PUFFFIN: an ultra-bright, customisable, single-plasmid system for labelling cell neighbourhoods.

Cell communication coordinates developmental processes, maintains homeostasis, and contributes to disease. Therefore, understanding the relationship between cells in a shared environment is crucial. Here we introduce Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a cell neighbour-labelling system based upon secretion and uptake of positively supercharged fluorescent protein s36GFP. We fused s36GFP to mNeonGreen or to a HaloTag, facilitating ultra-bright, sensitive, colour-of-choice labelling. Secretor cells transfer PUFFFIN to neighbours while retaining nuclear mCherry, making identification, isolation, and investigation of live neighbours straightforward. PUFFFIN can be delivered to cells, tissues, or embryos on a customisable single-plasmid construct composed of interchangeable components with the option to incorporate any transgene. This versatility enables the manipulation of cell properties, while simultaneously labelling surrounding cells, in cell culture or in vivo. We use PUFFFIN to ask whether pluripotent cells adjust the pace of differentiation to synchronise with their neighbours during exit from naïve pluripotency. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour.

Origin and Emergence of Microglia in the CNS-An Interesting (Hi)story of an Eccentric Cell.

Microglia belong to tissue-resident macrophages of the central nervous system (CNS), representing the primary innate immune cells. This cell type constitutes ~7% of non-neuronal cells in the mammalian brain and has a variety of biological roles integral to homeostasis and pathophysiology from the late embryonic to adult brain. Its unique identity that distinguishes its "glial" features from tissue-resident macrophages resides in the fact that once entering the CNS, it is perennially exposed to a unique environment following the formation of the blood-brain barrier. Additionally, tissue-resident macrophage progenies derive from various peripheral sites that exhibit hematopoietic potential, and this has resulted in interpretation issues surrounding their origin. Intensive research endeavors have intended to track microglial progenitors during development and disease. The current review provides a corpus of recent evidence in an attempt to disentangle the birthplace of microglia from the progenitor state and underlies the molecular elements that drive microgliogenesis. Furthermore, it caters towards tracking the lineage spatiotemporally during embryonic development and outlining microglial repopulation in the mature CNS. This collection of data can potentially shed light on the therapeutic potential of microglia for CNS perturbations across various levels of severity.

Androgens show sex-dependent differences in myelination in immune and non-immune murine models of CNS demyelination.

Neuroprotective, anti-inflammatory, and remyelinating properties of androgens are well-characterized in demyelinated male mice and men suffering from multiple sclerosis. However, androgen effects mediated by the androgen receptor (AR), have been only poorly studied in females who make low androgen levels. Here, we show a predominant microglial AR expression in demyelinated lesions from female mice and women with multiple sclerosis, but virtually undetectable AR expression in lesions from male animals and men with multiple sclerosis. In female mice, androgens and estrogens act in a synergistic way while androgens drive microglia response towards regeneration. Transcriptomic comparisons of demyelinated mouse spinal cords indicate that, regardless of the sex, androgens up-regulate genes related to neuronal function integrity and myelin production. Depending on the sex, androgens down-regulate genes related to the immune system in females and lipid catabolism in males. Thus, androgens are required for proper myelin regeneration in females and therapeutic approaches of demyelinating diseases need to consider male-female differences.

New oligodendrocytes exhibit more abundant and accurate myelin regeneration than those that survive demyelination.

Oligodendrocytes that survive demyelination can remyelinate, including in multiple sclerosis (MS), but how they do so is unclear. In this study, using zebrafish, we found that surviving oligodendrocytes make few new sheaths and frequently mistarget new myelin to neuronal cell bodies, a pathology we also found in MS. In contrast, oligodendrocytes generated after demyelination make abundant and correctly targeted sheaths, indicating that they likely also have a better regenerative potential in MS.

Selective vulnerability of inhibitory networks in multiple sclerosis.

