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.

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.

Neuregulin 1 type III improves peripheral nerve myelination in a mouse model of congenital hypomyelinating neuropathy.

Myelin sheath thickness is precisely regulated and essential for rapid propagation of action potentials along myelinated axons. In the peripheral nervous system, extrinsic signals from the axonal protein neuregulin 1 (NRG1) type III regulate Schwann cell fate and myelination. Here we ask if modulating NRG1 type III levels in neurons would restore myelination in a model of congenital hypomyelinating neuropathy (CHN). Using a mouse model of CHN, we improved the myelination defects by early overexpression of NRG1 type III. Surprisingly, the improvement was independent from the upregulation of Egr2 or essential myelin genes. Rather, we observed the activation of MAPK/ERK and other myelin genes such as peripheral myelin protein 2 and oligodendrocyte myelin glycoprotein. We also confirmed that the permanent activation of MAPK/ERK in Schwann cells has detrimental effects on myelination. Our findings demonstrate that the modulation of axon-to-glial NRG1 type III signaling has beneficial effects and improves myelination defects during development in a model of CHN.

Synergistic PXT3003 therapy uncouples neuromuscular function from dysmyelination in male Charcot-Marie-Tooth disease type 1A (CMT1A) rats.

Charcot-Marie-Tooth disease 1 A (CMT1A) is caused by an intrachromosomal duplication of the gene encoding for PMP22 leading to peripheral nerve dysmyelination, axonal loss, and progressive muscle weakness. No therapy is available. PXT3003 is a low-dose combination of baclofen, naltrexone, and sorbitol which has been shown to improve disease symptoms in Pmp22 transgenic rats, a bona fide model of CMT1A disease. However, the superiority of PXT3003 over its single components or dual combinations have not been tested. Here, we show that in a dorsal root ganglion (DRG) co-culture system derived from transgenic rats, PXT3003 induced myelination when compared to its single and dual components. Applying a clinically relevant ("translational") study design in adult male CMT1A rats for 3 months, PXT3003, but not its dual components, resulted in improved performance in behavioral motor and sensory endpoints when compared to placebo. Unexpectedly, we observed only a marginally increased number of myelinated axons in nerves from PXT3003-treated CMT1A rats. However, in electrophysiology, motor latencies correlated with increased grip strength indicating a possible effect of PXT3003 on neuromuscular junctions (NMJs) and muscle fiber pathology. Indeed, PXT3003-treated CMT1A rats displayed an increased perimeter of individual NMJs and a larger number of functional NMJs. Moreover, muscles of PXT3003 CMT1A rats displayed less neurogenic atrophy and a shift toward fast contracting muscle fibers. We suggest that ameliorated motor function in PXT3003-treated CMT1A rats result from restored NMJ function and muscle innervation, independent from myelination.

Localization and dynamic changes of neuregulin-1 at C-type synaptic boutons in association with motor neuron injury and repair.

C-type synaptic boutons (C-boutons) provide cholinergic afferent input to spinal cord motor neurons (MNs), which display an endoplasmic reticulum (ER)-related subsurface cistern (SSC) adjacent to their postsynaptic membrane. A constellation of postsynaptic proteins is clustered at C-boutons, including M2 muscarinic receptors, potassium channels, and σ-1 receptors. In addition, we previously found that neuregulin (NRG)1 is associated with C-boutons at postsynaptic SSCs, whereas its ErbB receptors are located in the presynaptic compartment. C-bouton-mediated regulation of MN excitability has been implicated in MN disease, but NRG1-mediated functions and the impact of various pathologic conditions on C-bouton integrity have not been studied in detail. Here, we investigated changes in C-boutons after electrical stimulation, pharmacological treatment, and peripheral nerve axotomy. SSC-linked NRG1 clusters were severely disrupted in acutely stressed MNs and after tunicamycin-induced ER stress. In axotomized MNs, C-bouton loss occurred in concomitance with microglial recruitment and was prevented by the ER stress inhibitor salubrinal. Activated microglia displayed a positive chemotaxis to C-boutons. Analysis of transgenic mice overexpressing NRG1 type I and type III isoforms in MNs indicated that NRG1 type III acts as an organizer of SSC-like structures, whereas NRG1 type I promotes synaptogenesis of presynaptic cholinergic terminals. Moreover, MN-derived NRG1 signals may regulate the activity of perineuronal microglial cells. Together, these data provide new insights into the molecular and cellular pathology of C-boutons in MN injury and suggest that distinct NRG1 isoform-mediated signaling functions regulate the complex matching between pre- and postsynaptic C-bouton elements.-Salvany, S., Casanovas, A., Tarabal, O., Piedrafita, L., Hernández, S., Santafé, M., Soto-Bernardini, M. C., Calderó, J., Schwab, M. H., Esquerda, J. E. Localization and dynamic changes of neuregulin-1 at C-type synaptic boutons in association with motor neuron injury and repair.

