repair. If sutures could not be sutured, meniscus plasty was performed.After all repairs were completed, the physical tests were performed to check the stability of the knee joint again. The operation was terminated after confirming that there was no knee instability.Antibiotics were used for 2 days after surgery to prevent infection, and other treatment such as pain killer and anticoagulation were also used. On the first day after surgery, bed joint function activities were started. After 2 weeks, partial weight-bearing functional training was allowed under the guidance of doctor.Regular follow-up was performed in 1, 2, 3, 6, 9, and 12 months after operation. The knee x-ray examination was performed every time, the knee CT examination was performed when necessary, and we checked the joint pain and joint mobility. We summarized the operation time, intraoperative blood loss, fracture healing time, fracture reduction, knee function score, knee mobility, and postoperative complications. Fracture healing time was judged according to clinical standards and imaging standards, Rasmussen scoring system  was usedt test. We used ROC curve (receiver operator characteristic curve) analysis to find the best cutoff value of pTSA degree change connected with the ligament injury. P < 0.05 was considered significant. The SPSS software  was used to make statistical analyze.The knee Rasmussen score and knee range motion were compared between no ligament injury patients and ligament injury patients by the independent A total of 25 patients were collected in the study. The follow-up time was 12–24 months (average 16.4 months). The operative time was 65–180 min (average 124 min), and the blood loss was 20–200 ml (average 106 ml). All patients were treated with open reduction and internal fixation operation. According to the fracture characteristics, targeted reduction of the articular surface and restoration of the lower limb force line were performed. The fixed plate was placed anteriorly to fix the fracture fragment. The patients with simple medial plateau fracture were treated with anterior medial single plate Fig. . The posP = 0.06), and the knee joint mobility in no repaired patients was 113.3° compared with repaired patients which was 118.8° (P = 0.54). The Rasmussen score in no ligament injury patients was 25.5 compared with ligament injury patients which was 24.0 (P = 0.51), and the knee joint mobility in no ligament injury patients was 119.3° compared with ligament injury patients which was 115.5°(P = 0.46). We used ROC curve (receiver operator characteristic curve) analysis to find the best cutoff value of pTSA degree change. In our results, 10° of pTSA change made the significant meaning. The area under curve was nearly 1 and P < 0.001. Vascular recanalization was good in the patient with popliteal artery injury, and no vascular embolism and limb ischemic necrosis occurred. Patients with compartment syndrome had no obvious muscle necrosis, and the function of the affected limbs recovered well , and excellent fracture reduction rate of 100%. Internal fixation was as follows: 4 cases with simple lateral plate, 2 cases with simple medial plate, 15 cases with medial + lateral plates, 1 case with medial plate + lateral suture anchor, 1 case with medial plate + posterior screw, 1 case with lateral plate + fibular screw, and 1 case with medial and lateral plates + suture anchor. All fracture achieved bone healing at the last follow-up, and the fracture healing time was 3–6 months (average 3.3 months). Postoperative Rasmussen score was 18–29 (mean 24.9), and postoperative knee joint mobility was 90–130° (mean 118°). There were 3 patients who had PLC injury, and 1 patient had PCL injury repaired, because the joint could not achieve stability after bone reconstruction. Other 5 patients had MCL injury, and 1 patient had PCL injury who did not receive ligament repair. The Rasmussen score in no repaired patients was 24.2 compared with repaired patients which was 23.8 . These data are very similar to the 27% incidence reported in previous literature . We routThe fracture collapse of this type of injury is located in the front, so the approach should be slightly forward, and the surgical approaches should be decided according to the site of plateau involvement. For a single plateau fracture, the medial/lateral approach can be used to obtain satisfactory exposure. For a fracture involving the medial and lateral plateau or the total anterior plateau, both medial and lateral incisions often require for surgical treatment and this time should pay attention to the distance between skin bridges, generally more than 10 cm which will not cause skin healing problems. The anterior middle approach of the knee joint is also an option . This meThe main imaging characteristic of “non-dislocated hyperextension tibial plateau fracture” is the change of tibial plateau posterior slope angle. The injury of single anteromedial column/anterolateral column fracture is easy to combine with “diagonal” injury, and when the tPSA changes more than 10°, it is easy to be combined with ligament injury. By reducing the joint articular surface and lower limb force line, repairing the soft tissue structure, and reconstructing the knee joint stability, we can get satisfactory results.


