This study aims to explore the effects and potential mechanisms of Modified Shuyu Pills in ameliorating depressive behaviors in the mouse model of vascular dementia(VaD). Seventy-two three-month-old male C57BL/6 mice were assigned into six groups: sham, model, low-, medium-, and high-dose Modified Shuyu Pills, and fluoxetine. The other five groups except the sham group underwent bilateral common carotid artery stenosis combined with chronic unpredictable stress. Depressive behaviors were assessed by the sucrose preference test and tail suspension test. Cerebral blood flow was measured by laser speckle imaging. Protein levels of low density lipoprotein receptor(LDLR), mitogen-activated protein kinase kinase(MEK), phosphorylated(p)-MEK, extracellular signal-regulated kinase(ERK), and p-ERK in the ventral tegmental area(VTA) and nucleus accumbens(NAc) were determined by Western blot. The fluorescence intensity of myelin basic protein(MBP) in the VTA and NAc were measured by immunofluorescence. Myelin sheath morphology in the VTA and NAc was observed by luxol fast blue staining, and the ultrastructure of myelin sheath in the VTA and NAc was examined by transmission electron microscopy. In the tail suspension test, the immobility time of the model group was longer than that of the sham group(P<0.01). In the sucrose preference test, the sucrose preference rate of the model group was lower than that of the sham group(P<0.01). After intervention with Modified Shuyu Pills, the immobility time in the tail suspension test was shortened(P<0.01), and the sucrose preference rate increased(P<0.01). Laser speckle imaging results showed that compared with the sham group, the model group showed reduced cerebral blood flow(P<0.01), and the reduction was reversed by medium-and high-dose Modified Shuyu Pills(P<0.01). Western blot results indicated that the relative expression levels of LDLR, p-MEK/MEK, and p-ERK/ERK in the VTA and NAc of the model group were lower than those in the sham group(P<0.01). Medium-and high-dose Modified Shuyu Pills reversed this trend(P<0.01). Immunofluorescence results showed that the fluorescence intensity of MBP in the VTA and NAc of the model group was lower than that of the sham group(P<0.01). The medium-and high-dose Modified Shuyu Pills groups showed increased fluorescence intensity of MBP in the VTA compared with the model group(P<0.01). In the NAc, the fluorescence intensity of MBP in all the groups of Modified Shuyu Pills increased to varying degrees compared with that in the model group(P<0.01). Luxol fast blue staining results showed that the model group presented lighter staining intensity and looser arrangement of myelin fibers than the sham group, indicating significant demyelination in the model group. However, after intervention with medium-and high-dose Modified Shuyu Pills, the staining intensity and myelin sheath structure in the VTA and NAc were improved. Transmission electron microscopy results revealed that the myelin sheath in the VTA and NAc of the sham group was intact and dense, while the model group exhibited extensive myelin loss, with myelin sheath degeneration and disintegration. After intervention with Modified Shuyu Pills, the myelin sheath loss in the VTA and NAc of mice was reduced, and the proportion of myelinated tissue increased. In summary, Modified Shuyu Pills may promote myelination via the VTA-NAc circuit by upregulating the LDLR/MEK/ERK signaling pathway, thereby ameliorating depressive-like behaviors in VaD mice.
Animals ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Ventral Tegmental Area/metabolism* ; Mice, Inbred C57BL ; Disease Models, Animal ; Depression/genetics* ; Receptors, LDL/genetics* ; Dementia, Vascular/psychology* ; MAP Kinase Signaling System/drug effects* ; Nucleus Accumbens/metabolism* ; Behavior, Animal/drug effects* ; Humans ; Myelin Sheath/drug effects* ; Extracellular Signal-Regulated MAP Kinases/genetics*

