Myelin Debris Research Articles

The Protective Effect of Ginkgolide K on Astrocytes Exposed to Myelin Debris in vitro and Demyelination in vivo

Background Ginkgolide K ( GK) has a protective effect on neurons and myelin sheath, and the function of astrocytes in phagocytizing myelin debris has attracted extensive attention in remyelination. Objectives This study focuses on the impact of GK on the phagocytosis of myelin debris by astrocytes and explores the possibility of treating demyelination. Materials and Methods Male C57BL/6 mice were used to establish a cuprizone (CPZ)-induced demyelination model. After being fed a normal or CPZ diet for 4 weeks, mice were intraperitoneally injected with PEG400 or GK for 14 consecutive days. GL261 and primary astrocytes were exposed to myelin debris. Immunohistochemistry/immunocytochemistry staining, western blot, RT-PCR, and other methods were used to detect the relevant indicators. Results Astrocytes engulfed myelin debris, leading to astrocyte reactivity with increased p-NF-kB/p65 and ATF6 expression and decreased nuclear factor erythroid 2-related factor 2 (Nrf2) expression, which was reversed by GK. Subsequently, astrocytes stimulated by myelin debris underwent self-apoptosis with increased expression of caspase-3 and BCL2-associated X (Bax), which was inhibited by GK. Simultaneously, GK efficiently promoted astrocytes to increase the production of neurotrophic ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (bFGF), speculating that increased CNTF/bFGF, decreased p-NF-kB/p65/ATF6, and up-regulated Nrf2 should participate in the protection of astrocyte apoptosis and build a beneficial microenvironment for myelin regeneration. Conclusion The results suggest that GK may have the potential to treat demyelination by promoting debris clearance and improving the brain microenvironment. However, further studies are needed to understand the physiological and pathological consequences of astrocytic phagocytosis and to investigate the possibility of using GK as a therapeutic application.

Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro

Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer’s disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Trem2 Enhances Demyelination in the Csf1r+/- Mouse Model of Leukoencephalopathy.

Colony-stimulating factor-1 receptor (CSF-1R)-related leukoencephalopathy (CRL) is a neurodegenerative disease that triggers early demyelination, leading to an adult-onset dementia. Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial receptor that promotes the activation of microglia and phagocytic clearance of apoptotic neurons and myelin debris. We investigated the role of Trem2 in the demyelination observed in the Csf1r+/- mouse model of CRL. We show that elevation of Trem2 expression and callosal demyelination occur in 4-5-month-old Csf1r+/- mice, prior to the development of symptoms. Absence of Trem2 in the Csf1r+/- mouse attenuated myelin pathology and normalized microglial densities and morphology in the corpus callosum. Trem2 absence also prevented axonal degeneration and the loss of cortical layer V neurons observed in Csf1r+/- mice. Furthermore, the absence of Trem2 prevented the accumulation of myelin-derived lipids in Csf1r+/- macrophages and reduced the production of TNF-α after myelin engulfment. These data suggest that TREM2 contributes to microglial dyshomeostasis in CRL.

Ablation of microglia following infection of the central nervous system with a neurotropic murine coronavirus infection leads to increased demyelination and impaired remyelination

Intracranial inoculation of susceptible mice with a glial-tropic strain of mouse hepatitis virus (JHMV), a murine coronavirus, results in an acute encephalomyelitis followed by viral persistence in white matter tracts accompanied by chronic neuroinflammation and demyelination. Microglia are the resident immune cell of the central nervous system (CNS) and are considered important in regulating events associated with neuroinflammation as well as influencing both white matter damage and remyelination. To better understand mechanisms by which microglia contribute to these immune-mediated events, JHMV-infected mice with established demyelination were treated with the small molecular inhibitor of colony stimulating factor 1 receptor (CSF1R), PLX5622, to deplete microglia. Treatment with PLX5622 did not affect viral replication within the CNS yet the severity of demyelination was increased and remyelination impaired compared to control mice. Gene expression analysis revealed that targeting microglia resulted in altered expression of genes associated with immune cell activation and phagocytosis of myelin debris. These findings indicate that microglia are not critical in viral surveillance in persistently JHMV-infected mice yet restrict white matter damage and remyelination, in part, by influencing phagocytosis of myelin debris.

