Betacellulin regulates schwann cell proliferation and myelin formation in the injured mouse peripheral nerve.

When a nerve fiber is cut or crushed, the axon segment that is separated from the soma degenerates distal from the injury in a process termed Wallerian degeneration (WD). C57BL/6OlaHsd-WldS (WldS ) mutant mice exhibit significant delays in WD. This results in considerably delayed Schwann cell and macrophage responses and thus in impaired nerve regenerations. In our previous work, thousands of genes were screened by DNA microarrays and over 700 transcripts were found to be differentially expressed in the injured sciatic nerve of WldS compared with wild-type (WT) mice. One of these transcripts, betacellulin (Btc), was selected for further analysis since it has yet to be characterized in the nervous system, despite being known as a ligand of the ErbB receptor family. We show that Btc mRNA is strongly upregulated in immature and dedifferentiated Sox2+ Schwann cells located in the sciatic nerve distal stump of WT mice, but not WldS mutants. Transgenic mice ubiquitously overexpressing Btc (Tg-Btc) have increased numbers of Schmidt-Lantermann incisures compared with WT mice, as revealed by Coherent anti-Stokes Raman scattering (CARS). Tg-Btc mice also have faster nerve conduction velocity. Finally, we found that deficiency in Btc reduces the proliferation of myelinating Schwann cells after sciatic nerve injury, while Btc overexpression induces Schwann cell proliferation and improves recovery of locomotor function. Taken together, these results suggest a novel regulatory role of Btc in axon-Schwann cell interactions involved in myelin formation and nerve repair. GLIA 2017 GLIA 2017;65:657-669.

Neuroinflammation in white matter tracts of Cnp1 mutant mice amplified by a minor brain injury.

Oligodendrocytes make myelin for rapid impulse propagation and contribute to the long-term survival of myelinated axons. The mechanisms by which oligodendroglial dysfunction(s) contribute to slowly progressive neurodegeneration are not well understood. Here, we demonstrate in Cnp1 mutant mice that secondary axonal degeneration in the subcortical white matter is associated with an age-dependent activation of both, innate and adaptive immune responses, including an expansion of infiltrating CD8+ T cells. While the detrimental role of lymphocytes in inherited myelin diseases is known, the role of activated microglia for the hypothetical cycle of inflammation/degeneration is unclear. We used a mild standardized cryolesion of the right parietal cortex to activate microglia at the vulnerable age of mouse puberty (postnatal day (P) 28). When applied to Cnp1 mutant mice, analyzed more than 3 months later, minor brain injury had acted as a "second hit" and significantly enhanced astrogliosis, microgliosis and axon degeneration, but not T cell infiltration. Interestingly, exacerbated neuropathological changes were also reflected by specific deterioration of working memory on top of an essentially normal basic behavior. We propose a model in which oligodendroglial dysfunctions can trigger a vicious cycle of neurodegeneration and low-grade inflammation that is amplified by nonspecific activators of the innate immune system. This interaction of genetic and environmental factors may be relevant for neuropsychiatric diseases associated with secondary neuroinflammation.

Uncoupling of neuroinflammation from axonal degeneration in mice lacking the myelin protein tetraspanin-2.

Deficiency of the major constituent of central nervous system (CNS) myelin, proteolipid protein (PLP), causes axonal pathology in spastic paraplegia type-2 patients and in Plp1(null) -mice but is compatible with almost normal myelination. These observations led us to speculate that PLP's role in myelination may be partly compensated for by other tetraspan proteins. Here, we demonstrate that the abundance of the structurally related tetraspanin-2 (TSPAN2) is highly increased in CNS myelin of Plp1(null) -mice. Unexpectedly, Tspan2(null) -mutant mice generated by homologous recombination in embryonic stem cells displayed low-grade activation of astrocytes and microglia in white matter tracts while they were fully myelinated and showed no signs of axonal degeneration. To determine overlapping functions of TSPAN2 and PLP, Tspan2(null) *Plp1(null) double-mutant mice were generated. Strikingly, the activation of astrocytes and microglia was strongly enhanced in Tspan2(null) *Plp1(null) double-mutants compared with either single-mutant, but the levels of dysmyelination and axonal degeneration were not increased. In this model, glial activation is thus unlikely to be caused by axonal pathology, and vice versa does not potentiate axonal degeneration. Our results support the concept that multiple myelin proteins have distinct roles in the long-term preservation of a healthy CNS, rather than in myelination per se.