Turbulence has long been known to drive phytoplankton fitness and species succession: motile species dominate in calmer environments and non-motile species in turbulent conditions. Yet a mechanistic understanding of the effect of turbulence on phytoplankton migratory behavior and physiology is lacking. By combining a method to generate turbulent cues, quantification of stress accumulation and physiology, and a mathematical model of stress dynamics, we show that motile phytoplankton use their mechanical stability to sense the intensity of turbulent cues and integrate these cues in time via stress signaling to trigger switches in migratory behavior. The stress-mediated warning strategy we discovered provides a paradigm for how phytoplankton cope with turbulence, thereby potentially governing which species will be successful in a changing ocean. Heterosigma akashiwo is the integration of reactive oxygen species (ROS) signaling generated by turbulent cues. Within timescales as short as tens of seconds, the emergent downward-migrating subpopulation exhibited a twofold increase in ROS, an indicator of stress, 15% lower photosynthetic efficiency, and 35% lower growth rate over multiple generations compared to the upward-migrating subpopulation. The origin of the behavioral split as a result of a bistable oxidative stress response is corroborated by the observation that exposure of cells to exogenous stressors , in lieu of turbulence, caused comparable ROS accumulation and an equivalent split into the two subpopulations. By providing a mechanistic link between the single-cell mechanics of swimming and physiology on the one side and the emergent population-scale migratory response and impact on fitness on the other, the ROS-mediated early warning response we discovered contributes to our understanding of phytoplankton community composition in future ocean conditions.Turbulence is an important determinant of phytoplankton physiology, often leading to cell stress and damage. Turbulence affects phytoplankton migration both by transporting cells and by triggering switches in migratory behavior, whereby vertically migrating cells can actively invert their direction of migration upon exposure to turbulent cues. However, a mechanistic link between single-cell physiology and vertical migration of phytoplankton in turbulence is currently missing. Here, by combining physiological and behavioral experiments with a mathematical model of stress accumulation and dissipation, we show that the mechanism responsible for the switch in the direction of migration in the marine raphidophyte  Turbulence regulates the distribution of dissolved and particulate matter in the ocean , and, al58Heterosigma akashiwo, Chattonella subsalsa, and Prorocentrum minimum, can alter their direction of vertical migration when exposed to the periodic changes of orientation relative to gravity caused by turbulent eddies, leading to the emergence of a downward-migrating subpopulation among cells originally migrating upward  signaling. We used time-lapse imaging to track the migration of individual cells of H. akashiwo in a small (12 mm × 4 mm × 1.6 mm) rotating chamber  occurred within the first 10 overturning events  to 300 (r = 0.09 ± 0.06) reorientations . The upward bias index, r = (f↑ − f↓)/(f↑ + f↓), measures the relative proportions of upward-migrating (f↑) and downward-migrating (f↓) cells, and is quantified once flipping ceases and the population has had time to reach a stationary distribution inside the chamber (−1 ≤ Ω ≤ 1 rad · s−1) for a fixed number of 10 reorientations with no resting time (τW = 0 s) changed the proportion of downward-migrating cells (= 1 rad · s−1), the highest concentration of downward-migrating cells was observed (r = 0.07 ± 0.10), while at the slowest rotation rate tested (Ω = 0.08 rad · s−1), the upward bias index (r = 0.44 ± 0.05) was not different from the nonrotating control experiment   