Construction and optimization of a coculture system of mouse brain microvascular endothelial cells and myelin debris

Microvascular endothelial cells are a newly discovered cell type involved in the phagocytosis of myelin debris, which play a key role in the repair of spinal cord injuries. Several methods for the preparation of myelin debris and parameters for constructing a coculture system of microvascular endothelial cells and myelin debris are available, but no systematic studies have yet been conducted, which hinders further exploration of the mechanisms of demyelinating disease repair. Herein, we aimed to develop a standardized method for this process. Myelin debris of different sizes was obtained from the brains of C57BL/6 mice by stripping the brains under aseptic conditions, multiple grinding, gradient centrifugation, etc. Transmission electron microscopy and nanoparticle size analysis were used to characterize myelin debris. Microvascular endothelial cells were cultured on a matrix gel, and myelin debris of different sizes (fluorescently labeled using CFSE) was placed in coculture after forming a vascular-like structure. Subsequently, myelin debris of different concentrations was cocultured in the vascular-like structure, and phagocytosis of myelin debris by microvascular endothelial cells was detected using immunofluorescence staining and flow cytometry. We found that myelin debris could be successfuly obtained from the mouse brain with secondary grinding and other steps and cocultured with microvascular endothelial cells at a concentration of 2 mg/mL, which promoted the phagocytosis of microvascular endothelial cells. In conclusion, we provide a reference for the protocol of a coculture system of microvascular endothelial cells and myelin debris.

How does Nogo receptor influence demyelination and remyelination in the context of multiple sclerosis?

Multiple sclerosis (MS) can progress with neurodegeneration as a consequence of chronic inflammatory mechanisms that drive neural cell loss and/or neuroaxonal dystrophy in the central nervous system. Immune-mediated mechanisms can accumulate myelin debris in the disease extracellular milieu during chronic-active demyelination that can limit neurorepair/plasticity and experimental evidence suggests that potentiated removal of myelin debris can promote neurorepair in models of MS. The myelin-associated inhibitory factors (MAIFs) are integral contributors to neurodegenerative processes in models of trauma and experimental MS-like disease that can be targeted to promote neurorepair. This review highlights the molecular and cellular mechanisms that drive neurodegeneration as a consequence of chronic-active inflammation and outlines plausible therapeutic approaches to antagonize the MAIFs during the evolution of neuroinflammatory lesions. Moreover, investigative lines for translation of targeted therapies against these myelin inhibitors are defined with an emphasis on the chief MAIF, Nogo-A, that may demonstrate clinical efficacy of neurorepair during progressive MS.

Quantitative biochemical phenotypic heterogeneity of macrophages after myelin debris phagocytosis at a single cell level by synchrotron radiation fourier transform infrared microspectroscopy

During the immuno-inflammatory pathophysiological process of spinal cord injury, traumatic brain injury, and ischemic stroke, macrophages play an important role in phagocytizing and clearing degenerated myelin debris. After phagocytizing myelin debris, the biochemical phenotypes related to the biological function of macrophages show vast heterogeneity; however, it is not fully understood. Detecting biochemical changes after myelin debris phagocytosis by macrophages at a single-cell level is helpful to characterize phenotypic and functional heterogeneity. In this study, based on the cell model of myelin debris phagocytosis by macrophages in vitro, the biochemical changes in macrophages were investigated using Synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy. Infrared spectrum fluctuations, principal component analysis, and cell-to-cell Euclidean distance statistical analysis of specific spectrum regions revealed dynamic and significant changes in proteins and lipids within macrophages after myelin debris phagocytosis. Thus, SR-FTIR microspectroscopy is a powerful identification toolkit for exploring biochemical phenotype heterogeneity transformation that may be of great importance to providing an evaluation strategy for studying cell functions related to cellular substance distribution and metabolism.

