PMP2 regulates myelin thickening and ATP production during remyelination.

It is well established that axonal Neuregulin 1 type 3 (NRG1t3) regulates developmental myelin formation as well as EGR2-dependent gene activation and lipid synthesis. However, in peripheral neuropathy disease context, elevated axonal NRG1t3 improves remyelination and myelin sheath thickness without increasing Egr2 expression or activity, and without affecting the transcriptional activity of canonical myelination genes. Surprisingly, Pmp2, encoding for a myelin fatty acid binding protein, is the only gene whose expression increases in Schwann cells following overexpression of axonal NRG1t3. Here, we demonstrate PMP2 expression is directly regulated by NRG1t3 active form, following proteolytic cleavage. Then, using a transgenic mouse model overexpressing axonal NRG1t3 (NRG1t3OE) and knocked out for PMP2, we demonstrate that PMP2 is required for NRG1t3-mediated remyelination. We demonstrate that the sustained expression of Pmp2 in NRG1t3OE mice enhances the fatty acid uptake in sciatic nerve fibers and the mitochondrial ATP production in Schwann cells. In sum, our findings demonstrate that PMP2 is a direct downstream mediator of NRG1t3 and that the modulation of PMP2 downstream NRG1t3 activation has distinct effects on Schwann cell function during developmental myelination and remyelination.

Hyperactivity is a Core Endophenotype of Elevated Neuregulin-1 Signaling in Embryonic Glutamatergic Networks.

The neuregulin 1 (NRG1) ErbB4 module is at the core of an "at risk" signaling pathway in schizophrenia. Several human studies suggest hyperstimulation of NRG1-ErbB4 signaling as a plausible pathomechanism; however, little is known about the significance of stage-, brain area-, or neural cell type-specific NRG1-ErbB4 hyperactivity for disease-relevant brain endophenotypes. To address these spatiotemporal aspects, we generated transgenic mice for Cre recombinase-mediated overexpression of cystein-rich domain (CRD) NRG1, the most prominent NRG1 isoform in the brain. A comparison of "brain-wide" vs cell type-specific CRD-NRG1 overexpressing mice revealed that pathogenic CRD-NRG1 signals for ventricular enlargement and neuroinflammation originate outside glutamatergic neurons and suggests a subcortical function of CRD-NRG1 in the control of body weight. Embryonic onset of CRD-NRG1 in glutamatergic cortical networks resulted in reduced inhibitory neurotransmission and locomotor hyperactivity. Our findings identify ventricular enlargement and locomotor hyperactivity, 2 main endophenotypes of schizophrenia, as specific consequences of spatiotemporally distinct expression profiles of hyperactivated CRD-NRG1 signaling.

Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin.

Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT4 (5-HT4R) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HT4R-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HT4R/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning use-dependent functional remodeling of excitatory synaptic connections.

Reprogramming of DNA methylation at NEUROD2-bound sequences during cortical neuron differentiation.

The characteristics of DNA methylation changes that occur during neurogenesis in vivo remain unknown. We used whole-genome bisulfite sequencing to quantitate DNA cytosine modifications in differentiating neurons and their progenitors isolated from mouse brain at the peak of embryonic neurogenesis. Localized DNA hypomethylation was much more common than hypermethylation and often occurred at putative enhancers within genes that were upregulated in neurons and encoded proteins crucial for neuronal differentiation. The hypomethylated regions strongly overlapped with mapped binding sites of the key neuronal transcription factor NEUROD2. The 5-methylcytosine oxidase ten-eleven translocation 2 (TET2) interacted with NEUROD2, and its reaction product 5-hydroxymethylcytosine accumulated at the demethylated regions. NEUROD2-targeted differentially methylated regions retained higher methylation levels in Neurod2 knockout mice, and inducible expression of NEUROD2 caused TET2-associated demethylation at its in vivo binding sites. The data suggest that the reorganization of DNA methylation in developing neurons involves NEUROD2 and TET2-mediated DNA demethylation.

