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Astrocytic expression of the RNA regulator HuR accentuates spinal cord injury in the acute phase
Neuroscience Letters 651 (2017) 140–145
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Astrocytic expression of the RNA regulator HuR accentuates spinal cord injury in the acute phase Thaddaeus Kwan a , Candace L. Floyd b , Jason Patel a , Amanda Mohaimany-Aponte b , Peter H. King a,c,d,∗ a
Department of Neurology, University of Alabama, Room 545 Civitan Building, 7th Ave South, Birmingham, AL 35233-0017, United States Department of Physical Medicine and Rehabilitation, University of Alabama, Rm SRC 529c, 1717 6th Ave South, Birmingham, AL 35249, United States c Department of Genetics, University of Alabama, Birmingham, AL, United States d Birmingham Veterans Affairs Medical Center, Birmingham, AL, United States b
h i g h l i g h t s • Transgenic HuR in astrocytes (Tg-HuR) translocates to the cytoplasm in spinal cord injury (SCI). • Tg-HuR mice have decreased neuronal survival in acute SCI. • Tg-HuR mice have increased astrocyte activation and vascular permeability in acute SCI.
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Article history: Received 24 February 2017 Received in revised form 30 April 2017 Accepted 2 May 2017 Available online 6 May 2017 Keywords: HuR Spinal cord injury (SCI) Post-transcriptional regulation Blood spinal cord barrier Astroglial activation
a b s t r a c t We recently showed that the RNA regulator, HuR, is translocated to the cytoplasm in astrocytes in the acute phase of spinal cord injury (SCI), consistent with its activation. HuR positively modulates expression of many pro-inflammatory factors, including IL-1, TNF-␣, and MMP-12, which are present at high levels in the early phase of SCI and exacerbate tissue damage. Knockdown of HuR in astrocytes blunts expression of these factors in an in vitro stretch injury model of CNS trauma. In this report, we further investigate the impact of HuR in early SCI using a mouse model in which human HuR is transgenically expressed in astrocytes. At 24 h following a mid-thoracic contusion injury, transgenic HuR translocated to the cytoplasm of astrocytes, similar to endogenous HuR, and consistent with its activation. Compared to littermate controls, the transgenic mice showed a global increase in astrocyte activation at the level of injury and a concomitant increase in vascular permeability. There was a significant decrease in neuronal survival at this time interval, but no differences in white matter sparing. Long term behavioral assessments showed no difference in motor recovery. In summary, transgenic expression of HuR in astrocytes accentuated neuronal injury and other secondary features of SCI including increased vascular permeability and astrocyte activation. These findings underscore HuR as a potential therapeutic target in early SCI. Published by Elsevier Ireland Ltd.
1. Introduction Traumatic spinal cord injury (SCI) is a severe clinical condition with an annual incidence of 20–40 cases per million in developed
Abbreviations: BMS, Basso Mouse Scale; GFAP, glial fibrillary acidic protein; RBP, RNA binding protein; SCI, spinal cord injury; Tg, transgenic; WT, wild type littermate controls. ∗ Corresponding author at: Department of Neurology, University of Alabama at Birmingham Medical Center, 1720-7th Ave South, Birmingham, AL, 35233-0017, United States. E-mail addresses: [email protected] (T. Kwan), candacefl[email protected] (C.L. Floyd), [email protected] (J. Patel), [email protected] (A. Mohaimany-Aponte), [email protected] (P.H. King). http://dx.doi.org/10.1016/j.neulet.2017.05.003 0304-3940/Published by Elsevier Ireland Ltd.
countries [33]. The immediate sequelae of SCI, including ischemia with infarction, edema and hemorrhage, and loss of autoregulation, lead to biochemical and pathological changes in the cord that contribute to secondary injury and clinical worsening [13,33]. A rapid inflammatory response is elicited by glial cells (astrocytes and microglia) in the vicinity of injury, characterized by the release of inflammatory mediators such as tumor necrosis factor (TNF)-␣, interleukin (IL)-1, IL-6, matrix metalloproteases (e.g. 2,3, 9 and 12), nitric oxide and other free radicals [8,10]. These mediators recruit and activate additional glia and other immune cells, leading to an accumulation of toxic substances and an accentuation of vascular permeability, ischemia and edema [3,10,14,16,26,27]. HuR is an RNA regulator that binds to adenine- and uridinerich elements (ARE) in non-coding regions of the mRNAs of many
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pro-inflammatory mediators that are upregulated in early SCI. While predominantly located in the nucleus, HuR translocates to the cytoplasm when activated, and transports the bound mRNA to polysomes to promote translation and RNA stabilization [1,4]. We recently showed that HuR translocates to the cytoplasm in astrocytes during the early phase of SCI. Using an in vitro injury model, we showed that HuR plays a positive regulatory role in astrocytic expression of inflammatory mediators detected in early SCI, including IL1-, TNF-␣ and MMP-12 [19]. Given the association of these inflammatory mediators to secondary injury following SCI, we hypothesized that overexpression of HuR in astrocytes would further exacerbate the neuropathological and clinical features of SCI. 2. Materials and methods 2.1. Spinal cord contusion injury in mice All animal procedures were reviewed and approved by the UAB Institutional Animal Care and Use Committee in compliance with the National Research Council Guide for the Care and Use of Laboratory Animals. The generation of HuR transgenic (HuR-Tg) mice is described elsewhere [35]. Eight week old, female HuR-Tg or littermate controls (WT) were administered a bilateral contusion injury at thoracic level T10 as previously described [19]. Sham control animals received laminectomies only. Sterile Ringers solution (1CC) was applied twice daily for a week as post-operative fluid replacement. Pain management consisted of subcutaneous injection of buprenorphine (0.05 mg/kg) prior to surgery and twice daily for 3 days following surgery. Bladders were manually expressed after surgery and twice daily until euthanasia. 