Remyelination in multiple sclerosis from the miRNA perspective.

Remyelination relies on the repair of damaged myelin sheaths, involving microglia cells, oligodendrocyte precursor cells (OPCs), and mature oligodendrocytes. This process drives the pathophysiology of autoimmune chronic disease of the central nervous system (CNS), multiple sclerosis (MS), leading to nerve cell damage and progressive neurodegeneration. Stimulating the reconstruction of damaged myelin sheaths is one of the goals in terms of delaying the progression of MS symptoms and preventing neuronal damage. Short, noncoding RNA molecules, microRNAs (miRNAs), responsible for regulating gene expression, are believed to play a crucial role in the remyelination process. For example, studies showed that miR-223 promotes efficient activation and phagocytosis of myelin debris by microglia, which is necessary for the initiation of remyelination. Meanwhile, miR-124 promotes the return of activated microglia to the quiescent state, while miR-204 and miR-219 promote the differentiation of mature oligodendrocytes. Furthermore, miR-138, miR-145, and miR-338 have been shown to be involved in the synthesis and assembly of myelin proteins. Various delivery systems, including extracellular vesicles, hold promise as an efficient and non-invasive way for providing miRNAs to stimulate remyelination. This article summarizes the biology of remyelination as well as current challenges and strategies for miRNA molecules in potential diagnostic and therapeutic applications.

Lentinan alleviates sciatic nerve injury by promoting autophagy to remove myelin fragments.

Lentinan, a natural drug with wide-ranging pharmacological activities, can regulate autophagy-the process through which Schwann cells (SCs) eliminate myelin fragments after peripheral nerve injury (PNI). However, the effect of lentinan after PNI and the role of accelerated myelin debris removal via autophagy in this process are unclear. This study examined the effect of lentinan on rat sciatic nerve repair following crush injury and the underlying mechanisms. After the successful establishment of the sciatic nerve compression injury model, group-specific treatments were performed. The treatment group received 20 mg/kg lentinan via intraperitoneal injection, while the model group was treated with normal saline. The recovery in each group was then evaluated. Further, a rat SC line (RSC96) was cultured in medium with/without lentinan after supplementation with homogenous myelin fractions to evaluate the removal of myelin particles. Our results showed that lentinan promotes autophagic flux in vivo via the AMPK/mTOR signaling pathway, accelerates the clearance of myelin debris by SCs, and inhibits neuronal apoptosis, thereby promoting neurological recovery. Similarly, in vitro experiments showed that lentinan promotes the phagocytosis of myelin debris by SCs. In conclusion, our results suggest that lentinan primarily promotes nerve regeneration by accelerating the autophagic clearance of myelin debris in SCs, and this process is likely regulated by the AMPK/mTOR signaling pathway. Therefore, this study provides compelling evidence that lentinan may be a cost-effective and natural treatment agent for PNI.

Astrocytic CXCL5 hinders microglial phagocytosis of myelin debris and aggravates white matter injury in chronic cerebral ischemia

