Dorsally-derived oligodendrocytes in the spinal cord contribute to axonal myelination during development and remyelination following focal demyelination

In the developing spinal cord, the majority of oligodendrocytes are derived from the ventral ventricular zone. Several recent studies suggested that a small number of oligodendrocyte precursor cells (OPCs) can also be generated in the dorsal spinal cord. However, it is not clear whether these dorsal oligodendrocyte precursor cells participate in myelination and remyelination. To investigate the fate and potential function of these dorsally-derived oligodendrocytes (dOLs) in the adult spinal cord, Cre-lox genetic fate mapping in transgenic mice was employed. We used the Pax3(Cre) knock-in mouse to drive Cre expression in the entire dorsal epithelium and the Rosa26-lacZ or Z/EG reporter line to trace their spatial distribution and population dynamics in the spinal cord. The dorsal OPCs generated from the Pax3-expressing domains migrate into all regions of spinal cord and subsequently undergo terminal differentiation and axonal myelination. In response to a focal demyelination injury, a large number of newly differentiated oligodendrocytes originated from dOLs, suggesting that dOLs may provide an important source of OPCs for axonal remyelination in multiple sclerosis or spinal cord injury.     

Platelet-derived growth factor-responsive neural precursors give rise to myelinating oligodendrocytes after transplantation into the spinal cords of contused rats and dysmyelinated mice

Spinal cord injury (SCI) results in substantial oligodendrocyte death and subsequent demyelination leading to white-matter defects. Cell replacement strategies to promote remyelination are under intense investigation; however, the optimal cell for transplantation remains to be determined. We previously isolated a platelet-derived growth factor (PDGF)-responsive neural precursor (PRP) from the ventral forebrain of fetal mice that primarily generates oligodendrocytes, but also astrocytes and neurons. Importantly, human PRPs were found to possess a greater capacity for oligodendrogenesis than human epidermal growth factor- and/or fibroblast growth factor-responsive neural stem cells. Therefore, we tested the potential of PRPs isolated from green fluorescent protein (GFP)-expressing transgenic mice to remyelinate axons in the injured rat spinal cord. PRPs were transplanted 1 week after a moderate thoracic (T9) spinal cord contusion in adult male rats. After initial losses, PRP numbers remained stable from 2 weeks posttransplantation onward and those surviving cells integrated into host tissue. Approximately one-third of the surviving cells developed the typical branched phenotype of mature oligodendrocytes, expressing the marker APC-CC1. The close association of GFP cells with myelin basic protein as well as with Kv1.2 and Caspr in the paranodal and juxtaparanodal regions of nodes of Ranvier indicated that the transplanted cells successfully formed mature myelin sheaths. Transplantation of PRPs into dysmyelinated Shiverer mice confirmed the ability of PRP-derived cells to produce compact myelin sheaths with normal periodicity. These findings indicate that PRPs are a novel candidate for CNS myelin repair, although PRP-derived myelinating oligodendrocytes were insufficient to produce behavioral improvements in our model of SCI.     

Identification of major S‐nitrosylated proteins in murine experimental autoimmune encephalomyelitis

Nitrosative stress has been implicated in the pathophysiology of several CNS disorders, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have recently shown that protein nitrosothiols (PrSNOs) accumulate in the brain of MS patients, and there is indirect evidence that PrSNO levels are also increased in EAE. In this study we sought to identify the major PrSNOs in the spinal cord of EAE animals prepared by active immunization of C57/BL6 mice with MOG_35–55 peptide. For this purpose, PrSNOs from control and EAE mice at various disease stages were derivatized with HPDP‐biotin, and the biotinylated proteins were isolated with streptavidin‐agarose. Proteins from total and streptavidin‐bound fractions were then analyzed by Western blotting using antibodies against the major S‐nitrosylated substrates of CNS tissue. With this approach we found that the proportion of S‐nitrosylated neurofilament proteins, NMDA receptors, α/β‐tubulin, β‐actin, and GAPDH is increased in EAE. Other potential substrates either were not S‐nitrosylated in vivo (HCN3, HSP‐72, CRMP‐2, γ‐actin, calbindin) or their S‐nitrosylation levels were unaltered in EAE (Na/K ATPase, hexokinase, glycogen phosphorylase). We also discovered that neuronal specific enolase is the major S‐nitrosylated protein in acute EAE. Given that S‐nitrosylation affects protein function, it is likely that the observed changes are significant to the pathophysiology of inflammatory demyelination. © 2009 Wiley‐Liss, Inc.     

