Nicolas Vallières

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-1β 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.

Requirement of myeloid cells for axon regeneration.

The role of CD11b+ myeloid cells in axonal regeneration was assessed using axonal injury models and CD11b-TKmt-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-TKmt-30 mice. Axonal regeneration and locomotor function recovery were severely compromised in ganciclovir-treated CD11b-TKmt-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.

Mutant Huntingtin Is Present in Neuronal Grafts in Huntington Disease Patients

Huntington disease (HD) is caused by a genetically encoded pathological protein (mutant huntingtin [mHtt]), which is thought to exert its effects in a cell‐autonomous manner. Here, we tested the hypothesis that mHtt is capable of spreading within cerebral tissue by examining genetically unrelated fetal neural allografts within the brains of patients with advancing HD.

Systemic injections of lipopolysaccharide accelerates myelin phagocytosis during Wallerian degeneration in the injured mouse spinal cord

The phagocytic cell response within the injured spinal cord is inefficient, allowing myelin debris to remain for prolonged periods of time within white matter tracts distal to the injury. Several proteins associated with this degenerating myelin are inhibitory to axon growth and therefore prevent severed axons from regenerating. Inflammatory agents such as lipopolysaccharide (LPS) can stimulate both the migration and phagocytic activity of macrophages. Using in situ hybridization, we found that the expression of the LPS membrane receptor, CD14, was enhanced in the mouse dorsal column following a dorsal hemisection. Double labeling studies showed that microglia and macrophages are the two major cell types expressing CD14 mRNA following spinal cord injury (SCI). We therefore tested whether systemic injections of LPS would increase the number and phagocytic activity of macrophages/microglia in the ascending sensory tract (AST) of the mouse dorsal column following a dorsal hemisection. Mice were treated daily via intraperitoneal injections of either LPS or phosphate‐buffered saline (PBS). At 7 days post‐SCI, greater numbers of activated mononuclear phagocytes were present in the AST undergoing Wallerian degeneration (WD) in LPS‐treated animals compared with controls. Animals treated with LPS also exhibited greater Oil Red O staining, which is specific for degenerating myelin and macrophages phagocytosing myelin debris. Myelin clearance was confirmed at 7 days using Luxol Fast Blue staining and on toluidine blue‐stained semi‐thin sections. These results indicate that it is possible to manipulate the innate immune response to accelerate myelin clearance during WD in the injured mouse spinal cord.

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.

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.

IL-1α Gene Deletion Protects Oligodendrocytes after Spinal Cord Injury through Upregulation of the Survival Factor Tox3

Spinal cord injury (SCI) causes the release of danger signals by stressed and dying cells, a process that leads to neuroinflammation. Evidence suggests that inflammation plays a role in both the damage and repair of injured neural tissue. We show that microglia at sites of SCI rapidly express the alarmin interleukin (IL)-1α, and that infiltrating neutrophils and macrophages subsequently produce IL-1β. Infiltration of these cells is dramatically reduced in both IL-1α−/− and IL-1β−/− mice, but only IL-1α−/− mice showed rapid (at day 1) and persistent improvements in locomotion associated with reduced lesion volume. Similarly, intrathecal administration of the IL-1 receptor antagonist anakinra restored locomotor function post-SCI. Transcriptome analysis of SCI tissue at day 1 identified the survival factor Tox3 as being differentially regulated exclusively in IL-1α−/− mice compared with IL-1β−/− and wild-type mice. Accordingly, IL-1α−/− mice have markedly increased Tox3 levels in their oligodendrocytes, beginning at postnatal day 10 (P10) and persisting through adulthood. At P10, the spinal cord of IL-1α−/− mice showed a transient increase in mature oligodendrocyte numbers, coinciding with increased IL-1α expression in wild-type animals. In adult mice, IL-1α deletion is accompanied by increased oligodendrocyte survival after SCI. TOX3 overexpression in human oligodendrocytes reduced cellular death under conditions mimicking SCI. These results suggest that IL-1α-mediated Tox3 suppression during the early phase of CNS insult plays a crucial role in secondary degeneration. SIGNIFICANCE STATEMENT The mechanisms underlying bystander degeneration of neurons and oligodendrocytes after CNS injury are ill defined. We show that microglia at sites of spinal cord injury (SCI) rapidly produce the danger signal interleukin (IL)-1α, which triggers neuroinflammation and locomotor defects. We uncovered that IL-1α−/− mice have markedly increased levels of the survival factor Tox3 in their oligodendrocytes, which correlates with the protection of this cell population, and reduced lesion volume, resulting in unprecedented speed, level, and persistence of functional recovery after SCI. Our data suggest that central inhibition of IL-1α or Tox3 overexpression during the acute phase of a CNS insult may be an effective means for preventing the loss of neurological function in SCI, or other acute injuries such as ischemia and traumatic brain injuries.

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.

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

FoxJ1 regulates spinal cord development and is required for the maintenance of spinal cord stem cell potential

ABSTRACT Development of the spinal cord requires dynamic and tightly controlled expression of numerous transcription factors. Forkhead Box protein J1 (FoxJ1) is a transcription factor involved in ciliogenesis and is specifically expressed in ependymal cells (ECs) in the adult central nervous system. However, using FoxJ1 fate‐mapping mouse lines, we observed that FoxJ1 is also transiently expressed by the progenitors of other neural subtypes during development. Moreover, using a knock‐in mouse line, we discovered that FoxJ1 is essential for embryonic progenitors to follow a normal developmental trajectory. FoxJ1 loss perturbed embryonic progenitor proliferation and cell fate determination, and resulted in formation of adult ECs having impaired stem cell potential and an inability to respond to spinal cord injury in both male and female animals. Thus, our study uncovers unexpected developmental functions of FoxJ1 in cell fate determination of subsets of neural cells and suggests that FoxJ1 is critical for maintaining the stem cell potential of ECs into adulthood.