ORIGINAL RESEARCH published: 21 June 2021 doi: 10.3389/fneur.2021.662235 Angiogenin in the Neurogenic Subventricular Zone After Stroke Marina Gabriel-Salazar 1† , Ting Lei 1† , Alba Grayston 1 , Carme Costa 2 , Esperanza Medina-Gutiérrez 1 , Manuel Comabella 2 , Joan Montaner 1 and Anna Rosell 1* 1 Neurovascular Research Laboratory and Neurology Service, Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Servei de Neurologia-Neuroimmunologia, Centre d’Esclerosi Múltiple de Catalunya (Cemcat) and Vall d’Hebron Research Institute, Hospital Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain Ischemic stroke is a leading cause of death and disability worldwide with effective acute thrombolytic treatments. However, brain repair mechanisms related to spontaneous or rehabilitation-induced recovery are still under investigation, and little is known about Edited by: the molecules involved. The present study examines the potential role of angiogenin Johannes Boltze, (ANG), a known regulator of cell function and metabolism linked to neurological disorders, University of Warwick, United Kingdom focusing in the neurogenic subventricular zone (SVZ). Angiogenin expression was Reviewed by: examined in the mouse SVZ and in SVZ-derived neural stem cells (NSCs), which Maria Adele Rueger, were exposed to exogenous ANG treatment during neurosphere formation as well University Hospital of as in other neuron-like cells (SH-SY5Y). Additionally, male C57Bl/6 mice underwent Cologne, Germany Michael Schroeter, a distal permanent occlusion of the middle cerebral artery to study endogenous and University Hospital of exercise-induced expression of SVZ-ANG and neuroblast migration. Our results show Cologne, Germany that SVZ areas are rich in ANG, primarily expressed in DCX+ neuroblasts but not *Correspondence: Anna Rosell in nestin+NSCs. In vitro, treatment with ANG increased the number of SVZ-derived anna.rosell@vhir.org NSCs forming neurospheres but could not modify SH-SY5Y neurite differentiation. Finally, † These authors have contributed physical exercise rapidly increased the amount of endogenous ANG in the ipsilateral SVZ equally to this work and share first niche after ischemia, where DCX-migrating cells increased as part of the post-stroke authorship neurogenesis process. Our findings position for the first time ANG in the SVZ during post-stroke recovery, which could be linked to neurogenesis. Specialty section: This article was submitted to Keywords: stroke, angiogenin, neural stem/progenitor cells, neurogenesis, neurorepair, exercise Stroke, a section of the journal Frontiers in Neurology INTRODUCTION Received: 31 January 2021 Accepted: 17 May 2021 Stroke affects 15 million people worldwide annually, and it is a leading cause of long-term disability Published: 21 June 2021 in industrialized countries (1, 2). Thrombolytic and endovascular thrombectomy are the only Citation: available treatments during the acute phase of ischemic stroke to reduce mortality and minimize Gabriel-Salazar M, Lei T, Grayston A, functional and motor disabilities (3–5). However, the narrow time window limits these strategies, Costa C, Medina-Gutiérrez E, and only a small number of patients benefit from them. Although these vessel-recanalization Comabella M, Montaner J and Rosell A (2021) Angiogenin in the strategies are effective, a large percentage of stroke survivors still suffer from motor disabilities and Neurogenic Subventricular Zone After neurological deficits. With this scenario, the only proven effective treatment for disabled stroke Stroke. Front. Neurol. 12:662235. patients is rehabilitation, which aims to compensate for the affected sensory-motor function and doi: 10.3389/fneur.2021.662235 improve life quality and independence for daily activities (6, 7). In spite of the proven benefits Frontiers in Neurology | www.frontiersin.org 1 June 2021 | Volume 12 | Article 662235 Gabriel-Salazar et al. SVZ Angiogenin After Stroke of multidisciplinary rehabilitation programs, these do not Mouse Habituation and Permanent Focal guarantee complete recovery for all patients, and individuals Cerebral Ischemia Model exhibit variable responses to similar treatments (8). In this Briefly, C57BL/6 mice were purchased from Janvier Laboratories regard, the biological responses responsible for the individual (Saint Berthevin, France). Mice were housed in a temperature- functional improvements have been investigated to identify /humidity-controlled room and maintained on a 12-h light–dark brain plasticity mechanisms and