Gli1 regulates the postnatal acquisition of peripheral nerve architecture
Peripheral nerves are organized into discrete compartments. Axons, Schwann cells (SCs), and endoneurial fibroblasts (EFs) reside within the endoneurium and are surrounded by the perineurium - a cellular sheath comprised of layers of perineurial glia (PNG). SC secretion of Desert Hedgehog (Dhh) regulates this organization. In Dhh nulls, the perineurium is deficient and the endoneurium is subdivided into small compartments termed minifascicles. Human Dhh mutations cause a neuropathy with similar defects. Here we examine the role of Gli1, a canonical transcriptional effector of hedgehog signaling, in regulating peripheral nerve organization in mice of both genders. We identify PNG, EFs, and pericytes as Gli1-expressing cells by genetic fate mapping. Although expression of Dhh by SCs and Gli1 in target cells is coordinately regulated with myelination, Gli1 expression unexpectedly persists in Dhh null EFs. Thus, Gli1 is expressed in EFs non-canonically i.e., independent of hedgehog signaling. Gli1 and Dhh also have non-redundant activities. Unlike Dhh nulls, Gli1 nulls have a normal perineurium. Like Dhh nulls, Gli1 nulls form minifascicles, which we show likely arise from EFs. Thus, Dhh and Gli1 are independent signals: Gli1 is dispensable for perineurial development but functions cooperatively with Dhh to drive normal endoneurial development. During development, Gli1 also regulates endoneurial extracellular matrix production, nerve vascular organization, and has modest, non-autonomous effects on SC sorting and myelination of axons. Finally, in adult nerves, induced deletion of Gli1 is sufficient to drive minifascicle formation. Thus, Gli1 regulates the development and is required to maintain the endoneurial architecture of peripheral nerves.SIGNIFICANCE STATEMENTPeripheral nerves are organized into distinct cellular/ECM compartments: the epineurium, perineurium and endoneurium. This organization, with its associated cellular constituents, are critical for the structural and metabolic support of nerves and their response to injury. Here, we show Gli1 - a transcription factor normally expressed downstream of hedgehog signaling - is required for the proper organization of the endoneurium but not the perineurium. Unexpectedly, Gli1 expression by endoneurial cells is independent of, and functions non-redundantly with, Schwann Cell-derived Desert Hedgehog in regulating peripheral nerve architecture. These results further delineate how peripheral nerves acquire their distinctive organization during normal development and highlight mechanisms that may regulate their reorganization in pathologic settings including peripheral neuropathies and nerve injury.
Transcriptomic analysis of loss of Gli1 in neural stem cells responding to demyelination in the mouse brain
In the adult mammalian brain, Gli1 expressing neural stem cells reside in the subventricular zone and their progeny are recruited to sites of demyelination in the white matter where they generate new oligodendrocytes, the myelin forming cells. Remarkably, genetic loss or pharmacologic inhibition of Gli1 enhances the efficacy of remyelination by these neural stem cells. To understand the molecular mechanisms involved, we performed a transcriptomic analysis of this Gli1-pool of neural stem cells. We compared murine NSCs with either intact or deficient Gli1 expression from adult mice on a control diet or on a cuprizone diet which induces widespread demyelination. These data will be a valuable resource for identifying therapeutic targets for enhancing remyelination in demyelinating diseases like multiple sclerosis.
Dual Color, Live Imaging of Vesicular Transport in Axons of Cultured Sensory Neurons
The function of neurons in afferent reception, integration, and generation of electrical activity relies on their strikingly polarized organization, characterized by distinct membrane domains. These domains have different compositions resulting from a combination of selective targeting and retention of membrane proteins. In neurons, most proteins are delivered from their site of synthesis in the soma to the axon via anterograde vesicular transport and undergo retrograde transport for redistribution and/or lysosomal degradation. A key question is whether proteins destined for the same domain are transported in separate vesicles for local assembly or whether these proteins are pre-assembled and co-transported in the same vesicles for delivery to their cognate domains. To assess the content of transport vesicles, one strategy relies on staining of sciatic nerves after ligation, which drives the accumulation of anterogradely and retrogradely transported vesicles on the proximal and distal side of the ligature, respectively. This approach may not permit confident assessment of the nature of the intracellular vesicles identified by staining, and analysis is limited to the availability of suitable antibodies. Here, we use dual color live imaging of proteins labeled with different fluorescent tags, visualizing anterograde and retrograde axonal transport of several proteins simultaneously. These proteins were expressed in rat dorsal root ganglion (DRG) neurons cultured alone or with Schwann cells under myelinating conditions to assess whether glial cells modify the patterns of axonal transport. Advantages of this protocol are the dynamic identification of transport vesicles and characterization of their content for various proteins that is not limited by available antibodies.