Measuring broader wellbeing in mental health services: validity of the German language OxCAP-MH capability instrument.

PURPOSE: The OxCAP-MH capabilities questionnaire was developed and validated in the UK for outcome measurement in mental health clinical studies. Its broader wellbeing construct or validity in routine mental health services so far has not been assessed. The objectives were to investigate the extent the OxCAP-MH measures broader wellbeing compared to the EQ-5D-5L and to test psychometric properties of the German language OxCAP-MH in routine mental health services in Austria. METHODS: Study sample consisted of patients in socio-psychiatric services (n = 159) assessed at baseline and 6-month follow-up. Underlying factors associated with quality-of-life/wellbeing concepts measured by the OxCAP-MH and EQ-5D-5L were identified in exploratory factor analysis (EFA). Responsiveness was assessed using anchor questionnaires and standardised response mean (SRM). For discriminant validity, subgroups of respondents were compared using t test and one-way ANOVA. Test-retest analysis was assessed for a period of maximum 30 days from the baseline assessment with intra-class correlation coefficient (ICC). RESULTS: EFA identified a two-factor structure. All EQ-5D-5L items and seven OxCAP-MH items loaded on one factor and nine remaining OxCAP-MH items loaded on a separate factor. Responsiveness was found for patients who improved in anchor questionnaire scores with large or moderate SRM statistics. OxCAP-MH discriminated between various groups in univariable and multivariable analyses. Reliability of the German language OxCAP-MH was confirmed by ICC of 0.80. CONCLUSIONS: Besides providing evidence that the OxCAP-MH measures broader wellbeing constructs beyond traditional health-related quality of life, the study also confirms the validity of the instrument for implementation in routine evaluation of mental health services.

Neuregulin1 displayed on motor axons regulates terminal Schwann cell-mediated synapse elimination at developing neuromuscular junctions.

Synaptic connections in the nervous system are rearranged during development and in adulthood as a feature of growth, plasticity, aging, and disease. Glia are implicated as active participants in these changes. Here we investigated a signal that controls the participation of peripheral glia, the terminal Schwann cells (SCs), at the neuromuscular junction (NMJ) in mice. Transgenic manipulation of the levels of membrane-tethered neuregulin1 (NRG1-III), a potent activator of SCs normally presented on motor axons, alters the rate of loss of motor inputs at NMJs during developmental synapse elimination. In addition, NMJs of adult transgenic mice that expressed excess axonal NRG1-III exhibited continued remodeling, in contrast to the more stable morphologies of controls. In fact, synaptic SCs of these adult mice with NRG1-III overexpression exhibited behaviors evident in wild type neonates during synapse elimination, including an affinity for the postsynaptic myofiber surface and phagocytosis of nerve terminals. Given that levels of NRG1-III expression normally peak during the period of synapse elimination, our findings identify axon-tethered NRG1 as a molecular determinant for SC-driven neuromuscular synaptic plasticity.

Yeast-based assays for screening 11ß-HSD1 inhibitors.

