ABSTRACT Spinal cord injury (SCI) is a major cause of paralysis, and involves multiple cellular and tissular responses including demyelination, inflammation, cell death and axonal

degeneration. Recent evidence suggests that perturbation on the homeostasis of the endoplasmic reticulum (ER) is observed in different SCI models; however, the functional contribution of

this pathway to this pathology is not known. Here we demonstrate that SCI triggers a fast ER stress reaction (1–3 h) involving the upregulation of key components of the unfolded protein

response (UPR), a process that propagates through the spinal cord. Ablation of X-box-binding protein 1 (XBP1) or activating transcription factor 4 (ATF4) expression, two major UPR

mice after SCI. Local expression of active XBP1 into the spinal cord using adeno-associated viruses enhanced locomotor recovery after SCI, and was associated with an increased number of

oligodendrocytes. Altogether, our results demonstrate a functional role of the UPR in SCI, offering novel therapeutic targets to treat this invalidating condition. SIMILAR CONTENT BEING

VIEWED BY OTHERS RESTORATION OF ER PROTEOSTASIS ATTENUATES REMOTE APOPTOTIC CELL DEATH AFTER SPINAL CORD INJURY BY REDUCING AUTOPHAGOSOME OVERLOAD Article Open access 20 April 2022

CYSTINE–GLUTAMATE ANTIPORTER DELETION ACCELERATES MOTOR RECOVERY AND IMPROVES HISTOLOGICAL OUTCOMES FOLLOWING SPINAL CORD INJURY IN MICE Article Open access 09 June 2021 TARGETING

ANXA7/LAMP5-MTOR AXIS ATTENUATES SPINAL CORD INJURY BY INHIBITING NEURONAL APOPTOSIS VIA ENHANCING AUTOPHAGY IN MICE Article Open access 24 August 2023 MAIN Spinal cord injury (SCI) due to

mechanical trauma, ischemia, or tumor invasion leads to invalidating locomotor impairment. SCI often affects individuals in their productive age, having an enormous social and economic

impact. The contribution of the various subcellular compartments to the process of tissue damage triggered by SCI has not been clearly elucidated. A number of pathological conditions

affecting the nervous system, including SCI, can interfere with the function of a specific subcellular organelle, the endoplasmic reticulum (ER), resulting in a cellular condition termed ER

stress.1, 2 To alleviate ER stress, cells activate an integrated signaling pathway known as the unfolded protein response (UPR), which reestablish homeostasis by decreasing the extent of

protein misfolding.2 Conversely, chronic or irreversible ER stress triggers apoptosis to eliminate nonfunctional cells.3 The UPR is initiated by several stress sensors, including

inositol-requiring enzyme-1 (IRE1)_α_, which is a kinase and endoribonuclease that upon activation, initiates the splicing of the mRNA encoding the transcriptional factor X-box-binding

protein 1 (XBP1),2, 4 converting it into a potent activator of multiple UPR-responsive genes (termed XBP1s). XBP1s controls the expression of genes involved in protein folding, secretion,

and protein quality control.5 IRE1_α_ also regulates other signaling events including the activation of JNK (c-Jun N-terminal kinases), modulating apoptosis and autophagy levels, in addition

to degrading a subset of mRNA through its RNAse activity (reviewed in Hetz2). Other important UPR effects are mediated by the stress sensor PKR-like endoplasmic reticulum kinase (PERK),

which phosphorylates the eukaryotic translation initiation factor 2_α_ (eIF2_α_), inhibiting protein translation into the ER, and reducing the overload of misfolded proteins.6 eIF2_α_

phosphorylation also triggers the specific translation of activating transcription factor 4 (ATF4), which is essential for the upregulation of different foldases and the regulation of the

redox and metabolic status of the cell.7 Under prolonged ER stress, ATF4 also controls the expression of pro-apoptotic components such as CCAAT/enhancer-binding protein (C/EBP)

