Discovery of novel post-translational modifications provides new insights into changes in protein function, localization, and stability. They are also key elements in understanding disease mechanisms and developing therapeutic strategies. We have previously reported that ubiquitin-like 3 (UBL3) serves as a novel post-translational modifier that is highly expressed in the cerebral cortex and hippocampus, in addition to various other organs, and that 60% of proteins contained in small extracellular vesicles (sEVs), including exosomes, are influenced by UBL3. In this study, we generated transgenic mice expressing biotinylated UBL3 in the forebrain under control of the alpha-CaMKII promoter (Ubl3Tg/+). Western blot analysis revealed that the expression of UBL3 in the cerebral cortex and hippocampus was 6- to 7-fold higher than that in the cerebellum. Therefore, we performed immunoprecipitation of protein extracts from the cerebral cortex of Ubl3+/+ and Ubl3Tg/+ mice using avidin beads to comprehensively discover UBL3 interacting proteins, identifying 35 new UBL3 interacting proteins. Nine proteins were annotated as extracellular exosomes. Gene Ontology (GO) analysis suggested a new relationship between sEVs and RNA metabolism in neurodegenerative diseases. We confirmed the association of endogenous UBL3 with the RNA-binding proteins FUS and HPRT1-both listed in the Neurodegenerative Diseases Variation Database (NDDVD)-and with LYPLA1, which is involved in Huntington\'s disease, using immunoprecipitation (IP)-western blotting analysis. These UBL3 interacting proteins will accelerate the continued elucidation of sEV research about proteins regulated by novel post-translational modifications by UBL3 in the brain.

Analyzing changes in gene expression within specific brain regions of individuals with Type 2 Diabetes (T2DM) who do not exhibit significant cognitive deficits can yield valuable insights into the mechanisms underlying the progression towards a more severe phenotype. In this study, transcriptomic analysis of the cortex and hippocampus of mice with long-term T2DM revealed alterations in the expression of 28 genes in the cerebral cortex and 15 genes in the hippocampus. Among these genes, six displayed consistent changes in both the cortex and hippocampus: Interferon regulatory factor 7 (Irf7), Hypoxia-inducible factor 3 alpha (Hif-3α), period circadian clock 2 (Per2), xanthine dehydrogenase (Xdh), and Transforming growth factor β-stimulated clone 22/TSC22 (Tsc22d3) were upregulated, while Claudin-5 (Cldn5) was downregulated. Confirmation of these changes was achieved through RT-qPCR. At the protein level, CLDN5 and IRF7 exhibited similar alterations, with CLDN5 being downregulated and IRF7 being upregulated. In addition, the hippocampus and cortex of the T2DM mice showed decreased levels of IκBα, implying the involvement of NF-κB pathways as well. Taken together, these results suggest that the weakening of the blood-brain barrier and an abnormal inflammatory response via the Interferon 1 and NF-κB pathways underlie cognitive impairment in individuals with long-standing T2DM.

Scale-free statistics of coordinated neuronal activity, suggesting a universal operating mechanism across spatio-temporal scales, have been proposed as a necessary condition of healthy resting-state brain activity. Recent studies have focused on anesthetic agents to induce distinct neural states in which consciousness is altered to understand the importance of critical dynamics. However, variation in experimental techniques, species, and anesthetics, have made comparisons across studies difficult. Here we conduct a survey of several common anesthetics (isoflurane, pentobarbital, ketamine) at multiple dosages, using calcium wide-field optical imaging of the mouse cortex. We show that while low-dose anesthesia largely preserves scale-free statistics, surgical plane anesthesia induces multiple dynamical modes, most of which do not maintain critical avalanche dynamics. Our findings indicate multiple pathways away from default critical dynamics associated with quiet wakefulness, not only reflecting differences between these common anesthetics but also showing significant variations in individual responses. This is suggestive of a non-trivial relationship between criticality and the underlying state of the subject.

Oligodendrocyte precursor cells (OPCs) give rise to myelinating oligodendrocytes of the brain. This process persists throughout life and is essential for recovery from neurodegeneration. To better understand the cellular checkpoints that occur during oligodendrogenesis, we determined the mitochondrial distribution and morphometrics across the oligodendrocyte lineage in mouse and human cerebral cortex. During oligodendrocyte generation, mitochondrial content expands concurrently with a change in subcellular partitioning towards the distal processes. These changes are followed by an abrupt loss of mitochondria in the oligodendrocyte processes and myelin, coinciding with sheath compaction. This reorganization and extensive expansion and depletion take 3 days. Oligodendrocyte mitochondria are stationary over days while OPC mitochondrial motility is modulated by animal arousal state within minutes. Aged OPCs also display decreased mitochondrial size, volume fraction, and motility. Thus, mitochondrial dynamics are linked to oligodendrocyte generation, dynamically modified by their local microenvironment, and altered in the aging brain.

