Biology subjects

Zandee, S.

Publications and source records attributed to Zandee, S..

11 recordsLinked to original sources

bioRxiv2025

Cerebrospinal fluid-driven ependymal motile cilia defects are implicated in multiple sclerosis pathophysiology

BackgroundMultiple sclerosis is a neurodegenerative autoimmune disorder of the central nervous system (CNS) in which autoreactive immune cells migrate through a damaged blood brain barrier, resulting in focal demyelinating lesions of both the white and grey matter. Of increasing interest is the repeated observation that beyond focal lesions, there are also diffuse, surface-in gradients of pathology in MS, wherein damage is most severe directly adjacent to cerebrospinal fluid (CSF)-contacting surfaces, such as the subpial and periventricular areas. This observation suggests that toxic factors within MS CSF may be contributing to the emergence and/or evolution of surface-in gradients. Directly separating the CSF from the periventricular parenchyma are ependymal cells - a glial epithelium that are equipped with tufts of motile cilia which are critical for circulating CSF solutes and regulating local fluid flow. While damage to ependymal cilia has the potential to drastically modify CSF homeostasis and thus contribute to the damage of CSF exposed regions, these motile cellular structures have yet to be investigated in the context of MS. MethodsWe first conducted single cell RNA sequencing of fresh human periventricular brain tissue containing ependymal cells from MS patients and non-MS disease controls. We subsequently collected CSF from MS patients and exposed cultured rodent ependymal cells to this CSF in order to evaluate impact on ependymal ciliary function. To complement our direct evaluation of cilia in the context of MS, we also confirmed whether cilia were altered in a classic animal model of MS, experimental autoimmune encephalomyelitis (EAE), and also designed a novel transgenic animal model to evaluate the cellular and behavioural effect(s) of adult ependymal ciliary disruption. ResultsSingle cell RNA sequencing analysis of human ependymal cells in MS demonstrated largescale dysregulation of ciliary genes and in situ stains of MS brain tissue confirmed a loss of ependymal cilia. Exposure of ependymal cells to MS CSF led to transcriptional modification of ciliary gene and protein expression and reduced ciliary beating frequency. Likewise, analysis of ependymal cells in EAE also demonstrated altered cilia gene and protein expression. Conditional knockout in adult mice, of the critical cilia-associated gene Ccdc39 in ependymal cells led to transient ventricular enlargement, increased periventricular microglial density, and alterations in nesting behaviour. ConclusionThese data suggest that motile cilia in ependymal cells are dysregulated in CNS autoimmunity. More importantly, however, they provide evidence to suggest that ependymal cilia disruption could play an active role in the development of periventricular pathology in MS and can lead to behavioural deficits that may underlie aspects non-motor MS symptomatology.

neuroscience↗

bioRxiv2025

An MRI-informed histo-molecular analysis implicates ependymal cells in the pathogenesis of periventricular pathology in multiple sclerosis

It is now widely recognized that the cerebrospinal fluid (CSF)-adjacent brain surfaces - namely the subpial cortical region and the ependyma-adjacent periventricular region - are uniquely susceptible to a distinct, diffuse form of pathology in multiple sclerosis. So-called surface-in gradients of pathology predict future disease relapses independent of classical white matter lesions and are thought to occur as a result of cytotoxic factors in the CSF. Given the underlying mechanisms driving surface-in gradients appear to be distinct, they represent a novel treatment target. However, exactly how cytotoxic factor entry into the brain is regulated at these CSF-facing borders is not understood, particularly at the ventricular interface. Indeed, although studies have indicated that ependymal cells may be damaged in MS, there has yet to be a comprehensive assessment of cell health in the disease. We employed ultra-high-field MRI-guided immunohistochemistry, electron microscopy, and multiomic single nucleus RNA/ATAC sequencing to deeply phenotype human ependymal cells in MS. Our data revealed that ependymal cell pathology is a direct correlate of periventricular surface-in gradients of pathology in MS, and that the immune-responsive, reactive state assumed by ependymal cells is associated with widespread transporter and junctional protein gene dysregulation. We then further defined the gene regulatory networks underpinning the MS ependymal state, predicted ligands known to be enriched in MS CSF that could drive the emergence of this state, and tested one candidate in vivo. We found that IFN{gamma} increased murine ependymal permeability and that conditional knockout of ependymal interferon gamma receptor 1 (Ifngr1) was sufficient to reverse this effect. Our data directly implicate ependymal cell dysregulation in the emergence of periventricular pathology in MS. More widely, we denote the modulatory capacity of CSF ligands on ependymal cell function and how this may influence the inflammatory status of the periventricular region. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/633055v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@d76aadorg.highwire.dtl.DTLVardef@9cb480org.highwire.dtl.DTLVardef@e4805org.highwire.dtl.DTLVardef@19a50b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