BACKGROUND: Intracellular metabolism of glucocorticoid hormones plays an important role in the pathogenesis of metabolic syndrome and regulates, among many physiological processes, collagen metabolism in skin. At the peripheral level the concentration of active glucocorticoids is mainly regulated by the 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) enzyme, involved in the conversion of cortisone into the biologically active hormone cortisol. Cortisol interacts with the glucocorticoid receptor and regulates the expression of different classes of genes within the nucleus. Due to its implication in glucocorticoid metabolism, the inhibition of 11ß-HSD1 activity has become a dominant strategy for the treatment of metabolic syndrome. Moreover, inhibitors of this target enzyme can be used for development of formulations to counteract skin ageing. Here we present the construction of two yeast cell based assays that can be used for the screening of novel 11ß-HSD1 inhibitors. RESULTS: The yeast Saccharomyces cerevisiae is used as a host organism for the expression of human 11ß-HSD1 as well as a genetically encoded assay system that allows intracellular screening of molecules with 11ß-HSD1 inhibitory activity. As proof of concept the correlation between 11ß-HSD1 inhibition and fluorescent output signals was successfully tested with increasing concentrations of carbenoxolone and tanshinone IIA, two known 11ß-HSD1 inhibitors. The first assay detects a decrease in fluorescence upon 11ß-HSD1 inhibition, whereas the second assay relies on stabilization of yEGFP upon inhibition of 11ß-HSD1, resulting in a positive read-out and thus minimizing the rate of false positives sometimes associated with read-outs based on loss of signals. Specific inhibition of the ABC transporter Pdr5p improves the sensitivity of the assay strains to cortisone concentrations by up to 60 times. CONCLUSIONS: Our yeast assay strains provide a cost-efficient and easy to handle alternative to other currently available assays for the screening of 11ß-HSD1 inhibitors. These assays are designed for an initial fast screening of large numbers of compounds and enable the selection of cell permeable molecules with target inhibitory activity, before proceeding to more advanced selection processes. Moreover, they can be employed in yeast synthetic biology platforms to reconstitute heterologous biosynthetic pathways of drug-relevant scaffolds for simultaneous synthesis and screening of 11ß-HSD1 inhibitors at intracellular level.

Neuronal basic helix-loop-helix proteins Neurod2/6 regulate cortical commissure formation before midline interactions.

Establishment of long-range fiber tracts by neocortical projection neurons is fundamental for higher brain functions. The molecular control of axon tract formation, however, is still poorly understood. Here, we have identified basic helix-loop-helix (bHLH) transcription factors Neurod2 and Neurod6 as key regulators of fasciculation and targeted axogenesis in the mouse neocortex. In Neurod2/6 double-mutant mice, callosal axons lack expression of the cell adhesion molecule Contactin2, defasciculate in the subventricular zone, and fail to grow toward the midline without forming Probst bundles. Instead, mutant axons overexpress Robo1 and follow random trajectories into the ipsilateral cortex. In contrast to long-range axogenesis, generation and maintenance of pyramidal neurons and initial axon outgrowth are grossly normal, suggesting that these processes are under distinct transcriptional control. Our findings define a new stage in corpus callosum development and demonstrate that neocortical projection neurons require transcriptional specification by neuronal bHLH proteins to execute an intrinsic program of remote connectivity.

Identification of the Campanian Ignimbrite in the Dead Sea and consequent time-transgressive hydroclimatic shifts in the Eastern Mediterranean.

Robust chronologies and time equivalent tephra markers are essential to better understand spatial palaeoenvironmental response to past abrupt climatic changes. Identification of well-dated and widely dispersed volcanic ash by tephra and cryptotephra (microscopic volcanic ash) provides time synchronous tie-points and strongly reduces chronological uncertainties. Here, we present the major, minor and trace element analyses of cryptotephra shards in the Dead Sea Deep Drilling sedimentary record (DSDDP 5017-1A) matching the Campanian Ignimbrite (CI). This geochemical identification expands the known dispersal range of the CI to the southeastern Mediterranean, over 2300 km from the volcanic source. Due to the CI eruption occurring near-synchronous with North Atlantic ice surge of Heinrich Event 4 (HE4), this tephra provides insights into regional responses to large-scale climatic change in the Mediterranean. In the Dead Sea, the CI layer is associated with wetter climatic conditions. This contrasts with the contemporaneous occurrence of the CI deposition and dry conditions in the central and eastern Mediterranean suggesting a possible climate time-transgressive expansion of HE4. Our finding underscores the temporal and spatial complexity of regional climate responses and emphasises the importance of tephra as a time marker for studying large-scale climatic changes verses regional variations.

In vivo imaging and noninvasive ablation of pyramidal neurons in adult NEX-CreERT2 mice.

To study the function of individual neurons that are embedded in a complex neural network is difficult in mice. Conditional mutagenesis permits the spatiotemporal control of gene expression including the ablation of cells by toxins. To direct expression of a tamoxifen-inducible variant of Cre recombinase (CreERT2) selectively to cortical neurons, we replaced the coding region of the murine Nex1 gene by CreERT2 cDNA via homologous recombination in embryonic stem cells. When injected with tamoxifen, adult NEX-CreERT2 mice induced reporter gene expression exclusively in projection neurons of the neocortex and hippocampus. By titrating the tamoxifen dosage, we achieved recombination in single cells, which allowed multiphoton imaging of neocortical neurons in live mice. When hippocampal projection neurons were genetically ablated by induced expression of diphteria toxin, within 20 days the inflammatory response included the infiltration of CD3+ T cells. This marks a striking difference from similar studies, in which dying oligodendrocytes failed to recruit cells of the adaptive immune system.

