Rapid transient isoform-specific neuregulin1 transcription in motor neurons is regulated by neurotrophic factors and axon-target interactions.

The neuregulins (NRGs) are a family of alternatively spliced factors that play important roles in nervous system development and disease. In motor neurons, NRG1 expression is regulated by activity and neurotrophic factors, however, little is known about what controls isoform-specific transcription. Here we show that NRG1 expression in the chick embryo increases in motor neurons that have extended their axons and that limb bud ablation before motor axon outgrowth prevents this induction, suggesting a trophic role from the developing limb. Consistently, NRG1 induction after limb bud ablation can be rescued by adding back the neurotrophic factors BDNF and GDNF. Mechanistically, BDNF induces a rapid and transient increase in type I and type III NRG1 mRNAs that peak at 4h in rat embryonic ventral spinal cord cultures. Blocking MAPK or PI3K signaling or blocking transcription with Actinomycin D blocks BDNF induced NRG1 gene induction. BDNF had no effect on mRNA degradation, suggesting that transcriptional activation rather than message stability is important. Furthermore, BDNF activates a reporter construct that includes 700bp upstream of the type I NRG1 start site. Protein synthesis is also required for type I NRG1 mRNA transcription as cycloheximide produced a super-induction of type I, but not type III NRG1 mRNA, possibly through a mechanism involving sustained activation of MAPK and PI3K. These results reveal the existence of highly responsive, transient transcriptional regulatory mechanisms that differentially modulate NRG1 isoform expression as a function of extracellular and intracellular signaling cascades and mediated by neurotrophic factors and axon-target interactions.

Autoimmune-induced preferential depletion of myelin-associated glycoprotein (MAG) is genetically regulated in relapsing EAE (B6 x SJL) F1 mice.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is commonly used to investigate mechanisms of autoimmune-mediated damage to oligodendrocytes, myelin, and axons in multiple sclerosis (MS). Four distinct autoimmune mechanisms with subsequently distinct patterns of demyelination have been recognized in acute MS lesions. EAE correlates for those distinct patterns of MS lesions are unknown. An excessive loss of myelin-associated glycoprotein (MAG), as a result of distal oligodendrogliopathy, is found exclusively in the subtype III lesion. We sought to answer if types of demyelination in acute lesions during onset and relapse of EAE can replicate the specific patterns observed in MS acute lesions. METHODS: In parental H-2b (C57BL/6, B6) and hybrid H-2b/s [(B6 x SJL) F1] EAE mice, we examined spinal cord levels of MOG, MAG, and myelin basic protein (MBP), and compared to levels of axonal neurofilament (NF160) to assess axonal function, and levels of PARPp85 as an indicator of irreversible apoptosis. RESULTS: During disease onset, levels of MOG significantly dropped in both strains, although more profoundly in H-2b/s mice. Levels of MOG recovered in relapsing mice of both strains. Regulation of MAG was dissimilar to MOG. Modest loss of MAG was found at disease onset in both strains of mice. Unexpectedly, in relapsing H-2b/s mice, a major depletion of MAG and NF160, accompanied with sharp elevation of PARPp85 levels, was measured. PARPp85 immunoreactivity was observed in cytoplasm and nuclei of some MBP containing cells. CONCLUSION: Taken together, our results show genetically controlled distinct patterns of MOG and MAG depletion, in MOG35-55 induced EAE in H-2b and H-2b/s mice. The data also suggest distinctive immune regulation of acute lesions that develop in relapsing compared to disease onset. A profound depletion of MAG, concomitant with marked depletion of axonal NF160, and sharp elevation of PARPp85 levels, occurred exclusively in relapsing H-2b/s mice. Our findings suggest concurrence of sharp decrease of MAG levels, axonal dysfunction and irreversible apoptosis with severe relapsing disease in H-2b/s mice. We propose that MOG-induced EAE in H-2b/s mice may prove as a useful model in studying mechanisms, which govern autoimmune-induced preferential loss of MAG, and its impact on oligodendroglial pathology.