Anxiety disorder is a major symptom of autism spectrum disorder (ASD) with a comorbidity rate of ~40%. However, the neural mechanisms of the emergence of anxiety in ASD remain unclear. In our study, we found that hyperactivity of basolateral amygdala (BLA) pyramidal neurons (PNs) in Shank3 InsG3680 knock-in (InsG3680^+/+) mice is involved in the development of anxiety. Electrophysiological results also showed increased excitatory input and decreased inhibitory input in BLA PNs. Chemogenetic inhibition of the excitability of PNs in the BLA rescued the anxiety phenotype of InsG3680^+/+ mice. Further study found that the diminished control of the BLA by medial prefrontal cortex (mPFC) and optogenetic activation of the mPFC-BLA pathway also had a rescue effect, which increased the feedforward inhibition of the BLA. Taken together, our results suggest that hyperactivity of the BLA and alteration of the mPFC-BLA circuitry are involved in anxiety in InsG3680^+/+ mice.
Animals ; Prefrontal Cortex/metabolism* ; Basolateral Nuclear Complex/metabolism* ; Mice ; Anxiety/metabolism* ; Nerve Tissue Proteins/genetics* ; Male ; Gene Knock-In Techniques ; Pyramidal Cells/physiology* ; Mice, Transgenic ; Neural Pathways/physiopathology* ; Mice, Inbred C57BL ; Microfilament Proteins

Oligodendrocyte lineage cells, including oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLs), are essential in establishing and maintaining brain circuits. Autophagy is a conserved process that keeps the quality of organelles and proteostasis. The role of autophagy in oligodendrocyte lineage cells remains unclear. The present study shows that autophagy is required to maintain the number of OPCs/OLs and myelin integrity during brain aging. Inactivation of autophagy in oligodendrocyte lineage cells increases the number of OPCs/OLs in the developing brain while exaggerating the loss of OPCs/OLs with brain aging. Inactivation of autophagy in oligodendrocyte lineage cells impairs the turnover of myelin basic protein (MBP). It causes MBP to accumulate in the cytoplasm as multimeric aggregates and fails to be incorporated into integral myelin, which is associated with attenuated endocytic recycling. Inactivation of autophagy in oligodendrocyte lineage cells impairs myelin integrity and causes demyelination. Thus, this study shows autophagy is required to maintain myelin quality during aging by controlling the turnover of myelin components.
Animals ; Autophagy/physiology* ; Oligodendroglia/metabolism* ; Myelin Sheath/physiology* ; Aging/pathology* ; Myelin Basic Protein/metabolism* ; Cell Lineage/physiology* ; Mice ; Oligodendrocyte Precursor Cells ; Mice, Inbred C57BL ; Brain/cytology* ; Cells, Cultured ; Cell Count

Acute mitochondrial damage and the energy crisis following axonal injury highlight mitochondrial transport as an important target for axonal regeneration. Syntaphilin (Snph), known for its potent mitochondrial anchoring action, has emerged as a significant inhibitor of both mitochondrial transport and axonal regeneration. Therefore, investigating the molecular mechanisms that influence the expression levels of the snph gene can provide a viable strategy to regulate mitochondrial trafficking and enhance axonal regeneration. Here, we reveal the inhibitory effect of microRNA-146b (miR-146b) on the expression of the homologous zebrafish gene syntaphilin b (snphb). Through CRISPR/Cas9 and single-cell electroporation, we elucidated the positive regulatory effect of the miR-146b-snphb axis on Mauthner cell (M-cell) axon regeneration at the global and single-cell levels. Through escape response tests, we show that miR-146b-snphb signaling positively regulates functional recovery after M-cell axon injury. In addition, continuous dynamic imaging in vivo showed that reprogramming miR-146b significantly promotes axonal mitochondrial trafficking in the pre-injury and early stages of regeneration. Our study reveals an intrinsic axonal regeneration regulatory axis that promotes axonal regeneration by reprogramming mitochondrial transport and anchoring. This regulation involves noncoding RNA, and mitochondria-associated genes may provide a potential opportunity for the repair of central nervous system injury.
Animals ; Zebrafish ; MicroRNAs/genetics* ; Nerve Regeneration/physiology* ; Mitochondria/metabolism* ; Zebrafish Proteins/genetics* ; Axons/metabolism* ; Signal Transduction/physiology* ; Axonal Transport/physiology* ; Nerve Tissue Proteins/genetics*