(CHOP)/GADD153 and several BCL-2 family members, including BCL-2, BIM, and PUMA, among others.3 Correlative studies indicate that markers of ER stress are observed in different models of SCI

triggered by trauma (by contusion and hemisection) and ischemia.8, 9, 10, 11, 12 Using a contusion model, ER stress responses were observed in the damaged zone, including XBP1 mRNA

splicing, in addition to the induction of CHOP and the ER chaperone BiP.10 Neuronal cells presented a fast and transient activation of UPR markers, whereas oligodendrocytes and astroglia

showed sustained activation for several days.13 Interestingly, signs of ER stress were also detected at some distance from the injury site,8 suggesting a spread of the pathological stress

process. Similarly, damage to the spinal cord by transient ischemia correlates with increased ER stress levels.12, 14 A recent study indicated that deletion of _chop_ enhances motor recovery

after moderate levels of SCI induced by contusion,13 whereas CHOP deficiency did not have any effect under conditions of severe SCI.15 As CHOP expression is one of dozens downstream targets

of the UPR,5 and the ATF4/CHOP pathway is activated by a variety on non-ER stress-related stimuli including DNA damage,16 oxidative stress,17 metabolic changes,18 inflammation, among other

effects,19 direct manipulation of proximal UPR components is required to directly define the contribution of ER stress to SCI. In this study, we confirm a massive and early upregulation of

key ER stress markers in the injury zone after spinal cord hemisection, including a rapid activation of XBP1 and ATF4, in addition to other important UPR downstream target genes. To address

the possible impact of ER stress in the functional recovery after experimental SCI, we analyzed the susceptibility of ATF4- and XBP1-deficient mice to experimental SCI. Remarkably, both

mouse models presented significant impairment of locomotor recovery after SCI compared with wild-type animals. These effects were associated with drastic changes at the level of

oligodendrocyte number, microglia activation, inflammation markers, and axonal degeneration. Furthermore, treatment of wild-type mice with Adeno-associated viruses (AAV2) to deliver an

active from of XBP1 locally into the spinal cord enhanced fine locomotor movements and increased oligodendrocyte content in the injury zone. Overall, our results indicate a detrimental

impact of ER stress in SCI, identifying an unanticipated signaling target to treat this pathological condition affecting motor and sensory function. RESULTS EARLY AND SUSTAINED ACTIVATION OF

THE UPR AFTER SPINAL CORD HEMISECTION To monitor the occurrence of ER stress after SCI, we first validated the possible activation of UPR components in wild-type mice after spinal cord

hemisection. A significant and progressive upregulation of XBP1 mRNA splicing was detected by RT-PCR 6 h after SCI in the injured region, and this change was sustained for several days

(Figure 1a). We also observed a significant increase in XBP1 mRNA splicing in rostral regions far from the injury zone (Figure 1b). Similarly, we found a significant increase in ATF4 protein

levels 6 h after SCI in the injury site, in addition to distant regions of the spinal cord (Figure 1c). These changes were observed as early as 1 h after SCI (Supplementary Figure S1A). In

agreement with these findings, we detected a sustained upregulation of classical UPR-target genes after SCI, including _bip_ and _chop_ (Figure 1d). In addition, we detected the upregulation

of additional markers including BIM (Figure 1e), _a_ pro-apoptotic BCL-2 family member modulated by ER stress,20 and increased accumulation of polyubiquitinated proteins (Supplementary

Figure S1B) after SCI. In summary, we were able to confirm previous findings indicating an early and sustained activation of the UPR after SCI, reinforcing the idea that protein-folding

stress is a major cellular reaction under this condition. ATF4 AND XBP1 DEFICIENCY ATTENUATES LOCOMOTOR RECOVERY AFTER SCI Activation of the UPR has two paradoxical consequences, adaptation

to stress, improving cell survival, or the induction of apoptosis programs to eliminate the chronically damaged cell. To determine the possible functional role of proximal UPR signaling

components in locomotor recovery after SCI, we subjected ATF4 knockout mice (_atf4_−/−) and littermate controls to spinal cord hemisection. Analysis of locomotor activity using the Basso

Mouse Scale (BMS) open field test21 revealed a delayed and reduced recovery of _atf4_−/− mice when compared with hemisected wild-type mice (Figure 2a left panel). A similar result was

obtained in the BMS subscore (Figure 2a right panel), which can detect differences in the fine details of locomotion that may not be apparent in the overall BMS score.21 No differences in

locomotor capacity were observed between non-injured control and _atf4_−/− mice by BMS or by monitoring rotarod performance (Figure 2a and Supplementary Figure S2A), indicating that basal

locomotor capacity is not compromised in _atf4_−/− mice. Under ER stress conditions, ATF4 and XBP1 have distinct effects on ER homeostasis because of the transcriptional regulation of

differential subsets of target genes.22 As ATF4 is also activated by many processes that are not related to ER stress,19 we decided to further analyze the possible contribution of the UPR in

