Nucleolin inhibitor GroA triggers reduction in epidermal growth factor receptor activation: Pharmacological implication for glial scarring after spinal cord injury.

Glial scarring, formed by reactive astrocytes, is one of the major impediments for regeneration after spinal cord injury (SCI). Reactive astrocytes become hypertrophic, proliferate and secrete chondroitin sulphate proteoglycans into the extracellular matrix (ECM). Many studies have demonstrated that epidermal growth factor receptors (EGFR) can mediate astrocyte reactivity after neurotrauma. Previously we showed that there is crosstalk between nucleolin and EGFR that leads to increased EGFR activation followed by increased cell proliferation. Treatment with the nucleolin inhibitor GroA (AS1411) prevented these effects in vitro and in vivo. In this study, we hypothesized that similar interactions may mediate astrogliosis after SCI. Our results demonstrate that nucleolin and EGFR interaction may play a pivotal role in mediating astrocyte proliferation and reactivity after SCI. Moreover, we demonstrate that treatment with GroA reduces EGFR activation, astrocyte proliferation and chondroitin sulphate proteoglycans secretion, therefore promoting axonal regeneration and sprouting into the lesion site. Our results identify, for the first time, a role for the interaction between nucleolin and EGFR in astrocytes after SCI, indicating that nucleolin inhibitor GroA may be used as a novel treatment after neurotrauma. A major barrier for axonal regeneration after spinal cord injury is glial scar created by reactive and proliferating astrocytes. EGFR mediate astrocyte reactivity. We showed that inhibition of nucleolin by GroA, reduces EGFR activation, which results in attenuation of astrocyte reactivity and proliferation in vivo and in vitro. EGFR, epidermal growth factor receptor.

Rapamycin increases neuronal survival, reduces inflammation and astrocyte proliferation after spinal cord injury.

Spinal cord injury (SCI) frequently leads to a permanent functional impairment as a result of the initial injury followed by secondary injury mechanism, which is characterised by increased inflammation, glial scarring and neuronal cell death. Finding drugs that may reduce inflammatory cell invasion and activation to reduce glial scarring and increase neuronal survival is of major importance for improving the outcome after SCI. In the present study, we examined the effect of rapamycin, an mTORC1 inhibitor and an inducer of autophagy, on recovery from spinal cord injury. Autophagy, a process that facilitates the degradation of cytoplasmic proteins, is also important for maintenance of neuronal homeostasis and plays a major role in neurodegeneration after neurotrauma. We examined rapamycin effects on the inflammatory response, glial scar formation, neuronal survival and regeneration in vivo using spinal cord hemisection model in mice, and in vitro using primary cortical neurons and human astrocytes. We show that a single injection of rapamycin, inhibited p62/SQSTM1, a marker of autophagy, inhibited mTORC1 downstream effector p70S6K, reduced macrophage/neutrophil infiltration into the lesion site, microglia activation and secretion of TNFα. Rapamycin inhibited astrocyte proliferation and reduced the number of GFAP expressing cells at the lesion site. Finally, it increased neuronal survival and axonogenesis towards the lesion site. Our study shows that rapamycin treatment increased significantly p-Akt levels at the lesion site following SCI. Similarly, rapamycin treatment of neurons and astrocytes induced p-Akt elevation under stress conditions. Together, these findings indicate that rapamycin is a promising candidate for treatment of acute SCI condition and may be a useful therapeutic agent.

EphA4 is associated with multiple cell types in the marmoset primary visual cortex throughout the lifespan.

Ephs form the largest family of receptor tyrosine kinases. They interact with the membrane-bound ligands - ephrins - to control crucial aspects of brain development. EphA4 is the most prominent member of the family in terms of versatility and ability to bind most ephrin ligands. EphA4 regulates brain development by modulating neuronal migration and connectivity. In the present study, we address the involvement of EphA4 in patterning the primary visual cortex (V1) of the marmoset monkey by characterizing the cellular expression profile of EphA4 from late embryonic stages to adulthood. We identified continuous expression on neurons in the cortical plate and mature neocortical layers, similar to that described in the mouse, excluding a role for EphA4 in the formation of borders between visual areas in the marmoset neocortex. In addition to neurons, we also report expression of EphA4 on glial populations, including radial glia and astrocytes. In contrast to what is seen in the mouse, EphA4 expression on astrocytes persists in the adult marmoset V1, including around blood vessels and in the white matter. Robust expression by glial populations, which retain neurogenic properties in the postnatal marmoset, indicates that EphA4 may have acquired additional roles during evolution, with important implications for the benefits of EphA4-blocking therapies following brain injury.

