Nuc-ErbB3 regulates H3K27me3 levels and HMT activity to establish epigenetic repression during peripheral myelination.

Nuc-ErbB3 an alternative transcript from the ErbB3 locus binds to a specific DNA motif and associates with Schwann cell chromatin. Here we generated a nuc-ErbB3 knockin mouse that lacks nuc-ErbB3 expression in the nucleus without affecting the neuregulin-ErbB3 receptor signaling. Nuc-ErbB3 knockin mice exhibit hypermyelination and aberrant myelination at the paranodal region. This phenotype is attributed to de-repression of myelination associated gene transcription following loss of nuc-ErbB3 and histone H3K27me3 promoter occupancy. Nuc-ErbB3 knockin mice exhibit reduced association of H3K27me3 with myelination-associated gene promoters and increased RNA Pol-II rate of transcription of these genes. In addition, nuc-ErbB3 directly regulates levels of H3K27me3 in Schwann cells. Nuc-ErbB3 knockin mice exhibit significant decrease of histone H3K27me3 methyltransferase (HMT) activity and reduced levels of H3K27me3. Collectively, nuc-ErbB3 is a master transcriptional repressor, which regulates HMT activity to establish a repressive chromatin landscape on promoters of genes during peripheral myelination.

Regulation of Peripheral Myelination through Transcriptional Buffering of Egr2 by an Antisense Long Non-coding RNA.

Precise regulation of Egr2 transcription is fundamentally important to the control of peripheral myelination. Here, we describe a long non-coding RNA antisense to the promoter of Egr2 (Egr2-AS-RNA). During peripheral nerve injury, the expression of Egr2-AS-RNA is increased and correlates with decreased Egr2 transcript and protein levels. Ectopic expression of Egr2-AS-RNA in dorsal root ganglion (DRG) cultures inhibits the expression of Egr2 mRNA and induces demyelination. In vivo inhibition of Egr2-AS-RNA using oligonucleotide GapMers released from a biodegradable hydrogel following sciatic nerve injury reverts the EGR2-mediated gene expression profile and significantly delays demyelination. Egr2-AS-RNA gradually recruits H3K27ME3, AGO1, AGO2, and EZH2 on the Egr2 promoter following sciatic nerve injury. Furthermore, expression of Egr2-AS-RNA is regulated through ERK1/2 signaling to YY1, while loss of Ser184 of YY1 regulates binding to Egr2-AS-RNA. In conclusion, we describe functional exploration of an antisense long non-coding RNA in peripheral nervous system (PNS) biology.

Sustained Local Release of Methylprednisolone From a Thiol-Acrylate Poly(Ethylene Glycol) Hydrogel for Treating Chronic Compressive Radicular Pain.

STUDY DESIGN: A preclinical animal model of chronic ligation of the sciatic nerve was used to compare the effectiveness of a slow-release hydrogel carrying methylprednisolone to methylprednisolone injection alone, which simulates the current standard of care for chronic compressive radiculopathy (CR). OBJECTIVE: To extend the short-term benefits of steroid injections by using a nonswelling, biodegradable hydrogel as carrier to locally release methylprednisolone in a regulated and sustained way at the site of nerve compression. SUMMARY OF BACKGROUND DATA: CR affects millions worldwide annually, and is a cause of costly disability with significant societal impact. Currently, a leading nonsurgical therapy involves epidural injection of steroids to temporarily alleviate the pain associated with CR. However, an effective way to extend the short-term effect of steroid treatment to address the chronic component of CR does not exist. METHODS: We induced chronic compression injury of the sciatic nerves of rats by permanent ligation. Forty-eight hours later we injected our methylprednisolone infused hydrogel and assessed the effectiveness of our treatment for 4 weeks. We quantified mechanical hyperalgesia using a Dynamic Plantar Aesthesiometer (Ugo Basile, Stoelting Co., IL, USA), whereas gait analysis was conducted using the Catwalk automated gait analysis platform (Noldus, Leesburg, VA, USA). Macrophage staining was performed with immunohistochemistry and quantification of monocyte chemoattractant protein-1 in sciatic nerve lysates was performed with multiplex immunoassay using a SECTOR Imager 2400A (Meso Scale Discovery, Rockville, MA, USA). RESULTS: We demonstrate that using the hydrogel to deliver methylprednisolone results in significant (P < 0.05) reduction of hyperalgesia and improvement in the gait pattern of animals with chronic lesions as compared with animals treated with steroid alone. In addition, animals treated with hydrogel plus steroid showed significant reduction in the number of infiltrating macrophages at the sciatic nerve and reduced expression of the neuroinflammatory chemokine monocyte chemoattractant protein-1 (P < 0.05). CONCLUSION: Use of hydrogels as carriers for sustained local release of steroids provides significantly better control of pain in an animal model of chronic CR. Our steroid-infused hydrogel could be an effective extender of the short-term benefits of epidural steroid injections for patients with chronic compression-induced radicular pain. LEVEL OF EVIDENCE: N/A.