Activated microglia drive demyelination via CSF1R signaling
Microgliosis is a prominent pathological feature in many neurological diseases including multiple sclerosis (MS), a progressive auto-immune demyelinating disorder. The precise role of microglia, parenchymal central nervous system (CNS) macrophages, during demyelination, and the relative contributions of peripheral macrophages are incompletely understood. Classical markers used to identify microglia do not reliably discriminate between microglia and peripheral macrophages, confounding analyses. Here, we use a genetic fate mapping strategy to identify microglia as predominant responders and key effectors of demyelination in the cuprizone (CUP) model. Colony-stimulating factor 1 (CSF1), also known as macrophage colony-stimulating factor (M-CSF) - a secreted cytokine that regulates microglia development and survival-is upregulated in demyelinated white matter lesions. Depletion of microglia with the CSF1R inhibitor PLX3397 greatly abrogates the demyelination, loss of oligodendrocytes, and reactive astrocytosis that results from CUP treatment. Electron microscopy (EM) and serial block face imaging show myelin sheaths remain intact in CUP treated mice depleted of microglia. However, these CUP-damaged myelin sheaths are lost and robustly phagocytosed upon-repopulation of microglia. Direct injection of CSF1 into CNS white matter induces focal microgliosis and demyelination indicating active CSF1 signaling can promote demyelination. Finally, mice defective in adopting a toxic astrocyte phenotype that is driven by microglia nevertheless demyelinate normally upon CUP treatment implicating microglia rather than astrocytes as the primary drivers of CUP-mediated demyelination. Together, these studies indicate activated microglia are required for and can drive demyelination directly and implicate CSF1 signaling in these events.
Relative Levels of Gli1 and Gli2 Determine the Response of Ventral Neural Stem Cells to Demyelination
Enhancing repair of myelin is an important therapeutic goal in many neurological disorders characterized by demyelination. In the healthy adult brain, ventral neural stem cells (vNSCs) in the subventricular zone, marked by GLI1 expression, do not generate oligodendrocytes. However, in response to demyelination, their progeny are recruited to lesions where they differentiate into oligodendrocytes and ablation of GLI1 further enhances remyelination. GLI1 and GLI2 are closely related transcriptional activators but the role of GLI2 in remyelination by vNSCs is not clear. Here, we show that genetic ablation of Gli1 in vNSCs increases GLI2 expression and combined loss of both transcription factors decreases the recruitment and differentiation of their progeny in demyelinated lesions. These results indicate that GLI1 and GLI2 have distinct, non-redundant functions in vNSCs and their relative levels play an essential role in the response to demyelination.