targets to modulate the cycle and given water and food ad libitum. The habituation natural evolution of brain repair by rehabilitation (9–11), but protocol for the treadmill was conducted to all mice before are not fully elucidated. In this regard, a few pre-clinical ischemia to avoid neophobic behaviors during exercise therapy. studies have associated rehabilitation with restorative brain Briefly, the week before ischemia and for 3 consecutive days, all plasticity, including mechanisms of neuroangiogenesis (12–16); mice were placed in a stationary treadmill apparatus 10 min/day however, current knowledge on the molecules modulated by (from 3:00 to 6:00 p.m.). rehabilitation and potentially associated with brain plasticity The distal occlusion of the middle cerebral artery (MCAo) is incomplete. was conducted under body temperature and cortical cerebral To further explore the molecular implications of tissue flood flow (CBF) monitoring as described (25). Animals were repair, we have focused on angiogenin (ANG), a ribonuclease anesthetized with isoflurane (Abbot Laboratories, Spain) for a protein that promotes cell proliferation and migration (17, 18) maximum of 30 min via face mask (4% for induction and 1– and related to excitotoxic motoneuron death in angiogenin 2% for maintenance in Medicinal Air, 79% N2 /21% O2 ), and loss-of-function mutations associated with ALS (19). We have eyes were protected using an ophthalmic ointment (LipolacTM , previously shown ANG role in secretome-based therapies on Angelini Farmaceutica, Barcelona, Spain). A small craniotomy brain endothelial cells (20), demonstrated the ANG upregulation was performed between the eye and ear area to expose the distal in the blood of stroke patients under rehabilitation related part of the MCA after temporal muscle retraction. The MCA was to better outcomes at long term, and the angiogenin mRNA compressed using a 30-G needle and indirectly electrocauterized overexpression in the infarct tissue of ischemic mice also after by heating the compressing needle. CBF was monitored using rehabilitation (21). However, previous studies reported that a laser-doppler flowmetry (Moor Instruments, Devon, UK), and neamine treatment (a blocking agent of the ANG activity) was only animals with a reduction in CBF below 80% were included. neuroprotective after stroke in a rat model of type I diabetic rats Buprenorphine (0.05 mg/kg) was administered subcutaneously and that the failure of bone-marrow-derived cell therapy after during surgery, the skin was sutured, and mice were allowed to stroke in the same model of diabetic rats was potentially linked recovery from anesthesia under body temperature control. to an increase in periinfarct and vascular ANG in infiltrating macrophages (22, 23). Non-diabetic animals did not show ANG expression linked to vascular dysfunction (23). Its implications in Pre-clinical Treadmill Rehabilitation neurogenesis are still unknown. In the present study, we aimed For the study of SVZ neurogenesis, rehabilitation began 48 h at investigating ANG in the subventricular zone (SVZ) niche after MCAo and consisted of 12 days of treadmill exercise or after stroke by studying its tissue expression, effects on SVZ- non-exercise (No-RHB). For treadmill, mice received 30 min of derived neural stem cells (NSC), and its modulation after cerebral exercise by increasing the speed every 10 min (10, 15, and 20 ischemia and rehabilitation. cm/s) without any aversive stimulus (such as the electric shock), and a plastic barrier was placed between the shock grid and the treadmill line to prevent animals from resting on the top of the MATERIALS AND METHODS grid during the rehabilitation protocol. The No-RHB group was placed at the treadmill apparatus (0 cm/s) for 30 min the same Brain Tissue Samples days of treatment, but only free movements were allowed. All animals used in the present investigation are C57BL/6 male For the ANG molecular analysis of the SVZ, a new group mice (6–12 weeks old). To investigate SVZ neurogenesis and of mice were habituated to the treadmill as described above, ANG cellular expression, we used brain tissue slices from a and 48 h after pMCAO, mice received treadmill rehabilitation or previous protocol in which cerebral ischemia was induced also no rehabilitation for 3 consecutive days. The day after the last by the permanent electrocauterization of the distal branch of session, mice were euthanized for brain processing. the middle cerebral artery and mice were submitted to physical exercise