Leukodystrophies are a heterogeneous group of rare genetic neurologic disorders characterized by white matter degeneration resulting from mutations affecting glial cells. This review focuses on the primary subtypes-astroglial, oligodendroglial, and microglial leukodystrophies-offering a detailed description of their neuropathologic features and clinical manifestations. It delves into key aspects of the pathogenesis, emphasizing the distinct cellular mechanisms that drive white matter damage. Advances in disease modeling, including the development of animal models with pathologic gene expressions and patient-derived iPS-cell models, have significantly enhanced our understanding of these rare disorders. Insights into the roles of different glial cell types highlight the complexity of leukodystrophies and provide a foundation for the development of targeted therapeutic strategies.

The nervous tissue is composed of neurons and neuroglia, which by working in a tightly coordinated manner, define the function of the nervous system. Neuroglia, defined as homeostatic and defensive cells of the nervous system, are highly heterogeneous in form and function and are endowed with a remarkable plasticity that allows life-long adaptation to environmental challenges. Neuroglia of the peripheral nervous system are represented by myelinating, nonmyelinating, perisynaptic, and cutaneous Schwann cells, satellite glia of sensory and sympathetic ganglia and enteric glia of the enteric nervous system. Neuroglia of the central nervous system (CNS) are classified into macroglia and microglia. Macroglia in turn are represented by astroglia and oligodendroglia. Astroglia represent an extended class of homeostatic glial cells, which include astrocytes (protoplasmic, fibrous, velate, and marginal), radial astrocytes (Bergmann glial cells, glia-like nervous stem cells, and tanycytes), and ependymoglia. The oligodendroglial lineage is mainly responsible for myelination and support of central axons and is represented by oligodendrocytes and oligodendrocyte precursor cells. Microglia are the cells of nonneural, myeloid origin that invade the neural tube early in embryonic development. These cells are tissue macrophages adapted to the nervous system requirements. Microglia contribute to physiology of the nervous tissue and to the innate immunity and defense of the CNS.

BACKGROUND:Survivors of aneurysmal subarachnoid hemorrhage (aSAH) often have cognitive impairment, which may be caused by long-term inflammation. We aimed to determine whether long-term neuroinflammation or microstructural brain damage is associated with cognitive impairment after aSAH. METHODS:In this prospective cohort study, we included patients >3 years after aSAH between 2020 and 2022. Patients underwent neuropsychological evaluation, translocator protein 18 kDA (TSPO) positron emission tomography (PET) imaging using [18F]DPA-714 to determine neuroinflammation, and brain diffusion kurtosis imaging (DKI) to determine microstructural damage. We compared TSPO PET binding potential, mean kurtosis (MK), kurtosis anisotropy (KA), axial kurtosis (AK), and radial kurtosis (RA) between groups and determined which metric was correlated with individual cognitive tests. RESULTS:We included 27 patients with aSAH; 14 with and 13 without cognitive impairment. Whole-brain TSPO binding potential was similar between groups (mean BPND: -0.046 [95% confidence interval (CI): -0.105; 0.013] vs -0.047 [95% CI -0.108; 0.014], p = 0.98) and there were no regional differences. Those with cognitive impairment had a lower whole-brain MK (mean MK 0.70 [95% CI: 0.69-0.72] vs 0.73 [95% CI: 0.72-0.74], p = 0.03) and whole-brain AK (mean AK 0.81 [95% CI: 0.78-0.83] vs 0.86 [0.84-0.87], p = 0.04). Left thalamic MK and AK were correlated with tests of verbal memory (r = 0.60-0.67, p < 0.01), while other correlation tests were non-significant. CONCLUSION:Our results do not support the hypothesis that long-term cognitive impairment after aSAH is caused by long-term neuroinflammation. Instead, microstructural damage may play a role.