2.2. Immunohistochemistry and western blot Spinal cord tissue was processed and sectioned into 30 m serial sections spaced 450 m apart as previously described [19]. Sections were blocked with 10% goat serum in PBS before incubation with anti-Flag (F4042) antibody (1:100, Sigma, St. Louis, MO) and antiGFAP (Z0334) antibody (1:500, Dako, Carpinteria, CA) overnight at 4◦ C. Sections were washed with PBS and incubated with an Alexafluor 555-conjugated secondary antibody (A-21428) for Flag-HuR and an Alexafluor 488-conjugated secondary antibody (A32731) for GFAP at 1:1000 (Thermofisher, Waltham, MA). Slides were treated with Autofluorescence Eliminator Reagent (EMD Millipore, Billerica, MA) before mounting of coverslips using ProLong Diamond Antifade Mountant with DAPI (ThermoFisher, Waltham, MA). Photomicrographs of serial sections spanning the epicenter of injury were taken and relative fluorescence intensity of GFAP immunoreactivity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD). Background was subtracted from the measured integrated density to give relative fluorescence intensity (RFI). RFI was averaged over four random high powered fields for each of four serial sections spanning 1.35 mm across the epicenter of injury. The total RFI for the four serial sections was then calculated. 2.3. Quantification of vascular permeability Twenty-one hours after injury, 2% Evans Blue in PBS was intravenously injected into mice (8 L/gram) and allowed to circulate for 3 h. After euthanasia, mice were exsanguinated by intracardiac perfusion of PBS over two minutes and a one cm section of spinal cord centered on the epicenter of injury was carefully removed from the spinal column. After removal of the dura, the spinal cord tissue was frozen at −80◦ C for 5 min and homogenized. The homogenate was
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added to a trichloroacetic acid (TCA) solution for a final concentration of 50% TCA and spun down at 1000 g for 30 min. Evans Blue fluorescence in the supernatant was measured at 620 nm excitation/690 nm emission and compared against a standard curve. 2.4. Neuronal counting Nissl staining for neurons using Cresyl Violet was performed as described previously [11]. Stereo Investigator software (MBF Bioscience, Williston, VT) was used to determine stereological estimates of neuronal counts using the Optical Fractionator probe. The criteria for neuron counting consisted of Cresyl Violet stained cells greater than 10 m in diameter with distinct cell bodies, and clearly defined nuclei/nucleoli. 2.5. White matter sparing Serial sections were stained using eriochrome cyanine R to determine white-matter sparing in the injured spinal cord. Spinal cord serial sections were dehydrated through ascending ethanol concentrations (70%–100%), defatted in xylene, and then rehydrated in ethanol solutions in descending concentrations. Sections were stained in a 0.2% eriochrome cyanine R for 10 min and differentiated in 0.5% ammonium hydroxide for 1 min. Tracing of eriochrome cyanine R-stained tissue was performed using Stereo Investigator software (MBF Bioscience, Williston, VT) to determine the areas of the entire cord, gray matter, as well as areas of damaged white matter. Percentage white matter area was calculated against total area of the cord in four serial sections spanning 1.35 mm across the epicenter of injury (based on lowest percent white matter among the entire set of serial sections). 2.6. Basso mouse scale Functional recovery of spinal cord injured mice was assessed weekly over a four-week period. Briefly, two independent observers naïve to the genotype of the animal scored hind-limb locomotor ability of injured mice in an open-field for four minutes using the Basso Mouse Scale as described previously [5]. 2.7. Statistics Statistical significance was determined by two-tailed Student’s t-test using Graphpad Prism 7 Software (Graphpad Software, La Jolla, CA). 3. Results 3.1. SCI induces translocation of transgenic HuR to the cytoplasm in astrocytes We previously developed a transgenic mouse in which Flagtagged human HuR is expressed in astrocytes under the control of the GFAP promoter [35]. The Flag fusion protein is detected in astrocytes throughout the spinal cord of the HuR transgenic (HuR-Tg) mouse and, as with endogenous HuR, is predominantly nuclear in location. In our previous work, we showed that HuR translocates from the nucleus to the cytoplasm in astrocytes following SCI consistent with its activation [1,4,6,19]. To determine whether transgenically expressed HuR recapitulates this pattern, we subjected HuR-Tg mice to a mid-thoracic contusion SCI and subsequently assessed the intracellular location of Flag-HuR (Fig. 1). In sham-injured mice, Flag-HuR was predominantly nuclear in location. At 24 h post SCI, there was translocation of Flag-HuR to the cytoplasm, as gauged by a strongly merged signal with GFAP, in the epicenter of injury. At levels rostral or caudal to the injury,
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Fig. 1. Transgenically expressed human HuR is translocated to the cytoplasm after SCI. HuR-Tg mice underwent a mid-thoracic spinal cord injury or sham injury. At 24 h post SCI, spinal cords were assessed by immunohistochemistry to detect expression of Flag-tagged HuR. Antibodies are shown at the top. Arrows highlight some areas of merged GFAP and Flag signal. The lower set of panels is an enlarged view of the areas highlighted by asterisks. For sham mice, sections were obtained from the epicenter of the laminectomy. Photomicrographs are representative of one injured and one sham-injured mouse, but a similar pattern was observed in three additional mice per group. Scale bar, 50 m, upper panels; 10 m, lower panels.