Lck tyrosine kinase mediates ß1-integrin signalling to regulate Schwann cell migration and myelination.

The interaction between laminin and ß1-integrin on the surface of Schwann cells regulates Schwann cell proliferation, maturation and differentiation. However, the signalling mediators that fine-tune these outcomes are not fully elucidated. Here we show that lymphoid cell kinase is the crucial effector of ß1-integrin signalling in Schwann cells. Lymphoid cell kinase is activated after laminin treatment of Schwann cells, while downregulation of ß1-integrin with short interfering RNAs inhibits lymphoid cell kinase phosphorylation. Treatment of Schwann cells with a selective lymphoid cell kinase inhibitor reveals a pathway that involves paxillin and CrkII, which ultimately elevates Rac-GTP levels to induce radial lamellipodia formation. Inhibition of lymphoid cell kinase in Schwann cell-dorsal root ganglion cocultures and dorsal root ganglions from Lck(-/-) mice show a reduction of Schwann cell longitudinal migration, reduced myelin formation and internode length. Finally, Lck(-/-) mice exhibit delays in myelination, thinner myelin with abnormal g-ratios and aberrant myelin outfoldings. Our data implicate lymphoid cell kinase as a major regulator of cytoskeletal dynamics, migration and myelination in the peripheral nervous system.

Identification of oligodendrocytes in experimental disease models.

The ability to identify oligodendrocytes in culture, in fixed tissue, and in vivo using unique markers is a requisite step to understanding their responses in any damage, recovery, or developmental process. Their nuclei are readily seen in histological preparations of healthy white and gray matter, and their cell bodies can be reliably identified with a variety of immunocytochemical markers. However, there is little consensus regarding optimal methods to assess oligodendrocyte survival or morphology under experimental injury conditions. We review common approaches for histological and immunocytochemical identification of these cells. Transgenic and viral methods for cell type-selective transfer of genes encoding fluorescent proteins offer promising new approaches for manipulating and visualizing oligodendrocytes in models of health and disease.

Insulin-like growth factor I, but not neurotrophin-3, sustains Akt activation and provides long-term protection of immature oligodendrocytes from glutamate-mediated apoptosis.

Glutamate toxicity is a major contributor to death of oligodendroglia in diverse CNS disorders. The goal of these studies was to investigate the mechanisms of glutamate toxicity and trophic factor protection of the immature pro-oligodendroblast (pro-OL). Glutamate induced time- and dose-dependent DNA fragmentation and caspase-3 activation in pro-OLs. IGF-I or NT-3, but not CNTF, prevented apoptosis of pro-OLs by 24 h via a PI3-kinase-dependent pathway; however, only IGF-I protected pro-OLs from glutamate toxicity through 48 h. Long-term protection of pro-OLs by IGF-I was correlated with sustained activation of Akt while NT-3 activation of Akt was transient. The differential ability of IGF-I and NT-3 to maintain Akt activation was due to differences in receptor activation and stability. In the presence of NT-3, TrkC phosphorylation and protein expression decreased significantly while activation of the IGF-IR was maintained in the pro-OLs in the presence of IGF-I.