SCI with a direct genetic manipulation. We evaluated the role of XBP1, which is one of the key components of the UPR and represents the most conserved ER stress signaling branch in

evolution.2 We recently generated a viable conditional knockout mouse model to delete _xbp1_ in the nervous system using the Nestin promoter to express Cre (XBP1Nes−/−), which do not present

basal motor phenotypes.23 Nestin-Cre system is predicted to induce deletion of oligodendrocytes and astrocytes, in addition to neurons.24 Remarkably, XBP1Nes−/− animals showed a marked

reduction in locomotor recovery after SCI by both the BMS score and subscore analysis (Figure 2b). Together, the side-by-side comparison of these two independent UPR knockout mice revealed a

clear functional role for ER stress in SCI. ENHANCED AXONAL DEGENERATION AND ALTERED CELLULAR ENVIRONMENT IN ATF4-DEFICIENT MICE AFTER SCI We then evaluated the impact of ATF4 deficiency on

the cellular alterations classically associated with SCI. In agreement with the negative impact of ATF4 deficiency on locomotor recovery after SCI, _atf4_−/− mice presented fewer axons with

normal morphology after SCI compared with control mice, as measured 2 mm caudal to the injury zone in toluidine blue-stained tissue (Figure 3a). No differences were observed in axonal

density in _atf4_−/− on the contralateral, non-injured side (Figure 3a). As a control, we performed eriochrome/cyanine staining to monitor the extent of the mechanical damage in both

wild-type and _atf4_−/− mice, which was virtually identical (Figure 3b), also indicating that the surgery protocol was highly reproducible. In addition to axonal degeneration, many different

cellular processes are triggered by SCI, and involve multiple cell types. To define the impact of the UPR on the cellular environment in the injury zone of _atf4_−/− mice, we monitored

glial reactions 5 and 14 days after surgery, at the beginning of the locomotor recovery phase according to the BMS score (Figure 2). Increased accumulation of oligodendrocytes was observed

in the area surrounding the injury zone of wild-type mice, as monitored by counting the number of olig2-positive cells by immunofluorescence (Figures 3c and d and Supplementary Figure S2B).

This increase was ablated in _atf4_−/− mice (Figure 3c). No significant differences were observed in the total number of neurons in the injury area by neuronal nuclei (NeuN) staining in

either control or _atf4_−/− mice after 14 days of spinal cord hemisection (Supplementary Figure S2C). We also monitored the reaction of microglia and astrocytes after genetically targeting

ATF4. Activation of microglia was assessed using Cd11b staining. The classical microglial reaction observed after SCI was drastically reduced in _atf4_−/− mice 5 days after SCI (Figure 4a).

In sharp contrast, analysis of astrocyte distribution by glial fibrillary acidic protein (GFAP) immunostaining did not reveal differences between control and _atf4_−/− mice at 5 or 14 days

after SCI (Figure 4b and Supplementary Figure S2D). Cell density was assessed by quantifying the number of cell nuclei after Hoechst staining, which possibly represents cellular infiltration

and proliferation of macrophages. This measurement was also enhanced in _atf4_−/− mice after SCI (Supplementary Figure S2E). We also monitored the levels of several pro-inflammatory

cytokines that are induced upon SCI including IL-1_β_, TNF_α_ and IL-6. Real-time PCR analysis revealed an induction of these three cytokines in the spinal cord area surrounding the injury

zone (Figure 4c). A fivefold increase in IL-6 mRNA levels was observed in _atf4_−/− mice 6 h after hemisection when compared with _atf4_+/+ mice (Figure 4c). TNF_α_ levels were slightly

decreased in _atf4_−/−, whereas IL-1_β_ was not altered (Figure 4c). Taken together, our results indicate that ATF4 expression has a broad impact on the cellular and functional alterations

observed after SCI. XBP1S GENE TRANSFER INTO THE SCI SITE ENHANCES LOCOMOTOR RECOVERY Our studies using XBP1-deficient animals indicate that the UPR has a functional role in alleviating ER

stress in SCI. To test the possible therapeutic impact of further enhancing UPR responses after SCI, we developed a gain of function approach to artificially activate the UPR. We generated

serotype 2 AAV-based vector to locally deliver the spliced form of XBP1 into the damaged area of the spinal cord. The vector was bicistonic and expressed green fluorescent protein (GFP) to

monitor efficiency of transduction. We performed spinal cord hemisection and immediately delivered 2 _μ_l (1012 DRP/ml) of AAV2, expressing either GFP alone or the XBP1s transgene into the

injury zone. This strategy leads to a local and partial transduction of cells closely surrounding the injury site (Figure 5a). Although the area transduced by AAV was small, we observed a

slight, but significant, improvement of locomotor performance after 28 days of SCI in animals injected with AAV2-XBP1s when compared with animals injected with the control virus (Figure 5b).