In multiple sclerosis (MS), a chronic demyelinating disease of the central nervous system, neurodegeneration is detected early in the disease course and is associated with the long-term disability of patients. Neurodegeneration is linked to both inflammation and demyelination, but its exact cause remains unknown. This gap in knowledge contributes to the current lack of treatments for the neurodegenerative phase of MS. Here we ask if neurodegeneration in MS affects specific neuronal components and if it is the result of demyelination. Neuropathological examination of secondary progressive MS motor cortices revealed a selective vulnerability of inhibitory interneurons in MS. The generation of a rodent model of focal subpial cortical demyelination reproduces this selective neurodegeneration providing a new preclinical model for the study of neuroprotective treatments.

Cryogel scaffolds for regionally constrained delivery of lysophosphatidylcholine to central nervous system slice cultures: A model of focal demyelination for multiple sclerosis research.

The pathology of multiple sclerosis (MS) is typified by focal demyelinated areas of the brain and spinal cord, which results in axonal degeneration and atrophy. Although the field has made much progress in developing immunomodulatory therapies to reduce the occurrence of these focal lesions, there is a conspicuous lack of licensed effective therapies to reduce axonal degeneration or promote repair. Remyelination, carried out by oligodendrocytes, does occur in MS, and is protective against axonal degeneration. Unfortunately, remyelination is not very efficient, and ultimately fails and so there is a research focus to generate new therapeutics to enhance remyelination leading to neuroprotection. To develop these therapies, we need preclinical models that well reflect remyelination in MS. We have previously characterized an ex vivo model that uses lysophosphatidylcholine (LPC) to cause acute and global demyelination of tissue slices, followed by spontaneous remyelination, which has been widely used as a surrogate for in vivo rodent models of demyelination. However, this ex vivo model lacks the focal demyelinated lesions seen in MS, surrounded by normal tissue from which the repairing oligodendrocytes are derived. Therefore, to improve the model, we have developed and characterized small macroporous cryogel scaffolds for controlled/regional delivery of LPC with diameters of either 0.5, 1 or 2 mm. Placement of LPC loaded scaffolds adjacent to ex vivo cultured mouse brain and spinal cord slices induced focal areas of demyelination in proximity to the scaffold. To the best of our knowledge, this is the first such report of spatial mimicry of the in vivo condition in ex vivo tissue culture. This will allow not only the investigation into focal lesions, but also provides a better platform technology with which to test remyelination-promoting therapeutics. STATEMENT OF SIGNIFICANCE: This manuscript is the first report of using macroporous hydrogels (cryogels) as a research tool for lysophosphatidylcholine (LPC) delivery, in order to create an ex vivo model of focal demyelination in the brain and spinal cord, which is of great relevance to multiple sclerosis research. Here, we transform an existing ex vivo model of demyelination by delivering LPC to focal regions of brain and spinal cord slice cultures. We have developed an easy-to-handle cylindrical and macroporous PEG-based sponge-like scaffold material (cryogel) that can deliver LPC only to a small area of the slice. Such cryogels are ideal as a delivery system in this culture model as they exhibit a soft but robust nature, with high mechanical deformability in their dry and swollen state, with no need to stay permanently hydrated. In addition, the synthesis of these cryogels is simple and easy to reproduce via photochemical cryopolymerisation using a PEG-diacrylate monomer and a photoinitiator, which are both commercially available. This more accurate model of demyelination will not only allow researchers to gain a better understanding of the CNS remyelination process in diseases such as MS, but also provides a platform technology, which could be utilized to screen and test pro-remyelination compounds which may help to find new therapeutics for progressive MS.

Alkyne-Tagged PLGA Allows Direct Visualization of Nanoparticles In Vitro and Ex Vivo by Stimulated Raman Scattering Microscopy.

Polymeric nanoparticles (NPs) are attractive candidates for the controlled and targeted delivery of therapeutics in vitro and in vivo. However, detailed understanding of the uptake, location, and ultimate cellular fate of the NPs is necessary to satisfy safety concerns, which is difficult because of the nanoscale size of these carriers. In this work, we show how small chemical labels can be appended to poly(lactic acid-co-glycolic acid) (PLGA) to synthesize NPs that can then be imaged by stimulated Raman scattering microscopy, a vibrational imaging technique that can elucidate bond-specific information in biological environments, such as the identification of alkyne signatures in modified PLGA terpolymers. We show that both deuterium and alkyne labeled NPs can be imaged within primary rat microglia, and the alkyne NPs can also be imaged in ex vivo cortical mouse brain tissue. Immunohistochemical analysis confirms that the NPs localize in microglia in the mouse brain tissue, demonstrating that these NPs have the potential to deliver therapeutics selectively to microglia.

