Protease activated receptor 2 controls myelin development,resiliency and repair

Oligodendrocytes are essential regulators of axonal energy homeostasis and electrical conduction and emerging target cells for restoration of neurological function. Here we investigate the role of protease activated receptor 2 (PAR2), a unique protease activated G protein‐coupled receptor, in myelin development and repair using the spinal cord as a model. Results demonstrate that genetic deletion of PAR2 accelerates myelin production, including higher proteolipid protein (PLP) levels in the spinal cord at birth and higher levels of myelin basic protein and thickened myelin sheaths in adulthood. Enhancements in spinal cord myelin with PAR2 loss‐of‐function were accompanied by increased numbers of Olig2‐ and CC1‐positive oligodendrocytes, as well as in levels of cyclic adenosine monophosphate (cAMP), and extracellular signal related kinase 1/2 (ERK1/2) signaling. Parallel promyelinating effects were observed after blocking PAR2 expression in purified oligodendrocyte cultures, whereas inhibiting adenylate cyclase reversed these effects. Conversely, PAR2 activation reduced PLP expression and this effect was prevented by brain derived neurotrophic factor (BDNF), a promyelinating growth factor that signals through cAMP. PAR2 knockout mice also showed improved myelin resiliency after traumatic spinal cord injury and an accelerated pattern of myelin regeneration after focal demyelination. These findings suggest that PAR2 is an important controller of myelin production and regeneration, both in the developing and adult spinal cord.     

Critical role for PAR1 in kallikrein 6‐mediated oligodendrogliopathy

Kallikrein 6 (KLK6) is a secreted serine protease preferentially expressed by oligodendroglia in CNS white matter. Elevated levels of KLK6 occur in actively demyelinating multiple sclerosis (MS) lesions and in cases of spinal cord injury (SCI), stroke, and glioblastoma. Taken with recent evidence establishing KLK6 as a CNS‐endogenous activator of protease‐activated receptors (PARs), we hypothesized that KLK6 activates a subset of PARs to regulate oligodendrocyte physiology and potentially pathophysiology. Here, primary oligodendrocyte cultures derived from wild type or PAR1‐deficient mice and the murine oligodendrocyte cell line, Oli‐neu, were used to demonstrate that Klk6 (rodent form) mediates loss of oligodendrocyte processes and impedes morphological differentiation of oligodendrocyte progenitor cells (OPCs) in a PAR1‐dependent fashion. Comparable gliopathy was also elicited by the canonical PAR1 agonist, thrombin, as well as PAR1‐activating peptides (PAR1‐APs). Klk6 also exacerbated ATP‐mediated oligodendrogliopathy in vitro, pointing to a potential role in augmenting excitotoxicity. In addition, Klk6 suppressed the expression of proteolipid protein (PLP) RNA in cultured oligodendrocytes by a mechanism involving PAR1‐mediated Erk1/2 signaling. Microinjection of PAR1 agonists, including Klk6 or PAR1‐APs, into the dorsal column white matter of PAR1^+/+ but not PAR1^?/? mice promoted vacuolating myelopathy and a loss of immunoreactivity for myelin basic protein (MBP) and CC‐1⁺ oligodendrocytes. These results demonstrate a functional role for Klk6‐PAR1 signaling in oligodendroglial pathophysiology and suggest that antagonists of PAR1 or its protease agonists may represent new modalities to moderate demyelination and to promote myelin regeneration in cases of CNS white matter injury or disease.     

PLP overexpression perturbs myelin protein composition and myelination in a mouse model of Pelizaeus-Merzbacher disease

Duplication of PLP1, an X‐linked gene encoding the major myelin membrane protein of the human CNS, is the most frequent cause of Pelizaeus‐Merzbacher disease (PMD). Transgenic mice with extra copies of the wild type Plp1 gene, a valid model of PMD, also develop a dysmyelinating phenotype dependant on gene dosage. In this study we have examined the effect of increasing Plp1 gene dosage on levels of PLP/DM20 and on other representative myelin proteins. In cultured oligodendrocytes and early myelinating oligodendrocytes in vivo, increased gene dosage leads to elevated levels of PLP/DM20 in the cell body. During myelination, small increases in Plp1 gene dosage (mice hemizygous for the transgene) elevate the level of PLP/DM20 in oligodendrocyte soma but cause only minimal and transient effects on the protein composition and structure of myelin suggesting that cells can regulate the incorporation of proteins into myelin. However, larger increases in dosage (mice homozygous for the transgene) are not well tolerated, leading to hypomyelination and alteration in the cellular distribution of PLP/DM20. A disproportionate amount of PLP/DM20 is retained in the cell soma, probably in autophagic vacuoles and lysosomes whereas the level in myelin is reduced. Increased Plp1 gene dosage affects other myelin proteins, particularly MBP, which is transitorily reduced in hemizygous mice but consistently and markedly lower in homozygotes in both myelin and naïve or early myelinating oligodendrocytes. Whether the reduced MBP is implicated in the pathogenesis of dysmyelination is yet to be established. © 2006 Wiley‐Liss, Inc.     