Intramuscular AAV delivery of NT‐3 alters synaptic transmission to motoneurons in adult rats

We examined whether elevating levels of neurotrophin‐3 (NT‐3) in the spinal cord and dorsal root ganglion (DRG) would alter connections made by muscle spindle afferent fibers on motoneurons. Adeno‐associated virus (AAV) serotypes AAV1, AAV2 and AAV5, selected for their tropism profile, were engineered with the NT‐3 gene and administered to the medial gastrocnemius muscle in adult rats. ELISA studies in muscle, DRG and spinal cord revealed that NT‐3 concentration in all tissues peaked about 3 months after a single viral injection; after 6 months NT‐3 concentration returned to normal values. Intracellular recording in triceps surae motoneurons revealed complex electrophysiological changes. Moderate elevation in cord NT‐3 resulted in diminished segmental excitatory postsynaptic potential (EPSP) amplitude, perhaps as a result of the observed decrease in motoneuron input resistance. With further elevation in NT‐3 expression, the decline in EPSP amplitude was reversed, indicating that NT‐3 at higher concentration could increase EPSP amplitude. No correlation was observed between EPSP amplitude and NT‐3 concentration in the DRG. Treatment with control viruses could elevate NT‐3 levels minimally resulting in measurable electrophysiological effects, perhaps as a result of inflammation associated with injection. EPSPs elicited by stimulation of the ventrolateral funiculus underwent a consistent decline in amplitude independent of NT‐3 level. These novel correlations between modified NT‐3 expression and single‐cell electrophysiological parameters indicate that intramuscular administration of AAV(NT‐3) can exert long‐lasting effects on synaptic transmission to motoneurons. This approach to neurotrophin delivery could be useful in modifying spinal function after injury.     

Expression and localization of insulin receptor in rat dorsal root ganglion and spinal cord

The expression and localization of the insulin receptor (IR) was examined in rat dorsal root ganglia (DRG) and spinal cord using Western blotting, in situ hybridization and immunocytochemistry. Western blotting showed that the molecular weight of the IR beta subunit was higher in PNS than that found in CNS. Both IR mRNA and protein expressions were highest in small-sized sensory DRG neurons and myelinated sensory root fibers expressed higher levels of IR protein than myelinated anterior root fibers. In the spinal cord, IR immunoreactive neurons were present in lateral lamina V and in lamina X, suggesting the presence of IR in nociceptive pathways. Electronmicroscopy of DRGs revealed a polarized localization of the IR in abaxonal Schwann cell membranes, outer mesaxons in close vicinity to tight junctions of both myelinating and non-myelinating Schwann cells and to plasma membranes of sensory neurons. From these findings, we speculate that insulin may play a role in sensory fibers involved in nociceptive function often perturbed in diabetic neuropathy. The high expression of IR localizing to tight junctions of dorsal root mesaxons of DRGs may suggest a regulatory role on barrier functions compensating for the lack of a blood-nerve barrier in dorsal root ganglia. This is consistent with the colocalization of IR with tight junctions of the paranodal barrier and endoneurial endothelial cells in peripheral nerve.     

Concurrent isolation and characterization of oligodendrocytes, microglia and astrocytes from adult human spinal cord

A cellular preparation of highly enriched oligodendrocytes was obtained from adult human spinal cord by Percoll gradient centrifugation followed by either differential adhesion or fluorescence-activated cell sorting after immunostaining with an antibody against galactocerebroside (Ol). The adherent and O1-negative cell fractions were 96% microglia. The non-adherent and O1-positive fractions were 96% positive for the oligodendrocyte markers O4 and O1, 0–2% positive for glial fibrillary acidic protein, and were devoid of neuronal or microglial markers. If the oligodendrocyte fraction was co-cultured with purified dissociated rat dorsal root ganglion neurons, the oligodendrocytes adhered to the axons and their numbers increased over a 4 week period. However, myelin sheaths were not produced around axons in these cultures. In contrast, if the oligodendrocyte cell fraction was grown alone in culture for 3 weeks, the number of oligodendrocytes decreased and a layer of astrocytes developed underneath the oligodendrocytes. The oligodendrocytes could be eliminated from these cultures by subsequent passaging, thus producing cultures of pure astrocytes. The astrocytes accumulated both K⁺ and glutamate with kinetic properties similar to those reported for rodent astrocytes. We suggest that these astrocytes arose in part from an O4/O1-positive precursor which did not initially express glial fibrillary acidic protein. These results define a relatively simple method by which highly enriched populations of oligodendrocytes, astrocytes and microglia can be obtained from adult human spinal cord.     