IGF-I prevents glutamate-mediated bax translocation and cytochrome C release in O4+ oligodendrocyte progenitors.

Oligodendroglial death due to overactivation of the AMPA/kainate glutamate receptors is implicated in white matter damage in multiple CNS disorders. We previously demonstrated that glutamate induces caspase-3 activation and death of the late oligodendrocyte progenitor known as the pro-oligodendroblast (pro-OL) via activation of the AMPA/kainate glutamate receptors. We also demonstrated that IGF-I had the unique ability to sustain activation of Akt in the pro-OL and provide long-term protection of these cells from glutamate-mediated apoptosis. The goal of these studies was to investigate the mechanisms of glutamate toxicity and IGF-I-mediated survival in the pro-OL. IGF-I prevented glutamate-induced loss of mitochondrial membrane potential, cytochrome c release, and caspase-9 activation. In contrast to IGF-I mediated survival mechanisms in neurons, IGF-I had no effect on the influx or recovery of intracellular calcium levels or on levels of major pro- and anti-apoptotic molecules including Bax or Bcl-xL. Rather, IGF-I prevented the glutamate-induced translocation of Bax to the mitochondria. Moreover, IGF-I prevented caspase-3 activation in pro-OLs as long as 8 h after exposure of the cells to glutamate, suggesting that delayed activation of IGF-I-mediated survival pathways can block glutamate-mediated apoptosis in pro-OLs. The results of these experiments define the mechanisms by which glutamate kills oligodendrocyte progenitor cells and by which IGF-I blocks glutamate-induced apoptosis in these cells. The data also demonstrate that IGF-I disrupts the glutamate-mediated apoptotic pathway in the pro-OL through mechanisms that are distinct from its survival-promoting actions in neurons.

IGF-I and NT-3 signaling pathways in developing oligodendrocytes: differential regulation and activation of receptors and the downstream effector Akt.

A previous study from our laboratory demonstrated differences in the ability of insulin-like growth factor-I (IGF-I) and neurotrophin-3 (NT-3) to promote survival of pro-oligodendroblasts (pro-OLs) against glutamate-mediated apoptosis. In the current study, we tested whether submaximal concentrations of NT-3 would maintain receptor tyrosine kinase TrkC activation and Akt phosphorylation and thus promote long-term survival of the pro-OL against glutamate. Our results demonstrate that NT-3 at any concentration sufficient to activate the TrkC receptor results in a transient phosphorylation of the receptor and of Akt due, in part, to downregulation of the Trk receptor. In contrast, even submaximal IGF-I concentrations maintain long-term Akt activation and prevent glutamate-mediated apoptosis in pro-OLs. In addition, we also present data showing that IGF-I and NT-3 differentially activate their receptors and Akt depending on the maturational stage of the oligodendrocyte.

Astrocytes produce CNTF during the remyelination phase of viral-induced spinal cord demyelination to stimulate FGF-2 production.

Multiple sclerosis is characterized by multiple lesions with selective loss of myelin and oligodendrocytes, leading to deficits of sensation and movement, as well as cognitive disabilities. Consequently, a major research endeavor is to identify strategies to enhance oligodendrocyte regeneration and remyelination. FGF-2 is a potent mitogen for OPCs, and it is induced in astrocytes in animal models of demyelinating diseases in conjunction with successful remyelination. However, the factors responsible for inducing FGF-2 after demyelination in astrocytes are unknown. Here we show that CNTF mRNA and protein increase coincident with spinal cord remyelination in mice recovering from MHV-induced demyelination. We identify CNTF within astrocytes surrounding and within remyelinating lesions, and show that CNTF increases FGF-2 ligand and receptor mRNAs in spinal cord after direct application. Furthermore, we show that CNTF increases FGF-2 mRNA approximately 2.5-fold in cultured mouse spinal cord astrocytes. Altogether, these results strongly implicate CNTF as an important cytokine in demyelinating disease and as an upstream regulator of FGF-2 production in astrocytes during early remyelination.