A DEAD‐box RNA helicase Ddx54 protein in oligodendrocytes is indispensable for myelination in the central nervous system

We recently reported that a new monoclonal antibody, 4F2, which labels oligodendroglial lineage cells, recognizes a DEAD‐box RNA helicase Ddx54 and that Ddx54 binds to myelin basic protein (MBP) in brain and cultured oligodendrocytes. To elucidate the biological function of Ddx54, we generated a recombinant adenovirus, Ad‐shRNA:Ddx54, expressing a short hairpin RNA to silence endogenous Ddx54 protein. The virus was intraventricularly injected into the brains of mice on postnatal day (PD) 2. The brains at PD 9 were then analyzed by immunohistochemistry. In untreated normal brain sections, as well as control brains that had been injected with Ad‐β‐Gal, myelination of axons occurred in the corpus callosum with filamentous patterns of immunosignals of myelin‐associated glycoprotein (MAG) and MBP. In Ad‐shRNA:Ddx54‐injected brain, substantial amounts of MAG and MBP immunosignals were present, but MBP immunosignals accumulated in the subplate layer and did not intrude into the emerging white matter. Immunoblot analysis revealed that Ddx54 knockdown caused a significant decrease in the level of 21.5 kDa MBP isoform and Ddx54, but the amount of Olig2; 2′,3′‐cyclic nucleotide 3′ phosphodiesterase; MAG; three MBP isoforms (14, 17.5, and 18 kDa); and QKI‐5, QKI‐6, and QKI‐7 proteins remained unchanged. Transfection of the Ddx54 expression vector into luciferase reporter‐introduced neuroepithelial cells resulted in upregulated MBP promoter activity. Immunoprecipitation of Ddx54 protein in MBP‐transfected HEK293 cells indicated that Ddx54 may directly interact with MBP mRNA. These results suggest that Ddx54 protein play an important role in central nervous system myelination, presumably in myelin sheath formation after the differentiation of oligodendrocytes. © 2012 Wiley Periodicals, Inc.     

IL‐1β induces hypomyelination in the periventricular white matter through inhibition of oligodendrocyte progenitor cell maturation via FYN/MEK/ERK signaling pathway in septic neonatal rats

Neuroinflammation elicited by microglia plays a key role in periventricular white matter (PWM) damage (PWMD) induced by infectious exposure. This study aimed to determine if microglia‐derived interleukin‐1β (IL‐1β) would induce hypomyelination through suppression of maturation of oligodendrocyte progenitor cells (OPCs) in the developing PWM. Sprague‐Dawley rats (1‐day old) were injected with lipopolysaccharide (LPS) (1 mg/kg) intraperitoneally, following which upregulated expression of IL‐1β and IL‐1 receptor 1 (IL‐1R₁) was observed. This was coupled with enhanced apoptosis and suppressed proliferation of OPCs in the PWM. The number of PDGFR‐α and NG2‐positive OPCs was significantly decreased in the PWM at 24 h and 3 days after injection of LPS, whereas it was increased at 14 days and 28 days. The protein expression of Olig1, Olig2, and Nkx2.2 was significantly reduced, and mRNA expression of Tcf4 and Axin2 was upregulated in the developing PWM after LPS injection. The expression of myelin basic protein (MBP) and 2',3'‐cyclic‐nucleotide 3"‐phosphodiesterase (CNPase) was downregulated in the PWM at 14 days and 28 days after LPS injection; this was linked to reduction of the proportion of myelinated axons and thinner myelin sheath as revealed by electron microscopy. Primary cultured OPCs treated with IL‐1β showed the failure of maturation and proliferation. Furthermore, FYN/MEK/ERK signaling pathway was involved in suppression of maturation of primary OPCs induced by IL‐1β administration. Our results suggest that following LPS injection, microglia are activated and produce IL‐1β in the PWM in the neonatal rats. Excess IL‐1β inhibits the maturation of OPCs via suppression of FYN/MEK/ERK phosphorylation thereby leading to axonal hypomyelination. GLIA 2016;64:583–602     

Rumpshaker: an X-linked mutation causing hypomyelination: developmental differences in myelination and glial cells between the optic nerve and spinal cord.