BackgroundChronic cerebral ischemia induces white matter injury (WMI) contributing to cognitive decline. Both astrocytes and microglia play vital roles in the demyelination and remyelination processes, but the underlying mechanism remains unclear. This study aimed to explore the influence of the chemokine CXCL5 on WMI and cognitive decline in chronic cerebral ischemia and the underlying mechanism.MethodsBilateral carotid artery stenosis (BCAS) model was constructed to mimic chronic cerebral ischemia in 7–10 weeks old male mice. Astrocytic Cxcl5 conditional knockout (cKO) mice were constructed and mice with Cxcl5 overexpressing in astrocytes were generated by stereotactic injection of adeno-associated virus (AAV). WMI was evaluated by magnetic resonance imaging (MRI), electron microscopy, histological staining and western blotting. Cognitive function was examined by a series of neurobehavioral tests. The proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), phagocytosis of microglia were analyzed via immunofluorescence staining, western blotting or flow cytometry.ResultsCXCL5 was significantly elevated in the corpus callosum (CC) and serum in BCAS model, mainly expressed in astrocytes, and Cxcl5 cKO mice displayed improved WMI and cognitive performance. Recombinant CXCL5 (rCXCL5) had no direct effect on the proliferation and differentiation of OPCs in vitro. Astrocytic specific Cxcl5 overexpression aggravated WMI and cognitive decline induced by chronic cerebral ischemia, while microglia depletion counteracted this effect. Recombinant CXCL5 remarkably hindered microglial phagocytosis of myelin debris, which was rescued by inhibition of CXCL5 receptor C-X-C motif chemokine receptor 2 (CXCR2).ConclusionOur study revealed that astrocyte-derived CXCL5 aggravated WMI and cognitive decline by inhibiting microglial phagocytosis of myelin debris, suggesting a novel astrocyte-microglia circuit mediated by CXCL5-CXCR2 signaling in chronic cerebral ischemia.

Remyelination and Ageing: Ethical Considerations of Using Surgically Joined Animals in Research.

Remyelination is a key repair process that ensures neurons remain protected following injury. This process is mediated by remyelinating oligodendrocytes in vertebrates, however, similarly to other neurobiological processes, the rate and efficiency of remyelination decreases across age and under pathological conditions. This has largely been attributed to two main contributors: 1) decreased exogenous signals supporting remyelination; and 2) aging of precursor cells that no longer differentiate into remyelinating oligodendrocytes. Here we discuss a key paper by Ruckh et al. (2012) who presented novel evidence that exposure to soluble bloodstream factors of young mice significantly rescues remyelination in old mice following a demyelinating insult. In this paper, a parabiosis approach was used where young and old mice were surgically joined for three weeks before and then left as a pair throughout the experiment. Ruckh and colleagues also offer novel insight into the role played by immune system cells, specifically macrophages, in clearance of myelin debris, a further contributor to remyelination. This paper is a good tool to expose undergraduate neuroscience students to basic molecular processes underlying conduction and transmission, helping them link cellular and network components. It also offers a platform for introducing the practicalities of in vivo research and debating ethical controversies that arise in animal research.

Progranulin deficiency results in sex-dependent alterations in microglia in response to demyelination.

Heterozygous mutations in the granulin (GRN) gene, resulting in the haploinsufficiency of the progranulin (PGRN) protein, is a leading cause of frontotemporal lobar degeneration (FTLD). Complete loss of the PGRN protein causes neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder. Polymorphisms in the GRN gene have also been associated with several other neurodegenerative diseases, including Alzheimer's disease (AD), and Parkinson's disease (PD). PGRN deficiency has been shown to cause myelination defects previously, but how PGRN regulates myelination is unknown. Here, we report that PGRN deficiency leads to a sex-dependent myelination defect with male mice showing more severe demyelination in response to cuprizone treatment. This is accompanied by exacerbated microglial proliferation and activation in the male PGRN-deficient mice. Interestingly, both male and female PGRN-deficient mice show sustained microglial activation after cuprizone removal and a defect in remyelination. Specific ablation of PGRN in microglia results in similar sex-dependent phenotypes, confirming a microglial function of PGRN. Lipid droplets accumulate in microglia specifically in male PGRN-deficient mice. RNA-seq analysis and mitochondrial function assays reveal key differences in oxidative phosphorylation in male versus female microglia under PGRN deficiency. A significant decrease in myelination and accumulation of myelin debris and lipid droplets in microglia were found in the corpus callosum regions of FTLD patients with GRN mutations. Taken together, our data support that PGRN deficiency leads to sex-dependent alterations in microglia with subsequent myelination defects.

Neural cell isolation from adult macaques for high-throughput analyses and neurosphere cultures.