Furthermore, improvement of locomotor performance was evident when the BMS subscore was analyzed, which reveals finer aspects of locomotor function, observing a significant difference in

the locomotor recovery of mice injected with XBP1s AAVs between 14 and 35 days after SCI (Figure 5c and Supplementary Figure S3a). The late effects of AAV2-mediated delivery of XBP1s

transgene are consistent with the reported delay of around 7–14 days for gene expression after AAV transduction.25, 26 Most of the cells transduced with AAV in the spinal cord were neurons

and oligodendrocytes, as predicted for the serotype 2 of the viral particles (Figure 5d and Supplementary Figure S3a). Global analysis of cellularity in the injury zone by Hoechst staining

indicated a reduction of cellular density after delivering XBP1s-expressing AAVs (Figures 5e and g). In addition, the accumulation of Olig2-positive cells was significantly increased in the

injury zone of AAV-XBP1s-treated mice when compared with mice injected with control viruses (Figures 5f and g). In summary, these results suggest that although the pathway is already

activated in SCI, forced expression of active XBP1s locally into the spinal cord can still improve locomotor performance after experimental SCI. DISCUSSION SCI is a major cause of locomotor

impairment and paralysis in the world. However, the molecular events underlying the cellular and functional alterations associated with SCI are not fully understood. Identifying primary

signaling events initiated by SCI is essential for future therapeutic interventions aiming to (i) restore the locomotor capacity of the affected individual or to (ii) attenuate the

pathological events associated with spinal cord damage. Recent studies indicate that ER stress is an early tissue reaction after SCI in different models, including mechanical trauma and

hypoxia.8, 10, 11, 12, 13, 27 Here we show a fast activation of a range of UPR responses after SCI, including the expression of the proximal components ATF4 and XBP1, major transcription

factors governing this pathway, in addition to the upregulation of downstream target genes. Remarkably, UPR activation was also observed in regions distant from the primary injury zone,

suggesting the activation of a broad ER stress response in the spinal cord after damage. Interestingly, a recent report in cancer models suggested that under ER stress conditions, a

transmissible signal related to pro-inflammatory events propagates the protein-folding stress response to the surrounding tissue,28 a phenomena that may also operate after SCI. Altered

axonal trafficking due to spinal cord hemisection may also contribute to the activation of the UPR at distal regions because of a traffic jam in vesicular transport. We also analyzed

available microarray data sets of SCI-contusion models in mice (Gene Expression Omnibus, NCBI) that confirmed a strong induction of sustained ER stress responses (Supplementary Figure S4).

Using genetic manipulation of the UPR, we demonstrated a functional role of this stress pathway in locomotor recovery after SCI. A recent study described the contribution of the

pro-apoptotic transcription factor CHOP in models of mild SCI.13 CHOP deficiency enhanced motor recovery after SCI, correlating with enhanced content of oligodendrocyte and white-matter

sparing.13 CHOP is one of a large group of downstream targets of proximal UPR transcription factors that controls the expression of many late-phase genes related to apoptosis and protein

synthesis.5 In contrast, in models of severe SCI, ablation of _chop_ did not have an effect on motor recovery.15 CHOP expression is mostly controlled by the eIF2_α_/ATF4 signaling branch,

which is activated not only by the UPR sensor PERK but also by the kinases GCN2, HRI, and PKR2 that are non-ER stress-dependent. Therefore, our genetic studies with XBP1-deficient animals,

and the development of an AAV-XBP1s gene therapy approach, represent the first direct proof for an involvement of the ER stress in SCI. We speculate that the UPR may enhance neuronal

plasticity events and functional recovery during the recovery phase after SCI related to neuronal functionality (cell autonomous events). Axonal degeneration was enhanced in _atf4_−/− mice,

correlating with impaired motor recovery after experimental SCI. Axonal degeneration might be an indicator of oligodendrocyte dysfunction, having a direct impact on locomotion recovery.

