A reverse genetics and genomics approach to gene paralog function and disease: Myokymia and the juxtaparanode.

The leucine-rich glioma-inactivated (LGI) family consists of four highly conserved paralogous genes, LGI1-4, that are highly expressed in mammalian central and/or peripheral nervous systems. LGI1 antibodies are detected in subjects with autoimmune limbic encephalitis and peripheral nerve hyperexcitability syndromes (PNHSs) such as Isaacs and Morvan syndromes. Pathogenic variations of LGI1 and LGI4 are associated with neurological disorders as disease traits including familial temporal lobe epilepsy and neurogenic arthrogryposis multiplex congenita 1 with myelin defects, respectively. No human disease has been reported associated with either LGI2 or LGI3. We implemented exome sequencing and family-based genomics to identify individuals with deleterious variants in LGI3 and utilized GeneMatcher to connect practitioners and researchers worldwide to investigate the clinical and electrophysiological phenotype in affected subjects. We also generated Lgi3-null mice and performed peripheral nerve dissection and immunohistochemistry to examine the juxtaparanode LGI3 microarchitecture. As a result, we identified 16 individuals from eight unrelated families with loss-of-function (LoF) bi-allelic variants in LGI3. Deep phenotypic characterization showed LGI3 LoF causes a potentially clinically recognizable PNHS trait characterized by global developmental delay, intellectual disability, distal deformities with diminished reflexes, visible facial myokymia, and distinctive electromyographic features suggestive of motor nerve instability. Lgi3-null mice showed reduced and mis-localized Kv1 channel complexes in myelinated peripheral axons. Our data demonstrate bi-allelic LoF variants in LGI3 cause a clinically distinguishable disease trait of PNHS, most likely caused by disturbed Kv1 channel distribution in the absence of LGI3.

Cellular senescence in progenitor cells contributes to diminished remyelination potential in progressive multiple sclerosis.

Cellular senescence is a form of adaptive cellular physiology associated with aging. Cellular senescence causes a proinflammatory cellular phenotype that impairs tissue regeneration, has been linked to stress, and is implicated in several human neurodegenerative diseases. We had previously determined that neural progenitor cells (NPCs) derived from induced pluripotent stem cell (iPSC) lines from patients with primary progressive multiple sclerosis (PPMS) failed to promote oligodendrocyte progenitor cell (OPC) maturation, whereas NPCs from age-matched control cell lines did so efficiently. Herein, we report that expression of hallmarks of cellular senescence were identified in SOX2+ progenitor cells within white matter lesions of human progressive MS (PMS) autopsy brain tissues and iPS-derived NPCs from patients with PPMS. Expression of cellular senescence genes in PPMS NPCs was found to be reversible by treatment with rapamycin, which then enhanced PPMS NPC support for oligodendrocyte (OL) differentiation. A proteomic analysis of the PPMS NPC secretome identified high-mobility group box-1 (HMGB1), which was found to be a senescence-associated inhibitor of OL differentiation. Transcriptome analysis of OPCs revealed that senescent NPCs induced expression of epigenetic regulators mediated by extracellular HMGB1. Lastly, we determined that progenitor cells are a source of elevated HMGB1 in human white matter lesions. Based on these data, we conclude that cellular senescence contributes to altered progenitor cell functions in demyelinated lesions in MS. Moreover, these data implicate cellular aging and senescence as a process that contributes to remyelination failure in PMS, which may impact how this disease is modeled and inform development of future myelin regeneration strategies.

After Nerve Injury, Lineage Tracing Shows That Myelin and Remak Schwann Cells Elongate Extensively and Branch to Form Repair Schwann Cells, Which Shorten Radically on Remyelination.

