Distinct axo-protective and axo-destructive roles for Schwann cells after injury in a novel compartmentalised mouse myelinating coculture system.

Myelinating Schwann cell (SC)- dorsal root ganglion (DRG) neuron cocultures have been an important technique over the last four decades in understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. While methods using rat SCs and rat DRG neurons are commonplace, there are no established protocols in the field describing the use of mouse SCs with mouse DRG neurons in dissociated myelinating cocultures. There is a great need for such a protocol as this would allow the use of cells from many different transgenic mouse lines. Here we describe a protocol to coculture dissociated mouse SCs and DRG neurons and induce robust myelination. Use of microfluidic chambers permits fluidic isolation for drug treatments, allows cultures to be axotomised to study injury responses, and cells can readily be transfected with lentiviruses to permit live imaging. We used this model to quantify the rate of degeneration after traumatic axotomy in the presence and absence of myelinating SCs and axon aligned SCs that were not induced to myelinate. We find that SCs, irrespective of myelination status, are axoprotective and delay axon degeneration early on. At later time points after injury, we use live imaging of cocultures to show that once axonal degeneration has commenced SCs break up, ingest, and clear axonal debris

Hyperosmotic stress induces cell-dependent aggregation of α-synuclein.

The aggregation of alpha-synuclein (α-syn) is a pathological feature of a number of neurodegenerative conditions, including Parkinson's disease. Genetic mutations, abnormal protein synthesis, environmental stress, and aging have all been implicated as causative factors in this process. The importance of water in the polymerisation of monomers, however, has largely been overlooked. In the present study, we highlight the role of hyperosmotic stress in inducing human α-syn to aggregate in cells in vitro, through rapid treatment of the cells with three different osmolytes: sugar, salt and alcohol. This effect is cell-dependent and not due to direct protein-osmolyte interaction, and is specific for α-syn when compared to other neurodegeneration-related proteins, such as Tau or Huntingtin. This new property of α-syn not only highlights a unique aspect of its behaviour which may have some relevance for disease states, but may also be useful as a screening test for compounds to inhibit the aggregation of α-syn in vitro.Funding was by the DDPDgenes, Rosetree Trust, Wellcome Trust PhD Program for Clinicians, the Danish Council for Independent Research | Natural Sciences (FNU-11-113326), the Stem Cell Institute and Wellcome Trust-MRC funded Cambridge Stem Cell Institute and an NIHR award of a Biomedical Research Centre for Addenbrooke’s Hospital/University of Cambridge. RA Barker is an NIHR Senior Investigator

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

SARM1 detection in myelinating glia: sarm1/Sarm1 is dispensable for PNS and CNS myelination in zebrafish and mice

Since SARM1 mutations have been identified in human neurological disease, SARM1 inhibition has become an attractive therapeutic strategy to preserve axons in a variety of disorders of the peripheral (PNS) and central nervous system (CNS). While SARM1 has been extensively studied in neurons, it remains unknown whether SARM1 is present and functional in myelinating glia? This is an important question to address. Firstly, to identify whether SARM1 dysfunction in other cell types in the nervous system may contribute to neuropathology in SARM1 dependent diseases? Secondly, to ascertain whether therapies altering SARM1 function may have unintended deleterious impacts on PNS or CNS myelination? Surprisingly, we find that oligodendrocytes express sarm1 mRNA in the zebrafish spinal cord and that SARM1 protein is readily detectable in rodent oligodendrocytes in vitro and in vivo. Furthermore, activation of endogenous SARM1 in cultured oligodendrocytes induces rapid cell death. In contrast, in peripheral glia, SARM1 protein is not detectable in Schwann cells and satellite glia in vivo and sarm1/Sarm1 mRNA is detected at very low levels in Schwann cells, in vivo, in zebrafish and mouse. Application of specific SARM1 activators to cultured mouse Schwann cells does not induce cell death and nicotinamide adenine dinucleotide (NAD) levels remain unaltered suggesting Schwann cells likely contain no functionally relevant levels of SARM1. Finally, we address the question of whether SARM1 is required for myelination or myelin maintenance. In the zebrafish and mouse PNS and CNS, we show that SARM1 is not required for initiation of myelination and myelin sheath maintenance is unaffected in the adult mouse nervous system. Thus, strategies to inhibit SARM1 function to treat neurological disease are unlikely to perturb myelination in humans.CM was funded by a Medical Research Council (UK) studentship (2251399). PA-F (206634/Z/17/Z), AL (210904/Z/18/Z), CC (220027/Z/19/Z), RB (203151/Z/16/Z) and MC (220906/Z/20/Z) were funded by the Wellcome Trust (UK). BS was supported by a Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (109408/Z/15/Z). KM was funded by the National Institute of Neurological Disorders and Stroke Awards (R01NS079445). JG-S was funded by a Miguel Servet Fellowship (CP22/00078) from the Instituto de Salud Carlos III and the Millennium Nucleus for the Study of Pain (MiNuSPain), Santiago, Chile. HC was funded by the Spanish “Ministerio de Economía y Competitividad” (BFU2016-75864R and PID2019-109762RB-I00), ISABIAL (UGP18-257 and UGP-2019-128), and Generalitat Valenciana (PROMETEO 2018/114). Y-PH and C-YC were funded by Academia Sinica, AS-IA-106-L04 to Y-PH.Peer reviewe