Transgenic overexpression of the type I isoform of neuregulin 1 affects working memory and hippocampal oscillations but not long-term potentiation.

Neuregulin 1 (NRG1) is a growth factor involved in neurodevelopment and plasticity. It is a schizophrenia candidate gene, and hippocampal expression of the NRG1 type I isoform is increased in the disorder. We have studied transgenic mice overexpressing NRG1 type I (NRG1(tg-type I)) and their wild-type littermates and measured hippocampal electrophysiological and behavioral phenotypes. Young NRG1(tg-type I) mice showed normal memory performance, but in older NRG1(tg-type I) mice, hippocampus-dependent spatial working memory was selectively impaired. Hippocampal slice preparations from NRG1(tg-type I) mice exhibited a reduced frequency of carbachol-induced gamma oscillations and an increased tendency to epileptiform activity. Long-term potentiation in NRG1(tg-type I) mice was normal. The results provide evidence that NRG1 type I impacts on hippocampal function and circuitry. The effects are likely mediated via inhibitory interneurons and may be relevant to the involvement of NRG1 in schizophrenia. However, the findings, in concert with those from other genetic and pharmacological manipulations of NRG1, emphasize the complex and pleiotropic nature of the gene, even with regard to a single isoform.

Persistent NRG1 Type III Overexpression in Spinal Motor Neurons Has No Therapeutic Effect on ALS-Related Pathology in SOD1G93A Mice.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting upper and lower motor neurons (MNs). Neuregulin-1 (NRG1) is a pleiotropic growth factor that has been shown to be potentially valuable for ALS when supplemented by means of viral-mediated gene therapy. However, these results are inconsistent with other reports. An alternative approach for investigating the therapeutic impact of NRG1 on ALS is the use of transgenic mouse lines with genetically defined NRG1 overexpression. Here, we took advantage of a mouse line with NRG1 type III overexpression in spinal cord α motor neurons (MN) to determine the impact of steadily enhanced NRG1 signalling on mutant superoxide dismutase 1 (SOD1)-induced disease. The phenotype of SOD1G93A-NRG1 double transgenic mice was analysed in detail, including neuropathology and extensive behavioural testing. At least 3 animals per condition and sex were histopathologically assessed, and a minimum of 10 mice per condition and sex were clinically evaluated. The accumulation of misfolded SOD1 (mfSOD1), MN degeneration, and a glia-mediated neuroinflammatory response are pathological hallmarks of ALS progression in SOD1G93A mice. None of these aspects was significantly improved when examined in double transgenic NRG1-SOD1G93A mice. In addition, behavioural testing revealed that NRG1 type III overexpression did not affect the survival of SOD1G93A mice but accelerated disease onset and worsened the motor phenotype.

Boosting neuregulin 1 type-III expression hastens SMA motor axon maturation.

Intercellular communication between axons and Schwann cells is critical for attaining the complex morphological steps necessary for axon maturation. In the early onset motor neuron disease spinal muscular atrophy (SMA), many motor axons are not ensheathed by Schwann cells nor grow sufficiently in radial diameter to become myelinated. These developmentally arrested motor axons are dysfunctional and vulnerable to rapid degeneration, limiting efficacy of current SMA therapeutics. We hypothesized that accelerating SMA motor axon maturation would improve their function and reduce disease features. A principle regulator of peripheral axon development is neuregulin 1 type III (NRG1-III). Expressed on axon surfaces, it interacts with Schwann cell receptors to mediate axon ensheathment and myelination. We examined NRG1 mRNA and protein expression levels in human and mouse SMA tissues and observed reduced expression in SMA spinal cord and in ventral, but not dorsal root axons. To determine the impact of neuronal NRG1-III overexpression on SMA motor axon development, we bred NRG1-III overexpressing mice to SMA∆7 mice. Neonatally, elevated NRG1-III expression increased SMA ventral root size as well as axon segregation, diameter, and myelination resulting in improved motor axon conduction velocities. NRG1-III was not able to prevent distal axonal degeneration nor improve axon electrophysiology, motor behavior, or survival of older mice. Together these findings demonstrate that early SMA motor axon developmental impairments can be ameliorated by a molecular strategy independent of SMN replacement providing hope for future SMA combinatorial therapeutic approaches.