Neuroglia in the CNS, represented by astroglia, oligodendroglia, and microglia, are responsible for the homeostatic support and protection of the nervous tissue. Neuroglia are intimately involved in the pathogenesis of all neurologic diseases, and neuroglial changes to a large extent define the progression of these diseases and their neurologic outcome. In contrast to neurons, neuroglia are capable of mounting an evolutionary conserved response to pathology known as reactive gliosis. Reactive gliosis is initially protective and allostatic, and it is aimed at preserving the nervous tissue function and integrity. However, in many diseases, neuroglial cells undergo atrophy and functional asthenia, contributing to nervous tissue damage.

Neural stem cells (NSCs) in the subventricular zone (SVZ) of the mammalian brain become increasingly quiescent with aging, which correlates to increased inflammatory signals in the SVZ. Targeting cells that secrete inflammatory signals, such as microglia, could potentially re-activate NSCs. In this study, we characterized CD11b-positive microglia isolated from post-mortem SVZ from non-demented control (Aged), Alzheimer's disease (AD), and Parkinson's disease (PD) by single-cell and bulk RNA sequencing. Our transcriptome data revealed changes in gene signature in SVZ microglia from PD and AD, highlighting a disease-dependent response. Culture of iPSC-derived NSCs with supernatant from Aged, PD, and AD SVZ microglia showed an increase in proliferation and neuronal differentiation in the PD condition. Furthermore, we identified NR4A2, a transcription factor that promotes an anti-inflammatory microglia state, as a potential molecular mechanism that promotes a pro-neurogenic microglia phenotype. Altogether, our work identified a pro-neurogenic subpopulation of SVZ microglia that could be a novel target to promote repair in neurodegenerative diseases.

Here, we present a protocol for generating long-term microglia-containing air-liquid-interface cortical organoid (MG-ALI-CO) cultures. This approach minimizes necrotic core formation, a common limitation of extended organoid cultures, favoring microglia survival and homeostasis. We describe steps for generating air-liquid-interface cortical organoids (ALI-COs), integrating macrophage precursors, and maintaining MG-ALI-COs. Additionally, we outline several experimental analyses of MG-ALI-COs, including immunostaining, imaging, and patch-clamp electrophysiological recordings. This model provides a physiologically relevant system to investigate human neuroimmune interactions in a 3D brain-like environment.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia, characterized by deposition of extracellular amyloid-beta (A beta) aggregates and intraneuronal hyperphosphorylated Tau. Many AD risk genes, identified in genome-wide association studies (GWAS), are expressed in microglia, the innate immune cells of the central nervous system. Specific subtypes of microglia emerged in relation to AD pathology, such as disease-associated microglia (DAMs), which increased in number with age in amyloid mouse models and in human AD cases. However, the initial transcriptional changes in these microglia in response to amyloid are still unknown. Here, to determine early changes in microglia gene expression, hippocampal microglia from male APPswe/PS1dE9 (APP/PS1) mice and wild-type littermates were isolated and analyzed by RNA sequencing (RNA-seq). By bulk RNA-seq, transcriptomic changes were detected in hippocampal microglia from 6-months-old APP/PS1 mice. By performing single-cell RNA-seq of CD11c-positive and negative microglia from 6-months-old APP/PS1 mice and analysis of the transcriptional trajectory from homeostatic to CD11c-positive microglia, we identified a set of genes that potentially reflect the initial response of microglia to A beta.

Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAP(R239C) mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research.

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed in specific types of glial cells in the nervous system. The expression of GFAP is highly regulated during brain development and in neurological diseases. The presence of distinct GFAP-isoforms in various cell types, developmental stages, and diseases indicates that GFAP (post-)transcriptional regulation has a role in glial cell physiology and pathology. GFAP-isoforms differ in sub-cellular localisation, IF-network assembly properties, and IF-dynamics which results in distinct molecular interactions and mechanical properties of the IF-network. Therefore, GFAP (post-)transcriptional regulation is likely a mechanism by which radial glia, astrocytes, and glioma cells can modulate cellular function.