D. These A = (2B)−1, where B is the characteristic time for the cell to rotate back to its vertical equilibrium orientation once perturbed from it. An analysis of the stability of a cell in an eddy with rotation rate Ω predicts that if |Ω| < |A|, the cell will swim with a constant angle relative to gravity (of angle θeq = arcsin(ΩA−1) for an upward-migrating cell or θeq = π – arcsin(ΩA−1) for a downward-migrating cell), whereas if |Ω| > |A|, the cell will tumble in a periodic orbit with period TB = 2π (Ω2 – A2)−1/2 (A452 = 0.09 ± 0.21 s−1). From the distribution of the cells’ initial stability parameter, our stability model correctly predicts the fraction of downward-migrating cells that emerge for each reorientation treatment . To further support our prediction that downward migration emerges when cells become destabilized by the fluid flow, we performed experiments on a second H. akashiwo strain (CCMP3374), which has higher mean stability than CCMP452 . We observed that CCMP3374 cells shift to downward swimming at higher rotation rates compared to CCMP452 cells  but likewise where |Ω| > |A| . The cell’s mechanical stability thus imposes an amplitude filter on the local vorticity of the turbulence signal, whereby only reorientations with rotation rates Ω faster than the stability parameter A cause an upward-migrating cell to tumble  and can thus trigger the emergence of downward migration.The initial stability of a cell regulates how the cell is affected by reorientations. The mechanical stability of a cell, which is typically produced by an asymmetry in the cell shape or a nonuniform distribution of cell density , 19, all A2)−1/2  (Materia A2)−1/2 D and E. W, a measure of the signal’s intermittency (−1), which induce a population split in CCMP452 in the absence of resting time . A threshold response is characteristic of stress responses in eukaryotes  that forms a fluorescent compound in the presence of ROS, which are signaling molecules that mediate the perception of diverse environmental stress conditions . After just 1 min of rolling (Ω = 1 rad · s−1), downward-migrating cells were found to have accumulated twofold more ROS compared to upward-migrating cells, and the difference in the accumulated stress between the two subpopulations was consistently detected also after 5 min and 20 min of rolling  .To test this hypothesis, we performed experiments with cells stained using a marker (CM-Hnditions . Intrace6 = 2.5) A. A simi−1, faster rotations caused a progressively higher ROS accumulation in the cells harvested at the top  and part of the cells with low mechanical stability (A < Ω) that accumulated ROS below the stress threshold for the switch in migratory direction. For rotation rates such that Ω ≥ 1 rad · s−1, the cells with low mechanical stability fully undertake the behavioral switch and accumulate progressively higher stress compared to upward-migrating cells  to the medium. H2O2 diffuses across the cell membrane, mimicking the physiological intracellular accumulation of ROS caused by the reorientations. We determined the ROS levels induced by H2O2 for both H. akashiwo strains, and we compared those levels with the ROS levels generated by exposure to 20 min of rolling. The results show that the stress levels generated in the upward- and downward-migrating subpopulations after exposure to H2O2 (at a concentration C = 33 µM) quantitatively match the ROS accumulation observed upon rolling, where we observed a bistable stress response , a concentration range that coincides with the H2O2 concentration causing the population split (−2 · s−1 or to full-spectrum light (320 to 800 nm) at intensities greater than 650 µmol photons m−2 · s−1 . Finally, we repeated the overturning experiments for cells pretreated with the ROS scavenger potassium iodide . No emergence of a downward-migrating subpopulation was observed in this case. To summarize, this suite of experiments, in which we exposed cells to ROS scavengers and inducers, showed that the behavioral response could be blocked by adding a ROS scavenger (KI) to the medium and that the behavioral response could be triggered by applying external ROS (in the form of H2O2 and high irradiance) that activated the response downstream in the signaling cascade. Taken together, these experiments demonstrate the causal link between intracellular stress accumulation mediated by ROS and the behavioral switch in migration direction.To further support the finding that ROS affects migration behavior, we observed the migration of cells exposed to different exogenous stressors known to cause ROS accumulation. In a