Molecular triggers of neuroinflammation in mouse models of demyelinating diseases.

Myelinating cells wrap axons with multi-layered myelin sheaths for rapid impulse propagation. Dysfunctions of oligodendrocytes or Schwann cells are often associated with neuroinflammation, as observed in animal models of leukodystrophies and peripheral neuropathies, respectively. The neuroinflammatory response modulates the pathological changes, including demyelination and axonal injury, but also remyelination and repair. Here we discuss different immune mechanisms as well as factors released or exposed by myelinating glia in disease conditions. The spectrum of inflammatory mediators varies with different myelin disorders and has a major impact on the beneficial or detrimental role of immune cells in keeping nervous system integrity.

Transcriptional profiling of the injured sciatic nerve of mice carrying the Wld(S) mutant gene: identification of genes involved in neuroprotection, neuroinflammation, and nerve regeneration.

Wallerian degeneration (WD) involves the fragmentation of axonal segments disconnected from their cell bodies, segmentation of the myelin sheath, and removal of debris by Schwann cells and immune cells. The removal and downregulation of myelin-associated inhibitors of axonal regeneration and synthesis of growth factors by these two cell types are critical responses to successful nerve repair. Here, we analyzed the transcriptome of the sciatic nerve of mice carrying the Wallerian degeneration slow (Wld(S)) mutant gene, a gene that confers axonal protection in the distal stump after injury, therefore causing significant delays in WD, neuroinflammation, and axonal regeneration. Of the thousands of genes analyzed by microarray, 719 transcripts were differentially expressed between Wld(S) and wild-type (wt) mice. Notably, the Nmnat1, a transcript contained within the sequence of the Wld(S) gene, was upregulated by five to eightfold in the sciatic nerve of naive Wld(S) mice compared with wt. The injured sciatic nerve of wt could be further distinguished from the one of Wld(S) mice by the preferential upregulation of genes involved in axonal processes and plasticity (Chl1, Epha5, Gadd45b, Jun, Nav2, Nptx1, Nrcam, Ntm, Sema4f), inflammation and immunity (Arg1, Lgals3, Megf10, Panx1), growth factors/cytokines and their receptors (Clcf1, Fgf5, Gdnf, Gfrα1, Il7r, Lif, Ngfr/p75(NTR), Shh), and cell adhesion and extracellular matrix (Adam8, Gpc1, Mmp9, Tnc). These results will help understand how the nervous and immune systems interact to modulate nerve repair, and identify the molecules that drive these responses.

Kinesin Kif2C in regulation of DNA double strand break dynamics and repair.

DNA double strand breaks (DSBs) have detrimental effects on cell survival and genomic stability, and are related to cancer and other human diseases. In this study, we identified microtubule-depolymerizing kinesin Kif2C as a protein associated with DSB-mimicking DNA templates and known DSB repair proteins in Xenopus egg extracts and mammalian cells. The recruitment of Kif2C to DNA damage sites was dependent on both PARP and ATM activities. Kif2C knockdown or knockout led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. Interestingly, Kif2C depletion, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Taken together, our study established Kif2C as a new player of the DNA damage response, and presented a new mechanism that governs DSB dynamics and repair.