Anti-lysophosphatidic acid antibodies improve traumatic brain injury outcomes.

BACKGROUND: Lysophosphatidic acid (LPA) is a bioactive phospholipid with a potentially causative role in neurotrauma. Blocking LPA signaling with the LPA-directed monoclonal antibody B3/Lpathomab is neuroprotective in the mouse spinal cord following injury. FINDINGS: Here we investigated the use of this agent in treatment of secondary brain damage consequent to traumatic brain injury (TBI). LPA was elevated in cerebrospinal fluid (CSF) of patients with TBI compared to controls. LPA levels were also elevated in a mouse controlled cortical impact (CCI) model of TBI and B3 significantly reduced lesion volume by both histological and MRI assessments. Diminished tissue damage coincided with lower brain IL-6 levels and improvement in functional outcomes. CONCLUSIONS: This study presents a novel therapeutic approach for the treatment of TBI by blocking extracellular LPA signaling to minimize secondary brain damage and neurological dysfunction.

Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish.

Adult zebrafish show a remarkable capacity to regenerate their spinal column after injury, an ability that stands in stark contrast to the limited repair that occurs within the mammalian CNS post-injury. The reasons for this interspecies difference in regenerative capacity remain unclear. Here we demonstrate a novel role for Fgf signaling during glial cell morphogenesis in promoting axonal regeneration after spinal cord injury. Zebrafish glia are induced by Fgf signaling, to form an elongated bipolar morphology that forms a bridge between the two sides of the resected spinal cord, over which regenerating axons actively migrate. Loss of Fgf function inhibits formation of this "glial bridge" and prevents axon regeneration. Despite the poor potential for mammalian axonal regeneration, primate astrocytes activated by Fgf signaling adopt a similar morphology to that induced in zebrafish glia. This suggests that differential Fgf regulation, rather than intrinsic cell differences, underlie the distinct responses of mammalian and zebrafish glia to injury.

Rho/ROCK pathway is essential to the expansion, differentiation, and morphological rearrangements of human neural stem/progenitor cells induced by lysophosphatidic acid.

We previously reported that lysophosphatidic acid (LPA) inhibits the neuronal differentiation of human embryonic stem cells (hESC). We extended these studies by analyzing LPA's effects on the expansion of neural stem/progenitor cells (NS/PC) derived from hESCs and human induced pluripotent stem cells (iPSC), and we assessed whether data obtained on the neural differentiation of hESCs were relevant to iPSCs. We showed that hESCs and iPSCs exhibited comparable mRNA expression profiles of LPA receptors and producing enzymes upon neural differentiation. We demonstrated that LPA inhibited the expansion of NS/PCs of both origins, mainly by increased apoptosis in a Rho/Rho-associated kinase (ROCK)-dependent mechanism. Furthermore, LPA inhibited the neuronal differentiation of iPSCs. Lastly, LPA induced neurite retraction of NS/PC-derived early neurons through Rho/ROCK, which was accompanied by myosin light chain (MLC) phosphorylation. Our data demonstrate the consistency of LPA effects across various sources of human NS/PCs, rendering hESCs and iPSCs valuable models for studying lysophospholipid signaling in human neural cells. Our data also highlight the importance of the Rho/ROCK pathway in human NS/PCs. As LPA levels are increased in the central nervous system (CNS) following injury, LPA-mediated effects on NS/PCs and early neurons could contribute to the poor neurogenesis observed in the CNS following injury.

Blockage of lysophosphatidic acid signaling improves spinal cord injury outcomes.

Evidence suggests a proinflammatory role of lysophosphatidic acid (LPA) in various pathologic abnormalities, including in the central nervous system. Herein, we describe LPA as an important mediator of inflammation after spinal cord injury (SCI) in zebrafish and mice. Furthermore, we describe a novel monoclonal blocking antibody raised against LPA that potently inhibits LPA's effect in vitro and in vivo. This antibody, B3, specifically binds LPA, prevents it from interacting with its complement of receptors, and blocks LPA's effects on the neuronal differentiation of human neural stem/progenitor cells, demonstrating its specificity toward LPA signaling. When administered systemically to mice subjected to SCI, B3 substantially reduced glial inflammation and neuronal death. B3-treated animals demonstrated significantly more neuronal survival upstream of the lesion site, with some functional improvement. This study describes the use of anti-LPA monoclonal antibody as a novel therapeutic approach for the treatment of SCI.

Species-specific microRNA roles elucidated following astrocyte activation.

MicroRNAs (miRNAs) are short non-coding RNAs that play a central role in regulation of gene expression by binding to target genes. Many miRNAs were associated with the function of the central nervous system (CNS) in health and disease. Astrocytes are the CNS most abundant glia cells, providing support by maintaining homeostasis and by regulating neuronal signaling, survival and synaptic plasticity. Astrocytes play a key role in repair of brain insults, as part of local immune reactivity triggered by inflammatory or pathological conditions. Thus, astrocyte activation, or astrogliosis, is an important outcome of the innate immune response, which can be elicited by endotoxins such as lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-γ). The involvement of miRNAs in inflammation and stress led us to hypothesize that astrogliosis is mediated by miRNA function. In this study, we compared the miRNA regulatory layer expressed in primary cultured astrocyte derived from rodents (mice) and primates (marmosets) brains upon exposure to LPS and IFN-γ. We identified subsets of differentially expressed miRNAs some of which are shared with other immunological related systems while others, surprisingly, are mouse and rat specific. Of interest, these specific miRNAs regulate genes involved in the tumor necrosis factor-alpha (TNF-α) signaling pathway, indicating a miRNA-based species-specific regulation. Our data suggests that miRNA function is more significant in the mechanisms governing astrocyte activation in rodents compared to primates.

Treatment with Pulsed Extremely Low Frequency Electromagnetic Field (PELF-EMF) Exhibit Anti-Inflammatory and Neuroprotective Effect in Compression Spinal Cord Injury Model.

BACKGROUND: Spinal cord injury (SCI) pathology includes both primary and secondary events. The primary injury includes the original traumatic event, and the secondary injury, beginning immediately after the initial injury, involves progressive neuroinflammation, neuronal excitotoxicity, gliosis, and degeneration. Currently, there is no effective neuroprotective treatment for SCI. However, an accumulating body of data suggests that PELF-EMF has beneficial therapeutic effects on neurotrauma. The purpose of this study was to test the efficacy of the PELF-EMF SEQEX device using a compression SCI mouse model. METHODS: C57BL/6 mice were exposed to PELF-EMF for 4 h on a daily basis for two months, beginning 2 h after a mild-moderate compression SCI. RESULTS: The PELF-EMF treatment significantly diminished inflammatory cell infiltration and astrocyte activation by reducing Iba1, F4/80, CD68+ cells, and GAFP at the lesion borders, and increased pro-survival signaling, such as BDNF, on the neuronal cells. Moreover, the treatment exhibited a neuroprotective effect by reducing the demyelination of the axons of the white matter at the lesion's center. CONCLUSIONS: Treatment with SEQEX demonstrated significant anti-inflammatory and neuroprotective effects. Considering our results, this safe and effective rehabilitative device, already available on the market, may provide a major therapeutic asset in the treatment of SCI.

Electroporation-based proteome sampling ex vivo enables the detection of brain melanoma protein signatures in a location proximate to visible tumor margins.

A major concern in tissue biopsies with a needle is missing the most lethal clone of a tumor, leading to a false negative result. This concern is well justified, since needle-based biopsies gather tissue information limited to needle size. In this work, we show that molecular harvesting with electroporation, e-biopsy, could increase the sampled tissue volume in comparison to tissue sampling by a needle alone. Suggested by numerical models of electric fields distribution, the increased sampled volume is achieved by electroporation-driven permeabilization of cellular membranes in the tissue around the sampling needle. We show that proteomic profiles, sampled by e-biopsy from the brain tissue, ex vivo, at 0.5mm distance outside the visible margins of mice brain melanoma metastasis, have protein patterns similar to melanoma tumor center and different from the healthy brain tissue. In addition, we show that e-biopsy probed proteome signature differentiates between melanoma tumor center and healthy brain in mice. This study suggests that e-biopsy could provide a novel tool for a minimally invasive sampling of molecules in tissue in larger volumes than achieved with traditional needle biopsies.

Regenerating the Injured Spinal Cord at the Chronic Phase by Engineered iPSCs-Derived 3D Neuronal Networks.

Cell therapy using induced pluripotent stem cell-derived neurons is considered a promising approach to regenerate the injured spinal cord (SC). However, the scar formed at the chronic phase is not a permissive microenvironment for cell or biomaterial engraftment or for tissue assembly. Engineering of a functional human neuronal network is now reported by mimicking the embryonic development of the SC in a 3D dynamic biomaterial-based microenvironment. Throughout the in vitro cultivation stage, the system's components have a synergistic effect, providing appropriate cues for SC neurogenesis. While the initial biomaterial supported efficient cell differentiation in 3D, the cells remodeled it to provide an inductive microenvironment for the assembly of functional SC implants. The engineered tissues are characterized for morphology and function, and their therapeutic potential is investigated, revealing improved structural and functional outcomes after acute and chronic SC injuries. Such technology is envisioned to be translated to the clinic to rewire human injured SC.

Blood glutamate scavengers increase pro-apoptotic signaling and reduce metastatic melanoma growth in-vivo.

Inhibition of extracellular glutamate (Glu) release decreases proliferation and invasion, induces apoptosis, and inhibits melanoma metastatic abilities. Previous studies have shown that Blood-glutamate scavenging (BGS), a novel treatment approach, has been found to be beneficial in attenuating glioblastoma progression by reducing brain Glu levels. Therefore, in this study we evaluated the ability of BGS treatment to inhibit brain metastatic melanoma progression in-vivo. RET melanoma cells were implanted in C56BL/6J mice to induce brain melanoma tumors followed by treatment with BGS or vehicle administered for fourteen days. Bioluminescent imaging was conducted to evaluate tumor growth, and plasma/CSF Glu levels were monitored throughout. Immunofluorescence staining of Ki67 and 53BP1 was used to analyze tumor cell proliferation and DNA double-strand breaks. In addition, we analyzed CD8, CD68, CD206, p-STAT1 and iNOS expression to evaluate alterations in tumor micro-environment and anti-tumor immune response due to treatment. Our results show that BGS treatment reduces CSF Glu concentration and consequently melanoma growth in-vivo by decreasing tumor cell proliferation and increasing pro-apoptotic signaling in C56BL/6J mice. Furthermore, BGS treatment supported CD8+ cell recruitment and CD68+ macrophage invasion. These findings suggest that BGS can be of potential therapeutic relevance in the treatment of metastatic melanoma.

LPA receptor expression in the central nervous system in health and following injury.

Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA(1-3)) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA(1) was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA(2) showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA(3) showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA(1-3) are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.

Blood glutamate scavengers and exercises as an effective neuroprotective treatment in mice with spinal cord injury.

OBJECTIVE: Excitotoxicity due to neuronal damage and glutamate release is one of the first events that leads to the progression of neuronal degeneration and functional impairment. This study is based on a paradigm shift in the therapeutic approach for treating spinal cord injury (SCI). The authors tested a new treatment targeting removal of CNS glutamate into the blood circulation by injection of the blood glutamate scavengers (BGSs) recombinant enzyme glutamate-oxaloacetate transaminase (rGOT1) and its cosubstrate oxaloacetic acid (OxAc). Their primary objective was to investigate whether BGS treatment, followed by treadmill exercises in mice with SCI, could attenuate excitotoxicity, inflammation, scarring, and axonal degeneration and, at a later time point, improve functional recovery. METHODS: A pharmacokinetic experiment was done in C57BL/6 naive mice to verify rGOT1/OxAc blood activity and to characterize the time curve of glutamate reduction in the blood up to 24 hours. The reduction of glutamate in CSF after BGS administration in mice with SCI was confirmed by high-performance liquid chromatography. Next, SCI (left hemisection) was induced in the mice, and the mice were randomly assigned to one of the following groups at 1 hour postinjury: control (underwent SCI and received PBS), treadmill exercises, rGOT1/OxAc treatment, or rGOT1/OxAc treatment followed by treadmill exercises. Treatment started 1 hour postinjury with an injection of rGOT1/OxAc and continued for 5 consecutive days. Starting 1 week after SCI, the exercises and the combined treatment groups recommenced the treadmill exercise regimen 5 days a week for 3 months. Locomotor function was assessed for 3 months using the horizontal grid walking test and CatWalk. Axonal anterograde and wallerian degenerations were evaluated using tetramethylrhodamine dextran. Tissue sections were immunofluorescently stained for Iba1, GFAP, GAP-43, synaptophysin, and NeuN. RESULTS: BGS treatment decreased the CSF glutamate level up to 50%, reduced axonal wallerian degeneration, and increased axonal survival and GAP-43 expression in neuronal cells. Combined treatment reduced inflammation, scarring, and lesion size. Additionally, the combination of BGS treatment and exercises increased synapses around motor neurons and enhanced axonal regeneration through the lesion site. This resulted in motor function improvement 3 months post-SCI. CONCLUSIONS: As shown by biochemical, immunohistochemical, and functional analysis, BGSs exhibit a substantial neuroprotective effect by reducing excitotoxicity and secondary damage after SCI. Furthermore, in combination with exercises, they reduced axonal degeneration and scarring and resulted in improved functional recovery.

Correction: Mesenchymal stem cells derived extracellular vesicles improve behavioral and biochemical deficits in a phencyclidine model of schizophrenia.

In the original Article, Dr. Angela Ruban's name was misspelled as "Aangela Ruban". This has been corrected in the PDF, HTML, and XML versions of this Article.

Mesenchymal stem cells derived extracellular vesicles improve behavioral and biochemical deficits in a phencyclidine model of schizophrenia.

Schizophrenia is a debilitating psychiatric disorder with a significant number of patients not adequately responding to treatment. Phencyclidine (PCP) is used as a validated model for schizophrenia, shown to reliably induce positive, negative and cognitive-like behaviors in rodents. It was previously shown in our lab that behavioral phenotypes of PCP-treated mice can be alleviated after intracranial transplantation of mesenchymal stem cells (MSC). Here, we assessed the feasibility of intranasal delivery of MSCs-derived-extracellular vesicles (EVs) to alleviate schizophrenia-like behaviors in a PCP model of schizophrenia. As MSCs-derived EVs were already shown to concentrate at the site of lesion in the brain, we determined that in PCP induced injury the EVs migrate to the prefrontal cortex (PFC) of treated mice, a most involved area of the brain in schizophrenia. We show that intranasal delivery of MSC-EVs improve social interaction and disruption in prepulse inhibition (PPI) seen in PCP-treated mice. In addition, immunohistochemical studies demonstrate that the EVs preserve the number of parvalbumin-positive GABAergic interneurons in the PFC of treated mice. Finally, MSCs-EVs reduced glutamate levels in the CSF of PCP-treated mice, which might explain the reduction of toxicity. In conclusion, we show that MSCs-EVs improve the core schizophrenia-like behavior and biochemical markers of schizophrenia and might be used as a novel treatment for this incurable disorder.

Correction: Mesenchymal stem cells derived extracellular vesicles improve behavioral and biochemical deficits in a phencyclidine model of schizophrenia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Altered mitochondrial dynamics and function in APOE4-expressing astrocytes.

APOE4 is a major risk factor for sporadic Alzheimer's disease; however, it is unclear how it exerts its pathological effects. Others and we have previously shown that autophagy is impaired in APOE4 compared to APOE3 astrocytes, and demonstrated differences in the expression of mitochondrial dynamics proteins in brains of APOE3 and APOE4 transgenic mice. Here, we investigated the effect of APOE4 expression on several aspects of mitochondrial function and network dynamics, including fusion, fission, and mitophagy, specifically in astrocytes. We found that APOE3 and APOE4 astrocytes differ in their mitochondrial dynamics, suggesting that the mitochondria of APOE4 astrocytes exhibit reduced fission and mitophagy. APOE4 astrocytes also show impaired mitochondrial function. Importantly, the autophagy inducer rapamycin enhanced mitophagy and improved mitochondrial functioning in APOE4 astrocytes. Collectively, the results demonstrate that APOE4 expression is associated with altered mitochondrial dynamics, which might lead to impaired mitochondrial function in astrocytes. This, in turn, may contribute to the pathological effects of APOE4 in Alzheimer's disease.

Treadmill training after spinal cord hemisection in mice promotes axonal sprouting and synapse formation and improves motor recovery.

Treadmill training with weight-support is a therapeutic strategy used in human patients after spinal cord injury (SCI). Exercise leads to locomotor improvement in a variety of animal models; however, the effect of exercise on axonal regrowth has not been directly examined. This study used several locomotor tests, including kinematic gait analysis, to analyze the differences between treadmill-trained and untrained mice in the usage of their paretic hindlimb following a low thoracic hemisection. Analysis of muscle atrophy, anterograde axonal tracing and expression of the synaptic markers synaptophysin and PSD95 were used to correlate observed behavioural changes with anatomical data. Treadmill trained mice showed significant improvement in use of their paretic hindlimb after 4 weeks of exercise compared to untrained mice in an open field locomotor test (Basso-Beattie-Bresnahan [BBB] scale), grid walking and climbing and inter-limb coordination tests. Movement of their hip joint started to approximate the pattern of intact mice, with concomitant use of their ankle. Unlike untrained mice, exercised mice showed decreased muscle atrophy, increased axonal regrowth and collateral sprouting proximal to the lesion site, with maintenance of synaptic markers on motor neurons in the ventral horn. However, there was no axonal regeneration into or across the lesion site indicating that the improved behaviour may have been, at least in part, due to enhanced neural activity above the lesion site.

Blood Glutamate Scavenger as a Novel Neuroprotective Treatment in Spinal Cord Injury.

Neurotrauma causes immediate elevation of extracellular glutamate (Glu) levels, which creates excitotoxicity and facilitates inflammation, glial scar formation, and consequently neuronal death. Finding factors that reduce the inflammatory response and glial scar formation, and increase neuronal survival and neurite outgrowth, are of major importance for improving the outcome after spinal cord injury (SCI). In the present study, we evaluated a new treatment aiming to remove central nervous system (CNS) Glu into the systemic blood circulation by intravenous (IV) administration of blood Glu scavengers (BGS) such as the enzyme recombinant glutamate-oxaloacetate transaminase 1 (rGOT1) and its co-substrate. In this study we induced in mice an SCI (hemisection), and 1 h post-injury started administering BGS treatment for 5 consecutive days. The treatment reduced the expression levels of p-p38, which regulates apoptosis and increased the expression of p-Akt, which mediates cell survival. Moreover, this treatment decreased pro-inflammatory cytokine expression and microglia activation, reduced astrocytes' reactivity, and facilitated expression of radial glia markers such as Pax6 and nestin. BGS treatment increased the survival of neurons at lesion site and enabled axonal regeneration into the injury site. These effects were correlated with improved functional recovery of the left paretic hindlimb. Thus, early pharmacological intervention with BGS following SCI may be neuroprotective and create a pro-regenerative environment by modulating glia cell response. In light of our results, the availability of the method to remove excess Glu from CNS without the need to deliver drugs across the blood-brain barrier (BBB) and with minimal or no adverse effects may provide a major therapeutic asset.