A subset of human choroid plexus epithelial cells exhibit mitochondrial eccentricity and distinct expression of the pigmentation-associated enzyme TYRP1

For decades, ultrastructural evaluation of epithelial cells in diverse organ systems has demonstrated the existence of two subtypes identified by stark differences in cytoplasmic electron density - so-called light and dark epithelial cells. Choroid plexus (CP) epithelial cells are key regulators of CSF homeostasis and are one of many specialized epithelial linings that exhibit this bimodal phenotype. Despite longstanding acknowledgement, it has been difficult to assess the potential significance of adult human light and dark CP epithelial cells due to a lack of characterization beyond electron microscopy (EM). We present the first transcriptomic analysis of adult human CP epithelial cells and denote the existence of four epithelial subpopulations, one of which is defined by elevated expression of TYRP1 - a melanocyte-associated tyrosine-related protein involved in cellular pigmentation and proliferation. TYRP1-high cells also downregulate genes related to cilia function (which is consistent with observations of dark cell identity in organoids) and upregulate genes associated with pathways related to cell cycling, stress, and iron regulation. Our data provide an explanation of the molecular underpinning of adult human light and dark cell identity and serve as a resource for investigations of epithelial heterogeneity in the CP and other organs where dark cells are found.

neuroscience↗

bioRxiv2024

Uncovering immune characteristics of neurons in the context of multiple sclerosis and experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an immune-mediated disease characterized by chronic inflammation and damage to the central nervous system, and substantial characterization of molecular signatures of glial and immune cells in the disease has been conducted. However, comparatively less well characterized is the molecular signature of pathologically inflamed neurons. Here, we accessed multi-omic high-throughput transcriptomic and epigenomic data to investigate the molecular signature of neurons from progressive MS patients and from mice subjected to experimental autoimmune encephalomyelitis (EAE). Our results indicate a shared and consistent neuronal gene expression signature in MS and EAE samples including a notable upregulation of immune system related genes and processes. Analysis of immune-enriched pathways in retinal ganglion cells (RGCs) and motor neurons from mice subjected to EAE revealed enrichment for the major histocompatibility complex class I pathway and interferon response genes in both types of neurons, and ATAC-seq analysis confirmed the accessibility of these genes. Parallel analyses of neurons from MS patients identified a signature of 48 commonly upregulated immune genes in neurons. Prediction of transcriptional regulators of these genes identified key upstream regulators including signal transducer and activation of transcription 1 (STAT1) and interferon response factor 5 (IRF5), highly involved in immune-related processes, with significant gene expression changes in both RGCs and motor neurons. Protein-protein interaction analyses among the transcriptional regulators unveiled intricate interactions among transcription factors, especially from the IRF and STAT families, suggesting their importance in regulating immune gene expression in inflamed neurons. Small molecule inhibition of IRF5 in inflamed neurons in vitro promoted cell survival and reduced the expression of the immune gene signature, supporting the biological relevance of IRF5 in mediating the neuronal inflammatory response. Together, this study identifies regulated immune processes in neurons from EAE mice and from MS patient samples suggesting potential roles for neuronal immune molecules in disease progression, as revealed by transcriptomic, epigenomic, and experimental approaches. Further, the results strongly implicate active interferon signaling in neurons driving immune related changes in gene expression through IRF5 and STAT1 transcription factor activity, potentially impacting downstream neuronal survival through IRF5 activity.