DNA can be damaged in many ways, and a double strand break is one of the most dangerous. This occurs when both strands of the double helix snap at the same time, leaving two broken ends. When cells detect this kind of damage, they race to get it fixed as quickly as possible. Fixing these double strand breaks is thought to involve the broken ends being moved to 'repair centers' in the nucleus of the cell, but it was unclear how the broken ends were moved. One possibility was that the cells transport the broken ends along protein filaments called microtubules. Cells can assemble these track-like filaments on-demand to carry cargo attached to molecular motors called kinesins. However, this type of transport happens outside of the cell's nucleus, and while there are different kinesin proteins localized inside the nucleus, their roles are largely unknown. In an effort to understand how broken DNA ends are repaired, Zhu, Paydar et al. conducted experiments that simulated double strand breaks and examined the proteins that responded. The first set of experiments involved mixing cut pieces of DNA with extracts taken from frog eggs or human cells. Zhu, Paydar et al. found that one kinesin called Kif2C stuck to the DNA fragments, and attached to many proteins known to play a role in DNA damage repair. Kif2C had previously been shown to help separate the chromosomes during cell division. To find out more about its potential role in DNA repair, Zhu, Paydar et al. then used a laser to create breaks in the DNA of living human cells and tracked Kif2C movement. The kinesin arrived within 60 seconds of the DNA damage and appeared to transport the cut DNA ends to 'repair centers'. Getting rid of Kif2C, or blocking its activity, had dire effects on the cells' abilities to mobilize and repair breaks to its DNA. Without the molecular motor, fewer double strand breaks were repaired, and so DNA damage started to build up. Defects in double strand break repair happen in many human diseases, including cancer. Many cancer treatments damage the DNA of cancer cells, sometimes in combination with drugs that stop cells from building and using their microtubule transport systems. Understanding the new role of Kif2C in DNA damage repair could therefore help optimize these treatment combinations.

Requirement of myeloid cells for axon regeneration.

The role of CD11b+ myeloid cells in axonal regeneration was assessed using axonal injury models and CD11b-TK(mt-30) mice expressing a mutated HSV-1 thymidine kinase (TK) gene regulated by the myeloid-specific CD11b promoter. Continuous delivery of ganciclovir at a sciatic nerve lesion site greatly decreased the number of granulocytes/inflammatory monocytes and macrophages in the distal stump of CD11b-TK(mt-30) mice. Axonal regeneration and locomotor function recovery were severely compromised in ganciclovir-treated CD11b-TK(mt-30) mice. This was caused by an unsuitable growth environment rather than an altered regeneration capacity of neurons. In absence of CD11b+ cells, the clearance of inhibitory myelin debris was prevented, neurotrophin synthesis was abolished, and blood vessel formation/maintenance was severely compromised in the sciatic nerve distal stump. Spinal cord-injured axons also failed to regenerate through peripheral nerve grafts in the absence of CD11b+ cells. Therefore, myeloid cells support axonal regeneration and functional recovery by creating a growth-permissive milieu for injured axons.

Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury.

Toll-like receptors (TLRs) bind specific components conserved among microorganisms as well as endogenous ligands produced by necrotic cells, injured axons, and the extracellular matrix. Here, we investigated whether TLRs are involved in regulating the immune response, Wallerian degeneration (WD), and nerve regeneration after sciatic nerve lesion. Early expression of interleukin-1beta and monocyte chemoattractant protein-1 was compromised in the sciatic nerve distal stump of mice deficient in TLR signaling. In addition, significantly fewer macrophages were recruited and/or activated in the sciatic nerve distal stump of TLR2-, TLR4-, and MyD88-deficient mice compared with wild-type littermates, whereas WD, axonal regeneration, and recovery of locomotor function were impaired. In contrast, animals that received a single microinjection of TLR2 and TLR4 ligands at the site of sciatic nerve lesion had faster clearance of the degenerating myelin and recovered earlier than saline-injected control rats. Finally, rats that had altered innate immune response through dexamethasone treatment exhibited three times more myelin debris in their sciatic nerve distal stump and a significant delay in recovery of locomotor function. Our results provide strong evidence that TLR signaling plays a critical role in orchestrating the innate immune response leading to efficient and rapid clearance of inhibitory myelin debris and nerve regeneration.

Dietary cholesterol promotes repair of demyelinated lesions in the adult brain.

Multiple Sclerosis (MS) is an inflammatory demyelinating disorder in which remyelination failure contributes to persistent disability. Cholesterol is rate-limiting for myelin biogenesis in the developing CNS; however, whether cholesterol insufficiency contributes to remyelination failure in MS, is unclear. Here, we show the relationship between cholesterol, myelination and neurological parameters in mouse models of demyelination and remyelination. In the cuprizone model, acute disease reduces serum cholesterol levels that can be restored by dietary cholesterol. Concomitant with blood-brain barrier impairment, supplemented cholesterol directly supports oligodendrocyte precursor proliferation and differentiation, and restores the balance of growth factors, creating a permissive environment for repair. This leads to attenuated axon damage, enhanced remyelination and improved motor learning. Remarkably, in experimental autoimmune encephalomyelitis, cholesterol supplementation does not exacerbate disease expression. These findings emphasize the safety of dietary cholesterol in inflammatory diseases and point to a previously unrecognized role of cholesterol in promoting repair after demyelinating episodes.