The low number of neural progenitor cells (NPCs) present in the adult and aged primate brains represents a challenge for generating high-yield and viable in vitro cultures of primary brain cells. Here we report a step-by-step approach for the fast and reproducible isolation of high-yield and viable primary brain cells, including mature neurons, immature cells and NPCs, from adult and aged macaques. We describe the anesthesia, transcardial perfusion and brain tissue preparation; the subsequent microdissection of the regions of interest and their enzymatic dissociation, leading to the separation of single cells. The cell isolation steps of our protocol can also be used for routine cell culturing, in particular for NPC expansion and differentiation, suitable for studies of hippocampal neurogenesis in the adult macaque brain. The purified primary brain cells are largely free from myelin debris and erythrocytes, paving the way for multiple downstream applications in vitro and in vivo. When combined with single-cell profiling techniques, this approach allows an unbiased and comprehensive mapping of cell states in the adult and aged macaque brain, which is needed to advance our understanding of human cognitive and neurological diseases. The neural cell isolation protocol requires 4 h and a team of four to six users with expertize in primary brain cell isolation to avoid tissue hypoxia during the time-sensitive steps of the procedure.

TREM2 deficiency inhibits microglial activation and aggravates demyelinating injury in neuromyelitis optica spectrum disorder

Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disorder of the central nervous system (CNS) triggered by autoimmune mechanisms. Microglia are activated and play a pivotal role in response to tissue injury. Triggering receptor expressed on myeloid cells 2 (TREM2) is expressed by microglia and promotes microglial activation, survival and phagocytosis. Here, we identify a critical role for TREM2 in microglial activation and function during AQP4-IgG and complement-induced demyelination. TREM2-deficient mice had more severe tissue damage and neurological impairment, as well as fewer oligodendrocytes with suppressed proliferation and maturation. The number of microglia clustering in NMOSD lesions and their proliferation were reduced in TREM2-deficient mice. Moreover, morphology analysis and expression of classic markers showed compromised activation of microglia in TREM2-deficient mice, which was accompanied by suppressed phagocytosis and degradation of myelin debris by microglia. These results overall indicate that TREM2 is a key regulator of microglial activation and exert neuroprotective effects in NMOSD demyelination.Graphical

Microglial aryl hydrocarbon receptor enhances phagocytic function via SYK and promotes remyelination in the cuprizone mouse model of demyelination

Multiple sclerosis (MS) is an inflammatory-mediated demyelinating disease of the central nervous system (CNS). Although studies have demonstrated that microglia facilitate remyelination in demyelinating diseases, the underlying mechanisms are still not fully characterized. We found that aryl hydrocarbon receptor (AhR), an environment sensor, was upregulated within the corpus callosum in the cuprizone model of CNS demyelination, and upregulated AhR was mainly confined to microglia. Deletion of AhR in adult microglia inhibited efficient remyelination. Transcriptome analysis using RNA-seq revealed that AhR-deficient microglia displayed impaired gene expression signatures associated with lysosome and phagocytotic pathways. Furthermore, AhR-deficient microglia showed impaired clearance of myelin debris and defected phagocytic capacity. Further investigation of target genes of AhR revealed that spleen tyrosine kinase (SYK) is the downstream effector of AhR and mediated the phagocytic capacity of microglia. Additionally, AhR deficiency in microglia aggravated CNS inflammation during demyelination. Altogether, our study highlights an essential role for AhR in microglial phagocytic function and suggests the therapeutic potential of AhR in demyelinating diseases.Graphical

IFN-Induced Protein with Tetratricopeptide Repeats 2 Limits Autoimmune Inflammation by Regulating Myeloid Cell Activation and Metabolic Activity.