first set of experiments, we added hydrogen peroxide . Before rolling, cells in early exponential phase presented a higher baseline ROS production rate compared to cells in mid-exponential phase. After rolling, the accumulated stress and the value of the threshold were higher in early exponential phase . These results are the consequence of a less-efficient scavenging machinery and an increased endogenous ROS production, in line with the literature on ROS production patterns in raphidophytes . This analysis allowed us to identify the threshold h = s/s0 = 2.3 ± 0.6 for the emergence of the migratory switch, because below this value of relative stress, cells still perform upward swimming after exposure to rolling.To determine the dependence of the stress threshold above which downward-swimming emerges on cell physiology, we conducted rolling experiments with cell cultures under different conditions. Because ROS production patterns in raphidophytes vary across different growth phases –35, we idophytes –35. HoweSI Appendix, Figs. S1C and S2A), thus creating resting times between periods during which the cell is overturned . Accordingly, a cell accumulates ROS whenever it is tumbled by an eddy and dissipates stress by means of its intracellular antioxidant capacity (S (SI Appendix). During a tumbling event, a cell is rapidly reoriented relatively to gravity, and it experiences an impulsive force of typical magnitude Fg ∼ 1 pN (SI Appendix). We assumed that, when tumbling, intracellular stress is generated in the cell in the form of a nearly instantaneous release  of ROS of amplitude Δs . These experiments showed that stress decays exponentially over time with a timescale τS = 87 ± 32 s  for the same range of resting times τW  above which a cell would switch its migratory strategy, we find that the theoretical prediction for the maximum resting time in the flipping experiments  associated with the emergence of downward-migrating cells . For the subpopulation performing upward swimming even after exposure to strong turbulence or high concentrations of H2O2, we implemented a smaller dissipation timescale. This follows from the observation of a bistable stress response upon induction with H2O2 without turbulence, in which the stability parameter does not play any role (SI Appendix). Using the same fitting parameter Δs/s0 = 0.40 estimated through the accumulated stress at the population scale .In order to model stress accumulation while capturing the effect of intrinsic variability in the mechanical stability and in the ROS scavenging efficiency, we developed an analytical model of stress accumulation during navigation under fluid rotations that accounts for heterogeneity in these two phenotypic traits , similarly to the stress saturation dynamics observed in the experiments . The model reveals a criterion for the emergence of downward migration: when τW/τS < 1, the stress increases hyperbolically as a function of the ratio τW/τS , because the ROS scavenging machinery of a cell is too slow in dissipating stress relative to the rate at which stress accumulates owing to the short interval between reorientations by turbulence, and the accumulated stress induces the switch in migratory behavior.Our model further predicts the temporal dynamics for the saturation of the stress response in which 98% of the total stress is accumulated within 5 min of rolling (at a rotation rate Ω = 1 rad · seriments A. The ti−1) had 15% lower photosystem (PS) II photosynthetic quantum yields (Fv/Fm) compared to upward-migrating cells . This reduction may stem directly from endogenous ROS, which can reduce photosynthetic quantum yields , with the downward-migrating subpopulation exhibiting a 35% lower growth rate over 4 d (g↓ = 0.47 ± 0.03 d−1) than the upward-migrating subpopulation (g↑ = 0.74 ± 0.02 d−1) (g = 0.69 ± 0.06 d−1) . We also estimated the instantaneous growth rate for the two subpopulations between 72 h and 96 h using log(n96/n72), where n72 and n96 are the cell concentrations at 72 h and 96 h after exposure to rolling. Within the time period 72 h to 96 h after exposure to turbulent cues, we observed a comparable growth rate in the two subpopulations (g↑ = 0.83 ± 0.39 d−1 for the upward-migrating subpopulation and g↓ = 1.01 ± 0.21 d−1 for the downward-migrating subpopulation). The growth reduction over multiple generations (approximately three) indicates that the reorientation-induced ROS accumulation has systemic consequences for H. akashiwo and suggests the potential presence of a transgenerational stress memory, akin to epigenetic effects observed in plants  B, with tn plants . This rend yeast , where mnd yeast .