Consistent with these findings, the accumulation of oligodendrocytes near the injury zone was reduced after deleting _atf4_ and enhanced after overexpressing XBP1s. Cell-type-dependent ER

stress responses have been previously observed in SCI models.10, 12, 13 It has been described that the presence of NG2-positive cells in the injury zone is associated with the stabilization

and regeneration of dystrophic axons after SCI.29 In addition, increased oligodendrocyte number may also enhance the functional recovery of spared axons that were not directly ruptured and

altered by the pro-inflammatory/oxidative environment. Remarkably, Keirstead _et al._30 reported a functional improvement of locomotion when human patients were transplanted with human

embryonic cell-derived oligodendrocyte progenitor cells. We also observed differential microglial activation 5 days after injury in _atf4_−/− mice, which may contribute to the attenuated

motor recovery in these mice after SCI. Interestingly, activated microglia has been suggested to attenuate oligodendrocyte precursor cell proliferation _in vitro,_31 providing a possible

link between both cell type responses following SCI. The differences in cytokine levels observed in _atf4_−/− mice could alter different cellular processes including proliferation,

differentiation, and cell death associated with SCI.29 On the basis of our observations, we speculate that the UPR may be involved in controlling oligodendrocyte-dependent protective

functions in the injury site that could contribute to locomotion recovery after SCI. ER stress is proposed to be a relevant factor in the pathogenesis of many neurodegenerative diseases

involving abnormal protein folding.1 We have previously investigated the impact of the UPR in neurodegenerative diseases. Ablation of XBP1 in a model of amyotrophic lateral sclerosis (ALS)

has a contrasting effect on spinal cord from the results presented here in SCI models. Despite expectations that XBP1 deficiency would enhance the pathogenesis of mutant SOD1-mediated ALS, a

dramatic neuroprotection was observed owing to an enhanced clearance of mutant SOD1 aggregates by macroautophagy.32 In sharp contrast, PERK haplo-insufficiency enhances ALS progression.33

Besides, XBP1 deficiency did not affect prion misfolding or pathogenesis _in vivo._23 Other reports indicate that XBP1 has a protective effect in animal models of Parkinson's disease,

Alzheimer's disease, and autosomal dominant retinitis pigmentosa.1 ATF4 deficiency results in resistance to oxidative stress in models of brain ischemia.34, 35 PERK/eIF2_α_ signaling

has protective effects on axonal remyelination in models of experimental autoimmune encephalomyelitis, possibly because of the control of oligodendrocyte viability and function.33 Together

with the current study, these reports suggest an intriguing scenario where specific UPR signaling branches may have distinct and contrasting consequence depending on the nature of the

pathological stimuli. A variety of mechanisms underlying modifications of ER homeostasis may take place in specific disease contexts, and could include inhibition of ER-associated

degradation, altered vesicular trafficking, and altered protein-folding networks among others.1 In addition, alterations in lipid, cholesterol, or calcium metabolism may also affect ER

function in many diseases affecting the nervous system. Interestingly, the physiological role of the UPR has been mostly attributed to maintaining an efficient rate of protein synthesis and

secretion in specialized secretory cells including B lymphocytes, pancreatic _β_ cells, salivary glands, and many others.5 Neuronal and glial populations with higher secretory requirements

might display increased sensitivity to genetic and environmental factors that disrupt ER function. In this context, oligodendrocytes and neuropeptide-secretory neurons are of particular

relevance for future studies in order to uncover the mechanisms underlying the impact of the UPR in SCI. In fact, correlative studies suggest that the protection of valproate11, 36 or sodium

4-phenylbutyrate14 against SCI models is associated with attenuated levels of ER stress markers. The partial improvement obtained after XBP1s gene transfer over a wild-type condition where

the UPR is already activated suggests that locomotor improvements can be achieved with treatments of days after damage (considering the delay of >1 week for gene expression after AAV gene

transfer). The improvements in locomotor recovery observed in the gene therapy experiments may be actually important in the context of a patient that could progress from a state of full

paralysis to gaining partial control of some movements, having significant impact on the quality of life. Overall, this study together with recent findings, identifies the UPR pathway as a

potential target to treat SCI. Importantly, the use of small molecules or gene therapy strategies to attenuate ER stress or artificially engage UPR responses may have a beneficial impact to

attenuate tissue damage and enhance locomotor recovery in conditions affecting the function of the spinal cord. MATERIALS AND METHODS EXPERIMENTAL ANIMALS AND SURGICAL PROCEDURES Animals of