Besides antiviral functions, type I IFN expresses potent anti-inflammatory properties and is being widely used to treat certain autoimmune conditions, such as multiple sclerosis. In a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis, administration of IFN-β effectively attenuates the disease development. However, the precise mechanisms underlying IFN-β-mediated treatment remain elusive. In this study, we report that IFN-induced protein with tetratricopeptide repeats 2 (Ifit2), a type I and type III IFN-stimulated gene, plays a previously unrecognized immune-regulatory role during autoimmune neuroinflammation. Mice deficient in Ifit2 displayed greater susceptibility to experimental autoimmune encephalomyelitis and escalated immune cell infiltration in the CNS. Ifit2 deficiency was also associated with microglial activation and increased myeloid cell infiltration. We also observed that myelin debris clearance and the subsequent remyelination were substantially impaired in Ifit2-/- CNS tissues. Clearing myelin debris is an important function of the reparative-type myeloid cell subset to promote remyelination. Indeed, we observed that bone marrow-derived macrophages, CNS-infiltrating myeloid cells, and microglia from Ifit2-/- mice express cytokine and metabolic genes associated with proinflammatory-type myeloid cell subsets. Taken together, our findings uncover a novel regulatory function of Ifit2 in autoimmune inflammation in part by modulating myeloid cell function and metabolic activity.

A novel phenotype of B cells associated with enhanced phagocytic capability and chemotactic function after ischemic stroke.

Accumulating evidence has demonstrated the involvement of B cells in neuroinflammation and neuroregeneration. However, the role of B cells in ischemic stroke remains unclear. In this study, we identified a novel phenotype of macrophage-like B cells in brain-infiltrating immune cells expressing a high level of CD45. Macrophage-like B cells characterized by co-expression of B-cell and macrophage markers, showed stronger phagocytic and chemotactic functions compared with other B cells and showed upregulated expression of phagocytosis-related genes. Gene Ontology analysis found that the expression of genes associated with phagocytosis, including phagosome- and lysosome-related genes, was upregulated in macrophage-like B cells. The phagocytic activity of macrophage-like B cells was verified by immunostaining and three-dimensional reconstruction, in which TREM2-labeled macrophage-like B cells enwrapped and internalized myelin debris after cerebral ischemia. Cell-cell interaction analysis revealed that macrophage-like B cells released multiple chemokines to recruit peripheral immune cells mainly via CCL pathways. Single-cell RNA sequencing showed that the transdifferentiation to macrophage-like B cells may be induced by specific upregulation of the transcription factor CEBP family to the myeloid lineage and/or by downregulation of the transcription factor Pax5 to the lymphoid lineage. Furthermore, this distinct B cell phenotype was detected in brain tissues from mice or patients with traumatic brain injury, Alzheimer's disease, and glioblastoma. Overall, these results provide a new perspective on the phagocytic capability and chemotactic function of B cells in the ischemic brain. These cells may serve as an immunotherapeutic target for regulating the immune response of ischemic stroke.

Renal-friendly Li+-doped carbonized polymer dots activate Schwann cell autophagy for promoting peripheral nerve regeneration.

Activation of autophagy in Schwann cells (SCs) has emerged as a powerful trigger for peripheral nerve injury (PNI) repair. Lithium ion (Li+) is a classical autophagy activator that plays an important role in promoting axonal extension and remyelination. However, the therapeutic window of existing lithium drugs is extremely narrow, and the adverse side effects, especially nephrotoxicity, severely limit their therapeutic value. Herein, Li+-doped carbonized polymer dots (Li-CPDs) was synthesized for the first time to change the pharmacokinetics of Li+ from occupying epithelial sodium channels to lipid raft-mediated endocytosis. The in-vivo results confirmed that Li-CPDs could accelerate the removal of myelin debris and promote nerve regeneration via activating autophagy of SCs. Moreover, Li-CPDs exhibited almost no renal toxicity compared to that of raw lithium drugs. Thus, Li-CPDs could serve as a promising Li+-based nanomedicine for PNI regeneration with improved biosafety. STATEMENT OF SIGNIFICANCE: Regardless of the fact that lithium drugs have been used in treatment of mental illness such as manic depression, the systemic side effects and renal metabolic toxicity still seriously restrict their clinical application. Since Li+ and Na+ compete for ion channels of cell membrane, the cell entry efficiency is extremely low and easily affected by body fluctuations, which seems to be an unsolvable problem. Herein, we rationally exploited the endocytotic features of CPDs to develop Li-CPDs. The Li-CPDs improved the entry pathway, greatly reduced nephrotoxicity, and inherited the biological function of Li+ to activate autophagy for promoting peripheral nerve regeneration. Due to the BBB-crossing property of Li-CPDs, it also showed application prospects in future research on central nervous system diseases.