Expression of QKI proteins and MAP1B identifies actively myelinating oligodendrocytes in adult rat brain

We have studied developing oligodendrocytes in tissue sections as they initiate myelination and have found that the transition from premyelinating oligodendrocytes into myelin-bearing cells is accompanied by a dramatic upregulation in expression of the RNA binding QKI proteins. We show that in mature oligodendrocytes in culture, the localization of cytoplasmic QKI isoforms requires an intact cytoskeleton. Together with previous observations, this indicates that cytoplasmic QKI proteins facilitate movement of mRNAs to myelin via the cytoskeleton. In the adult rat brain, we found that a subset of oligodendrocytes displays characteristics of actively myelinating cells seen during development, i.e., connections to myelin sheaths and elevated levels of QKI proteins and also MAP1B. These observations suggest that instead of merely maintaining myelin, oligodendrocytes in the normal adult CNS are capable of responding to demands for new myelin sheaths. This has important implications for the prospect of repair of myelin in demyelinating conditions such as multiple sclerosis.     

Differential expression of neuropsin and protease M/neurosin in oligodendrocytes after injury to the spinal cord

Neuropsin and protease M/neurosin are serine proteases expressed by neurons and glial cells, and serve a variety of functions in the central nervous system (CNS). The current study demonstrates changes in the expression of these proteases following hemisection of the mouse spinal cord. Within unlesioned spinal cord, neuropsin mRNA expression was occasionally observed in the gray but not white matter, while the level of protease M/neurosin mRNA was higher in the white matter. After injury to the spinal cord, neuropsin mRNA expression was induced in the white matter in the area immediately adjacent to the lesion, peaking at 4 days post-injury and disappearing by 14 days. Enhanced expression of protease M/neurosin mRNA was observed throughout the white and gray matter surrounding the lesion, peaking at 4 days and persisting for 14 days. Neuropsin mRNA was expressed predominantly by CNPase-positive oligodendrocytes. Furthermore, most of these cells were also associated with immunoreactivity for protease M/neurosin protein. Within unlesioned spinal cord, most protease M/neurosin mRNA-expressing cells were CNPase-positive oligodendrocytes, and a substantial fraction of these cells also showed immunoreactivity for NG2, a marker for oligodendrocyte progenitors. After injury, protease M/neurosin mRNA expression within NG2-positive cells was significantly decreased, while the constitutive expression in CNPase-positive oligodendrocytes appeared to be preserved. These findings suggest that each subpopulation of oligodendrocytes based on the expression of neuropsin and protease M/neurosin has different roles in the response of the spinal cord to injury as well as in normal homeostasis.     

Neuropathology in classical and variant ataxia‐telangiectasia

Ataxia‐telangiectasia (A‐T) is classically characterized by progressive neurodegeneration, oculocutaneous telangiectasia, immunodeficiency and elevated α‐fetoprotein levels. Some patients, classified as variant A‐T, exhibit a milder clinical course. In the latter patients extrapyramidal symptoms, instead of cerebellar ataxia, tend to be the dominating feature and other classical disease hallmarks, like telangiectasia, appear later or even may be absent. Some patients with variant disease have clinically pronounced anterior horn cell degeneration. Neuropathological studies of genetically proven A‐T patients are lacking. The aims of our study were to describe the neuropathology of three A‐T patients; in two of them the diagnosis was genetically confirmed. The neuropathological findings were compared with those of all known published autopsy findings in A‐T patients up to now. Two classical A‐T patients aged 19 and 22 and a 33‐year‐old patient with variant disease were autopsied. In line with previous reports, our patients had severe cerebellar atrophy, less pronounced degeneration of the dentate nucleus and inferior olive, degeneration of the posterior columns and neurogenic muscular atrophy. In addition, all three had anterior horn cell degeneration, which was most prominent at the lumbar level. Compared to the literature, the degenerative changes in the brain stem of the variant A‐T patient were somewhat less than anticipated for his age. Degenerative changes in the cerebellum and spinal cord were comparable with those in the literature. Progeric changes were lacking. In conclusion, compared to classical A‐T, the variant A‐T patient showed essentially the same, only slightly milder neuropathological abnormalities, except for anterior horn degeneration.     