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Fig. 2. HuR-Tg mice show increased astroglial activation and vascular permeability at the epicenter of SCI. A) 24 h post SCI, spinal cord sections at the epicenter of injury in HuR-Tg or littermate controls (WT) were probed with a GFAP antibody in parallel. Representative photomicrographs are shown above the graph. Immunoreactivity was quantitated in serial sections spanning the epicenter using ImageJ software (graph below). B) Evans Blue Dye was injected in the tail veins 24 h post SCI. Spinal cords were harvested and extravasation of dye into tissues was quantitated by a colorimetric assay. The number of mice for each cohort is shown in parentheses. *, P < 0.05. Scale bar, 50 m.
HuR was predominantly nuclear, indicating that the translocation was specific to the epicenter of injury. In summary, transgenically expressed HuR recapitulated the pattern of translocation observed with endogenous HuR in SCI. 3.2. HuR-Tg mice show increased glial activation and vascular permeability post SCI We recently reported astrocytic HuR regulates expression of factors that govern astroglial activation, including IL-1, TNF-␣, IL-6, and LIF [28]. Using GFAP as a marker of astrocyte activation, we examined tissue sections at the epicenter 24 h after SCI. Immunofluorescence was quantitated using ImageJ software. Overall there was a 2–3-fold increase in GFAP immunoreactivity in injured tissue as compared to tissue from sham-injured mice (Fig. 2A). In HuR-Tg mice, however, GFAP immunoreactivity was 1.6 fold higher compared to WT mice (P < 0.05). Since several factors regulated by HuR in astrocytes have been linked to increased blood-spinal cord barrier permeability, including MMP-12, we measured Evans Blue dye extravasation into spinal cord tissue after SCI (Fig. 2B) [19]. Compared to sham injured mice, there was an overall 2–3 fold increase in dye detection after SCI in wild type mice. In HuR-Tg mice, there was an approximate 1.8 fold increase in extravasated dye compared to the WT group (P < 0.05). In summary, there was an increase in astroglial activation and vascular permeability in HuR-Tg mice compared to WT mice in the acute phase following SCI. 3.3. HuR transgenic mice show increased loss of neurons but no difference in white matter sparing at the level of spinal cord injury At 24 h post SCI, sections from the epicenter of injury were stained for Nissl substance and remaining neurons were quantified by non-biased stereology. When comparing sham-injured HuR-Tg and sham-injured littermate control mice (WT), there was no sig-
nificant difference in neuronal counts (Fig. 3A). In injured wild-type mice, there was an 83% reduction in numbers of neurons in the epicenter whereas a 91% reduction was observed in HuR-Tg mice (P < 0.05). White matter sparing, as determined by the percent area of preserved white matter in the epicenter, was similar in sham controls (WT, 65.2%; HuR-Tg, 66.3%) and injured mice. (WT, 42.4%; HuR-Tg, 38.4%) (Fig. 3B). HuR-Tg mice showed similar recovery patterns to controls as determined by the Basso Mouse Scale for locomotion. No behavioral defects were detected in either sham WT or sham HuR-Tg mice. Injured WT and HuR-Tg mice scored similarly (mean BMS 1.4 and 1.8 respectively) after one week post injury, recovering to scores of 3.1 and 3.6 after the fourth week (Fig. 3C).
4. Discussion Based on our previous findings that HuR positively modulates astrocytic expression of key inflammatory cytokines linked to SCI, we hypothesized that transgenic expression of HuR in astrocytes would augment the clinicopathological features following acute SCI [19]. Here we showed that transgenically expressed HuR in spinal cord astrocytes recapitulates the pattern of endogenous HuR with a predominant nuclear localization at baseline and translocation to the cytoplasm in the acute phase of SCI. We showed that ectopic HuR accentuates several of the deleterious features in early SCI including global astrocyte activation, blood spinal cord barrier compromise and reduced neuronal survival. Astrocytes can play harmful or protective roles in SCI, depending on the time interval from the initial injury [22,29]. In the acute phase of SCI, astrocytes become quickly activated in the region of injury and secrete soluble factors, such as cytokines (e.g. IL-1 and TNF-␣), matrix metalloproteases (e.g. MMP-2, 9 and 12), reactive oxygen and reactive nitrogen species which, taken together, can promote cytotoxicity, blood spinal cord barrier dysfunction, edema,
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Fig. 3. HuR-Tg mice have greater neuronal loss at the epicenter of spinal cord injury but show no difference in white matter sparing. HuR-Tg or WT littermates were subjected to a mid-thoracic spinal cord contusion or sham injury. A) At 24 h post injury, sections were stained with cresyl violet and neurons were counted at the epicenter by nonbiased stereology. Representative photomicrographs are shown. B) White matter injury in the epicenter and nearby levels was assessed in luxol fast blue-stained sections by stereology. Representative photomicrographs at the epicenter are shown. C) Results of motor function testing post SCI using the Basso Mouse Scale for locomotion. The number of mice in each cohort is shown in parentheses. *, P < 0.05.