NRG1 type I dependent autoparacrine stimulation of Schwann cells in onion bulbs of peripheral neuropathies.

In contrast to acute peripheral nerve injury, the molecular response of Schwann cells in chronic neuropathies remains poorly understood. Onion bulb structures are a pathological hallmark of demyelinating neuropathies, but the nature of these formations is unknown. Here, we show that Schwann cells induce the expression of Neuregulin-1 type I (NRG1-I), a paracrine growth factor, in various chronic demyelinating diseases. Genetic disruption of Schwann cell-derived NRG1 signalling in a mouse model of Charcot-Marie-Tooth Disease 1A (CMT1A), suppresses hypermyelination and the formation of onion bulbs. Transgenic overexpression of NRG1-I in Schwann cells on a wildtype background is sufficient to mediate an interaction between Schwann cells via an ErbB2 receptor-MEK/ERK signaling axis, which causes onion bulb formations and results in a peripheral neuropathy reminiscent of CMT1A. We suggest that diseased Schwann cells mount a regeneration program that is beneficial in acute nerve injury, but that overstimulation of Schwann cells in chronic neuropathies is detrimental.

Investigation of Neuregulin-1 and Glial Cell-Derived Neurotrophic Factor in Rodent Astrocytes and Microglia.

Growth factors play a crucial role during de- and remyelination of the central nervous system (CNS) due to their neurotrophic functions. We have previously shown that the growth factors neuregulin-1 (Nrg-1) and glial cell-derived neurotrophic factor (Gdnf) are upregulated during the first 2 weeks after induction of toxic demyelination in the CNS. Nevertheless, the factors responsible for Nrg-1/Gdnf upregulation and their effects on glia cells are unknown. We investigated the effect on Nrg-1 and Gdnf expressions after stimulation of primary mouse microglia or astrocytes with various pro- and anti-inflammatory factors. Additionally, primary cells were incubated with NRG-1 and/or GDNF followed by determining the gene expression level of their receptors, chemokines, and other growth factors. We demonstrate that inflammatory stimuli have a distinct impact on the expression of Gdnf, Nrg-1, and their receptors in astrocytes and microglia. In microglia, LPS or simultaneous treatment with IFNγ plus TNFα led to downregulation of Nrg-1, whereas LPS treatment slightly increased Nrg-1 expression in astrocytes. Furthermore, Gdnf was slightly upregulated after TFG-ß treatment in microglia, while Gdnf was significantly upregulated after LPS treatment in astrocytes. In contrast, treatment with GDNF or/and NRG-1 did not alter any measured gene expression in microglia or astrocytes. Taken together, our in vitro studies show that Nrg-1, Gdnf, and their receptors are differently regulated in astrocytes and microglia upon inflammatory stimuli. The lack of response of astrocytes and microglia to NRG-1 and GDNF suggests that both factors exert their effects directly on neurons.

Enhanced axonal neuregulin-1 type-III signaling ameliorates neurophysiology and hypomyelination in a Charcot-Marie-Tooth type 1B mouse model.

Charcot-Marie-Tooth (CMT) neuropathies are a group of genetic disorders that affect the peripheral nervous system with heterogeneous pathogenesis and no available treatment. Axonal neuregulin 1 type III (Nrg1TIII) drives peripheral nerve myelination by activating downstream signaling pathways such as PI3K/Akt and MAPK/Erk that converge on master transcriptional regulators of myelin genes, such as Krox20. We reasoned that modulating Nrg1TIII activity may constitute a general therapeutic strategy to treat CMTs that are characterized by reduced levels of myelination. Here we show that genetic overexpression of Nrg1TIII ameliorates neurophysiological and morphological parameters in a mouse model of demyelinating CMT1B, without exacerbating the toxic gain-of-function that underlies the neuropathy. Intriguingly, the mechanism appears not to be related to Krox20 or myelin gene upregulation, but rather to a beneficial rebalancing in the stoichiometry of myelin lipids and proteins. Finally, we provide proof of principle that stimulating Nrg1TIII signaling, by pharmacological suppression of the Nrg1TIII inhibitor tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17), also ameliorates the neuropathy. Thus, modulation of Nrg1TIII by TACE/ADAM17 inhibition may represent a general treatment for hypomyelinating neuropathies.

