Isolation and Culture of Mouse Primary Microglia and Oligodendrocyte Precursor Cells.

Microglia and oligodendrocyte precursor cells (OPCs) are critical glia subsets in the central nervous system (CNS) and are actively engaged in a body of diseases, such as stroke, Alzheimer's disease, multiple sclerosis, etc. Microglia and OPC serve as compelling tools for the study of CNS diseases as well as the repair and damage of myelin sheath in vitro. In this protocol, we summarized a method which is capable of using the same batch of new-born mice to isolate and culture microglia and OPCs. It integrates the characteristics of practicality, convenience, and efficiency, providing a convenient, easy, and reliable technique for research.

Morphological correlates of pyramidal cell axonal myelination in mouse and human neocortex.

The axons of neocortical pyramidal neurons are frequently myelinated. Heterogeneity in the topography of axonal myelination in the cerebral cortex has been attributed to a combination of electrophysiological activity, axonal morphology, and neuronal-glial interactions. Previously, we showed that axonal segment length and caliber are critical local determinants of fast-spiking interneuron myelination. However, the factors that determine the myelination of individual axonal segments along neocortical pyramidal neurons remain largely unexplored. Here, we used structured illumination microscopy to examine the extent to which axonal morphology is predictive of the topography of myelination along neocortical pyramidal neurons. We identified critical thresholds for axonal caliber and interbranch distance that are necessary, but not sufficient, for myelination of pyramidal cell axons in mouse primary somatosensory cortex (S1). Specifically, we found that pyramidal neuron axonal segments with a caliber < 0.24 µm or interbranch distance < 18.10 µm are rarely myelinated. Moreover, we further confirmed that these findings in mice are similar for human neocortical pyramidal cell myelination (caliber < 0.25 µm, interbranch distance < 19.00 µm), suggesting that this mechanism is evolutionarily conserved. Taken together, our findings suggest that axonal morphology is a critical correlate of the topography and cell-type specificity of neocortical myelination.

Interaction between Oligodendrocytes and Interneurons in Brain Development and Related Neuropsychiatric Disorders.

A variety of neurological and psychiatric disorders have recently been shown to be highly associated with the abnormal development and function of oligodendrocytes (OLs) and interneurons. OLs are the myelin-forming cells in the central nervous system (CNS), while interneurons are important neural types gating the function of excitatory neurons. These two types of cells are of great significance for the establishment and function of neural circuits, and they share similar developmental origins and transcriptional architectures, and interact with each other in multiple ways during development. In this review, we compare the similarities and differences in these two cell types, providing an important reference and further revealing the pathogenesis of related brain disorders.

Functional myelin in cognition and neurodevelopmental disorders.

In vertebrates, oligodendrocytes (OLs) are glial cells of the central nervous system (CNS) responsible for the formation of the myelin sheath that surrounds the axons of neurons. The myelin sheath plays a crucial role in the transmission of neuronal information by promoting the rapid saltatory conduction of action potentials and providing neurons with structural and metabolic support. Saltatory conduction, first described in the peripheral nervous system (PNS), is now generally recognized as a universal evolutionary innovation to respond quickly to the environment: myelin helps us think and act fast. Nevertheless, the role of myelin in the central nervous system, especially in the brain, may not be primarily focused on accelerating conduction speed but rather on ensuring precision. Its principal function could be to coordinate various neuronal networks, promoting their synchronization through oscillations (or rhythms) relevant for specific information processing tasks. Interestingly, myelin has been directly involved in different types of cognitive processes relying on brain oscillations, and myelin plasticity is currently considered to be part of the fundamental mechanisms for memory formation and maintenance. However, despite ample evidence showing the involvement of myelin in cognition and neurodevelopmental disorders characterized by cognitive impairments, the link between myelin, brain oscillations, cognition and disease is not yet fully understood. In this review, we aim to highlight what is known and what remains to be explored to understand the role of myelin in high order brain processes.

7T MP2RAGE for cortical myelin segmentation: Impact of aging.