rehabilitation (n = 6) or non-rehabilitation (n = 6) (21). New mice were also used to obtain SVZ tissues in naive Infarct Volume Assessment animals (n = 4) or after ischemia/rehabilitation (treadmill, n = During the euthanasia procedure, brains were removed by 6) or ischemia/non-rehabilitation (n = 6). Finally, frozen NSC intracardiac perfusion with cold saline and under deep anesthesia obtained from mouse SVZ cultures following published protocols as described. Brains were cut into 1-mm-thick coronal sections (24) were also used. The experimental protocols were approved and stained with 2.5% of 2,3,4-triphenyl-2H-tetrazolium chloride (Protocol Number 21.16) and supervised by the Animal Ethics (TTC; Sigma, St. Louis, MO, USA) for 10–15 min at room Committee of Vall d’Hebron Institut de Recerca according with temperature when TTC solution was replaced by cold saline, and the Spanish legislation and the Directives of the European Union. images were acquired for infarct quantification by the ImageJ free ARRIVE guidelines were followed. software as described previously correcting for brain edema (21). Frontiers in Neurology | www.frontiersin.org 2 June 2021 | Volume 12 | Article 662235 Gabriel-Salazar et al. SVZ Angiogenin After Stroke Angiogenin ELISA fluorescence and double-positive cells (DCX+/BrdU+) was To determine the angiogenin levels in the SVZ, we dissected calculated using ImageJ software by an investigator who was two middle sections of TTC-stained brains corresponding to the blinded to the treatment group. SVZ area along the wall of the lateral ventricles of the ipsilateral and contralateral hemispheres. Tissues were snap frozen in dry Neural Stem Cells ice and stored at −80◦ C. Brain homogenates were prepared NSCs were obtained from the SVZ as previously described (24), with 150 µl ice-cold lysis buffer (50 mM Tris–HCl, 150 mM Figure 1A. Frozen NSCs were thaw at 37◦ C and cultured in a NaCl, 5 mM CaCl2 , 0.05% BRIJ-35, 0.02% NaN3 , 1% Triton X- mixture of 1:1 Dulbecco’s modified Eagle’s medium (DMEM) and 100, 1% phenylmethanesulfonyl fluoride, and 0.5% aprotinin), F12 (Gibco, Thermo Fisher, Waltham, MA, USA) supplemented and protein content was collected from the supernatant after with 0.25% of P/S, 8 µg/ml of heparin (H-3149; Sigma-Aldrich, centrifugation at 15,000 g for 12 min at 4◦ C. Total protein was St. Louis, MO, USA), 0.02 µg/ml of hFGF-B (PHG0024; Thermo determined by duplicate in each sample by the bicinchoninic Fisher, Waltham, MA, USA), 0.02 µg/ml of hEGF (PHG0314; acid (BCA) assay (Thermo Fisher Scientific Inc., Waltham, Thermo Fisher, Waltham, MA, USA), 2% of B27 (12587010; MA, USA). Finally, Mouse Angiogenin SimpleStep ELISA R Thermo Fisher, San Jose, CA, USA), and 1% of L-glutamine Kit (ab208349, Abcam, Cambridge, UK) was used following (25030149; Thermo Fisher, San Jose, CA, USA). After 2 days manufacturer’s instructions (sample dilution 1/5, and coefficient in culture, 3D-proliferating structures known as neurospheres of variation of replicates <25%). Data are expressed as picograms were observed (Figure 1C). of angiogenin per microgram of total protein per sample. For phenotyping purposes, growing neurospheres were stained for nestin and doublecortin (DCX) at day 2. Briefly, Immunohistochemistry neurospheres were fixed with 4% PFA for 10 min followed Mice of the SVZ neurogenesis study received daily by three washes with 1× Dulbecco’s phosphate-buffered saline ′ intraperitoneal injections of 5-bromo-2 -deoxyuridine (BrdU, (DPBS) and blocking with DPBS–Tween with 1% BSA (Sigma- 50 mg/kg in saline, B9285, Sigma-Aldrich, St. Louis, MO, Aldrich, St. Louis, MO, USA) for 1 h. The primary antibodies USA) beginning 48 h after MCAo until euthanasia to label mouse anti-nestin (1:200, BD-556309, BD Biosciences, San proliferating cells. For euthanasia, transcardial perfusion with Jose, CA, USA) and rabbit anti-DCX (1:200, ab18723, Abcam, cold paraformaldehyde (4% PFA) was performed under deep Cambridge, UK) were incubated overnight. Cells were washed anesthesia (isoflurane). Brains were removed and fixed with three times, and the secondary antibodies Alexa Fluor 568 4% PFA for 2 h, followed by 30% sucrose for cryoprotection, goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG, embedded in optimal cutting temperature (OCT) (Tissue-Tek, respectively, were added for 1 h. Finally, cells were mounted in Fisher Scientific, Waltham, MA, USA), and frozen at −80◦ C until FluoroshieldTM with DAPI, and pictures were acquired using the use. Slices (12-µm