8 weeks of age, with body weight between 20 and 25 g were used in this study. ATF4 knockout mice and XBP-1 conditional knockout mice were previously described.23, 37 All animals were used on

a pure C57bl6 genetic background. Mice were anesthetized with a single dose of 330 mg/kg of 2-2-2 Tribromoethanol (Sigma, St. Louis, MO, USA) intraperitoneally. Then, the dorsal zone of the

spinal cord was incised along the midline; the T12 vertebra was laminectomized to expose the spinal cord. At this level, the spinal cord was hemitransected on the right side using a vannas

micro scissor (RS-5658, ROBOZ, Gaithersburg, MD, USA). Sham animals include the complete surgical procedure without hemitransection of the spine. During recovery, mice were placed in a

temperature-controlled chamber. At different days after surgery, animals were euthanized by an overdose of anesthesia. Surgical interventions and animal care follows the Institutional Review

Board's Animal Care of the University of Chile (CBA # 0305 FMUCH). WESTERN BLOT ANALYSIS OF SPINAL CORD EXTRACTS A 5-mm spinal cord tissue containing the hemitransected region

(medial), in addition to rostral and caudal regions of the same size, were collected and homogenized in 0.1 M phosphate buffered saline (pH 7.4) containing a protease inhibitor cocktail

(PIC, Roche, Basel, Switzerland). Half of the homogenized volume was used for protein extraction and the other half was used for RNA extraction (see below). The first volume was

re-homogenized by sonication in RIPA buffer (20 mM Tris at pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, and 0.5% Triton X-100) plus PIC and then samples were analyzed by SDS-PAGE. The

following antibodies and dilutions were used; anti-HSP90, 1 : 5000 (sc-7947, H114, Santa Cruz, Santa Cruz, CA, USA), anti-ATF4, 1 : 3000 (sc-200, C-20, Santa Cruz), anti-BIM, 1 : 2000

(sc-8267, M-20, Santa Cruz), and anti-ubiquitin 1 : 1000 (sc-8017, P4D1, Santa Cruz). RNA EXTRACTION AND RT-PCR Total RNA was prepared from spinal cord tissue previously homogenized in

saline phosphate buffer (PBS) using TRIzol (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized by iScript cDNA Synthesis (Bio-Rad, Hercules, CA, USA) using random primers p(dN)6 (Roche,

Basel, Switzerland). Quantitative real-time PCR was performed in an ABI PRISM7700 system (Applied Biosystems, Foster City, CA, USA) employing SYBRgreen fluorescent reagent (Applied

Biosystems) using the following primers: _bip_ forward 5′-TCATCGGACGCACTTGGAA-3′; _bip_ reverse 5′-CAACCACCTTGAATGGCAAGA-3′; chop forward 5′-TGGAGAGCGAGGGCTTTG-3′; chop reverse

5′-GTCCCTAGCTTGGCTGACAGA-3′; _actin_ forward 5′-CTCAGGAGGAGCAATGATCTTGAT-3′; actin reverse 5′-TACCACCATGTACCCAGGCA-3′; _IL-1β_ forward 5′-CAACCAACAAGTGATATTCTCCATG-3′; _IL-1β_ reverse

5′-GATCCACACTCTCCAGCTGCA-3′; IL-6 forward 5′-GAGGATACCACTCCCAACAGACC-3′; _IL-6_ reverse, 5′-AAGTGCATCATCGTTGTTCATACA-3′; and _TNFα_ forward 5′-CATCTTCTCAAAATTCGAGTGACAA-3′; _TNFα_ reverse

5′-TGGGAGTAGACAAGGTACAACCC-3′. XBP1 mRNA splicing assay was performed as previously described using PstI digestion of PCR products using the following primers: mXBP1.3S

(5′-AAACAGAGTAGCAGCGCAGACTGC-3′) and mXBP1.2AS: (5′-GGATCTCTAAAACTAGAGGCTTGGTG-3′). LOCOMOTOR FUNCTION Locomotor recovery was evaluated in an open-field test using the nine-point Basso Mouse