BACKGROUND: Myelin and iron are major contributors to the cortical MR signal. The aim of this study was to investigate 1. Can MP2RAGE-derived contrasts at 7T in combination with k-means clustering be used to distinguish between heavily and sparsely myelinated layers in cortical gray matter (GM)? 2. Does this approach provide meaningful biological information? METHODS: The following contrasts were generated from the 7T MP2RAGE images from 45 healthy controls (age: 19-75, f/m = 23/22) from the ATAG data repository: 1. T1 weighted image (UNI). 2. T1 relaxation image (T1map). 3. INVC/T1map ratio (RATIO). K-means clustering identified 6 clusters/tissue maps (csf, csf/gm-transition, wm, wm/gm transition, heavily myelinated cortical GM (dGM), sparsely myelinated cortical GM (sGM)). These tissue maps were then processed with SPM/DARTEL (volume-based analyses) and Freesurfer (surface-based analyses) and dGM and sGM volume/thickness of young adults (n = 27, 19-27 years) compared to those of older adults (n = 18, 42-75 years) at p<0.001 uncorrected. RESULTS: The resulting maps showed good agreement with histological maps in the literature. Volume- and surface analyses found age-related dGM loss/thinning in the mid-posterior cingulate and parahippocampal/entorhinal gyrus and age-related sGM losses in lateral, mesial and orbitofrontal frontal, insular cortex and superior temporal gyrus. CONCLUSION: The MP2RAGE derived UNI, T1map and RATIO contrasts can be used to identify dGM and sGM. Considering the close relationship between cortical myelo- and cytoarchitecture, the findings reported here indicate that this new technique might provide new insights into the nature of cortical GM loss in physiological and pathological conditions.

Ionic plasmon-polariton in application to neurosignaling.

The diffusive ion current is insufficient to explain the fast saltatory conduction observed in myelinated axons and in pain-sensing C fibers in the human nervous system, where the stimulus signal exhibits a velocity two orders of magnitude greater than the upper limit of ion diffusion velocity, even when the diffusion is accelerated by myelin, as in the discrete cable model including the Hodgkin-Huxley mechanism. The agreement with observations has been achieved in a wave-type model of stimulus signal kinetics via synchronized ion local density oscillations propagating as a wave in axons periodically corrugated by myelin segments in myelinated axons, or by periodically distributed rafts with clusters of Na^{+} channels in C fibers. The resulting so-called plasmon-polariton model for saltatory conduction reveals also the specific role of myelin, which is different from what was previously thought. This can be important for identifying a new target for the future treatment of demyelination diseases.

Conversion of Astrocyte Cell Lines to Oligodendrocyte Progenitor Cells Using Small Molecules and Transplantation to Animal Model of Multiple Sclerosis.

Astrocytes, the most prevalent cells in the central nervous system (CNS), can be transformed into neurons and oligodendrocyte progenitor cells (OPCs) using specific transcription factors and some chemicals. In this study, we present a cocktail of small molecules that target different signaling pathways to promote astrocyte conversion to OPCs. Astrocytes were transferred to an OPC medium and exposed for five days to a small molecule cocktail containing CHIR99021, Forskolin, Repsox, LDN, VPA and Thiazovivin before being preserved in the OPC medium for an additional 10 days. Once reaching the OPC morphology, induced cells underwent immunocytofluorescence evaluation for OPC markers while checked for lacking the astrocyte markers. To test the in vivo differentiation capabilities, induced OPCs were transplanted into demyelinated mice brains treated with cuprizone over 12 weeks. Two distinct lines of astrocytes demonstrated the potential of conversion to OPCs using this small molecule cocktail as verified by morphological changes and the expression of PDGFR and O4 markers as well as the terminal differentiation to oligodendrocytes expressing MBP. Following transplantation into demyelinated mice brains, induced OPCs effectively differentiated into mature oligodendrocytes. The generation of OPCs from astrocytes via a small molecule cocktail may provide a new avenue for producing required progenitors necessary for myelin repair in diseases characterized by the loss of myelin such as multiple sclerosis.

BTK inhibition limits microglia-perpetuated CNS inflammation and promotes myelin repair.

