Sujata Saraswat Ohri

Attenuating the Endoplasmic Reticulum Stress Response Improves Functional Recovery After Spinal Cord Injury

Activation of the unfolded protein response (UPR) is involved in the pathogenesis of numerous CNS myelin abnormalities; yet, its direct role in traumatic spinal cord injury (SCI)‐induced demyelination is not known. The UPR is an evolutionarily conserved cell defense mechanism initiated to restore endoplasmic reticulum homeostasis in response to various cellular stresses including infection, trauma, and oxidative damage. However, if uncompensated, the UPR triggers apoptotic cell death. We demonstrate that the three signaling branches of UPR including the PERK, ATF6, and IRE1α are rapidly initiated in a mouse model of contusive SCI specifically at the injury epicenter. Immunohistochemical analyses of the various UPR markers revealed that in neurons, the UPR appeared at 6 and 24‐h post‐SCI. In contrast, in oligodendrocytes and astroglia, UPR persisted at least for up to 3 days post‐SCI. The UPR‐associated proapoptotic transcriptional regulator CHOP was among the UPR markers upregulated in neurons and oligodendrocytes, but not in astrocytes, of traumatized mouse spinal cords. To directly analyze its role in SCI, WT and CHOP null mice received a moderate T9 contusive injury. Deletion of CHOP led to an overall attenuation of the UPR after contusive SCI. Furthermore, analyses of hindlimb locomotion demonstrated a significant functional recovery that correlated with an increase in white‐matter sparing, transcript levels of myelin basic protein, and Claudin 11 and decreased oligodendrocyte apoptosis in CHOP null mice in contrast to WT animals. Thus, our study provides evidence that the UPR contributes to oligodendrocyte loss after traumatic SCI.

Restoring endoplasmic reticulum homeostasis improves functional recovery after spinal cord injury

The endoplasmic reticulum (ER) stress response (ERSR) is activated to maintain protein homeostasis or induce apoptosis in the ER in response to distinct cellular insults including hypoxia, inflammation, and oxidative damage. Recently, we showed ERSR activation in a mouse model of a contusive spinal cord injury (SCI) and an improved hindlimb locomotor function following SCI when the pro-apoptotic arm of ERSR was genetically inhibited. The objective of the current study was to explore if the pharmacological enhancement of the homeostatic arm of the ERSR pathway can improve the functional outcome after SCI. Salubrinal enhances the homeostatic arm of the ERSR by increasing phosphorylation of eIF2α. Salubrinal significantly enhanced the levels of phosphorylated eIF2α protein and modulated the downstream ERSR effectors assessed at the lesion epicenter 6h post-SCI. Hindlimb locomotion showed significant improvement in animals treated with salubrinal. Treadmill-based-gait assessment showed a significant increase in maximum speed of coordinated walking and a decrease in rear stance time and stride length in salubrinal-treated animals. This improved functional recovery corresponded with increased white matter sparing and decreased oligodendrocyte apoptosis. In addition, salubrinal protected cultured mouse oligodendrocyte progenitor cells against the ER stress-inducing toxin tunicamycin. These data suggest that boosting the homeostatic arm of the ERSR reduces oligodendrocyte loss after traumatic SCI and support the contention that pharmacological targeting of the ERSR after CNS trauma is a therapeutically viable approach.

Isolation of cortical mouse oligodendrocyte precursor cells

The reliable isolation of primary oligodendrocyte progenitors cells (OPCs) holds promise as both a research tool and putative therapy for the study and treatment of central nervous system (CNS) disease and trauma. Stringently characterized primary mouse OPCs is of additional importance due to the power of transgenics to address mechanism(s) involving single genes. In this study, we developed and characterized a reproducible method for the primary culture of OPCs from postnatal day 5-7 mouse cerebral cortex. We enriched an O4(+) OPC population using Magnetic Activated Cell Sorting (MACS) technology. This technique resulted in an average yield of 3.68×10(5)OPCs/brain. Following isolation, OPCs were glial fibrillary acidic protein(-) (GFAP(-)) and O4(+). Following passage and with expansion, OPCs were O4(+), A2B5(+), and NG2(+). Demonstrating their bi-potentiality, mouse OPCs differentiated into either more complex, highly arborized O4(+) or O1(+) oligodendrocytes (OLs) or GFAP(+) astrocytes. This bi-potentiality is lost, however, in co-culture with rat embryonic day 15 derived dorsal root ganglia (DRG). Following 7-14 days of OPC/DRG co-culture, OPCs aligned with DRG neurites and differentiated into mature OLs as indicated by the presence of O1 and myelin basic protein (MBP) immunostaining. Addition of ciliary neurotrophic factor (CNTF) to conditioned media from OPC/DRG co-cultures improved OPC differentiation into mature O1(+) and MBP(+) OLs. This method allows for the study of primary mouse cortical OPC survival, maturation, and function without relying on oligosphere formation or the need for extensive passaging.