Immunohistochemical localization of μ-opioid receptors in the central nervous system of the rat

Of the three major types of opioid receptors (μ, δ κ) in the nervous system, μ-opioid receptor shows the highest affinity for morphine that exerts powerful effects on nociceptive, autonomic, and psychological functions. So far, at least two isoforms of μ-opioid receptors have been cloned from rat brain. The present study attempted to examine immunohistochemically the distribution of μ-opioid receptors in the rat central nervous system with two kinds of antibodies to recently cloned μ-opioid receptors (MOR1 and MOR1B). One antibody recognized a specific site for MOR1, and the other bound to a common site for MOR1 and MOR1B. Intense MOR1-like immunoreactivity (LI) was seen in the ‘patch’ areas and subcallosal streak in the striatum, medial habenular nucleus, medial terminal nucleus of the accessory optic tract, interpeduncular nucleus, median raphe nucleus, parabrachial nuclei, locus coeruleus, ambiguus nucleus, nucleus of the solitary tract, and laminae I and II of the medullary and spinal dorsal horns. Many other regions, including the cerebral cortex, amygdala, thalamus, and hypothalamus, also contained many neuronal elements with MOR1-LI. The distribution pattern of the immunoreactivity revealed with the antibody to the common site for MOR1 and MOR1B (MOR1/1B-LI) was almost the same as that of MOR1-L1. Both MOR1-LI and MOR1/1B-LI were primarily located in neuronal cell bodies and dendrites. However, the immunoreactivities were observed in the accessory optic tract, fasciculus retroflexus, solitary tract, and primary afferent fibers in the superficial layers of the medullary and spinal dorsal horns. The presynaptic location of MOR1-LI and MOR1/1B-LI was confirmed by lesion experiments: Enucleation, placing a lesion in the medial habenular nucleus, removal of the nodose ganglion, or dorsal rhizotomy resulted in a clear reduction of the immunoreactivities, respectively, in the nuclei of the accessory optic tract, some subnuclei of the interpeduncular nucleus, nucleus of the solitary tract, or laminae I and II of the spinal dorsal horn. The results indicate that the μ-opioid receptors are widely distributed in the brain and spinal cord, mainly postsynaptically and occasionally presynaptically. Opioids, including morphine, may inhibit the excitation of neurons via the postsynaptic μ-opioid receptors, and also suppress the release of neurotransmitters and/or neuromodulators from axon terminals through the presynaptic μ-opioid receptors. © 1996 Wiley-Liss, Inc.     

Overexpression of Bcl‐XL in human neural stem cells promotes graft survival and functional recovery following transplantation in spinal cord injury

Transplantation of neural stem cells (NSCs) has shown promise for improving functional recovery after spinal cord injury (SCI). The inhospitable milieu of injured spinal cord, however, does not support survival of grafted NSCs, reducing therapeutic efficacy of transplantation. The present study sought to examine whether overexpression of antiapoptotic gene Bcl‐X_L in NSCs could promote graft survival and functional recovery following transplantation in rat contusive SCI model. A human NSC line (HB1.F3) was transduced with a retroviral vector encoding Bcl‐X_L to generate Bcl‐X_L‐overexpressing NSCs (HB1.F3.Bcl‐X_L). Overexpression of Bcl‐X_L conferred resistance to staurosporine‐mediated apoptosis. The number of HB1.F3.Bcl‐X_L cells was 1.5‐fold higher at 2 weeks and 10‐fold higher at 7 weeks posttransplantation than that of HB1.F3 cells. There was no decline in the number of HB1.F3.Bcl‐X_L cells between 2 and 7 weeks, indicating that Bcl‐X_L overexpression completely blocked cell death occurring between these two time points. Transplantation of HB1.F3.Bcl‐X_L cells improved locomotor scores and enhanced accuracy of hindlimb placement in a grid walk. Approximately 10% of surviving NSCs differentiated into oligodendrocytes. Surviving NSCs produced brain‐derived neurotrophic factor (BDNF), and the level of BDNF was significantly increased only in the HB1.F3.Bcl‐X_L group. Transplantation of HB1.F3.Bcl‐X_L cells reduced cavity volumes and enhanced white matter sparing. Finally, HB1.F3.Bcl‐X_L grafts enhanced connectivity between the red nucleus and the spinal cord below the lesion. These results suggest that enhancing graft survival with antiapoptotic gene can potentiate therapeutic benefits of NSC‐based therapy for SCI. © 2009 Wiley‐Liss, Inc.