and worsening inflammation through the recruitment/activation of additional glial and immune cells [10,15,20,34]. While these soluble factors represent structurally and functionally diverse proteins, the mRNAs that encode them share regulatory elements in the 3 UTR, referred to as AREs, which regulate their stability and translational efficiency through interactions with HuR and other RNA binding proteins (RBPs) [1,2,4]. HuR belongs to the ELAV family of RNA-binding proteins and its impact in human disease was first described in cancers of the brain and colon, where it was overexpressed and found to promote the expression of cytokines and other factors associated with tumor progression [9,24,25]. HuR mainly serves as a positive regulator of RNA stability and translational efficiency whereas other RBPs such as KH-type splicing regulatory protein (KSRP) or tristetraprolin (TTP) bind to the same ARE and exert negative regulation [1,4,7,18]. The impact of this level of gene regulation in glial cells is underscored by several previous reports showing significant and dramatic changes in the expression of target mRNAs when these RBPs are genetically silenced or chemically inhibited. In an in vitro stretch-injury model of CNS trauma, where SCI-relevant cytokine mRNAs such as IL-1, TNF-␣, CXCL1, IL-6, CCL2 and LIF are all upregulated in astrocytes, there is a significant attenuation of these mRNAs and a reduction in secreted proteins with HuR knock-
down [19]. IL-1 and MMP-12 show the most dramatic changes, decreasing by 5–10 fold. Similar targets were also affected in activated microglia with HuR knockdown [23]. In contrast, when KSRP is genetically deleted in astrocytes, IL-1 and TNF-␣ expression increase by at least 15-fold after lipopolysaccharide activation, indicating that this level of gene regulation is related, in part, to the balance between positive and negative RNA regulators [21]. This balance can also be altered by forced overexpression of RBPs as previously observed in cancer models. In vitro overexpression of HuR, for example, results in increased TNF-␣, IL-8 and/or VEGF levels in glioma and colon cancer cells, whereas TTP overexpression in glioma cells attenuates VEGF, IL-8 and IL-6 levels [9,25,31,32]. Other determinants of the RBP balance include post-translational modifications which can affect the RBP binding affinity for mRNA and the RBP location in relationship to the mRNA [36]. One hallmark of HuR activation is its translocation to the cytoplasm where it facilitates association of the bound mRNA to the polysome [1,4,6]. This shift was previously found in astrocytes during the acute phase of SCI and was also observed with transgenically expressed HuR in this report (Fig. 1) [19]. Thus, HuR transgene overexpression and its cytoplasmic localization are two factors that could tip the balance toward increased expression of key inflammatory factors in early SCI.
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The milieu of an acutely injured spinal cord is complex, with cellular debris and released intracellular contents, hemorrhage, ischemia, and edema all present and contributing to the phenotypes observed in this report. Moreover, SCI represents a cascade of interconnected events that amplify overtime. Thus, recruitment and activation of additional cells such as astrocytes, microglia or peripheral immune cells may have produced neuronal toxicity or disruption of the blood spinal cord barrier in the HuR-Tg mouse independent of transgenic HuR. In this report, HuR-Tg mice did not show any differences in motor recovery over time compared to WT which is consistent with the finding that the white matter sparing was no different between the two groups (Fig. 3) [5]. However, if the injury occurred in the cervical cord, for example, a similar increased loss of neurons in the HuR-Tg mice may have a greater functional impact on recovery. The lack of difference in white matter sparing despite increased neuron loss may reflect a differential vulnerability of cell populations to the inflammatory/toxic microenvironment in the injured spinal cords of HuR-Tg mice. Alternatively, some factors that are modulated by HuR in astrocytes may be salutary. LIF, for example, is positively regulated by HuR in astrocytes and promotes oligodendrocyte survival after SCI by blocking apoptosis [17]. Clinically, there is a distinct absence of early pharmacological interventions in SCI that either block the deleterious inflammatory response or facilitate neuroprotection in the contused spinal cord. Our findings indicate that transgenic expression of HuR in astrocytes exacerbated tissue injury in acute SCI. This finding, coupled with our previous finding that HuR positively regulates a number of inflammatory mediators in astrocytes (and microglia) that are linked to secondary tissue injury, suggests that HuR could be a therapeutic target in acute SCI [19,23]. Since HuR regulates a number of cell survival and growth factor mRNAs, including XIAP, LIF, Bcl2, HIF-1␣ and EGF in other cell systems (e.g. cancer), this RBP may be necessary for survival and/or recovery of neurons and oligodendroglia over time [12,19,30]. Further studies are necessary, including cell-specific knockout or inhibition of HuR, to assess these possibilities. Funding sources This work was supported by funding from the UAB Comprehensive Neuroscience Center and a grant from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Program (Merit Review BX001148) References [1] K. Abdelmohsen, K. Yuki, H.H. Kim, M. Gorospe, Posttranscriptional gene regulation by RNA-binding proteins during oxidative stress: implications for cellular senescence, Biol. Chem. 389 (2008) 243–255. [2] T. Bakheet, B.R.G. Williams, K.S.A. Khabar, ARED 3.0: the large and diverse AU-rich transcriptome, Nucl. Acids Res. 34 (2006) D111–114. [3] G. Barbagallo, F. Certo, V. Albanese, M. Visocchi, The impact of complications following cervical spine surgery: a systematic review, J. Neurosurg. Sci. 58 (2014) 55–64. [4] C. Barreau, L. Paillard, H.B. Osborne, AU-rich elements and associated factors: are there unifying principles? Nucleic Acids Res. 33 (2006) 7138–7150. [5] D.M. Basso, L.C. Fisher, A.J. Anderson, L.B. Jakeman, D.M. McTigue, P.G. Popovich, Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains, J. Neurotrauma 23 (2006) 635–659. [6] C.M. Brennan, J.A. Steitz, HuR and mRNA stability, Cell. Mol. Life Sci. 58 (2001) 266–277. [7] P. Briata, C.Y. Chen, A. Ramos, R. Gherzi, Functional and molecular insights into KSRP function in mRNA decay, Biochim. Biophys. Acta 1829 (2013) 689–694. [8] S. David, A. Kroner, Repertoire of microglial and macrophage responses after spinal cord injury, Nat. Rev. Neurosci. 12 (2011) 388–399. [9] D.A. Dixon, N.D. Tolley, P.H. King, L.B. Nabors, T.M. McIntyre, G.A. Zimmerman, S.M. Prescott, Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells, J. Clin. Invest. 108 (2001) 1657–1665.