In multiple sclerosis (MS), persisting disability can occur independent of relapse activity or development of new central nervous system (CNS) inflammatory lesions, termed chronic progression. This process occurs early and it is mostly driven by cells within the CNS. One promising strategy to control progression of MS is the inhibition of the enzyme Bruton's tyrosine kinase (BTK), which is centrally involved in the activation of both B cells and myeloid cells, such as macrophages and microglia. The benefit of BTK inhibition by evobrutinib was shown as we observed reduced pro-inflammatory activation of microglia when treating chronic experimental autoimmune encephalomyelitis (EAE) or following the adoptive transfer of activated T cells. Additionally, in a model of toxic demyelination, evobrutinib-mediated BTK inhibition promoted the clearance of myelin debris by microglia, leading to an accelerated remyelination. These findings highlight that BTK inhibition has the potential to counteract underlying chronic progression of MS.

Neddylation orchestrates the complex transcriptional and posttranscriptional program that drives Schwann cell myelination.

Myelination is essential for neuronal function and health. In peripheral nerves, >100 causative mutations have been identified that cause Charcot-Marie-Tooth disease, a disorder that can affect myelin sheaths. Among these, a number of mutations are related to essential targets of the posttranslational modification neddylation, although how these lead to myelin defects is unclear. Here, we demonstrate that inhibiting neddylation leads to a notable absence of peripheral myelin and axonal loss both in developing and regenerating mouse nerves. Our data indicate that neddylation exerts a global influence on the complex transcriptional and posttranscriptional program by simultaneously regulating the expression and function of multiple essential myelination signals, including the master transcription factor EGR2 and the negative regulators c-Jun and Sox2, and inducing global secondary changes in downstream pathways, including the mTOR and YAP/TAZ signaling pathways. This places neddylation as a critical regulator of myelination and delineates the potential pathogenic mechanisms involved in CMT mutations related to neddylation.

MYRF: A unique transmembrane transcription factor- from proteolytic self-processing to its multifaceted roles in animal development.

The Myelin Regulator Factor (MYRF) is a master regulator governing myelin formation and maintenance in the central nervous system. The conservation of MYRF across metazoans and its broad tissue expression suggest it has functions extending beyond the well-established role in myelination. Loss of MYRF results in developmental lethality in both invertebrates and vertebrates, and MYRF haploinsufficiency in humans causes MYRF-related Cardiac Urogenital Syndrome, underscoring its importance in animal development; however, these mechanisms are largely unexplored. MYRF, an unconventional transcription factor, begins embedded in the membrane and undergoes intramolecular chaperone mediated trimerization, which triggers self-cleavage, allowing its N-terminal segment with an Ig-fold DNA-binding domain to enter the nucleus for transcriptional regulation. Recent research suggests developmental regulation of cleavage, yet the mechanisms remain enigmatic. While some parts of MYRF's structure have been elucidated, others remain obscure, leaving questions about how these motifs are linked to its intricate processing and function.

Expanding the Clinical Spectrum of DRP2-Associated Charcot-Marie-Tooth Disease.

BACKGROUND AND OBJECTIVES: Germline truncating variants in the DRP2 gene (encoding dystrophin-related protein 2) cause the disruption of the periaxin-DRP2-dystroglycan complex and have been linked to Charcot-Marie-Tooth disease. However, the causality and the underlying phenotype of the genetic alterations are not clearly defined. METHODS: This cross-sectional retrospective observational study includes 9 patients with Charcot-Marie-Tooth disease (CMT) with DRP2 germline variants evaluated at 6 centers throughout Spain. RESULTS: We identified 7 Spanish families with 4 different DRP2 likely pathogenic germline variants. In agreement with an X-linked inheritance, men harboring hemizygous DRP2 variants presented with an intermediate form of CMT, whereas heterozygous women were asymptomatic. Symptom onset was variable (36.6 ± 16 years), with lower limb weakness and multimodal sensory loss producing a mild-to-moderate functional impairment. Nerve echography revealed an increase in the cross-sectional area of nerve roots and proximal nerves. Lower limb muscle magnetic resonance imaging confirmed the presence of a length-dependent fatty infiltration. Immunostaining in intradermal nerve fibers demonstrated the absence of DRP2 and electron microscopy revealed abnormal myelin thickness that was also detectable in the sural nerve sections. DISCUSSION: Our findings support the causality of DRP2 pathogenic germline variants in CMT and further define the phenotype as a late-onset sensory and motor length-dependent neuropathy, with intermediate velocities and thickening of proximal nerve segments.