−9–10−6 W · kg−1, SI Appendix, Fig. S12A). By responding to the ROS-mediated early warning upon first encountering a region of turbulence, cells with weaker mechanical stability will avoid swimming into the “eye of the storm” where they could get trapped (S) and/or a decreased baseline stress rate. The increased scavenging machinery in part of the population could originate as natural variability , in the absence of turbulent cues, shows that ROS accumulation is the cause of the migratory response but at the same time begs the question of how specificity of ROS signaling , UV-A radiation (300 µmol photons m−2 · s−1), and full-spectrum light (650 µmol photons m−2 · s−1) negatively impacted motility . Exposure to excessively high levels of irradiance can cause photoinhibition /exp(g↑t). A reduction in the growth rate of the emergent downward-migrating subpopulation indicates that even brief exposure to strong turbulence, in the order of hundreds of seconds, can induce lasting physiological changes similar to those induced by longer exposure to other environmental factors such as intense light , the physiological ramifications and acclimation of plankton to environmental signals in the ocean can be long term . Our finse light , 36, 37,se light , or nutrse light , 45. Finse light , 45. We se light . One exaH. akashiwo (−2 · s−1) in f/2 (minus silica) medium, at 21 °C (CCMP452) or 18 °C (CCMP3374). For propagation of the cell cultures, 2 mL of the parent culture was inoculated into 25 mL of fresh medium every 2 wk. Experiments with CCMP452 were carried out with monoclonal cell cultures that were grown from a single parent cell isolated using an inoculation loop (diameter of ∼100 μm) developed in-house. The loop was dipped into a culture to trap a thin liquid layer, and microscopy was used to select the cases with only a single CCMP452 in the layer. Each individual cell was transferred to a separate well in a 36-well plate containing fresh growth medium. Experiments were conducted between 96 h and 120 h after inoculation, which corresponds to the early exponential growth phase of CCMP452  Rolling). Prior to harvesting the subpopulations, the cell culture was allowed to attain the postturbulence stationary vertical distribution driven by their migration behavior . The harvested cells were introduced into the supernatant of the initial cell culture (from which the 2-mL suspension had been taken) in a 1:12 ratio and allowed to grow over 96 h  from a 2-mL cell culture vial that had been exposed to turbulent cues in the form of rolling for 5 min . CM-H2DCFDA is a chloromethyl derivative of H2DCFDA that enables the detection of low concentrations of ROS. The marker is a suitable indicator for long-term quantification of ROS as it passively diffuses into live cells and forms a highly stable fluorescent adduct when oxidized. After the 30-min incubation period, fluorescence intensities of single cells were quantified using a flow cytometer , in the FITC-A channel (Ex/Em: ∼488/520 nm). Single-cell oxidative stress levels are represented as (relative) fluorescence units. To obtain the stress levels, we subtracted the FITC-A values for the control in the absence of CM-H2DCFDA staining from the FITC-A values for the stained cells. This additional step ensured the subtraction from the stress measurement of the autofluorescence of H. akashiwo over the green portion of the spectrum, characteristic of raphidophytes /Fm, was measured after a 15-min dark incubation. Photosynthetically active radiation  was provided by coalescing red, green and blue (RGB) light-emitting diodes (LEDs), which were calibrated before each experiment using a PAR light-sensor  connected to a universal light meter . All measurements were performed using coalesced RGB LEDs (“white light”).PAM was used to quantify the photosynthetic performance of cells after exposure to turbulent cues. Microscopic multicolor-variable chlorophyll fluorescence imaging  was used to quantify the photosynthetic activity of individual cells of CCMP452. A detailed technical description of the microscope system can be found elsewhere , 53, whiA swimming at low Reynolds numbers in a fluid under solid body rotation in the vertical plane at a constant rotation rate Ω = π/τR (rad · s−1), which here exemplifies the characteristic reorientation rate 1/τK by Kolmogorov-scale turbulent eddies in the ocean, the equation of motion in the laboratory frame of reference readsIn Stokes flow regime (Reynolds numbers <1), the direction in which a gravitactic cell swims is at any instant determined by