neuroscience↗

bioRxiv2023

Gaining Biological Insights through Supervised Data Visualization

Dimensionality reduction-based data visualization is pivotal in comprehending complex biological data. The most common methods, such as PHATE, t-SNE, and UMAP, are unsupervised and therefore reflect the dominant structure in the data, which may be independent of expert-provided labels. Here we introduce a supervised data visualization method called RF-PHATE, which integrates expert knowledge for further exploration of the data. RF-PHATE leverages random forests to capture intricate featurelabel relationships. Extracting information from the forest, RF-PHATE generates low-dimensional visualizations that highlight relevant data relationships while disregarding extraneous features. This approach scales to large datasets and applies to classification and regression. We illustrate RF-PHATEs prowess through three case studies. In a multiple sclerosis study using longitudinal clinical and imaging data, RF-PHATE unveils a sub-group of patients with non-benign relapsingremitting Multiple Sclerosis, demonstrating its aptitude for time-series data. In the context of Raman spectral data, RF-PHATE effectively showcases the impact of antioxidants on diesel exhaust-exposed lung cells, highlighting its proficiency in noisy environments. Furthermore, RF-PHATE aligns established geometric structures with COVID-19 patient outcomes, enriching interpretability in a hierarchical manner. RF-PHATE bridges expert insights and visualizations, promising knowledge generation. Its adaptability, scalability, and noise tolerance underscore its potential for widespread adoption.

bioinformatics↗

bioRxiv2023

Cellular rejuvenation protects neurons from inflammation mediated cell death.

In multiple sclerosis (MS), the invasion of the central nervous system by peripheral immune cells is followed by the activation of resident microglia and astrocytes. This cascade of events results in demyelination, which triggers neuronal damage and death. The molecular signals in neurons responsible for this damage are not yet fully characterized. In MS, retinal ganglion cell neurons (RGCs) of the central nervous system (CNS) undergo axonal injury and cell death. This phenomenon is mirrored in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. To understand the molecular landscape, we isolated RGCs from mice subjected to the EAE protocol. RNA-sequencing and ATAC-sequencing analyses were performed. Pathway analysis of the RNA-sequencing data revealed that RGCs displayed a molecular signature, similar to aged neurons, showcasing features of senescence. Single-nucleus RNA-sequencing analysis of neurons from human MS patients revealed a comparable senescence-like phenotype., which was supported by immunostaining RGCs in EAE mice. These changes include alterations to the nuclear envelope, modifications in chromatin marks, and accumulation of DNA damage. Transduction of RGCs with an Oct4-Sox2-Klf4 transgene to convert neurons in the EAE model to a more youthful epigenetic and transcriptomic state enhanced the survival of RGCs. Collectively, this research uncovers a previously unidentified senescent-like phenotype in neurons under pathological inflammation and neurons from MS patients. The rejuvenation of this aged transcriptome improved visual acuity and neuronal survival in the EAE model supporting the idea that age rejuvenation therapies and senotherapeutic agents could offer a direct means of neuroprotection in autoimmune disorders.

neuroscience↗

bioRxiv2021

The role of complex IV activity in the axonal response of mitochondria to demyelination in experimental disease models of multiple sclerosis

Axon loss is a cardinal neuropathological feature of multiple sclerosis (MS). Axonal injury in MS and experimental disease models is most frequently detected in acutely demyelinating regions. Furthermore, acute axonal injury consistently correlates with the extent of inflammatory demyelination. Following lysolecithin-induced demyelination, we recently reported a compensatory response in neurons, where mitochondria move from the cell body to the acutely demyelinated axon and increase the mitochondrial content. We termed this energetics phenomenon, that is also evident in MS, the axonal response of mitochondria to demyelination (ARMD). In the present study, we assessed axonal mitochondrial content as well as axonal mitochondrial respiratory chain complex IV activity (COX) of axons and related these to axonal injury in nine different experimental disease models. We consistently found ARMD in all experimental disease models. However, the increase in mitochondrial content within demyelinated axons was not always accompanied by a proportionate increase in COX activity, particularly in experimental autoimmune encephalomyelitis (EAE). Axonal COX activity inversely correlated with the extent of axonal injury in experimental disease models. Our findings indicate that ARMD is a consistent and prominent finding and emphasises the need to preserve axonal mitochondrial COX activity in inflammatory demyelination, paving the way for the development of novel neuroprotective therapies.

neuroscience↗

bioRxiv2020

Age-Associated Insolubility of Parkin in Human Midbrain is Linked to Redox Balance and Sequestration of Reactive Dopamine Metabolites