A novel method for multiple labeling combining in situ hybridization with immunofluorescence.

In situ hybridization (ISH) is a particularly useful method to investigate de novo mRNA expression in tissue sections. High specificity and sensitivity of this technique combined with the great preservation of tissue and cellular morphology conferred by fixatives such as 4% paraformaldehyde, pH 9.5, make ISH a tool of choice for detecting genes of interest in individual cells in the central nervous system (CNS). Here we describe a novel method that combines radioactive ISH with immunofluorescence on the same tissue section to identify cell populations expressing selected mRNA transcripts. This novel method has several major advantages over previously described double-labeling light microscopic methods combining enzymatic immunohistochemistry and ISH including (1) complete protection against loss of hybridization signal that normally occurs during the immunoenzymatic reaction, (2) improved immunolabeling sensitivity due to the proteinase K digestion step during ISH, (3) detection of several proteins specific for different cell populations on the same tissue section, and (4) counterstaining of tissue sections without affecting visualization of immunolabeling. This new method will be particularly useful for investigators looking to identify cell populations producing mRNAs expressed in low abundance such as cytokines, chemokines, and growth factors in the intact and/or injured mammalian CNS.

Therapy of Pelizaeus-Merzbacher disease in mice by feeding a cholesterol-enriched diet.

Duplication of PLP1 (proteolipid protein gene 1) and the subsequent overexpression of the myelin protein PLP (also known as DM20) in oligodendrocytes is the most frequent cause of Pelizaeus-Merzbacher disease (PMD), a fatal leukodystrophy without therapeutic options. PLP binds cholesterol and is contained within membrane lipid raft microdomains. Cholesterol availability is the rate-limiting factor of central nervous system myelin synthesis. Transgenic mice with extra copies of the Plp1 gene are accurate models of PMD. Dysmyelination followed by demyelination, secondary inflammation and axon damage contribute to the severe motor impairment in these mice. The finding that in Plp1-transgenic oligodendrocytes, PLP and cholesterol accumulate in late endosomes and lysosomes (endo/lysosomes), prompted us to further investigate the role of cholesterol in PMD. Here we show that cholesterol itself promotes normal PLP trafficking and that dietary cholesterol influences PMD pathology. In a preclinical trial, PMD mice were fed a cholesterol-enriched diet. This restored oligodendrocyte numbers and ameliorated intracellular PLP accumulation. Moreover, myelin content increased, inflammation and gliosis were reduced and motor defects improved. Even after onset of clinical symptoms, cholesterol treatment prevented disease progression. Dietary cholesterol did not reduce Plp1 overexpression but facilitated incorporation of PLP into myelin membranes. These findings may have implications for therapeutic interventions in patients with PMD.

Expression profile of receptors for myelin-associated inhibitors of axonal regeneration in the intact and injured mouse central nervous system.

Although CNS neurons have the potential to regenerate their axons after injury, myelin debris carrying axon growth inhibitors rapidly induce growth cone collapse. Receptors (NgR1, NgR2) and coreceptors (LINGO-1, p75(NTR), TROY) for these inhibitors have been characterized and transduction pathways partially identified. However, little is known about the expression of these receptors in intact and lesioned supraspinal projection neurons. Using in situ hybridization, immunohistochemistry and neuronal tract-tracing, we found that NgR1, NgR2 and LINGO-1 are strongly expressed in several neuronal populations of the adult mouse brain projecting to the spinal cord, including neurons projecting through the corticospinal, rubrospinal, caerulospinal, reticulospinal, raphespinal and vestibulospinal tracts. As expected, p75(NTR) expression was restricted to neuronal descending pathways from the brainstem. TROY was absent from most brain regions and from all neuronal projection systems, suggesting that additional signal-transducing coreceptors exist. Qualitative and quantitative analyses revealed that brain expression for these receptors was not affected by a severe T10 spinal cord contusion.