the balance of viscous and gravitational torques on the cell. For gravitactic cells characterized by a stability parameter SI Appendix, Figs. S1C and S2A). We have used the relation between the vorticity and the rotation rate for a fluid in a continuous (clockwise) solid body rotation in the vertical plane performed by the flipping chamber (and the rolling device), with the strain rate set to E = 0 (eq = arcsin(ΩA−1) for upward-migrating cells (and θeq = π – arcsin(ΩA−1) for downward-migrating cells) relative to the vertical if |Ω| < |A| with 0 < θeq < π/2, or it may tumble if Ω is sufficiently large (|Ω| > |A|) and thus perform a periodic orbit with periodwhere θ measures the cell orientation to the vertical (to E = 0 . A graviB = (2A)−1 is the stability timescale  for two different stability parameters corresponding to the two strains CCMP452  and CCMP3374 . By applying the condition for tumbling |Ω| > |A| for the distribution of the stability parameter fA, measured experimentally (f↑ and downward f↓ can be extracted (SI Appendix), where we assumed, based on the saturation of the behavioral response after 10 flips only . An effect of rotation is that the gravitational acceleration g appears to be rotating in the rotating frame of reference. During a tumbling event, we assumed that intracellular stress is generated in the cell in the form of a nearly instantaneous release  of ROS whenever the cell is being reoriented relative to gravity (SI Appendix). In our model, the ROS spike specifically occurs at times ti whenever the cell swims in a direction θ = π/2, that is, in the direction perpendicular to the gravity vector. This particular choice of swimming direction is arbitrary, and we could choose any value between π/2 < θ < π without changing our results. The intracellular scavenging machinery of the cells dissipates the accumulated stress s with a characteristic timescale τS . The resulting intracellular stress accumulation–dissipation dynamics are captured by the following differential equationAbove some critical value of rotation rate Ωt − ti) records the stress spikes Δsi  occurring at times ti for a given swimming trajectory and c0 is the baseline stress rate. where the Dirac delta function δ(t − ti) is the Heaviside function and s0 = c0 τS is the baseline stress level before the fluid rotation. The population stress levels were measured with a flow cytometer for controls (no rotations) and after rolling  a continuous solid body rotation  with rotation rate Ω = π/τR , and 2) multiple, fast reorientations of amplitude π  occurring at a rate Ω >> A, alternating with resting phases captured by the timescale τW (SI Appendix).where the sequence of times scale τW B. To takSupplementary File


In Earth’s low atmosphere, hurricanes are destructive due to their great size, strong spiral winds with shears, and intense rain/precipitation. However, disturbances resembling hurricanes have not been detected in Earth’s upper atmosphere. Here, we report a long-lasting space hurricane in the polar ionosphere and magnetosphere during low solar and otherwise low geomagnetic activity. This hurricane shows strong circular horizontal plasma flow with shears, a nearly zero-flow center, and a coincident cyclone-shaped aurora caused by strong electron precipitation associated with intense upward magnetic field-aligned currents. Near the center, precipitating electrons were substantially accelerated to ~10 keV. The hurricane imparted large energy and momentum deposition into the ionosphere despite otherwise extremely quiet conditions. The observations and simulations reveal that the space hurricane is generated by steady high-latitude lobe magnetic reconnection and current continuity during a several hour period of northward interplanetary magnetic field and very low solar wind density and speed. Hurricanes in the Earth’s low atmosphere are known, but not detected in the upper atmosphere earlier. Here, the authors show a long-lasting hurricane in the polar ionosphere and magnetosphere with large energy and momentum deposition despite otherwise extremely quiet conditions. They are characterized by a low-pressure center (hurricane eye), strong winds and flow shears, and a spiral arrangement of towering clouds with heavy rains3. In space, astronomers have spotted hurricanes on Mars, and Saturn, and Jupiter5, which are similar to terrestrial hurricanes in the low atmosphere. There are also solar gases swirling in monstrous formations deep within the sun’s atmosphere, called solar tornadoes with widths of several Earth radii (RE)6. However, hurricanes have not been reported