The Lysosomal Transcription Factor TFEB Represses Myelination Downstream of the Rag-Ragulator Complex.

Myelin allows for fast and efficient axonal conduction, but much remains to be determined about the mechanisms that regulate myelin formation. To investigate the genetic basis of myelination, we carried out a genetic screen using zebrafish. Here, we show that the lysosomal G protein RagA is essential for CNS myelination. In rraga-/- mutant oligodendrocytes, target genes of the lysosomal transcription factor Tfeb are upregulated, consistent with previous evidence that RagA represses Tfeb activity. Loss of Tfeb function is sufficient to restore myelination in RagA mutants, indicating that hyperactive Tfeb represses myelination. Conversely, tfeb-/- single mutants exhibit ectopic myelin, further indicating that Tfeb represses myelination during development. In a mouse model of de- and remyelination, TFEB expression is increased in oligodendrocytes, but the protein is localized to the cytoplasm, and hence inactive, especially during remyelination. These results define essential regulators of myelination and may advance approaches to therapeutic remyelination.

The function of contactin-2/TAG-1 in oligodendrocytes in health and demyelinating pathology.

The oligodendrocyte maturation process and the transition from the pre-myelinating to the myelinating state are extremely important during development and in pathology. In the present study, we have investigated the role of the cell adhesion molecule CNTN2/TAG-1 on oligodendrocyte proliferation, differentiation, myelination, and function during development and under pathological conditions. With the combination of in vivo, in vitro, ultrastructural, and electrophysiological methods, we have mapped the expression of CNTN2 protein in the oligodendrocyte lineage during the different stages of myelination and its involvement on oligodendrocyte maturation, branching, myelin-gene expression, myelination, and axonal function. The cuprizone model of central nervous system demyelination was further used to assess CNTN2 in pathology. During development, CNTN2 can transiently affect the expression levels of myelin and myelin-regulating genes, while its absence results in reduced oligodendrocyte branching, hypomyelination of fiber tracts and impaired axonal conduction. In pathology, CNTN2 absence does not affect the extent of de- and remyelination. However during remyelination, a novel, CNTN2-independent mechanism is revealed that is able to recluster voltage gated potassium channels (VGKCs) resulting in the improvement of fiber conduction.

Inhibitory axons are targeted in hippocampal cell culture by anti-Caspr2 autoantibodies associated with limbic encephalitis.

Contactin-associated protein-like 2 (Caspr2), also known as CNTNAP2, is a cell adhesion molecule that clusters voltage-gated potassium channels (Kv1.1/1.2) at the juxtaparanodes of myelinated axons and may regulate axonal excitability. As a component of the Kv1 complex, Caspr2 has been identified as a target in neuromyotonia and Morvan syndrome, but also in some cases of autoimmune limbic encephalitis (LE). How anti-Caspr2 autoimmunity is linked with the central neurological symptoms is still elusive. In the present study, using anti-Caspr2 antibodies from seven patients affected by pure LE, we determined that IgGs in the cerebrospinal fluid of four out seven patients were selectively directed against the N-terminal Discoïdin and LamininG1 modules of Caspr2. Using live immunolabeling of cultured hippocampal neurons, we determined that serum IgGs in all patients strongly targeted inhibitory interneurons. Caspr2 was highly detected on GAD65-positive axons that are surrounding the cell bodies and at the VGAT-positive inhibitory presynaptic contacts. Functional assays indicated that LE autoantibodies may induce alteration of Gephyrin clusters at inhibitory synaptic contacts. Next, we generated a Caspr2-Fc chimera to reveal Caspr2 receptors on hippocampal neurons localized at the somato-dendritic compartment and post-synapse. Caspr2-Fc binding was strongly increased on TAG-1-transfected neurons and conversely, Caspr2-Fc did not bind hippocampal neurons from TAG-1-deficient mice. Our data indicate that Caspr2 may participate as a cell recognition molecule in the dynamics of inhibitory networks. This study provides new insight into the potential pathogenic effect of anti-Caspr2 autoantibodies in central hyperexcitability that may be related with perturbation of inhibitory interneuron activity.