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Research article
Astrocytic expression of the RNA regulator HuR accentuates spinal cord injury in the acute phase Thaddaeus Kwan a , Candace L. Floyd b , Jason Patel a , Amanda Mohaimany-Aponte b , Peter H. King a,c,d,∗ a
Department of Neurology, University of Alabama, Room 545 Civitan Building, 7th Ave South, Birmingham, AL 35233-0017, United States Department of Physical Medicine and Rehabilitation, University of Alabama, Rm SRC 529c, 1717 6th Ave South, Birmingham, AL 35249, United States c Department of Genetics, University of Alabama, Birmingham, AL, United States d Birmingham Veterans Affairs Medical Center, Birmingham, AL, United States b
h i g h l i g h t s • Transgenic HuR in astrocytes (Tg-HuR) translocates to the cytoplasm in spinal cord injury (SCI). • Tg-HuR mice have decreased neuronal survival in acute SCI. • Tg-HuR mice have increased astrocyte activation and vascular permeability in acute SCI.
a r t i c l e
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Article history: Received 24 February 2017 Received in revised form 30 April 2017 Accepted 2 May 2017 Available online 6 May 2017 Keywords: HuR Spinal cord injury (SCI) Post-transcriptional regulation Blood spinal cord barrier Astroglial activation
a b s t r a c t We recently showed that the RNA regulator, HuR, is translocated to the cytoplasm in astrocytes in the acute phase of spinal cord injury (SCI), consistent with its activation. HuR positively modulates expression of many pro-inflammatory factors, including IL-1, TNF-␣, and MMP-12, which are present at high levels in the early phase of SCI and exacerbate tissue damage. Knockdown of HuR in astrocytes blunts expression of these factors in an in vitro stretch injury model of CNS trauma. In this report, we further investigate the impact of HuR in early SCI using a mouse model in which human HuR is transgenically expressed in astrocytes. At 24 h following a mid-thoracic contusion injury, transgenic HuR translocated to the cytoplasm of astrocytes, similar to endogenous HuR, and consistent with its activation. Compared to littermate controls, the transgenic mice showed a global increase in astrocyte activation at the level of injury and a concomitant increase in vascular permeability. There was a significant decrease in neuronal survival at this time interval, but no differences in white matter sparing. Long term behavioral assessments showed no difference in motor recovery. In summary, transgenic expression of HuR in astrocytes accentuated neuronal injury and other secondary features of SCI including increased vascular permeability and astrocyte activation. These findings underscore HuR as a potential therapeutic target in early SCI. Published by Elsevier Ireland Ltd.
1. Introduction Traumatic spinal cord injury (SCI) is a severe clinical condition with an annual incidence of 20–40 cases per million in developed
Abbreviations: BMS, Basso Mouse Scale; GFAP, glial fibrillary acidic protein; RBP, RNA binding protein; SCI, spinal cord injury; Tg, transgenic; WT, wild type littermate controls. ∗ Corresponding author at: Department of Neurology, University of Alabama at Birmingham Medical Center, 1720-7th Ave South, Birmingham, AL, 35233-0017, United States. E-mail addresses: [email protected] (T. Kwan), candacefl[email protected] (C.L. Floyd), [email protected] (J. Patel), [email protected] (A. Mohaimany-Aponte), [email protected] (P.H. King). http://dx.doi.org/10.1016/j.neulet.2017.05.003 0304-3940/Published by Elsevier Ireland Ltd.
countries [33]. The immediate sequelae of SCI, including ischemia with infarction, edema and hemorrhage, and loss of autoregulation, lead to biochemical and pathological changes in the cord that contribute to secondary injury and clinical worsening [13,33]. A rapid inflammatory response is elicited by glial cells (astrocytes and microglia) in the vicinity of injury, characterized by the release of inflammatory mediators such as tumor necrosis factor (TNF)-␣, interleukin (IL)-1, IL-6, matrix metalloproteases (e.g. 2,3, 9 and 12), nitric oxide and other free radicals [8,10]. These mediators recruit and activate additional glia and other immune cells, leading to an accumulation of toxic substances and an accentuation of vascular permeability, ischemia and edema [3,10,14,16,26,27]. HuR is an RNA regulator that binds to adenine- and uridinerich elements (ARE) in non-coding regions of the mRNAs of many
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pro-inflammatory mediators that are upregulated in early SCI. While predominantly located in the nucleus, HuR translocates to the cytoplasm when activated, and transports the bound mRNA to polysomes to promote translation and RNA stabilization [1,4]. We recently showed that HuR translocates to the cytoplasm in astrocytes during the early phase of SCI. Using an in vitro injury model, we showed that HuR plays a positive regulatory role in astrocytic expression of inflammatory mediators detected in early SCI, including IL1-, TNF-␣ and MMP-12 [19]. Given the association of these inflammatory mediators to secondary injury following SCI, we hypothesized that overexpression of HuR in astrocytes would further exacerbate the neuropathological and clinical features of SCI. 2. Materials and methods 2.1. Spinal cord contusion injury in mice All animal procedures were reviewed and approved by the UAB Institutional Animal Care and Use Committee in compliance with the National Research Council Guide for the Care and Use of Laboratory Animals. The generation of HuR transgenic (HuR-Tg) mice is described elsewhere [35]. Eight week old, female HuR-Tg or littermate controls (WT) were administered a bilateral contusion injury at thoracic level T10 as previously described [19]. Sham control animals received laminectomies only. Sterile Ringers solution (1CC) was applied twice daily for a week as post-operative fluid replacement. Pain management consisted of subcutaneous injection of buprenorphine (0.05 mg/kg) prior to surgery and twice daily for 3 days following surgery. Bladders were manually expressed after surgery and twice daily until euthanasia. 