Scale (BMS).21 The BMS analysis of hindlimb movements and coordination for lateral hemisection SCI model was performed by two independent investigators blinded to the experimental condition

as described before.38 Our laboratory was certified during the 2009 Spinal Cord Injury Research Training Program at Ohio State University, USA, to perform BMS assays. BMS analysis involved

measuring the following parameters and scores: (0) no ankle movement; (1) slight ankle movement; (2) extensive ankle movement; (3) plantar placing of the paw with or without weight support,

or occasional, frequent or consistent dorsal stepping but no plantar stepping; (4) occasional plantar stepping; (5) frequent or consistent plantar stepping, no coordination or frequent, or

consistent plantar stepping, some coordination, paws rotated at initial contact and lift off; (6) frequent or consistent plantar stepping, some coordination, paws parallel at initial

contact, or frequent or consistent plantar stepping, mostly coordinated, paws rotated at initial contact and lift off; (7) frequent or consistent plantar stepping, mostly coordinated, paws

parallel at initial contact and rotated at lift off, or frequent or consistent plantar stepping, mostly coordinated, paws parallel at initial contact and lift off, and severe trunk

instability; (8) frequent or consistent plantar stepping, mostly coordinated, paws parallel at initial contact and lift off, and mild trunk instability, or frequent or consistent plantar

stepping, mostly coordinated, paws parallel at initial contact and lift off, and normal trunk stability, and tail down or up and down; (9) frequent or consistent plantar stepping, mostly

coordinated, paws parallel at initial contact and lift off, and normal trunk stability and tail always up. In general, the score for individual hindlimb ranged from 0 to 5 points (parameters

that do not involve coordination); and parameters involving the use of both hind limbs reached between 6 and 9 points. The 11-point BMS subscore was used to evaluate finer aspects of

locomotor capacity, which are not revealed by the BMS.21 BMS subscore included the following interval of scores: plantar stepping (0–2); coordination (0–2); paw position (0–4); trunk

stability (0–2); and tail position (0–1). To control the surgery, the following criteria of exclusion were employed: (i) at 1 day after surgery, mice with <5 points in its contralateral

hindlimb were excluded for the study (around 1 of 16 animals). (ii) If the BMS score for the ipsilateral hindlimb was >1 point at 1 day after surgery, the mouse was excluded for the study

(around 1 of 16 animals). The final score is presented as mean±S.E.M. HISTOLOGICAL ANALYSIS At 5 or 14 days after surgery, mice were perfused transcardially with 4% paraformaldehyde in 0.1 

M PBS. A 5-mm region of the spinal cord containing the lesion site was removed and post-fixed for 3 h in 4% paraformaldehyde. The spinal tissue was subjected to a sucrose gradient (5, 10,

and 30% sucrose in PBS), cryoprotected with optimal cutting temperature compound (Tissue-Tek, Alphen aan den Rijn, The Netherlands), and fast frozen using liquid nitrogen. The tissue was

longitudinally sectioned (5 _μ_m-thick slices) using a cryostat microtome (Leica, Nussloch, Germany). Sections were immunostained using antibodies anti-Cd11b, 1 : 100 (MCA74G, Serotec,

Morphosys, Oxford, UK), NeuN 1 : 300 (MAB377, Millipore Bioscience Research Reagents, Billerica, MA, USA), anti-Olig-2 1 : 200 (ab9610, Millipore Bioscience Research Reagents), and GFAP 1 : 

1000 (N1506, Dako, Glostrup, Denmark). Tissue sections were viewed with an Olympus IX71 microscope (Olympus, Center Valley, PA, USA) and images were captured using a QImaging QICAM Fast 1394

camera (Surrey, BC, Canada). Olig2-positive cells surrounding the injury zone were analyzed using a matrix of 50 _μ_m-separated concentric semicircles. GFAP staining intensity was

calculated by creating an integrated intensity profile along the spinal cord with its center located in the injury site. Neuronal numbers were assessed by counting NeuN-positive cells in the

spinal tissue, excluding the mechanically injured region. Cd11b-positive cells were determined in the region surrounding the mechanically injured zone. Longitudinal SC slices were also used

for eriochrome/cyanine staining. Slices were imaged using an OLYMPUS CX31 microscope fitted with a CCD camera. All quantifications were done using ImageJ software (NIH, Bethesda, MD, USA).