in the upper atmosphere of the planets in our heliosphere. Although vortex structures of aurora, called auroral spirals, often appear to evolve from arc-like auroras to a train of two or more spirals of diameter ~50 km in the Earth’s nightside auroral oval (about 65–75° magnetic latitude)8, they are not unusually intense and do not have similar features of a typical hurricane. In the Earth’s polar cap region (about 75–90° magnetic latitude), high-latitude dayside auroral (HiLDA) spots, but without spiral or hurricane features, have been reported to be caused by precipitating electrons predominantly during northward interplanetary magnetic field (IMF) with a strongly positive IMF By component13.Hurricanes often cause loss of life and property through high winds and flooding resulting from the coastal storm surge of the ocean and the torrential rains16 and it directly brings solar wind energy and plasma into the magnetosphere20. Under a northward IMF condition, magnetic reconnection is limited to a small high latitude region and K–H instability becomes important in bringing solar wind energy and plasma into the magnetosphere when the solar wind density and speed are high27. It is generally believed that transfer of solar wind energy and plasma into the magnetosphere and ionosphere is very weak when geomagnetic activity is extremely quiet (such as during a long period of strongly northward IMF with very low solar wind density and speed).A hurricane is clearly associated with strong energy and mass transportation, so a hurricane in Earth’s upper atmosphere must be violent and efficiently transfer solar wind/magnetosphere energy and momentum into the Earth’s ionosphere. It is well known that magnetic reconnection and Kelvin–Helmholtz (K–H) instability are the most important and fundamental processes for coupling solar wind energy into the magnetosphere-ionosphere system and similar coupling occurs in other astrophysical, space, and laboratory plasmas. For a southward IMF , magnetic reconnection occurs at the low-latitude dayside magnetopauseHere, we present an observation of a long-lasting, large and energetic space hurricane in the northern polar ionosphere that deposited solar wind/ magnetosphere energy and momentum into the ionosphere during a several hour period of northward IMF and very low solar wind density and speed.Bz > 0 for more than 8 h) occurred with a large duskward component (IMF By ~13 nT), and roughly stable interplanetary conditions with low solar wind speed and density  and number density (around 2 cm−3) indicates a very low dynamic pressure of around 0.5 nPa  and auroral electrojet AL and AU indices show non-storm and quiet auroral oval geomagnetic activity during the interval of interest  over the Northern Hemisphere. Around the north magnetic pole, a cyclone-like auroral spot (diameter over 1000 km) with multiple arms and a trend of anti-clockwise rotation is analogically named as space hurricane hereafter  with comparable solar wind number density (Nsw = ~4 cm) to the conditions described above  was generally quiet in the dawn sector while strong arcs persisted in the dusk sector. The field-aligned current (FAC) along the satellite track calculated from the magnetic field measurements of DMSP F16 indicates that the space hurricane was associated with an upward FAC.Figure 32, also shows a spot-like strong upward FAC  within a negative electric potential cell  global FAC map Fig. , estimatThe drift vectors (perpendicular to the spacecraft orbit) in Fig. 14 eV/(cm2·sr) from 16:16:58 to 16:18:51 UT, which is about 91.49% of the ΣJE (2.71 × 1014 eV/(cm2·sr)) for the whole polar pass (see Tables 14 eV/(cm2·sr)) is about 10 times higher than that for a polar pass without a space hurricane also under a geomagnetic quiet condition (see Tables 12 eV/(cm2·s·sr)) is much higher than that during substorm expansion34, but is comparable to that during super storms (sometimes exceeding 1013 eV/(cm2·s·sr) during strikingly super storms)36. Note that the large electron precipitation flux during substorms and storms is within the auroral oval, which is located at much lower latitudes than the space hurricane. Table 12 eV/(cm2·s·sr)) is about 28.8 times higher than that in the auroral oval (7.8 × 1010 eV/(cm2·s·sr)) and 71.7 times higher than that in the diffuse aurora region (3.1 × 1010 eV/(cm2·s·sr)) during the same pass 39, which uses the measured interplanetary conditions as inputs. Figure E. The Sun is on the right. The magnetopause boundary is characterized by a narrow downward FAC belt (purple) on the dayside, and by a narrow upward FAC belt (red) on