Anti-IL-16 therapy reduces CD4+ T-cell infiltration and improves paralysis and histopathology of relapsing EAE.

Infiltration of the central nervous system (CNS) by CD4+ Th1 cells precedes onset and relapses of experimental autoimmune encephalomyelitis (EAE). We reported that (B6xSJL) F1 (H-2b/s) mice with severe relapsing-remitting disease had extensive infiltration by CD4+ T cells compared to that in C57BL/6 (B6) (H-2b) mice, which developed mild low-relapsing disease in response to myelin oligodendrocyte peptide 35-55 (MOG(35-55)). This observation led us to search for mechanisms that specifically regulate trafficking of CD4+ cells in relapsing H-2b/s mice. We show that the CD4+ cell chemoattractant cytokine interleukin (IL)-16 has an important role in regulation of relapsing EAE induced by MOG(35-55) in the (B6xSJL) F1 (H-2b/s) mice. We found production of IL-16 in the CNS of mice with EAE. IL-16 levels in the CNS correlated well with the extent of CD4+ T-cell and B-cell infiltration during acute and relapsing disease. Infiltrating CD4+ T cells, B cells, and to a lesser extent CD8+ T cells all contained IL-16 immunoreactivity. Treatment with neutralizing anti-IL-16 antibody successfully reversed paralysis and ameliorated relapsing disease. In treated mice, diminished infiltration by CD4+ T cells, less demyelination, and more sparing of axons was observed. Taken together, our results show an important role for IL-16 in regulation of relapsing EAE. We describe a novel therapeutic approach to specifically impede CD4+ T cell chemoattraction in EAE based on IL-16 neutralization. Our findings have high relevance for the development of new therapies for relapsing EAE and potentially MS.

Distinct immune regulation of the response to H-2b restricted epitope of MOG causes relapsing-remitting EAE in H-2b/s mice.

To find immune mechanisms underlying relapse regulation, we developed a model of relapsing-remitting experimental autoimmune encephalomyelitis (EAE) in (B6xSJL) F1 (H-2(b/s)) mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG(35-55)) and compared with low/non-relapsing B6 (H-2(b)) mice. In relapsing H-2(b/s) mice, inflammatory lesions scattered throughout the white matter with extensive demyelination, consisted of CD4(+) T and B220(+) B cells with fewer Mac3(+) macrophages. Memory T cell proliferation to MOG(35-55) was significantly enhanced. Switch of macrophage chemoattractant protein-1 (MCP-1) production from GFAP(+) astrocytes to CD3(+) T cells was observed. Distinct patterns of inflammation and demyelination, MOG(35-55) memory T cell response and regulation of MCP-1 are associated with relapsing H-2(b/s) phenotype.

Neuregulin expression at neuromuscular synapses is modulated by synaptic activity and neurotrophic factors.

The proper formation of neuromuscular synapses requires ongoing synaptic activity that is translated into complex structural changes to produce functional synapses. One mechanism by which activity could be converted into these structural changes is through the regulated expression of specific synaptic regulatory factors. Here we demonstrate that blocking synaptic activity with curare reduces synaptic neuregulin expression in a dose-dependent manner yet has little effect on synaptic agrin or a muscle-derived heparan sulfate proteoglycan. These changes are associated with a fourfold increase in number and a twofold reduction in average size of synaptic acetylcholine receptor clusters that appears to be caused by excessive axonal sprouting with the formation of new, smaller acetylcholine receptor clusters. Activity blockade also leads to threefold reductions in brain-derived neurotrophic factor and neurotrophin 3 expression in muscle without appreciably changing the expression of these same factors in spinal cord. Adding back these or other neurotrophic factors restores synaptic neuregulin expression and maintains normal end plate band architecture in the presence of activity blockade. The expression of neuregulin protein at synapses is independent of spinal cord and muscle neuregulin mRNA levels, suggesting that neuregulin accumulation at synapses is independent of transcription. These findings suggest a local, positive feedback loop between synaptic regulatory factors that translates activity into structural changes at neuromuscular synapses.