2.2. Immunohistochemistry and western blot Spinal cord tissue was processed and sectioned into 30 m serial sections spaced 450 m apart as previously described [19]. Sections were blocked with 10% goat serum in PBS before incubation with anti-Flag (F4042) antibody (1:100, Sigma, St. Louis, MO) and antiGFAP (Z0334) antibody (1:500, Dako, Carpinteria, CA) overnight at 4◦ C. Sections were washed with PBS and incubated with an Alexafluor 555-conjugated secondary antibody (A-21428) for Flag-HuR and an Alexafluor 488-conjugated secondary antibody (A32731) for GFAP at 1:1000 (Thermofisher, Waltham, MA). Slides were treated with Autofluorescence Eliminator Reagent (EMD Millipore, Billerica, MA) before mounting of coverslips using ProLong Diamond Antifade Mountant with DAPI (ThermoFisher, Waltham, MA). Photomicrographs of serial sections spanning the epicenter of injury were taken and relative fluorescence intensity of GFAP immunoreactivity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD). Background was subtracted from the measured integrated density to give relative fluorescence intensity (RFI). RFI was averaged over four random high powered fields for each of four serial sections spanning 1.35 mm across the epicenter of injury. The total RFI for the four serial sections was then calculated. 2.3. Quantification of vascular permeability Twenty-one hours after injury, 2% Evans Blue in PBS was intravenously injected into mice (8 L/gram) and allowed to circulate for 3 h. After euthanasia, mice were exsanguinated by intracardiac perfusion of PBS over two minutes and a one cm section of spinal cord centered on the epicenter of injury was carefully removed from the spinal column. After removal of the dura, the spinal cord tissue was frozen at −80◦ C for 5 min and homogenized. The homogenate was
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added to a trichloroacetic acid (TCA) solution for a final concentration of 50% TCA and spun down at 1000 g for 30 min. Evans Blue fluorescence in the supernatant was measured at 620 nm excitation/690 nm emission and compared against a standard curve. 2.4. Neuronal counting Nissl staining for neurons using Cresyl Violet was performed as described previously [11]. Stereo Investigator software (MBF Bioscience, Williston, VT) was used to determine stereological estimates of neuronal counts using the Optical Fractionator probe. The criteria for neuron counting consisted of Cresyl Violet stained cells greater than 10 m in diameter with distinct cell bodies, and clearly defined nuclei/nucleoli. 2.5. White matter sparing Serial sections were stained using eriochrome cyanine R to determine white-matter sparing in the injured spinal cord. Spinal cord serial sections were dehydrated through ascending ethanol concentrations (70%–100%), defatted in xylene, and then rehydrated in ethanol solutions in descending concentrations. Sections were stained in a 0.2% eriochrome cyanine R for 10 min and differentiated in 0.5% ammonium hydroxide for 1 min. Tracing of eriochrome cyanine R-stained tissue was performed using Stereo Investigator software (MBF Bioscience, Williston, VT) to determine the areas of the entire cord, gray matter, as well as areas of damaged white matter. Percentage white matter area was calculated against total area of the cord in four serial sections spanning 1.35 mm across the epicenter of injury (based on lowest percent white matter among the entire set of serial sections). 2.6. Basso mouse scale Functional recovery of spinal cord injured mice was assessed weekly over a four-week period. Briefly, two independent observers naïve to the genotype of the animal scored hind-limb locomotor ability of injured mice in an open-field for four minutes using the Basso Mouse Scale as described previously [5]. 2.7. Statistics Statistical significance was determined by two-tailed Student’s t-test using Graphpad Prism 7 Software (Graphpad Software, La Jolla, CA). 3. Results 3.1. SCI induces translocation of transgenic HuR to the cytoplasm in astrocytes We previously developed a transgenic mouse in which Flagtagged human HuR is expressed in astrocytes under the control of the GFAP promoter [35]. The Flag fusion protein is detected in astrocytes throughout the spinal cord of the HuR transgenic (HuR-Tg) mouse and, as with endogenous HuR, is predominantly nuclear in location. In our previous work, we showed that HuR translocates from the nucleus to the cytoplasm in astrocytes following SCI consistent with its activation [1,4,6,19]. To determine whether transgenically expressed HuR recapitulates this pattern, we subjected HuR-Tg mice to a mid-thoracic contusion SCI and subsequently assessed the intracellular location of Flag-HuR (Fig. 1). In sham-injured mice, Flag-HuR was predominantly nuclear in location. At 24 h post SCI, there was translocation of Flag-HuR to the cytoplasm, as gauged by a strongly merged signal with GFAP, in the epicenter of injury. At levels rostral or caudal to the injury,
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Fig. 1. Transgenically expressed human HuR is translocated to the cytoplasm after SCI. HuR-Tg mice underwent a mid-thoracic spinal cord injury or sham injury. At 24 h post SCI, spinal cords were assessed by immunohistochemistry to detect expression of Flag-tagged HuR. Antibodies are shown at the top. Arrows highlight some areas of merged GFAP and Flag signal. The lower set of panels is an enlarged view of the areas highlighted by asterisks. For sham mice, sections were obtained from the epicenter of the laminectomy. Photomicrographs are representative of one injured and one sham-injured mouse, but a similar pattern was observed in three additional mice per group. Scale bar, 50 m, upper panels; 10 m, lower panels.