There is consensus that, distal to peripheral nerve injury, myelin and Remak cells reorganize to form cellular columns, Bungner's bands, which are indispensable for regeneration. However, knowledge of the structure of these regeneration tracks has not advanced for decades and the structure of the cells that form them, denervated or repair Schwann cells, remains obscure. Furthermore, the origin of these cells from myelin and Remak cells and their ability to give rise to myelin cells after regeneration has not been demonstrated directly, although these conversions are believed to be central to nerve repair. Using genetic lineage-tracing and scanning-block face electron microscopy, we show that injury of sciatic nerves from mice of either sex triggers extensive and unexpected Schwann cell elongation and branching to form long, parallel processes. Repair cells are 2- to 3-fold longer than myelin and Remak cells and 7- to 10-fold longer than immature Schwann cells. Remarkably, when repair cells transit back to myelinating cells, they shorten ∼7-fold to generate the typically short internodes of regenerated nerves. The present experiments define novel morphological transitions in injured nerves and show that repair Schwann cells have a cell-type-specific structure that differentiates them from other cells in the Schwann cell lineage. They also provide the first direct evidence using genetic lineage tracing for two basic assumptions in Schwann cell biology: that myelin and Remak cells generate the elongated cells that build Bungner bands in injured nerves and that such cells can transform to myelin cells after regeneration.SIGNIFICANCE STATEMENT After injury to peripheral nerves, the myelin and Remak Schwann cells distal to the injury site reorganize and modify their properties to form cells that support the survival of injured neurons, promote axon growth, remove myelin-associated growth inhibitors, and guide regenerating axons to their targets. We show that the generation of these repair-supportive Schwann cells involves an extensive cellular elongation and branching, often to form long, parallel processes. This generates a distinctive repair cell morphology that is favorable for the formation of the regeneration tracks that are essential for nerve repair. Remyelination, conversely, involves a striking cell shortening to form the typical short myelin cells of regenerated nerves. We also provide evidence for direct lineage relationships between: (1) repair cells and myelin and Remak cells of uninjured nerves and (2) remyelinating cells in regenerated nerves.

Graded Elevation of c-Jun in Schwann Cells In Vivo: Gene Dosage Determines Effects on Development, Remyelination, Tumorigenesis, and Hypomyelination.

Schwann cell c-Jun is implicated in adaptive and maladaptive functions in peripheral nerves. In injured nerves, this transcription factor promotes the repair Schwann cell phenotype and regeneration and promotes Schwann-cell-mediated neurotrophic support in models of peripheral neuropathies. However, c-Jun is associated with tumor formation in some systems, potentially suppresses myelin genes, and has been implicated in demyelinating neuropathies. To clarify these issues and to determine how c-Jun levels determine its function, we have generated c-Jun OE/+ and c-Jun OE/OE mice with graded expression of c-Jun in Schwann cells and examined these lines during development, in adulthood, and after injury using RNA sequencing analysis, quantitative electron microscopic morphometry, Western blotting, and functional tests. Schwann cells are remarkably tolerant of elevated c-Jun because the nerves of c-Jun OE/+ mice, in which c-Jun is elevated ∼6-fold, are normal with the exception of modestly reduced myelin thickness. The stronger elevation of c-Jun in c-Jun OE/OE mice is, however, sufficient to induce significant hypomyelination pathology, implicating c-Jun as a potential player in demyelinating neuropathies. The tumor suppressor P19ARF is strongly activated in the nerves of these mice and, even in aged c-Jun OE/OE mice, there is no evidence of tumors. This is consistent with the fact that tumors do not form in injured nerves, although they contain proliferating Schwann cells with strikingly elevated c-Jun. Furthermore, in crushed nerves of c-Jun OE/+ mice, where c-Jun levels are overexpressed sufficiently to accelerate axonal regeneration, myelination and function are restored after injury.SIGNIFICANCE STATEMENT In injured and diseased nerves, the transcription factor c-Jun in Schwann cells is elevated and variously implicated in controlling beneficial or adverse functions, including trophic Schwann cell support for neurons, promotion of regeneration, tumorigenesis, and suppression of myelination. To analyze the functions of c-Jun, we have used transgenic mice with graded elevation of Schwann cell c-Jun. We show that high c-Jun elevation is a potential pathogenic mechanism because it inhibits myelination. Conversely, we did not find a link between c-Jun elevation and tumorigenesis. Modest c-Jun elevation, which is beneficial for regeneration, is well tolerated during Schwann cell development and in the adult and is compatible with restoration of myelination and nerve function after injury.

c-Jun activation in Schwann cells protects against loss of sensory axons in inherited neuropathy.