Alterations of juxtaparanodal domains in two rodent models of CNS demyelination.

The segregation of myelinated fibers into distinct domains around the node of Ranvier--the perinodal areas--is crucial for nervous system homeostasis and efficient nerve conduction. Perinodal areas are formed by axo-glial interactions, namely the interaction of molecules between the axon and the myelinating glia. In a variety of demyelinating pathologies including multiple sclerosis, the molecular architecture of the myelinated fiber is disrupted, leading to axonal degeneration. In this study we have analyzed the alterations of TAG-1, Caspr2, and voltage-gated potassium channels (VGKCs), forming the juxtaparanodal tripartite complex, in relation to adjacent paranodal and nodal molecules, in two different models of CNS demyelination, the experimental autoimmune encephalomyelitis (EAE) and the cuprizone model of toxic demyelination. We found extensive alterations of the juxtaparanodal molecular architecture under de- and remyelinating conditions. Inflammation alone was sufficient to disrupt the borders between the domains leading to the diffusion of juxtaparanodal components to the adjacent paranodal area. EAE induction and cuprizone-induced demyelination resulted initially in paranodal domain elongation with subsequent diffusion of the juxtaparanodal components and the reduction of their expression levels. At later stages, with decreasing inflammation and spontaneous remyelination there was a partial restoration of the paranodal domain but not sufficient re-organization of the juxtaparanodes. The latter were re-formed only when complete remyelination was allowed in the cuprizone model, indicating that juxtaparanodal domain reorganization is a later event that may remain incomplete in a hostile inflammatory milieu.

Axons and myelinating glia: An intimate contact.

The coordination of the vertebrate nervous system requires high velocity signal transmission between different brain areas. High speed nerve conduction is achieved in the myelinated fibers of both the central and the peripheral nervous system where the myelin sheath acts as an insulator of the axon. The interactions between the glial cell and the adjacent axon, namely axo-glial interactions, segregate the fiber in distinct molecular and functional domains that ensure the rapid propagation of action potentials. These domains are the node of Ranvier, the paranode, the juxtaparanode and the internode and are characterized by multiprotein complexes between voltage-gated ion channels, cell adhesion molecules, members of the Neurexin family and cytoskeletal proteins. In the present review, we outline recent evidence on the key players of axo-glial interactions, depicting their importance in myelinated fiber physiology and disease.

The expression of TAG-1 in glial cells is sufficient for the formation of the juxtaparanodal complex and the phenotypic rescue of tag-1 homozygous mutants in the CNS.

Myelinated fibers are organized into specialized domains that ensure the rapid propagation of action potentials and are characterized by protein complexes underlying axoglial interactions. TAG-1 (Transient Axonal Glycoprotein-1), a cell adhesion molecule of the Ig superfamily, is expressed by neurons as well as by myelinating glia. It is essential for the molecular organization of myelinated fibers as it maintains the integrity of the juxtaparanodal region through its interactions with Caspr2 and the voltage-gated potassium channels (VGKCs) on the axolemma. Since TAG-1 is the only known component of the juxtaparanodal complex expressed by the glial cell, it is important to clarify its role in the molecular organization of juxtaparanodes. For this purpose, we generated transgenic mice that exclusively express TAG-1 in oligodendrocytes and lack endogenous gene expression (Tag-1(-/-);plp(Tg(rTag-1))). Phenotypic analysis clearly demonstrates that glial TAG-1 is sufficient for the proper organization and maintenance of the juxtaparanodal domain in the CNS. Biochemical analysis shows that glial TAG-1 physically interacts with Caspr2 and VGKCs. Ultrastructural and behavioral analysis of Tag-1(-/-);plp(Tg(rTag-1)) mice shows that the expression of glial TAG-1 is sufficient to restore the axonal and myelin deficits as well as the behavioral defects observed in Tag-1(-/-) animals. Together, these data highlight the pivotal role of myelinating glia on axonal domain differentiation and organization.