The X-linked mutation rumpshaker (rsh), which is probably an allele of jimpy (jp), causes hypomyelination in the CNS of mice. This study examines the developmental expression of the morphology, glial cells, and immunostaining of myelin proteins in the optic nerve and spinal cord. The optic nerve contains varying numbers of amyelinated and myelinated fibres. The majority of such sheaths are of normal thickness whereas in the spinal cord most axons are associated with a disproportionately thin sheath which changes little in thickness during development. In the optic nerve glial cell numbers are elevated in mutants during early and peak myelination but then fall slightly below normal in adults. In contrast, the number of glial cells is consistently elevated after 16 days of age in the spinal cord. The majority of the alterations to total glial cells are due to corresponding changes in the oligodendrocyte population. Immunostaining intensity is somewhat reduced for myelin basic protein (MBP) and the C-terminal common to proteolipid protein (PLP) and DM-20 and profoundly decreased for the PLP-specific peptide. Glial fibrillary acidic protein (GFAP) is increased in rsh. It is probable that some of the variation in myelination between optic nerve and cord in rsh is related to the difference in axon diameter in the two locations, as there are adequate numbers of oligodendrocytes at the time of myelination. However, the effect of the mutation on cell development in the brain and the spinal cord may be different. The immunostaining indicates a marked deficiency in PLP in myelin but suggests that DM-20 levels may be relatively normal. rsh shows several major differences from jp and other X-linked myelin mutants, particularly in relation to oligodendrocyte numbers, and will be useful to elucidate the role of the PLP gene in influencing oligodendrocyte differentiation and survival.     

A functional role of NMDA receptor in regulating the differentiation of oligodendrocyte precursor cells and remyelination

Differentiation of oligodendrocyte precursor cells (OPCs) is the most important event for the myelination of central nervous system (CNS) axons during development and remyelination in demyelinating diseases, while the underlying molecular mechanisms remain largely unknown. Here we show that NMDA receptor (NMDAR) is a functional regulator of OPCs differentiation and remyelination. First, GluN1, GluN2A, and GluN2B subunits are expressed in oligodendrocyte lineage cells (OLs) in vitro and in vivo by immunostaining and Western blot analysis. Second, in a purified rat OPC culture system, NMDARs specially mediate OPCs differentiation by enhancing myelin proteins expression and the processes branching at the immature to mature oligodendrocyte transition analyzed by a serial of developmental stage‐specific antigens. Moreover, pharmacological NMDAR antagonists or specific knockdown of GluN1 by RNA interference in OPCs prevents the differentiation induced by NMDA. NMDA can activate the mammalian target of rapamycin (mTOR) signal in OPCs and the pro‐differentiation effect of NMDA is obstructed by the mTOR inhibitor rapamycin, suggesting NMDAR exerts its effect through mTOR‐dependent mechanism. Furthermore, NMDA increases numbers of myelin segments in DRG‐OPC cocultures. Finally, NMDAR specific antagonist MK801 delays remyelination in the cuprizone model examined by LFB‐PAS, immunofluorescence and electron microscopy. This effect appears to result from inhibiting OPCs differentiation as more NG2⁺ OPCs but less GST‐π⁺ mature oligodendrocytes are observed. Together, these results indicate that NMDAR plays a critical role in the regulation of OPCs differentiation in vitro and remyelination in cuprizone model which may provide potential target for the treatment of demyelination disease.     

A selective thyroid hormone β receptor agonist enhances human and rodent oligodendrocyte differentiation

Nerve conduction within the mammalian central nervous system is made efficient by oligodendrocyte‐derived myelin. Historically, thyroid hormones have a well described role in regulating oligodendrocyte differentiation and myelination during development; however, it remains unclear which thyroid hormone receptors are required to drive these effects. This is a question with clinical relevance since nonspecific thyroid receptor stimulation can produce deleterious side‐effects. Here we report that GC‐1, a thyromimetic with selective thyroid receptor β action and a potentially limited side‐effect profile, promotes in vitro oligodendrogenesis from both rodent and human oligodendrocyte progenitor cells. In addition, we used in vivo genetic fate tracing of oligodendrocyte progenitor cells via PDGFαR‐CreER;Rosa26‐eYFP double‐transgenic mice to examine the effect of GC‐1 on cellular fate and find that treatment with GC‐1 during developmental myelination promotes oligodendrogenesis within the corpus callosum, occipital cortex and optic nerve. GC‐1 was also observed to enhance the expression of the myelin proteins MBP, CNP and MAG within the same regions. These results indicate that a β receptor selective thyromimetic can enhance oligodendrocyte differentiation in vitro and during developmental myelination in vivo and warrants further study as a therapeutic agent for demyelinating models. GLIA 2014;62:1513–1529     