Charcot-Marie-Tooth disease type 1A is the most frequent inherited peripheral neuropathy. It is generally due to heterozygous inheritance of a partial chromosomal duplication resulting in over-expression of PMP22. A key feature of Charcot-Marie-Tooth disease type 1A is secondary death of axons. Prevention of axonal loss is therefore an important target of clinical intervention. We have previously identified a signalling mechanism that promotes axon survival and prevents neuron death in mechanically injured peripheral nerves. This work suggested that Schwann cells respond to injury by activating/enhancing trophic support for axons through a mechanism that depends on upregulation of the transcription factor c-Jun in Schwann cells, resulting in the sparing of axons that would otherwise die. As c-Jun orchestrates Schwann cell support for distressed neurons after mechanical injury, we have now asked: do Schwann cells also activate a c-Jun dependent neuron-supportive programme in inherited demyelinating disease? We tested this by using the C3 mouse model of Charcot-Marie-Tooth disease type 1A. In line with our previous findings in humans with Charcot-Marie-Tooth disease type 1A, we found that Schwann cell c-Jun was elevated in (uninjured) nerves of C3 mice. We determined the impact of this c-Jun activation by comparing C3 mice with double mutant mice, namely C3 mice in which c-Jun had been conditionally inactivated in Schwann cells (C3/Schwann cell-c-Jun(-/-) mice), using sensory-motor tests and electrophysiological measurements, and by counting axons in proximal and distal nerves. The results indicate that c-Jun elevation in the Schwann cells of C3 nerves serves to prevent loss of myelinated sensory axons, particularly in distal nerves, improve behavioural symptoms, and preserve F-wave persistence. This suggests that Schwann cells have two contrasting functions in Charcot-Marie-Tooth disease type 1A: on the one hand they are the genetic source of the disease, on the other, they respond to it by mounting a c-Jun-dependent response that significantly reduces its impact. Because axonal death is a central feature of much nerve pathology it will be important to establish whether an axon-supportive Schwann cell response also takes place in other conditions. Amplification of this axon-supportive mechanism constitutes a novel target for clinical intervention that might be useful in Charcot-Marie-Tooth disease type 1A and other neuropathies that involve axon loss.

CRISPR-edited human ES-derived oligodendrocyte progenitor cells improve remyelination in rodents.

In Multiple Sclerosis (MS), inflammatory demyelinated lesions in the brain and spinal cord lead to neurodegeneration and progressive disability. Remyelination can restore fast saltatory conduction and neuroprotection but is inefficient in MS especially with increasing age, and is not yet treatable with therapies. Intrinsic and extrinsic inhibition of oligodendrocyte progenitor cell (OPC) function contributes to remyelination failure, and we hypothesised that the transplantation of 'improved' OPCs, genetically edited to overcome these obstacles, could improve remyelination. Here, we edit human(h) embryonic stem cell-derived OPCs to be unresponsive to a chemorepellent released from chronic MS lesions, and transplant them into rodent models of chronic lesions. Edited hOPCs display enhanced migration and remyelination compared to controls, regardless of the host age and length of time post-transplant. We show that genetic manipulation and transplantation of hOPCs overcomes the negative environment inhibiting remyelination, with translational implications for therapeutic strategies for people with progressive MS.

Brain matters: unveiling the distinct contributions of region, age, and sex to glia diversity and CNS function.

The myelinated white matter tracts of the central nervous system (CNS) are essential for fast transmission of electrical impulses and are often differentially affected in human neurodegenerative diseases across CNS region, age and sex. We hypothesize that this selective vulnerability is underpinned by physiological variation in white matter glia. Using single nucleus RNA sequencing of human post-mortem white matter samples from the brain, cerebellum and spinal cord and subsequent tissue-based validation we found substantial glial heterogeneity with tissue region: we identified region-specific oligodendrocyte precursor cells (OPCs) that retain developmental origin markers into adulthood, distinguishing them from mouse OPCs. Region-specific OPCs give rise to similar oligodendrocyte populations, however spinal cord oligodendrocytes exhibit markers such as SKAP2 which are associated with increased myelin production and we found a spinal cord selective population particularly equipped for producing long and thick myelin sheaths based on the expression of genes/proteins such as HCN2. Spinal cord microglia exhibit a more activated phenotype compared to brain microglia, suggesting that the spinal cord is a more pro-inflammatory environment, a difference that intensifies with age. Astrocyte gene expression correlates strongly with CNS region, however, astrocytes do not show a more activated state with region or age. Across all glia, sex differences are subtle but the consistent increased expression of protein-folding genes in male donors hints at pathways that may contribute to sex differences in disease susceptibility. These findings are essential to consider for understanding selective CNS pathologies and developing tailored therapeutic strategies.