For EM analyses, spinal cord tissue was processed as described.39 Resin slices measuring 1 _μ_m were cut and toluidine blue stained as described.39 Slices were imaged using an Olympus IX71

microscope fitted with a QImaging QICAM Fast 1394 camera. AAV PRODUCTION AND INJECTION OF VIRAL PARTICLES INTO THE SPINAL CORD The whole Xbp-1 expression cassette was excised from

pcDNA3XBP-1S as a MfeI/SphI fragment and inserted into a proviral plasmid pAAVsp70 containing AAV2-inverted terminal repeats (ITRs). The vector is bicistronic and carries a GFP expression

cassette that serves as a fluorescent marker for transduced cells. Recombinant AAV2.XBP1S was produced by triple transfection of 293 cells using a rep/cap plasmid and pHelper (Stratagene, La

Jolla, CA, USA) and purified by column affinity chromatography, as previously described.40 Viral titers were determined using a real-time TaqMan PCR assay (ABI Prism 7700; Applied

Biosystems) with primers that were specific for the BGH polyA sequence. For animal treatment, 2 _μ_l of AAVs (1012 DRP/ml) were injected slowly over the injury site, right after performing

the hemisection using a 10-_μ_l Hamilton syringe fitted with a 34G needle. We confirmed the expression of XBP1s protein and upregulation of the target genes EDEM1, ERp72, and Sec61 after the

stereotaxis injection of AAV-XBP1 into the brain followed by real time PCR analysis (not shown). STATISTICAL ANALYSIS Data are shown as mean±S.E.M. Statistical analyses were performed by

using Student's _t_ test or two-way repeated-measures ANOVA, followed by a Bonferroni _post hoc_ test for multiple comparisons. ABBREVIATIONS * ER: endoplasmic reticulum * UPR: unfolded

protein response * SCI: spinal cord injury * ATF4: activating transcription factor 4 * XBP1: X-box-binding protein 1 * XBP1s: spliced XBP1 * IRE1: inositol-requiring enzyme-1 * PERK:

PKR-like endoplasmic reticulum kinase * CHOP: CCAAT/enhancer-binding protein (C/EBP) * eIF2a: eukaryotic translation initiation factor 2A * BiP: binding immunoglobulin protein * BMS: Basso

Mouse Scale * JNK: c-Jun N-terminal kinases * AAV: Adeno-associated virus * GFP: green fluorescent protein * GFAP: glial fibrillary acidic protein * NeuN: neuronal nuclei * Olig2:

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adeno-associated virus (rAAV). _J Gene Med_ 2000; 2: 444–454. Article  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Monica Perez for excellent EM processing and Alejandra

Catenaccio for technical assistance. This work was primarily supported by a North American Spine Society (NASS) Award. We also thank for the financial support from FONDECYT No. 1100176,

FONDAP No. 15010006, Millennium Institute No. P09-015-F, Alzheimer's Association, Muscular Dystrophy Association, The Michael J. Fox Foundation for Parkinson's Research, and ICGEB

(to CH). CONICYT Doctoral fellowship (EC) and FONDECYT No. 1110987, Millennium Nucleus No. P07-011-F (FC). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Biomedical Neuroscience Institute,

Faculty of Medicine, University of Chile, Santiago, Chile V Valenzuela & C Hetz * Millennium Nucleus for Regenerative Biology, Faculty of Biology, P. Catholic University of Chile,

Santiago, Chile V Valenzuela, E Collyer & F A Court * Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, University of Chile, Santiago, Chile V Valenzuela & 

C Hetz * Department of Molecular Biology, Genzyme Corporation, 49 New York Avenue, Framingham, 01701, MA, USA D Armentano & G B Parsons * Neurounion Biomedical Foundation, Santiago,

Chile F A Court & C Hetz * Department of Immunology and Infectious diseases, Harvard School of Public Health, Boston, MA, USA C Hetz Authors * V Valenzuela View author publications You

can also search for this author inPubMed Google Scholar * E Collyer View author publications You can also search for this author inPubMed Google Scholar * D Armentano View author

publications You can also search for this author inPubMed Google Scholar * G B Parsons View author publications You can also search for this author inPubMed Google Scholar * F A Court View

author publications You can also search for this author inPubMed Google Scholar * C Hetz View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING

AUTHORS Correspondence to F A Court or C Hetz. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION Edited by A Verkhratsky

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protein response enhances motor recovery after spinal cord injury. _Cell Death Dis_ 3, e272 (2012). https://doi.org/10.1038/cddis.2012.8 Download citation * Received: 04 January 2012 *

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content-sharing initiative KEYWORDS * spinal cord injury * unfolded protein response * endoplasmic reticulum stress * XBP1 * motor dysfunction