thick) were cut in a cryostat, placed at room Leica DM IRBE (Leica Microsystems, Wetzlar, Germany). temperature for 30 min, washed three times [0.1% phosphate- buffered saline (PBS)-Tween, 0.3% PBS–Triton X-100, and 0.1% NSC Counts and Neurosphere Growth PBS–Tween] and further incubated for 1 h with 2 M HCl-PBS Cell cultures of NSCs forming neurospheres were assessed using followed by 10 min in 0.1 M borate buffer and 5 min in 0.1% the trypan blue exclusion method and a hemocytometer to PBS–Tween for the detection of nuclear BrdU of dividing cells. quantify NSCs numbers after angiogenin treatment (Figure 2A). Sections were blocked using 0.1% PBS–Tween containing 1% Single NSCs were seeded at a density of 30,000 cells/ml BSA (Sigma-Aldrich, St. Louis, MO, USA) and 5% goat serum and cultured in uncoated 12-well plates to allow neurosphere (Merck Millipore, Billerica, MA, USA) for 1 h. Then, slices were formation. Cells were treated on day 0, day 3, or both with 100 incubated with the following antibodies, namely, 1:400 rabbit or 200 ng/ml of angiogenin. We also treated cells with 100 µM anti-DCX (ab18723, Abcam, Cambridge, UK), 1:100 mouse neomycin (a selective inhibitor of angiogenin, which blocks its anti-DCX (sc-271390, Santa Cruz Biotechnology, Santa Cruz, nuclear translocation) on day 3. Three images per well were CA, USA), 1:100 rat anti-BrdU (ab6326, Abcam, Cambridge, captured at ×100 magnification on day 5 of treatment using UK) or 1:100 rabbit anti-angiogenin (NBP2-41185, Novus, the Olympus IX71 microscope. ImageJ software was used to Centennial, CO, USA), or 1:100 mouse anti-nestin (556309; BD measure the neurosphere diameter. Afterwards, the neurospheres Biosciences, San Jose, CA, USA), and washed three times with were collected and centrifuged at 1,500 rpm for 5 min; the pellet 0.1% PBS–Tween prior to secondary antibody incubation. Alexa was resuspended in 300 µl of NSCs media and pipetted to fluor 488 goat anti-rabbit IgG, Alexa fluor 488 goat anti-rat IgG, obtain single NSCs, which were quantified by the trypan blue Alexa fluor 647 goat anti-rabbit IgG, or Alexa fluor 633 goat method with a hemocytometer. To evaluate the effect of each anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) were used as independent experiment, data are expressed as a percentage of secondary antibodies at 1:500 for 1 h at room temperature and the experimental control condition. washed with 0.1% PBS–Tween. Finally, sections were mounted ′ in VectashieldTM with 4 ,6-diamidino-2-phenylindole (DAPI) SH-SY5Y Culture and Neurite Outgrowth (Vector Laboratories, Burlingame, CA, USA) and visualized The human neuroblastoma cell line SH-SY5Y was purchased using an Olympus BX61 (Olympus, Tokyo, Japan) or confocal from ATCC (ATCC R CRL-2266) since they exhibit a neuron- laser scanning (FV1000, Olympus, Japan) microscopes. like phenotype with outgrowth neurites in the presence of Two brain slices per animal were imaged for the entire Retinoic Acid (RA). Cells were maintained in complete medium dorsolateral SVZ for the analyses. The total area of DCX+ containing DMEM/F-12 (Gibco, Thermo Fisher, San Jose, Frontiers in Neurology | www.frontiersin.org 3 June 2021 | Volume 12 | Article 662235 Gabriel-Salazar et al. SVZ Angiogenin After Stroke FIGURE 1 | Angiogenin expression in the SVZ niche: (A) schematic figure of the SVZ dissection for NSCs isolation. (B) Representative image of Western blot proving the presence of angiogenin in SVZ niche of different naïve mice and in primary NSCs in culture derived from the adult SVZ (see online Supplementary Material for the full-length Western blot film); (C) Neurosphere immunocytochemistry phenotyping showing expected nestin and DCX markers. ANG, angiogenin; C–, negative control (NSC culture media); DCX, doublecortin; NSCs, neural stem cells; SVZ, subventricular zone. CA, USA), 10% fetal bovine serum (FBS), 1% non-essential USA) to visualize the chemiluminescence signal with Fujifilm amino acids (NEA), and 1% penicillin–streptomycin (P/S). We FPM-100A films. Molecular weight markers were also run for seeded 12,500 SH-SY5Y cells onto collagen type-I-coated 24- reference values. well plates with complete media for differentiation. The medium was replaced with differentiation media after 24 h for neurite Statistical Analysis outgrowth: DMEM/F-12 (Gibco, Thermo Fisher, San Jose, CA, The SPSS 20.0 package was used for all statistical analyses, USA), 1% FBS, 1% NEA, and 1% P/S supplemented with ANG and GraphPad Software was used for graph representations. (100/200 