AxoDetect: an automated nerve image segmentation and quantification workflow for computational nerve modeling.

Objective.Bioelectronic treatments targeting near-organ innervation have unprecedented clinical applications. Particularly in the spleen, the inhibition of the cholinergic inflammatory response by near-organ nerve stimulation has potential to replace pharmacological treatments in chronic and autoimmune diseases. A caveat is that the optimization of therapeutic stimulation parameters relies onin vivoexperimentation, which becomes challenging due to the small nerve diameters (2 µm), complex anatomy, and mixed axon type composition of the autonomic nerves. Effective development ofin silicomodels requires tools which allow for fast and efficient quantification of axonal composition of specific nerves. Current approaches to generate such information rely on manual image segmentation and quantification.Approach.We developed a combined image-segmentation and model-generation software called AxoDetect: a target- and format-agnostic computer vision algorithm which can segment myelin, endo/epineurium, and both myelinated and unmyelinated fibers from a nerve image without training.Main results.AxoDetect is over 10 times faster on average when compared with current automatic methods while maintaining flexibility through the use of tunable pixel threshold filters to detect different types of tissue. When compared to a distribution-based and a manually segmented model of the splenic nerve terminal branch 1, the model generated with AxoDetect had comparable threshold prediction and was able to accurately detect an increase in activation threshold caused by the addition of surrounding fat tissue to the modeled nerve.Significance.AxoDetect contributes to the acceleration of neuromodulation treatment development through faster model design and iteration without requiring training. Furthermore, the computer vision approach and tunable nature of the filters in our method allow for its use in a variety of histological applications. Our approach will impact not only the study of nerves but also the design of implantable neural interfaces to enhance bioelectronic therapeutic options.

Ageing impairs the regenerative capacity of regulatory T cells in mouse central nervous system remyelination.

Myelin regeneration (remyelination) is essential to prevent neurodegeneration in demyelinating diseases such as Multiple Sclerosis, however, its efficiency declines with age. Regulatory T cells (Treg) recently emerged as critical players in tissue regeneration, including remyelination. However, the effect of ageing on Treg-mediated regenerative processes is poorly understood. Here, we show that expansion of aged Treg does not rescue age-associated remyelination impairment due to an intrinsically diminished capacity of aged Treg to promote oligodendrocyte differentiation and myelination in male and female mice. This decline in regenerative Treg functions can be rescued by a young environment. We identified Melanoma Cell Adhesion Molecule 1 (MCAM1) and Integrin alpha 2 (ITGA2) as candidates of Treg-mediated oligodendrocyte differentiation that decrease with age. Our findings demonstrate that ageing limits the neuroregenerative capacity of Treg, likely limiting their remyelinating therapeutic potential in aged patients, and describe two mechanisms implicated in Treg-driven remyelination that may be targetable to overcome this limitation.

Association between T1w/T2w ratio in white matter and cognitive function in Alzheimer's disease.

Loss of myelin in the brain may lead to cognitive decline in Alzheimer's disease (AD). The ratio of T1 weighted/T2 weighted (T1w/T2w) on magnetic resonance imaging has been used as a proxy for myelin content in the brain. Using this approach, we investigated the correlation between the white matter (WM) T1w/T2w ratio and both cognitive scores and disease progression in AD. A total of 93 participants who were cognitively unimpaired or diagnosed with mild cognitive impairment or AD dementia were recruited between March 2021 and November 2022. All participants were assessed using neuropsychological tests, and a subset of the participants was assessed every 1 year to monitor disease progression. We observed significant positive associations between the WM T1w/T2w ratio and executive function within the fornix, sagittal stratum, anterior internal capsule, and body of the corpus callosum (False discovery rate [FDR]-corrected P-value < 0.05). There was a marginal interaction between the WM T1w/T2w ratio of the left anterior internal capsule and the longitudinal change in sum of boxes of the Clinical Dementia Rating Scale (FDR-corrected P-value = 0.05). The present study demonstrated that the WM T1w/T2w ratio was associated with executive function and disease progression, suggesting that it may be a novel neuroimaging marker for AD.