The human hypothalamus modulates mental health by balancing interactions between hormonal fluctuations and stress responses. Stress-induced progesterone release activates progesterone receptors (PR) in the human brain and triggers alterations in neuropeptides/neurotransmitters. As recent epidemiological studies have associated peripheral progesterone levels with suicide risks in humans, we mapped PR distribution in the human hypothalamus in relation to age and sex and characterized its (co-) expression in specific cell types. The infundibular nucleus (INF) appeared to be the primary hypothalamic structure via which progesterone modulates stress-related neural circuitry. An elevation of the number of pro-opiomelanocortin+ (POMC, an endogenous opioid precursor) neurons in the INF, which was due to a high proportion of POMC+ neurons that co-expressed PR, was related to suicide in patients with mood disorders (MD). MD donors who died of legal euthanasia were for the first time enrolled in a postmortem study to investigate the molecular signatures related to fatal suicidal ideations. They had a higher proportion of PR co-expressing POMC+ neurons than MD patients who died naturally. This indicates that the onset of endogenous opioid activation in MD with suicide tendency may be progesterone-associated. Our findings may have implications for users of progesterone-enriched contraceptives who also have MD and suicidal tendencies.

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD, even before symptom onset. Because these early disease phenotypes remain incompletely understood, we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients, presymptomatic C9ORF72-HRE (C9-HRE) carriers, and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition, single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids, including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further, molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly, organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology, whereas the extent to which more downstream cellular defects, as found in C9-ALS/FTD models, were detected varied for the different presymptomatic C9-HRE cases. Together, these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies.

Background: The major histocompatibility complex type II is downregulated in glioblastoma (GB) due to the silencing of the major transcriptional regulator class II transactivator (CIITA). We investigated the pro-immunogenic potential of CIITA overexpression in mouse and human GB. Methods: The intracerebral growth of wildtype GL261-WT cells was assessed following contralateral injection of GL261-CIITA cells or flank injections with GL261-WT or GL261-CIITA cells. Splenocytes obtained from mice implanted intracerebrally with GL261-WT, GL261-CIITA cells or phosphate buffered saline (PBS) were transferred to other mice and subsequently implanted intracerebrally with GL261-WT. Human GB cells and (syngeneic) GB-infiltrating immune cells were isolated from surgical samples and co-cultured with GB cells expressing CIITA or not, followed by RT-qPCR assessment of the expression of key immune regulators. Results: Intracerebral vaccination of GL261-CIITA significantly reduced the subsequent growth of GL261-WT cells implanted contralaterally. Vaccination with GL261-WT or -CIITA subcutaneously, however, equivalently retarded the intracerebral growth of GL261 cells. Adoptive cell transfer experiments showed a similar antitumor potential of lymphocytes harvested from mice implanted intracerebrally with GL261-WT or -CIITA. Human GB-infiltrating myeloid cells and lymphocytes were not activated when cultured with CIITA-expressing GB cells. Tumor-infiltrating NK cells remained mostly inactivated when in co-culture with GB cells, regardless of CIITA. Conclusion: these results question the therapeutic potential of CIITA-mediated immunotherapy in glioblastoma.

Glial fibrillary acidic protein (GFAP) is a well-established biomarker of reactive astrogliosis in the central nervous system because of its elevated levels following brain injury and various neurological disorders. The advent of ultra-sensitive methods for measuring low-abundant proteins has significantly enhanced our understanding of GFAP levels in the serum or plasma of patients with diverse neurological diseases. Clinical studies have demonstrated that GFAP holds promise both as a diagnostic and prognostic biomarker, including but not limited to individuals with Alzheimer's disease. GFAP exhibits diverse forms and structures, herein referred to as its proteoform complexity, encompassing conformational dynamics, isoforms and post-translational modifications (PTMs). In this review, we explore how the proteoform complexity of GFAP influences its detection, which may affect the differential diagnostic performance of GFAP in different biological fluids and can provide valuable insights into underlying biological processes. Additionally, proteoforms are often disease-specific, and our review provides suggestions and highlights areas to focus on for the development of new assays for measuring GFAP, including isoforms, PTMs, discharge mechanisms, breakdown products, higher-order species and interacting partners. By addressing the knowledge gaps highlighted in this review, we aim to support the clinical translation and interpretation of GFAP in both CSF and blood and the development of reliable, reproducible and specific prognostic and diagnostic tests. To enhance disease pathology comprehension and optimise GFAP as a biomarker, a thorough understanding of detected proteoforms in biofluids is essential.