ng/ml) or 10 µm RA to induce neurite differentiation. The normality of continuous variables was assessed using the The media and treatments were changed after 2 days, and cells Shapiro–Wilk-test (N < 30). Normally distributed variables were imaged (three fields/well) on day 6 using an Olympus were analyzed using ANOVA (followed by Tukey post-hoc), IX71 (×100 magnification), Figure 3A. Finally, WimNeuron and the Mann–Whitney U-test or Kruskal–Wallis-tests were automated analysis software (Wimasis Image Analysis R ) was used for non-normally distributed variables. For the analysis of used for quantification by measuring the circuitry length and the repeated measures, the Wilcoxon test was used in non-normal total thin neurite length. Data are expressed as a percentage of the distributions. Graphs represent means ± SEM or medians control condition of each independent experiment. (interquartile range, IQR) according to the normal or non- normal distribution of the represented variable, respectively. Western Blot Extreme values were excluded prior to data analyses of cell Brain dissections from the SVZ of naive C57Bl/6 mice and cell cultures using the mean ± 2SD criteria. The results with a p < pellets from cultured NSCs were homogenized and lysed with 0.05 were considered statistically significant. freshly prepared ice-cold lysis buffer containing 50 mM Tris– HCl, 150 mM NaCl, 5 mM CaCl2 , 0.05% BRIJ-35, 0.02% NaN3 , 1% Triton X-100, 1% phenylmethanesulfonyl fluoride (PMSF; RESULTS Sigma-Aldrich, St. Louis, MO, USA), and 0.5% aprotinin (Sigma- Aldrich, St. Louis, MO, USA). Homogenates were centrifuged at Angiogenin Is Expressed in the SVZ 12,000 rpm for 10 min at 4◦ C and the protein fraction in the Neurogenic Niche and Increases NSCs supernatants assessed by the bicinchoninic acid assay (Thermo Yields in Free-Floating Neurosphere ScientificTM , Rockford, IL, USA). A total amount of 10 µg protein First, we examined for the first time the presence of ANG in was mixed with Laemmli buffer and 5% of 2-mercaptoethanol, this SVZ neurogenic site with the hypothesis that angiogenin heated for 5 min at 95◦ C, run into 12% polyacrylamide could be involved in the regulation of neural precursors, which electrophoresis gels, and transferred into polyvinylidene fluoride are known to respond to brain injury or physical exercise. As (PVDF) membranes (Thermo ScientificTM , Rockford, IL, USA). shown in Figure 1B, SVZ-naive tissues were rich in angiogenin Then, membranes were blocked for 1 h with 10% non-fat as well as SVZ-derived NSC pools used in the present study for milk (in PBS, 0.1% Tween 20, Sigma-Aldrich, St. Louis, MO, neutrospheres formation, which, in culture, showed typical nestin USA) and incubated overnight at 4◦ C on a shaker with and DCX markers (Figure 1C). the following antibodies: anti-angiogenin (1:500, NBP2-41185, In vitro experiments with primary cell cultures of SVZ-derived Novus, Centennial, CO, USA) or β-actin (1:5,000, A5316, mouse NSCs exposed to exogenous ANG were conducted as Sigma-Aldrich, St. Louis, MO, USA). The membrane was then indicated in Figure 2A. Only the treatment with the highest washed three times (PBS−0.1% Tween 20) and incubated with dose of angiogenin (200 ng/ml) after the neurospheres were corresponding secondary antibodies at 1:2,000 for 1 h at RT with formed at day 3 significantly increased NSCs yields (p < 0.001), gentle agitation. Finally, membranes were washed three times whereas cotreatment with neomycin (a well-known angiogenin (PBS−0.1%Tween 20) and briefly incubated with Pierce R ECL activity inhibitor) completely abolished this proliferation (p < Western Blotting Substrate (Thermo ScientificTM , Rockford, IL, 0.001); Figure 2B. Indeed, no toxic evidence of neomycin was Frontiers in Neurology | www.frontiersin.org 4 June 2021 | Volume 12 | Article 662235 Gabriel-Salazar et al. SVZ Angiogenin After Stroke FIGURE 2 | SVZ-derived NSCs responses to angiogenin: (A) timeline of the experimental procedure of neurosphere cultures and representative images; (B,C) box plots showing quantification of the NSC-forming neurospheres after angiogenin stimulation (100 or 200 ng/ml) at different time points, the inhibition with neomycin, and the largest neurosphere diameters obtained with angiogenin treatment; n = 4–9; **p < 0.001 and ***p < 0.001 vs. control. Box plots represent median (IQR) of the percentage vs. control values of each independent experiment. ANG/A, angiogenin; NSCs, neural stem cells; NMC, neomycin; NF, neurospheres. observed on