Age-related loss of axonal regeneration is reflected by the level of local translation.

Regeneration capacity is reduced as CNS axons mature. Using laser-mediated axotomy, proteomics and puromycin-based tagging of newly-synthesized proteins in a human embryonic stem cell-derived neuron culture system that allows isolation of axons from cell bodies, we show here that efficient regeneration in younger axons (d45 in culture) is associated with local axonal protein synthesis (local translation). Enhanced regeneration, promoted by co-culture with human glial precursor cells, is associated with increased axonal synthesis of proteins, including those constituting the translation machinery itself. Reduced regeneration, as occurs with the maturation of these axons by d65 in culture, correlates with reduced levels of axonal proteins involved in translation and an inability to respond by increased translation of regeneration promoting axonal mRNAs released from stress granules. Together, our results provide evidence that, as in development and in the PNS, local translation contributes to CNS axon regeneration.

Failures of nerve regeneration caused by aging or chronic denervation are rescued by restoring Schwann cell c-Jun.

After nerve injury, myelin and Remak Schwann cells reprogram to repair cells specialized for regeneration. Normally providing strong regenerative support, these cells fail in aging animals, and during chronic denervation that results from slow axon growth. This impairs axonal regeneration and causes significant clinical problems. In mice, we find that repair cells express reduced c-Jun protein as regenerative support provided by these cells declines during aging and chronic denervation. In both cases, genetically restoring Schwann cell c-Jun levels restores regeneration to control levels. We identify potential gene candidates mediating this effect and implicate Shh in the control of Schwann cell c-Jun levels. This establishes that a common mechanism, reduced c-Jun in Schwann cells, regulates success and failure of nerve repair both during aging and chronic denervation. This provides a molecular framework for addressing important clinical problems, suggesting molecular pathways that can be targeted to promote repair in the PNS.

Reprogramming of Fibroblasts to Oligodendrocyte Progenitor-like Cells Using CRISPR/Cas9-Based Synthetic Transcription Factors.

Cell lineage reprogramming via transgene overexpression of key master regulatory transcription factors has been well documented. However, the poor efficiency and lack of fidelity of this approach is problematic. Synthetic transcription factors (sTFs)-built from the repurposed CRISPR/Cas9 system-can activate endogenous target genes to direct differentiation or trigger lineage reprogramming. Here we explored whether sTFs could be used to steer mouse neural stem cells and mouse embryonic fibroblasts toward the oligodendrocyte lineage. We developed a non-viral modular expression system to enable stable multiplex delivery of pools of sTFs capable of transcriptional activation of three key oligodendrocyte lineage master regulatory genes (Sox10, Olig2, and Nkx6-2). Delivery of these sTFs could enhance neural stem cell differentiation and initiated mouse embryonic fibroblast direct reprograming toward oligodendrocyte progenitor-like cells. Our findings demonstrate the value of sTFs as tools for activating endogenous genes and directing mammalian cell-type identity.

Multicellular rosette formation during cell ingression in the avian primitive streak.

Cell movements are a fundamental feature during the development of multi-cellular organisms. In amniote gastrulation, cells ingress through the primitive streak, which identifies the anterior-posterior axis of the embryo. We investigated the cytoskeletal architecture during these morphogenetic processes and characterized microtubule organisation in whole chick embryos. This revealed the distribution of cells with polarized and radial microtubule (MT) arrays across different regions of the embryo. Cells in the epiblast usually displayed radial MT-arrays, while the majority of cells in the primitive streak had polarized MT-arrays. Within the primitive streak, many cells organized into groups and were arranged in rosette-like structures with a distinct centre characterized by an accumulation of actin. Extended confocal microscopy and three-dimensional image reconstruction identified tips of polarized cells that were protruding from the plane of rosettes, usually from the centre. We propose that organization into higher order structures facilitates cell ingression during gastrulation.