Myelin insulation as a risk factor for axonal degeneration in autoimmune demyelinating disease.

Axonal degeneration determines the clinical outcome of multiple sclerosis and is thought to result from exposure of denuded axons to immune-mediated damage. Therefore, myelin is widely considered to be a protective structure for axons in multiple sclerosis. Myelinated axons also depend on oligodendrocytes, which provide metabolic and structural support to the axonal compartment. Given that axonal pathology in multiple sclerosis is already visible at early disease stages, before overt demyelination, we reasoned that autoimmune inflammation may disrupt oligodendroglial support mechanisms and hence primarily affect axons insulated by myelin. Here, we studied axonal pathology as a function of myelination in human multiple sclerosis and mouse models of autoimmune encephalomyelitis with genetically altered myelination. We demonstrate that myelin ensheathment itself becomes detrimental for axonal survival and increases the risk of axons degenerating in an autoimmune environment. This challenges the view of myelin as a solely protective structure and suggests that axonal dependence on oligodendroglial support can become fatal when myelin is under inflammatory attack.

Adipo-glial signaling mediates metabolic adaptation in peripheral nerve regeneration.

The peripheral nervous system harbors a remarkable potential to regenerate after acute nerve trauma. Full functional recovery, however, is rare and critically depends on peripheral nerve Schwann cells that orchestrate breakdown and resynthesis of myelin and, at the same time, support axonal regrowth. How Schwann cells meet the high metabolic demand required for nerve repair remains poorly understood. We here report that nerve injury induces adipocyte to glial signaling and identify the adipokine leptin as an upstream regulator of glial metabolic adaptation in regeneration. Signal integration by leptin receptors in Schwann cells ensures efficient peripheral nerve repair by adjusting injury-specific catabolic processes in regenerating nerves, including myelin autophagy and mitochondrial respiration. Our findings propose a model according to which acute nerve injury triggers a therapeutically targetable intercellular crosstalk that modulates glial metabolism to provide sufficient energy for successful nerve repair.

NeuroD factors regulate cell fate and neurite stratification in the developing retina.

Members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to control critical aspects of development in many tissues. To identify bHLH genes that might regulate specific aspects of retinal cell development, we investigated the expression of bHLH genes in single, developing mouse retinal cells, with particular emphasis on the NeuroD family. Two of these factors, NeuroD2 and NeuroD6/NEX, had not been previously reported as expressed in the retina. A series of loss- and gain-of-function experiments was performed, which suggested that NeuroD genes have both similarities and differences in their activities. Notably, misexpression of NeuroD genes can direct amacrine cell processes to two to three specific sublaminae in the inner plexiform layer. This effect is specific to cell type and NeuroD gene, as the AII amacrine cell type is refractory to the effects of NeuroD1 and NeuroD6, but uniquely sensitive to the effect of NeuroD2 on neurite targeting. Additionally, NeuroD2 is endogenously expressed in AII amacrine cells, among others, and loss of NeuroD2 function results in a partial loss of AII amacrine cells. The effects of misexpressing NeuroD genes on retinal cell fate determination also suggested shared and divergent functions. Remarkably, NeuroD2 misexpression induced ganglion cell production even after the normal developmental window of ganglion cell genesis. Together, these data suggest that members of the NeuroD family are important for neuronal cell type identity and may be involved in several cell type-specific aspects of retinal development, including fate determination, differentiation, morphological development, and circuit formation.

Fas/CD95 regulatory protein Faim2 is neuroprotective after transient brain ischemia.

Death receptor (DR) signaling has a major impact on the outcome of numerous neurological diseases, including ischemic stroke. DRs mediate not only cell death signals, but also proinflammatory responses and cell proliferation. Identification of regulatory proteins that control the switch between apoptotic and alternative DR signaling opens new therapeutic opportunities. Fas apoptotic inhibitory molecule 2 (Faim2) is an evolutionary conserved, neuron-specific inhibitor of Fas/CD95-mediated apoptosis. To investigate its role during development and in disease models, we generated Faim2-deficient mice. The ubiquitous null mutation displayed a viable and fertile phenotype without overt deficiencies. However, lack of Faim2 caused an increase in susceptibility to combined oxygen-glucose deprivation in primary neurons in vitro as well as in caspase-associated cell death, stroke volume, and neurological impairment after cerebral ischemia in vivo. These processes were rescued by lentiviral Faim2 gene transfer. In summary, we provide evidence that Faim2 is a novel neuroprotective molecule in the context of cerebral ischemia.