NSCs cultures (p > 0.05; Supplementary Figure 1, No-RHB contralateral SVZs), as seen in Figures 4A,B. This result Figure 1). We also treated NSCs with the highest angiogenin dose was not influenced by potential differences in the ischemic lesion, (200 ng/ml) at the beginning of the culture prior to neurosphere since infarct volumes were similar between groups (treadmill formation (day 0), during neurosphere growing (day 3 after 18.03 ± 3.2 mm3 vs. No-RHB 16.23 ± 3.6 mm3 ; p = 0.72, see seeding), or both, and again, all treatment conditions showed a Supplementary Figures 2A,B. significant increase in NSCs yields vs. the control (day 0, p = We also evaluated the presence of DCX+ cells in the SVZ, 0.002; day 3, p = 0.003; and day 0–3, p = 0.004) with a visible since neurogenesis was expected at later time points (12 days of increase in the neurosphere diameter; Figure 2C. In this regard, treadmill exercise, Figures 4A,C). Our results show significant the maximum diameter achieved occurred when angiogenin was differences among the studied areas (p = 0.015) with larger added to formed neurospheres on day 3 (p < 0.01 vs. control) DCX+ signal in the ipsilateral SVZ of treadmill-exercised mice (Figure 2C). (p = 0.08 vs. No-RHB ipsilateral and p = 0.013 vs. No-RHB Finally, we investigated whether ANG could also trigger contralateral, as shown in Figure 4C) but not in BrdU+ nuclei differentiation of a neuron-like cell line (SH-SY5Y cells) by (p = 0.06, not shown). its neurite outgrowth (Figure 3A). However, the capacity of The immunohistochemistry study showed that axonal/neurite sprouting was only confirmed in the presence of the ANG increase detected by ELISA in the SVZ of retinoic acid, as expected (p < 0.001), but ANG did not show this treadmill-exercised ischemic mice was localized in mature neuron-like phenotype at any of the tested concentrations DCX+ neuroblasts emerging from the SVZ (Figure 4D), (Figure 3B). in the vicinity of nestin+ cells, which did not present ANG colocalizations (see magnifications in Physical Exercise Increases SVZ Figure 4D). Angiogenin After Ischemia in Areas of Active Neurogenesis DISCUSSION Three days after ischemia (Figures 4A,B), brain NAD in the SVZ was altered among the studied areas (p = 0.0014), showing the The present investigation focuses on describing angiogenin largest amount in the ipsilateral SVZ of the treadmill exercise expression in the adult SVZ and its putative effects on neurogenic group (p = 0.0032 vs. treadmill contralateral and p = 0.0034 vs. responses after stroke. Specifically, we describe that (i) for Frontiers in Neurology | www.frontiersin.org 5 June 2021 | Volume 12 | Article 662235 Gabriel-Salazar et al. SVZ Angiogenin After Stroke FIGURE 3 | SH-SY5Y neurite differentiation: (A) timeline of the experimental procedure. (B) Box plots and representative images showing axonal/neurite outgrowth in the presence of Retinoic Acid (RA) as expected, but not with angiogenin stimulation; n = 5–6. The insert in RA shows a micrograph representative of the WimNeuron analysis. Box plots represent median (IQR) of the percentage vs. control values of each independent experiment, ***p < 0.001 vs. control. the first time, angiogenin is expressed in the adult SVZ, (ii) studies in rodents under exercise conditions showed enhanced angiogenin increases the NSC yields in SVZ-derived neurosphere neurogenesis in the hippocampus (13, 33, 34) related to memory cultures, (iii) angiogenin is overexpressed after physical exercise recovery and in the SVZ of ischemic brains (35, 36). According in the SVZ of the ischemic hemisphere during neurogenesis, to these data, our post-stroke recovery treadmill moderate and (iv) SVZ angiogenin is mainly expressed in DCX+ exercise also enhanced the DCX pools in the SVZ of the neuroblasts. Our results position angiogenin in the neurogenic ipsilateral hemispheres. In this pos-stroke SVZ niche, we report SVZ during stroke recovery, suggesting potential therapeutic for the first time the presence of a unique ribonuclease and interventions in neurorepair beyond the known actions on potent trophic factor, angiogenin, in migrating neuroblasts in angiogenesis. the active SVZ closely associated with other neural stem cell Many people survive stroke but exhibit physical and motor pools. Angiogenin is a ribonuclease protein that promotes cell deficits that limit functional independence and quality of life. proliferation and migration, and it is known to be secreted Current rehabilitation programs are implemented in