Depletion of procathepsin D gene expression by RNA interference – A potential therapeutic target for breast cancer

Elevated level of procathepsin D (pCD), a zymogen of lysosomal aspartic proteinase cathepsin D, is associated with highly invasive neoplasms that include breast cancer. Independent studies have established that secreted pCD functions as a growth factor acting both in an autocrine and paracrine manner. Therefore, to explore whether pCD can be employed as a therapeutic target, the present study evaluates the impact of pCD knockdown using RNA interference technology. Of the three siRNA oligos tested, siRNA-3 exhibited a 90% inhibitory effect on pCD gene expression. Stable attenuation of pCD in breast cancer cells MDA-MB-231 was achieved by using a plasmid vector-based shRNA system. Pronounced suppression of pCD expression was accompanied by a significant reduction in invasion and proliferation of MDA-MB-231 cells stably transfected with functional shRNA. Importantly, in the athymic nude mice model, downregulation of pCD in breast cancer cells significantly reduced their metastatic potential. In addition, we observed a reduction in Cdc42 and NF-κB2 expression in MDA-MB-231 cells with decreased pCD expression. When combined, our in vitro and in vivo experiments demonstrate that targeting pCD through RNAi technology represents a potential therapeutic tool for developing a therapy against breast cancer.

Deletion of the Pro-Apoptotic Endoplasmic Reticulum Stress Response Effector CHOP Does Not Result in Improved Locomotor Function after Severe Contusive Spinal Cord Injury

Manipulation of various components of the endoplasmic reticulum (ER) stress response (ERSR) has led to functional recovery in diabetes, cancer, and several neurodegenerative diseases, indicating its use as a potential therapeutic intervention. One of the downstream pro-apoptotic transcription factors activated by the ERSR is CCAAT enhancer binding protein (C/EBP) homologous protein (CHOP). Recently, we showed significant recovery in hindlimb locomotion function after moderate contusive spinal cord injury (SCI) in mice null for CHOP. However, more than 40% of human SCI are complete. Thus the present study examined the potential therapeutic modulation of CHOP in a more severe SCI injury. Contused wild-type spinal cords showed a rapid activation of PERK, ATF6, and IRE-1, the three arms of the ERSR signaling pathway, specifically at the injury epicenter. Confocal images of phosphorylated EIF2α, GRP78, CHOP, ATF4, and GADD34 localized the activation of the ERSR in neurons and oligodendrocytes at the injury epicenter. To directly determine the role of CHOP, wild-type and CHOP-null mice with severe contusive SCI were analyzed for improvement in hindlimb locomotion. Despite the loss of CHOP, the other effectors in the ERSR pathway were significantly increased beyond that observed previously with moderate injury. Concomitantly, Basso Mouse Scale (BMS) scores and white matter sparing between the wild-type and CHOP-null mice revealed no significant differences. Given the complex pathophysiology of severe SCI, ablation of CHOP alone is not sufficient to rescue functional deficits. These data raise the caution that injury severity may be a key variable in attempting to translate preclinical therapies to clinical practice.

Pleiotropic Effects of Cathepsin D

Over the past decades, the paradigm that lysosomal enzymes participate only in non-specific protein degradation during cell death has changed. Studies conducted both in cell cultures and in animals defined the role of these enzymes that includes cathepsin D (CD). Knockout mice revealed the role of CD in postnatal tissue homeostasis and remodeling. Mutations that abolish the CD enzymatic activity have been implicated in neural ceroid lipofuscinosis. Recent studies suggested a differential role of CD in regulation of apoptosis. The zymogen of CD, procathepsin D (pCD), is secreted by various cancer cells. Extensive studies showed that it acts as a mitogen on both cancer and stromal cells by stimulating their invasive and metastatic properties. Additional studies suggested that procathepsin D/CD is an independent prognostic factor in various cancers, leading to the investigations of pCD/CD as a potential target for designing anti-cancer therapy. In this review, we described the various forms of CD and their implications in numerous physiological as well as pathological conditions.