Autism Spectrum Disorder (ASD) is marked by early-onset neurodevelopmental anomalies, yet the temporal dynamics of genetic contributions to these processes remain insufficiently understood. This study aimed to elucidate the role of the Shank3 gene, known to be associated with monogenic causes of autism, in early developmental processes to inform the timing and mechanisms for potential interventions for ASD. Utilizing the Shank3B knockout (KO) mouse model, we examined Shank3 expression and its impact on neuronal maturation through Golgi staining for dendritic morphology and electrophysiological recordings to measure synaptic function in the anterior cingulate cortex (ACC) across different postnatal stages. Our longitudinal analysis revealed that, while Shank3B KO mice displayed normal neuronal morphology at one week postnatal, significant impairments in dendritic growth and synaptic activity emerged by two to three weeks. These findings highlight the critical developmental window during which Shank3 is essential for neuronal and synaptic maturation in the ACC.
Animals ; Nerve Tissue Proteins/metabolism* ; Mice, Knockout ; Dendrites/metabolism* ; Mice ; Synapses/metabolism* ; Gyrus Cinguli/metabolism* ; Male ; Mice, Inbred C57BL ; Autism Spectrum Disorder/genetics* ; Microfilament Proteins

Accurate timing of myelination is crucial for the proper functioning of the central nervous system. Here, we identified a de novo heterozygous mutation in TMEM63A (c.1894G>A; p. Ala632Thr) in a 7-year-old boy exhibiting hypomyelination. A Ca²⁺ influx assay suggested that this is a loss-of-function mutation. To explore how TMEM63A deficiency causes hypomyelination, we generated Tmem63a knockout mice. Genetic deletion of TMEM63A resulted in hypomyelination at postnatal day 14 (P14) arising from impaired differentiation of oligodendrocyte precursor cells (OPCs). Notably, the myelin dysplasia was transient, returning to normal levels by P28. Primary cultures of Tmem63a^-/- OPCs presented delayed differentiation. Lentivirus-based expression of TMEM63A but not TMEM63A_A632T rescued the differentiation of Tmem63a^-/- OPCs in vitro and myelination in Tmem63a^-/- mice. These data thus support the conclusion that the mutation in TMEM63A is the pathogenesis of the hypomyelination in the patient. Our study further demonstrated that TMEM63A-mediated Ca²⁺ influx plays critical roles in the early development of myelin and oligodendrocyte differentiation.
Animals ; Cell Differentiation/physiology* ; Oligodendroglia/metabolism* ; Mice, Knockout ; Mice ; Male ; Myelin Sheath/metabolism* ; Humans ; Child ; Cells, Cultured ; Oligodendrocyte Precursor Cells/metabolism*

Infiltration and activation of peripheral immune cells are critical in the progression of multiple sclerosis and its experimental animal model, experimental autoimmune encephalomyelitis (EAE). This study investigates the role of high mobility group box 1 (HMGB1) in oligodendrocyte precursor cells (OPCs) in modulating pathogenic T cells infiltrating the central nervous system through the blood-brain barrier (BBB) by using OPC-specific HMGB1 knockout (KO) mice. We found that HMGB1 released from OPCs promotes BBB disruption, subsequently allowing increased immune cell infiltration. The migration of CD4+ T cells isolated from EAE-induced mice was enhanced when co-cultured with OPCs compared to oligodendrocytes (OLs). OPC-specific HMGB1 KO mice exhibited lower BBB permeability and reduced immune cell infiltration into the CNS, leading to less damage to the myelin sheath and mitigated EAE progression. CD4+ T cell migration was also reduced when co-cultured with HMGB1 knock-out OPCs. Our findings reveal that HMGB1 secretion from OPCs is crucial for regulating immune cell infiltration and provides insights into the immunomodulatory function of OPCs in autoimmune diseases.
Animals ; Encephalomyelitis, Autoimmune, Experimental/metabolism* ; HMGB1 Protein/deficiency* ; Mice, Knockout ; Oligodendrocyte Precursor Cells/immunology* ; Mice, Inbred C57BL ; CD4-Positive T-Lymphocytes/immunology* ; Cell Movement ; Blood-Brain Barrier/immunology* ; Mice ; Myelin Sheath/pathology* ; Disease Models, Animal ; Coculture Techniques ; Oligodendroglia/metabolism* ; Female ; Cells, Cultured