developed by endothelial cells (17, 20, 37). The actions of angiogenin countries to reduce stroke-related disabilities to ultimately were first described in tumor angiogenesis (38), but it also compensate for the impaired functions (6–8). Several studies acts as a neuroprotectant in neurodegenerative diseases in vitro demonstrated that exercise improved functional recovery and and in vivo (39). Furthermore, angiogenin is present during activated cerebral-repair-associated processes within a plastic mouse embryogenesis and neuroectodermal differentiation (40), brain (11, 26). These data are supported by pre-clinical and it is also localized in axonal growth cones and neurites, rehabilitation models as emerging strategies to investigate where its inhibition impacts neural pathfinding (but not in underlying mechanisms during the recovery phase of stroke embryonic cell differentiation). Among the multiple implications and elucidate the molecular and cellular pathways activated of angiogenin, the most important function described so during the rehabilitation therapies received in the clinical setting far is its regulation of angiogenic-related routes in multiple (27–30). Previous investigations in experimental models have experimental cell lines (17). Its role in neurogenic mechanisms described neurogenesis as a key mechanism regulated after is still unknown, although a close RNase (RNase A) has been stroke by showing increased neurogenesis in the SVZ and recently described to induce NPC proliferation in embryonic- the subgranular zone (SGZ) of the hippocampus or reporting derived neurogenic cultures (41). In this argument line, other that neuroblasts from the SVZ migrated to infarct boundaries authors recently suggested that angiogenin, together with other in response to the ischemic injury (31, 32). Additionally, proteins, participated in the prevention of neural differentiation Frontiers in Neurology | www.frontiersin.org 6 June 2021 | Volume 12 | Article 662235 Gabriel-Salazar et al. SVZ Angiogenin After Stroke FIGURE 4 | Post-stroke SVZ neurogenesis and angiogenin expression: (A) timeline schemes describing the pre-clinical stroke and treadmill rehabilitation protocols. (B) Bar graph representing angiogenin after 3 days of treadmill rehabilitation or no rehabilitation in the SVZ area, measured by ELISA (n = 6/group); (C) bar graph representing DCX+ quantification in the immunohistochemistry study of the SVZ (n = 5/group) and representative immunostains of the ipsilateral hemisphere identifying the neuroblasts (DCX+). (D) Representative immunostains of the SVZ after stroke and treadmill exercise showing the distribution of nestin+ and DCX+ NSCs in the SVZ (left panel); notice that only neuroblasts (DCX+) had the most angiogenin expression, which is specifically shown in the insert magnifications and in white arrows. Bar graphs represent mean ± SEM, *p < 0.05 and **p < 0.01 as indicated by the horizontal lines. ANG, angiogenin; CL, contralateral; DAPI, ′ 4 ,6-diamidino-2-phenylindole; DCX, doublecortin; IP, ipsilateral; RHB, rehabilitation; SVZ, subventricular zone. The white * indicates the position of the lateral ventricle with contiguous dorsolateral SVZ. of neuroepithelial stem cells (42) or demonstrated increased survival actions of angiogenin treatment. Angiogenin has been neurosphere formation in an embryonic carcinoma cell line recently found to be present in the secretome of EPCs (20), after the addition of angiogenin in culture (43). Our study and several authors reported that culturing NSCs from the SVZ confirms the neurogenic actions of angiogenin protein in NSC with endothelial cells or its secretome maintained the stem-like primary cell cultures from adult SVZ niches for the first time characteristics and enhanced the proliferation of these cells by increasing the number of cell yields in growing neurosphere (44, 45). However, the same studies demonstrated that NSCs in structures, which was further confirmed by adding neomycin culture with endothelial cells under ischemic conditions migrated (inhibitor of angiogenin) and suppressing the NSC responses. and differentiated to a neuroblast-like phenotype, which suggest Importantly, we observe a dose–response effect that should that this mechanism serves as a repair response for neuronal be considered in any future therapeutic study design as well replacement after injury. as the fact that the increased number of NSC yields forming SH-SY5Y cells are a subclone of a human neuroblastoma neurospheres could respond to