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Fig. 2. HuR-Tg mice show increased astroglial activation and vascular permeability at the epicenter of SCI. A) 24 h post SCI, spinal cord sections at the epicenter of injury in HuR-Tg or littermate controls (WT) were probed with a GFAP antibody in parallel. Representative photomicrographs are shown above the graph. Immunoreactivity was quantitated in serial sections spanning the epicenter using ImageJ software (graph below). B) Evans Blue Dye was injected in the tail veins 24 h post SCI. Spinal cords were harvested and extravasation of dye into tissues was quantitated by a colorimetric assay. The number of mice for each cohort is shown in parentheses. *, P < 0.05. Scale bar, 50 m.
HuR was predominantly nuclear, indicating that the translocation was specific to the epicenter of injury. In summary, transgenically expressed HuR recapitulated the pattern of translocation observed with endogenous HuR in SCI. 3.2. HuR-Tg mice show increased glial activation and vascular permeability post SCI We recently reported astrocytic HuR regulates expression of factors that govern astroglial activation, including IL-1, TNF-␣, IL-6, and LIF [28]. Using GFAP as a marker of astrocyte activation, we examined tissue sections at the epicenter 24 h after SCI. Immunofluorescence was quantitated using ImageJ software. Overall there was a 2–3-fold increase in GFAP immunoreactivity in injured tissue as compared to tissue from sham-injured mice (Fig. 2A). In HuR-Tg mice, however, GFAP immunoreactivity was 1.6 fold higher compared to WT mice (P < 0.05). Since several factors regulated by HuR in astrocytes have been linked to increased blood-spinal cord barrier permeability, including MMP-12, we measured Evans Blue dye extravasation into spinal cord tissue after SCI (Fig. 2B) [19]. Compared to sham injured mice, there was an overall 2–3 fold increase in dye detection after SCI in wild type mice. In HuR-Tg mice, there was an approximate 1.8 fold increase in extravasated dye compared to the WT group (P < 0.05). In summary, there was an increase in astroglial activation and vascular permeability in HuR-Tg mice compared to WT mice in the acute phase following SCI. 3.3. HuR transgenic mice show increased loss of neurons but no difference in white matter sparing at the level of spinal cord injury At 24 h post SCI, sections from the epicenter of injury were stained for Nissl substance and remaining neurons were quantified by non-biased stereology. When comparing sham-injured HuR-Tg and sham-injured littermate control mice (WT), there was no sig-
nificant difference in neuronal counts (Fig. 3A). In injured wild-type mice, there was an 83% reduction in numbers of neurons in the epicenter whereas a 91% reduction was observed in HuR-Tg mice (P < 0.05). White matter sparing, as determined by the percent area of preserved white matter in the epicenter, was similar in sham controls (WT, 65.2%; HuR-Tg, 66.3%) and injured mice. (WT, 42.4%; HuR-Tg, 38.4%) (Fig. 3B). HuR-Tg mice showed similar recovery patterns to controls as determined by the Basso Mouse Scale for locomotion. No behavioral defects were detected in either sham WT or sham HuR-Tg mice. Injured WT and HuR-Tg mice scored similarly (mean BMS 1.4 and 1.8 respectively) after one week post injury, recovering to scores of 3.1 and 3.6 after the fourth week (Fig. 3C).
4. Discussion Based on our previous findings that HuR positively modulates astrocytic expression of key inflammatory cytokines linked to SCI, we hypothesized that transgenic expression of HuR in astrocytes would augment the clinicopathological features following acute SCI [19]. Here we showed that transgenically expressed HuR in spinal cord astrocytes recapitulates the pattern of endogenous HuR with a predominant nuclear localization at baseline and translocation to the cytoplasm in the acute phase of SCI. We showed that ectopic HuR accentuates several of the deleterious features in early SCI including global astrocyte activation, blood spinal cord barrier compromise and reduced neuronal survival. Astrocytes can play harmful or protective roles in SCI, depending on the time interval from the initial injury [22,29]. In the acute phase of SCI, astrocytes become quickly activated in the region of injury and secrete soluble factors, such as cytokines (e.g. IL-1 and TNF-␣), matrix metalloproteases (e.g. MMP-2, 9 and 12), reactive oxygen and reactive nitrogen species which, taken together, can promote cytotoxicity, blood spinal cord barrier dysfunction, edema,
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Fig. 3. HuR-Tg mice have greater neuronal loss at the epicenter of spinal cord injury but show no difference in white matter sparing. HuR-Tg or WT littermates were subjected to a mid-thoracic spinal cord contusion or sham injury. A) At 24 h post injury, sections were stained with cresyl violet and neurons were counted at the epicenter by nonbiased stereology. Representative photomicrographs are shown. B) White matter injury in the epicenter and nearby levels was assessed in luxol fast blue-stained sections by stereology. Representative photomicrographs at the epicenter are shown. C) Results of motor function testing post SCI using the Basso Mouse Scale for locomotion. The number of mice in each cohort is shown in parentheses. *, P < 0.05.