Emerging role of HDACs in regeneration and ageing in the peripheral nervous system: Repair Schwann cells as pivotal targets

This article belongs to the Special Issue Neuroepigenetic: From Bench to Bedside.The peripheral nervous system (PNS) has a remarkable regenerative capacity in comparison to the central nervous system (CNS), a phenomenon that is impaired during ageing. The ability of PNS axons to regenerate after injury is due to Schwann cells (SC) being reprogrammed into a repair phenotype called Repair Schwann cells. These repair SCs are crucial for supporting axonal growth after injury, myelin degradation in a process known as myelinophagy, neurotropic factor secretion, and axonal growth guidance through the formation of Büngner bands. After regeneration, repair SCs can remyelinate newly regenerated axons and support nonmyelinated axons. Increasing evidence points to an epigenetic component in the regulation of repair SC gene expression changes, which is necessary for SC reprogramming and regeneration. One of these epigenetic regulations is histone acetylation by histone acetyl transferases (HATs) or histone deacetylation by histone deacetylases (HDACs). In this review, we have focused particularly on three HDAC classes (I, II, and IV) that are Zn2+-dependent deacetylases. These HDACs are important in repair SC biology and remyelination after PNS injury. Another key aspect explored in this review is HDAC genetic compensation in SCs and novel HDAC inhibitors that are being studied to improve nerve regeneration.This work has been funded by grants from the Ministerio de Economía y Competitividad (BFU2016-75864R and PID2019-109762RB-I00), ISABIAL (UGP18-257 and UGP-2019-128) to H. Cabedo and Conselleria Educació Generalitat Valenciana (PROMETEO 2018/114) to H. Cabedo. Medical Research Council (UK) studentship (2251399) to C. Mutschler.Peer reviewe

Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell–neuron cocultures

Peer reviewed: TrueAcknowledgements: We thank Karin Müller, Martin Lenz, and Filomena Gallo at the Cambridge Centre for Advanced Imaging for assistance with microscopy. We thank Aviva Tolkovsky and Alex Clark for technical suggestions. We thank Peter Brophy for the gift of the periaxin antibody. We thank Claire Jacob for helpful suggestions.Funder: Belgian National Fund for Scientific Research F.R.S. - FNRS; doi: http://dx.doi.org/10.13039/501100002661Funder: University of Cambridge; doi: http://dx.doi.org/10.13039/501100000735Myelinating Schwann cell (SC)–dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell–cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. Although methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here, we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury and used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest and clear axonal debris

Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell–neuron cocultures

Peer reviewed: TrueAcknowledgements: We thank Karin Müller, Martin Lenz, and Filomena Gallo at the Cambridge Centre for Advanced Imaging for assistance with microscopy. We thank Aviva Tolkovsky and Alex Clark for technical suggestions. We thank Peter Brophy for the gift of the periaxin antibody. We thank Claire Jacob for helpful suggestions.Funder: Belgian National Fund for Scientific Research F.R.S. - FNRS; doi: http://dx.doi.org/10.13039/501100002661Funder: University of Cambridge; doi: http://dx.doi.org/10.13039/501100000735Myelinating Schwann cell (SC)–dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell–cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. Although methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here, we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury and used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest and clear axonal debris

Emerging Role of HDACs in Regeneration and Ageing in the Peripheral Nervous System: Repair Schwann Cells as Pivotal Targets

The peripheral nervous system (PNS) has a remarkable regenerative capacity in comparison to the central nervous system (CNS), a phenomenon that is impaired during ageing. The ability of PNS axons to regenerate after injury is due to Schwann cells (SC) being reprogrammed into a repair phenotype called Repair Schwann cells. These repair SCs are crucial for supporting axonal growth after injury, myelin degradation in a process known as myelinophagy, neurotropic factor secretion, and axonal growth guidance through the formation of Büngner bands. After regeneration, repair SCs can remyelinate newly regenerated axons and support nonmyelinated ax-ons. Increasing evidence points to an epigenetic component in the regulation of repair SC gene ex-pression changes, which is necessary for SC reprogramming and regeneration. One of these epigenetic regulations is histone acetylation by histone acetyl transferases (HATs) or histone deacetylation by histone deacetylases (HDACs). In this review, we have focused particularly on three HDAC classes (I, II, and IV) that are Zn2+-dependent deacetylases. These HDACs are important in repair SC biology and remyelination after PNS injury. Another key aspect explored in this review is HDAC genetic compensation in SCs and novel HDAC inhibitors that are being studied to improve nerve regeneration.Ministerio de Economía y Competitividad (BFU2016-75864R and PID2019-109762RB-I00) ISABIAL (UGP18-257 and UGP-2019-128) Conselleria Educació Generalitat Valenciana (PROMETEO 2018/114) Medical Research Council (UK) studentship (2251399

Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell/neuron cocultures.

Myelinating Schwann cell (SC)- dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. While methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury, used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest, and clear axonal debris