both proliferation and cell cell line and exhibit a neuroblast-like phenotype. These cells Frontiers in Neurology | www.frontiersin.org 7 June 2021 | Volume 12 | Article 662235 Gabriel-Salazar et al. SVZ Angiogenin After Stroke express a marker of stem cell characteristics (46), namely, ETHICS STATEMENT nestin, under undifferentiated conditions and differentiate into neurons in the presence of retinoic acid (47), which allows The animal study was reviewed and approved by Ethics investigations on neuronal differentiation via the addition of Committee of Animal Experimentation of the Vall d’Hebron drugs or molecules. In our study, we could not prove the Research Institute. differentiation of SH-SY5Y cells to a mature neuronal phenotype with neurite outgrowth when treated with angiogenin at the AUTHOR CONTRIBUTIONS tested doses since neither circuit or neurite length were enhanced in the presence of angiogenin. These results are consistent MG-S, TL, AG, MC, JM, and AR conceptualized and designed with a previous report (41) showing that angiogenin was the studies. MG-S, AG, TL, CC, EM-G, and AR performed the involved in the prevention of neural precursor maturation. in vitro and in vivo experiments/analyzed the data. MG-S and AR Here, we should also acknowledge the existence of biphasic contributed to writing of the manuscript. All authors critically actions of this particular ribonuclease molecule. Initially, stress- reviewed and approved the manuscript. induced responses have been extensively described in response to angiogenin actions leading to the cleavage of non-coding FUNDING transfer RNA (tRNA) anticodons and producing tRNA halves (tiRNA) with cytoprotective actions involving cell survival MG-S was supported by a pre-doctoral fellowship from the and antiapoptotic mechanisms (48, 49); however, some recent Vall d’Hebron Research Institute, AR was supported by the studies point at existing cytotoxic actions of ANG (50) Miguel Servet program and AG holds a pre-doctoral fellowship linked to the absence of the ribonuclease inhibitor protein, both from Instituto Carlos III (CPII15/00003 and FI17/00073, RNH1 (51). respectively), and TL was supported by the China Scholarship In conclusion, the present study identifies angiogenin in Council (CSC-201706170048). This research has been funded by the neurogenic SVZ and shows its potential actions on grants PI16/00981, PI19/00186, and RD16/0019/0021 (RETICS- NSCs during neurogenesis. Additionally, in the context of INVICTUS PLUS) from the Instituto de Salud Carlos III co- stroke, ANG is overexpressed in the ipsilateral SVZ after financed by the European Regional Development Funds, and by pos-stroke exercise coexisting with the migration of SVZ- the Generalitat de Catalunya (2017SGR 1427). derived neuroblasts. Overall, our results support further investigations on the molecular mechanisms activated by ACKNOWLEDGMENTS post-stroke neurorehabilitation and the role of ANG as a therapeutic target, which should be explored in vivo The content of this manuscript has been partially published in pre-clinical study designs of overexpression/exogenous as part of the thesis of MG-S: Modulació de marcadors de therapeutic administration of ANG considering the potential neuroreparació en teràpies rehabilitadores després de l’ictus i en interaction with comorbid conditions such as diabetes, age, un model pre-clínic d’isquèmia cerebral, Universitat Autònoma or hyperglycemia. de Barcelona, 2018; ISBN: 9788449080289. 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(2012) 227:2822– Copyright © 2021 Gabriel-Salazar, Lei, Grayston, Costa, Medina-Gutiérrez, 6. doi: 10.1002/jcp.23051 Comabella, Montaner and Rosell. This is an open-access article distributed under the 50. Rashad S, Niizuma K, Tominaga T. tRNA cleavage: a new insight. Neural terms of the Creative Commons Attribution License (CC BY). The use, distribution Regen Res. (2020) 15:47–52. doi: 10.4103/1673-5374.264447 or reproduction in other forums is permitted, provided the original author(s) and 51. Thomas SP, Hoang TT, Ressler VT, Raines RT. Human angiogenin is a potent the copyright owner(s) are credited and that the original publication in this journal cytotoxin in the absence of ribonuclease inhibidor. RNA. (2018) 24:1018– is cited, in accordance with accepted academic practice. No use, distribution or 27. doi: 10.1261/rna.065516.117 reproduction is permitted which does not comply with these terms. Frontiers in Neurology | www.frontiersin.org 10 June 2021 | Volume 12 | Article 662235