The Impact of Sample Storage on Blood Methylation: Towards Assessing Myelin Gene Methylation as a Biomarker for Progressive Multiple Sclerosis.

One of the major challenges in multiple sclerosis (MS) is to accurately monitor and quantify disability over time. Thus, there is a pressing need to identify new biomarkers for disease progression. Peripheral blood DNA methylation has been demonstrated to be an easily accessible and quantifiable marker in many neurodegenerative diseases. In this study, we aimed to investigate whether methylation patterns that were previously determined in chronic inactive white matter lesions of patients with progressive MS are also reflected in the blood, and whether the latter can serve as a biomarker for disease progression in MS. While our initial analysis revealed differences in the blood methylation state of important myelin-related genes between patients with progressive MS and controls, these findings could not be validated in other independent patient cohorts. Subsequent investigation suggests that sample storage can selectively influence DNA methylation patterns, potentially hindering accurate epigenetic analysis. Therefore, sample storage time should be taken into consideration during the initial sample selection stage in biomarker studies.

PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

Oligodendrocyte progenitor cells (OPCs) differentiate into myelin-producing cells and modulate neuronal activity. Defects in OPC development are associated with neurological diseases. N6-methyladenosine (m6A) contributes to neural development; however, the mechanism by which m6A regulates OPC development remains unclear. Here, we demonstrate that PRRC2B is an m6A reader that regulates OPC development and myelination. Nestin-Cre-mediated Prrc2b deletion affects neural stem cell self-renewal and glial differentiation. Moreover, the oligodendroglia lineage-specific deletion of Prrc2b reduces the numbers of OPCs and oligodendrocytes, causing hypomyelination and impaired motor coordination. Integrative methylated RNA immunoprecipitation sequencing, RNA sequencing, and RNA immunoprecipitation sequencing analyses identify Sox2 as the target of PRRC2B. Notably, PRRC2B, displaying separate and cooperative functions with PRRC2A, stabilizes mRNA by binding to m6A motifs in the coding sequence and 3' UTR of Sox2. In summary, we identify the posttranscriptional regulation of PRRC2B in OPC development, extending the understanding of PRRC2 family proteins and providing a therapeutic target for myelin-related disorders.

Testing SIPA1L2 as a modifier of CMT1A using mouse models.

Charcot-Marie-Tooth disease type 1A (CMT1A) is a demyelinating peripheral neuropathy caused by the duplication of peripheral myelin protein 22 (PMP22), leading to muscle weakness and loss of sensation in the hands and feet. A recent case-only genome-wide association study of CMT1A patients conducted by the Inherited Neuropathy Consortium identified a strong association between strength of foot dorsiflexion and variants in signal induced proliferation associated 1 like 2 (SIPA1L2), indicating that it may be a genetic modifier of disease. To validate SIPA1L2 as a candidate modifier and to assess its potential as a therapeutic target, we engineered mice with deletion of exon 1 (including the start codon) of the Sipa1l2 gene and crossed them to the C3-PMP22 mouse model of CMT1A. Neuromuscular phenotyping showed that Sipa1l2 deletion in C3-PMP22 mice preserved muscular endurance assayed by inverted wire hang duration and changed femoral nerve axon morphometrics such as myelin thickness. Gene expression changes suggest involvement of Sipa1l2 in cholesterol biosynthesis, a pathway that is also implicated in C3-PMP22 mice. Although Sipa1l2 deletion did impact CMT1A-associated phenotypes, thereby validating a genetic interaction, the overall effect on neuropathy was mild.