The control of reactive oxygen species production by SHP‐1 in oligodendrocytes

We have previously described reduced myelination and corresponding myelin basic protein (MBP) expression in the central nervous system of Src homology 2 domain‐containing protein tyrosine phosphatase 1 (SHP‐1) deficient motheaten (me/me) mice compared with normal littermate controls. Deficiency in myelin and MBP expression in both brains and spinal cords of motheaten mice correlated with reduced MBP mRNA expression levels in vivo and in purified oligodendrocytes in vitro. Therefore, SHP‐1 activity seems to be a critical regulator of oligodendrocyte gene expression and function. Consistent with this role, this study demonstrates that oligodendrocytes of motheaten mice and SHP‐1‐depleted N20.1 cells produce higher levels of reactive oxygen species (ROS) and exhibit corresponding markers of increased oxidative stress. In agreement with these findings, we demonstrate that increased production of ROS coincides with ROS‐induced signaling pathways known to affect myelin gene expression in oligodendrocytes. Antioxidant treatment of SHP‐1‐deficient oligodendrocytes reversed the pathological changes in these cells, with increased myelin protein gene expression and decreased expression of nuclear factor (erythroid‐2)‐related factor 2 (Nrf2) responsive gene, heme oxygenase‐1 (HO‐1). Furthermore, we demonstrate that SHP‐1 is expressed in human white matter oligodendrocytes, and there is a subset of multiple sclerosis subjects that demonstrate a deficiency of SHP‐1 in normal‐appearing white matter. These studies reveal critical pathways controlled by SHP‐1 in oligodendrocytes that relate to susceptibility of SHP‐1‐deficient mice to both developmental defects in myelination and to inflammatory demyelinating diseases. GLIA 2015;63:1753–1771     

PLP/DM20 Expression and turnover in a transgenic mouse model of pelizaeus‐merzbacher disease

The most common cause of Pelizaeus‐Merzbacher (PMD) is due to duplication of the PLP1 gene but it is unclear how increased gene dosage affects PLP turnover and causes dysmyelination. We have studied the dynamics of PLP/DM20 in a transgenic mouse model of PMD with increased gene dosage of the proteolipid protein gene (Plp1). The turnover of PLP/DM20 were investigated using an ex‐vivo brain slice system and cultured oligodendrocytes. Homozygous mice have reduced PLP translation, markedly enhanced PLP degradation, and markedly reduced incorporation of PLP into myelin. Proteasome inhibition (MG132) prevented the enhanced degradation. Numerous autophagic vesicles are present in homozygous transgenic mice that may influence protein dynamics. Surprisingly, promoting autophagy with rapamycin decreases the degradation of nascent PLP suggesting autophagic vacuoles serve as a cellular storage compartment. We suggest that there are multiple subcellular fates of PLP/DM20 when overexpressed: the vast majority being degraded by the proteasome, a proportion sequestered into autophagic vacuoles, probably fused with endolysosomes, and only a small proportion entering the myelin sheath, where its association with lipid rafts is perturbed. Transgenic oligodendrocytes have fewer membrane sheets and this phenotype is improved with siRNA‐mediated knockdown of PLP expression that promotes the formation of MBP+ myelin‐like sheets. This finding suggests that RNAi technology is in principle applicable to improve CNS myelination when compromised by PLP/DM20 overexpression. © 2010 Wiley‐Liss, Inc.     