The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions, which are reflected in its posttranslational modifications. We found that in human control brain, including the S. nigra, parkin is largely insoluble after age 40 years, which is linked to its oxidation, e.g., at Cys95 and Cys253. In mice, oxidative stress increases posttranslational modifications at parkin cysteines and reduces its solubility. Oxidation of recombinant parkin also promotes insolubility and aggregate formation, but in parallel, lowers hydrogen peroxide (H2O2). This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. Intriguingly, in parkin-deficient human brain H2O2 concentrations are elevated. In prkn-null mice, H2O2 levels are dysregulated under oxidative stress conditions, such as acutely by MPTP-toxin exposure or chronically due to a second genetic hit. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic M17 cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at primate-specific Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation. In sections of normal, adult human midbrain, parkin specifically co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these redox effects may augment oxidative stress in dopamine producing neurons of mutant PRKN allele carriers, thereby contributing to neurodegeneration.

neuroscience↗

bioRxiv2020

Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

The mechanisms by which Parkinson disease-linked parkin confers neuroprotection of human dopamine cells remain elusive. We hypothesized that its cysteines mediate multiple anti-oxidant effects in the midbrain. By studying >60 control specimens, we found that in adult human brain - but not in skeletal muscle- parkin is mostly aggregated and insoluble due to oxidative modifications, such as at C253. In vitro, parkins oxidation directly reduces hydrogen peroxide (H2O2) to water. In parkin-deficient human brain, H2O2 concentrations are elevated. In dopamine toxicity studies, wild-type parkin -but not disease-associated mutants-prevents neural death by lowering H2O2 and sequestering radicals within insoluble aggregates. Parkin conjugates dopamine metabolites at the human-specific residue C95 and augments melanin formation in vitro. Using epitope-mapped antibodies, we found that in adult Substantia nigra neurons parkin localizes to neuromelanin within LAMP-3/CD63-positive lysosomes. We conclude that parkins own oxidation, previously considered a loss-of-function event, underlies three neuroprotective effects in adult midbrain: its cysteines participate in H2O2 reduction, dopamine radical conjugation and the formation of neuromelanin.

neuroscience↗

bioRxiv2019

RNA-binding protein altered expression and mislocalization in multiple sclerosis

ObjectiveNuclear depletion and mislocalization of RNA-binding proteins (RBPs) trans-activation response DNA-binding protein of 43 kDa (TDP-43) and fused in sarcoma (FUS) are thought to contribute to the pathogenesis of a number of disorders, including amyotrophic lateral sclerosis (ALS). We recently found that TDP-43 as well as polypyrimidine tract binding protein (PTB) have decreased expression and mislocalization in oligodendrocytes in demyelinated lesions in an experimental mouse model of multiple sclerosis (MS) caused by Theilers murine encephalomyelitis virus infection.\n\nMethodsThe latter finding prompted us to investigate TDP-43, FUS, and PTB in the demyelinated lesions of MS and in in vitro cultured human brain-derived oligodendrocytes.\n\nResultsWe found: i) mislocalized TDP-43 in oligodendrocytes in active lesions in some MS patients; ii) decreased PTB1 expression in oligodendrocytes in mixed active/inactive demyelinating lesions; iii) decreased nuclear expression of PTB2 in neurons in cortical demyelinating lesions; iv) nuclear depletion of TDP-43 in oligodendrocytes under metabolic stress induced by low glucose/low nutrient conditions compared to optimal culture conditions.\n\nConclusionTDP-43 has been found to have a key role in oligodendrocyte function and viability, while PTB is important in neuronal differentiation, suggesting that altered expression and mislocalization of these RBPs in MS lesions may contribute to the pathogenesis of demyelination and neurodegeneration. Our findings also identify nucleocytoplasmic transport as a target for treatment.

neuroscience↗

bioRxiv2019

Multiplexed imaging of immune cells in staged multiple sclerosis lesions by mass cytometry

Multiple Sclerosis (MS) is characterized by demyelinated and inflammatory lesions in the brain and spinal cord. Lesions contain immune cells with variable phenotypes and functions. Here we use imaging mass cytometry (IMC) to enable the simultaneous imaging of 15+ proteins within 11 staged MS lesions. Using this approach, we demonstrated that the majority of demyelinating macrophage-like cells in active lesions were derived from the resident microglial pool. Although CD8+ T cells predominantly infiltrated the lesions, CD4+ T cells were also abundant but localized closer to blood vessels. B cells with a predominant switched memory phenotype were enriched across all lesion stages and were found to preferentially infiltrate the tissue as compared to unswitched B cells which localized to the vasculature. We propose that IMC will enable a comprehensive analysis of single-cell phenotypes, their functional states and cell-cell interactions in relation to lesion morphometry and demyelinating activity in the MS brain.

immunology↗