Spironolactone is an antagonist of NRG1-ERBB4 signaling and schizophrenia-relevant endophenotypes in mice.

Enhanced NRG1-ERBB4 signaling is a risk pathway in schizophrenia, and corresponding mouse models display several endophenotypes of the disease. Nonetheless, pathway-directed treatment strategies with clinically applicable compounds have not been identified. Here, we applied a cell-based assay using the split TEV technology to screen a library of clinically applicable compounds to identify modulators of NRG1-ERBB4 signaling for repurposing. We recovered spironolactone, known as antagonist of corticosteroids, as an inhibitor of the ERBB4 receptor and tested it in pharmacological and biochemical assays to assess secondary compound actions. Transgenic mice overexpressing Nrg1 type III display cortical Erbb4 hyperphosphorylation, a condition observed in postmortem brains from schizophrenia patients. Spironolactone treatment reverted hyperphosphorylation of activated Erbb4 in these mice. In behavioral tests, spironolactone treatment of Nrg1 type III transgenic mice ameliorated schizophrenia-relevant behavioral endophenotypes, such as reduced sensorimotor gating, hyperactivity, and impaired working memory. Moreover, spironolactone increases spontaneous inhibitory postsynaptic currents in cortical slices supporting an ERBB4-mediated mode-of-action. Our findings suggest that spironolactone, a clinically safe drug, provides an opportunity for new treatment options for schizophrenia.

Dysregulated expression of neuregulin-1 by cortical pyramidal neurons disrupts synaptic plasticity.

Neuregulin-1 (NRG1) gene variants are associated with increased genetic risk for schizophrenia. It is unclear whether risk haplotypes cause elevated or decreased expression of NRG1 in the brains of schizophrenia patients, given that both findings have been reported from autopsy studies. To study NRG1 functions in vivo, we generated mouse mutants with reduced and elevated NRG1 levels and analyzed the impact on cortical functions. Loss of NRG1 from cortical projection neurons resulted in increased inhibitory neurotransmission, reduced synaptic plasticity, and hypoactivity. Neuronal overexpression of cysteine-rich domain (CRD)-NRG1, the major brain isoform, caused unbalanced excitatory-inhibitory neurotransmission, reduced synaptic plasticity, abnormal spine growth, altered steady-state levels of synaptic plasticity-related proteins, and impaired sensorimotor gating. We conclude that an "optimal" level of NRG1 signaling balances excitatory and inhibitory neurotransmission in the cortex. Our data provide a potential pathomechanism for impaired synaptic plasticity and suggest that human NRG1 risk haplotypes exert a gain-of-function effect.

Neuregulin-1/ErbB signaling serves distinct functions in myelination of the peripheral and central nervous system.

Understanding the control of myelin formation by oligodendrocytes is essential for treating demyelinating diseases. Neuregulin-1 (NRG1) type III, an EGF-like growth factor, is essential for myelination in the PNS. It is thus thought that NRG1/ErbB signaling also regulates CNS myelination, a view suggested by in vitro studies and the overexpression of dominant-negative ErbB receptors. To directly test this hypothesis, we generated a series of conditional null mutants that completely lack NRG1 beginning at different stages of neural development. Unexpectedly, these mice assemble normal amounts of myelin. In addition, double mutants lacking oligodendroglial ErbB3 and ErbB4 become myelinated in the absence of any stimulation by neuregulins. In contrast, a significant hypermyelination is achieved by transgenic overexpression of NRG1 type I or NRG1 type III. Thus, NRG1/ErbB signaling is markedly different between Schwann cells and oligodendrocytes that have evolved an NRG/ErbB-independent mechanism of myelination control.

The role of neuregulin-ErbB4 interactions on the proliferation and organization of cells in the subventricular zone.