Demyelination and remyelination play key roles in spinal cord injury (SCI), affecting the recovery of motor and sensory functions. Research in rodent models is extensive, but the study of these processes in non-human primates is limited. Therefore, our goal was to thoroughly study the histological features of demyelination and remyelination after contusion injury of the cervical spinal cord in Macaca fascicularis. In a previous study, we created an SCI model in M. fascicularis by controlling the contusion displacement. We used Eriochrome Cyanine staining, immunohistochemical analysis, and toluidine blue staining to evaluate demyelination and remyelination. The results showed demyelination ipsilateral to the injury epicenter both rostrally and caudally, the former mainly impacting sensory pathways, while the latter primarily affected motor pathways. Toluidine blue staining showed myelin loss and axonal distension at the injury site. Schwann cell-derived myelin sheaths were only found at the center, while thinner myelin sheaths from oligodendrocytes were seen at the center and surrounding areas. Our study showed that long-lasting demyelination occurs in the spinal cord of M. fascicularis after SCI, with oligodendrocytes and Schwann cells playing a significant role in myelin sheath formation at the injury site.
Animals ; Spinal Cord Injuries/physiopathology* ; Demyelinating Diseases/etiology* ; Remyelination/physiology* ; Macaca fascicularis ; Disease Models, Animal ; Myelin Sheath/pathology* ; Oligodendroglia/pathology* ; Schwann Cells/pathology* ; Female ; Spinal Cord/pathology* ; Axons/pathology*

Myelin is an essential structure that facilitates rapid saltatory conduction in the nervous system. Discrepancies in myelin microstructure are a hallmark of numerous neurological disorders, rendering the assessment of myelin integrity and content an indispensable tool in clinical diagnostics and neuroscience research. Extensive research has been dedicated to scrutinizing its biochemical makeup and morphology under normal, pathological, and experimental conditions over the years. In this review, we present an updated summary of the myelin sheath's structure, composition, and developmental trajectory. We systematically enumerate and contrast eight prevalent myelin staining techniques across dimensions of sensitivity, specificity, and resolution, delving into their underlying staining principles. With an initial application of myelin histology on the mouse demyelination model, our review accentuates the accurate delineation of myelination and the microstructural analysis of the myelin sheath. Such insights are anticipated to significantly contribute to the evaluation and understanding of white matter pathologies.
Myelin Sheath/metabolism* ; Animals ; Humans ; Demyelinating Diseases/pathology* ; Staining and Labeling/methods*

Neuropathic pain, a debilitating condition caused by dysfunction of the somatosensory nervous system, remains difficult to treat due to limited understanding of its molecular mechanisms. Bioinformatics analysis identified cerebellin 2 (CBLN2) as highly enriched in human and murine proprioceptive and nociceptive neurons. We found that CBLN2 expression is persistently upregulated in dorsal root ganglia (DRG) following spinal nerve ligation (SNL) in mice. In addition, transcription factor SOX11 binds to 12 cis-regulatory elements within the Cbln2 promoter to enhance its transcription. SNL also induced SOX11 upregulation, with SOX11 and CBLN2 co-localized in nociceptive neurons. The siRNA-mediated knockdown of Sox11 or Cbln2 attenuated SNL-induced mechanical allodynia and thermal hyperalgesia. High-throughput sequencing of DRG following intrathecal injection of CBLN2 revealed widespread gene expression changes, including upregulation of numerous NF-κB downstream targets. Consistently, CBLN2 activated NF-κB signaling, and inhibition with pyrrolidine dithiocarbamate reduced CBLN2-induced pain hypersensitivity, proinflammatory cytokines and chemokines production, and neuronal hyperexcitability. Together, these findings identified the SOX11/CBLN2/NF-κB axis as a critical mediator of neuropathic pain and a promising target for therapeutic intervention.
Animals ; Neuralgia/metabolism* ; Ganglia, Spinal/metabolism* ; Up-Regulation ; Mice ; NF-kappa B/metabolism* ; SOXC Transcription Factors/genetics* ; Male ; Neuroinflammatory Diseases/metabolism* ; Mice, Inbred C57BL ; Nerve Tissue Proteins/genetics* ; Hyperalgesia/metabolism* ; Signal Transduction ; Spinal Nerves