Does the preclinical evidence for functional remyelination following myelinating cell engraftment into the injured spinal cord support progression to clinical trials

This article reviews all historical literature in which rodent-derived myelinating cells have been engrafted into the contused adult rodent spinal cord. From 2500 initial PubMed citations identified, human cells grafts, bone mesenchymal stem cells, olfactory ensheathing cells, non-myelinating cell grafts, and rodent grafts into hemisection or transection models were excluded, resulting in the 67 studies encompassed in this review. Forty five of those involved central nervous system (CNS)-derived cells, including neural stem progenitor cells (NSPCs), neural restricted precursor cells (NRPs) or oligodendrocyte precursor cells (OPCs), and 22 studies involved Schwann cells (SC). Of the NSPC/NPC/OPC grafts, there was no consistency with respect to the types of cells grafted and/or the additional growth factors or cells co-grafted. Enhanced functional recovery was reported in 31/45 studies, but only 20 of those had appropriate controls making conclusive interpretation of the remaining studies impossible. Of those 20, 19 were properly powered and utilized appropriate statistical analyses. Ten of those 19 studies reported the presence of graft-derived myelin, 3 reported evidence of endogenous remyelination or myelin sparing, and 2 reported both. For the SC grafts, 16/21 reported functional improvement, with 11 having appropriate cellular controls and 9/11 using proper statistical analyses. Of those 9, increased myelin was reported in 6 studies. The lack of consistency and replication among these preclinical studies are discussed with respect to the progression of myelinating cell transplantation therapies into the clinic.

Inhibition of GADD34, the Stress-Inducible Regulatory Subunit of the Endoplasmic Reticulum Stress Response, Does Not Enhance Functional Recovery after Spinal Cord Injury

Activation of the endoplasmic reticulum stress response (ERSR) is a hallmark of various pathological diseases and/or traumatic injuries. Restoration of ER homeostasis can contribute to improvement in the functional outcome of these diseases. Using genetic and pharmacological inhibition of the PERK-CHOP arm of the ERSR, we recently demonstrated improvements in hindlimb locomotion after spinal cord injury (SCI) and implicated oligodendrocyte survival as a potential mechanism. Here, we investigated the contribution of stress-inducible PPP1R15A/GADD34, an ERSR signaling effector downstream of CHOP that dephosphorylates eIF2α, in the pathogenesis of SCI. We show that although genetic ablation of GADD34 protects oligodendrocyte precursor cells (OPCs) against ER stress-mediated cell death in vitro and results in differential ERSR attenuation in vivo after SCI, there is no improvement in hindlimb locomotor function. Guanabenz, a FDA approved antihypertensive drug, was recently shown to reduce the burden of misfolded proteins in the ER by directly targeting GADD34. Guanabenz protected OPCs from ER stress-mediated cell death in vitro and attenuated the ERSR in vivo after SCI. However, guanabenz administration failed to rescue the locomotor deficits after SCI. These data suggest that deletion of GADD34 alone is not sufficient to improve functional recovery after SCI.

Histone deacetylase inhibition is cytotoxic to oligodendrocyte precursor cells in vitro and in vivo.

Histone deacetylase (HDAC) inhibition mediated by small molecule HDAC inhibitors (HDACi) has demonstrated divergent effects including toxicity towards transformed cell lines, neuroprotection in neurological disease models, and inhibition of oligodendrocyte precursor cell (OPC) differentiation to mature oligodendrocytes (OL). However, it remains unknown if transient HDAC inhibition may promote OPC survival. Using mouse cortical OPC primary cultures, we investigated the effects of the FDA approved pan‐HDACi suberoylanilide hydroxamic acid (SAHA) on OPC survival. Initial studies showed differences in the HDAC expression pattern of multiple HDAC isoforms in OPCs relative to their terminally differentiated progeny cells, OLs and astrocytes. Treatment of OPCs with SAHA for up to 72 h using a maximum concentration either at or lower than those necessary for cytotoxicity in most transformed cell lines resulted in over 67% reduction in viability relative to vehicle‐treated OPCs. This was at least partly due to increased apoptosis as SAHA‐treated cells displayed activated caspase 3 and were protected by the general caspase inhibitor Q‐VD‐OPH. Additionally, SAHA treatment of whole mice at postnatal day 5 induced apoptosis of cortical OPCs. These results suggest that SAHA negatively impacts OPC survival and may be detrimental to the myelinating brain and spinal cord. Such toxicity may be relevant in a clinical context as SAHA is currently involved in numerous clinical trials and is in consideration for use in the treatment of psychiatric and neurodegenerative conditions.

Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione.

The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10−8 M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter.