Axon regrowth after spinal cord injury (SCI) is inhibited by several types of inhibitory extracellular molecules in the central nervous system (CNS), including chondroitin sulfate proteoglycans (CSPGs), which also are components of perineuronal nets (PNNs). The axons of lampreys regenerate following SCI, even though their spinal cords contain CSPGs, and their neurons are enwrapped by PNNs. Previously, we showed that by 2 weeks after spinal cord transection in the lamprey, expression of CSPGs increased in the lesion site, and thereafter, decreased to pre-injury levels by 10 weeks. Enzymatic digestion of CSPGs in the lesion site with chondroitinase ABC (ChABC) enhanced axonal regeneration after SCI and reduced retrograde neuronal death. Lecticans (aggrecan, versican, neurocan, and brevican) are the major CSPG family in the CNS. Previously, we cloned a cDNA fragment that lies in the most conserved link-domain of the lamprey lecticans and found that lectican mRNAs are expressed widely in lamprey glia and neurons. Because of the lack of strict one-to-one orthology with the jawed vertebrate lecticans, the four lamprey lecticans were named simply A, B, C, and D. Using probes that distinguish these four lecticans, we now show that they all are expressed in glia and neurons but at different levels. Expression levels are relatively high in embryonic and early larval stages, gradually decrease, and are upregulated again in adults. Reductions of lecticans B and D are greater than those of A and C. Levels of mRNAs for lecticans B and D increased dramatically after SCI. Lectican D remained upregulated for at least 10 weeks. Multiple cells, including glia, neurons, ependymal cells and microglia/macrophages, expressed lectican mRNAs in the peripheral zone and lesion center after SCI. Thus, as in mammals, lamprey lecticans may be involved in axon guidance and neuroplasticity early in development. Moreover, neurons, glia, ependymal cells, and microglia/macrophages, are responsible for the increase in CSPGs during the formation of the glial scar after SCI.
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Myelin Debris Stimulates NG2/CSPG4 Expression in Bone Marrow-Derived Macrophages in the Injured Spinal Cord
Although the increased expression of members of the chondroitin sulfate proteoglycan family, such as neuron-glial antigen 2 (NG2), have been well documented after an injury to the spinal cord, a complete picture as to the cellular origins and function of this NG2 expression has yet to be made. Using a spinal cord injury (SCI) mouse model, we describe that some infiltrated bone marrow-derived macrophages (BMDMΦ) are early contributors to NG2/CSPG4 expression and secretion after SCI. We demonstrate for the first time that a lesion-related form of cellular debris generated from damaged myelin sheaths can increase NG2/CSPG4 expression in BMDMΦ, which then exhibit enhanced proliferation and decreased phagocytic capacity. These results suggest that BMDMΦ may play a much more nuanced role in secondary spinal cord injury than previously thought, including acting as early contributors to the NG2 component of the glial scar.
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- Award ID(s):
- 1661727
- NSF-PAR ID:
- 10263900
- Date Published:
- Journal Name:
- Frontiers in Cellular Neuroscience
- Volume:
- 15
- ISSN:
- 1662-5102
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract This study investigates the response to spinal cord injury in the gray short‐tailed opossum (
Monodelphis domestica ). In opossums spinal injury early in development results in spontaneous axon growth through the injury, but this regenerative potential diminishes with maturity until it is lost entirely. The mechanisms underlying this regeneration remain unknown. RNA sequencing was used to identify differential gene expression in regenerating (SCI at postnatal Day 7, P7SCI) and nonregenerating (SCI at Day 28, P28SCI) cords +1d, +3d, and +7d after complete spinal transection, compared to age‐matched controls. Genes showing significant differential expression (log2FC ≥ 1,Padj ≤ 0.05) were used for downstream analysis. Across all time‐points 233 genes altered expression after P7SCI, and 472 genes altered expression after P28SCI. One hundred and forty‐seven genes altered expression in both injury ages (63% of P7SCI data set). The majority of changes were gene upregulations. Gene ontology overrepresentation analysis in P7SCI gene‐sets showed significant overrepresentations only in immune‐associated categories, while P28SCI gene‐sets showed overrepresentations in these same immune categories, along with other categories such as “cell proliferation,” “cell adhesion,” and “apoptosis.” Cell‐type–association analysis suggested that, regardless of injury age, injury‐associated gene transcripts were most strongly associated with microglia and endothelial cells, with strikingly fewer astrocyte, oligodendrocyte and neuron‐related genes, the notable exception being a cluster of mostly downregulated oligodendrocyte‐associated genes in the P7SCI + 7d gene‐set. Our findings demonstrate a more complex transcriptomic response in nonregenerating cords, suggesting a strong influence of non‐neuronal cells in the outcome after injury and providing the largest survey yet of the transcriptomic changes occurring after SCI in this model. -
The ability to manipulate specific neuronal populations of the spinal cord following spinal cord injury (SCI) could prove highly beneficial for rehabilitation in patients through maintaining and strengthening still existing neuronal connections and/or facilitating the formation of new connections. A non-invasive and highly specific approach to neuronal stimulation is bioluminescent-optogenetics (BL-OG), where genetically expressed light emitting luciferases are tethered to light sensitive channelrhodopsins (luminopsins, LMO); neurons are activated by the addition of the luciferase substrate coelenterazine (CTZ). This approach utilizes ion channels for current conduction while activating the channels through the application of a small chemical compound, thus allowing non-invasive stimulation and recruitment of all targeted neurons. Rats were transduced in the lumbar spinal cord with AAV2/9 to express the excitatory LMO3 under control of a pan-neuronal or motor neuron-specific promoter. A day after contusion injury of the thoracic spine, rats received either CTZ or vehicle every other day for 2 weeks. Activation of either neuron population below the level of injury significantly improved locomotor recovery lasting beyond the treatment window. Utilizing histological and gene expression methods we identified neuronal plasticity as a likely mechanism underlying the functional recovery. These findings provide a foundation for a rational approach to spinal cord injury rehabilitation, thereby advancing approaches for functional recovery after SCI. Summary Bioluminescent optogenetic activation of spinal neurons results in accelerated and enhanced locomotor recovery after spinal cord injury in rats.more » « less
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Abstract Multiple therapies have been studied to ameliorate the neuroinhibitory cues present after traumatic injury to the central nervous system. Two previous in vitro studies have demonstrated the efficacy of the Food and Drug Administration‐approved cardiovascular therapeutic, protamine (PRM), to overcome neuroinhibitory cues presented by chondroitin sulfates; however, the effect of a wide range of PRM concentrations on neuronal and glial cells has not been evaluated. In this study, we investigate the therapeutic efficacy of PRM with primary cortical neurons, hippocampal neurons, mixed glial cultures, and astrocytes. The threshold for PRM toxicity is shown to be at or above 10 µg mL−1depending on the cell population, that 10 µg mL−1PRM enables neurons to overcome the inhibitory cues presented by chondroitin sulfate type A, and that soluble PRM allows neurons to more effectively overcome inhibition compared to a PRM coating. Changes in gene expression of reactive astrocytes is also assessed with soluble PRM and it is determined that PRM does not increase their neurotoxic phenotype and that PRM may reduce brevican and serpin transcription in cortical and spinal cord astrocytes. This is the first study to thoroughly assess the toxicity threshold of PRM with neural cells and study astrocyte response after acute exposure to PRM in vitro.
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Irrespective of the many strategies focused on dealing with spinal cord injury (SCI), there is still no way to restore motor function efficiently or an adequate regenerative therapy. One promising method that could potentially prove highly beneficial for rehabilitation in patients is to re-engage specific neuronal populations of the spinal cord following SCI. Targeted activation may maintain and strengthen existing neuronal connections and/or facilitate the reorganization and development of new connections. BioLuminescent-OptoGenetics (BL-OG) presents an avenue to non-invasively and specifically stimulate neurons; genetically targeted neurons express luminopsins (LMOs), light-emitting luciferases tethered to light-sensitive channelrhodopsins that are activated by adding the luciferase substrate coelenterazine (CTZ). This approach employs ion channels for current conduction while activating the channels through treatment with the small molecule CTZ, thus allowing non-invasive stimulation of all targeted neurons. We previously showed the efficacy of this approach for improving locomotor recovery following severe spinal cord contusion injury in rats expressing the excitatory luminopsin 3 (LMO3) under control of a pan-neuronal and motor-neuron-specific promoter with CTZ applied through a lateral ventricle cannula. The goal of the present study was to test a new generation of LMOs based on opsins with higher light sensitivity which will allow for peripheral delivery of the CTZ. In this construct, the slow-burn Gaussia luciferase variant (sbGLuc) is fused to the opsin CheRiff, creating LMO3.2. Taking advantage of the high light sensitivity of this opsin, we stimulated transduced lumbar neurons after thoracic SCI by intraperitoneal application of CTZ, allowing for a less invasive treatment. The efficacy of this non-invasive BioLuminescent-OptoGenetic approach was confirmed by improved locomotor function. This study demonstrates that peripheral delivery of the luciferin CTZ can be used to activate LMOs expressed in spinal cord neurons that employ an opsin with increased light sensitivity.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fncel.2021.651827