E‐Cadherin enhances neuregulin signaling and promotes Schwann cell myelination

In myelinating Schwann cells, E‐cadherin is a component of the adherens junctions that stabilize the architecture of the noncompact myelin region. In other cell types, E‐cadherin has been considered as a signaling receptor that modulates intracellular signal transduction and cellular responses. To determine whether E‐cadherin plays a regulatory role during Schwann cell myelination, we investigated the effects of E‐cadherin deletion and over‐expression in Schwann cells. In vivo, Schwann cell‐specific E‐cadherin ablation results in an early myelination delay. In Schwann cell‐dorsal root ganglia neuron co‐cultures, E‐cadherin deletion attenuates myelin formation and shortens the myelin segment length. When over‐expressed in Schwann cells, E‐cadherin improves myelination on Nrg1 type III^+/? neurons and induces myelination on normally non‐myelinated axons of sympathetic neurons. The pro‐myelinating effect of E‐cadherin is associated with an enhanced Nrg1‐erbB receptor signaling, including activation of the downstream Akt and Rac. Accordingly, in the absence of E‐cadherin, Nrg1‐signaling is diminished in Schwann cells. Our data also show that E‐cadherin expression in Schwann cell is induced by axonal Nrg1 type III, indicating a reciprocal interaction between E‐cadherin and the Nrg1 signaling. Altogether, our data suggest a regulatory function of E‐cadherin that modulates Nrg1 signaling and promotes Schwann cell myelin formation. GLIA 2015;63:1522–1536     

Modulation of rumpshaker phenotype with wild‐type PLP/DM20 suggests several pathogenic mechanisms

The rumpshaker mutation of the murine myelin proteolipid protein 1 (Plp1) gene generates misfolded PLP/DM20 protein, resulting in dysmyelination, increased oligodendrocyte apoptosis, and death prior to P40 when expressed on the C57 BL/6 background. In this study, we used transgenic complementation to normalize the levels of PLP/DM20 in myelin with wild‐type protein to determine whether loss of normal PLP function or gain of toxic function is responsible for dysmyelination in the rumpshaker. Restoring myelin PLP/DM20 levels extended the survival time to at least P60, significantly reduced the density of apoptotic cells, increased myelin volume, and restored normal periodicity of myelin. Biochemical analysis found that several myelin proteins that are reduced in rumpshaker, including MAG, CNP, and SirT2, are markedly elevated at peak myelination (P20) in the rumpshaker transgenic mouse. Myelin basic protein, however, remained low at peak myelination but was restored at P60 when myelin had matured and entered into a maintenance phase. Markers of the unfolded protein response (UPR), BiP and XBP1, remained activated with the introduction of wild‐type PLP. These data demonstrate that restoring wild‐type PLP/DM20 levels in rumpshaker improves the phenotype and the integrity of myelin, but hypomyelination persists and stress pathways remain activated. This suggests that both gain‐ and loss‐of‐function mechanisms are involved in the pathogenesis of the rumpshaker. © 2010 Wiley‐Liss, Inc.     

BRCA1/BRCA2-containing complex subunit 3 controls oligodendrocyte differentiation by dynamically regulating lysine 63-linked ubiquitination

Oligodendrocytes (OLs) provide the myelin sheath surrounding axons that propagates action potentials in the central nervous system (CNS). The metabolism of myelinated membranes and proteins is strictly regulated in the OLs and is closely associated with OL differentiation and maturation. The ubiquitination-associated proteasome and endosomal system have not yet been well studied during OL differentiation and maturation. Here, we determined the functions of the Lys63-linked ubiquitination (K63Ub) and K63-specific deubiquitination (DUB) systems regulated by BRCA1/BRCA2-containing complex subunit 3 (BRCC3) during OL differentiation. The competitive inhibition of K63Ub by overexpression of mutant ubiquitin (K63R) in oligodendrocyte precursor cells (OPCs) indicated that the two major CNS myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), were upregulated in OLs derived from K63R OPCs. In contrast, the knockdown of BRCC3 (BRCC3-KD) through the application of lentivirus-mediated shRNA delivery system into OPCs suppressed OL differentiation by decreasing MBP expression and PLP production. Further immunoprecipitation assays revealed higher levels of sphingolipid GalC, MBP, and PLP, which were associated with K63Ub-immunoprecipitants and detected in endosome/lysosomal compartments, in BRCC3-KD OLs than those in OLs transfected with the scrambled shRNA (scramble OLs). The differentiation of OLs from BRCC3-KD OPCs was impaired in the demyelinating corpus callosum of rats receiving a cuprizone-containing diet. In the demyelinating tissues from human patients suffering from multiple sclerosis, we detected a decreased number of BRCC3-expressing OLs at the lesion site, accompanied by a greater number of OLs expressing EEA1 and K63Ub at high levels. Altogether, the counterbalance of the K63Ub machinery and BRCC3-triggered DUB machinery are important for the cellular trafficking of myelin proteins and OL differentiation.