and worsening inflammation through the recruitment/activation of additional glial and immune cells [10,15,20,34]. While these soluble factors represent structurally and functionally diverse proteins, the mRNAs that encode them share regulatory elements in the 3 UTR, referred to as AREs, which regulate their stability and translational efficiency through interactions with HuR and other RNA binding proteins (RBPs) [1,2,4]. HuR belongs to the ELAV family of RNA-binding proteins and its impact in human disease was first described in cancers of the brain and colon, where it was overexpressed and found to promote the expression of cytokines and other factors associated with tumor progression [9,24,25]. HuR mainly serves as a positive regulator of RNA stability and translational efficiency whereas other RBPs such as KH-type splicing regulatory protein (KSRP) or tristetraprolin (TTP) bind to the same ARE and exert negative regulation [1,4,7,18]. The impact of this level of gene regulation in glial cells is underscored by several previous reports showing significant and dramatic changes in the expression of target mRNAs when these RBPs are genetically silenced or chemically inhibited. In an in vitro stretch-injury model of CNS trauma, where SCI-relevant cytokine mRNAs such as IL-1, TNF-␣, CXCL1, IL-6, CCL2 and LIF are all upregulated in astrocytes, there is a significant attenuation of these mRNAs and a reduction in secreted proteins with HuR knock-
down [19]. IL-1 and MMP-12 show the most dramatic changes, decreasing by 5–10 fold. Similar targets were also affected in activated microglia with HuR knockdown [23]. In contrast, when KSRP is genetically deleted in astrocytes, IL-1 and TNF-␣ expression increase by at least 15-fold after lipopolysaccharide activation, indicating that this level of gene regulation is related, in part, to the balance between positive and negative RNA regulators [21]. This balance can also be altered by forced overexpression of RBPs as previously observed in cancer models. In vitro overexpression of HuR, for example, results in increased TNF-␣, IL-8 and/or VEGF levels in glioma and colon cancer cells, whereas TTP overexpression in glioma cells attenuates VEGF, IL-8 and IL-6 levels [9,25,31,32]. Other determinants of the RBP balance include post-translational modifications which can affect the RBP binding affinity for mRNA and the RBP location in relationship to the mRNA [36]. One hallmark of HuR activation is its translocation to the cytoplasm where it facilitates association of the bound mRNA to the polysome [1,4,6]. This shift was previously found in astrocytes during the acute phase of SCI and was also observed with transgenically expressed HuR in this report (Fig. 1) [19]. Thus, HuR transgene overexpression and its cytoplasmic localization are two factors that could tip the balance toward increased expression of key inflammatory factors in early SCI.
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The milieu of an acutely injured spinal cord is complex, with cellular debris and released intracellular contents, hemorrhage, ischemia, and edema all present and contributing to the phenotypes observed in this report. Moreover, SCI represents a cascade of interconnected events that amplify overtime. Thus, recruitment and activation of additional cells such as astrocytes, microglia or peripheral immune cells may have produced neuronal toxicity or disruption of the blood spinal cord barrier in the HuR-Tg mouse independent of transgenic HuR. In this report, HuR-Tg mice did not show any differences in motor recovery over time compared to WT which is consistent with the finding that the white matter sparing was no different between the two groups (Fig. 3) [5]. However, if the injury occurred in the cervical cord, for example, a similar increased loss of neurons in the HuR-Tg mice may have a greater functional impact on recovery. The lack of difference in white matter sparing despite increased neuron loss may reflect a differential vulnerability of cell populations to the inflammatory/toxic microenvironment in the injured spinal cords of HuR-Tg mice. Alternatively, some factors that are modulated by HuR in astrocytes may be salutary. LIF, for example, is positively regulated by HuR in astrocytes and promotes oligodendrocyte survival after SCI by blocking apoptosis [17]. Clinically, there is a distinct absence of early pharmacological interventions in SCI that either block the deleterious inflammatory response or facilitate neuroprotection in the contused spinal cord. Our findings indicate that transgenic expression of HuR in astrocytes exacerbated tissue injury in acute SCI. This finding, coupled with our previous finding that HuR positively regulates a number of inflammatory mediators in astrocytes (and microglia) that are linked to secondary tissue injury, suggests that HuR could be a therapeutic target in acute SCI [19,23]. Since HuR regulates a number of cell survival and growth factor mRNAs, including XIAP, LIF, Bcl2, HIF-1␣ and EGF in other cell systems (e.g. cancer), this RBP may be necessary for survival and/or recovery of neurons and oligodendroglia over time [12,19,30]. Further studies are necessary, including cell-specific knockout or inhibition of HuR, to assess these possibilities. Funding sources This work was supported by funding from the UAB Comprehensive Neuroscience Center and a grant from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Program (Merit Review BX001148) References [1] K. Abdelmohsen, K. Yuki, H.H. Kim, M. Gorospe, Posttranscriptional gene regulation by RNA-binding proteins during oxidative stress: implications for cellular senescence, Biol. Chem. 389 (2008) 243–255. [2] T. Bakheet, B.R.G. Williams, K.S.A. Khabar, ARED 3.0: the large and diverse AU-rich transcriptome, Nucl. Acids Res. 34 (2006) D111–114. [3] G. Barbagallo, F. Certo, V. Albanese, M. Visocchi, The impact of complications following cervical spine surgery: a systematic review, J. Neurosurg. Sci. 58 (2014) 55–64. [4] C. Barreau, L. Paillard, H.B. Osborne, AU-rich elements and associated factors: are there unifying principles? Nucleic Acids Res. 33 (2006) 7138–7150. [5] D.M. Basso, L.C. Fisher, A.J. Anderson, L.B. Jakeman, D.M. McTigue, P.G. Popovich, Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains, J. Neurotrauma 23 (2006) 635–659. [6] C.M. Brennan, J.A. Steitz, HuR and mRNA stability, Cell. Mol. Life Sci. 58 (2001) 266–277. [7] P. Briata, C.Y. Chen, A. Ramos, R. Gherzi, Functional and molecular insights into KSRP function in mRNA decay, Biochim. Biophys. Acta 1829 (2013) 689–694. [8] S. David, A. Kroner, Repertoire of microglial and macrophage responses after spinal cord injury, Nat. Rev. Neurosci. 12 (2011) 388–399. [9] D.A. Dixon, N.D. Tolley, P.H. King, L.B. Nabors, T.M. McIntyre, G.A. Zimmerman, S.M. Prescott, Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells, J. Clin. Invest. 108 (2001) 1657–1665.
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