the dawn flank and in the high-latitude lobe region. In the center of Fig. 41) that precipitate into the polar ionosphere and generate the hurricane structure in the aurora. Note that a pair of current sheets can be seen on the duskside of the spot, at ~18–21 MLT, which appear to correspond with the duskside auroral arcs seen in the DMSP SSUSI images. These consistencies provide strong evidence that the PPMLR-MHD model captures the key physical processes for these northward IMF conditions.The formation of space hurricane is further investigated by simulation using a high-resolution 3-D global magnetohydrodynamics (MHD) code, piecewise parabolic method42.Figure By component, magnetic reconnection occurs between IMF and the Earth’s open-magnetic field lines tailward of the cusp in the afternoon sector30 . In order to maintain current continuity in the ionosphere, the system sets up an upward FAC with a parallel potential that accelerates the existing electrons into the ionosphere and creates an arm of the auroral spot33 observed by DMSP F16 in Fig. During a northward IMF with a dominant 41 and create the auroral spot with multiple arms as observed by DMSP F16 in Fig. By component. The footprint of these field line trajectories forms a circular large ionospheric convection lobe-cell with strong embedded circular horizontal plasma flow inside the normal afternoon convection cell42. Within this lobe-cell, strong radial electric fields point toward the cell center leading to a strong upward FAC that maintains current continuity in the ionosphere41. Strong magnetic field-aligned electric fields are required to give the strong FAC, accelerating electrons up to ~10 keV that precipitate and form the auroral signature of the space hurricane41. These observations indicate that there is a significant difference between the drivers of atmospheric and space hurricanes. Hurricanes or tropical cyclones require strong driving from below (latent heat flux due to rising moist air over a warm ocean), while space hurricanes occur under an extremely quiet interplanetary condition . The extremely quiet interplanetary condition results in efficient lobe reconnection which leads to the formation of the space hurricane. The space hurricane opens a rapid energy transfer channel from space to the ionosphere and thermosphere, and would be expected to lead to important space weather effects like increased satellite drag, disturbances in High Frequency (HF) radio communications, and increased errors in over-the-horizon radar location, satellite navigation, and communication systems43. The space hurricane is likely a universal phenomenon, occurring at other magnetized bodies in the universe . The process may also be important for the interaction between interstellar winds and other solar systems throughout the universe.When the lobe reconnection is pulsed or quasi-steady for an extended period of time , the reconnected open field lines will gradually return to their previous positions and participate in a new cycle of magnetic reconnection (Supplementary Movie 37 with a Lagrangian remap to MHD39. It is designed particularly for the solar wind–magnetosphere–ionosphere system24. The model possesses a high resolution for capturing MHD shocks and discontinuities and a low numerical dissipation for examining possible instabilities inherent in the system22.The PPMLR-MHD model is a 3-D MHD model, which is based on an extension of the piecewise parabolic methodE to –100 RE along the Sun-Earth line and from −50 RE to 50 RE in Y and Z directions, with 320 × 320 × 320 grid points and a minimum grid spacing of 0.15 RE. In order to avoid the complexities associated with the plasmasphere and large MHD characteristic velocity from the strong magnetic field, an inner boundary of radius 3 RE is set for the magnetosphere24. For allowing an electrostatic coupling process introduced between the ionosphere and the magnetospheric inner boundary, the model imbeds an electrostatic ionosphere shell with height-integrated conductance. An approximately dipole field has been used as the Earth’s magnetic field with a dipole moment of 8.06 × 1022 A/m in magnitude. For the current event, the model is run to solve the whole system by using the measured interplanetary conditions as inputs.A Cartesian coordinate system has been used in the model with the Earth’s center at the origin with X-axis pointing towards the Sun, Y-axis towards the dawn-dusk direction, and Z-axis towards the north. The size of the numerical box extends from 30 RSupplementary informationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4