The maturation of cerebral cortical networks during early life involves a major reorganization of long-range axonal connections. In a recent study, Bragg-Gonzalo, Aguilera, et al. discovered that in mice, the interhemispheric connections sent by S1L4 callosal projection neurons are pruned via the tight control of their ipsilateral synaptic integration, which relies on the early activity of specific interneurons.

BACKGROUND: Structural and functional abnormalities in the cortical-striatal network (CSN) are hypothesised to play a key role in the pathogenesis of neurological disease-associated fatigue. Some small-scale functional MRI (fMRI) studies have suggested that poststroke fatigue (PSF) is related to focal functional connectivity (FC) changes. To date, there has been no published large-scale fMRI study on PSF. This planned study will examine the role of the CSN FC on PSF.
METHODS: The planned study will be a prospective cohort study conducted at the Neurology Unit of the Prince of Wales Hospital. We will recruit 738 participants. The project duration will be 36 months. A psychiatrist will administer the Fatigue Severity Scale (FSS) at 3 months (P1) following the index stroke. PSF is defined as an FSS Score≥4.0. PSF severity will be defined by the FSS total score at P1. Participants with PSF at P1 will undergo two follow-up assessments at 9 (P2) and 15 (P3) months post stroke. PSF remission at P2 or P3 will be defined as a 50% reduction in FSS. Participants will undergo MRI examinations within 2 weeks of the 3-month poststroke assessment. Structural MRI, resting-state fMRI and diffusion tensor imaging will be performed. FC, structural connectivity, infarcts, cerebral microbleeds and white matter hyperintensities will be analysed. For the primary analysis, the effect of PSF on the FC, structural connectivity and diffusion metrics of CSN of stroke survivors, voxel-wise two-sample t-tests will be performed with FDR correction for multiple comparison and significance level set at p<0.05.
BACKGROUND: Ethical approval was obtained from the Joint Chinese University of Hong Kong-New Territories East Cluster clinical research ethics committee. The study findings will be shared through peer-reviewed journal publications, national and international conferences and social media platforms.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

This chapter describes basic principles and key findings regarding the development and maturation of the human brain, the former referring to the pre-natal and early post-natal periods and the latter concerning childhood and adolescence. In both cases, we focus on brain structure as revealed in vivo with multi-modal magnetic resonance imaging (MRI). We begin with a few numbers about the human brain and its cellular composition and a brief overview of a number of MRI-based metrics used to characterize age-related variations in grey and white matter. We then proceed with synthesizing current knowledge about developmental and maturational changes in the cerebral cortex (its thickness, surface area, and intra-cortical myelination) and the underlying white matter (volume and structural properties). To facilitate biological interpretations of MRI-derived metrics, we introduce the concept of virtual histology. We conclude the chapter with a few notes about future directions in the study of factors shaping the human brain from conception onwards.

BACKGROUND: Bisphenol A (BPA) is a widely used chemical with a harmful effect on animal and human. The neonatal and juvenile period is a highly risky neurodevelopmental period.
OBJECTIVE: This study aimed to determine how male albino rat pups\' cerebral cortex was altered by low doses of BPA given to mothers and the role of the oxidative stress.
METHODS: Thirty pregnant rats were randomly split into three equal groups, negative control, and positive control: received 1 cc of corn oil once a day through gastric tube and BPA treated: a dose of 200 µg/kg/day (dissolved in 1 cc corn oil). The male rat pups of each group were sacrificed at 1 week, 3 weeks and 6 weeks. The cerebra were then separated from the brain for histological and biochemical studies.
RESULTS: Rats administered BPA had raised levels of lipid peroxidation marker (MDA), lower levels of enzymatic antioxidants (SOD and CAT) with decreased body, cerebral weights, and decreased levels of non-enzymatic antioxidant defense (GSH). Histo-pathologically, shrunken pyramidal cells with congested blood vessels appeared. GFAP displayed increased number of positive immune-reactive astrocytes with high statistically significant increase in the area % in BPA treated group when compared to the control groups, on contrary to MBP. Semi-thin and ultra-thin BPA-sections revealed degenerative changes in myelinated axons with tiny nucleus and broken nuclear membranes. Lysosomes, dilated endoplasmic reticulum cisternae with noticeable increase in unmyelinated nerve fibers were also observed.
CONCLUSIONS: The structure of the developing cerebral cortex is negatively impacted by BPA due to oxidative stress.

ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.