Accumulation of Neurofascin at nodes of Ranvier is regulated by a Paranodal Switch
The paranodal junctions flank mature nodes of Ranvier and provide a barrier between ion channels at the nodes and juxtaparanodes. These junctions also promote node assembly and maintenance by mechanisms that are poorly understood. Here, we examine their role in the accumulation of NF186, a key adhesion molecule of PNS and CNS nodes. We previously showed NF186 is initially targeted/accumulates via its ectodomain to forming PNS (hemi)nodes by diffusion trapping whereas it is later targeted to mature nodes by a transport-dependent mechanism mediated by its cytoplasmic segment. To address the role of the paranodes in this switch, we compared accumulation of NF186 ectodomain and cytoplasmic domain constructs in wild type vs. paranode defective, i.e. Caspr-null mice. Both pathways are affected in the paranodal mutants. In the PNS of Caspr-null mice, diffusion trapping mediated by the NF186 ectodomain aberrantly persists into adulthood whereas the cytoplasmic domain/transport-dependent targeting is impaired. In contrast, accumulation of NF186 at CNS nodes does not undergo a switch - it is predominantly targeted to both forming and mature CNS nodes via its cytoplasmic domain and requires intact paranodes. FRAP analysis indicates the paranodes provide a membrane diffusion barrier that normally precludes diffusion of NF186 to nodes. Linkage of paranodal proteins to the underlying cytoskeleton likely contributes to this diffusion barrier based on 4.1B and Î²II spectrin expression in Caspr-null mice. Together, these results implicate the paranodes as membrane diffusion barriers that regulate targeting to nodes and highlight differences in the assembly of PNS and CNS nodes.SIGNIFICANCE STATEMENTNodes of Ranvier are essential for effective saltatory conduction along myelinated axons. A major question is how the various axonal proteins that comprise the multimeric nodal complex accumulate at this site. Here we examine how targeting of NF186, a key nodal adhesion molecule, is regulated by the flanking paranodal junctions. We show the transition from diffusion-trapping to transport-dependent accumulation of NF186 requires the paranodal junctions. We also demonstrate that these junctions are a barrier to diffusion of axonal proteins into the node and highlight differences in PNS and CNS node assembly. These results provide new insights into the mechanism of node assembly and the pathophysiology of neurological disorders in which impaired paranodal function contributes to clinical disability.
Control of Channel Clustering by Cleavage
Enrichment of sodium channels at nodes of Ranvier, a hallmark of myelinated axons, underlies effective saltatory conduction. In this issue of Neuron, Eshed-Eisenbach etÂ al. (2020) demonstrate that proteolysis of gliomedin, which drives initial channel clustering, provides a novel mechanism to ensure fidelity of channel localization to nodes.
Independent anterograde transport and retrograde cotransport of domain components of myelinated axons
Neurons are highly polarized cells organized into functionally and molecularly distinct domains. A key question is whether the multiprotein complexes that comprise these domains are preassembled, transported, and inserted as a complex or whether their components are transported independently and assemble locally. Here, we have dynamically imaged, in pairwise combinations, the vesicular transport of fluorescently tagged components of the nodes of Ranvier and other myelinated axonal domains in sensory neurons cultured alone or together with Schwann cells at the onset of myelination. In general, most proteins are transported independently in the anterograde direction. In contrast, there is substantial cotransport of proteins from distinct domains in the retrograde direction likely due to coendocytosis along the axon. Early myelination did not substantially change these patterns of transport, although it increased the overall numbers of axonal transport vesicles. Our results indicate domain components are transported in separate vesicles for local assembly, not as preformed complexes, and implicate endocytosis along axons as a mechanism of clearance.
An unfolding role for ankyrin-G at the axon initial segment
Necl-4/Cadm4 recruits Par-3 to the Schwann cell adaxonal membrane
Interactions between axons and Schwann cells are essential for the acquisition of Schwann cell radial and longitudinal polarity and myelin sheath assembly. In the internode, the largest of these longitudinal domains, axon-Schwann cell interactions are mediated by the Nectin-like (Necl) cell adhesion proteins, also known as SynCAMs or Cadms. In particular, Necl-1/Cadm3 expressed on the axon surface binds to Necl-4/Cadm4 expressed along the adaxonal membrane of myelinating Schwann cells. Necl-4 promotes myelination in vitro and is required for the timely onset of myelination and the fidelity of the organization of the myelin sheath and the internode in vivo. A key question is the identity of the downstream effectors of Necl-4 that mediate its effects. The cytoplasmic terminal region (CTR) of Necl-4 contains a PDZ-domain binding motif. Accordingly, we used the CTR of Necl-4 in an unbiased proteomic screen of PDZ-domain proteins. We identify Par-3, a multi-PDZ domain containing protein of the Par-aPKC polarity complex previously implicated in myelination, as an interacting protein. Necl-4 and Par-3 are colocalized along the inner Schwann cell membrane and coprecipitate from Schwann cell lysates. The CTR of Necl-4 binds to the first PDZ domain of Par-3 thereby recruiting Par-3 to sites of Necl-4/Necl-1 interaction. Knockdown of Necl-4 perturbs Par-3 localization to the inner membrane of Schwann cells in myelinating co-cultures. These findings implicate interactions of Necl-1/Necl-4 in the recruitment of Par-3 to the Schwann cell adaxonal membrane and the establishment of Schwann cell radial polarity.