Coordinated regulation of neuronal progenitor differentiation in the subventricular zone (SVZ) is a fundamental feature of adult neurogenesis. However, the molecular control of this process remains mostly undeciphered. Here, we investigate the role of neuregulins (NRGs) in this process and show that a NRG receptor, ErbB4, is primarily expressed by polysialylated neural cell adhesion molecule immature neuroblasts but is also detected in a subset of GFAP+ astroglial cells, ependymal cells, and Dlx2+ precursors in the SVZ. Of the NRG ligands, both NRG1 and -2 are expressed by immature polysialylated neural cell adhesion molecule neuroblasts in the SVZ. NRG2 is also expressed by some of the GFAP+ putative stem cells lining the ventricles. Infusion of exogenous NRG1 leads to rapid aggregation of Dlx2+ cells in the SVZ and affects the initiation and maintenance of organized neuroblast migration from the SVZ toward the olfactory bulb. In contrast, the infusion of NRG2 increased the number of Sox2 and GFAP+ precursors in the SVZ. An outcome of this NRG2 effect is an increase in the number of newly generated migrating neuroblasts in the rostral migratory stream and GABAergic interneurons in the olfactory bulb. The analysis of conditional null mice that lack NRG receptor, ErbB4, in the nervous system revealed that the observed activities of NRG2 require ErbB4 activation. These results indicate that different NRG ligands affect distinct populations of differentiating neural precursors in the neurogenic regions of the mature forebrain. Furthermore, these studies identify NRG2 as a factor capable of promoting SVZ proliferation, leading to the formation of new neurons in vivo.

Receptor tyrosine kinase ErbB4 modulates neuroblast migration and placement in the adult forebrain.

Neural progenitor proliferation, differentiation and migration are continually active in the rostral migratory stream of the adult brain. Here, we show that the receptor tyrosine kinase ErbB4 is expressed prominently by the neuroblasts present in the subventricular zone and the rostral migratory stream. The neuregulins (NRG1-NRG3), which have been identified as ErbB4 ligands, are detected either in the stream or in adjacent regions. Mice deficient in ErbB4 expressed under the control of either the nestin or the hGFAP promoter have altered neuroblast chain organization and migration and deficits in the placement and differentiation of olfactory interneurons. These findings suggest that ErbB4 activation helps to regulate the organization of neural chains that form the rostral migratory stream and influences the differentiation of olfactory interneuronal precursors.

Neuregulin-2 is synthesized by motor neurons and terminal Schwann cells and activates acetylcholine receptor transcription in muscle cells expressing ErbB4.

Acetylcholine receptor (AChR) genes are transcribed selectively in synaptic nuclei of skeletal muscle fibers, leading to accumulation of the mRNAs encoding AChR subunits at synaptic sites. The signals that regulate synapse-specific transcription remain elusive, though Neuregulin-1 is considered a favored candidate. Here, we show that motor neurons and terminal Schwann cells express neuregulin-2, a neuregulin-1-related gene. In skeletal muscle, Neuregulin-2 protein is concentrated at synaptic sites, where it accumulates adjacent to terminal Schwann cells. Neuregulin-2 stimulates AChR transcription in cultured myotubes expressing ErbB4, as well as ErbB3 and ErbB2, but not in myotubes expressing only ErbB3 and ErbB2. Thus, Neuregulin-2 is a candidate for a signal that regulates synaptic differentiation.

Axonal neuregulin-1 regulates myelin sheath thickness.

In the nervous system of vertebrates, myelination is essential for rapid and accurate impulse conduction. Myelin thickness depends on axon fiber size. We use mutant and transgenic mouse lines to show that axonal Neuregulin-1 (Nrg1) signals information about axon size to Schwann cells. Reduced Nrg1 expression causes hypomyelination and reduced nerve conduction velocity. Neuronal overexpression of Nrg1 induces hypermyelination and demonstrates that Nrg1 type III is the responsible isoform. We suggest a model by which myelin-forming Schwann cells integrate axonal Nrg1 signals as a biochemical measure of axon size.