Background: Previous works have shown that the expression of Class-II-Transactivator (CIITA) in tumor cells reduces the growth of glioblastoma (GB) in animal models, but immune effects cannot solely explain this. Here, we searched for immune-independent effects of CIITA on the proliferation of GB. Methods: Murine GL261 and human U87, GM2 and GM3 malignant glioma cells were transfected with CIITA. NSG (immunodeficient) and nude (athymic) mice were injected in the striatum with GL261-wildtype (-WT) and -CIITA, and tumor growth was assessed by immunohistology and luminescence reporter genes. Clonogenic, sphere-formation, and 3D Matrigel-based in vitro growth assays were performed to compare the growth of WT versus CIITA-expressing murine and human cells. Bulk RNA sequencing and RT2 qRT-PCR profiler arrays were performed on these four cell lines to assess RNA expression changes following CIITA transfection. Western blot analysis on several proliferation-associated proteins was performed. Results: The intracerebral growth of murine GL261-CIITA cells was drastically reduced both in immunodeficient and athymic mice. Tumor growth was reduced in vitro in three of the four cell types. RNA sequencing and RT2 profiler array experiments revealed a modulation of gene expression in the PI3-Akt, MAPK- and cell-cycle regulation pathways following CIITA overexpression. Western blot analysis showed an upregulation of p27 in the growth-inhibited cells following this treatment. PDGFR-beta was downregulated in all cells. We did not find consistent regulation of other proteins involved in GB proliferation. Conclusions: Proliferation is drastically reduced by CIITA in GB, both in vivo and in vitro, notably in association with p27-mediated inhibition of cell-cycle pathways.

Glial Fibrillary Acidic Protein (GFAP) and its isoforms are intermediate filament cytoskeletal proteins expressed in astrocytes. Reactive astrogliosis, hallmarked by an increase in GFAP expression, is part of the pathogenesis of Alzheimer’s disease (AD). A reliable and globally available method for measurement of GFAP in CSF and plasma is required as a pharmacodynamic biomarker of astrocyte reactivity in AD drug development programs. Here we describe the specificity and performance of a monoclonal antibody (mAb) based colorimetric ELISA for measurement of GFAP in human CSF and plasma while comparing with existing benchmarked measuring methods. A prototype colorimetric ELISA was designed using a 96-well microplate with capture mAb ADx RD-089 paired with the biotinylated detection mAb ADx RD-090. High specificity for GFAP was verified by testing recombinantly expressed intermediate filaments like vimentin, nestin, and synemin up to a concentration of 10,000 pg/mL. A method comparison study was performed using a set of 15 CSF and 13 plasma samples (commercially sourced, no clinical typing). Finally the prototype ELISA was compared to a commercially available QTX Simoa GFAP assay (Cat# 102336). The assay design is a 3-step sequential incubation (2-1-0.5 hrs) procedure while shaking with a respective 20- and 4-fold dilution of CSF and EDTA plasma. The assay calibration ranges between 25 and 5,000 pg/mL and GFAP levels of the CSF and plasma samples tested were above LLOQ and within the measuring range. Furthermore, reproducibility was between 2.0 – 13.6% for CSF, and between 5.4 – 11.4% for EDTA plasma. An appropriate parallelism was obtained with back-calculated concentrations between 80-120% across the measuring range. Finally, the assays’ GFAP levels correlated well with QTX Simoa for both CSF Pearson r = 0.98 (0.94 – 0.99) and plasma Pearson r = 0.97 (0.90 - 0.99). This colorimetric ELISA prototype assay enables the quantification of GFAP in both CSF and plasma. Alternatively it is a new accessible immunoassay method to support clinical trials where reactive astrogliosis needs to be assessed.

[This corrects the article DOI: 10.3389/fncel.2023.1173200.].