Lysosomal dysfunction in Schwann cells is involved in bortezomib-induced peripheral neurotoxicity.

Bortezomib (BTZ) is a proteasome inhibitor serves as a first-line drug for multiple myeloma treatment. BTZ-induced peripheral neuropathy (BIPN) is the most common adverse effect of BTZ with an incidence as high as 40-60%. However, the pathological mechanisms underlying BIPN remain largely unclear. BTZ leads to dramatic Schwann cell demyelination in sciatic nerves. Previous studies implied that myelin debris was predominantly degraded via autophagy-lysosome pathway in Schwann cells. However, the association of autophagy with BIPN has not been made. Mice were treated with BTZ (2mg/kg, i.v.) on Day1 and Day4 each week for continuous 4weeks. BTZ-treated mice showed enhanced mechanical hyperalgesia, decreased tail nerve conduction and sciatic nerve demyelination. Unexpectedly, BTZ led to the accumulation of autophagic vesicles, LC3-II and p62 in the sciatic nerve. Moreover, BTZ blocked autophagic flux in RSC96 Schwann cells as determined by mcherry-GFP-LC3 assay, suggesting BTZ may impair lysosomal function rather than inducing autophagy in Schwann cells. BTZ significantly reduced the lysosomal activity in Schwann cells as determined by reduced LysoTracker Red and DQ-Red-BSA staining and increased the level of immature Cathepsin B (CTSB). Remarkably, lysosomal activators PP242 and Torin1, significantly reversed the blockage of autophagic flux by BTZ. We further verified that Torin1 rescued the demyelination, nerve conduction and reduced the mechanical hyperalgesia in BIPN mice. Additionally, Torin1 did not compromise the efficacy of BTZ in suppressing multiple myeloma RPMI8226 cell. Taken together, we identified that lysosomal dysfunction in Schwann cells caused by BTZ is involved in the BIPN pathology. Improved lysosomal function in Schwann cells can be a promising strategy for BIPN treatment.

CD11c+ microglia promote white matter repair after ischemic stroke

Ischemic stroke leads to white matter damage and neurological deficits. However, the characteristics of white matter injury and repair after stroke are unclear. Additionally, the precise molecular communications between microglia and white matter repair during the stroke rehabilitation phase remain elusive. In this current study, MRI DTI scan and immunofluorescence staining were performed to trace white matter and microglia in the mouse transient middle cerebral artery occlusion (tMCAO) stroke model. We found that the most serious white matter damage was on Day 7 after the ischemic stroke, then it recovered gradually from Day 7 to Day 30. Parallel to white matter recovery, we observed that microglia centered around the damaged myelin sheath and swallowed myelin debris in the ischemic areas. Then, microglia of the ischemic hemisphere were sorted by flow cytometry for RNA sequencing and subpopulation analysis. We found that CD11c+ microglia increased from Day 7 to Day 30, demonstrating high phagocytotic capabilities, myelin-supportive genes, and lipid metabolism associated genes. CD11c+ microglia population was partly depleted by the stereotactic injecting of rAAV2/6M-taCasp3 (rAAV2/6M-CMV-DIO-taCasp3-TEVp) into CD11c-cre mice. Selective depletion of CD11c+ microglia disrupted white matter repair, oligodendrocyte maturation, and functional recovery after stroke by Rotarod test, Adhesive Removal test, and Morris Water Maze test. These findings suggest that spontaneous white matter repair occurs after ischemic stroke, while CD11c+ microglia play critical roles in this white matter restorative progress.